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Ion movements during contraction of the guinea pig ileum longitudinal smooth muscle James, Marilyn Rosamond 1977

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ION  MOVEMENTS  DURING  CONTRACTION  GUINEA P I G ILEUM LONGITUDINAL  OF  SMOOTH  THE MUSCLE  by MARILYN B.Sc,  State  ROSAMOND  University  A THESIS  SUBMITTED  THE  o f New  York  IN P A R T I A L  REQUIREMENT  DOCTOR  JAMES  FOR  OF  at B u f f a l o ,  F U L F I L L M E N T OF  THE D E G R E E  OF  PHILOSOPHY  in THE THE  GRADUATE S T U D I E S in F A C U L T Y OF P H A R M A C E U T I C A L S C I E N C E S  We  F A C U L T Y OF  accept to  this  thesis  the required  as  conforming  standard  /  THE  U N I V E R S I T Y OF B R I T I S H June,  1977  <6) Marilyn Rosamond James  COLUMBIA  1972  In  presenting  an  advanced  the L i b r a r y I  further  for  of  this  written  thesis  degree shall  agree  scholarly  by h i s  this  at  the U n i v e r s i t y  make  that  it  purposes  for  freely  permission may  representatives. thesis  in p a r t i a l . f u l f i l m e n t  financial  is  of  British  by  gain  Columbia  shall  the  that  not  requirements  Columbia,  I  agree  r e f e r e n c e and copying  t h e Head o f  understood  Depa r t m e n t  2075 Wesbrook Place Vancouver, Canada V6T 1W5  for  for extensive  permission.  The U n i v e r s i t y  British  available  be g r a n t e d  It  of  of  of  this  be a l l o w e d  or  that  study. thesis  my D e p a r t m e n t  copying  for  or  publication  without  my  i ION MOVEMENTS DURING CONTRACTION OF THE GUINEA PIG ILEUM LONGITUDINAL SMOOTH MUSCLE by MARILYN ROSAMOND JAMES The excitation-contraction-relaxation cycle of the guinea pig ileum l o n g i tudinal smooth muscle was studied i n muscles contracted by a muscarinic agent, cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide (CD) and by 60 mM KC1.  Aspects of the cycle were investigated by analyzing the active transport  enzyme a c t i v i t i e s i n the sarcolemma, the tissue Ca depots which could  release  Ca for contraction and the s e n s i t i v i t y of the c o n t r a c t i l e responses to extrac e l l u l a r ion changes.  E s s e n t i a l l y net changes of i n t r a c e l l u l a r Ca, Mg, Na and  K content during contractions were measured by a modified  'La method'.  The  tissues were washed for 30 min i n 160 mM Tris-HCl solution (pH 7.4) containing 10 mM LaCl3 at 4°C i n order to seal the i n t r a c e l l u l a r ions i n the c e l l and d i s place e x t r a c e l l u l a r ions. stance by reducing  A method to loosen the ' i n t e r c e l l u l a r cementing' sub-  the tissue Ca and Mg was developed as an adjunct to the pre-  paration of a sarcolemmal enriched microsomal f r a c t i o n .  The method reduced the  tenacity of the tissue and made the tissue easy to disrupt by a mild homogenizing procedure. proteins.  The method also appeared to a i d the extraction of c o n t r a c t i l e  The microsomal f r a c t i o n was not detectably contaminated by mitochon-  d r i a and was enriched with v e s i c l e s of sarcolemma, probably o r i g i n a t i n g from the muscle caveolae.. The sarcolemma enriched microsomal f r a c t i o n had a Ca-ATPase a c t i v i t y  that  -7 -4 2+ was progressively stimulated by 10 to 2.4 x 10 M free Ca , did not require Mg and was i n h i b i t e d by La. contamination by actomyosin.  The microsomal Ca-ATPase a c t i v i t y was not due to The actomyosin Ca-r-ATPase i n the soluble f r a c t i o n  had a higher a f f i n i t y than the microsomal Ca-ATPase for Ca and for La. microsomal Ca-ATPase a c t i v i t y was postulated  The  to be associated with an active Ca  ii pump thought to he located ;Ln the cayeolae,  xhe microsomal f r a c t i o n had a  Mg-dependent ATPase that could Be stimulated by Na, but K and ouabain had very l i t t l e additional e f f e c t .  The addition of an a c t i v a t i n g factor i n the soluble  f r a c t i o n conferred some K and ouabain s e n s i t i v i t y to the Mg-dependent NaATPase, which indicated that a Na,K^ATPase was present i n this tissue.  Low  doses of ouabain contracted the l o n g i t u d i n a l ileum but the responses were not antagonized  by r a i s i n g the external K concentration f i v e f o l d , as would be  expected i f ouabain acted by i n h i b i t i n g the Na,K-ATPase.  However, the ouabain  response was rapidly l o s t when e x t r a c e l l u l a r Ca was removed from,the medium and the decline of the response followed the same time course as the loss of e x t r a c e l l u l a r Ca.  The peak of the ouabain contraction coincided with  signifi-  cant increases of i n t r a c e l l u l a r Ca and Na, but K loss was not apparent u n t i l relaxation ensued.  The results suggested that ouabain has an early d i r e c t  e f f e c t on membrane permeability before i t i n h i b i t e d the Na,K-ATPase. CD (2 x 10 ^ M) and 60 mM KC1 induced phasic and tonic contractions of the longitudinal muscle of the ileum.  The phasic contraction declined from 100%  to 7% over 10 min when Ca was omitted from the p h y s i o l o g i c a l medium. decline followed the time course of the loss of e x t r a c e l l u l a r Ca.  This  This, to-  gether with the fact that low concentrations of LaCl^ inhibited the phasic component, indicated that Ca bound to the outer aspect of the c e l l was respons i b l e for the phasic component.  The tonic component was l o s t more rapidly  than the phasic component when the Ca was removed from the Tyrode's solution. The tonic component seemed activated by free Ca mobilized from the e x t r a c e l l u l a r space.  The e x t r a c e l l u l a r o r i g i n of the Ca f o r contraction was consistent  with the observed small net gain of i n t r a c e l l u l a r Ca that occurred during the phasic and tonic contractions.  The minimal volume of the sarcoplasmic  reti-  culum and the abundance of caveolae was also consistent with the high sensit i v i t y of the tissue to e x t r a c e l l u l a r Ca concentrations.  The i n t r a c e l l u l a r Ca  gained during contraction wa,s extruded within 30 sec after the CD or 60 mM  KC1  were washed out of the tissue bath, Following washout of CD, 'equilibration' phase. tension was  quiescent  Spontaneous a c t i v i t y was  below baseline.  to CD or to 60 mM KC1  the muscle was  min  absent during this phase and  After a maximal CD contraction, a second response  induced during the ' e q u i l i b r a t i o n ' phase had an altered  or desensitized biphasic appearance.  Responses of the muscle to CD for 10  were accompanied by a cytoplasmic 1 oss of K. regained  for the 20 to 30  slowly over 20 to 30 min.  After washout of CD,  the K  min  was  Stimulation of the tissue by 60 mM KC1  did  not cause a loss of K from the muscle nor did i t cause desensitization of the muscle.  Higher e x t r a c e l l u l a r K concentrations  decreased the time required  a f t e r CD contractions for the return of spontaneous a c t i v i t y and prevented muscle desensitization to repeated doses of CD, return of i n t r a c e l l u l a r K l e v e l s to normal.  probably by accelerating the  I t was  proposed that during con-  traction, elevated i n t r a c e l l u l a r Ca activated K channels, thereby increasing K permeability and causing the 'after-hyperpolarization' and subsequent desens i t i z a t i o n which follows muscarinic induced contractions.  TABLE OF CONTENTS page ABSTRACT  i  LIST OF TABLES  vii  LIST OF FIGURES  viii  LIST OF MICROGRAPH PLATES  xii  LIST OF ABBREVIATIONS  xiii  INTRODUCTION I. II.  The Reasons f o r S t u d y i n g the Guinea P i g Ileum L o n g i t u d i n a l Muscle.  Smooth  1  Methods o f S u b c e l l u l a r F r a c t i o n a t i o n o f Smooth Muscle Relevant to the P r e s e n t Study  3  a. Homogenization t e c h n i q u e s f o r smooth muscle  3  b. Common procedures f o r s e p a r a t i o n  5  c III.  1  Identification  of s u b c e l l u l a r f r a c t i o n s  of s u b c e l l u l a r f r a c t i o n s  S t u d i e s o f the Na,K-ATPase  6  i n Smooth Muscle  '  IV. S t u d i e s o f Ca-ATPases i n Smooth Muscle  7  V. Methods f o r Measuring Ion Movements i n Smooth Muscle  8  a. C a l c u l a t i o n o f i n t r a c e l l u l a r i o n l e v e l s f o r t o t a l i o n measurements and f l u x s t u d i e s b. The 'La Method'  13  C o u p l i n g (General)  V I I I . Sources o f C a l c i u m f o r E x c i t a t i o n - C o n t r a c t i o n Guinea P i g Ileum L o n g i t u d i n a l Smooth Muscle  14 C o u p l i n g i n the  17  IX. Receptor Theory and the D e s e n s i t i z a t i o n Phenomenon  21  X. Aims o f the P r e s e n t Study METHODS  9 10  VI. The Mechanism o f A c t i o n o f Ouabain VII. Excitation-Contraction  6  22  '  .  I.  D i s s e c t i o n o f the L o n g i t u d i n a l  II.  C o n t r a c t i l e Force Measurements  23  a. I s o m e t r i c f o r c e measurements  23  b. Normal Tyrode's s o l u t i o n  23  c. T r i s Tyrode's s o l u t i o n  26  d. ' G l y c e r o l shock' treatment  26  Measurement o f Muscle Na, K, Mg and Ca  27  a. T o t a l i o n measurement  27  b. M o d i f i e d 'La method' f o r measuring e s s e n t i a l l y i n t r a c e l l u l a r Na, K, Mg and Ca  27  III.  L a y e r o f the Guinea P i g Ileum  23 23  page 28  c. Extraction of ions from tissues ( t o t a l and i n t r a c e l l u l a r ion measurements) and atomic absorption measurements IV. Preparation of Sarcolemmal Enriched.!Microsomes  30  V. Measurement of ATPase and Membrane Marker Enzyme A c t i v i t i e s  30  a. Mg dependent Na,K-ATPase  30  b. Microsomal Ca-ATPase  31  c. Actomyosin Ca-ATPase  31  d. 5'-Nucleotidase  31  e. Acetylcholinesterase  ,  32  f. NADH Oxidase  32  g. Succinic Dehydrogenase  32  VI. Inorganic Phosphate Determination  33  VII. Protein Determination  33  VIII. Electron Microscopy  33  a. Electron microscopy - whole tissues  33  b. Electron microscopy - microsomal and mitochondrial p e l l e t s  34  c. Sectioning and s t a i n i n g  34  MATERIALS  36  RESULTS AND DISCUSSION  3?  I. Procedure for I s o l a t i n g Sarcolemmal Enriched Microsomes I I . Mg dependent Na,K-ATPase I I I . Ca-ATPases  •  37 51 73  a. Microsomal Ca-ATPase  73  b. Actomyosin Ca-ATPase  75  c. Comparison of the divalent cation stimulated ATPase a c t i v i t i e s  75  IV. The Structure of the Guinea Pig Ileum Longitudinal Smooth Muscle V. The Modified 'La Method' VI. The E f f e c t of Ouabain on the Intact Longitudinal Smooth Muscle of the Guinea Pig Ileum V l t . Possible Sources of Ca for the Phasic and Tonic Contractions of the Guinea Pig Ileum Longitudinal Smooth Muscle  89 108 121 138  a. The biphasic contraction  _3 9  b. S e n s i t i v i t y of the phasic and tonic contractions to Ca-free medium  _3 9  c. S e n s i t i v i t y of the phasic and tonic contractions to LaCl^  141  d. Measurements of i n t r a c e l l u l a r ion contents during contractions induced by CD and 60 mM KC1  143  VIII. The 'Desensitization' Phenomenon  '  vi page SUIVMARY AND CONCLUSIONS  223 •  REFERENCES  22 6 •  APPENDIX  2.4-0  Tables for Figures, (see L i s t of Figures) 2AO—259 2+ Appendix Table 1. Compilation of free Ca concentrations 260 2+ Calculation of the free Ca concentrations under various conditions 261 2+ A. Calculation of the free Ca concentration i n the presence of 261 3 mM MgCl and 3 mM ATP (no EGTA) 2+ B. Calculation of the free Ca concentration i n the presence of 263 3 mM ATP (no MgCl-, no EGTA) 2+ C. Calculation of the free Ca concentration i n the presence of 264 0.1 mM EGTA 2+ a) Calculation of the free Ca concentration i n the presence of 264 3 mM MgCl_, 3 mM ATP, and 0.1 mM EGTA 2+ b) Calculation of the free Ca concentration i n the presence of 26'5 3 mM ATP and 0.1 mM EGTA  vii LIST OF TABLES Table  Title  . page  1  Atomic^absorption standards and calculations for u g element i n tissue samples  29  2  Location of c e l l marker enzymes, s p e c i f i c a c t i v i t i e s and % recoveries i n each c e l l f r a c t i o n  47  3  Subcellular d i s t r i b u t i o n of ATPase a c t i v i t i e s  48  4  Cation levels i n the microsomal f r a c t i o n  58  5  Calculation of i n t r a c e l l u l a r and e x t r a c e l l u l a r ion concentrations  117  6.  Calculation of the resting membrane p o t e n t i a l  118  viii LIST OF FIGURES Fig.  Appendix page page 20  1.  Superimposed t r a c i n g s o f time c o u r s e s o f c o n t r a c t i o n s showing the e f f e c t o f d i f f e r e n t Ca c o n c e n t r a t i o n s f o r e q u i l i b r a t i o n [ C a g ^ r - ] - . and s t i m u l u s [ C a ^ _ J _ d u r i n g e x c i t a t i o n by CD (2 x 10~^M) (reproduced from Chang and T r i g g l e 1972).  2.  D i s s e c t i o n o f i n n e r v a t e d and denervated guinea p i g i l e u m l o n g i t u d i n a l smooth muscle  25  3.  C a t i o n l e v e l s o f the guinea p i g i l e u m l o n g i t u d i n a l . s m o o t h muscle a f t e r d e p l e t i o n o f the t i s s u e i n Ca,Mg-free Tyrode's s o l u t i o n as used i n the p r o c e d u r e f o r p r e p a r i n g sarcolemmal e n r i c h e d m i c r o somes  42  4.  Sucrose d e n s i t y g r a d i e n t treatment o f the microsomes  50  5.  S t i m u l a t i o n o f microsomal ATPase by Mg,  57  241  6.  The e f f e c t o f Mg, Na, K, ouabain and a s o l u b l e a c t i v a t i n g f a c t o r on the microsomal ATPases  60  242  7.  Upper  S u b s t r a t e dependence  62  Lower  Bar graph comparing the u t i l i z a t i o n o f ADP w i t h ATP  Na and K  curve o f microsomal ATPase  Na or 100 mM  240  62  8.  Lineweaver Burk p l o t o f the e f f e c t of 100 mM 3 mM K on the Mg-ATPase  Na p l u s  9.  Assay of the Mg-ATPase w i t h ( y - P ) l a b e l l e d ATP  66  10.  The e f f e c t o f Ca on the Mg dependent Na,K-ATPase  68  11.  P h a r m a c o l o g i c a l t e s t s o f the t i s s u e v i a b i l i t y under the b i o c h e m i - 70 c a l c o n d i t i o n s used to p r e p a r e sarcolemmal e n r i c h e d microsomes, as a check f o r the p o s s i b l e l o s s o f Na,K-ATPase  12.  P h a r m a c o l o g i c a l t e s t o f the muscle v i a b i l i t y a f t e r treatment i n Ca,Mg-free Tyrode's s o l u t i o n  72  13.  Sarcolemmal e n r i c h e d microsomal Ca-ATPase a c t i v i t y i n response to free C a  80  14.  The e f f e c t s o f ions on the microsomal Ca-ATPase  82  15.  The e f f e c t o f L a on the microsomal Ca-ATPase and the actomyosin Ca-ATPase  84  Top S u b s t r a t e dependence t i o n by L a C l 3  84  245  84  245  3 2  64  243  244  244  2 +  o f the microsomal Ca-ATPase and i n h i b i -  Bottom Response o f the s o l u b l e f r a c t i o n ATPase increasing free C a  (actomyosin) to  2 +  16.  C h a r a c t e r i s t i c s o f the actomyosin Ca-ATPase  86  Top ATP  86  The c o n c e n t r a t i o n dependence  Bottom  o f the actomyosin Ca-ATPase on  Actomyosin Ca-ATPase a c t i v i t y i n d i f f e r e n t b u f f e r systems  17.  Lineweaver Burk p l o t o f the s u b s t r a t e dependence and actomyosin Ca-ATPases  18.  An example o f the e f f e c t o f ' g l y c e r o l shock' treatment on the guinea p i g i l e u m l o n g i t u d i n a l smooth muscle  86  o f the microsomal 88 .. 107  246  ix  Appendix Fig. 19.  page Top Measurement o f the time r e q u i r e d f o r 10 mM L a C l 3 i n Caf r e e Tyrode's s o l u t i o n (pH/,7.4) a t 4°C to d i s p l a c e e x t r a c e l l u l a r Ca  114  Bottom Measurement of Ca, Mg, Na and K l e v e l s o f the guinea p i g i l e u m l o n g i t u d i n a l smooth muscle over time i n 160 mM T r i s - H C l (pH 7.4) c o n t a i n i n g 10 mM L a C l a t 4°C  114  20.  Measurement o f the t i s s u e i o n c o n t e n t and e s t i m a t i o n o f the r a t i o of the t i s s u e wet weight to the t i s s u e dry weight  116  21.  Measurement o f the t i s s u e i o n c o n t e n t s by the m o d i f i e d 'La method' a f t e r exposure o f the guinea p i g i l e u m l o n g i t u d i n a l smooth muscle s t r i p s to T r i s - T y r o d e ' s s o l u t i o n c o n t a i n i n g 3.6 mM CaCl„ f o r 0.5 min (B) and f o r 5 min (C) as compared t o c o n t r o l s (A; e q u i l i b r a t e d i n T r i s - T y r o d e ' s s o l u t i o n (1.8 mM CaCl-)  120  22.  The e f f e c t o f ouabain on i n n e r v a t e d and denervated guinea p i g i l e u m l o n g i t u d i n a l smooth muscle  129  23.  The e f f e c t o f i n c r e a s e d e x t r a c e l l u l a r K on c o n t r a c t i o n , 5 yM ouabain  131  24.  Top Responses to 60 mM KC1 a t the peak o f the e x c i t a t o r y ponse to ouabain and d u r i n g the i n h i b i t o r y phase  page  247  3  Bottom The e f f e c t o f 10 uM ouabain on a 60 mM and a 0.2 uM response to CD  KC1  by res-  response  248  133 133  25.  I n t r a c e l l u l a r i o n l e v e l s d u r i n g the course o f an ouabain r e s ponse  135  249  26.  The s e n s i t i v i t y o f the ouabain response to Ca removal  137  250  27.  Log dose response curve to cis-2-methyl-4-dimethylaminomethyl1,3-dioxolane m e t h i o d i d e (CD)  152  250  28.  The responses o f the guinea p i g i l e u m l o n g i t u d i n a l smooth muscle to i n c r e a s i n g doses of CD  154  29.  The response o f l o n g i t u d i n a l i l e a l muscle  156  30.  Top A comparison o f p h a s i c and t o n i c responses i n normal Tyrode's s o l u t i o n and a f t e r s w i t c h i n g to C a - f r e e Tyrode's s o l u t i o n f o r 5 sec b e f o r e the a d d i t i o n of 2 x 1 0 ^ M CD or 60 mM KC1  158  Bottom The l o s s o f the p h a s i c component when CD and h i g h KC1 are added a f t e r v a r i o u s times i n C a - f r e e Tyrode's s o l u t i o n  158  31.  The s e n s i t i v i t y o f responses to 60 mM KC1 and to 2 x 1 0 ~ M CD to the removal o f Ca from the l o n g i t u d i n a l i l e a l smooth muscle f o r 10 min and r e s t o r a t i o n o f Ca f o r 30 sec  160  32.  The e f f e c t o f i n c u b a t i o n o f t i s s u e s i n C a - f r e e Tyrode's s o l u t i o n f o r v a r i o u s times on t o t a l and e s s e n t i a l l y i n t r a c e l l u l a r t i s s u e ion levels  162  252  33.  The r a t e o f l o s s o f the p h a s i c component compared to the r a t e of l o s s of e s s e n t i a l l y i n t e r n a l and e x t e r n a l Ca  164  253  34.  The e f f e c t o f L a C l (10~ to 10~ M) (6.0 mM) i n T r i s - T y r o d e " s s o l u t i o n  166  to methacholine  251  -  7  3  on responses t o h i g h KC1  X  Fig.  page  Appendix page  35.  Comparison of the effect of LaCl-^ on the phasic and tonic component of responses induced by 60 mM KC1 and 2 x 10*""'' M CD  168  254  36.  I n t r a c e l l u l a r ion levels during the course of a contraction induced by 60 mM KC1 and the e q u i l i b r a t i o n i n Tris-Tyrode's solution  170  255  37.  I n t r a c e l l u l a r ion levels during the course of a contraction induced by 2 x 10~7 M CD and the e q u i l i b r a t i o n phase i n T r i s Tyrode 's solution  182  256  38.  The effect of reducing e x t r a c e l l u l a r Na on the longitudinal i l e a l muscle a c t i v i t y  184  39.  Changes of the biphasic c o n t r a c t i l e pattern when contractions to CD were induced at shorter e q u i l i b r a t i o n time intervals between contractions  192  40.  Graphical representation of the e f f e c t of the e q u i l i b r a t i o n time on contractions by CD  194  257  41.  The effect of the e q u i l i b r a t i o n time allowed a f t e r a 10 min exposure to CD  196  258  42.  The effect of a very short e q u i l i b r a t i o n time (30 sec) on a CD response after a long exposure to CD (35 min)  198  43.  The lack of effect of the e q u i l i b r a t i o n time allowed between responses to 60 mM KC1 on the biphasic shape of a series of responses  200  44.  The effect of the e q u i l i b r a t i o n time allowed after a 10 min response to carbachol on a second contraction induced by carbachol  202  45.  ..The effect of a 5 min or 10 min e q u i l i b r a t i o n time after a response of the muscle to methacholine on the biphasic appearance of a second response to methacholine  204  46.  'The e f f e c t of the duration of exposure to CD on the the time required for e q u i l i b r a t i o n , monitored by the return of spontaneous a c t i v i t y and the subsequent return of the normal biphasic c o n t r a c t i l e pattern  206  47.  The effect of a.: 10.'.min..exposure:"to..CD..otioa'.'response to 60 mM KC1, 2, 4, 6, 8 and 12 min after the response to CD was washed out  208  48.  Graphical representation of the e f f e c t of the time allowed for e q u i l i b r a t i o n after a response to CD on a response to 60 mM KC1  210  49.  The effect of a simultaneous addition of 60 mM KC1 and / x 10" M CD on the e q u i l i b r a t i o n phase of the muscle  212  50.  The e f f e c t of a l t e r i n g the concentration of KC1 i n the Tyrode's solution on the rate of return of spontaneous a c t i v i t y after a response to CD  214  51.  The s p e c i f i c i t y of increased external KC1 f o r accelerating the return of the usual biphasic c o n t r a c t i l e pattern compared to the lack of e f f e c t of higher e x t r a c e l l u l a r NaCl concentration  216  258  7  259  xi  Fig.  page  52.  The e f f e c t of various agents on the time required by the muscle for the return of spontaneous a c t i v i t y after a 10 min response to CD  218  53.  The effect of r a i s i n g the e x t r a c e l l u l a r Ca and Mg concentrations on the time required by the muscle to regain spontaneous a c t i vity  220  54.  The effect of depriving the muscle of dextrose and oxygen on the time required for the return of spontaneous a c t i v i t y  222  Appendix page  X I X  LIST OF MICROGRAPH PLATES Plate #  page  1  1  Mitochondria, 27,000 x g p e l l e t  44  1  2  Mitochondria, 27,000 x g p e l l e t  44  1  3  Mitochondria, 27,000 x g p e l l e t  44  2  4  Microsomes, 105,000 x g p e l l e t  46  2  5  Microsomes, 105,000 x g p e l l e t  46  2  6  Microsomes, 105,000 x g p e l l e t  46  3  7  Rolled l o n g i t u d i n a l ileum s t r i p  101  3 3  8 9  Auerbach's Myenteric nerve plexus Longitudinal section of l o n g i t u d i n a l i l e a l smooth muscle c e l l s (light microscope)  101 101  3  10  Longitudinal section (electron microscope)  101  3  11  Cross section of longitudinal i l e a l smooth muscle c e l l s ( l i g h t microscope)  101  3  12  Cross section (electron microscope)  10jl  4  13  Nucleus, sarcoplasmic reticulum, golgi apparatus  (perinuclear  103  region) 4  14  Enlargement of the perinuclear region  103  4  15  Nuclei and n u c l e o l i  103  4  16  S p i r a l indentations of n u c l e i i n a contracted x e l l  103  4  17  Nucleus i n contracted c e l l  103  4  18  Mitochondria drawn into folded nucleus  103  5  19  Subsarcolemmal membrane sacs  105  5  20  Subsarcolemmal sacs between sarcolemma and mitochondria  105  5  21  Masses of caveolae  105  5  22  Aggregate of caveolae  105  5 5  23 24  Enlargement of caveolae Rough endoplasmic reticulum, mitochondria and agranular membranes  105 105  5  25  Smooth subsarcolemmal sacs (sarcoplasmic reticulum)  105  .xiii  LIST  OF ABBREyIATONS  Acha s e  acetylcholine esterase  ADP  adenosine. 5 ' - d i p h o s p h a t e  5'-AMP  adenosine  5'-monophosphate  ATP  adenosine  5'-triphosphate  Ca-free Tyrode's  solution  Tyrode's  solution  from w h i c h Ca was  solution  from w h i c h Ca and  omitted Ca,Mg-free Tyrode's  s o l u t i o n Tyrode's  Mg were o m i t t e d Cch  carbachol  CD  cis-2-methyl-4-dimethylaminomethyl1,3-dioxolane  methiodide  DTNB  5,5'  EDTA  ethylenediamine  EGTA  ethyleneglycol-bis-(g-aminoethyl  dithio  bis-(2-nitro  tetraacetic  N,N'-tetraacetic La-Tris  solution  160  mM  Tris-HCl  10 mM L a C l  3  Mch  methacholine  NADH  nicotinamide (reduced  Na,K-ATPase  +  benzoic acid) acid ether)  acid  (pH 7.4) c o n t a i n i n g  a t 4°C  adenine  dinucleotide  form)  +  Na and K stimulated adenosine t r i p h o s p h a t a s e . (ATP p h o s p h o h y d r o l a s e E.G.3.6.1.3)  neo  neo 3feiggiirie:  nic  nicotine.  NT  normal  ouab  ouabain  P.  inorganic  X  Tyrode's  solution  phosphate  TCA  trichloroacetic  TT  Tris  buffered  Tris  Tris  (hydroxymethyl)  W  washout  acid  Tyrode's  solution  (pH 7.4)  aminomethane  o f s t i m u l a n t from muscle  bath  ACKNOWLEDGEMENT  I wish t o thank Dr.  B a s i l D.  Roufogalis  f o r h i s i n t e r e s t i i i the p r o j e c t  and  h i s a d v i c e , as w e l l as h i s f r i e n d s h i p . I am  g r a t e f u l to the M e d i c a l R e s e a r c h C o u n c i l of Canada f o r t h e i r f i n a n c i a l .  support  of the p r o j e c t and  I appreciate  the time and  (Dr. B. R o u f o g a l i s ,  myself. e f f o r t g i v e n by my  s u p e r v i s o r ; Dr.  F. Abbott, Dr.  Dr. J . M c N e i l l , Dean B. R i e d e l , Dr. M. examiner) d u r i n g meetings and  and Mr.  S u t t e r , Dr.  S. K a t z , Dr.  G. Kracke were g r e a t l y  I have enjoyed my  Helen  S u t t e r and Dr.  T r i g g l e , Dr. C R .  E. D a n i e l ,  Dr.  S.  Katz,  external  T r i g g l e , Dr.  V.  Palaty,  Wolowyk, Mr.  L. Veto  appreciated.  graduate student  days a t the U n i v e r s i t y of  British  concern of many f r i e n d s .  t h a n k f u l f o r the t y p i n g and p r o o f r e a d i n g Butt.  G. B e l l w a r d ,  Z. Chmielewicz, Dr. M.  Columbia thanks to the warmth and I am  committee members  f o r the r e a d i n g of the f i n a l t h e s i s .  H e l p f u l d i s c u s s i o n s w i t h Dr. D.J. Dr. M.  research  a s s i s t a n c e of June Lam  and  DEDICATION  To Mum, me  Dad,  J a n e t and  and, above a l l , f o r  Ian f o r t h e i r their  l o v e and  confidence i n encouragement.  1 INTRODUCTION i  Knowledge has steadily been accumulating about the sequence of events leading to contraction of smooth muscle.  Controversy s t i l l exists about the nature  of the events and their exact order.  Perhaps some of the controversy i s due to  the fact that d i f f e r e n t types of smooth muscles exhibit quite d i f f e r e n t characteristics.  I t now appears that many drugs a l t e r more than one phase of smooth  muscle a c t i v i t y .  When we have a better knowledge of the muscle, perhaps we w i l l  be better able to understand how drugs affect contraction. The reasons  why  diseased muscles work imperfectly may be resolved when we a t t a i n a better knowledge of the normal working of the muscle.  In recent years more attention has  been paid to the effects of various stimulants on membrane permeability and c e l l u l a r ion d i s t r i b u t i o n . on enzymatic processes.  Others have been searching for effects of stimulants  Perhaps stimulants cause a combination of permeability  changes and enzymatic a l t e r a t i o n s , each of these actions having an effect on the other.  The objective of this study was  to investigate certain aspects of  the guinea pig ileum longitudinal smooth muscle contracture, structure and biochemistry i n order to attempt to answer a few key questions.  I.  The Reasons for Studying the Guinea Pig Ileum Longitudinal Smooth Muscle The guinea p i g ileum longitudinal smooth muscle preparation (mainly one  muscle layer and Auerbach's nerve plexus) has several advantages over the whole ileum preparation (three muscle layers, two nerve plexuses and a mucosal membrane) by v i r t u e of being s t r u c t u r a l l y less complex.  Most other struc-  t u r a l l y complex organs, composed of smooth muscle, can not be separated as easily into layers.  The longitudinal layer of the ileum i s c h a r a c t e r i s t i c a l l y  d i f f e r e n t from many other smooth muscle tissues i n that each c e l l i s not i n d i v i d u a l l y innervated (Holman 1970).  However there are non-adrenergic arid non-  cholinergic neurons which may penetrate i n t e s t i n a l muscle layers (Furness and Costa 1973).  Nevertheless, e s s e n t i a l l y denervated s t r i p s which do not respond  2 to f i e l d stimulation can be obtained (Paton and Zar 1965) but the d i f f i c u l t y of the dissection procedure l i m i t s t h e i r use to special experiments. layer i s very thin ( i n the order of 50;,u  The muscle  thick, Paton 1975a) and i t s thinness  presents very l i t t l e impediment to the d i f f u s i o n of pharmacological agents through the tissue (Paton and Rang 1965).  Because the e x t r a c e l l u l a r f l u i d  rapidly equilibrates with physiological solutions of various compositions, the guinea pig ileum longitudinal smooth muscle has proven useful i n studies of muscle i o n i c fluxes (Weiss et a l . 1961; Lullman and Siegfriedt 1968; Hurwitz and Joiner 1969; Lullman and Mohn 1971; Weiss 1972).  The longitudinal muscle  s t r i p s , as usually prepared, are composed of 75% longitudinal smooth muscle c e l l s , approximately 18% nervous tissue and serosa (primarily fibrous l a r material) and the remaining  noncellu-  7% i s connective tissue and a few adhering .  c i r c u l a r muscle layer c e l l s (Weiss et a l . 1961).  Therefore changes i n ion levels  of the tissue can be assumed to r e f l e c t changes i n i o n levels of the longitudinal smooth muscle c e l l s .  One of the disadvantages of the preparation i s that the  tissue can be damaged during the f a i r l y extensive dissection procedure and increases  i n c e l l Ca and Na and losses of K down their concentration gradients  could result from c e l l damage rather than from agonist induced changes.  How-  ever, despite what appears to be harsh treatment of the muscle during the dissection procedure, the i s o l a t e d longitudinal ileum contracts very well to muscarinic agonists, high concentrations of KC1 and various other s t i m u l i and remains viable for many hours.  Weiss et a l . (1961) found that potassium fluxes  induced by acetylcholine were s i m i l a r and usually greater i n the isolated longitudinal layer than i n the whole ileum.  Lullman and Siegfriedt (1968)  observed that the Ca content of longitudinal i l e a l c e l l s varied s l i g h t l y , depending on the location along the i n t e s t i n e .  This i s another disadvantage that  might obscure changes of Ca levels between control and experimental s t r i p s . These disadvantages make i t necessary to study many muscle s t r i p s to obtain representative  control and experimental values, but one of the advantages of  3 the preparation i s that 16 to 20 muscle .strips can be obtained from each animal. Longitudinal i l e a l c e l l s are one of the smaller fibres of the smooth muscle type and the volume of each c e l l has been estimated to be that of a cylinder 5 p i n diameter and 50 u i n length (Paton and Rang 1965).  Most of the contrac-  t i l e filaments are not much farther than 1-2 u from the e x t r a c e l l u l a r space. The sarcoplasmic reticulum i s probably less than 2% of the t o t a l c e l l u l a r volume (similar to taenia c o l i c e l l s which are estimated to have 2% sarcoplasmic r e t i culum) (Devine et a l . 1972). surface area. ditions) may  The c e l l volume i s small compared to the large  Permeable or transiently permeable ions (under stimulation conexchange rapidly across the large surface area making longitudinal  i l e a l c e l l s very sensitive to i o n i c changes i n their i n t e r s t i t i a l fore these c e l l s may  space.  There-  serve as possible models for testing the hypothesis that  excitation-contraction coupling i n some types of smooth muscle i s regulated by trans-sarcolemmal storage s i t e s .  fluxes of Ca rather than by release of Ca from i n t r a c e l l u l a r  An advantage of the longitudinal layer of the ileum for i s o l a -  tion of a sarcolemmal f r a c t i o n i s that i t has less connective tissue than other types of smooth muscle and a r e l a t i v e lack of sarcoplasmic reticulum, mitochond r i a and lysosomes (Burnstock 1970).  Therefore i s o l a t i o n of a sarcolemmal frac-  tion, without major contamination by i n t r a c e l l u l a r organelles may be more feasible.  II.  Methods of Subcellular Fractionation of Smooth Muscle Relevant to the Present Study. a.  Homogenization techniques for smooth muscle  One of the f i r s t , and perhaps one of the most c r i t i c a l . s t e p s i n preparing c e l l fractions for biochemical analysis i s homogenization. can be extremely d i f f i c u l t  This routine step  i n tissues such as smooth muscles that are i n t e r -  woven with large amounts of connective tissue.  The various procedures for  homogenizing smooth muscle c e l l s have been c r i t i c a l l y  reviewed by Kidwai  (1975).  Although the Potter-Elvehjem homogenizer i s the most widely used because i t  .  4  causes the least organelle breakage, i t disrupts only a small percentage of the strongly cohesive smooth muscle c e l l s .  Prolonged homogenization increases the  c e l l breakage but also breaks mitochondria and n u c l e i .  A l l - g l a s s homogenizers  disrupt the tissue faster but glass p a r t i c l e s may contaminate the homogenate. The S o r v a l l Omnimixer or mortar and pestle methods can thoroughly disrupt the tissue for extraction of soluble proteins but are generally less suitable for studies leading to subcellular fractionation since they break the i n t r a c e l l u l a r organelles.  The Polytron homogenizer, when used at c a r e f u l l y chosen speeds for  short time i n t e r v a l s , may y i e l d good recoveries of c e l l fractions while minimizing, although not eliminating, breakage of subcellular organelles. Various adjuncts to these procedures have been devised although each has i t s disadvantage.  Short exposures to proteases (nagarse, collagenase) weaken  the tissue by digesting connective tissue thereby easing disruption (Stephens and Wrogemann 1970).  The use of these- enzymes can be advantageous for i s o l a t i o n  of i n t r a c e l l u l a r organelles but may be inappropriate for i s o l a t i o n of sarcolemmal enriched fractions because the surface membranes would also be susceptible to p r o t e o l y t i c damage.  Membrane fractions contaminated by c o n t r a c t i l e proteins  have higher non-specific protein contents. High i o n i c strength KC1 solutions s o l u b i l i z e and extract c o n t r a c t i l e proteins which i f not removed, gel the homogenate and the resuspended p e l l e t s .  Unfortunately, high ionic strength washing  solutions have been observed to reduce the s p e c i f i c a c t i v i t i e s of c e r t a i n membrane enzymes (Hui et a l . 1976).  EDTA solutions have been used to extract pro-  teins loosely bound to the membrane by divalent ions but they may also denature membrane embedded enzymes (Wolf 1972).  After many of the above d i f f i c u l t i e s  were experienced i n preliminary experiments a method was devised i n an attempt to avoid most of these problems.  Before homogenization, the muscles were washed  extensively with Ca,Mg-free Tyrode's solution (for 10 min at 37°C and 3 hr and 50 min at 4°C) to soften the i n t e r c e l l u l a r cement (Anderson 1953) and to make the plasma membranes leaky (Palaty 1974).  The reduction of temperature to 4°C  5 after the f i r s t 10 min was  designed to l i m i t lysozymal a c t i v i t y i f any lysozymes  were released during the f i r s t 10 min divalent cation depletion.  I t was  hoped that softening of the i n t e r c e l l u l a r cement would continue at 4°C i n t r a c e l l u l a r organelles would not be further damaged. disruption by a Potter-Elvehiem  also  while  The ease and extent of  homogenizer increased noticeably without any  apparent increase i n damage to i n t r a c e l l u l a r organelles.  At the same time the  reduction of Ca and Mg l e v e l s prevented the g e l l i n g and aided the extraction of c o n t r a c t i l e proteins.  The a c t i v i t y and c h a r a c t e r i s t i c s of the sarcolemmal en-  riched f r a c t i o n that resulted after the use of this modified method w i l l  be  described i n the Results- and Discussion. b.  Common procedures for separation of s u b c e l l u l a r fractions  Once homogenized, the c e l l can be separated d i f f e r e n t i a l centrifugation.  into three main fractions by  The heaviest components ( c e l l debris,  connective  tissue, n u c l e i and larger sheets of plasma membrane, depending on the homogenization procedure) are sedimented at low speeds (less than 3,000 x g).  Mito-  chondria, lysosomes and some rough endoplasmic reticulum and plasma membrane sheets can usually be'pelleted between 10,000 x g and 27,000 x g.  Ultracentri-  fugation at 105,000 x g separates v e s i c l e s of endoplasmic reticulum and plasma membrane.  The supernatant remaining a f t e r centrifugation at these high speeds  i s composed of the c o n t r a c t i l e filaments and soluble cytoplasmic proteins that are not attached to any membranous material.  I t i s obvious that there i s consi-  derable overlap i n the c e l l components sedimented at various speeds.  A finer  separation of each f r a c t i o n into bands based on their small differences of density and size can be achieved by recentrifugation on sucrose density gra-: dients.  Unfortunately, hyperosmotic sucrose solutions can damage properties of  the c e l l components and a considerable amount of the y i e l d has to be s a c r i f i c e d to obtain greater purity because the borders of each band of membranes are not c l e a r l y defined.  Even when separated on the basis of density, bands can contain  more than one c e l l component since the banding c h a r a c t e r i s t i c s also depend on  6 the size of the v e s i c l e which varies with:the osmotic strength of the sucrose. Although sucrose density gradients and other methods have improved the purity of subcellular fractions greatly, elaborate techniques  for separation of sub-  c e l l u l a r fractions can not be used to compensate for extremely destructive homogenization procedures (Kidwai 1975). c.  I d e n t i f i c a t i o n of subcellular fractions  The purity of a f r a c t i o n i s d i f f i c u l t about i t are usually made.  to determine, and subjective decisions  The degree of purity should be established before  major conclusions are made about the properties of the subcellular organelle under study.  Organelles are e a s i l y i d e n t i f i a b l e i n the intact c e l l but once  broken, a l l types of membranes appear similar i n the electron microscope. isolated mitochondria  are s t i l l e a s i l y d i s c e r n i b l e and, to some extent, so are  endoplasmic reticulum membranes i f they r e t a i n their ribosomes. plasmic reticulum, broken mitochondria, look very similar. plasma membranes.  Intact  Smooth endo-  plasma membrane and Golgi apparatus  A fuzzy basement membrane may  help to v i s u a l l y i d e n t i f y  A c t i v i t i e s of enzymes s p e c i f i c a l l y located on one type of  c e l l membrane have been used to i d e n t i f y c e l l components i n fractions.  However,  smooth muscle studies are complicated by the lack of well established marker enzymes for each component, especially for endoplasmic reticulum and Golgi apparatus (Kidwai 1975;  III.  Wei et a l . 1976;  Janis and Daniel 1977).  Studies of the Na,K-ATPase i n Smooth Muscle The cytoplasm of smooth muscle c e l l s ( l i k e that of most c e l l s ) normally  has a higher concentration of Kand a lower concentration of Na than the extracellular fluid  (Casteels 1970).  The sarcolemma i s permeable to K and to a lesser  extent to Na.  Cells would lose K and gain Na down their concentration gradients  i f they did not have a means by which to a c t i v e l y maintain these concentration gradients.  The Mg dependent Na,K-ATPase, a plasma membrane enzyme, has generally  been accepted  to be responsible for pumping K into and Na out of the c e l l , by  7 using the energy released during the hydrolysis of ATP  (Dahl and Hokin 1974).  Elevated levels of i n t r a c e l l u l a r Na or e x t r a c e l l u l a r K seem to stimulate this enzymatic pump. (Hoffman 1962;  Ouabain and K free medium lower the a c t i v i t y of most Na,K-ATPases  Whittam and Ager 1964;  and K free medium do not completely (Daniel et a l . 1971c).  Brinley and Mullins 1968).  But ouabain  block Na e f f l u x i n smooth muscle c e l l s  Daniel et a l . (1971c), A l l e n and Daniel (1970) and  Wolowyk et a l . (1971) have described a Na stimulated Mg-ATPase a c t i v i t y that i s independent of K and i n s e n s i t i v e to ouabain i n rat myometrial and rabbit vascul a r smooth muscle. and water content  This enzyme has been postulated to regulate i n t r a c e l l u l a r Na (Daniel and Robinson 1971a).  In addition to having an extra  sodium transport system, the rat uterus also has an ouabain s e n s i t i v e Na,K-pump but according to Daniel and Robinson (1971a) i t s c h a r a c t e r i s t i c s deviate from the accepted views about i t s operation.  Descriptions of the ATPase a c t i v i t y i n  sarcolemmal fractions of the guinea pig ileum l o n g i t u d i n a l smooth muscle by Godfraind and Verbeke (1973), Hurwitz et a l . (1973), O l i v i e r a and Holzhacker (1974) and Godfraind et a l . (1976) gave no i n d i c a t i o n of any abnormal behavior of this enzyme i n this smooth muscle. whether the stimulation was simultaneously.  due to Na alone or whether Na and K were required  The extent of i n h i b i t i o n by ouabain of the Na and K stimulation  was not evident from these studies. by Mg,  The nature of the ATPase a c t i v i t y stimulated  Na and K i n the guinea pig ileum l o n g i t u d i n a l smooth muscle and i t s  sensitivity IV.  These studies do not c l e a r l y indicate  to ouabain were re-investigated i n the present study.  Studies of Ca-ATPases i n Smooth Muscle 2+ F i l o et a l . (1965) measured the free Ca  concentration that would be re-  quired to maintain a relaxed state of vascular smooth muscle and found that i t -7 2+ must be less than 1.8 x 10 M. Maximal tension developed when the free Ca concentration was  s l i g h t l y greater than 10 ^ M.  The ATPase a c t i v i t y associated -7  with the c o n t r a c t i l e filaments increased over the range of 10  -5 to 10  2+ M Ca  8 (Mrwa and Ruegg 1976).  Since generally the i n t r a c e l l u l a r Ca content would give  a cytoplasmic concentration f a r greater (up to 5 mM) than the above values i f i t was evenly distributed throughout  the c e l l (Goodford 1970; Van Breemen et a l .  1966), the Ca must be concentrated i n various subcellular components of the c e l l (Batra and Daniel 1971; Somlyo et a l . 1974; Hess and Ford 1974).  Energy i n the  form of ATP i s required to concentrate the Ca i n sarcoplasmic -reticulum and mitochondria.  Electron probe X-ray microanalysis has confirmed that the cytoplasmic  Ca concentration i s extremely low i n vascular smooth muscle and that Ca i s concentrated i n mitochondria and sarcoplasmic reticulum (Somlyo et a l . 1976). ing  Dur-  excitation Ca flows into smooth muscle c e l l s from surface binding s i t e s and  the e x t r a c e l l u l a r space (Grundfest 1976; Tomita 1970; Hurwitz and Suria 1971). This Ca can be temporarily accumulated by sarcoplasmic reticulum and mitochondria but, since over time the c e l l Ca content i s r e l a t i v e l y stable, the Ca gained during excitation must eventually be returned to the e x t r a c e l l u l a r space.  This  might also require an active pump mechanism because the e x t r a c e l l u l a r Ca concent r a t i o n (approximately 2 mM) i s greater than the free cytoplasmic l e v e l s (Van Breemen et a l . 1966).  Recently, Janis et a l . (1977) provided the f i r s t  d i r e c t evidence for a role f o r the plasma membrane as well as mitochondria and sarcoplasmic reticulum i n the regulation of Ca a c t i v i t y of r a t uterine smooth muscle. act  The authors suggest that plasma membrane and endoplasmic reticulum may  to control Ca levels at cytoplasmic Ca concentrations of 3 x 10 ^ M or less  and that mitochondria may play an important role when the cytoplasmic Ca concent r a t i o n exceeds 10 ^ M.  Ca transport by subcellular fractions from smooth  muscle has recently been reviewed by Janis and Daniel (1977).  V.  Methods f o r Measuring Ion Movements i n Smooth Muscle Contraction may possibly be coupled to excitation by mobilization of Ca  from any one of the tissue Ca depots.  Methods do not exist yet to measure move-  ments from each one of these depots separately but with refinements methods to  9 )  measure small movements of Ca from outside the c e l l to the cytoplasm are becoming more workable.  Therefore the easiest hypothesis to test i s that e x c i t a t i o n -  contraction coupling i n smooth muscles may be regulated by trans-sarcolemmal ion movements. a.  Calculation of i n t r a c e l l u l a r ion levels from t o t a l ion measurements and flux studies  There are many obstacles to the study of trans-sarcolemmal ion fluxes i n smooth muscle (Daniel 1975; Van Breemen . et a l . 1973). :  below.  Some of these are outlined  The e x t r a c e l l u l a r space i s large and the e l a s t i n , collagen and  bind large quantities of ions, especially Ca.  mucoproteins  Ions dissociate from e x t r a c e l l u l a r  binding s i t e s at varying rates, sometimes more slowly than their exchange rates from i n t r a c e l l u l a r s i t e s .  Because of these and other reasons, .'  ..<_..;  tracer ion exchange curves plotted semilogarithmically can not be f i t t e d unequivocally to a few straight l i n e s from which f i r s t order rate constants can be calculated.  The e f f l u x rates are d i f f i c u l t to r e l a t e to losses of ion pools  sequestered i n c e l l organelles and bound to membranes.  The quantity of Ca needed  to activate a smooth muscle contraction i s about 100 times less than the t o t a l amount of exchangeable Ca.  Damaged c e l l s , on the surface of a dissected muscle  s t r i p , gain Ca and Na and lose K and Mg quite extensively.  Depending on the pro-  portion of c e l l s damaged, the f i n e r changes i n these ion levels during responses to excitatory and i n h i b i t o r y agents may be obscured. I n t r a c e l l u l a r ion content has often been calculated from the difference between t o t a l (measured by atomic absorption spectrophotometry) and e x t r a c e l l u l a r amounts.  The e x t r a c e l l u l a r content was estimated from the product of the physio-  l o g i c a l s a l t solution concentration and the experimentally measured volume of the tissue e x t r a c e l l u l a r space.  As yet, no universally accepted method has been  described for measuring the e x t r a c e l l u l a r space (Paton 1975b). Small molecules may permeate the unit membrane, large molecules may be excluded from small invaginated regions of the c e l l and charges, dipole moments or hydrophobic areas of  10 the marker molecule may membrane or collagen.  cause them to adhere to the plasma membrane, basement The accuracy of measuring the e x t r a c e l l u l a r space volume  i s very uncertain and the scatter of values, using various marker has been considerable (Goodford drugs may  and Leach 1966;  Setekleiv 1970).  substances, In addition,  change the e x t r a c e l l u l a r space volume e.g. by inducing contraction.  Small and rapid v a r i a t i o n s of i n t r a c e l l u l a r ion content were d i f f i c u l t to detect and generally the method was  inadequate.  Measurements of tracer ion fluxes were  more s e n s i t i v e but again, changes of the s i z e of the e x t r a c e l l u l a r space and also the r a t i o of the c e l l surface to volume could a f f e c t the r e s u l t s . mer  The  for-  i s d i f f i c u l t to monitor during muscle a c t i v i t y and the l a t t e r can hot be  monitored (Setekleiv 1970; b.  The  Daniel 1975).  'La Method'  A simple but s e n s i t i v e method was needed to measure net changes i n i n t r a c e l l u l a r ion l e v e l s that would be independent of e x t r a c e l l u l a r space changes and surface to volume ratios and would not require i n t e r p r e t a t i o n of flux k i n e t i c s . Most of the aforementioned obstacles, at least for measuring i n t r a c e l l u l a r Ca 45 levels with  Ca, can be circumvented by blocking Ca i n f l u x , i n h i b i t i n g Ca e f f l u x  and displacing the e x t r a c e l l u l a r bound Ca with La (Van Breemen and McNaughton 1970) .  Approximate net i n t r a c e l l u l a r Ca changes during rat myometrial contrac-  tions were measured by Marshall and Kroeger (1973) using a modification of the 'La method' and measuring the Ca by atomic absorption spectrophotometry.  Experi-  mental v e r i f i c a t i o n of the 'La method' has been presented by Van Breemen!, et a l . (1973) but imperfections of the method have been pointed but by Hodgson et a l . (1972), Hodgson and Daniel (1972), Freeman and Daniel (1973) and Weiss (1974). The l a t t e r author also points out that although not perfect, the 'La method' has aided studies of this type greatly.  Hodgson et a l . (1972) found that  and sarcoplasmic reticulum fractions isolated from LaCl- treated c e l l s La.  mitochondria contained  They also calculated that more La could bind to rat myometrial c e l l s than  could possibly t i g h t l y pack on their surface which, i f correct, would mean that  11 La gained access to the c e l l i n t e r i o r and perhaps could displace some i n t r a c e l l u l a r Ca. Using LaCl- rather than c o l l o i d a l La as an e x t r a c e l l u l a r s t a i n for electron microscopy, Langer and Frank (1972) did not observe La to penetrate plasma membrane of cultured heart c e l l s .  the unit  They reported that La stained the three  dimensional volume of the basement membrane (0.06 to 0.1 u thick) which would weaken the argument of Hodgson et a l . (1972) that La penetrated a two dimensional planar binding c a l c u l a t i o n .  the c e l l based on  Recently Ma and Bose (1977) ob-  served that when 10 mM LaCl-. was used to seal c e l l s , La did not appear i n the cytoplasm, except for a few spots which appeared s i m i l a r i n s i z e to caveolae (ves i c l e s opening into the e x t r a c e l l u l a r space) which might have o v e r l a i n the plane of the section.  Casteels et a l . (1972) investigated the La concentrations r e -  quired to cause various e f f e c t s and concluded that 10 mM LaCl- blocked  trans-  membrane calcium movements whereas the 2 mM LaCl-, as previously used i n the 'La method', was not quite s u f f i c i e n t .  Ca e f f l u x was i n h i b i t e d by 87% by 2 mM  LaCl-. and Van Breemen : et a l . (1973) attributed the residual 13% Ca loss from vascular smooth muscle to displacement from damaged c e l l s .  Others f e e l that La  does not block e f f l u x e f f e c t i v e l y at 37°C (Freeman and Daniel 1973; V. Palaty, personal communication) but that 10 mM LaCl- i n a washing solution at 4°C i s more effective,(V. Palaty, personal communication). er temperature (Goodford et a l . 1965).  Ca e f f l u x i s reduced at low-  In fact a l l active transport a c t i v i t y  would be greatly reduced at this temperature and passive leakages of ions across the membrane should also be reduced (Setekleiv 1970; Tomita 1970) e s p e c i a l l y with the additional.membrane s t a b i l i z i n g e f f e c t of 10 mM LaCl- (Weiss 1974).  Daniel  (1963a) and Setekleiv (1967) observed that K e f f l u x was reduced at lower temperatures  (Q"LQ  =  1-6 and 1.94, respectively) but Daniel (1963a) observed a tran-  s i e n t l y faster rate of K e f f l u x i n i t i a l l y when the temperature was reduced to 7°C. Some but not a l l of the faster e f f l u x was thought to have been due to a rapid loss of e x t r a c e l l u l a r K during cold contracture.  Cold contracture could be  prevented by removal of e x t r a c e l l u l a r Ca (Daniel 1964)... .  The rate of K e f f l u x  was reduced by 55% i n K free medium (Daniel 1963b).  At 5°C Na e f f l u x was very  markedly reduced (Daniel and Robinson 1971b)., Mg e f f l u x was shown to be dependent on metabolism and on exchange f o r e x t r a c e l l u l a r Ca (Moawad and Daniel 1971). A l t e r n a t i v e l y Mg e f f l u x may u t i l i z e the energy released i n the course of Na i n f l u x (Palaty 1974).  LaCl- (1 mM) did not reduce passive K e f f l u x i n tumor  c e l l s at 22°C but i t did i n h i b i t Na e f f l u x by 48% (Smith 1976).  Although LaCl-  s t a b i l i z e s membranes, i t also predisposes the membrane to damage by mechanical stress (Levinson et a l . 1972).  Therefore during any procedure using high LaCl-  concentrations, extra care must be taken not to stretch or crush the c e l l s .  If  La does slowly permeate c e l l s at 37°C, i t s permeability at 4°C would probably be 2+ markedly reduced since the uptake of Zn  , a heavy metal cation more permeable  3+ than La , was reduced at 5°C (Daniel et a l . 1971a). Based on the above reason\  ing,  i t was f e l t that at 4°C, an isotonic T r i s HC1 solution (pH 7.4) containing  10 mM LaCl- could be used to wash o f f e x t r a c e l l u l a r ions while r e t a i n i n g most of the i n t r a c e l l u l a r ions.  These could be extracted from the muscle and measured  by atomic absorption spectrophotometry. which the modified  In summary, the general theories upon  'La method' was based were:  1) active transport w i l l be reduced at 4°C 2) passive fluxes w i l l be reduced at 4°C 3) La w i l l s t a b i l i z e the membrane and reduce passive fluxes, especially for Ca and Na 4) cold contracture w i l l be minimized i n Ca free medium containing 10 mM LaCl5) passive K e f f l u x w i l l be further reduced i n K free medium 6) loss of i n t r a c e l l u l a r Mg w i l l be reduced by i n h i b i t i o n of metabolism at 4°C and by the absence of e x t r a c e l l u l a r Ca and Na to exchange f o r i t 7) La w i l l displace e x t r a c e l l u l a r l y bound Ca and probably Mg and Na also 8) K i s not t i g h t l y bound e x t r a c e l l u l a r l y and w i l l d i s s o c i a t e from i t s binding s i t e s i n K free medium 9) free e x t r a c e l l u l a r Ca, Mg, Na and K w i l l d i f f u s e out of the e x t r a c e l l u l a r space  in a large volume of isotonic T r i s HC1  containing no added Ca, Mg,  Na or K  10) La permeability i s very low and w i l l be even less at 4°C and therefore i t should not displace i n t r a c e l l u l a r ions. This modified .'La method' gave at least good q u a l i t a t i v e comparisons of i n t r a c e l l u l a r Ca, Na, K and Mg l e v e l s i n r e s t i n g muscles to these cation l e v e l s at various stages of muscle a c t i v i t y .  Because of the uncertain v a l i d i t y of the  'La method', the r e s u l t s from the modified  'La method' should be accepted cau-  t i o u s l y u n t i l a better method becomes available to check the v a l i d i t y of the results obtained by the 'La method'. VI.  The Mechanism of Action of Ouabain A considerable amount of evidence suggests that ouabain can produce an i n -  creased  force of contraction by i n h i b i t i n g the Na,K-ATPase and subsequently, af-  f e c t i n g membrane ion transport (Hadju and Leonard 1959; Schwartz 1976).  Akera and Brody  However the d e t a i l s are by no means c l e a r .  1976;  Although ouabain  i n h i b i t s the K-dependent step i n the reaction sequence of the Na,K-ATPase, i t does not show simple competition  with K activation>. of the enzyme (Glynn 1964).  The expected decrease i n cardiac i n t r a c e l l u l a r K due to Na,K-ATPase i n h i b i t i o n was  observed only a f t e r the p o s i t i v e inotropic response was  et a l . 1961;  Lee et a l . 1961;  Steiness and Valentin 1976).  completed (Tuttle Murthy et a l . (1974a)  dissociated ouabain binding by the t i s s u e from i t s c o n t r a c t i l e e f f e c t by demons t r a t i n g that the contraction of the rabbit myometrium was for 10 min whereas binding persisted a f t e r 10 min.  relaxed a f t e r washing  Tissue Na and K l e v e l s were  not changed a f t e r a 10 min exposure to 5 x 10 ^ M ouabain when i t s potentiating e f f e c t on acetylcholine contractions was maximal.  Ouabain increased K e f f l u x  from r a t u t e r i , but contrary to i t s proposed mechanism, ouabain did K i n f l u x (Daniel and Robinson 1971a).  not i n h i b i t  Ouabain produces an i n i t i a l depolarization  of taenia c o l i c e l l s , accompanied by an increase i n spike frequency and l a t e r a depolarization block, but the decrease i n i n t r a c e l l u l a r K and C l and increase i n i n t r a c e l l u l a r Na are not s u f f i c i e n t to explain the changes i n the membrane poten-  t i a l (Casteels 1966).  Reduction  of the Ca concentration of the medium lowers  the increase i n cardiac tension developed by ouabain (Holland and Sekul 1961)  and  2+ Mn  competitively blocks the postive inotropic effect of ouabain (Sabatini-Smith  and Holland 1969).  Several studies have shown that there i s an increased itttra45  c e l l u l a r uptake of Sekul 1961;  Ca during the increase i n tension to ouabain (Holland and  Casteels and Raeymaker 1976)  crease i n membrane permeability to Ca may  and the authors suggested that an i n be the primary effect of ouabain.  In  the present study a d d i t i o n a l data are presented i n d i c a t i n g .that ouabain may  cause  contraction of the guinea pig ileum l o n g i t u d i n a l smooth muscle by increasing the membrane permeability to Na and Ca before i n h i b i t i n g the Na,K-ATPase. VII.  Excitation-Contraction Coupling  (General)  Authors discussing smooth muscle e x c i t a t i o n contraction coupling almost i n variably try to draw comparisons to s k e l e t a l and cardiac muscle because more i s known about these c e l l s .  I r o n i c a l l y each comparison accentuates  so much their  differences and althoiigh_all muscle c e l l s produce contraction, the modes by which they can affect contraction seem to bervery d i f f e r e n t .  Skeletal and cardiac  muscle c e l l s have specialized continuations of t h e i r plasma membranes, called transverse tubules, which conduct depolarizations rapidly into the c e l l i n t e r i o r . When the depolarization arrives at the locus of the terminal cisternae of the sarcoplasmic reticulum, Ca i s released from them to the c o n t r a c t i l e filaments (Porter and Palade 1957;  Huxley and Taylor 1958;  Katz 1970;  Langer 1968).  The  sarcoplasmic reticulum volume i s greater i n s k e l e t a l than i n cardiac muscle c e l l s . Skeletal musclencells r e t a i n their a b i l i t y to contract during long periods i n Ca-free a r t i f i c i a l e x t r a c e l l u l a r f l u i d . dependent on e x t r a c e l l u l a r Ca.  In contrast, cardiac c e l l s are much more  Although they have a sarcotubular system and are  r i c h l y supplied with mitochondria,  Ca stored i n these s i t e s does not seem to be  releasable from them i n the absence of e x t r a c e l l u l a r Ca (Langer 1976).  Smooth  muscle c e l l s appear to have the least specialized i n t r a c e l l u l a r organization of  the muscle c e l l class.  They are smaller (6-10 u i n diameter or 1/3 - 1/30  diameter of a s k e l e t a l muscle f i b r e , Gabella 1971) tubules.  the  and devoid of transverse  Recently Devine et a l . (1972) and Gabella (1973) have indicated that  the sarcoplasmic reticulum i s more abundant than e a r l i e r reports indicated (Burnstock 1970).  Small sacs and tubules were not recognized as being sarco-  plasmic reticulum because their form and organization was l e t a l muscle.  so unlike that of ske-  Sarcoplasmic reticulum and mitochondria have been shown to accu-  mulate strontium and calcium (Somlyo and Somlyo 1976;  Somlyo et a l . 1976)  but so  far to my knowledge, there does not appear to be direct evidence that strontium N  and calcium can be released from them during contraction.  Indirect evidence for  Ca release from isolated s k e l e t a l muscle sarcoplasmic reticulum v e s i c l e s , either by reversal of the Ca pump, stimulation by " t r i g g e r calcium"', depolarization or the a p p l i c a t i o n of ionophores and caffeine has been described by Inesi and Malan (1976) but only the d i r e c t e l e c t r i c a l effects on the sarcoplasmic reticulum ves i c l e s seemed to occur under p h y s i o l o g i c a l conditions. approximately  6.6%  Mitochondria constitute  of the smooth muscle c e l l volume (Gabella 1973)  plasmic reticulum comprises 2 -  7.5%  of the cytoplasm  and sarco-  (Somlyo and Somlyo 1976).  The volume of sarcoplasmic reticulum i s greater i n the t o n i c a l l y contracting large e l a s t i c a r t e r i e s than i n the phasic spike generating smo.oth muscles such as taenia c o l i and p o r t a l anterior mesenteric vein (Somlyo and Somlyo 1975).  Smooth  muscle c e l l s contract much more slowly than s k e l e t a l muscles, which tends to reduce any obligatory requirement  for i n t r a c e l l u l a r Ca.  Indeed smooth muscle c e l l s  are highly dependent on e x t r a c e l l u l a r Ca and their degree of dependence seems to be related to their volume of sarcoplasmic reticulum (Devine et a l . 1972). I r o n i c a l l y the faster contracting (phasic) smooth muscles seem to have the least volume of sarcoplasmic reticulum.  Smooth muscle c e l l s have an abundance of sur-  face v e s i c l e s , c a l l e d caveolae, which are not c h a r a c t e r i s t i c of most other muscle cell-types (Burnstock 1970).  However, large numbers of caveolae were found i n  rat a t r i a l and l i z a r d v e n t r i c u l a r c e l l s but only i n those c e l l s which lacked a  transverse tubule system (Forssman and Girardier 1970; 1971).  Forbes and  Sperelakis  These v e s i c l e s increase the o v e r a l l surface area by more than 70% i n  guinea pig taenia c o l i c e l l s (Goodford 1970)  and by 25% i n l o n g i t u d i n a l smooth  muscle of the mouse intestine,(Rhodin 1962).  I t should be noted that much of  the sarcoplasmic  reticulum i s located near the smooth muscle c e l l surface, close-  l y associated to caveolae  (Gabella 1973).  The surface to volume r a t i o of these  c e l l s i s very large and therefore smooth muscle c e l l s could possibly use Ca from s u p e r f i c i a l s i t e s for at least part of their c o n t r a c t i l e cycle.  Upon stimulation,  smooth muscle c e l l s appear to u t i l i z e Ca rather than Na as the depolarizing i n ward current c a r r i e r .  Their e l e c t r i c a l spikes seem to be i n s e n s i t i v e to the extra-  c e l l u l a r Na concentration  (Holman 1958;  c e l l u l a r Ca levels (Holman 1958,  Sakamoto 1971), very s e n s i t i v e to extra-  Bulbring and Kuriyama 1963,  Tomita 1970), i n h i -  2+ bited by Mn 1969)  (Brading et a l . 1969), Nonamura et a l . 1966,  Bulbring and Tomita  and i n s e n s i t i v e to tetrodotoxin, a s p e c i f i c blocker of the Na channel  (Tomita 1970,  Nonamura et a l . 1966).  contraction (Goodford 1970;  This Ca current  Lullman and Mohn 1969;  may  contribute some Ca for  C o l l i n s et a l . 1972).  l a r i z a t i o n or Ca entering the c e l l during depolarization may  Depo-  release Ca bound to  the inside of the c e l l membrane or i n i t i a t e the release of Ca from sarcoplasmic reticulum or possibly from mitochondria  (Bianchi 1969).  Reduction of the cyto-  plasmic Ca to i t s resting levels (relaxation) i s usually attributed to sarcoplasmic r e t i c u l a r and mitochondrial re-sequestration of Ca. ternal sources may  be extruded immediately to cause relaxation or i t may  temporarily buffered by mitochondria 1974;  Devine et a l . 1973;  Price 1976)  Ca gained from ex-  and sarcoplasmic  V a l l i e r e s et a l . 1975;  from where i t may  reticulum (Hess and  Janis et a l . 1977;  be Ford  Huddart and  be extruded from the c e l l , by some hitherto unknown  mechanism, to maintain a f a i r l y constant i n t r a c e l l u l a r l e v e l of Ca.  There may  be a passive exchange of i n t r a c e l l u l a r Ca for e x t r a c e l l u l a r Na u t i l i z i n g the energy of the Na gradient which i n turn must be a c t i v e l y maintained (Bohr et a l . 1969; Reuter et a l . 1973).  An alternative and more d i r e c t means would be a sarco-  lemmal ATP requiring active transport system to prevent an i n t r a c e l l u l a r b u i l d up of Ca ( F i t z p a t r i c k et a l . 1972; Casteels et a l . 1973b; Janis et a l . 1977). Kirkpatrick et a l .  (1975) have pointed out that the agonists used i n such stu-  dies may produce effects i n unusual ways under certain circumstances but these may not be d i r e c t l y related to the mechanism involved i n the normal process of excitation-contraction coupling. Advocating generalities about smooth muscle behavior seems f u t i l e since each smooth muscle appears variably dependent on the r a t i o of the sizes of i t s i n t e r n a l and external Ca pools (Chang and Triggle 1972).  Each i s variably  sensitive to Ca removal from the physiological medium indicating that their sarcoplasmic reticulum volume (Devine et a l . 1972), surface to volume r a t i o (Bianchi 1969) and Ca a f f i n i t y at storage s i t e s (McGuffee and Bagby 1976) may be unique for each type of smooth muscle.  For the above reasons, the experimen-  t a l results obtained i n t h i s study have been most often compared to previously reported c h a r a c t e r i s t i c s of guinea p i g ileum longitudinal smooth muscle and to the c h a r a c t e r i s t i c s of guinea p i g taenia c o l i c e l l s which are f a i r l y similar i n o r i g i n and nature.  VIII.  Sources of Calcium for Excitation-Contraction Coupling i n the Guinea P i g Ileum Longitudinal Smooth Muscle Those studying the longitudinal smooth muscle of the guinea pig ileum often  imply that i t sequesters activator calcium i n a manner quite d i f f e r e n t from skel e t a l and cardiac muscle c e l l s (Hurwitz and Joiner 1969).  Perhaps this smooth  muscle does not need to sequester as much Ca i n t r a c e l l u l a r l y because, taking the differences of c e l l volume i n consideration, a 'volume-unit' of the guinea p i g ileum longitudinal smooth muscle takes up 65 times more Ca per stimulus than a 'volume-unit' of s k e l e t a l muscle (Lullman and Mohn 1969).  Lullman and Siegfriedt  (1968) (through Lullman 1970) described three fractions of ^ C a e f f l u x from guinea pig ileum longitudinal smooth muscle with half-times of 1 min (Fraction 1, 34% of  18 t o t a l ) , 4.5 mln (Fraction 2, 50% of total) and 25 min (Fraction 3, 16% of total) when the e x t r a c e l l u l a r Ca concentration was 1.8 mM. as the Ca i n the e x t r a c e l l u l a r f l u i d .  Fraction 1 was designated  Weiss (1972) found only two c l e a r l y deline-  45 ated components of  Ca e f f l u x .  The fast component had a half-time of 1 min and  was attributed to both free e x t r a c e l l u l a r Ca and s u p e r f i c i a l l y bound Ca. The slow component had a half-time of 9 min and contained s u p e r f i c i a l l y bound Ca and t i g h t l y bound Ca.  The presence of s u p e r f i c i a l l y bound Ca i n both the fast and  slow components was i d e n t i f i e d by the s h i f t i n g of part of the slow compartment to the fast component, probably by exchange at membrane s i t e s , when nonradioactive Ca was added. Joiner  After the e x t r a c e l l u l a r Ca was washed o f f (5 min) Hurwitz and  (1969) observed that Ca migrated into the e x t r a c e l l u l a r f l u i d from two  compartments, similar to fast Fraction 2 and slow Fraction and  Siegfriedt  (1968).  Hurwitz and Joiner  3 described by Lullman  (1969) reported that the plots of the  reciprocal of the c o n t r a c t i l e response as a function of the reciprocal of the ext r a c e l l u l a r Ca concentration or as a function of the reciprocal of the Ca i n the fast component were l i n e a r and had the same intercept, denoting the same, maximum response.  The l i n e a r relationship between Ca i n the fast compartment and the  e x t r a c e l l u l a r space was not compatible with the concept that calcium ions i n the bathing medium equilibrate with activator ions bound to some saturable group of l o c i within the f i b r e .  Hurwitz and Joiner  (1969) theorized that  "extracellular  calcium equilibrates with activator ions that are i n solution i n some biophase of the muscle f i b r e , perhaps the f i b r e membrane." The has  contraction  of the longitudinal ileum to muscarinic agents and high KC1  a biphasic appearance.  There i s a fast (phasic) contraction  slower tonic maintenance of tension.  followed by a  Ca for the phasic and tonic components, i n -  duced by d i f f e r e n t s t i m u l i , may not be supplied by the same Ca pool. mobilized by a muscarinic stimulus was studied by Chang and Triggle 1973)  (Fig. 1). They equilibrated  cle with 1.8 mM CaCl  ?  The Ca (1972 and  the guinea pig ileum longitudinal smooth mus-  and then abruptly changed the Ca concentration of the  Tyrode's solution (0 - 1.8 mM) at the same time that the muscle was stimulated with a muscarinic agonist, methiodide (CD).  cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane  The phasic component remained f a i r l y constant but the tonic  component decreased markedly when the Ca concentration was decreased ment a).  (experi-  When the muscle was equilibrated with Ca concentrations from 0 - 1 . 8  mM and stimulated with CD at the same time that the Ca concentration was abruptly changed to 0.2 mM, the phasic component of the muscle steadily increased with the increasing e q u i l i b r a t i n g Ca concentration (experiment  b).  I f the e q u i l i b r a t i n g  Ca concentration was held constant at a low concentration of 0.1 mM and the stimulus Ca l e v e l was varied from 0 to 1.8 mM, both the phasic and tonic com-, ponents increased with increasing stimulating levels of Ca (experiment  c).  Their  interpretation of the results was that the phasic component of the contraction was dependent on a membrane bound Ca pool that loads to a certain capacity depending on the e x t r a c e l l u l a r Ca concentration used to e q u i l i b r a t e the muscle (experiment  a).  of Ca (experiment  This pool slowly equilibrates to a lower capacity at low levels b) but can be rapidly reloaded when the stimulating l e v e l of  Ca i s raised (experiment  c).  Because the phasic component reloads almost instan-  taneously and i s i n h i b i t e d by low concentrations of La (thought to displace Ca bound to the external surface of the sarcolemma), mobilization of Ca bound to the outer aspect of the c e l l was considered to be most l i k e l y responsible for the phasic component.  The magnitude of the tonic component was d i r e c t l y related to  the free Ca concentration of the e x t r a c e l l u l a r space (experiment  a, b and c ) .  The magnitude of the high KC1 induced tonic contraction also increased hyperb o l i c a l l y with increasing Ca concentrations i n the external medium (Hurwitz and Suria 1971).  The phasic and tonic components appear to be dependent on two d i s -  t i n c t l y separate Ca pools but there i s rapid exchange of Ca between these two pools.  This hypothesis deserves  further investigation.  F i g . 1.  Superimposed tracings of time courses of contractions showing the effect of d i f f e r e n t Ca concentrations f o r e q u i l i b r a t i o n [Ca„„ ] m  iiiAl  CD (2 x 10~  M).  r  i-  and stimulus [Ca | |  _,]  _A1  b  during e x c i t a t i o n by  The muscle was equilibrat'ed i n [Ca_ _,]_, f o r 30 min. v  At the time of the  introduction of the agonist (CD) the Ca concentration was abruptly altered to [ C _ x T _ S " a  (reproduced from Chang and T r i g g l e 1972).  21 IX.  Receptor Theory and the Desensitization Phenomenon Chang and Triggle (1972) have proposed that the acetylcholine receptor  exists i n a Ca-associated  state which, when combined with an agonist,  the associated Ca into the c e l l to generate the phasic component.  propels  Their model  i s a modification of the model proposed by Hurwitz and Suria (1971).  The sub-  sequent Ca-dissociated active state can be permeated by free e x t r a c e l l u l a r Ca to generate the tonic component.  By their calculations, only a s m a l l f r a c t i o n N  of the number of Ca ions bound to s i t e s on the r e l a t i v e l y large surface of each longitudinal i l e a l f i b r e would, i f shunted inwards, be s u f f i c i e n t to raise the Ca concentration  of the cytoplasm to maximum c o n t r a c t i l e l e v e l s .  small f r a c t i o n i s associated with s p e c i f i c receptor s i t e s . the receptor  'active' state to a 'desensitized' state was  Perhaps this  A transformation  of  proposed to account  for the often observed but poorly understood desensitization phenomenon that occurs after prolonged exposure to high concentrations  of agonists.  But the phase  of decreased c o n t r a c t i l e responsiveness following a maximal contraction of i n t e s t i n a l muscle may  not be a s p e c i f i c receptor desensitization because there i s  a cross depression of responses to acetylcholine, histamine and a variety of smooth muscle stimulants  (Cantoni and Eastman 1946).  A maximal response of  one  agonist does not depress a second response to an agonist i f the f i r s t response i s washed out i n a medium containing a higher K concentration, whether or not the K concentration  i s s u f f i c i e n t to cause a contraction (Cantoni and Eastman 1946).  Born and Bulbring (1956) observed that acetylcholine caused a rapid e f f l u x of K from taenia c o l i c e l l s .  Several mechanisms for muscle desensitization have been  proposed but desensitization i s s t i l l not p r e c i s e l y understood.  Cantoni and  Eastman (1946) and Rand (1956) proposed that the responsiveness of a muscle i s determined i n part by the [K. ]/[Ca xn out J  Paton and Rothschild  and  [K. ]/[K 1 ratios respectively. xn out >  (1965) stated that although acetylcholine consistently  caused the guinea pig ileum longitudinal smooth muscle to lose K, desensitization did not correlate to K loss but rather to Na gain.  Based on new  experimental data  obtained i n the present study, an explanation w i l l be offered for the altered responsiveness of muscles previously contracted by optimal doses of muscarinic agents and for the protective effect of K. X.  Aims of the Present Study In the present study various biochemical and pharmacological aspects of the  contraction and relaxation of the longitudinal muscle of the guinea pig ileum have been examined.  S p e c i f i c a l l y , the aims of the study were to:  1) devise a method to fractionate smooth muscle c e l l s without using harsh, ing procedures  denatur-  thereby minimizing damage to i n t r a c e l l u l a r organelles and mini-  mizing changes i n enzyme c h a r a c t e r i s t i c s . 2) prepare a sarcolemmal enriched f r a c t i o n . 3) ascertain whether there i s a Na,K-ATPase i n the guinea pig ileum longitudinal smooth muscle that conforms to c h a r a c t e r i s t i c s described from studies of other c e l l s . 4) determine i f there i s a Ca-ATPase i n the sarcolemma which might be capable of transporting Ca. 5) explore, by electron microscopy, the various c e l l u l a r depots where Ca could be stored and from which Ca could be mobilized during excitation-contraction coupling. 6) devise a method for measuring net i n t r a c e l l u l a r changes i n Na, K, Mg and Ca during various stages of a c t i v i t y of muscle s t r i p s . 7) investigate the mechanism of action of ouabain by measuring i t s e f f e c t on tension and ion movements. 8) study the c o n t r a c t i l e a c t i v i t y i n response to muscarinic and high K stimulations under various conditions to determine the i o n i c movements responsible for the biphasic contraction. 9) investigate the ion movements which might be responsible.for muscle desensitization.  23 METHODS  I.  Dissection of the Longitudinal Layer of the Guinea Pig Ileum The ileum of male guinea pigs (250-350 g) was removed and divided into 5  segments.  The lumen of the segments was cleaned with 5 ml of Tyrode's solution  which was allowed to flow from a pipet through the segments under gravitational force. 37°C.  The segments were equilibrated i n normal Tyrode's solution for 15 min at The longitudinal layer was removed e s s e n t i a l l y by the method of Rang  (1964).  Each ileum segment (approximately 15 cm long) was drawn over a pipet.  A blunt s c a l p e l was run along both sides of the mesentery and the outer layer was brushed o f f with a cotton swab while the ileum was kept moist i n a trough containing Tyrode's solution at 37°C (see F i g . 2, innervated). For a few experiments, the dissection was altered to eliminate Auerbach's nerve plexus by the method of Paton and Zar (1965) (see F i g . 2, denervated).  The tissues were  tested for the presence of the nerve plexus by checking their responsiveness to nicotine and neostigmine (see Results F i g . 22).  II.  Contractile Force Measurements a.  Isometric force measurements  Muscle s t r i p s (1-2 cm long) were t i e d to stainless s t e e l tissue .hooks and r  attached to Grass FT.03C transducers i n 10 ml water jacketed baths at 37°C under 350 mg basal tension.  Isometric force was recorded on a 4 channel Grass 79 D  polygraph. b.  Normal Tyrode's Solution  The Tyrode's solution (pH 7.2) normally contained 136 mM NaCl, 2.6 mM KC1, 1.8 mM C a C l , 1 mM MgCl , 0.36 mM NaH-P0 , 11.9 mM NaHCO- plus 5.55 mM glucose 2  2  4  (Tyrode 1910) and was gassed with 95% 0^ - 5% CO2 through a sintered disc floor of the bath.  Ca-free and Ca,Mg-free Tyrode's solution refers to Tyrode's solu-  tion from which C a C l  2  or C a C l  2  and MgCl were omitted. 2  Drug solutions were add-  ed to the baths i n small volumes (less than 0.2 ml) with a Hamilton fixed needle syringe.  24  Fig.  2.  Dissection of the guinea p i g ileum longitudinal smooth muscle (innervated and denervated muscle s t r i p s ) . 1.  Strips of ileum (10-15 cm long) were equilibrated for 10-15 37°C i n Tyrode's solution to a t t a i n muscle tone.  2.  Each s t r i p was drawn over a pipet (0.1 ml) i n a 37°C jacketed trough f i l l e d with Tyrode's solution. A blunt s c a l p e l was drawn along either side of the mesenteric attachments.  3.  The scalpel was intended to cut j u s t through the longitudinal layer but i t also penetrates the Auerbach's nerve plexus and may p a r t i a l l y cut into the c i r c u l a r layer. Very l i t t l e pressure should be applied to the blunt s c a l p e l . A l i t t l e of the c i r c u l a r layer usually adheres to the longitudinal muscle s t r i p during the r o l l ing off procedure. Innervated  min  at  Denervated  A cotton swab was used to r o l l ' back the longitudinal layer and the adhering nerve plexus and a few c i r c u l a r muscle c e l l s .  A cotton-swab was used to loosen one end of the longitudinal layer and nerve plexus and r o l l i t to the other side of the ileum.  The longitudinal layer was r o l l e d ( l i k e a s c r o l l ) to the side of the ileum opposite the mesenteric attachments.  The loosened portion was tied with a piece of surgical thread.  One end of the r o l l e d l o n g i tudinal muscle layer was held with tweezers while a cotton swab gently brushed the layer free from the underlying c i r cular layer. The layer was placed i n another water jacketed tissue bath as i n part 1 u n t i l tied to tissue hooks.  The longitudinal layer was held s t e a d y ( n o t drawn backwards) by the s u r g i c a l thread and the c i r c u l a r layer was drawn forward over the p i pet. The shearing force broke the nerve plexus leaving i t attached to the c i r c u l a r layer and the l a t t e r portion of the longitudinal layer was free of the nerve plexus. The longitudinal layer was placed i n another water jacketed tissue bath as in part 1.  26 'High KC1'  contractions were induced by adding 0.5 ml of 1.16 M KC1 to bring the  f i n a l concentration of KC1 to 60 c.  mM.  Tris-Tyrode's Solution  For experiments that measured the i n h i b i t i o n of contraction by La or the i n t r a c e l l u l a r ion content by the modified 'La method', the phosphate and ate were omitted from the Tyrode's solution.  carbon-  The r e s u l t i n g solution was buffered  with 24 mM T r i s adjusted to pH 7.4 with HC1 at 37°C and then oxygenated with 100% 0^ to prevent lanthanum p r e c i p i t a t i o n . d.  'Glycerol shock! treatment  Glycerol (400 mM)  i n physiological solution has been shown to cause d i s -  ruption of transverse tubular membranes and loss of excitation-contraction coupl i n g i n s k e l e t a l muscle, upon returning the muscle to normal physiological solution (Howell and Jenden 1967; Eisenberg and Eisenberg 1968).  Ninety percent of  the transverse tubules are disconnected from the external solution (shown by the absence of e x t r a c e l l u l a r s t a i n by electron microscopy) (Franzini-Armstrong et a l . 1973).  Dulhunty and Gage (1973) f e l t that the excitation-coupling effect  was not related to the disconnection of transverse tubules because the former effect occurred before the l a t t e r . l i t y after this treatment was  In cardiac muscle, loss of twitch c o n t r a c t i -  associated with a s i g n i f i c a n t decrease i n the ex-  t r a c e l l u l a r space, an increase i n c e l l water, a decrease i n c e l l K and an i n crease i n c e l l Ca (Katzung and Teitelbaum 1974). that the washout of 800 mM  Frank and Hemker (1976) found  glycerol i n Krebs-Ringer  solution caused a period of  contracture i n rat hearts and they speculated that glycerol treatment unphysiological Ca fluxes.  caused  After this period the hearts were viable i f the  recovery solution contained 2 mM CaC^-  Eisenberg and Eisenberg (1968) cautioned  against interpretation of the effect i n muscles other than s k e l e t a l muscles. This does not appear to be a widely used procedure i n smooth muscle studies. 'Glycerol shock' was used i n the present study to see whether caveolae invaginations of the plasma membrane could be severed from the plasma membrane and  whether the densities sometimes connecting sarcoplasmic reticulum  sacs to the  caveolae (Somlyo and Somlyo 1976) might be necessary for direct transmission of the depolarization  to sarcoplasmic reticulum  for Ca release.  Normal Tyrode's  solution was made hypertonic by the i n c l u s i o n of 400 mM g l y c e r o l .  The muscles  (suspended as usual) were equilibrated with the hypertonic Tyrode's solution for 30 min and then quickly washed twice with the normal Tyrode's solution.  After  30 min, muscles were retested f o r their response to CD and high KC1. One hr treatments and washes are required  to disrupt 90% of the triads ( s t r u c t u r a l l y  d i s t i n c t regions where transverse tubules and terminal cisternae of the sarcoplasmic reticulum  come together) of s k e l e t a l muscle (Franzini-Armstrong et. al.1973)  A time of 30 min was f e l t to be s u f f i c i e n t i h longitudinal i l e a l muscle s t r i p s , since these are much thinner.  III.  '  "  '  Measurement of Muscle Na, K, Mg and Ca Tissues were equilibrated f o r 30 min before any control or experimental ion  levels were determined. a.  Total ion measurement  For the measurement of t o t a l ion content i n control tissues.the were equilibrated, blotted, dried and weighed. sues as described  below.  Ions were extracted  tissues  from the t i s -  For measurement of t o t a l i o n content, the experimental  tissues were equilibrated and then washed with various solutions  (e.g. Ca-free  Tyrode's solution or Ca,Mg-free Tyrode's solution) for certain time intervals and then blotted, dried and weighed. b.  Ions were extracted  as described below.  Modified 'La method' for measuring e s s e n t i a l l y i n t r a c e l l u l a r Na, K, Mg and Ca  I n t r a c e l l u l a r ion contents were determined by a modification method' (Van Breemen and McNaughton 1970).  of the 'La  Muscles used f o r determining control  i n t r a c e l l u l a r ion levels were equilibrated and then were quickly released  from  the transducers and plunged within 2 to 3 sec into 200 ml of i c e cold 10 mM LaCl^ and 160 mM Tris-HCl solution, pH 7.4 (La-Tris solution).  For determining ex-  perimental i n t r a c e l l u l a r ion levels muscles were equilibrated, then treated under experimental conditions (e.g. contraction by CD for 10 min) and plunged into La-Tris solution, as described above.  Muscles were cut free from the t i s -  sue hooks while they were submerged i n the solution and transferred (by means of  the s u r g i c a l thread that had connected them to the transducers) to 800 ml of  La-Tris solution i n an i c e bath for a t o t a l time of 30 min. ted,  dried and weighed.  Tissues were b l o t -  Ions were extracted from the muscles as described  below. c.  Extraction of ions from tissues (total and i n t r a c e l l u l a r ion measurements) and atomic absorption measurements  For  t o t a l and i n t r a c e l l u l a r ion measurements the muscles were blotted bet-  ween f i l t e r papers and then dried i n preweighed at 120°C.  2 ml volumetric flasks for 3 hr  Care was taken to ensure that the volumetric flasks were at room tem-  perature and positioned the same way on the balance pan for each weighing.  The  muscle weights were accurately determined by difference to +0.01 mg on a Mettler H20 T balance.  Muscles were dissolved i n 0.2 ml of a 1:1 mixture of g l a c i a l  acetic acid and 3 M TCA (Sparrow and Johnstone 1964) i n a b o i l i n g water bath. Addition of 1 ml of H-O and reheating caused protein aggregation.  LaCl-. (2 rimol<  in 0.2 ml) was added (to prevent Ca and Mg complexation with phosphate) and the volumes were made up to 2 ml with H^O.  Protein was removed by centrifugation at  maximum speed for 10 min i n an IEC model EXD centrifuge equipped with 6 tube adaptor blocks.  Aliquots (1 ml) of these deproteinized extracted muscle solu-  tions were diluted with 1 ml of a KC1 solution ( 5 mg K/ml, to suppress i o n i z a tion) to measure Ca and Mg ( f i n a l volume = 2 ml). Aliquots (0.4 ml) of the deproteinized extracted muscle solutions were diluted with 3.6 ml of IL-O to measure Na and K ( f i n a l volume = 4 ml). Standard solutions of these ions contained similar amounts of LaCl-, KC1, acetic acid and.TCA.  Blanks made up of a l l r e -  agents but without the muscle were subtracted from each reading (see Table 1 for description of standards and c a l c u l a t i o n s ) .  Samples were analyzed i n a Varian  IA3LE 1 . Atomic a b s o r p t i o n s t a n d a r d s and  c a l c u l a t i o n s f o r ug element i n t i s s u e samtiles  p r e p a r a t i o n of 500 ml o f s t a n d a r d s o l u t i o n element standard ' and stock' concentration solution (ml)  acetic TCA  3  a c i d : LaCl  samples and d i l u t i o n s KC1  -  (ml)  (nig)  (g)  Ca 1 ug/ml  0.5  25  88.3  4.77  Mg 0. 6 ug/ml.  0.3  25  88.3  4.77  Na 2 ug/ml  1.0  5  18  K 1.5  0.75  5  18  -  final sample volume (ml)  1/2  (1 ml)  2  1/2  {1 ml)  2  1/5  (.4  ml)  1/5  (.4  ml)  calculation d. b l a n k  factor  (ug) = (absorbance  of sample w i t h no  , . , , , _ ug element i n t i s s u e sample/mg d r y wt. ' e  v  6  =  calculation factor  («)  (ml/ug)  average blank  d.  (ug)  1  4  0.098± .008 n = 33  4  1.0  0.5  40  1.12 'n =  4  0.5  0.333  60  1.96 - .156 n = 43  .  1,0  Small a l i q u o t s o f t h e s e w e r e used to p r e p a r e  2-ml)  tissue)(factor)  (absorbance of t i s s u e sample) i'factor) - blank : : — — — t i s s u e d r y wt. c  c.  0.6  r  (a)(fraction cf i n i t i a l  3 .  . 4  a  :  absorptivity* value  .  and c o n t a i n e d 1 mg/ml of the iilement.  c  expanded absorbance scale setting  calculations  1  . expanded absorbance s c a l e s e t t i n g absorptivity = — , . c o n c e n t r a t i o n of STD (a) 1  e.  -  ug/nl  a . .AA standards were purchased from F i s h e r i n the ug/ml range of the samples. b.  fraction of i n i t i a l 2 ml sample  spectrometry  0.26 n =  .014 41  .156 42  accurate-standards  30 Atomic Absorption Spectrophotometer (AA-5) under the following conditions. element  IV.  wavelength (nm)  lamp current (mAmp.)  slit width  flame  CM)  Ca  422.9  4  100  Mg  285.2  4  95  Na  589.0  5  95  K  766.5  5  100  N-0acetylene airacetylene airacetylene airacetylene  Preparation of Sarcolemmal Enriched Microsomes The longitudinal layer of the ileum was dissected as described i n Methods  (section 1, innervated).  Long muscle s t r i p s were washed with twenty 10 ml por-  tions of oxygenated Ca,Mg-free Tyrode's solution for 10 min at 37°C and then washed f o r a further 3 hr and 50 min at 4°C.  The tissue was weighed (approxi-  mately 1.4 g from 2 animals) and washed twice with i c e cold 0.25 M sucrose (adjusted to pH 7.4 with h i s t i d i n e ) for 10 min, minced w e l l with scissors i n 20 volumes (by tissue wet weight) of 0.25 M cold sucrose and homogenized with 10^12 strokes at 300 rpm (Fisher Dyna-Mix) i n a Potter-Elvehjem homogenizer with t e f l o n pestle (.004-.006 inch clearance) i n ice.  The homogenate was centrifuged  at 2,700 x g at 4°C (IEC-B20A centrifuge) for 20 min.  The supernatant was cen-  trifuged twice at 27,000 x g for 20 min (IEC B20A centrifuge) and then at 105,000 x g (Beckman L2-65B centrifuge) for 1 hr at 4°C.  The microsomal and  other p e l l e t s were resuspended i n 5 volumes by tissue wet weight of cold sucrose (0.25 M) i n a Potter-Elvehjem homogenizer (150 rpm, 2-3 strokes).  V.  Measurement a.  of ATPases and Membrane Marker Enzyme A c t i v i t i e s  Mg dependent Na,K-ATPase was measured i n 0.6 ml of a medium containing  25 mM h i s t i d i n e (pH 7.4), 0.1 mM EGTA, 3 mM MgCl- and 15-25 p g of microsomal protein (0.05 ml of c e l l f r a c t i o n ) .  NaCl (100 mM),  3 mM KC1 and 3 mM ouabain  were added according to whether Na stimulation, K stimulation or ouabain i n h i b i -  31 ticm were b e i n g s t u d i e d . 16 x 98 mm)  Assay  tubes  ( p o l y p r o p y l e n e Kimble  centrifuge  tubes,  were p r e i n c u b a t e d w i t h s h a k i n g f o r 5 min a t 37°C (Dubnoff M e t a b o l i c  Shaking I n c u b a t o r ) .  The r e a c t i o n was s t a r t e d w i t h 3 mM ATP and was stopped  5 min w i t h 0.2 ml o f 20% TCA.  after  The tubes were c e n t r i f u g e d at 4°.C a t 10,000 x g  (IEC B20A c e n t r i f u g e ) and 0.5 ml a l i q u o t s were assayed f o r i n o r g a n i c phosphate by the method o f Lowry and Lopez (1949) (see Methods s e c t i o n V I ) . b.  Microsomal  Ca-ATPase was assayed  i n 0.6 ml of 100 mM  —6 —3 (pH 7.4) c o n t a i n i n g C a C l ^ (10 - 10 M).  Tris-HCl buffer  Some assays a l s o c o n t a i n e d 3 mM ii  MgCl for  or 0.1 mM EGTA as i n d i c a t e d .  2  Microsomal  protein  (15 - 25 ug) was  5 min at 37°C and the r e a c t i o n was s t a r t e d w i t h ATP  incubated  ( u s u a l l y 3 mM u n l e s s the  e f f e c t o f v a r y i n g the s u b s t r a t e c o n c e n t r a t i o n was b e i n g s t u d i e d ) and stopped 5 min  (unless otherwise s t a t e d ) w i t h 0.2 ml o f 20% TCA.  after  P r o t e i n was removed by  c e n t r i f u g a t i o n and P_. was assayed by the method o f Lowry and Lopez (1949) (see 2+ Methods s e c t i o n V I ) .  F r e e Ca  c o n c e n t r a t i o n s were c a l c u l a t e d a c c o r d i n g to the  method of Katz e t a l . (1970) (see Appendix). c.  Actomyosin  histidine  Ca-ATPase was assayed  (pH 7.6), 425 mM KC1, 4 mM  fraction protein  i n 0.6 ml o f a medium c o n t a i n i n g 50 mM  C a C l ~ , and a p p r o x i m a t e l y  (0.05 ml o f 105,000 x g s u p e r n a t a n t )  70 y g of s o l u b l e  (Wolowyk et a l . 1971).  A f t e r a 10 min p r e i n c u b a t i o n a t 37°C the r e a c t i o n was s t a r t e d w i t h ATP t r a t i o n g i v e n i n legends) and stopped t e i n was method d.  a f t e r 10 min w i t h 0.2 ml of 20% TCA.  removed by c e n t r i f u g a t i o n and P  was determined  Pro-  by the Lowry and Lopez  (1949) (see Methods s e c t i o n V I ) . 5'-Nucleotidase  (1973) i n 50 mM  Tris-HCl  was assayed e s s e n t i a l l y by the method, of Tanaka e t a l . (pH 8.6), 2 mM MgCl-. and 2 mM AMP.  Protein  (depending on the c e l l f r a c t i o n ) was s o l u b i l i z e d w i t h 3% T r i t o n X-100 b e f o r e adding i t to the assay medium ( f i n a l ,0.5%).  (concen-  (20 - 100 y g) f o r 10 min  c o n c e n t r a t i o n of T r i t o n X-100  T h i s amount o f T r i t o n X-100 i s i n excess over p r o t e i n f o r every  The medium, i n a f i n a l volume o f 0.6 ml, was r e a c t i o n was stopped w i t h 0.2 ml of 20% TCA.  was  fraction.  i n c u b a t e d a t 37°C f o r 1 h r and the The tubes were c e n t r i f u g e d at  32 10,000 x g f o r 10 min and 0.5 ml a l i q u o t s were assayed f o r P_^ by the method o f Lowry and Lopez (1949) (see Methods s e c t i o n V I ) . e. et  A c e t y l c h o l i n e s t e r a s e was determined by t h e c o l o r i m e t r i c method o f E l l m a n  a l . (1961).  Subcellular fractions  25°C i n 3 ml o f 0.1 M T r i s - H C l nitrobenzoic acid  (20 — 100 p g o f p r o t e i n ) were assayed a t  (pH 8.0), 0.4 M NaCl, 0.23 mM 5 , 5 ' d i t h i o b i s - 2 -  (DTNB) and 1 mM a c e t y l t h i o c h o l i n e i o d i d e , and r e a d a t 412 nm  on a double beam spectrophotometer f i t t e d w i t h a w a t e r - j a c k e t e d temperaturer e g u l a t e d c u v e t t e chamber. er.  R e s u l t s were r e c o r d e d on a P e r k i n Elmer c h a r t  Blanks d i d n o t c o n t a i n DTNB o r enzyme.  record-  A s u b s t r a t e b l a n k (DTNB p l u s a c e t y l -  t h i o c h o l i n e b u t no enzyme) was s u b t r a c t e d from each r e a d i n g to account f o r spontaneous h y d r o l y s i s o f t h e s u b s t r a t e . f.  NADH Oxidase was determined by the method o f S o t t o c a s a e t a l . (1967) by  f o l l o w i n g the r e d u c t i o n o f f e r r i c y a n i d e a t 420 nm w i t h time i n a H i t a c h i - P e r k i n Elmer double beam spectrophotometer. 20 mM potassium phosphate  buffer  The assay medium c o n t a i n e d 0.5 mM KCN,  (pH 7.4), 5 uM CaCl„, 0.5 mM NADH, 1 mM KFe (CN,) ,  and 40-200 Mg o f enzyme was assayed a t 25°C i n a f i n a l volume o f 3 ml.  A blank  c o n t a i n i n g NADH and KFe(CN^) b u t no enzyme was s u b t r a c t e d from each r e a d i n g . The r e a c t i o n was s t a r t e d w i t h enzyme. g.  S u c c i n i c Dehydrogenase was determined e s s e n t i a l l y as d e s c r i b e d by  Schoner e t a l . (1967) by f o l l o w i n g the r e d u c t i o n o f 2 , 6 - d i c h l o r o p h e n o l - i n d o p h e n o l (0.37 M) a t 578 nm. phate of  The r e a c t i o n medium a l s o c o n t a i n e d 0.01 M potassium  (pH 7.4), 3.75 uM C a C l , 0.01 M KCN, 0.01 M sodium 2  p r o t e i n i n a 3 ml volume a t 25°C.  phos-  s u c c i n a t e and 80-100 y g  I n t a c t m i t o c h o n d r i a have s t r o n g permeabi-  l i t y b a r r i e r s t o dyes b u t t h e i n c l u s i o n o f Ca, a t low c o n c e n t r a t i o n s , p e r m i t s f r e e permeation of dyes  (Bernath and S i n g e r 1962).  enzyme so t h a t t h e enzyme would be exposed  to the p r o t e c t i v e e f f e c t o f s u c c i n a t e  and would n o t be i n h i b i t e d by pre-exposure t o KCN. c a l c u l a t e d on the b a s i s o f the i n i t i a l  The r e a c t i o n was s t a r t e d w i t h  The s p e c i f i c a c t i v i t y was  (0-2 min) r e a c t i o n  rate.  33 VI.  Inorganic Phosphate Determination Inorganic phosphate was determined by the method of Lowry and Lopez (1949).  The assay medium (0.5 ml), containing 5% TCA a f t e r termination of the reaction, was d i l u t e d with 2 ml of 0.1 N sodium acetate i n 15 ml Corex centrifuge tubes to minimize s i l i c a t e interference of P^ determination. A 1:1  (v/v) mixture  (0.5 ml) of 1% ascorbic acid and 1% ammonium molybdate i n 0.05 N H~S0^  (mixed  just before using and discarded after 10 min) was added at room temperature by Vortex mixing and the colour was read a f t e r 30 min at 660 nm i n a Hitachi-Perkin Elmer double beam spectrophotometer.  Standards were prepared i n the same way as  samples except they did not contain ATP or enzyme but rather known amounts of KH^PO^ (0.1-0.5 ymoles P_./0.6 ml incubation volume).  Values for samples con-  taining ATP but no enzyme (0.01 - 0.02 absorbance) and enzyme but no ATP 0 absorbance) were subtracted from each reading.  (usually  Experimental samples were read  against blank samples containing no enzyme and no ATP.  VII.  Protein Determination Protein was determined according to Lowry et a l . (1951) using bovine serum  albumin (BSA) as a standard.  C e l l fractions (0.05 to 0.1 ml) (whole homogenates  and the 2,700 x g p e l l e t were diluted 5 fold) were mixed with 0.5 ml of 1 N NaOH for 30 min.  The volume was made up to 1 ml with H.O.  Five ml of a mixture of  2% Na C0„ (50 ml) and 0.5% CuSO. (0.5 ml) and 1% sodium, potassium tartrate I 3 4 o  (0.5 ml) was added by vortex mixing and was l e f t to stand for 10 min.  Phenol  reagent (Folin-Ciocalteau) (0.5 ml of IN) was added by rapid Vortex mixing and the solution was read at 660 nm after standing for 30 min at room temperature. Standards (25-100 ug BSA) were treated the same way. VIII.  Electron Microscopy a.  Electron microscopy - whole tissues  The ileum was dissected i n Tyrode's solution.  The longitudinal layer was  removed as usual and equilibrated for 10 min i n Tyrode's solution.  The s t r i p s  were cut into tiny pieces (approximately 1mm ) J  and fixed i n 10 ml of 2% glutar-  aldehyde i n Tyrode's solution for 10 min at 37°C and 1 hr and 50 min at 4°C. The samples were washed with Tyrode's solution 5 times during 5 min and postfixed i n 1% OsO^  i n Tyrode's solution for 2 hr.  during 5 min with Tyrode's solution.  They were again washed 5 times  Samples were dehydrated i n ethanol at 4°C  in 30% for 5 min, 50% for 5 min, 70% (containing saturated uranyl acetate) for 30 min, 90% for 30 min and 100% for 30 min.  They were equilibrated to room tem-  perature during a f i n a l 30 min i n 100% ethanol. 1:1  I n f i l t r a t i o n was commenced i n a  (v/v) mixture of 100% ethanol and Spurr's medium for 20 min, 1:3 for 20 min  and 100% Spurr's medium overnight.  Embedding was  completed  at 60°C f o r 7 hr  under vacuum. b.  Electron microscopy - microsomal and mitochondrial p e l l e t s  C e l l fractions were prepared as usual except that h i s t i d i n e was the isotonic sucrose homogenization  medium.  omitted from  P e l l e t s were loosened from the bot-  tom of the centrifuge tubes with a Pasteur pipet which was sealed and s l i g h t l y hooked at one end i n a Bunsen burner. pieces.  The p e l l e t s were broken into a few small  The pieces were transferred to small f i x a t i o n bottles and fixed at 4°C  with 1% glutaraldehyde i n a mixture of 8 ml of isotonic sucrose and 2 ml of 0.2 M Sorenson's phosphate buffer (pH 7.4)  for 5 min.  The samples were washed twice  with 0.25 M sucrose for 5 min and post-fixed with 1% OsO^ 2 hr.  i n 0.25 M sucrose for  The samples were washed twice with isotonic sucrose p r i o r to dehydration  i n ethanol at 4°C.  The dehydration and i n f i l t r a t i o n and embedding stages were  exactly the same as' for the whole tissue.  Spurr's medium contained v i n y l c y c l o -  hexane dioxide (10 g), d i g l y c i d y l ether of propylene glycol (6 g), nonenyl succinic anhydride (26 g) and dimethylaminoethanol  (0;4 g).  pleted at 60°C under 5 p of Hg vacuum i n a Marsh Instrument c.  Embedding was comCompany oven.  Sectioning and staining  Copper grids were coated with parlodian f i l m and then l i g h t l y coated with carbon.  The parlodian f i l m was dissolved i n acetone p r i o r to picking up the  sections.  Sections (60 - 100 nm thick) were cut with freshly prepared glass  knives, on a S o r v a l l Porter Blum microtome MT-1  (hand operated).  Sections were  stained with saturated uranyl acetate for 30 min more, washed with H^O for 10 min and stained with Reynold's lead c i t r a t e f o r 10 min and again washed for 10 min.  Electron micrographs were obtained on a P h i l i p s 75 or a Zeiss EM-10 elec-  tron microscope. Some thicker sections (2 u thick) were cut with the microtome, mounted on glass s l i d e s and stained with 1% toluidine blue for 5 min to be viewed under a light  microscope.  /  36  MATERIALS  The  c h e m i c a l s were p u r c h a s e d  Sigma: ATP ( d i s o d i u m s a l t ) , ouabain,  the f o l l o w i n g  5'-AMP, NADH, b o v i n e  EGTA, DTNB, h i s t i d i n e  chol, methacholine  from  serum  (free base), Trizma  and n e o s t i g m i n e .  Fisher:  companies: albumin,  base,  LaCl-,,  carba-  sucrose,  atomic  a b s o r p t i o n s t a n d a r d s , KCN, sodium a c e t a t e , d e x t r o s e ,  phenol  reagent,  Baker a. M g C ^ .  sodium, p o t a s s i u m  tartrate,  s o d i u m s u c c i n a t e , KC1.  lead  citrate.  M a l l i n c k r o d t : CaCl-,.  (  NaCl,  TCA, ammonium m o l y b d a t e ,  parlodian.  Baker  and Adamson:  KH-PO^, u r a n y l a c e t a t e , c o n c e n t r a t e d HC1.  British  nicotine,  KFe(CN)g,  ascorbic  M a t h e s o n and B e l l : C-I-L:glacial cyclohexane  acid,  CuS0^.5H-0.  2 ,6-dichlorop.henojj_ndS^  acetic  dioxide,  dimethylaminoethanol, glutaraldehyde.  NaHCO-., g l y c e r i n ,  Drug House:  acid.  .  Electron Microscopy  diglycidyl  Science:  ether of propylene  OsO^. T o u s i m i s  Research  glycol,  incorporation:  Rohn and Haas Co.: T r i t o n - X - 1 0 0 .  cis-2-methy1-4-dimethylaminomethy1-1,3-dioxolane was a g i f t at  Buffalo.  of  British  from  EDTA  P) ATP was a g i f t  were p r e p a r e d  demineralizer).  ion  -  State U n i v e r s i t y  methiodide of New  f r o m D r . S. K a t z ,  York  University  Columbia.  distilling  assay  Dr.'D.J. T r i g g l e , 32  (y  Solutions AG-3  vinyl-  or s t o r e  apparatus  fitted  Glassware solutions  i n a l l glass  with a Barnstead  and p o l y p r o p y l e n e t u b e s  used  f o r experiments  c o n t e n t were washed w i t h c h r o m i c solution  and d i s t i l l e d  distilled  water.  acid,  H-0  (Corning  Sybron used t o  concerned  tap water,  with  0.1%  RESULTS AND DISCUSSION  I.  Procedure for Isolating Sarcolemmal Enriched Microsomes A number of procedures for the preparation of sarcolemmal enriched f r a c -  tions were examined before the f i n a l procedure (described i n the methods) was developed.  In the procedure to i s o l a t e sarcolemmal enriched microsomes from  guinea pig ileum longitudinal smooth muscles described by Hurwitz et a l . (1973), the excised tissue was d i r e c t l y homogenized i n 0.25 M sucrose with a Potter Elvehjem homogenizer (similar to the method of Godfraind and Verbeke  (1973) who  used 0.32 M sucrose containing 0.08% deoxycholate and 0.1 mM cysteine as the homogenization medium).  In the present study the tissue was very tenacious un-  der these conditions which made i t d i f f i c u l t to homogenize.  The y i e l d of micro-  somes seemed very low and the resuspended p e l l e t invariably formed a g e l .  Sub-  s t i t u t i n g 0.65 M KC1 solution for the sucrose solution prevented the g e l l i n g but did not aid the homogenization.  The above procedure yielded a f r a c t i o n with  considerable ATPase a c t i v i t y when stimulated by Mg, but Na and K did not noticeably further stimulate the a c t i v i t y .  The appropriateness of homogenizing the  tissue i n deoxycholate (method of Godfraind and Verbeke 1973) seemed questionable since i n t r a c e l l u l a r organelles might p e l l e t at any c e n t r i f u g a l force depending on the extent to which they were s o l u b i l i z e d .  A low speed sarcolemmal .  f r a c t i o n prepared by the method of O l i v i e r a and Holzhacker (1974) apparently yielded 'ghosts and membrane fragments' of sarcolemma.  Following their proce-  dure, the longitudinal layer of the ileum was homogenized by a S o r v a l l Omni-mixer ( $ 6 r v a l i Omni-mixer was substituted for a V i r t i s homogenizer) i n a medium of 0.25 M sucrose, 1 mM EDTA and 20 mM Tris-maleate (pH 7.4), then f i l t e r e d and centrifuged at 650 x g.  The p e l l e t was resuspended with a Potter Elvehjem homo-  genizer and centrifuged at 164 x g (repeated 5 times) and incubated at 37°C for 30 min i n the i n i t i a l buffer system to extract 'cytoplasmic inclusions'. material was washed and centrifuged at 1,000 EDTA solution to extract actomyosin.  The  x g, 6 to 7 times more i n 0.01  mM  This preparation had v i r t u a l l y no ATPase  a c t i v i t y even when the number of washing procedures was  reduced,  A p i l o t study  using a pyrophosphate buffer to chelate divalent ions, thereby softening cohesive material between c e l l s , seemed to increase the tissue by the Potter-Elvehjem homogenizer. Ca,Mg-free Tyrode's solution for 10 min  the extent of disruption of  E q u i l i b r a t i o n of the tissue i n  at 37°C and  3 hr and 50 min at  (shorter times are also effective) without the addition of any (the method which was  chosen) was  the  4°C  chelating agents  observed to have the same e f f e c t .  Foaming  was  apparent on the top of the oxygenated tissue bath used for the depletion step at 37°C probably indicating the loss of i n t e r s t i t i a l connective proteins Ca,Mg-free Tyrode's solution.  Connective tissue proteins and  teins were assumed to be extracted  c o n t r a c t i l e pro-  from the tissues that were washed i n Ca,Mg-  free Tyrode's solution because the resuspended p e l l e t s did not gel and t e i n recovery i n the microsomal f r a c t i o n was  very low  (1%)  determined.  The e f f e c t of  the tissue i n Ca,Mg-free Tyrode's solution on t o t a l ion content  was  Following dissection of the tissues and e q u i l i b r a t i o n for 30 min  normal Tyrode's solution, the t o t a l Ca, Mg, a f t e r incubation 37°C and  the pro-  (Table 2), although  the s p e c i f i c a c t i v i t i e s of the microsomal enzymes were high. incubating  into the  Na and K contents were determined  of the tissues i n Ca,Mg-free Tyrode's solution for 10 min  then after various times at 4°C  in  (Fig. 3, s o l i d l i n e s ) .  at  The tissues l o s t  65% of their Ca content, 27% of their Mg content, 78% of t h e i r K content and gained 23% more Na after 4 hr, i n d i c a t i n g that the tissues had become leaky. Two  additional washes of the leaky tissues with isotonic sucrose further removed  tissue Ca, Mg and K to 22, 54 and 13% of t h e i r i n i t i a l amounts respectively washed away 53% of the e x t r a c e l l u l a r Na  (Fig. 3, dotted l i n e ) .  and  At this point,  the f r a g i l e c e l l s s t i l l held together as a tissue but they were very f l a c c i d about 4 strokes with a Potter-Elvehjem homogenizer e a s i l y dispersed c e l l u l a r material into the medium. more strokes to increase  Homogenization was  the extent of disruption.  most of  and the  continued for about 10  Electron micrographs of  27,000 x g p e l l e t confirmed that the majority of the mitochondria were l e f t  the  39 intact although they were swollen and mostly i n the condensed configuration (Somlyo et a l . 1975) (see plate 1, #1, 2 and 3). Cristae-without the outer mitochondrial membrane also sedimented at 27,000 x g. At this speed, v e s i c l e s of endoplasmic reticulum and sarcolemma also may have p e l l e t e d .  Indeed, endoplas-  mic reticulum and sarcolemmal marker enzyme a c t i v i t i e s were present i n this fraction (Table 2) but s a c r i f i c i n g some of the microsomal y i e l d to the 'mitochond r i a l ' p e l l e t seemed j u s t i f i a b l e for the sake of having a p a u c i t y of mitochondria i n the microsomal f r a c t i o n .  The microsomal f r a c t i o n contained amorphous small  v e s i c l e s but d i d not contain mitochondria  or mitochondrial dark s t a i n i n g parts  (Plate 2, #4 and 5) although the centre of plate 2, #6 has an unvesiculated dark membrane which looks suspiciously l i k e a c r i s t a e .  Some uneven fuzziness of the  membrane (plate 2, #5) could be i n d i c a t i v e of the basement membrane.  Subcellu-  l a r fractions were characterized by the d i s t r i b u t i o n of marker enzymes. The d i s t r i b u t i o n of 5'-nucleotidase  and acetylcholinesterase i n the subcellular,  fractions (Table 3) indicated that plasma membrane was concentrated somal f r a c t i o n .  i n the micro-  A Na stimulated Mg-ATPase a c t i v i t y was also concentrated  i n the  microsomal f r a c t i o n (Table 3) and i n the presence of a small volume of soluble activating factor, an ouabain i n h i b i t a b l e K stimulation could be demonstrated (see Fig. 6, C and D).  NADH oxidase was used as an endoplasmic reticulum marker  enzyme i n guinea p i g l o n g i t u d i n a l i l e a l and r a t a o r t i c smooth muscle c e l l s by Hurwitz et a l . (1973) although there i s no indisputable endoplasmic reticulum marker enzyme for smooth muscle studies (_Wei et a l . .1976).  Twice as much NADH  oxidase was recovered i n the 27,000 x g 'mitochondrial' p e l l e t than i n the microsomal f r a c t i o n although the s p e c i f i c a c t i v i t y was just s l i g h t l y greater i n the microsomal f r a c t i o n .  Succinic dehydrogenase a c t i v i t y , an inner mitochon-  d r i a l membrane marker enzyme of highest s p e c i f i c a c t i v i t y i n the 27,000 x g p e l l e t , was not detectable i n the microsomal f r a c t i o n (Table 2). Surprisingly, a considerable amount of the s u c c i n i c dehydrogenase a c t i v i t y was released from the mitochondria  and appeared i n the soluble f r a c t i o n .  The 27,000 x g 'mito-  chondrial' p e l l e t a l s o contained h i g h s p e c i f i c a c t i v i t i e s marker enzymes but as expected,  f o r plasma membrane  a l s o c o n t a i n e d the h i g h e s t s p e c i f i c a c t i v i t y of  the m i t o c h o n d r i a l marker enzyme. An attempt was  made t o s e p a r a t e the endoplasmic r e t i c u l u m from the s a r c o -  lemmal v e s i c l e s on a s u c r o s e d e n s i t y g r a d i e n t ( F i g . 4).  Although  p r o t e i n separ-  ated i n t o 4 peaks, the NADH o x i d a s e cosedimented w i t h the 5 ' - n u c l e o t i d a s e a c e t y l c h o l i n e s t e r a s e at a s p e c i f i c g r a v i t y of a p p r o x i m a t e l y dehydrogenase a c t i v i t y which was  absent  d e t e c t a b l e i n the g r a d i e n t f r a c t i o n s .  caveolae  u i n diameter;  to b e i n g pinched  still  T h e r e f o r e the microsomal p e l l e t was  unused  further purification.  t h a t i n c r e a s e the s u r f a c e a r e a of smooth muscle c e l l s are not  c h a r a c t e r i s t i c of other muscle t y p e s . than 0.1  Succinic  i n the microsomal p e l l e t , was  to study plasma membrane ATPase a c t i v i t i e s without The  1.122.  and  Perhaps t h e i r v e s i c u l a r - l i k e shape  (less  see R e s u l t s and D i s c u s s i o n , s e c t i o n IV) l e n d themselves  o f f i n t o microsomal s i z e d v e s i c l e s  d i a m e t e r ) , which might e x p l a i n why  t h e r e was  (approximately  0.085 u i n  a good r e c o v e r y of sarcolemmal mark-  er enzymes i n the microsomes as compared to o t h e r t i s s u e s i n which plasma membranes a r e u s u a l l y i s o l a t e d (1964) observed,  acids.  port processes.  and  Barrnett  t r i - p h o s p h a t e s but not monophosphates or other  T h i s i m p l i e s t h a t they may I f the microsomes a r e mainly  microsomes would be expected enzymes.  Rostgaard  by e l e c t r o n dense l e a d s t a i n i n g , t h a t c a v e o l a e e n z y m a t i c a l l y  h y d r o l y z e n u c l e o s i d e d i - and phosphoric  from low speed f r a c t i o n s .  be i n v o l v e d i n a c t i v e i o n t r a n s pinched  o f f c a v e o l a e , then  to have h i g h s p e c i f i c a c t i v i t i e s  the  for ion transport  41  F i g . 3.  C a t i o n l e v e l s of the guinea p i g i l e u m l o n g i t u d i n a l smooth muscle a f t e r d e p l e t i o n of the t i s s u e i n Ca,Mg-free Tyrode's s o l u t i o n as used i n the procedure f o r p r e p a r i n g sarcolemmal e n r i c h e d microsomes. C o n t r o l i o n c o n t e n t s (zero time on s o l i d l i n e ) were measured a f t e r e q u i l i b r a t i o n of the t i s s u e f o r 30 min i n T r i s - T y r o d e ' s s o l u t i o n . T i s s u e i o n cont e n t s were measured a f t e r Ca and Mg d e p l e t i o n f o r 10 min a t 37°C and a f t e r v a r i o u s times up to 4 hr at 4°C i n Ca,Mg-free Tyrode's s o l u t i o n (solid lines). The d o t t e d l i n e s i n d i c a t e i o n content of e q u i l i b r a t e d t i s s u e s washed twice f o r 10 min each time w i t h i c e c o l d i s o t o n i c s u c rose (zero time) and washed twice w i t h c o l d i s o t o n i c s u c r o s e s o l u t i o n a f t e r Ca and Mg d e p l e t i o n f o r 10 min a t 37°C and f o r f u r t h e r times a t 4°C up to 4 h r . Each p o i n t r e p r e s e n t s the mean + S. E.  43  P l a t e 1.  1.  27,000 x g p e l l e t . The f r a c t i o n c o n t a i n s i n t a c t m i t o c h o n d r i a , a l t h o u g h abnormal i n appearance. Some are l i g h t l y and o t h e r s darkly stained. The i s o l a t e d m i t o c h o n d r i a are l a r g e r than they are i n the i n t a c t c e l l (see P l a t e s 3-5). (14,700 x m a g n i f i c a t i o n Z e i s s EM-10)  2.  L i g h t and dark s t a i n e d m i t o c h o n d r i a . L i g h t l y stained mitoc h o n d r i a e x h i b i t double membranes w h i l e the i n n e r membrane and c r i s t a e of dark s t a i n e d m i t o c h o n d r i a aggregate i n the c e n t r e . (24,000 x m a g n i f i c a t i o n Z e i s s EM-10)  3.  S e c t i o n of 27,000 x g p e l l e t showing more microsomal v e s i c l e s t r u c t u r e s mixed w i t h m i t o c h o n d r i a . T h i s s e c t i o n was probably cut near the s u r f a c e of the p e l l e t where a w h i t e l a y e r i s seen to o v e r l a y the dark brown m i t o c h o n d r i a l p e l l e t . Arrow i n d i c a t e s ribosomes on a v e s i c l e . (14,700 x m a g n i f i c a t i o n Z e i s s EM-10)  45  Plate 2.  4.  Amorphous membrane v e s i c l e s i n 105,000 x g microsomal p e l l e t . None of the membrane structures resemble mitochondria. (75,000 x magnification Zeiss EM-10)  5.  Microsomal vesicles occasionally appear to encase other membrane fragments. (120,000 x magnification Zeiss EM-10)  6.  Lower power overview showing amorphous v e s i c l e s with no apparent mitochondria. Note magnification i s 1.56 times greater than Picture 2 of mitochondria. Therefore, i f mitochondria were present, they should appear as very large structures at this magnification. (37,500 x magnification Zeiss EM-10)  TABLE 2.. Location of c e l l marker: enzymes, s p e c i f i c a c t i v i t i e s and % r e c o v e r i e s i n each c e l l Fraction  . t o t a l.^recovery . o.ce Q  whole . homogenate 2,700 x g pellet  91.6 '  2,700 x g supernatant 27,000 x g pellet [mitochondria) 27 ,00.0 x g supernatant  61.3  acetylcholinesterase  5'- •AMPase  Protein-  3  umoles •Pi_i -•1 mg ~hr  recovery de %  100  3.18  100 66.9  • 0.470  54.6  5.8  56.9  •40. 6  30.2  0.435  30.9  4.7  28.4  2.610  10.8  15.0  5.2  ' 0.236  15.2  5.5  30.3  2.2  2.4  9.08  6.8  3 7.0  •1.82  •105 , 000' x g supernatant (soluble)  31.3  •  34.1  nmoles recovery -1 , -1 . mg mm % 100 6.8  61.1  29. 9.  0.9 "••  recovery umoles -1 . -1. • a mg mm 100 0.576  50.3  2.32  0.9  S u c c i n i c Dehydrogenase  NADH oxidase  58.4 .  40.9 ;. ^  105,000 x g pellet (microsomes)  5  2.77  37.5  3 3.9  •n'mo Les recovery - L .mg mm 100 55.0  fraction  19. 09  0. 51  .  ' -150.0 .  6.5  21.2  3 5.3  23.6  6 .'7  259.0  4.4  3.190  5.2  0.0  0.0  5.8  28.2  17.5.  0.115  6.8  6.2  31.1  b. t o t a l p r o t e i n = rr.g/.05 ml x f r a c t i o n volume. c. % recovery p r o t e i n = t o t a l p r o t e i n / t o t a l p r o t e i n homogenate x 100. d. % recovery enzyme a c t i v i t y = s p e c i f i c a c t i v i t y x. t o t a l p r o t e i n of c e l l f r a c t i o n / s p e c i f i c a c t i v i t y x t o t a l p r o t e i n of whole homogenate x 100. e. % recovery i n supernatant and p e l l e t f r a c t i o n f o r each c e n t r i f u g a t i o n f o r c e should equal % recovery from the previous supernatant f r a c t i o n . f . values represent one experiment i n order to show t o t a l a c t i v i t y and % recovery based on t o t a l p r o t e i n measurements. 3 other experiments showed s i m i l a r r e s u l t s .  TABLE " 3 .  S u b c e l l u l a r d i s t r i b u t i o n o f ATPase a c t i v i t i e s  Fraction ° ab  Mg ATPase umoles P i  recovery  mg" min" '•. whole ' homogenate •  0.263  2,700 x g pellet  0.151  2,700 x g supernatant  0.300  27,000 x g pellet [mitochondria)  '  g  0  Mg Na ATPase umoles P i  %  mg~ min~  100  0.410  3 8.5  0. 270  46.4  0.461 '  Mg Na K ATPase  h  recovery . % .'; 1 0 0  ;  umoles P i  recovery  mg~ min~ 0.395  44.1  0. 261  4 6.0  0.464  Mg Na K ATPase^ • + ouabain  1  umoles P i  recovery  %  mg~ min"  %  100  0.429  100  44.2  0.257  40.1  48.1  0.464  44.3.  •  1.714 •  1.  15.5  3.123  20.9  0. 214  .  •' 18. .2  3.013  18.2  2.965  IS.4  '19.3  0.207  19.4  0.205  .17.7  27,00 0 x g supernatant  0.140  10 5,000 x g pellet (microsomes)  2.029  7..4 .  2.974.  6.9  3.037  7.3  3.138  7.0  105,000 x g supernatant (soluble)  O.Otl  5.3  0.048  3.4  0.041  3,5  0.03 8  3.1  •;.  aJbcde s e e t a b l e 2 f. r e s u l t s are the average 1of d u p l i c a t e s . 3 other experiments showed similar r e s u l t s . g. 3 mK MgCl, h. 3 mM MgCl, + 100 mM NaCl i . 3 mM MgCl, + 10 0 mM NaCl + 3 mM KC1 j . 3 mM MgClj + 100 mM NaCl + 3 mM KC1 + 3 mM ouabain. 1  49  Fig. 4.  Sucrose density gradient treatment of the microsomal f r a c t i o n . Seven step discontinuous gradients (1.5 ml of 1.4 to 0.4 M sucrose) were prepared approximately 16 hr before the addition of 1.2 ml of freshly prepared microsomes i n 0.25 M sucrose. Fractions (0.65 ml each) were collected at 4°C using a Brinkman STA multipurpose p e r i s t a l t i c pump and a Gilson Microfractionator. Fractions 1, 5, 9 or 10, 13 and 16 which corresponded with the protein peaks (•) were assayed for a c e t y l cholinesterase ( A ) , succinic dehydrogenase (O)» NADH oxidase (Mi, and 5'-nucleotidase (5'-AMPase) (•) . 1  51 II.  Mg  dependent Na,K-ATPase  The  d i s t r i b u t i o n of v a r i o u s Mg  dependent ATPase a c t i v i t i e s i n the sub-  c e l l u l a r f r a c t i o n s i s shown i n T a b l e i n the microsomal f r a c t i o n . s t i m u l a t e d Mg-ATPase was natant  (Table 3 ) .  Na  found i n every  h y d r o l y s i s r e q u i r e d Mg.  Na  f r a c t i o n except the 105,000 x g super-  None o f the f r a c t i o n s r e q u i r e d K f o r Na  s i m i l a r Na-ATPase a c t i v i t y was and  The h i g h e s t s p e c i f i c a c t i v i t i e s appear  s t i m u l a t i o n o f ATP  were f u r t h e r s t i m u l a t e d by K nor  D a n i e l 1970)  3.  s t i m u l a t i o n and none  i n h i b i t e d by ouabain to any  previously reported  great e x t e n t .  i n r a t myometrial  (Allen  r a b b i t v a s c u l a r muscle f r a c t i o n s (Wolowyk e t a l . 1971).  and  The  degree of Na s t i m u l a t i o n found i n the p r e s e n t  study was  Wolowyk e t a l . (1971) and was  the n e c e s s i t y o f d e o x y c h o l a t e or  ageing creased (3 mM)  treatments  as MgCl. was was  and  mM,  Daniel  d i d not  i n c r e a s e d from 1 to 4 mM 3 mM  ATP  which i s s i m i l a r to the Na (1970).  K  (3 - 12 mM)  ( F i g . 5, upper graph).  f o r t e s t i n g of Na  ATPase a c t i v i t i e s  concentrations mately one  MgCl-^  stimulation.  NaCl  c o n c e n t r a t i o n dependence observed by A l l e n  i n the presence of 3 mM  MgCl-. and  100  mM  NaCl  a f u r t h e r c o n s i s t e n t c o n c e n t r a t i o n dependent a c t i v a t i o n ( F i g . 5,  lower graphs).  not due  by  the a c t i v i t y began to l e v e l o f f  The  reason why  the K s t i m u l a t i o n d i d not  c o n c e n t r a t i o n dependent i n the range of 1 - 12 mM,  K, was  than r e p o r t e d  The microsomal ATPase a c t i v i t y i n -  i n c r e a s e d the a c t i v i t y l i n e a r l y and  induce  upper and  D a n i e l 1970).  r o u t i n e l y used w i t h  (20 - 80 mM) at 100  ( A l l e n and  evident without  higher  A  (Skou 1974)  i s not  clear.  to s i g n i f i c a n t K c o n t a m i n a t i o n present  The  seem to be  i n c o n t r a s t to o t h e r Na  Na,K-  s t i m u l a t i o n , without  i n the membrane f r a c t i o n .  i n the f i n a l microsomal f r a c t i o n  (Table 4) are  added The  ion  approxi-  thousandth o f those r e q u i r e d to o p t i m a l l y a c t i v a t e ATPase a c t i v i t i e s  (Skou 1974).  T h i s agrees w i t h  tetraphenylboron  the f i n d i n g s of Wolowyk et a l . (1971) who  used  as a K c h e l a t o r to demonstrate t h a t the Na a c t i v a t i o n without  added K.wasLnot due-to K;.c on t amina t i bh-of the microsomes. A f u r t h e r a c t i v a t i o n o f the Na  s t i m u l a t e d Mg-ATPase by K r e q u i r e d  a d d i t i o n o f a s m a l l volume o f the s o l u b l e f r a c t i o n  the  (105,000 x g s u p e r n a t a n t ) to  ____  the microsomal f r a c t i o n , as previously reported by Wolowyk et a l , 0-971') i n rabbit vascular smooth muscle.  In the presence of a soluble a c t i v a t i n g factor,  K predominantly kept the ATPase a c t i v i t y of the microsomal f r a c t i o n above that with Mg and Na alone (Fig. 5, lower graph).  However the a c t i v i t y s t i l l  c l e a r l y not a function of the increasing K concentration.  was  Without the addition  of the soluble supernatant K appeared to cause i n h i b i t i o n . The Na stimulation of the Mg-ATPase was the ouabain concentration was s i t e was  inaccessible was  3 mM  examined.  not inhibited by ouabain, even when  (Fig. 6A).  The p o s s i b i l i t y that the ouabain  Ouabain (0.1 mM)  was  solution, the Ca,Mg-free Tyrode's solution and the 0.25 for  added to the Tyrode's  M sucrose medium used  the preparation of the tissue, the homogenization of the tissue and  resus-  pension of the microsomes.  Despite this treatment, Na stimulation was  evident  and the further addition of 3 mM ouabain did not  (greater than 100%)  s i g n i f i c a n t l y reduce the a c t i v i t y (Fig. 6B).  Therefore  still  the resistance of the  Na stimulation to ouabain was not due to i n a c c e s s i b i l i t y of the ouabain s i t e . The small K stimulation i n the presence of soluble f r a c t i o n was sensitive to ouabain (Fig. 6C).  The reversal of the s l i g h t K i n h i b i t i o n of the microsomal  ATPase to a small stimulation of the a c t i v i t y i n the presence of 0.01  ml of  soluble f r a c t i o n could not be accounted for by the small amount of ATPase a c t i v i t y (K independent) i n the soluble f r a c t i o n (Fig. 6C). tion by K was  Therefore  the a c t i v a -  not d i r e c t l y due to the soluble f r a c t i o n but rather was  of a component of the soluble f r a c t i o n on the microsomal a c t i v i t y .  an effect  Numerous  studies of the e f f e c t of the soluble f r a c t i o n on the K a c t i v a t i o n have indicated that the K stimulation was  usually s l i g h t but consistent (Fig. 6D).  The  fluc-  tuations between d i f f e r e n t batches of enzyme made i t d i f f i c u l t to demonstrate a s t a t i s t i c a l l y s i g n i f i c a n t K stimulation unless a paired t - t e s t was  used.  The  difference between Na and K stimulation and Na stimulation alone for every microsomal preparation was  s i g n i f i c a n t l y greater than zero at the 99% l e v e l and  ouabain i n h i b i t i o n of the K stimulation was  also s i g n i f i c a n t at the 99%  the  level.  A r e g u l a t o r y component of, the NajK^ATPase was r e p o r t e d the supernatants  t o be c o n c e n t r a t e d i n  o f homogenized mouse plasmocytoma a s c i t i c  Na,K-ATPase t h a t i s r e s i s t a n t  t o ouabain  c e l l s which have a  ( L e l i e v r e e t a l . 1976a).  Reconstitu-  t i o n o f the r e g u l a t o r y component w i t h t h e membranes r e q u i r e d the a d d i t i o n of Ca and Mg.  Experiments on t h e e f f e c t o f t h e r e g u l a t o r y component on t h e Na,K-ATPase  of mutant c e l l l i n e s l e d L e l i e v r e e t a l . (1976b) to h y p o t h e s i z e  t h a t t h e Na,K-  ATPase-ouabain i n t e r a c t i o n was modulated by a n o n s p e c i f i c membrane s t r u c t u r a l component. Although c o n t r o l experiments i n d i c a t e d t h a t t h e ATPase a c t i v i t y was l i n e a r w i t h enzyme c o n c e n t r a t i o n lower ATP c o n c e n t r a t i o n s  ( i . e . pseudo zero o r d e r w i t h r e s p e c t t o s u b s t r a t e ) , a t the umoles o f P^ l i b e r a t e d  exceeded t h e umoles o f ATP  added t o the assay medium when t h e i n c u b a t i o n was allowed ( F i g . 7, upper graph).  At f i r s t  to proceed f o r 30 min  t h e microsomes were suspected' o f c o n t a i n i n g an  ADPase a c t i v i t y b u t L u t h r a e t a l . (1976) have suggested t h a t an adenosine a c t i v i t y c o u l d a l s o e x p l a i n t h e r e l e a s e o f P_^ from ADP.  kinase  ADP (3 mM) was 1/6 as  a c t i v e as a s u b s t r a t e , compared t o ATP, f o r the microsomal p r o d u c t i o n  o f P.  s  ( F i g . 7, lower graph).  When t h e i n c u b a t i o n time was 5 min,  v e r y l i t t l e ADP  would be produced from ATP h y d r o l y s i s and t h e r e f o r e P^ p r o d u c t i o n negligible.  Under these  the umoles o f ATP added  c o n d i t i o n s , t h e umoles o f P^ produced d i d not exceed ( F i g . 8, i n s e r t ) .  Mg-ATPase a c t i v i t y decreased was  ( F i g . 8, i n s e r t ) and t h e d e c l i n e o f the a c t i v i t y  The a d d i t i o n o f Na and K prevented  Na and K i n c r e a s e d the maximal v e l o c i t y  t h a t t h e r e was a lower a f f i n i t y  (Dahl and Hokin 1974).  (data n o t  the s u b s t r a t e i n h i b i t i o n .  (V ) , t h e K was i n c r e a s e d max m  Whereas suggesting  f o r . ATP i n t h e presence o f Na and K ( F i g . 8 ) .  T h i s r e d u c t i o n o f t h e enzyme a f f i n i t y  f o r ATP by K had been r e p o r t e d by o t h e r s  A r a d i o a c t i v e assay o f t h e ATPase i n d i c a t e d t h a t the  gamma phosphate r e l e a s e was l i n e a r w i t h and  A t h i g h e r ATP c o n c e n t r a t i o n s , t h e  apparent even when t h e Mg c o n c e n t r a t i o n was r a i s e d t o 7.5 mM  shown).  from ADP i s  the s p e c i f i c a c t i v i t y  t h e amount o f microsomal p r o t e i n added  was' ,'in^.i.the'... same range as t h e a c t i v i t y c a l c u l a t e d  by the Lowry and Lopez method (Fig. 9.),  Na alone (with no K) caused an 81%  stimulation of the Mg-ATPase a c t i v i t y . The addition of Ca did not convert the Na-ATPase to a K dependent form (Fig. 10).  Ca alone activated an ATPase i n the microsomes but did not induce any  change i n the Mg dependent Na,K-ATPase pattern of a c t i v i t y observed i n previous experiments.  The Ca and Mg stimulations of the a c t i v i t y were not additive.  Na  nearly doubled the Mg-ATPase a c t i v i t y and K, i n the presence of the soluble act i v a t i n g factor, caused a small further stimulation which was antagonized by 3 mM ouabain. A possible explanation for the unusual behavior of the ATPases i n this tissue could have been that the Na,K-ATPase was changed to a K-independent ouabain-insensitive conformation during the i s o l a t i o n procedure. muscles were tested under the biochemical conditions employed of  The intact  for the isolation,,  the microsomes (except, of course, that the tissues were not homogenized or  centrifuged).  Muscles and the nerve plexus were v i a b l e over 3 days after they  were stored i n cold Tyrode's solution (Fig. 11, Top). the  The f i r s t responses on  second and t h i r d days were abnormal, probably because the tissues had be-  come Na r i c h and K poor when the pump a c t i v i t i e s were depressed at 4°C (Setekleiv 1970).  After e q u i l i b r a t i n g the muscles for longer times, the muscle responses  were normal.  This i s compatible with the view that cold temperature does not  i r r e v e r s i b l y destroy the normal a c t i v i t y of the muscle Na,K-ATPase.  The muscles  also functioned normally i n the physiological medium buffered with 25 mM dine instead of carbonate and phosphate  (data not shown).  histi-  As storage of the  muscles overnight i n isotonic cold sucrose, corrected to pH 7.4 with h i s t i d i n e (i.e.  the homogenization medium), also did not destroy their v i a b i l i t y  (Fig. 11,  lower experiment) the Na,K-ATPase can be assumed to be s t i l l functional. r e - e q u i l i b r a t i o n of the muscles i n normal Tyrode's solution, the muscles  After still  responded to ouabain on the second day, i n d i c a t i n g that the ouabain receptor was not  damaged by the homogenization medium.  The f i n a l test was to see i f the mus-  cle could regain a c t i v i t y and remain responsive to ouabain a f t e r treatment with Ca,Mg-free Tyrode's solution under exactly the same conditions as used to weaken the muscle f o r homogenization  ( i . e . 10 min at 37°C and 3 hr and 50 min at 4°C) .  Surprisingly 61% of the phasic and 33% of the tonic control responses to the muscarinic agent cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide (CD) could be regained after r e - e q u i l i b r a t i o n of the muscles i n normal Tyrode's solution (Fig. 12). Ouabain responses of the muscle were also 33% of their control responses (before Ca,Mg-free Tyrode's solution treatment), but even without Ca,Mg depletion a second response to ouabain was always less than the f i r s t response of the muscle to 'ouabain (Table f o r F i g . 12, 3rd Row) (see also Bolton 1973a; Casteels 1966).  I t seems more l i k e l y that the Ca,Mg-free Tyrode's  solution treatment weakened the muscle c o n t r a c t i l e apparatus and series e l a s t i c components thereby reducing CD and ouabain responses, rather than reducing the responses by destruction of the muscarinic and ouabain receptors.  Since the  muscles remained viable under the above conditions and responded f a i r l y normally, i t seemed reasonable to -assume that the Na,K-ATPase enzyme was not destroyed. The ouabain receptor was not destroyed because the muscles s t i l l responded to ouabain after such treatments.  But i s the Na,K-ATPase the ouabain receptor?  56  Fig. 5.  Stimulation of microsomal ATPases by Mg, Na and K. Points are the average of duplicates. Specific a c t i v i t y was calculated per mg microsomal protein. Incubation time at 37°C was 15 min. Upper Graph. 75 y l of the microsomal f r a c t i o n containing 28.5 yg of protein was assayed i n the presence of 25 y1 of the soluble f r a c t i o n containing 29 yg of protein. (O) MgCl • (•) 3 mM MgCl. plus NaCl; ( A ) 3 mM MgCl plus 100 mM NaCl plus KC1. 2  Lower Graph. 75 p l of the microsomal f r a c t i o n containing 23.2" yg of protein was assayed with 25 y l of soluble supernatant containing 41 yg of protein (#) or without soluble supernatant (•) i n the presence of 3 mM MgCl- and 100 mM NaCl and various K concentrations. The dotted l i n e represents Mg dependent Na-ATPase a c t i v i t y without K addition.  TABLE 4.  Cation l e v e l s i n the microsomal  fraction  Cation concentration (mM) Mg Sucrose (0.25 M )  a  Microsomes prepared^ i n 0.25 M sucrose""  Na  K  Ca  .0034  .747  .0306  .018  .046+.009  .789+.107  .063+.005  .028+.005  .0039  .066  .0053  .0024  0  Microsomes (see method) after d i l u t i o n i n assay Concentration of ions added for assay  3  100  3  av.isotonic sucrose (0.6 ml) was assayed to determine ion content of the suspend ing medium. b. samples of microsomes (0.6 ml) were assayed by the same method used to analyz tissue ion content (see methods). c. n = 6 d. microsomes were diluted 12-fold i n the assay medium.  59  F i g . 6.  The e f f e c t of Mg, Na, K, ouabain and a s o l u b l e a c t i v a t i n g the microsomal ATPases.  factor  on  A.  E f f e c t of Na, K and ouabain on the.Mg-ATPase a c t i v i t i e s i n the microsomal f r a c t i o n . The a c t i v i t i e s r e p r e s e n t the mean + S. E. M = MgCl, (3 mM), N = NaCl (100 mM), K = KC1 (3 mM), 0 = Ouabain (3 mM).  B.  ATPase a c t i v i t y i n the microsomal plasma membrane e n r i c h e d f r a c t i o n i n the presence of 0.1 mM ouabain (see R e s u l t s f o r more details). The f i n a l c o n c e n t r a t i o n o f ouabain i n - e a c h assay tube was 8.33 uM except i n the p r e s e n c e o f MNKO where an a d d i t i o n a l 3 mM ouabain was p r e s e n t .  C.  (1)  Assay of microsomal f r a c t i o n  (19  mg).  (2)  Assay of 19 mg microsomal p r o t e i n p l u s soluble fraction protein.  13.8 mg  (0.01 ml) o f  (2) - (1) r e p r e s e n t s the d i f f e r e n c e between microsomal a c t i v i t y i n the presence o f s o l u b l e f r a c t i o n and the microsomal a c t i v i t y alone. (3) D.  Assay o f s o l u b l e f r a c t i o n p r o t e i n  (69 mg)  i n 0.05  ml.  Assay of the microsomal f r a c t i o n i n the presence of s o l u b l e f r a c t i o n as i n F i g . 6C except t h a t the i n c u b a t i o n time was 15 min i n s t e a d o f 5 min. The a s t e r i s k i n d i c a t e s t h a t the p a i r e d t v a l u e s f o r i n d i v i d u a l a c t i v i t i e s of the means shown were s i g n i f i c a n t l y d i f f e r e n t a t the 99% l e v e l f o r 10 degrees of freedom.  61  Fig. 7.  Upper Graph. Substrate dependence curve of microsomal ATPase. The microsomal f r a c t i o n '(29.5 ug of protein) was incubated at 37°C for 30 min. More P. was liberated than ATP added at ATP concentrations lower than 1.125 mM (0.675 umoles/0.6 ml assay). Slope of the dotted l i n e (ATP/P.) = 1 (•) '3 mM MgCl, (•) 3 mM MgCl and 100 mM NaCl ( A ) 3 mM MgCl^, 100 mM NaCl and 3 mM KC1 1  Lower Graph. Bar graph comparing the u t i l i z a t i o n of ADP and ATP. Microsomal enzymes were incubated for 5 min at 37°C. Two possible mechanisms by which ADP allows production of P. are shown (ADPase and adenylate kinase). M = 3 mM MgCl,, N = 100 mM NaCl, and K = 3 mM KC1  62  63  F i g . 8.  Lineweaver Burk p l o t o f the e f f e c t o f 100 mM Na (•) or 100 mM Na p l u s 3 mM K ( A ) on the Mg-ATPase ( O ) • A l l assays c o n t a i n e d 3 mM MgC^. The i n s e r t e d graph i n d i c a t e s t h a t the l i b e r a t e d P. does not exceed t h e ATP added d u r i n g the 5 min i n c u b a t i o n . P o i n t s a r e t h e average o f d u p l i c a t e d e t e r m i n a t i o n s . Vmax = 2 . ymoles 1 P. mg  -1  min  -1  and Km = 0.23 mM  f o r the Mg-ATPase.  Na and K a b o l i s h e d the s u b s t r a t e i n h i b i t i o n . Na and K i n c r e a s e d the V t o 3.12 ymoles P i m g min b u t t h e K a l s o i n c r e a s e d t o 0.55 mM i n d i c a t i n g a lower a f f i n i t y f o r ATP than w i t h Mg a l o n e . -x  m a x  - x  m  ^moles Pj liberated  65  F i g . 9.  Assay of the Mg-ATPase with (Y-^^P) l a b e l l e d ATP. Data points are the average of duplicate assays containing 25 mM h i s t i d i n e buffer (pH 7.4), 0.1 mM EGTA, 3 mM MgCl (•) or 3 mM MgCl2 and 100 mM NaCl ( • ) . Microsomal protein which i s indicated on the abscissa and 1.8 umoles of (y-32~p) ATP (1.13 x 10^) cpm/mole were incubated at 37°C for 5 min i n 0.6 ml. The f i n a l ATP concentration was 3 mM. (y-32p) ATP of high s p e c i f i c a c t i v i t y and free of polyphosphate contaminants was prepared by Dr. S. Katz by the method of Glynn and Chappel (1964) as modified by Post and Sen (1967). The reaction was stopped with 0.2 ml of 20% TCA and 0.4 ml of charcoal (Norit A 1.5 gm/10 ml 5% TCA) was added. After 1 hr with occasional Vortex mixing, the charcoal was pelleted and a 0.3 ml aliquot was counted i n Bray's s c i n t i l l a t i o n c o c k t a i l . M = MgCl (3 mM); N = NaCl (100 mM) . 2  2  \  66  67  Fig. 10.  The e f f e c t of Ca on the Mg dependent Na,K-ATPase i n the microsomal f r a c t i o n i n the presence of soluble activating factor. The assay medium contained 3 mM ATP i n 0.6 ml of 25 mM h i s t i d i n e pH 7.4, 0.1 mM EGTA, 50 p l of microsomes and 10 p1 of soluble f r a c t i o n . Results are the average of^4 assays on 2 batches of enzymes. Error bars were omitted for c l a r i t y (see Appendix Table for F i g . 12). (O) (#) (•) (A)  No Mg 3 mM MgCl 3 mM MgCl and 100 mM NaCl 3 mM MgCl , 100 mM NaCl and 3 mM  (4)  3 mM MgCl , 100 mM NaCl, 3 mM KC1 and 3 mM  2  2  2  KC1 ouabain  69  Fig.  11.  P h a r m a c o l o g i c a l t e s t s o f the t i s s u e v i a b i l i t y under the b i o c h e m i c a l c o n d i t i o n s used to p r e p a r e sarcolemmal e n r i c h e d microsomes, as a check f o r the p o s s i b l e l o s s o f Na,K-ATPase a c t i v i t y .  Upper  The experiment demonstrates t h a t the muscle and the nerve p l e x u s remained v i a b l e over 3 days a f t e r 2 o v e r n i g h t s t o r a g e s i n c o l d Tyrode's s o l u t i o n . A f t e r r e - e q u i l i b r a t i o n i n normal Tyrode's s o l u t i o n (NT) a t 37°C the f i r s t response to 60 mM KC1 a f t e r s t o r a g e i n c o l d Tyrode's s o l u t i o n had a depressed p h a s i c but a normal t o n i c component, but the second responses (day 2 and day 3) were normal. The p r e p a r a t i o n s t i l l responded to 60 mM KC1 and 18.5 uM n i c o t i n e ( n i c ) a f t e r 3 days.  Lower  The experiment demonstrates t h a t the muscles were v i a b l e a f t e r 1 h r treatment i n c o l d i s o t o n i c s u c r o s e c o r r e c t e d t o pH 7.35 w i t h h i s t i d i n e ( i . e . the medium used f o r h o m o g e n i z a t i o n ) . Responses to 60 mM KC1 and CD (2 x 1 0 M) were s t i l l n e a r l y 100% of control. Muscles were s t i l l v e r y a c t i v e a f t e r o v e r n i g h t s t o r a g e i n c o l d i s o t o n i c s u c r o s e and they s t i l l responded t o ouabain (ouab) (10~5 M) i n d i c a t i n g that the i s o t o n i c s u c r o s e d i d not i n t e r f e r e w i t h the mechanism of a c t i o n of ouabain. 7  KCl responses a f t e r overnight s t o r a g e i n 0.2 5 M s u c r o s e ,°C pK 7.3 5 w i t h h i s t i d i n e day  1  (gm)  phasic  tonic  1.89  -1.41  5.2  2  day  Dhasic  (%•  tonic  73  t  5  cay  98 '  7  1)  30  71  Fig.  12.  Pharmacological test of the muscle v i a b i l i t y a f t e r treatment i n Ca, Mg-free Tyrode's solution. Control responses to CD (2 x 1 0 M) and ouabain (ouab) (10 ^ M) were recorded and the muscles were a l lowed to completely equilibrate (i.e. return of spontaneous a c t i v i t y , which requires approximately 1 hr) and then they were switched to Ca,Mg-free Tyrode's solution for 10 min at 37°C, which had a rapid d e s t a b i l i z i n g e f f e c t . Muscles were detached from the transducers and washed i n Ca,Mg-free Tyrode for an additional 3 hr and 50 min at 4°C. Muscles were reattached to transducers and set at their normal baselines and re-equilibrated i n normal Tyrode's solution. Resting basal tension increased as Ca and Mg were regained by the muscles. After an i n i t i a l dose of CD, the muscle would not relax after washing with normal Tyrode's solution (NT). This was probably due to the gain of Ca after the setting of basal tension when the muscles were f l a c c i d . Tension was reduced to the normal baseline by lowering the transducers. Testing the muscle again with CD indicated that 61% of the phasic and 33% of the tonic control tension was attained. The tissue responded to ouabain but only to 33% of the control response to ouabain. - 7  -  Table for F i g . 12. 1st  Row  Average results from 4 experiments similar to F i g . 12. E q u i l i b r a t i o n time refers to the time required for the muscle to regain i t s spontaneous a c t i v i t y following washout of the ouabain. A  2nd Row  Average r e s u l t s of 4 experiments s i m i l a r to F i g . 12 except that the time i n Ca,Mg-free Tyrode's solution was reduced to 1 hr.  3rd  Comparison of a f i r s t ouabain response of a muscle to a second ouabain response. The muscle was allowed to regain spontaneous a c t i v i t y before they were tested a second time. The second response was never as strong as the f i r s t .  Row  CD MT  CD  10 M ouabain e q u i l i b r a t i o n 5  2 x  10 ( g r i )  1 +•  1  i 7 2  ' M CD" tor  g 2  37'C 10 m i n OCaMg T y r ( ™ )  •i n  1. 2 H; 1 •1  •  1. 0 t .02 1.9 t. 2 .96  t . 08  57 t'4  0.7 T.05  57 2  '0. 79 : .14  t  74 *-3  -  ouab  NT a f t e r C a Kg a d d i t i o n 37"C OCaMg 4" C (grn) timp (min) 230  60  -  0.69  i . 05 0.31 + .C6 -  2 x 10 M CD % o f b e f o r e OCaKg phasic tonic 7  60.9 2  •3 3 •2.5  118 i 8„. 4  70.2 23.6  tl.  -  -  10 M ouabain % of before 0Ca'-';S 3 3.1 '  44. 9 -5.5  ' • t  86.0 9.7  n=4  N3  III.  Ca-ATPases a.  Microsomal Ca-ATPase  The p u r i t y of the sarcolemmal enriched microsomal f r a c t i o n becomes of major importance when discussing i t s Ca-ATPase a c t i v i t y because sarcoplasmic reticulum, mitochondria and actomyosin also have Ca-ATPases.  Wei et a l . (1976)  demonstrated that fractionated rat mesenteric arteries had four times more plasma membrane vesicles than endoplasmic reticulum v e s i c l e s (based on t o t a l protein quantities rather than recovery studies) which they f e l t was consistent with the minimal volume of endoplasmic reticulum that they and others (Devine et a l . 1972) observed i n electron micrographs of this tissue.  Similarly,  longitudinal i l e a l c e l l s have a minimal volume of sarcoplasmic reticulum and, i f NADH oxidase can be used as i t s marker, i t s recovery was least i n the microsomal p e l l e t and s l i g h t l y less than the sarcolemmal recovery i n the microsomes.  Mitochondria were not detectable i n the microsomes nor were the micro-  somes contaminated by actomyosin Ca-ATPase, as w i l l be demonstrated later Lin Results and Discussion I l l b ) .  Although some of the microsomal Ca-ATPase a c t i -  v i t y could have been from the endoplasmic reticulum, for the reasons explained above, the majority of the microsomal Ca-ATPase was assumed to be of sarcolemmal origin. The microsomal f r a c t i o n had ATPase a c t i v i t i e s that could be stimulated by Ca or by Mg but their stimulations of the the ATPase a c t i v i t y were never additive ( F i g . 13, see also F i g . 12 and F i g . 14, 2nd Row). inhibited the Mg-ATPase a c t i v i t y (Fig. 13).  Low amounts of Ca often  Since 1 mM Ca and 3 mM Mg stimu-  lated the a c t i v i t y to the same extent as 3 mM MgCl  2  alone, Mg may i n h i b i t some  of the Ca activation and Ca may prevent some of the Mg stimulation of the micro2+ somal ATPases.  The threshold free  ATPase a c t i v i t y was about 10  7  2.5 x 10  concentration for a c t i v a t i o n of the  M and the a c t i v i t y increased l i n e a r l y up to con-  centrations s l i g h t l y greater than 10 -4  Ca  5  M.  Maximal a c t i v a t i o n occurred at about  2+ M free Ca  (Fig. 14, 1st Row).  Increasing the MgCl  2  concentration  in the presence of 1 mM  Ca progressively- increased the ATPase a c t i v i t y but the  two a c t i v i t i e s were not additive (Fig, 14, 2nd Row), and 3 mM MgC^  Whereas both 1 mM  stimulated the enzymatic a c t i v i t y to 2 umoles P^/mg  CaC^  protein/min  a c t i v i t y , together they activated the ATPase a c t i v i t y to only 2.75 umoles P_^/ mg protein/min.  Na caused only a small gradual increase i n a c t i v i t y which might  have been due to an increase i n osmolarity or ionic strength (Fig. 14, 3rd but this effect was very small.  Row)  It i s u n l i k e l y that the Mg-ATPase and Ca-ATPase  a c t i v i t i e s are from the same enzyme because the Mg-ATPase i s very noticeably activated by Na whereas Na had l i t t l e effect on the Ca-ATPase. effect on the Ca-ATPase over the-,concentration range 2 - 8 mM KC1 may  KC1 had  little  (Fig. 14, 4th  Row)  have caused a s l i g h t i n h i b i t i o n of the microsomal Ca-ATPase. -3  ATPase a c t i v i t y , i n the presence of 10 concentration was decreased.  raised to 1 mM  (Fig. 15, Top).  The decrease i n a c t i v i t y may 2+  or reduction of the free Ca Ca-ATPase was  M t o t a l Ca, increased as the Above 1 mM ATP,  ATP  the a c t i v i t y  have been due to substrate i n h i b i t i o n  concentration by the excess ATP.  not inhibited by 1 mM LaCl^ when the ATP  The microsomal  concentration was  However, at lower ATP concentrations, La i n h i b i t i o n was very apparent.  3  mM.  LaCl^  changed the normal substrate dependent hyperbolic increase of the Ca-ATPase a c t i v i t y to a sigmoidal function of the ATP  concentration (Fig. 17).  Substrate  l i m i t a t i o n can be an explanation of the sigmoidicity of the Michaelis Menten plot and the nonlinearity of the double r e c i p r o c a l plot If a l l of. the LaCl^ was would be 0.5 mM, min.  chelated by 1.5 mM ATP,  (Fig. 17)  the e f f e c t i v e ATP  protein/min  (Fig. 15, Top).  concentration  only 0.875  Therefore substrate l i m i t a t i o n  not f u l l y explain the La i n h i b i t i o n of the Ca-ATPase a c t i v i t y . though there may  1969)  which should have an a c t i v i t y of 1.37 umoles P^ /mg protein/  However, the La treated enzyme a c t i v i t y at 1.5 mM ATP was  ymoles P^/mg  (Westley  may  It seems as  also be a direct i n h i b i t i o n by La at the Ca a c t i v a t i o n s i t e .  75 b,  Actomyosin Ca-ATPase  The concentration threshhold for a c t i v a t i o n of the Ca-ATPase i n the 105,000 x g soluble f r a c t i o n (which has been assumed to belong to the contrac-7 t i l e filaments) may be below 10  2+ M Ca  (Fig. 15, Bottom).  a c t i v i t y even when the ATP concentration was 3 mM.  LaCl^ i n h i b i t e d the  The inhibiton by La of the  actomyosin Ca-ATPase i s not due to substrate l i m i t a t i o n , by the chelation of La with ATP, because even i f LaCl^ complexed with a l l of the ATP, there would s t i l l be 2 mM ATP l e f t and the actomyosin Ca-ATPase was f u l l y activated by 1 mM ATP or above (Fig. 16, Top).  Unlike the microsomal Ca-ATPase, the actomyosin Ca-ATPase  did not exhibit substrate i n h i b i t i o n (Fig. 16, Top).  High i o n i c strength KC1  solution d e f i n i t e l y improved the actomyosin Ca-ATPase a c t i v i t y i n the presence of 4 mM C a C ^ compared to the. other lower ionic strength buffer systems found to be near optimal for the microsomal Ca-ATPase and the Mg dependent Na,K-ATPase assays.(Fig. 16, Bottom). c.  Comparison of the divalent cation stimulated ATPase a c t i v i t i e s  The substrate a f f i n i t i e s of the actomyosin and microsomal Ca-ATPase (from the tops of F i g . 15 and 16) are compared i n F i g . 17.  The microsomal  Ca-ATPase and the actomyosin Ca-ATPase have the same a f f i n i t i e s for ATP (Km = 0.5 mM). La treatment of the microsomal Ca-ATPase affected the a c t i v i t y so that the double r e c i p r o c a l plot was no longer l i n e a r , i n d i c a t i n g that there was some interaction between the substrate and the i n h i b i t o r , the effect being p a r t i c u l a r l y evident at lower ATP concentrations.  The nonlinearity makes  i t d i f f i c u l t to determine the mechanism of La inhibiton.  Nevertheless,  unlikely that the La i n h i b i t i o n i s competitive, as drawn i n F i g . 17.  i t is  Sulakhe  et a l . (1973) observed that La did not i n h i b i t Ca binding to microsomal v e s i c l e s or oxalate accumulation  i n the v e s i c l e s unless the La concentration exceeded the  ATP concentration.  La i n h i b i t i o n of Ca-ATPases may be due to (1) l i m i t a t i o n of  the free substrate  (2) prevention of the Ca-ATP complex which may serve as a  substrate or (3) direct competition for Ca binding s i t e s .  In this study of the  76' microsomal Ca-ATPase, a l l three p o s s i b i l i t i e s or a combination of Cl)  and  (3)  or (2) and (3) could have been the answer, depending on the substrate concentration.  The i n h i b i t i o n of the actomyosin Ca-ATPase by La seems to have been  by the third mechanism.  Actomyosin e f f e c t i v e l y binds La with greater a f f i n i t y  than the a f f i n i t y of ATP f o r La.  Ca seems to associate with actomyosin more  than with ATP thereby avoiding a c t i v i t y reduction at higher ATP concentrations. Divalent cations are known to associate with ATP and the association constants f o r Mg-ATP and Ca-ATP complexes are approximately 8.8 x 10 4 -1 (0*Sullivan and P e r r i n 1964) and 3.15 x 10 Table below and Appendix).  M  4-1 M  (Ogawa 1968) respectively (see  If the cation-ATP complex ±s\ the substrate and  determinant of the enzyme a c t i v i t y , then the a c t i v i t y should show saturation kinetics.  But the microsomal Ca-ATPase and the Mg-ATPase a c t i v i t i e s decreased  above 1 mM ATP and the i n h i b i t i o n can be explained i n two ways.  Either higher  free ATP concentrations i n h i b i t the a c t i v i t y or the a c t i v i t y i s less when the concentration of free divalent cation i s decreased. concentrations would not be expected to i n h i b i t any physiological  total [cation] mM  active transport a c t i v i t y for  purpose whereas an i n t r a c e l l u l a r loss of Ca or Mg would be  expected to turn o f f pump function. cation  Higher cytoplasmic ATP  total [ATP] mM  The decline i n  K •. ass  a c t i v i t y as the ATP i s  [cation-ATP] mM  f ree~ [cation] mM  Mg  3  1  8.8 x 10  4  0.983  2.027  0.027  Mg  3  3  8.8 x 10  4  2.79  0.21  0.21  Ca  1  1  3.15  x 10  4  .813  Ca  1  3  3.15 x 10  4  .978  .187 ,  .022  increased from 1 -,3 mM coincides with a large decrease i n free Mg o  Ca  free [ATP] mM  .187 2.022 and free  2+ concentrations whereas free ATP or cation-ATP complex increase (see Table  above).  Therefore, the decline i n a c t i v i t y which appears to be substrate i n h i -  b i t i o n may ing  actually be due to a reduction i n the free cation l e v e l .  evidence, Iso (1975) has shown that higher ATP  concentrations  As support-  reduce Mg  binding to longitudinal i l e a l microsomes and.would have reduced Ca binding i f excess Mg had not been present.  This implies that the K  of the Ca-[microso-  cLSS  mal Ca-ATPase] complex and the K  of the Mg-[microsomal Mg-ATPase] complex  cLSS  are less than the K  of Ca-ATP and Mg-ATP complexes respectively.  et a l . (1973) found that ATP  Sulakhe  up to 1.7 mM increased Ca binding but higher concen-  trations were i n h i b i t o r y . McNamara et a l . (1974) reported that ATP  i n excess  of the divalent cation concentration i n h i b i t e d the Ca-ATPase and Mg-ATPase activity. The sarcoplasmic a l . 1970)  reticulum Ca pump requires Mg  Katz at  but since the Ca-ATPase i n the l o n g i t u d i n a l i l e a l microsomes observed  i n this study and i n others did  (MacLennan 1970,  not require Mg,  (1971 and 1974)  (Godfraind et a l . 1976,  O l i v i e r a and Holzacker  it.would seem to be of sarcolemmal o r i g i n .  1974)  McNamara et a l .  found that the Ca-ATPase a c t i v i t i e s i n hamster s k e l e t a l and  dog  cardiac muscle sarcolemmal enriched fractions did not require Mg and were not stimulated by high i o n i c strength K solutions.  Therefore  attributed to the sarcolemma rather than to sarcoplasmic  this Ca-ATPase was reticulum or actomyosin.  They also found that the Ca-ATPase and Mg-rATPase had similar s p e c i f i c a c t i v i t i e s and both.-were i n h i b i t e d by excess ATP.  In their experiments and i n the  study the Ca-ATPase had a higher K for ATP m  (0.23 mM;  F i g . 8).  F i g . 17) than the Mg-ATPase  The Mg and Ca stimulations are not additive and Mg seems to  i n h i b i t the Ca-ATPase and v i c e versa. was  (0.5 mM;  present  The microsomal Mg-ATPase i n rat uterus  also observed to be inhibited.by Ca (Daniel et a l . 1971b). Na  powerfully  stimulated the Mg-ATPase but had l i t t l e e f f e c t on the Ca-ATPase . (McNamara"et - ;i a l . 1974  and the present  study).  These two ATPase a c t i v i t i e s probably originate  from two separate enzymes with d i f f e r e n t functions, but t h i s can not be stated conclusively without further evidence.  It i s clear that the Ca-ATPase i n the  microsomal membrane v e s i c l e s i s not due to adhering actomyosin f i b r i l s because  7 t h e i r properties of La i n h i b i t i o n , Ca a f f i n i t y , . s u b s t r a t e ponse to increasing i o n i c strength are so s t r i k i n g l y  i n h i b i t i o n and  different.  \  res-  8  79  Fig. 13.  Sarcolemmal enriched microsomal Ca-ATPase a c t i v i t y i n response to tree Ca2+. Approximately ,25 ug.of microsomal protein was assayed i n 20 mM T r i s HC1 (pH 7.4) at 37°C f o r 15 min with 3 mM ATP (no EGTA) ( s o l i d l i n e ) . The dotted l i n e i s the response to free C a ^ (no EGTA) i n the presence of 3 mM MgC^. Points represent the mean + S. E. (n = 4) +  81  F i g . 14.  The effect of ions on the microsomal Ca-ATPase. Each assay contained 10" M t o t a l Ca (2.0 x 10" M free C a ) indicated by the i n i t i a l bar graph i n Rows 1 - 4. More C a C l was added (Row 1). C a C l concentrations on the abscissa indicate t o t a l Ca concentration (see Appendix for free C a C l concentrations). MgCl (Row 2), NaCl (Row 3) and KC1 (Row 4) were added as indicated on the abscissa. MgCl added i n Row 2 would increase the free C a concentration compared to that i n d i cated above. 3  5  2 +  2  2  2  2  2  2 +  83  F i g . 15.  The e f f e c t of La on the microsomal Ca-ATPase and the Actomyosin CaATPase . Top. Substrate dependence of the microsomal Ca-ATPase (solid l i n e ) (10~ M t o t a l Ca) and i n h i b i t i o n by L a C l (10~ Mi);, (dotted l i n e ) . Insert indicates the LaCl3 i n h i b i t i o n i n r e l a t i o n to the umoles of ATP added. Data points are the average of duplicates. The assay conditions were, 25 ug microsomal protein i n 100 mM Tris-HCl (pH 7.4) and 0.1 mM EGTA incubated.for 5 min at 37°C. • The enzyme was pre-incubated with La for 5.min at 37°C before ATP was added. 3  3  3  Bottom. Response of the soluble f r a c t i o n ATPase (actomyosinjl to increasing free Ca? . 50 u l samples of.the soluble supernatant containing 69 ug of protein was assayed i n 50 mM h i s t i d i n e (pH 7.6), 425 mM KC1 and 3 mM ATP ( # ) . The open c i r c l e s (Q,) indicate the inhibition.by La of the.Ca a c t i v a t i o n . Data points are the average of duplicates. The enzyme was pre-incubated with La for 5 min at 37°C and the reaction was started with ATP and incubated for an additional 5 .min. Free Ca .concentrations of the La.treated enzyme are not corrected for the e f f e c t of La. +  2+  85  Fig. 16.  Characteristics of the Actomyosin Ca-ATPase. Top. The concentration dependence of actomyosin.Ca-ATPase on ATP (see methods for assay conditions).. .Results are:the average of duplicates. Bottom. Actomyosin Ca-ATPase a c t i v i t y i n d i f f e r e n t buffer systems. Results are the average of duplicates (ATP.= 4 mM, CaCl2 = 4 mM).  87  F i g . 17.  Lineweaver Burk plot of the substrate dependence of the microsomal and actomyosin Ca-ATPases; The graph also-indicates the e f f e c t of La ( 1 0 M) on the substrate dependence.of.the microsomal Ca-ATPase. The plots are from the data.in F i g . 15,TTopand F i g . 16, Top. -3  89 IV.  The Structure of the Guinea pig Ileum Longitudinal Smooth Muscle Aspects" of the structure of the longitudinal muscle were discussed to some  extent in'the Introduction.  The structure w i l l be described again here, i n  more d e t a i l , i n an attempt to correlate the biochemical results (previous three sections of Results and Discussion) with the c o n t r a c t i l e work and ion movement studies which w i l l be outlined i n the following sections. The dissection technique for removing the longitudinal layer of the ileum yields the muscle s t r i p r o l l e d up l i k e a s c r o l l . of the r o l l can be seen i n Plate 3, #7.  A cross section of one part  Auerbach's nerve plexus and a few  c i r c u l a r layer muscle c e l l s can be seen adhering to the longitudinal layer. V i s c e r a l smooth muscle c e l l s are not i n d i v i d u a l l y innervated. axon bundles and muscle membranes i s greater than 100 nm  The gap between  (Holman 1970)  and  there i s an absence of circumscribed end plate regions (Paton and Rang 1965). If acetylcholine receptors are evenly distributed over the entire large surface area, then a c t i v a t i o n of receptors could probably mobilize Ca from s i t e s l o cated anywhere on the entire surface area.  The loose innervation allows the  longitudinal layer to be separated from the nerve plexus.  This technique  was  useful to test whether ouabain acted on the muscle c e l l s d i r e c t l y or whether ouabain caused extensive release of neurotransmitters from the nerve plexus. The r e l a t i v e l y empty e x t r a c e l l u l a r space between longitudinal i l e a l c e l l s i s about 500 nm wide. which decreases  Collagen f i b r e s i n the e x t r a c e l l u l a r space are sparse,  the problems associated with i s o l a t i n g subcellular fractions  from these smooth muscles.  The wide spaces between the c e l l s and the thinness  of the muscle s t r i p s allows rapid d i f f u s i o n of ions and pharmacological agents throughout the tissue. The general c e l l shape i s long and narrow (Plate 3, #9).  The surface to  volume r a t i o i s increased by the unevenness of the c e l l surface and by the surface vesicles or caveolae that can be seen i n Plate 3, #12, Plate 5, #19,  21, 22, 23 and 24.  Plate 4, #14  The c e l l s appear s t e l l a t e i n cross section  and  (Plate 3, #11 and 121 c e l l surface may the tissue.  (see also Gabella 1971),  Some of the uneyenness of the  have been due to shrinkage of the c e l l s during the f i x a t i o n of  The c e l l s send long protrusions towards their neighbours.  Occa-  s i o n a l l y , the protrusions fuse with, or intrude into, other c e l l s to form what appear to be nexuses (Plate 3, #12 - arrow) or 'peg and socket structures' (Plate 3, #11).  Since the c e l l s are not i n d i v i d u a l l y innervated, there may  be  a morphological basis for the e l e c t r i c a l interaction between these smooth muscle c e l l s which enables them to behave as a synctia (Holman 1970).  Previously, i t  was proposed that tight junctions or nexuses might be low resistance pathways for ion movements between c e l l s , which might serve as an i n t e r c e l l u l a r communication system.  More recently, the work of Gabella (1973) and Daniel et a l .  (1976) demonstrated that true nexuses are absent or very rare i n the l o n g i t u dinal layer of the guinea pig and the dog intestine.  The l o n g i t u d i n a l layer of  the ileum works as a synctium even though nexal regions are absent.  Calcium  oxalate deposits were found at points of close i n t e r c e l l u l a r contact between taenia c o l i c e l l s which demonstrated a 7 layered membrane structure apparently with p a r t i a l fusing of the adjoining membranes ('gap' junction) (Popescu 1974).  et a l .  I t i s s t i l l not clear which, i f any, close membrane contact regions are  s i t e s of high permeability between c e l l s (Holman 1973;  Daniel et a l . 1976).  The function of these junctions i s also of interest to this study since, i f excitation-contraction coupling occurred at l o c a l i z e d points and i s communicated between c e l l s at low resistance i n t e r c e l l u l a r s i t e s , the associated ion movements would not be detectable by the modified  'La method' which measures essen-  t i a l l y net changes of i n t r a c e l l u l a r ion l e v e l s .  Under the experimental  condi-  tions of a drug i n a tissue bath, e x c i t a t i o n i s not l o c a l i z e d and we should be able to use the modified  'La method'.  Also i t was  hoped that loosening of  these i n t e r c e l l u l a r junctions would occur after incubation of muscles i n Ca,Mgfree Tyrode's solution.  91 Each, c e l l hap a f a i r l y large long and narrow nucleus. p a r a l l e l to the long axis of the c e l l .  I t s long axis i s  At each of i t s ends or poles, there i s  an aggregation of mitochondria, rough endoplasmic reticulum and a Golgi apparatus (Plate 4, #13 and 14),  A Golgi apparatus can be seen between two f a i r l y  large sacs of rough endoplasmic reticulum i n Plate 4, #14.  The membrane vacu-  oles of the Golgi complex are usually not accounted f o r i n c e l l f r a c t i o n a t i o n procedures as there i s no established membrane marker enzyme for the Golgi apparatus membranes (Janis and Daniel 1977).  Although very l i t t l e was known  about their function previously, i t now seems that the Golgi organelle i s a c t i v e l y involved i n the transfer of carbohydrate and s u l f a t e moieties to acid mucopolysaccharides and glycoproteins which are e s s e n t i a l for many membrane functions (Somlyo et a l . 1975).  These negatively charged substances can act as  Ca retaining material i n c e l l u l a r depots.  Since there are many aspects of  smooth muscle a c t i v i t y for which we have no answers, some of the less studied c e l l u l a r organelles may play more major roles than i s now r e a l i z e d . In the r e s t i n g c e l l , the nucleus i s elongated and indented  slightly.  Some  n u c l e i can be seen to contain 1 or 2 n u c l e o l i (Plate 4, #15). The nucleus i s encased i n a perinuclear sac of rough endoplasmic reticulum (Plate 4, #14). The perinuclear sac, i n the p i g coronary artery, accumulates Ca as does the rest of the endoplasmic reticulum (Jonas and Zelck 1974).  The calcium i n the perinuc-  lear sac i s i n the range of that required by the taenia c o l i c e l l for maximal contraction (Popescu et a l . 1974).  The calcium i n the nucleus i s 60 times higher  than that required by.the c e l l for maximum contraction.  The nuclear membrane  has s p i r a l indentations which become more marked when the c e l l contracts (Burnstock 1970; Plate 4, #16 and 17).  Mitochondria  seem to be pulled into the  folds of the nucleus when the c e l l s contract (Plate 4, #14 and 18).  The sub-  c e l l u l a r components at the nuclear poles would seem to be obligated to move with the nucleus as i t changes shape during contraction.  Popescu et a l . (1974) have  shown that there are 'close r e l a t i o n s h i p s ' between the mitochondria and the  92 perinuclear sac and between mitochondria and sarcoplasmic  reticulum,  Therefore,  unless these 'close r e l a t i o n s h i p s ' are broken when the c e l l contracts, the whole perinuclear region must move when the nucleus contorts.  Such concerted move-  ments may be responsible for the release of i n t e r n a l Ca, but the exact role of nuclear Ca i n contraction, i f any, i s not understood. Mitochondria  are crowded at the nuclear poles  (Plate 4, #13, 14 and 18)  and many are located peripherally, often near caveolae (Plate 3, #12). Mitochondria accumulate Ca and store large amounts of i t . Only 5% of the mitochond r i a l store of Ca i n one c e l l would be s u f f i c i e n t to r a i s e the cytoplasmic Ca concentration  to maximal c o n t r a c t i l e l e v e l s (Popescu et a l . 1974).  Excess  cations accumulated by mitochondria may be extruded into the e x t r a c e l l u l a r space through the surface vesicle-mitochondrial contacts  (Somlyo and Somlyo 1976).  When taenia c o l i c e l l u l a r Ca depots were mapped by Ca oxalate c r y s t a l staining, densely aggregated c r y s t a l s were observed at points where mitochondria were situated very near to caveolae,  sarcoplasmic  sarcolemma (Popescu'et a l . 1974).  Therefore,  reticulum, perinuclear sac and the they are not just physically near  to each other, but are probably functionally 'associated . 1  There i s no apparent  pattern to the mitochondrial positioning i n the guinea p i g longitudinal i l e a l c e l l s that might suggest that they are c r i t i c a l l y positioned for even dispersement of Ca from the mitochondria to the c o n t r a c t i l e filaments. Most of the rough sarcoplasmic region.  reticulum i s present  Ca i s accumulated i n the central sarcoplasmic  Ca i s s u f f i c i e n t to activate maximal contractions  i n the nuclear  pole  reticulum and 33% of the  (Popescu et a l . 1974). Mem-  branes with ribosomes are also apparent at the c e l l periphery but they are much more sparse there than i n the perinuclear region. at points where myofilaments are attached contract (Burnstock 1970).  The c e l l surface  to the sarcolemma when the c e l l s  Narrow membrane sacs are present  regions, just beneath the sarcolemma.  invaginates  i n these concave  These membrane sacs may be smooth sarco-  plasmic reticulum (Devine et a l . 1972) (Plate 5, #25, Plate 4, #14 and 16, and  Plate 3, #10). Only 20% of the Ca stored In the peripheral sarcoplasmic r e t i culum would be required to cause maximal contraction of the guinea p i g taenia c o l i (Popescu et a l . 1974),  Sarcoplasmic reticulum sacs are often associated  with caveolae (Plate 3, #10, Plate 5, #19, 23 and 25) and are quite often seen tucked between mitochondria and the sarcolemma (Plate 5, #20).  The presence of  sacs of sarcoplasmic reticulum between mitochondria and the sarcolemma have been previously reported by Gabella (1973) i n the guinea pig ileum longitudinal smooth muscle and i n guinea p i g taenia c o l i c e l l s (M. Wolowyk, personal communication) .  The physical closeness of the sarcoplasmic reticulum, mitochondria  and sarcolemma to each other suggests that they may form an area a c t i v e l y i n volved i n ion transport but direct evidence f o r such a function i s lacking. The pattern of d i s t r i b u t i o n of peripheral smooth sarcoplasmic reticulum membranes i s l i k e a maze between and around the caveolae.  The smooth sarco-  plasmic reticulum seldom extends conspicuously into the c e l l (Gabella 1971). There i s no apparent continuity between the peripheral and central sarcoplasmic reticulum (Plate 4, #13). Perhaps there i s no need f o r transverse extensions of these membrane sacs i n c e l l s that are only 2 - 5 p i n diameter.  A stenosis  of the small intestine can cause the c e l l s to a t t a i n diameters of 10 u.  Under  these conditions, the sarcoplasmic reticulum sacs have been observed to extend deeper into the c e l l thereby adapting the c e l l to having a smaller surface to volume r a t i o .  I f an extensive sarcoplasmic reticulum i s an adaption to a large  f i b r e size (as i s found i n s k e l e t a l muscle) then normal longitudinal i l e a l c e l l s should not require very much sarcoplasmic reticulum.  There i s more than twice  as much Ca i n the peripheral sarcoplasmic reticulum of taenia c o l i c e l l s as would be required to cause maximum contraction (Popescu et a l . 1974).  A trigger  of e x t r a c e l l u l a r Ca from the caveolae or a l o c a l potential f i e l d change may cause the mitochondria and the sarcoplasmic reticulum, located near the caveolae, to release their Ca.  The release of Ca by l o c a l f i e l d changes may only occur i n  c e l l s where the sarcoplasmic reticulum v e s i c l e s form d i r e c t contacts with the  plasma membrane,  Vascular sjnooth_muscles have electron dense physical couplings  spanning the 1Q ~ 12 nm gap separating sarcoplasmic reticulum and the caveolae (Somlyo and Somlyo 1976) but taenia c o l i c e l l s seem to lack these physical 2+ couplings (Somlyo and Somlyo 1972).  For this reason, the flow of Ca  through  the sarcolemma has been postulated to couple excitation to contraction of taenia c o l i c e l l s (Pogadeav and Timins 1976).  Gabella (1973) indicated that the sarco-  plasmic reticulum of i n t e s t i n a l muscle always makes contact with the sarcolemma at some point, although i t i s d i f f i c u l t to see such contacts i n the micrographs he presents as proof of t h i s .  Such contacts are much more apparent i n the taenia  c o l i c e l l s that have had their c e l l u l a r Ca depots mapped by the formation of Ca oxalate crystals (Popescu et a l . 1974).  The micrographs  of the sarcoplasmic  reticulum sacs that Gabella (1973) shows of i n t e s t i n a l muscle are from the c i r cular layer while the examples of caveolae are from the longitudinal layer.  The  biased choice of examples was probably unintentional but the claim that the sarcoplasmic reticulum i s quite extensive i n the longitudinal layer of the ileum would be more convincing i f examples of extensive sarcoplasmic reticulum were from micrographs  of the longitudinal layer rather than from the c i r c u l a r muscle  layer. Occasionally smooth sarcoplasmic reticulum membranes are observed to be continuous with rough sarcoplasmic reticulum (Plate 4, #18, arrows).  Plate 5, #24 - at  The volume of the sarcoplasmic reticulum i n smooth muscle c e l l s  based on the t o t a l of rough and smooth endoplasmic  was  reticulum (Devine et a l .  1972) because both types of sarcoplasmic reticulum have been observed to accumulate Ca.  In p u r i f i e d microsomal fractions of rough and smooth endoplasmic  reticulum from rat l i v e r , Moore et a l . (1975) reported that smooth endoplasmic reticulum accumulated  Ca more rapidly than the rough endoplasmic reticulum.  Popescu et a l . (1974) reported that the size of the microsomal v e s i c l e s from taenia c o l i c e l l s were mainly of two populations, 0.04 in diameter.  Approximately  - 0.08 u and 0.9 - 1.6 u  65% of the v e s i c l e s were of the small size corres-  ponding to the size of; cayeqlae (0.Q6. u diameter), and 35% seemed to be from V  the sarcoplasmic reticulum because the mean diameter of the larger v e s i c l e s  was  equal to microsomes of rat l i v e r , composed mainly of endoplasmic reticulum. Popescu et a l . (1974) obtained the 110,000 x g microsomal p e l l e t from the 10,000 x g supernatant while i n the present study, a 105,000 x g p e l l e t was from the 27,000 x g supernatant.  obtained  Based on the results obtained by Popescu e_t  a l . (1974), i t would be expected that the microsomes prepared i n the present study consist of greater than 65% caveolae, since many of the larger type of v e s i c l e s may have sedimented  at 27,000 x g.  Therefore the Ca-ATPase a c t i v i t y  observed i n the microsomes i n the present study, was almost c e r t a i n l y from the caveolae. Caveolae are perhaps the most s t r i k i n g feature of these c e l l s . to be distributed over the entire surface area of the c e l l .  They seem  Caveolae increase  the surface area by 25% i n mouse i n t e s t i n e to 70% i n taenia c o l i (Rhodin Goodford 1970).  1962;  Caveolae are most abundant at the ends of the i l e a l c e l l s , as  can be seen i n Plate 5, #21 and 22.  What appear to be large holes i n the c e l l  may be fused aggregates of caveolae ( C o l t o f f - S c h i l l e r et a l . 1976).  A caveolae  fusing process seems to have occurred i n the c e l l pole shown i n Plate 5,  #21.  Smooth muscle caveolae are smaller and more uniform i n size than micropinocytot i c v e s i c l e s of endothelial c e l l s (Gabella 1973).  In the present study, a ran-  dom sampling of 17 caveolae gave an average diameter of 8 0 + 4 nm and a depth of 9 5 + 5 nm, which i s about the same size as the sarcolemmal enriched microsomal v e s i c l e s i n Plate 2. (Gabella 1973).  The neck of the caveolae i s about 20 nm wide  The entire surface of the smooth muscle c e l l i s covered by a  basement lamina which does not penetrate the caveolae and which does not d i f f e r s t r u c t u r a l l y over the neck of the caveolae (Gabella 1973, Plate 5, #22 and 23). Impermeant e x t r a c e l l u l a r stains, used for electron microscopy, penetrate into the caveolae, indicating that the caveolae are open to the e x t r a c e l l u l a r space. L a C l , (Ma and Bose 1976) and c o l l o i d a l lanthanum (Gabella 1973) have been ob-  served to be retained i n the caveolae but not i n the e x t r a c e l l u l a r space after f i x a t i o n i n the absence of La,  This indicates that there may be some substance  retaining cations i n the caveolae,  Hyaluronidase has been demonstrated to cause  the elimination of caveolae from smooth muscle c e l l s (Gabella 1973).  Hyaluroni-  dase treatment also increased the i n u l i n measurement of the volume of e x t r a c e l l u l a r space of the i n t e s t i n a l muscle from 34% to 38% (Goodford and Leach 1966). Therefore the caveolar volume ( i f i t can be estimated from the 4% increase i n e x t r a c e l l u l a r space due to hyaluronidase) i s about 10% of the e x t r a c e l l u l a r space.  Gabella (1973) suggested that the caveolar lumen represents a tissue  compartment with properties intermediate between the cytoplasmic and e x t r a c e l l u l a r compartments and that the ionic composition of the caveolar compartment be controlled by the cytoplasm.  This may  may  explain why measurements of the amounts  of tracer K*~ i n the taenia c o l i , exchanging  from the fast exchanging  c e l l u l a r ) space, are considerably larger than the amount of tracer K be i n solution i n the e x t r a c e l l u l a r f l u i d (Brading 1973).  (extra+  that could  EDTA solutions also  caused the loss of caveolae from smooth muscle c e l l s (Higgs and Wolowyk 1974). It i s possible that caveolae have higher Ca and other cation concentrations than the e x t r a c e l l u l a r space.  These cations may be required to maintain the shape  of the caveolae (Higgs and Wolowyk 1976).  Quantitative estimation of the Ca i n  the caveolae indicates that they would contain enough Ca to i n i t i a t e contraction J  i f their Ca concentration was  equal to the e x t r a c e l l u l a r f l u i d .  However, Popescu  et a l . (1974) consistently found very dense Ca oxalate c r y s t a l s i n caveolae, often equal to the density of such crystals i n sarcoplasmic reticulum.  There-  fore caveolae probably contain f a r more Ca than i s required for contraction, which l e d Popescu et a l . (1974) to support the hypothesis that Ca i n caveolae plays a role i n the translocation of Ca during the contraction relaxation cycle. In addition, the caveolae have been demonstrated by histochemical methods to contain an ATPase that can a c t i v e l y accumulate Ca i n caveolae (Lane 1967).  If  microsomes are derived from pinched o f f caveolae, as postulated i n the present  97 study and previously  suggested by Popescu et a l . 0-974), then the a b i l i t y of Ca  to stimulate an ATPase i n the microsomes i s i n agreement with the results of Lane (1967). 'Glycerol shock' treatment i s used to uncouple excitation from for electrophysiological  studies of s k e l e t a l muscle (see Methods).  contraction The uncoup-  l i n g e f f e c t seems to be due to disruption of the sarcotubular system (FranziniArmstrong et al. 1973). Anexperiment based on the ' glycerol shock' treatment of skel e t a l muscles, was attempted i n the present study to see i f this treatment would have a similar effect on these smooth muscle c e l l s .  I t seemed possible  that the  hypertonic glycerol solution might a l t e r the caveolae and disconnect them from sarcoplasmic reticulum sacs i f they were physically coupled.  The 'glycerol shock'  treatment did not abolish contractions induced by 60 mM KC1 or CD (Fig. 18), which could either mean that excitation does not require physical coupling to the i n t r a c e l l u l a r Ca stores, or that the interconnection between the caveolae and the i n t e r c e l l u l a r Ca stores are insensitive to hypertonic solutions.  The muscle tension  increased transiently when Tyrode's solution containing 400 mM glycerol was added to the bath and they contracted again when the muscles were returned to normal Tyrode's solution (Fig. 18, 2nd Row).  Therefore, the treatment had some effect  on factors c o n t r o l l i n g the muscle tension, but i t did not uncouple excitation of the c e l l s from contraction  (Fig. 18, compare 1st and 3rd Rows).  The e f f e c t of  'glycerol shock' treatment on the structure of the c e l l was not investigated by electron microscopy.  Without s t r u c t u r a l v e r i f i c a t i o n , the f a i l u r e to uncouple  excitation of these c e l l s from contraction mechanism of excitation-contraction  can only be inferred to mean that the  coupling i n longitudinal i l e a l smooth muscles  i s very d i f f e r e n t from the coupling i n s k e l e t a l muscle c e l l s .  Perhaps this i s  because the c e l l s are small and hence may u t i l i z e s u p e r f i c i a l or e x t r a c e l l u l a r Ca pools for contractions. peripheral  Generally,  i t i s not readily apparent how small  sarcoplasmic reticulum sacs and mitochondria  over e x t r a c e l l u l a r Ca for delivering  Ca to the central  have an advantage c o n t r a c t i l e proteins of  the guinea p i g ileum when the c e l l i s excited at i t s surface.  In summary, the  general f e e l i n g amongst those studying smooth muscle structure and function i s that the sarcoplasmic reticulum, the sarcolemma and caveolae seem to be s i t e s for the release of activator Ca and that mitochondria and the nucleus seem to be involved i n Ca sequestration but the importance of each of these s i t e s vary from one type of smooth muscle to another.  may  Surface caveolae contain c a l -  cium extrusion pumps which may balance the accumulation of Ca i n the c e l l .  99  SYMBOLS FOR ELECTRON MICROGRAPHS OF WHOLE TISSUE ( P l a t e 3, 4, and 5)  Subsarcolemmal >  Caveolae  N  Nuclei  M G  membrane sacs  Mitochondria G o l g i apparatus N u c l e a r membrane  R  Rough endoplasmic  reticulum  100  Plate 3.  7.  A l i g h t micrograph of a thick tangential section of l o n g i t u d i n a l ileum folded a f t e r d i s s e c t i o n a l " r o l l i n g o f f " technique. Light and dark staining c e l l s are present and some n u c l e i are v i s i b l e i n the l i g h t staining c e l l s . (2,300 x magnification Nikon S-Kt)  8.  Thick section showing the Auerbach's nerve plexus running as a band between c i r c u l a r and l o n g i t u d i n a l layers. C e l l s are not i n d i v i d u a l l y innervated. (1,500 x magnification Nikon S-Kt)  9.  A l o n g i t u d i n a l section demonstrating the extreme length compared to width of the longitudinal i l e a l f i b e r s . The c e l l surface appears smooth i n longitudinal section r e l a t i v e to the cross section i n photograph 11. (2,700 x magnification Nikon S-Kt)  10.  An electron micrograph of a l o n g i t u d i n a l section showing the f l u t e d c e l l surface, caveolae and subsarcolemmal membrane sacs. An arrow points to a protrusion of one c e l l into a groove of another ("peg and socket" s t r u c t u r e s ) . (16,000 x magnification P h i l i p s 75-C)  11.  A cross s e c t i o n a l view which c l e a r l y shows the increased surface to volume r a t i o produced by the uneven c e l l surface. C e l l s evaginate towards t h e i r neighbours and points of contact may be s i t e s of i n t e r c e l l u l a r communication. (4,400 x magnification Nikon S-Kt)  12.  A cross sectional view of l o n g i t u d i n a l i l e a l c e l l s demonstrating the protrusion of c e l l s between and around other c e l l s , but the c e l l packing i s loose. The e x t r a c e l l u l a r space i s sparsely occupied by collagen f i b e r s . An arrow indicates a nexus l i k e structure between two c e l l s . The c e l l surface v e s i c l e s (caveolae) often aggregate near peripheral mitochondria. (11,500 x magni- f i c a t i o n Zeiss EM-10)  102  Plate 4.  13.  Organelles are concentrated near the nucleus and are interwoven by rough endoplasmic reticulum (R). Rough endoplasmic reticulum can be seen near the c e l l surface but continuity between central and peripheral endoplasmic reticulum i s not apparent i n the plane of the section. (11,350 x magnification Zeiss EM-10)  14.  Enlargement of the nuclear pole area i n photograph 13. The nucleus i s encased by a perinuclear sac of rough endoplasmic reticulum. Two closed sacs of rough endoplasmic reticulum can be seen near the bottom of the micrograph and a Golgi apparatus (G) i s present between them. (23,850 x magnification Zeiss EM10)  15.  Nuclei of two c e l l s containing n u c l e o l i . The nuclear surface i s f a i r l y smooth indicating that the c e l l s are relaxed. (17,143 x magnification P h i l i p s 75-C)  16.  S p i r a l indentations of the nuclear membrane are prominent i n a contracted cell.< (22,000 x magnification P h i l i p s 75-C)  17.  Indentations of the nucleus i n a contracted c e l l which makes the nucleus appear divided i n the plane of the section. (20,000 x magnification P h i l i p s 75-C)  18.  Contorted nucleus of contracted c e l l with c e l l organelles at the nuclear poles pulled into the pockets. Rough endoplasmic reticulum runs through the long axis of the c e l l and the arrow indicates i t s continuity with what could be smooth endoplasmic reticulum. (17,600 xmagnification P h i l i p s 75-C)  104  Plate 5.  19. A view of a longitudinal section demonstrating the r e l a t i v e l y smooth c e l l outline. Arrow heads indicate caveolae and dashes indicate subsarcolemmal membrane sacs (possibly sarcoplasmic reticulum). (18,000 x magnification P h i l i p s 75-C) 20:  An enlargement of the lower right portion of photograph 19, showing membrane sacs between sarcolemma and peripheral mitochondria.  21.  Masses of caveolae at c e l l pole which could possibly combine to form large evacuated areas i n the c e l l s . (17,777 x magnif i c a t i o n P h i l i p s 75-C)  22.  An aggregate of caveolae showing continuity of caveolae with the plasma membrane. Other surface v e s i c l e s may be caveolae whose necks do not appear i n the plane of the section. (43,120 x magnification Zeiss EM-10)  23.  Enlargement of caveolae showing that they are s l i g h t l y elongated. Random sampling of 17 caveolae gave a calculated diameter of 80 + 4 nm and a depth of 95 + 5 nm. (120,000 x magnification Zeiss EM-10)  24.  Rough endoplasmic reticulum and mitochondria can be seen running through the middle of a longitudinal section showing continuity with agranular membranes (see arrows) which could be smooth sarcoplasmic reticulum. (17,600 x magnification P h i l i p s 75-C)  j 25.  A cross sectional view of longitudinal i l e a l c e l l s that shows many d i s t i n c t smooth subsarcolemmal sacs indicated by dashes. The dash nearest the right side indicates smooth membrane sacs i n close apposition to caveolae. (29,333 x magnification P h i l i p s 75-C)  106  Fig. 18.  An example of the e f f e c t of 'glycerol shock' treatment on the guinea pig ileum longitudinal smooth muscle. 1st Row  Control responses to 60 mM  KC1  and  2 x 10~  2nd Row  After e q u i l i b r a t i o n the solution was changed to Tyrode's solution, containing 400 mM g l y c e r o l which caused a transient increase in tension. After 30 min, the muscles were washed twice with normal Tyrode's solution (NT) which again caused an increase i n tension.  3rd Row  The phasic responses to CD and 60 mM KC1 a f t e r 'glycerol shock' treatment were 101.5% + 16.5 and 97.5% +6.3 of control phasic responses, respectively. The tonic responses to CD and 60 mM KC1 a f t e r 'glycerol shock' t r e a t ment were 127.2% +14.4 and 86.2% + 4.3 of control responses, respectively (n = 4).  7  M  CD.  108 V.  The Modified 'La Method' The c a p a b i l i t y of the modified 'La method' to measure i n t r a c e l l u l a r ion  levels w i l l be analyzed i n this section before proceeding to describe  the results  of the studies of ion movements during contraction, relaxation and e q u i l i b r a t i o n using this method.  The  term 'La resistant Ca' and  'La displaceable  Ca' have  been used to denote the amount of Ca remaining i n the tissue and removed from the tissue with a La solution (Sutter and Kromer 1975). described  Under the  i n the present study, 'La-Tris-resistant Ca' and  are nearly equal.  Although the term'La-Tris-resistant  term ' i n t r a c e l l u l a r Ca' i s more convenient.  conditions  ' i n t r a c e l l u l a r Ca'  Ca' i s more exact, the  Therefore the Ca remaining in the  tissue a f t e r washing with La-Tris solution w i l l be referred to as ' i n t r a c e l l u l a r Ca'.  S i m i l a r l y , Na, K and Mg levels in the tissue a f t e r washing with La-Tris  solution at 4°C are also termed ' i n t r a c e l l u l a r ' although 'La-Tris-resistant 'La-Tris-resistant K' and  'La-Tris-resistant Mg'  Na',  would be more exact.  Van Breemen and McNaughton (1970) reported that the use of the  'La method' 45  c l e a r l y demonstrated that rabbit a o r t i c c e l l s take up e x t r a c e l l u l a r contractions  induced when 160 mM  KC1  or 160 mM L i C l was  i n a Tris-buffered physiological medium.  substituted  of the Ca content i n control a o r t i c muscle s t r i p s . to high KC1,  cause contraction.  for the NaCl  Using the same method, Van Breemen 45  et a l . (1972) observed that noradrenaline did not cause a  that i n contrast  Ca during  Ca uptake i n excess  Therefore, i t was  suggested  noradrenaline mainly mobilized i n t r a c e l l u l a r Ca to  Since the results agreed with those of Hinke (1965), i t  seemed that the 'La method' was  able to d i f f e r e n t i a t e between various agonists  and changes i n e x t r a c e l l u l a r i o n i c conditions  that mobilized e x t r a c e l l u l a r Ca  versus those that mobilized i n t r a c e l l u l a r Ca to cause contraction. buf f ered physiological medium containing method', displaced e x t r a c e l l u l a r  The  Tris-  2 mM LaCl^, o r i g i n a l l y used i n the  'La  Ca while preventing i n f l u x and e f f l u x of  45 Ca during 1 hr.  Marshall and Kroeger (1973) used a similar solution for 1 hr  but measured the net i n t r a c e l l u l a r Ca content with atomic absorption  spectro-  109 photometry.  They observed s i g n i f i c a n t increas.es of i n t r a c e l l u l a r Ca a f t e r  treatment of the rat myometrium with noradrenaline.  A similar procedure to  that of Marshall and Kroeger (1973) was t r i e d at f i r s t i n the present study to follow Ca movements during contractions of the guinea p i g ileum longitudinal smooth muscle induced by 60 mM KC1 and 2 x 10  7  M CD.  A control experiment  indicated that La displaced most of the tissue Ca i n 25 min ( F i g . 19, Top). Later, i n order to examine the mode of action of ouabain, the method was extended i n an attempt to measure Na, Mg and K i n addition to Ca. i s described i n the Introduction and Methods.  The procedure  A control experiment indicated  that 30 min was s u f f i c i e n t to remove most of the e x t r a c e l l u l a r Ca, Na, K and Mg ions without much loss of i n t r a c e l l u l a r ions (Fig. 19, Bottom).  The method  caused retention of 90% of the c e l l u l a r K whereas 81% of the tissue Na was l o s t , consistent with the high i n t r a c e l l u l a r K and low i n t r a c e l l u l a r Na concentrations expected i n this tissue.  Normally i n smooth muscle c e l l s , K has the highest ion  permeability (Brading 1971; Casteels 1969) and i f the La-Tris solution did not decrease this permeability, -the c e l l s should have l o s t considerably more than 10% of the c e l l K i n 30 min.  The r e s u l t s of many measurements of t o t a l (n =  32) and i n t r a c e l l u l a r (n = 79) cation levels -of control tissues using the modif i e d 'La method' are compiled i n Table 5.  The results were expressed i n nano-  moles/mg dry weight because a more precise measurement of tissue weight could be made a f t e r drying, as b l o t t i n g removed a variable amount of H 2 O .  Strips of  longitudinal ileum ( 1 - 2 cm) averaged 23.4 mg i n t i a l l y (wet weight) and 3.5 mg when dry (Fig. 20).  The tissues thus contained 85% rL^O and the r a t i o of the  wet weight/dry weight was 6.64.  This r a t i o can be used to convert the results  given as nanomoles/mg dry weight into nanomoles/mg wet weight.  The i n u l i n  space of the guinea p i g ileum longitudinal smooth muscle was calculated by Blowers et a l . (1977) and Burton and Godfraind (1973) to be 470 ml/kg wet weight and 435 ml/kg wet weight, respectively.  The average value i s 452 ml/kg .wet  14 weight.  The  C-sucrose space was 590 ml/kg wet weight (Blowers et a l . 1977).  110 These values and the measurement of the t o t a l tissue water (Fig, 20) have been used to calculate the range of Ca, K, Na and Mg i o n concentrations present i n the e x t r a c e l l u l a r and i n t r a c e l l u l a r spaces (Table 5). Although the levels of Ca and Mg are similar to those i n Tyrode's solution, i t i s readily apparent that the e x t r a c e l l u l a r K l e v e l i s about 3.6 to 4.7 times higher than can be accounted f o r as being i n solution i n the e x t r a c e l l u l a r space. ' Daniel (1963a) observed that a substantial portion of uterine potassium (13%) appeared to be located s u p e r f i c i a l l y . c e l l u l a r space are calculated  Since the quantities of ions i n the extra-  from the differences of the t o t a l and the i n t r a -  c e l l u l a r ion measurements, i t i s possible that some i n t r a c e l l u l a r K ions have escaped, making the i n t r a c e l l u l a r l e v e l of K too low and therefore the extrac e l l u l a r l e v e l too high.  On the other hand, i t may be that some of the excess  rapidly exchanging e x t r a c e l l u l a r K (Goodford 1970; Brading 1973; Palaty and Friedman 1975) was retained i n the caveolae at a higher concentration, more l i k e that of the i n t r a c e l l u l a r space (Gabella 1973).  The same reasoning can be ap-  p l i e d to explain the lower than expected e x t r a c e l l u l a r Na concentration. -4 If the e x t r a c e l l u l a r space i s 4.52 x 10 ml/mg wet weight and 10% of the e x t r a c e l l u l a r space i s i n the caveolae (Section IV), the caveolar volume would be « -4 0.452 x 10 ml/mg wet weight of tissue and the rest of the e x t r a c e l l u l a r space -4 (non-caveolar space) would be 4.07 x 10  ml/mg wet weight.  I f the ion concen-  trations i n the caveolae are more l i k e those i n the i n t r a c e l l u l a r space and the i n t r a c e l l u l a r K and Na concentrations are about 100 mM and 40 mM, then 45.2 x 10  moles of K/mg wet weight and 18.1 x 10 ^  weight are i n the caveolar space.  respectively,  moles of Na/mg wet  I f the rest of the e x t r a c e l l u l a r space con-  tains 2.7 mM K and 136 mM Na, as i n the Tyrode's solution, then 11 x 10 ^ of K/mg wet weight and 553 x 10 ^ noncaveolar e x t r a c e l l u l a r space.  moles of Na/mg wet weight would be i n the Together the caveolar e x t r a c e l l u l a r space and  the noncaveolar e x t r a c e l l u l a r space would contain 56.2 x 10 wet weight and 571 x 10  moles  moles of Na/mg wet weight.  moles of K/mg  In a t o t a l e x t r a c e l l u l a r  volume of 4.52  x 10  ml/mg wet weight, the e x t r a c e l l u l a r K concentration would  be equal to 12.4 mM and the e x t r a c e l l u l a r Na concentration would be 126  mM,  which are nearer to the levels calculated to be i n the e x t r a c e l l u l a r space (Table 5).  Although  the caveolae concentrations might be expected  to be i n t e r -  mediate between the i n t r a c e l l u l a r and e x t r a c e l l u l a r concentrations ( i . e . the concentrations estimated above are probably too high), at least part of the discrepancies found i n measurements of the e x t r a c e l l u l a r concentrations of ions may be due to a caveolar compartment of  10%.  Siegel et a l . (1976) have reported that i n the carotid artery, a f r a c t i o n of the e x t r a c e l l u l a r K, Na, Ca and Mg i s bound to connective tissue (14, 30, 30 and 4.5 nanomoles/mg dry weight, r e s p e c t i v e l y ) .  These values cannot be used  to correct for the binding of e x t r a c e l l u l a r ions i n the guinea pig ileum l o n g i tudinal smooth muscle because i t has less connective tissue than the carotid artery.  However, correcting for bound amounts of cations may bring the extra-  c e l l u l a r ion concentration measured by atomic absorption nearer to the  expected  concentrations i n the Tyrode's solution. -3 Since the calculated i n t r a c e l l u l a r Ca concentration i s 2 x 10 2+ resting free Ca  M and the  -7 concentration of the muscle should be 10  centrated 20,000 f o l d i n i n t r a c e l l u l a r s i t e s .  M, then Ca i s con-  Undoubtedly, there i s s u f f i c i e n t  i n t r a c e l l u l a r Ca to r a i s e the cytoplasmic Ca concentration far beyond that required for maximal contraction.  The freeing of 1/200  would cause maximal contraction. and i f so, how  i s i t released?  of the i n t r a c e l l u l a r Ca  But i s i n t r a c e l l u l a r Ca used for contraction  The extracelluar' Ca concentration was calculated  to be s l i g h t l y higher than the Ca concentration i n Tris-Tyrode's solution. the a d d i t i o n a l e x t r a c e l l u l a r Ca was concentration would be 7 mM.  located i n the caveolae, the caveolar-  If Ca  However, i t i s u n l i k e l y that a l l of the a d d i t i o n a l  e x t r a c e l l u l a r Ca would be exclusively i n the caveolae. The values for the i n t r a c e l l u l a r and e x t r a c e l l u l a r Na and K concentrations (calculated by the modified 'La method') were used to solve the Goldman constant  f i e l d equation to see whether the r e s u l t i n g values would give a reasonably good approximation of membrane p o t e n t i a l observed i n v i s c e r a l smooth muscle.  The  resting membrane p o t e n t i a l of rabbit small intestine i s -55 mV (El-Sharkawy and Daniel 1975).  The range of r e s t i n g membrane potentials of smooth muscles,  measured by microelectrodes i s 35 - 64 mV, according to the review by Casteels (1970).  Brading  (1971) and Casteels (1969) have calculated the r e s t i n g membrane  p o t e n t i a l of taenia  c o l i c e l l s to be -57 mV and -37 mV, respectively. The  observed concentration gradients of Na and K, as measured by the modified 'La method' y i e l d calculated r e s t i n g membrane p o t e n t i a l values i n the range of  '  .47 - 57 mV; (Table 6). 45 Sutter and Kromer (1975) observed that the La r e s i s t a n t tissue  Ca content  increased with time when the e x t r a c e l l u l a r Ca concentration was increased from 2.5 to 5 mM i n the rabbit mesenteric p o r t a l vein.  This could mean that the La  technique was not capable of d i s p l a c i n g a l l of the e x t r a c e l l u l a r Ca when the Ca concentration was raised.  However, i t could also mean that r a i s i n g the extra45  c e l l u l a r Ca concentration drives more of the l a b e l l e d where i t can not be as readily displaced.  Ca into the c e l l s from  Whether or not such an i n f l u x could  be balanced by an e f f l u x of nonradioactive Ca might be ascertained by measuring net Ca levels by atomic absorption spectrophotometry.  The a b i l i t y of the La-  T r i s solution to displace more e x t r a c e l l u l a r Ca i n 30 min was investigated. The e x t r a c e l l u l a r Ca concentration was increased from 1.8 to 3.6 mM for 30 sec or 5 min before using the modified  'La method' to measure i n t r a c e l l u l a r ion  l e v e l s . ( F i g . 21). The t o t a l levels of Ca increased as expected but the i n t r a c e l l u l a r Ca levels remained constant.  The t o t a l and i n t r a c e l l u l a r levels of  Na, K and Mg were not noticeably affected by r a i s i n g the e x t r a c e l l u l a r Ca concentration either.  Therefore the La-Tris solution was able to displace the addi-  t i o n a l e x t r a c e l l u l a r Ca i n the same amount of time and should be applicable to a variety of experiments  that examine the effects of changes i n e x t r a c e l l u l a r ion  levels on the i n t r a c e l l u l a r i o n l e v e l s .  113  Fig. 19. Top. Measurement of the time required for 10 mM LaCl in Ca free Tyrode's solution (pH 7.4) at 4°C to displace extracellular Ca. Ca was measured by atomic absorption spectrophotometry. Values are the average of duplicates. 3  Bottom. Measurement of Ca, Mg, Na and K levels of the guinea pig ileum longitudinal smooth muscle over time i n 160 mM Tris-HCl (pH 7.4) containing 10 mM LaC^ at 4°C. Tissue levels were expressed as a % of the control total tissue levels. (#) = Ca (^) = Mg, (•) = Na and (•) = K. Values are the mean + S. E. (n = 4)  115  Fig. 20.  Measurement of the tissue water content and estimation of the r a t i o of tissue wet weight to the tissue dry weight. Two s t r i p s of guinea pig ileum longitudinal smooth muscle (equilibrated i n normal Tyrode's solution) were blotted and weighed at i n t e r v a l s u n t i l they reached a constant weight at room temperature.  116  117  Table 5.  Calculation;of  the i n t r a c e l l u l a r and e x t r a c e l l u l a r i o n concentrations VOLUME/mg wet weight  t o t a l tissue water = 8.5 x 10" 3  ml  4  e x t r a c e l l u l a r space = 4.52 x l C T i n t r a c e l l u l a r space = 3.98 x 10"  4  ml  b  to 5.9 x 1 0  4  ml  d  to 2.6 x 10"  I n t r a c e l l u l a r Space nanomoles ion/mg^ dry wt Ca  0.8  2 - 3  32.0  80.4 - 123  38.6  38.9 - 59.6  309.7  212.3  '  Na  102.6  15.5  Mg  21.9  3.3  c  ml  e  nanomoles ion/mg^ concentration (mM)  K  4  ml  E x t r a c e l l u l a r Space  wet wt  5.26  - 4  8.3 -12.7  dry wt 7.61 *  3.4  concentration (mM)  wet wt  Tyrode's (mM) '  1.15  2.5 - 1.9  1.8  5.81  12.8 - 9.8  2.7  46.6  103.0 - 79  .51 .  1.1 - 0.09  a.  calculated from 85% tissue water, 1 mg tissue = .85 mg water = 8.5 x 10  b.  calculated from the average i n u l i n space measurements of Blowers et a l .  136 1 ml.  (1977) (470 ml/kg wet wt) and Burton and Godfraind (1973) (435 ml/kg wet wt) in the guinea p i g ileum longitudinal smooth muscle. c.  >  calculated from the ^ C-sucrose space, Blowers et a l . (1977) (590 ml/kg wet 4  wt) . d. e. f.  calculated from the difference of the t o t a l water - i n u l i n space water. 14 calculated from the difference of the t o t a l water C-sucrose space water. results from control tissues i n F i g . 36 and 37. _  118  Table 6.  Calculation of the resting membrane p o t e n t i a l  PT  P  iJ ] • K  +  P  M [Na]. .+ P_- [Cl]  F - M r? ^^ i „ „ K in Na m C1 out m " F § P [K]' • + P [Na] + P . [ C l ] . K out Na out Cl in. E  C 2  L  J  L  J  3 )l o  M  p  J  J  where, RT —F (2.3) at 37°C = 61 mV  (R = Gas Constant, T = absolute temperature, and F = Faraday's constant)  _ o  and  P  R  = 6.71 x 10  (Brading 1971)  P.. = 0.066 x 10~ Na P = 4.4 x 10"  8  8  cl  [Cl]. = 25 mM in [Cl]  out  = 144 mM  (Tris Tyrode's solution)  E m when  ^out  =  [Na]  out  [K]. in  2  #  7  (  = 136 mM  T r l s  ' Tyrode's- solution) " -57 mV  = 80.4 mM (Table 5)  [Na]. = 38.9 mM in  when  [K] = 12.8 mM (Table 5) out [Na] = 103 mM " out [K]. in  -47 mV  = 80.4 mM  [Na] . = 38.9 mM m  "  119  Fig. 21.  Measurement of tissue ion contents by the modified 'La method' after exposure of the guinea p i g ileum longitudinal smooth muscle s t r i p s to Tris-Tyrode's solution containing 3.6 mM CaCl2 f o r 0.5 min (B) and f o r 5 min (C) as compared to controls (A) equilibrated i n T r i s Tyrode's solution (1.8 mM CaCl2). The i n t r a c e l l u l a r contents of Ca, Na, K and Mg were independent of the CaCl2 concentration i n the Tyrode's solution. Tissues that were washed i n the La-Tris solution represent i n t r a c e l l u l a r l e v e l s . Tissues that were not washed with La-Tris solution 5^ represent t o t a l tissue ion l e v e l s .  121 VI.  The Effect of Ouabain on the Intact Longitudinal Smooth Muscle of the Guinea Pig Ileum The primary mechanism of action of ouabain has been attributed to the i n h i -  b i t i o n of the Na,K-ATPase (Akera and Brody 1976; Schwartz 1976) but i n the present study, most of the microsomal ATPase a c t i v i t y , stimulated by Mg and Na, was i n s e n s i t i v e to K and to 3 mM ouabain (Results and Discussion, Section I I ) . -6 However, the muscle s t r i p s were contracted by 5 x 10 (Fig.  22, F i g . 23, F i g . 24).  -4 M -to 10  M ouabain  Therefore, an attempt was made to determine whe-  ther the mechanism by which ouabain caused contraction of the guinea pig ileum longitudinal smooth muscle  was by i n h i b i t i o n of the Na,K-ATPase.  The contraction due to ouabain could have been due to an i n d i r e c t effect on the nerve plexus.  Denervated muscle s t r i p s of the longitudinal ileum were  prepared by the method of Paton and Zar (1965) to see i f the response to ouabain remained when the majority of the nerve terminals were removed.  The dener--  vated muscle s t r i p s did not respond to nicotine or neostigmine but did respond to CD and ouabain (Fig. 22, denervated) while the innervated s t r i p s were very sensitive to a l l of these agents (Fig. 22, innervated).  The denervated muscle  s t r i p s were not tested for responses to f i e l d stimulation so the complete absence of a l l nerve terminals (non-adrenergic, non-cholinergic) could not be ascertained.  Since muscle s t r i p s , as prepared by this method (Paton and Zar  1965) do not respond to f i e l d stimulation (Paton 1975) i t i s probable that ouabain acted d i r e c t l y on the smooth muscle.  This had been previously shown  by Daniel (1964) i n rabbit uterine smooth muscle.  In this l a t t e r tissue, the  s e l e c t i v e blockade of adrenaline, serotonin, histamine and acetylcholine-induced contractions f a i l e d to a f f e c t the responses to ouabain. Responses of muscle s t r i p s to ouabain were tested at a higher e x t r a c e l l u l a r K concentration to see i f K could antagonize the ouabain response, as would be expected i f ouabain acted by i n h i b i t i n g the Na,K-ATPase ( F i g . 23).  Muscle  s t r i p s were used only once because they equilibrated slowly after washout of  122 the ouabain and the second ouabain response was always less than the f i r s t (see Table for F i g . 12, 3rd Row; Bolton 1973a; Casteels 1966).  In order to compare  contractions of d i f f e r e n t tissues, the ouabain responses were expressed both as an increase i n tension above baseline and a percent of a control high KC1 (60 mM) response.  Five uM ouabain, a concentration f i f t y times lower than that  required for complete blockade of the electrogenic pump (Bolton 1973a), contracted the muscle equally well i n Tyrode's solution containing 14.3 mM K or 2.6 mM K (Fig. 23). Daniel (1964) observed that the response of the rat uterus to ouabain was enhanced when the e x t r a c e l l u l a r K concentration was increased from 5.8 to 9 or 18 mM while above 20 mM KC1, the ouabain response was diminished or abolished.  K had no effect on the binding of  [H]-ouabain to r a t myometrium  (Murthy et a l . 1974b), the contraction of this tissue seemed to be dissociable from the i n h i b i t i o n of the Na,K-ATPase (Murthy et a l . 1974a).  The lack of K  antagonism of the ouabain response i n the longitudinal ileum i s i n contrast to the antagonism by K of the ouabain p o s i t i v e inotropic response i n the heart (Caprio and Farah 1967).  K also competes f o r the binding of ouabain and the  ouabain i n h i b i t i o n of the Na,K-ATPase i n the heart (Matsui and Schwartz  1968).  This suggests that ouabain may have a d i f f e r e n t e f f e c t i n smooth muscle than i n heart. Ouabain (10 uM) i n i t i a l l y caused a small rapid increase i n tension i n the longitudinal i l e a l s t r i p s and the tension continued to increase for 2 to 3 min (Fig. 22, innervated; F i g . 24, Top; F i g . 25, Bottom).  At longer times, the  tension relaxed slowly to baseline, even though ouabain was not washed out of the bath.  The phase of r i s i n g tension due to ouabain has been c a l l e d the 'exci-  tatory' phase and the f a l l i n g phase of the tissue tension has been c a l l e d the 'inhibitory' phase.  The response to ouabain of taenia c o l i muscle s t r i p s  (Matthews and Sutter 1967) followed a similar time course to that observed i n longitudinal muscle of the ileum.  Although ouabain immediately reduced the  membrane potential i n taenia c o l i c e l l s , the depolarization was s l i g h t .  The  123 depolarization was accompanied by a 2 to 3 f o l d increase i n spike frequency (3 - 7 min) and a f t e r 15 min, by a depolarization block and Sutter 1967).  (Casteels 1966; Matthews  Complete depolarization was never reached.  That depolariza-  tion was s l i g h t during the i n i t i a l 10 min of ouabain exposure, was consistent with the observation  i n the present study  that a f u l l phasic response to depo-  l a r i z a t i o n by 60 mM KC1 could be attained at the peak of the ouabain response (Fig.  24, Top - top tracing)  was relaxed  and even a f t e r 10 min when the ouabain response  (Fig. 24, Top - lower tracing).  Therefore the muscle must have  been s u f f i c i e n t l y polarized i n order to be able to depolarize and contract when stimulated by 60 mM KC1.  The tonic component of a 60 mM KC1 induced contraction  was not maintained i n the presence of ouabain (Fig. 24, 2nd Row).  Ouabain,  when added during the tonic contraction, caused the longitudinal i l e a l muscles to relax (Fig. 24, 3rd Row) as has previously been observed by Bose (1975). i n taenia c o l i .  This e f f e c t i s not a s e l e c t i v e i n h i b i t i o n of 60 mil KC1, responses  as ouabain also quickly relaxed the tonic response to CD.  A transient  increase  i n tension was observed i n both cases before relaxation began. Changes of the i n t r a c e l l u l a r cation l e v e l s of the longitudinal ileum smooth muscle during ouabain contraction and relaxation were monitored using the modif i e d 'La method' (Fig. 25). I n t r a c e l l u l a r l e v e l s of Ca, Na, K and Mg of the resting muscles were 6.09 +0.22, 82.2 + 3.91, 249.6 + 8.6 and 22.47 + 0.49 nanomoles/mg dry weight, respectively.  The contraction of the muscle to 10 uM  ouabain reached a maximum between 2 - 3 min (Fig. 25, Bottom). I n t r a c e l l u l a r Mg content remained stable over 15 min (see also Palaty 1974). I n t r a c e l l u l a r Ca and Na concentrations  increased s i g n i f i c a n t l y (99% l e v e l ) at the peak of  the contraction (at 3 min, Ca was 7.48 + 0.35 and Na was 124.4 +7.7 nanomoles/ mg dry weight) and then declined as the muscle relaxed.  The increase of i n t r a -  c e l l u l a r Ca was probably not caused by an i n h i b i t i o n of a Ca pump by ouabain because the extra Ca gained was removed during the i n h i b i t o r y phase perhaps by a stimulation of a Ca pump at higher cytoplasmic Ca concentrations.  The i n t r a -  124 c e l l u l a r K concentration was nqt s i g n i f i c a n t l y reduced (95% l e v e l ) u n t i l a f t e r 10 min.  After 7 min,  the K loss was  5.5%  and thereafter the i n t r a c e l l u l a r K  content declined and i n t r a c e l l u l a r Na l e v e l s rose again.  After 15 min the t i s -  sue had l o s t 68.3 nanomoles/mg dry weight of K while only 32.2  nanomoles/mg dry  weight of Na were gained, even though K loss lagged behind the Na gain i n i t i a l l y . -The greater K loss than Na gain between 7 and 15 min was  consistent with the  reported greater permeability of smooth muscle membranes to K compared to Na (Casteels 1969;  Brading 1971).  The early increase i n tissue Ca and Na  content,  without apparent loss of K, was not consistent with a primary e f f e c t of ouabain on the Na,K-ATPase.  I t appeared that ouabain increased the permeability of the  membrane to Ca and Na and perhaps l a t e r also to K.  Daniel and Robinson (1971a)  observed that ouabain did not i n h i b i t K i n f l u x as one might expect.  Instead,  ouabain increased the rate of K e f f l u x from rat uterus which might explain why " K was  l o s t from guinea pig l o n g i t u d i n a l ileum a f t e r 7 min.  But the i l e a l s t r i p s ,  contracted by ouabain, did not exhibit a loss of K during the 'excitatory' phase. Steiness and Valentin (1976) also found that increased myocardial  force  preceded any loss of K i n hearts from intact dogs treated with digoxin and biopsied sequentially.  The temporal d i s s o c i a t i o n of the early onset of p o s i -  tive inotropism and the delayed i n h i b i t i o n of the Na,K-ATPase indicated to them that the p o s i t i v e inotropic effect of d i g i t a l i s glycosides was mediated by a d i f f e r e n t binding s i t e than the s i t e i n h i b i t i n g the Na,K-ATPase. ouabain may  Therefore  not act by i n h i b i t i o n of the Na,K-ATPase i n the heart.  Various reasons for the early 'excitatory' and l a t e r 'inhibitory' (relaxation) action of ouabain have been proposed.  Casteels (1966) attributed the  'inhibitory' e f f e c t of ouabain to the depolarization block, although tension began to decrease while spike frequency was  s t i l l elevated.  Bose (1975) a t t r i -  buted the 'inhibitory' effect of ouabain to a b u i l d up of i n t r a c e l l u l a r Na because the relaxation and the increased rate of K loss was prevented free medium (Pfaffman and Holland 1969).  i n Na  In the present study, Na accumulated  i n the muscle during the 'excitatory' phase to a higher l e v e l than when the 'inhibitory' phase had completely  relaxed the muscle.  Therefore the gain of  i n t r a c e l l u l a r Na can not t o t a l l y explain the i n h i b i t o r y stage.  Tsuda et a l .  (1975) have attributed the ouabain induced relaxation of K contractures to a decrease of the increased NADH linked 0 traction.  2  consumption produced by a high KC1 con-  This may explain why the muscles were unable to maintain a tonic  tension i n the presence of ouabain (Fig. 24). The Ca gained during the ouabain contraction (Fig. 25) may have entered from the e x t r a c e l l u l a r surface of the membrane, or from the lumen of the caveolae or from the e x t r a c e l l u l a r f l u i d .  The time dependence of the loss of the  ouabain response after removal of the e x t r a c e l l u l a r Ca was studied (Fig. 26). The ouabain response was reduced to 50% after 30 sec i n Ca-free Tyrode's solution and thereafter, the response declined gradually u n t i l i t was barely detectable after 10 min.  This time course of dependence of ouabain on Ca follows the  loss of e x t r a c e l l u l a r Ca i n Ca-free Tyrode's solution over 10 min, as measured by the difference between t o t a l and i n t e r n a l Ca by atomic absorption spectrophotometry (see F i g . 33). The loss of the response over 10 min indicates that Ca bound to the outer surface of the c e l l rather than free e x t r a c e l l u l a r Ca was responsible for the ouabain response. As the reduction of the membrane p o t e n t i a l by ouabain can not be explained on the basis of changes i n the Na and K gradients, Draper et a l . (1963) surmised that a Ca current could be responsible f o r the immediate reduction of the membrane p o t e n t i a l and the tension development i n s k e l e t a l muscle by ouabain.  A  s t a t i s t i c a l l y s i g n i f i c a n t (95% l e v e l ) increase of the i n t r a c e l l u l a r Ca content was observed at the peak of contraction i n this study of the l o n g i t u d i n a l i l e a l response to ouabain.  The increase of the i n t r a c e l l u l a r Ca concentration at the  peak of the contraction i s approximately cient for maximal contraction.  -4 5.3 x 10 M which i s more than s u f f i 45  This confirms the  Ca uptake seen during oua-  bain induced tension increases i n rabbit a t r i a (Holland and Sekul 1961) and  guinea pig taenia c q l i (Casteels and Raeymaker 1976),  The uptake of Ca would  not have been observed i f Ca had been mobilized from i n t r a c e l l u l a r compartments to the c o n t r a c t i l e proteins.  The excess i n t r a c e l l u l a r Ca was  removed at a time  coinciding with the onset of the ' i n h i b i t o r y ' phase, perhaps r e f l e c t i n g stimulation"? of the Ca pump by the elevated i n t r a c e l l u l a r Ca The mechanism by which ouabain may  concentration.  increase the membrane permeability to Na  and Ca i s not known. Ouabain i s amphipathic and has a steroid nucleus s i m i l a r to cholesterol, a major component of mammalian plasma membranes.  Cholesterol and  i t s analogues have been shown to influence membrane functions such as passive transport, c a r r i e r mediated transport and the a c t i v i t y of membrane bound enzymes (Demel and Dekruyff 1976).  Enzymatic oxidation of cholesterol causes i n h i b i t i o n  of the Na,K-ATPase (Seiler and Feihn 1976). t e r o l 's precursor)  Accumulation of desmeosterol (choles-  i n sarcolemma resulted i n i n t r a c e l l u l a r Na loss without a  change of the i n t r a c e l l u l a r K l e v e l s (Campion et a l . 1976).  I t i s also relevant  that desoxycorticosterol, although not an i n h i b i t o r of Na,K-ATPase competitively opposes the action of ouabain i n the guinea pig ileum at low concentrations mimics ouabain at higher concentrations  but  (Godfraind and Godfraind-DeBecker 1961).  Using fluorescent probes, Charnock and Bashford (1975) observed that the physical state of the membrane l i p i d s seemed to modulate the Na,K-ATPase a c t i v i t y .  Inhi-  b i t i o n by ouabain was p a r t i c u l a r l y s e n s i t i v e to the physical state of membrane l i p i d s , whereas cation a c t i v a t i o n was  less affected (Charnock et a l . 1975).  It i s possible that ouabain incorporates, l i k e cholesterol, into the plasma membrane and secondarily moves l a t e r a l l y through the b i l a y e r towards more polar molecules embedded i n the membrane, perhaps the Na,K-ATPase.  I t probably disso-  ciates from these regions very slowly because the e f f e c t s of ouabain on smooth muscle are exerted long a f t e r i t i s removed from the e x t r a c e l l u l a r medium ( G r i f f i n et a l . 1972).  Cholesterol tightens the packing of l i p i d s i n the f l u i d  b i l a y e r of the membrane and i t i s possible that i f ouabain incorporated into the b i l a y e r , ouabain would oppose this packing e f f e c t due to a greater p o l a r i t y than  cholesterol and a d i f f e r e n t stereochemistry. High Ca concentrations also s t a b i l i z e membranes by tightening packing (Csapo and Kuriyama 1963; Shanes 1958) and thus would be expected to oppose the incorporation of ouabain into the membrane and oppose the d e s t a b i l i z i n g effect of ouabain. expected at lower Ca concentrations.  The opposite would be  Indeed, rabbit a t r i a are less sensitive  to ouabain at higher Ca concentrations and more sensitive to lower doses of ouabain when e x t r a c e l l u l a r Ca i s reduced, even though i n t r i n s i c a c t i v i t y i s l e s under these conditions (Caprio and Farah 1967). If the primary action of ouabain was s p e c i f i c i n h i b i t i o n of the Na,K-ATPas an early loss of K would be expected because of the high permeability of the sarcolemma to K.  Since K loss lagged well behind Ca and Na gain, even though  they are much less permeant than K, there seems to be a primary increase of the membrane permeability to Ca and Na by ouabain.  128  Fig. 22.  The e f f e c t of ouabain on innervated and denervated guinea p i g ileum longitudinal smooth muscle. The responses to a s e l e c t i v e muscarinic agent (CD, 2 x 10~ M), ouabain (ouab, 10~ M, denervated; 5 x 1 0 M, innervated), nicotine (nic, 18.5 x 10"^ M) and neostigmine (neo, 16.5 x 10 ^ M) i n denervated guinea pig ileum longitudinal smooth muscle s t r i p s were compared to the responses of the innervated muscle s t r i p s to these agents. 7  -  4  -5  13 0  Fig. 23.  The effect of increased extracellular K on contraction by 5 uM ouabain. After equilibration following a high K control response, ouabain was added (top tracing) or the K concentration was increased to 14.3 mM for 5 min and ouabain was added (lower tracing). The response was expressed as the increase in tension and as a percentage of the fast phasic and sustained tonic control responses. Results represent the mean + S. E. of 10 determinations. ¥-2.b * represent normal Tyrode's solution and Tyrode's solution containing 14.3 mM K, respectively. a n c  132  Fig. 24.  Top. Responses to 60 mM KC1 at the response (top tracing) and during (lower tracing). Arrows indicate 10 uM ouabain and the washout (w)  peak excitatory ouabain (ouab) the ouabain i n h i b i t o r y phase the addition of 60 mM KC1 and i n normal Tyrode's solution.  Bottom. The e f f e c t of 10 UM ouabain on a 60 mM KC1 response (top tracing) and a 0.2 uM CD response (lower tracing) during the tonic component. Ouabain was added 7 min a f t e r the tonic response began.  13 3  134  Fig. 25.  I n t r a c e l l u l a r ion levels (top) during the course of an ouabain response (bottom). Control t o t a l tissue ion l e v e l s are indicated to the l e f t of time zero. Control i n t r a c e l l u l a r ion l e v e l s are i n d i cated at time zero. Points represent the mean + S. E. of n determinations indicated i n brackets above time markers.  (27)06)  n  (19)  W  06)  03)  I  (10)  (9)  10  time in the presence of ouabain (min)  (8)  136  Fig. 26.  The s e n s i t i v i t y of the ouabain response to Ca removal. Each ouabain response was calculated as a percent of i t s control phasic high KC1 response and was then expressed as a percent of the mean ouabain response i n normal Ca-containing Tyrode's solution. Each point represents the mean + S. E. of n determinations indicated i n brackets above the time markers. Examples of responses are included. S o l i d arrows indicate the addition of 10 uM ouabain. Dotted arrows indicate the change to Ca-free Tyrode's solution (omission of Ca C l from Tyrode's solution). 2  138 VII.  Possible Sources of Ca for the Phasic arid Tonic Contractions of the Guinea P i g Ileum Longitudinal Smooth Muscle a.  The biphasic contraction  The excitation-contraction-relaxation cycle of the guinea pig ileum l o n g i tudinal smooth muscle was  studied i n muscles contracted by a muscarinic  agent,  cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide (CD) and by 60 KC1.  CD was  the muscarinic  agent chosen for most of the studies of muscarinic  induced events because i t i s very potent, s e l e c t i v e for muscarinic and stable i n solution. f a m i l i a r muscarinic  receptors  The chemical structure i s given i n F i g . 27.  agents, carbachol  (Cch) and methacholine (Mch)  lar biphasic contractions of the l o n g i t u d i n a l ileum smooth muscle. dose of muscarinic  mM  Other more  induced simiAn  optimal  agent (2 x 10 ^ M CD, 5 x 10 ^ M methacholine, or 10 ^ M  carbachol) f i r s t induces a rapid tension increase, which peaks i n 10 sec (Fig. 28, 29 and 44).  This component i s c a l l e d the phasic contraction.  The  phasic  tension declines quickly but as i t declines, a 'shoulder' appears during the f a l l i n g phase of the phasic contraction.  The sudden r i s e of tension at the  'shoulder' would seem to indicate the s t a r t of the slower component of the b i phasic contraction c a l l e d the tonic contraction.  At f i r s t , the r i s i n g tonic  tension increases the net tension but a f t e r the 'shoulder',  the rate of decline  of the phasic tension exceeds the rate of increase of the tonic tension. causes the p a r t i a l relaxation which c l e a r l y separates mum  tensions.  the phasic and tonic maxi-  When the tonic tension increases faster than the phasic component  declines, the o v e r a l l tension increases. min.  This  The tonic component usually peaks af-  ter  5-8  The sudden appearance of a 'shoulder' of tension a f t e r the maxi-  mum  phasic tension i s attained suggests that there i s a sequential turning on of  the phasic and then the tonic component.rather than a simultaneous commencement of both components upon e x c i t a t i o n . Although the 'shoulder' i s not a d e f i n i t e proof that the i n i t i a t i o n of the tonic component lags behind the phasic component by approximately 15 sec, another explanation for the 'shoulder' i s not easy  139 to f i n d .  At lower doses of a muscarinic agent, l o n g i t u d i n a l i l e a l muscles do  not relax between phasic and tonic components, nor i s a 'shoulder' apparent. If the tonic contraction can s t i l l be assumed to lag behind  the phasic compo-r-  nent, then the phasic component can be estimated as the increase i n tension i n 10 sec.  Based on this assumption (which may  n  o  t be v a l i d at very low doses)  the maximum phasic and tonic responses to CD were monitored separately and the tonic component was  observed to be maximal at a lower dose than the phasic  —8  —7  component (5 x 10  M and 2 x 10  M, respectively) (Fig. 27).  tude of the tonic component induced by 2 x 10 —8 lower dose of 5 x 10  M.  7  M CD was  The magni-  less than i t was  at a  The tonic component rose at a slower rate than the  rate of relaxation of the phasic component which could account for the p a r t i a l relaxation between the components.  At lower doses, the tonic component rose  more quickly than the phasic component relaxed, therefore; tension between the components was  sustained.  An explanation for the change i n shape of the b i -  phasic contraction with dose w i l l be offered after the basis f o r the explanation has been presented  (see Results and Discussion, Section VIII).  A dose of 2 x 10  7  M CD was  r o u t i n e l y used to give a maximal phasic compo-  nent e a s i l y distinguishable from the s l i g h t l y submaximal tonic component. p o l a r i z a t i o n of a muscle by 60 mM KC1  De-  stimulated a phasic and tonic contraction,  s i m i l a r i n magnitude to a c o n t r o l response to 2 x 10  7  M CD  (99 + 5% of phasic  response to CD and 94 + 2% of the tonic response to CD; n = 16) i n the same muscle.  The  'shoulder' was  not apparent i n most responses to 60 mM KC1 because  the phasic seemed to f a l l so quickly r e l a t i v e to the rate of r i s e of the tonic component. b.  S e n s i t i v i t y of the phasic and tonic contractions to Ca-free medium  The phasic and tonic responses appear to have d i f f e r e n t s e n s i t i v i t i e s to Ca removal from the p h y s i o l o g i c a l medium.  When a response to CD was  induced 5 sec  a f t e r changing the p h y s i o l o g i c a l medium i n the tissue bath to Ca-free Tyrode's solution, the phasic component attained the same force as i t did i n normal  140 Tyrode's solution but i t was not followed by a 'shoulder' c h a r a c t e r i s t i c of the ensuing tonic component or the tonic component i t s e l f (Fig. 30, Top). I f the 'shoulder' indicates the s t a r t of the tonic response (15 sec after addition of CD),  then the apparent tonic response was l o s t i n 20 sec or less after removal  of e x t r a c e l l u l a r Ca. The tonic component of the response to 60 mM KC1 was l o s t equally as fast as the tonic response to CD.  The phasic component was not no-  ticeably reduced when the muscle was stimulated of e x t r a c e l l u l a r Ca.  by 60 mM KC1, 5 sec after removal  The tonic component would seem to be due to a mobilization  of free e x t r a c e l l u l a r Ca because most of the free e x t r a c e l l u l a r Ca should d i f f u s e out of the e x t r a c e l l u l a r space i n 20 sec. The residual l e v e l of Ca may not be able to i n i t i a t e the tonic component rapidly or to a noticeable  extent, above  the f a l l i n g tension of the phasic component i n Ca-free Tyrode's solution.  The  slower t r a i l i n g o f f of the phasic component conceivably may be due to a minor tonic component under these  conditions.  The induction of a 60 mM KC1 or CD response from 15 sec to 10 min after removing the e x t r a c e l l u l a r Ca caused a reduction  of the peak phasic tension as  a function of the time i n Ca-free Tyrode's solution (Fig. 30, Bottom).  The pha-  s i c component was reduced to 50% i n 2 min and was barely detectable after 10 min.  The difference i n the rates of loss and restoration of the phasic and  tonic components upon switching to Ca-free Tyrode s solution and returning to normal Tyrode's solution i s i l l u s t r a t e d i n F i g . 31. to be l o s t very rapidly and could be restored  The tonic component appeared  i n 30 sec or l e s s .  The phasic  component also could be completely restored after addition of Ca f o r 30 sec, which implies a reloading of high a f f i n i t y s u p e r f i c i a l Ca binding  sites.  The  results were e s s e n t i a l l y the same f o r high KC1 and CD responses. Reduction of the e x t r a c e l l u l a r Ca concentration can increase l i t y of the membrane (Shanes 1958).  the permeabi-  Total and i n t r a c e l l u l a r Ca, Mg, Na and K  ion l e v e l s were measured i n muscle s t r i p s equilibrated i n Ca-free Tyrode's solution for 0 - 1 0 min to see i f changes of* the tissue ion levels could be corre-  141 lated with the time course of the loss of c o n t r a c t i l i t y i n Ca-free medium (Fig.  32).  Ca were 110,  After 10 min  i n Ca-free medium, i n t e r n a l tissue Na, Mg,  87, 86 and 52% of t h e i r normal i n t e r n a l l e v e l s , respectively.  Although the muscle f i b e r s had  gained Na and l o s t K and Mg,  which would be  expected i f the c e l l s were more permeable, the major change was and i n t e r n a l Ca concentrations.  i n the  total  External Ca could be calculated from the d i f -  ference between the t o t a l and i n t e r n a l Ca l e v e l s . 10 min  K and  i n Ca-free Tyrode's solution was  The loss of tissue Ca over  plotted for F i g . 32 as the percent of  the control i n t e r n a l and external Ca contents of tissues equilibrated i n normal Tyrode's solution.  This was  component from F i g . 30.  compared with the rate of the loss of the phasic  There appeared to be a c o r r e l a t i o n between the loss of  external Ca and the loss of the phasic component (Fig. 33).  Since the free  e x t r a c e l l u l a r Ca should d i f f u s e out of the e x t r a c e l l u l a r space i n a far shorter time than 10 min,  the e x t r a c e l l u l a r Ca that correlates with the phasic  might be bound or retained i n the e x t r a c e l l u l a r space by some means.  tension The  surface of the sarcolemma may  r e t a i n Ca that can be transferred into the  when activated by CD or 60 mM  KC1.  c.  Perhaps caveolae serve i n this  S e n s i t i v i t y of the phasic and  tonic contractions  outer cell  respect,  to LaCl-^  LaCl^ has previously been shown to i n h i b i t the phasic component more than the tonic component of the guinea pig ileum longitudinal smooth muscle (Chang and Triggle 1972).  I t has not been s e t t l e d whether or not LaCl^ acts only on  the e x t r a c e l l u l a r surface of the c e l l (see Introduction,  Section V.b).  In the  present study, treatment of the muscle with LaCl^ 10 ^ to 10 ^ M for 5. min, s e l e c t i v e l y i n h i b i t e d the phasic component of the high KC1 The  contraction  tonic component of the La-inhibited response to 60 mM KC1  force but required a longer time to reach i t s maximum.  attained  (Fig. 34). the same  In contrast, both the  phasic and the tonic components induced by CD were i n h i b i t e d by La (Fig. 35). -4 Higher concentrations of La (10 the response to  CD.  M) are required  for complete i n h i b i t i o n of  142 The conditions under which La i s used are important.  At La concentrations  i n the uM range, Ca and Mg concentrations should be l e f t at their normal levels for maintenance of membrane i n t e g r i t y i n order to confine La to the e x t r a c e l l u l a r space, basement membrane and sarcolemmal surface.  At higher concentrations  (.10 mM), La has a powerful membrane s t a b i l i z i n g effect (Casteels et a l . 1972). Under these conditions, Ca and Mg can be omitted from the physiological medium without weakening membrane i n t e g r i t y and without allowing La to enter into c e l l s . La should not permeate intact c e l l s i n 5 min, at these concentrations, i n the presence of normal amounts of Ca and Mg to maintain membrane i n t e g r i t y . i n h i b i t i o n of actomyosin  The  Ca-ATPase by La (Fig. 15, Bottom) would indicate that  La would prevent c o n t r a c t i l e force production i f i t penetrated the c e l l .  La  could not replace Ca for the c o n t r a c t i l e response when allowed to enter c e l l s depleted of Ca and treated with A23187, an ionophore allowing i t s passage through the membrane (Triggle et a l . 1975).  In the present study, La completely  inhi-  bited the phasic component of a high KC1 response while the magnitude of the tonic contraction was unaffected (Fig. 34 and 35).  Therefore La remained on the out-  side of the c e l l because i f i t had entered  the c e l l s , the c e l l s would^nQt  havej. been able to contract t o n i c a l l y  The tonic component i n response to  60 mM KC1 i s not inhibited by La, but merely delayed, probably because there are at least 180 free Ca ions per La molecule the channels.  (at 10  M) to compete for access to  Since La probably displaces Ca bound to the outer aspect of the  c e l l , this provides a d d i t i o n a l evidence that at least part of the Ca responsible for the phasic component could be located on the outside of the* plasma membrane. It i s d i f f i c u l t to explain why La s e l e c t i v e l y blocks the phasic component of the contraction induced by 60 mM KC1 but blocks both the phasic and the tonic components of the contraction induced by CD.  The i n h i b i t i o n of both the phasic and  tonic components could be caused by La i n h i b i t i o n of the binding of CD to .the receptor rather than blockade of Ca movements.  Although La binds more t i g h t l y  to most anionic s i t e s than the acetylcholine quaternary ammonium cation, (Hauser  143 et a l . 1976) the concomitant tonic and phasic i n h i b i t i o n may not r e f l e c t receptor  antagonism i f the muscarinic receptor i s a more discriminating membrane  anionic receptor s i t e than most of the surface anionic s i t e s .  The magnitude  of the tonic component induced by CD may depend on the magnitude of the phasic component.  I f CD inwardly directs Ca from s p e c i f i c receptor associated s i t e s ,  causing a Ca.depolarizing current, (Chang  and T r i g g l e 1972) these calcium ions  may be less readily displaced by La and require a higher La concentration to displace them than the Ca mobilized by high KC1.  The magnitude of the CD tonic  component may r e f l e c t the degree of depolarization allowed when the 'phasic' Ca current was p a r t i a l l y blocked.  High e x t r a c e l l u l a r KC1 concentrations would  depolarize a c e l l whether or not there was a phasic Ca current ahd.iif the tonic component only requires a depolarization of the sarcolemma, this would explain why i t s tonic component i s independent of phasic blockade.  I f the explanation  above i s valid , i t might account f o r the d i f f e r e n t La s e n s i t i v i t i e s df the phasic: :  and tonic components induced by 60 mM KC1 or CD without necessarily implicating d i f f e r e n t u t i l i z a t i o n of Ca pools for phasic and tonic responses to d i f f e r e n t stimuli.  The higher valence and s i m i l a r ionic radius of La to Ca causes La to  bind at s u p e r f i c i a l anionic s i t e s with greater a f f i n i t y than Ca. T r i g g l e and 3+ Triggle (1976) have calculated that the density to which Tm  (the most potent  of the lanthanides) must bind to the surface of the guinea p i g ileum longitudinal ?2 smooth muscle c e l l s , to i n h i b i t the phasic component, i s only 1 per 4,000 A . Therefore the replacement of only a small f r a c t i o n of the t o t a l membrane bound 3+ Ca by lanthanides prevents the induction of the phasic component.  Perhaps Tm  displaces a s p e c i f i c Ca pool or blocks s p e c i f i c Ca s i t e s or channels that are c r i t i c a l for excitation-contraction coupling. d.  Measurements of i n t r a c e l l u l a r ion contents during contractions induced by CD and 60 mM KC1  Although  the c o n t r a c t i l e studies yielded ample information upon which to  theorize about the changes i n i n t e r n a l ion content during and following a con-  t r a c t i o n , i t was f e l t that the best way to determine the events responsible f o r the observed behavior was to measure i n t e r n a l l e v e l s of Ca, Mg, Na and K, using the modified 'La method' (Fig. 36 and 37).  Contractions induced by 60 mM KCl  were accompanied by a net gain of Ca, a loss of Mg, very l i t t l e change i n Na and a gain of K (Fig. 36). The gain of K was the only q u a l i t a t i v e difference between the response of high K and the response to CD.  Relaxation was accompanied by a  rapid loss of i n t r a c e l l u l a r Ca to below normal l e v e l s , a gain of Mg, a loss of Na and a sustained higher K content.  E q u i l i b r a t i o n was accompanied by a return  to normal of Ca, a fluctuating but generally returning to normal Mg, maintained lower Na and a sustained higher K content. The net increase of i n t r a c e l l u l a r Ca induced by 60 mM KCl i n 10 sec indicated that a mobilization of e x t r a c e l l u l a r Ca gave r i s e to the phasic tension.  From the experiments^ discussed e a r l i e r i n this section, i t i s apparent  that the phasic component i s not due to free e x t r a c e l l u l a r Ca because the muscle retains some of i t s phasic responsiveness over 10 min i n Ca-free Tyrode's solution.  Therefore, the Ca responsible f o r the phasic response i s bound to an  e x t r a c e l l u l a r s i t e , perhaps the outer surface of the membrane or bound i n caveolae.  Of a l l the experiments, each pointing to e x t r a c e l l u l a r membrane bound Ca as  the source of Ca for the high KCl phasic contraction, this evidence i s the most direct.  These findings support the e a r l i e r observations by T r i g g l e and Triggle  (1976) that high KCl induced a s i g n i f i c a n t uptake of ^Ca after 30 sec i n the 4  guinea p i g ileum longitudinal muscle.  In the present study, the high K depolari-  zation causes more than s u f f i c i e n t Ca to enter the c e l l for maximal contraction -4 -4 (2.4 x 10 M Ca at 10 min [phasic] and 6.4 x 10 M at 10 min [tonic]). 45 Ukrawa and Holland (1964) observed  that there was an enhanced  Ca uptake  during the phasic and tonic response of taenia c o l i contractions induced by 40 mM KCl.  The uptake during the tonic phase was greater than during the phasic  contraction. High K contractions were reported by Weiss (1972) to cause a de45 45 crease i n Ca e f f l u x , an increased uptake of Ca and a greater t o t a l Ca con-  145 tent.  Triggle and Triggle 0-976) observed  45 significant  Ca uptake i n guinea p i g  ileum longitudinal smooth muscles 15 sec (phasic) and 30 min l a t i o n of the muscle s t r i p s with 80 mM KCl.  (tonic) after stimul-  Lullman and Mohn (1969) reported that  guinea p i g ileum l o n g i t u d i n a l smooth muscle c e l l s , depolarized by e l e c t r i c a l s t i mulation rather than by high KCl, took up Ca during each stimulus. Their results 6 —17 indicate that 5 x 10 atoms (8.33 x 10 moles) of Ca are gained per c e l l per e l e c t r i c a l stimulus and i f the c e l l volume i s 9.81 x 10 5u, Paton and Rang 1965)  ml (cylinder 50y x  then the i n t r a c e l l u l a r Ca concentration would be raised  —6 to 8.5 x 10  M, which might be able to induce a maximal contraction.  Collins  et a l . (1972) have calculated that a single action potentialcouiddraise the i n t r a c e l l u l a r Ca concentration by 6 x 10 ^ M and 8 x 10 ^ M depending on whether the rabbit anterior mesenteric-portal vein c e l l i s assumed to be a cylinder or a double cone.  These values are above the threshhold Ca concentration required for -4  contraction.  In the present study, high KCl induced a Ca uptake of 2.4 x 10  M  during the phasic response which i s i n excess of that required to depolarize the c e l l by a Ca current or to maximally contract the c e l l .  The net uptake of Ca  after 10 min of sustained depolarization and sustained contraction by 60 mM  KCl  -4 was  6.4 x 10  M.  The contraction by CD was accompanied by a small gain of Ca, a loss of Mg, no apparent change i n Na and a loss of K (Fig. 36).  Relaxation i n 30 sec was  accompanied by a rapid loss of Ca to below normal l e v e l s , a rapid gain of Mg, no change i n Na and no change i n K.  E q u i l i b r a t i o n was accompanied by a gradual  regaining of Ca, an unsteady return to normal of Mg, no s t r i k i n g change i n Na and a gradual gain of K to normal l e v e l s .  The divalent cations, Ca and  changed i n opposite directions but the i n t r a c e l l u l a r changes  i n the monovalent  cation, K, were not accompanied by changes i n i n t r a c e l l u l a r Na l e v e l s . phasic component may ing s i t e s . (4.3 x 10  Mg,  The  CD  also be due to a mobilization of Ca from s u p e r f i c i a l bind-  A small increase i n i n t r a c e l l u l a r Ca occurred after 10 sec i n CD moles/mg dry weight or approximately  6.5 x 10  moles/mg wet  weight),  146 -4 which i s s u f f i c i e n t to raise the i n t r a c e l l u l a r Ca concentration to 1,6 x 10 M, -12 This i s much nearer to the predicted 3 ^ 20 x 10  moles of Ca per mg wet weight  (Blowers et a l . 1977: Freeman and Daniel 1973; Van Breemen and McNaughton 1970) necessary ^Ca  for maximal a c t i v a t i o n of the c o n t r a c t i l e proteins than the reported  uptake values of 1.3 - 4 x 10  moles per mg wet weight that these authors -12 obtained. A net increase of 4 x 10 moles per mg wet weight would raise the -4 -5 Ca concentration of the i n t r a c e l l u l a r compartment (4 x 10 ml) to 10 M. I t probably would have been impossible to prove the s i g n i f i c a n c e of an uptake of 4 x 10  moles of Ca per mg wet weight since even the increase of 6.5 x 10~^  moles of Ca per mg wet weight, a f t e r 10 sec of CD, was not quite s i g n i f i c a n t . -12 The t h e o r e t i c a l value of 4 x 10  moles/mg wet weight assumes that a l l the  Ca entering i s i n free form but since some of i t must bind to c o n t r a c t i l e proteins i n order to cause contraction, more Ca would be needed to make the free cytoplasmic Ca concentration 10 ^ M.  In the guinea pig ileum longitudinal smooth  muscle, the r a t i o of mg protein per mg wet weight i s 0,1 (Table 2: t o t a l whole homogenate protein = 91,6 mg/900 mg wet weight of t i s s u e ) .  I f approximately 50%  of this protein i s c o n t r a c t i l e filament and 10% of myofilaments bind Ca (troponinl i k e component), then 5 yg protein per mg wet weight w i l l bind Ca. This troponin-12 l i k e component binds 1.3 x 10 mole of Ca per yg c o n t r a c t i l e protein (Fuchs and -12 Briggs 1968), therefore 6.5 x 10 mole Ca are needed per mg wet weight of t i s -12 sue to saturate these s i t e s with Ca. Together the free cytoplasmic Ca (4 x 10 -12 mple/mg wet weight) and the bound Ca (6.5 x 10 mole Ca/mg wet weight) would -12 require the c e l l to take up 10.5 x 10  mole of Ca per mg wet weight of tissue  or 6.2 times less than was observed during the maximal phasic response induced by CD. 45 As long as  Ca was e q u i l i b r a t e d with surface binding s i t e s of l o n g i t u d i n a l 45 i l e a l s t r i p s for 10 to 60 min, CD induced a s i g n i f i c a n t Ca i n f l u x i n 30 sec (phasic response) above controls (Blowers et a l . 1977), Without p r i o r e q u i l i 45 45 bration (simultaneous addition of Ca and CD), Ca uptake was not seen. A  147 comparison of the two statements above indicates that the phasic component i s not due to an i n f l u x of free e x t r a c e l l u l a r Ca, but rather i t i s due to an i n f l u x 45 of membrane bound Ca which requires time to e q u i l i b r a t e with be detectable.  Ca tracer ions to  In a l l comparisons of high K and neurotransmitter induced res-  ponses, high K induces a greater uptake of Ca although i t induces the same tension increase as an optimal dose of the transmitter agent.  K seems to induce Ca  uptake i n excess of that which can be u t i l i z e d by the c o n t r a c t i l e filaments but 45 the  Ca measurements exaggerate this increase (especially at longer times) by  ignoring the nonradioactive Ca e f f l u x as i t i s replaced by ^Ca from the medium. 4  The atomic absorption method developed here yielded more r e a l i s t i c net changes in i n t r a c e l l u l a r ions.  The measured Ca uptakes during contractions of.the l o n g i -  tudinal ileum by CD, high K and ouabain more than s u f f i c e to activate the cont r a c t i l e apparatus.  Freeing of i n t r a c e l l u l a r Ca from sarcoplasmic reticulum  or mitochondria would only further increase the excess Ca already present. Yet Ca i s present i n these organelles i n smooth muscles (Devine et a l . 1973; Jonas and Zelck 1974;  Somlyo and Somlyo 1976)  and must serve some purpose.  The experi-  ments reported so far i n guinea pig ileum longitudinal smooth muscle suggest that a mobilization of a bound e x t r a c e l l u l a r Ca pool may be a l l that i s needed for contraction and i s almost c e r t a i n l y a prerequisite for any i n t e r n a l Ca release. The sarcolemmal enriched microsomal f r a c t i o n contains a Ca-ATPase that could possibly pump Ca out of the c e l l but the a b i l i t y of the microsomes to transport Ca was not investigated.  Rapid losses of i n t r a c e l l u l a r Ca were observed 30 sec  a f t e r termination of high K and CD contractions by washout.  I f Ca had been  temporarily stored i n i n t e r n a l organelles, i n t r a c e l l u l a r Ca would have remained high subsequent to relaxation but i t did not.  On the other hand, much can happen  i n 30 sec at the molecular l e v e l so the data i s not p o s i t i v e proof that the sarcoplasmic reticulum sacs do not accumulate the Ca before i t i s pumped out of the cells.  A sarcolemmal Ca pump i s probably activated by the higher than normal  i n t r a c e l l u l a r l e v e l s of Ca to remove the extra Ca since net i n t r a c e l l u l a r Ca  148 l e v e l s did not increase unreasonably oyer 10 min,  This suggests a cycling of  Ca into and out of the c e l l s during stimulation and contraction only when the rate of Ca i n f l u x exceeds Ca e f f l u x . "The Ca pump a c t i v i t y may catch up to or overtake the rate of i n f l u x of Ca during responses induced by CD for longer than 10 min (tension begins to decline).  Triggle and Triggle (1976) did not observe  45 Ca uptake by longitudinal i l e a l c e l l s a f t e r 30 min exposures to CD, although uptake was apparent a f t e r 30 sec exposures perhaps because the extrusion rate of the  pump caught up to the rate of Ca i n f l u x . The emphasis on the importance of the Ca d i s t r i b u t i o n for smooth muscle elec-  t r i c a l and mechanical events has obscured the importance of the Na, K and Mg gradients.  Since others have suggested that Ca i s pumped out of the c e l l by a  Na:Ca exchange mechanism (Reuter et a l . 1973; Bohr et a l . 1969), the effect of the  Na gradient on the;muscle tension and the a b i l i t y of the muscle to relax was  investigated.  Responses to high KCl were found to sustain tonic tension only  when the Tyrode's solution contained a normal amount of NaCl.  I f NaCl was r e -  duced to compensate for the increased osmolarity of an extra 58 mM KCl the tonic response was transient (data not shown).  Therefore, .-the  induced i n the presence of a normal amount of NaCl.  60 mM KCl responses were  The e f f e c t of reducing extra-  c e l l u l a r Na on the guinea pig ileum longitudinal smooth muscle i s shown i n F i g . 38. The normal Tyrode's s o l u t i o n containing NaCl, NaHCO^, NaH^PO^ and KCl was changed to contain NaCl, KHCO^ and KTL^PO^ which increased the K concentration from 2.7 to 12.3 mM,  therefore KCl was omitted.  The NaCl was gradually reduced to zero by  substituting T r i s t i t r a t e d to pH 7.4 with HC1.  The higher external K concentra-  t i o n at f i r s t increased the amplitude and frequency of spontaneous but the baseline remained at the same l e v e l (Fig. 38, 1st Row).  contractions  When Na was re-  duced from 149 mil to 104, 69.4, 34.7, 17.4 mM and f i n a l l y to zero, there was an i n i t i a l rapid gain of tension at each change which s e t t l e d down to a higher than normal baseline.  The amplitude of the spontaneous a c t i v i t y gradually declined  but s t i l l existed at 11% of normal Na, as was also observed by Holman (1957) i n  )  149 taenia c o l i smooth muscle . When the Na concentration was zero, spontaneity ceased i n the guinea pig l o n g i t u d i n a l ileum (Fig, 38, 1st Row) and i n the taenia c o l i (Holman 1957).  Spontaneous a c t i v i t y returned after 12 min i n normal Tyrode's  solution and the muscle responded to CD almost as i t had responded before Na deprivation (Fig. 38, 2nd Row).  Switching abruptly to Na free solution caused  a rapid gain i n tension and a gradual loss of spontaneity  (Fig. 38, 2nd Row).  The response appeared i d e n t i c a l to that observed by Holman ' (1957) when she s u b s t i tuted choline for Na.  Control experiments were performed maintaning KCl at i t s  normal 2.7 mM i n a Tris-buffered Tyrode's solution to see i f a sudden change to zero Na content of the Tyrode's s o l u t i o n s t i l l induced a contraction. were p o s i t i v e (Fig. 33, 4th Row).  The r e s u l t s  Removal of Na for 10 sec p r i o r to adding CD '  allowed the muscle to develop a normal phasic spike but the tonic component was weakened.  The washout with a normal Na-containing  Tyrode's solution caused  a sudden transient r i s e i n tension (Fig. 38, 4th Row * ) , intimating a release of the depression of the tonic response when Na was re-added.  Sucrose, rather than  Tris-HCl, s u b s t i t u t i o n for NaCl for 5 sec also altered CD and high K responses compared to controls (Fig, 38, Insert). a f t e r a prolonged  A response to CD could not be invoked  treatment of the muscle i n zero Na medium (Fig. 38, 3rd Row)  indicating that the muscle needs some Na to u t i l i z e Ca for contraction. Casteels (1970) has pointed out that osmotically equivalent substitution of Na by sucrose decreases i o n i c strength and reduces external chloride concentration, thereby reducing K permeability. sons. 7.4,  Its use may not be v a l i d for p h y s i o l o g i c a l rea-  T r i s chloride i s a better but not a perfect substitute for NaCl.  At pH  30% of the T r i s i s unionized and penetrates c e l l s thereby eventually d i s -  placing i n t r a c e l l u l a r K.  Therefore, the experiments i n F i g . 38 should be i n t e r -  preted bearing this i n mind. CD contractions could be relaxed i n the absence of e x t r a c e l l u l a r Na apparentl y at the same rate as i n the presence of e x t r a c e l l u l a r Na (Fig. 38, 3rd Row). The secondary increase i n tension was due to the higher than normal K (see F i g .  150 52, 3rd Row - washout In 5 times normal K).  Although e x t r a c e l l u l a r Na would not  be reduced to zero immediately, the r e s u l t s imply that contracted longitudinal i l e a l muscles do not reduce i n t r a c e l l u l a r Ca to r e s t i n g levels by a Na:Ca exchange mechanism.  The lack of sustained tonic tension for high K or CD responses  in reduced Na medium also argues against a Na:Ca exchange mechanism for relaxation.  151  F i g . 27.  Log dose response curve to cis-2-methyl-4-dimethylaminomethyl-l,3dioxolane methiodide (CD). The s o l i d l i n e represents the response of the phasic component to dosel The dotted l i n e indicates the response of the tonic component to dose. See:Fig. 28.for the actual polygraph tracings. CD (2 x 10~7 M) was chosen as an optimal dose for phasic and tonic components.. Points represent the mean + S. E. (n = 4)  153  F i g . 28.  The responses of the guinea p i g ileum longitudinal smooth muscle to increasing doses of CD. . The tonic, component ..increases more at lower doses than the phasic component. .Maximal responses are at 5 x 10 ^ M. f o r the tonic and 2 x 1 0 M f o r the phasic. At 2 x 1 0 M, there i s a d i s t i n c t shape change.i.e; the phasic and tonic components are separated by a p a r t i a l relaxation.'.At higher doses.the tonic component declines i n magnitude. -  - 7  i  - 7  154  155  F i g . 29.  Theiresponse of longitudinal i l e a l muscle to methacholine. Similar dose dependent shape changes as the biphasic shape changes to CD are observed. The biphasic shape change: i s not-peculiar to CD but rather i s a general muscarinic agonist phenomenon.  156  157  F i g . 30. Top. A comparison of phasic and tonic responses i n normal Tyrode's solution (control) and after switehMgg to Ca-free TyrodeSs solution for 5 sec prior to 2 x 10~ M CD or 60 mM KCl addition. The phasic component i s unaffected at 5 sec but the tonic component i s absent. 7  Bottom?. The loss of the phasic component when CD and high KCl are added a f t e r various times i n Ca-free Tyrode's.solution. The dotted l i n e represents the 2 x.10~7 M CD phasic response.and the s o l i d l i n e represents; the high ,K..phasic response. Points represent mean + S. E. Tabl'eiof n Values Time (min)  CD  -CKC1  0.25  8  12  0.50  16  7  1.00  12  8  1.50  12 ..  -  2.00  12  8  4.00  12  -  5.00  -  4  6.00  8  10200  4  10  \  159  F i g . 31.  The s e n s i t i v i t y of high K responses (top) and 2 x 10 ' M CD responses (bottom) to the removal of Ca from the longitudinal i l e a l smooth muscle for 10 min and restoration of Ca for 30 sec. Open bars represent the phasic components and shaded bars represent the tonic components.- Points represent - the mean + S. E. -  Table of n Values Treatment  CD  Ca-free 30 sec  12  Ca-free 10 min  4  10  Ca-free 10 min normal Tyrode's solution 30 sec  8  6  KCl 1  7  X  160  f  if  Ca free Tyrode  rriormai Tyrode  10 min  imsn normal 2u Tyrojae 3  0  r*  normal Tyrode  Ca jfree  Tyrode 30 S sec  Ca free Tyrode min . 1 0  Ca [free] Tyrode r norma! Tyrode 3 0 fsec  161  F i g . 32.  The effect of incubation of tissues i n Ca-free Tyrode's solution f o r various times on t o t a l ( s o l i d . l i n e ) and e s s e n t i a l l y i n t e r n a l (dotted l i n e ) tissue ion l e v e l s . At time 0, n = 50 f o r i n t e r n a l and n = 13 for t o t a l measurements of.ion.contents. A l l other experimental data points are the mean + S. E. (n = 6)  163  F i g . 33.  The rate of loss of the phasic component ( s o l i d l i n e s ) compared to the rate of loss of e s s e n t i a l l y i n t e r n a l and external Ca (dotted l i n e s ) . External Ca was calculated from the difference between the average t o t a l and the average i n t e r n a l Ca (n = 6) from F i g . 32. 100% of external Ca-was taken as the difference between t o t a l (n = 13) and i n t e r n a l (n = 50) Ca l e v e l s . External and i n t e r n a l Ca l e v e l s at various times a f t e r Ca-free Tyrode's solution were calculated as a percent.of the control.levels i n normal Tyrode's solution. The percent loss of the.phasic component..was.replotted from F i g . 32. Error bars were omitted f o r c l a r i t y .  165  F i g . 34.  The effect of L a C l (10~ t o . 1 0 M) on high KCl (60 mM) responses i n . T r i s Tyrode's solution. Each dose of.LaCl3 was.tested on a d i f ferent muscle because LaCl^ i n h i b i t i o n i s not.readily reversible. Percent i n h i b i t i o n was.calculated.from, a control response ( l e f t side). La was added 5 mimibefore• inducing a second .responseww;fcth6;60 mM KCl (right side). 6  -5  3  ;  167  F i g . 35.  Comparison of the effect of LaCl3 on the phasic (solid l i n e ) and tonic (dotted l i n e ) component of responses induced by 60 mM KCl and 2 x 10"? M CD. LaCl3 was added 5 min before the.test.response. Muscle s t r i p s were used only once. Points represent the mean + S. E. Table of n Values LaCl3 Concentration 10 2.5 x l O  - 6  - 6  5.0 x 10-6 7.5 x 10~§= 10  - 5  5.0 x 1 0  - 5  CD  KCl  M  11  4  M  12  4  12  4  7  3  23  4  8  4  4  7-  % inhibition of high KCl T  contractions  % inhibition of CD contractions  M 0 3  169  Fig.  36.  I n t r a c e l l u l a r ion levels during the course of a contraction induced by 60.mM KCl (from.10 sec to 10 min).and the e q u i l i b r a t i o n phase (from 0.5 to.30.min) i n Tris-Tyrode's s o l u t i o n . Control t o t a l tissue ion l e v e l s are indicated to the l e f t . o f time zero-(i.e. l e f t of the dotted l i n e s ) . Control i n t r a c e l l u l a r . i o n levels, are indicated at time zero. I n t r a c e l l u l a r ion.levels.at.10 sec and.10 min are indicated along the v e r t i c a l dotted l i n e s . I n t r a c e l l u l a r . i o n l e v e l s during e q u i l i b r a t i o n are to t h e , r i g h t . o f . t h e v e r t i c a l . d o t t e d l i n e s . The symbols represent (#).Ca..and (•) Mg on the right ordinate and ( A ) Na and (•) K on the l e f t ordinate.. Points represent the mean + S. E; of n determinations .indicated..in.brackets above the time markers. A representative contraction and.equilibration phase i s drawn at the bottom. ;  170  3.71  Fig.  37.  I n t r a c e l l u l a r ion l e v e l s during the course of a contraction induced by 2 x 1 0 M CD (fr om 10 sec to 10.min) and the e q u i l i b r a t i o n phase (from 0.5 to 30 min) i n Tris.Tyrode's solution (TT). Control t o t a l tissue ion levels are indicated to the l e f t of time zero ( i . e . l e f t of the v e r t i c a l dotted l i n e s ) . Control i n t r a c e l l u l a r ion l e v e l s are indicated at time zero. I n t r a c e l l u l a r ion l e v e l s during the contraction are indicated between.the .vertical dotted l i n e s . Intrac e l l u l a r ion l e v e l s during the.equilibration phase are indicated to the r i g h t of the dotted l i n e s . The symbols represent (•) Ca and (••) Mg on the right ordinate and. ( A ) Na.and (•) K on the l e f t ordinate. Points represent the mean,+ .S. E. of n.determinations indicated i n brackets above the time.markers. A representative cont r a c t i o n and e q u i l i b r a t i o n phase are:drawn at the bottom. - 7  s  172  time (min)  173  F i g . 38. The effect of reducing e x t r a c e l l u l a r Na on the longitudinal i l e a l muscle a c t i v i t y . The results represent 1 of 4 muscle s t r i p s run simultaneously. 1st Row  Control response to CD (2 x 1 0 M) i n Tyrode's solution containing 149 mM Na and 2.7 mM K. NaHC03 and NaHPO-4 were replaced by KHCO3 and KH2PO4 to maintain the pH but allow eventual reduction of Na to zero.. KCl was omitted because the K concentration due to KHCO3 and KH^PO^ was 12.3 mM. Raising K but maintaining 149 mM Na increased the amplitude and frequency of spontaneous contractions but did not increase basal tension.. T r i s HC1 pH 7.4 was substituted for NaCl a s J a was gradually reduced to 0 mM. Solution changes.are marked by dark bars. Tension .increased.after.each reduction i n Na. Spontaneous a c t i v i t y disappeared when.Na was reduced to zero. Muscles were returned to normal Tyrode's solution (NT) containing 149 mM Na. - 7  1  2nd Row  After returning.Na (NT) to the muscle, spontaneous a c t i v i t y took.12.min to return. A test does of CD (2 x 10~ M).indicated that the muscle had nearly.returned to normal (compare to control, 1st Row). A f t e r washout (NT) and e q u i l i b r a t i o n of the.muscle, a sudden change of Na from 149 to 0 mM.caused a large increase i n tension which decayed gradually and relaxed immediately when washed with NT. 7  3rd Row  Another muscle was stimulated with CD and l a t e r the CD was washed out with Tyrode's solution containing no Na and. 12.3 mM K.. Relaxation did not require Na. Increased basal tension a f t e r the CD responses i s probably due to the increased K (see also F i g . 51, 3rd Row). A second dose of CD did not induce a very noticeable response.  4th Row • Similar increases i n tension.due to reducing e x t r a c e l l u l a r Nawere observed i n Tris-Tyrode's solution at a normal K l e v e l . Even.a 10.sec removal of Na prior to CD stimul a t i o n altered the CD response.by preventing a sustained tension. Insert  The e f f e c t of reducing Na was not due to T r i s s u b s t i tution since CD and high K responses were altered after.5 sec i n Na-free.medium iso-osmotically s u b s t i tuted with sucrose.  175 VIII.  The 'Desensitization' Phenomenon When i n s u f f i c i e n t time was allowed between contractions induced by a maxi-  mal dose of CD, the shape of the biphasic contraction changed.  The muscles  relaxed immediately a f t e r wash out of CD but were not ready to respond i d e n t i c a l l y again for about 20 to 30 min. taneous a c t i v i t y .  Subsequent  During this time, the muscles lacked spon-  responses induced during the 'quiescent' phase did  not have a p a r t i a l relaxation between the phasic and tonic components and the contractions appeared more l i k e those induced by lower concentrations of CD (10  7  M or less) (see F i g . 28).  The peak phasic response was i n d i s t i n c t and i t s  magnitude had to be estimated as the .increase i n tension after 20 sec.  The pha-  s i c tension decreased when less time was allowed for the muscle to equilibrate between contractions induced by CD (Fig. 39).  The magnitude and rate of r i s e of  the tonic component of these desensitized contractions was increased above that in the control responses to CD.  This was more apparent when the magnitude of the  phasic and tonic responses were graphed as functions of the time allowed for equil i b r a t i o n (Fig. 40, L e f t ) .  The time to reach the peak tonic response markedly  decreased when the e q u i l i b r a t i o n time was shortened (Fig. 40, Right).  Tonic ten-  sion rose so soon a f t e r the phasic response when the time allowed f o r e q u i l i b r a tion was shortened that the p a r t i a l relaxation between the phasic and tonic maxima was  eliminated. For the experiments shown i n F i g . 39 and 40, CD was washed out as soon as  the maximal tonic tension of the muscle was attained.  Consequently, the duration  of exposure of the muscle to CD was shorter when contractions were induced at shorter intervals because the tonic component rose so quickly.  The effect of a  constant 10 min exposure of the muscle to CD on a second contraction induced by CD (Fig. 41) shortly a f t e r the f i r s t response, generally caused the second response to have an increased phasic and a decreased tonic response. pattern appeared s i m i l a r to those observed i n F i g . 39 and 40.  The biphasic  The effect of a long (35 min) exposure to CD on a second response to CD was examined.  The second response of the muscle to CD after a 30 sec e q u i l i b r a t i o n  between the two responses  appeared to be a continuation of the o r i g i n a l response  as i f the drug had never been washed out (Fig. 42, L e f t ) .  One can almost v i s u -  a l i z e the desensitized state of the muscle p a r t i a l l y returning towards equilibrium during the 1 min washout period, to where i t was a f t e r approximately 34 min of contraction, and the second response resuming from that point (Fig. 42, L e f t ) . The question arose as to whether :the rapid r i s e i n tension of the muscle by the second dose of CD to the tonic l e v e l then p r e v a i l i n g  actually represented a  phasic component or was the desensitized muscle only able to respond t o n i c a l l y . This was answered by repeating a similar experiment (as i n F i g . 42, Left) except that the muscle was washed for 60 sec with Ca-free Tyrode's solution following an extended (30 min followed by 5 min) exposure to CD (Fig. 42, Right).  An i n i -  t i a l , rapid increase of tension was s t i l l observed as would be expected i f the Increase of tension represented a true phasic component.  The reduced magnitude  of the phasic and tonic responses ( F i g . 42, Left) may have reflected the p r e v a i l ing  i o n i c conditions of the muscle just p r i o r to the washout of CD a f t e r the f i r s t  35 min exposure. A change of the biphasic shape of the response to 60 mM KCl was not observed when the e q u i l i b r a t i o n time was shortened.  These contractions have d i s t i n c t  phasic and tonic responses even i f only 2 min are allowed between contractions and the magnitudes of the phasic and tonic components are independent of the e q u i l i b r a t i o n time ( F i g . 43).  The second of two carbachol contractions induced  at 5 min intervals also had a d i f f e r e n t shape, which confirmed the previous observations with CD and indicated that the biphasic shape was not a unique charact e r i s t i c of CD induced contractions (Fig. 44).  Test pairs (a f i r s t exposure for  10 i n followed, a f t e r a set time, by a second exposure) of methacholine responses behaved s i m i l a r l y (Fig. 45).  177 An attempt was made to see i f the slowly reversible event responsible the shape change occurred during the phasic or the tonic component. of spontaneous a c t i v i t y was used as the c r i t e r i o n for estimating the equilibrated muscle state.  for  The return  the return of  CD was washed out after the maximum tonic res-  ponse was attained, at the point of least tension between phasic and tonic maxima, and just after the peak of the phasic component (Fig. 46, 1st Row). Spontaneous a c t i v i t y returned more quickly i f CD was washed out before the tonic component.  The graph at the bottom of Fig. 46 i l l u s t r a t e s that the time  required for e q u i l i b r a t i o n , measured by the return of spontaneous a c t i v i t y , i s a function of the exposure time to an optimal dose of CD.  The shape of the second  response of a test pair of contractions, 4 min apart, was also used as a c r i t e r i o n for  determining i f equilibrium had been restored.  When the f i r s t response of the  test pair was allowed to reach i t s maximum tonic response, the second response was  abnormal (Fig. 46, 2nd Row).  I f the f i r s t response was washed out before  the tonic component rose, the second response was nearly normal (Fig. 46, 2nd Row),  except that the tonic component peaked e a r l i e r than the control response.  When the contraction was terminated just after the phasic peak, the second response matched i t s control (Fig. 46, 3rd Row).  The effect of a CD exposure on a sub-  sequent high KCl contraction was also determined.  After the muscle attained  maximum tonic tension, the CD was washed out and 60 mM KCl was added a f t e r 4 min. The response to 60 mM KCl was very unusual (Fig. 46, 3rd Row) and was reminiscent of the high KCl responses of muscles a f t e r cold storage (Fig. 11). The phasic component was reduced and f e l l completely to baseline for approximately 1 min before the tonic component rose.  Therefore.the e q u i l i b r a t i o n phenomenon not only  affected subsequent CD responses but also affected subsequent high KCl responses, which ruled out a s p e c i f i c receptor desensitization and suggested a nonspecific muscle desensitization.  As expected from F i g . 43, subsequent responses to high  K at 4 min intervals were completely normal (Fig. 46, 3rd Row).  178 The e f f e c t of a response to CD on a subsequent response induced by 60 mM KCl at 2, 4, 6, 8 and 12 min intervals was investigated further (Fig. 47). The graphed r e s u l t s of the experiment outlined i n F i g . 47 demonstrated that the 60 mM K induced tonic component was b a s i c a l l y unaffected but the phasic component was reduced to 60% of i t s control (Fig. 48). The most dramatic e f f e c t of the response to CD shortly before the response to 60 mM KCl was the complete loss of tension of the desensitized muscles between the phasic and tonic components of the response to 60 mM KCl.  Although the muscles should be depolarized, they were com-  p l e t e l y relaxed between the phasic and tonic components for at least one min. This experiment ( F i g . 48, 2nd Row) c l e a r l y demonstrates that the phasic and tonic components of a high KCl response are completely separate events which probably mobilize two d i f f e r e n t Ca pools for contraction. Since high K responses e q u i l i b r a t e quickly and CD responses do not, i t was of interest to see what would happen to the contractions and the rate of e q u i l i b r a t i o n i f 60 mM K and 2 x 10  7  M CD were added simultaneously (Fig. 49).  The contraction shape of the combined stimulus (Fig. 49, 2nd Row) was i n t e r mediate to that of either alone ( F i g . 49, 1st Row).  Surprisingly, although the  responses to either agent alone relaxed immediately upon washout, with 60 mM KCl and CD together, the muscles relaxed very slowly and required about 12 min f o r their tensions to f a l l to baseline.  In f a c t , tension climbed instead of de-.  c l i n i n g just a f t e r washout of the high KCl and CD. Fig. 46, 3rd Row.  This can also be seen i n  Although relaxation was slow, the muscles gave separate  phasic and tonic responses when CD was added again after 10 min i n normal Tyrode's solution.  A subsequent response to CD a f t e r a 10 min e q u i l i b r a t i o n had  a separate phasic component but i t was very reduced and the tonic component maximized e a r l i e r .  The presence of 60 mM KCl during the response to the com-  bined s t i m u l i seemed to afford a faster return of the muscle to homeostasis with respect to the shape of a subsequent response but delayed return of homeostasis with respect to relaxation a f t e r the 60 mM KCl and CD were washed out.  The presence of higher than normal KCl concentrations i n the Tyrode's solution used to wash out the' response to> CD;,alsoenabled' theimusjEle to^quickly regain spontaneous a c t i v i t y (Fig. 50).  A contraction was induced with 2 x 10  7  M CD  for exactly 10 min and was washed out with Tyrode's solutions containing 1/2 to 5 times the normal amount of KCl (1.35 to 13.5 mM) . With 1/2 the normal K concentration, spontaneity did not return for more than 1 hr.  At twice the normal  KCl concentration, spontaneity of the muscle returned i n half the time (about 12 min) taken i n normal Tyrode's solution (25 min).  With 5 times the normal K  concentration, spontaneous a c t i v i t y returned i n about 5 min (see also F i g . 52, 3rd and 4th Rows).  When the spontaneous a c t i v i t y returned, i t appeared increased  i n magnitude and rate.  At higher external K concentrations, the r e s t i n g membrane  p o t e n t i a l would be decreased and would be nearer to i t s ' c r i t i c a l potential' above which sustained contracture would develop (Csapo 1956). The s p e c i f i c i t y of the K hastening e f f e c t on e q u i l i b r a t i o n , measured by the return of the usual biphasic appearance of the response to 2 x 10 ^ M CD, was ascertained by r a i s i n g NaCl by an equivalent amount (8 mM) (Fig. 51). The muscles were washed out for 10 min (8 min with the increased Na or K and 2 additional min i n normal Tyrode's solution) before the second dose of CD was administered to the tissue.  An additional 8 mM NaCl (Fig. 51, 3rd Row) and even  an a d d i t i o n a l 70 mM NaCl i . e . one and a h a l f times the normal Tyrode's solution concentration of NaCl (Fig. 51, 4th Row), did not hasten the return of the usual pattern of the response compared to the effectiveness i n this regard of an extra 8 mM KCl (Fig. 51, 2nd Row). Therefore the effect of K i s not due to increasing i o n i c strength but i s a s p e c i f i c effect of K.  Lowering Na to 1/2 of i t s normal  concentration hindered the e q u i l i b r a t i o n process although the rate of e q u i l i b r a tion was normal at 3/4 i t s normal concentration (Fig. 52, 1st Row). When the response to CD was washed out with Tyrode's solution containing 5 x.10  7  M oua-  bain, a concentration which i s i n s u f f i c i e n t to induce contraction, spontaneity had not returned after 40 min (Fig. 52, 2nd Row).  When 2 x 10 ^ M CD was added,  180 both the phasic and tonic contractions were very oddly reduced as i f the muscle had not only been unable to e q u i l i b r a t e but had been taken further from e q u i l i brium.  New muscles were tested to see i f the presence of 5 x 10  M ouabain for  7  30 min a f t e r complete e q u i l i b r a t i o n would prevent the d i s t i n c t i o n between the phasic and tonic components of a response to CD (Fig. 52, 2nd Row - to the right of  the dark l i n e ) .  The ouabain reduced the magnitude of the response somewhat  but had l i t t l e e f f e c t on i t s biphasic appearance: and other than a reduced tonic component (expected because of the experiment i n F i g . 24), the contraction was f a i r l y similar to the control response.  Therefore the odd response to CD after  e q u i l i b r a t i n g a previous response to CD i n the presence of 5 x 10  7  M ouabain,  was due mainly to the effect of ouabain,to prevent e q u i l i b r a t i o n of the post-CD contracted condition. not  certain.  Whether t h i s was due to i n h i b i t i o n of the Na,K-ATPase i s  The more marked effect of ouabain on the e q u i l i b r a t i o n process  implies that CD had given ouabain a lead on a l t e r i n g the i n t r a c e l l u l a r K, Ca, Mg and Na l e v e l s .  The relaxation of the tonic response to CD by ouabain was  more rapid than relaxation of the high K-induced tonic component by ouabain, perhaps because the loss of K at that point predisposes the muscle to relaxation by ouabain (Fig. 24). Raising the e x t r a c e l l u l a r Ca concentration to 5 times i t s normal l e v e l (1.8 mM)  and Mg to 3 times i t s normal concentration did not a l t e r the e q u i l i b r a -  tion time noticeably (Fig. 53). lar  The lack of e f f e c t of r a i s i n g the e x t r a c e l l u -  Ca concentration would indicate that the rate of relaxation by a Ca pump  and the rate of the return of spontaneous a c t i v i t y by other ion transport enzymes are  independent of the free Ca concentration gradient across the sarcolemma. Determination of the energy requirements for e q u i l i b r a t i o n was  difficult.  Lowering the temperature to halt the a c t i v i t y of active transport enzymes prevented spontaneity and increased tissue tension, thereby eliminating the 2 c r i t e r i a used to monitor e q u i l i b r a t i o n .  N  Instead, the energy sources of the muscle  were limited by omitting dextrose arid 0^ i n a T r i s buffered Tyrode's solution  181 (Fig.  54).  Spontaneity remained a f t e r 30 min i n this N  no dextrose was monitored. dextrose,  added (Fig. 54, Row  gassed medium to which  1) therefore this c r i t e r i o n could s t i l l be  After r e - e q u i l i b r a t i o n themuscles with 0  2  i n a medium containing  the muscles were contracted with CD for 10 min.  ponse to CD,  a washing out solution without dextrose or 0  muscle to regain 2nd Row).  2  After a 10 min ress t i l l allowed the  2  spontaneous a c t i v i t y i n the normal length of time (Fig. 54,  The next response had enough energy for the phasic spike but  unable to sustain a tonic tension.  was  After r e - e q u i l i b r a t i n g , the muscles were  tested for their a b i l i t y to contract following a much shorter (5 min)  period  of 0  A normal  2  and dextrose deprivation.  The muscles fatigued rapidly again.  response could be produced a f t e r supplying energy raw materials again.  New  muscles were tested to see i f they also fatigued rapidly a f t e r a 1 min dextrose and oxygen depletion  (Fig. 54, 3rd Row).  tonic response declined rapidly. for If  e q u i l i b r a t i o n was  I t was  Compared to the control response, the concluded that the energy requirement  far less than the energy expenditure during contraction.  energy i s required for e q u i l i b r a t i o n , the store of ATP,  and production  creatine phosphate  of ATP by gluconeogenesis coupled to g l y c o l y s i s i n the muscle was  s u f f i c i e n t to permit i t .  Desensitization of the muscle was  probably not due to  a depletion of high energy stores because i n that case, the high K tonic component would also be expected to decline. creased  tension caused an increase i n ADP  stimulated 0 creased  Tsuda et a l . (1975) observed that i n -  2  l e v e l s which i n turn, progressively  consumption i n taenia c o l i .  Contraction by CD and high KCl i n -  the energy demand of the muscle and increased the a c t i v i t i e s of enzymes  involved i n the Krebs cycle and oxidative phosphorylation et  a l . 1973), so that anabolism was  Therefore,  i n taenia c o l i (Bostrom  stimulated to keep up with  desensitization or tachyphylaxis  catabolsim.  i s u n l i k e l y to be due to metabolic  insufficiency. The measured i n t r a c e l l u l a r Ca, Mg,  Na and K levels during the contraction  induced by CD and the relaxation and e q u i l i b r a t i o n phases following washout of  182 CD are also relevant to the present discussion (Fig. 37), of the results follows.  A brief repetition  The responses to CD were accompanied by a small gain  of Ca, a loss of Mg, no apparent change i n Na and a loss of K.  Relaxation i n  30 sec was accompanied by a rapid loss of Ca to below normal l e v e l s , a rapid gain of Mg,  no change i n Na and no change i n K.  of the muscle to CD over 20 - 30 min was an jinsteady return to normal of Mg, of K to normal l e v e l s .  E q u i l i b r a t i o n of the responses  accompanied by a gradual gain of Ca,  no s t r i k i n g change i n Na and a gradual gain  The above results  w i l l be compared to the ion l e v e l  changes induced by 60 mM KCl which produces a contraction that does not require. v  a long time to e q u i l i b r a t e and does not desensitize the muscle to subsequent doses of CD or 60 mM KCl.  High KCl contractions were accompanied by a net gain  of Ca, a loss of Mg, no r e a l change i n Na and a gain of K (Fig. 36). of i n t r a c e l l u l a r K was  The gain  the only q u a l i t i a t i v e difference between the high K res-  ponse and the response to CD.  Relaxation was  accompanied by a rapid loss of  Ca to below normal l e v e l s , a gain of Mg and a loss of Na and the K content remained higher than usual.  E q u i l i b r a t i o n was  accompanied by a return to normal  of Ca l e v e l s , a fluctuating but generally returning to normal Mg content, maintained lower Na l e v e l s and a maintained  higher K concentration.  and loss of Mg were quickly reversed upon relaxation. l i m i t i n g factors for e q u i l i b r a t i o n .  They are probably not rate  On the other hand, the loss of K during a  response to CD and the slow re-accumulation bration.  The gain of Ca  of K may  l i m i t the rate of e q u i l i -  Muscles depolarized by 60 mM K did not lose K but rather gained i t  and these muscles equilibrated very quickly.  This would explain why  higher  ex-  ternal K concentrations increased the rate of e q u i l i b r a t i o n after contractions induced by CD (Fig. 49) and also would explain why rapidly (Fig. 42).  high KCl responses e q u i l i b r a t e  A r t e r i a l muscles made K poor by cold storage, recover their  a b i l i t y to respond to stimuli more quickly i n solutions with higher K tions (Barr et a l . 1962).  Barr et a l . (1962) concluded  concentra-  that the i n t r a c e l l u l a r  K concentration determined the c o n t r a c t i l t i t y and the rate of relaxation and  183 that the r a t i o of the i n t r a c e l l u l a r and e x t r a c e l l u l a r K concentrations  deter-  mine the e x c i t a b i l i t y and extent of the tonic shortening. The s i g n i f i c a n t loss of K i s a well established phenomenon of contractions induced by muscarinic  agents i n smooth muscles (Born and Bulbring 1956; Durbin  and Jenkinson 1961; Hurwitz et a l . 1960; Weiss et a l . 1961; Burgen arid Spero 1968;  and Hodgson and Daniel 1972).  At f i r s t , i t was thought that the loss of  K was required i n order f o r the muscle to contract, while others thought that the K loss during contraction might be an a r t i f a c t caused by mechanical deformation during tension development (reviewed by Setekleiv 1970).  The e f f l u x of K  from rat uterus i s increased by acetylcholine but not by oxytocin while both agents cause contraction and the acetylcholine-induced-K-efflux an a r t i f a c t of contraction (Hodgson and Daniel 1972).  i s therefore not  A f t e r 1 hr i n Ca-free  medium, pilocarpine w i l l not induce a contraction but a moderate additional increase i n potassium e f f l u x i s s t i l l apparent (Hurwitz et a l . 1960). sociation of contraction from K loss i s apparent i n the present the same force was developed by 2 x 10  7  The d i s -  study because  M CD and 60 mM KCl induced contractions  but only the muscles contracted by CD l o s t K.  The loss of K therefore does not  seem to be necessary f o r contraction but desensitization of muscles to repeated doses of muscarinic  agents may be related to an i n t r a c e l l u l a r deficiency of K.  Burgen and Spero (1968) observed that the K e f f l u x induced by carbachol was minimal at submaximal doses.  With higher doses of carbachol, causing near  maximal or maximal contractions, the induced K e f f l u x increased  dramatically.  At lower doses of CD (Fig. 28) spontaneous a c t i v i t y returned more quickly, i n d i cating that desensitization of the muscle was less at lower doses and may be related to.the smaller loss of K at these doses. of the biphasic contraction at 10  7  M to 2 x 10  7  The sudden change i n the shape M CD (Fig. 28) may be related  to the increased K e f f l u x beginning at these higher doses.  Paton and Rothschild  (1965) f e l t that desensitization was present during the action of a maximal dose of acetylcholine, not j u s t to a subsequent response to acetylcholine, because the  184 degree of desensitization was greater when the time i n t e r v a l between responses was shorter. Weiss et a l . 1961 observed that acetylcholine caused a larger K e f f l u x than pilocarpine and also the contraction induced by acetylcholine was l e s s well maintained than contraction induced by pilocarpine.  In the present study, the  tonic tension of maximal responses to CD was less well maintained than at lower doses and c e r t a i n l y , the responses to 60 mM KCl had more sustained tonic components probably because under the two l a t t e r conditions, the tissue would not lose K.  The slower rate of r i s e of the tonic component and therefore the p a r t i a l r e -  laxation between the phasic and tonic components probably was due to progressive desensitization during the response to CD.  Subsequent responses to doses of  —7 2 x 10  —8 M CD appeared more l i k e responses to doses of 5 x 10  —7 or 10  i f the muscle only recognized 1/4 to 1/2 the number of CD molecules.  M CD as But the  response to these lower doses had a faster r i s i n g tonic component which was greater i n magnitude than the usual response to 2 x 10  7  M CD (Fig. 40, L e f t ) .  I t was  d i f f i c u l t to reconcile a greater response occurring more rapidly with a desensitization.  Perhaps the slow, suppressed tonic component of a response to 2 x 10 ^  M CD was due to an opposition of an inward Ca i n f l u x by a rapid opposing K e f f l u x . When the i n t r a c e l l u l a r K concentration has been reduced by previous doses to CD, subsequent doses of CD may induce Ca i n f l u x during the tonic component unopposed by a s i g n i f i c a n t outwardly directed K e f f l u x . the  The unopposed Ca i n f l u x may  tonic component to r i s e more quickly and to a greater magnitude.  cause  Indeed, Burgen  and Spero (1968) observed that the K e f f l u x was not stimulated by subsequent maximal doses of carbachol up to 1 hr. Tachyphylaxis of the guinea pig taenia c o l i and the ileum longitudinal smooth muscle to stimulants was also studied by Bulbring and Burnstock (1960) and Paton and Rothschild (1965). mum  The onset of the second response was delayed but the maxi-  force was reached e a r l i e r .  The rate at which maximum force was attained de-  pended on the dose of the muscarinic agent used, the duration of the f i r s t a p p l i cation and the i n t e r v a l allowed for recovery.  The delayed second response of the  185 muscle was thought to be due to i t s depolarization being opposed by the faster r e p o l a r i z a t i o n of the f i r s t response.  Bolton  (1973b) observed that  termination  of acetylcholine exposure to guinea pig ileum longitudinal smooth muscle was followed not only by repolarization but also by an 'after-hyperpolarization'. The  Vafter-hyperpolarization' was prevented by ouabain, K-free solution, or Na  d e f i c i e n t solutions and therefore the acetylcholine induced 'after-hyperpolarization' was explained  on the basis of an electrogenic pump (electrogenicity based  on a 3 Na:2 K pumping r a t i o ) .  In the present study, responses of the muscle to  CD which were washed out with K or Na d e f i c i e n t Tyrode's solution or i n Tyrode's solution containing 5 x 10  7  M ouabain were prevented from r e - e q u i l i b r a t i n g .  These results agree with the observations Na,  Ca or Mg concentrations  by Bolton  (1973b).  Raising e x t r a c e l l u l a r  had no e f f e c t on the e q u i l i b r a t i o n phase but r a i s i n g  the e x t r a c e l l u l a r K concentration  accelerated the recovery.  c e l l u l a r K concentration hyperpolarized  Raising the extra-  a K d e f i c i e n t muscle and was attributed  to a stimulation of the electrogenic pump  (Bolton  1973a).  However, i t was hard  to reconcile the 'after-hyperpolarization' with a stimulation of an electrogenic Na,K  pump i n the present study because the reduced i n t r a c e l l u l a r K l e v e l s were  not rapidly regained.  The K e f f l u x induced by higher doses of carbachol was  refractory to subsequent doses of carbachol 1968)  for up to 1 hr (Burgen and Spero  which also indicates that the regaining of i n t r a c e l l u l a r K takes a long  time and may be inconsistent with a high rate of Na,K-ATPase pumping of K into the  cell. Kehoe and Ascher (1970) evaluated  the a c t i v a t i o n of an electrogenic sodium  pump to account for p o t e n t i a l changes and demonstrated that a hyperpolarizing synaptic p o t e n t i a l was the r e s u l t of an increased potassium permeability. ing  Quot-  these authors, "there i s no reason to doubt that the Na-K pump might be  activated after a synaptic p o t e n t i a l , insofar as a change i n cationic permeabil i t i e s w i l l lead to a change i n i n t e r n a l cation concentrations.  I t appears doubt-  f u l , however, that the minute changes involved i n most synaptic potentials are  186 s u f f i c i e n t to trigger a detectable  'pump' p o t e n t i a l change,"  Romero and Whittam  (1971) observed that red blood c e l l ghosts with, higher i n t r a c e l l u l a r Ca concent r a t i o n l o s t K and suggested that the permeability of K and Na could be regulated by i n t e r n a l Ca which i n turn could be regulated by a calcium pump.  Similar  experiments i n red blood c e l l s , with modifications to test other hypotheses, yielded r e s u l t s that indicated that the i n t r a c e l l u l a r Ca control of K permeability might be mediated through the Na-K pump because of i t s i n h i b i t i o n by ouabain (Blum and Hoffman 1972), but Lew (1974) had doubts about this because ouabain was without e f f e c t on the Ca dependent K loss at ATP concentrations 10 ^ M.  lower than  Reed (1976) found that A23187, a divalent cation ionophore, induced up-  take of Ca and Sr and subsequently produced potassium loss from erythrocytes. Mg uptake was i n e f f e c t i v e i n this regard.  Knauf et a l . (1975) noted that increas-  ing K above 4 mM on the outside of red blood c e l l s i n h i b i t e d the e f f l u x . activated K channel (sensitive to Ca antagonists)  A Ca-  generated an 'after-hyperpolari-  zation' i n cat, frog and s n a i l neurones and was d i s t i n c t from the K channel (tetraethylammonium sensitive) which controlled r e p o l a r i z a t i o n of the action pot e n t i a l (Krnjevic et a l . 1975; Barrett and Barrett 1976;  Heyer and Lux 1976).  Bursting pacemaker c e l l s of the Aplysia R15 neuron, which have an inward Ca current, gained i n t r a c e l l u l a r Ca during each burst and the increase of i n t r a c e l l u l a r Ca was s u f f i c i e n t to cause a hyperpolarization (Thomas and Gorman 1977). Internal Ca levels are high i n contracting smooth muscles and they have an increased K permeability  (Born and Bulbring 1956;  through Ca-activated K channels. out of an acetylcholine-induced  Burgen and Spero 1968)  perhaps  The 'after-hyperpolarization' following washcontraction might be better explained by an a c t i -  vation of a K channel rather than by a stimulation of an electrogenic Na pump. Bulbring and Kuriyama (1973) have put f o r t h a s i m i l a r hypothesis to explain the hyperpolarizing e f f e c t of a adrenergic agents on the guinea pig taenia c o l i . They f e e l that Ca bound to the inner surface of the sarcolemma regulates the membrane permeability to K which i n turn determines the membrane p o l a r i z a t i o n  187 and consequently  the degree of spontaneous a c t i v i t y .  The a adrenergic agents  may increase the binding of Ca at negative s i t e s on the inner surface of the v plasma membrane and according to Tomita and Watanabe (1973), this would increase the membrane permeability to K and thereby hyperpolarize the c e l l .  The amount  of Ca bound to the inner surface of the membrane would be regulated to some extent by the a c t i v i t y of an active Ca extrusion mechanism i n the sarcolemma.  A  s i m i l a r e f f e c t of membrane bound Ca has been postulated to explain why excess Ca bound to the plasma membrane, hyperpolarizes arid s t a b i l i z e s the membrane but also causes a decreased membrane resistance (Bulbring and Tomita 1969).  The  decreased membrane resistance of a s t a b i l i z e d , hyperpolarized membrane i s comp l i c a t e d i n view of current theories of the effect of Ca on membranes.  Excess  external Ca was found to hyperpolarize the membrane and increase the K conductance (Bulbring and Tomita 1969). decreases the K conductance.  Low external Ca depolarizes the membrane and  When the external Ca concentration i s decreased  to zero, the c e l l depolarizes and the membrane resistance becomes very low, indicatirig an increased Na conductance (Bulbring and Tomita 1969). A possible contradiction of the existence of a Ca-activated-K-channel  i s the  observation that pilocarpine s t i l l causes a moderately increased K e f f l u x i n the guinea pig ileum l o n g i t u d i n a l smooth muscle c e l l s that have been bathed i n Ca-free medium for 1 hr even though contraction was prevented 1960;  Weiss et a l . 1961).  (Hurwitz et a l .  A closer examination of the data indicates that the  K e f f l u x induced by pilocarpine a f t e r the muscle was i n Ca-free medium for 1 hr, was reduced to 1/10 the normal amount. Another apparent contradiction to the theory i s that oxytocin caused contraction of the rat uterus and therefore higher cytoplasmic Ca concentrations but did not cause an increased K e f f l u x while acetylcholine caused contraction and increased K e f f l u x (Hodgson and Daniel 1972). The model can be accommodated to account f o r these anomalies.  The a c t i v i t y of  the channel might depend on the amount of Ca bound at either end of the channel. Perhaps the K channel can only be activated when Ca, from some s i t e on the ex-  188 ternal surface of the membrane i s removed and relocated on the inner surface. Agents such as acetylcholine, by r e l o c a t i n g Ca bound on the outer aspect-of the c e l l to the inside of the membrane, might e f f e c t i v e l y activate the K channel f a r better than agents that only add Ca to the inside of the membrane (agents that release i n t e r n a l Ca) or conditions which only remove Ca from the outside of the membrane, such as i n Ca-free medium.  However, conditions that remove extra-  c e l l u l a r Ca or increase i n t r a c e l l u l a r Ca might be expected to act s y n e r g i s t i c a l l y with acetylcholine to activate the K channel to a greater extent.  Indeed, Chang  and T r i g g l e (1973b) have observed that the desensitization process to CD i s enhanced at lower external Ca concentrations.  The K e f f l u x induced by p i l o -  carpine i n Ca-free medium (Hurwitz et a l . 1960 and Weiss et a l . 1961) could also be accounted for by a greater s e n s i t i v i t y to a smaller r e a l l o c a t i o n of Ca from external receptor s i t e s to the i n t e r n a l surface.  Smaller doses of acetylcholine  cause smaller phasic than tonic responses (Fig. 27) and probably relocate less Ca from outside to inside the membrane.  These smaller doses would therefore  not be as e f f e c t i v e for a c t i v a t i n g the K channel at normal external Ca concentrations.  They may be more e f f e c t i v e for a c t i v a t i n g a K channel i f the external  Ca concentration was reduced.  In addition, the Ca mobilized by low doses of CD  may be attracted to the c o n t r a c t i l e filaments more than to the Ca s i t e s on the inner surface of the plasma membrane, leaving no extra Ca free to activate a K channel.  At higher doses, enough Ca may be allowed to enter the c e l l s to more  than saturate the c o n t r a c t i l e filaments and the extra Ca may activate K channels. This may account f o r the displacement  of the log dose-K-efflux-response  curve to  the right of the l o g dose-contractile-response curve induced by carbachol (Burgen and Spero 1968).  Chang and Triggle (1973a) observed that p a r t i a l agonists con-  tract the guinea pig ileum l o n g i t u d i n a l smooth muscle at a slower rate than f u l l agonists and desensitization of the muscle did not occur following exposure of the muscle to p a r t i a l agonists.  These results may indicate that only Ca which v  can be rapidly mobilized from membrane bound s i t e s to the inside of the c e l l f o r  189 the phasic component  activates a K channel,  Ca released from any other s i t e may  not be able to increase K permeability (e.g. Ca released by oxytocin i n uterus; Hodgson and Daniel 1972),  However, the experiments i n Fig. 46 seem to indicate  that the tonic component i s mainly responsible for the desensitization.  When the  results of this study and of the l i t e r a t u r e are analyzed, i t would appear that a K channel activated during the phasic component i s maintained component.  during the tonic  The extent of the loss of K during the tonic component, the degree  of desensitization and the time required for re-accumulation  of normal i n t r a c e l -  l u l a r K levels after washout of CD may be related to the time that the K channel i s held open i n the presence of CD (or any other f u l l muscarinic agonist) (Fig. 46, Graph). Desensitized muscles seem to mobilize Ca from the same locations as they do usually ( i . e . phasic rose i n Ca-free medium but the tonic did not; F i g . 42, Right). The second response to a dose of 2 x 10  7  M CD may mobilize as much Ca as i t  usually does but this Ca may be used i n e f f i c i e n t l y for tension development i n the extremely desensitized state of the muscle (hyperpolarized or K poor) (Fig. 46, Left).  On the other hand, i t i s possible that the desensitized muscles may not  be able to mobilize as much Ca.  The unusual shape of the response to 60 mM KCl  in densensitized muscles i s not as easy to explain.  I t i s similar i n shape to  the contractions induced by 60 mM KCl i n K poor tissues a f t e r cold storage. In both these cases, the relaxation between the phasic and tonic responses i s accentuated muscles.  instead of being absent as i n the responses to CD i n desensitized  One would not expect a hyperpolarizing-outward-K-current  the face of a 60 mM e x t r a c e l l u l a r K concentration. reversed temporarily u n t i l the c e l l regains K.  to occur i n  The K gradient may even be  Any further explanation  about  the responses to 60 mM KCl i n a desensitized muscle at this point would be unwarranted speculation. Although Ca i s immediately pumped out upon relaxation, K does not return to i t s normal l e v e l for some time after a CD response (Fig. 37). The increased  19 K permeability may counteract  the pump f o r some time a f t e r relaxation despite  the removal of the higher i n t r a c e l l u l a r Ca l e v e l s .  Though net i n t r a c e l l u l a r  Ca l e v e l s are lower a f t e r relaxation, more Ca could s t i l l be bound to the inside of the plasma membrane and sustain the hyperpolarizing K current.  On the other  hand, the Na,K-ATPase pump may not be adequate to restore K l e v e l s immediately. The data presented i n the Results and Discussion i n section I I tend to indicate that a K stimulated pump i s a minor component of the t o t a l cation  stimulated  ATPase a c t i v i t i e s that might be responsible f o r ion transport i n these longitud i n a l i l e a l muscles.  The Na,K-ATPase a c t i v i t y may be rate l i m i t i n g f o r the r e -  versal of the desensitized muscle state.  191  Fig. 39,. Changes of the biphasic c o n t r a c t i l e pattern when contractions to CD were induced at shorter e q u i l i b r a t i o n time intervals between contractions. Responses to CD (2 x 10~7 M) were terminated by washout (W) of the CD after the peak of the tonic component with normal Tyrode's solution. The tonic response peaked e a r l i e r as the e q u i l i b r a t i o n times was shortened—therefore exposures to CD were shortened accordingly. The tracing i s an example of one of the four experiments graphed i n F i g . 40.  \  193  Fig. 40.  Graphical representation of the effect of e q u i l i b r a t i o n time on contractions by CD. The results were obtained from experiments exemplif i e d i n F i g . 39. In the l e f t hand graph the dotted l i n e represents the tonic component and the s o l i d l i n e represents the phasic component. The right hand diagram i s a measure of the time required f o r the tonic component to peak after the addition of CD to the muscle. E q u i l i b r a t i o n time refers to the time allowed f o r e q u i l i b r a t i o n i n normal Tyrode's solution between CD responses. Points represent the mean + S. E. (n = 4)  194  5»  0)  • acorn  195  Fig.  41.  The e f f e c t of the e q u i l i b r a t i o n time allowed after a 10 min exposure to CD on a subsequent response to CD. The f i r s t exposure to CD (2 x 10 M) was kept constant at 10 min. The effect of this response was tested on a second response to CD, 2 to 8 min l a t e r after washing out (W) the f i r s t response with normal Tyrode's solution. The f i r s t and second responses constitute a test pair. Muscles were e q u i l i brated for at least 30 min between each test pair. The phasic and tonic components of the second response were measured as a percent of the paired control response. The dotted l i n e represents the tonic and the s o l i d l i n e represents the phasic component. Points represent the mean + S. E. (n = 7) - 7  196  197  Fig.  42.  Left The effect of a very short e q u i l i b r a t i o n time (30 sec) on a CD response.after a long exposure to CD (35 min). The CD concentration was 2 x 10" M. 7  Right E f f e c t of a short e q u i l i b r a t i o n time i n Ca-free Tyrode's solution on a second response to CD after a long f i r s t exposure to CD (30 min). A rapid response to CD after a 60 sec e q u i l i b r a t i o n time i n Ca-free Tyrode's solution was obtained which was followed by relaxation. Therefore the rapid f i r s t response would seem to be a true phasic response and the second response was c h a r a c t e r i s t i c of a tonic response.  198  199  F i g . 43.  The lack of e f f e c t of the e q u i l i b r a t i o n time allowed between responses to 60 mM KCl on the biphasic shape- of a series of responses. Muscles were washed out (W) with normal Tyrode's solution between stimulations by 60 mM KCl added as indicated by the arrows. The magnitudes of the phasic and tonic responses to 60 mM KCl were independent of the time allowed to e q u i l i b r a t e between contractions (graph at bottom). Points represent the mean + S. E. Cn = 4)  201  Fig. 44.  The e f f e c t of the e q u i l i b r a t i o n time allowed after a 10 min response to carbachol (Cch) on a second contraction induced by carbachol. An appropriate concentration of carbachol to give d i s t i n c t phasic and tonic components was found to be 1 0 ^ M (see l e f t s i d e ) . Washing (W) for 5 min was i n s u f f i c i e n t to allow a second response to 10 ^ M carbachol to respond the same as the f i r s t response (control) of the test pair (right side). -  202  203  Fig. 45.  The effect of a 5 or 10 min e q u i l i b r a t i o n time after a response of the muscle to methacholine on the biphasic appearance of a second response to methacholine (Men.) . The muscles were stimulated by 10' M methacholine and washed out (W) i n normal Tyrode's solution.  204  205  Fig. 46.  The e f f e c t of the duration of exposure to CD (2 x 10 ' M) on the time required for e q u i l i b r a t i o n , monitored by the return of spontaneous a c t i v i t y and the subsequent return of the normal biphasic c o n t r a c t i l e pattern. -  1st Row  The time required for the return of spontaneous a c t i v i t y was measured when CD was washed (W) out (1) after the tonic maximum, (2) at the point of least tension between the phasic and tonic components, and (3) after the phasic peak (See Graph at bottom).  2nd and 3rd Rows The biphasic appearance of the second response of a test pair was monitored as a means of determining the equil i b r a t i o n state. Four min was allowed between contractions to CD of d i f f e r e n t durations. The f i r s t contractions to CD were allowed to continue (1) u n t i l the tonic component was maximal, (2) u n t i l the point of least tension between the phasic and tonic components was reached and (3) u n t i l the phasic component peaked. Complete e q u i l i b r a t i o n was allowed between the test pairs. 1  3rd Row  After a phasic and tonic response to CD, 60 mM KCl was added after 4 min and the response was abnormal. High KCl was added twice more at 4 min intervals between responses and the responses were normal.  Graph  The results from 4 muscle s t r i p s run simultaneously i n the experiment represented i n the 1st Row were graphed. Points represent the mean + S. E.  K3 O  as  207  Fig. 47.  The e f f e c t of a 10 min exposure to CD on a response to 60 mM KCl, 2, 4, 6, 8 and 12 min a f t e r the response to CD was washed out (W). The responses to 2 x 1 0 M CD of these longitudinal i l e a l s t r i p s had d i f f e r e n t biphasic patterns than were usually observed. The tracings are one example of 4 muscle s t r i p s run simultaneously. - 7  1st Row  Two control responses to 60 mM KCl.  2nd Row  Test pairs of 10 min responses to CD followed by washout with normal Tyrode's solution (W) for 2 or 4 min and a stimulat ion of the muscle by 60 mM KCl. The phasic and tonic responses to 60 mM KCl are completely separated.  3rd Row  Similar test pairs as described i n the 2nd Row except that 6 and 8 min were allowed for e q u i l i b r a t i o n between the responses to CD and 60 mM KCl.  4th  A test pair with 12 min between the CD and 60 mM KCl responses followed by a control response to 60 mM KCl.  Row  208  209  Fig. 48.  Graphical representation of the e f f e c t of the time allowed f o r e q u i l i bration a f t e r a response to CD on a response to 60 mM KCl. Experiments are of the type shown i n F i g . 47. Results are p l o t t e d as a percent of the f i r s t control responses to 60 mM KCl. The time allowed to equilibrate refers to the time between the washout of CD and the addition of high KCl. The least tension between phasic and tonic responses means the magnitude of the unusually prolonged low tension (nearly to baseline) between the phasic and tonic components of the response to 60 mM KCl. Points represent the mean + S. E. (n = 4)  210  211  Fig. 49.  The effect of a simultaneous addition of 60 mM KCl and 2 x 10 CD on the e q u i l i b r a t i o n phase of the muscle.  M  1st Row  Control responses to CD and 60 mM KCl determined separately.  2nd Row  Simultaneous addition of CD and 60 mM KCl gave a contraction shape intermediate between the separate responses. Washout (W) i n normal Tyrode's solution did not cause immediate relaxation, which was d i f f e r e n t than either response alone.  3rd Row  The shape of a CD response a f t e r a 10 min washout after the simultaneous addition o f 60 mM KCl and 2 x 10 M CD to the muscle. The subsequent response to CD had separate phasic and tonic components. Again the CD was washed out after i t s tonic component reached maximum. Another response to CD alone, a f t e r 10 min, was abnormal. 7  213  Fig. 50.  The effect of a l t e r i n g the concentration of KCl i n the Tyrode's solution on the rate of return of spontaneous a c t i v i t y a f t e r a response to CD. The KCl concentration was varied from 1/2 (1.35 mM) to 5 times (13.5 mM) i t s normal concentration (2.7 mM) i n the Tyrode's solution used to wash.out (W) a 10 min response to CD. The e q u i l i bration time (ordinate) was measured by the time required for the muscle to regain spontaneous a c t i v i t y . Examples of the experiment are included on the graph. Points represent the mean + S. E. Chart of n Values KCl Cone. 1.35  mM  n 3  2.7  11  5.4  8  6.7  4  8.1  8  10.7  8  13.5  4  ,214  215  Fig. 51.  The s p e c i f i c i t y of increased external KCl f o r accelerating the return of the usual biphasic c o n t r a c t i l e pattern compared to the lack of effect of higher e x t r a c e l l u l a r NaCl concentrations. The experiment shown i s one of four run simultaneously. 1st Row  A control test pair of CD responses (2 x 10 M) with a 10 min e q u i l i b r a t i o n i n normal Tyrode's solution between the responses.  2nd Row  The f i r s t response of a test pair of responses to CD was equilibrated i n Tyrode's solution containing four times the normal KCl concentration for 8 min and 2 min i n normal Tyrode's solution before the second response. The e x t r a c e l l u l a r KCl concentration was raised 8 mM (2.7 to 10.7 mM).  3rd Row  A test pair of responses to CD equilibrated i n Tyrode's solution containing 8 mM more NaCl (136 - 144 mM NaCl) for 8 min and 2 min in.normal Tyrode's solution.  4th Row  A test pair of responses to CD equilibrated i n 205 mM NaCl (lh times normal) f o r 8 min and 2 min i n normal Tyrode's solution.  7  217  Fig. 52.  The e f f e c t of various agents on the time required by the muscle f o r the return of spontaneous a c t i v i t y a f t e r a 10 min response to CD (2 x 1 0 M). Each row of contractions represents one of four experiments . - 7  1st Row  The e f f e c t of reducing the e x t r a c e l l u l a r NaCl concentration on the time required by the muscle to regain spon-: taneous a c t i v i t y . Tris-HCl was used to maintain the osmolarity. An e x t r a c e l l u l a r NaCl concentration of 3/4 the normal l e v e l was s u f f i c i e n t to allow the muscle to regain spontaneous a c t i v i t y i n the usual length of time, but at 1/2 the normal e x t r a c e l l u l a r NaCl concentration, the spontaneous a c t i v i t y of the muscle did not return for more than 1 hr a f t e r a response to CD.  2nd Row  The e f f e c t of a low dose of ouabain (5 x 10 M) (which i s i n s u f f i c i e n t to cause contraction) on the rate of return of spontaneous a c t i v i t y a f t e r a 10 min response to CD. Spontaneous a c t i v i t y d i d not return a f t e r 40 min and a subsequent response to CD was abnormal. The second half of the,2nd Row shows a control experiment i n d i c a t i n g that the presence of 5 x 1 0 M ouabain for 30 min d i d not extensively a l t e r the phasic and tonic components of a response to CD i f i t was added to a f u l l y equilibrated muscle. 7  - 7  3rd Row > The e f f e c t of a l t e r i n g the e x t r a c e l l u l a r KCl concentration on the time required f o r the return of spontaneous a c t i v i t y of the muscle. These Rows are a complete tracing of the experiment shown i n F i g . 50.  0.75  24  AM  CD  NT  >4i  0.5 xNa  x Na i  24'  CD  . no spontaneous activity aftar i.hr.  W  5X10" M  5X10" M  ?  7  28 CD  ouab  CD  Ns  NT  NT C D  / CD  ouab 'CD  NT  CD  /3XK  4xK  .5XK  7.6  7.2  5.2  CD  w  NT  CO  0.5 xK  CD  NT  30  CD  W  "CD  w  ^  219  Fig. 53.  The e f f e c t of r a i s i n g the e x t r a c e l l u l a r Ca and Mg concentrations on the time required by the muscle to regain spontaneous a c t i v i t y . The experiments shown are one of four which were run simultaneously. 1st and 2nd Rows The e f f e c t of a f i v e - f o l d increase of the extrac e l l u l a r Ca concentration on the time required for the muscle to regain spontaneous a c t i v i t y following a 10 min response to 2 x 1 0 M CD. The muscle was returned to the normal Tyrode's solution after the return of spontaneous a c t i v i t y and before the induction of the next 10 min response to CD. The muscles regained spontaneous a c t i v i t y at about the same rate i n higher e x t r a c e l l u l a r Ca concentrations as they did i n 1.8 mM CaC^. - 7  3rd Row  The effect of a three-fold increase of the e x t r a c e l l u l a r MgCl2 concentration on the rate of return of spontaneous a c t i v i t y . Raising the e x t r a c e l l u l a r MgCl concentration did not change the rate of return of spontaneous a c t i v i t y compared to controls. 2  «J2™=.'ixCa ig  fwXi CD  28.4  2xCa  fi.5 x C a  264  bxCa 29.6  29.6  m 4xCa  i A.  5xCa ZOA  X  30  C  a  26'  ,ixMg 18  19.2'  1^ ' \  '  16  N3-  O  221  Fig. 54.  The effect of depriving the muscle of dextrose and oxygen on the time required for the return of spontaneous a c t i v i t y . T r i s buffered Tyrode's solution (TT) was used so that 95% 0 - 5% C0 would not be required to maintain pH. N was bubbled through the tissue chambers to drive o f f 0 from the Tyrode's solution. 2  2  2  2  1st Row  The time required for the muscle to regain spontaneous a c t i v i t y a f t e r a control response to 2 x 10 ' M CD was approximately 30 min. After 30 min i n Tris-Tyrode's solution without dextrose and N , the spontaneous a c t i v i t y (the test parameter) of the muscle was retained. The muscle was re-equilibrated with dextrose and 0 . -  2  2  2nd Row  After a 10 min response to CD, the muscle was e q u i l i b r a ted i n the absence of dextrose and 0 . Spontaneous a c t i v i t y returned i n the normal amount of time. A second response to CD had only enough energy for a phasic response but could not maintain a tonic tension. The muscles were re-equilibrated with 0 and dextrose. Deprivation of 0 and dextrose for 5 min also caused a rapid decline of tension after the phasic component. 2  2  2  3rd Row  the muscle was not permanently damaged as can be seen by the response i n the 3rd Row of the same muscle a f t e r ree q u i l i b r a t i o n i n Tris-Tyrode's solution. New muscles were tested f o r t h e i r a b i l i t y to contract a f t e r 1 min of dextrose and 0 deprivation. Even after 1 min the tonic tension dropped o f f f a r more quickly than the control response. 2  to to  223 SUMMARY AND  CONCLUSIONS  Ion movements"during and after contraction of the guinea pig ileum l o n g i tudinal smooth muscle were studied i n an attempt to examine some of the factors c o n t r o l l i n g the excitation-contraction-relaxation cycle.  The structure,  contrac-  t i l e c h a r a c t e r i s t i c s and ion movements were investigated i n the intact tissue and a sarcolemmal enriched  f r a c t i o n of the muscle was  related to ion transport.  analyzed for ATPase a c t i v i t i e s  Net changes of i n t r a c e l l u l a r Ca, Mg,  were measured using a modified  Na and K contents  'La method' that displaced e x t r a c e l l u l a r ions from  the tissue i n an isotonic Tris-HCl solution containing 10 mM LaCl^ at 4°C. hypothesis that was  tested was  of smooth muscle c e l l s may  The  that excitation-contraction coupling i n some types  be regulated by trans-sarcolemmal fluxes of  The following r e s u l t s were consistent with the above hypothesis.  Ca. (1) Guinea  pig ileum longitudinal smooth muscle c e l l s are very small and t h e i r surface areas are greatly increased by caveolae.  The large surface to volume r a t i o suggest that  they might use e x t r a c e l l u l a r Ca for contraction.  (2) Contractions  induced by  60 mM KCl and cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide were very sensitive to omission of Ca from the Tyrode's solution. component of.the contraction was  (CD)  (3) The tonic  l o s t a f t e r 20 sec i n Ca-free Tyrode's solution at  about the same rate as the free e x t r a c e l l u l a r Ca would be expected to be l o s t . (4) The phasic component decreased over 10 min i n Ca-free Tyrode's solution and followed the rate of removal of a bound f r a c t i o n of Ca from the e x t r a c e l l u l a r space. (5) Readdition  of Ca to the Tyrode's solution restored both the phasic and  components i n l e s s than 30 sec.  Therefore i t seems that the Ca used for contraction  i s located s u p e r f i c i a l l y . (6) Ca mobilized by La.  for the phasic component can be displaced  Consequently, Ca u t i l i z e d for the phasic component may  side of the plasma membrane or perhaps may 1  be bound to the out-  be stored i n caveolae. (7) Stimulation of  the ileum longitudinal smooth muscle, by 60 mM uptake (>6.5  tonic  KCl or by 2 x 10  7  M CD,  induced Ca  x 10 "^moles/mg wet weight) i n excess of the amount postulated to be  required for maximal contraction (1.05 x 10  moles/mg wet weight).  The  224 2+ free cytoplasmic Ca., concentration was••.;caleulated;,to....be;r_increas'ed . to • ;  i  v  -4 approximately .- 2 x 10 M by 60 mM KCl or. CD. A Ca-ATPase a c t i v i t y was  found i n a sarcolemmal enriched microsomal f r a c t i o n . 2+  The Ca-ATPase a c t i v i t y was stimulated by free Ca -7 of 10  concentrations over the range  -4 to 2.4 x 10  M.  The elevated Ca levels i n the tissue during contraction  were reduced i n 30 sec, perhaps by a sarcolemmal Ca-pump associated with the CaATPase a c t i v i t y found i n the sarcolemmal enriched f r a c t i o n . v e s i c l e s were postulated to be pinched o f f caveolae. tion as active transport centers for ion pumping.  The microsomal  The caveolae may  also func-  The sarcolemmal-enriched  micro-  somal f r a c t i o n had high s p e c i f i c a c t i v i t i e s for a Na-stimulated Mg-ATPase but the stimulation by K which could be i n h i b i t e d by ouabain was only a small compo- • nent of the t o t a l ATPase a c t i v i t y . A f t e r stimulation of the muscle by a muscarinic agent, the smooth muscle c e l l s not only gained Ca but l o s t K rapidly. v i a a Ca-activate-K-channel.  The loss of K was postulated to be  When the muscarinic stimulus was washed out, the  excess Ca was pumped out but K levels were not regained quickly. immediately  The muscle  relaxed a f t e r the stimulus was washed out, but basal tension was  lower than usual and the musclet.did not spontaneously 20 min by which time the tissue had regained the K.  contract for approximately An after-hyperpolarization' 1  was previously reported to follow the washout of a muscarinic stimulation of the guinea p i g ileum longitudinal smooth muscle. 'after-hyperpolarization' e f f e c t was a Ca-activated-K-channel  In the present study, the  explained by a greater K permeability through  rather than by the stimulation of an electrogenic Na,K  pump because K was not regained quickly a f t e r the contraction was relaxed.  De-  s e n s i t i z a t i o n of the muscle appeared to be correlated with the rate of loss of i n t r a c e l l u l a r K.  The tonic component increased more slowly under conditions  which would open or leave open the Ca-activate-K-channel.  By elevating the extra-  c e l l u l a r K concentrations after the contraction, the reversal of the desensitized state of the muscle could be accelerated.  Muscles stimulated by high KCl did not  225 lose K and were not deaensitized to subsequent s t i m u l i ,  The rate of re-accumula-  tion of K by the muscle c e l l s may l i m i t the rate of e q u i l i b r a t i o n of the muscle following contraction because the d e f i c i t of i n t r a c e l l u l a r K i s large and the a c t i v i t y of the Na,K-ATPase i s low.  f  The contraction induced by ouabain occurred before any loss of i n t r a c e l l u l a r K was detectable.  Therefore i t s action seemed to be dissociated from i n h i -  b i t i o n of the Na,K-ATPase.  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CGation l e v e l s after depletion i n Ca and Mg free Tyrode nanomoles/mg dry wt. treatment  Ca  Mg  Na  K  t o t a l ions  20.6 + .92  25.9 + .58  506.7 + 48.7  211.2 + 16.8  (D{)CaMg] 10 min 37°C  9.83 + .67  19.5 + .95  499.6 + 44.4  188.2 + 17.1  7.98  22.7 + .90  481.1 + 49/1  152.4 + 14.3  8.00 ± -  21.6 +1100  519.8 + 58.7  105.9 + 6.1  6.76 + .25  19.6 + .53  611.6 + 95.7  +  67.0 9.9  6.84 + .57  19.8 +2.1  604.2 + 45.7  +  61.6 7.3  7.06 +1.29  19.1 +1.3  621.6 + 76.5  +  47.6 4.1  2 (lOnimin) .,25M sucrose^4°C  11.4 + .97  20.0 +1.4  191.4 + 24.2  149.9 + 11.8  0[CaMg] 10 min 37°C 2(10 min) •.25 M sucrose 4°C  6.91 +1.19  15.3 +1.9  +  168.3 9.6  121.0 + 25.3  0[CaMg] 10 min 37°C 50 min 4°C 2(10 min) . 25 M sucrose 4°C  5.71 +5.70  17.3 +7.95  222.7 +236.2  75.2 + 74.7  0[CaMg] 10 min 37°C 3 hr 50 min 4°C 2(10 min) . 25 M sucrose 4°C  4.44 +1.92  13.9 + .82  269.7 + 29.1  +  a  0[CaMg] 10 min 37°C 20 min 4°C  ± 0[CaMg] 10 min 37°C 50 min 4°C  2 7  7 5  0[CaMg] 10 min 37°C 1 hr 50 min 4°C 0[CaMg] 10 min 37°C 2 hr 50 min 4°C 0[CaMg] 10 min 37°C 3 hr 50 min 4°C  a. 0[CaMg] = Ca and Mg free Tyrode b. .25 M sucrose was corrected to pH 7.4 with h i s t i d i n e c. values are means + S.E. n=4  28.3 3.7  • ' 241  Table f o r F i g . 5. Top.  Concentration dependence of the stimulation of microsomal ATPase by Mg, Na and K 0 .123  Mg (mM) pmoles Pi/mg/min 3 mM Mg + Na (mM) pmoles Pi/mg/min 3 mM Mg + 100 mM Na + K pinoles Pi/mg/min  (mM)  1 .708  2 .991  3 1.39  4 1.58  0 1.39  20 1.42  40 1.61  60 1.73  80 1.92  0 1.98  3 2.26  6 2.08  9 2.13  12 2.31  100 1.98  Table f o r F i g . §: Bottom.Lack of clear concentration dependence effect of K on the Mg dependent Na-ATPase 3 mM Mg + 100 mM Na + K 0 a microsomes Microsomes + s o l .  .75  (mM)  1.5  2.25  3  6  9  12  1.61  1.56  1.37  1.41  1.42  1.35  1.49  1.48  1.61  1.60  1.68  1.58  1.65  1.77  1.56  1.67  a. umoles Pi/mg protein/min b. umoles Pi/mg microsomal protein/min  Table f o r F i g . 6. Effect of Na, K, ouabain and a soluble activating factor on the Mg-ATPase a c t i v i t y  M  A. microsomes  1.302 + .162  50 u1 microsomes + 10 y l soluble f r a c t i o n  M N K 0  2.237 + .134  2.194 + .147  2.199 + .2141  1.426 + . 016  1.424 + .034  1.274 + .009  1.928  2.710  2.605  2.763  2.303  3.026  3.158  2.987  0.042  0.056  0.063  0.061  0.917 + .060  1.59 + .066  1.704 + .065  MN -M  MNK -MN  MNK -MNKO  MN -MNKO  .681 + . 04 17.35  .119 +.016 7.37  .052 +.011 4.76  -.063 +.022 2.90  B. microsomes prepared i n the 0.676 presence of 0.1 mM ouabain + . 030 C. 50 y l microsomes  ymoles Pi/mg protein/min M M N N K  5  50 y l soluble f r a c t i o n D. microsomes + s o l .  paired t-value (10 d e g r e e s o f freedom) mean + S.E.  1.658 + .065  Table for Fig, ff. Lineweaver Burk plot of the effect of 100 mM Na and 3 mM K on the Mg-ATPase (3 mM) mM ATP zumoles P i mg/min 1/V  Mg  5.33  .375 .225 2.67  .75 .45 1.33 .187  1.5 .9  1.875 1.125  2.25 1.35  2.625 1.578  3 1.8  .67  .53  .44  .38  .333  .183  .171  .175  .200  .159  .077  .109  .153  umoles P i mg/min  .620  .875  1.225  1.50  1.46  1.37  1.14  .816  .66  .68  .73  .714  .625  .787  .155  .222  .272  .247  .255  .246  .262  1.775  2.175  1.61  umoles  .052  .09  pmoles P i mg/min  .420  .720  1/V  Mg Na K  10.64  .1875 .1125  pmoles  1/V  Mg Na  .094 .0564  2.38  1.39  ymoles  .05  .114  umoles P i mg/min  .4  .91  2.5  1.09  1/V  1.24  1.4  1.6  1.97  2.04  1.96  1.27  2.09  .806  .563  .46  .51  .49  .51  .478  .128  .220  .258  .277  .258  .279m  .251  1.025 .976  1.76 .568  2.06  2.22  .48  .45  2.06 .485  2.23 .49  2.00 .5  average of duplicates  So Co  Table f o r F i g . jQ. The effect of Ca on the Mg dependent Na,K-ATPase t o t a l CaCl. (M) lO"  0  assay medium  lO"  4  3  25 mM Hist., .1 mM EGTA  .066 +.024  .088 +.029  .153 +.046  .256 +.093  25 mM Hist., .1 mM EGTA 3 mM MgCl  .485 + .127  .497 +.121  .513 + .118  .625 +.168  25 mM H i s t . , .1 mM EGTA 3 mM MgCl , 100 mM NaCl  .921 + .216  .960 + .231  1.057 +.260  1.025 +.259  25 mM H i s t . , .1 mM EGTA 3 mM MgCl-, 108 mM NaCl 3 mM KCl  .975 +.236  .976 + . 241  .976 + .239  1.122 +.295  25 mM H i s t . , .1 mM EGTA 3 mM MgCl„, 100 mM NaCl 3 mM KCl, 3 mM ouabain  .933 + .227  .950 + .238  1.100 + . 319  1.010 + . 242  2  2  mean + S.E. n = 4 experiments and each experiment was done i n duplicate Table f o r F i g . 13. Top Sarcolemmal. enriched microsomal Ca-ATPase a c t i v i t y t o t a l CaCl assay medium  8  20 mM T r i s HC1 PH 7.4 20 mM T r i s HC1 PH 7.4 3 mM MgCl 2  mean + S.E. n = 4  .866 +.099  lO"  6  lO"  (M) 5  lO"  4  lO"  3  .039 +.008  .103 + . 012  .352 +.037  .695 + .063  .820 +.152  ;.;705+.094  .,713 +.099  .840 +.173  245  Table f o r F i g . 15. Bottom  I n h i b i t i o n by,La of the actomyosin Ca-ATPase + 10  CaCl  pCa t o t a l  •oo  umoles P i mg/min  3  M LaCl  umoles P i mg/min  lO"  7  7  .156  .042  lO"  6  6  .154  .080  5.3  .148  .074  5  .150  .069  4.3  .187  .103  4  .210  .100  3.3  .254  .128  3  .287  .150  - 3  2.7  .324  .174  _ J  2.4  .346  .156  5 x 10'  lO" 5 x 10  5  - 5  lO" 5 x 10  6  4  - 4  lO'  3  2 x 10 4 x 10 ^ 4 x 10  3  average of duplicates Table f o r F i g . 1|. 'afoip. Substrate dependence of the sarcolemmal enriched microsomal Ca-ATPase and i t s effect on La i n h i b i t i o n 10  - 3  M LaCl„  ATP (mM)  ATP umoles  1/ATP (M )  .125 -2 .25  .075  8,000  .475  .15  4,000  ' .590  .50  .30  2,0000  1.37  .73  .249  4.02  i:o  .60  1,000  1.57  .64  .520  1.92  1.5  .9  666  1.36  .74  .875  1.14  2.0  1.2  5000  1.4  .71  1.31  .76  2.5  1.5  400  1.39  .72  1.14  .88  3.0  1.8  333  1.34  .75  1.33  .75  1 .ft  average of duplicates  X  umoles P i mg/min  1/V  umoles P i mg/min  2.1  0  1.69  .06  1/V  16.7  246 i  Table f o r F i g . 16 Top  Actomyosin Ca-ATPase dependence on ATP  ATP (mM)  1/ATP (M )  umoles P i mg/min  .125  8,000  .082  12.20  .25  4,000  .138  7.25  .5  2,000  .255  3.92  .75  1,333  .295  3.39.  1.0  1,000  .294  3.40  2.0  500  .328  3.05  4.0  250  .353  2.83  X  1/V  -average of duplicates Table f o r F i g . 23. E f f e c t of K on contraction by 5 uM ouabain  tension (g)  5 uM ouabain % of control high K phasic  % of control high K tonic  normal Tyrode 2.6 mM KCl  .55 + .098  43.4 + 4.76  69.5 + 8.84  Tyrode 14.3 mM KCl  .55 + .046  43.6 + 3.38  68.7 + 7.13  mean + S.E. n=10  \  Table for F i g . 19. Bottom.  Time course of Ca, Na, Mg and K loss i n 160 mM T r i s Tyrode pH 7.4 and 10 mM LaCl at 4°C % of t o t a l control l e v e l s time (min)  mean + S.E.  Na  Mg  K  :I5'  +  26.5 2.8  87.5 +21.7  +  95.7 6.98  30'  +  11.6 2.2  89.8 +10.1  90.6 + 7.9  45'  +  12.2 2.7  +  82 8.97  +  79.5 5.05  60'  4.4 +.1.48  +  74:9. 7.24  +  82.9 4.04  Ca +  24.7 5.1  - 16.2 + 5.03  +  15.4 1.5 4.5  n = 4  to  Table for F i g . 21.  Control experiment to see i f the L a C l ^ T r i s HC1 solution (pH 7.4, 4°C) could remove twice the amount of Ca i n the regular wash time (30 min) and whether Na, K and Mg values would be unaffected  control equilibrated with 1.8 mM CaCl„ 3  Ca  total  interval  14.64 + .873  6.09 + .22  equilibrated with 1.8 mM CaCl„ then 3.6 mM C a C l for 30 sec 0  total 25.9 + 1.15  equilibrated with 1.8 mM C a C l then 3.6 mM CaCl„ for 5 min  interval 6.81 + .92  total 24.8 + 3.57  interval 5.94 + .89  Na  439.4 + 28.4  82.2 + 3.91  519.8 + 23.0  84.8 + 3.5  465.4 + 61.7  92.7 +17.5  K  289.2 + 16.4  249.6 + 8.64  271.6 + 16.5  234.3 + 3.55  259.8 + 23.2  260.4 + 7.7  Mg  +  26.25 .451  +  22.47 .491  +  25.2 1.85  +  19.8 .70  Control levels are those from the ouabain experiments mean + S.E.  n = 4  i n Fig.  +  23.1 1.27  19.8 + 7.2  2  Table f o r F i g . 25. I n t r a c e l l u l a r ion levels during the course of an ouabain response. time with 10 Element Ca  total 14.64 + .873  internal 6.09 + . .22  1 6.49 + .44 •  3 7.48 + .351  5 7.06 + .45  Na  439.4 +28.4  82.2 + 3.91  94.39 + 7.74  124.4 + 7.66  139.2 +26.1  K  289.2 +16.4  249.6 + 8.64  262.6 +13.1  255.5 + 5.52  234.8 + 7.57  '22.43 + .321  21.44 ++ .37  Mg  26.25 + .451  222.47 + .491  21.77 + .586  M ouabain (min) 7 6.53 + .47  10 5.79 + .50  15  30  5.49 + .40  115.3 + 8.7  114.4 + 5.52  179.6 +29.4  235.8. +10.74  219.7 +10.79  181.3 +13.09  114.3 +22.0  21.98 + .66  22.34 + .37  20.58 + .49  1105.3 + 9.7  mean + S.E.  So  250  Table f o r F i g . 26.  S e n s i t i v i t y of the ouabain response to Ca removal normal ^ , Tyrode 0  . • „ ,. , . ^ time xn Ca free Tyrode (mm) ^ • .5 1 2.5 5 10 m  z  % of control high K phasic response  50.8 + 2.37  25.3 + 3.49  19.0. 12.8 +2.4 + 4.8  11.4 + 4.0  % of mean response i n normal Tyrode ( i . e . of 50.8%)  100 + 4.65  49.8 + 6.88  37.3 25.3 + 4.48 +9.43  22.4 + 5.5  12.5  4  2  n =  28  4  4  4  6.35  mean + S.E.  Table f o r F i g . 2r7*. Log dose response f o r cis-2-methyl-4-dimethylaminomethyl -1,3-dioxolane methiodide r  dose (M) log dose  -9 5 x 10 -8.31  10" -8  8  2.5 x 10 -7.6  8  5 x 10 -7.31  - 8  lO"  7  ~?7  2 x 10  - 7  -6.7  5 x 10~ -6.31  phasic  .375 + . 052  .487 +. 059  .812 +. 08  1.35 +.139  1.76 +.167  1.96 +.12  1.85 +.205  tonic  .325 + .063  .625 + .136  1.47 +. 145  1.99 +.094  2.01 +.124  1.86 +.159  1.45 +.147  g tension above basal 350 mg. mean + S.E. n=4  7  Table f o r F i g . 107. Bottom The loss of the phasic component when CD and high KCl are added a f t e r various times i n Ca free Tyrode . . .25  ._5  time i n Ca free Tyrode (min) 1  1_5  CD  75.7 +6.8  85.0 +4.6  70.1 +5.36  63.0 +4.94  high K  73.6 + 8.1  60.4 +11.5  51.6 + 5.05  +  mean + S.E.  -  2  4  5  6  10_  47.7 +4.45  19.26 +2.38  -  8.9 +2.15  8.5 +1.53  27.5 +3.5  -  15.4 + 5.0  -  4.93 +1.41  Table f o r F i g . 319.  The effect of Ca free Tyrode for various times on t o t a l and and internal  ion levels  time i n Ca free Tyrode (min) 0  .614  internal  +.200  Ca total  internal  =  total  internal K total  7 5 1  5.56  5.09  5.76  4.81  4.92  +.45  +.324  +.599  +.574  +..549  10.95  8.81  7.44  7.21  +..798  +.998  +.499  +.3499  +.848  +.452  Na  5JQ  11.32  26.25  internal  2^5  +.873  +.37  total  1J)  14.64  21.5  Mg  0_J>  20.5 +.823 26.38 +.41  88.73  93.08  +4.17  +16.09  19.26  19.2  20.53  +1.27  +1.48  +1.48  23.4  23.3  25.06  +.987 94.4 +10.35  +.226 113.9 +8.65  +.864  18.44 +.741 23.67 +2.18  10  3.14 +.374  6.14 +.699  18.68 +.699 23.12 *..535  99.6  94.39  97.4  +9.39<rr,  +6.83  +11.83  439.3  432.4  443.2  430.2  442.4  508.0  435.0  +28.4  +42.2  ±43.5  +23.4  +27.2  +39.5  +21.7  244.5  193.4  227.6  197.4  188.0  205.4  212.3  +  +16.3  +17.4  +15.3  +29.7  +20.8  +26.1  6.7  289.2  246.0  239.1  280.3  271.1  252.2  234.0  +16.4  +11.35  +17.1  +16.95  +37.6  +36.3  +16.3  nanomoles/mg dry wt. mean + S.E.  Ul  Table f o r F i g . 331. % of control i n normal Tyrode  Time, course of loss of the phasic component to the time course of loss of i n t e r n a l and external Ca. time i n Ca free Tyrode (min) 2.5  0.5  1.0  1.5  2.0  Ca i n t e r n a l  90.6  82.9  -  -  93.9  Ca external  67.9  69.1  -  -  35.9  CD phasic  85.0  70.1  63.0  47.7  -  High K phasic  60.4  51.6  -  27.5  -  ouabain  49.8  37.3  -  -  25.3  3  .  4.0  5.0  6.0  7.5  10.0  -  78.4  -  80.1  51.9  -  30.9  -  27.0  28.2  19.3  -  8.9  -  8.5  - -  15.4  -  -  4.9  -  22.4  -  -  12.5  a. data for ouabain i s not included on F i g . 30. but i s included here f o r comparison to the loss of e x t r a c e l l ular and i n t r a c e l l u l a r Ca.  K3 Co  Table for F i g . 35'.  The e f f e c t of L a C l responses.  on the phasic and tonic components of high K and 2 x 10  3  :  LaCl„ 3  lO"  72.4 4.5  +  41.2 5.6  +  83.2 5.0  +  55.9 4.7  tonic  high KCl  2.5 x 10  +  phasic CD  6  M CD  6  5 x  10  -6  (M)  7.5 x  10  io"  - 6  5  5 x 10  +  38.6 5.7  +  26.5 6.4  +  13.2 1.9  + u&  +  26.1 2.3  +  48.9 7.5  +  30.6 1.9  + —  33.7 4.9 41.7  lO"  5  0  +  13.0 4.5  phasic  76.3 +12.6  23.9 + 5.2  21.0 + 4.8  9.9 + 2.4  11.0 + 2.6  8.9 + 1.4  -  tonic  110.4 +11.4  +  89.6 2.2  105.4 + 5.0  104.8 +11.9  100.6 + 3.0  +  88.9 8.7  -  % of control mean +  S.E.  4  Table f o r F i g . 3I63.  I n t r a c e l l u l a r ion levels during the course of a high KCl contraction and the e q u i l i b r a t i o n phase contraction  equilibration  time i n the presence of high K 10 sec  10 min  5.26 + .17 (83)  5.91 + .60 (18)  +  K  250.9 +11.0 (33)  212.3 ++6.1 (74)  229.4 + 8.4 (18)  250.9 +12.8 (16)  272.9 +11.4 (12)  Na Na  412.3 +17.4 (30)  102.6 + 3.5 (83)  93.5 + 6.6 (18)  107.0 + 4.3 (16)  +  Mg  25.3 + .29 (32)  21.9 + .25 (77)  20.4 + .49 (17)  20.9 + .49 (17)  total  internal  Ca  12.87 + .60 (32)  nanomoles/mg dry wt. mean + S.E. (n)  ~  time after washout  +  30 sec  *  6.96 .45 (17)  +  4.94 .45 (12)  5 min 5.64 + . .77 :ai)  15 min +  30 min  5.24 .60 (8)  +  2.62 .32 (4)  (ID  249.9 +14.1 (8)  289.8 + 6.4 (4)  92.6 6.2 (12)  91.8 + 9.4 (11)  72.2 + 6.2 (8)  +  23.3 + .49 (11>>  19.9 + .58  18.6 + .78 (8)  20.8 + .66 (4)  l e v e l of significance  272.4 +16.5  (ID  9.01 s i % 9.7.5 %  V f  99.95 %  *  68.3 5.3 (4)  Table f o r F i g . ..3(55.  Ca  K  Na  Na  Mg  Intracellular ion levels during the course of a CD contraction and the e q u i l i b r a t i o n phase. 1 contraction  equilibration  time i n the presence of CD  time after washout  internal  10 sec  1 min  12.87 + .60 (32)  5.26 + .17 (83)  5.69" + .25 (20)  6.11 + .45 (19)  5.56" + .57 (12)  192.0 8.9 (20)  188.0 7.9 (13)  +  94.8 + 3.9 (13)  +  250.9 + 11.0 (33)  +  212.3 6.1 (74)  412.3 + 17.4 (30)  +  102.6 3.5 (83)  +  25.3 .29 (32)  nanomoles/mg dry wt. mean + S.E. (n)  +  21.9 .25 (77).  t  +  189.0 7.7 (28)  96.6 + 5.6 (28)  +  19.7 .74 (26)  ,t  +  +  93.9 + 5.6 (20) +  20.5 .58 (19)  10 min  30 sec  5.88 + .37 (43)  4.64 + .17 (25)  5 min  total  •k *  +  +  +  18.6 .78 (12)  +  109.6 4.3 (56)  +  170.3 6.5  170.3 7.0 (55),  +  19.7 .33 (52)  l e v e l of significance  104.8 4.2 (29) +  21.1 .41 (25)  20 min  30 min  6.11 + .22 (4)  4.39 + .45 (12)  5 min 4.29 + .17 (15) 166.0 +_12.8 (15) +  106.6 5.7 18.4 .49 (15)  not s i g n i f i c a n t 90 % 95 % 97.5 % 99.95 %  m  +  (ID +  204.8 + 29.7  ? V t *  107.0 9.0 (4) +  21.4 .66 (4)  +  210.2 9.4  90.2 + 5.9 (12) +  20.9 .49 (12)  Table for F i g . 4"Qf.  CD biphasic contraction  shape changes after various e q u i l i b r a t i o n times i n normal Tyrode. e q u i l i b r a t i o n time allowed  20  12  15  96.8 2.9  phasic % of control  100.9 + 5.9  105.7 + 6.4  +  tonic % of control  114.4 + 4.3  124.6 + 8.4  134.1 + 4.6  time to reach peak tonic (min)  +  7.55 .46  6.30  ± -  49  +  4.95 .16  8  10 +  91.6 4.9  128.0 + 5.0 +  4.10 .22  (min)  +  83.6 6.7  123.5 + 6.6 +  2.95 .10  6 +  81.1 7.6  119.8  ±  5  *  2  4  -  5  2.0 .65  +  82.5 7.7  ±  108.1 + 4.7  7  -  5  106.4 + 5.1  0.95  ± ±-  85.9  69  0.8 +. 01  i  to  258  Table for F i g . 41. Effect of a previous 10 min CD contraction on the contraction to a second dose of CD after various . times. e q u i l i b r a t i o n time after a 10 min CD contraction (min) 2  4  6  8  12  phasic  79.8 + 9.1  72.3 + 7.5  79.8 + 5.2  89.3 + 9.3  80.5 + 4.0  tonic  94.1 + .5.6  113.4 + 7.0  116.3 + 3.9  114.6 + 5.8  110.8 + 1.6  mean + S.E.  Table for F^g^48jr_The effect of a 10 min exposure of CD on4 a subsequent . high KCl response. e q u i l i b r a t i o n time after a 10 min CD contraction (min) 2  4  6  8  12  phasic  61.4 + 2.0  57.7 + 3.8  61.3 +13,5  68.2 + 1.66  83.2 + 1.4  tonic  86.7 + 4.2  93.4 +3355  92.5 + 3.9  89.2 + 4.5  85.0 + 4.5  least tension between phasic and tonic  11.6 +4.5  26.4 + 9.2  36.4 + 9.3  64.5 + 8.5  91.6 + 3.3  Table for F i g . 5.0>.  The effect of a l t e r i n g the Tyrodes's KCl concentration on the time required for equilibration. extracellular KCl concentration during e q u i l i b r a t i o n MM  .  times normal e q u i l i b r a t i o n time (min) mean—r S-.E.  1.34  2.68  5.36  6.70  8.05  0.5  1  2  2.5  3  70.2 + 3.2  25.3 + 1.0  12.8 + 0.6  10.4 +0.6  8.65 +0.39  10.73  13.41  4  5  7.05 +0.30  6.00 +0.43  APPENDIX Table 1.  Compilation of free Ca  concentrations.  -  ATP (M) 3 x 10 II  Ca (M)  Mg  - 3  EGTA (M)  free C a (M)  pfree Ca  2 +  l(f  3  -  1.75  II  lO"  4  -  1.75 -x i o "  II  l(f  5  -  1.75 x 10  II  lO"  6  -  1.75  x  io"  7  6.75  3 xl(f  3  x  IO"  4  3.75  5  4.75 5.75  6  •>  -  5  X  lO"  3  -  2.06  x  IO"  3  2.69  »  -  4  X  lO"  3  -  1.12  x  io"  3  2.95  »  -  3  X  lO"  3  -  2.43 x 10  »  -  2  X  lO"  3  -  7.3  •II  -  lO"  3  -  2.2 x i o "  5  4.66  »  -  lO"  4  -  1.8 x i o "  5  4.74  »  -  lO"  4  -  1.5 x i o "  6  5.82  lO"  5  -  6.1 x i o " ( 1.6 x i o "  7  6.21  7  6.80  io"  7  6.95  io"  7  7.00  „  5  -  5  X  X  4  .io"  x  5  3.61 4.14  1  »  -  »  -  »  io-  lO"  6  -  io"  6  -'  IO"  7  »  -  io"  3  »  -  IO"  4  »  -  io"  5  IO"  3  io"  4  io"  5  I,  3 x IO II  3 x 10  5  -  5  - 3  - 3  3 x lCf  3  X  -  1.1  X  -  .  -  -  io"  4  2.00xxl00  io"  4  3.0 x IO"  7  6.52  io"  4  8.6 x i o "  9  8.76  io"  4  1.67x IO"  4  3.80  io"  4  1.1  io"  6  5.96  io"  4  7.0 x i o "  8  7.15  X  55  4.70'  -2+ CALCULATION OF THE FREE Ca CONCENTRATION UNDER VARIOUS  CONDITIONS  A. Calculation of the free Ca concentration In the presence of 3 mM MgCl,, and 3 mM ATP (no EGTA) according to the method of Katz et a l . (1970) constants  references  K,. ATP = 1.0715 x 1 0 diss K  MgATP = 8.8 x 10  - 7  M  O'Sullivan and P e r r i n (1964)  M  -1  O'Sullivan and P e r r i n (1964)  K CaATP = 3.15 x 10 M ass  _1  Ogawa (1968) °  ctS  4  S 4  v  4Since Mg and Ca complex with ATP  primarily i t i s necessary to c a l c u l a t e the  f r a c t i o n of ATP that i s completely dissociated at pH 7.4. + 4K,. = 1.0715 x 10" j . n A T P ] [ATP "] 7  H  B  d  l  S  S  ( 1 )  3  7.4 = - l o g [ H ] , therefore  [H ] = 3.98 x 1 0  +  +  (2)  _ 8  3_ Substituting equation (2) into equation (1) and solving for ATP 4in terms of ATP gives ATP = 0.3714 ATP " 2+ 42+ Since Mg complexes with ATP more strongly than Ca , i t has an 3_  4overriding e f f e c t on the amount of ATP l e f t to bind Ca. -3 t o t a l ATP concentration = 3 x 10 M, then ATP  (3)  4  + ATP  4-  3_  + [MgATP ] = 3 x 1 0 2-  ,.  Since the  (4)  _ 3  -3 Since the t o t a l Mg concentration = 3 x 10  M  [Mg ] + [MgATP "] = 3 x 10~ M 2+  2  3  and, rearranging [Mg ] = 3 x 1 0 2+  - 3  - [MgATP ] ^  (5)  2-  Combining equation (3) and (4), then 1.3714 A T P + [MgATP ] = 3 x 1 0 42and ATP , i n terms of [MgATP ] from the above equation i s 4-  [ATP ] = 2.186 x 1 0 4-  2-  - 3  _ 3  -0.7308 [MgATP ] 2+  The association constant f o r Mg  (6)  2-  4to ATP  equals  2 62 t g ~] 2+ 4[Mg ][ATP ] M  = 8.8 x 10 M"  A T p 2  Z+  A  ' (7)  1  4  Substituting equation (5) f o r Mg  2+  and equation (6) for ATP  4-  into  equation (7), f (3 x 10  g  -[MgATP  " ' ])(2.186 x 10 A  T  p  2  ]  y= 8.8 x 10 -.7308[MgATP ])  4  2and solving f o r [MgATP  ] yields  [MgATP ] = 2.786 x 10 M  (8)  Substituting equation (8) into equation (6) y i e l d s A T P = 0.15 x I O M 42+ This i s the ATP l e f t to bind Ca . The association constant f o r  (9)  2-  _3  4-  - 3  2CaATP  gives  2JJ = 3.15 x 10 [Ca. ][ATP ] The known amount of Ca added to the assay medium = [Ca] .. and total C  a  A  T  P  (10)  4  1  [Ca]  = [Ca ]>+ 2+  t o t a l  [CaATP ] 2-  and, rearranging [Ca ] = [ C a ] 2+  - [CaATP ]  '  2-  t o t a l  (11)  Substituting equation (9) and (11) into equation {10) y i e l d s t ~] = .15xl0 ([Ca] - [ C a A T P " ] ) ( . 1 5 x 10 ) 2and solving f o r [CaATP ] i n terms of [Ca] total C a A T p 2  4  3  2  3  total  n  [CaATP ] = 0,825 [Ca]  (12)  2-  From equation (11) and equation (12) [ C a ] = [Ca] , - .825[Cal total total 2+ 2+  1  J  1  J  1  J  See appendix Table 1 for [Ca both 3 mM.  '  ] at various [ C a J ^ ^ i when ATP and MgCl are 2  2 63 2+ Calculation of the free Ca concentration i n the presence of 3 mM ATP  B.  (no MgCl , no EGTA) 2  •34(3) ATP = .3714 ATP  From section A. equation  -3 The t o t a l ATP concentration = 3 x 10  M  ATP " + ATP " + [CaATP "] = 3 x 10~ M 4  3  2  Substituting equation  (13)  3  (3) into equation (13)  ATP " + .3714 ATP " + [CaATP ] = 3 x 10~ 4and solving f o r ATP A T P = 2.186 x 10~ - .7308 [CaATP ] -3 4  4  2_  4_  T  3  3  (1.4)  2-  When the t o t a l CaCl^ concentration i s 10 [ C a ] + [CaATP ] = 1 0 and 2+  2-  [ C a ] = 10" 2+  M  _ 3  - [CaATP "]  3  (15)  2  From the CaATP association ' " [Ca^ ][ATP ] 9  j  C  a  M  A  T  P  constant  = 3.15 x 10  ]  (16)  4  Substituting equation ^-14) and (15) into equation ___  [CaATP ]  (16) y i e l d s  2-  (10  - [CaATP  =  ])(2.186 x 10  Subsituting equation [Ca ] = 10 2+  - 3  3  (1?) into equation  - .978 x 1 0  - 3  1 Q  4  - .7308[CaATP; ])  and solving for [CaATP ] = .978 x 10~ 2-  _  = .022 x 1 0  M  (13)  (15) gives - 3  M  At any desired t o t a l Ca concentration, equations  (18) (15), (10) and (lZ) can  be solved to give the free Ca concentration. See Appendix Table 1 f o r a compilation of free Ca concentrations under various  conditions.  2 64 2+  C. Calculation.of the free Ca  concentration i n the presence of 0.1 mM EGTA  The d i s s o c i a t i o n constant f o r CaEGTA depends on the pH.  K  pH IA  K =  T l + K [E ]  d ± S S  +  K  +  ±  ^  H  diss  V  +  (19)  K  ^  K  ^  ]  +•  3  K K K K [H ] +  1  2  3  4  4  10 where  = 4.47 x 10 K  when the four carboxyl groups of EGTA are completely dissociated-' (Schatzmann (1973) .  = 2.89 x 10  K  2  K  3  Portzehl et a l . (1964)  9  = 7.09 x 10  8  = 4.78 x 10  2  "  2  K.  = about  [H ]  10  "  = 3.98 x 10"  +  at pH 7.4  8  c i • ' /-.ON. i^P Solving equation (19), K *. H7  d  l  S  oo x m10 = i1.33  4  7  S  =  [GaEGTA][Ca^] [EGTA]  (20) ' v  2+ C. a) Calculation of the free Ca  concentration i n the presence of 3 mM M g C l  2>  3 mM ATP and 0.1 mM EGTA From equations (9) and (10), 2[CaATP  ]  [Ca ] [CaATP ]  =  (  3  <  1  5  x  1 0  4  ) ( 0 < 1 5  x  1 0  4  }  =  4  >  7  2  /+  2-  = 4.72[Ca ]  (21)  2+  -4 Since the t o t a l EGTA concentration i s 10 [EGTA] + [CaEGTA] = 1 0 [EGTA]  M  _ 4  = 10~  4  - [CaEGTA]  (22) -3  If the known .feot-aitCaClC^aS&ded to the assay medium i s e.g. 10  M then  [Ca ]-+[CaATP ] + [CaEGTA] = 10~ M 2+  2_  3  (23)  Substituting equation (21) into equation i('23) and c o l l e c t i n g l i k e terms 5.7-2[Ca ] + [CaEGTA] = 10~ M 2+ - -3 and rearranging [Ca ] = 10 - [CaEGTA] 5.72 [ C a ] = .175 x I O - .175[CaEGTA] 2+  3  2+  - 3  (24)  (25)  Substituting equations (22) and  (24) into equation (20) gives  [CaEGTA]  _ =  (.175  x 10-3 _ .175  [CaEGTA])(10  7 1.33 x 10  - [CaEGTA]) -4  and solving for [CaEGTA], then [CaEGTA] = very nearly 10  M  (26)  Substituting equation (26) into equation (24) and solving 5.72  [ C a ] + 10" 2+  =  10" 2+ [Ca ] = 1.57  4  (27)  3  x 10  -4  In l i k e manner, equations (23) through (27) can be solved for t o t a l Ca -4 concentrations of 10  -5 and 10  M, etc.  See Appendix Table 1 for the r e s u l t s .  2+ C. b) Calculation of the free Ca  concentration i n the presence of 3 mM  ATP  and 0.1 mM EGTA 2+ In order to express [Ca ] i n terms of [CaEGTA] for equation (23), we need to know the r a t i o of [CaATP] to [Ca]. In the absence of EGTA at 10 18) and  [CaATP  Therefore,  2-  -3  ] = .978 x 10  „.M C a  -3  t o t a l  ,  2+ [Ca. ] = 0.22  x 10  -3  M (see equation  M (see equation 17).  [CaATP ] =44.7 [ C a ] 2-  (28)  2+  2+ Substituting equation (28) into equation (23) and solving for [Ca [Ca ]  =  2+  .022 x 1 0  - 3  - .022  ]  [CaEGTA]  (29)  Substituting equations (29) and (22) into equation (20), .then (.022  C^^y x 10-3 _ .022 [CaEGTA])(10  = -4  1.33  xlO  7  - [CaEGTA])  and solving for [CaEGTA], then [CaEGTA] = .095 x 10~  3  M  (30) 2+  Substituting equation (30) into equation (29) and solving for [Ca gives  [ C a ] = 1.99 2+  x 10  5  ]  M.  2+ In l i k e manner, the free Ca -4 trations of 10  concentration can be solved f o r t o t a l Ca concen-  -5 and 10  M i n the presence of 3 mM ATP and 0.1 mM EGTA.  

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