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UBC Theses and Dissertations

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

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ION MOVEMENTS DURING CONTRACTION OF THE GUINEA PIG ILEUM LONGITUDINAL SMOOTH MUSCLE by MARILYN ROSAMOND JAMES B . S c , S t a t e U n i v e r s i t y o f New Y o r k at B u f f a l o , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES i n THE FACULTY OF PHARMACEUTICAL SCIENCES We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d / THE UNIVERSITY OF BRITISH COLUMBIA June, 1977 <6) Marilyn Rosamond James In p r e s e n t i n g t h i s t h e s i s in p a r t i a l . f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 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 longi-tudinal smooth muscle was studied in 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 activities in the sarcolemma, the tissue Ca depots which could release Ca for contraction and the sensitivity of the contractile responses to extra-cellular ion changes. Essentially net changes of intracellular Ca, Mg, Na and K content during contractions were measured by a modified 'La method'. The tissues were washed for 30 min in 160 mM Tris-HCl solution (pH 7.4) containing 10 mM LaCl3 at 4°C in order to seal the intracellular ions in the c e l l and dis-place extracellular ions. A method to loosen the 'intercellular cementing' sub-stance by reducing the tissue Ca and Mg was developed as an adjunct to the pre-paration of a sarcolemmal enriched microsomal fraction. The method reduced the tenacity of the tissue and made the tissue easy to disrupt by a mild homogeniz-ing procedure. The method also appeared to aid the extraction of contractile proteins. The microsomal fraction was not detectably contaminated by mitochon-dria and was enriched with vesicles of sarcolemma, probably originating from the muscle caveolae.. The sarcolemma enriched microsomal fraction had a Ca-ATPase activity that -7 -4 2+ was progressively stimulated by 10 to 2.4 x 10 M free Ca , did not require Mg and was inhibited by La. The microsomal Ca-ATPase activity was not due to contamination by actomyosin. The actomyosin Ca-r-ATPase in the soluble fraction had a higher af f i n i t y than the microsomal Ca-ATPase for Ca and for La. The microsomal Ca-ATPase activity was postulated to be associated with an active Ca i i pump thought to he located ;Ln the cayeolae, xhe microsomal fraction had a Mg-dependent ATPase that could Be stimulated by Na, but K and ouabain had very l i t t l e additional effect. The addition of an activating factor in the soluble fraction conferred some K and ouabain sensitivity to the Mg-dependent Na-ATPase, which indicated that a Na,K^ATPase was present in this tissue. Low doses of ouabain contracted the longitudinal ileum but the responses were not antagonized by raising the external K concentration five fold, as would be expected i f ouabain acted by inhibiting the Na,K-ATPase. However, the ouabain response was rapidly lost when extracellular Ca was removed from,the medium and the decline of the response followed the same time course as the loss of extracellular Ca. The peak of the ouabain contraction coincided with s i g n i f i -cant increases of intracellular Ca and Na, but K loss was not apparent unt i l relaxation ensued. The results suggested that ouabain has an early direct effect on membrane permeability before i t inhibited 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 physiological medium. This decline followed the time course of the loss of extracellular Ca. 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 respon-sible for the phasic component. The tonic component was lost 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 extracellu-lar space. The extracellular origin of the Ca for contraction was consistent with the observed small net gain of intracellular Ca that occurred during the phasic and tonic contractions. The minimal volume of the sarcoplasmic r e t i -culum and the abundance of caveolae was also consistent with the high sensi-t i v i t y of the tissue to extracellular Ca concentrations. The intracellular 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, the muscle was quiescent for the 20 to 30 min 'equilibration' phase. Spontaneous activity was absent during this phase and tension was below baseline. After a maximal CD contraction, a second response to CD or to 60 mM KC1 induced during the 'equilibration' phase had an altered or desensitized biphasic appearance. Responses of the muscle to CD for 10 min were accompanied by a cytoplasmic 1 oss of K. After washout of CD, the K was regained slowly over 20 to 30 min. 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 extracellular K concentrations decreased the time required after CD contractions for the return of spontaneous activity and prevented muscle desensitization to repeated doses of CD, probably by accelerating the return of intracellular K levels to normal. It was proposed that during con-traction, elevated intracellular Ca activated K channels, thereby increasing K permeability and causing the 'after-hyperpolarization' and subsequent desen-sitization which follows muscarinic induced contractions. TABLE OF CONTENTS page ABSTRACT LIST OF TABLES LIST OF FIGURES LIST OF MICROGRAPH PLATES LIST OF ABBREVIATIONS INTRODUCTION I. The Reasons for Studying the Guinea Pig Ileum Longitudinal Smooth 1 Muscle. I I . Methods of Subcellular Fractionation of Smooth Muscle Relevant to 3 the Present Study a. Homogenization techniques f or smooth muscle 3 b. Common procedures for separation of s u b c e l l u l a r f r a c t i o n s 5 c I d e n t i f i c a t i o n of s u b c e l l u l a r f r a c t i o n s 6 I I I . Studies of the Na,K-ATPase i n Smooth Muscle ' 6 IV. Studies of Ca-ATPases i n Smooth Muscle 7 V. Methods f o r Measuring Ion Movements i n Smooth Muscle 8 a. Calculation of i n t r a c e l l u l a r ion l e v e l s for t o t a l ion measurements 9 and f l u x studies b. The 'La Method' 10 VI. The Mechanism of Action of Ouabain 13 VII. Excitation-Contraction Coupling (General) 14 VIII. Sources of Calcium for Excitation-Contraction Coupling i n the 17 Guinea Pig Ileum Longitudinal Smooth Muscle IX. Receptor Theory and the Desensitization Phenomenon 21 X. Aims of the Present Study 22 METHODS ' . 23 I. Dissection of the Longitudinal Layer of the Guinea Pig Ileum 23 II. C o n t r a c t i l e Force Measurements 23 a. Isometric force 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. 'Glycerol shock' treatment 26 II I . Measurement of Muscle Na, K, Mg and Ca 27 a. Total ion measurement 27 b. 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 27 Na, K, Mg and Ca i v i i v i i i x i i x i i i 1 page c. Extraction of ions from tissues (total and intracellular ion 28 measurements) and atomic absorption measurements IV. Preparation of Sarcolemmal Enriched.!Microsomes 30 V. Measurement of ATPase and Membrane Marker Enzyme Activities 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 pellets 34 c. Sectioning and staining 34 MATERIALS 36 RESULTS AND DISCUSSION 3? I. Procedure for Isolating Sarcolemmal Enriched Microsomes • 37 II. Mg dependent Na,K-ATPase 51 III. Ca-ATPases 73 a. Microsomal Ca-ATPase 73 b. Actomyosin Ca-ATPase 75 c. Comparison of the divalent cation stimulated ATPase activities 7 5 IV. The Structure of the Guinea Pig Ileum Longitudinal Smooth Muscle 89 V. The Modified 'La Method' 108 VI. The Effect of Ouabain on the Intact Longitudinal Smooth Muscle of 121 the Guinea Pig Ileum Vlt. Possible Sources of Ca for the Phasic and Tonic Contractions of 138 ' the Guinea Pig Ileum Longitudinal Smooth Muscle a. The biphasic contraction _3 9 b. Sensitivity of the phasic and tonic contractions to Ca-free _3 9 medium c. Sensitivity of the phasic and tonic contractions to LaCl^ 141 d. Measurements of intracellular ion contents during contractions 143 induced by CD and 60 mM KC1 VIII. The 'Desensitization' Phenomenon v i page SUIVMARY AND CONCLUSIONS 223 • REFERENCES 22 6 • APPENDIX 2.4-0 Tables for Figures, (see List 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 in the presence of 261 3 mM MgCl and 3 mM ATP (no EGTA) 2+ B. Calculation of the free Ca concentration in the presence of 263 3 mM ATP (no MgCl-, no EGTA) 2+ C. Calculation of the free Ca concentration in the presence of 264 0.1 mM EGTA 2+ a) Calculation of the free Ca concentration in the presence of 264 3 mM MgCl_, 3 mM ATP, and 0.1 mM EGTA 2+ b) Calculation of the free Ca concentration in the presence of 26'5 3 mM ATP and 0.1 mM EGTA v i i LIST OF TABLES Table T i t l e . page 1 Atomic^absorption standards and calculations for ug element 29 in tissue samples 2 Location of c e l l marker enzymes, specific activities and % 47 recoveries in each c e l l fraction 3 Subcellular distribution of ATPase activities 48 4 Cation levels in the microsomal fraction 58 5 Calculation of intracellular and extracellular ion concentrations 117 6. Calculation of the resting membrane potential 118 v i i i LIST OF FIGURES Fig . Appendix page page 1. Superimposed tracings of time courses of contractions showing 20 the e f f e c t 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 [Cag^r-] - . and stimulus [Ca^_J_ during 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. Dissection of innervated and denervated guinea p i g ileum l o n g i t u - 25 di n a l smooth muscle 3. Cation l e v e l s of the guinea p i g ileum longitudinal.smooth muscle 42 240 a f t e r depletion of the tis s u e i n Ca,Mg-free Tyrode's s o l u t i o n as used i n the procedure for preparing sarcolemmal enriched micro-somes 4. Sucrose density gradient treatment of the microsomes 50 5. Stimulation of microsomal ATPase by Mg, Na and K 57 241 6. The e f f e c t of Mg, Na, K, ouabain and a soluble a c t i v a t i n g factor 60 242 on the microsomal ATPases 7. Upper Substrate dependence curve of microsomal ATPase 62 Lower Bar graph comparing the u t i l i z a t i o n of ADP with ATP 62 8. Lineweaver Burk p l o t of the e f f e c t of 100 mM Na or 100 mM Na plus 64 243 3 mM K on the Mg-ATPase 9. Assay of the Mg-ATPase with (y- 3 2P) l a b e l l e d ATP 66 10. The e f f e c t of Ca on the Mg dependent Na,K-ATPase 68 244 11. Pharmacological tests of the tissue v i a b i l i t y under the biochemi- 70 c a l conditions used to prepare sarcolemmal enriched microsomes, as a check for the possible loss of Na,K-ATPase 12. Pharmacological test of the muscle v i a b i l i t y a f t e r treatment i n 72 Ca,Mg-free Tyrode's s o l u t i o n 13. Sarcolemmal enriched microsomal Ca-ATPase a c t i v i t y i n response to 80 244 free C a 2 + 14. The e f f e c t s of ions on the microsomal Ca-ATPase 82 15. The e f f e c t of La on the microsomal Ca-ATPase and the actomyosin 84 Ca-ATPase Top Substrate dependence of the microsomal Ca-ATPase and i n h i b i - 84 245 tio n by LaCl3 Bottom Response of the soluble f r a c t i o n ATPase (actomyosin) to 84 245 increasing free C a 2 + 16. C h a r a c t e r i s t i c s of the actomyosin Ca-ATPase 86 Top The concentration dependence of the actomyosin Ca-ATPase on 86 246 ATP 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 86 17. Lineweaver Burk p l o t of the substrate dependence of the microsomal 88 and actomyosin Ca-ATPases 18. An example of the e f f e c t of 'gl y c e r o l shock' treatment on the .. 107 guinea p i g ileum l o n g i t u d i n a l smooth muscle i x Appendix F i g . page page 19. Top Measurement of the time required for 10 mM LaCl3 i n Ca- 114 free Tyrode's s o l u t i o n (pH/,7.4) at 4°C to displace e x t r a c e l l u l a r Ca Bottom Measurement of Ca, Mg, Na and K l e v e l s of the guinea 114 247 p i g ileum l o n g i t u d i n a l smooth muscle over time i n 160 mM Tris-HCl (pH 7.4) containing 10 mM L a C l 3 at 4°C 20. Measurement of the tissue ion content and estimation of the r a t i o 116 of the tissue wet weight to the tis s u e dry weight 21. Measurement of the t i s s u e ion contents by the modified 'La 120 248 method' a f t e r exposure of the guinea p i g ileum l o n g i t u d i n a l smooth muscle s t r i p s to Tris-Tyrode's s o l u t i o n containing 3.6 mM CaCl„ for 0.5 min (B) and for 5 min (C) as compared to controls (A; e q u i l i b r a t e d i n Tris-Tyrode's s o l u t i o n (1.8 mM CaCl-) 22. The e f f e c t of ouabain on innervated and denervated guinea p i g 129 ileum l o n g i t u d i n a l smooth muscle 23. The e f f e c t of increased e x t r a c e l l u l a r K on contraction, by 131 5 yM ouabain 24. Top Responses to 60 mM KC1 at the peak of the excitatory res- 133 ponse to ouabain and during the i n h i b i t o r y phase Bottom The e f f e c t of 10 uM ouabain on a 60 mM KC1 response 133 and a 0.2 uM response to CD 25. I n t r a c e l l u l a r ion l e v e l s during the course of an ouabain res- 135 249 ponse 26. The s e n s i t i v i t y of the ouabain response to Ca removal 137 250 27. Log dose response curve to cis-2-methyl-4-dimethylaminomethyl- 152 250 1,3-dioxolane methiodide (CD) 28. The responses of the guinea p i g ileum l o n g i t u d i n a l smooth muscle 154 to increasing doses of CD 29. The response of l o n g i t u d i n a l i l e a l muscle to methacholine 156 30. Top A comparison of phasic and tonic responses i n normal 158 251 Tyrode's s o l u t i o n and a f t e r switching to Ca-free Tyrode's so l u t i o n for 5 sec before the addition of 2 x 10 -^ M CD or 60 mM KC1 Bottom The loss of the phasic component when CD and high KC1 158 are added a f t e r various times i n Ca-free Tyrode's s o l u t i o n 31. The s e n s i t i v i t y of responses to 60 mM KC1 and to 2 x 10~ 7 M CD 160 to the removal of Ca from the l o n g i t u d i n a l i l e a l smooth muscle for 10 min and r e s t o r a t i o n of Ca for 30 sec 32. The e f f e c t of incubation of tissues i n Ca-free Tyrode's s o l u t i o n 162 252 for various 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 l e v e l s 33. The rate of loss of the phasic component compared to the rate 164 253 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 34. The e f f e c t of L a C l 3 (10~ to 10~ M) on responses to high KC1 166 (6.0 mM) i n Tris-Tyrode" s s o l u t i o n X Appendix Fig. page page 35. Comparison of the effect of LaCl-^ on the phasic and tonic 168 254 component of responses induced by 60 mM KC1 and 2 x 10*""'' M CD 36. Intracellular ion levels during the course of a contraction 170 255 induced by 60 mM KC1 and the equilibration in Tris-Tyrode's solution 37. Intracellular ion levels during the course of a contraction 182 256 induced by 2 x 10~7 M CD and the equilibration phase in Tris-Tyrode 's solution 38. The effect of reducing extracellular Na on the longitudinal 184 i l e a l muscle activity 39. Changes of the biphasic contractile pattern when contractions 192 to CD were induced at shorter equilibration time intervals between contractions 40. Graphical representation of the effect of the equilibration 194 257 time on contractions by CD 41. The effect of the equilibration time allowed after a 10 min 196 258 exposure to CD 42. The effect of a very short equilibration time (30 sec) on a 198 CD response after a long exposure to CD (35 min) 43. The lack of effect of the equilibration time allowed between 200 responses to 60 mM KC1 on the biphasic shape of a series of responses 44. The effect of the equilibration time allowed after a 10 min 202 response to carbachol on a second contraction induced by carbachol 45. ..The effect of a 5 min or 10 min equilibration time after a 204 response of the muscle to methacholine on the biphasic appearance of a second response to methacholine 46. 'The effect of the duration of exposure to CD on the the time 206 required for equilibration, monitored by the return of spontaneous activity and the subsequent return of the normal biphasic contractile pattern 47. The effect of a.: 10.'.min..exposure:"to..CD..otioa'.'response to 60 mM 208 KC1, 2, 4, 6, 8 and 12 min after the response to CD was washed out 48. Graphical representation of the effect of the time allowed for 210 258 equilibration after a response to CD on a response to 60 mM KC1 49. The effect of a simultaneous addition of 60 mM KC1 and 212 / x 10"7 M CD on the equilibration phase of the muscle 50. The effect of altering the concentration of KC1 in the 214 259 Tyrode's solution on the rate of return of spontaneous activity after a response to CD 51. The specificity of increased external KC1 for accelerating 216 the return of the usual biphasic contractile pattern compared to the lack of effect of higher extracellular NaCl concentration xi Appendix Fig. page page 52. The effect of various agents on the time required by the 218 muscle for the return of spontaneous activity after a 10 min response to CD 53. The effect of raising the extracellular Ca and Mg concentrations 220 on the time required by the muscle to regain spontaneous a c t i -vity 54. The effect of depriving the muscle of dextrose and oxygen on 222 the time required for the return of spontaneous activity X I X LIST OF MICROGRAPH PLATES Plate # page 1 1 Mitochondria, 27,000 x g pellet 44 1 2 Mitochondria, 27,000 x g pellet 44 1 3 Mitochondria, 27,000 x g pellet 44 2 4 Microsomes, 105,000 x g pellet 46 2 5 Microsomes, 105,000 x g pellet 46 2 6 Microsomes, 105,000 x g pellet 46 3 7 Rolled longitudinal ileum strip 101 3 8 Auerbach's Myenteric nerve plexus 101 3 9 Longitudinal section of longitudinal i l e a l smooth muscle cells 101 (light microscope) 3 10 Longitudinal section (electron microscope) 101 3 11 Cross section of longitudinal i l e a l smooth muscle cells 101 (light microscope) 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 nucleoli 103 4 16 Spiral indentations of nuclei in a contracted x e l l 103 4 17 Nucleus in 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 23 Enlargement of caveolae 105 5 24 Rough endoplasmic reticulum, mitochondria and agranular 105 membranes 5 25 Smooth subsarcolemmal sacs (sarcoplasmic reticulum) 105 . x i i i LIST OF ABBREyIATONS Acha s e a c e t y l c h o l i n e e s t e r a s e ADP adenosine. 5 '-diphosphate 5'-AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate Ca-free Tyrode's s o l u t i o n Tyrode's s o l u t i o n from which Ca was omitted Ca,Mg-free Tyrode's s o l u t i o n Tyrode's s o l u t i o n from which Ca and Mg were omitted Cch carbachol CD cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide DTNB 5,5' d i t h i o b i s - ( 2 - n i t r o benzoic a c i d ) EDTA ethylenediamine t e t r a a c e t i c a c i d EGTA e t h y l e n e g l y c o l - b i s - ( g - a m i n o e t h y l ether) N , N ' - t e t r a a c e t i c a c i d L a - T r i s s o l u t i o 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 3 at 4°C Mch methacholine NADH nico t i n a m i d e adenine d i n u c l e o t i d e (reduced form) Na,K-ATPase + + Na and K s t i m u l a t e d adenosine t r i -phosphatase. (ATP phosphohydrolase E.G.3.6.1.3) neo neo 3 fe iggiirie: n i c n i c o t i n e . NT normal Tyrode's s o l u t i o n ouab ouabain P . X i n o r g a n i c phosphate TCA t r i c h l o r o a c e t i c a c i d TT T r i s b u f f e r e d Tyrode's s o l u t i o n (pH 7.4) T r i s T r i s (hydroxymethyl) aminomethane W washout of stimulant from muscle bath ACKNOWLEDGEMENT I wish to 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 project and h i s advice, as well as his f r i e n d s h i p . I am g r a t e f u l to the Medical Research Council of Canada f o r t h e i r f i n a n c i a l . support of the project and myself. I appreciate the time and e f f o r t given by my research committee members (Dr. B. Roufogalis, supervisor; Dr. F. Abbott, Dr. G. Bellward, Dr. S. Katz, Dr. J . McNeill, Dean B. Riedel, Dr. M. Sutter and Dr. E. Daniel, external examiner) during meetings and for the reading of the f i n a l t h e s i s . Helpful discussions with Dr. D.J. T r i g g l e , Dr. CR. T r i g g l e , Dr. V. Palaty, Dr. M. Sutter, Dr. S. Katz, Dr. Z. Chmielewicz, Dr. M. Wolowyk, Mr. L. Veto and Mr. G. Kracke were greatly appreciated. I have enjoyed my graduate student days at the U n i v e r s i t y of B r i t i s h Columbia thanks to the warmth and concern of many f r i e n d s . I am thankful for the typing and proofreading assistance of June Lam and Helen Butt. DEDICATION To Mum, Dad, Janet me and, above a l l , and Ian f o r t h e i r confidence i n f o r t h e i r love and encouragement. 1 INTRODUCTION i Knowledge has steadily been accumulating about the sequence of events lead-ing 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 is due to the fact that different types of smooth muscles exhibit quite different charac-te r i s t i c s . It now appears that many drugs alter more than one phase of smooth muscle activity. 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 attain a better know-ledge 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 cellular ion distribution. Others have been searching for effects of stimulants on enzymatic processes. Perhaps stimulants cause a combination of permeability changes and enzymatic alterations, 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 bio-chemistry 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 pig 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 virtue of being structurally less complex. Most other struc-turally complex organs, composed of smooth muscle, can not be separated as easily into layers. The longitudinal layer of the ileum is characteristically different from many other smooth muscle tissues in that each c e l l i s not indi-vidually innervated (Holman 1970). However there are non-adrenergic arid non-cholinergic neurons which may penetrate intestinal muscle layers (Furness and Costa 1973). Nevertheless, essentially denervated strips 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 limits their use to special experiments. The muscle layer is very thin (in the order of 50;,u thick, Paton 1975a) and i t s thinness presents very l i t t l e impediment to the diffusion of pharmacological agents through the tissue (Paton and Rang 1965). Because the extracellular f l u i d rapidly equilibrates with physiological solutions of various compositions, the guinea pig ileum longitudinal smooth muscle has proven useful in studies of muscle ionic fluxes (Weiss et a l . 1961; Lullman and Siegfriedt 1968; Hurwitz and Joiner 1969; Lullman and Mohn 1971; Weiss 1972). The longitudinal muscle strips, as usually prepared, are composed of 75% longitudinal smooth muscle cells , approximately 18% nervous tissue and serosa (primarily fibrous noncellu-lar material) and the remaining 7% i s connective tissue and a few adhering . circular muscle layer cells (Weiss et a l . 1961). Therefore changes in ion levels of the tissue can be assumed to reflect changes in ion levels of the longitudinal smooth muscle ce l l s . One of the disadvantages of the preparation is that the tissue can be damaged during the f a i r l y extensive dissection procedure and increases in 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 isolated longitudinal ileum contracts very well to muscarinic agonists, high concentrations of KC1 and various other stimuli and remains viable for many hours. Weiss et a l . (1961) found that potassium fluxes induced by acetylcholine were similar and usually greater in the isolated lon-gitudinal layer than in the whole ileum. Lullman and Siegfriedt (1968) observed that the Ca content of longitudinal i l e a l cells varied slightly, de-pending on the location along the intestine. This i s another disadvantage that might obscure changes of Ca levels between control and experimental strips. These disadvantages make i t necessary to study many muscle strips to obtain representative control and experimental values, but one of the advantages of 3 the preparation is that 16 to 20 muscle .strips can be obtained from each animal. Longitudinal i l e a l cells 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 in diameter and 50 u in length (Paton and Rang 1965). Most of the contrac-t i l e filaments are not much farther than 1-2 u from the extracellular space. The sarcoplasmic reticulum i s probably less than 2% of the total cellular volume (similar to taenia c o l i cells which are estimated to have 2% sarcoplasmic r e t i -culum) (Devine et a l . 1972). The c e l l volume is small compared to the large surface area. Permeable or transiently permeable ions (under stimulation con-ditions) may exchange rapidly across the large surface area making longitudinal i l e a l cells very sensitive to ionic changes in their i n t e r s t i t i a l space. There-fore these cells may serve as possible models for testing the hypothesis that excitation-contraction coupling in some types of smooth muscle i s regulated by trans-sarcolemmal fluxes of Ca rather than by release of Ca from intracellular storage sites. An advantage of the longitudinal layer of the ileum for i s o l a -tion of a sarcolemmal fraction i s that i t has less connective tissue than other types of smooth muscle and a relative lack of sarcoplasmic reticulum, mitochon-dria and lysosomes (Burnstock 1970). Therefore isolation of a sarcolemmal frac-tion, without major contamination by intracellular 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 critical.steps in preparing c e l l fractions for biochemical analysis i s homogenization. This routine step can be extremely d i f f i c u l t in tissues such as smooth muscles that are inter-woven with large amounts of connective tissue. The various procedures for homogenizing smooth muscle cells have been c r i t i c a l l y reviewed by Kidwai (1975). Although the Potter-Elvehjem homogenizer is 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 nuclei. All-glass homogenizers disrupt the tissue faster but glass particles may contaminate the homogenate. The Sorvall 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 intracellular organelles. The Polytron homogenizer, when used at carefully chosen speeds for short time intervals, may yield good recoveries of c e l l fractions while minimiz-ing, 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 isolation of intracellular organelles but may be inappropriate for isolation of sarco-lemmal enriched fractions because the surface membranes would also be susceptible to proteolytic damage. Membrane fractions contaminated by contractile proteins have higher non-specific protein contents. High ionic strength KC1 solutions solubilize and extract contractile proteins which i f not removed, gel the homo-genate and the resuspended pellets. Unfortunately, high ionic strength washing solutions have been observed to reduce the specific activities of certain mem-brane 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 in preliminary experiments a method was devised in 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 intercellular 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 limit lysozymal activity i f any lysozymes were released during the f i r s t 10 min divalent cation depletion. It was also hoped that softening of the intercellular cement would continue at 4°C while intracellular organelles would not be further damaged. The ease and extent of disruption by a Potter-Elvehiem homogenizer increased noticeably without any apparent increase in damage to intracellular organelles. At the same time the reduction of Ca and Mg levels prevented the gelling and aided the extraction of contractile proteins. The activity and characteristics of the sarcolemmal en-riched fraction that resulted after the use of this modified method w i l l be described in the Results- and Discussion. b. Common procedures for separation of subcellular fractions Once homogenized, the c e l l can be separated into three main fractions by differential centrifugation. The heaviest components (c e l l debris, connective tissue, nuclei and larger sheets of plasma membrane, depending on the homogeni-zation 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 vesicles of endoplasmic reticulum and plasma membrane. The supernatant remaining after centrifugation at these high speeds is composed of the contractile filaments and soluble cytoplasmic proteins that are not attached to any membranous material. It is obvious that there is consi-derable overlap in the c e l l components sedimented at various speeds. A finer separation of each fraction 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 yield has to be sacrificed to obtain greater purity because the borders of each band of membranes are not clearly defined. Even when separated on the basis of density, bands can contain more than one c e l l component since the banding characteristics also depend on 6 the size of the vesicle 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-cellular fractions can not be used to compensate for extremely destructive homogenization procedures (Kidwai 1975). c. Identification of subcellular fractions The purity of a fraction is d i f f i c u l t to determine, and subjective decisions about i t are usually made. The degree of purity should be established before major conclusions are made about the properties of the subcellular organelle under study. Organelles are easily identifiable in the intact c e l l but once broken, a l l types of membranes appear similar in the electron microscope. Intact isolated mitochondria are s t i l l easily discernible and, to some extent, so are endoplasmic reticulum membranes i f they retain their ribosomes. Smooth endo-plasmic reticulum, broken mitochondria, plasma membrane and Golgi apparatus look very similar. A fuzzy basement membrane may help to visually identify plasma membranes. Activities of enzymes specifically located on one type of c e l l membrane have been used to identify c e l l components in 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; Wei et a l . 1976; Janis and Daniel 1977). III. Studies of the Na,K-ATPase in Smooth Muscle The cytoplasm of smooth muscle cells (like that of most cells) normally has a higher concentration of Kand a lower concentration of Na than the extra-cellular fluid (Casteels 1970). The sarcolemma is 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 actively 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 intracellular Na or extracellular K seem to stimulate this enzymatic pump. Ouabain and K free medium lower the activity of most Na,K-ATPases (Hoffman 1962; Whittam and Ager 1964; Brinley and Mullins 1968). But ouabain and K free medium do not completely block Na efflux in smooth muscle cells (Daniel et a l . 1971c). Daniel et a l . (1971c), Allen and Daniel (1970) and Wolowyk et a l . (1971) have described a Na stimulated Mg-ATPase activity that is independent of K and insensitive to ouabain in rat myometrial and rabbit vascu-lar smooth muscle. This enzyme has been postulated to regulate intracellular Na and water content (Daniel and Robinson 1971a). In addition to having an extra sodium transport system, the rat uterus also has an ouabain sensitive Na,K-pump but according to Daniel and Robinson (1971a) i t s characteristics deviate from the accepted views about i t s operation. Descriptions of the ATPase activity in sarcolemmal fractions of the guinea pig ileum longitudinal smooth muscle by Godfraind and Verbeke (1973), Hurwitz et a l . (1973), Oliviera and Holzhacker (1974) and Godfraind et a l . (1976) gave no indication of any abnormal behavior of this enzyme in this smooth muscle. These studies do not clearly indicate whether the stimulation was due to Na alone or whether Na and K were required simultaneously. The extent of inhibition by ouabain of the Na and K stimulation was not evident from these studies. The nature of the ATPase activity stimulated by Mg, Na and K in the guinea pig ileum longitudinal smooth muscle and i t s sensitivity to ouabain were re-investigated in the present study. IV. Studies of Ca-ATPases in Smooth Muscle 2+ Filo 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 slightly greater than 10 ^ M. The ATPase activity associated -7 -5 2+ with the contractile filaments increased over the range of 10 to 10 M Ca 8 (Mrwa and Ruegg 1976). Since generally the intracellular Ca content would give a cytoplasmic concentration far 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 in various subcellular components of the c e l l (Batra and Daniel 1971; Somlyo et a l . 1974; Hess and Ford 1974). Energy in the form of ATP is required to concentrate the Ca in sarcoplasmic -reticulum and mito-chondria. Electron probe X-ray microanalysis has confirmed that the cytoplasmic Ca concentration i s extremely low in vascular smooth muscle and that Ca is con-centrated in mitochondria and sarcoplasmic reticulum (Somlyo et a l . 1976). Dur-ing excitation Ca flows into smooth muscle cells from surface binding sites and the extracellular 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 is relatively stable, the Ca gained during excitation must eventually be returned to the extracellular space. This might also require an active pump mechanism because the extracellular Ca concen-tration (approximately 2 mM) is greater than the free cytoplasmic levels (Van Breemen et a l . 1966). Recently, Janis et a l . (1977) provided the f i r s t direct evidence for a role for the plasma membrane as well as mitochondria and sarcoplasmic reticulum in the regulation of Ca activity of rat uterine smooth muscle. The authors suggest that plasma membrane and endoplasmic reticulum may act 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 concen-tration exceeds 10 ^  M. Ca transport by subcellular fractions from smooth muscle has recently been reviewed by Janis and Daniel (1977). V. Methods for Measuring Ion Movements in 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 is that excitation-contraction coupling in smooth muscles may be regulated by trans-sarcolemmal ion movements. a. Calculation of intracellular ion levels from total ion measurements and  flux studies There are many obstacles to the study of trans-sarcolemmal ion fluxes in smooth muscle (Daniel 1975; Van Breemen:. et a l . 1973). Some of these are outlined below. The extracellular space is large and the elastin, collagen and mucoproteins bind large quantities of ions, especially Ca. Ions dissociate from extracellular binding sites at varying rates, sometimes more slowly than their exchange rates from intracellular sites. Because of these and other reasons, .' ..<_..; tracer ion exchange curves plotted semilogarithmically can not be fit t e d unequi-vocally to a few straight lines from which f i r s t order rate constants can be calculated. The efflux rates are d i f f i c u l t to relate to losses of ion pools sequestered in c e l l organelles and bound to membranes. The quantity of Ca needed to activate a smooth muscle contraction is about 100 times less than the total amount of exchangeable Ca. Damaged ce l l s , on the surface of a dissected muscle strip, gain Ca and Na and lose K and Mg quite extensively. Depending on the pro-portion of cells damaged, the finer changes in these ion levels during responses to excitatory and inhibitory agents may be obscured. Intracellular ion content has often been calculated from the difference bet-ween total (measured by atomic absorption spectrophotometry) and extracellular amounts. The extracellular content was estimated from the product of the physio-logical salt solution concentration and the experimentally measured volume of the tissue extracellular space. As yet, no universally accepted method has been described for measuring the extracellular space (Paton 1975b). Small molecules may permeate the unit membrane, large molecules may be excluded from small inva-ginated regions of the c e l l and charges, dipole moments or hydrophobic areas of 10 the marker molecule may cause them to adhere to the plasma membrane, basement membrane or collagen. The accuracy of measuring the extracellular space volume is very uncertain and the scatter of values, using various marker substances, has been considerable (Goodford and Leach 1966; Setekleiv 1970). In addition, drugs may change the extracellular space volume e.g. by inducing contraction. Small and rapid variations of intracellular 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 sensitive but again, changes of the size of the extracellular space and also the ratio of the c e l l surface to volume could affect the results. The for-mer is d i f f i c u l t to monitor during muscle activity and the latter can hot be monitored (Setekleiv 1970; Daniel 1975). b. The 'La Method' A simple but sensitive method was needed to measure net changes in intra-cellular ion levels that would be independent of extracellular space changes and surface to volume ratios and would not require interpretation of flux kinetics. Most of the aforementioned obstacles, at least for measuring intracellular Ca 45 levels with Ca, can be circumvented by blocking Ca influx, inhibiting Ca efflux and displacing the extracellular bound Ca with La (Van Breemen and McNaughton 1970) . Approximate net intracellular 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 verification 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 latter 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 mitochondria and sarcoplasmic reticulum fractions isolated from LaCl- treated cells contained La. They also calculated that more La could bind to rat myometrial cells than could possibly tightly pack on their surface which, i f correct, would mean that 11 La gained access to the c e l l interior and perhaps could displace some intracellu-lar Ca. Using LaCl- rather than colloidal La as an extracellular stain for elec-tron microscopy, Langer and Frank (1972) did not observe La to penetrate the unit plasma membrane of cultured heart ce l l s . 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 the c e l l based on a two dimensional planar binding calculation. 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 in the cy-toplasm, except for a few spots which appeared similar in size to caveolae (ve-sicles opening into the extracellular space) which might have overlain the plane of the section. Casteels et a l . (1972) investigated the La concentrations re-quired to cause various effects and concluded that 10 mM LaCl- blocked trans-membrane calcium movements whereas the 2 mM LaCl-, as previously used in the 'La method', was not quite sufficient. Ca efflux was inhibited 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 cell s . Others feel that La does not block efflux effectively at 37°C (Freeman and Daniel 1973; V. Palaty, personal communication) but that 10 mM LaCl- in a washing solution at 4°C is more effective,(V. Palaty, personal communication). Ca efflux is reduced at low-er temperature (Goodford et a l . 1965). In fact a l l active transport activity would be greatly reduced at this temperature and passive leakages of ions across the membrane should also be reduced (Setekleiv 1970; Tomita 1970) especially with the additional.membrane stabilizing effect of 10 mM LaCl- (Weiss 1974). Daniel (1963a) and Setekleiv (1967) observed that K efflux was reduced at lower temper-atures (Q"LQ = 1-6 and 1.94, respectively) but Daniel (1963a) observed a tran-siently faster rate of K efflux 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 efflux was thought to have been due to a rapid loss of extracellular K during cold contracture. Cold contracture could be prevented by removal of extracellular Ca (Daniel 1964)... . The rate of K efflux was reduced by 55% in K free medium (Daniel 1963b). At 5°C Na efflux was very markedly reduced (Daniel and Robinson 1971b)., Mg efflux was shown to be depen-dent on metabolism and on exchange for extracellular Ca (Moawad and Daniel 1971). Alternatively Mg efflux may u t i l i z e the energy released in the course of Na influx (Palaty 1974). LaCl- (1 mM) did not reduce passive K efflux in tumor cells at 22°C but i t did inhibit Na efflux by 48% (Smith 1976). Although LaCl-stabilizes 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 cell s . If La does slowly permeate cells 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 Tris HC1 solution (pH 7.4) containing 10 mM LaCl- could be used to wash off extracellular ions while retaining most of the intracellular ions. These could be extracted from the muscle and measured by atomic absorption spectrophotometry. In summary, the general theories upon which the modified '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 stabilize the membrane and reduce passive fluxes, especially for Ca and Na 4) cold contracture w i l l be minimized in Ca free medium containing 10 mM LaCl-5) passive K efflux w i l l be further reduced in K free medium 6) loss of intracellular Mg w i l l be reduced by inhibition of metabolism at 4°C and by the absence of extracellular Ca and Na to exchange for i t 7) La w i l l displace extracellularly bound Ca and probably Mg and Na also 8) K is not tightly bound extracellularly and w i l l dissociate from i t s binding sites in K free medium 9) free extracellular Ca, Mg, Na and K w i l l diffuse out of the extracellular space in a large volume of isotonic Tris HC1 containing no added Ca, Mg, Na or K 10) La permeability is very low and w i l l be even less at 4°C and therefore i t should not displace intracellular ions. This modified .'La method' gave at least good qualitative comparisons of intracellular Ca, Na, K and Mg levels in resting muscles to these cation levels at various stages of muscle activity. Because of the uncertain validity of the 'La method', the results from the modified 'La method' should be accepted cau-tiously u n t i l a better method becomes available to check the validity 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 inhibiting the Na,K-ATPase and subsequently, af-fecting membrane ion transport (Hadju and Leonard 1959; Akera and Brody 1976; Schwartz 1976). However the details are by no means clear. Although ouabain inhibits 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 in cardiac intracellular K due to Na,K-ATPase inhibition was observed only after the positive inotropic response was completed (Tuttle et a l . 1961; Lee et a l . 1961; Steiness and Valentin 1976). Murthy et a l . (1974a) dissociated ouabain binding by the tissue from i t s contractile effect by demon-strating that the contraction of the rabbit myometrium was relaxed after washing for 10 min whereas binding persisted after 10 min. Tissue Na and K levels were not changed after a 10 min exposure to 5 x 10 ^ M ouabain when i t s potentiating effect on acetylcholine contractions was maximal. Ouabain increased K efflux from rat uteri, but contrary to i t s proposed mechanism, ouabain did not inhibit K influx (Daniel and Robinson 1971a). 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 in spike frequency and later a depolarization block, but the decrease in intracellular K and Cl and increase in intracellular Na are not sufficient to explain the changes in the membrane poten-t i a l (Casteels 1966). Reduction of the Ca concentration of the medium lowers the increase in 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 itttra-45 cellular uptake of Ca during the increase in tension to ouabain (Holland and Sekul 1961; Casteels and Raeymaker 1976) and the authors suggested that an i n -crease in membrane permeability to Ca may be the primary effect of ouabain. In the present study additional data are presented indicating .that ouabain may cause contraction of the guinea pig ileum longitudinal smooth muscle by increasing the membrane permeability to Na and Ca before inhibiting the Na,K-ATPase. VII. Excitation-Contraction Coupling (General) Authors discussing smooth muscle excitation contraction coupling almost i n -variably try to draw comparisons to skeletal and cardiac muscle because so much more is known about these cel l s . Ironically each comparison accentuates their differences and althoiigh_all muscle cells produce contraction, the modes by which they can affect contraction seem to bervery different. Skeletal and cardiac muscle cells have specialized continuations of their plasma membranes, called transverse tubules, which conduct depolarizations rapidly into the c e l l interior. When the depolarization arrives at the locus of the terminal cisternae of the sarcoplasmic reticulum, Ca i s released from them to the contractile filaments (Porter and Palade 1957; Huxley and Taylor 1958; Katz 1970; Langer 1968). The sarcoplasmic reticulum volume is greater in skeletal than in cardiac muscle cel l s . Skeletal musclencells retain their a b i l i t y to contract during long periods in Ca-free a r t i f i c i a l extracellular f l u i d . In contrast, cardiac cells are much more dependent on extracellular Ca. Although they have a sarcotubular system and are richly supplied with mitochondria, Ca stored in these sites does not seem to be releasable from them in the absence of extracellular Ca (Langer 1976). Smooth muscle cells appear to have the least specialized intracellular organization of the muscle c e l l class. They are smaller (6-10 u in diameter or 1/3 - 1/30 the diameter of a skeletal muscle fibre, Gabella 1971) and devoid of transverse tubules. Recently Devine et a l . (1972) and Gabella (1973) have indicated that the sarcoplasmic reticulum is more abundant than earlier reports indicated (Burnstock 1970). Small sacs and tubules were not recognized as being sarco-plasmic reticulum because their form and organization was so unlike that of ske-l e t a l muscle. 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 toN be direct evidence that strontium and calcium can be released from them during contraction. Indirect evidence for Ca release from isolated skeletal muscle sarcoplasmic reticulum vesicles, either by reversal of the Ca pump, stimulation by "trigger calcium"', depolarization or the application of ionophores and caffeine has been described by Inesi and Malan (1976) but only the direct e l e c t r i c a l effects on the sarcoplasmic reticulum ve-sicles seemed to occur under physiological conditions. Mitochondria constitute approximately 6.6% of the smooth muscle c e l l volume (Gabella 1973) and sarco-plasmic reticulum comprises 2 - 7.5% of the cytoplasm (Somlyo and Somlyo 1976). The volume of sarcoplasmic reticulum is greater in the tonically contracting large elastic arteries than in the phasic spike generating smo.oth muscles such as taenia c o l i and portal anterior mesenteric vein (Somlyo and Somlyo 1975). Smooth muscle cells contract much more slowly than skeletal muscles, which tends to re-duce any obligatory requirement for intracellular Ca. Indeed smooth muscle cells are highly dependent on extracellular Ca and their degree of dependence seems to be related to their volume of sarcoplasmic reticulum (Devine et a l . 1972). Ironically the faster contracting (phasic) smooth muscles seem to have the least volume of sarcoplasmic reticulum. Smooth muscle cells have an abundance of sur-face vesicles, called caveolae, which are not characteristic of most other muscle cell-types (Burnstock 1970). However, large numbers of caveolae were found in rat a t r i a l and lizard ventricular cells but only in those cells which lacked a transverse tubule system (Forssman and Girardier 1970; Forbes and Sperelakis 1971). These vesicles increase the overall surface area by more than 70% in guinea pig taenia c o l i cells (Goodford 1970) and by 25% in longitudinal smooth muscle of the mouse intestine,(Rhodin 1962). It should be noted that much of the sarcoplasmic reticulum is located near the smooth muscle c e l l surface, close-ly associated to caveolae (Gabella 1973). The surface to volume ratio of these cells is very large and therefore smooth muscle cells could possibly use Ca from superficial sites for at least part of their contractile cycle. Upon stimulation, smooth muscle cells appear to u t i l i z e Ca rather than Na as the depolarizing i n -ward current carrier. Their e l e c t r i c a l spikes seem to be insensitive to the extra-cellular Na concentration (Holman 1958; Sakamoto 1971), very sensitive to extra-cellular Ca levels (Holman 1958, Bulbring and Kuriyama 1963, Tomita 1970), inhi-2+ bited by Mn (Brading et a l . 1969), Nonamura et a l . 1966, Bulbring and Tomita 1969) and insensitive to tetrodotoxin, a specific blocker of the Na channel (Tomita 1970, Nonamura et a l . 1966). This Ca current may contribute some Ca for contraction (Goodford 1970; Lullman and Mohn 1969; Collins et a l . 1972). Depo-larization or Ca entering the c e l l during depolarization may 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) is usually attributed to sarco-plasmic reticular and mitochondrial re-sequestration of Ca. Ca gained from ex-ternal sources may be extruded immediately to cause relaxation or i t may be temporarily buffered by mitochondria and sarcoplasmic reticulum (Hess and Ford 1974; Devine et a l . 1973; Vallieres et a l . 1975; Janis et a l . 1977; Huddart and Price 1976) from where i t may be extruded from the c e l l , by some hitherto unknown mechanism, to maintain a f a i r l y constant intracellular level of Ca. There may be a passive exchange of intracellular Ca for extracellular Na u t i l i z i n g the ener-gy of the Na gradient which in turn must be actively maintained (Bohr et a l . 1969; Reuter et a l . 1973). An alternative and more direct means would be a sarco-lemmal ATP requiring active transport system to prevent an intracellular build up of Ca (Fitzpatrick et a l . 1972; Casteels et a l . 1973b; Janis et al . 1977). Kirkpatrick et a l . (1975) have pointed out that the agonists used in such stu-dies may produce effects in unusual ways under certain circumstances but these may not be directly related to the mechanism involved in 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 ratio of the sizes of i t s internal and external Ca pools (Chang and Triggle 1972). Each is variably sensitive to Ca removal from the physiological medium indicating that their sarcoplasmic reticulum volume (Devine et a l . 1972), surface to volume ratio (Bianchi 1969) and Ca affinity at storage sites (McGuffee and Bagby 1976) may be unique for each type of smooth muscle. For the above reasons, the experimen-t a l results obtained in this study have been most often compared to previously reported characteristics of guinea pig ileum longitudinal smooth muscle and to the characteristics of guinea pig taenia c o l i cells which are f a i r l y similar in origin and nature. VIII. Sources of Calcium for Excitation-Contraction Coupling i n the Guinea Pig  Ileum Longitudinal Smooth Muscle Those studying the longitudinal smooth muscle of the guinea pig ileum often imply that i t sequesters activator calcium in a manner quite different from ske-l e t a l and cardiac muscle cells (Hurwitz and Joiner 1969). Perhaps this smooth muscle does not need to sequester as much Ca intracellularly because, taking the differences of c e l l volume in consideration, a 'volume-unit' of the guinea pig ileum longitudinal smooth muscle takes up 65 times more Ca per stimulus than a 'volume-unit' of skeletal muscle (Lullman and Mohn 1969). Lullman and Siegfriedt (1968) (through Lullman 1970) described three fractions of ^Ca efflux from guinea pig ileum longitudinal smooth muscle with half-times of 1 min (Fraction 1, 34% of 18 total), 4.5 mln (Fraction 2, 50% of total) and 25 min (Fraction 3, 16% of total) when the extracellular Ca concentration was 1.8 mM. Fraction 1 was designated as the Ca in the extracellular f l u i d . Weiss (1972) found only two clearly deline-45 ated components of Ca efflux. The fast component had a half-time of 1 min and was attributed to both free extracellular Ca and superficially bound Ca. The slow component had a half-time of 9 min and contained superficially bound Ca and tightly bound Ca. The presence of superficially bound Ca in both the fast and slow components was identified by the shifting of part of the slow compartment to the fast component, probably by exchange at membrane sites, when nonradioactive Ca was added. After the extracellular Ca was washed off (5 min) Hurwitz and Joiner (1969) observed that Ca migrated into the extracellular f l u i d from two compartments, similar to fast Fraction 2 and slow Fraction 3 described by Lullman and Siegfriedt (1968). Hurwitz and Joiner (1969) reported that the plots of the reciprocal of the contractile response as a function of the reciprocal of the ex-tracellular Ca concentration or as a function of the reciprocal of the Ca in the fast component were linear and had the same intercept, denoting the same, maximum response. The linear relationship between Ca in the fast compartment and the extracellular space was not compatible with the concept that calcium ions in the bathing medium equilibrate with activator ions bound to some saturable group of lo c i within the fibre. Hurwitz and Joiner (1969) theorized that "extracellular calcium equilibrates with activator ions that are in solution in some biophase of the muscle fibre, perhaps the fibre membrane." The contraction of the longitudinal ileum to muscarinic agents and high KC1 has a biphasic appearance. There i s a fast (phasic) contraction followed by a slower tonic maintenance of tension. Ca for the phasic and tonic components, in-duced by different stimuli, may not be supplied by the same Ca pool. The Ca mobilized by a muscarinic stimulus was studied by Chang and Triggle (1972 and 1973) (Fig. 1). They equilibrated the guinea pig ileum longitudinal smooth mus-cle with 1.8 mM CaCl ? 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, cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide (CD). The phasic component remained f a i r l y constant but the tonic component decreased markedly when the Ca concentration was decreased (experi-ment a). 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 equilibrating Ca concentration (experiment b). If the equilibrating Ca concentration was held constant at a low concentration of 0.1 mM and the stimulus Ca level 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 de-pending on the extracellular Ca concentration used to equilibrate the muscle (experiment a). This pool slowly equilibrates to a lower capacity at low levels of Ca (experiment b) but can be rapidly reloaded when the stimulating level of Ca is raised (experiment c). Because the phasic component reloads almost instan-taneously and i s inhibited 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 likely responsible for the phasic component. The magnitude of the tonic component was directly related to the free Ca concentration of the extracellular space (experiment a, b and c). The magnitude of the high KC1 induced tonic contraction also increased hyper-bolically with increasing Ca concentrations in the external medium (Hurwitz and Suria 1971). The phasic and tonic components appear to be dependent on two dis-tinctly separate Ca pools but there i s rapid exchange of Ca between these two pools. This hypothesis deserves further investigation. Fig. 1. Superimposed tracings of time courses of contractions showing the effect of different Ca concentrations for equilibration [Ca„„m]r and stimulus [Ca _,] during excitation by i i iAl i- | | _A1 b CD (2 x 10~ M). The muscle was equilibrat'ed i n [Ca_v_,]_, for 30 min. At the time of the introduction of the agonist (CD) the Ca concentration was abruptly altered to [C a _xT_S" (reproduced from Chang and Triggle 1972). 21 IX. Receptor Theory and the Desensitization Phenomenon Chang and Triggle (1972) have proposed that the acetylcholine receptor exists in a Ca-associated state which, when combined with an agonist, propels the associated Ca into the c e l l to generate the phasic component. Their model is a modification of the model proposed by Hurwitz and Suria (1971). The sub-sequent Ca-dissociated active state can be permeated by free extracellular Ca to generate the tonic component. By their calculations, only a small Nfraction of the number of Ca ions bound to sites on the relatively large surface of each longitudinal i l e a l fibre would, i f shunted inwards, be sufficient to raise the Ca concentration of the cytoplasm to maximum contractile levels. Perhaps this small fraction is associated with specific receptor sites. A transformation of the receptor 'active' state to a 'desensitized' state was 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 contractile responsiveness following a maximal contraction of i n -testinal muscle may not be a specific receptor desensitization because there is 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 is washed out in a medium containing a higher K concentration, whether or not the K concentration is sufficient to cause a contraction (Cantoni and Eastman 1946). Born and Bulbring (1956) observed that acetylcholine caused a rapid efflux of K from taenia c o l i cells. Several mechanisms for muscle desensitization have been proposed but desensitization is s t i l l not precisely understood. Cantoni and Eastman (1946) and Rand (1956) proposed that the responsiveness of a muscle is determined in part by the [K. ]/[Ca and [K. ]/[K 1 ratios respectively. J xn out xn out > Paton and Rothschild (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 in 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. Specifically, the aims of the study were to: 1) devise a method to fractionate smooth muscle cells without using harsh, denatur-ing procedures thereby minimizing damage to intracellular organelles and mini-mizing changes in enzyme characteristics. 2) prepare a sarcolemmal enriched fraction. 3) ascertain whether there is a Na,K-ATPase in the guinea pig ileum longitudinal smooth muscle that conforms to characteristics described from studies of other cells. 4) determine i f there is a Ca-ATPase in the sarcolemma which might be capable of transporting Ca. 5) explore, by electron microscopy, the various cellular depots where Ca could be stored and from which Ca could be mobilized during excitation-contraction coupling. 6) devise a method for measuring net intracellular changes in Na, K, Mg and Ca during various stages of activity of muscle strips. 7) investigate the mechanism of action of ouabain by measuring i t s effect on tension and ion movements. 8) study the contractile activity in response to muscarinic and high K stimu-lations under various conditions to determine the ionic movements responsible for the biphasic contraction. 9) investigate the ion movements which might be responsible.for muscle desensi-tization. 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. The segments were equilibrated in normal Tyrode's solution for 15 min at 37°C. The longitudinal layer was removed essentially by the method of Rang (1964). Each ileum segment (approximately 15 cm long) was drawn over a pipet. A blunt scalpel was run along both sides of the mesentery and the outer layer was brushed off with a cotton swab while the ileum was kept moist in a trough containing Tyrode's solution at 37°C (see Fig. 2, innervated). For a few ex-periments, the dissection was altered to eliminate Auerbach's nerve plexus by the method of Paton and Zar (1965) (see Fig. 2, denervated). The tissues were tested for the presence of the nerve plexus by checking their responsiveness to nicotine and neostigmine (see Results Fig. 22). II. Contractile Force Measurements a. Isometric force measurements Muscle strips (1-2 cm long) were tied to stainless steel tissue .hooksrand attached to Grass FT.03C transducers in 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 CaCl 2, 1 mM MgCl 2, 0.36 mM NaH-P04, 11.9 mM NaHCO- plus 5.55 mM glucose (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 CaCl 2 or CaCl 2 and MgCl 2 were omitted. Drug solutions were add-ed to the baths in small volumes (less than 0.2 ml) with a Hamilton fixed needle syringe. 24 Fig. 2. Dissection of the guinea pig ileum longitudinal smooth muscle (inner-vated and denervated muscle strips). 1. Strips of ileum (10-15 cm long) were equilibrated for 10-15 min at 37°C in Tyrode's solution to attain muscle tone. 2. Each strip was drawn over a pipet (0.1 ml) in a 37°C jacketed trough f i l l e d with Tyrode's solution. A blunt scalpel was drawn along either side of the mesenteric attachments. 3. The scalpel was intended to cut just through the longitudinal layer but i t also penetrates the Auerbach's nerve plexus and may parti-ally cut into the circular layer. Very l i t t l e pressure should be applied to the blunt scalpel. A l i t t l e of the circular layer usually adheres to the longitudinal muscle strip during the r o l l -ing off procedure. Innervated Denervated A cotton swab was used to r o l l ' back the longitudinal layer and the adhering nerve plexus and a few circular muscle cells. A cotton-swab was used to loosen one end of the longi-tudinal layer and nerve plexus and r o l l i t to the other side of the ileum. The longitudinal layer was rolled (like a scroll) to the side of the ileum opposite the mesenteric attachments. The loosened portion was tied with a piece of surgi-cal thread. One end of the rolled longi-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 in another water jac-keted tissue bath as in part 1 until tied to tissue hooks. The longitudinal layer was held steady(not drawn back-wards) by the surgical thread and the circular layer was drawn forward over the p i -pet. The shearing force broke the nerve plexus leav-ing i t attached to the circu-lar layer and the latter portion of the longitudinal layer was free of the nerve plexus. The longitudinal layer was placed in 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 fi n a l concentration of KC1 to 60 mM. c. Tris-Tyrode's Solution For experiments that measured the inhibition of contraction by La or the intracellular ion content by the modified 'La method', the phosphate and carbon-ate were omitted from the Tyrode's solution. The resulting solution was buffered with 24 mM Tris adjusted to pH 7.4 with HC1 at 37°C and then oxygenated with 100% 0^ to prevent lanthanum precipitation. d. 'Glycerol shock! treatment Glycerol (400 mM) in physiological solution has been shown to cause dis-ruption of transverse tubular membranes and loss of excitation-contraction coup-ling in skeletal muscle, upon returning the muscle to normal physiological solu-tion (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 extracellular stain by electron microscopy) (Franzini-Armstrong et  al . 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 latter. In cardiac muscle, loss of twitch contracti-l i t y after this treatment was associated with a significant decrease in the ex-tracellular space, an increase in c e l l water, a decrease in c e l l K and an i n -crease in c e l l Ca (Katzung and Teitelbaum 1974). Frank and Hemker (1976) found that the washout of 800 mM glycerol in Krebs-Ringer solution caused a period of contracture in rat hearts and they speculated that glycerol treatment caused unphysiological Ca fluxes. 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 in muscles other than skeletal muscles. This does not appear to be a widely used procedure in smooth muscle studies. 'Glycerol shock' was used in the present study to see whether caveolae invagina-tions 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 inclusion of 400 mM glycerol. 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 for their response to CD and high KC1. One hr treatments and washes are required to disrupt 90% of the triads (structurally distinct regions where transverse tubules and terminal cisternae of the sarco-plasmic reticulum come together) of skeletal muscle (Franzini-Armstrong et. al.1973) A time of 30 min was f e l t to be sufficient i h longitudinal i l e a l muscle strips, since these are much thinner. ' " ' III. Measurement of Muscle Na, K, Mg and Ca Tissues were equilibrated for 30 min before any control or experimental ion levels were determined. a. Total ion measurement For the measurement of total ion content in control tissues.the tissues were equilibrated, blotted, dried and weighed. Ions were extracted from the t i s -sues as described below. For measurement of total ion 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. Ions were extracted as described below. b. Modified 'La method' for measuring essentially intracellular Na, K, Mg  and Ca Intracellular ion contents were determined by a modification of the 'La method' (Van Breemen and McNaughton 1970). Muscles used for determining control intracellular ion levels were equilibrated and then were quickly released from the transducers and plunged within 2 to 3 sec into 200 ml of ice cold 10 mM LaCl^ and 160 mM Tris-HCl solution, pH 7.4 (La-Tris solution). For determining ex-perimental intracellular 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 surgical thread that had connected them to the transducers) to 800 ml of La-Tris solution in an ice bath for a total time of 30 min. Tissues were blot-ted, dried and weighed. Ions were extracted from the muscles as described below. c. Extraction of ions from tissues (total and intracellular ion measure- ments) and atomic absorption measurements For total and intracellular ion measurements the muscles were blotted bet-ween f i l t e r papers and then dried in preweighed 2 ml volumetric flasks for 3 hr at 120°C. 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 in 0.2 ml of a 1:1 mixture of glacial acetic acid and 3 M TCA (Sparrow and Johnstone 1964) in a boiling 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 in 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 ioniza-tion) to measure Ca and Mg (final volume = 2 ml). Aliquots (0.4 ml) of the de-proteinized extracted muscle solutions were diluted with 3.6 ml of IL-O to mea-sure Na and K (final 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 re-agents but without the muscle were subtracted from each reading (see Table 1 for description of standards and calculations). Samples were analyzed in a Varian IA3LE 1 . Atomic absorption standards and c a l c u l a t i o n s f o r ug element i n tissue samtiles preparation of 500 ml of standard s o l u t i o n samples and d i l u t i o n s spectrometry c a l c u l a t i o n s element standard 3' a c e t i c a c i d : LaCl KC1 f r a c t i o n f i n a l expanded absorptivity* 3 . c a l c u l a t i o n c. d. average and stock' TCA - of i n i t i a l sample absorbance value f a c t o r blank concentration s o l u t i o n 2 ml sample volume scale («) (ml) (ml) (nig) (g) (ml) set t i n g (ml/ug) (ug) Ca 0.5 25 88.3 4.77 1/2 (1 ml) 2 . 1,0 1 . 4 0.26 - .014 1 ug/ml - n = 41 Mg 0.3 25 88.3 4.77 1/2 {1 ml) 2 0.6 1 4 0.098± .008 0. 6 ug/ml. n = 33 Na 1.0 5 18 1/5 (.4 ml) 4 1.0 0.5 40 1.12 - .156 2 ug/ml ' n = 42 K 0.75 5 18 - 1/5 (.4 ml) 4 0.5 0.333 60 1.96 - .156 1.5 ug/nl . n = 43 a . .AA standards were purchased from Fisher and contained 1 mg/ml of the iilement. Small a l i q u o t s of these w e r e used to p r e p a r e accurate-standards i n the ug/ml range of the samples. b. . expanded absorbance scale s e t t i n g a b s o r p t i v i t y = — c : r a , . 1 concentration of STD (a) c a l c u l a t i o n f a c t o r ( a ) ( f r a c t i o n c f i n i t i a l 2-ml) d. blank (ug) = (absorbance of sample with no t i s s u e ) ( f a c t o r ) , . , , , _ (absorbance of t i s s u e sample) i'factor) - blank e. ug element i n t i s s u e sample/mg dry wt. = : : — c — — -e v 6 ' t i s s u e dry wt. 30 Atomic Absorption Spectrophotometer (AA-5) under the following conditions. element wavelength (nm) lamp current (mAmp.) s l i t width CM) flame Ca 422.9 4 100 N-0-acetylene Mg 285.2 4 95 a i r -acetylene Na 589.0 5 95 ai r -acetylene K 766.5 5 100 a i r -acetylene IV. Preparation of Sarcolemmal Enriched Microsomes The longitudinal layer of the ileum was dissected as described in Methods (section 1, innervated). Long muscle strips 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 for 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 ice cold 0.25 M sucrose (adjusted to pH 7.4 with histidine) for 10 min, minced well with scissors in 20 volumes (by tissue wet weight) of 0.25 M cold sucrose and homogenized with 10^12 strokes at 300 rpm (Fisher Dyna-Mix) in a Potter-Elvehjem homogenizer with teflon pestle (.004-.006 inch clearance) in 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 pellets were resuspended in 5 volumes by tissue wet weight of cold sucrose (0.25 M) in a Potter-Elvehjem homogenizer (150 rpm, 2-3 strokes). V. Measurement of ATPases and Membrane Marker Enzyme Activities a. Mg dependent Na,K-ATPase was measured in 0.6 ml of a medium containing 25 mM histidine (pH 7.4), 0.1 mM EGTA, 3 mM MgCl- and 15-25 pg of microsomal protein (0.05 ml of c e l l fraction). NaCl (100 mM), 3 mM KC1 and 3 mM ouabain were added according to whether Na stimulation, K stimulation or ouabain inhibi-31 ticm were being studied. Assay tubes (polypropylene Kimble centrifuge tubes, 16 x 98 mm) were preincubated with shaking for 5 min at 37°C (Dubnoff Metabolic Shaking Incubator). The reaction was started with 3 mM ATP and was stopped a f t e r 5 min with 0.2 ml of 20% TCA. The tubes were centrifuged at 4°.C at 10,000 x g (IEC B20A centrifuge) and 0.5 ml aliquots were assayed f o r inorganic phosphate by the method of Lowry and Lopez (1949) (see Methods section VI). b. Microsomal Ca-ATPase was assayed i n 0.6 ml of 100 mM Tris-HCl buffer —6 —3 (pH 7.4) containing CaCl^ (10 - 10 M). Some assays also contained 3 mM ii MgCl 2 or 0.1 mM EGTA as indicated. Microsomal protein (15 - 25 ug) was incubated for 5 min at 37°C and the reaction was started with ATP (usually 3 mM unless the e f f e c t of varying the substrate concentration was being studied) and stopped a f t e r 5 min (unless otherwise stated) with 0.2 ml of 20% TCA. Protein was removed by ce n t r i f u g a t i o n and P_. was assayed by the method of Lowry and Lopez (1949) (see 2+ Methods section VI). Free Ca concentrations were calculated according to the method of Katz et a l . (1970) (see Appendix). c. Actomyosin Ca-ATPase was assayed i n 0.6 ml of a medium containing 50 mM h i s t i d i n e (pH 7.6), 425 mM KC1, 4 mM CaCl~, and approximately 70 yg of soluble f r a c t i o n protein (0.05 ml of 105,000 x g supernatant) (Wolowyk et a l . 1971). After a 10 min preincubation at 37°C the reaction was started with ATP (concen-t r a t i o n given i n legends) and stopped a f t e r 10 min with 0.2 ml of 20% TCA. Pro-t e i n was removed by ce n t r i f u g a t i o n and P was determined by the Lowry and Lopez method (1949) (see Methods section VI). d. 5'-Nucleotidase was assayed e s s e n t i a l l y by the method, of Tanaka et a l . (1973) i n 50 mM Tris-HCl (pH 8.6), 2 mM MgCl-. and 2 mM AMP. Protein (20 - 100 y g) (depending on the c e l l fraction) was s o l u b i l i z e d with 3% T r i t o n X-100 for 10 min before adding i t to the assay medium ( f i n a l concentration of T r i t o n X-100 was ,0.5%). This amount of T r i t o n X-100 i s i n excess over p r o t e i n for every f r a c t i o n . The medium, i n a f i n a l volume of 0.6 ml, was incubated at 37°C for 1 hr and the reaction was stopped with 0.2 ml of 20% TCA. The tubes were centrifuged at 32 10,000 x g f o r 10 min and 0.5 ml aliquots were assayed for P_^  by the method of Lowry and Lopez (1949) (see Methods section VI). e. Acetylcholinesterase was determined by the col o r i m e t r i c method of Ellman et a l . (1961). Subcellular f r a c t i o n s (20 — 100 pg of protein) were assayed at 25°C i n 3 ml of 0.1 M Tris-HCl (pH 8.0), 0.4 M NaCl, 0.23 mM 5,5'dithiobis-2-nitrobenzoic acid (DTNB) and 1 mM ac e t y l t h i o c h o l i n e iodide, and read at 412 nm on a double beam spectrophotometer f i t t e d with a water-jacketed temperature-regulated cuvette chamber. Results were recorded on a Perkin Elmer chart record-er. Blanks did not contain DTNB or enzyme. A substrate blank (DTNB plus a c e t y l -thiocholine but no enzyme) was subtracted from each reading to account for spon-taneous hydrolysis of the substrate. f. NADH Oxidase was determined by the method of Sottocasa et a l . (1967) by following the reduction of fe r r i c y a n i d e at 420 nm with time i n a Hitachi-Perkin Elmer double beam spectrophotometer. The assay medium contained 0.5 mM KCN, 20 mM potassium phosphate bu f f e r (pH 7.4), 5 uM CaCl„, 0.5 mM NADH, 1 mM KFe (CN,) , and 40-200 Mg of enzyme was assayed at 25°C i n a f i n a l volume of 3 ml. A blank containing NADH and KFe(CN^) but no enzyme was subtracted from each reading. The reaction was started with enzyme. g. Succinic Dehydrogenase was determined e s s e n t i a l l y as described by Schoner et a l . (1967) by following the reduction of 2,6-dichlorophenol-indophenol (0.37 M) at 578 nm. The reaction medium also contained 0.01 M potassium phos-phate (pH 7.4), 3.75 uM Ca C l 2 , 0.01 M KCN, 0.01 M sodium succinate and 80-100 yg of protein i n a 3 ml volume at 25°C. Intact mitochondria have strong permeabi-l i t y b a r r i e r s to dyes but the i n c l u s i o n of Ca, at low concentrations, permits free permeation of dyes (Bernath and Singer 1962). The reaction was started with enzyme so that the enzyme would be exposed to the prot e c t i v e e f f e c t of succinate and would not be i n h i b i t e d by pre-exposure to KCN. The s p e c i f i c a c t i v i t y was calculated on the basis of the i n i t i a l (0-2 min) reaction 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 after termination of the reaction, was diluted 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 in 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 after 30 min at 660 nm in a Hitachi-Perkin Elmer double beam spectrophotometer. Standards were prepared in 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 (usually 0 absorbance) were subtracted from each reading. 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. Cell fractions (0.05 to 0.1 ml) (whole homogenates and the 2,700 x g pellet 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% NaoC0„ (50 ml) and 0.5% CuSO. (0.5 ml) and 1% sodium, potassium tartrate I 3 4 (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 in Tyrode's solution. The longitudinal layer was removed as usual and equilibrated for 10 min in Tyrode's solution. The strips were cut into tiny pieces (approximately 1mmJ) and fixed in 10 ml of 2% glutar-aldehyde in 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 post-fixed in 1% OsO^ in Tyrode's solution for 2 hr. They were again washed 5 times during 5 min with Tyrode's solution. Samples were dehydrated in 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 in 100% ethanol. Inf i l t r a t i o n was commenced in a 1:1 (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 for 7 hr under vacuum. b. Electron microscopy - microsomal and mitochondrial pellets Cell fractions were prepared as usual except that histidine was omitted from the isotonic sucrose homogenization medium. Pellets were loosened from the bot-tom of the centrifuge tubes with a Pasteur pipet which was sealed and slightly hooked at one end in a Bunsen burner. The pellets were broken into a few small pieces. The pieces were transferred to small fixation bottles and fixed at 4°C with 1% glutaraldehyde in 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^ in 0.25 M sucrose for 2 hr. The samples were washed twice with isotonic sucrose prior to dehydration in 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 vinylcyclo-hexane dioxide (10 g), diglycidyl ether of propylene glycol (6 g), nonenyl succinic anhydride (26 g) and dimethylaminoethanol (0;4 g). Embedding was com-pleted at 60°C under 5 p of Hg vacuum in a Marsh Instrument Company oven. c. Sectioning and staining Copper grids were coated with parlodian film and then lightly coated with carbon. The parlodian film was dissolved in acetone prior to picking up the sections. Sections (60 - 100 nm thick) were cut with freshly prepared glass knives, on a Sorvall 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 citrate for 10 min and again washed for 10 min. Electron micrographs were obtained on a Philips 75 or a Zeiss EM-10 elec-tron microscope. Some thicker sections (2 u thick) were cut with the microtome, mounted on glass slides and stained with 1% toluidine blue for 5 min to be viewed under a light microscope. / 36 MATERIALS The chemicals were purchased from the f o l l o w i n g companies: Sigma: ATP (disodium s a l t ) , 5'-AMP, NADH, bovine serum albumin, ouabain, EGTA, DTNB, h i s t i d i n e ( f r e e base), Trizma base, carba-c h o l , methacholine and neostigmine. F i s h e r : LaCl-,, sucrose, atomic a b s o r p t i o n standards, KCN, sodium a c e t a t e , dextrose, phenol reagent, sodium, potassium t a r t r a t e , l e a d c i t r a t e . Baker a. MgC^. sodium s u c c i n a t e , KC1. M a l l i n c k r o d t : CaCl-,. ( NaCl, TCA, ammonium molybdate, p a r l o d i a n . Baker and Adamson: KH-PO^, u r a n y l a c e t a t e , concentrated HC1. B r i t i s h Drug House: n i c o t i n e , a s c o r b i c a c i d , NaHCO-., g l y c e r i n , KFe(CN)g, CuS0^.5H-0. Matheson and B e l l : 2 ,6-dichlorop.henojj_ndS^ . C - I - L : g l a c i a l a c e t i c a c i d . E l e c t r o n Microscopy Science: v i n y l -cyclohexane d i o x i d e , d i g l y c i d y l ether of propylene g l y c o l , dimethylaminoethanol, OsO^. Tousimis Research i n c o r p o r a t i o n : g l u t a r a l d e h y d e . Rohn and Haas Co.: Triton-X-100. cis-2-methy1-4-dimethylaminomethy1-1,3-dioxolane methiodide was a g i f t from Dr.'D.J. T r i g g l e , State U n i v e r s i t y of New York 32 at B u f f a l o . ( y - P) ATP was a g i f t from Dr. S. Katz, U n i v e r s i t y of B r i t i s h Columbia. S o l u t i o n s were prepared i n a l l g l a s s d i s t i l l e d H-0 (Corning AG-3 d i s t i l l i n g apparatus f i t t e d with a Barnstead Sybron d e m i n e r a l i z e r ) . Glassware and polypropylene tubes used to assay or st o r e s o l u t i o n s used f o r experiments concerned with ion content were washed with chromic a c i d , tap water, 0.1% EDTA s o l u t i o n and d i s t i l l e d water. RESULTS AND DISCUSSION I. Procedure for Isolating Sarcolemmal Enriched Microsomes A number of procedures for the preparation of sarcolemmal enriched frac-tions were examined before the f i n a l procedure (described in the methods) was developed. In the procedure to isolate sarcolemmal enriched microsomes from guinea pig ileum longitudinal smooth muscles described by Hurwitz et a l . (1973), the excised tissue was directly homogenized in 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 yield of micro-somes seemed very low and the resuspended pellet invariably formed a gel. Sub-stituting 0.65 M KC1 solution for the sucrose solution prevented the gelling but did not aid the homogenization. The above procedure yielded a fraction with considerable ATPase activity when stimulated by Mg, but Na and K did not notice-ably further stimulate the activity. The appropriateness of homogenizing the tissue in deoxycholate (method of Godfraind and Verbeke 1973) seemed question-able since intracellular organelles might pellet at any centrifugal force depending on the extent to which they were solubilized. A low speed sarcolemmal . fraction prepared by the method of Oliviera 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 Sorvall Omni-mixer ($6 r v a l i Omni-mixer was substituted for a V i r t i s homogenizer) in a medium of 0.25 M sucrose, 1 mM EDTA and 20 mM Tris-maleate (pH 7.4), then fi l t e r e d and centrifuged at 650 x g. The pellet 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 in the i n i t i a l buffer system to extract 'cytoplasmic inclusions'. The material was washed and centrifuged at 1,000 x g, 6 to 7 times more in 0.01 mM EDTA solution to extract actomyosin. This preparation had virtually no ATPase activity even when the number of washing procedures was reduced, A pilot study using a pyrophosphate buffer to chelate divalent ions, thereby softening the cohesive material between ce l l s , seemed to increase the extent of disruption of the tissue by the Potter-Elvehjem homogenizer. Equilibration of the tissue in Ca,Mg-free Tyrode's solution for 10 min at 37°C and 3 hr and 50 min at 4°C (shorter times are also effective) without the addition of any chelating agents (the method which was chosen) was observed to have the same effect. 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 into the Ca,Mg-free Tyrode's solution. Connective tissue proteins and contractile pro-teins were assumed to be extracted from the tissues that were washed in Ca,Mg-free Tyrode's solution because the resuspended pellets did not gel and the pro-tein recovery in the microsomal fraction was very low (1%) (Table 2), although the specific activities of the microsomal enzymes were high. The effect of incubating the tissue in Ca,Mg-free Tyrode's solution on total ion content was determined. Following dissection of the tissues and equilibration for 30 min in normal Tyrode's solution, the total Ca, Mg, Na and K contents were determined after incubation of the tissues in Ca,Mg-free Tyrode's solution for 10 min at 37°C and then after various times at 4°C (Fig. 3, solid lines). The tissues lost 65% of their Ca content, 27% of their Mg content, 78% of their K content and gained 23% more Na after 4 hr, indicating 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 their i n i t i a l amounts respectively and washed away 53% of the extracellular Na (Fig. 3, dotted li n e ) . At this point, the fragile cells s t i l l held together as a tissue but they were very flaccid and about 4 strokes with a Potter-Elvehjem homogenizer easily dispersed most of the cellular material into the medium. Homogenization was continued for about 10 more strokes to increase the extent of disruption. Electron micrographs of the 27,000 x g pellet confirmed that the majority of the mitochondria were l e f t 39 intact although they were swollen and mostly in the condensed configuration (Somlyo et a l . 1975) (see plate 1, #1, 2 and 3). Cristae-without the outer mito-chondrial membrane also sedimented at 27,000 x g. At this speed, vesicles of endoplasmic reticulum and sarcolemma also may have pelleted. Indeed, endoplas-mic reticulum and sarcolemmal marker enzyme activities were present in this frac-tion (Table 2) but sacrificing some of the microsomal yield to the 'mitochon-d r i a l ' pellet seemed justifiable for the sake of having apaucity of mitochondria in the microsomal fraction. The microsomal fraction contained amorphous small vesicles but did not contain mitochondria or mitochondrial dark staining parts (Plate 2, #4 and 5) although the centre of plate 2, #6 has an unvesiculated dark membrane which looks suspiciously like a cristae. Some uneven fuzziness of the membrane (plate 2, #5) could be indicative of the basement membrane. Subcellu-lar fractions were characterized by the distribution of marker enzymes. The distribution of 5'-nucleotidase and acetylcholinesterase in the subcellular, fractions (Table 3) indicated that plasma membrane was concentrated in the micro-somal fraction. A Na stimulated Mg-ATPase activity was also concentrated in the microsomal fraction (Table 3) and in the presence of a small volume of soluble activating factor, an ouabain inhibitable K stimulation could be demonstrated (see Fig. 6, C and D). NADH oxidase was used as an endoplasmic reticulum marker enzyme in guinea pig longitudinal i l e a l and rat aortic smooth muscle cells by Hurwitz et a l . (1973) although there is no indisputable endoplasmic reticulum marker enzyme for smooth muscle studies (_Wei et a l . .1976). Twice as much NADH oxidase was recovered in the 27,000 x g 'mitochondrial' pellet than in the microsomal fraction although the specific activity was just slightly greater in the microsomal fraction. Succinic dehydrogenase activity, an inner mitochon-dr i a l membrane marker enzyme of highest specific activity in the 27,000 x g pellet, was not detectable in the microsomal fraction (Table 2). Surprisingly, a considerable amount of the succinic dehydrogenase activity was released from the mitochondria and appeared in the soluble fraction. The 27,000 x g 'mito-chondrial' p e l l e t also contained high s p e c i f i c a c t i v i t i e s f o r plasma membrane marker enzymes but as expected, also contained the highest s p e c i f i c a c t i v i t y of the mitochondrial marker enzyme. An attempt was made to separate the endoplasmic reticulum from the sarco-lemmal v e s i c l e s on a sucrose density gradient (Fig. 4). Although protein separ-ated into 4 peaks, the NADH oxidase cosedimented with the 5'-nucleotidase and acetylcholinesterase at a s p e c i f i c g r a v i t y of approximately 1.122. Succinic dehydrogenase a c t i v i t y which was absent i n the microsomal p e l l e t , was s t i l l un-detectable i n the gradient f r a c t i o n s . Therefore the microsomal p e l l e t was used to study plasma membrane ATPase a c t i v i t i e s without further p u r i f i c a t i o n . The caveolae that increase the surface area 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 types. Perhaps t h e i r v e s i c u l a r - l i k e shape (less than 0.1 u i n diameter; see Results and Discussion, section IV) lend themselves to being pinched o f f into microsomal sized v e s i c l e s (approximately 0.085 u i n diameter), which might explain why there was a good recovery of sarcolemmal mark-er enzymes i n the microsomes as compared to other tissues i n which plasma mem-branes are usually i s o l a t e d from low speed f r a c t i o n s . Rostgaard and Barrnett (1964) observed, by electron dense lead s t a i n i n g , that caveolae enzymatically hydrolyze nucleoside d i - and tri-phosphates but not monophosphates or other phosphoric acids. This implies that they may be involved i n a c t i v e ion trans-port processes. I f the microsomes are mainly pinched o f f caveolae, then the microsomes would be expected to have high s p e c i f i c a c t i v i t i e s for ion transport enzymes. 41 Fig. 3. Cation l e v e l s of the guinea p i g ileum l o n g i t u d i n a l smooth muscle a f t e r depletion of the tissue i n Ca,Mg-free Tyrode's s o l u t i o n as used i n the procedure for preparing sarcolemmal enriched microsomes. Control ion contents (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 for 30 min i n Tris-Tyrode's s o l u t i o n . Tissue ion con-tents were measured a f t e r Ca and Mg depletion for 10 min at 37°C and a f t e r various times up to 4 hr at 4°C i n Ca,Mg-free Tyrode's s o l u t i o n ( s o l i d l i n e s ) . The dotted l i n e s i n d i c a t e ion content of e q u i l i b r a t e d tissues washed twice for 10 min each time with i c e cold i s o t o n i c suc-rose (zero time) and washed twice with cold i s o t o n i c sucrose s o l u t i o n a f t e r Ca and Mg depletion for 10 min at 37°C and for further times at 4°C up to 4 hr. Each point represents the mean + S. E. 43 Plate 1. 1. 27,000 x g p e l l e t . The f r a c t i o n contains i n t a c t mitochondria, although abnormal i n appearance. Some are l i g h t l y and others darkly stained. The i s o l a t e d mitochondria are larger than they are i n the i n t a c t c e l l (see Plates 3-5). (14,700 x magnification Zeiss EM-10) 2. Light and dark stained mitochondria. L i g h t l y stained mito-chondria exhibit double membranes while the inner membrane and c r i s t a e of dark stained mitochondria aggregate i n the centre. (24,000 x magnification Zeiss EM-10) 3. Section of 27,000 x g p e l l e t showing more microsomal v e s i c l e structures mixed with mitochondria. This section was probably cut near the surface of the p e l l e t where a white layer i s seen to overlay the dark brown mitochondrial p e l l e t . Arrow indicates ribosomes on a v e s i c l e . (14,700 x magnification Zeiss EM-10) 45 Plate 2. 4. Amorphous membrane vesicles i n 105,000 x g microsomal pellet. 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 vesicles with no apparent mitochondria. Note magnification is 1.56 times greater than Picture 2 of mitochondria. Therefore, i f mitochondria were pre-sent, they should appear as very large structures at this magni-fication. (37,500 x magnification Zeiss EM-10) TABLE 2.. Location of c e l l marker: enzymes, spe c i f i c a c t i v i t i e s and % recoveries in each c e l l f r a c t i o n 5 Fraction 3 Protein- 5'-•AMPase acetylcholinesterase NADH oxidase Succinic Dehydrogenase . to t a l .^recovery . o.ce Q umoles _ i -mg ~hr •Pi-•1 recovery %de •n'mo Les - L . -mg mm recovery umoles -1 . -1. mg mm recovery • a nmoles recovery -1 , -1 . mg mm % whole . 91.6 100 3.18 100 55.0 100 0.576 100 6.8 100 homogenate 2,700 x g ' 61.3 66.9 2.77 58.4 . 50.3 61.1 • 0.470 54.6 5.8 56.9 pel l e t 2,700 x g 37.5 40.9 ;. 2.32 29. 9. •40. 6 30.2 0.435 30.9 4.7 28.4 supernatant ^ 27,000 x g 2.2 2.4 9.08 6.8 ' -150.0 . 6.5 2.610 10.8 15.0 5.2 pel l e t [mitochondria) 27 ,00.0 x g 3 3.9 3 7.0 •1.82 . 21.2 3 5.3 23.6 ' 0.236 15.2 5.5 30.3 supernatant 105,000 x g 0.9 0.9 "•• 19. 09 6 .'7 259.0 4.4 3.190 5.2 0.0 0.0 p e l l e t • (microsomes) •105 , 000' x g 31.3 34.1 0. 51 5.8 28.2 17.5. 0.115 6.8 6.2 31.1 supernatant (soluble) b. t o t a l protein = rr.g/.05 ml x fraction volume. c. % recovery protein = t o t a l protein/total protein 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 protein 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 protein of whole homogenate x 100. e. % recovery in supernatant and p e l l e t fraction for each centrifugation force 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 protein measurements. 3 other experiments showed similar results. TABLE " 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 Fraction a b° Mg ATPase g ' Mg Na ATPase h Mg Na K ATPase 1 Mg Na K ATPase^ • + ouabain umoles Pi recovery umoles Pi recovery umoles Pi recovery umoles Pi recovery mg" min" '•. % mg~ min~ . % mg~ min~ % mg~ min" % whole ' 0.263 100 0.410 .'; 100 0.395 100 0.429 100 homogenate • 2,700 x g 0.151 3 8.5 0. 270 44.1 0. 261 44.2 0.257 40.1 pell e t 2,700 x g 0.300 0 46.4 0.461 ' ; 4 6.0 0.464 48.1 0.464 44.3. supernatant • 27,000 x g 1.714 • 1 . 15.5 3.123 . •' 18. .2 3.013 18.2 2.965 IS.4 pellet [mitochondria) 27,00 0 x g 0.140 •;. 20.9 0. 214 '19.3 0.207 19.4 0.205 .17.7 supernatant 10 5,000 x g 2.029 7..4 . 2.974. 6.9 3.037 7.3 3.138 7.0 pelle t (microsomes) 105,000 x g O.Otl 5.3 0.048 3.4 0.041 3,5 0.03 8 3.1 supernatant (soluble) aJbcde see table 2 f. results are the average 1 of duplicates . 3 other experiments showed similar 1 results. 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. 49 Fig. 4. Sucrose density gradient treatment of the microsomal fraction. 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 in 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 acetyl-cholinesterase (A), succinic dehydrogenase 1 (O)» NADH oxidase (Mi, and 5'-nucleotidase (5'-AMPase) (•) . 51 I I . Mg dependent Na,K-ATPase The d i s t r i b u t i o n of various 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 Table 3. The highest s p e c i f i c a c t i v i t i e s appear i n the microsomal f r a c t i o n . Na stimulation of ATP hydrolysis required Mg. Na stimulated Mg-ATPase was found i n every f r a c t i o n except the 105,000 x g super-natant (Table 3). None of the f r a c t i o n s required K for Na stimulation and none were further stimulated by K nor i n h i b i t e d by ouabain to any great extent. A s i m i l a r Na-ATPase a c t i v i t y was previously reported i n rat myometrial (Allen and Daniel 1970) and rabbit vascular muscle f r a c t i o n s (Wolowyk et a l . 1971). The degree of Na stimulation found i n the present study was higher than reported by Wolowyk et a l . (1971) and was evident without the necessity of deoxycholate or ageing treatments (Allen and Daniel 1970). The microsomal ATPase a c t i v i t y i n -c r e a s e d as MgCl. was increased from 1 to 4 mM (Fig. 5, upper graph). MgCl-^ (3 mM) was routinely used with 3 mM ATP for t e s t i n g of Na stimulation. NaCl (20 - 80 mM) increased the a c t i v i t y l i n e a r l y and the a c t i v i t y began to l e v e l o f f at 100 mM, which i s s i m i l a r to the Na concentration dependence observed by A l l e n and Daniel (1970). K (3 - 12 mM) i n the presence of 3 mM MgCl-. and 100 mM NaCl did not induce a further consistent concentration dependent a c t i v a t i o n (Fig. 5, upper and lower graphs). The reason why the K stimulation did not seem to be concentration dependent i n the range of 1 - 12 mM, i n contrast to other Na,K-ATPase a c t i v i t i e s (Skou 1974) i s not c l e a r . The Na stimulation, without added K, was not due to s i g n i f i c a n t K contamination i n the membrane f r a c t i o n . The ion concentrations present i n the f i n a l microsomal f r a c t i o n (Table 4) are approxi-mately one thousandth of those required to optimally activate ATPase a c t i v i t i e s (Skou 1974). This agrees with the findings of Wolowyk et a l . (1971) who used tetraphenylboron as a K chelator to demonstrate that 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 further a c t i v a t i o n of the Na stimulated Mg-ATPase by K required the addition of a small volume of the soluble f r a c t i o n (105,000 x g supernatant) to the microsomal fraction, as previously reported by Wolowyk et a l , 0-971') in rabbit vascular smooth muscle. In the presence of a soluble activating factor, K predominantly kept the ATPase activity of the microsomal fraction above that with Mg and Na alone (Fig. 5, lower graph). However the activity s t i l l was clearly not a function of the increasing K concentration. Without the addition of the soluble supernatant K appeared to cause inhibition. The Na stimulation of the Mg-ATPase was not inhibited by ouabain, even when the ouabain concentration was 3 mM (Fig. 6A). The possibility that the ouabain site was inaccessible was examined. Ouabain (0.1 mM) was added to the Tyrode's solution, the Ca,Mg-free Tyrode's solution and the 0.25 M sucrose medium used for the preparation of the tissue, the homogenization of the tissue and resus-pension of the microsomes. Despite this treatment, Na stimulation was s t i l l evident (greater than 100%) and the further addition of 3 mM ouabain did not significantly reduce the activity (Fig. 6B). Therefore the resistance of the Na stimulation to ouabain was not due to inaccessibility of the ouabain site. The small K stimulation in the presence of soluble fraction was sensitive to ouabain (Fig. 6C). The reversal of the slight K inhibition of the microsomal ATPase to a small stimulation of the activity in the presence of 0.01 ml of soluble fraction could not be accounted for by the small amount of ATPase a c t i -vity (K independent) in the soluble fraction (Fig. 6C). Therefore the activa-tion by K was not directly due to the soluble fraction but rather was an effect of a component of the soluble fraction on the microsomal activity. Numerous studies of the effect of the soluble fraction on the K activation have indicated that the K stimulation was usually slight but consistent (Fig. 6D). The fluc-tuations between different 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 significant K stimulation unless a paired t-test was used. The difference between Na and K stimulation and Na stimulation alone for every micro-somal preparation was significantly greater than zero at the 99% level and the ouabain inhibition of the K stimulation was also significant at the 99% level. A regulatory component of, the NajK^ATPase was reported to be concentrated i n the supernatants of homogenized mouse plasmocytoma a s c i t i c c e l l s which have a Na,K-ATPase that i s r e s i s t a n t to ouabain (Lelievre et a l . 1976a). Reconstitu-t i o n of the regulatory component with the membranes required the addition of Ca and Mg. Experiments on the e f f e c t of the regulatory component on the Na,K-ATPase of mutant c e l l l i n e s led L e l i e v r e et a l . (1976b) to hypothesize that the Na,K-ATPase-ouabain i n t e r a c t i o n was modulated by a nonspecific membrane s t r u c t u r a l component. Although control experiments indicated that the ATPase a c t i v i t y was l i n e a r with enzyme concentration ( i . e . pseudo zero order with respect to substrate), at lower ATP concentrations the umoles of P^ li b e r a t e d exceeded the umoles of ATP added to the assay medium when the incubation was allowed to proceed for 30 min (Fig. 7, upper graph). At f i r s t the microsomes were suspected' of containing an ADPase a c t i v i t y but Luthra et a l . (1976) have suggested that an adenosine kinase a c t i v i t y could also explain the release of P_^  from ADP. ADP (3 mM) was 1/6 as active as a substrate, compared to ATP, for the microsomal production of P. s (Fig. 7, lower graph). When the incubation time was 5 min, very l i t t l e ADP would be produced from ATP hydrolysis and therefore P^ production from ADP i s n e g l i g i b l e . Under these conditions, the umoles of P^ produced did not exceed the umoles of ATP added (Fig. 8, i n s e r t ) . At higher ATP concentrations, the Mg-ATPase a c t i v i t y decreased (Fig. 8, ins e r t ) and the decline of the a c t i v i t y was apparent even when the Mg concentration was r a i s e d to 7.5 mM (data not shown). The addition of Na and K prevented the substrate i n h i b i t i o n . Whereas Na and K increased the maximal v e l o c i t y (V ), the K was increased suggesting max m that there was a lower a f f i n i t y for. ATP i n the presence of Na and K (Fig. 8). This reduction of the enzyme a f f i n i t y for ATP by K had been reported by others (Dahl and Hokin 1974). A radioactive assay of the ATPase indicated that the gamma phosphate release was l i n e a r with the amount of microsomal protein added and the s p e c i f i c a c t i v i t y was' ,'in .^i.the'... same range as the a c t i v i t y calculated by the Lowry and Lopez method (Fig. 9.), Na alone (with no K) caused an 81% stimulation of the Mg-ATPase activity. The addition of Ca did not convert the Na-ATPase to a K dependent form (Fig. 10). Ca alone activated an ATPase in the microsomes but did not induce any change in the Mg dependent Na,K-ATPase pattern of activity observed in previous experiments. The Ca and Mg stimulations of the activity were not additive. Na nearly doubled the Mg-ATPase activity and K, in the presence of the soluble ac-tivating factor, caused a small further stimulation which was antagonized by 3 mM ouabain. A possible explanation for the unusual behavior of the ATPases in this tissue could have been that the Na,K-ATPase was changed to a K-independent ouabain-insensitive conformation during the isolation procedure. The intact muscles were tested under the biochemical conditions employed for the isolation,, of the microsomes (except, of course, that the tissues were not homogenized or centrifuged). Muscles and the nerve plexus were viable over 3 days after they were stored in cold Tyrode's solution (Fig. 11, Top). The f i r s t responses on the second and third days were abnormal, probably because the tissues had be-come Na rich and K poor when the pump activities were depressed at 4°C (Setekleiv 1970). After equilibrating the muscles for longer times, the muscle responses were normal. This i s compatible with the view that cold temperature does not irreversibly destroy the normal activity of the muscle Na,K-ATPase. The muscles also functioned normally in the physiological medium buffered with 25 mM h i s t i -dine instead of carbonate and phosphate (data not shown). As storage of the muscles overnight in isotonic cold sucrose, corrected to pH 7.4 with histidine (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. After re-equilibration of the muscles in normal Tyrode's solution, the muscles s t i l l responded to ouabain on the second day, indicating 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 activity and remain responsive to ouabain after treatment with Ca,Mg-free Tyrode's solution under exactly the same conditions as used to weaken the muscle for 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 re-equilibration of the muscles in 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 for Fig. 12, 3rd Row) (see also Bolton 1973a; Casteels 1966). It seems more lik e l y that the Ca,Mg-free Tyrode's solution treatment weakened the muscle contractile apparatus and series elastic 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 is 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 activity was calculated per mg micro-somal protein. Incubation time at 37°C was 15 min. Upper Graph. 75 y l of the microsomal fraction containing 28.5 yg of protein was assayed in the presence of 25 y1 of the soluble fraction containing 29 yg of protein. (O) MgCl • (•) 3 mM MgCl. plus NaCl; (A) 3 mM MgCl 2 plus 100 mM NaCl plus KC1. Lower Graph. 75 p l of the microsomal fraction 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 line represents Mg dependent Na-ATPase activity without K addition. TABLE 4. Cation levels in the microsomal fraction Cation concentration (mM) Mg Na K Ca Sucrose (0.25 M) a .0034 .747 .0306 .018 Microsomes prepared^ 0 in 0.25 M sucrose"" .046+.009 .789+.107 .063+.005 .028+.005 Microsomes (see method) after dilution in assay .0039 .066 .0053 .0024 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 in the assay medium. 59 F i g . 6. The e f f e c t of Mg, Na, K, ouabain and a soluble a c t i v a t i n g factor on the microsomal ATPases. 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 represent 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 enriched f r a c -t i o n i n the presence of 0.1 mM ouabain (see Results for more d e t a i l s ) . The f i n a l concentration of ouabain in-each assay tube was 8.33 uM except i n the presence of MNKO where an a d d i t i o n a l 3 mM ouabain was present. C. (1) Assay of microsomal f r a c t i o n (19 mg). (2) Assay of 19 mg microsomal protein plus 13.8 mg (0.01 ml) of soluble f r a c t i o n p r o t e i n . (2) - (1) represents 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 of soluble f r a c t i o n and the microsomal a c t i v i t y alone. (3) Assay of soluble f r a c t i o n p rotein (69 mg) i n 0.05 ml. D. Assay of the microsomal f r a c t i o n i n the presence of soluble f r a c t i o n as i n F i g . 6C except that the incubation time was 15 min instead of 5 min. The a s t e r i s k indicates that the paired t values for 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 at 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 fraction '(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 dot-ted line (ATP/P.) = 1 (•) '3 mM MgCl, 1 (•) 3 mM MgCl and 100 mM NaCl ( A ) 3 mM MgCl^, 100 mM NaCl and 3 mM KC1 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 6 3 F i g . 8. Lineweaver Burk pl o t of the e f f e c t of 100 mM Na (•) or 100 mM Na plus 3 mM K ( A ) on the Mg-ATPase ( O ) • A l l assays contained 3 mM MgC^. The inserted graph indicates that the l i b e r a t e d P. does not exceed the ATP added during the 5 min incubation. Points are the average of duplicate determinations. -1 -1 V =2 ymoles P. mg min and K = 0.23 mM for the Mg-ATPase. max . 1 m Na and K abolished the substrate i n h i b i t i o n . Na and K increased the V m a x to 3.12 ymoles P i mg - x m i n - x but the K m also increased to 0.55 mM i n d i c a t i n g a lower a f f i n i t y for ATP than with Mg alone. ^moles Pj liberated 65 Fig. 9. Assay of the Mg-ATPase with (Y-^^P) labelled ATP. Data points are the average of duplicate assays containing 25 mM histidine buffer (pH 7.4), 0.1 mM EGTA, 3 mM MgCl 2 (•) or 3 mM MgCl2 and 100 mM NaCl (•). Microsomal protein which is 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 in 0.6 ml. The f i n a l ATP concentration was 3 mM. (y-32p) ATP of high specific activity 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 in Bray's s c i n t i l l a t i o n cocktail. M = MgCl 2 (3 mM); N = NaCl (100 mM) . \ 66 67 Fig. 10. The effect of Ca on the Mg dependent Na,K-ATPase in the microsomal fraction in the presence of soluble activating factor. The assay medium contained 3 mM ATP in 0.6 ml of 25 mM histidine pH 7.4, 0.1 mM EGTA, 50 pl of microsomes and 10 p1 of soluble fraction. Results are the average of^4 assays on 2 batches of enzymes. Error bars were omitted for clarity (see Appendix Table for Fig. 12). (O) No Mg (#) 3 mM MgCl (•) 3 mM MgCl 2 and 100 mM NaCl ( A ) 3 mM MgCl 2, 100 mM NaCl and 3 mM KC1 (4) 3 mM MgCl 2, 100 mM NaCl, 3 mM KC1 and 3 mM ouabain 69 F i g . 11. Pharmacological tests of the tis s u e v i a b i l i t y under the biochemical conditions used to prepare sarcolemmal enriched microsomes, as a check for the possible loss of Na,K-ATPase a c t i v i t y . Upper The experiment demonstrates that the muscle and the nerve plexus remained v i a b l e over 3 days a f t e r 2 overnight storages i n cold Tyrode's s o l u t i o n . After 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) at 37°C the f i r s t response to 60 mM KC1 a f t e r storage i n cold Tyrode's s o l u t i o n had a depressed phasic but a normal tonic component, but the second responses (day 2 and day 3) were normal. The preparation s t i l l responded to 60 mM KC1 and 18.5 uM n i c o t i n e (nic) a f t e r 3 days. Lower The experiment demonstrates that the muscles were v i a b l e a f t e r 1 hr treatment i n cold i s o t o n i c sucrose corrected to pH 7.35 with h i s t i d i n e ( i . e . the medium used for homogenization). Res-ponses to 60 mM KC1 and CD (2 x 10 7 M) were s t i l l nearly 100% of con t r o l . Muscles were s t i l l very ac t i v e a f t e r overnight storage i n cold i s o t o n i c sucrose and they s t i l l responded to 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 sucrose did not i n t e r f e r e with the mechanism of action of ouabain. K C l r e s p o n s e s a f t e r o v e r n i g h t 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)  p h a s i c t o n i c d a y 2 (%• c a y 1) D h a s i c t o n i c 1.89 5.2 -1.41 73 t 5 98 ' 7 30 71 Fig. 12. Pharmacological test of the muscle v i a b i l i t y after treatment in Ca, Mg-free Tyrode's solution. Control responses to CD (2 x 10 - 7 M) and ouabain (ouab) (10-^ M) were recorded and the muscles were a l -lowed to completely equilibrate (i.e. return of spontaneous activity, 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 destabilizing effect. Muscles were detached from the transducers and washed in 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 in 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 flaccid. 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. Table for Fig. 12. 1st Row Average results from 4 experiments similar to Fig. 12. Equilibration time refers to the time required for the muscle to regain i t s spontaneous activity following washout of the ouabain. A 2nd Row Average results of 4 experiments similar to Fig. 12 except that the time in Ca,Mg-free Tyrode's solution was reduced to 1 hr. 3rd Row 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 activity before they were tested a second time. The second response was never as strong as the f i r s t . CD MT CD NT ouab 2 x 10 ' M ( g r i ) t o r CD" •i n 10 5M • o u a b a i n e q u i l i b r a t i o n 37'C 10 min OCaMg T y r ( ™ ) OCaMg 4" C timp (min) a f t e r Ca a d d i t i o n (grn) Kg 37"C 2 x 10 7M CD % o f b e f o r e OCaKg p h a s i c t o n i c 10 M o u a b a i n % o f b e f o r e 0Ca'-';S 1 +• i 1. H; 2 1 1. 0 t .02 57 t'4 0.7 T.05 230 0.69 i . 05 60.9 tl. 2 •3 3 •2.5 3 3.1 ' 1 7 2 •1 g 2 1.9 t . 2 57 t 2 '0. 79 : .14 60 0.31 + .C6 118 i 8„. 4 70.2 23.6 44. 9 ' -5.5 .96 t . 08 74 *-3 - - - - - • 86.0 t 9.7 n=4 N3 III. Ca-ATPases a. Microsomal Ca-ATPase The purity of the sarcolemmal enriched microsomal fraction becomes of major importance when discussing i t s Ca-ATPase activity 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 vesicles (based on total 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 in electron micrographs of this tissue. Similarly, longitudinal i l e a l cells 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 in the microsomal pellet and slightly less than the sarcolemmal recovery in the micro-somes. Mitochondria were not detectable in 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 -vity 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 fraction had ATPase activities that could be stimulated by Ca or by Mg but their stimulations of the the ATPase activity were never addi-tive (Fig. 13, see also Fig. 12 and Fig. 14, 2nd Row). Low amounts of Ca often inhibited the Mg-ATPase activity (Fig. 13). Since 1 mM Ca and 3 mM Mg stimu-lated the activity to the same extent as 3 mM MgCl 2 alone, Mg may inhibit some of the Ca activation and Ca may prevent some of the Mg stimulation of the micro-2+ somal ATPases. The threshold free Ca concentration for activation of the ATPase activity was about 10 7 M and the activity increased linearly up to con-centrations slightly greater than 10 5 M. Maximal activation occurred at about -4 2+ 2.5 x 10 M free Ca (Fig. 14, 1st Row). Increasing the MgCl 2 concentration in the presence of 1 mM Ca progressively- increased the ATPase activity but the two activities were not additive (Fig, 14, 2nd Row), Whereas both 1 mM CaC^ and 3 mM MgC^ stimulated the enzymatic activity to 2 umoles P^ /mg protein/min activity, together they activated the ATPase activity to only 2.75 umoles P_^/ mg protein/min. Na caused only a small gradual increase in activity which might have been due to an increase in osmolarity or ionic strength (Fig. 14, 3rd Row) but this effect was very small. It is unlikely that the Mg-ATPase and Ca-ATPase activities are from the same enzyme because the Mg-ATPase is very noticeably activated by Na whereas Na had l i t t l e effect on the Ca-ATPase. KC1 had l i t t l e effect on the Ca-ATPase over the-,concentration range 2 - 8 mM (Fig. 14, 4th Row) KC1 may have caused a slight inhibition of the microsomal Ca-ATPase. -3 ATPase activity, in the presence of 10 M total Ca, increased as the ATP concentration was raised to 1 mM (Fig. 15, Top). Above 1 mM ATP, the activity decreased. The decrease in activity may have been due to substrate inhibition 2+ or reduction of the free Ca concentration by the excess ATP. The microsomal Ca-ATPase was not inhibited by 1 mM LaCl^ when the ATP concentration was 3 mM. However, at lower ATP concentrations, La inhibition was very apparent. LaCl^ changed the normal substrate dependent hyperbolic increase of the Ca-ATPase activity to a sigmoidal function of the ATP concentration (Fig. 17). Substrate limitation can be an explanation of the sigmoidicity of the Michaelis Menten plot and the nonlinearity of the double reciprocal plot (Fig. 17) (Westley 1969) If a l l of. the LaCl^ was chelated by 1.5 mM ATP, the effective ATP concentration would be 0.5 mM, which should have an activity of 1.37 umoles P^ /mg protein/ min. However, the La treated enzyme activity at 1.5 mM ATP was only 0.875 ymoles P^ /mg protein/min (Fig. 15, Top). Therefore substrate limitation may not fully explain the La inhibition of the Ca-ATPase activity. It seems as though there may also be a direct inhibition by La at the Ca activation site. 75 b, Actomyosin Ca-ATPase The concentration threshhold for activation of the Ca-ATPase in the 105,000 x g soluble fraction (which has been assumed to belong to the contrac--7 2+ t i l e filaments) may be below 10 M Ca (Fig. 15, Bottom). LaCl^ inhibited the activity even when the ATP concentration was 3 mM. The inhibiton by La of the actomyosin Ca-ATPase is not due to substrate limitation, 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 le f t and the actomyosin Ca-ATPase was fu 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 inhibition (Fig. 16, Top). High ionic strength KC1 solution definitely improved the actomyosin Ca-ATPase activity in the presence of 4 mM CaC^ 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 activities The substrate a f f i n i t i e s of the actomyosin and microsomal Ca-ATPase (from the tops of Fig. 15 and 16) are compared in Fig. 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 (K = 0.5 mM). La treatment of the microsomal Ca-ATPase affected the activity m so that the double reciprocal plot was no longer linear, indicating that there was some interaction between the substrate and the inhibitor, the effect be-ing particularly 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, i t is unlikely that the La inhibition is competitive, as drawn in Fig. 17. Sulakhe et a l . (1973) observed that La did not inhibit Ca binding to microsomal vesicles or oxalate accumulation in the vesicles unless the La concentration exceeded the ATP concentration. La inhibition of Ca-ATPases may be due to (1) limitation of the free substrate (2) prevention of the Ca-ATP complex which may serve as a substrate or (3) direct competition for Ca binding sites. 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 concen-tration. The inhibition of the actomyosin Ca-ATPase by La seems to have been by the third mechanism. Actomyosin effectively binds La with greater a f f i n i t y than the af f i n i t y of ATP for La. Ca seems to associate with actomyosin more than with ATP thereby avoiding activity reduction at higher ATP concentrations. Divalent cations are known to associate with ATP and the association con-4 - 1 stants for Mg-ATP and Ca-ATP complexes are approximately 8.8 x 10 M 4 -1 (0*Sullivan and Perrin 1964) and 3.15 x 10 M (Ogawa 1968) respectively (see Table below and Appendix). If the cation-ATP complex ±s\ the substrate and determinant of the enzyme activity, then the activity should show saturation kinetics. But the microsomal Ca-ATPase and the Mg-ATPase acti v i t i e s decreased above 1 mM ATP and the inhibition can be explained in two ways. Either higher free ATP concentrations inhibit the activity or the activity is less when the concentration of free divalent cation i s decreased. Higher cytoplasmic ATP concentrations would not be expected to inhibit active transport activity for any physiological purpose whereas an intracellular loss of Ca or Mg would be expected to turn off pump function. The decline in activity as the ATP is cation total [cation] mM total [ATP] mM K •. ass [cation-ATP] mM f ree~ [cation] mM free [ATP] 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 .187 .187 Ca 1 3 3.15 x 10 4 .978 , .022 2.022 increased from 1 -,3 mM coincides with a large decrease in free Mg and free o 2+ Ca concentrations whereas free ATP or cation-ATP complex increase (see Table above). Therefore, the decline in activity which appears to be substrate inhi-bition may actually be due to a reduction in the free cation level. As support-ing evidence, Iso (1975) has shown that higher ATP concentrations 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. Sulakhe et a l . (1973) found that ATP up to 1.7 mM increased Ca binding but higher concen-trations were inhibitory. McNamara et a l . (1974) reported that ATP in excess of the divalent cation concentration inhibited the Ca-ATPase and Mg-ATPase activity. The sarcoplasmic reticulum Ca pump requires Mg (MacLennan 1970, Katz at a l . 1970) but since the Ca-ATPase in the longitudinal i l e a l microsomes observed in this study and in others (Godfraind et a l . 1976, Oliviera and Holzacker 1974) did not require Mg, it.would seem to be of sarcolemmal origin. McNamara et a l . (1971 and 1974) found that the Ca-ATPase activities in hamster skeletal and dog cardiac muscle sarcolemmal enriched fractions did not require Mg and were not stimulated by high ionic strength K solutions. Therefore this Ca-ATPase was attributed to the sarcolemma rather than to sarcoplasmic reticulum or actomyosin. They also found that the Ca-ATPase and Mg-rATPase had similar specific activities and both.-were inhibited by excess ATP. In their experiments and in the present study the Ca-ATPase had a higher K m for ATP (0.5 mM; Fig. 17) than the Mg-ATPase (0.23 mM; Fig. 8). The Mg and Ca stimulations are not additive and Mg seems to inhibit the Ca-ATPase and vice versa. The microsomal Mg-ATPase in rat uterus was 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 effect on the Ca-ATPase . (McNamara"et - ;i a l . 1974 and the present study). These two ATPase activities probably originate from two separate enzymes with different functions, but this can not be stated conclusively without further evidence. It is clear that the Ca-ATPase in the microsomal membrane vesicles is not due to adhering actomyosin f i b r i l s because 7 8 their properties of La inhibition, Ca affinity,.substrate inhibition and res-ponse to increasing ionic strength are so strikingly different. \ 79 Fig. 13. Sarcolemmal enriched microsomal Ca-ATPase activity in response to tree Ca2+. Approximately ,25 ug.of microsomal protein was assayed in 20 mM Tris HC1 (pH 7.4) at 37°C for 15 min with 3 mM ATP (no EGTA) (solid l i n e ) . The dotted line i s the response to free Ca^ + (no EGTA) in the presence of 3 mM MgC^. Points represent the mean + S. E. (n = 4) 81 Fig. 14. The effect of ions on the microsomal Ca-ATPase. Each assay contained 10"3 M total Ca (2.0 x 10"5 M free Ca 2 +) indicated by the i n i t i a l bar graph in Rows 1 - 4. More CaCl 2 was added (Row 1). CaCl 2 concentra-tions on the abscissa indicate total Ca concentration (see Appendix for free CaCl 2 concentrations). MgCl 2 (Row 2), NaCl (Row 3) and KC1 (Row 4) were added as indicated on the abscissa. MgCl 2 added in Row 2 would increase the free C a 2 + concentration compared to that ind i -cated above. 83 Fig. 15. The effect of La on the microsomal Ca-ATPase and the Actomyosin Ca-ATPase . Top. Substrate dependence of the microsomal Ca-ATPase (solid line) (10~ 3 M total Ca) and inhibition by LaCl 3 (10~ 3 Mi);, (dotted li n e ) . Insert indicates the LaCl3 inhibition in relation to the umoles of ATP added. Data points are the average of duplicates. The assay conditions were, 25 ug microsomal protein in 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. Bottom. Response of the soluble fraction ATPase (actomyosinjl to increasing free Ca? +. 50 u l samples of.the soluble supernatant containing 69 ug of protein was assayed in 50 mM histidine (pH 7.6), 425 mM KC1 and 3 mM ATP (#). The open circles (Q,) i n d i -cate the inhibition.by La of the.Ca activation. 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 incu-bated for an additional 5 .min. Free Ca 2 +.concentrations of the La.treated enzyme are not corrected for the effect of La. 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 activity in different buffer systems. Results are the average of duplicates (ATP.= 4 mM, CaCl2 = 4 mM). 87 Fig. 17. Lineweaver Burk plot of the substrate dependence of the microsomal and actomyosin Ca-ATPases; The graph also-indicates the effect of La (10 - 3 M) on the substrate dependence.of.the microsomal Ca-ATPase. The plots are from the data.in Fig. 15,TTopand Fig. 16, Top. 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, in more detail, in an attempt to correlate the biochemical results (previous three sections of Results and Discussion) with the contractile 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 strip rolled up like a s c r o l l . A cross section of one part of the r o l l can be seen in Plate 3, #7. Auerbach's nerve plexus and a few circular layer muscle cells can be seen adhering to the longitudinal layer. Visceral smooth muscle cells are not individually innervated. The gap between axon bundles and muscle membranes is greater than 100 nm (Holman 1970) and there is an absence of circumscribed end plate regions (Paton and Rang 1965). If acetylcholine receptors are evenly distributed over the entire large surface area, then activation of receptors could probably mobilize Ca from sites lo-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 cells directly or whether ouabain caused extensive release of neurotransmitters from the nerve plexus. The relatively empty extracellular space between longitudinal i l e a l cells is about 500 nm wide. Collagen fibres in the extracellular space are sparse, which decreases the problems associated with isolating subcellular fractions from these smooth muscles. The wide spaces between the cells and the thinness of the muscle strips allows rapid diffusion of ions and pharmacological agents throughout the tissue. The general c e l l shape is long and narrow (Plate 3, #9). The surface to volume ratio is increased by the unevenness of the c e l l surface and by the surface vesicles or caveolae that can be seen in Plate 3, #12, Plate 4, #14 and Plate 5, #19, 21, 22, 23 and 24. The cells appear stellate in cross section (Plate 3, #11 and 121 (see also Gabella 1971), Some of the uneyenness of the c e l l surface may have been due to shrinkage of the cells during the fixation of the tissue. The cells send long protrusions towards their neighbours. Occa-sionally, the protrusions fuse with, or intrude into, other cells to form what appear to be nexuses (Plate 3, #12 - arrow) or 'peg and socket structures' (Plate 3, #11). Since the cells are not individually innervated, there may be a morphological basis for the electrical interaction between these smooth muscle cells 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 intercellular communi-cation system. More recently, the work of Gabella (1973) and Daniel et a l . (1976) demonstrated that true nexuses are absent or very rare in the longitu-dinal layer of the guinea pig and the dog intestine. The longitudinal layer of the ileum works as a synctium even though nexal regions are absent. Calcium oxalate deposits were found at points of close intercellular contact between taenia c o l i cells which demonstrated a 7 layered membrane structure apparently with partial fusing of the adjoining membranes ('gap' junction) (Popescu et a l . 1974). It is s t i l l not clear which, i f any, close membrane contact regions are sites of high permeability between cells (Holman 1973; Daniel et a l . 1976). The function of these junctions is also of interest to this study since, i f excitation-contraction coupling occurred at localized points and is communicated between cells at low resistance intercellular sites, the associated ion move-ments would not be detectable by the modified 'La method' which measures essen-t i a l l y net changes of intracellular ion levels. Under the experimental condi-tions of a drug in a tissue bath, excitation is not localized and we should be able to use the modified 'La method'. Also i t was hoped that loosening of these intercellular junctions would occur after incubation of muscles in Ca,Mg-free Tyrode's solution. 91 Each, c e l l hap a f a i r l y large long and narrow nucleus. Its long axis i s parallel to the long axis of the c e l l . At each of it s ends or poles, there is an aggregation of mitochondria, rough endoplasmic reticulum and a Golgi appara-tus (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 in Plate 4, #14. The membrane vacu-oles of the Golgi complex are usually not accounted for in c e l l fractionation procedures as there is 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 actively involved in the transfer of carbohydrate and sulfate moieties to acid mucopolysaccharides and glycoproteins which are essential for many membrane functions (Somlyo et a l . 1975). These negatively charged substances can act as Ca retaining material in cellular depots. Since there are many aspects of smooth muscle activity for which we have no answers, some of the less studied cellular organelles may play more major roles than is now realized. In the resting c e l l , the nucleus i s elongated and indented slightly. Some nuclei can be seen to contain 1 or 2 nucleoli (Plate 4, #15). The nucleus i s encased in a perinuclear sac of rough endoplasmic reticulum (Plate 4, #14). The perinuclear sac, in the pig coronary artery, accumulates Ca as does the rest of the endoplasmic reticulum (Jonas and Zelck 1974). The calcium in the perinuc-lear sac i s in 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 in the nucleus is 60 times higher than that required by.the c e l l for maximum contraction. The nuclear membrane has spiral 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 cells contract (Plate 4, #14 and 18). The sub-cellular 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 relationships' between the mitochondria and the 92 perinuclear sac and between mitochondria and sarcoplasmic reticulum, Therefore, unless these 'close relationships' 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 internal Ca, but the exact role of nuclear Ca in contraction, i f any, is 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). Mito-chondria accumulate Ca and store large amounts of i t . Only 5% of the mitochon-dr i a l store of Ca in one c e l l would be sufficient to raise the cytoplasmic Ca concentration to maximal contractile levels (Popescu et a l . 1974). Excess cations accumulated by mitochondria may be extruded into the extracellular space through the surface vesicle-mitochondrial contacts (Somlyo and Somlyo 1976). When taenia c o l i cellular Ca depots were mapped by Ca oxalate crystal staining, densely aggregated crystals were observed at points where mitochondria were situated very near to caveolae, sarcoplasmic reticulum, perinuclear sac and the sarcolemma (Popescu'et a l . 1974). Therefore, they are not just physically near to each other, but are probably functionally 'associated 1. There is no apparent pattern to the mitochondrial positioning in the guinea pig longitudinal i l e a l cells that might suggest that they are c r i t i c a l l y positioned for even disperse-ment of Ca from the mitochondria to the contractile filaments. Most of the rough sarcoplasmic reticulum is present in the nuclear pole region. Ca is accumulated in the central sarcoplasmic reticulum and 33% of the Ca is sufficient to activate maximal contractions (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 in the perinuclear region. The c e l l surface invaginates at points where myofilaments are attached to the sarcolemma when the cells contract (Burnstock 1970). Narrow membrane sacs are present in these concave regions, just beneath the sarcolemma. 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 pig 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) in the guinea pig ileum longitudinal smooth muscle and in guinea pig taenia c o l i cells (M. Wolowyk, personal communi-cation) . The physical closeness of the sarcoplasmic reticulum, mitochondria and sarcolemma to each other suggests that they may form an area actively i n -volved in ion transport but direct evidence for such a function is lacking. The pattern of distribution of peripheral smooth sarcoplasmic reticulum membranes is like 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 is no need for transverse extensions of these membrane sacs in cells that are only 2 - 5 p in diameter. A stenosis of the small intestine can cause the cells to attain 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 ratio. If an extensive sarcoplasmic reticulum is an adaption to a large fibre size (as is found in skeletal muscle) then normal longitudinal i l e a l cells should not require very much sarcoplasmic reticulum. There is more than twice as much Ca in the peripheral sarcoplasmic reticulum of taenia c o l i cells as would be required to cause maximum contraction (Popescu et a l . 1974). A trigger of extracellular Ca from the caveolae or a local 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 local f i e l d changes may only occur in cells where the sarcoplasmic reticulum vesicles form direct 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 cells 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 cells (Pogadeav and Timins 1976). Gabella (1973) indicated that the sarco-plasmic reticulum of intestinal muscle always makes contact with the sarcolemma at some point, although i t is d i f f i c u l t to see such contacts in the micrographs he presents as proof of this. Such contacts are much more apparent in the taenia c o l i cells that have had their cellular 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 intestinal 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 sar-coplasmic reticulum is quite extensive in 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 circular muscle layer. Occasionally smooth sarcoplasmic reticulum membranes are observed to be continuous with rough sarcoplasmic reticulum (Plate 4, #18, Plate 5, #24 - at arrows). The volume of the sarcoplasmic reticulum in smooth muscle cells was based on the total of rough and smooth endoplasmic reticulum (Devine et a l . 1972) because both types of sarcoplasmic reticulum have been observed to accu-mulate Ca. In purified 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 vesicles from taenia c o l i cells were mainly of two populations, 0.04 - 0.08 u and 0.9 - 1.6 u in diameter. Approximately 65% of the vesicles were of the small size corres-ponding to the size of; cayeqlae (0.VQ6. u diameter), and 35% seemed to be from the sarcoplasmic reticulum because the mean diameter of the larger vesicles 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 pellet from the 10,000 x g supernatant while in the present study, a 105,000 x g pellet was obtained from the 27,000 x g supernatant. Based on the results obtained by Popescu e_t a l . (1974), i t would be expected that the microsomes prepared in the present study consist of greater than 65% caveolae, since many of the larger type of vesicles may have sedimented at 27,000 x g. Therefore the Ca-ATPase activity observed in the microsomes in the present study, was almost certainly from the caveolae. Caveolae are perhaps the most striking feature of these c e l l s . They seem to be distributed over the entire surface area of the c e l l . Caveolae increase the surface area by 25% in mouse intestine to 70% in taenia c o l i (Rhodin 1962; Goodford 1970). Caveolae are most abundant at the ends of the i l e a l c e l l s , as can be seen in Plate 5, #21 and 22. What appear to be large holes in the c e l l may be fused aggregates of caveolae (Coltoff-Schiller et a l . 1976). A caveolae fusing process seems to have occurred in the c e l l pole shown in Plate 5, #21. Smooth muscle caveolae are smaller and more uniform in size than micropinocyto-t i c vesicles of endothelial cells (Gabella 1973). In the present study, a ran-dom sampling of 17 caveolae gave an average diameter of 80+4 nm and a depth of 95+5 nm, which is about the same size as the sarcolemmal enriched micro-somal vesicles in Plate 2. The neck of the caveolae is about 20 nm wide (Gabella 1973). The entire surface of the smooth muscle c e l l is covered by a basement lamina which does not penetrate the caveolae and which does not differ structurally over the neck of the caveolae (Gabella 1973, Plate 5, #22 and 23). Impermeant extracellular stains, used for electron microscopy, penetrate into the caveolae, indicating that the caveolae are open to the extracellular space. LaCl, (Ma and Bose 1976) and colloidal lanthanum (Gabella 1973) have been ob-served to be retained in the caveolae but not in the extracellular space after fixation in the absence of La, This indicates that there may be some substance retaining cations in the caveolae, Hyaluronidase has been demonstrated to cause the elimination of caveolae from smooth muscle cells (Gabella 1973). Hyaluroni-dase treatment also increased the inulin measurement of the volume of extracellu-lar space of the intestinal muscle from 34% to 38% (Goodford and Leach 1966). Therefore the caveolar volume (if i t can be estimated from the 4% increase in extracellular space due to hyaluronidase) is about 10% of the extracellular space. Gabella (1973) suggested that the caveolar lumen represents a tissue compartment with properties intermediate between the cytoplasmic and extracellu-lar compartments and that the ionic composition of the caveolar compartment may be controlled by the cytoplasm. This may explain why measurements of the amounts of tracer K*~ in the taenia c o l i , exchanging from the fast exchanging (extra-cellular) space, are considerably larger than the amount of tracer K + that could be in solution in the extracellular f l u i d (Brading 1973). EDTA solutions also caused the loss of caveolae from smooth muscle cells (Higgs and Wolowyk 1974). It i s possible that caveolae have higher Ca and other cation concentrations than the extracellular space. These cations may be required to maintain the shape of the caveolae (Higgs and Wolowyk 1976). Quantitative estimation of the Ca in the Jcaveolae indicates that they would contain enough Ca to i n i t i a t e contraction i f their Ca concentration was equal to the extracellular f l u i d . However, Popescu et a l . (1974) consistently found very dense Ca oxalate crystals in caveolae, often equal to the density of such crystals in sarcoplasmic reticulum. There-fore caveolae probably contain far more Ca than is required for contraction, which led Popescu et a l . (1974) to support the hypothesis that Ca in caveolae plays a role in 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 actively accumulate Ca in caveolae (Lane 1967). If microsomes are derived from pinched off caveolae, as postulated in the present 97 study and previously suggested by Popescu et a l . 0-974), then the ab i l i t y of Ca to stimulate an ATPase in the microsomes i s in agreement with the results of Lane (1967). 'Glycerol shock' treatment i s used to uncouple excitation from contraction for electrophysiological studies of skeletal muscle (see Methods). The uncoup-ling effect seems to be due to disruption of the sarcotubular system (Franzini-Armstrong et al. 1973). Anexperiment based on the ' glycerol shock' treatment of ske-l e t a l muscles, was attempted in the present study to see i f this treatment would have a similar effect on these smooth muscle cel l s . It seemed possible that the hypertonic glycerol solution might alter 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 intra-cellular Ca stores, or that the interconnection between the caveolae and the in-tercellular 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 controlling the muscle tension, but i t did not uncouple excitation of the cells from contraction (Fig. 18, compare 1st and 3rd Rows). The effect of 'glycerol shock' treatment on the structure of the c e l l was not investigated by electron microscopy. Without structural verification, the failure to uncouple excitation of these cells from contraction can only be inferred to mean that the mechanism of excitation-contraction coupling in longitudinal i l e a l smooth muscles is very different from the coupling in skeletal muscle cel l s . Perhaps this is because the cells are small and hence may u t i l i z e superficial or extracellular Ca pools for contractions. Generally, i t i s not readily apparent how small peripheral sarcoplasmic reticulum sacs and mitochondria have an advantage over extracellular Ca for delivering Ca to the central contractile proteins of the guinea pig ileum when the c e l l i s excited at i t s surface. In summary, the general feeling amongst those studying smooth muscle structure and function is that the sarcoplasmic reticulum, the sarcolemma and caveolae seem to be sites for the release of activator Ca and that mitochondria and the nucleus seem to be involved in Ca sequestration but the importance of each of these sites may vary from one type of smooth muscle to another. Surface caveolae contain ca l -cium extrusion pumps which may balance the accumulation of Ca in the c e l l . 99 SYMBOLS FOR ELECTRON MICROGRAPHS OF WHOLE TISSUE (Plate 3, 4, and 5) Subsarcolemmal membrane sacs > Caveolae N Nuclei M Mitochondria G Golgi apparatus Nuclear membrane R Rough endoplasmic reticulum 100 Plate 3. 7. A light micrograph of a thick tangential section of longitudinal ileum folded after dissectional " r o l l i n g off" technique. Light and dark staining cells are present and some nuclei are visible in the light staining ce l l s . (2,300 x magnification Nikon S-Kt) 8. Thick section showing the Auerbach's nerve plexus running as a band between circular and longitudinal layers. Cells are not individually innervated. (1,500 x magnification Nikon S-Kt) 9. A longitudinal section demonstrating the extreme length compared to width of the longitudinal i l e a l fibers. The c e l l surface appears smooth in longitudinal section relative to the cross section in photograph 11. (2,700 x magnification Nikon S-Kt) 10. An electron micrograph of a longitudinal section showing the fluted 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" structures). (16,000 x magnification Philips 75-C) 11. A cross sectional view which clearly shows the increased surface to volume ratio produced by the uneven c e l l surface. Cells eva-ginate towards their neighbours and points of contact may be sites of intercellular communication. (4,400 x magnification Nikon S-Kt) 12. A cross sectional view of longitudinal i l e a l cells demonstrating the protrusion of cells between and around other c e l l s , but the c e l l packing is loose. The extracellular space i s sparsely occu-pied by collagen fibers. An arrow indicates a nexus like struc-ture between two cell s . The c e l l surface vesicles (caveolae) often aggregate near peripheral mitochondria. (11,500 x magni-- fication 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 is not apparent in the plane of the section. (11,350 x magnification Zeiss EM-10) 14. Enlargement of the nuclear pole area in photograph 13. The nucleus is 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) is present between them. (23,850 x magnification Zeiss EM-10) 15. Nuclei of two cells containing nucleoli. The nuclear surface is f a i r l y smooth indicating that the cells are relaxed. (17,143 x magnification Philips 75-C) 16. Spiral indentations of the nuclear membrane are prominent in a contracted cell.< (22,000 x magnification Philips 75-C) 17. Indentations of the nucleus in a contracted c e l l which makes the nucleus appear divided in the plane of the section. (20,000 x magnification Philips 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 Philips 75-C) 104 Plate 5. 19. A view of a longitudinal section demonstrating the relatively smooth c e l l outline. Arrow heads indicate caveolae and dashes indicate subsarcolemmal membrane sacs (possibly sarcoplasmic reticulum). (18,000 x magnification Philips 75-C) 20: An enlargement of the lower right portion of photograph 19, showing membrane sacs between sarcolemma and peripheral mito-chondria. 21. Masses of caveolae at c e l l pole which could possibly combine to form large evacuated areas in the cell s . (17,777 x magni-fication Philips 75-C) 22. An aggregate of caveolae showing continuity of caveolae with the plasma membrane. Other surface vesicles may be caveolae whose necks do not appear in the plane of the section. (43,120 x magnification Zeiss EM-10) 23. Enlargement of caveolae showing that they are slightly 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 j sarcoplasmic reticulum. (17,600 x magnification Philips 75-C) 25. A cross sectional view of longitudinal i l e a l cells that shows many distinct smooth subsarcolemmal sacs indicated by dashes. The dash nearest the right side indicates smooth membrane sacs in close apposition to caveolae. (29,333 x magnification Philips 75-C) 106 Fig. 18. An example of the effect of 'glycerol shock' treatment on the guinea pig ileum longitudinal smooth muscle. 1st Row Control responses to 60 mM KC1 and 2 x 10~7 M CD. 2nd Row After equilibration the solution was changed to Tyrode's solution, containing 400 mM glycerol which caused a tran-sient increase in tension. After 30 min, the muscles were washed twice with normal Tyrode's solution (NT) which again caused an increase in tension. 3rd Row The phasic responses to CD and 60 mM KC1 after 'glycerol shock' treatment were 101.5% + 16.5 and 97.5% +6.3 of control phasic responses, respectively. The tonic res-ponses to CD and 60 mM KC1 after 'glycerol shock' treat-ment were 127.2% +14.4 and 86.2% + 4.3 of control responses, respectively (n = 4). 108 V. The Modified 'La Method' The capability of the modified 'La method' to measure intracellular ion levels w i l l be analyzed in this section before proceeding to describe the results of the studies of ion movements during contraction, relaxation and equilibration using this method. The term 'La resistant Ca' and 'La displaceable Ca' have been used to denote the amount of Ca remaining in the tissue and removed from the tissue with a La solution (Sutter and Kromer 1975). Under the conditions described in the present study, 'La-Tris-resistant Ca' and 'intracellular Ca' are nearly equal. Although the term'La-Tris-resistant Ca' is more exact, the term 'intracellular Ca' is more convenient. Therefore the Ca remaining in the tissue after washing with La-Tris solution w i l l be referred to as 'intracellular Ca'. Similarly, Na, K and Mg levels in the tissue after washing with La-Tris solution at 4°C are also termed 'intracellular' although 'La-Tris-resistant Na', 'La-Tris-resistant K' and 'La-Tris-resistant Mg' would be more exact. Van Breemen and McNaughton (1970) reported that the use of the 'La method' 45 clearly demonstrated that rabbit aortic cells take up extracellular Ca during contractions induced when 160 mM KC1 or 160 mM L i C l was substituted for the NaCl in a Tris-buffered physiological medium. Using the same method, Van Breemen 45 et a l . (1972) observed that noradrenaline did not cause a Ca uptake in excess of the Ca content in control aortic muscle strips. Therefore, i t was suggested that in contrast to high KC1, noradrenaline mainly mobilized intracellular Ca to cause contraction. Since the results agreed with those of Hinke (1965), i t seemed that the 'La method' was able to differentiate between various agonists and changes in extracellular ionic conditions that mobilized extracellular Ca versus those that mobilized intracellular Ca to cause contraction. The Tris-buf f ered physiological medium containing 2 mM LaCl^, originally used in the 'La method', displaced extracellular Ca while preventing influx and efflux of 45 Ca during 1 hr. Marshall and Kroeger (1973) used a similar solution for 1 hr but measured the net intracellular Ca content with atomic absorption spectro-109 photometry. They observed significant increas.es of intracellular Ca after treatment of the rat myometrium with noradrenaline. A similar procedure to that of Marshall and Kroeger (1973) was tried at f i r s t in the present study to follow Ca movements during contractions of the guinea pig 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 in 25 min (Fig. 19, Top). Later, in order to examine the mode of action of ouabain, the method was exten-ded in an attempt to measure Na, Mg and K in addition to Ca. The procedure is described in the Introduction and Methods. A control experiment indicated that 30 min was sufficient to remove most of the extracellular Ca, Na, K and Mg ions without much loss of intracellular ions (Fig. 19, Bottom). The method caused retention of 90% of the cellular K whereas 81% of the tissue Na was lost, consistent with the high intracellular K and low intracellular Na concentrations expected in this tissue. Normally in 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 cells should have lost considerably more than 10% of the c e l l K in 30 min. The results of many measurements of total (n = 32) and intracellular (n = 79) cation levels -of control tissues using the modi-fied 'La method' are compiled in Table 5. The results were expressed in nano-moles/mg dry weight because a more precise measurement of tissue weight could be made after drying, as blotting 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 ratio of the wet weight/dry weight was 6.64. This ratio can be used to convert the results given as nanomoles/mg dry weight into nanomoles/mg wet weight. The inulin space of the guinea pig 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 total tissue water (Fig, 20) have been used to calculate the range of Ca, K, Na and Mg ion concentrations present in the extracellular and intracellular spaces (Table 5). Although the levels of Ca and Mg are similar to those in Tyrode's solution, i t is readily apparent that the extracellular K level is about 3.6 to 4.7 times higher than can be accounted for as being in solution in the extracellular space. ' Daniel (1963a) observed that a substantial portion of uterine potassium (13%) appeared to be located superficially. Since the quantities of ions in the extra-cellular space are calculated from the differences of the total and the intra-cellular ion measurements, i t i s possible that some intracellular K ions have escaped, making the intracellular level of K too low and therefore the extra-cellular level too high. On the other hand, i t may be that some of the excess rapidly exchanging extracellular K (Goodford 1970; Brading 1973; Palaty and Friedman 1975) was retained in the caveolae at a higher concentration, more like that of the intracellular space (Gabella 1973). The same reasoning can be ap-plied to explain the lower than expected extracellular Na concentration. -4 If the extracellular space i s 4.52 x 10 ml/mg wet weight and 10% of the extracellular space is in 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 extracellular space -4 (non-caveolar space) would be 4.07 x 10 ml/mg wet weight. If the ion concen-trations in the caveolae are more like those in the intracellular space and the intracellular K and Na concentrations are about 100 mM and 40 mM, respectively, then 45.2 x 10 moles of K/mg wet weight and 18.1 x 10 ^  moles of Na/mg wet weight are in the caveolar space. If the rest of the extracellular space con-tains 2.7 mM K and 136 mM Na, as in the Tyrode's solution, then 11 x 10 ^  moles of K/mg wet weight and 553 x 10 ^  moles of Na/mg wet weight would be in the noncaveolar extracellular space. Together the caveolar extracellular space and the noncaveolar extracellular space would contain 56.2 x 10 moles of K/mg wet weight and 571 x 10 moles of Na/mg wet weight. In a total extracellular volume of 4.52 x 10 ml/mg wet weight, the extracellular K concentration would be equal to 12.4 mM and the extracellular Na concentration would be 126 mM, which are nearer to the levels calculated to be in the extracellular space (Table 5). Although the caveolae concentrations might be expected to be inter-mediate between the intracellular and extracellular concentrations (i.e. the concentrations estimated above are probably too high), at least part of the discrepancies found in measurements of the extracellular concentrations of ions may be due to a caveolar compartment of 10%. Siegel et a l . (1976) have reported that in the carotid artery, a fraction of the extracellular K, Na, Ca and Mg is bound to connective tissue (14, 30, 30 and 4.5 nanomoles/mg dry weight, respectively). These values cannot be used to correct for the binding of extracellular ions in the guinea pig ileum longi-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-cellular ion concentration measured by atomic absorption nearer to the expected concentrations in the Tyrode's solution. -3 Since the calculated intracellular Ca concentration is 2 x 10 M and the 2+ -7 resting free Ca concentration of the muscle should be 10 M, then Ca is con-centrated 20,000 fold in intracellular sites. Undoubtedly, there is sufficient intracellular Ca to raise the cytoplasmic Ca concentration far beyond that re-quired for maximal contraction. The freeing of 1/200 of the intracellular Ca would cause maximal contraction. But is intracellular Ca used for contraction and i f so, how is i t released? The extracelluar' Ca concentration was calculated to be slightly higher than the Ca concentration in Tris-Tyrode's solution. If the additional extracellular Ca was located in the caveolae, the caveolar- Ca concentration would be 7 mM. However, i t is unlikely that a l l of the additional extracellular Ca would be exclusively in the caveolae. The values for the intracellular and extracellular 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 resulting values would give a reasonably good approximation of membrane potential observed in visceral smooth muscle. The resting membrane potential of rabbit small intestine is -55 mV (El-Sharkawy and Daniel 1975). The range of resting 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 resting membrane potential of taenia c o l i cells to be -57 mV and -37 mV, respectively. The observed concentration gradients of Na and K, as measured by the modified 'La method' yield calculated resting membrane potential values in the range of ' .47 - 57 mV; (Table 6). 45 Sutter and Kromer (1975) observed that the La resistant tissue Ca content increased with time when the extracellular Ca concentration was increased from 2.5 to 5 mM in the rabbit mesenteric portal vein. This could mean that the La technique was not capable of displacing a l l of the extracellular Ca when the Ca concentration was raised. However, i t could also mean that raising the extra-45 cellular Ca concentration drives more of the labelled Ca into the cells from where i t can not be as readily displaced. Whether or not such an influx could be balanced by an efflux 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-Tris solution to displace more extracellular Ca in 30 min was investigated. The extracellular 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 intracellular ion levels.(Fig. 21). The total levels of Ca increased as expected but the intra-cellular Ca levels remained constant. The total and intracellular levels of Na, K and Mg were not noticeably affected by raising the extracellular Ca con-centration either. Therefore the La-Tris solution was able to displace the addi-tional extracellular Ca in the same amount of time and should be applicable to a variety of experiments that examine the effects of changes in extracellular ion levels on the intracellular ion levels. 113 Fig. 19. Top. Measurement of the time required for 10 mM LaCl 3 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. Bottom. Measurement of Ca, Mg, Na and K levels of the guinea pig ileum longitudinal smooth muscle over time in 160 mM Tris-HCl (pH 7.4) containing 10 mM LaC^ at 4°C. Tissue levels were ex-pressed 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 ratio of tissue wet weight to the tissue dry weight. Two strips of guinea pig ileum longitudinal smooth muscle (equilibrated in normal Tyrode's solution) were blotted and weighed at intervals u n t i l they reached a constant weight at room temperature. 116 117 Table 5. Calculation;of the intracellular and extracellular ion concentrations total tissue water 3 extracellular space intracellular space VOLUME/mg wet weight = 8.5 x 10"4 ml = 4.52 x lCT 4 ml b to 5.9 x 10 - 4 ml c = 3.98 x 10"4 ml d to 2.6 x 10"4 ml e Intracellular Space nanomoles ion/mg^ dry wt wet wt concentration (mM) Extracellular Space nanomoles ion/mg^ dry wt wet wt concentration Tyrode's (mM) (mM) ' Ca 5.26 0.8 2 - 3 7.61 * 1.15 2.5 - 1.9 K 212.3 ' 32.0 80.4 - 123 38.6 5.81 12.8 - 9.8 Na 102.6 15.5 38.9 - 59.6 309.7 46.6 103.0 - 79 Mg 21.9 3.3 8.3 -12.7 3.4 .51 . 1.1 - 0.09 1.8 2.7 136 1 a. calculated from 85% tissue water, 1 mg tissue = .85 mg water = 8.5 x 10 ml. b. calculated from the average inulin space measurements of Blowers et a l . (1977) (470 ml/kg wet wt) and Burton and Godfraind (1973) (435 ml/kg wet wt) in the guinea pig ileum longitudinal smooth muscle. > c. calculated from the ^ 4C-sucrose space, Blowers et a l . (1977) (590 ml/kg wet wt) . d. calculated from the difference of the total water - inulin space water. 14 e. calculated from the difference of the total water - C-sucrose space water. f. results from control tissues in Fig. 36 and 37. _ 118 Table 6. Calculation of the resting membrane potential P T P i J K ] • + P M [Na]. .+ P_- [Cl] F - M r? ^ ^ i „ „ K in NaL Jm C1L Jout Em " F C 2 - 3 ) l o § P [K]' • + P M [Na] + P p.[Cl]. K out Na Jout Cl Jin. where, F RT — (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 10~8 Na P c l= 4.4 x 10"8 [Cl]. = 25 mM in [Cl] = 144 mM (Tris Tyrode's solution) out E m when ^ o u t = 2 # 7 ( T r l s ' Tyrode's- solution) [Na] = 136 mM " out [K]. = 80.4 mM (Table 5) in [Na]. = 38.9 mM in -57 mV when [K] = 12.8 mM (Table 5) out [Na] = 103 mM " out [K]. = 80.4 mM in [Na] . = 38.9 mM " m -47 mV 119 Fig. 21. Measurement of tissue ion contents by the modified 'La method' after exposure of the guinea pig ileum longitudinal smooth muscle strips to Tris-Tyrode's solution containing 3.6 mM CaCl2 for 0.5 min (B) and for 5 min (C) as compared to controls (A) equilibrated in Tris Tyrode's solution (1.8 mM CaCl2). The intracellular contents of Ca, Na, K and Mg were independent of the CaCl2 concentration in the Tyrode's solution. Tissues that were washed in the La-Tris solu-tion represent intracellular levels. Tissues that were not washed with La-Tris solution 5^ represent total tissue ion levels. 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 inhi-bition of the Na,K-ATPase (Akera and Brody 1976; Schwartz 1976) but in the pre-sent study, most of the microsomal ATPase activity, stimulated by Mg and Na, was insensitive to K and to 3 mM ouabain (Results and Discussion, Section II). -6 -4 However, the muscle strips were contracted by 5 x 10 M -to 10 M ouabain (Fig. 22, Fig. 23, Fig. 24). 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 inhibition of the Na,K-ATPase. The contraction due to ouabain could have been due to an indirect effect on the nerve plexus. Denervated muscle strips of the longitudinal ileum were prepared by the method of Paton and Zar (1965) to see i f the response to oua-bain remained when the majority of the nerve terminals were removed. The dener--vated muscle strips did not respond to nicotine or neostigmine but did respond to CD and ouabain (Fig. 22, denervated) while the innervated strips were very sensitive to a l l of these agents (Fig. 22, innervated). The denervated muscle strips were not tested for responses to f i e l d stimulation so the complete ab-sence of a l l nerve terminals (non-adrenergic, non-cholinergic) could not be ascertained. Since muscle strips, 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 directly on the smooth muscle. This had been previously shown by Daniel (1964) in rabbit uterine smooth muscle. In this latter tissue, the selective blockade of adrenaline, serotonin, histamine and acetylcholine-induced contractions failed to affect the responses to ouabain. Responses of muscle strips to ouabain were tested at a higher extracellu-lar K concentration to see i f K could antagonize the ouabain response, as would be expected i f ouabain acted by inhibiting the Na,K-ATPase (Fig. 23). Muscle strips 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 Fig. 12, 3rd Row; Bolton 1973a; Casteels 1966). In order to compare contractions of different tissues, the ouabain responses were expressed both as an increase in 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 in 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 oua-bain was enhanced when the extracellular 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 rat myometrium (Murthy et a l . 1974b), the contraction of this tissue seemed to be dissociable from the inhibition of the Na,K-ATPase (Murthy et a l . 1974a). The lack of K antagonism of the ouabain response in the longitudinal ileum is in contrast to the antagonism by K of the ouabain positive inotropic response in the heart (Caprio and Farah 1967). K also competes for the binding of ouabain and the ouabain inhibition of the Na,K-ATPase in the heart (Matsui and Schwartz 1968). This suggests that ouabain may have a different effect in smooth muscle than in heart. Ouabain (10 uM) i n i t i a l l y caused a small rapid increase in tension in the longitudinal i l e a l strips and the tension continued to increase for 2 to 3 min (Fig. 22, innervated; Fig. 24, Top; Fig. 25, Bottom). At longer times, the tension relaxed slowly to baseline, even though ouabain was not washed out of the bath. The phase of rising tension due to ouabain has been called the 'exci-tatory' phase and the f a l l i n g phase of the tissue tension has been called the 'inhibitory' phase. The response to ouabain of taenia c o l i muscle strips (Matthews and Sutter 1967) followed a similar time course to that observed in longitudinal muscle of the ileum. Although ouabain immediately reduced the membrane potential in taenia c o l i c e l l s , the depolarization was slight. The 123 depolarization was accompanied by a 2 to 3 fold increase in spike frequency (3 - 7 min) and after 15 min, by a depolarization block (Casteels 1966; Matthews and Sutter 1967). Complete depolarization was never reached. That depolariza-tion was slight during the i n i t i a l 10 min of ouabain exposure, was consistent with the observation in the present study that a f u l l phasic response to depo-larization by 60 mM KC1 could be attained at the peak of the ouabain response (Fig. 24, Top - top tracing) and even after 10 min when the ouabain response was relaxed (Fig. 24, Top - lower tracing). Therefore the muscle must have been sufficiently polarized in 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 in 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). in taenia c o l i . This effect i s not a selective inhibition of 60 mil KC1, responses as ouabain also quickly relaxed the tonic response to CD. A transient increase in tension was observed in both cases before relaxation began. Changes of the intracellular cation levels of the longitudinal ileum smooth muscle during ouabain contraction and relaxation were monitored using the modi-fied 'La method' (Fig. 25). Intracellular levels 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 nano-moles/mg dry weight, respectively. The contraction of the muscle to 10 uM ouabain reached a maximum between 2 - 3 min (Fig. 25, Bottom). Intracellular Mg content remained stable over 15 min (see also Palaty 1974). Intracellular Ca and Na concentrations increased significantly (99% level) 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 intra-cellular Ca was probably not caused by an inhibition of a Ca pump by ouabain because the extra Ca gained was removed during the inhibitory phase perhaps by a stimulation of a Ca pump at higher cytoplasmic Ca concentrations. The intra-124 cellular K concentration was nqt significantly reduced (95% level) u n t i l after 10 min. After 7 min, the K loss was 5.5% and thereafter the intracellular K content declined and intracellular Na levels rose again. After 15 min the t i s -sue had lost 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 in tissue Ca and Na content, without apparent loss of K, was not consistent with a primary effect of ouabain on the Na,K-ATPase. It appeared that ouabain increased the permeability of the membrane to Ca and Na and perhaps later also to K. Daniel and Robinson (1971a) observed that ouabain did not inhibit K influx as one might expect. Instead, ouabain increased the rate of K efflux from rat uterus which might explain why " K was lost from guinea pig longitudinal ileum after 7 min. But the i l e a l strips, 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 in hearts from intact dogs treated with digoxin and biopsied sequentially. The temporal dissociation of the early onset of posi-tive inotropism and the delayed inhibition of the Na,K-ATPase indicated to them that the positive inotropic effect of d i g i t a l i s glycosides was mediated by a different binding site than the site inhibiting the Na,K-ATPase. Therefore ouabain may not act by inhibition of the Na,K-ATPase in the heart. Various reasons for the early 'excitatory' and later 'inhibitory' (relaxa-tion) action of ouabain have been proposed. Casteels (1966) attributed the 'inhibitory' effect 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 build up of intracellular Na because the relaxation and the increased rate of K loss was prevented in Na free medium (Pfaffman and Holland 1969). In the present study, Na accumulated in the muscle during the 'excitatory' phase to a higher level than when the 'inhibitory' phase had completely relaxed the muscle. Therefore the gain of intracellular Na can not totally explain the inhibitory stage. Tsuda et a l . (1975) have attributed the ouabain induced relaxation of K contractures to a decrease of the increased NADH linked 0 2 consumption produced by a high KC1 con-traction. This may explain why the muscles were unable to maintain a tonic tension in the presence of ouabain (Fig. 24). The Ca gained during the ouabain contraction (Fig. 25) may have entered from the extracellular surface of the membrane, or from the lumen of the caveo-lae or from the extracellular f l u i d . The time dependence of the loss of the ouabain response after removal of the extracellular Ca was studied (Fig. 26). The ouabain response was reduced to 50% after 30 sec in Ca-free Tyrode's solu-tion and thereafter, the response declined gradually un t i l i t was barely detec-table after 10 min. This time course of dependence of ouabain on Ca follows the loss of extracellular Ca in Ca-free Tyrode's solution over 10 min, as measured by the difference between total and internal Ca by atomic absorption spectro-photometry (see Fig. 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 extracellular Ca was responsible for the ouabain response. As the reduction of the membrane potential by ouabain can not be explained on the basis of changes in the Na and K gradients, Draper et a l . (1963) surmised that a Ca current could be responsible for the immediate reduction of the mem-brane potential and the tension development in skeletal muscle by ouabain. A st a t i s t i c a l l y significant (95% level) increase of the intracellular Ca content was observed at the peak of contraction in this study of the longitudinal i l e a l response to ouabain. The increase of the intracellular Ca concentration at the -4 peak of the contraction is approximately 5.3 x 10 M which is more than s u f f i -45 cient for maximal contraction. This confirms the Ca uptake seen during oua-bain induced tension increases in rabbit atria (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 intracellular compartments to the contractile proteins. The excess intracellular Ca was removed at a time coinciding with the onset of the 'inhibitory' phase, perhaps reflecting stimu-lation"? of the Ca pump by the elevated intracellular Ca concentration. The mechanism by which ouabain may increase the membrane permeability to Na and Ca is not known. Ouabain is amphipathic and has a steroid nucleus similar 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, carrier mediated transport and the activity of membrane bound enzymes (Demel and Dekruyff 1976). Enzymatic oxidation of cholesterol causes inhibition of the Na,K-ATPase (Seiler and Feihn 1976). Accumulation of desmeosterol (choles-terol 's precursor) in sarcolemma resulted in intracellular Na loss without a change of the intracellular K levels (Campion et a l . 1976). It is also relevant that desoxycorticosterol, although not an inhibitor of Na,K-ATPase competitively opposes the action of ouabain in the guinea pig ileum at low concentrations but mimics ouabain at higher concentrations (Godfraind and Godfraind-DeBecker 1961). Using fluorescent probes, Charnock and Bashford (1975) observed that the physical state of the membrane lipids seemed to modulate the Na,K-ATPase activity. Inhi-bition by ouabain was particularly sensitive to the physical state of membrane lip i d s , whereas cation activation was less affected (Charnock et a l . 1975). It i s possible that ouabain incorporates, like cholesterol, into the plasma membrane and secondarily moves laterally through the bilayer towards more polar molecules embedded in the membrane, perhaps the Na,K-ATPase. It probably disso-ciates from these regions very slowly because the effects of ouabain on smooth muscle are exerted long after i t is removed from the extracellular medium (Griffin et a l . 1972). Cholesterol tightens the packing of lipids in the fl u i d bilayer of the membrane and i t is possible that i f ouabain incorporated into the bilayer, ouabain would oppose this packing effect due to a greater polarity than cholesterol and a different stereochemistry. High Ca concentrations also stabi-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 destabilizing effect of ouabain. The opposite would be expected at lower Ca concentrations. Indeed, rabbit atria are less sensitive to ouabain at higher Ca concentrations and more sensitive to lower doses of ouabain when extracellular Ca i s reduced, even though i n t r i n s i c activity i s les under these conditions (Caprio and Farah 1967). If the primary action of ouabain was specific inhibition 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 effect of ouabain on innervated and denervated guinea pig ileum longitudinal smooth muscle. The responses to a selective muscarinic agent (CD, 2 x 10~7 M), ouabain (ouab, 10~4 M, denervated; 5 x 10 - 5 M, innervated), nicotine (nic, 18.5 x 10"^ M) and neostigmine (neo, 16.5 x 10 -^ M) in denervated guinea pig ileum longitudinal smooth muscle strips were compared to the responses of the innervated muscle strips to these agents. 13 0 Fig. 23. The effect of increased extracellular K on contraction by 5 uM oua-bain. 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 percen-tage of the fast phasic and sustained tonic control responses. Re-sults represent the mean + S. E. of 10 determinations. ¥-2.b a n c * represent normal Tyrode's solution and Tyrode's solution containing 14.3 mM K, respectively. 132 Fig. 24. Top. Responses to 60 mM KC1 at the peak excitatory ouabain (ouab) response (top tracing) and during the ouabain inhibitory phase (lower tracing). Arrows indicate the addition of 60 mM KC1 and 10 uM ouabain and the washout (w) in normal Tyrode's solution. Bottom. The effect 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 after the tonic res-ponse began. 13 3 134 Fig. 25. Intracellular ion levels (top) during the course of an ouabain res-ponse (bottom). Control total tissue ion levels are indicated to the l e f t of time zero. Control intracellular ion levels are indi -cated at time zero. Points represent the mean + S. E. of n determina-tions indicated in brackets above time markers. (27)06) (19) 06) 03) (10) (9) (8) n W I 10 time in the presence of ouabain (min) 136 Fig. 26. The sensitivity 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 in normal Ca-containing Tyrode's solution. Each point repre-sents the mean + S. E. of n determinations indicated in brackets above the time markers. Examples of responses are included. Solid arrows indicate the addition of 10 uM ouabain. Dotted arrows indicate the change to Ca-free Tyrode's solution (omission of Ca C l 2 from Tyrode's solution). 138 VII. Possible Sources of Ca for the Phasic arid Tonic Contractions of the  Guinea Pig Ileum Longitudinal Smooth Muscle a. The biphasic contraction The excitation-contraction-relaxation cycle of the guinea pig ileum longi-tudinal smooth muscle was studied in muscles contracted by a muscarinic agent, cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide (CD) and by 60 mM KC1. CD was the muscarinic agent chosen for most of the studies of muscarinic induced events because i t is very potent, selective for muscarinic receptors and stable in solution. The chemical structure is given in Fig. 27. Other more familiar muscarinic agents, carbachol (Cch) and methacholine (Mch) induced simi-lar biphasic contractions of the longitudinal ileum smooth muscle. An optimal dose of muscarinic 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 in 10 sec (Fig. 28, 29 and 44). This component is called 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 rise of tension at the 'shoulder' would seem to indicate the start of the slower component of the b i -phasic contraction called the tonic contraction. At f i r s t , the rising tonic tension increases the net tension but after the 'shoulder', the rate of decline of the phasic tension exceeds the rate of increase of the tonic tension. This causes the partial relaxation which clearly separates the phasic and tonic maxi-mum tensions. When the tonic tension increases faster than the phasic component declines, the overall tension increases. The tonic component usually peaks af-ter 5 - 8 min. The sudden appearance of a 'shoulder' of tension after the maxi-mum phasic tension is attained suggests that there is a sequential turning on of the phasic and then the tonic component.rather than a simultaneous commencement of both components upon excitation. Although the 'shoulder' is not a definite proof that the i n i t i a t i o n of the tonic component lags behind the phasic compo-nent by approximately 15 sec, another explanation for the 'shoulder' is not easy 139 to find. At lower doses of a muscarinic agent, longitudinal i l e a l muscles do not relax between phasic and tonic components, nor is 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 in tension in 10 sec. Based on this assumption (which may n o t be valid 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). The magni-tude of the tonic component induced by 2 x 10 7 M CD was less than i t was at a —8 lower dose of 5 x 10 M. The tonic component rose at a slower rate than the rate of relaxation of the phasic component which could account for the partial 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 in shape of the b i -phasic contraction with dose w i l l be offered after the basis for the explanation has been presented (see Results and Discussion, Section VIII). A dose of 2 x 10 7 M CD was routinely used to give a maximal phasic compo-nent easily distinguishable from the slightly submaximal tonic component. De-polarization of a muscle by 60 mM KC1 stimulated a phasic and tonic contraction, similar in magnitude to a control 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) in the same muscle. The 'shoulder' was not apparent in most responses to 60 mM KC1 because the phasic seemed to f a l l so quickly relative to the rate of rise of the tonic component. b. Sensitivity of the phasic and tonic contractions to Ca-free medium The phasic and tonic responses appear to have different sensitivities to Ca removal from the physiological medium. When a response to CD was induced 5 sec after changing the physiological medium in the tissue bath to Ca-free Tyrode's solution, the phasic component attained the same force as i t did in normal 140 Tyrode's solution but i t was not followed by a 'shoulder' characteristic of the ensuing tonic component or the tonic component i t s e l f (Fig. 30, Top). If the 'shoulder' indicates the start of the tonic response (15 sec after addition of CD), then the apparent tonic response was lost in 20 sec or less after removal of extracellular Ca. The tonic component of the response to 60 mM KC1 was lost equally as fast as the tonic response to CD. The phasic component was not no-ticeably reduced when the muscle was stimulated by 60 mM KC1, 5 sec after removal of extracellular Ca. The tonic component would seem to be due to a mobilization of free extracellular Ca because most of the free extracellular Ca should diffuse out of the extracellular space in 20 sec. The residual level of Ca may not be able to in 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 in Ca-free Tyrode's solution. The slower t r a i l i n g off 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 extracellular 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-sic component was reduced to 50% in 2 min and was barely detectable after 10 min. The difference in 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 is illustrated in Fig. 31. The tonic component appeared to be lost very rapidly and could be restored in 30 sec or less. The phasic component also could be completely restored after addition of Ca for 30 sec, which implies a reloading of high a f f i n i t y superficial Ca binding sites. The results were essentially the same for high KC1 and CD responses. Reduction of the extracellular Ca concentration can increase the permeabi-l i t y of the membrane (Shanes 1958). Total and intracellular Ca, Mg, Na and K ion levels were measured in muscle strips equilibrated i n Ca-free Tyrode's solu-tion 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 contractility in Ca-free medium (Fig. 32). After 10 min in Ca-free medium, internal tissue Na, Mg, K and Ca were 110, 87, 86 and 52% of their normal internal levels, respectively. Although the muscle fibers had gained Na and lost K and Mg, which would be expected i f the cells were more permeable, the major change was in the total and internal Ca concentrations. External Ca could be calculated from the d i f -ference between the total and internal Ca levels. The loss of tissue Ca over 10 min in Ca-free Tyrode's solution was plotted for Fig. 32 as the percent of the control internal and external Ca contents of tissues equilibrated in normal Tyrode's solution. This was compared with the rate of the loss of the phasic component from Fig. 30. There appeared to be a correlation between the loss of external Ca and the loss of the phasic component (Fig. 33). Since the free extracellular Ca should diffuse out of the extracellular space in a far shorter time than 10 min, the extracellular Ca that correlates with the phasic tension might be bound or retained in the extracellular space by some means. The outer surface of the sarcolemma may retain Ca that can be transferred into the c e l l when activated by CD or 60 mM KC1. Perhaps caveolae serve in this respect, c. Sensitivity of the phasic and tonic contractions to LaCl-^ LaCl^ has previously been shown to inhibit the phasic component more than the tonic component of the guinea pig ileum longitudinal smooth muscle (Chang and Triggle 1972). It has not been settled whether or not LaCl^ acts only on the extracellular 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, selectively inhibited the phasic component of the high KC1 contraction (Fig. 34). The tonic component of the La-inhibited response to 60 mM KC1 attained the same force but required a longer time to reach i t s maximum. In contrast, both the phasic and the tonic components induced by CD were inhibited by La (Fig. 35). -4 Higher concentrations of La (10 M) are required for complete inhibition of the response to CD. 142 The conditions under which La i s used are important. At La concentrations in the uM range, Ca and Mg concentrations should be l e f t at their normal levels for maintenance of membrane integrity in order to confine La to the extracellu-lar space, basement membrane and sarcolemmal surface. At higher concentrations (.10 mM), La has a powerful membrane stabilizing effect (Casteels et a l . 1972). Under these conditions, Ca and Mg can be omitted from the physiological medium without weakening membrane integrity and without allowing La to enter into ce l l s . La should not permeate intact cells in 5 min, at these concentrations, in the presence of normal amounts of Ca and Mg to maintain membrane integrity. The inhibition of actomyosin Ca-ATPase by La (Fig. 15, Bottom) would indicate that La would prevent contractile force production i f i t penetrated the c e l l . La could not replace Ca for the contractile response when allowed to enter cells 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 cell s , the cells would^nQt havej. been able to contract tonically The tonic component in response to 60 mM KC1 is not inhibited by La, but merely delayed, probably because there are at least 180 free Ca ions per La molecule (at 10 M) to compete for access to the channels. Since La probably displaces Ca bound to the outer aspect of the c e l l , this provides additional evidence that at least part of the Ca responsible for the phasic component could be located on the outside of the* plasma membrane. It is d i f f i c u l t to explain why La selectively blocks the phasic component of the contraction induced by 60 mM KC1 but blocks both the phasic and the tonic compo-nents of the contraction induced by CD. The inhibition of both the phasic and tonic components could be caused by La inhibition of the binding of CD to .the receptor rather than blockade of Ca movements. Although La binds more tightly to most anionic sites than the acetylcholine quaternary ammonium cation, (Hauser 143 et a l . 1976) the concomitant tonic and phasic inhibition may not reflect recep-tor antagonism i f the muscarinic receptor i s a more discriminating membrane anionic receptor site than most of the surface anionic sites. The magnitude of the tonic component induced by CD may depend on the magnitude of the phasic component. If CD inwardly directs Ca from specific receptor associated sites, causing a Ca.depolarizing current, (Chang and Triggle 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 reflect the degree of depolarization allowed when the 'phasic' Ca current was partially blocked. High extracellular 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. If the explanation above is valid :, i t might account for the different La sensitivities df the phasic: and tonic components induced by 60 mM KC1 or CD without necessarily implicating different u t i l i z a t i o n of Ca pools for phasic and tonic responses to different stimuli. The higher valence and similar ionic radius of La to Ca causes La to bind at superficial anionic sites with greater a f f i n i t y than Ca. Triggle 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 pig ileum longitudinal ?2 smooth muscle cell s , to inhibit the phasic component, i s only 1 per 4,000 A . Therefore the replacement of only a small fraction of the total membrane bound 3+ Ca by lanthanides prevents the induction of the phasic component. Perhaps Tm displaces a specific Ca pool or blocks specific Ca sites or channels that are c r i t i c a l for excitation-contraction coupling. d. Measurements of intracellular ion contents during contractions induced  by CD and 60 mM KC1 Although the contractile studies yielded ample information upon which to theorize about the changes in internal ion content during and following a con-traction, i t was f e l t that the best way to determine the events responsible for the observed behavior was to measure internal levels 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 in Na and a gain of K (Fig. 36). The gain of K was the only qualitative difference between the response of high K and the response to CD. Relaxation was accompanied by a rapid loss of intracellular Ca to below normal levels, a gain of Mg, a loss of Na and a sustained higher K content. Equilibration 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 intracellular Ca induced by 60 mM KCl in 10 sec indicated that a mobilization of extracellular Ca gave rise to the phasic ten-sion. From the experiments^ discussed earlier in this section, i t is apparent that the phasic component is not due to free extracellular Ca because the muscle retains some of i t s phasic responsiveness over 10 min in Ca-free Tyrode's solu-tion. Therefore, the Ca responsible for the phasic response i s bound to an extracellular site, perhaps the outer surface of the membrane or bound in caveo-lae. Of a l l the experiments, each pointing to extracellular 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 earlier observations by Triggle and Triggle (1976) that high KCl induced a significant uptake of 4^Ca after 30 sec in the guinea pig ileum longitudinal muscle. In the present study, the high K depolari-zation causes more than sufficient 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 de-45 45 crease in Ca efflux, an increased uptake of Ca and a greater total Ca con-145 45 tent. Triggle and Triggle 0-976) observed significant Ca uptake in guinea pig ileum longitudinal smooth muscles 15 sec (phasic) and 30 min (tonic) after stimul-lation of the muscle strips with 80 mM KCl. Lullman and Mohn (1969) reported that guinea pig ileum longitudinal smooth muscle c e l l s , depolarized by el 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 el e c t r i c a l stimulus and i f the c e l l volume is 9.81 x 10 ml (cylinder 50y x 5u, Paton and Rang 1965) then the intracellular 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 intra-cellular 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 is in 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 in Na and a loss of K (Fig. 36). Relaxation in 30 sec was accompanied by a rapid loss of Ca to below normal levels, a rapid gain of Mg, no change in Na and no change in K. Equilibration was accompanied by a gradual regaining of Ca, an unsteady return to normal of Mg, no striking change in Na and a gradual gain of K to normal levels. The divalent cations, Ca and Mg, changed in opposite directions but the intracellular changes in the monovalent cation, K, were not accompanied by changes in intracellular Na levels. The CD phasic component may also be due to a mobilization of Ca from superficial bind-ing sites. A small increase in intracellular Ca occurred after 10 sec in CD (4.3 x 10 moles/mg dry weight or approximately 6.5 x 10 moles/mg wet weight), 146 -4 which i s sufficient to raise the intracellular 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 for maximal activation of the contractile proteins than the reported ^Ca 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 intracellular compartment (4 x 10 ml) to 10 M. It probably would have been impossible to prove the significance 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, after 10 sec of CD, was not quite significant. -12 The theoretical value of 4 x 10 moles/mg wet weight assumes that a l l the Ca entering is in free form but since some of i t must bind to contractile proteins in order to cause contraction, more Ca would be needed to make the free cyto-plasmic Ca concentration 10 ^  M. In the guinea pig ileum longitudinal smooth muscle, the ratio of mg protein per mg wet weight i s 0,1 (Table 2: total whole homogenate protein = 91,6 mg/900 mg wet weight of tissue). If approximately 50% of this protein i s contractile filament and 10% of myofilaments bind Ca (troponin-like component), then 5 yg protein per mg wet weight w i l l bind Ca. This troponin--12 like component binds 1.3 x 10 mole of Ca per yg contractile 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 sites 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 equilibrated with surface binding sites of longitudinal 45 i l e a l strips for 10 to 60 min, CD induced a significant Ca influx in 30 sec (phasic response) above controls (Blowers et a l . 1977), Without prior 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 influx of free extracellular Ca, but rather i t is due to an influx 45 of membrane bound Ca which requires time to equilibrate with Ca tracer ions to be detectable. 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 ten-sion increase as an optimal dose of the transmitter agent. K seems to induce Ca uptake in excess of that which can be ut i l i z e d by the contractile filaments but 45 the Ca measurements exaggerate this increase (especially at longer times) by ignoring the nonradioactive Ca efflux as i t i s replaced by 4^Ca from the medium. The atomic absorption method developed here yielded more r e a l i s t i c net changes in intracellular ions. The measured Ca uptakes during contractions of.the longi-tudinal ileum by CD, high K and ouabain more than suffice to activate the con-tr a c t i l e apparatus. Freeing of intracellular Ca from sarcoplasmic reticulum or mitochondria would only further increase the excess Ca already present. Yet Ca is present in these organelles in 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 in guinea pig ileum longitudinal smooth muscle suggest that a mobilization of a bound extracellular Ca pool may be a l l that is needed for contraction and is almost certainly a prerequisite for any internal Ca release. The sarcolemmal enriched microsomal fraction contains a Ca-ATPase that could possibly pump Ca out of the c e l l but the ab i l i t y of the microsomes to transport Ca was not investigated. Rapid losses of intracellular Ca were observed 30 sec after termination of high K and CD contractions by washout. If Ca had been temporarily stored in internal organelles, intracellular Ca would have remained high subsequent to relaxation but i t did not. On the other hand, much can happen in 30 sec at the molecular level so the data i s not positive proof that the sarco-plasmic reticulum sacs do not accumulate the Ca before i t is pumped out of the cells. A sarcolemmal Ca pump is probably activated by the higher than normal intracellular levels of Ca to remove the extra Ca since net intracellular Ca 148 levels did not increase unreasonably oyer 10 min, This suggests a cycling of Ca into and out of the cells during stimulation and contraction only when the rate of Ca influx exceeds Ca efflux. "The Ca pump activity may catch up to or overtake the rate of influx 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 cells after 30 min exposures to CD, although uptake was apparent after 30 sec exposures perhaps because the extrusion rate of the pump caught up to the rate of Ca influx. The emphasis on the importance of the Ca distribution 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 is 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. If NaCl was re-duced to compensate for the increased osmolarity of an extra 58 mM KCl the tonic response was transient (data not shown). Therefore, .-the 60 mM KCl responses were induced in the presence of a normal amount of NaCl. The effect of reducing extra-cellular Na on the guinea pig ileum longitudinal smooth muscle is shown in Fig. 38. The normal Tyrode's solution 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 Tris titrated to pH 7.4 with HC1. The higher external K concentra-tion at f i r s t increased the amplitude and frequency of spontaneous contractions but the baseline remained at the same level (Fig. 38, 1st Row). 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 settled down to a higher than normal baseline. The amplitude of the spontaneous activity gradually declined but s t i l l existed at 11% of normal Na, as was also observed by Holman (1957) in ) 149 taenia c o l i smooth muscle . When the Na concentration was zero, spontaneity ceased in the guinea pig longitudinal ileum (Fig, 38, 1st Row) and in the taenia c o l i (Holman 1957). Spontaneous activity returned after 12 min in 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 in tension and a gradual loss of spontaneity (Fig. 38, 2nd Row). The response appeared identical to that observed by Holman ' (1957) when she substi-tuted choline for Na. Control experiments were performed maintaning KCl at i t s normal 2.7 mM in a Tris-buffered Tyrode's solution to see i f a sudden change to zero Na content of the Tyrode's solution s t i l l induced a contraction. The results were positive (Fig. 33, 4th Row). Removal of Na for 10 sec prior 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 rise in 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, substitution for NaCl for 5 sec also altered CD and high K responses compared to controls (Fig, 38, Insert). A response to CD could not be invoked after a prolonged treatment of the muscle in 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 ionic strength and reduces external chloride concentration, thereby reducing K permeability. Its use may not be valid for physiological rea-sons. Tris chloride i s a better but not a perfect substitute for NaCl. At pH 7.4, 30% of the Tris is unionized and penetrates cells thereby eventually dis-placing intracellular K. Therefore, the experiments in Fig. 38 should be inter-preted bearing this in mind. CD contractions could be relaxed in the absence of extracellular Na apparent-ly at the same rate as in the presence of extracellular Na (Fig. 38, 3rd Row). The secondary increase in tension was due to the higher than normal K (see Fig. 150 52, 3rd Row - washout In 5 times normal K). Although extracellular Na would not be reduced to zero immediately, the results imply that contracted longitudinal i l e a l muscles do not reduce intracellular Ca to resting levels by a Na:Ca ex-change 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 relaxa-tion. 151 Fig. 27. Log dose response curve to cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide (CD). The solid line represents the response of the phasic component to dosel The dotted line indicates the res-ponse 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 Fig. 28. The responses of the guinea pig 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. for the tonic and 2 x 10 - 7 M for the phasic. At 2 x 10 - 7 M, there is a distinct shape change.i.e; the phasic and tonic components are separated by a partial relaxation.'.At higher doses.the tonic compo-nent declines in magnitude. i 154 155 Fig. 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: is not-peculiar to CD but rather is a general muscarinic agonist phenomenon. 156 157 Fig. 30. Top. A comparison of phasic and tonic responses in normal Tyrode's solution (control) and after switehMgg to Ca-free TyrodeSs solu-tion for 5 sec prior to 2 x 10~7 M CD or 60 mM KCl addition. The phasic component i s unaffected at 5 sec but the tonic component i s absent. Bottom?. The loss of the phasic component when CD and high KCl are added after various times in Ca-free Tyrode's.solution. The dotted line represents the 2 x.10~7 M CD phasic response.and the solid line 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 Fig. 31. The sensitivity of high K responses (top) and 2 x 10 -' M CD res-ponses (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 KCl Ca-free 30 sec 12 1 7 X Ca-free 10 min 4 10 Ca-free 10 min normal Tyrode's solution 30 sec 8 6 160 f if rriormai Tyrode Ca free Tyrode 10 min normal Tyrode Ca jfree Tyrode 30 S sec Ca free Tyrode 1 0 min . imsn normal Tyrojae 3 0 2u r* [free] Ca Tyrode r norma! Tyrode 3 0 fsec 161 Fig. 32. The effect of incubation of tissues in Ca-free Tyrode's solution for various times on total (solid.line) and essentially internal (dotted line) tissue ion levels. At time 0, n = 50 for internal and n = 13 for total measurements of.ion.contents. A l l other experimental data points are the mean + S. E. (n = 6) 163 Fig. 33. The rate of loss of the phasic component (solid lines) compared to the rate of loss of essentially internal and external Ca (dotted lines). External Ca was calculated from the difference between the average total and the average internal Ca (n = 6) from Fig. 32. 100% of external Ca-was taken as the difference between total (n = 13) and internal (n = 50) Ca levels. External and internal Ca levels at various times after Ca-free Tyrode's solution were calculated as a percent.of the control.levels in normal Tyrode's solution. The percent loss of the.phasic component..was.replotted from Fig. 32. Error bars were omitted for c l a r i t y . 165 Fig. 34. The effect of LaCl 3 (10~ 6 to.10 - 5 M) on high KCl (60 mM) responses in.Tris Tyrode's solution. Each dose of.LaCl3 was.tested on a d i f -ferent muscle because LaCl^ inhibition i s not.readily reversible. Percent inhibition was.calculated.from, a control response (left side). La was added 5 mimibefore• inducing a second .responseww;fc;th6;60 mM KCl (right side). 167 Fig. 35. Comparison of the effect of LaCl3 on the phasic (solid line) and tonic (dotted line) component of responses induced by 60 mM KCl and 2 x 10"? M CD. LaCl3 was added 5 min before the.test.response. Muscle strips were used only once. Points represent the mean + S. E. Table of n Values LaCl3 Concentration CD KCl 10 - 6 M 11 4 2.5 x l O - 6 M 12 4 5.0 x 10-6 12 4 7.5 x 10~§= 7 3 10 - 5 23 4 5.0 x 10 - 5 8 4 4 7-% inhibition of high KCl % inhibition of CD T contractions contractions M 0 3 169 Fig. 36. Intracellular ion levels during the course of a contraction induced by 60.mM KCl (from.10 sec to 10 min).and the equilibration phase (from 0.5 to.30.min) in Tris-Tyrode's solution. Control total tissue ion levels are indicated to the left.of time zero-(i.e. l e f t of the dotted lines). Control intracellular.ion levels, are indicated at time zero. Intracellular ion.levels.at.10 sec and.10 min are indicated along the vertical dotted lines. Intracellular.ion levels during equilibration are to the,right.of.the ;vertical.dotted lines. 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 is drawn at the bottom. 170 3.71 Fig. 37. Intracellular ion levels during the course of a contraction induced by 2 x 10 - 7 M CD (fr om 10 sec to 10.min) and the equilibration phase (from 0.5 to 30 min) i n Tris.Tyrode's solution (TT). Control total tissue ion levels are indicated to the l e f t of time zero (i.e. l e f t of the vertical dotted lines). Control intracellular ion levels are indicated at time zero. Intracellular ion levels during the contraction are indicated between.the .vertical dotted lines. Intra-cellular ion levels during the.equilibration phase are indicated to the right of the dotted lines. The symbols represent (•) Casand (••) 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 con-traction and equilibration phase are:drawn at the bottom. 172 time (min) 173 Fig. 38. The effect of reducing extracellular Na on the longitudinal i l e a l muscle activity. The results represent 1 of 4 muscle strips run simultaneously. 1st Row Control response to CD (2 x 10 - 7 M) in 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.. Tris 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. Ten-sion .increased.after.each reduction in Na. Spontaneous activity disappeared when.Na was reduced to zero. Mus-cles were returned to normal Tyrode's solution (NT) con-taining 149 mM Na. 1 2nd Row After returning.Na (NT) to the muscle, spontaneous a c t i -vity took.12.min to return. A test does of CD (2 x 10~7 M).indicated that the muscle had nearly.returned to nor-mal (compare to control, 1st Row). After washout (NT) and equilibration of the.muscle, a sudden change of Na from 149 to 0 mM.caused a large increase in tension which decayed gradually and relaxed immediately when washed with NT. 3rd Row Another muscle was stimulated with CD and later 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 after the CD responses is probably due to the increased K (see also Fig. 51, 3rd Row). A second dose of CD did not induce a very noticeable response. 4th Row • Similar increases in tension.due to reducing extracellu-lar Nawere observed in Tris-Tyrode's solution at a normal K level. Even.a 10.sec removal of Na prior to CD stimu-lation altered the CD response.by preventing a sustained tension. Insert The effect of reducing Na was not due to Tris substi-tution since CD and high K responses were altered after.5 sec in Na-free.medium iso-osmotically substi-tuted with sucrose. 175 VIII. The 'Desensitization' Phenomenon When insufficient time was allowed between contractions induced by a maxi-mal dose of CD, the shape of the biphasic contraction changed. The muscles relaxed immediately after wash out of CD but were not ready to respond identi-cally again for about 20 to 30 min. During this time, the muscles lacked spon-taneous activity. Subsequent responses induced during the 'quiescent' phase did not have a partial relaxation between the phasic and tonic components and the contractions appeared more like those induced by lower concentrations of CD (10 7 M or less) (see Fig. 28). The peak phasic response was indistinct and i t s magnitude had to be estimated as the .increase in tension after 20 sec. The pha-sic tension decreased when less time was allowed for the muscle to equilibrate between contractions induced by CD (Fig. 39). The magnitude and rate of rise 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 equi-libration (Fig. 40, Left). The time to reach the peak tonic response markedly decreased when the equilibration time was shortened (Fig. 40, Right). Tonic ten-sion rose so soon after the phasic response when the time allowed for equilibra-tion was shortened that the partial relaxation between the phasic and tonic maxima was eliminated. For the experiments shown in Fig. 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 after the f i r s t response, generally caused the second res-ponse to have an increased phasic and a decreased tonic response. The biphasic pattern appeared similar to those observed in Fig. 39 and 40. 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 equilibration between the two responses appeared to be a continuation of the original response as i f the drug had never been washed out (Fig. 42, Left). One can almost visu-alize the desensitized state of the muscle partially returning towards equilibrium during the 1 min washout period, to where i t was after approximately 34 min of contraction, and the second response resuming from that point (Fig. 42, Left). The question arose as to whether :the rapid rise in tension of the muscle by the -second dose of CD to the tonic level then prevailing actually represented a phasic component or was the desensitized muscle only able to respond tonically. This was answered by repeating a similar experiment (as in Fig. 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 (Fig. 42, Left) may have reflected the prevail-ing ionic conditions of the muscle just prior to the washout of CD after 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 equilibration time was shortened. These contractions have distinct 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 equilibration time (Fig. 43). The second of two carbachol contractions induced at 5 min intervals also had a different shape, which confirmed the previous ob-servations with CD and indicated that the biphasic shape was not a unique charac-t e r i s t i c of CD induced contractions (Fig. 44). Test pairs (a f i r s t exposure for 10 in followed, after a set time, by a second exposure) of methacholine responses behaved similarly (Fig. 45). 177 An attempt was made to see i f the slowly reversible event responsible for the shape change occurred during the phasic or the tonic component. The return of spontaneous activity was used as the criterion for estimating the return of the equilibrated muscle state. CD was washed out after the maximum tonic res-ponse was attained, at the point of least tension between phasic and tonic maxi-ma, and just after the peak of the phasic component (Fig. 46, 1st Row). Spontaneous activity returned more quickly i f CD was washed out before the tonic component. The graph at the bottom of Fig. 46 illustrates that the time required for equilibration, measured by the return of spontaneous activity, is 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 criterion 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). If 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 earlier 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 after 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 after 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 equilibration phenomenon not only affected subsequent CD responses but also affected subsequent high KCl responses, which ruled out a specific receptor desensitization and suggested a nonspecific muscle desensitization. As expected from Fig. 43, subsequent responses to high K at 4 min intervals were completely normal (Fig. 46, 3rd Row). 178 The effect 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 results of the experiment outlined i n Fig. 47 demonstrated that the 60 mM K induced tonic component was basically unaffected but the phasic compo-nent was reduced to 60% of i t s control (Fig. 48). The most dramatic effect 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-pletely relaxed between the phasic and tonic components for at least one min. This experiment (Fig. 48, 2nd Row) clearly demonstrates that the phasic and tonic components of a high KCl response are completely separate events which probably mobilize two different Ca pools for contraction. Since high K responses equilibrate quickly and CD responses do not, i t was of interest to see what would happen to the contractions and the rate of equilibration 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 inter-mediate to that of either alone (Fig. 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 for their tensions to f a l l to baseline. In fact, tension climbed instead of de-. clining just after washout of the high KCl and CD. This can also be seen in Fig. 46, 3rd Row. Although relaxation was slow, the muscles gave separate phasic and tonic responses when CD was added again after 10 min in normal Tyrode's solution. A subsequent response to CD after a 10 min equilibration had a separate phasic component but i t was very reduced and the tonic component maximized earlier. The presence of 60 mM KCl during the response to the com-bined stimuli 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 after the 60 mM KCl and CD were washed out. The presence of higher than normal KCl concentrations in the Tyrode's solu-tion used to wash out the' response to> CD;,alsoenabled' theimusjEle to^quickly regain spontaneous activity (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 con-centration, spontaneity did not return for more than 1 hr. At twice the normal KCl concentration, spontaneity of the muscle returned in half the time (about 12 min) taken in normal Tyrode's solution (25 min). With 5 times the normal K concentration, spontaneous activity returned in about 5 min (see also Fig. 52, 3rd and 4th Rows). When the spontaneous activity returned, i t appeared increased in magnitude and rate. At higher external K concentrations, the resting membrane potential 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 specificity of the K hastening effect on equilibration, measured by the return of the usual biphasic appearance of the response to 2 x 10 ^  M CD, was ascertained by raising 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 in 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 additional 70 mM NaCl i.e. one and a half 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 in this regard of an extra 8 mM KCl (Fig. 51, 2nd Row). Therefore the effect of K is not due to increasing ionic strength but is a specific effect of K. Lowering Na to 1/2 of i t s normal concentration hindered the equilibration process although the rate of equilibra-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 is insufficient 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 equilibrate 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 7 M ouabain for 30 min after complete equilibration would prevent the distinction between the phasic and tonic components of a response to CD (Fig. 52, 2nd Row - to the right of the dark line). The ouabain reduced the magnitude of the response somewhat but had l i t t l e effect on i t s biphasic appearance: and other than a reduced tonic component (expected because of the experiment in Fig. 24), the contraction was f a i r l y similar to the control response. Therefore the odd response to CD after equilibrating a previous response to CD in the presence of 5 x 10 7 M ouabain, was due mainly to the effect of ouabain,to prevent equilibration of the post-CD contracted condition. Whether this was due to inhibition of the Na,K-ATPase is not certain. The more marked effect of ouabain on the equilibration process implies that CD had given ouabain a lead on altering the intracellular K, Ca, Mg and Na levels. The relaxation of the tonic response to CD by ouabain was more rapid than relaxation of the high K-induced tonic component by ouabain, per-haps because the loss of K at that point predisposes the muscle to relaxation by ouabain (Fig. 24). Raising the extracellular Ca concentration to 5 times i t s normal level (1.8 mM) and Mg to 3 times i t s normal concentration did not alter the equilibra-tion time noticeably (Fig. 53). The lack of effect of raising the extracellu-lar Ca concentration would indicate that the rate of relaxation by a Ca pump and the rate of the return of spontaneous activity by other ion transport enzymes are independent of the free Ca concentration gradient across the sarcolemma. Determination of the energy requirements for equilibration was d i f f i c u l t . Lowering the temperature to halt the activity of active transport enzymes pre-vented spontaneity and increased tissue tension, thereby eliminating the 2 c r i -teria used to monitor equilibration. N Instead, the energy sources of the muscle were limited by omitting dextrose arid 0^  in a Tris buffered Tyrode's solution 181 (Fig. 54). Spontaneity remained after 30 min in this N 2 gassed medium to which no dextrose was added (Fig. 54, Row 1) therefore this criterion could s t i l l be monitored. After re-equilibration themuscles with 0 2 in a medium containing dextrose, the muscles were contracted with CD for 10 min. After a 10 min res-ponse to CD, a washing out solution without dextrose or 0 2 s t i l l allowed the muscle to regain spontaneous activity in the normal length of time (Fig. 54, 2nd Row). The next response had enough energy for the phasic spike but was unable to sustain a tonic tension. After re-equilibrating, the muscles were tested for their a b i l i t y to contract following a much shorter (5 min) period of 0 2 and dextrose deprivation. The muscles fatigued rapidly again. A normal response could be produced after supplying energy raw materials again. New muscles were tested to see i f they also fatigued rapidly after a 1 min dextrose and oxygen depletion (Fig. 54, 3rd Row). Compared to the control response, the tonic response declined rapidly. It was concluded that the energy requirement for equilibration was far less than the energy expenditure during contraction. If energy is required for equilibration, the store of ATP, creatine phosphate and production of ATP by gluconeogenesis coupled to glycolysis in the muscle was sufficient to permit i t . Desensitization of the muscle was probably not due to a depletion of high energy stores because in that case, the high K tonic compo-nent would also be expected to decline. Tsuda et a l . (1975) observed that i n -creased tension caused an increase in ADP levels which in turn, progressively stimulated 0 2 consumption in taenia c o l i . Contraction by CD and high KCl i n -creased the energy demand of the muscle and increased the activities of enzymes involved in the Krebs cycle and oxidative phosphorylation in taenia c o l i (Bostrom et a l . 1973), so that anabolism was stimulated to keep up with catabolsim. Therefore, desensitization or tachyphylaxis is unlikely to be due to metabolic insufficiency. The measured intracellular Ca, Mg, Na and K levels during the contraction induced by CD and the relaxation and equilibration phases following washout of 182 CD are also relevant to the present discussion (Fig. 37), A brief repetition of the results follows. The responses to CD were accompanied by a small gain of Ca, a loss of Mg, no apparent change in Na and a loss of K. Relaxation in 30 sec was accompanied by a rapid loss of Ca to below normal levels, a rapid gain of Mg, no change in Na and no change in K. Equilibration of the responses of the muscle to CD over 20 - 30 min was accompanied by a gradual gain of Ca, an jinsteady return to normal of Mg, no striking change in Na and a gradual gain  of K to normal levels. The above results w i l l be compared to the ion level changes induced by 60 mM KCl which produces a contraction that does not require v. a long time to equilibrate 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 real change in Na and a gain of K (Fig. 36). The gain of intracellular K was the only qualitiative difference between the high K res-ponse and the response to CD. Relaxation was accompanied by a rapid loss of Ca to below normal levels, a gain of Mg and a loss of Na and the K content re- mained higher than usual. Equilibration was accompanied by a return to normal of Ca levels, a fluctuating but generally returning to normal Mg content, main-tained lower Na levels and a maintained higher K concentration. The gain of Ca and loss of Mg were quickly reversed upon relaxation. They are probably not rate limiting factors for equilibration. On the other hand, the loss of K during a response to CD and the slow re-accumulation of K may limit the rate of eq u i l i -bration. 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 equilibration after contractions induced by CD (Fig. 49) and also would explain why high KCl responses equilibrate rapidly (Fig. 42). Arterial muscles made K poor by cold storage, recover their a b i l i t y to respond to stimuli more quickly in solutions with higher K concentra-tions (Barr et a l . 1962). Barr et a l . (1962) concluded that the intracellular K concentration determined the contractiltity and the rate of relaxation and 183 that the ratio of the intracellular and extracellular K concentrations deter-mine the excitability and extent of the tonic shortening. The significant loss of K is a well established phenomenon of contractions induced by muscarinic agents in 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 in order for the muscle to contract, while others thought that the K loss during contraction might be an artifact caused by mechanical deforma-tion during tension development (reviewed by Setekleiv 1970). The efflux of K from rat uterus is increased by acetylcholine but not by oxytocin while both agents cause contraction and the acetylcholine-induced-K-efflux i s therefore not an artifact of contraction (Hodgson and Daniel 1972). After 1 hr in Ca-free medium, pilocarpine w i l l not induce a contraction but a moderate additional increase in potassium efflux i s s t i l l apparent (Hurwitz et a l . 1960). The dis-sociation of contraction from K loss i s apparent in the present study because the same force was developed by 2 x 10 7 M CD and 60 mM KCl induced contractions but only the muscles contracted by CD lost K. The loss of K therefore does not seem to be necessary for contraction but desensitization of muscles to repeated doses of muscarinic agents may be related to an intracellular deficiency of K. Burgen and Spero (1968) observed that the K efflux induced by carbachol was minimal at submaximal doses. With higher doses of carbachol, causing near maximal or maximal contractions, the induced K efflux increased dramatically. At lower doses of CD (Fig. 28) spontaneous activity returned more quickly, indi-cating that desensitization of the muscle was less at lower doses and may be related to.the smaller loss of K at these doses. The sudden change in the shape of the biphasic contraction at 10 7 M to 2 x 10 7 M CD (Fig. 28) may be related to the increased K efflux 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 just to a subsequent response to acetylcholine, because the 184 degree of desensitization was greater when the time interval between responses was shorter. Weiss et a l . 1961 observed that acetylcholine caused a larger K efflux than pilocarpine and also the contraction induced by acetylcholine was less 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 certainly, the responses to 60 mM KCl had more sustained tonic compo-nents probably because under the two latter conditions, the tissue would not lose K. The slower rate of rise of the tonic component and therefore the partial re-laxation between the phasic and tonic components probably was due to progressive desensitization during the response to CD. Subsequent responses to doses of — 7 —8 —7 2 x 10 M CD appeared more like responses to doses of 5 x 10 or 10 M CD as i f the muscle only recognized 1/4 to 1/2 the number of CD molecules. But the response to these lower doses had a faster rising tonic component which was greater in magnitude than the usual response to 2 x 10 7 M CD (Fig. 40, Left). It was d i f f i c u l t to reconcile a greater response occurring more rapidly with a desensi-tization. Perhaps the slow, suppressed tonic component of a response to 2 x 10 ^ M CD was due to an opposition of an inward Ca influx by a rapid opposing K efflux. When the intracellular K concentration has been reduced by previous doses to CD, subsequent doses of CD may induce Ca influx during the tonic component unopposed by a significant outwardly directed K efflux. The unopposed Ca influx may cause the tonic component to rise more quickly and to a greater magnitude. Indeed, Burgen and Spero (1968) observed that the K efflux was not stimulated by subsequent maxi-mal 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). The onset of the second response was delayed but the maxi-mum force was reached earlier. 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 appli-cation and the interval allowed for recovery. The delayed second response of the 185 muscle was thought to be due to it s depolarization being opposed by the faster repolarization 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 deficient solutions and therefore the acetylcholine induced 'after-hyperpolari-zation' was explained on the basis of an electrogenic pump (electrogenicity based on a 3 Na:2 K pumping ratio). In the present study, responses of the muscle to CD which were washed out with K or Na deficient Tyrode's solution or in Tyrode's solution containing 5 x 10 7 M ouabain were prevented from re-equilibrating. These results agree with the observations by Bolton (1973b). Raising extracellular Na, Ca or Mg concentrations had no effect on the equilibration phase but raising the extracellular K concentration accelerated the recovery. Raising the extra-cellular K concentration hyperpolarized a K deficient 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 in the present study because the reduced intracellular K levels were not rapidly regained. The K efflux induced by higher doses of carbachol was refractory to subsequent doses of carbachol for up to 1 hr (Burgen and Spero 1968) which also indicates that the regaining of intracellular K takes a long time and may be inconsistent with a high rate of Na,K-ATPase pumping of K into the c e l l . Kehoe and Ascher (1970) evaluated the activation of an electrogenic sodium pump to account for potential changes and demonstrated that a hyperpolarizing synaptic potential was the result of an increased potassium permeability. Quot-ing these authors, "there is no reason to doubt that the Na-K pump might be activated after a synaptic potential, insofar as a change in cationic permeabi-l i t i e s w i l l lead to a change in internal cation concentrations. It appears doubt-f u l , however, that the minute changes involved in most synaptic potentials are 186 sufficient to trigger a detectable 'pump' potential change," Romero and Whittam (1971) observed that red blood c e l l ghosts with, higher intracellular Ca concen-tration lost K and suggested that the permeability of K and Na could be regula-ted by internal Ca which in turn could be regulated by a calcium pump. Similar experiments in red blood c e l l s , with modifications to test other hypotheses, yielded results that indicated that the intracellular Ca control of K permeabili-ty might be mediated through the Na-K pump because of i t s inhibition by ouabain (Blum and Hoffman 1972), but Lew (1974) had doubts about this because ouabain was without effect on the Ca dependent K loss at ATP concentrations lower than 10 ^ M. 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 ineffective in this regard. Knauf et a l . (1975) noted that increas-ing K above 4 mM on the outside of red blood cells inhibited the efflux. A Ca-activated K channel (sensitive to Ca antagonists) generated an 'after-hyperpolari-zation' in cat, frog and snail neurones and was distinct from the K channel (tetraethylammonium sensitive) which controlled repolarization of the action po-tential (Krnjevic et a l . 1975; Barrett and Barrett 1976; Heyer and Lux 1976). Bursting pacemaker cells of the Aplysia R15 neuron, which have an inward Ca cur-rent, gained intracellular Ca during each burst and the increase of intracellular Ca was sufficient to cause a hyperpolarization (Thomas and Gorman 1977). Internal Ca levels are high in contracting smooth muscles and they have an increased K permeability (Born and Bulbring 1956; Burgen and Spero 1968) perhaps through Ca-activated K channels. The 'after-hyperpolarization' following wash-out of an acetylcholine-induced contraction 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 forth a similar hypothesis to explain the hyperpolarizing effect of a adrenergic agents on the guinea pig taenia c o l i . They feel that Ca bound to the inner surface of the sarcolemma regulates the membrane permeability to K which in turn determines the membrane polarization 187 and consequently the degree of spontaneous activity. The a adrenergic agents may increase the binding of Ca at negative sites 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 ex-tent by the activity of an active Ca extrusion mechanism in the sarcolemma. A similar effect of membrane bound Ca has been postulated to explain why excess Ca bound to the plasma membrane, hyperpolarizes arid stabilizes the membrane but also causes a decreased membrane resistance (Bulbring and Tomita 1969). The decreased membrane resistance of a stabilized, hyperpolarized membrane is com-plicated in view of current theories of the effect of Ca on membranes. Excess external Ca was found to hyperpolarize the membrane and increase the K conduc-tance (Bulbring and Tomita 1969). Low external Ca depolarizes the membrane and decreases the K conductance. 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 is the observation that pilocarpine s t i l l causes a moderately increased K efflux in the guinea pig ileum longitudinal smooth muscle cells that have been bathed in Ca-free medium for 1 hr even though contraction was prevented (Hurwitz et a l . 1960; Weiss et a l . 1961). A closer examination of the data indicates that the K efflux induced by pilocarpine after the muscle was in 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 high-er cytoplasmic Ca concentrations but did not cause an increased K efflux while acetylcholine caused contraction and increased K efflux (Hodgson and Daniel 1972). The model can be accommodated to account for these anomalies. The activity 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 site on the ex-188 ternal surface of the membrane i s removed and relocated on the inner surface. Agents such as acetylcholine, by relocating Ca bound on the outer aspect-of the c e l l to the inside of the membrane, might effectively activate the K channel far better than agents that only add Ca to the inside of the membrane (agents that release internal Ca) or conditions which only remove Ca from the outside of the membrane, such as in Ca-free medium. However, conditions that remove extra-cellular Ca or increase intracellular Ca might be expected to act synergistically with acetylcholine to activate the K channel to a greater extent. Indeed, Chang and Triggle (1973b) have observed that the desensitization process to CD is enhanced at lower external Ca concentrations. The K efflux induced by pi l o -carpine in Ca-free medium (Hurwitz et a l . 1960 and Weiss et a l . 1961) could also be accounted for by a greater sensitivity to a smaller reallocation of Ca from external receptor sites to the internal 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 effective for activating the K channel at normal external Ca concen-trations. They may be more effective for activating 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 contractile filaments more than to the Ca sites 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 cells to more than saturate the contractile filaments and the extra Ca may activate K channels. This may account for the displacement of the log dose-K-efflux-response curve to the right of the log dose-contractile-response curve induced by carbachol (Burgen and Spero 1968). Chang and Triggle (1973a) observed that par t i a l agonists con-tract the guinea pig ileum longitudinal 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 partial agonists. These results may indicate vthat only Ca which can be rapidly mobilized from membrane bound sites to the inside of the c e l l for 189 the phasic component activates a K channel, Ca released from any other site may not be able to increase K permeability (e.g. Ca released by oxytocin in uterus; Hodgson and Daniel 1972), However, the experiments in 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 literature are analyzed, i t would appear that a K channel activated during the phasic component is maintained during the tonic component. The extent of the loss of K during the tonic component, the degree of desensitization and the time required for re-accumulation of normal intracel-lular K levels after washout of CD may be related to the time that the K channel is held open in 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 in Ca-free medium but the tonic did not; Fig. 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 inefficiently for tension development in 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. It i s similar in shape to the contractions induced by 60 mM KCl in K poor tissues after cold storage. In both these cases, the relaxation between the phasic and tonic responses i s accentuated instead of being absent as in the responses to CD in desensitized muscles. One would not expect a hyperpolarizing-outward-K-current to occur in the face of a 60 mM extracellular K concentration. The K gradient may even be reversed temporarily u n t i l the c e l l regains K. Any further explanation about the responses to 60 mM KCl in a desensitized muscle at this point would be un-warranted speculation. Although Ca i s immediately pumped out upon relaxation, K does not return to i t s normal level for some time after a CD response (Fig. 37). The increased 19 K permeability may counteract the pump for some time after relaxation despite the removal of the higher intracellular Ca levels. Though net intracellular Ca levels are lower after 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 levels immediately. The data presented in the Results and Discussion in section II tend to indicate that a K stimulated pump is a minor component of the total cation stimulated ATPase activities that might be responsible for ion transport in these longitu-dinal i l e a l muscles. The Na,K-ATPase activity may be rate limiting for the re-versal of the desensitized muscle state. 191 Fig. 39,. Changes of the biphasic contractile pattern when contractions to CD were induced at shorter equilibration time intervals between contrac-tions. 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 earlier as the equilibration times was shortened—therefore exposures to CD were shortened accord-ingly. The tracing is an example of one of the four experiments graphed in Fig. 40. \ 193 Fig. 40. Graphical representation of the effect of equilibration time on con-tractions by CD. The results were obtained from experiments exempli-fied in Fig. 39. In the l e f t hand graph the dotted line represents the tonic component and the solid line represents the phasic compo-nent. The right hand diagram i s a measure of the time required for the tonic component to peak after the addition of CD to the muscle. Equilibration time refers to the time allowed for equilibration in normal Tyrode's solution between CD responses. Points represent the mean + S. E. (n = 4) 194 5» 0) • acorn 195 Fig. 41. The effect of the equilibration 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 - 7 M) was kept constant at 10 min. The effect of this response was tested on a second response to CD, 2 to 8 min later 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 eq 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 per-cent of the paired control response. The dotted line represents the tonic and the solid line represents the phasic component. Points represent the mean + S. E. (n = 7) 196 197 Fig. 42. Left The effect of a very short equilibration time (30 sec) on a CD response.after a long exposure to CD (35 min). The CD concentra-tion was 2 x 10"7 M. Right Effect of a short equilibration time in Ca-free Tyrode's solu-tion 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 equilibration time in 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 characteristic of a tonic response. 198 199 Fig. 43. The lack of effect of the equilibration 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 equilibrate between contractions (graph at bottom). Points represent the mean + S. E. Cn = 4) 201 Fig. 44. The effect of the equilibration time allowed after a 10 min response to carbachol (Cch) on a second contraction induced by carbachol. An appropriate concentration of carbachol to give distinct phasic and tonic components was found to be 10^ M (see l e f t side). Washing (W) for 5 min was insufficient 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 equilibration 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) in normal Tyrode's solution. 204 205 Fig. 46. The effect of the duration of exposure to CD (2 x 10 -' M) on the time required for equilibration, monitored by the return of spontaneous activity and the subsequent return of the normal biphasic contractile pattern. 1st Row The time required for the return of spontaneous activity was measured when CD was washed (W) out (1) after the tonic maxi-mum, (2) at the point of least tension between the phasic and tonic components, and (3) after the phasic peak (See Graph at bottom). 2nd1 and 3rd Rows The biphasic appearance of the second response of a test pair was monitored as a means of determining the equi-libration state. Four min was allowed between contractions to CD of different durations. The f i r s t contractions to CD were allowed to continue (1) unt i l the tonic component was maximal, (2) until the point of least tension between the phasic and tonic components was reached and (3) unt i l the phasic component peaked. Complete equilibration was allowed between the test pairs. 3rd Row After a phasic and tonic response to CD, 60 mM KCl was add-ed 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 strips run simultaneously in the experiment represented in the 1st Row were graphed. Points represent the mean + S. E. K 3 O as 207 Fig. 47. The effect of a 10 min exposure to CD on a response to 60 mM KCl, 2, 4, 6, 8 and 12 min after the response to CD was washed out (W). The responses to 2 x 10 - 7 M CD of these longitudinal i l e a l strips had different biphasic patterns than were usually observed. The tracings are one example of 4 muscle strips run simultaneously. 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 in the 2nd Row except that 6 and 8 min were allowed for equilibration between the responses to CD and 60 mM KCl. 4th Row A test pair with 12 min between the CD and 60 mM KCl responses followed by a control response to 60 mM KCl. 208 209 Fig. 48. Graphical representation of the effect of the time allowed for equ i l i -bration after a response to CD on a response to 60 mM KCl. Experi-ments are of the type shown i n Fig. 47. Results are plotted 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 M CD on the equilibration phase of the muscle. 1st Row Control responses to CD and 60 mM KCl determined separ-ately. 2nd Row Simultaneous addition of CD and 60 mM KCl gave a contrac-tion shape intermediate between the separate responses. Washout (W) in normal Tyrode's solution did not cause immediate relaxation, which was different than either response alone. 3rd Row The shape of a CD response after a 10 min washout after the simultaneous addition o f 60 mM KCl and 2 x 10 7 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, after 10 min, was abnormal. 213 Fig. 50. The effect of altering the concentration of KCl in the Tyrode's solu-tion on the rate of return of spontaneous activity after 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) in 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 activity. Examples of the experiment are included on the graph. Points represent the mean + S. E. Chart of n Values KCl Cone. n 1.35 mM 3 2.7 11 5.4 8 6.7 4 8.1 8 10.7 8 13.5 4 ,214 215 Fig. 51. The specificity of increased external KCl for accelerating the return of the usual biphasic contractile pattern compared to the lack of effect of higher extracellular NaCl concentrations. The experiment shown is one of four run simultaneously. 1st Row A control test pair of CD responses (2 x 10 7 M) with a 10 min equilibration in 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 in Tyrode's solution containing four times the normal KCl concentration for 8 min and 2 min in nor-mal Tyrode's solution before the second response. The extracellular KCl concentration was raised 8 mM (2.7 to 10.7 mM). 3rd Row A test pair of responses to CD equilibrated in 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 in 205 mM NaCl (lh times normal) for 8 min and 2 min in normal Tyrode's solution. 217 Fig. 52. The effect of various agents on the time required by the muscle for the return of spontaneous activity after a 10 min response to CD (2 x 10 - 7 M). Each row of contractions represents one of four experi-ments . 1st Row The effect of reducing the extracellular NaCl concentra-tion on the time required by the muscle to regain spon-: taneous activity. Tris-HCl was used to maintain the osmolarity. An extracellular NaCl concentration of 3/4 the normal level was sufficient to allow the muscle to regain spontaneous activity in the usual length of time, but at 1/2 the normal extracellular NaCl concentration, the spon-taneous activity of the muscle did not return for more than 1 hr after a response to CD. 2nd Row The effect of a low dose of ouabain (5 x 10 7 M) (which is insufficient to cause contraction) on the rate of return of spontaneous activity after a 10 min response to CD. Spontaneous activity did not return after 40 min and a subsequent response to CD was abnormal. The second half of the,2nd Row shows a control experiment indicating that the presence of 5 x 10 - 7 M ouabain for 30 min did not extensively alter 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. 3rd Row > The effect of altering the extracellular KCl concentration on the time required for the return of spontaneous a c t i -vity of the muscle. These Rows are a complete tracing of the experiment shown in Fig. 50. AM C D N T 28 C D N s / C D N T 24 >4i 0.75 x Na 24' CD W 5X10"7M ouab CD N T CD NT N T C D ' C D N T /3XK 7.6 w 0.5 xNa i . no spontaneous activity aftar i.hr. CD 0.5 xK C D W 5X10"?M ouab 30 4xK 7.2 C D .5XK C O w 5.2 "CD ^ 219 Fig. 53. The effect of raising the extracellular Ca and Mg concentrations on the time required by the muscle to regain spontaneous activity. The experiments shown are one of four which were run simultaneously. 1st and 2nd Rows The effect of a five-fold increase of the extra-cellular Ca concentration on the time required for the muscle to regain spontaneous activity following a 10 min response to 2 x 10 - 7 M CD. The muscle was returned to the normal Tyrode's solution after the return of spon-taneous activity and before the induction of the next 10 min response to CD. The muscles regained spontaneous activity at about the same rate in higher extracellular Ca concentrations as they did in 1.8 mM CaC^. 3rd Row The effect of a three-fold increase of the extracellular MgCl2 concentration on the rate of return of spontaneous activity. Raising the extracellular MgCl 2 concentration did not change the rate of return of spontaneous activity compared to controls. i g «J2™=.'ixCa fwXi 28.4 fi.5 xCa 264 CD m 2xCa 29.6 bxCa 29.6 i A. 4xCa ZOA 5xCa 30 X C a 26' ,ixMg 18 19.2' 1^ ' \ 16' N 3 -O 221 Fig. 54. The effect of depriving the muscle of dextrose and oxygen on the time required for the return of spontaneous activity. Tris buffered Tyrode's solution (TT) was used so that 95% 0 2 - 5% C0 2 would not be required to maintain pH. N 2 was bubbled through the tissue chambers to drive off 0 2 from the Tyrode's solution. 1st Row The time required for the muscle to regain spontaneous activity after a control response to 2 x 10 -' M CD was approximately 30 min. After 30 min in Tris-Tyrode's solution without dextrose and N 2, the spontaneous a c t i -vity (the test parameter) of the muscle was retained. The muscle was re-equilibrated with dextrose and 0 2. 2nd Row After a 10 min response to CD, the muscle was equilibra-ted in the absence of dextrose and 0 2. Spontaneous activity returned in 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 2 and dextrose. De-privation of 0 2 and dextrose for 5 min also caused a rapid decline of tension after the phasic component. 3rd Row the muscle was not permanently damaged as can be seen by the response in the 3rd Row of the same muscle after re-equilibration in Tris-Tyrode's solution. New muscles were tested for their a b i l i t y to contract after 1 min of dextrose and 0 2 deprivation. Even after 1 min the tonic tension dropped off far more quickly than the control res-ponse. to to 223 SUMMARY AND CONCLUSIONS Ion movements"during and after contraction of the guinea pig ileum longi-tudinal smooth muscle were studied in an attempt to examine some of the factors controlling the excitation-contraction-relaxation cycle. The structure, contrac-t i l e characteristics and ion movements were investigated in the intact tissue and a sarcolemmal enriched fraction of the muscle was analyzed for ATPase activities related to ion transport. Net changes of intracellular Ca, Mg, Na and K contents were measured using a modified 'La method' that displaced extracellular ions from the tissue in an isotonic Tris-HCl solution containing 10 mM LaCl^ at 4°C. The hypothesis that was tested was that excitation-contraction coupling in some types of smooth muscle cells may be regulated by trans-sarcolemmal fluxes of Ca. The following results were consistent with the above hypothesis. (1) Guinea pig ileum longitudinal smooth muscle cells are very small and their surface areas are greatly increased by caveolae. The large surface to volume ratio suggest that they might use extracellular Ca for contraction. (2) Contractions induced by 60 mM KCl and cis-2-methyl-4-dimethylaminomethyl-l,3-dioxolane methiodide (CD) were very sensitive to omission of Ca from the Tyrode's solution. (3) The tonic component of.the contraction was lost after 20 sec in Ca-free Tyrode's solution at about the same rate as the free extracellular Ca would be expected to be lost. (4) The phasic component decreased over 10 min in Ca-free Tyrode's solution and followed the rate of removal of a bound fraction of Ca from the extracellular space. (5) Readdition of Ca to the Tyrode's solution restored both the phasic and tonic components in less than 30 sec. Therefore i t seems that the Ca used for contraction is located superficially. (6) Ca mobilized for the phasic component can be displaced by La. Consequently, Ca utilized for the phasic component may be bound to the out-side of the plasma membrane or perhaps may be stored in caveolae. (7) Stimulation of the ileum 1longitudinal smooth muscle, by 60 mM KCl or by 2 x 10 7 M CD, induced Ca uptake (>6.5 x 10 "^moles/mg wet weight) in 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;i,to.v...be;r_increas'ed . to • -4 approximately .- 2 x 10 M by 60 mM KCl or. CD. A Ca-ATPase activity was found in a sarcolemmal enriched microsomal fraction. 2+ The Ca-ATPase activity was stimulated by free Ca concentrations over the range -7 -4 of 10 to 2.4 x 10 M. The elevated Ca levels in the tissue during contraction were reduced in 30 sec, perhaps by a sarcolemmal Ca-pump associated with the Ca-ATPase activity found in the sarcolemmal enriched fraction. The microsomal vesicles were postulated to be pinched off caveolae. The caveolae may also func-tion as active transport centers for ion pumping. The sarcolemmal-enriched micro-somal fraction had high specific a c t i v i t i e s for a Na-stimulated Mg-ATPase but the stimulation by K which could be inhibited by ouabain was only a small compo- • nent of the total ATPase activity. After stimulation of the muscle by a muscarinic agent, the smooth muscle cells not only gained Ca but lost K rapidly. The loss of K was postulated to be via a Ca-activate-K-channel. When the muscarinic stimulus was washed out, the excess Ca was pumped out but K levels were not regained quickly. The muscle immediately relaxed after the stimulus was washed out, but basal tension was lower than usual and the musclet.did not spontaneously contract for approximately 20 min by which time the tissue had regained the K. An 1after-hyperpolarization' was previously reported to follow the washout of a muscarinic stimulation of the guinea pig ileum longitudinal smooth muscle. In the present study, the 'after-hyperpolarization' effect was explained by a greater K permeability through a Ca-activated-K-channel rather than by the stimulation of an electrogenic Na,K pump because K was not regained quickly after the contraction was relaxed. De-sensitization of the muscle appeared to be correlated with the rate of loss of intracellular K. The tonic component increased more slowly under conditions which would open or leave open the Ca-activate-K-channel. By elevating the extra-cellular 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 stimuli, The rate of re-accumula-tion of K by the muscle cells may limit the rate of equilibration of the muscle following contraction because the de f i c i t of intracellular K i s large and the activity of the Na,K-ATPase is low. f The contraction induced by ouabain occurred before any loss of intracellu-lar K was detectable. Therefore i t s action seemed to be dissociated from inhi-bition of the Na,K-ATPase. Instead, the contraction induced by ouabain coincided with a gain of intracellular Ca and Na. 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CGation levels after depletion in Ca and Mg free Tyrode nanomoles/mg dry wt. treatment Ca Mg Na K total ions 20.6 + .92 25.9 + .58 506.7 + 48.7 211.2 + 16.8 (D{)CaMg]a10 min 37°C 9.83 + .67 19.5 + .95 499.6 + 44.4 188.2 + 17.1 0[CaMg] 10 min 37°C 20 min 4°C 7.98 ± - 2 7 22.7 + .90 481.1 + 49/1 152.4 + 14.3 0[CaMg] 10 min 37°C 50 min 4°C 8.00 ± - 7 5 21.6 +1100 519.8 + 58.7 105.9 + 6.1 0[CaMg] 10 min 37°C 1 hr 50 min 4°C 6.76 + .25 19.6 + .53 611.6 + 95.7 67.0 + 9.9 0[CaMg] 10 min 37°C 2 hr 50 min 4°C 6.84 + .57 19.8 +2.1 604.2 + 45.7 61.6 + 7.3 0[CaMg] 10 min 37°C 3 hr 50 min 4°C 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 2(10 min) • min 37°C .25 M sucrose 4°C 6.91 +1.19 15.3 +1.9 168.3 + 9.6 121.0 + 25.3 0[CaMg] 10 2(10 min) . min 37°C 50 min 4°C 25 M sucrose 4°C 5.71 +5.70 17.3 +7.95 222.7 +236.2 75.2 + 74.7 0[CaMg] 10 2(10 min) . min 37°C 3 hr 50 min 25 M sucrose 4°C 4°C 4.44 +1.92 13.9 + .82 269.7 + 29.1 28.3 + 3.7 a. 0[CaMg] = Ca and Mg free Tyrode b. .25 M sucrose was corrected to pH 7.4 with histidine c. values are means + S.E. n=4 • ' 241 Table for Fig. 5. Top. Concentration dependence of the stimulation of microsomal ATPase by Mg, Na and K  Mg (mM) 0 1 2 3 4 pmoles Pi/mg/min .123 .708 .991 1.39 1.58 3 mM Mg + Na (mM) 0 20 40 60 80 100 pmoles Pi/mg/min 1.39 1.42 1.61 1.73 1.92 1.98 3 mM Mg + 100 mM Na + K (mM) 0 3 6 9 12 pinoles Pi/mg/min 1.98 2.26 2.08 2.13 2.31 Table for Fig. §: Bottom.Lack of clear concentration dependence effect of K on the Mg dependent Na-ATPase  3 mM Mg + 100 mM Na + K (mM) 0 .75 1.5 2.25 3 6 9 12 a microsomes 1.61 1.56 1.37 1.41 1.42 1.35 1.49 1.48 Microsomes + sol. 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 for Fig. 6. Effect of Na, K, ouabain and a soluble activating factor on the Mg-ATPase activity  A. microsomes B. microsomes prepared in the presence of 0.1 mM ouabain C. 50 y l microsomes 50 u1 microsomes + 10 y l soluble fraction 5 50 y l soluble fraction D. microsomes + sol. M N K 0 2.199 + .2141 1.274 + .009 2.763 2.987 0.061 1.658 + .065 MN MNK MNK MN -M -MN -MNKO -MNKO .119 .052 -.063 +.016 +.011 +.022 7.37 4.76 2.90 .681 + . 04 paired t-value 17.35 (10 degrees of freedom)  ymoles Pi/mg protein/min M M M N N K 1.302 2.237 2.194 + .162 + .134 + .147 0.676 1.426 1.424 + . 030 + . 016 + .034 1.928 2.710 2.605 2.303 3.026 3.158 0.042 0.056 0.063 0.917 1.59 1.704 + .060 + .066 + .065 mean + S.E. 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 Pi .094 .0564 .1875 .1125 .375 .225 .75 .45 1.5 .9 1.875 1.125 2.25 1.35 2.625 1.578 3 1.8 mg/min 1/V 10.64 5.33 2.67 1.33 .67 .53 .44 .38 .333 pmoles .077 .109 .153 .187 .183 .171 .175 .200 .159 Mg umoles Pi mg/min .620 .875 1.225 1.50 1.46 1.37 1.4 1.6 1.27 1/V 1.61 1.14 .816 .66 .68 .73 .714 .625 .787 umoles .052 .09 .155 .222 .272 .247 .255 .246 .262 Mg Na pmoles Pi mg/min .420 .720 1.24 1.775 2.175 1.97 2.04 1.96 2.09 1/V 2.38 1.39 .806 .563 .46 .51 .49 .51 .478 Mg Na ymoles .05 .114 .128 .220 .258 .277 .258 .279m .251 umoles Pi .4 .91 1.025 1.76 2.06 2.22 2.06 2.23 2.00 K mg/min 1/V 2.5 1.09 .976 .568 .48 .45 .485 .49 .5 average of duplicates So Co Table for Fig. jQ. The effect of Ca on the Mg dependent Na,K-ATPase total CaCl. (M) assay medium 0 lO" 4 lO" 3 25 mM Hist., .1 mM EGTA .066 .088 .153 .256 +.024 +.029 +.046 +.093 25 mM Hist., .1 mM EGTA .485 .497 .513 .625 3 mM MgCl 2 + .127 +.121 + .118 +.168 25 mM Hist., .1 mM EGTA .921 .960 1.057 1.025 3 mM MgCl 2, 100 mM NaCl + .216 + .231 +.260 +.259 25 mM Hist., .1 mM EGTA .975 .976 .976 1.122 3 mM MgCl-, 108 mM NaCl +.236 + . 241 + .239 +.295 3 mM KCl 25 mM Hist., .1 mM EGTA .933 .950 1.100 1.010 3 mM MgCl„, 100 mM NaCl + .227 + .238 + . 319 + . 242 3 mM KCl, 3 mM ouabain mean + S.E. n =4 experiments and each experiment was done in duplicate Table for Fig. 13. Top Sarcolemmal. enriched microsomal Ca-ATPase activity assay medium 8 total lO" 6 CaCl (M) lO" 5 lO" 4 lO" 3 20 mM Tris HC1 PH 7.4 .039 .103 .352 .695 +.008 + . 012 +.037 + .063 20 mM Tris HC1 PH 7.4 .866 .820 ;.;705- .,713 .840 3 mM MgCl 2 +.099 +.152 +.094 +.099 +.173 mean + S.E. n = 4 245 Table for Fig. 15. Bottom Inhibition by,La of the actomyosin Ca-ATPase CaCl •oo pCa total umoles Pi mg/min + 10 3 M LaCl 3 umoles Pi mg/min lO" 7 7 .156 .042 lO" 6 6 .154 .080 5 x 1 0 ' 6 5.3 .148 .074 lO" 5 5 .150 .069 5 x 1 0 - 5 4.3 .187 .103 lO" 4 4 .210 .100 5 x 1 0 - 4 3.3 .254 .128 l O ' 3 3 .287 .150 2 x 1 0 - 3 4 x 10 ^ 4 x 1 0 _ J 2.7 2.4 .324 .346 .174 .156 average of duplicates Table for Fig. 1|. 'afoip. Substrate dependence of the sarcolemmal enriched microsomal Ca-ATPase and i t s effect on La inhibition 1 0 - 3 M LaCl„ ATP (mM) ATP umoles 1/ATP (M X) umoles Pi mg/min 1/V umoles Pi mg/min 1/V .125 -2 .25 .075 8,000 .475 2.1 0 .15 4,000 ' .590 1.69 .06 16.7 .50 .30 2,0000 1.37 .73 .249 4.02 1 .ft 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 average of duplicates 246 i Table for Fig. 16 Top Actomyosin Ca-ATPase dependence on ATP ATP (mM) 1/ATP (M X) umoles Pi mg/min 1/V .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 -average of duplicates Table for Fig. 23. Effect of K on contraction by 5 uM ouabain 5 uM ouabain tension (g) % 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 Fig. 19. Bottom. Time course of Ca, Na, Mg and K loss in 160 mM Tris Tyrode pH 7.4 and 10 mM LaCl at 4°C % of total control levels time (min) Na K Mg Ca :I5' 26.5 + 2.8 87.5 +21.7 95.7 + 6.98 24.7 + 5.1 30' 11.6 + 2.2 89.8 +10.1 90.6 + 7.9 - 16.2 + 5.03 45' 12.2 + 2.7 82 + 8.97 79.5 + 5.05 15.4 + 1.5 60' 4.4 +.1.48 74:9. + 7.24 82.9 + 4.04 4.5 mean + S.E. n = 4 to Table for Fig. 21. Control experiment to see i f the La C l ^ T r i s HC1 solution (pH 7.4, 4°C) could remove twice the amount of Ca in the regular wash time (30 min) and whether Na, K and Mg values would be unaffected control 3 equilibrated equilibrated with 1.8 mM CaCl„ equilibrated with 1.8 mM CaCl 2 with 1.8 mM CaCl„ then 3.6 mM CaCl 0 for 30 sec then 3.6 mM CaCl„ for 5 min total interval total interval total interval 14.64 6.09 25.9 6.81 24.8 5.94 Ca + .873 + .22 + 1.15 + .92 + 3.57 + .89 439.4 82.2 519.8 84.8 465.4 92.7 Na + 28.4 + 3.91 + 23.0 + 3.5 + 61.7 +17.5 289.2 249.6 271.6 234.3 259.8 260.4 K + 16.4 + 8.64 + 16.5 + 3.55 + 23.2 + 7.7 26.25 22.47 25.2 19.8 23.1 19.8 Mg + .451 + .491 + 1.85 + .70 + 1.27 + 7.2 Control levels are those from the ouabain experiments in Fig. mean + S.E. n = 4 Table for Fig. 25. Intracellular ion levels during the course of an ouabain response. time with 10 M ouabain (min) Element total internal 1 3 5 7 10 15 30 Ca 14.64 6.09 6.49 7.48 7.06 6.53 5.79 5.49 + .873 + . .22 + .44 • + .351 + .45 + .47 + .50 + .40 Na 439.4 82.2 94.39 124.4 139.2 1105.3 115.3 114.4 179.6 +28.4 + 3.91 + 7.74 + 7.66 +26.1 + 9.7 + 8.7 + 5.52 +29.4 K 289.2 249.6 262.6 255.5 234.8 235.8. 219.7 181.3 114.3 +16.4 + 8.64 +13.1 + 5.52 + 7.57 +10.74 +10.79 +13.09 +22.0 Mg 26.25 222.47 21.77 '22.43 21.44 21.98 22.34 20.58 + .451 + .491 + .586 + .321 ++ .37 + .66 + .37 + .49 mean + S.E. So 250 Table for Fig. 26. Sensitivity of the ouabain response to Ca removal normal . • „ ,- m . , . ^ ^ , time xn Ca free Tyrode (mm) Tyrode z ^ 0 • .5 1 2.5 5 10 % of control high K 50.8 25.3 19.0. 12.8 11.4 6.35 phasic response + 2.37 + 3.49 +2.4 + 4.8 + 4.0 % of mean response in 100 49.8 37.3 25.3 22.4 12.5 normal Tyrode + 4.65 + 6.88 + 4.48 +9.43 + 5.5 (i.e. of 50.8%) n = 28 4 4 4 4 2 mean + S.E. Table for Fig. 2r7r*. Log dose response for cis-2-methyl-4-dimethylaminomethyl -1,3-dioxolane methiodide  dose (M) log dose -9 5 x 10 10"8 2.5 x 10 8 5 x 10 - 8 lO" 7 2 x 10 - 7 5 x 10~ -8.31 -8 -7.6 -7.31 ~?7 -6.7 -6.31 phasic .375 .487 .812 1.35 1.76 1.96 1.85 + . 052 +. 059 +. 08 +.139 +.167 +.12 +.205 tonic .325 .625 1.47 1.99 2.01 1.86 1.45 + .063 + .136 +. 145 +.094 +.124 +.159 +.147 7 g tension above basal 350 mg. mean + S.E. n=4 Table for Fig. 107. Bottom The loss of the phasic component when CD and high KCl are added after various times in Ca free Tyrode . . time in Ca free Tyrode (min)  .25 ._5 1 1_5 2 4 5 6 10_ CD 75.7 85.0 70.1 63.0 47.7 19.26 - 8.9 8.5 +6.8 +4.6 +5.36 +4.94 +4.45 +2.38 +2.15 +1.53 high K 73.6 60.4 51.6 - 27.5 - 15.4 - 4.93 + 8.1 +11.5 + 5.05 + +3.5 + 5.0 +1.41 mean + S.E. Table for Fig. 319. The effect of Ca free Tyrode for various times on total and and internal ion levels Ca time in Ca free Tyrode (min) 0 0_J> 1 J ) 2^5 5 J Q 7 1 5 1 0 internal . 6 1 4 5 . 5 6 5 . 0 9 5 . 7 6 4 . 8 1 4 . 9 2 3 . 1 4 + . 2 0 0 + .45 + . 3 2 4 + . 5 9 9 + . 5 7 4 + . . 5 4 9 + . 3 7 4 total 1 4 . 6 4 1 1 . 3 2 1 0 . 9 5 8 . 8 1 7 . 4 4 7 . 2 1 6 . 1 4 + . 8 7 3 + . . 7 9 8 + .998 + . 4 9 9 + . 3 4 9 9 + .848 + . 6 9 9 Mg internal 2 1 . 5 2 0 . 5 1 9 . 2 6 1 9 . 2 2 0 . 5 3 1 8 . 4 4 1 8 . 6 8 + .37 + . 8 2 3 + 1 . 2 7 + 1 . 4 8 + 1 . 4 8 + .741 + . 6 9 9 Na K total 2 6 . 2 5 2 6 . 3 8 2 3 . 4 2 3 . 3 2 5 . 0 6 2 3 . 6 7 2 3 . 1 2 + . 4 5 2 + .41 + .987 + . 2 2 6 + . 8 6 4 + 2 . 1 8 * . . 5 3 5 internal 8 8 . 7 3 9 3 . 0 8 9 4 . 4 1 1 3 . 9 9 9 . 6 9 4 . 3 9 9 7 . 4 + 4 . 1 7 + 1 6 . 0 9 + 1 0 . 3 5 + 8 . 6 5 +9.39<rr, + 6 . 8 3 + 1 1 . 8 3 = total 4 3 9 . 3 4 3 2 . 4 4 4 3 . 2 4 3 0 . 2 4 4 2 . 4 5 0 8 . 0 4 3 5 . 0 + 2 8 . 4 + 4 2 . 2 ± 4 3 . 5 + 2 3 . 4 + 2 7 . 2 + 3 9 . 5 + 2 1 . 7 internal 2 4 4 . 5 1 9 3 . 4 2 2 7 . 6 1 9 7 . 4 1 8 8 . 0 2 0 5 . 4 2 1 2 . 3 + 6 . 7 + 1 6 . 3 + 1 7 . 4 + 1 5 . 3 + 2 9 . 7 + 2 0 . 8 + 2 6 . 1 total 2 8 9 . 2 2 4 6 . 0 2 3 9 . 1 2 8 0 . 3 2 7 1 . 1 2 5 2 . 2 2 3 4 . 0 + 1 6 . 4 + 1 1 . 3 5 + 1 7 . 1 + 1 6 . 9 5 + 3 7 . 6 + 3 6 . 3 + 1 6 . 3 nanomoles/mg dry wt. mean + S.E. U l Table for Fig. 331. Time, course of external Ca. loss of the phasic component to the time course of loss of internal and % of control in normal Tyrode 0.5 1.0 1.5 time 2.0 in Ca free 2.5 Tyrode 4.0 (min) 5.0 6.0 7.5 10.0 Ca internal 90.6 82.9 - - 93.9 . - 78.4 - 80.1 51.9 Ca external 67.9 69.1 - - 35.9 - 30.9 - 27.0 28.2 CD phasic 85.0 70.1 63.0 47.7 - 19.3 - 8.9 - 8.5 High K phasic 60.4 51.6 - 27.5 - - - 15.4 - - 4.9 ouabain 3 49.8 37.3 - - 25.3 - 22.4 - - 12.5 a. data for ouabain is not included on Fig. 30. but is included here for comparison to the loss of extracell-ular and intracellular Ca. K3 Co Table for Fig. 35'. The effect of LaCl 3 on the phasic and tonic components of high K and 2 x 10 M CD responses. :  LaCl„ (M) lO" 6 2.5 x 10 6 5 x 10 - 6 3 7.5 x 10 - 6 i o " 5 5 x 10 5 lO" 4 phasic 72.4 41.2 33.7 38.6 26.5 13.2 0 + 4.5 + 5.6 + 4.9 + 5.7 + 6.4 + 1.9 CD — tonic 83.2 55.9 41.7 26.1 48.9 30.6 13.0 + 5.0 + 4.7 + u& + 2.3 + 7.5 + 1.9 + 4.5 phasic 76.3 23.9 21.0 9.9 11.0 8.9 -high +12.6 + 5.2 + 4.8 + 2.4 + 2.6 + 1.4 KCl tonic 110.4 89.6 105.4 104.8 100.6 88.9 -+11.4 + 2.2 + 5.0 +11.9 + 3.0 + 8.7 % of control mean + S.E. Table for Fig. 3I63. Intracellular ion levels during the course of a high KCl contraction and the equilibration phase  contraction equilibration time in the presence of high K time after washout total internal 10 sec 10 min 30 sec 5 min 15 min 30 min 12.87 5.26 5.91 * 6.96 4.94 5.64 5.24 2.62 Ca + .60 + .17 + .60 + .45 + .45 + . .77 + .60 + .32 (32) (83) (18) (17) (12) : a i ) (8) (4) 250.9 212.3 229.4 250.9 272.9 272.4 249.9 289.8 K +11.0 ++6.1 + 8.4 +12.8 +11.4 +16.5 +14.1 + 6.4 (33) (74) (18) (16) (12) (ID (8) (4) Na 412.3 102.6 93.5 107.0 92.6 91.8 72.2 68.3 Na +17.4 + 3.5 + 6.6 + 4.3 + 6.2 + 9.4 + 6.2 + 5.3 (30) (83) (18) (16) (12) (11) (8) (4) 25.3 21.9 + 20.4 20.9 23.3 19.9 18.6 20.8 Mg + .29 + .25 + .49 + .49 + .49 + .58 + .78 + .66 (32) (77) (17) (17) (11>> (ID (8) (4) nanomoles/mg dry wt. level of significance 9.01 s i % V mean + S.E. 9.7.5 % f (n) ~ 9 9 . 95 % * Table for Fig. ..3(55. Intracellular ion levels during the course of a CD contraction and the equilibration phase. 1 contraction time in the presence of CD equilibration time after washout total internal 10 sec 1 min 5 min 10 min 30 sec 5 min 20 min 30 min Ca K Na Na Mg 12.87 + .60 (32) 250.9 + 11.0 (33) 412.3 + 17.4 (30) 25.3 + .29 (32) 5.26 + .17 (83) 212.3 + 6.1 (74) 102.6 + 3.5 (83) 21.9 + .25 (77). 5.69" + .25 (20) ,t t 189.0 + 7.7 (28) 96.6 + 5.6 (28) 19.7 + .74 (26) + 6.11 + .45 (19) 192.0+ 8.9 (20) 93.9 + 5.6 (20) 20.5 + .58 (19) + 5.56" + .57 (12) 188.0+ 7.9 (13) 94.8 + 3.9 (13) 18.6 + .78 (12) 5.88 + .37 (43) •k * + 170.3 7.0 (55), 109.6 + 4.3 (56) 19.7 + .33 (52) 4.64 + .17 (25) 170.3 + 6.5 104.8 + 4.2 (29) 21.1 + .41 (25) 4.29 + .17 (15) 166.0 +_12.8 (15) 106.6 + 5.7 (ID 18.4 + .49 (15) 6.11 + .22 (4) 204.8 + 29.7 m 107.0 + 9.0 (4) 21.4 + .66 (4) 4.39 + .45 (12) 210.2 + 9.4 90.2 + 5.9 (12) 20.9 + .49 (12) nanomoles/mg dry wt. mean + S.E. (n) level of significance not significant 90 % 95 % 97.5 % 99.95 % ? V t * Table for Fig. 4"Qf. CD biphasic contraction shape changes after various equilibration times in normal Tyrode. equilibration time allowed (min) 20 15 12 10 8 6 4 2 phasic 100.9 105.7 96.8 91.6 83.6 81.1 82.5 85.9 % of control + 5.9 + 6.4 + 2.9 + 4.9 + 6.7 + 7.6 + 7.7 ± 7 - 5 tonic 114.4 124.6 134.1 128.0 123.5 119.8 108.1 106.4 % of control + 4.3 + 8.4 + 4.6 + 5.0 + 6.6 ± 5 - 5 + 4.7 + 5.1 time to reach peak tonic 7.55 6.30 4.95 4.10 2.95 2.0 0.95 0.8 (min) + .46 ± - 4 9 + .16 + .22 + .10 * .65 ± ± - 6 9 +. 01 i to 258 Table for Fig. 41. Effect of a previous 10 min CD contraction on the contraction to a second dose of CD after various . times. equilibration 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.  equilibration 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 11.6 between phasic +4.5 and tonic 26.4 + 9.2 36.4 + 9.3 64.5 + 8.5 91.6 + 3.3 Table for Fig. 5.0>. The effect of altering the Tyrodes's KCl concentration on the time required for equilibration.  extracellular KCl concentration during equilibration MM . 1.34 2.68 5.36 6.70 8.05 10.73 13.41 times normal 0.5 1 2 2.5 3 4 5 70.2 25.3 12.8 10.4 8.65 7.05 6.00 + 3.2 + 1.0 + 0.6 +0.6 +0.39 +0.30 +0.43 equilibration time (min) mean—r S-.E. APPENDIX Table 1. Compilation of free Ca concentrations. -ATP Mg Ca EGTA free C a 2 + pfree Ca (M) (M) (M) (M) 3 x 10 - 3 3 x l ( f 3 l ( f 3 - 1.75 x IO" 4 3.75 I I I I lO" 4 - 1.75 -x i o " 5 4.75 I I l ( f 5 - 1.75 x 10 6 5.75 I I lO" 6 - 1.75 x i o " 7 6.75 •> - 5 X lO" 3 - 2.06 x IO" 3 2.69 » - 4 X lO" 3 - 1.12 x i o " 3 2.95 » - 3 X lO" 3 - 2.43 x 10 4 3.61 » - 2 X lO" 3 - 7.3 x . i o " 5 4.14 • I I - lO" 3 - 2.2 x i o " 5 4.66 » - 5 X lO" 4 - 1.8 x i o " 5 4.74 » - lO" 4 - 1.5 x i o " 6 5.82 „ - 5 X lO" 5 - 6.1 x ( i o " 7 6.21 » - i o - 5 -1 1.6 x i o " 7 6.80 » - 5 X lO" 6 - 1.1 X i o " 7 6.95 » - i o " 6 - i o " 7 7.00 I , - ' IO"7 - - -» - i o " 3 i o " 4 2.00xxl00 5 5 4.70' » - IO" 4 i o " 4 3.0 x IO" 7 6.52 » - i o " 5 i o " 4 8.6 x i o " 9 8.76 3 x IO - 3 IO" 3 i o " 4 1.67x IO" 4 3.80 I I 3 x 10 - 3 i o " 4 . i o " 4 1.1 X i o " 6 5.96 3 x l C f 3 i o " 5 i o " 4 7.0 x i o " 8 7.15 -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 10 - 7 M O'Sullivan and Perrin (1964) diss K MgATP = 8.8 x 10 4 M - 1 O'Sullivan and Perrin (1964) c t S S K CaATP = 3.15 x 10 4 M _ 1 Ogawa (1968) ass ° v 4-Since Mg and Ca complex with ATP primarily i t is necessary to calculate the fraction of ATP that i s completely dissociated at pH 7.4. + 4-K,. = 1.0715 x 10"7 B j H.nATP ] ( 1 )  d l S S [ATP3"] 7.4 = -log [H +], therefore [H+] = 3.98 x 10 _ 8 (2) 3_ Substituting equation (2) into equation (1) and solving for ATP 4-in terms of ATP gives ATP 3 _ = 0.3714 ATP4" (3) 2+ 4- 2+ Since Mg complexes with ATP more strongly than Ca , i t has an , . 4-overriding effect on the amount of ATP l e f t to bind Ca. Since the -3 total ATP concentration = 3 x 10 M, then ATP 4 - + ATP 3 _ + [MgATP2-] = 3 x 10 _ 3 (4) -3 Since the total Mg concentration = 3 x 10 M [Mg2+] + [MgATP2"] = 3 x 10~3 M and, rearranging [Mg2+] = 3 x 10 - 3 - [MgATP2-] ^ (5) Combining equation (3) and (4), then 1.3714 ATP 4 - + [MgATP2-] = 3 x 10 _ 3 4- 2-and ATP , in terms of [MgATP ] from the above equation is [ATP 4 -] = 2.186 x 10 - 3 -0.7308 [MgATP2-] (6) 2+ 4-The association constant for Mg to ATP equals 2 62 t M g A T p 2 ~ ] = 8.8 x 10 A M"1 ' (7) 2+ 4-[Mg Z +][ATP 4 ] 2+ 4-Substituting equation (5) for Mg and equation (6) for ATP into equation (7), f g A T p 2 " ] ' y - = 8.8 x 10 4 (3 x 10 -[MgATP ])(2.186 x 10 -.7308[MgATP ]) 2-and solving for [MgATP ] yields [MgATP2-] = 2.786 x 10 _ 3M (8) Substituting equation (8) into equation (6) yields ATP 4 - = 0.15 x IO - 3 M (9) 4- 2+ This is the ATP l e f t to bind Ca . The association constant for 2-CaATP gives 2-J - C a A T P J = 3.15 x 10 4 (10) [Ca.1 ][ATP ] The known amount of Ca added to the assay medium = [Ca] .. and total [ C a ] t o t a l = [Ca2+]>+ [CaATP2-] and, rearranging [Ca 2 +] = [ C a ] t o t a l - [CaATP2-] ' (11) Substituting equation (9) and (11) into equation {10) yields t C a A T p 2 ~ ] = 3 . 1 5 x l 0 4 ([Ca] t o t a l-[CaATP 2"])(.15 x 10 3) 2-and solving for [CaATP ] in terms of [Ca] n total [CaATP2-] = 0,825 [Ca] (12) From equation (11) and equation (12) [Ca 2 +] = [Ca] , - .825[Cal ' 1 J 1 J t o t a l 1 J t o t a l 2+ See appendix Table 1 for [Ca ] at various [ C a J ^ ^ i when ATP and MgCl 2 are both 3 mM. 2 63 2+ B. Calculation of the free Ca concentration in the presence of 3 mM ATP (no MgCl2, no EGTA) •3- 4-From section A. equation (3) ATP = .3714 ATP -3 The total ATP concentration = 3 x 10 M ATP4" + ATP3" + [CaATP2"] = 3 x 10~3M (13) Substituting equation (3) into equation (13) ATP4" + .3714 ATP4" + [CaATP2_] = 3 x 10~3 T 4-and solving for ATP ATP 4 _ = 2.186 x 10~3 - .7308 [CaATP2-] (1.4) -3 When the total CaCl^ concentration is 10 M [Ca 2 +] + [CaATP2-] = 10 _ 3 M and [Ca 2 +] = 10"3 - [CaATP2"] (15) From the CaATP association constant 9 j C a A T P ' " ] = 3.15 x 10 4 (16) [Ca^ ][ATP ] Substituting equation -^14) and (15) into equation (16) yields _ _ _ [CaATP2-] = _ 1 Q4 (10 - [CaATP ])(2.186 x 10 - .7308[CaATP; ]) and solving for [CaATP2-] = .978 x 10~3 M (13) Subsituting equation (1?) into equation (15) gives [Ca 2 +] = 10 - 3 - .978 x 10 - 3 = .022 x 10 - 3 M (18) At any desired total Ca concentration, equations (15), (10) and (lZ) can be solved to give the free Ca concentration. See Appendix Table 1 for a compilation of free Ca concentrations under various conditions. 2 64 2+ C. Calculation.of the free Ca concentration in the presence of 0.1 mM EGTA The dissociation constant for CaEGTA depends on the pH. KpH IA = K d i s s (19) d ± S S T l + K±[E+] + K ^ H V + K ^ K ^ ] 3 +• K 1 K 2 K 3 K 4 [ H + ] 4 10 where = 4.47 x 10 when the four carboxyl groups of EGTA are completely dissociated-' (Schatzmann (1973) . K = 2.89 x 10 9 Portzehl et a l . (1964) K 2 = 7.09 x 10 8 K 3 = 4.78 x 10 2 " 2 K . = about 10 " [H+] = 3.98 x 10"8 at pH 7.4 c i • ' / - . O N. i^PH 7 - 4 i oo m 7 [GaEGTA] (20) Solving equation (19), K* . = 1.33 x 10 = - v ' d l S S [Ca^] [EGTA] 2+ C. a) Calculation of the free Ca concentration in the presence of 3 mM MgCl 2 > 3 mM ATP and 0.1 mM EGTA From equations (9) and (10), 2-[CaATP ] = ( 3 < 1 5 x 1 0 4 ) ( 0 < 1 5 x 1 0 4 } = 4 > 7 2 [Ca / +] [CaATP2-] = 4.72[Ca2+] (21) -4 Since the total EGTA concentration i s 10 M [EGTA] + [CaEGTA] = 10 _ 4 [EGTA] = 10~4 - [CaEGTA] (22) -3 If the known .feot-aitCaClC^aS&ded to the assay medium is e.g. 10 M then [Ca2+]-+[CaATP2_] + [CaEGTA] = 10~3 M (23) Substituting equation (21) into equation i('23) and collecting like terms 5.7-2[Ca2+] + [CaEGTA] = 10~3M (24) 2+ - -3 and rearranging [Ca ] = 10 - [CaEGTA] 5.72 [Ca 2 +] = .175 x IO - 3 - .175[CaEGTA] (25) Substituting equations (22) and (24) into equation (20) gives [CaEGTA] _ 7 - = 1.33 x 10 (.175 x 10-3 _ .175 [CaEGTA])(10 - [CaEGTA]) -4 and solving for [CaEGTA], then [CaEGTA] = very nearly 10 M (26) Substituting equation (26) into equation (24) and solving 5.72 [Ca 2 +] + 10"4 = 10" 3 (27) 2+ -4 [Ca ] = 1.57 x 10 In like manner, equations (23) through (27) can be solved for total Ca -4 -5 concentrations of 10 and 10 M, etc. See Appendix Table 1 for the results. 2+ C. b) Calculation of the free Ca concentration in the presence of 3 mM ATP  and 0.1 mM EGTA 2+ In order to express [Ca ] in terms of [CaEGTA] for equation (23), we need to know the ratio of [CaATP] to [Ca]. -3 2+ -3 In the absence of EGTA at 10 „.M C a t o t a l , [Ca. ] = 0.22 x 10 M (see equation 2- -3 18) and [CaATP ] = .978 x 10 M (see equation 17). Therefore, [CaATP2 -] =44.7 [Ca 2 +] (28) 2+ Substituting equation (28) into equation (23) and solving for [Ca ] [Ca 2 +] = .022 x 10 - 3 - .022 [CaEGTA] (29) Substituting equations (29) and (22) into equation (20), .then C ^ ^ y = 1.33 x l O 7 (.022 x 10-3 _ .022 [CaEGTA])(10-4 - [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 [Ca 2 +] = 1.99 x 10 5 M. 2+ In lik e manner, the free Ca concentration can be solved for total Ca concen--4 -5 trations of 10 and 10 M in the presence of 3 mM ATP and 0.1 mM EGTA. 

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