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Arterial hydration during vasoconstriction Holtby, Mark Ernest 1970

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ARTERIAL HYDRATION DURING VASOCONSTRICTION by MARK ERNEST HOLTBY B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1964  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Anatomy  We accept t h i s t h e s i s as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May 1970  In p r e s e n t i n g t h i s t h e s i s an advanced degree at the L i b r a r y I  the U n i v e r s i t y  s h a l l make i t  f u r t h e r agree tha  in p a r t i a l  freely  f u l f i l m e n t o f the of B r i t i s h  available  for  requirements f o r  Columbia, I agree  that  reference and study.  p e r m i s s i o n f o r e x t e n s i v e copying o f t h i s  thesis  f o r s c h o l a r l y purposes may be granted by. the Head o f my Department o r by h i s of  representatives.  It  this thesis for financial  i s understood that copying o r p u b l i c a t i o n gain s h a l l  written permission.  Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  Date  0Y 9 rf?0  not be allowed without my  ii  ABSTRACT  The r e l a t i o n s h i p between v a s o c o n s t r i c t i o n and the hydration of the a r t e r y w a l l was examined using the t a i l a r t e r y of the r a t . Freeze s u b s t i t u t i o n was used to prepare h i s t o l o g i c a l sections of a r t e r i e s f i x e d i n a known s t a t e of c o n s t r i c t i o n . Measurements of w a l l dimensions showed that the more c o n s t r i c t e d a r t e r i e s had smaller w a l l and media c r o s s - s e c t i o n a l areas than the l e s s c o n s t r i c t e d a r t e r i e s .  The con-  stant length of the c o n s t r i c t i n g a r t e r y meant that the w a l l volume decreased by 14%. Considering the vascular smooth muscle c e l l as a double cone enabled formulation of r e l a t i o n s h i p s between the c e l l r a d i u s , length, surface area, and volume.  Radius and length measurements of the smooth muscle c e l l s of  the f r e e z e - s u b s t i t u t e d a r t e r i e s demonstrated that the c e l l radius doubled and the length decreased by h a l f during v a s o c o n s t r i c t i o n . revealed  These measurements  that the surface area of the double cone model of the c e l l remained  constant, while the volume increased during v a s o c o n s t r i c t i o n .  This suggested  that water entered the contracting vascular smooth muscle c e l l s . Water and i o n content determination of paired c o n t r o l and c o n s t r i c t e d i n v i t r o a r t e r i e s i n d i c a t e d that the a r t e r y w a l l l o s t 16% of i t s water. This represented a 13% decrease i n the w a l l volume.  The associated de-  creases i n the Na and CI contents and i n the i n u l i n space, as w e l l as the constant K content implied that the water was expelled from the e x t r a c e l l u l a r space of the c o n s t r i c t i n g a r t e r y .  While t h i s was true f o r a r t e r i e s con-  s t r i c t e d w i t h both norepinephrine and high K s o l u t i o n s , i t seemed that water l o s t from a r t e r i e s c o n s t r i c t e d with PLV-2, a s y n t h e t i c vasopressin,  may have  iii come from i n u l i n - i n a c c e s s i b l e phases of the w a l l .  The s i z e of the water l o s s  depended upon the d u r a t i o n of v a s o c o n s t r i c t i o n : the l o s s e s were l a r g e s t 30 seconds a f t e r the s t a r t of c o n s t r i c t i o n . Perfused r a t t a i l a r t e r i e s e x h i b i t e d pressure-flow c h a r a c t e r i s t i c s during v a s o c o n s t r i c t i o n which suggested that the p e r m e a b i l i t y of the w a l l had increased.  I t was discovered that the changes i n p e r m e a b i l i t y induced by  v a s o c o n s t r i c t i o n were d r a s t i c a l l y a f f e c t e d by changes i n the i n t r a v a s c u l a r pressure.  I n a t h i r d p e r f u s i o n experiment, the d i l u t i o n of Evans blue dye  passing through the lumen of a c o n s t r i c t i n g a r t e r y a l s o i n d i c a t e d a permeab i l i t y increase during v a s o c o n s t r i c t i o n . A r t e r i e s were incubated i n one of f i v e isosmotic s o l u t i o n s of d i f f e r ent i o n i c composition.  Comparison of a r t e r i a l contents and p e r f u s i o n  pressures showed that the absence of monovalent ions i n the bathing media r e s u l t e d i n a decrease i n a r t e r i a l water, an increase i n d i v a l e n t i o n content, and higher p e r f u s i o n pressures.  These observations can be explained by  changes i n the tension of the vascular smooth muscle c e l l s and p o s s i b l y by an i o n exchange process i n the p a r a c e l l u l a r matrix which caused conformat i o n a l changes i n the matrix, i n turn causing an a l t e r e d w a l l hydration. A r t e r i e s , cooled overnight a t 2°C, were rewarmed i n one of three s o l u t i o n s of d i f f e r e n t Na concentration.  The a r t e r i e s were t r a n s f e r r e d from  a s o l u t i o n a t 2°C to one a t a temperature between 2° and 37°C f o r 15 minutes. Comparison of the a r t e r i a l contents showed that a small amount of K was gained while l a r g e amounts of water and Na were l o s t from the a r t e r y w a l l during these short rewarming periods.  P o s t u l a t i o n of a 1:1 exchange of K  f o r Na f o r the c e l l metabolic Na-K pump means that a f a s t Na component was  iv  extruded, independent of K, from the rewarming a r t e r y w a l l .  The e x t r u s i o n  of the w a l l water may have been r e l a t e d to the extrusion of t h i s extra Na component, because they both had the same temperature and e x t e r n a l Na conc e n t r a t i o n dependencies.  The monitoring of the i n t r a v a s c u l a r pressure of  perfused rewarmed a r t e r i e s revealed pressure changes with the same temperature and e x t e r n a l Na concentration changes.  dependencies as the above water content  C a l c u l a t i o n s i n d i c a t e d that changes i n w a l l volume caused by  changes i n water content could p a r t i a l l y e x p l a i n the i n t r a v a s c u l a r pressure changes during  rewarming.  The w a l l water l o s s , the permeability  changes, and the c e l l water  increase associated with v a s o c o n s t r i c t i o n , are discussed  i n terms of an  osmotic and h y d r o s t a t i c pressure balance between the a r t e r y w a l l and i t s surroundings which i s upset by v a s o c o n s t r i c t i o n .  V  TABLE OF CONTENTS  CHAPTER I. II.  Page INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . .  1  CHANGES IN VASCULAR DIMENSIONS DURING CONSTRICTION. . . . .  6  A.  Changes i n Dimensions of A r t e r y Wall During Constrxction.  B.  ....so  . . . . . . .  . . . . « ' . .  1. 2. 3.  Methods . . . . . . . . . . . . . . . . . . . . . . Results E f f e c t of F i x a t i o n and Embedding on A r t e r i a l  4. 5.  Discussion Summary  6 7 9 23 24  . . . . . . . . . . .  Changes i n Dimensions of Smooth Muscle C e l l s During V a s o c o n s t r i c t i o n . . . . . . . . . . . . . . . 1. 2. 3. 4. 5.  III.  25  Mathematical Model f o r the Smooth Muscle C e l l . . . Measurement of C e l l Dimensions. . . . . . . . . . . F i t t i n g the Experimental Data to the Model. . . . . Discussion. . Summary . . . . . . . . . . . . .  26 28 30 31 38  CHANGES IN WATER CONTENT OF ARTERY WALL DURING CONSTRICTION. . .  i  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  39  A.  Methods . . . . . . . . . . . . . . . . . . . . . . . .  39  B.  Norepinephrine Induced V a s o c o n s t r i c t i o n . . . . . . . .  42  1. Water Content . . . . . . . . 2. Water and Ion Content 3. Varying Duration of V a s o c o n s t r i c t i o n . . . . . . . . 4. Discussion. . . . . . . . . . . . . . . 5. I n u l i n Space. . . . . . . . . . . . . . . 6. Chloride Content 7. Summary of Results of Norepinephrine Induced Vasoconstriction. . . . ......  42 43 48 50 54 58  C.  High K Induced V a s o c o n s t r i c t i o n  ......  61  D.  Synthetic Vasopressin,  PLV-2, Induced V a s o c o n s t r i c t i o n  63  60  vi  CHAPTER III.  Page (Continued) E.  Discussion. . . . . . . . . . . . . . . . . . . . . . . .  F.  Error i n C a l c u l a t i n g Inner Radius from  Isovolumetric  Assumption. . . . . . . . . . . . . . . . . . . . . . . G.  Examination of Possible Experimental A r t i f a c t s 1.  69 70  E f f e c t of Relaxation of Constricted A r t e r i e s on Water Content . . . . . . . . . . . . . . . . . . . . Water Remaining i n the Lumen a f t e r Gaseous Perfusion E f f e c t of Gaseous Perfusion on A r t e r i a l Water  71 73  HEMODYNAMIC AND PERMEABILITY CHANGES DURING VASOCONSTRICTION  78  A.  Pressure, Flow and Lumen Volume . . . . . . . . . . . . . .  78  1. 2. 3. 4.  79 80 82 83  2. 3.  IV.  64  B.  E f f e c t of Intravascular Pressure.  83  Methods . . . . . . . . . . . . . . . . . . . . . . . Results Discussion Summary  84 85 88 90  Dye D i l u t i o n . . . . . . . . . . . . . . . . . . . . . . .  90  1. 2. 3. 4. D.  . . .  . . . . . . . . . . . .  1. 2. 3. 4. C.  Methods Results Discussion Summary . . . . . . . . . . . . .  Methods Results Discussion. Summary . . . . . . . . . . .  Summary . . . . . . . . . . .  . . 91 91 93 . . . . 94 95  vii CHAPTER V.  Page EXPERIMENTALLY INDUCED ALTERATIONS IN THE HYDRATION OF THE ARTERY WALL. .  96  A.  96  E f f e c t of Varying E x t e r n a l Ionic Composition 1. Methods 2. Results 3. Discussion 4. Summary  B.  97 98 101 . . . . . . . . . . 103  E f f e c t of Cooling and Rewarming . . . . . . . . . . . . . . 103 1. 2.  VI.  . . . . . . . . . . . .  On the Water and Ion Content of the Vascular Wall . . . 103 On the I n t r a v a s c u l a r Pressure 118  SUMMARY AND DISCUSSION  128  A.  Loss of Wall Water During V a s o c o n s t r i c t i o n  . . . . 129  B.  A l t e r e d Vascular Permeability During V a s o c o n s t r i c t i o n . . . 138  C.  Increased  Smooth Muscle C e l l Volume During  Vasoconstriction. . . . . . . .  . . . . . . 143  APPENDIX I. II. III.  DIRECT MEASUREMENT OF ARTERY WALL RADII. . . . . . . . . . . . 162 SPIRAL ANGLE OF ARTERIAL SMOOTH MUSCLE CELLS . .  .162  WEIGHING AN ARTERY WITH AN ELECTROBALANCE. . . . . . . . . . . 163  viii LIST OF TABLES  TABLE I. II. III. IV.  V. VI. VII.  VIII.  IX.  X.  XI.  XII.  Page Dimensions of w a l l of r a t t a i l a r t e r y i n various s t a t e s of c o n s t r i c t i o n , c a l c u l a t e d from h i s t o l o g i c a l c r o s s - s e c t i o n s . . .  13  Outer radius and c r o s s - s e c t i o n a l area of the w a l l of f u l l y c o n s t r i c t e d middle segments of the r a t t a i l a r t e r y . . . . . .  20  Outer radius of the r a t t a i l a r t e r y from i n s i t u photographs, i n s i t u observations, and h i s t o l o g i c a l s e c t i o n s . . . . . . . . .  21  The c r o s s - s e c t i o n area of the r a t t a i l a r t e r y w a l l plus lumen from i n s i t u photographs, i n s i t u observations, and h i s t o l o g i c a l sections  22  H a l f - l e n g t h s and r a d i i of relaxed and contracted smooth muscle c e l l s of the r a t t a i l a r t e r y  29  Surface area and volume of relaxed and contracted smooth muscle c e l l s of the r a t t a i l a r t e r y . . . . . . . . .  30  Water contents of r a t t a i l a r t e r i e s removed at peak of norepinephrine induced c o n s t r i c t i o n a f t e r 3 hours of aerobic Water contents, sodium and potassium contents of r a t t a i l a r t e r i e s removed from s o l u t i o n at the peak of NE c o n s t r i c t i o n a f t e r 3 hours of aerobic i n c u b a t i o n . . . . . . . . . . . . . .  44  Water content, sodium and potassium contents of r a t t a i l a r t e r i e s c o n s t r i c t e d w i t h norepinephrine f o r s i x t y seconds a f t e r three hours of aerobic i n c u b a t i o n . . . .  46  Water contents, sodium and potassium contents of r a t t a i l a r t e r i e s c o n s t r i c t e d w i t h norepinephrine f o r 120 seconds a f t e r three hours of aerobic i n c u b a t i o n . . . . . . . . . . . . . . .  47  Water content and sodium content of a r t e r i e s a f t e r 15, 30, 60, and 120 seconds of norepinephrine-induced c o n s t r i c t i o n i n v i t r o during p e r f u s i o n w i t h O2/CO2 a f t e r 3 hours of aerobic incubation . . . . . . . . . . . . .  49  Water and i n u l i n space of r a t t a i l a r t e r i e s c o n s t r i c t e d f o r two minutes w i t h norepinephrine a f t e r t h i r t y minutes of aerobic incubation . . . . . . . . . . . . . . . .  56  ix TABLE XIII.  XIV.  XV.  XVI.  XVII.  XVIII. XIX.  Page Water and i n u l i n space of r a t t a i l a r t e r i e s c o n s t r i c t e d f o r two minutes w i t h norepinephrine a f t e r t h i r t y minutes of aerobic incubation . . . . . . . . . . . . . . . . .  57  Water and i o n content of r a t t a i l a r t e r i e s c o n s t r i c t e d w i t h norepinephrine f o r L5 minutes a f t e r 90 minutes of aerobic incubation . . . . . . . . . . . . . . . . .  59  O v e r a l l average water and sodium contents of r a t t a i l a r t e r i e s c o n s t r i c t e d i n v i t r o w i t h norepinephrine. The r a t i o of c o n s t r i c t e d t e s t a r t e r i e s w i t h smaller water or sodium than t h e i r corresponding c o n t r o l halves . . . . . . . .  60  Water content of r a t t a i l a r t e r i e s c o n s t r i c t e d f o r two minutes i n high potassium s o l u t i o n and the r a t i o of cons t r i c t e d t e s t a r t e r i e s with smaller water content than t h e i r corresponding non-constricted c o n t r o l halves a f t e r aerobic incubation . . . . . . . . . . . . . . . . . . .  63  Water content and i n u l i n space of r a t t a i l a r t e r i e s cons t r i c t e d f o r two minutes w i t h PLV-2 and r a t i o of c o n s t r i c t e d t e s t a r t e r i e s w i t h smaller amounts than t h e i r corresponding c o n t r o l halves a f t e r three hours of aerobic i n c u b a t i o n . . . .  64  I n t r a v a s c u l a r pressure, water and i o n contents of r a t t a i l a r t e r i e s incubated i n one of f i v e isosmotic s o l u t i o n s . . . . .  99  Changes i n water, sodium and potassium contents of r a t t a i l a r t e r i e s at d i f f e r e n t rewarming temperatures i n three s o l u t i o n s w i t h 140, 100, or 0 meq N a / l i t e r . . . . . . . . . . . .  107  X  LIST OF FIGURES FIGURE  Page  1.  Four degrees of c o n s t r i c t i o n of the r a t t a i l a r t e r y . . . . .  11  2.  R e l a t i o n between w a l l to lumen r a t i o , inner and outer r a d i i , and the degree of v a s o c o n s t r i c t i o n of the r a t t a i l artery  15  3.  R e l a t i o n between the c r o s s - s e c t i o n a l areas of the w a l l and media of the r a t t a i l a r t e r y and the w a l l to lumen r a t i o . .  16  4.  R e l a t i o n between the c r o s s - s e c t i o n a l area of the a r t e r y media and the w a l l to lumen r a t i o f o r the d i s t a l segments of the rat t a i l a r t e r y . . . . . . . . . . . . . . . . 19  5- 8.  9.  10.  11. 12.  13.  14. 15.  The changes i n surface area and volume of the double cone model of the vascular smooth muscle c e l l as functions of the changes i n c e l l h a l f - l e n g t h and radius . . . . . . . . .  32-35  Changes i n water content of r a t t a i l a r t e r y w a l l during norepinephrine induced c o n s t r i c t i o n i n v i t r o with O2/CO2 perfusion  51  Changes i n sodium content of rat t a i l a r t e r y w a l l during norepinephrine induced c o n s t r i c t i o n i n v i t r o with O2/CO2 perfusion. . . . . . . . . . . .  53  Varying durations of norepinephrine induced c o n s t r i c t i o n s of r a t t a i l a r t e r i e s perfused jin v i t r o with O2/CO2 . . . . .  55  Inner radius of a c o n s t r i c t e d a r t e r y c a l c u l a t e d from the c o n s t r i c t e d outer radius and the relaxed inner and outer r a d i i , w i t h and without a 13% decrease i n the volume o f the constricted artery wall  70  I n t r a v a s c u l a r pressure of a r a t t a i l a r t e r y c o n s t r i c t e d i n v i t r o with norepinephrine, then allowed to r e l a x , while being perfused with O2/CO2 V •  72  E f f l u e n t flow r a t e and pressure gradient f o r a r a t t a i l a r t e r y c o n s t r i c t e d i n s i t u with norepinephrine . . . . . . .  81  Pressure gradient down r a t t a i l a r t e r i e s c o n s t r i c t e d i n s i t u w i t h norepinephrine a t high and low i n t r a v a s c u l a r pressure ,  86  xi Page  FIGURE 16.  E f f l u e n t flow r a t e f o r r a t t a i l a r t e r i e s c o n s t r i c t e d i n s i t u w i t h norepinephrine at high and low i n t r a v a s c u l a r pressure . . 87  17.  I n t r a v a s c u l a r pressure and % transmission of the e f f l u e n t from a r a t t a i l a r t e r y i n s i t u a f t e r the a d d i t i o n of norepinephrine . . . . . . .  92  18.  Water content of r a t t a i l a r t e r i e s e q u i l i b r a t e d i n 1 of 5 isosmotic s o l u t i o n s of d i f f e r e n t i o n i c composition .  19-  E f f e c t of temperature during rewarming on the H2O content of the r a t t a i l a r t e r y cooled f o r 18 hours a t 2°C . . . . . . . . 108  20.  E f f e c t of temperature during rewarming on the Na content of the r a t t a i l a r t e r y cooled f o r 18 hours at 2°C . . . . . . . . 109  21.  E f f e c t of temperature during rewarming on the K content of the r a t t a i l a r t e r y cooled f o r 18 hours at 2°C . . . . . . .  22.  E f f e c t of e x t e r n a l Na concentration on the change i n water content and on the s i z e of the e x t r a , non-K linked* Na component extruded from the r a t t a i l a r t e r y during 15 minute rewarming periods between 2° and 37°C . . . . . . . . .  23-25.  26.  100  . 110  114  I n t r a v a s c u l a r pressure of r a t t a i l a r t e r i e s during rewarming.. . . . . . . . . . . . . . . . 121— 123 Schematic r e p r e s e n t a t i o n of the h y d r o s t a t i c pressure and the osmolarity across the w a l l of a d i s t r i b u t i n g a r t e r y . . . . 133  xii  ACKNOWLEDGEMENTS  For advice, patience, support, t e c h n i c a l a s s i s t a n c e , c r i t i c i s m , and encouragement, I would very much l i k e to thank:  the Canadian Heart Foundation,  Dr. P. Constantinides, Mr. B. Cox, Miss M. Drummond, Mrs. C.L. Friedman, Dr. S.M. Friedman, Miss B. Gustafson, Dr. J.A.M. Hinke, Mrs. R. Holtby, the l a t e Mr. C.G. Lemon, Mr. J . Lewis, Dr. C. Loeser, Mrs. C. MacDonald, Miss M. Mar, Dr. V. P a l a t y , the l a t e Dr. G.H. Scott, Mrs. G. Spieckermann, Dr. M.C. S u t t e r , and Mrs. M. Tanaka.  CHAPTER I INTRODUCTION  The artery i s a l i v i n g , working tissue, supplied by i t s own blood vessels and by blood flowing through i t s lumen.  The bulk of the wall of the  artery consists of s p i r a l l y oriented smooth muscle c e l l s immersed i n a network of collagen, e l a s t i n , and protein-polysaccharides.  The i n s i d e of the  wall i s l i n e d with endothelial c e l l s butted against an e l a s t i c lamina and the outside i s a loose coating of collagen f i b e r s , the a d v e n t i t i a .  The  volume of the artery lumen can be passively a l t e r e d by changes i n the i n t r a luminal pressure.  In addition, very f i n e nervous and humoral controls can  change the degree of tension i n the artery wall by changing the a c t i v i t y of the smooth muscle c e l l s , r e s u l t i n g i n an a c t i v e change i n lumen volume. This means that the artery wall regulates blood flow by varying the volume it  encloses. Vasoconstriction i s a decrease i n the s i z e of the a r t e r i a l lumen due  to an increase i n active tension: while the wall thickness increases.  both r a d i i of the artery wall decrease By possessing  sufficient  elasticity,  e l a s t i n acts i n cooperation with the smooth muscle to allow the t o t a l wall tension to automatically change as the artery changes i t s s i z e ( 1 ) . means that the artery i s capable of very f i n e gradations constriction (1).  Consequently, there are a multitude  This  i n i t s degree of  of physical states  of the wall during the t r a n s i t i o n from relaxed to f u l l y constricted states of the artery.  The wall can thus undergo very s l i g h t or very d r a s t i c  physical changes between these d i f f e r e n t states (see 2).  These changes  2 occur q u i c k l y and are thus much more d i f f i c u l t to study than the very slow changes that occur w i t h age  (3) and disease (4,5).  An understanding of the p h y s i c a l changes i n the a r t e r y w a l l assoc i a t e d w i t h c o n s t r i c t i o n could lead to an understanding of how of the a r t e r y w a l l work together. been considered:  the components  I s o l a t e d e f f e c t s of v a s o c o n s t r i c t i o n have  morphological changes (2,6), i o n movements w i t h respect to  the a r t e r y w a l l (7,8) and the smooth muscle c e l l s (9,10), membrane p o t e n t i a l changes (11), v i s c o - e l a s t i c changes (12,13), and hemodynamic changes (14). In a d d i t i o n , the causes of c o n s t r i c t i o n have been examined i n terms of  the  c o n t r a c t i l e proteins of the smooth muscle c e l l s (15,16), the i n i t i a t i o n of c o n s t r i c t i o n (17,18), and the actions of v a s o c o n s t r i c t i v e agents (19). However, a synthesis of these i s o l a t e d f i n d i n g s i s not yet p o s s i b l e .  One  of  the problems i s that few of the studies attempt to r e l a t e the observed e f f e c t s of c o n s t r i c t i o n to the a c t i o n of the whole w a l l .  The dynamic nature  of the a r t e r y w a l l has r e a l l y only been v i s u a l i z e d i n studies on the propagation of pulse pressures down the a r t e r i a l tree (20), i n some of  the  discussions on the question of h y p e r - r e a c t i v i t y of the hypertensive a r t e r y w a l l (21,22,23), and i n various models d e p i c t i n g the i n t e r a c t i o n s of  the  d i f f e r e n t w a l l components (1,24,25). One  component which has received l i t t l e a t t e n t i o n i n regard to vaso-  c o n s t r i c t i o n i s the water i n the a r t e r y w a l l . 75% of i t s weight i s water (26):  For a muscular a r t e r y , about  water f i l l s the smooth muscle c e l l s  the e x t r a c e l l u l a r spaces between them.  I t seems u n l i k e l y that t h i s water  simply remains a s t a t i c solvent during the profound changes i n the of the c o n s t r i c t i n g a r t e r y w a l l .  and  structure  3 There have been observations of losses of water from the a r t e r y w a l l during v a s o c o n s t r i c t i o n .  From electronmicrographs, Rhodin (27), found  smaller i n t e r c e l l u l a r spacings and e x t r a c e l l u l a r space i n contracted mouse i n t e s t i n a l smooth muscle than i n the relaxed muscle.  An increase i n the  percentage dry weight of the r a t aorta exposed to norepinephrine was observed by Rorive et a l . (28).  Daniel (29) found that e x t r a c e l l u l a r f l u i d was  squeezed out of contracted  u t e r i n e muscle.  Turker et a l . (30) observed that  angiotensin-induced c o n s t r i c t i o n of both c a r o t i d a r t e r y and uterus s t r i p s r e s u l t e d i n a l a r g e decrease i n the i n u l i n space.  Small i n s i g n i f i c a n t losses  of water from the c o n s t r i c t i n g a r t e r y w a l l have a l s o been reported  (7,31,32).  However, the only d i s c u s s i o n i n these 7 papers of the i m p l i c a t i o n s of t h i s f i n d i n g was Rorive's comment that the water l o s s may have been due t o the d i s t r i b u t i o n of ions between the d i f f e r e n t t i s s u e compartments (28). The reluctance  to consider  the i m p l i c a t i o n s of f l u i d l e a v i n g the con-  s t r i c t i n g a r t e r y w a l l may a r i s e from the assumption that the w a l l has a constant volume during v a s o c o n s t r i c t i o n (13,33). was c a r r i e d over from Lawton's observation  However, t h i s assumption  on the i n c o m p r e s s i b i l i t y of the  a r t e r y w a l l subjected to small s t r a i n s (34) which has nothing t o do w i t h vasoconstriction.  There are i n f a c t two d i s t i n c t processes:  s t r e t c h , to  which the a r t e r y w a l l responds p a s s i v e l y without the expenditure of energy (1), and v a s o c o n s t r i c t i o n , which i s an a c t i v e process a r i s i n g w i t h i n the w a l l i t s e l f , r e q u i r i n g energy, and i n v o l v i n g l a r g e s t r a i n s .  [This d i s t i n c -  t i o n , of course, i s f o r s t r e t c h without a myogenic response (see 35).] Examination of the e f f e c t of c o n s t r i c t i o n on a r t e r i a l water can lead to a b e t t e r understanding of how the a r t e r y c o n s t r i c t s .  Models of the  a r t e r y w a l l consider i t as a network of smooth muscle c e l l s and e x t r a c e l l u l a r  4 s o l i d s which a c t as a u n i t to a l t e r the degree of c o n s t r i c t i o n  (1,24,25).  There may be an analogy between the a r t e r y w a l l and a g e l or i o n exchange resin:  the degree of s w e l l i n g , i . e . the water content, i s a f f e c t e d by the  e l a s t i c forces w i t h i n the network of the g e l (36,37). e l a s t i c forces a l t e r the hydration of the g e l .  A l t e r a t i o n s i n the  S i m i l a r l y , changes i n a r t e r -  i a l water content may imply a l t e r a t i o n s i n the e l a s t i c forces w i t h i n the artery wall.  This point i s discussed more f u l l y i n Chapter V I .  The increased water content of hypertensive a r t e r i e s (21,26) may be r e l a t e d to the increased Na and mucopolysaccharide  contents (26,38).  Know-  ledge of the f a c t o r s a f f e c t i n g normal a r t e r i a l hydration may help i n understanding some of the p h y s i c a l changes associated w i t h hypertension. In t h i s study, the r e l a t i o n s h i p between v a s o c o n s t r i c t i o n and a r t e r i a l hydration was examined from s e v e r a l d i f f e r e n t experimental angles. the aspects considered were:  Some o f  the constancy of the volume of the c o n s t r i c t i n g  a r t e r y w a l l (Chapter I I ) , changes i n s i z e and shape of the c o n t r a c t i n g vascular smooth muscle c e l l s (Chapter I I ) , a r t e r i a l hydration (Chapter I I I ) ,  the e f f e c t of c o n s t r i c t i o n on  the r e l a t i o n s h i p between the c o n s t r i c t i n g  a r t e r y w a l l and i t s pressure-flow c h a r a c t e r i s t i c s (Chapter I V ) , and the r o l e of the p a r a c e l l u l a r matrix i n a r t e r i a l hydration during i o n exchange and cooling-rewarming the extremes:  procedures  (Chapter V).  The i n i t i a l experiments compared  relaxed and c o n s t r i c t e d a r t e r i e s , while the l a t e r  experiments  considered the process of v a s o c o n s t r i c t i o n . Since the whole vascular system, perfused with blood, was much too complex f o r t h i s i n v e s t i g a t i o n , i s o l a t e d a r t e r i e s perfused w i t h a physiol o g i c a l s o l u t i o n were used.  A l l the information was gathered from i n v i t r o  and i n s i t u preparations of the r a t t a i l a r t e r y .  V a s o c o n s t r i c t i o n was  5  induced by d i f f e r e n t agents which apparently have d i f f e r e n t mechanisms of a c t i o n ( f o r example, 19). Since t h i s study was concerned w i t h the e f f e c t s , not the causes of c o n s t r i c t i o n , the d i f f e r e n c e s between the agents were only b r i e f l y considered.  Adult male a l b i n o r a t s of a s p e c i f i c pathogen-free  Wistar s t r a i n (SPF, Woodlyn Farms) were used.  The r a t s were anesthetized  w i t h 3.33mg sodium p e n t o b a r b i t a l per 100 g body weight administered i n t r a p e r i t o n e a l l y , and 6 mg sodium phenobarbitone per 100 g administered subcutaneous l y . The methods and l i t e r a t u r e f o r each of the experiments are given i n the separate chapters.  The f i n a l chapter contains a d i s c u s s i o n which r e l a t e s  the i n d i v i d u a l f i n d i n g s to the o v e r a l l problem of v a s o c o n s t r i c t i o n and a r t e r i a l hydration. to the chapters.  The references i n the B i b l i o g r a p h y are arranged according  6 CHAPTER I I CHANGES IN VASCULAR DIMENSIONS DURING CONSTRICTION  A r t e r i a l dimensions have been measured i n s i t u (1-4) and from h i s t o l o g i c a l s e c t i o n s (5,6,7).  By p r o v i d i n g information on the appearance of the  a r t e r y i n a given p h y s i o l o g i c a l s t a t e , these methods are a good complement to the usual biochemical and p h y s i o l o g i c a l approaches.  Baez has examined  relaxed and c o n s t r i c t e d microvessels jLn v i v o (2) but there i s very l i t t l e data on dimensional changes of c o n s t r i c t i n g muscular a r t e r i e s .  The present  study attempts to provide some of t h i s information. Several approaches were taken to t h i s problem of changing of a c o n s t r i c t i n g a r t e r y :  dimensions  (a) d i r e c t measurements of the a r t e r i a l r a d i i  using a TV scan technique (1) (see Appendix I ) , (b) measurements of the s p i r a l angle of vascular smooth muscle c e l l s from h i s t o l o g i c a l s e c t i o n s (see Appendix I I ) , (c) h i s t o l o g i c a l procedures to f i x the a r t e r y i n a known functional state.  I t was found that the best method f o r determining vascu-  l a r dimensions was to use h i s t o l o g i c a l s e c t i o n s of f r e e z e - s u b s t i t u t e d arteries. A.  CHANGES IN DIMENSIONS OF ARTERY WALL DURING CONSTRICTION I t i s u s u a l l y assumed that the c r o s s - s e c t i o n a l area of a c o n s t r i c t i n g  a r t e r y i s constant (6,7). be so (8,9). examined:  However, there are i n d i c a t i o n s that t h i s may not  Using f r e e z e - s u b s t i t u t e d a r t e r i a l s e c t i o n s , the present study  (a) the w a l l c r o s s - s e c t i o n a l area and other a r t e r i a l  dimensions  during c o n s t r i c t i o n , and (b) the e f f e c t s of d i f f e r e n t v a s o c o n s t r i c t i v e agents on these dimensions.  7 1.  Methods The v e n t r a l t a i l a r t e r y of the r a t was exposed a t the base of the t a i l  for  cannulation.  from the r a t .  To remove neurogenic i n f l u e n c e s , the whole t a i l was removed  The t a i l a r t e r y was then perfused w i t h Krebs s o l u t i o n (see  Chapter I I I f o r composition) at 37°C using a constant i n f u s i o n pump. The i n t r a v a s c u l a r pressure was monitored with a Statham transducer proximal to the cannula.  A segment of the a r t e r y was exposed, the c o l l a t e r a l s t i e d , and  a s t r i p of f o i l placed under the segment.  Through a microscope, the a r t e r y  was photographed and i t s outer diameter measured using a micrometer eyepiece. The a r t e r y was then frozen i n l e s s than 1.5 seconds by d i r e c t l y applying a s i l v e r probe which had been cooled to -180°C i n l i q u i d nitrogen.  The a r t e r y  was cut f r e e and plunged w i t h the attached f o i l and probe i n t o absolute a l c o h o l a t -80°C.  F i x a t i o n was by freeze s u b s t i t u t i o n (5,10).  The a r t e r y  was t r a n s f e r r e d to a s o l u t i o n of 1% osmic a c i d and absolute a l c o h o l a t -80°C for  7 days, then allowed to warm up to room temperature over 48 hours.  During t h i s rewarming period, the a r t e r i e s were washed w i t h absolute a l c o h o l at the same temperature as the rewarming artery.  I n t h i s manner the problem  of r e f r e e z i n g any water remaining i n the a r t e r y was at l e a s t p a r t i a l l y solved.  The a r t e r i e s were embedded i n p a r a f f i n , cut i n c r o s s - s e c t i o n , and  stained w i t h Mallory trichrome.  The above procedure was done i n cooperation  with the l a t e Dr. G. Scott. Colour photographs of the a r t e r y cross-sections were projected onto a table top.  From these p r o j e c t i o n s , the dimensions of the cross-sections  were measured w i t h a planimeter.  The measurements were c a l i b r a t e d using the  p r o j e c t i o n of a micrometer scale photograph.  The t o t a l m a g n i f i c a t i o n was  458 x f o r most sections and 284 x f o r the l a r g e r s e c t i o n s .  The photographs  8 of the a r t e r i e s i n s i t u were used to determine the outer r a d i i f o r comparison w i t h those measured i n s i t u through the microscope and w i t h those c a l c u l a t e d from the h i s t o l o g i c a l s e c t i o n s . Three a r t e r i a l segments, each about 1.5 cm long, were taken s e r i a l l y from each r a t t a i l a r t e r y at 8, 5, and 2 cm from the base of the t a i l r e s pectively.  The three segments were treated i n the manner described above.  The middle a r t e r y segments were perfused w i t h Krebs s o l u t i o n to which vasoc o n s t r i c t i v e drugs had been added. used f o r the p e r f u s i o n .  A constant i n f u s i o n pump (B. Braun) was  These middle segments were photographed before and  a f t e r the drug a p p l i c a t i o n . Two  spots of I n d i a ink placed on these segments  enabled the photographs to be used f o r determining any changes i n the lengths of the segments.  The f o l l o w i n g 7 groups of 6 r a t s , d i f f e r i n g only i n the  treatment of the middle a r t e r y segment, were used: 1.  untreated  2.  norepinephrine ( l e v a r t e r e n o l b i t a r t r a t e , maximal pressor dose (2 ug)  3.  PLV-2 (phenylalanine^ - l y s i n e - vasopressin, Sandoz), non-pressor dose (10 mU)  4.  PLV-2, pressor dose (80  5.  angiotensin (angiotensin I I amide, Hypertensin, C i b a ) , non-pressor dose (20 ug)  6.  angiotensin, pressor dose (150  7.  low Na s o l u t i o n (Krebs s o l u t i o n w i t h a l l but 1.5 N a / l i t e r replaced by l a c t o s e ) .  Winthrop),  mU)  ug) meq  There was no increase i n the i n t r a v a s c u l a r pressure of a r t e r y segments treated w i t h non-pressor doses of PLV-2 or angiotensin. a r t e r y segments were processed.  A l t o g e t h e r , 126  9 2.  Results The a r t e r i a l dimensions measured from the h i s t o l o g i c a l sections were: A  Q  =  c r o s s - s e c t i o n a l area of lumen + w a l l  A  E  =  c r o s s - s e c t i o n a l area of lumen + media  =  c r o s s - s e c t i o n a l area of lumen  A^  There was some d i f f i c u l t y measuring AQ because the outside of the a d v e n t i t i a was very i r r e g u l a r and o c c a s i o n a l l y t o r n or broken o f f . measurements of A  E  Quite accurate  were made because of the sharp colour contrast between  the media (red) and the a d v e n t i t i a (blue). s e c t i o n s , A^ was e a s i l y measured.  Except f o r the very c o n s t r i c t e d  From these 3 c r o s s - s e c t i o n a l areas, the  f o l l o w i n g c a l c u l a t i o n s were made: Aw  =  c r o s s - s e c t i o n a l area of the a r t e r y w a l l  =  A  Q  &m  =  c r o s s - s e c t i o n a l area of the a r t e r y media =  A  E  A  ad  =  c r o s s - s e c t i o n a l area of the a d v e n t i t i a = A  r  o  =  outer radius of the w a l l = (AO/TT)  r  i  =  inner radius of  =  outer radius of the media = (A /ir)  R  =  w a l l to lumen r a t i o = &/2r±  6  =  thickness of the w a l l  =  r  Q  - r^  d  =  thickness of the media =  r  e  - r^  A  =  thickness of a d v e n t i t i a =  r  e  Q  - Ai -  - A  A  i  (includes intima)  e  h the w a l l = (A±/u) e  r  Q  - r  e  The f i l m s of the a r t e r i a l cross-sections were observed, without i d e n t i f y i n g the frames, and the sections were ranked as 1 , 2 , 3 , or 4 according to the degree of c o n s t r i c t i o n . the a r t e r y cross-sections  The ranking was based upon the appearance of  (see 5 ) :  ( 1 ) relaxed a r t e r i e s :  thin w a l l with a  10 smooth i n t i m a l surface and long t h i n smooth muscle c e l l s and n u c l e i , (2) s l i g h t l y c o n s t r i c t e d a r t e r i e s :  s l i g h t l y thickened w a l l w i t h  occasional  s l i g h t w r i n k l i n g s i n the intima, (3) moderately c o n s t r i c t e d a r t e r i e s : t h i c k e r w a l l w i t h a wrinkled intima, (4) f u l l y c o n s t r i c t e d a r t e r i e s :  small  lumen and very t h i c k w a l l with a very convoluted intima and shortened smooth muscle c e l l s and n u c l e i .  An example of each of these four states of con-  s t r i c t i o n i s shown i n F i g . 1. The errors i n the planimeter measurements and c a l c u l a t i o n s were q u i t e small.  To estimate the e r r o r , an a r t e r i a l s e c t i o n was photographed a t the  high and low m a g n i f i c a t i o n s — f o r culated values.  maximum d i f f e r e n c e s i n the measured and c a l -  The l a r g e s t d i f f e r e n c e f o r the areas, r a d i i , and  thicknesses  was 1.6%. The lengths of the a r t e r i a l segments remained constant during constriction.  Before and a f t e r the a p p l i c a t i o n of the v a s o c o n s t r i c t i v e agent,  the separation of the 2 India i n k spots on the middle segment was measured. The d i f f e r e n c e s between the f i n a l and i n i t i a l separations  were zero f o r  13/33 measurements, p o s i t i v e f o r 8/33 (maximum 1.5%), and negative f o r 12/33 (maximum -2.2%). (19.9  The average separation before the drug was 1.87 i 0.006 mm  cm on the p r o j e c t i o n ) .  The average length change associated w i t h the  a p p l i c a t i o n of the drug was e s s e n t i a l l y zero:  -0.0006 ~t 0.0004 mm.  This  value was smaller than the e r r o r i n the measurements which was about i 0.1 cm on the p r o j e c t i o n s , or i 0.01 mm on the a r t e r i a l segments.  Of these 33  middle a r t e r y segments, 3 were classed as relaxed, 9 s l i g h t l y c o n s t r i c t e d , 9 moderately c o n s t r i c t e d , and 12 were f u l l y c o n s t r i c t e d .  These s t a t i c  measurements i n d i c a t e that the length of these a r t e r i e s d i d not change.  Fig. 1  Four degrees of c o n s t r i c t i o n of the r a t t a i l a r t e r y : 1. relaxed, 2. s l i g h t l y c o n s t r i c t e d , 3. moderately c o n s t r i c t e d , 4. f u l l y c o n s t r i c t e d .  12 Since the a r t e r y segments were tethered at e i t h e r end, t h i s r e s u l t was not unexpected (see 11). Constant length means that the c r o s s - s e c t i o n a l area of the a r t e r y w a l l w i l l r e f l e c t any changes i n the volume of the w a l l . The dimensions of the a r t e r i a l cross-sections are given i n Table I f o r the three a r t e r y segments and the four states of c o n s t r i c t i o n .  The d i s t a l  and proximal segments were not treated with the v a s o c o n s t r i c t i v e agents while the middle segments were. ted than the other two.  However, the middle segments were not more c o n s t r i c There was simply a spread of the various degrees of  c o n s t r i c t i o n f o r a l l three segments.  In f a c t , most of the d i s t a l segments  were f u l l y c o n s t r i c t e d and many of the proximal segments were relaxed.  The  states of these segments before the experimental treatment were not determined, but the "relaxed" proximal segments may have been distended i n r e a c t i o n to the treatment of the middle segment.  The c o n s t r i c t e d appearance of the d i s t a l  segments suggests that they may have been hyper-reactive artery i s usually f a i r l y constricted.  or that the r a t t a i l  In any case, f o r the primary a n a l y s i s ,  the 126 sections were grouped together, regardless of the cause of c o n s t r i c tion. The r e l a t i o n s h i p s between the ranked degree of c o n s t r i c t i o n , 1,2,3,4, and the r a d i i , r  Q  and r ^ , and the w a l l to lumen r a t i o , R, of a l l 126 sections  are shown i n F i g . 2.  The 3 curves i n d i c a t e that the w a l l to lumen r a t i o i s  a good index of v a s o c o n s t r i c t i o n (see 5,12,13).  F i g . 2 also shows that the  greatest increase i n c o n s t r i c t i o n was between states 3 and 4.  Fig. 3 illus-  t r a t e s the r e l a t i o n between the w a l l to lumen r a t i o and the c r o s s - s e c t i o n a l areas of the w a l l and media, using the average values f o r the 4 states of c o n s t r i c t i o n rather than a l l 126 p a i r s of values.  F i g . 3 also shows that  TABLE I . Dimensions (±S.E.) of w a l l of r a t t a i l a r t e r y i n various states of c o n s t r i c t i o n , c a l c u l a t e d from h i s t o l o g i c a l c r o s s - s e c t i o n s . The number of sections i s given i n parenthesis. Overall  r  Q  1 relaxed  2  «**8htly constricted  3 moderately constricted  4 fully constricted  ( y ) outer radius  a l l 3 segments distal middle proximal  (126) (42) (42) (42)  (19) 321 ± 8 (0) (4) 335 ± 19 (15) 318 ± 9  (30) (3) (13) (14)  292 254 285 300  ± ± ± ±  7 22 10 8  (22) (3) (10) (9)  242 236 230 258  ± ± ± ±  7 11 11 11  (55) (36) (15) (4)  194 186 203 237  135 ± 8 54.1 ± 7 146 ± 12 204 ± 9  (19) 255 ± 8 (0) (4) 276 ± 21 (15) 250 ± 8  (30) 211 (3) 180 (13) .211 (14) 218  ± ± ± ±  6 24 10 8  (22) (3) (10) (9)  131 107 121 149  ± ± ± ±  9 2 14 15  (55) (36) (15) (4)  52.7 ± 4. 1 39.2 ± 3. 2 71.8 ± 5. 3 103 ± 23  110 ± 3 140 ± 4 101 ± 5 87.6 ± 4  (19) 66.3 ± 2.5 (0) (4) 58.5 ± 4.0 (15) 68.4 ± 2.8  (30) (3) (13) (14)  77.3 73.3 73.5 81.7  ± ± ± ±  1.8 5.9 2.4 2.4  (22) (3) (10) (9)  111 130 108 109  ± ± ± ±  4 9 6 6  (5) (36) (15) (4)  142 147 132 133  (30) (3) (13) (14)  43.8 40.3 42.1 46.1  ± ± ± ±  1.3 0.7 1.2 2.3  (22) (3) (10) (9)  68.9 82.0 67.2 66.4  (55) (36) (15) (4)  90.3 95.3 81.8 77.3  244 194 247 291  ± ± ± ±  5 5 8 7  ± ± ± ±  4 4 6 25  r^ ( p ) inner radius a l l 3 segments distal middle proximal  (126) (42) (42) (42)  6 ( y ) w a l l thickness a l l 3 segments distal middle proximal  (126) (42) (42) (42)  ± ± ± ±  2 3 3 8  d ( p ) media thickness a l l 3 segments distal middle proximal  (126) (42) (42) (42)  67.4 90.4 61.2 50.6  ± ± ± ±  2. 2 (19) 36.8 ± 1.4 2.9 (0) 3.2 (4) 31.3 ± 2.2 2. 5 (15) 38.3 ± 1.4)  +: 2.8 ±; 7.5 ±: 3.4 ± 4.3  ± ± ± ±  1.8 2.0 2.8 3. 6  TABLE I.  (Continued)  1 relaxed  Overall  A  4 fully constricted  2  a l l 3 segments distal middle proximal  (126) (42) (42) (42)  3 2 Am (10 y ) media area (126) a l l 3 segments distal (42) middle (42) proximal (42) R =  3 moderately constricted  ( 1 0 y ) w a l l area 3  w  2 slightly constricted  118 107 115 131  ± 2  ± 4 ± 3 ± 4  61.4 54.0 60.7 69.5  ± ± ± ±  1.6 2.5 2.3 2.8  ± ± ± ±  (55) (36) (15) (4)  55.1 52.4 58.2 68.0  4 4 5 25  122 ± 4 99.1 ± 12 115 ± 6 133 ± 6  (22) (3) (10) (9)  128 141 117 136  (19) 63.8 ± 3.6 (0) (4) 57.0 ± 4.6 (15) 65.0 ± 4.2  (30) (3) (13) (14)  64.9 50.7 61.3 71.3  (22) (3) (10) (9)  70.3 76.4 65.0 74.1  (19) 0.132 ± 0.006  (30) 0.189 ± 0.008  (22) 0.500 ± 0.052  (55) 1.867 ± 0.158  (3) 0.220 ± 0.044  (3) 0.606 ± 0.036  (36) 2.353 ± 0.195  3.3 6.2 3.2 5.6  5 15 7 7  110 106 114 143  (30) (3) (13) (14)  ± ± ± ±  ± ± ± ±  (55) (36) (15) (4)  (19) 121 ± 6 (0) (4) 111 ± 8 (15) 123 ± 7  ± ± ± ±  3.5 9.2 5.9 3.9  ± ± ± ±  2.1 2.5 3.7 9.6  6/2r^ w a l l to lumen r a t i o  a l l 3 segments  (126) 0.968 ± 0.100  distal  (42) 2.075 ± 0.199  (0)  middle  (42) 0.548 ± 0.063  (4) 0.110 ± 0.013  (13) 0.179 ± 0.011  (10) 0.545 ± 0.094  (15) 0.987 ± 0.068  proximal  (42) 0.278 ± 0.038  (15) 0.138 ± 0.006  (14) 0.191 ± 0.008  (9) 0.416 ± 0.062  (4) 0.801 ± 0.195  4^  15  2.0 r  DEGREE OF VASOCONSTRICTION  Fig. 2  Relation between wall to lumen r a t i o , R, inner and outer r a d i i , r ^ and r , and the degree of vasoc o n s t r i c t i o n , 1, 2, 3, or 4, f o r the r a t t a i l artery. The number of artery sections i s given by n. Q  160 •  140 •  40 -  20 •  i i i i i i i i i i 0.2  0.4  0.6  0.8  1.0  1.2  1.4  i i i  1.6  1.8  2.0  WALL TO LUMEN RATIO R  . 3  R e l a t i o n between the c r o s s - s e c t i o n a l areas of the r a t t a i l a r t e r y w a l l , A^,, and media, A , and the w a l l to lumen r a t i o , R. m  17 the main increase i n c o n s t r i c t i o n , i . e . the greatest  increase i n R,  was  associated with a decrease i n both the w a l l area and the media area.  This  decrease was  126)  also r e f l e c t e d i n the c o r r e l a t i o n c o e f f i c i e n t s ( f o r n =  between: 1.  A„ and R:  2.  A  w  ^  r = -0.412 (p < 0.001);  and R:  r = -0.444 (p < 0.001)  and states 1,2,3,4:  r = -0.218 (p <  0.02)  and states 1,2,3,4:  r = -0.171 (p <  0.05)  3.  Ay and r :  r = +0.392 (p < 0.001); Am and r :  r = +0.466 (p < 0.001)  4.  A„ and r :  r = +0.609 (p < 0.001);  r = +0.616 (p < 0.001)  ±  Q  ±  and r : Q  These s i g n i f i c a n t c o r r e l a t i o n s mean that the more c o n s t r i c t e d a r t e r i e s (with l a r g e r R, greater degree of c o n s t r i c t i o n , and smaller inner and outer r a d i i ) had smaller w a l l and media c r o s s - s e c t i o n a l areas.  The media decreased i t s  3 2 area by 8.7  x 10 u , or 14%, between relaxed and f u l l y c o n s t r i c t e d  and by 15.2  3 2 x 10 u , or 22%, between the moderately and f u l l y 3 2  states.  Although i t s o v e r a l l decrease of 11 x 10 u , or 9%,  laxed and f u l l y c o n s t r i c t e d states was  of these segments was  Over 80% of t h i s decrease i n the w a l l  constant, the decrease i n the  area means that v a s o c o n s t r i c t i o n was volume of the a r t e r y w a l l of about  between the r e -  between the moder-  area can be accounted f o r by the decrease i n the media area. length  constricted  not s i g n i f i c a n t l y d i f f e r e n t from zero, 3 2  the whole w a l l decreased i t s area by 18 x 10 u , or 14%, a t e l y and f u l l y c o n s t r i c t e d s t a t e s .  states,  Since the cross-sectional  associated with a decrease i n the 14%.  As shown i n the columns of Table I , the a r t e r y decreases i n s i z e distally.  Since the d i s t a l segments were more c o n s t r i c t e d than the proximal  segments, i t might be thought that t h i s taper i s the cause of the observed decrease i n w a l l and media areas.  However, the separate consideration  of  18 the 3 segments i n d i c a t e s that the decrease i n the w a l l area was  associated  with v a s o c o n s t r i c t i o n , not with the w a l l taper. Fig. A, m  4 shows the inverse r e l a t i o n s h i p between the area of the media,  and the w a l l to lumen r a t i o , R, f o r the d i s t a l segments.  These segments  were obtained from the t a i l a r t e r y before any drugs were a p p l i e d .  The  values of R were d e f i n i t e l y associated w i t h the smaller values of A  m  r = -0.479, so p < 0.01). A  w  A s i g n i f i c a n t c o r r e l a t i o n was  vs R f o r the d i s t a l segments, and f o r A  (On the other hand, there was proximal a r t e r y segments.  (n = 42,  also obtained f o r  vs R f o r the middle segments.  no c o r r e l a t i o n between R and the areas f o r the  The distended appearance of many of these proximal  segments suggested that they may segments.)  m  larger  have reacted to the treatment of the middle  These r e l a t i o n s h i p s mean that the taper of the t a i l a r t e r y  was  not the cause of the observed decrease i n the w a l l c r o s s - s e c t i o n a l area. Rather, the w a l l s of the more c o n s t r i c t e d a r t e r i e s had smaller  cross-sectional  areas, and hence, smaller volumes. The d i f f e r e n t v a s o c o n s t r i c t i v e agents did not have the same e f f e c t on the dimensions of the middle a r t e r i a l segments.  Having only 6 segments i n  each group meant that trends and not h i g h l y s i g n i f i c a n t d i f f e r e n c e s were observed.  Two  of the 7 groups of middle segments seemed d i f f e r e n t i n t h e i r  geometrical response to c o n s t r i c t i o n : 1. unique.  The group treated w i t h a non-pressor dose of angiotensin I t was  was  the only group f o r which the c o r r e l a t i o n c o e f f i c i e n t was  p o s i t i v e f o r R vs A  w  and f o r R vs A « m  This suggested that a f t e r a  pressor dose of angiotensin, a r t e r i e s which had c o n s t r i c t e d had c r o s s - s e c t i o n a l areas than a r t e r i e s which were l e s s opposite to the observation  non-  larger  constricted—the  f o r a l l 126 a r t e r i e s and f o r two of the segments.  19  \R = 4.l47-0.038A  0.5  1.5  2.0  m  2.5  3.0  35  4.0  4.5  5.0  5.5  WALL TO LUMEN RATIO R  Fig. 4  2.  R e l a t i o n between the c r o s s - s e c t i o n a l area of the a r t e r y media, A^, and the w a l l to lumen r a t i o , R, f o r the d i s t a l segments of the r a t t a i l a r t e r y .  Some o f the a r t e r i e s were f u l l y c o n s t r i c t e d even though they  showed no pressure r i s e to a non-pressor dose of PLV-2. unusual.  These a r t e r i e s were  Of the 15 f u l l y c o n s t r i c t e d middle segments., 3 were given PLV-2 i n  a non-pressor dose.  Table I I shows that the outer r a d i i and w a l l area of  these 3 were l a r g e r than those f o r the other 12 segments.  The d i f f e r e n c e  between these 2 groups was s i g n i f i c a n t only a t p < 0.05. These d i f f e r e n c e s mean that the f u l l y c o n s t r i c t e d a r t e r i e s which were given a non-pressor dose of PLV-2 had l a r g e r w a l l volumes than expected.  20 TABLE I I .  3 2 Outer radius ( y ) and c r o s s - s e c t i o n a l area (10 y ) of the w a l l of f u l l y c o n s t r i c t e d middle segments of the rat t a i l artery.  PLV-2 (non-pressor) segments  A l l segments  Aw  (15)  203 ± 6  (15)  114  t  5  other 12 segments  (3)  225 + 8  (12)  198 + 6  (3)  135 I 7  (12)  109 - 5  These two points suggest that non-pressor doses of angiotensin  and  PLV-2 a l t e r the geometry of c o n s t r i c t e d a r t e r i e s i n a manner d i f f e r e n t than other v a s o c o n s t r i c t i v e agents.  There i s a suggestion that they increase the  volume of the c o n s t r i c t e d a r t e r y w a l l .  I n s p i t e of t h i s f a c t , o m i t t i n g one  or both of these groups of 6 a r t e r i e s d i d not s i g n i f i c a n t l y a l t e r the R vs A  w  (and Apj) r e l a t i o n s h i p s f o r the 126 sections or f o r the middle a r t e r i a l  segments. 3.  E f f e c t of F i x a t i o n and Embedding on A r t e r i a l Dimensions Since the above f i n d i n g s depended upon measurements made from h i s t o l o -  g i c a l s e c t i o n s , i t i s important to know the e f f e c t of preparative on the a r t e r i a l dimensions.  handling  The only measureable f a c t o r was the e f f e c t on  the outer r a d i i of the v e s s e l s .  This determination l e d to an estimate of  the shrinkage caused by the f r e e z i n g , s u b s t i t u t i o n , and embedding processes (see 14). The outer r a d i i of the a r t e r i e s i n s i t u were measured:  (a) from pro-  j e c t i o n s of photographs, arid (b) through a microscope with a micrometer scale.  These values were compared to those c a l c u l a t e d from the h i s t o l o g i c a l  21 sections. and  These 3 outer r a d i i w i l l be l a b e l l e d as:  r (histology), respectively.  The 3 values of r  Q  r ( f i l m ) , r (observed), 0  Q  Q  can be compared s i n c e  the measurements were a l l made i n approximately the same spot on the a r t e r y segments:  r ( f i l m ) was measured i n the middle of the photographs, 0  r (observed) Q  was determined f o r the middle of the segment, and r ( h i s t o l o g y ) was c a l c u Q  l a t e d f o r s e c t i o n s cut from the middle of the segments. i n these values were:  The maximum e r r o r s  ± 10 u i n . r ( f i l m ) , ± 2 5 u i n r ( o b s e r v e d ) , and 0  i 2 u i n r (histology). Q  Q  The average values of the 3 outer r a d i i are pre-  sented i n Table I I I . TABLE I I I .  Outer r a d i u s , r ( i n u ) , of the r a t t a i l a r t e r y from i n s i t u photographs, i n s i t u observations, and h i s t o l o g i c a l s e c t i o n s . Q  overall  proximal segments  middle segments*  distal segments  r (film)  (157)** 310 ± 6  (40) 373 + 8  (41) 317 ± 9  (41) 232 ± 5  r (observed)  (159)** 290 + 5  (40) 355 + 7  (41) 287 ± 10  (42) 223 + 5  r (histology)  (126)  (42) 291 ± 7  (42) 247 ± 8  (42) 194 + 5  Q  Q  Q  244 + 5  * post-drug measurements ** includes pre-drug measurements of the middle segments  1.  The d i f f e r e n c e s between r ( f i l m ) and r (observed) were s i g n i f i c a n t (by Q  Q  the Student's t - t e s t ) only f o r the proximal a r t e r y segments (which had the l a r g e s t diameters).  That these 2 i n s i t u values should be d i f f e r e n t was not  unexpected, but why the average values of r ( f i l m ) were l a r g e r than those of 0  r (observed) can not be explained. Q  2.  The values of r ( h i s t o l o g y ) were s i g n i f i c a n t l y smaller than those of Q  e i t h e r r ( f i l m ) or r (observed). 0  The s i z e of the d i f f e r e n c e s i n d i c a t e d  22 that the a r t e r i e s d i d i n f a c t shrink during preparation  of the h i s t o l o g i c a l  sections. The extent of the shrinkage i n the sections can be estimated best by comparing the c r o s s - s e c t i o n a l areas of the a r t e r y w a l l plus lumen, A , before Q  and a f t e r the h i s t o l o g i c a l preparation.  The average values of A , Q  calcu-  l a t e d from the corresponding values of r , are given i n Table IV. 0  TABLE IV.  The c r o s s - s e c t i o n a l area of the r a t t a i l a r t e r y w a l l plus lumen, AQ ( i n 10-^p^), from i n s i t u photographs, i n s i t u observations, and h i s t o l o g i c a l s e c t i o n s .  overall  "  proximal segments  middle segments  distal segments  A (film)  (157)  318 t 11  (40) 444 ± 18  (41) 327 + 19  (41) 173 ± 8  AQ(observed)  (159)  280 ± 10  (40) 403 + 16  (41) 273 ± 19  (42) 160 + 7  AQ(histology)  (126) 199 ± 9  (42) 273 ± 12  (42) 201 + 14  (42) 122 ± 7  Q  The d i f f e r e n c e s between a l l 3 values were s i g n i f i c a n t ( i . e . p < 0.02): 3 2 A ( h i s t o l o g y ) - A ( f i l m ) = -119 x 10 u , a change of -37% 3 2 Q  AQ(histology)  Q  - AQ(observed) = -81 x 10 p , a change of -29%  The average decrease i n the c r o s s - s e c t i o n a l area of the a r t e r y w a l l plus lumen a f t e r f r e e z i n g , dehydration, and embedding was 33%. I t i s to be remembered that t h i s shrinkage of 1/3 was i n the c r o s s s e c t i o n a l area of the whole s e c t i o n , not j u s t i n the a r t e r y w a l l .  However,  i t was p o s s i b l e to measure the inner radius of the a r t e r y w a l l , r ^ , from the photographs of 3 segments. A  W  =  Since the c r o s s - s e c t i o n a l area of the w a l l i s  i r ( r ^ - v±^), i t was p o s s i b l e to compare A 0  W  with Ay c a l c u l a t e d from the h i s t o l o g i c a l s e c t i o n s .  f o r the a r t e r i e s i n s i t u The changes i n c r o s s -  23 3 2 s e c t i o n a l area ( i n 10 y ) a f t e r h i s t o l o g i c a l preparation of these 3 a r t e r i e s were:  (a) A^: + 1.8, A : + 59.3, (b) A : -24.2, A„: +42.7, (c) A : -40.4, w  A : -15.8. w  <3  Q  While i t i s not p o s s i b l e to make a d e f i n i t i v e statement about 3  a r t e r i e s , these values do i n d i c a t e that there need not be a c o r r e l a t i o n between the decrease i n AQ and the decrease i n A^.  I n a d d i t i o n , since the  lengths of the a r t e r y segments were not measured a f t e r f i x a t i o n and embedding, any changes i n length could not be determined.  Consequently, the shrinkage  i n the c r o s s - s e c t i o n a l area of the a r t e r y sections can not be extended to a decrease i n the volume of the s e c t i o n , or of the w a l l i t s e l f . 4.  Discussion The c r o s s - s e c t i o n a l areas of the w a l l s of relaxed and c o n s t r i c t e d  a r t e r i e s have been measured before.  However, e i t h e r no change i n w a l l area  with v a s o c o n s t r i c t i o n was observed, or the changes were not mentioned.  Using  h i s i m a g e - s p l i t t i n g technique, Baez measured the w a l l r a d i i of 14 microvessels i n vivo (2).  Although he found no consistent change i n w a l l area a f t e r  c o n s t r i c t i o n w i t h epinephrine or norepinephrine, he d i d observe an increase i n w a l l area a f t e r v a s o d i l a t i o n w i t h a c e t y l c h o l i n e .  Wiederhielm  reported  that 43 measurements (S.D. 1 10%) from photographs showed no s i g n i f i c a n t d i f f e r e n c e between the w a l l areas of relaxed and c o n s t r i c t e d a r t e r i o l e s ( 7 ) . On the other hand, there i s some support f o r a decrease i n a r t e r i a l w a l l volume during v a s o c o n s t r i c t i o n .  From X-ray photographs, Ticker and Sacks  (9) measured the r a d i i and lengths of human b r a c h i a l and dog t h o r a c i c a r teries  i n f l a t e d w i t h a i r . With  volume decreased.  increased  i n t r a l u m i n a l pressure, the w a l l  From electronmicrographs of mouse i n t e s t i n a l smooth  muscle, Rhodin (8) measured the i n t e r c e l l u l a r spacing and the e x t r a c e l l u l a r space.  The values were 1500 A and 11% f o r the relaxed muscles and 600 A and  24 5% f o r contracted muscles.  Hinke (6) measured the c r o s s - s e c t i o n a l areas of  perfused and non-perfused r a t t a i l a r t e r i e s from h i s t o l o g i c a l s e c t i o n s . Although not mentioned by him, the w a l l to lumen r a t i o s of these v e s s e l s show that the non-perfused a r t e r i e s , which were more c o n s t r i c t e d ( r a t i o = 3 2 1.22), had a smaller c r o s s - s e c t i o n a l w a l l area (46.5 x 10 u ) than the per3 2 fused a r t e r i e s ( r a t i o = 0.35 and area = 56.5 x 10 u ). Recently, Phelps and L u f t (15) compared the appearances of relaxed and c o n s t r i c t e d f r o g arterioles.  Two of t h e i r electronmicrographs show relaxed and c o n s t r i c t e d  pdrtions of the same a r t e r i o l e . Planimeter measurements of these f i g u r e s reveal that the c o n s t r i c t e d v e s s e l ( w a l l to lumen r a t i o = 0.738) had an 18% smaller w a l l c r o s s - s e c t i o n a l area than the relaxed v e s s e l ( r a t i o = 0.218). Baez suggested that the inner radius decreased more than the outer radius during c o n s t r i c t i o n because of a " t e t h e r i n g a c t i o n afforded by the t i s s u e s t r u c t u r e s surrounding the microvessel"  ( 2 ) . Since the same observa-  t i o n was made i n t h i s study f o r a r t e r i e s separated from t h e i r surroundings, t h i s suggestion seems u n l i k e l y .  The increase i n w a l l thickness i s more  l i k e l y due to the mechanism of c o n s t r i c t i o n of the vascular w a l l (see 16). A decrease i n the c r o s s - s e c t i o n a l area and volume of the a r t e r y w a l l during c o n s t r i c t i o n suggests that the w a l l l o s t some of i t s water when i t constricted. 5.  This suggestion w i l l be examined i n the next chapter.  Summary 1.  Freeze s u b s t i t u t i o n was a good method f o r f i x i n g a r t e r i e s i n v a r -  ious states of c o n s t r i c t i o n . 2.  The process of f r e e z i n g , dehydrating, and embedding the a r t e r i e s  was associated w i t h a decrease of 1/3 i n the t o t a l c r o s s - s e c t i o n a l area of the a r t e r y w a l l plus lumen.  25 3.  From a c l a s s i f i c a t i o n of 4 states of c o n s t r i c t i o n , the average  outer diameter decreased from 642 y f o r the relaxed r a t t a i l a r t e r y to 388 y f o r the f u l l y c o n s t r i c t e d a r t e r y — a decrease of 40%. the lumen decreased from 510 to 105 y — a decrease of 4.  The w a l l to lumen r a t i o was  The diameter of  80%.  a good index of the s t a t e of con-  s t r i c t i o n of these a r t e r i e s . 5.  In a l l states of c o n s t r i c t i o n , the media accounted f o r about 50%  of the a r t e r y w a l l c r o s s - s e c t i o n a l area. loose, the % by weight was 6.  Since the a d v e n t i t i a was  very  probably higher.  The length of these tethered a r t e r i a l segments d i d not change a f t e r  v a s o c o n s t r i c t i v e agents were a p p l i e d .  Thus, changes i n the a r t e r y w a l l volume  were r e f l e c t e d i n changes i n the c r o s s - s e c t i o n a l area. 7.  The more c o n s t r i c t e d a r t e r i e s had s i g n i f i c a n t l y smaller w a l l and  media c r o s s - s e c t i o n a l areas than l e s s c o n s t r i c t e d a r t e r i e s . was  Vasoconstriction  associated with a decrease i n the volume of the a r t e r y w a l l of about 8.  14%.  Such a decrease i n w a l l volume suggests that there are movements  of f l u i d out of the c o n s t r i c t i n g a r t e r y w a l l . 9.  Non-pressor doses of angiotensin and PLV-2 a l t e r e d the geometry  of c o n s t r i c t e d a r t e r i e s i n a manner d i f f e r e n t than other v a s o c o n s t r i c t i v e agents.  B.  CHANGES IN DIMENSIONS OF SMOOTH MUSCLE CELLS DURING VASOCONSTRICTION Very l i t t l e has been done on c h a r a c t e r i z i n g the geometrical changes of  contracting smooth muscle c e l l s .  A few dimensional measurements have been  made (8,15,17), but comparison of relaxed and contracted  c e l l s has been  mainly q u a l i t a t i v e . Baez (17) found a 32 to 70% increase i n the  thickness  26 of smooth muscle c e l l s of the c o n s t r i c t i n g r a t a r t e r i o l e — t h e greater i n crease f o r the greater dose of norepinephrine.  Using a model f o r the smooth  muscle c e l l and measurements from h i s t o l o g i c a l s e c t i o n s , a more d e t a i l e d study of the changes which the vascular smooth muscle c e l l undergoes during c o n s t r i c t i o n was  attempted.  1.  Model f o r the Smooth Muscle C e l l  Mathematical  As a model f o r the smooth muscle c e l l , consider 2 r i g h t c i r c u l a r cones j o i n e d at t h e i r bases:  For the dimensional parameters of the c e l l , l e t : length = 2h  2 2 volume = V = y irr h  diameter at center = 2r  surface area = A = 27rr(r + h ^ j = 27rrh  The approximation  2  f o r the surface area i s j u s t i f i e d since the dimensions of  the smooth muscle c e l l s of a small a r t e r y are such that h^>> approximation w i l l be examined l a t e r .  r^(8).  This  During c o n t r a c t i o n of the smooth  muscle c e l l these v a r i a b l e s w i l l change. variables.  5  Let A r e f e r to the changes i n these  For x, any c e l l u l a r v a r i a b l e , Ax = x ( a f t e r c o n t r a c t i o n ) -  x (before contraction) .  Also the average value of x i s then x =  hi2x + Ax) .  With these d e f i n i t i o n s , i t i s p o s s i b l e to express the changes i n the c e l l surface area and volume as functions of the c e l l u l a r radius and h a l f - l e n g t h . Change i n c e l l surface area: AA  =  A(2frrh)  =  2-rr(rAh + hAr) = 2iT(rAh + hAr + ArAh)  27 This equation i s f o r the % change i n the c e l l surface area as a f u n c t i o n of the % changes i n the c e l l radius and length. Change i n c e l l volume: AV  =  A(|irr h) = 2TT_  Ah(r  3 AV  '•• T  " y ( r A h + 2rhAr)  2  Ah  • T  . 2Ar  * =?  2  2  + 2rAr + ( A r ) ) +  Ar(2rh + hAr)  2  . 2 Ar  *  Ah  1  r  A  -  1 2  • [T] [ ' • ¥ ]  <•'  S u b s t i t u t i n g equation (1) i n t o equation ( 2 ) : AV V  2AA A  i2  Ah h  This equation shows that i f the % change i n the c e l l surface area i s zero, the % change i n the c e l l volume need not be. As an a l t e r n a t i v e model of the smooth muscle c e l l , consider a r i g h t circular cylinder:  9  T 2r  H  Let the c e l l parameters be: 2 length  =  H  volume  diameter = 2r Change i n c e l l surface area: AA So, '  A  AA —  =  A(2TrrH) = =  AH — H  , Ar + — r  2ir(rAH + HAr) , Ar + — r  V  =  irr H  surface area  =  A  =  =  2irrH  2TT (rAH + HAr + ArAH)  AH — H  ' - • «.u This equation i• s the same as equation ,v\ (1) s i•n c e : — H  ™ .  =  A H  = A(2h) —rr-— zn  Ah = -rh  28 S i m i l a r l y f o r the change i n c e l l volume:  AV V  _ AH H  r  2Ar  2Ar AH  r  H  r^I."| r 2  L rJ  |_  i +  Ml  H J  This equation i s the same as equation (2). For the 2 models, AA and AV are d i f f e r e n t , but the % changes i n A and V are the same.  This can be shown  by s u b s t i t u t i n g 2h f o r H i n the above 2 equations, so that: A ( c y l ) = 2 A(cones) and  AA(cyl) =  V ( c y l ) = 3 V(cones), and  M  m  V V /cyl  AV(cyl)  2AA(cones), so that:  =  3AV(cones), so t h a t :  /AV\ \ V J cones  In a d d i t i o n to g i v i n g the magnitude of the changes i n the c e l l area and volume, equations (1) and (2) contain i n f o r m a t i o n on the signs of these changes.  Since  Ar > 0 and Ah < 0, the signs of AA and AV w i l l depend on the  r e l a t i v e magnitudes of r , h,  Ar, and Ah.  But f o r a given a r t e r y , the values  of the radius and length of the smooth muscle c e l l s i n the relaxed s t a t e ( i . e . r and h) are f i x e d , at l e a s t w i t h i n a f a i r l y narrow range. magnitude of Ar and Ah determine the signs of AA and AV. construct graphs r e l a t i n g these v a r i a b l e s . and h have been chosen, AA/A  Thus, the .  I t i s p o s s i b l e to  For example, once values f o r r  from equation (1) can be p l o t t e d against Ah/h  for a f a m i l y of curves of d i f f e r e n t Ar values, or against Ar/r f o r a f a m i l y of d i f f e r e n t Ah values.  S i m i l a r l y f o r AV/V  from equation ( 2 ) . Values f o r  r and h are determined i n the f o l l o w i n g s e c t i o n . 2.  Measurement of C e l l Dimensions  Methods To obtain the values of the relaxed smooth muscle c e l l r a d i u s , r , and  29 length, 2h, f o r the r a t t a i l a r t e r y , and to estimate the p h y s i o l o g i c a l range of Ar and Ah ( i . e . the d i f f e r e n c e between the relaxed and contracted  values),  s e v e r a l of the h i s t o l o g i c a l sections from the study described above were used. The stained a r t e r i a l cross-sections were photographed and projected onto a table top where the t o t a l m a g n i f i c a t i o n was 2900 x. The c e l l h a l f - l e n g t h (from the middle of the n u c l e i to the end of the c e l l ) and maximum width through the n u c l e i of relaxed and contracted c e l l s were measured w i t h a r u l e r . C e l l s without n u c l e i i n the plane of the s e c t i o n and c e l l s cut i n c r o s s s e c t i o n were ignored.  Some of the sections contained both relaxed and con-  t r a c t e d c e l l s so that comparison of the dimensions i n the 2 s t a t e s was more meaningful.  I n a d d i t i o n , the areas of i n d i v i d u a l c e l l s i n the a r t e r i a l  c r o s s - s e c t i o n were measured with a planimeter double cones or c y l i n d e r .  to determine the b e t t e r model:  The area of 1/2 of a c e l l was compared w i t h the  c a l c u l a t e d areas of a t r i a n g l e (rh) and a rectangle (2rh), since they are the p r o j e c t i o n s onto 2 dimensions of a cone and a c y l i n d e r , to see which was the c l o s e s t f i t . Results The dimensions (± S.E.) of the relaxed and contracted smooth muscle c e l l s are given i n Table V. TABLE V.  Half-lengths and r a d i i ( i n y ) of relaxed and contracted smooth muscle c e l l s of the r a t t a i l a r t e r y . relaxed c e l l s  contracted  A  cells  half-length, h  (24) 40.3 + 1.4  (70) 20.9 + 0.5  Ah = -19.4  c e l l radius, r  (87) 1.76 ± 0.03  (83) 3.46 + 0.07  Ar = +1.70 i  From these values ,  4r n  x  100 = -48%  and  — r  x 100 =  + 97%.  30 The area of the h a l f - c e l l was best approximated by the area of a t r i a n g l e f o r both relaxed and contracted c e l l s .  So, the double cone model  i s the b e t t e r f i t f o r these smooth muscle c e l l s .  The appearance of the c e l l s  agreed w i t h t h i s , although the taper at the e x t r e m i t i e s of the c e l l s was more pronounced i n the relaxed than the contracted c e l l s . 3.  F i t t i n g the Experimental Data to the Model With the understanding  that the r e s u l t s are only approximate, the  values of the c e l l r a d i u s and h a l f - l e n g t h were s u b s t i t u t e d i n t o the equations f o r the surface area and volume of the double cone model.  The values  obtained  are given i n Table V I . TABLE VI.  Surface area ( u ) and volume ( u ) of relaxed and contracted smooth muscle c e l l s of the r a t t a i l a r t e r y . J  relaxed c e l l s  A  contracted c e l l s  surface area, A  446  454  AA = +8  volume, V  261  524  AV = +263  AA  From these values,  x  1 0 0  = + 2%  and  —  x 100  =  + 101%.  A So, the model i n d i c a t e s that c o n t r a c t i o n of these vascular smooth muscle c e l l s was associated w i t h no change i n the c e l l surface area but a doubling of i t s volume. 2  The approximation made f o r the surface area of the double ? J'  cone [ i . e . (h + r )  2  = h] i s poorest f o r the contracted c e l l s .  values were only 1.5% too s m a l l , so the approximation  But the  seems j u s t i f i e d .  If 2  d i f f e r e n t models were used to represent the relaxed and contracted c e l l s (considering the contracted c e l l s as e l l i p s o i d s i s the only other r e a l i s t i c p o s s i b i l i t y ) , then the % changes i n A and V become even l a r g e r .  31 The model p r e d i c t s the % changes i n the surface area and volume of the muscle c e l l s as functions of the % changes i n c e l l length and radius during v a s o c o n s t r i c t i o n . F i g s . 5, 6, 7, and 8 show these changes. curves were obtained from equations  The  (1) and (2) by s u b s t i t u t i o n of the ex-  perimental values f o r the relaxed c e l l h a l f - l e n g t h , h = 40.3 y , and r a d i u s , r = 1.76  y i n t o these equations.  from Table VI.  The p o i n t s on the graphs are those derived  C o n s t r i c t i o n i n the r a t t a i l a r t e r y would be represented  by  a s t r a i g h t l i n e drawn from the mid-axes point to the point derived from the data.  The graphs show, as mentioned above, that the signs of AA and AV are  determined by the signs of Ar and Ah.  F i g s . 5 and 6 show:  (a) For a given  decrease i n c e l l length, the greater the c e l l t h i c k e n i n g , the more p o s i t i v e the change i n c e l l surface area.  (b) For a given increase i n c e l l t h i c k n e s s ,  the greater the c e l l shortening, the more negative the change i n c e l l surface area.  F i g . 7 and 8 show:  (a) For a given decrease i n c e l l length, the  greater the c e l l t h i c k e n i n g , the more p o s i t i v e the change i n c e l l volume, (b) For a given increase i n c e l l thickness, the greater the c e l l shortening, the more negative the change i n c e l l volume.  (c) An increase i n the c e l l  volume i s not n e c e s s a r i l y accompanied by c o n t r a c t i o n , or v i c e v e r s a . 4.  Discussion These c e l l s are about the same s i z e as other v a s c u l a r smooth muscle  cells.  Baez (17) found that smooth muscle c e l l s i n relaxed r a t a r t e r i o l e s  were from 2.08  to 2.78  y thick.  Using f r o g a r t e r i o l e s , Phelps and L u f t (15)  reported that the relaxed c e l l s were 100 y long and 9 y at the widest p o i n t . Keatinge  (18) found the smooth muscle c e l l s of sheep c a r o t i d a r t e r i e s were  60 to 100 y long and 2.3 y wide.  Rhodin (8) noted that " . . .  muscle f i b e r s  32 F i g s . 5 to 8. The % changes i n surface area, AA/A, and volume, AV/V, of the double cone model of the vasular smooth muscle c e l l as functions of the % changes i n c e l l h a l f - l e n g t h , Ah/h, and r a d i u s , A r / r . These graphs were c a l culated from equations (1) and (2) i n the t e x t . The values f o r the h a l f length and radius of the relaxed smooth muscle c e l l s of the r a t t a i l a r t e r y (h = 40.3 u and r = 1.76 u ) were s u b s t i t u t e d i n t o these equations to obtain the 4 f a m i l i e s of curves. Note that the s c a l e of the h o r i z o n t a l a x i s i s d i f f e r e n t i n F i g s . 6 and 8 than i n F i g s . 5 and 7. The points on the graphs are those derived from Table VI.  Fig.  5  C e l l surface area changes vs c e l l length changes  33  -2.00  Fig. 6  -1.50  C e l l surface area changes vs c e l l radius changes  Fig. 7  C e l l volume changes vs c e l l length changes  -2.00  Fig. 8  -1.50  C e l l volume changes vs c e l l radius changes  36 of small a r t e r i e s have a cross diameter i n the c e n t r a l c e l l region of about 1.5  to 2.5 u and an average length of about 60 y. . . the t o t a l surface area  of the c e l l i s about 400 y  2  plus another 100 y  7  f o r numerous inpocketings  and p i n o c y t o t i c v e s i c l e s . " The double cone model was a b e t t e r f i t f o r these r a t t a i l a r t e r y smooth muscle c e l l s than the c y l i n d e r model. that the " . . .  This i s i n contrast to Rhodin's f i n d i n g  smooth muscle c e l l i n the relaxed s t a t e has the shape of a  c y l i n d e r rather than that of a double cone" (8).  However, Phelps and L u f t  (15) observed that the relaxed c e l l s of a f r o g a r t e r i o l e were s p i n d l e shaped and the contracted c e l l s had "a roughly cubic shape." The constant surface area during c o n t r a c t i o n of vascular smooth muscle c e l l s r e f e r s to the surface area of the double cone model—not n e c e s s a r i l y to that of the c e l l s themselves.  The model does not include the inpocketings  i n the smooth muscle c e l l membranes.  Changing the number of inpocketings  could a l t e r the surface area—depending on the mechanism:  (a) i f an  inpock-  e t i n g were closed o f f , then the surface area of the c e l l would be decreased, while that of the model would be constant, (b) on the other hand, i f a l l the c e l l membrane i n the inpocketing came to the outside of the c e l l , then the surface area of the model would be increased while that of the c e l l would be constant.  Since changes i n the surface area of the smooth muscle  c e l l would probably i n v o l v e the inpocketings and p i n o c y t o t i c v e s i c l e s ,  this  observation of constant surface area of the model during v a s o c o n s t r i c t i o n , r e f e r s to the "envelope" of the c e l l , not i t s a c t u a l surface area.  To i n -  clude the inpocketings, measurements of the surface areas of vascular smooth muscle c e l l s would have to be made from e l e c t r o n micrographs.  37 The increase i n the c e l l volume seems e x t r a o r d i n a r i l y l a r g e .  But the  model r e a l l y only q u a l i t a t i v e l y i n d i c a t e d that v a s o c o n s t r i c t i o n was a s s o c i ated w i t h an increase i n the volume of the smooth muscle c e l l .  It is  assumed that the quantity of s o l i d s i n the c e l l s remained constant  (19). I f  the density of bound water were l e s s than that of free water, an increase i n the amount of bound water i n the c o n t r a c t i n g smooth muscle c e l l would i n crease i t s volume, but only s l i g h t l y .  This means that a movement of water  i n t o the c o n t r a c t i n g muscle c e l l was the cause of i t s volume increase. Reasons why water would move i n t o the c o n t r a c t i n g c e l l w i l l be discussed i n Chapter V I . An increase i n the volume of c o n t r a c t i n g smooth muscle c e l l s would agree with the e f f e c t s of non-isosmotic s o l u t i o n s on vascular t i s s u e . s o l u t i o n s cause c e l l s w e l l i n g and v a s o c o n s t r i c t i o n (19), while s o l u t i o n s cause c e l l shrinkage and v a s o d i l a t i o n (20).  Hypotonic hypertonic  This i n t e r r e l a t i o n s h i p  between c e l l volume and the degree of vascular tension suggests that the same r e l a t i o n may hold f o r drug induced tension changes.  To e x p l a i n i o n i c move-  ments, Friedman (21) has suggested that the smooth muscle c e l l volume may i n crease during v a s o c o n s t r i c t i o n .  A 5% increase i n c e l l water was observed f o r  the r a t t a i l a r t e r y c o n s t r i c t e d with norepinephrine  (22),  In a d d i t i o n , a l -  though Jonsson (19) suggested that a c t i v e tension i n the r a t p o r t a l v e i n opposed c e l l s w e l l i n g i n hypotonic s o l u t i o n s , he found 7 to 11% increases i n the c e l l water of veins c o n s t r i c t e d w i t h norepinephrine.  I t should be noted  that these small increases were determined from i n u l i n and sucrose d i s t r i b u t i o n s which may not accurately r e f l e c t e x t r a c e l l u l a r space changes during v a s o c o n s t r i c t i o n (see Chapter I I I ) .  38 These p r e d i c t i o n s from geometric models and experimental measurements are f i r s t approximations.  The changes i n volume or surface area of the  vascular smooth muscle c e l l may t u r n out to be d i f f e r e n t than those p r e d i c t e d . I f so, i t would s t i l l remain to e x p l a i n e x a c t l y how the changes i n geometry of the c o n t r a c t i n g c e l l d i f f e r from those of the models. 5.  Summary 1.  Geometrical models of the v a s c u l a r smooth muscle c e l l provide a  good framework f o r understanding dimensional changes of the c o n t r a c t i n g c e l l . 2.  During c o n s t r i c t i o n of the r a t t a i l a r t e r y , the smooth muscle c e l l  length decreased by h a l f and the radius at the c e l l center doubled:  the  relaxed c e l l s were 80 y long and 3.5 y wide w h i l e the contracted c e l l s were 40 y long and 7 y wide. 3.  These c e l l s were best approximated by 2 r i g h t c i r c u l a r cones  joined a t t h e i r bases. 4.  This model of the c e l l provided a means of r e l a t i n g the changes  i n c e l l volume and surface area to the changes i n c e l l length and r a d i u s . 5.  The model i n d i c a t e d that the surface area of the smooth muscle  c e l l s was constant w h i l e the volume of the c e l l s increased during vasoconstriction. 6.  The increase i n volume i m p l i e d that the c o n t r a c t i n g muscle c e l l s  gained water.  39 CHAPTER I I I CHANGES IN WATER CONTENT OF ARTERY WALL DURING CONSTRICTION  The decrease i n the c r o s s - s e c t i o n a l area of the c o n s t r i c t e d a r t e r y w a l l , discussed i n the previous chapter, suggests the existence of a decrease i n the water content of the c o n s t r i c t i n g a r t e r y w a l l .  A few studies have  demonstrated such a water l o s s from contracted smooth muscle, but they d e a l t mainly w i t h changes i n Na and K and were not followed up (1,2).  In addition,  an e x t r a c e l l u l a r decrease associated w i t h v a s o c o n s t r i c t i o n has been measured (3).  Considerable e f f o r t has been expended i n analyses of the i o n i c content  of a r t e r i e s (4,5,6) and t h e i r exchanges during c o n s t r i c t i o n (1,7,8).  On the  other hand, comparison of the water content of a r t e r i e s i n two d i f f e r e n t s t a t e s , whether normotensive  and hypertensive or relaxed and c o n s t r i c t e d , i s  q u i t e a d i f f i c u l t procedure.  Extreme care i n handling the a r t e r i e s and ade-  quate c o n t r o l s are required.  This study was designed to c l e a r l y e s t a b l i s h  any changes i n the hydration of the c o n s t r i c t i n g a r t e r y w a l l .  The study o f  i o n i c and e x t r a c e l l u l a r changes was secondary to t h i s main g o a l .  A.  METHODS A f t e r i n c u b a t i o n , f l u i d trapped i n the a r t e r y lumen i n t e r f e r e s with  the determination of the water content.  The c o n t r i b u t i o n of t h i s f l u i d v a r i e s  w i t h the lumen s i z e , i . e . the degree of c o n s t r i c t i o n .  This v a r i a b l e was  eliminated by p e r f u s i n g i n t r a l u m i n a l l y w i t h an O2/CO2 mixture.  Use of a  p a r t i c u l a r a r t e r y from an inbred s t r a i n of r a t s s t i l l r e s u l t s i n a l a r g e i n d i v i d u a l v a r i a t i o n i n water content.  This problem was overcome by c u t t i n g  the t a i l a r t e r i e s i n t o proximal and d i s t a l halves so the c o n t r o l and t e s t  40 samples were from the same a r t e r y .  The t e s t samples were a l t e r n a t e l y p r o x i -  mal or d i s t a l halves. The  f o l l o w i n g incubation  experiments d i f f e r i n the treatment o f the  t e s t samples and the analyses a f t e r incubation. common to most of the experiments.  The i n i t i a l procedure was  The v e n t r a l t a i l a r t e r y of the anesthe-  t i z e d r a t was exposed and cannulated w i t h P.E. 50 polyethylene tubing a t i t s midpoint.  The d i s t a l h a l f - a r t e r y was then flushed w i t h Krebs s o l u t i o n ,  removed from the t a i l bed and t r a n s f e r r e d w i t h i t s cannula to e i t h e r a t e s t or c o n t r o l t e s t tube. proximal end,  The proximal h a l f - a r t e r y was next cannulated a t i t s  flushed w i t h Krebs s o l u t i o n , removed from i t s bed,  the other t e s t tube.  and placed i n  Thus each r a t i n an experiment contributed  1 control half-artery.  1 t e s t and  Both t e s t tubes contained Krebs s o l u t i o n aerated w i t h  95% O2 and 5% CO2 at 37°C.  A f t e r a h a l f - a r t e r y had been incubated f o r 3  hours, the polyethylene tubing c a r r y i n g the gas mixture i n t o the s o l u t i o n was  connected to the cannula of that a r t e r y .  w i t h O2/CO2 f o r 7 to 10 minutes.  The a r t e r y was then perfused  Intravascular  pressure was monitored w i t h  a Statham transducer connected at a T - j o i n t to the tubing c a r r y i n g the gas mixture to the a r t e r y .  The pressure was only an estimate of the w a l l tension  since an open perfusion  system was used.  a r t e r i e s were removed f o r a n a l y s i s .  A f t e r gaseous perfusion  the c o n t r o l  The t e s t a r t e r i e s , handled s i m i l a r l y ,  were induced to c o n s t r i c t w i t h various vasoactive agents f o r various times, then removed f o r a n a l y s i s . s t r i c t i v e agents were used:  Maximal pressor doses of the f o l l o w i n g vasoconnorepinephrine ( l e v a r t e r e n o l b i t a r t r a t e ,  Winthrop), high K s o l u t i o n (55 meq K / l i t e r , i d e n t i c a l to Krebs s o l u t i o n 2 except 50 mM KCL replaced 50 mM NaCl), and PLV-2 (phenylalanine vasopressin, Sandoz).  - lysine -  41 The  chemical composition of the Krebs s o l u t i o n , i n m e q / l i t e r , was:  Na 150, K 5.0, Ca 4.2, Mg 2.4, CI 124, HCO3 25.0,  H P0 2  4  1.2.  I t a l s o con-  tained 2 g / l i t e r of dextrose and 40 g / l i t e r of p o l y v i n y l p y r r o l i d i n e ( a plasma p r o t e i n s u b s t i t u t e ) .  The osmolarity of the Krebs s o l u t i o n was 295  mosmoles/liter. The water content of the a r t e r i e s was determined from the wet and dry weights (9) and expressed as ml water/100 g f a t f r e e dry weight.  As d e f a t t i n g  the a r t e r i e s decreased t h e i r weight by a n e g l i g i b l e amount, i t was omitted on occasion.  Some of the experiments involved analysing the Na and K contents  of wet ashed a r t e r i e s (9); these r e s u l t s were expressed as meq/100 g f a t free dry weight.  The d i s t r i b u t i o n of i n u l i n i n the a r t e r y , as an estimation of the  e x t r a c e l l u l a r space, was determined i n two ways: s o l u t i o n f o r incubation  (a) chemical; the Krebs  contained 300 mg% p u r i f i e d i n u l i n .  The i n u l i n space  of the a r t e r i e s was measured by the method of Friedman et al_. (9) . (b) isotope; the a r t e r i e s were incubated i n Krebs s o l u t i o n s containing 0.01  14 and 0.06 ucuries/ml of C - Inulin.  determined by l i q u i d s c i n t i l l a t i o n .  between  14 • The C content of the a r t e r i e s was  CI was determined by potentiometric  t i t r a t i o n with s i l v e r n i t r a t e . These experiments involved d i f f e r e n c e s  between the s t a t i c water content  determinations of a r t e r i e s i n non-constricted (control) and c o n s t r i c t e d states.  (The c o n t r o l a r t e r i e s could not be classed as relaxed  was no method of a s c e r t a i n i n g than the t e s t a r t e r i e s . )  (test)  since there  t h i s , but they were d e f i n i t e l y l e s s  constricted  Although no dynamic measurements of the hydration of  a c o n s t r i c t i n g a r t e r y were made, the d i f f e r e n c e s  i n water content are r e f e r r e d  to as "changes'.', f o r e a s i e r understanding of what was happening to the a r t e r y  w a l l as i t c o n s t r i c t e d .  I n the r e s u l t s that f o l l o w , A r e f e r s to the change  i n a parameter with v a s o c o n s t r i c t i o n , i . e . the t e s t value minus the c o n t r o l value.  Using c o n t r o l and t e s t samples from the same a r t e r y meant that the  r e s u l t s could be s i g n i f i c a n t i n two ways:  (a) the number of d i f f e r e n c e s f o r  the p a i r s of h a l f - a r t e r i e s which had the same s i g n ( + or - ) can be s i g n i f i 2 c a n t — c a l c u l a t e d from the X  d i s t r i b u t i o n corrected f o r c o n t i n u i t y ; (b) the  d i f f e r e n c e , A, between the average content of t e s t and c o n t r o l a r t e r i e s can be s i g n i f i c a n t l y d i f f e r e n t from z e r o — c a l c u l a t e d from the Student's t t e s t . This d i f f e r e n c e was included i n the tables when i t was s i g n i f i c a n t at l e v e l s of p < 0.02 or p < 0.05. B.  NOREPINEPHRINE INDUCED VASOCONSTRICTION Norepinephrine was selected as the v a s o c o n s t r i c t i v e agent f o r the  f i r s t s e t of experiments because i t e a s i l y induces f a i r l y  strong, s h o r t - l i v e d  c o n s t r i c t i o n s i n muscular a r t e r i e s , and because i t s a c t i o n on these a r t e r i e s has been w e l l studied (10-14).  Five groups of experiments were done:  (1)  Water Content, (2) Water and Ion Content, (3) Varying Durations of C o n s t r i c t i o n , and (4) I n u l i n Space, and (5) Chloride Content.  The f i r s t two groups  were rather p r e l i m i n a r y while the t h i r d forms the bulk of the r e s u l t s . The d i f f e r e n t experimental conditions i n these experiments meant that the water content of the c o n t r o l a r t e r i e s i n the d i f f e r e n t groups cannot be compared. The d i f f e r e n c e s between the absolute values provide the most i n t e r e s t i n g i n formation. 1.  Water Content A f t e r the a d d i t i o n of norepinephrine (NE) to the incubating s o l u t i o n ,  a pressure increase could be observed when O2/CO2 passed through the a r t e r y segment with i t s branches patent (see F i g . 11). The f i r s t few a r t e r i e s were  43 treated w i t h 2 ug NE/ml, the remainder, 3 ug NE/ml.  The t e s t a r t e r i e s were  removed from the Krebs s o l u t i o n when the peak of c o n s t r i c t i o n was observed on the pressure recording, and b l o t t e d between 2 pieces of f i l t e r paper. The average duration of c o n s t r i c t i o n (+ S.E.) f o r the 6 t e s t a r t e r i e s i n t h i s group was 127 i 38 seconds.  Five of the 6 t e s t h a l f - a r t e r i e s had smaller  t o t a l water contents than t h e i r corresponding c o n t r o l halves (5/6 i s not s t a t i s t i c a l l y s i g n i f i c a n t according to the chi-square t e s t ) .  The average  values f o r the two groups are shown i n Table V I I . TABLE V I I .  Water contents (ml/100 g dry wt) of r a t t a i l a r t e r i e s removed at peak of norepinephrine induced c o n s t r i c t i o n a f t e r 3 hours of aerobic incubation.  control arteries total H 0  (6)  283 + 11  % H0  (6)  73.8  2  2  ( ) number of h a l f - a r t e r i e s 1.  t  7  t e s t (NE) a r t e r i e s  A  tp < 0.05  254 + 1 2  - 29 + l i t  71.6 ± 0.9  -2.1 ± 0.8  + S.E.  This experiment showed i t was p o s s i b l e to observe a d i f f e r e n c e i n water  contents of non-constricted c o n t r o l and c o n s t r i c t e d t e s t a r t e r i e s w i t h t h i s procedure. 2.  C o n s t r i c t i o n induced by norepinephrine was associated w i t h a decrease of  about 10% i n the water content of the a r t e r y w a l l . 2.  Water and Ion Content This study involves a more d e t a i l e d chemical a n a l y s i s of the r a t t a i l  artery.  The experiments d i f f e r only i n the extent of handling and the dura-  t i o n of c o n s t r i c t i o n .  A f t e r removal from s o l u t i o n the a r t e r i e s were cut i n t o  +  44 small pieces a l t e r n a t e l y placed i n two groups i n a weighing b o t t l e .  One of  the groups was analysed f o r the water, Na, and K contents, the other f o r the i n u l i n space by the chemical method.  These i n u l i n space determinations were  u n s a t i s f a c t o r y i n a l l 3 experiments since the amount of t i s s u e a v a i l a b l e , about 4 mg wet weight, was too s m a l l . Test a r t e r i e s removed at peak of NE c o n s t r i c t i o n The f i r s t few t e s t a r t e r i e s were treated w i t h 3 yg NE/ml s o l u t i o n , the remainder, 4 yg/ml.  Upon removal from s o l u t i o n , the a r t e r i e s were b l o t t e d  between two pieces of f i l t e r paper.  Nine r a t s were used i n t h i s experiment.  The average d u r a t i o n of c o n s t r i c t i o n (+ S.E.) was 97 ± 15 seconds.  A l l 9 con-  s t r i c t e d t e s t h a l f - a r t e r i e s had smaller water contents than t h e i r corresponding c o n t r o l halves ( a l l 9 d i f f e r e n c e s negative i s s i g n i f i c a n t at p < 0.004).  Of  the 8 p a i r s of Na and K values, a l l 8 t e s t a r t e r i e s had l e s s Na (p < 0.008) but only 4 of the 8 had l e s s K (not s i g n i f i c a n t by the chi-square t e s t ) .  The aver-  age r e s u l t s of t h i s experiment are given i n Table V I I I . TABLE V I I I .  Water contents (ml/100 g dry w t ) , Na and K contents (meq/100 g dry wt) of r a t t a i l a r t e r i e s removed from s o l u t i o n at the peak of NE c o n s t r i c t i o n a f t e r 3 hours of aerobic incubation.  control arteries  t e s t (NE) a r t e r i e s  t  A *(p < 0.02)  (9)  203 + 6  152  % H0 2  (9)  67.2 + 0.5  59.3 + 1.9  - 7.5 ± 1.9*  t o t a l Na  (8)  41.1 ± 2.2  29.5 + 2.0  -11.9 ± 3.3*  total K  (8)  11.8 + 1.1  11.6 ± 1.3  total  H0 2  10  - 50 ± 9 *  45 1.  Water  V a s o c o n s t r i c t i o n induced  by norepinephrine f o r about 100 seconds  was associated w i t h a 25% decrease i n the a r t e r i a l water content.  The de-  crease was s i g n i f i c a n t both between the i n d i v i d u a l halves and between the averages f o r the halves. 2.  The Na and K contents of the non-constricted c o n t r o l a r t e r i e s were about  5 meq/100 g dry wt higher and lower r e s p e c t i v e l y , than the values found i n the l i t e r a t u r e f o r the r a t t a i l a r t e r y (15,16).  This suggests that there  was a 1:1 exchange of c e l l u l a r K f o r Na during the 3 hours of i n c u b a t i o n , i . e . the smooth muscle c e l l metabolic Na-K pump was operating a t l e s s than i t s "normal" l e v e l of a c t i v i t y . 3.  Sodium  V a s o c o n s t r i c t i o n was associated w i t h a 29% decrease i n the Na  content of the a r t e r y w a l l .  An i n d i c a t i o n of the source of t h i s Na l o s t  during c o n s t r i c t i o n can be obtained by assuming a l l the l o s t water came from the free ECS ( i . e . the e x t r a c e l l u l a r compartment which i s i n chemical e q u i l i b r i u m w i t h the e x t e r n a l medium and i s roughly equivalent to the i n u l i n space).  This assumption would mean that the 50 ml water l e a v i n g the f r e e  ECS would have " c a r r i e d " w i t h i t about 7.5 meq Na/100 g dry wt. A minimum of 11.9 - 7.5 = 4.4 meq Na then, must have come from other compartments. I f not a l l the l o s t water came from the f r e e ECS, then more than 4.4 meq Na must have come from compartments other than the f r e e ECS. This assumption and c a l c u l a t i o n says nothing about the movement of N a i n t o the c o n t r a c t i n g +  smooth muscle c e l l s , but deals only w i t h the movement of Na out of the whole a r t e r y w a l l during v a s o c o n s t r i c t i o n . 4.  Potassium  Changes i n the K content of the a r t e r y w a l l during c o n s t r i c -  t i o n could not be measured w i t h t h i s technique.  46 Test a r t e r i e s removed a f t e r 60 seconds of NE c o n s t r i c t i o n In t h i s and a l l the subsequent experiments there was an attempt to obtain l a r g e r and more "normal" water contents (15,17) by g e n t l e r handling of the a r t e r i e s .  Instead of being b l o t t e d , the a r t e r y was simply placed on  f i l t e r paper while i t s cannula was removed. incubating s o l u t i o n s of the t e s t a r t e r i e s .  Four yg NE/ml was added to the Eight r a t s were used i n each of  these two experiments. Of the 8 t e s t h a l f - a r t e r i e s , 7 had l e s s t o t a l water (not s i g n i f i c a n t ) , a l l 8 had l e s s t o t a l Na (p < 0.008), and 6 had l e s s t o t a l K (not s i g n i f i c a n t ) than t h e i r corresponding c o n t r o l halves.  The average r e s u l t s ( t S.E.) of  t h i s experiment are given i n Table IX. TABLE IX.  Water content (ml/100 g dry wt), Na and K contents (meq/100 g dry wt) of r a t t a i l a r t e r i e s c o n s t r i c t e d w i t h norepinephrine f o r 60_ seconds a f t e r 3 hours of aerobic i n c u b a t i o n . control arteries  t e s t (NE) a r t e r i e s  A  *(p < 0.02)  total H 0  (8)  292 + 9  231 ± 15  - 61 ± 13*  %  H0 2  (8)  74.4 + 0.6  69.3 ± 1.3  - 5.0 + 1.1*  t o t a l Na  (8)  43.7 + 1.8  34.8 + 1.8  - 8.9 + 1.7*  total K  (8)  13.9  t  13.5 + 1.2  2  1.0  A f t e r 60 seconds of c o n s t r i c t i o n , the a r t e r y w a l l l o s t about 21% of i t s water, about 20% of i t s Na, and none of i t s K.  I f i t i s again assumed  that a l l of the l o s t water came from the free ECS, then the movement of 61 ml water should have c a r r i e d with i t 9.1 meq Na.  Since the observed d i f f e r -  ence was -8.9 meq Na, t h i s assumption i n d i c a t e s that a l l of the l o s t Na  47 could have come from the free ECS.  I t should be noted that 61 ml water  l e a v i n g the free ECS would have a l s o c a r r i e d about 0.3 meq K along w i t h i t . Test a r t e r i e s removed a f t e r 120 seconds of NE c o n s t r i c t i o n Of the 8 t e s t h a l f - a r t e r i e s , 6 had l e s s t o t a l water and l e s s t o t a l Na (not s i g n i f i c a n t ) than t h e i r corresponding c o n t r o l halves.  Four of the 7  t e s t K values were l e s s than t h e i r c o n t r o l values (not s i g n i f i c a n t ) . The average contents are given i n Table X. TABLE X.  Water contents (ml/100 g dry w t ) , Na and K contents (meq/100 g dry wt) of r a t t a i l a r t e r i e s c o n s t r i c t e d w i t h norepinephrine f o r 120 seconds a f t e r 3 hours of aerobic incubation. control arteries  t e s t (NE) a r t e r i e s  total H 0  (8)  264 + 14  219 + 18  % H0  (8)  72.2 + 1.0  67.7 + 2.0  t o t a l Na  (8)  44.0 ± 1.6  42.4 ± 1.4  total K  (7)  13.5  t  11.9 + 1.0  2  2  0.8  A  *(p < 0.02) - 45 + 19 *  A f t e r 120 seconds of c o n s t r i c t i o n , the a r t e r y w a l l l o s t about 17% of i t s water.  The decreases i n the Na and K contents were not s i g n i f i c a n t .  Summary 1.  The water losses a f t e r 60, 97, and 120 seconds of v a s o c o n s t r i c t i o n  were 61, 50, and 45 ml water/100 g dry wt r e s p e c t i v e l y .  That i s , the amount  of water l o s t from the c o n s t r i c t i n g a r t e r y w a l l decreased between 1 and 2 minutes.  This suggests that a f t e r 1 minute of c o n s t r i c t i o n , water returned  to the w a l l , i . e . the o r i g i n a l water content was being restored. ing experiment i s an attempt to confirm t h i s suggestion.  The f o l l o w -  48 2.  There was no p a t t e r n to the l o s s of Na from the c o n s t r i c t i n g  artery w a l l .  The movement of Na involved 20 to 30% of the t o t a l w a l l Na.  Most of t h i s l o s s could be due to the movement of the l o s t water from the free ECS. 3.  The s l i g h t decrease i n the K content of the c o n s t r i c t i n g a r t e r y  was not s t a t i s t i c a l l y s i g n i f i c a n t . 3.  Varying Duration of  Vasoconstriction  A more extensive study of the changes i n a r t e r i a l water and i o n i c contents a f t e r d i f f e r e n t durations of c o n s t r i c t i o n was performed.  Since no  i n u l i n spaces were measured, the a r t e r i e s were not cut i n t o small pieces as i n the previous experiments.  Four experiments were performed i n which the  t e s t a r t e r i e s were treated w i t h 4 yg NE/ml s o l u t i o n and allowed to c o n s t r i c t f o r 15, 30, 60, or 120 seconds.  Eight r a t s were used i n each experiment.  The averaged r e s u l t s of these 4 timed experiments are given i n Table X I . 1.  Water  A l l 32 c o n s t r i c t e d t e s t h a l f - a r t e r i e s had l e s s t o t a l water than  t h e i r corresponding non-constricted c o n t r o l halves (p < 0.001). water l o s s was at 30 seconds of c o n s t r i c t i o n .  The greatest  The average % decreases i n  water at 15, 30, 60, and 120 seconds of c o n s t r i c t i o n were 18%, 23%, 17% and 13% r e s p e c t i v e l y . 2.  Sodium  The o v e r a l l average water l o s s was 18%.  Of the 30 p a i r s of Na values, 27 of the t e s t a r t e r i e s had l e s s  Na than t h e i r c o n t r o l s  (p < 0.001).  seconds of c o n s t r i c t i o n . respectively. 3.  Potassium  The greatest Na l o s s was also at 30  The average % Na losses were 13%, 16%, 9%, and 13%  The o v e r a l l average Na l o s s was 13%. The K contents were determined only f o r the 8 p a i r s of a r t e r -  i e s i n the 30 sec. experiment.  For 5 of these 8 p a i r s , the K content was l e s s  TABLE XI.  Water content (ml/100 g dry wt) and Na content (meq/100 g dry wt) of a r t e r i e s a f t e r 15, 30, 60, and 120 seconds of norepinephrine-induced c o n s t r i c t i o n i n v i t r o during perfusion with O2/CO2 a f t e r 3 hours of aerobic incubation.  total H 0 control test  A  2  time (sec)  % H0 test 2  control  A  control  t o t a l Na test  A  15  (8)  359 ± 14  292 ± 11  -66 ± 18  78.0 ± 0.7  74.3 ± 0.8  -3.7 ± 1.1  38.2 ± 0.7  34.1 ± 0.7  -4.8 ± 1.0  30  (8)  349 ± 12  270 ± 11  -80 ±22  77.6 ± 0.6  72.8 ± 0.9  -4.9 ± 1.4  41.7 ± 1.1  35.0 ± 1.4  -6.7 ± 1.9  60  (8)  381 9  317 ± 7  -64 ± 8  79.2 ± 0.3  76.0 ± 0.4  -3.3 ± 0.3  37.6 ± 1.1  34.1 ± 0.9  -3.5 ± 1.7t  377 ± 12  327 ± 11  -50 ± 10  78.9 ± 0.5  76.5 ± 0.6  -2.5 ± 0.5  39.2 ± 0.7  34.1 ± 0.7  -5.1 ± 0.8  367 ± 6  302 6  -65  78.5 ± 0.3  74.9 ± 0.4  -3.6  39.2 ± 0.6  34.3 ± 0.5  -4.9  ±  ± 120  (8)  average (32)  p < 0.05, a l l other A are s i g n i f i c a n t a t p < 0.01.  50 i n the c o n s t r i c t e d t e s t halves than i n the c o n t r o l s (not s i g n i f i c a n t ) .  There  was a l s o no s i g n i f i c a n t d i f f e r e n c e between the average K contents (- S.E.): c o n t r o l a r t e r i e s , 15.8 t. 0.7 meq K/100 g dry wt; t e s t (NE) a r t e r i e s , t 1.0 meq K/100 g dry wt.  13.4  The r e s u l t s of the water and Na analyses are d i s -  cussed i n more d e t a i l i n the f o l l o w i n g s e c t i o n . 4.  Discussion  Changes i n water content 1.  Comparison of Tables IX, X and XI shows that the water l o s s was  r e l a t i v e l y constant at 60 seconds (-64 and -61 ml water/100 g dry wt) and at 120 seconds (-50 and -45) of v a s o c o n s t r i c t i o n , even though the absolute water content of the c o n t r o l a r t e r i e s was q u i t e d i f f e r e n t .  This suggests that the  s i z e of the water l o s s from the w a l l was dependent on the duration of cons t r i c t i o n , not on the o r i g i n a l water content.  I t should be noted that the  s e v e r i t y of c o n s t r i c t i o n could not be estimated since the p e r f u s i o n pressures recorded during c o n s t r i c t i o n were mainly dependent upon the route of O2/CO2 escape from the lumen. 2.  Averaging the values f o r the 2 experiments i n v o l v i n g 60 seconds  and 120 seconds of v a s o c o n s t r i c t i o n y i e l d s water losses (+ S.E.) of 63 ± 7 ml at 60 seconds and 48 + 10 ml at 120 seconds. 3.  The pattern of water l o s s from the c o n s t r i c t i n g a r t e r y w a l l i s  shown i n F i g . 9.  The values f o r 15 and 30 seconds are from Table X I , while  the values f o r 60 and 120 seconds are the above averages. water l o s s are f o r the c o n t r o l a r t e r i e s .  The values at zero  Although there were no s i g n i f i c a n t  d i f f e r e n c e s between the 4 water l o s s e s , a f t e r 30 seconds of c o n s t r i c t i o n , there seems to be a smaller amount of water l o s t .  This suggests that water  l e f t the a r t e r y w a l l during the f i r s t 30 seconds of NE induced v a s o c o n s t r i c t i o n , then slowly returned to the w a l l .  51  -90>  NE(4.0yu/ml)  Fig. 9  Changes i n H 2 O content of r a t t a i l a r t e r y w a l l during norepinephrine induced c o n s t r i c t i o n i n v i t r o w i t h O 2 / C O 2 perfusion. The bars represent 1 S.E. The p o i n t s w i t h zero A H 2 O are the c o n t r o l values. The numbers i n parenthesis are the number of a r t e r i e s used.  52 Changes i n Na content 1.  The t o t a l Na contents of the 60 and 120 second c o n t r o l a r t e r i e s  i n Table XI are smaller than the previous values i n Tables IX and X.  This  f a c t , plus the higher t o t a l K content i n t h i s 30 second experiment, suggest that the a r t e r i e s were more v i a b l e i n these l a t e r experiments. 2.  Although the Na losses i n the two 60 second and the two 120 second  experiments were not s i m i l a r , f o r consistency the values were averaged(i S.E.): -6.2 JT 1.3 meq Na a t 60 seconds and -3.4 t 0.9 meq Na a t 120 seconds of constriction. 3.  These averaged values and the values from Table XI f o r 15 and 30  seconds of c o n s t r i c t i o n are given i n the lower curve of F i g . 10 which shows the pattern of Na l o s s from the c o n s t r i c t i n g a r t e r y w a l l .  There i s no s i g n i -  f i c a n t d i f f e r e n c e between any of the 4 values of ANa, but there i s a suggest i o n that the Na movements are s i m i l a r to the water movements:  the a r t e r y  w a l l loses Na f o r the f i r s t 30 seconds of c o n s t r i c t i o n , then slowly regains it. 4.  The upper curve i n F i g . 10, A(Na)  , i s a c a l c u l a t e d curve.  The  values on t h i s curve represent the Na losses which would have occurred i f the observed water losses from F i g . 9 were a l l from the f r e e ECS. c u l a t i o n s were the same as those made above.  The c a l -  In F i g . 10, the observed Na  l o s s i s smaller than the c a l c u l a t e d Na l o s s . 5.  The 2 curves i n F i g . 10 can best be understood by using the o v e r a l l  average values (n = 48) f o r the H2O and Na l o s s e s . -9.2 meq Na/100 g dry wt.  The average ANa^g =  That i s , the l o s s of 61 ml of water from the f r e e  ECS would have c a r r i e d 9.2 meq Na with i t . l e f t the c o n s t r i c t i n g a r t e r y w a l l .  However, only 4.8 meq Na a c t u a l l y  There are 2 possible explanations of t h i s  53  \  A(Na)ECS  MS)  (167  30 NEWO/i/ml)  Fig.  10  I  45  60  75  90  105  1  120  DURATION OF CONSTRICTION (seconds)  Changes i n Na content of r a t t a i l a r t e r y w a l l during norepinephrine induced c o n s t r i c t i o n In v i t r o with O2/CO2 perfusion. The bars represent 1 S.E. The points w i t h zero ANa are the c o n t r o l values. The number i n parenthesis i s the number of a r t e r i e s used. See the text f o r an explanation of the A(Na)„„ curve. 0  situation:  (a) Half the Na i n the 61 ml of f r e e ECS water which l e f t the  w a l l , moved from the f r e e ECS to other w a l l compartments. Na  +  which entered the contracting smooth muscle c e l l s .  This might include  (b) Some of the water  which l e f t the c o n s t r i c t i n g w a l l came from compartments other than the f r e e ECS.  To carry out 4.8 meq Na, about h a l f the observed water l o s s , 32 ml free  water/100 g dry wt, would have been required.  A combination of these 2 ex-  planations would also e x p l a i n the s i t u a t i o n . This d i s c u s s i o n i s , of n e c e s s i t y ,  54 s i m p l i s t i c , since the compartments of the a r t e r y w a l l are not i s o l a t e d and c e r t a i n l y there would be osmotic adjustments of water between them. 6.  The Na concentration  of the f l u i d l e a v i n g the c o n s t r i c t i n g a r t e r y  w a l l was 70 to 100 meq/liter. Changes i n i n t r a v a s c u l a r pressure 1.  The pattern of water and Na losses from the c o n s t r i c t i n g a r t e r y  w a l l was s i m i l a r to the p r o f i l e of the i n t r a v a s c u l a r pressure during NE i n duced v a s o c o n s t r i c t i o n : 2.  a r a p i d r i s e followed by a slow d e c l i n e .  The pressure recordings, however, showed that the i n t r a v a s c u l a r  pressure had reached a plateau only i n some of the 120 second experiments (see F i g . 11). This suggests that the water and Na had l e f t the a r t e r y w a l l and were returning while the pressure was s t i l l i n c r e a s i n g i n the lumen of the perfused c o n s t r i c t i n g a r t e r y . 3.  There was no c o r r e l a t i o n between the f i n a l pressure or the increase  i n pressure and the t o t a l water content or the decrease i n water content. 5.  I n u l i n Space I n u l i n i s generally regarded as the best marker f o r estimating the  volume of f l u i d i n the e x t r a c e l l u l a r space (ECS) (18-23).  For t h i s study, the  f l u i d assumed to be i n chemical e q u i l i b r i u m with the e x t e r n a l medium i s designated as the free ECS.  I t w i l l be assumed that the free ECS i s approximated  by the volume of d i s t r i b u t i o n of the i n u l i n molecule at the same concentrat i o n as i n the external medium. The above experiments have i n d i c a t e d that water was l o s t from the constricting artery wall.  To substantiate these experiments and to i n d i c a t e  the source of the l o s t water, estimates of the e x t r a c e l l u l a r space were r e quired.  The previous attempts to determine t h i s space by the d i s t r i b u t i o n of  i n u l i n f a i l e d because the a r t e r y samples were to'o small.  Using l a r g e r t i s s u e  55  15 SECONDS  100  X  E E  75  75  50  50  25 0  t  0  -NE—_  .NE.  120 SECONDS  60 SECONDS  <2 100 GO UJ  30 SECONDS  25  -rl  UJ or  or  100  75  t  .NE. NOREPINEPHRINE 4.0 /ig/ml  mm.  F i g . 11  Varying durations of norepinephrine induced c o n s t r i c t i o n of r a t t a i l a r t e r i e s perfused i n v i t r o with 02/C02»  samples, two i n u l i n space experiments were performed.  To permit the determiri-  14 a t i o n of the a r t e r i a l water content, C space marker. determined.  - I n u l i n was used as the e x t r a c e l l u l a r  This method uses dry t i s s u e so that wet and dry weights can be The a r t e r i e s were incubated f o r 30 minutes instead of 3 hours.  In a s e r i e s of experiments w i t h Miss M. Mar, r a t t a i l a r t e r i e s were incubated 14 in C - I n u l i n Krebs s o l u t i o n f o r 20, 40,60, and 120 minutes. There was no d i f f e r e n c e between the i n u l i n spaces or the water contents of these 4 groups of 14 arteries.  Thus, e q u i l i b r a t i o n of the r a t t a i l artery i n the C  s o l u t i o n has apparently occurred w i t h i n 30 minutes. e q u i l i b r a t i o n time f o r other t i s s u e s (22).  - I n u l i n Krebs  This agrees w i t h the  56 Whole a r t e r i e s There were 2 other procedural d i f f e r e n c e s  i n t h i s experiment,  (a) Only  small r a t s (250 g) were a v a i l a b l e , so the whole t a i l a r t e r y was required enough t i s s u e f o r the a n a l y s i s . about 400 g were used.)  for  (In the previous experiments, r a t s weighing  This meant that no comparison of paired t e s t and  c o n t r o l halves was p o s s i b l e and 16 r a t s were used f o r the 8 t e s t and 8 c o n t r o l arteries.  (b) The a r t e r i e s were not perfused w i t h the O2/CO2 mixture i n order  to determine the e f f e c t on the d i f f e r e n c e i n water content between the cons t r i c t e d t e s t and non-constricted c o n t r o l a r t e r i e s . a r t e r i e s were not cannulated.  Consequently, the  As before, the t e s t a r t e r i e s were allowed to  c o n s t r i c t f o r 2 minutes a f t e r the a d d i t i o n of 4 ug NE/ml to the t e s t a r t e r y solutions.  The averaged r e s u l t s of t h i s experiment are given i n Table X I I .  The % i n u l i n space i s the % of the t o t a l H^O, not the % of wet weight, as used by some authors ( f o r example, 22). TABLE X I I .  Water and i n u l i n space (ml/100 g dry wt) of r a t t a i l a r t e r i e s c o n s t r i c t e d f o r 2 minutes w i t h norepinephrine a f t e r 30 minutes of aerobic incubation.  control arteries  t e s t (NE) a r t e r i e s  total H 0  (8)  268+6  252 ± 5  i n u l i n space  (8)  90.4 ± 3.6  76.1 + 3.5  % i n u l i n space  (8)  33.4+1.2  30.8+1.6  2  1.  A  *p < 0.02  -14.3*  There was no s i g n i f i c a n t d i f f e r e n c e between the t e s t and c o n t r o l  water contents.  This suggests that the absence of the O2/CO2 perfusion  did  allow varying amounts of water to be l e f t i n the lumen, obscuring the d i f f e r ence between the 2 groups.  57 2.  The % i n u l i n space of the c o n t r o l a r t e r i e s , 33.4%, agrees w i t h  the values i n the l i t e r a t u r e ( 6 ) . 3.  The i n u l i n space decreased by about 16% during NE induced vaso-  constriction. Half-arteries This experiment followed the above procedure except that the a r t e r i e s were d i v i d e d i n h a l f so paired a n a l y s i s could be done.  In a d d i t i o n , the  a r t e r i e s were perfused w i t h  O 2 / C O 2  so that a s i g n i f i c a n t water content d i f -  ference could be observed.  Seven r a t s weighing about 400 g were used. A l l  7 t e s t h a l f - a r t e r i e s had l e s s water than t h e i r c o n t r o l s (p < 0.02), and 5 t e s t a r t e r i e s had smaller i n u l i n spaces than t h e i r c o n t r o l halves (not significant).  The average r e s u l t s of t h i s experiment are given i n Table  XIII. Water and i n u l i n space (ml/100 g dry wt) of r a t t a i l a r t e r i e s c o n s t r i c t e d f o r 2 minutes w i t h norepinephrine a f t e r 30 minutes of aerobic i n c u b a t i o n .  TABLE X I I I .  control arteries  t e s t (NE) a r t e r i e s  total H 2 O  (7)  298 + 4  261 ± 8  i n u l i n space  (7)  70.8 + 2.2  65.9 + 2.6  % i n u l i n space  (7)  24.9 + 0.9  25.0 ± 1.0  1.  A  *p < 0.02  -37 ± 9*  The f a c t that the water l o s s was s i g n i f i c a n t i n t h i s experiment,  but not i n the previous one, i n d i c a t e s that i n a c c u r a c i e s due to water trapped i n the lumen may o b l i t e r a t e d i f f e r e n c e s .  58 2.  The water l o s s associated with the NE induced c o n s t r i c t i o n  was  about 12% of the water content of the c o n t r o l a r t e r i e s . Discussion 1.  There are i n d i c a t i o n s that the i n u l i n space decreased during  repinephrine induced v a s o c o n s t r i c t i o n . t e s t a r t e r i e s had (p < 0.1,  Of 18 p a i r s of a r t e r i e s , 13 of  smaller i n u l i n spaces than t h e i r corresponding  not s i g n i f i c a n t according to the X  2  test).  the i n u l i n space f o r these 18 p a i r s of a r t e r i e s was wt  (not s i g n i f i c a n t l y d i f f e r e n t from zero).  The -17.3  This 20% ECS  no-  the  controls  average change i n 1 9.5  ml/100 g dry  decrease agrees  w i t h the observations of Turker et_ a l . (3). 2.  The  large standard errors of the i n u l i n space values meant that  decreases i n the i n u l i n space smaller than about 15% could not be observed. 3.  The  % i n u l i n space was  constant during c o n s t r i c t i o n :  p a i r s of values, 9 test a r t e r i e s had  smaller % i n u l i n spaces than t h e i r  This means that the % decrease i n the non-(free ECS) 4.  of the  In a d d i t i o n to the usual problem of how  was  controls.  also constant.  i n u l i n i s distributed in  the e x t r a c e l l u l a r space, there are other problems using i n u l i n as the marker during v a s o c o n s t r i c t i o n .  18  Conformational changes i n the  ECS  extracellular  s o l i d s or permeability changes i n the smooth muscle c e l l membranes (24) assoc i a t e d w i t h c o n s t r i c t i o n may  r e s u l t i n an a l t e r e d d i s t r i b u t i o n of i n u l i n .  f l u i d changes associated with the onset of c o n s t r i c t i o n may for the i n u l i n molecule to assume a new  equilibrium  occur too  distribution.  The  quickly  These pos-  s i b i l i t i e s imply that the decreases i n i n u l i n space during c o n s t r i c t i o n  are  only q u a l i t a t i v e measurements. 6.  Chloride Content Since the c o n s t r i c t i n g a r t e r y underwent a decrease i n ECS  and a l o s s  of Na unaccompanied by K, i t seemed that measurement of the predominately  59 e x t r a c e l l u l a r w a l l CI content would give a d d i t i o n a l information t r i b u t i o n of the H 2 O l o s s associated  with  on the d i s -  vasoconstriction.  Rat t a i l a r t e r i e s were incubated f o r 90 minutes i n Krebs s o l u t i o n aerated with  These a r t e r i e s were not divided i n h a l f , cannulated  O 2 / C O 2 .  or perfused w i t h were not made.  Consequently, i n t r a v a s c u l a r pressure measurements  O 2 / C O 2 .  Two  groups of 6 a r t e r i e s were used:  (a)  non-constricted  c o n t r o l a r t e r i e s which were removed f o r a n a l y s i s a f t e r pre-incubation,  and  (b) c o n s t r i c t e d t e s t a r t e r i e s which had 2 ug/ml of norepinephrine added to the media and were allowed to c o n s t r i c t f o r 15 minutes.  The averaged r e s u l t s  are given i n Table XIV. TABLE XIV.  Water (ml/100 g dry wt) and i o n content (meq/100 g dry wt) of r a t t a i l a r t e r i e s c o n s t r i c t e d with norepinephrine f o r 15 minutes a f t e r 90 minutes of aerobic incubation.  non-constricted total H 0  342  t o t a l Na  41.0  total K t o t a l CI  2  n = 6 for 1.  6  -59*  ± 0 .4  32.8  -8.2*  23.3  ± 0 .5  22.3 ±  0.5  34.9  ± 0 .5  29.1 +  0.6  ±  The  ±0.5  *p <  0.02  -5.8*  S.E.  c o n s t r i c t e d a r t e r i e s had 17% l e s s  i n K, and 17% l e s s CI than the non-constricted 2.  A  283 ± 4  each value The  t  c o n s t r i c t e d (NE)  H 0, 20% l e s s Na, no change 2  arteries.  r a t i o of Na l o s s to CI l o s s , 1.41,  i s approximately equal to  the r a t i o of Na and CI concentrations i n the e x t e r n a l media, 150/124 =1.21. 3. meq  The Na c a r r i e d out with 59 ml of ECS  f l u i d would have been  Na/100 g dry wt, only s l i g h t l y l a r g e r than the observed Na l o s s .  8.9  60 4.  The above 2 points suggest that the water l o s s associated w i t h  c o n s t r i c t i o n i s a movement of water and e l e c t r o l y t e s , mainly NaCl, from the e x t r a c e l l u l a r f l u i d of the c o n s t r i c t i n g a r t e r y w a l l . 7.  Summary of Results of Norepinephrine Induced V a s o c o n s t r i c t i o n 1.  The  and Na losses from the r a t t a i l a r t e r y c o n s t r i c t e d i n  v i t r o w i t h norepinephrine are shown i n Table XV.  The % H2O  l o s s agrees  w i t h that found f o r the contracted uterus (1) and c o n s t r i c t e d aorta ( 2 ) . Most of the observed changes i n Na content associated w i t h c o n s t r i c t i o n have not been s i g n i f i c a n t (2, 10, 25), although Daniel found a s i g n i f i c a n t l o s s of Na and water from the contracted uterus (26). TABLE XV.  O v e r a l l average H 2 O (ml/100 g dry wt) and Na (meq/100 g dry wt) contents of r a t t a i l a r t e r i e s c o n s t r i c t e d i n v i t r o w i t h norepinephrine. The r a t i o of c o n s t r i c t e d t e s t a r t e r i e s w i t h smaller H 2 O or Na than t h e i r corresponding c o n t r o l halves.  control  t e s t (NE)  A  % change  ratio  signif.  H0  (78)  307  256  -51  -16%  66/70  p < 0.001  Na  (55)  40.9  34.9  -6.0  -15%  49/54  p < 0.001  2  2.  In Table V I I I , the observed Na l o s s was greater than the ANa  c a l c u l a t e d from the water l o s s . while i n F i g .  In Tables IX and XIV, they were the same,  10, the observed Na l o s s was l e s s than the ANa  . The i n u l i n  space data suggested that only part of the water l o s s was from the ECS, while the  CI data suggested a l l the water l o s s could have been from the ECS.  It  thus seems p o s s i b l e that most of the H 0 l o s t from an a r t e r y w a l l c o n s t r i c t e d 2  with norepinephrine, came from the e x t r a c e l l u l a r space as a predominately NaCl s o l u t i o n .  The remainder of the Na present i n t h i s volume of e x t r a -  c e l l u l a r f l u i d may have entered the c o n t r a c t i n g smooth muscle c e l l s .  61 3.  There may have been a s l i g h t decrease i n the a r t e r i a l K content  during NE c o n s t r i c t i o n .  Less K than t h e i r c o n t r o l s was observed f o r 19/31  t e s t a r t e r i e s (not s i g n i f i c a n t ) .  The o v e r a l l average K values (± S.E.) f o r  the c o n t r o l (n = 33) and t e s t a r t e r i e s (n = 31) were 13.7 Z 0.5 and 12.6 i 0.6 meq K/100 g dry wt, r e s p e c t i v e l y ( n o n - s i g n i f i c a n t d i f f e r e n c e ) .  These  n o n - s i g n i f i c a n t changes i n the K content of the c o n s t r i c t i n g a r t e r y are i n agreement with some f i n d i n g s (2,27) but i n c o n f l i c t with others i n which large K decreases during c o n s t r i c t i o n were observed (10,25). 4.  The p a t t e r n of water, Na and i n t r a v a s c u l a r pressure f o r the NE  c o n s t r i c t e d a r t e r i e s shows that the H2O and Na losses had occurred and e q u i l i b r i u m was being restored before the i n c r e a s i n g i n t r a v a s c u l a r pressure had reached a plateau.  This suggests that the i o n and water changes were  associated with the onset of v a s o c o n s t r i c t i o n . C.  HIGH K INDUCED VASOCONSTRICTION Norepinephrine induced c o n s t r i c t i o n i s associated  from the a r t e r y w a l l .  The question now a r i s e s :  with a l o s s of water  I s t h i s water l o s s  associated  only with the a c t i o n of norepinephrine, o r i s i t common to a l l v a s o c o n s t r i c t i v e processes?  A commonly used pressor agent i s a s o l u t i o n with a high potassium  concentration,  c a l l e d a high K s o l u t i o n , made by r e p l a c i n g some of the NaCl  i n a p h y s i o l o g i c a l s o l u t i o n with e i t h e r KCL or ^SO^. s o l u t i o n s on vascular  The a c t i o n of high K  t i s s u e has been w e l l studied and appears to have a  d i f f e r e n t mode of a c t i o n than norepinephrine (14,27-34).  This experiment was  performed to determine i f high K induced c o n s t r i c t i o n s were also  associated  with a l o s s of water from the w a l l of the r a t t a i l a r t e r y . As i n the NE experiments, the a r t e r i e s were divided i n t o t e s t and c o n t r o l halves and e q u i l i b r a t e d i n Krebs s o l u t i o n .  The t e s t a r t e r i e s were  62 perfused w i t h C^/CC^ f o r 5 to 10 minutes while the pressure was monitored, t r a n s f e r r e d w h i l e s t i l l recording to high K s o l u t i o n f o r 2 minutes, then removed f o r a n a l y s i s . and removed.  The c o n t r o l a r t e r i e s were then s i m i l a r l y perfused  The high K s o l u t i o n was the same as the Krebs s o l u t i o n except  that 50 meq N a C l / l i t e r were replaced by 50 meq K C l / l i t e r , so the concent r a t i o n s were 100 meq N a / l i t e r and 55 meq K / l i t e r .  The osmolarity was 328  mosm/liter f o r the high K s o l u t i o n and 332 f o r the Krebs s o l u t i o n .  The con-  14 c e n t r a t i o n of C  - I n u l i n f o r the e x t r a c e l l u l a r space determination was the  same i n both s o l u t i o n s .  The a r t e r i e s were analysed f o r water content and  i n u l i n space.  Consequently, not enough t i s s u e was a v a i l a b l e f o r Na and K  measurements.  Three experiments were performed:  were p r e - e q u i l i b r a t e d  (1) A r t e r i e s from 8 r a t s  f o r 30 minutes i n normal Krebs s o l u t i o n .  Four of the  8 t e s t a r t e r i e s d i d not c o n s t r i c t i n the high K s o l u t i o n — a s observed from the pressure r e c o r d i n g s — a n d were grouped w i t h the c o n t r o l a r t e r i e s as nonconstricted arteries.  (2) The same procedure as i n (1) was used except the  14 C  - I n u l i n concentration was doubled.  (3) A r t e r i e s from 12 r a t s were  e q u i l i b r a t e d f o r 3 hours i n normal Krebs s o l u t i o n instead of 30 minutes as i n the above two experiments. The water content r e s u l t s of these 3 experiments are averaged i n Table XVI.  The only s a t i s f a c t o r y i n u l i n space values (± S.E.) were from  experiment ( 1 ) , i n ml/100 g dry wt: non-constricted a r t e r i e s (12), 77.1 ± 3.6, i . e . 26.5 t 1.1% and c o n s t r i c t e d a r t e r i e s ( 4 ) , 67.7 ± 5.8, i . e . 27.4 - 3.7%.  The d i f f e r e n c e s between the two groups were not s i g n i f i c a n t .  The  standard e r r o r s were so l a r g e , the i n u l i n space would have to decrease by more than 25% to be observed.  The % i n u l i n space was e s s e n t i a l l y constant.  Table XVI shows that the water losses associated w i t h high K induced v a s o c o n s t r i c t i o n were about the same s i z e (-14%) as those associated with  63 norepinephrine induced c o n s t r i c t i o n (-16%). TABLE XVI.  Water content (ml/100 g dry wt) of r a t t a i l a r t e r i e s c o n s t r i c t e d f o r 2 minutes i n high K s o l u t i o n and the r a t i o of c o n s t r i c t e d t e s t a r t e r i e s w i t h smaller water content than t h e i r corresponding non-constricted c o n t r o l halves a f t e r aerobic incubation.  Experiment  non-constricted  constricted  A  % change  1.  (12)  291 + 6  (4)  254 + 13  -37*  2.  (8)  323 + 8  (8)  247 + 12  -76  3.  (12)  308 + 9  (12)  279 + 9  -29  average  (32)  306  (24)  264  -42  ( ) number of h a l f - a r t e r i e s  D.  I S.E.  ratio  -13%  4/4  ± 10*  -23%  8/8  ± 12t  - 97,  9/12  -14%  21/24  *p<0.02  t  signif. none p < 0. 008 none p < 0. 001  p<0.05  SYNTHETIC VASOPRESSIN, PLV-2, INDUCED VASOCONSTRICTION As a f u r t h e r t e s t of the r e l a t i o n s h i p between v a s o c o n s t r i c t i o n  water l o s s from the a r t e r y w a l l , a synthetic vasopressin, PLV-2, was c o n s t r i c t the r a t t a i l a r t e r i e s .  and used to  Some work has been done on the e f f e c t s of  PLV-2 on the i o n i c exchanges i n the r a t aorta  (35).  The h a l f - a r t e r i e s were incubated f o r 3 hours i n Krebs s o l u t i o n  (332  14 mosm/liter) containing  C  - Inulin.  During the O2/CO2 perfusion  t e s t a r t e r i e s at the end of t h i s incubation pressure was  period, while the  of  the  intravascular  recorded, 40 m i l l i p r e s s o r - u n i t s PLV-2/ml were added to the media  of the t e s t a r t e r i e s .  They were allowed to c o n s t r i c t f o r 2 minutes before  removal f o r a n a l y s i s of t h e i r water and i n u l i n contents.  The  i e s were treated s i m i l a r l y but. were not induced to c o n s t r i c t . were used i n t h i s experiment.  control arterTwelve r a t s  The averaged r e s u l t s are given i n Table XVII.  64 TABLE XVII.  Water content and i n u l i n space (ml/100 g dry wt) of r a t t a i l a r t e r i e s c o n s t r i c t e d f o r 2 minutes w i t h PLV-2 and r a t i o of c o n s t r i c t e d t e s t a r t e r i e s w i t h smaller amounts than t h e i r corresponding c o n t r o l halves a f t e r 3 hours of aerobic incubation.  control  t e s t (NE)  A -38 ± 14*  total H 0  (12)  293 t 9  255 + 11  i n u l i n space  (12)  105 ± 4  102 ± 7  2  % i n u l i n space(12)  35.9  ± 1.0  ( ) number of h a l f - a r t e r i e s  1. was  39.7  ± 1.7  % change -13%  ratio  signif.  10/12  p <  8/12 +3.8  ts.E.  ± 1.1*  +11%  0.04  none  1/12  p < 0.006  *p<0.02  The water loss associated w i t h PLV-2 induced  vasoconstriction  i n the same range as the water l o s s associated w i t h high K and  norepine-  phrine induced c o n s t r i c t i o n s . 2. tion.  There was no change i n the i n u l i n space during PLV-2 c o n s t r i c -  In a d d i t i o n , the % i n u l i n space increased by more than 10%.  Such  an increase would be expected i f the water l o s t from the c o n s t r i c t i n g a r t e r y w a l l o r i g i n a t e d i n compartments other than the free ECS,  so that the amount  of water i n the i n u l i n space r e l a t i v e to the t o t a l water would be increased. This suggests that the e f f e c t of PLV-2 induced c o n s t r i c t i o n on the w a l l i s q u i t e d i f f e r e n t than the e f f e c t of high K and  E.  artery  norepinephrine.  DISCUSSION The  r a t t a i l a r t e r y w a l l l o s t water during c o n s t r i c t i o n induced by  norepinephrine, high K, or PLV-2. the c o n s t r i c t e d test a r t e r i e s had  Of the 106 p a i r s of h a l f - a r t e r i e s , 97 of l e s s water than t h e i r corresponding  c o n s t r i c t e d c o n t r o l halves (p < 0.001).  The  o v e r a l l average H 0 2  non-  contents  65 were (n = 118): i e s , 258.  c o n t r o l a r t e r i e s , 306 ml  H2O/IOO  g dry wt, and t e s t a r t e r -  The average H 2 O l o s s associated w i t h v a s o c o n s t r i c t i o n was  48 ml, or 16% of the c o n t r o l water content.  thus,  There have been s e v e r a l studies  which i n d i c a t e d there was a l o s s of water from the c o n s t r i c t i n g a r t e r y w a l l . In 1956, Tobian and Fox (10) found a s l i g h t , n o n - s i g n i f i c a n t decrease i n the water content of femoral a r t e r i e s c o n s t r i c t e d i n s i t u with  norepinephrine.  Daniel's e a r l y studies showed no s i g n i f i c a n t change i n hydration of the c o n s t r i c t e d r a t aorta (25, 36).  However, he observed water l o s s e s , or  decreases i n water gain, i n u t e r i n e s t r i p s contracted with vasoactive agents or metabolic i n h i b i t o r s (1,26,37,38). t o t a l water.  These s t r i p s l o s t 10 to 17% of t h e i r  D a n i e l suggested that " c o n t r a c t i o n causes l o s s of s u b s t a n t i a l  q u a n t i t i o n of i n t e r s t i t i a l f l u i d i n smooth muscle allowed to contract i s o t o n i c a l l y " (1).  Headings and Rondell (27) found dog c a r o t i d a r t e r i e s given  epinephrine l o s t 2.3% H 2 O . The data of Friedman et a l . (35) show that perf u s i o n of a s y n t h e t i c vasopressin, PLV-2, was associated with a s l i g h t nons i g n i f i c a n t decrease i n the water content of the r a t a o r t a .  On the other  hand, Henry et^ a l . (39) found a 25% increase i n the i n u l i n space of the r a b b i t aorta i n v i t r o a f t e r the a p p l i c a t i o n of adrenaline.  Similar i n u l i n  space increases were observed by h i s associates f o r the r a t diaphragm (40) and t r o u t d o r s a l muscle (41) given adrenaline.  The Somlyos (24) suggested  that since Henry's work c o n f l i c t e d with that of Daniel and that of Rorive (discussed below), "epinephrine may i n u l i n i n the r a b b i t aorta".  increase i n t r a c e l l u l a r penetration of  Although t h i s suggestion may  be true, i t i s  p o s s i b l e that the data of Henry et_ a l . do not apply to the c o n s t r i c t e d aorta.  They were i n t e r e s t e d i n the e f f e c t of adrenaline on the  turnover  r a t e of free n u c l e o t i d e s , not c o n s t r i c t i o n , and used extremely small doses  66 of adrenaline:  0.006 ug/ml s o l u t i o n and 0.04  ug/ml.  Supporting t h i s p o s s i -  b i l i t y i s the observation by Turker e_t a l . of a 10 to 30% decrease i n i n u l i n space of c a r o t i d a r t e r y and uterus s t r i p s c o n s t r i c t e d with angiotensin (3). They suggested that since the s t r i p s contracted, the decrease i n e x t r a c e l l u l a r space could be due to a "squeezing provides a c l e a r demonstration tion.  out e f f e c t " .  The work of Rorive et a l . (2)  of a water l o s s associated w i t h v a s o c o n s t r i c -  They found an increase i n the percentage dry weight of the r a t a o r t a  given norepinephrine  (5 ug/ml bath) or angiotensin.  c o n s t r i c t e d aorta l o s t 10 to 16% of i t s water.  Their data show the  They suggested that the l o s s  of water may have been due to " r e p a r t i t i o n des ions entre l e s d i f f e r e n t compartments t i s s u l a i r e s " .  Recently, Rorive (42) observed a s i g n i f i c a n t per-  cent dry weight increase and an i n u l i n space decrease f o r the r a t aorta c o n s t r i c t e d i n a high K s o l u t i o n (20 meq K / l i t e r ) . about 3/4 of the H 0 2  Why  His values show that  l o s s could be explained by the i n u l i n space decrease.  c o n s t r i c t i o n i s associated w i t h a l o s s of a r t e r i a l water w i l l be discussed  i n Chapter VI.  I t i s of some i n t e r e s t to consider the changes, caused by  c o n s t r i c t i o n , i n the p h y s i c a l parameters of the e n t i r e r a t t a i l a r t e r y w a l l . These changes can be estimated from the average water content and average water l o s s . (a) wet weight of c o n t r o l h a l f - a r t e r y (n = 149):  13.8 1 0.3  (b) wet weight of whole non-constricted a r t e r y :  2 x 13.8 =  27.6  (c) % water i n c o n t r o l a r t e r y (n = 118):  100(306/406)  75.5%  (d) water content of non-constricted a r t e r y :  0.755 x  20.8  27.6  mg  mg  (e) a l o s s of 16% of the water i n the a r t e r y w a l l meant a change of:  -0.16  x  20.8  -3.3 mg water  mg  67 (f) % change i n weight of whole a r t e r y : .  100(-3.3/27.6) =  (g) wet weight of c o n s t r i c t e d a r t e r y :  27.6-3.3  =  -12% 24.3 mg  (h) i f density of l o s t f l u i d was 1.00 g/cc,  then volume change of  c o n s t r i c t i n g a r t e r y was:  -3.3 u l  ( i ) density of a r t e r y (43,44):  1.06 g/cc  (j) volume of c o n t r o l . a r t e r y , V :  27.6/1.06 =  26.0  (k) volume of c o n s t r i c t e d a r t e r y :  26.0 - 3.3 =  22.7 y l  100(-3.3/26.0) =  -13%  w  u  l  (1) % change i n volume of a r t e r y w a l l during c o n s t r i c t i o n : (m) % volume change from h i s t o l o g i c a l sections i n Chapter I I :  -14%  (n) density of c o n s t r i c t e d a r t e r y :  24.3/22.7 =  1.07 g/cc  (o) c o n s t r i c t i o n of t a i l a r t e r y by NE, high K, or PLV-2 i s associated w i t h a change i n the density of the w a l l o f :  +0.01 g/cc  There was a small n o n - s i g n i f i c a n t l o s s of K from the norepinephrine constricted r a t t a i l artery.  Tobian and Fox found a large K l o s s from the  dog femoral a r t e r y c o n s t r i c t e d with NE (10), as d i d Daniel et_ al_. f o r the rat  aorta (25). On the other hand, Rorive et a l . found no change i n K f o r  the r a t aorta c o n s t r i c t e d with NE (2), and Headings and Rondell observed a s l i g h t gain i n K f o r the dog c a r o t i d a r t e r y c o n s t r i c t e d with epinephrine (27).  I t should be noted that the proportion of smooth muscle c e l l s i n the  aorta i s smaller than i n muscular a r t e r i e s or the uterus.  Consequently,  changes i n K, assumed to be predominately i n t r a c e l l u l a r , would be much more  68 d i f f i c u l t to observe i n the aorta.  However, there have been i n d i c a t i o n s  that epinephrine  may cause c o n s t r i c t i o n without membrane  and norepinephrine  d e p o l a r i z a t i o n (27,30,45), or even w i t h membrane h y p e r p o l a r i z a t i o n (46). This means that the l o s s of c e l l u l a r K u s u a l l y associated with d e p o l a r i z a t i o n and c o n t r a c t i o n i n s t r i a t e d muscle, need not occur i n NE induced vasoconstriction.  Shibata and Briggs (46) explained the h y p e r p o l a r i z a t i o n and the  large increases i n K e f f l u x and i n f l u x which t h e i r group had observed i n the r a b b i t aorta given epinephrine  (28), as due to an increase i n membrane per-  m e a b i l i t y to K caused by epinephrine, while " c o n t r a c t i o n r e s u l t s from an a c t i o n of epinephrine which involves Ca". I n short, i f norepinephrine  causes  c o n s t r i c t i o n without d e p o l a r i z i n g the vascular smooth muscle c e l l membrane, i t i s not s u r p r i s i n g that no change i n K was observed i n t h i s study. The norepinephrine  induced c o n s t r i c t i o n of the r a t t a i l a r t e r y was  associated with a l o s s of 15% of the a r t e r i a l Na.  I n the l i t e r a t u r e ,  there  i s no c l e a r p i c t u r e of changes i n Na content of the c o n s t r i c t i n g a r t e r y w a l l . N o n - s i g n i f i c a n t changes i n Na content were observed i n the r a t aorta (2,25) ' and dog femoral a r t e r y (10) c o n s t r i c t e d w i t h norepinephrine.  The s l i g h t  increases i n Na found (10,25) d i d not c o r r e l a t e w i t h the l o s s of K observed simultaneously.  On the other hand, Daniel found a l o s s of Na from the con-  t r a c t e d uterus (1,26,37) and commented that "contractions i n smooth muscle were accompanied by a decrease i n Na, CI and water i n some instances obscurr i n g c e l l u l a r uptake of Na" (1).  Without entering the 'Ca vs Na as the  current c a r r y i n g i o n ' debate, i t seems that even i f Na d i d enter  vascular  smooth muscle c e l l s during NE c o n s t r i c t i o n , the quantity of Na involved would be q u i t e small (36). rat  I n t h i s study, the Na l o s t from the c o n s t r i c t i n g  t a i l a r t e r y apparently accompanied the l o s s of e x t r a c e l l u l a r water as  suggested by Daniel (1).  69 F.  ERROR IN CALCULATING INNER RADIUS FROM ISOVOLUMETRIC ASSUMPTION Frequently i n the l i t e r a t u r e , the inner and outer r a d i i of an a r t e r y  w a l l are measured i n the relaxed s t a t e and the c r o s s - s e c t i o n a l area of wall i s calculated.  the  The a r t e r y i s then c o n s t r i c t e d and i t s outer radius  measured, but not the inner radius since i t i s not e a s i l y d i s c e r n i b l e i n the constricted state.  The inner radius f o r t h i s c o n s t r i c t e d s t a t e i s then c a l -  culated from the outer radius and the c r o s s - s e c t i o n a l area, using assumption that c o n s t r i c t i o n i s isovolumetric  the  ( f o r example, see 47,48).  the l i g h t of the i n d i c a t i o n of a l o s s of about 13% of the volume of  In  the  a r t e r y w a l l during c o n s t r i c t i o n , i t i s of i n t e r e s t to see what e r r o r i s introduced  i n t o the c a l c u l a t i o n of the inner radius by t h i s  assumption, AV  W  isovolumetric  = 0.  I f ( )" r e f e r s to v a r i a b l e s i n the c o n s t r i c t e d s t a t e and to v a r i a b l e s i n the V so  w  >  = V  *L(r w  ~ w V  rearranging:  ( ) refers  relaxed s t a t e , then: "  2 G  "  and  r ) 2  ±  AV  -  w  r^  =  The s i z e of the error introduced  V  ^(r^ \x' Jo  2  2  - r^  - r  2  =  W  TrL(rg -  - r  2  + r.  O  2  r^ )  2  2 Q  +  2  r ) 2  ±  - AV /TT L  .......... (1)  W  1  w  by assuming that the l a s t term i n equation  (1) i s zero can be observed i n F i g . 12.  The  from equation (1) by a r b i t r a r i l y considering which the outer radius decreased by 50%,  two curves were c a l c u l a t e d the case of c o n s t r i c t i o n s i n  i.e.'  =  assumed, f o r the sake of these c a l c u l a t i o n s , that: of the outer radius from 200 to 1000  u.  h o' r  It  r ^ = 7/8  w a s  r  Q  also f o r values  The lower curve i s f o r i s o v o l u -  metric c o n s t r i c t i o n , the upper curve f o r a 13% decrease i n the w a l l v o l u m e — which means that f o r a constant length of the a r t e r i a l segment, there was  a  70  250|-  0  200  400  600  800  RELAXED OUTER RADIUS r (AL)  1000  c  F i g . 12  13%  Inner r a d i u s o f a c o n s t r i c t e d a r t e r y c a l c u l a t e d from the c o n s t r i c t e d o u t e r r a d i u s and the r e l a x e d i n n e r and o u t e r r a d i i , w i t h and w i t h o u t a 13% d e c r e a s e i n the volume o f t h e c o n s t r i c t e d a r t e r y w a l l .  d e c r e a s e i n the c r o s s - s e c t i o n a l a r e a o f the w a l l .  assumption always r e s u l t s i n a c a l c u l a t e d artery Fig.  that  12.  i s too s m a l l :  T h i s means t h a t  than i n d i c a t e d by t h i s  G.  EXAMINATION  The contrary  inner  radius  The  isovolumetric  o f the c o n s t r i c t e d  the e r r o r i s about 42% f o r the example shown i n the a r t e r y r e a l l y c o n s t r i c t e d considerably  less  assumption.  OF POSSIBLE EXPERIMENTAL ARTIFACTS  f i n d i n g of decreased a r t e r i a l hydration  t o the g e n e r a l l y  during  a c c e p t e d view o f i s o v o l u m e t r i c  t h i s f i n d i n g to be a c c e p t a b l e , the p o s s i b i l i t y  that  constriction i s constriction.  For  the observed l o s s o f  71 water was an experimental a r t i f a c t has to be dismissed.  I n t h i s regard, there  are 3 objections which can be r a i s e d to claim the observed water l o s s was due to:  (1) changes i n the a r t e r y a f t e r 3 hours i n v i t r o , (2) water l e f t i n the  lumen, and (3) the e f f e c t of the  O 2 / C O 2  perfusion on the a r t e r i a l water con-  tent. 1.  E f f e c t of Relaxation  of Constricted A r t e r i e s on Water Content  There i s some i n d i c a t i o n that incubation of a r t e r i e s causes them to gain water slowly, i . e . e i t h e r they imbibe water or are recovering trauma of e x c i s i o n .  from the  I f i t i s assumed that v a s o c o n s t r i c t i o n causes only an  extra load of water, acquired during e q u i l i b r a t i o n , to be expelled from the w a l l , then subsequent r e l a x a t i o n w i l l leave them, a t l e a s t f o r a time, with l e s s water than t h e i r c o n t r o l halves.  On the other hand, i f the water l o s s  i s d i r e c t l y a f u n c t i o n of v a s o c o n s t r i c t i o n ,  then a r t e r i e s c o n s t r i c t e d then  relaxed should have the same amount of water as the untreated c o n t r o l s .  To  resolve t h i s question, a r t e r i e s were c o n s t r i c t e d and then allowed to r e l a x . Rat t a i l a r t e r i e s were divided i n h a l f and incubated f o r 3 hours. t e s t h a l f - a r t e r i e s were perfused with  0 2 / C 0 2 >  treated with 4 ug/ml of  norepinephrine, and allowed to c o n s t r i c t f o r 2 minutes.  These c o n s t r i c t e d  t e s t a r t e r i e s were then t r a n s f e r r e d to f r e s h Krebs s o l u t i o n and allowed to r e l a x f o r 25 minutes while the i n t r a v a s c u l a r pressure was monitored. c o n t r o l s were perfused with water measurement.  O 2 / C O 2  The  and removed with the t e s t a r t e r i e s f o r  Fourteen a r t e r i e s were used i n t h i s experiment.  For most c o n s t r i c t e d t e s t a r t e r i e s , the i n t r a v a s c u l a r pressure r e turned to normal a f t e r about 10 minutes i n the f r e s h Krebs s o l u t i o n (see Fig.  13), so c e r t a i n l y the test a r t e r i e s were relaxed a f t e r 25 minutes.  Seven of the 14 t e s t h a l f - a r t e r i e s had l e s s water than t h e i r corresponding  The  72  x!50  T R A N S F E R TO FRESH KREBS SOLUTION  NE4.0/i/ml  Fig.  13  ies,  REMOVAL FOR ANALYSIS  i  TIME  I n t r a v a s c u l a r pressure of a r a t t a i l a r t e r y c o n s t r i c t e d i n v i t r o w i t h norepinephrine, then allowed to r e l a x , w h i l e being perfused w i t h 02/C02»  c o n t r o l halves. difference:  lmin  The averaged values (± S.E.) showed a s i m i l a r n o n - s i g n i f i c a n t  c o n t r o l a r t e r i e s , 314 - 6 ml water/100 g dry wt, and t e s t a r t e r -  301 i 7, i . e . both had the same amount of water.  This means that the  l o s s of water during c o n s t r i c t i o n was not an a r t i f a c t due to simple removal of some acquired e x t r a water.  I t should be noted that the t e s t a r t e r i e s  could not have gained back any loaded water i n the 15 minutes of i n c u b a t i o n a f t e r they were r e l a x e d , since 15 minutes i s too short a time f o r t h i s to happen.  The experiment mentioned above showed there was no d i f f e r e n c e i n  water content of r a t t a i l a r t e r i e s incubated f o r 20 or 120 minutes.  In  a d d i t i o n , there was no c o r r e l a t i o n between the average i n c u b a t i o n times and the water contents of the c o n t r o l a r t e r i e s from Table X I .  In conclusion,  73  the  l o s s o f water from the a r t e r y w a l l d u r i n g c o n s t r i c t i o n and i t s subsequent  return  2.  d u r i n g r e l a x a t i o n were independent o f any i n c u b a t i o n  Water Remaining i n the Lumen a f t e r Gaseous  artifacts.  Perfusion  Could water l e f t i n the lumen a f t e r O2/CO2 p e r f u s i o n loss during vasoconstriction?  This  e i t h e r a c o n s t a n t volume o f f l u i d  r e m a i n i n g water c o u l d  or a l a y e r o f f l u i d  explain  the water  be c o n s i d e r e d as  of constant  thickness.  Here, c o n s t a n t r e f e r s to comparison o f the n o n - c o n s t r i c t e d c o n t r o l and the constricted  test arteries.  A c o n s t a n t volume  of f l u i d  l e f t i n the lumen  would not a f f e c t the c o n t r o l and t e s t a r t e r i e s d i f f e r e n t l y , so i t c o u l d be the cause o f the o b s e r v e d water l o s s . stant  thickness containing  less fluid  A boundary l a y e r of f l u i d  i n the c o n s t r i c t e d  not  of con-  s t a t e , due to the  s m a l l e r lumen, might g i v e the i m p r e s s i o n o f l e s s water i n the c o n s t r i c t e d arteries.  However, t h i s p o s s i b i l i t y does not seem too  Volume o f boundary l a y e r where:  L = length  r ^ = lumen Assuming then,  AV  fluid  of artery  =  V^ =  segment;  likely:  2ITL y r ^  y = boundary l a y e r  thickness;  radius.  Ay = 0 = AL, where A i s the change caused by c o n s t r i c t i o n , b  =  2TTL y  Ar^  Since,  q u e s t i o n i s t h e n reduced t o :  A^  < 0, then  AV  < 0.  b  The  Can a d e c r e a s e i n the boundary  layer  volume a c c o u n t f o r the observed l o s s o f water by the c o n s t r i c t i n g artery?  The r ^ v a l u e s from T a b l e I can be used f o r a rough i n d i c a -  t i o n o f Ar-p constricted cm, for If  AV  b  r ^ was  255 u and 55 u f o r the r e l a x e d  arteries, respectively.  = -1.20  y  a 10 cm l e n g t h  ( f o r AV  D  So, Ar-j. = -200  i n ml and y i n cm).  o f r a t t a i l a r t e r y was  the change i n boundary l a y e r volume,  fully  u.  For L =  The water  calculated  AVj, = -3.3  and  10  loss  to be 3.3 u l .  u l , then the  74  thickness  of the l a y e r , y = 27.5  of f l u i d 27.5 water l o s s .  u.  That i s , a boundary l a y e r  u t h i c k would be required  to e x p l a i n the observed  But t h i s would mean that the c o n s t r i c t i n g a r t e r y ,  with a lumen diameter of 110 u, would contain only a core of O 2 / C O 2 ,  55 u i n diameter, f i l l i n g only h a l f of the lumen.  the humidity of the  O 2 / C O 2  was  Since  zero, t h i s i s wholly u n r e a l i s t i c .  A v a r i a t i o n i n the amount of f l u i d l e f t i n the lumen thus cannot be used to r a t i o n a l i z e the water l o s s observed i n c o n s t r i c t e d a r t e r i e s . There i s a d d i t i o n a l support f o r t h i s conclusion:  i f a significantly  large layer of f l u i d remains i n the lumen, i t would e v i d e n t l y be smaller  in  the d i s t a l than i n the proximal h a l f - a r t e r i e s . The values f o r the c o n t r o l water content of the two halves were accordingly with a water content ( i S.E.) ted:  compared.  Control a r t e r i e s  i n the range of 300 ml/100 g dry wt were s e l e c -  proximal c o n t r o l a r t e r i e s (n = 32), 295 1 4 ml water/100 g dry wt,  d i s t a l c o n t r o l a r t e r i e s (n = 31), 313 - 6. cant.  The 18 ml d i f f e r e n c e was  and  signifi-  C e r t a i n l y then, the d i s t a l c o n t r o l a r t e r i e s do not have l e s s water  than the proximal c o n t r o l a r t e r i e s . 3.  E f f e c t of Gaseous Perfusion on A r t e r i a l Water Content Although adequate controls were used, the perfusion of a r t e r i e s with  0  2  / C 0  2  was  hardly p h y s i o l o g i c a l and some comment i s necessary to show that  i t d i d not produce the observed water l o s s during Evaporation from the a r t e r y w a l l during the vapour pressure of the w a l l f l u i d . i s about 47 mm Hg  (49).  are l e s s than 1% (50).  The  vasoconstriction.  O 2 / C O 2  perfusion depends on  At 37°C the vapour pressure of water  e f f e c t s of solutes and pressure on t h i s value  The w a l l f l u i d vapour pressure i s a f f e c t e d by  meshwork of e n d o t h e l i a l c e l l s and i n t e r n a l e l a s t i c lamina separating  the the  75 w a l l from the lumen.  The extent of t h i s e f f e c t depends e s s e n t i a l l y on the  shape of the meniscus of the f l u i d i n the "cracks" of the meshwork.  For a  convex/concave surface, the vapour pressure i s a l t e r e d by about + 10% 51,52).  (see  An a r t e r i a l f l u i d vapour pressure between 42 and 52 mm Hg would  cause some evaporation from the a r t e r y w a l l during  O 2 / C O 2  perfusion.  The  f o l l o w i n g i s a maximal e s t i m a t i o n of t h i s evaporation. Rate of evaporation from a r a t t a i l a r t e r y A non-constricted a r t e r y was l e f t to evaporate i n a weighing b o t t l e on a 6-place M e t t l e r balance and i t s weight was recorded every 1 or 2 minutes. The a r t e r y l o s t 3.5 ml water/100 g dry wt/minute f o r the f i r s t 40 minutes. This evaporation occurred from the a d v e n t i t i a l and luminal surfaces, but the i n v i t r o a r t e r y was exposed to  O 2 / C O 2  only on i t s luminal surface.  From  Table I , the inner and outer r a d i i of a relaxed r a t t a i l a r t e r y were 255 and 321 u, so f o r a 10 cm length of a r t e r y , the inner and outer surface areas 9  would be 1.6 and 2.0 cm , r e s p e c t i v e l y . A c t u a l l y , the surface area of the porous a d v e n t i t i a would be considerably l a r g e r . The maximum evaporation r a t e from the luminal surface during 02/C0 p e r f u s i o n might thus be e s t i 2  mated as 2 ml/100 g dry wt/minute.  The p e r f u s i o n time f o r the c o n t r o l  a r t e r i e s was about 4 or 5 minutes, compared to 5 or 6 minutes f o r the t e s t arteries.  This time d i f f e r e n c e could r e s u l t i n a hydration d i f f e r e n c e of no  more than 4 ml water/100 g dry wt—much l e s s than the observed 48 ml water difference.  In r e a l i t y , the evaporation l o s s would probably be even l e s s  because of osmotic replacement of any evaporated water.  The a r t e r i e s were  incubated i n Krebs s o l u t i o n and the a r t e r y w a l l i s q u i t e permeable to water (see Chapter I V ) , ensuring no dehydration of the luminal surface'of the gas perfused  arteries.  76 Another f a c t o r diminishing  the p o s s i b l e r o l e of evaporation i s that  evaporation depends upon the area exposed to the C^/CC^.  The  constricted  t e s t a r t e r i e s have a smaller luminal surface area than the non-constricted control arteries.  [ I f r ^ was  255 u and 55 u f o r the relaxed and  constricted arteries respectively  fully  (from Table I ) , then c o n s t r i c t i o n  associated w i t h an 80% decrease i n luminal surface area.]  was  So, the t e s t  a r t e r i e s should lose l e s s water per minute by evaporation than the  control  a r t e r i e s , r e s u l t i n g i n a higher water content than the c o n t r o l a r t e r i e s — the opposite of what was In general, i f  observed.  O 2 / C O 2  perfusion  did s i g n i f i c a n t l y a f f e c t the a r t e r i a l  h y d r a t i o n , then presumably the longer the perfusion effect.  However: (1) There was  the perfusion  time, the greater the  no c o r r e l a t i o n between the water content and  time f o r e i t h e r the c o n t r o l or t e s t a r t e r i e s .  (2) In  the  r e l a x a t i o n experiment, the t e s t a r t e r i e s were perfused f o r 30 minutes and the c o n t r o l s f o r 5, yet both had  the same water content.  tent of c o n t r o l a r t e r i e s perfused w i t h  O 2 / C O 2 ,  (3) The water con-  306 ml/100 g dry wt, was  the same as that obtained f o r a r t e r i e s perfused w i t h Krebs s o l u t i o n  H.  about  (17).  SUMMARY 1.  Vasoconstriction,  high K, or PLV-2, was  induced by strong pressor doses of norepinephrine,  associated with a 16% decrease i n the ^ 0  content of  rat t a i l artery w a l l . 2.  In the norepinephrine induced c o n s t r i c t i o n s , t h i s f i n d i n g of a  water l o s s was  supported by a decrease i n the Na and CI contents and  i n u l i n space of the a r t e r y w a l l .  the  the  (a) Most of the R^O  l o s s was  due  to a decrease i n the  extracellular  f l u i d volume. (b) The  s i z e of the  l o s s depended upon the duration of c o n s t r i c t i o n ,  not upon the o r i g i n a l water content. (c) The H^O  and Na losses were greatest 30 seconds a f t e r vasocon-  s t r i c t i o n began. (d) Before the plateau of the increasing  intravascular  pressure  was  reached, the water and Na began to return to the a r t e r y w a l l , i . e . the H^O of  and Na movements were associated w i t h the onset  vasoconstriction.  (e) The K content and during NE 3.  the % i n u l i n space remained e s s e n t i a l l y constant  constriction.  In the PLV-2 induced c o n s t r i c t i o n s , the i n u l i n space remained  constant w h i l e the % i n u l i n space increased.  This i n d i c a t e s that, i n contrast  w i t h the other 2 agents, almost a l l the l o s t water came from i n u l i n inaccessible 4.  compartments of the a r t e r y w a l l .  About the same water l o s s was  associated w i t h  vasoconstriction  induced by a l l 3 agents, although no comparison could be made between t h e i r vasoconstrictive  actions.  78 CHAPTER IV HEMODYNAMIC AND PERMEABILITY CHANGES DURING VASOCONSTRICTION  The morphological and biochemical studies i n the previous two chapters have demonstrated a l o s s of a few m i c r o l i t e r s of f l u i d from the c o n s t r i c t i n g rat t a i l artery w a l l .  Experiments were designed to determine i f t h i s a l t e r e d  w a l l h y d r a t i o n would a f f e c t the hydrodynamics of the c o n s t r i c t i n g a r t e r y . In a d d i t i o n to the expected observations, i t was discovered that the permeab i l i t y of the a r t e r y w a l l was considerably a l t e r e d during c o n s t r i c t i o n . Three experiments were performed during v a s o c o n s t r i c t i o n induced by norepinephrine:  pressure and flow measurements, varying i n t r a v a s c u l a r pressure,  and dye p e r f u s i o n .  Although constant pressure p e r f u s i o n i s o f t e n used i n  hydrodynamic experiments ( f o r example, 1,2), i t was decided to f o l l o w Burton's advice to use constant flow p e r f u s i o n ( 3 ) .  A.  PRESSURE, FLOW AND LUMEN VOLUME Hemodynamics has been the subject of numerous reviews (4-9).  Some work  has been done comparing the e l a s t i c and pressure-flow p r o p e r t i e s of relaxed and c o n s t r i c t e d a r t e r i e s (1,2,10-14).  There have also been observations on  the e f f e c t of v a s o c o n s t r i c t i v e agents on blood flow and pressure ( f o r example, 15-18).  However, these studies have been concerned w i t h the a l t e r e d  hemodynamics of the system once v a s o c o n s t r i c t i o n was e s t a b l i s h e d . They have not been concerned w i t h flow and pressure during the onset of c o n s t r i c t i o n . The major problem has been to obtain accurate estimates of flow.  The present  study used a flowmeter which was very s e n s i t i v e to small flow changes i n the  79 hope of d e t e c t i n g the a d d i t i o n of w a l l f l u i d to the lumen during c o n s t r i c t i o n . 1.  Methods A 12 cm segment of the t a i l a r t e r y was exposed and i t s c o l l a t e r a l s  tied.  I t was tested f o r leaks by p e r f u s i o n w i t h Krebs s o l u t i o n to which  Evans blue dye had been added. artery:  There were two subsequent procedures f o r t h i s  (a) i t remained i n s i t u with i t s exposed surface kept moist w i t h  Krebs s o l u t i o n , or (b) i t was removed from the t a i l bed and placed i n a p e r spex chamber a t 37°C. w i t h two syringes: norepinephrine  P e r f u s i o n was by a constant i n f u s i o n pump (B. Braun)  both contained Krebs s o l u t i o n , and one a l s o contained  (4 ug/ml)(NE).  V a s o c o n s t r i c t i o n was induced using a 4-way  switch t o change the p e r f u s i o n from Krebs s o l u t i o n to NE-Krebs s o l u t i o n . T o p i c a l a p p l i c a t i o n of NE was a l s o used f o r some i n s i t u a r t e r i e s .  The  pressure gradient down the a r t e r y was monitored w i t h two Statham transducers connected by T - j o i n t s to the P.E. 50 polyethylene tubing proximal and d i s t a l to the a r t e r y .  The flow r a t e of the e f f l u e n t was determined using a s p e c i a l  photocell-flowmeter.  The e f f l u e n t passed through c o i l s of polyethylene  tubing wound around l i g h t pipes attached to a p h o t o c e l l . The only path f o r l i g h t from a DC lamp to reach the p h o t o c e l l was through a t h i n l i n e along the c o i l s .  A small bubble was i n s e r t e d i n t o the system between the a r t e r y  and the flowmeter.  Each time the dark meniscus of the l e a d i n g edge of the  bubble crossed the l i n e along the c o i l s of the flowmeter, i t s passage was recorded as a v e r t i c a l d e f l e c t i o n on the polygraph output from the p h o t o c e l l . The r a t e of flow out of the a r t e r y was determined as f o l l o w s : The separation of the polygraph d e f l e c t i o n s , representing the passage of the bubble through 1 c o i l of the flowmeter, was measured during a c o n t r o l run ( i . e . without NE).  From the polygraph chart speed and the pump r a t e , the volume of one  80 c o i l was determined.  For measurements during v a s o c o n s t r i c t i o n , the number  of c o i l s , i n c l u d i n g f r a c t i o n s , traversed i n 15 seconds was determined.  From  the volume of 1 c o i l , the flow r a t e f o r t h i s 15 second i n t e r v a l was then calculated.  The flow measurements were accurate to + 0.02 u l / s e c , or ~t 0.6%.  For t h i s method to be that accurate, the v e l o c i t y of the bubble had to be equal to the average v e l o c i t y of the f l u i d .  The s i z e of the bubble used was  not too s m a l l , s i n c e i t would move i n the a x i a l stream at greater than the average v e l o c i t y , or too l a r g e , since i t would have a d i f f e r e n t v i s c o s i t y than the p e r f u s i n g s o l u t i o n (5).  I t has been estimated that the maximum  d e v i a t i o n s of the bubble v e l o c i t y from the blood flow, f o r a wide range of flows, are T 5% (19). The presence of t h i s flowmeter i n the system meant that the i n t r a v a s c u l a r pressure was f a i r l y high, about 44 mm Hg a t the midpoint of the a r t e r y , w h i l e the pressure gradient down the a r t e r y was about 8 mm Hg during the c o n t r o l runs.  The pressure and flow c h a r a c t e r i s t i c s of  the NE induced c o n s t r i c t i o n s were determined i n 14 r a t t a i l a r t e r i e s . 2.  Results The pressure gradient and the e f f l u e n t flow r a t e f o r a t y p i c a l a r t e r y  c o n s t r i c t e d w i t h norepinephrine are shown i n F i g . 14. At 30 t o 90 seconds a f t e r the onset of c o n s t r i c t i o n there was an increase i n the otherwise contant flow r a t e and an increase i n the i n c r e a s i n g pressure gradient.  These  anomalies were observed f o r a l l the c o n s t r i c t i o n s , although t h e i r s i z e s v a r i e d considerably.  The source of the anomalies l i e s i n the a r t e r i e s them-  selves because the flow r a t e of the i n f u s i o n pump was constant during these experiments.  The pump operated through a s e r i e s of gears which prevented  any "backlash" from the experimental system of the a r t e r y , tubing and f l o w meter onto the i n f u s i o n pump.  81  r»l Of  •  •  •  30  NOREPINEPHRINE  F i g . 14  i  60  i  i  90  1 1 1 1 1 1 1 1 120  150  180  210  1  1 1 1  240  '  270  TIME (seconds)  E f f l u e n t flow r a t e , F, and pressure gradient, P, f o r a r a t t a i l a r t e r y c o n s t r i c t e d iri s i t u w i t h norepinephrine.  i  '0  82 3.  Discussion Could f l u i d "squeezed out" of the lumen of the c o n s t r i c t i n g a r t e r y  have been the source of the increase i n flow r a t e and the "hump" i n the pressure r i s e ?  The concept of "squeezing out" i m p l i e s that there was a  r e l a t i v e l y s t a t i c amount of f l u i d stored i n the lumen which was expelled during c o n s t r i c t i o n .  But, the time course f o r c o n s t r i c t i o n (a few minutes)  was much longer than the time required f o r f l u i d to move through the lumen (a few seconds), so there could be no sudden s h i f t of f l u i d out of the lumen.  Over the few minutes of c o n s t r i c t i o n , the decrease i n the c r o s s -  s e c t i o n a l area of the lumen, A^> was constantly balanced by the increase i n the v e l o c i t y through the lumen, v, since the flow r a t e , F = A^v, constant.  remained  When the a r t e r y c o n s t r i c t e d , the volume of the whole system  ( a r t e r y lumen plus polyethylene tubing) decreased, so l e s s time was required for t r a n s i t through the system.  But the increase i n v e l o c i t y was only  through the a r t e r y lumen, not through the r e s t of the system.  Changes i n s i d e  the system could a l t e r the c r o s s - s e c t i o n a l areas, the v e l o c i t i e s , and the volumes, but not the flow r a t e s . The anomalies i n the curves of F i g . 14 can be explained by a movement of f l u i d i n t o the c o n s t r i c t i n g lumen. been:  The source of t h i s f l u i d could have  (a) the a r t e r y w a l l — w h i c h would agree w i t h the r e s u l t s of the  previous two chapters, or (b) the f l u i d surrounding the a r t e r y — w h i c h would i n d i c a t e that the p e r m e a b i l i t y of the a r t e r y w a l l was a l t e r e d during cons t r i c t i o n , to allow f l u i d to pass through i t i n t o the lumen. these two p o s s i b i l i t i e s are not mutually e x c l u s i v e .  Of course,  The s i z e of the  increase i n flow i n d i c a t e s that f l u i d passed i n t o the lumen from the surroundings.  The maximum increase i n flow i n F i g . 14 was 0.25 p l / s e c — a b o u t  83 10 x that expected from the l o s s of w a l l f l u i d .  A l s o , the area under the  flow curve i n F i g . 14 i n d i c a t e s that over 20 u l of f l u i d was added to the lumen.  The whole a r t e r y w a l l had only 20 to 30 u l of water. The increase i n flow i n F i g . 14 was accompanied by a "hump" i n the  i n c r e a s i n g pressure gradient.  This i s to be expected from P o i s e u i l l e ' s 2  equation f o r laminar flow through r i g i d tubes, by which P °= F/V , where: P = pressure gradient down the a r t e r y segment, F = outflow from the a r t e r y , and V = lumen volume.  Although P o i s e u i l l e ' s equation i s f o r equal i n f l o w  and outflow, the above r e l a t i o n s h i p i s probably s t i l l true as a f i r s t approximation when there i s a small amount of f l u i d added to or removed from the perfusate.  This r e l a t i o n means that a decrease i n V or an increase  i n F would cause an increase i n P.  A combination  of these two events during  v a s o c o n s t r i c t i o n would r e s u l t i n the observed pressure p a t t e r n i n F i g . 14. 4.  Summary 1.  There was a s l i g h t increase i n the outflow from the perfused  a r t e r y c o n s t r i c t e d w i t h norepinephrine.  This increase was associated w i t h  a "hump" i n the i n c r e a s i n g pressure gradient. 2.  These changes i n d i c a t e that there was an increase i n w a l l perme-  a b i l i t y during v a s o c o n s t r i c t i o n which allowed f l u i d to enter the lumen from the f l u i d surrounding B.  the a r t e r y .  EFFECT OF INTRAVASCULAR PRESSURE The previous s e c t i o n suggested that w a l l tension changes caused an  a l t e r a t i o n - i n the p e r m e a b i l i t y of the a r t e r y w a l l .  Sawyer and Valmont have  reported that the aorta and vena cava are permeable to Na and CI (20). I t  84 has been suggested that since the net i o n movements are i n d i f f e r e n t d i r e c t i o n s f o r these 2 v e s s e l s , the i o n movements may depend on the luminal pressure  (21). I t thus seemed reasonable  to examine the e f f e c t of the i n t r a -  vascular pressure on vascular p e r m e a b i l i t y . Most studies i n v o l v i n g v a r i a b l e i n t r a l u m i n a l pressure have been concerned with demonstration of a vascular myogenic response (22,23).  The  studies which considered the e f f e c t of pressure on permeability have been concerned with c a p i l l a r y f i l t r a t i o n (see 24), or transport through other membranes (25-28).  There has a l s o been some work on the e f f e c t of l u m i n a l  pressure on c o n s t r i c t i o n (29,30). Pressure and flow were measured during norepinephrine  induced con-  s t r i c t i o n i n the r a t t a i l a r t e r y at e i t h e r "high" or "low" i n t r a v a s c u l a r pressure. 1.  Methods The above procedure f o r perfusing r a t t a i l a r t e r i e s i n s i t u was used.  The a r t e r i e s were c o n s t r i c t e d by the a d d i t i o n of 0.2 ug norepinephrine i n a 10 second perfusion of NE-Krebs s o l u t i o n at 0.200 ml/min while the pressure gradient was monitored.  The flow r a t e of the e f f l u e n t was determined by  c o l l e c t i n g the e f f l u e n t every 20 seconds i n weighing b o t t l e s and measuring the volumes with a 100 u l syringe.  This method was l e s s accurate than that  of the photocell-flowmeter:  the volume readings were ± 1 u l and the time  recordings were ~t 1 second.  Consequently, the e r r o r i n an average flow of  0.180 ml/min f o r a 20 second i n t e r v a l i n which 60 u l was c o l l e c t e d was ± 0.012 ml/min or about 6.7%.  When m u l t i p l e c o n s t r i c t i o n s were induced i n  the same a r t e r y , at l e a s t .30 minutes e q u i l i b r a t i o n was allowed between constrictions .  85 The a r t e r i e s were c o n s t r i c t e d at two d i f f e r e n t b a s a l i n t r a v a s c u l a r pressures:  low and high.  (The i n t r a v a s c u l a r pressure, taken as the pressure  i n the lumen at the midpoint of the a r t e r y segment, should not be confused w i t h the pressure gradient, the d i f f e r e n c e i n the pressure between the two ends of the a r t e r y segment, used i n P o i s e u i l l e ' s equation.)  For low i n t r a -  vascular pressures, the polyethylene tubing d i s t a l to the a r t e r y was a t the same l e v e l as the a r t e r y .  For high i n t r a v a s c u l a r pressures, the d i s t a l  tubing was r a i s e d about 50 cm above the l e v e l of the a r t e r y .  This increased  the i n t r a v a s c u l a r pressure without producing a l a r g e change i n the pressure gradient.  The average i n t r a v a s c u l a r pressures were:  low, 3mm Hg, and high,  41 mm Hg. 2.  Results The pressure gradient patterns f o r the c o n s t r i c t i n g t a i l a r t e r y pre-  parations w i t h low and high i n t r a v a s c u l a r pressures are presented i n F i g . 15.  These curves represent the average r e s u l t s of 4 norepinephrine-induced  constrictions.  There was a s i g n i f i c a n t l y  l a r g e r increase i n the pressure  gradient during p e r f u s i o n a t the low i n t r a v a s c u l a r pressure (+ 140 mm Hg) than at the high i n t r a v a s c u l a r pressure (+ 84 mm Hg).  The time courses of  the pressure increases were the same but the r e l a x a t i o n was f a s t e r f o r the high pressure case.  The same r e s u l t was obtained on 1 a r t e r y when con-  s t r i c t i o n s at high and low i n t r a v a s c u l a r pressures were induced. The average flow patterns f o r 8 low and 4 high i n t r a v a s c u l a r pressure c o n s t r i c t i o n s are given i n F i g . 16. I t shows that: 1.  The "base l i n e " flow before and a f t e r c o n s t r i c t i o n was s i g n i f i -  c a n t l y l e s s f o r p e r f u s i o n at high i n t r a v a s c u l a r pressure than at low pressure. The average flow r a t e s (Z S.E.) before c o n s t r i c t i o n were:  (a) low i n t r a -  86  450  F i g . 15  Pressure gradient, P, down r a t t a i l a r t e r i e s c o n s t r i c t e d i n s i t u with norepinephrine (NE) a t high and low i n t r a v a s c u l a r pressure. The v e r t i c a l bars represent the S.E.  vascular pressure (n = 44), 0.182 + 0.002 ml/min, and (b) high i n t r a v a s c u l a r pressure (n = 24), 0.171 ± 0.001 ml/min.  In a d d i t i o n both e f f l u e n t flow  rates were l e s s than that of the i n f u s i o n pump: (n = 8 ) , 0.200 ± 0.002 ml/min — f r o m c a l i b r a t i o n s a t the 0.200 ml/min s e t t i n g of the pump. 2.  The changes i n the e f f l u e n t flow rates were remarkably  different  during v a s o c o n s t r i c t i o n . (a) The flow during the low pressure c o n s t r i c t i o n s decreased  con-  s i d e r a b l y , reaching i t s lowest point when the pressure was one-half i t s peak  87  INFUSION PUMP RATE  » P  V  »LOW INTRAVASCULAR P R E S S U R E n=8  HIGH INTRAVASCULAR PRESSURE n=4  T I M E (seconds)  Fig.  value.  16  E f f l u e n t flow r a t e , F, f o r r a t t a i l a r t e r i e s c o n s t r i c t e d i n s i t u with norepinephrine (NE) a t high and low i n t r a v a s c u l a r pressure. The v e r t i c a l bars represent the S.E.  The flow d i d not r e t u r n to i t s previous value u n t i l a f t e r the peak  of c o n s t r i c t i o n .  The area under the flow curve revealed that 55ul l e s s  f l u i d passed through the a r t e r y over 150 seconds. (b) The flow during the high pressure c o n s t r i c t i o n s was b i p h a s i c : the flow increased during the i n i t i a l r i s e i n pressure, returned t o i t s previous value when the pressure was one-half i t s peak value, and decreased to i t s lowest point when the peak pressure gradient was a t t a i n e d .  The  88 i n i t i a l increase i n flow represented about 25 u l more f l u i d i n the e f f l u e n t i n 50 seconds (5 u l of which, over 30 seconds, could not have come from the pump), while f o r the l a t e r decrease i n flow, there was about 40 u l l e s s f l u i d i n the e f f l u e n t over 110 seconds.  These losses and gains of f l u i d  could not be explained by leaks i n the system.  They were r e a l and repeatable  i n each of the c o n s t r i c t i o n s . 3.  Discussion The smaller peak pressure gradient f o r higher i n t r a v a s c u l a r pressures  was also observed by Nicholas and Hughes (30).  They found an inverse r e -  l a t i o n s h i p between the pressor response to norepinephrine and the r e s t i n g blood pressure.  On the other hand, Sparks and Bohr (31) found c o n t r a c t i o n  of a r t e r y s t r i p s , i n response to a standard e l e c t r i c a l stimulus, increased with s t r e t c h u n t i l an optimal length, a f t e r which the response decreased. The high i n t r a v a s c u l a r pressure r e s u l t s were obtained i n circumstances to those i n the above photocell-flowmeter experiment.  similar  The i n t r a v a s c u l a r  pressures i n the incubation experiments of Chapter I I I were between those i n the high and low pressure cases. The flow r e s u l t s can be explained only i f the In s i t u r a t t a i l a r t e r y preparation was permeable to the p e r f u s i n g Krebs s o l u t i o n .  The a r t e r i e s were  a l l tested f o r leaks from the t i e d c o l l a t e r a l s and there were no leaks from the constant flow pump assembly.  The p e r m e a b i l i t y of the a r t e r y w a l l was  a f f e c t e d by the i n t r a v a s c u l a r pressure and by v a s o c o n s t r i c t i o n , 1.  Before c o n s t r i c t i o n , f l u i d passed out of the lumen, through  a r t e r y w a l l and i n t o the surrounding f l u i d . 0.182  = 0.018  ml/min = 0.3'ul/sec  These losses were 0.200 -  during p e r f u s i o n at low i n t r a v a s c u l a r  the  89 pressures and 0.5 ul/sec a t high pressures.  (That the outflow was l e s s than  the i n f l o w was suspected from the r e s u l t s of the previous s e c t i o n , but the c o n t r o l runs were not of a s u f f i c i e n t length to comment.)  This i n d i c a t e s that  there was a simple f i l t r a t i o n process through the a r t e r y w a l l which increased when the pressure i n the a r t e r y lumen was increased.  A s i m i l a r observation  was made by Wilens and McClusky f o r excised human i l i a c a r t e r i e s and veins (32) . 2.  During the NE-induced v a s o c o n s t r i c t i o n and the r e s u l t i n g increase  i n the pressure gradient, the p e r m e a b i l i t y of the a r t e r y w a l l was d r a s t i cally altered,  (a) At low i n t r a v a s c u l a r pressures, the p e r m e a b i l i t y increased  so that even l a r g e r amounts of f l u i d l e f t the lumen.  (b) At high i n t r a -  vascular pressures, i n i t i a l l y the p e r m e a b i l i t y not only decreased but 5 p i of the f l u i d was added to the lumen, and then the p e r m e a b i l i t y increased so ' that l a r g e amounts of f l u i d l e f t the lumen.  In both cases, as the a r t e r y  r e l a x e d , the p e r m e a b i l i t y returned to i t s p r e - c o n s t r i c t i o n l e v e l and f l u i d continued to pass from the lumen to the surroundings. 3.  There was no simple inverse c o r r e l a t i o n between the i n t r a v a s c u l a r  pressures and the flow r a t e before and during the c o n s t r i c t i o n s .  The s i t u -  a t i o n was made complex by the opposite behavior of the flow i n the two pressure cases during the i n i t i a l r i s e i n pressure. There i s no d i r e c t evidence from t h i s experiment, f o r p o s s i b l e changes i n the hydration of the a r t e r y w a l l during c o n s t r i c t i o n .  The l a r g e movements  of f l u i d r i g h t through the a r t e r y w a l l completely obscured any small movements out of the w a l l .  A p o s s i b l e exception was the a d d i t i o n of about 5 y l  of f l u i d to the lumen i n the i n i t i a l phase of the pressure r i s e w i t h the high i n t r a v a s c u l a r pressure.  The d i r e c t i o n of the f l u i d movement through  90 the w a l l could have reversed o r the f l u i d could have come from the a r t e r y w a l l itself.  These two p o s s i b i l i t i e s cannot be d i s t i n g u i s h e d from t h i s experiment. P o s s i b l e causes of the changes i n a r t e r y w a l l permeability are d i s -  cussed i n Chapter V I . 4.  Summary 1.  The higher the i n t r a v a s c u l a r pressure, the smaller the pressure  response to norepinephrine. 2.  The w a l l of the perfused t a i l a r t e r y was q u i t e permeable:  fluid  passed out of the lumen, through the a r t e r y w a l l , and i n t o the surrounding fluid. 3.  This f r e e passage of f l u i d was very much a f f e c t e d by the pressure  i n the a r t e r y lumen and by the s t a t e of tension i n the w a l l . 4.  The higher the i n t r a v a s c u l a r pressure, the greater the permeability  of the w a l l , i . e . the greater the l o s s of f l u i d out through the a r t e r y w a l l . 5.  When the a r t e r y c o n s t r i c t e d at low i n t r a v a s c u l a r pressures, the  permeability  increased, but at high i n t r a v a s c u l a r pressures, i t decreased then  increased. 6.  There may have been a decrease i n the hydration of the a r t e r y w a l l  during c o n s t r i c t i o n at high i n t r a v a s c u l a r pressures.  C.  DYE DILUTION The w a l l water l o s s and the permeability  changes during  t i o n suggested a more d i r e c t estimation of f l u i d movement.  vasoconstric-  P e r f u s i o n of a  dye during c o n s t r i c t i o n would provide a medium which would "amplify" to an observable l e v e l any movements of f l u i d i n or out of the lumen.  The best  and most commonly used dye f o r vascular perfusion i s T 1824, Evans blue  91 (33,34,35).  This dye has s e v e r a l advantages f o r t h i s p a r t i c u l a r study:  (a) I t leaves the blood stream only slowly (35); (b) For concentration c a l c u l a t i o n s from o p t i c a l density measurements (see 36), Evans blue obeys the Lambert-Beer Law f o r s t r i c t p r o p o r t i o n a l i t y between o p t i c a l d e n s i t y and concentration (34); (c) In view of the Na movement accompanying the w a l l water l o s s , i t i s important that the o p t i c a l density of Evans blue i s not a f f e c t e d by v a r i a t i o n s i n NaCl concentration (34). The % transmission of Evans blue i n a Krebs s o l u t i o n perfusing the rat  t a i l a r t e r y was continuously monitored during c o n s t r i c t i o n using a  flow-through m i c r o c e l l on a Zeiss spectrophotometer. 1.  Methods The same i n s i t u preparation of the r a t t a i l a r t e r y was used as i n the  above two experiments.  The a r t e r i e s were perfused w i t h Krebs s o l u t i o n to  which Evans blue dye had been added (12.5 mg/liter)(282 mosm/liter). Norepinephrine  was added to the a r t e r y e i t h e r i n t e r n a l l y or t o p i c a l l y .  The  e f f l u e n t passed through a 20 u l flowthrough c e l l i n a Z e i s s spectrophotometer, set at 600 my,  the absorption peak f o r Evans blue (35).  pressure proximal to the a r t e r y was monitored.  The i n t r a v a s c u l a r  This pressure and the %  transmission of the e f f l u e n t were displayed on a polygraph.  F i f t y - o n e con-  s t r i c t i o n s were induced i n 13 t a i l a r t e r i e s . 2.  Results One of the responses of t h i s i n s i t u a r t e r y preparation to the a d d i t i o n  of norepinephrine i s shown i n F i g . 17.  The pressure increase was accompanied  by an increase i n the % transmission (% T) of the e f f l u e n t . the Evans blue i n the perfusate must have been d i l u t e d .  This meant that  The extent of the  92  F i g . 17  I n t r a v a s c u l a r pressure and % transmission of the e f f l u e n t from a r a t t a i l a r t e r y i n s i t u a f t e r the a d d i t i o n of norepinephrine.  dye d i l u t i o n was c a l c u l a t e d from the % T curve; Over the 500 seconds of the % T increase, the average % T was 54.7%.  From Beer's law f o r t h i s s o l u t i o n and  spectrophotometer  c e l l , the corresponding concentration was 42 log(l/0.547) = 11.0 u m o l e s / l i t e r .  The concentration before the NE was added  was 11.8 u m o l e s / l i t e r . In 500 seconds, the volume of f l u i d normally passing through the lumen would be 0.200 ml/min x 500/60 min = 1.667  ml.  I t i s assumed that the amount of dye  i n t h i s volume was constant.  Thus, the true volume of f l u i d  i n the 500 seconds must have been 1.667  x 11.8/11.0 = 1.912  ml.  So, the amount of water added to the perfusant was about 121 u l . From a c a l i b r a t i o n run, t h i s method of determining the added volume from the area under the curve was accurate to about Z 10%.  93 A l l the other NE-induced c o n s t r i c t i o n s showed % T patterns s i m i l a r to that i n F i g . 17. The changes i n % T were not a r t i f a c t s . did not enter the a r t e r y w a l l .  The Evans blue dye  In f a c t , the % T decreased s l i g h t l y during  the runs, i . e . the s o l u t i o n s became s l i g h t l y more concentrated, not l e s s . The % T was not s i g n i f i c a n t l y a f f e c t e d by changes i n the flow r a t e through the spectrophotometer 3.  cell.  Discussion As w i t h the previous experiments, the a d d i t i o n of over 100 p i of f l u i d  to the lumen of the c o n s t r i c t i n g a r t e r y can only mean that the p e r m e a b i l i t y of the a r t e r y w a l l was a l t e r e d during c o n s t r i c t i o n .  This p e r m e a b i l i t y change  can be explained i n 1 of 3 ways: 1.  Before the NE was added, f l u i d was l e a v i n g the lumen and passing  i n t o the f l u i d surrounding the a r t e r y .  C o n s t r i c t i o n was then associated w i t h  a decrease i n the p e r m e a b i l i t y of the w a l l , so that l e s s f l u i d passed out of the lumen, r e s u l t i n g i n a l e s s concentrated e f f l u e n t .  (This was the cause of  the i n i t i a l flow change i n the high pressure c o n s t r i c t i o n i n the previous experiment.) 2.  Before the NE was added, f l u i d was entering the lumen from the  surroundings.  C o n s t r i c t i o n then increased the w a l l p e r m e a b i l i t y so that even  more f l u i d entered the lumen, r e s u l t i n g i n a more d i l u t e d e f f l u e n t . 3. the w a l l .  Before the NE was added, no f l u i d entered or l e f t the lumen through C o n s t r i c t i o n then increased the w a l l p e r m e a b i l i t y and f l u i d moved  i n t o the lumen, r e s u l t i n g i n a d i l u t e d e f f l u e n t . There i s evidence that explanation #2 a p p l i e s to the changes observed i n F i g . 17. C a l c u l a t i o n of the molar e x t i n c t i o n c o e f f i c i e n t ( i n Beer's law) for the Evans blue-Krebs s o l u t i o n i n the absence of the a r t e r y , gave dye  94 concentrations which were too low when the a r t e r y was present.  (The dye  concentration was 12.5 rag/liter = 13.0 u m o l e s / l i t e r , compared to 11.8 umoles / l i t e r f o r F i g . 17.) This i n d i c a t e s that even before the NE was added, f l u i d was passing through the a r t e r y w a l l and entering the lumen.  Constriction i n -  creased t h i s passage. In the previous experiment, before c o n s t r i c t i o n f l u i d moved out of the lumen, while i n t h i s experiment, before c o n s t r i c t i o n f l u i d moved i n t o the lumen.  The d i r e c t i o n of flow through the w a l l was probably a f f e c t e d by the  dsmolarity  of the s o l u t i o n perfusing  the lumen and bathing the e x t e r i o r of  the a r t e r y and by the i n t r a v a s c u l a r pressure i n the two experiments. ( I n each experiment, the same s o l u t i o n was used i n s i d e and outside the a r t e r y . ) In any case, i t seems u n l i k e l y that there are large transmural f l u i d movements before c o n s t r i c t i o n f o r a r t e r i e s i n v i v o .  These experiments were not  designed to determine the "normal" w a l l permeability, which v a s o c o n s t r i c t i o n had on the w a l l 4.  but rather the e f f e c t  permeability.  Summary 1.  The Evans blue perfusing  the a r t e r i e s was d i l u t e d during vaso-  constriction. 2.  Before the a d d i t i o n of norepinephrine, f l u i d passed through the  a r t e r y w a l l from the surroundings i n t o the lumen. 3.  During c o n s t r i c t i o n , the permeability  increased—allowing  f l u i d to pour i n t o the lumen.  of the w a l l was d r a s t i c a l l y  95  D. SUMMARY  1. had  N o r e p i n e p h r i n e - i n d u c e d v a s o c o n s t r i c t i o n i n the r a t t a i l  p r o f o u n d e f f e c t s on i t s hemodynamic p r o p e r t i e s and 2.  There was  decreased during  a suggestion  t h a t the h y d r a t i o n  permeability.  of the a r t e r y w a l l  c o n s t r i c t i o n at " h i g h " i n t r a v a s c u l a r  pressure.  3.  The  w a l l s of t h e s e a r t e r i e s were q u i t e permeable to  4.  The  i n t r a v a s c u l a r pressure  permeability very 5.  and  artery  fluid,  vasoconstriction affected  this  drastically.  Higher i n t r a v a s c u l a r pressures  increased  the p e r m e a b i l i t y o f  the  artery wall. 6.  V a s o c o n s t r i c t i o n e i t h e r caused an i n c r e a s e i n w a l l  permeability,  or a d e c r e a s e f o l l o w e d by an i n c r e a s e i n p e r m e a b i l i t y . 7.  How  t y p i c a l are  a r t e r i e s can be argued. example, m o n i t o r i n g this question 8. pressure  these r a t t a i l  But  distributing  s t u d i e s on p e r f u s e d  arteries (for  c e r t a i n l y any  the c o n c e n t r a t i o n  of p e r m e a b i l i t y  a r t e r i e s o f muscular  of i o n s i n the e f f l u e n t ) ,  take  i n t o account.  That the p e r m e a b i l i t y of the w a l l was i n the a r t e r y lumen and  the  determine the p e r m e a b i l i t y o f the  the steady s t a t e which the w a l l m a i n t a i n s w i t h upon the i n t r a v a s c u l a r p r e s s u r e  a f f e c t e d both by  t e n s i o n i n the a r t e r y w a l l s u g g e s t s  the b a l a n c e of f o r c e s t h a t n o r m a l l y  vasoconstriction.  should  and  was  that wall—  i t s s u r r o u n d i n g s — w a s dependent  upset by  the changes a s s o c i a t e d  with  96 CHAPTER V EXPERIMENTALLY INDUCED ALTERATIONS IN THE HYDRATION OF THE ARTERY WALL  The experiments discussed  above have i n d i c a t e d that the hydration as  w e l l as the transmural permeability of the a r t e r y w a l l are considerably a l tered when the a r t e r y c o n s t r i c t s under the i n f l u e n c e of several agents.  vasoactive  The a r t e r y w a l l can be viewed as a network of components which are  a l l i n t i m a t e l y connected and act as a u n i t to regulate the volume of blood that passes through the a r t e r y lumen.  This network c o n s i s t s of e n d o t h e l i a l  and smooth muscle c e l l s and the e x t r a c e l l u l a r macromolecules: e l a s t i n , and protein-polysaccharide  complexes.  collagen,  The above experiments ex-  amined the e f f e c t s of a c t i v e changes i n the w a l l produced by v a s o c o n s t r i c t i o n . The contents of the vascular w a l l can also be a l t e r e d p a s s i v e l y .  In t h i s  study, passive changes were induced i n the a r t e r y w a l l by (a) varying the composition of the e x t e r n a l ions and by (b) c o o l i n g then rewarming the arteries. A.  EFFECT OF VARYING EXTERNAL IONIC COMPOSITION The anionic groups of the e x t r a c e l l u l a r m a t e r i a l (the p a r a c e l l u l a r  matrix) of the a r t e r y w a l l bind Na and other ions (1-7).  I t i s p o s s i b l e to  change the counter-ion bound to these groups by changing the i o n i c composit i o n of the s o l u t i o n i n which the a r t e r y i s incubated (6). Such an i o n exchange may a l t e r the c o n f i g u r a t i o n of the p a r a c e l l u l a r matrix and cohsequently a l t e r the hydration of the a r t e r y w a l l ( 7 ) .  To t e s t the i o n exchange p r o p e r t i e s of the r a t t a i l a r t e r y and t h e i r e f f e c t on the p h y s i c a l properties of the a r t e r y , a s e r i e s of experiments were performed using the techniques of Chapter I I I . A r t e r i e s were incubated i n isosmotic s o l u t i o n s of d i f f e r e n t i o n i c composition while t h e i r i n t r a v a s c u l a r pressure was monitored, then analysed f o r t h e i r water and i o n content. 1.  Methods A f t e r the v e n t r a l t a i l a r t e r y of the r a t was exposed, i t s d i s t a l and  proximal halves were cannulated by i n s e r t i n g polyethylene tubing a t the midpoint and proximal end of the a r t e r y .  The d i s t a l h a l f - a r t e r y was flushed  with  the s o l u t i o n i n which i t was to be incubated, removed from the t a i l bed and placed w i t h i t s cannula i n that p a r t i c u l a r s o l u t i o n .  The proximal h a l f -  a r t e r y was s i m i l a r l y flushed, removed and placed i n a second s o l u t i o n . F i v e s o l u t i o n s of d i f f e r e n t i o n i c composition were used as incubation media f o r the a r t e r i e s : 2.4, C a C l  MgS04  2  i n m e q / l i t e r , s o l u t i o n 1: NaCl 143, KC1 5.0,  4.2; s o l u t i o n 2: KC1 5.0, MgS04 2.4, C a C l  s o l u t i o n 3: MgSO^ 2.4, C a C ^ 4.2, l a c t o s e ; s o l u t i o n 4: s o l u t i o n 5: numerically isosmotic  lactose. before i t .  Tris-HCl.  CaCl  2  4.2, l a c t o s e ;  Solutions 2 to 5 had one c a t i o n l e s s than the s o l u t i o n The amounts of l a c t o s e added made the s o l u t i o n s  (290 mosm/liter).  a conventional  4.2, l a c t o s e ;  2  Since zero sodium s o l u t i o n s preclude the use of  NaHCO-j-Nal^PO^ b u f f e r system, the s o l u t i o n s were buffered  One-tenth the usual T r i s concentration,  with  0.7 g / l i t e r (5 mM), was  used to minimize both the c o n t r i b u t i o n of c h l o r i d e ions and the unknown e f f e c t of the T r i s c a t i o n on the e x t r a c e l l u l a r matrix of the a r t e r i a l w a l l . This low b u f f e r i n g capacity meant that the s o l u t i o n s could not be aerated w i t h 0 /C0 ; instead 100% oxygen was used, r e s u l t i n g i n a pH of 7.0 at 37°C. 2  2  Four experiments were performed, using a d i f f e r e n t p a i r of s o l u t i o n s i n each:  s o l u t i o n s 1 and 2, 2 and 3, 3 and 4, 4 and 5.  Eight r a t s were used i n  98 each experiment.  Both s o l u t i o n s i n an experiment  proximal h a l f - a r t e r i e s .  contained 4 d i s t a l and 4  A f t e r an a r t e r y had been incubated f o r 3 hours, i t  was perfused with oxygen f o r 7 to 10 minutes to remove f l u i d from the lumen and produce a b a s a l tension i n the a r t e r y w a l l . c a r r y i n g oxygen i n t o the s o l u t i o n was connected  The polyethylene tubing to the cannula of the a r t e r y .  The i n t r a v a s c u l a r pressure during t h i s gas flow was monitored by a Statham transducer connected j u s t proximal to the a r t e r y .  (The gas p e r f u s i o n was  e s s e n t i a l l y constant f o r a l l the a r t e r i e s so the pressures recorded f o r a r t e r i e s i n d i f f e r e n t s o l u t i o n s could be compared.)  The a r t e r y was  then  removed from s o l u t i o n , b l o t t e d , and analysed f o r i t s water and i o n contents as described i n Chapter I I I .  A Techtron atomic absorption spectrometer  was  used f o r the i o n a n a l y s i s . 2.  Results Table XVIII shows the water (see F i g . 18), i o n content, and i n t r a v a s c u l a r  pressure f o r a r t e r i e s incubated i n one of f i v e s o l u t i o n s .  Arteries i n solutions  3 and 4 (with no monovalent cations) had a greater d i v a l e n t i o n content, smaller water content and greater i n t r a v a s c u l a r pressure than a r t e r i e s i n s o l u t i o n 1. The only s i g n i f i c a n t d i f f e r e n c e between the a r t e r i e s i n s o l u t i o n s 3 and 4 was the gain i n Ca, equal to one-half the l o s s of Mg.  In s o l u t i o n 5, where H  +  was  the only c a t i o n , the water and pressure values were about the same as i n s o l u t i o n 1.  I t should be noted that these f i v e s o l u t i o n s were isosmotic.  So,  f o r example, a r t e r i e s i n s o l u t i o n 2 which l o s t Na and presumably CI, would not have l o s t water because of an osmotic  imbalance.  There was an inverse r e l a t i o n s h i p between the water content and the Ca + Mg content:  f o r the 60 a r t e r i e s i n the f i v e s o l u t i o n s , the c o r r e l a t i o n  c o e f f i c i e n t was -0.495 (p < 0.001).  This suggests that the quantity of  TABLE XVIII.  Intravascular, pressure, water and i o n contents (±S.E.) of r a t t a i l a r t e r i e s incubated i n one of 5 isosmotic s o l u t i o n s .  Solution  Pressure (mm Hg) 65 ± 5  1. ( a l l ions) (8)* A+  2. (no Na ) (12) +  3. (no Na , K ) (16) +  +  1^0  Na  (ml/100 g dry wt) 286 ± 13  + 62  -33  127 ± 12  253 ± 5  +32  -24  159 ± 8  229 ± 2  K  Mg^ " 1  +  -72 5.-(lactose) (8) *  76 ± 18  253 ± 3 +55 290 ± 8  Number of a r t e r i e s Difference between a r t e r i a l contents (p < 0.02),  1  (meq/100 g dry weight) 43.6 ± 1.7  12.5 ± 1.2  -41.4  -2.4  2.20 ± 0.28  10.1 ± 0.4  -1.17  -3.9  1.03 ± 0.14  6.19 ± 0.36  3.39 ± 0.15 +0.81  0.78 ± 0.08  5.67 ± 1.29  1.28 ± 0.15  6.01 ± 0.32  +0.80 2.99 ± 0.12  4.27 ± 0.10  3.24 ± 0.09  1.54 ± 0.04 +0.50  +0.50  2.19 ± 0.11  4.20 ± 0.09  -2.73 4. (no Na+, K , Mg++) 148 ± 14 (16)  Ca  2.03 ± 0.07  +1.46 4.70 ± 0.14 -3.73 0.97 ± 0.07  100  Na* 143.0 300 r  K*  290  Ca**  5.0  mEq  Mg** 2.4 - T 8 ANIMALS  280  IONS REPLACED BY LACTOSE SOLUTIONS ISOOSMOTIC AT 2 9 0 mosmoles  g 270 o>  O O  ^ 2 6 0 E  I _j  4.2  12  250  <  to  y—  240  *  6  230  220  F i g . 18  Na* K*  K*  Mg**Ca**  Mg**Ca**  Mg**Ca**  rPf Ca*  NO IONS  Water content of r a t t a i l a r t e r i e s e q u i l i b r a t e d i n 1 of 5 isosmotic s o l u t i o n s of d i f f e r e n t i o n i c composition.  101 d i v a l e n t cations was associated with changes i n the hydration of the a r t e r y wall.  An inverse r e l a t i o n s h i p was also observed between the water content  and the i n t r a v a s c u l a r pressure. e f f i c i e n t was -0.59 (p < 0.001).  For a l l 60 a r t e r i e s , the c o r r e l a t i o n coI n a d d i t i o n , the i n t r a v a s c u l a r pressure  was d i r e c t l y r e l a t e d to the Ca + Mg content Ca content  ( r = 0.46, p < 0.001).  ( r = 0.40, p < 0.01) and to the  I t was noted that the proximal h a l f -  a r t e r i e s had smaller water contents than the d i s t a l halves i n a l l f i v e s o l u t i o n s , but the d i f f e r e n c e was only s i g n i f i c a n t (p < 0.01) f o r the a r t e r i e s i n s o l u t i o n 5. 3.  Discussion There are two p o s s i b l e explanations f o r the observed r e s u l t s which are  not mutually e x c l u s i v e . 1.  The d i f f e r e n t i o n i c composition of the s o l u t i o n s caused changes i n  the membrane p o t e n t i a l of the vascular smooth muscle c e l l s .  The c e l l s of  a r t e r i e s i n s o l u t i o n 2 (with no Na), compared to those i n s o l u t i o n 1 ( a l l i o n s ) , were contracted and probably depolarized (8,9 and c a l c u l a t i o n s from the Goldman equation).  This c o n s t r i c t i o n might thus e x p l a i n :  (a) the i n -  creased content of Ca, since Ca apparently enters the c o n t r a c t i n g smooth muscle c e l l (10), (b) the increased i n t r a v a s c u l a r pressure, and (c) the decreased H 2 O content associated w i t h c o n s t r i c t i o n (see Chapter I I I ) .  It  i s u n l i k e l y that a l l of the 1 meq increase i n t o t a l Ca between s o l u t i o n s 1 and 3 was i n t r a c e l l u l a r , since that would mean the c e l l Ca concentration had increased by about 6 m e q / l i t e r c e l l H 2 O — a s s u m i n g the c e l l s contained 2/3 of the t o t a l water.  I n a d d i t i o n , since the i n u l i n space decreases i n low Na  s o l u t i o n s (7), the increase i n c e l l Ca would be even l a r g e r .  However, even  a small increase i n i n t r a c e l l u l a r Ca could probably account f o r a l l of the constriction.  102 2.  Some of the gain i n a r t e r i a l Ca i n s o l u t i o n 2 could have been  bound e x t r a c e l l u l a r l y .  Using an i o n exchange process, P a l a t y e_t a l . found  that the sum of the increases i n the Ca and Mg a r t e r i a l contents i n the low Na s o l u t i o n s was approximately  equal to the amount of Na bound e x t r a -  c e l l u l a r l y — a b o u t 5 meq/100 g dry wt f o r the r a t t a i l a r t e r y (6).  I n the  present study, the t o t a l d i v a l e n t i o n increase between s o l u t i o n s 1 and 2 was 1.61 meq/100 g dry wt, w e l l w i t h i n the estimates of P a l a t y e_£ a l . (6) and others (1,4,11,12) of the e x t r a c e l l u l a r l y bound Na. This suggests that changes i n the p a r a c e l l u l a r matrix of the a r t e r y w a l l may have played a r o l e i n the changes i n Table XVIII.  These changes can be q u a l i t a t i v e l y ex-  plained by considering the p a r a c e l l u l a r matrix as an i o n exchanger. I n general, the s w e l l i n g of a given i o n exchange r e s i n i s dependent upon the degree of c r o s s - l i n k i n g i n the r e s i n , and upon the valency, s i z e and e x t e r n a l concentration of the counter-ion (13).  I f the counter-ions are mainly  monovalent, then the e x t r a c e l l u l a r matrix of the a r t e r y w a l l i s extended and r e l a t i v e l y s w o l l e n — a s were the a r t e r i e s i n s o l u t i o n s 1 and 5.  With d i v a l e n t  counter-ions, the degree of c r o s s - l i n k i n g i s increased so the matrix becomes t i g h t e r and the increased e l a s t i c forces cause the e x t r a c e l l u l a r matrix to shrink.  The same r e s u l t s have been shown f o r p o l y e l e c t r o l y t e gels  (14,15).  Bozler has found that the s t i f f n e s s and opacity of f r o g stomach muscle i n d i l u t e s o l u t i o n s of CaCl2 and MgCl2 are increased s t r o n g l y (16,17).  For  the r a t t a i l a r t e r i e s , the 20% decrease i n w a l l water content between s o l u t i o n s 1 and 3 may be p a r t l y due to an i o n exchange process that replaces monovalent ions w i t h d i v a l e n t ions as the counter-ions to the a n i o n i c groups of the protein-polysaccharide complexes i n the p a r a c e l l u l a r matrix.  The  increased i n t r a v a s c u l a r pressure f o r a r t e r i e s i n the d i v a l e n t s o l u t i o n s could be explained i f the i o n exchange process, which r e s u l t e d i n conformational  103 changes i n the p a r a c e l l u l a r matrix and hydration changes i n the a r t e r y w a l l , a l s o profoundly a f f e c t e d the p h y s i c a l response of the a r t e r y to p e r f u s i o n . These r e s u l t s cannot be compared to those f o r hypertensive  arteries  i n which an increase i n water content (18,19) i s associated w i t h an increase i n the e l a s t i c s t i f f n e s s (20), since i n hypertensive a r t e r i e s the polysaccharide matrix i s a c t u a l l y increased (21). 4.  Summary 1.  ionic  Rat t a i l a r t e r i e s were incubated i n isosmotic s o l u t i o n s of d i f f e r e n t  composition. 2.  Removing monovalent ions from the s o l u t i o n r e s u l t e d i n a greater  d i v a l e n t i o n content, a smaller water content and a greater i n t r a v a s c u l a r pressure of the a r t e r i e s . 3.  The water content of the a r t e r i e s was smaller when:  (a) the Ca  + Mg content was greater, and (b) the i n t r a v a s c u l a r pressure was greater. 4.  In a d d i t i o n to d e f i n i t e changes i n the tension of the v a s c u l a r  smooth muscle c e l l s , there may have been changes i n the p a r a c e l l u l a r matrix of the a r t e r y w a l l :  an i o n exchange process may have a l t e r e d the c o n f i g u r -  a t i o n of the matrix and the hydration of the w a l l .  B.  EFFECT OF COOLING AND REWAKMING  1.  On the Water and Ion Content of the Vascular Wall Cold acts as an i n h i b i t o r of c e l l u l a r metabolism.  Tissues cooled to  2°C f o r an extended period of time gain sodium and water and lose potassium (22).  I f the t i s s u e s are rewarmed to 37°C, the processes are reversed as  the metabolism of the c e l l i s r e a c t i v a t e d . The e f f e c t s of c o o l i n g and rewarming on sodium and potassium exchanges have been studied using many  104 smooth muscle t i s s u e s (23,24), i n c l u d i n g v a s c u l a r smooth muscle (5,25-29). Friedman et a l . found that the curve of the Na e f f l u x from a rewarmed a r t e r y could be divided i n t o 2 components:  a f a s t (complete w i t h i n 15 minutes of  rewarming) component, apparently unaccompanied  by K and unaffected by idodo-  acetate, and a slower metabolic component coupled 1:1 w i t h K.  They suggested  that t h i s temperature s e n s i t i v e f a s t Na e f f l u x represents the e f f e c t of temperature on e i t h e r Na bound to the anionic groups of the e x t r a c e l l u l a r matrix or Na l e a v i n g the smooth muscle c e l l s through channels independent of the Na-K pump—perhaps accompanied by CI. Although c o o l i n g and rewarming i s an a r t i f i c i a l procedure, i t has provided considerable information on the normal behavior of Na and K i n the artery wall.  I t might a l s o help i n understanding the behavior of water i n  the a r t e r y w a l l ; i n p a r t i c u l a r the e f f e c t of various f a c t o r s on v a s c u l a r hydration.  I n t h i s regard, a s e r i e s of experiments was  performed i n an  attempt to answer several questions: 1.  What i s the e f f e c t of temperature on the e x t r u s i o n of water from the rewarming artery?  2.  What e f f e c t does a sodium gradient between the a r t e r i a l w a l l and the rewarming s o l u t i o n have on t h i s e x t r u s i o n of water?  3.  I s there any r e l a t i o n s h i p between the water e x t r u s i o n and the f a s t non-K l i n k e d sodium extrusion?  Since water can not be studied using the isotope or flow-through electrodes techniques other workers have employed i n t h e i r cooling-rewarming e x p e r i ments, i t was decided to simply incubate the a r t e r i e s at various temperatures and determine t h e i r water contents from wet and dry weights. (a) Methods The r a t t a i l a r t e r i e s were cooled overnight a t 2°C, then incubated f o r  105 15 minutes i n s o l u t i o n s of d i f f e r e n t sodium concentration a t a given temperature between 2° and 37°C.  This procedure allowed the water and i o n content  of the a r t e r i e s to be determined at d i s t i n c t points during the rewarming process.  F i f t e e n minutes was chosen as the incubation time, rather than a  few hours, so the changes that occurred during the e x t r u s i o n of the non-K l i n k e d , temperature-sensitive sodium component would not be obscured by the e f f e c t of the slower metabolic exchanges of Na and K. The t a i l a r t e r i e s of the r a t s were excised a f t e r being flushed w i t h Krebs 140 s o l u t i o n (140 r e f e r s to the Na concentration i n m e q / l i t e r — s i m i l a r l y f o r the other s o l u t i o n s ) .  The a r t e r i e s were cut i n h a l f , placed i n f l a s k s of  Krebs 140 s o l u t i o n and kept i n the r e f r i g e r a t o r overnight at 2°C.  The a r t e r -  i e s were t r a n s f e r r e d to f r e s h Krebs 140 solutions,.aerated w i t h oxygen, and incubated f o r a f u r t h e r 2 hours a t 2°C.  F i f t e e n h a l f - a r t e r i e s were removed  f o r a n a l y s i s of water and ions a t t h i s point.  F i v e groups of 8 h a l f - a r t e r i e s  were then t r a n s f e r r e d to Krebs 140 s o l u t i o n s , aerated w i t h O2, a t 8 ° , 10°, 20°, 30°, or 37°C f o r 15 minutes of incubation and then analysed. Another group of 8 h a l f - a r t e r i e s was incubated f o r 3 hours a t 37°C so that the e f f e c t of the complete rewarming process could be determined.  The Na and K contents  of the a r t e r i e s were determined using a Techtron absorption spectrophotometer. To examine the e f f e c t s of the sodium gradient, a f t e r overnight c o o l i n g i n Krebs 140 s o l u t i o n , the a r t e r i e s were:  (a) t r a n s f e r r e d to Krebs 100  s o l u t i o n and the above procedure repeated using Krebs 100 throughout the r e warming, (b) t r a n s f e r r e d to Krebs 0 s o l u t i o n at 5°, 8 ° , 10°, or 37°C f o r 15 minutes.  (Solutions 1 and 2 from the previous s e c t i o n were used f o r the  Krebs 140 and Krebs 0 s o l u t i o n s i n t h i s experiment.) aerated w i t h oxygen.  The s o l u t i o n s were  Lactose replaced the NaCl i n these s o l u t i o n s .  106 (b)  Results  E f f e c t of Temperature The changes i n the a r t e r i a l water and i o n content during rewarming i n Krebs 140 s o l u t i o n are shown i n F i g s . 19 to 21 and Table XIX.  Arteries re-  warmed i n Krebs 140 s o l u t i o n d i d not l o s e a s i g n i f i c a n t amount of water during the 15 minute rewarming periods between 2° and 37°C, although they l o s t 34 ml water/100 g dry weight during the 3 hours a t 37°C. sequent water and i o n values w i l l be per 100 g dry weight.) l o s t Na and gained K when rewarmed i n Krebs 140 s o l u t i o n .  ( A l l the sub-  The a r t e r i e s  Since the a r t e r i e s  were e q u i l i b r a t e d f o r 2 hours, these changes were due s o l e l y to the e f f e c t of temperature. I n the 15 minute rewarming periods between 2° and 37°C, there was an increase of 2.86 meq K.  I t w i l l be assumed that t h i s increase  i n K was due to the e f f e c t of temperature on the metabolic Na-K pump of the vascular smooth muscle c e l l s . the  The a c t i v i t y of the pump increased w i t h  temperature, r e s u l t i n g i n a s l i g h t r e s t o r a t i o n of the i o n i c gradients  during these 15 minute rewarming periods. during these periods.  The a r t e r i e s l o s t 6.08 meq Na  The exchange of Na and K by the pump i n v a s c u l a r  smooth muscle c e l l s i s probably i n a 1:1 r a t i o (5,29).  The d i f f e r e n c e be-  tween the Na l o s t and the K gained shows there was 3.22 meq of extra Na l o s t from the a r t e r y w a l l between 2° and 37°C.  This extra l o s t Na was not accom-  panied by K, and was presumably independent of the metabolic Na-K pump, although q u i t e dependent upon the temperature. Table I I a l s o shows that most of the Na extruded from the a r t e r y w a l l during the 15 minute rewarming periods l e f t between 10° and 20°C, while most of the K returned between 20° and 30°C.  So, the l o s s of t h i s extra Na component was not only independent  of K, but a l s o occurs a t a lower temperature than the metabolic Na-K exchanges. During the 15 minute rewarming periods only 1/3 of the t o t a l regained K r e -  107 TABLE XIX.  Changes i n H 0 (ml/100 g dry wt), Na and K (meq/100 g dry wt) contents of r a t t a i l a r t e r i e s at d i f f e r e n t rewarming temperatures i n 3 s o l u t i o n s w i t h 140, 100, or 0 meq N a / l i t e r . A i s given when p < 0.05. 2  Temperature range  A H  2°  ANa  AK  Krebs 140 2° 10° 20° 30° Subtotal Total Krebs 100  Subtotal Total Krebs 0  Subtotal  * 8° to 20°C  to to to to  10° 20° 30° 37°  -10.1*  -0.739 +1.56 +2.40  2° to 37° 37° to 37°(3 hr)  -23.6  -6.08 -5.48  +2.86 +7.06  2° to 37°(3 hr)  -34.0  -11.6  +9.91  -15.5 +19.2 -26.1  -6.00  2° to 37° 37° to 37°(3 hr)  -19.4  -8.79 -6.09  +3.27 +5.24  2° to 37°(3 hr)  -30.2  -14.9  +8.50  -7.59  +1.86  -8.23  +2.53  2° 10° 20° 30°  to to to to  10° 20° 30° 37°  5° to 8° 8° to 10° 10° to 37°  -44.5  5° to 37°  -67.6  |+0.51 +2.15  108  380  K  360  340  |  o< O O  Krebs 0  320  S300 E O  CM  j  280  o 260 Krebs 100""-^240  220. 0 I 2 TIME (hrs) at 2 ° C  F i g . 19  10  i  i 20  TEMPERATURE (degrees C)  30  1 2 3 37 0 TIME (hrs) at 37° C  E f f e c t of temperature during rewarming on the 1^0 content of the r a t t a i l a r t e r y cooled f o r 18 hours a t 2 C. The a r t e r i e s were t r a n s f e r r e d from the s o l u t i o n a t 2°C to one of the s o l u t i o n s between 2°C and 37°C f o r 15 minutes of rewarming. Three rewarming s o l u t i o n s with d i f f e r e n t Na concentrations were used. Each point represents 8 a r t e r i e s except a t 2°C where n = 15.  109  60  55  50  •° 45  a>  O O  •I  <35  o  t31 14  £  -5.  Krebs 0  10  0 I 2 TIME (hrs) at 2 ° C  F i g . 20  10  20 TEMPERATURE (degrees C)  30  13. 37  0 1 2 3 TIME (hrs) at 37° C  E f f e c t of temperature during rewarming on the Na content of the r a t t a i l a r t e r y cooled f o r 18 hours a t 2°C. The a r t e r i e s were t r a n s f e r r e d from the s o l u t i o n a t 2°C to one of the s o l u t i o n s between 2° and 37°C f o r 15 minutes of rewarming. Three rewarming s o l u t i o n s with d i f f e r e n t Na concentrations were used. Each point represents 8 a r t e r i e s except at 2°C where n = 15.  i  110  25 • — • K r e b s 140 • — - • K r e b s 100 o o Krebs 0  £20 >.  T3  O 15 o CT  E 10 o. -Hi • -o  0 1 2 TIME (hrs) at2°C  F i g . 21  10  20 30 TEMPERATURE (degrees C)  37 0 1 2 3 TIME (hrs) at 37° C  E f f e c t of temperature during rewarming on the K content of the r a t t a i l a r t e r y cooled f o r 18 hours a t 2°C. The a r t e r i e s were t r a n s f e r r e d from the s o l u t i o n a t 2°C to one of t h e . s o l u t i o n s between 2° and 37°C f o r 15 minutes of rewarming. Three rewarming s o l u t i o n s with d i f f e r e n t Na concentrations were used. Each point represents 8 a r t e r i e s except at 2°C where n = 15.  turned, while 1/2 of the t o t a l extruded Na l e f t .  This i n d i c a t e s that the  Na-K pump requires the extended incubation period a t 37°C to become f u l l y operating.  I t a l s o i n d i c a t e s that the 15 minute rewarming periods at tem-  peratures between 2° and 37°C are not long enough periods of time f o r the metabolic Na-K pump to r e s t o r e the i o n i c gradients upset by overnight c o o l i n g . During the 15 minute rewarming periods i n Krebs 100 s o l u t i o n , the a r t e r i e s l o s t about 19 ml water/100 g dry wt. At 30°C, there was a s i g n i f i c a n t  Ill r e l a t i v e maximum, or "hump" i n the water content. (This maximum was a l s o present f o r a r t e r i e s i n Krebs 140 s o l u t i o n , but was not s i g n i f i c a n t . )  As shown  i n Table XIX, compared to changes i n the a r t e r i e s rewarmed i n Krebs 140 s o l u t i o n , the K changes were about the same, while there was more Na l o s t from a r t e r i e s i n Krebs 100  solution.  A r t e r i e s rewarmed i n Krebs 0 s o l u t i o n showed a dramatic l o s s of 45 ml water between 8° and 10°C.  Between the 15 minute incubations at 5°C and at  37°C, there was a l o s s of about 68 ml water. the r e l a t i v e maximum at 30°C.  No information was obtained  on  These a r t e r i e s a l s o gained about the same  amount of K and l o s t more Na than the a r t e r i e s rewarmed i n Krebs 140.  Because  there was no e q u i l i b r a t i o n period f o r these a r t e r i e s , the Na l o s s on rewarming may  simply represent Na extruded from the a r t e r y i n 0 Na s o l u t i o n .  E f f e c t of the E x t e r n a l Na  Concentration  The a r t e r i a l hydration was very dependent upon the temperature and t h i s temperature dependence was r a d i c a l l y a f f e c t e d by the e x t e r n a l Na concentration.  The 3 d i f f e r e n t i n i t i a l values of the water content i n F i g . 19 are  probably due to the d i f f e r e n t o s m o l a r i t i e s of the 3 s o l u t i o n s : Krebs 0, Krebs 140, 313; Krebs 100, 329 mosm/liter.  290;  The i n i t i a l values are i n v e r s e l y  r e l a t e d to the osmolarity as would be expected.  The temperature at which  most of the water was extruded from the rewarming a r t e r y was lower when the e x t e r n a l Na concentration was lower.  F i g . 22 shows that the amount of  water l o s t during the 15 minute rewarming periods (2° to 37°C) was when the e x t e r n a l Na concentration was lower.  Since the Na-K  greater  pump was  only  minimally e f f e c t i v e during these short rewarming periods, t h i s suggests that some other mechanism was mainly responsible f o r the changes i n the hydration of the rewarming a r t e r y w a l l .  112 The d i f f e r e n t i n i t i a l values of the Na content f o r a r t e r i e s i n the 3 s o l u t i o n s i n F i g . 20 simply r e f l e c t the d i f f e r e n t Na concentrations of t h e i r bathing media.  The greater l o s s of a r t e r i a l Na when the e x t e r n a l Na concen-  t r a t i o n was lower w i l l be discussed below.  There does not seem to be any  e f f e c t of e x t e r n a l Na concentration on the a c t i v i t y or the temperature of operation of the metabolic Na-K pump.  The K gained between 2° and 37°C i n  15 minute rewarming periods was e s s e n t i a l l y the same f o r a l l 3 s o l u t i o n s . There was no r e a l d i f f e r e n c e  between the percentages of the t o t a l Na and K  movements during the 15 minute rewarming periods i n Krebs 140 and Krebs 100 solutions.  Nor d i d the e x t e r n a l Na concentration a f f e c t the temperature a t  which the Na-K pump "came i n t o a c t i o n " . E x t r u s i o n of the Non-K l i n k e d Na Component The e x t e r n a l Na concentration a f f e c t e d the e x t r u s i o n of the e x t r a non-K l i n k e d Na component.  I t was t h i s e f f e c t that r e s u l t e d i n the d i f f e r e n t  losses of Na i n the 3 s o l u t i o n s , and not any e f f e c t on the Na-K pump.  The  s i z e of t h i s e x t r a Na component released during the 15 minute rewarming periods between 2° and 37°C i s presumably the d i f f e r e n c e between the Na l o s t and the K gained i n the i n t e r v a l (see Table XIX):  Krebs 140 s o l u t i o n :  6.08 - 2.86 = 3.22 meq Na; Krebs 100 s o l u t i o n : 8.79 - 3.27 = 5.52 meq Na; Krebs 0 s o l u t i o n : 8.23 - 2.53 = 5.70 meq Na.  However, the true s i z e of t h i s  extra Na component i s smaller than these values because of the accompanying e x t r u s i o n of water from the rewarmed a r t e r y w a l l .  Some of t h i s extruded  water probably came from the free e x t r a c e l l u l a r space (ECS) which i s i n e q u i l i b r i u m w i t h the e x t e r n a l s o l u t i o n and consequently has the same Na concentration.  I n u l i n space measurements were not done, so the amount of  water that came from the free ECS i s not known.  For the purpose of c a l c u -  l a t i o n , suppose that a l l of the extruded water came from the free ECS.  113 Subtraction of the amount of Na that would have been c a r r i e d out with t h i s water w i l l give the maximum c o r r e c t i o n to the s i z e of the e x t r a non-K l i n k e d Na component.  The values thus obtained w i l l be the minimum amounts, i . e .  the amounts of Na released, unaccompanied by K, from compartments of the rewarming a r t e r y other than the f r e e ECS.  The above uncorrected  the maximum amounts, i . e . they include Na l o s t from the f r e e Krebs 140 s o l u t i o n ;  values  are  ECS.  The n o n - s i g n i f i c a n t decrease i n the a r t e r i a l water  content of 284 - 272 = 12 ml water between 2° and 37°C (see F i g . 19) w i l l considered  as a r e a l decrease f o r these rough c a l c u l a t i o n s . Twelve ml of f r e e  ECS water would have c a r r i e d out 0.14 0.06  meq  K.  6.08  - 1.68  x 12 = 1.68  - 2.86  - 0.06  = 1.43  meq  Na and 0.005 x 12 =  Na.  The 19.4 ml of l o s t water would have c a r r i e d out  Na and 0.097 meq K from the free ECS.  was 5.52  meq  The minimum s i z e of the e x t r a Na component would have been  Krebs 100 s o l u t i o n : meq  be  - 1.94  - 0.097 = 3.48 meq  Krebs 0 s o l u t i o n :  1.94  The minimum s i z e of the e x t r a Na  Na.  Since i t i s the minimum s i z e of the e x t r a Na component  that i s of i n t e r e s t , assume that the maximum trace Na i n t h i s s o l u t i o n was 2 meq N a / l i t e r . was  8.23  The minimum amount of Na i n the e x t r a component released  - (0.002 x 67.6)  - 2.53  - (0.005 x 67.6)  = 5.23  meq  Na.  These minimum and maximum values of the e x t r a non-K l i n k e d Na component released during rewarming are shown i n F i g . 22. I t i s i n t e r e s t i n g to note that the s i z e of the e x t r a Na component extruded i n Krebs 0 s o l u t i o n , 5.23  to 5.70  meq  Na/100 g dry weight, i s about  the same s i z e as Palaty's estimate of Na bound to the a n i o n i c s i t e s i n the p a r a c e l l u l a r matrix of the r a t t a i l a r t e r y ( 6 ) — a l t h o u g h , experimental  procedure was q u i t e d i f f e r e n t .  of course, t h e i r  114  F i g . 22  E f f e c t of e x t e r n a l Na concentration, [ N a ] , on the change i n water content, AH2O, and on the s i z e of the e x t r a , non-K l i n k e d Na component extruded from the r a t t a i l a r t e r y during 15 minute rewarming periods between 2° and 37°C. The m i n i mum e x t r a Na component i s the d i f f e r e n c e between the measured maximum e x t r a Na component and the Na c a r r i e d out i f a l l the extruded water were from the free e x t r a c e l l u l a r space. +  0  (c) Discussion The hydration changes during rewarming have to be explained i n terms of:  (a) the o v e r a l l water decrease, (b) the peak a t 30°C, and (c) the e f f e c t  of e x t e r n a l Na.  115 The metabolic Na-K pump i s not a s a t i s f a c t o r y mechanism to e x p l a i n these changes.  C e r t a i n l y the pump i s responsible f o r removing Na and water  from and r e s t o r i n g K t o the vascular smooth muscle c e l l s during rewarming. But 15 minute rewarming periods were not long enough f o r s u b s t a n t i a l pump a c t i vation.  D i s c o n t i n u i t i e s have been observed (30,31; also see 32) i n the  Arrhenius p l o t of the a c t i v i t y of the ATPase system presumably associated w i t h the Na-K pump (33) . But a t 30°C, there was not a simple change of the rate of water decrease, but an increase. a t i n g i n reverse. at 30°C.  This would imply that the pump was oper-  But, there was no corresponding decrease i n the K content  And f i n a l l y , the e x t e r n a l Na concentration d i d not a f f e c t the  Na-K pump during the 15 minute rewarming periods, although i t d r a s t i c a l l y a f f e c t e d the water l o s t from the rewarming a r t e r y . A clue f o r an explanation of the water changes may l i e i n a r e l a t i o n ship between the water and the Na content of the a r t e r y w a l l .  Both the  amount of water and the amount of e x t r a non-K l i n e d Na extruded from the rewarming a r t e r y v a r i e d i n v e r s e l y w i t h the e x t e r n a l Na concentration. I n a d d i t i o n , there were r e l a t i v e maxima i n both the Na content (although riot s i g n i f i c a n t ) and the water content a t 30°C.  There are 3 p o s s i b l e explana-  t i o n s of the o v e r a l l water decrease during the 15 minute rewarming periods which are r e l a t e d to t h i s f a s t non-K l i n k e d Na component: 1.  Channels i n the smooth muscle membranes, not connected to the  Na-K pump, might provide the mechanism of e x t r u s i o n of t h i s e x t r a Na component, perhaps accompanied by CI (29,34). The decreased water content of the a r t e r y w a l l could be due to a decrease i n the c e l l water accompanying the l o s s of c e l l u l a r NaCl to maintain c e l l u l a r osmolarity.  This NaCl l o s s might  be the r e s u l t of temperature dependent changes i n the s t a t e of the c o n t r a c t i l e proteins.  116 2.  An e l e c t r o g e n i c pump which operated between 10° and 20°C and  pumped out c e l l u l a r Na and water without the e x t r u s i o n of CI or the entry of K would e x p l a i n both the Na and water losses during rewarming.  This concept  i s supported by the membrane h y p e r p o l a r i z a t i o n observed by Taylor e_t al. (35) at the s t a r t of rewarming of the pregnant r a t uterus.  Upon immersion i n t o  s o l u t i o n at 38°C, precooled r a t l i v e r s l i c e s showed an immediate l o s s of Na and Ca, w h i l e the gain i n K was delayed 10 to 15 minutes (36).  I t was sug-  gested that the high c e l l Ca concentration decreased the membrane permeability to K.  Ca moving out of the c e l l s could be the source of the h y p e r p o l a r i z a -  tion.  Presumably there would be a l o s s of c e l l water accompanying  the Na  and Ca l o s s e s . 3.  I t has a l s o been suggested that the e x t r u s i o n of the f a s t Na com-  ponent during rewarming might represent an e f f e c t of temperature on i o n binding to anionic groups i n the p a r a c e l l u l a r matrix (5). The r e l e a s e of Na from the binding s i t e s might be accompanied by the binding of d i v a l e n t ions. Such an exchange would be accompanied by conformational changes i n the matrix r e s u l t i n g i n the expulsion of water from the e x t r a c e l l u l a r space. be noted that P a l a t y et_ a l . (7)  I t should  found a p o s i t i v e c o r r e l a t i o n between the  water content of the r a t t a i l a r t e r y and the amount of Na bound to the protein-polysaccharide complexes. In each of these cases, lowering the e x t e r n a l Na concentration would augment the removal of the extra non-K l i n k e d Na from the rewarming a r t e r y wall.  The presence of the peak i n water content at 30°C might be explained  by a r e v e r s a l , f o r reasons unknown, of these processes. There i s another p o s s i b l e source of the hydration changes during r e warming.  There may be anomalies i n the temperature dependence of the p h y s i c a l  117 p r o p e r t i e s of water (37-42, compare 43).  A l s o , i t i s known that "orthowater,"  grown i n c a p i l l a r y tubes and found at water-quartz i n t e r f a c e s has d i f f e r e n t p h y s i c a l p r o p e r t i e s than normal water (44). At 2°C, the water s t r u c t u r e i n the immediate v i c i n i t y of macromolecules, i n s i d e and outside the smooth muscle c e l l s , might be d i f f e r e n t than the water s t r u c t u r e i n more open spaces. I t i s also p o s s i b l e that rewarming a f f e c t s water next to macromolecules i n a d i f f e r e n t manner than water i n the open spaces.  I t could be speculated that  a l t e r a t i o n s i n the amount of water able to act as solvent, due to unequal s t r u c t u r a l changes i n the w a l l water during rewarming, could r e s u l t i n osmotic imbalances that produce the observed hydration changes.  Or, s t r u c t u r a l  changes i n the water during rewarming could cause conformational changes i n the matrix causing i t to shrink and s w e l l at various temperatures. (d) Summary 1.  The incubated rewarmed r a t t a i l a r t e r y gained K, and l o s t Na and  2.  There was only a s l i g h t gain of K during the 15 minute rewarming  water.  periods between 2° and 37°C.  This gain and the temperature at which i t  occurred were not a f f e c t e d by the e x t e r n a l Na concentration.  This suggests  that during these 15 minute rewarming periods the c e l l u l a r metabolic Na-K pump was:  (a) not f u l l y operating, and (b) not a f f e c t e d by the e x t e r n a l Na  concentration. 3.  The a r t e r y l o s t more Na than i t gained K during rewarming.  If a  1:1 exchange i s postulated f o r the Na-K pump, the d i f f e r e n c e represents an extra Na component extruded at lower temperatures than the metabolic Na-K exchanges.  The, s i z e of t h i s extra non-K l i n k e d Na component increased w i t h  decreasing e x t e r n a l Na.  118 4.  The amount of water extruded during rewarming was greater and  the  temperature at which most of the l o s s occurred was lower when the e x t e r n a l Na concentration was lower.  There was an increase i n the water content at  30°C. 5.  The Na-K  pump cannot f u l l y e x p l a i n these changes i n the hydration  of the rewarming a r t e r y .  The water changes may be r e l a t e d to the e x t r u s i o n  of the f a s t non-K l i n k e d Na component. 2.  On the I n t r a v a s c u l a r Pressure In some of the c o o l i n g and rewarming experiments p r e v i o u s l y mentioned,  the e f f e c t s of vascular tension and the response to some vasoactive agents have been examined (25,28).  Barr found that s p i r a l s t r i p s of the dog c a r o t i d  a r t e r y contracted when they were placed i n s o l u t i o n s at 37°C, then progress i v e l y relaxed as the i n t r a c e l l u l a r K increased during the rewarming process (25) . On the other hand, Friedman e_t a l . (28) found that slower rewarming of the r a t t a i l a r t e r y r e s u l t e d i n a r e l a x a t i o n which began w e l l before there were s i g n i f i c a n t movements of c e l l u l a r Na and K.  They suggested that the  temperature-sensitive tension changes were caused by changes i n the membrane permeability f o r Na or K, or by changes i n the e l a s t i c p r o p e r t i e s of the e x t r a c e l l u l a r matrix of the a r t e r y w a l l . Tobian (16) has suggested that the increased blood pressure i n hypertensive a r t e r i e s could be due to waterlogging of the a r t e r y w a l l .  In view  of t h i s suggestion and the observed changes i n the water content of the r e warming a r t e r y , i t seemed that a study of tension changes during rewarming might support these observations, and more important, provide some information  i on the r e l a t i o n s h i p between a r t e r i a l tension and a r t e r i a l hydration.  There  were s e v e r a l questions to be examined experimentally using the i n t r a v a s c u l a r pressure as the measure of a r t e r i a l tension:  119 1.  What i s the e f f e c t of temperature on the i n t r a v a s c u l a r pressure of  a rewarming artery? 2.  What i s the e f f e c t of the e x t e r n a l Na concentration on the i n t r a -  vascular pressure during rewarming? 3.  I s there any r e l a t i o n s h i p between the h y d r a t i o n of the a r t e r y w a l l  and the i n t r a v a s c u l a r pressure during rewarming? (a) Methods This experiment involved simultaneous monitoring of the i n t r a v a s c u l a r pressure and the temperature during rewarming.  A 12 cm segment of the r a t  v e n t r a l t a i l a r t e r y was exposed, i t s c o l l a t e r a l s t i e d , and both ends cannulated.  Removed from the t a i l bed, the a r t e r y was tested f o r l e a k s , placed i n  a t h i n channel i n a perspex chamber (the bottom of which was part of a c i r c u l a t i n g temperature-control system), f i l l e d w i t h Krebs 150 s o l u t i o n , and placed i n the r e f r i g e r a t o r at 2°C overnight (about 18 hours). During r e warming the artery was connected to a r o t a r y pump f o r open c i r c u i t p e r f u s i o n . By passing the perfusing s o l u t i o n through tubing i n the bottom of the a r t e r y chamber before i t entered the a r t e r y , i t was always a t the same temperature as the chamber and the a r t e r y .  The temperature was monitored w i t h a thermo-  couple i n the a r t e r y channel and the i n t r a v a s c u l a r pressure was monitored proximal to the a r t e r y w i t h a Statham transducer. The cooled a r t e r y was rewarmed: the  (a) q u i c k l y by allowing water from a bath at 40°C to pass through  chamber, (b) i n steps of 1 or 2 degrees every 3 or 4 minutes—about 30  seconds was required f o r each step, (c) i n steps of 10°C every 20 to 90 m i n u t e s — f r o m 3 to 15 minutes (depending upon the type of pump) were required for  the 10° increase.  During rewarming, 9 a r t e r i e s were perfused w i t h Krebs  100 s o l u t i o n and 4 w i t h Krebs 150 s o l u t i o n .  120 (b) Results The patterns of i n t r a v a s c u l a r pressure changes during rewarming w i t h Krebs 100 s o l u t i o n are shown i n F i g s . 23 to 25. pattern f o r a f a s t rewarming.  F i g . 23 shows the pressure  There are 3 features to n o t i c e :  (a) the  s l i g h t dip i n pressure near the s t a r t of the rewarming, (b) the increase i n pressure u n t i l about 30°C, and (c) the l a t e r decrease i n pressure to a l e v e l at 37°C below that before rewarming. almost a l l the a r t e r i e s .  These 3 changes were observed f o r  Since there was probably a l a g between the tempera-  ture of the a r t e r y and that on the record during t h i s f a s t rewarming, step increases i n temperature were required to determine the exact temperatures at which the pressure changes were o c c u r r i n g .  F i g . 24 shows the pressure  pattern f o r an a r t e r y e q u i l i b r a t e d f o r 1 hour then rewarmed i n steps of 1 or 2 degrees every 3 or 4 minutes.  The p a t t e r n i s the same as that i n F i g . 23  except that the dip and the increase i n pressure occur at lower temperatures. Two  separate increases i n pressure can be e l i c i t e d , as shown i n F i g . 25, i f  the a r t e r y i s kept at 11°C 21°C.  f o r only about 30 minutes before being r a i s e d to  F i g . 25 i s f o r an a r t e r y rewarmed i n Krebs 100 s o l u t i o n containing  -3 10  M iodoacetate.  E x a c t l y the same p a t t e r n was obtained f o r an a r t e r y  (not shown) rewarmed without iodoacetate f o r the same time periods.  This  i n d i c a t e s that iodoacetate, which blocks ATP production under these condit i o n s i n the smooth muscle c e l l s , has no e f f e c t on the p a t t e r n of i n t r a vascular pressure during rewarming.  (Of course, there may have been s u f f i -  c i e n t ATP stored i n the c e l l s to render the a c t i o n of the iodoacetate inconsequential.)  The pressure changes f o r a r t e r i e s rewarmed with Krebs 150  s o l u t i o n were much smaller than those f o r the a r t e r i e s rewarmed with Krebs 100  solution.  121  4 0 T E M P E R A T U R E (°C) r  20  I 0 0 P R E S S U R E mm Hg r  50-  TIME  F i g . 23  Temperature and i n t r a v a s c u l a r pressure during rewarming. Rat t a i l a r t e r i e s were cooled f o r 18 hours a t 2°C i n Krebs 150 s o l u t i o n , then rewarmed during perfusion w i t h Krebs 100 s o l u t i o n at 0.2 ml/min.  The average changes i n the i n t r a v a s c u l a r pressure were as f o l l o w s : Dips:  12/13 a r t e r i e s had s l i g h t dips i n the pressure below 10°C.  There was  no d i f f e r e n c e between the s i z e or the temperature a t which the dip occurred f o r the Krebs 100 or Krebs 150 s o l u t i o n s .  The average pressure change i n the  dip was -2.5 mm Hg, a t an average temperature of 8°C. Peaks: 30°C.  11/13 a r t e r i e s had increases i n the i n t r a v a s c u l a r pressure below The average pressure changes were:  Krebs 100, +44 mm Hg, and Krebs  150, +4 mm Hg. O v e r a l l changes: pressure.  12/13 a r t e r i e s had an o v e r a l l decrease i n the i n t r a v a s c u l a r  The average o v e r a l l changes were:  Krebs 150, -4 mm Hg.  Krebs 100, -10 mm Hg, and  122  F i g . 24  E f f e c t of time and temperature on i n t r a v a s c u l a r pressure during rewarming. Rat t a i l a r t e r i e s were cooled f o r 18 hours at 2°C i n Krebs 150 s o l u t i o n , then rewarmed during p e r f u s i o n with Krebs 100 s o l u t i o n at 0.2 ml/min.  These r e s u l t s show that the temperature dependence of the i n t r a v a s c u l a r pressure during rewarming was a f f e c t e d by the e x t e r n a l Na concentration.  The  o v e r a l l decrease i n the pressure and the pressure peak at 20° to 25°C were both greater when the e x t e r n a l Na concentration was smaller.  This was so even  though a r t e r i e s rewarmed i n Krebs 100 s o l u t i o n were e q u i l i b r a t e d i n Krebs 100  123  TIME (minutes)  F i g . 25  I n t r a v a s c u l a r pressure a t d i f f e r e n t temperatures during rewarming. Rat t a i l a r t e r i e s were cooled f o r 18 hours a t 2°C i n Krebs 150 s o l u t i o n , then rewarmed during p e r f u s i o n w i t h Krebs 100 s o l u t i o n containing 10""-* M iodoacetate, a t 0.2 ml/min.  s o l u t i o n f o r about 1 hour before rewarming.  The e x t e r n a l Na concentration  did not seem to a f f e c t the s i z e of the s l i g h t dip i n the pressure, or the temperature a t which i t occurred.  124 (c) Discussion During rewarming of the a r t e r y , the c e l l u l a r Na-K pump restores the Na and K gradients and the a r t e r y r e l a x e s .  I t was thus expected that the  i n t r a v a s c u l a r pressure of the perfused a r t e r y would decrease between 2° and 37°C.  However, since the a r t e r i e s were never a t temperatures c l o s e to 37°C  for very long, the pump was never f u l l y operating.  I n a d d i t i o n , the presence  of iodoacetate d i d not a f f e c t the o v e r a l l decrease.  Friedman eX a l . (28)  a l s o noticed that the o v e r a l l r e l a x a t i o n of the a r t e r y began w e l l before there were movements of c e l l u l a r Na or K.  For these perfused a r t e r i e s , no  ion measurements were made so the a c t i v i t y of the Na-K pump was not known. However, i t seems that the metabolic Na-K pump does not f u l l y e x p l a i n the o v e r a l l decrease i n the i n t r a v a s c u l a r pressure.  The peak i n the pressure a t  20°C to 25°C cannot be explained a t a l l i n terms of the Na-K pump. The s i z e and shape of the peak pressure were q u i t e dependent upon the length of time the a r t e r y remained a t a given temperature.  This might suggest the i n v o l v e -  ment of the Na-K pump since i t s a c t i v i t y i s l e s s a t lower temperatures so i t requires longer times t o r e s t o r e the i o n i c gradients.  But: (a) the r e s t o r -  a t i o n of the Na and K gradients relaxes the a r t e r y , i . e . decreases the i n t r a vascular pressure, not increases i t , and (b) iodoacetate d i d not a f f e c t the s i z e and shape of the pressure peak.  I t i s p o s s i b l e that the e x t r u s i o n or  storage of c e l l u l a r Ca during rewarming might play a r o l e i n the o v e r a l l r e l a x a t i o n and/or the pressure peak of the rewarming a r t e r y . There seems to be q u i t e a c l o s e l i n k between the pressure changes f o r the perfused rewarming:  a r t e r y and the hydration changes of the incubated  a r t e r y during  (a) Both the pressure and the water content decreased o v e r a l l  125 between 2° and 37°C, and both increased between 20° changes i n the pressure and hydration were increased Na concentration.  and 30°C, (b) Both these by lowering  Tobian (16) suggested that the increased  the  external  intravascular  pressure associated with hypertension might be explained by an increase i n the hydration of the a r t e r y w a l l and a r e s u l t a n t t h i c k e r w a l l and lumen.  smaller  The same phenomenon might p a r t l y e x p l a i n the o v e r a l l decrease and  peak i n the i n t r a v a s c u l a r pressure during rewarming.  Rough c a l c u l a t i o n s  support t h i s suggestion: From P o i s e u i l l e ' s equation, with constant v i s c o s i t y , length and P^/P^  = (V^/V^) where:  flow:  P = pressure gradient down the a r t e r y seg-  2  ment, V = lumen volume, and states 1 and 2 r e f e r to the a r t e r y before and a f t e r the changes i n w a l l water content r e s p e c t i v e l y . Knowing P-^ from the pressure recordings mating V-j^ and V~2 enables  during rewarming and  esti-  the pressure gradient a f t e r the w a l l  water change, to be c a l c u l a t e d .  V-^ was  c a l c u l a t e d from either:,  (a) P o i s e u i l l e ' s equation (knowing P-^ and the flow, and  estimating  the a r t e r y length and v i s c o s i t y of the Krebs s o l u t i o n ) , or (b) the inner radius values i n Table I i n Chapter I I . determined from  - V^,  V"2  was  the change i n lumen volume associated  with the changes i n a r t e r y w a l l volume r e s u l t i n g from the w a l l water changes.  I t was  assumed f o r the c a l c u l a t i o n s that  the  changes i n water content of the rewarmed perfused a r t e r y were the same as those determined i n the above s e c t i o n f o r the rewarmed incubated a r t e r y .  The lumen volume changes were c a l c u l a t e d from  the w a l l volume changes by assuming e i t h e r : increase was  1/2  the s i z e of the w a l l volume decrease, or (b)  increase i n inner radius was decrease.  (a) the lumen volume  the same s i z e as the outer radius  the  126 The o v e r a l l decrease i n the hydration of the rewarming a r t e r y could account for 80% of the o v e r a l l decrease i n the i n t r a v a s c u l a r pressure.  The increase  i n the water content a t 30°C could account f o r about 30% of the observed increase i n the pressure a t 20°C to 25°C. These estimates, as w e l l as the c o r r e l a t i o n between t h e i r temperature dependencies and e x t e r n a l Na concent r a t i o n dependencies, i n d i c a t e that the i n t r a v a s c u l a r pressure changes can be a t l e a s t p a r t i a l l y explained i n terms of the hydration changes of the rewarming a r t e r y . There i s no comparable c o r r e l a t i o n between the d i p i n pressure a t 8°C and the water content.  The dip may have been part of the o v e r a l l pressure  decrease and only appeared because of the subsequent peak i n the i n t r a v a s c u l a r pressure.  I t i s a l s o p o s s i b l e that the pressure d i p was simply due to the  a r t e r y w a l l volume being smallest a t 4°C when the density of water i s greatest. (d) Summary 1.  The perfused rewarmed r a t t a i l a r t e r y showed:  (a) a s l i g h t d i p  i n the i n t r a v a s c u l a r pressure at 8°C, (b) a peak i n the pressure a t 20° to 25°C, and (c) an o v e r a l l decrease i n the pressure between 2° and 37°C. 2.  When the e x t e r n a l Na concentration was lower:  (a) the d i p i n  the pressure was not affected,(b) the peak i n the pressure was greater, and (c) the o v e r a l l decrease i n the i n t r a v a s c u l a r pressure was greater. 3.  The shape and s i z e of the pressure peak a t 20° to 25°C was s t r o n g l y  a f f e c t e d by the length of time the a r t e r y was kept a t a given  temperature.  4. The c e l l u l a r metabolic Na-K pump cannot e x p l a i n the increase i n pressure a t 20°C to 25°C, and can only p a r t l y e x p l a i n the o v e r a l l decrease i n the i n t r a v a s c u l a r pressure.  127 5.  There seems to be a l i n k between the i n t r a v a s c u l a r pressure  changes and the hydration changes during rewarming.  Both have the same  temperature and e x t e r n a l Na concentration dependencies. 6.  On the b a s i s of a smaller a r t e r y w a l l and a l a r g e r lumen, the  o v e r a l l decrease of w a l l water could account f o r most of the o v e r a l l decrease i n the i n t r a v a s c u l a r pressure during rewarming.  The increased water content  at 30°C could account f o r only part of the increased i n t r a v a s c u l a r pressure at 20° to 25°C.  128 CHAPTER VI SUMMARY AND  1. The  DISCUSSION  E x t r a c e l l u l a r f l u i d i s expelled from the c o n s t r i c t i n g a r t e r y w a l l .  f o l l o w i n g points support t h i s conclusion: (a) Constricted a r t e r i e s l o s t 16% of t h e i r water, 15% of t h e i r Na, and 17% of t h e i r CI. (b) C o n s t r i c t e d a r t e r i e s had smaller i n u l i n spaces. (c) Constricted a r t e r i e s had a smaller w a l l c r o s s - s e c t i o n a l area than non-constricted  arteries.  Constant length meant the  volume of the c o n s t r i c t i n g a r t e r y w a l l decreased by  14%—  compared to 13% c a l c u l a t e d from the w a l l water l o s s . (d) The flow pattern of a c o n s t r i c t e d a r t e r y suggested that f l u i d was 2.  added to the lumen from the c o n s t r i c t i n g w a l l .  Contracting  smooth muscle c e l l s increase t h e i r volume due to the  entry of water. 3.  The a r t e r y w a l l i s permeable to f l u i d s .  increased by an increase i n i n t r a v a s c u l a r pressure.  The permeability  is  Vasoconstriction  also  increases the permeability with or without a short l i v e d i n i t i a l decrease. 4.  Isosmotic a l t e r a t i o n s i n e x t e r n a l i o n composition a l t e r the  hydration of the a r t e r y w a l l presumably by i o n exchange and/or v a s o c o n s t r i c t i o n . 5.  The hydration of rewarming a r t e r i e s depends on the temperature and  the e x t e r n a l Na concentration. (a) may and  The extrusion of w a l l water during rewarming:  be r e l a t e d to the extrusion of a f a s t non-K l i n k e d Na component,  (b) p a r t l y explains the i n t r a v a s c u l a r pressure changes during rewarming.  129 These studies have demonstrated that c o n s t r i c t i o n of the r a t t a i l a r t e r y i s associated with a l o s s of water, a change i n w a l l permeability, and an increase i n smooth muscle c e l l volume.  How do these changes occur?  Are there causal r e l a t i o n s h i p s between these 3 e f f e c t s of v a s o c o n s t r i c t i o n ? What are the consequences of these a l t e r a t i o n s ? Although no d e f i n i t e answers can be given, some p o s s i b l e explanations w i l l be examined.  A.  LOSS OF WALL WATER DURING VASOCONSTRICTION The l o s s of water from the c o n s t r i c t i n g r a t t a i l a r t e r y was due to  the expulsion of e x t r a c e l l u l a r f l u i d .  S i m i l a r losses were observed f o r  c o n t r a c t i n g gels (1,2), muscle homogenates (3), f r o g stomach muscle (4,5), u t e r i n e muscle (6,7), r a t aorta (8), and c a r o t i d a r t e r i e s (9).  To understand  why t h i s water l o s s occurred, i t may be u s e f u l to extend Bozler's explanation of f r o g stomach muscle behavior  to the a r t e r y w a l l :  i t behaves " l i k e a  c r o s s - l i n k e d g e l i n which osmotic balance i s determined i n part by h y d r o s t a t i c pressure a r i s i n g from e l a s t i c forces w i t h i n the f i b e r s " (4). Water moves down i t s chemical p o t e n t i a l gradient.  A movement of water  i n the absence of g r a v i t a t i o n a l f o r c e s , i n d i c a t e s the presence of osmotic or h y d r o s t a t i c pressure gradients.  I t i s d i f f i c u l t to imagine why ions or other  molecules would leave the a r t e r y w a l l f i r s t , causing a subsequent water s h i f t to balance osmolarity.  In a d d i t i o n , the f a c t that the f l u i d which l e f t the  c o n s t r i c t i n g a r t e r y was not too hypotonic probably not i n v o l v e d .  suggests that osmotic forces were  I f t h i s were so, then how could c o n s t r i c t i o n be  associated with a h y d r o s t a t i c pressure d i f f e r e n c e f o r c i n g water from the artery wall?  To attempt to answer t h i s question, the forces that  normally  determine the a r t e r i a l water content w i l l f i r s t be considered; then how v a s o c o n s t r i c t i o n could a l t e r these f o r c e s .  130 When the a r t e r i a l h y d r a t i o n i s i n e q u i l i b r i u m ,  the osmotic and  h y d r o s t a t i c forces determining the h y d r a t i o n w i l l be balanced. involved are e s s e n t i a l l y those f o r c a p i l l a r y f i l t r a t i o n .  The forces  There must be a  balance between the osmotic pressure of the blood and the h y d r o s t a t i c pressure of the a r t e r y w a l l f l u i d on one hand, and the osmotic pressure of the w a l l f l u i d and the h y d r o s t a t i c pressure of the p e r f u s i n g blood on the other.  There must be a s i m i l a r e q u i l i b r i u m between the w a l l and the surroun-  ding t i s s u e s :  the t i s s u e osmotic and the w a l l h y d r o s t a t i c pressures must  balance the w a l l osmotic and the t i s s u e h y d r o s t a t i c pressures.  The c o n s i d -  e r a t i o n of these 2 balance sheets leads to some i n t e r e s t i n g conclusions. The only complex f a c t o r i s the h y d r o s t a t i c pressure i n the a r t e r y wall.  This pressure i s a consequence of the tension i n the w a l l , a c t i v e  and e l a s t i c , which balances the transmural pressure (10). For a g e l or an ion exchange r e s i n t h i s r e l a t i o n s h i p can be e a s i l y seen.  As the r e s i n  s w e l l s , the e l a s t i c forces between the components of i t s matrix increase u n t i l the r e s u l t a n t h y d r o s t a t i c pressure i n the r e s i n balances the pressure i n the surroundings and e q u i l i b r i u m i s a t t a i n e d (11,12).  In the a r t e r y w a l l  the h y d r o s t a t i c pressure i s a complex f u n c t i o n of the w a l l tension (13). E s s e n t i a l l y , the t i s s u e pressure decreases from the l e v e l of the i n t r a vascular pressure to that i n the t i s s u e s surrounding the a r t e r y .  [The c l a i m  that the i n t e r s t i t i a l f l u i d pressure i s negative (14,15) has been f a i r l y w e l l disputed (16-19).]  An increase i n t i s s u e pressure from the a d v e n t i t i a l  to the i n t i m a l layers of the aorta was observed by Brinkman et a l . (20). This t i s s u e pressure gradient apparently determines the extent of p e n e t r a t i o n of vasa vasorum (21) and the d i r e c t i o n of net transport of ionsi across the vascular w a l l (21,22).  131 To prevent water movements i n the presence of the t i s s u e pressure gradient across the a r t e r y w a l l , there must a l s o be an osmotic gradient across the w a l l .  These gradients are shown i n F i g . 26.  For a steady s t a t e  of the water i n the w a l l , lumen and surrounding t i s s u e s , the s i t u a t i o n might resemble the f o l l o w i n g .  I f i t i s assumed that the e n d o t h e l i a l l a y e r i s f r e e l y  permeable to ions and small molecules but not to the plasma p r o t e i n s , then the osmotic pressure i n the lumen i s about 25 mm Hg higher than that i n the w a l l . (The vascular smooth muscle c e l l s w i l l be temporarily ignored.)  To prevent  water l e a v i n g the a r t e r y w a l l there must be a lower t i s s u e pressure i n the innermost l a y e r of the w a l l .  For an i n t r a v a s c u l a r pressure of 90 mm  the t i s s u e pressure need only be 65 mm Hg j u s t i n s i d e the w a l l .  Hg,  [This p o i n t  was ignored i n Burton's c o n s i d e r a t i o n of w a l l t i s s u e pressure (13).] I f , i n the a r t e r y w a l l f l u i d there are s o l u b l e molecules which cannot enter the lumen, then the d i s c o n t i n u i t i e s i n h y d r o s t a t i c pressure.and osmolarity at the lumen-wall i n t e r f a c e would be smaller.  In the w a l l l a y e r adjacent to  the innermost l a y e r , the t i s s u e pressure must be even lower (see F i g . 26). I f the chemical p o t e n t i a l of water i n these 2 l a y e r s were equal, the osmolarity of t h i s adjacent l a y e r must a l s o be lower.  This decrease  i n t i s s u e pressure and i n osmolarity continues to the a d v e n t i t i a l surface where the t i s s u e pressure equals that of the surrounding f l u i d .  Since  the manner i n which the t i s s u e pressure decreases across the w a l l depends on the tension i n the d i f f e r e n t w a l l l a y e r s true f o r the osmotic decrease.  (13),  the same w i l l be  I t should be noted that the exact shape of  the transmural pressure and osmolarity gradients are not known and apparently are q u i t e complex (see 13)-.  I f the innermost l a y e r had a t i s s u e pressure  of 65 mm Hg and the outer l a y e r was at 5 mm Hg, the o v e r a l l decrease i n concentration of the w a l l f l u i d across the w a l l w i l l be about 3 mM.  This  132 means that the w a l l f l u i d i s more d i l u t e i n the loose a d v e n t i t i a l l a y e r s than i n the inner layers of the a r t e r y w a l l . How i s t h i s s i t u a t i o n a l t e r e d when, i n response to nervous or humoral i n f l u e n c e s , the a r t e r y c o n s t r i c t s ?  The a c t i v e tension i n the w a l l  increases  and the e l a s t i c tension i s decreased so that the t o t a l t a n g e n t i a l w a l l tension, T, balances the transmural pressure, P, according to the law of Laplace:  T = P r (see 10,13,23,24,25).  The changes during  vasoconstriction  with a constant transmural pressure are s t r a i g h t forward and have been described by Burton (10).  However, when the i n t r a v a s c u l a r pressure  increases,  the e l a s t i c tension must adjust to both the decreasing radius and the i n creasing transmural pressure i n order that the sum of the a c t i v e and e l a s t i c tensions balances the pressure.  I t i s p o s s i b l e that during c o n s t r i c t i o n ,  the actions of the i n c r e a s i n g a c t i v e tension and the decreasing e l a s t i c tension might cause an increased h y d r o s t a t i c pressure i n the w a l l , upsetting the pressure balance with the lumen.  Such an increase could r e s u l t i n a  movement of f l u i d out of the c o n s t r i c t i n g a r t e r y w a l l . which might produce a pressure imbalance are:  Some of the f a c t o r s  d i f f e r e n t rates of development  of w a l l tension changes and i n t r a v a s c u l a r pressure changes, delays i n t r a n s l a t i n g a c t i v e tension i n t o a decreasing r a d i u s , or delays i n adjusting the e l a s t i c tension to the a c t i v e tension changes.  Since a water l o s s would  a f f e c t the s i z e of the a r t e r y w a l l , the volume of the lumen and thus the i n t r a v a s c u l a r pressure, the movement of the water i t s e l f might a c t to correct the pressure imbalance. f l u i d to return to the w a l l .  A r e v e r s a l of the imbalance would cause  Although t h i s d i s c u s s i o n i s t o t a l l y  conjecture,  some changes of t h i s s o r t are required to e x p l a i n the l o s s of w a l l water during  vasoconstriction.  133  £ E ElOOr  F i g . 26  BLOOD IN LUMEN  A R T E R Y WALL  SURROUNDING FLUID  Schematic r e p r e s e n t a t i o n of the h y d r o s t a t i c pressure and the osmolarity across the w a l l of a d i s t r i b u t i n g a r t e r y . The numerical values chosen are only f o r demonstration of the p r o f i l e s .  i  134 Imbalances between the a r t e r y w a l l and the surrounding t i s s u e s are probably not too important.  Van C i t t e r s e t - a l .  noted that i n the c o n s t r i c t e d  femoral a r t e r y , the "smooth muscle c e l l s adjacent to the e l a s t i c membrane were most severely deformed, and there was a diminution i n c y t o a r c h i t e c t u r a l a l t e r a t i o n s as the a d v e n t i t i a was reached" noted i n the r a t t a i l a r t e r y .  (26).  The same phenomenon was  This suggests that not only the t i s s u e  pressure but also the a c t i v e tension changes are greatest i n the inner l a y e r s of the w a l l . The law of Laplace was used i n the above d i s c u s s i o n of pressuretension r e l a t i o n s and not the equation suggested by Peterson (27) which i n cludes the w a l l thickness:  T = P r/6.  This l a t t e r equation r e a l l y describes  the s t r e s s on the v e s s e l w a l l , not i t s t a n g e n t i a l t e n s i o n , and was put f o r ward by Frank i n 1920 (21,28).  Many authors ignore both t h i s d i f f e r e n c e and  the f a c t that tension i s i n dynes/cm while s t r e s s , Peterson's " t e n s i o n " , i s i n dynes/cm . 2  The important r o l e played by the law of Laplace i n a r t e r i a l behavior can be seen i n the e f f e c t s of a l t e r i n g the pressure i n t i s s u e s surrounding arteries.  This a l t e r s the transmural pressure and thus the tension i n the  artery wall.  A l t e r e d hydrodynamics i n vascular beds have been observed  during elevated u r e t e r a l pressure (29), a l t e r e d c e r e b r o s p i n a l f l u i d pressure (30), and s t r i a t e d muscle c o n t r a c t i o n (31-34). Studies to test the e f f e c t of the a r t e r i a l pulse pressure have est a b l i s h e d that the a r t e r y i s incompressible when subjected to small s t r a i n s (35,36,37, compare 38).  This means that the r a t i o of transverse to l o n g i -  t u d i n a l s t r a i n , Poisson's r a t i o , i s 0.5 (36).  [This may not be s t r i c t l y  135 true f o r large s t r a i n s (39).]  I t i s i n c o r r e c t l y assumed that the same i s o -  volumetric s i t u a t i o n a p p l i e s during v a s o c o n s t r i c t i o n (25).  However, passive  s t r e t c h i n g , by applying a load or pressure, occurs without the a c t i v e tens i o n and expenditure of energy of v a s o c o n s t r i c t i o n (23).  E x t e r n a l forces  are required to s t r e t c h the a r t e r y , while the forces involved i n c o n s t r i c t i o n a r i s e w i t h i n the a r t e r y i t s e l f . Could the l o s s of water from the c o n s t r i c t i n g a r t e r y w a l l have an e f f e c t on the v i s c o - e l a s t i c properties of the c o n s t r i c t e d artery? a r t e r i e s decreases t h e i r d i s t e n s i b i l i t y (23,40).  Stretching  On the other hand, i t has  been reported that c o n s t r i c t i o n increases (23,25,41-44), decreases (27,45-48), or does not change (49) a r t e r i a l d i s t e n s i b i l i t y .  Apparently,  the f i n d i n g s  depend on the degree to which the a r t e r i e s are stretched (49) .  [The e l a s t i c  modulus used f o r these studies may be a deceptive index of w a l l e x t e n s i b i l i t y f o r large s t r a i n s (50) as are involved i n v a s o c o n s t r i c t i o n . ] of the c o n s t r i c t e d a r t e r y i s apparently increased studies considered  (27,51).  the water content of the a r t e r i e s .  The v i s c o s i t y None of these  I t i s p o s s i b l e that  the l o s s of e x t r a c e l l u l a r f l u i d during v a s o c o n s t r i c t i o n may play a r o l e i n these p h y s i c a l changes. There i s some i n d i c a t i o n that i n s t a t e s of the a r t e r y other than cons t r i c t i o n , hydration changes are associated with p h y s i c a l changes.  Aortic  s t r i p s immersed i n hypertonic s a l i n e undergo a great increase i n a r t e r i a l v i s c o s i t y (52,53).  I t i s known that age (54), hypertension  (55), and  increased distance from the heart (56,57) r e s u l t i n s t i f f e r a r t e r i e s .  In  a d d i t i o n to changes i n content and composition of the w a l l s o l i d s (58,59, 60), there are hydration changes associated with these a r t e r i e s (55,61,62).  136 I t i s p o s s i b l e that i n t e r r e l a t i o n s h i p s e x i s t between the s t r u c t u r a l and hydration changes and the observed p h y s i c a l changes of these a r t e r i e s . One i n t e r e s t i n g aspect of e x t r a c e l l u l a r vascular water changes i s the presence of n e g a t i v e l y charged mucopolysaccharides i n the p a r a c e l l u l a r matrix of e x t r a c e l l u l a r s o l i d s (see 63).  These mucopolysaccharides bind  water (21,58,64), and ions (63,65-68), and form complexes w i t h e x t r a c e l l u l a r  ++ p r o t e i n s (58,63).  The present study suggested that exchanging Ca  + f o r Na  as the main counter-ions to the a n i o n i c groups of the protein-polysaccharides r e s u l t e d i n conformational changes i n the matrix, an increased r e s i s t a n c e to p e r f u s i o n , and a l o s s of water from the a r t e r y w a l l (see 63,68).  Similarly,  the rewarming of a cooled a r t e r y r e s u l t e d i n a l o s s of w a l l w a t e r — p o s s i b l y r e l a t e d to the e x t r u s i o n of a f a s t , non-K l i n k e d  Na component, which may be  released during rewarming because of a temperature e f f e c t on the binding of Na to the p a r a c e l l u l a r matrix (67,69,70).  These experiments suggest a  p o s s i b l e r e l a t i o n s h i p between conformational changes of the p a r a c e l l u l a r matrix and l o s s of water from the a r t e r y w a l l .  The imbalance of h y d r o s t a t i c  f o r c e s , suggested as causing the w a l l water l o s s during v a s o c o n s t r i c t i o n , may i n v o l v e tension developed i n the p a r a c e l l u l a r matrix.  The i o n exchange  and rewarming processes may simply have a r t i f i c i a l l y produced changes i n the matrix which normally occur during v a s o c o n s t r i c t i o n . There are 2 p o s s i b l e ways the p a r a c e l l u l a r matrix could a f f e c t the hydration of the a r t e r y during constriction: (a) The c o n t r a c t i o n of the vascular smooth muscle c e l l s might compress the matrix, r e s u l t i n g i n an expulsion of e x t r a c e l l u l a r f l u i d from the a r t e r y w a l l . (b) The c o n t r a c t i o n of the smooth muscle c e l l s might be accompanied by changes i n the matrix counter-ions.  This would a l t e r the charge density of the  137 matrix, perhaps r e s u l t i n g i n shrinkage of the matrix and expulsion of e x t r a cellular  fluid.  These 2 explanations could be combined i f conformational the matrix during v a s o c o n s t r i c t i o n a l t e r e d the charge density or  changes i n counter-ion  s e l e c t i v i t y of the p a r a c e l l u l a r matrix, causing shrinkage of the matrix  and  f l u i d expulsion. Other authors have mentioned the p o s s i b l e r o l e of the mucopolysaccharides i n a r t e r i a l hydration:  Bader noted that "the ground substance has  the p r o p e r t i e s of a c o l l o i d — i t i s water i n s o l u b l e , but can bind water.  It  c o n s i s t s of mucopolysaccharides ... i s a very viscous m a t e r i a l and i t probably contributes to the t y p i c a l v i s c o - e l a s t i c behavior of d i s t e n s i b l e v e s s e l s " (21).  In h i s study of a combined s t r u c t u r e of hyaluronic a c i d ,  water and c o l l a g e n f i b e r s which had a d e f i n i t e r e s i s t a n c e to compression, F e s s l e r suggested that mucopolysaccharides have a mechanical f u n c t i o n (71). Frasher commented that " i t seems reasonable  to assume that [the ground  substance] i s involved i n changes i n water content of the w a l l and also that i t s p h y s i c a l s t a t e may  determine the mechanical r e s u l t a n t of the linkages  of the other components" (52) .  Zugibe and Brown demonstrated a t i g h t band  of a c i d mucopolysaccharides i n the subendothelial l a y e r of the aorta (72). I t i s i n t e r e s t i n g that i n t h i s l a y e r the w a l l h y d r o s t a t i c pressure i s l a r g e s t (13) and most of the tension i s developed during v a s o c o n s t r i c t i o n (26). Further studies which could be performed to demonstrate and e x p l a i n the l o s s of w a l l water during v a s o c o n s t r i c t i o n include:  138 1.  measuring the density of relaxed and c o n s t r i c t e d a r t e r i e s  dropping a r t e r i a l segments i n t o a s e r i e s of s o l u t i o n s with  by  densities  between 1.05 and 1.08 g/cc (see 73)I. 2.  a n a l y s i s of the water content of incubated a r t e r i e s every 30  seconds throughout c o n s t r i c t i o n and r e l a x a t i o n . 3.  examination of a r e l a t i o n s h i p between the w a l l water l o s s and  the extent of v a s o c o n s t r i c t i o n by varying the doses of v a s o c o n s t r i c t i v e agents over wide ranges. 4.  determination of inner and outer w a l l r a d i i , transmural pressure  and water content during v a s o c o n s t r i c t i o n i n order that the average w a l l tension (see 25) can be r e l a t e d to the water l o s t from the c o n s t r i c t i n g artery w a l l . 5.  i s o l a t i o n of a r t e r i a l protein-polysaccharides  f o r a study of  the r e l a t i o n between t h e i r hydration and v i s c o - e l a s t i c properties  B.  (see 74).  ALTERED VASCULAR PERMEABILITY DURING VASOCONSTRICTION The w a l l of the r a t t a i l a r t e r y was quite permeable to f l u i d .  Increased i n t r a v a s c u l a r pressure increased  the permeability  as d i d vaso-  c o n s t r i c t i o n with or without an i n i t i a l s h o r t - l i v e d decrease.  The permea-  b i l i t y of the a r t e r y w a l l w i l l f i r s t be considered, then the e f f e c t s of i n t r a v a s c u l a r pressure and v a s o c o n s t r i c t i o n . I t i s u s u a l l y assumed that a l l f l u i d exchange between blood and the t i s s u e s occurs across the c a p i l l a r i e s . that l a r g e r vessels are permeable.  However, there are some i n d i c a t i o n s  Zweifach has commented that "there i s  good evidence that movement of gases, water and small water-soluble  139 molecules occurs even across the w a l l s of terminal a r t e r i o l e s and prec a p i l l a r i e s " (75).  Jennings noted that "even p a r t i c l e s seem to be able to  leave normal blood v e s s e l s " (76).  Wilens and McClusky observed the passage  of blood serum through excised i l i a c a r t e r i e s and veins (77), although the vessels may not have been v i a b l e .  Sawyer and Valmont observed a net t r a n s -  port of Na and CI from the i n s i d e to the outside of the aorta and i n the opposite d i r e c t i o n f o r the vena cava (22). the a r t e r y w a l l by electro-osmosis  (78).  Water can be transported  across  I t was suggested that "both i o n  and water flow take place l a r g e l y through [the] e x t r a c e l l u l a r spaces (pores), which are the source of l e a s t r e s i s t a n c e to water and i o n movements" (78). While i t i s p o s s i b l e that the l a r g e transmural f l u i d movements observed f o r the r a t t a i l a r t e r y occur i n v i v o ,  t h i s seems u n l i k e l y . The  permeability of a r t e r i a l and c a p i l l a r y endothelium are probably not the same.  Duff found that dyes entered l a r g e a r t e r i e s because the w a l l of the  vasa vasorum was more permeable than the intima of the large blood (79).  vessels  In a d d i t i o n , the removal or separation of the r a t t a i l a r t e r i e s from  t h e i r surroundings meant that the p h y s i c a l e q u i l i b r i u m of the i n v i v o s i t u a t i o n was no longer present.  Large f l u i d movements across the a r t e r y w a l l  would c e r t a i n l y a f f e c t the h y d r o s t a t i c and osmotic pressure balance desc r i b e d above.  I t i s a l s o p o s s i b l e that the l a r g e vascular permeability was  due to anoxia, which i s known to increase c a p i l l a r y permeability (80). However, the presence of the normal pattern of norepinephrine s t r i c t i o n s i n the a r t e r i e s argues against t h i s (see 81).  induced con-  In any case, the  s i g n i f i c a n t f i n d i n g was that f l u i d could pass f r e e l y through the r a t t a i l a r t e r y w a l l — t h e d i r e c t i o n of flow depending on the experimental  conditions.  140 A r t e r i a l p e r m e a b i l i t y i s increased i n hypertension  (82,83), i n f l a m -  mation (84), and treatment with histamine or serotonin (85,86).  I t has  been suggested that the areas of blood v e s s e l s which r e a d i l y become s i t e s of a r t e r i o s c l e r o s i s have, even under normal c o n d i t i o n s , a higher permeability than other p a r t s of the v e s s e l (87). I t i s to be expected that increased i n t r a v a s c u l a r pressure caused an increase i n flow out of the lumen through the w a l l of the perfused r a t t a i l artery.  Landis showed that an increase i n c a p i l l a r y pressure  f i l t r a t i o n (88,89) as suggested by S t a r l i n g (90).  increased  This increased permea-  b i l i t y may be r e l a t e d to openings i n the c a p i l l a r y w a l l which close when the pressure decreases (88).  Increased i n t r a v a s c u l a r pressure increased  the  passage of blood serum through the vascular w a l l , although the pressure no e f f e c t when the v e s s e l was not allowed to d i l a t e (77).  had  This means that  s t r a i n , not pressure, determines the permeability of the vascular w a l l . S t r e t c h alone has been shown to increase the permeability of g e l a t i n f i l m s to hemoglobin (91) and the absorption of dyes by e l a s t i c membranes (92). The e f f e c t of c o n s t r i c t i o n on a r t e r i a l permeability has not been considered.  There i s , however, some information on the e f f e c t of vasocon-  s t r i c t i o n upon c a p i l l a r y f i l t r a t i o n and upon the whole c i r c u l a t o r y system where the e f f e c t s were assumed to have occurred at the c a p i l l a r i e s .  Renkin  observed that f o r a given blood flow, the clearance of t e s t molecules was much smaller during v a s o c o n s t r i c t i o n (93).  I t i s also known that prolonged  intravenous a d m i n i s t r a t i o n of catecholamines can deplete the blood volume (94-97,  compare 98).  In t h e i r studies on shock and reduced blood volume,  Freeman et a l . commented that " c a p i l l a r y permeability i s probably during v a s o c o n s t r i c t i o n produced by adrenaline" (95).  increased  However, Haddy et a l .  141 suggested  that the d e p l e t i o n of blood was instead due to the d i f f e r e n t  e f f e c t s of adrenaline on veins and a r t e r i e s so the r a t i o of these 2 pressures, hence the c a p i l l a r y h y d r o s t a t i c pressure, i s a l t e r e d , so the t i s s u e s gain weight (97).  This suggestion may w e l l apply to Renkin's f i n d i n g s .  A l s o , the  usefulness of epinephrine i n t r e a t i n g edema i s not due to the e f f e c t of cons t r i c t i o n on p e r m e a b i l i t y , but to the reduced blood flow during vasocons t r i c t i o n (89).  This a c t i o n of vasoactive agents on the c a p i l l a r y pressure  thus makes i t very d i f f i c u l t to assess any d i r e c t e f f e c t s of c o n s t r i c t i o n on vascular p e r m e a b i l i t y . However, there i s i n d i r e c t evidence to suggest a r e l a t i o n s h i p between v a s o c o n s t r i c t i o n and a l t e r e d v a s c u l a r p e r m e a b i l i t y : 1.  Vasopressin ( p i t r e s s i n ) i s a v a s o c o n s t r i c t o r (99) and increases  i n t e s t i n a l water absorption (100), the permeability of f r o g s k i n (101), and a c t i v e transport of Na by frog s k i n or bladder 2.  (102).  Serotonin i s a v a s o c o n s t r i c t o r (103) and increases c a p i l l a r y per-  m e a b i l i t y (104) . 3.  Histamine i s a v a s o d i l a t o r of small a r t e r i o l e s and a vasocon-  s t r i c t o r of l a r g e r a r t e r i o l e s and small a r t e r i e s (105) and increases c a p i l l a r y permeability (104). 4.  Norepinephrine  i s a v a s o c o n s t r i c t o r (106) and increases l i v e r  membrane p e r m e a b i l i t y to K (107). 5.  Epinephrine i s a c o n s t r i c t o r of most v a s c u l a r smooth muscle (106)  and increases the p e r m e a b i l i t y of f r o g s k i n to Na (108) and gut to sugars (109).  142 6.  Bradykinin i s a v a s o d i l a t o r (110) and increases the permeability  of s k i n blood vessels  (111).  In short, many vasoactive agents increase permeability.  In l i g h t of the  f i n d i n g s of the present study, the whole r e l a t i o n s h i p between these two p r o p e r t i e s should be i n v e s t i g a t e d . The f a c t that the changes i n permeability during v a s o c o n s t r i c t i o n depended upon the i n t r a v a s c u l a r pressure, suggests that the above a n a l y s i s for the w a l l water l o s s may also apply to t h i s a l t e r e d permeability ation.  Transmural f l u i d movements were observed.  situ-  Altered hydrostatic  pressures i n the w a l l and lumen of the c o n s t r i c t i n g a r t e r y could r e s u l t i n the lumen and extravascular f l u i d being e s s e n t i a l l y "connected" so that the osmotic and h y d r o s t a t i c pressure d i f f e r e n c e s between them would cause f l u i d to move through the w a l l . conformational  I n a d d i t i o n , the e x t r a c e l l u l a r s o l i d s undergo  changes during v a s o c o n s t r i c t i o n .  These changes would a l t e r  the path of flow ( i . e . the e x t r a c e l l u l a r space) across the c o n s t r i c t i n g a r t e r y w a l l and would a f f e c t vascular permeability.  I t was pointed out  above that pressure a l t e r a t i o n s during c o n s t r i c t i o n could cause the w a l l water l o s s which i n turn would decrease the i n t r a v a s c u l a r pressure. may be a s i m i l a r system operating here.  There  A l t e r a t i o n s i n the transmural  balance of osmotic and h y d r o s t a t i c pressures r e s u l t i n a l t e r e d w a l l permea b i l i t y , which i n turn a f f e c t s the osmotic and h y d r o s t a t i c developed w i t h i n the a r t e r y w a l l .  pressures  That i s , an increased permeability would  decrease the pressure gradients, the a l t e r a t i o n s i n which were the cause of the increased permeability.  The explanations  could consequently be turned around:  f o r the a l t e r e d permeability  an increase i n the path f o r flow  a f t e r c o n f i g u r a t i o n a l changes could cause transmural  f l u i d movements which  would upset the osmotic and h y d r o s t a t i c pressures across the a r t e r y w a l l  143 r e s u l t i n g i n an expulsion of e x t r a c e l l u l a r f l u i d .  I t i s i n t e r e s t i n g that  Brinkman et^ a l . found that X - i r r a d i a t i o n caused a decrease i n i n j e c t i o n pressure i . e . an increase i n water p e r m e a b i l i t y , i n the w a l l of the aorta due to "a s l i g h t depolymerization of the mucopolysaccharide connectivet i s s u e matrix" (20,112).  Measurements of D 0 f l u x across the c o n s t r i c t i n g 2  a r t e r y w a l l (see 113) with c o n t r o l l e d i n t r a - and extravascular  pressures  could supply some data to t e s t these s p e c u l a t i o n s .  C.  INCREASED SMOOTH MUSCLE CELL VOLUME DURING VASOCONSTRICTION The geometrical and h i s t o l o g i c a l studies of Chapter I I showed that  the a r t e r i a l smooth muscle c e l l can be represented as a double cone which had a constant surface area and an i n c r e a s i n g volume, due to the entry of water, during v a s o c o n s t r i c t i o n . This f i n d i n g agrees w i t h the c e l l volume increases observed during v a s o c o n s t r i c t i o n (114,115,116) and with the e f f e c t s of anisosmotic s o l u t i o n s : vascular smooth muscle c e l l s s w e l l and contract i n hypotonic s o l u t i o n s (116) and s h r i n k and r e l a x i n hypertonic s o l u t i o n s (117). I t i s g e n e r a l l y assumed that c e l l s have the same osmolarity as the f l u i d which surrounds them (see 118 f o r a f u l l d i s c u s s i o n ) .  However, t h i s  might not be so i n the a r t e r y w a l l i f the i n t r a c e l l u l a r tension caused a h y d r o s t a t i c pressure d i f f e r e n c e across the smooth muscle c e l l membrane. I n a d d i t i o n , the pressure and osmotic gradient across the a r t e r y w a l l might mean that the smooth muscle c e l l s i n the d i f f e r e n t l a y e r s of the media have d i f f e r e n t o s m o l a r i t i e s and h y d r o s t a t i c pressures, and hence d i f f e r e n t tensions.  This might be r e l a t e d t o the f a c t that the inner smooth muscle c e l l s  undergo the most d r a s t i c contractions during v a s o c o n s t r i c t i o n (26).  144 Movement of water i n t o the a r t e r i a l smooth muscle c e l l s means that one or more of the f o l l o w i n g occurred during c o n s t r i c t i o n : (a) c e l l u l a r osmolarity increased, (b) e x t r a c e l l u l a r space (ECS) osmolarity decreased, (c) c e l l u l a r h y d r o s t a t i c pressure decreased or (d) ECS h y d r o s t a t i c increased.  pressure  The explanation chosen must not c o n f l i c t with the f a c t that  e x t r a c e l l u l a r f l u i d was expelled from the c o n s t r i c t i n g a r t e r y w a l l , although of course, the c e l l u l a r s w e l l i n g may the ECS  fluid loss.  not have occurred at the same time as  Ion movements, tension changes and permeability changes  could a l l have caused these proposed osmotic and h y d r o s t a t i c  pressure  changes. The only explanation which can be r u l e d out i s ( c ) :  a decrease i n  c e l l h y d r o s t a t i c pressure i s u n l i k e l y since the tension w i t h i n the t i n g c e l l would increase.  contrac-  Jonsson suggested that the increase i n a c t i v e  tension i n the smooth muscle c e l l s of the c o n s t r i c t i n g r a t p o r t a l v e i n does not generate an i n t r a c e l l u l a r h y d r o s t a t i c pressure l a r g e enough to f i l t e r f l u i d out of the c e l l s (116).  While t h i s may  e x t r a c e l l u l a r h y d r o s t a t i c pressure may increase.  be t r u e , the increase i n  have balanced some of the c e l l u l a r  Of the other 3 explanations, there i s l i t t l e or no data a v a i l a b l e  e i t h e r to r e f u t e them or enable a choice to be made between them: (a) An increase i n c e l l u l a r osmolarity could be caused by a net gain i n c e l l u l a r i o n s , by bound c e l l u l a r ions becoming f r e e , or by free c e l l water becoming bound during c o n t r a c t i o n .  Nothing i s known about changing  amount of bound and f r e e ions and water i n c o n t r a c t i n g a r t e r i a l smooth muscle c e l l s .  During c o n t r a c t i o n , K leaves (119), while Na (73) and Ca  enter the smooth muscle c e l l s . t i c a l l y , Na may  These i o n s h i f t s may  (120)  not be balanced osmo-  be accompanied by CI, and an unknown amount of Ca may  be  145  free or bound i n the c e l l .  I f much of the smooth muscle c e l l ATP were i n -  volved i n a c t i v e muscle c o n t r a c t i o n , i t i s p o s s i b l e that the membrane i o n pumps could be a f f e c t e d , i n c r e a s i n g the permeability of the smooth muscle c e l l to ions and perhaps water.  An increase i n c e l l u l a r osmolarity  alone,  however, could not e x p l a i n the expulsion of e x t r a c e l l u l a r f l u i d from the constricting artery wall. (b) A decrease i n ECS osmolarity could occur i f the e n d o t h e l i a l permeability were a l t e r e d , i f ECS ions became bound, or i f water bound to e x t r a c e l l u l a r macromolecules became f r e e .  A l t e r a t i o n s i n the distance be-  tween the f i x e d charges on the e x t r a c e l l u l a r mucopolysaccharides (63) during c o n s t r i c t i o n might r e s u l t i n an increase i n bound e x t r a c e l l u l a r ions.  A  decrease i n ECS osmolarity would cause water to move from the ECS i n t o the c e l l s , lumen and surrounding  t i s s u e s to r e s t o r e osmotic e q u i l i b r i u m .  However, i t seems u n l i k e l y that the expelled ECS f l u i d would have  contained  70 to 100 meq N a / l i t e r i f t h i s were the only explanation of the w a l l water loss. (d) An increase i n e x t r a c e l l u l a r h y d r o s t a t i c pressure would r e s u l t when the network of c e l l s and e x t r a c e l l u l a r macromolecules acts to c o n s t r i c t the a r t e r y w a l l .  The existence of the transmural pressure gradient (13)  means that the i n c r e a s i n g i n t r a v a s c u l a r pressure would be accompanied by an i n c r e a s i n g w a l l h y d r o s t a t i c pressure.  Decreasing the e n d o t h e l i a l permeability  to water would a l s o increase the w a l l h y d r o s t a t i c pressure.  As with a de-  creased ECS osmolarity, an increased ECS h y d r o s t a t i c pressure, r e s u l t i n g i n an increased e x t r a c e l l u l a r chemical p o t e n t i a l of water , would d r i v e f l u i d i n t o the smooth muscle c e l l s and i n t o the lumen and surrounding  tissues.  146 No matter what the cause, the increase i n c e l l water during vasoc o n s t r i c t i o n would decrease c e l l u l a r i o n concentrations.  Associated with a  l o s s of c e l l K and a gain of c e l l Na, t h i s means the [ K ] ^ / [ K ] +  +  o  gradient  would decrease r e s u l t i n g i n d e p o l a r i z a t i o n of the vascular smooth muscle c e l l membrane.  The change i n Na gradient would depend on the r e l a t i v e s i z e s  of the c e l l Na and water gains.  I t i s not known whether the water entry i s  associated with a l t e r e d c e l l membrane permeability and thus aids i n membrane d e p o l a r i z a t i o n or whether the water entry occurs a f t e r the i n i t i a t i o n of contraction. The most important conclusion to be drawn from t h i s study i s that the hydration of the a r t e r y w a l l cannot be treated as a s t a t i c component of the wall.  In p a r t i c u l a r , during v a s o c o n s t r i c t i o n , water moves through the  a r t e r y w a l l , water enters the smooth muscle c e l l s and water i s expelled from the e x t r a c e l l u l a r space.  147  BIBLIOGRAPHY  CHAPTER I  1.  Burton, A.C.  P h y s i o l . Rev. 34: 619, 1954.  2.  Van C i t t e r s , R.L., Wagner, B.M., and Rushmer, R.F.  C i r c u l a t . Res.  10: 668, 1962. 3.  M i l c h , R.A.  4.  Moyer, J.H.(ed.).  5.  Constantinides, P. Experimental A t h e r o s c l e r o s i s . E l s e v i e r , Amsterdam, 1965. 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Pathol. 8: 219, 1932. Am. J . P h y s i o l . 83: 528, 1928.  and Spiro, D.  1  Am. J . Pathol.  160 83.  Olsen, F.  Acta Pathol. M i c r o b i o l . Scand. 74: 325, 1968.  84.  Marchesi, V.T.  85.  Majno, G. and Palade, G.E.  86.  R i c h t e r , W.  87.  A l t s c h u l , R.  Endothelium, MacMillan, New York, 1954.  88.  Landis, E.M.  Am. J . P h y s i o l . 82: 217, 1927.  89.  Landis, E.M.  P h y s i o l . Rev. 14: 404, 1934.  90.  S t a r l i n g , E.H.  91.  Florey, H.A.  92.  Lunseth, J.H.  93.  Renkin, E.M.  94.  Lamson, P.D. and K e i t h , N.M.  95.  Freeman, N.E.,  Proc. Roy. Soc.(London) Ser. B. 156: 550, 1962. J . Biophys. Biochem. C y t o l . 11: 571, 1961.  Acta Pharmacol. T o x i c o l . 27: 331, 1969.  J . P h y s i o l . 19: 312, 1896. J . P h y s i o l . 61: i P , 1926. Science 141: 438, 1963. Am. J . P h y s i o l . 197: 1205, 1959. J . Pharmacol. Exp. Therap. 8: 247, 1916.  Freeman, H., and M i l l e r , C.C.  Am. J . P h y s i o l . 131:  545, 1941. 96.  Rosenthal, M.E.  97.  Haddy, F.J., Molnar, J . I . , Borden, C.W., and Texter, E.C.,Jr. C i r c u l a t i o n 25: 239, 1962. Werner, M., Bock, K.D., Brandenburger, J . , and Sack, H. Clin.  98.  and D i Palma, J.R.  Fed. Proc. 18: 440, 1959.  Pharmacol. Therap. 9: 162, 1968. 99.  Hodge, R.L. and Dornhorst, A.C.  C l i n . Pharmacol. Therap. 7: 639, 1966.  100.  Sawyer, W.H.  Pharmacol. Rev. 13: 225, 1961.  101.  Ussing, H.H.  J . Gen. P h y s i o l . 43: 135, 1960.  102.  Maetz, J . , M o r e l , F., and Lahlouh, B.  103.  G i n z e l , K.H. and Kottegoda, S.R.  104.  Buckley, I.K. and Ryan, G.B.  105.  Dale, H.H.  106.  Furchgott, R.F.  107.  H a y l e t t , D.G.  Nature 184: 1236, 1959.  B r i t . J . Pharmacol. 8: 348, 1953.  Am. J . Pathol. 55: 329, 1969.  Lancet 1: 1179, 1233, and 1285, 1929. Pharmacol. Rev. 7: 183, 1955.  and Jenkinson, D.H.  Nature 224: 80, 1969.  161 108.  Ussing, H.H.  Acta P h y s i o l . Scand. 17: 1,  109.  G e l l h o r n , E.  Ann. I n t e r n . Med.  110.  Erdos, E.G.  111.  Oyvin, I.A., Gaponiuk, P.Y.,  112.  Brinkman, R., Lamberts, H.B., Wadel, J . , and Zuideveld, J . Rad. B i o l . 3: 205, 1961.  113.  V i s s c h e r , M.B., Fetcher, E.S.,Jr., Carr, C.W., Gregor, H.P., Bushey, M.S., and Barker, D.E. Am. J . P h y s i o l . 142: 550, 1944.  114.  Friedman, S.M. and Friedman, C.L. I I : 1135, 1963.  115.  Friedman, S.M. and Friedman, C.L. In E l e c t r o l y t e s and Cardiovascular Diseases. Ed. by E. Bajusz, S. Karger, Basel/New York, 1965, p. 323.  116.  Johnsson, 0.  117.  Mellander, S., Johansson, B., Gray, S., Jonsson, 0., L u n d v a l l , J . ,  (Ed.).  Ann. N.Y.  7: 33,  1949.  1933.  Acad. S c i . 104: 1, and Oyvin, V.I.  A n g i o l o g i c a 4: 310,  118.  Robinson, J.R.  119.  Tobian, L. and Fox, A.  120.  Hiraoka, M., 1968.  E x p e r i e n t i a 23: 925,  1967.  Int. J.  Handbook of Physiology, C i r c u l a t i o n  Acta P h y s i o l . Scand. 77: 201,  and Ljung, B.  1963.  P h y s i o l . Rev. 40: 112,  1969.  1967. 1960.  J . C l i n . Invest. 35: 297,  Yamagishi,. S., and Sano, T.  1956.  Am. J . P h y s i o l . 214:  1084,  162 APPENDIX I DIRECT MEASUREMENT OF ARTERY WALL RADII  Determination of the inner and outer r a d i i of the a r t e r y w a l l enables the w a l l c r o s s - s e c t i o n a l area to be c a l c u l a t e d (1,2).  To t e s t the assumption  of isovolumetric c o n s t r i c t i o n , Wiederhielm's TV scan method (3) was used with a considerably  l e s s s o p h i s t i c a t e d means f o r d i s p l a y i n g the video s i g n a l  information onto the cathode ray o s c i l l o s c o p e (CRO). rat  A 3 cm length of the  t a i l a r t e r y was enclosed i n a t h i n p l a s t i c chamber while being bathed  and perfused with Krebs s o l u t i o n .  Using a TV camera - microscope assembly,  the a r t e r y was displayed on a TV monitor. screen was then displayed on the CRO. a r t e r y dimensions.  The voltage from 1 l i n e on the  This voltage d i s p l a y represents the  This experiment was terminated when accurate measurements  of the relaxed and c o n s t r i c t e d a r t e r y dimensions could not be made. The e s s e n t i a l problem was m a g n i f i c a t i o n vs f i e l d of view. cluding the whole w a l l meant that the magnifications  The necessity of i n used were too low f o r  determination of small dimensional changes.  1.  Wiederhielm, C.A.  2.  Baez, S.  3.  Wiederhielm, C.A.  Fed. Proc. 24: 1075, 1965.  C i r c u l a t . Res. 25: 315, 1969. J . Appl. P h y s i o l . 18: 1041, 1963.  APPENDIX I I SPIRAL ANGLE OF ARTERIAL SMOOTH MUSCLE CELLS Although the smooth muscle s p i r a l angle has been measured i n various a r t e r i e s (1-6), very l i t t l e i s known about the s p i r a l angle during vasoconstriction.  L o n g i t u d i n a l sections of the r a t t a i l a r t e r y were used:  163 (a) Epon s e c t i o n s , cut 1.5 u t h i c k , were stained with t o l u i d i n e blue, (b) f r e e z e - s u b s t i t u t e d  a r t e r i e s with known s t a t e of c o n s t r i c t i o n (see  Chapter I I ) , were embedded i n p a r a f f i n , sectioned trichrome.  and stained with  Mallory  The smooth muscle c e l l s i n the relaxed a r t e r i e s were at 90° to  the a r t e r y a x i s , except c e l l s near the a d v e n t i t i a which were at 70° to 80° to the a x i s .  The c e l l s i n the c o n s t r i c t e d a r t e r i e s were s i m i l a r except the  outer c e l l s deviated even f u r t h e r from 90°. A l s o , some groups of c e l l s near the lumen were at 30° and 160° to the a x i s .  In short, no one s p i r a l angle  could be assigned to these vascular smooth muscle c e l l s and the c e l l arrangement was more random i n c o n s t r i c t e d than i n relaxed a r t e r i e s .  1.  Strong, K.C.  Anat. Rec. 72: 151, 1938.  2.  Schultze Jena, B.S.  3.  G o e r t t l e r , K.  4.  Rhodin, J .  Physiol.Rev.  5.  Rhodin, J .  J . U l t r a s t r u c t . Res. 18: 181, 1967.  6.  Phelps, P.C. and L u f t , J.H.  Geg. Morphol. Jahrb. 83: 230, 1939.  Geg. Morphol. Jahrb. 91: 368, 1951. 42: suppl. 5, 48, 1962.-  Am. J . Anat. 125: 399, 1969.  APPENDIX I I I WEIGHING AN ARTERY WITH AN ELECTROBALANCE The r a t t a i l a r t e r y was suspended i n Krebs s o l u t i o n from a Cahn electrobalance  (see 1 ) .  The change i n a r t e r y w a l l volume, AV , during conW  striction i s : AV  W  =  AW / &  p  f  - p  s  (1)  164 where:  AW  g  =  change during c o n s t r i c t i o n of the recorded weight of the  system, pf = density of the f l u i d l e a v i n g the a r t e r y , and p the  s o l u t i o n i n which the system i s suspended.  s  = density of  Although weights were r e -  corded f o r the r a t t a i l a r t e r y , i t never proved p o s s i b l e to add a vasocons t r i c t i v e agent such as norepinephrine to the s o l u t i o n without considerably d i s t u r b i n g the weighing.  Dripping s o l u t i o n down the a r t e r y suspended i n a i r  proved equally u n s a t i s f a c t o r y .  Consequently, t h i s experiment was terminated.  D e r i v a t i o n of equation ( 1 ) : The system i s the a r t e r y , the suspending wire and a small weight. For W  = weight of system i n a i r , and W  a  = weight of system i n s o l u t i o n ,  g  the buoyancy, B, i s the weight of s o l u t i o n displaced by the system: W  a  - W. s  So, AB = AW  a  - AW  S  during c o n s t r i c t i o n .  placed s o l u t i o n , i . e . V = B/p , so AB = p AV. s  s  Let V =• volume of d i s -  Thus, AW  S  = AW  a  - p AV. s  Since only the a r t e r y changes volume during c o n s t r i c t i o n , AV = AV . W  movement of f l u i d from the w a l l means AW  a  equations i n t o that f o r AW  S  1.  = pfAV . w  B =  The  S u b s t i t u t i n g these l a s t 2  gives equation ( 1 ) .  Lindemann, B. and Solomon, A.K.  J . Gen. P h y s i o l . 45: 801, 1962.  

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