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Arterial hydration during vasoconstriction 1970

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ARTERIAL HYDRATION DURING VASOCONSTRICTION by MARK ERNEST HOLTBY B.Sc, University 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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1970 In presenting th i s thes is in pa r t i a l f u l f i lmen t o f the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y ava i l ab le for reference and study. I fur ther agree tha permission for extensive copying of th i s thes is fo r scho la r l y purposes may be granted by. the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t ion of th i s thes is fo r f inanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Depa rtment The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date 0Y 9 rf?0 i i ABSTRACT The relationship between vasoconstriction and the hydration of the artery w a l l was examined using the t a i l artery of the r a t . Freeze substitution was used to prepare h i s t o l o g i c a l sections of art e r i e s fixed i n a known state of c o n s t r i c t i o n . Measurements of w a l l dimensions showed that the more constricted a r t e r i e s had smaller w a l l and media cross-sectional areas than the less constricted a r t e r i e s . The con- stant length of the co n s t r i c t i n g artery meant that the wa l l volume decreased by 14%. Considering the vascular smooth muscle c e l l as a double cone enabled formulation of relationships between the c e l l radius, length, surface area, and volume. Radius and length measurements of the smooth muscle c e l l s of the freeze-substituted a r t e r i e s demonstrated that the c e l l radius doubled and the length decreased by half during vasoconstriction. These measurements revealed that the surface area of the double cone model of the c e l l remained constant, while the volume increased during vasoconstriction. This suggested that water entered the contracting vascular smooth muscle c e l l s . Water and ion content determination of paired control and constricted i n v i t r o a r t e r i e s indicated that the artery w a l l l o s t 16% of i t s water. This represented a 13% decrease i n the wa 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 artery. While t h i s was true for a r t e r i e s con- s t r i c t e d with both norepinephrine and high K solutions, i t seemed that water l o s t from a r t e r i e s constricted with PLV-2, a synthetic vasopressin, may have i i i 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 size of the water loss depended upon the duration of vasoconstriction: the losses were largest 30 seconds after the st a r t of c o n s t r i c t i o n . Perfused rat t a i l a r t e r i e s exhibited pressure-flow c h a r a c t e r i s t i c s during vasoconstriction which suggested that the permeability of the wal l had increased. I t was discovered that the changes i n permeability induced by vasoconstriction were d r a s t i c a l l y affected by changes i n the intravascular pressure. In a t h i r d perfusion experiment, the d i l u t i o n of Evans blue dye passing through the lumen of a co n s t r i c t i n g artery also indicated a permea- b i l i t y increase during vasoconstriction. Ar t e r i e s were incubated i n one of f i v e isosmotic solutions 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 perfusion pressures showed that the absence of monovalent ions i n the bathing media resulted i n a decrease i n a r t e r i a l water, an increase i n divalent ion content, and higher perfusion pressures. These observations can be explained by changes i n the tension of the vascular smooth muscle c e l l s and possibly by an ion exchange process i n the paracellular matrix which caused conforma- t i o n a l changes i n the matrix, i n turn causing an altered w a l l hydration. A r t e r i e s , cooled overnight at 2°C, were rewarmed i n one of three solutions of d i f f e r e n t Na concentration. The ar t e r i e s were transferred from a solution at 2°C to one at a temperature between 2° and 37°C for 15 minutes. Comparison of the a r t e r i a l contents showed that a small amount of K was gained while large amounts of water and Na were l o s t from the artery w a l l during these short rewarming periods. Postulation of a 1:1 exchange of K for Na for the c e l l metabolic Na-K pump means that a fast Na component was i v extruded, independent of K, from the rewarming artery w a l l . The extrusion of the wal l water may have been related to the extrusion of t h i s extra Na component, because they both had the same temperature and external Na con- centration dependencies. The monitoring of the intravascular pressure of perfused rewarmed ar t e r i e s revealed pressure changes with the same tempera- ture and external Na concentration dependencies as the above water content changes. Calculations indicated that changes i n wal l volume caused by changes i n water content could p a r t i a l l y explain the intravascular 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 vasoconstriction, are discussed i n terms of an osmotic and hydrostatic pressure balance between the artery w a l l and i t s surroundings which i s upset by vasoconstriction. V TABLE OF CONTENTS CHAPTER Page I. INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . 1 I I . CHANGES IN VASCULAR DIMENSIONS DURING CONSTRICTION. . . . . 6 A. Changes i n Dimensions of Artery Wall During Constrxction. . . . . s o . . . . . . . . . . . « ' . . 6 1. Methods . . . . . . . . . . . . . . . . . . . . . . 7 2. Results 9 3. Effect of Fix a t i o n and Embedding on A r t e r i a l 4. Discussion 23 5. Summary . . . . . . . . . . . 24 B. Changes i n Dimensions of Smooth Muscle C e l l s During Vasoconstriction . . . . . . . . . . . . . . . 25 1. Mathematical Model for the Smooth Muscle C e l l . . . 26 2. Measurement of C e l l Dimensions. . . . . . . . . . . 28 3. F i t t i n g the Experimental Data to the Model. . . . . 30 4. Discussion. . 31 5. Summary . . . . . . . . . . . . . 38 I I I . CHANGES IN WATER CONTENT OF ARTERY WALL DURING CONSTRICTION. .. i . . . . . . . . . . . . . . . . . . . . 39 A. Methods . . . . . . . . . . . . . . . . . . . . . . . . 39 B. Norepinephrine Induced Vasoconstriction . . . . . . . . 42 1. Water Content . . . . . . . . 42 2. Water and Ion Content 43 3. Varying Duration of Vasoconstriction. . . . . . . . 48 4. Discussion. . . . . . . . . . . . . . . 50 5. I n u l i n Space. . . . . . . . . . . . . . . 54 6. Chloride Content 58 7. Summary of Results of Norepinephrine Induced Vasoconstriction. . . . . . . . . . 60 C. High K Induced Vasoconstriction . . . . . . 61 D. Synthetic Vasopressin, PLV-2, Induced Vasoconstriction 63 v i CHAPTER Page III. (Continued) E. Discussion. . . . . . . . . . . . . . . . . . . . . . . . 64 F. Error i n Calculating Inner Radius from Isovolumetric Assumption. . . . . . . . . . . . . . . . . . . . . . . 69 G. Examination of Possible Experimental Artifacts 70 1. Effect of Relaxation of Constricted Arteries on Water Content . . . . . . . . . . . . . . . . . . . . 71 2. Water Remaining in the Lumen after Gaseous Perfusion 73 3. Effect of Gaseous Perfusion on Art e r i a l Water IV. HEMODYNAMIC AND PERMEABILITY CHANGES DURING VASOCONSTRICTION 78 A. Pressure, Flow and Lumen Volume . . . . . . . . . . . . . . 78 1. Methods 79 2. Results 80 3. Discussion 82 4. Summary . . . . . . . . . . . . . . . . 83 B. Effect of Intravascular Pressure. . . . . . . . . . . . . 83 1. Methods . . . . . . . . . . . . . . . . . . . . . . . 84 2. Results 85 3. Discussion 88 4. Summary 90 C. Dye Dilution. . . . . . . . . . . . . . . . . . . . . . . 90 1. Methods . . 91 2. Results 91 3. Discussion. 93 4. Summary . . . . . . . . . . . . . . . 94 D. Summary . . . . . . . . . . . 95 v i i CHAPTER Page V. EXPERIMENTALLY INDUCED ALTERATIONS IN THE HYDRATION OF THE ARTERY WALL. . 96 A. Effect of Varying External Ionic Composition 96 1. Methods . . . . . . . . . . . . 97 2. Results 98 3. Discussion 101 4. Summary . . . . . . . . . . 103 B. Effect of Cooling and Rewarming . . . . . . . . . . . . . . 103 1. On the Water and Ion Content of the Vascular Wall . . . 103 2. On the Intravascular Pressure 118 VI. SUMMARY AND DISCUSSION 128 A. Loss of Wall Water During Vasoconstriction . . . . 129 B. Altered Vascular Permeability During Vasoconstriction . . . 138 C. Increased Smooth Muscle C e l l Volume During Vasoconstriction. . . . . . . . . . . . . . 143 APPENDIX I. DIRECT MEASUREMENT OF ARTERY WALL RADII. . . . . . . . . . . . 162 I I . SPIRAL ANGLE OF ARTERIAL SMOOTH MUSCLE CELLS . . .162 I I I . WEIGHING AN ARTERY WITH AN ELECTROBALANCE. . . . . . . . . . . 163 v i i i LIST OF TABLES TABLE Page I. Dimensions of w a l l of rat t a i l artery i n various states of co n s t r i c t i o n , calculated from h i s t o l o g i c a l cross-sections. . . 13 I I . Outer radius and cross-sectional area of the w a l l of f u l l y constricted middle segments of the rat t a i l artery . . . . . . 20 I I I . Outer radius of the rat t a i l artery 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. . . . . . . . . 21 IV. The cross-section area of the rat t a i l artery 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 V. Half-lengths and r a d i i of relaxed and contracted smooth muscle c e l l s of the rat t a i l artery 29 VI. Surface area and volume of relaxed and contracted smooth muscle c e l l s of the rat t a i l artery . . . . . . . . . 30 VII. Water contents of rat 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 V I I I . Water contents, sodium and potassium contents of rat t a i l a r t e r i e s removed from solution at the peak of NE c o n s t r i c t i o n after 3 hours of aerobic incubation. . . . . . . . . . . . . . 44 IX. Water content, sodium and potassium contents of rat t a i l a r t e r i e s constricted with norepinephrine for s i x t y seconds af t e r three hours of aerobic incubation. . . . 46 X. Water contents, sodium and potassium contents of rat t a i l a r t e r i e s constricted with norepinephrine for 120 seconds after three hours of aerobic incubation. . . . . . . . . . . . . . . 47 XI. 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 perfusion with O2/CO2 after 3 hours of aerobic incubation . . . . . . . . . . . . . 49 XII. Water and i n u l i n space of rat t a i l a r t e r i e s constricted for two minutes with norepinephrine after t h i r t y minutes of aerobic incubation . . . . . . . . . . . . . . . . 56 i x TABLE Page XI I I . Water and i n u l i n space of rat t a i l a r t e r i e s constricted for two minutes with norepinephrine after t h i r t y minutes of aerobic incubation . . . . . . . . . . . . . . . . . 57 XIV. Water and ion content of rat t a i l a r t e r i e s constricted with norepinephrine for L5 minutes a f t e r 90 minutes of aerobic incubation . . . . . . . . . . . . . . . . . 59 XV. Overall average water and sodium contents of rat t a i l a r t e r i e s constricted i n v i t r o with norepinephrine. The r a t i o of constricted test a r t e r i e s with smaller water or sodium than t h e i r corresponding control halves . . . . . . . . 60 XVI. Water content of rat t a i l a r t e r i e s constricted for two minutes i n high potassium solution and the r a t i o of con- s t r i c t e d test a r t e r i e s with smaller water content than t h e i r corresponding non-constricted control halves after aerobic incubation . . . . . . . . . . . . . . . . . . . 63 XVII. Water content and i n u l i n space of rat t a i l a r t e r i e s con- s t r i c t e d for two minutes with PLV-2 and r a t i o of constricted test a r t e r i e s with smaller amounts than t h e i r corresponding control halves after three hours of aerobic incubation . . . . 64 XVIII. Intravascular pressure, water and ion contents of rat t a i l a r t e r i e s incubated i n one of f i v e isosmotic solutions. . . . . 99 XIX. Changes i n water, sodium and potassium contents of rat t a i l a r t e r i e s at di f f e r e n t rewarming temperatures i n three solu- tions with 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 rat t a i l artery. . . . . 11 2. Relation between wa l l to lumen r a t i o , inner and outer r a d i i , and the degree of vasoconstriction of the rat t a i l artery 15 3. Relation between the cross-sectional areas of the wal l and media of the rat t a i l artery and the w a l l to lumen r a t i o . . 16 4. Relation between the cross-sectional area of the artery media and the w a l l to lumen r a t i o for the d i s t a l segments of the rat t a i l artery. . . . . . . . . . . . . . . . 19 5- 8. 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 half-length and radius . . . . . . . . . 32-35 9. Changes i n water content of rat t a i l artery 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 10. Changes i n sodium content of rat t a i l artery 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 11. Varying durations of norepinephrine induced constrictions of rat t a i l a r t e r i e s perfused jin v i t r o with O2/CO2 . . . . . 55 12. Inner radius of a constricted artery calculated from the constricted outer radius and the relaxed inner and outer r a d i i , with and without a 13% decrease i n the volume of the constricted artery w a l l 70 13. Intravascular pressure of a rat t a i l artery constricted i n v i t r o with norepinephrine, then allowed to relax, while being perfused with O2/CO2 V • 72 14. Effluent flow rate and pressure gradient for a rat t a i l artery constricted i n s i t u with norepinephrine . . . . . . . 81 15. Pressure gradient down rat t a i l a rteries constricted i n s i t u with norepinephrine at high and low intravascular pressure , 86 x i FIGURE 16. 17. 18. 19- 20. 21. 22. 23-25. 26. Page Effluent flow rate for rat t a i l a r t e r i e s constricted i n s i t u with norepinephrine at high and low intravascular pressure . . 87 Intravascular pressure and % transmission of the effluent from a rat t a i l artery i n s i t u after the addition of norepine- phrine . . . . . . . 92 Water content of rat t a i l a r t e r i e s equilibrated i n 1 of 5 isosmotic solutions of d i f f e r e n t i o n i c composition . 100 Effect of temperature during rewarming on the H2O content of the rat t a i l artery cooled for 18 hours at 2°C . . . . . . . . 108 Effect of temperature during rewarming on the Na content of the rat t a i l artery cooled for 18 hours at 2°C . . . . . . . . 109 Effect of temperature during rewarming on the K content of the rat t a i l artery cooled for 18 hours at 2°C . . . . . . . . 110 Effect of external Na concentration on the change i n water content and on the si z e of the extra, non-K linked* Na component extruded from the rat t a i l artery during 15 minute rewarming periods between 2° and 37°C . . . . . . . . . 114 Intravascular pressure of rat t a i l a r t e r i e s during rewarming.. . . . . . . . . . . . . . . . 121— 123 Schematic representation of the hydrostatic pressure and the osmolarity across the w a l l of a d i s t r i b u t i n g artery. . . . 133 x i i ACKNOWLEDGEMENTS For advice, patience, support, technical assistance, 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 la t e Mr. C.G. Lemon, Mr. J . Lewis, Dr. C. Loeser, Mrs. C. MacDonald, Miss M. Mar, Dr. V. Palaty, the l a t e Dr. G.H. Scott, Mrs. G. Spieckermann, Dr. M.C. Sutter, 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 spirally oriented smooth muscle ce l l s immersed in a net- work of collagen, elastin, and protein-polysaccharides. The inside of the wall i s lined with endothelial c e l l s butted against an elastic lamina and the outside i s a loose coating of collagen fibers, the adventitia. The volume of the artery lumen can be passively altered by changes i n the intra- luminal pressure. In addition, very fine nervous and humoral controls can change the degree of tension i n the artery wall by changing the activity of the smooth muscle c e l l s , resulting i n an active change i n lumen volume. This means that the artery wall regulates blood flow by varying the volume i t encloses. Vasoconstriction i s a decrease in the size of the a r t e r i a l lumen due to an increase i n active tension: both r a d i i of the artery wall decrease while the wall thickness increases. By possessing sufficient e l a s t i c i t y , elastin acts in cooperation with the smooth muscle to allow the total wall tension to automatically change as the artery changes i t s size (1). This means that the artery i s capable of very fine gradations i n i t s degree of constriction (1). Consequently, there are a multitude of physical states of the wall during the transition from relaxed to f u l l y constricted states of the artery. The wall can thus undergo very slight or very drastic physical changes between these different states (see 2). These changes 2 occur quickly and are thus much more d i f f i c u l t to study than the very slow changes that occur with age (3) and disease (4,5). An understanding of the physical changes i n the artery w a l l asso- ciated with c o n s t r i c t i o n could lead to an understanding of how the components of the artery w a l l work together. Isolated effects of vasoconstriction have been considered: morphological changes (2,6), ion movements with respect to the artery w a l l (7,8) and the smooth muscle c e l l s (9,10), membrane potential changes (11), v i s c o - e l a s t i c changes (12,13), and hemodynamic changes (14). In addition, 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 vasoconstrictive agents (19). However, a synthesis of these i s o l a t e d findings i s not yet possible. One of the problems i s that few of the studies attempt to r e l a t e the observed effects of c o n s t r i c t i o n to the action of the whole w a l l . The dynamic nature of the artery 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 pro- pagation 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 hyper-reactivity of the hypertensive artery w a l l (21,22,23), and i n various models depicting the interactions 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 attention i n regard to vaso- c o n s t r i c t i o n i s the water i n the artery w a l l . For a muscular artery, about 75% of i t s weight i s water (26): water f i l l s the smooth muscle c e l l s and 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 structure of the c o n s t r i c t i n g artery w a l l . 3 There have been observations of losses of water from the artery w a l l during vasoconstriction. 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 rat 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 uterine muscle. Turker et a l . (30) observed that angiotensin-induced c o n s t r i c t i o n of both carotid artery and uterus s t r i p s resulted i n a large 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 co n s t r i c t i n g artery w a l l have also been reported (7,31,32). However, the only discussion i n these 7 papers of the implications of t h i s finding was Rorive's comment that the water loss may have been due to 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 tissue compartments (28). The reluctance to consider the implications of f l u i d leaving the con- s t r i c t i n g artery w a l l may arise from the assumption that the w a l l has a constant volume during vasoconstriction (13,33). However, t h i s assumption was carried over from Lawton's observation on the incompressibility of the artery w a l l subjected to small strains (34) which has nothing to do with vasoconstriction. There are i n fact two d i s t i n c t processes: stretch, to which the artery w a l l responds passively without the expenditure of energy (1), and vasoconstriction, which i s an active process a r i s i n g within the wall i t s e l f , requiring energy, and involving large s t r a i n s . [This d i s t i n c - t i o n , of course, i s for stretch without a myogenic response (see 35).] Examination of the effect 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 better understanding of how the artery c o n s t r i c t s . Models of the artery 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 sol i d s which act as a unit to a l t e r the degree of co n s t r i c t i o n (1,24,25). There may be an analogy between the artery w a l l and a gel or ion exchange res i n : the degree of swelling, i . e . the water content, i s affected by the e l a s t i c forces within the network of the gel (36,37). Alterations i n the e l a s t i c forces a l t e r the hydration of the gel. S i m i l a r l y , changes i n ar t e r - i a l water content may imply al t e r a t i o n s i n the e l a s t i c forces within the artery w a l l . This point i s discussed more f u l l y i n Chapter VI. The increased water content of hypertensive a r t e r i e s (21,26) may be related to the increased Na and mucopolysaccharide contents (26,38). Know- ledge of the factors affecting normal a r t e r i a l hydration may help i n under- standing some of the physical changes associated with hypertension. In t h i s study, the relationship between vasoconstriction and a r t e r i a l hydration was examined from several d i f f e r e n t experimental angles. Some of the aspects considered were: the constancy of the volume of the c o n s t r i c t i n g artery w a l l (Chapter I I ) , changes i n size and shape of the contracting vascular smooth muscle c e l l s (Chapter I I ) , the effect of c o n s t r i c t i o n on a r t e r i a l hydration (Chapter I I I ) , the relationship between the c o n s t r i c t i n g artery 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 IV), and the role of the paracel l u l a r matrix i n a r t e r i a l hydration during ion exchange and cooling-rewarming procedures (Chapter V). The i n i t i a l experiments compared the extremes: relaxed and constricted a r t e r i e s , while the l a t e r experiments considered the process of vasoconstriction. Since the whole vascular system, perfused with blood, was much too complex for this i n v e s t i g a t i o n , isolated a r t e r i e s perfused with a physio- l o g i c a l solution 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 rat t a i l artery. Vasoconstriction was 5 induced by di f f e r e n t agents which apparently have di f f e r e n t mechanisms of action (for example, 19). Since t h i s study was concerned with 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 differences between the agents were only b r i e f l y considered. Adult male albino rats 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 rats were anesthetized with 3.33mg sodium pentobarbital per 100 g body weight administered i n t r a - p e ritoneally, and 6 mg sodium phenobarbitone per 100 g administered sub- cutaneous l y . The methods and l i t e r a t u r e for each of the experiments are given i n the separate chapters. The f i n a l chapter contains a discussion which relates the i n d i v i d u a l findings to the o v e r a l l problem of vasoconstriction and a r t e r i a l hydration. The references i n the Bibliography are arranged according to the chapters. 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 sections (5,6,7). By providing information on the appearance of the artery i n a given physiological state, these methods are a good complement to the usual biochemical and physiological approaches. Baez has examined relaxed and constricted microvessels jLn vivo (2) but there i s very l i t t l e data on dimensional changes of co 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 dimensions of a co n s t r i c t i n g artery: (a) dir 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 sections (see Appendix I I ) , (c) h i s t o l o g i c a l procedures to f i x the artery i n a known functional state. I t was found that the best method for determining vascu- l a r dimensions was to use h i s t o l o g i c a l sections of freeze-substituted a r t e r i e s . A. CHANGES IN DIMENSIONS OF ARTERY WALL DURING CONSTRICTION I t i s usually assumed that the cross-sectional area of a con s t r i c t i n g artery i s constant (6,7). However, there are indications that t h i s may not be so (8,9). Using freeze-substituted a r t e r i a l sections, the present study examined: (a) the wall cross-sectional 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 effects of dif f e r e n t vasoconstrictive agents on these dimensions. 7 1. Methods The ventral t a i l artery of the rat was exposed at the base of the t a i l for cannulation. To remove neurogenic influences, the whole t a i l was removed from the rat. The t a i l artery was then perfused with Krebs solution (see Chapter I I I for composition) at 37°C using a constant infusion pump. The intravascular pressure was monitored with a Statham transducer proximal to the cannula. A segment of the artery 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 artery was photographed and i t s outer diameter measured using a micrometer eyepiece. The artery was then frozen i n less 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 artery was cut free and plunged with the attached f o i l and probe into absolute alcohol at -80°C. Fi x a t i o n was by freeze substitution (5,10). The artery was transferred to a solution of 1% osmic acid and absolute alcohol at -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 with absolute alcohol at the same temperature as the rewarming artery. In th i s manner the problem of refreezing any water remaining i n the artery was at least 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 cross-section, and stained with Mallory trichrome. The above procedure was done i n cooperation with the l a t e Dr. G. Scott. Colour photographs of the artery cross-sections were projected onto a table top. From these projections, the dimensions of the cross-sections were measured with a planimeter. The measurements were calibrated using the projection of a micrometer scale photograph. The t o t a l magnification was 458 x for most sections and 284 x for the larger sections. 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 for comparison with those measured i n s i t u through the microscope and with those calculated from the h i s t o l o g i c a l sections. 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 rat t a i l artery at 8, 5, and 2 cm from the base of the t a i l res- pectively. The three segments were treated i n the manner described above. The middle artery segments were perfused with Krebs solution to which vaso- c o n s t r i c t i v e drugs had been added. A constant infusion pump (B. Braun) was used for the perfusion. These middle segments were photographed before and after the drug application. Two spots of India ink placed on these segments enabled the photographs to be used for determining any changes i n the lengths of the segments. The following 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 artery segment, were used: 1. untreated 2. norepinephrine (levarterenol b i t a r t r a t e , Winthrop), 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 mU) 5. angiotensin (angiotensin I I amide, Hypertensin, Ciba), non-pressor dose (20 ug) 6. angiotensin, pressor dose (150 ug) 7. low Na solution (Krebs solution with a l l but 1.5 meq N a / l i t e r replaced by lactose). There was no increase i n the intravascular pressure of artery segments treated with non-pressor doses of PLV-2 or angiotensin. Altogether, 126 artery segments were processed. 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 = cross-sectional area of lumen + wal l A E = cross-sectional area of lumen + media A^ = cross-sectional area of lumen There was some d i f f i c u l t y measuring AQ because the outside of the adventitia was very i r r e g u l a r and occasionally torn or broken o f f . Quite accurate measurements of A E were made because of the sharp colour contrast between the media (red) and the adventitia (blue). Except for the very constricted sections, A^ was e a s i l y measured. From these 3 cross-sectional areas, the following calculations were made: Aw = cross-sectional area of the artery w a l l = A Q - A i &m = cross-sectional area of the artery media = A E - A i (includes intima) Aad = cross-sectional area of the adventitia = A Q - A e ro = outer radius of the w a l l = (AO/TT) r i = inner radius of h the w a l l = (A±/u) r e = outer radius of the media = (A e/ir) R = wa 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 adventitia = r Q - r e The films 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 ranking was based upon the appearance of the artery cross-sections (see 5 ) : ( 1 ) relaxed a r t e r i e s : t h i n w a l l with a 10 smooth in t i m a l surface and long thin smooth muscle c e l l s and n u c l e i , (2) s l i g h t l y constricted a r t e r i e s : s l i g h t l y thickened w a l l with occasional s l i g h t wrinklings i n the intima, (3) moderately constricted a r t e r i e s : thicker w a l l with a wrinkled intima, (4) f u l l y constricted a r t e r i e s : small lumen and very thick 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 calculations were quite small. To estimate the error, an a r t e r i a l section was photographed at the high and low ma g n i f i c a t i o n s — f o r maximum differences i n the measured and c a l - culated values. The largest difference for 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 con- s t r i c t i o n . Before and after the application of the vasoconstrictive agent, the separation of the 2 India ink spots on the middle segment was measured. The differences between the f i n a l and i n i t i a l separations were zero for 13/33 measurements, p o s i t i v e for 8/33 (maximum 1.5%), and negative for 12/33 (maximum -2.2%). The average separation before the drug was 1.87 i 0.006 mm (19.9 cm on the projection). The average length change associated with the application 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 error i n the measurements which was about i 0.1 cm on the projections, or i 0.01 mm on the a r t e r i a l segments. Of these 33 middle artery segments, 3 were classed as relaxed, 9 s l i g h t l y constricted, 9 moderately constricted, and 12 were f u l l y constricted. These s t a t i c measurements indicate that the length of these a r t e r i e s did not change. Fig. 1 Four degrees of constriction of the rat t a i l artery: 1. relaxed, 2. s l i g h t l y constricted, 3. moderately constricted, 4. f u l l y constricted. 12 Since the artery segments were tethered at either end, t h i s result was not un- expected (see 11). Constant length means that the cross-sectional area of the artery 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 for the three artery 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 vasoconstrictive agents while the middle segments were. However, the middle segments were not more co n s t r i c - ted than the other two. There was simply a spread of the various degrees of co n s t r i c t i o n for 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 constricted 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 reaction to the treatment of the middle segment. The constricted appearance of the d i s t a l segments suggests that they may have been hyper-reactive or that the rat t a i l artery i s usually f a i r l y constricted. In any case, for the primary analysis, the 126 sections were grouped together, regardless of the cause of c o n s t r i c - t i o n . The relationships 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 indicate that the w a l l to lumen r a t i o i s a good index of vasoconstriction (see 5,12,13). Fig. 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. F i g . 3 i l l u s - trates the r e l a t i o n between the w a l l to lumen r a t i o and the cross-sectional areas of the wa l l and media, using the average values for the 4 states of co n s t r i c t i o n rather than a l l 126 pairs of values. F i g . 3 also shows that TABLE I. Dimensions (±S.E.) of wal l of rat t a i l artery i n various states of cons t r i c t i o n , calculated from h i s t o l o g i c a l cross-sections. The number of sections i s given i n parenthesis. Overall 1 relaxed 2 «**8htly 3 moderately 4 f u l l y constricted constricted constricted r Q (y) outer radius a l l 3 segments (126) 244 ± 5 (19) 321 ± 8 (30) 292 ± 7 (22) 242 ± 7 (55) 194 ± 4 d i s t a l (42) 194 ± 5 (0) (3) 254 ± 22 (3) 236 ± 11 (36) 186 ± 4 middle (42) 247 ± 8 (4) 335 ± 19 (13) 285 ± 10 (10) 230 ± 11 (15) 203 ± 6 proximal (42) 291 ± 7 (15) 318 ± 9 (14) 300 ± 8 (9) 258 ± 11 (4) 237 ± 25 r^ (p) inner radius a l l 3 segments (126) 135 ± 8 (19) 255 ± 8 (30) 211 ± 6 (22) 131 ± 9 (55) 52.7 ± 4. 1 d i s t a l (42) 54.1 ± 7 (0) (3) 180 ± 24 (3) 107 ± 2 (36) 39.2 ± 3. 2 middle (42) 146 ± 12 (4) 276 ± 21 (13) .211 ± 10 (10) 121 ± 14 (15) 71.8 ± 5. 3 proximal (42) 204 ± 9 (15) 250 ± 8 (14) 218 ± 8 (9) 149 ± 15 (4) 103 ± 23 6 (y) wall thickness a l l 3 segments (126) 110 ± 3 (19) 66.3 ± 2.5 (30) 77.3 ± 1.8 (22) 111 ± 4 (5) 142 ± 2 d i s t a l (42) 140 ± 4 (0) (3) 73.3 ± 5.9 (3) 130 ± 9 (36) 147 ± 3 middle (42) 101 ± 5 (4) 58.5 ± 4.0 (13) 73.5 ± 2.4 (10) 108 ± 6 (15) 132 ± 3 proximal (42) 87.6 ± 4 (15) 68.4 ± 2.8 (14) 81.7 ± 2.4 (9) 109 ± 6 (4) 133 ± 8 d (p) media thickness a l l 3 segments (126) 67.4 ± 2. 2 (19) 36.8 ± 1.4 (30) 43.8 ± 1.3 (22) 68.9 + : 2.8 (55) 90.3 ± 1. 8 d i s t a l (42) 90.4 ± 2. 9 (0) (3) 40.3 ± 0.7 (3) 82.0 ± ; 7.5 (36) 95.3 ± 2. 0 middle - (42) 61.2 ± 3. 2 (4) 31.3 ± 2.2 (13) 42.1 ± 1.2 (10) 67.2 ± : 3.4 (15) 81.8 ± 2. 8 proximal (42) 50.6 ± 2. 5 (15) 38.3 ± 1.4) (14) 46.1 ± 2.3 (9) 66.4 ± 4.3 (4) 77.3 ± 3. 6 TABLE I. (Continued) Overall 1 relaxed 2 s l i g h t l y constricted 3 moderately constricted 4 f u l l y constricted A w (10 3y 2) wall area a l l 3 segments d i s t a l middle proximal (126) 118 ± 2 (42) 107 ± 4 (42) 115 ± 3 (42) 131 ± 4 3 2 Am (10 y ) media area a l l 3 segments d i s t a l middle proximal (126) 61.4 ± 1.6 (42) 54.0 ± 2.5 (42) 60.7 ± 2.3 (42) 69.5 ± 2.8 R = 6/2r^ w a l l to lumen r a t i o a l l 3 segments d i s t a l middle proximal (126) 0.968 ± 0.100 (42) 2.075 ± 0.199 (42) 0.548 ± 0.063 (42) 0.278 ± 0.038 (19) 121 ± 6 (0) (4) 111 ± 8 (15) 123 ± 7 (19) 63.8 ± 3.6 (0) (4) 57.0 ± 4.6 (15) 65.0 ± 4.2 (19) 0.132 ± 0.006 (0) (4) 0.110 ± 0.013 (15) 0.138 ± 0.006 (30) 122 ± 4 (3) 99.1 ± 12 (13) 115 ± 6 (14) 133 ± 6 (30) 64.9 ± 3.3 (3) 50.7 ± 6.2 (13) 61.3 ± 3.2 (14) 71.3 ± 5.6 (30) 0.189 ± 0.008 (3) 0.220 ± 0.044 (13) 0.179 ± 0.011 (14) 0.191 ± 0.008 (22) 128 ± 5 (3) 141 ± 15 (10) 117 ± 7 (9) 136 ± 7 (22) 70.3 ± 3.5 (3) 76.4 ± 9.2 (10) 65.0 ± 5.9 (9) 74.1 ± 3.9 (22) 0.500 ± 0.052 (3) 0.606 ± 0.036 (10) 0.545 ± 0.094 (9) 0.416 ± 0.062 (55) 110 ± 4 (36) 106 ± 4 (15) 114 ± 5 (4) 143 ± 25 (55) 55.1 ± 2.1 (36) 52.4 ± 2.5 (15) 58.2 ± 3.7 (4) 68.0 ± 9.6 (55) 1.867 ± 0.158 (36) 2.353 ± 0.195 (15) 0.987 ± 0.068 (4) 0.801 ± 0.195 4^ 15 2.0 r DEGREE OF VASOCONSTRICTION Fig. 2 Relation between wall to lumen ratio, R, inner and outer r a d i i , r^ and r Q , and the degree of vaso- constriction, 1, 2, 3, or 4, for the rat t a i l artery. The number of artery sections i s given by n. 160 • 140 • 4 0 - 20 • i i i i i i i i i i i i i 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 WALL TO LUMEN RATIO R . 3 Relation between the cross-sectional areas of the rat t a i l artery w a l l , Â ,, and media, A m, and the wa l l to lumen r a t i o , R. 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 also reflected 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 (for n = 126) between: 1. A„ and R: r = -0.412 (p < 0.001); and R: r = -0.444 (p < 0.001) 2. A w 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 Q : r = +0.609 (p < 0.001); and r Q : r = +0.616 (p < 0.001) These s i g n i f i c a n t correlations mean that the more constricted a r t e r i e s (with larger 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 cross-sectional 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 constricted states, 3 2 and by 15.2 x 10 u , or 22%, between the moderately and f u l l y constricted 3 2 states. Although i t s o v e r a l l decrease of 11 x 10 u , or 9%, between the r e - laxed and f u l l y constricted states was 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%, between the moder- ately and f u l l y constricted states. Over 80% of t h i s decrease i n the w a l l area can be accounted for by the decrease i n the media area. Since the length of these segments was constant, the decrease i n the cross-sectional area means that vasoconstriction was associated with a decrease i n the volume of the artery w a l l of about 14%. As shown i n the columns of Table I, the artery decreases i n s i z e d i s t a l l y . Since the d i s t a l segments were more constricted 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 indicates that the decrease i n the wall area was associated with vasoconstriction, not with the w a l l taper. Fig . 4 shows the inverse relationship between the area of the media, A m, and the w a l l to lumen r a t i o , R, for the d i s t a l segments. These segments were obtained from the t a i l artery before any drugs were applied. The larger values of R were d e f i n i t e l y associated with the smaller values of A m (n = 42, r = -0.479, so p < 0.01). A s i g n i f i c a n t c o r r e l a t i o n was also obtained for A w vs R for the d i s t a l segments, and for A m vs R for the middle segments. (On the other hand, there was no c o r r e l a t i o n between R and the areas for the proximal artery segments. The distended appearance of many of these proximal segments suggested that they may have reacted to the treatment of the middle segments.) These relationships mean that the taper of the t a i l artery was not the cause of the observed decrease i n the w a l l cross-sectional area. Rather, the walls of the more constricted 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 vasoconstrictive agents did not have the same effect 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 highly s i g n i f i c a n t differences 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. The group treated with a non-pressor dose of angiotensin was unique. I t was the only group for 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 positive for R vs A w and for R vs Am« This suggested that a f t e r a non- pressor dose of angiotensin, a r t e r i e s which had constricted had larger cross-sectional areas than arte r i e s which were less c o n s t r i c t e d — t h e opposite to the observation for a l l 126 a r t e r i e s and for two of the segments. 19 \R = 4.l47-0.038Am 0.5 1.5 2.0 2.5 3.0 35 WALL TO LUMEN RATIO R 4.0 4.5 5.0 5.5 F i g . 4 Relation between the cross-sectional area of the artery media, A^, and the w a l l to lumen r a t i o , R, for the d i s t a l segments of the rat t a i l artery. 2. Some of the ar t e r i e s were f u l l y constricted even though they showed no pressure r i s e to a non-pressor dose of PLV-2. These arte r i e s were unusual. Of the 15 f u l l y constricted 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 wal l area of these 3 were larger than those for the other 12 segments. The difference between these 2 groups was s i g n i f i c a n t only at p < 0.05. These differences mean that the f u l l y constricted a r t e r i e s which were given a non-pressor dose of PLV-2 had larger w a l l volumes than expected. 20 3 2 TABLE I I . Outer radius (y) and cross-sectional area (10 y ) of the w a l l of f u l l y constricted middle segments of the rat t a i l artery. A l l segments PLV-2 (non-pressor) segments other 12 segments Aw (15) 203 ± 6 (15) 114 t 5 (3) 225 + 8 (3) 135 I 7 (12) 198 + 6 (12) 109 - 5 These two points suggest that non-pressor doses of angiotensin and PLV-2 a l t e r the geometry of constricted a r t e r i e s i n a manner d i f f e r e n t than other vasoconstrictive agents. There i s a suggestion that they increase the volume of the constricted artery w a l l . In spite of this f a c t , omitting one or both of these groups of 6 a r t e r i e s did 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) relationships for the 126 sections or for the middle a r t e r i a l segments. 3. Effect of Fixation and Embedding on A r t e r i a l Dimensions Since the above findings depended upon measurements made from h i s t o l o - g i c a l sections, i t i s important to know the effect of preparative handling on the a r t e r i a l dimensions. The only measureable factor was the effect on the outer r a d i i of the vessels. This determination led to an estimate of the shrinkage caused by the freezing, substitution, and embedding processes (see 14). The outer r a d i i of the ar t e r i e s i n s i t u were measured: (a) from pro- jections of photographs, arid (b) through a microscope with a micrometer scale. These values were compared to those calculated from the h i s t o l o g i c a l 21 sections. These 3 outer r a d i i w i l l be labelled as: r 0 ( f i l m ) , r Q(observed), and r Q ( h i s t o l o g y ) , respectively. The 3 values of r Q can be compared since the measurements were a l l made i n approximately the same spot on the artery segments: r 0 ( f i l m ) was measured i n the middle of the photographs, r Q(observed) was determined for the middle of the segment, and r Q ( h i s t o l o g y ) was calcu- lated for sections cut from the middle of the segments. The maximum errors i n these values were: ± 10 u i n . r 0 ( f i l m ) , ± 25u i n r Q(observed), and i 2 u i n r Q ( h i s t o l o g y ) . 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 radius, r Q ( i n u), of the rat t a i l artery 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. o v e r a l l proximal middle d i s t a l segments segments* segments r Q ( f i l m ) (157)** 310 ± 6 (40) 373 + 8 (41) 317 ± 9 (41) 232 ± 5 r Q(observed) (159)** 290 + 5 (40) 355 + 7 (41) 287 ± 10 (42) 223 + 5 r Q ( h i s t o l o g y ) (126) 244 + 5 (42) 291 ± 7 (42) 247 ± 8 (42) 194 + 5 * post-drug measurements ** includes pre-drug measurements of the middle segments 1. The differences between r Q ( f i l m ) and r Q(observed) were s i g n i f i c a n t (by the Student's t-test) only for the proximal artery segments (which had the largest diameters). That these 2 i n s i t u values should be di f f e r e n t was not unexpected, but why the average values of r 0 ( f i l m ) were larger than those of r Q(observed) can not be explained. 2. The values of r Q ( 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 either r 0 ( f i l m ) or r (observed). The size of the differences indicated 22 that the ar t e r i e s did i n fact 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 cross-sectional areas of the artery w a l l plus lumen, A Q, before and after the h i s t o l o g i c a l preparation. The average values of A Q, calcu- lated from the corresponding values of r 0 , are given i n Table IV. TABLE IV. The cross-sectional area of the rat t a i l artery 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 sections. o v e r a l l " proximal middle d i s t a l segments segments segments (157) 318 t 11 (40) 444 ± 18 (41) 327 + 19 (41) 173 ± 8 (159) 280 ± 10 (40) 403 + 16 (41) 273 ± 19 (42) 160 + 7 (126) 199 ± 9 (42) 273 ± 12 (42) 201 + 14 (42) 122 ± 7 A Q ( f i l m ) AQ(observed) AQ(histology) The differences 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 Q ( h i s t o l o g y ) - A Q ( f i l m ) = -119 x 10 u , a change of -37% 3 2 AQ(histology) - AQ(observed) = -81 x 10 p , a change of -29% The average decrease i n the cross-sectional area of the artery w a l l plus lumen after freezing, 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 cross- sectional area of the whole section, not just i n the artery w a l l . However, i t was possible to measure the inner radius of the artery w a l l , r ^ , from the photographs of 3 segments. Since the cross-sectional area of the wa l l i s A W = i r ( r 0 ^ - v±^), i t was possible to compare A W for the a r t e r i e s i n s i t u with Ay calculated from the h i s t o l o g i c a l sections. The changes i n cross- 23 3 2 sectional area ( i n 10 y ) after 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 w: + 59.3, (b) A Q: -24.2, A„: +42.7, (c) A<3: -40.4, A w: -15.8. While i t i s not possible 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 indicate 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^. In addition, since the lengths of the artery segments were not measured af 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 cross-sectional area of the artery sections can not be extended to a de- crease i n the volume of the section, or of the wal l i t s e l f . 4. Discussion The cross-sectional areas of the walls of relaxed and constricted ar t e r i e s have been measured before. However, either no change i n w a l l area with vasoconstriction was observed, or the changes were not mentioned. Using his image-splitting 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 with epinephrine or norepinephrine, he did observe an increase i n w a l l area after vasodilation with acetylcholine. Wiederhielm reported that 43 measurements (S.D. 1 10%) from photographs showed no s i g n i f i c a n t difference between the w a l l areas of relaxed and constricted a r t e r i o l e s (7). On the other hand, there i s some support for a decrease i n a r t e r i a l w a l l volume during vasoconstriction. From X-ray photographs, Ticker and Sacks (9) measured the r a d i i and lengths of human brachial and dog thoracic ar- teries i n f l a t e d with a i r . With increased intraluminal pressure, the wal l volume decreased. 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% for the relaxed muscles and 600 A and 24 5% for contracted muscles. Hinke (6) measured the cross-sectional areas of perfused and non-perfused rat 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 sections. Although not mentioned by him, the wal l to lumen r a t i o s of these vessels show that the non-perfused a r t e r i e s , which were more constricted ( r a t i o = 3 2 1.22), had a smaller cross-sectional wall area (46.5 x 10 u ) than the per- 3 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 Luft (15) compared the appearances of relaxed and constricted frog a r t e r i o l e s . Two of t h e i r electronmicrographs show relaxed and constricted pdrtions of the same a r t e r i o l e . Planimeter measurements of these figures reveal that the constricted vessel (wall to lumen r a t i o = 0.738) had an 18% smaller w a l l cross-sectional area than the relaxed vessel ( 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 "tethering action afforded by the tissue structures surrounding the microvessel" (2). Since the same observa- t i o n was made i n th i s study for ar t e r i e s separated from t h e i r surroundings, th i s suggestion seems u n l i k e l y . The increase i n wal l thickness i s more l i k e l y due to the mechanism of co n s t r i c t i o n of the vascular w a l l (see 16). A decrease i n the cross-sectional area and volume of the artery 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. This suggestion w i l l be examined i n the next chapter. 5. Summary 1. Freeze substitution was a good method for f i x i n g a r t e r i e s i n var- ious states of c o n s t r i c t i o n . 2. The process of freezing, dehydrating, and embedding the a r t e r i e s was associated with a decrease of 1/3 i n the t o t a l cross-sectional area of the artery 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 for the relaxed rat t a i l artery to 388 y for the f u l l y constricted a r t e r y — a decrease of 40%. The diameter of the lumen decreased from 510 to 105 y —a decrease of 80%. 4. The w a l l to lumen r a t i o was a good index of the state 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 for about 50% of the artery w a l l cross-sectional area. Since the adventitia was very loose, the % by weight was probably higher. 6. The length of these tethered a r t e r i a l segments did not change af t e r vasoconstrictive agents were applied. Thus, changes i n the artery w a l l volume were refl e c t e d i n changes i n the cross-sectional area. 7. The more constricted 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 cross-sectional areas than less constricted a r t e r i e s . Vasoconstriction was associated with a decrease i n the volume of the artery w a l l of about 14%. 8. 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 artery w a l l . 9. Non-pressor doses of angiotensin and PLV-2 altered the geometry of constricted a r t e r i e s i n a manner di f f e r e n t than other vasoconstrictive agents. B. CHANGES IN DIMENSIONS OF SMOOTH MUSCLE CELLS DURING VASOCONSTRICTION Very l i t t l e has been done on characterizing 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 rat a r t e r i o l e — t h e greater i n - crease for the greater dose of norepinephrine. Using a model for the smooth muscle c e l l and measurements from h i s t o l o g i c a l sections, a more detailed 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. Mathematical Model for the Smooth Muscle C e l l As a model for the smooth muscle c e l l , consider 2 ri g h t c i r c u l a r cones joined at t h e i r bases: For the dimensional parameters of the c e l l , l e t : 2 2 length = 2h volume = V = y irr h diameter at center = 2r surface area = A = 27rr(r 2 + h ^ j 5 = 27rrh The approximation for 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 artery are such that ĥ >> r ^ ( 8 ) . This approximation w i l l be examined l a t e r . During contraction of the smooth muscle c e l l these variables w i l l change. Let A refer to the changes i n these variables. For x, any c e l l u l a r variable, Ax = x (after contraction) - 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 possible 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 half-length. Change i n c e l l surface area: A A = A(2frrh) = 2-rr(rAh + hAr) = 2iT(rAh + hAr + ArAh) 27 This equation i s for the % change i n the c e l l surface area as a function of the % changes i n the c e l l radius and length. Change i n c e l l volume: AV = A(|irr 2h) = "y(r 2Ah + 2rhAr) 2TT_ 3 Ah(r 2 + 2rAr + (Ar) 2) + Ar(2rh + hAr) AV Ah . 2Ar . 2 Ar Ah 1 A - 1 2 '•• T • T * =? * r • [T] [ ' • ¥ ] <•' Substituting equation (1) into equation (2): i2 AV 2AA Ah V A 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 al 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 c i r c u l a r cylinder: T H 2r 9 Let the c e l l parameters be: 2 length = H volume = V = irr H diameter = 2r surface area = A = 2irrH Change i n c e l l surface area: AA = A(2TrrH) = 2ir(rAH + HAr) = 2TT (rAH + HAr + ArAH) AA AH , Ar , Ar AH So, — = — + — + — — ' A H r r H ™ . ' - • • «.u ,v\ • A H A(2h) Ah This equation i s the same as equation (1) since: — = — r r - — = -r- H zn h 28 Si m i l a r l y for the change i n c e l l volume: AV _ AH 2Ar 2Ar AH r ^ I . " | 2 r i + M l V H r r H L r J |_ 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 substituting 2h for H i n the above 2 equations, so that: A(cyl) = 2 A(cones) and AA(cyl) = 2AA(cones), so that: V(cyl) = 3 V(cones), and AV(cyl) = 3AV(cones), so that: M m / A V \ V V /cyl \ V J cones In addition to giving the magnitude of the changes i n the c e l l area and volume, equations (1) and (2) contain information 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 for a given artery, the values of the radius and length of the smooth muscle c e l l s i n the relaxed state ( i . e . r and h) are f i x e d , at least within a f a i r l y narrow range. Thus, the . magnitude of Ar and Ah determine the signs of AA and AV. I t i s possible to construct graphs r e l a t i n g these variables. For example, once values for r and h have been chosen, AA/A from equation (1) can be plotted against Ah/h for a family of curves of d i f f e r e n t Ar values, or against Ar/r for a family of d i f f e r e n t Ah values. S i m i l a r l y for AV/V from equation (2). Values for r and h are determined i n the following section. 2. Measurement of C e l l Dimensions Methods To obtain the values of the relaxed smooth muscle c e l l radius, r, and 29 length, 2h, for the rat t a i l artery, and to estimate the physiological range of Ar and Ah ( i . e . the difference between the relaxed and contracted values), several 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 magnification was 2900 x. The c e l l half-length (from the middle of the nucle i to the end of the c e l l ) and maximum width through the nuclei of relaxed and contracted c e l l s were measured with a r u l e r . C e l l s without nuc l e i i n the plane of the section and c e l l s cut i n cross- section were ignored. Some of the sections contained both relaxed and con- tracted c e l l s so that comparison of the dimensions i n the 2 states was more meaningful. In addition, 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 cross-section were measured with a planimeter to determine the better model: double cones or cylinder. The area of 1/2 of a c e l l was compared with the calculated areas of a tr i a n g l e (rh) and a rectangle (2rh), since they are the projections onto 2 dimensions of a cone and a cylinder, to see which was the closest 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 rat t a i l artery. relaxed c e l l s contracted c e l l s A 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 From these values , 4r x 100 = -48% n and — x 100 = + 97%. r i 30 The area of the h a l f - c e l l was best approximated by the area of a triangle for both relaxed and contracted c e l l s . So, the double cone model i s the better f i t for these smooth muscle c e l l s . The appearance of the c e l l s agreed with t h i s , although the taper at the extremities 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 res u l t s are only approximate, the values of the c e l l radius and half-length were substituted into the equations for the surface area and volume of the double cone model. The values obtained are given i n Table VI. TABLE VI. Surface area (u ) and volume ( u J ) of relaxed and contracted smooth muscle c e l l s of the rat t a i l artery. relaxed c e l l s contracted c e l l s A surface area, A 446 454 AA = +8 volume, V 261 524 AV = +263 From these values, AA x 1 0 0 = + 2% A and — x 100 = + 101%. So, the model indicates that contraction of these vascular smooth muscle c e l l s was associated with no change i n the c e l l surface area but a doubling of i t s volume. The approximation made for the surface area of the double 2 ? J' cone [ i . e . (h + r ) 2 = h] i s poorest for the contracted c e l l s . But the values were only 1.5% too small, so the approximation seems j u s t i f i e d . I f 2 dif 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 larger. 31 The model predicts 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 vasoconstriction. Figs. 5, 6, 7, and 8 show these changes. The curves were obtained from equations (1) and (2) by substitution of the ex- perimental values for the relaxed c e l l half-length, h = 40.3 y, and radius, r = 1.76 y into these equations. The points on the graphs are those derived from Table VI. Constriction i n the rat t a i l artery would be represented by a straight 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. Figs. 5 and 6 show: (a) For a given decrease i n c e l l length, the greater the c e l l thickening, 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 thickness, 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 thickening, the more pos 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 necessarily accompanied by contraction, or vice versa. 4. Discussion These c e l l s are about the same size as other vascular smooth muscle c e l l s . Baez (17) found that smooth muscle c e l l s i n relaxed rat a r t e r i o l e s were from 2.08 to 2.78 y thick. Using frog a r t e r i o l e s , Phelps and Luft (15) reported that the relaxed c e l l s were 100 y long and 9 y at the widest point. Keatinge (18) found the smooth muscle c e l l s of sheep carotid 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 Figs. 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 half-length, Ah/h, and radius, Ar/r. These graphs were c a l - culated from equations (1) and (2) i n the text. The values for the h a l f - length and radius of the relaxed smooth muscle c e l l s of the rat t a i l artery (h = 40.3 u and r = 1.76 u) were substituted into these equations to obtain the 4 families of curves. Note that the scale of the horizontal axis i s di f f e r e n t i n Figs. 6 and 8 than i n Figs. 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 -1.50 F i g . 6 C e l l surface area changes vs c e l l radius changes F i g . 7 C e l l volume changes vs c e l l length changes -2.00 -1.50 Fig. 8 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 central 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 2 7 of the c e l l i s about 400 y plus another 100 y for numerous inpocketings and pinocytotic v e s i c l e s . " The double cone model was a better f i t for these rat t a i l artery smooth muscle c e l l s than the cylinder model. This i s i n contrast to Rhodin's finding that the " . . . smooth muscle c e l l i n the relaxed state has the shape of a cylinder rather than that of a double cone" (8). However, Phelps and Luft (15) observed that the relaxed c e l l s of a frog a r t e r i o l e were spindle shaped and the contracted c e l l s had "a roughly cubic shape." The constant surface area during contraction of vascular smooth muscle c e l l s refers to the surface area of the double cone model—not necessarily 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- eting 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 involve the inpocketings and pinocytotic v e s i c l e s , this observation of constant surface area of the model during vasoconstriction, refers to the "envelope" of the c e l l , not i t s actual 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 electron micrographs. 37 The increase i n the c e l l volume seems extraordinarily large. But the model r e a l l y only q u a l i t a t i v e l y indicated that vasoconstriction was associ- ated with an increase i n the volume of the smooth muscle c e l l . I t i s assumed that the quantity of solids i n the c e l l s remained constant (19). I f the density of bound water were less than that of free water, an increase i n the amount of bound water i n the contracting 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 into the contracting muscle c e l l was the cause of i t s volume increase. Reasons why water would move into the contracting c e l l w i l l be discussed i n Chapter VI. An increase i n the volume of contracting smooth muscle c e l l s would agree with the effects of non-isosmotic solutions on vascular tissue. Hypotonic solutions cause c e l l swelling and vasoconstriction (19), while hypertonic solutions cause c e l l shrinkage and vasodilation (20). 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 for drug induced tension changes. To explain 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 vasoconstriction. A 5% increase i n c e l l water was observed for the rat t a i l artery constricted with norepinephrine (22), In addition, a l - though Jonsson (19) suggested that active tension i n the rat p o r t a l vein opposed c e l l swelling i n hypotonic solutions, he found 7 to 11% increases i n the c e l l water of veins constricted with 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 - tions 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 vasoconstriction (see Chapter I I I ) . 38 These predictions 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 turn out to be di f f e r e n t than those predicted. I f so, i t would s t i l l remain to explain exactly how the changes i n geometry of the contracting c e l l d i f f e r from those of the models. 5. Summary 1. Geometrical models of the vascular smooth muscle c e l l provide a good framework for understanding dimensional changes of the contracting c e l l . 2. During c o n s t r i c t i o n of the rat t a i l artery, the smooth muscle c e l l length decreased by half 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 while 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 right c i r c u l a r cones joined at th 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 radius. 5. The model indicated that the surface area of the smooth muscle c e l l s was constant while the volume of the c e l l s increased during vasocon- s t r i c t i o n . 6. The increase i n volume implied that the contracting 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 cross-sectional area of the constricted artery w a l l , discussed i n the previous chapter, suggests the existence of a decrease i n the water content of the cons t r i c t i n g artery w a l l . A few studies have demonstrated such a water loss from contracted smooth muscle, but they dealt mainly with 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 with vasoconstriction 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 their 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 di f f e r e n t states, whether normotensive and hypertensive or relaxed and constricted, i s quite a d i f f i c u l t procedure. Extreme care i n handling the ar t e r i e s and ade- quate controls 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 co n s t r i c t i n g artery w a l l . The study of io 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 goal. A. METHODS After incubation, f l u i d trapped i n the artery lumen interferes with the determination of the water content. The contribution of th i s f l u i d varies with the lumen s i z e , i . e . the degree of co n s t r i c t i o n . This variable was eliminated by perfusing intraluminally with an O2/CO2 mixture. Use of a par t i c u l a r artery from an inbred s t r a i n of rats s t i l l r e sults i n a large 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 cutting the t a i l a r t e r i e s into proximal and d i s t a l halves so the control and test 40 samples were from the same artery. The test samples were alternately p r o x i - mal or d i s t a l halves. The following incubation experiments d i f f e r i n the treatment of the test samples and the analyses after incubation. The i n i t i a l procedure was common to most of the experiments. The ventral t a i l artery of the anesthe- tiz e d rat was exposed and cannulated with P.E. 50 polyethylene tubing at i t s midpoint. The d i s t a l h a l f - a r t e r y was then flushed with Krebs solution, removed from the t a i l bed and transferred with i t s cannula to either a test or control test tube. The proximal hal f - a r t e r y was next cannulated at i t s proximal end, flushed with Krebs solution, removed from i t s bed, and placed i n the other test tube. Thus each rat i n an experiment contributed 1 test and 1 control h a l f - a r t e r y . Both test tubes contained Krebs solution aerated with 95% O2 and 5% CO2 at 37°C. After a half - a r t e r y had been incubated for 3 hours, the polyethylene tubing carrying the gas mixture into the solution was connected to the cannula of that artery. The artery was then perfused with O2/CO2 for 7 to 10 minutes. Intravascular pressure was monitored with a Statham transducer connected at a T-joint to the tubing carrying the gas mixture to the artery. The pressure was only an estimate of the w a l l tension since an open perfusion system was used. After gaseous perfusion the control a r t e r i e s were removed for analysis. The test a r t e r i e s , handled s i m i l a r l y , were induced to con s t r i c t with various vasoactive agents for various times, then removed for analysis. Maximal pressor doses of the following vasocon- s t r i c t i v e agents were used: norepinephrine (levarterenol b i t a r t r a t e , Winthrop), high K solution (55 meq K / l i t e r , i d e n t i c a l to Krebs solution 2 except 50 mM KCL replaced 50 mM NaCl), and PLV-2 (phenylalanine - l y s i n e - vasopressin, Sandoz). 41 The chemical composition of the Krebs solution, i n meq/liter, was: Na 150, K 5.0, Ca 4.2, Mg 2.4, CI 124, HCO3 25.0, H 2P0 4 1.2. I t also 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 protein substitute). The osmolarity of the Krebs solution was 295 mosmoles/liter. The water content of the art e r i e s was determined from the wet and dry weights (9) and expressed as ml water/100 g fat free dry weight. As defatting the a r t e r i e s decreased t h e i r weight by a ne 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 results 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 artery, as an estimation of the ex t r a c e l l u l a r space, was determined i n two ways: (a) chemical; the Krebs solution for incubation 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 ar t e r i e s was measured by the method of Friedman et al_. (9) . (b) isotope; the ar t e r i e s were incubated i n Krebs solutions containing between 14 14 • 0.01 and 0.06 ucuries/ml of C - I n u l i n . The C content of the a r t e r i e s was determined by l i q u i d s c i n t i l l a t i o n . 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 differences between the s t a t i c water content determinations of ar t e r i e s i n non-constricted (control) and constricted (test) states. (The control a r t e r i e s could not be classed as relaxed since there was no method of ascertaining t h i s , but they were d e f i n i t e l y less constricted than the test arteries.) Although no dynamic measurements of the hydration of a c o n s t r i c t i n g artery were made, the differences i n water content are referred to as "changes'.', for easier understanding of what was happening to the artery w a l l as i t constricted. In the results that follow, A refers to the change i n a parameter with vasoconstriction, i . e . the test value minus the control value. Using control and test samples from the same artery meant that the results could be s i g n i f i c a n t i n two ways: (a) the number of differences for the pairs of h a l f - a r t e r i e s which had the same sign ( + 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 for continuity; (b) the difference, A, between the average content of test and control 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 te s t . This difference 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 vasoconstrictive agent for the f i r s t set of experiments because i t e a s i l y induces f a i r l y strong, short-lived constrictions i n muscular a r t e r i e s , and because i t s action 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 Constric- 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 preliminary while the t h i r d forms the bulk of the r e s u l t s . The dif f e r e n t experimental conditions i n these experiments meant that the water content of the control 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 differences between the absolute values provide the most in t e r e s t i n g i n - formation. 1. Water Content After the addition of norepinephrine (NE) to the incubating solution, a pressure increase could be observed when O2/CO2 passed through the artery segment with i t s branches patent (see Fi g . 11). The f i r s t few arter i e s were 43 treated with 2 ug NE/ml, the remainder, 3 ug NE/ml. The test a r t e r i e s were removed from the Krebs solution when the peak of c o n s t r i c t i o n was observed on the pressure recording, and blotted 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.) for the 6 test a r t e r i e s i n this group was 127 i 38 seconds. Five of the 6 test 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 control 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 for the two groups are shown i n Table VII. TABLE VII. Water contents (ml/100 g dry wt) of rat 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 after 3 hours of aerobic incubation. control a r t e r i e s test (NE) a r t e r i e s A tp < 0.05 t o t a l H20 (6) 283 + 11 254 +12 - 29 + l i t % H20 (6) 73.8 t 7 71.6 ± 0.9 -2.1 ± 0.8+ ( ) number of h a l f - a r t e r i e s + S.E. 1. This experiment showed i t was possible to observe a difference i n water contents of non-constricted control and constricted test a r t e r i e s with this procedure. 2. Constriction induced by norepinephrine was associated with a decrease of about 10% i n the water content of the artery w a l l . 2. Water and Ion Content This study involves a more detailed chemical analysis of the rat 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 . After removal from solution the arte r i e s were cut into 44 small pieces alte 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 for the water, Na, and K contents, the other for the i n u l i n space by the chemical method. These i n u l i n space determinations were unsatisfactory i n a l l 3 experiments since the amount of tissue a v a i l a b l e , about 4 mg wet weight, was too small. 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 test a r t e r i e s were treated with 3 yg NE/ml sol u t i o n , the remainder, 4 yg/ml. Upon removal from solution, the arte r i e s were blotted between two pieces of f i l t e r paper. Nine rats were used i n t h i s experiment. The average duration 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 test h a l f - a r t e r i e s had smaller water contents than t h e i r corresponding control halves ( a l l 9 differences negative i s s i g n i f i c a n t at p < 0.004). Of the 8 pairs of Na and K values, a l l 8 test a r t e r i e s had less Na (p < 0.008) but only 4 of the 8 had less K (not s i g n i f i c a n t by the chi-square t e s t ) . The aver- age results of t h i s experiment are given i n Table VIII. TABLE VIII. Water contents (ml/100 g dry wt), Na and K contents (meq/100 g dry wt) of rat t a i l a r t e r i e s removed from solution 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 a r t e r i e s test (NE) ar t e r i e s A *(p < 0.02) t o t a l H 20 (9) 203 + 6 152 t 10 - 50 ± 9 * % H 20 (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* t o t a l K (8) 11.8 + 1.1 11.6 ± 1.3 45 1. Water Vasoconstriction induced by norepinephrine for about 100 seconds was associated with 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 for the halves. 2. The Na and K contents of the non-constricted control a r t e r i e s were about 5 meq/100 g dry wt higher and lower respectively, than the values found i n the l i t e r a t u r e for the rat t a i l artery (15,16). This suggests that there was a 1:1 exchange of c e l l u l a r K for Na during the 3 hours of incubation, i . e . the smooth muscle c e l l metabolic Na-K pump was operating at less than i t s "normal" l e v e l of a c t i v i t y . 3. Sodium Vasoconstriction was associated with a 29% decrease i n the Na content of the artery w a l l . An i n d i c a t i o n of the source of this 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 equi- librium with the external medium and i s roughly equivalent to the i n u l i n space). This assumption would mean that the 50 ml water leaving the free ECS would have "car r i e d " with 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 free ECS, then more than 4.4 meq Na must have come from compartments other than the free ECS. This assumption and c a l c u l a t i o n says nothing about the movement of Na + into the contracting smooth muscle c e l l s , but deals only with the movement of Na out of the whole artery w a l l during vasoconstriction. 4. Potassium Changes i n the K content of the artery wall during c o n s t r i c - tion could not be measured with t h i s technique. 46 Test arte r i e s removed after 60 seconds of NE c o n s t r i c t i o n In this and a l l the subsequent experiments there was an attempt to obtain larger and more "normal" water contents (15,17) by gentler handling of the a r t e r i e s . Instead of being blotted, the artery was simply placed on f i l t e r paper while i t s cannula was removed. Four yg NE/ml was added to the incubating solutions of the test a r t e r i e s . Eight rats were used i n each of these two experiments. Of the 8 test h a l f - a r t e r i e s , 7 had less t o t a l water (not s i g n i f i c a n t ) , a l l 8 had less t o t a l Na (p < 0.008), and 6 had less t o t a l K (not s i g n i f i c a n t ) than the i r corresponding control halves. The average results ( 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 rat t a i l a r t e r i e s constricted with norepinephrine for 60_ seconds a f t e r 3 hours of aerobic incubation. control a r t e r i e s test (NE) a r t e r i e s A *(p < 0.02) t o t a l H20 (8) 292 + 9 231 ± 15 - 61 ± 13* % H20 (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* t o t a l K (8) 13.9 t 1.0 13.5 + 1.2 After 60 seconds of c o n s t r i c t i o n , the artery 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 carried 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 indicates 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 leaving the free ECS would have also carried about 0.3 meq K along with i t . Test a r t e r i e s removed after 120 seconds of NE c o n s t r i c t i o n Of the 8 test h a l f - a r t e r i e s , 6 had less t o t a l water and less 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 control halves. Four of the 7 test K values were less than t h e i r control 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 wt), Na and K contents (meq/100 g dry wt) of rat t a i l a r t e r i e s constricted with norepinephrine for 120 seconds a f t e r 3 hours of aerobic incubation. control a r t e r i e s test (NE) a r t e r i e s A *(p < 0.02) t o t a l H 20 (8) 264 + 14 219 + 18 - 45 + 19 * % H20 (8) 72.2 + 1.0 67.7 + 2.0 t o t a l Na (8) 44.0 ± 1.6 42.4 ± 1.4 t o t a l K (7) 13.5 t 0.8 11.9 + 1.0 After 120 seconds of c o n s t r i c t i o n , the artery 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 after 60, 97, and 120 seconds of vasoconstriction were 61, 50, and 45 ml water/100 g dry wt respectively. That i s , the amount of water l o s t from the cons t r i c t i n g artery w a l l decreased between 1 and 2 minutes. This suggests that after 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. The follow- ing experiment i s an attempt to confirm t h i s suggestion. 48 2. There was no pattern to the loss 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 loss 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 co n s t r i c t i n g artery 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 after 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 ar t e r i e s were not cut into small pieces as i n the previous experiments. Four experiments were performed i n which the test a r t e r i e s were treated with 4 yg NE/ml solution and allowed to c o n s t r i c t for 15, 30, 60, or 120 seconds. Eight rats were used i n each experiment. The averaged res u l t s of these 4 timed experiments are given i n Table XI. 1. Water A l l 32 constricted test h a l f - a r t e r i e s had less t o t a l water than th e i r corresponding non-constricted control halves (p < 0.001). The greatest water loss was at 30 seconds of c o n s t r i c t i o n . 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% respectively. The o v e r a l l average water loss was 18%. 2. Sodium Of the 30 pairs of Na values, 27 of the test a r t e r i e s had less Na than t h e i r controls (p < 0.001). The greatest Na loss was also at 30 seconds of c o n s t r i c t i o n . The average % Na losses were 13%, 16%, 9%, and 13% respectively. The o v e r a l l average Na loss was 13%. 3. Potassium The K contents were determined only for the 8 pairs of a r t e r - ies i n the 30 sec. experiment. For 5 of these 8 pa i r s , the K content was less TABLE XI. Water content (ml/100 g dry wt) and Na content (meq/100 g dry wt) of arteries after 15, 30, 60, and 120 seconds of norepinephrine-induced constriction i n v i t r o during perfusion with O2/CO2 afte r 3 hours of aerobic incubation. t o t a l H20 % H20 t o t a l Na time (sec) control test A control test A control test A 15 (8) 359 292 -66 78.0 74.3 -3.7 38.2 34.1 -4.8 ± 14 ± 11 ± 18 ± 0.7 ± 0.8 ± 1.1 ± 0.7 ± 0.7 ± 1.0 30 (8) 349 270 -80 77.6 72.8 -4.9 41.7 35.0 -6.7 ± 12 ± 11 ±22 ± 0.6 ± 0.9 ± 1.4 ± 1.1 ± 1.4 ± 1.9 60 (8) 381 317 -64 79.2 76.0 -3.3 37.6 34.1 -3.5 ± 9 ± 7 ± 8 ± 0.3 ± 0.4 ± 0.3 ± 1.1 ± 0.9 ± 1.7t 120 (8) 377 327 -50 78.9 76.5 -2.5 39.2 34.1 -5.1 ± 12 ± 11 ± 10 ± 0.5 ± 0.6 ± 0.5 ± 0.7 ± 0.7 ± 0.8 average (32) 367 302 -65 78.5 74.9 -3.6 39.2 34.3 -4.9 ± 6 ± 6 ± 0.3 ± 0.4 ± 0.6 ± 0.5 p < 0.05, a l l other A are s i g n i f i c a n t at p < 0.01. 50 i n the constricted test halves than i n the controls (not s i g n i f i c a n t ) . There was also no s i g n i f i c a n t difference between the average K contents (- S.E.): control a r t e r i e s , 15.8 t. 0.7 meq K/100 g dry wt; test (NE) a r t e r i e s , 13.4 t 1.0 meq K/100 g dry wt. The res 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 following section. 4. Discussion Changes i n water content 1. Comparison of Tables IX, X and XI shows that the water loss 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 vasoconstriction, even though the absolute water content of the control a r t e r i e s was quite d i f f e r e n t . This suggests that the size of the water loss from the w a l l was dependent on the duration of con- s 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 severity of c o n s t r i c t i o n could not be estimated since the perfusion 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 for the 2 experiments involving 60 seconds and 120 seconds of vasoconstriction 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 loss from the c o n s t r i c t i n g artery w a l l i s shown i n Fig. 9. The values for 15 and 30 seconds are from Table XI, while the values for 60 and 120 seconds are the above averages. The values at zero water loss are for the control a r t e r i e s . Although there were no s i g n i f i c a n t differences between the 4 water losses, after 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 artery w a l l during the f i r s t 30 seconds of NE induced vasoconstric- t i o n , then slowly returned to the w a l l . 51 -90> NE(4.0yu/ml) F i g . 9 Changes i n H 2 O content of rat t a i l artery 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 O 2 / C O 2 perfusion. The bars represent 1 S.E. The points with zero A H 2 O are the control values. The numbers i n parenthesis are the number of art 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 control 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 fa c t , plus the higher t o t a l K content i n t h i s 30 second experiment, suggest that the ar t e r i e s were more viable 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 , for consistency the values were averaged(i S.E.): -6.2 JT 1.3 meq Na at 60 seconds and -3.4 t 0.9 meq Na at 120 seconds of con- s t r i c t i o n . 3. These averaged values and the values from Table XI for 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 loss from the co n s t r i c t i n g artery w a l l . There i s no s i g n i - f i c a n t difference between any of the 4 values of ANa, but there i s a sugges- ti o n that the Na movements are si m i l a r to the water movements: the artery w a l l loses Na for the f i r s t 30 seconds of c o n s t r i c t i o n , then slowly regains i t . 4. The upper curve i n F i g . 10, A(Na) , i s a calculated curve. The values on th i s curve represent the Na losses which would have occurred i f the observed water losses from Fig. 9 were a l l from the free ECS. The c a l - culations were the same as those made above. In F i g . 10, the observed Na loss i s smaller than the calculated 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) for the H2O and Na losses. The average ANa^g = -9.2 meq Na/100 g dry wt. That i s , the loss of 61 ml of water from the free ECS would have carried 9.2 meq Na with i t . However, only 4.8 meq Na act u a l l y l e f t the c o n s t r i c t i n g artery w a l l . There are 2 possible explanations of th i s 53 \ A(Na) ECS MS) I 30 45 60 75 90 105 DURATION OF CONSTRICTION (seconds) (167 1 120 NEWO/i/ml) F i g . 10 Changes i n Na content of rat t a i l artery 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 with zero ANa are the control values. The number i n parenthesis i s the number of art e r i e s used. See the text for an explanation of the A(Na)„„0 curve. s i t u a t i o n : (a) Half the Na i n the 61 ml of free ECS water which l e f t the wa l l , moved from the free ECS to other w a l l compartments. This might include Na + which entered the contracting smooth muscle c e l l s . (b) Some of the water which l e f t the constricting wall came from compartments other than the free ECS. To carry out 4.8 meq Na, about half 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 explain the s i t u a t i o n . This discussion i s , of necessity, 54 s i m p l i s t i c , since the compartments of the artery w a l l are not is o l a t e d and cer 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 leaving the co n s t r i c t i n g artery wall was 70 to 100 meq/liter. Changes i n intravascular pressure 1. The pattern of water and Na losses from the co n s t r i c t i n g artery w a l l was s i m i l a r to the p r o f i l e of the intravascular pressure during NE i n - duced vasoconstriction: a rapid r i s e followed by a slow decline. 2. The pressure recordings, however, showed that the intravascular 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 artery w a l l and were returning while the pressure was s t i l l increasing i n the lumen of the perfused c o n s t r i c t i n g artery. 3. There was no correla 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. Inulin Space Inulin i s generally regarded as the best marker for 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 th i s study, the f l u i d assumed to be i n chemical equilibrium with the external medium i s desig- nated 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 concentra- tion as i n the external medium. The above experiments have indicated that water was l o s t from the con s t r i c t i n g artery w a l l . To substantiate these experiments and to indicate 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 re- 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 artery samples were to'o small. Using larger tissue 55 X E E 100 75 50 25 0 UJ or <2 100 GO UJ or 75 15 SECONDS - r l -NE—_ 60 SECONDS mm. 100 75 50 25 0 30 SECONDS t .NE. 120 SECONDS t .NE. NOREPINEPHRINE 4.0 /ig/ml F i g . 11 Varying durations of norepinephrine induced c o n s t r i c t i o n of rat 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 ation of the a r t e r i a l water content, C - In u l i n was used as the e x t r a c e l l u l a r space marker. This method uses dry tissue so that wet and dry weights can be determined. The arteri e s were incubated for 30 minutes instead of 3 hours. In a series of experiments with Miss M. Mar, rat t a i l a r t e r i e s were incubated 14 i n C - Inu l i n Krebs solution for 20, 40,60, and 120 minutes. There was no difference between the i n u l i n spaces or the water contents of these 4 groups of 14 a r t e r i e s . Thus, e q u i l i b r a t i o n of the rat t a i l artery i n the C - In u l i n Krebs solution has apparently occurred within 30 minutes. This agrees with the eq u i l i b r a t i o n time for other tissues (22). 56 Whole a r t e r i e s There were 2 other procedural differences i n th i s experiment, (a) Only small rats (250 g) were available, so the whole t a i l artery was required for enough tissue for the analysis. (In the previous experiments, rats weighing about 400 g were used.) This meant that no comparison of paired test and control halves was possible and 16 rats were used for the 8 test and 8 control a r t e r i e s . (b) The art e r i e s were not perfused with the O2/CO2 mixture i n order to determine the effect on the difference i n water content between the con- s t r i c t e d test and non-constricted control a r t e r i e s . Consequently, the arte r i e s were not cannulated. As before, the test a r t e r i e s were allowed to constr i c t for 2 minutes after the addition of 4 ug NE/ml to the test artery solutions. The averaged r e s u l t s of t h i s experiment are given i n Table XII. The % i n u l i n space i s the % of the t o t a l Ĥ O, not the % of wet weight, as used by some authors (for example, 22). TABLE XII. Water and i n u l i n space (ml/100 g dry wt) of rat t a i l a r t e r i e s constricted for 2 minutes with norepinephrine after 30 minutes of aerobic incubation. control a r t e r i e s test (NE) ar t e r i e s A *p < 0.02 t o t a l H20 (8) 268+6 252 ± 5 i n u l i n space (8) 90.4 ± 3.6 76.1 + 3.5 -14.3* % i n u l i n space (8) 33.4+1.2 30.8+1.6 1. There was no s i g n i f i c a n t difference between the test and control 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 control a r t e r i e s , 33.4%, agrees with 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- c o n s t r i c t i o n . H a l f - a r t e r i e s This experiment followed the above procedure except that the a r t e r i e s were divided i n half so paired analysis could be done. In addition, the ar t e r i e s were perfused with 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 rats weighing about 400 g were used. A l l 7 test h a l f - a r t e r i e s had less water than thei r controls (p < 0.02), and 5 test a r t e r i e s had smaller i n u l i n spaces than thei r control halves (not s i g n i f i c a n t ) . The average results of this experiment are given i n Table X I I I . TABLE XI I I . Water and i n u l i n space (ml/100 g dry wt) of rat t a i l a r t e r i e s constricted for 2 minutes with norepinephrine after 30 minutes of aerobic incubation. control a r t e r i e s test (NE) a r t e r i e s A *p < 0.02 t o t a l H 2 O (7) 298 + 4 261 ± 8 -37 ± 9* 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. The fact that the water loss 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, indicates that inaccuracies due to water trapped i n the lumen may o b l i t e r a t e differences. 58 2. The water loss 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 control a r t e r i e s . Discussion 1. There are indications that the i n u l i n space decreased during no- repinephrine induced vasoconstriction. Of 18 pairs of a r t e r i e s , 13 of the test a r t e r i e s had smaller i n u l i n spaces than t h e i r corresponding controls (p < 0.1, not s i g n i f i c a n t according to the X 2 t e s t ) . The average change i n the i n u l i n space for these 18 pairs of a r t e r i e s was -17.3 1 9.5 ml/100 g dry wt (not s i g n i f i c a n t l y different from zero). This 20% ECS decrease agrees with 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 : of the 18 pairs of values, 9 test a r t e r i e s had smaller % i n u l i n spaces than the i r controls. This means that the % decrease i n the non-(free ECS) was also constant. 4. In addition to the usual problem of how i n u l i n i s d i s t r i b u t e d i n 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 ECS marker during vasoconstriction. Conformational changes i n the e x t r a c e l l u l a r s o l i d s or permeability changes i n the smooth muscle c e l l membranes (24) asso- ciated with c o n s t r i c t i o n may r e s u l t i n an altered d i s t r i b u t i o n of i n u l i n . The f l u i d changes associated with the onset of c o n s t r i c t i o n may occur too quickly for the i n u l i n molecule to assume a new equilibrium d i s t r i b u t i o n . 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 artery underwent a decrease i n ECS and a loss 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 additional information on the d i s - t r i b u t i o n of the H 2 O loss associated with vasoconstriction. Rat t a i l a r t e r i e s were incubated for 90 minutes i n Krebs solu t i o n aerated with O 2 / C O 2 . These a r t e r i e s were not divided i n h a l f , cannulated or perfused with O 2 / C O 2 . Consequently, intravascular pressure measurements were not made. Two groups of 6 a r t e r i e s were used: (a) non-constricted control a r t e r i e s which were removed for analysis a f t e r pre-incubation, and (b) constricted test 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 for 15 minutes. The averaged res u l t s are given i n Table XIV. TABLE XIV. Water (ml/100 g dry wt) and ion content (meq/100 g dry wt) of rat t a i l a r t e r i e s constricted with norepinephrine for 15 minutes after 90 minutes of aerobic incubation. non-constricted constricted (NE) A *p < 0.02 t o t a l H 20 342 t 6 283 ± 4 -59* t o t a l Na 41.0 ± 0 .4 32.8 ±0.5 -8.2* t o t a l K 23.3 ± 0 .5 22.3 ± 0.5 t o t a l CI 34.9 ± 0 .5 29.1 + 0.6 -5.8* n = 6 for each value ± S.E. 1. The constricted a r t e r i e s had 17% less H20, 20% less Na, no change i n K, and 17% less CI than the non-constricted a r t e r i e s . 2. The r a t i o of Na loss 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 external media, 150/124 =1.21. 3. The Na carried out with 59 ml of ECS f l u i d would have been 8.9 meq Na/100 g dry wt, only s l i g h t l y larger than the observed Na loss. 60 4. The above 2 points suggest that the water loss associated with 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 con s t r i c t i n g artery w a l l . 7. Summary of Results of Norepinephrine Induced Vasoconstriction 1. The and Na losses from the rat t a i l artery constricted i n v i t r o with norepinephrine are shown i n Table XV. The % H2O loss agrees with that found for the contracted uterus (1) and constricted aorta (2). Most of the observed changes i n Na content associated with 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 loss of Na and water from the contracted uterus (26). TABLE XV. Overall average H 2 O (ml/100 g dry wt) and Na (meq/100 g dry wt) contents of rat t a i l a r t e r i e s constricted i n v i t r o with nore- pinephrine. The r a t i o of constricted test a r t e r i e s with smaller H 2 O or Na than t h e i r corresponding control halves. control test (NE) A % change r a t i o s i g n i f . H20 (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. In Table V I I I , the observed Na loss was greater than the ANa calculated from the water loss. In Tables IX and XIV, they were the same, while i n Fig. 10, the observed Na loss was less than the ANa . The i n u l i n space data suggested that only part of the water loss was from the ECS, while the CI data suggested a l l the water loss could have been from the ECS. I t thus seems possible that most of the H20 l o s t from an artery w a l l constricted with norepinephrine, came from the e x t r a c e l l u l a r space as a predominately NaCl solution. The remainder of the Na present i n this volume of extra- c e l l u l a r f l u i d may have entered the contracting 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 the i r controls was observed for 19/31 test 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.) for the control (n = 33) and test 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, respectively (non-significant difference). These non-significant changes i n the K content of the c o n s t r i c t i n g artery are i n agreement with some findings (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 pattern of water, Na and intravascular pressure for the NE constricted a r t e r i e s shows that the H2O and Na losses had occurred and equilibrium was being restored before the increasing intravascular pressure had reached a plateau. This suggests that the ion and water changes were associated with the onset of vasoconstriction. C. HIGH K INDUCED VASOCONSTRICTION Norepinephrine induced c o n s t r i c t i o n i s associated with a loss of water from the artery w a l l . The question now arises: Is t h i s water loss associated only with the action of norepinephrine, or i s i t common to a l l vasoconstrictive processes? A commonly used pressor agent i s a solution with a high potassium concentration, c a l l e d a high K solution, made by replacing some of the NaCl i n a physiological solution with either KCL or ̂ SO^. The action of high K solutions on vascular tissue has been w e l l studied and appears to have a dif f e r e n t mode of action than norepinephrine (14,27-34). This experiment was performed to determine i f high K induced constrictions were also associated with a loss of water from the w a l l of the rat t a i l artery. As i n the NE experiments, the ar t e r i e s were divided into test and control halves and equilibrated i n Krebs solution. The test a r t e r i e s were 62 perfused with C^/CC^ for 5 to 10 minutes while the pressure was monitored, transferred while s t i l l recording to high K solution for 2 minutes, then removed for analysis. The control a r t e r i e s were then s i m i l a r l y perfused and removed. The high K solution was the same as the Krebs solution 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 concen- trations 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 for the high K solution and 332 for the Krebs solution. The con- 14 centration of C - I n u l i n for the e x t r a c e l l u l a r space determination was the same i n both solutions. The ar t e r i e s were analysed for water content and i n u l i n space. Consequently, not enough tissue was available for Na and K measurements. Three experiments were performed: (1) Arteries from 8 rats were pre-equilibrated for 30 minutes i n normal Krebs solution. Four of the 8 test a r t e r i e s did not cons t r i c t i n the high K s o l u t i o n — a s observed from the pressure recordings—and were grouped with the control a r t e r i e s as non- constricted a r t e r i e s . (2) The same procedure as i n (1) was used except the 14 C - I n u l i n concentration was doubled. (3) Arteries from 12 rats were equilibrated for 3 hours i n normal Krebs solution instead of 30 minutes as i n the above two experiments. The water content results of these 3 experiments are averaged i n Table XVI. The only satisfactory 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 constricted a r t e r i e s (4), 67.7 ± 5.8, i . e . 27.4 - 3.7%. The differences between the two groups were not s i g n i f i c a n t . The standard errors were so large, 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 with high K induced vasoconstriction were about the same size (-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 rat t a i l a r t e r i e s constricted for 2 minutes i n high K solution and the r a t i o of constricted test a r t e r i e s with smaller water content than their corresponding non-constricted control halves after aerobic incubation. Experiment non-constricted constricted A % change r a t i o s i g n i f . 1. (12) 291 + 6 (4) 254 + 13 -37* -13% 4/4 none 2. (8) 323 + 8 (8) 247 + 12 -76 ± 10* -23% 8/8 p < 0. 008 3. (12) 308 + 9 (12) 279 + 9 -29 ± 12t - 97, 9/12 none average (32) 306 (24) 264 -42 -14% 21/24 p < 0. 001 ( ) number of h a l f - a r t e r i e s I S.E. * p < 0 . 0 2 t p < 0 . 0 5 D. SYNTHETIC VASOPRESSIN, PLV-2, INDUCED VASOCONSTRICTION As a further test of the relationship between vasoconstriction and water loss from the artery w a l l , a synthetic vasopressin, PLV-2, was used to c o n s t r i c t the rat t a i l a r t e r i e s . Some work has been done on the effects of PLV-2 on the i o n i c exchanges i n the rat aorta (35). The h a l f - a r t e r i e s were incubated for 3 hours i n Krebs solution (332 14 mosm/liter) containing C - I n u l i n . During the O2/CO2 perfusion of the test a r t e r i e s at the end of t h i s incubation period, while the intravascular pressure was 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 test a r t e r i e s . They were allowed to c o n s t r i c t for 2 minutes before removal for analysis of their water and i n u l i n contents. The control a r t e r - ies 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 . Twelve rats were used i n t h i s experiment. The averaged res 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 rat t a i l a r t e r i e s constricted for 2 minutes with PLV-2 and r a t i o of constricted test a r t e r i e s with smaller amounts than the i r corresponding control halves after 3 hours of aerobic incubation. control test (NE) A % change r a t i o s i g n i f . t o t a l H20 (12) 293 t 9 255 + 11 -38 ± 14* -13% 10/12 p < 0.04 i n u l i n space (12) 105 ± 4 102 ± 7 8/12 none % i n u l i n space(12) 35.9 ± 1.0 39.7 ± 1.7 +3.8 ± 1.1* +11% 1/12 p < 0.006 ( ) number of h a l f - a r t e r i e s t s . E . * p < 0 . 0 2 1. The water loss associated with PLV-2 induced vasoconstriction was i n the same range as the water loss associated with high K and norepine- phrine induced con s t r i c t i o n s . 2. 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 - t i o n . In addition, 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 artery w a l l originated 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 effect of PLV-2 induced c o n s t r i c t i o n on the artery wall i s quite d i f f e r e n t than the effect of high K and norepinephrine. E. DISCUSSION The rat t a i l artery 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. Of the 106 pairs of h a l f - a r t e r i e s , 97 of the constricted test a r t e r i e s had less water than t h e i r corresponding non- constricted control halves (p < 0.001). The o v e r a l l average H20 contents 65 were (n = 118): control a r t e r i e s , 306 ml H 2 O/IOO g dry wt, and test a r t e r - i e s , 258. The average H 2 O loss associated with vasoconstriction was thus, 48 ml, or 16% of the control water content. There have been several studies which indicated there was a loss of water from the c o n s t r i c t i n g artery w a l l . In 1956, Tobian and Fox (10) found a s l i g h t , non-significant decrease i n the water content of femoral a r t e r i e s constricted i n s i t u with norepinephrine. Daniel's early studies showed no s i g n i f i c a n t change i n hydration of the constricted rat aorta (25, 36). However, he observed water losses, or decreases i n water gain, i n uterine 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). These s t r i p s l o s t 10 to 17% of t h e i r t o t a l water. Daniel suggested that "contraction causes loss of substantial quantition 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 carotid 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 per- fusion of a synthetic vasopressin, PLV-2, was associated with a s l i g h t non- s i g n i f i c a n t decrease i n the water content of the rat aorta. 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 rabbit aorta i n v i t r o a f ter the application of adrenaline. Similar i n u l i n space increases were observed by his associates for the rat diaphragm (40) and trout dorsal muscle (41) given adrenaline. The Somlyos (24) suggested that since Henry's work co n f l i c t e d with that of Daniel and that of Rorive (discussed below), "epinephrine may increase i n t r a c e l l u l a r penetration of i n u l i n i n the rabbit aorta". Although t h i s suggestion may be true, i t i s possible that the data of Henry et_ a l . do not apply to the constricted aorta. They were interested i n the effect of adrenaline on the turnover rate of free nucleotides, 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 solution and 0.04 ug/ml. Supporting t h i s p o ssi- 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 carotid artery and uterus s t r i p s constricted 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 out e f f e c t " . The work of Rorive et a l . (2) provides a clear demonstration of a water loss associated with vasoconstric- t i o n . They found an increase i n the percentage dry weight of the rat aorta given norepinephrine (5 ug/ml bath) or angiotensin. Their data show the constricted aorta l o s t 10 to 16% of i t s water. They suggested that the loss of water may have been due to " r e p a r t i t i o n des ions entre les d i f f e r e n t com- partments 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 for the rat aorta constricted i n a high K solution (20 meq K / l i t e r ) . His values show that about 3/4 of the H20 loss could be explained by the i n u l i n space decrease. Why c o n s t r i c t i o n i s associated with a loss 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 interest to consider the changes, caused by c o n s t r i c t i o n , i n the physical parameters of the entire rat t a i l artery w a l l . These changes can be estimated from the average water content and average water lo s s . (a) wet weight of control half-artery (n = 149): 13.8 1 0.3 mg (b) wet weight of whole non-constricted artery: 2 x 13.8 = 27.6 mg (c) % water i n control artery (n = 118): 100(306/406) 75.5% (d) water content of non-constricted artery: 0.755 x 27.6 20.8 mg (e) a loss of 16% of the water i n the artery w a l l meant a change of: -0.16 x 20.8 -3.3 mg water 67 (f) % change i n weight of whole artery: . 100(-3.3/27.6) = -12% (g) wet weight of constricted artery: 27.6-3.3 = 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 con s t r i c t i n g artery was: -3.3 u l (i ) density of artery (43,44): 1.06 g/cc (j) volume of control.artery, V w: 27.6/1.06 = 26.0 u l (k) volume of constricted artery: 26.0 - 3.3 = 22.7 y l (1) % change i n volume of artery w a l l during c o n s t r i c t i o n : 100(-3.3/26.0) = -13% (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 constricted artery: 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 artery by NE, high K, or PLV-2 i s associated with a change i n the density of the w a l l of: +0.01 g/cc There was a small non-significant loss of K from the norepinephrine constricted rat t a i l artery. Tobian and Fox found a large K loss from the dog femoral artery constricted with NE (10), as did Daniel et_ al_. for the rat aorta (25). On the other hand, Rorive et a l . found no change i n K for the rat aorta constricted with NE (2), and Headings and Rondell observed a s l i g h t gain i n K for the dog carotid artery constricted 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 arte 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 indications that epinephrine and norepinephrine may cause c o n s t r i c t i o n without membrane depolarization (27,30,45), or even with membrane hyperpolarization (46). This means that the loss of c e l l u l a r K usually associated with depolarization and contraction i n s t r i a t e d muscle, need not occur i n NE induced vasocon- s t r i c t i o n . Shibata and Briggs (46) explained the hyperpolarization 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 rabbit aorta given epinephrine (28), as due to an increase i n membrane per- meability to K caused by epinephrine, while "contraction r e s u l t s from an action of epinephrine which involves Ca". In short, i f norepinephrine causes co n s t r i c t i o n without depolarizing the vascular smooth muscle c e l l membrane, i t i s not surprising that no change i n K was observed i n th i s study. The norepinephrine induced c o n s t r i c t i o n of the rat t a i l artery was associated with a loss of 15% of the a r t e r i a l Na. In the l i t e r a t u r e , there i s no clear picture of changes i n Na content of the co n s t r i c t i n g artery w a l l . Non-significant changes i n Na content were observed i n the rat aorta (2,25) ' and dog femoral artery (10) constricted with norepinephrine. The s l i g h t increases i n Na found (10,25) did not correlate with the loss of K observed simultaneously. On the other hand, Daniel found a loss of Na from the con- tracted 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 obscur- ring c e l l u l a r uptake of Na" (1). Without entering the 'Ca vs Na as the current carrying ion' debate, i t seems that even i f Na did 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 quite small (36). In t h i s study, the Na l o s t from the co n s t r i c t i n g rat t a i l artery apparently accompanied the loss 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 artery w a l l are measured i n the relaxed state and the cross-sectional area of the w a l l i s calculated. The artery i s then constricted and i t s outer radius measured, but not the inner radius since i t i s not e a s i l y discernible i n the constricted state. The inner radius for t h i s constricted state i s then c a l - culated from the outer radius and the cross-sectional area, using the assumption that c o n s t r i c t i o n i s isovolumetric (for example, see 47,48). In the l i g h t of the i n d i c a t i o n of a loss of about 13% of the volume of the artery w a l l during c o n s t r i c t i o n , i t i s of interest to see what error i s introduced into the c a l c u l a t i o n of the inner radius by t h i s isovolumetric assumption, AV W = 0. I f ( )" refers to variables i n the constricted state and ( ) refers to variables i n the relaxed V w = * L ( r G 2 " r ± 2 ) s o> Vw ~ Vw " A Vw rearranging: r ^ The s i z e of the error introduced 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 two curves were calculated from equation (1) by a r b i t r a r i l y considering the case of constrictions i n which the outer radius decreased by 50%, i.e.' = hro' I t w a s also assumed, for the sake of these calc u l a t i o n s , that: r ^ = 7/8 r Q for values of the outer radius from 200 to 1000 u. The lower curve i s for isovolu- metric c o n s t r i c t i o n , the upper curve for a 13% decrease i n the w a l l volume— which means that for a constant length of the a r t e r i a l segment, there was a state, then: and V W = TrL(rg 2 - r^ 2) - ^ ( r ^ 2 - r ^ 2 - r Q 2 + r ± 2 ) = \x'2 - r 2 + r . 2 - AVW/TT L .......... (1) Jo O 1 w 70 250|- 0 200 400 600 800 1000 RELAXED OUTER RADIUS rc (AL) F i g . 12 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 , with and without a 13% decrease i n 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 . 13% decrease i n the c r o s s - s e c t i o n a l area of the w a l l . The isovolumetric assumption always r e s u l t s i n a ca l c u l a t e d inner radius of the c o n s t r i c t e d artery that i s too small: the erro r i s about 42% f o r the example shown i n F i g . 12. This means that the artery r e a l l y c o n s t r i c t e d considerably l e s s than indicated by t h i s assumption. G. EXAMINATION OF POSSIBLE EXPERIMENTAL ARTIFACTS The f i n d i n g of decreased a r t e r i a l hydration during c o n s t r i c t i o n i s contrary to the generally accepted view of isovolumetric c o n s t r i c t i o n . For t h i s f i n d i n g to be acceptable, the p o s s i b i l i t y that the observed l o s s of 71 water was an experimental a r t i f a c t has to be dismissed. In th i s regard, there are 3 objections which can be raised to claim the observed water loss was due to: (1) changes i n the artery 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 effect of the O 2 / C O 2 perfusion on the a r t e r i a l water con- tent. 1. Effect of Relaxation of Constricted Arteries on Water Content There i s some in 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 . either they imbibe water or are recovering from the trauma of excision. I f i t i s assumed that vasoconstriction 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 relaxation w i l l leave them, at least for a time, with less water than t h e i r control halves. On the other hand, i f the water loss i s d i r e c t l y a function of vasoconstriction, then a r t e r i e s constricted then relaxed should have the same amount of water as the untreated controls. To resolve t h i s question, a r t e r i e s were constricted and then allowed to relax. Rat t a i l a r t e r i e s were divided i n half and incubated for 3 hours. The test 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 co n s t r i c t for 2 minutes. These constricted test a r t e r i e s were then transferred to fresh Krebs solution and allowed to relax for 25 minutes while the intravascular pressure was monitored. The controls were perfused with O 2 / C O 2 and removed with the test a r t e r i e s for water measurement. Fourteen a r t e r i e s were used i n t h i s experiment. For most constricted test a r t e r i e s , the intravascular pressure re- turned to normal a f t e r about 10 minutes i n the fresh Krebs solution (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 after 25 minutes. Seven of the 14 test h a l f - a r t e r i e s had less water than their corresponding 72 x ! 5 0 NE4 .0 / i/ml TRANSFER TO FRESH KREBS SOLUTION lmin i TIME REMOVAL FOR ANALYSIS F i g . 13 Intravascular pressure of a rat t a i l artery constricted i n v i t r o with norepinephrine, then allowed to relax, while being perfused with 02/C02» control halves. The averaged values (± S.E.) showed a si m i l a r non-significant difference: control a r t e r i e s , 314 - 6 ml water/100 g dry wt, and test a r t e r - i e s , 301 i 7, i . e . both had the same amount of water. This means that the loss 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 extra water. I t should be noted that the test a r t e r i e s could not have gained back any loaded water i n the 15 minutes of incubation after they were relaxed, since 15 minutes i s too short a time for t h i s to happen. The experiment mentioned above showed there was no difference i n water content of rat t a i l a r t e r i e s incubated for 20 or 120 minutes. In addition, there was no cor r e l a t i o n between the average incubation times and the water contents of the control a r t e r i e s from Table XI. In conclusion, 73 the l o s s of water from the arte r y w a l l during c o n s t r i c t i o n and i t s subsequent return during r e l a x a t i o n were independent of any incubation a r t i f a c t s . 2. Water Remaining i n the Lumen a f t e r Gaseous Perfusion Could water l e f t i n the lumen a f t e r O2/CO2 perfusion explain the water loss during vasoconstriction? This remaining water could be considered as e i t h e r a constant volume of f l u i d or a l a y e r of f l u i d of constant thickness. Here, constant r e f e r s to comparison of the non-constricted c o n t r o l and the c o n s t r i c t e d t e s t a r t e r i e s . A constant 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 could not be the cause of the observed water l o s s . A boundary layer of f l u i d of con- stant thickness containing l e s s f l u i d i n the c o n s t r i c t e d state, due to the smaller lumen, might give the impression of l e s s water i n the c o n s t r i c t e d a r t e r i e s . However, t h i s p o s s i b i l i t y does not seem too l i k e l y : Volume of boundary la y e r f l u i d = V^ = 2ITL y r ^ where: L = length of arte r y segment; y = boundary la y e r thickness; r ^ = lumen radius. Assuming Ay = 0 = AL, where A i s the change caused by c o n s t r i c t i o n , then, AV b = 2TTL y A r ^ Since, A ^ < 0, then AV b < 0. The question i s then reduced to: Can a decrease i n the boundary l a y e r volume account f o r the observed loss of water by the c o n s t r i c t i n g artery? The r ^ values from Table I can be used f o r a rough i n d i c a - t i o n of Ar-p r ^ was 255 u and 55 u for the relaxed and f u l l y c o n s t r i c t e d a r t e r i e s , r e s p e c t i v e l y . So, Ar-j. = -200 u. For L = 10 cm, AV b = -1.20 y (for AV D i n ml and y i n cm). The water l o s s f o r a 10 cm length of r a t t a i l artery was c a l c u l a t e d to be 3.3 u l . If the change i n boundary layer volume, AVj, = -3.3 u l , then the 74 thickness of the layer, y = 27.5 u. That i s , a boundary layer of f l u i d 27.5 u thick would be required to explain the observed water lo s s . But t h i s would mean that the c o n s t r i c t i n g artery, 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 half of the lumen. Since the humidity of the O 2 / C O 2 was 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 loss observed i n constricted a r t e r i e s . There i s additional support for t h i s conclusion: i f a s i g n i f i c a n t l y large layer of f l u i d remains i n the lumen, i t would evidently be smaller i n 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 for the control water content of the two halves were accordingly compared. Control a r t e r i e s with a water content ( i S.E.) i n the range of 300 ml/100 g dry wt were selec- ted: proximal control a r t e r i e s (n = 32), 295 1 4 ml water/100 g dry wt, and d i s t a l control a r t e r i e s (n = 31), 313 - 6. The 18 ml difference was s i g n i f i - cant. Certainly then, the d i s t a l control a r t e r i e s do not have less water than the proximal control a r t e r i e s . 3. Effect 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 physiological and some comment i s necessary to show that i t did not produce the observed water loss during vasoconstriction. Evaporation from the artery w a l l during O 2 / C O 2 perfusion depends on the vapour pressure of the w a l l f l u i d . At 37°C the vapour pressure of water i s about 47 mm Hg (49). The effects of solutes and pressure on t h i s value are less than 1% (50). The w a l l f l u i d vapour pressure i s affected by the meshwork of endothelial c e l l s and i n t e r n a l e l a s t i c lamina separating the 75 w a l l from the lumen. The extent of this effect 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 altered by about + 10% (see 51,52). 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 artery w a l l during O 2 / C O 2 perfusion. The following i s a maximal estimation of t h i s evaporation. Rate of evaporation from a rat t a i l artery A non-constricted artery was l e f t to evaporate i n a weighing b o t t l e on a 6-place Mettler balance and i t s weight was recorded every 1 or 2 minutes. The artery l o s t 3.5 ml water/100 g dry wt/minute for 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 artery 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 rat t a i l artery were 255 and 321 u, so for a 10 cm length of artery, the inner and outer surface areas 9 would be 1.6 and 2.0 cm , respectively. Actually, the surface area of the porous adventitia would be considerably larger. The maximum evaporation rate from the luminal surface during 02/C02 perfusion might thus be e s t i - mated as 2 ml/100 g dry wt/minute. The perfusion time for the control arte r i e s was about 4 or 5 minutes, compared to 5 or 6 minutes for the test a r t e r i e s . This time difference could r e s u l t i n a hydration difference of no more than 4 ml water/100 g dry wt—much less than the observed 48 ml water difference. In r e a l i t y , the evaporation loss would probably be even less because of osmotic replacement of any evaporated water. The a r t e r i e s were incubated i n Krebs solution and the artery w a l l i s quite permeable to water (see Chapter IV), ensuring no dehydration of the luminal surface'of the gas perfused a r t e r i e s . 76 Another factor diminishing the possible r o l e of evaporation i s that evaporation depends upon the area exposed to the C^/CC^. The constricted test a r t e r i e s have a smaller luminal surface area than the non-constricted control a r t e r i e s . [If r ^ was 255 u and 55 u for the relaxed and f u l l y constricted a r t e r i e s respectively (from Table I ) , then c o n s t r i c t i o n was associated with an 80% decrease i n luminal surface area.] So, the test a r t e r i e s should lose less 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 control a r t e r i e s — the opposite of what was observed. In general, i f O 2 / C O 2 perfusion did s i g n i f i c a n t l y affect the a r t e r i a l hydration, then presumably the longer the perfusion time, the greater the e f f e c t . However: (1) There was no c o r r e l a t i o n between the water content and the perfusion time for either the control or test a r t e r i e s . (2) In the relaxation experiment, the test a r t e r i e s were perfused for 30 minutes and the controls for 5, yet both had the same water content. (3) The water con- tent of control a r t e r i e s perfused with O 2 / C O 2 , 306 ml/100 g dry wt, was about the same as that obtained for a r t e r i e s perfused with Krebs solution (17). H. SUMMARY 1. Vasoconstriction, induced by strong pressor doses of norepinephrine, high K, or PLV-2, was associated with a 16% decrease i n the ^ 0 content of the rat t a i l artery w a l l . 2. In the norepinephrine induced co n s t r i c t i o n s , this finding of a water loss was supported by a decrease i n the Na and CI contents and the i n u l i n space of the artery w a l l . (a) Most of the R̂ O loss was due to a decrease i n the e x t r a c e l l u l a r f l u i d volume. (b) The size of the loss 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 Ĥ O and Na losses were greatest 30 seconds af 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 artery w a l l , i . e . the Ĥ O and Na movements were associated with the onset of vasoconstriction. (e) The K content and the % i n u l i n space remained e s s e n t i a l l y constant during NE c o n s t r i c t i o n . 3. In the PLV-2 induced co n s t r i c t i o n s , the i n u l i n space remained constant while the % i n u l i n space increased. This indicates that, i n contrast with the other 2 agents, almost a l l the l o s t water came from i n u l i n - inaccessible compartments of the artery w a l l . 4. About the same water loss was associated with vasoconstriction induced by a l l 3 agents, although no comparison could be made between thei 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 loss of a few m i c r o l i t e r s of f l u i d from the co 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 altered w a l l hydration would affect the hydrodynamics of the co n s t r i c t i n g artery. In addition to the expected observations, i t was discovered that the permea- b i l i t y of the artery w a l l was considerably altered during c o n s t r i c t i o n . Three experiments were performed during vasoconstriction induced by nore- pinephrine: pressure and flow measurements, varying intravascular pressure, and dye perfusion. Although constant pressure perfusion i s often used i n hydrodynamic experiments (for example, 1,2), i t was decided to follow Burton's advice to use constant flow perfusion (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 properties of relaxed and constricted a r t e r i e s (1,2,10-14). There have also been observations on the effect of vasoconstrictive agents on blood flow and pressure (for example, 15-18). However, these studies have been concerned with the altered hemodynamics of the system once vasoconstriction was established. They have not been concerned with 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 sensitive to small flow changes i n the 79 hope of detecting the addition of wal 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 artery was exposed and i t s c o l l a t e r a l s t i e d . I t was tested for leaks by perfusion with Krebs solution to which Evans blue dye had been added. There were two subsequent procedures for t h i s artery: (a) i t remained i n s i t u with i t s exposed surface kept moist with Krebs so l u t i o n , or (b) i t was removed from the t a i l bed and placed i n a per- spex chamber at 37°C. Perfusion was by a constant infusion pump (B. Braun) with two syringes: both contained Krebs solution, and one also contained norepinephrine (4 ug/ml)(NE). Vasoconstriction was induced using a 4-way switch to change the perfusion from Krebs solution to NE-Krebs solution. Topical application of NE was also used for some i n s i t u a r t e r i e s . The pressure gradient down the artery was monitored with two Statham transducers connected by T-joints to the P.E. 50 polyethylene tubing proximal and d i s t a l to the artery. The flow rate of the effluent was determined using a special photocell-flowmeter. The effluent passed through c o i l s of polyethylene tubing wound around l i g h t pipes attached to a photocell. The only path for l i g h t from a DC lamp to reach the photocell was through a t h i n l i n e along the c o i l s . A small bubble was inserted into the system between the artery and the flowmeter. Each time the dark meniscus of the leading 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 photocell. The rate of flow out of the artery was determined as follows: The separation of the polygraph deflections, representing the passage of the bubble through 1 c o i l of the flowmeter, was measured during a control run ( i . e . without NE). From the polygraph chart speed and the pump rate, the volume of one 80 c o i l was determined. For measurements during vasoconstriction, the number of c o i l s , including 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 rate for this 15 second i n t e r v a l was then calculated. The flow measurements were accurate to + 0.02 ul/sec, 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 size of the bubble used was not too small, since 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 large, since i t would have a di f f e r e n t v i s c o s i t y than the perfusing solution (5). I t has been estimated that the maximum deviations of the bubble v e l o c i t y from the blood flow, for a wide range of flows, are T 5% (19). The presence of th i s flowmeter i n the system meant that the intravascular pressure was f a i r l y high, about 44 mm Hg at the mid- point of the artery, while the pressure gradient down the artery was about 8 mm Hg during the control runs. The pressure and flow c h a r a c t e r i s t i c s of the NE induced constrictions were determined i n 14 rat t a i l a r t e r i e s . 2. Results The pressure gradient and the effluent flow rate for a t y p i c a l artery constricted with norepinephrine are shown i n F i g . 14. At 30 to 90 seconds af 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 rate and an increase i n the increasing pressure gradient. These anomalies were observed for a l l the constr i c t i o n s , although t h e i r sizes varied considerably. The source of the anomalies l i e s i n the ar t e r i e s them- selves because the flow rate of the infusion pump was constant during these experiments. The pump operated through a series of gears which prevented any "backlash" from the experimental system of the artery, tubing and flow- meter onto the infusion pump. 81 r»l • • • i i i 1 1 1 1 1 1 1 1 1 1 1 1 ' ' 0 Of 30 60 90 120 150 180 210 240 270 NOREPINEPHRINE TIME (seconds) F i g . 14 Effluent flow rate, F, and pressure gradient, P, for a rat t a i l artery constricted i r i s i t u with norepine- phrine. i 82 3. Discussion Could f l u i d "squeezed out" of the lumen of the con s t r i c t i n g artery have been the source of the increase i n flow rate and the "hump" i n the pressure rise? The concept of "squeezing out" implies 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 for c o n s t r i c t i o n (a few minutes) was much longer than the time required for 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 cross- sectional area of the lumen, Â > was constantly balanced by the increase i n the v e l o c i t y through the lumen, v, since the flow rate, F = A^v, remained constant. When the artery constricted, the volume of the whole system (artery lumen plus polyethylene tubing) decreased, so less 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 artery lumen, not through the rest of the system. Changes inside the system could a l t e r the cross-sectional areas, the v e l o c i t i e s , and the volumes, but not the flow rates. The anomalies i n the curves of Fig. 14 can be explained by a movement of f l u i d into the con s t r i c t i n g lumen. The source of t h i s f l u i d could have been: (a) the artery wall—which would agree with the resu l t s of the previous two chapters, or (b) the f l u i d surrounding the artery—which would indicate that the permeability of the artery w a l l was altered during con- s t r i c t i o n , to allow f l u i d to pass through i t into the lumen. Of course, these two p o s s i b i l i t i e s are not mutually exclusive. The size of the increase i n flow indicates that f l u i d passed into the lumen from the sur- roundings. The maximum increase i n flow i n Fig. 14 was 0.25 pl/sec—about 83 10 x that expected from the loss of w a l l f l u i d . Also, the area under the flow curve i n F i g . 14 indicates that over 20 u l of f l u i d was added to the lumen. The whole artery w a l l had only 20 to 30 u l of water. The increase i n flow i n Fig. 14 was accompanied by a "hump" i n the increasing pressure gradient. This i s to be expected from P o i s e u i l l e ' s 2 equation for laminar flow through r i g i d tubes, by which P °= F/V , where: P = pressure gradient down the artery segment, F = outflow from the artery, and V = lumen volume. Although P o i s e u i l l e ' s equation i s for equal inflow and outflow, the above relationship 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 vasoconstriction would r e s u l t i n the observed pressure pattern 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 artery constricted with norepinephrine. This increase was associated with a "hump" i n the increasing pressure gradient. 2. These changes indicate that there was an increase i n w a l l perme- a b i l i t y during vasoconstriction which allowed f l u i d to enter the lumen from the f l u i d surrounding the artery. B. EFFECT OF INTRAVASCULAR PRESSURE The previous section suggested that wall tension changes caused an a l t e r a t i o n - i n the permeability of the artery 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 ion movements are i n d i f f e r e n t d i r e c - tions for these 2 vessels, the ion movements may depend on the luminal pressure (21). I t thus seemed reasonable to examine the effect of the i n t r a - vascular pressure on vascular permeability. Most studies involving variable intraluminal pressure have been con- cerned with demonstration of a vascular myogenic response (22,23). The studies which considered the effect 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 also been some work on the effect of luminal 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 rat t a i l artery at either "high" or "low" intravascular pressure. 1. Methods The above procedure for perfusing rat 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 constricted by the addition of 0.2 ug norepinephrine i n a 10 second perfusion of NE-Krebs solution at 0.200 ml/min while the pressure gradient was monitored. The flow rate of the effluent was determined by c o l l e c t i n g the effluent every 20 seconds i n weighing bottles and measuring the volumes with a 100 u l syringe. This method was less accurate than that of the photocell-flowmeter: the volume readings were ± 1 u l and the time recordings were ~t 1 second. Consequently, the error i n an average flow of 0.180 ml/min for a 20 second i n t e r v a l i n which 60 u l was collected was ± 0.012 ml/min or about 6.7%. When multiple constrictions were induced i n the same artery, at least .30 minutes e q u i l i b r a t i o n was allowed between con- s t r i c t i o n s . 85 The ar t e r i e s were constricted at two di f f e r e n t basal intravascular pressures: low and high. (The intravascular pressure, taken as the pressure i n the lumen at the midpoint of the artery segment, should not be confused with the pressure gradient, the difference i n the pressure between the two ends of the artery 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 artery was at the same l e v e l as the artery. For high intravascular pressures, the d i s t a l tubing was raised about 50 cm above the l e v e l of the artery. This increased the intravascular pressure without producing a large change i n the pressure gradient. The average intravascular pressures were: low, 3mm Hg, and high, 41 mm Hg. 2. Results The pressure gradient patterns for the con s t r i c t i n g t a i l artery pre- parations with low and high intravascular pressures are presented i n F i g . 15. These curves represent the average results of 4 norepinephrine-induced constrictions. There was a s i g n i f i c a n t l y larger increase i n the pressure gradient during perfusion at the low intravascular pressure (+ 140 mm Hg) than at the high intravascular pressure (+ 84 mm Hg). The time courses of the pressure increases were the same but the relaxation was faster for the high pressure case. The same result was obtained on 1 artery when con- s t r i c t i o n s at high and low intravascular pressures were induced. The average flow patterns for 8 low and 4 high intravascular pressure constrictions are given i n F i g . 16. I t shows that: 1. The "base l i n e " flow before and after c o n s t r i c t i o n was s i g n i f i - cantly less for perfusion at high intravascular pressure than at low pressure. The average flow rates (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 rat t a i l a r t e r i e s constricted i n s i t u with norepinephrine (NE) at high and low intravascular 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 intravascular pressure (n = 24), 0.171 ± 0.001 ml/min. In addition both effluent flow rates were less than that of the infusion 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 at the 0.200 ml/min setting of the pump. 2. The changes i n the effluent flow rates were remarkably d i f f e r e n t during vasoconstriction. (a) The flow during the low pressure constrictions decreased con- siderably, reaching i t s lowest point when the pressure was one-half i t s peak 87 INFUSION » PUMP RATE P V » L O W INTRAVASCULAR PRESSURE n=8 HIGH INTRAVASCULAR PRESSURE n=4 TIME (seconds) F i g . 16 Effluent flow rate, F, for rat t a i l a r t e r i e s constricted i n s i t u with norepinephrine (NE) at high and low intravascular pressure. The v e r t i c a l bars represent the S.E. value. The flow did not return to i t s previous value u n t i l a f ter the peak of c o n s t r i c t i o n . The area under the flow curve revealed that 55ul less f l u i d passed through the artery over 150 seconds. (b) The flow during the high pressure constrictions was biphasic: the flow increased during the i n i t i a l r i s e i n pressure, returned to 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 attained. 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 effluent i n 50 seconds (5 u l of which, over 30 seconds, could not have come from the pump), while for the l a t e r decrease i n flow, there was about 40 u l less f l u i d i n the effluent 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 con s t r i c t i o n s . 3. Discussion The smaller peak pressure gradient for higher intravascular pressures was also observed by Nicholas and Hughes (30). They found an inverse r e - lationship between the pressor response to norepinephrine and the resting blood pressure. On the other hand, Sparks and Bohr (31) found contraction of artery 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 stretch u n t i l an optimal length, after which the response decreased. The high intravascular pressure results were obtained i n circumstances s i m i l a r to those i n the above photocell-flowmeter experiment. The intravascular pressures i n the incubation experiments of Chapter I I I were between those i n the high and low pressure cases. The flow results can be explained only i f the In s i t u rat t a i l artery preparation was permeable to the perfusing Krebs solution. The a r t e r i e s were a l l tested for leaks from the tied c o l l a t e r a l s and there were no leaks from the constant flow pump assembly. The permeability of the artery w a l l was affected by the intravascular pressure and by vasoconstriction, 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 the artery w a l l and into the surrounding f l u i d . These losses were 0.200 - 0.182 = 0.018 ml/min = 0.3'ul/sec during perfusion at low intravascular 89 pressures and 0.5 ul/sec at high pressures. (That the outflow was less than the inflow was suspected from the results of the previous section, but the control runs were not of a s u f f i c i e n t length to comment.) This indicates that there was a simple f i l t r a t i o n process through the artery w a l l which increased when the pressure i n the artery lumen was increased. A s i m i l a r observation was made by Wilens and McClusky for excised human i l i a c a r t e r i e s and veins (32) . 2. During the NE-induced vasoconstriction and the re s u l t i n g increase i n the pressure gradient, the permeability of the artery w a l l was d r a s t i - c a l l y altered, (a) At low intravascular pressures, the permeability increased so that even larger 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 permeability not only decreased but 5 p i of the f l u i d was added to the lumen, and then the permeability increased so ' that large amounts of f l u i d l e f t the lumen. In both cases, as the artery relaxed, the permeability returned to i t s pre-constriction 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 intravascular pressures and the flow rate before and during the constrictions. The s i t u - ation 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 dir e c t evidence from this experiment, for possible changes i n the hydration of the artery w a l l during c o n s t r i c t i o n . The large movements of f l u i d r i g h t through the artery w a l l completely obscured any small move- ments out of the w a l l . A possible exception was the addition 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 with the high intravascular pressure. The di 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 or the f l u i d could have come from the artery w a l l i t s e l f . These two p o s s i b i l i t i e s cannot be distinguished from this experiment. Possible causes of the changes i n artery w a l l permeability are d i s - cussed i n Chapter VI. 4. Summary 1. The higher the intravascular pressure, the smaller the pressure response to norepinephrine. 2. The wal l of the perfused t a i l artery was quite permeable: f l u i d passed out of the lumen, through the artery w a l l , and into the surrounding f l u i d . 3. This free passage of f l u i d was very much affected by the pressure i n the artery lumen and by the state of tension i n the w a l l . 4. The higher the intravascular pressure, the greater the permeability of the w a l l , i . e . the greater the loss of f l u i d out through the artery w a l l . 5. When the artery constricted at low intravascular pressures, the permeability increased, but at high intravascular pressures, i t decreased then increased. 6. There may have been a decrease i n the hydration of the artery w a l l during c o n s t r i c t i o n at high intravascular pressures. C. DYE DILUTION The w a l l water loss and the permeability changes during vasoconstric- t i o n suggested a more direct estimation of f l u i d movement. Perfusion 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 for vascular perfusion i s T 1824, Evans blue 91 (33,34,35). This dye has several advantages for 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 - culations from o p t i c a l density measurements (see 36), Evans blue obeys the Lambert-Beer Law for s t r i c t proportionality between o p t i c a l density 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 affected by variations i n NaCl concentration (34). The % transmission of Evans blue i n a Krebs solution perfusing the rat t a i l artery 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 rat t a i l artery was used as i n the above two experiments. The a r t e r i e s were perfused with Krebs solution to which Evans blue dye had been added (12.5 mg/liter)(282 mosm/liter). Norepinephrine was added to the artery either i n t e r n a l l y or t o p i c a l l y . The effluent passed through a 20 u l flowthrough c e l l i n a Zeiss spectrophotometer, set at 600 my, the absorption peak for Evans blue (35). The intravascular pressure proximal to the artery was monitored. This pressure and the % transmission of the effluent were displayed on a polygraph. Fifty-one 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 artery preparation to the addition of norepinephrine i s shown i n Fig. 17. The pressure increase was accompanied by an increase i n the % transmission (% T) of the effluent. This meant that the Evans blue i n the perfusate must have been di l u t e d . The extent of the 92 Fi g . 17 Intravascular pressure and % transmission of the effluent from a rat t a i l artery i n s i t u a f t e r the addition of norepinephrine. dye d i l u t i o n was calculated from the % T curve; Over the 500 seconds of the % T increase, the average % T was 54.7%. From Beer's law for t h i s solution and spectrophotometer c e l l , the corresponding concentration was 42 log(l/0.547) = 11.0 umoles/liter. The concentration before the NE was added was 11.8 umoles/liter. 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 constrictions showed % T patterns si 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 . The Evans blue dye did not enter the artery w a l l . In f a c t , the % T decreased s l i g h t l y during the runs, i . e . the solutions 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 affected by changes i n the flow rate through the spectrophotometer c e l l . 3. Discussion As with the previous experiments, the addition of over 100 p i of f l u i d to the lumen of the cons t r i c t i n g artery can only mean that the permeability of the artery w a l l was altered during c o n s t r i c t i o n . This permeability change can be explained i n 1 of 3 ways: 1. Before the NE was added, f l u i d was leaving the lumen and passing into the f l u i d surrounding the artery. Constriction was then associated with a decrease i n the permeability of the w a l l , so that less f l u i d passed out of the lumen, re s u l t i n g i n a less concentrated effluent. (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. Constriction then increased the wall permeability so that even more f l u i d entered the lumen, re s u l t i n g i n a more diluted e f f l u e n t . 3. Before the NE was added, no f l u i d entered or l e f t the lumen through the w a l l . Constriction then increased the wal l permeability and f l u i d moved into the lumen, r e s u l t i n g i n a diluted effluent. There i s evidence that explanation #2 applies to the changes observed i n F i g . 17. Calculation of the molar extinction c o e f f i c i e n t ( i n Beer's law) for the Evans blue-Krebs solution i n the absence of the artery, gave dye 94 concentrations which were too low when the artery was present. (The dye concentration was 12.5 rag/liter = 13.0 umoles/liter, compared to 11.8 umoles / l i t e r for Fig. 17.) This indicates that even before the NE was added, f l u i d was passing through the artery 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 th i s experiment, before c o n s t r i c t i o n f l u i d moved into the lumen. The d i r e c t i o n of flow through the w a l l was probably affected by the dsmolarity of the solution perfusing the lumen and bathing the exterior of the artery and by the intravascular pressure i n the two experiments. (In each experiment, the same solution was used inside and outside the artery.) In any case, i t seems un l i k e l y that there are large transmural f l u i d move- ments before c o n s t r i c t i o n for ar t e r i e s i n vivo. These experiments were not designed to determine the "normal" w a l l permeability, but rather the effect which vasoconstriction had on the w a l l permeability. 4. Summary 1. The Evans blue perfusing the ar t e r i e s was di l u t e d during vaso- c o n s t r i c t i o n . 2. Before the addition of norepinephrine, f l u i d passed through the artery w a l l from the surroundings into the lumen. 3. During c o n s t r i c t i o n , the permeability of the w a l l was d r a s t i c a l l y increased—allowing f l u i d to pour into the lumen. 95 D. SUMMARY 1. Norepinephrine-induced 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 a r t e r y had profound e f f e c t s on i t s hemodynamic properties and permeability. 2. There was a suggestion that the hydration of the a r t e r y wall decreased 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 pressure. 3. The walls of these a r t e r i e s were quite permeable to f l u i d , 4. The 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 a f f e c t e d t h i s permeability very d r a s t i c a l l y . 5. Higher i n t r a v a s c u l a r pressures increased the permeability of the a r t e r y w a l l . 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 increase i n w a l l permeability, or a decrease followed by an increase i n permeability. 7. How t y p i c a l are these r a t t a i l a r t e r i e s of muscular d i s t r i b u t i n g a r t e r i e s can be argued. But c e r t a i n l y any studies on perfused a r t e r i e s (for example, monitoring the concentration of ions i n the e f f l u e n t ) , should take t h i s question of permeability i n t o account. 8. That the permeability of the w a l l was a f f e c t e d both by the pressure i n the a r t e r y lumen and tension i n the a r t e r y w a l l suggests that the balance of forces that normally determine the permeability of the w a l l — the steady state which the w a l l maintains with i t s surroundings—was dependent upon the i n t r a v a s c u l a r pressure and was upset by the changes associated with v a s o c o n s t r i c t i o n . 96 CHAPTER V EXPERIMENTALLY INDUCED ALTERATIONS IN THE HYDRATION OF THE ARTERY WALL The experiments discussed above have indicated that the hydration as we l l as the transmural permeability of the artery w a l l are considerably a l - tered when the artery constricts under the influence of several vasoactive agents. The artery w a l l can be viewed as a network of components which are a l l intimately connected and act as a unit to regulate the volume of blood that passes through the artery lumen. This network consists of endothelial and smooth muscle c e l l s and the e x t r a c e l l u l a r macromolecules: collagen, e l a s t i n , and protein-polysaccharide complexes. The above experiments ex- amined the effects of active changes i n the wal l produced by vasoconstriction. The contents of the vascular w a l l can also be altered passively. In t h i s study, passive changes were induced i n the artery w a l l by (a) varying the composition of the external ions and by (b) cooling then rewarming the ar t e r i e s . 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 material (the pa r a c e l l u l a r matrix) of the artery w a l l bind Na and other ions (1-7). I t i s possible to change the counter-ion bound to these groups by changing the i o n i c composi- ti o n of the solution i n which the artery i s incubated (6). Such an ion exchange may a l t e r the configuration of the paracellular matrix and cohse- quently a l t e r the hydration of the artery w a l l (7). To test the ion exchange properties of the rat t a i l artery and th e i r effect on the physical properties of the artery, a series of experiments were performed using the techniques of Chapter I I I . Arteries were incubated i n isosmotic solutions of di f f e r e n t i o n i c composition while t h e i r intravascular pressure was monitored, then analysed for thei r water and ion content. 1. Methods After the ventral t a i l artery of the rat was exposed, i t s d i s t a l and proximal halves were cannulated by in s e r t i n g polyethylene tubing at the mid- point and proximal end of the artery. The d i s t a l h a l f - a r t e r y was flushed with the solution i n which i t was to be incubated, removed from the t a i l bed and placed with i t s cannula i n that p a r t i c u l a r solution. The proximal h a l f - artery was s i m i l a r l y flushed, removed and placed i n a second solution. Five solutions of diff e r e n t i o n i c composition were used as incubation media for the a r t e r i e s : i n meq/liter, solution 1: NaCl 143, KC1 5.0, MgS04 2.4, CaCl 2 4.2; solution 2: KC1 5.0, MgS04 2.4, CaCl 2 4.2, lactose; solution 3: MgSO^ 2.4, CaC^ 4.2, lactose; solution 4: CaCl 2 4.2, lactose; solution 5: lactose. Solutions 2 to 5 had one cation less than the solution numerically before i t . The amounts of lactose added made the solutions isosmotic (290 mosm/liter). Since zero sodium solutions preclude the use of a conventional NaHCO-j-Nal^PO^ buffer system, the solutions were buffered with Tris-HCl. One-tenth the usual T r i s concentration, 0.7 g / l i t e r (5 mM), was used to minimize both the contribution of chloride ions and the unknown effect of the T r i s cation 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 buffering capacity meant that the solutions could not be aerated with 0 2/C0 2; instead 100% oxygen was used, re s u l t i n g i n a pH of 7.0 at 37°C. Four experiments were performed, using a dif f e r e n t pair of solutions i n each: solutions 1 and 2, 2 and 3, 3 and 4, 4 and 5. Eight rats were used i n 98 each experiment. Both solutions i n an experiment contained 4 d i s t a l and 4 proximal h a l f - a r t e r i e s . After an artery had been incubated for 3 hours, i t was perfused with oxygen for 7 to 10 minutes to remove f l u i d from the lumen and produce a basal tension i n the artery w a l l . The polyethylene tubing carrying oxygen into the solution was connected to the cannula of the artery. The intravascular pressure during t h i s gas flow was monitored by a Statham transducer connected just proximal to the artery. (The gas perfusion was e s s e n t i a l l y constant for a l l the a r t e r i e s so the pressures recorded for ar t e r i e s i n d i f f e r e n t solutions could be compared.) The artery was then removed from solution, blotted, and analysed for i t s water and ion contents as described i n Chapter I I I . A Techtron atomic absorption spectrometer was used for the ion analysis. 2. Results Table XVIII shows the water (see F i g . 18), ion content, and intravascular pressure for a r t e r i e s incubated i n one of f i v e solutions. Arteries i n solutions 3 and 4 (with no monovalent cations) had a greater divalent ion content, smaller water content and greater intravascular pressure than a r t e r i e s i n solution 1. The only s i g n i f i c a n t difference between the a r t e r i e s i n solutions 3 and 4 was the gain i n Ca, equal to one-half the loss of Mg. In solution 5, where H + was the only cation, the water and pressure values were about the same as i n solution 1. I t should be noted that these f i v e solutions were isosmotic. So, for example, ar t e r i e s i n solution 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 relationship between the water content and the Ca + Mg content: for the 60 a r t e r i e s i n the f i v e solutions, 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 ion contents (±S.E.) of rat t a i l arteries incubated i n one of 5 isosmotic solutions. Solution Pressure 1^0 (mm Hg) (ml/100 g dry wt) Na K Mg^1" (meq/100 g dry weight) Ca 1 1. ( a l l ions) (8)* A+ 2. (no Na +) (12) 3. (no Na +, K +) (16) 65 ± 5 + 62 127 ± 12 +32 159 ± 8 4. (no Na+, K +, Mg++) 148 ± 14 (16) 5.-(lactose) (8) -72 76 ± 18 286 ± 13 -33 253 ± 5 -24 229 ± 2 253 ± 3 +55 290 ± 8 43.6 ± 1.7 12.5 ± 1.2 3.39 ± 0.15 2.19 ± 0.11 -41.4 -2.4 +0.81 -2.73 +0.50 +0.50 +0.80 2.20 ± 0.28 10.1 ± 0.4 4.20 ± 0.09 2.99 ± 0.12 -1.17 -3.9 1.03 ± 0.14 6.19 ± 0.36 4.27 ± 0.10 3.24 ± 0.09 +1.46 0.78 ± 0.08 5.67 ± 1.29 1.54 ± 0.04 4.70 ± 0.14 -3.73 1.28 ± 0.15 6.01 ± 0.32 2.03 ± 0.07 0.97 ± 0.07 * Number of a r t e r i e s Difference between a r t e r i a l contents (p < 0.02), 100 300 r 2 9 0 280 g 270 o> O O ^ 2 6 0 E I _j 250 < t-o y— 2 4 0 2 3 0 2 2 0 Na* 143.0 K* 5.0 mEq Mg** 2.4 - T Ca** 4.2 8 ANIMALS IONS REPLACED BY LACTOSE SOLUTIONS ISOOSMOTIC AT 2 9 0 mosmoles 12 *6 rPf Na* K* K* Mg**Ca** Mg**Ca** Mg**Ca** Ca* NO IONS F i g . 18 Water content of rat t a i l a r t e r i e s equilibrated i n 1 of 5 isosmotic solutions of di f f e r e n t i o n i c composition. 101 divalent cations was associated with changes i n the hydration of the artery w a l l . An inverse relationship was also observed between the water content and the intravascular pressure. For a l l 60 a r t e r i e s , the co r r e l a t i o n co- e f f i c i e n t was -0.59 (p < 0.001). In addition, the intravascular pressure was d i r e c t l y related to the Ca + Mg content (r = 0.40, p < 0.01) and to the Ca content (r = 0.46, p < 0.001). 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 solu- tions, but the difference was only s i g n i f i c a n t (p < 0.01) for the a r t e r i e s i n solution 5. 3. Discussion There are two possible explanations for the observed r e s u l t s which are not mutually exclusive. 1. The d i f f e r e n t i o n i c composition of the solutions caused changes i n the membrane potential of the vascular smooth muscle c e l l s . The c e l l s of art e r i e s i n solution 2 (with no Na), compared to those i n solution 1 ( a l l ions), were contracted and probably depolarized (8,9 and calculations from the Goldman equation). This c o n s t r i c t i o n might thus explain: (a) the i n - creased content of Ca, since Ca apparently enters the contracting smooth muscle c e l l (10), (b) the increased intravascular pressure, and (c) the decreased H 2 O content associated with c o n s t r i c t i o n (see Chapter I I I ) . I t 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 solutions 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 meq/liter 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. In addition, since the i n u l i n space decreases i n low Na solutions (7), the increase i n c e l l Ca would be even larger. However, even a small increase i n i n t r a c e l l u l a r Ca could probably account for a l l of the co n s t r i c t i o n . 102 2. Some of the gain i n a r t e r i a l Ca i n solution 2 could have been bound e x t r a c e l l u l a r l y . Using an ion exchange process, Palaty 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 solutions was approximately equal to the amount of Na bound extra- c e l l u l a r l y — a b o u t 5 meq/100 g dry wt for the rat t a i l artery (6). In the present study, the t o t a l divalent ion increase between solutions 1 and 2 was 1.61 meq/100 g dry wt, wel l within the estimates of Palaty 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 paracellular matrix of the artery w a l l may have played a role 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 paracellular matrix as an ion exchanger. In general, the swelling of a given ion exchange re s i n i s dependent upon the degree of cross-linking i n the r e s i n , and upon the valency, s i z e and external 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 artery w a l l i s extended and r e l a t i v e l y swollen—as were the arter i e s i n solutions 1 and 5. With divalent counter-ions, the degree of cross-linking i s increased so the matrix becomes tighter 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 results have been shown for polyelectrolyte gels (14,15). Bozler has found that the s t i f f n e s s and opacity of frog stomach muscle i n d i l u t e solutions of CaCl2 and MgCl2 are increased strongly (16,17). For the rat t a i l a r t e r i e s , the 20% decrease i n w a l l water content between solu- tions 1 and 3 may be pa r t l y due to an ion exchange process that replaces monovalent ions with divalent ions as the counter-ions to the anionic groups of the protein-polysaccharide complexes i n the paracellular matrix. The increased intravascular pressure for a r t e r i e s i n the divalent solutions could be explained i f the ion exchange process, which resulted i n conformational 103 changes i n the paracellular matrix and hydration changes i n the artery w a l l , also profoundly affected the physical response of the artery to perfusion. These results cannot be compared to those for hypertensive a r t e r i e s i n which an increase i n water content (18,19) i s associated with 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 poly- saccharide matrix i s actually increased (21). 4. Summary 1. Rat t a i l a r t e r i e s were incubated i n isosmotic solutions of d i f f e r e n t i o n i c composition. 2. Removing monovalent ions from the solution resulted i n a greater divalent ion content, a smaller water content and a greater intravascular pressure of the a r t e r i e s . 3. The water content of the ar t e r i e s was smaller when: (a) the Ca + Mg content was greater, and (b) the intravascular pressure was greater. 4. In addition to d e f i n i t e changes i n the tension of the vascular smooth muscle c e l l s , there may have been changes i n the paracellular matrix of the artery w a l l : an ion exchange process may have altered the configur- ation 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 for an extended period of time gain sodium and water and lose potassium (22). I f the tissues are rewarmed to 37°C, the processes are reversed as the metabolism of the c e l l i s reactivated. The effects of cooling and rewarming on sodium and potassium exchanges have been studied using many 104 smooth muscle tissues (23,24), including vascular 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 artery could be divided into 2 components: a fast (complete within 15 minutes of rewarming) component, apparently unaccompanied by K and unaffected by idodo- acetate, and a slower metabolic component coupled 1:1 with K. They suggested that t h i s temperature sensitive fast Na ef f l u x represents the effect of temperature on either Na bound to the anionic groups of the e x t r a c e l l u l a r matrix or Na leaving the smooth muscle c e l l s through channels independent of the Na-K pump—perhaps accompanied by CI. Although cooling 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 w a l l . I t might also help i n understanding the behavior of water i n the artery w a l l ; i n pa r t i c u l a r the effect of various factors on vascular hydration. In t h i s regard, a series of experiments was performed i n an attempt to answer several questions: 1. What i s the effect of temperature on the extrusion of water from the rewarming artery? 2. What effect does a sodium gradient between the a r t e r i a l wall and the rewarming solution have on th i s extrusion of water? 3. Is there any relationship between the water extrusion and the fast non-K linked sodium extrusion? Since water can not be studied using the isotope or flow-through electrodes techniques other workers have employed i n their cooling-rewarming experi- ments, i t was decided to simply incubate the ar t e r i e s at various temperatures and determine thei r water contents from wet and dry weights. (a) Methods The rat t a i l a r t e r i e s were cooled overnight at 2°C, then incubated for 105 15 minutes i n solutions of diff e r e n t sodium concentration at a given tempera- ture between 2° and 37°C. This procedure allowed the water and ion content of the ar t e r i e s to be determined at d i s t i n c t points during the rewarming process. Fifteen minutes was chosen as the incubation time, rather than a few hours, so the changes that occurred during the extrusion of the non-K linked, temperature-sensitive sodium component would not be obscured by the effect of the slower metabolic exchanges of Na and K. The t a i l a r t e r i e s of the rats were excised after being flushed with Krebs 140 solution (140 refers to the Na concentration i n m e q / l i t e r — s i m i l a r l y for the other solutions). The ar t e r i e s were cut i n h a l f , placed i n flasks of Krebs 140 solution and kept i n the refrig e r a t o r overnight at 2°C. The a r t e r - ies were transferred to fresh Krebs 140 solutions,.aerated with oxygen, and incubated for a further 2 hours at 2°C. Fifteen h a l f - a r t e r i e s were removed for analysis of water and ions at th i s point. Five groups of 8 h a l f - a r t e r i e s were then transferred to Krebs 140 solutions, aerated with O2, at 8°, 10°, 20°, 30°, or 37°C for 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 for 3 hours at 37°C so that the effect of the complete rewarming process could be determined. The Na and K contents of the ar t e r i e s were determined using a Techtron absorption spectrophoto- meter. To examine the effects of the sodium gradient, after overnight cooling i n Krebs 140 solution, the arter i e s were: (a) transferred to Krebs 100 solution and the above procedure repeated using Krebs 100 throughout the re- warming, (b) transferred to Krebs 0 solution at 5°, 8°, 10°, or 37°C for 15 minutes. (Solutions 1 and 2 from the previous section were used for the Krebs 140 and Krebs 0 solutions i n th i s experiment.) The solutions were aerated with oxygen. Lactose replaced the NaCl i n these solutions. 106 (b) Results Effect of Temperature The changes i n the a r t e r i a l water and ion content during rewarming i n Krebs 140 solution are shown i n Figs. 19 to 21 and Table XIX. Arteries re- warmed i n Krebs 140 solution did not lose 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 at 37°C. ( A l l the sub- sequent water and ion values w i l l be per 100 g dry weight.) The ar t e r i e s l o s t Na and gained K when rewarmed i n Krebs 140 solution. Since the a r t e r i e s were equilibrated for 2 hours, these changes were due sol e l y to the effect of temperature. In 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 effect of temperature on the metabolic Na-K pump of the vascular smooth muscle c e l l s . The a c t i v i t y of the pump increased with the temperature, r e s u l t i n g i n a s l i g h t restoration of the io n i c gradients during these 15 minute rewarming periods. The art e r i e s l o s t 6.08 meq Na during these periods. The exchange of Na and K by the pump i n vascular smooth muscle c e l l s i s probably i n a 1:1 r a t i o (5,29). The difference 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 artery 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 quite dependent upon the temperature. Table I I also shows that most of the Na extruded from the artery wall 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 loss of th i s extra Na component was not only independent of K, but also occurs at 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 re- 107 TABLE XIX. Changes i n H20 (ml/100 g dry wt), Na and K (meq/100 g dry wt) contents of rat t a i l a r t e r i e s at di f f e r e n t rewarming temperatures i n 3 solutions with 140, 100, or 0 meq N a / l i t e r . A i s given when p < 0.05. Temperature A H2° ANa AK range Krebs 140 2° to 10° -0.739 10° to 20° -10.1* +1.56 20° to 30° +2.40 30° to 37° Subtotal 2° 37° to to 37° 37°(3 hr) -23.6 -6.08 -5.48 +2.86 +7.06 Total 2° to 37°(3 hr) -34.0 -11.6 +9.91 Krebs 100 2° to 10° |+0.51 10° to 20° -15.5 -6.00 20° to 30° +19.2 +2.15 30° to 37° -26.1 Subtotal 2° 37° to to 37° 37°(3 hr) -19.4 -8.79 -6.09 +3.27 +5.24 Total 2° to 37°(3 hr) -30.2 -14.9 +8.50 Krebs 0 5° to 8° 8° to 10° -44.5 10° to 37° -7.59 +1.86 Subtotal 5° to 37° -67.6 -8.23 +2.53 * 8° to 20°C 108 380 360 340 | 320 o< O O S 3 0 0 E O CM j 280 o 260 240 220. 0 I 2 TIME (hrs) at 2 °C K Krebs 0 Krebs 100""-^- i i 10 20 30 37 TEMPERATURE (degrees C) 0 1 2 3 TIME (hrs) at 37° C F i g . 19 Effect of temperature during rewarming on the 1^0 content of the rat t a i l artery cooled for 18 hours at 2 C. The arte r i e s were transferred from the solution at 2°C to one of the solutions between 2°C and 37°C for 15 minutes of rewarming. Three rewarming solutions with d i f f e r e n t Na concentrations were used. Each point represents 8 arte r i e s except at 2°C where n = 15. 109 60 55 50 •° 45 a> O O < 3 5 o t- 31 14 10 •I 0 I 2 TIME (hrs) at 2 °C -5. £ Krebs 0 10 20 30 TEMPERATURE (degrees C) 13. 37 0 1 2 3 TIME (hrs) at 37° C F i g . 20 Effect of temperature during rewarming on the Na content of the rat t a i l artery cooled for 18 hours at 2°C. The arte r i e s were transferred from the solution at 2°C to one of the solutions between 2° and 37°C for 15 minutes of rewarming. Three rewarming solutions with d i f f e r e n t Na concentrations were used. Each point represents 8 art e r i e s except at 2°C where n = 15. i 110 25 £20 >. T3 O 15 o CT E o. 10 -Hi • -o 0 1 2 TIME (hrs) at2°C • — • K r e b s 140 •—-•Krebs 100 o o Krebs 0 10 20 30 TEMPERATURE (degrees C) 37 0 12 3 TIME (hrs) at 37° C F i g . 21 Effect of temperature during rewarming on the K content of the rat t a i l artery cooled for 18 hours at 2°C. The arte r i e s were transferred from the solution at 2°C to one of the.solutions between 2° and 37°C for 15 minutes of rewarming. Three rewarming solutions with d i f f e r e n t Na concentrations were used. Each point represents 8 art 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 indicates that the Na-K pump requires the extended incubation period at 37°C to become f u l l y operating. I t also indicates that the 15 minute rewarming periods at tem- peratures between 2° and 37°C are not long enough periods of time for the metabolic Na-K pump to restore the i o n i c gradients upset by overnight cooling. During the 15 minute rewarming periods i n Krebs 100 solution, the ar- teri 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 I l l r e l a t i v e maximum, or "hump" i n the water content. (This maximum was also pre- sent for a r t e r i e s i n Krebs 140 solution, 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 solu- t i o n , the K changes were about the same, while there was more Na l o s t from ar t e r i e s i n Krebs 100 solution. Arteries rewarmed i n Krebs 0 solution showed a dramatic loss of 45 ml water between 8° and 10°C. Between the 15 minute incubations at 5°C and at 37°C, there was a loss of about 68 ml water. No information was obtained on the r e l a t i v e maximum at 30°C. These ar t e r i e s also 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 for these a r t e r i e s , the Na loss on rewarming may simply represent Na extruded from the artery i n 0 Na solution. Effect of the External 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 affected by the external Na concen- t r a t i o n . 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 osmolarities of the 3 solutions: Krebs 0, 290; Krebs 140, 313; Krebs 100, 329 mosm/liter. The i n i t i a l values are inversely related to the osmolarity as would be expected. The temperature at which most of the water was extruded from the rewarming artery was lower when the external 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 greater when the external Na concentration was lower. Since the Na-K pump was only minimally e f f e c t i v e during these short rewarming periods, this suggests that some other mechanism was mainly responsible for the changes i n the hydration of the rewarming artery 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 for a r t e r i e s i n the 3 solutions i n Fi g . 20 simply r e f l e c t the d i f f e r e n t Na concentrations of th e i r bathing media. The greater loss of a r t e r i a l Na when the external 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 effect of external 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 for a l l 3 solutions. There was no r e a l difference 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 did the external Na concentration a f f e c t the temperature at which the Na-K pump "came into action". Extrusion of the Non-K linked Na Component The external Na concentration affected the extrusion of the extra non-K linked Na component. I t was th i s effect that resulted i n the di f f e r e n t losses of Na i n the 3 solutions, and not any effect on the Na-K pump. The size of this extra Na component released during the 15 minute rewarming periods between 2° and 37°C i s presumably the difference 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 solution: 6.08 - 2.86 = 3.22 meq Na; Krebs 100 solution: 8.79 - 3.27 = 5.52 meq Na; Krebs 0 solution: 8.23 - 2.53 = 5.70 meq Na. However, the true size of this extra Na component i s smaller than these values because of the accompanying extrusion of water from the rewarmed artery w a l l . Some of th 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 equilibrium with the external solution 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 calcu- 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 carried out with t h i s water w i l l give the maximum correction to the size of the extra non-K linked 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 artery other than the free ECS. The above uncorrected values are the maximum amounts, i . e . they include Na l o s t from the free ECS. Krebs 140 solution; The non-significant 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 be considered as a r e a l decrease for these rough calculations. Twelve ml of free ECS water would have carried out 0.14 x 12 = 1.68 meq Na and 0.005 x 12 = 0.06 meq K. The minimum size of the extra Na component would have been 6.08 - 1.68 - 2.86 - 0.06 = 1.43 meq Na. Krebs 100 solution: The 19.4 ml of l o s t water would have carried out 1.94 meq Na and 0.097 meq K from the free ECS. The minimum size of the extra Na was 5.52 - 1.94 - 0.097 = 3.48 meq Na. Krebs 0 solution: Since i t i s the minimum size of the extra Na component that i s of i n t e r e s t , assume that the maximum trace Na i n this solution was 2 meq N a / l i t e r . The minimum amount of Na i n the extra component released was 8.23 - (0.002 x 67.6) - 2.53 - (0.005 x 67.6) = 5.23 meq Na. These minimum and maximum values of the extra non-K linked 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 size of the extra Na component extruded i n Krebs 0 solution, 5.23 to 5.70 meq Na/100 g dry weight, i s about the same size as Palaty's estimate of Na bound to the anionic s i t e s i n the paracellular matrix of the rat t a i l artery (6)—although, of course, the i r experimental procedure was quite d i f f e r e n t . 114 Fig . 22 Effect of external Na concentration, [ N a + ] 0 , on the change i n water content, AH2O, and on the size of the extra, non-K linked Na component extruded from the rat t a i l artery during 15 minute rewarming periods between 2° and 37°C. The mini- mum extra Na component i s the difference between the measured maximum extra Na component and the Na carried 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. (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 at 30°C, and (c) the effect of external Na. 115 The metabolic Na-K pump i s not a sati s f a c t o r y mechanism to explain these changes. Certainly the pump i s responsible for removing Na and water from and restoring K to the vascular smooth muscle c e l l s during rewarming. But 15 minute rewarming periods were not long enough for substantial pump a c t i - vation. Discontinuities have been observed (30,31; also see 32) i n the Arrhenius plot of the a c t i v i t y of the ATPase system presumably associated with the Na-K pump (33) . But at 30°C, there was not a simple change of the rate of water decrease, but an increase. This would imply that the pump was oper- ating i n reverse. But, there was no corresponding decrease i n the K content at 30°C. And f i n a l l y , the external Na concentration did not affect the Na-K pump during the 15 minute rewarming periods, although i t d r a s t i c a l l y affected the water l o s t from the rewarming artery. A clue for 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 artery w a l l . Both the amount of water and the amount of extra non-K lined Na extruded from the rewarming artery varied inversely with the external Na concentration. In addition, 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 at 30°C. There are 3 possible explana- tions of the o v e r a l l water decrease during the 15 minute rewarming periods which are related to this fast non-K linked Na component: 1. Channels i n the smooth muscle membranes, not connected to the Na-K pump, might provide the mechanism of extrusion of t h i s extra Na compon- ent, perhaps accompanied by CI (29,34). The decreased water content of the artery wall could be due to a decrease i n the c e l l water accompanying the loss of c e l l u l a r NaCl to maintain c e l l u l a r osmolarity. This NaCl loss might be the result of temperature dependent changes i n the state of the c o n t r a c t i l e proteins. 116 2. An electrogenic pump which operated between 10° and 20°C and pumped out c e l l u l a r Na and water without the extrusion of CI or the entry of K would explain both the Na and water losses during rewarming. This concept i s supported by the membrane hyperpolarization observed by Taylor e_t al. (35) at the sta r t of rewarming of the pregnant rat uterus. Upon immersion into solution at 38°C, precooled rat l i v e r s l i c e s showed an immediate loss of Na and Ca, while 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 hyperpolariza- t i o n . Presumably there would be a loss of c e l l water accompanying the Na and Ca losses. 3. I t has also been suggested that the extrusion of the fast Na com- ponent during rewarming might represent an effect of temperature on ion binding to anionic groups i n the paracellular matrix (5). The release of Na from the binding s i t e s might be accompanied by the binding of divalent ions. Such an exchange would be accompanied by conformational changes i n the matrix re 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. I t should be noted that Palaty et_ a l . (7) found a positive c o r r e l a t i o n between the water content of the rat t a i l artery and the amount of Na bound to the protein-polysaccharide complexes. In each of these cases, lowering the external Na concentration would augment the removal of the extra non-K linked Na from the rewarming artery w a l l . The presence of the peak i n water content at 30°C might be explained by a reversal, for reasons unknown, of these processes. There i s another possible source of the hydration changes during re- warming. There may be anomalies i n the temperature dependence of the physical 117 properties of water (37-42, compare 43). Also, 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 interfaces has d i f f e r e n t physical properties than normal water (44). At 2°C, the water structure i n the immediate v i c i n i t y of macromolecules, inside and outside the smooth muscle c e l l s , might be dif f e r e n t than the water structure i n more open spaces. It i s also possible that rewarming affects water next to macromolecules i n a di f f e r e n t manner than water i n the open spaces. I t could be speculated that alt e r a t i o n s i n the amount of water able to act as solvent, due to unequal st r u c t u r a l changes i n the wa l l water during rewarming, could res 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 swell at various temperatures. (d) Summary 1. The incubated rewarmed rat t a i l artery gained K, and l o s t Na and water. 2. There was only a s l i g h t gain of K during the 15 minute rewarming periods between 2° and 37°C. This gain and the temperature at which i t occurred were not affected by the external 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 affected by the external Na concentration. 3. The artery l o s t more Na than i t gained K during rewarming. If a 1:1 exchange i s postulated for the Na-K pump, the difference represents an extra Na component extruded at lower temperatures than the metabolic Na-K exchanges. The, size of this extra non-K linked Na component increased with decreasing external Na. 118 4. The amount of water extruded during rewarming was greater and the temperature at which most of the loss occurred was lower when the external 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 explain these changes i n the hydration of the rewarming artery. The water changes may be related to the extrusion of the fast non-K linked Na component. 2. On the Intravascular Pressure In some of the cooling and rewarming experiments previously mentioned, the effects 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 carotid artery contracted when they were placed i n solutions at 37°C, then progres- s 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 rat t a i l artery resulted i n a relaxation which began we 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 for Na or K, or by changes i n the e l a s t i c properties of the e x t r a c e l l u l a r matrix of the artery w a l l . Tobian (16) has suggested that the increased blood pressure i n hyper- tensive art e r i e s could be due to waterlogging of the artery w a l l . In view of t h i s suggestion and the observed changes i n the water content of the re- warming artery, i t seemed that a study of tension changes during rewarming might support these observations, and more important, provide some information i on the relationship between a r t e r i a l tension and a r t e r i a l hydration. There were several questions to be examined experimentally using the intravascular pressure as the measure of a r t e r i a l tension: 119 1. What i s the effect of temperature on the intravascular pressure of a rewarming artery? 2. What i s the effect of the external Na concentration on the i n t r a - vascular pressure during rewarming? 3. Is there any relationship between the hydration of the artery w a l l and the intravascular pressure during rewarming? (a) Methods This experiment involved simultaneous monitoring of the intravascular pressure and the temperature during rewarming. A 12 cm segment of the rat ventral t a i l artery was exposed, i t s c o l l a t e r a l s t i e d , and both ends cannu- lated. Removed from the t a i l bed, the artery was tested for leaks, placed i n a thin channel i n a perspex chamber (the bottom of which was part of a c i r - culating temperature-control system), f i l l e d with Krebs 150 solution, and placed i n the ref 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 rotary pump for open c i r c u i t perfusion. By passing the perfusing solution through tubing i n the bottom of the artery chamber before i t entered the artery, i t was always at the same temperature as the chamber and the artery. The temperature was monitored with a thermo- couple i n the artery channel and the intravascular pressure was monitored proximal to the artery with a Statham transducer. The cooled artery was rewarmed: (a) quickly by allowing water from a bath at 40°C to pass through the chamber, (b) i n steps of 1 or 2 degrees every 3 or 4 minutes—about 30 seconds was required for each step, (c) i n steps of 10°C every 20 to 90 minutes—from 3 to 15 minutes (depending upon the type of pump) were required for the 10° increase. During rewarming, 9 ar t e r i e s were perfused with Krebs 100 solution and 4 with Krebs 150 solution. 120 (b) Results The patterns of intravascular pressure changes during rewarming with Krebs 100 solution are shown i n Figs. 23 to 25. Fig. 23 shows the pressure pattern for a fast rewarming. There are 3 features to notice: (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. These 3 changes were observed for almost a l l the a r t e r i e s . Since there was probably a lag between the tempera- ture of the artery and that on the record during t h i s fast rewarming, step increases i n temperature were required to determine the exact temperatures at which the pressure changes were occurring. F i g . 24 shows the pressure pattern for an artery equilibrated for 1 hour then rewarmed i n steps of 1 or 2 degrees every 3 or 4 minutes. The pattern 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 artery i s kept at 11°C for only about 30 minutes before being raised to 21°C. Fig. 25 i s for an artery rewarmed i n Krebs 100 solution containing -3 10 M iodoacetate. Exactly the same pattern was obtained for an artery (not shown) rewarmed without iodoacetate for the same time periods. This indicates that iodoacetate, which blocks ATP production under these condi- tions i n the smooth muscle c e l l s , has no effect on the pattern of i n t r a - vascular pressure during rewarming. (Of course, there may have been s u f f i - cient ATP stored i n the c e l l s to render the action of the iodoacetate inconsequential.) The pressure changes for a r t e r i e s rewarmed with Krebs 150 solution were much smaller than those for the a r t e r i e s rewarmed with Krebs 100 solution. 121 40 r TEMPERATURE (°C) 20 I00 r PRESSURE mm Hg 50- TIME F i g . 23 Temperature and intravascular pressure during rewarming. Rat t a i l a r t e r i e s were cooled for 18 hours at 2°C i n Krebs 150 solution, then rewarmed during perfusion with Krebs 100 solution at 0.2 ml/min. The average changes i n the intravascular pressure were as follows: Dips: 12/13 ar t e r i e s had s l i g h t dips i n the pressure below 10°C. There was no difference between the size or the temperature at which the dip occurred for the Krebs 100 or Krebs 150 solutions. The average pressure change i n the dip was -2.5 mm Hg, at an average temperature of 8°C. Peaks: 11/13 ar t e r i e s had increases i n the intravascular pressure below 30°C. The average pressure changes were: Krebs 100, +44 mm Hg, and Krebs 150, +4 mm Hg. Overall changes: 12/13 art e r i e s had an ov e r a l l decrease i n the intravascular pressure. The average o v e r a l l changes were: Krebs 100, -10 mm Hg, and Krebs 150, -4 mm Hg. 122 F i g . 24 Effect of time and temperature on intravascular pressure during rewarming. Rat t a i l a r t e r i e s were cooled for 18 hours at 2°C i n Krebs 150 solution, then rewarmed during perfusion with Krebs 100 solution at 0.2 ml/min. These results show that the temperature dependence of the intravascular pressure during rewarming was affected by the external Na concentration. The ov e r a l l decrease i n the pressure and the pressure peak at 20° to 25°C were both greater when the external Na concentration was smaller. This was so even though a r t e r i e s rewarmed i n Krebs 100 solution were equilibrated i n Krebs 100 123 TIME (minutes) F i g . 25 Intravascular pressure at dif f e r e n t temperatures during rewarming. Rat t a i l a r t e r i e s were cooled for 18 hours at 2°C i n Krebs 150 solution, then rewarmed during perfusion with Krebs 100 solution containing 10""-* M iodoacetate, at 0.2 ml/min. solution for about 1 hour before rewarming. The external Na concentration did not seem to affe c t the size of the s l i g h t dip i n the pressure, or the temperature at which i t occurred. 124 (c) Discussion During rewarming of the artery, the c e l l u l a r Na-K pump restores the Na and K gradients and the artery relaxes. I t was thus expected that the intravascular pressure of the perfused artery would decrease between 2° and 37°C. However, since the ar t e r i e s were never at temperatures close to 37°C for very long, the pump was never f u l l y operating. In addition, the presence of iodoacetate did not affect the o v e r a l l decrease. Friedman eX a l . (28) also noticed that the o v e r a l l relaxation of the artery 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 explain the ov e r a l l decrease i n the intravascular pressure. The peak i n the pressure at 20°C to 25°C cannot be explained at a l l i n terms of the Na-K pump. The size and shape of the peak pressure were quite dependent upon the length of time the artery remained at a given temperature. This might suggest the involve- ment of the Na-K pump since i t s a c t i v i t y i s less at lower temperatures so i t requires longer times to restore the ionic gradients. But: (a) the restor- ation of the Na and K gradients relaxes the artery, i . e . decreases the i n t r a - vascular pressure, not increases i t , and (b) iodoacetate did not aff e c t the size and shape of the pressure peak. I t i s possible that the extrusion or storage of c e l l u l a r Ca during rewarming might play a role i n the o v e r a l l relaxation and/or the pressure peak of the rewarming artery. There seems to be quite a close l i n k between the pressure changes for the perfused artery and the hydration changes of the incubated artery during rewarming: (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° and 30°C, (b) Both these changes i n the pressure and hydration were increased by lowering the external Na concentration. Tobian (16) suggested that the increased intravascular pressure associated with hypertension might be explained by an increase i n the hydration of the artery w a l l and a resultant thicker w a l l and smaller lumen. The same phenomenon might partly explain the o v e r a l l decrease and peak i n the intravascular pressure during rewarming. Rough calculations 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 flow: P^/P^ = (V^/V^) 2 where: P = pressure gradient down the artery seg- ment, V = lumen volume, and states 1 and 2 refer to the artery before and after the changes i n w a l l water content respectively. Knowing P-̂  from the pressure recordings during rewarming and e s t i - mating V-ĵ  and V~2 enables the pressure gradient a f t e r the w a l l water change, to be calculated. V-̂  was calculated from either:, (a) P o i s e u i l l e ' s equation (knowing P-̂  and the flow, and estimating the artery 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 . V"2 was determined from - V^, the change i n lumen volume associated with the changes i n artery wall volume re s u l t i n g from the w a l l water changes. I t was assumed for the calculations that the changes i n water content of the rewarmed perfused artery were the same as those determined i n the above section for the rewarmed incubated artery. The lumen volume changes were calculated from the w a l l volume changes by assuming either: (a) the lumen volume increase was 1/2 the size of the wall volume decrease, or (b) the increase i n inner radius was the same size as the outer radius decrease. 126 The o v e r a l l decrease i n the hydration of the rewarming artery could account for 80% of the o v e r a l l decrease i n the intravascular pressure. The increase i n the water content at 30°C could account for about 30% of the observed increase i n the pressure at 20°C to 25°C. These estimates, as well as the corr e l a t i o n between thei r temperature dependencies and external Na concen- t r a t i o n dependencies, indicate that the intravascular pressure changes can be at least p a r t i a l l y explained i n terms of the hydration changes of the rewarming artery. There i s no comparable co r r e l a t i o n between the dip i n pressure at 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 intravascular pressure. I t i s also possible that the pressure dip was simply due to the artery w a l l volume being smallest at 4°C when the density of water i s greatest. (d) Summary 1. The perfused rewarmed rat t a i l artery showed: (a) a s l i g h t dip i n the intravascular pressure at 8°C, (b) a peak i n the pressure at 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 external Na concentration was lower: (a) the dip 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 intravascular pressure was greater. 3. The shape and si z e of the pressure peak at 20° to 25°C was strongly affected by the length of time the artery was kept at a given temperature. 4. The c e l l u l a r metabolic Na-K pump cannot explain the increase i n pressure at 20°C to 25°C, and can only partl y explain the o v e r a l l decrease i n the intravascular pressure. 127 5. There seems to be a l i n k between the intravascular pressure changes and the hydration changes during rewarming. Both have the same temperature and external Na concentration dependencies. 6. On the basis of a smaller artery w a l l and a larger lumen, the o v e r a l l decrease of wal l water could account for most of the o v e r a l l decrease i n the intravascular pressure during rewarming. The increased water content at 30°C could account for only part of the increased intravascular pressure at 20° to 25°C. 128 CHAPTER VI SUMMARY AND DISCUSSION 1. 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 artery w a l l . The following 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) Constricted 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 cross-sectional area than non-constricted a r t e r i e s . Constant length meant the volume of the c o n s t r i c t i n g artery w a l l decreased by 14%— compared to 13% calculated from the w a l l water lo s s . (d) The flow pattern of a constricted artery suggested that f l u i d was added to the lumen from the c o n s t r i c t i n g w a l l . 2. Contracting smooth muscle c e l l s increase t h e i r volume due to the entry of water. 3. The artery w a l l i s permeable to f l u i d s . The permeability i s increased by an increase i n intravascular pressure. 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 alterations i n external ion composition a l t e r the hydration of the artery w a l l presumably by ion exchange and/or vasoconstriction. 5. The hydration of rewarming a r t e r i e s depends on the temperature and the external Na concentration. The extrusion of wall water during rewarming: (a) may be related to the extrusion of a fast non-K linked Na component, and (b) p a r t l y explains the intravascular pressure changes during rewarming. 129 These studies have demonstrated that c o n s t r i c t i o n of the rat t a i l artery i s associated with a loss of water, a change i n wal l permeability, and an increase i n smooth muscle c e l l volume. How do these changes occur? Are there causal relationships between these 3 effects of vasoconstriction? What are the consequences of these alterations? Although no d e f i n i t e answers can be given, some possible explanations w i l l be examined. A. LOSS OF WALL WATER DURING VASOCONSTRICTION The loss of water from the co n s t r i c t i n g rat t a i l artery was due to the expulsion of e x t r a c e l l u l a r f l u i d . Similar losses were observed for contracting gels (1,2), muscle homogenates (3), frog stomach muscle (4,5), uterine muscle (6,7), rat aorta (8), and carotid a r t e r i e s (9). To understand why t h i s water loss occurred, i t may be useful to extend Bozler's explanation of frog stomach muscle behavior to the artery w a l l : i t behaves " l i k e a cross-linked gel i n which osmotic balance i s determined i n part by hydrostatic pressure a r i s i n g from e l a s t i c forces within the f i b e r s " (4). Water moves down i t s chemical potential gradient. A movement of water i n the absence of g r a v i t a t i o n a l forces, indicates the presence of osmotic or hydrostatic 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 artery wall f i r s t , causing a subsequent water s h i f t to balance osmolarity. In addition, the fact that the f l u i d which l e f t the cons t r i c t i n g artery was not too hypotonic suggests that osmotic forces were probably not involved. If 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 hydrostatic pressure difference forcing water from the artery wall? To attempt to answer th 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 vasoconstriction could a l t e r these forces. 130 When the a r t e r i a l hydration i s i n equilibrium, the osmotic and hydrostatic forces determining the hydration w i l l be balanced. The forces involved are e s s e n t i a l l y those for c a p i l l a r y f i l t r a t i o n . There must be a balance between the osmotic pressure of the blood and the hydrostatic pressure of the artery 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 hydrostatic pressure of the perfusing blood on the other. There must be a s i m i l a r equilibrium between the w a l l and the surroun- ding tissues: the tissue osmotic and the w a l l hydrostatic pressures must balance the wa l l osmotic and the tissue hydrostatic pressures. The consid- eration of these 2 balance sheets leads to some in t e r e s t i n g conclusions. The only complex factor i s the hydrostatic pressure i n the artery w a l l . This pressure i s a consequence of the tension i n the w a l l , active and e l a s t i c , which balances the transmural pressure (10). For a gel or an ion exchange r e s i n this relationship can be e a s i l y seen. As the r e s i n swells, the e l a s t i c forces between the components of i t s matrix increase u n t i l the resultant hydrostatic pressure i n the r e s i n balances the pressure i n the surroundings and equilibrium i s attained (11,12). In the artery w a l l the hydrostatic pressure i s a complex function of the w a l l tension (13). Es s e n t i a l l y , the tissue pressure decreases from the l e v e l of the i n t r a - vascular pressure to that i n the tissues surrounding the artery. [The claim 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 tissue pressure from the a d v e n t i t i a l to the in t i m a l layers of the aorta was observed by Brinkman et a l . (20). This tissue pressure gradient apparently determines the extent of penetration 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 tissue pressure gradient across the artery w a l l , there must also be an osmotic gradient across the w a l l . These gradients are shown i n Fig. 26. For a steady state of the water i n the w a l l , lumen and surrounding tissues, the s i t u a t i o n might resemble the following. I f i t i s assumed that the endothelial layer i s freely permeable to ions and small molecules but not to the plasma proteins, 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 leaving the artery w a l l there must be a lower tissue pressure i n the innermost layer of the w a l l . For an intravascular pressure of 90 mm Hg, the tissue pressure need only be 65 mm Hg j u s t inside the w a l l . [This point was ignored i n Burton's consideration of w a l l tissue pressure (13).] I f , i n the artery w a l l f l u i d there are soluble 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 hydrostatic pressure.and osmolarity at the lumen-wall interface would be smaller. In the w a l l layer adjacent to the innermost layer, the tissue pressure must be even lower (see F i g . 26). If the chemical potential of water i n these 2 layers were equal, the osmolarity of t h i s adjacent layer must also be lower. This decrease i n tissue pressure and i n osmolarity continues to the a d v e n t i t i a l surface where the tissue pressure equals that of the surrounding f l u i d . Since the manner i n which the tissue 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 layers (13), the same w i l l be true for the osmotic decrease. I t should be noted that the exact shape of the transmural pressure and osmolarity gradients are not known and apparently are quite complex (see 13)-. I f the innermost layer had a tissue pressure of 65 mm Hg and the outer layer was at 5 mm Hg, the o v e r a l l decrease i n concentration of the wall f l u i d across the wall w i l l be about 3 mM. This 132 means that the wal 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 layers than i n the inner layers of the artery w a l l . How i s t h i s s i t u a t i o n altered when, i n response to nervous or humoral influences, the artery constricts? The active 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 tangential 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 straight forward and have been des- cribed by Burton (10). However, when the intravascular 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 active and e l a s t i c tensions balances the pressure. I t i s possible that during c o n s t r i c t i o n , the actions of the increasing active tension and the decreasing e l a s t i c tension might cause an increased hydrostatic 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 cons t r i c t i n g artery w a l l . Some of the factors which might produce a pressure imbalance are: d i f f e r e n t rates of development of w a l l tension changes and intravascular pressure changes, delays i n trans l a t i n g active tension into a decreasing radius, or delays i n adjusting the e l a s t i c tension to the active tension changes. Since a water loss would affect the size of the artery w a l l , the volume of the lumen and thus the intravascular pressure, the movement of the water i t s e l f might act to correct the pressure imbalance. A reversal of the imbalance would cause f l u i d to return to the w a l l . Although t h i s discussion i s t o t a l l y conjecture, some changes of th i s sort are required to explain the loss of wal l water during vasoconstriction. 133 £ BLOOD IN LUMEN A R T E R Y WALL SURROUNDING E FLUID ElOOr Fig. 26 Schematic representation of the hydrostatic pressure and the osmolarity across the w a l l of a d i s t r i b u t i n g artery. The numerical values chosen are only for demonstration of the p r o f i l e s . i 134 Imbalances between the artery w a l l and the surrounding tissues are probably not too important. Van C i t t e r s e t - a l . noted that i n the constricted femoral artery, 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 cytoarchitectural alterations as the adventitia was reached" (26). The same phenomenon was noted i n the rat t a i l artery. This suggests that not only the tissue pressure but also the active tension changes are greatest i n the inner layers of the w a l l . The law of Laplace was used i n the above discussion of pressure- tension relations 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 stress on the vessel w a l l , not i t s tangential tension, and was put f o r - ward by Frank i n 1920 (21,28). Many authors ignore both t h i s difference and the fact that tension i s i n dynes/cm while stress, Peterson's "tension", i s i n dynes/cm2. The important role played by the law of Laplace i n a r t e r i a l behavior can be seen i n the effects of a l t e r i n g the pressure i n tissues surrounding a r t e r i e s . This a l t e r s the transmural pressure and thus the tension i n the artery w a l l . Altered hydrodynamics i n vascular beds have been observed during elevated ureteral pressure (29), altered cerebrospinal f l u i d pressure (30), and s t r i a t e d muscle contraction (31-34). Studies to test the effect of the a r t e r i a l pulse pressure have es- tablished that the artery i s incompressible when subjected to small strains (35,36,37, compare 38). This means that the r a t i o of transverse to l o n g i - tudinal 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 for large strains (39).] I t i s in 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 applies during vasoconstriction (25). However, passive stretching, by applying a load or pressure, occurs without the active ten- sion and expenditure of energy of vasoconstriction (23). External forces are required to stretch the artery, while the forces involved i n c o n s t r i c t i o n arise within the artery i t s e l f . Could the loss of water from the co n s t r i c t i n g artery w a l l have an effect on the v i s c o - e l a s t i c properties of the constricted artery? Stretching 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). 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 findings depend on the degree to which the ar t e r i e s are stretched (49) . [The e l a s t i c modulus used for 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 for large strains (50) as are involved i n vasoconstriction.] The v i s c o s i t y of the constricted artery i s apparently increased (27,51). None of these studies considered the water content of the a r t e r i e s . I t i s possible that the loss of e x t r a c e l l u l a r f l u i d during vasoconstriction may play a role i n these physical changes. There i s some i n d i c a t i o n that i n states of the artery other than con- s t r i c t i o n , hydration changes are associated with physical changes. A o r t i c s t r i p s immersed i n hypertonic saline 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 addition 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 possible that i n t e r r e l a t i o n s h i p s exist between the s t r u c t u r a l and hydration changes and the observed physical 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 negatively charged mucopolysaccharides i n the paracellular 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 with e x t r a c e l l u l a r ++ + proteins (58,63). The present study suggested that exchanging Ca for Na as the main counter-ions to the anionic groups of the protein-polysaccharides resulted i n conformational changes i n the matrix, an increased resistance to perfusion, and a loss of water from the artery w a l l (see 63,68). S i m i l a r l y , the rewarming of a cooled artery resulted i n a loss of w a l l water—possibly related to the extrusion of a f a s t , non-K linked Na component, which may be released during rewarming because of a temperature effect on the binding of Na to the paracellular matrix (67,69,70). These experiments suggest a possible relationship between conformational changes of the paracellular matrix and loss of water from the artery w a l l . The imbalance of hydrostatic forces, suggested as causing the w a l l water loss during vasoconstriction, may involve tension developed i n the paracellular matrix. The ion 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 vasoconstriction. There are 2 possible ways the paracellular matrix could affect the hydration of the artery during c o n s t r i c t i o n : (a) The contraction 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 artery w a l l . (b) The contraction 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 re s u l t i n g i n shrinkage of the matrix and expulsion of extra- c e l l u l a r f l u i d . These 2 explanations could be combined i f conformational changes i n the matrix during vasoconstriction altered the charge density or counter-ion s e l e c t i v i t y of the paracellular matrix, causing shrinkage of the matrix and f l u i d expulsion. Other authors have mentioned the possible r o l e of the mucopolysacch- arides i n a r t e r i a l hydration: Bader noted that "the ground substance has the properties of a c o l l o i d — i t i s water insoluble, but can bind water. I t consists of mucopolysaccharides ... i s a very viscous material 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 vessels" (21). In his study of a combined structure of hyaluronic acid, water and collagen f i b e r s which had a d e f i n i t e resistance to compression, Fessler suggested that mucopolysaccharides have a mechanical function (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 wall and also that i t s physical state may determine the mechanical resultant of the linkages of the other components" (52) . Zugibe and Brown demonstrated a tight band of acid mucopolysaccharides i n the subendothelial layer of the aorta (72). I t i s i n t e r e s t i n g that i n this layer the w a l l hydrostatic pressure i s largest (13) and most of the tension i s developed during vasoconstriction (26). Further studies which could be performed to demonstrate and explain the loss of w a l l water during vasoconstriction include: 138 1. measuring the density of relaxed and constricted a r t e r i e s by dropping a r t e r i a l segments into a series of solutions with densities between 1.05 and 1.08 g/cc (see 73)I. 2. analysis 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 relaxation. 3. examination of a relationship between the w a l l water loss and the extent of vasoconstriction by varying the doses of vasoconstrictive 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 vasoconstriction i n order that the average w a l l tension (see 25) can be related to the water l o s t from the co 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 for 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 (see 74). B. ALTERED VASCULAR PERMEABILITY DURING VASOCONSTRICTION The w a l l of the rat t a i l artery was quite permeable to f l u i d . Increased intravascular pressure increased the permeability as did vaso- c o n s t r i c t i o n with or without an i n i t i a l short-lived decrease. The permea- b i l i t y of the artery w a l l w i l l f i r s t be considered, then the effects of intravascular pressure and vasoconstriction. I t i s usually assumed that a l l f l u i d exchange between blood and the tissues occurs across the c a p i l l a r i e s . However, there are some indications that larger vessels are permeable. Zweifach has commented that "there i s good evidence that movement of gases, water and small water-soluble 139 molecules occurs even across the walls of terminal a r t e r i o l e s and pre- c 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 vessels" (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 trans- port of Na and CI from the inside to the outside of the aorta and i n the opposite d i r e c t i o n for the vena cava (22). Water can be transported across the artery w a l l by electro-osmosis (78). I t was suggested that "both ion and water flow take place largely through [the] e x t r a c e l l u l a r spaces (pores), which are the source of least resistance to water and ion movements" (78). While i t i s possible that the large transmural f l u i d movements observed for the rat t a i l artery occur i n vivo, 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 large a r t e r i e s because the wal l of the vasa vasorum was more permeable than the intima of the large blood vessels (79). In addition, the removal or separation of the rat t a i l a r t e r i e s from their surroundings meant that the physical equilibrium of the i n vivo s i t u - ation was no longer present. Large f l u i d movements across the artery w a l l would c e r t a i n l y affect the hydrostatic and osmotic pressure balance des- cribed above. I t i s also possible that the large 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 induced con- 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). In any case, the s i g n i f i c a n t finding was that f l u i d could pass fr e e l y through the rat t a i l artery 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 permeability i s increased i n hypertension (82,83), inflam- mation (84), and treatment with histamine or serotonin (85,86). I t has been suggested that the areas of blood vessels which readily 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 conditions, a higher permeability than other parts of the vessel (87). I t i s to be expected that increased intravascular pressure caused an increase i n flow out of the lumen through the w a l l of the perfused rat t a i l artery. Landis showed that an increase i n c a p i l l a r y pressure increased f i l t r a t i o n (88,89) as suggested by S t a r l i n g (90). This increased permea- b i l i t y may be related 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 intravascular pressure increased the passage of blood serum through the vascular w a l l , although the pressure had no effect when the vessel was not allowed to d i l a t e (77). This means that s t r a i n , not pressure, determines the permeability of the vascular w a l l . Stretch alone has been shown to increase the permeability of g e l a t i n films to hemoglobin (91) and the absorption of dyes by e l a s t i c membranes (92). The effect 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 effect 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 effects were assumed to have occurred at the c a p i l l a r i e s . Renkin observed that for a given blood flow, the clearance of test molecules was much smaller during vasoconstriction (93). I t i s also known that prolonged intravenous administration of catecholamines can deplete the blood volume (94-97, compare 98). In th 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 increased during vasoconstriction produced by adrenaline" (95). However, Haddy et a l . 141 suggested that the depletion of blood was instead due to the diff e r e n t effects of adrenaline on veins and ar 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 hydrostatic pressure, i s altered, so the tissues gain weight (97). This suggestion may w e l l apply to Renkin's findings. Also, the usefulness of epinephrine i n treating edema i s not due to the effect of con- s t r i c t i o n on permeability, but to the reduced blood flow during vasocon- s t r i c t i o n (89). This action 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 direct effects of c o n s t r i c t i o n on vascular permeability. However, there i s i n d i r e c t evidence to suggest a relationship between vasoconstriction and altered vascular permeability: 1. Vasopressin (pitressin) i s a vasoconstrictor (99) and increases i n t e s t i n a l water absorption (100), the permeability of frog skin (101), and active transport of Na by frog skin or bladder (102). 2. Serotonin i s a vasoconstrictor (103) and increases c a p i l l a r y per- meability (104) . 3. Histamine i s a vasodilator of small a r t e r i o l e s and a vasocon- s t r i c t o r of larger 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 vasoconstrictor (106) and increases l i v e r membrane permeability to K (107). 5. Epinephrine i s a constrictor of most vascular smooth muscle (106) and increases the permeability of frog skin to Na (108) and gut to sugars (109). 142 6. Bradykinin i s a vasodilator (110) and increases the permeability of skin blood vessels (111). In short, many vasoactive agents increase permeability. In l i g h t of the findings of the present study, the whole relationship between these two properties should be investigated. The fact that the changes i n permeability during vasoconstriction depended upon the intravascular pressure, suggests that the above analysis for the wall water loss may also apply to this altered permeability s i t u - ation. Transmural f l u i d movements were observed. Altered hydrostatic pressures i n the wal l and lumen of the cons t r i c t i n g artery 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 hydrostatic pressure differences between them would cause f l u i d to move through the w a l l . In addition, the e x t r a c e l l u l a r s o l i d s undergo conformational changes during vasoconstriction. 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 co n s t r i c t i n g artery wall and would affect vascular permeability. I t was pointed out above that pressure alterations during c o n s t r i c t i o n could cause the w a l l water loss which i n turn would decrease the intravascular pressure. There may be a s i m i l a r system operating here. Alterations i n the transmural balance of osmotic and hydrostatic pressures result i n altered w a l l perme- a b i l i t y , which i n turn affects the osmotic and hydrostatic pressures developed within the artery w a l l . That i s , an increased permeability would decrease the pressure gradients, the alterations i n which were the cause of the increased permeability. The explanations for the altered permeability could consequently be turned around: an increase i n the path for flow after configurational changes could cause transmural f l u i d movements which would upset the osmotic and hydrostatic pressures across the artery wall 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 interesting 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 permeability, i n the w a l l of the aorta due to "a s l i g h t depolymerization of the mucopolysaccharide connective- tissue matrix" (20,112). Measurements of D20 flu x across the c o n s t r i c t i n g artery w a l l (see 113) with controlled i n t r a - and extravascular pressures could supply some data to test these speculations. 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 increasing volume, due to the entry of water, during vasoconstriction. This finding agrees with the c e l l volume increases observed during vasoconstriction (114,115,116) and with the effects of anisosmotic solutions: vascular smooth muscle c e l l s swell and contract i n hypotonic solutions (116) and shrink and relax i n hypertonic solutions (117). I t i s generally assumed that c e l l s have the same osmolarity as the f l u i d which surrounds them (see 118 for a f u l l discussion). However, t h i s might not be so i n the artery w a l l i f the i n t r a c e l l u l a r tension caused a hydrostatic pressure difference across the smooth muscle c e l l membrane. In addition, the pressure and osmotic gradient across the artery w a l l might mean that the smooth muscle c e l l s i n the di f f e r e n t layers of the media have dif f e r e n t osmolarities and hydrostatic pressures, and hence d i f f e r e n t ten- sions. This might be related to the fact that the inner smooth muscle c e l l s undergo the most dr a s t i c contractions during vasoconstriction (26). 144 Movement of water into the a r t e r i a l smooth muscle c e l l s means that one or more of the following 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 hydrostatic pressure decreased or (d) ECS hydrostatic pressure increased. The explanation chosen must not c o n f l i c t with the fact 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 artery w a l l , although of course, the c e l l u l a r swelling may not have occurred at the same time as the ECS f l u i d l o s s . Ion movements, tension changes and permeability changes could a l l have caused these proposed osmotic and hydrostatic pressure changes. The only explanation which can be ruled out i s (c): a decrease i n c e l l hydrostatic pressure i s u n l i k e l y since the tension within the contrac- ting c e l l would increase. Jonsson suggested that the increase i n active 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 rat portal vein does not generate an i n t r a c e l l u l a r hydrostatic pressure large 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 be true, the increase i n e x t r a c e l l u l a r hydrostatic pressure may have balanced some of the c e l l u l a r increase. Of the other 3 explanations, there i s l i t t l e or no data available either to refute 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 ions, by bound c e l l u l a r ions becoming free, or by free c e l l water becoming bound during contraction. Nothing i s known about changing amount of bound and free ions and water i n contracting a r t e r i a l smooth muscle c e l l s . During contraction, K leaves (119), while Na (73) and Ca (120) enter the smooth muscle c e l l s . These ion s h i f t s may not be balanced osmo- t i c a l l y , Na may be accompanied by CI, and an unknown amount of Ca may be 145 free or bound i n the c e l l . If much of the smooth muscle c e l l ATP were i n - volved i n active muscle contraction, i t i s possible that the membrane ion pumps could be affected, increasing 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 explain the expulsion of e x t r a c e l l u l a r f l u i d from the const r i c t i n g artery w a l l . (b) A decrease i n ECS osmolarity could occur i f the endothelial permeability were altered, i f ECS ions became bound, or i f water bound to ex t r a c e l l u l a r macromolecules became free. Alterations i n the distance be- tween the fixed 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 res 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 into the c e l l s , lumen and surrounding tissues to restore osmotic equilibrium. 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 wall water los s . (d) An increase i n e x t r a c e l l u l a r hydrostatic pressure would res 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 cons t r i c t the artery w a l l . The existence of the transmural pressure gradient (13) means that the increasing intravascular pressure would be accompanied by an increasing w a l l hydrostatic pressure. Decreasing the endothelial permeability to water would also increase the wal l hydrostatic pressure. As with a de- creased ECS osmolarity, an increased ECS hydrostatic pressure, resulting i n an increased e x t r a c e l l u l a r chemical potential of water , would drive f l u i d into the smooth muscle c e l l s and into the lumen and surrounding tissues. 146 No matter what the cause, the increase i n c e l l water during vaso- co n s t r i c t i o n would decrease c e l l u l a r ion concentrations. Associated with a loss of c e l l K and a gain of c e l l Na, th i s means the [ K + ] ^ / [ K + ] o gradient would decrease r e s u l t i n g i n depolarization 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 sizes of the c e l l Na and water gains. 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The voltage from 1 l i n e on the screen was then displayed on the CRO. This voltage display represents the artery dimensions. This experiment was terminated when accurate measurements of the relaxed and constricted artery dimensions could not be made. The essential problem was magnification vs f i e l d of view. The necessity of i n - cluding the whole wa l l meant that the magnifications used were too low for determination of small dimensional changes. 1. Wiederhielm, C.A. Fed. Proc. 24: 1075, 1965. 2. Baez, S. C i r c u l a t . Res. 25: 315, 1969. 3. Wiederhielm, C.A. J . Appl. Physiol. 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 ar 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 vaso- co n s t r i c t i o n . Longitudinal sections of the rat t a i l artery were used: 163 (a) Epon sections, cut 1.5 u thick, were stained with toluidine blue, (b) freeze-substituted a r t e r i e s with known state 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 and stained with Mallory trichrome. 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 artery axis, except c e l l s near the adventitia which were at 70° to 80° to the axis. The c e l l s i n the constricted 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 further from 90°. Also, some groups of c e l l s near the lumen were at 30° and 160° to the ax 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 constricted 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. Geg. Morphol. Jahrb. 83: 230, 1939. 3. Goerttler, K. Geg. Morphol. Jahrb. 91: 368, 1951. 4. Rhodin, J. Physiol.Rev. 42: suppl. 5, 48, 1962.- 5. Rhodin, J. J. U l t r a s t r u c t . Res. 18: 181, 1967. 6. Phelps, P.C. and Luft, J.H. Am. J. Anat. 125: 399, 1969. APPENDIX I I I WEIGHING AN ARTERY WITH AN ELECTROBALANCE The rat t a i l artery was suspended i n Krebs solution from a Cahn electrobalance (see 1). The change i n artery w a l l volume, AVW, during con- s t r i c t i o n i s : AV W = AW&/ p f - p s (1) 164 where: AWg = 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 leaving the artery, and p s = density of the solution i n which the system i s suspended. Although weights were re- corded for the rat t a i l artery, i t never proved possible to add a vasocon- s t r i c t i v e agent such as norepinephrine to the solution without considerably disturbing the weighing. Dripping solution down the artery suspended i n a i r proved equally unsatisfactory. Consequently, this experiment was terminated. Derivation of equation (1): The system i s the artery, the suspending wire and a small weight. For Wa = weight of system i n a i r , and Wg = weight of system i n solution, the buoyancy, B, i s the weight of solution displaced by the system: B = Wa - Ws. So, AB = AWa - AWS during c o n s t r i c t i o n . Let V =• volume of d i s - placed solution, i . e . V = B/ps, so AB = psAV. Thus, AWS = AWa - psAV. Since only the artery changes volume during c o n s t r i c t i o n , AV = AVW. The movement of f l u i d from the wal l means AWa = pfAV w. Substituting these l a s t 2 equations into that for AWS gives equation (1). 1. Lindemann, B. and Solomon, A.K. J. Gen. Physiol. 45: 801, 1962.

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