UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Sodium and vascular smooth muscle reactivity Ragheb, Mohamed A. 1973

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1973_A6_7 R33_4.pdf [ 8.92MB ]
Metadata
JSON: 831-1.0101558.json
JSON-LD: 831-1.0101558-ld.json
RDF/XML (Pretty): 831-1.0101558-rdf.xml
RDF/JSON: 831-1.0101558-rdf.json
Turtle: 831-1.0101558-turtle.txt
N-Triples: 831-1.0101558-rdf-ntriples.txt
Original Record: 831-1.0101558-source.json
Full Text
831-1.0101558-fulltext.txt
Citation
831-1.0101558.ris

Full Text

SODIUM AND VASCULAR SMOOTH MUSCLE REACTIVITY by MOHAMED A. RAGHEB M.B., B.Ch., Cairo U n i v e r s i t y , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Pharmacology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1973 In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference and study. I further agree that permission for extensive copying of th i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia Vancouver 8 , Canada Date /fi/S//??* i i ABSTRACT Different drugs which can affect various aspects of sodium trans-port were tested on contractile activity of a variety of vascular smooth muscles. In the rabbit anterior mesenteric-portal vein, diphenylhydantoin sodium (DPH) attenuated the contractile responses to noradrenaline (NA) and the inhibition was reversible by washing. The p-hydroxy derivative, 5-p-hydroxyphenyl)-5-phenylhydantoin (DPHOH), which was reported to lack the anticonvulsant activity and the inhibitory effect of DPH on insulin secretion was without any effect on the rabbit anterior mesenteric vein. The inhibitory effect of DPH on contractile responses to NA was almost abolished by prior treatment with ouabain and i n K-free solutions. Evi-dence of a Na-Ca interaction i s provided by the finding that both low Na and high Ca Krebs attenuates the inhibitory effect of DPH while low Ca Krebs accentuates i t . Electrolyte studies using rat t a i l arteries showed that DPH could counteract the K loss and Na gain produced by either ouabain containing or K-deficient solutions. Prior cooling of the tissue for one hour at 1° C. and then testing the effect of DPH i n K-deficient solutions at 1° C , abolished the a b i l i t y of DPH to counteract the electrolyte changes i n K-deficient solutions. DPH i n normal Krebs solution did not significantly affect the Na con-tent of rat t a i l arteries. Under this condition, there was a slight diminu-tion i n the K content. The results suggest that DPH can stimulate the Na pump in rat t a i l arter-ies under conditions In which Na and Rare going along their electrochemical gradient, and/or under conditions simulating the depolarized state. In normal i i i Krebs, DPH does not seem to stimulate the Na pump of r a t t a i l a r t e r i e s . Under such experimental conditions, our data are more suggestive of an i n h i b i t i o n of the Na-K ATPase. Evidence i s also provided that the attenuation of c o n t r a c t i l e responses to NA i n the r a b b i t a n t e r i o r mesenteric v e i n , might be r e l a t e d to st i m u l a t i o n of the Na pump. Further studies were done to o u t l i n e the e f f e c t of a l t e r a t i o n of other parameters of Na transport i n vascular smooth muscles. Ouabain and ethacrynic a c i d , both i n h i b i t o r s of the Na-K ATPase, produced a c h a r a c t e r i s t i c pattern of response i n the r a b b i t a n t e r i o r mesenteric v e i n . There was at f i r s t a contr a c t i o n , followed by r e l a x a t i o n , which was followed by more p e r s i s t e n t contractures at higher doses. The d i u r e t i c agent, amiloride, which i s rep o r t -ed to i n h i b i t the passive Na i n f l u x i n a v a r i e t y of t i s s u e s , was found to attenuate c o n t r a c t i l e responses to NA i n r a b b i t a o r t i c s t r i p s , A.M.V., and r a t t a i l a r t e r i e s . In a o r t i c s t r i p s i t s e f f e c t was s p e c i f i c on the ra p i d phase of NA con t r a c t i o n . Since the ra p i d phase i s believed to be the r e s u l t I | of the release of a more t i g h t l y bound Ca pool , the l a t t e r e f f e c t suggests that amiloride a f f e c t s mainly the a v a i l a b i l i t y of a bound Ca pool to the con-t r a c t i l e p r o t e i n . Our r e s u l t s so f a r suggest that a l t e r a t i o n of d i f f e r e n t parameters of I | the Na transport might be involved i n a l t e r i n g the amount of i o n i z e d Ca a v a i l a b l e to the c o n t r a c t i l e p r o t e i n . More studies on t h i s subject are needed and might open the way f o r a b e t t e r understanding of the Na-Ca i n t e r a c t i o n i n vascular smooth muscles and t h e i r p o s s i b l e r e l a t i o n to the hypertensive s t a t e . Further experiments were done to study the r e l a x a t i o n of the r a b b i t a n t e r i o r mesenteric v e i n following c o n t r a c t i l e responses. I t seems that e x t r a c e l l u l a r Na i s involved i n the r e l a x a t i o n of the ra b b i t a n t e r i o r mesenteric v e i n following c o n t r a c t i l e responses, since t h i s i v tissue, when contracted i n low Na solutions, would not relax unless some of the Na is returned to the Krebs solution. L i could not sub-stitute for Na i n this function since the contracture occurred i n low Na solutions irrespective of whether the substitute was Tris-HCl or L i . the latte r was even more effective than Tris-HCl i n inducing con-tractures i n low Na Krebs. Moreover, relaxation of the rabbit anterior mesenteric vein, following contractile responses to NA was delayed i n low Na Krebs irrespective of whether the substitute was L i or Tris-HCl. Preliminary electron microscopic studies were carried on rabbit anterior mesenteric veins to evaluate the effect of the inhibitors of the ATPase, ethacrynic acid and ouabain, on ultrastructure. Both drugs induced disruption of the myofibrillar structures i n doses of 1 mM and 10 ^ M, respectively, when the tissue was exposed to the drug for 2 hours. An impressive finding was the dramatic diminution i n the number of plasma-lemmal vesicles i n the ethacrynic acid treated tissue. This finding raises the p o s s i b i l i t y that the formation of these vesicles might be an energy dependent process and opens the way for further studies to evaluate their possible importance i n active ionic transport processes. Supervisor TABLE OF CONTENTS Page ABSTRACT 1 INTRODUCTION 4 Ca and Smooth Muscle Contraction 5 A. C o n t r a c t i l e p r o t e i n 5 B. Membrane phenomena 8 (1) Electrogenic sodium pump i n smooth muscles 9 (2) Action p o t e n t i a l and graded d e p o l a r i z a t i o n 12 of smooth muscles C. E x c i t a t i o n contraction coupling 14 The Na-Ca I n t e r a c t i o n i n Smooth Muscles 17 Sodium and the Hypertensive Blood Vessel 20 STATEMENT OF THE PROBLEM 22 MATERIAL AND METHODS 26 D i s s e c t i o n of the Rabbit A n t e r i o r Mesenteric Vein 26 D i s s e c t i o n of A o r t i c S t r i p s 26 D i s s e c t i o n of Rat T a i l A r t e r i e s 27 P h y s i o l o g i c a l S a l t Solutions 28 Normal Krebs So l u t i o n 28 Phosphate-free Solution 28 Low Na Solutions 29 Na-free Krebs 29 K-free Solution 29 K - d e f i c i e n t S o l u t i o n 29 Estimation of the E l e c t r o l y t e Content of Rat T a i l A r t e r i e s 30 v i Page MATERIAL AND METHODS (continued) Calculation of H 20 and Electrolyte Content 30 Organ Bath Experiments 31 Apparatus for Perfusion of the Rat T a i l Artery 33 Apparatus for Incubation of the Rat T a i l Artery 35 Apparatus for Cooling Experiments 35 Preparation of Histological Sections 37 Preparation and Sources of the Drugs 37 St a t i s t i c a l Evaluation of Results 39 RESULTS 40 1. The inhibitory effect of DPH on spontaneous activity 40 and NA contractions of the rabbit anterior mesenteric vein 2. Comparison of the effect of DPH and DPHOH on NA 43 contractile responses 3. Effect of ouabain on the inhibitory action of DPH 45 4. Effect of K-free solutions on the inhibitory effect 46 of DPH on NA contractions 11 5. Effect of different Ca concentrations on the inhibitory 54 effect of DPH on NA contractions 6. Effect of low Na Krebs on the inhibitory action of DPH 56 7. Effect of DPH on the electrolyte content of rat t a i l 62 arteries i n normal Krebs solutions 8. Effect of DPH on the electrolyte content of rat t a i l 64 arteries i n the presence of ouabain 9. Effect of DPH on the electrolyte content of rat t a i l 65 arteries i n K-deficient solutions v i i Page RESULTS (continued) 10. E f f e c t of DPH on the e l e c t r o l y t e content of r a t t a i l 66 a r t e r i e s i n K - d e f i c i e n t s o l u t i o n s at 1° C. A l t e r a t i o n of Other Parameters of Na Transport and C o n t r a c t i l e 68 A c t i v i t y i n Vascular Smooth Muscle 1. E f f e c t of ouabain and ethacrynic a c i d on the r a b b i t 68 a n t e r i o r mesenteric v e i n 2. E f f e c t of amiloride on the c o n t r a c t i l e responses of 70 NA i n vascular smooth muscles Na and the Relaxation of the Rabbit A n t e r i o r Mesenteric Vein 77 1. E f f e c t of Na ions on r e l a x a t i o n of the r a b b i t a n t e r i o r 77 mesenteric v e i n 2. E f f e c t of DPH on r e l a x a t i o n following ouabain, procaine, 79 and NA contractions The I n h i b i t o r s of the ATPase and U l t r a s t r u c t u r e of the 88 Rabbit A n t e r i o r Mesenteric Vein 1. The e f f e c t of ethacrynic a c i d on u l t r a s t r u c t u r e of 88 r a b b i t a n t e r i o r mesenteric v e i n 2, The e f f e c t of ouabain on u l t r a s t r u c t u r e of the r a b b i t 89 a n t e r i o r mesenteric v e i n DISCUSSION 97 Diphenylhydantoin Sodium and Sodium Transport 97 DPH and C o n t r a c t i l e Responses to Drugs 100 The I n h i b i t o r s of the ATPase and C o n t r a c t i l e A c t i v i t y of 103 the Rabbit Anterior Mesenteric Vein Amiloride and C o n t r a c t i l e Responses to NA 104 Sodium and the Relaxation of the Rabbit A n t e r i o r Mesenteric Vein 105 BIBLIOGRAPHY 109 v i i i LIST OF TABLES Page Table 1. Effect of DPH on the electrolyte contents 63 of rat t a i l arteries Table 2. Effect of DPH on t. »_ following NA contractions 85 LIST OF FIGURES T i t l e Apparatus f o r organ bath experiments Apparatus f o r perfusion of r a t t a i l a r t e r i e s Apparatus f o r incubation of r a t t a l a r t e r i e s I n h i b i t o r y e f f e c t of DPH on spontaneous a c t i v i t y and c o n t r a c t i l e response of NA i n the ra b b i t A.M.V. In h i b i t o r y e f f e c t of 20 ug/cc DPH on NA contractions i n the r a b b i t A.M.V. -4 Comparison of the e f f e c t of 10 M DPH and DPHOH i n the r a b b i t A.M.V. on the c o n t r a c t i l e response of 5 x 10" 8 g/cc NA Comparison of the i n h i b i t o r y e f f e c t of 2 x 10 M DPH on 10~^ g/cc NA i n the ra b b i t A.M.V. i n normal Krebs and ouabain containing Krebs Comparison of the e f f e c t of DPH i n normal Krebs and ouabain containing Krebs Legend to F i g . 8 -4 Comparison of the i n h i b i t o r y e f f e c t of 2 x 10 M DPH on the c o n t r a c t i l e response of 10 ^  g/cc NA i n the ra b b i t A.M.V. i n normal Krebs and i n K-free Krebs Comparison of the e f f e c t of DPH i n normal Krebs and i n K-free Krebs Legend to F i g . 10 In h i b i t o r y e f f e c t of 10" 4 M DPH i n the ra b b i t A.M.V. on c o n t r a c t i l e responses of 10 ^  g/cc NA i n Krebs I | containing d i f f e r e n t Ca concentration X LIST OF FIGURES (continued) Fig. No. T i t l e Page Legend to Fig. 11 56 12 Comparison of the inhibitory effect of 15 yg/cc DPH 59 —8 on the contractile response of 3.2 x 10 g/cc NA in normal Krebs and i n 25 mM Na Krebs (Tris-HCl substitution) i n the rabbit A.M.V. 13 Comparison of the inhibitory effect of 15 Mg/cc DPH 60 —8 on the contractile response of 3.2 x 10 g/cc NA i n normal Krebs and i n 60 mM Na Krebs (Li substitution) i n the rabbit A.M.V. 14 Response of the rabbit A.M.V. to 5 x 10~6 M and 10~5 M 69 ouabain -4 -3 15 Response of the rabbit A.M.V. to 10 and 10 M 71 ethacyrnic acid 16 Response of the rabbit A.M.V. to repeated applications 72 of ethacrynic acid 17 Effect of amiloride on contractile responses of NA 74 in the rabbit A.M.V. 18 Effect of amiloride on contractile responses of NA In 75 rabbit aortic strips 19 Contractile response of the rabbit A.M.V. In normal 78 Krebs and i n 25 mM Na Krebs (Tris-HCl substitution) 20 Contractile response of the rabbit A.M.V. in normal 80 Krebs and i n 25 mM Na Krebs (Li substituted) Legend to Fig. 20 82 LIST OF FIGURES (continued) F i g . No. T i t l e Page 21 E f f e c t of DPH on r e l a x a t i o n of the ra b b i t A.M.V. 82 following ouabain c o n t r a c t i o n 84 22 E f f e c t of DPH on r e l a x a t i o n of the ra b b i t a n t e r i o r mesenteric v e i n following procaine con t r a c t i o n 23 & 24 Lon g i t u d i n a l s e c t i o n i n the ra b b i t A.M.V. following 90 & 91 -3 incubation i n Krebs containing 10 M ethacrynic a c i d 25 L o n g i t u d i n a l s e c t i o n of c o n t r o l r a b b i t A.M.V. incubated 92 i n normal Krebs s o l u t i o n 26 & 27 Longitudinal s e c t i o n i n the r a b b i t A.M.V. following 93 & 94 incubation i n Krebs containing 10 ouabain f o r 2 h r s . 28 Longitudinal s e c t i o n of c o n t r o l r a b b i t A.M.V. incubated 95 i n normal Krebs s o l u t i o n INTRODUCTION 4 I t has long been known that a l t e r a t i o n i n the concentration of s p e c i f i c e l e c t r o l y t e s a l t e r s p r e d i c t a b l y the mechanical response of muscles (Ringer, 1883). A proper understanding of the e f f e c t of a l t e r -a t i o n of Na transport on vascular smooth muscle r e a c t i v i t y would be of s p e c i a l i n t e r e s t i n view of the i m p l i c a t i o n of t h i s ion i n the produc-t i o n of the hypertensive state ( S a p i r s t e i n et a l . , 1950; Dahl, 1961; Friedman and Friedman, 1961; Friedberg, 1966), the p o s s i b i l i t y of a l t e r i n g vascular r e s i s t a n c e (Tobian, 1967) and the course of the hyper-tensive state by the amount of ingested sodium (Kempner, 1948; Corcoran et a l . , 1961), the well-known antihypertensive e f f e c t s of s a l u r e t i c agents (Tobian, 1967; Brest, 1969) and the various reports denoting an increase i n the sodium content (Tobian and Binion, 1962; Tobiaiy et a l . , 1961; Hagemeijer et a l . , 1966; S c h o f f e n i e l s , 1969) and vascular r e a c t i v i t y of the hypertensive blood v e s s e l (Bandick and Sparks, 1970; Bohr and S i t r i n , 1970; B e i l i n and Ziakus, 1971). In studying the e f f e c t of Na on vascular r e a c t i v i t y of smooth muscles, one has to e x p l a i n the r e s u l t s w i t h i n the context that Ca i s e s s e n t i a l f o r the a c t i v a t i o n of the c o n t r a c t i l e machinery and that a l t e r a t i o n i n the vas-c u l a r r e a c t i v i t y of the smooth muscle i s to be u l t i m a t e l y c o r r e l a t e d with an a l t e r a t i o n i n the amount of i o n i z a b l e Ca a v a i l a b l e to the c o n t r a c t i l e pro-t e i n ( F i l o et a l . , 1965; Murphy 1969; Sparrow et a l . , 1970). I t i s proper at t h i s p o i n t , therefore, to s t a r t a b r i e f review on the importance of Ca f o r smooth muscle cont r a c t i o n before discussing the present knowledge of the e f f e c t of Na on vascular smooth muscle r e a c t i v i t y . .../5 5 ++ Ca and Smooth Muscle Contraction Contraction of smooth muscles i s the result of excitation of the I | membrane leading to the supply of the stimulus Ca to the contractile protein; while under certain conditions contraction can be init i a t e d i n certain types of smooth muscles seemingly by the so-called pharmaco-mechanical coupling (Somlyo and Somlyo, 1968). For convenience of discussion, the matter w i l l be described under the following items: A. Contractile protein B. Membrane phenomena C. Excitation contraction coupling A. Contractile protein. Szent-Gydrgi (1951) found that treatment of fresh bundles of skeletal muscles with aqueous glycerol destroys the c e l l membrane and extracts c e l l components of metabolic importance, leaving the actin and myosin filaments intact. These glycerinated muscle fibers are relatively inextensible when placed i n buffered KC1 solution, but the exten-s i b i l i t y recurs by the addition of ATP. The phenomenon i s referred to as the "plasticizing action" of ATP and requires magnesium ions. Initiation of contraction can then be induced by minute amounts of calcium i n the presence of an active ATPase i n the contractile protein (Portzel, 1962). Contraction i s accompanied by ATP hydrolysis u n t i l a l l the l a t t e r has been hydrolyzed when the muscle becomes again inextensible (Szent-GyBrgi, 1951, F i l o et a l . , 1965). It seemed, therefore, that ATP had a dual effect on the myofilaments and that contraction i s the result of some form of interaction between action and myosin ++ i n the presence of Ca resulting i n activation of the ATPase. Purification of the contractile protein i n smooth muscles gave further information about the interaction between actin and myosin filaments. .../6 6 The p r o t e i n extracted from smooth muscles contain an actomyosin I | resembling that occurring i n s k e l e t a l muscles. I t shows a Ca and I | Mg a c t i v a t e d ATPase a c t i v i t y , s i m i l a r v i s c o s i t y p r o p e r t i e s , and s u p e r p r e c i p i t a t i o n reactions a f t e r the a d d i t i o n of 0.5 mM ATP ( F i l o et a l . . 1963). However, actomyosin of smooth muscles could be extracted i n s o l u -tions of low i o n i c strength (0.05 KC1), while that of s k e l e t a l muscle required a higher i o n i c strength (0.6 M KC1) and i s more e a s i l y e x t r a c t -able at pH 7 that at pH 6 (Shirmer, 1965). The a r t e r i a l c o n t r a c t i l e p r o t e i n ATPase has a b i p h a s i c pH dependence with an optimum at pH 5.2 and a small peak at pH 7 i n contrast to a s i n g l e optimum at pH 6.5 f o r s k e l e t a l muscle actomyosin (Murphy, 1969). A r t e r i a l actomyosin has a l s o a low ATPase a c t i v i t y (Sparrow et a l . , 1970),but generates more tension per u n i t amount of actomyosin when compared to actomyosin of s k e l e t a l muscles. Myosin was demonstrated i n smooth muscles by biochemical methods (Needham, 1962). However, there has been much controversy whether t h i c k filaments corresponding to myosin do e x i s t under p h y s i o l o g i c a l conditions i n smooth muscles. While s e v e r a l authors have v i s u a l i z e d t h i c k filaments i n recent years (Yamauchi and Burnstock, 1969; Cooke et a l . , 1970; Rice et a l . r 1970; Devine and Somlyo, 1971), i t was argued that only t h i n filaments corresponding to a c t i n could be i d e n t i f i e d under p h y s i o l o g i c a l conditions, and that the demonstration of t h i c k filaments i n some studies might have been due to an experimental a r t i f a c t r e s u l t i n g from the c o a l e s -cence of t h i n filaments during the preparation of the s e c t i o n (Panner and Honig, 1970). Several explanations were attempted to e x p l a i n the d i s c r e p -ancy of r e s u l t s presented by various authors. Pease (1968) could demon-.../7 7 s t r a t e t h i c k filaments when smooth muscles are prepared by i n e r t dehydration instead of chemical f i x a t i o n . In view of the marked s o l u b i l i t y of myosin, even i n media of low i o n i c strength, the lack of demonstration of t h i c k filaments i n some studies might have been due to de s t r u c t i o n of the l a t t e r during the method of preparation of the s e c t i o n . The explanation that myosin organizes i n t o t h i c k f i l a -ments only at the moment of e x c i t a t i o n c o n t r a c t i o n coupling does not seem p l a u s i b l e since t h i c k filaments have been shown i n approximately the same number i n vascular smooth muscles relaxed by theophylline, i n Ca - f r e e s o l u t i o n , or contracted by norepinephrine (Devine and Somlyo, 1971), I t i s p o s s i b l e , however, that s t r e t c h i n g and pre i n c u -bation of the smooth muscle i n p h y s i o l o g i c a l s a l t s o l u t i o n p r i o r to f i x a t i o n f a c i l i t a t e s the demonstration of t h i c k filaments (Devine and Somlyo, 1971), The existence of t h i c k filaments i n smooth muscles f i n d s a l s o some support from X-ray d i f f r a c t i o n studies (Lowy et a l , , 1970). Actomyosin extracted from muscle i s d i s s o c i a t e d to i t s i n d i v i d u a l components when Mg and ATP are added i n the absence of Ca (Weber, 1956), The r e a c t i o n i s associated with reduction i n v i s c o s i t y and double r e -f r a c t i o n of flow and i s analogous to the " p l a s t i c i z i n g a c t i o n " of ATP i n ++ g l y c e r i n a t e d muscle. The a d d i t i o n of Ca to the ATP actomyosin system r e s u l t s i n shrinkage with s u p e r p r e c i p i t a t i o n i f actomyosin was i n a s o l , and syneresis i f i t was a g e l , together with vigorous s p l i t t i n g of ATP (Szent-GyBrgi, 1947). 11 R e l a t i v e l y crude preparations of actomyosin required both Ca and I | ATP to produce s u p e r p r e c i p i t a t i o n , but the requirement f o r Ca was l o s t i n actomysosin obtained from p u r i f i e d a c t i n and myosin (Ebashi, 1963), . . . / 8 8 The Ca s e n s i t i v i t y was regained when another p r o t e i n , tropomyosin, obtained from the muscle was added to the experimental system. I t was, therefore, reasonable to conclude that tropomyosin exerted an i n h i b i t o r y a c t i o n on a c t i n and myosin and that the a d d i t i o n of Ca removed t h i s i n h i b i t i o n . Further p u r i f i c a t i o n of tropomyosin showed that i t i s a c t u a l l y composed of two prote i n s ; troponin, a globular p r o t e i n which binds I | four Ca ions/molecule, and tropomyosin B, a more filamentous p r o t e i n , I | with no a f f i n i t y f o r Ca , but which binds strongly to a c t i n (Ebashi et a l . , 1967). I t i s presently believed that tropomyosin i s present wi t h i n the -2 -4 h e l i c e s of n a t u r a l a c t i n r e p e l l i n g the Mg ATP and ATP attached to the myosin partner. By t h i s i n t e r a c t i o n tropomyosin can prevent the events leading to s p l i t t i n g of ATP and onset of co n t r a c t i o n . Ca binds to negatively charged groups of troponin, n e u t r a l i z i n g i t s charge and allowing the i n t e r a c t i o n between a c t i n and myosin to occur which would I | lead to c o n t r a c t i o n . The r o l e of Ca , therefore, could be described as derepressor of the i n t e r a c t i o n between a c t i n and myosin. While e a r l y biochemical evidence has been mostly derived from s k e l e -t a l muscles, smooth muscles seem to share t h i s property since tropomyosin was extracted from the smooth muscle of chicken g i z z a r d (Ebashi, 1966). However, i t appears that tropomyosin i s i n a c t i v a t e d with time and that t h i s i n a c t i v a t i o n could be prevented by keeping i t s s u l f h y d r y l (SH) groups i n the reduced form (Sparrow et a l . , 1970), B. Membrane phenomena. While the membrane of smooth muscles share many properties to those of other e x c i t a b l e t i s s u e s , there are some p e c u l i a r i t i e s to t h i s type of muscle. The permeability of the smooth 9 muscle membrane i s higher to Na ions (Kuriyama, 1963), v/hile that to K i s lower (Goodford and Hermansen, 1961) compared to s k e l e t a l muscles. This may contribute to the lower membrane p o t e n t i a l of smooth muscles compared to that of s k e l e t a l muscles. Chloride does not seem to be d i s -t r i b u t e d p a s s i v e l y since i n t r a c e l l u l a r c h l o r i d e concentration i s too high to f i t a passive d i f f u s i o n process (Casteels, 1970), and the K concentra-t i o n gradient across the membrane seems to be the main determinant of the membrane p o t e n t i a l . (1) Electrogenic sodium pump i n smooth muscles There i s ample evidence from recent studies that an e l e c t r o -genic sodium pump contributes to the r e s t i n g membrane p o t e n t i a l and could be stimulated under some conditions by the r i s e of i n t r a c e l l u -l a r sodium (Thomas, 1972). Casteels (1969) measured the Ionic fluxes of Na, K, and C l and estimated the i n t r a c e l l u l a r concentration of these ions. By using the constant f i e l d equation, he a r r i v e d at a value of membrane poten-t i a l of -37 mV, which was mainly due to a high PNa:PK of 0.16. However, the measured membrane p o t e n t i a l was 20 mV higher. The r e -s u l t was in t e r p r e t e d as a c o n t r i b u t i o n of an elec t r o g e n i c Na pump. The c o n t r i b u t i o n of the el e c t r o g e n i c Na pump i n t h i s study might have been exaggerated since Casteels used two exponential functions to analyze the Na e f f l u x data assuming that the slow component was from an i n t r a c e l l u l a r compartment. Brading (1971) found that the Na e f f l u x data could be better described as a three exponential, f u n c t i o n . Using a model i n which only the slowest exponential function was assumed to be representing transmembrane f l u x e s , she a r r i v e d at a c a l -culated membrane p o t e n t i a l of -57 mV with a PNarPK of 0.0098. .../10 10 However, s e v e r a l types of evidence are accumulating pointing to the importance of the Na pump i n the maintenance of membrane p o t e n t i a l . Tomita and Yamamoto (1971) found that removal of K depolarized the mem-brane of the guinea p i g taenia c o l i by 5 mV, while ouabain had a s i m i -l a r , but slower, e f f e c t . The d e p o l a r i z a t i o n occurring by removing was not a f f e c t e d by the presence of tetrodotoxin, atropine or i n low C l s o l u t i o n s . However, p r i o r cooling of the t i s s u e below 23° C. or t r e a t -ment with ouabain frequently r e s u l t e d i n hyp e r p o l a r i z a t i o n a f t e r K removal. Casteels et al.(1971) measured the i o n content, membrane p o t e n t i a l , and permeability of guinea p i g taenia c o l i on exposure to K-free s o l u t i o n s . They found that removal of K caused an i n i t i a l d e p o l a r i z a t i o n of about 10 mV followed by hyp e r p o l a r i z a t i o n r e s u l t i n g from an increase i n K permea-b i l i t y . They concluded that the e a r l y d e p o l a r i z a t i o n was due to an I n h i b i -t i o n of an ele c t r o g e n i c Na pump that was cont r i b u t i n g to the r e s t i n g mem-brane p o t e n t i a l . Matthews and Sutter (1967) found that ouabain depolarized the r a b b i t A.M.V. and guinea p i g taenia c o l i . Kuriyama at al.(1971) found that ouabain or Na-free s o l u t i o n s reduced the membrane p o t e n t i a l of guinea pi g p o r t a l veins from about -50 mV to -24 mV, and that cooling lowered i t to about -31 mV. The membrane p o t e n t i a l was analyzed i n i s o t o n i c and hypertonic s o l u t i o n s i n t o two components: metabolic (dependent on an ele c t r o g e n i c Na pump), and i o n i c (dependent on an e l e c t r i c a l d i f f u s i o n p o t e n t i a l ) . The con-t r i b u t i o n of the el e c t r o g e n i c sodium pump to the membrane p o t e n t i a l was estimated i n the range of -10 to -20 mV. Evidence i s accumulating that the Na pump may also become elec t r o g e n i c under c e r t a i n conditions favouring high I n t r a c e l l u l a r sodium. 11 Taylor et a l . (1970) loaded myometrial tissue with Na by cooling for 18 hours in K-free solutions. On warming the tissue the recorded membrane potential was -15 mV. The addition of K hyper-polarized the tissue to -70 mV. This hyperpolarization exceeded the EK potential, was more prominent in high external K solutions and was blocked by ouabain. Tomita and Yamamoto (1971) reported that in guinea pig taenia coli restoration of K after about 1 hour exposure to K-free solution at 37° C. caused a hyperpolarization of -10 to -15 mV which was abolished in the presence of ouabain or cooling to 23° C. Comparable results have been found by Casteels et a l . (1971b) in the same preparation and by Bolton (1971) in guinea pig ileum. It is also interesting to note that following acetylcholine application the smooth muscle of guinea pig ileum shows a response similar to post tetanic potentiation accompanied by hyperpolarization. This was attributed to activation of an electrogenic sodium pump (Bolton, 1971). The slow electrical waves seen in intestinal smooth muscles may also be linked to an oscilla-tory activity of an electrogenic sodium pump (Daniel, 1965), since there is a phasic correlation of Na efflux with their occurrence (Job, 1969) and they are ab olished in Na-free Ringer solution or in the presence of ouabain (Liu et al.,1969). The technical difficulties arising from getting valuable infor-mation from intracellular recording techniques and the complexity of the Na transport in smooth muscles, have made the quantitation of the contribution of an electrogenic sodium pump in the membrane potential of smooth muscles rather d i f f i c u l t . .../12 12 (2) Action p o t e n t i a l and graded d e p o l a r i z a t i o n of smooth muscles Smooth muscles behave d i f f e r e n t l y i n response to e x c i t a n t agents. They also may or may not have ac t i o n p o t e n t i a l s . Spon-taneously a c t i v e preparations l i k e guinea pig taenia c o l i , anter-i o r mesenteric v e i n and p o r t a l v e i n show a c t i o n p o t e n t i a l s that are myogenic i n nature since a concentration of l o c a l anaesthetic that blocks conduction, hexamethionium, atropine, or tetrodotoxin do not a b o l i s h spontaneous a c t i v i t y (Kuriyama et a l . , 1967; Johansson and Ljung, 1967; Tomita, 1970) and n e i t h e r phenoxy-benzamine nor propranolol a l t e r e d the e l e c t r i c a l and mechanical a c t i v i t y of r a t p o r t a l veins (Johansson et a l . , 1967). However, a strong neurogenic component has been described l n i n t e s t i n a l smooth muscles since atropine and tetrodotoxin modify t h e i r spontaneous a c t i v i t y by reducing the slow changes i n the membrane p o t e n t i a l (Kuriyama et a l . , 1967), Hukura and Fukuda, 1968). The i o n i c basis of the a c t i o n p o t e n t i a l seems to favour that I | they are mainly Ca spikes while the Na c o n t r i b u t i o n i s small (Brad-ing et a l . , 1969; BUlbring and Tomita, 1970), but q u a n t i t a t i v e v a r i a t i o n s about the importance of the Na current seems to e x i s t i n various types of smooth muscles and g e n e r a l i z a t i o n from studying a given type of smooth muscle Is not f e a s i b l e . Voltage clamp data (Anderson et a l . , 1971) seems to i n d i c a t e that neither Na nor Ca alone i s capable of supporting e x c i t a t i o n i n oestrogen dominated uter i n e smooth muscle. The vascular smooth muscle c e l l s of the common c a r o t i d a r t e r i e s seem to have predominantly Na-based a c t i o n p o t e n t i a l s at l e a s t under c e r t a i n experimental conditions since i t s c e l l s become e l e c t r i c a l l y a c t i v e i n Ca-free s a l i n e s o l u t i o n s and i n e x c i t a b l e ./13 13 when Na i n such s o l u t i o n s i s replaced by T r i s or choline and t h e i r e l e c t r i c a l a c t i v i t y i s associated with an increase i n Na i n f l u x (Keatingue, 1968a,b). Moreover, spike a c t i v i t y could be recorded i n t h i s t i s s u e i n the presence of 12.5 mM. EDTA at 0-10° C. f o r at l e a s t 2 hours (Graham and Keatings, 1970). In guinea p i g taenia c o l i , a preparation which has been exten-s i v e l y used f o r e l e c t r o p h y s i o l o g i c a l s t u d i e s , there i s strong e v i d -I | ence that Ca plays the predominant r o l e i n the genesis of the spike 11 p o t e n t i a l . With 2-5 mM Ca i n the incubating media, and a c e l l u l a r concentration of Ca l e s s than 10~ -10~ M (Caldwell, 1968), one would expect an equ i l i b r i u m p o t e n t i a l of more than +100 mV, Reduction of the external Na to 10 mM decreases the i n t e r n a l Na concentration from 35 mM to 24 mM and the sodium eq u i l i b r i u m p o t e n t i a l from 34 mV to -22 mV. However, the overshoot of the spike i s increased from +10 to +20 mV. (Brading and Tomita, 1968; Brading et a l . , 1969) This i s d i f f i c u l t to explain unless one assumes that another c a t i o n contributes s i g n i f i c a n t l y to the spike mechanism. Moreover, i n low Na s o l u t i o n s , Ca d e f i c i e n c y causes a s l i g h t d e p o l a r i z a t i o n , but decreases the amplitude and r a t e of r i s e of the spike p o t e n t i a l f a r i n excess of what can be expected from the s h i f t of the membrane p o t e n t i a l . This f i n d i n g suggests an increase I | i n membrane permeability to Ca during the genesis of the spike p o t e n t i a l (Brading et a l . , 1969). I t seems l i k e l y that the bound membrane Ca may contribute to the supply of Ca current f o r the a c t i o n p o t e n t i a l (Bttlbring and Tomita, 1970; Lammel and Golenhofen, 1971) and controls the Na permeability of the membrane (Bttlbring and Tomita, 1970). The p o s s i b i l i t y that smooth muscles may have a v e r s a t i l e means f o r u t i l i z i n g d i f f e r e n t ions as charge c a r r i e r s under d i f f e r e n t experimental conditions has not been excluded. Golenhofen and Petranyi (1969) found .../14 14 that the spikes of guinea pig taenia c o l i were r e s i s t a n t to t e t r o -dotoxin i n media containing normal Ca and Na. However, i n Ca - f r e e media and preferably with no Mg and sodium f l u o r i d e added, the spike i n i t i a l l y disappeared or diminished i n amplitude, but l a t e r returned to t h e i r i n i t i a l l e v e l , the new spikes were then s e n s i t i v e to t e t r o -dotoxin. Caution i s , however, to be taken i n making conclusions from such experiments i n view of the questioned s p e c i f i c i t y of tetrodotoxin i n blocking the Na channel (Kao, 1966; Keatings, 1968a). Large conduit vessels l i k e the aorta and pulmonary a r t e r i e s do not show a c t i o n p o t e n t i a l s , but rather respond to e x c i t a n t agents by graded d e p o l a r i z a t i o n (Somlyo and Somlyo, 1968). C. E x c i t a t i o n c o n t r a c t i o n coupling. Some aspects dealing with ex-c i t a t i o n of the membrane of smooth muscles and contraction of the contrac-t i l e p r o t e i n have been already discussed. The question now a r i s e s as to how these processes are l i n k e d together. In s k e l e t a l muscles e x c i t a t i o n -c o n t r a c t i o n coupling i s c a r r i e d by d e p o l a r i z a t i o n of the periphery of the muscle f i b e r , leading to propagation of an impulse through the transverse tubular membrane which i n turn releases sequestered Ca from the termin-a l c i s t e r n a e . Relaxation probably occurs by a c t i v e l y pumping back the Ca to the l o n g i t u d i n a l part of the sarcoplasmic reticulum and then r e d i s -t r i b u t i o n to the l a t e r a l c i s t e r n a e (Bianchi, 1968). In smooth muscles there appear to be q u a n t i t a t i v e d i f f e r e n c e s between d i f f e r e n t types of smooth muscles i n the manner these processes are l i n k e d I | together since the source of Ca f o r c o n t r a c t i o n and the degree of organiz-a t i o n of the sarcoplasmic reticulum shows q u a n t i t a t i v e d i f f e r e n c e s among d i f f e r e n t smooth muscles. I | Evidence i s accumulating that Ca i s a v a i l a b l e from more than one f u n c t i o n a l pool at l e a s t i n some types of smooth muscles. Hinke (1965) .../15 15 observed that the c o n t r a c t i l e responses of r a t t a i l a r t e r i e s to K de-c l i n e d more r a p i d l y than that to NA i n Ca-free media. This d i f f e r e n c e was s t i l l p e r s i s t e n t when EDTA was added to the perfusion f l u i d . Com-parable r e s u l t s have been subsequently shown i n a v a r i e t y of smooth muscle preparations (Sparrow and Simmons, 1965; Hiraoka et a l . , 1968; Hodgins and Weiss, 1968), The r e s u l t s suggest that drugs l i k e NA and ac e t y l c h o l i n e might be able to mobilize a more t i g h t l y bound store of ++ Ca , More d i r e c t evidence to these assumptions was provided from studies I | using lanthanum to eliminate e x t r a c e l l u l a r bound Ca from Ca i n f l u x measurements (Van Breeman, 1970; Goodman and Weiss, 1971), Van Breeman et a l . (1972) showed that d e p o l a r i z a t i o n by high K and Na replacement i n -11 11 creased Ca i n f l u x . However, norepinephrine f a i l e d to increase Ca i n f l u x , although i t was s t i l l able to induce con t r a c t i o n under the same experimental condi t i o n s . While d e p o l a r i z a t i o n of the membrane and release of Ca are involved i n the induction of contr a c t i o n , there are a number of observations which suggest that under c e r t a i n conditions c o n t r a c t i o n of the vascu l a r smooth muscle can occur independent of e l e c t r i c a l phenomena, Cuthbert and Sutter (1965) observed that c o n t r a c t i l e responses to NA i n A.M.V. induced an i n i t i a l increase i n spike a c t i v i t y which c o r r e l a t e d with an increase i n tension. However, a f t e r a few minutes the e l e c t r i c a l a c t i v i t y returned to normal, although the tension remained elevated. More s t r i k i n g i s the fi n d i n g that the smooth muscle can s t i l l contract when depolarized by K-r i c h s o l u t i o n s (Edman and S c h i l d , 1962) or ouabain (Matthews and Sutter, 1967). Somlyo and Somlyo (1968) used the term pharmacomechanical coupl-ing f o r drugs inducing c o n t r a c t i o n by passing the step of membrane depolar-i z a t i o n and the term electromechanical f o r those r e q u i r i n g i t . .../16 16 I t was generally assumed that the sarcoplasmic reticulum was poorly developed i n smooth muscles (Bumstock, 1970). The problem then remained to explain how the function of t h i s system could be accomplished i n smooth muscles. In recent years a well-developed sarcoplasmic reticulum has been described i n guinea p i g ileum (Gabella, 1971). Further semi-quantitative studies have revealed the presence of a sarcoplasmic reticulum i n a v a r i e t y of smooth muscles and that d i f f e r e n c e s i n the a b i l i t y of various smooth muscle preparations to maintain c o n t r a c t i l e responses to drugs i n Ca - f r e e media might be r e l a t e d i n part to q u a n t i t a t i v e d i f f e r e n c e s i n the volume of the sarcoplasmic reticulum (Somlyo et a l . , 1971; Devine et a l . , 1972). I t seems, therefore, that the sarcoplasmic reticulum of smooth muscles can act as a Ca store that would supply a f u n c t i o n -I | a l pool of Ca to the c o n t r a c t i l e p r o t e i n . The importance of t h i s I [ system i n taking up Ca a f t e r c o n t r a c t i o n f i n d s i n d i r e c t evidence by the demonstration of an accumulation of strontium i n the sarcoplasmic reticulum of spontaneously a c t i v e r a b b i t and guinea p i g a n t e r i o r mesen-t e r i c veins and the quiescent main pulmonary a r t e r y only a f t e r d e p o l a r i z -a t i o n or g l y c e r i n a t i o n (Somlyo, 1971,b). The surface area of the smooth muscle membrane i s g r e a t l y increased by surface invaginations v a r i a b l y known as micropiriocytotic v e s i c l e s , plasmalemmal v e s i c l e s or caveolae i n t r a c e l l u l a r e s . These structures com-municate with the e x t r a c e l l u l a r space as shown by t h e i r a b i l i t y to i n c o r -porate lanthanum (Devine and Sonlyo, 1970) and f e r r i t i n (Devine et a l . , 1972). In view of the close r e l a t i o n s h i p between the plasmalemmal v e s i c l e s and sarcoplasmic reticulum (Gabella, 1971; Devine et a l . , 1972) and the demon-s t r a t i o n of e l e c t r o n opaque connections i n between them (Devine et al.,1972), .../17 17 i t was suggested that these areas might be the probable s i t e s of electromechanical coupling i n smooth muscles (Devine et a l . , 1972). I t i s of some i n t e r e s t to note that the microsomal f r a c t i o n separated from the 100,000 g p e l l e t of r a b b i t sortas i s v e s i c u l a r i n nature and derived from micropinocytotic v e s i c l e s , c e l l membranes and sarcoplasmic reticulum ( V e r i t y and Bevan, 1969). Studies on t h i s f r a c t i o n showed a d e f i n i t e Ca binding a b i l i t y that was enhanced by ATP and d i b u t y r y l -c y c l i c AMP and i n h i b i t e d by angiotensin (D'auriaC et a l . , 1972). In view of the large surface area to volume r a t i o r e s u l t i n g from the pre-sence of micropinocytotic v e s i c l e s i n smooth muscles, i t remains to be studied i n more d e t a i l the p o s s i b l e c o n t r i b u t i o n of these s t r u c t u r e s i n -H-providing and uptaking Ca during the e x c i t a t i o n c o n t r a c t i o n c y c l e and I | i t s p o s s i b l e r e l a t i o n s h i p with the Ca s e n s i t i v e ATPase described i n membrane functions (Schatzman and V i n c e n z i , 1969). I t i s p e r t i n e n t at t h i s point to note that the calcium uptake system of a o r t i c microsomes separated from the i n t i m a l medial l a y e r i s enhanced i n the presence of Mg-ATP and that Mg-ADP cannot s u b s t i t u t e f o r the l a t t e r i n t h i s f u n c t i o n ( F i t z p a t r i c k et a l . , 1973). These properties are comparable to the ATPase a c t i v i t y dependent on Mg and Ca separated from s k e l e t a l muscle microsomes and which can p o t e n t i a l l y contribute s i g n i f i c a n t l y to the calcium sequestration system (Martosi and Feretos, 1964). The Na-Ca I n t e r a c t i o n i n Smooth Muscles Several studies were conducted to e l u c i d a t e the nature of the Na-Ca i n t e r a c t i o n since the demonstration by Ltlttgau and Nidergerke(1958) that tension development i n heart muscles was r e l a t e d to the r a t i o of 18 I | _j_ 2 [Ca ]:[Na ] i n the bathing f l u i d . The r e s u l t s were f i r s t i n t e r p r e t e d I | _|  as a simple competition between Ca and Na ions f o r an anionic group R at the c e l l surface according to the r e a c t i o n : 2Na + Ca R ^ Na~ R + Ca 2 where compound Ca R can induce a c o n t r a c t i l e response and Na2 R i s i n a c t -iv e i n t h i s respect. S i m i l a r work on vascular smooth muscles (Nash et a l . , 1966) revealed the Na-Ca antagonism, but f a i l e d to a t t r i b u t e tension develop-ment to a simple [Ca ]:[Na ] b a s i s . The antagonism of Na and Ca i n smooth muscles seemed, therefore, more complicated than a simple Na-Ca competition. However, an i n t e r a c t i o n of cations i n smooth muscles has been repeatedly demonstrated and t h i s can f i t the assumption that the c e l l membrane has f i x e d anionic groups on which d i f f e r e n t cations can compete with each other (Goodford, 1966; Sparrow, 1969), Further e l e c t r o n microscopic s t u d i e s , using uranyl c a t i o n s t a i n i n g i n smooth muscles,localized these s i t e s on both the outer and inner membrane surfaces of smooth muscles (Wolowyk, 1971; Goodford and Wolowyk, 1971), Evidence of i n t e r a c t i o n between d i f f e r e n t cations at these s i t e s was a l s o provided by the demonstration that the s t a i n i n g i n t e n s i t y of these s i t e s was dependent on the concentration of cations i n the r i n s i n g f l u i d (Goodford and Wolowyk, 1971; Goodford and Wolowyk, 1972), Baker et a l . (1969) found that i n squid axons Ca i n f l u x could be increased by i n c r e a s i n g the i n t r a c e l l u l a r sodium or diminishing the e x t r a -c e l l u l a r sodium concentration. The diminution of external sodium was accompanied by an increased Na e f f l u x which was p a r t l y ouabain s e n s i t i v e and p a r t l y ouabain i n s e n s i t i v e , but Ca-dependent. These experiments pro-vided, therefore, strong evidence f o r the existence of a Na-Ca exchange i n ,../19 19 squid axons i n which Na e f f l u x i s coupled with Ca i n f l u x . I t i s also suggested that the i n f l u x system carrying Na can be occupied by Ca (Baker et a l . , 1969; Baker, 1970). A s i m i l a r system was l a t e r described i n guinea pig a u r i c l e s ( G l i t s c h et a l . , 1970) and evidence was provided that the increased Ca i n f l u x r e s u l t i n g from r a i s i n g the i n t r a c e l l u l a r sodium concentration can make more Ca a v a i l a b l e to the c o n t r a c t i l e p r o t e i n ( G l i t s c h et a l . , 1970). I t was then tempting to speculate that a s i m i l a r system e x i s t s i n smooth muscles and may c o n t r o l smooth muscle r e a c t i v i t y . Bohr et a l . (1969) found that c o n t r a c t i l e responses to NA were potentiated i n Na-free media and that these media i n i t i a t e a c o n t r a c t i o n i n the mesenteric a r t e r y . E a r l i e r studies showing that diminution of external Na increased the Ca i n f l u x In Na-free media can be taken i n favour of the existence of t h i s system i n smooth muscle. S i m i l a r l y , the p o t e n t i a t i o n of NA responses i n the presence of ouabain as w e l l as the c o n t r a c t i l e responses of ouabain has been explained by a c t i v a t i o n of a Na-Ca exchange system r e s u l t i n g from the increased i n t r a c e l l u l a r sodium (Bohr et a l . , 1969). There i s a marked i n d i v i d u a l i t y i n between smooth muscles as f a r as the Na-Ca i n t e r a c t i o n i s concerned and the evidence f o r the existence of a Na-Ca exchange i s before a l l very I n d i r e c t and Inconclusive. The f i n d -ings at present a v a i l a b l e denote that there are two sources f o r a c t i v a t o r I [ Ca , which may be d i f f e r e n t l y a f f e c t e d by changes i n the Na transport. I | The r e l a t i v e importance of the two Ca pools i n d i f f e r e n t smooth muscles may a f f e c t the r e s u l t s obtained by manipulating the external sodium and calcium ( S i t r i n and Bohr, 1971). There i s evidence that c o n t r a c t i l e 20 responses i n guinea p i g taenia c o l i p a r a l l e l changes i n i n t r a c e l l u l a r Na and K by a mechanism independent of metabolic energy supply, d i r e c t e f f e c t on c o n t r a c t i l e components, or passive mechanical p r o p e r t i e s (Axelsson and Holmberg, 1971), They suggested that reduced i n t r a -c e l l u l a r K or Na excess i n t h i s t i s s u e may i n t e r f e r e with the normal functions of Ca i n the e x c i t a t i o n - c o n t r a c t i o n coupling, Burks et a l , (1971) showed, however, that there are c r i t i c a l l e v e l s of c e l l u l a r Na and K to obtain an optimum c o n t r a c t i l e response i n dog mesenteric arter-r i e s . I f the t i s s u e l e v e l s of e i t h e r ion f a l l s below these l e v e l s , the a r t e r y i s hyporesponsive. I f the a r t e r y contains minimal K and excess of Na, i t may be hyperresponsive (Burks et a l , , 1971). A coupling of Na i n f l u x and Ca e f f l u x has been described i n squid axons (Blaustein and Hodgin, 1969) and cardiac muscles (Reuter and S e i t z , 1968) and i t s existence i n smooth muscles has been suggested by Bohr et a l , (1969), There i s some evidence that Na may be important f o r Ca removal (Biamino and Johansson, 1970; Katase and Tomita, 1971), but the assumption of the presence of a coupling between Na i n f l u x and Ca e f f l u x i n smooth muscles l i e s mostly on i n d i r e c t evidence. More d i s c u s s i o n of t h i s matter w i l l be presented i n a l a t e r s e c t i o n a f t e r the presentation of our r e s u l t s . Sodium and the Hypertensive Blood Vessel Several studies were conducted since Tobian and Binion (1952) found an increased Na content of the hypertensive blood v e s s e l . A c o r r e l a t i o n between the changes i n e l e c t r o l y t e and vascular r e a c t i v i t y was hampered, however, by the questions as to the l o c a l i z a t i o n of t h i s increase In Na content ( S c h o f f e n i e l s , 1969) and the c o n f l i c t i n g r e s u l t s obtained by various authors studying the vascular r e a c t i v i t y of the hypertensive v e s s e l s . .•./21 21 There i s evidence that at l e a s t part of the increased sodium i n hyper-tensive vessels i s e x t r a c e l l u l a r and associated with the p r o t e i n muco-polysaccharides of the a r t e r i a l w a l l (Palaty et a l . , 1969a,b). H a r r i s (1970) suggested that t h i s f r a c t i o n may, i n part, be responsible f o r a r t e r i a l h y p e r r e a c t i v i t y . However, while some reports observed an increased responsiveness of hypertensive vessels to a v a r i e t y of agents (Bandick and Sparks, 1970; Bohr and S i t r i n , 1970; B e i l i n and Ziakus, 1971; Bohr and S i t r i n , 1971), others were not able to observe such changes ( F l e i s c h et a l . , 1969; Hallbach et a l . , 1971). Di f f e r e n c e i n preparations and experimental techniques might have been i n part r e l a t e d to the discrepancy of the r e s u l t s a v a i l a b l e . I t i s pertinent to note at t h i s point that the r e a c t i v i t y of the blood v e s s e l to various agonists might be a f f e c t e d by the age of the hypertensive s t a t e (Bohr, 1961; Bandick and Sparks, 1970). A r t e r i e s from r a t s hypertensive f o r more than 15 days develop l e s s c o n t r a c t i l e f o r c e than do normal blood vessels (Bandick and Sparks, 1970). .../22 22 STATEMENT OF THE PROBLEM From the introductory note, i t seemed c l e a r that there Is an i n t e r a c t i o n between Na and Ca and that t h i s might a f f e c t contract-i l e responses of smooth muscles. The nature of t h i s i n t e r a c t i o n i s , however, to a great extent unclear. We s t a r t e d a s e r i e s of e x p e r i -ments i n which a l t e r a t i o n s of Na transport were induced e i t h e r by drugs or changes i n the Na content of the incubating media. The idea was to t e s t the e f f e c t of such changes on vascular r e a c t i v i t y and the a v a i l a -11 b i l i t y of Ca to the c o n t r a c t i l e p r o t e i n . The r a b b i t a n t e r i o r mesenteric v e i n was chosen as a preparation which i s most s u i t a b l e f o r c o n t r a c t i l e studies since i t s p r o p e r t i e s resemble those of r e s i s t a n c e v e s s e l s and an ample amount of work has been already done to e l u c i d a t e i t s properties (Cuthbert and Sutter, 1965; Matthews and Sutter, 1967; C o l l i n s et a l . , 1972a,b). In s p e c i a l experiments i t was of some s i g n i f i c a n c e to d i f f e r e n t i a t e the e f f e c t of c e r t a i n drugs on the two phases of NA c o n t r a c t i o n s . For t h i s type of experiments, the r a b b i t a o r t i c s t r i p was used as a prepara-t i o n that has a t o n i c component dependent on e x t r a c e l l u l a r calcium and a phasic component presumably due to l i b e r a t i o n of a bound Ca pool (Bohr, 1963; Godfraind, 1969). In view of the heterogenity of the r a b b i t a n t e r i o r mesenteric v e i n , experiments designed to analyze changes i n e l e c t r o l y t e s of smooth muscles vascular w a l l were c a r r i e d on r a t t a i l a r t e r i e s , a preparation that has been extensively used f o r such a purpose. There i s an ample amount of evidence that diphenylphydantoin sodium (DPH) a l t e r s sodium transport (Woodbury and Kemp, 1971), but there are .../23 23 discrepancies i n r e s u l t s on the way t h i s a l t e r a t i o n of sodium trans-port i s produced. Woodbury (1969) and Woodbury and Kemp (1971) sug-gested that i t was due to s t i m u l a t i o n of the Na pump. However, i n v i t r o biochemical studies showed that DPH i s rather i n h i b i t o r y to t h i s enzyme (Pincus and Giarman, 1967; Rawson and Pincus, 1968; Formby, 1969). Later reports showed the drug to be i n h i b i t o r y when the Na-K r a t i o i s low and stimulatory when the Na-K r a t i o i s high (Festoff and Appel, 1968; Lewin and Bleck, 1971; Spain and Chidsey, 1971; S i e g e l and Goodwin, 1972), I t was suggested by Pincus that DPH i n h i b i t s the Na i n f l u x rather than stimu-l a t e s the sodium pump, since the e f f e c t was prominent on the Na ions and i n s i g n i f i c a n t on the K ions (Pincus, 1970). Later Pincus (1972), using r a d i o a c t i v e isotopes, showed that DPH i n h i b i t s the Na i n f l u x i n l o b s t e r nerves while having no e f f e c t on Na e f f l u x . While the discrepancy i n r e -s u l t s may be due i n part to d i f f e r e n c e s i n the t i s s u e examined (Williams et a l . , 1971) and v a r i a b l e approaches used by d i f f e r e n t authors, i t i s pertinent at t h i s point to note that the e f f e c t of the drug i s dependent on the dose used. B i h l e r and Sawh (1971) found that concentrations higher than 0.5 mM were i n h i b i t o r y to the sodium pump, although smaller concentra-tions seemed to produce a stimulatory e f f e c t under the same experimental co n d i t i o n s . In preliminary experiments we found that DPH i n h i b i t s c o n t r a c t i l e responses to NA, procaine and ouabin i n the r a b b i t a n t e r i o r mesenteric v e i n . We proceeded to analyze t h i s e f f e c t . (1) I f the a c t i o n of the drug i s rather s p e c i f i c to t h i s molecular s t r u c t u r e , then i t s P-hydroxy d e r i v a t i v e (5-p-hydroxyphenyl)-5-phenylhydantoin, which seemingly lacks the anticonvulsant and i n h i b i t o r y a c t i o n on i n s u l i n s e c r e t i o n from the pancreas (Butler, 1957; .../24 K i z e r e t a l . , 1970) should have no i n h i b i t o r y e f f e c t on c o n t r a c t i l e responses to NA. (2) I f the a c t i o n of DPH i s dependent on st i m u l a t i o n of the Na pump, then p r i o r i n h i b i t i o n of t h i s pump by ouabain or K-free s o l u t i o n s should a b o l i s h or attenuate the a b i l i t y of DPH to antagonize contract-i l e responses of NA. (3) I f DPH can stimulate the Na pump and there i s some form of competition between sodium and calcium, then in c r e a s i n g the calcium concentration or lowering the sodium concentration of the Krebs s o l u t i o n would be expected to attenuate the i n h i b i t o r y e f f e c t s of DPH on NA responses. On the other hand, lowering the calcium concentration would have the opposite e f f e c t . (4) Having tested the a b i l i t y of DPH to attenuate the c o n t r a c t i l e responses of NA under these experimental conditions, i t would be of i n t e r e s t to f i n d an i o n i c b a s i s f o r t h i s phenomenon by detecting the e l e c t r o l y t e changes produced by DPH i n the t i s s u e i n normal Krebs, i n the presence of ouabain and i n K - d e f i c i e n t Krebs. (-) I f these e l e c t r o l y t e changes show evidence of stimula-t i o n of the Na pump, then i t would be of i n t e r e s t to t e s t the e f f e c t of the drug at low temperatures i n a co n d i t i o n where metabolic processes have been damped down by c o o l i n g . .../25 25 Further work was done to t e s t the e f f e c t of other drugs a l t e r i n g d i f f e r e n t parameters of sodium transport on c o n t r a c t i l e a c t i v i t y of vascular smooth muscles. I f ouabain and ethacrynic a c i d induce a c o n t r a c t i l e response p r i m a r i l y by i n h i b i t i n g the sodium pump, then one would expect that the pattern of response induced by e i t h e r drug would be s i m i l a r . Recently the drug amiloride was shown to i n h i b i t the passive Na i n f l u x i n a v a r i e t y of t i s s u e s (DBrge and Nagel, 1970; Salako and Smith, 1970,a,b; Gatzy, 1971). I t was then of i n t e r e s t to t e s t whether i t would have any e f f e c t on vascular r e a c t i v i t y and to analyze i f t h i s 4+ would be more prominent on a p a r t i c u l a r Ca poo l . Further studies were conducted to evaluate the e f f e c t of Na on the r e l a x a t i o n of the a n t e r i o r mesenteric v e i n using drugs that a l t e r the Na transport and v a r i a t i o n of the sodium content of the incubating media. In view of the i m p l i c a t i o n of plasmalemmal v e s i c l e s i n many functions (Burnstock et a l . , 1970) and the complexity of the Na transport i n smooth muscles (Brading, 1971; Daniel and Robinson, 1970a,b,c) a preliminary e l e c t r o n microscopic study was conducted to evaluate the e f f e c t of the i n -h i b i t o r s of the ATPase, ouabain and ethacrynic a c i d , on the u l t r a s t r u c t u r e of the r a b b i t a n t e r i o r mesenteric v e i n . o .../26 26 MATERIAL AND METHODS In t h i s s e c t i o n , the b a s i c experimental procedures w i l l be descr-ibed, but f u r t h e r experimental d e t a i l s w i l l be presented i n appropriate s e c t i o n s . D i s s e c t i o n of the Rabbit A n t e r i o r Mesenteric Vein New Zealand white r a b b i t s , of e i t h e r sex, weighing 2,5 to 3.5 kg were k i l l e d by a blow on the head. The abdomen was cut open, the anter-i o r mesenteric v e i n was r a p i d l y freed from adjacent t i s s u e s and dissected from i t s p o i n t of formation to the point of entry i n the l i v e r . The v e i n was immediately placed i n a p e t r i d i s h containing Krebs s o l u t i o n and any adjacent t i s s u e was c a r e f u l l y removed. The v e s s e l was then bise c t e d l o n g i t u d i n a l l y and a loop of s u r g i c a l s i l k was passed on e i t h e r s i d e of the b i s e c t e d end i n preparation f o r organ bath experiments. D i s s e c t i o n of A o r t i c S t r i p s New Zealand white r a b b i t s of e i t h e r sex, weighing 2,0 to 3,5 kg were k i l l e d by a blow on the head. The chest was cut open. A s t r i p of the descending th o r a c i c aorta, about 2-3 cms long was r a p i d l y freed from adjac-ent t i s s u e and t r a n s f e r r e d to a p e t r i d i s h containing normal Krebs s o l u t i o n . Any adjacent t i s s u e was gently removed. H e l i c a l s t r i p s were prepared by c u t t i n g with s c i s s o r s through the v e s s e l and a loop of s u r g i c a l thread was passed through e i t h e r end of each s t r i p i n preparation f o r organ bath experiments. .../27 27 D i s s e c t i o n of Rat T a i l A r t e r i e s Male, Wistar s t r a i n r a t s , weighing 350-500 g, were anaesthesized with an i n t r a p e r i t o n e a l i n j e c t i o n of 6 mg/100 g weight pentobarbital, A v e n t r a l i n c i s i o n i n the middle of the r a t t a i l was followed by care-f u l c u t t i n g of the deep f a s c i a with s c i s s o r s under the d i s s e c t i n g microscope. An a r t e r y s t r i p of about 9-10 cms long was exposed, begin-ning with a point near the root of the t a i l to a d i s t a l one. In experiments destined f o r e l e c t r o l y t e estimation, the s t r i p was immediately separated and t r a n s f e r r e d to a p e t r i d i s h containing normal Krebs s o l u t i o n . Further cleaning from adjacent t i s s u e was then c a r r i e d out under the d i s s e c t i n g microscope. In other experiments destined f o r p e r f u s i o n techniques, a l l the t r i b u t a r i e s of the a r t e r i e s were traced under the d i s s e c t i n g microscope and l i g a t e d . At the completion of t h i s stage, the a r t e r y was covered with wet sponges, soaked i n normal Krebs s o l u t i o n , and l e f t undisturbed f o r a period of at l e a s t 1/2 hour to allow the t i s s u e to recover from the handling and to f a c i l i t a t e subsequent cannulation. Both ends of the a r t e r y were then cannulated with a number 25 cannula. Assessment of l i g a -t i o n of a l l t r i b u t a r i e s was checked at the end of the procedure by a drop of Evans blue, d i s s o l v e d i n the Krebs s o l u t i o n , and i n j e c t e d i n the catheter, at one end of the a r t e r y . In the case of p e r f u s i o n experiments, a minimum period of 45 mins was allowed to perfuse the a r t e r i e s with normal Krebs s o l u t i o n , before any drug was added. During t h i s time the a r t e r y was perfused at a constant r a t e and f i x e d temperature of 37° C. to s t a b i l i z e i t s i n t r a l u m i n a l pressure. . . ./28 28 A r t e r i e s destined f o r e l e c t r o l y t e a n a l y s i s were incubated f o r a period of at l e a s t 1-1/2 hours i n normal Krebs before any f u r t h e r manipulations. Unless otherwise s p e c i f i e d , a l l experiments were car-r i e d at a standard temperature of 37° C. P r i o r incubation i n normal Krebs f o r at l e a s t 1-1/2 hours, was e s s e n t i a l to allow the t i s s u e to recover from the trauma of d i s s e c t i o n and obtain consistent r e s u l t s i n a given experiment. P h y s i o l o g i c a l S a l t Solutions Several p h y s i o l o g i c a l s a l t s o l u t i o n s were used, depending on the aim of the experimental procedure. A b r i e f note on the use of these sol u t i o n s i s presented i n t h i s s e c t i o n , but whenever necessary, f u r -ther experimental d e t a i l w i l l be given i n appropriate s e c t i o n s . Normal Krebs S o l u t i o n A s l i g h t l y modified Krebs s o l u t i o n was mainly used; f o r convenience, i t w i l l be simply r e f e r r e d to as normal Krebs s o l u t i o n . At the s t a r t of each experiment, the blood v e s s e l was f i r s t incubated i n t h i s s o l u t i o n . A l l experiments not i n v o l v i n g a change i n the composition of the Krebs s o l u t i o n used the normal Krebs s o l u t i o n . The composition i n mM was as follows: NaCI—118; KC1—4.6; MgS0 4—125; C a C l 2 — 2 . 5 ; NaHC03-~25; ^ 2 P < ^ 4 — i » 2 » g l u c o s e — 5 . 6 . This s o l u t i o n was bubbled with a mixture of 95% 0 2 and 5% C0 2. Phosphate-free Solution This s o l u t i o n was used i n experiments i n v o l v i n g change i n Ca concen-t r a t i o n s , to prevent the p r e c i p i t a t i o n of Ca phosphates i n high Ca 29 s o l u t i o n s . The composition i n mM was as follows: NaCl—118; KC1—4 . 7 ; C a C l 2 — 2 . 5 ; MgSO^—1.25, NaHCC>3—25, glucose 5.6. This s o l u t i o n was bubbled with a mixture of 95% 0^ and 5% C0 2 and adjusted to a pH of 7.4 by adding HC1. Low Na Solutions The composition was the same as the normal Krebs s o l u t i o n except that the NaCl was e i t h e r substituted with an equivalent amount of L i - C l or T r i s - H C l buffered to a pH of 7.4. The bicarbonate b u f f e r was l e f t i n t a c t . This s o l u t i o n was bubbled with a mixture of 95% 0^ and 5% CC^. Na-free Krebs In a few experiments, we have used a Na-free s o l u t i o n , i n which a l l the NaCl and NaHCO^ was replaced by an equivalent amount i f T r i s - H C l buf-fered to a pH of 7.4. This s o l u t i o n was bubbled with 100% 0 2« K-free S o l u t i o n This s o l u t i o n was s i m i l a r to the normal Krebs s o l u t i o n , except that a l l the K was omitted and su b s t i t u t e d with an equivalent amount of NaCl. This s o l u t i o n was bubbled with a maxture of 95% 0 2 and 5% C0 2. K - d e f i c i e n t S o l u t i o n The composition of t h i s s o l u t i o n was s i m i l a r to normal Krebs, except that KC1 was omitted,leaving 1.2 mM of KH^O^ b u f f e r . No osmotic s u b s t i t u -t i o n was done since the change i n osmolarity i s not appreciable and the a r t e r i e s incubated i n such so l u t i o n s had t h e i r own c o n t r o l . This s o l u t i o n was bubbled with a mixture of 95% 0 o and 5% C0„. ,../30 30 Estimation of the E l e c t r o l y t e Content of Rat T a i l A r t e r i e s At the end of the experiment, the a r t e r y was gently b l o t t e d i n between 2 f i l t e r papers, and immediately t r a n s f e r r e d to a small weigh-ing c y l i n d e r to obtain the f r e s h weight. Weighing c y l i n d e r s had w e l l -f i t t e d stoppers. They were always kept i n a dryer, and manipulated by s p e c i a l forceps to avoid weight changes from moisture. The a r t e r y was d r i e d i n an oven at a temperature of 110° C. over-night and defatted the next day by anhydrous ether. This was followed by f u r t h e r drying of the v e s s e l i n the oven, at the same temperature, u n t i l a constant weight was obtained. Heating r a t t a i l a r t e r i e s f o r one more day a f t e r d e f a t t i n g at a temperature of 90-100° C. i s more than s u f f i c i e n t to evaporate a l l i t s water content. A f t e r drying, the artery was t r a n s f e r r e d from i t s weighing c y l i n d e r to a 4 ml volumetric f l a s k . I t was stored f o r at l e a s t one week i n 0.75 N HNO3. Storage of the a r t e r y f o r at l e a s t 6-7 days was found necessary to obtain consistent r e s u l t s and a low experimental e r r o r . Readings of Na and K were obtained from an atomic absorption spectrophotometer (Perkins Model 303), using the s p e c i a l l y designed lamp and standards of adequate concentrations. The instrument was prepared f o r use i n the usual manner described In the i n s t r u c t i o n manual. Adequate blanks containing the same concentration of solvent i n the standard and specimens of a r t e r y to be de-termined were prepared i n the process of s e t t i n g the instrument f o r e l e c t r o -l y t e estimation. C a l c u l a t i o n of H^O and E l e c t r o l y t e Content The water content of the preparation was obtained as the d i f f e r e n c e between the f r e s h and dry weight. The e l e c t r o l y t e content of the a r t e r y .../31 31 was obtained by matching the e l e c t r o l y t e content of the di s s o l v e d a r t e r y , i n the 4 ml f l a s k , with standards of known e l e c t r o l y t e con-centrations. Organ Bath Experiments Following the d i s s e c t i o n of r a b b i t a n t e r i o r mesenteric veins or a o r t i c s t r i p s , the t i s s u e was tr a n s f e r r e d to an organ bath ( F i g . 1 ) . I t i s an 18 ml muscle bath obtained from Glass Appliances L t d . , Aber-deen, Scotland. I t has a warming c o i l (A) of an 18 ml capacity, so that complete change of bathing f l u i d can be e f f e c t e d without change of temperature. The temperature of the bathing f l u i d was f i x e d at 37° C. by a the r m o s t a t i c a l l y c o n t r o l l e d pump. Warm water ejected by the pump came through an i n l e t (B) and was returned to the pump r e s e r -v o i r through the o u t l e t (C). Continuous aeration of the Krebs s o l u t i o n was maintained throughout the experiment by a small diameter p o l y e t h y l -ene tube (D), passed through the bottom d r a i n tube (E), to the c e n t r a l compartment (F), containing the b i o l o g i c a l t i s s u e (G). The end of the polyethylene tube was crushed with p l i e r s and then pierced with a needle to provide a f i n e flow of bubbles over the experimental t i s s u e . The t i s s u e was anchored at one end by a loop of s u r g i c a l thread, passed through the t i s s u e and over a glass hook (H). The other end was connected to a Grass FTO 3C fo r c e displacement transducer which was, i n turn, connected to a Grass Model 5 or 7 Polygraph. Isometric contractions were recorded at a standard tension of 500 mg i n the r a b b i t a n t e r i o r mesen-t e r i c v e i n and 5 gm i n r a b b i t a o r t i c s t r i p s . Tension on the t i s s u e could be adjusted by r a i s i n g or lowering the force transducer, using a rack and pi n i o n . ../32 32 F i g . 1 Apparatus f o r organ bath experiments F R O M P U M P TO P U M P .../33 33 Before adding any drug, experiments were always started by incu-bation of the tissue in normal Krebs solution for a period of at least 45 mins. This allows adequate equilibration of the tissue with the normal Krebs solution and helps the preparation to recover from the trauma of dissection. Within this period, the base line tension has stabilized and in the case of the anterior mesenterior vein, the spon-taneous activity has started and became regular. Drugs were added in the Krebs solution, using a 1 ml tuberculin syringe and a #25 needle. Washing the drug was carried by emptying the bath from the lower drain (E), and refilling, using the stop coks (I). Results were taken after the tissue gave a consistent quantitative response to the drug used. Tissues adequately dissected and continuously aerated in normal Krebs solution at 37° C. remained viable for a period of at least 8 hours. Apparatus for Perfusion of the Rat Tail Artery The apparatus (Fig. 2) consists of a chamber (A) connected to a thermostatically controlled pump through an inlet (B) and outlet (C). Chamber (A) contain a coiled polyethlene tube connected at one end with a perfusion pump (D), and the other end with a T-connection (E). Krebs solution ejected by the Harvard infusion pump at a constant rate of 0.5 ml/min was warmed out in chamber (A) and through one side of the T-connection, circulated in the polyethylene cannula, inserted on one end of the artery. The other side of the T-connection was con-nected to a Statham Model P23 AC pressure transducer for recording the intraluminal pressure. The thermostatic pump was adjusted to give a .../34 34 Fig. 2 Apparatus for perfusion of rat t a i l arteries y \ ARJERY TO TRANSDUCER FROM PUMP TO PUMP 35 Krebs s o l u t i o n of approximately 37° C. The cannulated arte r y was placed i n i t s chamber (F), bathed i n Krebs s o l u t i o n that was continuously bubbled with a mixture of 95% 02 and 5% C0 2 through the i n l e t s (G) and ( I ) . Drugs were dissol v e d i n the Krebs s o l u t i o n perfused through the art e r y by the constant pressure pump. Apparatus f o r Incubation of the Rat T a i l Artery The apparatus ( F i g . 3) consists of a simple bath with an i n l e t (A) connected to a th e r m o s t a t i c a l l y c o n t r o l l e d pump. Warm water ejected by the pump was adjusted to give a constant temperature i n the bath of about 37° C. and was returned through the o u t l e t (B) and the overflow opening (C). A rack holding the t e s t tubes, containing a r t e r i e s , was placed i n the bath. The bathing s o l u t i o n of the a r t e r i e s was continuously bubbled by small polyethylene tubes emerging from a main U-shaped tube (D) con-nected to the tank s u p p l i e r . Both t h i s apparatus and the apparatus f o r perfu s i o n of r a t t a i l a r t e r i e s were provided In the workshop of the Department of Pharmacology at The U n i v e r s i t y of B r i t i s h Columbia. The Author wishes to thank s p e c i f i c a l -l y Mr. Eddy Ingo and Mr. J e r r y Kent f o r t h e i r t e c h n i c a l assistance i n pro-v i d i n g t h i s apparatus. Apparatus f o r Cooling Experiments This was e s s e n t i a l l y an incubation bath i n which the temperature was kept constant at 1° C. by means of an Eberbach cooling thermostat. A rack i n the bath held the tubes containing the r a t t a i l a r t e r i e s . The bathing Krebs s o l u t i o n was continuously bubbled through polyethylene catheters Immersed i n the tubes. ../36 Fig. 3 Apparatus for incubation of rat t a i l arteries PUMP 37 Preparation of Histological Sections At the end of the experimental procedure, rabbit anterior mesen-teric veins destined to histological examination were removed from the organ bath. They were quickly tied by both ends to a wooden stick so that the tension remained the same as i t was before, and then immediate-ly immersed in 1% buffered osmic acid, pK 7.4, for one hour. The tissues were then dehydrated in alcohol and acetone arid embedded in maraglass. Micron sections were stained with toluidine blue for examination in the light microscope. Ultra-thin sections were stained with 1% uranyl acetate and 1% lead citrate and examined in a Siemens-El Mishop I Electron micro-scope. Pictures were taken at a standard magnification of 17,500 and were enlarged by 4.5 times. The Author wishes to thank at this point Dr. B. Chase of the Depart-ment of Pathology at The University of British Columbia for his help in providing the sections and electron micrographs. Preparation and Sources of the Drugs 1. The sodium salt of diphenylhydantoin was used in the present study (DPH). The drug Is practically insoluble in water and solu-tions had to be alkalinized. We used NaOH to solubilized the drug _2 and the pH of stock solutions of 1 to 4 x 10 M was slightly above 11.5. Storage of the drug at room temperature made DPH crystallize and come out of solutions within a few days. This process was asso-ciated with loss of potency. Our drug solutions were immediately refrigerated at a temperature below freezing, and the drug was left to dissolve again at room temperature on the day of the experiment. Enough drug was prepared to last only for a period of about one week. .../38 38 With t h i s method of p r e p a r a t i o n , s o l u t i o n s of the drug remained c l e a r and no c r y s t a l l i z a t i o n or p r e c i p i t a t e occurred upon a d d i t i o n of DPH to our Krebs s o l u t i o n . DPH was s u p p l i e d by Parke Davis Co. 2. 5-(P-hydroxyphenyl)-5-phenythydantoin (DPHOH) was pr a c -t i c a l l y i n s o l u b l e i n water. Stock s o l u t i o n s were prepared by a manner s i m i l a r to t h a t d e s c r i b e d f o r DPH, usin g NaOH f o r a l -k a l i n i z a t i o n of the s o l u t i o n . DPHOH was syn t h e s i z e d i n Dr. T. H. Brown's l a b o r a t o r y , F a c u l t y of Pharmaceutical Sciences, The U n i v e r s i t y of B r i t i s h Columbia, u s i n g the method of B u t l e r ( B u t l e r , 1957) o r purchased from Parke Davis Co. 3. E t h a c r y n i c a c i d was e i t h e r d i s s o l v e d i n T r i s b u f f e r , o r s u p p l i e d as the Na s a l t i n v i a l . S o l u t i o n s were prepared f r e s h on the day of the experiment to av o i d l o s s of potency t h a t occurs on storage of t h i s drug i n n e u t r a l o r a l k a l i n e s o l u t i o n s . E t h a c r y n i c a c i d was provided by Merck, Sharp and Dohm Co. 4. Ouabain, a m i l o r i d e , p r o c a i n e , n o r a d r e n a l i n e and phentolamine were a l l d i s s o l v e d i n demine r a l i z e d water. I n some experiments, usi n g h i g h c o n c e n t r a t i o n s of ouabain, the l a t t e r was d i r e c t l y d i s -s o l v e d i n the Krebs s o l u t i o n . Ouabain was obtained from Mann Research L a b o r a t o r i e s . A m i l o r -i d e was k i n d l y g i v e n as a g i f t from Dr. Dorian i n Merck, Sharp and Dohm Co., Mo n t r e a l . L - n o r a d r e n a l i n e - D - b i t a r t r a t e monohydrate was s u p p l i e d by Mann Research L a b o r a t o r i e s . P r o c a i n e h y d r o c h l o r i d e was obtained from Poulenc L t d . , M o n t r e a l , Quebec. Phentolamine was s u p p l i e d by C i b a . .../39 39 S t a t i s t i c a l Evaluation of Results whenever p o s s i b l e the c o n t r o l values were compared with e x p e r i -mentally treated value, using the student's t e s t . A(P) value of l e s s than 0.05 was considered to be s t a t i s t i c a l l y s i g n i f i c a n t . A l l r e s u l t s are presented as mean ± S.E. 40 RESULTS The i n h i b i t o r y e f f e c t of DPH on spontaneous a c t i v i t y and NA  contractions of the r a b b i t a n t e r i o r mesenteric v e i n . Non-cumulative dose response curves were obtained, using NA as an agonist. The e f f e c t of DPH was then tested i n a con-c e n t r a t i o n of 20 ug/cc (7,9 x IO-"* M), The drug was l e f t i n the organ bath f o r a standard period of 2-1/2 mins and then the agonist was added. Following a maximum response, given by an agonist concentration, the drug was washed and the t i s s u e was allowed s u f f i c i e n t time to recover, A s i m i l a r experimental design was followed i n other experiments i n v o l v i n g the study of the I n h i b i t o r y e f f e c t of DPH on c o n t r a c t i l e responses to agonists, unless s p e c i f i c a l l y c i t e d . In t h i s experiment, the peak tension, given by a c e r t a i n agonist concentration, was measured i n the absence and presence of DPH and p l o t t e d as the percent of maximum response. The a d d i t i o n of DPH i n a concetration of 20 ug/cc r e s u l t e d i n a gradual cessation of spontaneous a c t i v i t y . Within 2-1/2 mins, the rhythmic c o n t r a c t i l e a c t i v i t y of the ra b b i t a n t e r i o r mesenteric v e i n has markedly diminished i n both frequency and amplitude and i n many preparations had almost disappeared by the end of t h i s p e r i o d . The e f f e c t was r e v e r s i b l e f o llowing washing out the drug ( F i g , 4 ) . Dose response curves showed an i n h i b i t o r y e f f e c t of DPH on NA contractions and that the antagonism i s of a non-competitive nature ( F i g , 5), Fig. 4 Inhibitory effect of DPH on spontaneous activity and contractile response of NA i n the rabbit A. M. V. 1 2 5 m 9 JUJJJULUJ J NA(5xlO- 8g/ c. c., 2.5 min. •^ AAAJUXUUJluIuuX D P H / A  20ug/c.c. ( 5 x , ° 9/CC. DPH was f i r s t added and l e f t for a standard period of 2-1/2 mins. The agonist was then added and the peak response measured. The preparation was allowed sufficient time to recover. NOTE: The gradual diminution of spontaneous activity both i n frequency and amplitude following the addition of 20 vg/cc DPH and the re v e r s i b i l i t y of the effect. t -+ " NA -Arrows point to the time of application of the drug. R points to the time the drug was washed from the incubation medium Noradrenaline F i g . 5 Inhibitory e f f e c t of 20 ug/cc DPH on NA contractions i n the rabbit A.M.V. • • • • C O N T R O L L O G C O N C E N T R A T I O N OF N A I N G/C.C. 43 Comparison of the e f f e c t of DPH and DPHOH on NA c o n t r a c t i l e  responses. The i n h i b i t o r y e f f e c t of equimolar concentrations of DPH and DPHOH were compared, using a standard concentration of NA — 8 of 5 x 10 g/cc. Both drugs were dissolved i n an equal amount of NaOH to r a i s e the pH of the s o l u t i o n to above 11. The idea was to t e s t whether the i n h i b i t o r y e f f e c t i s r e s t r i c t e d to DPH and not to i t s p-hydroxy d e r i v a t i v e . The l a t t e r i s a metabolite of DPH and was found to be la c k i n g the pharmacological actions of DPH i n i n t a c t animals. I t was al s o a way to exclude the p o s s i b i l -i t y of an e f f e c t of the solvent on the r e s u l t s obtained. 0.1 mM DPHOH had no e f f e c t on spontaneous a c t i v i t y and was p r a c t i c a l l y devoid of i n h i b i t o r y e f f e c t compared to an equimolar amount of DPH ( F i g . 6). While the % reduction of the c o n t r a c t i l e responses to the standard concentration of NA was 43 ± 3.3 i n the case of DPH, i t was only 1.2 ± 2.5 i n the case of i t s P-hydroxy-d e r i v a t i v e . I t i s , therefore, concluded that the i n h i b i t o r y e f f e c t on NA responses i s r e s t r i c t e d to DPH and i s not shared by i t s P-hydroxy d e r i v a t i v e . The p o s s i b i l i t y of an experimental a r t i f a c t r e s u l t i n g from using NaOH as the solvent of the drug could be adequately ex-cluded by t h i s experiment. In some experiments the pH of the Krebs s o l u t i o n was monitored during a v a r i e t y of experiments. Within the concentration of 0.1 to 0.4 mM DPH used i n the present study, we could not detect any change of pH r e s u l t i n g from the a d d i t i o n of the drug. F i g . 6 Comparison of the e f f e c t of 10 M DPH and DPOH i n the rabbit A.M.V. on the c o n t r a c t i l e response of 5 x 10 g/cc NA I Results obtained from 6 ve i n s t r i p s of 4 d i f f e r e n t animals. 45 Effect of ouabain on the inhibitory action of DPH. As already cited, i n the previous section, one way by which the inhibitory effect of DPH could be mediated i s through stimula-tion of the Na pump. It was then of some interest to test whether an intact Na-K ATPase i s necessary for the inhibitory effect of DPH. Ouabain i s a well-known inhibitor of the Na-K ATPase. If the a c t i -v i t y of DPH i s i n some way related to the Na pump, then prior i n h i -bition of the pump should abolish or attenuate the inhibitory effect of DPH. -4 The inhibitory effect of 2 x 10 M DPH was tested i n normal Krebs solution on a standard concentration of 10 ^  g/cc NA and the results were expressed as % inhibition of the control. The same tissue was then exposed to the same Krebs i n which 5 x 10 ^  M oua-bain was added. The drug was l e f t to act for a period of about 2 hours. The same concentration of DPH was retested on the standard concentration of NA and the results were expressed as % inhibition of the control i n the same Krebs solution. Exposure of the tissue —6 to a concentration of 5 x 10 M ouabain resulted i n a small con-traction followed by relaxation within a period of about 15 mins. In the Krebs solution containing ouabain (5 x 10 ^  M), there was a rapid loss of the inhibitory effect of DPH on NA responses. Results were taken when a series of NA responses before and after the addition of DPH gave consistently stable quantitative readings. There was always an attenuation of NA responses in the ouabain treated tissue. But the responses to NA contractions stayed con-sistent after a few alternate responses to that drug i n the pre-sence and absence of DPH. 46 -4 While the % i n h i b i t o r y e f f e c t of 2 x 10 M DPH i n normal Krebs s o l u t i o n of 10 7 g/cc NA was In the order of 55.8 ± 1.9%, i t dropped to about 3.7 ± 1.2% following treatment of the t i s -sue f o r 2 hours with 5 x 10 ^ M ouabain ( F i g . 7). In some of these experiments the same preparation was tra n s f e r r e d again to a normal Krebs s o l u t i o n and l e f t to recover from ouabain f o r about 1-1/2 hours. As shown i n ( F i g . 8), removal of ouabain from the Krebs s o l u t i o n was associated with the return of the i n h i b i -tory e f f e c t of DPH on NA contrac t i o n s . I t seems, therefore, from t h i s experiment that the i n h i b i t o r y e f f e c t of DPH on c o n t r a c t i l e responses to NA i s i n some way r e l a t e d to the pump a c t i v i t y and that the e f f e c t i s f a i r l y r e v e r s i b l e . E f f e c t of K-free s o l u t i o n s on the i n h i b i t o r y e f f e c t of DPH on NA  contract i o n s. Since we had some evidence i m p l i c a t i n g the Na pump i n the i n h i b i t i o n e f f e c t of DPH, we proceeded to f u r t h e r t e s t t h i s p o s s i -b i l i t y . In a K-free Krebs one i s expecting to i n h i b i t the Na pump and a b o l i s h the concentration gradient of Na and K through the mem-brane. I f the i n h i b i t o r y e f f e c t of DPH i s i n some way r e l a t e d to the Na pump, then i n h i b i t i o n of t h i s pump by K-free s o l u t i o n s should give the same q u a l i t a t i v e r e s u l t s i n v o l v i n g the use of ouabain to i n h i b i t the Na-K ATPase. -4 The i n h i b i t o r y e f f e c t of 2 x 10 M DPH on a standard concen-t r a t i o n of 10 7 g/cc NA was tested i n normal Krebs s o l u t i o n . The ti s s u e was then exposed to a K-free s o l u t i o n f o r a period of 1 hour. Alt e r n a t e concentrations of agonists and antagonists were tested i n the same concentrations used i n normal Krebs s o l u t i o n . Results were F i g . 7 Comparison of the i n h i b i t o r y e f f e c t of 2 x 10~ 4 M DPH on 1 0 " 7 g/cc NA i n the r a b b i t A.M.V. i n normal Krebs and ouabain containing Krebs Results obtained from 8 v e i n s t r i p s of 6 d i f f e r e n t animals 48 .../49 49 LEGEND TO FIG. 8 -4 A = The i n h i b i t o r y e f f e c t of 2 x 10 M DPH was tested i n normal Krebs s o l u t i o n on the c o n t r a c t i l e response of 10~7 g/cc NA i n the rabbit A.M.V. B = The same concentration of DPH was retested on the c o n t r a c t i l e response of the same concentration of NA —6 i n a Krebs containing 5 x 10 M ouabain. C = The t i s s u e was reincubated i n normal Krebs and l e f t to recover. The i n h i b i t o r y e f f e c t of the same con-ce n t r a t i o n of DPH was retested again on the same con-centration of NA. ./50 50 taken when consistent responses were obtained. In the r a b b i t a n t e r i o r mesenteric v e i n , the s h i f t from a normal to a K-free Krebs, i n which the K was replaced by an equi-valent amount of NaCI to compensate f o r the small change i n osmo-l a r i t y , r e s u l t e d i n a gradual l o s s of spontaneous a c t i v i t y , but there were no c o n t r a c t i l e responses as obtained i n some other smooth muscle preparations (Brecht et a l , , 1969; Axelsson and Holmberg, 1971). Testing a l t e r n a t i v e l y the responses to NA i n the presence and absence of DPH, we found a r a p i d l o s s of the i n h i b i t o r y e f f e c t of DPH i n K-free s o l u t i o n s . The pattern of response was comparable to that obtained i n the presence of ouabain. While the i n h i b i t o r y - 4 - 7 e f f e c t of 2 x 10 M DPH on 10 g/cc NA was i n the order of 56.9 ± 5.4% i n normal Krebs, i t dropped to 12.3 ± 4.2% i n K-free Krebs ( F i g . 9 ) . In some of these experiments, the same preparation was r e - e q u i l i b r a t e d with normal Krebs s o l u t i o n f o r a period of 1 hour. S h i f t to the normal Krebs s o l u t i o n r e s u l t e d i n the reappear-ance of the i n h i b i t o r y e f f e c t of DPH ( F i g . 10). This experiment gives, therefore, some evidence about the i m p l i c a t i o n of the Na pump i n the i n h i b i t o r y e f f e c t of DPH. I t also shows that the l o s s of a c t i v i t y of t h i s drug i n K-free s o l u t i o n i s r e v e r s i b l e following r e - e q u i l i b r a t i o n of the t i s s u e with normal Krebs s o l u t i o n . In smooth muscles as i n other t i s s u e s there i s evidence of some form of i n t e r a c t i o n between Na and Ca Ions. While the pattern of the Na/Ca i n t e r a c t i o n i n smooth muscles i s not yet c l e a r , i t would be of some i n t e r e s t to t e s t the e f f e c t of d i f f e r e n t Ca concentrations and low Na s o l u t i o n s on the i n h i b i t o r y e f f e c t of DPH. I f the i n h i b i -51 F i g . 9 -4 Comparison of the i n h i b i t o r y e f f e c t of 2 x 10 M DPH on the c o n t r a c t i l e response of 10 7 g/cc NA i n the r a b b i t A.M.V. i n normal Krebs and i n K-free Krebs Results obtained from 8 v e i n s t r i p s of 4 d i f f e r e n t animals. 53 LEGEND TO FIG. 10 -4 A = The i n h i b i t o r y e f f e c t of 2 x 10 M DPH was tested on the c o n t r a c t i l e response of 10 7 g/cc NA i n the r a b b i t A.M.V. B = The same concentration of DPH was retested on the c o n t r a c t i l e response of 10 7 g/cc NA i n K-free s o l u t i o n . C = The preparation was e q u i l i b r a t e d with normal Krebs again, the t i s s u e was l e f t to recover, and the same concentration of DPH was retested on the c o n t r a c t i l e response of 10 7 g/cc NA. /54 54 tory e f f e c t of t h i s drug i s i n some way re l a t e d to the a c t i v a t i o n of the Na pump and there i s some form of competition between Na and Ca ions, one would expect an attenuation of the i n h i b i t o r y e f f e c t of DPH i n high Ca sol u t i o n s and p o t e n t i a t i o n of such an e f f e c t i n low Ca s o l u t i o n s . On the other hand low Na s o l u t i o n s would have the same e f f e c t as high Ca s o l u t i o n s . For t h i s reason we proceeded to t e s t the i n h i b i t o r y e f f e c t of DPH i n d i f f e r e n t Ca and low Na s o l u t i o n s . E f f e c t of d i f f e r e n t Ca concentrations on the i n h i b i t o r y e f f e c t of DPH on NA contractions. A standard concentration of 10 7 g/cc NA was used as the agonist. -4 DPH was used as the antagonist i n a concentration of 10 M. The ti s s u e was f i r s t e q u i l i b r a t e d i n normal Krebs s o l u t i o n f o r a period of at l e a s t 1/2 hour. Within t h i s time spontaneous a c t i v i t y had r e -sumed and the v i a b i l i t y of the preparation was tested by adding a —8 concentration of 2 x 10 g/cc NA to the incubation medium. The Krebs s o l u t i o n was then changed to a 1 mM Ca, 2.5 mM Ca, 5 mM Ca and 10 mM Ca, phosphate-free Krebs, to prevent the p r e c i p i t a t i o n of Ca phosphate i n high Ca s o l u t i o n s . Experiments were begun i n the low [Ca] Krebs or [Ca], then was ra i s e d to higher concentrations. Aperiod of 1/2 hour e q u i l i b r a t i o n was allowed a f t e r changing to a new Krebs s o l u t i o n and r e s u l t s were taken a f t e r g e t t i n g consistent r e s u l t s to a s e r i e s of NA responses. -4 The i n h i b i t o r y e f f e c t of 10 M DPH on responses to the standard con-—7 ++ cent r a t i o n of 10 g/cc NA i n a Krebs s o l u t i o n of a given Ca concen-t r a t i o n was p l o t t e d against the Ca concentration of the Krebs s o l u t i o n . This avoided the d i f f i c u l t y i n comparing the i n h i b i t o r y e f f e c t when the response to NA i n the low Ca concentration was d i s t i n c t l y attenuated. F i g . 11 -4 -7 I n h i b i t o r y e f f e c t of 10 M DPH i n the rabbit A.M.V. on c o n t r a c t i l e responses of 10 g/cc I | NA i n Krebs containing d i f f e r e n t Ca concentration u i U l 56 LEGEND TO FIG. 11 -4 The i n h i b i t o r y e f f e c t of 10 M DPH was tested i n the same preparation of the ra b b i t A.M.V. on the con-t r a c t i l e response of 10 7 g/cc NA. Phosphate-free Krebs s o l u t i o n was used throughout the experiment to avoid the p r e c i p i t a t i o n of calcium phosphate i n high calcium con-t a i n i n g Krebs s o l u t i o n . .../57 57 The i n h i b i t o r y e f f e c t of DPH i s to some extent dependent on the Ca concentration ( F i g . 11). In 1 mM Ca Krebs the i n h i b i -tory e f f e c t of DPH was greater than i n 2.5 mM (P < 0.005) and the l a t t e r s t i l l greater than i n 10 mM Ca Krebs (P < 0.0005); w i t h i n 1 to 5 mM, the p l o t i s almost l i n e a r . 6. E f f e c t of low Na Krebs on the i n h i b i t o r y a c t i o n of DPH. L i - C l and T r i s - H C l buffered to a pH of 7.4 were used as sub-s t i t u t e s f o r Na. In both s e r i e s of experiments the t i s s u e was f i r s t e q u i l i b r a t e d i n normal Krebs s o l u t i o n . I t was then tr a n s f e r r e d to e i t h e r 25 mM Na Krebs (T r i s - H C l substituted) or a 60 mM Na Krebs ( L i s u b s t i t u t e d ) . We could not go to lower concentrations of L i i n our experiments since t h i s manoeuvre r e s u l t e d i n contractures of the a n t e r i o r mesenteric v e i n which added f u r t h e r d i f f i c u l t i e s i n evaluating our r e s u l t s . This was not a problem i n 25 mM Na Krebs T r i s - H C l s u b s t i t u t e d . The i n h i b i t o r y e f f e c t of 15 ug/cc DPH (5.9 x 10 ^ M) on the response to a standard concentration of —8 3.2 x 10 g/cc NA was f i r s t tested i n normal Krebs s o l u t i o n . The t i s s u e was then l e f t to e q u i l i b r a t e f o r a period of at l e a s t 45 mins i n the low Na Krebs. The i n h i b i t o r y e f f e c t of the same dose of DPH was then retested on the same standard dose of NA. S h i f t to e i t h e r 25 mM Na T r i s s u b s t i t u t e d or 60 mM L i s u b s t i t u t e d r e s u l t e d i n an obvious p o t e n t i a t i o n of NA contractions. However, the l a t t e r e f f e c t was consistent only i n the T r i s s u b s t i t u t e d s o l u t i o n . In the L i s u b s t i t u t e d s o l u t i o n the p o t e n t i a t i o n wore o f f gradually a f t e r a l t e r n a t e a p p l i c a t i o n s of NA with and without the presence of DPH. .../58 58 While the i n h i b i t o r y e f f e c t of 15 yg/cc DPH (5.9 x 10"5 M) on the standard concentration of NA was 36 ± 2.7% i n normal Krebs, i t dropped to 20.2 ± 2.4% i n 25 mM Na T r i s - H C l substituted ( F i g . 12). The d i f f e r e n c e was s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t (P < 0.0025). However, with 60 mM Na, L i substituted Krebs the i n h i b i t o r y e f f e c t was 37.4 ± 2.4 and 33.5 ± 3.6% i n normal and L i subs t i t u t e d Krebs r e s p e c t i v e l y ( F i g . 13). The d i f f e r e n c e 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 (P > 0.05). At t h i s point, we had s u f f i c i e n t evidence to suggest the i m p l i -c a t i o n of the Na pump i n the mechanism by which DPH attenuates con-t r a c t i l e responses to drug. This l e d us to go fu r t h e r and attack the problem by more d i r e c t means. I f t h i s drug a l t e r s the a c t i v i t y of the Na pump, then i t should be p o s s i b l e to detect e l e c t r o l y t e changes induced by DPH. The question then a r i s e s : what are the conditions under which t h i s drug a f f e c t s the pump a c t i v i t y and how the changes i n pump a c t i v i t y could a f f e c t the Ca a v a i l a b l e to the c o n t r a c t i l e protein? In planning to study e l e c t r o l y t e changes induced by drugs i n the vascular w a l l , one has to consider a preparation i n which the smooth muscles c o n s t i t u t e i t s main bulk and where the c o n t r i b u t i o n of other elements to the vascular w a l l i s not appreciable. A s u f f i -c i e n t amount of l i t e r a t u r e on e l e c t r o l y t e studies should be i d e a l l y present to help the i n t e r p r e t a t i o n of r e s u l t s , and r e l i a b l e techniques f o r estimation of e l e c t r o l y t e changes should be a v a i l a b l e to detect small, but consistent, changes of e l e c t r o l y t e s induced by drugs. The r a b b i t a n t e r i o r mesenteric v e i n proved to be an u n s a t i s -factory preparation f o r t h i s purpose. The c o n t r i b u t i o n of f a t to the F i g . 12 Comparison of the i n h i b i t o r y e f f e c t of 15 ug/cc DPH on the c o n t r a c t i l e response of 3.2 x 10~ 8 g/cc NA i n normal Krebs and i n 25 mM Na Krebs (Tris-HCl s u b s t i t u t i o n ) i n the r a b b i t A.M.V. .NQRMAL__Krebs -A5_rnM_Na^K Results obtained from 8 v e i n s t r i p s of 5 d i f f e r e n t animals. Fig. 13 Comparison of the inhibitory effect of 15 pg/cc DPH on the contractile —8 response of 3.2 x 10 g/cc NA i n normal Krebs and i n 60 mM Na Krebs (Li substitution) i n the rabbit A.M.V. I Results obtained from 7 vein strips of 4 different animals. 61 bulk properties i s appreciable (about 50% of the dry weight i s l o s t following d e f a t t i n g with anhydrous ether compared to 1-2% i n r a t t a i l a r t e r i e s and guinea p i g taenia c o l i ) . Attempts of s t r i p p i n g t h i s f a t under the d i s s e c t i n g microscope has r e s u l t e d i n our hands i n important e l e c t r o l y t e changes, and the e l e c t r o -l y t e readings so reached were quite i n c o n s i s t e n t . Because of these inconvenient properties of the r a b b i t a n t e r i o r mesenteric v e i n , we chose the r a t t a i l a r t e r y as one of the most s a t i s f a c t -ory preparations f o r t h i s kind of study. I t i s an example of a small a r t e r y which bears more resemblance to re s i s t a n c e vessels than some other preparations l i k e a o r t i c s t r i p s . More than h a l f of the whole t i s s u e consists of muscle, the c o n t r i b u t i o n of other c e l l u l a r types to the bulk properties of the w a l l i s not appr e c i -able, and r e l i a b l e techniques to estimate the e l e c t r o l y t e changes i n the vascular w a l l are r e a d i l y a v a i l a b l e . In a l l subsequent experiments i n v o l v i n g e l e c t r o l y t e s t u d i e s , the r a t t a i l a r t e r y was used and r e s u l t s , unless s p e c i f i c a l l y mentioned, are expressed as IL^ O i n ml/100 g; dry defatted weight or meq/100 g; dry defatted weight f o r Na and K determinations. In perfusion experiments of the r a t t a i l a r t e r i e s we found that 0.2 mM DPH d i d not a f f e c t s i g n i f i c a n t l y the i n t r a l u m i n a l pressure when l e f t i n the perfusate f o r a period of 10 mins. While the c o n t r o l i n t r a l u m i n a l pressure was 50.5 ± 2.5 mm Hg, the in t r a l u m i n a l pressure following a d d i t i o n of 0.2 mM DPH to the Krebs s o l u t i o n was 48.6 ± 3.9. The d i f f e r e n c e 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 (P > 0.05). However, DPH was s t i l l able to antagonize the 62 c o n t r a c t i l e response of 0.4 ug/cc NA (38.9 ± 6.3% i n h i b i t i o n ) and t h i s compares to the i n h i b i t o r y e f f e c t i n the r a b b i t a n t e r i o r mesenteric v e i n . We proceeded, therefore, i n a s e r i e s of e x p e r i -ments to study the e l e c t r o l y t e changes induced by DPH i n the vascu-l a r w a l l of r a t t a i l a r t e r i e s under a v a r i e t y of experimental con-d i t i o n s . 7. E f f e c t of DPH on the e l e c t r o l y t e content of r a t t a i l a r t e r i e s i n  normal Krebs s o l u t i o n s . In s t a r t i n g t h i s study we f i r s t evaluated the e f f e c t of DPH i n normal Krebs s o l u t i o n . Follox^ing a standard incubation i n nor-mal Krebs s o l u t i o n f o r a period of 90 mins, one group of a r t e r i e s -4 was treated with DPH i n a concentration of 4 x 10 M f o r 1 hour. The other group of a r t e r i e s served as a c o n t r o l and was l e f t f o r a s i m i l a r period of time i n Krebs s o l u t i o n to which we added the same concentration of the NaOH used to d i s s o l v e the drug. Under t h i s experimental cond i t i o n (Table 1), there was no s i g n i f i c a n t d i f f e r e n c e on the Na content of c o n t r o l and DPH treated a r t e r i e s (P > 0.05). While the Na content of DPH treated a r t e r i e s was 36.84 ± 1.6, i t was 36.8 ± 1.2 i n c o n t r o l . Indeed, i f there has been any change, i t was rather a s l i g h t diminution i n the K content of DPH treated a r t e r i e s (20.57 ± 0.95 and 22.98 ± 0.65 f o r DPH and co n t r o l a r t e r i e s r e s p e c t i v e l y ) . This i s s t a t i s t i c a l l y s i g n i f i c a n t (P < 0.05). I t i s , therefore, apparent that under these experimental conditions, i f there i s any e f f e c t of DPH, i t Is compatible with i n h i -b i t i o n rather than s t i m u l a t i o n of the Na pump. .../63 TABI.P! 1 Experiment li^O ml/100 g Na meq/100 g K meq/100 g D.D.A. D.D.A. D.D.A. Normal Kreb3 (9): Control 295.43 ± 11.93 36.81 ± 1.60 22.98 i 0.65 (N.S.) (N.S.) (P < 0.05) DPH (4 x 10~* M 291.13 * 12.26 36.84 • 1.20 20.57 t 0.95 In the presence of ouabain (6): Control 282.94 ± 11.93 49.77 t 1.00 9.80 t 0.30 (N.S.) (P > 0.05) (P < 0.001) DPH (4 x 10** K 312.12 ± 12.58 46.23 1 1.69 12.20 t 0.02 In K-deflcient  solution (10)I Control 288.59 1 9.50 39.46 ± 1.08 13.10 t 0.30 (N.S.) (N.S.) (P < 0.001) DPH (2 x 10** M) 289.16 ± 7.90 38.86 1 1.17 14.90 • 0.20 In K-deflclent  solution at 1* C. (8): Control 330.74 ± 6.34 43.66 • 1.10 12.72 ± 0.53 (N.S.) (N.S.) (N.S.) DPH (2 x 10"* M) 318.84 5.84 43.55 1.22 12.60 0.59 N.B. Number l n between brackets indicate nunber of arteries in each control and drug treated experiment. D.D.A. - means dry defatted artery 64 E f f e c t of DPH on the e l e c t r o l y t e content of r a t t a i l a r t e r i e s  i n the presence of ouabain. Several reports, using a v a r i e t y of t i s s u e s , have already demonstrated an i n h i b i t i o n of the Na-K ATPase by DPH when tested i n incubation media, containing a low Na/K r a t i o (Pincus and Giarman, 1967; Rawson and Pincus, 1968; F e s t o f f and Appel, 1968; Formby, 1969). However, a v a r i e t y of other studies have i n d i c a t e d a s t i m u l a t i o n of the same enzyme under conditions of high Na/K r a t i o s (Festoff and Appel, 1968; Lewin and Bleck, 1971; Spain and Chidsey, 1971; S i e g e l and Goodwin, 1972). T h i s , together with the s e v e r a l reports i n d i c a t i n g an antagonism of the e f f e c t of oua-bain by DPH i n a v a r i e t y of t i s s u e s (Pincus, 1970; Woodbury and Kemp, 1971), and our studies on the r a b b i t a n t e r i o r mesenteric v e i n , suggesting the p o s s i b l e i m p l i c a t i o n of the Na pump i n the mechanism of a c t i o n of DPH, prompted us to study more c l o s e l y the e f f e c t of t h i s drug i n the presence of ouabain and i n K - d e f i c i e n t s o l u t i o n s . Following the standard incubation of 90 mins, one group of a r t -e r i e s was exposed to normal Krebs containing 2 mM ouabain and 0.4 mM DPH f o r a period of 3 hours. The other group was l e f t f o r a s i m i l a r period of time i n a Krebs containing 2 mM ouabain i n which NaOH was added i n the same concentration used to d i s s o l v e DPH. Under t h i s experimental condition, there was a h i g h l y s i g n i f i -cant d i f f e r e n c e i n the K content of ouabain treated (9.8 ± 0.3) com-pared to ouabain + DPH treated group (12.2 ± 0.2) (P < 0.001). I t i s , therefore, evident that DPH could counteract the l o s s of K i n a r t e r i e s exposed to ouabain. The e f f e c t on the Na gain was much l e s s evident under t h i s experimental c o n d i t i o n , but i t i s conceivable that the .../65 65 changes might have been i n part masked by a l t e r a t i o n i n the s i z e of the e x t r a c e l l u l a r space. The Na content of DPH + ouabain treated group was 46.23 ± 1.69 compared to 49.77 ± 1.00 f o r the ouabain treated group (P > 0.05) (Table 1 ) . I t i s , therefore, conceivable to suggest at t h i s point that, under the present experimental con-d i t i o n s , DPH can counteract both the Na gain and K l o s s induced by ouabain and to proceed f u r t h e r to i n v e s t i g a t e the e f f e c t of t h i s drug i n K - d e f i c i e n t s o l u t i o n s . 9. E f f e c t of DPH on the e l e c t r o l y t e content of r a t t a i l a r t e r i e s i n K- d e f i c i e n t s o l u t i o n s . In K - d e f i c i e n t s o l u t i o n s , one would expect an i n h i b i t i o n of the Na pump. Under t h i s condition r a t t a i l a r t e r i e s would gain Na and l o s e K. The experimental c o n d i t i o n would, therefore, simulate that r e s u l t i n g from the presence of ouabain. I f DPH acts by stimu-l a t i o n of the Na pump i n conditions where these e l e c t r o l y t e s are going along t h e i r electrochemical gradient, one would expect r e s u l t s i n K - d e f i c i e n t s o l u t i o n s , comparable to that obtained i n the presence of ouabain. Following the standard preincubation i n normal Krebs f o r 90 mins, one group of a r t e r i e s was tr a n s f e r r e d to a K - d e f i c i e n t s o l u t i o n con-t a i n i n g 0.2 mM DPH f o r a period of 2 hours. The other group of a r t e r i e s was incubated i n a K - d e f i c i e n t s o l u t i o n , containing the solvent of the drug, f o r a s i m i l a r period of time. Under these experimental conditions, DPH could counteract the K l o s s induced by incubation of the a r t e r i e s i n K - d e f i c i e n t s o l u t i o n s (Table 1 ) . The K content of c o n t r o l was 13.1 ± 0.1 compared to .../66 66 14.9 ± 0.2 i n DPH treated group. The d i f f e r e n c e i s s t a t i s t i c a l l y h ighly s i g n i f i c a n t (P < 0.001). As i n the case with the previous experiment, the e f f e c t on Na gain was much l e s s evident (39.46 ± 1.08 and 38.86 ± 1.17 i n c o n t r o l and DPH treated group r e s p e c t i v e -l y ) . I t i s , therefore, evident that DPH can counteract to some extent the e l e c t r o l y t e changes i n K - d e f i c i e n t s o l u t i o n s . Our r e -s u l t s under t h i s experimental cond i t i o n are comparable to those ob-tained i n the presence of ouabain (Table 1 ) . E f f e c t of DPH on the e l e c t r o l y t e content of r a t t a i l a r t e r i e s i n  K - d e f i c i e n t s o l u t i o n s at 1° C. In previous experiments i t was shown that DPH can counteract the K l o s s and Na gain of r a t t a i l a r t e r i e s incubated i n K - d e f i c i e n t s o l u t i o n s , and i n the presence of ouabain at 37° C. The r e s u l t s would suggest that the mechanism involves an energy dependent process, p o s s i -b l y through s t i m u l a t i o n of the Na pump. However, other studies i n crab nerves (Pincus, 1972) showed that DPH i n h i b i t e d the Na i n f l u x i n t h i s t i s s u e . I f the mechanism by which the s i z e a b l e e l e c t r o l y t e changes, described i n the present study, i s m e t a b o l i c a l l y dependent, then p r i o r cooling of the t i s s u e , which would damp down metabolic processes, would be expected to attenuate or a b o l i s h the a b i l i t y of DPH to act i n K-defi-c i e n t s o l u t i o n s . Rat t a i l a r t e r i e s were f i r s t incubated as usual i n normal Krebs s o l u t i o n at 37° C. f o r 90 mins. The a r t e r i e s were then t r a n s f e r r e d to a normal Krebs s o l u t i o n cooled to 1° C. A f t e r one hour e q u i l i b r a t i o n i n t h i s cooled s o l u t i o n , one group of a r t e r i e s was t r a n s f e r r e d to a K - d e f i c i e n t s o l u t i o n at 1° C , and was l e f t f o r 2 hours. This s o l u t i o n .../67 67 contained DPH l n a concentration of 0.2 mM. The other group of a r t e r i e s was tra n s f e r r e d to a s i m i l a r s o l u t i o n i n which the NaOH solvent was added to give the same solvent concentration as i n the s o l u t i o n of DPH, and was l e f t f o r a s i m i l a r period of time. In both groups, the temperature of the Krebs s o l u t i o n was kept constant at 1° C. throughout a l l the incubation period i n K - d e f i -c i e n t s o l u t i o n s . Cooling of the t i s s u e abolished the a b i l i t y of DPH to counter-act the e l e c t r o l y t e changes i n K - d e f i c i e n t s o l u t i o n s (Table 1 ) . The Na content i n c o n t r o l and drug treated groups were 43.66 ± 1.1 and 43.55 ± 1,22 and the K content 12.72 ± 0.53 and 12.60 ± 0.59 i n c o n t r o l and drug treated groups r e s p e c t i v e l y . The di f f e r e n c e s i n the Na and K content of both groups i s i n s i g n i f i c a n t (P > 0.05). This experiment gives, therefore, evidence that the mechanism by which DPH acts i n the presence of ouabain and i n K - d e f i c i e n t s o l u t i o n s i s m e t a b o l i c a l l y dependent. This r e s u l t , together with the f a c t that the K content of the DPH + ouabain and DPH i n K - d e f i c i e n t s o l u t i o n s was s i g n i f i c a n t l y higher than that of the c o n t r o l group treated with ouabain or i n K - d e f i c i e n t s o l u t i o n s , makes us conclude that DPH can stimulate the Na pump of r a t t a i l a r t e r i e s under experimental conditions favour-ing the downhill movements of Na and K along t h e i r electrochemical gradient. . . / 6 8 68 A l t e r a t i o n of Other Parameters of Na Transport and C o n t r a c t i l e A c t i v i t y  i n Vascular Smooth Muscles Since we had some evidence denoting that a c c e l a e r a t i o n of the pump a c t i v i t y might a f f e c t the c o n t r a c t i l e responses to drugs, we proceeded to evaluate drugs reported to a f f e c t d i f f e r e n t parameters of Na tra n s -port. 'The idea was to t e s t whether a l t e r a t i o n s of other parameters of the Na transport might a f f e c t c o n t r a c t i l e a c t i v i t y of vascu l a r smooth muscles. 1. E f f e c t of ouabain and ethacrynic a c i d on the r a b b i t a n t e r i o r mesen- t e r i c v e i n . Ouabain and ethacrynic a c i d are well-known i n h i b i t o r s of the Na-K ATPase (Wolowyk et a l . , 1971; Daniel et a l . , 1970). Ouabain was e s s e n t i a l l y reported to contract the r a b b i t A.M.V. followed by i t s r e l a x a t i o n (Matthews and Sutter, 1967). In t h i s study we have seen that the pattern of response i s somewhat dependent on the con-centration used. At concentrations of 5 x 10 ^ M ouabain, the rab-b i t a n t e r i o r mesenteric v e i n showed a weak co n t r a c t i o n followed by a r e l a x a t i o n . When higher concentration of 10 ^ or 5 x 10 ^ M were used, t h i s i n i t i a l phase was followed by severe contractures that l a s t e d f o r a period of 2 hours before the v e i n eventually relaxed again ( F i g . 14). -4 Ethacrynic a c i d was tested i n concentrations of 10 M and 1 mM. -4 At 10 M, ethacrynic a c i d r e s u l t e d i n a small c o n t r a c t i o n (about 100 mg) followed also by r e l a x a t i o n . The pattern of response was f a i r l y s i m i l a r to that induced by 5 x 10 ^ M ouabain. At higher concentrations (1 mM) the i n i t i a l small c o n t r a c t i o n and r e l a x a t i o n was soon followed .../69 F i g . 14 Response of the rabbit A.M.V. to 5 x 10~6 M and 10~5 M ouabain 70 by a severe contracture that l a s t e d f o r about 2 hours ( F i g . 15). The pattern of response was q u a l i t a t i v e l y f a i r l y s i m i l a r to that produced by 10 "* M ouabain. An important property of ethacrynic a c i d i s i t s t i g h t binding to receptor s i t e s which seems not to be a f f e c t e d even by homogeniza-t i o n i n ut e r i n e smooth muscles (Daniel et a l . , 1971). I f t h i s pattern of i r r e v e r s i b l e binding i s i n some way r e l a t e d to the c o n t r a c t i l e r e -sponses of ethacrynic a c i d , then one would expect l e s s of e f f e c t f o l l o w -ing repeated a p p l i c a t i o n of the drug. Indeed, when we retested etha-c r y n i c a c i d on the same preparation, there was hardly any response to the drug ( F i g . 16). 2. E f f e c t of amiloride on the c o n t r a c t i l e responses of NA i n vascular  smooth muscles. Several recent reports have appeared i n the l i t e r a t u r e denoting that the drug amiloride blocks the passive sodium i n f l u x i n c e l l s of a v a r i e t y of t i s s u e s (Salako and Smith, 1970a,b; Gatzy, 1971). I t would be, therefore, of i n t e r e s t to t e s t whether t h i s drug would have any e f f e c t on vascular smooth muscles. Three types of vascular smooth muscles were tested. The r a b b i t a n t e r i o r mesenteric v e i n as an example of a spontaneously a c t i v e preparation, possessing a c t i o n p o t e n t i a l s , the r a t t a i l a r t e r y as an example of a small a r t e r y which can be per-fused and r e s i s t a n c e through the v e s s e l measured, and a o r t i c s t r i p s i n which two d i s t i n c t phases on c o n t r a c t i l e responses to NA can be detected. A f a s t phase (phasic) presumably due to m o b i l i z a t i o n of bound Ca and I | a slow or t o n i c component dependent on the e x t r a c e l l u l a r Ca . In the r a b b i t a n t e r i o r mesenteric v e i n , concentrations i n between 0.1 to 0.2 mM amiloride d i d not have s i g n i f i c a n t e f f e c t on spontaneous a c t i v i t y of the preparation. However, the same concentration was able / 7 1 F i g . 15 ETHACRYNIC ACID IO4 M 50mg ETHACRYNIC ACID ia-3M 250mg A = Response of the rabbit A.M.V. to 10~ 4 M ethacrynic a c i d . B & C = Response of the rabbit A.M.V. to 10 M ethacrynic a c i d . Thick l i n e at the bottom of the f i g u r e denotes base l i n e . F i g . 16 B R 125mg ETHACRYNIC ACID IO"4 M JETHACRYIMIC / ACID |O"4M -4 A - Ethacrynic a c i d was applied to the r a b b i t A.M.V. at a concentration of 10 M and then washed (R). -4 B =» Ethacrynic a c i d was reapplied at the same concentration of 10 M to the same v e i n 73 to attenuate the c o n t r a c t i l e responses to 5 x 10 7 g/cc NA (32.5 ± 4.2%) ( F i g . 17) when l e f t f o r 5 mins before the a p p l i c a t i o n of the agonist. Perfusion of the r a t t a i l a r t e r y with 0.2 mM amiloride d i d not m a t e r i a l l y change the i n t r a l u m i n a l pressure of the perfused a r t e r y . The i n t r a l u m i n a l pressure was 51.8 ± 4.7 and 48.8 ± 6.8 mm Hg i n c o n t r o l and drug treated t i s s u e r e s p e c t i v e l y . The d i f f e r e n c e 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 (P > 0.05). However, when the a r t e r y was f i r s t perfused with 0.2 mM amiloride f o r 10 mins, followed by the same concentration of amiloride + 0.4 yg NA, there was almost complete i n h i b i t i o n of the c o n t r a c t i l e response of NA (94.5 ± 1.2%). In r a b b i t a o r t i c s t r i p s , p r i o r exposure of the t i s s u e to 0.2 mM amiloride f o r 15 mins, followed by adding a standard dose of 5 x 10 7 g/cc NA, g r e a t l y attenuated the response of the t i s s u e to the agonist compared to the c o n t r o l . A p e c u l i a r i t y i n t h i s i n h i b i t o r y e f f e c t of amiloride i s the almost s e l e c t i v e a c t i o n on the f a s t component of NA c o n t r a c t i o n . The l a t t e r had been almost completely abolished by p r i o r a p p l i c a t i o n of the drug ( F i g . 18). While no report i s yet a v a i l a b l e on the e f f e c t of amiloride on i o n i c fluxes i n smooth muscles, i t would be of great i n t e r e s t to proceed i n t o d e t a i l e d k i n e t i c studies of the e f f e c t of t h i s drug on Na and Ca transport. Our r e s u l t s suggest that i n a o r t i c s t r i p s amiloride I | more s e l e c t i v e l y a f f e c t s the a v a i l a b i l i t y of a bound Ca pool to the c o n t r a c t i l e p r o t e i n . The f a c t that t h i s drug can attenuate c o n t r a c t i l e responses to NA i n doses that do not s i g n i f i c a n t l y impair spontaneous a c t i v i t y i n the a n t e r i o r mesenteric v e i n may be taken i n favour that .../74 F i g . 17 E f f e c t of amiloride on c o n t r a c t i l e resposes of NA i n the r a b b i t A.M.V. 5 0 0 M G IMA / 5 X I Q - 7 G / C . C •i NA / A M I L O R I D E / 5X I0 - 5 G/CC 2 X I O - 4 M 1 2 Min. F i g . 18 E f f e c t of amiloride on c o n t r a c t i l e responses of NA i n ra b b i t a o r t i c s t r i p s 05G /NA fAMIL. / NA , 5X107G/CC 2xv>*M 5X10^G/CC -4 ON 76 spike a c t i v i t y i n t h i s t i s s u e i s mainly dependent on e x t r a c e l l u l a r Ca . Further e l e c t r o p h y s i o l o g i c a l studies and i o n i c fluxes measure-ments, comparing the a c t i o n of amiloride on spontaneously a c t i v e and quiescent type of smooth muscles seem to be a promising l i n e f o r f u r -ther i n v e s t i g a t i o n . I t i s a l s o suggested that t h i s drug might o f f e r a u s e f u l pharmacological t o o l to d i f f e r e n t i a t e the two Ca pools described i n a v a r i e t y of smooth muscle preparations. .../77 77 Na and the Relaxation of the Rabbit Anterior Mesenteric Vein The preceding s e c t i o n concentrated on various drugs a f f e c t i n g the Na transport i n respect to t h e i r e f f e c t on c o n t r a c t i l e a c t i v i t y and/or response to agonists i n a v a r i e t y of smooth muscle prepara-t i o n s , with p a r t i c u l a r reference to DPH. We proceeded i n t h i s sec-t i o n i n studying the r e l a x a t i o n of the r a b b i t a n t e r i o r mesenteric v e i n following c o n t r a c t i l e responses. 1. E f f e c t of Na ions on r e l a x a t i o n of the r a b b i t a n t e r i o r mesenter- i c v e i n . I t was already mentioned that a t e c h n i c a l d i f f i c u l t y arose while evaluating the e f f e c t of DPH i n low Na s o l u t i o n s . We could not go below 60 mM Na, L i s u b s t i t u t i o n , i n these experiments because the a n t e r i o r mesenteric v e i n went in t o contractures that could be arrested by r a i s i n g the Na i n the Krebs s o l u t i o n . In attempts to t e s t the i n h i b i t o r y e f f e c t of DPH i n Na-free s o l u t i o n s , T r i s - H C l s u b s t i t u t e d , the r a b b i t a n t e r i o r mesenteric v e i n went i n t o severe contractures that would promptly stop following the r e t u r n of the Na to the Krebs s o l u t i o n . The r e l a x a t i o n of the r a b b i t a n t e r i o r mesenteric v e i n , f o l l o w -ing c o n t r a c t i l e responses to NA, was a l s o delayed i n low Na Krebs. In 25 mM Na (Tris-HCl s u b s t i t u t e d Krebs), the c o n t r a c t i l e response to NA was potentiated together with a delay i n the r e l a x a t i o n f o l l o w -ing washing of the agonist ( F i g . 19). In 60 mM Na ( L i substituted Krebs), the i n i t i a l p o t e n t i a t i o n of NA responses wore o f f gradually a f t e r a few a p p l i c a t i o n s of DPH. F i g . 19 5 M g 2.5MILM. R t N A 3 .2X10 -8G^ .C . -8 C o n t r a c t i l e response of the ra b b i t A.M.V. to a concentration of 3.2 x 10 g/cc NA i n normal Krebs s o l u t i o n . C o n t r a c t i l e response of the rabbit A.M.V. to the same concentration of NA i n 25 nM Na Krebs (Tris-HCl s u b s t i t u t e d ) . 79 However, the r e l a x a t i o n following washing of the agonist was s t i l l prolonged at a time when no p o t e n t i a t i o n of NA contractions was detected ( F i g . 20). We further quantitated the r e l a x a t i o n time following c o n t r a c t i l e responses of NA i n 60 mM Na, L i s u b s t i t u t i o n , at a time when no p o t e n t i a t i o n of NA contractions was detected. While the ^f2 ^ 0 r a d o s e °^ ^.2 x g/cc NA was 1.48 ± 0.3 sees i n normal Krebs, i t was r a i s e d to 6.72 ± 0.4 i n 60 mM Na, L i sub-s t i t u t e d Krebs. The f a c t that the r e l a x a t i o n of the ra b b i t a n t e r i o r mesenteric v e i n , following Na contractions, i s delayed i n low Na so l u t i o n s , and that the contractures occurring i n such s o l u t i o n s could not be stopped, except by r a i s i n g the Na i n the bathing f l u i d , gives evidence of the importance of e x t r a c e l l u l a r Na i n the mechanism of r e l a x a t i o n of the a n t e r i o r mesenteric v e i n following c o n t r a c t i l e responses. E f f e c t of DPH on r e l a x a t i o n following ouabain, procaine, and NA  contractions. Ouabain was used i n a concentration of 10"""* M and 5 x IO-"* M, When the r a b b i t a n t e r i o r mesenteric v e i n reached the peak of the i n i t i a l c o n t r a c t i o n , DPH was immediately added i n a concentration -4 of 10 M. A c o n t r o l response of the same dose of ouabain was used i n order to compare the response i n the presence and absence of DPH. Immediately following the a d d i t i o n of DPH, there was a rapid r e l a x a -t i o n of the a n t e r i o r mesenteric v e i n ( F i g . 21). The r e s u l t was a c l e a r cut one. _3 A s i m i l a r procedure was used, using a dose of 10 g/cc procaine -4 as the agonist. The a d d i t i o n of 10 M DPH on the top of procaine con-t r a c t i o n s r e s u l t e d i n a prompt r e l a x a t i o n of the ra b b i t a n t e r i o r ./81 LEGEND TO FIG. 20 C o n t r a c t i l e response of the rab b i t A.M.V. to a concen-—8 t r a t i o n of 3.2 x 10 g/cc NA i n normal Krebs. C o n t r a c t i l e response of the same v e i n to the same con-cen t r a t i o n of NA a f t e r r e - e q u i l i b r a t i o n with 60 mM Na Krebs ( L i s u b s t i t u t i o n ) . C o n t r a c t i l e response of the same v e i n to the same concen-t r a t i o n of NA following a few a p p l i c a t i o n of DPH i n 60 mM Na ( L i s u b s t i t u t e d ) . Response i s taken i n the absence of DPH from the incubation medium. F i g . 21 r e l a x a t i o n of the rabbi t A.M.V. following ouabain c o n t r a c t i o n OUABAIN 5XICT5M (1 Jll DPH IO-4M' ' /R 2 Min. /OUABAIN 5XIO sM 83 mesenteric v e i n ( F i g . 22).' The r e s u l t was also a c l e a r cut one. A s i m i l a r procedure was used to t e s t the e f f e c t of DPH on NA c o n t r a c t i o n s . With t h i s methodology i t was d i f f i c u l t to assess the a c t i o n of DPH since the r e s u l t was not a c l e a r cut one. The a d d i t i o n of 1 mM ascorbic a c i d to the Krebs s o l u t i o n , to prevent the auto-oxidation of NA, could not s l v e t h i s problem. For t h i s reason, we proceeded i n more s e n s i t i v e methods to quantitate the time needed f o r r e l a x a t i o n of the a n t e r i o r mesenteric v e i n f o l l o w -ing c o n t r a c t i l e responses to NA. Two groups of experiments were designed. Group I. The v e i n was f i r s t placed i n Krebs containing —8 5 x 10 g/cc phentolamine and the w a s measured, using a standard dose of 10 ^ g/cc NA as the agonist. Phentolamine was used as a s p e c i f i c antagonist to NA to allow manipulations of doses of agonists and antagonists that would give the same peak response following the a p p l i c a t i o n of NA (see below). The same preparation -4 was then placed i n a Krebs containing 10 M DPH and the same agonist dose was used to obtain the ^fl' T ^ i e P e a ^ °^ a g ° n i s t r e -—8 sponse obtained i n the presence of 5 x 10 g/cc phentolamine and -4 10 M DPH was nearly the same. In each case the t ^ 2 . w a s measured as the time required f o r the v e i n to reach h a l f the maximal tension, s t a r t i n g the timing immediately a f t e r the agonist has been washed from the bathing f l u i d . While the c o n t r o l using t h i s experimental design, was 19.5 ± 2.2 sees, i t was 27 ± 4.9 sees i n the DPH treated group (Table 2). Therefore, i f there i s any e f f e c t of DPH on r e l a x a t i o n F i g . 22 85 TABLE 2 No. of group t l / 2 *"n c o n t r 0 - ' - 8 r o u P ^1/2 i n D P H treated group I (4) 19.5 ± 2.2 27.1 ± 4.9 II (4) 13.2 ± 0.9 13.9 ± 0.63 N.B. Number In brackets i n d i c a t e the number of experiments. ../86 86 following NA contractions, i t i s a c t u a l l y a d e c e l e r a t i o n rather than a c c e l e r a t i o n while using t h i s experimental design, but t h i s 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 (P > 0.05). Group I I . Half the v e i n was bathed i n a Krebs containing —8 5 x 10 g/cc phentolamine and served as a c o n t r o l . The other h a l f of the bisec t e d v e s s e l was bathed i n a Krebs s o l u t i o n con--4 t a i n i n g 10 M DPH. NA was used as an agonist i n a concentra-t i o n of 5 x 10 7 g/cc and 2 x 10 7 g/cc f o r the v e i n bathed i n the Krebs containing phentolamine and DPH r e s p e c t i v e l y . The agon-i s t gave approximately the same peak of response i n both cases. The t ^ y 2 *-n both c o n t r o l and DPH treated t i s s u e was obtained as previously described. While the c o n t r o l t i s s u e gave a °^ 13.2 ± 0.9 sees, the DPH treated t i s s u e had a t ^ 2 of 13.9 ± 0.63 sees (Table 2). From both groups of experiments, i t was then concluded that the r e l a x a t i o n of the r a b b i t a n t e r i o r mesenteric, following NA contractious, i s not accelerated by DPH. I f there i s any e f f e c t , i t Is an increase rather than a decrease i n the t^/2* These r e s u l t s d i f f e r , therefore, from those obtained when ouabain or procaine was used to induce a c o n t r a c t i l e e f f e c t i n the r a b b i t a n t e r i o r mesenteric v e i n . The present study shows the complexity of the phenomena i n -volved i n the r e l a x a t i o n of the r a b b i t a n t e r i o r mesenteric v e i n following d i f f e r e n t agonist responses. I t i s reasonable to sug-gest that NA can make a v a i l a b l e to the c o n t r a c t i l e p r o t e i n addi-I | t i o n a l Ca which cannot be mobilized by e i t h e r procaine or oua-bain, and which i s not a f f e c t e d by DPH treatment. Substantiating . . . / 8 7 8 7 t h i s assumption i s the f a c t that procaine cannot contract the a n t e r i o r mesenteric v e i n i n the presence of ouabain, while the c o n t r a c t i l e responses of NA i n the presence of ouabain are not af f e c t e d by DPH. .../88 88 The I n h i b i t o r s of the ATPase and U l t r a s t r u c t u r e of the Rabbit  An t e r i o r Mesenteric Vein The proper understanding of mechanical and e l e c t r i c a l phenomena i n vascular smooth muscles implies a better understanding of i t s u l t r a s t r u c t u r e c h a r a c t e r i s t i c s . Smooth muscles show d i s t i n c t features not shared by other types of muscles. Perhaps, the most important i s the presence of abundance of plasmalemmal v e s i c l e s and the generally l e s s elaborate sarcoplasmic reticulum when compared to s k e l e t a l and cardiac muscles. In view of the complexity of the Na transport i n smooth muscles, the l o c a l i z a t i o n of a high ATPase a c t i v i t y i n the v i c i n i t y of plasma-lemmal v e s i c l e s , and the s e v e r a l claims i m p l i c a t i n g these v e s i c l e s i n ( a v a r i e t y of functions, i t was decided to s t a r t a preliminary e l e c t r o n microscopic study to evaluate the e f f e c t of the i n h i b i t o r s of the ATPase on u l t r a s t r u c t u r e of the a n t e r i o r mesenteric v e i n . Ethacrynic a c i d and ouabain were selected as two d i f f e r e n t i n h i b i t o r s of the ATPase. 1. The e f f e c t of ethacrynic a c i d on u l t r a s t r u c t u r e of r a b b i t a n t e r i o r  mesenteric v e i n . Each a n t e r i o r mesenteric v e i n used i n these experiments was b i s e c t e d l o n g i t u d i n a l l y . Following the usual incubation of the preparation i n normal Krebs s o l u t i o n f o r 45 mins, one h a l f of the v e i n was exposed to 1 mM ethacrynic a c i d f o r 2 hours. The other h a l f of the bisected v e i n was l e f t f o r a s i m i l a r period of time i n normal Krebs s o l u t i o n . Both v e i n s t r i p s were subjected to the same tension of 500 mg. Longitudinal sections were prepared i n both c o n t r o l and drug treated t i s s u e following the same standard procedure previously described. 89 Examination of the ethacrynic a c i d treated t i s s u e showed a d i s r u p t i o n of the m y o f i b r i l l a r a r c h i t e c t u r e . The spaces between the m y o f i b r i l s were increased. There was a d i s r u p t i o n of the s a r -colemma and mitochondrial membranes at some s i t e s . The whole ar c h i t e c t u r e of the t i s s u e appeared as i f i t was oedematous and swollen ( F i g s . 23, 24). An outstanding feature r e s u l t i n g from t h i s treatment i s the dramatic diminution i n the number of plasmalemma v e s i c l e s when compared to the c o n t r o l h a l f , the former had few v e s i c l e s l e f t . A c a r e f u l examination of the c o n t r o l t i s s u e , that was l e f t Incubated i n normal Krebs s o l u t i o n showed a normal a r c h i -tecture with an abundance of plasmalemma v e s i c l e s ( F i g . 25). The e f f e c t of ouabain on u l t r a s t r u c t u r e of the r a b b i t a n t e r i o r  mesenteric v e i n . A s i m i l a r procedure as described i n the preceding ethacrynic a c i d experiment was followed. The t i s s u e was exposed to 10 ^ M ouabain f o r a period of 2 hours. Ouabain treated t i s s u e showed a r c h i t e c t u r a l changes compar-able to that of the ethacrynic a c i d treated v e s s e l i n some, but not a l l , f i b e r s . There was a great v a r i a b i l i t y i n the magnitude of changes detected from one block to another and from one f i e l d to the other ( F i g s . 26, 27). A c a r e f u l examination of the c o n t r o l t i s -sue showed a normal a r c h i t e c t u r e ( F i g . 28). The dramatic diminution i n the number of plasmalemma v e s i c l e s following treatment with ethacrynic a c i d r a i s e s the p o s s i b i l i t y that the formation and maintenance of f u n c t i o n of these v e s i c l e s might be an energy dependent process r e q u i r i n g an i n t a c t ATPase system. F i g . 23 F i g . 24 Longitudinal s e c t i o n i n the rabbit a n t e r i o r mesenteric vein following Incubation i n Krebs Fig. 25 Longitudinal s e c t i o n of c o n t r o l r a b b i t a n t e r i o r mesenteric v e i n incubated i n normal Krebs s o l u t i o n F i g . 26 Longitudinal s e c t i o n i n the r a b b i t a n t e r i o r mesenteric v e i n following incubation i n Krebs containing 10 5 M ouabain f o r 2 h r s . F i g . 27 Longitudinal s e c t i o n i n the r a b b i t a n t e r i o r mesenteric vein following incubation i n Krebs s o l u t i o n containing 10 5 M ouabain f o r 2 h r s . 4> F i g . 28 Longitudinal s e c t i o n os c o n t r o l r a b b i t a n t e r i o r mesenteric vein incubated i n normal Krebs s o l u t i o n 96 I t a l s o opens the question whether these v e s i c l e s might be i n some way involved i n the complexity of the Na transport i n smooth muscles. However, i t i s premature to draw conclusions at the pre-sent stage of study i n view of the severe s t r u c t u r a l changes r e s u l t -ing from such treatment. Quantitative studies, t r y i n g to d i s s i m i n -ate s t r u c t u r a l changes i n the m y o f i b r i l s and membrane from v a r i a t i o n s i n the number of v e s i c l e s , seem to be promising i n o f f e r i n g an u l t r a -s t r u c t u r a l basis of i o n i c transport i n smooth muscles. ,../97 97 DISCUSSION In the previous sections we have discussed some aspects of Na-Ca I | i n t e r a c t i o n . In i n t e r p r e t i n g our r e s u l t s , i t i s considered that Ca i s e s s e n t i a l f o r the a c t i v a t i o n of the c o n t r a c t i l e machinery and that whenever a l t e r a t i o n i n sodium transport a l t e r s the c o n t r a c t i l e responses of smooth muscles, i t should u l t i m a t e l y be c o r r e l a t e d with an a l t e r a t i o n of the amount of i o n i z e d Ca a v a i l a b l e to the c o n t r a c t i l e p r o t e i n . The bulk of the c o n t r a c t i l e studies have been performed, using the r a b b i t a n t e r i o r mesenteric v e i n , and i n t e r p r e t a t i o n of our r e s u l t s i s based on the properties of t h i s preparation and cannot be generalized to other types of smooth muscles. Smooth muscles show wide v a r i a t i o n s i n response to drugs and these i n d i v i d u a l i t i e s should be respected when fi n d i n g s are presented f o r a p a r t i c u l a r type of smooth muscle. Further ela b o r a t i o n on t h i s subject w i l l be presented i n appropriate s i t e s . Several points p e r t a i n i n g to our studies have been s u f f i c i e n t l y d i s -cussed i n the s e c t i o n of the RESULTS f o r the sake of c o n t i n u i t y and l o g i c a l progression of the i n v e s t i g a t i o n . Some of the important f i n d i n g s are d i s -cussed i n more d e t a i l i n t h i s s e c t i o n . Diphenylhydantoin Sodium and Sodium Transport Our e l e c t r o l y t e studies, using r a t t a i l a r t e r i e s , have shown that, i n normal Krebs s o l u t i o n , diphenylhydantoin sodium di d not a f f e c t s i g n i -f i c a n t l y the Na content of r a t t a i l a r t e r i e s . I f there has been any e f f e c t , i t was indeed a s l i g h t diminution of the K content of drug treated a r t e r i e s . I t cannot be excluded, however, that a small v a r i a t i o n i n the .../98 98 i n t r a c e l l u l a r sodium might have been masked by the f a c t that most of the sodium i s e x t r a c e l l u l a r or bound to the p a r a c e l l u l a r matrix. V a r i a t i o n s i n the volume of the e x t r a c e l l u l a r space and the l i m i t a t i o n of the s e n s i t i v i t y of our method i n face of the great heterogeneity of the vascular w a l l are points to be taken i n t o account i n such kinds of study. However, a s l i g h t diminution i n the K content of r a t t a i l a r t e r -i e s under t h i s c o n d i t i o n i s not compatible with the idea that DPH stimu-l a t e d the Na pump i n normal Krebs. Our r e s u l t s at t h i s point are s t i l l compatible with the fi n d i n g s of Pincus (1970) who showed that DPH d i d not s i g n i f i c a n t l y a f f e c t the e l e c t r o -l y t e content of l o b s t e r nerves and are comparable with those found by Watson and Woodbury i n guinea p i g car d i a c muscles (Woodbury and Kemp, 1971). The s l i g h t diminution of the K content i n normal Krebs i s more suggestive that DPH might have an i n h i b i t o r y e f f e c t on the sodium pump under such experimental c o n d i t i o n . This may be taken i n p a r a l l e l with the biochemical studies showing that DPH had an i n h i b i t o r y e f f e c t on the ATPase i n conditions of low Na-K r a t i o s i n the incubating media (Pincus and Giarman, 1967; Rawson and Pincus, 1968; F e s t o f f and Appel, 1968; Formby, 1969). When DPH was incubated with ouabain, the drug d i s t i n c t l y antagonized the K l o s s of r a t t a i l a r t e r i e s while presumably, f o r the same reasons already discussed, the e f f e c t on sodium gain was not prominent. The antagonism between DPH and ouabain i s s t r i k i n g and has been already described i n l o b s t e r nerves (Pincus, 1970), i n cardiac muscles (Woodbury and Kemp, 1971), on i n s u l i n release of pancreatic i s l e t s (Kizer et a l . , 1970), and on the ouabain-induced i n h i b i t i o n of s e c r e t i o n of eccrine glands (Sato et a l . , 1969). I t i s al s o evidenced c l i n i c a l l y by the sa l u t o r y e f f e c t of DPH on d i g i t a l i s induced arrhythmias (Conn, 1965; Rosen et a l . , 1967; .../99 99 Dreifus and Watanabe, 1970). That t h i s antagonism i s not s p e c i f i c to ouabain, but rather dependent on a condit i o n where the drug can a c t i -vate the sodium pump i n s i t u a t i o n s favouring the downhill movement of Na and K, i s furth e r substantiated by our studies which tested the e f f e c t of DPH i n K - d e f i c i e n t s o l u t i o n s . In these s o l u t i o n s the r e -s u l t s obtained were comparable to those found i n the presence of oua-ba i n . Under t h i s experimental c o n d i t i o n , DPH can s t i l l d i s t i n c t l y antagonize the K loss of r a t t a i l a r t e r i e s . Our r e s u l t s a t t h i s point are compatible with those of Escueta and Appel (1971) who found that DPH can enhance the K uptake of synapto-somes i n media containing a high Na-K r a t i o and i n the presence of oua-bain. Our r e s u l t s might a l s o be taken i n p a r a l l e l with the biochemical observations denoting that t h i s drug can stimulate the Na-K ATPase under conditions of a high Na-K r a t i o i n the incubating media (Festoff and Appel, 1968; Lewin and Bleck, 1971; Spain and Chidsey, 1971; S i e g e l and Goodwin, 1972). Further evidence that t h i s e f f e c t i s m e t a b o l i c a l l y dependent and r e l a t e d to s t i m u l a t i o n of the sodium pump i s provided by the i n a b i l i t y of DPH to counteract the e l e c t r o l y t e changes i n K - d e f i c i e n t s o l u t i o n s at 1° C. I t i s , therefore, concluded that i n r a t t a i l a r t e r i e s there are s p e c i a l requirements f o r DPH to be able to stimulate the sodium pump, i . e . , conditions favouring the downhill movements of Na and K and/or conditions simulating the d e p o l i a r i z e d s t a t e . In normal Krebs, DPH does not seem to stimulate the sodium pump. I f there has been any e f f e c t under the l a t t e r experimental c o n d i t i o n , i t i s more suggestive of an i n h i b i t i o n of the ATPase. 100 Obviously, our technique cannot exclude that DPH can i n h i b i t the sodium i n f l u x as was demonstrated i n l o b s t e r nerves (Pincus, 1972). However, i n view of the d i s t i n c t e f f e c t of DPH i n counteracting the K lo s s of r a t t a i l a r t e r i e s i n the presence of ouabain and i n K - d e f i c i e n t s o l u t i o n s , and the l a c k of the l a t t e r e f f e c t i n cooled t i s s u e , i t i s u n l i k e l y that our si z e a b l e e l e c t r o l y t e changes are the r e s u l t of a blocking e f f e c t on the passive sodium i n f l u x . DPH and C o n t r a c t i l e Responses to Drugs In the r a b b i t a n t e r i o r mesenteric v e i n we found DPH to be able to antagonize the c o n t r a c t i l e responses to NA. p-hydroxylation of one of the phenolic r i n g s , which i s accompanied by l o s s of the an t i c o n v u l -sant e f f e c t of the drug and i t s a b i l i t y to i n h i b i t i n s u l i n release from the pancreas (Butler, 1957; K i z e r e t _ a l . , 1970), r e s u l t e d a l s o i n the l o s s of the i n h i b i t o r y e f f e c t on NA responses. I t seems, therefore, that the m o d i f i c a t i o n of c o n t r a c t i l e responses to NA i s s p e c i f i c to the struc t u r e of DPH and bears i n t h i s respect some resemblance to the a n t i -convulsant and i n h i b i t o r y e f f e c t of t h i s drug on i n s u l i n release from the pancreas. P r i o r i n h i b i t i o n of the Na pump by e i t h e r ouabain or K-free s o l u t i o n s was al s o accompanied by a l o s s of the i n h i b i t o r y e f f e c t of DPH on NA contractions and t h i s was r e v e r s i b l e by leaving the preparation to recover i n normal Krebs. I t seemed, therefore, that an i n t a c t sodium pump was necessary f o r the i n h i b i t o r y e f f e c t of the drug. Our e l e c t r o l y t e studies i n r a t t a i l a r t e r i e s showed c l e a r l y that DPH can stimulate the sodium pump at l e a s t under conditions favouring the downhill movements of Na and K i o n s . The i n a b i l i t y of DPH to modify con-t r a c t i l e responses of NA i n the presence of ouabain and i n K-free s o l u t i o n s .../101 101 can be c o r r e l a t e d , therefore, with the lack of st i m u l a t i o n of t h i s pump under such experimental conditions. We made a furth e r assumption. I f DPH was to exert i t s stimula-tory e f f e c t by stimu l a t i o n of the sodium pump, there might be some form of competition between sodium and calcium ions at some anionic binding s i t e s . Evidence f o r the l a t t e r was provided by the f i n d i n g that high calcium and low sodium antagonized the i n h i b i t o r y e f f e c t of the drug while low calcium had the reverse e f f e c t . The second question now to answer i s : how sti m u l a t i o n of the sodium pump can a f f e c t the Ca a v a i l a b l e to the c o n t r a c t i l e protein? In t r y i n g to set up explanations f o r our r e s u l t s we should s t r e s s at t h i s point that the c o n t r a c t i l e studies were obtained from the r a b b i t a n t e r i o r mesenteric v e i n and t h i s does not mean that our explanations can be generalized to other preparations. The f i r s t p o s s i b i l i t y i s that DPH can stimulate the sodium pump and make i t e l e c t r o g e n i c . I f t h i s drug were to acc e l e r a t e the pumping of more sodium ions uncoupled with K ions, one would expect s t a b i l i z a -t i o n of the membrane and l e s s e f f e c t of an agonist response. Evidence has been provided i n the introductory note that an ele c t r o g e n i c sodium pump may contribute to the r e s t i n g p o t e n t i a l of smooth muscles and may be stimulated under a v a r i e t y of conditions favouring a high i n t r a c e l l u -l a r sodium (Thomas, 1972). I n d i r e c t evidence was a l s o provided i n smooth muscle of spleen capsule denoting that the pumping might be electrogenic i n conditions favouring the downhill movement of sodium and potassium (Bose and Innes, 1972). The unstable membrane p o t e n t i a l of spontaneously a c t i v e preparations might favour a c o n d i t i o n where DPH can stimulate the sodium pump and make i t e l e c t r o g e n i c . 102 In view of the f a c t that e l e c t r o p h y s i o l o g i c a l studies concerning the a c t i o n of t h i s drug on the membrane p o t e n t i a l of the r a b b i t a n t e r i o r mesenteric v e i n are s t i l l e n t i r e l y l a c k i n g , one cannot exclude at the present stage of the study that a part of the calcium a v a i l a b l e to the c o n t r a c t i l e p r o t e i n might be governed by the l e v e l of the i n t r a c e l l u l a r sodium and/or potassium. I t was already pointed out i n the introductory note that the responsiveness of the guinea pig taenia c o l i seems to be dependent i n part on the l e v e l of i n t r a c e l l u l a r sodium and potassium (Axelson and Holmberg, 1971), but there seems to be c r i t i c a l l e v e l s of c e l l u l a r sodium and potassium to obtain an optimum c o n t r a c t i l e response (Burks et a l , , 1971), In a recent study (Harris and Palmer, 1972), i t was shown that the r e a c t i v i t y of smooth muscles may depend, at l e a s t w i t h i n a c e r t a i n range, on the l e v e l of the sodium content of the prepara-t i o n . I t would be of i n t e r e s t to study the e f f e c t of DPH on membrane poten-t i a l i n a spontaneously a c t i v e preparation, l i k e the r a b b i t a n t e r i o r mesenteric v e i n , by means of i n t r a c e l l u l a r recording techniques and the double sucrose gap method. I f h y p e r p o l a r i z a t i o n occurs, i t would be of i n t e r e s t to t e s t the e f f e c t of ouabain and K-free s o l u t i o n s on the hyper-p o l a r i z a t i o n e f f e c t . U n t i l such records are a v a i l a b l e , i t i s reasonable to say that a mechanism by which DPH can attenuate c o n t r a c t i l e responses to agonists independent of e l e c t r i c a l phenomena and governed by the a l t e r a t i o n s i n the c e l l u l a r l e v e l of Na and/or K has not been excluded. The demonstration of an e f f e c t of DPH on smooth muscle and i n s u l i n release from the pancreas (Kizer et a l . , 1970)which are e v i d e n t l y calcium dependent systems and the p o s s i b l e r e l a t i o n of t h i s e f f e c t to the a n t i -convulsant a c t i v i t y of the drug as evidenced by the l a c k of e f f e c t of 1 0 3 DPHOH on these systems, r a i s e s the p o s s i b i l i t y that the anticonvulsant e f f e c t of the drug may be due i n part to an a l t e r a t i o n of the Ca transport. I t i s pertinent to note at t h i s point that the phenomenon of post t e t a n i c p o t e n t i a t i o n , which i s s p e c i f i c a l l y blocked by t h i s drug ( E s p l i n , 1957; Raines and Standaert, 1966), appears to be the r e s u l t of an increased transmitter release from presynaptic terminals and i t seems that t h i s release i s l i k e l y caused by the movement of c a l -cium i n t o the presynaptic area during r e p e t i t i v e s t i m u l a t i o n (Rosenthal, 1969). The r o l e of calcium ions i n the mechanism of anticonvulsant a c t i o n of DPH might have been underestimated. The I n h i b i t o r s of the ATPase and C o n t r a c t i l e A c t i v i t y of the Rabbit  A n t e r i o r Mesenteric Vein We proceeded f u r t h e r to evaluate the c o n t r a c t i l e responses of oua-bain and ethacrynic a c i d . The pattern of response of both drugs was remarkably s i m i l a r . Both drugs induced a c o n t r a c t i l e response followed by r e l a x a t i o n i n low doses and the l a t t e r was followed by secondary con-tractures i n higher doses. Davis (1970) using guinea p i g kidneys and Daniel et a l . (1971) using r a t u t e r i found an i r r e v e r s i b l e i n t e r a c t i o n between ethacrynic a c i d and i t s receptor s i t e . In the l a t t e r t i s s u e the drug remained bound a f t e r washing and homogenization (Daniel et a l . , 1971). I f the c o n t r a c t i l e response induced by ethacrynic a c i d i s p r i m a r i l y r e l a t e d to i n h i b i t i o n of the ATPase, one would expect l o s s of the p a r t i c u l a r e f f e c t of ethacrynic e f f e c t following repeated a p p l i c a t i o n of the drug, i n view of the apparent i r r e v e r s i b l e i n t e r a c t i o n between ethacrynic a c i d and the ATPase. Indeed, when ethacrynic a c i d was r e t e s t e d i n the same preparation a f t e r washing, there was hardly any response to the drug again. .../10A 104 Obviously, the remarkable s i m i l a r i t y between the pattern of r e -sponse induced by ouabain and ethacrynic a c i d and the l o s s of e f f e c t occurring with the l a t t e r c o n s t i t u t e no more than i n d i r e c t evidence that the c o n t r a c t i l e response to these drugs i s mediated p r i m a r i l y through i n h i b i t i o n of the sodium pump, Bohr et_al.(1969) suggested that the c o n t r a c t i l e response to ouabain may be r e l a t e d to a stimulated coupling between sodium e f f l u x and calcium i n f l u x r e s u l t i n g from the r a i s e d i n t r a c e l l u l a r sodium. U n t i l i o n i c fluxes are a v a i l a b l e to analyze the e l e c t r o l y t e changes occurring concommitant with the c o n t r a c t i o n -r e l a x a t i o n phase, i t i s too speculative to r e l a t e such responses to a l t e r a t i o n i n i n t r a c e l l u l a r sodium. However, these r e s u l t s s t i l l f i t ++ the assumption that part of the Ca a v a i l a b l e to the c o n t r a c t i l e p r o t e i n may be governed by the c e l l u l a r l e v e l s of sodium and/or potassium. Amiloride and C o n t r a c t i l e Responses to NA In view of the reported e f f e c t that amiloride can block the passive sodium i n f l u x i n a v a r i e t y of ti s s u e s (Ddrde and Nagel, 1970; Salako and Smith 1970a,b; Gatzy, 1971), we proceeded i n preliminary experiments to t e s t the e f f e c t of t h i s drug on NA responses. Two points then emerged: 1. Amiloride can a f f e c t the c o n t r a c t i l e responses to. NA i n concentrations that do not s i g n i f i c a n t l y a l t e r the spontaneous a c t i v i t y of the r a b b i t a n t e r i o r mesenteric v e i n . 2. The drug almost s p e c i f i c a l l y i n h i b i t e d the f a s t phase of the NA response i n the r a b b i t a o r t a . Bohr (1963) and Godfraind and Kaba (1969) provided evidence that the f a s t phase of NA i n a o r t i c s t r i p s i s dependent on a bound Ca while the slow or t o n i c component i s dependent on e x t r a c e l l u l a r calcium. Our r e s u l t s suggest, 105 therefore, that amiloride a f f e c t p r i m a r i l y the a v a i l a b i i l i t y of a bound calcium store to the c o n t r a c t i l e p r o t e i n , whether t h i s a c t i o n could be r e l a t e d to an i n h i b i t i o n of the passive sodium i n f l u x could not be answered at the present stage. Further i o n i c f l u x studies of sodium and calcum combined with e l e c t r o p h y s i o l o g i c a l studies of the e f f e c t of t h i s drug i n graded d e p o l a r i z a t i o n and a c t i o n p o t e n t i a l pro-ducing preparations seem to be a f r u i t f u l route f o r f u r t h e r i n v e s t i -gation. Cuthbert and Wong (1971) suggested that amiloride can block the entry of sodium v i a the transport system i n toad bladder epithelium by forming part of a ternary complex with calcium and the receptor. I t i s p o s s i b l e that the i n h i b i t o r y a c t i o n of amiloride on NA responses i s r e l a t e d to i t s t r a n s l o c a t i o n i n some binding s i t e s which can be occupied by calcium. I n d i r e c t evidence f o r t h i s i s our f i n d i n g that the i n h i b i t o r y e f f e c t was not completely reversed by washing. However, i f NA was reapplied and the t i s s u e then washed the c o n t r a c t i l e response of NA returned to i t s c o n t r o l l e v e l . Sodium and the Relaxation of the Rabbit A n t e r i o r Mesenteric Vein The diminution of the sodium i n the incubating Krebs s o l u t i o n r e -s u l t e d i n a contracture of the r a b b i t a n t e r i o r mesenteric v e i n . This contracture was obtained i r r e s p e c t i v e of whether the s u b s t i t u t e was L i or T r i s - H C l , and could be promptly stopped by r a i s i n g the sodium i n the Krebs s o l u t i o n . Lithium was more e f f e c t i v e than T r i s - H C l i n inducing such c o n t r a c t i l e responses, inasmuch as t h i s phenomenon i n t e r f e r e d with our evaluation of the i n h i b i t o r y a c t i o n of DPH i n media containing lower than 60 mM Na L i s u b s t i t u t e d Krebs, but was no problem i n 25 mM Na T r i s - H C l s u b s t i t u t e d Krebs. Bohr et al.(1969) suggested that t h i s e f f e c t f i t s the 106 assumption that a sodium-calcium exchange e x i s t s i n smooth muscles, i f one assumes that the i n f l u x system carrying Na can be occupied by Ca. I t i s s t i l l s p e c u lative to take t h i s f i n d i n g as favouring the existence of a Na-Ca exchange i n smooth muscles. An i n t e r a c t i o n of various cations on p o t e n t i a l binding s i t e s i n smooth muscles has been repeatedly described (Goodford, 1966) and was l o c a l i z e d on anionic s i t e s of membranes of smooth muscles (Wolowyk, 1971; Goodford and Wolowyk, 1971 and 1972). The f i n d i n g s can be equally explained i f one assumes a competition of various cations on these s i t e s and by which a diminution i n the concentration of Na can f a c i l i t a t e the a c t i o n of calcium. There was both a p o t e n t i a t i o n and a delay i n the r e l a x a t i o n of the r a b b i t a n t e r i o r mesenteric v e i n i n low sodium, T r i s - H C l s u b s t i t u t e d Krebs. In low sodium, L i s u b s t i t u t e d , the p o t e n t i a t i o n wore o f f gradually while proceeding our experiment u n t i l the c o n t r a c t i l e responses of NA gave a consistent r e s u l t . There are s e v e r a l points to be taken i n t o account when studying the e f f e c t of low sodium media on c o n t r a c t i l e responses (Bohr, 1964). However, i t i s generally believed that, i f a p e r s i s t e n t r e s u l t s t i l l occurs with d i f f e r e n t sodium s u b s t i t u t e s , then a p o s s i b l e experimental a r t i f a c t caused by the s u b s t i t u t e may be excluded. In our experiments with l i t h i u m s u b s t i t u t e d Krebs, there was frequently a continuous d e t e r i o r a t i o n i n the c o n t r a c t i l e responses to NA when tested i n media lower than 40 mM Na. I t cannot be excluded, however, that t h i s e f f e c t might have been i n part due to a t o x i c e f f e c t of l i t h i u m on the preparation. Lithium accumulates i n the c e l l and i s not pumped e f f e c t i v e l y by the sodium pump (Schou, 1957). I t i s p o s s i b l e that the d e t e r i o r a t i o n of the preparation /107 107 i n low Na media ( l i t h i u m substituted) i n part r e s u l t s from a c e l l u l a r t o x i c e f f e c t of the l i t h i u m ions. In 60 mM Na ( l i t h i u m substituted) t e s t i n g the e f f e c t of DPH showed that there was s t i l l a prolongation of the r e l a x a t i o n time a f t e r NA responses at a time when no p o t e n t i a -t i o n of these c o n t r a c t i l e responses could be detected. This suggests that e x t r a c e l l u l a r sodium plays a r o l e i n Ca removal following con-t r a c t i l e responses. Since the l a t t e r e f f e c t occurred i r r e s p e c t i v e of whether the s u b s t i t u t e i s l i t h i u m or T r i s - H C l i t can be concluded that i n the r a b b i t a n t e r i o r mesenteric v e i n l i t h i u m can not e n t i r e l y s u b s t i -tute f o r Na i n t h i s f u n c t i o n . Further evidence f o r t h i s i s provided by the f a c t that the c o n t r a c t i l e responses i n low sodium media occurred i r r e s p e c t i v e of whether the s u b s t i t u t e i s l i t h i u m or T r i s - H C l and that the threshold f o r these responses i s even lower i n l i t h i u m s u b s t i t u t e d Krebs. The phenomenon of r e l a x a t i o n of smooth muscles following c o n t r a c t -I | i l e responses may be c o r r e l a t e d with removal of Ca from i t s c e l l u l a r s i t e of a c t i o n . Our r e s u l t s suggested that e x t r a c e l l u l a r sodium may p a r t i c i p a t e i n t h i s function and support the previous observation of Biamino and Johansson (1970) and Katase and Tomita (1971). However, dif f e r e n c e s i n the behaviour of various preparations i n low sodium media and the r e l a t i v e importance of t h i s system i n d i f f e r e n t smooth muscles might be expected. The volume of the sarcoplasmic reticulum i n d i f f e r e n t smooth muscles i s v a r i a b l e (Devine et a l . , 1972). Since the uptake of 11 Ca can be accomplished i n part by these s t r u c t u r e s , i t i s not s u r p r i s i n g that v a r i a b i l i t y among d i f f e r e n t smooth muscle preparations might be explained i n part on an u l t r a s t r u c t u r e b a s i s . Our r e s u l t s i n L i s u b s t i t u t e d 108 Krebs are comparable to those obtained by Bohr et a l . (1969) who found a s i m i l a r delay i n the r e l a x a t i o n of mesenteric a r t e r i e s and a o r t i c s t r i p s i n Na-free, L i s u b s t i t u t e d , p h y s i o l o g i c a l s a l t s o l u t i o n . 109 BIBLIOGRAPHY Anderson, N. C., Ramon, F., and Snyder, A. (1971) Studies on calcium and sodium i n uter i n e smooth muscle e x c i t a t i o n under current-clamp and voltage-clamp condition, J . Gen. P h y s i o l . 58, 322-339. Axelsson, J . , and Holmberg, J . (1971) The e f f e c t s of K-free s o l u t i o n on tension development i n the smooth muscle taenia c o l i from guinea p i g , Acta P h y s i o l . Scand. 82, 322-332. Baker, P. F., Blaustein, M. P., Hodgin, A. L., and Steinhardt, R. A. (1969) The influence of calcium on sodium e f f l u x i n squid axons, J . P h y s i o l . 200, 431-458. Baker, P. F. (1970) Sodium-calcium exchange across the nerve c e l l mem-brane, i n Calcium and C e l l u l a r Function, Cuthbert, W. (Ed.), MacMillan Co., 99-107. Bandick, N. R. and Sparks, H. V. (1970) C o n t r a c t i l e response of vascular smooth muscle of re n a l hypertensive r a t s , Amer. J . P h y s i o l . 219, 340-344. B e i l i n , L. J . , and Ziakus, G. (1971) Vascular r e a c t i v i t y i n post doca hypertension, B r i t . J . Pharmacol. 43, 427P. Biamlno, G. and Johansson, B. (1970) E f f e c t s of calcium and sodium on contracture tension i n the smooth muscle of the r a t p o r t a l v e i n , PflUger Arch. 321, 143-158. Bianchi, C. P. (1968) Pharmacological actions on e x c i t a t i o n - c o n t r a c t i o n coupling i n s t r i a t e d muscle, Fed. Proc. 27, 126-131. BIhler, J . , and Sawh, P. C. (1971) E f f e c t s of diphenylhydantoin on the transport of Na and K and the r e g u l a t i o n of sugar transport i n muscle i n v i t r o , Biochem. Biophys. Acta 249, 240-251. 110 Bl a u s t e i n , M. P., and Hodgin, A. L. (1969) The e f f e c t of cyanide on the e f f l u x of calcium from squid axons, J . P h y s i o l , 200, 497-527, Bohr, D. F, (1961) R e a c t i v i t y i n aorta and a r t e r i o l e s i n re n a l hyperten-s i o n , Univ. Mich. Med. B u l l . 27, 196-197. Bohr, D. F. (1963) Vascular smooth muscle: dual e f f e c t of calcium, S c i . 139, 597-599. Bohr, D. F. (1964) E l e c t r o l y t e s and smooth muscle c o n t r a c t i o n , Pharmacol. Rev. 16, 85-111. Bohr, D. F., S e i d e l , C., and Sobieski, J . (1969) Possi b l e r o l e of sodium-calcium pumps i n tension development of vascular smooth muscles, Microv.  Res. 1, 335-343. Bohr, D. F., and S i t r i n , M. (1970) Regulation of vascular smooth muscle c o n t r a c t i o n : changes i n experimental hypertension, C i r c . Res. 26 (Supp. I I ) , I I 83-11 90, Bohr, D, F,, S i t r i n , M. D,, and Hansen, T. R. (1971) Vascular smooth muscle i n experimental hypertension, C l i n . S c i . 40, IP. Bolton, T. B. (1971) E l e c t r o p h y s i o l o g i c a l evidence of an e l e c t r o g e n i c sodium pump i n the l o n g i t u d i n a l muscle of guinea p i g ileum and i t s involvement i n the response to a c e t y l c h o l i n e , J . P h y s i o l . 218, 58P-59P. Bose, D., and Innes, T. R. (1972) Influence of sodium pumping on smooth muscle c o n t r a c t i o n , B r i t . J . Pharmacol. 45, 689-690. Brading, A. F. (1971) Analysis of the e f f l u x e s of sodium, potassium, and ch l o r i d e ions from smooth muscle i n normal and hypertonic s o l u t i o n s . J . P h y s i o l . 214, 393-416. Brading, A. F., BUlbring, E., and Tomita, T. (1969) The e f f e c t of sodium and calcium on the a c t i o n p o t e n t i a l of the smooth muscle of the guinea pig taenia c o l i , J . P h y s i o l . 200, 637-654. . . . / I l l I l l Brading, A. F., and Tomita, T. (1968) The a c t i o n p o t e n t i a l of the guinea pig taenia c o l i i n low sodium s o l u t i o n , J . P h y s i o l . 197, 30P-31P, Brecht, K,, Konold, P., and Gebert, G. (1969) The e f f e c t of potassium, catecholamines, and other vasoactive agents on i s o l a t e d a r t e r i a l seg-ments of the muscular type, P h y s i o l . Bohemoslov. 18, 15-21. Brest, A. N. (1969) C l i n i c a l pharmacology of anti-hypertensive drugs, i n Hypertensive Cardiovascular Disease, Brest, A. N. (Ed.), F. A. Davis Co., P h i l a d e l p h i a , 195-212. Briggs, A. H. (1963) C h a r a c t e r i s t i c of contraction i n gly c e r i n a t e d u t e r i n e smooth muscle, Amer. J . P h y s i o l . 204, 739-742. Briggs, A. H., and Melvin, S. (1961) Ion movements i n i s o l a t e d r a b b i t a o r t i c s t r i p s , Amer. J . P h y s i o l . 201, 365-368. BUlbring, E., and Tomita, T. (1970) E f f e c t of Ca removal on the smooth muscle of guinea pig taenia c o l i , J . P h y s i o l . 210, 217-232. Burke, T. F., Spalding, C. T., and Jones, V. D. (1971) Influence of sodium and potassium content on a r t e r i a l responsiveness, C i r c . Res. 39, 525-534. Burnstock, G. (1970) Structure of smooth muscle and i t s innervation, In Smooth Muscle, Bfllbring, E., Brading, A., Jones, A., and Tomita, T. (Eds.), Edward Arnold (Pub), 1-69. Butle r , T. C. (1957) The metabolic conversion of 5-5 diphenylhydantoin to 5-(P-hydroxyphenyl)-5-hydantoin, J . Pharmacol. Exp. Therap. 119, 1-11. Caldwell, P. C. (1968) Factors governing movement and d i s t r i b u t i o n of inorganic ions i n nerve and muscle, P h y s i o l . Rev. 48, 1-64. Casteels, R. (1969) C a l c u l a t i o n of the membrane p o t e n t i a l i n smooth muscle c e l l s of the guinea pig taenia c o l i by the Goldman equation, J . P h y s i o l . 205, 193-208. 112 Casteels, R. (1970) The r e l a t i o n s h i p between the membrane p o t e n t i a l and the i o n d i s t r i b u t i o n i n smooth muscles, i n Smooth Muscle, Blilbring E., Brading, A., Jones, A., and Tomita, T. (Eds.), Edward Arnold L t d . , London, 70-99. Casteels, R., Droogmans, G., and Hendricks, H. (1971a) Membrane p o t e n t i a l of smooth muscle c e l l s i n K-free s o l u t i o n s , J . P h y s i o l . 217, 281-295. Casteels, R., Droogmans, G., and Hendricks, H. (1971b), E l e c t r o g e n i c sodium pump i n smooth muscle c e l l s of the guinea p i g taenia c o l i . J . P h y s i o l . 217, 297-313. C o l l i n s , G. A., Sutter, M. C., and T e i s e r , J . C. (1972a) Calcium and con-t r a c t i o n i n the r a b b i t a n t e r i o r mesenteric p o r t a l v e i n , Canad. J . P h y s i o l . Pharmacol. 50, 289-299. C o l l i n s , G. A., Sutter, M. C., and T e i s e r , J . C. (1972b), The e f f e c t of manganese on the r a b b i t a n t e r i o r mesenteric p o r t a l v e i n , Canad. J . P h y s i o l . Pharmacol. 50, 300-309. Conn, R. D. (1965) Diphenylhydantoin sodium i n cardiac arrhythmics, New  Eng. J . Med. 272, 277-282. Cooke, P. H., Chase, R. H., and Cortes, J . M. (1970) Thick filaments resembling myosin i n e l e c t r o p h o r e t i c a l l y - e x t r a c t e d vertebrate smooth muscle, Exp. C e l l . Res. 60, 237-246. Corcoran, A. C , Taylor, R. D., and Page, T. H. (1951), Controlled obser-vations on the e f f e c t of low sodium dietotherapy i n e s s e n t i a l hyper-tension, C i r c . 3^ 1-16. Cuthbert, A. W., and Sutter, M. C. (1965) The e f f e c t s of drugs on the r e l a x a t i o n between the a c t i o n p o t e n t i a l discharge and tension i n a mammalian v e i n , B r i t . J . Pharmacol. Chemoth. 25, 592-601. Cuthbert, A. W., and Wong, P. Y. D. (1971) The amiloride receptor. B r i t . J . Pharmacol. 43, 416P-417P. .../113 113 Dahl, L. K. (1961) Possi b l e r o l e of chronic excess s a l t consumption i n the pathogenesis of e s s e n t i a l hypertension, Amer. J . C a r d i o l . _8, 571-575. D'auriac, G. A., Daudouin, M. and Meyer, P. (1972) Mechanism of ac t i o n of angiotensin i n smooth muscle: biochemical changes following i n t e r -a c t i o n of the hormone with i t s membrane receptors, C i r c . Res. 31  (Supp. I I ) , I I 151-11 157. Daniel, E. E. (1965) E f f e c t s of i n t r a - a r t e r i a l perfusions on e l e c t r i c a l a c t i v i t y and e l e c t r o l y t e contents of dog small i n t e s t i n e , Canad. J .  P h y s i o l . Pharmacol. 43, 551-577. Daniel, E. E., Kidwai, A. M., Robinson, K, Freeman, D., and F a i r , S. (1971) The mechanisms by which ethacrynic a c i d a f f e c t s i o n f l u x e s , volume and energy supply i n the r a t uterus, J . Pharmacol. Exp. Therap 0 176, 563-579. Dani e l , E. E., and Robinson, K. (1971a) E f f e c t s of i n h i b i t o r s of a c t i v e 22 42 transport on Na and K movements and on nucleotide l e v e l s i n r a t u t e r i at 25° C , Canad. J . P h y s i o l . Pharmacol. 49, 178-204. Daniel, E. E., and Robinson, K. (1971b) E f f e c t s of i n h i b i t o r s of metabolism 22 42 on adenine nucleotides and on Na and K and net movements i n r a t u t e r i at 25° C , Canad. J . P h y s i o l . Pharmacol. 49, 205-239. Daniel, E. E., and Robinson, K. (1971c) The e f f e c t of temperature on sodium movements i n r a t u t e r i and a model f o r c o n t r o l of t h e i r i o n content, Canad. J . P h y s i o l . Pharmacol. 49, 240-262. Devine, C. E., and Somlyo, A. P. (1970) U l t r a s t r u c t u r e of vascular smooth muscle studied with lanthanum, Fed. Proc. 29, 455. Devine, C. E., and Somlyo, A. 0. (1971) Thick filaments i n vascular smooth muscle, J . C e l l . B i o l . 49, 636-649. 114 Devine, C. E., Somlyo, A. V., and Somlyo, A. P. (1972) Sarcoplasmic reticulum and e x c i t a t i o n - c o n t r a c t i o n coupling i n mammalian smooth muscles, J . C e l l . B i o l . 52, 690-718. DOrge, A., and Nagel, W. (1970) E f f e c t of amiloride on sodium trans-port i n frog s k i n . I I . Sodium transport pool and u n i d i r e c t i o n a l f l u x e s , PflUger Arch. 321, 91-101. D r e i f u s , L. S., and Watanabe, Y. (1970) Current status of diphenyl-hydantoin, Am. Heart J . 80, 709-713. Ebashi, S. (1963) Third component p a r t i c i p a t i n g i n the s u p e r p r e c i p i -t a t i o n of n a t u r a l actomyosin, Nature 200, 1010. Ebashi, S., Ebashi, F., and Kodama, A. (1967) Troponin as the Ca receptive p r o t e i n i n the c o n t r a c t i l e system, J . Bioch. (Tokyo) 62, 137-138. Ebashi, S., Iwakura, H. Nakajima, H., Nakamura, R., and Choi, Y. (1966) New s t r u c t u r a l proteins from dog heart and chicken g i z z a r d , Biochem. Z. 345, 201-211. Edman, K. A. P., S c h i l d , H. 0. (1962) The need f o r calcium i n the con-t r a c t i l e responses induced by a c e t y l c h o l i n e and potassium i n the r a t uterus, J . P h y s i o l . 161, 424-441. Escueta, A. V., and Appel, S. (1971) Diphenylhydantoin and potassium transport i n i s o l a t e d nerve terminals, J . C l i n . Invest. 50, 1977-1984. E s p l i n , D. W. (1957) E f f e c t s of diphenylhydantoin on synaptic transmis-s i o n i n cat s p i n a l cord and s t e l l a t e ganglion, J . Pharmacol. Exp. Therap. 120, 301-323. F e s t o f f , B. W., and Appel, S. H. (1968) E f f e c t of diphenylhydantoin on synaptosome sodium-potassium ATPase. J . C l i n . Invest. 47, 2752-2758. 115 F i l o , R. S., Bohr, D. F., and Ruegg, J . C. (1965) Glycerinated s k e l e t a l and smooth muscle: calcium and magnesium dependence, S c i . 147, 1581-1583. F i l o , R. S., Ruegg, J . C., and Bohr, D. F. (1963) Actomyosin-like p r o t e i n of a r t e r i a l w a l l , Amer. J . P h y s i o l . 205, 1247-1252. F i t z p a t r i c k , D. F., Landon, E. J . , Debbas, G., and H u r i t z , L. (1972) A calcium pump i n vascular smooth muscle, S c i . 176, 305-306. F l e i s c h , J . H., Maling, H. M., and Brodie, B. B. (1969) Vascular smooth muscle r e a c t i v i t y i n normotensive and hypertensive r a t s , S c i . 166, 1300-1301. Formby, B. (1970) The i n v i v o and i n v i t r o e f f e c t of diphenylhydantoin and phenobarbitone on K + a c t i v a t e d phospholydrolase and (Na +, K +) activat e d ATPase i n p a r t i c u l a t e membrane f r a c t i o n s from r a t b r a i n , J . Pharm. Pharmacol. 22, 81-85. Friedberg, C. K. (1966) Sodium and hypertension, i n Disease of the Heart, Friedberg, C. K. (Ed.), Saunders Co., 1481-1482. Friedman, S. M., and Friedman, C. L. (1961) Sodium and potassium exchanges and p e r i p h e r a l v a s c u l a r r e s i s t a n c e , Amer. J . C a r d i o l . j$, 564-570. Gabella, G. (1971) Relationship between sarcoplasmic reticulum and carveolae i n t r a c e l l u l a r s i n the i n t e s t i n a l smooth muscle, J . P h y s i o l . 316, 42P-44P. Gatzy, J . T. (1971) The e f f e c t of K sparing d i u r e t i c s on ion transport across the excised toad bladder, J . Pharmacol. Exp. Therap. 76, 580-594. G l i t s c h , H. G., Reuter, H., and Scholz, H. (1970) the e f f e c t of i n t e r n a l sodium concentration on calcium fluxes i n i s o l a t e d guinea pig a u r i c l e s . J . P h y s i o l . 209, 25-43. Godfraind, T., and Kaba, A. (1969) Actions phasiques et tonique de l ' a d r e n a l -ine sur un muscle l i s s e v a s c u l a i r e et l e u r i n h i b i t i o n par des agents pharmacologiques, Arch. Int. Pharmacodyn. 178, 488-491. .../116 116 Golenhofen, K., and Petranyi, P. (1969) Spikes of smooth muscles i n calcium-free s o l u t i o n ( i s o l a t e d taenia c o l i of guinea p i g ) , Ex p e r i e n t i a 25, 271-273. Goodford, P. J . (1966) An i n t e r a c t i o n between potassium and sodium i n the smooth muscle of the guinea p i g taenia c o l i , J . P h y s i o l . 186, 11-26. Goodford, P. J . (1967) The calcium content of the smooth muscle of the guinea p i g taenia c o l i , J . P h y s i o l . 192, 145-157. Goodford, P. J . , and Hermansen, K. (1961) sodium and potassium movement i n the u n s t r i a t e d muscle of the guinea p i g taenia c o l i , J . P h y s i o l . 58, 426-448. Goodford, P. J . and Wolowyk, M.W. (1971) Counter-cation i n t e r a c t i o n at the smooth muscle c e l l membrane of guinea p i g taenia c o l i , J . P h y s i o l .  218, 36P-37P. Goodford, P. J . and Wolowyk, M. W. (1972) L o c a l i z a t i o n of c a t i o n i n t e r a c t i o n s i n the smooth muscle of the guinea p i g taenia c o l i , J . P h y s i o l . 224, 521-535. Goodman, F. R. and Weiss, G. B. (1971) D i s s o c i a t i o n by lanthanum of smooth muscle responses to potassium and acetylcholine,Amer.J. P h y s i o l . 220, 759-766. Graham, J . M., and Keatinge, W. R. (1970) sucrose-gap recording of prolonged e l e c t r i c i t y a c t i v i t y from a r t e r i e s i n Ca-free s o l u t i o n containing EDTA at low temperature, J . P h y s i o l . 208, 2P-3P. Hagemeijer, F., Rorive, G., and S c h o f f e n i e l s , E. (1966) Composition c a t i o n i -que de d i f f e r e n t s t i s s u s du r a t au cours de l'hypertension a r t e r i e l l e experimentale, Arch. Int. P h y s i o l . 74, 807-811. Hallback, M., Lundgren, J . , and Weiss, L. (1971) R e a c t i v i t y to noradrenaline of a o r t i c s t r i p s and p o r t a l veins from spontaneously hypertensive and normotensive r a t s , Acta P h y s i o l . Scand. 81, 176-181. .../117 117 H a r r i s , G. S. (1970) A r e l a t i o n s h i p among a r t e r i a l w a l l acid mucopoly-saccharides, c a t i o n binding, and a r t e r i a l r e a c t i v i t y , C i r c . Res. 2£, 1191-1197. H a r r i s , G. S., and Palmer, W. (1972) E f f e c t of increased sodium ion on a r t e r i a l sodium and r e a c t i v i t y , C l i n . S c i . 42, 301-309. Hinke, J . A. M. (1965) Calcium requirements f o r noradrenaline and high potassium i o n cont r a c t i o n i n a r t e r i a l smooth muscle, i n Muscle, Daniel, E. E., Kay, C. M., and Monkton, G. (Eds.), Pergamon Press (London), 269-285. Hiraoka, M., Yamagishi, S., and Sano, T. (1968) Role of calcium ions In the con t r a c t i o n of vascular smooth muscle, Amer. J . P h y s i o l . 214, 1084-1089. Holman, M. E. (1969) Electrophysiology of vascular smooth muscle, Ergbn. P h y s i o l . 61, 137-177. Hudgins, P. M., and Weiss, G. H. (1968) D i f f e r e n t i a l e f f e c t s of calcium removal upon vascular smooth muscle co n t r a c t i o n induced by norepine-phrine, histamine, and potassium, J . Pharmacol. Exp. Therap. 159, 91-97. Hukura, T., and Fukuda, H. (1968) the e l e c t r i c a l a c t i v i t y of guinea p i g small i n t e s t i n e with s p e c i a l reference to the slow wave, Jap. J . P h y s i o l . 18, 71-86. Job, D. D. (1969) Ionic basis of i n t e s t i n a l e l e c t r i c a l a c t i v i t y , Amer. J . P h y s i o l . 217, 1534-1541. Johansson, B., Jonsson, 0., Axelsson, J . , and Wahlstrom, B. (1967) E l e c t r i c a l and mechanical c h a r a c t e r i s t i c s of vascular smooth muscle response to norepinephrine and i s o p r o t e r e n o l , C i r c . Res. 21, 619-633. 118 Johansson, B., and Ljung, B. (1967) Spread of e x c i t a t i o n i n the smooth muscle of the r a t p o r t a l v e i n , Acta P h y s i o l . Scand, 70, 312-322. Kao, C. Y. (1966) Tetrodotoxin, s a x i t o n i n , and t h e i r s i g n i f i c a n c e i n the study of e x c i t a t i o n phenomena, Pharmacol. Rev. 18, 997-1050. Katase, T. and Tomita, T. (1971) Sodium p a r t i c i p a t i o n i n the recovery from K contracture i n the guinea p i g taenia c o l i . J . P h y s i o l . 218, 48P. Keatinge, W. R. (1968a) Tonic requirements f o r a r t e r i a l a c t i o n p o t e n t i a l , J . P h y s i o l . 194, 169-182. Keatinge, W. R. (1968b) Sodium f l u x and e l e c t r i c a l a c t i v i t y of a r t e r i a l muscle, J . P h y s i o l . 194, 183-200. Kempner, W. (1948) Treatment of hypertensive vascular disease with r i c e d i e t , Amer . J . Med. 4_, 545-577. K i z e r , S., Vargas-Cordon, M., Brendel, K., and B r e s s l e r , R. (1970) The i n v i t r o i n h i b i t i o n of i n s u l i n s e c r e t i o n by diphenylhydantoin. J . C l i n . Invest. 49, 1942-1948. Kuriyama, H. (1963) The in f l u e n c e of potassium, sodium and c h l o r i d e on the membrane p o t e n t i a l of the smooth muscle of taenia c o l i . J . P h y s i o l . 166, 15-28. Kuriyama, H., Oshima, K., and Sakamoto, J . (1971) The membrane properties of the smooth muscle of the guinea p i g p o r t a l v e i n i n i s o t o n i c and hypertonic s o l u t i o n s , J . P h y s i o l . 217, 179-199. Kuriyama, H., Osa, T., and Toida, N. (1967) Nervous f a c t o r s i n f l u e n c i n g the membrane a c t i v i t y of i n t e s t i n a l smooth muscles. J . P h y s i o l . 191, 257-270. 45 Lammel, E., and Golenhofen, K. (1971) Messungender Ca-Aufnahme on i n t e s t i n a l e r g l a t t e r musku-latur ziir hypothese eines von Ca-Lonen getragenen aktions stromes. Pfluger Arch. 329, 269-282. .../119 119 L i u , J . , Prosser, C. L., and Job, D, D. (1969) Ionic dependence of slow waves and spikes i n i n t e s t i n a l muscle,Amer.J. Physiol.217, 1542-1547. Lewin, E., and Black, V. (1971) The e f f e c t of diphenylhydantoin adminis-t r a t i o n on sodium potassium a c t i v a t e d ATPase i n cortex, Neurol. 21, 647-651. Lowy, J . Paulsen, F. R., V i b e r t , P. J . (1970) Myosin filaments i n v e r t e -brate smooth muscle, Nature 225, 1053-1054. LUttgau, H. C., and Niedergerke, R. (1958) The antagonism between Ca and Na ions on the frog's heart, J . P h y s i o l . 143, 486-505. Martosi, A., Feretos, R. (1964) Sarcoplasmic reticulum. I I . C o r r e l a t i o n I | between adenosine triphosphatase a c t i v i t y and Ca uptake, J . B i o l . Chem. 239, 659-668. Matthews, E. K., and Sutter, M. C. (1967) Ouabain induced changes i n the c o n t r a c t i l e and e l e c t r i c a l a c t i v i t y , potassium content and response to drugs of smooth muscle c e l l s , Canad. J . P h y s i o l . Pharmacol. 45, 509-520. Murphy, R. A. (1969) C o n t r a c t i l e proteins of vascular smooth muscle: e f f e c t s of hydrogen and a l k a l i metal cations on actomyosin adenosine-triphosphatase a c t i v i t y , Microv. Res. JL, 344-353. Nash, C. W., Luchka, E., and Jhamandas, K. H. (1966) An i n v e s t i g a t i o n of sodium and calcium competititon i n vascular smooth muscle Canad. J . P h y s i o l . Pharmacol. 44, 147-156. Needham, D. M. (1962) C o n t r a c t i l e proteins i n smooth muscle of the uterus, P h y s i o l . Rev.42 (Suppl. 5), 88-97. Palaty, V., Gustafson, B., and Friedman, S. (1969a) Sodium binding i n the a r t e r i a l w a l l , Canad. J . P h y s i o l . Pharmacol. 47, 763-770. Palaty, V., Gustafson, B., and Friedman, S. (1969b) The r o l e of p r o t e i n -polysaccarides i n hydration of the a r t e r i a l w a l l , Canad. J . P h y s i o l . Pharmacol. 48, 54-60. 120 Panner, B. J . , and Honig, C. R. (1970) Locus and state of aggregation of myosin i n t i s s u e sections of vertebrate smooth muscle. J . C e l l . B i o l . 44, 52-61. Pease, D. C. (1968) S t r u c t u r a l features of unfixed mammalian smooth and s t r i a t e d muscle prepared by g l y c o l dehydration, J . U l t r a s t r . Res. 23, 280-303. Pincus, J . H. (1970) Studies on the mechanism of a c t i o n of diphenylhy-dantoin, Arch. Neurol. 22, 566-571. Pincus, J . H. (1972) Diphenylhydantoin and i o n f l u x i n l o b s t e r nerve. Arch. Neurol. 26, 4-10. Pincus, J . H. and Giarman, N. Y. (1967) The e f f e c t of diphenylhydantoin on sodium potassium magnesium stimulated adenosine triphosphatase a c t i -v i t y of r a t b r a i n , Biochem. Pharmacol. 16, 600-603. P o r t z e l , H. (1962) Der Arbeitzykhus geordneter Aktomyosinsysterns, Z. Naturf 7b. 1-10. Raines, A., and Standaert, F. G. (1966) Pre- and p o s t - j u n c t i o n a l e f f e c t s of diphenylhydantoin at the soleus neuro-muscular j u n c t i o n . J . Pharmacol. Exp. Therap. 153, 361-366. Rawson, M. D., and Pincus, J . H. (1968) The e f f e c t of diphenylhydantoin on Na, K, Mg, a c t i v a t e d adenosine triphosphatase i n microsomal f r a c t i o n s of r a t and guinea p i g b r a i n and whole homogenates of human b r a i n . Biochem. Pharmacol. 17, 573-579. Rice, R. V., Moses, J . A., McManus, G. M., Brady, A. C. and B l a s i k , L. M. (1970) the organization of c o n t r a c t i l e filaments i n mammalian smooth muscle, J . C e l l . B i o l . 17, 183-196. Reuter, H., and S e i t z , N. (1968) The dependence of calcium e f f l u x from cardiac muscle on temperature and external i o n composition. J . P h y s i o l . 195. 451-470. .../121 121 Ringer, S. (1883) A fu r t h e r c o n t r i b u t i o n regarding the i n f l u e n c e of the d i f f e r e n t constituents of blood on the contraction of the heart. J . P h y s i o l . j>, 29-42. Rosen, M., Lisak, R., and Rubin, T. L. (1967) Diphenylhydantoin i n cardiac arrhythmias, Amer. J . C a r d i o l . 20, 674-678. Rosenthal, J . (1969) Post t e t a n i c p o t e n t i a t i o n at the neuromuscular j u n c t i o n of the f r o g . J . P h y s i o l . 203, 121-133. Salako, L, A. and Smith, A. J . (1970a) E f f e c t s of amiloride on a c t i v e sodium transport by the i s o l a t e d frog s k i n : evidence concerning s i t e of a c t i o n , B r i t . J . Pharmacol. 38, 702-718. Salako, D. A., and Smith, A. J . (1970b) Changes i n sodium pool and k i n e t i c s of sodium transport i n frog s k i n produced by amiloride. B r i t . J . Pharmacol. 39, 99-109. S a p i r s t e i n , L. A., Brandt, W, L., and Drury, D. R. (1950) Production of hypertension i n the r a t by s u b s t i t u t i n g hypertonic sodium c h l o r i d e s o l u t i o n s f o r drinking water. Proc. Soc. Exp. B i o l . Med. 73, 82-85. Sato, K., and Dobson, R. (1969) Reversal of ouabain-induced i n h i b i t i o n of human eccrine sweat gland f u n c t i o n by diphenylhydantoin. J . C l i n . Invest. 53, 283-288. Schatzman, H. J . , and V i n c e n z i , F. F. (1969) Calcium movements across the membrane of human red c e l l s , J . P h y s i o l . 201, 369-395. Shlrmer, R. H. (1965) Die besonderheiten des c o n t r a c t i l e n proteins der a r t e r l e n . Biochem. Z. 343, 269-282. Sc h o f f e n i e l s , E. (1969) Ionic composition of the a r t e r i a l w a l l . A n g i o l . 6^, 65-88 (1-24). Schou, M. (1957) Biology and pharmacology of the l i t h i u m i o n , Pharmacol. 122 S i e g e l , G. J . , and Goodwin, B. B, (1972) Sodium potassium a c t i v a t e d adenosine triphosphatase of b r a i n microsomes: m o d i f i c a t i o n of sodium i n h i b i t i o n by diphenylhydantoins, J . C l i n . Invest. 51, 1164-1169. S i t r i n , M. D., and Bohr, D. F. (1971) Ca and Na i n t e r a c t i o n i n vascular smooth muscle contraction, Amer. J . P h y s i o l . 220, 1124-1128. Somlyo, A. V., and Somlyo, A. P. (1968) Electromechanical and pharmaco-mechanical coupling i n vascular smooth muscle, J . Pharmacol. Exp. Therap. 159, 129-145. Somlyo, A. P., Devine, C. E., Somlyo, A. V., and North, S. R. (1971) Sarcoplasmic reticulum and the temperature dependent contraction of smooth muscle i n calcium-free s o l u t i o n s , J . C e l l . B i o l . 51, 722-741. Somlyo, A. V., and Somlyo, A. P. (1971) Strontium accumulation by sarco-plasmic reticulum and mitochondria i n vascular smooth muscle. S c i . 174, 955-957. Spain, R. and Chidsey, C. (1971) Myocardial Na/K adenosine triphosphates a c t i v i t y during r e v e r s a l of ouabain t o x i c i t y with diphenylhydantoin. J . Pharmacol. Exp. Therap. 179, 594-598. 28 Sparrow, M. 0. (1969) I n t e r a c t i o n of Mg with Ca and K i n the smooth muscle of the guinea p i g taenia c o l i , J . P h y s i o l . 205, 19-38. Sparrow, M. P., Maxwell, L. C., Ruegg, J . C., and Bohr, D. F. (1970) Preparation and properties of a calcium i o n - s e n s i t i v e actomyosin from a r t e r i e s , Amer. J . P h y s i o l . 219, 1366-1372. Sparrow, M. P. and Simmonds, W. J . (1965) The r e l a t i o n s h i p of the calcium content of smooth muscles to i t s c o n t r a c t i l i t y i n response to d i f f e r e n t modes of s t i m u l a t i o n , Biochem. Biophys. Acta 109, 503-511. ../123 123 Szent-Gybrgi, A. (1947) Chemistry of Muscular Contraction, Academic Press, New York. Szent-GyOrgi, A. (1951) i n Muscular Contraction (2nd Ed.), Academic Press, New York, 144-145. Taylor, G. S., Peton, D. M,, and Daniel, E. E. (1970) C h a r a c t e r i s t i c s of e l e c t r o g e n i c sodium pumping i n r a t myometrium, J . Gen. P h y s i o l . 56, 360-375. Thomas, R. C. (1972) Elec t r o g e n i c sodium pump i n nerve and muscle c e l l s . P h y s i o l . Rev. 52, 563-594. Tobian, L. (1967) Why do t h i a z i d e d i u r e t i c s lower blood pressure i n e s s e n t i a l hypertension, Ann. Rev. Pharmacol. T_t 399-408. Tobian, L., and Binion, J . T. (1952) Tissue cations and water i n a r t e r i a l hypertension, C i r c . 5, 754-758. Tobian, L, Janecek, J . , Tomboulian, A., and F e r r e i r a , 0. (1961) Sodium and potassium i n the walls of a r t e r i o l e s i n experimental hypertension, J . C l i n . Invest. 40, 1922-1925. Tomita, T. (1970) E l e c t r i c a l p roperties of mammalian smooth muscle, i n Smooth Muscle, BUlbring, E., Brading, A., Jones, A., and Tomita, T. (Eds.), Edward Arnold (Pub.), 197-243. Tomita, T. and Yamamoto, T. (1971) E f f e c t s of removing the external potassium on the smooth muscle of guinea p i g taenia c o l i . J . P h y s i o l . 212, 851-868. Van Breeman, C. (1970) Separation of c e l l membrane calcium transport from e x t r a c e l l u l a r calcium exchange i n vascu l a r smooth muscle, Biochem. Biophys. Res. Commun. 39, 567-574. Van Breeman, C., Farinas, B. R., Gerba, P., and E l i z a b e t h , D. (1972) E x c i t a t i o n - c o n t r a c t i o n coupling i n r a b b i t aorta studied by the lanthanum method f o r measuring c e l l u l a r calcium i n f l u x , C i r c . Res. 30, 44-53. .../124 124 V e r i t y , M. A., and•*Bevan, J . A. (1969) Membrane adenosine t r i p h o s p h a -tase a c t i v i t y of v a s c u l a r smooth muscle, Biochem. Pharmacol. 18, 327-338. Weber, A. (1956) The u l t r a c e n t r i f u g a l s e p a r a t i o n of L-myosin and a c t i n i n actomyosin s o l under the i n f l u e n c e of ATP, Biochem. Biophys. Acta 19, 345-351. W i l l i a m s , J . A., Whltrow, C. D., and Woodbury, D. M. (1971) E f f e c t s of ouabain and diphenylhydantoin on transmembrane p o t e n t i a l s , i n t r a c e l l u -l a r e l e c t r o l y t e s and c e l l pH of r a t muscle and l i v e r i n v i v o , J . P h y s i o l . 212, 101-115. Wolowyk, M. W. (1971) Membrane s t r a i n i n g by u r a n y l c a t i o n i n smooth muscle, J . P h y s i o l . 217, 3P-5P. Wolowyk, M. W., K i d u r a i , A. M., and D a n i e l , E. E.(1971) sodium-potassium s t i m u l a t e d adenosine trip h o s p h a t a s e of v a s c u l a r smooth muscle, Canad. J . Biochem. 49, 376-384. Woodbury, D. M. (1969) Mechanisms of a c t i o n of a n t i c o n v u l s a n t s , i n B a s i c  Mechanisms of the E p i l e p s i e s , J a s p e r , H. H., Ward, A. A., and Pope, A. (Eds.), L i t t l e Brown & Co., Boston, 647-681. Woodbury, D. M. and Kemp, Y. W. (1971) Pharmacology and mechanisms of a c t i o n of dip h e n y l h y d a n t o i n , P s y c h i a t . N e u r o l . Neurochir. 74, 91-115. Yamauchi, A., and Burnstock, G. (1969) Post n a t a l development of smooth muscle c e l l s i n the mouse vas deferens. A f i n e s t r u c t u r a l study, J . Anat. 104, 1-15. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0101558/manifest

Comment

Related Items