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Beta-adrenoceptor-induced relaxation and cyclic nucleotide levels in rat uterus Meisheri, Kaushik Damji 1979

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BETA-ADRENOCEPTOR-INDUCED RELAXATION and CYCLIC NUCLEOTIDE LEVELS IN RAT UTERUS by KAUSHIK DAMJI MEISHERI A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES In the Division of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA © December, I 9 7 8 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . i t i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Dep-a-rtmen-t- o f The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 ABSTRACT The cAMP-second messenger hypothesis f o r j}-adrenoceptor-induced r e l a x a t i o n of u t e r i n e smooth muscle was t e s t e d i n h l g h -K + d e p o l a r i z e d r a t u t e r u s . At 10" M c o n c e n t r a t i o n , I s o p r o t e r e n o l , a ^ - a d r e n e r g i c a g o n i s t , c o u l d cause r e l a x a t i o n of the d e p o l a r i z e d uterus without I n c r e a s i n g t i s s u e cAMP l e v e l s . F u r t h e r , although i n c r e a s e s i n cAMP l e v e l s were a s s o c i a t e d , i n some cases, w i t h -8 -il-l s o p r o t e r e n o l (10 M or 10 M)-Induced r e l a x a t i o n , there was no q u a n t i t a t i v e c o r r e l a t i o n between the Increases i n cAMP and r e l a x a t i o n . Pretreatment of the t i s s u e w i t h a phosphodiesterase I n h i b i t o r , RO 20-172*4- (10 M), d i d not p o t e n t i a t e the r e l a x a t i o n response to I s o p r o t e r e n o l . These r e s u l t s suggested t h a t there Is no simple cause and e f f e c t r e l a t i o n s h i p between ^-adrenoceptor-Induced Increases i n cAMP l e v e l s and r e l a x a t i o n i n u t e r i n e smooth muscle. The d i s s o c i a t i o n between cAMP and r e l a x a t i o n found l n the present study was a l s o extended to cGMP, s i n c e no changes i n cGMP l e v e l s were observed with i s o p r o t e r e n o l - i n d u c e d r e l a x a t i o n . I t i s g e n e r a l l y accepted t h a t the i o n i c environment o f the c e l l a f f e c t s the c e l l u l a r responses o f the t i s s u e . I t was demonstrated t h a t hlgh-K*" d e p o l a r i z a t i o n o f u t e r i n e smooth muscle caused an impairment o f the a b i l i t y of i s o p r o t e r e n o l to induce cAMP accumulation. T h i s was found to be r e l a t e d to Increased C a * * - I n f l u x known to occur d u r i n g d e p o l a r i z a t i o n . T h i s Is because pretreatment o f the t i s s u e w i t h 10~^M D-600, an I n h i b i t o r of C a + + - l n f l u x , r e s t o r e d the s t i m u l a t i o n of cAMP by I s o p r o t e r e n o l l n the d e p o l a r i z e d muscle to a l e v e l s i m i l a r to t h a t observed l n non-d e p o l a r i z e d muscle. Furthermore, there was an Inverse r e l a t i o n s h i p between fca +'^) ex l n the d e p o l a r i z i n g medium (range - I I I -0.9 to 7.2 mM) and increases i n cAMP produced by isoproterenol (10 M). It was also found that exposure of the rat uterus to a C a + + - d e f i c i e n t s o l u t i o n ( C a ^ - f r e e with 0.2 mM EGTA) accentu-ated the Increase of tissue cAMP content produced by isoproterenol ( I O - 8 M). The studies on ionic interactions demonstrated that the presence of Na +(80 mM) or high M g + + ( 2 . 5 mM) i n the depolarizing medium could overcome the blockade of lsoproterenol-induced increases i n cAMP level s by high-K + depolarization. The studies on the mechanism of this e f f e c t of Na + on the cAMP response reveal-ed that Na + exerted this e f f e c t probably by reducing the Increase In C a + + - i n f l u x occurring during depolarization. A sim i l a r type of inter a c t i o n between Mg + + and C a + + was also observed. These studies have pointed out a possible regulatory r o l e of Oa*"*" i n isoproterenol-lnduced Increases i n cAMP levels l n uterine smooth muscle. Since i t was also demonstrated that cAMP Is not an obligatory requirement In order for Isoproterenol to produce relaxation, these data have raised the question as to whether the Increases l n cAMP produced by ^-adrenoceptor stimulation Is an event secondary to the changes l n C a + + movements produced by the agonist. The e l e c t r o p h y s i o l o g i c a l studies showed that isoproterenol (10 M) could i n h i b i t spontaneous c o n t r a c t i l i t y of the r a t uterus without causing hyperpolarlzation. In hlgh-K + depolarized muscle, Isoproterenol (10~^M) produced relax a t i o n without any change i n membrane p o t e n t i a l . These data suggested that hyperpolarlzation of c e l l membranes Is not a prerequisite for ^-adrenoceptor-med-lated r e l a x a t i o n of uterine smooth muscle. - i v -TABLE OF CONTENTS Page ABSTRACT 1 ! LIST OF TABLES v l LIST OF FIGURES v i i I ACKNOWLEDGEMENTS x INTRODUCTION 1 Excitation-contraction coupling in smooth muscle. 1 cAMP and smooth muscle relaxation 4 cGMP and smooth muscle function 11 Influence of ionic environment on drug responses in smooth muscle..... 12 Relationship between hyperpolarization and relaxa-t i o n induced by catecholamines in smooth muscle.. 14 Use of depolarized preparation to study drug action on smooth muscle 16 S p e c i f i c alms 19 MATERIALS AND METHODS 20 Tissue preparation... 20 Protocol for individual studies 20 Cyc l i c nucleotide determinations 2h Computation of relaxation 27 Elec t r o p h y s i o l o g i c a l studies 2? RESULTS 31 C y c l i c nucleotide l e v e l s during spontaneous contractions......... 31 Isoproterenol-lnduced r e l a x a t i o n and c y c l i c nucleotide l e v e l s 31 - V -Studles with D-600 and EGTA 39 Calcium and isoproterenol-Induced Increases i n cAMP le v e l s and rel a x a t i o n ^5 Studies on Mg + +- C a + + - Na + interactions 57 Study with RO 20-172^, a phophodiesterase i n h i b i t o r 6 2 E f f e c t of C a + + d e f i c i e n c y 6 2 E l e c t r o p h y s i o l o g i c a l studies ^5 DISCUSSION 7 ^ Cyc l i c nucleotides i n spontaneous contractions .. 7^ Cyc l i c nucleotides In isoproterenol-induced rela x a t i o n 75 Ionic Interactions 8 0 Role of C a + + l n isoproterenol-lnduced increases In cAMP levels 8 6 Possible r o l e of cAMP l n smooth muscle relaxation Hyperpolarlzation and isoproterenol-lnduced rela x a t i o n • 91 SUMMARY 93 REFERENCES 9 5 - v i -LTST OF TABLES Table Page 1 . Composition of buffer solutions used , 2 2 2 . C y c l i c nucleotide l e v e l s i n rat uterus during spontaneous contraction cycle 3 3 3. Hlgh-K + tension and isoproterenol-Induced relaxation of rat uterus 3 5 4 . The ef f e c t of depolarization on cAMP levels 3 7 5. The ef f e c t of isoproterenol on cAMP levels in non-depolarized and depolarized, uterus ............ 3 8 6 . The effe c t of depolarization and Isoproterenol on cGMP le v e l s 40 7 . The ef f e c t of D - 6 0 0 on cAMP levels in non-depolarlzed and depolarized uterus 4 - 3 8. The effe c t of isoproterenol on cAMP level s in the presence of D - 6 0 0 9 . The ef f e c t of depolarization and Isoproterenol on cGMP level s In the presence of D - 6 0 0 46 1 0 . The effe c t of depolarization on r e s t i n g le v e l s of cAMP In the presence of varying {ca"*" ]^ ex 4 * 9 1 1 . The effe c t of isoproterenol on cAMP levels In the presence of varying [ k a ^ J e x , [Mg+"^|ex, and [ c a + * ] ex 5 8 1 2 . Hlgh-K + tension and isoproterenol-Induced relaxation i n the presence of varying [Na +]ex, [ M g ^ e x , and [ c a + ^ ex 6 0 1 3 . The Influence of RO 2 0 - 1 7 2 4 on hlgh-K + tension and Isoproterenol-Induced relaxation - v i l -of r a t uterus ,6^  14. The influence of a C a ^ - d e f i c i e n t s o l u t i o n on Isoproterenol-lnduced Increases In cAMP le v e l s 67 15 . The ef f e c t of Isoproterenol on membrane potent i a l in Isolated r a t myometrium 68 16. Hlgh-K + tension and isoproterenol-lnduced relaxation of Isolated r a t myometrium 72 17. The e f f e c t of Isoproterenol on membrane potential In depolarized myometrium 73 - v i i i -L I S T OF F I G U R E S F i g u r e P a g e 1. T e n s i o n , cAMP a n d cGMP l e v e l s d u r i n g s p o n t a n e o u s c o n t r a c t i o n - r e l a x a t i o n c y c l e i n r a t u t e r u s 32 2. T h e e f f e c t o f 127 mM KC1 a n d I s o p r o t e r e n o l o n u t e r i n e c o n t r a c t i o n 3^ 3. T h e e f f e c t o f D-600 o n s p o n t a n e o u s u t e r i n e c o n t r a c t i o n a n d h l g h - K + d e p o l a r i z a t i o n , . ^2 4. The e f f e c t o f C a + + - a n t a g o n l s t s o n I s o p r o t e r e n o l - l n d u c e d i n c r e a s e s i n cAMP l e v e l s I n n o n - d e p o l a r i z e d a n d d e p o l a r i z e d r a t u t e r u s ^7 5. The a n t a g o n i s m b y h i g h C a + + o f I s o p r o t e r e n o l -l n d u c e d I n c r e a s e s I n c A M P l e v e l s I n t h e p r e s e n c e o f 80 mM N a + 48 6. T h e e f f e c t o f [ca + H ^] e x o n I s o p r o t e r e n o l -l n d u c e d I n c r e a s e s i n cAMP l e v e l s i n d e p o l a r i z e d r a t u t e r u s 51 7. T h e I n f l u e n c e o f [ c a + ^ ] e x o n i s o p r o t e r e n o l -l n d u c e d r e l a x a t i o n o f d e p o l a r i z e d r a t u t e r u s 52 8. The I n f l u e n c e o f j c a ' ^ e x o n i s o p r o t e r e n o l -l n d u c e d r e l a x a t i o n ( A . T i n g) o f d e p o l a r i z e d r a t u t e r u s 53 9. T h e e f f e c t o f | c a + + ] e x o n h l g h - K + t e n s i o n I n r a t u t e r u s 54 10. The r e l a t i o n s h i p b e t w e e n % i n c r e a s e I n cAMP a n d % r e l a x a t i o n p r o d u c e d b y I s o p r o t e r e n o l - i x -l n d e p o l a r i z e d r a t uterus 55 11. The r e l a t i o n s h i p between % Increase i n cAMP and a b s o l u t e t e n s i o n reduced by i s o p r o t e r e n o l In d e p o l a r i z e d r a t uterus 56 12. The r e l a t i o n s h i p between % Increase i n cAMP & 13. and % r e l a x a t i o n or AT l n g produced by i s o p r o t e r e n o l i n d e p o l a r i z e d r a t uterus 61 14. The Infl u e n c e o f RO 20-1724 on cAMP l e v e l s l n the absence and presence of I s o p r o t e r e n o l i n d e p o l a r i z e d r a t uterus , 63 15. The e f f e c t o f a C a + + - d e f I c l e n t s o l u t i o n on spontaneous u t e r i n e c o n t r a c t i o n s 66 16. The e f f e c t of I s o p r o t e r e n o l on e l e c t r i c a l p r o p e r t i e s of r a t uterus 69 17. Hlgh-K +-induced membrane d e p o l a r i z a t i o n and t e n s i o n l n r a t uterus 70 18. A schematic diagram d e p i c t i n g i n t e r a c t i o n s i n v o l v i n g ^-adrenoceptor s t i m u l a t i o n , cAMP, and C a + + In smooth muscle c e l l 87 -X-ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Dr. John H. McNeill. His excellence in guiding me through academics and research has been equally matched by his constant encouragement to me as a fri e n d . Dr. V. Palaty's guidance throughout the course of this study as a teacher as well as a member of the advisory committee has been valuable to me. I am thankful to Drs. S. Katz and B. Roufogalls for t h e i r constructive c r i t i c i s m s of this work. The f i n a n c i a l assistance to the author from the University of B r i t i s h Columbia, The B r i t i s h Columbia Heart Foundation, and The Canadian Heart Foundation is g r a t e f u l l y acknowledged. I would l i k e to thank Pamela Livingstone who, as a f r i e n d , took typing this thesis in a short period as a challenge and won. F i n a l l y , I wish to recognize my Indebtedness to my parents, brothers, and s i s t e r s who provided continuous warmth and encour-agement during my ef f o r t s to reach this goal. - 1 -INTRQDUC TION I. Excitation-Contraction Coupling and Relaxation In Smooth  Muscle The term 'excitation-contraction coupling* (E.C. Coupling) was introduced by Sandow ( 1 9 5 2 ) to describe how changes In e l e c t r i c a l p o t e n t i a l of the sarcoplasmic membrane of the s k e l e t a l muscle induce contraction. The steps that are thought to comprise the E.C. Coupling have been proposed and are as follows (Ebashl, 1 9 7 6 ) J 1) The action potential at the sarcolemma is conducted into the i n t e r i o r of muscle f i b e r through the tubular system (T-system), 2 ) The depolarization of the T-system membrane releases stored C a + + ions from the terminal clsternae of the sarcoplasmic reticulum (SR), 3 ) C a + + Ions then reach troponin located In the t h i n filament to produce the contraction ( i . e . the i n t e r a c t i o n of the thick filament with the t h i n filament) by removing the depressive e f f e c t of Ca -free troponin located in the t h i n filament, 4 ) If the influence of depolarization ends, C a + + ions are reaccumulated by the whole surface of the SR u t i l i z i n g the energy of ATP; reduction of the Ca ion concentration releases Ca from troponin, and relaxa t i o n follows. Step 2 is considered to be an e s s e n t i a l and the most c r u c i a l one In the E.C. Coupling (Ebashl, 1 9 7 6 ; Endo, 1 9 7 7 ) . The term E.C. Coupling is also applied to smooth muscle assuming an analogous explanation for these contraction-relaxation phenomena. However, i n smooth muscle, the d i s t r i b u t i o n of the T-system i s not apparent, and a c h a r a c t e r i s t i c feature of the membrane i s the presence of numerous •lnpocketings' on the membrane surface (Kurlyama et a l , 1 9 7 7 ) . While In the s k e l e t a l muscle, the - 2 -Ca storage s i t e s are mainly located i n the SR, and not i n the T-system, i t is speculated that i n smooth muscle, microvesicles are important C a + + storage s i t e s (Somlyo, 1975). The sources of act i v a t o r C a + + have not been quanti t a t i v e l y characterized, although there is a general consensus of opinion that both the inf l u x of e x t r a c e l l u l a r C a + + and Its release from i n t r a c e l l u l a r compartments could contribute to a c t i v a t i o n of smooth muscle (Hlnke, 1965; Hurwltz and Suria, 1971; Somlyo and Somlyo, 1971). It has been pointed out that these two s i t e s do not play an equally Important physiological r o l e In a l l types of smooth muscles (Hurwltz, 1977). The studies performed to l o c a l i z e i n t r a c e l l u l a r pools of C a + + In smooth muscle have revealed three d i f f e r e n t s i t e s where the divalent ion can be concentrated; the endoplasmic reticulum, the mitochondria, and the surface membrane (see for example, Janis and Daniel, 1977). The ways In which excitatory agents induce the movement of Ca ions from storage s i t e s to the cytoplasm include at least two d i f f e r e n t mechanisms. One involves the generation of action potentials or a depolarization of the membrane (B'ulbring, 1955; Marshall, 1959; Somlyo and Somlyo, 1968) and the other operates In an i l l - d e f i n e d manner that is independent of any change i n membrane potential (Evans and S c h i l d , 1957; Kuriyama et a l , 1977). 4 . 4 . The removal of Ca from the cytoplasm and the subsequent I n i t i a t i o n of the relaxa t i o n process are probably accomplished ++ by energy dependent Ca transport systems i n smooth muscle (Batra 1973; Carsten, I969). Relaxation of smooth muscle presumably occurs when the cytoplasmic free C a + + concentration f a l l s below a c r i t i c a l l e v e l of 10~ 7 M (Bianchl, 1969). This - 3 -decrease In I n t r a c e l l u l a r Ca concentration Induced by smooth muscle relaxants could r e s u l t from an increase i n active C a + + extrusion by the c e l l membrane, a decrease in passive C a + + i n f l u x , rebinding of C a + + to i n t r a c e l l u l a r storage s i t e s or any com-bination of these processes (Van Breemen et a l , 1 9 7 3 ) . The i n t r a c e l l u l a r Ca concentration may be considered as a dynamic balance between supply and removal. The supply may be due to C a + + i n f l u x and release from such structures as the Inner plasmalemmal surface, SR, and mitochondria. The removal of C a + + is mediated by the processes mentioned above. Relaxation, then, w i l l r e s u l t from ++ a disturbance of this dynamic balance i n favor of Ca removal (Van Breemen, 1 9 7 5 ) . Catecholamines cause rela x a t i o n of smooth muscle by stimulation of ^ -adrenergic receptors ( M i l l e r and Marshall, 1 9 6 5 ; Furschgott, 1 9 7 0 ) . The mechanlsm(s) by which ^-adrenoceptor stimulation of smooth muscle leads to a decrease i n cytoplasmic free Ca concentration and subsequent relax a t i o n has been under intense investigation over a period of years. The discovery of c y c l i c 3 ' i 5 *-adenosine monophosphate (cAMP) and i t s possible r o l e In the mechanism of glycogenolytic action of adrenaline i n l i v e r (Sutherland and R a i l , 1 9 5 7 ) eventually led to the develop-ment of the concept that cAMP may play an Important r o l e in the In t r a c e l l u l a r events induced by the catecholamines l n a va r i e t y of systems. In the following section, cAMP-second messenger hypothesis postulated for catecholamine-Induced relaxation of smooth muscle w i l l be discussed with p a r t i c u l a r reference to uterine smooth muscle. -4-I I . cAMP and Smooth Muscle Relaxation Sutherland and R a i l ( i 9 6 0 ) postulated on the "basis of the • second messenger ' hypothesis that the relaxation produced by Jj-adrenergic receptor stimulation i n smooth muscle was mediated by I n t r a c e l l u l a r l e v e l s of cAMP. Sutherland and Roblson ( I 9 6 6 ) established c e r t a i n c r i t e r i a for the experimental evidence necessary to support the second messenger hypothesis for cAMP i n smooth muscle relaxation. Agents which are presumed to a l t e r uterine c o n t r a c t i l i t y through a change i n cAMP l e v e l s , a) should have a demonstrable e f f e c t on the adenylate cyclase and/or phosphodiesterase (PDE) a c t i v i t y of the tissue, b) should change the cAMP content of the tissue i n a d i r e c t i o n and with a time course consistent with the tissue response, c) should have pharmacological effects which are potentiated by drugs which i n h i b i t PDE and, d) th e i r effects should be mimicked by the addition of exogenous cAMP or i t s derivatives to the tissue preparation. Attempts to s a t i s f y these c r i t e r i a i n smooth muscle have been successful only to a li m i t e d extent and the matter is s t i l l a debatable issue as w i l l be clear from the following discussion. Triner e_t a l (1970a, 1971) were the f i r s t to demonstrate that adrenaline, noradrenaline and isoproterenol caused a dose-dependent increase in the a c t i v i t y of adenylate cyclase i n r a t uterus homogenates. A l l three agonists increased the act-i v i t y to about the same maximum. Propranolol, a ^-adrenergic blocking agent, antagonized the stimulatory effects of these agonists on the adenylate cyclase. When the stimulatory e f f e c t - 5 -of a low concentration of adrenaline (10"" M) was prevented by propranolol, there was a s i g n i f i c a n t decrease in the adenylate cyclase a c t i v i t y below control, i n d i c a t i n g an unmasked i n h i b i t o r y component of adrenaline action. In the presence of phentolamine, an o^-adrenergic blocking agent, the adenylate cyclase a c t i v i t y tended to be higher. When the ef f e c t of catecholamines on uterine c o n t r a c t i l i t y was investigated i n the above studies, i t was observed that the r a t i o of equipotent concentrations of adrenaline and noradrenaline xvlth regard to the i r Inhibitory e f f e c t on c o n t r a c t i l i t y was s i m i l a r to the r a t i o of the i r stimulatory e f f e c t on adenylate cyclase. Isoproterenol and phenylephrine-lnduced changes i n c o n t r a c t i l i t y could also be correlated with effects on adenylate cyclase (Triner et a l , 1971). Furthermore, propranolol, which was shown to antagonize the catecholamine-induced stimulation of adenylate cyclase also prevented the In h i b i t i o n by adrenaline of oxytocin-induced contraction. Poch and Kukovetz (1971) found that theophylline and papaverine were i n h i b i t o r s of phosphodiesterase i n uterus, and that doses which p a r t i a l l y i n h i b i t e d this enzyme i n homo-genates relaxed the uterus. Kroeger and Marshall (1974) demonstrated that papaverine s i g n i f i c a n t l y Increased the tissue cAMP content of the r a t myometrium and also caused I n h i b i t i o n of the spontaneous contractions of the muscle. Uruno et a l (1975) observed, i n isolated r a t uterus, a s i g n i f i c a n t increase in tissue cAMP concentration at a time when the muscle was just beginning to r e l a x . i n response to papaverine. - 6 -There are several reports showing changes in cAMP levels associated with catecholamine-Induced uterine relaxation. Dobbs and Roblson (1968) reported that isoproterenol relaxed the Isolated rabbit uterus and elevated cAMP levels. A subsequent study showed that isoproterenol caused both relaxation as well as Increases ln Intracellular levels of cAMP in rat uterus (Trlner et a l , 1 9 7 0 b ) . Both of the effects of Isoproterenol were blocked by propranolol. Marshall and Kroeger (1973) observed In pregnant rat myometrium, that isoproterenol Increased tissue cAMP levels with a time course paralleling that of relaxation both at 3 7 ° and 10°C. Papaverine showed similar actions. Vesln and Harbon (1974) reported a good correlation between the effect of adrenaline on adenylate cyclase stimulation, with a subsequent rise ln cAMP levels, and its relaxing effect on rat myometrium. Birnbaum et a l (1975) concluded, after studying the effects of enantlomers of isoproterenol on isolated rat uterus, that their data supported the postulate that cAMP was formed following interaction of isoproterenol with a receptor that is similar to the one activated to produce a mechanical effect. A dose-dependent effect of other synthetic^-adrenergic agonists, such as albuterol and terbutaline, on cAMP content in rat uterus has been demonstrated (Vulllemoz et a l , 1 9 7 5 ) . In an attempt to satisfy the third criterion regarding potentiation of the catecholamine effects by phosphodiesterase inhibition, most workers have employed theophylline and papaverine as the enzyme inhibitors. Dobbs and Roblson ( I 9 6 8 ) and Roblson et a l ( I 9 6 9 ) reported that theophylline potentiated the effects of isoproterenol on relaxation and Increases ln cAMP levels in -7-rat uterus. Addition of theophylline potentiated the inhibitory effects of adrenaline on vasopressin-Induced contraction in Isolated rat uterus (Mltznegg et a l , 1 9 7 1 ) . Regarding the fourth criterion, It has been shown that both cAMP and dlbutyryl cAMP (a more l i p i d soluble derivative of cAMP) could Inhibit oxytocin-induced contraction of rat uterus (Mltznegg et a l , 1970; Trlner et a l , 1971). Both cAMP and dlbutyryl cAMP have also been shown to reverslbly Inhibit spontaneous motility In isolated rat uterus, and the contractile response Induced by vasopressin (Mltznegg et a l , 1 9 7 1 ) . Marshall and Kroeger (1973) reported that dlbutyryl cAMP and Isoproterenol both relaxed hlgh-K*" contracted pregnant rat myometrium and both decreased tissue Ca* + content as measured by the Lanthanum technique. In estrogen-dominated rabbit uterus, dlbutyryl cAMP produced a relaxation which mimicked that produced by isoproterenol (Nesheim et a l , 1 9 7 5 ) . Similar lines of evidence have been presented to support the postulated role of cAMP In the drug-induced relaxation of smooth muscles other than uterus. These have been described at length In several reviews, (see Bar, 197^ and Andersson et a l , 1975 for Intestinal and tracheal smooth muscles; and Namm and Leader, 1976 for vascular smooth muscle). Thus, the evidence for the belief that cAMP functions as the intracellular mediator for the relaxant effects of ^-adrenoceptor activation in uterine smooth muscle can be summarized as follows 1 1 ) The a b i l i t y of various catecholamines to relax the uterus and to increase Its cAMP content is related to the - 8 -effectiveness of these amines as activators of^-adreno-ceptors, the order of potency being isoproterenol^ adrenaline ^noradrenaline. l i ) Both relaxation and increases in cAMP by the catecholamines are prevented by /^-adrenergic blockers. i l l ) There is a dose-response relationship between the ^-adrenergic amines and the Increases In tissue cAMP content and relaxation. iv) The Increases In cAMP levels produced by stimulation of ^-adrenoceptors correlates temporally with relaxation. v) Inhibitors of phosphodiesterase relax the muscle and potentiate the inhibitory effects of ^ -adrenoceptor activation. vi) The contractions produced by oxytocin and vasopressin are inhibited by exogenous cAMP or dibutyryl cAMPi Although there is a considerable amount of data available in support of the cAMP-second messenger hypothesis, the subject is not free from controversy. F i r s t reports of dissociation between isoproterenol and papaverine-Induced changes in cAMP levels and relaxation in rat uterus have been provided by Polacek and Daniel ( 1 9 ? 1 ) and Polacek et a l (1971). These Investigators showed that the relaxation of rat myometrium by adrenaline was associated with increased cAMP levels, but its reversal to contrac-tion by propranolol did not restore the cAMP concentration to basal levels. Further, the antagonism by adrenaline of oxytocin-lnduced contractions could be reversed by propranolol when cAMP levels were s t i l l elevated. They concluded that the increase ln cAMP was neither necessary nor sufficient for inhibition of contraction, Nesheim et a l ( 1 9 7 5 ) reported that isoproterenol (2xlO"8M) produced an inhibition of mechanical acti v i t y of -9-rabbit uterus without increasing cAMP levels, although at a higher concentration (2xlO~ 6M), Isoproterenol produced both Inhibition of mechanical a c t i v i t y and an Increase In cAMP levels. They concluded that cAMP does not seem to be an oblig-atory link between stimulation of ^ -adrenoceptor and relaxation. Diamond and Holmes (1975) concluded from their study on the effect of isoproterenol on depolarized rat myometrium that the relaxation and cAMP Increases caused by Isoproterenol are concurrent but not necessarily causally related events. These conclusions are supported by the findings of Verma and McNeill (1976) who reported that isoproterenol could cause dose-dependent (10~^ to 10"^ M) relaxation of the hlgh-K* depolarized rat uterus, but only 10"^ M Isoproterenol produced a significant Increase In cAMP levels. They suggested that an Increase In whole tissue levels of cAMP was not necessary in order for Isoproterenol to produce relaxation of the depolarized rat uterus. Harbon and Clauser (1971) demonstrated that cAMP levels In rat uterus were increased by relaxant doses of adrenaline as well as stimulant doses of prostaglandin E i (PGEi). This apparent discrepancy was f i r s t explained by suggesting that two drugs with opposite physiological effects Increased cAMP levels in separate intra-cellular compartments. However, In recent publications from the same laboratory (Harbon et a l , 1976 and Vesln et a l , 1978) the hypothesis of cAMP compartamentallzation was Invalidated since for a given concentration of Intracellular cAMP Induced by either the contracting agent (PGE^) or the relaxing agent (adrenaline), an Identical degree of saturation of Intracellular cAMP receptor proteins and equal activation of myometrlal cAMP- dependent protein kinases were obtained. These observations render the -10-role of cAMP In uterine smooth muscle relaxation more complex. Several observations in other smooth muscles have also been reported that are not consistent with the cAMP-second messenger concept, Andersson (1973) found that In the bovine mesenteric a r t e r y , ^ - adrenergic stimulation led to increases in cAMP levels. However, a rise In cAMP at the onset of relaxation, which would be expected l f cAMP triggered the relaxant response, was not observed. Daniel and Crankshaw (197 * 0 tested the cAMP hypothesis by studying relaxation In rabbit pulmonary artery. They found that exposure to isoproterenol for two minutes re-laxed the serotonin-contracted arteries without increasing cAMP levels. In their study, marked relaxation could occur with l i t t l e or no increase in cAMP and an increase in cAMP of a substantial magnitude produced only minimal relaxation. They concluded that there Is no simple relationship between cAMP levels and the degree of relaxation in smooth muscle. This interpretation Is supported by the work of Collins and Sutter (1975) In rabbit anterior mesenteric vein. It Is apparent from the foregoing discussion that no uniformity exists in the data concerning the role of cAMP In smooth muscle relaxation. Although the evidence for a role of cAMP as a mediator of smooth muscle relaxation appears to be strong for^-adrenergic agonists, some negative evidence has been reported and a causal relationship between cAMP increases and -adrenergic relaxation has not yet been established (Daniel and Janls, 1975; Diamond, 1 9 7 8 ) . Thus , one objective of this project was to define the interrelationship between cAMP and relaxation In uterine smooth muscle during^-adrenoceptor stim-ulation. -11-III. Cyclic GMP (cGMP) and Smooth Muscle Function Goldberg et a l (1975) proposed the concept of biological regulation through opposing actions of cAMP and cGMP. They stated this as Yin yang hypothesis, symbolizing a dualism between opposing natural forces but also taking into account that under certain circumstances the forces may enter into a mutual inter-action. The general hypothesis has been suggested that increases in cAMP levels promote smooth muscle relaxation, whereas increases in cGMP levels promote smooth muscle contraction. Thus, these two nucleotides are thought to function ln a reciprocal fashion ln the control of smooth muscle contractility. In support of the above concept are the findings of Hardman and Sutherland (I969) who demonstrated the presence of guanylate cyclase ln rat uterus. Goldberg et a l (1973» 197*0 demonstrated in Isolated rat uterus that methachollne and other stimulatory agents such as oxytocin, PGE2 could a l l enhance the accumulation of cGMP. Atropine prevented the methacholine-lnduced Increases ln cGMP levels. Furthermore, Johansson and Andersson (1975) reported that cAMP and cGMP levels fluctuated during various stages of contraction-relaxation cycle in spontaneously contracting rat uterus. It was suggested that these two nucleotides may also be involved in the normal physiological regulation of uterine motility. On the other hand, Diamond's laboratory has consistently made observations that do not support the above Interpretations. For example, Diamond and Hartle (197*+) could not find any significant changes ln the levels of either cAMP or cGMP at any stage ln the contraction cycle of rat uterus. Further, carbachol-induced contractions of estrogen-primed - 1 2 -rat uteri were not accompanied by changes in cGMP levels (Diamond and Hartle, 1976) . Even in tissues ln which large increases in cGMP levels could be detected during carbacol-ln-duced contractions (e.g. guinea pig myometrium), the contractions appeared to precede the cGMP increases by several seconds (Diamond, 1978). In view of the inconsistency ln the above data, we decided to reinvestigate the changes ln cAMP and cGMP levels during various stages of the contraction-relaxation cycle of rat uterus. It was also decided to determine whether the postulated reciprocal relationship between cAMP and cGMP could be demonstrated durlrrg isoproterenol-lnduced relaxation of uterine smooth muscle, IV. Influence of Ionic Environment on Drug Responses ln  Smooth Muscle It is generally accepted that the Ionic environment of the c e l l profoundly affects the cellular response of the tissue (Bohr, 1964). For example, the presence of Na"*" ions ln fehe extracellular medium is necessary for the maintenance of normal function in a variety of excitable tissues including uterus (Marshall, I963). Alterations in the concentration of extracell-ular ions such as Na + and Ca** have been shown to affect elec-t r i c a l as well as mechanical activities of smooth muscle (Osa, 1971; 1973)* In pregnant mouse myometrium, the effect of carb-achol was depressed ln low Na + solutions. In Na + free solutions, neither oxytocin nor carbachol was effective ln producing changes in membrane potential or in tension development (Osa and Taga, 1973)• Magaribuchi et a l (1971) showed that the removal of ex-ternal Na + abolished the effect of noradrenaline or isoproterenol -13-on the el e c t r i c a l and mechanical acti v i t i e s of guinea pig vas deferens. In another study,Magarlbuchi and Osa (1971) observed in pregnant mouse myometrium that isoproterenol restored to normal the depolarization and contracture evoked in zero Na+, and that the existence of external Ca** was essential to production of the repolarization. Edman and Schlld (1963) demonstrated a non-competitive . but relatively specific antagonism of Ca++ by adrenaline and isoproterenol in uterine smooth muscle. Osa and Taga (1973) showed the Inhibitory effect of excess C a + + on carbachol actions on pregnant mouse myometrium. Elevation of extracellular C a + + antagonized the relaxant actions of tetracaine, papaverine and nitroglycerine (Diamond and Marshall, 1 9 6 9 a ) . Further, in the presence of 5.4 mM Ca + +, adrenaline failed to reduce the spike frequency.„ Kroeger and Marshall (1973) demonstrated that the elimination of Ca** from the bathing medium abolished hyper-polarization produced by isoproterenol in rat myometrium. Exposure of rat myometrium to a Ca + +-free solution has been shown to accentuate the increase of tissue cAMP content produced by Isoproterenol (Polacek and Daniel, 1971$ Kroeger and Marshall, 197*0. Magnesium has been similarly shown to Influence smooth muscle responses to various drugs, Clegg et a l (1966) showed that an increase in extracellular Mg** inhibited the contractile response of the guinea pig myometrium to PGEi and PGEg. Altura et a l (1976) demonstrated that Mg** ions can modulate prosta-glandin- induced contraction and relaxation in vascular smooth muscle. The effect of Mg4"*" on isoproterenol-lnduced changes in mechanical responses has been demonstrated in vascular smooth -14-muscle (Turlapathy et a l , 1 9 7 5 ) . Mg** decreased the sensitivity of rabbit aortic strips ln response to isoproterenol and enhanced the degree of reserplne-Induced supersensitivity to Isoproterenol. Incubation in Mg**-free solutions reverslbly blocked the hyper-polarlzlng effect of cAMP on vascular smooth muscle (Somlyo e_t a l , 1 9 7 2 ) . In view of these ionic Interactions in smooth muscle, i t was decided to investigate the Influence of Ionic composition of the physiological salt solution on the mechanical relaxation and biochemical events (such as cAMP and cGMP) Induced by isoproterenol. It was thought that the study of ionic Influences on isoproter-enol-lnduced changes In the cyclic nucleotide levels and relax-ation, would also allow us to examine further the relationship between cAMP and relaxation under various experimental conditions. V. Relationship Between Membrane Hyperpolarlzation and Mechanical Relaxation by the Catecholamines ln Smooth Muscle The alterations ln the electrophysiological characteristics Induced by the adrenergic amines Is one of the mechanisms thought to be responsible for the Inhibitory effect of /^-adrenoceptor stimulation of smooth muscle (see review by Marshall, 1 9 7 3 ) . In a spontaneously contracting smooth muscle such as the uterus, activation is initiated from the pacemaker cells and the action potentials are then propagated to the rest of the muscle (Kurlyama and Csapo, I 9 6 I ) . Kumar et a l ( I 9 6 5 ) reported hyper-polarlzation of the c e l l membrane of Isolated human myometrium by adrenaline during Inhibition of spontaneous contractility. Marshall (1967) showed that ^ -adrenergic agents Inhibit spontan-- 1 5 -•eous contractions and action potentials of pregnant r a t myometrium. Diamond and Marshall (1969b) observed that adrenaline, noradren-a l l n e a n d Isoproterenol abolished spontaneous m o t i l i t y of Isolated r a t myometrium. This action of the catecholamines was accompanied by hyperpolarlzatlon of the c e l l membrane. The I n h i b i t i o n of m o t i l i t y and the hyperpolarlzatlon were correlated to the extent that both were beta-adrenergic effects as shown by studies with adrenergic blocking agents. Magarlbuchl and Osa (1971) s i m i l a r l y observed i n pregnant mouse myometrium that i n h i b i t i o n of spontaneous m o t i l i t y by adrenaline, noradrenaline and Isoproterenol was associated with hyperpolarlzatlon of the membrane. Kroeger and Marshall (1973) showed that Isoproterenol and exogenous dl b u t y r y l cAMP hyperpolarlzed r a t myometrial membrane and that amlnophylllne (a PDE i n h i b i t o r ) acted s i m i l a r l y . Furthermore, amlnophylllne potentiated the action of subthreshold doses of Isoproterenol. I t has been found that during exposure of r a t uterus to Isoproterenol the time course for the devel-opment of hyperpolarlzatlon followed that for the Increase i n tissue cAMP content (Marshall and Kroeger, 1 9 7 3 ) . Bulbrlng (195*0 and Bulbrlng and Tomita ( I 9 6 9 ) showed that the i n h i b i t o r y action of adrenaline on guinea pig taenia c o l l was associated with hyperpolarlzatlon and suppression of spon-taneous spike discharge. Shuba et a l (1976) reported s i m i l a r effects of noradrenaline In the same tissue. Somlyo and Somlyo (1968) have shown that /^-adrenergic agents under appropriate conditions hyperpolarlze the vascular smooth muscle membrane. Potentiation of ^-adrenergic hyperpolarlzatlon by theophylline and hyperpolarlzatlon by d l b u t y r y l cAMP have also been demonstrated -16-ln vascular smooth muscle (Somlyo e_t a l , 1970; Somlyo and Somlyo, 1 9 7 D . Although the alterations in electrical events seem to be an Integral part of drug action on smooth muscle, reports from several laboratories have Indicated that under certain conditions the catecholamines can produce contraction and relaxation of smooth muscles without changes, in membrane potential. Thus, the inhibitory action of the catecholamine Is usually, but not invariably, associated with membrane hyperpolarlzation ln uterus and other smooth muscles (Daniel et a l , 1970? Bulbring, 1 9 7 3 ) . Diamond and Marshall (1969b) observed that a low concentration of adrenaline (2xlO~8M), which reduced but did not abolish spontaneous contractions, did not cause hyperpolarlzation. In another study, i t was. shown that uterine strips which were de-polarized by hlgh-K* solutions could s t i l l be relaxed by adrenaline although the membrane potential was not altered by the drug under these conditions (Diamond and Marshall, 1 9 6 9 a ) . This appears to be in agreement with the original observation made by Evans ejt a l (1958) that rat uterus depolarized by an Isotonic potassium solution could s t i l l be relaxed by Isoproterenol. In view of the above data, i t was decided to reinvestigate the relation-ship between isoproterenol-lnduced relaxation and changes in membrane potential In normal and depolarized rat uterus. VI. Use of Depolarized Preparation to Study Drug Action on  Smooth Muscle One of the d i f f i c u l t i e s encountered ln studying smooth muscle relaxation is due to the problem of accurately determining the time of onset of relaxation induced by the agonist. The use -17-of depolarized muscle offers the advantage that the onset of relaxation induced by the agonist can be determined much more precisely than It can be in spontaneously contracting preparations, and accurate temporal correlations or dissociations can therefore be made. The depolarized preparation was f i r s t Introduced by Evans and Schlld in 1957. They showed that smooth muscle retains its a b i l i t y to contract in response to drugs when suspended in Ringer's solution in which the sodium Ions have been replaced by potassium Ions (Evans and Schild, 1 9 5 7 ) . Similar conclusions were reached Independently by Singh and Acharya ( 1 9 5 7 ) . It was suggested that this kind of preparation provides a means of studying the action of drugs on the contraction of smooth muscle under conditions uncomplicated by their effects on membrane polarization and electrical conduction (Edman and Schlld, 1962). The studies from Schlld's laboratory on the action of Isoproterenol on the high-K+ depolarized rat uterus Indicated that the KC1-depolarlzed rat uterus was highly sensitive to isoproterenol (Schild, 1966, 1967). Further studies with a ^-adrenergic blocking agent suggested that isoproterenol activates the same receptors in normal (polarized) and depolarized uterus. Since drugs produce the same effects in polarized and depolarized smooth muscle and act in similar concentrations and on the same receptors, It would appear that the same processes are activated (Schlld, 1967). As pointed out earlier, Diamond and Marshall (1969b) have employed a high-K* depolarized rat uterus preparation to study the relationship between the catecholamine-induced relaxation and -18-hyperpolarization. Diamond and Holmes (1975) were the f i r s t to use the hlgh-K + depolarized r a t myometrium to investigate the re l a t i o n s h i p between isoproterenol (and other smooth muscle relaxants)-Induced r e l a x a t i o n and changes i n cAMP l e v e l s . A study from our laboratory (Verma and McNeill, 1976) also employed the high-K + depolarized r a t uterus to test the cAMP-second mess-enger hypothesis for lsoproterenol-induced relaxation. Based on the above Information, i t was decided to use the hlgh-K + depolarized preparation as: the experimental model to further Investigate the r e l a t i o n s h i p between ^-adrenoceptor-induced relaxation and changes i n the c y c l i c nucleotide l e v e l s in uterine smooth muscle. -19-VII. Specific Alms The specific alms of this project were as follows» 1) To determine If the levels of cAMP and cGMP fluctuate during spontaneous contractions of rat uterus in a manner postulated by the Yin-Yang hypothesis (Goldberg et a l , 1975). 2) To investigate the relationship between Isoproterenol-lnduced relaxation and changes In cAMP and cGMP levels in hlgh-K+ depolarized rat uterus. 3) To study the influence of variation In ionic composition of the depolarizing medium on isoproterenol-lnduced relaxation and cAMP levels In rat uterus. Thus, i t was decided to study the influence of variation in external i r , Na , Ca , and Mg + + concentrations on the above two parameters. 4) To investigate the relationship between isoproterenol-lnduced relaxation and membrane hyperpolarlzatlon in normal and high-K + depolarized uterine preparations. -20-MATERIALS AND METHODS I. Tissue Preparation Female Wlstar rats, weighing up to 250 grams, were estrogen primed by injecting them with diethylstilbestrol (300 ug., l.p.) dissolved ln peanut o i l 24 hours before sacrifice. Rats were sacrificed by a sharp blow to the head and the excised uterine horns were placed in a physiological salt solution (Trls-buffer) of the following composition (ln mM)i NaCl, 125.0; CaCl2» 1.8; KC1, 2.4; MgCl2, 0.5; glucose, 11.0; and Tris [Tris(hydroxymethyl) aminomethane^J , 23.8. The pH was adjusted to 7.50 (* 0.05) with HCl, and the solution was continuously aerated with 100$ oxygen and kept at 37°C (* 0.5°C). The uterine strips were allowed to equilibrate ln Isolated muscle baths for about JO minutes and then Isometric tension was recorded on a Grass polygraph. Two strips were obtained from each of the two uterine horns from a given rat. A l l drug studies were carried out on a paired basis - i.e. one strip always served as the control for the other s t r i p . II. Protocol for Individual Studies 1) Study with spontaneously contracting uterus During equilibration period, the muscles developed rhythmic spontaneous contractions. The muscle strips were frozen during various stages of contraction and relaxation by means of tongs previously chilled ln a mixture of dry Ice and 2-Methylbutane, The tension achieved by the muscle at that point was recorded. The frozen samples were stored at -80°C u n t i l analyzed for cyclic nucleotides. - 2 1 -In order to study the effect of Isoproterenol, the drug was added to the "bath when the muscle was In the resting state and then the muscle was frozen after 30 seconds. A time-response study had Indicated that a maximum increase in cAMP levels after Isoproterenol occurred at 30 seconds (Verma and McNeill, 1 9 7 6 ) . The control stri p was frozen when the muscle was In the resting state. 2) Studies with depolarized uterus 2 a . influence of variation In [fit) ex a n d. jffa*] ex In order to study the influence of variation in the ionic composition of the depolarizing medium 6n Isoproterenol-lnduced relaxation and changes in cyclic nucleotides levels, three solutions were employed. The composition of these solutions is shown in Table 1. Solution I is the 'normal' Tris-buffer? solutions II, III and IV were used as the depolarizing media. In solution II, a l l the NaCl was replaced by KC1, thus bringing the KC1 concentration to 127.4 mM. In solution III, KC1 con-centration reduced to 47.5 mM with 0 NaCl and isosmolarity was maintained by the addition of sucrose. Solution IV contained 47.5 mM KC1 and 80 mM NaCl. The concentrationssof CaCl2» MgCl 2. glucose and Tris remained the same in a l l four solutions. Before changing the bathing solution to a depolarizing solution (solution II, III or IV), the muscle st r i p was allowed to contract spontaneously for 30 minutes. Addition of the depolarizing solution immediately produced a sustained contracture. The contracture stabilized at 15 minutes and at this time the mus-—8 —4 cle was either frozen as a control or isoproterenol ( 1 0 ~ or 10" Mj fi n a l bath concentration) was added in a 1 ml. volume into a 50 ml. bath. The 15 minute time period was chosen based on a TABLE 1. Composition ( i n mM) of b u f f e r s o l u t i o n s used. Ingredient 'Normal' 127 mM KC1 47.5 mM KC1 47.5 mM KC1 T r i s - b u f f e r (0 NaCl) (0 NaCl) + (I) ( I I ) ( I I I ) 80 mM NaCl (IV) NaCl 125.0 0.0 0.0 79.9 KC1 2.4 127.4 ^7.5 47.5 Sucrose 0.0 0.0 1 5 9 . 8 a 0.0 CaClg 1.8 1.8 1.8 1.8 MgCl 2 0.5 0.5 0.5 0.5 Glucose 11.0 11.0 11.0 11.0 Tr i s 23.8 23.8 23.8 23.8 159.8 mM sucrose was taken to be isosmolar w i t h 79.9 mM NaCl, Tr i s (hydroxymefchyl) amlnome thane. -23-prevlous study (Verma and McNeill, 1976) which showed that cAMP values in the depolarized strips were not significantly elevated relative to control values after 15 minutes of depolarization. Isoproterenol strips were frozen at 30 seconds after the addition of the drug. The frozen samples were used for cyclic nucleotide determinations. Isoproterenol-lnduced relaxation in these muscles was computed as w i l l be described in a later section. 2"b* Influence of variation in [ca**] ex Two depolarizing solutions were used in these experiments! 4-7.5 mM K C 1 ( 0 NaCl) and 47.5 mM K C 1 (+ 80mM NaCl). The following external Ca** concentrations were usedi zero Ca**, Ca**-free, 0.9 mM, 1 . 8 , 3 .6, 5.4, and 7 . 2 . The experimental protocol consisted of depolarizing the muscle by a depolarizing solution containing the indicated Ca** concentration. 1 . 8 mM was the normal Ca** concentration used. Ca**-free medium contained no added Ca** and thus was nominally Ca**-free. A zero Ca** condition was achieved by either using D - 6 0 0 or EGTA [j5thyleneglycol-bls-(^-amlnoethyl ether) N.N'Tetracetlc acid]. D-600, a calcium-antagonist, was used at a concentration of 10"^M In the presence of normal Ca** ( 1 . 8 mM). D-600 (10"^M, fi n a l bath concentration) was added 15 minutes before addition of a depolarizing medium. The depolarizing solution also contained 10"5M D - 6 0 0 . For the EGTA experiments, the experimental protocol consisted of incubating the muscle for 15 minutes In Ca**-free Tris-buffer containing 0 . 2 mM EGTA and then changing the bathing solution to a Ca**-free depolarizing solution containing 0 . 2 m M EGTA. In the experiments with EGTA, the concentration of Mg** in the solutions was increased from 0 . 5 to 0 . 7 mM in order to avoid chelation of Mg** ions. -24-As indicated i n section 2 a , the muscle was depolarized with hlgh-K + solutions for 15 minutes and then exposed to isoproterenol for 30 seconds and the tissues were frozen as described previously. Appropriate controls were also c o l l e c t e d . 2 c , Influence of v a r i a t i o n in rMg~] ex The influence of 0 . 5 m M (normal) and 2 . 5 m M Mg + + in the depolarizing s o l u t i o n on isoproterenol-lnduced relaxation and cAMP levels was studied in high-K + solution. Both mediaj 4-7.5mM KC1 ( 0 NaCl) and 4 7 . 5 m M KC1 (+ 80mM NaCl) were employed. The influence of 2 . 5 m M Mg + + was studied at 1 .8mM (normal) as well as 3.6mM C a + + concentration. The experimental procedure was s i m i l a r to that described In section 2 a . 2 d . Studies with a PDE i n h i b i t o r , RO 20-1724 Spontaneously contractive uterine s t r i p s were f i r s t exposed to 10~^M RO 2 0 - 1 7 2 4 (prepared l n 50% propylene glycol) for 1 5 minutes. At this time the bathing s o l u t i o n was changed to the hlgh-K depolarizing medium ( 4 7 . 5 m M KC1, zero Na ) containing - 4 * 10 M RO 2 0 - 1 7 2 4 . At 1 5 minutes a f t e r the high-K* depolarization, p 1 0 " M isoproterenol was added to the bath and the muscle frozen at 30 seconds - for cAMP determination. Isoproterenol-lnduced rel a x a t i o n was also computed. III. C y c l i c Nucleotide Determinations 1) Tissue extraction The method of extraction was modified from that of Gilman ( 1 9 7 0 ) as followsj A 5 0 - 8 0 mg. frozen sample of the uterus was r a p i d l y homogen-ized i n 3 ml. of 5% t r i c h l o r o a c e t i c acid (TCA) at 4°C. C e n t r i -fugation i n a bench centrifuge set at i t s maximum speed for 15 -25-mlnutes removed a l l the Insoluble proteins and the p e l l e t thus formed was discarded. The supernatant was extracted 5 times using 10 ml. of ether each time to remove TCA which otherwise interferes with the c y c l i c nucleotide assays. An appropriate control, containing 5% TCA without any tissue, was used l n every assay to e s t a b l i s h that the ether extraction removed a l l of the TCA. The residual ether was removed by flush i n g with nitrogen. An aliqu o t of the extract was used for c y c l i c AMP determination and the rest was l y o p h l l l z e d , recon-s t i t u t e d the next day and used for the determination of c y c l i c GMP. Ly o p h i l l z a t i o n was necessary because i t was not fea s i b l e to do both of the assays on the same day. 2) c'AMP determination 6AMP was determined by using an Amersham/Searle cAMP Assay K i t code TRK 4-32. The k i t is a commercial adaptation of a si m p l i f i e d competitive protein binding assay for cAMP i n plasma which has been previously described by Latner and Prudhoe ( 1 9 7 3 ) . The method is based on the competition between unlabeled cAMP and a fixed quantity of 3H-labeled cAMP for binding to a protein which has a high s p e c i f i c i t y for cAMP (Gllman, 1 9 7 0 ) . The amount of labeled cAMP-protein complex formed is inversely related to the amount of unlabeled cAMP present l n the assay. The concen-t r a t i o n of cAMP i n the unknown was determined by comparison with a l i n e a r standard curve. Separation of the protein bound cAMP from the unbound nucleotides was achieved by adsorption of the free nucleotide on charcoal, followed by centrifugation, as I n i t i a l l y described for this assay by Brown et a l ( 1 9 7 1 ) . The supernatant was then r e -moved for l i q u i d s c i n t i l l a t i o n counting by decanting. -26-A s t r a i g h t l i n e was computed from the experimental data by the method of le a s t squares on a Wang 600 programable calc u l a t o r . The amount of cAMP i n each unknown was determined by computer from the standard curve. This figure was corrected for d i l u t i o n and o r i g i n a l tissue weight. Preliminary experiments indicated that the recovery of 3H-cAMP during the extraction was approximately 90%, The values of cAMP reported here were not corrected for recovery. Throughout the text, cAMP levels are expressed as pmol./mg. tissue wet weight. 3) cGMP determination cGMP was determined by using a commercial radioimmunoassay k i t from Schwarz/Mann. The assay involves the competitive binding of succinyl cGMP tyrosine methyl ester [ f ^ l ] and endogenous cGMP for a lim i t e d number of binding s i t e s on an antibody s p e c i f i c for cGMP. The protocol upon which the Schwarz/Mann radioimmunoassay k i t i s based follows the procedure described by Stelner et a l (1972). The antibody In the k i t was produced In rabbits against a suc c i n y l cGMP-albumin conjugate and i s s p e c i f i c for cGMP. The labeled antigen i s a c y c l i c GMP der i v a t i v e , s u c c i n y l cGMP antibody from free antigen was achieved by p r e c i p i t a t i o n of the antigen-antibody complex with ammonium s u l f a t e . A f t e r cent-r i f u g a t i o n and decantation, the bound r a d i o a c t i v i t y i n the res-idual s o l i d s (the labeled antigen-antibody complex) was determined by counting in a s c i n t i l l a t i o n counter. The concentration of cGMP i n the unknown sample was deter-mined by comparison with a l i n e a r standard curve as described for the cAMP determination. Throughout the text, cGMP levels Separation of antigen bound to -27-are expressed as pmol./mg. tissue wet weight. IV. Computation of Relaxation Relaxation induced "by Isoproterenol was expressed ln two waysi 1) as the absolute tension (^ T ln g) reduced, and 2) as the % relaxation. The % relaxation by isoproterenol was calculated as a percent of the hlgh-K* tension as follows» T k -% Relaxation = — ~ x 100 ^k Where T k is the tension ln g after 15 minutes of hlgh-K +, and is the tension lngg after Isoproterenol for 30 seconds. In the study with spontaneous contractions, no attempt was made to compute Isoproterenol-lnduced relaxation. V. Electrophysiological Studies These studies were carried out in collaboration with Dr. lean M. Marshall at Brown University in Providence, R.I. 02912, U.S.A. Pregnant white rats (Charles River Breeding Laboratories Inc., Wilmington, Mass.) were used for these experiments. The uterine strips were obtained from rats on the 18th to 22nd day of their f i r s t pregnancy. The animals were anesthetized with diethyl ether, the uterine horns were quickly removed and the fetuses were expelled by gentle manipulation of the uterus. Small longitudinal strips (about 10x12 mm, average weight - kO mg.) were excised from the antimesometrial border of the uterus. The strips were suspended ln Luclte muscle chamber through which physiological salt solution flowed at a rate of about 6 ml./min. The pH of the solution was 7.5 at 37°C and 100% 0 2 was bubbled through the solution ln the muscle chamber and in the reservoir bottles. The composition of Trls-buffer has been given before -28-(see Table 1). Two reservoir bottles were maintained - one containing 'normal' Trls-buffer and the other containing the depolarizing solution being tested. The reservoir bottles.were arranged such that switching from the 'normal' Trls-buffer to the depolarizing solution could be accomplished rapidly. The three depolarizing solutions used in these experiments were the same as those described In section 2a (see Table 1). The muscles were equilibrated for about 30 minutes during which time they developed spontaneous rhythmic contractions. Isometric tension was recorded with a force transducer (Grass, PT - 03) connected to an Ink-writing oscillograph, (Brush, Mark 280). The resting tension was about 0.4 g. Measurement of membrane potential Ele c t r i c a l a c t i v i t y of the myometrlal c e l l was measured with Intracellular glass capillary electrodes. The procedure has been described at length by Kroeger and Marshall ( 1 9 7 3 ) . Briefly, glass mlcroelectrodes of 30-80 Mft resistance f i l l e d with 2M KC1 were used to penetrate the myometrlal c e l l s . After equilibration, measurements were made of resting membrane pot-ential (RMP; maximum level of membrane polarization between spontaneous contractions), action potential height and frequency during contraction. The c r i t e r i a used for an acceptable elec-trode penetration werei 1) an abrupt shift from extracellular reference potential to a negative resting potential when the electrode entered the c e l l , 2) a stable resting potential of the same magnitude before and after a spontaneous contraction, 3) action potentials of uniform height and of magnitude equal to or greater than the absolute value of RMP, 4) no change in tip potential or electrode resistance after withdrawal of the electrode - 2 9 -from the c e l l (Kroeger and Marshall, 1 9 7 3 ) . Whenever possible, measurements of membrane potential were made ln the same c e l l before, during and after exposure to the drug. During the studies with depolarizing media, It became impossible to remain in the same c e l l during the 15 minutes of depolarization. There-fore, during this period, different cells were penetrated and 2 to k successful penetrations were recorded. Thus, ln the RESULTS, the n value for the electrophysiological data is given as X/Y, where X = the total number of Observations made, and Y s the number of muscle strips used} each strip having been taken from a different rat. The term 'membrane potential' refers to the potential measured by an Intracellular electrode relative to the extra-cellular f l u i d , and hence the RMP has a negative sign. However, the absolute values of membrane potential are given without Indicating their sign in order to conform with previous publications in this f i e l d . Thus, an increase ln potential Indicates hyper-polarlzation, and a decrease indicates depolarization. Experimental procedure After the equilibration period, control values of RMP, action potential height, action potential frequency, and the amplitude of muscle contraction were obtained. Isoproterenol 8 6 (10 or 10 M, f i n a l bath concentration) was Injected into the bath In a 0.1 ml. volume ln a 10 ml, bath. During the time of exposure to the drug, measurements of membrane potential were again made. Since the bathing solution was constantly flowing, the drug was progressively diluted and eventually washed away. Within 15 to 25 minutes, the muscle developed spontaneous contractions again. After about 2 hours of study of the spontaneous contractions, - 3 0 -the "bathing solution was changed to a depolarizing solution. At 15 minutes after the depolarization, the muscle was exposed to 10~^ M isoproterenol. Membrane potential measurements were made during the last 10 minutes of the contracture "before addition of the drug and then up to about 5 minutes after addition of isopro-terenol to the bath. Relaxation induced by Isoproterenol was computed and expressed as described In section IV. VI. S t a t i s t i c a l Analysis S t a t i s t i c a l analysis was performed by using the students •t* test for paired or unpaired data. A probability of less than 0.05 (p<0.05) was chosen as the criterion of significance. VII. Chemicals Diethylstilbestrol, Tris(hydroxymethyl)aminomethane, EGTA, and D,L-isoproterenol - a l l from Sigma Chemical Company, St.Louis, Mo.* D-600 HCl (A.G. Knoll Co., West Germany); RO 20 - 1724 (Hoffmann - La Roche Ltd., Vaudreuil, Quebec). -31-RESULTS Cyclic Nucleotide Levels During the Spontaneous  Contraction-Relaxation Cycle ln Rat Uterus Changes In tension and cAMP and cGMP levels observed during the spontaneous uterine contraction cycle are shown In Figure 1 and Table 2. The resting tension was about 0.5 £• After Initiation of contraction, the muscle reached Its peak tension of 4,34 g In afeout 9 to 10 seconds and the cycle was completed within 25 seconds. cAMP levels appeared to Increase gradually from a resting value of O.436 * 0.05 pmol./mg. tissue wet weight and reached a peak value of 0.650 * 0.073 pmol./mg. tissue at around 17 seconds after i n i t i a t i o n of contraction. At this time period, the muscle was already ln Its later stage of relaxation. The difference between these two points was s t a t i s t i c a l l y significant (p^O.05). cGMP levels fluctuated from the resting value of 0.022 - 0.002 pmol./mg. tissue wet weight; however, no significant changes ln the nucleotide levels were detected at any of the stages of contraction studied. Isoproterenol-lnduced Relaxation and Cyclic Nucleotide Levels The relationship between isoproterenol-lnduced relaxation and changes ln cyclic nucleotide levels was studied ln depolarized uterus. A representative tracing of hlgh-K* contracture and Isoproterenol-lnduced relaxation is shown in Figure 2. Isoproterenol produced dose-dependent relaxation ln the depolarized rat uterus (Table 3)« The percent relaxation for 8 it-each concentration of isoproterenol (10 or io" M) was not - 3 2 -a | 0.01 -o I 1 I I I 1 L 0 5 10 15 20 25 TIME (IN SECONDS) AFTER INITIATION O F C O N T R A C T I O N Figure 1. Changes In t e n s i o n , cAMP and cGMP l e v e l s d u r i n g the spontaneous c o n t r a c t i o n - r e l a x a t i o n c y c l e In r a t uterus. The n value f o r each poi n t Is Indicated In p a r e n t h e s i s . TABLE 2. Cyclic nucleotide levels In rat uterus during spontaneous contraction cycle. Cyclic AMP Cyclic GMP Tension S t S t e Average % Average £ Average % p moi change from p moi change from g change from mg tissue resting mg tIssue resting e resting level level level I) Resting 0.436*0.05(20) 0 0.022*0.002(25) 0 0.50*0.02(20) 0 I T ) contraction 0.^5*0.033(20) +2.1 0.019*0.001(15) -13.6 2.40*0.!?**( 15) +380 m ) contraction 0.562*0.06o(20) +28.9 0.026*0.004(16) +18.2 4.34*0.22«*(14) +768 T V ) r t S x a t l o n 0.608^0.080(20) +39.5 0.023*0.003(12) +4.6 3.03*0.11**(14) +506 V ) r t l a x l t l o n 0.650*0.073*(20) +49.1 0.021*0.002(14) -4.6 1.12*0.05**(l6) +124 * S i g n i f i c a n t l y d i f f e r e n t ( p ^ O . 0 5 ) than the r e s t i n g c o n t r o l . * S i g n i f i c a n t l y d i f f e r e n t (p^O.05) than the r e s t i n g c o n t r o l . -34-KCI control(15min) 15min I 127mM KCI I s o p r o t e r e n o I 1 2 7 m M KCI Figure 2. A r e p r e s e n t a t i v e t r a c i n g showing the e f f e c t of 127 mM KCI and 10""4M i s o p r o t e r e n o l on r a t uterus. S t r i p s were d e p o l a r i z e d and placed i n contracture f o r 15 minutes (top panel) and Iso p r o t e r e n o l was added (bottom pan e l ) . The s t r i p t r e a t e d w i t h i s o p r o t e r e n o l was f r o z e n a t 30 seconds a f t e r a d d i t i o n of the drug. TABLE 3. Hlgh-K* tension and Isoproterenol-lnduced relaxation of rat uter u s . a Depolarizing Isoproterenol Hlgh-K* Tension Absolute tension % Relaxation medium concentration (g) reduced by Iso by Iso" ( A T In g) 12? mM KCI io" -8 M 3.01 + 0.3^ (7) 0.43 ± 0.04 (7) 14.4 + 1.1 (7) (0 NaCl) io" -4 M 3.10 0.29 (15) 1.0 + 0.2 (8) 46.9 + 4.0 (8) 47.5 mM KCI io" -8 M 4.83 + 0.40 (8) 0.59 + 0.06 (8) 12.9 + 1.6 (8) (0 NaCl) io" .4 M 3.70 + 0.40 (19) 1.0 + 0.1 (10) 41.8 + 1.3 (10) 47.5 mM KCI io" -8 M 5.24 + 0.61 (7) c 0.93 + 0.09 (7)° 18.4 + 2.1 (7) (80 mM NaCl) io" -4 M 4.60 + o.5l (18) C 1.40 + 0.10 ( l l ) c 38.7 + 3.8 (11) a i The values are given as Mean - S.E.M. (n) b The % relaxation at each concentration of Iso ( 1 0 ~ ^ M or 10"^ M) was not s i g n i f i c a n t l y d i f f e r e n t In any of the media. c S i g n i f i c a n t higher ( P 4 0.05) than value for 47.5 mM KCI (0 NaCl) for corresponding Iso concentration. - 3 6 -slgnificantly different ln any of the three depolarizing media. The absolute tension achieved after 15 minutes of hlgh-K"*" depolarization was different for different depolarizing solutions. In one case, that of 47,5 mM KC1 (0 Na4), the hlgh-K + tension value was different for the two groups of uterine strips used. The reason for this difference is not clear. The depolarizing solution containing 4?.5 mM KC1 and 80 mM Na+produced significantly greater tension as compared to 4-7.5 mM KC1 (OSMa*) medium. However, when the hlgh-K + tension was significantly greater, the absolute reduction ln tension (<^T in g) produced by Isoproterenol (10 or 10 M) was also significantly greater. Therefore, for a given concentration of the agonist, the degree of relaxation was the same in a l l of the depolarizing solutions. Since the percent relaxation after Isoproterenol was not dependent on the absolute tension after hlgh-K + depolarization, the percent relaxation could be used as an appropriate indicator of the effects of Isoproterenol on the relaxation responses of the depolarized uterus under the present experimental conditions. It Is apparent that neither the reduction ln extracellular K+ from 127 to 4-7.5 mM nor the presence of 80 mM Na + ln the medium with 47.5 mM KC1 influenced the degree of relaxation produced by Isoproterenol. cAMP Levels As a control, the effect of depolarization on cAMP levels was studied for each depolarizing solution. As shown in Table 4, only the solution containing 47.5 mM KC1 (0 Na) produced a significant Increase In cAMP levels at 15 minutes after depolar-ization. Table 5 summarizes the effect of Isoproterenol on cAMP TABLE 4. E f f e c t of d e p o l a r i z a t i o n on cAMP l e v e l s . a t b Medium 'Normal' T r I s - b u f f e r Levels a f t e r 15 min % change from (Resting Levels) of d e p o l a r i z a t i o n R e s t i n g l e v e l s 127 mM KC1 0.574 * 0.077 (10) 0.598 * 0.077 (10) 6.0 ± 2.3 do) (0 NaCl) 47.5 mM KC1 0.497 - 0.037 (9) 0.747 ± 0.067 (9) 49.1 ± 4.8 (9)° (0 NaCl) 47.5 mM KC1 0.521 4 0.026 (12) 0.483 * 0.032 (12) -4.3 * 6.9 (12) 80 mM NaCl a A l l values given as Mean - S.E.M. (n) b cAMP values expressed as pmol./mg. t i s s u e wet weight. S i g n i f i c a n t (p<o.o5) % change from r e s t i n g l e v e l s . TABLE 5. E f f e c t of Isoproterenol on cAMP l e v e l s l n non-depolarized and depolarized uterus. Medium. Isopro-t e r e n o l cone. ( M ) cAMP l e v e l s (pmol./mg. t i s s u e ) • Normal• T r l s - b u f f e r (Resting l e v e l s ; KC1 c o n t r o l Iso % Increase ln cAMP 127 mM KC1 10' -8 - 0.609*0.057(7) 0.647*0.075(7) (0 NaCl) io' -4 - 0.874*0.075(4) 1.813*0.159(7)a 47.5 mM KC1 10' -8 _ 0.666*0.074(8) 0.640*0.032(8) (0 NaCl) 11 0.937*0.078(5) 10' -*+ - 1.857±0.109(10) a 47.5 mM KC1 10' -8 - 0.495*0.04 (8) 1.114*0.049(8) a + (80 mM NaCl) 10' -4 - 0.576*0.069(7) 2 . 3 4 l * 0 . l 8 3 ( l l ) a 'Normal' 10' -8 0.401*0.042(13) - 0.746*0.087(13)a T r l s - b u f f e r 10' -4 0.720*0.089(7) - 4.682*0.557(7)a 6.9*9.0(7) 111.7*26.4(7) 6.0*8.1(9) 106.6*19.8(10) 126.2*8.5(8) 337.8±48.6(ll)b 93.7*18.5(13)° 575.6*71.7(7)d S i g n i f i c a n t l y d i f f e r e n t (p^0.05) from corresponding c o n t r o l . b S i g n i f i c a n t l y d i f f e r e n t (p^.0.05) from 127 mM KC1 and 47.5 mM KC1 (0 NaCl). c Not s i g n i f i c a n t l y d i f f e r e n t from 47.5 mM KC1 + 80 mM NaCl at 10~8M Iso. d S i g n i f i c a n t l y d i f f e r e n t (p^.0.05) from 47.5 mM KC1 + 80 mM NaCl. -39-levels. In the depolarizing solution containing 12? mM KCI, the a b i l i t y of isoproterenol to cause accumulation of cAMP was -4 impaired since 10 M Isoproterenol could increase cAMP levels only by about 111.7$ as opposed to the 575.6$ increase that was observed in 'normal' Trls-buffer. A similar (106.6$) increase in cAMP was seen in 47.5 mM KCI (0 Na) solution. However, when 80 mM Na+ was present with 47.5 mM KCI, 10~^M isoproterenol caused a 337.8$ Increase in cAMP which was significantly higher than that obtained with the other two depolarizing media. Thus, the presence of 80 mM Na + caused a partial recovery of the a b i l i t y of Isoproterenol to induce cAMP accumulation in depolarized uterus. Studies using 10 M isoproterenol produced qualitatively similar results. It should be noted that even though the increases in cAMP were different In different media, the percent relaxation remained the same as was shown earlier. This was the case 4 fi whether 10 or 10 M isoproterenol was used. cGMP Levels Table 6 summarizes the changes in cGMP levels Induced by depolarization as well as by isoproterenol. Depolarization produced significantly lower (3^$) cGMP levels in only one casej that of 47.5 mM KCI + 80 mM NaCl, when compared to corresponding -4 resting levels. Isoproterenol (10 M) did not have any influence on cGMP levels either in non-depolarized or in depolarized uterus. Studies with 10 M Isoproterenol were not carried out since no significant changes in cGMP levels were observed with -4 10 M isoproterenol. Studies With D-600 In order to determine the role of Ca^-influx in the TABLE 6. Effec t of depolarization and Isoproterenol (10"^M) on cGMP l e v e l s . a Medium •Normal* Trls-buffer (Resting Levels) KCI control Iso % change from KCI control 127 mM KCI (0 NaCl) 0.021 - 0.006(5) 0.016 - 0.003(4) 0.020 - 0.007(9) NSb 47.5 mM KCI (0 NaCl) 0.026 - 0.005(5) 0.026 *• 0.008(5) 0.022 - 0.004(10) NS 47.5 MM KCI + 80 mM NaCl 0.044 - 0.006(7) 0.029 t 0.004(7)° 0.029 - 0.003(12) .NS • Normal * Tr l s - b u f f e r 0.059 - 0.012(5) - 0.052 - 0.011(5) NS cGMP level s expressed as pmol./mg. tissue wet weight. NS =s Not s i g n i f i c a n t . S i g n i f i c a n t l y d i f f e r e n t ( p ^ 0 . 0 5 ) than corresponding r e s t i n g l e v e l s . -41-blockade of Isoproterenol-lnduced Increase ln cAMP in depolarized uterus, studies with a calcium-antagonist, D-600 were carried out. A representative tracing showing the effect of iO~^M D-600 on spontaneous contraction and hlgh-K* depolarization is shown in Figure 3. Table ? summarizes the results of a control experiment in which the effect of l O " ^ D-600 was studied on cAMP levels. In the presence of D-600, none of the three depolarizing solutions produced any significant changes in cAMP levels when compared with corresponding resting levels. Pretreatment with D-600 did not have any influence on the resting levels of cAMP. The effect of Isoproterenol on cAMP levels ln the presence of D-600 Is shown ln Table 8. In the presence of D-600, the Increase ln cAMP levels induced by 10~^M Isoproterenol in a l l three depolarizing media was similar and was not different from the Increase produced ln non-depolarlzed muscle. On comparison of the data ln Table 8 with the data ln Table 5, It Is evident that D-600 abolished the inhibitory Influence of the depolarizing solutions on Isoproterenol-lnduced cAMP accumulation. It is also seen, on comparison of the above two tables, that D-600 did not Influence isoproterenol-lnduced increases ln cAMP levels ln non-depolarized uterus. Similar results were obtained with -8 10 M isoproterenol. It should be noted that no additive effect of 80 mM Na+ and lo"^ M D-600 was seen on the cAMP accumulation induced by -4 -8 Isoproterenol ln depolarized uterus at either 10 or 10 M concentrat ion. cGMP levels In the presence of D-600 and D-600 + high-K* were not significantly different from the corresponding control -10 min-t D-600 (10"5 M) yy t high K (+D-600) Figure 3. A rep r e s e n t a t i v e t r a c i n g showingthe e f f e c t of 10 M D-600 on spontaneous u t e r i n e c o n t r a c t i o n and high-K* d e p o l a r i z a t i o n . The a d d i t i o n of D-600 o b l i t e r a t e d the spontaneous c o n t r a c t i o n s and the contracture produced by hlgh-K* s o l u t i o n ( a l s o c o n t a i n i n g D-600) was n e g l i g i b l e . TABLE 7. E f f e c t of D-600 (10"^ M) on cAMP l e v e l s i n non-depolarized and depola r i z e d uterus. Med ium •Normal' T r l s - b u f f e r (Resting Levels) Resting l e v e l s a f t e r D-600 Levels a f t e r c o n t a i n i n g D-KC1 -600 % change 127 mM KCI 0.552 1 0.07(5) - 0.556 * 0.072(5) NS (0 NaCl) 47.5 mM KCI 0.681 ± 0.122(5) - 0.868 * 0.117(5) NS (0 NaCl) 47.5 mM KCI 80 mM NaCl 0.552 - 0 . 1 3 5 W - 0.516 1 0.06(4) NS T r l s - b u f f e r 0.586 * 0.172(4) 0.5^1 * 0.096(4) a NS a NS = Not s i g n i f i c a n t . TABLE 8. E f f e c t of Isoproterenol on cAMP l e v e l s In non-depolarized and depolarized uterus In the presence of D-600 (10~-> M). Medlum + D-600 Isopro-terenol cone. (M) cAMP l e v e l s (pmol./mg. t i s s u e ) 127 mM KCI 10' (0 NaCl) 47.5 mM KCI 10 (0 NaCl) 10 47.5 mM KCI 10 + (80 mM NaCl) 10 'Normal * 10 T r l s - b u f f e r -4 -8 -4 -4 • Normal•TrIs-buffer (Resting Levels) KCI c o n t r o l Iso 0.541*0.096(4) 0.556*0.072(5) 0.749*0.046(4) 0.868*0.117(5) 0.578*0.044(5) 0.516*0.06(4) 4.00*0.704(5)a 1.397*0.094(4)a 6.126*0.764(5)a 1.107*0.15?(5)a 3.848*0.244(4)a 4.170*0.52(4)a % Increase In cAMP 627.5*83.3(5) 89.9*20.9(4) 647.4*99.9(5) 90.8*22.0(5^ 676.8*95.8(4) 755.8*206 (4)( l I a S i g n i f i c a n t l y d i f f e r e n t (p 0.05) from corresponding c o n t r o l . —8 b Not s i g n i f i c a n t l y d i f f e r e n t from 47.5 mM KCI (0 NaCl) a t 10" M Iso. -4 No t s i g n i f i c a n t l y d i f f e r e n t from any of the three d e p o l a r i z i n g media a t 10 M Iso. -45-values (Table 9). In the presence of D-600, as ln Its absence, Isoproterenol did not alter cGMP levels. Studies With EGTA In order to confirm the results with D-600, experiments with EGTA were carried out. As shown ln Figure 4, the % increases In cAMP levels by Isoproterenol in EGTA-containing depolarizing solutions (Ca^-free with 0.2 mM EGTA) were essentially similar to those obtained with D-600. Calcium and Isoproterenol-lnduced Increases ln cAMP Levels ln Depolarized Uterus The experiments with Ca + +-antagonists (D-600 and EGTA) indicated that the observed Influence of Na + on the cAMP response to isoproterenol might be due to Its interaction with C a + + Ions. It was decided to determine i f the observed effect of Na* on cAMP could be antagonized by increasing [pa*^ 6** T h e results of such -4 an experiment are shown ln Figure 5 . 10 M Isoproterenol pro-duced a 106% Increase ln cAMP ln a depolarizing solution ln the absence of Na+. When 80mM Na + was present, this increase was enhanced to 337#. When [ c a ^ ex was now increased from 1,8 to 3,6 mM in the medium containing 80 mM Na+, there was a significant drop ln the % Increase in cAMP to 169$. In view of the significant effect of [_Ca^Jex ln the above experiments, we decided to study the influence of a variety of [ca +^jex on isoproterenol-lnduced Increases in cAMP levels ln the depolarized uterus. The absolute values of cAMP levels after high-K* depolarization ln the presence of varying [ca +^j ex are list e d ln Table 10. None of the hlgh-K + cAMP values are signi-ficantly different from the corresponding resting values except TABLE 9. E f f e c t of depolarization and Isoproterenol ( 1 0 ~ M) on cGMP levels i n presence of D-600 ( 1 0 - 5 M). Medium Resting Resting levels Levels a f t e r KC1 Iso % change* levels a f t e r D-600 containing D-600 12? mM KC1 0.024 - 0.006(5) - 0.014 - 0.003(5) 0.017 - 0.002(5) NS b (0 NaCl) 47.5 mM KC1 (0 NaCl) 0.025 - 0.005(6) - 0.020 - 0.003(6) 0.027 1 0.004(6) NS 47.5 mM KC1 80 mM NaCl 0.031 - 0.008(6) - 0.023 " 0.008(6) 0.025 - 0.006(6) NS Tr iS-buffer 0.059 4 0.012(5) 0.062 4 0.029(5) - 0.049 4 0.014(5) NS a % change from KC1 control except for •Normal* Tr i s - b u f f e r where i t Is % change from Resting lev e l s after D-600. b NS = Not s i g n i f i c a n t - 4 7 -D-600 (10°M) Tris-Buffer (0 No) (80 mM No) Figure 4 . The e f f e c t of Ca - antagonists on isoproterenol (10 M)-Induced Increases In cAMP levels In non-depolarized and depolarized r a t uterus. The cAMP values for the D-600 study were obtained from Table 8 . For experimental protocol, see Materials and Methods section. -48-Flgure 5. The antagonism by high (3.6 mM> Ca of Isoproterenol (10-^M) Induced Increases In cAMP level s In the presence of 80 mM Na + In depolarized r a t uterus. The control le v e l s of cAMP i n d i f f e r e n t depolarizing solutions are given i n Table 10. - R O -TABLE 10. E f f e c t of hlgh-K* depolarization on r e s t i n g levels of cAMP In presence of varying e x t r a c e l l u l a r Ca 2+ concentratlon a t«»c ex cAMP Levels 'Normal' Tr l s - b u f f e r After hlgh-K+ (Resting Levels) depolarization I) C a 2 + - f r e e a) 47.5 mM KC1 b) 47.5 mM KC1 II) 0.9 mM C a 2 + a) 47.5 mM KC1 III) 1.8 mM C a 2 + a) 47.5 mM KC1 b) 47.5 mM KC1 IV) 3.6 mM C a 2 + a) 47.5 mM KC1 b) 47.5 mM KC1 V) 5.4 mM C a 2 + a) 47.5 mM KC1 b) 47.5 mM KC1 VI) 7.2 mM C a 2 + a) 47.5 mM KC1 b) 47.5 mM KC1 0 Na) 0.587 * 0.04(6) 80 Na) 0.502 * 0.03(7) 0 Na) 80 Na) 0 Na) 80 Na) 0 Na) 80 Na) 0 Na) 80 Na) 0.507*0*021(6) 0. 539*0.04(7) 0 Na) 0.757 - 0.068(6) 0.841*0.050(6) 0.497 * 0.037(9) 0 . 7 4 7 * 0 . 0 6 7 ( 9 ) d 0.521 * 0.026(12) 0.483*0.032(12) 0.480 * 0.053(8) 0.714 * 0.068(8) 0.820 * 0.13(6) 0.725 * 0.081(6) 0.461*0.036(8) 0.643*0.04(8) 0.813*0.09(6) 0.801*0.11(6) 0.461 * 0.131(7) 0.441*0.062(7) 0.614 * 0.034(6) 0.590*0.033(6) a A l l values given as Mean * S.E.M. (n) b cAMP values expressed as pmol./mg. tissue wet weight For experimental protocol, see Methods S i g n i f i c a n t l y higher than corresponding r e s t i n g levels ( p < 0 . 0 5 ) - 5 0 -in one case (1.8 mM Ca**, 4 7 . 5 mM KC1, 0 Na +). Figure 6 illustrates the Influence of [ca*^]ex o n isoproterenol (10"^M) -Induced Increases ln cAMP levels. The cAMP values are expressed here as % increase In cAMP over control in order to normalize the data. It Is evident from Figure 6 that there was an inverse relationship between [^ a +£] e 3 ! : a n d t n e a b i l i t y of isoproterenol to increase cAMP levels. This was true whether or not 80 mM Na+ was present in the depolarizing solution. In the presence of 80 mM Na+ in the depolarizing medium, significantly higher % increases in cAMP produced by Isoproterenol were obtained at the following pointsi Ca -free, 1.8 mM, 3 . 6 mM and 5 . 4 mM [ca +^]ex. Calcium and Isoproterenol-lnduced Relaxation of Depolarized Uterus Ca ex on isoproterenol-The Influence of variation ln induced relaxation was also studied. There was an lmsrerse relationship between {jCa"1 ]^ ex and isoproterenol-lnduced relaxation expressed either as % relaxation (Figure 7) or as-^T (ln g) reduced by Isoproterenol (Figure 8). The influence of the presence of 80 mM Na+ ln the medium was not as clear-cut as seen ln the case of the cAMP data. Isoproterenol produced a significantly different degree of relaxation only at some points when 80 mM Na + was present In the medium (see Figures 7 and 8). The effect of [ca +^]ex on the hlgh-K + tension Is Illustrated In Figure 9. In the presence of 80 mM Na + ln the medium, hlgh-K + tension values were significantly higher at only two [ca+_^]ext 1*8 mM and 3 . 6 mM. Figures 10 and 11 illustrate the relationship between % -51-700 r o'A) — i 1 1 1 I I C a 2 + 0.9 1.8 3.6 5.4 7.2 Free Extracellular ( C a 2 + ] ( m M ) Figure 6. The effect of ex on isoproterenol (lO'^E)-Induced increases in cAMP levels in depolarized rat uterus. Two depolarizing solutions were studled i 4?.5 mM KCI (zero Na+), and 5?.5 mM KCI (+ 80 mM Na+). The control levels of cAMP in different depolarizing solutions are given in Table 10. The points on the ordinate indicate % increase in cAMP by isoproterenol in the presence of D-600 (10~5 M). -52-- 1 ' . — L _ i i 0 9 1 8 3.6 5.4 7.2 Extracellular [ C a 2 + ] ( m M ) Figure 7. The Influence of jca. f} ex on i s o p r o t e r e n o l (10~^M)-Induced r e l a x a t i o n of de p o l a r i z e d r a t uteru s . Two d e p o l a r i z i n g s o l u t i o n s were used as Indicated i n the f i g u r e . The % r e l a x a t i o n was computed as described In the M a t e r i a l s and Methods s e c t i o n . -53-i 0 . 9 1.8 3.6 5.4 7.2 Extracellular [Co7+ ] (mM) Figure 8. The influence of [ca "3 ex on isoproterenol (10-**M)-induced relaxation of depolarized rat uterus. Two depolarizing solutions were used as indicated In the figure. The relaxation is expressed here as the absolute tension AT in g reduced by isoproterenol. 500 r 400 a-< Z 300 U J co < U J oc U 200 100 + + 10 20 30 __L_ 40 50 60 % RELAXATION Figure 10. The relationship between % Increase In cAMP and % relaxa t i o n produced by Isoproterenol In depolarized rat uterus under the experimental conditions of varying [Natl ex and [Ca 2 +Jex. This graph was produced by using the values from Figure 6 (for cAMP) and Figure 7 (for % re l a x a t i o n ) . A T (g) PRODUCED BY Iso Figure 11. The relationship between % Increase In cAMP and absolute tension reduced by isoproterenol In depolarized rat uterus under experimental conditions of varying ex and [£a2tJ ex. This graph was produced by using the values from Figure 6 (for cAMP) and Figure 8 (forAT In g). - 5 7 -lncrease ln cAMP and % relaxation of-^T in g produced by . Isoproterenol in depolarized uterine strips under the above experimental conditions of varying [ca +^]ex and [Na^ex. No linear relationship could be found between the two parameters ln either case. At significantly different levels of cAMP increases, a similar degree of relaxation was produced by isoproterenol. On the other hand, at similar increases in cAMP levels, iso-proterenol produced significantly varying degrees of relaxation. Studies on Mg4"*"- Ca**- Na+ Interactions The successful demonstration of a Na+- Ca++ Interaction using Isoproterenol-lnduced Increases ln cAMP levels as the parameter, encouraged us to search In the literature for similar ionic interactions. It soon became evident that not only Na* ions but other physiological cations such as Mg + + were also known to interact with extracellular Ca + +. It was therefore decided to test the influence of alteration ln {^Mg*^} ex on the cAMP responses to isoproterenol, in the absence and in the presence of 80 mM Na+ ln the medium. The results of a study on Mg++- Ca"*"*"- Na+ Interactions are summarized in Table 11. In spontaneously contracting uterine —8 strips, 10" M isoproterenol caused a 93»7# increase In cAMP levels over the resting levels (Table 11, solution VIII). This increase ln cAMP by isoproterenol was abolished when the uterus was depolarized with 4-7.5 mM K +(wlth zero Na+» normal Ca 4*, 1.8 mM; and normal Mg"1**, 0.5 mM) as shown in solution I. A five-fold increase ln Mg + + (solution II) or the addition of 80 mM Na + in the medium (solution IV) restored the stimulation of cAMP by Isoproterenol. ? Increasing [ c a ^ e x from 1.8 to 3.6 mM successfully T A B L E 1 1 . E f f e c t o f I s o p r o t e r e n o l ( 1 0 M) o n c A M P l e v e l I n n o n - d e p o l a r i z e d a n d d e p o l a r i z e d r a t u t e r u s . B u f f e r C o m p o s i t i o n cAMP l e v e l s ( p m o l . / m g . t i s s u e ) (mM) % I n c r e a s e i n K Na C a Mg K C l c o n t r o l . I s o cAMP D e p o l a r i z i n g M e d i u m I 47.5 0 . 0 1 . 8 0.5 0.666 ± 0.074 ( 8 ) 0.640 + 0.032 ( 8 ) 6 . 0 + 8 . 1 ( 8 ) I I 47.5 0 . 0 1 . 8 2.5 0 . 6 3 8 * 0.098(7) 1.176 ± 0 . 2 2 5 ( 7 ) 8 6 . 3 ± 25.7(7) I I I 47.5 0 . 0 3.6 2.5 0 . 6 0 6 ± 0.074(5) 0.626 ± 0 . 0 6 8 ( 5 ) 5 . 0 + 8 . 0 ( 5 ) I V 47.5 80 . 0 1 . 8 0.5 0 . 4 9 5 t 0.040 ( 8 ) 1.114 ± 0 . 0 4 9 ( 8 ) b 126 . 2 ± 8 . 5 ( 8 ) V 47.5 80 . 0 1 . 8 2.5 0 . 5 7 1 ± 0 . 0 9 9 ( 6 ) 1 . 0 5 3 ± 0.183(6) b 87.5 26 . 8(6) V I 47.5 80 . 0 3.6 2.5 O . 6 0 5 ± 0.07K6) 0.912 + 0 . 0 6 8 ( 6 ) b 5 0.4 + 7 . 2(6) V I I 47.5 80 . 0 3.6 0.5 0.614 ± 0 . 1 0 0 ( 5 ) 0.727 ± 0.148(5) 2 9.9 ± 9.9(5) N o n - D e p o l a r i z i n g M e d i u m R e s t i n g L e v e l V I I I 2.4 125 . 0 1 . 8 0.5 0.401 ± 0.042 ( 1 3 ) 0.746 ± 0.087 ( 1 3 ) 93.7 ± 18.5 ( 1 3 G l u c o s e ( 1 1 . 0 mM) a n d T r l s ( 2 3 . 8 mM) r e m a i n e d t h e same I n a l l s o l u t i o n s . S i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o r r e s p o n d i n g c o n t r o l a t p ^ 0 . 0 5 . - 5 9 -antagonized the observed effect of high-Mg"*** on the cAMP response (compare solutions II and III) and that of added Na + (compare solutions IV and VII). No additive effect of high-Mg + + and added Na + on the cAMP response to isoproterenol was observed (solution V). Further, an increase In [ca +^]ex to 3.6 mM did not antagonize the Influence exerted by high-Mg"*"*" and added Na + together on the cAMP response to isoproterenol (solution VI). The relaxation responses to Isoproterenol under the above conditions are summarized in Table 12. The tension achieved by the various hlgh-K + solutions used In this study were not slgni--A I ficantly different from each other. The presence of high-Mg (solution II) or added Na + (solution IV) did not have a signl-o fleant Influence on the % relaxation produced by 10 M Isoproterenol. Increasing [ca +^]ex from 1.8 to 3.6 mM signi-ficantly inhibited the<*VT as well as the % relaxation in the medium with hlgh-Mg (solution III) as well as in the medium with added Na + (solution VII). When the depolarizing solution containing both hlgh-Mg"*"*' and added Na + (solution V) was used, the % relaxation and the ^T produced by isoproterenol were significantly greater than that produced in the medium containing hlgh-Mg'*"*' alone (solution II), but remained similar to that produced In the medium containing added Na+ alone (solution IV). High [ca"*^ e x failed to influence the % relaxation and <^ T produced by isoproterenol in the medium containing both high-Mg + + and added Na+ (solution VI). Figures 12 and 13 illus t r a t e again the relationship between cAMP and relaxation under the above conditions. In this set of experiments, Isoproterenol produced relaxation of depolarized TABLE 12. Hlgh-K + tension and effect of Isoproterenol (10" M) ln rat uterus. Buffer Composition b Absolute Tension * _ _ . , (mM) Hlgh-K* Tension Reduced by Iso * Relaxation (g) / i by Iso K Na Ca Mg I 47.5 0.0 1.8 0.5 4.83 ± 0.4(8) 0.60 ± 0.06(8) 12.9 + 1.6(8) II 47.5 0.0 1.8 2.5 4.22 ± 0.18(12) 0.47 + 0.03(6) 10.3 0.3(6) III 47.5 0.0 3.6 2.5 3.33 ± 0.29(H) 0.20 + 0.04(6) c 4.0 + 0.6(6)c IV 47.5 80.0 1.8 0.5 5.24 ± 0.5K7) 0.93 ± 0.09(7) 18.4 ± 2.1(7) V 47.5 80.0 1.8 2.5 4.32 ± 0.38(12) 0.95 ± 0.10(6) 20.0 + 2.5(6) VI 47.5 80.0 3.6 2.5 4.05 + 0.36(10) O.83 ± 0.11(6) 19.0 + 3.4(6) VII 47.5 80.0 3.6 0.5 5.11 + 0.50(6) 0.53 ± 0.05(6)d 10.5 ± 0 .8(6) a Glucose (11.0 mM) and Trls (23.8 mM) remained the same l n a l l solutions. None of the values are s i g n i f i c a n t l y d i f f e r e n t from each other at p^.0.05. S i g n i f i c a n t l y d i f f e r e n t from the corresponding value for sol u t i o n II at p^.0.05. S i g n i f i c a n t l y d i f f e r e n t from the corresponding value for so l u t i o n IV at p^0.05. -61 -o IOOI s < UJ < UJ a. u z X 20t i n 10 15 % RELAXATION by ISO Figure 12. 140 r A T fg) REDUCED by ISO Figure 13. Figures 12 and 13. The relationship between % increase in cAMP and % relaxation or AT in g produced by isoproterenol in depolarized rat uterus. The values from Tables 11 and 12 were used to produce these graphs. •I -62-uterine strips without necessarily Increasing cAMP levels. Furthermore, although Increases In cAMP levels were associated, In some cases, with Isoproterenol-lnduced relaxation, the lack of quantitative correlation between the two parameters was apparent. Study With the Phosphodiesterase (PDE) Inhibitor - RO 20-1724 The observed Inhibitory influence of Ca** on the cAMP response in the depolarized uterus could have been due either to decreased generation of cAMP (i.e. inhibition of adenylate cyclase) or to increased breakdown of cAMP (I.e. activation of PDE) or both. In order to determine the contribution of the latter mechanism, i f any, towards the observed effect of Ca**, a study with a more selective PDE inhibitor, RO 20-1724 was carried out. The experiments were carried out using 47.5 mM KCI (0 NaCl) depolarizing medium. Pretreatment of the muscle with RO 20-1724 (10""^ M) caused about a 3-fold increase in the control levels of Q cAMP (Figure 14). 10~ M Isoproterenol failed to Increase cAMP levels in either the presence or absence of RO 20-1?24. Table 13 summarizes the tension data from the above study. The presence of RO 20-1724 failed to significantly influence either the high-K* tension or the degree of relaxation produced by —8 isoproterenol (10~ M). Effect of Ca**- Deficiency In order to study the effect of Ca** removal on isoproterenol-lnduced increases In cAMP in non-depolarized uterus, the following experiment was carried out. Spontaneously contracting uterine strips were exposed to a Ca**- deficient Trls-buffer (Ca**-free with 0.2 mM EGTA) for 30 minutes with 3 washings at regular - 6 3 -| | control 10"8M Iso 2.0r 3 1^.0 o E a a 5 o 47.5 mM KCI (Zero Na) 47.5 mM KCI (Zero Na) + RO 20-1724 (10"4M) Figure 14. The Influence of a phosphodiesterase Inhibitor, RO 20-1724 (10-%) on cAMP levels In the absence and presence of isoproterenol (10-°M) In ra t uterus depolarized with hlgh-K+. For experimental protocol see Materials and Methods. - 6 4 -TABLE 13. Influence of RO 20-1724 (10"^ M) on highgK"*" tension and Isoproterenol (10""B M)-induced relaxation. Treatment Hlgh-K + Tens Ion (g) £ » T Reduced by Iso, 10_fcSM Cg) % Relaxation by Iso, 10~ BM I. 4 7 . 5 mM KCI (0 Na) 4.47 * 0 . 3 K 1 2 ) 0 .40 * 0.03(6) 9.9 ± 0.7(6) I I . 4 7 . 5 mM KCI (0 Na) + RO 20-1724 ( i o - % ) 5.42 * 0 . 5 5(12) a 0.38 t 0 . 0 3 ( 6 ) a 9.5 ± 1 . 6 ( 6 ) a a Not s i g n i f i c a n t l y d i f f e r e n t from the corresponding value without R0 20-1724. -65-Intervals, At the end of this time period, the strips were p exposed to 10 M isoproterenol and frozen for cAMP determinations (Figure 1 5 ) . Exposure to a Ca + +- deficient solution did not alter the control levels of cAMP (Table 14), although It did abolish the Q spontaneous contractions (Figure 1 5 ) . 10" M isoproterenol Increased cAMP in muscles ln both the normal and Ca + +-deflclent solutions, the increase being significantly greater In the latter (Table 14). Electrophysiological Studies 'i The relationship between hyperpolarlzation and Isoproterenol-lnduced relaxation was studied ln non-depolarized and depolarized rat myometrium, p s Isoproterenol (10 M or 10~°M) inhibited the spontaneous uterine contractions and the muscle relaxed to the baseline p tension, 10 M isoproterenol failed to affect the resting membrane potential (Table 15). However, at the higher concentration (10"^M), isoproterenol caused a significant degree of hyperpolarlzation (Table 15 and Figure 16). Addition of high-K+ medium caused depolarization, initiated action potentials and resulted In phasic contraction. Within a few minutes, the membrane was depolarized to a level at which action potentials could no longer be generated. Figure 17(A) shows the i n i t i a l phase of membrane depolarization (upper tracing) and the muscle contraction (lower tracing). Figure 17(B) shows a representative tracing of the 15 minutes, of hlgh-K* contracture and the relaxation by 10"% isoproterenol. A l l three depolarizing solutions produced a 10 min-*-t Ca-deficient Tris-buffer * t Isoproterenol, 10 8 M *Ca- f ree Tris-buffer with 0.2 mM EGTA for 30 min with 3 washings Figure 15. A re p r e s e n t a t i v e t r a c i n g showing the e f f e c t of a C a 2 + - d e f l c l e n t T r l s b u f f e r on spontaneously c o n t r a c t i n g r a t uter u s . A f t e r 30 minute exposure, Isoproterenol (10~ BM) was added to the bath f o r 30 seconds and the muscle f r o z e n f o r cAMP determination. -67-TABLE 14. Influence of a Ca - d e f i c i e n t s o l u t i o n (Ca -free Trls«buffer with 0.2 mM EGTA) on isoproterenol (10 M)-Induced Increases i n cAMP l e v e l s . Treatment cAMP (pmol./mg. tissue) Control Iso, 10"8 M % Increase In cAMP •Normal' 0.401 ±0.042(13) 0.7^6 - 0.087(13)a 93.?~18«5(13) T r l s - b u f f e r C a 2 + - D e f i c l e n t 0.434 ±0.047(4) 1.559 1 0.202(4)a 285.0±87.0(4)b T r l s - b u f f e r S i g n i f i c a n t l y d i f f e r e n t (p^: 0 . 0 5 ) from the corresponding control. S i g n i f i c a n t l y d i f f e r e n t (p^ 0 . 0 5 ) from the % increase i n cAMP obtained in Normal T r i s - b u f f e r . TABLE 15. E f f e c t of Isoproterenol on membrane p o t e n t i a l In I s o l a t e d myometrium. RMP (mV) A c t i o n P o t e n t i a l Height (mV) Frequency (AP/min.) Tension (g) C o n t r o l (Spontaneous c o n t r a c t i o n s ) 40.2 ± 0.3 b (n = 35/16)D 44.6 * 0.8 (n = 19/16) 139.9 ± 5.7 (n = 3 V 1 6 ) 4.3 ± 0.2 (n = 3 V I 6 ) I s o p r o t e r e n o l a) 10~8 M b) 10"6 M 0.4 - 0.4 n = 14/8) 48.7 ± 0.7° (n = 13/10) R e l a x a t i o n R e l a x a t i o n R e s t i n g Membrane P o t e n t i a l . n value i s given as X/Y, where X = number of observations made and Y number of s t r i p s useds each s t r i p from a d i f f e r e n t r a t . S i g n i f i c a n t l y (p<0.05) d i f f e r e n t than the c o n t r o l value. -69-Figure 16.(A). Membrane potential and action potential frequency (top tracing) and tension (bottom tracing) during spontaneous contraction In normal solution. (B). Membrane hyperpolarlzatlon (top tracing) and relaxation (bottom tracing) produced by Isoproterenol (10- f cM). - 7 0 -(X) HIGH - K+DEPOLARIZATION ' 30 sec MEMBRANE POTENTIAL 40.4 19.8 17.4 (mV) Figure 17.(A). High-K+-induced membrane depolar-ization (top tracing) and tension (bottom tracing). The membrane was depolarized to about -20 mV; and at this level action potentials no longer occurred. (B). A representative tracing of high-K* contracture and isoproterenol (10~6M)-induced relaxation. Typical membrane potential values during different phases of the experiment are Indicated. -71-similar degree of tension (Table 16). Isoproterenol-lnduced relaxation (^T or % relaxation) was also similar in a l l three media. The degree of membrane depolarization produced was similar for a l l three depolarizing solutions (about 20 mV; Table 17). Isoproterenol (10~^M) was without any effect on the membrane potential in any of the three depolarizing media. TABLE 16. Hlgh-K + tension and Isoproterenol ( 1 0 " M) -induced r e l a x a t i o n . 3 . Depolarizing Hlgh-K + Tension* Absolute Tension % Relaxation 6 1 Medium (g) Reduced by I s o c (g) 127 mM KCI 3.9 ± 0.8 (6) 1.5 ± 0.5 (6) 32.5 + 4.5 (6) (0 NaCl) 47.5 mM KCI 3.8 ± 0.4 (5) 1.3 ± 0.3 (5) 35.9 ± 8.1 (5) (0 NaCl) 47.5 mM KCI 3.0 ± 0.2 (6) 1.1 + 0.2 (6) 36.0 + 5.0 (6) (80 mM NaCl) The values are given as Mean ±S.E.M. None of the values are s i g n i f i c a n t l y None of the values are s i g n i f i c a n t l y None of the values are s i g n i f i c a n t l y (n). d i f f e r e n t from each other (p^.0.05). d i f f e r e n t from each other (p.^0.05). d i f f e r e n t from each other ( p $ 0 . 0 5 ) . TABLE 17. Ef f e c t of isoproterenol (10~ M) on membrane potential l n depolarized myometrium. Depolarizing Med. ium Membrane Potential (mV) Control Isoproterenol 1 127 mM KC1 (0 NaCl) 20.3 (n = ± 0.6 14/6) 19.7 (n = ± 0.9 10/6) 4-7.5 mM KC1 (0 NaCl) 16.8 * 0.5(17/5) 17.2 ± 0.8(11/5) 4-7.5 mM KC1 (80 mM NaCl) 19.3 t 0.5(19/6) 18.3 ± 0.5(12/6,). a None of the values are s i g n i f i c a n t l y d i f f e r e n t from each other ( p $ 0 . 0 5 ) . b None of the values are s i g n i f i c a n t l y d i f f e r e n t from each other ( p $ 0 . 0 5 ) . -7*+-DISCUSSION I. Cyclic Nucleotides In Spontaneous Contraction Cycle In  Rat Uterus The results of the cyclic nucleotide determinations made during the contraction cycle of estrogen-primed rat uterus provide negative evidence for the role of cAMP and/or cGMP in the regulation of spontaneous contractility of uterine smooth muscle. This Is in agreement with the report of Diamond and Hartle ( 1 9 7 * 0 , but Is ln contrast to the report of Johansson and Andersson (1975)« Both of the above studies were carried out in estrogen-primed rat uterus. Johansson and Andersson (1975) observed that the highest cAMP levels occurred at the peak of contraction and therefore implied that these high levels of cAMP played a role ln the in i t i a t i o n of relaxation. However, our results show the highest cAMP levels ln the tissue at a time when It is nearlng the end of relaxation process, and thus do not support the Implied role of cAMP ln the in i t i a t i o n of relaxation process during the contraction cycle. Diamond and Hartle (197*0 found no significant changes ln the tissue content of cAMP at any of the stages of contraction studied. In the case of the cGMP levels, our results confirm the conclusion of Diamond and Hartle (197 *0 J that there are no detectable changes in the levels of this nucleotide. Again, ln contrast, Johansson and Andersson (1975) reported that the highest cGMP levels were found 5 seconds after the start of the spontaneous contraction. The reason for the apparent discrepancy among the results of these groups Is not clear. The variation in the data reported - 7 5 -by Johansson and Andersson ( 1 9 7 5 ) was quite high. The physiological s a l t s o l u t i o n used, in our study (Trls-buffer) was s i m i l a r to the one used, by Diamond, and Hartle, but d i f -ferent from the one used by Johansson and Andersson, who employed a bicarbonate buffer. Diamond and Hartle ( 1 9 7 4 ) reported that the resu l t s were e s s e n t i a l l y the same when the c y c l i c nucleotide levels were calculated both i n terms of pmol./mg. wet weight of tissue and. pmoL/mg. of protein. Furthermore, they have also shown that r e p e t i t i o n of some of the experiments on myometrial preparations (from which endometrium and c i r c u l a r muscle had been stripped away) produced, re s u l t s s i m i l a r to those obtained with whole uterine s t r i p s . Thus, our i n a b i l i t y to observe s i g n i f i c a n t a l t e r a t i o n s In the c y c l i c GMP levels does not seem to be related, to the type of tissue preparation used or to the mode of expression of the data. It should, be pointed out that although Johansson and Andersson ( 1 9 7 5 ) observed the o s c i l l a t i o n s In the c y c l i c nucleotide l e v e l s , the question of the causal r e l a t i o n s h i p between the observed changes ln the c y c l i c nucleotide levels and the contraction-relaxation cycle remained, unanswered. II. C y c l i c Nucleotides ln Isoproterenol-lnduced Relaxation In the present study, i t was found that isoproterenol could, relax depolarized r a t uterus without necessarily increasing cAMP l e v e l s . Furthermore, although increases In cAMP levels were associated In some cases with isoproterenol-lnduced relaxation, there did. not appear to be a quantitative r e l a t i o n s h i p between the two parameters. At s i m i l a r levels of cAMP increases, s i g n i f i c a n t l y d i f f e r e n t degrees of relaxation by Isoproterenol -76-could be obtained. On the other hand, at s i g n i f i c a n t l y d i f f e r e n t lev e l s of cAMP increases, Isoproterenol produced a s i m i l a r degree of r e l a x a t i o n . Thus, there does not appear to be a simple cause and e f f e c t r e l a t i o n s h i p between ^-adrenoceptor-induced changes in cAMP and relaxa t i o n i n uterine smooth muscle. These con-clusions are In agreement with the res u l t s of several workers in demonstrating a d i s s o c i a t i o n between cAMP and jj-adrenoceptor-induced relaxation of uterine smooth muscle (Polacek and Daniel, 1971; Polacek et a l , 1971; Diamond and Holmes, 1975; Verma and McNeill, 1 9 7 6 ) , The present study confirms a previous observation made i n —8 this laboratory that at a.low concentration ( 1 0 ~ M), isoproterenol could relax the depolarized uterus without increasing cAMP leve l s (Verma and McNeill, 1976). On the other hand, Diamond and Holmes (1975) reported that In t h e i r study with KCl-depolarized r a t myometrium, 5xlO~^M Isoproterenol caused a small but s i g n i f i c a n t percent Increase in cAMP leve l s (16.1$). The values of percent relaxation produced by isoproterenol were also higher than the values reported in the present study. We do not have an explanation for such a variance In the data. However, i t may be relevant to point out that the conclusions drawn by the authors in the above study (Diamond and Holmes, 1975) are not at odds with the present s;tudy, since they also found that the relaxation and cAMP Increases produced by Isoproterenol In depolarized r a t myometrium could not be causally r e l a t e d . Although there are a large number of studies carried out i n order to s a t i s f y the various c r i t e r i a established by Sutherland and Roblson ( I 9 6 6 ) (see Introduction), there Is l i t t l e d i r e c t evidence of a c o r r e l a t i o n between the response of the muscle and -77-a change In tissue concentration of cAMP. In fact, muscle tension has actually been monitored In only a few of the published reports dealing with the proposed role of cAMP and cGMP in drug-induced relaxation and contraction of smooth muscle (for example, Andersson, 1973; Marshall and Kroeger, 1973)* Polacek and Daniel (1971) measured total cAMP content of the rat uterus as a function of time and they reported that.an Increase in cAMP occurred some minutes after the onset of relaxation. In contrast, Marshall and Kroeger (1973) demonstrated a significant increase in tissue cAMP concentration In rat uterus at a time when the muscle was just beginning to relax in response to ^xlO'^M isoproterenol. It should be pointed out that although. cAMP levels have been shown to be elevated by selected high concentrations of Isoproterenol, Inhibition of contractility of uterine smooth muscle has been shown to occur at lower con-—8 centrations, such as 2x10*" M of the agonist, without any increase in cAMP (Nesheim et a l , 1 9 7 5 ) . The results of the present study as well as those of Verma and McNeill (19?6) have demonstrated relaxation of depolarized rat uterus by 10" M isoproterenol unaccompanied by any Increase in whole tissue levels of cAMP. The use of the so-called PDE Inhibitors, such as theophylline and papaverine, in supporting the role of cAMP in smooth muscle relaxation has been questioned because of some recent evidence (for example, Bowman and Hall, 1 9 7 0 ) . Polacek et a l (1971) observed that theophylline and papaverine in low concentrations relaxed the rat uterus with minimal or no elevation of cAMP levels. Mltznegg et a l (197*0 demonstrated that in Isolated rat uterus, low doses of theophylline (5x10 M) caused relaxing effect by a calcium-antagonistic activity. This dose of theophylline did - 7 8 -not affect the tissue levels of cAMP. It has been pointed out that these so-called PDE Inhibitors exert wide ranging depressant effects on the c e l l membrane, and also alter catecholamine metabolism (Namm and Leader, 1 9 7 6 ) . Thus, the results obtained with the use of these agents should be treated with some reser-vation. In the present study, a more selective PDE Inhibitor, RO 20-1724 was employed (Van Breemen, 1977; Newman et a l , 1 9 7 8 ) . No potentiation of the relaxation response to Isoproterenol in depolarized uterus was obtained, although RO 20-1724 increased the control cAMP levels by about 3-fold presumably by PDE inhibition. This evidence again does not support the role of cAMP in isoproterenol-lnduced relaxation of uterine smooth muscle. The fourth criterion requires the demonstration that exo-genously applied cAMP mimics those uterine effects for which i t Is presumed to be an Intracellular mediator. Attempts to satisfy this requirement have again led to considerable controversy. As early as I 9 6 8 , Kim and co-workers showed that a l l adenine nucleotides- adenosine, 5»-AMP, AMP, cAMP and ATP- Inhibited spontaneous motility of the Isolated Ileum within seconds of exposure and before and after adrenergic blockade. Their data tend to suggest a non-specific role for cAMP. Bertl et a l (1971) found divergent effects of cAMP and Its more l i p i d soluble analog, db-cAMP in vascular smooth muscle. We have found that db-cAMP caused relaxation of hlgh-K + depolarized uterus at mllllmolar concentrations (Melsheri and McNeill, unpublished observation' ). However, adenosine and 5'-AMP were also capable of causing relaxation ln the same preparation. A possibility has been raised that the effect of exogenous cyclic nucleotide could occur secondary to its catabollsm to either 5'-AMP or adenosine (Namm -79-and Leader, 1 9 7 6 ) . In conclusion, the hypothesis that cAMP mediates uterine relaxation induced "by ^-adrenergic agonist Is not supported by the present study u t i l i z i n g various c r i t e r i a established for such a mediator role. The quantitative differences between the levels of cAMP measured in the present study and that by Verma and McNeill (1976) can be attributed to a change in methodology. Preliminary studies demonstrated that a lyophfclization step used by Verma and McNeill (1976) but omitted in the present study could account for the higher cAMP values reported in the present study. It should be noted that rather large animal-to-anlmal variation In cAMP and cGMP levels was observed in this study. Such a variation in the cyclic nucleotide levels has been reported by other workers In the f i e l d as well (for example, Diamond and Hartle, 1 9 7 6 ) . Thus, the use of paired experiments is Important, at least in smooth muscle, since although the variation from animal to animal may be large, the variation between pairs from the same animal Is quite small. The dissociation found between cAMP and relaxation -in the present study was extended to cGMP, since no changes in cGMP levels were observed with Isoproterenol in non-depolarized as well as depolarized uterus. Thus, a reciprocal relationship between cAMP and cGMP (as postulated by Goldberg ejb a l , 1975) during Isoproterenol-lnduced relaxation of uterine smooth muscle could not be established. Diamond and Holmes (1975) have similarly reported a lack of the effect of isoproterenol on cGMP levels in rat myometrium., Diamond and Janis ( I 9 7 8 ) attempted to correlate cGMP levels and drug-induced relaxation in rat vas - 8 0 -deferens. They found that the KCl-depolarlzed smooth muscle could be relaxed by hydralazine and verapamil without any effect on cGMP levels. On the other hand, sodium nltroprusside failed to relax the depolarized muscle, while i t significantly Increased cGMP levels. These results do not support the conclusions of Schultz et a l (1977) who prematurely postulated that cGMP may be responsible for the smooth muscle relaxant effects of certain drugs, The work of Diamond and Hartle (1976) and Diamond (1978) provided results which do not support the notion that cGMP is responsible for the i n i t i a t i o n of drug-induced contractions ln smooth muscle. Various observations now Indicate that the motility of some types of smooth muscle can be altered without changing cyclic nucleotide levels. As has been pointed out. by Diamond and Holmes (1975)» the very lack of consistency In the cyclic nucleotide data from one muscle type to another is not supportive of a general theory involving changes ln cyclic nucleotide levels ln the physiological control of smooth muscle contractility. Furthermore, there is good evidence that the increases ln cGMP levels which accompany contractions din some types of smooth muscle are secondary to Increases in Intracellular ionized calcium (Diamond, 1978). Thus, with the current evidence in mind, It could be stated that the function of cGMP in smooth muscle contractility is unknown at this point. III. Ionic Interactions In the present study, i t was noted that 127 mM KC1 depolarizing medium inhibited the a b i l i t y of isoproterenol to Increase tissue levels of cAMP as compared to normal Trls-buffer. Thus, the question arose as to what influence the ionic composition -81-of the depolarizing medium might have on the physiological and biochemical events Induced by Isoproterenol. When the external K+ concentration was changed from 127 mM KCI (G NaCl) to 47.5 mM KCI (0 NaCl), the mechanical and biochemical (cAMP as well as cGMP) responses to isoproterenol remained essentially unaltered. The lowering of the C l " concentration in the extracellular f l u i d (127 mM to 47.5 mM) had no significant effect on either the mechanical or biochemical changes Induced by isoproterenol. It has been repeatedly shown in various physiological studies that substitution of NaCl with lithium chloride, choline chloride, or sucrose essentially produces similar results (Marshall, 1963; Osa, 1971; Mlnornneau, 1 9 7 6 ) . In preliminary studies, when a depolarizing solution containing 47.5 mM KCI with 80 mM choline chloride (with lO'-'M atropine) was employed, i t was found that -4 the percent relaxation produced by 10 M isoproterenol was similar to that produced in the three depolarizing media used. Thus, the contribution of the lowering of external C l " on depolarization is probably not great. The cAMP levels after 15 minutes of depolarization in the medium without Na+ (47.5 mM KCI) were significantly higher than the cAMP levels in the medium with Na + (47.5 mM KCI + 80 mM NaCl) (Table 4 ) . This increase in cAMP levels observed in the medium without Na + could have resulted from release of catecholamine, A time-dependent catecholamine release had been observed with 12$ mM KCI (0 NaCl) by Verma and McNeill ( 1 9 7 6 ) . The percent Increase in cAMP by Isoproterenol in the medium with Na + (47.5 mM KCI + 80 mM NaCl) was significantly higher than in the two media without Na + (Table 5 ) . The difference in percent Increase is partly due to the difference in the control cAMP levels. However, - 8 2 -the isoproterenol-lnduced increase l n cAMP le v e l s i n the medium containing Na + was s i g n i f i c a n t l y higher than that without Na +. The difference i n the control cAMP level s is thus not s u f f i c i e n t to a l t e r the i n t e r p r e t a t i o n that Na + can influence the a b i l i t y of isoproterenol to Increase cAMP, Furthermore, this e f f e c t of Na + P was reproducible and even more pronounced at 10 M isoproterenol. In an attempt to elucidate the mechanism of the Na + e f f e c t , experiments with D-600 were carried out. D-600, a methoxy derivative of verapamil, i s a well-known C a + + antagonist (Fleckenstein, 1977) and i t has been shown to p r e f e r e n t i a l l y block C a + + i n f l u x i n the uterus (Kroeger e_t a l , 1 9 7 5 ) . Since i n the present study, D-600 was able to restore the stimulation of cAMP by isoproterenol In the depolarized muscle to a l e v e l s i m i l a r to that observed in non-depolarIzed muscle, It would appear that the f a i l u r e of isoproterenol to elevate cAMP i n the depolarized uterus was due to Increased C a + + Influx known to occur during depolarization (Daniel and Janis, 1 9 7 5 ) . This observation is consistent with the findings of Verma and McNeill ( 1 9 ? 6 ) . In view of the known Na +-Ca + + interaction i n smooth muscle, i t was considered that Na + might be exerting i t s influence i n d i r e c t l y by i n t e r a c t i n g with C a + + . It has been suggested that there is a competition between Na + and C a + + for binding or trans-port s i t e s (Marshall, 1963; Osa, 1 9 ? 1 ) . There Is also a p o s s i b i l i t y of Na + Influx linked to C a + + e f f l u x ; however there Is no convin-cing evidence i n favor of such a mechanism, at l e a s t in uterine smooth muscle (Daniel and Janls, 1 9 7 5 ) . Thus i t was of interest to study i f both Na + and D-600 were exerting t h e i r influence through the i r e f f e c t on C a + + Influx. It was postulated that i f e x t r a c e l l --83-u l a r N a + and D-600 were "both w o r k i n g v i a independent mechanisms t o r e v e r s e the i n h i b i t i o n o f l s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP, then an a d d i t i v e e f f e c t w i t h N a + and D-600 t o g e t h e r would be ex p e c t e d . I n c o n t r a s t , l f t h e y were a c t i n g t h r o u g h the same mech-anism ( i . e . i n h i b i t i o n o f C a + + i n f l u x ) t h e n an a d d i t i v e e f f e c t would not be e x p e c t e d . The r e s u l t s from Table 8 show t h a t no a d d i t i v e e f f e c t o f N a + and D-600 was observed e i t h e r a t 10~ M o r 1 0 ~ % i s o p r o t e r e n o l . I t c o u l d be argued t h a t an a d d i t i v e e f f e c t on -4 cAMP a c c u m u l a t i o n a t 10 M I s o p r o t e r e n o l may n o t have been o b s e r -ved I f the system was m a x i m a l l y s t i m u l a t e d by t h i s c o n c e n t r a t i o n . However, the absence o f an a d d i t i v e e f f e c t even a t 10"" M i s o p r o -t e r e n o l i n d i c a t e d t h a t e x t r a c e l l u l a r N a + and D-600 a c t t h r o u g h a s i m i l a r mechanism. The expe r i m e n t s w i t h EGTA c o n f i r m e d t h i s c o n c l u s i o n . I t i s t h e r e f o r e proposed t h a t the e x t r a c e l l u l a r Na i n the d e p o l a r i z i n g medium can overcome the b l o c k a d e o f i s o p r o t e r e n o l -l n d u ced i n c r e a s e s I n cAMP by h i g h - K + and t h a t Na* produces t h i s e f f e c t by r e d u c i n g Ca I n f l u x o c c u r r i n g d u r i n g d e p o l a r i z a t i o n . The s t u d i e s w i t h D-600 and EGTA p r o v i d e e v i d e n c e f o r the p o s t u l a t e t h a t + +•+• e x t r a c e l l u l a r Na may d e c r e a s e Ca i n f l u x by competing w i t h ex-++ t r a c e l l u l a r Ca . T h i s Is f u r t h e r s u p p o r t e d by the o b s e r v a t i o n t h a t an i n c r e a s e i n jca"1"1^] ex c o u l d a n t a g o n i z e the obser v e d i n f l u e n c e o f Na + on the cAMP r e s p o n s e t o I s o p r o t e r e n o l . F u r t h e r m o r e , a s i g n i -f i c a n t and c o n s i s t e n t I n f l u e n c e o f N a + on the cAMP r e s p o n s e was obse r v e d a t a v a r i e t y o f | c a + " ^ ex used ( F i g u r e 6 ) . + ++ The p o s t u l a t e t h a t e x t r a c e l l u l a r Na may d e c r e a s e Ca i n f l u x by competing w i t h C a + + f i n d s s u p p o r t from v a r i o u s s t u d i e s I n a v a r i e t y o f smooth muscles ( M a r s h a l l , 1963. Bauer e t a l , 1965; S i t r i n and Bohr, 1971). I t has been shown t h a t the N a + i o n - 8 4 -may compete with C a + + required for excitation-contraction coupling (Osa, 1 9 7 1 ) . Osa (1973) showed that ln pregnant mouse myometrium, contracture and depolarization which are produced ln low Na + and maintained l n Na +-free solutions may be due, i n part, to an Increased Ca* + inflow across the membrane, > He also observed that excess C a + + (22mM) prevented depolarization and contracture i n Na +-free solutions. He suggested that C a + + acts to s t a b i l i z e the c e l l membrane by lowering permeability to Na + and other ions. Recently, Crankshaw e_t a l (1977) reported that the presence of lOOmM NaCl In the Incubation medium s i g n i f i c a n t l y reduced the accumulation of ^ C a + + "by the plasma membrane and the endoplasmic r e t i c u l a r fractions of r a t myometrium, suggesting competition between Na + and Ca . A preliminary piece of evidence concerning a s i m i l a r + + + 4 -Interaction between Mg and Ca was obtained In the present 4 * 4 ' 4*4* study. Such an interaction between Mg and Ca ions using a biochemical parameter such as the cAMP response has, to our + 4 -knowledge, not been demonstrated before. The e f f e c t of Mg on isoproterenol-lnduced changes l n mechanical responses has been reported l n vascular smooth muscle (Turlapathy et a l , 1975)• The effects of Mg + + were thought to be the r e s u l t of Its i n t e r a c t i o n 4-4* with Ca movements at the membrane or at I n t r a c e l l u l a r binding + 4 -s i t e s . Mg has been shown to antagonize the c o n t r a c t i l e e f f e c t 4 > + of Ca ln the depolarized r a t uterus (Edman and S c h l l d , I 9 6 2 ) . The postulate that C a + + and Mg"1"*" compete with each other for anionic s i t e s at the external and internal c e l l membrane surface of smooth muscle c e l l s i s supported by electron microscopic findings (Goodford and Wolowyk, 19 7 2 ) . It was observed i n the present study that the simultaneous -85-presence of 80mM Na + and 2.5mM Mg + + d i d not exert an additive e f f e c t on the cAMP responses to isoproterenol. Furthermore, an increase in [ c a + + ] ex could not antagonize the influence of added Na + and high Mg + + together on isoproterenol-lnduced Increases in cAMP levels whereas a si m i l a r increase in {ca +^j ex could e f f e c t i v e l y antagonize the influence on the cAMP response exerted by added Na or high Mg alone. These findings further + ++ support the postulate that e x t r a c e l l u l a r Na and/or Mg i n the depolarizing medium can overcome the blockade of isoproterenol-lnduced Increases In cAMP level s by hlgh-K + and that they do so by virtue of their a b i l i t y to i n h i b i t passive C a + + i n f l u x by competition for entry s i t e s . The lack of e f f e c t of e x t r a c e l l u l a r Na + and Mg + + on the hlgh-K* tension reported i n this study do not appear to be consistent with the concept of antagonism between these cations and Ca at the c e l l surface. Such an antagonism would be expected, to produce smaller contractures during depolarization with added Na + or high Mg + + or both i n the medium. It needs to be pointed out that changes in tension do not r e l i a b l y r e f l e c t c e l l membrane C a + + fluxes due to variable a c t i v i t y of i n t r a c e l l u l a r C a + + sources (Van Breemen, 1 9 7 5 ) . Mg was shown to have a s i g n i f i c a n t i n h i b i t o r y e f f e c t on Ca* + uptake in hlgh-K + solutions i n rabbit a o r t i c s t r i p s when d i r e c t measurements of Ca fluxes were carried out (Carrier et a l , 1 9 7 6 ) . In another study from the same laboratory (Turla-pathy et a l , 1975)» Mg + + f a i l e d to influence Isoproterenol-lnduced rela x a t i o n mediated by ^ -adrenergic receptors i n vascular smooth muscle, whereas i t affected the c o n t r a c t i l e responses mediated by c<-adrenergic receptors. One possible explanation for the present data is that the Ca that is affected by the presence of Na - 8 6 -and/or Mg has a d i f f e r e n t influence on the contraction-rela x a t i o n system as opposed to the cAMP system. IV. Role of Ca"**4, i n Isoproterenol-lnduced Increases in cAMP Levels It was found during the course of the study that the impairment of the a b i l i t y of Isoproterenol to elevate cAMP le v e l s l n depolarized uterus was d i r e c t l y related to the Increase l n Ca"1"1"-inf lux during depolarization. The dependency of the cAMP 4"4" response to isoproterenol on Ca became apparent when i t was observed that the a b i l i t y of isoproterenol to Increase cAMP le v e l s could be increased by lowering |ca + +] ex and decreased by incr e a s l n g [ c a + 4 J e x . Furthermore, manipulation of the Ca4"1" 4" 4-4-environment of the tissue by other cations such as Na and Mg also produced a s i g n i f i c a n t Influence on the cAMP response. The various interactions involving C a + + , cAMP and 'jj-adrenoceptor stimulation are depicted l n Figure 18. The jj-agonlst Interacts with the regulatory subunit a f t e r binding to Its receptor and activates the c a t a l y t i c subunit. The stimulation of adenylate cyclase then re s u l t s i n Increased i n t r a c e l l u l a r l e v e l s of cAMP. The various ways l n which Ca4"4, movements may occur across the smooth c e l l membrane are also depicted. 4 * 4 ' The dependence of the cAMP response on Ca has also been observed i n other c e l l systems. For example, Harary e_t a l (1976) observed, i n cultured heart c e l l s from r a t s , that the Increase i n cAMP by noradrenaline could be alt e r e d by alteringjca * + ] lex, Schwabe et a l (1978) demonstrated that the presence of e x t r a c e l l u l a r Ca4"*" was c r i t i c a l for noradrenaline and histamlne-ellclted accum-u l a t i o n of cAMP i n brain s l i c e s . As depicted i n Figure 18, the observed Inhibitory influence of Ca4™1" on the cAMP response to Isoproterenol could have been due OUTSIDE j3-agonist Ca 2 + i / - A T P r D-600 Na+, Mg 2 + N I \ ADP INSIDE t j8-agonist \ • cAMP I -* [ C o " ] , @ | Ca influx (g) • Ca efflux @ I Ca sequest-Relaxation Contraction Relaxation schematic diagram d e p i c t i n g various I n t e r a c t i o n s i n v o l v i n g beta-adrenoceptor s t i m u l a t i o n , cAMP and Ca z+ In smooth muscle c e l l . The Figure 18. A various ways In which C a 2 + movements may occur across the smooth muscle c e l l membrane are a l s o depicted. On the extreme r i g h t , a p o s s i b l e sequence of events presumed to occur a f t e r beta-adrenoceptor s t i m u l a t i o n as postulat e d by the second messenger hypothesis Is i n d i c a t e d . For d i s c u s s i o n of t h i s model see t e x t . -88-either to decreased generation of cAMP ( i . e . i n h i b i t i o n of adenylate cyclase) or to increased breakdown of cAMP ( i . e . a c t i v a t i o n of PDE). Kroeger (1975) has indicated that Ca"1"1" was capable of stimulating PDE i n r a t uterus. It was considered a p o s s i b i l i t y that, i n the depolarized uterus, although cAMP was produced i n response to Isoproterenol stimulation, It was broken down more r a p i d l y by PDE and the Increase in cAMP was:< thus not detectable. Our study with RO 20-1724, a PDE Inhibitor, provided negative evidence for such a p o s s i b i l i t y . Isoproterenol f a i l e d to Increase cAMP le v e l s i n the presence of RO 20-1724, although RO 20-1724 I t s e l f Increased cAMP level s presumably v i a PDE i n h i b i t i o n . Furthermore, the e f f e c t of Ca4"*" on the cAMP l e v e l was s p e c i f i c In that i t only affected the agonist-induced increases i n cAMP l e v e l s , but did not have any s i g n i f i c a n t Influence on the control le v e l s of cAMP. In the case of s i g n i f i c a n t a c t i v a t i o n of PDE, one would expect the control cAMP level s to be s i g n i f i c a n t l y lower. Thus, although not absolutely conclusive, the above evidence does not support PDE-actlvation as the possible mechanism'for the observed e f f e c t of C a + + on cAMP. It is therefore postulated that Ca4"* i n h i b i t s the generation of cAMP observed during the stimulation of j3-adrenoceptor by Isoproterenol. I n h i b i t i o n of adrenaline-stimulated adenylate cyclase by ionized Ca** was observed in turkey erythrocytes (Steer e t : a l , 1 9 7 5 ) . Lynch et a l (1976) concluded, based on th e i r study in rat brain s l i c e s , that the adenylate cyclase a c t i v i t y i n vivo could be modulated by the c e l l u l a r flux of Ca*4". Wlemer et a l (1978) observed in Immature erythrocytes from rats that Ca4"*", at concentrations above 1 0 " % , acted as a noncompetitive i n h i b i t o r of adenylate cyclase. - 8 9 -Steer and L e v i t z k i (1975) have proposed, based on the study of catecholamine-stimulated adenylate cyclase and Ca transport l n intact turkey erythrocytes, that the a c t i v a t i o n of adenylate cyclase by catecholamines occurs l n two phases. The f i r s t phase i s the increase of a net C a + 4 - e f f l u x from a c r u c i a l C a + + pool, thus removing C a + + from i t s i n h i b i t o r y s i t e s on the adenylate cyclase complex. The second phase is the a c t i v a t i o n of the deinhibited adenylate cyclase by the agonist. This hypothesis has not been tested l n smooth muscle. Although our data does not provide d i r e c t support for such a hypothesis, the question can be r a i s e d as to whether the increase i n cAMP produced by the catecholamines Is an event secondary to the changes i n Ca movements produced by the agonist. It was also observed i n the pre-sent study that exposure of the rat uterus to a Ca - d e f i c i e n t s o l u t i o n accentuated the increase of tissue cAMP content produced by isoproterenol. S i m i l a r e f f e c t s of C a + + - d e f l c l e n t s o l u t i o n on the cAMP level s l n r a t uterus have been reported by several investigators (Polacek and Daniel, 1971» Kroeger and Marshall, 1974; Johansson and Andersson, 1978). This would appear to Indicate that there was an e x i s t i n g Inhibitory influence of Ca 4 4" ++ on adenylate cyclase and removal of the Influence of Ca l n ++ Ca - d e f i c i e n t s o l u t i o n allowed an exaggerated cAMP response to isoproterenol. In conclusion, the question of a possible regulatory r o l e of C a + + in ^ -adrenoceptor-induced increases i n cAMP i n uterine smooth muscle has been r a i s e d . These studies have also indicated, as already discussed, that an increase l n cAMP i s not an obligatory requirement i n order for isoproterenol to produce relaxation. Thus, a more i n t r i c a t e r e l a t i o n s h i p between Ca and cAMP during - 9 0 -J2> - a d r e n o c e p t o r - i n d u c e d r e l a x a t i o n i n s m o o t h m u s c l e a p p e a r t o e x i s t r a t h e r t h a n a s i m p l e s e q u e n c e o f e v e n t s a s p o s t u l a t e d b y t h e s e c o n d m e s s e n g e r h y p o t h e s i s ( F i g u r e 1 8 ) . V . P o s s i b l e R o l e o f cAMP I n S m o o t h M u s c l e R e l a x a t i o n A s i m p l e I n t e r p r e t a t i o n o f t h e p r e s e n t d a t a , t h a t c A M P i s n o t i n v o l v e d I n ^ - a d r e n o c e p t o r - I n d u c e d r e l a x a t i o n o f s m o o t h m u s c l e , I s n o t c o n s i s t e n t w i t h t h e a v a i l a b l e I n f o r m a t i o n I n t h e l i t e r a t u r e ( K o r e n m a n a n d K r a l l , 1 9 7 7 ) . I t h a s b e e n d e m o n s t r a t e d t h a t cAMP a n d d l b u t y r y l cAMP c a n s t i m u l a t e C a + + b i n d i n g i n t h e r a t a n d r a b b i t a o r t i c m i c r o s o m e s ( B a u d o u i n - L e g r o s a n d M e y e r , 1 9 7 3 ) . V a n B r e e m e n (1977) h a s p r o v i d e d i n d i r e c t e v i d e n c e f o r cAMP s t i m u l a t i o n o f C a + + p u m p i n g b y S R I n v a s c u l a r s m o o t h m u s c l e . M a r s h a l l a n d K r o e g e r (1973) s h o w e d t h a t d l b u t y r y l cAMP c a u s e d a d e c r e a s e i n t i s s u e C a + + c o n t e n t i n r a t m y o m e t r i u m a n d c a u s e d r e l a x a t i o n , H a r b o n e t a l (1976) f o u n d t h a t e x p o s u r e o f r a t m y o m e t r l a l s t r i p s t o a d r e n a l i n e r e s u l t e d i n a n e l e v a t i o n o f c A M P l e v e l s a c c o m p a n i e d b y s a t u r a t i o n o f I n t r a c e l l u l a r c A M P r e c e p t o r p r o t e i n s a n d a c t i v a t i o n o f m y o m e t r l a l c A M P - d e p e n d e n t p r o t e i n k i n a s e . N l s h l k o r i e t a l ( I 9 7 8 ) r e p o r t e d t h a t c A M P e n h a n c e d C a u p t a k e b y t h e m i c r o s o m a l f r a c t i o n f r o m t h e r a t u t e r u s . F u r t h e r m o r e , t h e r e was a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n c A M P - d e p e n d e n t p r o t e i n A p h o s p h o r y l a t i o n a n d c A M P - s t l m -u l a t e d C a + + u p t a k e , K o r e n m a n a n d K r a l l (1977) d e m o n s t r a t e d t h a t t r e a t m e n t o f t h e r a t m y o m e t r i u m w i t h i s o p r o t e r e n o l r e s u l t e d i n a n I n c r e a s e I n p r o t e i n k i n a s e a c t i v i t y a s s o c i a t e d w i t h c e l l u l a r m e m b r a n e s a n d i n c r e a s e d A T P - d e p e n d e n t C a t r a n s p o r t b y t h e s e same m e m b r a n e s . F u r t h e r m o r e , c A M P was a b l e t o d u p l i c a t e t h e e f f e c t s o f - a g o n i s t s t i m u l a t i o n i n c e l l f r e e m e m b r a n e p r e p a r a t i o n s . T h u s , a l t h o u g h c A M P c a n n o t b e c o n s i d e r e d a s t h e e x c l u s i v e r e g u l a t o r o f s m o o t h m u s c l e r e l a x a t i o n , I t s t i l l may e x e r t a -91-modulatory a c t i v i t y a t the l e v e l of a l t e r n a t i v e mechanisms (e.g. r e g u l a t i o n of c e l l u l a r c o n c e n t r a t i o n s and t r a n s p o r t of Ca ) o p e r a t i n g i n the c o n t r o l of smooth muscle c o n t r a c t i l i t y ( L e i b e r et a l , 1978). Overweg and S c h l f f (1978) have suggested t h a t l n smooth muscle, there may be an e x i s t e n c e of a r a p i d ^ - a d r e n e r g i c mechanism independent of cAMP as w e l l as a slower cAMP-dependent mechanism f o r r e l a x a t i o n . Such a d u a l process model f o r J3 - a d r e n e r g i c e f f e c t s In the h e a r t has a l s o been suggested ( M c N e i l l , 1 9 7 9 ) . VI. H y p e r p o l a r l z a t i o n and I s o p r o t e r e n o l - l n d u c e d R e l a x a t i o n The r e s u l t s of the present study i n d i c a t e that ^ - a d r e n e r g i c s t i m u l a t i o n o f u t e r i n e smooth muscle can i n h i b i t spontaneous c o n t r a c t i l i t y without n e c e s s a r i l y c a u s i n g h y p e r p o l a r l z a t i o n , and that i s o p r o t e r e n o l - l n d u c e d r e l a x a t i o n of h l g h - K + d e p o l a r i z e d muscle can occur without any change i n membrane p o t e n t i a l . Such a d i s s o c i a t i o n between the catecholamine-induced r e l a x a t i o n and h y p e r p o l a r l z a t i o n thus confirms a previous o b s e r v a t i o n made on r a t myometrium (Diamond and M a r s h a l l , 1 9 6 9 a ) . However, q u a n t i -t a t i v e values f o r the degree o f r e l a x a t i o n and the e f f e c t on mem-brane p o t e n t i a l by a d r e n a l i n e l n d e p o l a r i z e d muscle were not r e p o r t e d l n the above study. A d r e n a l i n e was a l s o shown to be e f f e c t i v e i n r e d u c i n g the c o n t r a c t i o n s e l i c i t e d by supramaximal e l e c t r i c a l f i e l d s t i m u l a t i o n , a c o n d i t i o n i n which c o n t r a c t i o n s do not Involve spontaneous pacemaker d i s c h a r g e and propagation of e x c i t a t i o n . Kroeger and M a r s h a l l ( 1 9 7 3 ) , while s t u d y i n g the e f f e c t of e x t e r n a l potassium c o n c e n t r a t i o n s on the h y p e r p o l a r i z i n g a c t i o n of i s o p r o t e r e n o l demonstrated t h a t a t H 8 m M [ K + J e x , 4x10 M i s o p r o t e r e n o l f a i l e d to cause h y p e r p o l a r l z a t i o n i n the - 9 2 -pregnant rat myometrium. The relaxation response to isoproterenol was not reported in the above study. In a recent study, Nesheim and Slgurdsson (1978) demonstrated, using the sucrose gap method, that in the rabbit uterus pa r t i a l l y depolarized with 15mM K + the mechanical relaxation by Isoproterenol was independent of the membrane potential. Diamond and Marshall ( 1 9 6 9 a ) , using various smooth muscle relaxants, showed that resting membrane potential and resting tension were not necessarily related in rat myometrium. It was suggested that relaxant drugs inhibit motility probably by virtue of their depressant effect on the pacemaker acti v i t y of the uterus rather than hyperpolarlzatlon of the myometrlal c e l l . This conclusion is further substantiated by the present study since -8 10 M Isoproterenol caused inhibition of spontaneous contractions and abolished the generation of action potentials but did not cause hyperpolarlzatlon. It may be noted that although hyper-polarlzatlon appears neither necessary nor responsible for the relaxation, i t is possible that the hyperpolarlzatlon produced by the catecholamines may reinforce the depressant effects of these drugs on the mechanical acti v i t y of the rat uterus. The data presented here appear to be consistent with the prevailing concept that relaxation of the contractile mechanism by electrical events of the c e l l membrane is not the only physiological way of smooth muscle relaxation. -93-SUMMARY The study of the involvement of c y c l i c nucleotides i n the control of uterine smooth muscle c o n t r a c t i l i t y led to the following conclusions! 1. The postulated r o l e of cAMP and cGMP i n the control of the spontaneous uterine contraction-relaxation cycle was not supported by the data obtained i n this study. 2. A d i s s o c i a t i o n between isoproterenol-lnduced cAMP level s and relaxation i n depolarized uterus was obtained. Thus, there is no apparent simple cause and e f f e c t r e l a t i o n s h i p between ^-adrenoceptor-induced changes i n cAMP level s and relaxat i o n i n uterine smooth muscle. 3. A r e c i p r o c a l r e l a t i o n s h i p between cAMP and cGMP i n Isoproterenol-lnduced relaxation of r a t uterus could not be established. 4 . The a b i l i t y of Isoproterenol to Increase cAMP level s i n depolarized r a t uterus was found to be dependent on C a + + . + 4 . Thus, the question of a possible regulatory role of Ca In 'jj-adrenoceptor-induced Increases in cAMP was rai s e d . 5. It was proposed that e x t r a c e l l u l a r cations such as Na + and ++ Mg i n the depolarizing medium can overcome the blockade of isoproterenol-lnduced increases in cAMP levels induced by hlgh-K +. Na + and Mg + + produce this e f f e c t probably by virtue of t h e i r a b i l i t y to i n h i b i t passive C a + + - l n f l u x occurring during high-K + depolarization. -9k-The electrophysiological studies demonstrated that the hyperpolarlzation of c e l l membranes Is not a prerequisite for ^-adrenoceptor mediated relaxation of uterine smooth muscle. -95-REFERENCES Andersson, R.i Role of c y c l i c AMP and C a + + i n mechanical and metabolic events in isometrically contracting vascular smooth muscle. Acta. Physiol. Scand. 87i 8 4 - 9 5 , 1973. Andersson, R., Nllsson, K., Wikberg, J . , Johansson, S., and Lundholm, L. j C y c l i c nucleotides and the contraction of smooth muscle. Inj Advances in C y c l i c Nucleotide Research, eds. Drummond, G.I., Greengard, P., and Roblson, G.A., Raven Press, N.Y. 5 j 491-518, 1975. A l t u r a , B.M., A l t u r a , B.T. , and. 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