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Changes in protein phosphorylation during changes in vascular smooth muscle tone Marshall, Caroline Louise 1984

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CHANGES IN PROTEIN PHOSPHORYLATION DURING CHANGES IN VASCULAR SMOOTH MUSCLE TONE by Caroline Louise Marshall B . S c , The University of Strathclyde, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES 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 March 1984 Caroline Louise Marshall, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e 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 a g r e e t h a t t h e L i b r a r y s h a l l m a k e 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 a n d s t u d y . I f u r t h e r a g r e e 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 m a y b e g r a n t e d b y t h e h e a d o f m y d e p a r t m e n t o r b y h i s o r h e r 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 t h a t 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 n o t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f og^s*? , i x i r a l CSg3(51-rvOi<^ T h e 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 1 9 5 6 M a i n M a l l V a n c o u v e r , C a n a d a V 6 T 1 Y 3 E - 6 ( 3 / 8 1 ) i i ABSTRACT I t i s now g e n e r a l l y accepted that changes i n p r o t e i n phosphorylation play an important r o l e i n mediating smooth muscle tone. An attempt was made to determine whether nitrogen oxide c o n t a i n i n g v a s o d i l a t o r s such as sodium n i t r o p r u s s i d e and n i t r o g l y c e r i n exert t h e i r r e l a x ant e f f e c t v i a a phosphorylation r e a c t i o n . I s o e l e c t r i c focusing was i n i t i a l l y used as a method of d e t e c t i n g phosphorylation. However, due to the smaller percentage of p r o t e i n i n smooth muscle with a r e l a t i v e increase i n other c e l l c o n s t i t u e n t s , crude smooth muscle homogenates were deemed to be too complex f o r a n a l y s i s by t h i s technique alone. Phosphorylation changes are commonly studied by incubating the muscle i n l a b e l l e d i n o r g a n i c phosphate, thus l a b e l l i n g the ATP pools. P r o t e i n s are separated by SDS-polyacrylamide gel e l e c t r o p h o r e s i s . Following s t a i n i n g and d r y i n g , gels are exposed to X-ray f i l m and phosphorylation l e v e l s determined. By comparing a o r t i c s t r i p s relaxed i n Ca^-free, 5 mM EGTA Krebs with muscle s t r i p s contracted i n 1^(124 mM) Krebs, a s i g n i f i c a n t d i f f e r e n c e between the phosphorylation l e v e l s of myosin l i g h t chain was q u a n t i t a t e d . By reproducing t h i s well documented phenomena, we demonstrated that we had e s t a b l i s h e d a working methodology i n the l a b o r a t o r y . In view of the controversy i n the l i t e r a t u r e concerning sustained l e v e l s of myosin l i g h t chain phosphorylation during sustained K^-induced c o n t r a c t i o n s , a K"^time course study was performed. Levels of myosin l i g h t chain phosphorylation increased s i g n i f i c a n t l y w i t h i n the f i r s t 2 min of c o n t r a c t i o n and were maintained f o r 12 min, though a n o n - s i g n i f i c a n t decrease was observed a f t e r 2 min. Tension peaked at 4 min and t h e r e a f t e r remained constant. F inal ly , the effect of nitroglycerin (10~tVl) on \C-induced contractions was br ief ly examined. Nitroglycerin caused a 70% relaxation within 2 min and s ignif icant ly decreased myosin l ight chain phosphorylation levels . There also appeared to be an increase in phosphorylation of a 160kD protein with nitroglycerin treatment, which due to technical d i f f i cu l t i es could not be quantitated. i v TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i LIST OF ABBREVIATIONS i x INTRODUCTION 1 Evidence for a role of protein phosphorylation in the regulation of smooth muscle tone. a) Resting tension 3 b) Spontaneous contractions 4 c) Induced contractions 5 d) Calcium dependency 11 e) Relaxation 15 f) Vasodilators 2 0 OBJECTIVES 24 MATERIALS AND METHODS A. Materials 25 1) Animals 25 2) Chemicals 25 i) Radioisotopes i i ) Electrophoresis i i i ) Other reagents PAGE B. Methods 1) Tissue preparation 27 2) Sample preparation 28 3) Electrophoretic techniques a) Isoelectric focusing 28 b) SDS - polyacrylamide gel electrophoresis 31 4) Miscellaneous assays a) Protein assay 33 b) Stat is t ics 33 RESULTS : r. 1. Isoelectric focusing 34 2. SDS - Polyacrylamide gel electrophoresis i ) Calcium free studies 51 i i ) Potassium time course study 58 i i i ) Effect of nitroglycerin (10~6M) 68 DISCUSSION 69 i ) Effect of Calcium 73 i i ) Potassium time course study ^5 i i i ) Nitroglycerin induced relaxation 79 SUMMARY AND CONCLUSION 84 BIBLIOGRAPHY 85 v i LIST OF TABLES TABLE PAGE 1) Phosphorylation of 20,000 - dalton l ight chain of 14 myosin in intact arter ial smooth muscle under various conditions 2) Summary of methods used for sample preparation 37 prior to isoelectr ic focusing 3) % Phosphorylation of protein in t i b i a l i s anterior 38 samples prepared by various methods 4) Summary of experiments using isoelectr ic focusing 50 to detect changes in phosphorylation 5) Calcium dependency of phosphorylation of 23 kD 57 protein in rabbit aorta 6) Effect of nitroglycerin on K +-induced contractions 61 7) Effect of nitroglycerin (10~6M) on 3 2 P incorporation 64 of 23 kD protein during K +-induced contraction in rabbit aorta 8) Summary of experiments use SDS-polyacrylamide gel 67 electrophoresis to detect changes in phosphorylation v i i LIST OF FIGURES FIGURE PAGE 1 . Mechanical response f o r spontaneously c o n t r a c t i n g 6 r a t myometrium 2. R e l a t i o n s h i p between developed f o r c e and myosin l i g h t 9 cha in phosphory la t ion i n s t r i p s prepared from the hog c a r o t i d media 3 . S t e a d y - s t a t e C a 2 + dependence of s t r e s s and myosin 12 phosphory la t ion 4 . Hypothesis f o r r e g u l a t i o n of c r o s s br idges 16 5. Comparison of the d i s t r i b u t i o n of p r o t e i n s as r e s o l v e d 39 on i s o e l e c t r i c f o c u s i n g po l yac ry lamide ge l s i n 6 week t i b i a l i s a n t e r i o r 6 . Scans of 6 week t i b i a l i s a n t e r i o r 4 1 7. Comparison o f the d i s t r i b u t i o n o f p r o t e i n s as r e s o l v e d 43 on i s o e l e c t r i c f o c u s i n g po lyac ry lamide ge ls i n myometrial samples 8 . Scans of myometrium 46 9. Scans of myometrium 48 10 . Autoradiogram of a SDS -po l yac ry lamide g r a d i e n t gel 52 v i i i FIGURE PAGE 11. Scans of autoradiograms of SDS-polyacrylamide gradient gel 54 12. Phosphorylation of myosin l ight chain K-induced contraction 59 of rabbit aorta 13. Mechanical responses of rabbit aorta to 124 mM KC1 and 62 nitroglycerin (10~6M) 32 14. Scans of P autoradiograms of SDS-polyacrylamide 65 gradient gels 15. Autoradiograms of soluble and particulate fractions of 71 rat thoracic aorta after two-dimensional gel electrophoresis 16. A. Time course of myosin l ight chain phosphorylation 77 stress development, and shortening velocity in K +-stimulated carotid media tissues B. Myosin phosphorylation during methacholine induced contraction of tracheal smooth muscle i x L I S T OF ABBREVIATIONS ATP a d e n o s i n e 5 ' - t r i p h o s p h a t e A T P a s e a d e n o s i n e t r i p h o s p h a t a s e cAMP c y c l i c a d e n o s i n e 5 ' - m o n o p h o s p h a t e cGMP c y c l i c g u a n o s i n e 5 ' - m o n o p h o s p h a t e EGTA e t h y l e n e g l y c o l - b i s - ( £ - a m i n o e t h y l e t h e r ) N . N ' - t e t r a - a c e t i c a c i d IEF i s o e l e c t r i c f o c u s i n g MLC m y o s i n l i g h t c h a i n MLCK m y o s i n l i g h t c h a i n k i n a s e MLCP m y o s i n l i g h t c h a i n p h o s p h a t a s e NG n i t r o g l y c e r i n SDS-PAGE , s o d i u m d o d e c y l s u l p h a t e - p o l y a c r l y a m i d e g e l e l e c t r o p h o s e s i s SNP s o d i u m n i t r o p r u s s i d e TA t i b i a l i s a n t e r i o r TCA t r i c h l o r o a c e t i c a c i d X ACKNOWLEDGEMENT I am grateful to my supervisor, Dr. J . Diamond for his guidance and support throughout this study. I would especially l ike to thank Dr. L.G. Jasch for her appreciable assistance throughout the study. My thanks also go to the rest of my committee, Drs. K. MacLeod, J .H . McNeill and S. Katz for their constructive cr i t ic ism and suggestions during this study. I was supported throughout this study by the B.C. and Canadian Heart Foundations and this funding is gratefully acknowledged. My thanks to Mrs. J . Wayne for her assistance in the preparation of this thesis. I also wish to thank al l my friends within and outwith the faculty for having made this masters program most enjoyable. F inal ly , I wish to extend my sincere thanks to two people, Mrs. Evelyn Chu and Mr. Mike Bailey, without whose unfailing help and support this study would not have been completed. x i THIS THESIS IS DEDICATED TO MY PARENTS FOR THEIR CONTINUAL SUPPORT AND ENCOURAGEMENT THROUGHOUT THE YEARS 1 INTRODUCTION The calcium-dependent regulatory systems for the contractile proteins in smooth muscle have become the subject of intense research. The release of Ca^into the sarcoplasm is the primary event in excitation-contraction coupling in al l types of muscles. The subsequent binding of Ca^to specif ic high-affinty sites on proteins associated with, or acting on, the contractile apparatus ultimately results in contraction, which i s , in a l l types of muscle, the interaction of actin and myosin. The difference between muscles ( i . e . skeletal and smooth) is the mechanism by which calcium causes this interaction. Such differences are attributed to differences in regulatory proteins, which mediate the Ca'** effects and to isozymic differences in the myosin molecule, (Stull et a l r 1980). One of the Ca^dependent processes that has been the subject of much study is the obligatory in i t ia t ion of the actin-activated ATPase act iv i ty of myosin via the phosphorylation of the myosin l ight chain (MLC). This reaction is catalyzed by a calcium dependent myosin l ight chain kinase (MLCK), (Aksov et a l . 1976; Chacko et a l . 1977; DiSalvo et a l . 1978; Gorecka et a l . 1976). These studies have resulted in the hypothesis (Adelstein et a l , 1977; Hartshorne et a l . 1977) that calcium plays an important role in smooth muscle contraction by activating the MLCK, which in turn phosphorylates the MLC, which in i t iates an interaction between actin and myosin, resulting in contraction. According to the hypothesis, i t would follow that a decrease in cytoplasmic calcium would inactivate MLCK and the MLC would be dephosphorylated by an endogenous myosin l ight chain phosphatase (MLCP) or phosphatases, resulting in relaxation. 2 The above hypothesis based on biochemical data from isolated protein studies is not unchallenged.(Mikawa et a l t 1977) The main evidence against phosphorylation as the principal control mechanism for contraction is the apparent dissociation between phosphorylation and actin activated Mg-ATPase found by some workers. Studies by Mikawa et a l . (1977), using smooth muscle actomyosin fai led to detect any change in the act iv i ty of actomyosin ATPase with varying degrees of phosphorylation. Bose et a l . (1979), using chicken gizzard actomyosin examined i ts calcium sensi t iv i ty . The authors concluded from this work that calcium sensit iv i ty was altered by ATP concentrations, which appeared to regulate the degree of repression of myosin. These authors believe that at high levels of ATP myosin is repressed and can only interact with actin once i t is phosphorylated. At low levels of ATP myosin is derepressed. In this state myosin can interact with actin as long as Mg is present and subsequently develop tension. Alternative hypotheses proposed, include calcium binding to myosin, a new type of thin filament control system, which was named leiotonin and a troponin control (Marston, 1982) Inspite of these controversies, most investigators believe that phosphorylation of MLC is the main regulatory mechanism of contraction in smooth muscle. In addition to biochemical data obtained on studies in isolated proteins, correlations between increased levels of phosphorylation have been found in intact (Barron et a l . 1979; Driska et a l , 1979; Gualtieri et a l , 1977) and functionally skinned ( Hoar et a l , 1979) smooth muscle preparations. I.Evidence for a Role of Protein Phosphorylation in the Regulation of Smooth Muscle Tone 3 In reviewing some of the evidence for a role of l ight chain phosphorylation in smooth muscle regulation, i t seems logical to f i r s t discuss the basal level of phosphorylation in a resting muscle, from there moving on to a spontaneously contracting muscle (e.g. myometrium) then to chemically-induced contractions and f ina l l y completing the cycle with a look at relaxation and relaxants. This is by no means a complete review of the l i terature in these areas but some of the major theories are highlighted, and some of the problems and differences in interpreting data are discussed. a) Resting tension: There is general agreement in the l i terature that even a resting muscle has detectable levels of MLC phosphorylation. This can be attributed to a number of possible reasons. When studies are carried out on intact smooth muscles, the muscles are generally under some degree of resting tension, the magnitude of which varies between types of muscles and species or ig in . Barron et a l . (1979), demonstrated, using intact pig carotid arter ies, that MLC phosphorylation was dependent upon the passive tension applied to the muscle. A similar observation was made by Dil lon et a l . (1981), using pig carotid arteries and de Lanerolle and Stul1,(1980), using bovine trachea. It was proposed that stretching the muscle might cause an increase in cytoplasmic calcium concentration, which in turn might cause partial phosphorylation, (de Lanerolle and Stul1 , 1980). The degree of such phosphorylation might be commensurate with the tension of the muscle. A second possible reason for the detectable levels of MLC phosphorylation in resting muscles was proposed by Aksoy et a l , (1982). These authors suggested that there may be a sl ight artifactual increase in 4 MLC phosphorylation associated with the immersion freezing technique, however i t can be argued that this is due to prevention of dephosphorylation which may otherwise occur in some cases during homogenisation due to incomplete inactivation of phosphatases. Thirdly, i t is conceivable that in muscles exhibiting spontaneous electr ical act iv i ty (e.g. myometrium) this electr ical act iv i ty is associated with a small influx of Ca"^, which could be suff icient to cause partial MLC phosphorylation. The exact origin of Ca"*"*' required for such a reaction in resting muscle is unclear. Final ly i t has been suggested that a small (15%) systemic overestimation of phosphorylation wil l arise i f i soelct r ic focusing (IEF) is used as a method of detection. This results from the carbamylation of the polypeptide, which wil l increase i ts negative charge - this is a possible result of tissue preparation. Other possible chemical modifications which may occur include oxidation, sulphonation of tyrosine residues, acetylation of lysine or N-terminal amino acids or deamination by isocyanates in urea, (Dillon et a l . 1981). The use of IEF as a method of detection of protein phosphorylation stems from the observation that when a protein undergoes phosphorylation, i t s i soe lc t r ic point becomes signif icantly more acidic and this shift is detectable by IEF. Densitometry of the IEF gels is used to measure the changes, (Driska et a l . 1981). b) Spontaneous contractions: In a study by Janis et a l . (1980), using rat myometrium, these workers spec i f ica l ly looked at the levels of MLC phosphorylation during spontaneous contractions. Both one and two dimensional electrophoresis were used to determine phosphorylation and the results were presented as % 5 phosphorylation of relaxed controls, (Fig 1). These authors concluded that the results obtained support the hypothesis that contraction and relaxation of smooth muscle is part ia l ly regulated by phosphorylation and dephosphorylation of MLC. It also was observed that phosphorylation preceded maximal spontaneous contraction and dephosphorylation preceded maximal relaxation. c) Induced contractions: Not al l smooth muscles wil l contract spontaneously and many studies have been carried out on induced contractions. Contractions may be ini t iated by a number of agonists acting at a variety of s i tes . In general, most studies agree with the hypothesis that MLC phosphorylation wil l precede contraction and dephosphorylation will precede relaxation. Barron et a l , (1980), demonstrated reversible MLC phosphorylation in swine carotid arter ies, during the contraction-relaxation-contraction cycle using 100 mM KCl to contract the tissue and 10 mM theophylline to induce relaxation. The muscles were frozen after maximal tension (6 min treatment time) had been reached and there was an increase in phosphorylation in resting muscles from 0.48 mol phosphate / mol MLC to 0.65 mol / mol in contracted muscles. This level of MLC phosphorylation decreased to 0.31 mol / mol after 10 mM theophylline had caused complete relaxation (total treatment time 36 min). A similar phosphorylation of the MLC using noradrenaline as the contracti le agonist had also been noted by these workers, (Barron et a l , 1979). However, in contrast to these results, Murphy and co-workers have suggested that phosphorylation declined signif icantly from i ts peak values before steady-state force was attained. Levels continued to decline towards 6 FIGURE 1: Mechanical response of spontaneously contracting rat myometrium. Contractions occurred about one per minute. Also shown are the means for isotopic phosphorus incorporation at certain levels of isometric tension. Reproduced from Jam's et a l . (1980). 7 TIME (sec) 8 control values after 1 0 min of stimulation. The contractile agonist in this case was 1 2 4 mM KCl and swine carotid arteries were the tissue used, ( Driska et a l . 1 9 8 1 ) . Peak MLC phosphorylation, with K"*~-stimulation was about 65% and occurred about 3 0 s after the muscle was exposed to the stimulus. Active force subsequently increased and was maintained while the levels of MLC phosphorylation were f a l l i n g . After 1 2 min of stimulation the level of MLC phosphorylation was approximately 30%, (Fig 2 ) . The difference in the time course of MLC phosphorylation is not restricted to swine carotid arter ies. A study by de Lanerolle and Stu l1 , ( 1 9 8 0 ) , investigated MLC phosphorylation in canine tracheal smooth muscle and showed that the phosphorylation increased from a value of 0 . 5 0 to 1 . 1 mol phosphate / mol myosin, within 3 min after the addition of I O O ^ J M methacholine. These authors concluded that MLC phosphorylation coincided temporally with the increase in isometric tension. Aksoy et a l . ( 1 9 8 2 ) , attributed the thickness of the muscle preparation and the long agonist diffusion times as reasons for the fai lure to observe any rapid transient changes in these studies. For the formentioned reasons, Gerthoffer and Murphy, ( 1 9 8 3 a) , used rabbit tracheal is to study MLC phosphorylation. Results from their time course studies, using 1 0 5 M carbachol as a contractile agonist, were similar to those Driska et a l . ( 1 9 8 1 ) , had found with swine carotid arter ies. MLC phosphorylation was low in resting muscle, increased rapidly to 0 . 4 6 mol phosphate / mol MLC and subsequently declined toward resting levels prior to reaching steady-state active stress. Si lver and S tu l1 , ( 1 9 8 2 ) , also found a transient increase in MLC phosphorylation in a thin preparation of bovine tracheal muscle. However, Janis et a l t ( 1 9 8 0 ) , found that phosphorylation levels in rat uterine 9 FIGURE 2: Relationship between developed force and MLC phosphorylation in strips prepared from the hog carotid media. A: open c i rc les represent data from tissues frozen in the unstimulated state possessing low levels of tonic contracti le act iv i ty . The remaining points were obtained from tissues frozen during a contraction produced by a high K+ physiological salt solution. Phosphorylation increased rapidly during the f i r s t 60s of the response, reaching a maximum value when force had reached about half of i ts maximum level (solid c i r c l e s ) . After 2 min of stimulation, force was near maximum, although phosphorylation levels averaged somewhat lower (solid tr iangles) . Strips frozen after approximately 6 min (open squares) or 12 min of stimulation (solid squares) showed progressively lower levels of MLC phosphorylation. Maximum active force (±SE) averaged 3 .34±0.15x l0 5 h/m 2 (n=17), and, once developed, was maintained without s ignif icant changes in the presence of high K +. Reproduced from Driska et_ al_. (1981) 10 o O 80 CL o ro o + 60 h O OJ ( J CL o CO c t CL 40 2 0 P o A Controcting Tissues A A e • J 1 L 25 50 75 100 Active Force (% of Maximum) 11 smooth muscle were s t i l l elevated at 0.72 mol phosphate / mol MLC after 3 -5 min 100^uM carbachol. Resting levels were estimated at 0.4 mol / mol. There appear to be two schools of thought on the role of MLC phosphorylation, one that i t is associated with contraction and is maintained during contraction and the other that i t is more closely related to isotonic shortening velocit ies and cross-bridge cycling rates, (Aksoy et a l , 1982; Dil lon et a l , 1981). d) Calcium dependency: Calcium has long been implicated as a regulatory ion in muscle contraction. There are many examples in the l i terature showing a correlation between calcium concentration, tension and MLC phosphorylation. In the following example, (Chatterjee et a l t 1983), skinned carotid arteries were used to study the calcium requirements of MLC phosphorylation, (Fig 3). As the concentration of calcium increased, the MLC phosphorylation and tension increased. If calcium is omitted from the physiological salt solution and 1.0 mM EGTA added, there is a marked decrease in the MLC phosphorylation, (Table 1), (Barron et a l . 1979). Also shown in Table 1 is the effect of passive tension on phosphorylation as was previously discussed. It has been suggested that maximum phosphorylation will occur at calcium concentrations of lO~bM, (Driska et a l , 1981). However, Murphy and co-workers over the past few years, have produced a number of reports that suggest that contraction can be maintained without phosphorylation, ( Chatterjee et a l . 1983; Dil lon et a l . 1981; Aksoy et a l , 1982; Aksoy et a l . 1983). These workers summarise the hypothesis for the contraction-relaxation 12 FIGURE 3: Steady-state C a 2 + dependence of stress and myosin phosphorylation when C a 2 + was increased from 1.8xlO' 8M (means±standard errors, n=4 to 6). Maximum stress was 9.3±1.2x10" n/m2 (n=10). The calculated concentration of C a 2 + for half-maximal change in the response (K s o ) is shown with a 65% confidence interval (•). Reproduced from Chatterjee e_t al_. (1983). pC a 14 TABLE 1: Phosphorylation of 20,000 dalton LC of myosin in intact arter ial smooth muscle under various conditions. 32Phosphate/MLC mol/mol Resting, with passive tension Ca 2 + - f ree solD* 0.13 Resting, with no passive tension and C a 2 + 0.34 Resting, with passive tension and C a 2 + 0.55 Contracted, with norepinephrine 0.79 *Ca2 was omitted from the physiological salt solution and 1.0 roM EGTA was added. Barron et a l . , 1979 15 cycle for arterial smooth muscle as follows, (see Fig 4). Relaxed muscle contains thin actin filaments (A) and unattached myosin cross-bridges (M). A rise in calcium concentration on stimulation activates MLCK and phosphorylates free cross-bridges (M-P), which then can interact with act in. Evidence indicates that kinase-phosphatase systems can also act on attached cross-bridges. Dephosphorylation of attached cycling cross-bridges (AM-P) arrests cycling to form attached, noncycling cross-bridges (AM), termed latch bridges. Latch bridges constitute internal load on cycling cross-bridges and decrease shortening velocity (heavy arrow). Second, direct action of calcium on thin filaments or myosin is shown by dependence of stress (reflecting both AM and AM-P) on this ion. Dashed arrows indicate possibi l t ies that detachment of latch bridges may occur by mechanical breakage and that direct formation may occur in the presence of calcium, (Aksoy et a l , 1982). These latch bridges could explain why arter ies, which have a comparable ernergy usage to that of skeletal muscle during development of isometric tension, can maintain high degrees of tone inspite of a subsequent decrease in energy usage during steady-state maintaenance of tone, (Aksoy et a l , 1982). e) Relaxation: As has been previously discussed, MLC phosphorylation is reversible and dephosphorylation is often associated with relaxation, (Jam's et a l , 1980; Barron et a l . 1980; Chatterjee et a l , 1983; Aksov et a l . 1982; Driska et a l , 1981). However, Murphy and coworkers have produced evidence that phosphorylation is not necessarily sustained during contraction and i t 16 FIGURE 4 : Hypothesis for regulation of cross bridges. See text for explanation. Reproduced from Aksoy et al_. (1982). 17 (Co**) $k "MLCK (Reloxotion) A •M • i r A + M-P MLCPase/ /Crossbridg AM-P MLCPase (LofcK) (Cycling) 18 would f o l l o w that dephosphorylation might then not be so s t r i c t l y c o r r e l a t e d with r e l a x a t i o n . Therefore G e r t h o f f e r and Murphy, (1983 b ) , examined the r e l a t i o n s h i p between dephosphorylation and r e l a x a t i o n . Using i n t a c t swine c a r o t i d a r t e r i e s , the above authors contracted the t i s s u e with 110 mM KCl s o l u t i o n and then washed the agonist out. The r e l a x a t i o n f o l l o w i n g washout was analyzed as a dual exponential decay. The i n i t a l r a p i d phase (2 min) was associated with dephosphorylation and the decay ca p a c i t y of i s o t o n i c shortening v e l o c i t y . The second slow phase (45 min) was a t t r i b u t e d to the noncycling l a t c h bridges. The rate of decay of t h i s slow phase was enhanced by the removal of calcium and retarded by an increase i n calcium to 5 mM. These r e s u l t s tend to support the above hypothesis as proposed by Aksoy et a l , (1982), (see F i g 4 ) , i l l u s t r a t i n g the existence of two types of c r o s s - b r i d g e s , one population of which i s dephosphorylated, s e n s i t i v e to calcium and capable of maintaining s t r e s s . Taking t h i s one step f u r t h e r , how do rel a x a n t s a f f e c t phosphorylation l e v e l s ? Since the discovery by Perry, (1979), of the MLCK / MLCP system, a growing body of evidence has s t r o n g l y i m p l i c a t e d MLC phosphorylation i n the calcium-dependent r e g u l a t i o n of c o n t r a c t i o n of smooth muscle. This i s not the only enzyme system i n operation. A d e l s t e i n and Hathaway, (1979), described a system, which i s dependent on c y c l i c AMP (cAMP). The cAMP-dependent p r o t e i n kinase w i l l phosphorylate MLCK, decreasing i t s a f f i n i t y f o r the Ca^-calmodulin complex thus decreasing l e v e l s of MLC phosphorylation. This l e d A d e l s t e i n and Hathaway to postulate that increased cAMP l e v e l s would r e s u l t i n phosphorylation of MLCK, a decrease i n phosphorylation of MLC and r e l a x a t i o n . These workers proposed that beta-adrenergic a g o n i s t s , which cause r e l a x a t i o n of contracted vascular smooth muscle, exerted t h e i r e f f e c t s i n t h i s way. 19 Considering the theory presented by Murphy and colleagues concerning latch bridges, (Fig 4), (Aksoy et a l , 1982; Gerthoffer and Murphy, 1983 a, b), the above hypothesis proposed by Adelstein and Hathaway, (1979), seems lacking. How can relaxants induce relaxation via MLC dephosphorylation i f phosphorylation i s , as studies by Murphy and coworkers suggest, already at basal levels? A recent investigation by Gerthoffer and Murphy (in press) addresses this very question. Using swine carotid arteries as previously described, (Driska et a l , 1981), these workers examined the effects of various relaxants on K1"- and phenylephrine-induced contractions. Muscles were contracted for 15 min, after which the agonist was washed out and the relaxant added 2 min later , when phosphorylation levels were near control values, (Gerthoffer and Murphy, 1983 b). The relaxants chosen represented a variety of different classes, adenosine and forskol in , which are thought to act via cAMP-dependent mechanisms, 3 - isobuty l - l methyl xanthine as a phosphodiesterase inhibitor and sodium nitroprusside (SNP) and 8-bromo-cGMP, which are thought to act via cycl ic GMP (cGMP) dependent mechanisms. In al l cases the results were similar . The above agents al l enhanced the relaxation rate after MLC had been dephosphorylated. These results support the hypothesis, (Aksoy et a l . 1982), (see Fig 4), that there are two regulatory mechanisms governing vascular smooth muscle tone. 1) Init ial phosphorylation of MLC which results in phosphorylated cycling cross bridges. 2) Following dephosphorylation of the above, stress is maintained by non-cycling cross-bridges termed latch bridges, which are calcium dependent in an as yet unknown manner. 20 Cyclic AMP-induced dephosphorylation by beta adrenergic agonists would not appear to be the primary method of relaxation by these agents. f) Vasodilators: In the last section, various vasodilators were mentioned as well as their effects on cycl ic nucleotides. The relationship between cycl ic nucleotides, particularly cGMP, and mechanical act iv i ty of smooth muscle has been the subject of several disparate theories. Increases, (Lee et a l , 1972), as well as decreases in smooth muscle tension, (Diamond and Holmes, 1975), were observed to be associated with increased cGMP levels . Subsequently, i t was found that a number of c l i n i c a l l y useful vasodilators, including nitroglycerin (NG) and SNP, could increase cGMP levels in the absence of Cations in intact smooth muscle t issue, (Schultz et a l . 1977). The hypothesis, which is becoming widely accepted, was then put forward that these nitrogen oxide containing compounds exert their relaxant effect by virtue of their ab i l i t y to increase levels of cGMP in vascular smooth muscle. There are many observations in the l i terature consistent with this hypothesis. A variety of smooth muscle relaxants have been shown to increase levels of cGMP in various t issues, (Katsuki and Murad, 1977; Katsuki et a l , 1977 a, b; Bohme et a l , 1978; Janis and Diamond, 1979). Some of these vasodilators, namely NG and SNP, also activate guanylate cyclase di rect ly , (Gruetter et a l . 1980, 1981). There appears to be a close correlation between the degree of smooth muscle relaxation and the extent of cGMP formation by NG, SNP and other smooth muscle relaxing agents, (Katsuki and Murad, 1977; Axelsson et a l . 1979; Janis and Diamond, 1979; Kukovetz et a l , 1979). Another piece of evidence in favour of the 21 hypothesis is that the l ipophi l ic derivative of cGMP, 8Br-cGMP, is capable of relaxing smooth muscle, (Katsuki and Murad, 1977; Schultz et a l , 1979; Napoli et a l , 1980). Studies by Gruetter et a l , (1981), show that cGMP accumulation actually preceded the onset of relaxation induced by NG and was temporally correlated with relaxation induced by NG and SNP. This study also demonstrated that an inhibitor of guanylate cyclase, methylene blue, could simultaneously inhibit cGMP accumulation and relaxation by nitrogen oxide-containing vasodilators. The return of cGMP levels to control values preceded the return of arterial tone. These results were later confirmed by Keith et al.(1982). Furthermore, inhibition of vascular smooth muscle relaxation was seen with NG tolerance and there was a corresponding inhibition of cGMP generation, (Keith et a l , 1982). Subsequent action of cGMP does not appear to be impaired by the presence of NG tolerance or the inhibitor , methylene blue, since 8Br-cGMP is s t i l l able to relax vascular smooth muscle. This evidence would tend to suggest that the abi l i ty to generate cGMP may be an important step in the process by which NG and SNP relax vascular smooth muscle. It is thought that these nitrogen oxide containing compounds relax vascular smooth muscle via the reactive intermediate, n i t r i c oxide (NO) and that the biological action of NO is associated with the activation of guanylate cyclase, (Gruetter et a l , 1979). This would stimulate cGMP formation in vascular and non vascular smooth muscle, (Schultz et a l , 1977; Katsuki et a l , 1977 a, b). These increases in cGMP may then in i t iate relaxation in some, as yet unknown, way. Despite a l l the above evidence, results from this laboratory are not consistent with the hypothesis that cGMP and relaxation are causally 22 related at least in some types of smooth muscle. SNP was able to produce large increases in cGMP levels in K4"- or phenylephrine - contracted strips of rat vas deferens without causing relaxation, while hydralazine and verapamil caused relaxation without an increase in cGMP levels , (Diamond and Jam's, 1978). When other types of smooth muscle, such as guinea-pig taenia coli and vascular smooth muscle were examined, the results indicated that in these tissues also, the relaxant effects of hydralazine and verapamil were not associated with increased cGMP levels . Low concentrations of SNP were also able to relax teania coli without simultaneously elevating cGMP levels , however at concentrations greater than 1 i^M there appeared to be a correlation between relaxation and increases in cGMP, (Janis and Diamond, 1979). In more recent experiments from this laboratory i t was shown that both NG and SNP are able to cause an elevation in cGMP levels in the rat vas deferens and myometrium, though only NG can cause relaxation, despite the fact that the increase in cGMP levels was greater with SNP, (Diamond, 1983). This data s t i l l might be compatible with the hypothesis i f these tissues do not possess a cGMP-dependent mechanism of relaxation and therefore do not relax on increases in cGMP levels caused by such compounds as SNP. NG, however may have two mechanisms of relaxation (one cGMP-dependent and one cGMP-independent) and, at high concentrations of NG the latter mechanism could be responsible for the relaxation observed. However, as pointed out ear l ie r , the exogenous administration of 8Br-cGMP was capable of relaxing these preparations and is cited as strong evidence in favour of the proposed role for cGMP as a mediator of smooth 23 muscle relaxation, (Schultz et a l , 1979; Napoli et a l , 1980). If 8Br-cGMP can relax these muscles, presumably by increasing tissue levels of cGMP, why are the increases in cGMP levels caused by SNP not accompanied by relaxation? It is possible that in these tissues cGMP does not play a role in relaxation and that 8Br-cGMP can relax the muscle by a mechanism independent of cGMP. Alternatively, the increases in cGMP might occur in a compartment or pool which is not physiologically relevant. For example, i t is thought that cGMP exerts i ts effect via activation of a cGMP-dependent protein kinase. If this is a necessary step in smooth muscle relaxation i t is possible that in some tissues this kinase is inaccessible to the increased cGMP levels due to compartmentalization of the lat ter . Preliminary observations from this laboratory suggest that smooth muscle relaxation and activation of cGMP-dependent protein kinase may be correlated under certain conditions. The act iv i ty of the kinase is d i f f i cu l t to measure di rect ly , due to technical problems. If however, activation of cGMP-dependent protein kinase is involved in mediating vascular relaxation, a possible mechanistic pathway is via phosphorylation of specif ic protein(s), which are involved in the regulation of smooth muscle tension in the muscle. Reports in the l i terature, (Draznin et a l . 1982, Rapoport et a l , 1982), have shown increased phosphorylation of several proteins in rat aortic tissues exposed to 0.5 u^M SNP for 2-15 min. These authors suggested that one or more of these proteins may be involved in the SNP induced relaxation of vascular smooth muscle. 24 OBJECTIVES 1) The m a i n o b j e c t i v e o f t h i s p r o j e c t was t o e s t a b l i s h a w o r k i n g p r o t o c o l i n o u r l a b o r a t o r y f o r m e a s u r i n g p r o t e i n p h o s p h o r y l a t i o n i n s m o o t h m u s c l e . A s a m e a s u r e o f t h e s u i t a b i l i t y o f t h e m e t h o d we a t t e m p t e d t o q u a n t i t a t e p h o s p h o r y l a t i o n o f MLC d u r i n g c a l c i u m - i n d u c e d c o n t r a c t i o n s o f r a b b i t a o r t a . C a l c i u m - d e p e n d e n t p h o s p h o r y l a t i o n o f MLC i n s m o o t h m u s c l e i s a w e l l d o c u m e n t e d p h e n o m e n o n . 2) A s e c o n d o b j e c t i v e was t o e x a m i n e t h e t i m e c o u r s e o f MLC p h o s p h o r y l a t i o n d u r i n g p o t a s s i u m - i n d u c e d c o n t r a c t i o n s o f r a b b i t a o r t a . 3) A t h i r d o b j e c t i v e was t o d e m o n s t r a t e c h a n g e s i n p r o t e i n p h o s p h o r y l a t i o n d u r i n g r e l a x a t i o n o f p o t a s s i u m - c o n t r a c t e d a r t e r i e s by n i t r o g l y c e r i n . 25 MATERIALS AND METHODS A) MATERIALS: 1) Animals: Female Wistar rats were oestrogen primed 24hrs prior to sacr i f i ce . They were k i l led by cervical dislocation and two myometrial strips were prepared from each animal. White New Zealand rabbits of either sex (2 - 3kg) were k i l led by a blow to the base of the skull and bled. Descending thoracic aortas were then removed. 2) Chemicals: i) Radioisotopes ^ P as carr ier free H^PO^ (285 Ci / mg at 100% isotopic enrichment) was obtained from ICN. i i ) Electrophoresis: Sucrose and acetic acid were obtained from Analar. The following were purchased from Bio-Rad: ammonium persulphate, bisacrylamide, SDS-PAGE high and low molecular weight standards. Ampholines pH range 5-7 and 3.5-10 were purchased from LKB and Bio-Rad. Acrylamide and SDS were i n i t i a l l y purchased from Sigma chemical Co. and then Bio-Rad was used as the supplier due to increased purity. Phosphoric acid and sodium hydroxide were purchased from Fisher Sc ient i f i c . Urea was purchased from Mallinckrodt. Ethanol and methanol were purchased from T and B Westline. Al l other reagents for electrophoresis were purchased from Sigma Chemical Co. i i i ) Other reagents: Sodium molybdate was purchased from Al l ied Chemical Baker and Adamson. 26 Isopropyl acetate, sodium dihyrogen phosphate and sodium hydrogen carbonate were purchased from Analar. The following were purchased from Amachem: Calcium chloride, glucose, potassium chloride, potassium dihydrogen phosphate. Aqueous counting sc int i l la t ion f lu id and econfluor were purchased from Amersham Radiochemicals. Perchloric acid was purchased from G. Fredrick Smith Chemical Co. Magnesium sulphate and 2-methyl butane were purchased from MCB Manufacturing Chemists. Nitroglycerin was purchased from L i l l y . Al l other reagents were purchased from Sigma Chemical Co. 27 B) METHODS 1) Tissue preparation: The in i t i a l studies involving IEF ut i l ised rat myometrium. Female Wistar rats (250 - 300 g) were oestrogen primed 24 hours before sacr i f ice with a subcutaneous injection of 50jjg estradiol benzoate (dissolved in peanut o i l ) . Rats were k i l led by cervical dislocation and two myometrial strips were prepared from each animal as described by Diamond and Hartle, (1974). The incubation solution was (in mM): NaCl, 125; KCl, 2.4; MgClA, 0.5; CaClj, 1.8; glucose, 11; and Tr is -HCl , 23.8 (pH 7.0). The solution was aerated with 95% O T / 5% CO and maintained at 37°C. Strips were incubated •* a. for 30 min with a resting tension of 0.5 g. Isoproterenol was added directly to the baths and the muscles were frozen at predetermined times. Studies involving SDS polyacrylamide gel electrophoresis (SDS-PAGE) were conducted on rabbit aorta. Descending thoracic aortae were removed from white New Zealand rabbits of either sex ( 2 - 3 kg) and were stripped of superficial fa t . Helical s t r ips , approximately 3 x 10 mm, were prepared and suspended in 3 ml organ baths between stainless steel hooks for recording of isometric tension. Tension was monitered on a Grass model 7C polygraph.. The strips were equlibrated for 30 min at 37°C in a physiological salt solution with the following composition (mM): NaCl, 118; KCl, 5.7; MgS0+, 2.37; CaCl2 , 1.26; NaHaP0^, 1.17; NaHCO .^25 and glucose, 11. Preparations were oxygenated by bubbling with 95% 0 a / 5% CO^, which maintained the pH of the solution at 7.4. A resting tension of 2 g was applied to the str ip and readjusted frequently throughout the equilibration period. 200 uCi of carr ier f ree^P was then added direct ly to each bath and 28 the strips were labelled for a period of 60 min. The^P in the extracellular space was removed by a series of at least f ive r inses. Muscles strips were treated as indicated in the results and nitroglycerin was added direct ly to the baths to give a final concentration of lC^M. Aortic strips were frozen at appropriate times after drug addition by means of a Wollenberger-type clamp precooled to -80°C. 2. Sample preparation: The methods used in preparing samples are discussed in detail in the results with the IEF gels produced. Samples were prepared for SDS-PAGE by the method of Janis et a l . (1980). Frozen muscles were homogenised using an Eberbach homogeniser with a tight f i t t ing pestle, in 1 ml ice cold 12% TCA in water. The pestle was then rinsed in 1 ml 12% TCA. The homogenates were centrifuged at 6,000 x G for 10 min. The acid was extracted from the pellet with ether. The pellet was redissolved in a sample buffer, comprising 10% (w/v) SDS / 125 mM Tris/HCl (pH 6.8) / 20% (w/v) sucrose / 150 mM 2-mercaptoethanol. The resulting suspension was boiled for 5 min. Bromophenol blue was added to each sample and the sample respun at 6000 x G for 5 min. Aliquots containing approximately 150 mg protein were loaded onto SDS slab gels. 3. Electrophoretic techniques: a) IEF Solutions: Lysis buffer: 9.5 M urea, 2% w/v NP-40, 2% Ampholines (comprised of 1.6% pH range 5 to 7 and 0.4% pH range 3.5 to 10) and 5% beta mercaptoethanol - stored as frozen aliquots. Gel overlay solution: 8 mM 29 urea. Sample overlay solution: 9 M urea, 1% Ampholines (comprising of 0.8% pH 5 to 7 and 0.2% pH 3.5 to 10) - stored as frozen aliquots. Acrylamide stock solution: 7.1 g acrylamide and 0.405 g bisacrylamide in 25 ml d i s t i l l e d water, made fresh each time. 10% w/v NP-40 solution: 1 g / 10 ml was made up and stored at 4 C. Fixing solution: 150 ml methanol, 350 ml d i s t i l l ed water, 17.25 g sulphosalicyl ic acid, and 57.5 g tr ichloracet ic acid. Destaining solution: 500 ml ethanol, 160 ml acetic acid, diluted to 2 L with d i s t i l l e d water. Procedure: IEF gels were made in glass tubing (130 mm x 5 mm inside diameter) according to the method of 0 'Fa r re l l , (1975). The tubes were cleaned in chromic acid for 24 hrs, rinsed well in hot water and then soaked in IN K0H / methanol for 24 hrs to neutralise the acid. They were subsequently rinsed thoroughly in hot water followed by d i s t i l l ed water. It was very important to ensure that the tubes were clean as this avoided the gels slipping out during a run and made extrusion of the gels easier. The tubes were dried in an oven and the ends sealed with Parafilm, while s t i l l warm as this ensured a good seal . The gel mixture was made in a 125 ml flask from 22 g ultrapure, powdered urea, 5.2 ml acrylamide stock solution, 8 ml 10% NP-40 solution and 7.8 ml d i s t i l l e d water. The above were mixed for approximately 15 min and then given a short blast of heat (37°C) to aid dissolution of the urea. Since cyanate formation from urea is accelerated with increasing temperature, i t was important not to overheat or the urea. Cyanate reacts with amino groups forming carbamylated derivatives thus altering the charge on the protein, (Hames, 1982). Subdequently, 1.6 ml Ampholine pH 5-7 (40% commerical preparation), 0.4 ml Ampholine pH 3.5-10 (40% commerical 30 preparation) were added slowly. When adding ampholites the cap of the vial was wiped with 95% ethanol before and after insertion of the neddle to try to keep the solution reasonably s te r i l e . 40 ^ il 10% fresh ammonium persulphate (2 mg / 2 ml) were added, followed by 20 JJ! TEMED. The gel mixture was stirred well , avoiding the incorporation of a lot of bubbles. The gel would not set in the flask due to the amount of 0 2 that i t was exposed to. Using a long necked disposable pipette the tubes were carefully f i l l e d with gel mixture. Each tube had a notch at 120 nm and were f i l l e d past this mark and then enough gel was removed to drop the meniscus down to the notch. As the length of the gel affects the reproducibi l i ty, i t was important to keep this measurement consistent between samples that were to be compared. Once al l the gels were poured, some of the remaining gel mixture was drawn into the pipette and allowed to set. This was to determine i f the gel mixture polymerised in the tubes. The gel mixture was overlaid very carefully with 50 ^ul gel overlay solution and le f t to set for 1 - 2 hours. The gel overlay solution was removed and replaced with 100^il fresh lys is buffer, which was overlaid with d i s t i l l e d water and lef t for 1 hour. The Parafilm was removed and the gels placed in a standard tube gel electrophoresis chamber. The f lu id at the top of the gel was removed and 100 jjl fresh lys is buffer added, this was overlaid with 0.02 M NaOH. The upper reservoir was f i l l e d with 0.02 M NaOH and the lower with 0.01 M phosphoric acid. Any bubbles at the bottom of the tubes were removed with the curved pipette. The gels were prerun according to the following schedule a) 200 volts for 1/4 hour b) 300 volts for 1/2 hour c) 400 volts for 1/2 hour. The upper reservoir was emptied and the f luids removed from the top of 31 the gels. The samples were loaded and overlaid with 40yul of sample overlay. This was overlaid with 0.02 M NaOH. The upper reservoir was re f i l l ed with NaOH. The samples were focused for 17 hours at 400 volts and 1 hour at 800 volts. There was enough acid in the lower reservoir to cover most of the length of the tube, this acted as a heat sink and prevented the gels overheating with the voltage. Gels were f ixed, stained and destained according to the method of Jasch et a l , (1982). They were removed from the tubes by expulsion with a i r , direct ly into tubes (7 mm x 140 mm) containing a f ixing solution for 2 hours, rinsed in destain for 10 min and then stained for 30 min in a 60°C water bath in a stain comprised of 0.115 mg Coomassie Br i l l iant Blue R 250 and 100 ml destain, (staining solution was f i l tered before use). After staining gels were rinsed in destain and transferred to 16 x 150 mm capped culture tubes f i l l e d with destain. After 3 days of destaining in the dark, the gels were transferred back into the 7 mm diameter tubes with fresh destain and on day 4 were scanned on a Gilford spectrophotometer, f i t ted with a Gilford gel scanner. Normally gels were scanned by adjusting the fu l l scale absorbance or wavelength (from 560 nm to lower wavelengths) so that the highest peak of the scan reached the top of the chart paper. Gels were photographed on the day after scanning. b) SDS-PAGE Solutions: Stain; 1.375 g Coomassie Br i l lant Blue R 250, 500 ml methanol, 100 ml acetic acid and 500 ml d i s t i l l e d water. Destain I; 500 ml methanol, 500 ml d i s t i l l e d water and 100 ml acetic acid. Destain II; 100 ml acetic acid, 400 ml methanol and 1500 ml d i s t i l l e d water. 32 Procedure: As for the IEF gels - the glass plates for casting slab gels had to be scrupously cleaned before use, to avoid contamination of gels by exogenous proteins. The gels were cast according to the method of Laemmli and Favre, (1973). A Bio-Rad model 220 apparatus was used with a separating gel 180 nm wide, 120 mm high and 3 mm thick. Typical ly, 5-20% gradient gels were used as they gave better resolution of low molecular weight proteins than did 12% gels. Gradients were cast using a Pharmacia Gradient Mixer GM-1 and a LKB Varioperpex II per ista l t ic pump. No difference in resolution was found between gels which had been allowed to set overnight and those which were used within 3 hours of casting. Stacking gels were 40 mm high and consisted of 5% gel . 150yjg of protein was added per well and molecular weight standards were run concomitantly on each gel . The protein standards used for the estimation of molecular weight (in daltons) myosin (200,000),^ galactosides (116,250), phosphorylase b (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400). The gels were run at a constant current of 60 mA, using a Pharmacia power supply (Model EPS 500/400), until the bromophenol blue marker dye had reached the bottom of the gel . Cooling was necessary. Gels were stained for 30 min, the stain poured off and reused. The gels were rinsed in water and gently agitated in destain I until destaining was completed and rehydrated in destain II. They were dried by heat under vacumn using a Bio-Rad model 224 slab gel dryer. The dried gel was placed in contact with X-ray fi lm (Kodak Min-R), in some cases along with intensif ier screens (Cronex Lightning Plus) for 7 - 3 0 days, following which, the f i lm was developed. After both gels and autoradiograms had been 33 photographed, the autoradiograms were scanned at 560 nm in a Gelman DCD-16 gel scanner. The optical density was altered with each scan to ensure that the ta l lest peak was at the top of the chart paper. 4. Miscellaneous assays a) Protein assay: A protein assay was performed on the resuspended pellet before the bromophenol blue was added, by the method of Zaman and Verwilghen, (1979). It was a simple method devised for the determination of protein concentration in just such a solution as the one used above. The method was based on protein and Coomassie Br i l l iant Blue G-250 binding but i t involves an in i t i a l step, which precipitates excess SDS with 100 mM potassium phosphate buffer, pH 7.4 to 7.5. Bovine serum albumin was used as a standard. b) S tat is t ics : Al l data are presented as means ± S .D . , (Glantz, 1980). Differences between treatments were tested using a student "t" test or in the case of the K + -t ime course study by a modified "t" test and the Bonferroni method . of simultaneous multiple comparisons, (Wallenstein et_al , 1980). 34 RESULTS IEF Sample preparation; Preliminary experiments were carried out to determine the most suitable method of sample preparation for the type of muscle chosen - rat myometrium. The varieties of methods of sample preparation are as numerous as smooth muscle types to be studied. Five different methods were selected and tube gels of each sample were run in duplicate as indicated in Methods. The c r i t i ca l step in sample preparation in this type of study is the immediate and complete inactivation of the kinase and phosphatase. If these enzymes are not denatured at the onset of sample preparation, alterations in phosphorylation levels will occur. A muscle sample containing a phosphorylated protein was the t i b i a l i s anterior (TA) muscle of the mouse, (Dr. L.G. Jasch, personal communication). Experiments assessing the effect of sample preparation on the levels of phosphorylation could than be performed using this muscle. (No previous studies had reported an IEF profi le for rat myometrium. Thus, although this was the muscle of interest in this study i t could not be used to assess sample preparative techniques.). A. A previously reported method of mouse skeletal muscle sample preparation, (Jasch et a l . 1982), involved homogenisation in Tris buffer (pH 7.4, 4°C) . Aliquots of the homogenate containing the desired weight of muscle t issue, were withdrawn and lyophilised for 20 hours. Samples were rehydrated and part ia l ly solubil ised in lys is buffer, ( O 'Farre l l , 1975). The entire sample, which due to the high protein content was part ia l ly 35 gelled, was loaded onto the IEF ge l . This method was used as a control. TA and myometrium were prepared in this manner. B. Since Rapoport et a l , (1982), had carried out similar studies on protein phosphorylation, their method of sample preparation was also used. This involved homogenisation in an ice-cold stopping buffer (100 mM NaF / 80 mM sucrose / 10 mM EDTA / 10m mM NJtris(hydroxymethyl) methyl^ aminoethanesulphonic acid, pH 7.4,- Garrison, 1978). NaF and EDTA were added as inhibitors of kinases and phosphatases. Homogenates were spun at 105,000 x G for 60 min. The supernatant fraction was removed and the pellet resuspended in lysis buffer as previously described. This would y ie ld the particulate fraction only. C. Samples were homogenised in stopping buffer as in method B. The resulting homogenate was lyophilised as in method A. D. Janis et a l , (1980), when preparing myometrial samples for 2-D electrophoresis used the method of Garrison, (1978), as described above (method B) but for one-dimensional electrophoresis the muscle was homogenised in TCA solution and the precipitated protein spun down at 6,000 x G for 10 min, as described in the methods. E. Since the experimental protocol to be used would involve freezing the muscle, samples were f i r s t frozen, ground in TCA and prepared as described in D above. F. A report appeared in the l i terature, (Driska et a l , 1981), in which a study comparing the levels of phosphorylation in muscles, which had been freeze-clamped to samples which were frozen in part ia l ly frozen acetone slush. The frozen muscle samples were allowed to reach room temperature in the thawing acetone. These authors believe acetone would denature the endogenous enzymes and preserve levels of phosphorylation prevailing at the 36 time of freezing. The tissues were subsequently homogenised. Muscle samples were denatured in this manner and then homogenised in TCA solution as described for D. Table 2 repesents a summary of the methods used. TA and myometrial strips were run in para l le l . The degree of phosphorylation in TA samples was estimated from the ratio of peak height of the phosphorylated to non-phosphorylated protein. An alteration in this value was taken as an indication of the sui tabl i t iy of the method. Table 3 summarises the data from TA muscles and Fig 5 shows two of the scans obtained. From pilot studies i t appeared that there was no significant differences between methods D and E and therefore method D was omitted from subsequent experiments, for this reason i t does not appear in Table 3. Comparing the values given in Table3, i t appears that inhibition of enzyme act iv i ty at the onset of sample preparation is necessary. Unlike the studies conducted by Driska et a l , (1981), there did not appear to be any significant difference between freeze-clamping and f ixing in acetone slush, each yielding a value of 77% phosphorylation. The best y ields of phosphorylation appeared to be achieved when the stopping buffer of Garrison, (1978), was used, (83%). Unfortunately this method has disadvantages. If the sample is lyophilised with stopping buffer, (Fig 6, lower scan), there is a non-indentical shift in bands. When the sample is prepared as outlined by method B only the particulate fraction is used, eliminating the observation of possible changes in phosphorylation levels of proteins remaining in the soluble fract ion. This point is clearly indicated by the myometrial samples depicted in F ig . 7. It can be seen by comparing the scans that certain peaks appearing towards the basic end of the lower scan B, are missing in the upper scan A. 37 TABLE 2: Summary of methods used for sample preparation prior to IEF. A: Homogenisation in 1 mM T r i s , lyophi l isation and reconstitution in lys is buffer Jasch et a l . , 1982 B: Homogenisation in stopping buffer, centrifugation at 105,000 x G for 60 min and reconstitution of pellet in lys is buffer Garrison et a l . , 1978 C: Homogenisation in stopping buffer, lyophi l isat ion and reconstitution in lys is buffer D: Homogenisation in 12% TCA, centrifugation at 6,000 x G for 10 min and reconstitution of pellet in l ys is buffer Janis et a l . , 1980 E: Freeze clamp muscle - prepare as in D Driska et al_. , 1981 Janis et a l . , 1980 F. Denature in acetone slush prepare as in D Driska et a l . , 1981 38 TABLE 3: % Phosphorylation of protein in t i b i a l i s anterior samples prepared by various methods. Method of preparation % phosphorylation A: Homogenisation in 1 mM Tris 46 lyophi l isation B: Homogenisation in stopping buffer, 83 centrifugation at 105,000 x G for 1 hr C: Homogenisation in stopping 81 buffer, lyophi l isation E: Freeze clamp, homogenisation in 77 12% TCA, centrifugation at 6000 x G for 10 min F: Fix in acetone slush, homogenisation in 12% TCA, centrifugation at 6000 x G for 10 min 77 39 FIGURE 5: Comparison of the distr ibution of proteins as resolved on isoelectr ic focusing polyacrylamide gels in 6 week t i b i a l i s anterior samples; acidic end is to the right. Each gel contained proteins derived from the whole homogenate of 11 mg wet weight muscle. Approximately the top(at lef t ) centimeter of each gel and al l gels i l lustrated in subsequent plates was cut off to accommodate the gel in the 10 cm scanning cuvette. A: Muscle was homogenised in 1 mM Tris (pH 7 . 4 ) as per method A in text. B: Muscle was frozen, fixed in dry ice acetone and homogenised in 12% TCA as per method F in text. X And X-P represent a non-phosphorylated protein and i ts phosphorylated counterpart. 40 41 FIGURE 6: Scans of 6 week t i b i a l i s anterior. Orientation and labels as in Figure 5. / A. Muscle was prepared by method A (see text). B. Muscle was prepared by method E (see text). 42 X X-P Y f 1/ 43 FIGURE 7: Comparison of the distr ibution of proteins as resolved on isoelectr ic focusing polyacrylamide gels in myometrial samples. Gels A & B contained proteins derived from the whole homogenate of 15 mg and 12.5 mg wet weight muscle respectively. Acidic end is on the l e f t . A: Myometrium was prepared by method B - see text. B: Myometrium was prepared by method D - see text. 45 By virtue of the above pi lot study, freeze-clamping and subsequent homogenisation in 12% TCA was chosen as a method of sample preparation. In i t ia l l y a comparison between relaxed controls and strips contracted with a submaximal dose (40 mM KCl) for 10 min was made. Spontaneously contracting myometrial strips were suspended and incubated in Tyrodes as described in the methods. Relaxed controls were muscles frozen in the relaxed state between spontaneous contractions. Contracted myometrium were treated with 40 mM KCl Tyrodes, in which NaCl had been replaced with an equimolar concentration of KCl to give the desired f inal KCl concentration, (40 mM KCl). Fig 8 shows typical scans of the tube gels obtained. No consistant signif icant changes could be seen. In trying to prompt some kind of phosphorylation, K^-contracted st r ips , (10 min) were relaxed with isoproterenol 10 M, (1 min). Again there were no detectable changes in the scans (data not shown). Final ly myometrial strips were relaxed in Ca^" free Tyrodes and some were then contracted with 40 mM KCl and the resulting IEF gels compared. Once again there was no detectable difference, (Fig 9). IEF of myometrial s t r ips , (Table 4) , lacked the sensit iv ity required to visualize changes in the protein profi le of crude myometrial homogenates. The concentration of MLC present in these samples was too small to allow detection of changes in phosphorylation by Coomassie blue staining. For this reason the more senstive technique u t i l i s i n g 3 2 ? in conjunction with SDS-PAGE was adopted. SDS-PAGE was chosen as this one-dimensional technique allows for limited handling of radioactive material when compared to two-dimensional electrophoresis. It also results in a f la t gel ideal for autoradiography. Since myometrial strips appeared to be unresponsive to NG, one of the drugs of choice in this study, rabbit 46 FIGURE 8 : Scans o f myometrium. O r e i e n t a t i o n as i n F igure 5 . A: Myometrium was c o n t r a c t e d w i t h 40 mM KC1 (10 min) and f r o z e n . B: Myometrium was f rozen r e l a x e d . 47 48 FIGURE 9: Myometrium were either relaxed in a C a 2 + free Tyrodes (A) or contracted in 40 mM KC1 (B) as described in the text orientation as in Figure 5 . 50 TABLE 4: Summary of experiments using IEF to detect changes in phosphorylation. n_ I: Relaxed control vs. KC1 (40 mM) 10 min 6 II: KC1 (40 mM) 10 min vs. KC1 (40 mM) 10 min and ISO (10 _ 6 M, last 1 min) 2 III: C a 2 + free Tyrodes - vs. equi l ibrat ion in C a 2 + free Tyrodes then KC1 (40 mM) 5 min 4 No differences in the protein prof i les were noted. 51 aortic str ips were used. 2.SDS-PAGE i) Calcium free study: In order to show that the technique was working I chose to compare two extreme states. One set of aortic strips (n=6), was labelled with^P, and incubated in a Ca"*" free Krebs buffer containing 5 mM EGTA for 20 min. This ensured that the tissues were completely relaxed. The second set of muscle strips were contracted with 124 mM KC1 Krebs buffer in which NaCl had been replaced by KCl in equimolar proportions. This concentration of K + -depolarised the muscles and produced a maximal sustained contraction. The muscles were frozen after 30 s as th is , according to Aksoy et a l . (1982), is the point of peak phosphorylation. Contracted and relaxed muscle str ip samples were run on the same gradient gels, to allow easy comparison between the two types of treatments. A typical example of such an autoradiogram is shown in Fig 10. It is obvious from the autoradiogram that there is phoshorylation of a protein, 23kD, in the contracted tissues whereas those strips incubated in a Ca^free Krebs buffer appear to have very l i t t l e phosphorylation at this point. The molecular weight of the proteins were determined by the method of Weber et a l . (1972), by comparing the mobility of the proteins with those of marker proteins run concomitantly on each gel . The autoradiograms were scanned in an attempt to quantitate this change in phosphorylation. Fig 11 shows four such scans. The differance in degree of phosphorylation of the 23kD protein (assumed to be the myosin l ight chain) is well visualized by the scans. In order to quantitate the results the outline of the scans were cut out and the paper weighed, (DiSalvo et a l , 1978). The 52 FIGURE 10: Autoradiogram of a SDS polyacrylamide gradient gel . Tracks shown represent samples: a) Relaxed in Ca 2 + - f ree (5mM EGTA) Krebs, 20 min b) Contracted in 124 mM KCl Krebs, 30 s Preparation of samples, gel and autoradiogram were as described in Methods. Result shown is a typical auto-radiogram. a b 5 4 FIGURE U: Scans of autoradiograms of SDS - polyacrylamide gradient gels, as described in the text. A and B represent aortic str ips contracted in 124 mM KCl and frozen after 30 s. C and D represent aortic str ips relaxed in C a 2 + free (5 mM EGTA) buffer and frozen after 20 min. The 23 kD peak was assumed to be myosin l ight chain (MLC). For rationale see text. 56 peak at 23kD was expressed as a percentage of the total weight of the scan. The results shown in Table 5 indicate a significant difference between the treatment groups. This was taken as validation of the techniques used. This 23kD protein can not be def initely identif ied as MLC from these studies. However, i ts characteristics are similar to those documented for MLC. The protein is phosphorylated on contraction and dephosphorylated in Ca^free buffer, i t also appears as the only protein phosphorylated in that region, 15 - 30kD. For these reasons i t was assumed to be MLC. However there are obvious problems in evaluating the data in this manner. By expressing the change in phosphorylation of the 23kD protein as a percentage of the total phosphorylation the assumption is being made that there are no other increases or decreases in protein phosphorylation. If the scans in Fig 11, between contracted and relaxed muscle strips are compared, there are no other v is ible changes in phosphorylation levels . It was therefore concluded that although such a method of expressing protein phosphorylation would not produce figures that were an accurate representation of the degree of phosphorylation, signif icant differences would be apparent and trends in phosphorylation rather than absolute values could be expressed. Another problem encountered using this technique is the vertical streaking of proteins. This could be caused by several factors: a) It could be sample overload, in which case di lut ing the sample would be a solution. However, the proteins in the low molecular weight range are already faint and di lut ing the sample further would possibly dilute out the protein under study, b) Some native proteins form aggregates and may precipitate at the high concentrations reached in the sharply stacked zone in the stacking gel and fa i l to enter the resolving gel . This phenomenon of 57 TABLE 5: Ca2+-Dependency of phosphorylation of 23kD protein in rabbit aorta. % of total phosphorylation in 23kD protein band 1 .7±0.24(6) 4.6+0.68(6)* Each value represents the mean±SD of the number of experiments shown in parentheses. •Significantly different from relaxed phosphorylation values, p<0.05. Treatment C a 2 + f ree, 5 mM EGTA 20 min . 124 mM KCl, 1.26 mM C a 2 + , 30 s 58 streaking occurs when aggregated protein accumulates at the gel surface and then slowly enters the gel during electrophoresis, causing protein streaks running in the direction of migration. To ensure that al l insoluble material is removed prior to electrophoresis, samples were centrifuged before application. i i ) Potassium - time course study: Having established a working protocol to examine MLC phosphorylation levels and considering the controversy in the l i terature, i t seemed pertinent to determine the time point of peak MLC phosphorylation during a K^-induced contraction. In addition, before examining the effect of relaxants on phosphorylation i t was crucial to determine the degree of posphorylation at the time of addition of relaxant. A K^-time course study was therefore undertaken. The tissues were frozen after contraction with 124 mM K"4" Krebs buffer at the time points indicated, (Fig. 12). Muscle st r ips , which were not treated with this depolarising buffer served as relaxed controls. Tension in the muscle strips reached a maximum after 4 min and this degree of contraction was maintained throughout the duration of the time course. The phosphorylation peaked around 2 min. There then appears to be a trend towards dephosphorylation. The results from the CeTdependency study are included as the gels were comparable. It is worth noting that the levels of phosphorylation in Ca^free relaxed muscles (1.7% of total phosphorylation of 23kD protein: Table 5) are signif icantly lower than the relaxed controls (2.4% of total phosphorylation of 23kD protein: Fig 12). 59 FIGURE 12: Phosphorylation of MLC during K + - induced contraction of rabbit aorta. The solid l ine represents tension developed by a representative muscle contracted for 12 min. The columns represent the mean MLC phosphate content of muscles (expressed as a % of total phosphorylation) frozen at the indicated times. The error bars represent the standard deviation of the mean for 4 phosphate determinations. •significantly different from control p<0.05. ••significantly different from 30 s p<0.05. I TENSION (grams) — 1 to I ca ro ro ro - r - r 0> MLC PHOSPHORYLATION (% of total) 61 TABLE 6: E f f e c t of n i t r o g l y c e r i n ( I O - 6 ) on K + - i n d u c e d c o n t r a c t i o n s . Percent Maximal Treatment Tension  124 mM KCl f o r 7 min 100% (5) 124 mM KCl f o r 7 m i n , *30%±28 (5) NG(10" 6M) f o r l a s t 2 min The number o f exper iments i s shown i n parentheses . *Value r e p r e s e n t s the mean±SD. 6 2 FIGURE 13: Mechanical responses of rabbit aorta to 124 mM KCl and nitroglycerin (10"6M). Aortic strips were f i r s t contracted with 124 mM KCl, and in A, frozen after 7 min. B, nitroglycerin (10"6M) was added for last 2 min and then frozen. 63 A KCI 124mM 6 4 TABLE 7: Effect of nitroglycerin (10_ 6M) on 3 2 p incorporation of 23kD protein during K+-induced contraction in rabbit aorta. % of total phosphorylation Treatment in 23 kD protein band 124 mM KC1 for 7 min 3.2±0.66 (5) 124 mM KC1 for 7 min, *1.9±1.13 (5) NG (10-6M) last 2 min Each value represents the mean±SD of the number of experiments shown in parentheses. •Significantly different from contracted phosphorylation values, p<0.05. 65 FIGURE 14: Scans of P autoradiograms of SDS polyacrylamide gradient gels, as described in text. A and B representing aortae contracted in 124 mM KC1 , (7 min) C and D representing aortae contracted in 124 mM KC1 , (7 min) with nitroglycerin (10-6M) added in last 2 min. 6 7 TABLE 8: Summary of experiments using SDS-PAGE to detect changes in phosphorylation. I C a + + Free Krebs (5 mM EGTA), 20 min vs. 124 mM KCl, 30 s II 124 mM KCl, 2, 6 and 12 min vs. relaxed control III 124 mM KCl, 7 min vs. 124 mM KCl, 7 min plus nitroglycerin (10"6M) last 2 min 68 i i i )E f fec t of nitroglycerin (10 M) Since contraction appears to be correlated with phosphorylation, the relationship between tension and levels of phosphorylation was subsequently examined in muscles relaxed by NG (10~bM). The muscle strips were contracted in 124 mM KT Krebs, as previously described, and NG was added direct ly to the organ baths to produce a final concentration of 10^M. This produced a 70% relaxation, (Table 6) . Fig 13 shows typical traces obtained. The % phosphorylation values of the MLC, for the two treatments are given in Table 7. There is a significant decrease in the % phosphorylation of MLC (3.2% verses 1.9% of total phosphorylation of 23kD protein for 124 mM K"*" Krebs and nitroglycerin treatment respectively). If the scans of the autoradiograms are compared, (Fig 14), not only is the alteration in the phosphorylation of MLC seen, but there appears to be increased phosphorylation of a protein at approximately 160kD in the muscles treated with NG. In order to quantitate this difference, the sides of the peaks must be extrapolated to the base l ine . The bases of the peaks from the contracted muscles are wider than their relaxed counterparts. As a result , altough the 160kD peak of the NG-treated muscles appears to be elevated, the area under the peak is not s ignif icantly di f ferent . Due to the other background radioactivity on one-dimensional gels, the values for percent increase in phosphorylation may be somewhat underestimated, (Janis et a l . 1981). Table 8 represents a summary of the experiments using SDS-PAGE to detect" changes in phosphorylation. 69 DISCUSSION The major objective was the establishment of a suitable protocol for determining changes in protein phosphorylation in smooth muscle. Electrophoresis has become the preferred technique for studying protein phosphorylation in intact muscles. The number of reports in the l i terature using these techniques is immense, (for reviews see Marston, 1982; Stull et a l , 1980; Murphy, 1982; S t u l l , 1980). IEF is widely used as the f i r s t step in two-dimensional electrophoresis. It is not so common to find i t used alone in protein analysis of smooth muscle. Intact smooth muscle homogenates contain many other constituents besides contractile proteins. In these studies, i t was estimated that 11% of total wet weight of myometrial tissue and 8% of aortic tissue was protein. This is the total weight of protein in the muscle, of which only a small portion is contracti le proteins. Myosin has been estimated by some to be 7% of total protein content of porcine smooth muscle, (Cohen and Murphy, 1978). For this reason many workers have tended to purify their muscle preparations prior to 2-D electrophoresis. Looking at the IEF scans obtained using myometrium, (Fig 7,8,9) , i t can be seen that the base l ine at the basic end is elevated, causing differences in phosphorylation to appear less pronounced. From studies using SDS-PAGE, i t became apparent that the relative amount of MLC present in these samples was small. So with hindsight i t is not surprising that the changes in l ight chain phosphorylation are not detectable using IEF. For these reasons IEF, when used on i ts own, was deemed to be an inappropriate method of studying protein phosphorylation in these types of preparations. Using^P overcomes this problem, as autoradiography is much more 70 sensitive than scanning a stained protein spot. It should be remembered that using SDS-PAGE there is no detectable difference between a phosphorylated or non-phosphorylated protein, as separation is on the basis of size rather than charge. Charge will vary s ignif icantly on phosphorylation whereas size will not. SDS PAGE was more useful particularly when gradient gels were employed. Since there were no other proteins of the same size phosphorylated in that region, (15 - 25kD), the estimation of % phosphorylation of the MLC was easier. SDS-PAGE is limited in i ts use when proteins of a high molecular weight (150kD and heavier) are studied, (Fig 14). There appears from Fig 10 to be a high level of radioactivity associated with proteins in the higher molecular weight range. The exact nature of this act iv i ty cannot be determined from these autoradiograms. It may in part be due to the spreading of the spot on the f i lm due to the long exposure times ( 7 - 3 0 days) or i t may represent phosphorylated proteins in such low concentrations that they are not adequately resolved by this technique. Two-dimensional electrophoresis may not necessarily solve this problem. There may s t i l l be a large amount of streaking at the top of the gel , except that i t would be spread across the width of the gel . Fig 15 i l lustrates this and detection of phosphorylation in the high molecular weight region would be impossible. Possible solutions to this dilemma are discussed at a later point. A signif icant difference in the levels of phosphorylation between K"1--induced contracted and Ca*" - free relaxed aortic smooth muscle, was taken as an indication that the methodology was suitable and working. In the studies above, these changes were seen in a 23kD protein. The widely FIGURE 15: Autoradiograms of soluble (upper) and particulate (lower) fractions of rat thoracic aorta after two-dimensional gel electrophoresis. Samples were prepared as in method B, soluble fraction represents supernatant. Reproduced from Rapoport e_t al_. (1982). 73 accepted weight of the MLC of smooth muscle is 20kD. The MLC of skeletal muscle is quoted as 18.5kD, (Stull et a l , 1980). DiSalvo et a l , (1978), estimated the molecular weight of the two types of l ight chain in bovine aortic actomyosin to be 15kD and 13kD, which corresponds to values for chicken gizzard, (Sobieszek et a l . 1977; Aksoy et a l , 1976; Gorecka et a l , 1976) or platelets, (Chacko et a l , 1977) and this reflects the heterogenity of MLC isolated from different sources, (Mannherz et a l . 1976). As is indicated throughout both IEF and SDS-PAGE are sensitive to artifactual variation. Therefore the IEF point and the molecular size of proteins will vary from one laboratory to another. What is important is consistency of results within a laboratory. i) Effect of Calcium Perry and his colleagues f i r s t described the MLCK/MLCP system (Perry, 1979). However, since MLC phosphorylation had no apparent effect on actin-activated ATPase activ i ty of skeletal muscle, no significance was placed on the phosphorylation of MLC with respect to a possible regulatory role in muscle contraction, (Perry, 1979). The discovery that in smooth muscle MLC phosphorylation preceded an increase in actin-activated myosin ATPase act iv i ty led to the hypothesis that calcium plays an important role in smooth muscle contraction by activating the MLCK, which in turns phosphorylates the MLC, which in i t iates an interaction between actin and myosin, resulting in contraction, (Chacko et a l . 1977; Gorecka et a l , 1976; Sobieszek A, 1977). This model is based on the assumption that increased ATPase act iv i ty is due to increased interaction between a large number of myosin molecules with act in . Relaxation, according to the above hypothesis 74 would result from Ca^sequestration, MLCK inactivation and subsequent dephosphorylation of MLC by MLCP. If Ca4*"-induced phosphorylation mediates cross-bridge attachment in l iv ing smooth muscle, then active stress should be related to the level of MLC phosphorylation under al l conditions. This has been observed many times in the l i terature . In the study reported here, unstimulated t issue, in this case relaxed controls, had low levels of phosphorylation ( i . e . 2.4% of total phosphorylation observed, Fig 12). In the l i terature , levels range between 0.1 and 0.2 mol phosphate / mol MLC, (Aksov et al 1982; Barron et a l , 1979; Butler et_al,1983; de Lanerolle and S t u l l , 1980; Driska et a l . 1981; Gerthoffer and Murphy 1983 a, b; Si lver and S t u l l , 1981). These non zero values in relaxed tissues could be attributed to the presence of tone, (Barron et a l . 1979; Driska et a l , 1981), errors in phosphorylation determination, (Aksoy et a l , 1982; Driska et a l , 1981; Manning et a l , 1980), or cooperativity between heads of myosin, (Persechini et a l . 1981). During K*"-induced contractions there was a significant increase in MLC phosphorylation, (Table 5). Values in the l i terature appear as high as 0.7 mol phosphate / mol MLC either before or coincident with tension development, (Aksoy et a l . 1982; Barron et a l . 1979; Butler et a l . 1983; de Lanerolle and S t u l l , 1980; Gerthoffer and Murphy, 1983 a, b; Janis et a l , 1981; Si lver and S t u l l , 1981). Although maximal force is achieved, given values for phosphorylation are submaximal ( i . e . less than 1 mol phosphate / mol MLC). This could be caused by the fai lure of an agonist to completely activate the contracti le machinery, or to some threshold or cooperativity phenomenon, (Kerrick et a l , 1980). On removal of Ca"^ f rom the system using Ca*" - f ree Krebs and 5 mM EGTA 75 there was a further decrease in phosphorylation levels from relaxed controls (1.7% verses 2.4% of total phosphorylation of 23kD protein, Table 5). High basal levels may also be due to spontaneous action potentials within the muscle, which in turn would release Ca^from the t issue, resulting in a certain degree of phosphorylation. Total removal of Ca^from the system would eliminate this effect decreasing levels of phosphorylation, (de Lanerolle et a l . 1982). As previously discussed, these crude muscle preparations contain various ce l l s other than smooth muscles ce l l s (for example fibroblasts and endothelial ce l l s and a variety of enzmyes such as kinases and phosphatases, some of which are also involved in phosphorylation reactions). Fig 10 i l lustrates this point. Superimposed on the elevated level of phosphorylation, definite bands can be seen, with apparent molecular weights of 28, 37, 42, 78, 94, 137, 160 kD. The degree of phosphorylation of these bands does not seem to be altered by any of the treatments in this study, with the exception of the 160 kD protein, which appears to have increased phosphorylation with NG, (Fig 14). Aside from the two proteins which have already been discussed ( i . e . MLC and the 160kD protein), the identity of the other phosphorylated proteins,which appear on the gels, is as yet unknown. It should be stressed again that this is a crude muscle homogenate and so there are many proteins and enzmyes present and these phosphorylations may have no bearing on contraction. i i ) Potassium - time course study. Before the effect of relaxants on phosphorylation levels can be determined i t is important to establish the time course of MLC 76 phosphorylation during a K -induced contraction and to determine at what time point peak phosphorylation levels are attained. As was alluded to in the introduction, there is some controversy as to the time course of phosphorylation with respect to contraction. Fig 16 (a+b) summarises the two concepts presented in the l i terature. Fig 16a is the time course obtained by Aksoy et a l . (1982). This study was conducted in swine carotid arteries using K^as a stimulant. Phosphorylation was determined by 2D electrophoresis and scanning of the l ight chain spots. From this study the authors concluded that MLC phosphorylation levels rapidly increased to peak values with stimulation and then declined s igni f icant ly , although stress continued to rise to a maximium, (Aksoy et a l . 1982). In F ig . 16b, the study was conducted on canine tracheal muscle using methacholine as the stimulant. Phosphorylation was determined by IEF of purified myosin. The findings of this study were that the level of MLC phosphorylation rose with tension development to a maximium, where i t remained throught the study. There were a few differences between these two studies worth comment; a) Vascular and non-vascular smooth muscles are being compared. Vascular smooth muscle is known to have a high degree of tone, (Aksov et a l . 1982) Its physiological role is obviously different and thus different characteristics would be important. A method by which energy could be conserved during contraction ( i . e . via latch bridges) would be advantageous in the case of vascular smooth muscle, b) The two preparations used are of different thickness and this may alter the penetration of the agonist into the tissue and i t ' s effect with respect to time, c) Different stimulants were used, K + in the case of the former study and methacholine in the lat ter . Although they have at least one mode of action in common, that is 7 7 FIGURE 16: A. Time course of MLC phosphorylation s t r e s s development, and shortening v e l o c i t y i n K + - s t i m u l a t e d c a r o t i d media t i s s u e s . Each phosphorylation measurement (n=42) was obtained on s i n g l e t i s s u e frozen a t i n d i c a t e d time during i s o m e t r i c c o n t r a c t i o n (#>, standard f r e e z i n g protocol by immersion i n acetone-dry i c e s l u r r y ; * , quick frozen using metal clamps precooled i n l i q u i d n i t r o g e n ; 0 , immersion f r e e z i n g a f t e r step shortening. Mechanical data are reproduced from D i l l an et a l _ . , 1981. Reproduced from Aksoy e_t aJL (1982). B. Myosin phosphorylation during methacholine induced c o n t r a c t i o n of t r a c h e a l smooth muscle. The s o l i d l i n e represents tension developed by a r e p r e s e n t a t i v e muscle contracted f o r 15 min. The columns represent the mean myosin phosphate content of muscles frozen at the i n d i c a t e d times. The e r r o r bars represent the standard e r r o r o f the mean f o r f i v e to seven phosphate determinations. Reproduced from de L a n e r o l l e et aj_. (1980). MOLES PHOSPHATE PER MOLE MYOSIN O O O o -• -• 0» O M M o SHOTS L^^M^vLA^•nv^vvv^'>-, BE 8 m g KSCBB o Is 8 Meth. I O - * M g nsssnsssssKssroraro BED-i ssss» o 8 8 TENSION (grams) • 79 they cause depolarisation of smooth muscle, methacholine may be exerting other effects causing the differences noted. The results presented in this study fa l l between the two camps. Vascular muscle was used, as was K"1" as a stimulant, in an attempt to reproduce Murphy and colleagues' findings, (Aksoy et a l , 1982). However such a marked decrease was not seen, (Fig 12). There appeared to be a decreasing trend but this was not signif icant at the levels tested and without later time points, no firm conclusion can be made. A main disadvantage of the method of quantitating MLC phosphorylation as used in this study, is that results obtained cannot be compared with results quoted in the l i terature . i i i ) Nitroglycerin - induced relaxation The observed decrease in levels of phosphorylation of MLC on relaxation of K 4-induced contractions with NG (ld^M) were predicted by the original hypothesis that MLC phosphorylation in i t iates an interaction between actin and myosin resulting in contraction.. Whether the rate of MLC dephosphorylation is altered by NG is worth investigating as i t cannot be determined from this experiment. According to Driska et a l , (1981), i f swine carotid arterial strips are contracted with for 2 min and the stimulus washed out, the levels of phosphorylation have reached basal values before force has declined by 50%. In this study levels of phosphorylation decreased to basal levels with NG even though there was a degree of tension le f t in the muscle. However i t should also be noted that the values for the K + -contracted muscle s t r ips , which acted as a control , were lower than those expected from the time course. The reason for this is not c lear. It can be concluded that both stimulus washout and NG-induced 80 relaxation cause dephosphorylation. It is d i f f i c u l t to speculate whether a decrease in levels of phosphorylation seen with NG treatment is necessarily causing relaxation. The fact that there is substantial proof that muscles can maintain tone, with reduced levels of phosphorylation, complicates the data interpretation. Murphy and co-workers have analysed the relaxation of carotid arteries following agonist (110 mM K ) washout. It was found that the kinetics of relaxation could be described by a dual exponential decay. The in i ta l phase (about 2 min) was rapid and depended on the length of time of agonist stimulation. It also correlated with MLC dephosphorylation and isotonic shortening ve loc i t ies . It was concluded that this phase was associated with the phosphorylated rapid - cycling cross-bridges, which appeared to dephosphorylate with time during a sustained K +-induced contraction. Hence i t was dependent on the length of time of agonist stimulation. The second phase was much slower (estimated at 45 min) This phase was sensitive to Ca"*^concentration. Decreases in Ca^concentration (0 mM Ca~*^  0.1 mM EGTA) greatly reduce the duration of this phase. Increasing Ca"^ concentrations to 5 mM results in prolongation. It was concluded that this was a second regulatory mechanism present in smooth muscle, referred to as latch bridges which are dephosphorylated cross bridges maintaining stress, at a greatly reduced energy leve l , (Gerthoffer and Murphy, 1983 b). A recent study by Gerthoffer et al (in press) looked at the effect of SNP on this second phase of relaxation. SNP is thought to cause relaxation in a manner similar to NG. These two vasodilators activate guanylate cyclase direct ly via the reactive intermediate NO, (Gruetter et a l , 1979, 1980, 1981). There appears to be a close correlation between the degree of 81 smooth muscle relaxation and the extent of cGMP formation, (Katsuki and Murad, 1977, Axelsson et a l . 1979, Kukovetz et a l , 1979). The other conclusion drawn from the study by Gerthoffer and coworkers is that SNP will enhance the second phase of relaxation in carotid arteries without decreasing the already low levels of phosphorylation ( i . e . this vasodilator exerts i ts effect on the Ca^dependent stress maintained by latch bridges rather than on the MLCK/MLCP system). The other interesting feature noted in the NG study presented here was the apparent increase in phosphorylation of a 160kD protein in the muscles treated with NG. This increase was not s igni f icant , by the methods used, though a possible explanation for this has previously been discussed. NG causes an increase in cGMP levels and this protein phosphorylation could result from altered act iv i t ies of a protein kinase or a phosphoprotein phosphatase or both. What this alteration i s , what the 160kD protein is and whether this phosphorylation has pharmacological bearing are questions which merit further study. The nature of the 160kD protein is unknown. These preparations are crude muscle homogenates. One way of determining whether this is a contracti le protein would be to purify the homogenate and run an SDS-PAGE of the myofibrils only. One of the problems in dealing with a protein of this size is that proteins do not appear as discrete bands due to vertical streaking.. IEF may be a method of solving this i f the phosphorylation changes can be easily spotted. Due to the abundance of other cel lu lar material, smooth muscle homogenates result in gels with varying base l ine levels of staining due to incomplete focusing of certain proteins. The phosphorylation might be cGMP-dependent but that would have to be firmly established by treating aortic strips with other drugs known to 82 increase cGMP levels (e.g. SNP). Another way to test this would be with in vitro studies adding cGMP in a fashion similar to in vitro studies on the cAMP-dependent protein kinase system. Is this effect restricted to aortic smooth muscle or is i t seen in other vascular and non-vascular muscle? It would be a convenient answer to the controversial problem of why SNP produces large increases in cGMP levels in K +-contracted strips of vas deferens without causing relaxation. (Diamond, 1978.) Could i t be that this 160kD protein is involved in relaxation and is not phosphorylated by SNP in rat vas deferens? Comparative studies using vascular and non-vascular smooth muscle should provide valuable information regarding these poss ib i l i t i es . If phosphorylation occurs in both types of muscle this would indicate that either the protein phosphorylation is not responsible for relaxation or that some other factor is.missing in non-responsive t issues. One could speculate endlessly, but only further investigations will give the answers. A similar study with SNP and rat thoracic aorta was conducted by Rapoport et a l , (1982). This group found a concentration-dependent increase in the phosphorylation of nine proteins and a decrease in the phosphorylation of two proteins, with SNP. This effect appeared to be mimicked by cGMP analogues. However the 2-D gels published in this study do not visually demonstrate this finding and these changes were noted by using a video camera scanning system with an analog-to-digital converter and a microcomputer. Neither were any values for phosphorylation levels quoted. Al l of the proteins in the above study are in the molecular weight range 21-49kD and there was no report of a change in the higher molecular weight proteins. The phosphorylations observed may in fact be due to activation of a cGMP-dependent protein kinase by SNP and NG, but may have 83 nothing to do with the mechanisms of muscle relaxation. Conversely, proteins involved in the regulation of muscle relaxation may be phosphorylated in NG-relaxed muscles by kinases other than those which are cGMP-dependent. 84 SUMMARY AND CONCLUSION 1) Although IEF is used on its own as a method of protein separation in skeletal muscle studies, this technique was deemed unsuitable for the analysis of crude homogenates of smooth muscle. 2) A working technique was established in the laboratory in which changes in MLC phosphorylation could be examined. 3) The degree of MLC phosphorylation with respect to time during K +-induced contractions appears to correlate with tension. However, further time points are needed to determine i f the decreasing trend in phosphorylation is s igni f icant . 4) NG induces relaxation and a dephosphorylation of the MLC. There also appears to be an increase in the phosphorylation levels of a 160kD protein with NG treatment. This apparent increase merits further study and may lead to an explanation of possible anomalies found in the l i terature, with respect to vasodilators of this type and their methods of action. 85 BIBLIOGRAPHY Adelstein, R.S. , Conti, M.A., Scord i l i s , S . P . , Chacko, S . , Barylko, B. and Trotter. J . A . : In Excitation-Contraction coupling in smooth muscle, edited by R. Casteels, T. Godfraind and J .C . Ruegg, Amsterdam: Elsevier/ North Holland, 1977, p. 359-366. Adelstein, R.S. and Hathaway, D.R.: Role of calcium and cycl ic adenosine 3 ' , 5 ' monophosphate in regulating smooth muscle contraction. Am. J . Card io l . , 44:783-787, 1979. 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