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Relationship between cyclic AMP-dependent protein kinase activation and smooth muscle relaxation by cyclic… MacDonell, Karen Loraine 1991

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RELATIONSHIP BETWEEN CYCLIC AMP-DEPENDENT PROTEIN KINASE ACTIVATION AND SMOOTH MUSCLE RELAXATION BY CYCLIC AMP AND ANALOGS by KAREN LORAINE MAC DONELL B.Sc. (Pharm.), Dalhousie University, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT 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 February 1991 (c) Karen Loraine Mac Donell In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of 7% -^^ ^^<^^ -^ /c^/g^^y The University of British Columbia Vancouver, Canada DE-6 (2/88) To Janet, my sisters and brother, Ann Marie, Paula, and Stan, and, especially, with great love, my mother, Erma Dwyer for their care and support i i ABSTRACT It is generally held that adenosine 3',5'-cyclic monophosphate (cAMP) mediates smooth muscle relaxation by the activation of cAMP-dependent protein kinase (PKA). This hypothesis was tested in two intact smooth muscle preparations, the rat vas deferens and the bovine coronary artery, using exogenously applied cAMP and cAMP analogs. After 30 minutes of incubation, /V6,2'-0-dibutyryl-cAMP (dBu-cAMP) (1 - 100 fM) inhibited phenylephrine (PE)-induced tension generation in the rat vas deferens in a dose-dependent manner. This analog (10 juM) also activated the soluble fraction of PKA but did not activate the particulate fraction kinase. In contrast, 8-bromo-cAMP (8Br-cAMP) (10 -100 /JM) did not have any significant effect on inhibition of PE-induced tension after 30 minutes of incubation but, at a concentration of 10 juM, significantly activated both the soluble and particulate fractions of PKA. The time course of activation of soluble PKA activation by 8Br-cAMP (10 /JM) demonstrated that the kinase was significantly activated only after 30 minutes of exposure to the analog. In the bovine coronary artery, cAMP (10 - 100 /iM) relaxed potassium-depolarized helical strips and significantly activated soluble PKA in a dose-dependent manner. dBu-cAMP (10 - 100 JJM) affected neither tension nor soluble PKA activit y . 8Br-cAMP (10 - 100 /iM) did not affect the coronary artery tension but did activate soluble PKA. Both smooth muscle preparations were homogenized with charcoal prior to the determination of PKA activity in order to minimize artifactual assay results. As a further precaution, extracellularly associated cAMP and analogs were also washed from bovine coronary artery i i i s t r i p s a f t e r the i n c u b a t i o n p e r i o d . These c o n t r o l s allowed f o r a v a l i d assessment o f PKA a c t i v i t y i n the c y c l i c n u c l e o t i d e - t r e a t e d t i s s u e s . The r e s u l t s o f the t e n s i o n and kinase s t u d i e s demonstrate a l a c k o f c o r r e l a t i o n between a c t i v a t i o n o f PKA and i n h i b i t i o n o f r a t vas deferens c o n t r a c t i o n or r e l a x a t i o n o f bovine coronary a r t e r y . T h i s does not support the hypothesis that the kinase i s r e s p o n s i b l e f o r cAMP-induced r e l a x a t i o n o f v a s c u l a r and n o n - v a s c u l a r smooth muscle. While the mechanism by which exogenous cAMP and s p e c i f i c analogs induce r e l a x a t i o n in some smooth muscle p r e p a r a t i o n s remains u n c l e a r , i t can be suggested that PKA a c t i v a t i o n i s not n e c e s s a r i l y r e q u i r e d f o r the f i n a l f u n c t i o n a l e f f e c t . i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS v i i i ACKNOWLEDGMENTS ix INTRODUCTION 1 A. cAMP and Smooth Muscle Relaxation 1 B. Biochemical Features of PKA 6 C. Putative Mechanisms of Action Of PKA in Smooth Muscle 7 D. Measurement of PKA Activity in Intact Smooth Muscle Preparations 11 OBJECTIVES OF THE PRESENT INVESTIGATION 14 MATERIALS AND METHODS MATERIALS 15 METHODS 16 1. Separation of PKA Isozymes from Bovine Coronary Artery by DEAE-cellulose Anion Exchange Chromatography 16 2. Tissue Preparation and Tension Studies of Rat Vas Deferens and Bovine Coronary Artery 17 3. Washout of [^ H]cAMP from Bovine Coronary Artery 19 4. Tissue Extraction and PKA Activity Determination 19 a. Extraction of Intact Rat Vas Deferens and Bovine Coronary Artery 19 b. PKA Assay 20 5. Prevention of PKA Activation in Bovine Coronary Artery by Charcoal 22 6. Effect of Charcoal on Purified PKA Activity Ratio 22 7. Statistical Analysis 23 V RESULTS 1. P a r t i a l Determination o f Optimal Condit ions f o r PKA Assay a. PKA Isozyme Separation P r o f i l e o f Bovine Coronary A r t e r y b. Charcoal Adsorption and Washout of cAMP Analogs 2. cAMP Analog-Induced I n h i b i t i o n of Rat Vas Deferens C o n t r a c t i o n 3. PKA A c t i v i t y i n cAMP-Treated Rat Vas Deferens 4. Time Course o f PKA A c t i v a t i o n i n Rat Vas Deferens by 8Br-cAMP 5. E f f e c t s o f dBu-cAMP M e t a b o l i t e s on Rat Vas Deferens Tension and PKA A c t i v i t y 6. R e l a x a t i o n o f Bovine Coronary A r t e r y by cAMP and Analogs 7. PKA A c t i v i t y i n cAMP- and Analog-Treated Bovine Coronary A r t e r y 8. S p e c i f i c i t y o f the PKA Assay f o r cAMP-Dependent P r o t e i n Kinase A c t i v i t y DISCUSSION SUMMARY REFERENCES vi LIST OF TABLES Table Page E f f e c t s o f cAMP and Analogs on Bovine Coronary A r t e r y PKA A c t i v i t y Ratio 53 c LIST OF FIGURES Figure 1 Putative Mechanisms of Action of PKA in the Relaxation of Smooth Muscle 2 Isozyme Profile of Soluble Fraction of PKA in Bovine Coronary Artery 3 Washout of [3H]cAMP from Bovine Coronary Artery Helical Strips 4. Prevention of PKA Activation in Bovine Coronary Artery Homogenates Exposed to 8Br-cAMP by Activated Charcoal 5 Effect of Charcoal on Purified Type I PKA Activity Ratio 6 Rat Vas Deferens Soluble PKA Activity Ratio at Various Time Points After Exposure to Charcoal 7 Concentration-Response Curves for the Inhibition of PE (3 0)-Induced Rat Vas Deferens Contractions by cAMP Analogs 8 Effect of cAMP Analogs on PE (1/iM)-Induced Rat Vas Deferens Contractions 9 Effect of cAMP Analogs on Soluble and Particulate PKA Activity in Rat Vas Deferens 10 Time Course of PKA Activation by 8Br-cAMP in Rat Vas Deferens 11 Effect of cAMP and Analogs on Bovine Coronary Artery Tension and PKA Activity 12 Effect of Protein Kinase Inhibitor on Rat Vas Deferens and Bovine Coronary Artery Soluble PKA Activity vi i i LIST OF ABBREVIATIONS 8Br-cAMP 8-bromo-cAMP Ca^+i intracellular calcium cAMP adenosine 3',5'-cyclic monophosphate dBu-cAMP A/6,2'-0-dibutyryl-cAMP DEAE diethyl aminoethyl DTT dithiothreitol EDTA ethylenediamine tetraacetatic acid IBMX isobutyl methyl xanthine KC1 potassium chloride mBu-cAMP /V -^monobutyryl-cAMP MLCK myosin light chain kinase NaF sodium fluoride PE phenylephrine PGEj prostaglandin Ej PKA cAMP-dependent protein kinase PKG guanosine 3'-5'-cyclic monophosphate-dependent protein kinase PKI synthetic protein kinase inhibitor PMSF phenyl methylsulfonyl fluoride SEM standard error of the mean ix Acknowledgements I would l i k e to extend my a p p r e c i a t i o n to my s u p e r v i s o r , Dr. Jack Diamond, P h . D . , f o r h i s guidance, s c h o l a r l y example, and f i n a n c i a l a s s i s t a n c e d u r i n g the course o f my degree work. I thank the members o f my t h e s i s committee, D r s . G a i l B e l l w a r d , Kathleen MacLeod, Alan M i t c h e l l , and V l a d i m i r P a l a t y , f o r t h e i r c o n s t r u c t i v e c r i t i c i s m and suggestions. Thanks to Mr. Yong-jiang Hei f o r generously t a k i n g the time to teach l a b o r a t o r y techniques to me and f o r helping me i n understanding the work. F i n a l l y , thanks go to my col leagues John Langlands, Ashwin P a t e l , Yong-j iang H e i , and the members o f Dr. K. MacLeod's l a b o r a t o r y f o r h e l p i n g to put fun i n t o l a b work. 1 INTRODUCTION Cyclic adenosine 3',5'-monophosphate (cAMP) is generally believed to play an important role in smooth muscle relaxation. Since this role was first suggested (Sutherland and Rail, 1960), the cyclic nucleotide has been extensively studied in a host of tissues and species, resulting in a large body of evidence indicating temporal and concentration dependent correlations with smooth muscle relaxation (reviewed by Hardman, 1984). cAMP-dependent protein kinase (PKA) has been postulated to be the sole intracellular receptor for cAMP in mammalian cells (Kuo and Greengard, 1969) and is believed to mediate functional changes by phosphorylation of serine and/or threonine residues on specific proteins (for reviews, see Flockhart and Corbin, 1975; Edelman et al., 1987). I will briefly review some of the evidence for and against the cAMP-PKA-relaxation hypothesis and present the rationale for the present study which determined the PKA activity of intact smooth muscle preparations treated with exogenously applied cAMP and analogs as a test of the hypothesis. A. cAMP and Smooth Muscle Relaxation cAMP is generated by the catalytic action of plasma membrane-bound adenylate cyclase on ATP which results in the cyclization of the a-phosphoryl group at the 3' and 5' positions and release of pyrophosphate. Sutherland et al. (1968) suggested that the hormonally-stimulated presence of this soluble nucleotide may be causally associated with a functional effect i f certain criteria are met and, at 2 least to some extent, those criteria have been fulfilled for the role of cAMP in the mediation of smooth muscle relaxation. A modification of those criteria is as follows, with references germane to the case of smooth muscle relaxation: 1. Hormones which increase cAMP in intact tissue should activate adenylate cyclase in broken cell preparations. This was the case with such agonists as isoproterenol which stimulated bovine coronary artery adenylate cyclase in vitro in a dose-dependent manner (Kukovetz et al., 1978). 2. Hormonal stimulation should induce increases in the levels of cAMP in a manner which correlates with the functional effect. This was demonstrated in a temporal fashion by isoproterenol in rabbit anterior mesenteric-portal vein (Collins and Sutter, 1975). Isoproterenol (500 lM) relaxed the venous tissue and increased cAMP over time in a pattern which matched the time course of relaxation. The adenylate cyclase activator, forskolin, increased cAMP levels and relaxed rat aorta in a concentration- and dose-dependent manner. The elevation in cAMP preceded the change in smooth muscle tension as would be expected for a mediator of the tension reduction (Lincoln and Fisher-Simpson, 1983). 3. The effect associated with cAMP is potentiated by phosphodiesterase inhibitors. This was demonstrated by the inhibitor papaverine which shifted the dose-relaxation curve of isoproterenol and adenosine to the left in the bovine coronary artery (Kukovetz et al., 1978). 4. Exogenous cAMP or analogs should mimic the hormonally-induced physiological response. dBu-cAMP (1 mM) relaxed noradrenaline (0.3 JUM)-induced rabbit anterior mesenteric-portal vein tension to the same 3 extent as isoproterenol (0.05 /iM) (Collins and Sutter, 1975). By extension, these compounds should induce the same biochemical changes, such as PKA activation, in vitro and in vivo as does hormonal stimulation. There remains, however, evidence which mitigates against this association between smooth muscle relaxation and cAMP. A typical case in point is from the work of Harbon and associates (Do Khac et al., 1986). In keeping with the hypothesis, isoproterenol (0.05 /iM) and forskolin (10 /iM) relaxed carbachol-contracted rat myometrium and inhibited these contractions by, preincubation. The relaxation coincided with dose-dependent increases in cAMP. Upon closer examination, the correlation becomes more tenuous. Much larger increases in cAMP (10-fold) by forskolin were associated with a given degree of myometrial relaxation in comparison to isoproterenol. For example, isoproterenol (2 nM) inhibited myometrial contractions by 50% and significantly increased cAMP from 5.1 (± 0.5) pmol/mg protein to 8.4 (± 0.7) pmol/mg protein while an equi-relaxant dose of forskolin (1.5 /iM) elevated cAMP to 80.5 ± 9 pmol/mg protein. Low doses of forskolin (0.1 /iM) could additively increase cAMP in combination with isoproterenol (0.6 nM) but synergistically enhanced relaxation. Therefore, the magnitude of the effect of a particular degree of cAMP elevation by one agonist may not be comparable by that of another cAMP-elevating agent. Further dissociations between cAMP levels and relaxation were also noted using prostaglandin Ei (PGEj) in the rabbit aorta (Vegesna and Diamond, 1986). This hormone significantly increased cAMP levels but induced contractions in rabbit aorta and relaxation of this tension by isoproterenol resulted in no further increases in cAMP. Therefore, 4 hormonally-associated cAMP increases can be correlated with smooth muscle relaxation but it is not certain i f the presence of the nucleotide is actually necessary for mediation of the functional effect (for reviews, see Hardman, 1984; Diamond, 1990). It is also not known i f there is an interplay between cAMP-dependent and cAMP-7'ndependent processes which finally leads to the agonist-induced relaxation or i f functional compartmentalization of cAMP exists in some tissues (Diamond and Vegesna, 1984; Do Khac et a/., 1986). The fulfillment of the fourth criterion relating cAMP to smooth muscle relaxation through the use of exogenous cAMP or analogs is also debatable. This point was excellently reviewed by Kramer and Hardman (1980). In support of the hypothesis, the cAMP analog dBu-cAMP ( 1 - 1 0 #M) inhibited noradrenaline-induced contractions in vascular and non-vascular tissues, namely the rat aorta and rat vas deferens (Kreye and Schultz, 1972). dBu-cAMP (10 - 500 /iM) and cAMP (100 jfM) itself relaxed serotonin-contracted rat vascular smooth muscle preparations in a manner dependent upon the age of the animal (Cohen and Burkowitz, 1974). While the more lipid soluble analog proved to be the more potent relaxant in the latter case, as would be expected i f penetration of lipid bilayers is an important factor in the efficacy of exogenous cyclic nucleotides, the use of other analogs has complicated the apparent link between cAMP and relaxation. Schultz et al. (1978) reported that 8Br-cAMP (10 (M) was not capable of inhibiting noradrenaline-induced rat vas deferens contractions while dBu-cAMP (10 /zM) was a potent relaxant. This occurred even though dBu-cAMP is known to be less effective than 8Br-cAMP in activating PKA in vitro (Meyer and Miller, 1974). 8Br-cAMP is somewhat less lipid soluble than dBu-cAMP but they both demonstrate a far greater 5 tendency to partition into an aqueous phase than an oleaginous one. The octanol-water partition coefficient for 8Br-cAMP is approximately 0.0054 to 0.0036 and for dBu-cAMP is 0.079 to 0.0177 (Korth and Engels, 1987; Nakatsu and Diamond, 1989). Based on the potency of the 8Br-analog to activate PKA and the fairly comparable solubilities of the two compounds, the ineffectiveness of 8Br-cAMP in inhibiting rat vas deferens contractions in comparison to dBu-cAMP was somewhat surprising. While both analogs are resistant to phosphodiesterase hydrolysis, cAMP is relatively labile in the presence of the enzyme (Miller et al., 1980, Meyer and Miller, 1974). This instability of the parent compound, as well as its relatively low lipid solubility (octanol-water coefficient = 0.0011, Nakatsu and Diamond, 1989) apparently did not prove to be limiting factors in the relaxation of bovine coronary arteries (Napoli et a/., 1980). Of cAMP, 8Br-cAMP and dBu-cAMP, only cAMP (30 - 100 /tM) could relax the potassium-depolarized vascular strips. This again conflicts with the assumption that the most potent PKA activator or the one which is best able to penetrate the cell to access PKA would be the best relaxant amongst a series of cAMP analogs. Hypothetically, the relaxation by cAMP and its analogs is exclusively mediated by PKA activation but the actual activation state of the kinase was not determined in the above mentioned studies. It is this information which would most directly relate the observed functional effect with kinase activity. 6 B. Biochemical Features of PKA As reviewed by Flockhart and Corbin (1982) and Edelman et al. (1987), PKA holoenzyme exists as a ternary structure with two regulatory subunits and two catalytic subunits. cAMP binds with a high af f i n i t y (Kj = 10 nM) to the regulatory subunits, inducing a change in the holoenzyme such that the affinity of the regulatory subunits for the catalytic subunits decreases 104-fold. This leads to PKA activation by the reversible dissociation of catalytic monomers from regulatory dimers, simply described as: R2C2 (inactive) + 4cAMP — T R2CAMP4 + 2C (active) The free catalytic subunit transfers the -y-phosphoryl group from ATP, in the presence of Mg^+, to the serine , preferably, or threonine residues of specific proteins. The mechanism of this reaction probably involves an ordered sequential binding of ATP and then the protein substrate followed by the sequential release of phosphorylated product and ADP (Whitehouse et al., 1983). When intracellular cAMP levels decrease, cAMP dissociates from the regulatory subunits and catalytic activity is eliminated by reassociation of the constituent subunits of the holoenzyme. The domain on the regulatory subunit which interacts with the catalytic subunit is similar to that of substrates except that phosphorylation probably does not take place and, as such, i t is a pseudosubstrate (Taylor, 1989). An endogenous, specific PKA inhibitor protein (Walsh inhibitor) and a synthetic peptide (PKI), based on the amino acid sequence 5-22 of the inhibitor, also competitively bind the 7 catalytic subunit as pseudosubstrates (Cheng et al., 1986; Kemp et al., 1989). The endogenous inhibitor may function to inhibit basal activity of free catalytic subunits and enhance the sensitivity of the cell to hormonal stimulation of PKA (Beavo et al., 1975). At least two isozymes of PKA exist, types I and II, and can be separated by DEAE-cellulose anion exchange chromatography. These isoforms differ in their physical and functional properties as determined by the nature of the regulatory subunits. Differences include amino acid composition, tissue distribution, susceptibility to autophosphorylation and affinity for cAMP (Hofmann et al., 1975; Edelman et al., 1987). Changes in the association-dissociation equilibrium due to altered NaCl concentrations vary with the identity of the isozymes, as well. Type I isozyme tends to dissociate in the presence of high salt (e.g. 0.5 M), and type II will reassociate in its absence (Corbin et al, 1975). There is evidence for variable distribution of isozymes between subcellular compartments (Corbin et al., 1977). This, in turn, may have some bearing on the extent to which a particular isozyme participates in a PKA-associated function. Differential activation of PKA compartmentalized between soluble and particulate fractions has been suggested to account for the actions of hormones such as PGEj in the rat heart (Hayes et al., 1980). C. Putative Mechanisms of Action of PKA in Smooth Muscle While the range of proteins which bind PKA without phosphorylation is probably limited to the regulatory subunit and PKI, the specificity for PKA substrates is very broad indeed (Flockhart and Corbin, 1982). 8 Considering the crucial role of Ca2+ in smooth muscle contraction (Somlyo and Himpens, 1989), those phosphorylated proteins which may be associated with smooth muscle relaxation would most likely be involved in the reduction of free intracellular calcium (Ca2 +i) and/or the reduction of the sensitivity of the contractile apparatus to Ca2 +i. Several theories have been proposed but none have been fully substantiated . These include myosin light chain kinase (MLCK) phosphorylation, increased Ca2+ uptake into the sarcoplasmic reticulum (SR), reduced Ca2+ influx across the plasma membrane through voltage-operated channels, and enhanced Ca2+ efflux (Fig. 1). MLCK has a reduced affinity for its activators, calcium and calmodulin, when it is phosphorylated by PKA. This leads to inhibition of myosin light chain phosphorylation and, thereby, decreased ATPase activity of actomyosin (Adelstein et a7., 1982). Inhibition of MLCK activity may contribute to relaxation by cAMP-elevating compounds but such an effect is probably not an essential component of the tension reduction process. This was indicated by the work of Gerthoffer et a/. (1984) in which elevation of cAMP and relaxation of porcine carotid artery by forskolin occurred at basal levels of myosin phosphorylation. Decreased cytosolic calcium by translocation to intracellular stores could contribute to smooth muscle relaxation and PKA activation has been associated with processes which can achieve lowering of Ca2 +i. PKA phosphorylated proteins of, and enhanced Ca2+ sequestration into, bovine pulmonary artery SR vesicles in vitro (Raeymaekers et al., 1990). This parallels PKA-associated phospholamban phosphorylation and calcium uptake into SR seen in the heart (Kranias et al., 1988). The stimulation of calcium uptake into the SR by isoproterenol was demonstrated by the 9 Figure 1. Putative Mechanisms of Action of PKA in the Relaxation o f Smooth Muscle. Hormonal s t i m u l a t i o n o f s p e c i f i c plasmalemmal r e c e p t o r s (R) induces the conversion o f a s t i m u l a t o r y G p r o t e i n (G) to i t s a c t i v e form, r e s u l t i n g i n the a c t i v a t i o n o f adenylate c y c l a s e (AC). T h i s membrane bound enzyme c a t a l y z e s the conversion o f ATP to cAMP which i s then c a t a b o l i z e d by phosphodiesterases (PDE) or binds and a c t i v a t e s PKA. PKA may induce the e f f l u x o f C a 2 + by ( 1 ) i n h i b i t i o n o f a plasmalemmal v o l t a g e - o p e r a t e d C a 2 + channel (VOC), ( 2 ) a c t i v a t i o n o f a N a + - K + ATPase with ( 3 ) the r e s u l t a n t increase i n C a 2 + e f f l u x by a N a + - C a 2 + exchanger ( X ) , or ( 4 ) a c t i v a t i o n o f a C a 2 + ATPase. C a 2 + uptake i n t o the sarcoplasmic r e t i c u l u m (SR) by ( 5 ) the a c t i v a t i o n o f a C a 2 + ATPase may occur as well as ( 6 ) the i n h i b i t i o n o f C a 2 + - c a l m o d u l i n (CAM) b i n d i n g to myosin l i g h t chain k i n a s e . 10 11 enhanced subsequent caffeine-induced contractions of guinea pig mesenteric artery (Itoh et al., 1982). Since such contractions are dependent upon release of intracellular stores of calcium, PKA activation by the cAMP-elevating agent was believed to mediate loading of Ca2 + into the SR. Plasma membrane-associated proteins are also implicated as potential sites for PKA regulation and diminution of C a2 +i . PKA phosphorylated proteins in sarcolemmal inside-out vesicles from rat aorta and these vesicles demonstrated enhanced Ca2 + uptake and Na+-K+ ATPase activity (Brockbank and England, 1980). This was interpreted as an increased extrusion of Ca2 + by an associated Na+-Ca2+ exchanger. Potentiated removal of Ca2 + from the smooth muscle cell by a plasmalemmal Ca2+-Mg2+ ATPase has also been proposed as a device by which PKA can mediate relaxation (Kattenburg and Daniel, 1984). D. Measurement of PKA Activity in Intact Smooth Muscle Preparations Studies which actually measured hormonal activation of PKA during relaxation of intact smooth muscle preparations are not extensive in number. Among these studies, Silver et al. (1982, 1984) found a strong association between kinase activation and bovine coronary artery relaxation. The dose-dependent increase in type II PKA activity by isoproterenol and adenosine demonstrated a significant correlation (R > 0.93) with reduction in potassium-induced coronary artery tension. In other studies, this relationship was not always consistent. PGEj (10 /zM) activated PKA in rabbit aortic rings but induced an increase in tension (Vegesna and Diamond, 1986). When a relaxant dose of forskolin (1 /zM) 12 was used in addition to PGE], the PKA activation was synergistic but the tissue s t i l l contracted. This indicated that a correlation between hormonal activation of PKA and smooth muscle relaxation may not reflect a cause-effect relationship. As mentioned, exogenous cAMP and analogs have been used to bypass receptor- and cyclase-mediated events to elucidate the functional effects of elevated endogenous cAMP. The actual activation state of PKA in intact tissues after treatment with cAMP analogs had not been examined prior to the work of Hei et a7. (1991), probably due to the technical problem of minimizing the effects of nonspecifically trapped analog in the extracellular and intracellular spaces of the tissue. The free analog would spuriously activate the kinase after cell rupture during the homogenization process of the PKA assay. This problem was originally addressed by Corbin et al. (1975) as a result of efforts to limit the confounding effects of hormonally elevated endogenous cAMP. Activated charcoal (10 mg/mL) proved to be effective in preventing PKA activation beyond control levels when rat heart homogenates were exposed to cAMP (0.1 /iM). Palmer (1982) also strongly recommended the use of this adsorbent, demonstrating that its absence led to falsely high estimations of PKA activity in tissues treated with cAMP-elevating agents. The objective of the present study was to apply techniques to control for artifactual PKA activity determinations, including the use of charcoal, in the assessment of the relationship between PKA activity and smooth muscle relaxation in intact tissues treated with exogenous cAMP or analogs. As such, this would be a test of the hypothesis which states that PKA activity mediates cAMP-associated smooth muscle 13 r e l a x a t i o n . The r e s u l t s o f the study suggest that PKA a c t i v i t y can be a c c u r a t e l y assessed i n a n a l o g - t r e a t e d i n t a c t t i s s u e s and demonstrate t h a t PKA a c t i v a t i o n by analogs, such as 8Br-cAMP, does not n e c e s s a r i l y r e s u l t i n r e d u c t i o n o f t e n s i o n in r a t vas deferens or bovine coronary a r t e r y . Such a l a c k o f c o r r e l a t i o n between the two events c a l l s i n t o quest ion the r o l e o f PKA i n cAMP-induced smooth muscle r e l a x a t i o n . 14 SUMMARY of OBJECTIVES of the PRESENT INVESTIGATION 1. To determine the appropriate use of activated charcoal and tissue washout as controls for the determination of PKA activity in cAMP-and analog-treated smooth muscle preparations. 2. To assess the effect of activated charcoal on the activity ratio of PKA. 3. To confirm the isozyme profile of PKA in bovine coronary artery. 4. To compare the dose-dependent inhibition of PE-induced rat vas deferens tension by dBu-cAMP and 8Br-cAMP, or the dose-dependent relaxation of potassium-depolarized bovine coronary artery by cAMP, dBu-cAMP and 8Br-cAMP, with PKA activation. 5. To determine the time-course of PKA activation by kinase-activating analogs in the rat vas deferens. 6. To evaluate the specificity of the assay for cAMP-dependent protein kinase activity. 15 MATERIALS AND METHODS MATERIALS The following chemicals were purchased from Sigma Chemical Co., St. Louis, MO: activated neutralized charcoal (Pdt # C-5385), ATP (disodium salt), 8Br-cAMP (sodium salt), benzamidine, bovine serum albumin, cAMP (sodium salt), dithiothreitol (DTT), dBu-cAMP (sodium salt), ethylenediamine tetraacetic acid (EDTA), isobutyl methyl xanthine (IBMX), Kemptide, /V6-monobutyryl-cAMP (mBu-cAMP), PE, phenylmethylsulfonyl fluoride (PMSF), sodium butyrate, sodium fluoride (NaF), type I PKA (rabbit skeletal muscle), soybean trypsin inhibitor, synthetic protein kinase inhibitor (Code P-8140), Triton X-100, and Tween 20. Phosphoric acid was purchased from Fischer Scientific Co., Fair Lawn, NJ. [-y32P]ATP, [3H]cAMP and ACS scintillation fluid were obtained from Amersham, Oakville ONT. The following were purchased from BDH, Toronto, ONT: HC1, diethyl ether, 95% ethanol, potassium phosphate buffers, salts and d-glucose for buffers and depolarizing solutions. Wistar rats were purchased from Animal Care Unit, University of British Columbia and fresh bovine hearts were obtained from J. & L. Meats, Cloverdale, B.C. and Intercontinental Meat Packers, Vancouver, B.C. 16 METHODS 1. Separation of PKA Isozymes from Bovine Coronary Artery by DEAE-Cellulose Anion Exchange Chromatography The isozymic forms of PKA were separated by anion exchange chromatography as described by Giembycz and Diamond (1990). Briefly, approximately lOOmg of frozen bovine coronary artery, previously dissected free of loosely adhering adipose, connective tissue and myocardium, was homogenized with a Vari-Mix dental amalgam mixer in a liquid N2-cooled capsule (15s, high speed). The homogenization was repeated after the addition of 20 volumes of 5 mM potassium phosphate buffer (pH 6.8) containing EDTA (1 mM), DTT (10 mM), PMSF (1 mM), soybean trypsin inhibitor (20 nq/ml), and benzamidine (20 /zg/mL). The homogenate was centrifuged for 15 minutes at 31,000 x g in a Sorvall RC2-B centrifuge using a SM-24 rotor. The soluble fraction was applied to a 1.5- x 8- cm column of DEAE-cellulose (DE-52, Whatman). The column had been pre-equilibrated with the buffer described above containing potassium phosphate, EDTA, and DTT and was washed with a further 95 mL of this low conductivity buffer after the sample was loaded. This allowed the binding of the PKA isozymes to the resin and removal of unbound proteins. The buffer, supplemented with a linear NaCl gradient (0-400 mM), was pumped through the column at a rate of 0.180 mL/min and 54 x 1.5 mL fractions were collected. Every second fraction was examined for PKA activity. All steps in this chromatographic procedure were conducted at 4°C. 17 2. Tissue Preparation and Tension Studies of Rat Vas Deferens and Bovine Coronary Artery. Rat vasa deferentia were prepared as described by Schultz (1979). The tissue (25 mm long) was removed from Wistar rats (250-300 g) which had been killed by stunning and decapitation and was suspended longitudinally by silk thread (4-0) in 10 mL tissue baths containing a physiological salt solution (PSS) of the following composition (mM): KC1 (4.75), KH2P04 (1.19), MgS04 (1.19), CaCl2 (1.27), NaCl (118), NaHC03 (25), and d-glucose (11.12). The baths were warmed by water jackets to 37°C and bubbled with 5% CO2 in 02 which maintained the pH at about 7.4. The tissues were equilibrated for 90 minutes under 2 g of tension, applied by mechanically stretching the tissue. The buffer was replaced with fresh solution every 15 minutes during equilibration. The vas deferens was contracted with PE (1 or 3 /iM) while isometric tension was monitored by a force-displacement transducer (Grass FT03.C) coupled to a polygraph (Grass 7C). After peak tension was generated, the baths were washed with fresh buffer and tension was allowed to fall to baseline. These control contractions were repeated at least twice. Tissues were then incubated with various concentrations of dBu-cAMP, 8Br-cAMP, mBu-cAMP or butyrate for 30 minutes after which the PE contractions were repeated. cAMP analogs were re-added to the baths between each washout of PE. Tissue treated with 10/tM analogs were quick-frozen with liquid N2-cooled clamps and stored at -70°C until PKA activity was determined. Some vas deferens preparations were incubated with 8Br-cAMP (10/zM) for various time periods (1-30 minutes) without PE challenges to determine the time course for PKA activation. The tissues were quick-18 frozen, homogenized in the presence or absence of charcoal, and examined for PKA activity. Preparation of bovine coronary artery helical strips was based on the description by Napoli et a/. (1980). Bovine hearts were obtained from freshly slaughtered cattle at local abattoirs, immersed immediately in ice-cold PSS (with 5.56 mM d-glucose for all bovine coronary artery experiments) and transported on ice to the laboratory. Circumflex and left anterior descending coronary arteries were isolated and dissected free of visible adipose, connective and myocardial tissue. Helical strips (4mm x 20 mm) were suspended by silk thread in tissue baths as described above. Arterial tissue not used on the day of the receipt of the heart were stored in PSS at 4°C for up to 5 days. After two hours equilibration under 4 g resting tension, the bathing solution was replaced with isotonic modified PSS containing 124 mM KC1 and free of NaCl. After maximal tension was generated, the high potassium solution was washed out and tension was allowed to fall to baseline. The tissues were then rechallenged at least twice with modified PSS containing 30 mM KC1 and 88 mM NaCl. Tonic contractions induced by 30 mM KCL were exceptionally stable and typically generated greater than 10 g of tension. At steady state contracture, the arterial strips were treated with cAMP, dBu-cAMP or 8Br-cAMP (10, 30, 100 /iM) for 30 minutes and then were removed from the tension apparatus for 5 x 1 minute washes in 5mL ice-col PSS. Tissues were then quick-frozen in liquid N2 and stored at -70°C until PKA activity determination. 19 3. Washout of [JH]cAMP from Bovine Coronary Artery. Cleaned bovine coronary artery was cut into helical strips (4mm x 20mm), suspended by silk thread on aluminum supports and immersed in PSS (5mL). The tissues were warmed to 37°C in a shaking water bath and bubbled with 5% CO2 in 02 for two hours, at which time they were exposed to 10 /zM [3H]cAMP (100,000 cpm/mL, 4.5 /zCi//zmol). After 30 minutes, a washout protocol of the tissue was performed which entailed ten successive 1 minute washes followed by two successive 5 minute washes in ice-cold PSS (5mL). The washed strips were digested in 70% perchloric acid overnight at 37°C. Aliquots of 0.5 mL from the [3H]cAMP incubation medium, each washout solution and the perchloric acid digest were mixed with 2.5 mL ACS and the radioactivity was quantified by a liquid scintillation counter (Packard Tricarb 460 CD). A quench curve indicated an 85% counting efficiency of the aqueous samples while the perchloric acid in the tissue digest completely quenched any 3H radioactivity. 4. Tissue Extraction and PKA Activity Determination. 4a. Extraction of Intact Rat Vas Deferens and Bovine Coronary Artery. Frozen, intact rat vasa deferentia or bovine coronary artery helical strips, previously treated in tissue baths, were homogenized in liquid N2-cooled capsules using a Vari-Mix dental amalgam mixer (15 s, high speed) and then mixed with ice-cold homogenization buffer (20 volumes for vas deferens, 12.5 volumes for coronary artery) consisting of (mM): potassium phosphate buffer, pH 6.8 (10), EDTA (10), IBMX (0.5), 20 DTT (10) and NaCl (150) with or without charcoal (7.5 to 20 mg/mL). Homogenates were centrifuged at 31,000 x g for 15 minutes at 4°C in a Sorvall RC-2B centrifuge using a SM-24 rotor. The supernatant constituted the soluble fraction. The particulate fraction of rat vas deferens was prepared by the re-suspension of the pellet in the same volume of homogenization buffer, now supplemented with Triton X-100 (0.2%), a detergent used to separate membrane-bound PKA from phospholipid bilayers. The pellet was stirred for 60 minutes at 4°C and re-centrifuged as described above. The supernatant was used to determine PKA activity in the particulate fraction. 4b. PKA Assay. PKA activity was determined as described by Corbin and Reimann (1974) as modified by Giembycz and Diamond (1990). To initiate the assay, aliquots (25 /zL) of soluble or particulate fractions, purified PKA or chromatographic fractions were added to reaction buffer (65 /zL) in the presence or absence of cAMP (10 /iM). The reaction buffer consisted of potassium phosphate buffer (20 mM, pH 6.8), magnesium acetate (10 mM), IBMX (0.5 mM), NaF (10 mM), Kemptide (71 /iM), and [7-32P]ATP (0.1 mM, 100 cpm/pmol). Tween 20 (0.01%) was present in the reaction buffer of all assays except those for rat vas deferens in order to stabilize the catalytic subunit (Murray and Leigh, 1986). The mixture was incubated for 8 minutes at 30"C. All samples were tested in duplicate and blanks containing 25 /zL homogenization buffer were included. The reaction of vas deferens samples was stopped by the 21 spotting of 70 ul aliquots of the mixture onto phosphocellulose paper (2 x 2 cm, Whatman P81) and immersion of the papers into phosphoric acid (0.5%). The papers were washed in the acid four times for 5 minutes each time. All other samples were stopped by the addition of 10 ul HC1 (IN) and cooling on ice (Murray et al., 1990). This procedure made use of a repeater pipet which facilitated determination of PKA activity of more samples per assay. A 70 /uL aliquot was then spotted on phosphocellulose paper and washed as for the vas deferens samples. The papers were dehydrated by immersion in ethanol (95%) and diethyl ether for an additional 5 minutes each and air-dried in a fume hood. Quantitation of the bound radioactivity was determined by placement of the papers in 2.5 mL ACS and counting by a liquid scintillation counter (Packard Tricarb 460 CD). The activity of PKA was expressed as pmol of phosphate incorporated into Kemptide per minute per mg of protein. The activation of the kinase relative to the total potential PKA of the sample was expressed as the ratio of PKA activity in the presence and absence of a maximally activating concentration of cAMP (10 uV\). The specificity of the assay for the detection of Kemptide phosphorylation by a cAMP-dependent protein kinase was determined by the use of PKI, the synthetic, specific inhibitor of PKA (Cheng et al., 1986). The PKA activity of the soluble fractions of rat vas deferens and bovine coronary artery was determined as described above in the presence and absence of PKI (2.56 uq/ml). The incorporation of phosphate into Kemptide, with or without exogenous cAMP, in the presence of PKI represented cAMP-independent kinase activity and thereby reflected the specificity of the assay for PKA activity. 22 Protein concentrations were quantified using a commercially available dye-binding assay (Biorad). Bovine serum albumin was used as a standard. 5. Prevention of PKA Activation in Bovine Coronary Artery by Charcoal. In the determination of a useful charcoal concentration to bind extracellularly trapped cAMP and analogs, frozen bovine coronary artery was homogenized as described in Section 4a except 8Br-cAMP was added to the pulverized tissue along with the homogenization buffer. The 8Br-cAMP was contained in a volume equivalent to 70% of the tissue mass, reflecting an estimate of the extracellular space of the tissue. The 8Br-cAMP was in a concentration equal to 10% of that in which the tissue would have theoretically been incubated during tension studies, ie. 10% of 10, 30 and 100 /iM, based on the washout study described in Section 3. The homogenization buffer contained various concentrations of charcoal (0-20 mg/mL). The homogenate was centrifuged as described in Section 4a and the supernatant was assayed for PKA activity. 6. Effect of Charcoal on Purified PKA Activity Ratio. The effect of charcoal on the PKA activity ratio of purified kinase was determined using commercially available type I PKA. The purified PKA, reconstituted in distilled water as recommended by the supplier, was incubated at 37°C for 10 minutes with 8Br-cAMP (0.01 - 0.3 /iM) and then mixed with NaCl-free homogenization buffer in the presence or absence of charcoal (10 mg/mL). The suspension was vortexed for 10 seconds and centrifuged as described in Section 4a. The supernatant was assayed for PKA activity. 23 7. Statistical Analysis Differences between values were assessed as to their significance using one-way analysis of variance (ANOVA) followed by Neuman-Keul's multiple comparison test. A p value of 0.05 was used as the criterion for significant difference. Values are presented as the mean ± the standard error of the mean (SEM). 24 RESULTS 1. Partial Determination of Optimal Conditions for PKA Assay. The accurate estimation of PKA activity in cAMP analog-treated tissue is dependent upon several conditions including 1) a salt concentration in the homogenization buffer which would stabilize the association-dissociation equilibrium of the PKA isozymes and 2) the reduction of free cAMP analog released during the homogenization process. These conditions were optimized, as described below, through the selection of a physiological NaCl concentration based on the known and confirmed PKA isozyme profiles of rat vas deferens and bovine coronary artery, respectively, and the washing and homogenization of tissues with charcoal prior to PKA activity determination. la. PKA Isozyme Separation Profile of Bovine Coronary Artery. Since the isozyme profile of the rat vas deferens had previously been determined (Hei et al., 1991), the chromatographic evaluation was restricted to the confirmation of the isozyme profile of bovine coronary artery. This was previously described by Silver et a/. (1982) using hi stones as the assay substrate and a DEAE-Sephacel anion exchange resin. The determination described here utilized the PKA-specific substrate, Kemptide, as the phosphoryl acceptor and a DEAE-cellulose (DE-52, Whatman) anion exchange resin. 25 The elution of bovine coronary artery soluble fraction along a NaCl (0-400 mM) gradient yielded two peaks of activity in the presence of cAMP (lOjiM) (Fig. 2). The first peak eluted between approximately 3 to 11 mmhos/cm, corresponding to type I holoenzyme, and the second eluted between 18 to 22 mmhos/cm, corresponding to type II. In two separate experiments, the ratio of type I to II was approximately 40:60. No cAMP-^ dependent peak of activity (free catalytic subunit) was noted, probably a reflection of the loss of the catalytic subunit during the wash of the column with low ionic strength buffer (< 0.9 mmhos/cm) prior to elution. This isozyme profile, similar to that described by Silver et al. (1982), confirms the necessity for the use of 0.150 M NaCl during the homogenization of rat vas deferens and bovine coronary artery in order to prevent the dissociation of type I isozyme and the reassociation of type II during the assay procedure (Corbin et al., 1975; Palmer, 1980). lb. Charcoal Adsorption and Washout of cAMP Analogs. The use of cAMP and analogs as tools to activate PKA in intact tissue necessitates the determination of methodological controls to avoid false positive PKA activation results. The potential for these fallacious results arises from the release of extracellularly associated free cAMP or analogs during the disruption of frozen intact tissues by homogenization. This problem was approached in two ways. Firstly, free analog was washed from the extracellular space of treated bovine coronary artery after the 30 minute incubation period, prior to quick-freezing. Secondly, the addition of charcoal as an adsorbent to the 26 Figure 2. Isozyme Profile of Soluble Fraction of PKA in Bovine Coronary Artery. The soluble fraction of bovine coronary artery was prepared as described in Methods. Fractions were eluted from a DEAE-cellulose column with a NaCl gradient (0-400 mM). Fractions were assayed for PKA activity in the presence and absence of cAMP (10 J I M ) . 27 T 1 1 1 r 10 2 0 3 0 4 0 5 0 FRACTION 28 homogenization buffer removed free analog released from the disrupted tissue. Since rat vas deferens was exposed to 10/xM analog only and lOmg/mL charcoal effectively adsorbed the free analog (Hei et al., 1991), a washout procedure was not used for this tissue. In order to determine an appropriate washout procedure for bovine coronary artery, a control experiment was performed to follow the removal of tritiated cAMP from arterial helical strips. The tissue was incubated in [3H]cAMP for 30 minutes and successive 1 to 5 minute washes were performed. Approximately 90% of the total recovered radioactivity was removed from the strip in 5 minutes and was fully recovered in 20 minutes (Fig. 3). The total recovered radioactivity represented a volume equivalent to 40% to 80% of the tissue mass. In addition to washing the extracellular space of analog, optimal concentrations of charcoal were determined which would bind the remaining free analog at the time of homogenization of bovine coronary artery. Since the washout procedure would remove 90% of the cyclic nucleotides from the extracellular space in 5 minutes, one-tenth of the analog concentration to be used in the tension/PKA experiments was utilized in the following charcoal studies. Frozen untreated bovine coronary artery, when exposed to 1/zM or 3/zM 8Br-cAMP at the time of homogenization, required 7.5 mg/mL charcoal to prevent the activation of the kinase above control values using charcoal only (Fig. 4). The activity ratio was 0.13 (+ 0.012) for 1/xM 8Br-cAMP and 0.17 (± 0.049) for 3/zM 8Br-cAMP, similar to 0.13 (± 0.022) for control. These concentrations of analog (1/xM and 3/uM) represented that expected to be sequestered in the coronary artery extracellular 29 Figure 3. Washout of [3H]cAMP from Bovine Coronary Artery Helical Strips. Bovine coronary artery was incubated in [3H]cAMP and washed for 10 x 1 minutes and 2 x 5 minutes in ice-cold buffer as described in Methods. Points (•) indicate the percentage of the total radioactivity removed from the tissue during each wash. Values represent the mean ± SEM of 4 experiments. 30 100 i 80 A CD < 60 A LL. O 40 20 H 0 J i r 0 2 4 6 8 10 12 14 16 18 20 TIME (min) 31 Figure 4. Prevention of PKA Activation 1n Bovine Coronary Homogenates Exposed to 8Br-cAMP by Activated Charcoal. Frozen bovine coronary artery was homogenized with or without 8Br-cAMP (1, 3, 10 uH) and charcoal (7.5 or 20 mg/ml). The activity ratio in the soluble fraction was determined. See Methods for further details. Values in parentheses indicate the number of experiments. 32 CONTROL 8 B r - c A M P 0.4 0.3 A (5) (5) 0.2 A 0.1 A 0.0 (11) (14) 7.5 7.5 20 [CHARCOAL] (mg/mL) 1 3 10 [8Br-cAMP] (uM) 33 space after the 5 x 1 minute washout of tissue treated with 10/aM and 30/tM, respectively. Using one-tenth of the incubation concentration of 100/zM, a higher concentration of charcoal, 20mg/mL, was necessary to prevent the spurious activation of PKA. The PKA activity ratio of coronary artery treated with 20mg/mL charcoal was 0.08 (± 0.008) and was 0.10 (± 0.005) for 8Br-cAMP (10/iM)-treated tissue. These results indicate cAMP analog-treated intact bovine coronary artery strips could be examined for PKA activity without interference by the free exogenous cyclic nucleotides when an appropriate concentration of charcoal was added to the homogenization buffer, with a prior washout of the treated tissue. While charcoal is of great assistance in estimating PKA activity, the adsorbent was assessed as to its own potential to alter the activity of PKA. The basal PKA activity ratio of rat vas deferens was 0.16 (± 0.009) but was reduced to 0.11 (± 0.015) by charcoal (lOmg/mL) (these values are illustrated in Fig. 10, see zero minute time points). Bovine coronary artery strips, untreated with cAMP analogs, demonstrated a PKA activity ratio of 0.29 (± 0.050) in the absence of charcoal whereas this was reduced to 0.10 (± 0.004) and 0.08 (± 0.011) by 7.5 mg/mL and 20 mg/mL charcoal, respectively. Thus, charcoal appeared to diminish the basal activity ratio. This potential of charcoal to alter the activity of PKA was investigated further by the use of purified type I PKA holoenzyme. The kinase was incubated with increasing amounts of 8Br-cAMP and was then exposed to 10 mg/mL charcoal. After separation from the charcoal, the kinase demonstrated a reduced catalytic activity ratio, in comparison to the enzyme which was not treated with charcoal (Fig. 5). After exposure 34 Figure 5. Effect of Charcoal on Purified Type I PKA Activity Ratio. Purified type I PKA was activated with 8Br-cAMP (0.01 - 0.3 /JM) and exposed to charcoal (10 mg/mL). The PKA activity ratio was determined as described in Methods. Points indicate mean values from 1-2 experiments. 35 36 to 0.03/iM or 0.3 /iM 8Br-cAMP, the activity ratio was decreased at least two-fold in the presence of charcoal. The stability of the association-dissociation equilibrium of PKA between the time of the processing of the tissue with charcoal and the actual time of incubation with substrate in the assay was evaluated. This would lend some understanding to the question of whether charcoal altered the specific activity ratio of PKA very soon after exposure to the homogenate or if reassociation occurred gradually over the time required to prepare the homogenate for the final assay steps. The specific activity ratio of rat vas deferens tissue remained consistent over a period of 30 to 120 minutes between the homogenization process, with or without charcoal, and the final assay for activity (Fig. 6). Therefore, while charcoal can apparently reduce the activity ratio of PKA, it probably does so fairly rapidly, at least within 30 minutes. After that time point, the ratio is stable. 2. cAMP Analog-Induced Inhibition of Rat Vas Deferens Contraction. The range of concentrations of cAMP analogs used to examine the inhibition of rat vas deferens PE-induced contractions reported previously (Schultz et a/., 1979) was expanded to consider doses from 1 to 100/iM dBu-cAMP and 10 to 100/zM 8Br-cAMP (Fig. 7). dBu-cAMP demonstrated a distinct dose-response relationship over the concentrations considered and essentially eliminated tension development at 100/zM. 8Br-cAMP was not able to significantly inhibit contractions at any of the concentrations considered. For example, in a separate experiment done in collaboration with Yong-jiang Hei, dBu-cAMP (10 /iM) 37 Figure 6. Rat Vas Deferens Soluble PKA Activity Ratio at Various Time Points After Exposure to Charcoal. Rat vas deferens homogenate was incubated with 8Br-cAMP (0.6 /xM) for 10 minutes and briefly vortexed in the absence ( O ) or the presence of charcoal (10 mg/mL) ( • ) . The soluble fraction was assayed for PKA activity at various time points after exposure to charcoal. Points represent a single experiment. PKA ACTIVITY RATIO ( - / + cAMP) 39 Figure 7. Concentration-Response Curves for the Inhibition of PE (3 /JM)-Induced Rat Vas Deferens Contractions by cAMP Analogs . The tissues were contracted three times with PE, as control values. After incubation with dBu-cAMP ( O ) or 8Br-cAMP (•) for 30 minutes, the rat vasa deferentia were re-exposed to PE and responses were compared to controls. Values represent the mean ± SEM of at least 3 experiments. (*) indicates values significantly from control, p < 0.05. 40 120 n 10~ 7 10~ 6 10~ 5 10~ 4 10~ 3 CONCENTRATION (M) 41 reduced PE (1/xM)-induced tension by more than 87% of control while 8Br-cAMP (10 nM) had no significant effect (Fig. 8). Clearly, these analogs differ markedly in their potential to inhibit rat vas deferens contractions. The tension generated by PE is phasic in nature and attempts were made to evaluate the relaxant effects of these analogs on tonic contractures. Potassium chloride has been used previously to induce such tension in the epididymal portion of the rat vas deferens (Langton and Huddart, 1988) but stable contractures could not be reproducibly elicited in our laboratory. As a result, conclusions were limited to the alteration of phasic tension by cAMP analogs in this tissue. 3. PKA Activity in cAMP Analog-Treated Rat Vas Deferens. Since there was a clear difference between dBu-cAMP and 8Br-cAMP in their inhibitory effects at 10/xM on rat vas deferens contractions, the activity of PKA in these tissues was then compared. The PKA activity ratio of dBu-cAMP (10/zM)-treated rat vas deferens was elevated significantly in the soluble fraction to 0.22 (± 0.015) over a control value of 0.16 (+ 0.025) but was not significantly activated in the particulate fraction (Fig. 9). In comparison, vas deferens incubated in 8Br-cAMP (10/zM) demonstrated significantly increased PKA activity ratios in both soluble and particulate fractions, with values of 0.30 (± 0.023) and 0.36 (± 0.0.12), respectively. The particulate control value was 0.16 (± 0.005). These increases were even larger than that seen with the relaxant analog, dBu-cAMP. 42 Figure 8. Effect of cAMP Analogs on PE [luVl)-Induced Rat Vas Deferens Contractions. Rat vas deferens was treated as described in Figure 7 and incubated with 10 /iM of dBu-cAMP or 8Br-cAMP. Responses were compared to controls. Values represent the mean ± SEM of 5-7 experiments. (*) indicates values significantly different from control , p < 0.05. From Hei et a7. (1991). 43 I I CONTROL V7A d B u - c A M P 44 Figure 9. Effect of cAMP Analogs on Soluble and Particulate PKA Activity in Rat Vas Deferens. Tissues were treated with 10 /iM of dBu-cAMP or 8Br-cAMP as described in Figure 7 and freeze-clamped after the second set of PE contractions had been performed. PKA activity ratios in soluble and particulate fractions of rat vas deferens were determined by measurement in the presence and absence of cAMP (10 /xM). Values represent the mean ± SEM of 5-7 experiments. (*) indicates values significantly different from control. 45 CONTROL V7\ d B u - c A M P • 8 B r - c A M P 0.4 -, Soluble PKA Part icu late PKA 46 4. Time Course of PKA Activation in Rat Vas Deferens by 8Br-cAMP. The experimental evidence reported by Hei et al. (1991) demonstrated that 10 mg/mL charcoal prevented the spurious activation of PKA during the homogenization of cAMP (10/zM)-treated rat vas deferens. This procedure involved the homogenization of frozen tissue with analog in a charcoal-containing buffer. To ensure that these results were relevant to vasa deferentia which had actually been incubated at 37°C with the analogs, an experiment was designed to test the capability of charcoal to bind free 8Br-cAMP in tissues incubated at 378C with 10 uH of the analog for various lengths of time. This also provided an assessment of the pattern of PKA activation by this analog over time. Homogenization, in the absence of charcoal, of rat vas deferens which had been incubated in 8Br-cAMP (10/iM) for 1, 5, and 30 minutes demonstrated a significant and consistent degree of PKA activation (Fig. 10). The presence of charcoal (10 mg/mL) resulted in a PKA activation profile which reflected a gradual activation of the kinase over time, most likely a result of the slow penetration of the analog into cells. A significant increase in activity was apparent only after 30 minutes. This suggested that a major basis for the activation of PKA in rat vas deferens homogenized without charcoal, particularly at the 1 and 5 minute time points, was the release of extracellularly associated free 8Br-cAMP. This component was minimized in rat vas deferens homogenized with charcoal since the free analog was bound by the adsorbent and the activation profile probably reflected the state of the kinase as it existed in the intact treated tissue or, at least, an underestimate thereof. 47 Figure 1 0 . Time Course of PKA Activation by 8Br-cAMP in Rat Vas Deferens. Rat vas deferens was incubated with 8Br-cAMP (10/JM) for 3 0 minutes and freeze-clamped at the indicated times. Tissue was homogenized with (•) or without ( O ) charcoal ( 1 0 mg/mL), centrifuged and the soluble fraction was assayed for PKA activity. Values represent the mean ± S E M of 6 - 7 experiments. (*) indicates values significantly different from control (zero time point), p < 0 . 0 5 . 48 TIME (min) 49 5. Effects of dBu-cAMP Metabolites on Rat Vas Deferens Tension and PKA Activity. The activation of PKA by dBu-cAMP is believed to be the result of the intracellular deacylation of the nucleotide to the PKA-activator, mBu-cAMP. (Kaukel et al., 1972). The effects of mBu-cAMP and butyrate on inhibition of contraction and PKA activity were considered in an effort to gain understanding into the actions of the parent compound. mBu-cAMP (10/zM) reduced the contractile response of rat vas deferens to PE by approximately 25% (± 6.4%, n=7) after 30 minutes of incubation and significantly activated PKA from a control of 0.06 (± 0.01, n=5) to 0.12 (± 0.01, n=7). Butyrate did not alter tension, as reported previously (Kreye and Schultz, 1972), nor PKA activity (control=0.06 ± 0.01, n=5; butyrate= 0.07, n=2) 6. Relaxation of Bovine Coronary Artery by cAMP and Analogs Bovine coronary artery helical strips, depolarized by 30 mM KC1, maintained very stable contractures over extended periods of time (> 4 hours). Incubation of the strips with cAMP led to a small but significant and reproducible relaxation (Fig. 11). Using 30 /zM and 100 juM cAMP, loss in tension generally began within 1 minute and reached a nadir of 90.6% (± 4.07) and 92.2% (± 2.67) of control tension, respectively, in about 10 minutes. After a 30 minute incubation, the tension inhibition had reversed slightly to 92.9% (± 2.62) in the presence of 30 /iM cAMP and 93.5% (+ 2.58) in the presence of 100 juM 50 Figure 11. Effect of cAMP and Analogs on Bovine Coronary Artery Tension and PKA Activity. Bovine coronary artery helical strips were contracted with 30 mM KC1 and incubated with cAMP or analogs (10 - 100 /zM). Tension reduction and activation of soluble PKA was determined after 30 minutes of incubation as described in Methods. Points ( • ) indicate the mean tension reduction of 3-5 experiments. Bars indicate the mean percent increase in soluble PKA activity ratio ± SEM over control of 3-5 experiments. The control PKA activity ratio for samples treated with 10 and 30 /zM cAMP/analog was 0.10 (± 0.004) and that for treatments with 100 jzM was 0.08 (± 0.011). (*) indicates values significantly different from control, p < 0.05. 51 12 O X LxJ 9 -6 -3 -0 12 9 6 -3 -0 12 9 6 3 0 10 30 100 8 B r - c A M P * T 10 30 100 300 - 250 - 200 - 150 - 100 300 - 250 - 200 - 150 - 100 300 o —h o o O T J > > O —! < o 10 30 100 C O N C E N T R A T I O N (uM) 52 induced bovine coronary artery tension. These results confirm those reported by Napoli et a7.(1980). 7. PKA Activity in cAMP- and Analog-Treated Bovine Coronary Artery. As in the rat vas deferens studies, changes in potassium-depolarized bovine coronary artery tension were compared to the activation of PKA. The relaxant compound, cAMP, significantly activated the soluble fraction of the kinase at all concentrations examined (Table 1). Even at 10 /iM, a concentration which had essentially no effect on tension, cAMP increased the PKA activity ratio from 0.10 (± 0.004) to 0.17 (± 0.014). Higher concentrations of cAMP produced greater activation of PKA with a 2.30- and 2.25-fold increase over control levels at 30 /iM and 100 /iM, respectively. As in the rat vas deferens, 8Br-cAMP (10-100 /iM) was effective in activating the kinase in the bovine coronary artery. This non-relaxant analog activated the soluble fraction of PKA to approximately the same degree as equivalent concentrations of cAMP. Finally, not only did dBu-cAMP not induce relaxation, but it did not significantly alter PKA activity in concentrations of 10 /iM to 100 /iM. The PKA activation data for each nucleotide was replotted to allow for comparison with the change in coronary artery tension (Fig. 11). These results indicate that activation of PKA in both vascular and non-vascular tissues may occur without necessarily inducing relaxation. 53 Table 1: Effects of cAMP and Analogs on Bovine Coronary Artery PKA Activity Ratio. [analog] PKA ACTIVITY RATIO dBu-cAMP 8Br-cAMP cAMP 10 /iMa 0.10 (± 0.014) 0.16 (± 0.003)* 0.17 (± 0.014)* 30 /xMa 0.11 (± 0.003) 0.24 (± 0.009)* 0.23 (± 0.022)* 100 /iMb 0.08 (± 0.008) 0.20 (± 0.009)* 0.18 (± 0.029)* Bovine coronary artery were contracted with 30 mM KC1 and treated with various concentrations of cAMP or analogs. After 30 minutes, the tissues were frozen and examined for PKA activity as described in Methods. Values represent the mean ± SEM of 3-5 experiments. (*) indicates values significantly different from control, p <0.05. a = tissue was treated with 7.5 mg/ml charcoal during homogenization. Control value = 0.10 (± 0.004), n=7. b = tissue was treated with 20 mg/mL charcoal during homogenization.Control value = 0.08 (± 0.011), n=4. 54 8. Specificity of the PKA Assay for cAMP-Dependent Protein Kinase Activity. The assay procedure itself was tested as to its specificity for rat vas deferens and bovine coronary artery PKA activity in comparison to cAMP-//?dependent activity. Approximately 97% and 95% of the maximal incorporation of phosphate into Kemptide by vas deferens and coronary artery soluble fractions, respectively, was dependent upon PKA, using the PKA-specific inhibitor, PKI (Fig. 12). 55 Figure 12. Effect of Protein Kinase Inhibitor on Rat Vas Deferens and Bovine Coronary Artery Soluble PKA Activity. In rat vas deferens (Panel A) and bovine coronary artery (Panel B), basal (-cAMP) and maximal (+cAMP) PKA activity was determined in the presence and absence of PKI (2.5 /ig/mL). The bars represent values from two (Panel A) and one (Panel B) separate experiments. PKA ACTIVITY (pmol PO , i n c o r p . / m g / m i n ) o > Cn O O i o o o _ J _ Cn O o N) O O O rO Cn O O o o o CD > T J o o o _1_ r rO O O O O O O O O O cn o o o CD o o o —1 o > + o > + I T J T J 7^  7^  57 DISCUSSION The use of exogenous cAMP and analogs as tools to mimic the effects of intracellularly generated cAMP is a valid technique to investigate the physiological role of PKA. Sutherland et a7. (1968) designated the use of exogenous cyclic nucleotides as critical in implicating the cAMP-initiated cascade of biochemical events in a given functional effect. Exogenous cAMP analogs have been used to this end in a myriad of experiments over the past three decades (for review, see Kramer and Hardman, 1980). Since all intracellular actions associated with cAMP are generally believed to be mediated by PKA, a logical corollary is that functional effects, such as smooth muscle relaxation, induced by exogenous cAMP or analogs reflect the activity of PKA. This study went one step beyond indirectly assessing the role of PKA in smooth muscle relaxation based on the functional effects of putative kinase activators. The actual activation state of the kinase was evaluated and compared to the degree of relaxation. The results indicate that, while dBu-cAMP in the rat vas deferens and cAMP in the bovine coronary artery did activate PKA in a manner which correlated with relaxation, the activation of PKA by 8Br-cAMP in those tissues had no bearing on changes in tissue tension. In the absence of relaxation, 8Br-cAMP activated PKA to an equal or even greater extent than did the relaxant cyclic nucleotides, cAMP and dBu-cAMP, in the bovine coronary artery and rat vas deferens, respectively. A similar lack of association between PKA activation, assessed indirectly, and smooth muscle relaxation was noted by Francis et a/. (1988). They noted a very poor 58 correlation between the known potencies of cyclic nucleotides to activate PKA in vitro and the relaxation of porcine coronary artery and guinea pig trachealis. The validity of conclusions drawn from these results is contingent on the accuracy of the determination of PKA activity in cAMP analog-treated tissues. Adjustments in the treatment of the tissues after exposure to analogs and during the extraction process ensured that the PKA activity was not overestimated by dissociation of the kinase during homogenization. The NaCl concentration of the homogenization buffer was selected based on the isozyme profiles of the tissues. Both type I and type II were present in the soluble fractions of rat vas deferens (Hei et a/., 1991) and bovine coronary artery (Fig. 2, Silver et a7., 1982) and type II only was in the particulate fraction of rat vas deferens (Hei et al., 1991). Accordingly, an intermediate concentration of 0.150 M NaCl was selected to minimize changes in the PKA activity ratio during homogenization and extraction of these tissues, as previously suggested for tissues which contain a combination of the isozymes (Corbin et al., 1975; Palmer, 1980). Activated charcoal was another important device which minimized spurious activation of PKA during processing of the tissues. This large surface area adsorbent has been extensively used, especially in radioimmunoassays, for the separation of free fraction (hormone, etc.) from bound complexes (hormone-antibody complexes, etc.) (Binoux and Odell, 1973; Ratcliff, 1974). Concentrations of free small molecules such as cAMP, AMP, and ATP can be reduced essentially to zero by charcoal (Corbin et al, 1975). This is particularly advantageous because the release of extracellularly associated cAMP and analogs during 59 homogenization can then be neutralized by adsorption and the activation state of PKA present in the intact tissue can thereby be assessed. Charcoal had previously been used in the PKA assay of rat vas deferens treated with 8Br-cAMP (10 uV\) or dBu-cAMP (10 uVl) (Hei et al., 1991). This study extended these results to a time-course study of the activation of rat vas deferens PKA by 8Br-cAMP between 1 and 30 minutes of incubation, in the presence or absence of charcoal during homogenization. If charcoal blocked the spurious activation of PKA, a gradual activation of the kinase in the intact tissue would be expected as sufficient time elapsed for the fairly lipid-insoluble analog to penetrate the intact tissue. This was indeed the case (Fig. 10). The kinase was significantly activated only after 30 minutes of incubation with 8Br-cAMP if the tissue was homogenized in the presence of charcoal, whereas it was activated within 1 minute in the absence of charcoal. In the latter case, the activity ratio held steady for the next 30 minutes of incubation. The activation at the 1 minute time point without charcoal in the homogenization buffer was most likely due to dissociation of the kinase during tissue processing by the release of free analog from the homogenized tissue. It is apparent, therefore, that charcoal (10 mg/mL) was effective in the adsorption of free analog which would otherwise induce false-positive results in the determination of PKA activity in cAMP analog-treated rat vas deferens. While the optimal assay conditions for the assay of vas deferens were limited to the consideration of one incubation concentration of cAMP analog (10 /zM), a wider range of concentrations (10, 30 and 100 /iM) were used in the case of bovine coronary artery. The determination of PKA activity in the presence of 30 /iM and 100 /iM analog proved to be a 60 major methodological problem. Concentrations of charcoal necessary to bind these amounts of analog tended to lower protein concentrations and, concomitantly, kinase activity. Due to limitations in the sensitivity of the assay, the accuracy of the assay was compromised. A washout procedure was developed to diminish the free analog associated with the extracellular space of the treated coronary artery strips. The utility of this procedure was based on the likelihood that the extracellular fluid and matrix contained the vast majority of analog associated with the tissue after 30 minutes of incubation. This is probable due to the high water solubility and poor tissue penetrability of the compounds. The removal of [3H]cAMP from bovine coronary artery strips followed an exponential decline with respect to time and, based on this result, a standard washout procedure (5x1 minute ice-cold washes) was applied to all arterial tissue after analog treatment. This reduced the amount of adsorbent required to bind free analog during homogenization. A higher concentration of charcoal was necessary to bind 10 /iM 8Br-cAMP in control experiments with bovine coronary artery than was required with rat vas deferens, namely, 20 mg/mL in comparison to 10 mg/mL. This is probably due to the smaller volume of buffer used in the homogenization of coronary artery. Arterial tissue was homogenized in 12.5 volumes whereas vas deferens was mixed with 20 volumes. The absolute amount of charcoal was apparently important in the adsorption of the free analog, since approximately a two-fold higher concentration of charcoal would be required to deliver an amount of charcoal to the bovine coronary artery equivalent to that provided in a two-fold larger volume of buffer with 10 mg/mL charcoal in the case of rat vas deferens. 61 The selection of 8Br-cAMP in the control studies of charcoal binding with free analog was based on the high potency of the analog relative to dBu-cAMP and cAMP. The Ka for activation of PKA by 8Br-cAMP and cAMP are 0.06 /iM and 1 /iM, respectively (Flockhart and Corbin, 1982; Francis et al., 1988). dBu-cAMP itself does not activate PKA but its metabolite, mBu-cAMP, has a Ka for activation approximately equivalent to that of cAMP (Meyer and Miller, 1974). The charcoal concentrations which bind 8Br-cAMP were presumed to be more than sufficient to neutralize the less potent compounds if they demonstrated charcoal binding characteristics similar to 8Br-cAMP. The lack of specificity of charcoal for adsorption (Palimeri et a/., 1971) suggests that these structurally similar compounds interact comparably with charcoal. While it is apparent that charcoal prevented the spurious activation of PKA, the use of charcoal can, itself, affect PKA activity. Charcoal reduced the basal PKA activity ratio of rat vas deferens and bovine coronary artery control tissues as described in Results, section lb. Reduction in control activity ratios was also noted by Silver et al. (1982) who reported a control activity ratio of bovine coronary artery type II PKA equal to 0.54 ± 0.06 in the absence of charcoal and 0.44 ± 0.05 after homogenization with 2.5 mg/mL charcoal. Contrasting results were found in rat heart in which control values were identical with or without charcoal (10 mg/mL) (Corbin et a/., 1975). The reduction in control PKA activity ratios in this study could be interpreted as the removal by charcoal of endogenous cAMP sequestered into intracellular compartments, unavailable for PKA activation while the cell was intact. The compartmentalization of cAMP is a phenomenon which has been suggested to account for the lack of correlation of the elevation in 62 cAMP levels and activation of PKA by isoproterenol, forskolin and PGEi with relaxation of bovine coronary artery (Vegesna and Diamond, 1984). An alternative interpretation of the reduction in the PKA activity ratio by charcoal is that the adsorbent's depletion of free cAMP or analogs from the homogenate leads, even at 4"C, to a shift in the equilibrium of associated and dissociated regulatory and catalytic subunits such that net reassociation occurs. This would seem unlikely since cAMP and analogs have a very high affinity for PKA, for example, the K<j for dissociation of cAMP from PKA is approximately 10 nM (Flockhart and Corbin, 1982). Also, physico-chemical processes such as reassociation would be greatly inhibited at 4°C. The influence of potentially compartmentalized endogenous cAMP was circumvented using commercially available purified PKA holoenzyme to assess effects of charcoal. Activity ratios of 8Br-cAMP-activated purified PKA were appreciably diminished in the presence of 10 mg/mL charcoal (Fig. 5). This more clearly shows that charcoal itself can reduce the activity ratio of PKA, independently of binding endogenous cAMP. Of course, purified PKA, reconstituted in distilled water and devoid of cellular constituents which could enhance its stability, might more readily succumb to alterations in its association-dissociation equilibrium by charcoal than might PKA buffered in a tissue homogenate. In this way, changes in purified PKA activity by charcoal may exaggerate the actual situation with tissue homogenates. Nevertheless, it is important to note that charcoal can prevent a false positive PKA activation result in cAMP- and analog-treated tissues and its use may even lead to an underestimation of PKA activation. The extent of kinase activation determined in the assay procedure using charcoal is likely to 63 reflect an even larger PKA activity ratio which existed in the intact tissue. This suggests that the degree to which relaxation and PKA activation by 8Br-cAMP do not correlate is possibly even more profound in actuality. The lack of inhibitory effect by 8Br-cAMP on rat vas deferens contractions did not appear to result from a deficiency in affecting a particular isozyme or compartment of PKA. The analog significantly activated the kinase in both the soluble and particulate fractions of vas deferens homogenates. These fractions contained a mixture of type I and II and exclusively type II, respectively (Hei et al., 1991). The particulate fraction of bovine coronary artery was not examined for PKA activity and, therefore, it remains to be seen i f selective activation of this compartment could account for the relaxant effect of cAMP and the lack thereof by 8Br-cAMP and dBu-cAMP. It may be argued that the heterogeneity of cell types in intact rat vas deferens and bovine coronary artery precludes the conclusion that the PKA activity determined in the tissues was actually that which was present in smooth muscle cells. This leads to the possibility that the dissociation between activation of PKA and relaxation by 8Br-cAMP may actually be irrelevant since the PKA activity determined in the assay had had its source in non-smooth muscle cells. This argument is probably not applicable in this case since smooth muscle cells constitute a large fraction of the mass of these tissues, PKA is known to exist in smooth muscle cells, and it is improbable that the analogs would preferentially partition into non-smooth muscle cells. The PKA activity measured is, therefore, most likely a reasonable reflection of the kinase activation in smooth muscle cells. 64 Many different protein kinases exist in smooth muscle cells yet there is l i t t l e likelihood that the phosphorylating activity assessed in the assay was due to cAMP-Zndependent protein kinases. Approximately 95% of the total phosphorylation of Kemptide was inhibited by PKA-specific PKI as determined in the soluble fractions of rat vas deferens and bovine coronary artery. Abolition of activity by this inhibitor reflects the specificity of the assay for a cAMP-dependent kinase. It is therefore evident that the conditions of the assay were appropriate for the determination of PKA activity in cAMP- and analog-treated intact rat vas deferens and bovine coronary artery. As mentioned, the mediation of /3-agonist and other hormonally-induced relaxation of vascular and non-vascular smooth muscle by PKA activation and subsequent protein phosphorylation is a fairly well entrenched hypothesis (Hardman, 1984; Silver et a l . , 1982, 1984; Kukovetz, et al. , 1978). On the other hand, there is an increasing body of evidence which indicates that activation of PKA does not necessarily lead to events associated with relaxation. Felbel et al.(1988) demonstrated that 8Br-cAMP had variable effects on intracellular calcium levels. Carbachol-elevated calcium levels were not reduced by prior incubation (20 minutes) of isolated bovine tracheal smooth muscles with up to 100 /iM of 8Br-cAMP. The elevated calcium levels could only be diminished by application of the analog at or just prior to agonist stimulation. The authors suggested that PKA activation by 8Br-cAMP could augment intracellular calcium lowering by other PKA-activating compounds (e.g. isoproterenol) i f the agents were applied at similar time points but the reason for the inability of the analog to do so alone or with other PKA activators after 20 to 30 minutes of preincubation remained 65 unclear. Lincoln et al. (1990) demonstrated that the activation of PKA by forskolin and isoproterenol was not sufficient to reduce vasopressin-induced increases in calcium in cultured vascular smooth muscle cells, nor was the catalytic subunit of PKA itself, when introduced into the cells. Only in the presence of PKG could the adenylate cyclase activators reduce intracellular calcium levels. The relative importance of PKA and PKG in relaxation was also explored by Francis et al. (1988) using exogenously applied analogs and intact smooth muscle preparations. A much stronger correlation was noted between the Ka of cyclic nucleotide analogs for the in vitro activation of PKG, rather than PKA, and the relaxation of porcine coronary arteries and guinea pig tracheal is. PKG activation provides an interesting alternative explanation for the relaxation of bovine coronary artery by cAMP and the lack of this effect by 8Br-cAMP and dBu-cAMP. 8Br-cAMP, dBu-cAMP and its metabolite, mBu-cAMP, have about 3.5- to 1000-fold greater Ka values than cAMP for activation of PKG. While the Ka for PKG activation by cAMP is approximately 1 /iM (Lincoln et a/., 1977), the values for 8Br-cAMP, dBu-cAMP and mBu-cAMP are 3.5 /iM, 1200 /iM, and 34.4 /iM, respectively (Francis et al., 1988). Therefore, if relaxation of the bovine coronary artery by cyclic nucleotides is a result of PKG activation, cAMP would be, and was, the strongest relaxant amongst the compounds tested. While this correlation exists, it may be coincidental considering the temporal pattern of coronary artery relaxation by cAMP, as discussed below. Also, this association is not applicable in the case of inhibition of rat vas deferens contractions by dBu-cAMP. This analog was a relatively potent inhibitor of PE-induced contractions but it and its metabolite are considerably less capable of activating PKG than is 66 the non-relaxant analog, 8Br-cAMP, as indicated by the Ka values mentioned above. The conversion of the relaxant analogs to a variety of metabolites by extracellular and intracellular degradative enzymes is an important consideration in the analysis of their relaxant effects. dBu-cAMP is hydrolyzed by a deacylase within cells (O'Neill et al., 1975) and i t s monobutyryl derivative is believed to be it s i n t r a c e l l u l a r ^ active form with respect to PKA activation (Kaukel et al., 1972). While mBu-cAMP probably accounted for the PKA activation seen in dBu-cAMP treated rat vas deferens, the degree of relaxation seen with dBu-cAMP could only be partly accounted for by the actions of the monobutyryl form. The metabolite was a relatively weak inhibitor of vas deferens contractions in comparison to its parent compound. It is possible that mBu-cAMP was less able to penetrate the plasma membrane of rat vas deferens smooth muscle cells than was dBu-cAMP due to i t s single butyryl group and resulting lower 1 ipophilicity. This potential to penetrate membranes was considered an important factor in the inotropic potencies of ffi-substituted cyclic nucleotide analogs in the guinea pig heart (Kawada et al., 1989) but was noted as not being a factor in the correlation of cyclic nucleotide-induced relaxation of porcine coronary arteries (Francis et al., 1988). Even i f mBu-cAMP did not penetrate the vas deferens as well as dBu-cAMP, i t doubled the activity ratio of PKA from control values, as did dBu-cAMP, but had markedly inferior effects on tension reduction. This suggests that the mechanism of contraction inhibition by dBu-cAMP is not entirely related to PKA activation. The absence of any PKA activation by dBu-cAMP in bovine coronary artery may 67 be related to a paucity of the appropriate deacylase in this tissue to generate mBu-cAMP. Butyrate is the other product of dBu-cAMP deacylation and changes in cell function of dBu-cAMP-treated tissues, such as in neuroblastoma cells, have been attributed to its release (Yusta et a/., 1988). The lack of inhibition of rat vas deferens relaxation, also noted by Kreye and Schultz (1972), and PKA activation by butyrate indicates the release of this product had little impact on the actions associated with dBu-cAMP. Susceptibility of cAMP to enzymatic catabolism, for example, to phosphodiesterase-catalyzed hydrolysis, is well established (Francis et al., 1988) and has been the basis for the selection of more phosphodiesterase-resistant analogs in examining the effects of the cyclic nucleotide. The pattern of cAMP-induced bovine coronary artery relaxation indicates an extracellular event may be operative in the tension reduction and may possibly be related to a cAMP degradation product. The onset of relaxation is very rapid (less than 2 minutes), much faster than anticipated from the expected slow penetration of the water soluble compound into the muscle cells. An extracellular process would more likely explain this rapid onset, such as the perturbation of the smooth muscle plasma membrane and resulting changes in ion conductance through membrane-spanning channels. Evidence exists for direct effects such as these by cAMP. For instance, kinase-independent increases in sodium conductance through plasma membrane channels of olfactory cilia by cAMP have been reported (Gold and Nakamura, 1987). As well, cAMP may stimulate extracellularly associated receptors, such as adenosine receptors. The PKA activation by cAMP seen after 30 minutes 68 incubation may thereby be unrelated to the functional change. Complicating the suggestion that adenosine receptors may be involved is the strong correlation between adenosine activation of PKA and bovine coronary artery relaxation (Silver et a l . , 1984) but, as noted by Lincoln et al. (1990), hormonally induced elevations in cAMP can be sufficient to activate both PKA and PKG. This leads to the possibility that the final mediator of the vascular relaxation could be a cellular component other than PKA. Therefore, the direct or non-PKA actions of cAMP and dBu-cAMP may account for their relaxant effects but definitive evidence for these possibilities has not yet been described in smooth muscle preparations. In conclusion, the inhibition of rat vas deferens contraction and relaxation of bovine coronary artery by exogenous cAMP and analogs did not consistently correlate with PKA activation. Using an assay procedure specially modified to determine PKA activity in cAMP- and analog-treated intact tissue, the activation of the kinase by 8Br-cAMP did not result in smooth muscle relaxation, while a similar or lesser degree of activation was present in rat vas deferens and bovine coronary artery relaxed in a dose-dependent manner by dBu-cAMP and cAMP, respectively. This evidence implies that PKA activation by cyclic nucleotides is not sufficient for the relaxation of vascular and non-vascular smooth muscle. 69 SUMMARY The r e s u l t s o f t h i s i n v e s t i g a t i o n i n d i c a t e that the PKA a c t i v i t y r a t i o o f cAMP- or analog- t r e a t e d i n t a c t smooth muscle p r e p a r a t i o n s can be determined without spurious a c t i v a t i o n d u r i n g the homogenization process o f the assay. A c t i v a t e d charcoal was used to adsorb free exogenous c y c l i c n u c l e o t i d e s and t i s s u e washout was used to reduce the analog l o a d i n the e x t r a c e l l u l a r space. A c t i v a t e d charcoal appeared to reduce the a c t i v i t y r a t i o o f PKA. Using these c o n t r o l s , changes i n the PKA a c t i v i t y r a t i o o f r a t vas deferens and bovine coronary a r t e r y t i s s u e were compared to changes in t e n s i o n caused by cAMP and analogs. PKA a c t i v a t i o n by dBu-cAMP i n the s o l u b l e f r a c t i o n o f r a t vas deferens and by cAMP i n the s o l u b l e f r a c t i o n o f bovine coronary a r t e r y c o r r e l a t e d with i n h i b i t i o n o f t e n s i o n g e n e r a t i o n and r e l a x a t i o n o f t o n i c t e n s i o n , i n the r e s p e c t i v e t i s s u e s . In c o n t r a s t , 8Br-cAMP a c t i v a t e d s o l u b l e and p a r t i c u l a t e PKA i n r a t vas deferens and s o l u b l e PKA in bovine coronary a r t e r y without s i g n i f i c a n t l y a f f e c t i n g t e n s i o n i n e i t h e r smooth muscle p r e p a r a t i o n . These r e s u l t s demonstrate that a c t i v a t i o n of PKA by non-relaxant analogs may occur to an equal or g r e a t e r extent as that induced by r e l a x a n t n u c l e o t i d e s . 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