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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  degree  this  thesis  in  at the University of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department  this or  thesis by  for scholarly  his  publication of this thesis  or  her  of  for  an  purposes  may be  representatives.  7%^-^^^<^^^-  advanced  I further agree that permission for extensive  It  is  granted  by the head  understood  that  for financial gain shall not be allowed without  The University of British Columbia Vancouver, Canada  DE-6 (2/88)  requirements  I agree that the Library shall make it  permission.  Department of  the  /c^/g^^y  of  my  copying  or  my written  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  i s generally  (cAMP) mediates  held  that  smooth muscle  adenosine 3',5'-cyclic monophosphate  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. 6  After 30 minutes of incubation, /V ,2'-0-dibutyryl-cAMP (dBu-cAMP) (1 - 100 fM) inhibited phenylephrine (PE)-induced tension generation in the  r a t vas deferens  also  in a dose-dependent manner. This  activated the soluble  fraction  analog  (10 juM)  of PKA but did not activate the  particulate f r a c t i o n kinase. In contrast, 8-bromo-cAMP (8Br-cAMP) (10 100 /JM) d i d not have any s i g n i f i c a n t effect on i n h i b i t i o n of PE-induced tension a f t e r 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 s i g n i f i c a n t l y  activated  only  after 30 minutes of exposure to the analog. In  the bovine  coronary  artery,  cAMP  (10 -  100 /iM) relaxed  potassium-depolarized helical s t r i p s and s i g n i f i c a n t l y activated soluble PKA in a dose-dependent manner. dBu-cAMP (10 - 100 JJM) affected neither tension nor soluble PKA a c t i v i t y . 8Br-cAMP (10 - 100 /iM) d i d not affect the coronary artery tension but did activate soluble PKA. Both prior  smooth muscle  preparations  to the determination  artifactual  assay  results.  of PKA  were homogenized with activity  As a further  in order  precaution,  charcoal  to minimize  extracellularly  associated cAMP and analogs were also washed from bovine coronary artery  iii strips  after  assessment  the  i n c u b a t i o n p e r i o d . These  o f PKA a c t i v i t y  The r e s u l t s of  correlation  deferens not  of  the  between  the  allowed  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 tension  and k i n a s e  activation  c o n t r a c t i o n or r e l a x a t i o n  support  controls  hypothesis  that  of  PKA  studies and  for  tissues.  demonstrate  inhibition  of  o f bovine c o r o n a r y a r t e r y . the  kinase  is  a valid  responsible  a  lack  rat  vas  This  does  for  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 m u s c l e . While the mechanism by which exogenous cAMP and s p e c i f i c  analogs  i n some smooth muscle p r e p a r a t i o n s remains u n c l e a r , that  PKA  functional  activation effect.  is  not  necessarily  it  required  induce r e l a x a t i o n can be for  suggested the  final  iv TABLE OF CONTENTS  Page ABSTRACT  ii  LIST OF TABLES  vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS ACKNOWLEDGMENTS INTRODUCTION  viii ix 1  A. cAMP and Smooth Muscle Relaxation B. Biochemical Features of PKA C. Putative Mechanisms of Action Of PKA in Smooth Muscle D. Measurement of PKA Activity in Intact Smooth Muscle Preparations OBJECTIVES OF THE PRESENT INVESTIGATION  1 6 7 11 14  MATERIALS AND METHODS MATERIALS  15  METHODS 1. Separation of PKA Isozymes from Bovine Coronary Artery by DEAE-cellulose Anion Exchange Chromatography 2. Tissue Preparation and Tension Studies of Rat Vas Deferens and Bovine Coronary Artery  16 16 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 b. PKA Assay  19 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 D e t e r m i n a t i o n o f Optimal PKA Assay a.  PKA Isozyme S e p a r a t i o n Coronary A r t e r y  b.  Charcoal Analogs  Conditions  P r o f i l e of  for  Bovine  A d s o r p t i o n and Washout o f cAMP  2. cAMP Analog-Induced I n h i b i t i o n o f Rat Vas Deferens C o n t r a c t i o n 3. PKA A c t i v i t y Deferens  i n cAMP-Treated Rat Vas  4. Time Course o f PKA A c t i v a t i o n Deferens by 8Br-cAMP  i n Rat Vas  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 T e n s i o n and PKA A c t i v i t y  6.  Relaxation Analogs  7.  PKA A c t i v i t y i n cAMP- and A n a l o g - T r e a t e d Coronary A r t e r y  o f Bovine Coronary A r t e r y by cAMP and  8. S p e c i f i c i t y o f the PKA Assay Protein Kinase A c t i v i t y DISCUSSION SUMMARY REFERENCES  for  Bovine  cAMP-Dependent  vi L I S T 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 R a t i o  c  53  L I S T 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  L I S T OF ABBREVIATIONS  8Br-cAMP  8-bromo-cAMP  Ca^ i  intracellular calcium  cAMP  adenosine 3',5'-cyclic monophosphate  dBu-cAMP  A/ ,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  +  6  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 t o extend my a p p r e c i a t i o n t o my s u p e r v i s o r , Diamond,  Ph.D.,  assistance I  scholarly  example,  and  financial  Alan  o f my t h e s i s Mitchell,  and  committee, Vladimir  Drs. Gail Palaty,  Bellward, for  their  c r i t i c i s m and s u g g e s t i o n s .  Thanks  the  t h e members  MacLeod,  constructive  teach  h i s guidance,  d u r i n g t h e course o f my degree work.  thank  Kathleen  for  D r . Jack  t o Mr. Yong-jiang  laboratory  techniques  Hei f o r generously  t o me and f o r h e l p i n g  taking  t h e time  to  me i n u n d e r s t a n d i n g  work. Finally,  Yong-jiang  thanks  go t o my c o l l e a g u e s  H e i , and t h e members  h e l p i n g t o put fun i n t o l a b work.  of  John L a n g l a n d s ,  D r . K. MacLeod's  Ashwin  Patel,  laboratory  for  1 INTRODUCTION  Cyclic adenosine 3',5'-monophosphate (cAMP) is generally believed to play an important role in smooth muscle relaxation. Since this role was f i r s t suggested (Sutherland and Rail, 1960), the cyclic nucleotide has been extensively studied in a host of tissues and species, resulting in  a large  dependent  body of evidence  correlations with  indicating temporal smooth  muscle  and concentration  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-PKArelaxation 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 aphosphoryl  group  at  the 3'  pyrophosphate. Sutherland stimulated  presence  and  5'  positions  and  release  of  et al. (1968) suggested that the hormonally-  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 f u l f i l l e d 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).  extension, these compounds should induce the same biochemical such  as  PKA  activation,  in  vitro  and  in  vivo  as  By  changes,  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., In keeping with the hypothesis, isoproterenol (0.05 /iM) and  1986).  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  inhibited myometrial contractions by cAMP from 5.1  (± 0.5)  example, isoproterenol 50%  and  pmol/mg protein to 8.4  significantly (± 0.7)  (2  nM)  increased  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 muscle  relaxation  cAMP increases can  but  i t is not  certain  be  correlated i f the  with  presence  smooth 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 #M)  (1-10  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) i t s e l f 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 l i p i d soluble analog proved to be the more potent relaxant in the latter case, as would be expected i f penetration of l i p i d 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. not  capable  of  inhibiting  (1978) reported that 8Br-cAMP (10 (M) was 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 l i p i d 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  compounds, the ineffectiveness  comparable  solubilities  of 8Br-cAMP  of the two  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  i t s relatively  low  lipid  solubility  (octanol-water  coefficient = 0.0011, Nakatsu and Diamond, 1989) apparently did not prove  to be limiting  factors  arteries (Napoli et a/.,  in the relaxation  of bovine  coronary  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 i t s 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 effect with kinase activity.  functional  6  B. Biochemical  As  reviewed  by Flockhart  Features of PKA  and Corbin  (1982) and Edelman et  al.  (1987), PKA holoenzyme exists as a ternary structure with two regulatory subunits and two c a t a l y t i c subunits. cAMP binds with a high a f f i n i t y (Kj = 10 nM) to the regulatory subunits, inducing a change in the holoenzyme such that subunits  the a f f i n i t y  of the regulatory  4  decreases  10 -fold.  reversible dissociation  This  leads  of c a t a l y t i c  subunits to PKA  f o r the c a t a l y t i c activation  by the  monomers from regulatory  dimers,  simply described as:  R2C2 (inactive)  The ATP,  +  4cAMP  free c a t a l y t i c subunit  — T R2CAMP4  +  2C (active)  transfers the -y-phosphoryl group from  +  in the presence of Mg^ , to the serine , preferably, or threonine  residues of s p e c i f i c proteins. The mechanism of t h i s reaction probably involves  an ordered  substrate followed and  ADP  sequential  binding  of ATP and then  the protein  by the sequential release of phosphorylated product  (Whitehouse  et  al.,  1983). When  intracellular  decrease, cAMP dissociates from the regulatory  subunits  cAMP  levels  and c a t a l y t i c  a c t i v i t y i s eliminated by reassociation of the constituent subunits of the  holoenzyme. The domain on the regulatory  with the c a t a l y t i c subunit  subunit  which interacts  i s similar to that of substrates except that  phosphorylation  probably  pseudosubstrate  (Taylor, 1989). An endogenous, s p e c i f i c  protein  does not take  place  and, as such,  (Walsh i n h i b i t o r ) and a synthetic peptide  PKA  i t is a inhibitor  (PKI), based on the  amino acid sequence 5-22 of the i n h i b i t o r , 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 I I , and can be separated isoforms  by  DEAE-cellulose  differ  in their  anion  exchange  physical  and  chromatography.  functional  These  properties  as  determined by the nature of the regulatory subunits. Differences include amino  acid  composition,  tissue  distribution,  susceptibility  autophosphorylation and affinity for cAMP (Hofmann et al.,  to  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 i s 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 Ca  2+  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  reduction of the Several  sensitivity  theories  substantiated  .  have  2+  proposed  2+  but  myosin  none  light  have  chain  been  fully  kinase  uptake into the sarcoplasmic  (MLCK)  reticulum  influx across the plasma membrane through voltage-  operated channels, and enhanced Ca MLCK has  and/or the 2+  include  phosphorylation, increased Ca  (Ca i)  of the contractile apparatus to C a i .  been  These  (SR), reduced Ca  2+  of free intracellular calcium  a reduced  2+  affinity  efflux (Fig. 1). for i t s activators, calcium  and  calmodulin, when i t is phosphorylated by PKA. This leads to inhibition of myosin light chain phosphorylation activity of actomyosin activity may  and, thereby, decreased ATPase  (Adelstein et a7.,  1982). Inhibition of MLCK  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 2+  has been associated with processes which can achieve lowering of C a i . PKA  phosphorylated proteins of, and enhanced Ca  2+  sequestration  bovine pulmonary artery SR vesicles in vitro (Raeymaekers et al.,  into, 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  Smooth Muscle. (R)  induces  form,  Mechanisms of Action of  Hormonal s t i m u l a t i o n  in  the  membrane bound enzyme then c a t a b o l i z e d induce  with  (3)  the  (X),  or  (4)  sarcoplasmic o c c u r as w e l l  activation  catalyzes  of  the  the  efflux  Ca  of  channel  2 +  resultant  as  (6)  chain  of  (SR)  the  kinase.  by  2 +  (5)  Ca  receptors  (G) to  its  cyclase  o f ATP t o  inhibition of  (1) (2)  in C a  a  adenylate  of  active  (AC).  This  cAMP which  is  (PDE) o r binds and a c t i v a t e s PKA.  by  (VOC),  increase  activation reticulum  Ca  Relaxation  plasmalemmal  G protein  conversion  by p h o s p h o d i e s t e r a s e s  voltage-operated  myosin l i g h t  specific  the c o n v e r s i o n o f a s t i m u l a t o r y  resulting  PKA may  of  PKA in the  2 +  the  2 +  activation efflux  ATPase.  of  a Na -K +  by a N a - C a +  Ca  activation  of  2 +  uptake  2 +  a Ca  i n h i b i t i o n of Ca -calmodulin 2 +  a plasmalemmal  2 +  +  ATPase  exchanger into  the  ATPase may  (CAM) b i n d i n g  to  10  11  enhanced  subsequent  mesenteric  artery  dependent  upon  caffeine-induced et  (Itoh  release  al.,  of  contractions  1982). Since  intracellular  of  such  guinea pig  contractions are  stores  calcium, PKA  of  a c t i v a t i o n by the cAMP-elevating agent was believed to mediate loading of C a  2+  into the SR.  Plasma potential  membrane-associated  sites  phosphorylated  f o r PKA  proteins  proteins  are  regulation  and diminution  in sarcolemmal  inside-out  aorta and these vesicles demonstrated enhanced ATPase a c t i v i t y an  increased  also  Ca  implicated of C a  vesicles  2+  2+  as  i . PKA  from rat +  uptake and Na -K  +  (Brockbank and England, 1980). This was interpreted as  extrusion  Potentiated  removal  plasmalemmal  Ca -Mg  2+  of C a  of 2+  Ca  2+  2+  by an associated from  the  smooth  ATPase has also been  +  Na -Ca muscle  2+  exchanger. cell  by  a  proposed as a device by  which PKA can mediate relaxation (Kattenburg and Daniel, 1984).  D. Measurement of PKA A c t i v i t y in Intact Smooth Muscle Preparations  Studies which actually measured hormonal relaxation  of intact  smooth muscle preparations  number. Among these studies, S i l v e r et al. association relaxation.  between  activation of PKA during  kinase  activation  The dose-dependent  increase  are not extensive in  (1982, 1984) found a strong and  bovine  in type  coronary  artery  II PKA a c t i v i t y  isoproterenol and adenosine demonstrated a s i g n i f i c a n t c o r r e l a t i o n 0.93)  with  reduction  by (R >  in potassium-induced coronary artery tension. In  other s t u d i e s , t h i s 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 f o r s k o l i n  (1 /zM)  12  was used in addition to PGE], the PKA activation was synergistic but the tissue  still  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  estimations of PKA activity in tissues  absence led to falsely high treated  with  cAMP-elevating  agents. The control  objective  of the present study was to apply techniques to  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 relaxation. accurately that  The r e s u l t s assessed  PKA a c t i v a t i o n  result artery.  in  in  a lack  the  of  study  analog-treated  by a n a l o g s ,  reduction of  Such  of  tension  suggest  that  intact  PKA a c t i v i t y  tissues  such as 8Br-cAMP, does in rat  vas  c o r r e l a t i o n between  deferens the  two  can  be  and  demonstrate  not  necessarily  o r bovine events  coronary  calls  q u e s t i o n the r o l e o f PKA i n cAMP-induced smooth muscle r e l a x a t i o n .  into  14  SUMMARY of OBJECTIVES o f the PRESENT INVESTIGATION  1. To determine the appropriate use of activated charcoal and tissue washout as controls for the determination of PKA activity in cAMPand 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,  (disodium  MO: salt),  activated 8Br-cAMP  neutralized (sodium  charcoal  salt),  (Pdt # C-5385), ATP  benzamidine, bovine serum  albumin, cAMP (sodium salt), dithiothreitol  (DTT), dBu-cAMP (sodium  s a l t ) , ethylenediamine tetraacetic acid (EDTA), isobutyl methyl xanthine (IBMX),  Kemptide,  6  /V -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 32  3  Fair Lawn, NJ. [-y P]ATP, [ H]cAMP and ACS  scintillation  Co.,  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 DEAECellulose 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  dissected free  of  frozen  bovine  coronary  loosely adhering adipose,  artery,  connective  previously 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.  conducted at 4°C.  All steps in this chromatographic procedure were  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), KH P0 2  4  (1.19), MgS0 (1.19), CaCl 4  2  (1.27), NaCl (118), NaHC0  3  (25), and d-glucose (11.12). The baths were warmed by water jackets to 37°C and bubbled with 5% CO2 in 0 which maintained the pH at about 7.4. 2  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, mBucAMP 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 [H]cAMP from Bovine Coronary Artery. J  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 0 for two hours, at which time they were exposed 2  to 10 /zM [H]cAMP (100,000 cpm/mL, 4.5 /zCi//zmol). After 30 minutes, a 3  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 [H]cAMP incubation 3  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 H radioactivity. 3  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 volumes for vas deferens,  buffer  (20  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%),  separate  a  detergent  used  to  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  consisted  of potassium phosphate buffer  (20 mM, pH 6.8),  buffer  magnesium  acetate (10 mM), IBMX (0.5 mM), NaF (10 mM), Kemptide (71 /iM), and [732  P]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  duplicate  and blanks  at 30"C. All samples were tested in  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 dehydrated by immersion in ethanol  (95%)  samples. The papers were 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 reduction  of  free  equilibrium of the PKA isozymes and 2) the  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 coronary artery, respectively,  of rat vas deferens  and bovine  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/. hi stones as the assay substrate resin.  The determination  (1982) using  and a DEAE-Sephacel anion exchange  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  reassociation of type II during the assay procedure (Corbin et  the 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 quickfreezing. 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 JIM).  27  T  1  10  20  1  30  FRACTION  1  40  r  50  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 [H]cAMP for 30 3  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  [H]cAMP from Bovine Coronary Artery Helical 3  Strips. Bovine coronary artery was incubated in [H]cAMP and washed for 3  10 x 1 minutes and 2 x 5 Methods. Points ( • )  minutes in ice-cold buffer as described in  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  60  A  CD  <  40 LL. O  20 H  0 J i  r  0  2  4  6  8  10 12 14 16 18 2 0  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. number of experiments.  Values in parentheses indicate the  32  CONTROL 8Br-cAMP 0.4  0.3  A (5) (5)  0.2 A (11)  0.1  0.0  (14)  A  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 i f 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  demonstrated  a  10 to  distinct  100/zM 8Br-cAMP  dose-response  (Fig.  7).  relationship  dBu-cAMP 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  CONCENTRATION (M)  10~  3  41  reduced PE (1/xM)-induced tension by more than 87% of control while 8BrcAMP (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 8BrcAMP 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. from control.  (*)  indicates values significantly different  45  CONTROL V7\  dBu-cAMP  •  8Br-cAMP  0.4 -,  Soluble PKA  Particulate  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 37 C with 10 uH 8  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 minutes  and  freeze-clamped  homogenized with ( • )  at  the  indicated  times.  (10/JM)  for  Tissue  30  was  or without ( O ) charcoal ( 1 0 mg/mL), centrifuged  and the soluble fraction was assayed for PKA activity. Values represent the mean ± SEM 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 1 1 . Effect of cAMP and Analogs on Bovine Coronary Artery Tension and PKA Activity. Bovine coronary artery helical s t r i p s 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 tension increase  as described  reduction  in Methods. Points  of 3-5 experiments. Bars  in soluble  PKA a c t i v i t y  ratio  ( • ) indicate the mean  indicate  ± SEM over  the mean percent control  of 3-5  experiments. The control PKA a c t i v i t y r a t i o f o r samples treated with 10 and 30 /zM cAMP/analog was 0.10 (± 0.004) and that f o r treatments with 100 jzM was 0.08 (± 0.011). (*) indicates values s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , p < 0.05.  51  12  300  9 -  -  250  6 -  -  200  3 -  -  150  o  —h  0 12  10  30  8Br-cAMP  O  X  o o  100 300 * T  9  100  6 -  O  - 250  TJ  - 200  > O  3 -  -  150  0  -  100  > —! <  LxJ  10  30  o  100  12  300  9 6 3 0 10  30  100  CONCENTRATION  (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  depolarized  bovine  coronary  artery  studies, changes tension  were  in potassium-  compared  to the  activation of PKA. The relaxant compound, cAMP, significantly activated the soluble fraction of the kinase at a l l 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).  activation  Higher  of PKA with  concentrations  of cAMP  a 2.30- and 2.25-fold  produced  greater  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  concentrations relaxation,  to approximately  the same  of cAMP. Finally, not only  but i t did not significantly  degree  as  equivalent  did dBu-cAMP not induce 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  indicate  artery  tension  (Fig. 11). These  results  activation of PKA in both vascular and non-vascular without necessarily inducing relaxation.  that  tissues may occur  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 /iM  0.10 (± 0.014)  0.16 (± 0.003)*  0.17 (± 0.014)*  30 /xM  0.11 (± 0.003)  0.24 (± 0.009)*  0.23 (± 0.022)*  100 /iM  0.08 (± 0.008)  0.20 (± 0.009)*  0.18 (± 0.029)*  a  a  b  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 ( p m o l P O , Cn O O  o o o  i  _J_  Cn O  o  N) O O O  rO Cn O O  o o o  incorp./mg/min) o o o  rO O O O  O O O  O O O  cn o o o  _1_  CD  o o o —1  CD  o >  > TJ  o >  r  + o  >  +  I  TJ  TJ  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, 8BrcAMP 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 analogtreated 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 (Corbin et al.,  isozymes  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., This  study extended  these results  to  a time-course  1991).  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 i f 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  charcoal  in  the  homogenization  1 minute time point without  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 [H]cAMP from bovine coronary artery strips followed an 3  exponential decline with respect to time and, based on this result, a standard washout procedure ( 5 x 1 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 K for activation of PKA by 8Br-cAMP a  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 K for activation approximately equivalent a  to that of cAMP (Meyer and Miller, 1974). The charcoal concentrations which bind 8Br-cAMP were presumed to  be more than sufficient  neutralize the less potent compounds i f  to  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, equilibrium of  associated  even at 4"C, to a shift  and dissociated  in the  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  important to note that  homogenates.  Nevertheless,  charcoal can prevent a false  it  is  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 tissue. This  activity ratio which existed  suggests that the  degree to which  in the  relaxation  intact  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  contractions did not appear to result from a deficiency particular activated  isozyme or the  compartment of  PKA.  The  kinase in both the soluble and  deferens  in affecting a  analog significantly  particulate fractions of  vas deferens homogenates. These fractions contained a mixture of type I and  II and  exclusively  (Hei et  type II, respectively  al.,  1991). The  particulate fraction of bovine coronary artery was not examined for  PKA  activity and, therefore, i t 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 rat vas  be argued that the heterogeneity of cell types in intact  deferens and  bovine coronary artery  precludes the  that the PKA activity determined in the tissues was was  actually that which  present in smooth muscle c e l l s . This leads to the possibility that  the dissociation between activation of PKA may  conclusion  actually  be  irrelevant  since the  PKA  and  relaxation  by 8Br-cAMP  activity determined in  the  assay had  had  its source in non-smooth muscle c e l l s . This argument is  probably  not  applicable  in  this  case  since  smooth  muscle  constitute a large fraction of the mass of these tissues, PKA to exist in smooth muscle c e l l s , and  cells  is known  i t is improbable that the analogs  would preferentially partition into non-smooth muscle c e l l s . The  PKA  activity measured i s , therefore, most likely a reasonable reflection of the kinase activation in smooth muscle c e l l s .  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  induced relaxation of vascular and non-vascular activation  and subsequent protein  entrenched  hypothesis  smooth muscle by PKA  phosphorylation  (Hardman, 1984; Silver  hormonally-  is a fairly  well  et a l . , 1982, 1984;  Kukovetz, et a l . , 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 vasopressininduced 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 K of cyclic nucleotide analogs for the in vitro activation a  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 K values than cAMP for activation of PKG. While the K for PKG a  a  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, i f 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 K  a  values  mentioned above. The conversion of the relaxant analogs to a variety of metabolites by e x t r a c e l l u l a r and i n t r a c e l l u l a r degradative enzymes i s an important consideration  i n the analysis  of t h e i r  hydrolyzed by a deacylase within c e l l s  relaxant e f f e c t s . dBu-cAMP i s (O'Neill  et al.,  1975) and i t s  monobutyryl derivative i s believed to be i t 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 f o r the PKA activation seen i n dBu-cAMP treated r a t vas deferens, the degree of relaxation seen with dBu-cAMP could only be partly  accounted  f o r by the actions  of the monobutyryl  form. The  metabolite was a r e l a t i v e l y weak i n h i b i t o r of vas deferens contractions in comparison to i t s parent compound. It i s possible that mBu-cAMP was less able to penetrate the plasma membrane of r a t vas deferens smooth muscle c e l l s  than  was dBu-cAMP due to i t s single  butyryl group and  resulting lower 1 i p o p h i l i c i t y . This potential to penetrate membranes was considered  an  important  factor  i n the inotropic  potencies  of ffi-  substituted c y c l i c nucleotide analogs i n the guinea pig heart (Kawada et al.,  1989) but was noted as not being  cyclic (Francis  nucleotide-induced et  al.,  relaxation  1988). Even  a factor of  i n the correlation of  porcine  coronary  i f mBu-cAMP did not penetrate  arteries the vas  deferens as well as dBu-cAMP, i t doubled the a c t i v i t y r a t i o of PKA from control  values, as did dBu-cAMP, but had markedly i n f e r i o r effects on  tension  reduction. This  suggests  that  i n h i b i t i o n by dBu-cAMP i s not e n t i r e l y  the mechanism  of contraction  related to PKA a c t i v a t i o n . 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 dBucAMP. Susceptibility of cAMP to enzymatic catabolism, for example, to phosphodiesterase-catalyzed hydrolysis, is well established (Francis et al.,  1988)  and has  been  phosphodiesterase-resistant  the  basis  analogs  for  the  selection  in examining the effects  of more 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  conductance  muscle  plasma membrane and resulting  through membrane-spanning channels.  in  ion  Evidence exists  for  direct effects such as these by cAMP. For instance, increases  changes  kinase-independent  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 muscle.  for the  relaxation of vascular and non-vascular smooth  69 SUMMARY  The r e s u l t s  of  this  investigation  r a t i o o f cAMP- o r a n a l o g - t r e a t e d be  determined  process  of  without  the  assay.  exogenous c y c l i c analog  load  spurious  intact  charcoal  and t i s s u e  i n the  extracellular  reduce the a c t i v i t y  r a t i o o f PKA.  during was  washout  space.  that  the  the  used was  tension  and bovine caused  coronary a r t e r y  by cAMP and a n a l o g s .  s o l u b l e f r a c t i o n o f r a t vas deferens of  bovine  generation  coronary  and r e l a x a t i o n  In c o n t r a s t , deferens  Activated  tonic  were  tension  in  a c t i v a t i o n of  or greater  suggests t h a t to  either  with  tension,  charcoal  by dBu-cAMP  g e n e r a l l y held hypothesis PKA a c t i v a t i o n .  in  tension  respective  tissues.  PKA i n r a t  preparation.  thereby  the  of  placing  vas  significantly These  PKA by n o n - r e l a x a n t analogs  relaxation,  in  fraction  results  may o c c u r  extent as t h a t induced by r e l a x a n t n u c l e o t i d e s .  muscle  to  vas  changes  a c t i v a t i o n o f PKA by exogenous cAMP o r analogs  smooth  the  r a t i o of rat  and p a r t i c u l a t e  muscle  free  appeared  inhibition  i n the  can  reduce  and by cAMP i n the s o l u b l e  soluble  smooth  adsorb  compared t o  PKA a c t i v a t i o n  correlated  8Br-cAMP a c t i v a t e d  demonstrate t h a t  related  of  tissue  to  and s o l u b l e PKA i n bovine coronary a r t e r y without  affecting  an equal  artery  activity  homogenization  used t o  Using t h e s e c o n t r o l s , changes i n the PKA a c t i v i t y deferens  PKA  smooth muscle p r e p a r a t i o n s  activation  Activated  nucleotides  indicate  doubt  to  This  may not  be  upon  the  t h a t cAMP-associated r e l a x a t i o n i s mediated by  70 REFERENCES Adelstein, R. 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