UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effects of carbachol on cAMP and protein kinase A activity in rat ventricles Zhang, Jack Zhen 1994

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

Item Metadata

Download

Media
831-ubc_1994-0329.pdf [ 3.22MB ]
Metadata
JSON: 831-1.0087347.json
JSON-LD: 831-1.0087347-ld.json
RDF/XML (Pretty): 831-1.0087347-rdf.xml
RDF/JSON: 831-1.0087347-rdf.json
Turtle: 831-1.0087347-turtle.txt
N-Triples: 831-1.0087347-rdf-ntriples.txt
Original Record: 831-1.0087347-source.json
Full Text
831-1.0087347-fulltext.txt
Citation
831-1.0087347.ris

Full Text

THE EFFECTS OF CARBACHOL ON cAMP AND PROTEIN KINASE A ACTIVITY IN RAT VENTRICLES by JACK ZHEN ZHANG B.M., Sun-Yat Sen University of Medical Sciences_, i 9 S o M.Sc. (Pharm), Sun-Yat Sen University of Medical Sciences^ 19 & 3 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 Faculty of the Pharmaceutical Sciences We accept this thesis as conforming to the/eqDjred_standa'rd THE UNIVERSITY OF BRITISH COLUMBIA April 1994 © Jack Zhen Zhang, 1994 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. 1 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. (Signature) Department of The University of British Columbia Vancouver, Canada Date H&I ~Q~ 9<y DE-6 (2/88) ABSTRACT It has been shown that (CCH) antagonizes the positive inotropic response to forskolin (FSK) in rabbit ventricles without decreasing total cAMP levels elevated by FSK. Further reports proposed that only cAMP (Cyclic AMP) in the particulate fraction of myocardium determines the inotropic state and Ca 2 + transients of the cell in the presence of cAMP elevating agents. In this investigation, we studied the effects of CCH on compartmental cAMP and PKA (protein kinase A) activity changed by cAMP-generating agents in rat ventricles. The elevation of particulate cAMP content was correlated with the LVP (left ventricle pressure) increased by isoproterenol (ISO) (10-7 M), and FSK (10-6 M) (r=0.90). Although PGE1 (prostaglandin E-\, 3x10-5 M) increased the total cAMP content and soluble PKA activity, it neither significantly affected the particulate cAMP content and PKA activity nor the LVP. Pre-perfusion with CCH (3x10-6 M) for 1 min, in addition to antagonizing the LVP elevated by ISO (122.8 ± 12.4 to 60.3 ± 8.4 mm Hg), decreased the total and particulate cAMP (26.8 ± 1.3 to 18.2 ± 1.6 and 16.4 ± 1.2 to 12.1 ± 0.8 pmol/mg protein respectively), lowered the soluble PKA ratio (0.60 ± 0.02 to 0.42 ± 0.03) and increased the percentage of particulate PKA (19.46 ±1.15 to 26.22 ± 0.54 %). CCH had no effect on either total and particulate cAMP levels or soluble and particulate PKA activity in the presence of FSK although the positive inotropic response to FSK was abolished by CCH (93.1 ±8.1 to 54.2 ± 8.5 mm Hg). The results support the hypothesis that particulate cAMP and PKA may determine the inotropic state of myocardium after cAMP-generating agent stimulation. However, the ability of CCH to antagonize the positive inotropic effect of FSK does not appear to be associated with a reduction in particulate cAMP or PKA activity. i i TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS x ACKNOWLEDGMENTS xii DEDICATION xiii 1. INTRODUCTION 1 1.1 Overview 1 1.2 p-adrenergic receptor (P-ARs) activities 2 1.3 p-ARs in heart 3 1.4. p-AR structure and ligand binding 3 1.5 Interactions of p-ARs with G-proteins 4 1.6 Mechanisms of the p-ARs 5 1.6.1 cAMP-dependent pathway 5 1.6.2 cAMP-independent pathway 7 1.6.3 Compartmentation of cAMP 8 1.7 The interaction between PKA and cAMP 9 1.7.1 Biochemical characteristics and activation of PKA 9 1.7.2 The regulation of PKA by cAMP 10 1.8 Muscarinic receptor (MR) activities 12 1.9 MRs in Heart 13 1.10 MR structure and ligand binding 13 1.11 Interaction of MRs and G-proteins 14 1.12 Mechanisms of muscarinic receptors agonists(MRAs) 14 in Page 1.12.1 cAMP-dependent pathway 15 1.12.2 cAMP-independent pathway 17 1.12.2.1 Promotion of phosphoinositide turnover 17 1.12.2.2. Regulation of K+ and Ca 2 + channels 18 1.12.2.3. Elevation of cGMP levels 18 1.12.2.4. Activation of phosphatase 19 1.13 Summary of objectives of this study 20 2. MATERIALS AND METHODS 21 2.1 Materials 21 2.2 Main Equipment 22 2.3 Prereparation of Solutions 23 2.3.1 Physiologic solution for heart perfusion 23 2.3.2 Drug solutions 23 2.3.3 Solutions for the cAMP assay 23 2.3.3.1 Buffer for tissue homogenization 23 2.3.3.2 Water-saturated diethyl ether 24 2.3.4 Solutions for the PKA assay 24 2.3.4.1 Buffer for extraction of PKA 25 2.3.4.2 Reaction medium for PKA assay 24 2.3.4.3 Stop solution for PKA assay 25 2.4 Methods 25 2.4.1 Procedure of heart perfusion 25 2.4.2 Extraction procedure for cAMP determination. 26 2.4.3 PKA Assay 27 2.4.3.1 Extraction procedure for PKA estimation. 28 IV Page 2.4.3.2 PKA estimation 29 2.5 Statistical Analysis 29 3. RESULTS 31 3.1 Effects of ISO, FSK and PGEi on LVP 31 3.2 Effects of ISO, FSK and PGEi on total and particulate cAMP 31 3.3 Effects of ISO, FSK and PGEi on soluble and particulate PKA 32 3.4 Relation between compartments of cAMP and inotropic responses to ISO, FSK and PGEi. 32 3.5 The effects of CCH pre-perfusion on the response of ventricles to ISO perfusion. 33 3.6 Effects of CCH pre-perfusion on the responses of ventricles to FSK. 33 3.7 Effects of CCH on the response of ventricles to ISO and FSK. 34 4. DISCUSSION 70 4.1 The effects of ISO, PGE-) and FSK on inotropic responses, cAMP levels and PKA activities of rat hearts. 70 4.2 The effects of pre-perfusion with CCH on positive inotropic response, cAMP content and PKA activity induced by ISO 73 Page 3 The effects of pre-perfusion with CCH on positive inotropic response, cAMP content and PKA activity induced by FSK 74 4 The effects of CCH on positive inotropic response, cAMP content and PKA activity induced by ISO 76 5 Clinical meaning of our experiments 78 SUMMARY AND CONCLUSIONS 80 REFERENCES LIST OF TABLES Table Page 1 Effects and selective antagonists of MRs. 12 2 Relation between cardiac muscle tension and intracellular cAMP content in presence of MRAs and cAMP-elevating agents. 16 3 Effects of ISO, FSK and PGE-| on LVP, cAMP and PKA. 41 Vll LIST OF FIGURES Figure Page 1 Tracings showing the effects of ISO, FSK and PGEi on left ventricular pressure (LVP) of rat hearts. 35 2 Effects of ISO, FSK and PGEi on left ventricular pressure (LVP) of rat hearts. 37 3 Effects of ISO, FSK and PGEi on total and particulate cAMP of rat ventricles 39 4 Correlation between changes in left ventricular pressure (LVP) and cAMP content of rat ventricles in response to cAMP-elevating agents. 42 5 Soluble and particulate PKA activity in rat ventricles treated with different agents. 44 6 Tracings showing the effects of CCH pre-perfusion on the positive inotropic response to ISO. 46 7 The effects of CCH pre-perfusion on the positive inotropic response of rat ventricles to ISO. 48 8 The effects of CCH pre-perfusion on particulate and total cAMP increased by ISO. 50 9 The effects of CCH pre-perfusion on soluble and particulate PKA activity of rat ventricles in the presence of ISO. 52 10 Tracings showing the effects of CCH pre-perfusion on the positive inotropic response to FSK. 54 11 The effects of CCH pre-perfusion on the positive inotropic response of rat ventricles to FSK. 56 12 The effects of CCH pre-perfusion on particulate and total cAMP in the presence of FSK. 58 Vlll Page 13 The effects of CCH pre-perfusion on soluble and particulate PKA activity in the presence of FSK. 60 14 Tracing showing the effects of CCH on the positive inotropic responses to ISO or FSK. 62 15 The effects of CCH on the positive inotropic response of rat ventricles to ISO or FSK 64 16 The effects of CCH on particulate and total cAMP induced by ISO or FSK. 66 17 The effects of CCH on soluble and particulate PKA activity in the presence or absence of FSK or ISO. 68 IX LIST OF ABBREVIATIONS ACh p-AR p-ARA [7-32P] A T p 4-AP AC C-protein Ca2+ cAMP cAMP[1 2 5 l ] CCH cGMP cpm DAG DEAE-cellulose dpm DTT EDTA ER FSK Gpa Gj G i a Go G s Acetylcholine p-adrenergic receptor P adrenergic receptor agonist ATP where the 7-phosphate group is radioactive 4-Aminopyridine Adenylyl cyclase A muscle protein bound to myosin Calcium Adenosine 3',5' cyclic monophosphate 125lodine-labeledcAMP Carbachol Guanosine 3',5' cyclic monophosphate Counts per min Diacylglycerol O-(Diethylaminoethyl) cellulose, an anion exchanger Disintegrations per min Dithiothreitol Ethylenediamine tetra acetic acid Endoplasmic reticulum Forskolin (Adenylyl cyclase activator) Beta and gamma subunits of G-protein Inhibitory G-protein a-subunit of the inhibitory G-protein Other G-protein Stimulatory G-protein GTP IBMX IP3 ISO kDa LVP mM mAChR MR MRA NaF PDE PGE-i PKA PKC PLC pmol PPM Rl, Rll SEM SR TCA TX-100 Guanosine 5'-0-(3-thiotriphosphate) Isobutylmethyl xanthine (PDE inhibitor) lnositol-1,4,5-trisphosphate Isoproterenol Kilodalton Left ventricular pressure Millimolar Gene muscarinic receptor Muscarinic receptor Muscarinic receptor agonist Sodium fluoride Cyclic nucleotide phosphodiesterase Prostaglandin E-| cyclic AMP-dependent protein kinase Protein kinase C Phospholipase C picomole Protein phosphatase inhibitor 1 Regulatory subunits of cyclic AMP-dependent protein Standard error of the mean Sarcoplasmic reticulum Trichloroacetic acid Triton X-100, a surface-active agent XI ACKNOWLEDGMENTS I would like to express my appreciation to my supervisor, Dr. Kathleen M. MacLeod for her continuous support and scholarly guidance in my graduate studies. I would like to thank my thesis committee members, Dr. Helen M. Burt, Dr. Jack Diamond, Dr. John H. McNeill and Dr. Glen Tibbits for their constructive criticism and assistance. I am very grateful to my colleagues in laboratory. They are: Ms. Slavica Bosnjak, Ms. Lynn Weber, Ms. Yi Jia Bi, Dr. Abhijit Ray and Mr. Ali Tabatabaei for all their help and friendship. I would like to acknowledge the financial support of the Stroke and Heart Foundation of B.C. & Yukon. Finally, I would like to thank all the faculty members, staff and graduate students for making my M.Sc. training most enjoyable. Xll To Hattie, my wife, and my parents for their care and support X l l l INTRODUCTION 1.1 Overview It is well known that the sympathetic and parasympathetic branches of the autonomic nervous system modulate cardiac activities by activating adrenergic or muscarinic receptors (AR and MR) respectively. To cause positive inotropic and chronotropic responses in hearts, p receptors (p-ARs) activated by adrenergic hormones or p-adrenergic receptor agonists (p-ARAs) interact with a stimulatory guanine nucleotide-binding regulatory protein (Gs) which mediates the stimulation of adenylyl cyclase (AC). Cyclic AMP (cAMP) is synthesized by activated AC at the cytoplasmic surface of the plasma membrane. The intracellular receptor for cAMP is cyclic AMP-dependent protein kinase (PKA). When activated by cAMP, the kinase phosphorylates a variety of cellular proteins and regulates their activities. Although the antagonism by muscarinic receptor agonists (MRAs) of the effects of p-ARs involves the blockade of the cAMP-dependent pathway of p-ARs, the activation of K+ channels and phosphatase, the reduction of Ca 2 + currents and the elevation of intracellular cGMP by MR may also contribute to the MR antagonism. Cyclic AMP is an important regulator of phosphorylation of protein in cells and is related to the positive inotropic and chronotropic response to P-ARAs. It has been demonstrated that only the increase of cAMP in the particulate compartment of myocytes is correlated with the increase of Ca2 + transient and positive inotropic responses to p-ARAs or the AC activator, forskolin (FSK) (Hohl and Li, 1991). PKA is the major intracellular mediator of cAMP action in cardiac tissue. It has been reported that PKA is compartmentalized into soluble and particulate fractions and the activation of the holoenzyme by cAMP elevation results in translocation of the dissociated catalytic subunit from the particulate to soluble 1 portion of the cell and phosphorylation of cytoplasmic or other proteins (Flockhart et al., 1982; Edelman et al., 1987). The objectives of the present study were to investigate 1) the effects of MRAs on cAMP content in different compartments of the heart increased by various cAMP elevating agents, and 2) the effects of MRAs on PKA activity and translocation. In the following pages, I will briefly review (3-AR and MR distribution, activation and the consequent intracellular responses, focusing on cAMP and PKA activation. 1.2 p -adrenergic receptor (P -AR) activities p-ARs are found in various cardiac tissues, blood vessels, in the bronchi and intestine, and in the central nervous system. Lands and his coworkers (1967) first subdivided p-ARs into p-|-AR in the heart and P2-AR in the smooth muscles because noradrenaline and epinephrine are equally potent in the heart but epinephrine is about 10-30 fold more potent than noradrenaline in bronchial smooth muscle. Recently, the results of DNA sequencing of cloned receptors have led to the postulation of a p3-receptor which is about tenfold more sensitive to noradrenaline than to epinephrine and is relatively resistant to blockade by propranolol (Emorine et al., 1989). So far this concept has not been followed by functional data but it has been suggested to mediate responses to the catecholamine at sites such as adipose tissue. Stimulation of post synaptic p-ARs causes a variety of physiological and pharmacological effects and these actions have been summarized extensively (Stiles et al., 1984; Benovic et al., 1986; Gilman, 1987). 2 1.3. p -ARs in heart Based on the results derived from radioligand-binding studies, the coexistence of (3-|-and p2-ARs in one organ from different species of animals has been demonstrated (Carlsson, 1972; Daly et al., 1979; Brodde, 1989). This coexistence has been revealed in the hearts of rat, cats, dogs and rabbits (Stiles et al., 1984; Brodde, 1987, 1988). As a general rule, p-|-ARs predominate in ventricles, constituting 80% or more of total p-AR population. P2-ARS are present in larger amounts in atria than in ventricles, comprising 30-40% of the total p-ARs (Brodde, 1988). Although both p-j-and P2-AR stimulation cause positive inotropic effects in vivo and in vitro (Levine, et al., 1989; Lemoine et al., 1988), p-|-AR stimulation caused maximal positive inotropic effects, while p£-AR stimulation only resulted in submaximal positive inotropic effects. In the studies of positive chronotropic effects of p-ARs, isoproterenol (ISO)-induced tachycardia is mediated by both p-j-and P2-AR activation to about the same degree (Brodde et al., 1988; Motomura et al., 1990). 1.4. p -ARs structure and ligand binding According to the data obtained by hydropathy analysis, the primary sequences of p-ARs are characterized by seven hydrophobic transmembrane stretches of 20-25 amino acids, which are surrounded by eight hydrophilic regions of variable length. (Strader et al., 1989). Many data based on oligonucleotide-directed mutagenesis have shown that Asp113 residue in helix 3 is conserved among all of the G protein-linked receptors (see section 1.5) (Strader et al., 1988; Main, et al., 1985) and the carboxylate side chain of the residue is the location anchored by the amino group of agonist or antagonist (Findlay et al., 1986). In addition, hydrogen bonding between the catechol hydroxyl groups on the ligand 3 and Ser residues on helix 5, as well as hydrophobic interactions between residues on helix 6 and the aromatic portion of the ligand, appear to be involved in the ligand-p receptor binding (Strader et al., 1988). 1.5 Interactions of$-ARs with G-proteins Using cloning and sequencing techniques, at least 3 types of G-proteins have been identified in cardiovascular tissue on the basis of molecular weight of a-subunits of G-proteins (Robinshaw et al., 1989). 1. Gs (the stimulatory G-protein) which consists of 2 subtypes of oc-subunits. Their molecular weights are 45 and 52 kDa respectively, and Gsa is a substrate for ADP-ribosylation by cholera toxin. The function of Gs protein is related to the activation of p-AR and mediates stimulation of AC, production of cAMP and stimulation of voltage-dependent calcium channels (Brown 1990). 2. Gj (the inhibitory G-protein) which consists of three types of oc-subunit namely Gja-| to GJCC3. Gia-j and Gjcc3 possess a molecular weight of 40 kDa, while Gjoc2 is 41 kDa. G j a is the substrate for ADP-ribosylation by pertussis toxin and mediates the hormonal-inhibition of AC and activation of potassium channels mainly (McClue et al., 1992; Birnbaumeret al., 1990). 3. Go (the other G protein) which consists of two types of cc-subunits having the same molecular weight of 39 kDa. Similar to Gja> G o a is a substrate for ADP-ribosylation by pertussis toxin and its main function may be related to inhibition of the pacemaker current channel in the heart (Brown and Birnbaumer, 1990). The interaction between the activated p-AR and Gs has been shown to involve an intracellular region of the p-AR that is postulated to form an amphophilic cc-helix. It is confirmed that regions at both the amino and carboxyl ends of the putative third intracellular loop of the protein are critical for the coupling of the p-ARs to G s (Dixon 1987). In addition, a region of eight amino acids connecting the 4 carboxyl terminus of the fifth transmembrane helix and the third intracellular loop was determined to be absolutely required for AC activation in studies using mutant receptor expressed in oocytes (O'Dowd et al., 1988). The mechanism of p-AR agonist (p-ARAs) stimulation of AC has been explained in detail (Fleming et al., 1987; Birnbaumer et al., 1990). The Gs protein undergoes a regulatory cycle after combining with p-ARAs, which consists of binding of GTP to the Ga-subunit of the Gapy heterotrimeric complex, dissociation of the inhibitory Gp-y complex and interaction of Ga-GTP with the effectors or AC, leading to their activation (Birnbaumer 1990). Termination of the cycle occurs with the hydrolysis of GTP to GDP and reassociation of Ga-GDP with Gp-y to form the inactive complex. These features of the GTP regulatory cycle are similar for all of the signal-transducing G proteins, including the MR-coupled G-proteins mentioned below (Fleming et al., 1992). 1.6. Mechanisms of the fi-ARs 1.6.1. cAMP-dependent pathway Stimulation of both p-j-and P2-ARS by catecholamines results in the activation of Gsa which leads to the activation of AC. The latter causes accumulation of cAMP which serves as a second messenger in cell signal transduction pathways. Based on the extensive studies on this topic (Gilman 1987; Ikezono et al., 1987), the four important criteria for a second messenger role of cAMP (Sutherland et al. 1968, Tsien et al., 1977; Scholz et al., 1980) in the positive inotropic effects of p-ARAs on human hearts seem to be fulfilled: a) in broken cell preparations, AC responds to the same agents that are effective in intact tissues, and in both experimental systems, the order of potency of agonists is the same and the effectiveness of competitive antagonists is similar, b) the increase of cAMP 5 precedes the physiological responses, c) inhibition of breakdown of cAMP by PDE inhibitor enhances the effects of agonists on both cAMP and force of contraction; and d) dibutyryl cAMP mimics the effects of drugs or hormones that activate AC. Cyclic AMP synthesized from ATP by AC, in turn, activates PKA which phosphorylates protein in myocytes (Brown 1987). Some of the PKA interactions and the associated cellular responses include: 1) phosphorylation of troponin I, which decreases Ca 2 + affinity of the troponin complex and probably leads to an increase in the rate of relaxation of the heart induced by epinephrine (Adelstein et al., 1980). 2) phosphorylation of myofibrillar C-protein which is a constituent of the thick filament and located in the area of thick and thin filament overlap (Hartzell et al., 1982). It has been demonstrated that the phosphorylation of C-protein is well correlated with relaxation parameters measured in hearts responding to adrenergic agents (Hartzell et al., 1984) 3) phosphorylation of phospholamban. The protein regulates the activity of Ca2+-ATPase in sarcoplasmic reticulum membranes resulting in an increased uptake of calcium during diastole, contributing both to an increased rate of relaxation and an increased release of calcium during subsequent systole after phosphorylation (Ahmad, etal., 1989). 4) phosphorylation of voltage-sensitive Ca2+ channels for increased Ca2 + influx across the sarcolemma (Yatani et al., 1989). 5) phosphorylation and activation of inhibitor-1 of type 1 phosphatase. This action decreases the activity of type 1 phosphatase and in turn lowers the dephosphorylation of activated proteins (Neumann et al., 1991). 6) phosphorylation and activation of phosphorylase b kinase which in turn activates phosphorylase b, resulting in glycogen breakdown (Hayes et al., 1981; Insel, etal., 1988). 6 7) it has been shown that a CI" channel in the cardiac SR membrane is activated by the catalytic subunit of PKA (Kawanmo et al., 1992). It is postulated that the opening of CI" channels may possibly contribute to the neutralization of the potential across the SR membrane generated by Ca 2 + uptake through the calcium pump during relaxation. The above effects mainly facilitate the Ca2 + transient. With respect to contraction, some of the above effects facilitate the influx of Ca2 + , activate substantial contraction, trigger SR Ca 2 + release and serve the loading of Ca 2 + to SR for the next contraction. With respect to relaxation, some of the above effects accelerate the efflux of Ca 2 + from the cytoplasm via Na+/Ca2+ exchange or reaccumulation by the SR. This dynamic balance of intracellular Ca 2 + and the phosphorylation of proteins affected by B-agonists in the heart leads to the increase in contractility and rate of relaxation. 1.6.2. cAMP-independent pathway It has recently been shown that B-ARAs, through activation of the Gs protein, can directly activate cardiac Ca 2 + and Na+ channels. This process does not require the formation of cAMP (Bimbaumer 1990; Hartzell et al., 1991). Both cAMP-dependent and independent effects of B-ARAs result in large increases in the amplitude of the Ca 2 + transient since Ca 2 + influx into myocytes can serve to trigger SR Ca 2 + release and also contribute to the Ca 2 + loading of the SR for the next contraction. These changes induced by p-ARAs lead to the increase in contractility, conduction velocity and automaticity of hearts. 1.6.3. Compartmentation of cAMP Another interesting hypothesis for the mechanisms of B-ARAs is compartmentation of cAMP. Since Corbin (1977) introduced the hypothesis of 7 compartmentalized hormonal control, the evidence supporting this theory has been shown as follows. Firstly, England and Shahid (1987) have demonstrated that a low concentration of FSK can elevate cAMP levels to a much greater extent than does ISO; however the effects of FSK on PKA, phosphorylase a content and contractile force are less than ISO. Therefore, the authors suggested that cAMP is compartmentalized in perfused rat heart, and that much of the cAMP produced by FSK does not contribute to contraction. Secondly, the effects of cAMP-elevating agents on contractility of hearts is related to the cAMP in a specific pool, the particulate fraction, only. For example, PGE-j has no effect on contractility of hearts because it only increase the cAMP content in the soluble fraction instead of the particulate fraction (Hayes et al., 1980). Another similar example is prenalterol, a partial (3-ARA, which increased cAMP content to the same degree as did ISO in the particulate fraction of hearts at equieffective concentrations regarding the positive inotropic effect, although prenalterol produced a much less increase in total cAMP content than did ISO (Aass et al., 1988). Lastly, in isolated ventricular myocytes, the cAMP content increased in the particulate fraction is correlated to the free Ca 2 + transient promoted by FSK, IBMX and ISO, instead of total cAMP levels (Hohl and Li, 1992). In summary, according to this theory, different cAMP-elevating agents activate a specific pool of AC and lead to cAMP accumulation in the pool; only the cAMP in this particulate pool can activate the PKA and induce a positive inotropic response effectively according to this theory. Since PKA is a major receptor for cAMP in heart tissue, the finding of a compartmentation of cAMP suggested the existence of compartments of PKA as well (see below). In general, two methods were used to separate the particulate fraction of myocardium for cAMP and PKA measurement (see section 1.7 as well): A) homogenization of cardiac tissue first, then centrifugation (>20,000 x g ) for separation of particulate fraction (Brunton et al., 1981; Aass et al., 1988). B) 8 Separation of myocytes first, then dissolution of the sarcolemmal membrane of cells by a certain concentration of digitonin in order to release and remove the soluble cAMP, followed by rapid sedimentation of particulate fraction of myocyte ( Hohl and Li, 1992). It is worth noting that no correlation has yet been made between the centrifugation speed and subcellular fraction or structures (Hartzell, 1988). Hence, there may be variations between the results when different conditions and species of animal are used. 1.7. The interaction between PKA and cAMP As is the theory of compartmentation of cAMP which better explains the mechanisms of p-ARAs, the concept of subcompartments of PKA, and its activation and translocation is attractive as well. 1.7.1 Biochemical characteristics and activation of PKA. Since PKA was discovered in 1968, the role of PKA as the major intracellular mediator of cAMP action in mammalian systems has been widely accepted (Krebs et al., 1972). The inactive PKA holoenzyme is composed of two catalytic subunits and a dimer of regulatory subunits. Two major isozymic forms, type I and type II, exist according to their sequential chromatographic elution from DEAE-cellulose (Corbin et al., 1975). This distinction is conferred by two major classes of regulatory subunits called Rl (49 kDa) and Rll (55 kDa) through using SDS PAGE. These isomers differ in physical and functional properties as determined by the features of the regulatory subunits, such as: 1) the inhibitory domains of each regulatory subunit share little homology, except for an amino acid sequence that has been identified as a pseudosubstrate site in Rl and an autophosphorylation site in Rll (Kemp et al., 1989). This difference gives each regulatory subunit unique biochemical properties. 2) The two regulatory subunit 9 cAMP-binding sites have distinct properties. For example, cAMP dissociates 10 times more rapidly from site 2 than from site 1 (Rannels et al. 1981). 3) Type I isozyme tends to dissociate in the presence of high salt (e.g. 0.5 M), and type II will reassociate again in the absence of the salt (Corbin et al., 1975). PKA isozyme distribution varies from tissue to tissue in a given species and is species-dependent. For example, rat heart contains mostly the type I isozyme and bovine heart contains mostly the type II isozyme. There is evidence that PKA is compartmentalized in soluble and particulate fractions of hearts as is cAMP (section 1.6.1.) and these become accessible to each other in response to selected stimuli (Corbin et al., 1975; Bode et al., 1988). The activation of the holoenzyme by cAMP elevation results in translocation of the dissociated catalytic subunit from the particulate to soluble portion of the cell where it catalyzes phosphorylation of cytoplasmic or other proteins (Flockhart et al., 1982; Edelman, 1987). Differential activation of PKA compartmentalized between soluble and particulate fraction has been suggested to explain the divergent actions between ISO, FSK and PGE-| (Hayes et al., 1979; Keely, et al., 1979) as described below. 1.7.2. The regulation of PKA by cAMP A large percentage (about 50%) of the total PKA of heart tissue is associated with the particulate fraction of homogenates. The holoenzyme is attached to the cellular membrane (particulate fraction) by its regulatory subunit. As soon as cAMP, increased by ISO, activates PKA by binding to the regulatory subunits, it triggers a conformational change that allows the catalytic subunits to dissociate and phosphorylate protein substrate (Beavo et al., 1974; Ogreid et al., 1982). After the dissociation, the regulatory subunit remains bound to particulate material, but the catalytic subunit transfers to the soluble fraction. The cAMP-elevating agent, ISO, causes the accumulation of cAMP in both soluble and 10 particulate compartments and the accumulation of cAMP in the particulate compartment leads to the local activation of PKA and translocation of the catalytic subunits of particulate PKA to the soluble fraction. These events correlate with p-AR responses: activation of phospholamban, phosphorylase, Ca 2 + channels and increased inotropic state (see section 1.6.1.) because the catalytic subunits transferred from the particulate compartment may phosphorylate their specific targets or substrates in the soluble compartment or substrates close to the particulate compartment in cells (Hayes et a!., 1980). By contrast, another cAMP-elevating agent, PGE-| only activated the cAMP and PKA in the soluble compartment, which may not have the same access as particulate PKA to its substrates as mentioned above, and then caused none of the responses generally associated with intracellular cAMP elevated by ISO (Keely et al., 1980). On the other hand, activated PKA contributes to the lowering of cAMP in intact cells through phosphorylation of a cAMP-specific phosphodiesterase, seemingly like a negative feedback control (Gettys et al., 1988; MacPhee et al., 1987). When the free cAMP concentration is lowered sufficiently, the catalytic subunits of PKA reassociate with the regulatory subunit dimer, resulting in the release of bound cAMP and a return of the holoenzyme to the cAMP-free conformation. PKA is a major intracellular mediator of cAMP actions and possesses the feature of specific compartmentalized activation induced by cAMP-elevating agents. The study of the effect of MRAs on PKA activity changes in different compartments of the heart is one of research objectives of the research in this thesis. n 1.8. Muscarinic receptor (MRs) activities Muscarinic receptor actions are widely distributed in vertebrate organs and the diversity of MRs was first found in the late 1970's. Because the MR antagonist, pirenzepine, has been reported to bind selectively to MRs in brain and has much lower affinity for cardiac MRs, the predominant cardiac receptor has been classified as belonging to the M2 subtype. On the other hand, the MRs in brain are known as the M-| subtype of MR (Hammer et al., 1980). With the rapid development of molecular biological cloning techniques, 5 subtypes of MR (m AChR) have been revealed by cloning of complementary DNAs (cDNA) that encode MRs (Bonner 1987; Peralta et al., 1987). Pharmacologically distinguishable forms of the m AChR occur in different tissues and have been classified into M-j, M2 and M3 according to their amino acid sequences, structures and differences in apparent affinity for antagonists (Hammer et al., 1986; Birdsall and Hulme, 1983). The m4 and m5 species are not sufficiently characterized to be assigned to pharmacological M4 or M5 receptor subtypes (Bonner 1989). The effects and distribution of M-j, M2 and M3 subtypes are summarized in table 1. Table 1. Effects and selective antagonists of MRs. Receptor type Molecular sequence Organ/tissue Stimulation Selective antagonists Mi ml Neurons Excitation pirenzepine M2 m2 Heart Bradycardia Contractility I methoctramine M3 m3 Smooth muscle Contraction hexahydro-siladifenidol 12 1.9. MRs in Hearts Although M2 receptors predominate in the myocardium (Nathanson, 1987; Loffelholz and Papano, 1985), their distribution in atria and ventricles varies. It has been indicated that the density of ventricular MR expressed as percent of that of atria approximates 20% or less in the hearts of rats, guinea pigs, rabbit and dogs (Fields et al., 1978; Wei, 1978). In addition to the differences in distribution, the responses of atria and ventricle to MR activation are varied as well. Acetylcholine and vagal stimulation inhibit supraventricular electrical and mechanical activity markedly; however, they exert negative inotropic effects on the ventricle that are demonstrable only when the ventricle has been stimulated by sympathetic stimulation or by p-ARs agonists (Levy et al., 1983). 1.10. MR structure and ligand binding Hydropathy analysis indicated that all subtypes of MRs contain seven transmembrane segments and belongs to the class of hormone receptors that are structurally similar to rhodopsin (Dratz et al.,1983). The amino terminus, which contains potential sites for glycosylation, is located extracellularly; the carboxyl terminus is located intracellular^. Peptide mapping studies of 3H propylbenzilylcholine affinity-alkylated receptor from rat forebrain (Wheatley et al., 1988) indicated that an aspartate residue located in the second transmembrane segment is the most likely candidate for ion pairing with the quaternary ammonium group of muscarinic ligands. Furthermore, it has been shown that a muscarinic antagonist binds to Asp 105 (equivalent to Asp 113 of (3-AR) of the M-| receptor as well (Cheung et al., 1989). 13 1.11. Interaction of MRs and G-proteins Among the 3 G-proteins mentioned above (section 1.5), both Gj and G0 proteins are present in cardiovascular tissue and selectively activated by M2 receptor, but only Gj has been demonstrated to inhibit AC (Seamon, et al., 1982; Jakobs, et al., 1983; Ross and Berstein 1993) and directly couple cell membrane receptor ion channels (Christie et al., 1988). After binding MRAs, the pertussis sensitive Gj protein is activated and possesses the same regulatory cycle as Gs does (Robinshaw et al., 1988). The dissociated Gja and (3y subunits exerts the effects described below. G0 has been shown by Florio and Stemweis (1985) to regulate MR affinity for agonists in brain. Recently, Pang and Stemweis (1990) have identified a new G-protein, Gq, which is a 42-kDa protein and is absent of the site for ADP-ribosylation by bacterial toxin. Later, it was demonstrated that Gq purified from bovine brain and liver cells is seletively activated by human M-| receptor purified from cells called Sf9 (Parker et al., 1991, Smrcka et al., 1991, Ross and Berstein, 1993) and its activation led to stimulation of phospholipase C (PLC) which induced the promotion of phosphoinositide turnover. More details will be discussed in section 1.12.2.1. 1.12. Mechanism of muscarinic receptors agonists (MRAs) The physiological and biochemical consequences of muscarinic receptors are as yet somewhat fragmentary, however, two main pathways are currently appreciated: a cAMP-dependent pathway and a cAMP-independent pathway. They regulate phosphoinositide turnover, ion channels, phosphatase, cGMP, cAMP levels etc. 14 1.12.1 cAMP-dependent pathway This pathway for mediation of responses to MRAs is evoked by activation of M2 and M4 receptors. These receptors interact with Gj, resulting in inhibition of AC (Brown and Birnbaumer, 1990). When the activity of AC is inhibited, the content of the catalytic product, intracellular cAMP, declines, which results in decrease of cardiac contractility. This hypothesis is based on many demonstrations. Some researchers reported that stimulation of muscarinic receptors resulted in inhibition of AC activity in cardiac membrane vesicle preparations (Watanabe et al., 1978; Jakobs et al., 1979). In addition, muscarinic agonists have been shown to antagonize the tension and cAMP increased by ISO in rabbit papillary muscles (Inui et al., 1982). Later, Sorota (1985) demonstrated that the pertussis toxin-sensitive Gj was involved in the inhibition of AC caused by MRAs and this led to a reduction in the amount of cAMP generated by AC. However, this mechanism of MRs can not totally explain the inhibitory actions of MRs on positive inotropic drugs that act via increasing cAMP. Although muscarinic receptor agonists have been demonstrated to reduce the cAMP level increased by p-AR stimulation, the decreased tension produced by muscarinic receptor agonists, for example CCH, in presence of p-ARs is greater than can be explained by the reduction in total cAMP level alone (Endoh et. al., 1985; MacLeod, 1985). As well as the above reports, antagonism by CCH of positive inotropic response to FSK (AC stimulator) was not accompanied by lowering of the associated increase of cAMP in left atria and right ventricular papillary muscles of rabbit (MacLeod and Diamond, 1986; Ray and MacLeod, 1992 and 1993). Moreover, Ach reduced the positive inotropic effect induced by the phosphodiesterase inhibitor, isobutylmethyxanthine, without affecting the accumulation of cAMP induced by this agent (Biegon et al, 1980). The relation 15 between cardiac inotropic responses to MAs and intracellular cAMP content are shown in Table 2. Table 2. Relations between cardiac muscle tension and intracellular cAMP content in presence of MRAs and cAMP-elevating agents. ("-": no change) Effects of MR stimulation Tension cAMP In the presence of i i i or - (3 -ARAs i i - FSK (AC activator) U - IBMX(PDE inhibitor) Based on the above results and the hypothesis of compartmentation of cAMP (see section 1.6.3.), it was postulated that only the AC and/or cAMP linked to the inotropic response is susceptible to inhibition by MAs, and this inhibition is masked by the FSK-induced activation of all the AC units (MacLeod 1986; Hartzell 1988). Therefore, it is not clear if this tension-cAMP dissociation phenomenon in muscarinic antagonism of positive inotropic responses to cAMP-generating agents results from a total cAMP measurement which is not sensitive enough to determine cAMP content change in a certain compartment, or if it occurs via a cAMP-independent process (see the following section). Thus, investigating whether MRAs have effects on the cAMP content of specific compartments in the presence or absence of cAMP-generating agents is another research objective here. 16 1.12.2. cAMP-independent pathway: The finding that cAMP levels are not correlated with contractile force antagonized by MARs lead to the consideration of the cAMP-independent pathway although the hypothesis of compartmentation of cAMP and whether whole cell cAMP measurements reflect important changes in subcompartments of cAMP should not be ignored. This pathway involves IP3, cGMP, direct effects of G-protein on ion channels and activation of phosphatase mainly. 1.12.2.1 Promotion of phosphoinositide turnover MRs are coupled to phosphoinositide turnover through Gj or Gq, which are responsible for stimulation of phospholipase C activity (Berridge 1987, Robinshow et al., 1989, Ross and Berstein, 1993). After the activation of MRs, the immediate result is hydrolysis of phosphatidylinositol polyphosphates to form inositol polyphosphates. Some of the inositol phosphate isomers, e.g. inositol-1,4,5-trisphosphate (IP3) cause release of intracellular Ca 2 + from stores in the endoplasmic reticulum (ER). Thus, these receptors mediate such Ca2+-dependent phenomena as contraction of muscles and secretion. The second product of the phospholipase C reaction, diacylglycerol (DAG), activates protein kinase C which, in turn, phosphorylates and activates sarcoplasmic Ca 2 + channels (Robinshow 1989). High concentrations of MRA, 10 \iM or higher, have been demonstrated to increase phosphoinositide turnover in the hearts (Brown 1986; Pappano 1990). It is suggested that this effect elicits the positive inotropic and chronotropic response to high concentration of MRAs in the hearts. While it seems likely that phosphoinositide turnover is one of the mechanisms of MRAs in hearts, there is some controversy over the resulting effects. For example, Movsesian et al. (1985) found no effect of IP3 on isolated SR of canine heart. Fabiato (1990) showed IPs-induced SR Ca 2 + release that was much smaller and slower than that induced by 17 Ca2+-induced Ca2+-release in skinned rat myoctes. These different conclusions may lead to another consideration in which IP3 may be important in the increase of Ca2+-sensitivity of Ca2+-induced Ca2+-release (Suarez-lsla et al., 1988). 1.12.2.2 Regulation of K+ and Ca2 + channels It has been demonstrated that M2 receptors are linked directly to potassium channels by means of a pertussis toxin-sensitive G-protein (Pffafinger et al., 1985). It is believed that activation of K+ channels which shortens the duration of the atrial action potential, indirectly reduces the influx of Ca2 + during excitation (TenEick, et al., 1976; Cerbai et al., 1988). Uncoupling of MRs from K+ channels, by using pertussis toxin, results in attenuation of the outward potassium current and the negative inotropic response induced by MRA (Pffafinger et al., 1985; Urquhart et al. 1991; Ray and MacLeod 1992) Therefore these actions could account for the direct negative inotropic and chronotropic effects of MRA in atrial myocardium. 1.12.2.3 Elevation of cGMP levels It has been shown that the negative inotropic responses to MR stimulation were associated with an increase in cGMP levels in atria and ventricles (Fink et al., 1976). Moreover, Watanabe and Besh (1975) and Wahler (1985, 1986) demonstrated that cGMP and its analogs were capable of inducing MR stimulation-like responses in ventricles with or without cAMP-generating agents. A few studies also have shown cGMP increased the hydrolysis of cAMP by activating a cGMP-stimulated phosphosdiesterase (Fischmeister and Hartzell 1986; Fischmeister and Hartzell, 1987). However, the hypothesis that cGMP is involved in the negative inotropic response of the heart to MRAs has been controversial in other studies. Some reports showed that Ach reduced contractile force without changing the cGMP 18 levels (Linden and Brooker, 1979; Diamond et al., 1977). Others have shown that sodium nitroprusside, a guanylyl cyclase activator, did not attenuate the atrial contraction in the absence or presence of various cAMP-elevating agents (Diamond, et al., 1977; Linden and Brooker, 1979). In addition, MacLeod and Diamond, (1986) suggested that elevation of cGMP levels may only be related to the negative inotropic response to MRAs in ventricles because a cGMP lowering agent, LY 83583, did not affect the negative inotropic response of rabbit left atria to CCH stimulation in the absence or presence of the AC stimulator, FSK, although LY 83583 abolished the CCH-induced increase in cGMP. More recently, in preliminary experiments from Diamond's lab, it has been shown that cGMP levels were not correlated with the negative inotropic response of myocytes to CCH in presence of ISO (MacDonnell et al., 1994 ). In summary, the controversial data above may result from different conditions, animal species and preparations. In addition, the functional consequences of cGMP elevated by MRAs appear to be arguable and need to be studied further. 1.12.2.4 Activation of phosphatase Opposite to the inhibition of phosphatase induced by p-ARAs (mentioned in section 1.6.1.), the activity of phosphatase could be increased by MRAs in intact heart (Neumann et al., 1991). These authors have shown that the MRA, acetylcholine, antagonized the activating effects of ISO and FSK on inhibitor-1, which inhibited type-1 phosphatase. In addition, Ach alone stimulated type-1 phosphatase activity. These effects would be expected to decrease the state of phosphorylation of various proteins, including phospholamban (Watanabe et al., 1989). 19 1.13. Summary of objectives of this study As mentioned above, cAMP and PKA in the particulate compartment seem to be most closely related to the inotropic response of myocardium to cAMP-elevating agents. When positive inotropic responses of ventricles to cAMP-elevating agents are antagonized by MRAs significantly, the accompanying increase in total cAMP levels is either not affected or is partially reduced (Watanabe and Besch, 1975; Keely et al., 1978; MacLeod, 1986;). The purpose of the present work was to test the hypothesis that MRAs inhibit the cAMP generation and PKA activation induced by cAMP-elevating agents in the particulate compartment linked to the contractile mechanism. In order to test this hypothesis, the objectives of present study were set up as follows: A) To observe the effects of cAMP-elevating agents, ISO, FSK and PGE-|, on contractile force of rat ventricles in Langendorff heart model. B) To determine the cAMP content and PKA activity in soluble and particulate compartments of rat ventricles in the presence or absence of the above cAMP- elevating agents. C) To determine the correlation between contractile force of ventricles and cAMP content and PKA activity in different compartments of cardiac tissues in the presence and absence of the above cAMP-elevating agents. D) To investigate the effects of the MRA, CCH, on cAMP content and PKA activity in soluble and particulate compartments of ventricle in the presence or absence of the above cAMP-elevating agents. 20 MATERIALS AND METHODS 2.1 MATERIALS The materials used in the study were purchased from the following sources: Amersham Canada Ltd.. Canada [7-32 P] ATP cAMP [1251] Assay kit (Code RPA 508) ACS scintillation fluid BDH Chemical Co.. Canada Calcium chloride dihydrate d-Glucose Diethyl ether Dimethyl sulphoxide Hydrochloride acid Magnesium chloride hexahydrate Potassium chloride Sodium bicarbonate Sodium chloride Trichloroacetic acid BIO-RAD Laboratories. Ontario. Canada Protein assay kit Sigma Chemical Co.. St.Louis. U.S.A. cAMP sodium salt 21 cGMP Dithiothreitol (DTT) EDTA Forskolin Isobutylmethylxanthine ISO hydrochloride Kemptide Phenylephrine hydrochloride Prostagladin E-j Sodium fluoride (NaF) Triton X-100 Tween 20 Ascorbic acid Fisher Scientific Co.. Fair Lawn. NJ. Phosphoric acid 2.2. MAIN EQUIPMENT Polygraph: Grass Model 7E. Infusion pump: Harvard Apparatus, Model 975. Peristaltic pump: (Holter® pump) Extracorporeal Med. Spec. Inc. Liquid scintillation counter: Beckman LS 6000 Series Liquid Scintillation System. 7-counter: Packard® Crystal Multi-Detector System. Beckman L8-60M ultracentrifuge and its Type 65 rotor. Wistar rats were purchased from the Animal Care Unit, University of British Columbia. 22 2.3. PREPARATION OF SOLUTIONS 2.3.1. Physiologic solution for heart perfusion A twenty times concentrated stock solution was prepared by dissolving sodium chloride 139.1 g, potassium chloride 4.473 g, calcium chloride dihydrate 5.879 g, and magnesium sulphate heptahydrate 5.915 g in 1000 ml of distilled water. On the day of the experiment, sodium hydrogen carbonate 8.368 g, d-glucose 7.2 g, potassium dihydrogen orthophosphate 1.308 g and sodium ascorbate 0.008 g were dissolved into about 3500 ml distilled water, 200 ml of the stock solution was added slowly into the solution which was being stirred continuously and the volume was adjusted to 4000 ml volume with distilled water. The perfusion solution contained (mmol/L): NaCI 119.0, KCI 3.0, CaCl2 2.0, MgSC>4 1.2, KH2PO4 2.4, NaHCC>3 24.9, glucose 10, sodium ascorbate 0.01, and was aerated with 95% 02/5% CO2 for 20 min, at 30 °C, pH 7.4, (Aass et. a!., 1988). 2.3.2 Drug solutions CCH and ISO were dissolved in distilled water. PGE-| and forskolin were dissolved in 90 % ethanol to obtain stock solutions of 10"2 M (stored in 200 ml aliquots at -70 °C) from which the required dilution was made using the perfusion solution on the day of the experiment. 2.3.3. Solutions for the cAMP assay 2.3.3.1. Buffer for tissue homogenization 500 ml of potassium phosphate buffer (50 mM pH 7.0) contained 4 mM EDTA, 20% saturated U2SO4 (100% saturation^.4 M) was prepared. On the day 23 of the experiment, cGMP and 5-AMP were added into 30 ml buffer (enough for 12 samples) to obtain a final concentration of 5x10"5 M and 10"4 M, respectively, and kept on ice. 2.3.3.2. Water saturated diethyl ether The water saturated diethyl ether was prepared by mixing distilled water and diethyl ether thoroughly in a ratio of 1:5 and was kept standing overnight. The upper layer of solution was used to remove TCA in cAMP extraction. 2.3.4. Solutions for the PKA assay 2.3.4.1. Buffer for extraction of PKA The same buffer used in the tissue homogenization of cAMP assay was used (see 2.3.3.1 section). On the day of the experiment, cGMP and 5-AMP were added into 20 ml buffer (enough for 12 samples) to obtain a final concentration of 5x10"5 M and 10~4 M respectively, and kept on ice. 2.3.4.2. Reaction medium for PKA assay The reaction buffer consisted of potassium phosphate buffer 20 mM, pH 6.80, IBMX 0.5 mM, NaF 10 mM, Kemptide 71 ^M, ATP 100 \iM, stored at -70 °C. On the day of the experiment, 0.36 ml of a stock solution of magnesium acetate (100 mM) and 7-32P ATP (the amount of 7-32P ATP added was calculated according to the radioactivity on the date of experiment) were added into 2.074 ml of the reaction buffer in a final volume of 2.564 ml adjusted with distilled water. The final concentrations of magnesium acetate and 7-32P ATP were 10 mM and 100 cpm/pmol) respectively. 24 2.3.4.3. Stop solution for PKA assay Concentrated phosphoric acid 100 ml was diluted to 20:l with distilled water, to obtain a final concentration of 0.5%. 2.4. METHODS 2.4.1. Procedure of heart perfusion Male Wistar albino rats (300-400 g), were deeply anesthetized with 6.5 % sodium pentobarbital (65 mg/kg). The chests were opened and hearts were removed quickly and placed into the ice-cold oxygenated perfusion buffer. Having been gently squeezed several times between fingers to remove the blood left in chambers of the heart, the hearts were attached via the aorta to a perfusion cannula. After an initial 3 min washout period, hearts were retrogradely perfused by a peristaltic pump at a rate of 10 ml/min without recirculation. The perfusion buffer was kept at 30 °C and aerated with 95% 02/5% CO2 continuously. Contractile force of hearts were determined by measuring left ventricle pressure (LVP) by inserting a polypropylene tube attached to a pressure transducer into the pulmonary vein, with the tube tip reaching the left ventricle. Hearts were paced at 240 beats/min, (2-7 V, 5 ms duration) and equilibrated for 20 min, after which LVP was recorded via the pressure transducer attached to a polygragh. Perfusate containing either no drug or testing reagents was then introduced into the aorta by a Harvard infusion pump at a rate of not more than 0.1 ml/min. At appropriate times, the hearts were frozen with clamps pre-cooled in liquid nitrogen. The great vessels and atria were trimmed away, and the ventricle stored at -70 °C until assayed for cAMP and PKA activity. There was no loss in enzyme activity or change in cAMP levels after 3 months storage. 25 To determine the influence of cAMP-elevating agents on LVP of rat hearts, tissues received perfusion buffer for different periods, corresponding to a 1.5 min perfusion with ISO (10~7 M) or 2 and 5 min perfusion with FSK (10"6 M) or 2 min perfusion with PGE-| (3X10"^ M). For experiments involving the effect of CCH on the positive inotropic responses to ISO or FSK, tissues were perfused according to one of the following schedules: 1) Hearts were perfused with buffer containing CCH (3x10"6 M) and ISO (10-7M)for1.5min. 2) Hearts were perfused with buffer containing CCH (3x10"6 M) and FSK (10"6 M)for2 or 5 min. 3) Hearts were pre-perfused with CCH (3x10"^ M) for 1 min before the hearts were perfused with either ISO (10"7 M) for 1.5 min or FSK (10-6 M) for 2 or 5 min. 2.4.2. Extraction procedure for cAMP determination. The extraction of cAMP from hearts was done as described by Aass et al. (1988). Briefly, approximately 40 mg of frozen ventricle were homogenized in 2.0 ml of ice-cold homogenization buffer using a Vari-Mix dental amalgam mixer as follows: A. Each tissue was put into a capsule containing an iron bar cooled in liquid-nitrogen for 10 sec. B. 1 ml ice-cold homogenization buffer was put in the container, and the tissue was homogenized for 10 sec at middle speed. The upper layer of the homogenate was transferred from the capsule to a polypropylene tube (10x70 mm). C. Another 1 ml of ice-cold homogenization buffer was added to the capsule, and the tissue was homogenized for 10 sec again. This was thoroughly combined with the homogenate from B in the above polypropylene tube. 26 D. A 0.2 ml aliquot of the homogenate was withdrawn and combined with 0.5 ml of 7% TCA for extraction of total cAMP in the homogenate. Another 0.2 ml homogenate was withdrawn for protein assay in step G (see the followings). E. A 1 ml aliquot of homogenate was immediately centrifuged at 100,000 x g, 4 °C for 10 min in a Type 65 rotor, Beckman L8-60M ultracentrifuge. The resulting supernatant fraction (containing soluble PKA and cAMP, the latter was not measured in this study) was separated from the pellet (containing particulate PKA and cAMP). The surface of the pellet was rinsed once with 1 ml homogenization buffer per tube, then the pellet was re-suspended in 1 ml of the buffer and 2.5 ml of 7 % TCA was added for extraction of particulate cAMP. F. After the addition of TCA, all tubes were centrifuged at 8700 x g, for 40 min in Sorvall RC 2-B centrifuge. TCA was removed by extraction of the supernatant four times with 5 volumes of water-saturated ether. The pellet was used for the protein assay in step G (see the following). Total and particulate cAMP levels in the homogenate were determined by using a radio-immunoassay kit obtained from Amersham Canada Ltd., Canada. Values are expressed as picomoles of cAMP per mg of protein. G. 0.2 ml or 1 ml of 1N NaOH were added into the 0.2 ml of homogenate which was withdrawn in step C and the pellet which was separated on step F respectively. All of the above were mixed well and kept for 24 hours in order to determine protein content by using the Biorad dye-binding assay. 2.4.3. PKA Assay Soluble and particulate PKA activity were estimated essentially as outlined by Giembycz and Diamond (1990). 27 2.4.3.1. Extraction procedure for PKA estimation. The procedure is similar to the extraction procedure for cAMP but some steps were modified. Approximately 40 mg of frozen ventricle were homogenized in 1.0 ml of ice-cold homogenization buffer using a Vari-Mix dental amalgam mixer as follows: A. Each tissue was put into a capsule containing an iron bar cooled in liquid-nitrogen for 10 sec. B. 0.5 ml ice-cold homogenization buffer was put in the container, and the tissue was homogenized for 10 sec at middle speed. Upper layer of the homogenate was transferred from the capsule to a 10 ml centrifuge tube used in Beckman L8-60M ultracentrifuge. C. Another 0.5 ml of ice-cold homogenization buffer was added to the capsule, and the tissue left was homogenized for 10 sec again. The homogenate was transferred from the capsule to the above centrifuge tube as well. D. The homogenate combined above was immediately centrifuged at 100,000 x g, 4 °C for 10 min in a Type 65 rotor, Beckman L8-60M ultracentrifuge. The resulting supernatant fraction (containing the soluble PKA) separated from the pellet (containing particulate PKA and cAMP). E. The supernatant was transferred from the centrifuge tube to a polypropylene tube (10x70 mm) and kept on ice for PKA activity estimation except a 0.2 ml aliquot of supernatant was withdrawn for protein assay in step G (see the following) F. The surface of the pellet was rinsed once with 1 ml homogenization buffer per tube, then the pellet was resuspended by being stirred for 30 min at 4 °C in 1 ml of the buffer, now supplemented with Triton X-100 (0.2 %), a detergent used to separate membrane-bound PKA from phospholipid bilayers. The re-suspended pellet was centrifuged at 100,000 x g, 4 °C for 10 min in a Type 65 rotor, Beckman L8-60M ultracentrifuge again. The resulting supernatant fraction was transferred 28 from the centrifuge tube to a polypropylene tube (10x70 mm) and kept in ice for estimation of PKA activity in the particulate fraction while the pellet was used for determination of protein content in next step. G. 0.2 ml or 1 ml of 1N NaOH were added into the 0.2 ml of supernatant which was withdrawn in step E and the pellet which was separated on step F respectively. All of the above were mixed thoroughly and kept for 24 hours in order to determine protein content by using Biorad dye-binding assay. 2.4.3.2. PKA estimation To initiate the assay, aliquots (25 |il) of soluble or particulate fractions were added to reaction buffer (65 nl) in the presence or absence of cAMP (10 |j,M). The mixture was incubated for 8 min at 30 °C. Blank tubes containing 25 JLLI homogenization buffer were included. The reaction of the samples was stopped by the spotting of 70 JLLI aliquots of the mixture onto phosphocellulose paper (P81) and immersion of the paper into 0.5 % phosphoric acid. The papers were washed in the acid four times for 5 min each time and placed into 2.5 ml ACS and counted by liquid scintillation counting. The activity of PKA was expressed as pmol of phosphate incorporated into Kemptide per mg of protein. The PKA activity of the soluble fraction is expressed as the ratio of activity in the absence of cAMP to that in the presence of 10 \xM cAMP. Total activity of the particulate PKA was determined in the presence of 10 \iM cAMP and is expressed as a percentage of the total protein kinase activity after corrections for dilution as [particulate activity (+cAMP)]:[particulate activity (+cAMP) + soluble activity (+cAMP)]. 2.5. Statistical Analysis The results of contractile force studies were compared by one way analysis of variance followd by Neuman Keul's multiple range test. Data obtained from 29 different treated groups were compared by two way analysis of variance followed by Neuman Keul's multiple range test.. A p value of less than or equal to 0.05 was used as the criterion for a significant difference. Values are presented as the mean ± the standard error of the mean (SEM). The relationship between values was assessed using linear regression analysis (Pearson's). The correlation coefficient was considered to be significantly different than 0 at a p < 0.05. 30 RESULTS 3.1..Effects of ISO, FSK and PGE-f on L VP Hayes et al. (1980) reported that ISO and PGE-) both caused the elevation of cAMP but only ISO increased LVP in rabbit, guinea pig and rat hearts. We have compared the effects of cAMP-elevating agents, ISO, FSK and PGE-] on LVP (Fig. 1, 2 and Table 3.). LVP was significantly increased after perfusion with 10~7 M ISO for 1.5 min or 10"6 M FSK for 2 min. Perfusion with the same dose of FSK for 5 min induced a much greater increase in LVP than that after 2 min perfusion with FSK. PGE-) (3x10"5) did not cause a positive inotropic response, as Hayes et al. (1980) reported. 3.2. Effects of ISO, FSK and PGEf on total and particulate cAMP Aass et al (1988) suggested that only the cAMP in the particulate fraction of the myocardium determines the inotropic state, and that this could explain the different effects of ISO and prenalterol (a partial (3 agonist) on inotropic state and cAMP levels in rat ventricles. In order to observe the effects of different cAMP-elevating agents on cAMP levels in rat ventricles, total and particulate cAMP levels were measured after perfusion with ISO, FSK and PGE-|. The total cAMP level was increased to two times the control level after ISO perfusion, and to almost 3 times control level after 5 min perfusion with FSK. Two min perfusion with FSK, and PGE-| also elevated total cAMP significantly as well. However, particulate cAMP levels were raised significantly only by the perfusion with ISO and with FSK for 2 and 5 min, and not by PGEi perfusion (Fig. 3 and Table 3). 31 3.3. Relation between compartments of cAMP and inotropic responses to ISO^SKandPGE-/. Changes in the cAMP levels were plotted as a function of positive inotropic response to ISO, FSK and PGEi in Fig. 4. It can be seen that the elevation of total cAMP by these agents varies considerably, and a consistent relation between total cellular cAMP content and positive inotropic responses to these agents is not readily apparent (left panel of Fig. 4). This is because ISO, FSK and PGE-| all increased total cAMP level, but PGE-( did not have any effect on the LVP of rat hearts. Furthermore, perfusion with FSK for 5 min increased total cAMP levels to a much greater extent than did ISO, but enhanced the LVP to the same level as ISO. However, when the particulate cAMP levels are plotted against the corresponding values of LVP in the presence of the cAMP-elevating agents, a linear relationship between them emerges (right panel of Fig. 4.). 3.4. Effects of ISO, FSK and PGEf on soluble and particulate PKA Corbin et al (1977) reported that as much as 50% of the cAMP and PKA activity in rabbit heart homogenates is associated with the particulate fraction. Perfusion of rabbit hearts with epinephrine led to an increase in the cAMP content of the particulate fraction with a concomitant translocation of the catalytic subunit of PKA from the particulate to the supernatant fraction. We have assessed the functional importance of soluble and particulate pools of PKA activity by perfusing the cAMP-elevating agents, ISO, FSK and PGE-j. Although perfusion of rat hearts with all these agents increased the activity ratio of soluble PKA (Table 3 and Fig. 5), only ISO and FSK caused a significant decrease in the percentage of PKA activity in the particulate fraction. PGE-) did not affect the particulate protein kinase activity even though it caused the activation of soluble PKA. 32 3.5..The effects of CCH pre-perfusion on the response of ventricles to ISO perfusion. The effects of CCH on the positive inotropic response, cAMP content and protein kinase activity changed by ISO are shown in Fig. 6 to 9. The CCH (3x10"6 M) was pre-perfused for 1 min, then hearts were perfused with ISO (10~7 M) for 1.5 min in the presence of same dose of CCH. CCH produced a significant decrease of LVP from the value of ISO perfusion group (from 122.81 ± 12.43 to 60.32 ± 8.41 mm Hg) (Fig 6 and 7). In addition to decreasing the PKA activity in soluble fraction and increasing the percentage of PKA activity in particulate fraction significantly compared with those of ISO perfusion group (Fig. 9), CCH reduced the total and particulate cAMP levels elevated by ISO significantly as well (Fig. 8). 3.6. Effects of CCH pre-perfusion on the responses of ventricles to FSK. To investigate the antagonizing effects of CCH on the ventricles in the presence of FSK, LVP was measured in hearts pre-perfused with CCH (3x10"6 M) for 1 min, and then perfused with FSK (10~6 M) for 2 min or 5 min in the presence of same dose of CCH. LVP in the presence of CCH declined to 60.2 ± 3.2 % and 44.6 ± 2.6 % of the LVP values increased by perfusion with FSK for 2 and 5 min alone, respectively (Fig. 10 and 11). However, CCH had no effect on either the total and particulate cAMP increased by FSK (Fig. 12), or on the PKA activity in soluble and particulate fractions (Fig. 13). 3.7 Effects of CCH on the response of ventricles to ISO and FSK The interaction of CCH and ISO or FSK in rat ventricles was also studied in hearts perfused with CCH (3x10"6 M) and ISO (10"7 M) for 1.5 min or with the 33 same dose of CCH and FSK (10-6 M) for 2 min. LVP in the presence CCH declined to 60.0 % and 23.3 % of the LVP increased by perfusion with ISO for 1.5 and FSK for 2 min alone respectively (Fig. 14 and 15). However, CCH had no significant effect on the total and particulate cAMP levels, or on the PKA activity in soluble and particulate fractions of myocardium (Fig. 16 and 17). 34 Fig. 1. Tracings showing the effects of ISO, FSK and PGE-j on left ventricular pressure (LVP) of rat hearts. Rat hearts were perfused with 1) vehicle buffer, 2) ISO (10"7 M) for 1.5 min, 3) FSK (10"6 M) for 2 min, 4) FSK (10"6 M) for 5 min, and 5) PGE-i (3x10"5M)for2min. 35 70 o-1-LVP (mm Hg) 70 T 0 I min. |—f LVP (mm Hg) 7 0 T 0-L ( 3 ) FSK 2 MIN. 1 min. LVP (mm Hg) PGEt 2 MIN. 1 min ( 2 ) [SO 1.5 MIN. 1 min. ( 4 ) FSK 5 MIN. 1 min. I—i 36 Fig. 2. Effects of ISO, FSK and PGE-| on left ventricular pressure (LVP) of rat hearts. LVP before and after perfusion with ISO (1Cr7 M) for 1.5 min, FSK (10-6 M) for 2 or 5 min, or PGE-| (3x10"5 M) for 2 min. Each bar represents the mean ± SEM of 6-8 rat hearts except PGE-| group (n=3). (*) Represents significantly different from the values before treatment. (#) Represents significantly different from the value of FSK 2 min group. 37 THE EFFECTS OF cAMP-ELEVATING AGENTS ON LEFT VENTRICULAR PRESSURE OF RAT HEARTS _^^  Ofl 6 J, E> C73 CO CU « 3 ^ 1—1 K E-i Z W > E-& H H -J 140 -i 130 -120 -110 -100 -90 -80 -70 -60 -50 -40 -30 -20 -10 -0 I i. I x >* N K: X I T 4 BEFORE TREATMENT AFTER 2I CONTROL t ^ ISOPROTERENOL FSK 2 MIN. B FSK 5 MIN. PGE1 38 Fig. 3. Effects of ISO, FSK and PGE-| on total and particulate cAMP of rat ventricles. Hearts were equilibrated for 20 min and then perfused with ISO (10~7 M) for 1.5 min, FSK (10"6 M) for 2 or 5 min, and PGE-) (3x10"5 M) for 2 min. Each bar represents the mean ± SEM of 6-8 rat hearts except PGE-j group (n=3). (*) Represents significantly different from control values. Left panel shows the total cAMP content. Right panel shows the particulate cAMP content. 39 OP Total cAMP (pmol /mg prote in) o o o 25 GO o i—i W 3 ^ 0 1 - r . 2: -d Q o ho 0 CrJ O ->• O O l O o> 0 -vl 0 H - t * H * Part icula te cAMP (pmol /mg prote in) Table 3. Effects of ISO, FSK and PGE-j on LVP, cAMP and PKA. Rat hearts were perfused for 20 min with control buffer, then with the indicated agents. Hearts were frozen and assayed as described under "Materials and Methods". Soluble PKA activity is expressed as a ratio of activity in the absence of cAMP to that in the presence of 10 ^ M cAMP. Particulate PKA activity is expressed as per cent of total PKA activity (soluble + particulate). Total and particulate cAMP are expressed as pmol/mg protein. Values are the mean ± of determinations from 6-8 rat hearts except PGE-j group (n=3). (*) Represents significantly different from the control values by unpaired t test analysis. (#) Represents significantly different from the FSK 2 min perfusion group. CONTROL ISO FSK PGE-) 2 MIN 5 MIN LVP 67.7±4.2 122.8±12.4* 93.1 ±8.1* 128.8±9.6*# 78.3±2.2 (mm Hg) Total cAMP 15.3±1.5 26.8±1.3* 21.2±1.9* 55.6±7.6*# 25.6+2.1* (pmol/mg protein) Part. cAMP 11.2±0.8 16.4±1.2* 14.1±0.5* 17.7±0.4*# 12.2±0.5 (pmol/mg protein) Sol. PKA 0.24±0.01 0.60±0.02* 0.62±0.01* 0.61 ±0.02* 0.45±0.02* (-cAMP:+cAMP) Part. PKA 35.25±0.96 19.46±1.15* 18.97±1.51* 20.25±0.45* 32.43±1.19 (% Total) 41 Fig. 4. Correlation between changes in left ventricular pressure (LVP) and cAMP content of rat ventricles in response to cAMP-elevating agents. (O) control group (O) perfusion with ISO for 1.5 min, (O) perfusion with PGE-| for 2 min, (O) perfusion with FSK for 2 min and (O) perfusion with FSK for 5 min. Points represent the mean ± SEM of 6-8 rat hearts except PGE-| group (n=3). Agonist-induced increases in LVP are shown as a function of total cAMP content (left panel) or particulate cAMP (right panel), r: correlation coefficient, obtained by using linear correlation analysis (Pearson's). 42 J L LVP vs total cAMP r=0.52 I I I I 0 10 20 30 40 50 60 Total cAMP (pmol /mg protein) 160 140 120 a 100 B 8 ^ 80 > -J 60 40 20 0 LVP vs particulate cAMP r=0.90 0 10 20 30 40 Par t icula te cAMP (pmol /mg protein) 43 Fig. 5. Soluble and particulate PKA activity in rat ventricles treated with different agents. The PKA activity ratio in the soluble fraction and the activity as a percentage of the total PKA in the particulate fraction were determined. See "Methods" for further details. Each bar represents the mean ± SEM, values in parentheses indicates the number of hearts. (*) Represents significantly different from the control group. 44 PARTICULATE PKA {% TOTAL) o m m ^ o st- m n o to m CNI o CN in *— o *— in 0. W 2| s * R 3 £ 3 I D *h *l * h (S) NIW z iaod ( 9 ) N W 9 MS J ( 9 ) N I « z xsi * l -(<{,) NM 9'T OSI (6) IOHINOO 1 0 T -1 <J> O 1 CO O 1 r^  0 1 CO O 1 m 0 1 ^ 0 1 fO 0 1 CN O 1 O O O (dwv=>+/dwv3-) v>id nos Fig. 6. Tracings showing the effects of CCH pre-perfusion on the positive inotropic response to ISO. Rat hearts were perfused with (1) vehicle buffer. (2) ISO (10-7 M) alone for 1.5 min, (3) CCH (3x10'6 M) for 1 min, then ISO (10"7M) plus the above dose of CCH for another 1.5 min. 46 70 O-1-LVP (mm Hg) CONTROL 1 min ISO 1.5 MIN. 1 min. 70 T 0 CCHIMIN, CCH/tSO 1.5 MIN. LVP (mm Hg) 1 min. 47 Fig. 7. The effects of CCH pre-perfusion on the positive inotropic response of rat ventricles to ISO. Rat hearts were perfused with ISO (10~7 M) alone for 1.5 min or pre-perfused with CCH (3x10"6 M) for 1 min, then ISO (10"7 M) plus the above dose of CCH for another 1.5 min. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the basal values. 48 LEFT VENTRICULAR PRESSURE (mmHg) o o -3 S3 O t-za ux o ra o o X + o o -> M CJ o o o o CD > > - 3 S3 S3 T3 G w o o Ul Ol \ l CO ID o o o o o O -* N) U > o o o o o O 4^ Fig. 8. The effects of CCH pre-perfusion on particulate and total cAMP increased by ISO. Rat hearts were perfused with ISO (10"7 M) alone for 1.5 min or pre-perfused with CCH (3x10'6 M) for 1 min, then ISO (10"7 M) plus the above dose of CCH for another 1.5 min. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the control group. (#) Represents significantly different from the ISO group. 50 ( CYCLIC AMP CONTENT IN RAT LEFT VENTRICLES o K O O II Total cAMP P a r t i c u l a t e cAMP 51 Fig. 9. The effects of CCH pre-perfusion on soluble and particulate PKA activity of rat ventricles in the presence of ISO. Rat hearts were perfused with ISO (10 -7 M) alone for 1.5 min or pre-perfused with CCH (3x10"6 M) for 1 min, then ISO (10~7 M) plus the above dose of CCH for another 1.5 min. The activity ratio in the soluble and the activity as a percentage of total in the particulate fraction were determined. See "Methods" for further details. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the control group., (#) Represents significantly different from the ISO group. 52 m •VI PARTICULATE PKA (% TOTAL) o m m •sj- o Nf m ro o to in CM o CM LO *— o *-• u a D U < a. 9 m 3 o CO I D * (-* I-LO 'NIW S'T OSI/HDO + 'Nin I HOD NIK 9'I OSI 10HJ.NOD 1 o «-1 CD o 1 CO o i r^  o i CD O 1 m o i -a-o 1 m o i CM O 1 o o o (dkW3+/dm/3-) v>id nos ^ -Fig. 10. Tracings showing the effects of CCH pre-perfusion on the positive inotropic response to FSK. Rat hearts were perfused buffer without drugs (1) or with FSK (1(r6 M) alone for 2 min (2) and 5 min (3) or pre-perfused with CCH (3x10"6 M) for 1 min, then FSK (10"6 M) plus the above dose of CCH for 2 min (4) or 5 min (5). 54 ( 70 01-LVP (mm Hg) ( 1 ) 70 T 0-^ ( 2 ) LVP (mm Hg) 1 min. t-H FSK 5 MIN. 1 min. ( 3 ) LVP (mm Hg) 1 min. I—H 1 min. 70-r 0 ( 4 ) CCH I MIN. + CCH/FSK 2 MIN. LVP (mm Hg) 1 min. I—I T T CCH 1 MIN. + CCH/FSK 5 MIN. 1 min. 55 Fig. 11. The effects of CCH pre-perfusion on the positive inotropic response of rat ventricles to FSK. Rat hearts were perfused with FSK (10'6 M) alone for 2 or 5 min or pre-perfused with CCH (3x10"6 M) for 1 min, then FSK (10'6 M) plus the above dose of CCH for 2 or 5 min. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the basal values. (#) Represents significantly different from the corresponding FSK groups. IXX1 LEFT VENTRICULAR PRESSURE (mmHg) o o 2! » O 5S 5 CXI o 50 -9 > -3 -9 S3 - ' M t A i ^ U i O l s J O O l O O - ' M U i ^ O O O O O O O O O O O O O O O v W W W W W ^ ZZZZZZZZZZZZZh SSSSSEa \\\\\\\wv^^q-. / / / / / / / / / / / / / / / / / / ^ • H=fe - I * Fig. 12. The effects of CCH pre-perfusion on particulate and total cAMP in the presence of FSK. Rat hearts were perfused with FSK (10"6 M) alone for 2 or 5 min or pre-perfused with CCH (3x10"6 M) for 1 min, then FSK (10"6 M) plus the above dose of CCH for 2 or 5 min. Left and right panels showing the effects of CCH (3x10"6 M) on the total or particulate cAMP content induced by FSK (10'6 M) respectively. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the control group. 58 Total cAMP (pmol/mg protein) —* D O 1 M O 1 C-J o 1 • ^ O 1 Cn O 1 cn -. o c 1 , 1 CONTROL FSK 2 MIN. FSK 5 MIN. CCH+CCH/FSK 2 MIN. CCH+CCH/FSK 5 MIN. < * Particulate cAMP (pmol /mg protein) O cn o _! cn I to O cn o i . i CONTROL FSK 2 MIN. FSK 5 MIN. CCH+CCH/FSK 2 MIN. CCH + CCH/FSK 5 MIN. i Fig. 13. The effects of CCH pre-perfused on soluble and particulate PKA activity in the presence of FSK. Rat hearts were perfused with FSK (10~6 M) alone for 2 or 5 min or pre-perfused with CCH (3x10"6 M) for 1 min, then FSK (10"6 M) plus the above dose of CCH for 2 or 5 min. The activity ratio in the soluble and the activity as a percentage of total in the particulate fraction were determined. See "Methods" for further details. Values in parentheses indicates the number of hearts. Each bar represents the mean ± SEM (*) Represents significantly different from the control group. 60 SOL PKA (-C.AMP/+C.AMP) p b CONTROL ( g ) O p o O p CO p O CO i r o to o ~l FSK 2 MIN ( 6 ) •I * FSK 6 MIN ( 6 ) CCH+CCH/FSK (Q\ 2 MINUTES CCH+CCH/FSK ( 4 ) 5 MINUTES cn H * -\ * H * T" 1 1 r D I CO o f c COE 5 •0 ^ 2 o £ i r — » o — 1 Ol hJ o M cn OJ o OJ cn -I*-O 4=> cn cn O (ivioi %) v^d Bivinouavd a\ Fig. 14. Tracing showing the effects of CCH on the positive inotropic responses to ISO or FSK. Rat hearts were perfused with vehicle buffer (1), FSK (10'6 M) perfusion alone (2), or in the presence of CCH (3x10"6 M) for 2 min (3), and ISO (10"7 M) alone (4), or in the presence of CCH (3x10'6 M) for 1.5 min (5). 62 70 0 LVP (mm Hg) 70-r o-1-LVP (mm Hg) ESK 2MIN. lmin. ( 1 ) ( 2 ) ISO 1.5 MIN. I min. ( 4 ) 7 0 -0-L LVP (mm Hg) CCH/FSK 2 MIN. 1 min. I ( 3 ) CCH/ISO 1.5 MIN. 1 min. ( 5 ) 63 Fig. 15. The effects of CCH on the positive inotropic response of rat ventricles to ISO or FSK. Rat hearts were perfused with ISO (10"7 M) or FSK (10" 6 M) in the presence or absence of CCH (3x10"6 M) for 1.5 or 2 min, respectively. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the basal values. (#) Represents significantly different from the ISO or FSK group respectively. 64 CO CO O o K o o 2 ; SO O r1 E 00 O B3 00 O o LEFT VENTRICLE PRESSURE (mmHg) - M O J ^ U l O l s l C O l D O - ' M W ^ O O O O O O O O O O O O O O O cd > 00 \\\\\\\\\\\N3X1 TZZZZZZZZZZZZb ^SH^SSSS t?d 90 -a w c 00 p — 1 O 2! H sSSS\\s\S\V53-' V/////////////////////Z H * __H=te t-fl Fig. 16. The effects of CCH on particulate and total cAMP induced by ISO or FSK. Rat hearts were perfused with ISO (1CT7 M) or FSK (10~6 M) in the presence or absence of CCH (3x10"6 M) for 1.5 or 2 min respectively. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the basal values. 66 Total cAMP (pmol/mg protein) CONTROL ISO 1.5 MIN. en I o I en _ 1 _ M M C-J O cn O cn o I . I . _ _ J I 1 I I ISO + CCH 1.5 MIN. FSK 2 MIN. FSK + CCH 2 MIN. Particulate cAMP (pmol/mg protein) cn _| O _ l _ cn i O cn C*J o I L CONTROL ISO 1.5 MIN. ISO + CCH 1.5 MIN. FSK 2 MIN. FSK + CCH 2 MIN. 67 Fig. 17. The effects of CCH on soluble and particulate PKA activity in the presence or absence of FSK or ISO. Rat hearts were perfused with ISO (10~7 M) or FSK (10"6 M) in the presence or absence of CCH (3x10"6 M) for 1.5 or 2 min, respectively. The activity ratio in the soluble and the activity as a percentage of total in the particulate fraction were determined. See "Methods" for further details. Each bar represents the mean ± SEM of 6-8 rat hearts. (*) Represents significantly different from the basal values. 68 PKA IN SOLUBLE AND PARTICULATE FRACTIONS 1.0 r 0.9 J o a z o o • I PARTICULATE PKA I I SOLUBLE PKA Z 3 o z 2 •a X o \ o CO z 3 CM z 3 C\I K O 50 45 69 DISCUSSION 4.1. The effects of ISO, PGE-i and FSK on inotropic responses, cAMP levels and PKA activities of rat hearts. We have measured the cAMP content and PKA activities in different compartments of ventricles. In the present study, ISO and FSK not only increased the total and particulate cAMP levels but also elevated PKA activity in the soluble fraction and lowered PKA activity in the particulate fraction. The right panel of Fig. 4 shows the regression line for the relationship between inotropic effect (LVP) and the cAMP content bound in the particulate fraction after the perfusion with different cAMP- elevating agents. The correlation coefficient for the linearity of the response was 0.90 (P<0.05) for LVP versus particulate cAMP content. However, FSK elicited a greater increase in the total cAMP levels than ISO did at functionally equieffective concentrations, while PGE-| increased cAMP in this fraction but did not cause any positive inotropic response. The corresponding correlation coefficient for the linearity of LVP versus total cAMP content was only 0.52 (left panel of Fig. 4). These data are consistent with previous reports ( Hayes et al., 1980) and support the hypothesis that particulate cAMP is the most important factor for regulation of the cardiac contraction (Aass 1988). The possible reasons for FSK causing higher total cAMP than did ISO at the concentrations that were equieffective for inotropy may be that the former increases cAMP in a compartment inaccessible to PKA activation (Murray 1989) or that this direct AC activator activates all of the AC within the tissue, including that which is not linked to the contractile response (Seamon and Daly 1983). These hypotheses are supported by several studies in which FSK has been shown to increase total cAMP levels more markedly than PKA activity, in some cases, even producing a significant elevation in total cAMP levels with little or no activation of PKA (England 70 and Shahid, 1987; Do Khac et al., 1986). Our results are consistent with other observations as well, in which FSK (<1 mM) failed to potentiate the effects of ISO, despite increasing total cAMP levels in various muscle tissues ( Vegesna and Diamond, 1983; Bowman et al., 1985; Waldeck and Widmark, 1985). This suggests that FSK predominantly activates a subtraction of AC not activated by |3 agonists, particularly in low concentrations, and the cAMP produced is in a different functional compartment from PKA (Brunton et al., 1981). PGE-j led to increases in total cAMP and soluble PKA activity but did not alter the cAMP content and PKA activity in the particulate fraction, or the contractile force of myocardium. This is consistent with the previous results obtained in rat, guinea pig and rabbit myocardium (Hayes and Buxton, 1982). In addition, it has been shown that PGE-j neither increased troponin I phosphorylation (Brunton et al., 1979) nor affected the phosphorylation of some proteins as ISO did in cultured rat myocytes (Hayes and Brunton, 1982). Although our data here could not demonstrate whether the subcompartment of AC stimulated by PGE-| is the same as that stimulated by low concentrations of FSK, this interesting question may be solved in the future by using the more precise separation of subcompartments, or antibodies and other probes for localizing the activated AC in specific subcellular fractions (Jungmann et al., 1988; Byus and Fletcher, 1988). Our data support the hypothesis that elevation of particulate cAMP which binds to the regulatory subunit of PKA in the particulate fraction leads to translocation of the released catalytic subunit of PKA from the particulate to the soluble fraction of myocardium (Hayes et al., 1980). In our experiments, it was shown that accompanying the positive inotropic responses to ISO or FSK, soluble PKA activity and particulate cAMP content increased but PKA activity in particulate fraction decreased (Fig. 3 and 5). This translocation was not observed in response to PGE-), suggesting that the translocation of the released catalytic subunit of PKA 71 from the particulate to the soluble fraction of myocardium is necessary for the inotropic response. This supports another suggestion as well, which is that soluble PKA may not have access to its potential substrates (Murry et al., 1989). It is also worth mentioning that while increasing the perfusion time of FSK from 2 min to 5 min resulted in a further increase in particulate cAMP levels, there was no further decrease in particulate PKA activity. This suggests that particulate PKA was not activated and further translocated to the soluble fraction, although particulate cAMP levels still increased following a prolonged perfusion with FSK. The possibility that the lack of correlation results from the measurement of particulate cAMP and PKA in different fractions instead of in the same fraction does not seem likely since we extracted the particulate cAMP and PKA of myocardium by the same method. Furthermore, compared with the centrifugal speed (20,000 x g for 10 min.) used to separate the soluble and particulate fractions in Brunton's lab ( Brunton et al. 1981), we used a higher speed of centrifugation (Aass et al., 1988. see materials and methods for detail). Therefore, it does not seem possible that the particulate fractions of myocardium were only partially collected in our sample precipitation after centrifugation, leading to an under-evaluation of activity of particulate PKA after the 5 min perfusion with FSK. According to a report by England and Shahid (1987), phosphorylase a activity increased much faster than the cAMP level did in rat heart at the beginning of perfusion with FSK. Subsequently, phosphorylase a reached a plateau within 50 seconds, while cAMP content still increased and reached a peak after 5 min. A clear dissociation between activation of phosphorylase and increased cAMP content was observed when hearts were perfused with FSK. This may indicate that phosphorylase, as a substrate of cAMP, was activated maximally at a rather low concentration of cAMP. Therefore, after the maximal activation, phosphorylase a activity remained constant despite the continuous increase in cAMP level. 72 Because PKA is a substrate of cAMP as well and its activity is parallel to the activity of phosphorylase a (England and Shahid, 1987, Hayes et al., 1980), it is suggested that the dissociation between particulate cAMP and PKA activity on continuous perfusion with FSK may result from a similar mechanism which caused the dissociation between cAMP and phosphorylase a. 4.2. The effects of pre-perfusion with CCH on positive inotropic response, cAMP content and PKA activity induced by ISO. It is well known that CCH antagonizes the positive inotropic effects of ISO at least in part by lowering the total cAMP content in the heart (Brown et al., 1980; Endoh et al., 1985; Sorota et al., 1985; MacLeod 1986; MacLeod and Diamond 1986). In the present study, pre-perfusion of hearts with CCH (3x10"6) resulted in complete antagonism of the positive inotropic response to ISO and decreased cAMP and PKA activity significantly, in agreement with the previous reports. These results support the hypothesis that CCH antagonism of cAMP and PKA activation plays an important role in its inhibition of positive inotropic responses to ISO. Although the effects of ISO on contractile force were abolished by CCH, the ISO-induced changes in cAMP and PKA activity were not inhibited completely. For example, after CCH pre-perfusion, CCH reduced the effect of ISO on LVP from 81.3 % increase over control to -9.2 % in 6 hearts. However the total cAMP level antagonized by CCH in the presence of ISO declined from 75.2 % increase over control to 21.5 % and the particulate cAMP level from 43.6 % to 11.3 % while the activity of soluble PKA decreased from 145 % increase over control to 80 % and activity of particulate PKA which had decreased to 55.2 % of control value ws now decreased only to 73.6 %. These results do not support the hypothesis of Hartzell (1988) in which he postulated that the change of cAMP levels in the particulate compartment may 73 parallel the inhibitory effects of CCH on the contractility of myocardium (Keely et al. 1978, Watanabe et al., 1984). Thus, this inhibitory dissociation may involve cAMP-independent mechanisms instead of the previous postulation. The possible explanation for cAMP-independent mechanisms of CCH are mentioned in Introduction section (1.11) and the following discussion. Comparing Fig. 8 with Fig. 9, the inhibitory effects of CCH on cAMP levels increased by ISO were more obvious than those on the PKA activity. The possible reasons for this inhibitory dissociation may involve the following. PKA activity is the most sensitive index for evaluating cAMP-elevating agents among cAMP, PKA activity and phosphorylase activity measurements (Hayes et al., 1980). Therefore, although elevated cAMP levels were reduced significantly by perfusion with CCH, PKA activity was still kept in an activated state, reflecting the cAMP system was still activated slightly. This dissociation of antagonism by Ach between cAMP and PKA activity was shown in Keely's paper as well (Keely et al., 1978). 4.3. The effects of pre-perfusion with CCH on positive inotropic response, cAMP content and PKA activity induced by FSK. CCH reversed the positive inotropic response to FSK in the rat ventricle, but it did not antagonize either the FSK-induced elevation in total cAMP and PKA, or the effect of FSK on particulate cAMP and PKA. In addition, we obtained similar results when rat hearts were perfused with CCH and FSK simultaneously for 2 min (Fig. 14-17). This suggests that CCH may antagonize the positive inotropic effect of FSK by the following cAMP-independent pathways. Firstly, the cGMP pathway is considered by many workers to play a role in antagonising the positive inotropic rsponses to FSK or ISO. The most confirmed functions of cGMP may be the antagonism of calcium channels stimulated by cAMP (Hartzell and Fischmeister, 1986), which decreases 45Ca influx into the 74 myocardium (Nawrath, 1977), shortens action potential duration (Trautwein et al., 1982) and inhibits Ca2+-dependent action potentials (Kohlhardt and Haap, 1978, Mehegan et al., 1985). More recently, it has been demonstrated that 8-bromo-cGMP inhibited the basal calcium current in embryonic chick ventricular myocytes in a cAMP-independent way (Wahler et al., 1990). The proposed pathways of cGMP functions include activation of cGMP-dependent protein kinase (Lincoln and Keely, 1980; Lohmann et al., 1991), and activation of cGMP-stimulated cAMP-phosphodiesterase (cGS-PDE), which then increases the hydrolysis of cAMP (Fischmeister and Hartzell, 1987). The latter has to be demonstrated in mammalian myocardium. Secondly, in guinea pig ventricle, Ach has been demonstrated to activate a type-1 phosphatase which may dephosphorylate different proteins, including phosphalamban, phosphorylated by PKA (Ahmad et al., 1989 and see Introduction 1.12.2.4). Recently, Gupta et al. (1993) demonstrated that Ach and adenosine antagonized the stimulatory effect of ISO or FSK on protein phosphatase inhibitor (PPI-1) without affecting PKA activity. Therefore they speculated that MRAs and adenosine reduced PPI-1 activity by a cAMP-independent mechanism. This could be an alternative mechanism by which CCH antagonized the positive inotropic responses to FSK without affecting the cAMP levels. Thirdly, it is well known that atria, and to a lesser extent ventricles, exhibit the Ach-activated K+ channel. The activation of the time-dependent K+ current by Ach overlaps Ca2+-dependent action potentials during contraction and make it appear smaller (Sorota et al., 1985, TenEick et al., 1976). In atria, MRAs have been shown to be linked to K+ channels by a pertussis toxin sensitive G-protein and pertussis toxin pre-treatment of animals can be shown to attenuate the ability of MRAs both to activate the K+ current and to exert a direct negative response (Endoh et al., 1985, Birnbaumer, et al., 1990). More recently, Ray and MacLeod 75 (1992) demonstrated that in rabbit left atria, pre-treatment of rabbits with pertussis toxin only partially attenuated the inhibitory effect of CCH on FSK and IBMX-induced positive inotropy. However, 4-aminopyridine, a potassium channel antagonist, produced a concentration-dependent attenuation of the inhibitory effect of CCH on positive inotropic responses to FSK and IBMX in the left atria from both saline and pertussis toxin-pretreated rabbits. Although it can be argued that ventricle of rat has no MR-coupleed K+ channels, they are definitely present in the ventricles of some species such as frog and ferret (Hartzell and Simmons, 1987, Boyett et al., 1988). In the present study, we have not examined the changes of K+ channel activity in the same experiment, therefore, the contribution of this current should be conclusively ruled out in the future. Before we can suggest that CCH antagonized the effects of FSK by means of the cAMP-independent pathways above, some things should be borne in mind. FSK may have some positive inotropic mechanisms which do not involve increases in cAMP levels. For example, FSK blocks rectifying potassium channels in pancreatic p-cells without changing cAMP levels (Zunkler et al. 1988), and FSK desensitized the Ach receptor independently of cAMP (Wagoner et al. 1988). Although these mechanisms of FSK have been demonstrated, our results have confirmed that CCH antagonized the inotropic effects of FSK by means of a cAMP-independent pathway. 4.4. The effects of CCH on positive inotropic response, cAMP content and PKA activity induced by ISO We could not demonstrate that the changes in cAMP and PKA activity induced by ISO (10-6M) were antagonized by CCH (3x10-6 M) when both of them were perfused together for 1.5 min, although CCH abolished the positive inotropic response to ISO at the same time. In general, antagonism by CCH of the effects of 76 ISO has been examined by two kinds of administration, either administration of CCH and ISO together, or CCH first for a period of time then CCH and ISO together. Different conclusions could be found in various laboratories. For example, Watanabe and Besch (1975) have demonstrated that Ach produced negative inotropic effects without reducing cAMP levels in guinea pig hearts when perfused with ISO for 2 min. Gupta et al. (1993) reported that when Ach was perfused with ISO or FSK simultaneously for 1 min in guinea pig ventricles, the rate of tension development was decreased without reducing PKA activity ratio which is regulated by cAMP level. On the other hand, Keely (1978) reported that Ach antagonized all p-adrenergic effects of epinephrine in rat heart, including the increase in cAMP levels, PKA activity and contractile force when both were perfused at the same time. Recently, George (1991) described that CCH antagonized the above effects of ISO by incubating the rat myocytes in CCH for 60 sec first, then in CCH and ISO simultaneously for 30 sec. The different doses of CCH and ISO used in these experiments could be one of the reasons for the above different conclusions. It has long been known that there is a dissociation between the antagonism by MRAs of positive inotropic responses and change in cAMP or PKA activity in the presence of ISO (Loffelholz and Pappano, 1985; Watanabe et al., 1984). That is, the tension was antagonized to a greater extent than cAMP or PKA activity by CCH in the presence of ISO. Therefore, if researchers select a different dose of CCH perfused with a different amount of ISO to observe the antagonizing effects of CCH, the decrease in cAMP and PKA activity elevated by ISO could be missed. This possibility was demonstrated in Keely and his co-worker's report (1978) in which they showed that Ach (5x10"6 M) failed to reduce the PKA or phosphorylase activities increased by 10~5 M epinephrine although Ach antagonized all effects produced by 10~6 M epinephrine. Another reason for the different conclusions may involve the 77 perfusion time with CCH. According to the report of Katano and Endoh (1993), CCH antagonized the cAMP accumulation induced by ISO only after a 2 and 3 min administration of CCH and ISO. In addition, this effect was merely significant. Therefore in our experiments, 1.5 min of CCH perfusion time may not have been long enough to let CCH exert its antagonizing effects on ISO-induced changes in cAMP and PKA activity. It is not known why the inhibitory effects of CCH on ISO-induced changes in cAMP and PKA activity could be seen on the prolongation of CCH perfusion time. It is possible that it involves a mechanism by which CCH has been proposed to inhibit AC activation. CCH activates Gj protein, which then may inhibit the interaction between (3-receptor with GSj resulting in the reduction of adenylate cyclase activity (Levitski et al., 1986; Gillman, 1987). 4.5. Clinical meaning of our experiments The administration of exogenous adenosine or its analogs, or the increase in endogenous adenosine levels by an inhibition of its breakdown via adenosine deaminase have been shown to reduce the extent of ischaemic injury, including: preventing or delaying the ischaemic contracture, decreasing the ischaemia-induced loss of cellular ATP content and enhancing the recovery of function and ATP content (Ely et al., 1985, Lasley et al., 1990, Zhu et al., 1990 and Hendrikx et al., 1993). Recent studies suggest that the anti-ischaemic action of adenosine is mediated by A-j receptors (Lasley et al., 1990). MRAs share many electrophysiological and metabolic actions with adenosine (Belardinelli et al., 1989, Pappano and Mubagwa 1992) due to the similar effect on Gj protein after the activation of the respective receptors. Both agonists activate specific K+ channels and inhibit the accumulation of cAMP while having direct negative chronotropic and dromotropic (conductive) effects and antiadrenergic inotropic effect in the 78 ventricles. Numerous reports indicate that a high level of parasympathetic stimulation before or during myocardial ischaemia protects against arrhythmogenesis during myocardial ischaemia and reperfusion (Myers 1974, Vanoli et al., 1991 and Schwartz et al., 1988). Some studies have even shown that adenosine was less effective in attenuating ISO-induced phosphorylation of protein substrates (George 1991). Therefore, our findings that CCH antagonized the ISO-induced elevation of cAMP and PKA activation in the particulate compartment of myocardium have complemented the reports from above labs demonstrating MRAs have inhibitory effects on cAMP increased by ISO, as adenosine has, and supply clues for usage of CCH to treat ischaemia in the clinic. 79 SUMMARY AND CONCLUSIONS 1. The increase in particulate cAMP content was closely correlated with the LVP elevated by the 1.5 min perfusion of ISO, and the 2 and 5 min perfusion of FSK. 2. PGE-) raised the total cAMP and soluble PKA activity but affected neither the LVP nor the particulate cAMP and PKA. These results support the hypothesis that particulate cAMP and PKA may determine the inotropic state of myocardium after cAMP-generating agent stimulation. 3. The pre-perfusion with CCH abolished the positive inotropic effects of ISO by lowering the total and particulate cAMP, decreasing the soluble PKA activity and raising the percentage of particulate PKA partially. The cAMP-independent antagonism by CCH of the effects of ISO was not ruled out in this study. 4. Either pre-perfusion of CCH or perfusion of CCH in the presence of FSK diminished the positive inotropic effects of FSK without affecting the total and particulate cAMP or soluble PKA activity and percentage of particulate PKA. 5. The 1.5 min perfusion of CCH (3x10"6 M) had no effect on either the increases in the total and particulate cAMP, or soluble PKA activity and percentage of particulate PKA, while it eliminated the positive inotropic response induced by 10"7 M ISO. Taken with 4, this suggests that the inhibition of cAMP content and PKA activities by CCH was delayed behind the elimination of positive inotropic response to ISO. 6. These data suggest that the ability of CCH to antagonize the positive inotropic effect of FSK does not appear to be associated with a reduction in particulate cAMP or PKA activity. The dissociation of antagnizing effects by CCH 80 between contractile force and cAMP levels or PKA activity in ventricles need to be studied in the future. REFERENCES Aass, H., Skomedal, T., Osnes, J.B. (1988) Increase of cyclic AMP in subcellular fractions of rat heart muscle after B-adrenergic stimulation: Prenalterol and isoprenaline caused different distribution of bound cyclic AMP. J. Mol. Cell Cardiol. 20: 84-860. Adilstein, R.S. and Eisenberg, E. (1980) Regulation and kinetics of the actin-myosin-ATP interaction. Annu. Rev Biochem. 49: 921-956. Ahmad, Z., Green, F.J., Subuhi, H.S. and Watanabe A.M. (1989) Autonomic regulation of type 1 protein phosphatase in cardiac muscle. J. Biol. Chem. 264: 3859-3863. Arnold, S.S. and Burgen, D. (1989) Some consideration of receptor specificity. Trend Pharmacol. Sci. (suppl.) 1-10. Beavo, J.A., Bechtel, P.J. and Krebs, E.G. (1975) Mechanisms of control for cAMP-dependent protein kinase from skeletal muscle. Adv. Cyclic Nuceotide Res. 5: 241-251. Belardinelli, L, Linden J. and Berne, R.M. (1989) The cardiac effects of adenosine. Prog. Cardiovasc. Dis. 32: 73-97. Berridge, M.J. (1987) Inositol triphosphate and diacylglycerol: two interacting second messengers. Annu. Rev. Biochem. 56: 159-193. Biegon, R.L., Epstein, P.M. and Pappano, A.J. (1980) Muscarinic antagonism of the effects of a phosphodiesterase inhibitor (methyl isobutyl-xanthine) in embryonic chick ventricle. J. Pharmacol. Exp. Ther. 215: 348-356. Birdsall, N.J.M. (1986) Soluble and membrane-bound muscarinic acetylcholine receptors. Biochem. Soc. Symp. 52: 23-32. Birdsall, N.J.M. and Hulme, E.C. (1983) Muscarinic receptor subclasses. Trends Pharmacol. Sci. 4: 459-463. Bonner T.I. (1989) New subtypes of muscarinic acetylcholine receptors. Trends Pharmacol. Sci. suppl. 4:11-15. Bowman, W.C., Lam, F.Y., Rodger, I.W. and Shahid, M. (1985) Cyclic nucleotides and contractility of isolated soleus muscle. Br. J. Pharmcol. 84: 259-264. Boyett, M.R., Kirby, M.S., Orchard, C.H. and Roberts, A. (1988) The negative inotropic effect of acetylcholine on terret ventricular myocardium. J. Physiol. 404: 613-635. 82 Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Birnbaumer, L. (1990) G-protein in signal transduction. Ann. Rev. Pharmacol. Toxocol. 30: 675-705. Brodde O.E., Bisoprolol, P. (1986) a highly selective p-adrenoceptor antagonist: in vitro and in vivo studies. J. Cardiovas. Pharmacol. 8 (suppl.11): S29-S35. Brodde, O.E., Beckeringh, J. J. and Micheal, M.C. (1987) Human heart p-adrenoceptors: a fair comparison with lymphocyte p-adrenoceptors? Trends Pharmacol. Sci. 8: 403-407. Brodde, O.E., Daul, A., Wellstein, A., Palm, D., Micheal, M. C. and Beckeringh. J. J. (1988) Differentiation of p-|- and P2- adrenoceptors-mediated effects in humans. Am. J. Physiol. 254: H199-H206. Brodde, O.E., Zerkowski, H.R., Borst, H.G., Maier, W. and Micheal, M.C. (1989) Drug and disease-induced changes of human cardiac p-| and P2 adrenoceptors. Eur. Heart J. 10 (suppl. B): 38-44. Brown A.M. and Birnbaumer, L. (1990) Ionic channels and their regulation by G protein subunits. Annu. Rev. Physiol. 52: 197-213. Brown, B., Poison J.B., Krzanowski, J.J. and Wiggins, J.J. (1980) Influence of isoproterenol and methylisobutylxanthine on the contractile and cyclic nucleotide effects of methacholine in isolated rat atria. J. Pharmacol. Exp. Ther. 212: 325-332. Brown, J.H. (1979) Cholinergic inhibition of catecholamine stimulable cAMP accumulation in murine atria. J. Cyclic Nucl. Res. : 423-433. Brown, J.H. and Jones, L.G. (1986) Phosphoinositide metabolism in the heart. In "Phosphoinositides and receptor mechanisms", ed. Putney, J. (Jr), pp. 245-270, Alan R. Liss Inc., New York. Brunton, L.L., Hayes, J.S. and Mayer, S.E. (1979) Hormonally specific phosphorylation of cardiac troponin I and activation of glycogen phosphorylase. Nature (London) 280: 78-80. Brunton, L.L., Hayes, J.S. and Mayer, S.E. (1981) Functional compartmentation of cyclic AMP and protein kinase in heart. Adv. Cyclic Nucleotide Res. 14: 391-397. 83 Byus, C.V.and Fletcher, W.H. (1988) Direct cytochemical localization of the free catalytic subunit of cAMP-dependent protein kinase. Meth. Enzym. 159: 236-254. Carlsson, E., Ablad, C, Brandstrom, A. and Carlsson, B. (1972) Differentiated blockade of the chronotropic effects of various adrenergic stimuli in the cat heart. Life Sci. 11:953-958, Castagna, M., Palmer, W.K. and Walsh, D.A. (1975) Nuclear protein-kinase activity in perfused rat liver stimulated with dibutyryl-adenosine cyclic 3':5'-monophosphate. Eur. J. Biochem. 55:193-199. Cerbai, E., Klockner, U. and Isenberg, G. (1988) Ca-antagonistic effects of adenosine in guinea pig atrial cells. Am. J. Physiol. 255: H827-H878. Chad, J., and Echert, R. (1985) Calcineurin, a calcium-dependent phosphatase, enhances Ca-mediated inactivation of Ca current in perfused snail neurons. Biophys J 47: 266a. (abstract) Cheung, A.H., Sigal, I.S., Dixon, R.A.F. and Strader, CD. (1989) Agonist-promoted sequestration of the beta 2-adrenergic receptor requires regions involved in functional coupling with Gs. Mol. Pharmacol. 34: 132-138. Christie, M.J., North R.A. (1988) Control of ion conductance by muscarinic receptors . Trends Pharmacol Sci. 9 (suppl): 30-34. Corbin, J.D., Keely, S.L, Sodering, T.R., and Park, C.R. (1975) Hormonal regulation of adenosine 3", 5'-monophosphate-dependent protein kinase. Adv. Cycli Nucleotide Res. 5: 265-279. Costa, E., Kurosawa, A. and Guidotti, A. (1976) Activation and nuclear translocation of protein kinase during transsynaptic induction of tyrosion 3-monoxygenase. Proc. Natl. Acad. Sci. U.S.A. 73:1058-1062. Daly, M.J. and Levy, G.P. (1979) The subclassification of B-adrenocepotors: evidence in support of the dual B-adrenoceptor hypothesis. In trends in Autonomic Pharmacology, ed. by S. kalsner, vol. 1, pp. 347-385, Urban and Schwarzenberg, Bltimore. Diamond, J., TenEick, R. and Trapani, A.J. (1977) Are increases in cGMP levels responsible for the negative inotropic effects of acetylcholine in the heart? Biochem. Biophys. Res. Commun. 79: 912-918. Dixon, R.A.F. Sigal, I.S., Rands, E., Register, R.B., Candelore, M.R.M, Blake, A.D. and Strader, CD.(1987) Ligand binding to the B-adrenergic receptor involves its rhodopsin-like core. Nature (London) 326: 73-77. 84 Do Khac, L, Mokhtari, A. and Harbon, S. (1986) A re-evaluated role for cyclic AMP in uterine relaxation. Differential effect of isoproterenol and forskolin. J. Pharmacol. Exp. Ther. 239, 236- 242. Dratz, E.A., Hargrave, P.A. (1983) The structure of rhodopsin and the rod outersegmetn disc membrane. Trends Biochem. Sci. 8:128-31. Edelman, A.M., Blumenthal, D.K. and Krebs, E.G. (1987) Protein serine/threonine kinase. Ann. Rev. Biochem. 56: 567-613. Ely, S.W., Mentzer R.M., Lasley, R.D., Lee B.K. and Berne, R.M. (1985) Functional and metabolic evidence of enhanced myocardial tolerance to ischaemia and reperfusion with adenosine. J. Thorac Cardiovasc Surg. 90: 549-56. Emorire, L.J., Marullo, S., Brien, S., Patey, G., Tate, K., Delavier, K.C. and Stros B.D. (1989) Molecular characterization of human b3-adrenergic receptor. Science. 245:1118-1121. Endoh, M., Maruyama, M. and lijima, T. (1985) Attenuation of muscarinic cholinergic inhibition by islet activating protein. Am. J. Physiol. 249: H309-H320. Fabiato, A. (1992) Two kinds of calcium-induced release of calcium from the sarcoplasmic reticulum of skinned cardiac cells. Adv. Exp. Med. Biol. 311: 245-262. Fields, J. Z., Roeske, W. R., Morkin, E. and Yamamura, H. (1978) Cardiac muscarinic cholinergic receptors: Biochemical identification and characterization. J. Biol. Chem. 253:3251-3258. Findlay, J.B.C. and Pappin, J.B.C. (1986) The opsin family of proteins. Biochem. J. 238: 625-642. Fischmeister, R. and Hartzell, H.C. (1987) Cyclic guanosine 3', 5'-monophosphate regulates the calcium current in single cells from frog ventricle. J. Physiol. 387:453-472. Fleming, J. W., Strawbridge, R. A. and Watanabe, A. M. (1987) Muscarinic receptor regulation of cardiac adenylate cyclase activity. J. Mol. Cell. Cardiol. 19: 47-61. Fleming, J. W., Wisler, P.L. and Watanabe, A. M. (1992) Signal transduction by G proteins in cardiac tissues. Circulation 85: 420-433. 85 Flockhart, D.A. and Corhin, J.D. (1982) Regulatory mechanisms in the control of protein kinase, CRC Crit. Rev. Biochem. 12: 133-186. Florio, V.A. and Sternweis, P.C. (1985) Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins. J. Biol. Chem. 1985; 260: 3477-3483. Gautier, P., Bertrand, J.P. and Guiraudou, P. (1991) Effects of SR 44866, a potassium channel opener, on action potentials of rabbit, guinea pig and human heart fibers. J. Cardiovas. Pharmacol. 17: 692-700. George, E.E., Romano, F.D. and Dobson Jr, J.G. (1991) Adenosine and acetylcholine reduce isoproterenol-induced protein phosphorylation of rat myocytes. J. Mol. Cell Cardiol. 23: 749-764. Gettys, T.W., Vine, A.J., Simonds, M.F. and Corbin, J.D. (1988) Activation of the particulate low Km phosphodiesterase of adipocytes by addition of cAMP-dependent protein kinase. J. Biol. Chem. 263: 10359-10363. Gilman, A.G. (1987) G-protein: transducers of receptor-generated signals. Ann. Rev. Biochem. 56: 615-649. Gupta, R.C., Neumann, J. and Watanabe, A.M. (1993) Comparison adenosine and muscarinic receptor-mediated effects on protein phosphatase inhibitor-1 activity in the heart. J. Pharmacol. Exp. Therap. 266:16-22. Hammer, R., Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V. and Hulme, E.C. (1980) Pirenzepin distinguishes between different subclasses of muscarinic receptors. Nature (London) 283: 90-92. Hammer, R., Giraldo, E., Schiavi, G.B., Monferini, E. and Laninsky, H. (1986) Binding profile of a novel cardioselective muscarinic receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat. Life Sci. 38: 1653-1662. Hartzell H.C. (1988) Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systems. Prog. Biophys. Molec. Biol. 52: 165-247. Hartzell, H.C. and Fischmeister R. (1987) Effect of forskolin and acetylcholine on calcium current in single isolated cardiac myocytes. Mol. Pharmacol. 32: 639-645. Hartzell, H.C. and Simmon, M. (1987) Comparison of effects acetylcholine on calcium and potassium in frog atrium and ventricle. J. Physiol. 389: 411-422. 86 Hartzell, H.C. and Titus, L. (1982) Effects of cholinergic and adrenergic agonists of phosphorylation of a 165,000-dalton myofibrillar protein in intact cardiac muscle. J. Biol. Chem. 257:2111-2120. Hayes, J.S. and Brunton, L.L. (1982) Functional compartments in cyclic nucleotide action. J. Cyclic Nucleotide Res. 8:1-10. Hayes, J.S., Brunton, L.L. and Mayer, S.E. (1980) Selective activation of particulate cAMP-dependent protein kinase by isoproterenol and prostaglandin E-j. J. Biol. Chem. 255:5113-5119. Hayes, J.S., Brunton, L.L., Brown, J.H., Reese, J.B., and Mayer, S.E. (1979) Hormonally specific expression of cardiac protein kinase activity. Proc. Natl. Acad. Sci. U.S.A. 76:1570-1574 Hayes, J.S., Brunton, L.L., Brown, J.H., Reese, J.B., and Mayer, S.E. (1979) Hormonally specific expression of cardiac protein kinase activity. Proc. Natl. Acad. Sci. U.S.A. 76:1570-1574. Hendrikx, M., Toshima, Y., Mubagwa, K. and Flameng (1993) Muscarinic receptor stimulation by carbachol improves functional recovery in isolated, blood perfused transients. Cir. Res. 69: 1369-1379. Hescheler, J., Kameyama, M., Trautwein, W., Mieskes, G. and Soling H.D. (1987) Regulation of the cardiac calcium channel by protein phosphatase. Eur. J. Biochem. 165:261-266. Hohl, CM. and Li, Q (1991) Compartmentation of cAMP in adult canine ventricular myocytes relation to single-free Ca2 + transients. Circ. Res. 69:1369-1379. Ikezono, K., Micheal, M.C., Zerkowski, H.R., Beckeringh, J.J. and Brodde, O.E. (1987) The role of cyclic AMP in the positive inotropic effect mediated by p-|-and (^-adrenoceptors in isolated human right atrium. Naun. Sch. Archiv. Pharmacol. 335:561-566. Insel P.A. and Ransnas L.A. (1988) G protein and cardiovascular disease. Circulation 78:1511-1513. Inui, J., Brodde, O.E. and Schumman, H.J. (1982) Influence of acetylcholine on the positive inotropic effect evoked by p-adrenoceptor stimulation in the rabbit heart. Naun. Sch. Archiv. Pharmacol. 320: 152-159. Jakobs, K.H., Aktories, K. and Schultz, G. (1979) GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists. Naun. Sch. Archiv. Pharmacol. 310: 113-119. 87 Jakobs, K.H., Schultz, G., Gaugler B., Pferffer, T. (1983) Inhibition of Ns-protein-stimulated human-platelet adenylate cyclase by epinephrine and stable GTP analogs. Eur. J. Biochem. 134: 351-354. Jungmann, R.A., Hiestand, P.C. and Schweppe, J.S. (1974) Mechanism of action of gonadotropin IV Cyclic adenosine monophosphate-dependent translocation of ovarian cytoplasmic cyclic adenosine monophosphate-binding protein and protein kinase to nuclear acceptor sites. Endocrinology 94:168-183. Jungmann, R.A., Kuettel, M.R., Squinto, S.P. and Kwast-Welfeld, J. (1988) Using immunocolloidal gold electron microscope to investigate cAMP-dependent protein kinase cellular compartmentation. Meth. Enzym. 159: 225-235. Kawano, S., Nakamura, F., Tanaka, T. and Hiraoka, M. (1992) Cardiac sarcoplasmic reticulum chloride channels regulated by protein kinase A. Circ. Res. 71:585-589. Keely, S.L., (1979) Prostaglandin E-\ activation of heart cAMP-dependent protein kinase: apparent dissociation of protein kinase activation from increases in phosphorylase activity and contractile force. Mol. Pharmacol. 15: 235-245. Keely, S.L., Lincoln, T.M. and Corbin, J.D. (1978) Interaction of acetylcholine and epinephrine on heart cyclic AMP-dependent protein kinase. Am. J. Physiol. 234: H432-H438. Kemp, B.E., Pearson, R.B., House, C, Robinson, P.J. and Means, A.R. (1989) Regulation of protein kinases by pseudosubstrate prototypes. Cellular Signaling. 1:303-311. Krebs, E.G. (1972) Protein kinase. Curr. Top. Cell Regul. 5: 99-133. Lasley R.D., Rhee, J.W., Van Wylen, D.G.L. and Mentzer, R.M. (1990) Adenosine A1 receptors mediated protection of the globally ischaemic isolated heart. J. Mol. Cell Cardiol 22: 39-47. Lemoine, H., Schonell, H. and Kaumann, A.J. (1988) Contribution of b-|- and D2-adrenoceptors of human atrium and ventricle to the effects of noradrenaline and adrenaline as assessed with (- -atenolol. Br. J. Pharmacol. 95: 55-66. Levine, M.A.H., and Leenen, F.H.H. (1989) Role of (3-)-receptors and vagal tone in cardiac inotropic and chronotropic responses to a p2-agonist in humans. Circulation 79:107-115. 88 Levy, M. N. (1983) Neural control of cardiac rhythm and contraction. In Cardiac Therapy, ed. by M. R. Rosin and B. F. Hoffman, pp. 73-94, Martinus Nijhoff, Boston. Linden, J and Brooker, G. (1979) The questionable role of cyclic guanosine 3':5' monophosphate in heart. Biochem. Pharmacol. 28: 3353-3360. Lindermann, J.J., Jones, L.R., Hathaway, D.R., Henry, B.G. and Watanabe, A.M. (1983) Beta-adrenergic stimulation of phospholamban phosphorylation and Ca2+-ATPase activity in guinea pig ventricles. J. Biol. Chem. 258: 464-471. Loffelholtz, K and Pappano, A.J. (1985) The parasympathetic neuroeffector junction of the heart. Pharmacol. Rev. 37: 1-24. MacDonell, K., Tibbits, G. and Diamond, J. (1994) Effects of muscarinic receptor agonists on contractility and cGMP levels in rat ventricular cardiomyocytes. (Abstract) XIV International Congress of Pharmacology (Montreal, Canada). MacLeod, K.M. (1984) The interaction of carbachol and forskolin in rabbit papillary muscle. Eur. J. Pharmacol. 107: 95-99. MacLeod, K.M (1986) Adrenergic-cholinergic interactions in left atria: Interaction of carbachol with alpha- and beta- adrenoceptor agonists. Can. J. Pharmacol. 64: 597-601 MacLoed, K.M. and Diamond, J. (1986) Effects of cGMP lowering agent LY 83583 on the interaction of carbachol with carbachol with forskolin in rabbit isolated cardiac preparations. J. Pharmacol.Exp. Ther. 238: 313-318. MacPhee, C.H., Reifsnyder, D.H., Moore, T.A. and Beavo, J.A. (1987) Intact cell and cell-free phosphorylation and concomitant activation of low Km cAMP phosphodiesterase found in human platelets. J. Cyclic Nucleotide Protein Phosphorylation Res. 11: 487-496. Main, B.G,and Tucker, H. (1985) Recent advances in 8-adrenergic blocking agents. Prog. Med. Chem 22:122-143 Metzger, H. and Lindner, E. (1981) Forskolin: a novel adenylate cyclase activator. I.R.C.S. Med. Sci. (Biochem.) 9: 99. Movsesian, M.A., Nishikawa, M. and Adelstein, R.S. (1984) Phosphorylation of phospholamban by calcium-activated, phospholipid-dependent protein kinase. Stimulation of cardiac sarcoplasmic reticulum calcium uptake. J. Biol. Chem. 259: 8029-8032. 89 Motomura, S., Zerkowski, H.-R., Daul, A., and Brodde, O.-E. (1990) On the physiologic role of beta-2 adrenoceptors in the human heart: in vitro and in vivo studies. Am. Heart J. 119:608-619. Murry, M.J., Reeves, M.L. and England, P.J. (1989) Protein phosphorylation and compartments of cyclic AMP in the control of cardiac contraction. Mol. Cellular Biochem. 89: 175-179. Myers, R.W., Pearlman, A.S. and Hyman, R.M. et al. (1974) Beneficial effects of vagal stimulation and bradycardia during experimental acute myocardial ischaemia. Circulation 59: 943-7. Nathanson, N.M. (1987) Molecular properties of the muscarinic acetylcholine receptor. Annu. Rev. Neurosci. 10: 195-236. Neumann, R.C., Gupta, R.C., Schmitz, W., Scholz, H., Nairn, A.C. and Watanabe, A.M. (1991) Evidence for isoproterenol-induced phosphorylation of phosphatase inhibitor-1 in the intact heart. Circ. Res. 69:1450-1457. O'Dowd B.F., Hnatowich, M., Regan. J.W., Leader, W.M., Carron, M.G. and Lefkowitz, R.J. (1988) Site-directed muta-genesis of the cotoplasmic domains of the human p2-adrenergic receptor. J. Biol. Chem. 263: 15985-15992. Ogreid, D. and Doskeland, S.O. (1982) Activation of protein kinase isoenzymes under near physiological conditions: Evidence that both types (A and B) of cAMP binding sites are involved in the activation of protein kinase by cAMP and 8-N3-cAMP. FEBS Lett. 150:161-166. Pang I.H., Sternweis, P.C. (1990) Purification of unique subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized subunits. J. Biol. Chem. 265: 18707-18712. Pappano, A. J. (1990) Parasympathetic control of cardiac electrical activity. In "Cardiac electrophysiology: From cell to bed side" eds. Zipes, D., P. and Jalife, J. pp 271-276, W.B. Saunders Co. Pappano, A.J. and Mubagwa, K (1992) Actions of muscarinic agents and adenosine on the heart. In : Fozzard H.A., Haber, E., Jennings, R.B., Katz, A.M. and Morgan H., eds. The heart and cardiovascular system, 2nd ed. New York: Raven Press, 1765-1776. Parker, E.M., Kameyama, K., Higashijima, T. and Ross, E.M. (1991) Reconstitutively active G protein-coupled receptors purified from baculovirus-infected insect cells. J. Biol. Chem. 266: 519-527. 90 Peralta, E.G., Ashkenazi, A., Winslow, J.W., Smith, D.J., and Ramachandran, J. (1987) Distinct primary structures, ligand-binding properties and tissue specific expression of four human muscarinic acetylcholine receptors. EMBO. J. 6: 3923-3929. Pffafinger, P., Martin, J., Hunter, D.D. and Nathanson, N.M. (1985) GTP binding proteins couple muscarinic receptors to a K channel. Nature 317: 536-538. Rannels, S.R., and Corbin, J.D. (1981) Studies on the function of the two intrachain cAMP-binding sites of protein kinases. J. Biol. Chem., 256: 7871-7876. Ray, A. and MacLeod, KM. (1992) Role of cAMP in the function interaction of carbachol with different cAMP elevating agents in rabbit atrium. Life Sci. 51: 1411-1418. Ray, A. and MacLeod, K.M. (1993) A pharmacological investigation of the contribution of muscarinic receptor-linked potassium channels to the reversal by carbachol of positive inotropic responses of rabbit left atrium to cyclic AMP-generating agents. J. Pharmacol. Exp. Ther. 266:1594-1601. Ripoll, C, Lederer, W.J. and Nichols, C.G. (1990) Modulation of ATP-sensitive K+ channel activity and contractile behavior in mammalian ventricle by the potassium channel openers cromakalim and RP49356. J. Pharmcol. Expl. Therap. 255: 429-435. Roberds, S.L. and Tamkun, M.M. (1991) Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart. Proceedings of the National Academy of Sci. of the United States of America 88:1798-1802 Robinshaw JD, Foster KA. (1989) Role of G proteins in the regulation of the cardiovascular system. Ann. Rev. Physiol. 51:229-244. Ross, E.M. and Gerstein, G. (1993) Regulation of the M-| muscarinic receptor-Gq. phospholipase C-B pathway by nucleotide exchange and GTP hydrolysis. Life Sci. 52:413-419. Scholz, H. (1980) Effects of beta- and alpha-adrenoceptor activators and adrenergic transmitter releasing agents of the mechanical activity of the heart. In Hand-book of Experimental Pharmacology, vol. 54/I: Adrenergic Activators and Inhibitors, ed. by L. Szekeres, pp. 651-733, Springer-Verlag Berlin, Heidelberg,New York. Schubert B, VanDongen A.M.J., Kersch G.E., Brown A.M. (1989) B-Adrenergic inhibition of cardiac sodium channels by dual G-protein pathways. Science 245: 516-519. 91 Schwartz P.J., Vanoli E, Stramba-Badial M, De Ferrai G.M., Billman, G.E. and Foreman, R.D. (1988) Autonomic mechanisms and sudden death. New insights from analysis of baroreceptor reflexes in conscious dogs with and without a myocardial infarction. Circulation 78: 969-79. Seamon, K.B., Daly, J.W. (1982) Guanosine 5'- (p,7-imido) triphosphate inhibition of forskolin-activated adenylate cyclase is mediated by the putative inhibitory guanine nucleotide regulatory protein. J. Biol. Chem. 257:11591-11595. Smrcka, A.V., Hepler, J.R., Brown, K.O. and Sternwieis, P.C. (1991) Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science 251:804-807. Sorota, S., Tsuji, Y., Tajima, T. and Pappano, A.F. (1985) Pertussis toxin treatment blocks hyperpolarization by muscarinic agonists in chick atrium. Circ. Res. 57: 748-758. Stiles, G.L., Caron, M.G., and Lefkowitz, R.J. (1984) 8-adrenergic receptors: biochemical mechanisms of physiological regulation. Physiol. Rev. 64: 661-743. Strader, CD., Sigal, I.S., Candelore, M.R., Rands, E., Hill, W.S. and Dixon, R.A.F. (1988) Conservced aspartic acid residues 79 and 113 of the b-adrenergic receptor have different roles in receptor function. J. Biol. Chem. 263:10267-10271. Strader, C.S., Sigal, I.S. and Dixon R.A.F. (1989) Structural basis of b-adrenergic receptor function. FASEB J. 3: 1825-1832. Suarez-lsla, B.A., Irribarra, V., Oberhauser, A., Larralde, L, Bull, Rl, Hidalgo, C. and Jaimovich, E. (1988) Inositol (1,4,5,)-triphosphate activate a calcium channel in isolated sarcoplasmic reticulum membrane. Biophys. J. 54: 737-741. Sutherland, E. W., Robison, G. A. and Butcher, R. W. (1968) Some aspects of the biological role of adenosine 3',5'-monophosphate (cyclic AMP). Circulation 37: 279-306. Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) Phosphorylation of a 22,000-dalton component of the cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase. J. Biol. Chem. 250: 2640-2647. TenEick, R., Nawrath, H., McDonald, T. F. and Trautwein, W. (1976) On the mechanism of the negative inotropic effect of acetylcholine. Pfluger's Arch. 361: 207-213. 92 Tsien, R. W. (1977) Cyclic AMP and contractile activity in heart. In Advances in Cyclic Nucleotide Research, ed. by P. Greengard and G. A. Robison, vol. 8, pp. 363-420, Raven Press, New York. Urquhart, R.A., Rothaul, A.L. and Broadley, K. (1991) 86Rubidium efflux and negative inotropy induced by PI- and muscarinic receptor agonists in guinea pig left atria. Effects of potassium channel blockers. Biochem. Pharmacol. 42: 655-662. Vanoli, E., De Ferrari G.M., Stramba-Badiale, M., Hull, S.S., Foreman, R.D. and Schwartz, P.J. (1991) Vagal stimulation and prevention of sudden death in conscious dogs with a healed myocardial infarction. Circ Res. 68:1471-81. Vegesna, R.V. and Diamond, J. (1983) Comparison of the effects of forskolin and isoproterenol on cyclic AMP levels and tension in bovine coronary artery. Can. J. Physiol. Pharmacol. 61:1202-1205. Wagoner, P.K. and Pallotta, B.S. (1988) Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP. Science 240, 1655-1657. Wahler, G.M. and Sperelakis, J. (1986) Cholinergic attenuation of the electrophysiological effects of forskolin. J. Cyc. Nucl. Res. 11:1-10. Wahler, G.M. and Sperelakis, N. (1985) Intracellular injection of cyclic GMP depresses cardiac slow action potentials. J. Cyc. Nucl. Res. 10: 83-95. Waldeck, B. and Widmark, E. (1985) Comparison of the effects of forskolin and isoprenaline on tracheal, cardiac and skeletal from guinea-pig. Eur. J. Pharmacol. 112:349-353. Watanabe, A.M. and Besch, H.R. (1975) Interaction between cyclic adenosine monophosphate and cyclic guanosine monophosphate in guinea pig ventricular myocardium. Circ. Res. 37: 309-317. Watanabe, A.M., Ahmad, F.G. (1989) Studies on the cellular mechanisms of action of positive and negative inotropic agents, pp. 19-22. Inotropic stimulation and myocardial energetics. Eds. Scholz, H.H. Watanabe, A.M., Lindemann, J.P. and Fleming, J.W. (1984) Mechanisms of muscarinic modulation of protein phophorylation in intact ventricles. Fedn Proc. 43:2618-2623. Watanabe, A.M., McConnaughey, M.M., Strawbrige, R.R., Fleming, J.W., Jone, L.R. and Besch, H.R. (Jr) (1978) Muscarinic cholinergic receptor modulation of beta adrenergic receptor affinity for catecholamines. J. Biol. Chem. 253: 4833-4836. 93 Wei, J.E, and Sulakhe, P. (1978) Regional and subcelluair distribution of myocardial muscarinic cholinergic receptors. Eur. J. Pharmacol. 52: 235-238. Wheatley, M., Hulme, E.C., Birdsall, N.J.M., Curtis, C.A.M. and Eveleigh, P. (1988) Peptide mapping studies on muscarinic receptors; Receptor structure and localization of the ligand binding site. Trends Pharmacol. Sci. Suppl. 8:19-24. Yatani A. and Brown A.M. (1989) Rapid beta-adrenergic modulation of calcium channel currents by a fast G protein pathway. Science 245: 71-74. Zhu G-Y, Chen S-G, and Zou C-M (1990) Protective effects of an adenosine deaminase inhibitor on ischaemia-reperfusion injury in isolated perfused heart. Am. J. Physiol. 259 (Heart Circ Physiol 28):H835-838. Zunkler. B.J., Trube, G. and Ohno-Shosaku, T. (1988) Forskolin-induced block of delayed rectifying K+ channels in pancreatic b-cells is not modified by cAMP. Pflugers Arch. 411:613-619. 94 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0087347/manifest

Comment

Related Items