August M. Watanabe

Cyclic Adenosine Monophosphate Modulation of Slow Calcium Influx Channels in Guinea Pig Hearts

The relationship between cellular levels of cyclic adenosine monophosphate (AMP) and slow inward calcium ion (Ca2+) current was studied in isolated perfused guinea pig hearts in which fast sodium ion (Na+) channels had been inactivated by depolarization with potassium ions (K+) or blockade with tetrodotoxin. Perfusion with 22 mM K+ depolarized cardiac tissue to approximately −40 mv and rendered hearts inexcitable. Tetrodotoxin (3 × 10−5M) blocked excitability without altering resting membrane potential. Excitability and contractions could be restored to these hearts with a variety of inotropic agents that also raised the measured tissue levels of cyclic AMP or with high concentrations of Ca2+. The magnitude of steady-state tension developed by restored hearts was directly related to the external Ca2+ concentration as well as to the concentration of the restoring agent used. The tension of restored hearts was markedly reduced by Ca2+-channel antagonists. Elevation of cyclic AMP levels preceded restoration of excitability to inactivated hearts. A highly significant positive correlation was found between the magnitude of Ca2+-dependent tension developed by restored hearts and the level of cyclic AMP in those hearts. Glucagon and ouabain, inotropic drugs that do not elevate myocardial levels of cyclic AMP, failed to restore depolarized or tetrodotoxin-blocked hearts. Therefore, cyclic AMP appears to modulate slow Ca2+ influx channels in myocardial cells.

Signal transduction by G proteins in cardiac tissues.

The role of G proteins in mediating the responses of the heart to circulating catecholamines and to the influences of the autonomic nervous system is of special interest to cardiologists. It is evident that G proteins are essential links in the cascade of biochemical events that ensure when neurotransmitters and hormones interact with receptors on myocardial cells. It is likely [corrected] that dysfunction of G proteins plays a role in cardiovascular pathophysiology. With current methodologies, especially molecular biological and recombinant DNA techniques, and with transgenic animal models that can relate physiological function and specific gene dosage, some cardiovascular diseases may be traced to G protein-related defects.

Denervation supersensitivity of refractoriness in noninfarcted areas apical to transmural myocardial infarction.

Denervation supersensitivity was demonstrated in anesthetized dogs 5 to 10 days after transmural myocardial infarction produced by latex embolization of a diagonal branch of the left anterior descending coronary artery. Sympathetic efferent denervation in noninfarcted myocardium apical to the infarction was demonstrated by a 90% depletion of myocardial norepinephrine content in the apical (45 +/- 15 pg norepinephrine/g tissue) vs basal (437 +/- 76 pg/g tissue) regions and by the lack of effective refractory period (ERP) shortening during bilateral ansae subclaviae stimulation in 34% of sites apical to the infarction. Supersensitivity in the area apical to the infarction was manifested by an exaggerated shortening of the ERP during both norepinephrine and isoproterenol infusions, with an upward and leftward shift in the dose-response curves in the apical vs basal regions (p less than .001). The cellular mechanism for denervation supersensitivity did not involve detectable changes in the beta-adrenergic receptor adenylate cyclase system. There was no difference in the density of beta-adrenergic receptors ([125I]-cyanopindolol) in the apical (268.6 +/- 22.7 fmol/mg protein) vs the basal (253.5 +/- 24.8 fmol/mg protein) regions. Adenylate cyclase activity stimulated by guanosine triphosphate plus isoproterenol was slightly greater in the apical (58.7 +/- 17.4%) than in the basal (49.6 +/- 10.9%) region, but this difference did not reach statistical significance (p = .068). Muscarinic modulation of beta-receptor coupling (oxotremorine attenuation of guanosine triphosphate plus isoproterenol-stimulated adenylate cyclase activity) also was not significantly different at the apical (31.6 +/- 17.5% inhibition) and basal (21.4 +/- 20.9% inhibition) sites. These data show that a transmural myocardial infarction produces denervation supersensitivity in areas apical to the infarction, but in this preparation no differences in the total number or a redistribution of beta-adrenergic receptors or adenylate cyclase activity were detected.

Evidence for isoproterenol-induced phosphorylation of phosphatase inhibitor-1 in the intact heart.

The positive inotropic effect of the beta-adrenoceptor agonist isoproterenol is accompanied by inhibition of phosphatase type 1 activity in myocardium. Indirect assays suggest that this effect is due to activation of protein phosphatase inhibitor-1, which inhibits phosphatase activity only when phosphorylated. To test this hypothesis directly, electrically stimulated (3 Hz) guinea pig ventricular preparations were perfused according to the Langendorff method with physiological buffers with or without 5 mCi 32P/heart, and then various concentrations of isoproterenol were applied. Contractility was recorded. Hearts were freeze-clamped and cAMP and inhibitor-1 activities were measured. In 32P-labeled hearts a protein at about 26 kd on autoradiograms of 12% sodium dodecyl sulfate gels was detected. Isoproterenol (1 microM) increased rate of tension development to 238% of the predrug value, cAMP concentrations 1.5-fold, and inhibitor-1 activity threefold. Concomitantly, there was an increase in a 32P-labeled band at about 26 kd from 380 to 540 pmol 32P/mg protein. This protein at about 26 kd, after transfer to nitrocellulose, was recognized by an antiserum prepared against rabbit skeletal muscle inhibitor-1. More radioactive protein of about 26 kd could be immunoprecipitated by the antiserum from isoproterenol-treated than from untreated hearts. It is concluded that a protein, probably identical to phosphatase inhibitor-1, is phosphorylated in vivo in the heart in the presence of isoproterenol. Phosphorylation of inhibitor-1 with consequent modification of type 1 phosphatase activity may contribute to the effects of isoproterenol in the heart.

Characterization of [3H](+/-)carazolol binding to beta-adrenergic receptors. Application to study of beta-adrenergic receptor subtypes in canine ventricular myocardium and lung.

[3H](±)Carazolol, a newly available β-adrenergic receptor antagonist, has been used to characterize β-adrenergic receptor subtypes present in membrane vesicles derived from canine ventricular myocardium and canine lung. [3H](±)Carazolol binding is saturable, of high affinity, and in displaceable by β-adrenergic agents in accordance with their known pharmacological potencies. The interaction of carazolol with β-adrenergic receptors is stereospeclflc; the (−) stereoisomer demonstrates greater potency than the (+) stereoisomer. Kinetic analysis of [JH}(±)carazolol interaction with β-adrenergic receptors suggests the presence of two phases of interaction, consistent with initial rapidly reversible “low” affinity association of ligand with receptor, followed by Isomerization to form a high affinity, slowly reversible complex. Through use of a [3H](±)carazolol binding assay based on the high affinity complex, pharmacological specificities of £-adrenergic receptor populations of canine myocardium and lung were quantified. Analysis using computer-assisted techniques suggests a β1/β2 receptor ratio of approximately 85%/15% for canine myocardium and 5%/95% for canine lung. In the absence of added guanine nucleotides, comparison of potencies of β-adrenergic agonists in the two membrane systems suggests significant β, selectivity of l-isoproterenol and l-eplnephrine, and nonselectivity of norepinephrine. In the presence of saturating levels of guanine nucleotides, comparison of agonist potencies confirms the non-selectivity of l-isoproterenol and l-epinephrine, and β1 selectivity of norepinephrine. These results demonstrate that the state of guanine nucleotide regulation of receptors should be defined when examining agonist selectivities for β-adrenergic receptor subtypes in vitro.

Isolation and characterization of adult rat heart cells

Abstract Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca 2+ at concentrations greater than 10 −5 m .

Anticholinergic effects of disopyramide and quinidine on guinea pig myocardium. Mediation by direct muscarinic receptor blockade.

We studied the interaction of disopyramide, quinidine, and procainamide with cardiac muscarinic receptors. In electrophysiological experiments, the effects of disopyramide, quinidine, procainamide, and atropine were determined on spontaneously depolarizing guinea pig right atria (GPRA) both in the presence and absence of pharmacologically induced (physostigmine) cholinergic stimulation. All four agents demonstrated a concentration-dependent antagonism of the negative chronotropic effects of physostigmine. The order of anticholinergic potency was atropine » disopyr-amide > quinidine » procainamide. The ability of disopyramide to antagonize the physostigmine induced slowing was stereoselective, (+)disopyramide > (—)disopyramide. In contrast, the ability of quinidine to antagonize the negative chronotropic effects of physostigmine was non-stereoselective, quinidine = quinine. In parallel experiments, we studied the ability of disopyramide, quinidine, procainamide, and atropine to compete with the radiolabeled muscarinic receptor antagonist [3H] quinuclidinyl benzilate ([3H]QNB) for binding to muscarinic receptors in crude homogenates of GPRA and membrane vesicles from canine ventricular myocardium. All four agents inhibited [3H]QNB binding to muscarinic receptors. The order of anticholinergic potency determined by the receptor binding studies was identical to that determined by the physiological studies. The interaction of disopyramide with muscarinic receptors was stereoselective, (+)disopyramide > (−)disopyramide. Quinidine was only slightly more potent than quinine in inhibiting [3H]QNB binding to muscarinic receptors. Inter-action of antiarrhythmic drugs with muscarinic receptors satisfied criteria for a competitive interaction. The data from this study localize the anticholinergic effects of disopyramide and quinidine to the muscarinic receptor. Circ Res 47: 855-865, 1980

Thyroxine and propylthiouracil effects on alpha- and beta-adrenergic receptor number, ATPase activities, and sialic acid content of rat cardiac membrane vesicles

Membrane vesicle preparations from the hearts of euthyroid, hyperthyroid, and hypothyroid rats were analyzed in an attempt to identify biochemical changes which might correlate with known functional changes occurring in these thyroid states. Several sarcolemmal constituents which have been shown to influence myocardial contractility were measured in membrane vesicle preparations from the hearts of animals in the three thyroid states. These constituents included the apparent number of alpha- and beta-adrenergic receptors (judged from specific binding of radiolabeled adrenergic antagonists) and Na-, K--ATPase activity. As a control for the recovery of sarcolemma in the preparations, the sialic acid content was measured in all preparations. The activity of K-, Ca2--ATPase, a sarcoplasmic reticulum enzyme which regulates intracellular ionized Ca2- concentration, was also measured. Membrane vesicles of the thyroxine-treated hyperthyroid rats showed a decrease (41%, p < 0.01) in the total apparent number of alpha-adrenergic receptors relative to euthyroid controls and an increase in the total apparent number of beta-adrenergic receptors (43%, p < 0.001). Membrane vesicles prepared from propylthiouracil-treated hypothyroid rats showed a decrease relative to euthyroid controls in the total apparent number of both alpha-adrenergic receptors (29%, p < 0.01) and beta-adrenergic receptors (23%, p < 0.05). The ratio of beta- to alpha-receptors approximately doubled in the hyperthyroid animals but remained unchanged compared to controls in the membranes isolated from hypothyroid animals. In the same membrane vesicle preparations, total sialic acid content was similar regardless of thyroid state. In addition.

Muscarinic receptor regulation of cardiac adenylate cyclase activity.

Stimulation and inhibition of adenylate cyclase activity are mediated by the guanine nucleotide regulatory proteins Gs and Gi, respectively. Two general mechanisms have been proposed for the inhibition of activated adenylate cyclase: direct inhibition of the catalyst by Gi, and indirect inhibition of the activated catalyst mediated by Gi inhibition of Gs. We have assessed direct inhibition of adenylate cyclase by evaluating the ability of Gpp(NH)p to inhibit the forskolin-stimulated enzyme in the presence of various concentrations of magnesium ions and the guanine nucleotide. Gpp(NH)p inhibition of adenylate cyclase activity was only observed in the presence of forskolin and low concentrations of MgCl2. Muscarinic agonists did not increase Gpp(NH)p inhibition of the forskolin-stimulated enzyme, even in the presence of low concentrations of MgCl2 and guanine nucleotide (near the respective Kact or Ki). Whether in the absence or presence of muscarinic agonists, no concentration of Gpp(NH)p was found to inhibit basal adenylate cyclase activity in the absence of forskolin. In addition, muscarinic agonists had no effect on the rate constant (kon) for Gpp(NH)p activation of the enzyme. In contrast to these data, the muscarinic agonist methacholine stimulated the inactivation rate constant (koff) for isoproterenol plus GTP-activated adenylate cyclase activity 15-fold, and the increase in koff was blocked by atropine. Moreover, the sarcolemma displayed specific, high affinity GTP hydrolytic activity which was stimulated by methacholine activation of muscarinic receptors. These data further support our original hypothesis, indicating that although direct inhibition of the catalyst by Gi may occur in cardiac sarcolemma, physiologically relevant attenuation of adenylate cyclase activity by muscarinic agonists occurs by a mechanism linked to GTP hydrolysis.

Acetylcholine antagonism of the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers.

The purpose of these experiments was to determine whether or not acetylcholine modulated the electrophysiological effects of isoproterenol on canine cardiac Purkinje fibers. Conventional microelectrode techniques were used. Predictably, isoproterenol produced shortening of action potential duration; acetylcholine significantly blunted this effect of isoproterenol. Isoproterenol restored excitability to fibers exposed to 22 mM potassium solutions, and acetylcholine abolished this isoproterenol-restored excitability. Both of these antagonistic effects of acetylcholine were blocked by atropine. Acetylcholine alone did not affect action potential duration in polarized fibers or excitability in potassium-depolarized fibers. Furthermore, acetylcholine had no effect on the decrease in action potential duration induced by premature electrical stimulation or by acetylstrophanthidin administration, or on excitability of fibers exposed to a zero sodium, high calcium superfusant-These data demonstrate a direct cellular basis for cholinergic antagonism of the electrophysiological effects of β-adrenergic stimulation of canine cardiac specialized intraventricular conducting tissue. cire Res 44: 378-383, 1979