Norepinephrine down-regulates the activity of protein S on endothelial cells.

The adrenergic agonist norepinephrine is shown to stimulate endothelium to induce protein S release and degradation, leading to diminished anti-coagulant activity and to down-regulation of protein S cell surface-binding sites. Norepinephrine-induced release of intracellular protein S was blocked by the alpha 1-adrenergic antagonist prazosin (10(-7) M) but not by the alpha-adrenergic antagonist propranolol (10(-6) M) or the alpha 2-adrenergic antagonist yohimbine (10(-5) M) indicating that this response resulted from the specific interaction of norepinephrine with a class of alpha 1-adrenergic receptors not previously observed on endothelium. Attenuation of norepinephrine-induced release of protein S by pertussis toxin in association with the ADP-ribosylation of a 41,000-D membrane protein indicates that this intracellular transduction pathway involves a regulatory G protein. The observation that protein S was released from endothelium in response to maneuvers which elevate intracellular calcium or activate protein kinase C suggests that the response may be mediated via intermediates generated through the hydrolysis of phosphoinositides. Morphologic studies were consistent with a mechanism in which norepinephrine causes exocytosis of vesicles containing protein S. In addition to release of protein S, norepinephrine also induced loss of endothelial cell protein S-binding sites, thereby blocking effective activated protein C-protein S-mediated factor Va inactivation on the cell surface. Norepinephrine-mediated endothelial cell stimulation thus results in loss of intracellular protein S and suppression of cell surface-binding sites, modulating the anti-coagulant protein C pathway on the vessel wall. These studies define a new relationship between an anti-coagulant mechanism and the autonomic nervous system, and indicate a potential role for an heretofore unrecognized class of alpha 1-adrenergic receptors in the regulation of endothelial cell physiology.

Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the alpha 1-receptor.

During development, the chronotropic response of rat ventricular myocardium to alpha 1-adrenergic stimulation changes from positive to negative. The alpha 1-agonist phenylephrine increases the rate of contraction of neonatal rat myocytes cultured alone but decreases the rate of contraction when the myocytes are cultured with functional sympathetic neurons. The developmental induction of the inhibitory myocardial response to alpha 1-adrenergic stimulation in intact ventricle and in cultured myocytes was shown to coincide with the functional acquisition of a substrate for pertussis toxin. A 41-kilodalton protein from myocytes cultured with sympathetic neurons and from adult rat myocardium showed, respectively, 2.2- and 16-fold increases in pertussis toxin-associated ADP-ribosylation (ADP, adenosine diphosphate) as compared to controls. In nerve-muscle cultures, inhibition of the actions of this protein by pertussis toxin-specific ADP-ribosylation reversed the mature inhibitory alpha 1-adrenergic response to an immature stimulatory pattern. The results suggest that innervation is associated with the appearance of a functional pertussis toxin substrate by which the alpha 1-adrenergic response becomes linked to a decrease in automaticity.

Scaffolding proteins in cardiac myocytes.

Post-translational modification, such as protein phosphorylation, plays a critical role to reversibly amplify and modulate signaling pathways. Since kinases and phosphatases have broad substrate recognition motifs, compartmentalization and localization of signaling complexes are required to achieve specific signals. Scaffolds are proteins that associate with two or more binding partners and function to enhance the efficiency and/or specificity of cellular signaling pathways. The identification of scaffolding proteins that control the tempo and/or spatial organization of signaling pathways in cells has benefited enormously from recent technological advances that allow for the detection of protein-protein interactions, including in vivo in intact cells. This review will focus on scaffolding proteins that nucleate multi-protein complexes (and could represent novel entry points into signaling pathways that might be amenable to therapeutic manipulation) in cardiomyocytes.

Hypoxia alters the subcellular distribution of protein kinase C isoforms in neonatal rat ventricular myocytes.

Cardiac myocytes coexpress multiple protein kinase C (PKC) isoforms which likely play distinct roles in signaling pathways leading to changes in contractility, hypertrophy, and ischemic preconditioning. Although PKC has been reported to be activated during myocardial ischemia, the effect of ischemia/hypoxia on individual PKC isoforms has not been determined. This study examines the effect of hypoxia on the subcellular distribution of individual PKC isoforms in cultured neonatal rat ventricular myocytes. Hypoxia induces the redistribution of PKC alpha and PKC epsilon from the soluble to the particulate compartment. This effect (which is presumed to represent activation of PKC alpha and PKC epsilon) is detectable by 1 h, sustained for up to 24 h, and reversible within 1 h of reoxygenation. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) prevents the hypoxia-induced redistribution of PKC alpha and PKC epsilon, whereas chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) blocks the redistribution of PKC alpha, but not PKC epsilon; D609 and BAPTA do not influence the partitioning of PKC alpha and PKC epsilon in normoxic myocytes. Hypoxia, in contrast, decreases the membrane association of PKC delta via a mechanism that is distinct from the hypoxia-induced translocation/activation of PKC alpha/PKC epsilon, since the response is slower in onset, slowly reversible upon reoxygenation, and not blocked by D609 or BAPTA. The hypoxia-induced shift of PKC delta to the soluble compartment does not prevent subsequent 4-beta phorbol 12-myristate-13-acetate-dependent translocation/activation of PKC delta. Hypoxia does not alter the abundance of any PKC isoform nor does it alter the subcellular distribution of PKC lambda. The selective hypoxia-induced activation of PKC isoforms through a pathway involving phospholipase C (PKC alpha/PKC epsilon) and intracellular calcium (PKC alpha) may critically influence cardiac myocyte contractility, gene expression, and/or tolerance to ischemia.

Expression of protein kinase C beta in the heart causes hypertrophy in adult mice and sudden death in neonates.

Protein kinase C (PKC) activation in the heart has been linked to a hypertrophic phenotype and to processes that influence contractile function. To establish whether PKC activation is sufficient to induce an abnormal phenotype, PKCbeta was conditionally expressed in cardiomyocytes of transgenic mice. Transgene expression in adults caused mild and progressive ventricular hypertrophy associated with impaired diastolic relaxation, whereas expression in newborns caused sudden death associated with marked abnormalities in the regulation of intracellular calcium. Thus, the PKC signaling pathway in cardiocytes has different effects depending on the timing of expression and, in the adult, is sufficient to induce pathologic hypertrophy.

Coupling function of endogenous alpha(1)- and beta-adrenergic receptors in mouse cardiomyocytes.

Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. beta(1)-ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective beta(2)-AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. beta(2)-AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. alpha(1)-ARs fail to activate phospholipase C, the extracellular signal-regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated by Pasteurella multocida toxin (a G(q) alpha-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via beta-ARs in mouse cardiomyocytes, but beta-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in alpha(1)-AR signaling and a defect in beta(2)-AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.

Signaling properties and functions of two distinct cardiomyocyte protease-activated receptors.

Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal-regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal-regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH(2)-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by alpha(1)-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.

Sympathetic innervation modulates ventricular impulse propagation and repolarisation in the immature rat heart.

OBJECTIVE: When cardiac sympathetic innervation in neonatal rats is retarded by antiserum to nerve growth factor, there is a corresponding increase in the QT interval on ECG. Since the propagation of the cardiac impulse and the repolarisation of cardiac cells both contribute to the QT interval, the aim of this study was to determine the role of sympathetic innervation in modulating ventricular impulse propagation and repolarisation. METHODS: Neonatal rats were treated for the first 10 days of life with nerve growth factor (NGF), its antiserum (As), or placebo. Standard microelectrode techniques were used to study the transmembrane action potential characteristics of subendocardial (ventricular septal) and subepicardial ventricular myocardium. Bipolar surface electrograms were used to record the velocity of impulse propagation and electron microscopy to examine the intercalated discs. RESULTS: In the subendocardium, the phase 0 upstroke velocity of the action potential (dV/dtmax) was lowest in the As treated rats. The latter group also showed the slowest conduction velocity. There were no differences in control action potential durations in the endocardium among the three groups, but in the epicardial tissues, action potential duration was longest in the As treated group. Thus the dispersion in action potential duration was smallest in the As treated animals. Electron microscopic studies of the intercalated discs of ventricular myocytes showed significant enhancement of nexal junction formation in NGF treated rats, whereas As treated animals showed a retarded pattern of both nexal and desmosomal junction formation. CONCLUSIONS: The differences in ultrastructure, conduction, and repolarisation seen in As and NGF treated animals may explain the prolonged QT interval seen in the As treated group.

Deaminovasopressin has direct and modulatory effects on ventricular automaticity in the rat heart.

OBJECTIVE: Some neuropeptides have direct cardiac effects and also modulate the cardiac effects of catecholamines. Vasopressin is an abundantly available neuropeptide having well known interactions with catecholamines in vascular smooth muscle. The aim of this study was to determine the direct and modulatory effects of vasopressin on ventricular automaticity. METHODS: The cardiac effects of deaminovasopressin (dAVP), a long acting synthetic analogue of vasopressin, were tested on basal and alpha 1 agonist induced changes in automaticity in isolated ventricular septal preparations from adult and neonatal rats after chronic exposure (10 micrograms.kg-1 x d-1 subcutaneously for 10 d) and acute exposure (in vitro bath superfusion with 10(-8) M dAVP for 1 h). RESULTS: Chronic exposure to dAVP decreased basal ventricular automaticity in the adult and in 10-11 d old rats. Although alpha 1 agonists tended to decrease automaticity in adult rat heart, prior chronic dAVP exposure altered the chronotropic response to alpha 1 agonist so that only an increase in automaticity was observed. A similar result was seen in adult ventricular septal preparations upon acute superfusion with dAVP. Acute dAVP exposure reduced basal ventricular automaticity, and modified the alpha 1 adrenergic chronotropic response, such that only an increase in automaticity occurred. Acute dAVP exposure in adult ventricular septal preparations did not significantly change total alpha 1 adrenergic receptor density or antagonist affinity, alpha 1 adrenergic receptor subtype expression, or the amount of pertussis toxin sensitive G protein measured in an ADP ribosylation assay. CONCLUSION: dAVP not only exerted direct effects of chronotropy, but also influenced the expression of alpha 1 adrenergic chronotropic responsiveness. If vasopressin has a similar action, this may have important implications in instances where levels of this peptide are raised. For example, surgical stress and cardiopulmonary bypass are clinical situations associated with increases in both vasopressin and catecholamine levels. An interaction between the two may contribute to the development of tachyarrhythmias in these settings.

beta(1) and beta(2)-adrenergic receptor subtype effects in German shepherd dogs with inherited lethal ventricular arrhythmias.

OBJECTIVE: Delayed afterdepolarization-induced triggered activity originating in ventricular myocardium is a mechanism for some age-dependent, inherited ventricular tachycardias in a colony of German shepherd dogs. METHODS: We used standard microelectrode techniques to study beta-adrenergic receptor subtype modulation of the triggered activity in anteroseptal left ventricular myocardium from eleven of these dogs and seven unafflicted, age-matched German shepherd controls. RESULTS: During sustained stimulation at cycle lengths of 300-4000 ms, 10(-9)-10(-7) M isoproterenol concentration-dependently shortened action potential duration (APD) to 90% repolarization more in myocardium from afflicted than from unafflicted dogs. This shortening was prevented by a beta(1)-blocker CGP20712A (10(-7) M) while a beta(2)-blocker ICI118551 (10(-7) M) did not modify the effect of isoproterenol in either group. The beta(2)-agonist zinterol 10(-8)-10(-6) M had no effect on APD. Stimulation at a cycle length of 250 ms in the presence of 10(-7) M isoproterenol induced more triggered AP in myocardium from afflicted than unafflicted dogs. beta(1)-Blockade completely eliminated, while beta(2)-blockade facilitated, and the beta(2)-agonist zinterol did not induce triggered activity in the two groups. CONCLUSION: Isoproterenol effects on APD and triggered activity in the myocardium of dogs with inherited arrhythmias are due primarily to an abnormality of beta(1)-adrenoceptor mediated signaling that is subject to beta(2)-adrenergic modulation.

Abnormal cardiac repolarization and impulse initiation in German shepherd dogs with inherited ventricular arrhythmias and sudden death.

OBJECTIVE: We tested the hypothesis that delayed afterdepolarization (DAD)-associated rhythms in German shepherd dogs with reduced anteroseptal left ventricular (LV) sympathetic innervation derive from abnormal beta-adrenergic receptor effector coupling. METHODS AND RESULTS: In anteroseptal LV midmyocardium of afflicted dogs, beta-receptor density was greater than that in normal dogs (P < .05), with affinity being equal in both groups. Basal and maximum isoproterenol (ISO) stimulated adenylyl cyclase activity of anteroseptal LV of afflicted dogs was greater than that in normal dogs (P < .05). Isolated anteroseptal M cell preparations of afflicted dogs studied with microelectrodes showed abnormal lengthening, rather than shortening of action potential duration in response to ISO, as well as a 61% incidence of 10(-7) mol/l ISO-induced triggered activity as compared to 12% in normals (P < .05). In contrast, there was no difference between afflicted and control dogs in triggered activity, beta-receptors or adenylyl cyclase activity in a normally innervated region of the ventricles. CONCLUSION: In this model there is an increase in beta-receptor density and beta-adrenergic stimulation of adenylyl cyclase and of triggered activity in anteroseptal myocardium but not in a normally innervated region of the heart. Hence, abnormal beta-adrenergic signal transduction appears associated with the neural abnormality identified in dogs with inherited VT.

A pertussis toxin substrate regulates alpha 1-adrenergic dependent phosphatidylinositol hydrolysis in cultured rat myocytes.

The chronotropic response of the heart to alpha 1-adrenergic catecholamines influenced by pertussis toxin under certain conditions. In view of the fact that alpha 1-adrenergic action is mediated by the phosphatidylinositol pathway of hormone action in many cells, we examined the hypothesis that alpha-adrenergic agonists stimulate phosphatidylinositol hydrolysis in cardiomyocytes and that this effect is sensitive to pertussis toxin. Addition of norepinephrine to cultured rat ventricular myocytes prelabeled with myo-[2-3H]inositol resulted in rapid and significant accumulation of inositol phosphate (IP1) and inositol biphosphate. Norepinephrine-stimulated IP1 formation was not inhibited by propranolol, but was inhibited by alpha-adrenergic antagonists with an order of potency indicating alpha 1-adrenergic receptor subselectivity: prazosin (alpha 1; 3 nM) greater than yohimbine (alpha 2; 10 microM). The effect of norepinephrine to enhance IP1 formation was markedly attenuated in cells pretreated with pertussis toxin. Pertussis toxin also induced the transfer of ADP-ribose from NAD to a 41,000-dalton membrane protein in these cells. The concentration of pertussis toxin resulting in maximal inhibition of norepinephrine-stimulated IP1 formation correlated well with the concentration of pertussis toxin necessary to completely ADP-ribosylate a 41,000-dalton membrane protein (1 ng/ml). The range over which pertussis toxin inhibited norepinephrine-dependent IP1 formation and ADP-ribosylated the 41,000-dalton substrate was virtually identical. These observations establish a role for a 41,000-dalton pertussis toxin substrate in coupling the alpha 1-adrenergic receptor to phosphoinositol hydrolysis in myocardial cells.

Total and free propranolol levels in sensitive and resistant patients.

The clinical effects of the beta-adrenergic inhibitor, propranolol, are believed to correlate best with circulating levels of free drug, the component of total drug concentration that is not bound to plasma proteins and is therefore free to interact with tissue beta receptors. We used the radioreceptor assay for propranolol to determine total and free propranolol levels in 32 hospitalized patients receiving a wide range of doses (40 to 1280 mg/day). Free propranolol was determined by equilibrium dialysis and by the erythrocyte drug level. Erythrocyte levels were found to correlate strongly with free drug, as determined by equilibrium dialysis. Mean percent free propranolol was 16.9 +/- 2.4 ng/ml (SEM) by equilibrium dialysis and 18.8 +/- 2.3 ng/ml (SEM) by the red cell level. Despite considerable variability in the actual level achieved at any given dose, total and free propranolol concentrations were related to dose. A weaker correlation for free drug concentration resulted from variability in free drug among responsive patients (mean +/- SD free propranolol was 13.5 +/- 8.8 ng/ml), and suggested a threshold level of free drug required to achieve clinical effects. Six patients were resistant to high doses of propranolol. Excessive protein binding was noted in four, suggesting abnormalities at the level of protein-drug interaction. Several other possible mechanisms for propranolol resistance became apparent.

Therapy of hypercalcemia due to parathyroid carcinoma with intravenous dichloromethylene diphosphonate.

Dichloromethylene diphosphonate (Cl2MDP) a potent inhibitor of osteoclast-mediated bone resorption, lowers serum calcium in hypercalcemia associated wit malignancies and with primary hyperparathyroidism, and reduces excess calcium mobilization from bone in multiple myeloma and in Paget's disease. We have evaluated the effectiveness of intravenously administered Cl2MDP in five patients with parathyroid carcinoma, a disorder characterized by severe hypercalcemia, very high parathyroid hormone (PTH) levels, and marked osteoclast-mediated bone resorption. All patients had biopsy-proved metastatic parathyroid carcinoma and hypercalcemia which persisted after multiple surgical procedures and other attempts at management. During a three-day observation period, each patients continued to demonstrate stable or progressive hypercalcemia despite infusion with saline solution and furosemide. Cl2MDP was administered over 2 hours at 2.5 mg/kg on day 1 and 5 mg/kg on days 2 through 7. Response was noted in all five patients; there was a gradual decline in the average serum calcium from 16.0 +/- 1.1 mg/dl (SEM) to 11.1 +/- 0.9 mg/dl by the eighth day (p less than 0.01). There were concomitant reductions in urinary calcium excretion, from 798 +/- 153 mg/g creatinine to 350 +/- 96 mg/g creatinine (p less than 0.05) and in the urinary hydroxyproline excretion, from 155 +/- 38 mg/g creatinine to 94 +/- 29 mg/g creatinine (p less than 0.02). Serum PTH levels remained markedly elevated (460 +/- 141 micrograms eq/ml to 493 +/- 169 micrograms eq/ml). In three patients, all indices returned to pretreatment levels by 10 days after the last infusion. In two of these patients there was a response to retreatment with Cl2MDP with a fall in calcium from 16.9 +/- 0.5 mg/dl to 12.4 +/- 1.5 mg/dl. There was no response in one patient. No adverse reactions to Cl2MDP were observed. The decrease in serum calcium and concomitant declines in urinary calcium and hydroxyproline suggest that Cl2MDP can effectively inhibit the excessive bone resorption associated with parathyroid carcinoma.

Tissue-specific developmental regulation of protein kinase C isoforms.

The limited amount of available information regarding the developmental control of protein kinase C (PKC) isoform expression restricts our understanding of the role of these enzymes in normal physiology. Accordingly, this study investigated PKC isoform expression in selected tissues from fetal, neonatal, and adult rats. PKC beta immunoreactivity was prominent in brain tissue, whereas the expression of PKC alpha, PKC delta, PKC epsilon, and PKC zeta was found to be widespread. Although no developmental change in any PKC isoform was evident in liver, striking tissue-specific age-dependent differences in PKC isoform abundance were noted in other tissues. For example, age-dependent increases in PKC alpha, PKC beta, and PKC delta in brain contrasted with age-dependent decreases in PKC alpha and PKC delta in lung, kidney, and heart. Immunoreactivity for PKC epsilon was abundant in all fetal/neonatal tissues; PKC epsilon was detected in the adult brain, heart, and liver, but not the adult kidney and lung. Finally, PKC zeta was more abundant in fetal/neonatal than in adult brain, lung, kidney, and heart. These results indicate that the fetal/neonatal lung, kidney, and heart are enriched in PKC zeta, PKC alpha, PKC delta, and PKC epsilon, relative to the adult tissues. These age-dependent variations in the abundance of individual isoforms of PKC may critically influence tissue responsiveness to external stimuli. Moreover, the finding that PKC zeta is particularly abundant in fetal tissues as well as the liver, the only tissue included in this study which retains regenerative capacity in the adult animal, is consistent with the notion that PKC zeta may play a role in cell proliferation.

Stimulatory and inhibitory regulation of myocardial adenylate cyclase by 5'-guanylyl-imidodiphosphate.

Particulate and soluble rat myocardial adenylate cyclase enzymes were characterized with respect to their stimulatory and inhibitory regulation by Gpp(NH)p. Gpp(NH)p (60 microM) stimulated Mg2+- and Mn2+-dependent adenylate cyclase. High concentrations of Gpp(NH)p (600 microM) attenuated the maximal stimulatory response to Gpp(NH)p but only at low cation concentrations. The attenuating effects of 600 microM Gpp(NH)p resulted predominantly from the introduction of a prolonged lag in the kinetics of activation of adenylate cyclase. Steady-state rates of adenylate cyclase activities were similar with either 60 or 600 microM Gpp(NH)p. At any concentration of Gpp(NH)p, the lag was eliminated by Mg ions or isoproterenol. No antihysteretic property for free Mn ions was evident. Forskolin-sensitive particulate adenylate cyclase was not stimulated further by Gpp(NH)p. A 600 microM concentration of Gpp(NH)p inhibited particulate forskolin-sensitive adenylate cyclase at low Mg ion concentrations. In contrast, Gpp(NH)p at 60 microM consistently activated forskolin-sensitive adenylate cyclase after solubilization. The early transient inhibitory properties of 600 microM Gpp(NH)p which resulted in attenuation of adenylate cyclase by 600 microM Gpp(NH)p were diminished by detergent extraction, resulting in only a minor effect of 600 microM Gpp(NH)p to inhibit solubilized adenylate cyclase. These findings indicate that guanine nucleotides exert both stimulatory and inhibitory control upon the myocardial adenylate cyclase enzyme; that solubilization shifts the balance between the stimulatory and inhibitory properties of Gpp(NH)p to allow more dominant expression of the stimulatory response; and that Mg ions critically modify the nature of the myocardial adenylate cyclase response to Gpp(NH)p.

Sympathetic neural and alpha-adrenergic modulation of arrhythmias.

alpha 1-Adrenergic stimulation of the neonatal heart may induce either an increase or a decrease in ventricular automaticity, with the latter response predominating as age increases. We used isolated tissues from the hearts of neonatal and adult dogs and rats, as well as rat myocytes in tissue culture alone or in coculture with sympathetic nerves, to study the role of sympathetic innervation in modulating the alpha-adrenergic response. In the absence of sympathetic innervation, alpha-adrenergic stimulation uniformly increases automaticity. As the myocyte is innervated, an increased quantity of a GTP regulatory protein is detectable. That this protein is an essential transducer of alpha-adrenergic inhibition of automaticity is evidenced by the conversion of the alpha response from excitatory to inhibitory as the protein develops. ADP-ribosylation of the protein with pertussis toxin causes the alpha response to revert to excitation in both adult canine hearts and innervated myocytes in tissue culture. Hence, we have evidence for sympathetic modulation of cardiac rhythm via a regulatory protein whose function depends on normal neuronal development. Abnormal development of innervation may predispose to arrhythmogenesis via persistence of a primitive response to alpha stimulation.

Changes in adenylyl cyclase isoforms as a mechanism for thyroid hormone modulation of cardiac beta-adrenergic receptor responsiveness.

Although thyroid hormones are known to modulate cardiac beta-adrenergic receptor expression, the physiologic implications of these changes in the cardiac manifestations of altered thyroid hormone metabolism have been disputed. This study examined whether thyroid hormone modulates signaling via the cyclic adenosine monophosphate (cAMP) pathway by regulating cardiac adenylyl cyclase (AC) isoform expression. Northern blot analyses and AC enzyme assays were performed on preparations from hypothyroid, euthyroid, and hyperthyroid rat ventricles. Steady-state levels of cardiac AC mRNA types V and VI in hypothyroid ventricles were 173% +/- 8% and 149% +/- 12%, respectively, of the values in euthyroid ventricles (P < .01). This increase in AC mRNA isoforms was accompanied by a 1.5-fold increase (P < .05) in the activation of catalytic AC by forskolin and Mn. In contrast, the relative abundance of transcripts for types V and VI AC was similar in hyperthyroid and euthyroid ventricles, but catalytic AC activation by forskolin and Mn was significantly reduced by 35% in membranes obtained from hyperthyroid ventricles. AC activation through beta-adrenergic receptor stimulation by isoproterenol was not altered by thyroid hormone status. Thus, the effect of thyroid hormone to repress AC catalytic activity would be anticipated to offset the increase in beta-adrenergic receptor expression in hyperthyroidism. These studies identify cardiac AC enzymes as important targets for thyroid hormone-dependent regulation of signaling via the cAMP pathway, and support the finding that cardiac adrenergic responsiveness is unaltered in thyroid disease states.

Characterization of the alpha1-adrenergic chronotropic response in neuropeptide Y-treated cardiomyocytes.

The cardiac alpha1-adrenergic chronotropic response changes from stimulatory to inhibitory post-natally. The mature inhibitory response is mediated by the alpha1B-adrenoceptor and a pertussis toxin sensitive G protein. In vivo and in vitro studies identify sympathetic innervation as critical for the maturation of this inhibitory response. Additional experiments in a culture model indicate the effect of innervation is dependent on neurally released neuropeptide Y. The present study establishes that the individual signaling elements in the neuropeptide Y induced alpha1-adrenergic cascade are the same as those appearing during normal in vivo development. In addition, the data demonstrate that the effect of neuropeptide Y does not result from activation of the putative cardiac Y3 neuropeptide Y receptor subtype, since it is reproduced by the peptide fragment neuropeptide Y-(13-36) but not by [Leu31, Pro34]neuropeptide Y.

Endothelial cells contain beta adrenoceptors.

The direct identification of beta adrenoceptors in endothelial cell cultures has not been possible until the advent of a new beta-adrenergic radioligand, [125I]iodocyanopindolol ([125I]ICYP). Using [125I]ICYP, we report the successful identification of a beta adrenoceptor in cultured bovine aortic endothelial cells. At 37 degrees C, specific binding is saturable, stable and reversible. There is a single class of binding sites (21,500 +/- 2,900 sites/cell) with an equilibrium dissociation constant (Kd) of 109 +/- 26 pM. The rate constant of association, k1, is 1.22 X 10(9) M-1 min-1 and of dissociation, k-1, is 0.01 min-1. Binding studies on monolayers of endothelial cells grown in microtiter plates yield similar data (Kd = 53 +/- 9 pM, Bmax = 20,000 +/- 1,900 sites/cell). Stereoselectivity of binding for the (-)-isomer is demonstrable for both agonists and antagonists. A series of adrenergic agonists competes with [125I]ICYP for binding with an order of potency suggesting beta 2 subselectivity ; isoproterenol (0.73 microM) greater than epinephrine (15 microM) greater than norepinephrine (71 microM). Furthermore, the beta 2 inhibitor butoxamine is more potent than the beta 1 inhibitor practolol (7.7 microM vs 22 microM respectively). The GTP analogue, Gpp(NH)p, reduces isoproterenol affinity to 1.9 microM and increases the Hill coefficient from 0.62-0.90.