Dietary cholesterol influences cardiac beta-adrenergic receptor adenylate cyclase activity in the marmoset monkey by changes in membrane cholesterol status

The activity of the beta-adrenergic receptor/adenylate cyclase system of the marmoset monkey heart was investigated following dietary cholesterol supplementation (0.5%). After 22 weeks, plasma cholesterol levels in the cholesterol group were more than twice that of the control group. In the cholesterol-fed group, the affinity for ICYP binding to cardiac membranes was elevated more than 2-fold, while the receptor number was decreased by 31%. Isoproterenol, norepinephrine and sodium fluoride stimulated adenylate cyclase activity was significantly higher in the cholesterol-fed group although the fold stimulation over basal levels was not affected. The most prominent change in the cardiac membrane lipids was an increase in the cholesterol to phospholipid ratio in marmoset monkeys fed cholesterol. These results indicate that in the marmoset, membrane cholesterol is an important factor in determining various properties of the cardiac beta-adrenergic receptor particularly receptor affinity which may impact on the response of the beta-adrenergic receptor/adenylate cyclase system of the heart to catecholamines. This result is in agreement with dietary fatty acid supplements designed to increase cardiac membrane cholesterol in this animal species (McMurchie, E.J. et al. (1988) Biochim. Biophys. Acta 937, 347-358). Elevated membrane cholesterol enhances beta-adrenergic receptor affinity and certain aspects of adenylate cyclase activity. This is a likely mechanism whereby atherogenic diets could promote cardiac arrhythmia in non-human primates and indeed in man.     

Beta-adrenergic and antiadrenergic modulation of cardiac adenylyl cyclase is influenced by phosphorylation

Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A1 receptor (A1R). Because beta 1-adrenergic receptor (beta 1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the beta 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the beta 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140-120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51-54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29-50%, thereby yielding a total antiadrenergic effect of approximately 78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.     

Modification of beta-adrenoceptors and adenylyl cyclase in hearts perfused with hypochlorous acid

It is now well known that the signal transduction pathway involving beta-adrenoceptors and adenylyl cyclase is altered in ischemic heart disease. Since leukocytes accumulate in the ischemic heart and produce hypochlorous acid (HOCl), we investigated the effects of HOCl upon beta-adrenoceptors and adenylyl cyclase activities by perfusing rat hearts with 0.1 mM HOCl for 10 min and isolating cardiac membranes. Marked depressions in both the density and affinity of beta1-adrenoceptors were observed, whereas no significant change in the affinity or density of beta2-adrenoceptors was seen in hearts perfused with HOCl. After treatment of hearts with HOCl, competition curves using isoproterenol, a beta-adrenoceptor agonist, revealed a decrease in the proportion of high affinity binding sites. The adenylyl cyclase activities in the absence and presence of forskolin, NaF, Gpp(NH)p, or isoproterenol were depressed in hearts perfused with HOCl; however, the stimulatory effects of these agents on adenylyl cyclase were either unaltered or augmented. The presence of methionine in the perfusion medium prevented the HOCl-induced changes in beta1-adrenoceptors and adenylyl cyclase activity. These results suggest that HOCl may produce a defect in the beta-adrenoceptor linked signal transduction mechanism by affecting both beta1-adrenoceptors and adenylyl cyclase enzyme in the myocardium.     

Beta-adrenergic receptor in the brain: comparison of 3H-dihydroalprenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in cell free homogenates.

The properties of specific ³H-dihydroalprenolol binding sites resemble the properties of the beta-receptor regulating hormone-sensitive adenylyl cyclase activity in an homogenate of rabbit cerebellum. The rabbit cerebellum has 5 to 6 pmole per gm (wet weight) of high affinity (K_D=1.3 nM) specific binding sites for ³H-dihydroalprenolol. the interaction of several beta-adrenergic agonists and antagonists with the specific binding sites is rapid, reversible, and demonstrates stereospecificity which parallels the properties of the beta receptor. Beta-adrenergic agonists show a similar potency as agonists upon adenylyl cyclase activity and as inhibitors of ³H-dihydroalprenolol binding: i.e. l-isoproterenol > l-epinephrine > l-norepinephrine (suggesting a beta₂ adrenergic receptor). The binding affinities of several beta-adrenergic agonists and antagonists for the specific binding sites approximate the affinities of these compounds for the stimulation of adenylyl cyclase. Thus, the ³H-dihydroalprenolol binding sites have properties similar to the beta-adrenergic receptor regulating adenylyl cyclase activity in a rabbit cerebellar homogenate.     

The influence of dietary lipid supplementation on cardiac beta-adrenergic receptor adenylate cyclase activity in the marmoset monkey

Dietary lipid supplements high in either saturated fat derived from sheep kidney fat or unsaturated fat derived from sunflower seed oil, and a low mixed fat reference diet were fed to marmoset monkeys for 20 months and the effects on cardiac membrane lipid composition, and myocardial catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor binding activity were investigated. For cardiac membranes enriched for beta-adrenergic binding activity, the dietary lipid treatment resulted in small changes in the proportion of saturated to unsaturated fatty acids and substantial changes in the (n - 6) to (n - 3) series of unsaturated fatty acids in the membrane phospholipids. The sheep kidney fat diet increased the cholesterol-to-phospholipid ratio in cardiac membranes in comparison to the other diets. This diet also significantly elevated basal and isoproterenol-, epinephrine- and norepinephrine-stimulated adenylate cyclase activity. The value of the dissociation constant (Kd) and the receptor number (Bmax) for the binding of [125I]ICYP to the beta-adrenergic receptor was significantly reduced in marmosets fed the sheep kidney fat diet. These results suggest that dietary lipids can influence the activity of the beta-adrenergic/adenylate cyclase system of the heart. Modulation of this transmembrane signalling system may be induced by changes in the properties of the associated membrane lipids, particularly by alteration in the membrane cholesterol-to-phospholipid ratio. This effect may be limited to those animal species in which the nature of the dietary fatty acid intake may be influencing cardiac membrane cholesterol homeostasis, which is in agreement with previous results in rats following dietary cholesterol supplementation (McMurchie et al. (1987) Biochim. Biophys. Acta 898, 137-153). ICYP, (-)-iodocyanopindolol.     

Diabetes reduces right atrial beta-adrenergic signaling but not agonist stimulation of heart rate in swine

This study assessed the effects of streptozotocin diabetes in swine on the heart rate response to beta-adrenergic stimulation the adenylyl cyclase signal transduction pathway. Diabetic animals (n = 9) were hyperglycemic compared to the control group (n = 10) (12.6 +/- 1.0 vs. 3.53 +/- 0.29 mM). There were no significant differences between the diabetic and nondiabetic groups in the heart rate response to isoproterenol, however, there was a significant reduction (14%) in beta-adrenergic receptor density in the right atrium in the diabetic (61 +/- 3 fmol/mg protein) versus the nondiabetic group (71 +/- 3) (P < 0.05). The content of guanosine triphosphate binding regulatory proteins (Gs and Gi) in the right atrium was not affected by diabetes, nor was adenylyl cyclase activity under unstimulated conditions or with receptor-dependent stimulation with isoproterenol. On the other hand, adenylyl cyclase activity was 34% lower when directly stimulated with forskolin, and it was reduced by 23% when stimulated through Gs with Gpp(NH)p. In conclusion, beta-adrenergic stimulation of heart rate with isoproteronol and the receptor-dependent signal transduction pathway remained intact in the right atrium of diabetic swine despite reduced beta-adrenergic receptor density, G-protein content, and direct stimulation of adenylyl cyclase activity.     

Heart receptors for VIP, PHI and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology

We have assessed the presence of VIP/PHI/secretin receptors in heart by: (1) testing the ability of the corresponding peptides to activate adenylate cyclase in cardiac membranes from rat, dog, Cynomolgus monkey and man, and (2) examining the ability of the same peptides to exert inotropic and chronotropic effects on heart preparations from rat and Cynomolgus monkey in vitro. Based on their affinity for natural peptides and synthetic analogs, two types of VIP/PHI/secretin receptors were characterized: the relatively nonspecific "secretin/VIP receptor" of rat heart (that is "secretin-preferring" only in that secretin was more efficient than VIP in stimulating adenylate cyclase), and the "VIP/PHI-preferring" receptor of man, monkey and dog heart. Four physiopathological situations affecting secretin/VIP receptors in rat heart were explored: In male rats from the Okamoto strain and the Lyon strain, two strains presenting spontaneous hypertension, heart membranes exhibited a markedly decreased response of adenylate cyclase to secretin/VIP, with lesser alterations in the responses to isoproterenol and glucagon. This impairment developed in parallel with the occurrence of hypertension and was reproduced in normotensive rats submitted to chronic isoproterenol treatment (but not in Goldblatt hypertensive rats). These findings are consistent with a hyperactivity of norepinephrine pathways in spontaneously hypertensive rats, leading to a reduced number of cardiac post-junctional secretin/VIP receptors bound to adenylate cyclase. Heart membranes from genetically obese (fa/fa) Zucker rats also exhibited severely decreased responses to secretin/VIP with lesser alterations in the responses to glucagon and isoproterenol. These anomalies were specific for the heart, and developed in concomitance with obesity. The first anomaly could not be corrected by severe food restriction. Secretin stimulation of heart adenylate cyclase was also selectively altered in streptozotocin-diabetic rats. Thus, two types of diabetic cardiomyopathy were characterized by a severe local alteration of secretin/VIP receptors coupled to adenylate cyclase. Hypothyroidism, provoked in rat by thyroidectomy or propylthiouracil treatment, again induced a marked decrease in secretin-stimulated cardiac adenylate cyclase activity. In rat papillary muscle electrically stimulated in vitro, secretin exerted a positive inotropic effect. This effect was reduced in obese (fa/fa) Zucker rats. In rat right atrium, secretin also exerted a positive chronotropic effects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thyroid hormone differentially regulates development of beta-adrenergic receptors, adenylate cyclase and ornithine decarboxylase in rat heart and kidney.

In mature animals, thyroid hormone produces parallel up-regulation of beta-adrenergic receptor binding sites and their linkage to adenylate cyclase; during development, these same processes may be critical in establishing the set-point for subsequent adrenergic reactivity. In the current study, we administered triiodothyronine to neonatal rats for the first five days postpartum and evaluated [125I]pindolol binding capabilities and adenylate cyclase activity in membrane preparations from heart and kidney. In the heart, hyperthyroidism elicited an initial increase in receptor density, with subsequent deficits and an eventual return to normal values by young adulthood. In contrast, the ability of isoproterenol, a beta-adrenergic agonist, to stimulate adenylate cyclase was enhanced regardless of whether receptor numbers were increased or decreased; the same effects were also present for basal adenylate cyclase activity and non-receptor-mediated stimulation by forskolin. Enhanced cyclase activity involved both increases in the magnitude of response as well as accelerated onset of the postweaning peak of enzyme activity, results which suggest a direct impact of thyroid status on the ontogenetic expression of adenylate cyclase itself. The kidney, which possesses less efficient beta-receptor coupling to adenylate cyclase in the neonate, was less drastically affected by triiodothyronine for either beta-receptor binding sites or enzyme activity. As we had previously shown that neonatal hyperthyroidism uncouples beta-receptors from growth-related enzymes, such as ornithine decarboxylase, we also evaluated whether the promotion of adenylate cyclase responses was mechanistically linked to effect on ornithine decarboxylase; administration of cyclic AMP analogs to 5 days-old rats led to inhibition of the enzyme in the heart, whereas the same treatment in 9 days-old animals was ineffective. These data suggest that thyroid hormone differentially regulates the development of beta-receptors as well as adenylate cyclase and ornithine decarboxylase, with preferential effects on tissues, such as the heart, that already possess efficient linkage of the receptors to cell transduction mechanisms at birth.     

Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a beta-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of beta-adrenergic receptors: in fact, [125I]pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.     

The role of protein kinase C in insulin signal transduction via adenylyl cyclase signaling mechanism

Activation of proteinkinase C with diacylglycerol or phorbol-12-myristate-13-acetate in the rat muscle membrane or Anodonta cygnea mollusc blocks the insulin stimulating signal to adenylyl cyclase via tyrosinekinase type receptor. The same occurs with stimulating effect of biogenic amines to adenylyl cyclase via serpentine type receptor. Transduction of the inhibitory signal induced with isoproterenol to adenylyl cyclase remained unchanged in case of the proteinkinase C activation. The findings suggest that phorbol-sensitive proteinkinase C realizes a negative regulation of insulin-sensitive adenylyl cyclase signalling system. This negative regulation might prove a universal mechanism of the adenylyl cyclase system desensitisation.     

Alpha adrenergic drugs inhibit [3H]-QNB binding to muscarinic receptors of rat heart, brain and parotid gland membranes

Alpha adrenergic agonists and antagonists as clonidine, guanfacine, yohimbine, phenylephrine and prazosin inhibited the [3H]-QNB binding to rat brain cortex muscarinic acetylcholine receptor (mAChR, M-1 subtype), heart (M-2 subtype) and parotid gland homogenate (M-3 subtype) in a dose-dependent competitive fashion. Ki values were between 10(-6) and 10(-3) M. Hill coefficients were about 1. No correlation was found between mAChR inhibiting capacity of these drugs and their activity on alpha adrenergic receptors. In contrast, other transmitters, as dopamine, GABA, glutamic acid, histamine, serotonin, isoproterenol and platelet activating factor (PAF) did not affect the QNB binding.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Muscarinic cholinergic receptor mediated inhibitory transduction of adenylate cyclase activity in subcellular fractions from rat heart: improved detection in sodium phosphate buffer

Summary Cholinergic inhibition of myocardial adenylate cyclase activity in cell-free fractions has been known for many years, although the reported degrees of inhibition have been rather modest (20–30%), notably in rat heart fractions. The present study conducted with rat heart subcellular fractions document following major findings: (1) Myocardial adenylate cyclase activity and notably its cholinergic inhibition in cell-free fractions are notoriously labile to storage at 4°C whereas its stimulation by beta adrenergic receptor agonists or forskolin are reasonably well preserved during storage. (2) Among four buffers (Tris, glycylglycine, imidazole and sodium phosphate) examined, sodium phosphate buffer afforded the best preservation of cholinergic inhibitory response of adenylate cyclase. (3) The commonly used biochemical buffers, notably imidazole, exerted deleterious effect on the cholinergic inhibition of myocardial adenylate cyclase such that it was considerably attenuated or barely detectable; this explains, in part, the reported poor inhibition of myocardial enzyme by others. (4) Imidazole buffer, on the other hand, augmented beta adrenergic and forskolin stimulated adenylate cyclase activity. The likely significance of these findings is discussed from consideration that the observed differential influence of buffers results from differential actions on the interactions between the components (receptor/coupling G proteins/catalyst) comprising autonomic receptor coupled adenylate cyclase system in rat heart.     

Differential targeting of beta -adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway

Differential modes for beta(1)- and beta(2)-adrenergic receptor (AR) regulation of adenylyl cyclase in cardiomyocytes is most consistent with spatial regulation in microdomains of the plasma membrane. This study examines whether caveolae represent specialized subdomains that concentrate and organize these moieties in cardiomyocytes. Caveolae from quiescent rat ventricular cardiomyocytes are highly enriched in beta(2)-ARs, Galpha(i), protein kinase A RIIalpha subunits, caveolin-3, and flotillins (caveolin functional homologues); beta(1)-ARs, m(2)-muscarinic cholinergic receptors, Galpha(s), and cardiac types V/VI adenylyl cyclase distribute between caveolae and other cell fractions, whereas protein kinase A RIalpha subunits, G protein-coupled receptor kinase-2, and clathrin are largely excluded from caveolae. Cell surface beta(2)-ARs localize to caveolae in cardiomyocytes and cardiac fibroblasts (with markedly different beta(2)-AR expression levels), indicating that the fidelity of beta(2)-AR targeting to caveolae is maintained over a physiologic range of beta(2)-AR expression. In cardiomyocytes, agonist stimulation leads to a marked decline in the abundance of beta(2)-ARs (but not beta(1)-ARs) in caveolae. Other studies show co-immunoprecipitation of cardiomyocytes adenylyl cyclase V/VI and caveolin-3, suggesting their in vivo association. However, caveolin is not required for adenylyl cyclase targeting to low density membranes, since adenylyl cyclase targets to low buoyant density membrane fractions of HEK cells that lack prototypical caveolins. Nevertheless, cholesterol depletion with cyclodextrin augments agonist-stimulated cAMP accumulation, indicating that caveolae function as negative regulators of cAMP accumulation. The inhibitory interaction between caveolae and the cAMP signaling pathway as well as domain-specific differences in the stoichiometry of individual elements in the beta-AR signaling cascade represent important modifiers of cAMP-dependent signaling in the heart.     

Chronic Treatment of C6 Glioma Cells with Antidepressant Drugs Increases Functional Coupling Between a G Protein (GS) and Adenylyl Cyclase

Abstract: It has been reported that antidepressant treatment in rats results in a significant increase of G_s-mediated stimulation of adenylyl cyclase and this effect correlates well with the clinical therapeutic response. This increased activity occurs despite a down-regulation of several receptors linked normally to the stimulation of that enzyme. To distinguish between these effects and to determine whether presynaptic components of the cell are required, C6 glioma cells were treated with antidepressants. Tricyclic (amitriptyline and desipramine) or atypical (iprindole) antidepressant exposure to C6 cells for 5 days significantly increased guanylyl-5′-imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activity in membrane preparations in a manner similar to that seen for rat brain membranes after 21-day treatment. This effect was drug dose and exposure time dependent. Nevertheless, stimulation of adenylyl cyclase by isoproterenol was decreased after antidepressant treatment. By comparison, the antidepressant-induced β-receptor desensitization occurred earlier than the enhancement of Gpp(NH)p-activated adenylyl cyclase, and extensive desensitization of β receptors by isoproterenol treatment did not enhance the Gpp(NH)p-stimulated adenylyl cyclase activity. These results indicated that the antidepressant has a direct effect on cell signaling and this enhanced Gpp(NH)p-stimulated adenylyl cyclase activity is not correlated with desensitization of β-adrenergic receptor stimulated adenylyl cyclase. These data contribute to the suggestion that G proteins (especially G_s) are the target of antidepressant actions. Immunoblotting showed that neither the number of G protein subunits (α_s, α_i, α_o, and β) nor their association with the plasma membrane was changed after antidepressant treatment. Thus, these results are consistent with the hypothesis that chronic antidepressant treatment acts directly at the postsynaptic membrane to increase the coupling between G_s and adenylyl cyclase.     

LOW CONCENTRATIONS OF LITHIUM INHIBIT THE SYNTHESIS OF CYCLIC AMP AND CYCLIC GMP IN THE RAT PINEAL GLAND

Abstract— Lithium chloride (2 m m ) significantly inhibited the increases in cyclic AMP and in cyclic GMP caused by norepinephrine or high concentrations of potassium in intact rat pineal glands. Adenylyl cyclase activity in homogenates and its stimulation by isoproterenol, a β-adrenergic agonist, were also inhibited. Lithium reduced the apparent V _max of isoproterenol-stimulated adenylyl cyclase activity without significantly affecting the apparent affinity for isoproterenol. There was no effect on the binding of the antagonist [³H]dihydroalprenolol to the β-adrenergic receptors, nor on the competition for binding sites by isoproterenol. Inhibition of adenylyl cyclase activity by lithium was inversely related to the magnesium concentration in the reaction mixture. There was no differential effect of lithium on adenylyl cyclase activity from supersensitive vs subsensitive glands. Lithium may inhibit cyclic nucleotide synthesis by interfering with the role of divalent cations.     

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.     

Sympathetic potentiation of cyclic ADP-ribose formation in rat cardiac myocytes

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.     

Myometrial adenylyl cyclase protein decreases on the last day of pregnancy in the rat

To determine whether gestation-related changes in responsiveness of the rat uterus to beta-adrenergic agonists are mediated at the level of adenylyl cyclase, we measured myometrial adenylyl cyclase activity and protein quantities during pregnancy and labor. In rat myometrial membranes, basal adenylyl cyclase activity increased from the nonpregnant state to mid (Days 12-14) and then late (Days 18-20) gestation and then decreased intrapartum (Day 22). Stimulated adenylyl cyclase activity, at the level of the beta-adrenergic receptor (isoproterenol, 10(-4) M), the G protein (GTP, 10(-5) M), or the adenylyl cyclase enzyme (MnCl(2), 20 mM), was similarly altered during gestation. Total adenylyl cyclase protein was quantified by [(3)H]forskolin binding assay in myometrial membranes from nonpregnant and pregnant (Day 14, Day 20, Day 21, and intrapartum Day 22) rats. Adenylyl cyclase protein increased progressively from nonpregnant rats to pregnant rats at mid (Day 14) and late (Day 20) gestation, but it decreased abruptly to nonpregnant levels on Day 21, the day before parturition, and remained at similar levels on Day 22 (intrapartum). The gestation-related increase in expression of myometrial adenylyl cyclase protein may facilitate uterine quiescence during pregnancy, and the abrupt decrease of adenylyl cyclase protein on the last day of pregnancy may be a contributing mechanism for the initiation of labor.