Beta-adrenergic Receptor Levels Research Articles

Novel Insights into Beta 2 Adrenergic Receptor Function in the rd10 Model of Retinitis Pigmentosa.

Background: In retinitis pigmentosa (RP), inherited rod death is followed by cone loss and blindness. Why cones die is still a matter of consideration. Here, we investigate the pathogenic role of the sympathetic transmission in the rd10 mouse model of RP. Methods: Retinal levels of beta adrenergic receptor (BAR) 2 and norepinephrine (NE) were measured. After administration of the BAR1/2 blocker propranolol or the hypoxia-inducible factor (HIF)-1 activator dimethyloxalylglycine (DMOG), retinal levels of HIF-1α, BAR2 or proteins involved in BAR2 desensitization were also measured. In DMOG treated mice, expression and localization of BAR2, inflammatory markers and cone arrestin were determined. Finally, rd10 mice were subjected to electroretinogram (ERG) analysis to assess rod and cone function. Results: In the rd10 retina, BAR2 overexpression and NE accumulation were found, with BAR2 immunoreactivity localized to Müller cells. BAR2 overexpression was likely due to desensitization defects. Upregulated levels of BAR2 were drastically reduced by propranolol that also restored desensitization defects. Due to the low level of HIF-1 consequent to the hyperoxic environment in the rd10 retina, we hypothesized a link between HIF-1 and BAR2. HIF-1α stabilization with DMOG resulted in i. increased HIF-1α accumulation, ii. decreased BAR2 levels, iii. restored desensitization processes, iv. reduced expression of inflammatory markers and v. increased cone survival without improved retinal function. Conclusions: Our results support a pathogenic role of the sympathetic system in RP that might help to understand why rd10 mice show a positive response to BAR blockers.

Alcohol consumption combined with dietary low-carbohydrate/high-protein intake increased the left ventricular systolic dysfunction risk and lethal ventricular arrhythmia susceptibility in apolipoprotein E/low-density lipoprotein receptor double-knockout mice.

Alcohol abuse is positively associated with cardiovascular disease. Dietary low-carbohydrate/high-protein (LCHP) intake confers a greater mortality risk. Here, the impact of ethanol consumption in combination with dietary LCHP intake on left ventricular (LV) systolic function and lethal ventricular arrhythmia susceptibility were investigated in apolipoprotein E/low-density lipoprotein receptor double-knockout (AL) mice. The underlying mechanisms, cardiac sympathovagal balance, beta-adrenergic receptor (ADRB) levels, and gap junction channel protein connexin 43 (Cx43) expression, were examined. Male AL mice fed an LCHP diet with or without ethanol were bred for 16 weeks. Age-matched male AL and wild-type mice received standard chow diet and served as controls. The following were used to assess LV systolic function, lethal ventricular arrhythmia susceptibility, cardiac sympathovagal balance, Cx43 expression, and ADRB levels: The results demonstrated that ethanol consumption in combination with dietary LCHP intake worsened LCHP-induced LV systolic dysfunction in AL mice and enhanced their susceptibility in the ventricular arrhythmia-evoked test. There were concomitant increases in LV weight, LF/HF ratio shown by HRV, TH, ADRB1, ADRB2, and Cx43 expressions by LV fluorescence immunohistochemistry, and LV Cx43 messenger ribonucleic acid expression by PCR. In AL mice, alcohol consumption combined with dietary LCHP intake may thus promote a shift in cardiac sympathovagal balance toward sympathetic predominance, the increases in beta-adrenergic receptors (ADRB1 and ADRB2), and then affect the gap junction channel protein Cx43, which in turn could contribute to increased risks of LV systolic dysfunction and susceptibility to lethal ventricular arrhythmia.

Targeting of beta adrenergic receptors results in therapeutic efficacy against models of hemangioendothelioma and angiosarcoma.

Therapeutic targeting of the beta-adrenergic receptors has recently shown remarkable efficacy in the treatment of benign vascular tumors such as infantile hemangiomas. As infantile hemangiomas are reported to express high levels of beta adrenergic receptors, we examined the expression of these receptors on more aggressive vascular tumors such as hemangioendotheliomas and angiosarcomas, revealing beta 1, 2, and 3 receptors were indeed present and therefore aggressive vascular tumors may similarly show increased susceptibility to the inhibitory effects of beta blockade. Using a panel of hemangioendothelioma and angiosarcoma cell lines, we demonstrate that beta adrenergic inhibition blocks cell proliferation and induces apoptosis in a dose dependent manner. Beta blockade is selective for vascular tumor cells over normal endothelial cells and synergistically effective when combined with standard chemotherapeutic or cytotoxic agents. We demonstrate that inhibition of beta adrenergic signaling induces large scale changes in the global gene expression patterns of vascular tumors, including alterations in the expression of established cell cycle and apoptotic regulators. Using in vivo tumor models we demonstrate that beta blockade shows remarkable efficacy as a single agent in reducing the growth of angiosarcoma tumors. In summary, these experiments demonstrate the selective cytotoxicity and tumor suppressive ability of beta adrenergic inhibition on malignant vascular tumors and have laid the groundwork for a promising treatment of angiosarcomas in humans.

Beta-Adrenergic Receptor 1 Selective Antagonism Inhibits Norepinephrine-Mediated TNF-Alpha Downregulation in Experimental Liver Cirrhosis

BackgroundBacterial translocation is a frequent event in cirrhosis leading to an increased inflammatory response. Splanchnic adrenergic system hyperactivation has been related with increased bacterial translocation. We aim at evaluating the interacting mechanism between hepatic norepinephrine and inflammation during liver damage in the presence of bacterial-DNA.Animals and MethodsForty-six mice were included in a 16-week protocol of CCl4-induced cirrhosis. Laparotomies were performed at weeks 6, 10, 13 and 16. A second set of forty mice injected with a single intraperitoneal dose of CCl4 was treated with saline, 6-hydroxidopamine, Nebivolol or Butoxamine. After 5 days, mice received E. coli-DNA intraperitoneally. Laparotomies were performed 24 hours later. Liver bacterial-DNA, norepinephrine, TNF-alpha, IL-6 and beta-adrenergic receptor levels were measured.ResultsBacterial-DNA translocation was more frequent in CCl4-treated animals compared with controls, and increased as fibrosis progressed. Liver norepinephrine and pro-inflammatory cytokines were significantly higher in mice with vs without bacterial-DNA (319.7±120.6 vs 120.7±68.6 pg/g for norepinephrine, 38.4±6.1 vs 29.7±4.2 pg/g for TNF-alpha, 41.8±7.4 vs 28.7±4.3 pg/g for IL-6). Only beta-adrenergic receptor-1 was significantly increased in treated vs control animals (34.6±7.3 vs 12.5±5.3, p = 0.01) and correlated with TNF-alpha, IL-6 and norepinephrine hepatic levels in animals with bacterial-DNA. In the second set of mice, cytokine levels were increased in 6-hydroxidopamine and Nebivolol (beta-adrenergic receptor-1 antagonist) treated mice compared with saline. Butoxamine (beta-adrenergic receptor-2 antagonist) didn’t inhibit liver norepinephrine modulation of pro-inflammatory cytokines.ConclusionsBeta-adrenergic receptor-1 mediates liver norepinephrine modulation of the pro-inflammatory response in CCl4-treated mice with bacterial-DNA.

Sympathetic nerve activity in normal and cystic follicles from isolated bovine ovary: local effect of beta-adrenergic stimulation on steroid secretion

Cystic ovarian disease (COD) is an important cause of abnormal estrous behavior and infertility in dairy cows. COD is mainly observed in high-yielding dairy cows during the first months post-partum, a period of high stress. We have previously reported that, in lower mammals, stress induces a cystic condition similar to the polycystic ovary syndrome in humans and that stress is a definitive component in the human pathology. To know if COD in cows is also associated with high sympathetic activity, we studied isolated small antral (5mm), preovulatory (10mm) and cystic follicles (25mm). Cystic follicles which present an area 600 fold greater compared with preovulatory follicles has only 10 times less concentration of NE as compared with small antral and preovulatory follicles but they had 10 times more NE in follicular fluid, suggesting a high efflux of neurotransmitter from the cyst wall. This suggestion was reinforced by the high basal release of recently taken-up 3H-NE found in cystic follicles. While lower levels of beta-adrenergic receptor were found in cystic follicles, there was a heightened response to the beta-adrenergic agonist isoproterenol and to hCG, as measured by testosterone secretion. There was however an unexpected capacity of the ovary in vitro to produce cortisol and to secrete it in response to hCG but not to isoproterenol. These data suggest that, during COD, the bovine ovary is under high sympathetic nerve activity that in addition to an increased response to hCG in cortisol secretion could participate in COD development.

Diet‐induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high-fat (HF) diet-induced obese (DIO) mice were switched to a low-fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF-chow), but retained a greater amount of adiposity than chow-fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF-chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF-chow mice. Leptin administration was used to test whether reduced leptin level of HF-chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF-HF mice had lower mRNA levels of beta(3) adrenergic receptor (beta(3)-AR) in epididymal WAT (EWAT) compared to chow-fed mice, and diet change led to an increase in the WAT beta(3)-AR mRNA levels that were similar to the levels of chow-fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF-HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF-chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.

Diastolic dysfunction in alveolar hypoxia: a role for interleukin-18-mediated increase in protein phosphatase 2A

Chronic obstructive pulmonary disease with alveolar hypoxia is associated with diastolic dysfunction in the right and left ventricle (LV). LV diastolic dysfunction is not caused by increased afterload, and we recently showed that reduced phosphorylation of phospholamban at serine (Ser) 16 may explain the reduced relaxation of the myocardium. Here, we study the mechanisms leading to the hypoxia-induced reduction in phosphorylation of phospholamban at Ser16. In C57Bl/6j mice exposed to 10% oxygen, signalling molecules were measured in cardiac tissue, sarcoplasmic reticulum (SR)-enriched membrane preparations, and serum. Cardiomyocytes isolated from neonatal mice were exposed to interleukin (IL)-18 for 24 h. The beta-adrenergic pathway in the myocardium was not altered by alveolar hypoxia, as assessed by measurements of beta-adrenergic receptor levels, adenylyl cyclase activity, and subunits of cyclic AMP-dependent protein kinase. However, alveolar hypoxia led to a significantly higher amount (124%) and activity (234%) of protein phosphatase (PP) 2A in SR-enriched membrane preparations from LV compared with control. Serum levels of an array of cytokines were assayed, and a pronounced increase in IL-18 was observed. In isolated cardiomyocytes, treatment with IL-18 increased the amount and activity of PP2A, and reduced phosphorylation of phospholamban at Ser16 to 54% of control. Our results indicate that the diastolic dysfunction observed in alveolar hypoxia might be caused by increased circulating IL-18, thereby inducing an increase in PP2A and a reduction in phosphorylation of phospholamban at Ser16.

Effect of salbutamol on nasal symptoms and mast cell degranulation induced by adenosine 5′ monophosphate nasal challenge

In addition to its well-known functional agonism at the level of beta(2) adrenergic receptors on airways smooth muscle cells, salbutamol appears to have additional protective effects, possibly through an inhibition of mast cell activation. The aim of this study is to provide the first evidence in vivo of inhibition of human mast cell activation by salbutamol. Nine atopic subjects received placebo and salbutamol (5 mg/mL) 15 min before an adenosine 5' monophosphate (AMP) nasal provocation in a double-blind crossover study design. The nasal lavage was collected from these subjects prior to or 3, 5, 15 or 30 min after the AMP nasal challenge, and concentrations of histamine and tryptase in the nasal lavage were measured. AMP nasal provocation produced considerable sneezing and induced a transient increase in histamine and tryptase release with peak values achieved at 3 min after the challenge in all the subjects studied. Compared with placebo, salbutamol significantly attenuated the release of histamine and tryptase induced by AMP challenge (P=0.048 and 0.020, respectively). Moreover, the AMP-induced sneezing was also inhibited by pre-treatment with salbutamol (P=0.004). Intranasal salbutamol attenuates nasal symptoms and inhibits histamine and tryptase release caused by AMP nasal provocation thus supporting the hypothesis that salbutamol may play an additional protective role in the airways by inhibiting mast cell activation.

β3‐Adrenergic activation of adenylyl cyclase in mouse white adipocytes: modulation by GTP and effect of obesity

Lipolysis and adenylyl cyclase (AC) activation in response to beta-adrenergic agents are abnormally low in white epididymal adipose tissue (WAT) of the ob/ob mouse. The abundance of G-proteins (Gs alpha and Gi alpha) linked to AC is also abnormally low. By contrast, beta-adrenergic receptor (beta-AR) levels were previously found to be normal in WAT and elevated in liver. The relative importance of various forms of the beta-AR in mouse WAT was reassessed in view of the discovery of the beta 3-AR. The results show that (1) the beta 3-AR is mainly responsible for AC activation in lean-mouse WAT; (2) the beta 3-AR is only partly responsible for AC activation in obese mouse WAT; and (3) GTP modulates beta 3--but not beta 1--or beta 2-AR activation of AC in a biphasic manner. Therefore, the beta 3-AR appears responsible for the well-known bimodal effect of GTP on beta-adrenergic receptor-mediated AC activity in WAT.

The glucogenic capacity of the fetal pig: developmental regulation by cortisol

In the present study, the ontogenic changes in gluconeogenic enzyme activities and in hepatic glycogen and beta-adrenergic receptor levels were investigated in fetal pigs from 70 days of gestation until delivery at term (114 +/- 2 days). The values were compared with those observed in fetuses infused subcutaneously with cortisol for 6 days beginning at 82-84 or 92-94 days of gestation. Tissue glucose-6-phosphatase (G6Pase) activity increased with increasing gestational age in the liver, kidney and duodenum of control fetal pigs. At birth, there was a further increase in G6Pase activity in the liver but not in the kidney or duodenum. In the kidney, there was a similar gestational increase in phosphoenolpyruvate carboxykinase (PEPCK) activity. These changes in enzyme activities closely paralleled the prepartum increase in fetal plasma cortisol and were accompanied by increases in hepatic glycogen content and beta-adrenergic receptor density. At 98-100 days, there were significant increases in G6Pase activity in the liver, kidney and duodenum of the cortisol-infused fetuses, whereas at 88-90 days only renal G6Pase was significantly elevated by cortisol infusion. Cortisol infusion also increased hepatic beta-receptor density at 88-90 days and hepatic glycogen content at both gestational ages. There were no changes in hepatic PEPCK, hepatic or renal fructose diphosphatase and aspartate amino transferase activities during cortisol infusion or with increasing gestational age. When the data from all the piglets were combined, irrespective of age or treatment, there were significant positive correlations between log plasma cortisol and G6Pase activity in the liver, kidney and duodenum. Similar positive correlations were observed between hepatic beta-adrenoceptor density and log plasma cortisol and between the latter values and the hepatic glycogen content. These findings show that cortisol induces tissue G6Pase activity in the fetal pig and suggest that the prepartum rise in endogenous cortisol may be responsible for the increase in fetal glucogenic capacity observed towards term in this as in other species.

Decreased Levels of Adenylate Cyclase Contribute to the Down-Regulation of Beta-Adrenergic Signal Transduction in the Salivary Glands of the Non-Obese Diabetic (NOD) Mouse

Stimulation of salivary gland secretion by treatment of NOD mice with the beta-adrenergic receptor agonist, isoproterenol, was previously shown to cause a lower accumulation of intracellular cAMP than that found that in BALB/c controls (Hu et al., 1994, Am. J. Physiol. 266: G433). This was, in part, due to decreased levels of beta-adrenergic receptor localized in the plasma membrane. In the present investigation, basal levels of cAMP in the salivary glands of NOD mice were found to be similar to controls, while direct stimulation of adenylate cyclase by incubation of isolated membranes with forskolin showed a reduced cAMP accumulation in NOD mice versus BALB/c control mice. Using direct [3H]-forskolin binding, it was determined that the plasma membranes of NOD mice salivary glands contain reduced levels of membrane-associated adenylate cyclase.

Induction of beta 2-adrenergic receptor mRNA and ligand binding in HeLa cells.

HeLa cells express low levels of beta-adrenergic receptor (beta AR) of the beta 2-subtype. When exposed to sodium butyrate, receptor levels increased up to 4-fold in a time dependent manner, reaching a maximum after 12 to 15 h of treatment. Sodium butyrate treatment also caused a 3 to 4 fold increase in levels of beta 2AR mRNA determined by hybridization blot analysis. The induction of beta 2AR mRNA temporally preceded the increase in receptor binding activity, reaching a maximum after 4 to 6 h of treatment, and remaining elevated for up to 24 h. Prior exposure of the cells to the protein synthesis inhibitor cycloheximide prevented the butyrate-induced increase in receptor binding but had no effect on the increase in receptor mRNA. Blocking DNA synthesis and cell growth by excess thymidine did not increase beta 2AR mRNA or binding or prevent the effects of sodium butyrate. Thus, butyrate appears to induce beta 2AR mRNA by a mechanism independent of DNA and protein synthesis.

Myocardial catecholamine and neuropeptide Y depletion in failing ventricles of patients with idiopathic dilated cardiomyopathy. Correlation with beta-adrenergic receptor downregulation.

Myocardial adrenergic neurotransmitters and beta-adrenergic receptor levels were measured in left and right ventricular myocardial specimens obtained from 30 patients with biventricular failure resulting from idiopathic dilated cardiomyopathy. Nonfailing myocardium obtained from 12 organ donors provided control data. Norepinephrine, dopamine, and neuropeptide Y concentrations were significantly decreased in failing compared with nonfailing control hearts. The mean ratio of dopamine to norepinephrine and of dopamine to neuropeptide Y in failing hearts was also significantly decreased compared with nonfailing control hearts. Compared with nonfailing control hearts, Bmax and beta 1-receptor density were significantly decreased in failing hearts and there were weak but significantly positive correlations of Bmax and beta 1-adrenergic receptors with norepinephrine, dopamine, and neuropeptide Y. Norepinephrine and its cotransmitter neuropeptide Y are depleted in failing human ventricular myocardium. Decreased norepinephrine stores correlate weakly with beta 1-adrenergic receptor downregulation consistent with the hypothesis that norepinephrine depletion occurs in response to increased adrenergic drive. Decreased dopamine relative to norepinephrine implies that an abnormality of dopamine conversion to norepinephrine is not present in failing human heart.

Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells.

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.

Hormonal Regulation of β-Adrenergic Receptors in the Rat Mammary Gland during the Estrous Cycle and Lactation: Role of Sex Steroids and Prolactin

To gain further knowledge on the role of ovarian hormones in the regulation of mammary beta-adrenergic receptors, virgin animals were killed during the various phases of the estrous cycle as well as after ovariectomy and treatment with sex steroid. beta-Adrenergic receptor levels fluctuate in the rat mammary gland during the estrous cycle, with higher receptor numbers during the proestrous and estrous phases of the cycle. Ovariectomy caused an almost 50% loss of beta-adrenergic receptor concentration in the mammary gland of virgin rats. Treatment of ovariectomized animals with 17 beta-estradiol or progesterone alone or in combination for 3 weeks induced a marked increase in beta-adrenergic receptor concentration, while administration of the androgen dihydrotestosterone did not modify mammary beta-adrenergic binding sites. While levels of beta-adrenergic receptors in control lactating animals (10 days of lactation) were elevated, chronic treatment with the dopaminergic-mimetic agent 2 alpha-bromoergocryptine (CB-154; for 7 days) reduced beta-adrenergic receptor concentration. Castration of lactating animals decreased beta-adrenergic receptor number to approximately 30% of the value in intact controls, while combined withdrawal of circulating ovarian hormones and inhibition of plasma PRL levels caused an almost complete inhibition of beta-adrenergic receptor concentration. Scatchard analysis of the binding data revealed that the observed alterations in beta-adrenergic receptors resulted from changes in the number of beta-adrenergic binding sites, with no change in binding affinities. The present findings indicate that the beta-adrenergic receptor population of the rat mammary gland is under the control of ovarian hormones and PRL and suggest that circulating or locally released catecholamines could interact with sex steroids and PRL in the regulation of mammary gland growth, differentiation, and activity.

Beta-adrenergic receptors in DMBA-induced rat mammary tumors: Correlation with progesterone receptor and tumor growth

In order to gain further knowledge about the potential role of catecholamines in mammary carcinoma, we have used the potent beta-adrenergic antagonist cyanopindolol (CYP) as iodinated ligand to characterize beta-adrenergic receptors in membranes prepared from mammary tumors induced by dimethylbenz(a)anthracene (DMBA) administration in the rat. The binding of [125I]CYP to membrane preparations of DMBA-induced rat mammary tumors is rapid at room temperature, reaching half maximal specific binding at 30 min of incubation. Scatchard analysis of the data indicates that [125I]CYP binds to a single class of high affinity sites (114 +/- 2.1 fmoles/mg protein) at an apparent KD value of 38.0 +/- 0.3 pM. The order of potency of a series of agonists to compete for [125I]CYP binding is consistent with interaction with a beta 2-subtype receptor: zinterol greater than (-)isoproterenol greater than (-)epinephrine much greater than (-)norepinephrine. In addition, the potency of a series of specific beta 1 and beta 2 synthetic compounds to displace [125I]CYP in mammary tumors is similar to their potency in typical beta 2-adrenergic tissues. The binding of [125I]CYP to DMBA-induced rat mammary tumors shows a marked stereoselectivity, the (-)isomers of isoproterenol and propranolol being 150 and 80 times more potent, respectively, than their respective enantiomers. The autoradiographic localization of [125I]CYP performed on frozen sections revealed the presence of specific beta-adrenergic receptors in all the malignant cells. Spontaneous mammary tumors of aging (18-22 months) female rats have high levels of beta-adrenergic receptors. Castration decreased the concentration of [125I]CYP binding sites in DMBA-induced mammary tumors. A close correlation was observed between progressing, static, and regressing tumors after ovariectomy and beta-adrenergic receptor concentration. The presence of beta-adrenergic receptors in mammary tumors as well as the modulation of their level by ovarian hormones provides a mechanism for catecholaminergic influence in mammary cancer tissue.

Decreased bioactivity of the guanine nucleotide-binding protein that stimulates adenylate cyclase in hearts from cardiomyopathic Syrian hamsters.

We investigated regulation of cardiac adenylate cyclase in 29-d-old BIO 14.6 Syrian hamsters, which inherit cardiomyopathy as an autosomal recessive trait. Pharmacologic stimulation of adenylate cyclase in cardiac membranes with isoproterenol, fluoride ion, guanine nucleotide, forskolin, and manganous ion indicated that there was defective coupling of the guanine nucleotide-binding protein that stimulates adenylate cyclase (Gs) to adenylate cyclase. Cyc complementation assays revealed congruent to 50% less Gs activity in cardiac and skeletal muscle from cardiomyopathic hamsters. Despite this decrease in functional Gs, there were no changes in immunologic levels of the alpha-subunit of Gs (alpha Gs) or in levels of mRNA encoding alpha Gs. The defect in Gs bioactivity was limited to cardiac and skeletal muscle, occurred only in animals homozygous for the dystrophic trait, and was demonstrable before any cardiac abnormalities were evident on light microscopy. By contrast, cardiac levels of beta-adrenergic receptors were not different in cardiac membranes from BIO 14.6 hamsters. We conclude that a functional defect in alpha Gs may contribute to a contractile abnormalities in the cardiomyopathic BIO 14.6 hamster. However, the etiology of the alpha Gs defect remains obscure.

Effects of Low Density Lipoproteins and Mevinolin on Sympathetic Responsiveness in Cultured Chick Atrial Cells: Regulation of β-adrenergic receptors and αs

We have previously demonstrated that cultures of myocytes from embryonic chick atria grown in media supplemented with fetal calf serum from which lipoproteins have been removed demonstrate a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared with cells grown with control medium. This increased response to carbamylcholine was associated with a 1.4-fold increase in total cell cholesterol, a 2-fold increase in the number of muscarinic receptors which bind agonist with high affinity, and a 2-fold increase in the levels of the alpha subunits of Go and Gi (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In the studies reported here, we determined the responsiveness of cells grown in lipoprotein-depleted serum (LPDS) to beta-adrenergic stimulation. Isoproterenol stimulated a contractile response of 58% measured as an increase in amplitude of contraction with a half-maximal effect at 3 x 10(-7) M for cells grown in fetal calf serum, but had no significant effect on amplitude of contraction on cells grown in LPDS. In cells grown in media supplemented with fetal calf serum, isoproterenol (1 x 10(-3) M) stimulated adenylate cyclase activity 100% over basal with an EC50 of 7 x 10(-6) M compared with an increase of 32% in cells grown in media supplemented with LPDS. beta-Adrenergic receptor number as measured by the binding of 125I-pindolol decreased from 24 +/- 3 (+/- S.E., n = 6) fmol/mg protein in cells grown under control conditions to 12 +/- 2 (n = 6) fmol/mg protein in media supplemented with LPDS. The level of alpha s as measured both by ADP-ribosylation with cholera toxin in the presence of 32P-NAD and by immunoblotting with specific antibody to alpha s decreased by 3-fold in cells grown in media supplemented with LPDS compared with control. All of these effects of growth of cells in LPDS were reversed by incubating cells with LPDS plus 30 microM mevinolin, an inhibitor of endogenous cholesterol synthesis. These studies indicate that growth of cells in media supplemented with LPDS results in a coordinate decrease in the levels of beta-adrenergic receptors and alpha s. Taken together with our previous studies these data support the hypothesis that the receptors and guanine nucleotide-binding proteins which mediate sympathetic and parasympathetic responsiveness in the heart are reciprocally regulated.

Effect of Acute Ethanol Intake and Hangover on the Levels of Plasma and Urinary Catecholamines and Lymphocytic β‐Adrenergic Receptors

To determine whether acute ethanol administration affects the function of the adrenergic system the concentrations of plasma catecholamines and cyclic AMP (cAMP), the level of lymphocytic beta-receptors, the concentration of basal and isoproterenol-stimulated lymphocytic cAMP and the excretion of urinary catecholamine metabolites were studied in six healthy men. These parameters were also measured during the hangover, both under resting condition and during an anaerobic ergometer exercise. Acute intake of ethanol (1.5 g/kg body weight) had no statistically significant effect either on plasma adrenaline and noradrenaline concentrations or beta-adrenergic receptor levels. Ethanol consumption did neither change the urinary excretion of catecholamine metabolites (homovanillic acid, normetanephrine, metanephrine, and 3-methoxyhydroxymandelic acid). Exercise was associated with a 6-10-fold elevation in plasma adrenaline and noradrenaline concentrations and with a two- to threefold elevation on beta-adrenergic receptor levels. This effect of exercise was not modified by preceding alcohol intake and resulting hangover. These preliminary findings suggest that acute alcohol intake does not significantly alter the concentration and functioning of human beta-adrenergic receptors.

Hepatocyte beta-adrenergic responsiveness and guanine nucleotide-binding regulatory proteins

Hepatocytes isolated from hypothyroid, adrenalectomized, or partially hepatectomized rats display an enhanced beta-adrenergic responsiveness as compared with cells from control animals. The enhanced beta-adrenergic responsiveness is evidenced by both increased ureagenesis and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to isoproterenol. The role of stimulatory guanine nucleotide-binding protein (Gs) and inhibitory guanine nucleotide-binding protein (Gi) in the enhanced responsiveness was studied. It was observed, contrary to what would have been anticipated, that the level of Gs [as reflected by cholera toxin-catalyzed ADP ribosylation, 5'-guanosine gamma-thiotriphosphate (GTP gamma S)-stimulated adenylate cyclase activity, and a functional reconstitution assay] was decreased in liver membranes from adrenalectomized and partially hepatectomized rats as compared with the controls. Furthermore, the level of Gi was increased in these conditions as reflected by pertussis toxin-catalyzed ADP ribosylation. The data suggest that changes in beta-adrenergic receptor levels rather than the levels of guanine nucleotide-binding (G) regulatory proteins predominate in regulation of hepatic beta-adrenergic responses by hypothyroidism, adrenalectomy, or partial hepatectomy.