Isoproterenol-stimulated Activity Research Articles

The role of phospholipase in reduced beta-adrenergic responsiveness in experimental asthma.

This investigation was designed to elucidate the role of phospholipase (PLase) in relation to reduced beta-adrenergic responsiveness in guinea pigs subjected to experimental asthma. In the in vivo experiment, guinea pigs that had developed asthma-like symptoms after exposure to an aerosol of 2% ovalbumin for 7 to 8 min for 10 successive days were used as the experimental asthma group. The control group was exposed to saline. The endogenous PLase activity was determined by high performance liquid chromatography using ditridecanoyl phosphatidylcholine as a substrate. PLase activity of lung membranes in the experimental asthma group was significantly elevated by 50% compared with that in the control group. Phospholipid content of lung membranes in the experimental asthma group was decreased by 11% compared with that in the control group. The experimental asthma group showed a 37% decrease in the number of beta-adrenoceptors in lung membranes and a 54% decrease in isoproterenol-stimulated adenylate cyclase activity in lung membranes compared with the control group. Although forskolin-stimulated adenylate cyclase activity was also reduced by 24%, decreases in forskolin-stimulated activity were less than decreases in isoproterenol-stimulated activity. In the in vitro experiment phospholipids in lung membranes were degraded by pretreatment with 0.1 U of PLase A2. After pretreatment of lung membranes with PLase A2, the number of beta-adrenoceptors was reduced by 25% compared with that in the control group, and adenylate cyclase activity stimulated by isoproterenol and forskolin were also reduced by 67 and 28%, respectively. PLase A2 had a minor effect on forskolin-stimulated activity as compared with isoproterenol-stimulated activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear β-adrenergic receptors modulate gene expression in adult rat heart

Both β 1- and β 3-adrenergic receptors (β 1ARs and β 3ARs) are present on nuclear membranes in adult ventricular myocytes. These nuclear-localized receptors are functional with respect to ligand binding and effector activation. In isolated cardiac nuclei, the non-selective βAR agonist isoproterenol stimulated de novo RNA synthesis measured using assays of transcription initiation (Boivin et al., 2006 Cardiovasc Res. 71:69–78). In contrast, stimulation of endothelin receptors, another G protein-coupled receptor (GPCR) that localizes to the nuclear membrane, resulted in decreased RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the presence or absence of inhibitors of different mitogen-activated protein kinase (MAPK) and PI3K/PKB pathways. Components of p38, JNK, and ERK1/2 MAP kinase cascades as well as PKB were detected in nuclear preparations. Inhibition of PKB with triciribine, in the presence of isoproterenol, converted the activation of the βAR from stimulatory to inhibitory with regards to RNA synthesis, while ERK1/2, JNK and p38 inhibition reduced both basal and isoproterenol-stimulated activity. Analysis by qPCR indicated an increase in the expression of 18 S rRNA following isoproterenol treatment and a decrease in NFκB mRNA. Further qPCR experiments revealed that isoproterenol treatment also reduced the expression of several other genes involved in the activation of NFκB, while ERK1/2 and PKB inhibition substantially reversed this effect. Our results suggest that GPCRs on the nuclear membrane regulate nuclear functions such as gene expression and this process is modulated by activation/inhibition of downstream protein kinases within the nucleus.

Go controls the hyperpolarization-activated current in embryonic stem cell-derived cardiocytes

Hyperpolarization current (I(f)) is an important player in controlling heart rate and is stimulated by cAMP and inhibited by members of the pertussis toxin-sensitive G-protein G(i)/G(o) family. We have successfully derived cardiocytes from embryonic stem cells lacking G(o) or G(i2) and G(i3). We have established that both basal and isoproterenol-stimulated activities of I(f) in these cardiocytes have typical nodal-atrial characteristics and are unaffected by targeted gene inactivation of the G proteins G(o) or G(i2) and G(i3). Under basal conditions, both G(o) and G(i) are required for muscarinic inhibition of I(f) activity via a mechanism that involves the generation of nitric oxide, whereas, with prior stimulation by beta-agonists, only G(o) is required and G(i) and nitric oxide production are not. Our findings establish an essential role for G(o) in the antiadrenergic effect of muscarinic agent on I(f).

Abstract 373: A Novel Haplotype In The G(alpha)s Gene Alters Gene Expression And Responsiveness To Receptor Stimulation And Predicts Cardiac Performance In Humans

Background: Transgenic mice with cardiac Gαs overexpression exhibit enhanced inotropic and chronotropic responses to sympathetic stimulation, but develop cardiomyopathy with age. In contrast, chronic beta-adrenergic receptor blockade (beta-AR) mitigates cardiac dilation and depressed left ventricular function in these models. We tested the hypothesis that naturally occurring variants in regulatory regions of the human gene GNAS encoding Gαs result in altered Gαs expression and signal transduction properties in humans. Methods and results: Sequencing the promoter and intron 1 of GNAS revealed 11 single nucleotide polymorphisms resulting in three common haplotypes. In reporter assays haplotype *3 constructs exhibited significantly higher promoter activity compared to haplotype *1 and *2 constructs. In hearts from 54 patients under chronic beta-AR blockade scheduled for coronary artery bypass grafting (CABG) Gαs mRNA expression was more than 50% higher in homozygous *3 carriers (*3/*3) than in heterozygous (*3/−) and negative *3 (−/−) carriers (Gαs/Actin, 0.16±0.06 vs. 0.10±0.03 vs. 0.09±0.04; p=0.002). Consistent with increased Gαs expression, functional studies showed that *3/*3 carriers had higher basal activities of adenylyl cyclase (AC, 32.4±12.9 vs. 29.1±7.9 vs. 25.5±5.3 pmol/min/mg; p=0.019) and maximal isoproterenol-stimulated activities were markedly higher for the *3/*3 compared to *3/− and −/− carriers (79.3±29.7 vs. 66.7±16.2 vs. 57.3±11.1 pmol/min/mg; p=0.002). Direct AC stimulation with Forskolin was unaffected by GNAS haplotypes. Hemodynamic measurements in 137 bypass patients under chronic beta-AR blockade revealed a higher cardiac index (2.2±0.2 vs. 1.9±0.1 vs. 1.8±0.1 l min-1 m-2; p=0.025) in *3/*3 carriers. This was also in line with a lower NYHA functional class (1.8±0.2 vs. 2.2±0.1 vs. 2.3±0.1; p=0.040) and NT-proBNP levels (339±87 vs. 746±110 vs. 1365±259 pg/ml; p=0.002). Summary: Increased Gαs expression due to a functional haplotype results in altered signal transduction properties and may have salutary effects on cardiac performance in CABG patients under chronic beta-AR blockade. Our data therefore could help to translate results from transgenic mice to genotype-phenotype correlations in humans.

Expression of adenylyl cyclase V/VI mRNA and protein is upregulated in cyanotic infant human myocardium.

We have previously demonstrated that both basal and isoproterenol-stimulated activities of myocardial adenylyl cyclase were greater in cyanotic patients with tetralogy of Fallet (TOF) than those in acyanotic patients. However, it was not determined whether increased enzyme activity was related to a similar increase in adenylyl cyclase protein and mRNA expression. In the current study, we examined the mRNA and protein expression of cardiac adenylyl cyclase, types V and VI, in cyanotic and acyanotic patients with TOF. Ribonuclease protection assays and immunoblotting were performed on myocardial specimens obtained from cyanotic patients with TOF and acyanotic patients with TOF or ventricular septal defect. We demonstrated that in both cyanotic and acyanotic patients, there was more type V adenylyl cyclase mRNA than type VI. Types V and VI cardiac adenylyl cyclase mRNA were significantly increased in myocardium of the cyanotic group compared to the acyanotic group. Protein expression of both V and VI adenylyl cyclases was correspondingly upregulated in cyanotic patients compared to acyanotic patients. Our results indicate that gene and protein expression of cardiac adenylyl cyclases, types V and VI, is increased in the cyanotic myocardium. These results suggest that chronic hypoxemia may regulate the expression of adenylyl cyclase enzymes.

Differential alterations in cardiac adrenergic signaling in chronic hypoxia or norepinephrine infusion.

Norepinephrine (NE)-induced desensitization of the adrenergic receptor pathway may mimic the effects of hypoxia on cardiac adrenoceptors. The mechanisms involved in this desensitization were evaluated in male Wistar rats kept in a hypobaric chamber (380 Torr) and in rats infused with NE (0.3 mg. kg(-1). h(-1)) for 21 days. Because NE treatment resulted in left ventricular (LV) hypertrophy, whereas hypoxia resulted in right (RV) hypertrophy, the selective hypertrophic response of hypoxia and NE was also evaluated. In hypoxia, alpha(1)-adrenergic receptors (AR) density increased by 35%, only in the LV. In NE, alpha(1)-AR density decreased by 43% in the RV. Both hypoxia and NE decreased beta-AR density. No difference was found in receptor apparent affinity. Stimulated maximal activity of adenylate cyclase decreased in both ventricles with hypoxia (LV, 41%; RV, 36%) but only in LV with NE infusion (42%). The functional activities of G(i) and G(s) proteins in cardiac membranes were assessed by incubation with pertussis toxin (PT) and cholera toxin (CT). PT had an important effect in abolishing the decrease in isoproterenol-induced stimulation of adenylate cyclase in hypoxia; however, pretreatment of the NE ventricle cells with PT failed to restore this stimulation. Although CT attenuates the basal activity of adenylate cyclase in the RV and the isoproterenol-stimulated activity in the LV, pretreatment of NE or hypoxic cardiac membranes with CT has a less clear effect on the adenylate cyclase pathway. The present study has demonstrated that 1) NE does not mimic the effects of hypoxia at the cellular level, i.e., hypoxia has specific effects on cardiac adrenergic signaling, and 2) changes in alpha- and beta-adrenergic pathways are chamber specific and may depend on the type of stimulation (hypoxia or adrenergic).

Glucose modifies the cross-talk between insulin and the beta-adrenergic signalling system in vascular smooth muscle cells.

Abnormalities in the vascular function of insulin are observed in insulin resistance, and hyperglycaemia is one of the important factors inducing insulin resistance. To investigate the role of glucose in the interaction of insulin and beta-adrenergic signalling systems in vascular smooth muscle cells (VSMC). After cells were treated with D-glucose (525 mmol/l) and insulin (100 nmol/l), adenylyl cyclase activity was measured in the presence of isoproterenol, forskolin, and cholera toxin. Assays for insulin-induced activities of insulin receptor substrate (IRS)-1, phosphoinositide 3-kinase (PI3-K) and mitogen-activated protein kinase (MAPK) were performed. In the presence of low glucose concentrations (5 mmol/l), insulin enhanced isoproterenol-, forskolin- and cholera toxin-stimulated adenylyl cyclase activities. This stimulatory effect was abolished by PI3-K inhibitors, wortmannin, or LY294002. In contrast, in the presence of high glucose concentrations (25 mmol/l), insulin attenuated isoproterenol-stimulated activity but not cholera toxin- or forskolin-stimulated activity. Insulin-stimulated activities of IRS-1 and PI3-K, but not MAPK activity, were also attenuated in the presence of high concentrations of glucose. The MAPK kinase inhibitor, PD98059, abolished the inhibitory effect of insulin on the beta-adrenergic signalling system. Troglitazone and pioglitazone prevented this inhibitory effect of insulin by restoring IRS-1 and PI3-K activities. In the presence of low glucose concentrations, insulin stimulates the beta-adrenergic signalling system through the IRS-1/PI3-K pathway. However, in the presence of high glucose concentrations, the effect of insulin is switched to an inhibitory one, through the MAPK pathway. Our finding suggests that high glucose concentrations modify the cross-talk between insulin and the beta-adrenergic signalling systems in VSMC.

Purines, a new class of agonists in salivary glands?

The response of rat submandibular glands to extracellular purines was tested. In crude cellular suspensions, ATP increased the [Ca2+]i mostly by promoting uptake of extracellular calcium. ATP caused the pHi to drop, a response blocked by chloride channel inhibitors. ATP also inhibited the basal and isoproterenol-stimulated activity of the Na+-K+-2Cl-cotransporter. These effects were reproduced by benzoyl-ATP, an agonist of ionotropic purinoceptors. In pure ductal suspensions, ATP activated a metabotropic P2Y1 purinergic receptor coupled to phospholipase C and opened a non-specific cation channel coupled to a P2X7 receptor. Activation of these receptors stimulated a Ca2+-dependent and a Ca2+-independent phospholipase A2, the latter resulting in kallikrein secretion. We conclude that purinergic agonists can modulate the activity of both acinar and ductal phases of secretion. Activation of metabotropic receptors coupled to phospholipase C could lead to responses resembling those to muscarinic or adrenergic agonists. Activation of ionotropic receptors could stimulate new intracellular responses also involved in secretory function.

Purines, a new class of agonists in salivary glands?

The response of rat submandibular glands to extracellular purines was tested. In crude cellular suspensions, ATP increased the [Ca 2+] i mostly by promoting uptake of extracellular calcium. ATP caused the pH i to drop, a response blocked by chloride channel inhibitors. ATP also inhibited the basal and isoproterenol-stimulated activity of the Na +-K +-2Cl-cotransporter. These effects were reproduced by benzoyl-ATP, an agonist of ionotropic purinoceptors. In pure ductal suspensions, ATP activated a metabotropic P2Y 1 purinergic receptor coupled to phospholipase C and opened a non-specific cation channel coupled to a P2X 7 receptor. Activation of these receptors stimulated a Ca 2+-dependent and a Ca 2+-independent phospholipase A 2, the latter resulting in kallikrein secretion. We conclude that purinergic agonists can modulate the activity of both acinar and ductal phases of secretion. Activation of metabotropic receptors coupled to phospholipase C could lead to responses resembling those to muscarinic or adrenergic agonists. Activation of ionotropic receptors could stimulate new intracellular responses also involved in secretory function.

Atypical beta-adrenergic receptors in rat liver: evidence for transient expression during aging.

This study was designed to determine whether functional beta 3-adrenergic receptors exist in the rat liver and whether there are alterations in the beta 3-adrenergic response with age in a manner similar to that which occurs for beta 2-adrenergic receptors. The beta 3 stimulation of adenylyl cyclase activity was assessed using the novel beta 3-specific agonist, CGP-12177A. Adenylyl cyclase activation by CGP-12177A was seen only in the adults. Isoproterenol-stimulated adenylyl cyclase activity was high in the neonates, declined by 45% in the adults, and was high again in the senescent rats. ICI 118551, a beta 2-selective antagonist, attenuated the isoproterenol-stimulated activity by two-thirds but had no effect on the CGP-12177A-stimulated activity. These data demonstrated the presence of the beta 3-adrenergic receptor in the rat liver, although only at the adult stage of development. In addition, these data confirm earlier findings that beta 2-adrenergic activation of adenylyl cyclase is biphasic with age and indicate that the emergence of the beta 3-stimulated activity coincides with the attenuation of beta 2-stimulated activity.

Regulation of cardiac adenylyl cyclase by Epidermal Growth Factor (EGF): Role of EGF receptor protein tyrosine kinase activity

We have shown previously that the α subunit of the stimulatory GTP binding regulatory component of adenylyl cyclase (G sα) mediates epidermal growth factor (EGF)-elicited stimulation of rat cardiac adenylyl cyclase (Nair et al., J Biol Chem 265: 21317–21322, 1990). Employing purified protein phosphotyrosine phosphatase, and benzylidene derivatives (tyrphostins: compounds 11 and 12) that selectively inhibit EGF receptor protein tyrosine kinase (EGFRK) activity, the role of EGFRK in EGF-mediated stimulation of cardiac adenylyl cyclase was investigated. The ability of the tyrphostins to inhibit the EGFRK activity in cardiac membranes was determined by monitoring tyrosine phosphorylation of either the 170 kDa protein or immunoprecipitated EGF receptor at 0° and room temperature, respectively. Compounds 11 and 12, in a concentration-dependent manner, inhibited EGF receptor tyrosine kinase activity. In assays of adenylyl cyclase activity neither compound 11 nor compound 12 altered Gpp(NH)p- or isoproterenol-stimulated activity. However, both compounds, in a concentration-dependent manner, attenuated the ability of EGF to stimulate adenylyl cyclase activity without altering specific binding of [ 125I]EGF to cardiac membranes. Similarly, protein phosphotyrosine phosphatase obliterated the ability of EGF, but not isoproterenol, to stimulate adenylyl cyclase. Thus, we conclude that protein tyrosine kinase activity of the EGF receptor is essential for the stimulation of cardiac adenylyl cyclase by EGF.

β 1-Adrenergic and D 1 Dopaminergic Receptors in Human Neurotumor Cells: Differences in Spare Receptors and Desensitization of Adenylyl Cyclase

Haman SK-N-MC neurotumor cells express both β 1-adrenergic and D 1 dopaminergic receptors. Although there were sevenfold more β 1 than D 1 receptors, maximum stimulation of adenylyl cyclase activity by isoproterenol was only threefold more than that by dopamine. We had shown previously that the pattern of agonist-mediated densensitization was different for the two receptors. To compare the efficiency of the two receptors to couple to the same adenylyl cyclase and to explore the possible role of spare receptors, the cells were exposed to N -ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which irreversibly alkylates and inactivates catecholamine receptors. Loss of receptor binding activity was dependent on the concentration of EEDQ, D 1 receptors being more sensitive to EEDQ than β 1 receptors with IC 50 values of 1 and 30 μ M, respectively. β 1 receptors were more sensitive to the irreversible β-adrenergic antagonist N 8 -bromoacetyl- N 1-(3′-(4-indolyloxy)-2′-hydroxy-propyl)-[Z]-1,8-diamino- p-menthane (BIM) with an IC 50 of 1 n M. Inactivation of D 1 receptors parallelled the reduction in dopamine-stimulated adenylyl cyclase activity without a shift in K act, indicating the absence of spare D 1 receptors in SK-N-MC cells. By contrast, there appeared to be spare β1 receptors as the K act shifted before any reduction in isoproterenol-stimulated activity occurred in EEDQ- or BIM-treated cells. Inactivation of 70% of the β 1 receptors resulted in only a 20% decrease in isoproterenol-stimulated adenylyl cyclase activity, and the remaining β 1 receptors were as efficient as D 1 receptors in stimulating adenylyl cyclase. In addition, inactivation of the spare β 1 receptors did not alter the unique pattern of agonist-mediated desensitization. We conclude that human β 1 and D 1 receptors are equally efficient at stimulating adenylyl cyclase but differ in their mechanisms of agonist-mediated desensitization.

Desipramine desensitizes β-adrenergic signal transduction in rat salivary glands

The consequences of the tricyclic antidepressant drugs include sedation, orthostatic hypotension and salivary dysfunction. It was reported that administration of desipramine resulted in a decrease in the concentration of secreted salivary protein. Tricyclic antidepressants may indirectly alter salivary function as a result of their action on the central nervous system to decrease adrenergic neural transmission or, alternately, may act directly on salivary glands to modulate β-adrenergic signal transduction. To investigate the latter possibility, the effects of administration of desipramine (DMI) for 28 days was assessed on β-adrenergic and post-receptor signal transduction in the parotid and submandibular glands of the rat and the reversibility of this treatment following a washout period of 15 days. Both glands demonstrated desensitization of the isoproterenol-stimulated activity, and in the parotid gland treatment with DMI decreased the post-receptor signal transduction as well. The washout period of 15 days completely reversed the desensitization in the parotid gland and partially reversed the effects in the submandibular gland. These data suggest that desipramine-induced attenuation of β-adrenergic signal transduction is not limited to the brain and that these direct effects on salivary glands may explain the salivary dysfunction observed after administration of desipramine.

Inhibitory and stimulatory effects of neuropeptide Y(17-36) on rat cardiac adenylate cyclase activity. Structure-function studies.

Neuropeptide Y (NPY) inhibits cardiac adenylate cyclase activity by interacting with specific receptors coupled to a pertussis toxin-sensitive G protein. Structure-activity studies revealed that only C-terminal fragments can exhibit an NPY-like inhibitory effect on 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes. Although NPY(17-36) inhibited 125I-NPY binding with high potency, it produced a biphasic effect on basal (GTP, 10 and 100 microM or guanosine 5'-gamma-O-(thio)triphosphate (GTP gamma S, 10 microM) adenylate cyclase activity. Low concentrations (less than 1 nM) of NPY(17-36) inhibited the adenylate cyclase activity whereas high concentrations (greater than 1 nM) reversed this action. GTP gamma S (100 microM) reversed the biphasic effect of NPY(17-36). NPY(17-36) exhibited only a stimulatory effect in the membranes from pertussis toxin-treated rats and an inhibitory effect with membranes from cholera toxin-treated rats. Low concentrations (less than 1 nM) of NPY(17-36) inhibited isoproterenol-stimulated adenylate cyclase activity whereas high doses (greater than 1 nM) reversed this activity. The cardiac NPY receptor antagonist, NPY(18-36) (1 microM), completely blocked the biphasic effect of NPY(17-36) on isoproterenol-stimulated activity. The inhibitory dose-response curve of NPY on isoproterenol-stimulated adenylate cyclase activity was shifted parallel to the right by NPY(17-36) (1 microM), suggesting that it is an antagonist of NPY at high concentrations. N-alpha-acetylated and C-terminally deamidated analogs of NPY(17-36) had no effect on the adenylate cyclase activity. [im-DNP-His26] NPY exhibited a more pronounced biphasic effect whereas N-alpha-myristoyl-NPY(17-36) elicited only a stimulatory effect. These investigations suggest that: 1) the inhibitory and stimulatory effects of NPY(17-36) are mediated by high affinity NPY receptors coupled to a pertussis toxin-sensitive G protein and a distinct population of low affinity receptors coupled to a cholera toxin-sensitive G protein, respectively; and 2) the stimulatory effect of NPY(17-36) is dissociable.

The effects of benzodiazepines on basal and isoproterenol‐stimulated N‐acetyltransferase activity by the rat pineal gland, in vivo and in vitro

The present study examined the effects of "peripheral," "central," and "mixed" benzodiazepine agonists and antagonists on the nocturnal rise in rat pineal NAT activity in vivo and the isoproterenol-stimulated NAT activity of pineals in organ culture. Administration of the central agonist clonazepam or the mixed agonist-antagonist diazepam, 4 hr after dark, at a dose of 25 mg/kg each, inhibited nocturnally elevated NAT 20 min later, while this same dose of the peripheral agonist RO 54864 elevated NAT activity. In a second study these agents were administered in vivo 1 hr before dark, at a dose of 3, 10, or 25 mg/kg i.p. and tested 4 hr after dark for in vitro rat pineal NAT activity. None of these agents affected NAT activity at the 3- or 10-mg/kg dose, but RO 54864 25 mg/kg did induce elevated activity. In a third study, all of these agents prolonged the time period for NAT induction by isoproterenol in rat pineals cultured for 48 hr before stimulation. The data suggests that benzodiazepine stimulation of NAT activity in vitro is not specific to "central" or "peripheral" benzodiazepine receptors and that inhibition of melatonin production in vivo occurs either at some step before NAT induction or is involved with the inhibition of pineal HIOMT activity.

Adenylate cyclase agonist properties of CGP-12177A in brown fat: evidence for atypical beta-adrenergic receptors.

Thermogenesis in brown adipose tissue (BAT) is stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. Recently it was reported that the beta-adrenergic antagonist CGP-12177A stimulates oxygen consumption in BAT. To investigate the mechanism of action of CGP-12177A in BAT, we assessed the inhibitory and stimulatory affects of CGP-12177A on the adenylate cyclase system in myocardial and BAT membranes from rats. CGP-1277A inhibited isoproterenol-stimulated adenylate cyclase activity in a dose-dependent manner, with an inhibitory constant (Ki) of 1.94 +/- 0.18 microM in BAT and 0.49 +/- 0.11 microM in the heart. However, in the absence of isoproterenol, CGP-12177A stimulated adenylate cyclase in BAT with two components of activation, and half-maximal stimulation occurred at 1 microM and 1.5 mM. In contrast, CGP-12177A did not stimulate adenylate cyclase activity in heart membranes. Propranolol inhibited the isoproterenol-stimulated activity with a potency that was one log less in BAT compared with heart. Propranolol fully blocked the high-affinity component but only weakly blocked the low-affinity component of CGP-12177A-stimulated activity in BAT. Pindolol was also less potent in BAT but inhibited the CGP-12177A-stimulated activity in a manner similar to the inhibition of the isoproterenol-stimulated activity, suggesting the CGP-12177A activation was beta-receptor mediated. Binding curves of [125I]iodocyanopindolol ([125I]ICYP) in competition with CGP-12177A demonstrated a shift to lower affinity in the presence of beta,gamma-imidoguanosine 5'-triphosphate, indicating that CGP-12177A has agonist properties with respect to the [125I]ICYP binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prolonged hypoxia on adenylate cyclase activity and beta-adrenergic receptors in pulmonary and systemic arteries of the rat.

Prolonged hypoxia causes pulmonary hypertension but no change in systemic vasomotor tone. In an effort to define the mechanisms involved, we determined the effects of 3 and 7 days of hypoxia on adenylate cyclase activity and beta-adrenergic receptor binding characteristics in pulmonary and systemic arteries in an adult rat model of hypoxic pulmonary vasoconstriction produced by hypobaria. Basal and stimulated adenylate cyclase activity were measured in crude membrane preparations by radioimmunoassay for cyclic AMP. Basal enzyme activity in pulmonary arteries did not change with hypoxia, whereas in systemic arteries it increased 3.5- and 5.3-fold following 3 and 7 days of hypoxia, respectively. GTP-stimulated activity in pulmonary arteries also did not change, but in systemic arteries it increased 7.1- and 5.5-fold. Isoproterenol-stimulated activity in pulmonary arteries was decreased to 49% of control-stimulated activity following 3 days but was similar to control-stimulated activity after 7 days of hypoxia; in systemic arteries it increased 5.6- and 4.6-fold. Sodium fluoride-stimulated activity in pulmonary arteries was unchanged, whereas in systemic arteries it increased 3.8- and 5.3-fold. In contrast, forskolin-stimulated activity, which also was not altered in pulmonary arteries, was increased by only 85% and 71% in systemic arteries. beta-Adrenergic receptors were studied with [125I]iodocyanopindolol. Seven days of hypoxia decreased receptor density by 37% and 57% in the pulmonary and systemic arteries, respectively. Receptor affinity for agonists was not altered. Thus, despite downregulation of beta-adrenergic receptors in both artery types, prolonged hypoxia has no sustained effect on adenylate cyclase activity in pulmonary arteries, whereas enzyme activity in systemic arteries is markedly increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nonachlazine on the adenylate cyclase system of the rabbit heart

The influence of nonachlazine on the adenylate cyclase system of the rabbit heart was investigated. The faint beta-blocking activity (displacement of up to 20% of [3H]-dihydroalprenolol and inhibition of isoproterenol-stimulated activity) as well as the ability to stimulate basal activity in micromolar concentrations has been observed. Such combination of properties may be important in the realization of some positive effects of nonachlazine pharmacological activity.

A comparison of the properties of the cardiac beta-adrenergic receptor adenylyl cyclase system in the rat and the marmoset monkey

1. 1. A comparison was made between adrenergic receptor binding properties and catecholamine-stimulated adenylyl cyclase activity in cardiac membrane fractions from the rat and the marmoset monkey. 2. 2. [ 125I]HEAT and [ 125I]ICYP were used to determine respectively, the α- and β- adrenergic receptor binding in cardiac membrane fractions. 3. 3. Greatest adrenergic receptor density and degree of specific binding was evident using membranes sedimenting between 6000 and 46,000 g. 4. 4. In rat heart, the ratio of β- to α-adrenergic receptors was 57:43, while for the marmoset this ratio was 92:8. 5. 5. Basal, isoproterenol, sodium fluoride and forskolin-stimulated adenylyl cyclase activities in the rat and marmoset monkey were investigated in several different cardiac membrane fractions. 6. 6. The highest-fold stimulation of adenylyl cyclase activity was present in membranes sedimenting between 0 and 500 g. 7. 7. Adenylyl cyclase activities were higher in the marmoset heart membrane preparations, however the rat heart adenylyl cyclase exhibited greater sensitivity to isoproterenol; ED 50 3.8 × 10 −7 M compared with 7.5 × 10 −7 M for the marmoset. 8. 8. Differences between rat and marmoset catecholamine-sensitive adenylyl cyclase activity were apparent when a variety of adrenergic agonists and antagonists were tested. 9. 9. In the marmoset but not the rat, adrenergic antagonists alone stimulated basal adenylyl cyclase activity. 10. 10. Differences in the activation of cardiac adenylyl cyclase by GTP and GMP-PNP were also evident between the rat and the marmoset monkey, particularly with regard to basal and isoproterenol-stimulated activity.

Adrenergic and serotonergic regulation of skeletal muscle metabolism in the rat: specificity of the serotonin- and isoproterenol-stimulable adenylyl cyclase in sarcolemma

The specificity and kinetics of rat sarcolemmal serotonin- and isoproterenol-stimulable adenylyl cyclases were studied. The stimulation of adenylyl cyclase by serotonin was less than that by isoproterenol and required low concentrations of ATP (0.1 mM), 10 μM GTP, and free Mg ion concentrations of 10–20 μM. The isoproterenol-stimulated activity was readily detectable over a much wider range of MgCl 2 (1.0–5.0 mM) and ATP (0.1–3.0 mM). (±)-Oxprenolol and l(−)-propranolol inhibited isoproterenol stimulation with K B values of 6.3 × 10 −9 M and 1.9 × 10 −9 M, respectively. Interference by (±)-cyproheptadine, (±)-methysergide and d(±)-propranolol was not strictly competitive. However, these were much more potent inhibitors of serotonin stimulation than were oxprenolol and l-propranolol. The concentrations of antagonists producing 50% inhibition (IC 50) of the maximal adenylyl cyclase stimulation by serotonin were compared to the IC 50 obtained with isoproterenol; the ratios were 0.02 for oxprenolol, 0.50 for l-propranol, 295 for d-propranolol, 2438 for cyproheptadine, 5785 for methysergide and > 125 000 for lysergic acid diethylamide (LSD). These data indicate that rat skeletal muscle adenylyl is stimulated by discrete serotonergic and adrenergic receptors, and that there may be distinct conditions for optional stimulation.