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P A Insel

Publications and source records attributed to P A Insel.

At least 19 recordsLinked to original sources
PubMed1992

Plasma-membrane-independent pool of the alpha subunit of the stimulatory guanine-nucleotide-binding regulatory protein in a low-density-membrane fraction of S49 lymphoma cells.

We report that compartmentalisation of the stimulatory guanine-nucleotide-binding regulatory protein (Gs) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the alpha subunit of Gs (Gs alpha) [Ransnäs, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900-7903], three membrane-bound forms of Gs alpha were identified through rate-zonal centrifugation in sucrose density gradients, Gs alpha-specific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of Gs alpha in the high-density portion of the gradient (1.13-1.16 g/cm3) is identical with that of beta-adrenergic-receptor binding, Na/K-ATPase and adenylate cyclase activity, and may therefore be identified as plasma-membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09-1.11 g/cm3), contains a much lower total amount of Gs alpha and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH-cytochrome-c reductase and glucose-6-phosphatase. The identity of the third pool of Gs alpha located at the top of the gradient (1.06-1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose:N-acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high-density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K-ATPase activity indicates that this low-density (light) membrane form of Gs alpha is distinct from any plasma-membrane fragments. Furthermore, sedimentation at 100,000 x g proves its particulate (membrane) character. The light membrane form of Gs alpha subunit is functionally active in an adenylate cyclase complementation assay using cyc- membranes devoid of Gs alpha. Overall, our data indicates that a substantial portion of Gs alpha is localized in membrane pools other than plasma membrane.

Adenylyl Cyclases↗
PubMed1992

Beta-adrenergic-receptor-mediated dissociation and membrane release of the Gs protein in S49 lymphoma-cell membranes. Dependence on Mg2+ and GTP.

We reported [Ransnäs, Svoboda, Jasper & Insel (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900-7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, Gs, into its alpha s and beta gamma subunits, leading to redistribution of alpha s from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of Gs dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha s levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha s and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha s/mg of membrane protein and lacked significant dissociation of Gs and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of Gs in plasma membranes and for membrane release of alpha s. At a physiological concentration of free Mg2+ ions (100 microM), 100 microM-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of Gs/mg of membrane protein. A substantial increase in the dissociation rate of Gs was achieved by addition of 1 microM-isoprenaline and 100 microM-GTP; 18.4 +/- 0.9 pmol of Gs/mg of membrane protein was dissociated, with a kappa of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of Gs was completely blocked by the beta-adrenergic receptor antagonist propranolol. The concentration-response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a kappa act. of 16 +/- 5 nM, whereas the kappa act. for isoprenaline-induced membrane release was 10 nM. We conclude that release of alpha s from plasma membranes is likely to accompany Gs-subunit dissociation and constitutes a potentially important facet of Gs action.

Animals↗
PubMed1992

Evolving concepts of partial agonism. The beta-adrenergic receptor as a paradigm.

The exact mechanism of receptor activation at the molecular level are still not known, nor do we completely understand the precise factors that distinguish agonist- and partial agonist-induced activation. Nevertheless, recent years have brought forth an explosion of new information regarding beta-adrenergic receptor structure and ligand-induced activation. Partial agonists are likely intermediate in their ability to interact with crucial serine residues (Ser204 and Ser207) on the beta-adrenergic receptor; these interactions allow either incomplete stimulation of the entire receptor population, or full stimulation of only a portion of the entire receptor population. From the work presented by Tota and Schimerlik for the muscarinic cholinergic receptor (another G-protein coupled receptor), it is likely that partial agonists induce or stabilize receptor conformations that have a lower affinity for their G protein compared to receptors stimulated by a full agonist. Molecular cloning of beta-adrenergic receptors and analyses of mutated and chimeric receptors expressed in transfected systems have indicated that domains of the receptor that bind agonists may be different from those with which antagonists interact. Thus, the ability of a partial agonist to interact with these two different domains may be a determinant of efficacy. Agonists alter the sulfhydryl redox status of the beta-adrenergic receptors in the presence of Gs. Disulfide rearrangement has been postulated to provide a structural constraint which biases G-protein-linked receptors in the "ground state" and may be important for stabilizing the active state of the receptor and holding the agonist/receptor/Gs ternary complex in the high-affinity state. Partial agonists induce this state less efficaciously or are less capable of holding the receptor in the active conformation to allow disulfide exchange to take place. The extent of receptor stimulation may dictate which G proteins are activated by a particular receptor, and thus which cellular effectors are stimulated. Alternatively, the level of activation of a receptor may translate into varying states of activation of a particular G protein (stabilized in part by disulfide bonds). Techniques such as fluorescence energy transfer in reconstitution systems or nuclear magnetic resonance spectroscopy should prove useful in distinguishing among these possible mechanisms. Ultimately, as a long-term goal, X-ray crystallography of unoccupied receptors and receptors liganded by partial or full agonists may provide definitive insights. Although definitive answers are not yet possible, the rapid progress in understanding aspects of receptor structure allows a reformulation of ideas regarding the molecular basis of efficacy and partial agonism.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic beta-Agonists↗
PubMed1992

A substantial proportion of cardiac Gs is not associated with the plasma membrane.

The precise interactions between the subunits of Gs (alpha s, beta, gamma) and the plasma membrane remain to be established. If alpha s is associated loosely with the inner membrane, is labile during activation, or is always present to some extent in the cytoplasm, then it should fractionate to the supernatant of a high-speed centrifugation. We identified abundant alpha s (52-66% of total cellular) in the supernatant fraction of right atrial and left ventricular membrane preparations of porcine heart as shown by two distinct measures of alpha s (immunoblotting and ADP ribosylation by cholera toxin). However, functional assays utilizing reconstitution of cardiac alpha s with cyc- S49 membranes revealed that the supernatant fraction contained approximately 16% of total cellular alpha s activity. The alpha s present in the supernatant fraction did not result from contamination by sarcolemmal membrane fragments. We conclude that traditional methods for quantifying alpha s which utilize only detergent extracts from high-speed pellets do not account for a sizable proportion of total cellular alpha s, but that the majority of this population of cardiac alpha s may not be functional, at least with respect to adenylyl cyclase activation.

Adenosine Diphosphate Ribose↗
PubMed1992

Expression of renal alpha 1-adrenergic receptor subtypes in established hypertension.

Radioligand binding studies were undertaken in renal membranes of normotensive and hypertensive rats in order to test the hypothesis that there are alterations in renal alpha 1-adrenergic subtypes of genetic hypertensive animals. The highly selective competitive compound, (+)-niguldipine, was used to distinguish high-affinity (alpha 1a) from low-affinity (alpha 1b) sites, after initial studies demonstrated that this compound had greater selectivity than 5-methylurapidil in distinguishing alpha 1a and alpha 1b sites in rat renal membranes. In contrast to the significant difference in the blood pressure of the spontaneously hypertensive rats (delta BP = 74 mm Hg), there was no difference in the renal alpha 1-adrenergic receptor density. Membranes from the whole kidneys of spontaneously hypertensive rats (SHRs) possessed 31% alpha 1a and 69% alpha 1b sites with -log(Ki) values of 10.0 +/- 0.3 and 7.1 +/- 0.1, respectively, for (+)-niguldipine. However, these values were not different from those obtained from renal membranes of the normotensive Wistar-Kyoto (WKY) rats. These results indicate that in spite of the elevated blood pressure during the established phase of hypertension, the number, the affinity, and the ratio of the alpha 1a and alpha 1b appear not to be responsible for the manifestation of hypertension during this phase.

Animals↗
PubMed1992

GTP-binding proteins and post-receptor components in hypertension.

Alterations in neurohormonal response are a widely-observed feature in various forms of hypertension. Such responses depend not only on levels of hormones/neurotransmitters, but also on receptors and post-receptor components. With respect to G protein-coupled receptors, such as those for catecholamines, angiotensin II, and bradykinin, it is possible that G-proteins or G protein-coupled effector molecules are altered in hypertension. In this article, several classes of G alpha proteins and effectors which link to these proteins are briefly discussed. Evidence is presented in support of the concept that signal amplification in G protein-coupled receptor systems occurs at the level of receptor activation of the G proteins. Limited data are as yet available that directly assess whether changes in the amount or properties of particular G alpha proteins or G-protein-linked effectors, are altered in hypertension. The availability of antibody, cDNA and other genetic probes should prove highly useful in testing the hypothesis that such alterations are important for the pathogenesis and maintenance of the hypertensive state.

Animals↗
PubMed1992

Myocardial beta-adrenergic receptor expression and signal transduction after chronic volume-overload hypertrophy and circulatory congestion.

BACKGROUND: The volume-overload, high-output state induced by aortocaval fistula is unique because it is not generally associated with marked abnormalities of contractile function. Thus, changes in beta-adrenergic receptor (beta AR) expression should reflect more directly the influence of neurohumoral adrenergic tone, clarifying the manner in which peripheral (neurohumoral) versus primary myocardial factors are operative in decreased beta AR-dependent signal transduction. METHODS AND RESULTS: We examined the beta-adrenergic receptor-responsive adenylyl cyclase pathway in hearts from pigs subjected to volume-overload hypertrophy with circulatory congestion. Nine pigs underwent initial pharmacological and hemodynamic studies, and, 5 weeks after aortocaval fistula placement, when signs of circulatory congestion were evident, these measurements were repeated. Biochemical analyses of plasma and myocardium from these animals and seven normal animals were compared. Experimental animals showed signs of circulatory congestion (tachypnea, weight gain, pulmonary rales) within 3-4 weeks of fistula placement. Necropsy showed ascites and biventricular cardiac hypertrophy, but no fibrosis or inflammation was present on histological inspection. Heart rate responsiveness to beta AR stimulation was blunted, with ED50, for isoproterenol increased 133% (p less than 0.001) after development of circulatory congestion. Biochemical analyses of the beta AR-responsive adenylyl cyclase pathway showed uniform decreases in beta AR number in right atrium, right ventricle, and left ventricle (36-41% decreases, p less than 0.005). Downregulation was selective for beta 1-receptors, and remaining receptors in the right and left ventricles showed low-affinity agonist binding, suggesting an uncoupling from Gs. All measures of adenylyl cyclase activity were diminished significantly in membrane homogenates from the right atrium (mean reduction, 50 +/- 10%) and left ventricle (mean reduction, 44 +/- 8%) after volume overload. Finally, we found that amounts of cardiac Gs, as measured in reconstitution assays, were decreased in both the right atrium (p less than 0.02) and the left ventricle (p less than 0.01) of volume-overloaded animals but that levels of pertussis toxin substrate were unchanged. CONCLUSIONS: Biochemical findings occurred in the absence of myocardial inflammation or fibrosis and without pharmacological interventions, suggesting that circulatory congestion, with attendant elevation in plasma norepinephrine, may be a sufficient stimulus to induce such changes. The data are compatible with a catecholamine-driven beta AR pathway desensitization. Thus, a primary defect in intrinsic contractile function is not a necessary component for abnormalities of the myocardial beta AR-responsive adenylyl cyclase pathway.

Adenylyl Cyclases↗
PubMed1992

On the role of renal alpha-adrenergic receptors in spontaneously hypertensive rats.

We tested the hypothesis that a genetically determined increase in renal alpha-adrenergic receptor density might be a pathophysiologically important factor in the spontaneously hypertensive rat model of genetic hypertension. In a first study, we compared renal alpha 1 and alpha 2-adrenergic receptor density with systolic blood pressure in 45 rats of an F2 generation of Wistar-Kyoto x spontaneously hypertensive rat hybrids but were unable to detect significant cosegregation between either receptor density or blood pressure. In a second study, we determined renal alpha 1- and alpha 2-adrenergic receptor density in Wistar-Kyoto and spontaneously hypertensive rat kidneys that were transplanted into an F1 generation of Wistar-Kyoto x spontaneously hypertensive rat hybrids. Although Wistar-Kyoto kidneys lowered blood pressure in these animals and spontaneously hypertensive rat kidneys increased blood pressure, renal alpha-adrenergic receptor densities were similar in membranes from both types of kidneys. Since rat kidney coexpresses alpha 1A- and alpha 1B-adrenergic receptors, we also investigated whether differential regulation of these two subtypes might conceal ongoing alterations. The alpha 1A/alpha 1B-adrenergic receptor ratio, however, was similar in Wistar-Kyoto rats, spontaneously hypertensive rats, and F1 rats transplanted with a kidney from either strain. Taken together these data do not support the hypothesis that genetically determined alterations of renal alpha-adrenergic receptor numbers play an important role in the development of elevated blood pressure in the spontaneously hypertensive rat.

Animals↗
PubMed1991

Inhibition of subunit dissociation and release of the stimulatory G-protein, Gs, by beta gamma-subunits and somatostatin in S49 lymphoma cell membranes.

We examined the interaction between the stimulatory guanine-nucleotide-binding protein, Gs, and the inhibitory guanine-nucleotide-binding protein, Gi, in cell membranes of S49 lymphoma cells. In these cells, beta-adrenergic receptors stimulate the activity of adenylate cyclase via Gs, whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, Gi. Using an antibody that selectively recognizes alpha s, the monomeric, but not the heterotrimeric, alpha-subunit of Gs, we quantified the extent of dissociation of Gs in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 microM-isoprenaline, 100 microM free Mg2+ and 100 microM-GTP produced substantial subunit dissociation of Gs, which was reversible by addition of purified beta gamma-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated Gs (kinhib. for somatostatin = 51 +/- 12 nM) and in the activity of adenylate cyclase (kinhib. = 121 +/- 20 nM). By contrast, after addition of a 10-fold molar excess of beta gamma-dimer relative to alpha s, there was a 2-3 min lag, after which the beta gamma-dimer re-associated Gs. Isoprenaline-induced dissociation of Gs was accompanied by a release of alpha s from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of alpha s by the beta-agonist and reversal by somatostatin. Membrane release of Gs by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conclude that in native cell membranes somatostatin-induced activation of Gi dissociates Gi and interferes with the Gs activation cycle by providing beta gamma-dimer, which acts to prevent or reverse formation of monomeric alpha s. Because alpha s can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated alpha s is likely to be an important factor in modulating the activity of adenylate cyclase.

Animals↗
PubMed1991

Intracellular Ca2+ and protein kinase C interact to regulate alpha 1-adrenergic- and bradykinin receptor-stimulated phospholipase A2 activation in Madin-Darby canine kidney cells.

Alpha 1-Adrenergic receptors and bradykinin receptors are two distinct membrane receptors that stimulate phospholipid breakdown and arachidonic acid and arachidonic acid metabolite release. In the current studies, we have examined several mechanisms to assess their possible contribution to arachidonic acid release in the Madin-Darby canine kidney cell line by agonist stimulation of these receptors: 1) activation of phospholipase A2 (PLA2); 2) sequential activation of phospholipase C, diacylglycerol lipase, and monoacylglycerol lipase; and 3) inhibition of the sequential action of fatty acyl-CoA synthetase and lysophosphatide acyltransferase. Experiments were conducted to measure the stimulation of lysophospholipid production by epinephrine and bradykinin, the rate of incorporation of [3H]arachidonic acid into stimulated and unstimulated cells, and the effect on [3H]arachidonic acid release of treating cells with exogenous phospholipase C. The data indicate that stimulation of PLA2 activity is regulated by alpha 1-adrenergic and bradykinin receptors and that this stimulation is mediated, at least in part, by the activation of protein kinase C. We find that the role of diacylglycerol in arachidonic acid release is as an activator of protein kinase C and not as a substrate for a lipase. Moreover, the hormonal agonists do not appear to inhibit fatty acid reacylation. Experiments using the Ca2(+)-sensitive dye fura-2 and the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid suggest that bradykinin activates PLA2 by a transient elevation of intracellular Ca2+. This action appears to be less important for activation of PLA2 by epinephrine. Taken together, these data are consistent with the following conclusions. 1) Hormone-stimulated arachidonic acid release in Madin-Darby canine kidney-D1 cells occurs as a consequence of PLA2 activation. 2) The ability of an agonist both to mobilize Ca2+ and to activate protein kinase C contributes to its efficacy as a stimulator of PLA2-mediated arachidonic acid release.

Animals↗
PubMed1991

Stoichiometry of receptor-Gs-adenylate cyclase interactions.

Little is known about the relative stoichiometry of guanine nucleotide-binding (G) proteins relative to the effector systems to which they link. We addressed this question for the stimulatory G protein (Gs) linked to adenylate cyclase. Forskolin stimulates the catalytic subunit of adenylate cyclase (C), but it has a higher efficacy and potency when C also interacts with the G protein Gs. Accordingly, we measured high-affinity [3H]forskolin binding to intact cells to assay alpha s-C complexes. No high-affinity specific binding occurred with unstimulated cells. The beta-adrenergic agonist isoproterenol promoted the binding of [3H]forskolin to about 3000 sites per cell, suggesting that each receptor on average activates at least several Gs molecules. Activating Gs directly with cholera toxin maximally promoted [3H]forskolin binding to a similar number of sites, suggesting that this is the maximal number of alpha s-C complexes formed per cell. We conclude that each cell likely contains only a few thousand functional copies of C, and that the availability of C (rather than Gs, which exists in more than 100,000 copies per cell) is likely to be limiting for agonist stimulation of adenylate cyclase activity.

Adenylyl Cyclases↗
PubMed1991

Decreased beta 2-adrenergic receptor mRNA expression in receptor-deficient S49 lymphoma cells.

Many variants of the S49 mouse lymphoma cell have been isolated along the pathway of cyclic AMP generation and response. Two such variants, beta p and beta d, were isolated by Johnson and colleagues and described in 1979 [Mol. Pharmacol. 15:16-27 (1979)]. The beta p and beta d variants express one half and one quarter, respectively, of the wild-type number of beta 2-adrenergic receptors. This observation has now been extended through the use of DNA-excess solution hybridization. Using this exquisitely sensitive technique for quantitation of gene and mRNA, we have been able to demonstrate that the beta 2-adrenergic receptor-deficient variant cells contain the same quantity of the beta 2-adrenergic receptor gene as the wild-type cells. In contrast, the beta 2-adrenergic receptor-deficient variant cells express reduced quantities of beta 2-adrenergic receptor-specific mRNA. The amount of beta 2-adrenergic receptor-specific mRNA correlates very well with the reduction in receptor expression in these cells. Both gene and mRNA in the wild-type and variant cells appear to be the same size, as judged by Southern and Northern analysis. Thus, the diminution of beta 2-adrenergic receptors in the beta p and beta d variants appears to reflect primarily the relative paucity of gene transcripts in the variant cells. These data imply that variations in cellular content of beta 2-adrenergic receptor mRNA, which may occur among closely related cells, is one explanation for differences in receptor number.

Animals↗
PubMed1991

Renal alpha 1-adrenergic receptor subtypes: MDCK-D1 cells, but not rat cortical membranes possess a single population of receptors.

Recent work has demonstrated that alpha 1-adrenergic receptors are composed of at least two subtypes, termed alpha 1a and alpha 1b. It has been proposed that these subtypes may be linked to distinct second messenger systems. In the current studies, we have compared the properties of alpha 1-adrenergic receptors in rat renal cortical membranes with those in MDCK-D1 cells, a clonal cell line derived from distal tubule/collecting duct. Competitive binding studies with [3H]prazosin and compounds [5-methylurapidil, (+)-niguldipine, WB4101, and oxymetazoline] that distinguish high affinity (alpha 1a) and low affinity (alpha 1b) sites indicated that rat renal cortical membranes contain about 50% of each class of site. In contrast, MDCK-D1 cells contained a single population of low affinity sites. 5-Methylurapidil, but not the other compounds, recognized binding sites in these cells with a substantially lower affinity than has been observed for the low affinity site in other tissues and in parallel studies with renal cortical membranes. [3H]Prazosin binding sites in these cells, as well as alpha 1-adrenergic receptor-mediated arachidonic acid release and phosphoinositide and phosphatidylcholine hydrolysis, were sensitive to inactivation by chloroethylclonidine (IC50 approximately 0.7 microM), as expected for alpha 1b receptors. However, alpha 1-adrenergic receptors of MDCK-D1 cells required extracellular calcium for biological response, unlike what has been hypothesized for the alpha 1b receptor subtype. These data indicate that the population of alpha 1-adrenergic receptors of distal tubule/collecting duct cells likely consists of receptors of the alpha 1b subtype. The low affinity binding of 5-methylurapidil and the requirement for extracellular calcium for biological response in these cells suggest that this receptor may not be identical to the alpha 1b receptor that has been observed in other systems.

Animals↗
PubMed1990

Beta-adrenergic receptors and the Gs protein in myocardial ischemia and injury.

The purpose of this study was to explore alterations in the life cycle of adrenergic receptors and the Gs protein in the heart of ischemic animals. In initial experiments left anterior descending coronary artery occlusion was performed in guinea pigs. Sarcolemmal (SL) and light vesicle (LV) (presumably intracellular) fractions were prepared. Both fractions contained a substantial number of beta-adrenergic receptors and alpha 1-adrenergic receptors: the relative proportion of beta-adrenergic receptors in LV/SL was greater than for alpha 1-adrenergic receptors. Myocardial ischemia produced a rapid externalization of beta-adrenergic receptors from LV to SL. alpha 1-adrenergic receptors also increased in SL but without an apparent externalization from LV. Pretreatment of animals with either the non-selective beta-antagonist propranolol or the beta 1-selective antagonist atenolol increased the number of SL beta-receptors and blunted the ischemia-induced increase in SL beta-adrenergic receptors. Treatment with the partial agonist pindolol did not cause up-regulation of beta-receptors, and did not block ischemia-induced externalization. In the second part of this study, we have begun to examine post-receptor events in a rat model of myocardial ischemia. Ligation of the distal left main coronary artery in the rat led to an increase in SL beta-receptors. As G proteins play a pivotal role in transducing receptor occupancy to activation of effector molecules, we measured levels of Gs which stimulates adenylate cyclase activity, using an ELISA technique. In rat SL the amount of alpha s markedly decreased within 15 min of coronary occlusion. There was no transfer of Gs activity to the light vesicle fraction. These studies indicate the dynamic nature of adrenergic receptors and the alpha s protein in the sarcolemma in myocardial ischemia. Changes in adrenergic receptor number and in G protein expression may contribute to the altered pathophysiology of the ischemic heart.

Animals↗
PubMed1990

Beta-adrenoceptor metabolism in wild-type, Gs, and protein kinase A-variant S49 cells.

To determine the role of the stimulatory guanine nucleotide-binding protein, Gs, and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase in the basal metabolism of beta-adrenergic receptors in S49 lymphoma cells, we measured the return of receptor number and function after irreversible blockade of receptors. After inactivation of receptors with the irreversible ligand N8-(bromoacetyl)-N'-[3-(4-indolyoxy)-2-hydroxypropyl]-(2)-1,8-diam ino-p- methane (BIM), beta-adrenergic receptors (defined as [125I]iodocyanopindolol binding sites) reappeared in a biphasic manner, the faster phase having a half-time (t 1/2) of 3-8 h (approximately 50% of the sites) and the slower phase greater than 40 h. Although the slow phase is not readily explained, recovery of binding sites during the first 10 h matched recovery of receptor function after BIM treatment (as measured by stimulation of cAMP accumulation) and recovery of receptor sites after downregulation induced by the agonist isoproterenol. Thus quantifying receptor recovery during the first 10 h after BIM treatment appears to be a reasonable method for examining basal receptor metabolism in S49 cells. Measured in this way, metabolism of beta-adrenergic receptors is very similar in wild-type S49 and the following variant clones: cyc- (absent Gs alpha), UNC and H21a (defective Gs alpha), and kin- (lacking cAMP-dependent protein kinase activity). Although previous data have demonstrated that agonist-promoted downregulation of beta-adrenergic receptors requires functional receptor-Gs coupling, the current data suggest that neither Gs nor cAMP-dependent protein kinase activity plays an important role in the regulation of basal metabolism of beta-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1990

Amplification of cyclic AMP generation reveals agonistic effects of certain beta-adrenergic antagonists.

Some beta-adrenergic receptor (beta AR) antagonists, in addition to blocking receptor-mediated responses, possess agonistic properties or intrinsic sympathomimetic activity (ISA). In this study we describe several techniques for amplification of cAMP levels as a measure of agonistic activity, and we apply these techniques to the study of beta AR antagonists with ISA. We show that 1) a variety of beta AR antagonists with ISA, including alprenolol and cyanopindolol, enhance cyclic AMP accumulation in S49 lymphoma cells if cells are also incubated with the diterpene forskolin; 2) beta AR blockers with ISA stimulate cAMP accumulation in the presence of a water-soluble analog of forskolin but not in the presence of 9,11-dideoxyforskolin (which does not activate adenylyl cyclase); 3) the potentiation by forskolin is not unique to S49 cells but is also observed in BC3H1 smooth muscle-derived cells; 4) stimulation of cAMP accumulation by beta-blockers with ISA occurs in S49 cells in three additional settings that do not involve the use of forskolin, after pretreatment with pertussis toxin to inactivate the inhibitory guanine nucleotide binding protein, after pretreatment with [D-Trp8]-somatostatin to sensitize adenylyl cyclase, and using a radioimmunoassay to quantitate levels of cellular cAMP. We conclude that beta AR antagonists with ISA can weakly stimulate intracellular cAMP accumulation, but this stimulation is not easily detected. Elevation of cAMP levels may account for the agonistic effects of these drugs or, at least provides a measure of stimulatory guanine nucleotide-binding protein activation by these compounds.

Adenylate Cyclase Toxin↗