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Biomedical subjects

G L Stiles

Publications and source records attributed to G L Stiles.

At least 109 records · Page 6Linked to original sources
PubMed1984

Effect of pertussis toxin on alpha 2-adrenoceptors: decreased formation of the high-affinity state for agonists.

Administration of pertussis toxin to hamsters abolishes the alpha 2-adrenergic inhibition of adenylate cyclase in their adipocytes. The toxin did not modify the number of adipocyte alpha 2-adrenoceptors or their affinity for antagonists. In contrast, the affinity for agonists was significantly diminished in adipocyte membranes obtained from pertussis toxin-treated hamsters as compared to the controls. This decrease in affinity was due to a significant diminution in the proportion of sites displaying the high-affinity state for agonists. It is concluded that pertussis toxin induces a shift in the proportion of sites in high- and low-affinity states. for agonists towards the low-affinity conformation.

Adenylate Cyclase Toxin↗
PubMed1984

Cardiac adrenergic receptors.

Recently developed pharmacological and biochemical techniques have brought new insights about the structure, function, and regulation of beta-adrenergic receptors. This chapter focuses on the cardiac beta-adrenergic receptor and the possible clinical and physiological implications of this new information.

Adenylyl Cyclases↗
PubMed1984

Translocation and uncoupling of the beta-adrenergic receptor in rat lung after catecholamine promoted desensitization in vivo.

Beta-adrenergic agonists and antagonists are widely used in many clinical situations to regulate beta-adrenergic stimulation. However, responsiveness to beta-stimulation may be reduced by the process of desensitization. We have established an in vivo mammalian model system, the rat lung, to study the cellular and biochemical basis for beta-agonist induced desensitization. After in vivo administration of a beta-agonist [(-)isoproterenol] the adenylate cyclase becomes rapidly insensitive to further stimulation by beta-agonists with no change in basal or NaF-stimulated activity. The in vivo desensitization can be blocked by the simultaneous administration of a beta-antagonist [(+/-)propranolol] and the process displays the pharmacological characteristics typifying the beta 2 receptor of rat lung. This indicates that the in vivo desensitization is itself a receptor-mediated event. The processes of de- and resensitization are very rapid with onset within 5 min, maximal effect at 10 min, and complete reversal by 2-3 h. The change of adenylate cyclase sensitivity is paralleled by a translocation of approximately 40% of the beta-receptors from the plasma membrane fraction to a light membrane fraction, which has very low activities of plasma membrane marker enzymes. The receptors translocated to the light membrane fraction as well as those remaining in the plasma membranes are uncoupled with loss of their ability to form the high affinity, nucleotide sensitive, physiologically active state of the receptor. During resensitization the receptors in the plasma membrane fraction are recoupled before all the translocated receptors have returned. This suggests that translocation and uncoupling of the receptors are two distinct, probably independent processes. During the entire process of de- and resensitization no structural change of the receptor protein residing in the plasma membranes or light membrane fraction can be demonstrated as visualized by photoaffinity labeling.

5'-Nucleotidase↗
PubMed1984

Demonstration of beta adrenoceptor blockade by propafenone hydrochloride: clinical pharmacologic, radioligand binding and adenylate cyclase activation studies.

The postulated beta adrenoceptor blocking properties of the new antiarrhythmic drug propafenone were studied by in vivo comparison against placebo and propranolol in the antagonism of both exercise- and isoproterenol-induced tachycardia and by in vitro radioligand binding studies of animal and human left ventricular muscle membrane preparations. Interaction with frog erythrocyte membrane adenylate cyclase was also investigated. In the clinical studies, a double blind crossover comparison of oral propafenone (300 mg), propranolol (40 mg) and placebo indicated significant antagonism of chronotropic response to isoproterenol 2 hr postdose with dose ratios of 4.1 +/- 1.3 (mean +/- S.E.M.) for propafenone and 16.8 +/- 5.1 for propranolol. Chronotropic response to exercise was modestly reduced by propafenone. Analysis of the binding of [125I]iodocyanopindolol to human left ventricular membranes revealed specific beta adrenoceptor competition by propafenone with an EC50 of 111 +/- 13 nM. Propranolol EC50 was 2.4 +/- 0.2 nM in this system. Competitive inhibition of isoproterenol-stimulated frog erythrocyte membrane adenylate cyclase activity was also obtained with propafenone. The ratio of affinities (calculated from the apparent dissociation constant; KD) for propranolol-propafenone was 1:40 for the in vivo study and 1:50 for the in vitro system. Propafenone is a specific antagonist of the human beta adrenoceptor and this action can be demonstrated during in vivo study in human subjects. At clinical dosages it appears likely that it will achieve a modest degree of beta blockade which may contribute to its antiarrhythmic effect.

Adenylyl Cyclases↗
PubMed1983

The effect of reducing agents on the structure of mammalian beta-adrenergic receptors.

Under reducing conditions (5% beta-mercaptoethanol) the mammalian beta-adrenergic receptor binding site from both beta 1 (porcine heart membranes) and beta 2 receptors (hamster lung and rat erythrocyte membranes) appears to reside on peptides of Mr 62,000-65,000 as determined by photoaffinity labeling with p-azido-m-[125I]iodobenzylcarazolol and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When similar experiments are performed in these same systems under a variety of non-reducing conditions, there are minimal changes in the apparent molecular weight of both the beta 1- and beta 2-adrenergic receptor binding subunits and no specifically labeled higher molecular weight proteins are observed suggesting that there are no disulfide linked subunits in mammalian beta-adrenergic receptors.

Animals↗
PubMed1983

Mammalian beta-adrenergic receptors. Structural differences in beta 1 and beta 2 subtypes revealed by peptide maps.

Photoaffinity labeling techniques using p-azido-m-[125I]iodobenzylcarazolol have recently demonstrated that both the beta 1- and beta 2-adrenergic receptor-binding subunits from mammalian tissues including heart, lung, and erythrocytes reside on peptides of Mr approximately equal to 62,000-64,000. In this study, a two-dimensional gel electrophoresis method for peptide mapping was used to investigate and compare the structure of beta 1 - and beta 2-adrenergic receptor subtypes. When the photoaffinity labeled Mr approximately equal to 62,000 peptides from the beta 2-adrenergic receptors of rat lung and erythrocyte are subjected to simultaneous proteolysis using Staphylococcus aureus V8 proteinase or papain, exactly the same peptide fragments are generated from each subunit. In contrast, when the Mr approximately equal to 62,000 peptide containing the beta 1-adrenergic receptor-binding subunit derived from the rat heart is proteolyzed simultaneously with the Mr approximately equal to 62,000 peptide containing the beta 2-adrenergic receptors from either lung or erythrocyte, the peptide fragments generated are distinctly different. Peptide maps of beta 1-adrenergic receptors from the myocardial tissue of different species (pig versus rat) yield slightly different maps while the maps derived from the beta 2-adrenergic receptors of hamster lung and rat lung or erythrocytes reveal no interspecies differences. These data suggest: 1) alterations in the primary structure of the beta-adrenergic receptor may be responsible for the pharmacological specificities characteristic of beta 1- and beta 2-adrenergic receptor subtypes; and 2) alterations in the primary structure of similar beta-adrenergic receptor subtypes across different species may relate to the magnitude of their phylogenetic differences.

Affinity Labels↗
PubMed1983

Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding.

Human myocardial beta-adrenergic receptors were directly identified and characterized using the high affinity antagonist radioligand [125I]iodocyanopindolol. Beta 1 and beta 2 adrenergic receptors were found to coexist in both the left ventricle and right atrium. The relative proportions of the two receptor subtypes were determined by the use of competition radioligand binding and computer modelling techniques employing the subtype selective agents atenolol (beta 1 selective) and zinterol (beta 2 selective). The left ventricle contains 86 +/- 1% beta 1 and 14 +/- 1% beta 2 adrenergic receptors while the right atrium contains 74 +/- 6% beta 1 and 26 +/- 6% beta 2 adrenergic receptors. The direct demonstration of beta 2 adrenergic receptors in the human heart, with a higher proportion in the right atrium agrees with pharmacologic data and supports the notion that chronotropic effects of adrenergic agonists in man may be mediated by both beta 1 and beta 2 adrenergic receptors.

Adrenergic beta-Agonists↗
PubMed1983

The cardiac beta-adrenergic receptor. Structural similarities of beta 1 and beta 2 receptor subtypes demonstrated by photoaffinity labeling.

The beta-adrenergic receptor photoaffinity ligand p-azido-m-[125I]iodobenzylcarazolol has been used to covalently label the beta 1 and beta 2 adrenergic receptor binding subunits present in left ventricular myocardial membranes derived from mammalian (including human) and nonmammalian species. Covalent incorporation of the photoaffinity ligand into membrane proteins was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the case of the human, canine, porcine, rabbit, and rat left ventricle, all of which contain predominantly or exclusively beta 1-adrenergic receptors, two peptides of Mr approximately equal to 62,000 (major component) and Mr approximately equal to 55,000 (minor component) were specifically labeled and visualized by autoradiography. Photoincorporation into these two bands could be blocked with the appropriate drugs to display a beta 1-adrenergic receptor pharmacological specificity. Simultaneous sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples from each species revealed that all of the Mr = 62,000 peptides co-migrated suggesting similarity in the beta 1-adrenergic receptor binding subunit peptides in all of these species. The minor component Mr approximately equal to 55,000 appears to be a proteolytic degradation product of the Mr = to 62,000 peptide. Its formation could be decreased by proteinase inhibitors. This suggests that the heterogeneity of the labeling pattern observed in mammalian tissues in this and previous studies may be the result of proteolytic degradation of the receptor subunit which occurs during membrane preparation. Photoaffinity labeling of frog ventricular membranes which contain predominantly beta 2-adrenergic receptors also revealed two peptides of Mr approximately equal to 62,000 (major component) and 55,000 (minor component) with the pharmacological selectivity of a beta 2-adrenergic receptor. These data suggest marked similarities in the beta 1- and beta 2-adrenergic receptor binding subunits of different species and suggest that the pharmacological subtype might be determined by the detailed structure, i.e. amino acid sequence, at the ligand binding sites of the receptor peptide.

Adult↗
PubMed1983

Photoaffinity labelling of mammalian beta-adrenergic receptors: metal-dependent proteolysis explains apparent heterogeneity.

The beta-adrenergic receptors in membranes from rat and hamster lungs have been studies using the photoaffinity label p-azido-m-[125I]-iodobenzyl-carazolol. Previous work with several beta adrenergic photoaffinity probes has suggested heterogeneity of the labelled beta adrenergic receptor peptides with 2-3 receptor peptides generally being identified. We now report that rat and hamster lung membranes prepared either in the presence or absence of protease inhibitors reveal striking differences in the ratios of photoaffinity labelled peptides. In the rat lung the inclusion of protease inhibitors in the membrane preparation changes the ratios of the 64,000, 53,000 and 44,000 molecular weight peptides from 28:42:30 to 72:16:12. Similarly, in hamster lung membranes there is evidence of multiple photoaffinity labelled peptides in preparations without protease inhibitors while only one peptide (app. Mr = 64,000) is labelled in preparations with protease inhibitors. Of the inhibitors tested EDTA and EGTA were the most active in preventing appearance of multiple labelled peptides suggesting that metal-dependent proteolysis may be involved in the generation of apparent receptor peptide heterogeneity.

Affinity Labels↗
PubMed1981

Hypothyroidism modulates beta adrenergic receptor adenylate cyclase interactions in rat reticulocytes.

We have investigated alterations in beta adrenergic receptor binding sites of rat reticulocytes occurring in animals rendered hypothyroid by thyroidectomy. Beta adrenergic receptor interactions were assessed by measuring the number of (-)[3H]-dihydroalprenolol binding sites and the ability of an agonist to compete for occupancy of the receptors. The number of receptors was significantly reduced in cells from the hypothyroid animals. In addition, there were significant agonist-specific alterations in binding. Using computer assisted curve fitting techniques, it was found that the ability of (-)isoproterenol to stabilize a high affinity guanine nucleotide sensitive "coupled" form of the receptor was impaired. Reticulocytes from hypothyroid animals have, in addition, a reduction in the concentration of the nucleotide regulatory protein as assessed by the number of 42,000 Mr substrates for cholera toxin catalyzed ADP ribosylation. These alterations are associated with reductions in catecholamine and NaF stimulated adenylate cyclase activity. Diminished coupling of beta adrenergic receptors with other regulatory components of the adenylate cyclase system represents a mechanism by which altered thyroid states modulate beta adrenergic receptor function and beta adrenergic responsiveness of tissues.

Adenylyl Cyclases↗
PubMed1980

Clinical relevance of exercise-induced S-T segment elevation.

Patients with exercise-induced S-T elevation or S-T depression were evaluated with demographic, treadmill and angiographic data. When 541 patients with S-T depression were compared with 109 patients with S-T elevation, a greater proportion of the former had chest pain (71 versus 58 percent) and a normal-sized ventricle (86 versus 61 percent) with normal wall motion (54 versus 30 percent). A greater proportion of patients with S-T elevation had had a previous myocardial infarction (61 versus 33 percent). Among patients without prior infarction (360 with S-T depression and 42 with S-T elevation), these differences disappeared. In this group of 42 patients with S-T elevation, 83 percent had a normal-sized ventricle, 64 percent had normal contractility and none had a ventricular aneurysm; the severity of coronary disease and ventricular dysfunction did not differ from the severity in patients with S-T depression. Thus, in patients without prior myocardial infarction, the cause of the development of S-T elevation or S-T depression during exercise does not appear to be related to the severity of the coronary lesions, ventricular function or wall abnormalities at rest. In patients with prior myocardial infarction, exercise-induced S-T elevation appears to be a marker of depressed left ventricular function.

Adult↗