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Biomedical subjects

R J Lefkowitz

Publications and source records attributed to R J Lefkowitz.

At least 523 records · Page 29Linked to original sources
PubMed1978

Agonist-induced increase in apparent beta-adrenergic receptor size.

The properties of digitonin-solubilized beta-adrenergic receptors from frog erythrocyte membranes were studied by gel exclusion chromatography on AcA 34 Ultragel. beta-Adrenergic receptor binding activity in these membranes can be identified by both an agonist ligand, [(3)H]hydroxybenzylisoproterenol, and the antagonist ligands, [(3)H]dihydroalprenolol and (125)I-labeled hydroxybenzylpindolol. Occupancy of the beta-adrenergic receptors with the [(3)H]hydroxybenzylisoproterenol agonist prior to their solubilization from the membrane leads to an increase in apparent receptor size. Alterations in the molecular size of the receptor cannot be mimicked by occupancy of the binding site with the antagonist ligands. Exposure of frog erythrocyte membranes to [(3)H]hydroxybenzylisoproterenol agonist in the presence of 10 muM Gpp(NH)(p), a guanyl nucleotide analog that exerts multiple regulatory effects on the catecholamine-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] system, results in the elution of the [(3)H]hydroxybenzylisoproterenol radioligand in both the region characteristic of the agonist-receptor complex and the region characteristic of the antagonist-receptor complex. The precise molecular interactions responsible for the agonist-induced increase in apparent beta-adrenergic receptor size are still unresolved. However, the low concentrations of agonist that are capable of altering apparent receptor size and the sensitivity of this effect to guanyl nucleotides suggest that these phenomena may be intimately involved in eliciting the physiological effects of beta-adrenergic catecholamines at the molecular level.

Adenylyl Cyclases↗
PubMed1978

Alpha-adrenergic receptors in rat myocardium. Identification by binding of [3H]dihydroergocryptine.

[3H]Dihydroergocryptine ([3H]DHE) binds to sites in membranes derived from rat myocardium that have the characteristics expected of alpha-adrenergic receptors. The binding is saturable with 41 fmol [3H]DHE bound per mg of protein and of high affinity with KD = 2.9 nM. The binding is rapid and readily reversible. Adrenergic agonists compete with [3H]DHE for binding in the order: epinephrine greater than norepinephrine greater than isoproterenol; and adrenergic antagonists compete for binding in the order: phentolamine greater than propranolol. For comparison, (-)[3H]dihydroalprenolol [(-)[3h]dha] was used to bind to sites in the same membrane preparations having characteristics of beta-receptors. The number and affinity of beta-receptors were quite similar to those of the alpha-receptors with 46 fmol (-)[EH]DHA per mg protein bound at saturation and KD = 2.5 nM. These techniques allowed identification of both beta- and alpha-adrenergic receptors in membranes derived from isolated atria, right ventricular free walls, and left ventricles including interventricular septa. This is the first report documenting direct identification of myocardial alpha-receptors by radioligand-binding techniques and complements the literature previously reporting myocardial inotopic and electrophysiological responses to alpha-adrenergic stimulation.

Alprenolol↗
PubMed1978

Identification of alpha-adrenergic receptors in human platelets by [3H]dihydroergocryptine binding.

Binding of [(3)H]dihydroergocryptine to platelet lysates appears to have all the characteristics of binding to alpha-adrenergic receptors. At 25 degrees C binding reaches equilibrium within 20 min and is reversible upon addition of excess phentolamine. Binding is saturable with 183+/-22 fmol of [(3)H]dihydroergocryptine bound per mg of protein at saturation, corresponding to 220+/-26 sites per platelet. Kinetic and equilibrium studies indicate the dissociation constant of [(3)H]dihydroergocryptine for the receptors is 1-3 nM. The specificity of the binding sites is typical of an alpha-adrenergic receptor. Catecholamine agonists compete for occupancy of the [(3)H]dihydroergocryptine binding sites with an order of potency (-)epinephrine> (-)norepinephrine>> (-)isoproterenol. Stereospecificity was demonstrated inasmuch as the (+)isomers of epinephrine and norepinephrine were 10-20-fold less potent than the (-)isomers. The potent alpha-adrenergic antagonists phentolamine, phenoxybenzamine, and yohimbine competed potently for the sites, whereas beta-antagonists such as propranolol and dichlorisoproterenol were quite weak. Dopamine and serotonin competed only at high concentrations (0.1 mM). The [(3)H]dihydroergocryptine binding sites could also be demonstrated in intact platelets where they displayed comparable specificity, stereospecificity, and saturability. Saturation binding studies with the intact platelets indicated 220+/-45 receptors per platelet, in good agreement with the value derived from studies with platelet lysates. Ability of alpha-adrenergic agonists to inhibit adenylate cyclase and of alpha-adrenergic antagonists to antagonize this inhibitory effect directly paralleled ability to interact with the [(3)H]dihydroergocryptine binding sites. These data demonstrate the feasibility of directly studying alpha-adrenergic receptor binding sites in human platelets with [(3)H]dihydroergocryptine.

Adenylyl Cyclases↗
PubMed1978

Identification and regulation of alpha- and beta-adrenergic receptors.

Direct radioligand binding methods for studying the alpha- and beta-adrenergic receptors have been developed over the past several years. These techniques use radioactively labeled adrenergic antagonists and agonists to identify the receptors in appropriate membrane fractions from catecholamine-sensitive tissues. In the case of the beta-adrenergic receptors, confident receptor identification has been aided by the close correlation of binding data with data on adenylate cyclase activation. Such direct binding studies are providing new insights about the molecular characteristics and regulatory properties of the receptors.

Adenylyl Cyclases↗
PubMed1978

Multiple effects of N, N' dicyclohexyl carbodiimide on the beta-adrenergic receptor--adenylate cyclase system in frog erythrocytes.

Treatment of frog erythrocytes with N,N' dicyclohexylcarbodiimide (DCCD) leads to a loss of catecholamine stimulated adenylate cyclase activity without any decrease in fluoride or PGE1 stimulated cyclase. However, the concentrations of the reagent which inhibit catecholamine sensitive adenylate cyclase activity are 10 fold lower than those which inhibit specific [3H]dihydroalprenolol ([3H]DHA) beta-adrenergic receptor binding. By contrast binding of the readiolabeled beta-adrenergic agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) is considerably more sensitive than antagonist binding to the effects of DCCD. The data suggest that low concentrations of the reagent may modify the effector portion of the beta-adrenergic receptor leading to functional uncoupling of the beta-receptor adenylate cyclase system. At higher concentrations of the reagent the ligand bidning site of the beta-receptor appears also to be altered.

Adenylyl Cyclases↗
PubMed1977

Thyroid hormone regulation of beta-adrenergic receptor number.

The effects of exogenous thyroid hormones (thyroxine and triiodothyronine) on beta-adrenergic receptors in the rat myocardium were investigated. The potent beta-adrenergic antagonist, (-)-[3H]dihydroalprenolol, was used to directly estimate the number and affinity of beta-adrenergic receptors in rat heart membranes from control and hyperthyroid rats. Cardiac membranes from hyperthyroid rats contained 196 +/- 7 fmol of (-)-[3H]dihydroalprenolol binding sites/mg of protein which was significantly (p less than 0.005) greater than the number of binding sites (89 +/- 5 fmol/mg of protein) present in control membranes. The equilibrium dissociation constant (KD) for the interaction of receptors with dihydroalprenolol was the same (2 to 15 nM) in membranes from control and hyperthyroid rats. Similarly, there was no significant difference between the control and hyperthyroid membranes in the affinity of the beta-adrenergic receptor binding sites for the beta-adrenergic agonist isoproterenol. The results of this study demonstrate that thyroid hormones can regulate the number of cardiac beta-adrenergic receptors. The increased numbers of receptors may be responsible, at least in part, for the enhanced catecholamine sensitivity of beta-adrenergic-coupled cardiac responses in the hyperthyroid state.

Adrenergic beta-Antagonists↗