Showing papers by "Robert J. Lefkowitz published in 1981"

Pharmacological Characterizations of Adrenergic Receptors in Human Adipocytes

Abstract: Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [3H]dihydroalprenolol binding (beta adrenergic) Bmax 280 fmol/mg protein, KD 0.38 nM; [3H]para-aminoclonidine binding (alpha-2 adrenergic) Bmax 166 fmol/mg protein, KD 0.49 nM; [3H]WB 4101 binding (alpha-1 adrenergic) Bmax 303 fmol/mg protein, KD 0.86 nM. In adipocytes from subcutaneous adipose tissue, [3H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors. Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 μM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 μM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [32P]phosphate and epinephrine (10 μM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in 32P incorporation into phosphatidylinositol. Prazosin (0.1 μM) blocked this action whereas yohimbine (0.1 μM) was without effect. Thus, in a homogeneous cell preparation, the human adipocyte appears to have three different adrenergic receptors, each of which is coupled to a distinct biochemical response.

Agonist-Promoted High Affinity State of the β-Adrenergic Receptor in Human Neutrophils: Modulation by Corticosteroids

Abstract: β-Adrenergic agonists form high affinity complexes with receptors, resulting in activation of the associated adenylate cyclase. To examine the formation of the high affinity state of the receptor, curves were constructed for the competition of the full β-adrenergic agonist isoproterenol, partial agonists cobefrin and soterenol, and the antagonist propranolol for [3H]dihydroalprenolol binding to β-adrenergic receptors on human neutrophil membranes. Curve modeling by computer yielded a two-state binding model for the agonists, with distinct dissociation constants for the high (KH) and low (KL) affinity states. The ratio of dissociation constants (KL/KH) was found to be well correlated (P < 0.01) with the drug’s intrinsic activity for stimulation of adenylate cyclase. Thus, the degree of coupling of receptor occupation with adenylate cyclase activation is correlated with the magnitude of KL/KH. Administration of cortisone to humans resulted in a substantial rise in the proportion of receptors in the high aff...

Anomalous equilibrium binding properties of high-affinity racemic radioligands.

Abstract: In receptor binding studies, high-affinity racemic radioligands are often used as tracers, neglecting the difference in affinity of their stereoisomers. We present an experimental and theoretical study comparing the binding of (±)-[3H]carazolol and (±)-[125I]hydroxy-benzylpindolol (HYP) to their pure respective isomers in the frog erythrocyte beta-adrenergic system. Saturation binding curves with the racemic radioligands showed deviations from a binding isotherm for a single ligand which were accentuated at higher receptor concentrations. When different affinity constants for both isomers were considered, significant improvement in the fits of the data were obtained by computer-modeling procedures. The KDav (average dissociation constant) obtained by considering the racemic radioligand as a single ligand, as has generally been done in the literature, varied with the receptor concentration from approximately 2 KD(-) at low receptor concentrations to ≅0.5 KD(+) at high receptor concentrations. Thus the generally measured "KDav" of these racemic radioligands is really a hybrid of KD(-) and KD(+). These experimental findings are in very good agreement with Monte Carlo simulations and may help to explain the discrepancies in dissociation constants of high-affinity racemic radioligands reported in the literature. Experimental data and simulations also indicate that information about the KD(-) is greatest at low receptor concentrations, whereas that about KD(+) is greatest at high receptor concentrations. Simultaneous computer fitting of saturation curves from racemic [125I]HYP and the (+)-isomer, isolated by repeated incubations with frog erythrocyte membranes under appropriate conditions, indicates approximately a 20-fold ratio for the individual isomer KD values. Estimated KD values of the stereoisomers of [125I]HYP and [3H]carazolol were virtually identical, being KD(-) = 10-50 pM and KD(+) ≅ 400-2000 pM at 25°. Use of the KDav for a racemic radioligand resulted in up to 5-fold systematic underestimation of the affinity of nonracemic competitors. The KD values of all high-affinity competitors were also found to be misestimated by as much as 10-fold using the commonly employed Cheng and Prusoff [Biochem. Pharmacol.22: 3099-3108 (1973)] approximation when the affinity of the radioligand was significantly lower than that of the competitor. Under such circumstances, slope factors of ≅2 were obtained for competition curves in the absence of cooperativity.

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

Abstract: 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.

Regulation of Adrenergic Receptors

Abstract: Transmission of intercellular messages by the adrenergic mediators, epinephrine and norepinephrine, is a ubiquitous phenomenon in vertebrates which continues to invoke investigative effort after many years of research. It has become apparent that both the release and biological actions of these mediators are modulated very specifically by pharmacological and pathological states. An understanding of the mechanisms of modulation of adrenergic actions is fundamental to addressing the relationships between adrenergic responses and other hormonal or cellular responses.

Myocardial beta-adrenergic receptors from adrenalectomized rats: impaired formation of high-affinity agonist-receptor complexes.

Abstract: Adrenal steroid hormones potentiate beta-adrenergic actions on the heart. Accordingly, we investigated the effects of adrenalectomy on agonist and antagonist interactions with myocardial beta-adrenergic receptors and adenylate cyclase. The affinity and number of beta-adrenergic receptor sites, both defined by the antagonist (-) [3H]dihydroalprenolol, did not change after adrenalectomy. Computer modelling of agonist (-)-isoproterenol competition curves indicated the presence of two discrete receptor states with high and low affinities. After adrenalectomy, the agonist curves were shifted to the right, and the dissociation constant of the high-affinity state significantly rose from 12 to 48 nM (p < .001), but the dissociation constant of the low affinity state was unchanged. Although basal, maximal isoproterenol-stimulated and fluoride-stimulated adenylate cyclase activities were unaltered, the EC50 for isoproterenol stimulation was increased significantly from 490 to 1500 nM (p <.018). These results suggest that adrenal steroid hormones may regulate the ability of the beta-adrenergic receptors to form a high-affinity "coupled" state, presumably by modulating the interaction of the receptor with nucleotide regulatory proteins.

Subtype specificity of alpha-adrenergic receptors in rat heart.

Abstract: Recent studies suggest that two subtypes of alpha-adrenergic receptors (alpha 1 and alpha 2) can be distinguished on the basis of the differential affinities of certain adrenergic ligands for each subtype. We have investigated the binding characteristics of several such ligands for alpha-adrenergic receptors in membrane preparations derived from rat heart. alpha-Adrenergic antagonists competed for binding sites identified by the nonsubtype selective alpha-adrenergic ligand [3H]dihydroergocryptine (DHE) in the order of potency expected for alpha 1-receptors, namely, prazosin (EC50 = 0.35 nM) greater than phentolamine (EC50 = 37 nM) greater than yohimbine (EC50 = 918 nM). Furthermore, the nonsubtype selective radioligand [3H]DHE identified a quantitatively similar number of specific binding sites in rat cardiac membranes as the alpha 1-selective radioligand [3H]prazosin (33 and 36 fmoles/mg protein, respectively), while the alpha 2-selective ligand [3H]clonidine at concentrations up to 20 nM demonstrated negligible specific binding. We conclude that the alpha-adrenergic receptors of rat heart homogenates demonstrate binding characteristics typical of alpha 1-receptors. While we cannot exclude the presence of small numbers of alpha-2 receptors, the similar number of binding sites identified at saturation for the alpha 1-specific ligand [3H]prazosin and for the nonsubtype selective ligand [3H]DHE supports the hypothesis that the alpha-receptors of rat heart are predominantly of the alpha 1-subtype.

Alternative Explanation for the Apparent "Two-Step" Binding Kinetics of High-Affinity Racemic Antagonist Radioligands

Abstract: Recent studies of agonist and antagonist binding to the beta -adrenergic receptor and to other receptors have established the notion of agonist specific binding properties unshared by antagonists and reflecting the activation of the effector. However, previous reports on the dissociation kinetics of the widely used high-affinity beta -adrenergic antagonist (±)-[ 125 I]hydroxybenzylpindolol (HYP) have indicated complex binding kinetics which led to the proposal of a receptor isomerization model involving antagonist promoted transitions. We report here that the binding properties of two high-affinity beta -adrenergic antagonists can be fully explained by their racemic nature. (a) Binding data for the association and the dissociation kinetics of (±)-[ 125 I]HYP to frog erythrocyte membranes can be adequately fitted by computer modeling assuming different rate constants for each enantiomer. (b) In contrast, purified (+)-[ 125 I]HYP shows only uniphasic fast-dissociation kinetics. (c) Similarly, the antagonists (-)-[ 3 H]carazolol and (±)-[ 3 H]carazolol show uniphasic slow-dissociation kinetics and biphasic dissociation kinetics, respectively. These results demonstrate that the complex binding kinetics of these high-affinity racemic radioligands are not due to receptor state transitions but rather to simultaneous binding of both isomers.

α-Adrenergic Receptors in Human Adipocyte Membranes: Direct Determination by [3H]Yohimbine Binding

Abstract: The presence of α2-adrenergic receptors in membranes derived from human sc adipose tissue was directly demonstrated with a new α2-selective ligand, [3H]yohimbine. Binding of this radiolabeled antagonist to adipocyte membranes was of high affinity (Kd =3.9 ± 2.4 nin) and saturable. Computer modelling of [3H]yohimbine saturation curves demonstrated that it binds to a homogeneous class of sites with a density of 145.0 ± 33.8 fmol/mg protein. Adrenergic agonists competed with [3H]yohimbine in the order expected of α-receptors, and their binding was strongly influenced by guanine nucleotides. Competition of α-antagonists with this radioligand demonstrated yohimbine to be more potent than prazosin, indicative of α2-receptors. Antagonist binding was unaffected by guanine nucleotides. Paired saturation curves in these adipocyte membranes with the α2-selective [3H]yohimbine and the nonsubtype selective α-antagonist [3H]dihydroergocryptine demonstrated similar receptorconcentrations. [3H]Dihydroergocryptine has bee...

Determination of subtype selectivity of alpha-adrenergic antagonists: comparison of selective and nonselective radioligands.

Abstract: The alpha 1/ alpha 2-adrenergic receptor subtype selectivity of twelve adrenergic antagonists was assessed by two different radioligand binding approaches in rabbit uterine membranes which contain both receptor subtypes. In the first approach, a nonsubtype selective antagonist radioligand, [3H]dihydroergocryptine ([3H]DHE), was used to label all of the alpha -receptors. Selective competing ligands produced complex competition curves which could be analyzed by nonlinear least-squares curve-fitting methods to yield the Kd values of the competitor for the alpha 1- and alpha 2-receptors, respectively. The second approach utilized the radioligand antagonists [3H]prazosin and [3H]yohimbine. [3H]Prazosin was found to label alpha 1-receptors, whereas [3H]yohimbine labeled alpha 2-receptors. Competition experiments performed with subtype selective antagonists with these radioligands produced steep uniphasic competition curves in all cases. Dissociation constants of drugs for the [3H]prazosin and [3H]yohimbine sites correlated highly with the alpha 1 and alpha 2 components of the complex [3H]DHE competition curves, respectively. Good quantitative agreement between the two sets of data was obtained, indicating the validity of both approaches for the determination of receptor subtype affinities. Nonetheless, use of subtype selective radioligands offered several advantages. When the nonsubtype selective radioligand [3H]DHE was used in this system, 100-fold selectivity of a competitor was required in order to determine reliably alpha 1 and alpha 2 affinities. In contrast, use of the selective radioligands discriminated much smaller degrees of selectivity without the necessity for sophisticated computer analysis of the data. ACKNOWLEDGMENTS We would like to thank Drs. Frank E. Harrel and Andre De Lean for their helpful suggestions and statistical advice.

Catecholamine-induced desensitization in turkey erythrocytes: cAMP mediated impairment of high affinity agonist binding without alteration in receptor number.

Abstract: Desensitization of turkey erythrocyte adenylate cyclase by exposure of these cells to the beta-adrenergic agonist isoproterenol leads to a decrease in subsequent adenylate cyclase stimulation by isoproterenol, F-, or Gpp(NH)p without any apparent loss or down regulation of receptors (B.B. Hoffman et al. J. Cyclic Nucl. Res. 5: 363-366, 1979). We now report that the desensitization is associated with a functional "uncoupling" of the beta-adrenergic receptor. This is evidenced by an impaired ability of receptors to form a high affinity, guanine nucleotide sensitive complex with agonist as assessed by computer analysis of radioligand binding data. The changes in adenylate cyclase responsiveness as well as the alterations in receptor affinity for agonists are reproduced by incubation of turkey erythrocytes with the cAMP analog 8-Bromo-adenosine 3':5'- cyclic monophosphate. These findings suggest that one possible mechanism for the development of desensitization in adenylate cyclase systems may be a cAMP mediated alteration of a component(s) of the beta-adrenergic receptor-adenylate cyclase complex which results in impaired receptor-cyclase coupling.

Differential effects of cholera toxin on guanine nucleotide regulation of beta-adrenergic agonist high affinity binding and adenylate cyclase activation in frog erythrocyte membranes.

Abstract: The guanine nucleotide regulatory protein(s) regulates both adenylate cyclase activity and the affinity of adenylate cyclase-coupled receptors for hormones or agonist drugs. Cholera toxin catalyzes the covalent modification of the nucleotide regulatory protein of adenylate cyclase systems. Incubation of frog erythrocyte membranes with cholera toxin and NAD+ did not substantially alter the dose dependency for guanine nucleotide activation of adenylate cyclase activity. In contrast, toxin treated membranes demonstrated a 10 fold increase in the concentrations of guanine nucleotide required for a half maximal effect in regulating beta-adrenergic receptor affinity for the agonist (+/-) [3H]hydroxybenzylisoproterenol. The data emphasize the bifunctional nature of the guanine nucleotide regulatory protein and suggest that distinct structural domains of the guanine nucleotide regulatory protein may mediate the distinct regulatory effects on adenylate cyclase and receptor affinity for agonists.

Polymeric drugs by direct copolymerization: Polymer of β-adrenergic antagonist alprenolol and its binding to receptors and antibodies

Abstract: Alprenolol (1-(o-allylphenoxy)-3-isopropylamino-2-propanol), a drug which is used in the management of cardiovascular diseases, was copolymerized with acrylamide. The incorporation of alprenolol into the copolymer decreased its potency for binding to beta-adrenergic receptors located on membranes of frog erythrocytes a thousand times. However, this incorporation decreased the potency of alprenolol for binding to antibodies specific for catecholamines and alprenolol-related drugs only about three times. The incorporation of the drug into the polymer apparently results in steric strains preventing the approach of the drug residues to sterically constrained receptor binding sites, but allowing its approach to the accessible antibody binding sites. Copolymerization with acrylamide was used to prepare the polymer bound form of another drug which contains an allyl group, an alpha-adrenergic antagonist, azapetine (6-allyl-6,7-dihydro-5H-dibenz[c,e]azepine).