Showing papers by "Robert J. Lefkowitz published in 1976"

Identification of beta-adrenergic receptors in human lymphocytes by (-) (3H) alprenolol binding.

Abstract: Human lymphocytes are known to posessess a catecholamine-responsive adenylate cyclase which has typical beta-adrenergic specificity. To identify directly and to quantitate these beta-adenergic receptors in human lymphocytes, (-) [3H] alprenolol, a potent beta-adrenergic antagonist, was used to label binding sites in homogenates of human mononuclear leukocytes. Binding of (-) [3H] alprenolol to these sites demonstrated the kinetics, affinity, and stereospecificity expected of binding to adenylate cyclase-coupled beta-adrenergic receptors. Binding was rapid (t1/2 less than 30 s) and rapidly reversible (t1/2 less than 3 min) at 37 degrees C. Binding was a saturable process with 75 +/- 12 fmol (-) [3H] alprenolol bound/mg protein (mean +/- SEM) at saturation, corresponding to about 2,000 sites/cell. Half-maximal saturation occurred at 10 nM (-) [3H] alprenolol, which provides an estimate of the dissociation constant of (-) [3H] alprenolol for the beta-adrenergic receptor. The beta-adrenergic antagonist, (-) propranolol, potently competed for the binding sites, causing half-maximal inhibition of binding at 9 nM. beta-Adrenergic agonists also competed for the binding sites. The order of potency was (-) isoproterenol greater than (-) epinephrine greater than (-)-norepinephrine which agreed with the order of potency of these agents in stimulating leukocyte adenylate cyclase. Dissociation constants computed from binding experiments were virtually identical to those obtained from adenylate cyclase activation studies. Marked stereospecificity was observed for both binding and activation of adenylate cyclase. (-)Stereoisomers of beta-adrenergic agonists and antagonists were 9- to 300-fold more potent than their corresponding (+) stereoisomers. Structurally related compounds devoid of beta-adrenergic activity such as dopamine, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine did not effectively compete for the binding sites. (-) [3H] alprenolol binding to human mononuclear leukocyte preparations was almost entirely accounted for by binding to small lymphocytes, the predominant cell type in the preparations. No binding was detectable to human erythrocytes. These results demonstrate the feasibility of using direct binding methods to study beta-adrenergic receptors in a human tissue. They also provide an experimental approach to the study of states of altered sensitivity to catecholamines at the receptor level in man.

Alpha-Adrenergic Receptor Identification by [3H]Dihydroergocryptine Binding

Abstract: A radioactively labeled alpha-adrenergic antagonist, [3H]dihydroergocryptine, binds specifically to a site on rabbit uterine membranes. Binding is rapid, reaching equilibrium in less than 17 minutes at 25 degrees C. Adrenergic agonists compete for this binding site with an order of affinities identical to the pharmacological potency order of these agents as alpha-adrenergic agonists (epinephrine greater than norepinephrine greater than isoprotereonl). The (-) stereoisomers of epinephrine and norepinephrine are 30 times more potent in competing for the site than the corresponding (+) stereoisomers. alpha-Adrenergic antagonists, such as phentolamine and phenoxybenzamine, potently compete for the binding sites while the beta-adrenergic antagonist propranolol does not. Structural analogs of catecholamines that are devoid of alpha-adrenergic physiological activity do not compete for [3H]dihydroergocryptine binding sites. These data suggest that alpha-adrenergic receptors can be directly identified and studied by [3H]dihydroergocryptine binding.

Regulation of Adenylate Cyclase-Coupled Beta Adrenergic Receptor Binding Sites by Beta Adrenergic Catecholamines in Vitro

Abstract: Tolerance to catecholamines was studied using a frog erythrocyte model system in vitro. Incubation of cells with (-)-isoproterenol resulted in up to a 58% decline ( p epinephrine > norepinephrine. The beta antagonist propranolol, while having no effect of its own, blocked the desensitization caused by isoproterenol. In contrast, the alpha adrenergic antagonist phentolamine did not interfere with the phenomenon. The desensitized state was shown to be reversible. If isoproterenol was removed from desensitized cells by washing, and cells were further incubated with propranolol, receptor binding and enzyme activity returned to nearly normal levels. Incubation of cells with PGE1 produced desensitization to subsequent prostaglandin stimulation but had no effect on catecholamine sensitivity or beta receptor binding. Dibutyryl cAMP did not cause a decrease in binding or enzyme activity. It appears that catecholamines, through beta adrenergic receptor interactions, play a role in regulation of the number of functioning receptors at the cell surface. This may be a major mechanism for the induction of catecholamine tolerance.

Regulation of Adenylate Cyclase Coupled β-Adrenergic Receptors by β-Adrenergic Catecholamines

Abstract: Injection of frogs with β-adrenergiccatecholamines produced a selective desensitization(loss of responsiveness) of the erythrocyte membraneadenylate cyclase to subsequent stimulation in vitroby isoproterenol. Basal, prostaglandin E1-and fluoride-sensitive enzyme activitieswere unaffected. A 77% (P<0.001) decline in isoproterenol-responsiveenzyme activity in the cells from the treatedanimals was observed with no change in the KM forisoproterenol stimulation of the enzyme (concentrationcausing ½ maximal enzyme activation). The decrease in catecholamine-sensitive adenylate cyclasewas accompanied by a parallel 68%(P < 0.001)fall in the apparent number ofβ-adrenergic receptorsin the erythrocyte membranes,assessed by (−)[3H] alprenolol binding studies. There was no changein the affinity of the receptor binding sites. The catecholamine-induced desensitization and fall in the β-adrenergic receptor number were both concentrationand time-dependent and displayed β-adrenergicspecificity. Isoproterenol was more potent...

Desensitization of beta-adrenergic receptors by beta-adrenergic agonists in a cell-free system: resensitization by guanosine 5'-(beta, gamma-imino)triphosphate and other purine nucleotides.

Abstract: Incubation of purified frog erythrocyte membranes with beta-adrenergic agonists at 25 degrees produces relatively rapid (half-time about 10 min) desensitization (inactivation) of about 60% of the beta-adrenergic receptor binding sites. The desensitized receptors no longer bind the specific beta-adrenergic ligand (-)[3H]dihydroalprenolol. The decrease in the number of functional beta-adrenergic receptors is also manifest as a decreased ability of isoproterenol to stimulate the membrane-bound adenylate cyclase.

Adenylate cyclase-coupled beta adrenergic receptors effect of membrane lipid-perturbing agents on receptor binding and enzyme stimulation by catecholamines.

Abstract: Binding of (-)-[3H]dihydroalprenolol, a potent competitive beta adrenergic antagonist, to sites in frog erythrocyte membranes has previously been demonstrated to possess the essential properties expected of binding to adenylate cyclase-coupled beta adrenergic receptors. The present studies were designed to test the effects of a variety of membrane lipid-perturbing agents on both beta adrenergic receptor binding and catecholamine-responsive adenylate cyclase in frog erythrocyte membranes. Digestion of membranes with phospholipases A, C, and D causes a dose-dependent decline in receptor binding capacity without altering receptor affinity. Amphotericin B, a nondegradative membrane lipid perturbant, also causes a dose-dependent decrease in (-)-[3H]dihydroalprenolol binding. Decrements in catecholamine-stimulated adenylate cyclase activity caused by these agents are always greater than decreases in basal and fluoride-sensitive enzyme activities. Decreases in (-)-[3H]dihydroalprenolol binding parallel the disproportionate reduction in catecholamine responsiveness of adenylate cyclase. By contrast, the polyene antibiotic Filipin appears to "uncouple" receptor binding and enzyme activation, since a marked reduction in isoproterenol-stimulated adenylate cyclase is not accompanied by a decrease in specific (-)-[3H]dihydroalprenolol binding. ACKNOWLEDGMENTS The electron microscopic studies in consultation were performed by Dr. J. R. Sommer, Director, Veterans Administration EM Laboratory, Veterans Administration Hospital, Durham, N. C. The authors thank Dr. Mary Ellen Switzer for fibrinogen degradation studies and Dr. Patrick McKee for helpful discussion. The authors also acknowledge Mr. Michael Coverstone for technical assistance.

Structure-activity relationships of adenylate cyclase-coupled beta adrenergic receptors: determination by direct binding studies.

Abstract: Recently developed techniques for directly studying ligand binding to beta adrenergic receptors with (-)-[3H]alprenolol have been used to delineate in detail the binding specificity of the adenylate cyclase-coupled beta adrenergic receptors in a model system, the frog erythrocyte membrane. The abilities of 60 beta adrenergic agents to compete for the binding sites and to interact with the adenylate cyclase (as agonists or antagonists) were quantitated and compared. The specificity of the receptors determined by direct binding studies or by adenylate cyclase studies was comparable. The KD values of the agents as determined by inhibition of (-)-[3H]alprenolol binding correlated well ( r = 0.95) with their apparent dissociation constants determined by enzyme studies. The latter were determined as the concentrations of agonists necessary to cause 50% maximal enzyme stimulation, or the concentrations of antagonists necessary to produce a 2-fold rightward shift in the (-)-isoproterenol dose-response curve. Agonists and antagonists appeared to compete for the same set of receptor binding sites. Structure-activity relationships determined by the direct binding studies were in excellent agreement with those previously determined in more intact tissue preparations. For agonists the structural features which determined receptor affinity (assessed by direct binding studies) were distinct from those which determined intrinsic activity (maximum ability to stimulate adenylate cyclase). The affinity of agonists was increased by increasing the size of the substituent on the amino nitrogen, by a (-) configuration of the hydroxyl on the β-carbon, and by the presence of a catechol moiety. Methyl or ethyl substitution on the α-carbon had only a slight (generally inhibitory) effect on affinity. Intrinsic activity of agonists was determined primarily by the nature of the substituents on the phenyl ring. Full intrinsic activity requires the presence of hydroxyl groups on the ring at positions 3 and 4 as well as the β-carbon hydroxyl in the (-) configuration. Deletion of the β-carbon hydroxyl, as in compounds such as dopamine, dobutamine, and related agents, leads to substantial loss of intrinsic activity and affinity even in the presence of large amino nitrogen substituents. A methanesulfonamide group substituted for the hydroxyl in position 3 on the ring results in reduced intrinsic activity. Deletion of the ring hydroxyl at either position 3 or 4 or substitution by chlorine produces competitive antagonists. Structure-activity relationships of antagonists were similar to those of agonists, except that the catechol moiety was replaced by a single or double aromatic ring structure. Separation of this moiety from the ethanolamine side chain by an ether function significantly increased affinity. When a phenyl group was present, a single substituent at the para position was associated with reduced affinity. ACKNOWLEDGMENT We gratefully acknowledge the expert assistance of the analytical chemistry department of New England Nuclear Corporation in developing chromatographic procedures for (-)-alprenolol.

Beta-adrenergic receptors: recognition and regulation.

Abstract: Of the various endogenous hormones and exogenous drugs, few have more widespread and potent effects than the catecholamines. It is therefore not surprising that natural and synthetic catecholamines and a variety of compounds that antagonize or block their effects are among the most useful and versatile agents at the physician's disposal. Catecholamine Receptors More than 25 years ago Ahlquist1 used the disparate relative potency of catecholamines in stimulating a variety of physiologic processes to classify adrenergic effects into two main types, α and β (Table 1). Each type of response is characterized by a typical order of potency of agonist . . .

Regulation of adenylate cyclase coupled beta-adrenergic receptors.

Abstract: Publisher Summary The β -adrenergic receptor, of the two major types of adrenergic receptors α and β , is the only adrenergic receptor associated with the stimulation of adenylate cyclase activity. This chapter reviews few recent results derived from binding studies with the radioligand (±)[ 3 H) alprenolol, which is a potent competitive β -adrenergic antagonist. It emphasizes on three areas: (1) how binding of this radioligand to membranes from a variety of tissues meets the essential criteria expected of binding to β -adrenergic receptors; (2) the nature and properties of the β -adrenergic receptors as revealed by these techniques; and (3) studies of physiological, biochemical, and developmental regulation of the β -adrenergic receptors. The use of radioactively labeled β -adrenergic antagonists such as (-)[ 3 H) alprenolol makes it feasible to identify β -adrenergic receptor binding sites in membrane fractions. The binding sites identified with this ligand fulfill all the criteria of kinetics, affinity, specificity, and stereospecificity expected of the physiologically relevant adenylate cyclase-coupled β -adrenergic receptors. These new methods are applicable to a wide variety of mammalian and non mammalian tissues. These techniques provide an important tool for obtaining new insights into the biochemical nature and physiological regulation of the β -adrenergic receptors, apart from providing the assay methods necessary for purification of the β -adrenergic receptors.

Editorial: Smoking, catecholamines, and the heart.

Abstract: Ample, carefully documented evidence supports the familiar warning printed on each of the 30 billion packages of cigarettes sold in this country each year. Nonetheless, cigarette consumption in this country continues to rise at the rate of 2 to 3 per cent per year. Of the illnesses in which increased morbidity or mortality has been associated with cigarette smoking none are of greater public-health importance than coronary-artery disease.1 A great deal of epidemiologic and laboratory effort has been directed toward gaining a better understanding of the mechanisms by which smoking promotes the development and exacerbates the symptoms of atherosclerotic coronary-artery . . .

Pharmacological activity of nitroxide analogues of dichloroisoproterenol and propranolol.

Abstract: Spin-labeled analogues of dichloroisoproterenol and propranolol were synthesized. It was found that the KD's of both probes for the beta-adrenergic receptors of frog erythrocytes were about 30-fold higher than the KD's previously reported for the parent antagonists. Thus the introduction of a bulky nitroxide moiety in place of the isopropyl group on the amino nitrogen is associated with a decrease in affinity for the beta-adrenergic receptors. Nonetheless, the affinity of the spin-labeled propranolol would appear to be within a range compatible with EPR measurements.

Membrane-Bound Hormone Receptors

Abstract: Receptors are those cellular structures with which biologically active hormones and drugs first interact. Physiologically these receptors perform two essential functions. The first is that of recognition of a particular biologically active chemical structure presumably by some complimentarity in the structure of the hormone or drug and the receptor itself. This recognition is effected by specific binding of the hormone or drug to the cellular receptor structures. The second function of these receptors is that of activation of biological processes. This may be effected through a stimulation of an enzyme activity, a change in the membrane conductance for a specific ion, etc. For certain hormones, e.g., the steroid hormones, the receptors appear to be cytoplasmic in location (Baulieu et al., 1971). For many other hormones and drugs, however, the receptor structures appear to be located within the plasma membranes of the responsive target cells. This chapter will be confined to the latter class of hormone receptors.

Biochemical and Molecular Characteristics of β-Adrenergic Receptor Binding Sites

Abstract: The availability of radiolabeled hormones, drugs and analogs which retain their characteristic biological activity and selectivity has provided a valuable tool for the study of the interaction of these agents with their target cell receptors. Despite the major advances in the investigation of receptors for polypeptide (14, 37) and steroid hormones (13) and nicotinic cholinergic drugs (11) using these techniques, the confident identification of the β-adrenergic receptors for catecholamines has remained until recently an elusive goal. Initial studies employed tritium labeled β-adrenergic agonists to identify binding sites in membrane fractions containing catecholamine-sensitive adenylate cyclase. However, some of the characteristics of binding observed with these agents diverged from which might be expected of the physiological β-adrenergic receptor (3, 17, 18). Perhaps the difficulties in these studies can be attributed to: 1) the multiple potential binding sites in addition to the adenylate cyclase coupled β-adrenergic receptor for labeled native catecholamines, 2) the relatively low affinity for cata-cholamines in in vitro membrane preparations (KD ≅ 10−6M), 3) the unimpressive specific radioactivity of available tritiated ligands (≅ 1 Ci/mmol), or 4) the absence of tissue preparations which provided a high concentration of β-adrenergic receptors relative to other constituents. Even in the case of the high affinity β-adrenergic antagonist propranolol, which would not likely interact with degradative enzymes and uptake mechanisms directed at catecholamines, binding characteristics indicated that this ligand binds disproportionately to non β-adrenergic receptor sites (40), possibly those responsible for the so-called “anaesthetic” properties of this drug.