Beta adrenergic receptors of polymorphonuclear particulates in bronchial asthma.
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We have tested the beta adrenergic receptor theory of bronchial asthma by determining the number and affinity of binding sites of the beta adrenergic radioligand [(3)H]dihydroalprenolol (DHA) and the activity of adenylate cyclase in broken cell preparations of polymorphonuclear leukocytes (PMN). We studied 31 control subjects (group 1), 30 asthmatics receiving no systemic adrenergic medication (group 2), and 17 asthmatics receiving adrenergic agonists systemically (group 3). Control subjects and asthmatics taking no adrenergic drugs bound similar amounts of DHA at 0.5 nM and 30 nM DHA and had about 900 binding sites per PMN. In contrast, asthmatics receiving adrenergic agonists had a >70% decrease in their number of DHA binding sites per PMN (254+/-57). In a subset of our three groups of subjects (eight from group 1, six from group 2, and five from group 3) we measured DHA binding at several DHA concentrations and found similar values (0.4-0.7 nM) for the dissociation constant of DHA among these subjects. In further studies we examined the interaction of the agonist (-)-isoproterenol with beta adrenergic receptors in 8 normal subjects and 10 asthmatics not receiving adrenergic medication. We tested the ability of isoproterenol to compete for DHA binding sites and to stimulate adenylate cyclase in sonicates prepared from PMN and examined under identical conditions. The dissociation constants for the competition of isoproterenol for DHA binding sites in normal and asthmatic subjects were virtually identical ( approximately 1.0 muM). In addition, the (activation constant) values for stimulation of adenylate cyclase were similar (0.16-0.19 muM) in the two groups of subjects.Thus, these data suggest that asthma per se is not associated with alteration in either the number or affinity of beta adrenergic receptors in PMN. Our findings indicate that previous reports of abnormal beta adrenergic receptor function in asthmatic patients may in part be explained by prior treatment of such patients with adrenergic agonists. Because the asthmatics who received adrenergic agonists in our study tended to be more ill and to receive additional medication compared to subjects in group 2, we cannot rule out unequivocally that severe asthma may be associated with decreased binding to beta adrenergic receptors. Nevertheless, we conclude that beta adrenergic receptors on PMN from asthmatics are relatively normal unless such patients are treated with adrenergic agonists. (+info)
Biphasic adrenergic modulation of beta-adrenergic receptors in man. Agonist-induced early increment and late decrement in beta-adrenergic receptor number.
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beta-Adrenergic receptors in mononuclear leukocyte preparations were assessed with (-)[(3)H]-dihydroalprenolol binding studies during the infusion of adrenergic agonists into normal human subjects. During the infusion of isoproterenol into seven subjects, mean (+/-SE) (-)[(3)H]dihydroalprenolol binding increased from 25+/-3 fmol/mg protein to 47+/-8 fmol/mg protein (P < 0.02) at 0.5 h and 40+/-3 fmol/mg protein (P < 0.01) at 1 h and decreased to 12+/-1 fmol/mg protein (P < 0.01) at 4-6 h. During the infusion of epinephrine into three subjects, mean (-)[(3)H]dihydroalprenolol binding increased from 32+/-3 to 63+/-3 fmol/mg protein (P < 0.01) at 0.5-1 h. By Scatchard plot analysis, these changes were attributable to changes in the number of available binding sites rather than changes in binding affinity. The observed changes in the number of (-)[(3)H]dihydroalprenolol binding sites were not paralleled by changes in total mononuclear cell counts or in T lymphocyte, B lymphocyte, and monocyte distributions. Thus, we conclude that adrenergic agonists modulate the number of available beta-adrenergic receptors on circulating mononuclear cells in a biphasic manner, with an early increment and a late decrement, in man. Further, the finding that the increase in pulse rate in response to a "pulse" infusion of isoproterenol was significantly greater after 0.5-1 h of agonist infusion suggests that the observed early agonist-induced increment in beta-adrenergic receptor number on circulating cells is paralleled by increments in extra-vascular beta-adrenergic receptor sensitivity. (+info)
In vivo receptor binding of iodinated beta-adrenoceptor blockers.
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Six radiolabeled beta-adrenoceptor blocking agents with a range of affinity constants were evaluated as radioindicators for adrenoceptors in guinea-pig heart and lung. All concentrated in the heart and lung at levels in excess of 0.1% dose/g tissue. On the basis of displacement studies using propranolol, two of the six compounds showed beta-adrenoceptor binding in the lung, and one, H-3 carazolol, showed receptor binding in the heart. These results agree qualitatively with a bi-molecular reversible equilibrium model, and suggest that the beta-adrenoceptor blockers as a group will not be useful in vivo probes of receptor concentration in the heart because of the low affinity constants and high levels of nonreceptor binding associated with the present-day clinical beta blockers. Beta-adrenoceptor blocking agents with affinity constants in excess of 10(9) will be needed to give heart-to-blood ratios of 10. (+info)
Relationship between the (--)-[3H]-dihydroalprenolol binding to beta-adrenoceptors and transmembrane 86Rb efflux of the BC3H1 nonfusing muscle cell line.
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1 We have studied the binding properties of the beta-adrenoceptor antagonist [3H]-dihydroalprenolol ([3H]-DHA) on a membrane preparation of the non-fusing muscle cells BC3H1. 2 [3H]-DHA appears to bind to two classes of sites. The first site has a high affinity (KD = 0.53 nM) and a low capcity (Bmax = 58 fmol/mg of protein). The second site has a low affinity (KD = 110 nM) and a high capacity (Bmax = 1100 fmol/mg of protein). 3 The pharmacological properties of the high affinity low capacity site correspond to the known properties of the beta 2-adrenoceptors since the agonists inhibit [3H]-DHA binding following the series isoprenaline greater than adrenaline greater than noradrenaline greater than phenylephrine and the antagonists following the series alprenolol congruent to propranolol greater than butoxamine greater than practolol greater than phentolamine. 4 The binding properties of the beta-adrenoceptors were correlated with the effect of beta-adrenoceptor agonists and antagonists on 86Rb efflux rate from BC3H1 Cells. 5 There is very good correlation between the dissociation constants obtained by inhibition of [3H]-DHA binding by the antagonists alprenolol and propranolol, and the inhibition constants calculated from their antagonism of the 86Rb efflux rate stimulation by adrenaline. The ratio of the dissociation constants obtained by inhibition of [3H]-DHA binding by agonists and their EC50, calculated from 86Rb efflux curves, is higher than 1. This high KD/EC50 ratio indicates a high coupling efficiency between receptor occupancy by agonists and the biological effect measured. (+info)
Evidence against beta-adrenoceptor blocking activity of diltiazem, a drug with calcium antagonist properties.
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1 The isolated spontaneously beating atria of the rat, diltiazem (0.01 to 0.1 microM) shifted the atrial rate concentration-response curves to isoprenaline to the right in a non-parallel manner and depressed their maxima. Under the same experimental conditions, (+/-)-propranolol (0.03 to 0.1 microM) behaved as a competitive beta-adrenoceptor antagonist. 2 Whereas (+/-)-propranolol (IC50 = 12 nM) and isoprenaline (IC50 = 0.9 microM) inhibited (-)-[3H]-dihydroalprenolol binding to rat brain membrane preparations, diltiazem failed to do so in concentrations up to 10 microM. 3 Diltiazem but not (+/-)-propranolol, antagonized the positive chronotropic responses to calcium in spontaneously beating rat atria. 4 It is proposed that diltiazem inhibited the tachycardia induced by isoprenaline through an effect on calcium which may be an essential modulator of the sequence of events linking the beta-adrenoceptor activation and heart rate response. (+info)
Distinction between two subpopulations of beta 1-adrenergic receptors in human adipose cells.
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The beta-adrenergic receptors in human adipose membranes were identified by the specific and saturable binding of the beta-adrenergic antagonist (--)-[3H]dihydroalprenolol. The total number of sites in control membranes was 0.32 +/- 0.03 pmol/mg protein and the equilibrium dissociation constant for binding (Kd) was 2.6 nM and 2.5 nM as determined by Scatchard analysis of experiments on equilibrium binding and kinetics, respectively. The beta 1-adrenergic nature of the receptors was derived from the order of potencies of beta-adrenergic agonists (isoproterenol greater than norepinephrine greater than epinephrine) to complete with (--)-[3H]dihydroalprenolol for binding. Studies of saturation binding, kinetics and competition binding revealed the presence of a single class of beta 1-adrenergic receptors. Prolonged incubation of human adipose cells in the presence of (--)-norepinephrine decreases the lipolytic response to beta-adrenergic agonists, and reduces by 50% the concentration of beta-adrenergic receptors. The Kd values for (--)-[3H]dihydroalprenolol and the beta-adrenergic agonists remain unchanged. Catecholamines also produce a rapid conformational change of approximatively 50% of the receptors in control membranes as revealed by their increased sensitivity towards inactivation by the alkylating agent N-ethylmaleimide. This inactivation process is not observed in desensitized membranes, which indicates that desensitization and inactivation by agonists plus N-ethylmaleimide affect the same receptor population. The beta 1-adrenergic receptors in human adipocytes can thus be divided into two subpopulations on the basis of the different consequences of their interaction wtih agonist molecules. (+info)
Inhibitors of transglutaminase prevent agonist-mediated internalization of beta-adrenergic receptors.
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In frog erythrocytes, desensitization of beta-adrenergic receptors is characterized by a decrease in the beta-receptor recognition sites in the plasma membrane and a concomitant increase in the number of this receptor's binding sites in the cytosol. We have documented that this redistribution of the receptor recognition sites reflects the internalization of the surface-bound beta-adrenergic receptors. The present study was addressed to examine whether transglutaminase plays a role in the agonist-mediated internalization of beta-adrenergic receptor recognition sites. Pretreatment of cells with methylamine was found to decrease the internalization and the loss of membrane-bound beta-adrenergic receptors induced by isoproterenol. Methylamine appears to be equally potent in inhibiting transglutaminase activity and in preventing internalization and the receptor loss. The effect of methylamine on soluble and on membrane-bound beta-adrenergic receptors is due to a change in Bmax rather than Kd of these binding sites. Among eight inhibitors of transglutaminase tested, the rank order potency for blocking the enzyme can be correlated with that for preventing the receptor loss and receptor internalization. Moreover, these drug effects on beta-adrenergic receptors are unrelated to the inhibition of isoproterenol-sensitive adenylate cyclase or the binding of [3H]dihydroalprenolol to beta-receptors. These result may lend credence to the view that transglutaminase participates in the internalization and the decrease of membrane-bound receptors during desensitization of beta-adrenergic receptors. (+info)
An assay for beta-adrenergic receptors in isolated human fat cells.
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The beta-adrenergic receptors have been characterized in isolated human adipocytes using a potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Binding of (-)-[3H]dihydroalprenolol to isolated fat cells was stereospecific and saturable, the maximum number of binding sites calculated being 7.8 +/- 2.2 pmol of bound ligand/10(7) cells, corresponding to 450,000 binding sites/cell. The dissociation constant was estimated to be 2.7 +/- 1.1 nM. The results with competition-inhibition experiments using beta-adrenergic agonists and antagonists indicated that the binding sites in isolated adipocytes were predominantly of the beta1-subtype; about 80% of the receptors were of this type. With the present method, specific beta-adrenergic receptor number and affinity in isolated human adipocytes could be determined in about 1 g of human adipose tissue. (+info)