Influence of polymeric effectors on binding of 3H-dihydroalprenolol to beta-adrenergic receptor of rat brain.
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The significance of anionic and cationic charges of glycocalyx, phospholipid or protein, etc. on the cell surface of the rat brain was examined for beta-adrenoceptors using the radioligand binding assay method. Thus, this experiment was designed to assess the effects of polymeric effectors, DNA, heparin, polymyxin B, histone, gelatin, colominic acid and bovine serum albumin (BSA), on the affinity of beta-adrenoceptors. The rat brain was used and the beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol as a radioligand. Polymyxin B, DNA and heparin significantly caused a reduction in the maximum number of beta-adrenoceptors (Bmax), but only small changes were observed with histone, gelatin, BSA and colominic acid. Only DNA induced a decrease in the value of the dissociation constant (Kd) of beta-adrenoceptors. These results suggest that anionic or cationic charges in the environment of the receptor sites could have a crucial role in drug-receptor interaction. (+info)
Hepatic adenylate cyclase. Development-dependent coupling to the beta-adrenergic receptor in the neonate.
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Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear. (+info)
Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of lectins.
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The significance of the carbohydrate moieties of the beta-adrenergic receptor molecule in the rat brain was examined using the radioligand binding assay method. Thus, this experiment was designed to assess the effects of lectins, concanavalin A (Con A), Phaseolus vulgaris agglutinin (PHA), and wheat germ agglutinin (WGA) on the affinity of the beta-adrenoceptor. The rat brain was used and the beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol as a ligand. Con A and PHA significantly caused an increase in the values of the density of beta-adrenoceptor (Bmax) and a reduction in the values of the dissociation constant (Kd), but significant changes were not observed with WGA. These results strongly suggest that the carbohydrate moieties of the cell surface containing the beta-adrenoceptor molecule may have a crucial role in the drug-receptor interaction, and they imply that the beta-adrenoceptor molecule is a glycoprotein which contains N-linked carbohydrate chains. (+info)
Studies on the affinity and selectivity of denopamine (TA-064), a new cardiotonic agent, for beta-adrenergic receptors.
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Denopamine is a new orally active cardiotonic agent. The present experiment was carried out to characterize the binding affinity and selectivity of this drug for beta-adrenergic receptor subtypes. Binding studies were performed using 3H-dihydroalprenolol as the radioligand. Binding affinities of denopamine and some beta-agonists for rat heart membranes (KiH), which contain predominantly the beta 1-subtype, were in the order of isoproterenol (Iso, 14.1 nM) greater than prenalterol (158) greater than norepinephrine (Nor, 227) greater than or equal to epinephrine (Epi, 248) greater than denopamine (545) greater than or equal to dobutamine (645) greater than procaterol (1440) greater than terbutaline (6420). In rat lung membranes (predominantly beta 2-subtype), the order of potency (KiL) was Iso (20.6 nM) greater than procaterol (70.2) greater than Epi (136) greater than prenalterol (412) greater than dobutamine (735) greater than or equal to Nor (744) greater than denopamine (2205) greater than terbutaline (2500). The beta 1/beta 2-selectivity as judged from the KiL/KiH values was in the order of denopamine (4.1) greater than Nor (3.3) greater than prenalterol (2.6) greater than Iso (1.5) greater than dobutamine (1.1) greater than Epi (0.55) greater than terbutaline (0.39) greater than procaterol (0.05). Practolol, a beta 1-antagonist, showed a high beta 1-selectivity (KiL/KiH = 15.3). In the presence of guanine nucleotide (GTP), the denopamine radioligand competition curve showed a rightward shift, and its Hill coefficient increased like other agonists, although the degree of the shift was less than that observed with full agonists such as Iso. These results essentially correspond with the pharmacological and biochemical properties of denopamine and confirm the beta 1-selectivity and the agonist property of this compound. (+info)
Irreversible inactivation of the beta-adrenoreceptor by a partial agonist. Evidence for selective loss of the agonist high affinity binding sites.
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The catecholamine derivatives aminomenthylnorepinephrine (compound 1) and bromoacetylaminomenthylnorepinephrine (compound 2) were synthesized and their interaction with the rat lung beta-adrenoreceptor was characterized. Compared to (-)-isoproterenol, compounds 1 and 2 were 10 and 280 times less potent, respectively, at inhibiting (-)-[3H]dihydroalprenolol binding. At pH 7.4, all 3 compounds induced a loss of receptors (40-60%) which could be recovered by treatment with guanyl-5'-yl imidodiphosphate (Gpp(NH)p). However, at pH 8.1 Gpp(NH)p treatment did not recover those receptors lost by compound 2 only. The compound 2-induced receptor loss at pH 8.1 was time-dependent, prevented by propranolol but unaffected by Gpp(NH)p or after membrane heating at 50 degrees C which prevented the formation of the agonist high affinity binding state. Although, the maximal receptor loss as measured by [3H]dihydroalprenolol was 40-60%, more than 80% of the receptors were lost when measured by direct agonist binding, and the receptors left showed little agonist high affinity binding state formation. In rat reticulocyte membranes, compounds 1 and 2 stimulated adenylate cyclase activity with intrinsic activities of 0.55 and 0.31, respectively. However, at pH 8.1, compound 2 initially stimulated the enzyme followed by a blockade. These data indicated that both compounds 1 and 2 were partial beta-adrenoreceptor agonists and, at pH 8.1, compound 2 appeared to bind irreversibly only to those lung receptors able to form the agonist high affinity binding state. Furthermore, after irreversible binding, compound 2 appeared to act as an antagonist. (+info)
Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of exo- and endo-glycosidases and glycopeptidase.
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The significance of carbohydrate moieties containing the beta-adrenoceptor molecule in the rat brain was examined using radioligand binding assay methods. Thus, this experiment was designed to assess the effects of exoglycosidase (alpha-D-mannosidase and neuraminidase), endoglycosidase (endoglycosidase D and endoglycosidase H), and glycopeptidase A on the affinity of beta-adrenoceptor. The main reason why five kinds of enzymes were used in the present study is that they can hydrolyze different carbohydrate molecules from cell membranes. Rat brain was used and beta-adrenoceptor binding assay was carried out using 3H-dihydroalprenolol (3H-DHA) as a ligand. 3H-DHA binding to beta-adrenoceptors was sensitive to very low concentration of endoglycosidase H and glycopeptidase A, thus indicating that the treatments with these enzymes of rat brain membrane appear to decrease the number of beta-receptor binding sites. On the other hand, the treatment with neuraminidase, endoglycosidase H, and glycopeptidase A of the membrane induced lower values of the dissociation constant (Kd) than those of the control. alpha-D-mannosidase and endoglycosidase D are without effect in spite of the removal of hexose contents and total carbohydrate contents with these treatments, respectively. These results imply that complex type N-linked acidic carbohydrate chains containing neuraminic acid and high mannose type N-linked carbohydrate chains, which are hydrolyzed with endoglycosidase H and glycopeptidase A, of the rat brain membrane containing beta-adrenoceptor molecules play a crucial role in the drug-receptor interaction. (+info)
Effect of GTP on the affinity of denopamine, a new cardiotonic agent, for beta-adrenergic receptors of turkey erythrocytes and rat reticulocyte membranes.
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Affinities of denopamine, a new cardiotonic agent, and several beta-adrenergic drugs for turkey erythrocyte membranes (TEM) and rat reticulocyte membranes (RRM) which contain homogeneous beta 1- and beta 2-receptors, respectively, were studied by receptor binding. The order of potencies of denopamine and several beta-adrenergic agonists in displacing 3H-dihydroalprenolol binding (Ki, nM) in TEM was isoproterenol (Iso, 27) greater than norepinephrine (Nor, 360) greater than epinephrine (Epi, 860) greater than dobutamine (DB, 1380) greater than denopamine (1540) greater than dopamine (DA, 49500). The order in RRM was Iso (7.3) greater than Epi (58) greater than DB (750) greater than Nor (1090) greater than denopamine (2300) greater than DA (26800). In the presence of GTP, competition curves for full agonists like Iso, Epi and Nor shifted to the low affinity side (Ki values increased by 300-500% in TEM and 200-460% in RRM), and the slopes were steepened in both membrane preparations. The Ki value for denopamine increased only in TEM (70%) and that in RRM was not influenced by GTP. This suggests that denopamine has an agonist property at the beta 1-receptor but not at the beta 2-receptor and that the intrinsic activity at the beta 1-receptor of the drug is lower than full agonists. Affinities of DB and DA for TEM were influenced by GTP as well as those for RRM, although the extent of the rightward shift was less than full agonists. (+info)
Characterization of beta adrenergic receptors in human cerebral arteries and alteration of the receptors after subarachnoid hemorrhage.
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The nature of beta adrenergic receptors in human cerebral arteries was characterized and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. The specific 3H-dihydroalprenolol, a beta adrenergic antagonist, binding to human cerebral arteries was saturable and of high affinity (KD = 12.3 nM) with a Bmax of 790 fmol/mg protein. Ki values and Hill coefficients of adrenergic agents for 3H-dihydroalprenolol were as follows; propranolol, 4.1 X 10(-8)M, 1.01; isoproterenol, 1.7 X 10(-6)M, 0.80; epinephrine, 8.3 X 10(-6)M, 0.48; norepinephrine, 2.3 X 10(-5)M, 0.45; metoprolol, 6.8 X 10(-8)M and 7.9 X 10(-6)M, 0.62; butoxamine, 2.2 X 10(-8)M and 2.1 X 10(-6)M, 0.43. The analysis of inhibition of specific 3H-dihydroalprenolol binding by these adrenergic agents suggests that human cerebral arteries contain a high density of beta adrenergic receptors and that the receptors are classified into two types, namely beta 1 and beta 2 adrenergic receptors. The calculated beta 1/beta 2 ratio from Hofstee plots was approximately 4/6. KD and Bmax of 3H-dihydroalprenolol binding to the cerebral arteries after subarachnoid hemorrhage were compared with those of control group. KD and Bmax of 3H-dihydroalprenolol binding of subarachnoid hemorrhage group were 13.9 nM and 1140 fmol/mg protein, respectively. The calculated beta 1/beta 2 ratio was approximately 6/4. These data suggest that the density of total beta adrenergic receptors increased without any significant change in the affinity after subarachnoid hemorrhage and that the increase of beta 1 adrenergic receptors was dominant. (+info)