Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum. |
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Authors: | K Nishikori H Maeno |
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Affiliation: | Department of Pharmacology and Biochemistry, Central Research Laboratories, Yamanouchi Pharmaceutical Co. Ltd., Tokyo 174, Japan |
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Abstract: | Binding of (?)-[]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 108m?1 min?1 and 0.189 min?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (KD) of 5 to 7 nm. Bound (?)-[]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[]dihydroalprenolol were not affected by β1-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β1-responses in vitro, whereas β2-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β2-adrenergic receptor. |
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