Desensitization of the β-adrenoceptor-adenylate cyclase system of immature erythrocytes by in-vivo treatment of rats with isoprenaline
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Rats pretreated with 1-acetyl-2-phenylhydrazide, to induce reticulocytosis, were treated with (±)isoprenaline (4×30 mg/kg in 6 h intervals within 24 h before blood sampling) in order to desensitize the β-adrenoceptor-adenylate cyclase-system of circulating red cells.
In membrane preparations Vmax-values of (-)isoprenaline-sensitive adenylate cyclase activity declined by about 50% without significant alterations of the apparent Km-values. Basal activity, as well as enzyme activity stimulated maximally by guanylyl-imidodiphosphate and by fluoride were also decreased to the same extent.
In intact cells, maximal synthesis of cAMP stimulated by 10−5 M (-)isoprenaline was decreased by about 50% compared to cell suspensions from control animals in the presence of Ro 20-1724 or papaverine, inhibitors of phosphodiesterase. Without inhibition of phosphodiesterase, (-)isoprenaline stimulated cAMP synthesis in cell suspensions from desensitized animals exceeded that from control animals more than 10-fold. A 20–30% decrease of phosphodiesterase activity, measured in membrane and cytoplasmic fractions explains this unexpected result.
In membrane preparations from untreated animals, Bmax-values for the antagonist ligand [3H] dihydroalprenolol ([3H] DHA) were (0.98±0.16 pmoles/mg protein (n=7). For the agonist-ligand [3H] hydroxybenzylisoprenaline ([3H]-HBI) only 0.23±0.024 pmoles/mg protein (n=12) were obtained. After pretreatment of the animals with (±)isoprenaline, the Bmax-value for [3H] HBI was decreased by about 50%; whereas, that for [3H] DHA was decreased only by about 10%. No change in KD-values for both ligands occurred.
It is proposed that β-adrenergic desensitization in immature red cells, i.e. the decrease of (-)isoprenalinesensitive adenylate cyclase activity, results from loss of high affinity β-adrenoceptor sites. This might be induced by uncoupling the adrenoceptors from the nucleotide binding protein. A functional impairment or loss of this regulatory protein may be responsible for the apparent loss of adenylate cyclase activity. Since [3H] HBI, in contrast to [3H] DHA, labels predominantly β-adrenoceptors in the high affinity state it is a more sensitive marker to detect alterations at the adrenoceptor level following desensitization.
Key wordsDesensitization β-Adrenoceptors Adenylate cyclase Agonist binding Rat reticulocytes
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