Characterisation of alpha1B-adrenoceptors linked to inositol phosphate formation and calcium mobilisation in human astrocytoma U373 MG cells.

Abstract

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.