Characterisation of α_1B-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 α₁-adrenoceptors are present on these cells. The specific binding of [³H]prazosin to membranes of U373 MG cells (B_max 32±3 fmol mg^–1 protein, K_d 0.27±0.03 nM) was inhibited in a monophasic manner by α₁-antagonists that have different affinities for α_1A-, α_1B- and α_1D-adrenoceptors. Estimates for pK_i 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 µM chloroethylclonidine for 15 min.

In the presence of 30 mM LiCl, 100 µM 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 µM. Noradrenaline-induced [³H]IP₁ accumulation was also inhibited by α₁-antagonists. Estimates for pK_i 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 µM noradrenaline was not significantly affected by raising the extracellular Ca²⁺ concentration from 1.3 to 4 mM. Noradrenaline (100 µM) also produced an increase in intracellular Ca²⁺ (mean peak 86±5 nM above basal). Pretreatment with chloroethylclonidine (10 µM, 15 min) abolished noradrenaline-induced [³H]IP₁ accumulation and Ca²⁺ mobilisation. Activation of the α_1B-adrenoceptors by 10 µM 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 α₁-adrenoceptors, the α_1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to α₁-agonists.