CALCIUM-DEPENDENCE OF HISTAMINE-INDUCED AND CARBACHOL-INDUCED INOSITOL PHOSPHATE FORMATION IN HUMAN U373 MG ASTROCYTOMA-CELLS - COMPARISON WITH HELA-CELLS AND BRAIN-SLICES

1 Histamine (1 mM) induced an accumulation of inositol monophosphate ([H-3]-IP1) in the U373 MG human astrocytoma cell line which increased with time in the presence of 30 mM Li+, After a 30 min incubation period with 1 mM histamine [H-3]-IP1 was the major product detected (84 +/- 1% of total [H-3]-IPx) and was present at a level 11 (+/-1) fold of basal accumulation. 2 Concentration-response curves for histamine-induced [H-3]-IP1 accumulation in U373 MG cells (EC(50) 5.4 +/- 0.5 mu M) were shifted to the right in a parallel fashion by mepyramine (slope of a Schild plot 0.99 +/- 0.08), yielding a K-d for mepyramine of 3.5 +/- 0.3 nM, consistent with the involvement of histamine H-1-receptors. 3 The temelastine-sensitive binding of [H-3]-mepyramine to a membrane fraction from U373 MG cells was hyperbolic and had a mean K-d of 2.5 +/- 1.0 nM. The maximum amount of temelastine-sensitive binding was 86 +/- 19 pmol g(-1) membrane protein. 4 Carbachol also induced [H-3]-IP1 accumulation in U373 MG cells, 2,8 (+/- 0.1) fold of basal with 1 mM carbachol, with an EC(50) of 48 +/- 8 mu M. Pirenzepine shifted carbachol concentration-response curves to the right (slope of Schild plot 0.89 +/- 0.07) giving a K-d for pirenzepine of 0.10 +/- 0.01 mu M, suggesting that phosphoinositide hydrolysis in U373 MG cells is mediated by the M(3-), rather than the M(1-), muscarinic receptor subtype. 5 [H-3]-IP1 accumulation induced by both 1 mM histamine and by 1 mM carbachol increased when the Ca2+ concentration of the medium was increased from 'zero' (no added Ca2+) to 0.3 mM. Histamine-stimulated [H-3]-IP1 accumulation was further increased, although not so markedly, as the Ca2+ was raised to 4 mM. The same pattern was apparent with histamine-induced accumulations of [H-3]-IP2 and [H-3]-IP3. In contrast, [H-3]-IPx accumulation in response to carbachol increased between 0.3 and 1.3 mM, but thereafter remained unchanged ([H-3]-IP1) Or declined ([H-3]-IP2 and [H-3]-IP3). 6 In HeLa cells, [H-3]-IP1 accumulations induced by 1 mM histamine and 1 mM carbachol showed the same pattern of Ca2+ dependence and were independent of extracellular Ca2+ above 0.3 mM (histamine) or 1.3 mM (carbachol). The response to carbachol appeared to be mediated by an M(3)-muscarinic receptor (apparent K-d for pirenzepine 0.09 mu M). 7 In cross-chopped slices of guinea-pig cerebral cortex and guinea-pig cerebellum, [H-3]-IP1 accumulation induced by 1 mM histamine in the presence of 10 mM Li+ increased as the extracellular Ca2+ was increased from 0.3 to 2.5 mM, but a further increase to 4 mM had no further effect. In contrast the response to histamine iii rat cerebral cortex increased markedly between 1.3 and 4 mM Ca2+: Accumulations of [H-3]-IP1 induced by carbachol in guinea-pig or rat cerebral cortical slices were not increased as extracellular Ca2+ was raised from 0.3 to 4 mM. 8 Nimodipine (100 nM) and omega-conotoxin (3 mu M) had no significant effect on histamine-induced [H-3]-IP1 accumulation in rat cerebral cortical slices or in U373 MG cells.