Calcium‐dependence of histamine‐ 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 ([3H]‐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 [3H]‐IP1 was the major product detected (84 ± 1 % of total [3H]‐IPx) and was present at a level 11 (±1) fold of basal accumulation. 2 Concentration‐response curves for histamine‐induced [3H]‐IP1 accumulation in U373 MG cells (EC50 5.4 ± 0.5 μm) were shifted to the right in a parallel fashion by mepyramine (slope of a Schild plot 0.99 ± 0.08), yielding a Kd for mepyramine of 3.5 ± 0.3 nm, consistent with the involvement of histamine H1‐receptors. 3 The temelastine‐sensitive binding of [3H]‐mepyramine to a membrane fraction from U373 MG cells was hyperbolic and had a mean Kd 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 [3H]‐IP1 accumulation in U373 MG cells, 2.8 (± 0.1) fold of basal with 1 mm carbachol, with an EC50 of 48 ± 8 μm. Pirenzepine shifted carbachol concentration‐response curves to the right (slope of Schild plot 0.89 ± 0.07) giving a Kd for pirenzepine of 0.10 ± 0.01 μm, suggesting that phosphoinositide hydrolysis in U373 MG cells is mediated by the M3‐, rather than the M1‐, muscarinic receptor subtype. 5 [3H]‐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 [3H]‐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 [3H]‐IP2 and [3H]‐IP3. In contrast, [3H]‐IPx accumulation in response to carbachol increased between 0.3 and 1.3 mm, but thereafter remained unchanged ([3H]‐IP1) or declined ([3H]‐IP2 and [3H]‐IP3). 6 In HeLa cells, [3H]‐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 M3‐muscarinic receptor (apparent Kd for pirenzepine 0.09 μm). 7 In cross‐chopped slices of guinea‐pig cerebral cortex and guinea‐pig cerebellum, [3H]‐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 in rat cerebral cortex increased markedly between 1.3 and 4 mm Ca2+. Accumulations of [3H]‐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 ω‐conotoxin (3 μm) had no significant effect on histamine‐induced [3H]‐IP1 accumulation in rat cerebral cortical slices or in U373 MG cells. 9 We conclude that histamine‐induced [3H]‐IP1 accumulation in U373 MG cells does appear to have a component dependent on the extracellular Ca2+ concentration. The degree of Ca2+‐dependence approaches that observed in guinea‐pig cerebral cortex but is much less than in rat cerebral cortex. Whether U373 MG cells will be of use as a model system for the apparent Ca2+‐entry component observed in guinea‐pig or rat brain slices remains to be established.