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 microM) 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 microM. 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 microM, 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 microM). 7. In cross-chopped slices of guinea-pig cerebral cortex and guinea-pig cerebellum, [3H]-IPI 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 w-conotoxin (3 microM) had no significant effect on histamine-induced [3H]-IP1accumulation 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.
Arias-Montaño
J A
JA
Department of Pharmacology, University of Cambridge.
Berger
V
V
Young
J M
JM
D000818:Animals; D001254:Astrocytoma / Q000378:metabolism*; D001923:Brain Chemistry / Q000187:drug effects*; D001932:Brain Neoplasms / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism; D006168:Guinea Pigs; D006367:HeLa Cells; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000096:biosynthesis*; D008094:Lithium / Q000494:pharmacology; D008297:Male; D009553:Nimodipine / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011738:Pyrilamine / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D013045:Species Specificity; D014407:Tumor Cells, Cultured; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D007295:Inositol Phosphates; D010455:Peptides; D009553:Nimodipine; D006632:Histamine; D002217:Carbachol; D020866:omega-Conotoxin GVIA; D008094:Lithium; D011738:Pyrilamine; D002118:Calcium