Determination of the inositol (1,4,5) trisphosphate requirement for histamine- and substance P-induced Ca2+ mobilisation in human U373 MG astrocytoma cells.
In human U373 MG astrocytoma cells, histamine and substance P stimulated similar peak increases in intracellular free calcium concentrations ([Ca2+]i), as measured by single cell imaging of Fura-2 fluorescence. Best-fit EC50 values for the peak Ca2+ response were 1.86 microM for histamine and 0.93 nM for substance P. The histamine Ca2+ response was manifest as either a series of repetitive spikes, or, at higher concentrations, a peak followed by a lower plateau level of Ca2+. In contrast, the substance P response became more transient at higher agonist concentrations. Substance P (10 nM) stimulated a biphasic increase in levels of inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) with a peak of 97 +/- 5 pmoles/mg protein at 10 s. In contrast, the Ins(1,4,5)P3 response to 100 microM histamine was only marginally above basal levels of around 12 pmoles/mg protein. However, concentrations of histamine and substance P giving similar Ins(1,4,5)P3 responses produce similar peak increases in [Ca2+]i. HPLC analysis indicated that histamine stimulated the production of [3H]-Ins(1,4,5)P3 and its metabolites, although the magnitude of response was smaller than that observed with substance P. The initial Ca2+ responses to histamine and substance P did not require the presence of extracellular Ca2+. The Ca2+ response to histamine was unaffected by treatment with ryanodine, and was shifted to areas of lower agonist concentration by thimerosal. These results demonstrate that extremely small increases in Ins(1,4,5)P3 can stimulate large increases in [Ca2+]i in U373 MG cells, and suggest a marked redundancy for Ins(1,4,5)P3 production in the Ca2+ signalling pathway.
Young
K W
KW
Department of Cell Physiology and Pharmacology, University of Leicester, UK. KWY1@LE.AC.UK
Pinnock
R D
RD
Nahorski
S R
SR
D001254:Astrocytoma; D020013:Calcium Signaling / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D006632:Histamine / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D012433:Ryanodine / Q000494:pharmacology; D013373:Substance P / Q000378:metabolism*; D013696:Temperature; D013849:Thimerosal / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D012433:Ryanodine; D013849:Thimerosal; D013373:Substance P; D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate