DETERMINATION OF THE INOSITOL-(1,4,5) TRISPHOSPHATE REQUIREMENT FOR HISTAMINE-INDUCED AND SUBSTANCE-P-INDUCED CA2-CELLS( MOBILIZATION IN HUMAN U373 MG ASTROCYTOMA)

In human U373 MG astrocytoma cells, histamine and substance P stimulat ed similar peak increases in intracellular free calcium concentrations ([Ca2+](i)), as measured by single cell imaging of Fura-2 fluorescenc e. Best-fit EC50 values for the peak Ca2+ response were 1.86 mu M 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 conce ntrations, a peak followed by a lower plateau level of Ca2+. In contra st, the substance P response became more transient at higher agonist c oncentrations. Substance P (10 nM) stimulated a biphasic increase in l evels of inositol (1,4,5) trisphosphate (Ins(1,4,5)P-3) with a peak of 97 +/- 5 pmoles/mg protein at 10 s. In contrast, the Ins(1,4,5)P-3 re sponse to 100 mu M 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)P-3 responses produce similar pe ak increases in [Ca2+](i). HPLC analysis indicated that histamine stim ulated the production of [H-3]-Ins(1,4,5)P-3 and its metabolites, alth ough the magnitude of response was smaller than that observed with sub stance P. The initial Ca2+ responses to histamine and substance P did not require the presence of extracellular Ca2+. The Ca2+ response to h istamine was unaffected by treatment with ryanodine, and was shifted t o areas of lower agonist concentration by thimerosal. These results de monstrate that extremely small increases in Ins(1,4,5)P-3 can stimulat e large increases in [Ca2+](i) in U373 MG cells, and suggest a marked redundancy for Ins(1,4,5)P-3 production in the Ca2+ signalling pathway .