Abstract
In human U373 MG astrocytoma cells, histamine and substance P stimulated similar peak increases in intracellular free calcium concentrations ([Ca 2+] i), as measured by single cell imaging of Fura-2 fluorescence. Best-fit EC 50 values for the peak Ca 2+ response were 1.86 μM for histamine and 0.93 nM for substance P. The histamine Ca 2+ response was manifest as either a series of repetitive spikes, or, at higher concentrations, a peak followed by a lower plateau level of Ca 2+. 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)P 3) with a peak of 97 ± 5 pmoles/mg protein at 10 s. In contrast, the lns(1,4,5) P3 response to 100 μM 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 peak increases in [Ca 2+] i. HPLC analysis indicated that histamine stimulated the production of [ 3H]-Ins(1,4,5)P 3 and its metabolites, although the magnitude of response was smaller than that observed with substance P. The initial Ca 2+ responses to histamine and substance P did not require the presence of extracellular Ca 2+. The Ca 2+ 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)P 3 can stimulate large increases in [Ca 2+] i in U373 MG cells, and suggest a marked redundancy for Ins(1,4,5)P 3 production in the Ca 2+ signalling pathway.
Published Version
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