DUAL EFFECTS OF HISTAMINE AND SUBSTANCE-P ON INTRACELLULAR CALCIUM LEVELS IN HUMAN U373 MG ASTROCYTOMA-CELLS - ROLE OF PROTEIN-KINASE-C

1 In human U373 MG astrocytoma cells agonist-induced increases in intr acellular Ca2+ ([Ca2+](i)) are rapidly returned towards prestimulated levels. Examination of the effect of histamine and substance P on [Ca2 +](i) in thapsigargin-treated cells has allowed a mechanism contributi ng to this effect to be characterized. 2 Histamine and substance P sti mulated [H-3]-inositol monophosphate ([H-3]-IP1) accumulation in U373 MG cells. Concentration-response curves of [H-3]-IP, accumulation in s uspensions of U373 MG cells in HEPES buffer containing 30 mM Li+ yield ed best-fit EC50 values of 19.1+/-1.5 mu M for histamine and 5.7+/-1.3 nM for substance P. 3 In confluent monolayers of fura-2 loaded U373 M G cells perfusion with 100 mu M histamine resulted in a transient 597/-50 nM increase in [Ca2+](i). The best-fit EC50 for histamine was 4.6 +/-2.2 mu M. The initial. transient, histamine response was often foll owed by further small transient increases in [Ca2+](i). 4 Treatment of U373 MG cells with 5 mu M thapsigargin, followed by the readdition of 1.8 mM Ca2+ to the perfusion buffer, resulted in a steady-state level of [Ca2+](i) 97+/-5 nM above pretreated levels (measured 400 s after readdition of Ca2+). Perfusion of histamine (100 mu M, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca2](i). This effect of histamine was normally reversible upon washout. T he best-fit EC50 for the histamine response was 0.8 +/- 0.2 mu M. Subs tance P (10 nM, 100 s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca2+](i). 5 Neither 100 mu M histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn2+ in U373 MG cells pretreated with 5 mu M thapsigargin, indicating that the depressant effect on steady-state raised [Ca2+](i) was proba bly not due to a block of Ca2+ entry. 6 The depressant effect of hista mine on [Ca2+](i) was blocked by 1 mu M mepyramine, and was partially reduced by pre-incubation with 1 mu M staurosporine (61+/-7% reduction ) and with Ro 31-8220 (24 +/- 10% and 50+/-6% reduction by 1 and 10 mu M Ro 31-8220, respectively). Pre-incubation with H-89 did not alter t he depressant effect of histamine. 7 Neither 1 mu M staurosporine nor 10 mu M KN-62 inhibited the binding of [H-3]-mepyramine to guineapig c erebellar membranes, whereas it was reduced by 17+/-1% and 55+/-2% by 1 and 10 mu M Ro 31-8220, respectively. However, [H-3]-IP1 accumulatio n stimulated by histamine in U373 MG cells was not inhibited by 1 or 1 0 mu M Ro 31-8220 and in 2 out of 3 experiments there was a significan t potentiation of the response to histamine with both concentrations o f Ro 31-8220. Staurosporine, 1 mu M, similarly potentiated the respons e to 100 mu M histamine in 3 out of 4 experiments. KN-62 (10 mu M) did not stimulate histamine-induced [H-3]-IP1 accumulation. 8 In HEPES bu ffer to which no Ca2+ had been added, histamine stimulated a transient 451 +/- 107 nM increase in [Ca2+](i). Pretreatment with 1 mu M and 10 mu M Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increase s in [Ca2+](i) were returned to prestimulated levels. Pretreatment wit h KN-62 had no significant effect on the response to histamine, but co nsistently inhibited the secondary slower phase of the decline in [Ca2 +](i). H-89 did not alter the histamine response. 9 The effect of hist amine in stimulating Ca2+ extrusion was not confined to U373 MG cells, since 100 mu M histamine also caused a rapid decrease in steady-state levels of [Ca2+](i) in thapsigargin-treated human HeLa cells. 10 The results indicate that agonists which increase [Ca2+](i) via activation of phosphoinositide metabolism can also stimulate a homeostatic mecha nism which acts to reduce [Ca2+](i). The balance of the evidence indic ates that in U373 MG cells the latter effect most likely involves a PK C-mediated stimulation of a Ca2+-extrusion pump.