Dual effects of histamine and substance P on intracellular calcium levels in human U373 MG astrocytoma cells: role of protein kinase C

Abstract

1 In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca 2+ ([Ca 2+ ] i ) are rapidly returned towards prestimulated levels. Examination of the e ect of histamine and substance P on [Ca 2+ ] i in thapsigargin-treated cells has allowed a mechanism contributing to this e ect to be characterized. 4 Treatment of U373 MG cells with 5 mM thapsigargin, followed by the readdition of 1.8 mM Ca 2+ to the perfusion bu er, resulted in a steady-state level of [Ca 2+ ] i 97+5 nM above pretreated levels (measured 400 s after readdition of Ca 2+ ). Perfusion of histamine (100 mM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca 2+ ] i . This e ect of histamine was normally reversible upon washout. The best-®t EC 50 for the histamine response was 0.8+0.2 mM. Substance P (10 nM, 100 s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca 2+ ] i . 5 Neither 100 mM histamine nor 10 nM substance P inhibited the rate of quench of fura-2¯uorescence by Mn 2+ in U373 MG cells pretreated with 5 mM thapsigargin, indicating that the depressant e ect on steady-state raised [Ca 2+ ] i was probably not due to a block of Ca 2+ entry. 6 The depressant e ect of histamine on [Ca 2+ ] i was blocked by 1 mM mepyramine, and was partially reduced by pre-incubation with 1 mM staurosporine (61+7% reduction) and with Ro 31-8220 (24+10% and 50+6% reduction by 1 and 10 mM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant e ect of histamine. 7 Neither 1 mM staurosporine nor 10 mM KN-62 inhibited the binding of [ 3 H]-mepyramine to guineapig cerebellar membranes, whereas it was reduced by 17+1% and 55+2% by 1 and 10 mM Ro 31-8220, respectively. However, [ 3 H]-IP 1 accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 mM Ro 31-8220 and in 2 out of 3 experiments there was a signi®cant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 mM, similarly potentiated the response to 100 mM histamine in 3 out of 4 experiments. KN-62 (10 mM) did not stimulate histamine-induced [ 3 H]-IP 1 accumulation. 8 In HEPES bu er to which no Ca 2+ had been added, histamine stimulated a transient 451+107 nM increase in [Ca 2+ ] i . Pretreatment with 1 mM and 10 mM Ro 31-8220 did not signi®cantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca 2+ ] i were returned to prestimulated levels. Pretreatment with KN-62 had no signi®cant e ect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca 2+ ] i . H-89 did not alter the histamine response. 9 The e ect of histamine in stimulating Ca 2+ extrusion was not con®ned to U373 MG cells, since 100 mM histamine also caused a rapid decrease in steady-state levels of [Ca 2+ ] i in thapsigargin-treated human HeLa cells. 10 The results indicate that agonists which increase [Ca 2+ ] i via activation of phosphoinositide metabolism can also...

Characteristics of the Ca2+‐dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells

“…Histamine (10 mM) acting alone had no eect on cyclic AMP accumulation, consistent with our earlier observations on dissociated U373 MG cells (Young et al, 1998a), but produced a marked inhibition of the response to isoprenaline (Figure 2). The inhibitory eect of histamine was completely dependent on the presence of Ca 2+ in the extracellular medium.…”

“…U373 MG astrocytoma cells were cultured and dissociated as described previously (Young et al, 1998a). The cells were seeded onto 6-well culture plates at a density of approximately 7610 4 cells well 71 and grown to near con¯uence.…”

“…Measurements of the intracellular Ca 2+ concentration ( [Ca 2+ ] i ) in U373 MG cells grown on coverslips and loaded with fura-2 were made as described previously (Young et al, 1998a) using a Hitachi F-2000¯uorimeter. The coverslips were superfused with a modi®ed HEPES medium at 378C with or without added Ca 2+ and with or without La…”

“…Statistical comparison of parameters characterizing two concentration response curves was made by ®tting the curves simultaneously and assessing the increase in the residual sum of squares when parameters were constrained to be the same for both curves, as described previously (Young et al, 1998a). Where multiple comparisons of means were made within the same experiment the signi®cance of dierences between conditions was made using the Student ± Newman ± Keuls multiple range test, following one-way analysis of variance.…”

“…3 H]-Inositol phosphates were separated as described previously (Young et al, 1998a). Statistical comparison of parameters characterizing two concentration response curves was made by ®tting the curves simultaneously and assessing the increase in the residual sum of squares when parameters were constrained to be the same for both curves, as described previously (Young et al, 1998a).…”