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 intracellular 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 contributing to this effect to be characterized.
2. Histamine and substance P stimulated [³H]-inositol monophosphate ([³H]-IP₁) accumulation in U373 MG cells. Concentration-response curves of [³H]-IP₁ accumulation in suspensions of U373 MG cells in HEPES buffer containing 30 mM Li⁺ yielded best-fit EC₅₀ values of 19.1±1.5 μM for histamine and 5.7±1.3 nM for substance P.
3. In confluent monolayers of fura-2 loaded U373 MG cells perfusion with 100 μM histamine resulted in a transient 597±50 nM increase in [Ca²⁺]_i. The best-fit EC₅₀ for histamine was 4.6±2.2 μM. The initial, transient, histamine response was often followed by further small transient increases in [Ca²⁺]_i.
4. Treatment of U373 MG cells with 5 μM 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 μM, 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. The best-fit EC₅₀ for the histamine response was 0.8±0.2 μM. Substance P (10 nM, 100 s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca²⁺]_i.
5. Neither 100 μM histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn²⁺ in U373 MG cells pretreated with 5 μM thapsigargin, indicating that the depressant effect on steady-state raised [Ca²⁺]_i was probably not due to a block of Ca²⁺ entry.
6. The depressant effect of histamine on [Ca²⁺]_i was blocked by 1 μM mepyramine, and was partially reduced by pre-incubation with 1 μM staurosporine (61±7% reduction) and with Ro 31-8220 (24±10% and 50±6% reduction by 1 and 10 μM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant effect of histamine.
7. Neither 1 μM staurosporine nor 10 μM KN-62 inhibited the binding of [³H]-mepyramine to guinea-pig cerebellar membranes, whereas it was reduced by 17±1% and 55±2% by 1 and 10 μM Ro 31-8220, respectively. However, [³H]-IP₁ accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 μM Ro 31-8220 and in 2 out of 3 experiments there was a significant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 μM, similarly potentiated the response to 100 μM histamine in 3 out of 4 experiments. KN-62 (10 μM) did not stimulate histamine-induced [³H]-IP₁ accumulation.
8. In HEPES buffer to which no Ca²⁺ had been added, histamine stimulated a transient 451±107 nM increase in [Ca²⁺]_i. Pretreatment with 1 μM and 10 μM Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca²⁺]_i were returned to prestimulated levels. Pretreatment with KN-62 had no significant effect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca²⁺]_i. H-89 did not alter the histamine response.
9. The effect of histamine in stimulating Ca²⁺ extrusion was not confined to U373 MG cells, since 100 μM 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 mechanism which acts to reduce [Ca²⁺]_i. The balance of the evidence indicates that in U373 MG cells the latter effect most likely involves a PKC-mediated stimulation of a Ca²⁺-extrusion pump.