Article date: February 1998
By: K W. Young, R D. Pinnock, W J. Gibson, J M. Young, in Volume 123, Issue 3, pages 545-557
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
British Journal of Pharmacology (1998) 123, 545–557; doi:10.1038/sj.bjp.0701620
DOI: 10.1038/sj.bjp.0701620
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