Histamine H1-receptor-mediated calcium influx in DDT1MF-2 cells

Abstract

Undifferentiated monolayers of the hamster vas deferens smooth-muscle cell line, DDT1MF-2, were grown on glass coverslips and loaded with the Ca2+-sensitive fluorescent dye fura-2. Stimulation with histamine produced a rapid and maintained increase in intracellular free Ca2+ ([Ca2+](i)), with an EC50 of 7.0 ± 0.7 μM. The initial rise in [Ca2+](i) can be attributed to Ca2+ release from intracellular stores, whereas the maintained or plateau phase is due to influx of extracellular Ca2+. The Ca2+ influx associated with the plateau phase required the continued presence of histamine on the receptor, since the H1-antagonist mepyramine (10 μM) attenuated the rise in [Ca2+](i) observed when extracellular Ca2+ was re-applied after the cells had been stimulated with histamine, in experiments performed in nominally Ca2+-free buffer. Pretreatment with the inorganic Ca2+-channel blockers Ni2+ (1 mM) and Co2+ (1 mM) inhibited the influx component, whereas the organic voltage-operated Ca2+-channel antagonists nifedipine (10 μM) and PN-200-110 (10 μM) had no effect. These data suggest that histamine stimulates Ca2+ influx through an H1-receptor-activated Ca2+ channel. Experiments with Mn2+ indicated that the receptor-mediated Ca2+-influx pathway(s) is impermeable to Mn2+. Furthermore, the refilling of Ca2+ stores can occur independently of H1-receptor-mediated influx, since store refilling can be demonstrated even when the receptor-mediated Ca2+ entry is blocked by mepyramine. In conclusion, H1-receptor activation in the smooth-muscle cell line DDT1MF-2 stimulates both release of Ca2+ from intracellular stores [inositol 1,4,5-trisphosphate (InsP3)mediated] and Ca2+ influx through a receptor-activated Ca2+ channel. The subsequent refilling of the InsP3-sensitive intracellular Ca2+ store is independent of histamine H1-receptor stimulation (mepyramine-insensitive) and occurs without an observable rise in cytosolic free Ca2+.