HISTAMINE H(1)-RECEPTOR-MEDIATED CALCIUM INFLUX IN DDT(1)MF-2 CELLS

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-mu-M. 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 H-1-antagonist mepyramine (10-mu-M) 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-mu-M) and PN-200-110 (10-mu-M) had no effect. These data suggest that histamine stimulates Ca2+ influx through an H-1-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 H-1-receptor-mediated influx, since store refilling can be demonstrated even when the receptor-mediated Ca2+ entry is blocked by mepyramine. In conclusion, H-1-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 H-1-receptor stimulation (mepyramine-insensitive) and occurs without an observable rise in cytosolic free Ca2+.