Characterization of the substance P receptor-mediated calcium influx in cDNA transfected Chinese hamster ovary cells. A possible role of inositol 1,4,5-trisphosphate in calcium influx

Abstract

In Chinese hamster ovary cells expressing the substance P (SP) receptor clone (CHO-SPR cells), we examined SP-stimulated [Ca2+]i changes by microscopic fluorescence analysis and electrophysiological recordings. In fura-2-loaded cells, SP (1 microM) induced a prolonged elevation of [Ca2+]i, which comprised a rapid and transient Ca2+ mobilization and a prolonged phase of Ca2+ entry, but thrombin (1 unit/ml) induced only transient elevation of [Ca2+]i. The formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was stimulated to 230% above the control by SP but 10% by thrombin 10 s after stimulation. In whole cell clamp recordings, SP induced a long lasting inward current, whereas thrombin did not evoke an inward current. Gq alpha antibody applied intracellularly blocked the SP-induced current, but GS alpha antibody did not block it. Furthermore, decavanadate and heparin, inhibitors of Ins(1,4,5)P3 binding to its receptor, suppressed the SP-induced current. In cell-attached patch, bath-applied SP activated channel currents carried by Ba2+, Ca2+, or Na+. In inside-out patches, Ins(1,4,5)P3, but neither inositol 1,3,4-trisphosphate nor inositol 1,3,4,5-tetrakisphosphate, activated channel currents carried by Ba2+, Ca2+, or Na+. The channel activity induced by Ins(1,4,5)P3 was abolished by heparin. These results demonstrate that SP induces Ca2+ entry through activation of cation channels and suggest that Ins(1,4,5)P3 may regulate both SP-induced Ca2+ mobilization and Ca2+ entry in CHO-SPR cells.