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

In Chinese hamster ovary cells expressing the substance P (SP) recepto r 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 muM) 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 t ransient elevation of [Ca2+]i. The formation of inositol 1,4,5-trispho sphate (Ins(1,4,5)P3) was stimulated to 230% above the control by SP b ut 10% by thrombin 10 s after stimulation. In whole cell clamp recordi ngs, SP induced a long lasting inward current, whereas thrombin did no t evoke an inward current. G(qalpha) antibody applied intracellularly blocked the SP-induced current, but G(salpha) antibody did not block i t. Furthermore, decavanadate and heparin, inhibitors of Ins(1,4,5)P3 b inding to its receptor, suppressed the SP-induced current. In cell-att ached patch, bath-applied SP activated channel currents carried by Ba2 +, Ca2+, or Na+. In inside-out patches, Ins(1,4,5)P3, but neither inos itol 1,3,4-trisphosphate nor inositol 1,3,4,5-tetrakisphosphate, activ ated channel currents carried by Ba2+, Ca2+, or Na+. The channel activ ity induced by Ins(1,4,5)P3 was abolished by heparin. These results de monstrate that SP induces Ca2+ entry through activation of cation chan nels and suggest that Ins(1,4,5)P3 may regulate both SP-induced Ca2+ m obilization and Ca2+ entry in CHO-SPR cells.