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) 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.
Mochizuki-Oda
N
N
Department of Cell Biology, Osaka Bioscience Institute, Suita, Japan.
Nakajima
Y
Y
Nakanishi
S
S
Ito
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001464:Barium / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002999:Clone Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000276:immunology / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism* / Q000494:pharmacology; D007295:Inositol Phosphates / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D012964:Sodium / Q000494:pharmacology; D013373:Substance P / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D000906:Antibodies; D015220:Calcium Channels; D018076:DNA, Complementary; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D018040:Receptors, Neurokinin-1; C081655:inositol-1,3,4,5-tetrakisphosphate; D001464:Barium; D013373:Substance P; D014638:Vanadates; D015544:Inositol 1,4,5-Trisphosphate; D006493:Heparin; D012964:Sodium; D013917:Thrombin; D019204:GTP-Binding Proteins; D002118:Calcium