Molecular Pharmacological Studies on signal transduction cells reconstituted by gene transfer

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The transmembrane signaling mechanism were investigated using-Xenopus oocytes injected with rat brain mRNA and NIH 3T3 fibroblast cells transformed with v-ki-ras and v-src. The results obtained as followings:1) serotonin (5-HT)-evoked inward current under the voltage clamp condition holding at -60 mv was suppressed by mianserin, suggesting that the current is due to activation of 5-HT _<1c> receptors. Other antidepressant drugs (inipramine, desipramine, amoxapine, setiptilline) also inhibited the responses probably by blocking 5-HT _<1c> receptors. Outward transient current was observed by depolarization for 5 sec from -60 mv to 0 mv in mRNA-injected oocytes in Ca ^<2+>-containing medium.This reflects calmodulin-mediated opening of Cl channels in response to Ca^<2+> influx via Ca channels.The responses were greatly potentiated for more than 30 sin after brief stimulation of acetylcholine or 5-HT _<1c> recceptors. The long-lasting potentiation of Ca ^<2+>-dependent ion channel activity seems to due to Ca^<2+>-calmodulin but not to be protein kinase C.2) In NIH 3T3 cells transfected with v-src, isoproterenol-induced cAMP elevation was less than in normal NIH 3T3. Binding experiments using [^3 H]dhydroalprenolol showed the decrease in Bmax of the ng binding in transformants. In addition, phosphorylation of G_s by pp60 ^<v-src> which seems to cause uncoupling of receptors, effecters from G _s , is presumably involved in less sensitivity to isoproterenol in transformants than in normal cells as well.