ACTIVATION OF CA2-RAS( INFLUX BY TRANSFORMING HA)

The effect of an induction of transforming Ha-ras on Ca2+ influx into NIH3T3 cells was studied employing Fura-2 quenching by Mn2+. The expre ssion of transforming p21(Ha-ras) caused a significant increase in Mn2 + influx which was blocked by Cd2+, La3+, niguldipine and the Ca2+-cha nnel blocker SK&F96365, This effect was specific for transforming Ha-r as and was not seen after overexpression of the Ha-ras proto-oncogene or v-mos. In addition to the enhanced Mn2+ influx, transforming p21 (H a-ras) elicited an increased efflux of the K+-congener Rb-86(+) which was inhibitable by Ca2+-channel blockers and charybdotoxin, a selectiv e inhibitor of high and intermediate conductance Ca2+-dependent K+ cha nnels. Charybdotoxin did not reduce the increase in Mn2+ influx by ras , demonstrating that the activation of Ca2+-dependent K+ channels was not required for the sustained Mn2+/Ca2+ influx in the presence of tra nsforming Ha-ras. In ras-expressing cells, the bradykinin-induced Mn2 influx charybdotoxin sensitive Rb-86(+) efflux were markedly potentia ted. The increase in the inositol-1,4,5-trisphosphate and inositol-1,3 ,4,5-tetrakisphosphate levels by ras is not sufficient to explain the elevated Mn2+ influx. The mitogenic response to an expression of trans forming Ha-ras was inhibited by the Ca2+-channel blockers not, however , by charybdotoxin. These data suggest the existence of an agonist-ind ependent activation of a receptor- or second messenger-operated Ca2+ c hannel by transforming Ha-ras which is necessary for the mitogenic res ponse to the activation of the oncogene.