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Expression of a transforming Ha-ras by dexamethasone in NIH3T3 cells transfected with a glucocorticoid-inducible Ha-ras construct results in a rapid desensitization of the intracellular Ca(2+)-mobilizing system to bombesin. This effect precedes the down-modulation of inositol trisphosphate (IP3) formation by several hours and is, therefore, not explained by an uncoupling of phosphoinositidase C. It is demonstrated that expression of Ha-ras attenuates the Ca(2+)-release by IP3 in permeabilized cells. The IP3 concentration required for half-maximal Ca(2+)-release is doubled in Ha-ras expressing cells. Maximal Ca(2+)-release which is obtained with 2 microM IP3 in control cells requires 10 microM IP3 in cells expressing Ha-ras. The desensitization of the IP3 receptors coincides with the desensitization of the Ca(2+)-mobilizing system to bombesin. The results indicate that the Ha-ras mediated desensitization of the Ca(2+)-releasing system to bombesin is--at least in part--caused by a decrease in the affinity of the IP3 receptor to inositol trisphosphate.

Hexadecylphosphocholine (HePC) inhibits protein kinase C (PKC) from NIH3T3 cells in cell-free extracts with a 50% inhibitory concentration of about 7 microM. Inhibition is competitive with regard to phosphatidylserine with a Ki of 0.59 microM. In order to determine whether HePC affects PKC in intact cells, the bombesin or tetradecanoylphorbolacetate-induced, PKC-mediated activation of the Na+/H(+)-antiporter was determined. It is demonstrated that HePC causes a drastic inhibition of this enzyme indicating a similar sensitivity of PKC to HePC in intact cells compared to cell-free extracts. In addition to the effects on PKC, treatment of NIH3T3 cells with HePC depresses the bombesin-induced formation of inositol 1,4,5-trisphosphate and the concomitant mobilization of intracellular Ca2+. Dose-response curves for the inhibition of inositol 1,4,5-trisphosphate formation and Ca2+ mobilization reveal 50% inhibitory concentrations of 2 or 5 microM, respectively. Polyphosphorylated phosphoinositides accumulate in HePC-treated cells indicating that the depression of inositol 1,4,5-trisphosphate generation is not caused by an inhibition of phosphoinositide kinases. Addition of bombesin to HePC-treated cells in the presence of LiCl revealed no evidence for an accelerated rate of inositol 1,4,5-trisphosphate turnover by the phospholipid analogue. It is concluded that HePC inhibits phosphoinositidase C in intact cells. The data strongly suggest that the growth-inhibitory effect of HePC is at least in part explained by the interference with mitogenic signal transduction.

Nitrogen mustard (N-mustard) inhibits the ouabain-sensitive and the furosemide-sensitive Rb uptake of Ehrlich ascites tumor cells, whereas the transport, which is resistant to both inhibitors, is not affected by the alkylating agent. At N-mustard concentrations below 10 microM, the reduction in Rb uptake is predominantly due to an interference with the furosemide-sensitive system. The dose response curve for the inhibition by N-mustard of the furosemide-sensitive Rb uptake closely parallels the dose response curve for the anti-tumor activity of the alkylating drug. This is in contrast to the behaviour of the ouabain-sensitive Rb transport. The inhibition of the furosemide-sensitive Rb uptake is expressed much less in cells which are resistant to N-mustard. The recovery of the furosemide-sensitive transport system after a single exposure to N-mustard is relatively slow and characterized by an initial 4 h lag period, whereas the repair of DNA-interstrand cross-links starts immediately after removal of the drug. At mM concentrations furosemide blocks the multiplication of Ehrlich ascites tumor cells. However, lower concentrations of furosemide which cause a 50% reduction in the furosemide-sensitive Rb uptake do not interfere with cell proliferation. This is in contrast to the behaviour of N-mustard which exerts a clear-cut depression of cell growth at concentrations leading to a 50% inhibition of the furosemide-sensitive Rb transport. It is concluded, therefore, that the inhibition of the furosemide-sensitive system alone is not sufficient to explain the anti-tumor activity of the alkylating agent. The effect is discussed as part of a more extended N-mustard-induced membrane alteration which may be important for the growth inhibitory effect of the alkylating agent.