Activation of Na+/H(+)-exchanger by transforming Ha-ras requires stimulated cellular calcium influx and is associated with rearrangement of the actin cytoskeleton.
Expression of the Ha-ras oncogene in NIH 3T3 fibroblasts (+ ras cells) results in growth factor-independent proliferation and marked alteration of cytoskeletal architecture including breakdown of actin stress fiber network. Compared to identical cells not expressing the oncogene (-ras cells), + ras cells exhibit a more alkaline intracellular pH (pHi) and a larger cell volume (CV), both of which are important mitogenic elements. They are due to a set point shift for activation of the Na+/H(+)-exchanger. Moreover + ras cells respond to stimuli like 0.5% fetal calf serum or bradykinin with sustained oscillation of the cell membrane potential (PD) due to stimulated Ca2+ entry which triggers pulsatile release of calcium from internal stores and subsequent activation of calcium-sensitive K+ channels. 10 mumol/l bepridil inhibit oscillations of PD and protect + ras cells against actin stress fiber depolymerization. It is shown that bepridil blocks both cellular calcium entry as measured by Mn2+ quenching of fura-2 fluorescence and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene. Inhibition of the Na+/H(+)-exchange with 10 mumol/l HOE 694, on the other hand, does not significantly alter Ha-ras stimulated calcium entry or cytoskeletal rearrangement. In -ras cells ionomycin (0.1 mumol/l) leads to a transient increase in Cai. This effect is paralleled by a transient depolymerization of actin stress fiber network which cannot be inhibited by HOE 694. Disruption of the actin cytoskeleton in -ras cells by cytochalasin D does not alter steady state cell volume or Na+/ H(+)-exchange activity. However, stimulation of cytochalasin-treated -ras cells with bradykinin leads to cell swelling which can be blunted by HOE 694. The results show that both cytoskeletal rearrangement and activation of the Na+/H(+)-exchanger following expression of the Ha-ras oncogene require stimulated calcium influx and Cai oscillations. The depolymerization of the actin cytoskeleton is permissive for the Na+/ H(+)-exchanger to cause cell swelling upon stimulation with bradykinin.[1]References
- Activation of Na+/H(+)-exchanger by transforming Ha-ras requires stimulated cellular calcium influx and is associated with rearrangement of the actin cytoskeleton. Ritter, M., Wöll, E., Haller, T., Dartsch, P.C., Zwierzina, H., Lang, F. Eur. J. Cell Biol. (1997) [Pubmed]
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