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.
Ritter
M
M
Hospital for Internal Medicine, University of Innsbruck, Innsbruck/Austria.
Wöll
E
E
Haller
T
T
Dartsch
P C
PC
Zwierzina
H
H
Lang
F
F
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
D000199:Actins; D017923:Sodium-Hydrogen Exchangers; D015689:Oncogene Protein p21(ras); D001920:Bradykinin; D002118:Calcium