THE ROLE OF CALCIUM IN CELL SHRINKAGE AND INTRACELLULAR ALKALINIZATION BY BRADYKININ IN HA-RAS ONCOGENE EXPRESSING CELLS

In ras oncogene expressing cells, bradykinin leads to intracellular alkalinization by activation of the Na+/H+ exchanger. This effect is paralleled by oscillatory increase of intracellular calcium activity and cell shrinkage. Staurosporine (1 mumol/l) is not sufficient to prevent bradykinin induced intracellular alkalinization, thus pointing to a protein kinase C independent pathway for the activation of Na+/H+ exchange. The present study has been performed to elucidate, whether the increase of intracellular calcium contributes to cell shrinkage and activation of the Na+/H+ exchanger. To this end, the effects of the calcium ionophore ionomycin have been tested. Ionomycin leads to a dose dependent increase of intracellular calcium activity. At 100 nmol/l ionomycin intracellular calcium is increased from 114 +/- 17 nmol/l to 342 +/- 24 nmol/l (n = 9), a value within the range of intracellular calcium concentrations following application of bradykinin. The calcium increase is paralleled by a decrease of cell volume by 12 +/- 2% (n = 5) and an increase of intracellular pH from 6.78 +/- 0.02 to 6.90 +/- 0.03 (n = 11), values similar to those following application of bradykinin. The alkalinizing effect of ionomycin is completely abolished in the presence of the novel Na+/H+ exchange inhibitor HOE 694 (10 mumol/l), but is not inhibited by 1 mumol/l staurosporine. Inhibition of K+ and Cl- channels by barium (5 mmol/l) and ochratoxin-A (5 mumol/l) prevents both ionomycin induced cell shrinkage and protein kinase C independent intracellular alkalinization. It is concluded that bradykinin leads to intracellular alkalinization mainly by increasing intracellular calcium concentration. Calcium triggers calcium sensitive K+ channels, and presumably Cl-channels, the subsequent loss of cellular KCl leads to cell shrinkage which, in turn, activates Na+/H+ exchange.