Mechanism of the activation of arachidonic acid release by oxysterols in NRK 49F cells: role of calcium.

We previously demonstrated that oxysterols added to the culture medium of NRK 49F cells labelled with [14C] arachidonic acid potentiated arachidonic acid (AA) release and prostaglandin (PG) E2 biosynthesis induced by the activation of these cells with fetal calf serum (FCS). In the absence of FCS, oxysterols had no effect on AA release. As phospholipase (Plase) A2 activity is Ca2(+)-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca2+ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca2+ concentration; when external Ca2+ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca2+ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca2+ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 micrograms/ml), oxysterols are not able to increase, immediately or after a short time of contact (90 min) the concentration of intracellular free Ca2+ ([Ca2+])i measured by fluorescence of Quin-2; at very high concentration of oxysterol (25 micrograms/ml), [Ca2+]i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca2+ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect of oxysterols on cell Ca2+ level.