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) E 2 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) A 2 activity is Ca 2+-dependent, we investigated whether oxysterol potentiating effect on AA release was related to an effect of these compounds on cell Ca 2+ concentration. In this paper, we show that the intensity of potentiation by oxysterol varies with the external cell Ca 2+ concentration; when external Ca 2+ is chelated by EGTA, the oxysterol effect persists, though it is decreased. The Ca 2+ channel inhibitor nifedipine does not decrease the potentiating effect of 25-OH cholesterol, indicating that, if oxysterol favours Ca 2+ entry into the cell, the nifedipine inhibited channel is not involved. At the usual concentration (5 μm/ml), oxysterols are not able to increase, mimmediately or after a short time of contact (90 min) the concentration of intracellular free Ca 2+ ([Ca 2+]) i measured by fluorescence of Quinn-2; at very high concentration of oxysterol (25 μm/ml), [Ca 2+] i only slightly increases (+30%). The liberation of AA induced by cell activation with the Ca 2+ ionophore ionomycin is also potentiated by 25-OH cholesterol. All these observations are not in favour of a proper effect o oxysterols on cell Ca 2+ level.