Protein kinase C activation modulates arachidonic acid metabolism in cultured alveolar epithelial cells.

Abstract

Cultured alveolar type II cells can liberate esterified arachidonic acid (AA) and metabolize it predominantly via the cyclooxygenase pathway, and their capacity to do so increases as they alter their phenotype over time in culture. Little is known, however, about the regulation of AA metabolism in alveolar pneumocytes. We have examined the effects of protein kinase C (PKC) activation on arachidonate metabolism in primary cultures of rat alveolar epithelial cells studied at 2 and 7 days following isolation. The potent PKC activator phorbol myristate acetate (PMA) stimulated dose-dependent increases in free AA levels in both day 2 and day 7 cultures, with optimal stimulation at 50 nM. Greater stimulation was demonstrated for day 7 cells, and this was associated with greater prostanoid synthesis in response to PMA by day 7 than by day 2 cells. The capacity of PMA to "prime" epithelial cells for augmented AA liberation and metabolism in response to calcium ionophore A23187 (5 microM) was examined also. Significant priming by PMA was observed in both day 2 and day 7 cells; once again, augmentation of both free AA levels as well as prostaglandin E2 levels was greater for day 7 cells than for day 2 cells. That the capacity of PMA to modulate AA metabolism was mediated by activation of PKC was confirmed by demonstrating that (1) phorbol didecanoate, which lacks the ability to activate PKC, failed to activate AA metabolism; (2) pretreatment for 18 h with 1 microM PMA, which depletes cellular PKC, abolished subsequent AA metabolism activated by 50 nM PMA; and (3) the PKC inhibitor staurosporine abrogated increases in the quantities of both free AA and prostaglandin E2 in response to PMA. We conclude that activation of PKC increases the availability of AA for prostanoid synthesis in alveolar pneumocytes, and that this effect is more evident as type II cell differentiation is modeled during prolonged cultivation.