PKCα and MAP kinase are independently and obligatorily required for P_2U-purinergic receptor-coupled regulation of arachidonic acid release in MDCK-D₁ cells

Although involvement of protein kinase C (PKC) and MAP kinase (MAPK) has been implicated in the regulation of the 85-kDa cytosolic phospholipase A₂ (cPLA₂) and arachidonic acid (AA) release in many cells, whether the two kinases function sequentially or independently remains undefined. We found that blockade of MAPK stimulation by the MAPK kinase inhibitor PD098059 inhibited AA release promoted by P_2u, agonists ATP and UTP in MDCK-D₁ cells. Down-regulation of PKC inhibited both AA release and MAPK activation. The data thus suggest sequential activation of PKC then MAPK in the regulation of AA release induced by P_2U-purinergic receptors. On the other hand, the selective PKCα inhibitor GF109203 X, at < 3 μM, inhibited P_2U-purinergic-mediated AA release, but not MAPK activation. Similar differential effects of this inhibitor (at <1 μM) were observed for PMA-mediated responses. Furthermore, partial blockade of expression of PKCα with antisense transfection inhibited PMA-promoted AA release without affecting MAPK stimulation. Thus, PKCα is required for AA release but not for MAPK activation. Together with our previous results that P_2U receptors promote AA release through cPLA₂ in MDCK-D₁ cells, the present study suggests that in these cells PKCα and MAPK, acting independently, are both obligatorily required for the regulation of cPLA₂ activation and AA release by P_2U-purinergic receptors.