In the medullary thick ascending limb (MTAL) of the rat kidney, prostaglandin E-2 (PGE(2)) reverses inhibition of HCO3- absorption by arginine vasopressin (AVP). This effect of PGE(2) is blocked by chelerythrine or staurosporine and mimicked by phorbol ester, suggesting a critical role for protein kinase C (PKC). The present study was designed to examine directly regulation of PKC isoforms by PGE(2) in the inner stripe of the outer medulla and in microdissected MTALs. Immunoblots with isoform-specific anti-PKC antibodies detected alpha-, beta II-, delta-, epsilon-, and zeta-isoforms in both inner stripe and MTAL. The beta I- and gamma-isoforms were not detected. Translocation and activation of PKC were assessed by immunoblot analysis and by direct measurement of enzyme activity using an immune complex kinase assay. In inner stripe tissue incubated with 10(-10) M AVP, PGE(2) (10(-6) M for 20 min) induced translocation of PKC-delta from the cytosolic fraction to the membrane fraction. This translocation was associated with an 85% increase in PKC-delta activity in the membrane fraction and a 70% decrease in PKC-delta activity in the cytosolic fraction. PGE(2) had no effect on the subcellular distribution or the activities of the other isoforms. Activation of PKC-delta was confirmed directly in microdissected MTALs, in which PGE(2) caused a near complete loss of PKC-delta from the cytosolic fraction. PGE(2) did not induce translocation of PKC-delta in the absence of AVP. These results demonstrate that 1) the MTAL expresses Ca2+- dependent (alpha, beta II) and Ca2+-independent (delta, epsilon, zeta) PKC isoforms; 2) PGE(2) causes selective activation of PKC-delta, which likely mediates the action of PGE(2) to reverse AVP inhibition of HCO3- absorption; and 3) PGE(2) activation of PKC-delta requires the presence of AVP, which may explain the fact that PGE(2) influences HCO3- transport only when AVP is present.
