PKC regulation of organic anion secretion in perfused S2 segments of rabbit proximal tubules

To examine the role of protein kinase C (PKC) in organic anion (OA) secreti on, we used epifluorescence microscopy to study steady-state transepithelia l secretion of 1 mu M fluorescein (FL) by isolated perfused S2 segments of rabbit renal proximal tubules. Addition of 100 nM phorbol 12-myristate 13-a cetate (PMA), a known PKC activator, to the bathing medium decreased steady -state secretion of FL by similar to 30% after 25 min. This inhibition was irreversible and, indeed, increased to similar to 40% at 25 min following r emoval of PMA [10 mu M 1,2-dioctanoyl-sn-glycerol (DOG) produced a comparab le inhibition]. The inhibition produced by PMA was blocked when 100 nM of e ither staurosporine (ST) or bisin-dolylmaleimide I (BIM), both known PKC in hibitors, was added to the bath for a 20-min preexposure followed by the ad dition of PMA. ST or BIM alone had no significant effect on FL secretion, s uggesting that the basal FL secretion rate was not under influence of PKC. Addition of 1 mu M of either the peptide hormone bradykinin (BK) or the alp ha(1)-receptor agonist phenylephrine (PE), both of which stimulate PKC via a ligand-receptor-PKC coupling reaction, to the bath also inhibited FL secr etion by similar to 22 and similar to 27%, respectively. However, the inhib ition was completely reversible after removal of BK or PE. Pretreatment of tubules with 100 nM BIM eliminated the inhibition of FL secretion produced by exposure to PE. We conclude that PKC negatively regulates the net secret ion of OAs in rabbit renal proximal tubules. The data indicate that BK or c atecholamines can play a physiological role in regulating OA secretion via PKC activation.