Phorbol ester modulation of active ion transport across the rabbit conjunctival epithelium

Protein kinase C (PKC) activation elicits diverse cell-type specific effects on key epithelial transporters. The present work examined the influence of phorbol esters, which are known activators of PKC isoenzymes, on the short-circuit current (Isc), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the Iscmeasures a Na+-dependent, bumetanide-inhibitable Cl-transport in the basolateral-to-apical direction plus an amiloride-resistant Na+absorptive process in the opposite direction. Additions of phorbol 12-myristate-13-acetate (PMA) to the basolateral bathing media did not affect the transepithelial electrical parameters; but its introduction to the apical bath at 1 and 10 micrometers elicited a transient ( approximately 2 min duration) Iscspike followed by a sustained reduction relative to the control level. Such PMA-elicited Iscreductions were from 14. 0+/-2.0 to 3.1+/-0.8 microA cm-2(+/-s.e.m.'s, n =3) at 1 micrometer and from 16.5+/-1.9 to 4.6+/-0.7 microA cm-2(n =22) at 10 micrometers. The former concentration failed to produce extensive Iscreductions in 3 other experiments. Similar results were obtained with phorbol 12, 13-dibutyrate (PDBu). Its apical administration at 0.1 micrometer reduced the Iscfrom 18.5+/-4.1 to 7.8+/-2.0 (n =3), and from 16. 5+/-2.9 to 6.9+/-1.2 (n =7) when introduced at 1 micrometer. The phorbol-evoked Iscreductions occurred without a simultaneous change in transepithelial resistance (Rt). However, after about 15-20 min, Rtgradually declined by about 25%. In contrast to these results, treatment with a phorbol ester known not to activate PKC (4-alpha-PMA) did not affect the electrical parameters when added at 10 micrometers. PMA- and PDBu-evoked Iscreductions could be obtained with conjunctiva that were (1) pretreated with bumetanide, (2) bathed in Cl--free media, and (3) pretreated with amphotericin B, changes consistent with a likely inhibition of the basolateral Na+/K+pump. Such Iscinhibitions were attenuated with conjunctiva pre-exposed to 1 micrometer staurosporine, a nonselective kinase inhibitor known to suppress PKC activity. Staurosporine, in itself, produced a rapid 26% Iscinhibition (n =15) along with a 17% Rtincrease upon its apical introduction. These electrical responses were less extensive in Cl--free media and absent in amphotericin B-treated conjunctiva, suggesting the presence of a kinase-mediated regulation of apical channels for both Na+and Cl-. Overall, these results imply that in addition to previously demonstrated epinephrine-elicited, up-regulation of Cl-secretion, mechanisms may also exist, via PKC activation, to suppress Na+/K+pumping and consequently reduce transepithelial transport rates.