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

Protein kinase C (PKC) activation elicits diverse cell-type specific effect s on key epithelial transporters. The present work examined the influence o f phorbol esters, which are known activators of PKC isoenzymes, on the shor t-circuit current (I-sc), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the I-sc measur es 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 electri cal parameters; but its introduction to the apical bath at 1 and 10 mu M el icited a transient (approximate to 2 min duration) I-sc spike followed by a sustained reduction relative to the control level. Such PMA-elicited I-sc reductions were from 14.0 +/- 2.0 to 3.1 +/- 0.8 mu A cm(-2) (+/- S.E.M.'S, n = 3) at 1 mu M and from 16.5 +/- 1.9 to 4.6 +/- 0.7 mu A cm(-2) (n = 22) at 10 mu M. The former concentration failed to produce extensive I,, reduc tions in 3 other experiments. Similar results were obtained with phorbol 12 ,13-dibutyrate (PDBu), Its apical administration at 0.1 mu M reduced the I- sc from 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 mu M. The phorbol-eooked I-sc reduction s occurred without a simultaneous change in transepithelial resistance (R-t ). However, after about 15-20 min, R-t gradually declined by about 25%. In contrast to these results, treatment with a phorbol ester known not to acti vate PKC (4-alpha-PMA) did not affect the electrical parameters when added at 10 mu M. PMA- and PDBu-evoked I-sc. reductions could be obtained with co njunctiva that were (1) pretreated with bumetanide, (2) bathed in Cl--free media, and (3) pretreated with amphotericin B, changes consistent with a li kely inhibition of the basolateral Na+/K+ pump. Such I-sc inhibitions were attenuated with conjunctiva pre-exposed to 1 mu M staurosporine, a nonselec tive kinase inhibitor known to suppress PKC activity. Staurosporine, in its elf, produced a rapid 26% I-sc inhibition (n = 15) along with a 17% R-t inc rease upon its apical introduction. These electrical responses were less ex tensive 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 pre viously demonstrated epinephrine-elicited, up-regulation of Cl- secretion, mechanisms may also exist, via PKC activation, to suppress Nat/K+ pumping a nd consequently reduce transepithelial transport rates. (C) 1999 Academic P ress.