Phorbol Ester Modulation of Active Ion Transport across the Rabbit Conjunctival Epithelium

• Published in: Experimental eye research Vol. 69; no. 1; pp. 33 - 44
• Main Authors: ALVAREZ, LAWRENCE J., CANDIA, OSCAR A., TURNER, HELEN C., ZAMUDIO, ALDO C.
• Format: Journal Article
• Language: English
• Published: London Elsevier Ltd 01.07.1999; Elsevier
• Subjects: Animals; Biological and medical sciences; Biological Transport, Active - drug effects; Bumetanide - pharmacology; Chlorine - metabolism; Conjunctiva - drug effects; Conjunctiva - metabolism; Culture Techniques; electrolyte transport; Enzyme Inhibitors - pharmacology; Eye and associated structures. Visual pathways and centers. Vision; Female; Fundamental and applied biological sciences. Psychology; Ion Transport - drug effects; Ion Transport - physiology; Male; Ouabain - pharmacology; Phorbol 12,13-Dibutyrate - pharmacology; Phorbol Esters - pharmacology; protein kinase C; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - physiology; rabbit conjunctiva; Rabbits - metabolism; short-circuit current; staurosporine; Staurosporine - pharmacology; Tetradecanoylphorbol Acetate - pharmacology; Ussing chamber; Vertebrates: nervous system and sense organs; Ussing chamber; staurosporine; rabbit conjunctiva; short-circuit current; protein kinase C; electrolyte transport; Eye; Visual system; Vertebrata; Protein kinase C; Mammalia; Enzyme; Transferases; Rabbit; Ion transport; Lagomorpha; Conjunctiva; Epithelium
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1006/exer.1999.0676

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 μm elicited a transient (≈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 μA cm-2(±s.e.m.'s, n =3) at 1 μm and from 16.5±1.9 to 4.6±0.7 μA cm-2(n =22) at 10 μm . 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 μm 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 μm . 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-α-PMA) did not affect the electrical parameters when added at 10 μm . 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 μm 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.