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

Lawrence J. Alvarez, Oscar A. Candia, Helen C. Turner, Aldo C. Zamudio

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9 Scopus citations


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 (I(sc)), a direct measure of net transcellular electrolyte transport, of the rabbit conjunctiva. In this preparation, the I(sc) measures 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) 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 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 I(sc) reductions in 3 other experiments. Similar results were obtained with phorbol 12,13-dibutyrate (PDBu). Its apical administration at 0.1 μM 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 μM. The phorbol-evoked I(sc) reductions 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 activate PKC (4-α-PMA) did not affect the electrical parameters when added at 10 μM. PMA- and PDBu-evoked I(sc) reductions 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 I(sc) inhibitions 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% I(sc) inhibition (n = 15) along with a 17% R(t) increase 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.

Original languageEnglish
Pages (from-to)33-44
Number of pages12
JournalExperimental Eye Research
Issue number1
StatePublished - Jul 1999


  • Electrolyte transport
  • Protein kinase C
  • Rabbit conjunctiva
  • Short-circuit current
  • Staurosporine
  • Ussing chamber


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