Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Abstract

In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15 mM, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential difference Vsc from -54 to -18 mV (tear:reference) and the voltage divider ratio FRo decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10(-5) M amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC = 0), 1 X 10(-3) M diltiazem depolarized the Vsc from -78 to -9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1 X 10(-3) M) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Visualization of ouabain binding in bovine cornea

“…The results obtained in this study are in agreement with the reults found previously by measurements of electrophysiological properties of bovine cornea (Midelfart 1987), where a high sensitivity of the endothelium and only a marginal sensitivity of the epithelium to ouabain were detected. They also support the results achieved by a direct Na,K-ATPase activity measurement in bovine corneal epithelium (Langham & Kostelnik 1965;Huff & Reinach 1985) showing that only 10 to 20% of this enzyme is sensitive to ouabain, and are consistent with the conclusions achieved in many other transporting epithelia (Macknight & Leaf 1977), where existence of an ouabain-insensitive Na,K-ATPase has been postulated.…”

“…Several investigations indicate that the corneal epithelium also possesses NA,K-ATPase activity (Langham & Kostelnik 1965;Rogers 1968;Green 1970;Leuenberger & Novikoff 1974, Tervo & Palkama 1975Ruf & Ebel 1976;Huff & Reinach 1985) and is in fact capable of transporting ions and water, assisting the endothelium in the regulation of corneal transparency (Klyce & Crosson 1985;Beekhuis & McCarey 1986). However, as for the endothelium (Geroski et al 1984), a marked difference in ouabain sensitivity has been reported for the corneal epithelium from different species (Green 1970;Midelfart 1987).…”