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Chloride ion (Cl-) secretion by the rabbit corneal epithelium involves a catecholamine-stimulated Cl- conductance at the apical membrane, but the characteristics of the ion channel(s) responsible are unknown. A primary cell culture system was developed that induces stratified epithelial differentiation of rat and rabbit corneal epithelial cells, as detected by differential interference contrast light and transmission electron microscopy. In patch clamp studies, gigaOhm seal, were obtained easily during the first 1-4 days in vitro, and patches contained a voltage-dependent outwardly rectifying channel. Single-channel conductance at 24 degrees C was about 10, 29, and 72 pS in symmetric 150 mM NaCl at membrane potentials of -60, 0, and +60 mV, respectively. The current-voltage relationship measured with 75 mM NaCl and sucrose in the bath indicated anion selectivity. Increasing the temperature to 37 degrees C and replacing HEPES buffer with tricine, increased channel conductance and decreased rectification. Characteristics of this channel and a low conductance Cl- channel are compared with those of anion channels of other vertebrate epithelia.

Brook trout (Salvelinus fontinalis) urinary bladder in vitro had a low serosa-positive transepithelial potential (6.7 +/- 1.2 mV), low transmural conductance (0.23 +/- 0.03 mS. cm-2), and net absorptive transport of Na+ and Cl-. The net flux of Na+ was equivalent to that of Cl- and much larger than the membrane current, indicating neutral NaCl uptake. Na+ uptake was not coupled to that of Cl-, since absorptive Na+ transport continued in (Cl-)-free media. In the absence of Cl-, the net absorption of Na+ was accompanied by net secretion of titratable acidity, suggestive of Na+-H+ exchange on the luminal surface. Active Cl- transport persisted in dilute (2.0 mM) tetraethylammonium chloride with zero K+ and zero Na+, indicating full independence of the Cl- transport from cation coupling and suggesting the presence of Cl(-)-HCO-3 exchange. The kinetics of Cl- uptake (K1/2 = 35-37 mM, maximal transport rate = 3.0-3.4 mu eq . cm-2 . h-1) were not significantly affected by removal of mucosal Na+ . Cl- uptake was inhibited partially by 10(-4) M amiloride but not by 10(-4) M bumetanide. The results strongly support a model for active NaCl transport involving paired ion exchangers, likely located at the luminal membrane.

1. The effects of thiocyanate, ouabain, ion-substituted Ringer solution and electrochemical gradients on Na+ and Cl- transport were examined using the isolated skin of the marine teleost, Gillichthys mirabilis. 2. Bilateral replacement of Na+ with choline in the bathing solutions reduces net Cl- flux by 93%, indicating that active Cl- transport by the skin is Na-dependent. 3. Thiocyanate inhibits short-circuit current with an ED50 of 6.4 x 10(-4)M, and, at 10(-2)M, decreases Cl-efflux, influx, net flux and short-circuit current by 68, 33, 74 and 81%, respectively. 4. Ouabain (10(-5)M) reduces Cl- efflux and net flux by 56 and 86%, respectively, indicating that the Cl- transport requires Na,K-ATPase. 5. Subsequent addition of thiocyanate to ouabain-treated skin reduces Cl- efflux, net flux and short-circuit current, suggesting that the two agents operate at different sites involved in Cl- transport. 6. Unilateral substitution of gluconate for Cl- on the serosal side does not affect Cl- influx, indicating that Cl- passive transport is via Fickean diffusion, not Cl-Cl exchange diffusion. 7. The addition of NaCl to the mucosal side, which mimics the in vivo sea-water condition, increases Cl- influx and transepithelial potential and decreases tissue resistance. The net flux (secretion) of Cl- with hypertonic saline on the mucosal side (0.51 +/- 0.06 muequiv/cm2 . hr) demonstrates that the skin could secrete Cl- in vivo. 8. Na+ fluxes across the skin are passive, as the observed flux ration (efflux/influx) is similar to that predicted by the Ussing-Teorell equation under both closed- and open-circuit conditions. 9. The permeability ratio (PNa:PCl) in approximately 5.4:1.0, indicating that the skin is more permeable to Na+, and that at least part of the serosa-positive transepithelial potential may be a Na+ diffusion potential. 10. The results suggest that Cl- secretion by Gillichthys skin is secondary active transport involving Na,K-ATPase and serosal Na+.

The fluxes of 86Rb were measured across the isolated skin of the marine teleost Gillichthys. The efflux of 86Rb in Cl Ringer with 1.0 mM RbCl exceeded the influx by 8.0 +/- 1.2 neq . cm-2 . h-1 at closed circuit, indicating active transport of Rb+ in the secretory direction. However, the net Rb+ flux was not significantly different from zero in (Cl-free) gluconate Ringer, indicating that the active Rb+ transport was Cl dependent. The epithelium behaved as a single passive resistor to Rb+ in the absence of Cl-, and Rb+ movement under these conditions appeared to be by Fickean diffusion. Epinephrine (10(-5) M) inhibited partially the active Rb+ transport in Cl Ringer. These results suggest that there is active K+ transport across Gillichthys skin.

Evidence is presented that serotonin acts as a neurotransmitter in the cornea of the adult rabbit. Serotonin was localized to granules in a sparse population of subepithelial corneal nerves by an electron microscopic histochemical procedure. Significant endogenous levels of serotonin and its principal metabolite, 5-hydroxyindoleacetic acid, were detected in the central cornea by a fluorometric assay. Exogenous serotonin stimulated ion transport by corneal epithelium. This effect was potentiated by monoamine oxidase inhibition and was unaffected by an alpha-adrenergic receptor antagonist. Serotonin-stimulated ion transport was inhibited by the specific antagonist, methysergide, and by the replacement of Cl- with an impermeable anion. In tracer experiments, the serotonin-stimulated ion transport was shown to be caused by increased epithelial Cl- secretion. The serotonin response was partially inhibited by the beta-adrenergic antagonist, timolol. In a companion article, assay of corneal cyclic AMP showed stimulation of cyclic AMP synthesis by serotonin, inhibition by the specific antagonist, lysergic acid diethylamide, and potentiation by monoamine oxidase inhibition. We postulate that specific serotonergic receptors are present in the corneal epithelium and that activation of these receptors by serotonin released from serotonergic neurons increases the level of cyclic AMP, which stimulates active Cl- secretion by the corneal epithelium.