Cyclic AMP-dependent stimulation of basolateral K+ conductance in the rabbit conjunctival epithelium

The regulation of Na+ transport by cAMP in freshly isolated rabbit conjunct ival epithelium, a tissue exhibiting, both Cl- secretion and Na+ absorption , was examined. Bulbar-palpebral segments of conjunctiva were mounted betwe en Ussing-type hemichambers under short-circuit conditions in Cl- free medi a. In this situation, the short-circuit current (I-sc) measures an amilorid e-resistant Na+ absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 mu M) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na+-dependent I-sc by app roximate to 3.5-4.5 mu A cm(-2) (minimal 40% increase) and reduced transepi thelial resistance (R-t) by at least 7% (P < 0.05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 mu M), which in itself inhibi ted the I-sc by 0.5 mu A cm(-2), attenuated the I-sc responses of the cAMP- elevating agents (P < 0.05, unpaired data). In reverse, H-89 promptly decre ased the I-sc by 1.5-2.5 mu A cm(-2) and increased R-t by 5% (P < 0.05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX co mbination. Additions of epinephrine or rolipram to apically permeabilized p reparations using amphotericin B, increased the I-sc by 12 and 22% respecti vely over baseline and reduced R-t by 6% (P < 0.05). Similarly, in the pres ence of a transepithelial K+ gradient (apical to basolateral) and amphoteri cin B, cAMP elevation stimulated K diffusion across the preparation by at l east 1.8 mu A cm(-2) and decreased the R-t by 4% (P < 0.05), changes that w ere reversed by subsequent H-89 addition. Under Cl- rich conditions, pretre atment with 5 mM Ba2+ reduced the basal I-sc by 59% and blocked the cAMP-in duced I-sc stimulations typically seen in the presence of the anion. The re sults provide evidence for a PKA-regulated, Ba2+-inhibitable (voltage insen sitive) basolateral K+ conductance in rabbit conjunctival epithelial cells. The action of Cl- secretogogues acting via cAMP on basolateral K+ channel activity indicates that endogenous levels of cAMP may play a role in the re gulation of Cl- secretion and Na+ absorption in the conjunctiva. (C) 2000 A cademic Press.