Abstract
The regulation of Na+transport by cAMP in freshly isolated rabbit conjunctival epithelium, a tissue exhibiting both Cl−secretion and Na+absorption, was examined. Bulbar-palpebral segments of conjunctiva were mounted between Ussing-type hemichambers under short-circuit conditions in Cl−free media. In this situation, the short-circuit current (Isc) measures an amiloride-resistant Na+absorptive process in the apical-to-basolateral direction. Apical additions (each at 10 μm) of cAMP-elevating compounds, forskolin, rolipram, IBMX and epinephrine all stimulated the Na+-dependent Iscby ≈3.5–4.5 μA cm−2(minimal 40% increase) and reduced transepithelial resistance (Rt) by at least 7% (P<0.05). Pre-exposure (1 hr) to the protein kinase A (PKA) inhibitor H-89 (10 μm), which in itself inhibited the Iscby 0.5 μA cm−2, attenuated the Iscresponses of the cAMP-elevating agents (P< 0.05, unpaired data). In reverse, H-89 promptly decreased the Iscby 1.5–2.5 μA cm−2and increased Rtby 5% (P<0.05) in tissues pre-stimulated with either forskolin or an epinephrine plus IBMX combination. Additions of epinephrine or rolipram to apically permeabilized preparations using amphotericin B, increased the Iscby 12 and 22% respectively over baseline and reduced Rtby 6% (P<0.05). Similarly, in the presence of a transepithelial K+gradient (apical to basolateral) and amphotericin B, cAMP elevation stimulated K diffusion across the preparation by at least 1.8 μA cm−2and decreased the Rtby 4% (P<0.05), changes that were reversed by subsequent H-89 addition. Under Cl−rich conditions, pretreatment with 5 m m Ba2+reduced the basal Iscby 59% and blocked the cAMP-induced Iscstimulations typically seen in the presence of the anion. The results provide evidence for a PKA-regulated, Ba2+-inhibitable (voltage insensitive) 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 regulation of Cl−secretion and Na+absorption in the conjunctiva.
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