Adrenergic receptor activated ion transport in human fetal retinal pigment epithelium

To identify the apical and basolateral membrane mechanisms and intracellular signaling pathways in human fetal retinal pigment epithelium (HRPE) that mediate membrane voltage and resistance changes caused by apical membrane adrenergic receptor activation.

Intact sheets of RPE-choroid from human fetal eyes were mounted in a modified Ussing chamber. Ringer's solution composition changes on the retina-facing and choroid-facing sides of the tissue were separately controlled. Intracellular microelectrodes recorded the membrane voltage and resistance changes after the addition of pharmacologic agents to the apical or basal baths.

Apical adrenergic agonists, isoproterenol and epinephrine (10(-8) M), depolarized the basolateral membrane, decreased total tissue resistance (R:(t)) and increased the ratio of apical-to-basolateral membrane resistance (R:(A)/R:(B)). Experiments using antagonists for alpha(1) and ss adrenergic receptors, prazosin and propranolol, respectively, indicated that both receptor types were present. The epinephrine responses were inhibited by apical bumetanide and basal 4,4'-diisothiocyanostilbene-2,2' disulfonic acid (DIDS). A cocktail of cyclic adenosine monophosphate (cAMP)-elevating agents produced basolateral membrane voltage and resistance changes very similar to the isoproterenol responses. The cAMP-induced electrical responses were strongly inhibited by basal 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). Ionomycin (to elevate intercellular Ca(2+), [Ca(2+)](i)) produced electrical responses similar to those caused by epinephrine. The Ca(2+) responses were unaffected by NPPB but were inhibited by 3 mM DIDS in the basal bath.

The results provide evidence for two apical membrane adrenergic receptors, alpha(1) and ss, activated by epinephrine and isoproterenol, respectively. The membrane voltage and resistance changes produced by these two agonists mimic those produced by elevating [Ca(2+)](i) and [cAMP](i), suggesting that these ubiquitous signaling molecules activate separate basolateral membrane Cl channels inhibited by DIDS and NPPB, respectively. These two receptors, the apical membrane NaK2Cl cotransporters and the basolateral membrane Cl channels form a complex of proteins that help mediate fluid absorption across human RPE.