Adrenergic receptor activated ion transport in human fetal retinal pigmentepithelium

PURPOSE. To identify the apical and basolateral membrane mechanisms and int racellular signaling pathways in human fetal retinal pigment epithelium (HR PE) that mediate membrane voltage and resistance changes caused by apical m embrane adrenergic receptor activation. METHODS. Intact sheets of RPE-choroid from human fetal eyes were mounted in a modified Ussing chamber. Ringer's solution composition changes on the re tina-facing and choroid-facing sides of the tissue were separately controll ed. Intracellular microelectrodes recorded the membrane voltage and resista nce changes after the addition of pharmacologic agents to the apical or bas al baths. RESULTS. 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 beta adrenergic receptors, prazosin and propranolol, respectively, indicated that both rec eptor types were present. The epinephrine responses were inhibited by apica l bumetanide and basal 4,4'-diisothiocyanostilbene-2,2' disulfonic acid (DI DS). A cocktail of cyclic adenosine monophosphate (cAMP)-elevating agents p roduced basolateral membrane voltage and resistance changes very similar to the isoproterenol responses. The cAMP-induced electrical responses were st rongly inhibited by basal 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). Ionomycin (to elevate intercellular Ca2+, [Ca2+](i)) produced electrical re sponses similar to those caused by epinephrine. The Ca2+ responses were una ffected by NPPB but were inhibited by 3 mM DIDS in the basal bath. CONCLUSIONS. The results provide evidence for two apical membrane adrenergi c receptors, alpha (1), and beta, activated by epinephrine and isoprotereno l, respectively. The membrane voltage and resistance changes produced by th ese two agonists mimic those produced by elevating [Ca2+](i) and [cAMP](i), suggesting that these ubiquitous signaling molecules activate separate bas olateral membrane Cl channels inhibited by DIDS and NPPB, respectively. The se two receptors, the apical membrane NaK2Cl cotransporters and the basolat eral membrane Cl channels form a complex of proteins that help mediate flui d absorption across human RPE.