Adrenergic receptor activated ion transport in human fetal retinal pigment epithelium.

PURPOSE: 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. METHODS: 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. 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 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. CONCLUSIONS: 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.

Nonsteroidal anti-inflammatory drugs alter chloride and fluid transport in bovine retinal pigment epithelium.

Nonsteroidal anti-inflammatory drugs (NSAIDs) were added to the solutions bathing the apical membrane of bovine retinal pigment epithelium (RPE)-choroid explants. For example, niflumic acid (100 microM) depolarized the basolateral membrane voltage (VB) by approximately 12 mV, increased transepithelial potential by 4.5 mV, decreased intracellular Cl activity by 13 mM, decreased transepithelial resistance by 17 omega.cm2, and increased the ratio of apical to basolateral membrane resistance nearly threefold. All of these changes are consistent with an increase in basolateral membrane Cl conductance. In addition, niflumic acid caused intracellular Ca concentration to decrease by 16 nM and fluid transport rate to increase by 1.5 microliters.cm-2.h-1. Flufenamic acid, which is structurally very similar to niflumic acid, had the opposite effects on membrane voltage and resistance. Basal application of the Cl channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or current clamping VB to the reversal potential for Cl practically abolished the niflumic acid response. The niflumic acid results suggest that certain NSAIDs can directly alter Cl conductance in the bovine RPE, apparently independently of cyclooxygenase inhibition.

Comparative electrophysiological properties of NG108-15 cells in serum-containing and serum-free media.

The electrophysiological properties of NG108-15 neuroblastoma x glioma hybrids were compared after culture in serum-containing medium (SCM) versus serum-free media (SFM) containing N2 or B27 supplements. The excitability of cells was media dependent (B27 > N2 > SCM). Action potential profiles of SFM cells were characterized by slower activation and prolonged after hyperpolarization which predisposed SFM cells to fire repetitively. The presence of three types of inward calcium currents was also revealed in SFM cells. These differential effects were primarily attributable to the media used with a secondary enhancement by the chemical differentiating agents used (dB-cAMP and forskolin).

Measurement of acetylcholine receptor function in microcircuit-coupled myoblasts.

An electrophysiological measurement principle for long-term, noninvasive monitoring of the nicotinic acetylcholine receptor (nAChR) function is described. The measurement is based on the ability to record agonist-induced depolarizations of clonal myoblasts that have formed high impedance seals with extracellular microcircuit electrodes. The technique appears promising for several types of assays and environmental monitoring applications.