Catecholaminergic regulation of Na-K-Cl cotransport in pigmented ciliary epithelium: Differences between PE and NPE

Pigmented (PE) and nonpigmented (NPE) ciliary epithelial cells comprise the ciliary epithelium, the site of aqueous humor formation in the eye. In man , catecholamines increase the rate of aqueous humor formation, but the mech anism underlying these effects is not understood. Recent evidence suggests that Na-K-Cl cotransport plays a central role in blood-to-aqueous chloride transport across ciliary epithelium in cow and rabbit. We therefore investi gated whether catecholamines stimulate Na-K-CI cotransport in human PE cell s. Na-K-Cl cotransporter protein was detected as a 170 163a protein band on immunoblots. Immunofluorescence microscopy detected cotransporter on the b asolateral membranes of the PE layer of ciliary epithelium from a human don or. Cotransporter immunofluorescence was also detected in cultured PE cells . Na-K-Cl cotransport activity measured as ouabain-insensitive bumetanide-s ensitive Rb-86 uptake was stimulated by isoproterenol 1.6-fold, with an EC5 0 = 28 nM and maximal stimulation at 1 muM. Other transport mechanisms invo lved in Rb-86 uptake were not affected. Stimulation by 1 muM isoproterenol was blocked by 10 nM ICI 118,551, a beta (2)-specific receptor antagonist, whereas the receptor subtype-specific antagonists yohimbine (alpha (2)), pr azosin (alpha (1)) and atenolol (beta (1)) were ineffective. Norepinephrine stimulation (EC50 = 280 nM) was also blocked by ICI 118,551. Dopamine stim ulated Na-K-Cl cotransport 1.6-fold with an EC50 = 14 muM. The dopamine eff ect could not be blocked by 10 muM SCH 23390, a D1-antagonist, but was abol ished by ICI 118,551. Forskolin and CPT-cAMP stimulated Na-K-Cl cotransport 1.79- and 1.71-fold, respectively, whereas the inactive forskolin analogue 1,9-dideoxyforskolin had no effect. However, high concentrations of the PK A inhibitors PIU amide 14-22 and KT 5720 were needed to inhibit both PICA a ctivity in cell lysates and isoproterenol stimulation of cotransport. This finding may indicate the presence of a novel PICA isoform in PE cells. Inhi bitors of other protein kinases, including myosin light chain kinase, prote in kinase G, calmodulin-dependent kinase and tyrosine kinase, were without effect on stimulated Na-K-Cl cotransport. When EC(50)s for catecholaminergi c stimulations of Na-K-Cl cotransport in PE were compared to those in NPE, values within five-fold of one another were seen for isoproterenol and nore pinephrine. In contrast, dopamine was 28-fold more potent in NPE than in PE . The data suggest that both PE and NPE possess beta (2) adrenergic recepto rs, but only NPE cells possess dopamine D1 receptors linked to Na-K-Cl cotr ansport. (C) 2001 Academic Press.