Catecholaminergic Regulation of Na-K-Cl Cotransport in Pigmented Ciliary Epithelium: Differences Between PE and NPE

Abstract

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 mechanism 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 investigated whether catecholamines stimulate Na-K-Cl cotransport in human PE cells. Na-K-Cl cotransporter protein was detected as a 170kDa protein band on immunoblots. Immunofluorescence microscopy detected cotransporter on the basolateral membranes of the PE layer of ciliary epithelium from a human donor. Cotransporter immunofluorescence was also detected in cultured PE cells. Na-K-Cl cotransport activity measured as ouabain-insensitive bumetanide-sensitive86Rb uptake was stimulated by isoproterenol 1.6-fold, with an EC50=28n M and maximal stimulation at 1μM. Other transport mechanisms involved in86Rb uptake were not affected. Stimulation by 1 μM isoproterenol was blocked by 10n M ICI 118,551, a β2-specific receptor antagonist, whereas the receptor subtype-specific antagonists yohimbine (α2), prazosin (α1) and atenolol (β1) were ineffective. Norepinephrine stimulation (EC50=280n M) was also blocked by ICI 118,551. Dopamine stimulated Na-K-Cl cotransport 1.6-fold with an EC50=14μM. The dopamine effect could not be blocked by 10μM SCH 23390, a D1-antagonist, but was abolished 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 PKA inhibitors PKI amide 14–22 and KT 5720 were needed to inhibit both PKA activity in cell lysates and isoproterenol stimulation of cotransport. This finding may indicate the presence of a novel PKA isoform in PE cells. Inhibitors of other protein kinases, including myosin light chain kinase, protein kinase G, calmodulin-dependent kinase and tyrosine kinase, were without effect on stimulated Na-K-Cl cotransport. When EC50s for catecholaminergic 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 norepinephrine. In contrast, dopamine was 28-fold more potent in NPE than in PE. The data suggest that both PE and NPE possess β2adrenergic receptors, but only NPE cells possess dopamine D1 receptors linked to Na-K-Cl cotransport.