MECHANISMS OF REGULATORY VOLUME DECREASE IN NONPIGMENTED HUMAN CILIARY EPITHELIAL-CELLS

To study the net solute and water efflux pathways of the ciliary epithelium we employed a cultured human NPE cell line. Because of the possible relationship between transepithelial ion and water flux and cell volume regulation, the ion efflux pathways mediating regulatory volume decrease (RVD) were investigated. Osmotic swelling of NPE cells was followed by a volume recovery. Volume recovery was K+ dependent and inhibited by K+ channel blockers such as quinine (1 mM). After osmotic swelling, a Cl--dependent membrane depolarization occurred that was inhibited by Cl- channel blockers such as 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 mu M) or Ca2+ chelators such as ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 2.0 mM). Cell swelling was also accompanied by an increase in intracellular Ca2+ concentration ([Ca2+](i)) of similar to 200 nM. The swelling-induced rise in [Ca2+](i) and RVD were diminished in the presence of 10 mu M La3+, 50 nM 12-O-tetradecanoylphorbol 13-acetate, and nominally Ca2+-free medium. Near total blockage of RVD occurred after pretreatment of NPE cells with Ca2+-free EGTA-1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester-containing solutions. The inhibition of RVD by EGTA-BAPTA treatment was overcome by increasing K+ conductance with gramicidin. The above findings indicate that RVD in NPE cells is mediated by separate K+ and Cl- conductances (channels). These data also show that swelling-induced increases in [Ca2+]i help modulate net ion efflux during regulation.