Na+/H+ and Cl--/HCO3--antiporters of bovine pigmented ciliary epithelial cells

Medical therapy of glaucoma commonly aims at slowing aqueous humor formatio n by the ocular ciliary epithelial bilayer, but underlying mechanisms are p oorly understood. The first step in secretion is NaCl uptake from the strom a into the pigmented ciliary epithelial (PE) cell layer by electroneutral t ransporters. After crossing gap junctions into the nonpigmented ciliary epi thelial (NPE) cell layer, solute is released into the aqueous humor, publis hed data have indicated that both paired Na+/H+ and Cl-/HCO3 antiporters an d the Na+-K+-2Cl(-)symporter are involved in net uptake. The molecular iden tities of the paired antiporters have not been elucidated. We have studied continuously cultured bovine PE cells. Acid-activated Na-22(+) uptake was i nhibited by cariporide, EIPA (ethyl-isopropyl-amiloride) and amiloride, at concentrations characteristic of the NHE-1 isoform. Videomicroscopy of BCEC F-loaded PE cells verified the presence of an EIPA-inhibitable Na+/H+ antip orter. Removing external Cl- also triggered an alkalinization, which was Na +-independent and could be inhibited by 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid (DIDS). Application of hypotonicity followed by return to i sotonicity triggered a regulatory volume increase, which was pharmacologica lly similar to the uptake mechanisms de scribed for intact rabbit ciliary e pithelium. Reverse transcriptase polymerase chain reaction (RT-PCR) amplifi cation of RNA from the human ciliary body detected expression of the AE2 Cl -/HCO3- exchanger, but not of AE1, cAE3 or bAE3. Immunostaining of bovine P E cells also revealed the presence of AE2 epitope. We conclude that paired NHE-1 Na+/H+ and AE2 Cl-/HCO3- antiporters are important components in the initial step in aqueous humor formation.