The delayed basolateral membrane hyperpolarization of the bovine retinal pigment epithelium: mechanism of generation.

1. Conventional and ion‐selective double‐barrelled microelectrodes were used in an in vitro preparation of bovine retinal pigment epithelium (RPE)‐choroid to measure the changes in membrane voltage, resistance and intracellular Cl‐ activity (aCli) produced by small, physiological changes in extracellular potassium concentration ([K+]o). These apical [K+]o changes approximate those produced in the extracellular (subretinal) space between the photoreceptors and the RPE following transitions between light and dark. 2. Changing apical [K+]o from 5 to 2 mM in vitro elicited membrane voltage responses with three distinct phases. The first phase was generated by an apical membrane hyperpolarization, followed by a (delayed) basolateral membrane hyperpolarization (DBMH); the third phase was an apical membrane depolarization. The present experiments focus on the membrane and cellular mechanisms that generate phase 2 of the response, the DBMH. 3. The DBMH was abolished in the presence of apical bumetanide (100 microM); this response was completely restored after bumetanide removal. 4. Reducing apical [K+]o, adding apical bumetanide (500 mM), or removing apical Cl‐ decreased aCli by 25 +/‐ 6 (n = 8), 28 +/‐ 1 (n = 2) and 26 +/‐ 5 mM (n = 3), respectively; adding 100 microM apical bumetanide decreased aCli by 12 +/‐ 2 mM (n = 3). Adding apical bumetanide or removing apical bath Cl‐ hyperpolarized the basolateral membrane and decreased the apparent basolateral membrane conductance (GB). 5. DIDS (4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid) blocked the RPE basolateral membrane Cl‐ conductance and inhibited the DBMH and the basolateral membrane hyperpolarization produced by apical bumetanide addition or by removal of apical Cl‐o. The present results show that the DBMH is caused by delta[K]o‐induced inhibition of the apical membrane Na(+)‐K(+)‐2Cl‐ cotransporter; the subsequent decrease in aCli generated a hyperpolarization at the basolateral membrane Cl‐ channel.