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 (a(Cl)(i)) produced by small, physiological changes in extracellular potassium concentration ([K+](0)). These apical [K+](0) 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+](0) 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 mu M); this response was completely restored after bumetanide removal. 4. Reducing apical [K+](0), adding apical bumetanide (500 mM), or removing apical Cl- decreased a(Cl)(i) by 25 +/- 6 (n = 8), 28 +/- 1 (n = 2) and 26 +/- 5 mM (n = 3), respectively; adding 100 mu M apical bumetanide decreased a(Cl)(i) 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 (G(B)). 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-0(-). The present results show that the DBMH is caused by Delta[K](0)-induced inhibition of the apical membrane Na+-K+-2Cl(-) cotransporter; the subsequent decrease in a(Cl)(i) generated a hyperpolarization at the basolateral membrane Cl- channel.