THE DELAYED BASOLATERAL MEMBRANE HYPERPOLARIZATION OF THE BOVINE RETINAL-PIGMENT EPITHELIUM - MECHANISM OF GENERATION

1. Conventional and ion-selective double-barrelled microelectrodes wer e 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, physiolog ical changes in extracellular potassium concentration ([K+](0)). These apical [K+](0) changes approximate those produced in the extracellula r (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 distin ct phases. The first phase was generated by an apical membrane hyperpo larization, followed by a (delayed) basolateral membrane hyperpolariza tion (DBMH); the third phase was an apical membrane depolarization. Th e present experiments focus on the membrane and cellular mechanisms th at generate phase 2 of the response, the DBMH. 3. The DBMH was abolish ed 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- decrease d 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 b ath Cl- hyperpolarized the basolateral membrane and decreased the appa rent basolateral membrane conductance (G(B)). 5. DIDS (4,4'-diisothioc yanostilbene-2,2'-disulphonic acid) blocked the RPE basolateral membra ne Cl- conductance and inhibited the DBMH and the basolateral membrane hyperpolarization produced by apical bumetanide addition or by remova l 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 hyperp olarization at the basolateral membrane Cl- channel.