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.
Bialek
S
S
University of California, School of Optometry, Berkeley 94720, USA.
Joseph
D P
DP
Miller
S S
SS
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D008433:Mathematics; D008564:Membrane Potentials / Q000187:drug effects; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000502:physiology*; D011188:Potassium / Q000494:pharmacology*
D002712:Chlorides; D002034:Bumetanide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium