Control of apical membrane chloride permeability in the renal A6 cell line by nucleotides

1. The effect of extracellular nucleotides applied on the apical side of polarised BE cells grown on permeant filters was investigated by measuring the changes in (i) the Cl-36 efflux through the apical membranes, (ii) the intracellular chloride concentrations (aCl(i), measured with N-(6-methoxyquinolyl) acetoethyl ester, MQAE), (iii) I-Cl, the short-circuit current in the absence of Nat transport and (iv) the characteristics of the apical chloride channels using a patch-clamp approach. 2. ATP or UTP (0.1-500 mu M) transiently stimulated I-Cl. The sequence of purinergic agonist potencies was UTP = ATP > ADP >> the P2X-selective agonist beta,gamma-methylene ATP = the P2Y-selective agonist 2-methylthioATP. Suramin (100 mu M) as the P2Y antagonist. Reactive Blue 2 (10 mu M) had no effect on the UTP (or ATP)-stimulated current. These findings are consistent with the presence of P2Y(2)-like receptors located on the apical membranes of AB cells. Apical application of adenosine also transiently increased I-Cl. This effect was blocked by theophylline while the UTP-stimulated I-Cl was not. The existence of a second receptor, of the P1 type is proposed. 3. ATP (or UTP)-stimulated I-Cl was blocked by apical application of 200 mu M N-phenyl-anthranilic acid (DPC) or 100 mu M niflumic acid a while 100 mu M glibenclamide was ineffective. 4. Ionomycin and thapsigargin both transiently stimulated I-Cl; the nucleotide stimulation of I-Cl was not suppressed by pre-treatment with these agents. Chlorpromazin (50 mu M), a Ca2+-calmodulin inhibitor strongly inhibited the stimulation of I-Cl induced either by apical UTP or by ionomycin application. BAPTA-AM pre-treatment of AB cells blocked the UTP-stimulated I-Cl. Niflumic acid also blocked the ionomycin stimulated I-Cl 5. A fourfold increase in Cl-36 effluxes through the apical membranes was observed after ATP or UTP application. These increases of the apical chloride permeability could also be observed when following aCl(i) changes. Apical application of DPC (1 mM) or 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB; 500 mu M) produced an incomplete inhibition of Cl-36 effluxes through the apical membranes in ATP-stimulated and in untreated monolayers. 6. In single channel patch-clamp experiments, an apical chloride channel with a unitary single channel conductance of 7.3 +/- 0.6 pS (n = 12) was usually observed. ATP application induced the activation of one or more of these channels within a few minutes. 7. These results indicate that multiple purinergic receptor subtypes are present in the apical membranes of A6 cells and that nucleotides can act as modulators of Cl- secretion in renal cells.