Inhibition of the endogenous volume-regulated anion channel (VRAC) in HEK293 cells by acidic di-aryl-ureas.

The endogenous volume-regulated anion channel (VRAC) from HEK293 cells was pharmacologically characterized using the whole-cell patch-clamp technique. Under isotonic conditions a small (1.3 nS), Ca(2+)-independent Cl conductance was measured. However, swelling at 75% tonicity activated a VRAC identified as an outward-rectifying anion current ( P(l) > P(Cl) > P(gluconate)), which was ATP-dependent and showed inactivation at positive potentials. Activation of this current followed a sigmoid time course, reaching a plateau conductance of 42.6 nS after 12-15 min ( t(1/2) = 7 min). The pharmacology of this VRAC was investigated using standard Cl(-)-channel blockers (NPPB, DIDS, and tamoxifen) as well as a new group (acidic di-aryl ureas) of Cl(-)-channel blockers (NS1652, NS3623, NS3749, and NS3728). The acidic di-aryl ureas were originally synthezised for inhibition of the human erythrocyte Cl(-) conductance in vivo. NS3728 was the most potent VRAC blocker in this series ( IC(50) = 0.40 micro M) and even more potent than tamoxifen (2.2 micro M). NS3728 accelerated channel inactivation at positive potentials. These results show that acidic di-aryl ureas constitute a promising starting point for the synthesis of potent inhibitors of VRAC.

Cellular mechanisms for apical ATP effects on intracellular pH in human bronchial epithelium.

The effect of external ATP on intracellular pH (pH(i)) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o-) loaded with BCECF-AM. The steady-state pH(i) of 16HBE14o- epithelial monolayers was 7.137 +/- 0.027 (n = 46). Apical addition of ATP (10(-4) M) to epithelial monolayers induced a rapid and sustained pH(i) decrease of 0.164 +/- 0.024 pH units (n = 17; P < 0.001). The intracellular acidification was rapidly reversed upon removal of external ATP. In contrast, the non-hydrolysable ATP analogue AMP-PNP did not produce any significant change in pH(i). Inhibition of purinoreceptors by suramin did not affect the acidification induced by apical ATP. Inhibition of Na+-H+ exchange by apical Na+ removal or addition of amiloride (0.5 mM) reduced the apical ATP-induced pH(i) decrease, suggesting the involvement of a Na+-H+ exchanger or surface pH effects on the ATP-induced pH(i) response. Inhibitors of proton channels such as ZnCl2 (10(-4) M) also partially inhibited the ATP response. The pH(i) response to ATP was dependent on the external pH (pH(o)), with increasing acidification produced at lower pH(o) values. Neither the basal pH(i) nor the ATP-induced intracellular acidification was affected by thapsigargin (a Ca2+-ATPase inhibitor), chelerythrine chloride (a protein kinase C (PKC) inhibitor), RpcAMP (a protein kinase A (PKA) inhibitor) or PMA (a PKC activator). Therefore, the intracellular acidification of human bronchial epithelial cells induced by apical ATP does not involve signalling via Ca2+, PKC or PKA nor binding to a purinoreceptor. We interpret the effect of ATP to produce an intracellular acidification as a three step process: activation of H+ channels, inhibition of Na+-H+ exchange and influx of protonated ATP.