AMILORIDE-SENSITIVE SODIUM CONDUCTANCE IN HUMAN B-LYMPHOID CELLS

The Na+ conductance of RPMI 1640 and the pipette solution cells was examined using whole cell patch clamp. When the bath solution contained RPMI 1640 and the pipette solution contained (in mM) 100 potassium gluconate, 30 KCl, 10 NaCl, 0.5 ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). and 20 N-2-hydroxythylpiperazine-N'-2-ethanesulfonic acid (HEPES), as well as well as <10 nM free Ca2+, pH 7.25, the mean membrane potential was -58 +/- 4.5 mV (n = 18). Cells were voltage clamped from -160 to +40 mV in 20-mV increments. The inward conductance was 767 +/- 221 pS/10 pF, and the outward conductance was 1,212 +/- 272 pS/10 pF (n = 12). Superfusion with 2 muM amiloride significantly hyperpolarized the cells by 7.4 +/- 2.2 mV (P = 0.004), significantly reduced the inward conductance to 221 +/- 65 pS/10 pF (P = 0.028), but had no effect on the outward conductance (1.294 +/- 236 pS/10 pF, P = 0.820, after amiloride). Next, the pipette and bath solutions were changed to (in mM) 150 sodium gluconate, 0.5 EGTA, and 20 HEPES, as well as <10 nM free Ca2+, pH 7.25. Under these conditions amiloride significantly reduced (50%, P < 0.05; n = 7) the whole cell currents. When potassium gluconate was substituted for sodium gluconate, amiloride had no effect. Thus amiloride inhibited a Na+-specific conductance expressed by B lymphoid cells. Benzamil is also a potent inhibitor of Na+ conductance in B lymphoid cells (50% inhibitory concentration = 10 nM; n = 4); however, dimethyl amiloride and ethylisopropyl amiloride (2 muM) had no effect. 8-(4-Chlorophenylthio) adenosine 3',5'-cyclic monophosphate (400 muM) specifically increased inward Na+ conductance, which was completely and reversibly inhibited by 2 muM amiloride. The properties of the amiloride-sensitive component of the Na+ conductance expressed by human B lymphoid cells are indistinguishable from those expressed in the apical membranes of epithelial cells.