The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of alpha-, beta- and gamma-subunits and the alpha- and beta-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat alpha- (ralpha) or guinea-pig alpha-subunit (gpalpha) with Xenopus betagamma-subunits. The gpalphaxbetagamma was significantly stimulated by glibenclamide (100 microM) (184+/-15%), whereas the ralpha-combination was slightly down-regulated by the sulfonylurea (79+/-4%). The stimulating effect did not interfere with Na(+)-self-inhibition resulting from intracellular accumulation of Na(+)-ions. We exchanged cytosolic termini between both orthologs but the gpalpha-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-beta-S but not by ATP-gamma-S, when applied intracellularly. Intracellular loading with Na(+)-ions after inhibition of Na(+)/K(+)-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH(3)) slightly reduced stimulation of I(ami) (118+/-12%; control: 132+/-9%) while phosphatidylinositol-4,5-biphosphate (PIP(2)) significantly reduced the effect of glibenclamide to 101+/-3%.