Bumetanide blocks CFTR G(Cl) in the native sweat duct

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K +-2Cl(-) cotransporter. We were surprised in preliminary studies to find th at bumetanide in the contraluminal bath also inhibited NaCl absorption in t he human sweat duct, which is apparently poor in cotransporter activity. In hibition was accompanied by a marked decrease in the transepithelial electr ical conductance. Because the cystic fibrosis transmembrane conductance reg ulator (CFTR) Cl- channel is richly expressed in the sweat duct, we asked w hether bumetanide acts by blocking this anion channel. We found that bumeta nide 1) significantly increased whole cell input impedance, 2) hyperpolariz ed transepithelial and basolateral membrane potentials, 3) depolarized apic al membrane potential, 4) increased the ratio of apical-to-basolateral memb rane resistance, and 5) decreased transepithelial Cl- conductance (G(Cl)). These results indicate that bumetanide inhibits CFTR G(Cl) in both cell mem branes of this epithelium. We excluded bumetanide interference with the pro tein kinase A phosphorylation activation process by "irreversibly" phosphor ylating CFTR [by using adenosine 5'-O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We the n activated CFTR G(Cl) by adding 5 mM ATP. Bumetanide in the cytoplasmic ba th (10(-3) M) inhibited similar to 71% of this ATP-activated CFTR G(Cl) ind icating possible direct inhibition of CFTR G(Cl). We conclude that bumetani de inhibits CFTR G(Cl) in apical and basolateral membranes independent of p hosphorylation. The results also suggest that >10(-5) M bumetanide cannot b e used to specifically block the Na+-K+-2Cl(-) cotransporter.