G alpha i-3 regulates epithelial Na+ channels by activation of phospholipase A2 and lipoxygenase pathways

Polarized renal epithelial cells have pertussis toxin-sensitive Gi proteins at their apical membrane capable of modulating Na+ channel activity (Cantiello, H.F., Patenaude, C.R., and Ausiello, D.A. (1989) J. Biol. Chem. 264, 20867-20870). In this study, the patch clamp technique was used to assess if this Gi-mediated regulation of Na+ channels is a component of a phospholipid signal transduction pathway. In excised inside-out patches of apical membranes of A6 cells, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated Na+ channel activity (percent open time and channel number) was inhibited by the phospholipase inhibitor mepacrine (50 microM), which had no effect on single channel conductance. In contrast, Na+ channel activity increased in a Ca2(+)-dependent manner following the addition of 100 nM mellitin to untreated or pertussis toxin-treated patches. Addition of 10 microM arachidonic acid in the presence of mepacrine increased Na+ channel activity. Both percent open time and Na+ channel number induced by GTP gamma S, the exogenous alpha i-3 subunit, or arachidonic acid were inhibited by the addition of the 5-lipoxygenase inhibitor nordihydroguaiaretic acid. Na+ channel activity was restored with the addition of leukotriene D4 (100 nM) or the parental leukotriene substrate 5-hydroperoxyeicosatetraenoic acid (10 microM). Thus, Gi activation of apical membrane epithelial Na+ channels is mediated through the regulation of phospholipase and lipoxygenase activities. This apically located signal transduction pathway may be sensitive to, or independent of, classical second messengers generated at the basolateral membrane and known to be responsible for modulation of Na+ channel activity in epithelia.