NO inhibits Na+-K+-2Cl- cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1)

We determined the effect of NO on Na+-K+-2Cl(-) cotransport in a cell line (MMDD1) with properties of macula densa. Na+-K+-2Cl(-) cotransport was measured as bumetanide-sensitive Rb-86(+) uptake in the presence of ouabain. MMDD1 cells expressed mRNA for the neuronal isoform of nitric oxide synthase, as well as NKCC1 and NKCC2(B) isoforms of the Na+-K+-2Cl(-) cotransporter. Preincubation of cells with the NO donors sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP) caused concentration-dependent inhibition of Na+-K+-2Cl(-) cotransport. Both apical and basolateral Na+-K+-2Cl(-) cotransport was inhibited by NO donors. SNP or SNAP had no significant effect on cellular levels of cGMP, cAMP, cytosolic calcium, or phosphorylation of ERK1 and ERK2. In contrast, the inhibitors of cytochrome P-450, 1-aminobenzotriazole (ABT; 10(-3) M) or ketoconazole (1.5 x 10(-5) M), completely reversed the inhibitory effect of SNAP on apical or basolateral Na+-K+-2Cl(-) cotransport [apical: control 1.18 +/- 0.15 vs. SNAP (10(-4) M) 0.41 +/- 0.05 pmol.mg(-1).5 min(-1); P < 0.001; SNAP (10(-4) M) + ABT 1.32 +/- 0.10 pmol.mg(-1).5 min(-1); P = not significant vs. control; n = 5]. The cytochrome P-450 epoxyeicosatrienoic acid (EET) metabolite 14,15-EET (5 x 10(-7) M) inhibited both apical and basolateral cotransport, whereas 8,9-EET and 11,12-EET had no significant effect. Although 20-hydroxyeicosatetraenoic acid inhibited apical cotransport, the inhibitor of omega-hydroxylase activity HET0016 did not reverse SNAP-mediated inhibition of apical cotransport. These data indicate that NO inhibits apical and basolateral Na+-K+-2Cl(-) cotransport in MMDD1 cells. The results suggest that the inhibitory pathway is independent of cGMP and might involve stimulation of a cytochrome P-450-dependent pathway.