He Hao, Podymow Tiina, Zimpelmann Joseph, Burns Kevin D
Department of Medicine, Ottawa Hospital, and the Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6.
Am J Physiol Renal Physiol. 2003 Jun;284(6):F1235-44. doi: 10.1152/ajprenal.00192.2002. Epub 2003 Feb 11.
Nitric oxide (NO) exerts direct effects on nephron transport. 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 (86)Rb(+) 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 x mg(-1) x 5 min(-1); P < 0.001; SNAP (10(-4) M) + ABT 1.32 +/- 0.10 pmol x mg(-1) x 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.
一氧化氮（NO）对肾单位转运有直接影响。我们在具有致密斑特性的细胞系（MMDD1）中确定了NO对Na(+)-K(+)-2Cl(-)协同转运的影响。Na(+)-K(+)-2Cl(-)协同转运通过在哇巴因存在下测定布美他尼敏感的(86)Rb(+)摄取来衡量。MMDD1细胞表达一氧化氮合酶神经元亚型的mRNA，以及Na(+)-K(+)-2Cl(-)协同转运体的NKCC1和NKCC2(B)亚型。用NO供体硝普钠（SNP）或S-亚硝基-N-乙酰青霉胺（SNAP）对细胞进行预孵育会导致Na(+)-K(+)-2Cl(-)协同转运受到浓度依赖性抑制。顶端和基底外侧的Na(+)-K(+)-2Cl(-)协同转运均受到NO供体的抑制。SNP或SNAP对细胞内cGMP、cAMP、胞质钙水平或ERK1和ERK2的磷酸化没有显著影响。相反，细胞色素P-450抑制剂1-氨基苯并三唑（ABT；10(-3) M）或酮康唑（1.5 x 10(-5) M）完全逆转了SNAP对顶端或基底外侧Na(+)-K(+)-2Cl(-)协同转运的抑制作用[顶端：对照组1.18±0.15 vs. SNAP（10(-4) M）0.41±0.05 pmol x mg(-1) x 5 min(-(-1))；P < 0.001；SNAP（10(-4) M）+ ABT 1.32±0.10 pmol x mg(-1) x 5 min(-(-1))；与对照组相比P = 无显著性差异；n = 5]。细胞色素P-450环氧二十碳三烯酸（EET）代谢产物14,15-EET（5 x 10(-7) M）抑制顶端和基底外侧的协同转运，而8,9-EET和11,12-EET没有显著影响。尽管20-羟基二十碳四烯酸抑制顶端协同转运，但ω-羟化酶活性抑制剂HET0016并未逆转SNAP介导的顶端协同转运抑制。这些数据表明NO抑制MMDD1细胞顶端和基底外侧的Na(+)-K(+)-2Cl(-)协同转运。结果表明，抑制途径独立于cGMP，可能涉及对细胞色素P-450依赖性途径的刺激。
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