Carroll M A, Sala A, Dunn C E, McGiff J C, Murphy R C
Department of Pharmacology, New York Medical College, Valhalla 10595.
J Biol Chem. 1991 Jul 5;266(19):12306-12.
The medullary thick ascending limb of Henle's loop (mTALH) contributes importantly to the regulation of extracellular fluid volume and composition and metabolizes arachidonic acid (AA) chiefly by a cytochrome P450 monooxygenase pathway. Rabbit mTALH cells, when incubated with radiolabeled [14C]AA, form products that segregate into two peaks designated P1 and P2 based on their reverse-phase high pressure liquid chromatography retention times. We have now definitively identified their chemical structures. mTALH cells, isolated from the rabbit outer medulla, were homogenized and incubated with [14C]AA in the presence of NADPH. The AA metabolites in P1 and P2 were identified by gas chromatographic-mass spectrometric methods, including fast atom bombardment, negative ion electron capture, and electron ionization. All mass spectrometric data, the lack of UV chromophores, and comparisons with authentic standards were consistent with P1 containing two principal components: 19-hydroxy-5,8,11,14 eicosatetraenoic acid (19-HETE) and 20 - hydroxy - 5,8,11,14 - eicosatetraenoic acid (20-HETE), P2 contained primarily 1,20-eicosa-5,8,11,14-tetraenedioic acid (20-COOH-AA). The biological properties of P1 and P2 were compared with those of the authentic standards of 19- and 20-HETE and 20-COOH-AA. P1 dose dependently relaxed precontracted mesenteric arterial rings, as did authentic (19S)- and (19R)-HETE, whereas 20-HETE relaxed at lower and contracted at higher concentrations. As P1 contained a mixture of 19- and 20-HETE, each of these AA metabolites presumably contributed to the vascular relaxation produced by P1. Neither P2 nor 20-COOH-AA exhibited vasoactivity, but each demonstrated a similar potency in inhibiting rabbit medullary Na(+)-K(+)-ATPase activity. As previously reported, P2 was a more potent inhibitor of Na(+)-K(+)-ATPase than P1. The lesser inhibitory activity of P1 presumably reflects the presence of similar amounts of 19-HETE, the least active metabolite, and 20-HETE, which resembles 20-COOH-AA in its capacity to inhibit Na(+)-K(+)-ATPase. Thus, the biological activity of the less polar peak, P1, can be accounted for by 19- and 20-HETE, and that of P2, by 20-COOH-AA.
亨氏袢髓质厚升支（mTALH）对细胞外液体积和成分的调节起着重要作用，并且主要通过细胞色素P450单加氧酶途径代谢花生四烯酸（AA）。兔mTALH细胞在与放射性标记的[14C]AA孵育时，会形成根据反相高压液相色谱保留时间分为两个峰（分别命名为P1和P2）的产物。我们现已明确鉴定出它们的化学结构。从兔外髓质分离出的mTALH细胞经匀浆处理后，在存在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的情况下与[14C]AA一起孵育。通过气相色谱 - 质谱法（包括快原子轰击、负离子电子捕获和电子电离）鉴定P1和P2中的AA代谢产物。所有质谱数据、缺乏紫外发色团以及与标准品的比较均表明，P1含有两种主要成分：19 - 羟基 - 5,8,11,14 - 二十碳四烯酸（19 - HETE）和20 - 羟基 - 5,8,11,14 - 二十碳四烯酸（20 - HETE），P2主要含有1,20 - 二十碳 - 5,8,11,14 - 四烯二酸（20 - COOH - AA）。将P1和P2的生物学特性与19 - 和20 - HETE以及20 - COOH - AA的标准品进行比较。P1能剂量依赖性地舒张预收缩的肠系膜动脉环，标准的（19S） - 和（19R） - HETE也有同样的作用，而20 - HETE在较低浓度时舒张，在较高浓度时收缩。由于P1含有19 - 和20 - HETE的混合物，这些AA代谢产物可能各自促成了P1产生的血管舒张作用。P2和20 - COOH - AA均未表现出血管活性，但二者在抑制兔髓质钠钾ATP酶活性方面表现出相似的效力。如先前报道，P2是比P1更有效的钠钾ATP酶抑制剂。P1较小的抑制活性可能反映出存在相似量的活性最低的代谢产物19 - HETE和在抑制钠钾ATP酶能力上与20 - COOH - AA相似的20 - HETE。因此，极性较小的峰P1的生物学活性可由19 - 和20 - HETE来解释，而P2的生物学活性可由20 - COOH - AA来解释。
