Sasaki Shota, Siragy Helmy M, Gildea John J, Felder Robin A, Carey Robert M
Department of Medicine, University of Virginia School of Medicine, Charlottesville, USA.
Hypertension. 2004 Feb;43(2):286-91. doi: 10.1161/01.HYP.0000112421.18551.1e. Epub 2004 Jan 12.
The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3', 5' monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65+/-4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64+/-6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na+ concentration. In the presence of the Na+/K+ ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-4) M), increased both intracellular and extracellular cGMP (from 1.26+/-0.21 to 88.7+/-12.6 pmol/mg protein and from 0.58+/-0.10 to 9.24+/-1.9 pmol/mL, respectively, P<0.01) and decreased cellular Na+ uptake by 37.4+/-6.8% (P<0.05) compared with control. The effects of SNAP on cGMP production were similar in normotensive and hypertensive subjects. The increases in intracellular and extracellular cGMP concentration because of SNAP were blocked completely by soluble guanylyl cyclase inhibitor ODQ (1-H-[1,2,4] oxadiazolo [4,2-alpha] quinoxalin-1-one). Probenecid, an organic anion transport inhibitor, augmented the SNAP (10(-6) M)-induced increase in intracellular cGMP accumulation (from 4.9+/-0.9 to 9.8+/-1.5 pmol/mg protein, P<0.05), abrogated the SNAP-induced increase in extracellular cGMP extrusion (from 1.07+/-0.4 to 0.37+/-0.1 pmol/L, P<0.05) and blocked the SNAP-induced reduction in cellular Na+ uptake. Neither intracellular nor extracellular cGMP were influenced by l-arginine, the metabolic precursor of NO, or N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10(-5) M) or its membrane-permeable analogue 8-Br-cGMP (10(-5) M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36+/-0.19 to 289.7+/-29.4 pmol/mg protein, P<0.01). However, both cGMP and 8-Br-cGMP were effective in decreasing cellular Na+ uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain constitutive NO synthase, the NO-generating cGMP must be derived from sources outside the human RPT. The cGMP cellular export system is critical in the regulation of RPT cellular Na+ absorption in humans.
本研究旨在确定人肾近端小管（RPT）在一氧化氮（NO）供体直接刺激可溶性鸟苷酸环化酶时产生和输出鸟苷 3'，5'-环磷酸（cGMP）的能力。此外，我们研究了人RPT细胞中cGMP的外排是否是抑制细胞钠摄取所必需的。RPT细胞取自单侧肾切除术后的新鲜人肾（血压正常受试者，n = 4，平均年龄65±4.7岁，3名男性，1名女性；高血压患者，n = 6，平均年龄64±6.1岁，4名男性，2名女性）。使用荧光染料Sodium Green测定细胞质Na⁺浓度。在存在Na⁺/K⁺ ATP酶抑制剂哇巴因的情况下，在适当波长（激发波长485 nm，发射波长535 nm）下监测荧光。与对照组相比，NO供体S-亚硝基-N-乙酰青霉胺（SNAP，10⁻⁴ M）使细胞内和细胞外cGMP均增加（分别从1.26±0.21增至88.7±12.6 pmol/mg蛋白和从0.58±0.10增至9.24±1.9 pmol/mL，P<0.01），并使细胞钠摄取减少37.4±6.8%（P<0.05）。SNAP对cGMP产生的影响在血压正常和高血压受试者中相似。由于SNAP导致的细胞内和细胞外cGMP浓度增加被可溶性鸟苷酸环化酶抑制剂ODQ（1-H-[1,2,4]恶二唑并[4,2-α]喹喔啉-1-酮）完全阻断。丙磺舒，一种有机阴离子转运抑制剂，增强了SNAP（10⁻⁶ M）诱导的细胞内cGMP积累增加（从4.9±0.9增至9.8±1.5 pmol/mg蛋白，P<0.05）；消除了SNAP诱导的细胞外cGMP外排增加（从1.07±0.4降至0.37±0.1 pmol/L，P<0.05），并阻断了SNAP诱导的细胞钠摄取减少。细胞内和细胞外cGMP均不受NO的代谢前体L-精氨酸或NO合酶抑制剂N(G)-硝基-L-精氨酸甲酯的影响。在体外给予cGMP（10⁻⁵ M）或其膜通透性类似物8-溴-cGMP（10⁻⁵ M）后，只有8-溴-cGMP穿过细胞膜增加细胞内cGMP（从1.36±0.19增至289.7±29.4 pmol/mg蛋白，P<0.01）。然而，cGMP和8-溴-cGMP均能有效减少细胞钠摄取。总之，人RPT细胞含有可溶性鸟苷酸环化酶，并且能够响应NO产生和输出cGMP。由于人RPT细胞本身不含有组成型NO合酶，产生cGMP的NO必定来源于人RPT之外的来源。cGMP细胞外排系统在调节人RPT细胞钠吸收中起关键作用。
