氯噻嗪刺激小鼠远曲小管细胞钙转运的机制。
Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells.
作者信息
Gesek F A, Friedman P A
机构信息
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03756.
出版信息
J Clin Invest. 1992 Aug;90(2):429-38. doi: 10.1172/JCI115878.
Thiazide diuretics inhibit Na+ and stimulate Ca2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCT) cells to determine the mechanism underlying the dissociation of sodium from calcium transport and the stimulation of calcium absorption induced by thiazide diuretics. Control rates of 22Na+ uptake averaged 272 +/- 35 nmol min-1 mg protein-1 and were inhibited 40% by chlorothiazide (CTZ, 10(-4) M). Control rates of 36Cl- uptake averaged 340 +/- 50 nmol min-1 mg protein-1 and were inhibited 50% by CTZ. CTZ stimulated 45Ca2+ uptake by 45% from resting levels of 2.86 +/- 0.26 nmol min-1 mg protein-1. Bumetanide (10(-4) M) had no effect on 22Na+, 36Cl-, or 45Ca2+ uptake. Control levels of intracellular calcium activity ([Ca2+]i) averaged 91 +/- 12 nM. CTZ elicited concentration-dependent increases of [Ca2+]i to a maximum of 654 +/- 31 nM at 10(-4) M. CTZ reduced intracellular chloride activity ([Cl-]i), as determined with the chloride-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 10(-5) M) abolished the effect of CTZ on [Cl-]i. NPPB also blocked CTZ-induced increases of 45Ca2+. Resting membrane voltage, measured in cells loaded with the potential-sensitive dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)], averaged -72 +/- 2 mV. CTZ hyperpolarized cells in a concentration-dependent and reversible manner. At 10(-4) M, CTZ hyperpolarized MDCT cells by 20.4 +/- 7.2 mV. Reduction of extracellular Cl- or addition of NPPB abolished CTZ-induced hyperpolarization. Direct membrane hyperpolarization increased 45Ca2+ uptake whereas depolarization inhibited 45Ca2+ uptake. CTZ-stimulated 45Ca2+ uptake was inhibited by the Ca2+ channel blocker nifedipine (10(-5) M). We conclude that thiazide diuretics block cellular chloride entry mediated by apical membrane NaCl cotransport. Intracellular chloride, which under control conditions is above its equilibrium value, exits the cell through NPPB-sensitive chloride channels. This decrease of intracellular chloride hyperpolarizes MDCT cells and stimulates Ca2+ entry by apical membrane, dihydropyridine-sensitive Ca2+ channels.
噻嗪类利尿剂可抑制肾远曲小管中的Na⁺吸收并刺激Ca²⁺吸收。在永生化小鼠远曲小管（MDCT）细胞上进行了实验，以确定噻嗪类利尿剂引起的钠与钙转运解离以及钙吸收刺激背后的机制。22Na⁺摄取的对照速率平均为272±35 nmol·min⁻¹·mg蛋白⁻¹，氯噻嗪（CTZ，10⁻⁴ M）可抑制40%。36Cl⁻摄取的对照速率平均为340±50 nmol·min⁻¹·mg蛋白⁻¹，CTZ可抑制50%。CTZ使45Ca²⁺摄取从静息水平的2.86±0.26 nmol·min⁻¹·mg蛋白⁻¹增加了45%。布美他尼（10⁻⁴ M）对22Na⁺、36Cl⁻或45Ca²⁺摄取无影响。细胞内钙活性（[Ca²⁺]i）的对照水平平均为91±12 nM。CTZ引起[Ca²⁺]i浓度依赖性增加，在10⁻⁴ M时最高可达654±31 nM。用对氯敏感的荧光染料6-甲氧基-N-（3-磺丙基）喹啉鎓测定，CTZ降低了细胞内氯活性（[Cl⁻]i）。氯通道阻滞剂5-硝基-2-（3-苯丙基氨基）苯甲酸（NPPB，10⁻⁵ M）消除了CTZ对[Cl⁻]i的影响。NPPB也阻断了CTZ诱导的45Ca²⁺增加。用电压敏感染料3,3'-二己基氧杂羰花青碘化物[DiOC6(3)]加载的细胞中测量的静息膜电压平均为-72±2 mV。CTZ以浓度依赖性和可逆方式使细胞超极化。在10⁻⁴ M时，CTZ使MDCT细胞超极化20.4±7.2 mV。细胞外Cl⁻的减少或NPPB的添加消除了CTZ诱导的超极化。直接的膜超极化增加了45Ca²⁺摄取，而去极化抑制了45Ca²⁺摄取。CTZ刺激的45Ca²⁺摄取被Ca²⁺通道阻滞剂硝苯地平（10⁻⁵ M）抑制。我们得出结论，噻嗪类利尿剂阻断了由顶端膜NaCl共转运介导的细胞氯内流。在对照条件下高于其平衡值的细胞内氯，通过NPPB敏感的氯通道排出细胞。这种细胞内氯的减少使MDCT细胞超极化，并通过顶端膜、二氢吡啶敏感的Ca²⁺通道刺激Ca²⁺内流。
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