Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells.

Abstract

Thiazide diuretics inhibit Na+ and stimulateCa2+ absorption in renal distal convoluted tubules. Experiments were performed on immortalized mouse distal convoluted tubule (MDCIT) 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-' mg protein-' and were inhibited 40% by chlorothiazide (CTZ, 10'-M). Control rates of MCl-uptake averaged 340±50 nmol min' mg protein-' and were inhibited 50% by CTZ. CTZ stimulated 4"Ca2" uptake by 45% from resting levels of 2.86±0.26 nmol min' mg protein'. Bumetanide (10 -4 M) had no effect on 22Na +, -6CI -, or 45Ca 2+ uptake. Control levels of intracellular calcium activity ([Ca2+1I) averaged 91±12 nM. CTZ elicited concentration-dependent increases of ICa2+J1 to a maximum of 654±31 nM at 10-4 M. Reduction of extracellular Clor 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. (J. Clin. Invest. 1992. 90:429-438.) Key

Phosphorylation Decreases Ubiquitylation of the Thiazide-sensitive Cotransporter NCC and Subsequent Clathrin-mediated Endocytosis

“…Previous studies of NCC trafficking have been performed using immortalized DCT cell lines (33)(34)(35)(36), stably transfected NCC-expressing MDCKII cell lines (26,37), or transiently transfected cells such as COS and HEK (10,11,38). However, these cell lines are not ideal for studying trafficking events of NCC due to, for example, a lack of polarized delivery of NCC, absence of NCC complex glycosylation, and in the case of immortalized DCT cells the inability to assess the roles of sitedirected mutations for NCC activity.…”

Renal expression of parvalbumin is critical for NaCl handling and response to diuretics

“…The mechanism by which PV could regulate NCC expression was investigated in mDCT cells (16,17), which endogenously express PV and NCC (Fig. 5).…”

“…The immortalized mDCT cells were kindly provided by P. A. Friedman (University of Pittsburgh School of Medicine, Pittsburgh, PA). The cells have been previously characterized as a model for the thiazide-sensitive Na ϩ and Ca 2ϩ transport occurring in the DCT (16,17). The cells were grown in DMEM/Ham's F-12 media (Lonza, Verviers, Belgium) supplemented with 5% FCS (Lonza), 2 mM L-glutamine (Invitrogen), and a mixture of penicillin/streptomycin in a humidified atmosphere of 95% air-5% CO 2 at 37°C.…”

β1-Adrenergic Receptor Signaling Activates the Epithelial Calcium Channel, Transient Receptor Potential Vanilloid Type 5 (TRPV5), via the Protein Kinase A Pathway

“…Gesek and Friedman showed that isoproterenol stimulated both cAMP production and Na ϩ uptake, but not Ca 2ϩ influx into the cells (31). However, they selected thiazide-sensitive DCT1 cells, which contain no TRPV5, resulting in very low Ca 2ϩ transport rates (20,24,31). In contrast, in the present study, DCT2/CNT tubules were selectively sorted from TRPV5-expressing cells.…”