Characterization of the thiazide-sensitive Na(+)-Cl(-) cotransporter: a new model for ions and diuretics interaction.
The thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC) is the major pathway for salt reabsorption in the apical membrane of the mammalian distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both cotransported ions, with the Michaelis-Menten constant (K(m)) for Na(+) of 7.6 +/- 1.6 mM and for Cl(-) of 6.3 +/- 1.1 mM, and Hill coefficients for Na(+) and Cl(-) consistent with electroneutrality. The affinities of both Na(+) and Cl(-) were increased by increasing concentration of the counterion. The IC(50) values for thiazides were affected by both extracellular Na(+) and Cl(-). The higher the Na(+) or Cl(-) concentration, the lower the inhibitory effect of thiazides. Finally, rTSC function is affected by extracellular osmolarity. We propose a transport model featuring a random order of binding in which the binding of each ion facilitates the binding of the counterion. Both ion binding sites alter thiazide-mediated inhibition of transport, indicating that the thiazide-binding site is either shared or modified by both Na(+) and Cl(-). (+info)
Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome.
Gitelman's syndrome is an autosomal recessive renal tubular disorder characterized by hypokalemic metabolic alkalosis, hypomagnesemia, and hypocalciuria. This disorder results from mutations in the thiazide-sensitive NaCl cotransporter (NCC). To elucidate the functional implications of mutations associated with this disorder, metolazone-sensitive (22)Na(+) uptake, subcellular localization, and glycosidase-sensitive glycosylation of human NCC (hNCC) were determined in Xenopus laevis oocytes expressing FLAG-tagged wild-type or mutant hNCC. Injection of 10 ng of FLAG-tagged hNCC cRNA resulted in metolazone-sensitive (22)Na(+) uptake of 3.4 +/- 0.2 nmol Na(+)/oocyte per 2 h. Immunocytochemical analysis revealed sharp immunopositive staining at the plasma membrane. In agreement with this finding, a broad endoglycosidase H-insensitive band of 130 to 140 kD was present in Western blots of total membranes. The plasma membrane localization of this complex-glycosylated protein was confirmed by immunoblotting of purified plasma membranes. The mutants could be divided into two distinct classes. Class I mutants (G439S, T649R, and G741R) exhibited no significant metolazone-sensitive (22)Na(+) uptake. Immunopositive staining was present in a diffuse band just below the plasma membrane. This endoplasmic reticulum and/or pre-Golgi complex localization was further suggested by the complete absence of the endoglycosidase H-insensitive band. Class II mutants (L215P, F536L, R955Q, G980R, and C985Y) demonstrated significant metolazone-sensitive (22)Na(+) uptake, although uptake was significantly lower than that obtained with wild-type hNCC. The latter mutants could be detected at and below the oocyte plasma membrane, and immunoblotting revealed the characteristic complex-glycosylated bands. In conclusion, this study substantiates NCC processing defects as the underlying pathogenic mechanism in Gitelman's syndrome. (+info)
Pathophysiology of functional mutations of the thiazide-sensitive Na-Cl cotransporter in Gitelman disease.
Most of the missense mutations that have been described in the human SLC12A3 gene encoding the thiazide-sensitive Na(+)-Cl(-) cotransporter (TSC, NCC, or NCCT), as the cause of Gitelman disease, block TSC function by interfering with normal protein processing and glycosylation. However, some mutations exhibit considerable activity. To investigate the pathogenesis of Gitelman disease mediated by such mutations and to gain insights into structure-function relationships on the cotransporter, five functional disease mutations were introduced into mouse TSC cDNA, and their expression was determined in Xenopus laevis oocytes. Western blot analysis revealed immunoreactive bands in all mutant TSCs that were undistinguishable from wild-type TSC. The activity profile was: wild-type TSC (100%) > G627V (66%) > R935Q (36%) = V995M (32%) > G610S (12%) > A585V (6%). Ion transport kinetics in all mutant clones were similar to wild-type TSC, except in G627V, in which a small but significant increase in affinity for extracellular Cl(-) was observed. In addition, G627V and G610S exhibited a small increase in metolazone affinity. The surface expression of wild-type and mutant TSCs was performed by laser-scanning confocal microscopy. All mutants exhibited a significant reduction in surface expression compared with wild-type TSC, with a profile similar to that observed in functional expression analysis. Our data show that biochemical and functional properties of the mutant TSCs are similar to wild-type TSC but that the surface expression is reduced, suggesting that these mutations impair the insertion of a functional protein into the plasma membrane. The small increase in Cl(-) and thiazide affinity in G610S and G627V suggests that the beginning of the COOH-terminal domain could be implicated in defining kinetic properties. (+info)
Beneficial effects of metolazone in a rat model of preeclampsia.
Preeclampsia is a disorder that continues to exact a significant toll with respect to maternal morbidity and mortality as well as fetal wastage. Furthermore, the treatment of this disorder has not changed significantly in 50 years and is unsatisfactory. The use of diuretics in this syndrome is controversial because there is a concern related to potential baleful effects of volume contraction leading to a possible further decrement in the perfusion of the maternal-fetal unit. Metolazone is a diuretic/antihypertensive agent, which has a therapeutic effect on blood pressure (BP) in human essential hypertension without causing a natriuresis. We administered the drug in nondiuretic doses in a rat model of preeclampsia previously developed in this laboratory. The drug reduced BP without an accompanying natriuresis. Although there was a trend toward an improvement in intrauterine growth restriction, as determined by litter size and the number of pups demonstrating malformations, the values did not reach statistical significance. We conclude that metolazone, in low dosage, is an effective antihypertensive in this rat model. These studies have implications for the treatment of the human disorder. (+info)
Effects of diuretic treatment and of dietary sodium on renal binding of 3H-metolazone.
We report a series of experiments designed to determine if agents and conditions that have been reported to alter sodium reabsorption, Na-K-ATPase activity or cellular structure in the rat distal nephron might also regulate the density or affinity of binding of 3H-metolazone to the putative thiazide receptor in the distal nephron. Experimental conditions selected for study were acute (60-min) and chronic hydrochlorothiazide (HCTZ), acute acetazolamide, acute and chronic furosemide, and 14 days of varied intake of dietary sodium. The density of the binding of 3H-metolazone was increased 47% by acute HCTZ (P less than 0.001) and 39% (P less than 0.001) by acute furosemide. In contrast, acute acetazolamide produced no change in binding despite eliciting a dramatic diuresis. Chronic HCTZ (5 days) and chronic furosemide (7 days) increased binding of 3H-metolazone by 46% (P less than 0.001) and by 101% (P less than 0.001), respectively. Variation of dietary sodium intake over a range that allowed normal growth of the animal and that produced urinary excretion of Na varying from 0.28 to 2.62 mEq/100 g/day failed to alter the density of binding of 3H-metolazone. These studies are the first indication that the density of the thiazide receptor is regulated by a variety of both acute and chronic conditions that have previously been associated with changes in transport, ultrastructure or Na-K-ATPase activity in the distal nephron.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters.
Thiazide diuretic drug receptors in rat kidney: identification with [3H]metolazone.
Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. We report here that [3H]metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. [3H]Metolazone bound with high affinity (Kd = 4.27 nM) to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or no binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated with their clinical potency. Halide anions (Cl-, Br-, and I-) specifically inhibited high-affinity binding of [3H]metolazone to this site. [3H]Metolazone also bound with lower affinity (Kd = 289 nM) to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had Ki values of 0.6-10 microM for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity [3H]metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics. (+info)