ABSTRACT
SIGNIFICANCE STATEMENT: Thiazide diuretics (thiazides) are among the most widely prescribed drugs worldwide, but their use is associated with glucose intolerance and new-onset diabetes mellitus. The molecular mechanisms remain elusive. Our study reveals that thiazides attenuate insulin secretion through inhibition of the mitochondrial carbonic anhydrase isoform 5b (CA5b) in pancreatic ß cells. We furthermore discovered that pancreatic ß cells express only one functional carbonic anhydrase isoform, CA5b, which is critical in replenishing oxaloacetate in the mitochondrial tricarboxylic acid (TCA) cycle (anaplerosis). These findings explain the mechanism for thiazide-induced glucose intolerance and reveal a fundamental role of CA5b in TCA cycle anaplerosis and insulin secretion in ß cells. BACKGROUND: Thiazide diuretics are associated with glucose intolerance and new-onset diabetes mellitus. Previous studies demonstrated that thiazides attenuate insulin secretion, but the molecular mechanisms remain elusive. We hypothesized that thiazides attenuate insulin secretion via one of the known molecular thiazide targets in ß cells. METHODS: We performed static insulin secretion experiments with islets of wild-type, Sodium/chloride co-transporter (NCC) (SLC12A3), and sodium-driven chloride/bicarbonate exchanger (NDCBE) (SLC4A8) knock-out (KO) mice and with murine Min6 cells with individual knockdown of carbonic anhydrase (CA) isoforms to identify the molecular target of thiazides in ß cells. CA isoform 5b (CA5b) KO mice were then used to assess the role of the putative thiazide target CA5b in ß -cell function and in mediating thiazide sensitivity in vitro and in vivo . RESULTS: Thiazides inhibited glucose- and sulfonylurea-stimulated insulin secretion in islets and Min6 cells at pharmacologically relevant concentrations. Inhibition of insulin secretion by thiazides was CO 2 /HCO 3- -dependent, not additive to unselective CA inhibition with acetazolamide, and independent of extracellular potassium. By contrast, insulin secretion was unaltered in islets of mice lacking the known molecular thiazide targets NCC or NDCBE. CA expression profiling with subsequent knockdown of individual CA isoforms suggested mitochondrial CA5b as a molecular target. In support of these findings, thiazides significantly attenuated Krebs cycle anaplerosis through reduction of mitochondrial oxaloacetate synthesis. CA5b KO mice were resistant to thiazide-induced glucose intolerance, and thiazides did not alter insulin secretion in CA5b KO islets. CONCLUSIONS: Thiazides attenuate insulin secretion via inhibition of the mitochondrial CA5b isoform in ß cells of mice.
Subject(s)
Carbonic Anhydrases , Diabetes Mellitus , Glucose Intolerance , Insulin-Secreting Cells , Islets of Langerhans , Mice , Animals , Insulin Secretion , Thiazides/pharmacology , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporter Inhibitors/pharmacology , Chlorides/metabolism , Glucose/metabolism , Carbonic Anhydrases/metabolism , Sodium/metabolism , Insulin/metabolism , Insulin-Secreting Cells/metabolismABSTRACT
The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the mammalian distal convoluted tubule, and the inhibition of its function with thiazides is widely used for the treatment of arterial hypertension. In mammals and teleosts, NCC is present as one ortholog that is mainly expressed in the kidney. One exception, however, is the eel, which has two genes encoding NCC. The eNCCα is located in the kidney and eNCCß, which is present in the apical membrane of the rectum. Interestingly, the European eNCCß functions as a Na+-Cl- cotransporter that is nevertheless resistant to thiazides and is not activated by low-chloride hypotonic stress. However, in the Japanese eel rectal sac, a thiazide-sensitive NaCl transport mechanism has been described. The protein sequences between eNCCß and jNCCß are 98% identical. Here, by site-directed mutagenesis, we transformed eNCCß into jNCCß. Our data showed that jNCCß, similar to eNCCß, is resistant to thiazides. In addition, both NCCß proteins have high transport capacity with respect to their renal NCC orthologs and, in contrast to known NCCs, exhibit electrogenic properties that are reduced when residue I172 is substituted by A, G, or M. This is considered a key residue for the chloride ion-binding sites of NKCC and KCC. We conclude that NCCß proteins are not sensitive to thiazides and have electrogenic properties dependent on Cl-, and site I172 is important for the function of NCCß.
Subject(s)
Chlorides , Sodium Chloride Symporter Inhibitors , Animals , Chlorides/metabolism , Eels/metabolism , Mammals/metabolism , Sodium Chloride , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporter Inhibitors/pharmacology , Sodium Chloride Symporters/genetics , Sodium Chloride Symporters/metabolism , Solute Carrier Family 12, Member 3/genetics , Thiazides/pharmacologyABSTRACT
The thiazide-sensitive Na+-Cl- cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, state-of-the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics.
Subject(s)
Diuretics/metabolism , Imaging, Three-Dimensional , Kidney Tubules, Distal/metabolism , Sex Characteristics , Animals , Female , Imaging, Three-Dimensional/methods , Kidney Tubules, Distal/diagnostic imaging , Male , Mice , Phosphorylation , Sodium/metabolism , Sodium Chloride Symporter Inhibitors/metabolismABSTRACT
Luminal nucleotide stimulation is known to reduce Na(+) transport in the distal nephron. Previous studies suggest that this mechanism may involve the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), which plays an essential role in NaCl reabsorption in the cells lining the distal convoluted tubule (DCT). Here we show that stimulation of mouse DCT (mDCT) cells with ATP or UTP promoted Ca(2+) transients and decreased the expression of NCC at both mRNA and protein levels. Specific siRNA-mediated silencing of P2Y2 receptors almost completely abolished ATP/UTP-induced Ca(2+) transients and significantly reduced ATP/UTP-induced decrease of NCC expression. To test whether local variations in the intracellular Ca(2+) concentration ([Ca(2+)]i) may control NCC transcription, we overexpressed the Ca(2+)-binding protein parvalbumin selectively in the cytosol or in the nucleus of mDCT cells. The decrease in NCC mRNA upon nucleotide stimulation was abolished in cells overexpressing cytosolic PV but not in cells overexpressing either a nuclear-targeted PV or a mutated PV unable to bind Ca(2+). Using a firefly luciferase reporter gene strategy, we observed that the activity of NCC promoter region from -1 to -2,200 bp was not regulated by changes in [Ca(2+)]i. In contrast, high cytosolic calcium level induced instability of NCC mRNA. We conclude that in mDCT cells: (1) P2Y2 receptor is essential for the intracellular Ca(2+) signaling induced by ATP/UTP stimulation; (2) P2Y2-mediated increase of cytoplasmic Ca(2+) concentration down-regulates the expression of NCC; (3) the decrease of NCC expression occurs, at least in part, via destabilization of its mRNA.
Subject(s)
Kidney Tubules, Distal/metabolism , Receptors, Purinergic P2Y2/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporters/metabolism , Sodium Chloride/metabolism , Adenosine Triphosphate/metabolism , Animals , Calcium/metabolism , Cell Line , Cytosol/metabolism , Down-Regulation , Mice , Promoter Regions, Genetic , RNA, Messenger/genetics , Receptors, Purinergic P2Y2/genetics , Signal Transduction , Sodium Chloride Symporters/genetics , Solute Carrier Family 12, Member 3/genetics , Solute Carrier Family 12, Member 3/metabolism , Uridine Triphosphate/metabolismABSTRACT
The cation chloride cotransporters (CCCs) represent an important family of transporters that plays key roles in vectorial electrolyte movement across epithelia and in intracellular chloride homeostasis of neurons and muscle cells. The CCCs are composed of three broad groups, two of which include multiple isoforms: Na-Cl cotransporter (NCC; SLC12A3), Na-K-2Cl cotransporter (NKCC; SLC12A1-2), and K-Cl cotransporter (KCC; SLC12A4-7). The CCCs are inhibited by clinically relevant drugs, including loop diuretics that inhibit NKCC2 in the renal thick ascending limb and thiazide diuretics that inhibit NCC in the renal distal tubule. For many years, much research on this gene family has centered on understanding ion binding and inhibitor interaction which represent important features of the molecular operation of these transporters. Recently, high resolution structures of bacterial transport proteins related to the CCCs have become available, thus permitting structural context in which to evaluate previous ion and inhibitor studies of the CCCs. In this article, I review past molecular and structure-function studies that have provided key pieces of information about ion binding and inhibitor interaction primarily of NKCC for which we have the most information. I then place these findings into the structural context of recent homology models of NKCC based on the outward-facing open and occluded conformations of the related bacterial transporters. These homology models provide our first glimpse into the fine details of the molecular operation of the CCCs.
Subject(s)
Chlorides/metabolism , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Sodium-Potassium-Chloride Symporters/chemistry , Sodium-Potassium-Chloride Symporters/metabolism , Animals , Binding Sites , Cations , Humans , Kidney/metabolism , Models, Molecular , Muscle Cells/metabolism , Neurons/metabolism , Protein Conformation , Sodium Chloride Symporter Inhibitors/metabolism , Structure-Activity RelationshipABSTRACT
We are showing that chlorothiazide, a diuretic, is an ABCG2 substrate. It is a Biopharmaceutics Classification System/Biopharmaceutics Drug Distribution and Classification System (BCS/BDDCS) Class IV drug with low bioavailability. Therefore, we tested if chlorothiazide interacts with major apically located intestinal efflux transporters. Our data show that chlorothiazide is transported by ABCG2 with a Km value of 334.6 µM and does not interact with ABCB1 or ABCC2. The chlorothiazide-ABCG2 interaction results in a vectorial transport in MDCKII-BCRP and Caco-2 cells with efflux ratios of 36 and 8.1 respectively. Inhibition of ABCG2 in Caco-2 cells reduced the efflux ratio to 1.4, suggesting that ABCG2 plays a role in limiting chlorothiazide bioavailability in humans.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cell Membrane Permeability , Chlorothiazide/metabolism , Diuretics/metabolism , Enterocytes/metabolism , Neoplasm Proteins/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Animals , Biological Transport/drug effects , Caco-2 Cells , Cell Membrane Permeability/drug effects , Dogs , Enterocytes/drug effects , Estrone/analogs & derivatives , Estrone/metabolism , Humans , Intestinal Absorption/drug effects , Kinetics , Madin Darby Canine Kidney Cells , Membrane Transport Modulators/pharmacology , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Transport Vesicles/drug effects , Transport Vesicles/metabolismABSTRACT
In sports, thiazide diuretics are used to flush out previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. Thiazide diuretics include compounds which are very unstable and hydrolyse in aqueous media. Because information regarding the urinary detection of the hydrolysis products is limited, urinary excretion profiles for the hydrolysis product 4-amino-6-chloro-1,3-benzenedisulphonamide were established in 6 healthy volunteers after oral administration of altizide (15 mg per tablet) and hydrochlorothiazide (25mg per tablet). Additionally, the excretion profile of chlorothiazide, a metabolite of altizide and hydrochlorothiazide, was also determined. A quantitative liquid-chromatographic tandem mass spectrometric method to detect the 4 substances was developed and validated. The result of this work shows that altizide is eliminated within 48 h in urine whereas hydrochlorothiazide was detectable after 120 h. Chlorothiazide was determined to be a minor metabolite of altizide and hydrochlorothiazide and could be detected up to 120 h. The hydrolysis product, 4-amino-6-chloro-1,3-benzenedisulphonamide, was detectable 120 h after administration, with concentrations at least 10 times higher than the parent drug. Concentrations ranged between 41-239 and 60-287 ng/mL after altizide and hydrochlorothiazide administration, respectively. The study shows that 4-amino-6-chloro-1,3-benzenedisulphonamide is an important target compound for the long time detection of thiazide diuretics in urine.
Subject(s)
Benzothiadiazines/metabolism , Biomarkers, Pharmacological/urine , Doping in Sports , Hydrochlorothiazide/metabolism , Administration, Oral , Adult , Benzothiadiazines/administration & dosage , Chlorothiazide/urine , Chromatography, Liquid/methods , Diuretics/administration & dosage , Diuretics/metabolism , Female , Humans , Hydrochlorothiazide/administration & dosage , Least-Squares Analysis , Linear Models , Male , Reproducibility of Results , Sensitivity and Specificity , Sodium Chloride Symporter Inhibitors/administration & dosage , Sodium Chloride Symporter Inhibitors/metabolism , Sulfanilamides/urine , Tandem Mass Spectrometry/methodsABSTRACT
In sports, diuretics are used for two main reasons: to flush previously taken prohibited substances with forced diuresis and in sports where weight classes are involved to achieve acute weight loss. A common property observed for thiazides is hydrolysis in aqueous media resulting in the formation of the degradation product aminobenzenedisulphonamide. This degradation product can be observed for several thiazides. Because there is limited information regarding the effect of pH, temperature and light on the stability of thiazides, these parameters were investigated for chlorothiaizide, hydrochlorothiazide and altizide. For all three compounds the degradation product could be detected after incubation at pH 9.5 for 48h at 60 degrees C. At lower pH and temperature the degradation product could not be detected for all compounds. When samples were exposed to UV-light altizide and hydrochlorothiazide were photodegraded to chlorothiazide. When the degradation rate between the different compounds was compared for a given temperature and pH, altizide is the most unstable compound. This study confirms that thiazide degradation products can be formed in urine during transport. Hence doping control laboratories shall include them into their routine testing methods as required by WADA.
Subject(s)
Diuretics/metabolism , Diuretics/urine , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporter Inhibitors/urine , Aniline Compounds/metabolism , Buffers , Diuretics/chemistry , Drug Stability , Drug Storage , Humans , Hydrogen-Ion Concentration , Light , Linear Models , Models, Biological , Molecular Structure , Photochemistry/methods , Photolysis/radiation effects , Reference Standards , Sodium Chloride Symporter Inhibitors/chemistry , Specimen Handling , Spectrophotometry, Ultraviolet , Sulfonamides/metabolism , Temperature , Time Factors , Water/chemistryABSTRACT
The thiazide-sensitive Na+:Cl- cotransporter is the major salt transport pathway in the distal convoluted tubule of the kidney, and a role of this cotransporter in blood pressure homeostasis has been defined by physiological studies on pressure natriuresis and by its involvement in monogenic diseases that feature arterial hypotension or hypertension. Data base analysis revealed that 135 single nucleotide polymorphisms along the human SLC12A3 gene that encodes the Na+:Cl- cotransporter have been reported. Eight are located within the coding region, and one results in a single amino acid change; the residue glycine at the position 264 is changed to alanine (G264A). This residue is located within the fourth transmembrane domain of the predicted structure. Because Gly-264 is a highly conserved residue, we studied the functional properties of this polymorphism by using in vitro mutagenesis and the heterologous expression system in Xenopus laevis oocytes. G264A resulted in a significant and reproducible reduction ( approximately 50%) in (22)Na+ uptake when compared with the wild type cotransporter. The affinity for extracellular Cl- and for thiazide diuretics was increased in G264A. Western blot analysis showed similar immunoreactive bands between the wild type and the G264A cotransporters, and confocal images of oocytes injected with enhanced green fluorescent protein-tagged wild type and G264A cotransporter showed no differences in the protein surface expression level. These observations suggest that the G264A polymorphism is associated with reduction in the substrate translocation rate of the cotransporter, due to a decrease in the intrinsic activity. Our study also reveals a role of the transmembrane segment 4 in defining the affinity for extracellular Cl- and thiazide diuretics.
Subject(s)
Carrier Proteins/metabolism , Kidney/metabolism , Receptors, Drug/metabolism , Symporters , Adult , Amino Acid Sequence , Animals , Benzothiadiazines , Binding Sites/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Chlorides/metabolism , Diuretics , Female , Humans , Ion Transport , Male , Middle Aged , Molecular Sequence Data , Polymorphism, Single Nucleotide , Rats , Receptors, Drug/chemistry , Receptors, Drug/genetics , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporters , Solute Carrier Family 12, Member 3 , Structure-Activity RelationshipABSTRACT
BACKGROUND/AIMS: Several lines of evidence support that the kidney is involved in the increase of arterial blood pressure, and some genetic studies suggest that the thiazide-sensitive Na+:Cl- cotransporter could be implicated in the development of hypertension. In the present study, we analyzed the Na+:Cl- cotransporter mRNA levels in the kidney during the development of hypertension in three experimental models. METHODS: The first model included 18 spontaneously hypertensive rats studied at 4, 10, and 16 weeks of age. The second model included 28 Wistar rats with two-kidney, one-clip Goldblatt hypertension studied at 7, 14, 21, and 28 days. The third model included 6 Wistar rats treated with N(G)-nitro-L-arginine methyl ester during 10 days. Respective controls were studied for all models. At the end of each experimental period, the systolic blood pressure was measured in the tail by plethysmography. Individual renal cortex total RNA was extracted, and the mRNA levels of the thiazide-sensitive Na+:Cl- cotransporter were assessed following a semiquantitative RT-PCR strategy. RESULTS: All experimental models developed systemic hypertension. However, the level of mRNA expression of the Na+:Cl- cotransporter did not change in any of the models studied as compared with their respective controls. CONCLUSION: Our results suggest that a change in mRNA levels of the thiazide-sensitive Na+:Cl- cotransporter is not associated with the development of hypertension in spontaneously hypertensive rats, in rats with renovascular hypertension, nor in rats with hypertension induced by nitric oxide synthesis inhibition.
Subject(s)
Carrier Proteins/genetics , Hypertension/genetics , Receptors, Drug/genetics , Symporters , Animals , Benzothiadiazines , Carrier Proteins/metabolism , Disease Models, Animal , Diuretics , Gene Expression Regulation , Hypertension/metabolism , Hypertension/physiopathology , Hypertension, Renovascular/genetics , Hypertension, Renovascular/metabolism , Hypertension, Renovascular/physiopathology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Rats, Wistar , Receptors, Drug/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporters , Solute Carrier Family 12, Member 3ABSTRACT
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(-).
Subject(s)
Carrier Proteins/metabolism , Receptors, Drug/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporter Inhibitors/pharmacology , Sodium/metabolism , Symporters , Animals , Bendroflumethiazide/metabolism , Bendroflumethiazide/pharmacology , Binding Sites/drug effects , Biological Transport/drug effects , Carrier Proteins/genetics , Chlorides/metabolism , Chlorides/pharmacology , Diuretics , Hydrochlorothiazide/metabolism , Hydrochlorothiazide/pharmacology , Hydrogen-Ion Concentration , Inhibitory Concentration 50 , Kinetics , Metolazone/metabolism , Metolazone/pharmacology , Microinjections , Models, Biological , Oocytes/drug effects , Oocytes/metabolism , Osmolar Concentration , Polythiazide/metabolism , Polythiazide/pharmacology , Rats , Receptors, Drug/genetics , Sodium/pharmacology , Sodium Chloride Symporters , Solute Carrier Family 12, Member 3 , XenopusABSTRACT
Cl- apically enters the epithelium of rabbit gallbladder by a Na+-Cl- symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl- conductance (G(Cl)), the symport inhibition might be merely due to a short circuit of the symport by G(Cl) rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl- uptake, electrophysiological determinations of intracellular Cl- and Na+ activities (a(i,Cl) and a(i,Na)) with selective theta microelectrodes and molecular biology methods were used. The 13Cl- uptake proved to be a measurement of the apical unidirectional Cl- influx (Jmc) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by G(Cl) activation or abolition. After HCTZ treatment the decrease in a(i,Cl) (measured as the initial rate or in 3 min) was larger than the decrease in a(i,Na). The difference was reduced to one third in a group of epithelia in which the elicited G(Cl) was reduced to one third; moreover it was abolished in any case when G(Cl) was abolished with 10(-4) M SITS. The SITS-insensitive rate of a(i,Cl) decrease was equal to that of the a(i,Na) decrease in any case. Thus the a(i,Cl) decrease displays a component dependent on G(Cl) activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely G(Cl) activation and Na+-Cl- symport inhibition. This Na+-Cl- symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium.
Subject(s)
Benzothiadiazines , Carrier Proteins/metabolism , Gallbladder/metabolism , Receptors, Drug/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Symporters , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/genetics , Cells, Cultured , Chlorides/metabolism , DNA, Complementary , Diuretics , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gallbladder/chemistry , Humans , Intracellular Fluid/metabolism , Male , Molecular Sequence Data , Rabbits , Receptors, Drug/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Homology, Amino Acid , Sodium/metabolism , Sodium Chloride Symporters , Solute Carrier Family 12, Member 3ABSTRACT
The role of tubular accumulation in renal disposition and diuretic efficacy of hydrochlorothiazide was studied in the isolated perfused rat kidney. Hydrochlorothiazide resulted in a dose-dependent increase in the fractional excretion of sodium, chloride and potassium, and in urinary flow and pH. Renal clearance of the drug was low as a result of a low extraction ratio and extensive nonionic back-diffusion. Hydrochlorothiazide was subject to saturable tubular secretion, following Michaelis-Menten kinetics. Parameters obtained after nonlinear regression analysis were a maximum tubular transport velocity of 42 +/- 6 micrograms/min, a Michaelis-Menten constant of secretion of 38 +/- 11 micrograms/ml and a fraction of excreted drug reabsorbed passively of 0.49 +/- 0.03. The thiazide diuretic accumulated extensively in kidney tissue due to active cellular uptake (maximum capacity of renal accumulation of 500 +/- 270 micrograms/g; affinity constant of renal accumulation of 28 +/- 16 micrograms/ml) and passive diffusion. Plots were constructed of the sodium excretion rate versus hydrochlorothiazide perfusate concentration or the renal excretion rate. The perfusate plot could be described by the sigmoid Emax model, while a simplification of the model had to be used for the response curve in urine because a maximum effect was not observed. The apparent maximum effect resulting from the perfusate concentration-response curve and the discrepancy with the renal excretion rate-response curve indicates that the diuretic effect of hydrochlorothiazide is restricted by saturable accumulation and secretion.
Subject(s)
Hydrochlorothiazide/pharmacology , Kidney/drug effects , Sodium Chloride Symporter Inhibitors/pharmacology , Animals , Diuretics , Hydrochlorothiazide/metabolism , In Vitro Techniques , Kidney/metabolism , Kidney Function Tests , Kinetics , Perfusion , Rats , Sodium Chloride Symporter Inhibitors/metabolismABSTRACT
We report here the identification of a cDNA encoding a human thiazide-sensitive sodium-chloride cotransporter (hTSC) using a PCR-based method. The homology of the hTSC with rat TSC (rTSC) and rat bumetanide-sensitive sodium-potassium-chloride cotransporter (rBSC) was 86% and 64%, respectively, at the nucleotide level, and 92% and 61%, respectively, at the amino acid level. Using fluorescence in situ hybridization (FISH), the hTSC gene has been mapped to chromosome 16q13. Northern blot analysis using polyA+RNA from various human tissues, revealed a major 4.5 kb transcript and a minor 6.5 kb specifically in the kidney, but low level of expression was also observed in small intestine, placenta, prostate, colon, and spleen.
Subject(s)
Carrier Proteins/biosynthesis , Carrier Proteins/chemistry , Receptors, Drug/biosynthesis , Receptors, Drug/chemistry , Symporters , Transcription, Genetic , Amino Acid Sequence , Animals , Base Sequence , Benzothiadiazines , Carrier Proteins/genetics , Chromosome Mapping , Chromosomes, Human, Pair 16 , Cloning, Molecular , Colon/metabolism , DNA Primers , DNA, Complementary , Diuretics , Female , Flounder , Humans , In Situ Hybridization, Fluorescence , Intestine, Small/metabolism , Kidney/metabolism , Male , Molecular Sequence Data , Organ Specificity , Placenta/metabolism , Polymerase Chain Reaction , Pregnancy , Prostate/metabolism , RNA, Messenger/biosynthesis , Rats , Receptors, Drug/genetics , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Sodium Chloride Symporter Inhibitors/metabolism , Sodium Chloride Symporters , Solute Carrier Family 12, Member 3 , Spleen/metabolismABSTRACT
Cloned human metabotropic glutamate receptors, mGlu1 alpha and mGlu5 alpha, were functionally expressed in Xenopus oocytes. Cyclothiazide dose-dependently inhibited glutamate-stimulated human mGlu1 alpha responses (IC50 = 18 microM) in a non-competitive manner. In contrast, cyclothiazide slightly potentiated glutamate-stimulated human mGlu5 alpha responses. GYKI 52466 (1-(4-amino-phenyl)-4-methyl-7,8- methyl-endioxyl-5H-2,3-benzodiazepinehydrochloride) did not alter glutamate-stimulated human mGlu1 alpha or human mGlu5 alpha responses, either in the presence or absence of cyclothiazide. Thus, human metabotropic glutamate receptors coupled to phosphoinositide stimulation appear to contain sites sensitive to cyclothiazide but insensitive to GYKI 52466.
Subject(s)
Anti-Anxiety Agents , Benzothiadiazines/pharmacology , Phosphatidylinositols/metabolism , Receptors, Metabotropic Glutamate/drug effects , Sodium Chloride Symporter Inhibitors/pharmacology , Animals , Benzodiazepines/metabolism , Benzodiazepines/pharmacology , Benzothiadiazines/metabolism , Binding Sites , Diuretics , Dose-Response Relationship, Drug , Electrophysiology , Excitatory Amino Acid Antagonists/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Humans , Hydrolysis , Oocytes , Receptors, Metabotropic Glutamate/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Xenopus laevisABSTRACT
The influence of gender and gonadectomy on (1) the density of the renal thiazide-sensitive ion transporter, as quantitated by the ability of renal membranes to bind (3H)metolazone, and (2) the changes in the urinary excretion of electrolytes caused by maximal bendroflumethiazide (BFTZ) in Sprague-Dawley rats was determined. The density of the thiazide receptor was twofold higher (P < 0.001) in females than in males. Orchiectomy increased thiazide receptor significantly in one of two studies (P < 0.01). Ovariectomy decreased thiazide receptor by more than 20% (P < 0.01) in both studies. The rates of the urinary excretion of sodium and chloride after BFTZ and the increases in the urinary excretion of sodium, chloride, and ammonium caused by BFTZ were greater in intact females than in intact males; BFTZ decreased the urinary excretion of calcium 50% in intact females, but not in intact males. Regression analysis of the thiazide receptor (in intact and gonadectomized animals) versus the urinary excretion of electrolytes before and after BFTZ yielded a model in which one-third of the variation in thiazide receptor could be related to the change in the excretion of calcium and ammonium produced by BFTZ, raising the possibility that the density of the thiazide receptor might be related to calcium or acid-base homeostasis. It was concluded that the renal excretion of sodium, chloride, calcium, and ammonium are, in part, controlled by gender and sex hormones via their regulation of the renal density of the thiazide diuretic receptor.
Subject(s)
Carrier Proteins/metabolism , Kidney/metabolism , Receptors, Drug/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Symporters , Animals , Bendroflumethiazide/pharmacology , Carrier Proteins/drug effects , Diuresis/drug effects , Diuretics , Female , Gonadal Steroid Hormones/physiology , Kidney/anatomy & histology , Kidney/drug effects , Male , Metolazone/metabolism , Orchiectomy , Organ Size , Ovariectomy , Rats , Rats, Sprague-Dawley , Receptors, Drug/drug effects , Sex Characteristics , Sodium Chloride Symporter Inhibitors/pharmacology , Sodium Chloride Symporters , Solute Carrier Family 12, Member 3ABSTRACT
Thiazide diuretics increase urinary NaCl excretion primarily by inhibiting Na and Cl transport across the apical membrane of cells in the renal distal tubule. Although these diuretics bind to a membrane protein that couples transport of Na and Cl directly, the molecular nature of this transporter and its localization in the mammalian kidney remain controversial. The present experiments were designed to develop monoclonal antibodies to the high-affinity thiazide diuretic receptor to investigate its molecular characteristics and its cellular and subcellular localization in rabbit kidney. Mice were immunized with high-affinity thiazide diuretic receptors that had been partially purified from rabbit kidney cortex. Resulting hybridomas were screened for the ability to immunoprecipitate thiazide diuretic receptors that were labeled with the thiazide-like diuretic [3H]metolazone. A single hybridoma (MAb JM5) produced antibodies capable of immunoprecipitating up to 80% of the labeled thiazide receptors from solubilized renal cortical membranes. MAb JM5 reacted with a 125-kDa protein on Western blots of solubilized renal cortical apical membranes. It stained the apical membrane of cells in the distal convoluted and connecting tubule but did not stain proximal tubules, glomeruli, or interstitial structures. Less intense staining of apical membranes of principal cells in the collecting tubule and a subpopulation of cells in the thick ascending limb were also present. These results indicate that the high-affinity thiazide diuretic receptor comprises a 125-kDa protein that localizes to the apical membrane of cells in the renal distal tubule.
Subject(s)
Benzothiadiazines , Carrier Proteins , Kidney Cortex/metabolism , Receptors, Drug/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Symporters , Animals , Antibodies, Monoclonal , Binding, Competitive , Cell Membrane/metabolism , Diuretics , Immunohistochemistry , Kidney Tubules, Distal/metabolism , Rabbits , Receptors, Drug/immunology , Sodium Chloride SymportersABSTRACT
The metabolic side effects of thiazide diuretics are believed to be responsible for the failure of thiazide diuretics to reduce cardiovascular morbidity in patients with hypertension. However, the decrease in the incidence of osteoporotic fractures that are associated with thiazide administration may be relevant in elderly patients with arterial hypertension. Spironolactone (SP) appears not to influence the metabolic risk profile of the patient with hypertension, and no studies have examined its effect on calcium metabolism. Therefore, in 22 patients with mild to moderate essential hypertension, the authors performed a parallel, randomized, double-blind, placebo-controlled study that compared the effects on serum urate and lipid, potassium, magnesium, and calcium metabolism of hydrochlorothiazide (HC) (mean [+/- SD] dose, 72 +/- 26 mg/d) and SP (144 +/- 53 mg/d) during a 52-week period. As compared with placebo, HC significantly increased serum urate and total cholesterol concentrations, and decreased serum potassium levels. SP did not affect serum urate or cholesterol levels but increased serum potassium concentrations. Neither diuretic significantly modified magnesium metabolism. Little changes were seen in serum calcium levels during HC or SP treatment, whereas urinary calcium excretion was significantly decreased by HC (mean decrease, 45%; P less than .01) or SP (40%; P less than .01). The authors conclude that SP, in addition to its potassium-sparing properties, has a calcium-sparing effect that may be beneficial for patients in whom reduction of urinary calcium excretion has a therapeutic value.
Subject(s)
Hydrochlorothiazide/pharmacology , Hypertension/metabolism , Sodium Chloride Symporter Inhibitors/metabolism , Spironolactone/pharmacology , Adult , Aged , Blood Pressure/drug effects , Cholesterol/blood , Cholesterol/metabolism , Diuretics , Double-Blind Method , Female , Humans , Hydrochlorothiazide/metabolism , Hypertension/drug therapy , Lipids/blood , Male , Middle Aged , Potassium/blood , Potassium/metabolism , Sodium Chloride Symporter Inhibitors/administration & dosage , Sodium Chloride Symporter Inhibitors/therapeutic use , Spironolactone/metabolism , Time Factors , Uric Acid/blood , Uric Acid/metabolismABSTRACT
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.