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1.
Am J Physiol Cell Physiol ; 323(2): C385-C399, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35759442

RESUMO

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.


Assuntos
Cloretos , Inibidores de Simportadores de Cloreto de Sódio , Animais , Cloretos/metabolismo , Enguias/metabolismo , Mamíferos/metabolismo , Cloreto de Sódio , Inibidores de Simportadores de Cloreto de Sódio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Simportadores de Cloreto de Sódio/genética , Simportadores de Cloreto de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Tiazidas/farmacologia
2.
J Biol Chem ; 279(16): 16553-60, 2004 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-14766743

RESUMO

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.


Assuntos
Proteínas de Transporte/metabolismo , Rim/metabolismo , Receptores de Droga/metabolismo , Simportadores , Adulto , Sequência de Aminoácidos , Animais , Benzotiadiazinas , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cloretos/metabolismo , Diuréticos , Feminino , Humanos , Transporte de Íons , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Ratos , Receptores de Droga/química , Receptores de Droga/genética , Inibidores de Simportadores de Cloreto de Sódio/metabolismo , Simportadores de Cloreto de Sódio , Membro 3 da Família 12 de Carreador de Soluto , Relação Estrutura-Atividade
3.
Kidney Blood Press Res ; 24(1): 57-63, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11174008

RESUMO

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.


Assuntos
Proteínas de Transporte/genética , Hipertensão/genética , Receptores de Droga/genética , Simportadores , Animais , Benzotiadiazinas , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Diuréticos , Regulação da Expressão Gênica , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Hipertensão Renovascular/genética , Hipertensão Renovascular/metabolismo , Hipertensão Renovascular/fisiopatologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Wistar , Receptores de Droga/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/metabolismo , Simportadores de Cloreto de Sódio , Membro 3 da Família 12 de Carreador de Soluto
4.
Am J Physiol Renal Physiol ; 279(1): F161-9, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10894798

RESUMO

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(-).


Assuntos
Proteínas de Transporte/metabolismo , Receptores de Droga/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Sódio/metabolismo , Simportadores , Animais , Bendroflumetiazida/metabolismo , Bendroflumetiazida/farmacologia , Sítios de Ligação/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/genética , Cloretos/metabolismo , Cloretos/farmacologia , Diuréticos , Hidroclorotiazida/metabolismo , Hidroclorotiazida/farmacologia , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Metolazona/metabolismo , Metolazona/farmacologia , Microinjeções , Modelos Biológicos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Concentração Osmolar , Politiazida/metabolismo , Politiazida/farmacologia , Ratos , Receptores de Droga/genética , Sódio/farmacologia , Simportadores de Cloreto de Sódio , Membro 3 da Família 12 de Carreador de Soluto , Xenopus
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