RESUMEN
Isosymmetric structural phase transition (IPT, type 0), in which there are no changes in the occupation of Wyckoff positions, the number of atoms in the unit cell, and the space group symmetry, is relatively uncommon. Chlorothiazide, a diuretic agent with a secondary function as an antihypertensive, has been proven to undergo pressure-induced IPT of Form I to Form II at 4.2 GPa. For that reason, it has been chosen as a model compound in this study to determine if IPT can be predicted in silico using periodic DFT calculations. The transformation of Form II into Form I, occurring under decompression, was observed in geometry optimization calculations. However, the reverse transition was not detected, although the calculated differences in the DFT energies and thermodynamic parameters indicated that Form II should be more stable at increased pressure. Finally, the IPT was successfully simulated using ab initio molecular dynamics calculations.
Asunto(s)
Clorotiazida/farmacología , Teoría Funcional de la Densidad , Algoritmos , Antihipertensivos/farmacología , Simulación por Computador , Diuréticos/farmacología , Modelos Teóricos , Simulación de Dinámica Molecular , Transición de Fase , Presión , Teoría Cuántica , TermodinámicaRESUMEN
Calbindin-D28k (CBD-28k) is a calcium binding protein located in the distal convoluted tubule (DCT) and plays an important role in active calcium transport in the kidney. Loop and thiazide diuretics affect renal Ca and Mg handling: both cause Mg wasting, but have opposite effects on Ca excretion as loop diuretics increase, but thiazides decrease, Ca excretion. To understand the role of CBD-28k in renal Ca and Mg handling in response to diuretics treatment, we investigated renal Ca and Mg excretion and gene expression of DCT Ca and Mg transport molecules in wild-type (WT) and CBD-28k knockout (KO) mice. Mice were treated with chlorothiazide (CTZ; 50 mg · kg(-1) · day(-1)) or furosemide (FSM; 30 mg · kg(-1) · day(-1)) for 3 days. To avoid volume depletion, salt was supplemented in the drinking water. Urine Ca excretion was reduced in WT, but not in KO mice, by CTZ. FSM induced similar hypercalciuria in both groups. DCT Ca transport molecules, including transient receptor potential vanilloid 5 (TRPV5), TRPV6, and CBD-9k, were upregulated by CTZ and FSM in WT, but not in KO mice. Urine Mg excretion was increased and transient receptor potential subfamily M, member 6 (TRPM6) was upregulated by both CTZ and FSM in WT and KO mice. In conclusion, CBD-28k plays an important role in gene expression of DCT Ca, but not Mg, transport molecules, which may be related to its being a Ca, but not a Mg, intracellular sensor. The lack of upregulation of DCT Ca transport molecules by thiazides in the KO mice indicates that the DCT Ca transport system is critical for Ca conservation by thiazides.
Asunto(s)
Calbindina 1/metabolismo , Calcio/metabolismo , Clorotiazida/farmacología , Furosemida/farmacología , Túbulos Renales Distales/efectos de los fármacos , Magnesio/metabolismo , Eliminación Renal/efectos de los fármacos , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Animales , Western Blotting , Calbindina 1/deficiencia , Calbindina 1/genética , Calcio/orina , Canales de Calcio/genética , Canales de Calcio/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Genotipo , Túbulos Renales Distales/metabolismo , Magnesio/orina , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína G de Unión al Calcio S100/genética , Proteína G de Unión al Calcio S100/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
The main distal nephron segment sodium transporters are the distal tubule chlorothiazide-sensitive sodium chloride cotransporter (NCC) and the collecting duct amiloride-sensitive epithelial sodium channel (ENaC). The infusion of ghrelin into the renal interstitium stimulates distal nephron-dependent sodium reabsorption in normal rats, but the mechanism is unknown. Here we localize renal ghrelin receptors (GR) to the cortical collecting duct (CCD). Ghrelin significantly increased phosphorylated serum/glucocorticoid-regulated kinase-1 (pSGK1), a major upstream signaling intermediate regulating ENaC. To test whether increased apical membrane αENaC induced the antinatriuresis, ghrelin was infused in the presence of acute and chronic amiloride, a selective inhibitor of ENaC. In the presence of amiloride, renal interstitial ghrelin failed to reduce urine sodium excretion, suggesting that ghrelin-induced sodium reabsorption is dependent on intact ENaC activity. While the main sodium transporter of the CCD is ENaC, NCC is also present. In response to renal interstitial ghrelin infusion, neither total nor phosphorylated NCC levels are altered. Ghrelin-induced sodium reabsorption persisted in the presence of chlorothiazide (selective inhibitor of NCC), indicating that intact NCC activity is not necessary for ghrelin-induced antinatriuresis. Finally, renal interstitial ghrelin infusion significantly increased interstitial cAMP levels and adenylyl cyclase blockade abolished ghrelin-induced antinatriuresis. Thus, GRs expressed in the CCD regulate sodium reabsorption by cAMP-induced trafficking of ENaC to the apical membrane.
Asunto(s)
AMP Cíclico/fisiología , Canales Epiteliales de Sodio/fisiología , Túbulos Renales Colectores/fisiología , Receptores de Ghrelina/fisiología , Transducción de Señal/fisiología , Sodio/metabolismo , Amilorida/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Clorotiazida/farmacología , Femenino , Ghrelina/farmacología , Proteínas Inmediatas-Precoces/fisiología , Modelos Animales , Proteínas Serina-Treonina Quinasas/fisiología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
We report that the loop diuretic drugs bumetanide and furosemide used in the treatment of hypertension are GPR35 agonists. We utilized calcium flux, inositol phosphate accumulation, and dynamic redistribution assays to examine the pharmacology of these compounds on the human, mouse and rat GPR35. While potent on human GPR35, neither bumetanide nor furosemide were active against mouse or rat GPR35. Furthermore, the Na(+)-Cl(-) cotransporter inhibi- tors chlorothiazide and hydrochlorothiazide were inactive against GPR35 in all three species. We also demonstrate that GPR35 is expressed in human skin where it has been shown that loop diuretics inhibit histamine-induced flare and itch response. These findings suggest that GPR35 may play an important role in skin cell biology and be a potential target for the treatment of a variety of immune disorders.
Asunto(s)
Bumetanida/farmacología , Diuréticos/farmacología , Furosemida/farmacología , Receptores Acoplados a Proteínas G/agonistas , Animales , Células CHO , Calcio/metabolismo , Clorotiazida/farmacología , Cricetinae , Células HEK293 , Células HT29 , Humanos , Hidroclorotiazida/farmacología , Fosfatos de Inositol/metabolismo , Ratones , Ratas , Receptores Acoplados a Proteínas G/biosíntesis , Piel/metabolismo , Transfección/métodosRESUMEN
A recently reported stabilization ('splinting') of the resting membrane potential ( Em) observed in amphibian skeletal muscle fibres despite extracellular hyperosmotic challenge has been attributed to high resting ratios of membrane Cl- to K+ permeability ( P Cl/ P K) combined with elevations of their intracellular Cl- concentrations, [Cl-]i, above electrochemical equilibrium by diuretic-sensitive cation-Cl-, Na-Cl (NCC) and/or Na-K-2Cl (NKCC), co-transporter activity. The present experiments localized this co-transporter activity by investigating the effects of established detubulation procedures on Em splinting. They exposed fibres to introduction and subsequent withdrawal of 400 mM extracellular glycerol, high divalent cation concentrations, and cooling. An abolition of tubular access of extracellularly added lissamine rhodamine fluorescence, visualized by confocal microscopy, and of the action potential afterdepolarization together confirmed successful transverse (T-) tubular detachment. Fibre volumes, V , of such detubulated fibres, determined using recently introduced confocal microscope-scanning methods, retained the simple dependence upon 1/[extracellular osmolarity], without significant evidence of the regulatory volume increases described in other cell types, previously established in intact fibres. However detubulation abolished the Em splinting shown by intact fibres. Em thus varied with extracellular osmolarity in detubulated fibres studied in standard, Cl(-)-containing, Ringer solutions and conformed to simple predictions from such changes in assuming that intracellular ion content was conserved and membrane potential change DeltaEm was principally determined by the K+ Nernst potential. Furthermore, cation--Cl- co-transport block brought about by [Cl-]o or [Na+]o deprivation, or inclusion of bumetanide (10 microM) and chlorothiazide (10 microM) in the extracellular fluid gave similar results. When taken together with previous reports of significant Cl- conductances in the surface membrane, these findings suggest a model that contrastingly suggests a T-tubular location for cation--Cl- co-transporter activity or its regulation.
Asunto(s)
Potenciales de Acción/fisiología , Transporte Iónico/fisiología , Músculo Esquelético/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Bumetanida/farmacología , Cationes Bivalentes/química , Clorotiazida/farmacología , Frío , Glicerol/química , Transporte Iónico/efectos de los fármacos , Modelos Biológicos , Músculo Esquelético/efectos de los fármacos , Presión Osmótica , Rana temporariaRESUMEN
Thiazide diuretics are specific inhibitors of the Na-Cl cotransporter in the distal convoluted tubule (DCT). In addition to producing diuresis and natriuresis, they have a hypocalciuric effect. Recently, two apical calcium channels have been identified, transient receptor potential vanilloid 5 (TRPV5) and TRPV6; both are expressed in the DCT. We studied the effects of thiazides on mouse renal calcium handling and renal gene expression of TRPV5 and TRPV6, as well as calbindin-D(28k) and calbindin-D(9k), both of which are calcium transport facilitators located in the DCT. Upregulation of renal TRPV5 was found 4 h after intraperitoneal injection of chlorothiazide (CTZ) at both 25 and 50 mg/kg, but not at 100 mg/kg. Chronic treatment with CTZ at 25 mg/kg twice daily for 3 days, with or without salt supplementation of 0.8% NaCl and 0.1% KCl in the drinking water, caused hypocalciuria, but the gene expression patterns were different. Without salt supplementation, mice developed volume contraction and there were no changes in gene expression. When volume contraction was prevented by salt supplementation, there was a significant increase in gene expression of TRPV5, calbindin-D(28k), and calbindin-D(9k). Salt supplementation alone also induced significant upregulation of TRPV5, TRPV6, and both calbindins. The upregulation of TRPV5 by CTZ and salt supplementation and salt alone was further confirmed with immunofluorescent staining studies. Our studies suggest that thiazides induce hypocalciuria through different mechanisms depending on volume status. With volume contraction, increased calcium reabsorption in the proximal tubule plays the major role. Without volume contraction, hypocalciuria is probably achieved through increased calcium reabsorption in the DCT by the activation of a transcellular calcium transport system and upregulation of apical calcium channel TRPV5, calbindin-D(28k), and calbindin-D(9k).
Asunto(s)
Canales de Calcio/genética , Calcio/orina , Regulación de la Expresión Génica/efectos de los fármacos , Riñón/metabolismo , Proteína G de Unión al Calcio S100/genética , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Animales , Calbindinas , Calcio/metabolismo , Clorotiazida/administración & dosificación , Clorotiazida/efectos adversos , Clorotiazida/farmacología , Diuréticos , Técnica del Anticuerpo Fluorescente , Riñón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidores de los Simportadores del Cloruro de Sodio/efectos adversos , Canales Catiónicos TRPVRESUMEN
Glutamate receptor currents were examined in horizontal cells from cultured human retina using whole-cell recording procedures. Horizontal cells possess both AMPA and kainate receptors and both produce significant sustained currents. The kainate-induced current did not show significant desensitization and was not enhanced by concanavalin A. The sustained AMPA current was smaller than the kainate current, but the difference was almost entirely due to pronounced desensitization. The horizontal cell AMPA current was enhanced by cyclothiazide but not by PEPA, indicating the presence of the flip receptor variant. GYKI-52466 blocked the AMPA response (IC50 = 5 microM against 100 microM AMPA) but also blocked the kainate response (IC50 = 45 microM against 100 microM kainate). The diversity of glutamate receptors in human horizontal cells suggests that synaptic input to these neurons may be multiplexed through both kainate and AMPA channels.
Asunto(s)
Neuronas/metabolismo , Receptores AMPA/metabolismo , Receptores de GABA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Ácido Kaínico/metabolismo , Retina/metabolismo , Benzodiazepinas/farmacología , Clorotiazida/farmacología , Concanavalina A/farmacología , Técnicas de Cultivo , Conductividad Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Humanos , Ácido Kaínico/farmacología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/fisiología , Receptores de GABA/fisiología , Receptores de Glutamato/fisiología , Receptores de Ácido Kaínico/antagonistas & inhibidores , Receptores de Ácido Kaínico/fisiología , Retina/citología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Freshwater (FW) rainbow trout (Oncorhynchus mykiss) urinary bladders mounted in vitro under symmetrical saline conditions displayed electroneutral active absorption of Na(+) and Cl(-) from the mucosal side; the transepithelial potential (V(t)) was 0.1 mV, and the short-circuit current was less than 1 microA cm(-2). Removal of Na(+) from mucosal saline decreased Cl(-) absorption by 56% and removal of Cl(-) decreased Na(+) absorption by 69%. However, active net absorption of both Na(+) and Cl(-) was not abolished when Cl(-) or Na(+) was replaced with an impermeant ion (gluconate or choline, respectively). Under physiological conditions with artificial urine (¿Na(+) = 2.12 mM, ¿Cl(-) = 3.51 mM) bathing the mucosal surface and saline bathing the serosal surface, transepithelial potential (V(t)) increased to a serosal positive approximately +7.6 mV. Unidirectional influx rates of both Na(+) and Cl(-) were 10-20-fold lower but active absorption of both ions still occurred according to the Ussing flux ratio criterion. Replacement of Na(+) with choline, or Cl(-) with gluconate, in the mucosal artificial urine yielded no change in unidirectional influx of Cl(-) or Na(+), respectively. However, kinetic analyses indicated a decrease in maximum Na(+) transport rate (J(max)) of 66% with no change in affinity (K(m)) in the low Cl(-) mucosal solution relative to the control solution. Similarly, there was a 79% decrease in J(max) values for Cl(-), again with no change in K(m), in the low-Na(+) mucosal bathing. The mucosal addition of DIDS, amiloride or bumetanide (10(-4) M) had no effect on either Na(+) or Cl(-) transport, under either symmetrical saline or artificial urine/saline conditions. Addition of the three drugs simultaneously (10(-4) M), or chlorothiazide (10(-3) M), under symmetrical saline conditions also had no effect on Na(+) or Cl(-) transport rates. Cyanide (10(-3) M) addition to mucosal artificial urine caused a slowly developing decrease of Na(+) influx to 59% and Cl(-) influx to 50% in the period after drug addition. Na(+) and Cl(-) reabsorption appears to be a partially coupled process in the urinary bladder of O. mykiss; transport mechanisms are both dependent upon and independent of the other ion.
Asunto(s)
Cloruros/metabolismo , Oncorhynchus mykiss/metabolismo , Sodio/metabolismo , Vejiga Urinaria/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Amilorida/farmacología , Animales , Bumetanida/farmacología , Clorotiazida/farmacología , Electrofisiología , Técnicas In Vitro , Transporte Iónico , Cianuro de Sodio/farmacología , Vejiga Urinaria/efectos de los fármacosRESUMEN
In order to clarify the effects on sodium reabsorption in the loop of Henle of methazolamide (a carbonic anhydrase inhibitor), chlorothiazide and the loop diuretics frusemide and bumetanide, superficial loops were perfused in vivo in anaesthetized rats and the individual diuretics were included in the perfusate. Differentiation between effects in the pars recta and in the thick ascending limb of Henle (TALH) was achieved by comparing responses to the diuretics when using a standard perfusate, designed to mimic native late proximal tubular fluid, and a low-sodium perfusate, designed to block net sodium reabsorption in the pars recta. With the standard perfusate, methazolamide caused decreases in sodium reabsorption (J(Na)) and water reabsorption (J(V)); with the low-sodium perfusate, a modest effect on J(Na) persisted, suggesting that carbonic anhydrase inhibition reduces sodium reabsorption in both the pars recta and the TALH. The effects of chlorothiazide were very similar to those of methazolamide with both the standard and low-sodium perfusates, suggesting that chlorothiazide also inhibits sodium reabsorption in the pars recta and TALH, perhaps through inhibition of carbonic anhydrase. With the standard perfusate, both frusemide and bumetanide produced the expected large decreases in J(Na), but J(V) was also lowered. With the low-sodium perfusate, the inhibitory effects of the loop diuretics, particularly those of frusemide, were substantially reduced, while net potassium secretion was found. These observations indicate that a significant component of the effect of frusemide (and possibly of bumetanide) on overall sodium reabsorption is located in the pars recta, and that loop diuretics induce potassium secretion in the TALH.
Asunto(s)
Diuréticos/farmacología , Asa de la Nefrona/efectos de los fármacos , Absorción/efectos de los fármacos , Animales , Bumetanida/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Clorotiazida/farmacología , Furosemida/farmacología , Asa de la Nefrona/metabolismo , Masculino , Metazolamida/farmacología , Ratas , Ratas Sprague-Dawley , Sodio/farmacocinéticaAsunto(s)
Antihipertensivos/uso terapéutico , Diuréticos/uso terapéutico , Hipertensión/tratamiento farmacológico , Antihipertensivos/economía , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Clorotiazida/farmacología , Clorotiazida/uso terapéutico , Diuréticos/economía , Diuréticos/farmacología , Edema/tratamiento farmacológico , Furosemida/farmacología , Furosemida/uso terapéutico , Guías como Asunto , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/prevención & control , Humanos , Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Sodio/metabolismo , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Inhibidores de los Simportadores del Cloruro de Sodio/uso terapéutico , Triantereno/farmacología , Triantereno/uso terapéutico , Resistencia Vascular/efectos de los fármacosRESUMEN
Genetic analysis has demonstrated complete linkage between the human thiazide-sensitive sodium chloride cotransporter gene (NCCT or TSC) and Gitelman's syndrome (GS). Several genomic NCCT mutations have been reported. This study was performed to determine whether peripheral blood mononuclear cells (PBMC) express NCCT mRNA and whether defective PBMC NaCl cotransport could be demonstrated in GS. PBMC were isolated from two brothers with GS, their parents, and healthy control subjects. Northern analysis revealed that NCCT mRNA is expressed in PBMC. The sequence of full-length NCCT cDNA amplified from normal PBMC was identical to human renal NCCT cDNA. Two different mutations were detected in the patients' NCCT cDNA (compound heterozygote). In cDNA derived from the patient's maternal allele, exon 24 was deleted, resulting in a premature stop codon (after amino acid 920). cDNA derived from the patient's paternal allele had an additional 119-bp insertion between exons 3 and 4, generating a premature stop codon (after amino acid 187). The patient's genomic DNA had a previously described 5' splice site mutation in intron 24, GGT --> GTT (maternal allele), and a new 3' splice site mutation in intron 3, CAG --> CAA (paternal allele), which resulted in the activation of a nearby cryptic splice site in intron 3. The latter mutation was not present in 300 normal chromosomes. To determine the functional significance of these findings, chlorothiazide-inhibitable 22Na uptake was measured in PBMC from control subjects, the parents, and the patients with GS in the presence of bumetanide. In control PBMC, chlorothiazide inhibited 22Na uptake by approximately 9%. PBMC from the two patients with GS failed to respond to chlorothiazide. These results demonstrate that PBMC can be used for mutational analysis of NCCT mRNA in patients with GS. Furthermore, functional evidence is provided that the underlying cause of GS is defective NCCT NaCl cotransport.
Asunto(s)
Síndrome de Bartter/sangre , Síndrome de Bartter/genética , Proteínas Portadoras/genética , Monocitos/metabolismo , Mutación/genética , ARN Mensajero/metabolismo , Simportadores , Adulto , Secuencia de Bases , Proteínas Portadoras/efectos de los fármacos , Clorotiazida/farmacología , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Simportadores del Cloruro de SodioRESUMEN
In Ménière's disease, an inner ear disorder related to an endolymphatic hydrops, an alteration of the functioning of the endolymphatic sac has been proposed. The endolymphatic sac is assumed to be involved in the secretion/resorption of endolymph. The epithelial transport systems have been indirectly studied by the recording of the endolymphatic sac transepithelial potential (ESP) in control conditions and after the local injection of drugs such as diuretics that have been proposed in the treatment of Ménière's disease. The ESP was recorded, in vivo, in guinea pigs up to 150 minutes after the perisaccular injection of 5 microL of a 150 mmol/L (mM) NaCl solution containing various drugs known to inhibit ionic transport systems. The initial ESP was +8.4+/-0.3 mV (mean +/- SEM, n = 78). The basolateral injection of 5 microL of 150 mM NaCl induced an ESP decrease of 64%+/-6.0% (n = 12), 5 minutes after the end of the injection. Then ESP increased, returning to its initial value at 60 minutes and surpassing it at 120 minutes. Diuretics such as acetazolamide (10[-3] mol/L [M]), an inhibitor of carbonic anhydrase, and amiloride (10[-4] M), an inhibitor of Na channel or Na/H exchanger, decreased the ESP recovery. At variance, bumetanide (10[-6] M, 10[-4] M), the Na-K-Cl cotransport inhibitor, and chlorothiazide (10[-4] M), a Na-Cl cotransporter inhibitor, failed to alter the ESP as compared with the control group. Ouabain (10[-3] M), the Na+,K+-adenosine triphosphatase (ATPase) inhibitor, prevented the ESP recovery otherwise observed 60 minutes after the NaCl injection. Bafilomycin A1, the inhibitor of the vacuolar-type H+-ATPase, prevented the recovery of the ESP with a log-dose/effect (10[-5] M, 10[-6] M, 10[-8] M). Disulfonic acid stilbene (DIDS) (10[-4] M), an inhibitor of transporters involving HCO3-, also prevented the ESP recovery. These results suggest that the genesis of the ESP was highly dependent on acid-base transport systems including carbonic anhydrase, a vacuolar-type H+-ATPase, and an anionic transport system blocked by DIDS. Further studies are needed to confirm the alteration of the acid-base balance in this epithelium and its possible involvement in the pathogenesis of Ménière's disease.
Asunto(s)
Diuréticos/farmacología , Saco Endolinfático/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Macrólidos , Potenciales de la Membrana/efectos de los fármacos , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Acetazolamida/farmacología , Desequilibrio Ácido-Base/complicaciones , Desequilibrio Ácido-Base/fisiopatología , Amilorida/farmacología , Animales , Antibacterianos/farmacología , Bumetanida/farmacología , Carbonatos/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Canales de Cloruro/antagonistas & inhibidores , Clorotiazida/farmacología , Endolinfa/metabolismo , Hidropesía Endolinfática/etiología , Hidropesía Endolinfática/fisiopatología , Saco Endolinfático/metabolismo , Saco Endolinfático/fisiopatología , Inhibidores Enzimáticos/farmacología , Epitelio/metabolismo , Epitelio/fisiopatología , Cobayas , Masculino , Enfermedad de Meniere/etiología , Enfermedad de Meniere/fisiopatología , Ouabaína/farmacología , Bloqueadores de los Canales de Potasio , ATPasas de Translocación de Protón/antagonistas & inhibidores , Bloqueadores de los Canales de Sodio , Cloruro de Sodio , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Factores de TiempoRESUMEN
Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel alpha1 pore-forming subunit, alpha1C, and one isoform of the calcium channel beta accessory subunit, beta3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the alpha1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the beta3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i. beta3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.
Asunto(s)
Canales de Calcio/fisiología , Clorotiazida/farmacología , Células Epiteliales/fisiología , Túbulos Renales Distales/fisiología , Oligonucleótidos Antisentido/farmacología , Hormona Paratiroidea/farmacología , Animales , Calcio/metabolismo , Canales de Calcio/biosíntesis , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L , Células Cultivadas , Cartilla de ADN , Células Epiteliales/efectos de los fármacos , Cinética , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Metabolic alkalosis leads to renal magnesium conservation, whereas metabolic acidosis is associated with urinary magnesium wasting. Micropuncture studies suggest that these actions affect magnesium transport in the distal tubule. The cellular mechanisms of acid-base changes were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration ([Mg2+]i) was determined by microfluorescence using the Mg(2+)-responsive dye, mag-fura 2. Mg2+ transport was assessed as a function of change in [Mg2+]i with time following placement of Mg(2+)-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+, d([Mg2+]i)/dt, into Mg(2+)-depleted cells determined with a buffer solution of pH 7.4 was 178 +/- 21 nM/s. Mg2+ uptake at pH 8.0 was markedly increased 278 +/- 35 nM/s, whereas transport at pH 6.0 was significantly reduced to 121 +/- 15 nM/s. Mg2+ uptake at pH 7.4 was not stimulated with 20 or 40 mM bicarbonate, nor were the differences in Mg2+ uptake with pH associated with changes in membrane voltage. Mg2+ uptake was stimulated with membrane hyperpolarization at pH 6.0 but not at pH 8.0. Chlorothiazide (10(-4) M), which stimulates Mg2+ uptake by hyperpolarizing the membrane voltage, increased uptake at pH 6.0, 59 +/- 14%, but decreased it at alkaline pH of 8.0, -55 +/- 3%. Accordingly, MDCT cells become refractory to the stimulating effects of hyperpolarization at alkaline pH values. These studies show that protons may directly affect Mg2+ transport in MDCT cells.
Asunto(s)
Equilibrio Ácido-Base , Túbulos Renales Distales/metabolismo , Magnesio/farmacocinética , Animales , Línea Celular Transformada , Clorotiazida/farmacología , Electrofisiología , Concentración de Iones de Hidrógeno , Túbulos Renales Distales/citología , Túbulos Renales Distales/fisiología , Magnesio/antagonistas & inhibidores , Membranas/fisiología , Ratones , Nifedipino/farmacología , Bicarbonato de Sodio/farmacologíaRESUMEN
The use of the distally-acting diuretic, chlorothiazide, has been reported to have important effects on renal magnesium handling. The cellular mechanisms of chlorothiazide action on Mg2+ uptake was investigated in immortalized mouse distal convoluted tubule (MDCT) cells. Intracellular free Mg2+ concentration was determined by microfluorescence. Mg2+ transport was measured as a function of change in intracellular Mg2+ concentration with time following placement of Mg2+-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+ into Mg2+-depleted cells was 179 +/- 28 nM/second. Mg2+ uptake was dependent on the membrane voltage as membrane hyperpolarization enhanced uptake whereas depolarization diminished transport. Chlorothiazide increased Mg2+ uptake by 58%, from 179 +/- 28 to 283 +/- 23 nM/second. The ability of chlorothiazide to stimulate Mg2+ uptake in MDCT cells was concentration-dependent and related to the diuretic-induced hyperpolarization of the plasma membrane. These studies support the notion that acute chlorothiazide administration enhances renal magnesium conservation through its effects on Mg2+ transport within the distal convoluted tubule. Since chronic chlorothiazide administration may result in hypokalemia as well as hypomagnesemia, Mg2+ uptake was determined in potassium-depleted MDCT cells. Mg2+ uptake was diminished, 80 +/- 24 nM/second, in potassium depleted cells. Hyperpolarization of the plasma membrane with the cell permanent anion, SCN-, corrected Mg2+ uptake in potassium depleted cells suggesting that the basis for diminished uptake may, in part, be due to depolarization of the membrane voltage. In summary, acute chlorothiazide stimulates Mg2+ transport in MDCT cells. We postulate that chronic chlorothiazide use may lead to hypokalemia that in turn diminishes Mg2+ transport in the distal tubule resulting in urinary magnesium-wasting.
Asunto(s)
Clorotiazida/farmacología , Túbulos Renales Distales/efectos de los fármacos , Túbulos Renales Distales/metabolismo , Magnesio/metabolismo , Potasio/metabolismo , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Animales , Línea Celular , Diuréticos , Hipopotasemia/inducido químicamente , Hipopotasemia/metabolismo , Transporte Iónico/efectos de los fármacos , Túbulos Renales Distales/citología , Cinética , Potenciales de la Membrana , RatonesRESUMEN
The use of thiazide diuretics is associated with increased bone mineral density and, in some studies with reduced incidence of fractures, suggesting a potential role for these drugs in the treatment of osteoporosis. Our objective was to examine the effects of thiazides on osteoblast-like cells using the rat UMR-106 osteosarcoma cell line. Treatment of UMR-106 cells with chlorothiazide caused membrane depolarization and a rise of intracellular calcium but had no effect on adenosine 3,5'-cyclic monophosphate accumulation. The rise of intracellular calcium was partially inhibited by nifedipine and removal of extracellular calcium, indicating calcium uptake from the extracellular media, as well as by thapsigargin or dantrolene, indicating contributions from calcium release from intracellular stores. Reverse transcriptase-polymerase chain reaction was used to isolate a partial cDNA clone for the thiazide-sensitive sodium-chloride cotransporter from UMR-106 cells that hybridized to 5.0- and 11.0-kilobase mRNAs when Northern blot analysis was conducted. Antisense oligonucleotides to the sodium-chloride cotransporter specifically inhibited the chlorothiazide-induced depolarization and rise of intracellular calcium and reduced immunofluorescence staining for the sodium-chloride cotransporter protein in UMR-106 cells. We conclude that thiazide diuretics inhibit sodium-chloride cotransporter activity in UMR-106 cells, thereby altering intracellular calcium regulation. These results provide evidence for direct effects of thiazide diuretics on bone cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Simportadores , Animales , Calcio/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Clorotiazida/antagonistas & inhibidores , Clorotiazida/farmacología , ADN Complementario/aislamiento & purificación , Diuréticos , Electrofisiología , Técnica del Anticuerpo Fluorescente , Membranas Intracelulares/metabolismo , Oligonucleótidos Antisentido/farmacología , Concentración Osmolar , Osteoblastos/fisiología , Ratas , Simportadores del Cloruro de Sodio , Células Tumorales CultivadasRESUMEN
1. The effects of prolonged chlorothiazide treatment of left ventricular failure on cardiac hypertrophy, circulating vasoactive hormones and exchangeable body sodium were examined in rats with chronic myocardial infarction induced by left coronary artery ligation. Chlorothiazide therapy commenced either immediately or 2 weeks after infarction. For 4 weeks, the rats were given either chlorothiazide (50 mg day-1 kg-1) in their drinking water or drinking water alone. 2. Cardiac weight increased in untreated rats with infarction in comparison with sham-operated controls, indicating the presence of chronic left ventricular dysfunction, although exchangeable body sodium, plasma renin activity, plasma vasopressin and plasma osmolality remained unchanged. 3. Chlorothiazide raised haematocrit and plasma renin activity equally in rats with and without infarction, although exchangeable body sodium, plasma vasopressin and plasma osmolality were not changed by the treatment. Plasma atrial natriuretic peptide was 2-fold higher in rats with infarction and this response was not affected by chlorothiazide treatment. Chlorothiazide therapy did not prevent or reverse cardiac hypertrophy. 4. Chronic diuretic therapy in this experimental model of heart failure did not reduce extracellular sodium, plasma vasopressin or the extent of ventricular hypertrophy, possibly because the condition was associated with activation of the renin-angiotensin system.
Asunto(s)
Clorotiazida/farmacología , Diuréticos/farmacología , Infarto del Miocardio/tratamiento farmacológico , Sulfonamidas , Disfunción Ventricular Izquierda/tratamiento farmacológico , Animales , Arginina Vasopresina/metabolismo , Factor Natriurético Atrial/metabolismo , Cardiomegalia/patología , Enfermedad Crónica , Femenino , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Miocardio/patología , Tamaño de los Órganos , Ratas , Ratas Wistar , Factores de Tiempo , Disfunción Ventricular Izquierda/metabolismo , Disfunción Ventricular Izquierda/patologíaRESUMEN
The current experiments were designed to test the hypothesis that adrenal steroids increase thiazide-sensitive Na and Cl transport by the mammalian renal distal convoluted tubule (DCT). Male Sprague-Dawley rats were adrenalectomized and received steroid hormones by osmotic pumps. Six groups of animals were studied as follows: group I, no hormones; group II, replacement levels of dexamethasone only; group III, replacement levels of aldosterone only; group IV, replacement levels of both hormones; group V; replacement levels of aldosterone and high levels of dexamethasone; and group VI, replacement levels of dexamethasone and high levels of aldosterone. Circulating levels of both hormones were found to be in the high physiological range when infused at the high rate. In vivo microperfusion of distal tubules was performed to determine rates of Na and Cl transport. Chlorothiazide was used to assess the magnitude of electroneutral Na-Cl cotransport. Both aldosterone and dexamethasone stimulated thiazide-sensitive Na and Cl transport by the distal tubule by more than fivefold. [3H]metolazone binding was measured to assess the number of thiazide-sensitive Na-Cl cotransporters in renal cortex. Each steroid also increased the number of [3H]metolazone binding sites in kidney cortex more than threefold. The results are consistent with the presence of both mineralocorticoid and glucocorticoid receptors in the mammalian DCT. Physiological changes in circulating levels of adrenal steroids may affect renal NaCl excretion in part by regulating the rate of electroneutral Na-Cl absorption by the DCT.
Asunto(s)
Aldosterona/farmacología , Proteínas Portadoras/metabolismo , Clorotiazida/farmacología , Dexametasona/farmacología , Túbulos Renales Distales/metabolismo , Simportadores , Adrenalectomía , Animales , Sitios de Unión , Combinación de Medicamentos , Corteza Renal/metabolismo , Masculino , Metolazona/metabolismo , Ratas , Ratas Sprague-Dawley , Simportadores del Cloruro de SodioRESUMEN
Possible mechanisms of primary fluid formation by macropodine parotid glands were investigated in anaesthetized red kangaroos using ion transport inhibitors. Carotid plasma amiloride concentrations of 0.05-0.5 mmol.l-1 progressively reduced a stable acetylcholine-evoked half-maximal flow rate of 2.0 +/- 0.04 to 0.22 +/- 0.024 ml.min-1 (mean +/- SEM). Concurrently, saliva bicarbonate concentration and secretion fell (135 +/- 1.6 to 67 +/- 1.7 mmol.l-1 and 272 +/- 7.6 to 15 +/- 2.6 mumol.min-1, respectively); [phosphate], [chloride] and [sodium] rose and [potassium] and osmolality were unaltered. High-rate cholinergic stimulation did not increase saliva flow beyond 11 +/- 1.0% of that for equivalent pre-amiloride stimulation. Equipotent levels of amiloride and methazolamide given concurrently were no more effective at blocking flow and bicarbonate secretion than when given separately. Furosemide (up to 2 mmol.l-1), bumetanide (up to 0.2 mmol.l-1) and ethacrynate (1 mmol.l-1) in carotid plasma had no effect on salivary flow or ion concentrations. During methazolamide blockade, furosemide did not curtail the concurrent increase in salivary [chloride]. Chlorothiazide at 0.25-1.0 mmol.l-1 caused progressive depression of saliva flow and [bicarbonate], and elevation of [chloride]. 4-acetamido-4'-isothiocyanatostilbene-2,2'disulphonic acid at 0.1 mmol.l-1 was without effect, whereas at 0.5 mmol.l-1 it stimulated fluid secretion and increased saliva [protein], [sodium], [potassium], [bicarbonate] and osmolality. Concurrently, mean arterial blood pressure and pulse pressure fell and heart rate, haematocrit and carotid artery plasma flow rose. These responses were absent if saliva flow was kept constant by reduction in cholinergic stimulation during 4-acetamido-4-isothiocyanatostilbene-2,2'disulphonic acid administration. It is concluded that secretion of primary fluid by the kangaroo parotid is initiated mainly (> 90%) by secretion of bicarbonate which is formed in the endpiece cells from CO2 delivered by the circulation. No evidence was found for initiation of fluid secretion by chloride transport involving basolateral Na(+)-K(+)-2Cl- symports, Na(+)-Cl- symports or Cl-/HCO3- antiports.
Asunto(s)
Diuréticos/farmacología , Transporte Iónico/efectos de los fármacos , Glándula Parótida/metabolismo , Amilorida/metabolismo , Amilorida/farmacología , Animales , Bicarbonatos/metabolismo , Presión Sanguínea/fisiología , Bumetanida/metabolismo , Bumetanida/farmacología , Arterias Carótidas/fisiología , Clorotiazida/metabolismo , Clorotiazida/farmacología , Diuréticos/metabolismo , Electrólitos/sangre , Electrólitos/metabolismo , Ácido Etacrínico/metabolismo , Ácido Etacrínico/farmacología , Femenino , Furosemida/metabolismo , Furosemida/farmacología , Frecuencia Cardíaca/fisiología , Macropodidae/fisiología , Masculino , Metazolamida/metabolismo , Metazolamida/farmacología , Concentración Osmolar , Glándula Parótida/efectos de los fármacos , Flujo Sanguíneo Regional/fisiología , Saliva/efectos de los fármacos , Saliva/metabolismoRESUMEN
Sodium transport across apical membranes of distal convoluted tubules is thought to be mediated by Na-Cl cotransport and conductive Na entry. Immortalized mouse distal convoluted tubule cells were used to characterize Na entry pathways. Chlorothiazide, an inhibitor of Na-Cl cotransport, and amiloride, which blocks epithelial Na channels, reduced ouabain-suppressible oxygen consumption by 40 and 35%, respectively. In simple buffer solutions, free of bicarbonate, phosphate, or formate, chlorothiazide inhibited Na uptake by 44% and Cl uptake by 48%. Michaelis constants of 21 mM for Na and 14 mM for chloride were calculated. Amiloride inhibited Na uptake by 49% and had no effect on Cl uptake. The calculated Hill coefficient of 1.07 and the equivalence of chlorothiazide-sensitive Na and Cl uptake are consistent with the presence of Na-Cl cotransport. Na-Cl cotransport and amiloride-sensitive Na influx account for 85% of Na entry in distal convoluted tubule cells in the absence of phosphate and formate. The selective Na/H exchange inhibitor ethylisopropyl amiloride had no effect on Na uptake; however, it abolished formate-stimulated Na uptake. The anion exchange blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited Na uptake. These findings are consistent with parallel Na/H and Cl/formate exchange. Na uptake was inhibited 8% by the selective Na/Ca exchange inhibitor, dimethylbenzamil. An additional 7% of Na entry was phosphate dependent and was abolished by phosphonoformic acid, a competitive inhibitor of Na-Pi cotransport. In summary, the majority of Na entry into distal convoluted tubule cells occurs through Na-Cl cotransport and an amiloride-sensitive pathway (75% in presence of phosphate and formate). An additional 13% may enter by Na/H exchange, with the remainder mediated by Na/Ca exchange and Na-Pi cotransport.