RESUMEN
CLC-K1 is a kidney-specific chloride channel that mediates transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) in the inner medulla. Transport of NaCl in the tAL is thought to be a component of urinary concentration in a passive model of the countercurrent multiplication system, but there has been no direct evidence that CLC-K1 is involved in urine concentration. To analyse the physiological function of CLC-K1 in vivo, we generated mice lacking CLC-K1 by targeted gene disruption. Clcnk1-/- mice were physically normal appearance, but produced approximately five times more urine than Clcnk1+/- and Clcnk1+/+ mice. After 24 hours of water deprivation, Clcnk1-/- mice were severely dehydrated and lethargic, with a decrease of approximately 27% in body weight. Intraperitoneal injection of the V2 agonist 1-deamino-8-D-arginine vasopressin (dDAVP) induced a threefold increase in urine osmolarity in Clcnk1+/- and Clcnk1+/+ mice, whereas only a minimal increase was seen in Clcnk1-/- mice, indicating nephrogenic diabetes insipidus. After in vitro perfusion of the tAL, the lumen-to-bath chloride gradient did not produce a diffusion potential in Clcnk1-/- mice in contrast to Clcnk1+/+ and Clcnk1+/- mice. These results establish that CLC-K1 has a role in urine concentration, and that the countercurrent system in the inner medulla is involved in the generation and maintenance of hypertonic medullary interstitium.
Asunto(s)
Canales de Cloruro/fisiología , Diabetes Insípida Nefrogénica/etiología , Animales , Canales de Cloruro/genética , Diabetes Insípida Nefrogénica/sangre , Diabetes Insípida Nefrogénica/orina , Modelos Animales de Enfermedad , Femenino , Asa de la Nefrona/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The CIC family is the superfamily of voltage-gated Cl- channels. Although the CIC channels expressed in Xenopus oocytes have been characterized, their channel properties are still poorly understood. We recently cloned a unique member of the CIC family, CIC-3, that is expressed abundantly in neurons. Its channel activity was regulated by phorbol esters. Now, we have established a stably transfected somatic cell line expressing functional CIC-3 channels and examined the CIC-3 single-channel current by patch-clamp techniques. In inside-out patches from the stably transfected cells, a rise of bath Ca2+ concentration in the physiological range of intracellular Ca2+ concentrations inhibited the CIC-3 single-channel currents. This inhibition by Ca2+ was independent of phosphorylation and ATP. Thus, the CIC-3 channel is a Ca(2+)-sensitive Cl- channel localized in neuronal cells, and its Ca2+ sensitivity implies a physiological role in neuronal functions.
Asunto(s)
Canales de Cloruro/genética , Expresión Génica , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina Trifosfato/metabolismo , Animales , Células CHO , Calcio/metabolismo , Calcio/farmacología , Canales de Cloruro/fisiología , Cricetinae , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana , Técnicas de Placa-Clamp , Fosforilación , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
cDNA (CIC-3) encoding a protein kinase C-regulated chloride channel was cloned and characterized. The open reading frame encodes 760 amino acids, which possess significantly amino acid identity with previously cloned CIC chloride channels. The chloride currents expressed in Xenopus oocytes injected with CIC-3 cRNA were completely blocked by activation of protein kinase C by 12-O-tetradecanoylphorbol 13-acetate. Abundant expression of CIC-3 mRNA was observed in rat brain, especially in the olfactory bulb, hippocampus, and cerebellum. These findings suggest that CIC-3 may play an important role in neuronal cell function through regulation of membrane excitability by protein kinase C.
Asunto(s)
Encéfalo/metabolismo , Canales de Cloruro/metabolismo , Clonación Molecular , Neuronas/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/citología , ADN Complementario/genética , Sondas Moleculares/genética , Datos de Secuencia Molecular , Ratas , Distribución TisularRESUMEN
The effects of acidosis and mineralocorticoids on cellular H+/HCO3- transport mechanisms were examined in intercalated cells of the outer stripe of outer medullary collecting duct (OMCDo) from rabbit. Intracellular pH (pHi) of intercalated cells was monitored by fluorescence ratio imaging using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). pHi recovered from an acid load at 2.8 +/- 0.5 x 10(-3) pHU/s in the absence of ambient Na+. This pHi recovery rate was similar in chronic acidosis induced by NH4Cl loading, but it was enhanced (+111%) by treatment with deoxycorticosterone acetate (DOCA). In a DOCA-treated group, luminal 10 microM SCH28080 and 0.1 mM omeprazole, H(+)-K(+)-ATPase inhibitors, did not change the pHi recovery rate, while luminal 0.5 mM N-ethylmaleimide blocked the rate by 68%. DOCA, but not acidosis, increased (approximately 40%) initial pHi response to bath HCO3- or Cl- reduction in Na(+)-free condition. After an acid load in the absence of Na+ and HCO3-, pHi response to basolateral Na+ addition was stimulated (+66%) by acidosis, but not by DOCA. Our results suggest that (a) mineralocorticoids stimulate H+/HCO3- transport mechanisms involved in transepithelial H+ secretion, i.e., a luminal NEM-sensitive H+ pump and basolateral Na(+)-independent Cl(-)-HCO3- exchange; and (b) acidosis enhances the activity of basolateral Na(+)-H+ exchange that may be responsible for pHi regulation.
Asunto(s)
Equilibrio Ácido-Base , Acidosis/metabolismo , Bicarbonatos/metabolismo , Médula Renal/metabolismo , Túbulos Renales Colectores/metabolismo , Mineralocorticoides/fisiología , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Cloruros/metabolismo , Femenino , Técnicas In Vitro , Médula Renal/anatomía & histología , Túbulos Renales Colectores/citología , Conejos , Sodio/metabolismo , Intercambiadores de Sodio-HidrógenoRESUMEN
Recent studies have suggested a selective effect of atrial natriuretic peptide (ANP) in regulating NaCl reabsorption in juxtamedullary nephrons. We examined (a) functional differences between medullary thick ascending limbs from long and short loops of Henle (lMAL and sMAL, respectively) and (b) the interaction of ANP and arginine vasopressin (AVP) on Cl- transport (JCl) in these two segments. AVP-, glucagon-, and calcitonin-stimulated cAMP accumulation was higher in lMAL than in sMAL. 10(-10) M AVP increased JCl in lMAL but not in sMAL. ANP-stimulated cGMP production was higher in lMAL than in sMAL. 10(-10) and 10(-8) M ANP inhibited AVP-stimulated JCl in lMAL by 26-30% (from 70.3 +/- 11.4 to 51.7 +/- 13.6 pmol/mm per min and from 88.1 +/- 10.1 to 61.8 +/- 11.7 pmol/mm per min, respectively), and this effect was mimicked by 10(-5) to 10(-4) M cGMP. This effect of ANP in lMAL could account for a large part of the ANP-induced natriuresis and diuresis in vivo, in that the rate of NaCl reabsorption in MAL is the largest among distal nephron segments, providing the chemical potential energy for the renal countercurrent multiplication system.
Asunto(s)
Arginina Vasopresina/farmacología , Factor Natriurético Atrial/farmacología , Cloruros/metabolismo , Médula Renal/metabolismo , Asa de la Nefrona/metabolismo , Equilibrio Hidroelectrolítico/efectos de los fármacos , Animales , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Masculino , Ratas , Ratas Endogámicas , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
We investigated the tubular action of endothelin in rat nephron segments. The effects of endothelin on arginine vasopressin (AVP)-, parathyroid hormone-, glucagon-, calcitonin-, and isoproterenol-dependent cAMP accumulation were studied. The following nephron segments were microdissected: glomerulus (Gl), proximal convoluted tubule (PCT), cortical and medullary thick ascending limbs of Henle's loop (cTAL and mTAL, respectively), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD). Endothelin dose dependently (10(-8)-10(-10)M) inhibited AVP-dependent cAMP accumulation in CCD, OMCD, and IMCD. This effect was independent of the presence or absence of phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, Ca channel blocker nicardipine, or indomethacin, but was abolished in the presence of protein kinase C inhibitor H-7. Protein kinase C stimulator dioctanoyl glycerol mimicked the effect of endothelin. On the other hand, endothelin had no inhibitory effect on AVP-dependent cAMP accumulation in cTAL or mTAL, parathyroid hormone-dependent cAMP accumulation in Gl and PCT, or glucagon-, calcitonin-, and isoprotereol-dependent cAMP accumulation in OMCD. We conclude that endothelin specifically inhibits AVP-dependent cAMP accumulation in CCD, OMCD, and IMCD through activating protein kinase C. This effect possibly has a role in maintaining urine volume to counteract the decrease in GFR caused by endothelin itself.
Asunto(s)
AMP Cíclico/metabolismo , Túbulos Renales/metabolismo , Péptidos/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , 1-Metil-3-Isobutilxantina/farmacología , Animales , Arginina Vasopresina/farmacología , Calcitonina/farmacología , Calcio/fisiología , Endotelinas , Glucagón/farmacología , Técnicas In Vitro , Indometacina/farmacología , Isoproterenol/farmacología , Isoquinolinas/farmacología , Túbulos Renales/efectos de los fármacos , Asa de la Nefrona/fisiología , Nicardipino/farmacología , Hormona Paratiroidea/farmacología , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , RatasRESUMEN
Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl(2) inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca(++) inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl(2) reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E(1) (PGE(1) = 10(-7)m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10(-4)m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10(-7)m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE(1) modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed.
Asunto(s)
Adenilil Ciclasas/metabolismo , Calcio/farmacología , AMP Cíclico/metabolismo , Riñón/enzimología , Prostaglandinas/farmacología , Vasopresinas/antagonistas & inhibidores , Animales , Cloruros , Cricetinae , Depresión Química , Ácidos Grasos/farmacología , Femenino , Fluoruros/farmacología , Iones , Sodio/farmacología , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Recent studies have revealed that endothelins (ETs) have at least two types of receptors. One receptor has high affinity to ET-1 and ET-2 and low affinity to ET-3 (A type). The other receptor binds almost equally to ET-1, ET-2, and ET-3 (B type). In this study, microlocalization of mRNA coding for the A-type and B-type ET receptors was carried out in the rat kidney using a reverse transcription and polymerase chain reaction assay of individual microdissected renal tubule segments along the nephron, glomeruli, vasa recta bundle, and arcuate arteries. Large signals for the B-type receptor polymerase chain reaction product were detected in the initial and terminal inner medullary collecting duct and the glomerulus, while small signals were found in the cortical collecting duct and outer medullary collecting duct, vasa recta bundle, and arcuate artery. In contrast, A-type receptor mRNA was detected only in the glomerulus, vasa recta bundle, and arcuate artery. Thus, the two ET receptor subtypes are distributed differently along the nephron. This suggests that the two types of receptors and ET families may affect kidney functioning in different ways.
Asunto(s)
Endotelinas/metabolismo , Nefronas/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Receptores de Superficie Celular/genética , Animales , Secuencia de Bases , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas , Receptores de Superficie Celular/análisis , Receptores de Endotelina , Transcripción GenéticaRESUMEN
Stimulation of the release of nitric oxide (NO) in the kidney has been shown to result in renal hemodynamic changes and natriuresis. NO is a potent stimulator of soluble guanylate cyclase, leading to an increase of cyclic GMP. The precise localization of NO synthase and soluble guanylate cyclase in the renal structure is not known. In this study, the microlocalization of mRNAs coding for constitutive NO synthase and soluble guanylate cyclase was carried out in the rat kidney, using an assay of reverse transcription and polymerase chain reaction in individual microdissected renal tubule segments along the nephron, glomeruli, vasa recta bundle, and arcuate arteries. A large signal for constitutive NO synthase was detected in inner medullary collecting duct. Small signals were detected in inner medullary thin limb, cortical collecting duct, outer medullary collecting duct, glomerulus, vasa recta, and arcuate artery. Soluble guanylate cyclase mRNA is expressed largely in glomerulus, proximal convoluted tubule, proximal straight tubule, and cortical collecting duct, and in small amounts in medullary thick ascending limb, inner medullary thin limb, outer medullary collecting duct, inner medullary collecting duct, and the vascular system. Our data demonstrate that NO can be produced locally in the kidney, and that soluble guanylate cyclase is widely distributed in glomerulus, renal tubules, and the vascular system.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Guanilato Ciclasa/genética , Nefronas/enzimología , Animales , Secuencia de Bases , Expresión Génica , Datos de Secuencia Molecular , Óxido Nítrico Sintasa , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ADN Polimerasa Dirigida por ARN/metabolismo , RatasRESUMEN
By measurements of NO2-/NO3- (NOx) production and Northern blot analysis, we studied the effects of a membrane-permeable cAMP derivative, 8-bromo-cAMP, on the expression of inducible nitric oxide synthase (iNOS) gene and the synthesis of NOx in cultured rat vascular smooth muscle cells (VSMCs). 8-bromo-cAMP stimulated NOx production and increased steady-state levels of iNOS mRNA in rat VSMC in a time- and dose-dependent manner. NG-monomethyl-L-arginine, a NOS inhibitor, completely blocked the 8-bromo-cAMP-induced NOx production, whose effect was partially, but significantly reversed by an excess L-arginine, but not by D-arginine. Compounds that increase intracellular cAMP levels (cholera toxin, forskolin, and 3-isobutyl-1-methylxanthine), all stimulated NOx production. Dexamethasone inhibited the stimulated NOx production, as well as the induction of iNOS mRNA by cAMP. Both actinomycin D and cycloheximide completely blocked the stimulated NOx production by cAMP. Actinomycin D abolished the cAMP-induced iNOS mRNA, whereas cycloheximide remarkably increased iNOS mRNA levels in the presence and absence of 8-bromo-cAMP (superinduction). Actinomycin D, but not dexamethasone, completely abolished the cycloheximide-induced iNOS mRNA. The half-life of cAMP-induced iNOS mRNA was approximately 2 h, whereas no decay in the cycloheximide-induced iNOS mRNA was observed during 12 h. These results demonstrate that iNOS gene is upregulated by cAMP and the superinduction of iNOS mRNA is attributable to increased mRNA stability in rat VSMC.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Aminoácido Oxidorreductasas/biosíntesis , Aorta Torácica/enzimología , Arginina/análogos & derivados , AMP Cíclico/metabolismo , Músculo Liso Vascular/enzimología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Arginina/farmacología , Células Cultivadas , Toxina del Cólera/farmacología , Colforsina/farmacología , Cicloheximida/farmacología , Dactinomicina/farmacología , Dexametasona/farmacología , Inducción Enzimática , Cinética , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa , ARN Ribosómico/biosíntesis , ARN Ribosómico/aislamiento & purificación , Ratas , Ratas Wistar , omega-N-MetilargininaRESUMEN
Recent studies have revealed that arginine vasopressin (AVP) has at least two types of receptors in the kidney: V1a receptor and V2 receptor. In this study, microlocalization of mRNA coding for V1a and V2 receptors was carried out in the rat kidney using a reverse transcription and polymerase chain reaction. Large signals for V1a receptor PCR product were detected in the glomerulus, initial cortical collecting duct, cortical collecting duct, outer medullary collecting duct, inner medullary collecting duct, and arcuate artery. Small but detectable signals were found in proximal convoluted and straight tubules, inner medullary thin limbs, and medullary thick ascending limbs. Large signals for V2 receptor mRNA were detected in the cortical collecting duct, outer medullary collecting duct, and inner medullary collecting duct. Small signals for V2 receptor were found in the inner medullary thick limbs, medullary thick ascending limbs, and initial cortical collecting duct. Next, we investigated V1a and V2 receptor mRNA regulation in the dehydrated state. During a 72-h water restriction state, the plasma AVP level increased and V2 receptor mRNA decreased in collecting ducts. In contrast, V1a receptor mRNA did not change significantly. Thus, the two AVP receptor subtypes are distributed differently along the nephron, and these mRNAs are regulated differently in the dehydrated state.
Asunto(s)
Regulación de la Expresión Génica , Nefronas/metabolismo , ARN Mensajero/metabolismo , Receptores de Vasopresinas/genética , Animales , Secuencia de Bases , Disección , Glomérulos Renales/metabolismo , Túbulos Renales/metabolismo , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Agua/metabolismoRESUMEN
The kidney both produces and responds to endothelin. We examined the production and the expression of mRNA of endothelin-1 (ET-1) in tubule suspensions and microdissected nephron segments. ET-1 production was measured by RIA using an ET-1-specific antibody. We applied the reverse transcription and polymerase chain reaction (PCR) technique to detect ET-1 mRNA along the nephron segments. Stimulation of ET-1 production was observed in the presence of FCS and transforming growth factor-beta (TGF-beta) in inner medullary tubules but not in cortical or outer medullary tubule suspensions. Among dissected nephron segments, ET-1 production was observed in glomeruli and inner medullary collecting ducts (IMCD), whereas it was negligible in proximal convoluted tubules (PCT) and medullary thick ascending limbs (MAL). In addition, the PCR product of ET-1 mRNA was also higher in glomeruli and IMCD, whereas it was undetectable in PCT and MAL. Furthermore, FCS and TGF-beta increased ET-1 mRNA in microdissected glomeruli and IMCD. These data clearly demonstrated that the production sites of ET-1 are glomeruli and IMCD among the nephron segments. ET-1 is an autocrine factor in these sites.
Asunto(s)
Endotelinas/biosíntesis , Nefronas/metabolismo , ARN Mensajero/análisis , Animales , Secuencia de Bases , Endotelinas/genética , Sangre Fetal/fisiología , Técnicas In Vitro , Túbulos Renales Colectores/metabolismo , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conejos , Ratas , Ratas Endogámicas , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The regulation of osmotic water permeability (Pf) by vasopressin (VP) in kidney collecting tubule involves the exocytic-endocytic trafficking of vesicles containing water channels between an intracellular compartment and apical plasma membrane. To examine effects of transcellular water flow on vesicle movement, Pf was measured with 1-s time resolution in the isolated perfused rabbit cortical collecting tubule in response to addition and removal of VP (250 microU/ml) in the presence of bath greater than lumen (B greater than L), lumen greater than bath (L greater than B), and lumen = bath (L = B) osmolalities. With VP addition, Pf increased from 12 to 240-270 x 10(-4) cm/s (37 degrees C) in 10 min. At 1 min, Pf was approximately 70 x 10(-4) cm/s for B greater than L, L greater than B, and L = B conditions. At later times, Pf increased fastest for L greater than B and slowest for B greater than L osmolalities; at 5 min, Pf was 250 x 10(-4) cm/s (L greater than B) and 158 x 10(-4) cm/s (B greater than L). With VP removal, Pf returned to pre-VP levels at the fastest rate for B greater than L and the slowest rate for L greater than B osmolalities; at 30 min, Pf was 65 x 10(-4) cm/s (B greater than L) and 183 x 10(-4) cm/s (L greater than B). For a series of osmotic gradients of different magnitudes and directions, the rates of Pf increase and decrease were dependent upon the magnitude of transcellular volume flow; control studies showed that paracellular water flux, asymmetric transcellular water pathways, or changes in cell volume could not account for the data. VP-dependent endocytosis was measured by apical uptake of rhodamine-dextran; in paired studies where the same tubule was used for + and - gradients, B greater than L and L greater than B osmolalities gave 168% and 82% of uptake measured with no gradient. In contrast, endocytosis in proximal tubule was not dependent on gradient direction. These data provide evidence that transcellular volume flow modulates the vasopressin-dependent cycling of vesicles containing water channels, suggesting a novel driving mechanism to aid or oppose the targeted, hormonally directed movement of subcellular membranes.
Asunto(s)
Agua Corporal/metabolismo , Endocitosis , Exocitosis , Túbulos Renales Colectores/metabolismo , Animales , Técnicas In Vitro , Concentración Osmolar , Permeabilidad , Conejos , Vasopresinas/farmacologíaRESUMEN
We investigated the effects of endothelins on receptor-mediated cyclic nucleotide metabolism in rat glomerulus, inner medullary collecting duct (IMCD), and also in cultured rat glomerular mesangial cells. Endothelin (ET)-3 dose-dependently stimulated cGMP accumulation in glomerulus, which was higher than that of ET-1 or ET-2. ETB receptor agonist IRL 1620 produced cGMP in a dose-dependent manner, mimicking the effect of ET-3. ETA receptor antagonist BQ123-Na did not inhibit ET-3- or IRL 1620-stimulated cGMP generation. NG-monomethyl-L-arginine (L-NMMA) significantly inhibited ET-3- or IRL 1620-induced cGMP production, suggesting that ET-3- or IRL 1620-stimulated cGMP generation was mediated through nitric oxide (NO). Intracellular Ca chelator BAPTA/AM and calmodulin antagonist W-7, but not Ca channel blocker nicardipine, significantly inhibited ET-3- or IRL 1620-induced cGMP generation. In cultured rat mesangial cells, ET-3 stimulated cGMP generation through NO in the presence of fetal calf serum, which was not inhibited by addition of BQ123-Na. In IMCD, ET-3 had no stimulative effect on cGMP generation. We conclude that ET-3 stimulates NO-induced cGMP generation through ETB receptor in glomerulus. This effect seems to be mediated through intracellular Ca/calmodulin, but not through Ca influx via L-type Ca channel. Mesangial cells can be a source of NO coupled to ETB receptor activation in glomerulus. From these results, mesangial ETB receptor may work to counteract the vasoconstrictive effect of endothelin caused via ETA receptor in glomerulus.
Asunto(s)
1-Metil-3-Isobutilxantina/farmacología , GMP Cíclico/metabolismo , Endotelinas/farmacología , Mesangio Glomerular/metabolismo , Glomérulos Renales/metabolismo , Óxido Nítrico/biosíntesis , Receptores de Endotelina/fisiología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Calmodulina/antagonistas & inhibidores , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Antagonistas de los Receptores de Endotelina , Mesangio Glomerular/efectos de los fármacos , Guanilato Ciclasa/antagonistas & inhibidores , Hemoglobinas/farmacología , Técnicas In Vitro , Glomérulos Renales/efectos de los fármacos , Cinética , Masculino , Azul de Metileno/farmacología , Nicardipino/farmacología , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Endotelina/efectos de los fármacos , Sulfonamidas/farmacología , omega-N-MetilargininaRESUMEN
We have recently reported that endothelin-1 (ET-1) mediates angiotensin II-induced hypertrophy of cardiomyocytes as an autocrine/paracrine factor. In the present study, we examined whether endothelin-3 (ET-3) induces hypertrophy of cultured neonatal rat cardiomyocytes and whether endogenous ET-1 mediates this effect. ET-3 (10(-7) M) increased the cell surface area of cardiomyocytes after 48 h. ET-3 dose dependently (10(-9)-10(-7) M) stimulated protein synthesis as evaluated by [3H]leucine incorporation; the maximum response was 1.4-fold increase over the control at 10(-7) M. Since the response of cardiac hypertrophy is characterized by enhanced expression of fetal isoforms of muscle specific genes, the effect of ET-3 on steady state levels of mRNA for skeletal alpha-actin was evaluated by Northern blot analysis. ET-3 (10(-9)-10(-7) M) increased mRNA level for skeletal alpha-actin with a maximum response after 6 h. ET-3-induced [3H]leucine incorporation, skeletal alpha-actin mRNA and cell surface area were inhibited by a synthetic ETB receptor antagonist (BQ788). Interestingly, ET-3-induced skeletal alpha-actin gene expression and [3H]leucine incorporation were inhibited by a synthetic ETA receptor antagonist (BQ123) as well as by antisense oligonucleotides against peproET-1 mRNA. ET-3 (10(-7) M) transiently increased mRNA levels for ET-1 peaking at 30 min and stimulated the release of immunoreactive ET-1 from cardiomyocytes. These results suggest that endogenous ET-1 locally generated and secreted by cardiomyocytes may contribute to ET-3-induced cardiac hypertrophy as an autocrine/paracrine factor.
Asunto(s)
Endotelinas/fisiología , Miocardio/patología , Receptores de Endotelina/metabolismo , Actinas/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Unión Competitiva , Células Cultivadas , Antagonistas de los Receptores de Endotelina , Expresión Génica , Hipertrofia , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Oligonucleótidos Antisentido/química , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Piperidinas/farmacología , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
Mutations in beta or gamma subunit of the epithelial sodium channel (ENaC) have been found to cause a hereditary form of human hypertension, Liddle syndrome. Most of the mutations reported are either nonsense mutations or frame shift mutations which would truncate the cytoplasmic carboxyl terminus of the beta or gamma subunits of the channel, suggesting that these domains are important for the normal regulation of this channel. We sequenced ENaC in a family with Liddle syndrome and found a missense mutation in beta subunit which predicts substitution of Tyr by His at codon 618, 2 bp downstream from a missense mutation (P616L) that has been reported recently. Presence of this mutation correlates with the clinical manifestations (hypertension, hypokalemia, suppressed aldosterone secretion) in this kindred. Functional expression studies in the Xenopus oocytes revealed constitutive activation of the Y618H mutant indistinguishable from that observed for the deletion mutant (R564stop) identified in the original pedigree of Liddle. Our data suggest that the region between Pro616 and Tyr618 is critically important for regulation of ENaC activity.
Asunto(s)
Hipertensión/genética , Hipertensión/metabolismo , Mutación Puntual , Canales de Sodio/genética , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Análisis Mutacional de ADN , Cartilla de ADN/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Conformación Proteica , Canales de Sodio/química , SíndromeRESUMEN
Endothelins (ET) produce endothelium-dependent vasodilation through nitric oxide (NO) synthesis. The present study was designed to elucidate the cellular mechanism by which ET induces synthesis and release of endothelium-derived NO by cultured bovine endothelial cells (EC). Binding studies revealed that bovine EC membrane had the binding sites of a novel agonist (BQ3020) for non-isopeptide-selective receptor subtype (ETB). Affinity labeling studies showed a major labeled band with the apparent molecular mass of 50 kD. Northern blot analysis demonstrated the expression of mRNA for ETB receptor. BQ3020 rapidly and dose dependently induced formation of inositol-1,4,5-triphosphate and increased intracellular Ca2+ concentrations in fura-2-loaded cells. Concomitantly, BQ3020 dose dependently stimulated production of both nitrate/nitrite (NOx) and cyclic GMP; a highly significant correlation existed between NOx and cGMP production. The stimulatory effect on NOx and cGMP production by ETB agonist was inhibited by NO synthase inhibitor monomethyl-L-arginine; this effect was reversed by coaddition of L-arginine, but not D-arginine. NOx and cGMP production stimulated by BQ3020 was inhibited by pretreatment with pertussis toxin. ETB agonist-induced NOx production was blocked by a calmodulin inhibitor and an intracellular Ca2+ chelator, but not by an extracellular Ca2+ chelator or a Ca2+ channel blocker. These data suggest that endothelins stimulate ETB receptor-mediated phosphoinositide breakdown via pertussis toxin-sensitive G-protein(s), which triggers release of intracellular Ca2+, thereby activating Ca2+/calmodulin-dependent NO synthase in EC.
Asunto(s)
Aminoácido Oxidorreductasas/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/ultraestructura , Receptores de Endotelina/fisiología , Animales , Arginina/metabolismo , Northern Blotting , Calcio/análisis , Calcio/fisiología , Calmodulina/fisiología , Bovinos , GMP Cíclico/metabolismo , Endotelinas/farmacocinética , Endotelio Vascular/metabolismo , Activación Enzimática/efectos de los fármacos , Proteínas de Unión al GTP/fisiología , Inosina Trifosfato/metabolismo , Radioisótopos de Yodo , Nitratos/metabolismo , Óxido Nítrico Sintasa , Nitritos/metabolismo , Fragmentos de Péptidos/farmacocinética , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
To examine whether expression and distribution of aquaporin of collecting duct (AQP-CD) are regulated by vasopressin V2 receptor (V2R), we performed immunohistochemical studies with specific antibody against AQP-CD. Normal Wistar rats were divided into four groups and treated for 3 d; control, dehydration, vasopressin V1 receptor (V1R) antagonist (OPC-21268 120 mg/kg), V2R antagonist (OPC-31260 30 mg/kg). At time of death, urine osmolality (Uosm) in the dehydration group (1884 +/- 245 mOsm/kg) was significantly higher than that in the control (938 +/- 91). In the V2R antagonist group, Uosm was significantly decreased to 249 +/- 29, whereas V1R antagonist showed no effect on Uosm. In the control and V1R antagonist groups, immunofluorescence studies showed the AQP-CD staining of both apical membrane and subapical cytoplasm of CD cells of the cortex and the inner medulla. Dehydration increased the immunostaining of both apical membrane and subapical cytoplasm of CD cells of the inner medulla, and the degree of increase was dominant in apical membrane. In the V2R antagonist group, only faint staining of apical membrane and weak labeling of cytoplasm of CD cells of the inner medulla were observed. These changes in the localization and protein amount of AQP-CD by dehydration and V2R antagonist were quantitatively confirmed by immunogold studies and immunoblot analysis of the inner medulla. The present results indicate that the distribution and amount of AQP-CD in the CD cells are regulated by vasopressin V2 receptor.
Asunto(s)
Agua Corporal/metabolismo , Canales Iónicos/química , Túbulos Renales Colectores/química , Receptores de Vasopresinas/fisiología , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Immunoblotting , Inmunohistoquímica , Masculino , Permeabilidad , Piperidinas/farmacología , Quinolonas/farmacología , Ratas , Ratas WistarRESUMEN
We studied whether a novel vasoconstrictor peptide, endothelin-1 (ET-1), is synthesized by and released from human carcinoma cell lines, and whether ET-1 stimulates proliferation of these tumor cells. ET-1-like immunoreactivity was released from both HeLa and HEp-2 cells as a function of time. Reverse-phase HPLC of the conditioned media from HeLa cells revealed a major peak coeluting with standard ET-1. Northern blot analysis demonstrated the expression of mRNA for ET-1 precursor in both tumor cell lines. Both cell lines contained a single class of specific binding sites for ET-1. ET-1 dose-dependently induced increases in cytosolic free Ca2+ concentration in fura-2-loaded tumor cells, whose effect was completely abolished by chelating extracellular Ca2+ or by Ca(2+)-channel blocker. ET-1 stimulated proliferation of the quiescent cell lines in a dose-dependent manner, whose effect was inhibited by Ca(2+)-channel blocker. Polyclonal antibody for ET-1 inhibited proliferation of these cell lines, whereas nonimmune serum had no effect. These results demonstrate that ET-1 is synthesized by and released from human epithelial carcinoma cell lines, and that exogenous and endogenous ET-1 stimulates proliferation of the cells possibly through Ca2+ influx, suggesting its role as an autocrine/paracrine growth factor for certain tumor cells.
Asunto(s)
Endotelinas/farmacología , Sustancias de Crecimiento/farmacología , Calcio/metabolismo , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelinas/análisis , Endotelinas/genética , Células HeLa , Humanos , Células Tumorales CultivadasRESUMEN
We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype-1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the pairs of interferon-resistant and interferon-sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the COOH-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the COOH-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40 amino acid stretch of the NS5A region around codon 2218 (from codon 2209 to 2248) in interferon-sensitive HCV. On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype-1b (HCV-J, HCV-JTa, or HC-J4). We designated this region as the interferon sensitivity determining region. Thus, HCV genotype-1b with the prototype interferon sensitivity determining region appears to be interferon-resistant strains. The specific nature of these mutations might make it possible to predict prognostic effects of interferon treatment.