RESUMEN

This study investigated whether repeated administration of recombinant adeno-associated virus type 5 (rAAV5) to the airways induces inflammatory processes in the lungs of BALB/c-mice, with mechanical and histologic changes. Saline was instilled intratracheally in the control group, and rAAV5-green fluorescence protein (GFP) (4x10(11)particles) in the virus group (VR). These groups were subdivided into four subgroups: one dose analyzed 3 weeks later (VR1d3w) and two doses analyzed 1 (VR2d1w), 2 (VR2d2w) and 3 weeks (VR2d3w) after the second dose. Lung morphometry, mechanical parameters, airway responsiveness, rAAV5-GFP transduction and the expression of inflammatory cytokines were investigated. No significant differences in lung mechanics, airway responsiveness, and morphometry were observed. Re-administration of rAAV5 vector resulted in a decrease in GFP mRNA expression in the VR2d3w group. There was no evidence of inflammatory response or apoptosis in any group. rAAV5 did not induce an inflammatory process, mechanical or morphometric changes in the lungs. AAV5 may be an appropriate vector for lung gene therapy.

Asunto(s)

Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Neumonía/etiología , Neumonía/patología , Resistencia de las Vías Respiratorias , Análisis de Varianza , Animales , Apoptosis , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/genética , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/metabolismo , Mecánica Respiratoria/fisiología , Factores de Tiempo

RESUMEN

Pseudohypoaldosteronism type II (PHA II) is caused by mutations of two members of WNK ((with no lysine (k)) kinase family. WNK4 wild type (WT) has been shown to inhibit the activity and surface expression of sodium chloride cotransporter (NCC) when expressed in Xenopus oocytes. Here, we have studied NCC protein processing in mammalian cells in the presence or absence of WNK4 WT and its mutants, E562K and R1185C, by surface biotinylation, Western blot, co-immunoprecipitation (Co-IP) and immunostaining. WNK4 WT significantly reduced NCC surface expression in Cos-7 cells (58.9+/-6.8% vs 100% in control, P<0.001, n=6), whereas its mutant E562K has no significant effect on NCC surface expression (92.9+/-5.3% vs 100%, P=NS, n=6). Another mutant R1185C still partially reduces surface expression of NCC (76.2+/-11.8% vs 100%, P<0.05, n=6). The reduction of NCC surface expression by WNK4 WT (62.9+/-3.3% of control group) is not altered by WT dynamin ((61.8+/-3.7% (P=NS)) or its mutant K44A ((65.4+/-14.1% (P=NS)). A Co-IP study showed that both WNK4 WT and WNK4 E562K interact with NCC. Furthermore, a proton pump inhibitor, bafilomycin A1, partially reverses the inhibitory effect of WNK4 WT on NCC expression. Our data suggest that WNK4 WT significantly inhibits NCC surface expression, which is not owing to an increase in clathrin-mediated endocytosis of NCC, but likely results from enhanced degradation of NCC through a lysosomal pathway.

Asunto(s)

Células Epiteliales/fisiología , Riñón/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Seudohipoaldosteronismo/genética , Simportadores del Cloruro de Sodio/efectos de los fármacos , Simportadores del Cloruro de Sodio/fisiología , Animales , Biotinilación , Western Blotting , Células COS , Línea Celular , Chlorocebus aethiops , Dinamina II/farmacología , Dinaminas/farmacología , Células Epiteliales/química , Células Epiteliales/citología , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Inmunoprecipitación , Riñón/química , Riñón/citología , Lisosomas/efectos de los fármacos , Lisosomas/fisiología , Mutación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/farmacología , Estructura Terciaria de Proteína/fisiología , Seudohipoaldosteronismo/etiología , Seudohipoaldosteronismo/fisiopatología , Simportadores del Cloruro de Sodio/genética

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) is one of the most intensively investigated Cl- channels. Different mutations in the CFTR gene cause the disease cystic fibrosis (CF). CFTR is expressed in the apical membrane of various epithelial cells including the intestine. The major organ affected in CF patients is the lung, but it also causes an important dysfunction of intestinal ion transport. The modulation of CFTR mRNA expression by atrial natriuretic peptide (ANP) was investigated in rat proximal colon and in human intestinal CaCo-2 cells by RNase protection assay and semi-quantitative reverse transcriptase PCR techniques. Groups of rats subjected to volume expansion or intravenous infusion of synthetic ANP showed respective increases of 60 and 50% of CFTR mRNA expression in proximal colon. CFTR mRNA was also increased in cells treated with ANP, reaching a maximum effect at 10(-9) M ANP, probably via cGMP. ANP at 10(-9) M was also able to stimulate both the CFTR promoter region (by luciferase assay) and protein expression in CaCo-2 cells (by Western blot and immunoprecipitation/phosphorylation). These results suggested the involvement of ANP, a hormone involved with extracellular volume, in the expression of CFTR in rat proximal colon and CaCo-2 intestinal cells.

Asunto(s)

Factor Natriurético Atrial/administración & dosificación , Colon/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/análisis , Animales , Western Blotting/métodos , Células CACO-2 , GMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Expresión Génica/genética , Humanos , Inmunoprecipitación/métodos , Infusiones Intravenosas , Masculino , Fosforilación , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ribonucleasas/metabolismo

RESUMEN

Spironolactone, a diuretic antagonist of aldosterone has an unexplained side effect of amenorrhea which could be due to an angiogenesis inhibition. In this study we compared the effects of spironolactone, canrenone an active metabolite of spironolactone and eplerenone a more selective mineralocorticoid antagonist in rats implanted with a fibrin gel chamber. Perforated plexiglass chambers filled with rat fibrin, spironolactone (50 microM), canrenone (100 microM), eplerenone (500 microM), DMSO (0.05%) and control were implanted into the dorsal subcutaneous space of wistar rats. After 14 days of implantation, an invasion of the fibrin gel chamber by neovascularised buds had occurred through the holes. The number of vessels in the central field and in two or three peripheral fields covering the surface of the bud, were measured for each drug tested and compared to the control. In spironolactone treated chambers, the numbers of peripheral and central vessels were significantly reduced compared to control (p < 0.001). Canrenone, eplerenone and DMSO did not reduce the number of vessels (m +/- ESM, ANOVA followed by Newman-Keuls test). Spironolactone but not canrenone, nor eplerenone inhibited vessels formation in vivo. This antiangiogenic activity appeared to be not related to the antimineralocorticoid effect of spironolactone.

Asunto(s)

Amenorrea/inducido químicamente , Canrenona/efectos adversos , Canrenona/farmacología , Antagonistas de Receptores de Mineralocorticoides/efectos adversos , Antagonistas de Receptores de Mineralocorticoides/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Espironolactona/análogos & derivados , Espironolactona/efectos adversos , Espironolactona/farmacología , Amenorrea/fisiopatología , Animales , Canrenona/administración & dosificación , Eplerenona , Femenino , Fibrina , Humanos , Hipertensión/tratamiento farmacológico , Antagonistas de Receptores de Mineralocorticoides/administración & dosificación , Ratas , Espironolactona/administración & dosificación

RESUMEN

Angiogenesis requires endothelial cell proliferation and their vascular rearrangement. A report of inhibiting effect of spironolactone on smooth muscle cell proliferation led us to study in vitro the effects of this drug on the endothelial cell proliferation and migration. Spironolactone (10 to 100 microM) and one of its active metabolite, canrenone (10 to 100 microM), are added to human umbilical vein endothelial cells (HUVEC). Their effect on cellular proliferation is evaluated by measuring the amount of the cellular nucleic acids using a fluorometric assay (CyQuant). Cell migration is measured using a multiwell chamber assay (Transwell). In further experiments, we investigated their effect on the capillary-like tube formation in vitro generated by HUVEC seeded in a three-dimensional biological gel (Matrigel). The VEGF (10 ng/mL) and the bFGF (10 ng/mL) were used as mitotic and cell differentiation factors. Effect on cell cycle distribution is investigated by flow cytometry analysis. Spironolactone inhibits HUVEC proliferation but canrenone does not have any significant effect. The growth promoters VEGF or bFGF do not modify inhibiting effect of spironolactone. Spironolactone (50 microM) and canrenone (50 microM) are without effect on cell migration. Capillary-like networks on Matrigel is not modified by spironolactone or canrenone. Spironolactone inhibits progression through S phase of the cell cycle. Spironolactone inhibits the proliferation of the endothelial cells in vitro but shows no effect on their migration and their rearrangement in capillary-like structures. These data should be confirmed in models of angiogenesis in vivo.

Asunto(s)

Canrenona/farmacología , Antagonistas de Receptores de Mineralocorticoides/farmacología , Neovascularización Patológica , Espironolactona/farmacología , Técnicas de Cultivo de Célula , División Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/citología , Fluorometría , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Ácidos Nucleicos/análisis

RESUMEN

The contributions of amiloride-sensitive and -insensitive fractions of alveolar fluid clearance in adult ventilated rats were studied under control conditions and after beta-adrenergic stimulation. Rats were instilled with a 5% albumin solution containing terbutaline (10(-4) M) or dibutyryl-cGMP (DBcGMP; 10(-4) M) with or without the cyclic nucleotide-gated cation channel inhibitor l-cis-diltiazem (10(-3) M) and/or amiloride (10(-3) M). Alveolar fluid clearance over 1 h was 18 +/- 2% in controls. In controls, amiloride inhibited 46 +/- 15% of alveolar fluid clearance, whereas l-cis-diltiazem had no inhibitory effect. Terbutaline and DBcGMP stimulated alveolar fluid clearance by 85 +/- 3 and 36 +/- 5%, respectively. Amiloride and l-cis-diltiazem inhibited nearly equal fractions of terbutaline-stimulated alveolar fluid clearance when given alone. Amiloride and l-cis-diltiazem given together inhibited a significantly larger fraction of alveolar fluid clearance in terbutaline-stimulated rats and in DBcGMP-stimulated rats. Based on these data, terbutaline stimulation recruited both amiloride-sensitive and l-cis-diltiazem-sensitive pathways. In contrast, DBcGMP mainly recruited l-cis-diltiazem-sensitive pathways. Therefore, the amiloride-insensitive fraction of Na+-driven alveolar fluid clearance may be partly mediated through cyclic nucleotide-gated cation channels and activated by an increase in intracellular cGMP.

Asunto(s)

Amilorida/farmacología , Alveolos Pulmonares/fisiología , Agonistas Adrenérgicos beta/farmacología , Animales , Líquidos Corporales/efectos de los fármacos , Líquidos Corporales/fisiología , AMP Cíclico/fisiología , GMP Cíclico/fisiología , GMP Dibutiril Cíclico/farmacología , Diltiazem/farmacología , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Masculino , Alveolos Pulmonares/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Respiración Artificial , Terbutalina/farmacología , Vasodilatadores/farmacología

RESUMEN

Analogs of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] activate both genomic mechanisms via the nuclear vitamin D(3) receptor (nVDR) and nongenomic pathways via the plasma membrane vitamin D(3) receptor (pmVDR). Both of these pathways are normally activated by 1alpha,25(OH)(2)D(3), but as a result of synthesis of numerous analogs of 1alpha,25(OH)(2)D(3) these pathways can be distinguished. We used increasing doses of vitamin D(3) analogs to determine their potencies of action on these two distinct pathways, measuring calcium channel potentiation as an indicator of the nongenomic action and measuring increases in osteocalcin mRNA and protein release and bone resorption as indicators of genomic action. We found that both 25(OH)-16,23E-diene-D(3) (R) and 1alpha,25(OH)(2)-16,23E-diene-D(3) (A) are 10-fold more potent than 1alpha,25(OH)(2)D(3) for activation of the nongenomic pathway because double bonds in the side chain and the D ring increase the affinity for calcium channel potentiation. While the C-1alpha-hydroxyl group is not necessary for potentiation of calcium channels, methyl groups at this position can alter the affinity for calcium channel potentiation. On the other hand, 1000 fold higher concentrations of nongenomic analogs were needed compared to 1alpha,25(OH)(2)D(3) to increase osteocalcin mRNA or protein release. 1alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorovitamin D(3), (E) is an agent that is 10 fold more potent than 1alpha,25(OH)(2)D(3) at increasing osteocalcin mRNA and protein release, whereas 1alpha,25(OH)(2)-3-epi-D(3) increases osteocalcin mRNA and protein with a potency over 10 fold lower than 1alpha,25(OH)(2)D(3). These results suggest that double bonds in the side chain and the D ring stabilize action on the nongenomic pathway whereas F(6) on the terminal portion of the side chain increases potency for nVDR. On the other hand, while the C-1alpha-hydroxyl group is necessary for activation of genomic events via nVDR, the activation of nongenomic events occurs in the absence of this group.

Asunto(s)

Calcitriol/farmacología , Animales , Resorción Ósea/inducido químicamente , Resorción Ósea/metabolismo , Calcitriol/análogos & derivados , Calcitriol/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Radioisótopos de Calcio , Relación Dosis-Respuesta a Droga , Feto , Osteocalcina/efectos de los fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteosarcoma/patología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Activación Transcripcional/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

Nephrolithiasis (kidney stones) affects 5-10% of adults and is most commonly associated with hypercalciuria, which may be due to monogenic renal tubular disorders. One such hypercalciuric disorder is Dent's disease, which is characterized by renal proximal tubular defects that include low molecular weight proteinuria, aminoaciduria and glycosuria, together with rickets in some patients. Dent's disease is due to inactivating mutations of the renal-specific voltage-gated chloride channel, CLC-5, which is expressed in the proximal tubule, thick ascending limb and collecting duct. The subcellular localization of CLC-5 to the proximal tubular endosomes has suggested a role in endocytosis, and to facilitate in vivo investigations of CLC-5 in Dent's disease we generated mice lacking CLC-5 by targeted gene disruption. CLC-5-deficient mice developed renal tubular defects which included low molecular weight (<70 kDa) proteinuria, generalized aminoaciduria that was more pronounced for neutral and polar amino acids, and glycosuria. They also developed hypercalciuria and renal calcium deposits and some had deformities of the spine. Furthermore, endocytosis as assessed by horseradish peroxidase uptake in the proximal tubule was severely impaired in CLC-5-deficient mice, thereby demonstrating a role for CLC-5 in endosomal uptake of low molecular weight proteins. Thus, CLC-5-deficient mice provide a model for Dent's disease and this will help in elucidating the function of this chloride channel in endocytosis and renal calcium homeostasis.

Asunto(s)

Canales de Cloruro/genética , Endocitosis/fisiología , Cálculos Renales/genética , Animales , Calcio/metabolismo , Células Cultivadas , Canales de Cloruro/deficiencia , Canales de Cloruro/metabolismo , Modelos Animales de Enfermedad , Femenino , Homeostasis , Técnicas para Inmunoenzimas , Cálculos Renales/metabolismo , Cálculos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL

RESUMEN

Previous reports indicate that the mRNA for the cardiac isoform of the voltage-gated L-type calcium channel (alpha(1C)) is elevated in colon cancer. The aim of these experiments was to verify that the mRNA for alpha(1C) was significantly increased in tumors of two separate populations of patients when compared to normal adjacent mucosa. The second aim was to measure the distribution of alpha(1C) using immunocytochemistry in normal human colon and in colon cancer and to determine what might regulate the channel expression. Biopsies were taken from patients with various stages of colon cancer and nearby normal mucosa were used as control. RNA was prepared and mRNA level measured by semiquantitative reverse transcriptase-polymerase chain reaction. The mRNA of the calcium channel was compared with other markers including beta-actin. The mRNA for alpha(1C) was increased significantly in colon cancers compared to nearby adjacent mucosa. Using confocal microscopy alpha(1C) was localized mainly at the apical membrane in the surface epithelium of normal human colon with less distribution on the lateral and basal membranes. The channel was localized on the lateral and basal membranes in crypt cells. Calcium channel localization appeared to be nearer nuclei in colon cancer samples, in part because of the smaller size of the cells. Likewise, cultured Caco-2 and T84 cells showed a membrane distribution. Western blotting indicated that alpha(1C) protein was increased in nonconfluent cultures of colonic carcinoma cells compared to confluent cells and immunocytochemistry confirms that there is more calcium channel protein in cells that are nonconfluent. We conclude that the increase in mRNA of alpha(1) subunit of the cardiac isoform of the L-type calcium channel may be a useful marker of colon cancer compared to other markers because the increase is large and this increase can be documented on small samples using a simple semiquantitative reverse transcriptase-polymerase chain reaction. We found that alpha(1C) protein is increased when colonic cells are nonconfluent or dividing which may account for the increase in cancer.

Asunto(s)

Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , ARN Mensajero/metabolismo , Western Blotting , Línea Celular , Colon/metabolismo , Humanos , Inmunohistoquímica , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular

Asunto(s)

Amilorida/farmacología , Orthomyxoviridae/fisiología , Receptores Virales/fisiología , Mucosa Respiratoria/fisiología , Mucosa Respiratoria/virología , Canales de Sodio/fisiología , Animales , Orthomyxoviridae/patogenicidad , Canales de Sodio/efectos de los fármacos , Tráquea/fisiología , Tráquea/virología

RESUMEN

L-type calcium channels have been identified previously in both osteoblast-like osteosarcoma cell lines and primary cultures of osteoblasts using numerous techniques such as patch clamp recording, drug inhibited 45Ca2+ uptake, and Fura-2 measurements, but intact bone has not been investigated. Using reverse-transcription polymerase chain reaction (RT-PCR) we found that the three major isoforms of the alpha 1-subunit of L-type calcium channels, (alpha 1C, alpha 1D, and alpha 1S) are present in RNA extracted from ROS 17/2.8 osteosarcoma cells, rat femur, and rat skull. Sequencing of most of the alpha 1C-subunit from rat femur and ROS cells revealed that the splice variants in osteosarcoma cells and intact bone differ, but there are no unique sequence variations compared with those found in other tissues. Northern blot analysis of ROS cell RNA indicated that cyclic adenosine monophosphate (cAMP), but not 1 alpha, 25-dihydroxyvitamin D3, increased the messenger RNA (mRNA) of the alpha 1C-subunit. Western blot of ROS cell lysates revealed a band of more then 220 kDa, the amount of which increased in cells treated with cAMP. Using confocal microscopy combined with immunohistochemistry in ROS cells, intact bone, and cartilage, we found that the alpha 1C-subunit of this channel is expressed in osteoblasts and chondrocytes suggesting this channel may be a pathway for signal transduction in intact tissue, because it is in osteosarcoma cell lines and primary osteoblasts grown in tissue culture.

Asunto(s)

Huesos/metabolismo , Canales de Calcio Tipo L/genética , AMP Cíclico/farmacología , Miocardio/metabolismo , Biosíntesis de Proteínas , Transcripción Genética , Animales , Calcitriol/farmacología , Calcio/metabolismo , Cartílago/metabolismo , Células Cultivadas , Fémur/metabolismo , Osteoblastos/metabolismo , Osteosarcoma , Biosíntesis de Proteínas/efectos de los fármacos , Isoformas de Proteínas/genética , Subunidades de Proteína , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

The fetal lung actively transports chloride across the airway epithelium. ClC-2, a pH-activated chloride channel, is highly expressed in the fetal lung and is located on the apical surface of the developing respiratory epithelium. Our goal was to determine whether acidic pH could stimulate chloride secretion in fetal rat distal lung epithelial cells mounted in Ussing chambers. A series of acidic solutions stimulated equivalent short-circuit current (I(eq)) from a baseline of 28 +/- 4.8 (pH 7.4) to 70 +/- 5 (pH 6.2), 114 +/- 12.8 (pH 5.0), and 164 +/- 19.2 (pH 3.8) microA/cm(2). These changes in I(eq) were inhibited by 1 mM cadmium chloride and did not result in large changes in [(3)H]mannitol paracellular flux. Immunofluorescent detection by confocal microscopy revealed that ClC-2 is expressed along the luminal surface of polarized fetal distal lung epithelial cells. These data suggest that the acidic environment of the fetal lung fluid could activate chloride channels contributing to fetal lung fluid production and that the changes in I(eq) seen in these Ussing studies may be due to stimulation of ClC-2.

Asunto(s)

Cloruros/metabolismo , Hidrógeno/metabolismo , Pulmón/embriología , Animales , Transporte Biológico/efectos de los fármacos , Canales de Cloruro CLC-2 , Cloruro de Cadmio/farmacología , Canales de Cloruro/metabolismo , Canales de Cloruro/fisiología , Conductividad Eléctrica , Células Epiteliales/metabolismo , Feto/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Manitol/farmacología , Ratas , Ratas Sprague-Dawley , Distribución Tisular

RESUMEN

Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1-100 microM) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced OC release by these cells (maximal effect, -40-50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (-47.2+/-4.0%; p < 0.0001 by paired ANOVA with 100 microM 13.6+/-0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, -22.5+/-6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1-100 microM) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate.

Asunto(s)

Benzotiadiazinas , Osteoblastos/efectos de los fármacos , Osteocalcina/biosíntesis , Receptores de Calcitriol/efectos de los fármacos , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Transcripción Genética/efectos de los fármacos , Northern Blotting , Western Blotting , Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Diuréticos , Factores de Unión al ADN Específico de las Células Eritroides , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Factor de Transcripción YY1

RESUMEN

1. Late gestation fetal sheep were chronically catheterised in utero to allow measurement of the rate of production of lung liquid (Jv) from 132-143 days gestation (term, 147 days), and to test the hypothesis that cyclic nucleotide gated cation channels mediate a component of fetal lung liquid absorption. 2. In eight experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.005) reduction in Jv from +18. 12 +/- 3.52 to -10.27 +/- 5.26 ml h-1. Dichlorobenzamil (a blocker of cyclic nucleotide gated cation channels) at 1.5 x 10-5 M did not significantly inhibit the adrenaline-induced lung liquid absorption (Jv dichlorobenzamil, -5.77 +/- 2.78 ml h-1; P > 0.1) when the data were grouped, but did exert a significant gestational effect (r = 0. 90, P < 0.01). Subsequent addition of 10-4 M amiloride (a blocker of epithelial sodium channels) abolished the adrenaline-induced absorption of lung liquid (mean Jv amiloride, +6.45 +/- 1.59 ml h-1; P < 0.01 relative to Jv adrenaline and P < 0.005 relative to Jv dichlorobenzamil). 3. In seven experiments, 0.5 microg min-1 adrenaline caused a significant (P < 0.0005) reduction in Jv from +18.95 +/- 2. 98 to -10.08 +/- 3.75 ml h-1. Amiloride (10-4 M) inhibited the adrenaline response (Jv amiloride, +5.46 +/- 1.09 ml h-1; P < 0.005). However, subsequent addition of 1.5 x 10-5 M dichlorobenzamil had no additive effect to that of amiloride (Jv dichlorobenzamil, +4.58 +/- 0.93 ml h-1; P > 0.1). 4. In six experiments, the cGMP analogue 8-Br-cGMP at 10-4 M caused a significant (P < 0.05) reduction in Jv from +15.20 +/- 2.81 to +11.63 +/- 1.71 ml h-1. Amiloride (10-4 M) did not block the effect of 8-Br-cGMP (Jv amiloride, +14.00 +/- 2.49 ml h-1; not significantly different from 8-Br-cGMP). Subsequent addition of 1.5 x 10-5 M dichlorobenzamil also did not block the effect of 8-Br-cGMP (Jv dichlorobenzamil, +11.37 +/- 1.22 ml h-1; not significantly different from either Jv amiloride or Jv 8-Br-cGMP). 5. We conclude that, in fetal sheep, neither adrenaline nor cGMP stimulate lung liquid absorption by actions on cyclic nucleotide gated cation channels, and that the effect of cGMP on fetal lung liquid secretion is minor and does not involve epithelial sodium channels. The effect of dichlorobenzamil, when given before amiloride, was probably due to an action on amiloride sensitive epithelial sodium channels.

Asunto(s)

Agua Pulmonar Extravascular/metabolismo , Canales Iónicos/fisiología , Pulmón/embriología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Cateterismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Diuréticos/farmacología , Epinefrina/farmacología , Madurez de los Órganos Fetales/efectos de los fármacos , Madurez de los Órganos Fetales/fisiología , Canales Iónicos/antagonistas & inhibidores , Pulmón/efectos de los fármacos , Ovinos , Bloqueadores de los Canales de Sodio

RESUMEN

Mutations in the chloride channel, ClC-5, have been described in several inherited diseases that result in the formation of kidney stones. To determine whether ClC-5 is also involved in calcium homeostasis, we investigated whether ClC-5 mRNA and protein expression are modulated in rats deficient in 1alpha,25(OH)(2) vitamin D(3) with and without thyroparathyroidectomy. Parathyroid hormone (PTH) was replaced in some animals. Vitamin D-deficient, thyroparathyrodectomized rats had lower serum and higher urinary calcium concentrations compared with control animals as well as lower serum PTH and calcitonin concentrations. ClC-5 mRNA and protein levels in the cortex decrease in vitamin D-deficient, thyroparathyroidectomized rats compared with both control and vitamin D-deficient animals. ClC-5 mRNA and protein expression increase near to control levels in vitamin D-deficient, thyroparathyroidectomized rats injected with PTH. No significant changes in ClC-5 mRNA and protein expression in the medulla were detected in any experimental group. Our results suggest that PTH modulates the expression of ClC-5 in the kidney cortex and that neither 1alpha,25(OH)(2) vitamin D(3) nor PTH regulates ClC-5 expression in the medulla. The pattern of expression of ClC-5 varies with urinary calcium. Animals with higher urinary calcium concentrations have lower levels of ClC-5 mRNA and protein expression, suggesting that the ClC-5 chloride channel plays a role in calcium reabsorption.

Asunto(s)

Canales de Cloruro/metabolismo , Colecalciferol/deficiencia , Riñón/metabolismo , Hormona Paratiroidea/sangre , ARN Mensajero/metabolismo , Deficiencia de Vitamina D/metabolismo , Animales , Calcitonina/sangre , Calcio/sangre , Calcio/orina , Femenino , Masculino , Paratiroidectomía , Embarazo , Ratas , Ratas Wistar , Tiroidectomía

RESUMEN

We found mRNA for the three isoforms of the cyclic nucleotide-gated nonselective cation channel expressed in the mucosal layer of the rat intestine from the duodenum to the colon and in intestinal epithelial cell lines in culture. Because these channels are permeable to sodium and calcium and are stimulated by cGMP or cAMP, we measured 8-bromo-cGMP-stimulated sodium-mediated short-circuit current (I(sc)) in proximal and distal colon and unidirectional (45)Ca(2+) fluxes in proximal colon to determine whether these channels could mediate transepithelial sodium and calcium absorption across the colon. Sodium-mediated I(sc), stimulated by 8-bromo-cGMP, were inhibited by dichlorobenzamil and l-cis-diltiazem, blockers of cyclic nucleotide-gated cation channels, suggesting that these ion channels can mediate transepithelial sodium absorption. Sodium-mediated I(sc) and net transepithelial (45)Ca(2+) absorption were stimulated by heat-stable toxin from Escherichia coli that increases cGMP. Addition of l-cis-diltiazem inhibited the enhanced transepithelial absorption of both ions. These results suggest that cyclic nucleotide-gated cation channels simultaneously increase net sodium and calcium absorption in the colon of the rat.

Asunto(s)

Calcio/farmacocinética , Colon/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Canales Iónicos/metabolismo , Sodio/farmacocinética , Animales , Línea Celular , Clonación Molecular , Colon/química , Colon/citología , Cartilla de ADN , Expresión Génica/fisiología , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Canales Iónicos/genética , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley

RESUMEN

Previous studies have shown that the mRNA of cyclic-nucleotide-gated nonselective cation (CNG) channels is expressed in rat airway epithelia and that these channels contribute to sodium-mediated short-circuit currents in cultured rat tracheal epithelia. Patch-clamp studies from human A549 cells indicate that these channels contribute to cGMP-stimulated L-cis-diltiazem- and dichlorobenzamil-inhibited whole-cell sodium currents. This study demonstrates that mRNA for primary and secondary subunits of CNG channels, halphaCNG1 and hbetaCNG1 respectively, are expressed in several human airway cell lines, including normal and cystic fibrosis bronchial airway cells, in normal and cystic fibrosis tracheal airway cell lines and nasal polyp tissue from a cystic fibrosis patient. The mRNA of ralphaCNG1 in rat lung increased in response to increased circulating glucocorticoids and decreased in animals with lowered circulating glucocorticoids after aminoglutethimide treatment. Likewise the mRNA of halphaCNG1 increased in the presence of glucocorticoids in cultured alveolar airway cells. The mRNA of alphaCNG1 in rat lung was also increased in response to a low-salt diet and lowered in animals fed a high-salt diet. Likewise the mRNA of alphaCNG1 was increased in response to increased aldosterone and decreased in animals given spironolactone. These results suggest that mRNA for alphaCNG1 increases in response to elevated glucocorticoids or mineralocorticoids. Because alphaCNG1 is a functional sodium entry channel in both rat and human airway epithelial cells, if channel protein is also elevated this channel could mediate an increase in sodium absorption across lung epithelia in response to circulating hormones.

Asunto(s)

Canales Iónicos/genética , ARN Mensajero/metabolismo , Sistema Respiratorio/metabolismo , Animales , Bronquios/metabolismo , Línea Celular , Línea Celular Transformada , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Fibrosis Quística , Dieta Hiposódica , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Pulmón/metabolismo , Pólipos Nasales/metabolismo , Alveolos Pulmonares/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus 40 de los Simios , Espironolactona/farmacología , Tráquea/metabolismo

RESUMEN

1. Sheep lungs were artificially perfused in situ with warmed whole oxygenated sheep blood. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of net transepithelial liquid movement under various conditions. 2. Dichlorobenzamil (1.5 x 10-5 M), a blocker of cyclic nucleotide-gated cation channels, inhibited the resting absorption of lung liquid in sheep aged 6 months (n = 5) (from -36.47 +/- 4.62 to -4.36 +/- 5.27 ml h-1, means +/- s.e.m.; P < 0.005, paired t test). Amiloride (10-4 M), a blocker of epithelial sodium channels, had no additive effect to that of dichlorobenzamil. 3. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.21 +/- 8.57 to -11.05 +/- 4.91 ml h-1; P < 0.05, one-tailed paired t test), and dichlorobenzamil (1.5 x 10-5 M) exerted an additive effect to that of amiloride resulting in secretion at +6.29 +/- 3.05 ml h-1 (P < 0. 01, paired t test). 4. In the lungs of sheep aged 6 weeks (n = 3), amiloride (10-4 M) also inhibited the resting absorption of liquid (from -26.36 +/- 14.05 to -5.17 +/- 8.27 ml h-1; P < 0.05, one-tailed paired t test); however, dichlorobenzamil (1.5 x 10-5 M) did not exert an additive effect to that of amiloride. 5. In the lungs of sheep aged 6 months (n = 4), amiloride (10-4 M) partially inhibited the resting absorption of liquid (from -35.70 +/- 8.58 to -6.79 +/- 4.28 ml h-1; P < 0.05, paired t test), and pimozide (1.5 x 10-4 M), another blocker of cyclic nucleotide-gated cation channels, also exerted an additive effect to that of amiloride, resulting in secretion of lung liquid at +15.36 +/- 9.14 ml h-1 (P < 0.05, paired t test). 6. We conclude that cyclic nucleotide-gated cation channels mediate a component of lung liquid absorption in sheep aged 6 months (but not in sheep aged 6 weeks), and that a mechanism for lung liquid secretion (present in fetuses) is retained at 6 months of age.

Asunto(s)

Líquidos Corporales/fisiología , Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Pulmón/fisiología , Nucleótidos Cíclicos/fisiología , Absorción , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Animales Recién Nacidos , Líquidos Corporales/efectos de los fármacos , Diuréticos/farmacología , Antagonistas de Dopamina/farmacología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/antagonistas & inhibidores , Presión Osmótica , Pimozida/farmacología , Ovinos , Canales de Sodio/efectos de los fármacos , Canales de Sodio/fisiología

RESUMEN

Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with halphaCNC1. Whole cell currents stimulated by 8-Br-cGMP in HK293 cells transfected with halphaCNC1 or A549 cells are carried by inward sodium and outward potassium with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected with halphaCNC1 are inhibited by l-cis-diltiazem with an IC(50) of 154 microm, by 2',4'-dichlorobenzamil with an IC(50) of 50 microm and by amiloride with an IC(50) of 133 microm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an IC(50) of 87 microm, by 2'4'-dichlorobenzamil with an IC(50) of 38 microm and by amiloride with an IC(50) of 32 microm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both alphaCNC1 and betaCNC subunits are present in A549 cells and the presence of the betaCNC subunit, may as previously reported, increase the affinity of these channel blockers compared to the halphaCNC1 subunit alone. The mRNA of two other isoforms of this channel, CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC(50) of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption via cyclic nucleotide-gated cation channels in airway cells.

Asunto(s)

GMP Cíclico/análogos & derivados , Células Epiteliales/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/fisiología , Cationes , GMP Cíclico/farmacología , Humanos , Transporte Iónico , Pulmón , Técnicas de Placa-Clamp , Células Tumorales Cultivadas

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Evolución Molecular , Fibrosis Quística/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/microbiología , Humanos , Mucosa Intestinal/microbiología , Pulmón/microbiología , Mutación , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Salmonella typhi/patogenicidad , Salmonella typhi/fisiología