RESUMEN
Na(+)/H(+) exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3(-/-)) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3(-/-) mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Portadoras/fisiología , Riñón/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Adenina/análogos & derivados , Adenina/farmacocinética , Animales , Células HEK293 , Humanos , Proteínas de la Membrana , Ratones , Organofosfonatos/farmacocinética , Regulación hacia ArribaRESUMEN
Multi-drug resistance 1 (MDR1, ABCB1), also known as P-glycoprotein (P-gp), restricts intestinal uptake of many drugs, and contributes to cellular resistance to cancer chemotherapy. In this study, we examined the pharmacologic characteristics of HM30181, a newly developed MDR1 inhibitor, and tested its capacity to increase the oral bioavailability and efficacy of paclitaxel, an anti-cancer drug usually given by intravenous injection. In the ATPase assay using MDR1-enriched vesicles, HM30181 showed the highest potency (IC(50)=0.63nM) among several MDR1 inhibitors, including cycloporin A, XR9576, and GF120918, and effectively blocked transepithelial transport of paclitaxel in MDCK monolayers (IC(50)=35.4nM). The ATPase inhibitory activity of HM30181 was highly selective to MDR1. HM30181 did not inhibit MRP1 (ABCC1), MRP2 (ABCC2), and MRP3 (ABCC3), and partially inhibited BCRP (ABCG2) only at very high concentrations. Importantly, co-administration of HM30181 (10mg/kg) greatly increased oral bioavailability of paclitaxel from 3.4% to 41.3% in rats. Moreover, oral co-administration of paclitaxel and HM30181 showed a tumor-inhibitory strength equal or superior to that of intravenous paclitaxel in the xenograft model in nude mice. These results identify HM30181 as a highly selective and potent inhibitor of MDR1, which in combination with paclitaxel, may provide an orally effective anti-tumor regimen.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Benzopiranos/farmacología , Isoquinolinas/farmacología , Paclitaxel/farmacocinética , Paclitaxel/uso terapéutico , Tetrazoles/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Benzopiranos/administración & dosificación , Disponibilidad Biológica , Línea Celular , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/terapia , Perros , Humanos , Isoquinolinas/administración & dosificación , Masculino , Ratones , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Paclitaxel/administración & dosificación , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato , Tetrazoles/administración & dosificación , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Single nucleotide polymorphisms (SNPs) are the most common form of human genetic variation. Non-synonymous SNPs (nsSNPs) change an amino acid. Organic anion transporters (OATs) play an important role in eliminating or reabsorbing endogenous and exogenous organic anionic compounds. Among OATs, hOAT4 mediates high affinity transport of estrone sulfate and dehydroepiandrosterone sulfate. The rapid bone loss that occurs in post-menopausal women is mainly due to a net decrease of estrogen. In the present study we searched for SNPs within the exon regions of hOAT4 in Korean women osteoporosis patients. Fifty healthy subjects and 50 subjects with osteoporosis were screened for genetic polymorphism in the coding region of SLC22A11 (hOAT4) using GC-clamp PCR and denaturing gradient gel electrophoresis (DGGE). We found three SNPs in the hOAT4 gene. Two were in the osteoporosis group (C483A and G832A) and one in the normal group (C847T). One of the SNPs, G832A, is an nsSNP that changes the 278th amino acid from glutamic acid to lysine (E278K). Uptake of [3H] estrone sulfate by oocytes injected with the hOAT4 E278K mutant was reduced compared with wild-type hOAT4. Km values for wild type and E278K were 0.7 microM and 1.2 microM, and Vmax values were 1.8 and 0.47 pmol/oocyte/h, respectively. The present study demonstrates that hOAT4 variants can causing inter-individual variation in anionic drug uptake and, therefore, could be used as markers for certain diseases including osteoporosis.
Asunto(s)
Pueblo Asiatico/genética , Transportadores de Anión Orgánico Sodio-Independiente/genética , Osteoporosis/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Anciano , Animales , Composición de Base , Secuencia de Bases , Transporte Biológico , Estudios de Casos y Controles , Análisis Mutacional de ADN , Electroforesis en Gel de Agar , Exones/genética , Femenino , Genoma Humano/genética , Humanos , Intrones/genética , Corea (Geográfico) , Persona de Mediana Edad , Datos de Secuencia Molecular , Oocitos , Reacción en Cadena de la Polimerasa , XenopusRESUMEN
The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.
Asunto(s)
Riñón/metabolismo , Transportadores de Anión Orgánico/aislamiento & purificación , Transportadores de Anión Orgánico/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Clonación Molecular , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Oligopéptidos/inmunología , Filogenia , Isoformas de Proteínas/aislamiento & purificación , Estructura Terciaria de Proteína , Conejos , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The purpose of this study was to demonstrate the cellular localization of cyclooxygenase-2 (COX-2) and caveolin-3 (Cav-3) in primarily cultured rat chondrocytes. In normal rat chondrocytes, we observed relatively high levels of Cav-3 and a very low level of COX-2 mRNA and protein. Upon treating the chondrocytes with 5 microM of CdCl(2) (Cd) for 6 hr, the expressions of COX-2 mRNA and protein were increased with the decreased Cav-3 mRNA and protein expressions. The detergent insoluble caveolae-rich membranous fractions that were isolated from the rat chondrocytes and treated with Cd contained the both proteins of both COX-2 and Cav-3 in a same fraction. The immuno-precipitation experiments showed complex formation between the COX-2 and Cav-3 in the rat chondrocytes. Purified COX-2 with glutathione S-transferase-fused COX-2 also showed complex formation with Cav-3. Confocal and electron microscopy also demonstrated the co-localization of COX-2 and Cav-3 in the plasma membrane. The results from our current study show that COX-2 and Cav-3 are co-localized in the caveolae of the plasma membrane, and they form a protein-protein complex. The co-localization of COX-2 with Cav-3 in the caveolae suggests that the caveolins might play an important role for regulating the function of COX-2.
Asunto(s)
Caveolina 3/genética , Condrocitos/metabolismo , Ciclooxigenasa 2/genética , Animales , Animales Recién Nacidos , Western Blotting , Cloruro de Cadmio/farmacología , Caveolas/efectos de los fármacos , Caveolas/metabolismo , Caveolas/ultraestructura , Caveolina 3/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Expresión Génica , Inmunoprecipitación , Microscopía Confocal , Microscopía Electrónica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The kidney is an important organ for controlling the volume of body fluids, electrolytic balance and excretion/reabsorption of endogenous and exogenous compounds. Among these renal functions, excretion/reabsorption of endogenous and exogenous substance is very important for the maintenance of physiological homeostasis in the body. Recently discovered organic anion transporters (OAT or SLC22A) have important roles for renal functions. It is well known as drug transporter. Several isoforms belong to SLC22A family. They showed different transport substrate spectrums and different localizations within the kidney. Their gene expressions are changed by some stimulus. The functional transport properties are regulated by protein kinase C. In addition, the function of organic anion transporters are also regulated by protein-protein interaction, such as caveolin which is compositional protein of caveolae structure. In this review, we will give an introduction of organic anion transporters and its regulatory mechanisms.
RESUMEN
BACKGROUND: Cystinuria has been proposed as an inherited disease causing disorders in renal cystine and basic amino acid transport in the proximal tubules. Although cystinuria-related amino acid transporter gene related to b0,+-type amino acid transporter (rBAT1) and its substrate transport properties have been reported, the functional regulatory mechanisms remain to be elucidated. In this study, protein-protein interaction between rBAT1 and caveolin (Cav)-1 was investigated. METHODS: The renal distribution of rBAT1, rBAT and Cav-1 were demonstrated by employing reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Co-localization of rBAT1 and Cav-1 was observed by immunocytochemistry in primary cultured renal proximal tubule-derived cells using a confocal microscope. This result was confirmed by Western blot analysis of isolated caveolae-rich membrane fraction and immunoprecipitation experiments using respective antibodies. RESULTS: In the separated rat kidney tissues following the corticomedullary axis, Cav-1 mRNA and protein expressions were increased from the cortex to the inner medulla. rBAT1 mRNA and protein expression were detected mainly in the outer medulla. Confocal microscopic results showed rBAT1 and Cav-1 co-localization in the plasma membrane. This result was confirmed by Western blot analysis of caveolae-rich membrane fraction and immunoprecipitates by respective antibodies. The effect of Cav-1 on rBAT1 function was evaluated using Cav-1 antisense oligodeoxynucleotide (ODN). The [14C] arginine uptake by rBAT1 was unchanged by the treatment with antisense ODN. CONCLUSIONS: From these results, rBAT1 and Cav-1 share a cellular expression in the segregated caveolae structure. As caveolae are rich in signaling molecules, BAT1 could play a role in diverse pathophysiological processes.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Aminoácidos/metabolismo , Caveolina 1/metabolismo , Túbulos Renales Proximales/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Animales , Transporte Biológico Activo/fisiología , Western Blotting , Caveolina 1/genética , Células Cultivadas , Inmunohistoquímica , Túbulos Renales Proximales/citología , Microscopía Confocal , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The extracellular calcium sensing receptor (CaSR) belongs to the type III family of G-protein-coupled receptors, a family that comprises the metabotropic glutamate receptor and the putative vomeronasal organ receptors. The CaSR plays an important role for calcium homeostasis in parathyroid cells, kidney cells and other cells to directly 'sense' changes in the extracellular calcium ion concentration ([Ca2+]o). The mesangial cells are known to be involved in many pathologic sequences through the mediation of altered glomerular hemodynamics, cell proliferation, and matrix production. In this study, we examined the expression of the CaSR in the mouse mesangial cell lines (MMC, ATCC number CRL-1927). Reverse transcription-polymerase chain reaction (RT-PCR) was perform with CaSR-specific primers, and this was followed by nucleotide sequencing of the amplified product; this process identified the CaSR transcript in the MMCs. Moreover, CaSR protein was present in the MMCs as assessed by Western blot and immunocytochemical analysis using a polyclonal antibody specific for the CaSR. Functionally, [Ca2+]o induced the increment of the intracellular calcium concentration ([Ca2+]i) in a dose-dependent manner. This [Ca2+]i increment by [Ca2+]o was attenuated by the pretreatment with a phospholipase C inhibitor (U73122) and also by a pretreatment with a CaSR antagonist (NPS 2390). The similar results were also obtained in IP3 accumulation by [Ca2+]o. To investigate the physiological effect of the CaSR, the effect of the [Ca2+]o on cell proliferation was studied. The increased [Ca2+]o (up to 10 mM) produced a significant increase in the cell numbers. This mitogenic effect of [Ca2+]o was inhibited by the co-treatment with a CaSR antagonist. From these results, the [Ca2+]o-induced [Ca2+]i elevation in the MMC is coupled with the extracellular calcium sensing receptor. Furthermore, [Ca2+]o produces a mitogenic effect in MMCs.
Asunto(s)
Células Mesangiales/citología , Células Mesangiales/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Proliferación Celular , Inositol 1,4,5-Trifosfato/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Sensibles al Calcio/genéticaRESUMEN
A family of organic anion transporters (OAT) recently identified has important roles for the excretion or reabsorption of endogenous and exogenous compounds, and several new isoforms have been reported in this decade. Although the transepithelial transport properties of organic anions are gradually being understood, many portions of their functional characteristics in functions remain to be elucidated. A recently reported new cDNA encoding a mouse OAT5 (mOAT5) was constructed, using 3'-RACE PCR, with the total RNA isolated from a mouse kidney. When mOAT5 was expressed in Xenopus oocytes, mOAT5 transported estrone sulfate, dehydroepiandrosterone sulfate and ochratoxin A. Estrone sulfate uptake by mOAT5 displayed a time-dependent and sodium-independent manner. The Km values of estrone sulfate and dehydroepiandrosterone sulfate were 2.2 and 3.8 microM, respectively. mOAT5 interacted with chemically heterogeneous steroid or organic sulfates, such as nitrophenyl sulfate, methylumbelliferyl sulfate and estradiol sulfates. In contrast to the sulfate conjugates, mOAT5-mediated estrone sulfate uptake was not inhibited by the steroid or organic glucuronides. The mOAT5 protein having about 85 kDa molecular weight was shown to be mainly localized in the apical membrane of the proximal tubules of the outer medulla. These results suggest an important role of mOAT5 for the excretion or reabsorption of steroid sulfates in the kidney.
Asunto(s)
Riñón/metabolismo , Transportadores de Anión Orgánico/química , Sulfatos/metabolismo , Animales , Transporte Biológico , Western Blotting , Proteínas Portadoras/química , Clonación Molecular , ADN Complementario/metabolismo , Sulfato de Deshidroepiandrosterona/metabolismo , Relación Dosis-Respuesta a Droga , Estrona/análogos & derivados , Estrona/metabolismo , Glucurónidos/química , Immunoblotting , Inmunohistoquímica , Túbulos Renales/metabolismo , Cinética , Ratones , Ocratoxinas/metabolismo , Oocitos/metabolismo , ARN/metabolismo , Sulfatos/química , Factores de Tiempo , XenopusRESUMEN
The rat organic anion transporter 3 (rOAT3) has recently been identified as the third isoform of the OAT family. The mechanisms that regulate rOAT3's functions remain to be elucidated. rOAT3 contributes for moving a number of negatively charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role as a membrane transporter. To address the relationship of these two proteins, we investigated the protein-protein interaction between rOAT3 and Cav-1. The rOAT3 mRNA and protein expression were observed in the rat kidney, and the expressions of Cav-1 mRNA and protein were also detected in the kidney. Confocal microscopy of the immuno-cytochemistry experiments using primary cultured renal proximal tubular cells showed that rOAT3 and Cav-1 were co-localized at the plasma membrane. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions from the rat kidney and immuno-precipitation experimentation. When rOAT3's synthesized cRNA of rOAT3 along with the antisense oligo deoxynucleotide ofXenopusCav-1 were co-injected intoXenopusoocytes, the [(3)H] estrone sulfate uptake was significantly decreased. These findings suggest that rOAT3 and caveolin-1 share a cellular expression in the plasma membrane and Cav-1 up-regulates the organic anionic compound uptake via rOAT3 under normal physiological conditions.
Asunto(s)
Caveolinas/metabolismo , Riñón/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , ARN Mensajero/metabolismo , Ratas/metabolismo , Análisis de Varianza , Animales , Transporte Biológico Activo/fisiología , Western Blotting , Caveolina 1 , Membrana Celular/metabolismo , Cartilla de ADN , Estrona/análogos & derivados , Estrona/metabolismo , Inmunohistoquímica , Inmunoprecipitación , Transporte Iónico/fisiología , Microscopía Confocal , Oligonucleótidos Antisentido , Oocitos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tritio , Xenopus , Proteínas de XenopusRESUMEN
The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [(14)C]p-aminohippurate and [(3)H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.
Asunto(s)
Transporte Biológico Activo/fisiología , Caveolinas/metabolismo , Membrana Celular/metabolismo , Túbulos Renales Proximales/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Animales , Células CHO , Caveolina 2 , Células Cultivadas , Cricetinae , Inmunoprecipitación , Metotrexato/metabolismo , Microscopía Confocal , Oligonucleótidos Antisentido/farmacología , Oocitos/metabolismo , Proteína 1 de Transporte de Anión Orgánico/antagonistas & inhibidores , Proteína 1 de Transporte de Anión Orgánico/genética , ARN Complementario/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Xenopus laevis/metabolismo , Ácido p-Aminohipúrico/metabolismoRESUMEN
The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca(2+)](o)) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca(2+)](i)) was increased by an increment of [Ca(2+)](o). This [Ca(2+)](i) increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca(2+)](o)-induced [Ca(2+)](i) increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR-related disorders in the body.
Asunto(s)
Calcio/metabolismo , Caveolinas/metabolismo , Membrana Celular/metabolismo , Receptores Sensibles al Calcio/metabolismo , Neoplasias Óseas , Fraccionamiento Celular , Línea Celular Tumoral , Humanos , Microscopía Confocal , Oligorribonucleótidos Antisentido/farmacología , Osteosarcoma , Receptores Sensibles al Calcio/antagonistas & inhibidores , Regulación hacia ArribaRESUMEN
Corneal endothelial cells play an important role in maintaining the transparency and ionic balance of the cornea. Inflammation causes many changes in the intracellular and extracellular environment of the cornea, including acidosis. We examined the relationship between changes in extracellular pH and expression of cyclooxygenase-2 in cultured bovine corneal endothelial cells. When extracellular pH ([pH]o) was reduced to pH 6.4, COX-2 mRNA increased, with a peak at 2 h. This was blocked by pretreatment with actinomycin D and incubation with spermine NONOate (SPER/NO, a nitric oxide donor). Exposure to the H+ ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), also raised COX-2 mRNA levels. CCCP-induced COX-2 mRNA expression was also reduced by SPER/NO. These results were confirmed immuno-cytochemically. These data demonstrate that COX-2 expression is stimulated by the lowering of extracellular pH that could result from bacterial infection, and that this is countered by over-production of nitric oxide, which could also result from bacterial infection.
Asunto(s)
Acidosis/metabolismo , Epitelio Corneal/efectos de los fármacos , Óxido Nítrico/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Bovinos , Células Cultivadas , Ciclooxigenasa 2 , Dactinomicina/farmacología , Epitelio Corneal/enzimología , Concentración de Iones de Hidrógeno , Ionóforos/farmacología , Donantes de Óxido Nítrico/farmacología , Óxidos de Nitrógeno , Prostaglandina-Endoperóxido Sintasas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Espermina/análogos & derivados , Espermina/farmacologíaRESUMEN
The purpose of this study was to confirm protein-protein interaction between cyclooxygenase-1 (COX-1) and caveolins. The interaction of cyclooxygenase-1 and caveolins in the cultured human embryonic kidney (HEK 293) cells was investigated using immuno-precipitation and Western blot analysis. In HEK 293 cells, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Caveolae rich membranous fractions from the HEK 293 cells contained both COX-1 and caveolin-1 or caveolin-2 in same fractions. The experiments of immuno-precipitation showed complex formation between the COX-1 and caveolin-1 or caveolin-2 in the HEK 293 cells. Confocal microscopic results also support co-localization of COX-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with cylooxygenase-1 in caveolae suggested that caveolin would play an important role in regulating the function of COX-1.
Asunto(s)
Caveolinas/metabolismo , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Western Blotting , Caveolina 1 , Caveolina 2 , Membrana Celular/metabolismo , Células Cultivadas , Ciclooxigenasa 1 , Cartilla de ADN , Humanos , Inmunoprecipitación , Proteínas de la Membrana , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The purpose of this study is to understand the interaction of Na + -Ca2+ exchanger (NCX1), that is one of the essential regulators of Ca2+ homeostasis, with caveolin (Cav)-1 and Cav-2 in Cav-3 null cell (rat C6 glioma cell). Both mRNA and protein expression of NCX1, Cav-1 and Cav-2 was observed, but no expression of mRNA and protein of Cav-3 were observed in C6 glioma cells. In isolated caveolae-enriched membrane fraction, the NCX1, Cav-1 and Cav-2 proteins localized in same fractions. The experiment of immuno-precipitation showed complex formation between the NCX1 and Cavs. Confocal microscopy also supported co-localization of NCX1and Cavs at the plasma membrane. Functionally, sodium-free induced forward mode of NCX1 attenuated by Cav-1 antisense ODN. When treated cells with Cav-2 antisense ODN, both reverse and forward mode of NCX1 was attenuated. From these results, in the Cav-3 lacking cells, the function of NCX1 might be regulated by binding with Cavs. Considering the decrement of NCX1 activity by antisense ODNs, caveolins may play an important role in diverse of pathophysiological process of NCX1-related disorders in the body.