RESUMO
Rumen fermentation of plant-based forage in bovines is the major site for generation and absorption of short-chain fatty acids. Consequentially, the rumen is also the site for initial exposure to toxins released from diet. Accordingly, we have investigated the expression of bovine ABC transporters in the rumen associated with cytoprotection against xenobiotic exposure, namely MDR1 (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Bovine rumen samples from the ventral sac were obtained post-mortem from a commercial slaughterhouse after humane killing. Rumen papilla samples were then prepared for total RNA isolation for RT-PCR, SDS-PAGE/Western blotting and immunohistochemistry. PCR products of the predicted size were observed for both MDR1 and BCRP, but not for MRP2 using bovine-specific primers. ß-actin was used as a control transcript. Western blot analysis using C219 primary monoclonal antibody revealed MDR1 protein expression in bovine rumen (Mapp, of ~170-180 kD). Immunolocalisation of MDR1 using UIC2 monoclonal antibody within cryosections of bovine rumen showed extensive membrane staining in the cells of the stratum granulosum, stratum spinosum and stratum basale. MDR1 expression was absent from outer stratum corneum. Protein expression and immunolocalisation were also confirmed for BCRP, with prevalent staining in the stratum basale, becoming weaker in the stratum spinosum and stratum granulosum.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Rúmen/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Bovinos , Masculino , Proteínas de Neoplasias/genéticaRESUMO
Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human and rodent BCRP. An additional pathway for ciprofloxacin secretion exists in Caco-2 cells, which is unlikely to be MRP(4)-mediated. BCRP is likely to be the dominant transport mechanism for ciprofloxacin efflux in both rat and human jejunum.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antibacterianos/farmacocinética , Ciprofloxacina/farmacocinética , Mucosa Intestinal/metabolismo , Proteínas de Neoplasias/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Disponibilidade Biológica , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos WistarRESUMO
Facilitative UT-B urea transporters have been located in the gastrointestinal tract of numerous mammalian species. We have previously identified UT-B urea transporters within the epithelial layers of the bovine (b) rumen. The aim of this study was to test the hypothesis that ruminal bUT-B urea transporters are regulated by dietary intake. Six Limousine-cross steers (initial BW = 690 +/- 51 kg) were separated into 2 groups fed a basic silage-based diet (RS) or a concentrate-based diet (RC) for 37 d and compared for ruminal morphology, content, and bUT-B expression. Analysis by reverse transcription-PCR showed that ruminal bUT-B2 mRNA expression was greater in RC-fed than RS-fed animals. Utilizing an anti-bUT-B antibody, we also detected a significant increase in bUT-B2 protein expression in RC-fed rumen (P < 0.05, n = 3). In agreement with these findings, immunolocalization studies of RC-fed ruminal tissue showed strong bUT-B signals throughout all epithelial layers, in contrast to weaker staining in RS-fed rumen that was more localized to the stratum basale. This study therefore confirmed that ruminal bUT-B urea transporter expression and localization were indeed altered by changes in dietary intake. We conclude that UT-B transporters play a significant role in the dietary regulation of bovine nitrogen balance.
Assuntos
Ração Animal , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Rúmen/metabolismo , Animais , Bovinos , Dieta , Concentração de Íons de Hidrogênio , Immunoblotting/veterinária , Masculino , Proteínas de Membrana Transportadoras/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Rúmen/ultraestrutura , Transportadores de UreiaRESUMO
Intestinal induction of Pgp is known to limit the oral availability of certain drug compounds and give rise to detrimental drug-drug interactions. We have investigated the induction of P-glycoprotein (Pgp; MDR1) activity in a human intestinal epithelial cell line (T84) following pre-exposure to a panel of drug compounds, reported to be Pgp substrates, inhibitors or inducers. Human MDR1-transfected MDCKII epithelial monolayers were used to assess Pgp substrate interactions and inhibition of digoxin secretion by the selected drug compounds. The T84 cell line was used to assess induction of Pgp-mediated digoxin secretion following pre-exposure to the same compounds. Changes in gene expression (MDR1, MRP2, PXR and CAR) were determined by quantitative RT-PCR. Net transepithelial digoxin secretion was increased (1.3 fold, n=6, P<0.05) following pre-exposure to the PXR activator hyperforin (100nM, 72h), as was MDR1 mRNA expression (3.0 fold, n=4, P<0.05). A number of Pgp substrates (quinidine, amprenavir, irinotecan, topotecan, atorvastatin and erythromycin) induced net digoxin secretion, as did the non-Pgp substrate artemisinin. Various non-Pgp substrates demonstrated inhibition of digoxin secretion (verapamil, mifepristone, clotrimazole, mevastatin, diltiazem and isradipine) but did not induce Pgp-mediated digoxin secretion. Of the compounds that increased Pgp secretion, quinidine, topotecan, atorvastatin and amprenavir pre-exposure also elevated MDR1 mRNA levels, whereas erythromycin, irinotecan and artemisinin displayed no change in transcript levels. This indicates possible post-translational regulation of digoxin secretion. Finally, a strong correlation between drug modulation of MRP2 and PXR mRNA expression levels was evident.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Células Epiteliais/efeitos dos fármacos , Intestinos/citologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Xenobióticos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Receptor Constitutivo de Androstano , Digoxina/metabolismo , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND AND PURPOSE: Oral drug bioavailability is limited by intestinal expression of P-glycoprotein (MDR1, Pgp, ABCB1) whose capacity is regulated via nuclear receptors e.g. the pregnane X receptor (PXR, SXR, NR1I2). In order to study dynamic regulation of MDR1 transport capacity we have identified the T84 epithelial cell-line as a model for human intestine co-expressing MDR1 with PXR. The ability of rifampin, a known PXR agonist and digoxin, a model MDR1 substrate, to regulate MDR1 expression and transport activity has been tested, in these T84 cells. EXPERIMENTAL APPROACH: Transport was assayed by bi-directional [(3)H]-digoxin transepithelial fluxes across epithelial layers of T84 cells seeded onto permeable filter supports following pre-exposure to rifampin and digoxin. Quantitative real-time PCR, Western blotting and immunocytochemistry were used to correlate induction of MDR1 transcript and protein levels with transport activity. KEY RESULTS: Rifampin exposure (10 microM, 72 hours) increased MDR1 transcript levels (3.4 fold), MDR1 total protein levels (4.4 fold), apical MDR1 protein (2.7 fold) and functional activity of MDR1 (1.2 fold). Pre-incubation with digoxin (1 microM, 72 hours) potently induced MDR1 transcript levels (92 fold), total protein (7 fold), apical MDR1 protein (4.7 fold) and functional activity (1.75 fold). Whereas PXR expression was increased by rifampin incubation (2 fold), digoxin reduced PXR expression (0.3 fold). CONCLUSIONS AND IMPLICATIONS: Chronic digoxin pre-treatment markedly upregulates MDR1 expression and secretory capacity of T84 epithelia. Digoxin-induced changes in MDR1 levels are distinct from PXR-mediated changes resulting from rifampin exposure.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Antituberculosos/farmacologia , Digoxina/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Rifampina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/biossíntese , Actinas/biossíntese , Actinas/genética , Transporte Biológico Ativo/efeitos dos fármacos , Western Blotting , Células CACO-2 , Linhagem Celular , Receptor Constitutivo de Androstano , Humanos , Imuno-Histoquímica , Receptor de Pregnano X , RNA/biossíntese , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores de Esteroides/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/efeitos dos fármacosRESUMO
Extracellular pyrophosphate (PPi) plays a central role in the control of normal bone mineralization since it antagonizes inorganic phosphate in the promotion of hydroxyapatite deposition. Studies using knock-out mice have established the functional importance of PPi generation via nucleotide pyrophosphatase phosphodiesterases (NPP) and of PPi transmembrane transport by the progressive ankylosis (ANK) protein. Tissue non-specific alkaline phosphatase activity counteracts this by hydrolysis of PPi to inorganic phosphate. The molecular nature and transport function of ANK are reviewed. A close parallel is drawn between the controlled mineralization of bone and the prevention of abnormal calcium crystal deposition within the kidney, especially when concentrated urine is produced. Pyrophosphate is present in urine, and ANK is expressed in the cortical collecting duct where PPi transport to both the tubular lumen and the renal interstitium may occur. Pyrophosphate may also be generated here by nucleoside triphosphate diphosphohydrolases (NTPD2 and 3) together with NPP1. Alkaline phosphatase activity is restricted to the proximal nephron, remote from these sites of PPi generation, transport and function. The physiological importance of PPi generation and transport in preventing idiopathic calcium renal stone disease and nephrocalcinosis now needs to be established.
Assuntos
Calcificação Fisiológica/fisiologia , Cálculos Renais/fisiopatologia , Animais , Calcificação Fisiológica/efeitos dos fármacos , Difosfatos/metabolismo , Humanos , Articulações/crescimento & desenvolvimento , Articulações/fisiologia , Rim/metabolismo , Cálculos Renais/tratamento farmacológicoRESUMO
Monocarboxylate-H+ cotransporters, such as monocarboxylate transporter (MCT) SLC16A, have been suggested to mediate transruminal fluxes of short-chain fatty acids, ketone bodies, and lactate. Using an RT-PCR approach, we demonstrate expression of MCT1 (SLC16A1) and MCT2 (SLC16A7) mRNA in isolated bovine rumen epithelium. cDNA sequence from these PCR products combined with overlapping expressed sequence tag data allowed compilation of the complete open reading frames for MCT1 and MCT2. Immunohistochemical localization of MCT1 shows plasma membrane staining in cells of the stratum basale, with intense staining of the basal aspects of the cells. Immunostaining decreased in the cell layers toward the rumen lumen, with weak staining in the stratum spinsoum. Immunostaining in the stratum granulosum and stratum corneum was essentially negative. Since monocarboxylate transport will load the cytosol with acid, expression and location of Na+/H+ exchanger (NHE) family members within the rumen epithelium were determined. RT-PCR demonstrates expression of multiple NHE family members, including NHE1, NHE2, NHE3, and NHE8. In contrast to MCT1, immunostaining showed that NHE1 was predominantly localized to the stratum granulosum, with a progressive decrease toward the stratum basale. NHE2 immunostaining was observed mainly at an intracellular location in the stratum basale, stratum spinosum, and stratum granulosum. Given the anatomic localization of MCT1, NHE1, and NHE2, the mechanism of transruminal short-chain fatty acid, ketone body, and lactate transfer is discussed in relation to a functional model of the rumen epithelium comprising an apical permeability barrier at the stratum granulosum, with a cell syncitium linking the stratum granulosum to the blood-facing stratum basale.
Assuntos
Transportadores de Ácidos Monocarboxílicos/metabolismo , Rúmen/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Ácidos/metabolismo , Animais , Bovinos , Primers do DNA , Epitélio/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Dados de Sequência Molecular , Transportadores de Ácidos Monocarboxílicos/biossíntese , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trocadores de Sódio-Hidrogênio/biossínteseRESUMO
We have used the perforated patch clamp and fura-2 fluorescence techniques to study the effect of extracellular Zn(2+) on whole-cell Ca(2+)-activated Cl(-) currents (I (CLCA)) in mouse inner medullary collecting duct cells (mIMCD-3). I (CLCA) was spontaneously active in 74% of cells under basal conditions and displayed time and voltage-independent kinetics and an outwardly rectifying current/voltage relationship (I/V). Addition of zinc chloride (10-400 microM) to the bathing solution resulted in a dose-dependent increase in I (CLCA) with little change in Cl(-) selectivity or biophysical characteristics, whereas gadolinium chloride (30 microM) and lanthanum chloride (100 microM) had no significant effect on the whole-cell current. Using fura-2-loaded mIMCD-3 cells, extracellular Zn(2+) (400 microM) stimulated an increase in intracellular Ca(2+) to an elevated plateau. The Zn(2+)-stimulated [Ca(2+)](i) increase was inhibited by thapsigargin (200 nM), the IP(3) receptor antagonist 2-aminoethoxydiphenyl borate (10 microM) and removal of bath Ca(2+). Pre-exposure to Zn(2+) (400 microM) markedly attenuated the ATP (100 microM)-stimulated [Ca(2+)](i) increase. These data are consistent with the hypothesis that extracellular Zn(2+) stimulates an increase in [Ca(2+)](i) by a release of calcium from thapsigargin/IP(3) sensitive stores. A possible physiological role for a divalent metal ion receptor, distinct from the extracellular Ca(2+)-sensing receptor, in IMCD cells is discussed.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/fisiologia , Medula Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Zinco/farmacologia , Animais , Compostos de Boro/farmacologia , Células Cultivadas , Canais de Cloreto/metabolismo , Cloretos/farmacologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Fluorometria , Medula Renal/citologia , Medula Renal/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Tapsigargina/farmacologia , Compostos de Zinco/farmacologiaRESUMO
Mutations in CLCN5, which encodes the voltage-dependent Cl(-)/H(+)antiporter, CLC-5, cause Dent's disease. This disorder is characterized by low molecular-weight proteinuria, hypercalciuria, nephrocalcinosis and nephrolithiasis. Using a collecting duct cell model (mIMCD-3) in which endogenous clc-5 is disrupted by antisense clc-5 or overexpression of truncated clc-5, we demonstrate altered expression of the crystal adhesion molecule, annexin A2. Endogenously expressed annexin A2 is intracellular with limited plasma membrane localization. Following clc-5 disruption, there is both a marked increase in plasma membrane annexin A2 and an increase in cell surface crystal retention and agglomeration, which may be attenuated using pretreatment with anti-annexin A2 antibodies or wheat germ agglutinin lectin but not by concanavalin A. We hypothesize that in Dent's disease, endocytic failure leads to an accumulation at the plasma membrane of crystal-binding molecules that include annexin A2 leading to retention of calcium crystals and ultimately nephrocalcinosis and nephrolithiasis.
Assuntos
Anexina A2/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Nefropatias/metabolismo , Túbulos Renais Coletores/metabolismo , Animais , Anexina A2/genética , Linhagem Celular , Canais de Cloreto/genética , Cristalização , Endocitose , Células Epiteliais/metabolismo , Expressão Gênica , Nefropatias/fisiopatologia , Túbulos Renais Coletores/citologia , Camundongos , Mutação , Ácido N-Acetilneuramínico/metabolismo , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
The UT-A (SLC14a2) and UT-B (SLC14a1) genes encode a family of specialized urea transporter proteins that regulate urea movement across plasma membranes. In this report, we describe the structure of the bovine UT-B (bUT-B) gene and characterize UT-B expression in bovine rumen. Northern analysis using a full-length bUT-B probe detected a 3.7-kb UT-B signal in rumen. RT-PCR of bovine mRNA revealed the presence of two UT-B splice variants, bUT-B1 and bUT-B2, with bUT-B2 the predominant variant in rumen. Immunoblotting studies of bovine rumen tissue, using an antibody targeted to the NH2-terminus of mouse UT-B, confirmed the presence of 43- to 54-kDa UT-B proteins. Immunolocalization studies showed that UT-B was mainly located on cell plasma membranes in epithelial layers of the bovine rumen. Ussing chamber measurements of ruminal transepithelial transport of (14)C-labeled urea indicated that urea flux was characteristically inhibited by phloretin. We conclude that bUT-B is expressed in the bovine rumen and may function to transport urea into the rumen as part of the ruminant urea nitrogen salvaging process.
Assuntos
Bovinos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Rúmen/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Southern Blotting , Western Blotting , Epitélio/metabolismo , Feminino , Imuno-Histoquímica , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Oócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Xenopus , Transportadores de UreiaRESUMO
The functional organization of the bovine rumen epithelium has been examined by electron and light microscopy combined with immunocytochemistry to define a transport model for this epithelium. Expression of connexin 43, an integral component of gap junctions, the tight-junction molecules claudin-1 and zonula occludens 1 (ZO-1), and the catalytic alpha-subunit of Na(+)-K(+)-ATPase was demonstrated by SDS-PAGE and Western blotting. From the lumen surface, four cell layers can be distinguished: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale. Both claudin-1 and ZO-1 immunostaining showed plasma membrane staining, which was present at the stratum granulosum with decreasing intensity through the stratum spinosum to the stratum basale. The stratum corneum was negative for claudin-1 immunostaining. Transmission electron microscopy confirmed that occluding tight junctions were present at the stratum granulosum. Plasma membrane connexin 43 immunostaining was most intense at the stratum granulosum and decreased in intensity through stratum spinosum and stratum basale. There was intense immunostaining of the stratum basale for Na(+)-K(+)-ATPase, with weak staining of the stratum spinosum. Both the stratum granulosum and the stratum corneum were essentially negative. Stratum basale cells also displayed a high mitochondrial density relative to more apical cell layers. We conclude that epithelial barrier function may be attributed to the stratum granulosum and that cell-cell gap junctions allow diffusion to interconnect the barrier cell layer with the stratum basale where Na(+)-K(+)-ATPase is concentrated.
Assuntos
Rúmen/fisiologia , Animais , Transporte Biológico Ativo/fisiologia , Bovinos , Claudina-1 , Conexina 43/fisiologia , Epitélio/anatomia & histologia , Epitélio/fisiologia , Epitélio/ultraestrutura , Expressão Gênica , Proteínas de Membrana/fisiologia , Mitocôndrias/fisiologia , Rúmen/anatomia & histologia , Rúmen/ultraestrutura , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Defects in an intracellular chloride channel CLC-5 cause Dent's disease, an inherited kidney stone disorder. Using a collecting duct model, mIMCD-3 cells, we show expression of dimeric mCLC-5. Transient transfection of antisense CLC-5 reduces CLC-5 protein expression. Binding of both calcium phosphate (hydroxyapatite) and calcium oxalate monohydrate (COM) crystals overlaid onto mIMCD-3 cultures was affected by altered CLC-5 expression. Calcium phosphate crystal agglomerations (>10 microm) were minimal in control (9%) and sense (13%) CLC-5-transfected cells, compared to 66% of antisense CLC-5-transfected cells (P<0.001). Small calcium phosphate crystals (<10 microm) were found associated with 45% of sense CLC-5-treated cells, of which the majority (11/14 cells) appeared to be internalised within the cell. Calcium oxalate agglomerations (>10 microm) were also largely absent for controls or sense mCLC-5 transfectants (11% and 9% of cells, respectively) with COM crystal agglomerates predominating in antisense CLC-5 transfectants (66%, P<0.0001). We conclude that collecting duct cells with reduced CLC-5 expression lead to a tendency to form calcium crystal agglomeration, which may help explain the nephrocalcinosis and nephrolithiasis seen in Dent's disease.
Assuntos
Oxalato de Cálcio/química , Oxalato de Cálcio/metabolismo , Fosfatos de Cálcio/química , Fosfatos de Cálcio/metabolismo , Canais de Cloreto/metabolismo , Regulação da Expressão Gênica , Túbulos Renais Coletores/metabolismo , Animais , Western Blotting , Linhagem Celular , Canais de Cloreto/biossíntese , Canais de Cloreto/genética , Cristalização , Túbulos Renais Coletores/citologia , CamundongosRESUMO
Using the whole cell patch-clamp technique, a Ca2+-activated Cl- conductance (CaCC) was transiently activated by extracellular ATP (100 microM) in primary cultures of mouse inner medullary collecting duct (IMCD) cells and in the mouse IMCD-K2 cell line. ATP also transiently increased intracellular Ca2+ concentration ([Ca2+]i) from 100 nM to peak values of approximately 750 nM in mIMCD-K2 cells, with a time course similar to the ATP-induced activation and decay of the CaCC. Removal of extracellular Ca2+ had no major effect on the peak Cl- conductance or the increase in [Ca2+]i induced by ATP, suggesting that Ca2+ released from intracellular stores directly activates the CaCC. In mIMCD-K2 cells, a rectifying time- and voltage-dependent current was observed when [Ca2+]i was fixed via the patch pipette to between 100 and 500 nM. Maximal activation occurred at approximately 1 microM [Ca2+]i, with currents losing any kinetics and displaying a linear current-voltage relationship. From Ca2+-dose-response curves, an EC50 value of approximately 650 nM at -80 mV was obtained, suggesting that under physiological conditions the CaCC would be near fully activated by mucosal nucleotides. Noise analysis of whole cell currents in mIMCD-K2 cells suggests a single-channel conductance of 6-8 pS and a density of approximately 5,000 channels/cell. In conclusion, the CaCC in mouse IMCD cells is a low-conductance, nucleotide-sensitive Cl- channel, whose activity is tightly coupled to changes in [Ca2+]i over the normal physiological range.
Assuntos
Cálcio/farmacocinética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Animais , Bestrofinas , Células Cultivadas , Citosol/metabolismo , Proteínas do Olho/metabolismo , Ativação do Canal Iônico/fisiologia , Canais Iônicos , Medula Renal/citologia , Túbulos Renais Coletores/citologia , Cinética , Camundongos , Técnicas de Patch-ClampRESUMO
1. We have tested the hypothesis that the voltage-dependent Cl(-) channel, ClC-5 functions as a plasma membrane Cl(-) conductance in renal inner medullary collecting duct cells. 2. Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl(-) conductance that was unaffected by external DIDS. 3. Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl(-) currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca(2+)-activated Cl(-) conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4. A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5. Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6. Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl(-)-selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl(-) conductances. Transient transfection with sense mClC-5 failed to induce the Cl(-) conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca(2+)-activated Cl(-) conductance 24 h post-transfection. 8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9. These data discount a major role for ClC-5 as a plasma membrane Cl(-) conductance in mIMCD-3 cells but suggest a role in endosomal function.
Assuntos
Canais de Cloreto/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Algoritmos , Animais , Células CHO , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Canais de Cloreto/genética , Cricetinae , Primers do DNA , Eletrofisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Imuno-Histoquímica , Túbulos Renais Coletores/citologia , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Técnicas de Patch-Clamp , TransfecçãoRESUMO
Loop-diuretic-sensitive 86Rb+(K+) transmembrane fluxes were determined in cells of a mouse inner medullary collecting duct cell line (mIMCD-K2). The furosemide-sensitive (0.1 mM) influx was a substantial fraction of the total influx (0.39+/-0.04 or 0.42+/-0.03, n=5 in the presence or absence of ouabain, respectively). Furosemide also reduced 86Rb+(K+) efflux by a similar fraction (0.46). RT-PCR analysis revealed expression of mRNA for the Na+-K+-2Cl- cortransporter-1 (NKCC1), but not NKCC2. Loop-diuretic-sensitive 86Rb+(K+) influx was confined to the basolateral membrane, confirming its localisation there. The physiological properties of NKCC1 expressed in mIMCD-K2 cells, including the dependence upon medium Na+, K+ and Cl- and the relative sensitivity to loop diuretics as assessed by the concentration required for half-maximal inhibition (IC50) (bumetanide 3.3+/-1.4x10-7 M>piretanide 2.5+/-0.15x10-6 M>furosemide 2.3+/-1.2x10-5 M) were typical for NKCC1. Possible functions of NKCC1 were tested; furosemide did not inhibit the majority of forskolin-stimulated secretory short-circuit current (Isc) (83.5+/-5.3% of the maintained response at 5 min). Secondly, total 86Rb+(K+) influx was stimulated markedly when external osmolarity was increased to 600 mosmol/l by mannitol due to an increase via NKCC1 from 55+/-11 to 191+/-2 nmol/106 cells per 15 min, (both n=4, P<0.01). In contrast, 10-5 M forskolin did not stimulate total 86Rb+(K+) influx. Finally, the ability of both K+ and NH4+ to compete for ouabain-insensitive 86Rb+(K+) influx via NKCC1 was confirmed with similar concentrations for half-maximal influx reduction (K0.5). Apical exposure to NH4+ elicited rapid cytosolic alkalinisation in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded epithelial layers, consistent with selective permeability of the apical membrane to NH3. Conversely, NH4+ (5 mM) at the basal cell surface resulted in progressive acidification, the initial rate being reduced by 43% by furosemide. We conclude that NKCC1 participates in selective uptake of NH4+ at the basal surface, and that IMCD may function in direct NH4+ deposition to urine.
Assuntos
Expressão Gênica , Medula Renal/metabolismo , Túbulos Renais Coletores/metabolismo , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/fisiologia , Cloreto de Amônio/farmacologia , Animais , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Tamanho Celular/fisiologia , Colforsina/farmacologia , Diuréticos/farmacologia , Condutividade Elétrica , Células Epiteliais/metabolismo , Fluoresceínas/administração & dosagem , Furosemida/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Concentração Osmolar , Potássio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Radioisótopos de Rubídio/metabolismo , Membro 2 da Família 12 de Carreador de SolutoRESUMO
Unconjugated bile acids such as cholic acid cause diarrhoea, mucosal irritation and toxicity. We sought to define the mechanism of cholate permeation across intestinal mucosal cells to understand how cellular exposure and accumulation are deleterious to mucosal function. Human intestinal Caco-2 and T84 cell monolayers were prepared by high-density seeding and cultured for >14 days on permeable culture supports. Cholate transport and cellular accumulation were determined using [3H]cholic acid. Epithelial barrier function was assessed by measuring transepithelial electrical resistance (Rt) and [14C]mannitol fluxes. Exposure of Caco-2 epithelia to serosal cholate caused a dose- and time-dependent disruption of barrier function. Apical exposure was without disruptive effect. Similar responses were observed for T84 epithelia. Cholate was preferentially accumulated across the basolateral surfaces in both Caco-2 and T84 cells, but was subject to active transepithelial secretion in Caco-2 monolayers only. Net secretion was substantially reduced by ATP depletion, showed saturation kinetics, and was subject to competitive inhibition by other bile acids. Cholate secretion was also sensitive to inhibition by the leukotriene antagonist MK-571 but not by digoxin, suggesting that MRP2, not MDR1, was responsible. RT-PCR and Western blotting confirmed MRP2 expression in Caco-2 epithelia but indicated its apparent absence from T84 cells.
Assuntos
Ácido Cólico/farmacologia , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Absorção , Trifosfato de Adenosina/fisiologia , Ácidos e Sais Biliares/farmacologia , Transporte Biológico , Células CACO-2 , Linhagem Celular , Ácido Cólico/farmacocinética , Impedância Elétrica , Enterócitos/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Manitol/farmacocinética , Membranas/metabolismo , Permeabilidade/efeitos dos fármacosRESUMO
We have used perforated patch clamp and Fura-2 microfluorescence measurements to study Ca(2+)-activated Cl- currents in cultured mouse renal inner medullary collecting duct cells (mIMCD-3). The conductance was spontaneously active under resting conditions and whole cell currents were time and voltage-independent with an outwardly rectifying current-voltage relationship. The channel blockers DIDS, niflumic acid, glybenclamide and NPPB reversibly decreased the basal currents, whereas the sulfhydryl agent, DTT produced an irreversible inhibition. Increasing or decreasing extracellular calcium produced parallel changes in the size of the basal currents. Variations in external Ca2+ were associated with corresponding changes in free cytosolic Ca2+ concentration. Increasing cytosolic Ca2+ by extracellular ATP or ionomycin, further enhanced Cl- conductance, with whole cell currents displaying identical biophysical properties to the basal currents. However, the agonist-stimulated currents were now increased by DTT exposure, but still inhibited by the other channel blockers. Using RT-PCR, three distinct mRNA transcripts belonging to the CLCA family of Ca(2+)-activated Cl- channel proteins were identified, two of which represent novel sequences. Whether different channels underlie the basal and agonist-stimulated currents in mIMCD-3 cells is unclear. Our findings establish a novel link between alterations in external and internal Ca2+ and the activity of Ca(2+)-activated Cl- transport in these cells.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Medula Renal/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Canais de Cloreto/antagonistas & inibidores , Células Epiteliais , Medula Renal/citologia , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Swelling-activated Cl(-) currents (I(Cl,swell)) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I(-) > Br(-) > Cl(-) > Asp(-). NPPB (100 microm) inhibited the current in a voltage independent manner, as did exposure to 10 microm tamoxifen and 500 microm niflumic acid (NFA). In contrast, DIDS (100 microm) blocked the current with a characteristic voltage dependency. These characteristics of I(Cl, swell) in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I(Cl,swell) by hypotonicity. However, PDBu inhibition of I(Cl,swell) was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca(2+) activated Cl(-) conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I(Cl,swell). Control of I(Cl,swell) by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl(-) secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I(Cl,swell) is therefore capable of regulating transepithelial Cl(-) secretion and suggests that I(Cl,swell) is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl(-) secretion, but tamoxifen (100 microm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I(sc)). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line.
Assuntos
Canais de Cálcio/fisiologia , Canais de Cloreto/fisiologia , Medula Renal/fisiologia , Túbulos Renais Coletores/fisiologia , Animais , Canais de Cloro CLC-2 , Linhagem Celular , Canais de Cloreto/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Condutividade Elétrica , Expressão Gênica , Canais Iônicos , Medula Renal/citologia , Túbulos Renais Coletores/citologia , Camundongos , Proteína Quinase C/metabolismoRESUMO
1. The nature of Cl- conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD-K2 cell line) was investigated. 2. Extracellular ATP (100 microM) stimulated a transient increase in both whole-cell Cl- conductance and intracellular free Ca2+. In contrast, ionomycin (10-100 nM) caused a sustained increase in whole-cell Cl- conductance. Pre-loading cells with the Ca2+ buffer BAPTA abolished the ATP-dependent responses and delayed the onset of the increase observed with ionomycin. 3. The Ca2+-activated whole-cell Cl- current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady-state current-voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at -80 mV. In BAPTA-loaded cells, ionomycin-elicited whole-cell currents exhibited pronounced outward rectification with time-dependent activation/inactivation. 4. Ca2+-activated and forskolin-activated Cl- conductances co-exist since ATP activation of whole-cell current occurred during a maximal stimulation by forskolin in single cell recordings. 5. In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (Isc) dependent upon basal medium Na+ and Cl-/HCO3- but independent of the presence of apical bathing medium Na+ and Cl-/HCO3-. This was identical to forskolin stimulation and consistent with transepithelial anion secretion. 6. PCR amplification of reverse-transcribed mRNA using gene-specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca2+-activated Cl- channel (mCLCA1) mRNA in mIMCD-K2 cells. 7. Ca2+ and forskolin-activated Cl- conductances participate in anion secretion by IMCD.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Coletores/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Cálcio/farmacologia , Linhagem Celular , Canais de Cloreto/biossíntese , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Colforsina/farmacologia , AMP Cíclico/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/biossíntese , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Células Epiteliais/efeitos dos fármacos , Indicadores e Reagentes/farmacologia , Líquido Intracelular/metabolismo , Transporte de Íons/efeitos dos fármacos , Ionomicina/farmacologia , Ionóforos/farmacologia , Túbulos Renais Coletores/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , RNA Mensageiro/biossínteseRESUMO
1. Transintestinal absorption of gamma-aminobutyric acid (GABA) via a pH-dependent mechanism is demonstrated in the model human intestinal epithelial cell line Caco-2. 2. Experiments with BCECF [2',7',-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]-loaded Caco-2 cells demonstrate that GABA transport across the apical membrane is coupled to proton flow into the cell. 3. Short-circuit current (ISC) measurements using Caco-2 cell monolayers under voltage-clamped conditions demonstrate that pH-dependent GABA transport is a rheogenic process even in the absence of extracellular Na+, consistent with H+/GABA symport. 4. A range of GABA analogues were tested for their abilities to: (a) inhibit pH-dependent [3H]GABA uptake across the apical membrane; (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers; (c) increase inward ISC across voltage-clamped Caco-2 cell monolayers. 5. Nipecotic acid, isonipecotic acid, D,L-beta-aminobutyric acid, and 3-amino-1-propanesulphonic acid each caused a marked acidification of intracellular pH and an increase in ISC when superfused at the apical surface of Caco-2 cell monolayers. In contrast L-alpha-amino-n-butyric acid failed to induce proton flow or ISC. The ability of these compounds to induce proton or current flow across the apical surface of this intestinal epithelium was closely related to the relative inhibitory effects on [3H]GABA uptake. 6. These observations demonstrate H+/GABA symport and suggest that this transport mechanism may be accessible as a route for oral absorption of therapeutically-useful GABA analogues.