RESUMEN
RATIONALE: Circulating adiponectin levels are negatively associated with glucose intolerance, inflammation and central adiposity. Since these conditions are common in cystic fibrosis (CF), we examined whether adiponectin values are altered in these patients. AIM: To determine if CF patients have altered adiponectin levels and if these levels correlate with glucose tolerance categories (normal, impaired glucose tolerance (IGT) and cystic fibrosis-related diabetes (CFRD)), insulin resistance or inflammatory markers such as fibrinogen and C-reactive protein (CRP). METHODS: Oral glucose tolerance tests (OGTTs) were performed and adiponectin levels were measured in 90 CF patients not known to be diabetic and 15 healthy controls matched for age, sex and body mass index (BMI). Inflammatory markers, serum albumin concentrations and the clinical status of CF patients (i.e. pulmonary function) were also examined. RESULTS: CF pathology was characterized by a high prevalence (43.5%) of glucose tolerance abnormalities: 26.5% of IGT and 17.0% of newly diagnosed CFRD. CF patients also presented systemic inflammation as revealed by a significant increase of fibrinogen (P=0.029) in all patients and higher CRP levels in CFRD patients compared to the controls (P<0.05). On the other hand, CF and control subjects had similar albumin serum concentration. While CF patients and controls had similar serum adiponectin values, women had significantly higher hormone levels than men (P<0.001). Adiponectin levels did not correlate with glucose tolerance, inflammatory markers or insulin resistance. On the other hand, they correlated positively with both total and HDL-cholesterol (P<0.001). CONCLUSION: CF patients did not show any alterations in adiponectin levels despite insulin resistance, glucose intolerance and sub clinical chronic inflammation. Thus, CF appears to be one of the rare conditions in which discordance between adiponectin values and insulin resistance or inflammation is evident.
Asunto(s)
Adiponectina/sangre , Fibrosis Quística/sangre , Complicaciones de la Diabetes/sangre , Diabetes Mellitus/sangre , Intolerancia a la Glucosa/sangre , Adulto , Glucemia/metabolismo , Proteína C-Reactiva/metabolismo , Colesterol/sangre , Fibrosis Quística/complicaciones , Femenino , Fibrinógeno/metabolismo , Intolerancia a la Glucosa/complicaciones , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Lipoproteínas HDL/sangre , Masculino , Valores de Referencia , Triglicéridos/sangreRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.
Asunto(s)
AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Activación del Canal Iónico/fisiología , Oocitos/metabolismo , Técnicas de Placa-Clamp/métodos , Canales de Sodio/metabolismo , Animales , Canales Epiteliales de Sodio , Humanos , Potenciales de la Membrana/fisiología , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
We investigated the role of taurine in cell homeostasis and characterized the taurine transport pathway in cultured kidney cells (A6). The taurine concentration in A6 cells varies with the osmolarity of the culture medium, suggesting that taurine participates in cell osmolarity. Under isosmotic conditions, 14C-taurine efflux through the apical membranes (aJtaur) was 6-7 times lower than that through the basolateral membranes (bJtaur). Under hyposmotic conditions, aJtaur remained almost unchanged. On the contrary, bJtaur increased 8 times in comparison with isosmotic conditions. In hyposmotic conditions, bJtaur was inhibited by 500 microM DIDS, 50 microM NPPB, 10 microM of the two oxonol derivatives DISBAC(2)3 and WW-791, and 100 microM ketoconazole. Conversely, 100 microM 1,9-dideoxyforskolin, 10 microM tamoxifen, 100 microM niflumic acid and 50 microM verapamil had no inhibitory effects. Cell volume regulation upon hyposmotic stress was also found to be inhibited by DISBAC(2)3 (K0.5 of 5+/-1 microM) and by ketoconazole. Nystatin was used to permeabilize the apical membranes with the aim to further characterize bJtaur. 14C-taurine transepithelial fluxes in nystatin-treated cells were found to be linear over taurine concentrations ranging from 3.5 microM to 35 mM. Clamping the transepithelial voltage at positive values (serosal side) slightly stimulated the 14C-taurine transport. Similar time courses of 14C-taurine, 36Cl and 86Rb transepithelial fluxes were found under osmotic stimulation followed by DIDS inhibition in nystatin-treated cells. In whole cell patch-clamp experiments, DISBAC(2)3 application resulted in a strong and reversible decrease of the global Cl- current which was stimulated by hyposmotic stress. Our study indicates that taurine participates in the control of A6 cell osmolarity and that the transporting taurine pathway (efflux) is on the basolateral membranes. In addition to usual chloride channel blockers, oxonol was found to be a potent blocker of the taurine transport and of the swelling-activated chloride current. Using a pharmacological approach, we could not distinguish between a common or different pathway for Cl- and taurine.
Asunto(s)
Células Epiteliales/fisiología , Homeostasis/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Taurina/fisiología , Línea Celular , Tamaño de la Célula , Canales de Cloruro/fisiología , Células Epiteliales/efectos de los fármacos , Soluciones Hipotónicas/farmacología , Riñón/efectos de los fármacos , Riñón/fisiología , Presión Osmótica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Taurina/farmacocinéticaRESUMEN
In rabbit proximal convoluted tubules, an ATP-sensitive K(+) (K(ATP)) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na(+) transport and basolateral K(+) conductance. This K(+) conductance is inhibited by taurine. We sought to isolate this K(+) channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K(+) conductance, largely inhibited by extracellular Ba(2+) and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K(+) current which was Ba(2+)-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K(+) channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K(+) current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K(+) current. The possible involvement of AK in the K(ATP) channel's regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.
Asunto(s)
Adenilato Quinasa/biosíntesis , Túbulos Renales Proximales/metabolismo , Oocitos/metabolismo , Canales de Potasio/genética , Adenosina Trifosfato/análisis , Adenilato Quinasa/química , Adenilato Quinasa/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/química , Electroquímica , Biblioteca de Genes , Gliburida , Técnicas In Vitro , Manitol , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Canales de Potasio/química , Canales de Potasio/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Conejos , Alineación de Secuencia , Taurina , Transfección , XenopusRESUMEN
K channels are ubiquitous in animal cells, where they are involved in a variety of physiological functions. In epithelial cells of the kidney, K channels are primarily involved in maintaining membrane potential, recycling and secreting K and regulating cell volume. As many renal K channels have now been studied or identified at the molecular level by means of a variety of approaches, including patch-clamp recordings, cDNA cloning and immunohistochemistry, the purpose of this review is to summarize what is presently known about the molecular identity of renal K channels with an emphasis on their regulatory properties.
Asunto(s)
Túbulos Renales/fisiología , Canales de Potasio/química , Canales de Potasio/fisiología , Animales , Humanos , Canales de Potasio/clasificaciónRESUMEN
The cell-attached configuration of the patch-clamp technique was used to investigate the effects of taurine on the basolateral potassium channels of rabbit proximal convoluted tubule. In the absence of taurine, the previously reported ATP-blockable channel, K(ATP), was observed in 51% of patches. It is characterized by an inwardly rectifying current-voltage curve with an inward slope conductance of 49 +/- 5 pS (n = 15) and an outward slope conductance of 13 +/- 6 pS (n = 15). The K(ATP) channel open probability (P(o)) is low, 0.15 +/- 0.06 (n = 15) at a -V(p) = -100 mV (V(p) is the pipette potential), and increases slightly with depolarization. The gating kinetics are characterized by one open time constant (tau(o) = 5.0 +/- 1.9 ms, n = 6) and two closed time constants (tau(C1) = 5. 2 +/- 1.5 ms, tau(C2) = 140 +/- 40 ms; n = 6). In 34% of patches, a second type of potassium channel, sK, with distinct properties was recorded. Its current-voltage curve is characterized by a sigmoidal shape, with an inward slope conductance of 12 +/- 2 pS (n = 4). Its P(o) is voltage independent and averages 0.67 +/- 0.03 (n = 4) at -V(p) = -80 mV. Both its open time and closed time distributions are described by a single time constant (tau(o) = 96 +/- 19 ms, tau(C) = 10.5 +/- 3.6 ms; n = 4). Extracellular perfusion of 40 mM taurine fails to affect sK channels, whereas K(ATP) channel P(o) decreases by 75% (from 0.17 +/- 0.06 to 0.04 +/- 0.02, n = 7, P < 0.05). In conclusion, the absolute basolateral potassium conductance of rabbit proximal tubules is the resulting combination of, at least, two types of potassium channels of roughly equal importance: a high-conductance low-open probability K(ATP) channel and a low-conductance high-open probability sK channel. The previously described decrease in the basolateral absolute potassium conductance by taurine is, however, mediated by a single type of K channel: the ATP-blockable K channel.
Asunto(s)
Membranas Intracelulares/metabolismo , Túbulos Renales Proximales/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Taurina/farmacología , Adenosina Trifosfato/farmacología , Animales , Femenino , Activación del Canal Iónico/fisiología , Cinética , Canales de Potasio/clasificación , ConejosRESUMEN
1. The effect of extracellular nucleotides applied on the apical side of polarised A6 cells grown on permeant filters was investigated by measuring the changes in (i) the 36Cl efflux through the apical membranes, (ii) the intracellular chloride concentrations (aCli, measured with N-(6-methoxyquinolyl) acetoethyl ester, MQAE), (iii) ICl, the short-circuit current in the absence of Na+ transport and (iv) the characteristics of the apical chloride channels using a patch-clamp approach. 2. ATP or UTP (0.1-500 microM) transiently stimulated ICl. The sequence of purinergic agonist potencies was UTP = ATP > ADP >> the P2X-selective agonist beta,gamma-methylene ATP = the P2Y-selective agonist 2-methylthioATP. Suramin (100 microM) as the P2Y antagonist Reactive Blue 2 (10 microM) had no effect on the UTP (or ATP)-stimulated current. These findings are consistent with the presence of P2Y2-like receptors located on the apical membranes of A6 cells. Apical application of adenosine also transiently increased ICl. This effect was blocked by theophylline while the UTP-stimulated ICl was not. The existence of a second receptor, of the P1 type is proposed. 3. ATP (or UTP)-stimulated ICl was blocked by apical application of 200 microM N-phenylanthranilic acid (DPC) or 100 microM niflumic acid while 100 microM glibenclamide was ineffective. 4. Ionomycin and thapsigargin both transiently stimulated ICl; the nucleotide stimulation of ICl was not suppressed by pre-treatment with these agents. Chlorpromazin (50 microM), a Ca2+-calmodulin inhibitor strongly inhibited the stimulation of ICl induced either by apical UTP or by ionomycin application. BAPTA-AM pre-treatment of A6 cells blocked the UTP-stimulated ICl. Niflumic acid also blocked the ionomycin stimulated ICl. 5. A fourfold increase in 36Cl effluxes through the apical membranes was observed after ATP or UTP application. These increases of the apical chloride permeability could also be observed when following aCli changes. Apical application of DPC (1 mM) or 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB; 500 microM) produced an incomplete inhibition of 36Cl effluxes through the apical membranes in ATP-stimulated and in untreated monolayers. 6. In single channel patch-clamp experiments, an apical chloride channel with a unitary single channel conductance of 7.3 +/- 0.6 pS (n = 12) was usually observed. ATP application induced the activation of one or more of these channels within a few minutes. 7. These results indicate that multiple purinergic receptor subtypes are present in the apical membranes of A6 cells and that nucleotides can act as modulators of Cl- secretion in renal cells.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Canales de Cloruro/metabolismo , Riñón/metabolismo , Nucleótidos/farmacología , Animales , Calcio/metabolismo , Línea Celular , Canales de Cloruro/efectos de los fármacos , Cloruros/metabolismo , Inhibidores Enzimáticos/farmacología , Ionomicina/farmacología , Ionóforos/farmacología , Riñón/citología , Riñón/efectos de los fármacos , Cinética , Tapsigargina/farmacología , Uridina Trifosfato/farmacología , Xenopus laevisRESUMEN
The nature of the calcium-transporting mechanisms involved in A6 cell calcium homeostasis under iso- and hypo-osmotic conditions was investigated using fura-2 (AM) as a cell calcium indicator. Under steady-state conditions, intracellular calcium (Ca2+i) was increased by Bay K8644 or by gramicidin, an ionophore which depolarises A6 cell membranes. The Ca2+i increase following calcium addition (to calcium-depleted cells) or membrane depolarisation was blocked by nifedipine but not by verapamil or omega-conotoxin, indicating that the membrane calcium permeability may be mediated by voltage-dependent and dihydropyridine-sensitive calcium channels. Ca2+i could also be increased by a hypo-osmotic shock having a linear relationship with the osmolarity change. This osmotically induced Ca2+i increase had an extracellular origin since it was absent when cells were suspended in a calcium-free medium and it was not affected by thapsigargin or TMB-8 application. In addition, it was inhibited by the calcium channel inhibitor, nifedipine. Furthermore, under hypo-osmotic conditions, an additional Ca2+i increase, sensitive to nifedipine, was measured when cells were depolarised by gramicidin or K-gluconate addition. It is proposed that the hypo-osmotically induced cell calcium increase implies the activation of voltage-dependent and nifedipine-sensitive calcium channels, presenting the same pharmacological characteristics as those involved in cell calcium homeostasis under iso-osmotic conditions. The initial Ca2+i increase was transient and stabilised to a value nevertheless higher than the iso-osmotic level; this secondary and incomplete regulatory phase did not occur in the presence of thapsigargin or TMB-8, thus providing evidence of intracellular calcium storage.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Túbulos Renales Distales/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Antibacterianos/farmacología , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Gramicidina/farmacología , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Transporte Iónico , Túbulos Renales Distales/citología , Túbulos Renales Distales/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Concentración Osmolar , Tapsigargina/farmacología , Xenopus laevisRESUMEN
The permeability to Cl- of the basolateral membrane (blm) was investigated in renal (A6) epithelial cells, assessing their role in transepithelial ion transport under steady-state conditions (isoosmotic) and following a hypoosmotic shock (i.e. in a regulatory volume decrease, RVD). Three different complementary studies were made by measuring: (1) the Cl- transport rates (delta F/Fo s-1 (x10(-3))), where F is the fluorescence of N-(6-methoxyquinoyl) acetoethyl ester, MQAE, and Fo the maximal fluorescence (x10(-3)) of both membranes by following the intracellular Cl- activities (ai Cl-, measured with MQAE) after extracellular Cl- substitution (2) the blm 86Rb and 36Cl uptakes and (3) the cellular potential and Cl- current using the whole-cell patch-clamp technique to differentiate between the different Cl- transport mechanisms. The permeability of the blm to Cl- was found to be much greater than that of the apical membranes under resting conditions: aiCl- changes were 5.3 +/- 0.7 mM and 25.5 +/- 1.05 mM (n = 79) when Cl- was substituted by NO3(-) in the media bathing apical and basolateral membranes. The Cl- transport rate of the blm was blocked by bumetanide (100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 microM) but not by N-phenylanthranilic acid (DPC, 100 microM). 86Rb and 36Cl uptake experiments confirmed the presence of a bumetanide- and a NPPB-sensitive Cl- pathway, the latter being approximately three times more important than the former (Na/K/2Cl cotransporter). Appli-cation of a hypoosmotic medium to the serosal side of the cell increased delta F/Fo s-1 (x10(-3)) after extracellular Cl- substitution (1.03 +/- 0.10 and 2.45 +/- 0.17 arbitrary fluorescent units s-1 for isoosmotic and hypoosmotic conditions respectively, n = 11); this delta F/Fo s-1 (x10(-3)) increase was totally blocked by serosal NPPB application; on the other hand, cotransporter activity was decreased by the hypoosmotic shock. Cellular Ca2+ depletion had no effect on delta F/Fo s-1 (x10(-3)) under isoosmotic conditions, but blocked the delta F/Fo s-1 (x10(-3)) increase induced by a hypoosmotic stress. Under isotonic conditions the measured cellular potential at rest was -37.2 +/- 4.0 mV but reached a maximal and transient depolarization of -25.1 +/- 3.7 mV (n = 9) under hypoosmotic conditions. The cellular current at a patch-clamping cellular potential of -85 mV (close to the Nernst equilibrium potential for K+) was blocked by NPPB and transiently increased by hypoosmotic shock (≈50% maximum increase). This study demonstrates that the major component of Cl- transport through the blm of the A6 monolayer is a conductive pathway (NPPB-sensitive Cl- channels) and not a Na/K/2Cl cotransporter. These channels could play a role in transepithelial Cl- absorption and cell volume regulation. The increase in the blm Cl- conductance, inducing a depolarization of these membranes, is proposed as one of the early events responsible for the stimulation of the 86Rb efflux involved in cell volume regulation.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Animales , Agua Corporal/metabolismo , Bumetanida/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Tamaño de la Célula/fisiología , Canales de Cloruro/efectos de los fármacos , Diuréticos/farmacología , Colorantes Fluorescentes , Riñón/citología , Concentración Osmolar , Técnicas de Placa-Clamp , Radioisótopos , Radioisótopos de Rubidio , Simportadores de Cloruro de Sodio-Potasio , Xenopus laevisRESUMEN
The presence of a Na/Ca exchanger in A6 cells was investigated by measuring intracellular calcium (Cai) fluctuations and the 45Ca fluxes through the basolateral membranes (blm) of the cell monolayer. Removal of Na+ from the medium produced a transient increase in Cai followed by a regulatory phase returning Cai to control levels in 3-4 min, this phase being greatly accelerated (< 60 s) by NaCl addition (apparent Km of approximately 5 mM Na+). The Cai increase was only found with the Na(+)-free medium on the basolateral side of the cell monolayer. A twofold increase in the 45Ca influx was observed under these conditions. In Ca(2+)- depleted cells, the initial Cai increase after Ca2+ addition to the medium was greater when the putative Na/Ca exchanger was not functioning (i.e. in a Na(+)-free medium). 45Ca effluxes through the blm of the monolayer were greatly and transiently increased by a Na(+)-free medium on the serosal side and blocked by orthovanadate (1 mM). The Cai increased induced by a hypo-osmotic shock was greater in cells bathed in a Na(+)-medium, conditions expected to block the activity of the Na/Ca exchanger. These findings support the hypothesis that a Na/Ca exchanger is present on the blm of A6 cells and affirm its role in Cai homeostasis in steady-state conditions and following osmotic shock. In addition, a Ca2+ pump also located on the blm and Ca2+ stores sensitive to inositol 1,4,5-trisphosphate were found to be implicated in Cai homeostasis.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Sodio/metabolismo , Animales , Calcio/farmacocinética , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Proteínas Portadoras/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Células Cultivadas , Homeostasis/efectos de los fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Transporte Iónico/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/fisiología , Presión Osmótica , Sodio/farmacocinética , Intercambiador de Sodio-Calcio , Terpenos/farmacología , Tapsigargina , Xenopus laevisRESUMEN
To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-)-freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes.
Asunto(s)
Permeabilidad de la Membrana Celular , Cloruros/metabolismo , Túbulos Renales Distales/metabolismo , Sodio/metabolismo , Animales , Línea Celular , Polaridad Celular , Tamaño de la Célula , Ciclamatos/metabolismo , Epitelio/metabolismo , Colorantes Fluorescentes , Gluconatos/metabolismo , Líquido Intracelular/metabolismo , Túbulos Renales Distales/citología , ÓsmosisRESUMEN
The K+ permeabilities (86Rb(K) transport) of the basolateral membranes (JbK) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation. Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Cai (max < 1 min) and cell swelling (max at 3-5 min) followed by a regulatory volume decrease (5-30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the 86Rb(K) transport and the SCC were partially blocked by Ba2+, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3-6 min) stimulation of the 86Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca(2+)-free media or quinidine addition did not affect the initial osmotic stimulation of JbK but prevented its "secondary regulation", whereas TEA, glibenclamide and DIDS completely blocked the initial JbK increase. Under hypo-osmotic conditions, the initial JbK increase was enhanced by the presence of 1 mM of barium and delayed with higher concentrations (5 mM). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media. We propose that a TEA- and glibenclamide-sensitive but quinidine-insensitive increase in K+ permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K+ permeability mediated by the observed calcium rise.
Asunto(s)
Permeabilidad de la Membrana Celular , Potasio/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula , Gliburida/farmacología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Transporte Iónico/efectos de los fármacos , Riñón/citología , Riñón/metabolismo , Modelos Biológicos , Presión Osmótica , Quinidina/farmacología , Rubidio/farmacocinética , Sodio/metabolismo , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Xenopus laevisAsunto(s)
Cadmio/toxicidad , Euglena gracilis/efectos de los fármacos , Euglena gracilis/metabolismo , Lectinas/metabolismo , Acetilgalactosamina/fisiología , Adaptación Biológica , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Resistencia a Medicamentos/fisiología , Citometría de Flujo , Glucolípidos/metabolismo , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Microscopía FluorescenteRESUMEN
The relationship linking Na+ and H+ transports and exocytosis/endocytosis located in the apical membranes of the frog skin epithelium was investigated under various conditions of ion transport stimulation. The exocytosis process, indicating insertion of intracellular vesicles, which were preloaded with fluorescent FITC-dextran (FD), was measured by following the FD efflux in the apical bathing solution. Na+ transport stimulators such as serosal hypotonic shock (replacement of serosal Ringer solution by half-Ringer or 4/5-Ringer), apical PCMPS (10(-3) M) and amphotericin-B (20 micrograms/ml), were also found to stimulate the exocytotic rates of FD. Acidification of the epithelium by CO2 or post NH4 load, conditions which increase the proton secretion also stimulated the FD release in the apical bathing solution. On the other hand, alkalization of the epithelial cells increased the endocytosis rate. Hypotonic shock, acid load and PCMPS induced an increase in cell calcium which is probably the signal within the cell for exocytosis. In addition, quantitative spectrofluorimetric measurements of F-actin content after rhodamine-phalloidin staining, indicated a decrease in the F-actin content as a result of cell acidosis, hypotonic conditions and amphotericin additions. It is proposed that the insertion/retrieval of intracytoplasmic vesicles containing H+ pumps plays a key role in the regulation of proton secretion in tight epithelia. In addition, it is suggested that cytoskeleton depolymerization of F-actin filaments facilitates H+ pump insertion. A comparable working hypothesis for the control of Na+ transport is proposed.