RESUMO
In this study we use a theoretical approach to study the volumetric response of goldfish hepatocytes challenged by osmotic gradients and compared it with that of hepatocytes from another teleost (the trout) and a mammal (the rat). Particular focus was given to the multiple non-linear interactions of transport systems enabling hypotonically challenged cells to trigger a compensatory response known as volume regulatory decrease or RVD. For this purpose we employed a mathematical model which describes the rates of change of the intracellular concentrations of main diffusible ions, of the cell volume, and of the membrane potential. The model was fitted to experimental data on the kinetics of volume change of hepatocytes challenged by anisotonic media. In trout and rat hepatocytes, experimental results had shown that hypotonic cell swelling was followed by RVD, whereas goldfish cells swelled with no concomitant RVD (M.V. Espelt et al., 2003, J. Exp. Biol. 206, 513-522). A comparison between data predicted by the model and that obtained experimentally suggests that in trout and rat hepatocytes hypotonicity activates a sensor element and this, in turn, activates an otherwise silent efflux of KCl - whose kinetics could be successfully predicted - thereby leading to volume down-regulation. In contrast, with regard to the absence of RVD in goldfish hepatocytes the model proposed suggests that either a sensor element triggering RVD is absent or that the effector mechanism (the loss of KCl) remains inactive under the conditions employed. In line with this, we recently found that extracellular nucleotides may be required to induce RVD in these cells, indicating that our model could indeed lead to useful predictions.
Assuntos
Tamanho Celular , Hepatócitos/citologia , Modelos Biológicos , Vertebrados/metabolismo , Animais , Transporte Biológico , Peixes , Bombas de Íon/metabolismo , Soluções Isotônicas , Ligantes , Potenciais da Membrana , Osmose , Potássio/metabolismo , Ratos , Fatores de TempoRESUMO
The relationship between cell volume and K(+) transmembrane fluxes of goldfish (Carassius auratus) hepatocytes exposed to anisotonic conditions or energetic limitation was studied and compared with the response of hepatocytes from trout (Oncorhynchus mykiss) and rat (Rattus rattus). Cell volume was studied by video- and fluorescence microscopy, while K(+) fluxes were assessed by measuring unidirectional (86)Rb(+) fluxes. In trout and rat hepatocytes, hyposmotic (180 mosmoll(-1)) exposure at pH 7.45 caused cell swelling followed by a regulatory volume decrease (RVD), a response reported to be mediated by net efflux of KCl and osmotically obliged water. By contrast, goldfish hepatocytes swelled but showed no RVD under these conditions. Although in goldfish hepatocytes a net ((86)Rb(+))K(+) efflux could be activated by N-ethylmaleimide, this flux was not, or only partially, activated by hyposmotic swelling (120-180 mosmoll(-1)). Blockage of glycolysis by iodoacetic acid (IAA) did not alter cell volume in goldfish hepatocytes, whereas in the presence of cyanide (CN(-)), an inhibitor of oxidative phosphorylation, or CN(-) plus IAA (CN(-)+IAA), cell volume decreased by 3-7%. Although in goldfish hepatocytes, energetic limitation had no effect on ((86)Rb(+))K(+) efflux, ((86)Rb(+))K(+) influx decreased by 57-66% in the presence of CN(-) and CN(-)+IAA but was not significantly altered by IAA alone. Intracellular K(+) loss after 20 min of exposure to CN(-) and CN(-)+IAA amounted to only 3% of the total intracellular K(+). Collectively, these observations suggest that goldfish hepatocytes, unlike hepatocytes of anoxia-intolerant species, avoid a decoupling of transmembrane K(+) fluxes in response to an osmotic challenge. This may underlie both the inability of swollen cells to undergo RVD but also the capability of anoxic cells to maintain intracellular K(+) concentrations that are almost unaltered, thereby prolonging cell survival.
Assuntos
Carpa Dourada/fisiologia , Hepatócitos/metabolismo , Potássio/metabolismo , Truta/fisiologia , Anaerobiose , Animais , Transporte Biológico/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Cianetos/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Soluções Hipertônicas/farmacologia , Soluções Hipotônicas/farmacologia , Ácido Iodoacético/farmacologia , Masculino , Microscopia de Fluorescência , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Radioisótopos de Rubídio , Sódio/metabolismo , Água/fisiologiaRESUMO
Volume changes were studied in Beta vulgaris storage root vacuoles, using video microscopy, when exposed to hypotonic conditions. The osmotic gradient was either step-applied or progressively imposed in perfusion experiments. Preincubation at low pH (6.6) or with HgCl2 strongly reduced the vacuoles' water permeability, measured in step experiments. Furthermore, the volumetric response depended on the rate with which the aniso-osmotic condition was established. In perfusion experiments a "plateau value" (osmotic equilibrium or steady-state volume value) was observed, which was significantly lower than the theoretically expected one. Furthermore, if vacuoles were preincubated in presence of HgCl2 or at low pH and then the hypo-osmotic challenge was applied in perfusion experiments, a still lower "plateau value" was observed. This reduction was concentration-dependent and completely reversible. In these conditions, when HgCl2 concentration was 300 mM or medium pH was 6.6, the volume change was abolished. In other experiments, when urea iso-osmotically replaced mannitol, a reversible, pH-dependent volumetric response was observed. These results can be interpreted accepting that 1) mercury-sensitive water channels, present in the studied structure, were blocked by low pH during the hypo-osmotic challenge; 2) modification of water permeability prevents excessive swelling during the osmotic shock; 3) the effectiveness of this last mechanism depended on the osmotic challenge rate; and 4) additionally, urea reflection coefficients were also modified by reduced medium pH.
Assuntos
Beta vulgaris/metabolismo , Permeabilidade da Membrana Celular/fisiologia , Citoplasma/química , Água/metabolismo , Beta vulgaris/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Concentração de Íons de Hidrogênio , Manitol/metabolismo , Cloreto de Mercúrio/farmacologia , Concentração Osmolar , Pressão Osmótica , Raízes de Plantas , Ureia/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.
Assuntos
Aquaporinas/fisiologia , Colo/química , Absorção Intestinal/fisiologia , Adulto , Sequência de Aminoácidos , Animais , Aquaporina 3 , Aquaporinas/química , Aquaporinas/genética , Northern Blotting , Permeabilidade da Membrana Celular , Criança , Pré-Escolar , Células Epiteliais/química , Fluorimunoensaio , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Oócitos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Xenopus laevisRESUMO
Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98 percent identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells
Assuntos
Adulto , Pré-Escolar , Criança , Humanos , Aquaporinas/fisiologia , Colo/química , Células Epiteliais/química , Absorção Intestinal/fisiologia , Sequência de Aminoácidos , Aquaporinas/química , Aquaporinas/genética , Northern Blotting , Permeabilidade da Membrana Celular , Imunofluorescência , Fluorimunoensaio , Imuno-Histoquímica , Oócitos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/análise , Xenopus laevisRESUMO
The biophysical models describing the structure of water pores or channels have evolved, during the last forty years, from a pure 'black box' approach to a molecular based proposal. The initial 'sieving pore' in which water and other molecules were moving together was replaced by a more restrictive model, where water is moving alone in a 'single file' mode. Aquaporins discovery and cloning [G.M. Preston, T.P. Carroll, W.B. Guggino, P. Agre, Science 256 (1992) 365] leaded to the 'hour-glass model' and other alternative proposals, combining information coming from molecular biology experiments and two dimensional crystallography. Concerning water transfers in epithelial barriers the problem is quite complex, because there are at least two alternative pathways: paracellular and transcellular and three different driving forces: hydrostatic pressure, osmotic pressure or 'transport coupled' movements. In the case of ADH-sensitive epithelia it is more or less accepted that regulated water channels (AQP2), that can be inserted in the apical membrane, coexist with basolateral resident water channels (AQP3). The mechanism underlying the so-called 'transport associated water transfer' is still controversial. From the classical standing gradient model to the ion-water co-transport, different hypothesis are under consideration. Coming back to hormonal regulations, other than the well-known regulation by neuro-hypophysis peptides, a steroid second messenger, progesterone, has been recently proposed [P. Ford, G. Amodeo, C. Capurro, C. Ibarra, R. Dorr, P. Ripoche, M. Parisi, Am. J. Physiol. 270 (1996) F880].
RESUMO
The ovarian oocytes from Bufo arenarum (BAO) but not those from Xenopus laevis (XLO) would have water channels (WC). We now report that the injection of the mRNA from BAO into the oocytes from XLO increased their water osmotic permeability (Pi) (reduced by 0.3 mM HgCl2 and reversed by 5 mM beta-mercaptoethanol). A 30-min challenge with progesterone induced, 18 h later, a reduction of the mercury-sensitive fraction of Pf in the BAO (but not in XLO). The mRNA from BAO pretreated with progesterone lost its capacity to induce WC in the XLO, but the hormone did not affect the expression of the WC in XLO previously injected with the mRNA from BAO. Pf was also measured in urinary bladders of BAO. Eighteen hours after a challenge with progesterone, a reduction in the hydrosmotic response to oxytocin was observed. Finally, the mRNA from the urinary bladder of BAO was injected into XLO. An increase in Pf was observed. This was not the case if, before the mRNA extraction, the bladders were treated with progesterone. We conclude that the BAO WC share progesterone sensitivity with the oxytocin-regulated water channel present in the toad urinary bladder.