RESUMO
Using a rodent model of gut ischemia-reperfusion (I/R), we have previously shown that the induction of inducible nitric oxide synthase (iNOS) is harmful, whereas the induction of heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is protective. In the present study, we hypothesized that the luminal nutrients arginine and glutamine differentially modulate these molecular events in the postischemic gut. Jejunal sacs were created in rats at laparotomy, filled with either 60 mM glutamine, arginine, or magnesium sulfate (osmotic control) followed by 60 min of superior mesenteric artery occlusion and 6 h of reperfusion, and compared with shams. The jejunum was harvested for histology or myeloperoxidase (MPO) activity (inflammation). Heat shock proteins and iNOS were quantitated by Western blot analysis and PPAR-gamma by DNA binding activity. In some experiments, rats were pretreated with the PPAR-gamma inhibitor G9662 or with the iNOS inhibitor N-[3(aminomethyl)benzyl]acetamidine (1400W). iNOS was significantly increased by arginine but not by glutamine following gut I/R and was associated with increased MPO activity and mucosal injury. On the other hand, PPAR-gamma was significantly increased by glutamine but decreased by arginine, whereas heat shock proteins were similarly increased in all experimental groups. The PPAR-gamma inhibitor G9662 abrogated the protective effects of glutamine, whereas the iNOS inhibitor 1400W attenuated the injurious effects of arginine. We concluded that luminal arginine and glutamine differentially modulate the molecular events that regulate injurious I/R-mediated gut inflammation and injury. The induction of PPAR-gamma by luminal glutamine is a novel protective mechanism, whereas luminal arginine appears harmful to the postischemic gut due to enhanced expression of iNOS.
Assuntos
Arginina/administração & dosagem , Jejuno/irrigação sanguínea , Jejuno/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , PPAR gama/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Nutrição Enteral , Jejuno/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
It is well established, for all Na-absorbing epithelia, that an increase in the rate of transcellular Na+ absorption is accompanied by an increase in the conductance of the basolateral membrane to K+. For the case of small intestinal epithelial cells from the salamander Necturus maculosus, where the rate of transcellular Na+ absorption can be increased manyfold by the addition of sugars or amino acids to the luminal bathing solution, it appears that this parallelism between Na-K pump rate and basolateral membrane K+ conductance is closely related to volume regulation by the enterocyte. Recent studies have disclosed the presence of stretch-activated K+ channels, in a highly enriched basolateral membrane fraction isolated from these epithelial cells, whose activity is increased by an increase in vesicle volume and inhibited by a decrease in vesicle volume or ATP. The activity of this channel also appears to be regulated by the degree of organization of the cortical actin cytoskeleton; activity is increased by depolymerization of the actin cytoskeleton and decreased by repolymerization of that structure. We postulate that the inhibitory effect of ATP is related to its role in promoting the polymerization of G-actin to form F-actin. We propose that enterocyte swelling that results from the intracellular accumulation of sugars or amino acids in osmotically active forms brings about disorganization of the cortical actin cytoskeleton and activates these channels and is, at least in part, responsible for the "pump-leak parallelism" in this amphibian.
Assuntos
Enterócitos/citologia , Canais de Potássio/metabolismo , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Sódio/farmacocinética , Absorção/fisiologia , Alanina/química , Animais , Anuros , Impedância Elétrica , Enterócitos/metabolismo , Modelos Animais , Necturus maculosus , Sódio/metabolismo , XenopusRESUMO
We have previously reported that ATP-inhibitable K(+) channels, in vesicles derived from the basolateral membrane of Necturus maculosus small intestinal cells, exhibit volume regulatory responses that resemble those found in the intact tissue after exposure to anisotonic solutions. We now report that increases in K(+) channel activity can also be elicited by exposure of these vesicles to isotonic solutions containing glucose or alanine that equilibrate across these membranes. We also demonstrate that swelling after exposure to a hypotonic solution or an isotonic solution containing alanine or glucose reduces inhibition of channel activity by ATP and that this finding cannot be simply attributed to dilution of intravesicular ATP. We conclude that ATP-sensitive, stretch-activated K(+) channels may be responsible for the well-established increase in basolateral membrane K(+) conductance of Necturus small intestinal cells after the addition of sugars or amino acids to the solution perfusing the mucosal surface, and we propose that increases in cell volume, resulting in membrane stretch, decreases the sensitivity of these channels to ATP.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Enterócitos/metabolismo , Membranas Intracelulares/metabolismo , Necturus maculosus/metabolismo , Canais de Potássio/metabolismo , Alanina/farmacologia , Animais , Enterócitos/citologia , Enterócitos/fisiologia , Glucose/farmacologia , Membranas Intracelulares/fisiologia , Bloqueadores dos Canais de Potássio , Estresse MecânicoRESUMO
Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K(+) channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intravesicular solution increases channel activity whereas suspension in a solution that is hypertonic to the intravesicular solution decreases, and may abolish, channel activity. These volume regulatory responses appear to be mediated by the same K(ATP) channel and depend on an intact actin cytoskeletal network. The responses to both hypotonic and hypertonic challenge are abolished by cytochalasin D or by incubating the vesicles under conditions that are known to depolymerize actin. Phalloidin, which is known to stabilize actin filaments, partially prevents the action of cytochalasin D. Thus, the present results indicate that the K(ATP) channel activity of basolateral membrane vesicles from Necturus basolateral membranes respond to hypo- and hypertonic challenge monotonically around an isotonic "set point" and that these responses depend on an intact actin cytoskeleton.
Assuntos
Citoesqueleto/fisiologia , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Canais de Potássio/fisiologia , Actinas/efeitos dos fármacos , Actinas/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Tamanho Celular , Citocalasina D/farmacologia , Glibureto/farmacologia , Homeostase , Mucosa Intestinal/ultraestrutura , Cinética , Necturus , Concentração Osmolar , Faloidina/farmacologia , Canais de Potássio/efeitos dos fármacos , Rubídio/farmacocinética , Radioisótopos de RubídioRESUMO
Stanley G. Schultz received the seventh annual Arthur C. Guyton Physiology Teacher of the Year Award. The following is a speech he delivered as he was presented the award at Experimental Biology '99 in Washington, DC, in April 1999.
Assuntos
Fisiologia/educação , Distinções e Prêmios , Educação de Pós-Graduação em Medicina/história , Educação de Pós-Graduação em Medicina/tendências , Educação de Graduação em Medicina/história , Educação de Graduação em Medicina/tendências , História do Século XX , Fisiologia/história , Fisiologia/tendências , Pesquisa/tendências , Sociedades Científicas , Estados UnidosRESUMO
86Rb fluxes through ATP-regulated K+ (KATP) channels in membrane vesicles derived from basolateral membranes of Necturus small intestinal epithelial cells as well as the activity of single KATP channels reconstituted into planar phospholipid bilayers are inhibited by the presence of ADP plus phosphoenolpyruvate in the solution bathing the inner surface of these channels. This inhibition can be prevented by pretreatment of the membranes with 2, 3-butanedione, an irreversible inhibitor of pyruvate kinase (PK) and reversed by the addition of 2-deoxyglucose plus hexokinase. The results of additional studies indicate that PK activity appears to be tightly associated with this membrane fraction. These results, together with considerations of the possible ratio of Na+-K+ pumps to KATP channels in the basolateral membrane, raise the possibility that "cross talk" between those channels and pumps (i.e., the "pump-leak parallelism") may be mediated by local, functionally compartmentalized ATP-to-ADP ratios that differ from those in the bulk cytoplasm.
Assuntos
Trifosfato de Adenosina/fisiologia , Glicólise/fisiologia , Intestino Delgado/metabolismo , Membranas Intracelulares/metabolismo , Necturus/metabolismo , Canais de Potássio/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Desoxiglucose/farmacologia , Diacetil/farmacologia , Combinação de Medicamentos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Hexoquinase/farmacologia , Intestino Delgado/citologia , Bicamadas Lipídicas/metabolismo , Fosfoenolpiruvato/farmacologia , Bloqueadores dos Canais de Potássio , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/metabolismo , Distribuição TecidualAssuntos
Células Epiteliais/fisiologia , Transporte de Íons/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Sódio/metabolismo , Anfíbios , Animais , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Polaridade Celular , Tamanho Celular , Ativação do Canal Iônico , Potenciais da Membrana/efeitos dos fármacos , Potássio/metabolismo , Canais de Potássio/classificação , Canais de Potássio/fisiologia , CoelhosRESUMO
During the past century, the generally accepted view of the function of biological membranes has evolved from that of a static, inert lipoprotein envelope that simply separates the intracellular machinery from the outside milieu, to that of a dynamic structure that is, in fact, an integral part of that machinery actively involved in homeostasis and bioenergetics. This brief review traces that paradigm shift with special emphasis on the evolution of central concepts in epithelial transport physiology.
Assuntos
Clonagem Molecular , Células Epiteliais/fisiologia , Fisiologia/história , Animais , Transporte Biológico , História do Século XIX , História do Século XX , Modelos Biológicos , Publicações Periódicas como Assunto/história , Estados UnidosRESUMO
In all Na(+)-absorbing and Cl(-)-secreting epithelia, an increase in the activity of the Na+,K(+)-pump at the basolateral membrane is accompanied by an increase in the K+ conductance of that barrier and vice versa. We have recently identified an ATP-regulated K+ channel, K(ATP), in basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells that could be responsible for this parallelism between pump activity and leak. Thus, an increase in pump activity would result in a decrease in local ATP activity and an increase in local ADP activity and, in turn, an increase in the open-probability of the channel whereas a decrease in in pump activity would have the opposite effect. Further, the likelihood that the number of pumps far exceeds the number of leaks per unit area of membrane suggests that the ATP and ADP activities that influence K(ATP) channel activity may differ markedly from the "bulk" cytoplasmic values.
Assuntos
Trifosfato de Adenosina/fisiologia , Canais de Potássio/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Membrana Celular/fisiologia , Células Epiteliais/fisiologia , NecturusRESUMO
K+ channels present in basolateral membrane vesicles isolated from Necturus maculosa small intestinal cells and reconstituted into planar phospholipid bilayers are inhibited by MgATP and sulfonylurea derivatives, such as tolbutamide and glibenclamide, when these agents are added to the solution bathing the inner mouth of the channel. In addition, these channels possess an intrinsic "voltage gate" and are blocked when the electrical potential difference across the channel is oriented so that the inner solution is electrically positive with respect to the outer solution. We now show that increasing the concentration of permeant ions such as K+ or Rb+ in the outer solution reverses channel inhibition resulting from the addition of 50 microM glibenclamide to the inner solution and also inhibits intrinsic voltage gating; these effects are not elicited by increasing the concentrations of the relatively impermeant ions, Na+ or choline, in the outer solution. Furthermore, increasing the K+ concentration in the outer solution in the absence of glibenclamide inhibits voltage gating, and, under these conditions, the subsequent addition of glibenclamide to the inner solution is ineffective. These results are consistent with a model in which the voltage gate is an open-channel blocker whose action is directly reversed by elevating the external concentration of relatively permeant cations and where the action of glibenclamide is to stabilize the inactivated state of the channel, possibly through hydrophobic interactions.
Assuntos
Glibureto/farmacologia , Bloqueadores dos Canais de Potássio , Animais , Sistema Livre de Células , Colina/farmacologia , Epitélio/fisiologia , Mucosa Intestinal/fisiologia , Intestino Delgado , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Necturus , Potássio/farmacologia , Rubídio/farmacologia , Sódio/farmacologiaRESUMO
We have previously reported that basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells and incorporated into planar phospholipid bilayers display a highly selective "maxi"-conductance K+ channel whose open-time probability is affected by voltage. We now report that this channel is inhibited by MgATP in the solution bathing the intracellular face of the channel but not by Mg2+ or the Na+ or K+ salts of ATP; the effects of MgATP can be prevented or reversed by MgADP. The channel is also inhibited by the nonhydrolyzable ATP analogue magnesium adenosine 5'-O-(3-thiotriphosphate) and the sulfonylurea derivatives tolbutamide and glibenclamide; all of these agents are effective in the intracellular compartment but not when added to the extracellular compartment alone. Channel activity is stimulated by the "K+ channel opener," diazoxide, which also reverses the effect of glibenclamide but not of MgATP. The possible role of this channel as a mediator of the parallelism between basolateral membrane Na(+)-K+ pump activity and the macroscopic K+ conductance of that barrier is discussed.
Assuntos
Trifosfato de Adenosina/fisiologia , Intestino Delgado/metabolismo , Canais de Potássio/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Diazóxido/farmacologia , Glibureto/farmacologia , Intestino Delgado/citologia , Membranas/metabolismo , Necturus , Canais de Potássio/efeitos dos fármacos , Tolbutamida/farmacologiaAssuntos
Mucosa Intestinal/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células Epiteliais , Epitélio/enzimologia , Epitélio/metabolismo , Intestinos/citologia , Intestinos/enzimologia , Potenciais da Membrana/fisiologia , Necturus , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismoRESUMO
We have previously demonstrated that a synthetic peptide composed of the first 22 amino acids from the NH2-terminus of the Shaker B K+ channel protein deactivates a voltage-dependent K+ channel present in basolateral membrane of Necturus small intestinal epithelial cells reconstituted into planar lipid bilayers (Dubinsky et al. Proc. Natl. Acad. Sci. USA 89: 1770-1774, 1992). We now demonstrate that this peptide interacts with the inner surface of the Necturus channel only when it is in the open or conducting configuration and that this interaction is hindered by tetraethylammonium ion, a well-established blocker of this and other K+ channels. We conclude that this peptide is an open-pore blocker of the Necturus K+ channel as it appears to be in the case of the Shaker B K+ channel. We further demonstrate that trypsin, which abolishes the ability of this peptide to block both the Necturus and the Shaker K+ channels and inhibits spontaneous inactivation of the Shaker K+ channel, also impairs the voltage-gate of the Necturus K+ channel. These findings, and others to be reported in a companion paper, suggest structural homologies between the "inactivation peptide" of the Shaker B K+ channel and the voltage-gate of the Necturus K+ channel.
Assuntos
Drosophila melanogaster/metabolismo , Intestino Delgado/metabolismo , Necturus maculosus/metabolismo , Fragmentos de Peptídeos/farmacologia , Canais de Potássio/metabolismo , Tripsina/farmacologia , Animais , Drosophila melanogaster/genética , Eletrofisiologia , Imunoglobulina G/farmacologia , Intestino Delgado/citologia , Mutação , Canais de Potássio/genética , Canais de Potássio/fisiologia , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologiaRESUMO
We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsible for the inactivation of this A-type channel, blocks the inner, open mouth of a voltage-gated K+ channel present in the basolateral membrane of Necturus maculosa small intestinal enterocytes. We now demonstrate that antibodies to this "inactivating" peptide interact with proteins in solubilized and intact basolateral membranes from Necturus enterocytes. Asolectin vesicles reconstituted with the full complement of solubilized basolateral membrane proteins display 86Rb+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipitation with these antibodies. Furthermore, asolectin vesicles containing protein eluted from an antibody-affinity column display 86Rb+ uptake that is abolished by boiling. Finally, reconstitution of the immunoisolated protein into planar phospholipid bilayers disclosed a K+ channel whose single-channel properties are identical to those of the voltage-gated channel in the native basolateral membranes. Our data are consistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ channel activity and that this protein is antigenically similar to the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.
Assuntos
Imuno-Histoquímica/métodos , Intestino Delgado/metabolismo , Necturus maculosus/metabolismo , Canais de Potássio/metabolismo , Animais , Anticorpos/imunologia , Proteínas de Transporte/metabolismo , Intestino Delgado/ultraestrutura , Lipossomos/metabolismo , Membranas/metabolismo , Fragmentos de Peptídeos/imunologia , Potássio/metabolismo , Testes de PrecipitinaRESUMO
Exposure of the cytoplasmic side of calcium-activated, high (maxi)-conductance potassium [BK(Ca)] channels in basolateral membrane vesicles from rabbit colonocytes incorporated into planar phospholipid bilayers to trypsin rapidly reduces, but does not abolish, the sensitivity of this channel to activation by calcium without affecting its conductance or high selectivity for K+ over Cl-. The results of these studies also indicate that this BK(Ca) channel does not have intrinsic voltage-gating properties but that its voltage sensitivity is related to its ability to interact with calcium. This conclusion is consistent with the model proposed by Moczydlowski and Lattore (J. Gen. Physiol. 82: 511-542, 1983) for the role of membrane voltage in modulating the interaction between calcium and the BK(Ca) channel in rat skeletal muscle.
Assuntos
Cálcio/farmacologia , Bicamadas Lipídicas , Fosfolipídeos , Canais de Potássio/metabolismo , Tripsina/farmacologia , Animais , Colo/citologia , Colo/metabolismo , Colo/ultraestrutura , EstereoisomerismoRESUMO
A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.
Assuntos
Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Polaridade Celular , Drosophila melanogaster/fisiologia , Técnicas In Vitro , Mucosa Intestinal/fisiologia , Ativação do Canal Iônico , Bicamadas Lipídicas , Potenciais da Membrana , Dados de Sequência Molecular , Necturus/fisiologia , Peptídeos/química , Peptídeos/farmacologia , Canais de Potássio/químicaRESUMO
Using calmodulin-affinity chromatography, we have isolated a fraction of proteins from solubilized basolateral membranes of rabbit colonocytes which when reconstituted into planar phospholipid bilayers disclosed Ca(2+)-activated single K+ channel activities. The properties of the reconstituted channels are identical to those of native membrane vesicles incorporated into these bilayers with respect to their high selectivity for K+ over C-, high ("maxi") conductance, voltage gating, and inhibition by trifluoperazine. Two-dimensional sodium dodecyl sulfate gel electrophoresis of these proteins revealed three major protein species with molecular masses of 120, 60, and 35 kDa, which constituted 70, 10, and 20%, respectively, of the total protein. The results of other studies strongly suggest that the 35-kDa protein may be the Ca(2+)-activated K+ channel protein in these membranes.
Assuntos
Cálcio/farmacologia , Colo/química , Proteínas de Membrana/metabolismo , Canais de Potássio/metabolismo , Animais , Calmodulina , Cromatografia de Afinidade , Colo/citologia , Eletrofisiologia , Bicamadas Lipídicas , Fosfolipídeos , Canais de Potássio/fisiologia , CoelhosRESUMO
A highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989) J. Membrane Biol. 110:147-162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance. Ca2+-activated K+ channel. This channel is highly selective for K+ over Na+ and Cl-, displays voltage-gating similar to "maxi" K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K+ diffusion through the channel involves either the binding of a single K+ ion to a site within the channel or "single-filing" ("multi-ion occupancy"). Channel activity is inhibited by the venom from the scorpion Leiurus quinquestriatus, Ba2+, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed.