Journal of Experimental Zoology Part A: Comparative Experimental Biology, 2006
In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially sw... more In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially swell and then release small organic compounds and osmotically obligated water in what is called a regulatory volume decrease (RVD) to restore cell volume. One of the major intracellular solutes lost during this process is the non-metabolized beta amino acid taurine. This hypoosmoticinduced increase in cell taurine permeability requires the anion exchanger, skAE1. The abundance of this transporter increases on the surface plasma membrane by a process of exocytosis. The secondmessenger pathways involved in exocytosis of skAE1 were investigated with the use of inhibitors which affect membrane trafficking. Hypoosmotic-stimulated taurine uptake was significantly decreased by 42% with wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase) inhibitor. Additional evidence for the involvement of PI3K was obtained with a second inhibitor, LY294002,
Comparative Biochemistry and Physiology Part A: Physiology, 1997
Intestinal sodium transporters, such as the Na+/H+ exchanger (NHE) are important for Na+ conserva... more Intestinal sodium transporters, such as the Na+/H+ exchanger (NHE) are important for Na+ conservation in land birds. In mammals, at least five isoforms of the exchanger, NHEs 1-5, have been cloned, with NHE-1 occurring in epithelial basolateral and nonepithelial cell membranes and NHE-3 being restricted to epithelial apical/brush border membranes. We had demonstrated earlier that chicken intestinal brush border membranes possess NHE activity that functionally resembles mammalian NHE3. In this study, we used mammalian NHE-1 and NHE-3 probes to examine if chicken enterocytes possess these transporters. Antisera against rat NHE3 recognized a 97 kDa protein in chicken intestinal brush border membrane, while a NHE-3 cDNA probe failed to recognize any transcript. A NHE-1 antibody failed to recognize any protein in brush border or basolateral membrane, while a NHE-1 cDNA probe recognized a 3.9 kb transcript. Thus, there is more than one NHE isoform in chicken intestine, and our results suggest a novel avian NHE family. COMP BIOCHEM PHYSIOL
Translocation of bacteria and other luminal factors from the intestine following surgical injury ... more Translocation of bacteria and other luminal factors from the intestine following surgical injury can be a major driver of critical illness. Bile acids have been shown to play a key role in the loss of intestinal epithelial barrier function during states of host stress. Experiments to study the ability of nonionic block copolymers to abrogate barrier failure in response to bile acid exposure are described. In vitro experiments were performed with the bile salt sodium deoxycholate (SDC) on Caco-2 enterocyte monolayers using transepithelial electrical resistance (TER) to assay barrier function. A bisphenol-A coupled tri-block polyethylene glycol, PEG 15-20, was shown to prevent SDC-induced barrier failure. ELISA, LDH, and caspase-3 based cell death detection assays demonstrated that bile acid-induced apoptosis and necrosis were prevented with PEG 15-20. Immunofluorescence microscopic visualization of the tight junctional protein zonula occludens-1 (ZO-1) demonstrated that PEG 15-20 prevented significant changes in tight junction organization induced by bile acid exposure. Preliminary TER-based studies examining structure-function correlates of polymer protection against bile acid damage were performed with a small library of polyethylene glycol-based copolymers. Polymer properties associated with optimal protection against bile acid-induced barrier disruption were PEG-based compounds with a molecular weight > 10 kDa and amphiphilicity. The data demonstrate that PEG-based copolymer architecture is an important determinant that confers protection against bile acid injury of intestinal epithelia.
Volume expansion of cardiac cells from a wide variety of species stimulates the efflux of the bet... more Volume expansion of cardiac cells from a wide variety of species stimulates the efflux of the beta-amino acid taurine through an osmolyte channel. Previous studies have suggested that the osmolyte channel in epithelial cells is a swelling-activated anion channel (pICln). In skate heart, a 37-kDa protein is present which is recognized by a specific antibody to a protein characterized in MDCK cells as pICln. This protein is present predominantly in the cytosol (only 10% in the membrane fraction) of heart incubated under isotonic conditions. After transfer to hypotonic medium (one-half osmolarity), the distribution of this protein is markedly altered and significant amounts of the protein are found in the membrane fraction. After hypotonic exposure, the amount of the protein in the membrane fraction rises to 38 +/- 11% (range 18-53, n = 3). The translocation to the membrane fraction suggests that this protein may play a role in the taurine efflux in this tissue stimulated by hypotonic stress.
Journal of Experimental Zoology Part A: Comparative Experimental Biology, 2005
The aim of this study was to determine whether hypo-osmolarity, which activates taurine transport... more The aim of this study was to determine whether hypo-osmolarity, which activates taurine transport through the volume-sensitive organic osmolyte channel in skate (Raja erinacea) erythrocytes, also activates the organic osmolyte channel activity of skate AE1 (skAE1) expressed in oocytes. When Xenopus laevis oocytes expressing skAE1 were incubated in hypo-osmotic ND 96 (210 mOsm) media, taurine was transported at a significantly higher rate than when incubated in ND 96 (235 mOsm), which is iso-osmotic to Xenopus plasma. Therefore, hypo-osmotic stress is part of the activation mechanism of the organic osmolyte channel in skAE1 expressing oocytes.
Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabo... more Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC). A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied OPEN ACCESS cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose-and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC.
A variety of cells, including skate RBC, release osmolytes (e.g. taurine) when hypotonically swol... more A variety of cells, including skate RBC, release osmolytes (e.g. taurine) when hypotonically swollen as part of a regulatory volume decrease. In this study we show that skate RBC also release ATP into the incubation medium under the same conditions. Furthermore extracellular ATP as well as other nucleotides likely to be released from the RBC, inhibit the hypotonically activated transport of taurine. Therefore, ATP and other nucleotides released from hypotonically stressed RBC have the potential to act as modulators of osmolyte release during hyposmotic stress.
Volume expansion of erythrocytes of little skate, Raja erinacea, triggers the opening of an osmol... more Volume expansion of erythrocytes of little skate, Raja erinacea, triggers the opening of an osmolyte channel. We review this transport mechanism and further investigate the channel's physicochemical nature by probing the channel with a series of pyridoxine derivatives in skate RBC as well as in epithelial cells: MDCK and C6 glioma cells and in skate hepatocytes. The identity of the transport mechanism (band 3 vs. an anion channel) which mediates the swelling-activated efflux of osmolytes in fish RBC is controversial. Therefore, we compared taurine and Cl- effluxes in similar conditions. We found that there is significant Cl- loss from volume-expanded skate RBC. However, there was no effect of either hypotonicity or a number of taurine transport inhibitors on this loss. Utilizing changes in intracellular pH as a means of indirectly measuring H+/Cl- cotransport, we found that a rise in cell pH accompanied the loss of Cl-. This suggests that Cl- efflux could occur via a H+/Cl- cotransporter. To probe and compare the osmolyte channel (taurine efflux) of the skate RBC and three other cell types we used a family of pyridoxine inhibitors. The inhibitory patterns for the skate erythrocytes and hepatocytes differed from those for MDCK and C6 glioma cells and the two former cell types differed from each other. Therefore, the results show that the osmolyte channel in the skate differs from that in other epithelial cells with regard to pyridoxine derivative binding properties.
Hypoosmotic stress in skate RBC causes cell swelling followed by the release of solutes (osmolyte... more Hypoosmotic stress in skate RBC causes cell swelling followed by the release of solutes (osmolytes) to bring about a regulatory volume decrease (RVD). The p-amino acid, taurine, is a primary osmolyte in RVD of skate RBC. Taurine efflux is inhibited by stilbene disulfonates and other inhibitors of the anion exchanger, band 3, suggesting that band 3 is involved in the release of taurine during RVD in skate RBC. Since RBC of the cyclostomes, hagfish and lamprey, are reported to be deficient in band 3, we examined taurine efflux, quantity of band 3, and volume response in these RBC. There was no significant increase in taurine efflux when hagfish RBC were exposed to hypoosmotic medium, and the rate of taurine efflux in these RBC was markedly lower than that in skate RBC. Hagfish RBC plasma membranes show only 11% of the level of 3H2DIDS bound to skate RBC membranes. In addition, DIDS-sensitive sulfate exchange is much lower in hagfish compared with skate RBC. Similarly, lamprey RBC showed little taurine efflux compared with skate, and the amount of 3H2DIDS binding to these RBC membranes was 24% that of skate. Finally, although both hagfish and lamprey RBC swell in hypoosmotic medium, the lamprey cells show a n RVD while the hagfish cells do not. Thus, in the RBC of the two cyclostomes, one showing an RVD and the other not, low band 3 is associated with low volume-activated taurine efflux. These results support the hypothesis that band 3 is involved in volume-activated taurine eMux. o
A permanent cell line with inducible expression of the trout anion exchanger protein (trAE1) was ... more A permanent cell line with inducible expression of the trout anion exchanger protein (trAE1) was constructed in a derivative of human embryonic kidney cells . In the absence of the inducer, muristerone A, the new cell line had no detectable trAE1 protein by Western analysis, biotinylation, and 36 Cl À £ux.The amount of trAE1 protein increased with increasing dose and incubation time with muristerone A. Anion exchange inhibitors signi¢cantly inhibited the inducible £ux of anions (i.e., 36 chloride and 35 sulfate) and taurine in isotonic media. The transfected cells had the characteristics of trAE1-mediated transport in intact trout erythrocytes: (1) inhibition by anion transport inhibitors, (2) pH independence over the pH range of 6.5^7.5, and (3) activation of 35 sulfate e¥ux by external anions in the selective order of Cl > Br > IZF: These cells, in contrast to trout erythrocytes, were not sensitive to the anion exchange inhibitor, 4,4 0 -diisothiocyanostilbene-2,2 0 -disulfonic acid (DIDS), suggesting some di¡erence in the properties of the transfected AE1.These results demonstrate the inducible expression of new anion transport membrane protein in HEK-293 cells. This is the ¢rst expression of trAE1 in a mammalian system.
We have shown previously that the PA-I lectin of Pseudomonas aeruginosa plays a key role in gut-d... more We have shown previously that the PA-I lectin of Pseudomonas aeruginosa plays a key role in gut-derived sepsis during surgical stress. The aims of this study were to determine if the intestinal tract lumen of a stressed host contained soluble factors that could induce the expression of PA-I. Mice were subjected to either 30% surgical hepatectomy or sham-laparotomy, and P. aeruginosa was introduced into the cecum. Twenty-four hours later, feces were recovered, and PA-I and exotoxin A were determined by real-time polymerase chain reaction (PCR). In reiterative experiments, fecal filtrates from both hepatectomy and sham-operated mice were tested for their ability to induce PA-I expression in cultures of P. aeruginosa. Finally, the media from cultured human intestinal epithelial (Caco-2) cells stressed with excess glutamine was tested for its ability to induce the expression of PA-I in cultures of P. aeruginosa. Both PA-I and exotoxin A mRNA were increased in vivo in the intestinal tract of mice subjected to 30% hepatectomy. Soluble fecal filtrates from hepatectomy mice induced PA-I in vitro. Media from epithelial cells exposed to excess glutamine alone induced PA-I expression. The intestinal environment of a stressed host contains soluble factors capable of inducing lethal virulence traits in human opportunistic pathogen P. aeruginosa.
... Flavio Rocha BS, Robert Laughlin BA, Mark W. Musch PhD, Barbara A. Hendrickson MD, EugeneB. C... more ... Flavio Rocha BS, Robert Laughlin BA, Mark W. Musch PhD, Barbara A. Hendrickson MD, EugeneB. Chang MD and John Alverdy MD. ... Strains were tested for their ability to adhere to and alter the transepithelial electrical resistance (TEER) of cultured young adult mouse colon ...
Clinical trials have demonstrated that glutamine (GLN) supplementation can decrease infectious mo... more Clinical trials have demonstrated that glutamine (GLN) supplementation can decrease infectious morbidity and improve survival in a number of settings of critical illness. The mechanism of this protection remains unclear. The objective of this study was to evaluate the effect of GLN on cytokine release, organ injury, and survival from endotoxin-induced septic shock. Endotoxemia was induced in Male Sprague-Dawley rats by intravenous administration of 5 mg/kg Escherichia coli lipopolysaccharide (LPS). Concomitantly, animals were fluid resuscitated with a lactated ringers (LR) solution and given GLN (0.75 g/kg i.v.) or LR alone. Blood samples were obtained at multiple time points post-LPS injury for cytokine analysis. Survival rates were monitored for 72 h. Organ injury was evaluated in a separate set of animals via pathologic exam of tissues harvested 6 h post-LPS injury. A single dose of GLN significantly attenuated the release of TNF-alpha at 2 h (P < 0.005) and IL-1 beta at 4 h (P < 0.0001). This attenuation of cytokine release was associated with a significant decrease in mortality (P < 0.003). Pathologic exam demonstrated significant protection of both lung and small bowel tissue by GLN. Blood gas values 6-h post-LPS injury showed increased PaO2 and bicarbonate concentration in GLN treated animals. These data indicate that GLN can significantly attenuate pro-inflammatory cytokine release, protect against end-organ damage, and decrease mortality from endotoxemia. GLN confers protection even when administered at the onset of endotoxemia, rather then as pre-treatment. Thus, one explanation for the clinical benefits observed from GLN-supplementation may be related to the attenuation of pro-inflammatory cytokines.
Pfl�gers Archiv European Journal of Physiology, 1998
Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to acco... more Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to accomplish a regulatory volume decrease (RVD). One protein that may play a role in taurine efflux is the cytosolic protein ICln. In rat neonatal cardiac myocytes under isotonic conditions, ICln is found predominantly (greater than 90%) in the cytosol. However, after cell volume expansion by exposure to hypotonic medium, ICln rapidly translocates to the particulate fraction (the Triton X-114-insoluble fraction). After 2 min in hypotonic medium the percentage of ICln in the particulate fraction increases to 30%, 46% at 5 min, 40% at 10 min, and 25% at 30 min. The time course of this response is similar to that of hypotonicity-stimulated taurine efflux. Hypotonicity-stimulated taurine efflux as well as ICln translocation parallel the reduction in medium osmolarity. As osmolarity decreases, taurine efflux and ICln movement increase. The movement of ICln from the particulate back to the cytosolic fraction is accelerated when volume-expanded cells are returned to isotonic medium. When ICln is analyzed under non-denaturing conditions, a dimer is detected in the particulate fraction of volume-expanded cells, along with the monomer. This dimer is not detected in the cytosol. Treatment of the particulate fraction from volume-expanded cells with the lyotropic agent KSCN caused release of ICln but not Na-K-ATPase into the soluble fraction, indicating that translocated ICln associates with membranes in the particulate fraction rather than inserting into them.
Journal of Experimental Zoology Part A: Comparative Experimental Biology, 2006
In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially sw... more In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially swell and then release small organic compounds and osmotically obligated water in what is called a regulatory volume decrease (RVD) to restore cell volume. One of the major intracellular solutes lost during this process is the non-metabolized beta amino acid taurine. This hypoosmoticinduced increase in cell taurine permeability requires the anion exchanger, skAE1. The abundance of this transporter increases on the surface plasma membrane by a process of exocytosis. The secondmessenger pathways involved in exocytosis of skAE1 were investigated with the use of inhibitors which affect membrane trafficking. Hypoosmotic-stimulated taurine uptake was significantly decreased by 42% with wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase) inhibitor. Additional evidence for the involvement of PI3K was obtained with a second inhibitor, LY294002,
Comparative Biochemistry and Physiology Part A: Physiology, 1997
Intestinal sodium transporters, such as the Na+/H+ exchanger (NHE) are important for Na+ conserva... more Intestinal sodium transporters, such as the Na+/H+ exchanger (NHE) are important for Na+ conservation in land birds. In mammals, at least five isoforms of the exchanger, NHEs 1-5, have been cloned, with NHE-1 occurring in epithelial basolateral and nonepithelial cell membranes and NHE-3 being restricted to epithelial apical/brush border membranes. We had demonstrated earlier that chicken intestinal brush border membranes possess NHE activity that functionally resembles mammalian NHE3. In this study, we used mammalian NHE-1 and NHE-3 probes to examine if chicken enterocytes possess these transporters. Antisera against rat NHE3 recognized a 97 kDa protein in chicken intestinal brush border membrane, while a NHE-3 cDNA probe failed to recognize any transcript. A NHE-1 antibody failed to recognize any protein in brush border or basolateral membrane, while a NHE-1 cDNA probe recognized a 3.9 kb transcript. Thus, there is more than one NHE isoform in chicken intestine, and our results suggest a novel avian NHE family. COMP BIOCHEM PHYSIOL
Translocation of bacteria and other luminal factors from the intestine following surgical injury ... more Translocation of bacteria and other luminal factors from the intestine following surgical injury can be a major driver of critical illness. Bile acids have been shown to play a key role in the loss of intestinal epithelial barrier function during states of host stress. Experiments to study the ability of nonionic block copolymers to abrogate barrier failure in response to bile acid exposure are described. In vitro experiments were performed with the bile salt sodium deoxycholate (SDC) on Caco-2 enterocyte monolayers using transepithelial electrical resistance (TER) to assay barrier function. A bisphenol-A coupled tri-block polyethylene glycol, PEG 15-20, was shown to prevent SDC-induced barrier failure. ELISA, LDH, and caspase-3 based cell death detection assays demonstrated that bile acid-induced apoptosis and necrosis were prevented with PEG 15-20. Immunofluorescence microscopic visualization of the tight junctional protein zonula occludens-1 (ZO-1) demonstrated that PEG 15-20 prevented significant changes in tight junction organization induced by bile acid exposure. Preliminary TER-based studies examining structure-function correlates of polymer protection against bile acid damage were performed with a small library of polyethylene glycol-based copolymers. Polymer properties associated with optimal protection against bile acid-induced barrier disruption were PEG-based compounds with a molecular weight > 10 kDa and amphiphilicity. The data demonstrate that PEG-based copolymer architecture is an important determinant that confers protection against bile acid injury of intestinal epithelia.
Volume expansion of cardiac cells from a wide variety of species stimulates the efflux of the bet... more Volume expansion of cardiac cells from a wide variety of species stimulates the efflux of the beta-amino acid taurine through an osmolyte channel. Previous studies have suggested that the osmolyte channel in epithelial cells is a swelling-activated anion channel (pICln). In skate heart, a 37-kDa protein is present which is recognized by a specific antibody to a protein characterized in MDCK cells as pICln. This protein is present predominantly in the cytosol (only 10% in the membrane fraction) of heart incubated under isotonic conditions. After transfer to hypotonic medium (one-half osmolarity), the distribution of this protein is markedly altered and significant amounts of the protein are found in the membrane fraction. After hypotonic exposure, the amount of the protein in the membrane fraction rises to 38 +/- 11% (range 18-53, n = 3). The translocation to the membrane fraction suggests that this protein may play a role in the taurine efflux in this tissue stimulated by hypotonic stress.
Journal of Experimental Zoology Part A: Comparative Experimental Biology, 2005
The aim of this study was to determine whether hypo-osmolarity, which activates taurine transport... more The aim of this study was to determine whether hypo-osmolarity, which activates taurine transport through the volume-sensitive organic osmolyte channel in skate (Raja erinacea) erythrocytes, also activates the organic osmolyte channel activity of skate AE1 (skAE1) expressed in oocytes. When Xenopus laevis oocytes expressing skAE1 were incubated in hypo-osmotic ND 96 (210 mOsm) media, taurine was transported at a significantly higher rate than when incubated in ND 96 (235 mOsm), which is iso-osmotic to Xenopus plasma. Therefore, hypo-osmotic stress is part of the activation mechanism of the organic osmolyte channel in skAE1 expressing oocytes.
Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabo... more Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC). A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied OPEN ACCESS cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose-and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC.
A variety of cells, including skate RBC, release osmolytes (e.g. taurine) when hypotonically swol... more A variety of cells, including skate RBC, release osmolytes (e.g. taurine) when hypotonically swollen as part of a regulatory volume decrease. In this study we show that skate RBC also release ATP into the incubation medium under the same conditions. Furthermore extracellular ATP as well as other nucleotides likely to be released from the RBC, inhibit the hypotonically activated transport of taurine. Therefore, ATP and other nucleotides released from hypotonically stressed RBC have the potential to act as modulators of osmolyte release during hyposmotic stress.
Volume expansion of erythrocytes of little skate, Raja erinacea, triggers the opening of an osmol... more Volume expansion of erythrocytes of little skate, Raja erinacea, triggers the opening of an osmolyte channel. We review this transport mechanism and further investigate the channel's physicochemical nature by probing the channel with a series of pyridoxine derivatives in skate RBC as well as in epithelial cells: MDCK and C6 glioma cells and in skate hepatocytes. The identity of the transport mechanism (band 3 vs. an anion channel) which mediates the swelling-activated efflux of osmolytes in fish RBC is controversial. Therefore, we compared taurine and Cl- effluxes in similar conditions. We found that there is significant Cl- loss from volume-expanded skate RBC. However, there was no effect of either hypotonicity or a number of taurine transport inhibitors on this loss. Utilizing changes in intracellular pH as a means of indirectly measuring H+/Cl- cotransport, we found that a rise in cell pH accompanied the loss of Cl-. This suggests that Cl- efflux could occur via a H+/Cl- cotransporter. To probe and compare the osmolyte channel (taurine efflux) of the skate RBC and three other cell types we used a family of pyridoxine inhibitors. The inhibitory patterns for the skate erythrocytes and hepatocytes differed from those for MDCK and C6 glioma cells and the two former cell types differed from each other. Therefore, the results show that the osmolyte channel in the skate differs from that in other epithelial cells with regard to pyridoxine derivative binding properties.
Hypoosmotic stress in skate RBC causes cell swelling followed by the release of solutes (osmolyte... more Hypoosmotic stress in skate RBC causes cell swelling followed by the release of solutes (osmolytes) to bring about a regulatory volume decrease (RVD). The p-amino acid, taurine, is a primary osmolyte in RVD of skate RBC. Taurine efflux is inhibited by stilbene disulfonates and other inhibitors of the anion exchanger, band 3, suggesting that band 3 is involved in the release of taurine during RVD in skate RBC. Since RBC of the cyclostomes, hagfish and lamprey, are reported to be deficient in band 3, we examined taurine efflux, quantity of band 3, and volume response in these RBC. There was no significant increase in taurine efflux when hagfish RBC were exposed to hypoosmotic medium, and the rate of taurine efflux in these RBC was markedly lower than that in skate RBC. Hagfish RBC plasma membranes show only 11% of the level of 3H2DIDS bound to skate RBC membranes. In addition, DIDS-sensitive sulfate exchange is much lower in hagfish compared with skate RBC. Similarly, lamprey RBC showed little taurine efflux compared with skate, and the amount of 3H2DIDS binding to these RBC membranes was 24% that of skate. Finally, although both hagfish and lamprey RBC swell in hypoosmotic medium, the lamprey cells show a n RVD while the hagfish cells do not. Thus, in the RBC of the two cyclostomes, one showing an RVD and the other not, low band 3 is associated with low volume-activated taurine efflux. These results support the hypothesis that band 3 is involved in volume-activated taurine eMux. o
A permanent cell line with inducible expression of the trout anion exchanger protein (trAE1) was ... more A permanent cell line with inducible expression of the trout anion exchanger protein (trAE1) was constructed in a derivative of human embryonic kidney cells . In the absence of the inducer, muristerone A, the new cell line had no detectable trAE1 protein by Western analysis, biotinylation, and 36 Cl À £ux.The amount of trAE1 protein increased with increasing dose and incubation time with muristerone A. Anion exchange inhibitors signi¢cantly inhibited the inducible £ux of anions (i.e., 36 chloride and 35 sulfate) and taurine in isotonic media. The transfected cells had the characteristics of trAE1-mediated transport in intact trout erythrocytes: (1) inhibition by anion transport inhibitors, (2) pH independence over the pH range of 6.5^7.5, and (3) activation of 35 sulfate e¥ux by external anions in the selective order of Cl > Br > IZF: These cells, in contrast to trout erythrocytes, were not sensitive to the anion exchange inhibitor, 4,4 0 -diisothiocyanostilbene-2,2 0 -disulfonic acid (DIDS), suggesting some di¡erence in the properties of the transfected AE1.These results demonstrate the inducible expression of new anion transport membrane protein in HEK-293 cells. This is the ¢rst expression of trAE1 in a mammalian system.
We have shown previously that the PA-I lectin of Pseudomonas aeruginosa plays a key role in gut-d... more We have shown previously that the PA-I lectin of Pseudomonas aeruginosa plays a key role in gut-derived sepsis during surgical stress. The aims of this study were to determine if the intestinal tract lumen of a stressed host contained soluble factors that could induce the expression of PA-I. Mice were subjected to either 30% surgical hepatectomy or sham-laparotomy, and P. aeruginosa was introduced into the cecum. Twenty-four hours later, feces were recovered, and PA-I and exotoxin A were determined by real-time polymerase chain reaction (PCR). In reiterative experiments, fecal filtrates from both hepatectomy and sham-operated mice were tested for their ability to induce PA-I expression in cultures of P. aeruginosa. Finally, the media from cultured human intestinal epithelial (Caco-2) cells stressed with excess glutamine was tested for its ability to induce the expression of PA-I in cultures of P. aeruginosa. Both PA-I and exotoxin A mRNA were increased in vivo in the intestinal tract of mice subjected to 30% hepatectomy. Soluble fecal filtrates from hepatectomy mice induced PA-I in vitro. Media from epithelial cells exposed to excess glutamine alone induced PA-I expression. The intestinal environment of a stressed host contains soluble factors capable of inducing lethal virulence traits in human opportunistic pathogen P. aeruginosa.
... Flavio Rocha BS, Robert Laughlin BA, Mark W. Musch PhD, Barbara A. Hendrickson MD, EugeneB. C... more ... Flavio Rocha BS, Robert Laughlin BA, Mark W. Musch PhD, Barbara A. Hendrickson MD, EugeneB. Chang MD and John Alverdy MD. ... Strains were tested for their ability to adhere to and alter the transepithelial electrical resistance (TEER) of cultured young adult mouse colon ...
Clinical trials have demonstrated that glutamine (GLN) supplementation can decrease infectious mo... more Clinical trials have demonstrated that glutamine (GLN) supplementation can decrease infectious morbidity and improve survival in a number of settings of critical illness. The mechanism of this protection remains unclear. The objective of this study was to evaluate the effect of GLN on cytokine release, organ injury, and survival from endotoxin-induced septic shock. Endotoxemia was induced in Male Sprague-Dawley rats by intravenous administration of 5 mg/kg Escherichia coli lipopolysaccharide (LPS). Concomitantly, animals were fluid resuscitated with a lactated ringers (LR) solution and given GLN (0.75 g/kg i.v.) or LR alone. Blood samples were obtained at multiple time points post-LPS injury for cytokine analysis. Survival rates were monitored for 72 h. Organ injury was evaluated in a separate set of animals via pathologic exam of tissues harvested 6 h post-LPS injury. A single dose of GLN significantly attenuated the release of TNF-alpha at 2 h (P < 0.005) and IL-1 beta at 4 h (P < 0.0001). This attenuation of cytokine release was associated with a significant decrease in mortality (P < 0.003). Pathologic exam demonstrated significant protection of both lung and small bowel tissue by GLN. Blood gas values 6-h post-LPS injury showed increased PaO2 and bicarbonate concentration in GLN treated animals. These data indicate that GLN can significantly attenuate pro-inflammatory cytokine release, protect against end-organ damage, and decrease mortality from endotoxemia. GLN confers protection even when administered at the onset of endotoxemia, rather then as pre-treatment. Thus, one explanation for the clinical benefits observed from GLN-supplementation may be related to the attenuation of pro-inflammatory cytokines.
Pfl�gers Archiv European Journal of Physiology, 1998
Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to acco... more Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to accomplish a regulatory volume decrease (RVD). One protein that may play a role in taurine efflux is the cytosolic protein ICln. In rat neonatal cardiac myocytes under isotonic conditions, ICln is found predominantly (greater than 90%) in the cytosol. However, after cell volume expansion by exposure to hypotonic medium, ICln rapidly translocates to the particulate fraction (the Triton X-114-insoluble fraction). After 2 min in hypotonic medium the percentage of ICln in the particulate fraction increases to 30%, 46% at 5 min, 40% at 10 min, and 25% at 30 min. The time course of this response is similar to that of hypotonicity-stimulated taurine efflux. Hypotonicity-stimulated taurine efflux as well as ICln translocation parallel the reduction in medium osmolarity. As osmolarity decreases, taurine efflux and ICln movement increase. The movement of ICln from the particulate back to the cytosolic fraction is accelerated when volume-expanded cells are returned to isotonic medium. When ICln is analyzed under non-denaturing conditions, a dimer is detected in the particulate fraction of volume-expanded cells, along with the monomer. This dimer is not detected in the cytosol. Treatment of the particulate fraction from volume-expanded cells with the lyotropic agent KSCN caused release of ICln but not Na-K-ATPase into the soluble fraction, indicating that translocated ICln associates with membranes in the particulate fraction rather than inserting into them.
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