Biomedical subjects

E B Chang

Publications and source records attributed to E B Chang.

At least 19 recordsLinked to original sources

PubMed2001

Oxidants potentiate Ca(2+)- and cAMP-stimulated Cl(-) secretion in intestinal epithelial T84 cells.

BACKGROUND & AIMS: Diarrhea is one of the major complications of inflammatory bowel disease. The role of oxidants in promoting net intestinal secretion is important, but the cellular mechanisms underlying their effects are unclear. We examined the effects and defined the cellular actions of the oxidant monochloramine (NH(2)Cl) on anion secretion in human colonic T84 cells. METHODS: Effects of NH(2)Cl on basal and agonist-stimulated short-circuit current (Isc) of T84 monolayers were determined. Apical Cl(-) and basolateral K(+) conductances were measured by efflux of (125)I(-) and (86)Rb(+), respectively. RESULTS: NH(2)Cl alone had little effect on Isc and (125)I(-) efflux. However, pretreatment with NH(2)Cl led to a concentration-dependent potentiation of the Ca(2+)-mediated Isc and of submaximal cAMP-mediated responses. These effects were associated with increased basolateral K(+) channel conductance and were blocked by increasing cellular Ca(2+) buffering capacity with Quin-2. Whole-cell voltage clamp experiments showed that NH(2)Cl potentiated Ca(2+) activation of basolateral K(+) channel conductance. CONCLUSIONS: Oxidants potentiate both Ca(2+)- and cAMP-stimulated Cl(-) secretion by a direct effect on calcium-activated basolateral K(+) channel conductance, lowering its Ca(2+) activation threshold. This effect may play an important role in amplifying and prolonging the secretory response of inflamed intestinal mucosa and enhancing the severity of diarrhea.

Aminoquinolines↗

PubMed2001

Inhibition of Na+,K+-ATPase by interferon gamma down-regulates intestinal epithelial transport and barrier function.

BACKGROUND & AIMS: To determine how interferon (IFN)-gamma inhibits epithelial barrier and ion transport functions, intestinal T84 cells were studied. METHODS: Acute and chronic effects of IFN-gamma on T84 barrier function, Na+,K+-adenosine triphosphatase (ATPase) activity, and certain ion transport and tight junctional proteins were determined. To assess the role of Na+,K+-ATPase and intracellular Na+, similar studies with the Na+,K+-ATPase inhibitor ouabain and Na+ ionophore monensin were performed. To determine the role of nitric oxide (NO), the NO donor SPER-NO was used. RESULTS: IFN-gamma acutely (<6 hour) decreased cellular Na+,K+-ATPase activity, followed later (>24 hours) by decreases in expression of Na/K/2Cl, the alpha subunit of Na+,K+-ATPase, occludin, and ZO-1. In contrast, cystic fibrosis transmembrane conductance regulator or the Na+ pump beta subunit were unchanged. Ouabain and monensin caused nearly identical changes to IFN-gamma. Incubation in low Na+ media significantly blunted the chronic effects of IFN-gamma. Hypotonic-induced cell swelling, in contrast, had effects similar to IFN-gamma but did not alter the expression of the Na+ pump alpha subunit. The NO donor SPER-NO rapidly inhibited Na+,K+-ATPase and also down-regulated transport and barrier proteins. CONCLUSIONS: IFN-gamma inhibition of Na+,K+-ATPase activity acutely causes increases in intracellular Na(i) concentration and cell volume, which are distinct signaling events that ultimately result in a leaky and dysfunctional epithelium associated with chronic inflammation.

Biological Transport↗

PubMed2001

Short-chain fatty acids induce intestinal epithelial heat shock protein 25 expression in rats and IEC 18 cells.

BACKGROUND & AIMS: Because short-chain fatty acids (SCFAs) and heat shock proteins (hsps) confer protection to intestinal epithelia cells (IECs), we studied whether SCFAs modulate IEC hsp expression. METHODS: Hsp 25, hsp72, and hsc73 protein expression in rat intestinal tissues and IEC-18 cells were determined by Western blot and immunohistochemistry. Cell survival under conditions of oxidant stress (monochloramine) was determined using (51)Cr release in hsp25 cDNA anti-sense and sense-transfected cells expressing minimal and increased hsp25, respectively. RESULTS: Butyrate induces a time- and concentration-dependent increase in hsp25, but not hsp72 or hsc73, protein expression in rat IEC-18 cells but not 3T3 fibroblasts. Other SCFAs, including the poorly metabolized isobutyate, also induced selective expression of hsp25. Butyrate treatment significantly improved the ability of IEC-18 cells to withstand oxidant (monochloramine) injury. This effect could be blocked in cells in which hsp25 induction by butyrate was blocked by stable hsp25 antisense transfection. Additionally, hsp25-transfected overexpressing IEC-18 cells showed increased resistance to monochloramine. In vivo, increasing dietary fiber increased colonic, but not proximal, ileal hsp25 while having no effect on hsp72 or hsc73 expression. CONCLUSIONS: SCFAs, the predominant anions of colonic fluid derived from bacterial flora metabolism of luminal carbohydrates, protect IECs against oxidant injury, an effect mediated in part by cell-specific hsp25 induction.

3T3 Cells↗

PubMed2001

Surgical stress shifts the intestinal Escherichia coli population to that of a more adherent phenotype: role in barrier regulation.

BACKGROUND: We have shown that the combination of surgical stress and starvation in mice is associated with a defect in epithelial permeability and increased numbers of mucosa-associated Escherichia coli in the cecum. The aim of this study was to determine the specific role of mucosa-associated E coli on epithelial barrier dysfunction in this model. METHODS: Cecal E coli were harvested from mice 48 hours after a sham operation (control mice) or after a 30% surgical hepatectomy with only water provided ad libitum (short-term starvation) after the surgical procedure. Strains were tested for their ability to adhere to and alter the transepithelial electrical resistance (TEER) of cultured young adult mouse colon epithelial cells. TEER changes were further characterized by mannitol fluxes to confirm a defect in paracellular permeability. RESULTS: Strains of cecal E coli harvested from hepatectomy-starved mice adhered to and altered the permeability of young adult mouse colon cells, whereas E coli from the cecum of control mice were less adherent and had no effect on epithelial permeability. The effect of the strains harvested from mice after hepatectomy on the TEER of young adult mouse colon cells was inhibited by mannose and reversed by ciprofloxacin. CONCLUSION: The combination of surgical stress and short-term starvation is associated with a greater abundance of adherent and barrier-altering strains of E coli in the mouse cecum.

Animals↗

PubMed2001

Glutamine reduces cytokine release, organ damage, and mortality in a rat model of endotoxemia.

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.

Animals↗

PubMed2001

IFN-gamma downregulates expression of Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and human Caco-2/bbe cells.

Diarrhea associated with inflammatory bowel diseases has traditionally been attributed to stimulated secretion. The purpose of this study was to determine whether chronic stimulation of intestinal mucosa by interferon-gamma (IFN-gamma) affects expression and function of the apical membrane Na(+)/H(+) exchangers NHE2 and NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells expressing NHE2 and NHE3 were treated with IFN-gamma for 2, 24, and 48 h. Adult rats were injected with IFN-gamma intraperitoneally for 12 and 48 h. NHE2 and NHE3 activities were measured by unidirectional (22)Na influx across C2 cells and in rat brush-border membrane vesicles. NHE protein and mRNA were assessed by Western and Northern blotting. IFN-gamma treatment of C2 monolayers caused a >50% reduction in NHE2 and NHE3 activities and protein expression. In rats, region-specific, time- and dose-dependent reductions of NHE2 and NHE3 activities, protein expression, and mRNA were observed after exposure to IFN-gamma. Chronic exposure of intestinal epithelial cells to IFN-gamma results in selective downregulation of NHE2 and NHE3 expression and activity, a potential cause of inflammation-associated diarrhea.

Animals↗

PubMed2001

SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells.

Short-chain fatty acids (SCFA), produced by colonic bacterial flora fermentation of dietary carbohydrates, promote colonic Na absorption through mechanisms not well understood. We hypothesized that SCFA promote increased expression of apical membrane Na/H exchange (NHE), serving as luminal physiological cues for regulating colonic Na absorptive capacity. Studies were performed in human colonic C2/bbe (C2) monolayers and in vivo. In C2 cells exposed to butyrate, acetate, proprionate, or the poorly metabolized SCFA isobutyrate, apical membrane NHE3 activity and protein expression increased in a time- and concentration-dependent manner, whereas no changes were observed for NHE2. In contrast, no significant changes in brush-border hydrolase or villin expression were noted. Analogous to the in vitro findings, rats fed the soluble fiber pectin exhibited a time-dependent increase in colonic NHE3, but not NHE2, protein, mRNA, and brush-border activity. These changes were region-specific, as no changes were observed in the ileum. We conclude that luminal SCFA are important physiological cues for regulating colonic Na absorptive function, allowing the colon to adapt to chronic changes in dietary carbohydrate and Na loads.

Animals↗

PubMed2001

Glutamine induces heat shock protein and protects against endotoxin shock in the rat.

Enhanced expression of heat shock protein (HSP) has been shown to be protective against laboratory models of septic shock. Induction of HSPs to improve outcome in human disease has not been exploited because laboratory induction agents are themselves toxic and not clinically relevant. In this study, we demonstrate that a single dose of intravenous glutamine causes a rapid and significant increase in HSP25 and HSP72 expression in multiple organs of the unstressed Sprague-Dawley rat. With the utilization of a fluid-resuscitated rat model of endotoxemia, mortality was dramatically reduced by glutamine administration concomitant with the endotoxin injury. Endotoxin-treated animals given glutamine exhibited dramatic increases in tissue HSP expression and marked reduction of end-organ damage. These data suggest glutamine may protect against mortality and attenuate end-organ injury in endotoxemic shock via enhanced HSP expression. Furthermore, glutamine confers protection when administered at the initiation of sepsis, rather than as pretreatment. Thus glutamine appears to be a clinically viable enhancer of HSP expression and may prove beneficial in the therapy of sepsis and sepsis-induced organ injury.

Ammonia↗

PubMed2001

Role of increased basal expression of heat shock protein 72 in colonic epithelial c2BBE adenocarcinoma cells.

Although the expression of heat shock proteins (hsps) can be induced by a variety of stressful stimuli, certain neoplasms, including human intestinal T84, HT-29, and Caco2 adenocarcinoma cell lines, express constitutively high levels even under nonstress conditions. In this study, we examine the functional significance of increased hsp72 in spontaneously differentiating Caco2bbe (C2) cells. The expression of hsp72 in these cells was specifically inhibited by hsp72 antisense transfection. The loss of hsp72 expression did not affect growth rate, contact inhibition, morphological development, or functional differentiation. In contrast, these cells were significantly more sensitive to the injurious effects of oxidants and tumor necrosis factor (TNF) but not doxorubicin. To investigate potential mechanisms of action, a number of steps in the TNF-mediated cell death was measured. Antisense reduction of hsp72 did not alter activation of IkappaB. In contrast, mitochondrial cytochrome c release and activation of caspase 9 were significantly delayed in hsp72 antisense cells stimulated either with TNF or monochloramine. In conclusion, high endogenous expression of hsp72 by intestinal adenocarcinoma cells appears to confer selective survival advantage but does not affect their growth and differentiation.

Adenocarcinoma↗

PubMed2000

The key role of Pseudomonas aeruginosa PA-I lectin on experimental gut-derived sepsis.

OBJECTIVE: To examine the effect of Pseudomonas aeruginosa on intestinal barrier function and its lethal potential when introduced into the intestinal tract of mice. SUMMARY BACKGROUND DATA: The mere presence of P. aeruginosa in the intestinal tract of critically ill patients is associated with a threefold increase in death compared with matched cohorts without this pathogen. Whether this effect is a cause or a consequence of the critically ill state has not been previously addressed. METHODS: Transepithelial electrical resistance, a measure of tight junction permeability, was evaluated in Caco-2 intestinal epithelial cells cells apically inoculated with live P. aeruginosa, exotoxin A, or purified PA-I lectin, an adhesin of P. aeruginosa. Lethality studies to P. aeruginosa were carried out in mice undergoing 30% surgical hepatectomy by injecting the bacteria or its various components directly into the cecum. RESULTS: Only cells exposed to P. aeruginosa or its PA-I lectin developed alterations in barrier function. P. aeruginosa or the combination of PA-I and exotoxin A was lethal to mice when injected into the cecum after partial hepatectomy. Alterations in epithelial barrier function and death in mice were prevented when Pseudomonas was pretreated with N-acetyl D-galactosamine (GalNAc), a binder of PA-I. CONCLUSIONS: P. aeruginosa may act as a pathogen in the gastrointestinal tract, resulting in altered epithelial barrier function and death in a susceptible host. The PA-I lectin of P. aeruginosa may play a key role in its pathogenicity to the intestinal epithelium by inducing a permeability defect to its cytotoxic exoproducts such as exotoxin A.

Adhesins, Bacterial↗

PubMed1999

Regulation of intestinal epithelial brush border Na(+)/H(+) exchanger isoforms, NHE2 and NHE3, in C2bbe cells.

Until recently, studies to characterize the intestinal epithelial Na(+)/H(+) exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na(+)/H(+) exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH(i) only approximate Na(+)/H(+) exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used (22)[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca(++) involves changes to both K(Na) and V(max). In contrast, the same two second messengers inhibit NHE3 by a decrease in V(max) exclusively. Phorbol ester activation of protein kinase C alters both V(max) and K(Na) of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways.

Caco-2 Cells↗

PubMed1999

Heat-shock protein 72 protects against oxidant-induced injury of barrier function of human colonic epithelial Caco2/bbe cells.

BACKGROUND & AIMS: Barrier function of the inflamed intestinal mucosa can be compromised by reactive oxygen metabolites that increase mucosal permeability and disrupt the actin cytoskeleton, the integrity of which is important for maintaining tight epithelial junctions. Because heat-shock protein 72 (hsp72) protects intestinal epithelial cells against injury, we determined whether resistance of Caco2/bbe (C2) intestinal monolayer barrier function was related to their high endogenous hsp72 expression. METHODS: hsp72 anti-sense (C2/AS) and vector-only transfected C2 (C2/CEP4) clones, lines that exhibit low and high hsp72 expression, respectively, were studied. Permeability was assessed by measuring electrical resistance and mannitol fluxes and actin organization by confocal fluorescein isothiocyanate-phalloidin analysis. RESULTS: Basal transepithelial electrical resistance (TER) and mannitol fluxes were not significantly different between groups. However, the oxidant monochloramine rapidly decreased TER and increased mannitol permeability of C2/AS monolayers compared with C2/CEP4 (50% effective doses at 30 minutes were 0.53 +/- 0.11 and 2.06 +/- 0.34 mmol/L, respectively). Associated with these changes, decreased cell viability, dissociation and aggregation of perijunctional and stress actin filaments, loss of cell height, and increased intercellular separation were observed only in C2/AS cells treated with monochloramine. CONCLUSIONS: hsp72 protects intestinal epithelial barrier function against oxidant-induced stress, in part, by protecting the integrity of the actin cytoskeleton.

Actins↗

PubMed1999

Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells.

Several members of the Na+/H+ exchanger gene family (NHE1, NHE2, NHE3, and NHE4) with unique functional properties have been cloned from rat epithelial tissues. The present study examined the molecular and pharmacological properties of Na+/H+ exchange in rat parotid salivary gland cells. In acinar cells superfused with a physiological salt solution (145 mM Na+), Na+/H+ exchanger activity was inhibited by low concentrations of the amiloride derivative ethylisopropyl amiloride (EIPA; IC50 = 0.014 +/- 0.005 microM), suggesting the expression of amiloride-sensitive isoforms NHE1 and/or NHE2. Semiquantitative RT-PCR confirmed that NHE1 transcripts are most abundant in this cell type. In contrast, the intermediate sensitivity of ductal cells to EIPA indicated that inhibitor-sensitive and -resistant Na+/H+ exchanger isoforms are coexpressed. Ductal cells were about one order of magnitude more resistant to EIPA (IC50 = 0.754 +/- 0.104 microM) than cell lines expressing NHE1 or NHE2 (IC50 = 0.076 +/- 0.013 or 0.055 +/- 0.015 microM, respectively). Conversely, ductal cells were nearly one order of magnitude more sensitive to EIPA than a cell line expressing the NHE3 isoform (IC50 = 6.25 +/- 1.89 microM). Semiquantitative RT-PCR demonstrated that both NHE1 and NHE3 transcripts are expressed in ducts. NHE1 was immunolocalized to the basolateral membranes of acinar and ductal cells, whereas NHE3 was exclusively seen in the apical membrane of ductal cells. Immunoblotting, immunolocalization, and semiquantitative RT-PCR experiments failed to detect NHE2 expression in either cell type. Taken together, our results demonstrate that NHE1 is the dominant functional Na+/H+ exchanger in the plasma membrane of rat parotid acinar cells, whereas NHE1 and NHE3 act in concert to regulate the intracellular pH of ductal cells.

Amiloride↗

PubMed1999

A unique Na+/H+ exchanger, analogous to NHE1, in the chicken embryonic fibroblast.

We report the characterization of an Na+/H+ exchanger (NHE) in embryonic fibroblasts (SL-29 cells) of the chicken, a terrestrial vertebrate, where Na+ conservation is important. This exchanger is electroneutral, has a single Na+ binding site, and is highly sensitive to amiloride (IC50 2 microM), dimethyl amiloride (350 nM), and ethyl-isopropyl amiloride (25 nM). It is stimulated by serum, transforming growth factor-alpha, hypertonicity, and okadaic acid. Although these features make it resemble mammalian NHE1, other characteristics suggest distinct differences. First, in contrast to mammalian NHE1 it is inhibited by cAMP and shows a biphasic response to phorbol esters and a highly variable response to increased intracellular Ca2+ concentration. Second, whereas full-length human and rat NHE1 cDNA probes recognize a 4.8-kb transcript in rat tissues, they recognize only a 3.9-kb transcript in chicken tissues. An antibody against amino acids 631-746 of human NHE1 sequence fails to recognize a protein in SL-29 cells. Rat NHE2 and NHE3 probes do not recognize any transcript in chicken fibroblasts. The SL-29 exchanger differs markedly from the previously characterized chicken intestinal apical exchanger in its amiloride sensitivity and regulation by phorbol esters. These results suggest that a modified version of mammalian NHE1 is present in chicken tissues and imply that another functionally distinct Na+/H+ exchanger is expressed in aves.

Amiloride↗

PubMed1998

Dexamethasone protection of rat intestinal epithelial cells against oxidant injury is mediated by induction of heat shock protein 72.

Although the therapeutic actions of glucocorticoids are largely attributed to their anti-inflammatory and immunosuppressive effects, they have been implicated in enhancing tissue and cellular protection. In this study, we demonstrate that dexamethasone significantly enhances viability of IEC-18 rat small intestinal cells against oxidant-induced stress in a dose-dependent fashion. This protective action is mediated by induction of hsp72, the major inducible heat shock protein in intestinal epithelial cells. Dexamethasone stimulates a time- and dose-dependent response in hsp72 protein expression that parallels its effects on cell viability. Furthermore, the induction of hsp72 is tissue dependent, as nonintestinal epithelioid HeLa cells show differential induction of hsp72 expression in response to the same dexamethasone treatment. Antisense hsp72 cDNA transfection of IEC-18 cells abolishes the dexamethasone-induced hsp72 response, without significantly affecting constitutive expression of its homologue, hsc73. Dexamethasone treatment also significantly induces hsp72 protein expression in rat intestinal mucosal cells in vivo. These data demonstrate that glucocorticoids protect intestinal epithelial cells against oxidant-induced stress by inducing hsp72.

Animals↗

PubMed1998

Aldosterone stimulates intestinal Na+ absorption in rats by increasing NHE3 expression of the proximal colon.

Na+ retention by the colon in response to salt deprivation is mediated in part by the resulting secondary hyperaldosteronism. We show that experimental hyperaldosteronism, to levels seen with salt deprivation, causes an increase in the selective expression and activity of NHE3, an apically located isoform of the Na+/H+ exchange family that functions in transepithelial Na+ absorption. The effect of aldosterone on NHE3 expression is tissue specific, occurring in intestine and not in kidney. Within the intestine, these effects are regional, being observed only in proximal colon, and different in distribution from that observed with glucocorticoids, where the predominant effect occurs in ileum. Although glucocorticoids are well known to exert many effects via regulation of transcript levels, the present study demonstrates that aldosterone stimulates intestinal Na+ absorption by increasing cellular NHE3 expression, a response that is tissue and region specific.

Absorption↗

PubMed1998

Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe.

We examined the regulation of the Na+/H+ exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane 22Na+ influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-(N,N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.

Amiloride↗

PubMed1997

Inverse relationship between membrane lipid fluidity and activity of Na+/H+ exchangers, NHE1 and NHE3, in transfected fibroblasts.

This report presents a study of the effects of the membrane fluidizer, benzyl alcohol, on NHE isoforms 1 and 3. Using transfectants of an NHE-deficient fibroblast, we analyzed each isoform separately. An increase in membrane fluidity resulted in a decrease of approximately 50% in the specific activities of both NHE1 and NHE3. Only Vmax was affected; KNa was unchanged. This effect was specific, as Na+, K+, ATPase activity was slightly stimulated. Inhibition of NHE1 and NHE3 was reversible and de novo protein synthesis was not required to restore NHE activity after washout of fluidizer. Inhibition kinetics of NHE1 by amiloride, 5-(N,N-dimethyl)amiloride (DMA), 5-(N-hexamethyl)amiloride (HMA) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were largely unchanged. Half-maximal inhibition of NHE3 was also reached at approximately the same concentrations of amiloride and analogues in control and benzyl alcohol treated, suggesting that the amiloride binding site was unaffected. Inhibition of vesicular transport by incubation at 4 degrees C augmented the benzyl alcohol inhibition of NHE activity, suggesting that the fluidizer effect does not solely involve vesicle trafficking. In summary, our data demonstrate that the physical state of membrane lipids (fluidity) influences Na+/H+ exchange and may represent a physiological regulatory mechanism of NHE1 and NHE3 activity.

Amiloride↗