Mechanism of arsenic removal using brown seaweed derived impregnated with iron oxide biochar for batch and column studies.

Water contaminated with arsenic presents serious health risks, necessitating effective and sustainable removal methods. This article proposes a method for removing arsenic from water by impregnating biochar with iron oxide (Fe2O3) from brown seaweed (Sargassum polycystum). After the seaweed biomass was pyrolyzed at 400 °C, iron oxide was added to the biochar to increase its adsorptive sites and surface functional groups, which allowed the binding of arsenic ions. Batch studies were conducted to maximize the effects of variables, including pH, contact time, arsenic concentration, and adsorbent dosage, on arsenic adsorption. The maximum arsenic adsorption efficiency of 96.7% was achieved under optimal conditions: pH 6, the adsorbent dosage of 100 mg, the initial arsenic concentration of 0.25 mg/L, and a contact time of 90 min. Langmuir and Freundlich's isotherms favored the adsorption process, while the kinetics adhered to a pseudo-second-order model, indicating chemisorption as the controlling step. Column studies revealed complete saturation after 200 min, and the adsorption behavior fits both the Adams-Bohart and Thomas models, demonstrating the potential for large-scale application. The primary mechanism underlying the interaction between iron-modified biochar and arsenic ions is surface complexation, enhanced by increased surface area and porosity. This study highlights the significant contribution of iron-modified biochar derived from macroalgae as an effective and sustainable solution for arsenic removal from water.

Regulation of nutrient and electrolyte absorption in human organoid-derived intestinal epithelial cell monolayers.

Recently developed human intestinal epithelial 3D organoid cultures are a useful cell culture model to study intestinal transport physiology. From these, 2D monolayer cultures can be generated in which apical transporters are exposed to the medium, thereby better facilitating in vitro investigation of intestinal absorption processes. However, whether nutrient and electrolyte absorption can be physiologically regulated in human organoid-derived monolayers has not been determined. Constitutive nitric oxide (cNO) is known to regulate multiple gastrointestinal physiological functions. Previous studies using in vivo and in vitro mammalian animal models indicate that enhanced intracellular cNO differentially regulates the two primary apical Na transporters in small intestinal epithelial cells. Here, we generated human jejunal organoid-derived monolayers to determine whether apical nutrient and electrolyte transporter function is regulated by cNO in human enterocytes. Western blot analysis and immunocytochemical staining showed that organoid-derived 2D cultures express markers of enterocyte differentiation and form intact monolayers of apical-basal polarized epithelial cells. Uptake studies demonstrated that jejunal monolayers exhibit functional activity of Na-glucose cotransporter 1 (SGLT1; SLC5A1) and Na-H exchanger 3 (NHE3; SLC9A3). In response to physiological increases in cNO, the two primary apical Na transporters were differentially regulated in human intestinal organoid-derived monolayers, across multiple human specimens. An increase in cNO stimulated SGLT1, while NHE3 was inhibited. These results are similar to what is seen in vivo and in vitro in different animal intestinal models. Thus, human jejunal organoid-derived monolayers are an ideal in vitro model to better understand how intestinal nutrient absorption is regulated.

The Role of Gut Microbiota and Metabolites in Obesity-Associated Chronic Gastrointestinal Disorders.

The gut microbiota is a complex community of microorganisms that has become a new focus of attention due to its association with numerous human diseases. Research over the last few decades has shown that the gut microbiota plays a considerable role in regulating intestinal homeostasis, and disruption to the microbial community has been linked to chronic disease conditions such as inflammatory bowel disease (IBD), colorectal cancer (CRC), and obesity. Obesity has become a global pandemic, and its prevalence is increasing worldwide mostly in Western countries due to a sedentary lifestyle and consumption of high-fat/high-sugar diets. Obesity-mediated gut microbiota alterations have been associated with the development of IBD and IBD-induced CRC. This review highlights how obesity-associated dysbiosis can lead to the pathogenesis of IBD and CRC with a special focus on mechanisms of altered absorption of short-chain fatty acids (SCFAs).

The Adipose Tissue-Derived Secretome (ADS) in Obesity Uniquely Induces L-Type Amino Acid Transporter 1 (LAT1) and mTOR Signaling in Estrogen-Receptor-Positive Breast Cancer Cells.

Obesity increases the risk of postmenopausal breast cancer (BC). This risk is mediated by obesity-induced changes in the adipose-derived secretome (ADS). The pathogenesis of BC in obesity is stimulated by mTOR hyperactivity. In obesity, leucine might support mTOR hyperactivity. Leucine uptake by BC cells is through L-Type Amino Acid Transporter 1 (LAT1). Our objective was to link obesity-ADS induction of LAT1 to the induction of mTOR signaling. Lean- and obese-ADS were obtained from lean and obese mice, respectively. Breast ADS was obtained from BC patients. Estrogen-receptor-positive BC cells were stimulated with ADS. LAT1 activity was determined by uptake of 3H-leucine. The LAT1/CD98 complex, and mTOR signaling were assayed by Western blot. The LAT1 antagonists, BCH and JPH203, were used to inhibit LAT1. Cell migration and invasion were measured by Transwell assays. The results showed obese-ADS-induced LAT1 activity by increasing transporter affinity for leucine. Consistent with this mechanism, LAT1 and CD98 expression were unchanged. Induction of mTOR by obese-ADS was inhibited by LAT1 antagonists. Breast ADS from patients with BMIs > 30 stimulated BC cell migration and invasiveness. Collectively, our findings show that obese-ADS induction of LAT1 supports mTOR hyperactivity in luminal BC cells.

Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells.

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and ß1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and ß1 subunits, mediated via the Ca-activated PKC pathway.

Mast Cell Mediated Regulation of Small Intestinal Chloride Malabsorption in SAMP1/YitFc Mouse Model of Spontaneous Chronic Ileitis.

In Inflammatory Bowel Disease (IBD), malabsorption of electrolytes (NaCl) results in diarrhea. Inhibition of coupled NaCl absorption, mediated by the dual operation of Na:H and Cl:HCO3 exchangers on the brush border membrane (BBM) of the intestinal villus cells has been reported in IBD. In the SAMP1/YitFcs (SAMP1) mice model of spontaneous ileitis, representing Crohn's disease, DRA (Downregulated in Adenoma) mediated Cl:HCO3 exchange was shown to be inhibited secondary to diminished affinity of the exchanger for Cl. However, NHE3 mediated Na:H exchange remained unaffected. Mast cells and their secreted mediators are known to be increased in the IBD mucosa and can affect intestinal electrolyte absorption. However, how mast cell mediators may regulate Cl:HCO3 exchange in SAMP1 mice is unknown. Therefore, the aim of this study was to determine the effect of mast cell mediators on the downregulation of DRA in SAMP1 mice. Mast cell numbers and their degranulation marker enzyme (ß-hexosaminidase) levels were significantly increased in SAMP1 mice compared to control AKR mice. However, treatment of SAMP1 mice with a mast cell stabilizer, ketotifen, restored the ß-hexosaminidase enzyme levels to normal in the intestine, demonstrating stabilization of mast cells by ketotifen. Moreover, downregulation of Cl:HCO3 exchange activity was restored in ketotifen treated SAMP1 mice. Kinetic studies showed that ketotifen restored the altered affinity of Cl:HCO3 exchange in SAMP1 mice villus cells thus reinstating its activity to normal. Further, RT-qPCR, Western blot and immunofluorescence studies showed that the expression levels of DRA mRNA and BBM protein, respectively remained unaltered in all experimental conditions, supporting the kinetic data. Thus, inhibition of Cl:HCO3 exchange resulting in chloride malabsorption leading to diarrhea in IBD is likely mediated by mast cell mediators.

Mechanism of Na-K-ATPase Inhibition by PGE2 in Intestinal Epithelial Cells.

The primary means of intestinal absorption of nutrients by villus cells is via Na-dependent nutrient co-transporters located in the brush border membrane (BBM). These secondary active co-transport processes require a favorable transcellular Na gradient that is provided by Na-K-ATPase. In chronic enteritis, malabsorption of essential nutrients is partially due to inhibition of villus Na-K-ATPase activity mediated by specific immune inflammatory mediators that are known to be elevated in the inflamed mucosa. However, how Prostaglandin E2 (PGE2), a specific mediator of nutrient malabsorption in the villus BBM, may mediate the inhibition of Na-K-ATPase is not known. Therefore, this study aimed to determine the effect of PGE2 on Na-K-ATPase in villus cells and define its mechanism of action. In vitro, in IEC-18 cells, PGE2 treatment significantly reduced Na-K-ATPase activity, accompanied by a significant increase in the intracellular levels of cyclic Adenosine Monophosphate (cAMP). The treatment with cAMP analog 8-Bromo-cAMP mimicked the PGE2-mediated effect on Na-K-ATPase activity, while Rp-cAMP (PKA inhibitor) pretreatment reversed the same. The mechanism of inhibition of PGE2 was secondary to a transcriptional reduction in the Na-K-ATPase α1 and ß1 subunit genes, which was reversed by the Rp-cAMP pretreatment. Thus, the PGE2-mediated activation of the PKA pathway mediates the transcriptional inhibition of Na-K-ATPase activity in vitro.

Unique Regulation of Intestinal Villus Epithelial Cl-/HCO3- Exchange by Cyclooxygenase Pathway Metabolites of Arachidonic Acid in a Mouse Model of Spontaneous Ileitis.

Electrolytes (NaCl) and fluid malabsorption cause diarrhea in inflammatory bowel disease (IBD). Coupled NaCl absorption, mediated by Na+/H+ and Cl-/HCO3- exchanges on the intestinal villus cells brush border membrane (BBM), is inhibited in IBD. Arachidonic acid metabolites (AAMs) formed via cyclooxygenase (COX) or lipoxygenase (LOX) pathways are elevated in IBD. However, their effects on NaCl absorption are not known. We treated SAMP1/YitFc (SAMP1) mice, a model of spontaneous ileitis resembling human IBD, with Arachidonyl Trifluoro Methylketone (ATMK, AAM inhibitor), or with piroxicam or MK-886, to inhibit COX or LOX pathways, respectively. Cl-/HCO3- exchange, measured as DIDS-sensitive 36Cl uptake, was significantly inhibited in villus cells and BBM vesicles of SAMP1 mice compared to AKR/J controls, an effect reversed by ATMK. Piroxicam, but not MK-886, also reversed the inhibition. Kinetic studies showed that inhibition was secondary to altered Km with no effects on Vmax. Whole cell or BBM protein levels of Down-Regulated in Adenoma (SLC26A3) and putative anion transporter-1 (SLC26A6), the two key BBM Cl-/HCO3- exchangers, were unaltered. Thus, inhibition of villus cell Cl-/HCO3- exchange by COX pathway AAMs, such as prostaglandins, via reducing the affinity of the exchanger for Cl-, and thereby causing NaCl malabsorption, could significantly contribute to IBD-associated diarrhea.

Dietary Supplementation with Vitamin D, Fish Oil or Resveratrol Modulates the Gut Microbiome in Inflammatory Bowel Disease.

Gastrointestinal health is influenced by the functional genes and metabolites generated by the human microbiome. As the volume of current biomedical and translational research indicates, the importance and impact of this ecosystem of microorganisms, especially those comprising the gut microbiome on human health, has become increasingly apparent. Changes to the gut microbiome are associated with inflammatory bowel disease (IBD), which is characterized by persistent intestinal inflammation. Furthermore, the lifetime dietary choices of their host may positively or negatively affect both the gut microbiome and its impact on IBD. As such, "anti-inflammatory" dietary supplements, their impact, and mechanisms in restoring gut microbiota homeostasis during IBD is an area of intensive research. Dietary supplementation may represent an important adjuvant treatment avenue for limiting intestinal inflammation in IBD. Overall, this review addresses the development of the gut microbiome, the significance of the gut microbiome in IBD, and the use of dietary supplements such as vitamin D, fish oil, and resveratrol in the mitigation of IBD-associated gut dysbiosis and intestinal inflammation.

Inducible Nitric Oxide Regulates Na-Glucose Co-transport in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Ileitis.

In mammalian small intestine, glucose is primarily absorbed via Na-dependent glucose co-transporter (SGLT1) on the brush border membrane (BBM) of absorptive villus cells. Malabsorption of nutrients (e.g., glucose) leads to malnutrition, a common symptom of inflammatory bowel disease (IBD), where the mucosa is characterized by chronic inflammation. Inducible nitric oxide (iNO) is known to be elevated in IBD mucosa. SAMP1/YitFc (SAMP1) mouse is a spontaneous model of chronic ileitis that develops lesions in its terminal ileum, very similar to human IBD. How SGLT1 may be affected in SAMP1 model of chronic ileitis is unknown. Ten-week-old SAMP1 mice with AKR mice as control were treated with N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) to inhibit iNO production. Intracellular NO levels were found to be increased in villus cells from SAMP1 mice. Moreover, SGLT1 and Na+/K+-ATPase activities and BBM SGLT1 expression were significantly decreased. However, L-NIL treatment reduced the intracellular iNO production, and reversed both downregulated SGLT1 and Na+/K+-ATPase activities in SAMP1 mice. Inhibition of iNO by L-NIL treatment also significantly reversed the BBM SGLT1 protein expression in SAMP1 mice. L-NIL reversed the inflammation mediated downregulation of SGLT1 activity by restoring the BBM SGLT1 expression. Thus, regulation of SGLT1 in chronic ileitis is likely mediated by iNO.

Moderate Alcohol Consumption Uniquely Regulates Sodium-Dependent Glucose Co-Transport in Rat Intestinal Epithelial Cells In Vitro and In Vivo.

BACKGROUND: Chronic alcohol use often leads to malnutrition. However, how the intestinal absorption of nutrients such as glucose may be affected during moderate ethanol use has not been investigated. Glucose is absorbed via sodium (Na)-dependent glucose co-transport (SGLT1; SLC5A1) along the brush border membrane (BBM) of intestinal absorptive villus cells. OBJECTIVE: The aim of this study was to investigate how moderate alcohol consumption affects the absorption of glucose via SGLT1. METHODS: Intestinal epithelial cells (IEC-18; rat) were exposed to 8.64 mM ethanol over 1, 3, 6, and 12 h. Rats (16-wk-old, male, Sprague-Dawley) were administered 2 g/kg ethanol over 1, 3, and 6 h. Na-dependent 3H-O-methyl-d-glucose uptake was measured to assess SGLT1 activity. Na-K-ATPase activity was measured as a function of inorganic phosphate release. Protein expression was analyzed by Western blot analysis and immunohistochemical staining. RESULTS: Ethanol significantly decreased Na-dependent glucose absorption in enterocytes in vitro (ethanol treatment: 48.4% of controls at 1 h; P < 0.01) and in vivo (ethanol treatment: 60.0% of controls at 1 h; P < 0.01). Na-K-ATPase activity was significantly inhibited in vitro (ethanol treatment: 36.9% of controls at 1 h; P < 0.01) and in vivo (ethanol treatment: 42.1% of controls at 1 h; P < 0.01). Kinetic studies showed that the mechanism of inhibition of Na-glucose co-transport was secondary to a decrease in the affinity (1/Km) of the co-transporter for glucose both in vitro and in vivo. Western blots and immunohistochemistry further demonstrated unaltered amounts of SGLT1 after ethanol treatment. CONCLUSIONS: Moderate ethanol significantly decreases glucose absorption in IEC-18 cells and in villus cells of Sprague-Dawley rats. The inhibition of SGLT1 is secondary to an altered Na gradient at the cellular level and secondary to diminished affinity of the co-transporter for glucose at the protein level in the BBM. These observations may, at least in part, explain 1 possible mechanism of the onset of malnutrition associated with alcohol consumption.

Moderate Alcohol Consumption Inhibits Sodium-Dependent Glutamine Co-Transport in Rat Intestinal Epithelial Cells in Vitro and Ex Vivo.

Malnutrition is present in chronic alcoholics. However, how moderate alcohol consumption affects the absorption of nutrients like glutamine has not been investigated. Glutamine, an amino acid, is vital to gastrointestinal health. Glutamine is absorbed via sodium-dependent glutamine co-transport (B0AT1; SLC6A19) along the brush border membrane of absorptive villus cells. Rat intestinal epithelial cells (IEC-18) and sixteen-week-old Sprague Dawley rats were administered the equivalent of a 0.04% blood alcohol content of ethanol (8.64 mM; 2 g/kg) to investigate the effect of moderate alcohol on sodium-glutamine co-transport. Sodium-dependent 3H-glutamine uptakes were performed to measure B0AT1 activity. Inorganic phosphate was measured as a function of Na-K-ATPase activity. Protein expression was analyzed by immunohistochemical and Western blot analysis. Ethanol significantly inhibited sodium-dependent glutamine absorption and Na-K-ATPase activity in enterocytes in vitro and ex vivo. Kinetic studies suggested that the mechanism of inhibition was due to decreased maximal rate of uptake (Vmax) of the B0AT1 co-transporter, corresponding to decreased B0AT1 protein expression and secondary to an inhibited sodium-gradient at the cellular level in vitro and ex vivo. In all, moderate ethanol significantly inhibited glutamine absorption at the level of decreased B0AT1 expression at the brush border membrane and a reduced sodium gradient, which may contribute to malnutrition present in chronic alcoholics.

Unique Regulation of Na-K-ATPase during Growth and Maturation of Intestinal Epithelial Cells.

Na-K-ATPase on the basolateral membrane provides the favorable transcellular Na gradient for the proper functioning of Na-dependent nutrient co-transporters on the brush border membrane (BBM) of enterocytes. As cells mature from crypts to villus, Na-K-ATPase activity doubles, to accommodate for the increased BBM Na-dependent nutrient absorption. However, the mechanism of increased Na-K-ATPase activity during the maturation of enterocytes is not known. Therefore, this study aimed to determine the mechanisms involved in the functional transition of Na-K-ATPase during the maturation of crypts to villus cells. Na-K-ATPase activity gradually increased as IEC-18 cells matured in vitro from day 0 (crypts) through day 4 (villus) of post-confluence. mRNA abundance and Western blot studies showed no change in the levels of Na-K-ATPase subunits α1 and ß1 from 0 to 4 days post-confluent cells. However, Na-K-ATPase α1 phosphorylation levels on serine and tyrosine, but not threonine, residues gradually increased. These data indicate that as enterocytes mature from crypt-like to villus-like in culture, the functional activity of Na-K-ATPase increases secondary to altered affinity of the α1 subunit to extracellular K+, in order to accommodate the functional preference of the intestinal cell type. This altered affinity is likely due to increased phosphorylation of the α1 subunit, specifically at serine and tyrosine residues.

Stimulation of constitutive nitric oxide uniquely and compensatorily regulates intestinal epithelial cell brush border membrane Na absorption.

In the mammalian small intestine, sodium is primarily absorbed by Na+ /H+ exchange (NHE3) and Na-glucose cotransport (SGLT1) in the brush border membrane (BBM) of villus cells. However, how enhanced cellular constitutive nitric oxide (cNO) may affect NHE3 and SGLT1 remains unclear. Both in vivo in rabbit intestinal villus cells and in vitro IEC-18 cells, administration of NO donor, GSNAP, modestly increased cNO. GSNAP stimulated SGLT1 in villus and IEC-18 cells. The mechanism of stimulation was secondary to an increase in the affinity of SGLT1 for glucose. The change in SGLT1 was not secondary to altered Na-extruding capacity of the cell since Na+ /K+ -ATPase was decreased by GSNAP treatment. In contrast, GSNAP inhibited NHE3 activity in villus cell BBM. The mechanism of NHE3 inhibition was secondary to reduced BBM transporter numbers. These studies demonstrated that the physiological increase in cNO uniquely regulates mammalian small intestinal NHE3 and SGLT1 to maintain Na homeostasis.

Inhibition of intestinal villus cell Na/K-ATPase mediates altered glucose and NaCl absorption in obesity-associated diabetes and hypertension.

During obesity, diabetes and hypertension inevitably coexist and cause innumerable health disparities. In the obesity, diabetes, and hypertension triad (ODHT), deregulation of glucose and NaCl homeostasis, respectively, causes diabetes and hypertension. In the mammalian intestine, glucose is primarily absorbed by Na-glucose cotransport 1 (SGLT1) and coupled NaCl by the dual operation of Na-H exchange 3 (NHE3) and Cl-HCO3 [down-regulated in adenoma (DRA) or putative anion transporter 1 (PAT1)] exchange in the brush border membrane (BBM) of villus cells. The basolateral membrane (BLM) Na/K-ATPase provides the favorable transcellular Na gradient for BBM SGLT1 and NHE3. How these multiple, distinct transport processes may be affected in ODHT is unclear. Here, we show the novel and broad regulation by Na/K-ATPase of glucose and NaCl absorption in ODHT in multiple species (mice, rats, and humans). In vivo, during obesity inhibition of villus-cell BLM, Na/K-ATPase led to compensatory stimulation of BBM SGLT1 and DRA or PAT1, whereas NHE3 was unaffected. Supporting this new cellular adaptive mechanism, direct silencing of BLM Na/K-ATPase in intestinal epithelial cells resulted in selective stimulation of BBM SGLT1 and DRA or PAT1 but not NHE3. These changes will lead to an increase in glucose absorption, maintenance of traditional coupled NaCl absorption, and a de novo increase in NaCl absorption from the novel coupling of stimulated SGLT1 with DRA or PAT1. Thus, these novel observations provide the pathophysiologic basis for the deregulation of glucose and NaCl homeostasis of diabetes and hypertension, respectively, during obesity. These observations may lead to more efficacious treatment for obesity-associated diabetes and hypertension.-Palaniappan, B., Arthur, S., Sundaram, V. L., Butts, M., Sundaram, S., Mani, K., Singh, S., Nepal, N., Sundaram, U. Inhibition of intestinal villus cell Na/K-ATPase mediates altered glucose and NaCl absorption in obesity-associated diabetes and hypertension.

Direct and specific inhibition of constitutive nitric oxide synthase uniquely regulates brush border membrane Na-absorptive pathways in intestinal epithelial cells.

Pharmacological manipulations of constitutive nitric oxide (cNO) levels have been shown to have variable effects on Na absorption in vivo and in vitro in different tissues. Species differences, untoward in vivo effects (e.g. ENS, blood flow) and pharmacological non-specificity may account for these confounding observations. Thus, to directly and specifically determine the effect of cNO on brush border membrane Na/H exchange (NHE3) and Na-dependent glucose co-transport (SGLT-1), we inhibited cNO synthase (NOS3) with its siRNA in rat small intestinal epithelial cells (IEC-18) in vitro. As expected, intracellular cNO levels were reduced in siRNA NOS3 transfected cells. In these cells, SGLT-1 was significantly reduced compared to control. In contrast, NHE3 was significantly increased in siRNA NOS3 transfected cells. To determine if SGLT-1 changes were secondary to altered Na/K-ATPase, its activity was measured and found to be increased in NOS3 silenced cells. The mechanism of inhibition of SGLT-1 was secondary to diminished affinity of the co-transporter for glucose in NOS3 silenced cells. In contrast, the mechanism of stimulation of NHE3 is by increasing BBM exchanger numbers in siRNA NOS3 cells while the affinity was unaffected. Western blot studies of immunoreactive BBM proteins also confirmed the kinetic studies. All these data indicates that direct and specific inhibition of NOS3 with its siRNA inhibits SGLT-1 while stimulating NHE3 in the BBM. Thus, cNO uniquely and compensatorily regulates BBM NHE3 and SGLT-1 to maintain cellular Na homeostasis and these unique alterations by cNO are mediated by its intracellular 2nd messenger cGMP.

Mast cell regulation of Na-glutamine co-transporters B0AT1 in villus and SN2 in crypt cells during chronic intestinal inflammation.

BACKGROUND: In the chronically inflamed rabbit small intestine, brush border membrane (BBM) Na-glutamine co-transport is inhibited in villus cells (mediated by B0AT1), while it is stimulated in crypt cells (mediated by SN2/SNAT5). How mast cells, known to be enhanced in the chronically inflamed intestine, may regulate B0AT1 in villus and SN2/SNAT5 in crypt cell is unknown. Thus, the aim of the present study is to determine the regulation of B0AT1 and SN2/SNAT5 by mast cells during chronic enteritis. METHODS: Chronic intestinal inflammation was induced in male rabbits with intra-gastric inoculation of Eimeria magna oocytes. Rabbits with chronic inflammation were treated with ketotifen (10 mg/day) or saline (Placebo) for 2 days. Villus and crypts cells were isolated from the rabbit intestine using the Ca++ chelation technique. Na/K-ATPase activity was measured as Pi from cellular homogenate. BBM vesicles (BBMV) were prepared from villus and crypt cells and uptake studies were performed using rapid filtration technique with (3)H-Glutamine. Western blot analyses were done using B0AT1 and SN2 specific antibodies. RESULTS: In villus cells, Na-glutamine co-transport inhibition observed during inflammation was completely reversed by ketotifen, a mast cell stabilizer. In contrast, in crypt cells, Na-glutamine co-transport stimulation was reversed to normal levels by ketotifen. Kinetic studies demonstrated that ketotifen reversed the inhibition of B0AT1 in villus cells by restoring co-transporter numbers in the BBM, whereas the stimulation of SN2/SNAT5 in crypts cells was reversed secondary to restoration of affinity of the co-transporter. Western blot analysis showed that ketotifen restored immune-reactive levels of B0AT1 in villus cells, while SN2/SNAT5 levels from crypts cell remained unchanged. CONCLUSION: In the present study we demonstrate that mast cells likely function as a common upstream immune pathway regulator of the Na-dependent glutamine co-transporters, B0AT1 in villus cells and SN2 in crypts cells that are uniquely altered in the chronically inflamed small intestine.

Chronic and selective inhibition of basolateral membrane Na-K-ATPase uniquely regulates brush border membrane Na absorption in intestinal epithelial cells.

Na-K-ATPase, an integral membrane protein in mammalian cells, is responsible for maintaining the favorable intracellular Na gradient necessary to promote Na-coupled solute cotransport processes [e.g., Na-glucose cotransport (SGLT1)]. Inhibition of brush border membrane (BBM) SGLT1 is, at least in part, due to the diminished Na-K-ATPase in villus cells from chronically inflamed rabbit intestine. The aim of the present study was to determine the effect of Na-K-ATPase inhibition on the two major BBM Na absorptive pathways, specifically Na-glucose cotransport and Na/H exchange (NHE), in intestinal epithelial (IEC-18) cells. Na-K-ATPase was inhibited using 1 mM ouabain or siRNA for Na-K-ATPase-α1 in IEC-18 cells. SGLT1 activity was determined as 3-O-methyl-D-[(3)H]glucose uptake. Na-K-ATPase activity was measured as the amount of inorganic phosphate released. Treatment with ouabain resulted in SGLT1 inhibition at 1 h but stimulation at 24 h. To further characterize this unexpected stimulation of SGLT1, siRNA silencing was utilized to inhibit Na-K-ATPase-α1. SGLT1 activity was significantly upregulated by Na-K-ATPase silencing, while NHE3 activity remained unaltered. Kinetics showed that the mechanism of stimulation of SGLT1 activity was secondary to an increase in affinity of the cotransporter for glucose without a change in the number of cotransporters. Molecular studies demonstrated that the mechanism of stimulation was not secondary to altered BBM SGLT1 protein levels. Chronic and direct silencing of basolateral Na-K-ATPase uniquely regulates BBM Na absorptive pathways in intestinal epithelial cells. Specifically, while BBM NHE3 is unaffected, SGLT1 is stimulated secondary to enhanced affinity of the cotransporter.

Molecular mechanism of regulation of villus cell Na-K-ATPase in the chronically inflamed mammalian small intestine.

Na-K-ATPase located on the basolateral membrane (BLM) of intestinal epithelial cells provides a favorable intracellular Na+ gradient to promote all Na dependent co-transport processes across the brush border membrane (BBM). Down-regulation of Na-K-ATPase activity has been postulated to alter the absorption via Na-solute co-transporters in human inflammatory bowel disease (IBD). Further, the altered activity of a variety of Na-solute co-transporters in intact villus cells has been reported in animal models of chronic enteritis. But the molecular mechanism of down-regulation of Na-K-ATPase is not known. In the present study, using a rabbit model of chronic intestinal inflammation, which resembles human IBD, Na-K-ATPase in villus cells was shown to decrease. The relative mRNA abundance of α-1 and ß-1 subunits was not altered in villus cells during chronic intestinal inflammation. Similarly, the protein levels of these subunits were also not altered in villus cells during chronic enteritis. However, the BLM concentration of α-1 and ß-1 subunits was diminished in the chronically inflamed intestinal villus cells. An ankyrin-spectrin skeleton is necessary for the proper trafficking of Na-K-ATPase to the BLM of the cell. In the present study, ankyrin expression was markedly diminished in villus cells from the chronically inflamed intestine resulting in depolarization of ankyrin-G protein. The decrease of Na-K-ATPase activity was comparable to that seen in ankyrin knockdown IEC-18 cells. Therefore, altered localization of Na-K-ATPase as a result of transcriptional down-regulation of ankyrin-G mediates the down-regulation of Na-K-ATPase activity during chronic intestinal inflammation.

Protein kinase C-mediated phosphorylation of RKIP regulates inhibition of Na-alanine cotransport by leukotriene D(4) in intestinal epithelial cells.

Leukotriene D4 (LTD4) is an important immune inflammatory mediator that is known to be elevated in the mucosa of chronically inflamed intestine and alter nutrient absorption. LTD4 inhibits Na-alanine cotransport in intestinal epithelial cells by decreasing the affinity of the cotransporter ASCT1. LTD4 is known to increase intracellular Ca(++) and cAMP concentrations. However, the intracellular signaling mechanism of LTD4-mediated ASCT1 inhibition is unknown. In the present study, pretreatment with calcium chelator BAPTA-AM or inhibition of Ca(++)-dependent protein kinase C (PKC), specifically PKCα, resulted in the reversal of LTD4-mediated inhibition of ASCT1, revealing the involvement of the Ca(++)-activated PKC pathway. PKCα is known to phosphorylate Raf kinase inhibitor protein (RKIP), thus activating its downstream signaling pathway. Immunoblotting with anti-RKIP-Ser(153) antibody showed an increase in phosphorylation levels of RKIP in LTD4-treated cells. Downregulation of endogenous RKIP showed no decrease in ASCT1 activity by LTD4, thus confirming its involvement in ASCT1 regulation. Phosphorylation of RKIP by PKC is known to activate different signaling pathways, and in this study it was found to activate cAMP-activated protein kinase A (PKA) pathway. Although protein abundance of ASCT1 was not altered in any of the experimental conditions, there was an increase in the levels of phosphothreonine in ASCT1 protein, thus showing that phosphorylation changes were responsible for the altered affinity of ASCT1 by LTD4. In conclusion, LTD4 inhibits ASCT1 through PKC-mediated phosphorylation of RKIP, leading to the subsequent activation of PKA pathway, possibly through ß2-andrenergic receptor activation.