Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms

Methods available to assess therapeutic potential of fibrinolytic enzymes of microbial origin: a review

Abstract Fibrinolytic enzymes are agents administered for the treatment of myocardial infarctions, strokes, cardiac and respiratory failure. Although several microorganisms are known to produce these fibrinolytic enzymes, only a few of such enzymes, along with the age-old oral anticoagulants, have been employed in the clinical and therapeutic applications in humans. The use of these agents is associated with drawbacks such as allergic reactions and bleeding complications; therefore, it necessitates frequent monitoring of drug levels in the blood. Due to this, there is an impetus on the current effort to identify newer potential candidates from the novel microbial sources which show longer half-life, higher fibrin specificity, higher therapeutic index and lesser allergic reactions. Various methods are available for the preliminary evaluation of a potential drug candidate for the therapeutic use. Choosing the right combination of in vitro and in vivo methods would give crucial insight on the therapeutic potential of the chosen test compound. This article discusses various assay techniques, in vitro trails and in vivo models available, to help researchers in choosing right biological methods and its combinations to evaluate efficacy of potential drug candidate

Salmonella Typhimurium Infection Reduces the Ascorbic Acid Uptake in the Intestine

Salmonella Typhimurium infection of the gastrointestinal tract leads to damage that compromises the integrity of the intestinal epithelium and results in enterocolitis and inflammation. Salmonella infection promotes the expression of inflammasome NLRP3, leading to activation and release of proinflammatory cytokines such as IL-1β, and the infected host often displays altered nutrient levels. To date, the effect of Salmonella infection and proinflammatory cytokine IL-1β on the intestinal uptake of ascorbic acid (AA) is unknown. Our results revealed a marked decrease in the rate of AA uptake in mouse jejunum infected with Salmonella wild type (WT). However, the nonpathogenic mutant (Δ invA Δ spiB) strain did not affect AA uptake. The decrease in AA uptake due to Salmonella WT infection is accompanied by significantly lower expression of mouse (m)SVCT1 protein, mRNA, and hnRNA levels. NLRP3 and IL-1β expression levels were markedly increased in Salmonella-infected mouse jejunum. IL-1β-exposed Caco-2 cells displayed marked inhibition in AA uptake and significantly decreased hSVCT1 expression at both protein and mRNA levels. Furthermore, the activity of the SLC23A1 promoter was significantly inhibited by IL-1β exposure. In addition, GRHPR (a known SVCT1 interactor) protein and mRNA expression levels were significantly reduced in Salmonella-infected mouse jejunum. These results indicate that Salmonella infection inhibits AA absorption in mouse jejunum and IL-1β-exposed Caco-2 cells. The observed inhibitory effect may partially be mediated through transcriptional mechanisms

The location of pacemakers in the uteri of pregnant guinea pigs and rats

The pregnant uterus is a smooth muscle organ whose pattern of contraction is dictated by the propagation of electrical impulses. Such electrical activity may originate from one or more pacemakers, but the location of these sites has not yet been determined. To detect the location of the pacemaker in the gravid uterus, two approaches were used: 1) determine the site from where the contraction started using isolated uteri from the pregnant guinea pig, and videotape their contractions; and 2) record, in isolated uteri from pregnant term rats, with 240 extracellular electrodes simultaneously, and determine where the electrical bursts started. In both the contractile and electrophysiological experiments, there was not a single, specific pacemaker area. However, most contractions (guinea pig 87%) and bursts (rat 76%) started close to the mesometrial border (mean 2.7 ± 4.0 mm SD in guinea pigs and 1.3 ± 1.4 mm in rats). In addition, in the rat, most sites of initiations were located closer to the ovarial end of the horn (mean distance from the ovarial end 6.0 ± 6.2 mm SD), whereas such an orientation was not seen in the guinea pig. In both guinea pig and rat uteri at term, there is not one specific pacemaker area. Rather, contractile and electrical activity may arise from any site, with the majority starting close to the mesometrial border. Furthermore, in the rat, most activities started at the ovarial end of the horn. This may suggest a slightly different pattern of contraction in both species

Differential expression of human riboflavin transporters -1, -2, and -3 in polarized epithelia: A key role for hRFT-2 in intestinal riboflavin uptake

AbstractTransport of riboflavin (RF) across both the brush border membrane (BBM) and basolateral membrane (BLM) of the polarized enterocyte occurs via specific carrier-mediated mechanisms. Although, three human riboflavin transporters (hRFTs), i.e., hRFT-1, hRFT-2 and hRFT-3 are expressed in the intestine, little is known about the cell surface domain(s) at which these specific hRFTs are expressed. Here, we used live cell confocal imaging of intestinal epithelial Caco-2 and renal MDCK cells to show that the hRFT-1 is mainly expressed at the BLM, hRFT-2 is exclusively expressed at the apical membrane, while hRFT-3 is mostly localized inside intracellular vesicular structures (with some expression at the BLM). Further the level of hRFT-2 mRNA expression in Caco-2 cells and in native human intestine is significantly higher than that of hRFT-1 and -3; hRFT-2 was also more efficient in transporting 3H-RF than hRFT-1 and -3. These findings implied an important role for hRFT-2 in intestinal RF uptake, a conclusion that was further supported by findings of hRFT-2 gene-specific siRNA knockdown investigation. These results show that members of the hRFT family are differentially expressed in polarized epithelia, and that the apically expressed hRFT-2 plays a key role in intestinal RF accumulation