Fatty acids and epithelial permeability: effect of conjugated linoleic acid in Caco-2 cells.

Conjugated linoleic acid (CLA) is a collective term referring to the positional and geometric isomers of linoleic acid. This novel fatty acid has been shown to have a number of beneficial actions, including immunomodulatory, anticarcinogenic, and antiatherogenic effects. Tight junctions of epithelial cells determine epithelial membrane integrity and selective paracellular permeability to ions and macromolecules. Occludin and ZO-1 are integral structural components of the tight junction, which are involved in the biogenesis and functional integrity of the epithelial monolayer. This study investigated the effects of two isomers of CLA (cis-9 and trans-10 isomers) on Caco-2 cell transepithelial resistance (TER) development, paracellular epithelial permeability, and occludin and ZO-1 expression. Caco-2 cells were grown in media supplemented with 0.05 mM linoleic acid, cis-9 CLA, or trans-10 CLA for 21 days. The trans-10 CLA isomer delayed Caco-2 cell TER development, which is an in vitro measure of epithelial cell integrity, and increased paracellular epithelial permeability. Immunofluorescent staining of Caco-2 cell epithelial monolayers grown in media supplemented trans-10 CLA showed that the trans-10 CLA isomer altered distribution of occludin and ZO-1. The trans-10 CLA isomer delayed the acquisition of transepithelial resistance and altered the cellular distribution of occludin, which have important implications in relation to epithelial permeability.

Roles of glucitol in the GutR-mediated transcription activation process in Bacillus subtilis: tight binding of GutR to tis binding site.

Glucitol induction in Bacillus subtilis requires a transcription activator, GutR, and a sequence located upstream of the gut promoter. To understand the initial steps involved in the GutR-mediated transcription activation process and the physiological roles of glucitol, GutR was overproduced and purified. In the absence of glucitol, GutR exists as a monomer and binds directly to its binding site in the gut regulatory region. This binding site was mapped to a 29-base pair imperfect inverted repeat located between -78 and -50, and there is only one GutR binding site within the regulatory region. The kinetic parameters of the interaction between GutR and its binding site were monitored in real time using surface plasmon resonance. The half-life of the GutR-DNA complex in the absence of glucitol was estimated to be 6.8 min. In contrast, in the presence of glucitol, the half-life of the complex was extended to longer than 19 h by affecting only the off-rate but not the on-rate. This effect is glucitol-specific. These data indicate that glucitol binds to GutR and induces GutR to have an extremely tight binding at its binding site. The physiological relevance of this process in transcription activation is discussed.

Adenovirus-mediated transfer of an Na+/K+-ATPase beta1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats.

Pulmonary edema is cleared via active Na(+) transport by alveolar epithelial Na(+)/K(+)-ATPases and Na(+) channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na(+)/K(+)-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na(+)/K(+)-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 x 10(9) PFU of recombinant adenoviruses containing rat alpha(1) and beta(1) Na(+)/K(+)-ATPase subunit cDNAs (adalpha(1) and adbeta(1), respectively) to rat lungs 7 days prior to exposure to 100% O(2) for 64 hr. As compared with controls and ad alpha(1), AFC in the adbeta(1) rats was increased by >300%. Permeability for large solutes was less in the ad beta(1) than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adbeta(1) group. Survival through 14 days of hyperoxia was 100% in the adbeta(1) group but was not different from hyperoxic controls in animals given adalpha(1). Our data show that overexpression of a beta(1) Na(+)/K(+)-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na(+)/K(+)-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.

Evaluation of an accelerated Caco-2 cell permeability model.

An accelerated 3-7-day Caco-2 cell permeability model was examined and compared to the traditional 21-25-day model. Caco-2 cell permeability coefficients (P(Caco-2)) of 33 structurally diverse small molecular weight compounds from apical to basolateral (AP-->BL) direction in the accelerated model were approximately twice those in the traditional model. As observed with microscopy and transepithelial electrical resistance measurements, this difference was attributed to less confluent and differentiated Caco-2 cell monolayers in the accelerated model. However, there were no significant differences in rank ordering of the compounds. The expression of P-glycoprotein in the accelerated model was shown to be significantly less than that in the traditional model. This resulted in lower permeability directional ratios defined as the ratio between permeability coefficients from BL-->AP and from AP-->BL for compounds that were cellular efflux pump substrates. The accelerated model may not be suitable for studying cellular efflux pumps such as P-glycoproteins. However, it is a feasible alternative to the traditional model for rank ordering of compounds in the process of drug discovery and development by significantly improving the turnover time and labor efficiency. This makes it an excellent Caco-2 cell permeability model for high throughput screening.

[Intestinal permeability in the first year of life. The effect of diarrhea]. / Permeabilidad intestinal en el primer año de vida. Efecto de la diarrea.

OBJECTIVE: The permeability of the intestinal mucosa to lactulose and mannitol was explored longitudinally in infants at 1, 3-4 and 11-12 months of age. This was also evaluated during the episodes of diarrhea that they suffered during follow-up. PATIENTS AND METHODS: Sugar excretion was measured by gas chromatography in five-hour urine samples. RESULTS: A decrease in lactulose excretion was observed, which became significant at 11-12 months of age (p = 0.02). No changes were detected in mannitol excretion, although this showed a tendency to decrease. The lactulose/mannitol (L/M) ratio remained unchanged. During the 15 episodes of diarrhea observed in these infants during the 12 month follow-up, a considerable increase in this ratio was seen, due mainly to increased lactulose excretion. CONCLUSIONS: It is hypothesized that the decrease in lactulose excretion between one and 11-12 months of age is part of the maturational process of the intestinal barrier, while diarrhea results in increased permeability due to damage to the absorptive epithelium.

Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers.

PURPOSE: To determine whether the expression and activity of the P-glycoprotein (P-GP) drug efflux pump vary with the culture age of Caco-2 cell monolayers. METHODS: Caco-2 cell monolayers were grown for 3-27 days on tissue culture-treated Transwells. P-GP efflux function was determined by measuring transmonolayer fluxes of cyclosporin A (CsA) and verapamil, while P-GP expression level was evaluated by Western blot analysis using monoclonal antibody C219. RESULTS: The apparent permeability coefficient (Papp) of CsA (0.5 microM) in the basolateral-to-apical (B-->A) direction increased with culture age and was higher than the apical-to-basolateral (A-->B) direction at all times. Net secretory Papp significantly increased from day 17 onward compared to that observed during day 3 through 13. Verapamil (100 microM) significantly inhibited CsA transport in the B-->A direction from day 17 to 27, while elevating CsA transport in the A-->B direction from day 6 to 27. Interestingly, the Papp of verapamil (0.5 microM) in the B-->A direction was significantly higher than in the A-->B direction from day 6 to 27, rendering increases in net secretory Papp of verapamil with culture age. Western analysis revealed that P-GP expression level was in the order of 4 weeks approximately 1 week > 3 weeks > 2 weeks at equal loading of cell proteins. CONCLUSIONS: P-GP is continuously expressed throughout the culture period, but it may not be fully functional at an early age. Caco-2 cell monolayers of day 17 to 27 appear to be a good model to evaluate the functional role of P-GP in drug efflux.

Effects of diabetes, insulin treatment, and osmolality on contractility of isolated rat resistance arteries.

The effects of osmolality, diabetes, and insulin-treatment on microvascular contractility were examined in mesenteric resistance arteries (internal diameter approximately 250 microns) isolated from streptozotocin-induced diabetic rats, streptozotocin-induced diabetic rats treated with 1-3 U insulin/day during the week before being killed, and age- and sex-matched control rats. Vessels were mounted in a microvascular myograph for isometric tension recording and responses were generated in physiological salt solutions with varying amounts of glucose or mannitol added. The passive response (expressed as the diameter the vessels would maintain if relaxed and exposed to a transmural pressure of 100 mmHg), the maximal response to noradrenaline, and the response produced by partial depolarization with 50 mmol/l potassium were not dependent on glucose or mannitol concentrations of the bathing medium; also, these responses were not dissimilar in vessels from the three groups of rats tested. The sensitivity to noradrenaline, however, was inversely related to the concentration of glucose (P < 0.01) and mannitol (P < 0.01) of the bathing medium without significant differences in slopes of regression lines between rat groups. Moreover, vessels from streptozotocin-induced diabetic rats were less sensitive to noradrenaline than were vessels from control rats; vessels from insulin-treated streptozotocin-induced diabetic animals had the lowest sensitivity to noradrenaline. These data suggest that osmolality, diabetes, and insulin-treatment independently affect microvascular sensitivity to the endogenous neurotransmitter, noradrenaline.

Gut permeability in IgA nephropathy.

Gut permeability to small molecules was assessed by the differential absorption of cellobiose and mannitolin 18 patients with IgA nephropathy (IgAN). The urinary cellobiose:mannitol excretion ratio in patients did not differ (P = 0.42) from controls. These findings do not support the hypothesis that chronic increased mucosal permeability allows excessive antigen penetration to the mucosal immune system, predisposing to glomerular IgA deposition in IgA nephropathy. However, the patient with the greatest cellobiose:mannitol ratio developed macroscopic haematuria within 3 weeks of testing, raising the possibility of a transient abnormality in gut permeability.

Diuretic synergy in the treatment of acute experimental cerebral edema.

Acute cerebral edema was created in dogs by the intracarotid injection of sodium lauryl sulfate, a method that produces no structural or vascular disruption in the brain. Cerebrospinal fluid (CSF) pressure elevations were measured through subdural balloons, and ranged from 300 to 1500 mm H2O. The nature of the cerebral edema produced was studied with intravital Trypan blue, electrocorticography, and visual observation of cerebral circulation, and by postmortem histological sections and determinations of brain water content. Two dissimilar diuretic agents were studied: the osmotic diuretic, mannitol; and the renal diuretic, ethacrynic acid. As expected, mannitol reduced CSF pressure effectively, with no significant rebound overshoot. Ethacrynic acid, despite favorable reports, proved to have only a slight effect on CSF pressure but did prove to be a potent diuretic. Unexpectedly, the two agents were found to act synergistically. When both agents were administered, significantly greater reductions in pressure were obtained and pressure reductions were maintained for longer periods.