Thomas Y. Ma

Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: Role of cytoskeletal involvement

Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohns disease, NSAID‐associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco‐2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca++ modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter‐grown Caco‐2 intestinal monolayers in Ca++‐free solution (CFS), consisting of modified Krebs‐buffer solution containing 0 mM Ca++ and 1 mM EGTA, resulted in a rapid drop in Caco‐2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco‐2 TJ permeability was rapidly reversed by the re‐introduction of Ca++ (1.8 mM) into the incubation medium. The CFS‐induced increase in Caco‐2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO‐1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS‐induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri‐junctional actin and myosin filaments. The inhibition of CFS‐induced activation of Caco‐2 MLCK with MLCK inhibitor (ML‐7) prevented the CFS‐induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS‐induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco‐2 MLCK activation and the resultant contraction of the peri‐junctionally located actin‐myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg++‐myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS‐induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca++ is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca++ from the incubation medium causes activation of Caco‐2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability. Microsc. Res. Tech. 51:156–168, 2000.

Intestinal Epithelial Barrier Dysfunction in Crohn's Disease

Abstract Despite extensive research, the etiology of Crohns disease remains unknown. Accumulating evidence suggests the possibility that a primary defect of intestinal barrier function may be present in Crohns disease. In this review, the possible role of intestinal barrier defect in Crohns disease is discussed. It has been recognized for some time that Crohns patients have a defective intestinal epithelial barrier function manifested by an increase in intestinal permeability. Recent studies indicate that a subgroup of healthy first-degree relatives of Crohns patients (a population at high risk for developing Crohns disease) also have increased intestinal permeability. Additionally, this subgroup of patients have evidence of increased exposure to foreign antigens, suggesting a possible link between increase in intestinal permeability and increase in antigenic penetration. Furthermore, exacerbation of Crohns disease is produced by agents that disrupt intestinal epithelial barrier function, while remission of active disease is induced by decreasing intestinal antigenic load. A “leaky gut” hypothesis is advanced which proposes that a preexisting disorder of intestinal permeability is responsible for the intestinal inflammation of Crohns disease.

Mechanisms and sites of mannitol permeability of small and large intestine in the rat

Mannitol is commonly used as an intestinal permeability probe, yet the mechanisms of its penetration of the intestinal barrier are not entirely clear. Therefore, we studied mannitols permeability of different segments of the intestine and studied the kinetics and influence of intraluminal factors on mannitol permeabilityin vivo in perfused intestinal segments of rats. There was linear relationship between permeability rate of mannitol and its luminal concentration (y=7.2x+1.7;r=0.98), indicating that passive diffusion is involved in mannitols permeability. Increased luminal fluid osmolarity from 0.3 to 0.6 osmol/liter resulted in decreased net water flux with a corresponding decrease in mannitol permeability in both jejunum and colon (P<0.01), indicating the prominent influence of solvent drag on net mannitol permeability. The relationship between mannitol permeability and water absorption at different osmolarities was linear in the jejunum and colon. At luminal osmolarity of 0.3 osmol/liter, 34.6% of mannitol permeability was mediated by passive diffusion and 65.4% was mediated by solvent drag in the jejunum. Mannitol permeability was much more dependent on solvent drag in the colon (88.9%) than in the small intestine (65.4%). The net permeability rate of mannitol was similar in the jejunum and ileum but was much higher in the colon (P<0.01). Addition of chenodeoxycholate (5 mM) to the perfusate resulted in a significant decrease in absorption of water (P<0.01) with a corresponding decrease in mannitol permeability (P<0.01). These studies indicate that mannitol permeability of the intestinal barrier is mediated by passive diffusion and solvent drag, with the latter accounting for a greater fraction of the total permeability.

PEG 400, a hydrophilic molecular probe for measuring intestinal permeability.

There is a widely held misconception that low-molecular-weight polyethylene glycols are highly lipophilic permeability probes and therefore are transported across lipid cell membranes. The relative lipophilicity of polyethylene glycols 400 and 600 were examined by determining their partition coefficients (Kd) in water and organic solvents of increasing relative polarity. The Kd of polyethylene glycol 414 between hexane and water was 0.000015, indicating that there are only 1.5 parts of polyethylene glycol 414 in hexane for 100,000 parts of polyethylene glycol 414 in water. When the Kd was determined in organic solvents with increasing relative polarity or water character, there was a linear increase in Kd. The relative urinary recovery of individual molecular weight fractions of polyethylene glycol 400 in normal volunteers was analyzed. After oral ingestion, there was a progressive decrease in relative urinary recovery of increasing molecular weight fractions of polyethylene glycol 400 suggesting that increase in the molecular size limited polyethylene glycol intestinal permeability. There was excellent correlation between the relative urinary recovery and the hydrophilicity of the intravenously administered polyethylene glycol 400 fractions. It is concluded that polyethylene glycols 400 and 600 are strongly hydrophilic. Since partitioning of polyethylene glycol into lipid phase is negligible in lipid/water mixtures, they are unlikely to be transported via lipid pathways. The intestinal permeability of polyethylene glycols are governed by their molecular size, and once in circulation their urinary excretion appears to be governed in part by their plasma or water solubility.

Mechanisms of polyethylene glycol 400 permeability of perfused rat intestine

Abnormal permeability to polyethylene glycol 400 (PEG 400) has been demonstrated in various disorders with defective intestinal barrier functions. To understand the basic mechanisms of PEG 400 permeability, we compared PEG 400 permeation in different segments of the intestine and studied the kinetics and influence of intraluminal factors on PEG 400 absorption in vivo in perfused intestinal segments of the rat. The permeation rate of PEG 400 was dependent on the luminal concentration (y = 12.99x + 3.5; r = 0.97), indicating that passive movement is the mechanism involved in PEG 400 absorption. Changing the perfusate pH from 6 to 7.4 or modifying the unstirred water layer resistance by changing luminal flow rate did not affect PEG 400 absorption. When luminal osmolarity was varied from 0.225 to 0.6 osmol/L, higher osmolarity decreased both water and PEG 400 absorption (p greater than 0.01). The relationship between PEG 400 and water absorption at different osmolarities was linear (y = 0.9x + 5.7; r = 0.98). At a luminal osmolarity of 0.3 osmol/L 43% of PEG 400 permeation was mediated by passive diffusion and 57% was mediated by solvent drag. Increasing water absorption by decreasing luminal osmolarity resulted in proportional increase of PEG 400 permeation through solvent drag or convection. The solvent drag reflection coefficient (sigma f) for PEG 400 permeation of the jejunum was 0.1. Taurocholic acid (10 mM) alone or with oleic acid (2.5 mM) did not affect PEG 400 absorption. Permeabilities of 1 mM PEG 400 and water were similar in jejunum and ileum but were markedly increased in the colon (p greater than 0.01). These studies demonstrate that PEG 400 is absorbed by both passive diffusion and by solvent drag, with the latter accounting for a greater fraction of the absorptive drive under normal conditions. Polyethylene glycol 400 uses aqueous pathways for its permeation across the intestinal epithelium.

Human intestinal cell line Caco-2: a useful model for studying cellular and molecular regulation of biotin uptake.

The mechanisms of enterocyte and molecular regulation of biotin uptake are poorly understood. An intestinal cell line processing the transport characteristics of native intestinal cells is highly desirable to investigate the finer details of the cellular processing and molecular regulation of biotin transport. In the present study, we investigated the uptake of the water-soluble vitamin biotin by a human intestinal cell line Caco-2. Uptake of both low (4 nM) and high (20 microM) concentrations of biotin by confluent monolayers of Caco-2 cells was appreciable and linear for up to 10 min of incubation. Replacement of Na+ in the incubation medium with other monovalent cations--K+, choline, Li+ and NH4(+)--caused a significant inhibition of biotin uptake; a relatively lesser inhibition was seen with Li+. Initial rate of uptake of biotin was temperature-dependent and saturable as a function of concentration at 37 degrees C but not at 4 degrees C. The Vmax and apparent Km of the temperature-dependent saturable process were 520 pmol/mg protein per min and 9.5 microM, respectively. The addition of unlabeled biotin and the structural analogue desthiobiotin to the incubation media caused a significant inhibition of the uptake of [3H]biotin. The inhibitory effect of desthiobiotin was competitive in nature with an inhibition constant (Ki) of 41 microM. Biocytin, on the other hand, was a weak inhibitor and biotin methyl ester and diaminobiotin did not have any effect. Pretreatment of Caco-2 cells with the monovalent cation ionophore gramicidin and the Na+, K+(-)ATPase inhibitor ouabain caused significant inhibition of biotin uptake. Pretreatment with the K+ ionophore valinomycin did not affect biotin uptake. Using the Activation Method, the stoichiometric ratio of biotin- to Na+ coupling was found to be 1:1. Growing confluent Caco-2 cells in a biotin-deficient environment resulted in rapid up-regulation of biotin transport with a marked increase (258%) in the Vmax of biotin uptake. These findings demonstrate that biotin uptake by Caco-2 cells is via a carrier-mediated system. This system is temperature-dependent, driven by Na(+)-gradient and is regulated by the substrate level. These in-vitro findings are very similar to and further confirm previous findings in human and animal studies and dispute other findings previously reported for Caco-2 cells; the present study also demonstrates the suitability of this system for further characterization of the cellular and molecular regulation of biotin uptake.

Effect of aging and caloric restriction on intestinal permeability

Intestinal permeability is increased in several disorders such as Crohns disease or rheumatoid arthritis. Since aging leads to alteration of many biological functions, the effect of aging on intestinal permeability was studied by measuring the intestinal permeability in aging rats gavaged with different size permeability probes--mannitol, polyethylene glycol (PEG) 400, and inulin. In rats fed with control diet, there was a significant increase in intestinal permeability to medium size probes PEG 400 (14.8 +/- 0.4 and 21.0 +/- 1.1% at 3 and 28 months respectively, p less than .01) and mannitol (3.41 +/- 0.4 and 5.3 +/- 0.5% at 3 and 28 months, respectively, p less than .01). Intestinal permeability of the large macromolecule inulin did not change (0.42 +/- 0.03 and 0.38 +/- 0.02% at 3 and 28 months, respectively) with aging. There was no correlation between weight of the rats and their intestinal permeability. Because dietary caloric restriction has been found to prolong the life span, retard deterioration of several biological functions, and affect intestinal absorptive functions, we examined the effect of lifelong calorie restriction on intestinal permeability changes. Lifelong calorie-restricted diet did not affect age-related change in intestinal permeability. We conclude that intestinal permeability of medium size probes increases with aging and that lifelong caloric restriction does not prevent this change. We speculate that age-associated deterioration in intestinal barrier functions could permit increased systemic absorption of lumenal antigens and could perhaps contribute to the genesis of antigen-related age-associated diseases.

Oxygen free radical injury of IEC-18 small intestinal epithelial cell monolayers

Oxygen radicals can cause endothelial and epithelial permeability changes and mucosal injury of the small intestine. There is no clear consensus concerning the relative injurious potential of individual oxygen radicals. In this study, the small intestinal cell line IEC-18 was used as an in vitro model to study the relative injurious effects of reactive oxygen metabolites. By introducing different combinations of oxygen metabolite-producing enzymes, xanthine oxidase, superoxide dismutase, and catalase, and an iron chelator, deferoxamine, to the fully confluent monolayers and to proliferating IEC-18 cells, the differential injurious effects of the oxygen metabolites O2-, H2O2, and OH. could be evaluated. The extent of cellular injury was assessed using [3H]thymidine uptake, 51Cr release, and morphological evaluations. Our results suggest that OH. produced as a by-product of O2- and H2O2 via the Haber-Weiss reaction was the most injurious oxygen species involved in cellular injury of IEC-18 monolayers induced by xanthine oxidase. O2- produced by xanthine oxidase appeared to be only minimally injurious, and H2O2 produced by xanthine oxidase and as a result of conversion of O2- by superoxide dismutase was moderately injurious. Superoxide dismutase and deferoxamine at appropriate concentrations were protective against xanthine/xanthine oxidase-induced monolayer injury. H2O2 added directly or produced indirectly by glucose oxidase was very injurious to the intestinal monolayers, and this injury was mitigated by catalase.

Mechanism of colonic permeation of inulin: Is rat colon more permeable than small intestine?

BACKGROUND/AIMSnColonic epithelium is considered to be relatively tight. The colonic pore diameter is 6 A; therefore, colonic epithelium has generally been considered to be impermeable to hydrophilic probes with a cross-sectional diameter of > 6 A. This study examined whether rat colon is permeable to inulin, a large hydrophilic macromolecule having a molecular weight of 5000 g/mol and a cross-sectional diameter of 15 A (hydration diameter, 20 A).nnnMETHODSnThe colonic permeation of inulin (10 mumol/L) in vivo was investigated by perfusion of rat colonic segments.nnnRESULTSnThere was significant colonic permeation of inulin, but tissue retention of inulin was low. The net colonic flux of inulin was strongly dependent on net water flux, showing a strong solvent drag effect. Addition of 16,16-dimethyl prostaglandin E2 decreased water flux with a corresponding decrease in inulin flux; this process seemed to be mediated by 5-cyclic adenosine monophosphate because both the phosphodiesterase inhibitor aminophylline and dibutyryl adenosine 5-cyclic adenosine monophosphate decreased water and inulin flux in a parallel manner. Chenodeoxycholic and taurocholic acids decreased net mucosal-to-serosal water flux but increased inulin flux. The net colonic permeation rate of inulin was higher than the small intestinal permeation rate.nnnCONCLUSIONSnRat colon is permeable to inulin. The higher net colonic permeability may be caused by differences in mucosal surface, permselectivity, solvent drag effect, and differences in net water fluxes of the colon and small intestine.

Eicosanoids and the small intestine.

Prostaglandins play an important role in modulation of various physiologic processes in the small intestine. In this review, the involvement of prostaglandins in various small-intestinal functions including small-intestinal secretion, mucosal protection, epithelial and endothelial barrier function, and motility are discussed.