Enterocytes isolation

Blachier, F., M rabet-Touil, H., Darcy-Vrillon, B., Posho, I., and Duee, P.-H. (1991). Stimulation by D-glucose of the direct conversion of arginine to citrulline in enterocytes isolated from pig jejunum. Biochem. Biophys. Res. Commun. 177, 1171-1177. 

Initial reports on PTXs hepatotoxicity prompted the study of in vitro effects of these toxins on hepatocytes. There are several reports on PTX-1 and -2 in vitro cytotoxicity to hepatocytes. PTX-1 has been demonstrated to induce morphological changes and apoptosis in freshly prepared rat and sahnon hepatocytes [31,32]. PTX-1 toxic effects on primary cultures of liver cells have also been reported as disarrangement of actin and microtubule cytoskeleton [33]. Recently, in our laboratory, we found that actin cytoskeleton of primary rat hepatocytes was also altered by PTX-2 and -11 in the same way as PTX-1 but with a slightly higher potency [34]. Another primary cell type sensitive to PTXs is fresh enterocytes isolated from rabbit, in which PTX-6 induces a significant depolimeriza-tion of actin filaments [35]. 

Solute uptake can also be evaluated in isolated cell suspensions, cell mono-layers, and enterocyte membrane vesicles. In these preparations, uptake is normalized by enzyme activity and/or protein concentration. While the isolation of cells in suspension preparations is an experimentally easy procedure, disruption of cell monolayers causes dedifferentiation and mucosal-to-serosal polarity is lost. While cell monolayers from culture have become a popular drug absorption screening tool, differences in drug metabolism and carrier-mediated absorption [70], export, and paracellular transport may be cell-type- and condition-depen-dent. 

Mucosal brush border membrane vesicles and basolateral membrane vesicles can be isolated to study solute uptake across specific enterocyte boundaries. These more isolated vesicle systems allow for investigation of solute transport across a particular membrane barrier and permit separation of membrane trans-... 

The Caco-2 cell line was isolated from a human colon carcinoma, and has been characterized as one of the best in vitro models of intestinal epithelium. Indeed, in contrast to other intestinal cell lines, Caco-2 cells are able to constitute a homogenous monolayer and to spontaneously differentiate into polarized cells, highly similar to human mature enterocytes, after approximately 2 weeks of culture. Furthermore, the Caco-2 cells present microvillosities at the apical side and have a high transmembrane resistivity, which confirms the fact that the cells are confluent and link to one another via gap junctions. Finally, they can absorb different compounds, express many enzymes involved in intestinal metabolic pathways (Pinto et al. 1983, Musto et al. 1995, Salvini et al. 2002), and give reproducible in vitro results consistent with results obtained in in vivo studies (Artursson and Karlsson 1991). 

It had long been expected that an iron transporter would be found at the basolateral membrane of the enterocyte responsible for the exportation of iron from the enterocytes into the portal vein circulation, and it turns out that three groups appear to have made this discovery almost simultaneously (McKie et ah, 2000 Donovan et ah, 2000 Abboud and Haile, 2000). The first of these, IREG1, was originally identified by subtractive cloning techniques (McKie et ah, 1998), and isolated and... 

INTESTINE Characterization of a membrane potassium ion conductance in intestinal secretory cells using whole cell patch-clamp and calcium-sensitive dye techniques, 192, 309 isolation of intestinal epithelial cells and evaluation of transport functions, 192, 324 isolation of enterocyte membranes, 192, 341 established intestinal cell lines as model systems for electrolyte transport studies, 192, 354 sodium chloride transport pathways in intestinal membrane vesicles, 192, 389 advantages and limitations of vesicles for the characterization and the kinetic analysis of transport systems, 192, 409 isolation and reconstitution of the sodium-de-pendent glucose transporter, 192, 438 calcium transport by intestinal epithelial cell basolateral membrane, 192, 448 electrical measurements in large intestine (including cecum, colon, rectum), 192, 459... 

I.R. Falconer, M. Dornbusch, G. Moran and S.K. Yeung, Effect of the cyanobacterial (blue-green algal) toxins from Microcystis aeruginosa on isolated enterocytes from the chicken small intestine, Toxicon, 30 (1992) 790-793. 

Gross, C.J., L.M. Henderson, and D.A. Savaiano. 1986. Uptake of L-camitine, D-carnitine and acetyl-L-carnitine by isolated guinea-pig enterocytes. Biochim Biophys Acta 886 425. 

Calonge, M.L., Iludian. A., Bolufer, J. (1989). Ionic dependence of glycylsarcosine uptake by isolated chicken enterocytes. J. Cell. Physiol. 138,579-585. 

With the isolated perfused duodenum, there is a rapid increase in calcium transport in response to the addition of calcitriol to the perfusion medium. Isolated enterocytes and osteoblasts also show a rapid increase in calcium uptake in response to calcitriol. It is not associated with changes in mRNA or protein synthesis, but seems to be because of recruitment of membrane calcium transport proteins from intracellular vesicles to the cell surface. It is inhibited by the antimicrotubule compound colchicine. It can only be demonstrated in tissues from animals that are adequately supplied with vitamin D in vitamin D-deficient animals, the increase in intestinal calcium absorption occurs only more slowly, together with the induction of calbindin. 

Several different cell monolayer models that mimic in vivo barriers have been developed and are very popular models in industry as well as academia. The ideal model would be a monolayer of polarized normal human enterocytes, but attempts to isolate and grow them have failed because of the low viability and the complicated requirement for attachment, mono-layer formation, and differentiation. However, tumor cells grow... 

Spencer, R.J. and Chesson, A. 1994. The effect of Lactobacillus spp. in the attachment of enterotoxigenic Escherichia coli to isolated porcine enterocytes. J. Appl. Bacteriol. 77, 115-220. 

Use of freshly isolated enterocytes to evaluate uptake of NMEs is well documented. Various laboratories [48—50] have established isolation techniques. Advantages... 

The isolated brush border vesicles from the plasma membrane of the microvilU is the simplest in vitro system used so far. The interaction of lipid with rabbit intestinal brush border vesicles has been investigated by Proulx et al. [50] who found that PC, phosphatidylethanolamine, cholesterol, diglyceride as well as fatty acids were taken up by vesicles. Barsukov et al. [51] have shown that transfer of PC from PC vesicles to isolated brush border vesicles can occur in the presence of PC-exchange protein. The use of brush border vesicles is an interesting new approach that permits detailed studies of rate of transfer of specific lipids into the plasma membrane of the enterocyte. The model is seriously hmited by the fact that incubation with solutions containing bile salts at a concentration above the critical micellar concentration will result in partial or total solubilization of the membrane vesicles. 

The enterocytes of the small intestine can be isolated and used for study of intracellular aspects of intestinal lipid transport like triglyceride synthesis [52]. The disappearance of the mucus barrier during isolation of the epithelial cells results in plasma membrane disintegration and loss of cellular integrity when the cells are exposed to bile salts. As is the case for brush border vesicles, the system of isolated cells does not allow study of interaction between enterocytes and lipids dispersed in a form that resembles physiological conditions, i.e. solubilized in mixed bile salt micelles. 

A b55g cytochrome was isolated from brush border membranes of rabbit enterocytes (KnOpfel and SoLioz 2002). The purified haemoprotein exhibited ascorbate-stimulated reduction of iron(III) and copper(n). The rate constants, k, for these reactions were 1.3810.12 and 0.6410.16 min" , respectively. 

Both ischaemia (clamping of the superior mesenteric artery for 30,60, 90 min) and ischaemia/reperfusion affected respiratory function of isolated rat enterocyte mitochondria as compared to control (Madesh et al. 2000). Preconditioning with nitric oxide donor, sodium nitroprusside (1 mM, given into the proximal jejunal lumen at a rate of 1 ml/ min), significantly enhanced the recovery of the respiratory control rate. Mitochondrial lipid changes suggestive of activation of phospholipase... 

Gastaldi, G., Laforenza, U., Casirola, D., Ferrari, G., Tosco, M., and Rindi, G., 1999. Energy depletion differently affects membrane transport and intracellular metabolism of riboflavin taken up by isolated rat enterocytes. Journal of Nutrition. 129 406-409. 

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