Caco brush-border enzymes

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

Other cell lines used in permeability studies include the T84 human colonic adenocarcinoma colonic crypt cell model. This line has a reduced carrier expression, secrets mucus, and has very high resistance [31, 32], The IEC cell line is a rat fetal intestinal epithelium cell with higher permeabilities than Caco-2 cells [33], LLC PKi is a pig kidney epithelial cell line with low expression of efflux systems, but expression systems for transport proteins [32], 2/4/A1 cells are a conditionally immortalized rat fetal intestinal epithelium line with crypt cell-like morphology and temperature-sensitive differentiation [34], They form differentiated monolayers with tight junctions, increased brush border enzymes when grown on extracellular matrices with laminin. Transport of drugs with LP in 2/4/A1 monolayers was comparable to that in the human jejunum and up to 300 times faster than that in Caco-2 monolayers. In contrast, the permeability of HP drugs was comparable in both cell lines [34],... 

Caco-2 cells form tight junctions and express many of the brush border enzymes (hydrolases) that are found in the normal small intestine, for example, alkaline phosphatase, sucrase, and amino peptidases [26-29]. Cytochrome P450 (CYP450) isoenzymes and some phase II enzymes (e.g., glutathione-S-transferases, sulfotrans-ferase, and glucuronidase) have been identified [29-33] in these cells however, the level of CYP expression (e.g., CYP3A4) is low in the original cells under standard cell culture conditions [34]. 

The most widely used ceU line in drug transport studies is the Caco-2 cell line. This is an iimnortal ceU line derived from human colon carcinoma that can be grown to monolayer on porous support. Functionally, this cell line models the colon more than the small intestine. The Caco-2 model allows characterization of both mucosal-to-serosal and serosal-to-mucosal transport and can also be used to study transcellular and paracellular transport, hi addition to expressing small intestinal brush border enzymes, Caco-2 cells also express Phase I and Phase II enzymes and can be used to evaluate metabohsm of compounds during transport across the intestinal barrier. ... 

Drugs may also undergo hydrolysis by intestinal esterases (hydrolases), more specifically carboxylesterases (EC 3.1.1.1) in the intestinal lumen and at the brush border membrane [58, 59]. It has been shown that intestinal hydrolase activity in humans was closer to that of the rat than the dog or Caco-2 cells [60]. In these studies, six propranolol ester prodrugs and p-nitrophenylacetate were used as substrates, and the hydrolase activity found was ranked in the order human > rat Caco-2 cells > dog for intestinal microsomes. The rank order in hydrolase activity for the intestinal cytosolic fraction was rat > Caco-2 cells = human > dog. The hydrolase activity towards p-nitrophenylacetate and tenofovir disoproxil has also been reported in various intestinal segments from rats, pigs and humans. The enzyme activity in intestinal homogenates was found to be both site-specific (duodenum > jejunum > ileum > colon) and species-dependent (rat > man > Pig)-... 

Inhibition of riboflavin transport across the Caco-2 monolayer by 2,4-dini-trophenol (DNP), reducing the cellular ATP agent, points toward the necessity of delivering energy. However, the initial phase of whole absorption process i.e. transport across the epithelium membrane) does not require a supply of metabolic energy and is a consequence of riboflavin s association with carrier protein which is presented on the brush-border membrane. In the next step, some of the riboflavin absorbed inside the enterocytes cytosol is phosphory-lated by flavokinase and converted by FAD synthetase—enzymes which require ATP molecules for their action (Gastaldi et al. 1999). 

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