Intestine endocytosis

C1C-3, -4 and -5 form the second branch of the CLC gene family. These proteins are 80% identical, and with the exception of C1C-5, which is most highly expressed in kidney and intestine, show a broad expression pattern. C1C-3 to C1C-5 reside in intracellular membranes of the endocytotic pathway [4]. Disruption of C1C-5 leads to a defect in endocytosis in mouse... 

Attempts to study the entry of ES products into cells using markers of fluid phase endocytosis yielded unexpected results. When larvae browse resistant IEC-6 cells in the presence of extracellular fluorescent dextran, dextran enters the cytoplasm of a significant proportion of the cells in the mono-layer (Butcher et al., 2000). The parameters of dextran entry are most compatible with the conclusion that larvae wound the plasma membranes of IEC-6 cells that is, they create transient breaches in the membrane that allow impermeant markers to enter the cell (McNeil and Ito, 1989). Wounding is considered to be a common occurrence in intestinal epithelia (McNeil and Ito, 1989). Injured cells are able to heal their wounds by recruiting vesicles to seal the breach (Steinhardt et al., 1994). In an experimental system, healing allows the injured cell to retain cytoplasmic dextran. In epithelial cell cultures inoculated with T. spiralis larvae, the relationship between glycoprotein delivery and injury of plasma membranes is not clear, i.e. dextran-laden cells do not always stain with Tyv-specific antibodies and... 

However, the receptor-mediated endocytosis of iron-transferrin studies [63] does not explain the initial uptake of iron from nutrients in the intestinal (villus) cells, since apotransferrin is generally not available in the lumen, except in a limited amount from biliary excretion. Work on other iron transport mechanisms has mainly been reported in the last five years. 

The protein that transports iron around the body in blood and lymph, and indeed within the cell, is transferrin (500 kDa). It has two binding sites for iron (Fe " ) when no iron is bound, it is known as apotransferrin. Transferrin picks up not only the iron absorbed from the intestine but also that released from the macrophages and then transports it to the cells that require it, which is primarily the cells in the bone marrow but also other cells that are proliferating. For uptake into the cells, the transferrin binds to a receptor on the plasma membrane and then the complex enters the cell by endocytosis. The iron is released from the complex in the cytosol where it is bound by the intra-... 

There are four main compartments a soluble macromolecule can enter the central compartment (blood and lymphatic system), interstitium, intestinal lumen, and lysosomes [100, 101]. Minor compartments are primary urine, liquor, bile, etc. There is no experimental evidence that clearly indicates the penetration of synthetic macromolecules into the cytoplasm, i.e, into the intracellular compartment (inside the cell but outside the endosomes or lysosomes) [101]. The movements of soluble macromolecules between body compartments have been extensively reviewed [14, 20,100-104] and will not be covered in detail here. We shall concentrate on the discussion of main factors influencing the movement of soluble macromolecules when administered into the bloodstream. Depending on the structure and molecular weight distribution, part of the polymeric molecules are excreted in the urine. Simultaneously, the macromolecules are cleared from the bloodstream by endocytosis. It is important to note that nonspecific capture of soluble macromolecules by the specialized cells of the reticuloendothelial system is generally much less (orders of magnitude) when compared to vesicular carriers of a comparable structure. 

The liver eliminates proteins on first pass after oral administration and on each pass of hepatic blood flow. Hepatocytes, Kupffer cells, adipocytes, and endothelial cells can all be involved in proteolysis (Figure 5.6). Proteolysis can occur in lysosomes after endocytosis of a protein and lysosomal fusion. Endocytosis of a protein may be a nonspecific or receptor-mediated process. Proteolytic products are eliminated from the liver through biliary excretion, and subsequently digested further in the intestinal tract. 

Dietary cholesterol, together with triacylglycerols, is absorbed from the intestinal tract and enters the large lipoprotein chylomicrons (see Fig. 21-1). Absorption of cholesterol is incomplete, usually amounting to less than 40% of that in the diet. Absorption requires bile salts and is influenced by other factors.186 As it is needed cholesterol is taken from the plasma lipoproteins into cells by endocytosis. Much of the newly absorbed cholesterol is taken up by the liver. The liver also secretes cholesterol, in the form of esters with fatty acids, into the bloodstream. 

FIGURE 1.11 A scheme of the various absorption routes across the intestinal epithelium and cellular barriers to xenobiotics absorption. A, Transcellular absorption (plain diffusion) B, paracellular absorption C, carrier-mediated transcellular absorption D, facilitated diffusion E, the MDR and P-gp absorption barrier and F, endocytosis. (From Hunter, J. and Hirst, B.H., Adv. Drug Deliv. Rev., 25, 129, 1997. With permission.)... 

Physical barrier. Following oral administration of macromolecular drugs, their potential absorption pathways from the intestinal lumen to the bloodstream can be classified into transcellular transport associated with adsorptive or receptor-mediated endocytosis and paracellu-lar transport (Fig. 10.1). The GI tract surface consists of a tightly bound single layer of epithelial cells covered with thick and viscous mucus, which serves as a defensive deterrent against permeation of xenobi-otics and harmful pathogens. The epithelial cells in the GI tract are... 

Figure 10.1 Pathways for intestinal absorption of macromolecular drugs, (a) Paracellular transport of macromolecules can be achieved by altering or disrupting the tight junctions that exist between cells and are only permeable to small molecules (<100 to 200 Da). (b) Adsorptive enterocytes and (c) M cells of Peyer s patches allow transcellular transport of macromolecules involving transcytosis and receptor-mediated endocytosis.

The apoproteins of HDL are secreted by the liver and intestine. Much of the lipid comes from the surface monolayers of chylomicrons and VLDL during lipolysis. HDL also acquire cholesterol from peripheral tissues in a pathway that protects the cholesterol homeostasis of cells. In this process, free cholesterol is transported from the cell membrane by a transporter protein, ABCA1, acquired by a small particle termed prebeta-1 HDL, and then esterified by lecithin cholesterol acyltransferase (LCAT), leading to the formation of larger HDL species. The cholesteryl esters are transferred to VLDL, IDL, LDL, and chylomicron remnants with the aid of cholesteryl ester transfer protein (CETP). Much of the cholesteryl ester thus transferred is ultimately delivered to the liver by endocytosis of the acceptor lipoproteins. HDL can also deliver cholesteryl esters directly to the liver via a docking receptor (scavenger receptor, SR-BI) that does not endocytose the lipoproteins. 

Chylomicrons are synthesized in the intestine and transport dietary triacylglycerols to skeletal muscle and adipose tissue, and dietary cholesterol to the liver. At these target tissues the triacylglycerols are hydrolyzed by lipoprotein lipase on the surface of the cells and the released fatty acids are taken up either for metabolism to generate energy or for storage. The resulting cholesterol-rich chylomicron remnants are transported in the blood to the liver where they are taken up by receptor-mediated endocytosis. 

The second reason to consider the intracellular enzymes is because of the absorption mechanisms by which macromolecules may cross the intestinal mucosa. There are two possible mechanisms for relatively small macromolecules such as therapeutic peptides and oligonucleotides, they may be able to pass via the paracellular route between the cells, particularly if some absorption enhancers are present. For example, Tsutsumi et al. (2008) have shown in vitro that in the presence of chenodeoxycholate as an absorption enhancer modified oligonucleotides with molecular weights of nearly 3,700 and 7,400 Da could cross rat intestine via the paracellular route. In the case of the paracellular route the macromolecules will not be exposed to the intracellular enzymes and thus they will not be subject to intracellular hydrolysis. However, macromolecules, especially larger ones, will cross the epithelium via the transcellular mechanism of endocytosis. In this case they will be taken into the lysosomes that contain a formidable array of digestive enzymes (see later in Section 1.5.2). 

The lysosomal enzymes The lysosomes are membrane vesicles ubiquitous to mammalian cells and contain a panoply of hydrolytic enzymes, estimated to be over 60 in number, that function to digest practically any biological macromolecule. They are important to the discussion of oral macromolecular drug delivery for two reasons. First, any macromolecules that escape digestion by the pancreatic and brush border enzymes are likely to be taken up into the epithelial cells by the process of endocytosis. In this process, the apical membrane invaginates and the target molecules enter endocytic vesicles that then fuse with the lysosomes and are subjected to intracellular hydrolysis by the lysosomal enzymes. Second, the sloughing-off of the epithelial cells means that the lysosomal enzymes will be released into the lumen of the intestine. They may be... 

Simionescu N (1983) Cellular aspects of transcapillary exchange. Physiol Rev 63 1536-1579 Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387 569-572 Snoeck V, Goddeeris B, Cox E (2005) The role of enterocytes in the intestinal barrier function and antigen uptake. Microbes Infect 7 997-1004 Sugiyama Y, Kato Y (1994) Pharmacokinetic aspects of peptide delivery and targeting importance of receptor-mediated endocytosis. Drug Dev Ind Pharm 20 591-614 Takano M, Yumoto R, Murakami T (2006) Expression and function of efflux transporters in the intestine. Pharmacol Therap 109 137-161... 

Fig. 3.2. General mechanisms for the uptake and transport of macromolecules by an enterocyte. Intracellular uptake-, after absorption and endocytosis by the microvillous membrane, macromolecules are transported in small vesicles and larger phagosomes. Intracellular digestion occurs when lysosomes combine to form phagolysosomes. Intact molecules that remain after digestion are deposited in the intercellular space by exocytosis. Intercellular uptake-, alternatively, macromolecules may cross the tight junction barrier between cells and diffuse into the intercellular space. (After Walker, W. A. Isselbacher, K. J. Uptake and transport of macromolecules by the intestine possible role in clinical disorders. Gastroenterology, 6T. 531-50, by Williams Wilkins (1974).)...

Considerable evidence has accumulated indicating that macromolecules and microparticulates can be taken up by the intestinal enterocytes, generally via pinocytosis. In some cases, transcytosis, i.e. passage through the cells, has been observed, with microparticles subsequently gaining access to the lymphatics of the mucosa. For example, studies have shown that receptor-mediated endocytosis via enterocytes is a major pathway for the internalization of certain antisense oligonucleotides. 

Microtubules and microfilaments are ubiquitous components of almost all cells (red blood cells excluded). Microfilaments are polymers of actin, which in the non-muscle cell can exist in two forms /3-actin and T-actin. Microfilaments have a diameter of about 6 nm. In certain structures such as the microvilli of intestinal mucosal cells, microfilaments are associated with myosin, so that movement is possible given an appropriate stimulus. Endocytosis and exocytosis events are apparently mediated via microfilaments. Microfilaments are disrupted by the drug cytochalasin. 

In hepatocytes, vitamin E can take two routes. A fraction of it is packaged as VLDL and reenters the circulation, while excess is excreted in the bile. Plasma lipolysis of the VLDL particle again results in release not only of lipids, but also of vitamin E, with the remainder left with the LDL particles. This fraction can be further distributed to tissues via LDL receptor-mediated endocytosis or transferred between lipoproteins, mainly to HDL, by plasma lipid transfer proteins. Thus, mobilization of vitamin E from intestinal and liver... 

The second major obstacle of the oral delivery of proteins is the low permeability of proteins in the intestinal epithelium. The uptake of proteins is mediated by passive diffusion across the enterocytes (transcellular diffusion), paracellular diffusion (through intercellular spaces) and mostly by transcytosis (facilitated by receptor-mediated endocytosis). Erodible microcapsules and nanoparticles were shown to be absorbed intact through the GI tract and have opened the pos-... 

Chylomicrons are produced from dietary fat by the removal of resynthesised triglycerides from the mucosal cells of the small intestine into the intestinal lumen. These then enter the circulation via the thoracic dncts in the lymphatic system and enter into the subclavian veins, where triglyceride content is reduced by the action of lipoprotein lipases (LPL) on capillary endothelial surfaces in skeletal muscle and fat. The free fatty acids (FFA) from the triglycerides are used by the tissues as an energy source or stored as triglycerides. The chylomicron remnants, stripped of triglyceride and therefore denser, are then taken up by the liver by LDL receptor-mediated endocytosis, thereby delivering cholesterol to the liver. 

Figure 1 Pathways of the intestinal barrier. A paracellular passive diffusion, B transcellular passive diffusion, CF influx/efflux facilitated transport facilitated by membrane proteins, G transcytosis, and H endocytosis (reprinted from Reference 2 with kind permission from Dr. Jon Vabeno and Dr. Roy...

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