Endocytosis fluid-phase

Figure 41 -15. Two types of endocytosis. An endocytotic vesicle (V) forms as a result of invagination of a portion of the plasma membrane. Fluid-phase endocytosis (A) is random and nondirected. Receptor-mediated endocytosis (B) is selective and occurs in coated pits (CP) lined with the protein clathrin (the fuzzy material). Targeting is provided by receptors (black symbols) specific for a variety of molecules. This results in the formation of a coated vesicle (CV).

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... 

Hufnagel H, Hakim P, Lima A, Hollfelder F (2009) Fluid phase endocytosis contributes to transfection of DNA by PEI-25. Mol Ther 17 1411-1417... 

Wright KM, Oparka KJ. Uptake of Lucifer Yellow CH into plant-cell protoplasts a quantitative assessment of fluid-phase endocytosis. Planta 1989 179 257-264. 

Synncs, M., Prydz, K., Lovdal, T., Brech, A. and Berg, T. (1999). Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes, Biochim. Biophys. Acta-Biomembranes, 1421, 317-328. 

Raths, S., Rohrer, J., Crausaz, F. and Riezman, H., 1993, Two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J.Cell Biol., 120 55-65. 

Smaller peptides may be transported across the BBB either by nonspecific fluid-phase endocytosis or RMT mechanisms. It may also be possible for them to use a peptide-specific transporter protein, directly inserted into the cell membrane in a similar manner to the solute transporters, which flips them across the membrane [43,56]. In some cases the receptor that transduces the signal at the cell membrane may also act as the transporter for the peptide and is co-opted to initiate RMT, or another transport system in other cases the membrane transporter for a peptide may be quite distinct in structure from the receptor that transduces signals at the cell membrane from a signaling peptide [45]. 

Preston et al. [66] have shown that BBB permeability to the nonmetabolizable, but slowly BBB penetrant, tracer mannitol (182 Da), is between 0.19 and 0.22 pL/g/min in the brain of rats of 1 week of age and this permeability is identical to that of adult rats. The vascular space occupied by the tracer mannitol (the initial volume of distribution Fj) falls from 1.23 mL, at 1 week of age, to 0.75 mL per 100 g brain in the adult rat [66], indicating either a larger vascular volume, resulting from a greater capillary density or capillary diameter in the neonatal rat, or to a significant degree of internalization of the mannitol by the endothelium, possibly by fluid-phase endocytosis into the cerebral capillary endothelial cells in the newborn, compared to the adult. 

Fig. 6.2. Model for how FcRn rescues IgG from catabolism by recycling and transcytosis. IgG and many other soluble proteins are present in extracellular fluids. Vascular endothelial cells are active in fluid phase endocytosis of blood proteins. Material taken up by these cells enters the endosomes where FcRn is found as an integral membrane protein. The IgG then binds FcRn in this acidic environment. This binding results in transport of the IgG to the apical plasma membrane for recycling into the circulation, or to the basolateral membrane for transcytosis into the extracellular space. Exposure to a neutral pFI in both locations then results in the release of IgG. The remaining soluble proteins are channeled to the lysosomal degradation pathway.

As a nonspecific fluid-phase endocytosis marker, HRP was used to evaluate the transport characteristics across rat alveolar epithelia cell monolayers. HRP was transported relatively intact (about 50%) across the alveolar barrier via nonspecific fluid-phase endocytosis. The permeability coefficient of HRP was decreased upon lowering the temperature [51], but after conjugation with transferrin via a disulfide linkage, HRP uptake by alveolar cell monolayers was significantly increased. Receptor-mediated internalization of the conjugated HRP was verified by competition for the transferrin receptor [52]. 

Guillot FL, Audus KL. Angiotensin peptide regulation of fluid-phase endocytosis in brain microvessel endothelial cell monolayers. J Cereb Blood Flow Metab 1990 10 (6) 827-834. 

MDR cell lines exhibit several other changes in surface membrane properties. Often, the structural order is increased in resistant cells as analyzed by electron spin resonance (ESR) and fluorescence anisotropy studies [98]. In addition, an increase in intramembranous particles and the rate of fluid-phase endocytosis are reported for resistant cells [99, 100]. 

Padron, D. M.E. Bizeau, and J.R. Hazel (2000). Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures Am J. Physiol. (Regulatory Integrative Comp. Physiol.) 278 R529-R536. 

The concentration of NBP as well as the duration of drug exposure required to inhibit prenylation varies among cell types. The unique sensitivity of osteoclasts and macrophages to these agents is likely a consequence of enhanced cellular uptake by fluid-phase endocytosis [40]. Studies have also demonstrated that resistance to NBP-mediated inhibition of protein prenylation may be a consequence of elevated basal levels of FPP in certain cell types [41]. 

Thompson, K., Rogers, M.J., Coxon, F.P., and Crockett, J.C. (2006). Cytosohc entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis. Mol Pharmacol 69 1624—1632. 

Endocytosis The term endocytosis denotes the internalization (= encasing) of molecules inside vesicles. This can take place in the form ofpinocytosis (= fluid-phase endocytosis), whereby the molecules remain in a detached state with no membrane binding. It can also occur in the form of adsorptive endocytosis, whereby the molecules are bound to membrane receptors by a ligand and the whole complex is transported into the cell. 

Three processes are involved in transcellular transport across the intestinal epithelial cells simple passive trans-port, passive diffusion together with an efflux pump, and active transport and endocytosis. Simple passive transport is the diffusion of molecules across the membrane by thermodynamic driving forces and does not require direct expenditure of metabolic energy. In contrast, active transport is the movement of molecules across the mem-brane resulting directly from the expenditure of metabolic energy and transport against a concentration gradient. Endocytosis processes include three mechanisms fluid-phase endocytosis (pinocytosis), receptor-mediated endocytosis, and transcytosis (Fig. 6). Endocytosis processes are covered in detail in section Absorption of Polypeptides and Proteins, later. 

Fig. 6 Schematic depiction of cellular uptake mechanisms fluid-phase endocytosis, receptor-mediated endocytosis, and transc5dosis. (Illustration by Leigh A. Rondano, Boehringer Ingelheim Pharmaceuticals, Inc.)...

The epithelial membrane of the GI tract consists of a continuous barrier of cells, which allows the transport of low-molecular-weight molecules by simple diffusion or various carrier processes. Macromolecules such as proteins may be absorbed from the intestinal lumen by cellular vesicular processes, through fluid-phase endocytosis (pinocytosis), or by receptor-mediated endocytosis or transcytosis (Fig. 6). In pinocytosis, extracellular fluid is captured within an epithelial membrane vesicle. It begins with the formation of a pocket... 

One of the mechanisms of active reabsorption is endocytosis. Fluid phase endocytosis consists of the incorporation of fluid and solutes in vesicles formed at the base of the brush border membrane of the proximal tubular cells (Figure 1). A more efficient absorptive endocytosis involves first binding of a drug, such as the cationic aminoglycoside and/or may be cadmium [30, 31], to a carrier (phosphatidyhnositol) located in the luminal membrane of the wall of the pinocytotic vesicle occurs followed by endocytosis and lysosomal fusion [32, 33]. 

Takano M, Ohishi Y, Okuda M, Yasuhar M, Hori R, Transport of gentamicin and fluid-phase endocytosis markers in the LLC-PK1 kidney epithelial cell line, J Pharmacol ExpTherap, 1993,268(2) 669-74. 

Reversible binding to the target Target + mAb <- mAb.Target complex mAb clearance by fluid phase endocytosis mAb —> Sink... 

Another method of Pgp regulation has been demonstrated by adrenomedullin (AM) (89). It is produced by endothelial cells in the brain and acts as a vasodilator in the cerebral circulation. It was shown that AM antisense decreased the transendo-thelial electrical resistance across endothelial monolayers. Treatment of these cells with AM activated Pgp, suggesting that AM acts as an autocrine mediator in the regulation of the properties of BBB endothelial cells. In addition, AM incubation decreased BBB permeability for sodium fluorescein (376 Da) but not for Evan s blue albumin (67 kDa). An interesting observation was that it also attenuated fluid-phase endocytosis. 

In addition to the blood-brain barrier, two other barrier layers limit and regulate molecular exchange at the interface between the blood and the neural tissue and its fiuid spaces the choroid plexus epithelium between blood and ventricular CSF and the arachnoid epithelium between blood and subarachnoid CSF. These CNS barriers perform a number of functions such as the ionic homeostasis, the restriction of small molecule permeation, the specific transport of small molecules required to enter or leave the brain, the restriction and regulation of large molecule traffic by reducing the fluid-phase endocytosis via pinocytotic vesicles, the separation of peripheral and central neurotransmitter pools, and the immune privilege [16]. 

Gonatas, N.K., Stieber, A., Hickey, W.F, Herbert, S.H., and Gonatas, J.O. (1984) Endosomes and Golgi vesicles in adsorptive and fluid phase endocytosis. The Journal of Cell Biology, 99,1379-1390. 

---