Transport Of Epidermal Growth Factor Research Articles

Transport of epidermal growth factor in the stroke-injured brain

Stroke is a neurological disorder that currently has no cure. Intrathecal delivery of growth factors, specifically recombinant human epidermal growth factor (rhEGF), stimulates endogenous neural precursor cells in the subventricular zone (SVZ) and promotes tissue regeneration in animal models of stroke. In this model, rhEGF is delivered with an invasive minipump/catheter system, which causes trauma to the brain. A less invasive strategy is to deliver rhEGF from the brain cortex; however, this requires the protein to diffuse through the brain, from the site of injection to the SVZ. Although this method of delivery has great potential, diffusion is limited by rapid removal from the extracellular space and hence for successful translation into the clinic strategies are needed to increase the diffusion distance. Using integrative optical imaging we investigate diffusion of rhEGF vs. poly(ethylene glycol)-modified rhEGF (PEG-rhEGF) in brain slices of both uninjured and stroke-injured animals. For the first time, we quantitatively show that PEG modification reduces the rate of growth factor elimination by over an order of magnitude. For rhEGF this corresponds to a two to threefold increase in predicted brain penetration distance, which we confirm with in vivo data.

The cytotoxic action of diphtheria toxin and its degradation in intact Vero cells are inhibited by bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase.

The role of vacuolar-type H(+)-ATPase (V-ATPase) in the cytotoxic action of diphtheria toxin (DT) was studied by using bafilomycin A1, a specific inhibitor of V-ATPase. Studies with acridine orange showed that the acidification of intracellular acidic compartments was inhibited strongly when Vero cells were treated with 500 nM bafilomycin A1, indicating that bafilomycin effectively inhibits V-ATPase when it is added to the culture medium. The toxicity of DT to Vero cells, which was determined by the inhibition of protein synthesis by DT, was inhibited partially by bafilomycin at 10 nM and inhibited completely at 500 nM. Therefore, V-ATPase is involved in the expression of the toxicity of DT. Studies using 125I-labeled DT showed that bafilomycin inhibited the degradation of internalized DT, indicating that V-ATPase is also involved in this step. Subcellular fractionation revealed that 125I-DT accumulated mainly in the endosome fraction, and not in the lysosome fraction, when the cells were incubated with 125I-DT in the presence of bafilomycin. Under the cell fractionation conditions similar to those used for the DT-treated cells, we determined the location of 125I-labeled epidermal growth factor in the degradation pathway. The result suggests that bafilomycin A1 does not inhibit the transport of epidermal growth factor to lysosome.

Receptor-mediated disposition of polypeptides: Kinetic analysis of the transport of epidermal growth factor as a model peptide using in vitro isolated perfused organs and in vivo system

The concept of “transport receptor” is now well established and receptor-mediated endocytosis (RME) is known as a general mechanism in the uptake of biologically important peptides and proteins. Using epidermal growth factor (EGF) as a model polypeptide, we have developed in vivo and in vitro methods to define the kinetic characteristics of RME. Kinetic analysis of the tissue distribution of EGF in rats performed in vivo clarified the following points. (1) The uptake of EGF by the liver, kidney and some other tissues showed clear saturation, which may represent RME. (2) The hepatic extraction ratio of EGF at a low dose was 0.8 and its uptake was limited mostly by the hepatic plasma flow rate. (3) In the intact animal, the bulk (ca. 50%) of the removal of EGF from the circulation was accounted for by hepatic clearance, followed by kidney clearance (ca. 15%). We then analyzed the hepatic and renal handling of EGF by isolated perfused organs, and the kinetic parameters have been obtained. The analysis clarified that, in the overall receptormediated processing of EGF by rat liver, the ligand-receptor association process is rapid (mean time < 1 min), the degradation process is much slower (mean time 4.5 h) and the receptor internalization process is intermediate (mean time 4.5 min). An internalization rate constant comparable with that in the liver was obtained also in the kidney. The extent of the receptor downregulation and the recovery rate of the available receptors were determined in several organs after the bulk of the receptors had been down-regulated by injecting excess EGF in vivo. The mean time required for the recovery showed inter-organ differences and the value (ca. 30 min) in the liver was much shorter than those (2–5 h) in other organs. Such recovery was considered at least in the liver to reflect the processes of recycling of internalized EGF receptors to the cell surface. Finally, time profiles of EGF plasma concentrations and their surface receptors in the liver were simulated with a physiological pharmacokinetic model.

Uptake and transport of epidermal growth factor by the small intestinal epithelium of the fetal rat

Epidermal growth factor may play an important part in postnatal gastrointestinal development. However, little is known of its role prenatally. The aim of this study was to detect epidermal growth factor in amniotic fluid and to study its uptake and transfer across the epithelium of fetal rat small intestine. Anesthetized 20-day gestation rats underwent caesarian section. Three fetuses were exteriorized, their abdomens were opened, and ligated loops of proximal and distal small intestine were infused with 100 μg epidermal growth factor. Infused segments were removed 30 min later and processed for electron microscopy, and tissue was embedded in LR gold resin. Sections were treated with rabbit anti-rat epidermal growth factor antibody, followed by 5 nm gold-labeled goat anti-rabbit immunoglobulin before staining. Epidermal growth factor was measured in amniotic fluid by radioimmunoassay. In the proximal and distal small intestine epidermal growth factor was found membrane-associated along the luminal surface of microvilli, within apical invaginations and endosomal compartments, free from the membrane in multivesicular bodies, within large clear vesicles, basal vesicles and in association with the basolateral membrane and beyond. Epidermal growth factor was found in amniotic fluid at a concentration of 0.38 ± 0.07 (SD) ng/ml. This study shows that epidermal growth factor is present in amniotic fluid and is transported across the epithelium of fetal rats by an endocytotic process in both the upper and lower small intestine. It is likely that amniotic fluid epidermal growth factor plays a part in intestinal mucosal development, and may be active systemically after transepithelial passage in utero.

Decrease in the number of receptors for epidermal growth factor in the liver of d-galactosamine-intoxicated rats

Hepatic transport of epidermal growth factor (EGF) was studied in d-galactosamine-intoxicated rats by the multiple-indicator dilution (MID) method. The extraction ratio of 125I-labeled EGF in the intoxicated rats, obtained from a model-independent analysis of the dilution curves, decreased to 45% of the control values. A distributed two-compartment model was fitted to the dilution data by nonlinear least-squares regression, and the kinetic parameters, k on · P T (product of on-rate constant and receptor density), k off (off-rate constant) and k s (sequestration rate constant) were determined. The values of k on · P T and k s in the intoxicated rats decreased to approximately one-half and one-third of those in the control rats respectively. Similar decreases in the k on · P T and k s values in the intoxicated rats were also observed for the transport of 125I-labeled insulin, a positive control, into the liver. The 125I-labeled EGF binding experiment at equilibrium using liver homogenates revealed that the intoxication reduced the receptor density ( P T ) to one-third of the control values, whereas the equilibrium dissociation constant ( k d ) did not change significantly. The activities of Na +, K +-ATPase, cytochrome P-450 and glutathione S-transferase decreased in the intoxicated rats to 70–80% of the control values. The number of nuclei per unit area of tissue slices was also reduced to 70% of the control. Thus, the extent to which the enzyme activities and the number of nuclei decreased in the intoxicated liver was smaller than that of the number of EGF receptors. It is concluded that the reduction of EGF receptors cannot be explained by the “intact hepatocyte hypothesis” but rather by the functional change of hepatocytes induced by the administration of d-galactosamine.

Absorption of epidermal growth factor and insulin in rabbit renal proximal tubules

Epidermal growth factor (EGF), which stimulates the growth of a variety of tissues, was originally isolated from mouse submandibular glands. Human EGF (hEGF) has been isolated from the urine, and cDNA encoding for EGF has been isolated from the human kidney. Thus the kidney may represent an alternate source of EGF. Another potential explanation for the urinary content of EGF is a limited reabsorption of filtered EGF and/or a transtubular transport of EGF from the peritubular compartment to the lumen. Therefore, we exposed isolated and perfused rabbit proximal tubules to 125I-hEGF either in the perfusate or in the bath fluid. Luminal uptake of hEGF was compared with uptake of 125I-porcine insulin, which is known to be taken up with large efficiency. The results demonstrate that only 4% of the perfused 125I-hEGF load was taken up per millimeter of tubule length compared with 73% for insulin. Furthermore, hEGF is subject to a small transtubular transport from bath to lumen. Renal clearance of endogenous rabbit EGF was also measured and was comparable with that of creatinine. Thus in conclusion this study strongly suggests that filtered EGF to a large extent remains in the ultrafiltrate, unlike insulin, which is removed within the first few millimeters of proximal tubule. In addition filtered EGF in preurine is probably supplied by EGF transported intact across the proximal tubular epithelium.

Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum.

Epidermal growth factor (EGF), an acid-stable peptide present in rodent and human milk, is absorbed and promotes intestinal growth when fed to suckling rats. To determine whether absorptive cells of suckling rat ileum conduct selective transepithelial transport of EGF, we followed uptake of 125I-EGF from ileal loops by autoradiography and biochemical methods. Specific binding sites for 125I-EGF were localized by electron microscope autoradiography on apical membranes of ileal epithelial sheets in vitro. During uptake in vivo, radiolabeled molecules were concentrated in apical endosomal compartments and were also associated with lysosomal vacuoles, basolateral cell surfaces, and lamina propria. Excess cold EGF reduced basolateral label by 44% and TCA precipitable serum label by 38%. After 30 and 60 min of continuous uptake, radiolabeled molecules in epithelium, denuded mucosa, blood, and liver were analyzed under reducing conditions by reversed-phase high-pressure liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although considerable degradation of 125I-EGF occurred after uptake from the lumen, a portion of radiolabel in epithelium and mucosa represented 125I-EGF which eluted somewhat more rapidly from C18 HPLC columns and showed a slight decrease in apparent molecular weight by SDS-PAGE. All radiolabel in blood and liver represented breakdown products. Thus, EGF is selectively transported across the ileal epithelium in suckling rats but is modified during transport. Milk EGF may accumulate in the lamina propria where it could influence growth and maturation of the suckling intestine.

Transport of epidermal growth factor by rat liver: evidence for a nonlysosomal pathway.

Epidermal growth factor (EGF), circulating in the blood, is taken up by rat liver hepatocytes by means of specific and saturable receptor-mediated endocytosis. These experiments were undertaken to determine (a) the transport pathway(s) of EGF taken up by rat liver and (b) the effects of lysosomal inhibition on its transport. 125I-EGF was injected into rat portal veins, and bile samples were collected and analyzed for both total and immunoprecipitable radioactivity. In addition, the livers were examined by electron microscopic autoradiography. Some animals received injections of chloroquine before surgery, to disrupt lysosomal function. The results indicate that most of the EGF taken up by the hepatocytes is transported to lysosomes and degraded. However, a small but significant percentage of endocytosed EGF is transported by a pathway independent of the lysosomal system, resulting in secretion of intact EGF: (a) Both degraded and immunoprecipitable EGF are secreted into bile. (b) Immunoprecipitable radioactivity peaks at 20 min after EGF injection, whereas degradation-associated radioactivity does not peak until 40 min postinjection. (c) EGF isolated from bile is specifically taken up by isolated hepatocytes in monolayer culture, indicating that it is still recognizable by the EGF receptor. (d) When the lysosomal system is inhibited with chloroquine, secretion of degraded EGF is significantly inhibited, whereas the amount of intact EGF secreted into bile is unchanged. The utilization by liver of a dual transport process for EGF represents an unusual system of intracellular ligand processing, whose physiological significance has yet to be determined.