Abstract. Substantial amounts of epidermal growth factor (EGF) are cleared from the circulation by hepatocytes via receptor-mediated endocytosis and subsequently degraded within lysosomes. We have used a combined biochemical and morphological approach to examine the fate of the receptor after exposure to EGF. Polyclonal antibodies were prepared against the purified receptor and their specificity established by immunoprecipitation and immunoblotting techniques. The EGF receptor was then localized by immunofluorescence and immunoperoxidase techniques and quantified on immunoblots. In untreated livers, EGF receptor was restricted to the sinusoidal and lateral surfaces of hepatocytes. 2-4 min after exposure of cells to EGF, the receptor was found in small vesicles (i.e., coated vesicles) as well as larger vesicles and tubules at the cell periphery. By 15 min the receptor was found in multivesicular endosomes located near bile canaliculi. Exposure of hepatocytes to EGF also resulted in a rapid loss of receptor protein from total liver homogenates and a decrease in its half-life from 8.7 h in control livers to 2.5 h. This EGF-induced loss of receptors was not observed when lysosomal proteinases were inhibited by leupeptin or when endosome/lysosome fusion was prevented by low temperature (16°C). In the presence of leupeptin, receptor could be detected in structures identified as lysosomes using acid-phosphatase cytochemistry. All these results suggested rapid internalization of EGF receptors in response to ligand and degradation within lysosomes. However, four times more ligand was degraded at 8 h than the number of high-affinity (Kd of 8-15 nM) EGF-binding sites lost, suggesting either (a) high-affinity receptors were recycled, and/or (b) more than 300,000 receptors were available for EGF uptake. We identified and characterized a latent pool of-300,000 low-affinity receptors (Kd ~ 200 nM) that could be separated on sucrose gradients from the plasma membrane pool of ~300,000 high-affinity receptors (Kd of 8-15 nM). Despite the differences in their binding affinities, the high-and low-affinity receptors appeared to be structurally identical and were both EGF-dependent protein kinases. In addition, the dynamics of the low-affinity receptors were consistent with a functional role in EGF uptake and delivery to lysosomes.ECEPTOa-mediated endocytosis is well-recognized as a general mechanism used by many cells for uptake of biologically important molecules (reviewed in references 7 and 51). The fates of these ligands, once internalized, vary from degradation to intracellular storage to transport across the cell and subsequent release. The receptors also have diverse fates, some being re-utilized, others degraded, and a few released with their ligand. The principle cell in the liver, the parenchymal cell or hepatocyte, possesses many specific membrane receptors that mediate the internalization of different molecules from the circulation. The fates of these ligands and their receptors encompass most currently established fates...

Polymeric IgA (plgA) is transported by liver parenchymal cells (hepatocytes) from blood to bile via a receptor-mediated process. We have studied the intracellular pathway taken by a TEPC15 mouse myeioma plgA. When from 1 pg to 1 mg 12Sl-plgA was injected into the saphenous vein of a rat, 36% was transported as intact protein into the bile over a 3-h period. The concentration of transported 1251-plgA was maximal in bile 30-60 min after injection, and -80% of the total 12Sl-plgA ultimately transported had been secreted into bile by 90 min. A horseradish peroxidase-plgA conjugate (~251-plgA-HRP) was transported to a similar extent and with kinetics similar to that of unconjugated 1251-plgA and was therefore used to visualize the transport pathway. Peroxidase cytochemistry of livers fixed in situ 2.5 to 10 min after ~2Sl-plgA-HRP injection demonstrated a progressive redistribution of labeled structures from the sinusoidal area to intermediate and bile canalicular regions of the hepatocyte cytoplasm. Although conjugate-containing structures began accumulating in the bile canalicular region at these early times, no conjugate was present in bile until 20 min. From 7.5 to 45 min after injection ~30% of the labeled structures were in regions that contained Golgi complexes and lysosomes; however, we found no evidence that either organelle contained ~2Sl-plgA-HRP. At least 85% of all positive structures in the hepatocyte were vesicles of 110-160-nm median diameters, with the remaining structures accounted for by tubules and multivesicular bodies. Vesicles in the bile canalicular region tended to be larger than those in the sinusoidal region. Serial sectioning showed that the 1251-plgA-HRP-containing structures were relatively simple (predominantly vesicular) and that extensive interconnections did not exist between structures in the sinusoidal and bile canalicular regions.Receptor-mediated endocytosis is an important cellular process by which macromolecules enter cells. Liver parenchymal cells (hepatocytes) internalize a wide variety of molecules from the circulation via this mechanism; however, once endocytosed, the ligands exhibit diverse intracellular fates. Asialoglycoproteins (41) and epidermal growth factor (6) are taken up by rat hepatocytes and degraded in lysosomes, whereas polymeric leA (picA) 1 is transported from blood to Abbreviations used in this paper: HRP, horseradish peroxidase; PC25-BSA-Sepharose 4B, Sepharose 4B to which 2.9 mg phosphorylcholine25-bovine serum albumin per ml is attached; plgA, polymeric t2SlgA; ~25I-plgA-HRP, conjugate between plgA and HRP; SC, secretory component; TBS, Tris-HCl-buffered saline.bile without degradation (24,27,30,40). The fates of the surface receptors can also vary in hepatocytes. The asialoglycoprotein receptor is reutilized for further rounds of ligand internalization (33, 38), whereas the receptor for epidermal growth factor is degraded when cells are exposed to the hormone (6a). Secretory component (SC), the receptor for plgA, is transported across the cell ...

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BMS-663749, a phosphonooxymethyl prodrug 4 of the HIV-1 attachment inhibitor 2-(4-benzoyl-1-piperazinyl)-1-(4,7-dimethoxy-1H-pyrrolo[2,3-c]pyridin-3-yl)-2-oxoethanone (BMS-488043) (2) was prepared and profiled in a variety of preclinical in vitro and in vivo models designed to assess its ability to deliver parent drug following oral administration. The data showed that prodrug 4 had excellent potential to significantly reduce dissolution rate-limited absorption following oral dosing in humans. Clinical studies in normal healthy subjects confirmed the potential of 4, revealing that the prodrug significantly increased both the AUC and C(max) of 2 compared to a solid capsule formulation containing the parent drug upon dose escalation. These data provided guidance for further efforts to obtain an effective HIV-1 attachment inhibitor.

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