The surface activity of the same subpopulation of galactosyl receptors on isolated rat hepatocytes is modulated by colchicine, monensin, ATP depletion, and chloroquine

In this study, we characterized and compared the ligand-independent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes treated with monensin, chloroquine, microtubule depolymerizing agents, or NaN3 and NaF at 37 degrees C. Freshly isolated hepatocytes exhibit predominately one subset of surface Gal receptors, termed State 1 receptors (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). During equilibration at 37 degrees C, these cells also express a second subset of Gal receptors at the surface, termed State 2 receptors, and routinely double their total surface Gal receptor activity. Following equilibration at 37 degrees C and then inhibitor treatment, hepatocytes bound 40-60% less 125I-asialoorosomucoid (ASOR) at 4 degrees C than did untreated cells. Treated cells maintained a basal nonmodulated level of surface receptor activity regardless of temperature, perturbant concentration, or incubation time. Loss of surface Gal receptor activity on cells treated with multiple inhibitors simultaneously or sequentially was not additive. Thus, all treatments affected the same subpopulation of surface Gal receptors. None of these inhibitors decreased surface State 1 Gal receptor activity, but all prevented the normal appearance of State 2 Gal receptors on freshly isolated cells during incubation at 37 degrees C. The endocytic capability of residual surface State 1 Gal receptors on inhibitor-treated cells varied depending on the inhibitor. Hepatocytes treated first at 24 degrees C or with colchicine at 37 degrees C internalized greater than 85% of surface-bound 125I-ASOR. In contrast, monensin- or chloroquine-treated cells internalized approximately 50% of surface-bound 125I-ASOR. Azide-treated cells internalized less than 20% of surface-bound 125I-ASOR. We conclude that only surface State 2 Gal receptor activity is sensitive to these various perturbants. State 1 Gal receptor activity is not modulated. These data are consistent with the conclusion that only State 2 Gal receptors constitutively recycle.     

Loss of surface galactosyl receptor activity on isolated rat hepatocytes induced by monensin or chloroquine requires receptor internalization via a clathrin coated pit pathway

We studied the effect of hyperosmotic inhibition of the clathrin coated pit cycle on the monensin- and chloroquine-dependent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes. Cells treated for 60 min without ligand at 37 degrees C with 25 microM monensin or 300 microM chloroquine in normal medium (osmolality congruent to 275 mmol/kg) bound 40-60% less 125I-asialo-orosomucoid (ASOR) at 4 degrees C than untreated cells. Cells exposed to monensin or chloroquine retained progressively more surface Gal receptor activity, however, when the osmolality of the medium was increased above 400 mmol/kg (using sucrose as osmolite) 10 min prior to and during drug treatment. Cells pretreated for 10 min with hyperosmolal media (600 mmol/kg) alone internalized less than or equal to 10% of surface-bound 125I-ASOR. Thus, the ligand-independent loss of surface Gal receptor activity on monensin- and chloroquine-treated hepatocytes requires internalization of constitutively recycling receptors via a coated pit pathway.     

Monensin inhibits ligand dissociation only transiently and partially and distinguishes two galactosyl receptor pathways in isolated rat hepatocytes

Monensin has been shown to inhibit the dissociation of internalized asialoorosomucoid (ASOR) from galactosyl (Gal) receptors in hepatocytes (Harford et al., J. Cell. Biol., 96:1824, 1983). Examination of the long-term kinetics of dissociation of a single round of surface-bound 125I-ASOR in the presence of monensin revealed, however, that dissociation resumed after a lag of 30-40 min. Dissociation proceeded slowly with apparent first order kinetics (k = 0.006-0.022 min-1) and reached a plateau after 4 h, both in freshly isolated cells in suspension and in cells cultured for 24 h. Only a portion of the ligand bound to surface Gal receptors was capable of dissociating. The degree of dissociation was correlated with the expression of a subpopulation of receptors we have recently designated as state 1 Gal receptors (Weigel et al., Biochem. Biophys. Res. Commun. 140:43, 1986). The recovery and dissociation of a portion of 125I-ASOR-receptor complexes after the lag period is not due to a depletion of monensin, since a second addition of the drug has no affect once dissociation resumes. Furthermore, as assessed by the accumulation of the fluorescent dye acridine orange, cells have not recovered the ability to acidify intracellular compartments during the time that dissociation occurs. The results support a model for the hepatic Gal receptor system, in which there are two functionally different receptor populations, recycling pathways, and ligand processing pathways. Monensin blocks dissociation of 125I-ASOR from receptors in the major pathway completely. In the minor pathway dissociation proceeds to completion only after a lag. In this minor pathway monensin appears to temporarily delay a maturation or translocation process that must occur prior to dissociation. We conclude that the observed dissociation in the presence of monensin cannot be mediated by low pH, or by pH or pNa gradients.     

Effects of monensin on vesicular transport pathways in the perfused rat liver

In the rat hepatocyte, the internalization and degradation of asialoglycoproteins and the secretion of plasma and biliary proteins require specific intracellular sorting of vesicles. To aid in the biochemical characterization of these different vesicular pathways, we examined the effects of the ionophore monensin on the uptake and degradation of 125I-asialoorosomucoid (ASOR) and on the secretion of plasma and biliary proteins by the in situ perfused rat liver. In control livers, 77% of injected 125I-ASOR was extracted on first pass; 93% of the extracted radioactivity was released back into the circulation (totally degraded and some intact ASOR was found); and approximately 2% was recovered in the bile, some of which was intact. Monensin treatment decreased first pass uptake of 125I-ASOR to 57% and abruptly blocked the release of radioactivity into the perfusate and the bile. When hepatic proteins were biosynthetically labeled with 3H-leucine, monensin treatment dramatically reduced and delayed the secretion of newly synthesized proteins into both the perfusate and the bile. In contrast with control livers, in which secretion of protein into the perfusate preceded secretion of protein into the bile, TCA-precipitable 3H-protein appeared in bile about 20 min before TCA-precipitable 3H-protein appeared in the perfusate in monensin-treated livers. Thus, monensin treatment in the perfused liver blocked the degradation of asialoglycoproteins and inhibited the secretion of plasma proteins but had less effect on biliary protein secretion. These data document physiologic effects of monensin in an intact organ and suggest that biochemical distinctions between different vesicular pathways exist in the rat hepatocyte.     

The hepatic galactosyl receptor system: two different ligand dissociation pathways are mediated by distinct receptor populations

After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs.     

Low concentrations of primaquine inhibit degradation but not receptor-mediated endocytosis of asialoorosomucoid by HepG2 cells

Asialoorosomucoid (ASOR) is internalized and degraded by HepG2 cells after binding to the asialoglycoprotein (ASGP) receptor, internalization through the coated pit/coated vesicle pathway, and trafficking to lysosomes. Primaquine, an 8-aminoquinoline antimalarial compound, inhibits ASOR degradation at concentrations greater than 0.2 mM by neutralizing intracellular acid compartments. This leads to alterations in surface receptor number, receptor-ligand dissociation, and receptor recycling. We have investigated the effects of primaquine on 125I-ASOR uptake and degradation as a function of primaquine concentration and duration of exposure. Concentrations below those required for neutralization of acidic compartments block 125I-ASOR degradation in HepG2 cells and lead to intracellular ligand accumulation. This effect is maximal at 80 microM primaquine. The intracellular 125I-ASOR is undegraded, dissociated from the ASGP receptor, and contained within vesicular compartments distinct from lysosomes, plasma membrane, or endosomes. In addition, the effect of 80 microM primaquine on 125I-ASOR degradation is very slowly reversible (greater than 6 h), in contrast to primaquine's rapidly reversible effect on receptor recycling and ligand uptake (10 min). Furthermore, the effect is ligand-specific. 125I-asialofetuin, another ASGP receptor ligand, is internalized and degraded in lysosomes at normal rates in HepG2 cells exposed to 80 microM primaquine. These findings indicate that primaquine has multiple effects on the uptake and degradation of ligand occurring in the endosome-lysosome pathway. These effects of primaquine differ in their concentration-dependence, site of action, reversibility, and ligand selectivity.     

Vanadate modulates the activity of a subpopulation of asialoglycoprotein receptors on isolated rat hepatocytes: active surface receptors are internalized and replaced by inactive receptors

In the absence of ligand, sodium vanadate causes a time- and dose-dependent loss of up to approximately 50% of the surface galactosyl receptor (GalR) activity in rat hepatocytes at 37 degrees C. The effect on total (surface plus intracellular) GalR activity is also dependent on exposure time and vanadate concentration. At less than 1 mM, vanadate induces a transient decrease and then partial recovery of cell surface GalR activity. At greater than 3 mM vanadate, surface GalR activity decreases rapidly (t1/2 approximately 2 min). Lost surface activity is initially recovered in digitonin-permeabilized cells, indicating that active surface GalRs redistribute to the cell interior. However, an antibody assay for GalR protein showed that although surface activity decreased, there was no decrease in surface receptor protein. The active intracellular GalRs then slowly inactivate over 30-60 min. With 8 mM vanadate, the loss of both surface and total cellular GalR activity is more rapid and coincident; no lag is observed. Maximal activity loss, however, was still only approximately 50%. Again, no net change was seen in the distribution of GalR protein between the cell surface and the interior. These results indicate that vanadate causes active GalRs to move from the surface to the inside and be replaced by inactive receptors moving from the inside to the cell surface. The Gal receptor system is comprised of two functionally different receptor subpopulations that operate via two distinct intracellular pathways. Only the State 2 GalRs, which recycle constitutively, are sensitive to modulation by vanadate. Consistent with this, vanadate inhibits the endocytosis of 125I-asialoorosomucoid (ASOR) only partially. The rate of uptake and the steady state level of ASOR intracellular accumulation were maximally inhibited by 50 and 70%, respectively, at 0.2 mM vanadate. The rate and extent of degradation of 125I-ASOR were also inhibited by 50-70%. Residual ASOR uptake and degradation is accounted for by the minor vanadate-resistant State 1 Gal receptor pathway.     

Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Effects of hyperosmolarity on ligand processing and receptor recycling in the hepatic galactosyl receptor system

Binding, endocytosis, and degradation of asialo-orosomucoid (ASOR) mediated by the galactosyl (Gal) receptor were examined in isolated rat hepatocytes in complete media supplemented with an osmolite. The specific binding of 125I-ASOR to cells at 4 degrees C was unaffected by up to 0.4 M sucrose or NaCl. Unlike sucrose or NaCl, mannitol stimulated 125I-ASOR binding at low concentrations but inhibited binding at higher concentrations. Continuous internalization at 37 degrees C, which requires receptor recycling, was completely blocked at 0.2 M sucrose or 0.15 M NaCl, corresponding in each case to a total osmolality of about 550 mmol/kg. This effect was reversed and endocytic function was restored by washing the cells, indicating that cell viability was unaffected. The rate of degradation of internalized 125I-ASOR was also inhibited by increasing sucrose concentrations. This inhibition is due to a block in the delivery of ligand to lysosomes and not an effect on degradation per se. In the presence of 0.2 M sucrose, the rate and extent of endocytosis of surface-bound 125I-ASOR were, respectively, 33.0 +/- 8.1% and 69.4 +/- 10.5% (n = 8) of the control without sucrose. Under these conditions, the dissociation of internalized receptor-ASOR complexes was completely inhibited. When sucrose was added, the effect on the endocytosis of surface-bound 125I-ASOR was virtually immediate. Previous studies showed that about 40% of the surface-bound 125I-ASOR which is internalized can return to the cell surface still bound to receptor (Weigel and Oka: J Biol Chem 259:1150, 1984). If 0.2 M sucrose was added after endocytosis occurred, 125I-ASOR still returned to the cell surface, although the rate and extent of return were inhibited by more than 50%. Interestingly, hyperosmolarity is the only treatment we have found which can reversibly inhibit, although only partially, the endocytosis of surface-bound 125I-ASOR.     

Studies of the mechanism of cell intoxication by diphtheria toxin fragment A-asialoorosomucoid hybrid toxins. Evidence for utilization of an alternative receptor-mediated transport pathway

In order to study the variables important for determining the cytotoxicity of protein-protein hybrid toxins, the disulfide and thioiether conjugates between diphtheria toxin fragment A (DTA) and asialoorosomucoid (ASOR) were synthesized, purified, and tested for cytotoxicity toward isolated rat hepatocytes as monitored by the inhibition of protein synthesis. After 4-h incubation at 37 degrees C, both DTA-ASOR conjugates were nontoxic, but were highly toxic in the presence of an inhibitor of 125I-ASOR degradation. At concentrations inhibiting 125I-ASOR degradation to the same extent, the efficiency of inhibitors to enhance the toxicity of both conjugates varied with the following rank order: colchicine greater than chloroquine greater than NH4Cl greater leupeptin greater than cytochalasin B. In the absence of an inhibitor, both conjugates, after 10-h incubation, also exhibited measurable toxicity at concentrations as low as 10(-9) M, and DTA-S-S-ASOR was 60 times more toxic than DTA-S-ASOR. In a physiological salt solution containing energy source and bovine serum albumin, both conjugates also exhibited partial inhibition of protein synthesis after 4-h incubation and were enhanced by 2 microM colchicine toward complete inhibition with similar C0.5 of 2 X 10(-10) M. The toxicity of the conjugates were prevented in the presence of excess ASOR. These results not only indicate that intracellular degradation is an important factor restricting the toxicity of the conjugates, but also indicate that the toxic entry of DTA activity from these hybrids is independent of lysosomal degradation. Consequently, it appears that conjugate toxicity is dependent on the release of DTA activity from an extralysosomal compartment whose pool size is increased by the inhibitors and the change of medium. The effects of the inhibitors to inhibit 125I-ASOR uptake and degradation were also studied to facilitate interpretation of their enhancement of conjugate toxicity. Specifically, colchicine inhibited both internalization and degradation of the ligand in a saturable fashion with a C0.5 of 0.5 microM and a maximum inhibition of 65%. The data of the present study are interpreted so as to support a hypothesis that two receptor-mediated pathways are involved in the internalization and transport to lysosomes of ASOR and DTA-ASOR conjugates, only one of which is colchicine-sensitive.     

Recycling of the hepatic asialoglycoprotein receptor in isolated rat hepatocytes. Receptor-ligand complexes in an intracellular slowly dissociating pool return to the cell surface prior to dissociation

We recently reported that the dissociation of internalized receptor-125I-asialo-orosomucoid (ASOR) complexes by isolated hepatocytes is a biphasic process; most complexes dissociate rapidly but 25-50% dissociate slowly (Oka, J. A., and Weigel, P. H. J. Biol. Chem. 258, 10253-10262). Cells were allowed to endocytose a pulse of surface-bound 125I-ASOR, and were washed and then incubated at 37 degrees C in the presence or absence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Without EGTA, very little intact ASOR appeared in the medium. With EGTA present, a large amount of intracellular ligand appeared undegraded in the medium in a time-dependent manner. N-Acetylgalactosamine, but not ASOR, in the medium also caused release of intact 125I-ASOR. Within 15 min, more than 50% and by completion at least 80% of the internalized ligand in the slow dissociation compartment was released into the medium. If cells containing internalized ligand were incubated at 37 degrees C for increasing times before the addition of EGTA, then progressively less ligand accumulated in the medium. Experiments at 18 degrees C, a temperature at which neither degradation nor slow dissociation occurred, demonstrated that in the presence of EGTA the intracellular free 125I-ASOR pool did not change. The amount of receptor-bound ligand in the slowly dissociating pool decreased and the amount of intact ligand in the medium increased by essentially equal amounts. The temperature dependence for the return of internal 125I-ASOR to the cell surface was similar to that for endocytosis, with a cut-off temperature of about 12 degrees C. We conclude that a normal part of the endocytic process involves the return of receptor-ligand complexes to the cell surface from an internal slowly dissociating pool. This might reflect either an obligatory step or a reversible statistically random step in the endocytic/recycling pathway.     

Shunting of insulin from a retroendocytotic pathway to a degradative pathway by sodium vanadate

Adipocytes route internalized insulin through two major pathways, a degradative pathway and a retroendocytotic pathway. To examine whether sorting of incoming insulin-receptor complexes can be altered, we assessed the effect of vanadate on the intracellular processing of both insulin and insulin receptors. After cells were pretreated with vanadate (1 mM for 30 min at 37 degrees C), 125I-insulin was loaded into the cell interior. When the net efflux of insulin from cells into the medium was then monitored, vanadate was found to slow the efflux of insulin from a t1/2 of 6.2 min (controls) to 11 min. Since efflux reflects both the rapid extrusion of intact insulin and the slower release of degradative products, we proposed that vanadate diverts more insulin into the degradative pathway. Further evidence in support of this idea included the following: 1) when intracellular degradation of insulin was impaired by chloroquine, undegraded insulin accumulated faster within vanadate-treated cells, consistent with greater flux through a degradative pathway; 2) vanadate increased the percentage of degraded insulin released from cells from 61 and 72%; and 3) under steady-state binding conditions, more insulin resided in the cell interior of vanadate-treated cells (44.8% versus 34.5%), and the time required for the intracellular pool to reach equilibrium was prolonged (t1/2 of 5.5 min versus 4.0). Neither insulin internalization nor degradation was impaired by vanadate alone. In related studies Tris was found to inhibit insulin-mediated receptor recycling by only 10%, whereas in the presence of vanadate (plus Tris) almost all incoming insulin receptors were prevented from recycling. Vanadate alone had no effect on the ability of insulin receptors to recycle. Based on these results we conclude that: 1) vanadate shunts incoming insulin from a more rapid retroendocytotic pathway to a slower degradative pathway and diverts insulin receptors from a Tris-insensitive recycling pathway to one that can be completely inhibited by Tris; 2) these effects are selective, in that vanadate impairs neither insulin degradation nor receptor uptake and recycling. Considered together, these findings support the idea that a sorting mechanism exists for the intracellular routing of incoming insulin-receptor complexes.     

Total cellular activity and distribution of a subpopulation of galactosyl receptors in isolated rat hepatocytes are differentially affected by microtubule drugs,monensin, low temperature,and chloroquine

We studied the effects of low temperature (20–37°C), monensin, chloroquine, and microtubule drugs on the cellular distribution and activity of galactosyl (Gal) receptors in isolated rat hepatocytes. After equilibration at 37°C, hepatocytes were incubated at 37°C, 31°C, 25°C, or 20°C or treated with or without inhibitors at 37°C in the absence of ligand. The cells were then assayed at 4°C for ¹²⁵I-asialo-orosomucoid binding, to measure receptor activity, or ¹²⁵I-anti-Gal receptor IgG binding, to measure receptor protein. Surface or total (surface and intracellular) Gal receptor activity and protein were measured on intact or digitonin-permeabilized cells, respectively. These inhibitors fell into two categories. Type I inhibitors (sub-37°C temperatures or colchicine) induced receptor redistribution but not inactivation. Treated cells lost up to 40% of surface Gal receptor activity and protein. Lost surface receptors were recovered intracellularly with no loss of receptor activity. Type II inhibitors (monensin or chloroquine) induced receptor inactivation but not redistribution. Treated cells lost 50–65% of their surface Gal receptor activity but only ? 15% of their surface receptor protein. These cells lost up to 60% of total cellular Gal receptor activity with no loss of total receptor protein. Of the total inactive Gal receptors, up to 50% and75%, respectively, were present intracellularly in monensin-and chloroquine-treated cells. Loss of ligand binding to permeable treated cells was not due to changes in receptor affinity. A third category, Type III inhibitors (metabolic energy poisons that deplete ATP) induce both Gal receptor redistribution and inactivation (Biochemistry 27:2061, 1988). We conclude that only one of the two previously characterized subpopulations of Gal receptors on hepatocytes, termed State 2 receptors (J Biol Chem 265:629, 1990), recycles constitutively. The activity and distribution of State 2 but not State 1 Gal receptors are differentially affected by these specific drugs or treatments.     

Dual pathways for the intracellular processing of insulin. Relationship between retroendocytosis of intact hormone and the recycling of insulin receptors

Adipocytes process insulin through either of two pathways: a retroendocytotic pathway that culminates in the release of intact insulin, and a degradative pathway that terminates in the intracellular catabolism and release of degraded ligand. Mechanistically, these pathways were found to differ in several ways. First, temporal differences were found in the rate at which intact and degraded products were extruded. After 125I-insulin was preloaded into the cell interior, intact ligand was completely released during the first 10 min (t 1/2 = 2 min), whereas degraded insulin was released at a much slower rate over 1 h (t 1/2 greater than 8 min). Secondly, it was found that chloroquine profoundly inhibited the insulin degradative pathway, resulting in the intracellular accumulation of intact ligand and a reduction in the release of degraded products. In contrast, however, chloroquine was without effect on the retroendocytotic processing of insulin. Based on the known actions of chloroquine, it appears that retroendocytosis of insulin does not involve vesicular acidification or dissociation of the insulin-receptor complex and that insulin is most likely carried to the cell exterior in the same vesicles (either receptor-bound or free) as those mediating recycling receptors. Interestingly, accumulation of undergraded insulin within chloroquine-treated cells did not result in the release of additional intact ligand, suggesting that once insulin enters the degradative compartment it is committed to catabolism and cannot exit the cell through the retroendocytotic pathway. A third difference was revealed by the finding that extracellular unlabeled insulin (100 ng/ml) markedly accelerated the rate at which preloaded 125I-insulin was released from adipocytes (t 1/2 of 3 min versus 7 min in controls cells). Analysis of the composition of the released products revealed that extracellular insulin rapidly augmented (over 10 min) in a dose-dependent manner (5-200 ng/ml) the amount of insulin released intact (from 25 to 38% of preloaded counts; insulin ED50 = 10 ng/ml). Although extracellular insulin had no effect on the early extrusion of degraded insulin, the release of catabolized products was reduced at later times. The interpretation of these results is that the rate or amount of incoming insulin-receptor complexes can effect a sorting process (prior to bifurcation) such that a proportion of insulin is shunted from the slower degradative pathway to the more rapid retroendocytotic pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Asialoorosomucoid hepatobiliary transport is unaltered by the loss of liver asialoglycoprotein receptors in aged rats

The hepatobiliary transport of asialoorosomucoid (ASOR) was examined in aging male Fischer 344 rats. The time course of transport of 125I-ASOR from blood to bile was identical in both senescent and young adult rats. Peak secretion occurred at approximately 35 minutes after injection via the femoral vein. Total secretion of radiolabeled ASOR (3.6% of injected dose), bile secretion and rate of secretion of radiolabeled ligand (approximately 2% of administered dose/hr/gm bile/liver) were not significantly different for the two age groups. Determination of the binding capacity for 125I-ASOR with liver plasma membrane-enriched preparations showed the membranes from old animals capable of binding approximately 50% less radiolabeled ligand as the young adult animals. Analysis of the distribution of 125I-ASOR autoradiographic grains along the liver lobule indicated extensive uptake of ligand in Zone 2 and 3 cells in senescent animals, whereas uptake in young rats was essentially limited to Zone 1 parenchymal cells. These results indicate that, contrary to the age-related loss of hepatic receptors for dimeric IgA and the concomitant reduction in hepatobiliary secretion of IgA, loss of ASOR binding capacity on liver plasma membranes from old animals is not reflected in diminished hepatobiliary secretion of ASOR. The loss of ASOR binding capacity is offset by the recruitment of Zone 2 and 3 hepatocytes along the liver lobule. This result suggests that hepatic metabolism and hepatobiliary secretion of macromolecules which exhibit a lobular gradient of uptake (e.g. ASOR) will be relatively less affected by loss of receptors compared to ligands which do not display such a gradient (e.g. IgA).     

Influence of the N-linked oligosaccharides on the biosynthesis, intracellular routing, and function of the human asialoglycoprotein receptor

The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein of 46,000 Da which possesses two N-linked oligosaccharide chains (Schwartz, A. L., and Rup, D. (1983) J. Biol. Chem. 258, 11249-11255). In order to examine the role of N-linked oligosaccharides in the biosynthesis, intracellular routing, and function of the ASGP-R, we have used Hep G2 cells, which have a large number of ASGP-R, and two inhibitors of glycosylation, swainsonine and tunicamycin. In the presence of swainsonine, newly synthesized ASGP-R is a 43,000-Da species which is endoglycosidase H-sensitive, appears on the Hep G2 cell surface, and specifically binds 125I-asialoorosomucoid (ASOR). In the presence of tunicamycin newly synthesized ASGP-R is a 34,000-Da nonglycosylated species which appears on the Hep G2 cell surface where it specifically binds 125I-ASOR. There is no major effect on subsequent uptake and degradation of 125I-ASOR in cells whose ASGP-R was synthesized in the presence of tunicamycin. The turnover of ASGP-R synthesized in the presence of either swainsonine or tunicamycin is not significantly altered from that found for the normal 46,000-Da species. Thus, it appears that the two N-linked oligosaccharide chains of the human ASGP-R do not play a major role in the intracellular routing, turnover, or function of ASGP-R.     

ATP-dependent inactivation and reactivation of constitutively recycling galactosyl receptors in isolated rat hepatocytes

Isolated rat hepatocytes depleted of ATP with NaN3 without ligand lose galactosyl (Gal) receptors from the cell surface and accumulate inactive receptors within the cell [McAbee, D. D., & Weigel, P. H. (1987) J. Biol. Chem. 262, 1942-1945]. Here, we describe the kinetics of receptor redistribution and inactivation after ATP depletion with NaN3 and of receptor redistribution and reactivation after ATP recovery. Only intact cells (greater than 98% viable) isolated from Percoll gradients were assayed. Gal receptor activity and protein were measured by the binding of 125I-asialoorosomucoid (125I-ASOR) and 125I-anti-Gal receptor IgG (125I-IgGR), respectively, at 4 degrees C. Surface and total (surface and intracellular) cellular Gal receptors were measured in the absence or presence, respectively, of digitonin. Following ATP depletion, 60-70% of Gal receptor activity and protein were lost from cell surfaces with first-order kinetics (t1/2 = 6.5 min, k = 0.107 min-1) at an initial rate of 11,000 125I-ASOR binding sites cell-1 min-1. Lost cell-surface Gal receptors were transiently recovered still active inside the cell. After a short lag, total cellular receptor inactivation then proceeded with first-order kinetics (t1/2 = 13 min, k = 0.053 min-1) at an initial rate of 14,000 125I-ASOR binding sites cell-1 min-1. Up to half of all cellular Gal receptors were inactivated by 40 min. 125I-IgGR binding to NaN3-treated, permeable cells, however, was virtually constant. The distribution of total cellular receptors changed from 35% on the cell surface initially to 10% after 40 min of ATP depletion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.