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P H Weigel

Publications and source records attributed to P H Weigel.

At least 91 records · Page 5Linked to original sources
PubMed1987

Effects of hyaluronic acid and other glycosaminoglycans on fibrin polymer formation.

Previously, we reported that the glycosaminoglycan (GAG) hyaluronic acid (HA) specifically bound to the plasma protein fibrinogen [LeBoeuf, R. D., Raja, R. R., Fuller, G. M., & Weigel, P. H. (1986) J. Biol. Chem. 261, 12586]. The binding of other macromolecules to fibrinogen could influence the conversion of fibrinogen to fibrin. Therefore, we tested whether HA and other GAGs could alter the kinetics of fibrin polymer formation and the physical structure of the resulting gel. In this study, we present data showing that the GAGs HA and chondroitin sulfate (CS) affect fibrin formation in three specific ways: (i) they decreased the clotting time of fibrinogen 3-10-fold; (ii) both GAGs increase significantly the rate of fibrin polymer formation; and (iii) fibrin gels containing HA or CS had a final A450 that was greater than controls, indicating that these two glycosaminoglycans influence either the final size of fibrin fibrils or the extent of the lateral association between fibrils. These results demonstrate that the interactions of HA and CS with forming fibrin polymers can alter both the kinetics of formation and may produce structural changes in fibrin gels.

Animals↗
PubMed1987

ATP depletion causes a reversible redistribution and inactivation of a subpopulation of galactosyl receptors in isolated rat hepatocytes.

Isolated rat hepatocytes, treated with metabolic energy poisons such as NaN3 in the absence of exogenous ligand, lose surface galactosyl (Gal) receptor activity (Clarke, B. L., and Weigel, P. H. (1985) J. Biol. Chem. 260, 128-133). We have used 125I-labeled asialo-orosomucoid and affinity-purified anti-receptor IgG to quantitate, respectively, the activity and the amount of Gal receptor protein. Cells were treated with NaN3 at 37 degrees C and the surface or total (surface and intracellular) binding of these two probes was measured at 4 degrees C, respectively, in intact cells or in cells permeabilized with digitonin. As a function of NaN3 concentration, both surface receptor activity and protein decreased in parallel by 50-80%. Virtually all of the lost surface receptor protein was found inside the cell, but only about 50% of all cellular Gal receptors were active. As determined by equilibrium binding studies, this decreased receptor activity reflected an overall loss of ligand binding sites with little change in binding affinity of the remaining Gal receptors for asialo-orosomucoid. When ATP was restored, normal surface receptor activity and number completely recovered even in the absence of protein synthesis. We conclude that a subpopulation of Gal receptors constitutively recycles and undergoes an inactivation/reactivation cycle. In the absence of ligand, these receptors are normally internalized and then inactivated. Loss of cellular ATP blocks receptor reactivation, prevents the reappearance of receptors at the cell surface and redistributes Gal receptors as inactive receptors accumulate intracellularly.

Adenosine Triphosphate↗
PubMed1987

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Human fibrinogen specifically binds hyaluronic acid.

Fibrin and hyaluronic acid (HA) are macromolecules whose concentrations are elevated at the same time in the extracellular space of damaged tissues. We have investigated whether HA can bind to fibrinogen using solid phase and soluble assays. Purified human fibrinogen specifically bound to HA-Sepharose to a greater extent (greater than 5-fold) than did alpha 1-acid glycoprotein, DNaseI, ovalbumin, haptoglobin, or lysozyme. Fibrinogen did not bind to ethanolamine-Sepharose, a control chromatographic support. Treatment of HA-Sepharose containing bound 125I-fibrinogen with ovine testicular hyaluronidase released 44% of the 125I radioactivity, indicating that fibrinogen was specifically bound to HA. Moreover, 125I-fibrinogen bound to HA-Sepharose could be displaced by free HA but not by either of the monosaccharide components of this polymer, glucuronic acid, or N-acetylglucosamine. Chondroitin sulfate and polygalacturonic acid competed only weakly for bound 125I-fibrinogen. Bound 125I-fibrinogen was also not released by high concentrations of NaCl (up to 4 M), indicating that the interaction is not simply ionic. The apparent affinity of fibrinogen for HA covaried with the molecular weight of the HA. Small HA oligosaccharides (Mr = 3900) were only 50% as effective as larger HA (Mr = 8 X 10(5)) in eluting bound 125I-fibrinogen from HA-Sepharose. The optimal oligosaccharide size for displacement of bound 125I-fibrinogen was greater than or equal to 200 monosaccharides. Additionally, the amount of 125I-fibrinogen bound to HA-Sepharose was directly related to the size of the HA-amine linked to the affinity support. The affinity constant for fibrinogen binding to 125I-HA (approximately 150 monosaccharides) is estimated to be at least 2 X 10(7) M-1. These results demonstrate for the first time a specific, reversible binding between HA and fibrinogen.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Preparation and use of synthetic cell culture surfaces. A new reagent for the covalent immobilization of proteins and glycoproteins on a nonionic inert matrix.

To study cell interactions with external molecules immobilized on a chemically defined nonionic, inert matrix, we have prepared flat polyacrylamide matrices containing covalently attached carbohydrate or protein. A new acrylamide derivative, containing a terminal 1,2-dihydroxy group, was synthesized and then copolymerized with acrylamide and bisacrylamide to make 20% polyacrylamide matrices, which could be oxidized with NaIO4 to generate reactive aldehyde groups. Molecules containing a free amine (e.g. proteins or glycopeptides) can be coupled to the aldehyde-activated matrix by formation of a Schiff base and reduction with NaCNBH3 to form a stable -CH2-NH-bond. Unreacted aldehyde groups are reduced to hydroxyl groups with NaBH4. In order to immobilize polysaccharides on the activated surfaces, these molecules are first modified to contain a free amine. We have described a procedure to convert purified hyaluronic acid oligosaccharides to a reactive alkylamine derivative uniquely modified at the reducing end (Raja, R. H., LeBoeuf, R. D., Stone, G. W., and Weigel, P. H. (1984) Anal. Biochem. 139, 168-177). The covalent attachment of [3H]hyaluronate-amine, [14C]ethanolamine, or 125I-bovine serum albumin, to activated surfaces was complete within 5-24 h. The amount immobilized was directly proportional to the amine concentration and to the aldehyde content of the matrix and inversely proportional to the molecular weight of the amine. About 90% of the available aldehyde groups reacted with ethanolamine, whereas less than 0.01% reacted with albumin. Molecules larger than 3300 Da were excluded from the interior of the matrix and could therefore only be attached to the surface of the matrix. These synthetic surfaces can be used in long term culture experiments to study cellular interactions with virtually any type of immobilized molecule.

Acrylic Resins↗
PubMed1986

A model for the role of hyaluronic acid and fibrin in the early events during the inflammatory response and wound healing.

A model is presented outlining the molecular and cellular events that occur during the early stages of the wound healing process. The underlying theme is that there is a specific binding interaction between fibrin, the major clot protein, and hyaluronic acid (HA), a constituent of the wound extracellular matrix. This binding interaction, which could also be stabilized by other cross-linking components, provides the driving force to organize a three-dimensional HA matrix attached to and interdigitated with the initial fibrin matrix. The HA-fibrin matrix plays a major role in the subsequent tissue reconstruction processes. We suggest that HA and fibrin have both structural and regulatory functions at different times during the wound healing process. The concentration of HA in blood and in the initial clot is very low. This is consistent with the proposed interaction between HA and fibrin(ogen), which could interfere with either fibrinogen activation or fibrin assembly and cross-linking. We propose that an activator (e.g. derived from a plasma precursor, platelets or surrounding cells) is produced during the clotting reaction and then stimulates one or more blood cell types to synthesize and secrete HA into the fibrin matrix of the clot. We predict that HA controls the stability of the matrix by regulating the degradation of fibrin. The new HA-fibrin matrix increases or stabilizes the volume and porosity of the clot and then serves as a physical support, a scaffold through which cells trapped in the clot or cells infiltrating from the peripheral edge of the wound can migrate. The HA-fibrin matrix also actively stimulates or induces cell motility and activates and regulates many functions of blood cells, which are involved in the inflammatory response, including phagocytosis and chemotaxis. The secondary HA-fibrin matrix itself is then modified as cells continue to migrate into the wound, secreting hyaluronidase and plasminogen activator to degrade the HA and fibrin. At the same time these cells secrete collagen and glycosaminoglycans to make a more differentiated matrix. The degradation products derived from both fibrin and HA are, in turn, important regulatory molecules which control cellular functions involved in the inflammatory response and new blood vessel formation in the healing wound. The proposed model generates a number of testable experimental predictions.

Blood Coagulation↗
PubMed1986

Binding and spreading of hepatocytes on synthetic galactose culture surfaces occur as distinct and separable threshold responses.

Isolated rat hepatocytes bind to synthetic flat polyacrylamide matrices containing covalently attached galactose residues in a sugar-specific and concentration-dependent manner. Cell binding is mediated by the asialoglycoprotein receptor and occurs as a threshold response at or above a critical concentration of sugar in the matrix. Hepatocytes in the presence or absence of serum were able to spread on these synthetic galactose surfaces and were morphologically similar to cells on tissue culture plastic. Cell spreading also occurred as a threshold response but at a much higher critical concentration of sugar than for the cell-binding response. Above the critical concentration for spreading, the area occupied by a cell increased as the sugar concentration increased. By manipulating the galactose content of the matrix, cell spreading and cell binding can be differentiated as independent and separable threshold responses to the extracellular substratum.

Acrylic Resins↗
PubMed1985

A new competition assay for the solubilized hepatic galactosyl receptor.

The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total 125I-asialo-orosomucoid (125I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both 125I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the 125I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described.

Animals↗
PubMed1985

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.

Adenosine Triphosphate↗
PubMed1984

Preparation of alkylamine and 125I-radiolabeled derivatives of hyaluronic acid uniquely modified at the reducing end.

Present procedures to obtain radiolabeled hyaluronic acid derivatives are limited to low-specific-activity isotopes and small amounts of material, and often involve multiple points of chemical modification within the polymer. A synthesis has been developed which affords large quantities of a unique, chemically modified derivative of hyaluronic acid containing a single hydroxyphenyl group at the reducing end, which can be radioiodinated to high specific activity. Very little alteration in oligosaccharide structure is expected since only the terminal reducing sugar is modified. Oligosaccharides of hyaluronic acid, which have no free amino groups, were first converted to alkylamine derivatives to allow subsequent reaction with the Bolton-Hunter reagent, N-succinimidyl-3(4-hydroxyphenyl)propionate. Synthesis of the hyaluronate-amine was achieved by (i) reduction of the terminal reducing sugar with sodium borohydride, (ii) controlled sodium periodate oxidation to generate an aldehyde group only at the reduced end, and (iii) coupling this aldehyde to an alpha,omega- alkyldiamine (e.g., 1,6- hexanediamine ) in the presence of sodium cyanoborohydride. Purified hyaluronate-amine oligosaccharides were then reacted with the Bolton-Hunter reagent, and the hydroxyphenyl derivative thus obtained was radioiodinated with Na125I. Specific activities up to 8 X 10(9) cpm/nmol oligosaccharide can be obtained. This approach yields a uniquely modified, highly radioactive probe which will be useful in studies of cellular and extracellular matrix interactions with hyaluronic acid. In addition, the uniquely modified alkylamine derivative of hyaluronic acid has been used to prepare affinity chromatography media and synthetic cell culture surfaces.

Amines↗
PubMed1984

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.

Acetylgalactosamine↗
PubMed1983

Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes.

Microtubule-depolymerizing drugs, such as colchicine, vinblastine sulfate, colcemide and podophyllotoxin, cause an apparent inhibition of the ability of rat hepatocytes to degrade asialo-orosomucoid. However, the binding of asialo-orosomucoid to the cell surface at 0 degrees C, the endocytosis of pre-bound glycoprotein at 37 degrees C, and the dissociation of internal receptor-glycoprotein complexes are unaffected by these microtubule drugs. Receptor recycling is slowed but still occurs, although degradation is blocked. The rate of degradation is decreased by low concentrations of drugs. (For example, 0.25 microM vinblastine sulfate, colchicine and colcemide inhibited 93%, 79% and 26%, respectively.) Neither beta- nor gamma-lumicolchicine affected any of the processes examined. The degree of inhibition with colchicine could be enhanced by a brief treatment of the cells at low temperature to depolymerize microtubules. However, if cells were allowed to endocytose asialo-orosomucoid at 37 degrees C prior to addition of the microtubule drug, then the inhibition of protein degradation was greatly reduced. The decrease in the inhibition of degradation was proportional to the amount of time that cells were exposed to asialoglycoprotein before addition of the drug. The results indicate that the segregation of protein from receptor after they dissociate and/or the subsequent translocation of internalized asialoglycoprotein from the cell perimeter to the lysosomal region requires intact microtubules.

Animals↗
PubMed1983

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. Dissociation of internalized ligand from receptor occurs in two kinetically and thermally distinguishable compartments.

We have examined the rate of dissociation of internalized 125I-asialo-orosomucoid-receptor complexes in freshly isolated rat hepatocytes. Cell suspensions were washed with ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 0 degrees C to remove surface-bound ligand and then assessed for the retention of radioactive glycoprotein in the presence of digitonin, which permeabilized the cells and released the internal soluble contents. In cells which initially contained only surface-bound ligand, about 50% of the internalized ligand dissociated from receptor very rapidly (t1/2 less than or equal to 2.5 min, k greater than or equal to 0.28 min-1), at 37 degrees C, whereas the other 50% dissociated more slowly with apparent first order kinetics (t1/2 = 50 min, k = 0.014 min-1). This equal distribution of internalized ligand into two compartments, from which dissociation occurred with very different kinetics, did not depend on the extent of surface receptor occupancy and also occurred under non-steady state conditions of continuous exposure to ligand. Ligand entering both the rapid and slow dissociation compartments was eventually degraded with apparent first order kinetics (k = 0.0047 min-1), suggesting that the intracellular routing of ligand to lysosomes after dissociation from either compartment was via the same pathway. The fast and slow dissociation of receptor-ligand complexes were also distinguished by different temperature sensitivities; the slow dissociation process ceased below 18 degrees C, whereas the fast dissociation process still proceeded. The equal partition of internalized complexes into the two kinetic compartments did not change as a function of temperature but did change as cells continued to endocytose asialo-orosomucoid at 37 degrees C. As the internal receptor pool approached a steady state level of occupancy, there was an increase in the average time for receptor recycling and an increase in the fraction of incoming receptor-ligand complexes which dissociated rapidly (approximately 75%). In addition, under steady state conditions, the rate of the slow dissociation process increased (k = 0.026 min-1, t1/2 = 27 min).

Animals↗
PubMed1983

The surface content of asialoglycoprotein receptors on isolated hepatocytes is reversibly modulated by changes in temperature.

We have investigated the effect of temperature on the content of surface asialoglycoprotein receptors on isolated rat hepatocytes. Receptor was determined by measuring the specific binding of 125I- or [3H] asialo-orosomucoid at 0 degrees C. As reported previously, the receptor number/cell increases 2-3-fold within 30-60 min when freshly isolated cells are warmed from 0-37 degrees C (Weigel, P. H. (1980) J. Biol. Chem. 255, 6111-6120). This increase in receptor number is not inhibited by cycloheximide and also occurs on cells which have first been treated with EDTA to expose a population of cryptic receptors on the cell surface. The rate and extent of the receptor number increase on the cell surface are proportional to the temperature above about 17 degrees C. If cells are first equilibrated at 37 degrees C and then transferred to a lower temperature, the surface receptor number decreases at a rate and to an extent dependent on the temperature. The surface receptor number can be modulated up and down by successive temperature change cycles between 25 and 37 degrees C. In this temperature range, the number of surface receptors/cell is dependent on the final temperature but independent of the pathway to that temperature and is, therefore, a function of state with respect to temperature. The results demonstrate that temperature changes reversibly modulate the number of receptors on the hepatocyte surface. We conclude that, in the absence of ligand, surface receptors can either recycle or can be reversibly internalized or sequestered to prevent access to ligand. The results may also explain why different laboratories have reported a wide range of values for the number of receptors per hepatocyte.

Animals↗
PubMed1983

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes.

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.

Animals↗
PubMed1983

Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells.

The distribution of membrane-bound receptors and enzymes between the cell surface and the cell interior can be determined without solubilization or gross disruption of cell organelles in the presence of the nonionic detergent digitonin. This steroid glycoside permeabilizes cells, releases cytoplasmic proteins with subunit molecular weights up to 200,000, and allows exogenous molecules to gain access to intracellular receptors. All cell types examined were affected similarly by digitonin. Permeabilization was complete within 2 min at 0 degree C and did not require the continued presence of digitonin. A characteristic amount of protein (approximately 50%) was lost between 0.02 and 0.08% (w/v) digitonin. Three independent systems were examined: the insulin receptor in 3T3 fibroblasts and the asialoglycoprotein receptor and the Na+/K+-ATPase in rat hepatocytes. In each case an increase in the specific activity of enzyme/receptor occurred over a range of detergent concentration in which the retention of cell protein was constant and virtually no solubilization of membrane-bound activity occurred. The binding of 125I-asialo-orosomucoid to rat hepatocytes at 0 degree C in the presence of digitonin was linear with cell number and kinetically indistinguishable from binding to intact cells. Receptors exposed by digitonin were shown to be intracellular by light microscopic examination of permeabilized cells first treated with antiserum to the receptor and then with a second antibody horseradish peroxidase conjugate. The use of digitonin has many advantages over procedures which require total cell disruption or solubilization to assess intracellular receptors. The technique has already been valuable in studies on recycling and endocytosis mediated by the asialoglycoprotein receptor (P.H. Weigel and J.A. Oka (1983) J. Biol. Chem. 258, 5095-5102) and should also be useful in studies with other membrane-bound receptors and enzymes in other cell types.

Animals↗