BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
46,
152-168 (1991)
Glucocorticoid Hepatopathy: Effect on Receptor-Mediated Endocytosis of Asialoglycoproteins’ MARK S. KUHLENSCHMIDT,~‘~
WALTER
E.
HOFFMANN,~
AND MARIAN
K.
KLPPY
Department of Pathobiology, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Avenue, Urbana, Illinois 61801 Received December 26, 1990; and in revised form May 8, 1991 Histologic and electron microscopic examination of liver tissue from ghrcocorticoid-treated dogs (GT dogs) showed a markedly abnormal hepatocellular morphology which consisted of severe hepatocellular swelling, vacuolation, and peripheral displacement of subcellular organelles. The abnormal cell morphology was typical of that seen in clinical cases of canine Cushing’s Syndrome. The hepatocyte isolation procedure used here works equally well for the preparation of viable hepatocytes from both normal and GT dogs even though GT dogs displayed a pronounced hepatopathy. Cell yields (lo9 cells from a 30-cm3 section of liver) are similar to those reported for rat hepatocytes using whole liver in situ perfusion and cell viability is routinely greater than 85%. The isolation procedure preserved the “abnormal” state or swollen morphology of the hepatocytes from GT dogs and thus can be used in pathophysiological studies of glucocorticoid-induced hepatopathy. The isolated hepatocytes were 3.2 times greater in cell volume than normal hepatocytes. We also observed over a 12.3-fold increase in alkaline phosphatase activity and the appearance in both the liver and the serum of GT dogs of the unique, cortiocosteroid alkaline phosphatase isozyme (CALP). In spite of the obvious abnormal liver morphology and elevated serum and liver alkaline phosphatase activities, the function of the hepatic cell surface carbohydrate binding protein, the Gal/GalNAc or asialoglycoprotein receptor, was not impaired. We found a trend of about a 1.5fold increase in the initial rate of ligand uptake as well as l&fold more receptors on GT dog hepatocytes compared to normal hepatocytes. The ligand binding affinity of these receptors, as well as the rate of ligand degradation, was identical in hepatocytes isolated from normal and diseased dogs. When intestinal alkaline phosphatase (IALP) is used as the ligand, approximately 25% was exocytosed intact following endocytosis. These results demonstrate that dogs with ghrcocorticoid hepatopathy possess a normally functioning Gal/GalNAc receptor. Furthermore, these data are consistent with the hypothesis that structurally related IALP and CALP isozymes may also be metabolically related through the Gal/GalNAc receptor endocytosis pathway. That is, a portion of the IALP normally endocytosed through the Gal/GalNAc receptor pathway in ghrcocorticoid-treated dogs may be recycled and converted (hyperglycosylated) to the abnormal serum CALP isozyme rather than being degraded. Q 1991 Academic Press, Inc. ’ This project was supported by a Biomedical Research Support Grant (USPHS) and a grant from the Morris Animal Foundation. * Equal contribution of these authors. 3 To whom reprint requests should be addressed. 152 0885-4505/91 $3.08 Copyright 0 1991 by Academic Press.. Inc. All rights of reproduction in any form reserved.
ENDOCYTOSIS
IN STEROID
HEPATOPATHY
153
A common syndrome in dogs, glucocorticoid hepatopathy, is caused by an excess of endogenous or exogenous glucocorticoids. The disease is characterized by pronounced alterations in hepatocyte morphology involving severe hepatocellular swelling, vacuolation, accumulation of cytoplasmic glycogen, marked displacement of subcellular organelles, dramatic increases in serum alkaline phosphatase activity (l-3) and the appearance in the liver and the serum of a unique form of intestinal alkaline phosphatase, known as the corticosteroid alkaline phosphatase (CALP) (3-7). We have recently shown CALP to be a highly glycosylated form of the canine intestinal alkaline phosphatase isozyme (IALP) (8). Although some biochemical and hepatocellular morphological anomalies are well documented, their effects on hepatocyte function are not known. The asialoglycoprotein receptor (Gal/GalNAc binding protein), the major carbohydrate binding protein of liver, is uniquely located on the sinusoidal membrane of hepatocytes in a variety of mammalian species (9-11) but has not been characterized, to our knowledge, in dog hepatocytes. Various forms of liver disease are known to have deleterious effects on this receptor activity (12-17). However, its activity has not been examined in hepatocytes isolated from dogs with glucocorticoid hepathopathy. A known function of this receptor is to remove asialoglycoproteins from the circulation by receptor-mediated endocytosis, transport them intracellularly in endocytic vesicles, and ultimately deliver them to lysosomes for degradation while simultaneously preserving the receptor by recycling it back to the plasma membrane to continue the cycle (9,lO). The overall process of ligand sorting, routing, and receptor recycling is an intricate and complicated one involving multiple steps, several endocytic vesicles, and interactions with various subcellular organelles (18-20). Thus, by quantitative measurement of ligand flux through this receptor pathway we have been able to simultaneously evaluate the integrity of several hepatocyte functions in dogs with glucocorticoid hepatopathy. In this report we describe: (1) the isolation of large numbers of viable, functional hepatocytes from both normal and GT dogs, (2) an evaluation of the kinetic parameters of the asialoglycoprotein receptor in normal dogs and their comparison to those described in other species, (3) an evaluation of the asialoglycoprotein receptor mediated endocytosis pathway in hepatocytes from GT dogs using both synthetic neoglycoproteins and native dog IALP as ligands, and (4) measurement of the rate of in vivo and in vitro clearance of IALP by the asialoglycoprotein receptor pathway. MATERIALS AND METHODS All reagents were purchased from Sigma Chemical Co., St. Louis, Missouri, unless otherwise specified. Experimental induction of glucocorticoid hepatopathy. All dogs were housed in ACLAM-approved cages in the College of Veterinary Medicine, Small Animal Clinic, University of Illinois. All experiments involving dogs have conformed to both the National Research Council’s and the University of Illinois, College of Veterinary Medicine, Laboratory Animal Care Committee’s criteria for the care and use of laboratory animals in research. Glucocorticoid hepatopathy was induced in normal dogs (20- to 40-lb beagles presenting with normal serum chemistry
154
KUHLENSCHMIDT,
HOFFMANN,
AND RIPPY
profiles and liver function tests) by utilizing 2.0-ml Alzet osmotic pumps (Alza Corp., Palo Alto, CA) containing either 2 g of cortisol-21 phosphate in sterile water or sterile water alone. General anesthesia was induced by halothane mask and the osmotic pumps were surgically placed in a subcutaneous pocket and the output directed by intramedic tubing into the left branch of the jugular vein. The pumps delivered cortisol phosphate (n = 5) or sterile water (n = 4) at a constant rate of 2.7 ml/h for 30 days. All dogs were fed once daily (standard dry dog food, Wayne Foods, Inc.) and given water ad lib&m. Dogs treated for 30 days with the pumps containing cortisol phosphate displayed an average serum cortisol concentration of 223 + 49.1 rig/ml as compared to 20 -e 12.9 rig/ml for control dogs receiving sterile water. Measurement of serum cortisol. Cortisol determinations were performed using a solid-phase radioimmunoassay (Micromedic Systems, Inc., Horsham, PA) per the manufacturer’s instructions and using ‘251-cortisol as a standard. The sensitivity of the assay was 0.3 pg/dl with intraassay and interassay coefficients of variation of 4.2 and 3.9%, respectively. Isolation of hepatocytes. Hepatocytes were isolated by modifications of a previously described collagenase perfusion technique (21-24). Dogs were euthanized by an overdose of a sodium pentobarbital and their abdomens were clipped, surgically scrubbed, and then immediately opened. The entire liver was removed with care to ensure that the left lateral lobe was not injured. This lobe was cut free from the rest of the liver and packed in crushed ice for transport to the laboratory. The perfusion was begun within 30 min of removal of the liver. The hepatic vein was cannulated with G-in.-i.d. Tygon tubing and the lobe initially perfused at 80 ml/min with 800 ml of 10 mM Hepes buffer (pH 7.4) containing 142 mM NaCl, 6.7 mM KCl, and 2 mM EGTA (Buffer 1). All of this perfusate was allowed to run into a waste container. The lobe was then perfused with 120 mg of collagenase (CLS 2, 125 units/mg, Lot 67237M, Worthington Biochemical Corp.) in 150 ml of 100 mM Hepes buffer (pH 7.4) containing 67 mM NaCl, 6.7 mM KCl, 6.3 mM CaC12, and 15 g/liter bovine serum albumin (Buffer 2). This buffer was allowed to recirculate through the liver for 15 min or until the liver capsule began to tear; the lobe was perfused for an additional 2 min with Buffer 1. All buffers were maintained at 37°C throughout perfusion. Sections (approximately 30 cm’) were cut from portions with the softest texture and placed in a sterile dish in a laminar flow hood. The amount of liver used was determined by the number of cells desired. The isolation of single cells was accomplished by addition of Buffer 1 (25°C) containing 15 g/liter bovine serum albumin and the tissue gently shaken with forceps, but not minced, to allow the cells to float free. The cell suspension was poured through successive nylon nitex filters of 100 and 35 pm (Tetko, Inc., Elmsford, NJ) to reduce the number of cell aggregates. If a high percentage of single cells is desired the suspension should also be filtered through a 25-pm nitex filter. The cell suspension was centrifuged in 50-ml tubes at 60g for 2 min and the supernate gently decanted. This washing procedure was repeated three times or as needed to remove red blood cells, endothelial cells, Kupffer cells, and nonviable hepatocytes. Final cell preparations consisted of greater than 95% hepatocytes as judged by light microscopic obser-
ENDOCYTOSIS
IN STEROID
HEPATOPATHY
155
vation. This hepatocyte purity agrees well with that reported for other species (21-24). Cell viability was determined by exclusion of trypan blue and release of lactic acid dehydrogenase activity as previously described (25). The cell suspension was stored on ice until use (less than 3 hr). Although the use of EGTA is not necessary for the isolation of rat hepatocytes (23), we found it substantially improved the yield, viability, and percentage of single cells during the isolation of dog hepatocytes. Electron microscopy of liver tissue. Small liver sections (15-20 mg) were taken from the liver immediately after removal from the dogs. These specimens were fixed in 2% glutaraldehyde containing 0.2 M cacodylate buffer (pH 7.2) and imbedded in epoxy resin, thin sectioned, and stained with uranyl acetate and lead citrate. The specimens were examined at 100 kV using a CX JOEL transmission electron microscope. Measurement ofhepatocyte cell volume. Determination of hepatocyte cell volume was done by electronic particle counting using a Model ZM Coulter counter. Aliquots of the final purified hepatocytes were placed in a final volume of 100 ml of isotone (Coulter Electronics, Hialeah, FL) to achieve a final cell concentration of 7.5-8.0 x lo4 cells/ml. The loo-ml suspension was gently stirred, without creating a visible vortex at the surface of the liquid, throughout the sizing measurements. The Coulter counter was first calibrated with 19.1~pm-diameter latex beads. Determination of hepatocellulur DNA. Freshly excised liver tissue (about 0.1 g) was immediately homogenized in 1 ml, 1°C 10 mM Hepes buffer (pH 7.4) with a Dounce homogenizer and immersed into a dry ice-ethanol slurry and stored at -70°C until used for biochemical analyses. DNA analyses were performed on liver homogenates using a fluorescent-enhancement assay employing the DNA binding dye, bisbenzimidazole (Hoechst 33258, American Hoechst Corp., Domerville, NJ), and polymerized calf thymus DNA (type 1) as a standard (26). Measurement of ALP activity. Total ALP activity was measured in liver and serum samples by a calorimetric assay using 122 pmol p-nitrophenylphosphate as substrate in a final volume of 0.5 ml of 1.02 M diethanolamine-0.51 mM MgClz buffer (pH 9.8, Boehringer-Mannheim System Pack, Houston TX) at 37°C by automated analysis employing a Hitachi 705 chemistry analyzer (Hitachi Limited, Tokyo, Japan). The results are reported as international units per liter or per micrograms protein (liver homogenates). Measurement of CALP activity. CALP activity was measured in liver extracts and serum by an enzyme antigen immunoassay (EAIA) using a monoclonal antibody which recognizes either IALP or CALP but not LALP (8). Qualitative electrophoretic separation of ALP isozymes (27) did not demonstrate detectable amounts of IALP at any time in the liver and serum from either normal or GT dogs. Therefore, as utilized in these studies, the monoclonal antibody primarily measures CALP activity. The EAIA assay was performed as follows. Costar serocluster flat bottom well microtiter plates (Costar Co., Cambridge, MA) were coated with monoclonal antibody (1 mg/ml in 0.1 M Na2C03-NaHC03 buffer, pH 9.6) and stored at 4°C overnight. The next morning the wells were rinsed three times with phosphate-buffered saline (PBS)-0.05% Tween 20, the antigen
156
KUHLENSCHMIDT,
HOFFMANN,
AND
RIPPY
samples added, and the plates incubated for 2 h at 25°C. At the end of the incubation the fluid was removed from each of the wells by aspiration, the wells were rinsed three times with PBS-Tween 20, substrate (25 mmol p-nitrophenylphosphate/100 ml 0.1 M Na,C03-NaHC03 buffer, pH 9.6) was added, and the plates were incubated at 37°C for 30 min. The amount of p-nitrophenol produced was determined by measuring the absorbance at 410 nm using a MicroELISA autoreader (Dynatech Labs, Inc., Chantilly, VA). Absorbance values determined using the autoreader and pure IALP or CALP as antigen were directly correlated with values (units/liter) measured on duplicate samples by the autonanalyzer (see above). In this manner a standard curve was constructed such that CALP activity could be determined as units/liter from the EAIA and directly compared with total serum and liver ALP activities measured with the autoanalyzer. From linear regression analysis (? = 0.933 for a linear fit) an activity of one IU/liter on the autoanalyzer corresponded to an absorbance (410 nm) of 0.0036 on the microreader during the EAIA. Determination of Protein. Protein was determined calorimetrically in serum and liver homogenates (28) using bicinchoninic acid (Pierce Chemical Co., Rockford, IL) and employing a protein standard containing human serum albumin and yglobulin (Sigma Cat. No. 540-10) diluted to 0.96 mg/ml. Measurement of the Gal/GalNAc asialoglycoprotein receptor activity of isolated hepatocytes. The activity of the hepatic cell surface Gal/GalNAc receptor on dog hepatocytes in suspension was determined as previously reported (21,29,30). Briefly, the neoglycoprotein, galactose-BSA (GalBSA) (31), generously donated by Dr. Y. C. Lee, The Johns Hopkins University, was iodinated with [lZ]sodium iodide (carrier free, IMS30, Amersham Corp., Arlington Heights, IL) using chloramine-T as previously described (21,29). Purified IALP (8) was radioiodinated using modifications of the Bolton-Hunter procedure (32). Intestinal alkaline phosphatase (50 ~1, 0.68 mg/ml in 0.15 M potassium phosphate buffer, pH 7.5), purified from a GT dog as previously described (8), was added to 2 mCi of 125 labeled Bolton-Hunter reagent (ICN Radiochemicals, Irvine, CA, Cat. No. 65OOlS, 2000 Ci/mmol) at 1°C overnight. Excess free radioactivity was separated from protein-bound radioactivity by gel filtration using Sephadex G-10. The column was equilibrated and eluted in 0.15 M potassium phosphate buffer (pH 7.5) containing 1% (w/v) gelatin. The void volume peak was collected and passed over a monoclonal antibody affinity column as previously described (8) to further purify the iodinated IALP. The specific radioactivity of IALP following the above procedure was 4300 cpm/ng. For uptake experiments approximately 9.0 pg of lZI-GalBSA (1180 cpm/ng) or 1.7 pg of rZI-IALP (7 x lo6 cpm) was incubated with 4 ml of cells (4 x lo6 cell/ml) in medium A (21) at 37°C by end-over-end rotation as described above. At indicated times, 300 ~1 of the cell suspension was aliquoted in duplicate and placed on top of 500 ~1 of oil (4 parts Dow Coming 550 [William F. Nye, Inc., New Bedford, MA] and 1 part light mineral oil) in 1.5 -ml polypropylene microfuge tubes and immediately centrifuged (12,000g) for 30 s in a microfuge to separate the cells from the media (29). The tubes were inverted and their tips containing the cell pellets were cut off and placed in 10
ENDOCYTOSIS
IN STEROID
HEPATOPATHY
157
x 75 mm glass tubes, and radioactivity was determined in a gamma counter (Auto-Gamma 800, Packard Instrument Co.). Measurement of the number of cell surface asialogycoprotein receptors. Estimation of the number of cell surface receptors and their relative affinities in normal and GT dogs was done by steady-state binding experiments at 1°C essentially as described by Connolly et al. (33) for isolated rabbit hepatocytes. Briefly, freshly isolated hepatocytes (4 x lo6 from either normal or GT dogs) were incubated in medium A with various concentrations of ‘=I-GalBSA for 120 min. Duplicate samples for each GalBSA concentration were analyzed for cell associated radioactivity as described above. Receptor number and binding affinity were determined from these binding isotherm data using an iterative curve fitting computer program (34), obtained from the Biomedical Computing Technology Information Center, Vanderbilt Medical Center, Nashville, Tennessee. The best fit of the data was obtained with a one-site model (a single class of high affinity ligand sites exist on the hepatocyte surface). The data for receptor number are presented as ligand binding sites per cell and assume a ligand/receptor stoichiometry of one. The amount of nonspecifically bound radioactivity was determined using duplicate incubations conducted in the presence of 2 mM EGTA or a lofold mass excess of unlabeled GalBSA. The nonspecific binding value was then evaluated as a separate parameter from specific binding during computer-assisted curve fitting analyses (34). Statistical analysis was performed using an unpaired t test and pooled variance to calculate the t value. Measurement of the rate of degradation of GalBSA. Determination of the rate and extent of degradation of GalBSA by isolated hepatocytes from normal and GT dogs was performed as follows. Hepatocytes (4 x lo6 cells/ml) were preincubated at 37°C with ‘*-?-GalBSA (40 nM) for 60 min. The hepatocytes were then washed two times by centrifugation (SOOg, 2 min) in the presence of 2 mM EGTA in medium A to remove free and surface bound GalBSA. The washed hepatocytes were then incubated at 37°C for various times in the presence of 400 nM unlabeled GalBSA. Duplicate aliquots (300 ~1) were removed at each time point and centrifuged through oil (see above) to separate incubation media from cells. Aliquots (150 ~1) of the media on top of the oil were added to cold 10% (w/v) trichloroacetic acid (TCA) and allowed to stand overnight at 4°C. The TCA suspensions were centrifuged (12,OOOg, 2 min) and the amount of radioactivity in the supematants (degraded protein) and pellets (intact protein) was determined. Determination of the rate of clearance of dog IALP by the hepatic asialoglycoprotein receptor. Purified GT- dog IALP (unlabeled or radioiodinated) alone or in the presence of GalBSA or asialofetuin was injected iv into normal or GT dogs and the kinetics of serum disappearance monitored at various times postinjection. Serum ALP activity in the normal dogs injected with unlabeled IALP was 50-80 IU. The dose of injected IALP (0.4 mg) increased the serum ALP activity to about 400 +- 25 IU at 30 s postinjection. At various times, blood (2 ml) was collected from normal dogs injected with unlabeled IALP and the serum ALP activity determined by autoanalyzer as described above. For GT dogs IALP (7.0 pg [rZI]IALP, 3. x lo7 cpm + 12.5 pg unlabeled IALP) was injected in 4
158
KUHLENSCHMIDT,
HOFFMANN,
AND RIPPY
ml sterile, isotonic saline and at various times postinjection collected and assayed for radioactivity in a gamma counter. RESULTS
1 ml blood
was
AND DISCUSSION
Experimental induction of glucocorticoid hepatopathy. All GT dogs responded by developing severe, delocalized hepatocellular swelling (Fig. l), and clinical signs (alopecia, polyuria, polydipsia, depression, skeletal muscle wasting) typical of canine Cushing’s Syndrome. Serum cortisol concentrations in these dogs were increased an average of 9.3-fold (223 _+ 49.10 rig/ml) compared to that of control dogs (20.0 + 12.90 rig/ml) following 30 days of treatment. There was also a significant increase in total serum ALP (linear regression, p < 0.0002) and CALP (P < 0.001) following 30 dyas of GC treatment. By Day 30 treated dogs displayed an average 12.3-fold increase in ALP (515 2 212 IU compared to 41.7 2 12.1 IU for these dogs before treatment and a 3-fold increase in CALP (140 + 38 IU compared to 45 + 11 IU before treatment). Electron microscopic examination of liver. Biopsy tissue demonstrated extensive hepatocellular swelling and pronounced peripheral displacement of subcellular organelles in glucocorticoid-treated dogs (Figs. 1A and 1B). These morphological alterations were essentially identical to those previously reported in GT dogs W). Hepatocytes of GT dogs. Although
we observed extensive hepatocellular swelling in GT dogs the hepatocyte isolation procedure resulted in yields of viable hepatocytes similar to those in normal dogs and was similar to that reported for the rat using in situ perfusion (22-24). Hepatocytes isolated from either normal or GT dogs were routinely 75-80% single cells and greater than 85% viable. In addition, the abnormal, swollen hepatocyte morphology seen in vivo was preserved in the isolated hepatocytes from GT dogs. Coulter counter sizing analyses demonstrated that the hepatocytes isolated from GT dogs displayed markedly increased cell volumes as compared to normal hepatocytes (Fig. 2). The median volume of hepatocytes isolated from GT dogs was approximately 6600 pm3 compared to 2060 pm3 for normal hepatocytes. Characterization of the asialoglycoprotein receptor-mediated endocytosis pathway in GT dogs. Normal dog hepatocytes in suspension rapidly endocytosed lz51GalBSA (0.013 pmol/min/l x lo6 cells; Fig. 3). The rate and steady-state level of GalBSA uptake is comparable to those reported for rat (21,30) and rabbit (33) hepatocytes. Previous studies (for review see (9,lO)) have demonstrated that the mammalian asialoglycoprotein receptor displays an obligate requirement for calcium and binds only oligosaccharide or glycoprotein ligands which are terminated at the nonreducing end in galactose or N-acetylgalactosamine. Since the dog hepatocyte asialoglycoprotein receptor has not been extensively characterized and to ensure that the ligand uptake was indeed occurring through this receptor we examined the metal ion requirement and sugar specificity for uptake. The uptake of ‘*?-GalBSA demonstrated an obligate requirement for calcium ions, since uptake was blocked by EGTA, but could be restored to normal activity by the addition of calcium but not magnesium (Fig. 4). Furthermore, the uptake was specific for galactose. Uptake of ‘*?-GalBSA was competitively inhibited by
ENDOCYTOSIS
IN STEROID
HEPATOPATHY
159
FIG. 1. Electron photomicrographs of liver biopsy tissue obtained from normal and GT dogs. Biopsy tissue was collected and processed for electron microscopy as described under Materials and Methods. (A) Normal dog liver (B) GT dog liver. ~2171. The arrows indicate the typical pattern of peripherally displaced mitochondria that is seen within the swollen hepatocytes of GT dogs.
160
KUHLENSCHMIDT,
HOFFMANN,
AND RIPPY
16 e
14
P 3
12 10
Ii 8
a
5
6
Y E
4
CL
2 0
1.0
2.6
4.1
5.6
7.2
6.7
VOLUME
(CU.
MC.
x 1000)
FIG. 2. Sizing analysis of hepatocytes isolated and cell volume determinations performed using Methods. The figure represents a typical sizing GT dog hepatocytes. Normal dogs (open bars);
10.2
11.2
from normal and GT dogs. Hepatocytes were isolated a Coulter counter as described under Materials and profile of five separate comparisons of normal and GT dogs (closed bars).
excess unlabeled GalBSA but not by GlcNAcBSA (Fig. 5). In addition 12?GlcNAcBSA was not endocytosed by dog hepatocytes. These results confirm that GalBSA uptake is occurring through the hepatic asialoglycoprotein receptor. Surprisingly, we found that the rate and steady-state level of GalBSA uptake in the morphologically altered hepatocytes isolated from GT dogs were slightly greater than those seen in normal hepatocytes (Fig. 3). Although the results of binding isotherm experiments (Fig. 6) indicated that GT dog hepatocytes displayed slightly more (1.6-fold) cell surface receptors than normal hepatocytes, statistical analysis indicated this was not a significant difference. Computer-assisted analyses (34) of these data indicated that both normal and GT dog hepatocytes contained
g (D
::
1.2 -
GT DOGS
1.0.
0
20
60
40 WE
80
loo
(min)
FIG. 3. Comparison of the kinetics of ‘?-GalBSA uptake by normal and GT dog hepatocytes. Normal and GT dog hepatocytes were isolated and incubated with ‘=I-GaIBSA at 37°C and the rate of uptake was determined as described under Materials and Methods. These data are presented as the average + standard deviation of four normal dogs (0) and five GT dogs (0).
ENDOCYTOSIS
IN STEROID
161
HEPATOPATHY
+Ca/Mg / /
-EGTA
LEGTA
c
TIME (YIN) FIG. 4. Effect of divalent cations on uptake of ‘*‘I-GalBSA by isolated dog hepatocytes. Normal dog hepatocytes were isolated and incubated with ‘“I-GalBSA and uptake was determined at 37°C as described under Materials and Methods. (m) Normal medium A; (0) medium A plus 4 mru EGTA; (Cl) medium A plus mM EGTA and 6 mM CaCl,; (0) medium A plus 4 mM EGTA and 6 mM MgCl?.
a single class of high affinity GalBSA binding sites. Furthermore, the ligand binding affinities were the same (Kdapp = 1 nM) in both normal and GT dogs. A summary of these data is shown in Table 1. The values we obtained for receptor numbers and ligand binding affinities are in close agreement with previously published values for the hepatic lectins of chicken (29) and rabbit (33) hepatocytes and well within the range of the number of receptor sites reported for rat hepatocytes (for review see (9,lO)). Even though kinetic analyses revealed no differences in the activity of the asialoglycoprotein receptor in normal and GT dogs, we thought it possible that the rate of GalBSA degradation may be altered in the treated animals because Ti =:
0.9 0.6
us 2
0.7 0.6
>20 4 u" 2
05. 0.4 0.3 0.2 0.1
4
FIG. 5. Sugar specificity patocytes were isolated and as described under Materials Fg GlcNAcBSA; (W) 9.0 pg radioiodinated material.
Oo
20
40 TIME
60
60
100
120
IMIN) of neoglycoprotein uptake by isolated dog hepatocytes. Normal incubated with ‘%noeglycoprotein and uptake was determined and Methods. (0) 9.0 pg ‘=I-GalBSA; (0) 9.0 pg ‘?-GalBSA ‘=I-GalBSA plus 100 pg GalBSA; (0) 9.0 pg ‘?-GlcNAcBSA.
dog heat 37°C plus 100 *denotes
162
KUHLENSCHMIDT,
HOFFMANN,
AND RIPPY
GT-DOGS 0.25
1
NORMAL
0.10
0.00 J 0
10
20
30 Free GaLlXA
DOGS
40
50
60
(nM)
FIG. 6. Equilibrium binding of ‘2SI-GalBSA to dog hepatocytes at 1°C. Hepatocytes were isolated from normal and GT dogs and incubated with various concentrations of ?-GalBSA for 2 h and specifically bound ‘251-GalBSA was determined as described under Materials and Methods. These data are presented as the average * standard deviation of four normal dogs (0) and five GT dogs (0).
TABLE 1 Comparison of the Kinetic Constants of the Asialoglycoprotein GT Dog Hepatocytes
Receptors of Normal and
Receptors/cell (1000 ligands/cell)
K WPP) W
Normal dogs 1 2 3 4
Average GT dogs 1 2 3 4 5
Average
51,531 53,999 54,481 48,425
1.91 2.17 1.80
52,109 _’ 2,775
1.79
86,628 63,812 84,220
0.94 2.14
1.28
113,176
1.06 1.19
65,606
1.73
82,688
+ 19,972
f 0.37
1.41 + 0.51
Note. Hepatocytes were incubated at 1°C with ‘=I-GalBSA and the amount of radioactivity bound was determined as described under Materials and Methods. Cell surface receptor numbers and ligand binding affinities were determined using the computer program, Ligand, assuming a one-site model as described under Materials and Methods. The average receptor numbers in normal and GT dogs were not statistically different as analyzed by an unpaired t test using pooled variance to calculate
t(t
= 0.558,
P > 0.5).
ENDOCYTOSIS
IN STEROID
HEPATOPATHY
163
0
10 20 30 40 60 90 120 150 1 IO TIME (MINI FIG. 7. Rate of degradation of ‘251-GalBSA in normal and GT dog hepatocytes. Hepatocytes were preincubated for 60 min at 37°C with ‘2SI-GalBSA (40 r&r), washed two times with medium A containing EGTA (2 mM) to remove free and surface bound ligand, and incubated at 37°C for various times in the presence of 400 ntu GalBSA. The amount of ligand degraded at each time point was calculated as the percentage of total radioactivity incorporated into the preincubated, washed hepatocytes which was recovered in the cell-free media and soluble in 10% cold TCA (see Materials and Methods). (0) Normal dogs; (m) GT dogs.
of the severe hepatocellular swelling and peripheral displacement of subcellular organelles. However, we detected no difference in the rate of GalBSA degradation in GT dogs compared to normal animals (Fig. 7). Approximately, 70% of the cell-associated GalBSA was degraded by 180 min (lost from the cell as TCAsoluble radioactivity). The remaining 30% was found still associated with the cell and TCA precipitable. Hepatic endocytosis of IALP. Having demonstrated that the asialoglycoprotein receptor-mediated endocytosis pathway appears to function normally in GT dogs, we determined whether purified, native IALP could serve as a ligand for this receptor. Our initial experiments showed that iv-injected IALP was rapidly cleared from the circulation of either normal or GT dogs with a half-life of approximately 6-7 min and that the rate of clearance was inhibited by simultaneous injection of GalBSA (Fig. 8) or asialofetuin (data not shown). Similarly, IALP was also rapidly taken up by isolated dog hepatocytes and the uptake was completely blocked by simultaneous addition of 2 mM EGTA or a lo-fold mass excess of GalBSA (Fig. 9). Exocytosis of ZALP. In contrast to the results using GalBSA as the ligand, preliminary experiments demonstrated that approximately 25% of the total IALP endocytosed by GT dog hepatocytes at steady state was exocytosed into the medium in undegraded, TCA-precipitable form following a 60-min chase with excess unlabeled IALP (Fig. 10). A known function of the asialoglycoprotein receptor-mediated endocytosis pathway is to remove asialoglycoproteins from the circulation, transport them intracellularly in endocytic vesicles, and ultimately deliver them to lysosomes for degradation while simultaneously preserving the receptor by recycling it back to the
l&I
KUHLENSCHMIDT,
0
HOFPMANN,
20
AND RIPPY
40
TIME POST INJECTION(MINI FIG. 8. in viva clearance of IALP in dogs. Purified IALP was injected iv in normal (unlabeled IALP) and GT-treated dogs (radioiodinated IALP) and the amount of IALP remaining in the circulation at various times postinjection was determined by assaying enzymatic activity or radioactivity as described under Materials and Methods. (m) IALP (0.4 mg) injected in a normal dog; (0) IALP (0.4 mg plus 50 mg GalBSA) injected in a normal dog; (0) IALP (7.0 pg ‘=I-IALP plus 12.5 pg unlabled IALP) injected in a GT dog.
plasma membrane to continue the cycle (for review see (9,lO)). The overall process of ligand sorting, routing, and receptor recycling is an intricate and complicated one involving multiple steps, several endocytic vesicles, and interactions with various subcellular organelles (18-20). Accordingly, various forms of liver disease, diabetes, galactosamine toxicity, liver regeneration, and certain surgical manipulations are known to have a deleterious effect on this receptor activity (13-17). Therefore, it was surprising, in light of the striking derangement of the topography of subcellular organelles, that the rate and steady-state amount of GalBSA uptake,
0
5
1015202530354045505560 TIME (MN)
9. Uptake of ‘251-IALP by dog hepatocytes. Normal and GT dog hepatocytes were incubated with 1.7 pg “‘I-IALP for various times at 37°C and the amount of radioactivity taken up in the presence and absence of GalBSA or EGTA was determined as described for GalBSA (see Materials and Methods). (0) Normal dog; (0) GT dog; (A) uptake in presence of either 100 x mass excess of GalBSA or 4 mM EDTA. FIG.
ENDOCYTOSIS
IN STEROID
HEPATOPATHY
165
0
20 40 60 TIME OF CHASE MIN) FIG. 10. Rate of exocytosis of ‘Z51-IALP from dog hepatocytes. GT dog hepatocytes were incubated with iZSI-IALP for 120 min (as described in Fig. 6 except at 37°C) to achieve steady-state uptake. The hepatocytes were then washed three times in medium A to remove free ligand and then reincubated at 37°C with a 50-fold mass excess of unlabeled IALP as a chase. At the indicated times of the chase aliquots of the incubation mixture were removed, the cells pelleted through oil, and the cell-free media subjected to TCA precipitation as described under Materials and Methods for measurement of ligand degradation. The amount of ligand exocytosed is expressed as a percentage of the total IALP taken up at steady state. (m) Total exocytosed IALP; (0) IALP exocytosed intact (TCAprecipitable form).
the rate of GalBSA degradation, the cell surface receptor number, and ligand binding affinities were the same or greater in hepatocytes isolated from GT dogs. These data clearly demonstrate that the 3.2-fold increase in hepatocyte size and the pronounced delocalization of subcellular organelles induced by excess glucocorticoids do not adversely affect lysosomal function or receptor-mediated endocytosis through the complex asialoglycoprotein pathway. While these results cannot be extrapolated to the multitude of unrelated functions of hepatocytes it can be concluded that the numerous pathways and cellular functions involved in this receptor system are not impaired by the hepatic morphological alterations induced by GC. We have also shown by competition studies that IALP is recognized and removed from the blood or media (in vitro) by the asialoglycoprotein receptor as had previously been suggested (35). Interestingly, the clearance of rat IALP from rat plasma appears to be mediated by the N-acetylglucosamine/mannose receptor of nonparenchymal cells rather than the hepatic asialoglycoprotein receptor (36). These data indicate that the nonreducing terminal carbohydrate sequence of rat IALP is different from dog IALP. In agreement with our findings in dogs, earlier studies by Sholtens et al. (37) demonstrated that dog IALP was cleared in the isolated perfused rat liver and in the intact rat by the asialoglycoprotein receptor. The plasma disappearance curve showed an initial, rapid clearance of IALP followed by a slower, secondary phase which the authors suggested may be caused by exocytosis of a portion of the internalized enzyme. While we looked only at the initial rate of clearance of IALP from dog plasma, our preliminary experiments using GT dog hepatocytes indicate that in contrast to our results with the synthetic
166
KUHLENSCHMIDT,
HOFFMANN,
AND RIPPY
neoglycoprotein ligand, GalBSA, approximately 25% of the endocytosed lz51IALP is exocytosed intact from the cells rather than being degraded. We are currently characterizing the exocytosed radioactivity to determine whether any of the material is altered by further glycosylation as has been shown to occur in rat hepatocytes using asialotransferrin as the ligand (38). Preliminary data indicates that exocytosed IALP has a more anodal electrophoretic mobility on native PAGE gels than does the IALP used as the ligand. Although this electrophoretic mobility is indistinguishable from that of CALP, we have as yet no evidence that exocytosed IALP is identical to CALP. Albeit speculation, hyperglycosylation of IALP following endocytosis and exocytosis through the liver could represent a novel pathway for the direct production of serum CALP in glucocorticoid-treated dogs in lieu of de MVO protein synthesis. We have recently observed that IALP isolated from a GT-treated dog is also exocytosed from normal dog hepatocytes. These data, along with those demonstrating normal functioning of the Gal/GalNAc receptor in GT dogs, suggest the information necessary for exocytosis of IALP may be inherent in the oligosaccharide structure of GT dog IALP rather than resulting from a glucocorticoid-induced alteration in receptor-mediated endocytosis of asialoglycoproteins. ACKNOWLEDGMENTS We sincerely thank Dr. Y. C. Lee for his generous gifts of GalBSA and helpful advice during this investigation. We are grateful to Dr. Saul Roseman for his many helpful discussions. We also thank Mr. Keith Melton for his technical assistance and help in preparing photographs and Dr. Edward Bascall for his assistance with electron microscopy.
REFERENCES 1. Badylak SF, Van Vleet JF. Sequential morphologic and clinicopathologic alterations in dogs with experimentally induced glucocorticoid hepatopathy. Am J Vet Res 42:1310-1318, 1981. 2. Frittschen C, Bellamy JEC. Prednisone-induced morphologic and chemical changes in the liver of dogs. Vet Path01 21:399-406, 1984. 3. Rippy MK, Hoffmann WE, Kuhlenschmidt MS. Kinetics of biochemical and histologic changes associated with glucocorticoid-induced hepatopathy in the dog. Proc Am Co11 Vet Pathol 110, 1986. [Abstract] 4. Hoffmann WE, Dorner JL. A comparison of canine normal hepatic alkaline phosphatase and variant alkaline phosphatase of serum and liver. Clin Chim Actu 62~137-142, 1975. 5. Wellman ML, Hoffmann WE, Domer JL, Mock RE. A comparison of steroid-induced, intestinal and liver isoenxymes of alkaline phosphatase in the dog. Am J Vet Res 43:1204-1207, 1982. 6. Dorner JI, Hoffmann WE, Long GB. Corticosteroid induction of an isoenzyme of alkaline phosphatase in the dog. Am J Vet Res 35:1457-1458, 1974. 7. Hadley SP, Hoffmann WE, Kuhlenschmidt MS, Domer JL, Sanecki RK. Effect of glucocorticoids on alkaline phosphatase, alanine aminotransferase and gamma glutamyltranspeptidase in primary cultured dog hepatocytes Enzyme 43~89-98, 1990. 8. Sanecki RK, Hoffmann WE, Dorner JL, Kuhlenschmidt MS. Purification and comparison of corticosteroid-induced and intestinal isoenzymes of alkaline phosphatase in dogs. Am J Vet Res 51:1964-1968, 1990. 9. Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Ann Rev Biochem 51:531554, 1984. 10. Harford J, Ashwell, G., The hepatic receptor for asialo-glycoproteins. In The Glycoconjugates (Horowitz MIT, Ed.). New York: Academic Press, 1982, pp. 27-55. 11. Hubbard Al, Wilson, G, Ashwell G, Stukenbrok H. An electron microscope autoradiographic
ENDOCYTOSIS
IN STEROID
HEPATOPATHY
167
study of the carbohydrate recognition systems in rat liver. I. Distribution of 12%ligands among the liver cell types. J Cell Biol 83~47-64, 1979. 12. Huber BE, Glowinski IB, Thorgeirsson SS. Transcriptional and post-transcriptional regulation of the asialoglycoprotein receptor in normal and neoplastic rat liver. J Biol Chem 261:12,400-12,407, 1986. 13. Dodeur M, Cormoul S, Durand D. Diabetes induced variation in hepatic binding protein. Biochem Biophys
Res Commun
ll5:82-86,
1983.
14. Sawamura T, Kawasato S, Shiozaki Y. Decrease of an hepatic binding protein specific for asialoglycoproteins with accumulation of serum asialoglycoproteins in galactosamine treated rats. Gustroenterology 81:527-533, 1981. 15. Dillon AR, Sorjonen DC, Powers, RD, Spano S. Effects of dexamethasone and surgical hypotension on hepatic morphologic features and enzymes of dogs. Am J Vet Res 44:1966-1999, 1983. 16. Howard DJ, Stockert RJ, Morel1 AG. Asialoglycoprotein receptors in hepatic regeneration. J Biol Chem 257~2856-2858, 1982. 17. Serbource-Goguel N, Bore1 B, Dodeur M, Scarmato P, Bourel B, Feger J, Durand G. Endocytosis and binding of asialoorosomucoid by hepatocytes from rats with jejunoileal bypass. Hepatology 5~220-223,
1985.
18. Hubbard Al, Stukenbrok H. An electron microscope autoradio-graphic study of the carbohydrate recognition systems in rat liver. II. Intracellular fate of the 1251-ligands. J Cell Biol 83~65-81, 1979. 19. Weigel PH. Receptor recycling and ligand processing mediated by hepatic galactosyl receptors: A two pathway system. In Vertebrate Lectins (Olden K, Parent JB, Eds.), Van Nostrand-Reinhold Advanced Cell Biology Series. New York: Van Nostrand-Reinhold, 1987, pp. 65-91. 210. Wolkoff AW, Klausner RD, Ashwell G, Harford J. Intracellular segregation of asialoglycoprotiens and their receptor: A prelysosomal event subsequent to dissociation of the ligand-receptor complex. J Cell Biol98:375-381, 1984. 21. Kuhlenschmidt MS, Schmell E, Slife CW, Kuhlenschmidt TB, Sieber F, Lee YC, Roseman S. Studies on the intercellular adhesion of rat and chicken hepatocytes: Conditions affecting cellcell specificity. J Biol Chem 257:3157-3164, 1982. 22. Seglen PO. Preparation of rat liver cells. I. Effect of Ca” on enzymatic dispersion of isolated, perfused liver. Exp Cell Res 74~450-454, 1972. 23. Seglen PO. Preparation of rat liver cells. II. Effects of ions and chelators on tissue dispersion. Exp Cell Res 76~25-30, 1973. 24. Seglen PO. Preparation of rat liver cells. III. Enzymatic requirements for tissue dispersion. Exp Cell Res 82~391-398, 1973. 25. Schnaar RL, Weigel PH, Kuhlenschmidt MS, Lee YC, Roseman S. Adhesion of chicken hepatocytes to polyacrylamide gels derivatized with N-acetylglucosamine. J Biol Chem 253~7940-7951, 1978. 26. Downs TR, Wilfinger WW. Fluorometric quantification or DNA in cells and tissue. Anal Biochem 131:538-547,
1983.
27. Hoffmann WE, Dorner JL. Separation of isoenzymes of canine alkaline phosphatase by cellulose acetate electrophoresis. J Am Anim Hosp Assoc 11:283-285, 1975. 28. Smith, PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Bicinchoninic acid protein assay. Anal Biochem lSO:76-85, 1985. 29. Kuhlenschmidt TB, Kuhlenschmidt MS, Roseman S, Lee YC. Binding and endocytosis of glycoproteins by isolated chicken hepatocytes. Biochemistry 2X437-6444, 1984. 30. Kawaguchi K, Kuhlenschmidt MS, Roseman S, Lee YC. Differential uptake of n-galactosyl and o-glucosyl neoglycoproteins by isolated rat hepatocyte. J Biol Chem 256~2230-2234, 1981. 31. Stowell CP, Lee YC. Preparation of some new neoglycoproteins by amidination of bovine serum albumin using 2-methoxyethyl-1-thioglycosides. Biochemistry 19:4899-4904, 1980. 32. Bolton AE, Hunter WM. The labelling of proteins to high specific radioactivities by conjugation to a 1251-containing acylating agent. Biochem J l33:529-539, 197’3. 33. Connolly DT, Townsend RR, Kawaguchi K, Hobish MK, Bell WR, Lee YC. Binding and en-
168
34. 35. 36. 37. 38.
KUHLENSCHMIDT,
HOFFMANN,
AND RIPPY
docytosis of glycoproteins and neoglycoproteins by isolated rabbit hepatocytes. &o&m J 214:421431, 1983. Munson PJ, Rodbard D. LIGAND, A versatile computerized approach for characterization of ligand-binding systems. Anal Biochem 107:220-239, 1980. Hoffmann WE, Dorner JL. Serum half-life of intravenously injected intestinal and liver alkaline phosphatase isoenzymes. Am J Vet Res 38~1553-1556, 1977. Young, GP, Rose IS, Cropper S, Seetharam S, Alpers DH. Hepatic clearance of rat plasma intestinal alkaline phosphatase. Am J Physiol 247 (Gastrointest Liver Physiol lO):G419-G426, 1984. Scholtens HB, Hardonk MJ, Meijer DKF. A kinetic study of hepatic uptake of canine intestinal alkaline phosphatase in the rat. Liver 2~1-13, 1982. Regoeczi E, Chindemi PA, Debanne MT, Charlwood, PA. Partial resialylation of human asialotransfenin type 3 in the rat. Proc Nat1 Acad Sci U S A 79:2226-2230, 1982.