EXPERIMENTAL

CELL

RESEARCH

199,223-228

(19%)

Effects of Vasopressin on Receptor-Mediated Endocytosis of Asialoglycoprotein by Hepatocytes from Normal and Diabetic Rats SOPHIE GIL-FALGON, CHRISTOPHE LAMAZE, SALIMA HACEIN-BEY, AND JEANNE FEGER’ Laboratoire

de Biochimie, UFR aks Sciences Pharmaceutiques et Bialogiques, Universitk 5 rue JB Clement, 92296 Ch&enuy-Malabry, Ceder, France

quent trafficking

Paris-&d,

of ligand to lysosomes for degradation

The hepatic asialoglycoprotein receptor is a memwhile receptors recycle back to the cell surface. brane glycoprotein used as a model to study receptorIn vitro modulations of this receptor have been used mediated endocytosis. In order to examine the ability of to dissect the critical steps in the pathways involved and second messengers to modulate intracellular traffickto elucidate some aspects of their mechanisms. It has ing, we performed a comparative study on normal and been shown that drugs such as sodium arsenite [8], diabetic rat hepatocytes exploring the effects of an in monensin [g-14], colchicine [ll, 141, and metabolic envivo modulation, streptozotocin-diabetes, and an in viergy poisons [8, l&17] alter ligand internalization, distro modulator, vasopressin, which transduces signals sociation, degradation, and receptor recycling, respecvia the phosphoinositide pathway. We studied three tively. Such an approach has been used to evidence the main experimental aspects: (1) constitutive endocytorole of free thiol residues, acidic vesicles, cytoskeleton sis, (2) continuous ligand flux, and (3) a synchronous motility, and metabolic energy. However, little is known wave of ligand. In normal cells, vasopressin decreased of the regulatory mechanisms involved. ligand-binding capacity by 2070, without altering the Recently, substantial data have suggested that intramechanism of internalization, and decreased the level cellular protein phosphorylation by phorbol esters may of degradation, without affecting the distribution of deghave an effect on receptor movement, distribution, and radation products. Diabetic cells were characterized by ligand-binding affinity but that the effects vary accorda 50% decrease in cell-surface and intracellular receptor ligand-binding capacity, slowed internalization of a , ing to the receptor system, cell type, and duration of exposure [18-201. The cell’s ability to adapt to altered synchronous wave of ligand, and markedly reduced degsituations in uivo has also been explored in an attempt radation with an altered distribution of degraded products. Vasopressin had no additive effect on the modificato understand the regulatory mechanisms underlying tion induced by diabetes. These results suggest that secthese various intracellular pathways. For example, ond messengers generated by hormones play a role in chronic ethanol ingestion by rats decreases the number the regulation of receptor-mediated endocytosis. They of cell-surface ligand-binding receptors and alters the also confirm that receptors are subdivided into those internalization of cell surface-bound ligand [21]. susceptible to modulation of any kind and those insensiWe have shown that streptozotocin-induced diabetes tive to modulation, although the boundary between the in rats decreases the clearance of asialoorosomucoid in two subsets is variable. o 1992 AW&IU~C PBS, IJIC. vivo and induces a loss of binding capacity in vitro both

INTRODUCTION Receptor-mediated endocytosis is a process common to many cell types and is responsible for the selective uptake of extracellular molecules [l-5]. One of the best-characterized models is the hepatocyte receptor for desialylated glycoproteins [6, 71. The main steps are ligand binding by the receptor at the cell surface, internalization via coated pits and vesicles, dissociation in internal acidic compartments, and subse1To whom correspondenceand reprint requests should be addressed.Fax: 33-l- 46 83 13 03. 223

at the cell surface and within the cell [23, 241, although the number of immunoreactive receptors remains constant [25]. These results suggest that hormones may modulate receptor activity and movement, and point to the role of transduction and second messengers in this regulation. In addition, the results of most studies show that receptors can be divided into two apparent subsets, one of which, R2, is sensitive to modulation while the other, Rl, is not [26]. We suspected that in diabetes the inactive receptors may belong to the R2 type and that still-active receptors may belong to the Rl type, which is less (or not) sensitive to modulations by drugs. We therefore studied asialoglycoprotein-receptor (ASGP-R) endocytosis in nor0014-4827192

$3.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

224

GIL-FALCON

ma1 and diabetic rat hepatocytes in the presence or absence of ligand, using vasopressin, a physiological hormone, to generate second messengers of transduction [reviewed in 27, 281. To check whether the still-active receptors in diabetes were of the Rl type, we determined if the effects of the added modulator vasopressin were distinguishable from, or superimposable on, those effects already induced by the pathology dysregulations. Our results indicate that vasopressin induces a partial decrease in ligand binding by both cell-surface and intracellular receptors in normal hepatocytes and alters the mechanisms of degradation. Nonetheless, vasopressin was unable to superimpose a significant alteration of the binding or degradation of ASGP-R ligand onto diabetes-induced effects. MATERIALS

AND

METHODS

Human orosomucoid (Sigma Chemical Co., St. Louis, MO) was desialylated with agarose-immobilized neuraminidase type X-A (EC 3.2.1.18) (Sigma) and3H labeling was performed by reductive methylation according to Wilder et al. [29]. The specific radioactivity was 710 cpm/ng ASOR. [&Arginine]Vasopressin, collagenase (type IV), bovine serum albumin, reagents for hepatocyte incubation, and streptozotocin were from Sigma. Digitonin and reagents for the isolated rat hepatocyte preparation were from Merck & Co. (Rahway, NJ). [3H]KBH, (50-G Ci/mmol) was from CEA France. Male Sprague-Dawley rats (180-200 g) were obtained from Charles River Breeding Laboratories (Wilmington, MA). Treatment

of Rats

After an overnight fast with free access to water, rats were randomly divided into two groups. Animals in one group received streptozotocin (65 mg/kg body wt) which was dissolved in isotonic saline and acidified to pH 4.5 with citric acid, via the tail vein. After 11 days, rats with glycosuria and blood glucose levels above 25 mmol/liter were considered diabetic and used for experimentation. Animals in the second group were injected with the medium alone and considered normal. Hepatocyte

Preparation

Hepatocytes were prepared by collagenase perfusion according to the technique of Berry and Friend [30], as modified by Davy et al. [31]. Final cell pellets were suspended in ice-cold medium, were usually 85-95% viable as judged by 0.06% Trypan blue exclusion, and were composed of single cells. We used a Hepes-Tes-Tricin (HTT) medium as described by Seglen [32], with calcium and bovine serum albumin, pH 7.40. This medium contained 30 mM Hepes, 30 mM Tes, 36 mM Tricin, 68 mM NaCl, 5.36 mM KCl, 2.5 mM CaCl,, 0.64 mM MgCl,, 1.1 mM KH,PO,, 0.31 mM Na,SO,, and 0.1% BSA. Prior to all experiments, suspensions of freshly isolated hepatocytes (3 X 106/ ml) ware incubated at 37°C for 25 min in a gyratory water bath (60 rpm) to obtain an optimal number of surface receptors/cell [33]. Binding

Capacity of Cell-Surface

and Total Receptors

Cell-surface and total receptor-binding assays were performed by mixing the cells (3 X 106/ml) with saturating concentrations of [3H]ASOR (2 pglml). Incubation was carried out in duplicate at 4°C for 90 min. Total-binding studies were performed after permeabilization of the cells with 0.055% digitonin for 20 min at 4°C. Excess [aH]ASOR was then washed out three times with 0.15 M NaCl + 2.5 mM CaCl, and the radioactivity of the pellet was counted.

ET AL. Continuous

Endocytosis

of [“H]ASOR

Cells (3 X 106/ml) were allowed to internalize [“HIASOR (2 fig/ml) at 37°C for 75 min in the presence of vasopressin. At given incubation times, an aliquot of 4 ml was withdrawn from the flask, diluted with 2 ml of ice-cold HTT medium, and divided into aliquots of 1 ml. All of the following steps were carried out at 4°C. Treatment of the first aliquots. After centrifugation (7OOg, 45 s), 0.5 ml of the supernatant was blended in a Vortex mixer with an equal volume of 2.5% (w/v) phosphotungstic acid in 2 N HCl. After 20 min, the precipitates were centrifuged at 3000g for 10 min. The supernatant was counted to quantitate excreted degraded [3H]ASOR. The cell pellet was resuspended in 1 ml of 0.15 M NaCl with 0.02 M EDTA in order to remove surface-bound ASOR. After a 15.min incubation at 4°C and centrifugation (7OOg, 45 s), radioactivity was determined in 0.5 ml of the supernatant to quantitate cell surface-bound ligand. The cell pellet was washed with 3 ml of 0.15 M NaCl + 0.02 M EDTA and then counted to quantitate total intracellular ligand. Treatment of the second aliquots. Digitonin was immediately added to the cell suspension and incubated for 20 min. After centrifugation, 0.5 ml of the supernatant was treated as described for degraded ASOR (first aliquot), to quantitate total degraded ligand.

Internalization

of a Synchronous

Wave of Prebound

Cells (3 X 106/ml in HTT buffer) were allowed to bind [3H]ASOR (2 pg/ml) at 4°C for 60 min and were washed three times to eliminate unbound ligand. The amount of prebound [3H]ASOR at time 0 was measured in two l-ml aliquots after three washings with isotonic saline containing 2.5 mM CaCl, and was then treated for specific determination. They were then resuspended in a prewarmed 37°C buffer. The cells were kept in suspension in a gyratory water bath. At various incubation times an aliquot of 4 ml was withdrawn from the flask, diluted with 2 ml of ice-cold HTT medium, divided into l-ml aliquots, and treated as above to follow internalization by measuring surface binding and degradation.

General Specifications All parameters were determined in duplicate, and experiments were repeated five times. Aspecific values were assessed as above in the presence of 0.02 mM EDTA.

RESULTS

AND

DISCUSSION

The present study was undertaken to test the regulatory effect of the second messengers, generated by vasopressin, on ASGP-R activity and function. Vasopressin, a physiologic hormone, was used to generate second messengers of transduction; it is considered a good agonist to produce phosphoinositide-derived metabolites in hepatocytes, given the high number of Via receptors in these cells [33-361. We also compared the effects of diabetes, a pathological state characterized by an insulin deficiency and an increase in both glucagon and vasopressin [37, 381. We used isolated hepatocytes within the l-3 h following their isolation, conditions that have been widely used for metabolic investigations in normal and pathological situations such as diabetes [39].

EFFECT

OF VASOPRESSIN

ON ASGP-R

225

ENDOCYTOSIS

80 -

60 + 0

IO

20

I SO

Tfme (mln)

60 4 0

IO

20

1 SO

Tlme (mln)

FIG. 1. Time course of the effect of vasopressin on ligand-binding capacity by cell-surface receptors (Cl) and total receptors (ml in the presence of vasopressin compared to control cells in normal (A) and diabetic (B) rat hepatocytes. Cells (3 X 106/ml) were incubated at 37°C with and without vasopressin. At the indicated times, the cells were chilled rapidly to 4°C and incubated with 2 rg/ml of [3H]ASOR for 90 min, then washed three times. Radioactivity was counted to quantitate cell-surface binding. Total binding was similarly determined after permeabilization of the cells bv a 20-min incubation at 4°C with 0.055% dkitonin. Each data point represents the mean of five independent experiments performed in duplicate.

Ligand-Binding

Capacity

Freshly isolated hepatocytes were first incubated at 37°C for 25 min to stabilize the specific cell-surface ligand binding [40]. Mean maximal cell-surface binding was 23.25 + 1.81 ng/106 cells with normal rat hepatocytes and 9.25 f 0.99 ng/106 cells with diabetic rat hepatocytes. Total cell receptor binding was measured after preliminary permeabilization of the cells with 0.055% digitonin at 4°C for 20 min. By interacting with membrane cholesterol, this steroid glycoside facilitates the diffision of molecules with apparent molecular weights below 200 kDa, while receptor proteins, being exclusively membrane-associated, are retained within the cells [41]. Saturation bindings were determined at 4°C and Scatchard plots of the corresponding curves showed no variation of the affinity (data not shown). We found that at saturation, 67.5 f 6.6 and 27.6 + 4.2 ng of ASOR were bound by lo6 normal and diabetic cells, respectively. Some results are further expressed as a percentage of these binding values.

Effects of Vasopressin on Constitutive Receptor Endocytosis We next examined the effects of incubation with vasopressin at 37°C for different times with a concentration of low7 M, as in most metabolic studies involving hepatocytes. In the absence of added ligand, vasopressin induced a progressive loss of normal hepatocyte surface binding, which resulted in a maximal inactivation of 20.0 +- 1.6% within 20-30 min, with no further decline thereafter (Fig. 1A). The binding capacity of total cell receptors was decreased by about the same level (17.1 f 1.5%). We showed that this loss was not the result of a partial decrease in the affinity of all receptors, but of a total inactivation of some receptors, in saturation-binding

experiments at 4°C (data not shown). After a 20-min exposure to vasopressin, receptor-binding affinity did not vary, while the decrease in cell-surface and total receptor numbers was consistent with the above reduction in ligand-binding capacity. Incubation with vasopressin had no significant effect on either cell-surface or total receptor-binding activity in hepatocytes from diabetic rats (4.4 + 1.8 and 1.3 + 0.8%, respectively) (Fig. 1B). The fact that the inactivation was partial, reached a maximum within 20 min, and did not increase thereafter is compatible with the existence of two subsets of receptors [26], one of which was maximally inactivated during the first round of endocytosis in the presence of the modulator, while the other remained totally insensitive, even after prolonged contact. The similarity between the degree of inactivation of cell-surface and total receptors suggests that vasopressin did not affect the receptor distribution between the cell surface and the intracellular pool, and that receptor recycling continued in the presence of the modulator, independent of the changes in binding capacity. We then studied the behavior of receptors internalizing ligand in the presence of vasopressin.

Effects of Vasopressin on the Internalization Continuous Flux of Ligand

of a

Cells were incubated for 75 min with a saturating continuous flux of [3H]ASOR in the presence of 10e7 M vasopressin. In normal cells, the level of bound ligand at the cell surface remained practically constant during the incubation period (Fig. 2A). When vasopressin was added at the same time as the ligand, there was a rapid decrease in the amount of surface-bound ligand (19.5 f 3.1% at 5 min). This was similar to the level of vasopressin-in-

226

GIL-FALGON

ET AL.

C

_ 0

50

is

50

25

7s

Tlmt (mln)

76

Time (mln)

D

60

is

50

75

Tlme (mln)

25

so

75

Time (mid

FIG. 2. Effect of vasopressin on surface-bound (A,C) and internalized (B,D) [3H]ASOR in normal (A,B) and diabetic (C,D) rat hepatocytes. Cells (3 X 106/ml) were incubated at 37°C with a saturating concentration of [3H]ASOR (2 rg/ml), with (w) or without (A) vasopressin. At the indicated times, samples of the cell suspension were removed and centrifuged, and the medium was discarded. The cells were incubated in the presence of EDTA to remove cell surface-bound ligand and were then centrifuged. The medium was counted to determine surface-bound 13H]ASOR. The cells were washed three times and counted to determine internalized [3H]ASOR. Averages of duplicate determinations are shown from one representative of five similar experiments.

duced inactivation in the absence of ligand, but it occurred much more rapidly. Since binding capacity was stable after this time, this effect suggested the existence of continuous recycling and the return of the subset of still-active receptors to the cell surface. The plateau of internalized ligand was reached rapidly with both control and modulated cells, but was about 22.8 f 3.0% lower in the presence of vasopressin, a difference which persisted throughout the incubation period (Fig. 2B). These findings suggested that the decreased level of internalization was a consequence of rapid inactivation of some receptors but not of an alteration of the internalization step itself. In addition, the level of internalized ligand at the plateau, regardless of the presence or absence of vasopressin, suggested that all of the active receptors participated in ligand endocytosis. In diabetic rat cells (Fig. 2C), we again observed the impairment of ligand-surface receptor binding. As a consequence, the plateau of internalization was lower than in the controls (Fig. 2D). Vasopressin did not superimpose any alteration. Contrary to normal cells, the comparison of total binding capacity in diabetic cells with the height of the plateau suggested a slight defect in the internalization process. To better follow the internalization step, we used the ligand synchronous wave technique.

Effect of Vasopressin on the Internalization Synchronous Wave of Ligand

of a

Cell-surface receptors were loaded with [3H]ASOR. After warming to 37°C in the presence of vasopressin, the ligand was rapidly internalized (Fig. 3A) at a rate similar to control values: kl constant was 0.78 min-‘. We concluded that the receptor-ligand complex internalization mechanism was not altered by vasopressin. In diabetic rat cells (Fig. 3B), internalization was slower than in normal cells and some complexes remained at the cell surface. The kl constant value was 0.28 min-‘. Vasopressin had no apparent effect. Effect of Vasopressin on the Degradation Continuous Flux of Ligand

of a

We examined the level of degradation of internalized ligand and the distribution of degraded products between the intracellular compartment and the incubation medium. Digitonin was used to allow dissociated and degraded products to diffuse freely out of the cells. P. H. Weigel[41] has shown that in these conditions no detectable protease activity is discharged, suggesting that significant amounts of ligand are not lost. In normal cells, degraded products were present in the medium 30 min after the beginning of the ligand

EFFECT

OF VASOPRESSIN

ON ASGP-R

227

ENDOCYTOSIS

B

01

2

0

4

a

6

01

IO

0

4

2

6

a

IO

Time (mln)

Tlme (mln)

FIG. 3. Effect of vasopressin on the internalization of a synchronous wave of 13H]ASOR in normal (A) and diabetic (B) rat hepatocytes. of [3H]ASOR (2 pg/ml) for 90 min, then washed three times Cells (3 X 106/ml) were preincubated in duplicate with a saturating concentration to remove unbound ligand. They were resuspended in prewarmed buffer, with (B) or without (A) 10m7M vasopressin. At given times, two aliquots were withdrawn and washed. The pellet was resuspended, washed with 0.15 M NaCl + 0.02 MEDTA, and centrifuged before counting to determine surface-bound ligand. Data represent the average of duplicate determinations from one representative of five similar experiments.

endocytosis process. At 60 min, they appeared to be equally distributed between the intracellular and extracellular compartments, suggesting that they were able to move freely between the two compartments (Fig. 4A). In the presence of vasopressin, there was no change in the distribution of degraded products (Fig. 4B), but the total amount was decreased by about half (12.1 + 1.6 &IO6 cells at 60 min compared to 26.3 k 3.6 ng/lO” cells in controls).

A

40

1

45

This raised the question as to the effect of vasopressin on this step of the process; we addressed this issue by using the synchronous wave technique. In diabetic rat hepatocytes, the total amount of degraded ligand was very low compared to normal cells; a larger proportion of degraded products was found in the extracellular medium, as if they had been extruded (Fig. 4C). Vasopressin had no further apparent effect on the 4

c

1

60

45

Time (mln)

60

Time (min)

D

B

30

Time (mid

4s

60

75

Time hln)

FIG. 4. Effects of vasopressin on the degradation of r3H]ASOR by normal and diabetic rat hepatocytes after a 60-min endocytosis. Cells were treated as in Fig. 2 for continuous endocytosis. At given times, two aliquots were withdrawn. The first aliquot was centrifuged, and the supernatant was blended with an equal volume of 2.5% phosphotungstic acid in 2 N HCl. After 20 min the mixture was centrifuged again and the supernatant was counted to quantitate excreted degraded ligand (Cl). The second aliquot was chilled to 4”C, incubated with digitonin for 20 min, and centrifuged; the supernatant was treated as above to quantitate total degraded r3H]ASOR. Intracellular degraded ligand (U) was equal to the difference (total degraded - excreted degraded). Data represent the average value of duplicate determinations from one representative of five similar experiments.

228

GIL-FALGON

degradation mechanism in diabetic cells (Fig. 4D) and was unable to superimpose any alteration onto that already present as a consequence of the pathology dysregulations. (Note that on Figs. 4C and 4D the scale is one-tenth that of Figs. 4A and 4B.) Effect of Vasopressin on the Degradation Synchronous Wave of Ligand

ex-

1.

Anderson, R. G., and Kaplan, J. (1983) in Modern Cell Biology (Satir, B., Ed.), pp. l-52. A. R. Liss, New York. 2. Steinmann, R. M., Mellman, I. S., Muller, W., and Cohn, Z. (1983) J. Cell Biol. 96, l-27. 3. Goldstein, J. L., Anderson, R. G. W., and Brown, M. S. (1979) Nature 279,679-685. 4. Pastan, I. H., and Willingham, M. C. (1981) Anna. Rev. Physiol.

42,239-250. 5. Schwartz, A. L. (1990) Annu. Reu. Immunol. 8, 195-229. 6. Ashwell, G., and Harford, J. (1982) Annu. Reu. Biochem. 51, 531-557. I. Schwartz, A. L. (1984) CRC Crit. Reu. Biochem. 16, 207-233. 8. Scarmato, P., Durand, G., Agneray, J., and F&per, J. (1986) Biol. Cell. 56, 255-258. 9. Berg, T., Blomhoff, R., Naess, L., Tolleshaug, H., and Drevon, C. (1983) Exp. Cell Res. 148.319-330.

Received August 13,199l Revised version received November

18. 1991

R. (1985)

123, 243%

252. 13.

Clarke, B. L., and Weigel, P. H. (1985) J. Biol. Chem. 260,

128-

133. J. Biol. Chem. 265,629-635.

REFERENCES

M. D., and Baezinger,

12.

Berg, T., Kindberg, G. M., Ford, ‘I’., and Blomhoff, Exp. Cell Res. 161, 285-296. Oka, J. A., and Weigel, P. H. (1987) J. Cell. Physiol.

15. Tolleshaug,

This work was supported by grants from INSERM (Contrat terne) and Minis&e de 1’Education Nationale (rhseau).

Fiete, D., Brownell, Chem. 258,817-823.

11.

14. McAbee, D. D., Clarke, B. L., Oka, J. A., and Weigel, P. H. (1990)

of a

After 60 min of incubation there was a 48.7 c 2.8% inhibition of the degradation of a synchronous wave of ligand internalized by cells incubated with vasopressin, relative to controls (data not shown). These results are in good agreement with the hypothesis that, in the presence of the hormone, degradation was altered in two ways, not only as a logical consequence of the inactivation of 20% of the surface receptors, but also by some direct effect on the mechanism involved. Vasopressin had no further apparent effect on the degradation mechanism in diabetic cells. In conclusion, our results suggest that the second messengers which transduce the vasopressin signal play a role in the regulation of some steps of endocytosis, i.e., the degree of activity of endocytosed receptors, and some mechanism of degradation. The use of drugs with selective effects will be of help in determining the specific sites of hormone action. Only a subset of receptors was affected, in good agreement with the existence of two subpopulations of receptors differing by their sensitivity to modulations. In diabetic rat hepatocytes, there was no further inactivation or alteration, suggesting that the modulationsensitive receptors--the so-called R2 receptors-had already been altered by the pathology.

10.

ET AL.

J. U. (1983) J. Biol.

H., Kolset, S. O., and Berg, T. (1985) Biochem. Pharmacol. 34,1639-1645. 16. MC Abee, D. D., and Weigel, P. H. (1987) J. Biol. Chem. 262, 1942-1945.

17. MC Abee, D. D., and Weigel, P. H. (1988) Biochemistry27,20612069. 18. 19.

Fallon, R. J., and Schwartz, 15,081-15,089. Fallon, R. J., and Schwartz,

A. L. (1986) J. Biol. Chem. 261, A. L. (1987) Mol. Pharmacol.

32,

348-355. 20. Fallon,

R. J., and Schwartz, 13,159-13,166.

21.

A. L. (1988) J. Biol. Chem. 263,

Casey, C. A., Kragskow, S. L., Sorell, (1987) J. Biol. Chem. 262,2704-2710.

M. F., and Tuma,

22. Appel, M., Potrat, P., F&per, J., Mas-Chamberlin, and, G. (1986) Diabetologia

D. J.

C., and Dur-

29, 383-387.

23. Durand,

G., Dumont, J. P., Appel, M., Durand, D., Davy, J., FBger, J., and Agneray, J. (1980) Horm. Metab. Res. 12.247-251. 24. Dodeur, M., Durand, D., Dumont, J., Durand, G., Fbger, J., and Agneray, J. (1982) Eur. J. Biochem. 123, 383-387.

25. Slama, A., Zinbi, H., FBger, J., and Dodeur, M. (1988) Biol. Cell. 63,367-369. 26. Weigel, P. H. (1987) in Vertebrates Lectins (Olden, K., and Parent, J. B., Eds.), pp. 65-91, Van Nostrand

Reinhold,

New York.

27. Nishizuka, Y. (1986) Science 233,305-312. 28. Berridge, M. J. (1987) Annu. Reu. Biochem. 56,159-193. 29. Wilder, R. L., Yen, C. C., Subbarad, B., Woods, V. L., Alexander, C. B., and Mage, A. G. (1979) in Methods in Enzymology (Ginsburg, V., Ed.), Vol. 28, 255-263, Academic Press, San Diego. 30. Berry, M. N., and Friend, D. S. (1969) J. Cell. Biol. 43,506-520. 31. Davy, J., Appel, M., Biou, D., FBger, J., and Agneray, J. (1983) Biol. Cell. 48, 203-206. 32. Seglen, P. 0. (1973) Exp. Cell Res. 76, 25-30.

33. Fishman, J. B., Dikey, B. F., Bucher, N. L. R., and Fine, R. E. (1985) J. Biol. Chem. 260,

12,641-12,646. R. H. (1985) in Molecular Mechanisms of Transmembrane Signalling (Cohen, P., and Houslay, M. D., Eds.), pp. 3-56, Elsevier, New York. Exton, J. H. (1988) FASEB J. 2, 2670-2676. Garrison, J. C., Johnsen, D. E., and Campanile, C. P. (1984) J. Biol. Chem. 259,3283-3292. Vokes, T., and Robertson, G. L. (1985) in Vasopressin (Schrier, R. W., Ed.), pp. 271-279, Raven Press, New York. Charlton, J. A., Thompson, C. J., and Baylis, P. H. (1987) J. Endocrinol. 116, 343-348. Bushfield, M., Griffiths, S. L., Murphy, G. J., Pyne, N. J., Knowler, J. T., Milligan, G., Parker, P. J., Mollner, S., and Houslay, M. D. (1990) Biochem. J. 271, 365-372.

34. Downes, C. P., and Michell,

35. 36. 37. 38. 39.

40. Weigel, P. H. (1980) J. Biol. Chem. 255,6111-6120. 41. Weigel, P. H., Ray, D. A., and Oka, J. A. (1983) Anal. Biochem. 133.437-449.

Effects of vasopressin on receptor-mediated endocytosis of asialoglycoprotein by hepatocytes from normal and diabetic rats.

The hepatic asialoglycoprotein receptor is a membrane glycoprotein used as a model to study receptor-mediated endocytosis. In order to examine the abi...
674KB Sizes 0 Downloads 0 Views