Degradation of glucagon receptors by dispase treatment of isolated intact rat hepatocytes

218

Bioehimlca et Biopl~vsi('a A eta. 721 (1982) 218 222 Elsevier Biomedical Press

BBA Report BBA 10004

DEGRADATION OF GLUCAGON R E C E P T O R S BY DISPASE T R E A T M E N T OF ISOLATED INTACT RAT H E P A T O C Y T E S VFRA PINGOUD, T H E O D O R HAAS and IVAR T R A U ' I S C H O L D

Zenlrum Biochemie. Medizinische ttochschule ttannot~er, Kon.~tantv-(;utschow-Str. ,Y. D-3000 ttannot'er 61 (t'~R.G.) (Received February 8th, 1982) (Revised manuscript received June 15th, 1982)

Key wordY. Glucagon," Hormone receptor," Protein degradation," Dispase; (Rat hepatocvte)

Collagenase-isolated rat hepatoeytes were treated with dispase I1, a neutral proteolytic enzyme which is often used for the disintegration of neonatal cells. The treatment of hepatocytes with dispase !! caused a significant reduction of glueagon binding to the intact cells. The deleterious effect of this enzyme on the specific glucagon binding sites is accompanied by a reduction of the maximum intracellular cyclic A m P production.

Little is known about the structure of the glucagon receptor in the liver plasma membrane. As all other peptide hormone receptors that have been tested so far, it is susceptible to degradation by some proteolytic enzymes, suggesting that it has a protein component which is necessary for hormone recognition. In addition, an important role for membrane phospholipids has been suggested for the glucagon receptors, which exhibit decreased hormone binding and adenylate cyclase stimulating capacity when treated with phospholipase A [1]. The glucagon receptor, therefore, possibly is a lipoprotein. In the present study, we have investigated the effect of dispase treatment of isolated intact hepatocytes on glucagon binding. Dispase is an unspecific neutral protease which cleaves Phe-Val, His-Leu, Glu-Ala, Ala-Leu, LeuTyr, Leu-Val, Gly-Phe, Phe-Phe, Phe-Tyr, and Lys-Ala. The observed effects have been compared to those of collagenase and a combined collagenase/dispase treatment. The glucagon binding to hepatocytes was tested by competition experiments with ~25I-labelled and unlabelled glucagon, yielding the dissociation constants, Kj, and the concentration of binding sites R. In parallel, the levels of endogenous cyclic 0167-4889/82/0000 0000/$02.75 ' 1982 Elsevier Biomedical Press

AMP under glucagon-stimulated conditions were investigated. The results demonstrate that the glucagon receptor as judged by glucagon binding and adenylate cyclase stimulation, is highly sensitive to dispase and insensitive to collagenase. Consequently, dispase should not be used for the isolation of hepatocytes whenever the effect of glucagon is the object of the study in question. Crystalline porcine glucagon was obtained from NOVO, Copenhagen. Carrier free Na~25I was purchased from Amersham Buchler, Brunswick. ~25I-labelled glucagon was prepared with a specific activity of ( 4 - 7 ) - 1 0 5 C i / m o l (0.2-0.3 atoms of ~25I per molecule of glucagon) by the chloramine-T method [2,3]. Eagle's minimal essential medium, 2'-O-succinyltyrosylmethyl ester-adenosine-3',5'monophosphate and calf serum (the same batch throughout the experimental work) were obtained from Boehringer Mannheim. t25I-labelled succinyltyrosylmethyl ester-cAMP was prepared with a specific activity of (2-3). 10 5 C i / m o l by the chloramine-T method [4]. The specific cyclic AMP antibody was kindly supplied by Dr. W. Schmitz. Department of Pharmacology and Toxicology, Medizinische Hochschule Hannover. Collagenase (type I, spec. act., 160 U / m g , batch 68C-0595)

219

was obtained from Sigma, St. Lious; dispase II (spec. act., 0.5 U / m g , batch 1111413) and collagenase/dispase (spec. act., 0.8 U / m g , 0.1 U / m g , batch 1408101) from Boehringer-Mannheim; bacitracin and theophyllin from Serva, Heidelberg; silicone oil (AR20 and AR200) from Wacker-Chemie, Mimchen; and Percoll T M from Pharmacia, Uppsala. All other reagents used were obtained from Merck, Darmstadt and were of the highest available commercial grade. Hepatocytes of adult fed (3 months old, 160 200g) male Han:Wistar rats were prepared by the perfusion technique with 80 U / m l collagenase I or with 1.8, U / m l dispase according to Seglen [5]. The parenchymal cells were incubated in Eagle's minimal essential medium supplemented with 10% calf serum as control or with the addition of dispase (1.8 U / m l ) or collagenase (80 U / m l ) or dispase/collagenase (0.8 U / m l , 0.1 U/ml). The incubations were performed for 30 rain at 37°C. After the enzymatic treatment, the suspensions of liver cells were purified by isopycnic centrifugation through a continuous Percoll gradient [6]. The density gradient of 40.5% (v/v) Percoll in Eagle's minimal essential medium was generated by centrifugation at 15000 × g for 20 min in a fixedangle rotor (Beckman, J2-21). Cell suspensions containing 5. 10 6 cells/ml were layered on top of a 40 ml gradient and a further centrifugation was carried out for 30 min at 100 × g in a swing-bucket rotor (Haereus-Christ, U J1). Intact parenchymal cells banded at a density of 1.075 1.090 g / m l as determined refractometrically or using marker beads, whereas non-viable parenchymal cells, non-parenchymal cells, and cell fragments banded at a lower density, 1.030 1.060 g/ml. After removal from the gradient, the cells were washed with suspension buffer [5] and finally suspended in Eagle's minimal essential medium supplemented with 10% calf serum, 0.7 mM bacitracin and 5 mM theophyllin. 85-90% of the cells proved to be viable as judged by Trypan blue exclusion. Binding of 1~-SI-labelled glucagon to isolated hepatocytes was carried out by the following procedure: 400 /.tl of the cell suspension (1.8. 10 6 cells/ml) were added to 50/L1 125I-labelled glucagon (17 nmol/l) and 50 /xl Eagle's minimal essential medium or unlabelled glucagon (10 n m o l / l - 1 0 0 ~tmol/1). The incubation was carried

out at 37°C for 30 min (pH 7.4) and was terminated by transferring 100-/~1 aliquots of the incubation mixture in duplicate to microtubes containing 200 /~1 of a 1:1 mixture of silicone oils AR20 and AR200. The membrane-bound hormone was isolated by centrifugation through the silicone oil in an Eppendorf laboratory centrifuge at 10000 × g for 30 s. Separation of the cells from the incubation medium was complete within seconds after the centrifugation was begun. The microtubes were frozen in liquid nitrogen and cut through the oil layer. Cell bound and free hormone were counted in a gamma-counter (Berthold, LB MAG 510). Evaluation of the measured data was performed by a computerized curve-fitting procedure [10]. Endogenous cyclic AMP was determined by radioimmunoassay. For this purpose, 1 vol. of the cell suspension was extracted with 4 vol. of methanol. The extract was evaporated to dryness and solubilized in acetate buffer (50 mmol/I) [7,8].

4500

2.3

• °Oo z 2700

E

g 900

10-10

/

10-9

10-8 10-7 10-6 Glucagon (tool/I)

10-5

Fig. I. Effect of a 30 min dispase digestion of intact hepatocytes on the binding of glucagon. The binding data are expressed in two different ways: (1) The activity of bound 125i_ labelled glucagon (cpm/10/zg DNA) is plotted vs. the total glucagon concentration. The solid lines are computer generated curves fitting the points obtained assuming one class of binding sites. Points represent the control competition experinaent without enzyme ( 0 ) and squares the experiment with dispasetreated hepatocytes (m). (2) Dashed lines represent binding isotherms calculated from the theoretical solid curve, demonstrating the concentration dependence of glucagon binding (glucagonbound) to untreated ( O ) or dispase-treated hepatocytes (D). The numerical evaluation yields K d 38 nM, R 1.10 nM for glucagon binding to hepatocytes incubated without dispase and K d 30 nM. R 0.21 nM for glucagon binding to hepatocytes incubated with dispase.

220

The D N A content of the hepatocytes was assayed by a fluorimetric method similar to that described by Hesse et al. [9]. The influence of different enzymatic digestion methods on the specific glucagon binding to hepatocytes was tested on adult rat liver parenchymal cells initially isolated by collagenase treatment. The cells were incubated either with collagenase (80 U / m l ) , collagenase/dispase (0.1 U / m l , 0.8 U / m l ) or with dispase II (1.8 U / m l ) up to 30 min at 37°C. Equilibrium binding studies were performed by competition experiments at 37°C with 12~l-labelled and unlabelled glucagon over the concentration range of 1.7 - 10 9 m o l / l to 1 • 10 5 mol/l. The binding data were analyzed by a nonlinear least-squares curve fitting procedure as described previously [10]. The data were evaluated on the assumption of two different binding models: (1) one class of independent binding sites, (II) two classes of independent binding sites. The comparison of the best fitted theoretical curves to our binding data reveals that both binding models are compatible with the data within the range of error. Fig. 1 represents two typical competition experiments, one performed after the enzymatic treatment of hepatocytes with dispase II and the other one after a control incubation without enzyme for 30 min at 37°C. The displacement curves and the binding isotherms are theo-

retical curves according to the binding model of one class of independent receptors. We found that the collagenase-treated cells retained their full glucagon binding capacity, whereas exposure to collagenase/dispase or dispase caused a significant reduction of glucagon binding sites (Table I). We further investigated the influence of the enzymatic digestion on the intracellular response. The binding of glucagon to a specific receptor results in the activation of adenylate cyclase. Therefore, the extracellular glucagon binding and the intracellular cyclic A M P production was measured in parallel. The dose-response of glucagonstimulated cyclic A M P accumulation was studied with increasing concentrations of glucagon. Experiments were carried out such that the cyclic A M P content of hepatocytes was measured in the same vial and at the same time as the glucagon binding in order to minimize experimental errors and thereby to allow a meaningful correlation. The cyclic A M P levels were higher in untreated or collagenase-treated hepatocytes than in collagenase//dispase - or dispase-treated cells at concentrations of glucagon tested from 1.7-10 ~ m o l / l to 1 • 10 5 mol/l. The maximal cyclic A M P accumulation was achieved with glucagon concentrations of 75 nmol/1 in all experiments (Fig. 2). Untreated or collagenase-treated (30 min) cells

30-

TABLE 1

~c z

EFFECTS OF COLLAGENASE, C O L L A G E N A S E / D I S PASE, A N D DISPASE ON THE TOTAL G L U C A G O N RECEPTOR C O N C E N T R A T I O N IN ISOLATED INTACT RAT H EPATOCYTES

~ o

3 Data are given for three cell preparations from three different animals. Since binding parameters vary somewhat depending on the cell preparation, for each 30-rain experiment a control experiment was carried out with the same cell preparation without enzyme. This control experiment without enzyme represents the 100% value of receptor concentration.

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•

10-

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f

I

r

i

i

10-9

10-8

10-7

10-6

10-5

Glucagon ( m o l / I Receptor concn. (%) Minimal medium Collagenase Collagenase/Dispase Dispase

100 + 18 103 + 21 61 + 10 19~: 6%

•

o.

I

Fig. 2. Effect of dispase treatment on the glucagon-stimulated cyclic AMP production in isolated intact hepatocytes. After 30 min of incubation in Eagle's minimal essential medium with or without dispase, the cells were separated and incubated with glucagon. 0.2 ml aliquots were removed for the determination of cyclic AMP by radioimmunoassay. Untreated cells, O: dispase-treated cells, I I .

221 TABLE II EFFECTS OF COLLAGENASE, C O L L A G E N A S E / D I S PASE, A N D D1SPASE ON THE MAXIMAL CYCLIC AMP P R O D U C T I O N IN ISOLATED INTACT HEPATOCYTES A F T E R G L U C A G O N STIMULATION The maximal cyclic AMP production is expressed as the value in percent after subtraction of the basic cAMP content obtained in the absence of glucagon. Results are the average of three experiments. cAMP production/10 ~g DNA

MEM Collagenase Collagenase/Dispase Dispase

15 rain

30 min

100~7 98 + 5 93 + 5 68 + 6

100+6 102 +- 8 69 + 5 35 + 4

were stimulated to produce maximal 23 pmol c A M P / 1 0 /,g D N A over and above the basic cAMP content, whereas collagenase/dispasetreated (30 min) cells accumulated 16.5 and dispase-treated hepatocytes only 8.9 pmol cAMP,/10 /,g DNA (TAble II). The reduced cyclic AMP production in dispase II-treated cells correlates directly with a decreased binding of glucagon. However, the reduction in the binding is somewhat higher than the reduction of cyclic AMP production. We suggest that this slight discrepancy is due to the fact that the determination of receptor concentration is much more error prone than the measurement of the intracellular cyclic AMP concentration. Coilagenase and dispase, alone or in combination, are widely used for the disintegration of various tissues in order to obtain intact ceils for the study of, for example, the interaction between hormones and receptors. We have been specifically interested in the binding of glucagon to hepatocytes and the concomitant stimulation of the adenylate cylase. It is mandatory for such a study that the enzymes used for the isolation of the cells of interest do not affect the receptor or its immediate environment. This can most easily be checked by a mild digestion of liver using one of these enzymes followed by a further incubation with these enzymes mixtures during which the hormone binding and cAMP formation is monitored. A time-dependent decrease in hormone bi-

nding or cAMP formation then would indicate that the enzyme or enzyme mixture used has a deleterious effect on the integrity of the receptor or its surroundings, thereby affecting the accessibility of the hormone for its receptor and coupled reactions. Our data show that hepatocytes which have been isolated with collagenase exhibit a decrease in glucagon binding and cAMP formation when treated for 30 min with a commercial preparation of dispase (1.8 U / m l ) and collagenase/dispase (0.1 u / m l , 0.8 U / m l ) ; but not when incubated with collagenase alone. We conclude that collagenase does not significantly affect the binding of glucagon to hepatocytes and the concomitant adenylate cylase stimulation; but that dispase does. This result is further substantiated by experiment,; in which hepatocytes were isolated by dispase digestion: under these conditions a very low level of glucagon binding and cAMP formation is obtained which is further reduced by dispase but not by collagenase treatment. Although both collagenase and dispase are specifically recommended for tissue disintegration and cell isolation respectively, only collagenase can be used for the isolation of hepatocytes, when glucagon binding to these cells is studied, Possibly, similar problems arise when other hormone-receptor interactions are investigated. Therefore, experiments such as reported in this paper should be carried out prior to extensive investigations. We thank Ms. M. Petry for expert technical assistance. This work was supported by the Gesellschaft der Freunde der Medizinischen Hochschule, Hannover. References 1 Rodbell, M., Krans, H.M.J., Pohl, S.L. and Birnbaumer, L. (1971) J. Biol. Chem. 246, 1861-1871 2 Hunter, W.M. and Greenwood, F.C. (1962) Nature 194, 495-496 3 Pohl, S.L. (1976) in Methods in Molecular Biology (Blecher, M., ed.), pp. 159-174, Dekker, New York 4 Steiner. A.L. (1979) in Methods of Hormone Radioimmunoassay (Jaffe, B.M. and Behrmann, H.R.. eds.), 2nd Edn., pp. 3-17, Academic Press, New York 5 Seglen, P.O. (1973) Exp. Cell Res. 82, 391-398 6 Pertoft, H., Rubin, K., Kjellen, L., Laurent, T.C. and Klingeborn, B. (1977) Exp. Cell Res. 110, 449-457

222 7 Rosselin, G., Freychet, P., Fouchereau, M., Rancon, F. and Broer, Y. (1974) Horm. Metab. Res. Suppl. Series 5, 78- 86 8 Broer, Y. Freychet, P. and Rosselin, G. (1977) Endocrinology 101, 236 249

9 Hesse, G., Lindner, R. and Krebs. D. (1975) Z. Allg, Microbiol. 15, 9-1g 10 Peters, F. and Pingoud, A. (1979) lnt, J. Bio-Med. Computing 10, 401 415