Inactivation kinetics of horseradish peroxidase microinjected into hepatocytes from mice of various ages

Mechanisms of Ageing and Development, 46 f1988) 125-133

125

Elsevier Scientific Pubfishers Ireland Ltd.

[NACTIVATION KINETICS OF HORSERADISH PEROXIDASE MICROINJECTED INTO H E P A T O C Y T E S FROM MICE OF VARIOUS AGES

AKIHITO ISHIGAMI and SATARO GOTO* Department of Biochemisa'y, School o[ Pharmaceutical Science, Toho University, Miyama 2-2-1. Funabashi, Chiba 274 (Japan) (Received June 3rd, 1988) (Revision received August 6th, 1988)

SUMMARY

We have studied degradation of proteins in liver parenchymal cells in culture obtained from mice of various ages. Horseradish peroxidase (HRP) was microinjected into the cells by the hypertonic-hypotonic method of Okada and Rechsteine~~ (CeU, 29 (1982) 33-41). Rate ~ inactivation of HRP was examined biochemic=ally. Average functional half-lives of the enzyme were 50 h, 53 h and 75 h in the cells of young (3-8 months old), middle-aged (18-20 months old) and old (28-30 months old) mice, respectively. Thus, half-life of the inactivation of the enzyme in "old" ceiIs was about 50% longer than that in "young" or "middle-aged" cells, suggesting that rate of protein degradation is lowered in senescent animals.

Key words: Mouse hepatocyte; Aging; Microinjection; Protein turnover: Horseradish peroxidase INTRODUCTION

While functional deterioration is a general occurrence in cells and tissues of aging animals, no commoa mechanism of deterioration (if, indeed, there is one) is known. Accumulation of altered proteins in one candidate for such a mechanism because age-related alterations could occur in any cellular proteins such as metabolic enzymes, cytoskeletal proteins, receptors or membrane proteins, in fact, many enzymes are known to change with advancing age [2,3]. We have reported that the amount of heat-labile forms of enzymes involved in

*To whom all correspondence should be ad,~'essed. 004%6374]88/$03.50 Printed and Published in ire|and

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© 1988 Elsevier S~ientific Publishers Ireland Ltd.

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translation is increased in tissues of old mice and rats [4-6]. It should be noted that heat-labile aminoacyi tRNA synthetases decrease under catabolic conditions of dietary restriction [7]. Thus, although it is not known how altered proteins are generated and whether the rate of formation of these proteins is different between young and old animals, it possibly emerges that the turnover rate of proteins slows with age and that this can be reversed by nutritional and perhaps other means. Sharma et al. [8], Lavie et al. [9] and Reznick et ai. [10] have found by pulse-chase experiments that the rate of degradation of proteins in nematodes and mice is slower in old animals than in young ones. They have studied degradation of labeled proteins synthesized in vivo. In these instances one is not sure that the same proteins are being examined in young and old animals. Nor is it easy in such in vivo experiments to study degradation of altered proteins per se. We therefore examined the degradation rate of proteins microinjected into cells derived from young and old animals by the hypertonic-hypotonic method of Okada and Rechsteiner [1]. This method enabled us to check the turnover of the same proteins in different cells, and also to study the degradation of proteins modified in vitro [ 11,12]. Here, we report on inactivation kinetics of horseradish peroxidase (HRP) microinjected into mouse liver parenchymal cells in primary cultured derived from mice of various ages. MATERIALS AND METHODS

Animals

Male BDFI mice (FI hybrids of C57BL/6N and DBAI2N) were obtained from Charles River Japan, Inc. and maintained as described [4]. The mice had a mean life-span of about 27 months (data obtained from Charles River Japan, Inc.). Chemicals

Calf serum (CS) was obtained from Flow Laboratories, USA and medium 199 was obtained from Nissui Pharmaceutical Inc. Japan. Insulin, dexamethasone, horseradish peroxidase (HRP), glutaraldehyde, diaminobenzidine and o-dianisidine were from Sigma, USA. Sucrose, coUagenase and bovine serum albumin (BSA) were from Wako Pure Chemicals, Japan. Polyethylene glycol (PEG) 1000 was from Koch-Light Laboratories, Ltd. USA and plastic dishes (~ 60 mm) were from Corning Inc., USA. Isolation and culture of hepatocytes Hepatocytes were isolated by in situ perfusion of the liver with collagenase

[I 3]. Mice were lightly anesthetized by intraperitoaeal injection of Somnopentil {sodium pentobarbit~l 64~8 mg/ml, Pit~,lan-Moore, Inc., USA) and the livers were perfused with EGTA :~olution containing 0.5raM EGTA, 5.5ram glucose, 142 mM NaCI, 6.7 mM KCI, 10 mM HEPES (pH 7.4), 1 p.M insulin, 0.1 mg/ml

127 Kanamycin and then with coUagenase solution containing 0.05% collagenase, 5ram CaCI2, l l 7 m M NaCI, 6.7mM KCI, 30mM HEPES (pH 7.6), 5.SmM glucose, 0.005% trypsin inhibitor, 1/~M insulin, 0.1 mg/ml Kanamycin, 1% BSA at 15m l/rain for 10-20min. Dispersed cells were suspended in medium 199 containing 10% calf serum, 1/~M insulin and 10 ~tM dexamethasone and the suspension was centrifuged at 10 g for 5 min. Final cell pellets obtained after three cycles of suspension-centrifugation were suspended in medium 199 and the cells were plated in 60-ram plastic dishes at a concentration of 2 × 106 cells/dish. The cells were cultured in a CO2 incubator (95% 02 and 5% CO2). After 2 h of culture, the cells which did not attach on the dishes were washed out and very few dead cells remained during injection and chase. Cell viability was determined by trypan blue exclusion test just before the cells were plated.

Microinjection o[ HRP into hepatocytes HRP was injected into hepatocytes by osmotic lysis of pinosomes, a method developed by Okada and Rechsteiner [1]. The cells were exposed to a hypertonic medium containing 0.5 M sucrose, 10% polyethylene glycol 1000 and 4mg/ml HRP in medium 199 for 10min at 37°C. Next, they were incubated in a hypotonic medium (6:4 mixtule, of medium 199 and H20) for 2 min at 37°C. The plates were then washed three times with a standard isotonic 199 medium. To ensure elimination of any HRP still attached to the cell surface, they were incubated for 2 h before the first sample was taken. At various times the ceils were harvested and iysed by repeated freeze-thawing in 0~1 M phosphate buffer (pH 5.0) and stored at -20°C until use.

Assay [or HRP HRP activity in the cell lysates was determined using o-dianisidine as a substrate [14]. Four hundred microliters of the cell lysate was mixed with 2.1 ml of buffer containing 0.1 mM o-dianisidine, 0.8 mM H202, 0.1 M phosphate buffer (pH 5.0) in a quar~ ¢uvette in a spectrophotometer at room temperature and the rate of color development was recorded at 460 nm for 1-3 rain. For each assay, rates of color development in stalldard reaction mixtures containing 0-10 ng/mi of HRP were determined. Amount of HRP in celt lysates was calculated using a standard curve of the relation between the concentration of HRP and the rate of color development which turned out to be linear (Fig. 1).

Histochemicai detection of HRP The cells were washed once with PBS(-) and fixed with 3% paraformaldehyde in PBS(-) for 30 min at 37°C. After being washed with PB$(-) three times, the fixed cells were stained with 0.5% diaminobenzidine solution containing 50 mM Tris-HC! (pH 7.6) and 0.01% [']202 for 2 h at 37°C [15].

128

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Fig. I. Standard curve for the correlation between HRP concentration and the enzyme activity. Rate ,ff oxidation of o-dianisidine in the presence, of H202 was determined at various concentrations of HRP. Extent of oxidation was monitored by measuring the increase in the absorbance at 460 nm tA4~o) for 180 s. Ordinate: enzyme activity (A45J i 00 s), Abscissa: concentration of HRP.

RESULTS

Microi,#ction of HRP The viability of the cells obtained from young (3-8 months), middle-aged (18-20months) and old (28-30 months) mice was 55.24-6.1% ( n = 6 ) , 63.1 45.4% (n = 6) and 63.6+ 5.3% (n = 5), respectively. The viability seems to be low as compared to the case for hepatocytes isolated from rat liver, which is reported to be more than 85%. Viability of rat hepatocytes isolated by our hands using a method similar to the one employed in this study was also more than 85%. Thus, lower viability of the mouse hepatocyte seems to be due to some inherent properties of the cells or the liver of mouse but not due to the method or technique employed. However, we cannot explain the reason for the difference between rat and mouse. Amount of DNA per dish and hence the number of cells did not change for at least 5 days in culture (data not shown). Under our experimental conditions the hepatocytes did not divide. The cells continued to synthesize albumin and some other serum proteins and secrete them into the medium for at least 5 days (Ikeda, T. and Goto, S., in preparation). Proteins have been microinjected by osmotic procedure into cells in culture such as mouse L929 cells [l], rat hepatoma cells [16] and IMR-90 human fibroblasts [ 11]. We have applied this procedure to mouse liver parenchymal cells in primary culture. By the standard procedure described in Materials and Methods I-3 pg/10 cells (50-150 ngidish) of HRP was successfully loaded into

129 the hepatocytes. There was no significant difference of the amount of HRP injected between cells derived from young and old animals. The procedure was without any significant effects on cell viability as judged by trypan blue dye exclusion and activity of protein synthesis as determined by [3H]leucine incorporation into cellular proteins (data not shown). Thus, the osmotic procedure was demonstrated to be useful for injecting proteins in a given amount into mouse hepatocytes in primary culture without apparent harmful effects. Histochemical analysis by diaminobenzidine staining [15] revealed that the cytoplasm of most of the injected cells contained HRP, being evenly stained in brownish orange, but not the nucleus. Non-injected cells were not stained. The injected cells appeared to contain varying amounts of HRP as judged by the intensity of the color. To examine the possibility that HRP once incorporated in the cells is excreted into the medium and inactivated there, H R P was added to the culture and the enzyme activity was determined after 60 h of incubation under standard culture conditions. The enzyme activity remained the same as that before incubation (data not shown). Thus, even if excreted, H R P is not inactivated in the medium with hepatocytes nor re-incorporated into the cells during the chase period.

Assay of HRP Under standard assay conditions, an amount of HRP as low as I ng/ml can be reliably determined in the presence of hepatocyte extracts (Fig. I). Hepatocytes are considered to have high activities of catalase and glutathione peroxidase which might interfere with the assay of H R P microinjected. But virtually no peroxidase activity was detected under our assay conditions in the extracts prepared from non-injected hepatocytes. Thus, HRP can sez:ve as a good model enzyme to study protein degradation in hepatocytes as is true in established non-hepatic cells [1,1 i ,16]. lOO

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Fig. 2, The inactivationkinetics of HRP microinjectedinto (a) youngand (b) old mouse hepatocytes, Results of typical experiments are shown for the celts derived from two young and two old mice. Inactivation was regarded as to follow the first-order reaction and the regression lines were obtained by the ]east squares method. Unbroken lines are regressionlines for the closed circles (@) &,~,dbroken lines are those for the open circles (O). Half-liveso1 inactivationare shown.

130

TABLE I FUNCTIONAL HALF-LIFE OF HRP MICROINJECTED CULTURE FROM YOUNG, MIDDLE AND OLD MICE

~ge

(months) YOUNG

MIDDLE AGE

OLD AGE

3 3.5

HEPATOCYTES

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46 57

18 19

80

20 20 20

49 40 44

27 27 28 29 30

69 94 75 58 78

53 + 5 (6)

75 ± 5 (5)

aValues are expressed as mean :e standard error. bNumbers in parentheses indicate the number of mice examined.

Age-related change in the functional half-life of HRP Typical examples of inactivation kinetics of HRP in young and old hepatocytes in culture are shown in Figs. 2a,b. Half-lives of the enzyme activity were obtained by drawing regression lines by least squares method. Correlation coefficients were in the range of 0.768-0.966, 0.721-0.942 and 0.618-0.853 for the plots in the young, middle-aged and old mice, respectively. No correlation was found between the viability of the cells and half-life of HRP. Also, we did not find correlation between the amount of HRP and the half-life. Table I summarizes the half-life of HRP in the cells from mice of each age group. The differences between the young or middle-aged group and the old group are significant at P < 0.05 level as analyzed by Student's t-test. DISCUSSION

Microinjection of proteins into mammalian cells in culture by pinocytosis has been successfully applied to established cell lines [1,11,16]. Success of the method depends on the pinocytotic activity and osmotic resistance of the cells.

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We used this method to introduce HRP into mouse liver parenchymal cells in primary culture. Viability and protein synthetic activity did not appear to change after hypertoniclhypotonic treatment in either young or old cells, suggesting that the treatment does not harm these cells. Using erythrocyte gi~ost-raediated method of microinjection, Knowles et al. [17] found that HRP iajected into rat myoblasts, human diploid fibroblasts and HeLa cells has an approximate functional half-life of 12-18h. The functional half-life of HRP in mouse hepatocytes obtained in this work was much longer, i.e. about 5 0 h in young cells and 70h in old cells. We have not s~udieJ the degradation of HRP protein itself; it is therefore possible that the degradation of the protein is different from the decrease in the functional activity. Knowles et al. reported that half-life of the degradation of radioactively labeled HRP is the same as that of the emyme activity [17]. It is therefore not unreasonable to speculate that this is also true for the hepatocytes. We do not, however, know the mechanism accounting for the greater stability of HRP in the hepatocytes. Gershon's and Rothstein's groups have reported that half-lives of pulselabeled proteins in nematode [8] and mouse [9,10] increase with age. Ove et al. [18] studied turnover of total liver proteins and ferritin in young and old rats. While no difference was observed in the turnover rate of total proteins, half-life of ferritin in the old animals was two times longer than that of young animals. The latter observation was confirmed by Obenrader et al. [19]. As to an in vitro aging of cells, Dice [20] compared the rates of degradation of proteins microinjected into the early and late passage IMR-90 human fibroblasts. The half-lives of proteins increased significantly as the fibroblasts aged in culture. We have shown here that the functional half-life of HRP microinjected into mouse hepatocytes increases in old animals by about 50% over that of young and middle-aged counterparts (Table I). If the functional half-life of HRP corresponds to the half-life of the protein, our findings are consistent with those of in vivo experiments by the investigators cited above in that the turnover rate of proteins decreases with age. We recently found that the turnover rate of ovalbumin microinjected into mouse hepatocytes also decreases with age (Ishigami, A. and Ooto, S., in preparation). All these findings are consistent with the idea that the accumulation of altered enzymes in tissues of old animals [2-6] is at least partly due to decrease in the turnover of proteins. Burrows and Davison [21] studied degradation of pulse-labeled proteins of mouse hepatocytes in culture derived from mice of different ages. They determined the rate of protein degradation by measuring the release of a~id-soluble radioactivity from the cells during the first several hours of the experiment. No age-related difference in the rate was found. These findings are not necessarily inconsistent with our present results, because what Burrows and Davison studied is the turnover rate of relatively short-lived proteins, while injected HRP seems to be a long-lived protein in the hepatocytes. There are generally considered to be at least two pathways of protein

132

degradation in mammalian cells, i.e. lysosomal and non-lysosomal [22]. Abnormal proteins are known to be degraded in the non-lysosomal pathway. But it is not clear whether injected HRP is recognized as abnormal or is altered by the cellular systems of protein degradation. It is of interest to determine which system is affected in the older animals, causing a reduction in the turnover rate of injected proteins and probably endogenous altered proteins as well. Tissue culture is a better system by which to pursue this issue than use of the whole animal, because in the former system conditions which influence proteolysis in either of the two pathways can be manipulated in more detail. REFERENCES I C.Y. Okada and M. Rechsteiner, Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell, 29 (1982) 33-41. 2 A.Z. Reznick and D. Gershon, The effect of age on the protein degradation system in the nematoda Turbatrix aceti. Mech. Ageing. Dev., 11 (1979)403-415. 3 D. Gershon, Current status of age altered enzymes: Alternative mechanisms. Mech. Ageing. Dev., 9 (1979) 189-196. 4 R. Takahashi, N. Mori and S. Goto, Alteration of aminoacyl tRNA synthetases with age: Accumulation of heat-labile enzyme molecules in rat liver, kidney and brain. Mech. Ageing Dee., 33 (1985) 67-75. 5 R. Takahashi, N. Marl and S. Goto, Accumulation of heat-labile elongation factor 2 in the liver of mice and rats. Exp. Gemntol., 20 (1985) 325-331. 6 R. Takahashi and S. Goto, Age-associated accumulation of heat-labile aminoacyl-tRNA synthetases in mice and rats. Arch. Gemntol. Geriatr., 6 (1987) 73-82. 7 R. Takahashi and S. Goto, Influence of dietary restriction on accumulation of heat-labile enzyme molecules in the liver and brain of mice. Arch. Biochem. Biophys., 257 (1987) 200-206. 8 H.K. Sharma, H.R. Prasanna, R.S. Lane and M. Rothstein, The effect of age on enolase turnover in the free-living nematode, Turbatrix aceti. Arch. Biochem. Biophys., 194 (1979) 275-282. 0 L. Lavie, A Z. Reznick and D. f,3et3hon, Decreased protein and puromycinyl-peptide degradation in livers of senescent mice. Biochem. J., 202 (1982) 47-51. I0 A.Z. Reznick, L. Lavie, H.E. Gershon and D. Gershon, Age-associated accumulation of altered FDP aldolase B in mice. FEBS Lett., 128 (1981) 221-224. I I J.F. Dice, H.L. Chiang, E.P. Spencer and J.M. Backer, Regulation of catabolism of microinjected ribonuclease A. J. Biol. Chem., 26 ! (1986) 6853-6859. 12 J.M. Backer and J.F. Dice, Covalent linkage of ribonuclease S-peptide to microinjected proteins causes their intracellular degradation to be enhanced during serum withdrawal. Prec. Natl. Acad. Sci. USA, 83 (',986) 5830-5834. 13 P.O. Seglen, Preparation of isolated rat liver cells. Methods Cell. Biol., 13 (1976) 29-83. 14 R.L Steinman, JM. Silver and Z.A. Chon, Pinocytosis in fibroblasts. J. Cell. Biol., 63 (1974) 949-969. 15 R.L. Steinman and Z.A. Cohn, The interaction of soluble horseradish peroxidase with mouse peritoneal mactophages in vitro. J. Cell. Biol., 55 (1982) 186-204. 16 Kozui Shii and R.A. Roth, Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease. Proc. Natl. Acad. Sci. USA, 83 (1986) 4147-4151. 17 S.E. Knowles, M.F. Hopgood and F.J. Ballard, Degradation of horseradish peroxidase after microinjection into mammalian cells. Exp. Cell. Res., 174 (1988) 266-278. 18 P. Ore, M. Obenrader and A. Lansing, Synthesis and degradation of liver proteins in young and old rats. Biochim. Biophys. Acre, 277 (1972) 211-221. 19 M. Obenrader, J. Chem P. Ore and A.I. Lansing, Functional regeneration in liver of old rats after partial hepatectomy. Exp. Gemntoi., 9 (1974) 181-190.

133 20 J.F. Dice, Altered degradation of proteins microinjected into senescent human fibroblasts. Y. Biol. Chem., 257 (1982) 14624-14627. 21 R.B. Burrows and P.F. Davison, Protein catabolism in cultures of hepatocytes derived from mice of various ages. Mech. Ageing. Dev., 19 (1982) 85-94. 22 A. Hershko and A. Ciechanover, Mechanisms of intracellular protein breakdown. Annu. Rev. Biochem., 51 (1982) 335-364.