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Retention of lactate dehydrogenase cortisol binding and lack of enzyme induction in rat liver cell suspensions
0
332
Experimental
1966
by Academic
Cell Research
Press
44, 332-340
Inc
(1966)
RETENTION OF LACTATE DEHYDROGENASE CORTISOL BINDING AND LACK OF ENZYME INDUCTION IN RAT LIVER CELL SUSPENSIONS C. M. TSAI, Oklahoma
State
University,
N. BEST
and K. E. EBNER
Agricultural Experiment Station, Department Stillwater, Okla. 74074, U.S.A.
of Biochemistry,
Received June 10, 1966
THERE
has been considerable interest in the metabolic properties of dispersed rat liver cells and attempts have been made to demonstrate normal liver function in these cells since they would provide an attractive system for the study of liver function. Jacob and Bhargava [13] have examined the incorporation of 32P-phosphate and 14C-adenine into RNA of suspended liver cells. Exton [6] has shown that rat liver cell suspensions do not metabolize carbohydrates but do metabolize citric acid cycle intermediates and oxidize palmitate, largely to ketone bodies [7]. lchihara [la] has shown that 14C-acetate is incorporated into lipids of dispersed rat liver cells. Berry [2] has prepared mouse liver cell suspensions and shown that these cells are able to synthesize urea. A serious limitation of isolated cell preparations has been their failure to utilize glycolytic intermediates [6] and many of the soluble components of the cells, including enzymes, are lost during the preparation procedure. Such losses have been attributed to alterations in the permeability of the cell membranes as evidenced by electron microscopy [3]. The soluble enzymes; aldolase, lactate dehydrogenase, iditol dehydrogenase [6], threonine dehydrase, serine dehydrase, tryptophan pyrrolase [23] and phosphorylase [9] are lost from rat liver cells during the preparation of cell suspensions. Glutamic pyruvate transaminase [23], malate dehydrogenase [B], glutamic oxaloacetic transaminase [10, 23 ], glutamate dehydrogenase [ 10] and glucose-6-phosphatase [lo, 231 are enzymes associated with the particulate portion of the cells and are retained to varying degrees in dispersed liver cells. Nicotinamide nucleotides are essentially lost during the preparation of liver cells [6]. The potential use of isolated liver cells to study normal liver function especially the hormonal induction of enzymes, prompted the present studies. Cortisol and RNA were examined for their ability to induce enzyme formation in rat liver cell suspensions. The binding of 14C-coritsol and the retention of Experimental
Cell Research
44
Studies with
rat liver cell suspensions
333
liver enzymes in the cell suspensions were also examined. The studies showed that tyrosine transaminase and glucose-6-phosphatase were not induced by cortisol or RNA. Lactate dehydrogenase could be retained in isolated rat liver cells under certain conditions whereas other soluble enzymes were lost during the preparation procedure. METHODS Preparation of rat liver cell suspensions.-Liver cell suspensionswere prepared from male rats (Holtzman) weighing between 150 and 300 g. The animals were anesthetized with ether and the liver was perfused via the portal vein to remove the blood. Various perfusion media were used and perfusion was carried out at 37°C. The liver was removed, cut into small pieces and then gently forced through a tissuepress(Harvard Apparatus). Two grams of the liver were added to 9 ml of homogenizing medium (4°C) and gently homogenized by hand in a loosefitting teflon on glasshomogenizer (A. Thomas, type B, teflon pestle and a 19.5 mm internal diameter test tube was used). The crude cell suspensionwas filtered through 4 layers of cheese-cloth to remove connective tissue and large aggregates. The filtrate was centrifuged at 120 x g for IO min at 4°C. The supernatant solution was designated asthe “cell supernatant”. The cell pellet was washed twice with two volumes of the dispersion medium and the cells were suspendedin 5 ml of incubation medium. Enzymic assays.-The following enzymes were assayed by standard procedures: lactate dehydrogenase [181, tyrosine transaminase [ 191, 3-hydroxyanthranilate oxygenase [5], glucose-6-phosphatase [22]. In the tyrosine transaminase assay, 10 mM azide was used to replace the diethydithiocarbamate which formed insoluble chelates with Ca2+and MgZ+ present in the perfusion medium. The assayswere linear with time and rates were proportional to enzyme concentration. The assays were conducted at room temperature in a Cary Model 14 spectrophotometer. Binding of 14C-4-cortisoI.-14C-4-Cortis01,specific activity 45.0 mc/mM was a product of New England Nuclear. The W-4-cortisol was dissolved in ethanol and 1 to 10 ,IL~ were usedin each assay. An equal volume of ethanol was added to controls. Cells and cellular fractions, isolated by differential centrifugation [ll] were solubilized with nitric acid [21] and aliquots were counted at infinite thickness in a Baird Atomic Automatic Planchet Counter. RESULTS
Attempted induction of tyrosine transaminase and glucose-6-phosphatase In rats, tyrosine transaminase activity may be stimulated 3 to 4 fold by the administration of cortisol [ 151 and it has been shown by immunological techniques that the increase in enzymic activity is due to the induction of specific enzymic protein [14]. These observations prompted attempts to induce tyrosine transaminase in isolated liver cells, since such a system would be free from whole animal complications. Experimental
Cell Research
44
334
C. M. Tsai,
N. Best and K. E. Ebner
The rat livers were perfused and homogenized with a divalent ion-free solution containing in g/l NaCl8.0; KC1 0.2; Na,HPO, 1.15; K,HPO, 0.2; glucose 0.2 and 0.05 per cent EDTA at pH 7.4 and 37”. The cells were washed twice with Hank’s balanced salt solution. Tyrosine transaminase activity was unaffected by perfusion of the liver. \Vhen compared to whole liver, the isolated liver cells contained relatively lo\v amounts of tyrosine transaminase as shown in Table I which presents the distribution of tyrosine transaminase between the perfused liver and isolated liver cells. Essentially all of the enzymic activity was recovered in the distribution experiments, which indicated that there was a loss of enzyme from the cells to the medium. Similar results were obtained when livers were perfused in 0.25 M sucrose-mM EDTA and homogenized in a solution containing 0.4 per cent methylcellulose-0.2,5 mM sucrose and 20 mM Tris, pH 7.3. l?ven though the cells had lost the majority of their tyrosine transaminase, attempts were made to induce the enzyme by the addition of hydrocortisone hemisuccinate. Enzymic activity was not lost when the liver cells \vere incubated for 3 hr at 30°C in a medium containing 0.25 M sucrose-20 mM KCIL 20 mM Tris, pH 7.3. The addition of varying amounts of hydrocortisonehemisuccinate from zero to mM did not stimulate tyrosine transaminase activity. Further experiments showed that the cells were not metabolically inactive since they had an appreciable endogenous oxygen uptake as measured on a Warburg respirometer. Also, oxygen uptake was maximum with 10 mM succinate or glutamate and in the presence of these substrates, oxygen consumption was linear for 2 hr. The addition of substrates (10 mM succinate, 10 mM glutamate, mM ADP, mM NADf) and hydrocortisone did not stimulate tyrosine transaminase activity. TABLE
Hat 11 13 1s 21
I. Distribution
of
Cell supernatant
Perfused liver 2820’ 2075 3290 3540
tyrosine transaminase isolated liver cells.
(lOO)b (100) (100) (100)
2782 1690 2760 2690
(98) (82) (83) (75)
between
Unwashed cells 248 590 512 824
( 1) (28) (16) (23)
perfused
liver
and
2 x washed cells 124 221 24 115
(0.5) (1.0) (0.7) (3.0)
a Total units of tyrosine transaminase where 1 unit is equal to an absorbance change of 0.001 per minute. b The numbers in brackets are percentages with 100 equal to the activity in the perfused liver. Experimental
Cell Research
44
Studies with
335
rut liver cell suspensions
Experiments by Niu [20] have indicated that the addition of rat liver RNA to the medium of Ehrlich ascities cells induced the formation in situ of normal rat liver function. That is, in the presence of RNA the ascites cells synthesized serum albumin and have glucose-6-phosphatase and tryptophan pyrrolase activity. The addition of commercial preparations of RNA, yeast RNA from Schwarz and rat liver RNA from Sigma (1-4 mg/ml) in the absence of or in conjunction Jvith hydrocortisone (O-l mg/ml), under short term incubations (3 hr at 30”) or under the conditions described by Siu [20] failed to increase the activity of tprosine transaminase in the isolated liver cells. Rat liver RNA isolated by the procedure described by Kirby [17 I was also ineffective under similar conditions. Niu [20] has reported that glucose-6-phosphatase activity is induced in ascities liver cells by RNA, \\-hereas prior to the addition of RN-4 no enzymic activity was detectable in these cells. In contrast to tyrosine transaminase which is a soluble enzyme, glucose-6-phosphatase is a particulate enzyme and consequently does not readily leak out of the liver cells. In an enzyme partition experiment, 69 per cent of the glucose-6-phosphatase activity was recovered in twice-washed cells whereas only 1 to 3 per cent of the tyrosine transaminase was found in the same cells. The addition of RNA isolated from rat liver by the procedure described by Kirby [17] to isolated liver cells under the conditions described by Niu [20] failed to increase the activity of glucose-6-phosphatase. Cortisol and RN.4 in various combination were also ineffective. TABLE II.
No.
The effect of preparation media on the retention of lactic dehydrogenase in dispersed liver cells.
Preparation
Experimerits
medium
0.25 M sucrose
pH
7.3
0.25 M sucrose,
0.02 M Tris-HCl
0.25 M sucrose, pH 7.3
0.02 M potassillm
0.25 M sucrose,
0.4 % methylcellulose,
pH
0.25 M sucrose, 0.4 % methylcellulose, Tris-HCl, pH 7.3 Polyvinylpyrrolidone pH 7.3
(22 %) Sucrose
7.3
Av. % lactate dehydrogenase in cells
17
40.2
4
7.7
1
1.4
1
40.5
1
10.1
2
57.5
phosphate, pH
7.3
0.02 M (10 X),
Experimental
Cell Research
44
336 Incorporation
C. M. Tsai, N. Best and K. E. Ebner of cortisol-4-14C by isolated liver cells
Recent work has indicated that the induction of certain liver enzymes, including tyrosine transaminase, may be mediated by cortisol at the gene level [4]. Since the results of previous experiments on the induction of enzymes by cortisol were negative, experiments were designed to determine the cortisol uptake by liver cells and its intracellular distribution. Cells were prepared by the method of Berry [2] except that all solutions contained 22 per cent polyvinylpyrrolidone (w/v). The time course of 14C-cortisol uptake at 37” showed that maximum uptake occurred at 15 min. Incubation experiments were conducted for 20 min at 37”. There was a linear relationship between cortisol uptake and concentration in the range of 1 to 10 ,ug of corisol per ml of medium. There was also a linear relationship between the uptake of cortisol and the amount of cells in medium over an 8 fold range of cell concentration while the cortisol concentration was held at 0.7 ,ug/ml of medium. When isolated liver cells were incubated at 37” for 20 min with 14C-4-cortisol (0.1 ,ug/ml, specific activity 25 mc/mM) and were subsequently washed twice \vith medium, only 2 to 4 per cent of the 14C-4-cortisol was retained by the cells. Subcellular distribution of 14C-4-cortisol in isolated liver cells The isolated liver cells were incubated with 14C-4-cortisol as previously described and after incubation they were washed twice with medium and suspended in 9 vol of 0.25 M sucrose. The cells were homogenized in a teflon on glass motor-driven homogenizer (Eberbach) and the subcellular fractions were isolated bv differential centrifugation [ll]. The average distribution of 14C-4-cortisol in four experiments was: nuclear, 36.4 per cent (30.8-42.6); mitochondrial, 20.6 per cent (14.2-27.7); microsomal, 16.5 per cent (14.417.9) and soluble, 26.5 per cent (18.8-39.0). The values shown in the brackets are the observed ranges. Retention on lactate dehydrogenase in dispersed rat liver cells The studies reported above showed that tyrosine transaminase was lost during the preparation of the liver cells. Such losses of soluble cellular comTABLE III. Retention of enzymes in liver cellsprepared in 0.25 M sucrose,pH 7.3. % enzyme in cells
Enzyme Lactate dehydrogenase 3-Hydroxyanthranilate Tvrosine transaminase Experimental
Cell Research
44
oxygenase
40.2 2.1 3.8
Studies with rat liver cell suspensions
337
ponents have been attributed to alteration of the normal membrane permeability of the isolated cells. Since the cells lost most of their tyrosine transaminase and presumably other soluble components necessary for protein synthesis, efforts were made to find a preparation procedure that would result in cells with improved membrane permeability characteristics. Lactate dehydrogenase was chosen as an enzyme for determining whether the cells would have improved permeability characteristics since the enzymic activity is high in liver, the assay is rapid and between 90-95 per cent of the enzyme is reported to be lost during the preparation of isolated liver cells [6]. Cells were isolated in a variety of preparation media (Table II) and the percentage of lactate dehydrogenase retained in the cells was measured. The data presented in Table II show that 40 to 57 per cent of the lactate dehydrogenase is retained in the cells when 0.25 per cent sucrose alone or in conjunction with 0.4 per cent methylcellulose or 22 per cent polyvinylpyrrolidone is present in the preparation media. However, the presence of 20 mM Tris or phosphate at pH 7.3 in the media results in cells that retain only 1 to 10 per cent of their lactate dehydrogenase. The retention of other soluble enzymes was examined in order to determine if the preparation procedure did produce cells with more intact cell membranes or if the results were unique to lactate dehydrogenase. Cells were prepared in 0.25 it4 sucrose, pH 7.3 and lactate dehydrogenase, 3-hydroxyanthranilate oxygenase and tyrosine transaminase retention were measured. The results in Table III show that lactate dehydrogenase is retained some 40 per cent whereas only 2.1 per cent of the 3-hydroxyanthranilate oxygenase and 3.8 per cent of the tyrosine transaminase is retained under identical isolation conditions. The indication is that the retention of lactate dehydrogenase is unique to this enzyme and that the isolation TABLE
IV. Effect of KC1 in the 0.25 M sucrose preparation media on the retention of lactate dehydrogenase in dispersed rat liver cells. ?A retention KC1 (mM)
Experiment
of LDH I
in cells
Experiment
0 1
54 60
44 -
5 10 20 50
49 7 -
36 27 9 2
II
Experimental
Cell Research 44
338
C. M. Tsai,
N.
Best and K. E. Ebner
procedures did not give cells with improved permeability characteristics. The results given in Table II show that cells isolated in 6.25 M sucrose, 20 m&Z phosphate pH 7.3 retained less lactate dehydrogenase than cells isolated in 0.25 M sucrose, 20 mM Tris-HCl, pH 7.3. The possibility then existed that the retention of lactate dehydrogenase was a function of the ionic strength of the isolation medium since at comparable molarities, phosphate has a higher ionic strength than Tris. This possibility was examined by preparing cells in 0.25 M sucrose but with varying concentrations of KCl. These results, presented in Table I\‘, show that the retention of lactate dehydrogenase in isolated liver cells is dependent upon the concentration of KC1 in the isolation medium. The presence of 20 mM KC1 in the medium reduced the retention of lactate dehgdrogenase to that of 20 rn% Tris-HCl. Experiment 2 (Table 11:) clearly shows that the retention of lactate dehydrogenase is progressively reduced as the concentration of KC1 is increased. These data support the view that the retention of lactate dehydrogenase in isolated liver cells may be a function of ionic strength of the isolation media.
DISCUSSION
Tyrosine transaminase was not induced by cortisol and/or RNA in dispersed liver cells isolated under a variety of conditions, even though cortisol was bound to various portions of the cells. The failure to observe enzyme induction suggests that the cells as isolated do not exhibit normal liver function. Also, glucose-6-phosphatase was not induced by rat liver RNA or by RNA from other sources as observed by Niu [26] with Ehrlich ascities cells or Novikoff hepatomas. The failure of the isolated cells to exhibit enzyme induction is probably due to the loss of soluble components from the cells. The presence of ADP, phosphate or NAD+ stimulated respiration of succinate and glutamate but did not aid in inducing enzyme protein synthesis. Enzyme protein synthesis involves certain soluble components and losses to the medium would seriously impair synthesis. The binding of 14C-4-cortisol by isolated liver cells resembles the results of Ketchel and Garabedian [16] who examined the in vitro binding of l*C-4cortisol by human leucocytes. They found that about 4 per cent of the 14C-4cortisol was adsorbed from the medium and that the binding apparently followed a simple diffusion process since the rate of binding was directly proportional to the concentration of 14C-4-cortisol in the medium. Bellamy Experimental
Cell Research 44
Studies with rat liver cell suspensions
339
et al. [l ] showed that in rat liver slices cortisol enters the cell by simple diffusion and a favorable gradient is maintained by intracellular adsorption. In rat liver slices, about 85 per cent of the intracellular cortisol was associated with the particulate fractions. In the present study, about 73 per cent of the l*C-4-cortisol in the cell was associated with the particulate fractions. The nuclear fraction adsorbed slightly more cortisol than the other fractions which is of interest since it has been suggested that cortisol may mediate enzyme protein synthesis at the gene level. Tyrosine transaminase is a soluble liver enzyme and most of the enzyme was lost during isolation of the liver cells. Such observations have been made with other soluble liver enzymes including lactate dehydrogenase [6]. However, the data presented in Table II showed that lactate dehydrogenase may be retained in the isolated liver cells (40 to 51 per cent) when buffers were absent from the isolation media, suggesting that retention was dependent upon the ionic strength of the media. Under the same conditions where the isolation medium was 0.25 !V sucrose, pH i.3, lactate dehydrogenase \vas retained in the cells (40 per cent) whereas 3-hydroxyanthranilate oxygenase (2.1 per cent) and tyrosine transaminase (3.8 per cent) were lost to the medium. Increasing the concentration of KC1 in a 0.25 M sucrose isolation medium resulted in a progressive decrease in the retention of lactate dehydrogenase in the isolated liver cells. These data suggest that the retention of lactate dehydrogenase in isolated liver cells was related to the ionic strength of the medium. Lactate dehydrogenase exists as a mixture of isozymes and liver contains mainly one of the five common isozymes [a]. The fact that low concentrations of KC1 added to the isolation medium caused a release of lactate dehydrogenase to the medium would suggest that when cells are isolated in the presence of 0.25 n/r sucrose, lactate dehydrogenase may be bound in some manner to the particulate portion of the cell. The addition of KC1 or other salts of comparable ionic strength appears to result in dissociation and loss of lactate dehydrogenase to the medium. Takeda et al. [23] have observed that when rats are given glucocorticoids (deaamethasone) for several days prior to preparation ‘of isolated liver cells, these cells had an increased retention of lactate dehydrogenase (4 to 40 per cent). However, the leakage of tryptophan pyrrolase and serine and threonine dehydrase was not prevented by prior treatment of rats with steroids. Again, lactate dehydrogenase was the only soluble enzyme that was retained. The authors suggest that the glucocorticoids may influence the permeability of the cell membrane, thereby releasing other soluble enzymes but retaining lactate dehydrogenase. Experimental
Cell Research 44
340
C. M. Tsai, N. Best and K. E. Ebner
At the present time, the use of isolated liver cells for the study functions appears to be essentially limited to particulate enzyme Studies with soluble systems because of leakage of soluble cellular nents are limited until isolation procedures are developed which can cells with normal cell membrane characteristics.
of liver systems. compoproduce
SUMMARY
Cortisol or RNA, singly and in combination, were not able to induce the formation of tyrosine transaminase or glucose-6-phosphatase in isolated rat liver cells. The majority of 14C-4-cortisol was bound to the nuclear fraction of the isolated cells. Lactate dehydrogenase was retained to a large extent in isolated rat liver cells when they are prepared in 0.25 M sucrose or in a media of low ionic strength. The enzyme is lost from the cells when they are isolated in media of higher ionic strength. REFERENCES 1. BELLAMY,
D., PHILIPPS,
J. B., CHESTER-JONES,
I. and LEONARD,
R. A.,
Biochem. J. 85, 537
(1962). BERRY, M. N., J. Cell Biol. 15, 1 (1962). BERRY, M. N. and SIMPSON, F. O., J. Cell Biol. 15, 9 (1962). DAVIDSON, E. H., Sci. Am. 212 (6), 36 (1965). DECKER, R. H., KASG, H. H., LEACH, F. R. and HENDERSON, 3076 (1961). 6. EXTON, J. H., Biochem. J. 92, 457 (1964). 2. 3. 4. 5.
7. ~
L. M., J. Biol.
Chem. 236,
ibid. 92, 467 (1964).
FINE, I. H. and COSTELLO, L. A., Methods in Enzymol. 6, 958 (1963). GAJA, G. and BERNELLI-ZAZZERA, A., Ital. J. Biochem. 10, 283 (1961). HENLEY, K. S., SORENSEN, 0. and POLLARD, H. M., Nafure 184,140O (1959). HOGEBOOM, G. H., Methods in Enzymol. 1, 16 (1955). Y., J. Biochem. 57, 696 (1965). ICHIHARA, A., A., ADACHI, K., DAIKUHARA, Y. and TAKEDA, JACOB, S. T. and BHARGA~A, P. M., Biochem. J. 95, 568 (1965). KEP*‘NEY, F. T. and FLORA, R. M., ibid. 236, 2699 (1961). KENNEY, F. T., .Z. Biol. Chem. 237, 1610 (1962). KETCHEL, M. M. and GARABEDIAN, E., rlcta Endocrinol. 42, 12 (1963). KIRBY, K. S., Biochem. Biophys. Acta 55, 545 (1962). KORNBERG, A., Methods in Enzymol. 1, 441 (1955). LIN, E. C., PITT, B. M., CIVIN, 111. and KNOX, W. E., J. Biol. Chem. 233, 668 (1958). NIU, M. C., CORDOVA, C. C., NIU, L. C. and RADBILL, G. L., Proc. Nail Acad. Sci. U.S. 48, 1964 (1962). 21. O’BRIEN, R. D., Anal. Biochem. 7, 251 (1964). 22. SWANSON, M., Methods in Enzymol. 2, 541 (1955). 23. TAKEDA, Y., ICHIHARA, A., TANIOKA, H. and INOUE, H., J. Biol. Chem. 239, 3590 (1964). 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Experimental
Cell Research 44
332
Experimental
1966
by Academic
Cell Research
Press
44, 332-340
Inc
(1966)
RETENTION OF LACTATE DEHYDROGENASE CORTISOL BINDING AND LACK OF ENZYME INDUCTION IN RAT LIVER CELL SUSPENSIONS C. M. TSAI, Oklahoma
State
University,
N. BEST
and K. E. EBNER
Agricultural Experiment Station, Department Stillwater, Okla. 74074, U.S.A.
of Biochemistry,
Received June 10, 1966
THERE
has been considerable interest in the metabolic properties of dispersed rat liver cells and attempts have been made to demonstrate normal liver function in these cells since they would provide an attractive system for the study of liver function. Jacob and Bhargava [13] have examined the incorporation of 32P-phosphate and 14C-adenine into RNA of suspended liver cells. Exton [6] has shown that rat liver cell suspensions do not metabolize carbohydrates but do metabolize citric acid cycle intermediates and oxidize palmitate, largely to ketone bodies [7]. lchihara [la] has shown that 14C-acetate is incorporated into lipids of dispersed rat liver cells. Berry [2] has prepared mouse liver cell suspensions and shown that these cells are able to synthesize urea. A serious limitation of isolated cell preparations has been their failure to utilize glycolytic intermediates [6] and many of the soluble components of the cells, including enzymes, are lost during the preparation procedure. Such losses have been attributed to alterations in the permeability of the cell membranes as evidenced by electron microscopy [3]. The soluble enzymes; aldolase, lactate dehydrogenase, iditol dehydrogenase [6], threonine dehydrase, serine dehydrase, tryptophan pyrrolase [23] and phosphorylase [9] are lost from rat liver cells during the preparation of cell suspensions. Glutamic pyruvate transaminase [23], malate dehydrogenase [B], glutamic oxaloacetic transaminase [10, 23 ], glutamate dehydrogenase [ 10] and glucose-6-phosphatase [lo, 231 are enzymes associated with the particulate portion of the cells and are retained to varying degrees in dispersed liver cells. Nicotinamide nucleotides are essentially lost during the preparation of liver cells [6]. The potential use of isolated liver cells to study normal liver function especially the hormonal induction of enzymes, prompted the present studies. Cortisol and RNA were examined for their ability to induce enzyme formation in rat liver cell suspensions. The binding of 14C-coritsol and the retention of Experimental
Cell Research
44
Studies with
rat liver cell suspensions
333
liver enzymes in the cell suspensions were also examined. The studies showed that tyrosine transaminase and glucose-6-phosphatase were not induced by cortisol or RNA. Lactate dehydrogenase could be retained in isolated rat liver cells under certain conditions whereas other soluble enzymes were lost during the preparation procedure. METHODS Preparation of rat liver cell suspensions.-Liver cell suspensionswere prepared from male rats (Holtzman) weighing between 150 and 300 g. The animals were anesthetized with ether and the liver was perfused via the portal vein to remove the blood. Various perfusion media were used and perfusion was carried out at 37°C. The liver was removed, cut into small pieces and then gently forced through a tissuepress(Harvard Apparatus). Two grams of the liver were added to 9 ml of homogenizing medium (4°C) and gently homogenized by hand in a loosefitting teflon on glasshomogenizer (A. Thomas, type B, teflon pestle and a 19.5 mm internal diameter test tube was used). The crude cell suspensionwas filtered through 4 layers of cheese-cloth to remove connective tissue and large aggregates. The filtrate was centrifuged at 120 x g for IO min at 4°C. The supernatant solution was designated asthe “cell supernatant”. The cell pellet was washed twice with two volumes of the dispersion medium and the cells were suspendedin 5 ml of incubation medium. Enzymic assays.-The following enzymes were assayed by standard procedures: lactate dehydrogenase [181, tyrosine transaminase [ 191, 3-hydroxyanthranilate oxygenase [5], glucose-6-phosphatase [22]. In the tyrosine transaminase assay, 10 mM azide was used to replace the diethydithiocarbamate which formed insoluble chelates with Ca2+and MgZ+ present in the perfusion medium. The assayswere linear with time and rates were proportional to enzyme concentration. The assays were conducted at room temperature in a Cary Model 14 spectrophotometer. Binding of 14C-4-cortisoI.-14C-4-Cortis01,specific activity 45.0 mc/mM was a product of New England Nuclear. The W-4-cortisol was dissolved in ethanol and 1 to 10 ,IL~ were usedin each assay. An equal volume of ethanol was added to controls. Cells and cellular fractions, isolated by differential centrifugation [ll] were solubilized with nitric acid [21] and aliquots were counted at infinite thickness in a Baird Atomic Automatic Planchet Counter. RESULTS
Attempted induction of tyrosine transaminase and glucose-6-phosphatase In rats, tyrosine transaminase activity may be stimulated 3 to 4 fold by the administration of cortisol [ 151 and it has been shown by immunological techniques that the increase in enzymic activity is due to the induction of specific enzymic protein [14]. These observations prompted attempts to induce tyrosine transaminase in isolated liver cells, since such a system would be free from whole animal complications. Experimental
Cell Research
44
334
C. M. Tsai,
N. Best and K. E. Ebner
The rat livers were perfused and homogenized with a divalent ion-free solution containing in g/l NaCl8.0; KC1 0.2; Na,HPO, 1.15; K,HPO, 0.2; glucose 0.2 and 0.05 per cent EDTA at pH 7.4 and 37”. The cells were washed twice with Hank’s balanced salt solution. Tyrosine transaminase activity was unaffected by perfusion of the liver. \Vhen compared to whole liver, the isolated liver cells contained relatively lo\v amounts of tyrosine transaminase as shown in Table I which presents the distribution of tyrosine transaminase between the perfused liver and isolated liver cells. Essentially all of the enzymic activity was recovered in the distribution experiments, which indicated that there was a loss of enzyme from the cells to the medium. Similar results were obtained when livers were perfused in 0.25 M sucrose-mM EDTA and homogenized in a solution containing 0.4 per cent methylcellulose-0.2,5 mM sucrose and 20 mM Tris, pH 7.3. l?ven though the cells had lost the majority of their tyrosine transaminase, attempts were made to induce the enzyme by the addition of hydrocortisone hemisuccinate. Enzymic activity was not lost when the liver cells \vere incubated for 3 hr at 30°C in a medium containing 0.25 M sucrose-20 mM KCIL 20 mM Tris, pH 7.3. The addition of varying amounts of hydrocortisonehemisuccinate from zero to mM did not stimulate tyrosine transaminase activity. Further experiments showed that the cells were not metabolically inactive since they had an appreciable endogenous oxygen uptake as measured on a Warburg respirometer. Also, oxygen uptake was maximum with 10 mM succinate or glutamate and in the presence of these substrates, oxygen consumption was linear for 2 hr. The addition of substrates (10 mM succinate, 10 mM glutamate, mM ADP, mM NADf) and hydrocortisone did not stimulate tyrosine transaminase activity. TABLE
Hat 11 13 1s 21
I. Distribution
of
Cell supernatant
Perfused liver 2820’ 2075 3290 3540
tyrosine transaminase isolated liver cells.
(lOO)b (100) (100) (100)
2782 1690 2760 2690
(98) (82) (83) (75)
between
Unwashed cells 248 590 512 824
( 1) (28) (16) (23)
perfused
liver
and
2 x washed cells 124 221 24 115
(0.5) (1.0) (0.7) (3.0)
a Total units of tyrosine transaminase where 1 unit is equal to an absorbance change of 0.001 per minute. b The numbers in brackets are percentages with 100 equal to the activity in the perfused liver. Experimental
Cell Research
44
Studies with
335
rut liver cell suspensions
Experiments by Niu [20] have indicated that the addition of rat liver RNA to the medium of Ehrlich ascities cells induced the formation in situ of normal rat liver function. That is, in the presence of RNA the ascites cells synthesized serum albumin and have glucose-6-phosphatase and tryptophan pyrrolase activity. The addition of commercial preparations of RNA, yeast RNA from Schwarz and rat liver RNA from Sigma (1-4 mg/ml) in the absence of or in conjunction Jvith hydrocortisone (O-l mg/ml), under short term incubations (3 hr at 30”) or under the conditions described by Siu [20] failed to increase the activity of tprosine transaminase in the isolated liver cells. Rat liver RNA isolated by the procedure described by Kirby [17 I was also ineffective under similar conditions. Niu [20] has reported that glucose-6-phosphatase activity is induced in ascities liver cells by RNA, \\-hereas prior to the addition of RN-4 no enzymic activity was detectable in these cells. In contrast to tyrosine transaminase which is a soluble enzyme, glucose-6-phosphatase is a particulate enzyme and consequently does not readily leak out of the liver cells. In an enzyme partition experiment, 69 per cent of the glucose-6-phosphatase activity was recovered in twice-washed cells whereas only 1 to 3 per cent of the tyrosine transaminase was found in the same cells. The addition of RNA isolated from rat liver by the procedure described by Kirby [17] to isolated liver cells under the conditions described by Niu [20] failed to increase the activity of glucose-6-phosphatase. Cortisol and RN.4 in various combination were also ineffective. TABLE II.
No.
The effect of preparation media on the retention of lactic dehydrogenase in dispersed liver cells.
Preparation
Experimerits
medium
0.25 M sucrose
pH
7.3
0.25 M sucrose,
0.02 M Tris-HCl
0.25 M sucrose, pH 7.3
0.02 M potassillm
0.25 M sucrose,
0.4 % methylcellulose,
pH
0.25 M sucrose, 0.4 % methylcellulose, Tris-HCl, pH 7.3 Polyvinylpyrrolidone pH 7.3
(22 %) Sucrose
7.3
Av. % lactate dehydrogenase in cells
17
40.2
4
7.7
1
1.4
1
40.5
1
10.1
2
57.5
phosphate, pH
7.3
0.02 M (10 X),
Experimental
Cell Research
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336 Incorporation
C. M. Tsai, N. Best and K. E. Ebner of cortisol-4-14C by isolated liver cells
Recent work has indicated that the induction of certain liver enzymes, including tyrosine transaminase, may be mediated by cortisol at the gene level [4]. Since the results of previous experiments on the induction of enzymes by cortisol were negative, experiments were designed to determine the cortisol uptake by liver cells and its intracellular distribution. Cells were prepared by the method of Berry [2] except that all solutions contained 22 per cent polyvinylpyrrolidone (w/v). The time course of 14C-cortisol uptake at 37” showed that maximum uptake occurred at 15 min. Incubation experiments were conducted for 20 min at 37”. There was a linear relationship between cortisol uptake and concentration in the range of 1 to 10 ,ug of corisol per ml of medium. There was also a linear relationship between the uptake of cortisol and the amount of cells in medium over an 8 fold range of cell concentration while the cortisol concentration was held at 0.7 ,ug/ml of medium. When isolated liver cells were incubated at 37” for 20 min with 14C-4-cortisol (0.1 ,ug/ml, specific activity 25 mc/mM) and were subsequently washed twice \vith medium, only 2 to 4 per cent of the 14C-4-cortisol was retained by the cells. Subcellular distribution of 14C-4-cortisol in isolated liver cells The isolated liver cells were incubated with 14C-4-cortisol as previously described and after incubation they were washed twice with medium and suspended in 9 vol of 0.25 M sucrose. The cells were homogenized in a teflon on glass motor-driven homogenizer (Eberbach) and the subcellular fractions were isolated bv differential centrifugation [ll]. The average distribution of 14C-4-cortisol in four experiments was: nuclear, 36.4 per cent (30.8-42.6); mitochondrial, 20.6 per cent (14.2-27.7); microsomal, 16.5 per cent (14.417.9) and soluble, 26.5 per cent (18.8-39.0). The values shown in the brackets are the observed ranges. Retention on lactate dehydrogenase in dispersed rat liver cells The studies reported above showed that tyrosine transaminase was lost during the preparation of the liver cells. Such losses of soluble cellular comTABLE III. Retention of enzymes in liver cellsprepared in 0.25 M sucrose,pH 7.3. % enzyme in cells
Enzyme Lactate dehydrogenase 3-Hydroxyanthranilate Tvrosine transaminase Experimental
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oxygenase
40.2 2.1 3.8
Studies with rat liver cell suspensions
337
ponents have been attributed to alteration of the normal membrane permeability of the isolated cells. Since the cells lost most of their tyrosine transaminase and presumably other soluble components necessary for protein synthesis, efforts were made to find a preparation procedure that would result in cells with improved membrane permeability characteristics. Lactate dehydrogenase was chosen as an enzyme for determining whether the cells would have improved permeability characteristics since the enzymic activity is high in liver, the assay is rapid and between 90-95 per cent of the enzyme is reported to be lost during the preparation of isolated liver cells [6]. Cells were isolated in a variety of preparation media (Table II) and the percentage of lactate dehydrogenase retained in the cells was measured. The data presented in Table II show that 40 to 57 per cent of the lactate dehydrogenase is retained in the cells when 0.25 per cent sucrose alone or in conjunction with 0.4 per cent methylcellulose or 22 per cent polyvinylpyrrolidone is present in the preparation media. However, the presence of 20 mM Tris or phosphate at pH 7.3 in the media results in cells that retain only 1 to 10 per cent of their lactate dehydrogenase. The retention of other soluble enzymes was examined in order to determine if the preparation procedure did produce cells with more intact cell membranes or if the results were unique to lactate dehydrogenase. Cells were prepared in 0.25 it4 sucrose, pH 7.3 and lactate dehydrogenase, 3-hydroxyanthranilate oxygenase and tyrosine transaminase retention were measured. The results in Table III show that lactate dehydrogenase is retained some 40 per cent whereas only 2.1 per cent of the 3-hydroxyanthranilate oxygenase and 3.8 per cent of the tyrosine transaminase is retained under identical isolation conditions. The indication is that the retention of lactate dehydrogenase is unique to this enzyme and that the isolation TABLE
IV. Effect of KC1 in the 0.25 M sucrose preparation media on the retention of lactate dehydrogenase in dispersed rat liver cells. ?A retention KC1 (mM)
Experiment
of LDH I
in cells
Experiment
0 1
54 60
44 -
5 10 20 50
49 7 -
36 27 9 2
II
Experimental
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338
C. M. Tsai,
N.
Best and K. E. Ebner
procedures did not give cells with improved permeability characteristics. The results given in Table II show that cells isolated in 6.25 M sucrose, 20 m&Z phosphate pH 7.3 retained less lactate dehydrogenase than cells isolated in 0.25 M sucrose, 20 mM Tris-HCl, pH 7.3. The possibility then existed that the retention of lactate dehydrogenase was a function of the ionic strength of the isolation medium since at comparable molarities, phosphate has a higher ionic strength than Tris. This possibility was examined by preparing cells in 0.25 M sucrose but with varying concentrations of KCl. These results, presented in Table I\‘, show that the retention of lactate dehydrogenase in isolated liver cells is dependent upon the concentration of KC1 in the isolation medium. The presence of 20 mM KC1 in the medium reduced the retention of lactate dehgdrogenase to that of 20 rn% Tris-HCl. Experiment 2 (Table 11:) clearly shows that the retention of lactate dehydrogenase is progressively reduced as the concentration of KC1 is increased. These data support the view that the retention of lactate dehydrogenase in isolated liver cells may be a function of ionic strength of the isolation media.
DISCUSSION
Tyrosine transaminase was not induced by cortisol and/or RNA in dispersed liver cells isolated under a variety of conditions, even though cortisol was bound to various portions of the cells. The failure to observe enzyme induction suggests that the cells as isolated do not exhibit normal liver function. Also, glucose-6-phosphatase was not induced by rat liver RNA or by RNA from other sources as observed by Niu [26] with Ehrlich ascities cells or Novikoff hepatomas. The failure of the isolated cells to exhibit enzyme induction is probably due to the loss of soluble components from the cells. The presence of ADP, phosphate or NAD+ stimulated respiration of succinate and glutamate but did not aid in inducing enzyme protein synthesis. Enzyme protein synthesis involves certain soluble components and losses to the medium would seriously impair synthesis. The binding of 14C-4-cortisol by isolated liver cells resembles the results of Ketchel and Garabedian [16] who examined the in vitro binding of l*C-4cortisol by human leucocytes. They found that about 4 per cent of the 14C-4cortisol was adsorbed from the medium and that the binding apparently followed a simple diffusion process since the rate of binding was directly proportional to the concentration of 14C-4-cortisol in the medium. Bellamy Experimental
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Studies with rat liver cell suspensions
339
et al. [l ] showed that in rat liver slices cortisol enters the cell by simple diffusion and a favorable gradient is maintained by intracellular adsorption. In rat liver slices, about 85 per cent of the intracellular cortisol was associated with the particulate fractions. In the present study, about 73 per cent of the l*C-4-cortisol in the cell was associated with the particulate fractions. The nuclear fraction adsorbed slightly more cortisol than the other fractions which is of interest since it has been suggested that cortisol may mediate enzyme protein synthesis at the gene level. Tyrosine transaminase is a soluble liver enzyme and most of the enzyme was lost during isolation of the liver cells. Such observations have been made with other soluble liver enzymes including lactate dehydrogenase [6]. However, the data presented in Table II showed that lactate dehydrogenase may be retained in the isolated liver cells (40 to 51 per cent) when buffers were absent from the isolation media, suggesting that retention was dependent upon the ionic strength of the media. Under the same conditions where the isolation medium was 0.25 !V sucrose, pH i.3, lactate dehydrogenase \vas retained in the cells (40 per cent) whereas 3-hydroxyanthranilate oxygenase (2.1 per cent) and tyrosine transaminase (3.8 per cent) were lost to the medium. Increasing the concentration of KC1 in a 0.25 M sucrose isolation medium resulted in a progressive decrease in the retention of lactate dehydrogenase in the isolated liver cells. These data suggest that the retention of lactate dehydrogenase in isolated liver cells was related to the ionic strength of the medium. Lactate dehydrogenase exists as a mixture of isozymes and liver contains mainly one of the five common isozymes [a]. The fact that low concentrations of KC1 added to the isolation medium caused a release of lactate dehydrogenase to the medium would suggest that when cells are isolated in the presence of 0.25 n/r sucrose, lactate dehydrogenase may be bound in some manner to the particulate portion of the cell. The addition of KC1 or other salts of comparable ionic strength appears to result in dissociation and loss of lactate dehydrogenase to the medium. Takeda et al. [23] have observed that when rats are given glucocorticoids (deaamethasone) for several days prior to preparation ‘of isolated liver cells, these cells had an increased retention of lactate dehydrogenase (4 to 40 per cent). However, the leakage of tryptophan pyrrolase and serine and threonine dehydrase was not prevented by prior treatment of rats with steroids. Again, lactate dehydrogenase was the only soluble enzyme that was retained. The authors suggest that the glucocorticoids may influence the permeability of the cell membrane, thereby releasing other soluble enzymes but retaining lactate dehydrogenase. Experimental
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C. M. Tsai, N. Best and K. E. Ebner
At the present time, the use of isolated liver cells for the study functions appears to be essentially limited to particulate enzyme Studies with soluble systems because of leakage of soluble cellular nents are limited until isolation procedures are developed which can cells with normal cell membrane characteristics.
of liver systems. compoproduce
SUMMARY
Cortisol or RNA, singly and in combination, were not able to induce the formation of tyrosine transaminase or glucose-6-phosphatase in isolated rat liver cells. The majority of 14C-4-cortisol was bound to the nuclear fraction of the isolated cells. Lactate dehydrogenase was retained to a large extent in isolated rat liver cells when they are prepared in 0.25 M sucrose or in a media of low ionic strength. The enzyme is lost from the cells when they are isolated in media of higher ionic strength. REFERENCES 1. BELLAMY,
D., PHILIPPS,
J. B., CHESTER-JONES,
I. and LEONARD,
R. A.,
Biochem. J. 85, 537
(1962). BERRY, M. N., J. Cell Biol. 15, 1 (1962). BERRY, M. N. and SIMPSON, F. O., J. Cell Biol. 15, 9 (1962). DAVIDSON, E. H., Sci. Am. 212 (6), 36 (1965). DECKER, R. H., KASG, H. H., LEACH, F. R. and HENDERSON, 3076 (1961). 6. EXTON, J. H., Biochem. J. 92, 457 (1964). 2. 3. 4. 5.
7. ~
L. M., J. Biol.
Chem. 236,
ibid. 92, 467 (1964).
FINE, I. H. and COSTELLO, L. A., Methods in Enzymol. 6, 958 (1963). GAJA, G. and BERNELLI-ZAZZERA, A., Ital. J. Biochem. 10, 283 (1961). HENLEY, K. S., SORENSEN, 0. and POLLARD, H. M., Nafure 184,140O (1959). HOGEBOOM, G. H., Methods in Enzymol. 1, 16 (1955). Y., J. Biochem. 57, 696 (1965). ICHIHARA, A., A., ADACHI, K., DAIKUHARA, Y. and TAKEDA, JACOB, S. T. and BHARGA~A, P. M., Biochem. J. 95, 568 (1965). KEP*‘NEY, F. T. and FLORA, R. M., ibid. 236, 2699 (1961). KENNEY, F. T., .Z. Biol. Chem. 237, 1610 (1962). KETCHEL, M. M. and GARABEDIAN, E., rlcta Endocrinol. 42, 12 (1963). KIRBY, K. S., Biochem. Biophys. Acta 55, 545 (1962). KORNBERG, A., Methods in Enzymol. 1, 441 (1955). LIN, E. C., PITT, B. M., CIVIN, 111. and KNOX, W. E., J. Biol. Chem. 233, 668 (1958). NIU, M. C., CORDOVA, C. C., NIU, L. C. and RADBILL, G. L., Proc. Nail Acad. Sci. U.S. 48, 1964 (1962). 21. O’BRIEN, R. D., Anal. Biochem. 7, 251 (1964). 22. SWANSON, M., Methods in Enzymol. 2, 541 (1955). 23. TAKEDA, Y., ICHIHARA, A., TANIOKA, H. and INOUE, H., J. Biol. Chem. 239, 3590 (1964). 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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