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The uptake of ornithine and lysine by isolated hepatocytes and fibroblasts
Inr. J. Biochem.Vol. 16, No. 7, pp. 833-836, 1984 Printed in Great Britain. All rights reserved
Copyright 0
0020-711X/84 $3.00 +O.OO 1984 Pergamon Press Ltd
THE UPTAKE OF ORNITHINE AND LYSINE BY ISOLATED HEPATOCYTES AND FIBROBLASTS K. Department
of Cell and Molecular
METOKI
Biology,
and F. A.
Medical
HOMMES
College
of Georgia,
Augusta,
GA 30912, U.S.A.
(Received 5 October 1983) Abstract-l. The uptake of ornithine and lysine by isolated hepatocytes and cultured human skin fibroblasts were studied. Both types of cells can accumulate these amino acids via a saturable and a non-saturable process, the latter being active at high substrate concentration. 2. The apparent Km for omithine and lysine for the saturable process in hepatocytes were 0.26 and 0.94 mM respectively, those values for fibroblasts were found to be 0.43 and 0.57 mM respectively. 3. Fractionation of the cells by the digitonin technique into a cytosolic compartment and a particulate compartment, showed that a rapid equilibration occurs between the external medium and the cytosolic compartment of hepatocytes and fibroblasts.
INTRODUCTION
System y+ is a general amino
acids
(White
transport
system
the appropriate concentration of “C-labelled amino acid (0.2-0.3 PCi per ml) and [)H]dextran (2-3 PCi per ml). After the incubation the tube was centrifuged in an Eppendorf centrifuge for 1 min. 25 ~1 of the top layer was added to 5 ml Scintiverse (Fisher Scientific Co.) and counted. The remainder of the top layer was removed and the space was washed 3 x with distilled water. Most of the silicon oil was then removed as well and 100 ~1 of the bottom layer was added to 5ml of Scintiverse and counted. The uptake by the particulate fraction of the cells was measured by the digitonin fractionation technique (Zuurendonk and Tager, 1974), as described earlier (Hommes ef al., 1982). [‘HIsucrose was added to the incubation medium to correct for adhering medium in the pellet fraction in the experiments with hepatocytes. L-[l-‘4C]ornithine monohydrochloride (sp. act. 50 mCi/mmol) and L-[U-‘%Jlysine monohydrochloride (sp. act. 338 mCi/mmol) were purchased from New England Nuclear, Boston, Massachusetts, U.S.A. [jH]Dextran (av. mol. wt 80,000; sp. act. 185 mCi/g) and [‘HIsucrose (sp. act. lOCi/mmol) were obtained from Amersham, Arlington Height, Illinois, U.S.A. All chemicals used were of the highest purity grade available.
for cationic
et al., 1982). It has been shown
to be present in cultured human fibroblasts (Groth and Rosenberg, 1972; White et al., 1982), rabbit reticulocytes (Christensen et al., 1969), Ehrlich cells (Christensen, 1964), rat kidney (Segal et al., 1967), and mice brain (Blasberg and Lajtha, 1966). Uptake of the cationic amino acids by this transport system occurs via a saturable process. In addition to this saturable system, cationic amino acids can be taken up by a nonsaturable mechanism, at least by fibroblasts (White et aI., 1982) and isolated rat hepatocytes (White and Christensen, 1982). The saturable system could barely, if at all, be observed in cultured isolated rat hepatocytes, but is active in several hepatoma cell lines (White and Christensen, 1982). The present communication describes some experiments on the uptake of ornithine and lysine by isolated rat hepatocytes and human cultured fibroblasts.
RESULTS MATERIALS
AND METHODS
The quality of the isolated hepatocytes was evaluated by Trypan Blue exclusion and by measuring the rate of gluconeogenesis and ureogenesis. 85595% of the hepatocytes excluded Trypan Blue. The formation of glucose and urea from 1OmM alanine at 37°C was linear up to 60 min. The rates of synthesis were 102 pm/hr per g dry wt and 130 p m/hr per g dry wt respectively, which are similar to those reported by others (Williamson et al., 1974; Rognstad, 1977). Aminooxyacetate (AOA) as an inhibitor of amino transferases and rotenone as an inhibitor of NADH dehydrogenase were added to the media in all uptake studies to inhibit the metabolism of ornithine and lysine. The time course of ornithine and lysine uptake by isolated hepatocytes is shown in Fig. 1. Such curves allow the determination of initial rates of uptake at different substrate concentrations. Saturable uptake of ornithine and lysine could be observed at low substrate concentrations, as is illustrated in Fig. 2. An
Hepatocytes were obtained from 24 hr fasted female Wistar rats according to the method of Berry and Friend (1969), as modified bv Krebs ef al. (1974). The wet weight and the dry weight bf the cells were obtained from &II counts, using the conversion factors derived by Krebs et al. (1974). The hepatocytes were incubated in Ringer bicarbonate solution, pH_7.2, containing 2.5% defatted bovine serum albumin and gassed with 97X 0, + 3X CO, at 37°C. j The cells were used-within 4 hr a&r &olati&. Human skin fibroblasts, obtained from healthy males, were cultured in minimal Eagle’s medium, according to standard procedures (Sly and Grobb, 1979). The cells were harvested at confluency with 0.25% trypsin solution and used immediately for study. Hank’s balanced salt solution, without Ca”, supplemented with 1% defatted bovine serum albumin was used as medium. Cells were incubated in medium layered on top of 0.4 ml of a proper mixture of silicon oils, itself layered on top of 0.2 ml 1.5 N HCIO, in an Eppendorf tube. Dow Corning Fluid 550 (d = I .05) and Dow Corning Fluid 556 (d = 0.98) were used in a ratio of 10:3 for hepatocytes and 1: 1 for fibroblasts respectively. The incubation medium contained 833
K. METOKI and
F. A. HOMM-ES
0.06 0
.
0
0.05
?
i? =
0.03
0.02
II”
%
0.01
0.2
,4
r’
,’
2
0
0.040 i_/-
0
5
10
1s
*o
0 1 0
Seconds
Fig. 1. Time course of uptake of ornithine (A) and of lysine (B) by isolated hepatocytes. The concentration of ornithine and lysine were 0.22 and 1.OmM respectively. K,,, of 0.25 mM could be determined for ornithine uptake, while the apparent K,,, for lysine uptake was found to be 0.94mM. The maximum velocities of uptake were determined at 99.5 nmol/ min per g wet wt and 1.2 ~mol~min per g wet wt for ornithine and lysine respectively. White and Christensen (1982) were unable to detect a saturable uptake system (the y+ system) in cultured isolated hepatocytes, but suggested as a possible explanation that this system is relatively small as compared to the nonsaturable system. That seems indeed to be the case. It should be mentioned that the values for the kinetic constants of the saturable system are uncorrected for contribution by the nonsaturable system. Figure 3 extends the observations given in Fig. 2 to higher amino acid concentrations, showing the nonsaturable uptake system at these higher substrate levels. The effect of lysine on the uptake of ornithine by the saturable is given in Table 1. The saturable system is not inhibited by lysine. Similar results were obtained when the inhibition of lysine uptake by ornithine was studied. Here again, no inhibition could be observed. Preincubation of the hepatocytes
0
0.5
1.0
1.5
2.0
mM Om
5
10
15
I 20
mM Lys
Fig. 3. Concentration ornithine (A---a) hepatocytes at high dimensions
dependence of the rate of uptake of and of lysine (‘-0) by isolated substrate concentration. The inner refer to omithine uptake.
apparent
s
1 1
6
E
14 :
‘1
2
with lysine or ornithine followed by measurement of the uptake of ornithine or lysine showed a stimulation of the initial rates of uptake (Table 2). Transstimulation of cationic amino acid uptake can therefore take place in freshly isolated hepatocytes. In order to determine which fraction of the amino acid taken up by the hepatocytes remains in the Table 1. Effect of lysine on the uptake of ornithine by isolated hepatocytes by the saturable system Rate of Uptake (nmol~min per g wet wt) ~
Ornithine (oW -.-.~~.. 0.08 0.10 0.12 0.16 0.26
Ornithine
Lysine in preincubation medium (mW 0 2 4 8 20 Ornithine in p~~incubation medium (mW
0.0
x
sE
0.4
0
0.2
: 4 R
P
0.80 i-: 0
0.5
a.0
uptake
Rate (nmol~min per g wet wtf 0.44 0.47 0.50 0.55 0.66 Lwitxe uptake
mw
d .G E
25 34 49 63 83
Table 2. Effect of preincubation of isolated rat hepatocytes with lysine or ornithine on the uptake of ornithine or lysine
012345
i i
-.-. + I~mM Lys----
- Lys ~__. 31 41 50 67 92
1.5
2.0
mM
Fig. 2. Concentration dependence of the uptake of ornithine (A) and of lysine (B) by isolated hepatocytes. The inserts show the Lineweaver-Burk plots. Straight lines were drawn by the method of least squares. The correlation coefficients were 0.999 and 0.997 respectively.
Rate (nmoi/min per g wet wt) 0.75 0.90 1.02 I.14 1.16
Hepatocytes were preincubated with lysine or ornithine at the concentration indicated for 2 min at 37°C. centrifuged and resuspended in the medium without the amino acid of preincubation, but with the amino acid of uptake at a final concentration of 0.5 mM for ornithine and 1.0 mM for lysine.
Uptake
of Om and Lys by isolated
835
cells 300,
Seconds
Seconds
Fig. 4. Uptake of omithine (A) and of lysine (B) by the cytosol (W-W) and the particulate (0-O) fraction of isolated hepatocytes. Cells were incubated with either omithine (0.5mM) or lysine (1 mM). The fractionation
medium of the digitonin method contained 2 mg digitonin per ml and 25mM omithine or 50mM lysine to suppress non-specific adsorption of the lahelled amino acid to the particulate fraction. cytosol and which part is taken up by the particulate fraction of the hepatocytes, the digitonin method of Zuurendonk and Tager (1974) was applied. Figure 4 shows the result of such an experiment. It proved to be necessary to include a high concentration of the nonlabelled amino acid in the fractionation medium to suppress non-specific adsorption of the labelled amino acid to the particulate fraction. Without this precaution, the uptake by the particulate fraction was found to be 2-3 times higher than the uptake by whole cells during the first 40 sec. Despite the high concentration of amino acid in the fractionation medium the particulate fraction showed a considerable “uptake” at the first time point measured. Aronson and Diwan (1981) concluded that part of the ornithine taken up by mitochondria is distributed in the matrix space and part bound to solid structures of the mitochondria. It can therefore not be excluded that part of the uptake is indeed due to non-specific binding. Ornithine increased slowly in the particulate fraction, while no increase in that fraction could be observed for lysing over the time span measured. This is consistent with the much slower rate of uptake of lysine by rat liver mitochondria (Hommes et al., 1983). Similar experiments have been carried out with cultured human skin fibroblasts. Figure 5 shows the
mM
Fig. 6. Concentration dependence of the rate of uptake of omithine (O-0) and of lysine (W----W) by cultured human skin fibroblasts at high substrate concentrations.
Lineweaver-Burk plots for ornithine and lysine uptake. The apparent K,,, for ornithine uptake was found to be 0.43 mM, that for lysine uptake 0.57 mM, with maximum rates of uptake of 17.5 and 19.3 nmol/min per mg protein, respectively. A second uptake system becomes operative at higher substrate concentrations (Fig. 6). That system seems to be a non-saturable system, at least up to the concentrations tested (20 mM). Lysine inhibited the uptake of ornithine by the saturable system and vice versa (Table 3). The inhibition is of the mixed type. As was the case with isolated hepatocytes, most of the ornithine or lysine taken up by the fibroblasts was recovered in the cytosolic compartment of the cell when the fibroblasts were fractionated by the digitonin technique (Fig. 7).
DISCUSSION
The present study has demonstrated the existence of a saturable system for the uptake of ornithine and lysine in isolated hepatocytes. The capacity of this system is, however, low as compared with the nonsaturably system for the uptake of these amino acids. It should also be mentioned that the saturable system could not be observed in all preparations of isolated hepatocytes. This did not seem to correlate with either the percentage of trypan blue exclusion or the Table 3. Effect of lysine on the uptake of omitbine by cultured human skin fibroblasts and the effect of omithine on the uptake of lysine by these cells Rate of uptake (nmol/min per mg protein) (A)
-Lys
Substrate 0.2 mM 0.3 mM 0.5 mM 0.7 mM 1.O mM
Ornithine Ornithine Ornithine Ornithine Ornithine
3.8 4.6 8.2 11.3 13.2
+
I mM
Lys
2.3 3.2 5.9 11.4 13.0
Rate of uptake (nmol/min per mg protein) -1
-2
-3
0
1
2
3
4
5
6
7 mM-’
IIS
Fig. 5. Lineweaver-Burk plots for the uptake of omithine (O-O) and lysine (W-W) by cultured human skin fibroblasts. For experimental details see “Materials and Methods”.
(8)
-0m
Substrate 0.15 0.20 0.30 0.50 0.80
mM mM mM mM mM
Lysine Lysine Lysine Lysine Lysine
4.8 6.0 7.0 12.6 18.4
+2mM 1.9 2.7 4.0 6.4 14.5
Om
836
K. METOKI and F. A. HOMMES Acknowledgement-This research was supported Grant AM29691. Contribution No. 0794.
by NIH
REFERENCES
0-7 0 10
20
30
40
Seconds
50
60
OY 0 10
20
30
40
50
60
Seconds
Fig. 7. Uptake of ornithine (A) and of lysine (B) by the cytosolic (A--A) and the particulate (0-O) fraction of cultured human skin fibroblasts. Cells were incubated with either ornithine or lysine (1 mM). The fractionation medium of the digitonin method contained 0.6 mg digitonin per ml and 50mM omithine or lysine to suppress non-
specific adsorption of the labelled amino acid to the particulare fraction.
rate of gluconeogenesis and ureogenesis from alanine, i.e. the criteria applied to evaluate the quality of the cells. The saturable uptake system is apparently rather sensitive to proteolytic attack by the enzymes used in the isolation of the hepatocytes. However, the fact that the saturable uptake system can be observed in isolated hepatocytes demonstrates the existence of this system in the liver. The maximum rate of uptake as determined in the present study may have been underestimated, due to the lability of this uptake system. Most of the amino acids taken up by isolated hepatocytes or fibroblasts is recovered in the cytosolic compartment of the cell. It can be calculated from the data given in Fig. 4, the volume of the liver cell, the number of cells per gram wet weight (Krebs et al., 1974) and the fraction of the cell volume taken by the cytosolic compartment (Vergonet et al., 1970) that within the first 60 set after addition of ornithine to the cells, the concentration of ornithine reaches a value of 0.27 mM, versus an outside concentration of 0.5 mM in that experiment. Equilibration with the medium proceeds therefore rapidly for this amino acid, as it does for lysine, which reaches an inside concentration of 0.4 mM, versus an outside concentration of 1 mM. The same uias observed in fibroblasts (Fig. 7), in agreement with the observation of White et al. (1982). An inside concentration of 0.78 and 0.46 mM for ornithine and lysine, respectively, is reached after 60 set incubation in the presence of 1 mM ornithine or 1 mM lysine.
Aronson D. L. and Diwan J. J. (1981) Uptake of ornithine by rat liver mitochondria. Biochemistry 20, 7064-7068. Berry M. N. and Friend D. S. (1969) High yield preparation of isolated rat liver parenchymal cells. J. cell. Biol. 43, 506-520. Blasberg H. and Lajtha A. (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain. Brain Rex 1, 86-104. Christensen H. N. (1964) A transport system serving for mono- and diamino acids. Proc. natn. Acad. Sri. U.S.A. 51, 337-344. Christensen H. N. and Antonioli J. A. (1969) Cationic amino acid transport in the rabbit reticulocyte. J. Viol. Chem. 244, 1497-1504. Groth U. and Rosenberg L. E. (1972) Transport of dibasic amino acids, cystine and tryptophan by cultured human fibroblasts: Absence of a defect in cystinuria and Hartnup disease. J. clin. Invest. 51, 213&2142. Hommes F. A., Ho C. K., Roesel R. A., Coryell M. E. and Gordon B. A. (1982) Decreased transport of ornithine across the inner mitochondrial membrane as a cause of hyperornithinemia. J. Inter. Metab. Dis. 5, 41-47. Hommes F. A., Kitchings L. and Eller A. G. (1983) The uptake of omithine and lysine by rat liver mitochondria. Biochem. Med. 30, 313-322. Krebs H. A., Cornell N. W., Lund P. and Hems R. (1974) Isolated liver cells as experimental material. In Regulation of Hepatic Metabolism (Edited by Lundquist F. and Tygstrup N.) pp. 726-750. Munsgaard, Copenhagen. Rognstad R. (1977) Sources of ammonia for urea synthesis in isolated rat liver cells. Biochim. biophys. Actu 496, 249-254. Segal S., Schwartzman L., Blair A. and Bertolli 0. (1967) Dibasic amino acid transport in rat kidney cortex slices. Biochim. biophys. Acta 135, 127-I 35. Sly W. S. and Grobb J. (1979) Isolation of fibroblasts from patients. Meth. Enzym. 58, 444450. Vergonet C., Hommes F. A. and Molenaar I. (1970) A morphometric and biochemical study to fetal and adult rat liver cells with special reference to energy metabolism. Biol. Neonat. 16, 297-305. White M. F. and Christensen H. N. (1982) Cationic amino acid transport into cultured animal cells. II. Transport system barely perceptible in ordinary hepatocytes but active in hepatoma cell lines. J. biol. Gem. 257, 445&4457. White M. F., Gazzola G. C. and Christensen H. N. (1982) Cationic amino acid transport into cultured human fibroblasts. J. biol. Chem. 257, 44434449. Williamson J. R., Meyer A. J. and Okhawa K. (1974) Interrelations between anion transport, ureogenesis, and gluconeogenesis in isolated liver cells. In Regulation qf Heparic Metabolism (Edited by Lundquist F. and Tygstrup N.) pp. 457-479. Munsgaard, Copenhagen. Zuurendonk P. F. and Tager J. M. (1974) Rapid separation of particulate components and soluble cytoplasm of isolated rat liver cells. Biochim. biophys. Acra 333, 393-399.
Copyright 0
0020-711X/84 $3.00 +O.OO 1984 Pergamon Press Ltd
THE UPTAKE OF ORNITHINE AND LYSINE BY ISOLATED HEPATOCYTES AND FIBROBLASTS K. Department
of Cell and Molecular
METOKI
Biology,
and F. A.
Medical
HOMMES
College
of Georgia,
Augusta,
GA 30912, U.S.A.
(Received 5 October 1983) Abstract-l. The uptake of ornithine and lysine by isolated hepatocytes and cultured human skin fibroblasts were studied. Both types of cells can accumulate these amino acids via a saturable and a non-saturable process, the latter being active at high substrate concentration. 2. The apparent Km for omithine and lysine for the saturable process in hepatocytes were 0.26 and 0.94 mM respectively, those values for fibroblasts were found to be 0.43 and 0.57 mM respectively. 3. Fractionation of the cells by the digitonin technique into a cytosolic compartment and a particulate compartment, showed that a rapid equilibration occurs between the external medium and the cytosolic compartment of hepatocytes and fibroblasts.
INTRODUCTION
System y+ is a general amino
acids
(White
transport
system
the appropriate concentration of “C-labelled amino acid (0.2-0.3 PCi per ml) and [)H]dextran (2-3 PCi per ml). After the incubation the tube was centrifuged in an Eppendorf centrifuge for 1 min. 25 ~1 of the top layer was added to 5 ml Scintiverse (Fisher Scientific Co.) and counted. The remainder of the top layer was removed and the space was washed 3 x with distilled water. Most of the silicon oil was then removed as well and 100 ~1 of the bottom layer was added to 5ml of Scintiverse and counted. The uptake by the particulate fraction of the cells was measured by the digitonin fractionation technique (Zuurendonk and Tager, 1974), as described earlier (Hommes ef al., 1982). [‘HIsucrose was added to the incubation medium to correct for adhering medium in the pellet fraction in the experiments with hepatocytes. L-[l-‘4C]ornithine monohydrochloride (sp. act. 50 mCi/mmol) and L-[U-‘%Jlysine monohydrochloride (sp. act. 338 mCi/mmol) were purchased from New England Nuclear, Boston, Massachusetts, U.S.A. [jH]Dextran (av. mol. wt 80,000; sp. act. 185 mCi/g) and [‘HIsucrose (sp. act. lOCi/mmol) were obtained from Amersham, Arlington Height, Illinois, U.S.A. All chemicals used were of the highest purity grade available.
for cationic
et al., 1982). It has been shown
to be present in cultured human fibroblasts (Groth and Rosenberg, 1972; White et al., 1982), rabbit reticulocytes (Christensen et al., 1969), Ehrlich cells (Christensen, 1964), rat kidney (Segal et al., 1967), and mice brain (Blasberg and Lajtha, 1966). Uptake of the cationic amino acids by this transport system occurs via a saturable process. In addition to this saturable system, cationic amino acids can be taken up by a nonsaturable mechanism, at least by fibroblasts (White et aI., 1982) and isolated rat hepatocytes (White and Christensen, 1982). The saturable system could barely, if at all, be observed in cultured isolated rat hepatocytes, but is active in several hepatoma cell lines (White and Christensen, 1982). The present communication describes some experiments on the uptake of ornithine and lysine by isolated rat hepatocytes and human cultured fibroblasts.
RESULTS MATERIALS
AND METHODS
The quality of the isolated hepatocytes was evaluated by Trypan Blue exclusion and by measuring the rate of gluconeogenesis and ureogenesis. 85595% of the hepatocytes excluded Trypan Blue. The formation of glucose and urea from 1OmM alanine at 37°C was linear up to 60 min. The rates of synthesis were 102 pm/hr per g dry wt and 130 p m/hr per g dry wt respectively, which are similar to those reported by others (Williamson et al., 1974; Rognstad, 1977). Aminooxyacetate (AOA) as an inhibitor of amino transferases and rotenone as an inhibitor of NADH dehydrogenase were added to the media in all uptake studies to inhibit the metabolism of ornithine and lysine. The time course of ornithine and lysine uptake by isolated hepatocytes is shown in Fig. 1. Such curves allow the determination of initial rates of uptake at different substrate concentrations. Saturable uptake of ornithine and lysine could be observed at low substrate concentrations, as is illustrated in Fig. 2. An
Hepatocytes were obtained from 24 hr fasted female Wistar rats according to the method of Berry and Friend (1969), as modified bv Krebs ef al. (1974). The wet weight and the dry weight bf the cells were obtained from &II counts, using the conversion factors derived by Krebs et al. (1974). The hepatocytes were incubated in Ringer bicarbonate solution, pH_7.2, containing 2.5% defatted bovine serum albumin and gassed with 97X 0, + 3X CO, at 37°C. j The cells were used-within 4 hr a&r &olati&. Human skin fibroblasts, obtained from healthy males, were cultured in minimal Eagle’s medium, according to standard procedures (Sly and Grobb, 1979). The cells were harvested at confluency with 0.25% trypsin solution and used immediately for study. Hank’s balanced salt solution, without Ca”, supplemented with 1% defatted bovine serum albumin was used as medium. Cells were incubated in medium layered on top of 0.4 ml of a proper mixture of silicon oils, itself layered on top of 0.2 ml 1.5 N HCIO, in an Eppendorf tube. Dow Corning Fluid 550 (d = I .05) and Dow Corning Fluid 556 (d = 0.98) were used in a ratio of 10:3 for hepatocytes and 1: 1 for fibroblasts respectively. The incubation medium contained 833
K. METOKI and
F. A. HOMM-ES
0.06 0
.
0
0.05
?
i? =
0.03
0.02
II”
%
0.01
0.2
,4
r’
,’
2
0
0.040 i_/-
0
5
10
1s
*o
0 1 0
Seconds
Fig. 1. Time course of uptake of ornithine (A) and of lysine (B) by isolated hepatocytes. The concentration of ornithine and lysine were 0.22 and 1.OmM respectively. K,,, of 0.25 mM could be determined for ornithine uptake, while the apparent K,,, for lysine uptake was found to be 0.94mM. The maximum velocities of uptake were determined at 99.5 nmol/ min per g wet wt and 1.2 ~mol~min per g wet wt for ornithine and lysine respectively. White and Christensen (1982) were unable to detect a saturable uptake system (the y+ system) in cultured isolated hepatocytes, but suggested as a possible explanation that this system is relatively small as compared to the nonsaturable system. That seems indeed to be the case. It should be mentioned that the values for the kinetic constants of the saturable system are uncorrected for contribution by the nonsaturable system. Figure 3 extends the observations given in Fig. 2 to higher amino acid concentrations, showing the nonsaturable uptake system at these higher substrate levels. The effect of lysine on the uptake of ornithine by the saturable is given in Table 1. The saturable system is not inhibited by lysine. Similar results were obtained when the inhibition of lysine uptake by ornithine was studied. Here again, no inhibition could be observed. Preincubation of the hepatocytes
0
0.5
1.0
1.5
2.0
mM Om
5
10
15
I 20
mM Lys
Fig. 3. Concentration ornithine (A---a) hepatocytes at high dimensions
dependence of the rate of uptake of and of lysine (‘-0) by isolated substrate concentration. The inner refer to omithine uptake.
apparent
s
1 1
6
E
14 :
‘1
2
with lysine or ornithine followed by measurement of the uptake of ornithine or lysine showed a stimulation of the initial rates of uptake (Table 2). Transstimulation of cationic amino acid uptake can therefore take place in freshly isolated hepatocytes. In order to determine which fraction of the amino acid taken up by the hepatocytes remains in the Table 1. Effect of lysine on the uptake of ornithine by isolated hepatocytes by the saturable system Rate of Uptake (nmol~min per g wet wt) ~
Ornithine (oW -.-.~~.. 0.08 0.10 0.12 0.16 0.26
Ornithine
Lysine in preincubation medium (mW 0 2 4 8 20 Ornithine in p~~incubation medium (mW
0.0
x
sE
0.4
0
0.2
: 4 R
P
0.80 i-: 0
0.5
a.0
uptake
Rate (nmol~min per g wet wtf 0.44 0.47 0.50 0.55 0.66 Lwitxe uptake
mw
d .G E
25 34 49 63 83
Table 2. Effect of preincubation of isolated rat hepatocytes with lysine or ornithine on the uptake of ornithine or lysine
012345
i i
-.-. + I~mM Lys----
- Lys ~__. 31 41 50 67 92
1.5
2.0
mM
Fig. 2. Concentration dependence of the uptake of ornithine (A) and of lysine (B) by isolated hepatocytes. The inserts show the Lineweaver-Burk plots. Straight lines were drawn by the method of least squares. The correlation coefficients were 0.999 and 0.997 respectively.
Rate (nmoi/min per g wet wt) 0.75 0.90 1.02 I.14 1.16
Hepatocytes were preincubated with lysine or ornithine at the concentration indicated for 2 min at 37°C. centrifuged and resuspended in the medium without the amino acid of preincubation, but with the amino acid of uptake at a final concentration of 0.5 mM for ornithine and 1.0 mM for lysine.
Uptake
of Om and Lys by isolated
835
cells 300,
Seconds
Seconds
Fig. 4. Uptake of omithine (A) and of lysine (B) by the cytosol (W-W) and the particulate (0-O) fraction of isolated hepatocytes. Cells were incubated with either omithine (0.5mM) or lysine (1 mM). The fractionation
medium of the digitonin method contained 2 mg digitonin per ml and 25mM omithine or 50mM lysine to suppress non-specific adsorption of the lahelled amino acid to the particulate fraction. cytosol and which part is taken up by the particulate fraction of the hepatocytes, the digitonin method of Zuurendonk and Tager (1974) was applied. Figure 4 shows the result of such an experiment. It proved to be necessary to include a high concentration of the nonlabelled amino acid in the fractionation medium to suppress non-specific adsorption of the labelled amino acid to the particulate fraction. Without this precaution, the uptake by the particulate fraction was found to be 2-3 times higher than the uptake by whole cells during the first 40 sec. Despite the high concentration of amino acid in the fractionation medium the particulate fraction showed a considerable “uptake” at the first time point measured. Aronson and Diwan (1981) concluded that part of the ornithine taken up by mitochondria is distributed in the matrix space and part bound to solid structures of the mitochondria. It can therefore not be excluded that part of the uptake is indeed due to non-specific binding. Ornithine increased slowly in the particulate fraction, while no increase in that fraction could be observed for lysing over the time span measured. This is consistent with the much slower rate of uptake of lysine by rat liver mitochondria (Hommes et al., 1983). Similar experiments have been carried out with cultured human skin fibroblasts. Figure 5 shows the
mM
Fig. 6. Concentration dependence of the rate of uptake of omithine (O-0) and of lysine (W----W) by cultured human skin fibroblasts at high substrate concentrations.
Lineweaver-Burk plots for ornithine and lysine uptake. The apparent K,,, for ornithine uptake was found to be 0.43 mM, that for lysine uptake 0.57 mM, with maximum rates of uptake of 17.5 and 19.3 nmol/min per mg protein, respectively. A second uptake system becomes operative at higher substrate concentrations (Fig. 6). That system seems to be a non-saturable system, at least up to the concentrations tested (20 mM). Lysine inhibited the uptake of ornithine by the saturable system and vice versa (Table 3). The inhibition is of the mixed type. As was the case with isolated hepatocytes, most of the ornithine or lysine taken up by the fibroblasts was recovered in the cytosolic compartment of the cell when the fibroblasts were fractionated by the digitonin technique (Fig. 7).
DISCUSSION
The present study has demonstrated the existence of a saturable system for the uptake of ornithine and lysine in isolated hepatocytes. The capacity of this system is, however, low as compared with the nonsaturably system for the uptake of these amino acids. It should also be mentioned that the saturable system could not be observed in all preparations of isolated hepatocytes. This did not seem to correlate with either the percentage of trypan blue exclusion or the Table 3. Effect of lysine on the uptake of omitbine by cultured human skin fibroblasts and the effect of omithine on the uptake of lysine by these cells Rate of uptake (nmol/min per mg protein) (A)
-Lys
Substrate 0.2 mM 0.3 mM 0.5 mM 0.7 mM 1.O mM
Ornithine Ornithine Ornithine Ornithine Ornithine
3.8 4.6 8.2 11.3 13.2
+
I mM
Lys
2.3 3.2 5.9 11.4 13.0
Rate of uptake (nmol/min per mg protein) -1
-2
-3
0
1
2
3
4
5
6
7 mM-’
IIS
Fig. 5. Lineweaver-Burk plots for the uptake of omithine (O-O) and lysine (W-W) by cultured human skin fibroblasts. For experimental details see “Materials and Methods”.
(8)
-0m
Substrate 0.15 0.20 0.30 0.50 0.80
mM mM mM mM mM
Lysine Lysine Lysine Lysine Lysine
4.8 6.0 7.0 12.6 18.4
+2mM 1.9 2.7 4.0 6.4 14.5
Om
836
K. METOKI and F. A. HOMMES Acknowledgement-This research was supported Grant AM29691. Contribution No. 0794.
by NIH
REFERENCES
0-7 0 10
20
30
40
Seconds
50
60
OY 0 10
20
30
40
50
60
Seconds
Fig. 7. Uptake of ornithine (A) and of lysine (B) by the cytosolic (A--A) and the particulate (0-O) fraction of cultured human skin fibroblasts. Cells were incubated with either ornithine or lysine (1 mM). The fractionation medium of the digitonin method contained 0.6 mg digitonin per ml and 50mM omithine or lysine to suppress non-
specific adsorption of the labelled amino acid to the particulare fraction.
rate of gluconeogenesis and ureogenesis from alanine, i.e. the criteria applied to evaluate the quality of the cells. The saturable uptake system is apparently rather sensitive to proteolytic attack by the enzymes used in the isolation of the hepatocytes. However, the fact that the saturable uptake system can be observed in isolated hepatocytes demonstrates the existence of this system in the liver. The maximum rate of uptake as determined in the present study may have been underestimated, due to the lability of this uptake system. Most of the amino acids taken up by isolated hepatocytes or fibroblasts is recovered in the cytosolic compartment of the cell. It can be calculated from the data given in Fig. 4, the volume of the liver cell, the number of cells per gram wet weight (Krebs et al., 1974) and the fraction of the cell volume taken by the cytosolic compartment (Vergonet et al., 1970) that within the first 60 set after addition of ornithine to the cells, the concentration of ornithine reaches a value of 0.27 mM, versus an outside concentration of 0.5 mM in that experiment. Equilibration with the medium proceeds therefore rapidly for this amino acid, as it does for lysine, which reaches an inside concentration of 0.4 mM, versus an outside concentration of 1 mM. The same uias observed in fibroblasts (Fig. 7), in agreement with the observation of White et al. (1982). An inside concentration of 0.78 and 0.46 mM for ornithine and lysine, respectively, is reached after 60 set incubation in the presence of 1 mM ornithine or 1 mM lysine.
Aronson D. L. and Diwan J. J. (1981) Uptake of ornithine by rat liver mitochondria. Biochemistry 20, 7064-7068. Berry M. N. and Friend D. S. (1969) High yield preparation of isolated rat liver parenchymal cells. J. cell. Biol. 43, 506-520. Blasberg H. and Lajtha A. (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain. Brain Rex 1, 86-104. Christensen H. N. (1964) A transport system serving for mono- and diamino acids. Proc. natn. Acad. Sri. U.S.A. 51, 337-344. Christensen H. N. and Antonioli J. A. (1969) Cationic amino acid transport in the rabbit reticulocyte. J. Viol. Chem. 244, 1497-1504. Groth U. and Rosenberg L. E. (1972) Transport of dibasic amino acids, cystine and tryptophan by cultured human fibroblasts: Absence of a defect in cystinuria and Hartnup disease. J. clin. Invest. 51, 213&2142. Hommes F. A., Ho C. K., Roesel R. A., Coryell M. E. and Gordon B. A. (1982) Decreased transport of ornithine across the inner mitochondrial membrane as a cause of hyperornithinemia. J. Inter. Metab. Dis. 5, 41-47. Hommes F. A., Kitchings L. and Eller A. G. (1983) The uptake of omithine and lysine by rat liver mitochondria. Biochem. Med. 30, 313-322. Krebs H. A., Cornell N. W., Lund P. and Hems R. (1974) Isolated liver cells as experimental material. In Regulation of Hepatic Metabolism (Edited by Lundquist F. and Tygstrup N.) pp. 726-750. Munsgaard, Copenhagen. Rognstad R. (1977) Sources of ammonia for urea synthesis in isolated rat liver cells. Biochim. biophys. Actu 496, 249-254. Segal S., Schwartzman L., Blair A. and Bertolli 0. (1967) Dibasic amino acid transport in rat kidney cortex slices. Biochim. biophys. Acta 135, 127-I 35. Sly W. S. and Grobb J. (1979) Isolation of fibroblasts from patients. Meth. Enzym. 58, 444450. Vergonet C., Hommes F. A. and Molenaar I. (1970) A morphometric and biochemical study to fetal and adult rat liver cells with special reference to energy metabolism. Biol. Neonat. 16, 297-305. White M. F. and Christensen H. N. (1982) Cationic amino acid transport into cultured animal cells. II. Transport system barely perceptible in ordinary hepatocytes but active in hepatoma cell lines. J. biol. Gem. 257, 445&4457. White M. F., Gazzola G. C. and Christensen H. N. (1982) Cationic amino acid transport into cultured human fibroblasts. J. biol. Chem. 257, 44434449. Williamson J. R., Meyer A. J. and Okhawa K. (1974) Interrelations between anion transport, ureogenesis, and gluconeogenesis in isolated liver cells. In Regulation qf Heparic Metabolism (Edited by Lundquist F. and Tygstrup N.) pp. 457-479. Munsgaard, Copenhagen. Zuurendonk P. F. and Tager J. M. (1974) Rapid separation of particulate components and soluble cytoplasm of isolated rat liver cells. Biochim. biophys. Acra 333, 393-399.