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Stabilization of cholic acid uptake in primary cultures of hepatocytes by dexamethasone and tocopherol
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 214, No. 2, April 1, pp. 850-852, 1982
COMMUNICATION Stabilization
of Cholic Acid Uptake in Primary Cultures of Hepatocytes Dexamethasone and Tocopherol’
by
JOHN GALIVAN Division
of Laboratories
and Research, New York State Department
of Health, Albany, New York 1.~01
Received October 8, 1981
The carrier-mediated transport of cholic acid has been examined in primary monolayer cultures of rat hepatocytes. The capacity of the cells to concentrate cholate was reduced by 96% between 24 and 72 h in culture. Inclusion of dexamethasone and tocopherol in the medium stabilized this process (resulting in a 2-fold elevation in uptake after 48 h in culture and 3.4-fold elevation after ‘72 h). Dexamethasone alone had no effect and tocopherol caused a partial stabilization. The two additives completely stabilized bromosulfophthalein uptake for 72 h, which showed a 50% reduction in unsupplemented medium over the same time period. The uptake of cholic acid and bromosulfophthalein was reduced by 98 and 96%, respectively, in a stable, transformed hepatic cell line.
Bile acids are transported into the liver by a carrier-mediated process, after which they are restricted for the most part to enterohepatic circulation (1, 2). To investigate this transport process, preparations of freshly isolated hepatocytes have been used (1, 2). The results indicate that hepatic uptake of cholate and taurocholate is extremely complex and may be mediated by as many as three systems. The use of primary cultures of hepatocytes rather than fresh isolates to study such processes offers the advantage of longer periods of viability. This advantage may be offset by a selective instability in certain membrane-related functions (3-6). Here we show that cholic acid uptake by hepatocytes in culture is unstable but can be stabilized by including dexamethasone and tocopherol in the medium. MATERIALS
AND METHODS
Hepatocytes were isolated and cultured from male Lewis rats as previously described (5, 6) in either Swims S77 medium or Liebowitz L15 medium. When
i This study was supported by NIH Research Grants from NC1 (CA25933) and NIA (AG0020’7). PHS/DHHS. 0003-9861/82/040850-03$02.00/O Copyright 0 1982 by Academic Press, Inc. All righta of reproduction in any form reserved.
included, the concentration of dexamethasone was 0.1 PM, and that of DL-cu-tocopherol was 10 FM. [2,4‘H]Cholic acid (New England Nuclear) was diluted to 2 X lo4 dpm/nmol, and uptake by hepatocytes was conducted as previously described (3, 5, 6). When included, inhibitors were added to the medium 15 min before addition of rH]cholic acid. Culturing and measurement of transport with H35 cells were conducted as previously described (7). RESULTS Uptake of cholic acid by primary cultures of hepatocytes decreases by 78% at 48 h and by 96% at 72 h, compared to uptake at 24 h in culture (Fig. 1). The use of a medium (L15) enriched in amino acids causes a higher level of cholate uptake, but the relative loss over 3 days in culture nearly offsets it. Inclusion of dexamethasone and tocopherol stabilized the cholate uptake through 48 h and caused a partial retention in uptake after 72 h in culture. When the same experiment was conducted with the hepatodiagnostic dye bromosulfophthalein the results were qualitatively similar, but the latter system was more stable. Inclusion of additives with Swims S77 medium caused increases in cholate uptake similar to those observed in L15. We have previously reported that other hepatic-membrane-related functions, such 850
CHOLIC
ACID UPTAKE
IN CULTURED
851
HEPATOCYTES
mulation
of cholate at a medium concentration of 10 reduction (1) and ouabain (1,2) are potent inhibitors of the bile acid transport system. Inhibition of cholate uptake by chenodeoxycholate suggests carrier-mediated transport and testifies to the similarity of transport in freshly isolated (2) and cultured hepatocytes. Dexamethasone alone had no effect on the level of cholate uptake with 48-h cultured hepatocytes, but tocopherol caused partial stabilization, and the two in combination appeared to be synergistic (Fig. 3). The dependence of elevated cholate uptake on the concentration of each additive when the other is present at saturation was examined. Dexamethasone exerted a half-maximal effect at 3 nM and tocopherol at 3.5 pM; the effect of each was maximal at 10 nM and 10 pM, respectively. pM. Temperature
I
48 HOURS
72 IN
24
48
72
CULTURE
FIG. 1. Uptake of cholic acid and bromosulfophthalein by hepatocytes as a function of time in culture. Hepatocytes were cultured in Swims S77 medium (m), L15 medium alone (o), or L15 with dexamethasane and tocopherol (lB). [3H]Cholic acid was added to the medium at 10 pM and bromosulfophthalein at 50 j&M. After a 2-h incubation the cellular level of each was measured as described under Materials and Methods, and bromosulfophthalein was detected spectrophotometrically at 580 nm (16).
as a-aminoisobutyrate uptake and tyrosine aminotransferase induction, are stable and actually increase between 24 and ‘72 h in culture, indicating that there is no general deterioration in membrane function (5). The effects of inhibitors and analogs on the uptake of cholate by hormone-stabilized hepatocytes which have been cultured for 48 h (Fig. 2) indicates that this transport process is not greatly different from that observed in freshly isolated cells (1,2). Concentrative uptake is demonstrated by the ‘IO-fold accu-
DISCUSSION The loss of cholic acid transport in unsupplemented hepatic monolayer cultures precludes the use of this system for prolonged studies of uptake and metabolism. Including dexamethasone and tocopherol in the medium stabilizes the levels of cholate uptake for 48 h in culture. Bromosulfophthalein uptake is stabilized by these inclusions for 72 h in culture. The similar effects of stabilization are consistent with the observation that cholate and bromosulfophthalein utilize the same Na+-independent transport system in liver (8). The reason(s) for the selective loss in transport functions is not understood. Certain liver plasma membrane receptors have a much more rapid turnover than the total plasma membrane pool (9). Also, hepatocytes are in negative nitrogen balance due to
DEXAMETHA’SNE
MINUTES FIG. 2. Effect
of inhibitors on uptake of cholic acid. The uptake of cholic acid was conducted as described in Fig. 1, after 48 h in culture. The control cultures were incubated in L15 medium with dexamethasone and tocopherol. Omission of these two components is indicated by “-hormone.”
MINUTES FIG. 3. Effect
of dexamethasone and tocopherol on cholic acid uptake. Cholic acid uptake was measured with hepatocytes after 48 h in culture as described in Fig. 1. Control measurements were conducted on cells that were cultured in unsupplemented L15 medium.
852
JOHN GALIVAN TABLE I
UPTAKE
OFBROMOSULFOPHTHALEIN AND CHOLIC BY CULTUREDHEPATOCYTES~
ACID
Substrate Cholic acid, 10PM
Cells
Hepatocytes H35 Cells Bromosulfophthalein, Hepatocytes 50 *td H35 Cells
Uptake (pmol/g cell protein) 1.8 0.04 16 0.6
“Uptake was conductedas describedin Fig. 1 using hepatocytes that had been in culture for 48 b and confluent H35 cells. high proteolytic activity during the first 24 h in culture (10). It is possible that the enrichment of amino acids in L15 medium relative to Swims S77 results in elevated cholate uptake by restricting proteolysis (10). A comparison of the effects of additives on the stabilization of cholate and methotrexate (6) uptake substantiates other findings about the transport of these two compounds in hepatocytes. Both are stabilized by similar medium additives (6), and when dexamethasone replaces hydrocortisone, glucagon offers no additional stimulation (J. Galivan, unpublished data). The possibility that the uptake of bile acids and methotrexate share some aspect of the same transport system was first suggested by Strum et al. (11) and this was substantiated by the observation of competition for transport between bile acids (also other organic acids and bromosulfophthalein) and methotrexate (12). In addition ouabain and sodium azide exert similar effects on the accumulation of cholic acid (Fig. 2) and methotrexate (4, 6). We have detected four hepatic transport functions (methotrexate (6), asialoglycoprotein (4, 5), cholic acid, bromosulfophthalein), where loss in vitro precedes cell deterioration under the culture conditions that we employ. The effects of medium additives suggest that dexamethasone and tocopherol may play a role in stabilizing these functions in vivo although further studies are needed to determine the mechanism of stabilization. Removing the cells from the animal and placing them in tissue culture is not the only perturbation which causes loss of these functions. When a stable rat hepatocarcinoma cell line (H35) was examined for uptake of asialoglycoprotein, this activity was either virtually undetectable or extremely low (4, 5). Hickman and Ashwell have reported that the membrane asialoglycoprotein receptor was absent in several rat liver hepatomas that they examined (13). An examination of the capacity of H35 cells to accumulate cholic acid and bromosulfophthalein revealed that there is little uptake relative to hepatocytes (Table I). Thus, the organic anion transport system also shows a marked insta-
bility in cultured cells and loss in the transformed hepatic cell line. The inclusion of dexamethasone and tocopherol had no effect on these transport functions in H35 cells (J. Galivan, unpublished data). In contrast a-aminoisobutyrate uptake and tyrosine aminotransferase inducibility, which are extremely stable in cultured hepatocytes (5,14), are present at high levels in H35 cells (15). The possible relationship in hepatic cells between loss of function due to (a) isolation and cultivation and (b) transformation is at present ambiguous. It is interesting to speculate that these membrane changes, which precede hepatic cell deterioration in culture, may be predictive of pathologic changes in vivo. First, however, other transformed hepatic cell lines should be examined to determine if the absence of these transport systems is a general phenomenon. ACKNOWLEDGMENTS The author wishes to thank Pat Fox, Zenia Nimec, and Joe Katagiri for their excellent technical assistance. REFERENCES 1. SCHWARZ,L. R., BURR,R., SCHWENK,M., PFAFF, E., AND GRIEM, H. (1975) Eur. J. Biochem 55, 617-623. 2. ANWER,M. S., ANDHEGNER,D. (1978) Hoppe Seylet- Z. PhysioL Chem 359, 181-192.
3. GALIVAN, J. (1979) Res. Commun. Chem. PathoL PhamzacoL 24,571-582. 4. GALIVAN, J., TARENTINO,A., AND SAMSONOFF, W. (1980) Ann N. Y. Acad. Sci. 349, 332-345. 5. TARENTINO,A. L., ANDGALIVAN, J. (1980)In Vitro 16,833-845. 6. GALIVAN, J. (1981) Arch B&hem. Biophys. 206, 113-121. 7. GALIVAN, J. (1979) Cancer Res. 39,735-743. 8. ANWER,M. S., ANDHEGNER,D. (1978) Hoppe Seyler Z. PhysioL Chem 359, 1027-1030. 9. WARREN,R., AND DOYLE,D. (1981) J. BioL Chem. 256,1346-1355. 10. SCHWARZ,P. E., AND SEGLEN,P. 0. (1980) Exp. Cell Res. 130.185-190. 11. STRUM,W. B., LIEM, H. H., AND MULLER-EBERHARD, U. (1978) Clin Res. 26, 113A. 12. GEWIRTZ,D. A., RANDOLPH,J. K., AND GOLDMAN, I. D. (1980) Cancer Res. 40, 1852-1857. 13. HICKMAN, J., AND ASHWELL, G. (1974) Enzyme Therapy in Liposomal Storage Diseases (Tager, J. M., Hooghwinkel, G. J. M., and Daems, W., eds.), pp. 169-172,North-Holland, Amsterdam. 14. BONNEY,R. J. (1974) In Vitro 10,130-142. 15. KRAWITT, E. L., BARIL, E. F., BECKER,J. E., AND POITER, V. R. (1970) Science 169, 294-296. 16. ACOSTA, D., ANUFORO, D. C., AND SMITH, R. V. (1978) In Vitro 14, 428-436.
COMMUNICATION Stabilization
of Cholic Acid Uptake in Primary Cultures of Hepatocytes Dexamethasone and Tocopherol’
by
JOHN GALIVAN Division
of Laboratories
and Research, New York State Department
of Health, Albany, New York 1.~01
Received October 8, 1981
The carrier-mediated transport of cholic acid has been examined in primary monolayer cultures of rat hepatocytes. The capacity of the cells to concentrate cholate was reduced by 96% between 24 and 72 h in culture. Inclusion of dexamethasone and tocopherol in the medium stabilized this process (resulting in a 2-fold elevation in uptake after 48 h in culture and 3.4-fold elevation after ‘72 h). Dexamethasone alone had no effect and tocopherol caused a partial stabilization. The two additives completely stabilized bromosulfophthalein uptake for 72 h, which showed a 50% reduction in unsupplemented medium over the same time period. The uptake of cholic acid and bromosulfophthalein was reduced by 98 and 96%, respectively, in a stable, transformed hepatic cell line.
Bile acids are transported into the liver by a carrier-mediated process, after which they are restricted for the most part to enterohepatic circulation (1, 2). To investigate this transport process, preparations of freshly isolated hepatocytes have been used (1, 2). The results indicate that hepatic uptake of cholate and taurocholate is extremely complex and may be mediated by as many as three systems. The use of primary cultures of hepatocytes rather than fresh isolates to study such processes offers the advantage of longer periods of viability. This advantage may be offset by a selective instability in certain membrane-related functions (3-6). Here we show that cholic acid uptake by hepatocytes in culture is unstable but can be stabilized by including dexamethasone and tocopherol in the medium. MATERIALS
AND METHODS
Hepatocytes were isolated and cultured from male Lewis rats as previously described (5, 6) in either Swims S77 medium or Liebowitz L15 medium. When
i This study was supported by NIH Research Grants from NC1 (CA25933) and NIA (AG0020’7). PHS/DHHS. 0003-9861/82/040850-03$02.00/O Copyright 0 1982 by Academic Press, Inc. All righta of reproduction in any form reserved.
included, the concentration of dexamethasone was 0.1 PM, and that of DL-cu-tocopherol was 10 FM. [2,4‘H]Cholic acid (New England Nuclear) was diluted to 2 X lo4 dpm/nmol, and uptake by hepatocytes was conducted as previously described (3, 5, 6). When included, inhibitors were added to the medium 15 min before addition of rH]cholic acid. Culturing and measurement of transport with H35 cells were conducted as previously described (7). RESULTS Uptake of cholic acid by primary cultures of hepatocytes decreases by 78% at 48 h and by 96% at 72 h, compared to uptake at 24 h in culture (Fig. 1). The use of a medium (L15) enriched in amino acids causes a higher level of cholate uptake, but the relative loss over 3 days in culture nearly offsets it. Inclusion of dexamethasone and tocopherol stabilized the cholate uptake through 48 h and caused a partial retention in uptake after 72 h in culture. When the same experiment was conducted with the hepatodiagnostic dye bromosulfophthalein the results were qualitatively similar, but the latter system was more stable. Inclusion of additives with Swims S77 medium caused increases in cholate uptake similar to those observed in L15. We have previously reported that other hepatic-membrane-related functions, such 850
CHOLIC
ACID UPTAKE
IN CULTURED
851
HEPATOCYTES
mulation
of cholate at a medium concentration of 10 reduction (1) and ouabain (1,2) are potent inhibitors of the bile acid transport system. Inhibition of cholate uptake by chenodeoxycholate suggests carrier-mediated transport and testifies to the similarity of transport in freshly isolated (2) and cultured hepatocytes. Dexamethasone alone had no effect on the level of cholate uptake with 48-h cultured hepatocytes, but tocopherol caused partial stabilization, and the two in combination appeared to be synergistic (Fig. 3). The dependence of elevated cholate uptake on the concentration of each additive when the other is present at saturation was examined. Dexamethasone exerted a half-maximal effect at 3 nM and tocopherol at 3.5 pM; the effect of each was maximal at 10 nM and 10 pM, respectively. pM. Temperature
I
48 HOURS
72 IN
24
48
72
CULTURE
FIG. 1. Uptake of cholic acid and bromosulfophthalein by hepatocytes as a function of time in culture. Hepatocytes were cultured in Swims S77 medium (m), L15 medium alone (o), or L15 with dexamethasane and tocopherol (lB). [3H]Cholic acid was added to the medium at 10 pM and bromosulfophthalein at 50 j&M. After a 2-h incubation the cellular level of each was measured as described under Materials and Methods, and bromosulfophthalein was detected spectrophotometrically at 580 nm (16).
as a-aminoisobutyrate uptake and tyrosine aminotransferase induction, are stable and actually increase between 24 and ‘72 h in culture, indicating that there is no general deterioration in membrane function (5). The effects of inhibitors and analogs on the uptake of cholate by hormone-stabilized hepatocytes which have been cultured for 48 h (Fig. 2) indicates that this transport process is not greatly different from that observed in freshly isolated cells (1,2). Concentrative uptake is demonstrated by the ‘IO-fold accu-
DISCUSSION The loss of cholic acid transport in unsupplemented hepatic monolayer cultures precludes the use of this system for prolonged studies of uptake and metabolism. Including dexamethasone and tocopherol in the medium stabilizes the levels of cholate uptake for 48 h in culture. Bromosulfophthalein uptake is stabilized by these inclusions for 72 h in culture. The similar effects of stabilization are consistent with the observation that cholate and bromosulfophthalein utilize the same Na+-independent transport system in liver (8). The reason(s) for the selective loss in transport functions is not understood. Certain liver plasma membrane receptors have a much more rapid turnover than the total plasma membrane pool (9). Also, hepatocytes are in negative nitrogen balance due to
DEXAMETHA’SNE
MINUTES FIG. 2. Effect
of inhibitors on uptake of cholic acid. The uptake of cholic acid was conducted as described in Fig. 1, after 48 h in culture. The control cultures were incubated in L15 medium with dexamethasone and tocopherol. Omission of these two components is indicated by “-hormone.”
MINUTES FIG. 3. Effect
of dexamethasone and tocopherol on cholic acid uptake. Cholic acid uptake was measured with hepatocytes after 48 h in culture as described in Fig. 1. Control measurements were conducted on cells that were cultured in unsupplemented L15 medium.
852
JOHN GALIVAN TABLE I
UPTAKE
OFBROMOSULFOPHTHALEIN AND CHOLIC BY CULTUREDHEPATOCYTES~
ACID
Substrate Cholic acid, 10PM
Cells
Hepatocytes H35 Cells Bromosulfophthalein, Hepatocytes 50 *td H35 Cells
Uptake (pmol/g cell protein) 1.8 0.04 16 0.6
“Uptake was conductedas describedin Fig. 1 using hepatocytes that had been in culture for 48 b and confluent H35 cells. high proteolytic activity during the first 24 h in culture (10). It is possible that the enrichment of amino acids in L15 medium relative to Swims S77 results in elevated cholate uptake by restricting proteolysis (10). A comparison of the effects of additives on the stabilization of cholate and methotrexate (6) uptake substantiates other findings about the transport of these two compounds in hepatocytes. Both are stabilized by similar medium additives (6), and when dexamethasone replaces hydrocortisone, glucagon offers no additional stimulation (J. Galivan, unpublished data). The possibility that the uptake of bile acids and methotrexate share some aspect of the same transport system was first suggested by Strum et al. (11) and this was substantiated by the observation of competition for transport between bile acids (also other organic acids and bromosulfophthalein) and methotrexate (12). In addition ouabain and sodium azide exert similar effects on the accumulation of cholic acid (Fig. 2) and methotrexate (4, 6). We have detected four hepatic transport functions (methotrexate (6), asialoglycoprotein (4, 5), cholic acid, bromosulfophthalein), where loss in vitro precedes cell deterioration under the culture conditions that we employ. The effects of medium additives suggest that dexamethasone and tocopherol may play a role in stabilizing these functions in vivo although further studies are needed to determine the mechanism of stabilization. Removing the cells from the animal and placing them in tissue culture is not the only perturbation which causes loss of these functions. When a stable rat hepatocarcinoma cell line (H35) was examined for uptake of asialoglycoprotein, this activity was either virtually undetectable or extremely low (4, 5). Hickman and Ashwell have reported that the membrane asialoglycoprotein receptor was absent in several rat liver hepatomas that they examined (13). An examination of the capacity of H35 cells to accumulate cholic acid and bromosulfophthalein revealed that there is little uptake relative to hepatocytes (Table I). Thus, the organic anion transport system also shows a marked insta-
bility in cultured cells and loss in the transformed hepatic cell line. The inclusion of dexamethasone and tocopherol had no effect on these transport functions in H35 cells (J. Galivan, unpublished data). In contrast a-aminoisobutyrate uptake and tyrosine aminotransferase inducibility, which are extremely stable in cultured hepatocytes (5,14), are present at high levels in H35 cells (15). The possible relationship in hepatic cells between loss of function due to (a) isolation and cultivation and (b) transformation is at present ambiguous. It is interesting to speculate that these membrane changes, which precede hepatic cell deterioration in culture, may be predictive of pathologic changes in vivo. First, however, other transformed hepatic cell lines should be examined to determine if the absence of these transport systems is a general phenomenon. ACKNOWLEDGMENTS The author wishes to thank Pat Fox, Zenia Nimec, and Joe Katagiri for their excellent technical assistance. REFERENCES 1. SCHWARZ,L. R., BURR,R., SCHWENK,M., PFAFF, E., AND GRIEM, H. (1975) Eur. J. Biochem 55, 617-623. 2. ANWER,M. S., ANDHEGNER,D. (1978) Hoppe Seylet- Z. PhysioL Chem 359, 181-192.
3. GALIVAN, J. (1979) Res. Commun. Chem. PathoL PhamzacoL 24,571-582. 4. GALIVAN, J., TARENTINO,A., AND SAMSONOFF, W. (1980) Ann N. Y. Acad. Sci. 349, 332-345. 5. TARENTINO,A. L., ANDGALIVAN, J. (1980)In Vitro 16,833-845. 6. GALIVAN, J. (1981) Arch B&hem. Biophys. 206, 113-121. 7. GALIVAN, J. (1979) Cancer Res. 39,735-743. 8. ANWER,M. S., ANDHEGNER,D. (1978) Hoppe Seyler Z. PhysioL Chem 359, 1027-1030. 9. WARREN,R., AND DOYLE,D. (1981) J. BioL Chem. 256,1346-1355. 10. SCHWARZ,P. E., AND SEGLEN,P. 0. (1980) Exp. Cell Res. 130.185-190. 11. STRUM,W. B., LIEM, H. H., AND MULLER-EBERHARD, U. (1978) Clin Res. 26, 113A. 12. GEWIRTZ,D. A., RANDOLPH,J. K., AND GOLDMAN, I. D. (1980) Cancer Res. 40, 1852-1857. 13. HICKMAN, J., AND ASHWELL, G. (1974) Enzyme Therapy in Liposomal Storage Diseases (Tager, J. M., Hooghwinkel, G. J. M., and Daems, W., eds.), pp. 169-172,North-Holland, Amsterdam. 14. BONNEY,R. J. (1974) In Vitro 10,130-142. 15. KRAWITT, E. L., BARIL, E. F., BECKER,J. E., AND POITER, V. R. (1970) Science 169, 294-296. 16. ACOSTA, D., ANUFORO, D. C., AND SMITH, R. V. (1978) In Vitro 14, 428-436.