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Sources of reducing equivalents for the microsomal steroid 21-hydroxylation in isolated rat adrenal cells
ARCHIVES
OF
BIOCHEMISTRY
Sources
AND
BIOPHYSICS
173, 121-124
(1976)
of Reducing Equivalents for the Microsomal Steroid Hydroxylation in Isolated Rat Adrenal Cells
AJAI
HAKSAR,’
MING-TE
LIN,
AND
FERNAND
21-
G. PERON
With the Technical Assistance of William F. Robidoux, Jr., and Sara Seekings Worcester Foundation
for
Experimental Received
Biology, Shrewsbury, August
Massachusetts 01545
6, 1975
21-Hydroxylation of II@hydroxyprogesterone in the intact adrenal cells was stimulated by both glucose and pyruvate. Arsenite inhibited the basal as well as the pyruvatesupported reaction and also prevented the entry of pyruvate carbon into the Krebs cycle. Glucose-supported 21-hydroxylation was not inhibited by arsenite. It is proposed that NADPH for the microsomal21-hydroxylation is derived by at least two mechanisms: (1) metabolism of glucose via the pentose shunt and (2) a mechanism involving transfer of reducing equivalents from the mitochondria into the cytosol. The latter would involve the transfer of some Krebs cycle intermediate (or intermediates) from the mitochondria to the cytosol followed by its eventual metabolism in the cytosol via an NADPH-linked dehydrogenase. This mechanism may assume importance when the cell has a limited supply of glucose.
Biologically active corticosteroids possessa hydroxyl group in 21-position of the steroid side chain. Hydroxylation in this position is catalyzed by the enzyme 21hydroxylase, which is localized in the adrenal cortex microsomes and shows a requirement for NADPH and molecular oxygen (1, 2). Although the adrenal cortex has several NADPH-linked dehydrogenase in the cytosol, i.e., glucose-8phosphate and 6-phosphogluconate dehydrogenases of the pentose shunt, isocitrate dehydrogenase, and malic enzyme (3-6), the relative contribution of these towards the production of NADPH required for 21-hydroxylation is not known. Recently, we have shown that operation of the Krebs cycle is essential for the production of intramitochondrial NADPH required for 11/3-hydroxylation of deoxycorticosterone in the intact rat adrenal cells (7). Further, the data obtained by several investigators suggest that a constant supply of oxaloacetate, via the enzyme pyruvate carboxylase, may be essential for the optimum operation of the Krebs cycle in adrenal mitochondria (7-9). Normally, if there ’ Deceased
August
8, 1975. 121
Copyright
0 1976 by Academic
Press.
Inc.
is no drain on the Krebs cycle intermediates, the cycle should be able to provide sufficient oxaloacetate, via malate dehydrogenase, for its optimum operation. If, however, some Krebs cycle intermediate (or intermediates) is drawn away from the mitochondria into the cytosol, there would be a need for the replenishment of oxaloacetate via pyruvate carboxylase for proper functioning of the cycle. If some Krebs cycle acid does leave the mitochondria, its function in the cytosol may be to provide NADPH for the microsomal steroid 21-hydroxylase. Thus, malate and isocitrate could provide NADPH in the cytosol via NADP-linked malic enzyme and isocitrate dehydrogenase, respectively. Citrate and oxaloacetate, on the other hand, could provide NADPH via reaction 3 (catalyzed by malic enzyme) of the following scheme: Citrate
A oxaloacetate
5 malate
-% pyruvate
In this report, we present data that suggest that there is a transfer of reducing equivalents from the mitochondria into the cytosol and that this may be a mechanism in intact adrenal cells for providing some of the NADPH for the microsomal steroid 21-hydroxylase.
122
HAKSAR, MATERIALS
AND
LIN
AND
DISCUSSION
Figure 1 shows the effect of steroid substrate concentration on the rate of 21-hydroxylation supported by endogenous energy sources of the adrenal cells. Maximum basal production, obtained with 30 PM ll@hydroxyprogesterone, varied from l-2.5 nmol of corticosterone/105 cells/2 h on different days of experimentation. Figure 2 shows that the rate of corticosterone for-
114 ~Hyaroxrpr39estero”e.~~~
PERON
METHODS
Trypsin and lima bean trypsin inhibitor were purchased from Worthington Biochemical Corp. ; collagenase from Gallard Schlesinger Chemical Manufacturing Corp.; bovine serum albumin (fraction V powder, fatty acid poor) from Miles Laboratories; 11/3-hydroxyprogesterone from Sigma Chemical Co.; [3J4C1pyruvate, [l-‘4Clglucose, and 16J4C1glucose from New England Nuclear Corp. All other chemicals used were reagent grade. Suspensions of isolated adrenal cells were prepared by collagenase-trypsin treatment of rat adrenal sections (10, 11). Incubations were carried out in duplicate or triplicate in Krebs-Ringer phosphate buffer, pH 7.4, containing 5 mM bicarbonate (7). Procedures for the collection of ‘%02 and measurement of radioactivity have been described previously (7). For studying the Zl-hydroxylation reaction, the cells were incubated with exogenously added liphydroxyprogesterone and the product of the reaction, corticosterone, was measured by the method of Silber et al. (12) in the dichloromethane exact of the cell suspensions. The substrate, llphydroxyprogesterone, does not interfere in the corticosterone assay. RESULTS
AND
Time, m\n”tes
FIG. 1. Effect of substrate concentration on the 21-hydroxylation reaction in intact adrenal cells. Incubation time, 2 h. FIG. 2. Time course of 21-hydroxylation of liphydroxyprogesterone (30 PM). Control, 0; 4 m&f pyruvate, A; 8 mM glucose, 0.
0
06 Number
12 01 cells
1.8 x105 3
01 Glucose
10 0, pfruvcfe.
100 rnM
FIG. 3. Corticosterone production from 30 PM 11/3-hydroxyprogesterone versus number of cells. Control, 0; 4 mM pyruvate, A; 8 mM glucose, 0. FIG. 4. Effect of different concentrations of pyruvate (A) and glucose (0) on the Pl-hydroxylation of lip-hydroxyprogesterone. Concentration of the steroid, 30 PM.
mation remained constant during a 2-h incubation. Addition of 8 mM glucose increased the rate by more than twofold. A similar effect was observed with 4 mM pyruvate. Figure 3 shows that the production of corticosterone from llp-hydroxyprogesterone was proportional to the number of cells, at least up to 1.8 x lo5 cells/tube, regardless of whether pyruvate or glucose was present in the incubation medium. All other experiments reported in this paper were carried out with 1.5-2.0 x lo5 cells/tube. Figure 4 shows the effect of different concentrations of pyruvate and glucose on the conversion of 11/3-hydroxyprogesterone into corticosterone. The data are plotted semilogarithmically to compare pyruvate and glucose curves. Maximum effect of pyruvate was observed at 0.2-0.4 mM concentration, while that of glucose was observed at about 2 mM. Stimulation of 21-hydroxylation by glucose may be explained by the fact that metabolism of glucose in the cytosol yields NADPH via the pentose shunt. Pyruvate, on the other hand, is not known to undergo oxidative metabolism in the cytosol. It is, however, known to supply carbon for the operation of the Krebs cycle in the mitochondria of intact adrenal cells (7, 13). If the stimulation of 21-hydroxylation by pyruvate depended on its utilization by the mitochrondrial enzyme pyruvate dehydro-
NADPH
FOR
STEROID
genase, then inhibition of this enzyme by arsenite also should lead to a decrease in the conversion of lip-hydroxyprogesterone into corticosterone. Table I shows that the basal as well as pyruvate stimulated 21hydroxylation was inhibited by arsenite in a concentration dependent manner. This inhibition could not be reversed by increasing the concentration of pyruvate to 4 mM. Stimulation of the 21-hydroxylation reaction by glucose also appeared to be inhibited by arsenite. However, this inhibition is more apparent than real since it is all attributable to inhibition of the basal corticosterone production. As can be calculated (subtracting the arsenite values from the nonarsenite ones in the basal conditions and adding the differences to those values obtained at the same arsenite concentrations plus glucose), 1 mM arsenite which produced 55% inhibition of the pyruvate-supported 21-hydroxylation caused little or no inhibition of corticosterone formation when the incubations were carried out in the presence of 1, 2, and 8 mM glucose, respectively (Table I). Experimental data which could be used to explain the inhibition of the 21-hydroxylation reaction in the basal conditions are not available at present. Nevertheless, it can be conjectured that there is sufficient provenance of endogenous intracellular pyruvate to support the steroid hydroxylation reaction albeit at low levels. As can be seen in Table I, TABLE EFFECT
Expt
OF ARSENITE
between 1.4 and 1.9 nmol corticosterone are produced in the absence of arsenite in the basal conditions. The inhibition brought about by various arsenite concentrations in this situation is indeed striking and in these cases, as in those incubations carried out with exogenous pyruvate, arsenite is likely to act in a similar manner and inhibit endogenous pyruvate utilization via the complex pyruvate dehydrogenase enzyme. Table II shows that 1 mM arsenite almost completely inhibited the production TABLE EFFECT
[3-14C]P~~uv~~~,
Labelled
Substrate
1.0 rnM 4.0 rnM
8rnM
OF “C0,
PRODUCTION
[l-“TIGLUCOSE, %]GLUCOSE”
[6-
AND
‘%O. _ Formed,
dpm/lO” hours
- Arsenite [3-‘%lPyruvate [1-‘“ClGlucose [6-%lGlucose
+
cells/2
Arsenite (500 PM)
4,970 750 270
170 1,530 70
u Each tube contained 1.5 x lo” cells, 30 nmol of 11/3-hydroxyprogesterone and 3 pmol ofthe appropriate labeled substrate in a total volume of 1.5 ml. The specific activities of [3-‘%lpyruvate, [l-14Clglucose, and [6-“Vlglucose were 0.74, 0.35, and 0.30 x lo6 dpm/Fmol, respectively. The incubation procedure and measurement of ‘%O, has been described previously (7). I INTO
30
Arsenite 30 Corticosterone
None Pyruvate 0.5 rnM
II
ON THE
-
ON THE CONVERSION OF lip-HYDROXYPROCESTERONE (CONCENTRATION OF THE STEROID SUBSTRATE,
0
IrnM 2rnM
OF ARSENITE
FROM
Additions
None Glucose
123
21-HYDROXYLATION
CORTICOSTERONE
PM)
(KM) 300
100
formed
(nmol/lO”
cells/2
1000
h)
1.4
0.9
0.7
0.5
0.4
2.9 2.9 2.9 1.9
2.0 2.2 2.3 1.0
1.3 1.4 0.8
0.5 0.4 0.4 0.6
0.3 0.3 0.3 0.5
3.6 4.0 3.9
2.9 3.6 3.8
2.7 3.1 3.6
2.4 2.8 3.4
1.6 2.4 3.2
1.1
124
HAKSAR,
LIN
AND
PERON
importance when there is a limited supply of glucose to the cell. It may be pointed out here that in the adipose tissue only about 60% of the NADPH required to support fatty acid synthesis in the cytosol is generated via the pentose pathway (14, 15); the remainder being derived via malic enzyme and isocitrate dehydrogenase (16, 17). ACKNOWLEDGMENTS FIG. 5. Sources droxylation.
of NADPH
for microsomal
21-hy-
of 14C0, from [3-14Clpyruvate, indicating that the entry of pyruvate carbon into the Krebs cycle was prevented. As expected, arsenite also inhibited the production of 14C02 from [6-14C]glucose. The observation that the production of corticosterone from lip-hydroxyprogesterone depends to a certain extent on the activity of the Krebs cycle, therefore, suggests the possibility that some Krebs cycle acid may leave the mitochondria to provide NADPH for the microsomal 21-hydroxylation. The stimulation of 14C0, production from [l14C]glucose by arsenite, indicating an increase in the pentose shunt activity, was unexpected (Table II). It may be mentioned here that a similar effect of arsenite on the production of 14C0, from [l14Clglucose also has been observed in the intact cells obtained from Snell Adrenocortical Carcinoma 494 (P&on, unpublished observations). At present we do not have an explanation for this phenomenon. It is possible, however, that because of a decrease in the Krebs cycle activity in the presence of arsenite, the transfer of reducing equivalents from the mitochondria into the cytosol was also decreased, and therefore, the cell may have adapted by increasing the activity of the pentose shunt to maintain adequate levels of NADPH in the cytosol. In conjunction with our previous resu1t.s (71, the data reported now indicate that at least a portion of the NADPH required for the microsomal 21-hydroxylation may be derived by a mechanism involving the transfer of reducing equivalents from mitochondria into the cytosol via some Krebs cycle intermediate (Fig. 5). Also, it appears that this mechanism may assume
This work was supported tional Science Foundation tional Institute of Arthritis, tive Diseases (AM-04899).
by grants (GB-36246) Metabolism
from the Naand the Naand Diges-
REFERENCES 1. RYAN, K. J., AND ENGEL, L. L. (1957) J. Biol. Chem. 255,103-114. 2. COOPER, D. Y., NARASIMHULU, S., ROSENTHAL, O., AND ESTABROOK, R. W. (1968) in Functions of the Adrenal Cortex, (McKerns, K. W., ed.), pp. 897-942, Appleton-Century-Crofts, New York. 3. KELLY, T. L., NELSON, E. D., JOHNSON, R. B., AND VESTLING, C. S. (1955) J. Biol. Chem. 212, 545-554. 4. GLOCK, G. E., AND MCLEAN, P. (1954) B&hem. J. 56, 171-175. 5. STUDZINSKI, G. P., SYMINGTON, T., AND GRANT, J. K. (1962) Acta Endocrinol. 40, 232-246. 6. SIMPSON, E. R., CAMNER, W., AND ESTABROOK, R. W. (1968) Biochem. Biophys. Res. Commun. 31, 113-118. 7. TSANG, C. P. W., AND PBRON, F. G. (1970) Steroids 15, 251-265. 8. LIN, M. T., HAKSAR, A., AND P&RON, F. G. (1974) Arch. Biochem. Boiphys. 164,429-439. 9. SIMPSON, E. R., AND BOYD, G. S. (1971) Eur. J. B&hem. 22,489-499. 10. HAKSAR, A., AND P%RON, F. G. (1972) J. Steroid B&hem. 3,847-857. 11. HAKSAR, A., BANIUKIEWICZ, S., AND PBRON, F. G. (1973) Biochem. Biophys. Res. Commun. 52, 959-966. 12. SILBER, R. H., BUSCH, R. D., AND OSLAPAS, R. (1958) Clin. Chem. 4, 278-285. 13. LIN, M. T., HAKSAR, A., AND P&RON, F. G. (1974) Biochem. Biophys. Res. Commun. 58, 983989. 14. KATZ, J., LANDAU, B. R., AND BARTSCH, G. E. (1966) J. Biol. Chem. 241, 727-740. 15. FLATT, J. P., AND BALL, E. G. (1964) J. Biol. Chem. 239, 675-685. 16. YOUNG, J. W., SHRAGO, E., AND LARDY, H. A. (1964) Biochemistry 3, 1687-1692. 17. HANSON, R. W., PATEL, M. S., JOWAIN-BAUM, M., AND BALLARD, F. J. (1971) Metabolism 20, 27-42.
OF
BIOCHEMISTRY
Sources
AND
BIOPHYSICS
173, 121-124
(1976)
of Reducing Equivalents for the Microsomal Steroid Hydroxylation in Isolated Rat Adrenal Cells
AJAI
HAKSAR,’
MING-TE
LIN,
AND
FERNAND
21-
G. PERON
With the Technical Assistance of William F. Robidoux, Jr., and Sara Seekings Worcester Foundation
for
Experimental Received
Biology, Shrewsbury, August
Massachusetts 01545
6, 1975
21-Hydroxylation of II@hydroxyprogesterone in the intact adrenal cells was stimulated by both glucose and pyruvate. Arsenite inhibited the basal as well as the pyruvatesupported reaction and also prevented the entry of pyruvate carbon into the Krebs cycle. Glucose-supported 21-hydroxylation was not inhibited by arsenite. It is proposed that NADPH for the microsomal21-hydroxylation is derived by at least two mechanisms: (1) metabolism of glucose via the pentose shunt and (2) a mechanism involving transfer of reducing equivalents from the mitochondria into the cytosol. The latter would involve the transfer of some Krebs cycle intermediate (or intermediates) from the mitochondria to the cytosol followed by its eventual metabolism in the cytosol via an NADPH-linked dehydrogenase. This mechanism may assume importance when the cell has a limited supply of glucose.
Biologically active corticosteroids possessa hydroxyl group in 21-position of the steroid side chain. Hydroxylation in this position is catalyzed by the enzyme 21hydroxylase, which is localized in the adrenal cortex microsomes and shows a requirement for NADPH and molecular oxygen (1, 2). Although the adrenal cortex has several NADPH-linked dehydrogenase in the cytosol, i.e., glucose-8phosphate and 6-phosphogluconate dehydrogenases of the pentose shunt, isocitrate dehydrogenase, and malic enzyme (3-6), the relative contribution of these towards the production of NADPH required for 21-hydroxylation is not known. Recently, we have shown that operation of the Krebs cycle is essential for the production of intramitochondrial NADPH required for 11/3-hydroxylation of deoxycorticosterone in the intact rat adrenal cells (7). Further, the data obtained by several investigators suggest that a constant supply of oxaloacetate, via the enzyme pyruvate carboxylase, may be essential for the optimum operation of the Krebs cycle in adrenal mitochondria (7-9). Normally, if there ’ Deceased
August
8, 1975. 121
Copyright
0 1976 by Academic
Press.
Inc.
is no drain on the Krebs cycle intermediates, the cycle should be able to provide sufficient oxaloacetate, via malate dehydrogenase, for its optimum operation. If, however, some Krebs cycle intermediate (or intermediates) is drawn away from the mitochondria into the cytosol, there would be a need for the replenishment of oxaloacetate via pyruvate carboxylase for proper functioning of the cycle. If some Krebs cycle acid does leave the mitochondria, its function in the cytosol may be to provide NADPH for the microsomal steroid 21-hydroxylase. Thus, malate and isocitrate could provide NADPH in the cytosol via NADP-linked malic enzyme and isocitrate dehydrogenase, respectively. Citrate and oxaloacetate, on the other hand, could provide NADPH via reaction 3 (catalyzed by malic enzyme) of the following scheme: Citrate
A oxaloacetate
5 malate
-% pyruvate
In this report, we present data that suggest that there is a transfer of reducing equivalents from the mitochondria into the cytosol and that this may be a mechanism in intact adrenal cells for providing some of the NADPH for the microsomal steroid 21-hydroxylase.
122
HAKSAR, MATERIALS
AND
LIN
AND
DISCUSSION
Figure 1 shows the effect of steroid substrate concentration on the rate of 21-hydroxylation supported by endogenous energy sources of the adrenal cells. Maximum basal production, obtained with 30 PM ll@hydroxyprogesterone, varied from l-2.5 nmol of corticosterone/105 cells/2 h on different days of experimentation. Figure 2 shows that the rate of corticosterone for-
114 ~Hyaroxrpr39estero”e.~~~
PERON
METHODS
Trypsin and lima bean trypsin inhibitor were purchased from Worthington Biochemical Corp. ; collagenase from Gallard Schlesinger Chemical Manufacturing Corp.; bovine serum albumin (fraction V powder, fatty acid poor) from Miles Laboratories; 11/3-hydroxyprogesterone from Sigma Chemical Co.; [3J4C1pyruvate, [l-‘4Clglucose, and 16J4C1glucose from New England Nuclear Corp. All other chemicals used were reagent grade. Suspensions of isolated adrenal cells were prepared by collagenase-trypsin treatment of rat adrenal sections (10, 11). Incubations were carried out in duplicate or triplicate in Krebs-Ringer phosphate buffer, pH 7.4, containing 5 mM bicarbonate (7). Procedures for the collection of ‘%02 and measurement of radioactivity have been described previously (7). For studying the Zl-hydroxylation reaction, the cells were incubated with exogenously added liphydroxyprogesterone and the product of the reaction, corticosterone, was measured by the method of Silber et al. (12) in the dichloromethane exact of the cell suspensions. The substrate, llphydroxyprogesterone, does not interfere in the corticosterone assay. RESULTS
AND
Time, m\n”tes
FIG. 1. Effect of substrate concentration on the 21-hydroxylation reaction in intact adrenal cells. Incubation time, 2 h. FIG. 2. Time course of 21-hydroxylation of liphydroxyprogesterone (30 PM). Control, 0; 4 m&f pyruvate, A; 8 mM glucose, 0.
0
06 Number
12 01 cells
1.8 x105 3
01 Glucose
10 0, pfruvcfe.
100 rnM
FIG. 3. Corticosterone production from 30 PM 11/3-hydroxyprogesterone versus number of cells. Control, 0; 4 mM pyruvate, A; 8 mM glucose, 0. FIG. 4. Effect of different concentrations of pyruvate (A) and glucose (0) on the Pl-hydroxylation of lip-hydroxyprogesterone. Concentration of the steroid, 30 PM.
mation remained constant during a 2-h incubation. Addition of 8 mM glucose increased the rate by more than twofold. A similar effect was observed with 4 mM pyruvate. Figure 3 shows that the production of corticosterone from llp-hydroxyprogesterone was proportional to the number of cells, at least up to 1.8 x lo5 cells/tube, regardless of whether pyruvate or glucose was present in the incubation medium. All other experiments reported in this paper were carried out with 1.5-2.0 x lo5 cells/tube. Figure 4 shows the effect of different concentrations of pyruvate and glucose on the conversion of 11/3-hydroxyprogesterone into corticosterone. The data are plotted semilogarithmically to compare pyruvate and glucose curves. Maximum effect of pyruvate was observed at 0.2-0.4 mM concentration, while that of glucose was observed at about 2 mM. Stimulation of 21-hydroxylation by glucose may be explained by the fact that metabolism of glucose in the cytosol yields NADPH via the pentose shunt. Pyruvate, on the other hand, is not known to undergo oxidative metabolism in the cytosol. It is, however, known to supply carbon for the operation of the Krebs cycle in the mitochondria of intact adrenal cells (7, 13). If the stimulation of 21-hydroxylation by pyruvate depended on its utilization by the mitochrondrial enzyme pyruvate dehydro-
NADPH
FOR
STEROID
genase, then inhibition of this enzyme by arsenite also should lead to a decrease in the conversion of lip-hydroxyprogesterone into corticosterone. Table I shows that the basal as well as pyruvate stimulated 21hydroxylation was inhibited by arsenite in a concentration dependent manner. This inhibition could not be reversed by increasing the concentration of pyruvate to 4 mM. Stimulation of the 21-hydroxylation reaction by glucose also appeared to be inhibited by arsenite. However, this inhibition is more apparent than real since it is all attributable to inhibition of the basal corticosterone production. As can be calculated (subtracting the arsenite values from the nonarsenite ones in the basal conditions and adding the differences to those values obtained at the same arsenite concentrations plus glucose), 1 mM arsenite which produced 55% inhibition of the pyruvate-supported 21-hydroxylation caused little or no inhibition of corticosterone formation when the incubations were carried out in the presence of 1, 2, and 8 mM glucose, respectively (Table I). Experimental data which could be used to explain the inhibition of the 21-hydroxylation reaction in the basal conditions are not available at present. Nevertheless, it can be conjectured that there is sufficient provenance of endogenous intracellular pyruvate to support the steroid hydroxylation reaction albeit at low levels. As can be seen in Table I, TABLE EFFECT
Expt
OF ARSENITE
between 1.4 and 1.9 nmol corticosterone are produced in the absence of arsenite in the basal conditions. The inhibition brought about by various arsenite concentrations in this situation is indeed striking and in these cases, as in those incubations carried out with exogenous pyruvate, arsenite is likely to act in a similar manner and inhibit endogenous pyruvate utilization via the complex pyruvate dehydrogenase enzyme. Table II shows that 1 mM arsenite almost completely inhibited the production TABLE EFFECT
[3-14C]P~~uv~~~,
Labelled
Substrate
1.0 rnM 4.0 rnM
8rnM
OF “C0,
PRODUCTION
[l-“TIGLUCOSE, %]GLUCOSE”
[6-
AND
‘%O. _ Formed,
dpm/lO” hours
- Arsenite [3-‘%lPyruvate [1-‘“ClGlucose [6-%lGlucose
+
cells/2
Arsenite (500 PM)
4,970 750 270
170 1,530 70
u Each tube contained 1.5 x lo” cells, 30 nmol of 11/3-hydroxyprogesterone and 3 pmol ofthe appropriate labeled substrate in a total volume of 1.5 ml. The specific activities of [3-‘%lpyruvate, [l-14Clglucose, and [6-“Vlglucose were 0.74, 0.35, and 0.30 x lo6 dpm/Fmol, respectively. The incubation procedure and measurement of ‘%O, has been described previously (7). I INTO
30
Arsenite 30 Corticosterone
None Pyruvate 0.5 rnM
II
ON THE
-
ON THE CONVERSION OF lip-HYDROXYPROCESTERONE (CONCENTRATION OF THE STEROID SUBSTRATE,
0
IrnM 2rnM
OF ARSENITE
FROM
Additions
None Glucose
123
21-HYDROXYLATION
CORTICOSTERONE
PM)
(KM) 300
100
formed
(nmol/lO”
cells/2
1000
h)
1.4
0.9
0.7
0.5
0.4
2.9 2.9 2.9 1.9
2.0 2.2 2.3 1.0
1.3 1.4 0.8
0.5 0.4 0.4 0.6
0.3 0.3 0.3 0.5
3.6 4.0 3.9
2.9 3.6 3.8
2.7 3.1 3.6
2.4 2.8 3.4
1.6 2.4 3.2
1.1
124
HAKSAR,
LIN
AND
PERON
importance when there is a limited supply of glucose to the cell. It may be pointed out here that in the adipose tissue only about 60% of the NADPH required to support fatty acid synthesis in the cytosol is generated via the pentose pathway (14, 15); the remainder being derived via malic enzyme and isocitrate dehydrogenase (16, 17). ACKNOWLEDGMENTS FIG. 5. Sources droxylation.
of NADPH
for microsomal
21-hy-
of 14C0, from [3-14Clpyruvate, indicating that the entry of pyruvate carbon into the Krebs cycle was prevented. As expected, arsenite also inhibited the production of 14C02 from [6-14C]glucose. The observation that the production of corticosterone from lip-hydroxyprogesterone depends to a certain extent on the activity of the Krebs cycle, therefore, suggests the possibility that some Krebs cycle acid may leave the mitochondria to provide NADPH for the microsomal 21-hydroxylation. The stimulation of 14C0, production from [l14C]glucose by arsenite, indicating an increase in the pentose shunt activity, was unexpected (Table II). It may be mentioned here that a similar effect of arsenite on the production of 14C0, from [l14Clglucose also has been observed in the intact cells obtained from Snell Adrenocortical Carcinoma 494 (P&on, unpublished observations). At present we do not have an explanation for this phenomenon. It is possible, however, that because of a decrease in the Krebs cycle activity in the presence of arsenite, the transfer of reducing equivalents from the mitochondria into the cytosol was also decreased, and therefore, the cell may have adapted by increasing the activity of the pentose shunt to maintain adequate levels of NADPH in the cytosol. In conjunction with our previous resu1t.s (71, the data reported now indicate that at least a portion of the NADPH required for the microsomal 21-hydroxylation may be derived by a mechanism involving the transfer of reducing equivalents from mitochondria into the cytosol via some Krebs cycle intermediate (Fig. 5). Also, it appears that this mechanism may assume
This work was supported tional Science Foundation tional Institute of Arthritis, tive Diseases (AM-04899).
by grants (GB-36246) Metabolism
from the Naand the Naand Diges-
REFERENCES 1. RYAN, K. J., AND ENGEL, L. L. (1957) J. Biol. Chem. 255,103-114. 2. COOPER, D. Y., NARASIMHULU, S., ROSENTHAL, O., AND ESTABROOK, R. W. (1968) in Functions of the Adrenal Cortex, (McKerns, K. W., ed.), pp. 897-942, Appleton-Century-Crofts, New York. 3. KELLY, T. L., NELSON, E. D., JOHNSON, R. B., AND VESTLING, C. S. (1955) J. Biol. Chem. 212, 545-554. 4. GLOCK, G. E., AND MCLEAN, P. (1954) B&hem. J. 56, 171-175. 5. STUDZINSKI, G. P., SYMINGTON, T., AND GRANT, J. K. (1962) Acta Endocrinol. 40, 232-246. 6. SIMPSON, E. R., CAMNER, W., AND ESTABROOK, R. W. (1968) Biochem. Biophys. Res. Commun. 31, 113-118. 7. TSANG, C. P. W., AND PBRON, F. G. (1970) Steroids 15, 251-265. 8. LIN, M. T., HAKSAR, A., AND P&RON, F. G. (1974) Arch. Biochem. Boiphys. 164,429-439. 9. SIMPSON, E. R., AND BOYD, G. S. (1971) Eur. J. B&hem. 22,489-499. 10. HAKSAR, A., AND P%RON, F. G. (1972) J. Steroid B&hem. 3,847-857. 11. HAKSAR, A., BANIUKIEWICZ, S., AND PBRON, F. G. (1973) Biochem. Biophys. Res. Commun. 52, 959-966. 12. SILBER, R. H., BUSCH, R. D., AND OSLAPAS, R. (1958) Clin. Chem. 4, 278-285. 13. LIN, M. T., HAKSAR, A., AND P&RON, F. G. (1974) Biochem. Biophys. Res. Commun. 58, 983989. 14. KATZ, J., LANDAU, B. R., AND BARTSCH, G. E. (1966) J. Biol. Chem. 241, 727-740. 15. FLATT, J. P., AND BALL, E. G. (1964) J. Biol. Chem. 239, 675-685. 16. YOUNG, J. W., SHRAGO, E., AND LARDY, H. A. (1964) Biochemistry 3, 1687-1692. 17. HANSON, R. W., PATEL, M. S., JOWAIN-BAUM, M., AND BALLARD, F. J. (1971) Metabolism 20, 27-42.