AJH
1992;
5:800-805
Rama Natarajan, Hsin Yang, Peter F. Hall, and Jerry Nadler
We have examined the regulation of cytochrome P-450 side chain cleavage enzyme ( P - 4 5 0 ) and Ρ-45011β(18) hydroxylase (P-450 ) enzyme expres sion b y angiotensin I I (All), the major regulator of aldosterone biosynthesis, in cultured human adre nal glomerulosa cells. We also examined the effect of lipoxygenase products of arachidonic acid on the expression of these enzymes since it has been pre viously shown that the 12-lipoxygenase pathway plays a key role in All-induced aldosterone synthe sis in rat and human adrenal glomerulosa cells. A l l ( 1 0 " mol/L) induced a 3-fold stimulation of s c c
11)S
7
P-450
s c c
and over a 2 τ ί ο Μ increase in P - 4 5 0 ^
protein expression. T h e 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) caused a 2-fold increase in P-450 ^ levels without altering P-450 levels. These results show for the first time that A l l can directly increase the levels of P-450 and P - 4 5 0 ^ enzymes in glomerulosa cells. T h e results also suggest that 12-HETE may mediate long term effects of A l l action b y stimulating P - 4 5 0 ^ levels. Am J Hypertens 1992;5:800-805 n
s c c
s c c
KEY WORDS: Angiotensin I I , aldosterone, lipoxygen ase, Ρ-450 . 11)?
A
ysteroid dehydrogenase/isomerase to progesterone, which in turn, can be converted to 11-deoxycorticoster one by the 21-hydroxylase P - 4 5 0 i . Deoxycorticoster one is converted to aldosterone in the zona glomerulosa by hydroxylation at the 11 β and 18 positions followed by conversion of the primary alcohol at C to an alde hyde. This reaction, called the late step in aldosterone biosynthesis, is thought to be catalyzed by a single P-450 enzyme, or two separate enzymes depending on the species. Thus, in the bovine adrenal cortex, both corticosterone and aldosterone are obtained by the ac tion of a single enzyme, the cytochrome P - 4 5 0 hy Received March 13, 1992. Accepted July 14, 1992. droxylase ( P - 4 5 0 ^ ) , which is present in all three From the Department of Diabetes, Endocrinology, and Metabolism, zones of the adrenal cortex. In the adrenal cortex of the City of Hope Medical Center, Duarte, California (RN,HY,JN), and rat, however, two distinct enzymes, aldosterone synth Department of Endocrinology, University of New South Wales, Rand wick, New South Wales, Australia (PFH). ase cytochrome P-450 (P-450 aldo) and corticosterone This work was supported by a Grant-in-Aid from the American Heart Association, Los Angeles Affiliate (907-GI) (to RN) and by a synthesizing cytochrome P-450 (P-450 ) are involved grant from the National Institutes of Health, Bethesda, MD, (R01 in the biosynthesis of aldosterone and corticosterone, DK39721, SCOR HL 44404) (to JN). respectively. " Recent work has shown the presence of Address correspondence and reprint requests to Rama Natarajan, two similar enzymes in the human adrenal gland. MorPhD, Dept. of Diabetes, Endocrinology, & Metabolism, City of Hope net et al isolated and characterized two kinds of human National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010. ngiotensin II is one of the major regulators of aldosterone biosynthesis. The synthesis of al dosterone from cholesterol in the adrenal cortex requires the activation of several cy tochrome P-450 steroid hydroxylases. The initial or early step is cholesterol side chain cleavage reaction re sulting in the formation of pregnenolone from choles terol. This rate-limiting step is catalyzed by the cytochrome P-450 side chain cleavage enzyme (P4 5 0 S C C ) . Pregnenolone can be converted by 3/?-hydrox-
C2
1 8
1
11)?
u
2,3
11)ff
4
© 1992 by the American Journal of Hypertension, Inc.
6
0895-7061/92/$5.00
Downloaded from http://ajh.oxfordjournals.org/ at Florida Atlantic University on December 7, 2016
Angiotensin II Regulates Cytochrome P-450 Steroid Hydroxylase Enzyme Expression in Human Adrenal Glomerulosa Cells
AJH-NOVEMBER
1992-VOL
5, NO. 11
ANGIOTENSIN II AND STEROID HYDROXYLASE EXPRESSION
1 V
7
11)5
8
9
9,10
s c c
n
11
1,12
s c c
11)?
13,14
Ham's F12(DME/F12) medium containing 0.2% bovine serum albumin (BSA), penicillin G (100 U/mL), and streptomycin (100 //g/mL). Cell suspensions were prepared as described earlier. The zona fasciculata contamination in these zona glomerulosa cell cultures was assessed to be very minimal since Cortisol levels were very low (1.6 //g/dL compared to 55 //g/dL produced by fasciculata cultures prepared from the same adrenals) and by the very low expression of the 17a-hydroxylase gene. Glomerulosa cell suspensions in DME/F12 containing 1 0 % fetal calf serum (2 X 10 /mL) were plated on 6-well culture plates and medium changed every 48 h. Cells were in culture for 5 to 7 days before an experiment. Approximately 24 h prior to an experiment, the medium was replaced with antibiotic-free DME/F12 containing 0 . 5 % fetal calf serum and 0.2% BSA. Immediately prior to the incubations the low serum medium was freshly added, followed by addition of agents and vehicle controls ( 0 . 0 1 % DMSOfor HETEs). Agents were added again after 12 h. At the end of the 30 h incubation, the plates were cooled. Supernates were saved at — 20 °C for measurement of aldosterone by radioimmunoassay, which was carried out using a kit supplied by ICN (Costa Mesa, CA). The cell monolayers were washed twice with ice cold phosphate buffered saline (PBS) and finally harvested. Cell pellets were lysed in a lysis buffer containing PBS (pH 7.4), 1 % Triton X-100, phenylmethylsulfonyl fluoride (PMSF) (1 mmol/L), leupeptin (50 //mol/L), and 0 . 1 % sodium dodecyl sulfate (SDS). An aliquot of the lysate was put aside for protein estimation and the remainder saved at — 70 °C for Western Blot analysis. 14
19
5
Electrophoresis and Western Blotting Sodium dodecyl sulfate polyacrylamide gel electrophoresis ( 1 0 % running gel, 4 % stacking gel) was performed according to the method of Laemmli. Equal amounts of protein were loaded. For Western Blotting, gels were equilibrated in a transfer buffer (35 mmol/L Tris base, 192 mmol/L glycine, and 2 0 % methanol, pH 8.3) and then transferred to nitrocellulose (Hybond, Amersham Radiochemical, Amersham, Buckinghamshire, England) as described by Towbin et a l , in a semidry polyblot apparatus (American Bionetics, Inc., Emeryville, CA). The nonspecific sites were blocked with PBS containing 3 % BSA at 4°C overnight. The membranes were then washed twice with PBST (PBS + 0 . 0 5 % Tween-20) and incubated with primary antibody in PBST containing 1 % BSA and 2 0 % v/v fetal calf serum for 3 h at room temperature. The membranes were incubated with a second antibody (goat antirabbit) conjugated with alkaline phosphatase ( 1 : 5 0 0 0 , Promega Corp., Madison, WI). Color development was carried out using substrate mixture (nitroblue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate from Promega). Nonspecific bind15
MATERIALS AND M E T H O D S Materials Angiotensin II (human, synthetic) was obtained from Peninsula Laboratories (Belmont, CA). Synthetic ACTH _ (Cortrosyn) was obtained from Organon Inc. (West Orange, NJ). Both 12- and 15-hydroxyeicosatetraenoic acids (HETEs) were obtained from Biomol Research Laboratories (Philadelphia, PA). 1
24
Methods Culture and Incubation of Human Adrenal Glomerulosa Cells Fresh normal human adrenal tissue samples were obtained from surgeries involving nephrectomies for renal carcinoma or adrenalectomies during retroperitoneal lymph node dissections. Tissue samples were obtained with the City of Hope Institutional Review Board (IRB) approval. The outer adrenal cortical tissue was separated by a pathologist with microscopic assistance under sterile conditions. The outer glomerulosa tissue was minced in Dulbecco's modified Eagle/
16
Downloaded from http://ajh.oxfordjournals.org/ at Florida Atlantic University on December 7, 2016
P - 4 5 0 genes, termed CYP11B1 and CYP11B2, and suggested that the aldosterone synthase is a product of the CYP11B2 gene. Ogishima et al showed the presence, along with P - 4 5 0 , of a P-450 aldo with similar properties as the rat P-450 aldo in the mitochondria from adrenals of patients with primary aldosteronism. Curnow et al have used a sensitive assay based on a polymerase chain reaction to show that transcripts of CYP11B2 are present not only in high levels in aldosterone secreting adenomas, but also in low levels in normal adrenals. It has also been demonstrated that the product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Angiotensin II (All) enhances the activity of both the early ( P - 4 5 0 ) and the late (P-450 ^) pathways of aldosterone synthesis and it is clear that All-induced aldosterone synthesis is enhanced during states of renin-angiotensin system activation, such as during sodium deprivation. It has been previously shown that ACTH can induce new steroid synthesizing P-450 enzyme expression in the adrenal fasciculata. However, very few studies have addressed whether All can induce the synthesis of aldosterone synthetic P-450 enzymes. In the present study, we have examined the regulation of P - 4 5 0 and P - 4 5 0 enzyme expression by All in cultured human adrenal glomerulosa cells. Previous studies have shown that activation of the 12-lipoxygenase (LO) pathway of arachidonic acid plays a key role in mediating All-induced aldosterone synthesis in rat and human adrenal glomerulosa cells since All, but not ACTH or potassium, could stimulate the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE). Also, inhibitors of 12-HETE synthesis blocked A l l — b u t not ACTH or potassium—induced aldosterone synthesis. We therefore also examined whether the LO products alter the levels of these key aldosterone synthetic enzymes.
801
802
NATARAJAN ET AL
AJH-NOVEMBER
s c c
n
17
s c c
s c c
5, NO. 11
Regulation of P-450 Figure 3 shows a representative Western immunoblot of glomerulosa cell lysates using an antibody to the P - 4 5 0 enzyme. It is seen that All (10~ mol/L) produced a significant increase in P - 4 5 0 ^ (51K) levels at 30 h. All at 10" mol/L also produced a similar increase. ACTH (10~ mol/L) also stimulated Ρ - 4 5 0 £ enzyme levels. Further, as seen in the fourth lane of the Western blot, 12-HETE also caused a signifi cant increase in P - 4 5 0 ^ levels. Figure 4 is a graphical representation of several sets of Western blot data ob tained by analyzing blots by video densitometry. It clearly shows that All induced greater than a two-fold stimulation of Ρ - 4 5 0 ^ enzyme levels. Similarly, 12HETE (10~ mol/L) was nearly as effective as All in in creasing P - 4 5 0 £ levels. However, unlike the 12-LO product, 12-HETE, 15-HETE (10" mol/L) had no effect on inducing protein expression. 11)S
11)5
7
n
8
7
Π
n
η
7
U
7
RESULTS Regulation of P - 4 5 0 Figure 1 shows a Western immunoblot of cultured human adrenal glomerulosa cell lysates obtained from cells treated with various agents. It is clearly seen that All (10~ mol/L) caused a signifi cant increase in P - 4 5 0 (50K) levels at 30 h. This time period was chosen based on earlier reports showing peak responses with bovine fasciculata cells around this time period. Similar increase in P - 4 5 0 levels was seen with All 10~ mol/L (results not shown). We ob served that ACTH (10~ mol/L) was also a potent stimu lator. However, the 12-LO product 12-HETE ( 1 0 m o l / L) did not cause any significant change in P - 4 5 0 over basal. Figure 2 is a graphical representation of Western blots obtained by quantitative video densitometry. This shows that All induced greater than a three-fold in crease in P - 4 5 U S C C . In contrast, neither 12- nor 15-HETE (10~ mol/L) altered P - 4 5 0 protein expression. s c c
7
s c c
18
s c c
8
7
Effect of Agonists on Aldosterone Production at. 30 h Figure 5 shows the effect of All (10~ mol/L) and the HETE's (10~ mol/L) on aldosterone production at 30 h. It is seen that both All and 12-HETE caused a significant stimulation of aldosterone synthesis at this time period (All, 190 ± 1 0 % of control, Ρ < .001; 12-HETE, 169 ± 18%, P < . 0 2 ) . ACTH also had a significant effect (330 ± 32%, Ρ < .001). 15-HETE had a slight but non significant effect (128 ± 1 1 % , Ρ = .1). 7
7
_7
DISCUSSION
s c c
7
s c c
Angiotensin II is one of the major regulators of aldoster one synthesis. However, few studies have addressed the role of All in regulating long term expression of the key aldosterone synthesizing enzymes. The present studies show for the first time that All can increase the levels of
FIGURE 1. Regulation ofP-450 . Immunoblot analysis of the effect of various agents on the levels of P-450 in human adrenal glomeru losa cells. Blots were probed with a 1:200 dilution of a specific antibody to P-450sc and visualized by the alkaline phosphatase color reaction as described under Materials and Methods. scc
scc
C
12 HETE All (10" H) (10" M) T
7
ACTH (l
1992;
5:800-805
Rama Natarajan, Hsin Yang, Peter F. Hall, and Jerry Nadler
We have examined the regulation of cytochrome P-450 side chain cleavage enzyme ( P - 4 5 0 ) and Ρ-45011β(18) hydroxylase (P-450 ) enzyme expres sion b y angiotensin I I (All), the major regulator of aldosterone biosynthesis, in cultured human adre nal glomerulosa cells. We also examined the effect of lipoxygenase products of arachidonic acid on the expression of these enzymes since it has been pre viously shown that the 12-lipoxygenase pathway plays a key role in All-induced aldosterone synthe sis in rat and human adrenal glomerulosa cells. A l l ( 1 0 " mol/L) induced a 3-fold stimulation of s c c
11)S
7
P-450
s c c
and over a 2 τ ί ο Μ increase in P - 4 5 0 ^
protein expression. T h e 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid (12-HETE) caused a 2-fold increase in P-450 ^ levels without altering P-450 levels. These results show for the first time that A l l can directly increase the levels of P-450 and P - 4 5 0 ^ enzymes in glomerulosa cells. T h e results also suggest that 12-HETE may mediate long term effects of A l l action b y stimulating P - 4 5 0 ^ levels. Am J Hypertens 1992;5:800-805 n
s c c
s c c
KEY WORDS: Angiotensin I I , aldosterone, lipoxygen ase, Ρ-450 . 11)?
A
ysteroid dehydrogenase/isomerase to progesterone, which in turn, can be converted to 11-deoxycorticoster one by the 21-hydroxylase P - 4 5 0 i . Deoxycorticoster one is converted to aldosterone in the zona glomerulosa by hydroxylation at the 11 β and 18 positions followed by conversion of the primary alcohol at C to an alde hyde. This reaction, called the late step in aldosterone biosynthesis, is thought to be catalyzed by a single P-450 enzyme, or two separate enzymes depending on the species. Thus, in the bovine adrenal cortex, both corticosterone and aldosterone are obtained by the ac tion of a single enzyme, the cytochrome P - 4 5 0 hy Received March 13, 1992. Accepted July 14, 1992. droxylase ( P - 4 5 0 ^ ) , which is present in all three From the Department of Diabetes, Endocrinology, and Metabolism, zones of the adrenal cortex. In the adrenal cortex of the City of Hope Medical Center, Duarte, California (RN,HY,JN), and rat, however, two distinct enzymes, aldosterone synth Department of Endocrinology, University of New South Wales, Rand wick, New South Wales, Australia (PFH). ase cytochrome P-450 (P-450 aldo) and corticosterone This work was supported by a Grant-in-Aid from the American Heart Association, Los Angeles Affiliate (907-GI) (to RN) and by a synthesizing cytochrome P-450 (P-450 ) are involved grant from the National Institutes of Health, Bethesda, MD, (R01 in the biosynthesis of aldosterone and corticosterone, DK39721, SCOR HL 44404) (to JN). respectively. " Recent work has shown the presence of Address correspondence and reprint requests to Rama Natarajan, two similar enzymes in the human adrenal gland. MorPhD, Dept. of Diabetes, Endocrinology, & Metabolism, City of Hope net et al isolated and characterized two kinds of human National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010. ngiotensin II is one of the major regulators of aldosterone biosynthesis. The synthesis of al dosterone from cholesterol in the adrenal cortex requires the activation of several cy tochrome P-450 steroid hydroxylases. The initial or early step is cholesterol side chain cleavage reaction re sulting in the formation of pregnenolone from choles terol. This rate-limiting step is catalyzed by the cytochrome P-450 side chain cleavage enzyme (P4 5 0 S C C ) . Pregnenolone can be converted by 3/?-hydrox-
C2
1 8
1
11)?
u
2,3
11)ff
4
© 1992 by the American Journal of Hypertension, Inc.
6
0895-7061/92/$5.00
Downloaded from http://ajh.oxfordjournals.org/ at Florida Atlantic University on December 7, 2016
Angiotensin II Regulates Cytochrome P-450 Steroid Hydroxylase Enzyme Expression in Human Adrenal Glomerulosa Cells
AJH-NOVEMBER
1992-VOL
5, NO. 11
ANGIOTENSIN II AND STEROID HYDROXYLASE EXPRESSION
1 V
7
11)5
8
9
9,10
s c c
n
11
1,12
s c c
11)?
13,14
Ham's F12(DME/F12) medium containing 0.2% bovine serum albumin (BSA), penicillin G (100 U/mL), and streptomycin (100 //g/mL). Cell suspensions were prepared as described earlier. The zona fasciculata contamination in these zona glomerulosa cell cultures was assessed to be very minimal since Cortisol levels were very low (1.6 //g/dL compared to 55 //g/dL produced by fasciculata cultures prepared from the same adrenals) and by the very low expression of the 17a-hydroxylase gene. Glomerulosa cell suspensions in DME/F12 containing 1 0 % fetal calf serum (2 X 10 /mL) were plated on 6-well culture plates and medium changed every 48 h. Cells were in culture for 5 to 7 days before an experiment. Approximately 24 h prior to an experiment, the medium was replaced with antibiotic-free DME/F12 containing 0 . 5 % fetal calf serum and 0.2% BSA. Immediately prior to the incubations the low serum medium was freshly added, followed by addition of agents and vehicle controls ( 0 . 0 1 % DMSOfor HETEs). Agents were added again after 12 h. At the end of the 30 h incubation, the plates were cooled. Supernates were saved at — 20 °C for measurement of aldosterone by radioimmunoassay, which was carried out using a kit supplied by ICN (Costa Mesa, CA). The cell monolayers were washed twice with ice cold phosphate buffered saline (PBS) and finally harvested. Cell pellets were lysed in a lysis buffer containing PBS (pH 7.4), 1 % Triton X-100, phenylmethylsulfonyl fluoride (PMSF) (1 mmol/L), leupeptin (50 //mol/L), and 0 . 1 % sodium dodecyl sulfate (SDS). An aliquot of the lysate was put aside for protein estimation and the remainder saved at — 70 °C for Western Blot analysis. 14
19
5
Electrophoresis and Western Blotting Sodium dodecyl sulfate polyacrylamide gel electrophoresis ( 1 0 % running gel, 4 % stacking gel) was performed according to the method of Laemmli. Equal amounts of protein were loaded. For Western Blotting, gels were equilibrated in a transfer buffer (35 mmol/L Tris base, 192 mmol/L glycine, and 2 0 % methanol, pH 8.3) and then transferred to nitrocellulose (Hybond, Amersham Radiochemical, Amersham, Buckinghamshire, England) as described by Towbin et a l , in a semidry polyblot apparatus (American Bionetics, Inc., Emeryville, CA). The nonspecific sites were blocked with PBS containing 3 % BSA at 4°C overnight. The membranes were then washed twice with PBST (PBS + 0 . 0 5 % Tween-20) and incubated with primary antibody in PBST containing 1 % BSA and 2 0 % v/v fetal calf serum for 3 h at room temperature. The membranes were incubated with a second antibody (goat antirabbit) conjugated with alkaline phosphatase ( 1 : 5 0 0 0 , Promega Corp., Madison, WI). Color development was carried out using substrate mixture (nitroblue tetrazolium and 5-bromo-4-chloro3-indolyl phosphate from Promega). Nonspecific bind15
MATERIALS AND M E T H O D S Materials Angiotensin II (human, synthetic) was obtained from Peninsula Laboratories (Belmont, CA). Synthetic ACTH _ (Cortrosyn) was obtained from Organon Inc. (West Orange, NJ). Both 12- and 15-hydroxyeicosatetraenoic acids (HETEs) were obtained from Biomol Research Laboratories (Philadelphia, PA). 1
24
Methods Culture and Incubation of Human Adrenal Glomerulosa Cells Fresh normal human adrenal tissue samples were obtained from surgeries involving nephrectomies for renal carcinoma or adrenalectomies during retroperitoneal lymph node dissections. Tissue samples were obtained with the City of Hope Institutional Review Board (IRB) approval. The outer adrenal cortical tissue was separated by a pathologist with microscopic assistance under sterile conditions. The outer glomerulosa tissue was minced in Dulbecco's modified Eagle/
16
Downloaded from http://ajh.oxfordjournals.org/ at Florida Atlantic University on December 7, 2016
P - 4 5 0 genes, termed CYP11B1 and CYP11B2, and suggested that the aldosterone synthase is a product of the CYP11B2 gene. Ogishima et al showed the presence, along with P - 4 5 0 , of a P-450 aldo with similar properties as the rat P-450 aldo in the mitochondria from adrenals of patients with primary aldosteronism. Curnow et al have used a sensitive assay based on a polymerase chain reaction to show that transcripts of CYP11B2 are present not only in high levels in aldosterone secreting adenomas, but also in low levels in normal adrenals. It has also been demonstrated that the product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Angiotensin II (All) enhances the activity of both the early ( P - 4 5 0 ) and the late (P-450 ^) pathways of aldosterone synthesis and it is clear that All-induced aldosterone synthesis is enhanced during states of renin-angiotensin system activation, such as during sodium deprivation. It has been previously shown that ACTH can induce new steroid synthesizing P-450 enzyme expression in the adrenal fasciculata. However, very few studies have addressed whether All can induce the synthesis of aldosterone synthetic P-450 enzymes. In the present study, we have examined the regulation of P - 4 5 0 and P - 4 5 0 enzyme expression by All in cultured human adrenal glomerulosa cells. Previous studies have shown that activation of the 12-lipoxygenase (LO) pathway of arachidonic acid plays a key role in mediating All-induced aldosterone synthesis in rat and human adrenal glomerulosa cells since All, but not ACTH or potassium, could stimulate the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid (12-HETE). Also, inhibitors of 12-HETE synthesis blocked A l l — b u t not ACTH or potassium—induced aldosterone synthesis. We therefore also examined whether the LO products alter the levels of these key aldosterone synthetic enzymes.
801
802
NATARAJAN ET AL
AJH-NOVEMBER
s c c
n
17
s c c
s c c
5, NO. 11
Regulation of P-450 Figure 3 shows a representative Western immunoblot of glomerulosa cell lysates using an antibody to the P - 4 5 0 enzyme. It is seen that All (10~ mol/L) produced a significant increase in P - 4 5 0 ^ (51K) levels at 30 h. All at 10" mol/L also produced a similar increase. ACTH (10~ mol/L) also stimulated Ρ - 4 5 0 £ enzyme levels. Further, as seen in the fourth lane of the Western blot, 12-HETE also caused a signifi cant increase in P - 4 5 0 ^ levels. Figure 4 is a graphical representation of several sets of Western blot data ob tained by analyzing blots by video densitometry. It clearly shows that All induced greater than a two-fold stimulation of Ρ - 4 5 0 ^ enzyme levels. Similarly, 12HETE (10~ mol/L) was nearly as effective as All in in creasing P - 4 5 0 £ levels. However, unlike the 12-LO product, 12-HETE, 15-HETE (10" mol/L) had no effect on inducing protein expression. 11)S
11)5
7
n
8
7
Π
n
η
7
U
7
RESULTS Regulation of P - 4 5 0 Figure 1 shows a Western immunoblot of cultured human adrenal glomerulosa cell lysates obtained from cells treated with various agents. It is clearly seen that All (10~ mol/L) caused a signifi cant increase in P - 4 5 0 (50K) levels at 30 h. This time period was chosen based on earlier reports showing peak responses with bovine fasciculata cells around this time period. Similar increase in P - 4 5 0 levels was seen with All 10~ mol/L (results not shown). We ob served that ACTH (10~ mol/L) was also a potent stimu lator. However, the 12-LO product 12-HETE ( 1 0 m o l / L) did not cause any significant change in P - 4 5 0 over basal. Figure 2 is a graphical representation of Western blots obtained by quantitative video densitometry. This shows that All induced greater than a three-fold in crease in P - 4 5 U S C C . In contrast, neither 12- nor 15-HETE (10~ mol/L) altered P - 4 5 0 protein expression. s c c
7
s c c
18
s c c
8
7
Effect of Agonists on Aldosterone Production at. 30 h Figure 5 shows the effect of All (10~ mol/L) and the HETE's (10~ mol/L) on aldosterone production at 30 h. It is seen that both All and 12-HETE caused a significant stimulation of aldosterone synthesis at this time period (All, 190 ± 1 0 % of control, Ρ < .001; 12-HETE, 169 ± 18%, P < . 0 2 ) . ACTH also had a significant effect (330 ± 32%, Ρ < .001). 15-HETE had a slight but non significant effect (128 ± 1 1 % , Ρ = .1). 7
7
_7
DISCUSSION
s c c
7
s c c
Angiotensin II is one of the major regulators of aldoster one synthesis. However, few studies have addressed the role of All in regulating long term expression of the key aldosterone synthesizing enzymes. The present studies show for the first time that All can increase the levels of
FIGURE 1. Regulation ofP-450 . Immunoblot analysis of the effect of various agents on the levels of P-450 in human adrenal glomeru losa cells. Blots were probed with a 1:200 dilution of a specific antibody to P-450sc and visualized by the alkaline phosphatase color reaction as described under Materials and Methods. scc
scc
C
12 HETE All (10" H) (10" M) T
7
ACTH (l
