communication vol. 266, no. the 17, issue of june 15, of ... · 10731 . 10732 cytochrome p-450,~~...

Communication Vol. 266, No. 17, Issue of June 15, pp. 10731-10734,1991 THE JOURNAL OF BIOLOGICAL CHEMISTRV

0 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

Aldosterone Synthase Cytochrome P-450 Expressed in the Adrenals of Patients with Primary Aldosteronism*

(Received for publication, January 22, 1991) Tadashi Ogishima, Hirotaka ShibataS, Hidm Shimada, Fumiko Mitani, Hiromichi SuzukiS, Takao SarutaS, and Yuzuru Ishimurat From the Departments of Biochemistry and SZnternal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

A human cytochrome P-450 with aldosterone synthase activity was purified from the mitochondria of an aldosterone-producing adenoma. It was recognized by an anti-bovine cytochrome P-450118 IgG and by a specific antibody raised against a portion of the CYPllB2 gene product, one of the two putative proteins encoded by human cytochrome P-4SOl18-related genes (Mornet, E., Dupont, J., Vitek, A., and White, P. C. (1989) J. Biol. Chem. 264,20961-20967). A similar and probably the same aldosterone synthase cytochrome P-450 was detected in the adrenal of a patient with idiopathic hyperaldosteronism. These aldosterone synthases were distinguishable from cytochrome P- 45OIl8, the product of another cytochrome P-450118- related gene, i.e. CYP11B1, by their catalytic, molecular, and immunological properties and also by their localization. The latter enzyme was unable to produce aldosterone and did not react with the specific antibody against the CYPllB2 gene product. It was present both in tumor and non-tumor portions of the adrenals carrying the adenoma and in normal adrenal cortex. On the other hand, aldosterone synthase cytochrome P-450 localized in the tumor portion of the adrenals or in the adrenal of a patient with idiopathic hyperaldosteronism. Thus aldosterone synthase cytochrome P- 450, a distinct species from cytochrome P-45OIl8, is responsible for the biosynthesis of aldosterone in the human, at least in patients suffering from primary aldosteronism.

At least two types or modes of adrenocortical steroidogenesis are known to exist in mammals (1). One such is seen in the adrenal cortex of the rat, where two distinct enzymes, aldosterone synthase cytochrome P-450 (cytochrome P- 450alda) and corticosterone-producing cytochrome P-450 (cytochrome P-45011~) participate in the syntheses of aldosterone

* This work was supported in part by grants-in-aid for Scientific Research on Priority Areas (to Y. I.) and for Encouragement of Young Scientists (to T. 0.) from the Ministry of Education, Science and Culture of Japan, by a grant-in-aid from the Ministry of Health and Welfare (to T. S.), and by grants from the Ono Medical Research Foundation, from the Yamanouchi Foundation for Research on Met- abolic Disorders, and from Keio University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom all correspondence should be addressed.

and corticosterone, respectively (1,2). The former cytochrome P-450 localizes exclusively in the zona glomerulosa, while the latter is present mainly in the zonae fasciculata-reticularis but also in the zona glomerulosa though smaller in quantity. In bovine adrenal cortex, on the other hand, the only cytochrome P-450118 species is present throughout the three zones and is responsible for both corticosterone and aldosterone synthesis (3-5). In this case, cytochrome P-450118 in the zonae fasciculata-reticularis catalyzes formation of corticosterone but not that of aldosterone from 11-deoxycorticosterone (DOC),’ while the same enzyme in the zona glomerulosa produces aldosterone from the same substrate. It has been postulated, therefore, that a yet unknown mechanism(s) is operating to regulate the activity of cytochrome P-450110 in each zone (6) . Thus the enzymes involved in the adrenocortical steroidogenesis as well as their regulatory mechanisms are different from one another depending on the animal species (1, 3). Nevertheless, no information is available on the situation in human adrenal cortex despite its importance in maintaining the homeostasis of the body.

Recently, Mornet et al. (7) have isolated and characterized two kinds of human cytochrome P-450118-related genes and named them as CYPllBl and CYPllB2. The significance of these genes in the corticosteroidogenesis is, however, ambig- uous. They reported that the transcript of the former gene, presumably cytochrome P-45011p, was detectable in human adrenal mRNA, while that of the latter was found neither in the mRNA nor in the corresponding cDNA clones obtained from a human adrenal library. In view of these and the above mentioned situations, we attempted in this study to identify the enzyme responsible for aldosterone synthesis in the human using surgically removed adrenals including aldosterone- producing adenomas and those from a patient with idiopathic hyperaldosteronism. The results revealed that, in addition to cytochrome P-45OllP, the mitochondria from adrenals of patients with primary aldosteronism contained an aldosterone synthase cytochrome P-450, the properties of which are similar to those of rat. Evidence has also been presented that the aldosterone synthase is a product of the CYP11B2 gene (7). A preliminary report pertinent to a portion of this paper has been presented.*

EXPERIMENTAL PROCEDURES

Adrenal Glands and Preparation of Mitochondria-Surgically removed adrenals from the patients with Grawitz’s tumor (7 specimens), Cushing’s syndrome (2 specimens), pheochromocytoma (1 specimen), aldosterone-producing adenoma (14 specimens), and idiopathic hyperaldosteronism (1 specimen) were obtained from the patients with informed consent in accordance with institutional guidelines (School of Medicine, Keio University). The glands bearing tumors were bisected and separated into the tumor and non-tumor portions, and the non-tumor portions were further separated into the core and the capsular layers. The former contained zonae fasciculata-reticularis and medulla, while the latter contained the zona glomerulosa (1). Mitochondria were prepared from each portion as described elsewhere (8).

Enzyme Assay-Mitochondria were disrupted by freezing and

The abbreviations used are: DOC, 11-deoxycorticosterone; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

* Presented at the 63rd Annual Meeting of the Japanese Biochem- ical Society, September 13th, 1990, Osaka, Japan (21).

10731

10732 Cytochrome P - 4 5 0 , ~ ~ in Primary Aldosteronism

thawing. Aldosterone-synthesizing and corticosterone-producing (llp-hydroxylase) activities in the mitochondria and those in partially purified enzyme preparations were determined a t 37 "C using DOC as a substrate in a reconstituted assay system composed of NADPH, bovine adrenodoxin, and its reductase as described previously (1, 3). Activities of purified aldosterone synthase and llp-hydroxylase were determined by the method used for the bovine and rat enzymes with minor modifications (1, 3, 9). The reaction products were analyzed by either normal-phase or reversed-phase HPLC.

Enzyme Purification-Procedures for purification of aldosterone- synthesizing cytochrome P-450 and/or cytochrome P-45olIp ( l lp- hydroxylase) were essentially the same as described for rat (1). Briefly, mitochondria (1.5 mg as protein) from the tumor portion or those (4 mg) from the capsular layer of the non-tumor portion of a patient with aldosterone-producing adenoma (case 7 in Table I) were solubilized with 1.8% sodium cholate (Sigma). After partial purification with an o-aminohexyl-Toyopearl column (4 X 10 mm), the preparations were subjected to hydroxylapatite HPLC on a Biofine HAC-5CP column (7.5 X 100 mm, Jasco).

Preparation of an Anti-peptide Antibody-An oligopeptide corresponding to amino acid residues 80-90 of a presumed product of CYP11B2 gene reported by Mornet et al. (7) was chemically synthe- sized on a polyamide resin (10). The sequence, RYELGGBMVC, was considerably divergent from the corresponding portion of the CYPl lB l gene product, RYDLGGSMVC, and allowed us to raise an antibody specific to that sequence. Deprotection and removal of the peptide from the resin were achieved as described elsewhere (11). The peptide was purified by reversed-phase HPLC, and its purity was affirmed by fast atom bombardment mass spectrometry. It was then coupled to equine myoglobin (12), emulsified with Freund's complete adjuvant, and was injected subcutaneously to footpads of domestic rabbits twice at a 7-day interval. The rabbits were boosted with the peptide-myoglobin conjugates on the 28th day after the last injection and then bled on the 4th day after the booster.

Other Analytical Methods-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on a 7.5% gel (13), and protein bands were stained with silver (14). Immunoblot analysis (15) was performed with anti-bovine cytochrome P-45Ol1,, IgG (16) or the anti-peptide serum described above.

RESULTS

Aldosterone-synthesizing Activity in the Mitochondria of Human Adrenals-Aldosterone-synthesizing activity was detected in the mitochondria from adenomatous portions of the aldosterone-producing adenoma but not in those from the non-tumor portions of the same adrenals as depicted in Table I. The activity was also detected in those from hyperplastic portions of the adrenal from a patient suffering from idiopathic hyperaldosteronism. In the mitochondria from the healthy adrenals associated with a Grawitz's tumor, however, the aldosterone-synthesizing activities were below the limit of our detection system. The activities were also undetectable in the mitochondria of adrenals from the patients with Cush- ing's syndrome and pheochromocytoma (data not shown). On the other hand, strong activities for the synthesis of corticosterone from DOC (llp-hydroxylase activities) were always detectable in all the mitochondrial preparations so far examined.

Purification of Cytochrome P-450 with llp-Hydroxylase Ac- tivity-We then attempted to purify the aldosterone-synthesizing enzyme from the mitochondria of both tumor and non- tumor portions of an aldosterone-producing adenoma (from the patient of case 7 in Table I). Fig. 1 shows the final step of the purifications using hydroxylapatite HPLC, where elution profiles of the enzymes and proteins were followed by measuring the llp-hydroxylase activity and optical density at 417 nm due mainly to hemoproteins, respectively. It has been shown that aldosterone synthase cytochrome P-450 of rat has llp-hydroxylase activity as well (I), and we had assumed the aldosterone-synthesizing enzyme of the human to also be a cytochrome P-450. As seen on the left, a partially purified preparation from the tumor portion of the adenoma was

TABLE I Aldosterone-synthesizing and Ilp-hydroxylase activities of

mitochondria from various adrenal glands The mitochondria were isolated from zona glomerulosa of healthy

adrenals associated with Grawitz's tumor (control), from that of non- tumor portions of aldosterone-producing adenomas, from the tumor portions of the aldosterone-producing adenomas, and from a hyperplastic portion of an adrenal of idiopathic hyperaldosteronism. Al- dosterone-synthesizing and llp-hydroxylase (corticosterone-synthesizing) activities of the mitochondrial lysates were assayed using DOC as the substrate. Other details are described under "Experimental Procedures."

Source of mitochondria Product formed from DOC

Aldosterone Corticosterone

nrnollminlrng protein Control (healthy adrenal)

Case 1 <0.001" 1.7 Case 2 <0.001" 1.2 Case 3 <0.001" 1.9

Case 4 Aldosterone-producing adenoma

Non-tumor portion <0.001" 2.0 Tumor portion 0.025 3.0




Case 8 0.066 2.2

Case 5

Case 6

Case 7

Idiopathic hyperaldosteronism

The values were below the limit of our detection system, less than 0.001.

=. f r o m f r o m $ T u m o r P o r t i o n

E : .-

N o n - t u m o r P o r t i o n

E

.- * 9 Ql

s I A117= 0.006

2 % 60- 4 $

4.0-

I 0 20 40 60 0 20 4 0

/ / peak 2

i peak 3

60 T i m e ( m i n )

FIG. 1. Purification of aldosterone synthase cytochrome P- 450 from the tumor portion of aldosterone-producing adenoma. Partially purified preparations obtained by w-aminohexyl- Toyopearl column chromatography were applied to a Biofine HAC- 5CP column as described under "Experimental Procedures." The elution profiles were monitored by measuring the absorbance a t 417 run. Eluates were collected at appropriate time intervals (15-60 s) and were assayed for the llp-hydroxylase activity. Left, purification from the tumor portion; right, that from the non-tumor portion of an aldosterone-producing adenoma.

resolved into several fractions, of which four peak fractions had the llp-hydroxylase activity. On the right, three peak fractions with llp-hydroxylase activity were obtained from those of a non-tumor portion of the same adrenal. Among them, peaks 1-3 were common to the preparations both from

Cytochrome P-45OOld, in Primary Aldosteronism 10733

tumor and non-tumor portions and did not exhibit aldosterone-synthesizing activity. As will be described later, all these fractions reacted with an anti-bovine cytochrome P-45Olrd IgG but not with the specific antibody raised against a portion of the presumed CYPllB2 gene product. Thus they were regarded as llp-hydroxylase (cytochrome P-45oIln) fractions, where the peak 2 fraction is likely to be the native cytochrome P-45OIld, since it gave the same band in immunoblot analyses as that of cytochrome P-45OIls in intact mitochondria. The other fractions might be partially degraded or modified enzymes produced during the purification as judged by their apparent molecular mass (MI) in immunoblot analyses.

On the other hand, the peak 4 fraction, which was present only in the tumor portion, reacted with the specific antibody to the CYPllB2 gene product. It was homogeneous upon SDS-PAGE with M , of 48,500 (see Fig. 2) and was identified as a cytochrome P-450 by its absorption spectrum. The fraction exhibited a ferric high spin type of the spectrum char- acteristic to a cytochrome P-450 in the presence of DOC. A similar fraction with aldosterone-synthesizing activity was obtained from the hyperplastic portion of an adrenal of idiopathic hyperaldosteronism but not from the other kinds of adrenal glands such as non-tumor portions of adrenals with aldosterone-producing adenoma and healthy portions associated with the Grawitz's tumor. Then the largest peak fractions with intense absorptions a t 417 nm (solid lines) in both left and right were shown to be those of cytochrome P-45OsCc as judged by their cholesterol side-chain cleavage activity (17) and by their reactivities to an anti-bovine cytochrome P-45OsCc IgG (16).

Catalytic Activities of the Purified Enzymes-Table I1 sum- marizes the activities of purified enzymes from the tumor portion of the aldosterone-producing adenoma. When activities of various fractions in Fig. 1, left, i.e. fractions 1, 2, and 4, were assayed using DOC as a substrate, they all exhibited strong llp-hydroxylase activities to form corticosterone. Among them, however, only the fraction 4 enzyme had a significant activity to synthesize aldosterone from DOC. The enzyme also produced considerable amounts of 18-hydroxycorticosterone and 18-hydroxy-DOC, the metabolic interme- diates in the conversion of DOC to aldosterone. We have failed to determine the activity of the fraction 3 enzyme per mol of cytochrome P-450 quantitatively, and hence the results were omitted from the table. It was confirmed, however, that the fraction had no activity to produce aldosterone from DOC.

In a separate series of experiments, we have also examined the activities of fractions 1-3 from the non-tumor portion of the aldosterone-producing adenoma. They also had strong

Mr A B C (kDa) pmw

::.5: - N T P4 P4 P4 N T

FIG. 2. Immunoblot and SDS-PAGE analyses of mitochondrial lysates and purified aldosterone synthase cytochrome P- 450. A, immunoblot analyses of mitochondrial lysates (each 10 pg as protein) from a tumor and a non-tumor portion of an adrenal with an aldosterone-producing adenoma and the peak 4 fraction from the tumor portion (Fig. 1) with anti-bovine cytochrome P-4!5oI1,, IgG. B, analysis of the peak 4 fraction by SDS-PAGE with silver staining. C, immunoblot analyses of the same mitochondrial lysates (each 5 pg as protein) and the peak 4 fraction with the anti-synthetic peptide serum. N, non-tumor portion; T, tumor portion; 2'4, the peak 4 fraction.

TABLE I1 Activities of the enzymes purified

from aldosterone-producing adenoma The activities were measured using DOC as a substrate as described

under "Experimental Procedures." Amounts of the enzymes employed were 3.6, 3.8, and 2.8 pmol as cytochrome P-450 for peaks 1, 2, and 4 in Fig. 1, left, respectively. The amount of cytochrome P-4.!10~~~ in the peak 2 fraction was estimated by assuming that the fraction contained an equimolar cytochrome P-45Oscc to that of cytochrome P-45OIld a t 50% on the basis of SDS-PAGE analysis. Incubation time was 10 min a t 37 "C. Data represent mean values obtained from the duplicate assay.

Product formed from DOC Enzyme

COR" 18-OH-DOC" 18-OH-COR" ALDO" nmol/min/nmol

Peak 1 fraction 29 1.1 0.17 <0.02h Peak 2 fraction 31 1.2 0.19 <0.02h Peak 4 fraction 22 2.2 1.5 1.0 " COR, 18-OH-DOC, 18-OH-COR, and ALDO denote corticoster-

one, 18-hydroxy-ll-deoxycorticosterone, 18-hydroxycorticosterone, and aldosterone, respectively.

The values were less than 0.02.

A

b 480.5

1 2 3 4 5 6 7 FIG. 3. Analyses of mitochondrial proteins from

kinds of human adrenals with the anti-synthetic various peptide

serum. Mitochondria (each 10 p g as protein) from control adienals associated with Grawitz's tumors (lanes 1 and 2), from tumor portions of aldosterone-producing adenomas (lanes 3-61, and from a hyperplastic portion of an adrenal of idiopathic hyperaldosteronism (lane 7 ) were analyzed by immunoblotting with the anti-peptide serum (A) and with the anti-bovine cytochrome P-45OIlA IgG ( B ) . The mitochondria for lanes 3-7 were prepared from the patients of cases 4-8 in Table I, respectively.

llp-hydroxylase activities, but none of them showed aldosterone-synthesizing activity (data not shown). From these findings and those described earlier, the 4th fraction enzyme with aldosterone-synthesizing activity from the tumor portion of the aldosterone-producing adenoma is hereafter referred to as human aldosterone synthase cytochrome P-450 or cytochrome P-450aldo (1).

Immunoblot and SDS-PAGE Analyses of the Enzymes- Fig. 2A shows immunoblot analyses of the mitochondrial lysates from both tumor and non-tumor portions of the aldosterone-producing adenoma with an anti-bovine cytochrome P-45OlIp IgG. As seen, one having an M , of 50,000 was always found in the lysates both from tumor and non-tumor portions of the aldosterone-producing adenoma, while the other with M , of 48,500 was only present in those from the tumor portion. The antibody reacted also with the purified aldosterone synthase cytochrome P-450, i.e. the peak 4 fraction, giving the same M , of 48,500. Fig. 2B demonstrates that our purified aldosterone synthase is indeed homogeneous upon SDS-PAGE giving a single band at M , of 48,500 as visualized by silver staining. Furthermore, the specific antiserum raised against the CYPllB2 gene product recognized only the 48,500 protein in the mitochondrial lysate from the tumor portion and the purified aldosterone synthase (Fig. 2C). None of the other fractions from the tumor portion in Fig. 1 reacted with the specific antiserum. These results

10734 Cytochrome P-45OaMO in Primary Aldosteronism

indicate that the 48,500 and 50,000 proteins are the aldosterone synthase cytochrome P-450 and cytochrome P-45011P, respectively, and that the aldosterone synthase cytochrome P-450 is a product of the CYPllB2 gene.

Expression of Aldosterone Synthase Cytochrome P-450 in Adrenal Glands-Mitochondria from various kinds of human adrenals were now reexamined for the expression of aldosterone synthase cytochrome P-450 by using the specific antiserum against the CYP11B2 gene product. The enzyme was detectable both in the tumor portions of aldosterone-producing adenomas and the hyperplastic portion of the adrenal of idiopathic hyperaldosteronism, but not in the control adrenals (Fig. 3A). A similar blotting experiment with the anti-bovine cytochrome P-450110 IgG revealed that the 50,000 protein, i.e. cytochrome P-450118, distributed among all kinds of the mitochondria so far examined (Fig. 323). It appears likely therefore that aldosterone synthase cytochrome P-450, the product of CYP11B2 gene, is specifically expressed in the adrenals of patients with primary aldosteronism.

DISCUSSION

Aldosterone, the most potent mineralocorticoid in mammals, acts primarily on the distal and collecting tubules of the kidney to stimulate the reabsorption and excretion of electro- lytes such as Na+ and K', respectively (18). An abnormal production of the hormone in the adrenal cortex therefore leads to a variety of diseased states including hypokalemia and hypertension. However, the regulatory mechanisms of aldosterone biosynthesis, especially that in human, are not completely understood. As mentioned earlier, even the enzyme(s) responsible for the biosynthesis has not been identified.

In the present study, we purified and identified an aldosterone synthase from the adrenals of patients with primary aldosteronism. The enzyme was shown to be a cytochrome P- 450 but was distinct from cytochrome P-450110 responsible for the corticosterone synthesis from DOC. Available evidence indicated that cytochrome P-45OllP and aldosterone synthase cytochrome P-450 were the products of two closely related genes, CYPllBl and CYP11B2, respectively. In accordance with this interpretation, Kawamoto et al. (19) have isolated two cDNA clones corresponding to CYPllBl and CYPllB2 genes and expressed them in COS-7 cells, where their products exhibited ll@-hydroxylase and aldosterone synthase activities, respectively. Thus two distinct cytochrome P-450 species, which are under the control of two different genes, exist in human adrenals to produce the mineralocorticoid and the glucocorticoids, respectively, a t least in those of primary aldosteronism. In this respect, human corticosteroidogenesis resembles that of rat (1) and differs from that in cattle (3).

Recently we were able to show that rat aldosterone synthase cytochrome P-450 was an ultimate target of the regulation by angiotensin I1 and K+ (20). By analogy, therefore, the human

aldosterone synthase described here might also play a central role in the regulation of aldosterone biosynthesis in the human beings. It remains to be elucidated, however, whether the aldosterone synthase cytochrome P-450 is expressed only in the adrenals of patients with primary aldosteronism or it is also expressed in normal subjects but is overexpressed in such diseased states. Identification of the aldosterone-synthesizing enzyme system in normal subjects is currently under investi- gation.

Acknowledgments-We thank Drs. H. Tazaki, T. Hata, and S. Saitou (Department of Urology, School of Medicine, Keio University), T. Imafuku (Sizuoka Red Cross Hospital), and Y. Asou (Department of Urology, Faculty of Medicine, University of Tokyo) for their kind supply of surgically removed human adrenals.

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