AJH 1991; 4:280-282
Regulation of Aldosterone Biosynthesis:
A Continual Challenge
Jürg Müller and Markus Lauber
A l d o s t e r o n e s e c r e t i o n b y t h e z o n a g l o m e r u l o s a of t h e a d r e n a l cortex is d i r e c t l y i n f l u e n c e d b y a l a r g e , g r o w i n g n u m b e r of d i f f e r e n t p h y s i o l o g i c a g e n t s . T h e i r a c t i o n i n c l u d e s : 1) a c u t e s t i m u l a t i o n or i n h i b i t i o n of e a r l y b i o s y n t h e t i c s t e p s ; 2) l o n g - t e r m a c t i v a t i o n or s u p p r e s s i o n of l a t e b i o s y n t h e t i c s t e p s ; a n d 3) i n d u c t i o n of g r o w t h or a t r o p h y of z o n a g l o m e r u losa cells. I n r a t s , a d a p t a t i o n of l a t e s t e p s of a l d o s t e r o n e b i o s y n t h e s i s to s o d i u m a n d p o t a s s i u m i n t a k e is m e d i a t e d b y t h e i n d u c t i o n or r e p r e s s i o n of a sec o n d f o r m of c y t o c h r o m e P - 4 5 01 1 / ?, w h i c h differs from t h e m a i n f o r m of t h e e n z y m e b y a l o w e r m o l e c u l a r w e i g h t a n d a g r e a t e r r a n g e of catalytic p r o p e r t i e s . A m J H y p e r t e n s 1991;4:280-282 KEY W O R D S : S o d i u m i n t a k e , p o t a s s i u m i n t a k e , rat, c y t o c h r o m e P - 4 5 0n^ .
A
generation ago, t h e impact of t h e discovery of a close functional correlation b e t w e e n t h e r e n i n - angiotensin system a n d aldosterone secretion w a s so extensive t h a t angiotensin II is still widely considered to b e t h e p r e d o m i n a n t if n o t t h e only physiologically i m p o r t a n t regulator of aldosterone production. H o w e v e r , already in 1960, t h e direct influ ences of A C T H a n d t h e extracellular p o t a s s i u m a n d so d i u m concentrations o n t h e z o n a glomerulosa of t h e a d r e n a l cortex h a d b e e n clearly established. Today, at least 20 different substances of potential physiologic i m p o r t a n c e are k n o w n to directly stimulate aldosterone b i o s y n t h e s i s .1 Their list includes p e p t i d e s (angiotensin IIa n d III, corticotropin, α - M S H (melanocyte-stimulating h o r m o n e ) , a n d other P O M C derivatives, prolactin, v a s o
-From the Steroid Laboratory, Department of Medicine, University Hospital, Zürich, Switzerland.
Recent work from our laboratory has b e e n supported by Research Grant 3 2 . 8 5 7 2 . 8 5 of the S w i s s National Foundation for Scientific Re search.
Dr. Lauber is presently at Cecil H. and Ida Green Center for Repro ductive Biology Sciences, University of Texas Health Science Center, Dallas.
This article is based o n data presented at the American Society of Hypertension annual meeting, May 18 to 2 1 , 1990.
Address correspondence and reprint requests to Prof. Jürg Müller, Steroid Laboratory, University Hospital, C H - 8 0 9 1 Zürich, Switzer land.
pressin, oxytocin, vasoactive intestinal peptide, n e u r o p e p t i d e Y, endothelin), m o n o v a l e n t cations (K+, H+, N H4+ ) , a m i n e s (serotonin, histamine), acetylcholine,
a n d prostaglandins. There is also a steadily growing n u m b e r of substances reported to directly a n t a g o n i z e t h e action of aldosterone-stimulating agents. A m o n g t h e m , atrial natriuretic peptides are at present of particu lar interest, because of their h i g h inhibitory activity d e m onstrated in t h e presence of all types of aldosterone stimulators a n d b e c a u s e of t h e k n o w n d e p e n d e n c e of their secretion o n t h e s o d i u m a n d w a t e r b a l a n c e of the m a m m a l i a n organism. P e r h a p s , in t h e future, a o u a bain-like natriuretic h o r m o n e m i g h t t u r n out to b e an equally i m p o r t a n t physiologic aldosterone inhibitor.
M O D E O F A C T I O N O F R E G U L A T O R S T h e m e c h a n i s m of action of t h e m a i n aldosterone regu lators o n t h e z o n a glomerulosa cell h a s b e e n extensively studied d u r i n g t h e past 15 years a n d is still a n o p e n area of basic r e s e a r c h .1 - 5 In principle, different stimulators
interact w i t h different receptors, w h i c h in t u r n activate different second messenger systems: a d e n y l a t e cyclase, p h o s p h o l i p a s e C, or voltage-gated calcium c h a n n e l s . H o w e v e r , their final effect is t h e s a m e , ie, t h e rapid a n d sustained facilitation of a biosynthetic step early in the p a t h w a y to aldosterone, preceding t h e formation of p r e g n e n o l o n e . In addition, most of t h e stimulators h a v e
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b e e n s h o w n to h a v e a m i n o r s h o r t - t e r m stimulatory ef fect o n t h e conversion of corticosterone to aldosterone. Both of these effects can b e blocked b y atrial natriuretic peptides b y a still u n k n o w n m e c h a n i s m . These agents act t h r o u g h their o w n receptors a n d a p p e a r to block intracellular signal transmission at a site distal to t h e generation of second messengers.
L O N G - T E R M R E G U L A T I O N O F L A T E B I O S Y N T H E T I C S T E P S
In m a n as well as in experimental animals, t h e r e s p o n s e in aldosterone secretion to stimulators is variable a n d d e p e n d e n t o n s o d i u m a n d p o t a s s i u m intake. T h e activ ity of e n z y m e s regulating late steps in aldosterone bio synthesis plays a m a i n role in this l o n g - t e r m a d a p t a t i o n to alterations in s o d i u m or p o t a s s i u m intake (see Figure 1). T h u s , agents m a r k e d l y stimulating aldosterone p r o duction in capsular a d r e n a l s of normally fed rats did n o t stimulate aldosterone p r o d u c t i o n w h e n a d d e d to c a p s u lar a d r e n a l s of potassium-deficient, s o d i u m - l o a d e d , or mineralocorticoid-treated rats, b u t elicited strikingly greater i n c r e m e n t s in deoxycorticosterone o u t p u t .1 This
indicated t h a t t h e activity of late steps in aldosterone biosynthesis involved in t h e conversion of deoxycorti costerone to aldosterone w a s rate-limiting a n d subject to c h a n g e s in r e s p o n s e to alterations in s o d i u m a n d p o t a s sium intake. These c h a n g e s mainly c o n c e r n e d t h e con versions of corticosterone to 18-hydroxycorticosterone a n d of 18-hydroxycorticosterone to aldosterone, ie, t h e so-called corticosterone m e t h y l oxidations (CMO) 1 a n d 2, as well as t h e l l / ? h y d r o x y l a t i o n of 1 8 h y d r o x y l l -deoxycorticosterone. The l l / ? - h y d r o x y l a t i o n or
18-hy-ACTH ( chron. ) zona glornerulosâ cell f7\ cholesterol ANP [N*+] ! © ι pregnenolone ANGE ANGE
M
ACT H Cacufe) serotonindroxylation of deoxycorticosterone w e r e either n o t af fected or affected only to a m i n o r extent. T h e s e c h a n g e s in late steps of aldosterone biosynthesis occurred slowly, in t h e course of d a y s or at least several h o u r s , a n d w e r e characterized b y a d e l a y e d onset. According to indirect evidence, t h e l o n g - t e r m regulation of late steps of aldosterone biosynthesis is also multifactorial. W h e r eas c h a n g e s in p o t a s s i u m b a l a n c e are m a i n l y m e d i a t e d b y t h e extracellular p o t a s s i u m concentration, alterations in late steps in a d a p t a t i o n to t h e s o d i u m status are p a r tially m e d i a t e d b y t h e renin - angiotensin system, b u t additional u n k n o w n factors are also involved. Cortico tropin, w h i c h is a p o t e n t stimulator of a l d o s t e r o n e b i o synthesis in s h o r t - t e r m experiments, h a s a l o n g - t e r m inhibitory effect o n corticosterone m e t h y l o x i d a t i o n s .6
A M I T O C H O N D R I A L P R O T E I N D E P E N D E N T O N S O D I U M A N D P O T A S S I U M I N T A K E
R e s u m p t i o n of p o t a s s i u m intake b y p o t a s s i u m - d e p l e t e d rats resulted in t h e a p p e a r a n c e of a protein w i t h a molec ular w e i g h t of 49,000 in t h e m i t o c h o n d r i a of t h e z o n a glomerulosa, b u t n o t in t h o s e of t h e i n n e r z o n e s of t h e a d r e n a l cortex, as characterized b y a distinct b a n d r e vealed b y s o d i u m dodecyl sulfate-polyacrylamide gel electrophoresis.7 O v e r 48 h of p o t a s s i u m repletion, t h e
concentration of this p r o t e i n increased in parallel w i t h t h e final steps in a l d o s t e r o n e biosynthesis, as reflected b y increased p r o d u c t i o n of 18-hydroxycorticosterone (18-OH-B) a n d a l d o s t e r o n e (Aldo) a n d d e c r e a s e d o u t p u t s of deoxycorticosterone ( D O C ) , corticosterone (B), a n d 18hydroxy11deoxycorticosterone ( 1 8 O H -D O C ) b y i n c u b a t e d capsular a d r e n a l s . S o d i u m restric tion of rats also resulted in a m a r k e d increase in t h e concentration of t h e s a m e m i t o c h o n d r i a l p r o t e i n .8 This
protein crossreacted w i t h a m o n o c l o n a l a n t i b o d y raised against purified b o v i n e a d r e n a l c y t o c h r o m e P - 4 5 0n^ .9
T h e s a m e a n t i b o d y stained a protein b a n d w i t h a molec ular w e i g h t of 51,000 in m i t o c h o n d r i a of z o n a fascicu-lata cells a n d in m i t o c h o n d r i a of z o n a glomerulosa cells of rats in w h i c h a l d o s t e r o n e biosynthesis h a d b e e n s u p pressed b y p o t a s s i u m restriction a n d s o d i u m loading.
K+de ficien cy Na +intake nz A CT Η(chron.) corficosterone aldosterone Na+ deficiency • ANGE [K+] raP 'd (minutes) - r ^ slow (hours - days) FIGURE 1. Multifactoral regulation of aldosterone biosynthe sis at three different levels: 1) acute stimulation or inhibition of early biosynthetic steps; 2) long-term stimulation or suppression of late biosynthetic steps; and 3) growth or involution of zona glomerulosa cell. ANG II, angiotensin II; ANP, atrial natriuretic
peptides. From Müller J (1988).1
E N Z Y M O L O G Y O F T H E F I N A L S T E P S O F A L D O S T E R O N E B I O S Y N T H E S I S
T h e r e is l o n g - s t a n d i n g evidence t h a t t h e final t w o steps of aldosterone biosynthesis are catalyzed b y m i t o c h o n drial c y t o c h r o m e P - 4 5 0 . H o w e v e r , t h e identity of this e n z y m e h a s only recently b e e n elucidated. T w o differ e n t g r o u p s of investigators h a v e s h o w n t h a t p r e p a rations of b o v i n e or porcine c y t o c h r o m e P - 4 5 0n^ ,
r e m o v e d from m i t o c h o n d r i a a n d purified to electropho-retic h o m o g e n e i t y , catalyze n o t only l l / ? - h y d r o x y l a tion, b u t also t h e t w o - s t e p conversion of Β to Aldo, irrespective of t h e z o n a l origin of t h e reconstituted e n z y m e .1 0 1 1 If all three steps involved in t h e conversion of
282 MÜLLER A N D LAUBER AJH-MARCH 1991-VOL. 4, NO. 3, PART 1
D O C to Aldo are m e d i a t e d b y t h e s a m e e n z y m e ("one-e n z y m ("one-e " h y p o t h ("one-e s i s ; r("one-ef("one-er("one-enc("one-e 9), t h ("one-e variabl("one-e rat("one-es of corticosterone m e t h y l oxidations 1 a n d 2 in a d a p t a t i o n to t h e s o d i u m a n d p o t a s s i u m status are possibly regu lated b y u n k n o w n mitochondrial factors. Local inhibi tors m i g h t also b e responsible for t h e constant s u p p r e s sion of t h e i n h e r e n t aldosterone biosynthetic activity of c y t o c h r o m e P - 4 5 0n^ in zona fasciculata mitochondria
in t h e b o v i n e or porcine a d r e n a l cortex. H o w e v e r , nei ther t h e " o n e - e n z y m e " h y p o t h e s i s n o r a previously p o s t u l a t e d " t w o - e n z y m e " h y p o t h e s i s9 are valid for t h e
rat species, according to recent evidence o b t a i n e d in our l a b o r a t o r y1 2 a n d b y other i n v e s t i g a t o r s .1 3 T h e i m m u n o
reactive 5 I K protein w a s isolated from z o n a fasciculata mitochondria a n d purified to electrophoretic h o m o g e n e ity by c h r o m a t o g r a p h y o n octyl-Sepharose. In a recon stituted e n z y m e system (with b o v i n e a d r e n a l a d r e n o -doxin a n d a d r e n o d o x i n reductase), it converted D O C to Β a n d 1 8 - O H - D O C b u t n o t to 18-OH-B a n d Aldo. T h e i m m u n o r e a c t i v e 49 Κ protein w a s isolated from z o n a glomerulosa m i t o c h o n d r i a of rats k e p t on a l o w - s o d i u m , h i g h - p o t a s s i u m regimen. By c h r o m a t o g r a p h y o n octyl-S e p h a r o s e , it could b e s e p a r a t e d from t h e 5 I K protein b u t n o t from o t h e r mitochondrial proteins. A reconsti t u t e d eluate fraction containing t h e 49K protein con verted D O C to B, 1 8 - O H - D O C , 18-OH-B, a n d Aldo. These findings indicate t h a t t h e rat a d r e n a l cortex con tains t w o different forms of active cytochrome P - 4 5 0n^
(5IK a n d 49K), w i t h b o t h of t h e m capable of m o n o h y -droxylating D O C in either t h e 11/?- or t h e 18-position, b u t w i t h only o n e of t h e m (the 49K form) also capable of catalyzing t h e conversions of Β to 18-OH-B a n d Aldo ("one e n z y m e , t w o f o r m s " h y p o t h e s i s ; reference 9). T h e a p p e a r a n c e of t h e 49K form of t h e e n z y m e in z o n a glomerulosa m i t o c h o n d r i a is a crucial e l e m e n t in t h e a d a p t a t i o n of a l d o s t e r o n e biosynthesis to s o d i u m deficiency or p o t a s s i u m intake in rat. A n e n z y m e c o m p a r a b l e to t h e 49K form of c y t o c h r o m e P - 4 5 01 1 ) ?
h a s as yet n o t b e e n discovered in h u m a n a d r e n a l s or a d r e n a l s from a n y a n i m a l species o t h e r t h a n t h e r a t .1 1'1 4
T h e elucidation of t h e m e c h a n i s m s of zonal specificity a n d l o n g - t e r m a d a p t a t i o n of aldosterone biosynthesis to s o d i u m a n d p o t a s s i u m intake in these organisms re m a i n s a continual challenge for p r e s e n t a n d future investigators.
A C K N O W L E D G M E N T S
The expert technical assistance of Heidi Seiler, Carmen Matt, Gaby Haesler-Vetsch, and Dora Schmid is gratefully acknowl edged.
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