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Specificity and mechanism of fatty acid inhibition of aldosterone secretion
PROSTAGLANDINSLEUKOTRIENES AND ESSENTIALFATTYACIDS Prostaglandins Leukotrienes and Essential Fatty Acids (1995) 52, 145-149 © Pearson Professional Ltd 1995
Specificity and Mechanism of Fatty Acid Inhibition of Aldosterone Secretion T. L. Goodfriend, W-M. P. Lee, D. L. Ball and M. E. Elliott
William S. Middleton Memorial Veterans Hospital and Departments of Medicine and Pharmacology, University of Wisconsin, Madison, WI 53705, USA (Reprint requests to TLG) A B S T R A C T. We have shown that unesterified, unsaturated long-chain fatty acids inhibit angiotensin II (AII) binding to receptors in adrenal glomerulosa cells. In this report, we show that oleic and arachidonic acids are specific inhibitors of the AT 1 subtype of angiotensin receptor, and exert no effect on receptors of the AT 2 subtype. By contrast, decanoic acid is a weak inhibitor of the AT2 subtype only. Our previous work on a post-receptor locus of inhibition by fatty acids of aldosterone biosynthesis showed that the 18-oxidase step is uniquely sensitive. In brief, the first and last steps involved in angiotensin-stimulated aldosterone secretion are particularly sensitive to inhibition by fatty acids. These results suggest a specific role for unesterified fatty acids in regulation of salt and water metabolism.
Production of aldosterone by freshly isolated adrenal cells can be augmented by washing the cells with delipidated albumin. Albumin effects this augmentation by removing inhibitory fatty acids, specifically oleic, linoleic, and other long-chain unsaturates. Replacement of the fatty acids restores adrenal cells to a partially inhibited state (1). Fatty acids inhibit aldosterone secretion in vitro in two ways: they block binding of angiotensin to its receptors, and they inhibit aldosterone biosynthesis at a post-receptor site (2, 3). Since our original observation of fatty acid effects on angiotensin binding, it has become clear that there are at least two angiotensin receptors on adrenal glomerulosa cells, designated AT1 and AT 2 (4, 5). In addition, the adrenal glomerulosa responds to, and has receptors for, atrial natriuretic peptide (ANP), ACTH, and probably other hormones. We have shown that fatty acids do not affect binding of labeled ANP to adrenal cells (2). In this report, we show that long-chain unsaturated fatty acids affect only type 1 angiotensin receptors (AT1). We also discuss the postreceptor site of inhibition of steroidogenesis by fatty acids and the implications of these observations for the regulation of aldosterone secretion and salt and water balance.
medulla prepared from abattoir material. Methods of tissue homogenization and incubation have been published (6). Fatty acids were added from ethanol stock solutions. Receptors were assessed by measuring binding of labeled peptide ligands. The radioactive ligand lasISarl-Angiotensin II binds avidly to both types of angiotensin receptor. The peptide analogue designated CGP 42, 112 is specific for the type 2 receptor (AT2) (7). Bovine adrenal glomerulosa contains predominantly AT 1. To measure binding to that receptor, the non-specific radioactive ligand was used, and a small concentration of unlabeled CGP42,112 was added to saturate AT2 sites. To measure binding to AT 2 receptors, the ligand was ~25I-CGP42,112. Effects on non-saturable binding (sometimes called 'non-specific' binding) were measured in the presence of 1 micromolar unlabeled peptide corresponding to the labeled ligand. Steroid metabolism was measured in suspensions of adrenal cells prepared as needed from the outer slice (glomerulosa) or inner cortex (fasciculata) of bovine adrenals obtained from the abattoir (8). Aldosterone and cortisol were measured in incubation supernatants by radioimmunoassay. Effects of exogenous fatty acids on steroidogenesis were most evident when cells were first washed with de-lipidated albumin, which was then removed, and cells incubated in medium devoid of albumin.
METHODS
RESULTS
INTRODUCTION
Receptor-specific effects of fatty acids
All receptor studies were performed on subcellular fractions of bovine adrenal zona glomerulosa and adrenal
Figure 1A shows the effects of oleic acid on binding 145
146
Prostaglandins Leukotrienes and Essential Fatty Acids
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OLEIC ACID (M)
DECANOIC ACID (M)
Fig. 1 Panel A: Oleic acid inhibits angiotensin receptor subtype AT~ but not AT2; Panel B: Decanoic acid inhibits angiotensin receptor subtype AT2 but not AT 1. To measure receptors of the AT 1 subtype (Panel A), aliquots of the membrane fraction of bovine adrenal glomerulosa cells were incubated with 12SI-sarcosineLangiotensin II in the presence of 10~M CGP42,112. To measure receptors of the AT 2 subtype (Panel B), membrane particles were incubated with 125I-CGP42, 112. Oleic acid was added in graded concentrations. The incubation was terminated by centrifugation, and labeled ligand bound to the pelleted particles was counted in a well-type scintillation counter. Each point is the mean of triplicate tubes, and the brackets represent standard error of the mean. Data were calculated as a percentage of the maximum amount of each ligand bound in the absence of fatty acid. All results were corrected for unsaturable binding observed in the presence of excess unlabeled ligand.
of labeled ligands to the two types of angiotensin receptors from bovine adrenal glomerulosa cells. Oleic acid inhibited receptors of the AT~ subtype with an ICs0 of 2 x 104 M. There was no significant inhibition of AT 2 receptors at concentrations 10-times higher. Similar differentiation of the two receptor subtypes was observed with arachidonic acid and eicosatetraynoic acid (ETYA) (data not shown). Less specificity for AT1 receptors was shown by linoleic and palmitic acids. By contrast, shorter-chain saturated fatty acids showed the reverse specificity. Decanoic acid, for example, was approximately 20-times more potent against AT 2 than against AT1, although it was, at best, a weak inhibitor (Fig. 1B). We did not detect any effect of fatty acids on nonsaturable binding of either ligand; all inhibition was exerted against saturable sites.
Inhibition of steroidogenesis by fatty acids Figure 2 shows that linoleic acid was equipotent in inhibiting aldosterone synthesis whether stimulated by AII or dibutyryl cyclic AMP (dbcAMP). Inhibition of dbcAMP action indicates that step(s) other than AII receptor binding were blocked by fatty acids. We postulated that the other affected steps were beyond signal transduction and in the pathway of steroidogenesis. A1dosterone biosynthesis can be divided into two parts, the early and late pathways. The early pathway ends with side chain cleavage of cholesterol and the production of pregnenolone. The late pathway metabolizes pregnenolone to aldosterone via corticosterone (compound B)(9). Aldosterone and cortisol biosynthesis share the early pathway and some elements of the late pathway. The enzyme 18-hydroxylase/18-oxidase is unique to aldosterone biosynthesis by the zona glomerulosa.
We incubated a mixture of fasciculata and glomerulosa cells with a variety of steroid substrates in the presence and absence of fatty acids, and measured the production of aldosterone and cortisol to determine which biosynthetic steps were inhibited. Figure 3 shows that oleic acid is a potent inhibitor of the 18hydroxylase/18-oxidase function of P4501m, which converts corticosterone to aldosterone. P45021, which converts 1 l[~,17c~-dihydroxyprogesterone (21 deoxy-
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Linoleic Acid (~tM) Fig. 2 Linoleic acid inhibits aldosterone synthesis stimulated by angiotensin II (AII) or dibutyryl cyclic AMP (dbcAMP). Bovine adrenal glomerulosa cells were prepared by collagenase digestion of outer slices of fresh glands and incubated in balanced saline medium. Angiotensin II was added to a final concentration of 10-TM and dibutyryl cyclic AMP to a final concentration of 3 mM. Linoleic acid was added from an ethanol stock solution. After 3 h at 37°C, ceils were pelleted by centrifugation, and aldosterone in the supernatant was measured by direct radioimmunoassay. Data were calculated from the mean of 5 tubes + SEM, and presented as a percentage of the steroid produced in the presence of stimulus and the absence of fatty acid.
Specificity and Mechanism of Fatty Acid Inhibition of Aldosterone Secretion
Corticosterone "~Aldosterone
21-Deoxycortisol ~Cortisol
I I#,17oc,21TrihydroxyPregnenolone ,~ Cortisol
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147
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,.,.%,, ....%., %..,,.,
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Oleic Acid (juM) Fig. 3 Relative susceptibility to oleic acid of steroid metabolic transformations. Bovine adrenal cells were prepared as described in the legend to Figure 2, but the slices of adrenal were chosen so that the digest contained cells from both the gtomerulosa and fasciculata zones. At the end of a 3 h incubation, supernatants were assayed for aldosterone or cortisol by direct radioimmunoassay. Results are presented as the mean and standard error of the mean of five replicate tubes, then presented as a percentage of the increment in steroid product produced in the presence of the precursor and the absence of fatty acid.
cortisol) to cortisol, was relatively unaffected, as was 3~3-hydroxysteroid dehydrogenase/isomerase. Conversion of 1 l[]-hydroxyprogesterone to cortisol, which requires the actions of both P45021 and P45017~, was only weakly inhibited by oleic acid. The last step in aldosterone biosynthesis is corticosterone conversion to aldosterone, with 18-hydroxycorticosterone as an intermediate. Corticosterone requires both 18hydroxylation and oxidation of the hydroxyl to an aldehyde, whereas 18-hydroxycorticosterone requires only oxidation to form aldosterone. We showed that oleic acid inhibits corticosterone conversion to aldosterone and 18-hydroxycorticosterone conversion to aldosterone with equal potency (3). Thus, oleic acid blocks the very last step in aldosterone synthesis. Figure 4 is a summary diagram of the mechanisms by which AII acutely stimulates aldosterone synthesis. Long-chain fatty acids inhibit the first and last steps of this process, binding of AII to AT 1 receptors, and the conversion of corticosterone to aldosterone.
DISCUSSION We share a common fear of investigators studying fatty acid effects on biological systems - that the phenomena
we discover will prove to be 'non-specific', resulting from detergent action or some other physiologically irrelevant physico-chemical interaction. It was reassuring, therefore, to find that oleic and arachidonic acids display a high degree of specificity toward one isoform of the angiotensin receptor family. Amino acid sequences of AT1 and AT 2 angiotensin receptors have been determined (10, 11). The two subtypes show 33% homology and both of them have the hydrophobicity profiles of other receptors with seven membrane-spanning regions. Yet only one, the AT1 isoform, is inhibited by long-chain unsaturated fatty acids. We interpret this as strong evidence against nonspecificity of fatty acid effects on angiotensin receptors. These new observations supplement an earlier finding that also suggested fatty acid specificity - a lack of effect of fatty acids on receptors for atrial natriuretic peptide in the same adrenal glomerulosa cells where angiotensin receptors are inhibited (2). Since we do not know what biological actions are mediated by receptors of the AT2 subtype, we cannot corroborate with bioassays the lack of fatty acid inhibition of its interaction with angiotensin. We can, however, corroborate with bioassays fatty acid inhibition of AT 1 receptors, because that receptor mediates stimulation of aldosterone synthesis. We found that long-
148
Prostaglandins Leukotrienes and Essential Fatty Acids
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synthase' because it is specific to aldosterone biosynthesis. Other metabolic conversions in the pathway from cholesterol to adrenal steroid hormones, such as those leading to cortisol in the zona fasciculata, are at least 10-times more resistant to inhibition by fatty acids. It is difficult to imagine a biochemical basis for the shared fatty acid sensitivity of the angiotensin receptor and the steroid hydroxylase/oxidase. Aldosterone synthase has been sequenced. It is a P450 cytochrome, and there is no obvious amino acid sequence homology between this enzyme and the AT 1 receptor (12). One might postulate that fatty acids alter a common subcellular milieu that surrounds these two macromolecules, but the enzyme resides in mitochondrial membrane and the receptor in plasma membrane. Furthermore, other membrane receptors and other mitochondrial enzymes in adrenal glomerulosa cells are not sensitive to fatty acid inhibition. Finally, an effect on the milieu would not explain the most remarkable aspect of fatty acid inhibition of aldosterone synthase in bovine adrenals its relative specificity to the 18-hydroxylase/oxidase function of that enzyme compared to the 11-hydroxylase function• In bovine zona glomerulosa, those reactions are catalyzed by the same enzyme protein, yet we found ll-hydroxylation resistant to fatty acids (13). We are struck by the susceptibility of the first and last steps of angiotensin-stimulated aldosterone synthesis to fatty acid inhibition (Fig. 4). This dual effect suggests that fatty acids play an integral role in regulating aldosterone secretion, salt and water metabolism, and perhaps blood pressure. One might predict that situations encountered by animals and humans in which fatty acids are elevated would be accompanied by reduced aldosterone levels and sodium diuresis. One possibility is starvation, which induces a natriuresis that can be reversed by carbohydrate feeding (14). Although starvation elevates fatty acid levels and carbohydrate depresses them, the current explanation of starvation natriuresis invokes the action of glucagon, not unesterified fatty acids or aldosterone (14). Other situations in which unesterified fatty acids are elevated include sepsis and massive parasitic infections where lipolysis is mediated in part by tumor necrosis factor (15). Intravascular volume depletion in these conditions may have many causes, and the possible role of fatty acids and depressed aldosterone secretion has not been studied. When we studied humans with widely varying blood levels of unesterified fatty acids, we found that their plasma aldosterone was not noticeably affected until very high concentrations of fatty acids were achieved (16). There appeared to be an inhibitory threshold at approximately 150 micromolar oleic acid. It may be that the physiologic or pathologic correlates of our in vitro observations are limited to situations in which plasma fatty acids rise to extremely high levels. Another possible role of fatty acids is autocrine control, in which adrenal glomerulosa cells might regulate their own •
-I
Pig. 4 Scheme of an adrenal glomerulosa cell, showing the mechanism of stimulation of aldosterone synthesis by angiotensin II (AII) and the two sites where fatty acids inhibit this stimulation. The two types of angiotensin receptor are shown at the top of the figure, and the signal transduction mechanism coupled to type AT~ outlined below the receptor. Cholesterol side-chain cleavage enzyme (SCC) is depicted in a mitochondrion in the center of the figure, and the late pathway of steroidogenesis is shown in a mitochondrion at the bottom. Long chain unsaturated fatty acids inhibit binding of angiotensin to the coupled receptor, and inhibit the last enzymatic step in synthesis of aldosterone.
chain unsaturated fatty acids indeed blocked angiotensin stimulation of aldosterone synthesis, but they also blocked aldosteronogenesis stimulated by cyclic AMP and potassium, neither of which act through receptors (3). We concluded that fatty acids must have two effects on aldosterone biosynthesis, one at the AII receptor mad one distal to receptors and beyond the early steps of signal transduction specific to angiotensin. We have shown that the last step of aldosterone biosynthesis, oxidation at carbon atom 18, is exquisitely and uniquely sensitive to fatty acid inhibition. The 18hydroxylase/oxidase enzyme is also called 'aldosterone
.
,
I
Specificity and Mechanism of Fatty Acid Inhibition of Aldosterone Secretion aldosterone p r o d u c t i o n with intracellular fatty acids released during signal transduction. Inhibition by fatty acids of angiotensin receptors and actions is not restricted to the adrenal z o n a glomerulosa. U l l i a n f o u n d that polyunsaturated l o n g - c h a i n fatty acids added to the incubation m e d i u m o f v a s c u l a r s m o o t h m u s c l e cells inhibited b i n d i n g o f angiotensin to its receptors and release o f inositol phosphates (17). R e c e p t o r s o f the A T 1 subtype p r e d o m i n a t e in v a s c u l a r s m o o t h muscle. Others h a v e o b s e r v e d fatty acid inhibition of a n g i o t e n s i n - i n d u c e d contraction in arterial strips (18, 19). It should be n o t e d that our e x p e r i m e n t s w e r e perf o r m e d acutely w i t h unesterified fatty acids, and m a y h a v e limited r e l e v a n c e to the m a n y studies o f f e e d i n g described in the literature. In those experiments, specific fatty acids or oils w e r e administered o v e r l o n g periods of time, and b l o o d pressure was m e a s u r e d at the end o f that intervention. M o s t o f that literature c o n c l u d e s that f e e d i n g unsaturated fatty acids, especially those f o u n d in fish oil, reduces the acute r e s p o n s i v e n e s s o f b l o o d pressure to angiotensin (20). W h e t h e r or not that inhibition depends on release o f unesterified fatty acids and their direct interaction w i t h angiotensin receptors or steroidogenic e n z y m e s remains to be seen.
Acknowledgments This work was supported by the US Department of Veterans Affairs. It was presented, in part, at the 73rd Annual Meeting of The Endocrine Society, 1991, Abstract #667. We are indebted to Dr Marc de Gasparo of Ciba-Geigy, and Dr Andrew Chiu of DuPont/Merck for gifts of specific, non-peptide inhibitors of angiotensin receptors. Expert editorial advice was given by Ms Susi Nehls.
References 1. Goodfriend T L, Ball D L, Elliott M E, Morrison A R, Evenson M A. Fatty acids are potential endogenous regulators of aldosterone secretion. Endocrinology 1991; 128: 2511-2519. 2. Goodfriend T L, Ball D L. Fatty acid effects on angiotensin receptors. J Cardiovasc Pharmacol 1986; 8: 1276-1283. 3. Elliott M E, Goodfriend T L. Mechanism of fatty acid inhibition of aldosterone synthesis by bovine adrenal glomerulosa cells. Endocrinology 1993; 132: 2453-2460. 4. Chiu A T, Herblin W F, Ardecky R Jet al. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun 1989; 165: 196-203.
5. Bumpus F M, Catt K J, Chiu A T et al. Nomenclature for angiotensin receptors. Hypertension 1991; 17: 720-723. 6. Lin S-Y, Goodfriend T L. Angiotensin receptors. Am J Physiol 1970; 218: 1319-1328. 7. Whitebread S E, Taylor V, Bottari S P, Kamber B, de Gasparo M. Radioiodinated CGP 42112A; a novel high affinity and highly selective ligand for the characterization of angiotensin ATz receptors. Biochem Biophys Res Commun 1991; 1811 1365-1371. 8. Elliott M E, Goodfriend T L. Identification of the cycloheximide-sensitive site in angiotensin-stimulated aldosterone synthesis. Biochem Pharmacol 1984; 33: 1519-1524. 9. Miiller J. Regulation of aldosterone biosynthesis: physiological and clinical aspects. 2nd rev. ed. New York: Springer-Verlag, 1988: 364. 10. Sasaki K, Yamano Y, Bardhan Set al. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type 1 receptor. Nature 1991; 351: 230-232. 11. Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt R E, Dzan V J. Expression cloning of type-2 angiotensin II receptor reveals a unique class of seventransmembrane receptors. J Biol Chem 1993: 268: 24547-24550. 12. Kirita S, Morohashi K, Hashimoto T, Yoshioka H, FujiKuriyama Y, Omura T. Expression of two kinds of cytochrome P-450(1113) mRNA in bovine adrenal cortex. J Biochem 1988; 104: 683-686. 13. Yanagibashi K, Haniu M, Shively J E, Shen W H, Hall P. The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11 beta-18-hydroxylation, and aldehyde synthesis. J Biol Chem 1986; 261: 3556-3562. 14. Chinn R H, Brown J J, Fraser R et al. The natriuresis of fasting: relationship to changes in plasma renin and plasma aldosterone concentrations. Clin Sci 1970; 39: 437-455. 15. Sitprija V, Vongsthongsri M, Poshyachinda V, Arthachinta S. Renal failure in malaria: a pathophysiologic study. Nephron 1977; 18: 277-287. 16. Goodfriend T L, Ball D L, Elliott M E et al. Fatty acids may regulate aldosterone secretion and mediate some of insulin's effects on blood pressure. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 43-50. 17. Ullian M E. Fatty acid inhibition of angiotensin IIstimulated inositol phosphates in smooth muscle cells. Am J Physiol 1993; 264: H595-H603. 18. Juan H, Sametz W. Vasoconstriction induced by noradrenaline and angiotensin II is antagonized by eicosapentaenoic acid independent of formation of trienoic eicosanoids. Arch Pharmacol 1986; 332: 288-292. 19. Yoshimura T, Ito M, Matsui K, Fujisaki S. Effects of highly purified eicosapentaenoic acid on vascular reactivity to angiotensin II and norepinephrine in the rabbit. Prostaglandins 1986; 32: 179-188. 20. Kenny D, Warltier D C, Pleuss J A, Hoffman R G, Goodfriend T L, Egan B M. Effect of omega-3 fatty acids on the vascular response to angiotensin in normotensive men. Am J Cardiol 1992; 70: 1347-1352.
t49
Specificity and Mechanism of Fatty Acid Inhibition of Aldosterone Secretion T. L. Goodfriend, W-M. P. Lee, D. L. Ball and M. E. Elliott
William S. Middleton Memorial Veterans Hospital and Departments of Medicine and Pharmacology, University of Wisconsin, Madison, WI 53705, USA (Reprint requests to TLG) A B S T R A C T. We have shown that unesterified, unsaturated long-chain fatty acids inhibit angiotensin II (AII) binding to receptors in adrenal glomerulosa cells. In this report, we show that oleic and arachidonic acids are specific inhibitors of the AT 1 subtype of angiotensin receptor, and exert no effect on receptors of the AT 2 subtype. By contrast, decanoic acid is a weak inhibitor of the AT2 subtype only. Our previous work on a post-receptor locus of inhibition by fatty acids of aldosterone biosynthesis showed that the 18-oxidase step is uniquely sensitive. In brief, the first and last steps involved in angiotensin-stimulated aldosterone secretion are particularly sensitive to inhibition by fatty acids. These results suggest a specific role for unesterified fatty acids in regulation of salt and water metabolism.
Production of aldosterone by freshly isolated adrenal cells can be augmented by washing the cells with delipidated albumin. Albumin effects this augmentation by removing inhibitory fatty acids, specifically oleic, linoleic, and other long-chain unsaturates. Replacement of the fatty acids restores adrenal cells to a partially inhibited state (1). Fatty acids inhibit aldosterone secretion in vitro in two ways: they block binding of angiotensin to its receptors, and they inhibit aldosterone biosynthesis at a post-receptor site (2, 3). Since our original observation of fatty acid effects on angiotensin binding, it has become clear that there are at least two angiotensin receptors on adrenal glomerulosa cells, designated AT1 and AT 2 (4, 5). In addition, the adrenal glomerulosa responds to, and has receptors for, atrial natriuretic peptide (ANP), ACTH, and probably other hormones. We have shown that fatty acids do not affect binding of labeled ANP to adrenal cells (2). In this report, we show that long-chain unsaturated fatty acids affect only type 1 angiotensin receptors (AT1). We also discuss the postreceptor site of inhibition of steroidogenesis by fatty acids and the implications of these observations for the regulation of aldosterone secretion and salt and water balance.
medulla prepared from abattoir material. Methods of tissue homogenization and incubation have been published (6). Fatty acids were added from ethanol stock solutions. Receptors were assessed by measuring binding of labeled peptide ligands. The radioactive ligand lasISarl-Angiotensin II binds avidly to both types of angiotensin receptor. The peptide analogue designated CGP 42, 112 is specific for the type 2 receptor (AT2) (7). Bovine adrenal glomerulosa contains predominantly AT 1. To measure binding to that receptor, the non-specific radioactive ligand was used, and a small concentration of unlabeled CGP42,112 was added to saturate AT2 sites. To measure binding to AT 2 receptors, the ligand was ~25I-CGP42,112. Effects on non-saturable binding (sometimes called 'non-specific' binding) were measured in the presence of 1 micromolar unlabeled peptide corresponding to the labeled ligand. Steroid metabolism was measured in suspensions of adrenal cells prepared as needed from the outer slice (glomerulosa) or inner cortex (fasciculata) of bovine adrenals obtained from the abattoir (8). Aldosterone and cortisol were measured in incubation supernatants by radioimmunoassay. Effects of exogenous fatty acids on steroidogenesis were most evident when cells were first washed with de-lipidated albumin, which was then removed, and cells incubated in medium devoid of albumin.
METHODS
RESULTS
INTRODUCTION
Receptor-specific effects of fatty acids
All receptor studies were performed on subcellular fractions of bovine adrenal zona glomerulosa and adrenal
Figure 1A shows the effects of oleic acid on binding 145
146
Prostaglandins Leukotrienes and Essential Fatty Acids
lesZ- CGP
100 -
8O X <
. . . . . .
. . . . .
_
Ioo
A
oo
60
B
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2; 60
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(.9
(_9 Z
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F, 40
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20 I II
0
I
I
I
I
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10.5
3XlO-5
I0 -4
0
0
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I
I
I
I
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10-5
3xl0-5
10-4
OLEIC ACID (M)
DECANOIC ACID (M)
Fig. 1 Panel A: Oleic acid inhibits angiotensin receptor subtype AT~ but not AT2; Panel B: Decanoic acid inhibits angiotensin receptor subtype AT2 but not AT 1. To measure receptors of the AT 1 subtype (Panel A), aliquots of the membrane fraction of bovine adrenal glomerulosa cells were incubated with 12SI-sarcosineLangiotensin II in the presence of 10~M CGP42,112. To measure receptors of the AT 2 subtype (Panel B), membrane particles were incubated with 125I-CGP42, 112. Oleic acid was added in graded concentrations. The incubation was terminated by centrifugation, and labeled ligand bound to the pelleted particles was counted in a well-type scintillation counter. Each point is the mean of triplicate tubes, and the brackets represent standard error of the mean. Data were calculated as a percentage of the maximum amount of each ligand bound in the absence of fatty acid. All results were corrected for unsaturable binding observed in the presence of excess unlabeled ligand.
of labeled ligands to the two types of angiotensin receptors from bovine adrenal glomerulosa cells. Oleic acid inhibited receptors of the AT~ subtype with an ICs0 of 2 x 104 M. There was no significant inhibition of AT 2 receptors at concentrations 10-times higher. Similar differentiation of the two receptor subtypes was observed with arachidonic acid and eicosatetraynoic acid (ETYA) (data not shown). Less specificity for AT1 receptors was shown by linoleic and palmitic acids. By contrast, shorter-chain saturated fatty acids showed the reverse specificity. Decanoic acid, for example, was approximately 20-times more potent against AT 2 than against AT1, although it was, at best, a weak inhibitor (Fig. 1B). We did not detect any effect of fatty acids on nonsaturable binding of either ligand; all inhibition was exerted against saturable sites.
Inhibition of steroidogenesis by fatty acids Figure 2 shows that linoleic acid was equipotent in inhibiting aldosterone synthesis whether stimulated by AII or dibutyryl cyclic AMP (dbcAMP). Inhibition of dbcAMP action indicates that step(s) other than AII receptor binding were blocked by fatty acids. We postulated that the other affected steps were beyond signal transduction and in the pathway of steroidogenesis. A1dosterone biosynthesis can be divided into two parts, the early and late pathways. The early pathway ends with side chain cleavage of cholesterol and the production of pregnenolone. The late pathway metabolizes pregnenolone to aldosterone via corticosterone (compound B)(9). Aldosterone and cortisol biosynthesis share the early pathway and some elements of the late pathway. The enzyme 18-hydroxylase/18-oxidase is unique to aldosterone biosynthesis by the zona glomerulosa.
We incubated a mixture of fasciculata and glomerulosa cells with a variety of steroid substrates in the presence and absence of fatty acids, and measured the production of aldosterone and cortisol to determine which biosynthetic steps were inhibited. Figure 3 shows that oleic acid is a potent inhibitor of the 18hydroxylase/18-oxidase function of P4501m, which converts corticosterone to aldosterone. P45021, which converts 1 l[~,17c~-dihydroxyprogesterone (21 deoxy-
1oo A~
U ~ bcAMp-Stimulated
80 if} 60 .~
g, 40 co e
o -o <
20 o
1t
I
I
I
I
I
3
I0
30
Linoleic Acid (~tM) Fig. 2 Linoleic acid inhibits aldosterone synthesis stimulated by angiotensin II (AII) or dibutyryl cyclic AMP (dbcAMP). Bovine adrenal glomerulosa cells were prepared by collagenase digestion of outer slices of fresh glands and incubated in balanced saline medium. Angiotensin II was added to a final concentration of 10-TM and dibutyryl cyclic AMP to a final concentration of 3 mM. Linoleic acid was added from an ethanol stock solution. After 3 h at 37°C, ceils were pelleted by centrifugation, and aldosterone in the supernatant was measured by direct radioimmunoassay. Data were calculated from the mean of 5 tubes + SEM, and presented as a percentage of the steroid produced in the presence of stimulus and the absence of fatty acid.
Specificity and Mechanism of Fatty Acid Inhibition of Aldosterone Secretion
Corticosterone "~Aldosterone
21-Deoxycortisol ~Cortisol
I I#,17oc,21TrihydroxyPregnenolone ,~ Cortisol
P4502i
3/~HSD
¢.0 0
-0--,
0
P450t i#
V
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o~
(18-hydroxylose + 18-oxidose )
147
I I/~-HydroxyProgesterone -~ Cortisol
P45°iz= I + P45021
,.,.%,, ....%., %..,,.,
Or) "0 0 t~ (lO
60-
Or) t-
4O-
10
20-
¢m
:.:,:,:,
0
5
I0 20 30
0
5
I0 20 30
0
5
I0 20 30
0
5 I0 20 30
Oleic Acid (juM) Fig. 3 Relative susceptibility to oleic acid of steroid metabolic transformations. Bovine adrenal cells were prepared as described in the legend to Figure 2, but the slices of adrenal were chosen so that the digest contained cells from both the gtomerulosa and fasciculata zones. At the end of a 3 h incubation, supernatants were assayed for aldosterone or cortisol by direct radioimmunoassay. Results are presented as the mean and standard error of the mean of five replicate tubes, then presented as a percentage of the increment in steroid product produced in the presence of the precursor and the absence of fatty acid.
cortisol) to cortisol, was relatively unaffected, as was 3~3-hydroxysteroid dehydrogenase/isomerase. Conversion of 1 l[]-hydroxyprogesterone to cortisol, which requires the actions of both P45021 and P45017~, was only weakly inhibited by oleic acid. The last step in aldosterone biosynthesis is corticosterone conversion to aldosterone, with 18-hydroxycorticosterone as an intermediate. Corticosterone requires both 18hydroxylation and oxidation of the hydroxyl to an aldehyde, whereas 18-hydroxycorticosterone requires only oxidation to form aldosterone. We showed that oleic acid inhibits corticosterone conversion to aldosterone and 18-hydroxycorticosterone conversion to aldosterone with equal potency (3). Thus, oleic acid blocks the very last step in aldosterone synthesis. Figure 4 is a summary diagram of the mechanisms by which AII acutely stimulates aldosterone synthesis. Long-chain fatty acids inhibit the first and last steps of this process, binding of AII to AT 1 receptors, and the conversion of corticosterone to aldosterone.
DISCUSSION We share a common fear of investigators studying fatty acid effects on biological systems - that the phenomena
we discover will prove to be 'non-specific', resulting from detergent action or some other physiologically irrelevant physico-chemical interaction. It was reassuring, therefore, to find that oleic and arachidonic acids display a high degree of specificity toward one isoform of the angiotensin receptor family. Amino acid sequences of AT1 and AT 2 angiotensin receptors have been determined (10, 11). The two subtypes show 33% homology and both of them have the hydrophobicity profiles of other receptors with seven membrane-spanning regions. Yet only one, the AT1 isoform, is inhibited by long-chain unsaturated fatty acids. We interpret this as strong evidence against nonspecificity of fatty acid effects on angiotensin receptors. These new observations supplement an earlier finding that also suggested fatty acid specificity - a lack of effect of fatty acids on receptors for atrial natriuretic peptide in the same adrenal glomerulosa cells where angiotensin receptors are inhibited (2). Since we do not know what biological actions are mediated by receptors of the AT2 subtype, we cannot corroborate with bioassays the lack of fatty acid inhibition of its interaction with angiotensin. We can, however, corroborate with bioassays fatty acid inhibition of AT 1 receptors, because that receptor mediates stimulation of aldosterone synthesis. We found that long-
148
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synthase' because it is specific to aldosterone biosynthesis. Other metabolic conversions in the pathway from cholesterol to adrenal steroid hormones, such as those leading to cortisol in the zona fasciculata, are at least 10-times more resistant to inhibition by fatty acids. It is difficult to imagine a biochemical basis for the shared fatty acid sensitivity of the angiotensin receptor and the steroid hydroxylase/oxidase. Aldosterone synthase has been sequenced. It is a P450 cytochrome, and there is no obvious amino acid sequence homology between this enzyme and the AT 1 receptor (12). One might postulate that fatty acids alter a common subcellular milieu that surrounds these two macromolecules, but the enzyme resides in mitochondrial membrane and the receptor in plasma membrane. Furthermore, other membrane receptors and other mitochondrial enzymes in adrenal glomerulosa cells are not sensitive to fatty acid inhibition. Finally, an effect on the milieu would not explain the most remarkable aspect of fatty acid inhibition of aldosterone synthase in bovine adrenals its relative specificity to the 18-hydroxylase/oxidase function of that enzyme compared to the 11-hydroxylase function• In bovine zona glomerulosa, those reactions are catalyzed by the same enzyme protein, yet we found ll-hydroxylation resistant to fatty acids (13). We are struck by the susceptibility of the first and last steps of angiotensin-stimulated aldosterone synthesis to fatty acid inhibition (Fig. 4). This dual effect suggests that fatty acids play an integral role in regulating aldosterone secretion, salt and water metabolism, and perhaps blood pressure. One might predict that situations encountered by animals and humans in which fatty acids are elevated would be accompanied by reduced aldosterone levels and sodium diuresis. One possibility is starvation, which induces a natriuresis that can be reversed by carbohydrate feeding (14). Although starvation elevates fatty acid levels and carbohydrate depresses them, the current explanation of starvation natriuresis invokes the action of glucagon, not unesterified fatty acids or aldosterone (14). Other situations in which unesterified fatty acids are elevated include sepsis and massive parasitic infections where lipolysis is mediated in part by tumor necrosis factor (15). Intravascular volume depletion in these conditions may have many causes, and the possible role of fatty acids and depressed aldosterone secretion has not been studied. When we studied humans with widely varying blood levels of unesterified fatty acids, we found that their plasma aldosterone was not noticeably affected until very high concentrations of fatty acids were achieved (16). There appeared to be an inhibitory threshold at approximately 150 micromolar oleic acid. It may be that the physiologic or pathologic correlates of our in vitro observations are limited to situations in which plasma fatty acids rise to extremely high levels. Another possible role of fatty acids is autocrine control, in which adrenal glomerulosa cells might regulate their own •
-I
Pig. 4 Scheme of an adrenal glomerulosa cell, showing the mechanism of stimulation of aldosterone synthesis by angiotensin II (AII) and the two sites where fatty acids inhibit this stimulation. The two types of angiotensin receptor are shown at the top of the figure, and the signal transduction mechanism coupled to type AT~ outlined below the receptor. Cholesterol side-chain cleavage enzyme (SCC) is depicted in a mitochondrion in the center of the figure, and the late pathway of steroidogenesis is shown in a mitochondrion at the bottom. Long chain unsaturated fatty acids inhibit binding of angiotensin to the coupled receptor, and inhibit the last enzymatic step in synthesis of aldosterone.
chain unsaturated fatty acids indeed blocked angiotensin stimulation of aldosterone synthesis, but they also blocked aldosteronogenesis stimulated by cyclic AMP and potassium, neither of which act through receptors (3). We concluded that fatty acids must have two effects on aldosterone biosynthesis, one at the AII receptor mad one distal to receptors and beyond the early steps of signal transduction specific to angiotensin. We have shown that the last step of aldosterone biosynthesis, oxidation at carbon atom 18, is exquisitely and uniquely sensitive to fatty acid inhibition. The 18hydroxylase/oxidase enzyme is also called 'aldosterone
.
,
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Specificity and Mechanism of Fatty Acid Inhibition of Aldosterone Secretion aldosterone p r o d u c t i o n with intracellular fatty acids released during signal transduction. Inhibition by fatty acids of angiotensin receptors and actions is not restricted to the adrenal z o n a glomerulosa. U l l i a n f o u n d that polyunsaturated l o n g - c h a i n fatty acids added to the incubation m e d i u m o f v a s c u l a r s m o o t h m u s c l e cells inhibited b i n d i n g o f angiotensin to its receptors and release o f inositol phosphates (17). R e c e p t o r s o f the A T 1 subtype p r e d o m i n a t e in v a s c u l a r s m o o t h muscle. Others h a v e o b s e r v e d fatty acid inhibition of a n g i o t e n s i n - i n d u c e d contraction in arterial strips (18, 19). It should be n o t e d that our e x p e r i m e n t s w e r e perf o r m e d acutely w i t h unesterified fatty acids, and m a y h a v e limited r e l e v a n c e to the m a n y studies o f f e e d i n g described in the literature. In those experiments, specific fatty acids or oils w e r e administered o v e r l o n g periods of time, and b l o o d pressure was m e a s u r e d at the end o f that intervention. M o s t o f that literature c o n c l u d e s that f e e d i n g unsaturated fatty acids, especially those f o u n d in fish oil, reduces the acute r e s p o n s i v e n e s s o f b l o o d pressure to angiotensin (20). W h e t h e r or not that inhibition depends on release o f unesterified fatty acids and their direct interaction w i t h angiotensin receptors or steroidogenic e n z y m e s remains to be seen.
Acknowledgments This work was supported by the US Department of Veterans Affairs. It was presented, in part, at the 73rd Annual Meeting of The Endocrine Society, 1991, Abstract #667. We are indebted to Dr Marc de Gasparo of Ciba-Geigy, and Dr Andrew Chiu of DuPont/Merck for gifts of specific, non-peptide inhibitors of angiotensin receptors. Expert editorial advice was given by Ms Susi Nehls.
References 1. Goodfriend T L, Ball D L, Elliott M E, Morrison A R, Evenson M A. Fatty acids are potential endogenous regulators of aldosterone secretion. Endocrinology 1991; 128: 2511-2519. 2. Goodfriend T L, Ball D L. Fatty acid effects on angiotensin receptors. J Cardiovasc Pharmacol 1986; 8: 1276-1283. 3. Elliott M E, Goodfriend T L. Mechanism of fatty acid inhibition of aldosterone synthesis by bovine adrenal glomerulosa cells. Endocrinology 1993; 132: 2453-2460. 4. Chiu A T, Herblin W F, Ardecky R Jet al. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun 1989; 165: 196-203.
5. Bumpus F M, Catt K J, Chiu A T et al. Nomenclature for angiotensin receptors. Hypertension 1991; 17: 720-723. 6. Lin S-Y, Goodfriend T L. Angiotensin receptors. Am J Physiol 1970; 218: 1319-1328. 7. Whitebread S E, Taylor V, Bottari S P, Kamber B, de Gasparo M. Radioiodinated CGP 42112A; a novel high affinity and highly selective ligand for the characterization of angiotensin ATz receptors. Biochem Biophys Res Commun 1991; 1811 1365-1371. 8. Elliott M E, Goodfriend T L. Identification of the cycloheximide-sensitive site in angiotensin-stimulated aldosterone synthesis. Biochem Pharmacol 1984; 33: 1519-1524. 9. Miiller J. Regulation of aldosterone biosynthesis: physiological and clinical aspects. 2nd rev. ed. New York: Springer-Verlag, 1988: 364. 10. Sasaki K, Yamano Y, Bardhan Set al. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type 1 receptor. Nature 1991; 351: 230-232. 11. Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt R E, Dzan V J. Expression cloning of type-2 angiotensin II receptor reveals a unique class of seventransmembrane receptors. J Biol Chem 1993: 268: 24547-24550. 12. Kirita S, Morohashi K, Hashimoto T, Yoshioka H, FujiKuriyama Y, Omura T. Expression of two kinds of cytochrome P-450(1113) mRNA in bovine adrenal cortex. J Biochem 1988; 104: 683-686. 13. Yanagibashi K, Haniu M, Shively J E, Shen W H, Hall P. The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11 beta-18-hydroxylation, and aldehyde synthesis. J Biol Chem 1986; 261: 3556-3562. 14. Chinn R H, Brown J J, Fraser R et al. The natriuresis of fasting: relationship to changes in plasma renin and plasma aldosterone concentrations. Clin Sci 1970; 39: 437-455. 15. Sitprija V, Vongsthongsri M, Poshyachinda V, Arthachinta S. Renal failure in malaria: a pathophysiologic study. Nephron 1977; 18: 277-287. 16. Goodfriend T L, Ball D L, Elliott M E et al. Fatty acids may regulate aldosterone secretion and mediate some of insulin's effects on blood pressure. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 43-50. 17. Ullian M E. Fatty acid inhibition of angiotensin IIstimulated inositol phosphates in smooth muscle cells. Am J Physiol 1993; 264: H595-H603. 18. Juan H, Sametz W. Vasoconstriction induced by noradrenaline and angiotensin II is antagonized by eicosapentaenoic acid independent of formation of trienoic eicosanoids. Arch Pharmacol 1986; 332: 288-292. 19. Yoshimura T, Ito M, Matsui K, Fujisaki S. Effects of highly purified eicosapentaenoic acid on vascular reactivity to angiotensin II and norepinephrine in the rabbit. Prostaglandins 1986; 32: 179-188. 20. Kenny D, Warltier D C, Pleuss J A, Hoffman R G, Goodfriend T L, Egan B M. Effect of omega-3 fatty acids on the vascular response to angiotensin in normotensive men. Am J Cardiol 1992; 70: 1347-1352.
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