Printed in U. S. A. Regulation of Rat ..... hanced mRNA levels of enzymes implicated in the first step(s) .... John, M. E., John, M. C., Asley, P., McDonald, R. J., Simpson, ... Boyd, J. E., Palmore, W. D., and Mulrow, P. J. (1971) Endocri-. 42.
THEJOURNALOF (C) 1991 by
Vol. 266, No. 4, Issue of February 5, pp. 2245-2251, 1991 Printed in U.S.A.
BIOLOGICAL CHEMISTRY
The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Rat Adrenal Messenger RNA and ProteinLevels for Cytochrome P-450s andAdrenodoxin by Dietary Sodium Depletion or Potassium Intake* (Received for publication, March 30, 1990)
Andre TremblaySS, Michael R. Watermanll, Keith L. Parker**, and Jean-GuyLehouxlll From the $Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, QuebecJ1H 5N4, Canada, the TDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235, and the **Howard Hughes Medical Institute andthe Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
The effect of low sodium and high potassium intake terol side-chain cleavage cytochrome P-450 (P-450,,,)’ cataon rat adrenal zona glomerulosa (ZG) and zona fasci- lyzes the conversion of cholesterol to pregnenolone, which is culata-reticularis (ZFR) were studied during a 7-day the first and rate-limiting step in steroidogenesis (I). Cytoperiod, by analyzing mRNA and protein levels of var- chrome P-450,21 is responsiblefor the 21-hydroxylation of ious enzymes involved in aldosteronesynthesis. In ZG progesterone t o yield 11-deoxycorticosterone (DOC) (2). Cytochrome P-45OIlpis believed t o be involved in the last three significant increasesin cytochrome P-450,,, P-450,21, P-45011B,adrenodoxin mRNA and protein levels were enzymatic steps of aldosterone synthesis, namely the llpobserved after 2 days with either diet, and at day 7 hydroxylation of DOC, the 18-hydroxylation of corticosterthese levels were furtherincreased. The largestmRNA one, and the 18-methyl oxidationleading to aldosterone proinduction was observed at day 7 in sodium-depleted duction. rats for P-45OlIB,with a 4-fold increase, followed by However, the 18-methyl oxidase activity is known to occur 2.7-and 2.0-fold increases for P-450.,, and P-450c21, exclusively in the zona glomerulosa (ZG) of mammalian adrespectively. A pattern similar to those of P-450.,, and renalcortex(3) t o which aldosteronebiosynthesisisreP-45011Bwas observed for adrenodoxin with a 2.1-fold stricted, although both ZG and zona fasciculata-reticularis increase after 7 days of Na’ restriction. In K+-loaded (ZFR) possess the P-45Oliirenzyme (4). The basisof the cellrats mRNA levels for P-450,,, P-45011~, P-45oCz1, and specific production of aldosterone remains unclearat present. adrenodoxin were also increased by 2.2-, 2.1-, 1.5-, P-450,,,, as well as P-45olIp, arelocated in the inner memand 1.9-fold respectively. Protein levels of these en- brane of mitochondria and functionas terminal oxidases in a zymes were also measured in ZG and showed increases mitochondrial NADPH-dependent electron transport system similar to those of their respective mRNAs for both consisting of a flavoprotein, adrenodoxin reductase, and an treatments. On the other hand, mRNA levels of P- iron-sulfur protein, adrenodoxin. The role of adrenodoxin is 450,,, P-45OlIB,P-450,21, and adrenodoxin in ZFR to transfer electrons from adrenodoxin reductase toP-450,,, were found significantly lower than in ZG, although or P-450118 bya dissociation-reassociation process(5). In they wereslightly increased forboth treated groupsof contrast, cytochrome P-450,21 resides in the smooth endorats as compared with controls. In addition, ZFR pro- plasmicreticulumandacceptselectrons from a different tein levels of corresponding enzymes did not fluctuate electron transport system than that found in mitochondria significantly underboth ionic regimens. In conclusion, (6). both low sodium and high potassium intakes act priIt is well known that dietarysodium restriction and/or high marily on ZG. Their action on plasma aldosterone potassium intake markedly stimulate the production of aldosseems to be mediated by increasing both mRNA and terone. This regulation is a priori the result of increased protein andlevels of steroidogenic enzymes, especially cytochrome P-450 activities of both the early and late steps at the early step(cytochrome P-450,,) and even more in aldosterone biosynthesis. Indeed, it has been shown with at the late steps(cytochrome P-450110). In addition, a in vivo (7, 8 ) and in vitro methods using adrenaltissue close relationship appears to exist between the two incubations (9, 10) and isolated cells (11-13) that potassium mitochondrial P-450s and their electron donor adre- loading stimulated the productionof pregnenolone as well as nodoxin, since their mRNA and protein levels were the conversion of corticosterone into aldosterone and DOC. similarly enhanced for both diets used. Similarly, a number of investigators have documented that sodium depletion also increased activitiesof enzymes responsible for the early (14-16) and late steps (14-19) of steroidogenesis. The biosynthesis of aldosterone in the adrenal involves the In this study, we present conclusive evidence and confirm participation of various cytochrome P-450 enzymes. Cholesour preliminary reports (20, 21), indicating that dietary so* This work was supported by Grant MT-4653 from the Medical dium restriction or potassium loading in rats enhanced the Research Council of Canada. The costs of publication of this article levels of ZGadrenal mRNAsfor cytochromeP-450,,,, P-450,21, 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 solelyto indicate this fact. 5 Recipient of a studentship from the Canadian Heart Foundation. 11 T o whom all correspondence should be addressed: Dept. of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada.
The abbreviations used are: P-450,,, cholesterol side-chain cleavage cytochrome P-450; P-45OC2,,steroid 21-hydroxylase cytochrome P-450; P-450118, steroid llp-hydroxylase cytochrome P-450; DOC, 11deoxycorticosterone; ZG, zona glomerulosa; ZFR, zona fasciculatareticularis; PRA, plasma renin activity; AII, angiotensin 11; kb, kilobases.
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In Vivo Regulation of Aldosterone Synthesis
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and P-45OIl8, as well as for adrenodoxin. Furthermore, we sodium-deficient or high potassium diets, and remained elehave found that proteinlevels of these enzymes were concom- vated throughout the treatmentperiod (Fig. 1, center panel). itantly enhanced along with theirrespective mRNAs. Among Plasma corticosteroid levels (Fig. 1, upperpanel) were elevated all enzymes studied, the level of adrenal ZG P-45Ollp mRNA in both groups of rats but to a lesser extent than those of appears tobe the most up-regulated,especially during sodium plasma aldosterone. In general, the low sodium diet seemed depletion. t o have a greater effect than the high potassium diet on the two above mentionedparameters.Plasmareninactivity EXPERIMENTALPROCEDURES (PRA) was found to be markedly elevated in Na2+-depleted Animals and Diets-Male Long Evans rats (250 g) were obtained rats with significant differences after 2 days (17.9 ng of from our local breeding colony. "Sodium-depleted rats" were fed a angiotensin I/ml/h) and progressive increases up to the 7th low sodium diet (
