Peripheral and central angiotensin II regulates of genes of the renin-angiotensin system KATSUHIKO KOHARA, CARLOS M. FERRARIO,

expression

K. BRIDGET BROSNIHAN, AND AMY MILSTED

Department of Brain and Vascular Research, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 Kohara, Katsuhiko, K. Bridget Brosnihan, Carlos M. Ferrario, and Amy Milsted. Peripheral and central angiotensin II regulates expression of genes of the renin-angiotensin system. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E651E657, 1992.-We investigated whether angiotensin (ANG) II has the potential to regulate expressionof genesof the reninangiotensin system (RAS) in peripheral and central tissues. ANG II (0.1 or 6.0 nmol/h) was infused by osmotic minipump into male Sprague-Dawley rats (225-250 g) for 5 days, either intravenously or intracerebroventricularly. We measuredangiotensinogenmRNA in liver, adrenalglands,and brain (hypothalamus and lower brain stem), renin mRNA in the kidney, and angiotensin-converting enzyme (ACE) mRNA in the lung and testis by Northern blot analysis.We demonstratedthat plasma ANG II increasesthe levels of liver angiotensinogenmRNA, decreaseskidney renin mRNA, and decreaseslung ACE mRNA. Intracerebroventricular administration of ANG II resulted in a different pattern of responses of the peripheral RAS components.Liver angiotensinogenmRNA was increased,and kidney renin mRNA was decreasedby both dosesof ANG II, whereas lung ACE mRNA remained unresponsive at either dose.Centrally mediated influences of ANG II are most likely indirect since plasmaANG II concentration was not changed. This study has revealed that ANG II has profound diverse effects that influence the regulation of its formation. Further, results indicate that genesof the RAS respondedto exogenous ANG II in both tissue- and route-specific ways. messenger ribonucleic acid; angiotensinogen;renin; angiotensinconverting enzyme; generegulation; peptide infusions RENIN-ANGIOTENSIN SYSTEM (RAS) plays a major role in both hemodynamic and fluid balance homeostasis (9). Angiotensinogen is processed to angiotensin (ANG) I by the proteolytic enzyme renin. The further action by angiotensin-converting enzyme (ACE) yields the bioactive peptide ANG II. Negative feedback by ANG II on plasma renin activity (PRA) and renin release from juxtaglomerular cells (17) is a well-documented intrinsic mechanism for controlling production of the bioactive peptide. On the other hand, ANG II increases release of angiotensinogen from the liver (11, 28) and plasma levels of angiotensinogen (18), thereby exerting positive feedback regulation on angiotensinogen expression. Extensive evidence demonstrates that tissue RAS also play important roles as autocrine or paracrine regulators of tissue functions (8, 9). With the recent introduction of molecular biology techniques, analyses of regulation of mRNA expression are providing unique and valuable information that furthers our understanding of the physiological modulations of tissue RAS (9). Regarding the regulatory role of ANG II in the RAS, ANG II has been shown to exert positive actions on liver angiotensinogen mRNA (27) and negative effects on kidney renin mRNA (14, 27). However, no study to date has

THE

0193~1849/92

$2.00

examined the regulatory roles of ANG II on mRNA levels in tissues other than liver and kidney. Because tissues have their own components of the RAS independent of circulating RAS (8), investigation of the effects of ANG II on RAS mRNA expression in tissues provides new opportunities for understanding the molecular mechanisms at work in tissue RAS. Here, we applied molecular biological approaches to in vivo studies of peptidergic systems to begin to uncover the molecular mechanisms involved in regulation of expression of genes of the RAS. We examined the potential regulatory effects of exogenous ANG II on mRNA encoding the following three key components of the RAS: 1) angiotensinogen mRNA levels in liver, adrenal gland, and brain (hypothalamus and lower brain stem), 2) renin mRNA in kidney, and 3) ACE mRNA in lung and testis to explore whether ANG II regulates expression of those genes in a tissue-specific fashion. It is also well known that ANG II elicits different hemodynamic and humoral changes, depending on the route of its administration (10, 30). Therefore, we compared intravenous and intracerebroventricular administration of ANG II to determine whether route-specific modulations by ANG II extended to mRNA of the RAS in tissues. METHODS

AND

MATERIALS

Male Sprague-Dawleyrats (Hilltop Lab Animals, Scottsdale, PA) weighing 225-250 g were housedin individual cagesin a room maintained at constant temperature (22°C) with a 12:12-h dark-light cycle. Rats were allowedfree accessto tap water and were fed a standard rat chow (Purina Rat Chow, Fetzer Brothers, Bedford, OH). Animals were then placed into one of the following protocols: groups of six rats each received a 5-day intravenous infusion of either vehicle (0.9% NaCl) or ANG II given at dosesof 0.1 or 6.0 nmol/h. Another three groupsof rats were given 5-day infusions of either artificial cerebrospinalfluid [CSF (in mM) 133.3 NaCl, 3.4 KCl, 1.3 CaCl,, 1.2 MgC1,, 0.6 NaH2P04, 32 NaHC03, and 3.4 glucose(n = 8)] or ANG II into a lateral cerebral ventricle. ANG II was infused at either 0.1 nmol/h (n = 6) or 6.0 nmol/h (n = 8). Solutions were given via osmotic minipumps(model2001; Alza, Palo Alto, CA) that were calibrated to deliver the agentsat a rate of 1 pi/h. Surgical procedures. Under light halothane anesthesia,a polyethylene tube (PE-10 connected to PE-60; Clay Adams, Parsippany, NJ) wasplacedinto a jugular vein. The free end of the catheter wasconnectedto an osmotic minipump implanted within the subcutaneoustissueat the level of the interscapular region. Brain cannulaswere inserted into a lateral cerebralventricle of rats anesthetizedwith pentobarbital sodium (30 mg/kg ip). A 25-gaugestainless-steelneedlewas bent at a right angle; one end was connectedto a polyethylene tube while the beveled end was inserted into the brain ventricle using the following coordinates:0.8 mm posterior, 1.5 mm lateral from the bregma, and 3.8 mm ventral to the surfaceof the skull. The catheter was

Copyright 0 1992 the American Physiological

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E651

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fixed to the skull with dental cement and screws.The polyethylene end of the catheter was advanced into the interscapular region subcutaneously and was connected to an osmotic minipump. All surgical procedures were done under sterile conditions. Experimental protocol. On the 4th day after commencement of the infusion of either vehicle or ANG II, an arterial catheter was implanted into the abdominal aorta through a femoral artery under light halothane anesthesia.Baseline measuresof mean arterial pressure (MAP) and heart rate (HR) were obtained 24 h later. Arterial pressurewas recordedwith a solid strain transducer (MP-150; Micron Instruments, Los Angeles, CA) on a multichannel polygraph (model 2000; Gould, Cleveland, OH) in consciousfreely moving animals. Blood samples werewithdrawn through the arterial catheter for determination of plasmaangiotensinogen,PRA, and plasma ANG II immunoreactivity (irANG II). Rats were then killed quickly by decapitation, and the following tissueswere collected: liver, adrenal glands, kidney, lung, testis, hypothalamus, and lower brain stem. Tissue sampleswere snap-frozen on dry ice and stored at -80°C until RNA extraction. Biochemical determination. For plasmaangiotensinogenand PRA determinations, 1 ml of blood was collected into a prechilled glass tube containing 50 ~1 of 0.5 M NH,-EDTA solution. For plasmaANG II determination, 2 ml of blood were withdrawn into a prechilled glasstube containing 100~1of the following inhibitor cocktail solution (in mM): 25 NH,-EDTA (final concn in blood-collecting-tube), 0.44 o-phenanthroline, 0.12pepstatin A, and 14-chloromercuribenzoic acid. This cocktail was previously shown to eliminate both generation and degradationof ANG peptidesduring blood-samplingprocedures (20). After centrifugation at 4”C, plasmawas frozen on dry ice and stored at -20°C until assay.Plasma angiotensinogenand PRA were determined by radioimmunoassay(RIA) for ANG I generated,as reported previously (33). PlasmairANG II levels were measuredby a sensitive RIA describedin detail elsewhere (19). Sensitivity of the ANG II RIA is 0.5 pg, and intra- and interassayvariation of ANG II measurementsare 5.0 and 9.0%, respectively. Preparation of a- 32P-labeled probes. Hybridization probes were labeled with [a- 32P]dATP using a nick-translation kit from Boehringer Mannheim (Mannheim, FRG) to a specific activity of 3 x lo8 disintegrations min-’ l pg-l with the following cDNA: rat angiotensinogencDNA, rat renin cDNA, human endothelial ACE cDNA, and HeLa ,&actin cDNA. Total cellular RNA was extracted by the guanidine isothiocyanate-cesiumchloride procedure describedelsewhere(29). In brief, the tissueswere homogenizedin 5 M guanidine thiocyanate, 50 mM tris(hydroxymethyl)aminomethane HCl, 2 mM EDTA, 0.5% N-lauryl sarcosine,and 2 M CsCl solution. The homogenatewas placed on a cushion of 5.7 M CsCl in 0.1 M EDTA, pH 7.5, followed by centrifugation at 180,000g for at least 5 h. The pelleted RNA was resuspendedin 5 mM sodium citrate, 5 mM EDTA, and 1% sodiumdodecyl sulfate (SDS), pH 7.3. The resuspendedRNA was extracted with chloroform-lbutanol (4:1), followed by ethanol extraction. RNA was resuspendedin H,O and stored at -20°C. RNA concentrations were quantitated by absorption at 260 nm. Northern blot hybridization analysis. Electrophoresisof 20 pg total cellular RNA from each preparation was carried out in 0.8% agarose gels containing borate buffer and 3% formaldehyde. After electrophoresis,RNA was transferred to Gene Screen (New England Nuclear, Boston, MA). The blots wereprehybridized for at least 1 h at 42°C. Hybridization buffer wascomprisedof 50% deionized formamide, 0.02 M piperazineN,N’-bis(2-ethanesulfonic acid), 0.8 M NaCl, 2 mM EDTA, 100 pg/ml of denatured salmonspermDNA, and 0.5% SDS. Hybridl

BY ANG II

izations wereperformed for 12-18 h at 42°C with labeledcDNA probe (1 x lo6 counts min-’ ml-l of hybridization buffer). To remove the unhybridized probe, nylon membraneswere washedsuccessivelyin x2, xl, and x0.1 SSC. All washescontained 0.1% SDS. Stringencies of washesfor each probe were 60°C for angiotensinogencDNA and 50°C for renin, ACE, and @-actincDNA. Blots were exposedto XAR-5 X-ray film (Eastman Kodak, Rochester, NY) with two intensifying screens (Cronex; E.I. du Pont de Nemours,Wilmington, DE). Radiolabeled standards (ARC-146 carbon-14 standards; American RadiolabeledChemicals,St. Louis, MO) were included to determine relative optical densities (OD). Several exposureswere obtained to achieve optimal autoradiography signalsthat could be visualized within the rangeof optical gray densitiesprovided by carbon-14 standards.Video densitometry of autoradiographs wasperformed with the Image 1.28 analysisprogram (National Institutes of Health). For quantitation of relative levels of expression of mRNA, autoradiographic signals for angiotensinogen,renin, and ACE mRNA were standardized to signals determinedfrom unregulated“housekeeper”@-actinmRNA (23, l

25). Statistical

analyses. All valuesare expressedas meansk SE. One-way analysisof variance was usedto evaluate the effect of ANG II administration within intravenous or intracerebroventricular groups. Duncan’s multiple-range test was applied to determine the difference from vehicle-treated rats. Two-way analysisof variance wasemployedto assess differencesbetween intravenous and intracerebroventricular ANG II administration. The criterion for the statistical significancewasP < 0.05. Relationshipsbetween humoral factors and mRNA levelswere analyzed with univariant correlation analysis. RESULTS

Effect of ANG II on hemodynamic variables. Table 1 shows that the low-dose intravenous infusion of ANG II did not cause any significant changes in MAP or HR. In contrast, intravenous infusion of ANG II at a dose of 6.0 nmol produced increases in MAP of a magnitude that was comparable to that elicited by the administration of the high dose of ANG II into a brain ventricle. An intermediate elevation of blood pressure was found with the low dose of intracerebroventricular ANG II. In addition, ANG II given intracerebroventricularly caused increases in HR. Peripheral humoral changes. Intravenous infusion of

Table 1. Effects of 5-day infusions of ANG II

l

Intravenous

Cerebroventricular

MAP, mmHg

HR, beats/min

n

Vehicle

105t2

389k6

6

ANG II, nmol 0.1

105t3

385&14

6

141t4*

360t12

6

6.0

MAP, mmHg

102*2 (NW

HR beats/min

392t14 (NW

n

8

116&3* 433&7* 6 (P c 0.05) (P < 0.05)

136*4* 436t7” 8 (P < 0.01) W) Values are means t SE; n, no. of rats. Effect of 5-day infusion of angiotensin II (ANG II) on mean arterial pressure (MAP) and heart rate (HR) of conscious Sprague-Dawley rats. NS, not statistically different. *P < 0.01 compared with its own vehicle control. Both responses of MAP and HR to ANG II administration were significantly different between route of administration by two-way analysis of variance (P c 0.05 and P < 0.01, respectively). P values in parentheses are statistical differences compared with rats administered ANG II intravenously.

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E653

.--.I uose-uepenuem -1--- J~.~~.~J~..L uecreases J--..----- m 0.. rnA nil A anu -. -1 ANG II produceu increases in plasma angiotensinogen concentrations. Plasma irANG II was unchanged by the infusion of the lower ANG II dose but increased >lO-fold after intravenous administration of the higher dose of ANG II (Fig. 1). Cerebroventricular administration of the same doses of ANG II was associated with equivalent reductions in PRA. Plasma angiotensinogen concentrations increased in a dose-dependent manner after central administration of ANG II. In contrast, intracerebroventricular administration of ANG II did not change plasma ANG II levels at either dose. Effect of ANG II on expression of mRNA in peripheral and central tissues. Representative examples of Northern

blot analyses of angiotensinogen, renin, and ACE mRNA (Fig. 2) confirmed the size of angiotensinogen mRNA as 1.8 kilobases (kb) and that of renin mRNA as 1.6 kb. These hybridization patterns and sizes of mRNA species agree with those reported previously (4, 9, 16). Rat lung ACE mRNA separated on gel electrophoresis with a size of 4.3 kb, whereas rat testicular ACE mRNA was 3.0 kb in length. These findings agree with the sizes of human endothelial and testicular ACE mRNA reported by others (31). Blots were subsequently hybridized with P-actin cDNA. This procedure allowed each specific mRNA signal to be normalized relative to the amount of total RNA actually present in each sample. In our studies, for quantitation Iof relative levels of or rej nin mRNA, autoraexpression of angiotensinogen

Ye”trlC”l&r Fig. 1. Plasma renin activity, plasma angio tensinogent and plasma IrltraYeAOUS

Cerebra

immunoreactive aneiotensin (ANG II) after 5 clays of either intravenous or intracerebroventricular infusion of vehicle ‘---(open I‘-rs), oa. 0.1 nmol/h (hatched ham). or 6.0 nmol/h of ANG TT (filled- bars). _-._, VrJues are means with vehicle-treated rats.

Fig. 2. Representative examples of Northern blot analvsis. A: aneiotensinogen mRNA in adrenal gland after vehicle (V), low dose (0.1 nmol/h; L), or high dose (6.0 nmol/h; H) of ANG II administered centrally into different groups of rats (20 pg of RNA in H). ANG-converting enzyme (ACE) mRNA from lung (C) and testis (D) in rats administered ANG II centrally. Sizes of RNA markers are indicated. Bottom blot in each panel shows p-actin mRNA levels obtained from same blot.

diographic signals for angiotensinogen and renin were standardized to signals measured for fi-actin mRNA in each preparation to control for amounts of RNA loaded per lane. Under most circumstances, fl-actin mRNA expression does not vary significantly (l), and it is commonly used as an unregulated mRNA (26). However, as shown recently by Turla et al. (34), treatment of smooth muscle cell cultures with ANG II (1 PM) can increase levels of P-actin mRNA by as much as twofold (34). As part of another study in our laboratory in the aorticligated model of hypertension, where endogenous plasma ANG II levels are high, we calculated the expression of P-actin mRNA as relative OD units per milligram of tissue in sham-operated animals and in hypertensive animals. Six days after aortic ligation, when endogenous levels of plasma ANG II were elevated by approximately sevenfold, OD units of actin per milligram tissue were unchanged (M. Nishimura, C. M. Ferrario and A. Milsted, unpublished observations). In Fii. 3, changes in angiotensinogen mRNA levels in peripheral tissues-are shown. Intravenous ANG II administered at the dose of 6.0 nmol significantly increased angiotensinogen mRNA in the liver. In adrenal glands both intravenous ANG II doses reduced angiotensinogen mRNA levels. Central administration of ANG II at both doses elicited a significant increase in angiotensinogen mRNA in the liver and the adrenal glands. Changes in adrenal gland angiotensinogen mRNA were significantly

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different and opposite in direction between intravenous and intracerebroventricular administration by two-way analysis of variance [F(2,30) = 12.15, P < O.OOl]. Changes in brain angiotensinogen mRNA levels in ANG II-treated rats are expressed relative to those in vehicle-treated rats in Fig. 4. Peripheral ANG II administration did not alter the expression of angiotensinogen mRNA in either hypothalamus or the lower brain stem.

Intravenous

Cerebroventricular

Intravenous

Cerebroventricular

300 200 3 ; 6

100

E

0

-50 Fig. 3. Changes in angiotensinogen mRNA levels in liver (A) and adrenal gland (23) after 5 days of intravenous or intracerebroventricular infusion of 0.1 nmol/h (hatched bars) or 6.0 nmol/h of ANG II (filled bars). Changes are expressed as relative changes to those of vehicletreated rats. Values are means k SE. * P < 0.05 and ** P < 0.01 when compared with vehicle-treated rats.

% t"(0 6 E

25 0 -25

I

t t

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Intravenous

Cerebroventricular

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II

Likewise, intracerebroventricular administration of ANG II had no significant effect on the expression of angiotensinogen mRNA in the hypothalamus (+30.0 t 18.4 and +19.4 t 9.6%, low-dose and high-dose groups, respectively) or in the lower brain stem [ +21.4 t 7.2 and +33.1 t l&9%, low-dose and high-dose groups, respectively, = 2.70, P < 0.11. The absence of a statistical w,w difference may be due to the relatively small number of animals included in the sample since reanalysis combining rats treated with both doses of ANG II revealed that the expression of angiotensinogen mRNA was significantly increased in the lower brain stem [+28.1 t 9.44% as compared with vehicle-treated animals, F( 1,20) = 4.94, P < 0.051 but not in hypothalamus [+23.9 t 9.3% of vehicle-treated animals, F(1,20) = 3.7, P < 0.071 of rats given intracerebroventricular ANG II. The same analysis yielded no significant differences in angiotensinogen mRNA levels in brains of rats given intravenous infusions of ANG II. Changes in renal renin mRNA in response to peripheral or central administration of ANG II are illustrated in Fig. 5. Renin mRNA was significantly reduced by the higher dose of ANG II administered peripherally. After intracerebroventricular administration, both doses of ANG II significantly decreased the expression of renin mRNA in the kidney. ACE mRNA expression was examined in lung and testis (Fig. 6). Five days of peripheral administration of ANG II significantly decreased ACE mRNA expression in the lung. The low dose of ANG II was as effective as the high dose. On the other hand, intracerebroventricular administration had no effects on the expression of the ACE mRNA in the lungs. In testis, neither route of ANG II administration altered ACE mRNA levels. ACE mRNA expression in testis was -1.7 t 9.8 and -9.7 t 11.8% for low-dose and high-dose intravenous ANG IItreated rats and -9.9 t 18.4 and -12.7 t 15.1% of the vehicle-treated rats by low-dose and high-dose intracerebroventricular ANG II administration. Correlation analysis revealed a positive correlation between plasma irANG II and liver angiotensinogen mRNA (r = 0.65, P < 0.01) and a negative correlation between plasma irANG II and kidney renin mRNA (r = -0.70, P < 0.01) in intravenous ANG II-infused rats (n = 17). Positive correlations were also found between PRA and angiotensinogen mRNA in hypothalamus (r = 0.57, 0

z : 6 be ;

25 0 -25 -5o

1

t 1

-90 Intravenous

Cerebroventricular

Fig. 4. Changes in angiotensinogen mRNA levels in hypothalamus (A) and lower brain stem (medulla oblongata; B) after 5 days of intravenous or intracerebroventricular infusion of 0.1 nmol/h (hatched bars) or 6.0 nmol/h (filled bars) of ANG II. Changes are expressed as relative changes to those of vehicle-treated rats. Values are means t SE.

Intravenous

Cerebroventricular

Fig. 5. Renin mRNA levels in kidney after 5 days of intravenous or intracerebroventricular infusion of 0.1 nmol/h (hatched bars) or 6.0 nmol/h of ANG II (filled bars). Changes are expressed relative to changes in vehicle-treated rats. Values are means t SE. ** P < 0.01 when compared with vehicle-treated rats.

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A

-60

Intravenous

Cerebroventricular

Intravenous

Cerebroventricular

n 40 D I

-60' Fig. 6. ACE mRNA levels in lung (A) and testis (B) after 5 days of intravenous or intracerebroventricular infusion of 0.1 nmol/h (hatched bars) or 6.0 nmol/h (filled bars) of ANG II. Changes are expressed relative to those of vehicle-treated rats. Values are means k SE. * P < 0.05 when compared with vehicle-treated rats.

P < 0.05), adrenal gland (r = 0.52, P < 0.05), kidney renin mRNA (r = 0.71, P < O.Ol), and lung ACE mRNA (r = 0.58, P < 0.05) in rats given intravenous infusions of ANG II or vehicle (n = 16). Between PRA and liver

angiotensinogen mRNA, a negative correlation was found (r = -0.56, P < 0.05). MAP correlated positively with plasma irANG II (r = 0.86, P C O.Ol), plasma angiotensinogen (r = 0.57, P < 0.05), and liver angiotensinogen mRNA (r = 0.62, P < 0.01) whereas negative correlations were shown with PRA (r = -0.59, P < 0.05) and kidney renin mRNA (r = -0.84, P < 0.01, n = 17). Taken together, these correlations are consistent with the notion that effects of peripherally infused ANG II result from direct regulatory mechanisms. In contrast, in rats infused intracerebroventricularly with ANG II, analysis revealed a total lack of correlation of either PRA or plasma irANG II with changes in mRNA expression, except for kidney renin mRNA (PRA with renin mRNA: r = 0.77, P < 0.01; irANG II with renin mRNA: r = 0.51, P C 0.05, n = 16). These data support the concept that intracerebroventricularly infused ANG II acts through indirect mechanisms. DISCUSSION

These studies showed a differential effect of varying endogenous plasma and CSF concentration of ANG II on the expression of angiotensinogen mRNA in peripheral and central organs of normal rats. A key finding of this study is that intracerebroventricular infusion of ANG II was a potent stimulus of the expression of angiotensinoen mRNA in both the periphery and the brain. These effects were not associated with changes in plasma levels of ANG II, suggesting either that circulating levels of ANG II are not true indicators of the regulatory effec-

BY ANG

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tiveness of ANG II or that the observed changes are the results of central actions of ANG II. We favor the latter possibility because the association between increased expression of angiotensinogen mRNA and elevated blood pressure after central infusion of ANG II illustrates the apparent ability of brain ANG II to indirectly affect regulation of its own production in peripheral tissues. The liver is the main source of circulating angiotensinogen (4). Our finding that intravenous ANG II had a positive regulatory effect on liver angiotensinogen mRNA is consistent with a previous report (27). Moreover, our studies suggest that the effects of ANG II on angiotensinogen mRNA are dose dependent. Centrally administered ANG II increased both plasma angiotensinogen and liver angiotensinogen mRNA expression but not plasma levels of irANG II. These findings suggest that the mechanisms that account for the effects of intracerebroventricular ANG II are unrelated to direct actions of the peptide on expression of the angiotensinogen gene. This interpretation is consistent with the observation that central administration of ANG II causes marked rises in plasma cortisol (lo), which in turn may directly stimulate transcription of the angiotensinogen gene through glucocorticoid-responsive elements present in the regulatory region of this gene (4). Several studies have shown that glucocorticoids increase angiotensinogen mRNA in liver (4,16). In our experiments, both the low and high dose of ANG II stimulated the expression of angiotensinogen mRNA and decreased PRA. The two routes of ANG II administration had strikingly different effects on angiotensinogen mRNA expression in the adrenal glands. Adrenal angiotensinogen mRNA expression was increased by central infusions of ANG II. Concurrent measurements of plasma irANG II ruled out that these changes were induced by leakage of the peptide into the peripheral circulation. Therefore, we interpret these findings as suggesting the participation of other humoral factors in the control of adrenal gland angiotensinogen synthesis. Likely candidates include enhanced release of cortisol or increased sympathetic drive (10). On the other hand, the reduction of adrenal angiotensinogen mRNA expression by peripherally administered ANG II suggests that ANG II exerts a direct negative feedback action on adrenal angiotensinogen mRNA levels. In situ hybridization histocytochemistry has revealed that angiotensinogen mRNA is located in periadrenal fibroblast-like cells and brown adipose tissue, but whether or not it is actually contained with cells of the adrenal cortex, medulla, capsule, or vessels is not yet clear (3). Further study will be necessary to elucidate the mechanisms of ANG II regulation of adrenal angiotensinogen mRNA. To examine the molecular aspects of the role of brain ANG II on the regulation of angiotensinogen, we evaluated the action of the peptide on the expression of angiotensinogen mRNA in those areas of the brain that contain the pathways that participate in the central control of blood pressure (10). Others showed that the hypothalamus and the medulla oblongata are rich in angiotensinogen mRNA (22). Our studies showed that central but not peripheral infusions of ANG II augmented the

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expression of angiotensinogen mRNA in the lower brain stem. Centrally administered ANG II may reach both the hypothalamus and brain stem tissues, whereas peripherally administered ANG II accesses only those areas that are outside the blood-brain barrier (10). These differences may bear on the results obtained in our studies. Differences in regulation of the central vs. peripheral RAS have been shown by several other studies (10, 24, 32). In the present study, we showed that brain angiotensinogen mRNA expression changed less than liver angiotensinogen mRNA in response to exogenous ANG II. The stimulatory action of brain ANG II may result from a direct effect of the peptide on the angiotensinogen gene. Alternatively, it may be caused by stimulation of cortisol, catecholamines, or both (10, 12). Previous studies showed that brain angiotensinogen mRNA is increased by glucocorticoids (6, 16). Although inflammation (15) and bilateral nephrectomy (13) are conditions that result in increased liver angiotensinogen mRNA, these maneuvers had no effect on the expression of brain angiotensinogen mRNA. Differences in the expression of angiotensinogen mRNA among these experimental situations would suggest that brain angiotensinogen mRNA is under more complex regulation than liver angiotensinogen mRNA. Kidney renin and the mRNA encoding it are regulated by mechanisms that include the intrarenal baroreceptor (5), sympathetic nerves (5, 17, 18), and sodium intake (5, 17). Recently, Nakamura et al. (27) found that a shortterm systemic infusion of ANG II significantly decreased renin mRNA in the kidney. In the current study, we observed that the effect of 5-day intravenous ANG II infusion on renal renin mRNA expression was dependent on the dose of ANG II. Only the high dose of ANG II caused a significant decrease in renin mRNA expression. Although the low dose of ANG II administered intravenously had no detectable effect on kidney renin mRNA, it did significantly decrease PRA. This indicates that a low dose of circulating ANG II is more effective at a posttranscriptional level, such as secretion, than at the transcriptional level. A low dose of intracerebroventricular ANG II was as effective as the high dose of ANG II infused intravenously in decreasing kidney renin mRNA. Our results suggest that ANG II given intracerebroventricularly exerts effects on kidney renin mRNA regulation by indirect mechanisms, such as the intrarenal baroreceptor or the macula densa. Both of these mechanisms are known to regulate renin release (5). Another possible explanation for the effect of centrally infused ANG II on renal renin mRNA is that ANG II administered intracerebroventricularly stimulated centrally mediated pathways to suppress renin release (2). In our study, we detected two species of rat ACE mRNA in lung and testis, using a human ACE cDNA probe. This is in agreement with earlier observations that two forms of ACE isozymes exist in endothelial cells and testis (9, 31). By cloning and sequencing of ACE cDNA, it has been demonstrated that the single ACE gene is transcribed into two species of mRNA, a 4.3-kb mRNA in vascular endothelial cells and 3.0-kb mRNA in testis (31). We observed that peripherally administered ANG II decreased ACE mRNA in the lung but had no effect on

BY ANG II

the testicular ACE mRNA. We also demonstrated that centrally infused ANG II had no effect on either lung or testicular ACE mRNA. These results suggest that circulating ANG II regulates ACE mRNA expression in endothelial tissues, whereas central ANG II had neither direct nor indirect effects on endothelial ACE mRNA. In addition, because ACE has other substrates in addition to ANG I (such as bradykinin), additional regulatory effects may be accounted for by other biologically active peptide systems. The findings in our study also verify that testicular ACE mRNA is under separate control from lung ACE mRNA. From studies of structure-function relationships of the lung and testicular ACE mRNA transcripts and their promoters, it is now known that transcription of the two ACE mRNAs is regulated differently (21, 31). Another likely explanation is that the blood-testis barrier may prevent ANG II access to the testis (7), analogous to the blood-brain barrier that prevents circulating ANG II from modulating angiotensinogen mRNA expression in those brain regions within the blood-brain barrier (Fig. 3). In summary, chronic administration of ANG II either peripherally or centrally produced changes in mRNA levels of each of the significant RAS genes involved in its formation in liver, kidney, adrenal glands, lung, and brain. ANG II either directly or indirectly regulated expression of three key components of the RAS in tissuespecific and route-dependent manners. These results suggest that both circulating and central ANG II have influences on the expression of mRNA of the tissue RAS. This study reveals multiple-regulated components of the RAS that interact to result in finely tuned control of production of biologically active ANG II. Clones for rat angiotensinogen cDNA (pRAng 6) and rat renin cDNA (pRen 44.ceb) were generous gifts from Dr. K. R. Lynch (Charlottesville, VA). The clone for human endothelial ACE cDNA (pB35-19) was kindly provided by Dr. P. Corvol (Paris, France). HeLa fl-actin cDNA was a generous gift from Dr. J. H. Nilson (Cleveland, OH). We thank Laura L. Yoho for expert technical assistance. This work was supported in part by National Heart, Lung, and Blood Institute Grant POl-HL-6835, Grant-in-Aid no. 891061 from the American Heart Association, National Center, and by the George Storer Foundation of Miami, FL. Address for reprint requests: A. Milsted, Dept. of Brain and Vascular Research, Research Institute, NC-3, The Cleveland Clinic Foundation, 9500 Euclid Ave., 1 Clinic Ctr., Cleveland, OH 44195. Received 28 May 1991; accepted in final form 25 November 1991. REFERENCES 1. Anderson, B., A. Milsted, G. Kennedy, and J. H. Nilson. Cyclic AMP and phorbol esters interact synergistically to regulate expression of the chorionic gonadotropin genes. J. Biol. Chem. 263: 15578-15583, 1988. 2. Brosnihan, K. B., and C. M. Ferrario. Central regulation of renin release. In: Hypertension and the Brain, edited by G. P. Guthrie, Jr., and T. A. Kotchen. New York: Futura, 1984, p. 83-112. in situ 3. Campbell, D. J., and J. F. Habener. Hybridization studies of angiotensinogen gene expression in rat adrenal and lung. Endocrinology 124: 218-222, 1989. 4. Clauser, E., I, Gaillard, L. Wei, and P. Corvol. Regulation of angiotensinogen gene. Am. J. Hypertens. 2: 403-410, 1989. 5. Davis, J. O., and R. H. Freeman. Mechanisms regulating renin release. Physiol. Reu. 56: l-56, 1976. 6. Deschepper, C. F., and M. Flaxman. Glucocorticoid regulation of rat diencephalon angiotensinogen production. Endocrinology 126: 963-970, 1990.

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Peripheral and central angiotensin II regulates expression of genes of the renin-angiotensin system.

We investigated whether angiotensin (ANG) II has the potential to regulate expression of genes of the renin-angiotensin system (RAS) in peripheral and...
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