Vol. 179, No. 3, 1991

September 30, 1991

BIOCHEMICAL AND EIIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 1361-l 367

CHANGES IN EXPRESSION OF ANGIOTENSIN RECEPTOR SUBTYPES IN THE RAT AORTA DURING DEVELOPMENT

Mohan Viswanathan, Keisuke Tsutsumi, Fernando M.A. Correa, and Juan M. Saavedra

Section on Pharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bldg. 10; Rm. 2D-45, 9000 Rockville Pike, Bethesda, MD 20892

Received July 29, 1991

Quantitative autoradiography was used to characterize angiotensin AT, and AT, receptors, in the rat aorta at three developmental ages; embryonic day 18 (El8), and postnatal weeks 2 and 8. The expression of angiotensin receptors was higher in the aorta of El8 and 2-week-old rat. A major proportion of the angiotensin receptors expressed in the aorta at these two ages was AT, (84 and 81% respectively). Conversely, in the aorta of 8-week-old rats, AT, was the predominant angiotensin receptor subtype (71%). In 8-week-old rats, the AT, subtype was also present (28%). In pre- and postnatal rats, [‘251]Sar’-angiotensin II binding to AT, receptors was sensitive to GTPyS whereas binding to AT, receptors was not. AT, receptors may serve an important role during stages of rapid growth of the aorta, and also have a significant function in the adult vasculature. 0 1991 Academic Press, Inc.

Vasoconstriction, mediated through specific angiotensin (AT) receptors in the vascular smooth

muscle, is one of the well recognized physiological functions of angiotensin II (ANG) (1). AT receptors have

recently been classified into two distinct subtypes, AT, and AT,, based on their affinities for peptidic and

nonpeptidic ligands and also by their differential sensitivities to dithiothreitol (2). Vascular AT receptors are

currently believed to be exclusively AT,. Rat aortic smooth muscle cells maintained in culture express only

AT, receptors (3,4), and the AT, antagonist, DuP 753, inhibits ANG-induced contraction in the rabbit aorta

(5,6). We have used quantitative autoradiography to study AT receptors in the aorta of fetal (El 8) young

(2-week-old) and adult (8-week-old) rats. We report that both subtypes of AT receptors are present in the

rat aorta, and that during development, there is a dramatic change in the expression of AT, and AT, receptors

in this organ.

MATERIALS AND METHODS

Animals Sprague-Dawley rats from Zivic-Miller (Alliston Park, PA) were used in the study. Pregnant females, 1 O-day-old male pups along with lactating females, and 7-week-old males were maintained in a 12:12 h light-dark cycle. Food and water were provided ad libitum. Rat fetuses were removed from 18day pregnant females after decapitating them. Fetuses were decapitated and frozen in isopentane at -30 “C. Pieces of descending (thoracic) aorta (1 cm long) were taken from 2 and 8-week-old males killed by decapitation, and were also frozen in isopentane. The tissues were kept frozen at -70 ‘C.

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Vol. 179, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Bindina Assays Cross sections (16 pm) of the fetus at the level of the thorax containing ascending and descending aorta, and cross sections of the aorta from 2 and B-week-old rats (referred to as young and adult, respectively) were cut in a cryostat at -17 ‘C, thaw mounted on gelatin-coated glass slides and dried overnight in a desiccator at 4 ‘C.

The binding conditions used have been described (7,8). The AT agonist, [‘251]Sar’-ANG (Peninsula, Belmont, CA; iodinated by New England Nuclear, Wilmington, DE; specific activity, 2200 Ci/mmol) was used as the ligand. Tissue sections were preincubated for 15 min in 10 mM phosphate buffer (pH 7.4) containing 120 mM NaCI, 5 mM NqEDTA, 0.005% bacitracin (Sigma Chemical Co., St. Louis, MO), and 0.2% proteinase-free bovine serum albumin (Sigma), followed by incubation for 120 min in fresh buffer containing the appropriate concentration of the ligand. After incubation, the sections were washed 4 times, for 1 min each, in fresh ice-cold 50 mM Tris-HCI buffer (pH 7.6), followed by 30 set in distilled water at 0 ‘C.

To characterize ANG receptor subtypes (9), consecutive sections were incubated with [‘251]Sar’-ANG (5xlV” M) and increasing concentrations of ANG, AT, antagonist DuP 753 (2-n-butyl-4-chloro-5- hydroxymethyl-1-[2’(1 H-tetrazol-5-yl)biphenyl-4-yl-methyl]imidazole, from DuPont, Wilmington, DE) or AT, competitor PD 123177 [l-(4-amino-3-methyl-phenyl)methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-l H- imidazo[4,5-Clpyridine-6-carboxylic acid-2HCI, from Parke Davis, Ann Arbor, Ml).

The possible regulation of AT receptor subtypes by guanine nucleotide binding proteins was studied by incubating consecutive sections in the presence of increasing concentrations of a nonhydrolysable analogue of GTP, guanosine-5’-O-(3-thiotriphosphate) (GTPyS). These incubations were done while exposing the sections to either DuP 753 or PD 123177 (lo” M) in order to differentiate the effect of GTPyS on the specific receptor subtype. Preincubation and incubation buffers in these experiments consisted of 50 mM phosphate buffer (pH 7.4) containing 120 mM NaCI, 10 mM MgCI,, 0.005% bacitracin, and 0.2% proteinase- free bovine serum albumin. Quantitative Autoradioaraohv Sections were dried after washing and exposed to Hyperfilm-[3H] (Amersham Corporation, Arlington Heights, IL), along with 16 pm sections of [‘251]-labeled Micro-scale standards (Amersham). [‘251]Sar’-ANG binding was quantified as described using computerized microdensitometry (10). IC,, values were calculated with the GraphPad lnplot program (GraphPad, San Diego, CA).

RESULTS

Autoradiography revealed AT binding in the aorta of the fetal, young, and adult rats. ANG inhibited

[‘251]Sar’-ANG binding in a monophasic manner in the aorta at all three ages, with complete displacement at

1 O6 M (I&; fetus, 1.2 X 10.’ M; young, 3.6 X 10.’ M; adult, 3.2 X 10.’ M). Neither DuP 753 nor PD 123177

was able to completely inhibit ANG binding indicating the presence of more than one subtype of the receptor

in this tissue (Fig. 1). A major proportion of the binding in the aorta of fetus and young rats was displaced

s El8 150

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2 m -10 -8 -6 -4

2-Week-old 8-Week-old 150 150

100 100

50 50

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[Competitor]. log M

F~JJ. Competition of angiotensin II, DuP 753, and PD 123177 for specific [‘251]Sar’-ANG binding in the rat aorta. Competition curves were obtained from consecutive sections of aorta from El 8 fetuses, and 2 and 8-week-old rats, incubated in the presence of 5 X 10” M [‘251]Sar’-ANG and increasing concentrations of angiotensin II (circles) or AT receptor competitors, DuP 753 (squares) and PD 123177 (triangles). Results are expressed as the mean + S.E.M of groups of five rats, measured individually.

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Vol. 179, No. 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

El8 2 Heeks 8 Weeks El8 2 W&s E Heeks

Fiq. Angiotensin receptor subtypes in the rat aorta during development. A: apparent amount of AT receptors during development. AT, specific [‘251]Sar’-ANG binding; AT, and AT,, specific binding not displaced by 1W5 M PD 123177 or DuP 753, respectively. B: proportions of AT receptor subtypes in the aorta during development. Values are percentage of specific [‘?]Sar’-ANG binding.

by PD 123177, while that in the adult aorta was displaced by DuP 753. The apparent number of AT

receptors in the fetal aorta was 6 times higher than that in young rats and 20 times higher than that in adult

rats (Fig. 2A). The proportions of the two subtypes of AT receptors, when quantified, revealed that more than

60% of the receptors expressed in the aorta of the fetus and young rats and about 30% in the adult were AT,

(Fig. 28). That the rest of the AT binding was to AT, receptors was confirmed by the complete displacement

of binding when both DuP 753 and PD 123177 were used together (Fig. 3).

While AT, receptors showed a uniform distribution over the entire sonic wall of the fetal (Fig. 4),

young and adult rats, AT, receptors seemed to be restricted to the media layer (Fig. 5). This was especially

conspicuous in the aorta of adult rats (Fig. 5). The identification of the cell type(s) expressing these receptor

subtypes in the aortic wall was not possible with quantitative autoradiography because of the inherent

limitation in the resolution of the technique.

8-Week-old

'50t

100

50

0 P!k

G=T e -10 -8 -6 -4 0 -10 -8 -6 -4 0 -10 4 -6 -4

[PD 1231771 [PD 1231771 [DuP 7531

log M

Fiq. Competition of PD 123177 or DuP 753 for specific [“‘I]Sar’-ANG binding in rat aorta. In the aorta from El8 fetus and 2-week-old rats, AT, receptors were blocked by DuP 753 (10.’ M), whereas in 8- week-old rats AT, receptors were blocked by PD 123177 (IO’ M). Results are expressed as the mean f S.E.M. of groups of five rats, measured individually.

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lmm

F~J& Autoradiography of AT receptors in the fetal rat aorta. A: transverse section stained with hematoxylin and eosin. B-E: enlarged view (magnification X2) of area marked in A. B: stained with hematoxylin and eosin. C: consecutive section showing total ANG binding after incubation with 5 X 10” M [‘251]Sar’-ANG. D: consecutive section incubated as in C, in the presence of 1U5 M DuP 753. E: consecutive section incubated as in C, in the presence of IO5 M PD 123177. Arrow points to descending aorta, and arrow head to ascending aorta.

Incubation of sections with increasing concentrations of GTPyS decreased DuP-sensitive AT binding

(AT, receptors) from the fetal, young and adult aorta (IC,,; 3.8, 1.7, and 1 .O X 10.’ M respectively). PD

123177-sensitive AT binding (AT, receptors) was not affected by the guanine nucleotide (Fig. 6).

DISCUSSION

Characterization by autoradiography and displacement with selective competitors revealed for the

first time that the aorta contains both AT, and AT, receptors, and that in fetal and young rats, most of the AT

receptors belong to the AT, type. We also report that AT, receptors in the aorta at all ages are insensitive

to guanine nucleotides, a finding consistent with our observations in some of the other peripheral tissues of

the rat fetus (11). Aortic AT, receptors, like AT, receptors in other tissues (11,12) and AT receptors in

vascular smooth muscle cells in culture (13), are sensitive to guanine nucleotides, which indicates a possible

association to G-proteins (14), and that this association may already be present during the embryonic life.

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0.5mm Fiq. Autoradiography of AT receptors in young and adult rat aorta. A-E: consecutive sections of aorta from a 2-week-old rat. F-J: consecutive sections from an 8-week-old rat. A and F: sections stained with hematoxylin and eosin. 13 and G: Sections showing binding after incubation with 5 x 10 ” M [‘25f]Sar’- ANG (total binding). C and H: sectrons incubated as in B and G, in the presence of 1O-5 M DuP 753. D and I: sections incubated as In B and G, in the presence of 10 5 M PD 123177. E and J: sectrons incubated as in B and G, in the presence of 5 x 106 M ANG (nonspecific binding). Thin arrow pornts to tunica adventitia, and arrowhead to tunica media.

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Vol. 179, No. 3, 1991 BIOCHEMICAL AND BlOPHYSICAL RESEARCH COMMUNICATIONS

1 $c 150

F e

t 100 BP

E? G 2 50

2 .3 x ::

0 m -0 -7 -6 -5 -4 m -8 -7 -6 -5 -4

&Week-old

m -8 -7 -6 -5 -4

[GTPC5], log M

F~J& Effects of GTPy?? on ANG binding in rat aorta. Consecutive sections of El8 fetus and aorta from 2 and B-week-old rats were incubated with 5 X 10” M [‘251]Sar’-ANG in the presence of 1O-5 M DuP 753, to block AT, receptors (open circles) or 1 O5 PD 123177, to block AT, receptors (closed circles), and increasing concentrations of GTPyS. Results are expressed as the mean f S.E.M. of groups of five rats, measured individually.

The expression of AT receptors in the rat aorta is many times higher during fetal life than after birth,

and the AT receptor number is several times higher in immature, young animals when compared to adult rats.

Our findings are consistent with previous data of a marked activation of the renin-angiotensin system in the

late gestation fetus (1516) indicating a role for ANG during gestation. In cultured vascular smooth muscle

cells, ANG induces hypertrophy and/or hyperplasia (17) and stimulates the synthesis of type V collagen,

associated with cell growth and tissue structural remodeling (18). ANG has been suggested to mediate

vascular hypertrophy through a non-pressor related mechanism (19) and to promote vascular growth (20).

Most of the AT receptors in the aorta of the fetus and young rat belong to the AT, type. The functional

significance of AT, receptors, and specifically, the significance of the presence of large numbers of AT,

receptors in a rapidly growing structure, has not yet been clarified. However, their marked expression in fetal

aorta, and in other fetal tissues (11) indicates the possibility of a specialized role of AT, receptors during

organ development.

AT, receptors are responsible for the vascular contraction produced by ANG, since ANG-induced

aortic contractions can be completely inhibited by the AT, antagonist DuP 753 and not affected by AT,

competitor PD 123177 (5,6), and in hypertensive rats administration of DuP 753 reduces blood pressure (21).

In adult rats, the proliferative response to vascular injury is suppressed by angiotensin converting enzyme

inhibition (22), and ANG stimulates collagen synthesis (18). These data indicate participation of ANG in the

mechansims of vascular tissue repair. The hypertrophy of vascular smooth muscle produced by ANG is

antagonized by DuP 753 (23) suggesting the role of AT, receptors in this phenomenon. Our results

demonstrate, for the first time, that in addition of AT, receptors, AT, receptors are also expressed in the aorta

of the adult rat. Whether the role of ANG in vascular growth in adult life is solely dependent on stimulation

of AT, receptors, or depends on the interplay of AT, and AT, receptor stimulation, remains to be studied.

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5.

6.

7. 8. 9.

10. 11.

12.

13.

14. 15. 16. 17. 18.

19.

20.

21.

22.

23.

REFERENCES

Valloton, M.B. (1987) TIPS 8,69-74. Bumpus, F.M., Catt, K.J., Chiu, A.T., de Gasparo, M., Goodfriend, T., Husain, A., Peach, M.J., Taylor Jr., D.G., and Timmermans, P.B.M.W.M. (1991) Hypertension 17,720-721. Whitebread , S., Mele, M., Kamber, B., and de Gasparo, M. (1989) Biochem. Biophys. Res. Commun. 163,284-291. de Gasparo, M., Whitebread, S., Mele, M., Motani, AS., Whitcombe, P.J., Ramjoue, H.P. and Kamber, B. (1990) J. Cardiovasc. Pharmacol. 16,S31-S35. Wong, PC, Hart, SD., Zaspel, A.M., Chiu, A.T., Ardecky. R.J., Smith, RD., and Timmermans, P.B.M.W.M. (1990) J. Pharmacol. Exp. Ther. 255,584~592. Dudley, D.T., Panek, R.L., Major, T.C., Lu, G.H., Bruns, R.S., Klinkefus, B.A., Hodges, J.C., and Weishaar, R.E. (1990) Mol. Pharmacol. 38, 370-377. Tsutsumi, K., and Saavedra, J.M. (1991) J. Neurochem. 56,348-351. Tsutsumi, K., and Saavedra, J.M. (1991) Endocrinology 128:630-632. Chiu, A.T., Herblin, W.F., McCall, D.E., Ardecky, R.J., Carini, D.J., Duncia, J.V., Pease, L.J., Wong, P.C., Wexler, RR., Johnson, A.L., and Timmermans, P.B.M.W.M. (1989) Biochem. Biophys. Res. Commun. 165,196-203. Nazarali, A.J., Gutkind, J.S., and Saavedra, J.M. (1989) J. Neurosci. Meth. 30,247-253. Tsutsumi, K., Stromberg, C., Viswanathan, M., and Saavedra, J.M. (1991) Endocrinology (in press). Pucell, A.G., Hodges, J.C., Sen, I., Bumpus, F.M., and Husain, A. (1991) Endocrinology 128,1947-l 959. Griendling, K.K., Berk, B.C., Socorro, L., Tsuda, T., Delafontaine, P., and Alexander, R.W. (1988) Clin. Exp. Pharmacol. Physiol. 15,105-l 12. Birnbaumer, L. (1990) FASEB J. 4:3068-3078. Raimbach, S.J., and Thomas, A.L. (1990) J. Physiol. (Lond.) 423,441-451. Mott, J.C. (1975) Br. Med. Bull. 31,44-50. Schelling, P., Fischer, H., and Ganten, D. (1991) J. Hypertens. 9,3-15. Kato, H., Suzuki, H., Tajima, S., Ogata, Y., Tominaga, T., Sato, A., and Saruta, T. (1991) J. Hypertens. 9,17-22. Griffin, S.A., Brown, W.C.B., MacPherson, F., McGrath, J.C., Wilson, V.G., Korsgaard, N., Mulvany, M.J., and Lever, A.F. (1991) Hypertension 17,626-635. Le Noble, F.A.C., Hekking, J.W.M., Van Straaten, H.W.M., Slaaf, D.W., and Struyker Boudier, H.A.G. (1991) Eur. J. Pharmacol. 195,305-306. Wong, P.C., Price Jr., W.A., Chiu, A.T., Duncia, J.V., Carini, D.J., Wexler, R.R., Johnson, A.L., and Timmermans, P.B.M.W.M. (1990) Hypertension 15,459-468. Powell, J.S., Muller, R.K.M., Rouge, M., Kuhn, H., Hefti, F. and Baumgartner, H.R. (1990) J. Cardiovasc. Pharmacol. 16,S42-S49. Smith, R.D., Chiu, A.T., Roscoe, W.A., McCall, D.E., and Timmermans, P.B.M.W.M. (1990) Eur. J. Pharmacol. 183,1307.

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