- Email: [email protected]
Deferential roles of angiotensin receptor subtypes in adrenocortical function in mice
Life Sciences, Vol. 63, No. 18, pp. 1593-1598, 1998 Copyright Q1998 Elsevier Science Inc. Primed in the USA. All rights rcservcd 0024-3205/98 519.fm + .@I ELSEVIER
PI1 SOO24-3205(9&X)00428-7
DEFERENTIAL ROLES OF ANGIOTENSIN RECEPTOR SUBTYPES IN ADRENOCORTICAL FUNCTION IN MICE Mitsuhide Narusel, Akiyo Tanabe, Takeshi Sugayp, Kiyoko Nat-use, Takanobu Yoshimoto, Toshirou Seki, Toshihiro Imaki, Reiko Demura, Kazuo Murakamib and Hiroshi Demura. Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical University, Tokyo 162-8666, Tanabe Seiyaku Co., Ltd., Osaka 532, bInstitute of Applied Biochemistry, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305, Japan. (Reccivcd
in final form August
19, l-98)
Summary The functional significance of angiotensin II (Ang II) receptor subtypes in adrenals remains unknown. Ang II receptor type la (ATla) expression was localized by in situ hybridization to the zona glomerulosa and zona fasciculata, while ATlb was localized to the zona glomerulosa. Plasma aldosterone and corticosterone levels were measured after injection with Ang II or the type 2 receptor (AT2) agonist CGP-42112 in wild-type and ATla deficient mice. Aldosterone and corticosterone levels were lower in AT la deficient mice. Ang II increased plasma aldosterone levels in ATla deficient mice, but to a lesser extent in mice pretreated with nonselective AT la/AT 1b antagonist, CV-11974. CGP-42112 did not affect aldosterone levels. Ang II increased corticosterone levels in wild-type mice but not in ATla deficient mice. Results suggest Ang II stimulates aldosterone secretion via ATla and ATlb in the zona glomerulosa and corticosterone secretion via ATla in the zona fasciculata, and provide first evidence for differential roles of ATla and AT lb in the adrenals. Key Words: angiotensin II, aldosterone secretion, angiotensin
II receptor,
adrenal
cortex
Angiotensin II (Ang II) plays a major role in the regulation of water and electrolyte balance and blood pressure via specific receptors. Pharmacological studies using selective antagonists (1,2) followed by molecular cloning (3-5) demonstrated multiple receptor subtypes, type 1 (ATl) and type 2 (AT2), in various mammals. AT 1 was shown to mediate most of the major functions of Ang II including vasoconstriction (6), cell proliferation (7,8) and aldosterone (Aldo.) secretion (6), while AT2 was suggested to play counter-regulatory roles to AT1 (9). Two subtypes of AT1 have been identified as products of separate genes, and designated ATla and ATlb (10-13). Although these two AT1 receptor subtypes exhibit high nucleotide and amino acid sequence similarity (lo-14), the relative ratio of AT 1 subtype expression levels varies from tissue to tissue. ATla is predominantly expressed in many tissues including the liver, heart and lung (12), while ATlb is expressed in relatively high density only in a limited number of tissues such as the uterus and anterior pituitary (13). ATla and ATlb mRNA are equally expressed in the adrenals (15), one of the major target organs of Ang II, suggesting the ATlb subtype may have an important role in the regulation of adrenal function. However, the differential roles of the Ang II receptor subtypes in adrenals remain to be elucidated. ICorrespondence: Mitsuhide Naruse, MD., Department of Medicine, Tokyo Women’s Medical University, 8-l Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan Phone: 81-3-3359-5559, Fax: 81-3-3359-5559, E mail: [email protected]
1594
AT1 Receptor Subtypes in Adrenals
Vol. 63, No. 18, 1998
In the present study, we investigated the localization and functional significance of the Ang II receptor subtypes in the mouse adrenal cortex by the combined usage of an AT1 antagonist and an AT2 agonist in ATla deficient mice (16). Materials
and methods
Animals. Ten-week-old male homozygous and heterozygous ATla deficient mice (16) and agematched C57BU6 wild-type mice were used. The ATla receptor locus was replaced by the LacZ gene in the ATla deficient mice (16). In situ hybridization of ATZa and ATZb receptors. Tissue samples, immediately frozen at -60 C, were cut into 15pm-thick cryostat sections. The 490-bp Sac IlPmaC I fragment and the 520-bp Sac I/Spe I fragment, which correspond to 3’untranslated regions of ATla and ATlb, respectively, were used as subtype-specific RNA probes. Each subtype-specific region was subcloned into pBluescript II (Stratagene). Antisense and sense probes were made by in vitro transcription in the presence of digoxigenin-labeled dUTP. In situ hybridization using the digoxigenin-labeled probes was performed as described previously (17). Basal plasma Aldo. and corticosterone levels and plasma renin activity. Blood samples were obtained by cardiac puncture from lo-week-old male homozygous ATla deficient mice and agematched male wild-type mice under pentobarbital anesthesia. Blood was collected into microcentrifuge tubes containing Na2-EDTA and immediately centrifuged. Plasma samples were kept at -20C until assayed. Plasma Aldo. levels and plasma renin activity were determined by commercially available mdioimmunoassay kits (Dinabot RI Laboratories, Tokyo, Japan). Plasma corticosterone level was also determined by commercially available radioimmunoassay kits (ICN Biomedicals Inc., Costea Mesa, CA, USA). Plusma Aldo. and corticosterone levels after administration of Ang ZZor selective AT2 agonist. To assess the role of ATla in response to Ang II stimulation, Ang II (1 mg/kg, Peptide Institute, Osaka, Japan) or vehicle (200 ~1 saline) was administered intraperitoneally (i.p.) in lo-week-old male homozygous AT la deficient mice. To assess the effects of blocking both AT la and AT 1b, Ang II (1 mg/kg) or vehicle (200 ~1 saline) was also administered i.p. in lo-week-old male homozygous ATla deficient mice pretreated with the nonselective AT la/AT 1b antagonist CV11974 (1 mg/kg, i.p., Takeda Chemical Industries, Ltd., Osaka, Japan) (18) for seven days. To assess the role of AT2. selective AT2 agonist CGP-42112 (1 mg/kg, Peptide Institute) (19) was administered i.p. in IO-week-old ATla deficient mice pretreated with CV-11974 (1 mg/kg, i.p., n = 6) and age-matched male wild type mice (n = 7) for seven days. Blood samples were obtained as described above 20 min after administration, the point at which our preliminary experiments showed the plasma Aldo. and corticosterone levels reached peak levels. Plasma Aldo. and corticosterone levels were determined by radioimmunoassay as described above. Statistical analysis. The results were expressed as mean+SEM. Statistical analysis was performed using the Student’s t test or Mann Whitney U test, as appropriate. A p value less than 0.05 was considered statistically significant. Results
In situ hybridization demonstrated that ATla expression was localized in the zona glomerulosa, zona fasciculata, and adrenal medulla. By contrast, ATlb expression was localized exclusively in the zona glomerulosa in the adrenal cortex (Fig. la, b). No signals were detected with sense probes for ATla (Fig. lc) and ATlb (data not shown). Plasma renin activity was significantly higher in homozygous ATla deficient mice (16.252.6 ngAngI/ml/h, n = 8) compared to wild-type mice (6.8+ 1.1 ng AngI/ml/h, n = 8, p < 0.01). However, both basal plasma Aldo. (149.6 + 16.0 pg/ml, n = 4) and corticosterone (137.0 +25.1
Vol. 63, No. 18, 1998
ATI Receptor
Subtypes in Adrenals
1595
nglml, n = 4) levels in homozygous ATla deficient mice were significantly lower than that in wild type mice (Aldo.: 239.9 f 18.1 pglml, n = 8, p < 0.05; corticosterone: 219.7 k25.1 nglml, n = 8, p < 0.05) (Fig. 2).
Fig. 1 In situ hybridization using the antisense RNA probe for ATla (a) and ATlb (b) receptors in adrenals from heterozygous ATla deficient mice. No signal was detected using the sense probe for ATla (c). (x125)
666 , z
600-
a E 400-
*
2 6 3ws Q 200E 3 c looOL
vehicle Ang II
vehkle cv-11974
Wild type
Ang II (-)
vehkk
Ang II
cv-11974 (+)
ATia deficient .
Fig. 2 Effects of Ang II (lmg/kg, i.p., hatched column) or vehicle (200 ~1 saline, open column) on plasma Aldo. levels in wild-type mice (n=8) and homozygous ATla deficient mice with (n=4) and without (n=4) pretreatment with nonselective ATla/ATlb antagonist CV-11974. Values are expressed as mean +SEM. *p < 0.05 vs. vehicle; +p < 0.05 vs. vehicle in wild type; ++p
15%
AT1 Receptor Subtypes in Adrenals
Vol. 63, No. 18, WI8
Administration of Ang II resulted in a two fold increase in plasma Aldo. levels in ATla deficient mice, with or without pretreatment of CV-11974. However, plasma Aldo. levels, both before and after Ang II administration, were significantly lower in ATla deficient mice pretreated with CV11974 compared to untreated mice (Fig. 2). Administration of Ang II produced a significant increase in plasma corticosterone levels in wild type mice, but levels were not increased in ATla deficient mice. Although plasma corticosterone levels before and after Ang II administration tended to be lower in the ATla deficient mice with CV-11974 pretreatment than those without, the difference was not statistically significant (Fig. 3). Administration of AT2 agonist CGP-42112 did not increase plasma Aldo. and corticosterone levels in wild-type and ATla deficient mice, with or without CV-11974 pretreatment (data not shown).
*
-T
vehicle Ang II
vehicle Ang II cv-11974 (-)
Wild type
vehicle Ang II cv-11974(+)
ATla deficient
Fig. 3 Effects of Ang II (lmglkg, i.p., hatched column) or vehicle (200~1 saline, open column) on plasma corticosterone levels in wild-type mice (n= 8) and homozygous AT la deficient mice with (n=4) and without (n=4) pretreatment with nonselective ATlaIATl b antagonist CV-11974. Values are expressed as mean +SEM. *p < 0.05 vs. vehicle; +p < 0.05 vs. vehicle in wild type
Discussion The localization of ATla expression in the zona glomerulosa and zona fasciculata, and ATlb expression in the zona glomerulosa of the mouse adrenal cortex were clearly demonstrated by in situ hybridization. These results are in agreement with the studies in rats (10). The differential expression of ATla and ATlb suggests that they may have different biological roles in adrenocortical function. As the zona glomerulosa is the site of Aldo. production and the zona fasciculata is the site of corticosterone production, it is possible that ATla is involved both in Aldo. and corticosterone secretion, while ATlb is involved only in Aldo. secretion. This hypothesis was investigated by the combined usage of ATla deficient mice and pharmacological antagonists of the Ang II receptor. Basal plasma Aldo. levels were significantly decreased in ATla deficient mice compared to wild-type mice, suggesting that ATla is involved in Aldo. secretion. However, administration of Ang II produced a significant increase in plasma
Vol. 63, No. 18, 1998
AT1 Receptor Subtypes in Adrenals
1597
Aldo. levels in AT la deficient mice, suggesting that Ang II can stimulate Aldo. secretion through non-ATla receptors. When AT1 b function was blocked in ATla deficient mice using the nonselective ATla/ATlb antagonist CV-11974, plasma Aldo. levels were low both before and after Ang II administration. Thus it appears Ang II-dependent Aldo. secretion is also mediated by ATlb. Although compensatory enhancement of ATlb gene expression in ATla deficient mice cannot be completely excluded, parallel elevation of both ATla and ATlb gene expression during sodium depletion (20) supports involvement of ATlb as well as ATla in Ang II-induced Aldo. secretion. As Ang II significantly increased Aldo. levels even in the absence of AT la and AT 1b function, this suggests non-ATla/ATlb receptor(s) contribute to Aldo. secretion. However, the specific AT2 agonist did not increase Aldo. secretion in wild-type mice or mice with blocked ATla and ATlb function. Therefore our results suggest the existence of functionally significant nonATlIAT2 receptor(s) for Ang II. Whether the AT3 (21) and AT4 receptors (22) implied in neuronal cells play a part in Ang II-induced Aldo. secretion remains to be elucidated. Plasma corticosterone levels were also significantly decreased in ATla deficient mice compared to wild-type mice. However, in contrast to Aldo., Ang II did not show any increase in plasma corticosterone level in ATla deficient mice, indicating that corticosterone secretion is mediated mainly by the AT la receptor. The distinct functions of AT la and AT 1b in Aldo. secretion from the zona glomerulosa and corticosterone secretion from the zona fasciculata corresponds to the localization of the two subtypes of Ang II receptor in adrenal cortex. In conclusion, our results suggest that Ang II stimulates Aldo. secretion mainly through the ATla/AT lb receptors in the zona glomerulosa and corticosterone secretion through the AT la receptor in the zona fasciculata. Significant increase in Aldo. secretion by Ang II even without functional ATla/ATlb and lack of effects of AT2 agonist provide evidence for the existence of non-ATl/AT2 receptor(s). Acknowledgments We wish to thank Takeda Chemical Industries, Ltd. (Osaka, Japan) for providing CV-11974. This work was supported in part by research grants from the Yayoi Yoshioka Research Award of Tokyo Women’s Medical University for Akiyo Tanabe, the Japanese Ministry of Education, Science, and Culture, and the Japanese Ministry of Health and Welfare “Disorders of Adrenal Hormones” Research Committee. References 1. SE. WHITEBREAD, M. MELE, B. KAMBER AND M. DE GASPARO, B&hem. Biophys. Res. Commun. 163 284-291 (1989). 2. A.T. CHIU, W.F. HERBLIN, D.E. MCCALL, R.J. ARDECKY, D.J. CARINI, J.V. DUNCIA, L.J. PEASE, P.C. WONG, R.R. WEXLER, A.L. JOHNSON AND P.B.TIMMERMANS, B&hem. Biophys. Res. Commun. 165 196-203 (1989). 3. K. SASAKI, Y. YAMANO, S. BARDHAN, N. IWAI, J.J. MURRAY, M. HASEGAWA, Y. MATSUDA AND T. INAGAMI, Nature 351230-236 (1991). 4. S. TSUZUKI, T. ICHIKI, H. NAKAKUBO, Y. KITAMI, D-F. GUO, H. SHIRAI AND T. INAGAMI, Biochem. Biophys. Res. Commun. 200 1449-1454 (1994). 5. G. KOIKE, M. HORIUCHI, T. YAMADA, C. SZPIRER, H.J. JACOB AND V.J. DZAU, Biochem. Biophys. Res. Commun. 203 1842-1850 (1994). 6. G. AGUILERA, Mol. Cell. Endocrinol. 90 53-60 (1992). 7. A.M. KATZ, J. Mol. Cell. Cardiol. 22 739-747 (1990). 8. J.F. ACETO AND K.M. BAKER, Am. J. Physiol. 258 H806-H813 (1990). 9. T. ICHIKI, P.A. LABOSKY, C. SHIOTA, S. OKUYAMA, Y. IMAGAWA, A. FOGO, F. NIIMURA, I. ICHIKAWA, B.L. HOGAN AND T. INAGAMI, Nature 377 748-750 (1995). 10. J.M. GASC, S. SHANMUGAM, M. SIBONY AND P. CORVOL, Hypertension 24 531537 (1994). 11. N. IWAI AND T. INAGAMI, FEBS Letters 298 257-260 (1992).
1598
AT1 Receptor Subtypes in Adrenals
Vol. 63, No. 18, 1998
12. J.M. BURSON, G. AGUIERA. K.W. GROSS AND C.D. SIGMUND, Am. J. Physiol. 267 E260-E267 (1994). 13. H. KONISHI, S. KURODA, Y. INADA AND Y. FUJISAWA, Bicchem. Biophys. Res. Commun. 199 467-474 (1994). 14. H. SASAMURA, L. HEIN, J.E. KRIEGER, R.E. PRATT, B.K. KOBILKA AND V.J. DZAU, Biochem. Biophys. Res. Commun. 185 253-259 (1992). 15. C. LLORENS-CORTES, B. GREENBERG, H. HUANG AND P. CORVOL, Hypertension 24 x538-548 (1994). 16. T. SUGAYA, S. NISHIMATSU, K. TANIMOTO, E. TAKIMOTO, T. YAMAGISHI. K. IMAMURA, S. GOTO, K. IMAIZUMI, Y. HISADA AND A. OTSUKA, J. Biol. Chem. 270 18719-18722 (1995). 17. K. IMAIZUMI, M. TSUDA, A. WANAKA, M. TOHYAMA AND T. TAKAGI, Mol. Brain Res. 26 189-l% (1994). 18. Y. SHIBOUTA, Y. INADA. M. OJIMA, T. WADA, M. NODA, T. SANADA. K. KUBO, Y. KOHARA, T. NAKA AND K. NISHIKAWA, J. Pharmacol. Exp. Ther. 266 114-120 (1993). 19. B. BUISSON, S.P. BOTTARY, M. DE GASPARO, M. GALLO-PAYET AND M. PAYEI’, FEBS Lett. 309 161-164 (1992). 20. Y. DU, D.F. GUO, T. INAGAMI, R.C. SPETH AND D.H. WANG, Am. J. Physiol. 271 H440-H446 (19%). 21. S.S. CHAKI AND T. INAGAMI, Eur. J. Pharomacol. 225 355-356 (1992). 22. M.DE GASPARO, A. HUSAIN, W. ALEXANDER, K.J. CATT, A.T. CHIU, M. DREW, T. GOODFRIEND, J.W. HARDING, T. INAGAMI AND P.B. TIMMERMANS, Hypertension 25 924-927 (1995).
PI1 SOO24-3205(9&X)00428-7
DEFERENTIAL ROLES OF ANGIOTENSIN RECEPTOR SUBTYPES IN ADRENOCORTICAL FUNCTION IN MICE Mitsuhide Narusel, Akiyo Tanabe, Takeshi Sugayp, Kiyoko Nat-use, Takanobu Yoshimoto, Toshirou Seki, Toshihiro Imaki, Reiko Demura, Kazuo Murakamib and Hiroshi Demura. Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical University, Tokyo 162-8666, Tanabe Seiyaku Co., Ltd., Osaka 532, bInstitute of Applied Biochemistry, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305, Japan. (Reccivcd
in final form August
19, l-98)
Summary The functional significance of angiotensin II (Ang II) receptor subtypes in adrenals remains unknown. Ang II receptor type la (ATla) expression was localized by in situ hybridization to the zona glomerulosa and zona fasciculata, while ATlb was localized to the zona glomerulosa. Plasma aldosterone and corticosterone levels were measured after injection with Ang II or the type 2 receptor (AT2) agonist CGP-42112 in wild-type and ATla deficient mice. Aldosterone and corticosterone levels were lower in AT la deficient mice. Ang II increased plasma aldosterone levels in ATla deficient mice, but to a lesser extent in mice pretreated with nonselective AT la/AT 1b antagonist, CV-11974. CGP-42112 did not affect aldosterone levels. Ang II increased corticosterone levels in wild-type mice but not in ATla deficient mice. Results suggest Ang II stimulates aldosterone secretion via ATla and ATlb in the zona glomerulosa and corticosterone secretion via ATla in the zona fasciculata, and provide first evidence for differential roles of ATla and AT lb in the adrenals. Key Words: angiotensin II, aldosterone secretion, angiotensin
II receptor,
adrenal
cortex
Angiotensin II (Ang II) plays a major role in the regulation of water and electrolyte balance and blood pressure via specific receptors. Pharmacological studies using selective antagonists (1,2) followed by molecular cloning (3-5) demonstrated multiple receptor subtypes, type 1 (ATl) and type 2 (AT2), in various mammals. AT 1 was shown to mediate most of the major functions of Ang II including vasoconstriction (6), cell proliferation (7,8) and aldosterone (Aldo.) secretion (6), while AT2 was suggested to play counter-regulatory roles to AT1 (9). Two subtypes of AT1 have been identified as products of separate genes, and designated ATla and ATlb (10-13). Although these two AT1 receptor subtypes exhibit high nucleotide and amino acid sequence similarity (lo-14), the relative ratio of AT 1 subtype expression levels varies from tissue to tissue. ATla is predominantly expressed in many tissues including the liver, heart and lung (12), while ATlb is expressed in relatively high density only in a limited number of tissues such as the uterus and anterior pituitary (13). ATla and ATlb mRNA are equally expressed in the adrenals (15), one of the major target organs of Ang II, suggesting the ATlb subtype may have an important role in the regulation of adrenal function. However, the differential roles of the Ang II receptor subtypes in adrenals remain to be elucidated. ICorrespondence: Mitsuhide Naruse, MD., Department of Medicine, Tokyo Women’s Medical University, 8-l Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan Phone: 81-3-3359-5559, Fax: 81-3-3359-5559, E mail: [email protected]
1594
AT1 Receptor Subtypes in Adrenals
Vol. 63, No. 18, 1998
In the present study, we investigated the localization and functional significance of the Ang II receptor subtypes in the mouse adrenal cortex by the combined usage of an AT1 antagonist and an AT2 agonist in ATla deficient mice (16). Materials
and methods
Animals. Ten-week-old male homozygous and heterozygous ATla deficient mice (16) and agematched C57BU6 wild-type mice were used. The ATla receptor locus was replaced by the LacZ gene in the ATla deficient mice (16). In situ hybridization of ATZa and ATZb receptors. Tissue samples, immediately frozen at -60 C, were cut into 15pm-thick cryostat sections. The 490-bp Sac IlPmaC I fragment and the 520-bp Sac I/Spe I fragment, which correspond to 3’untranslated regions of ATla and ATlb, respectively, were used as subtype-specific RNA probes. Each subtype-specific region was subcloned into pBluescript II (Stratagene). Antisense and sense probes were made by in vitro transcription in the presence of digoxigenin-labeled dUTP. In situ hybridization using the digoxigenin-labeled probes was performed as described previously (17). Basal plasma Aldo. and corticosterone levels and plasma renin activity. Blood samples were obtained by cardiac puncture from lo-week-old male homozygous ATla deficient mice and agematched male wild-type mice under pentobarbital anesthesia. Blood was collected into microcentrifuge tubes containing Na2-EDTA and immediately centrifuged. Plasma samples were kept at -20C until assayed. Plasma Aldo. levels and plasma renin activity were determined by commercially available mdioimmunoassay kits (Dinabot RI Laboratories, Tokyo, Japan). Plasma corticosterone level was also determined by commercially available radioimmunoassay kits (ICN Biomedicals Inc., Costea Mesa, CA, USA). Plusma Aldo. and corticosterone levels after administration of Ang ZZor selective AT2 agonist. To assess the role of ATla in response to Ang II stimulation, Ang II (1 mg/kg, Peptide Institute, Osaka, Japan) or vehicle (200 ~1 saline) was administered intraperitoneally (i.p.) in lo-week-old male homozygous AT la deficient mice. To assess the effects of blocking both AT la and AT 1b, Ang II (1 mg/kg) or vehicle (200 ~1 saline) was also administered i.p. in lo-week-old male homozygous ATla deficient mice pretreated with the nonselective AT la/AT 1b antagonist CV11974 (1 mg/kg, i.p., Takeda Chemical Industries, Ltd., Osaka, Japan) (18) for seven days. To assess the role of AT2. selective AT2 agonist CGP-42112 (1 mg/kg, Peptide Institute) (19) was administered i.p. in IO-week-old ATla deficient mice pretreated with CV-11974 (1 mg/kg, i.p., n = 6) and age-matched male wild type mice (n = 7) for seven days. Blood samples were obtained as described above 20 min after administration, the point at which our preliminary experiments showed the plasma Aldo. and corticosterone levels reached peak levels. Plasma Aldo. and corticosterone levels were determined by radioimmunoassay as described above. Statistical analysis. The results were expressed as mean+SEM. Statistical analysis was performed using the Student’s t test or Mann Whitney U test, as appropriate. A p value less than 0.05 was considered statistically significant. Results
In situ hybridization demonstrated that ATla expression was localized in the zona glomerulosa, zona fasciculata, and adrenal medulla. By contrast, ATlb expression was localized exclusively in the zona glomerulosa in the adrenal cortex (Fig. la, b). No signals were detected with sense probes for ATla (Fig. lc) and ATlb (data not shown). Plasma renin activity was significantly higher in homozygous ATla deficient mice (16.252.6 ngAngI/ml/h, n = 8) compared to wild-type mice (6.8+ 1.1 ng AngI/ml/h, n = 8, p < 0.01). However, both basal plasma Aldo. (149.6 + 16.0 pg/ml, n = 4) and corticosterone (137.0 +25.1
Vol. 63, No. 18, 1998
ATI Receptor
Subtypes in Adrenals
1595
nglml, n = 4) levels in homozygous ATla deficient mice were significantly lower than that in wild type mice (Aldo.: 239.9 f 18.1 pglml, n = 8, p < 0.05; corticosterone: 219.7 k25.1 nglml, n = 8, p < 0.05) (Fig. 2).
Fig. 1 In situ hybridization using the antisense RNA probe for ATla (a) and ATlb (b) receptors in adrenals from heterozygous ATla deficient mice. No signal was detected using the sense probe for ATla (c). (x125)
666 , z
600-
a E 400-
*
2 6 3ws Q 200E 3 c looOL
vehicle Ang II
vehkle cv-11974
Wild type
Ang II (-)
vehkk
Ang II
cv-11974 (+)
ATia deficient .
Fig. 2 Effects of Ang II (lmg/kg, i.p., hatched column) or vehicle (200 ~1 saline, open column) on plasma Aldo. levels in wild-type mice (n=8) and homozygous ATla deficient mice with (n=4) and without (n=4) pretreatment with nonselective ATla/ATlb antagonist CV-11974. Values are expressed as mean +SEM. *p < 0.05 vs. vehicle; +p < 0.05 vs. vehicle in wild type; ++p
15%
AT1 Receptor Subtypes in Adrenals
Vol. 63, No. 18, WI8
Administration of Ang II resulted in a two fold increase in plasma Aldo. levels in ATla deficient mice, with or without pretreatment of CV-11974. However, plasma Aldo. levels, both before and after Ang II administration, were significantly lower in ATla deficient mice pretreated with CV11974 compared to untreated mice (Fig. 2). Administration of Ang II produced a significant increase in plasma corticosterone levels in wild type mice, but levels were not increased in ATla deficient mice. Although plasma corticosterone levels before and after Ang II administration tended to be lower in the ATla deficient mice with CV-11974 pretreatment than those without, the difference was not statistically significant (Fig. 3). Administration of AT2 agonist CGP-42112 did not increase plasma Aldo. and corticosterone levels in wild-type and ATla deficient mice, with or without CV-11974 pretreatment (data not shown).
*
-T
vehicle Ang II
vehicle Ang II cv-11974 (-)
Wild type
vehicle Ang II cv-11974(+)
ATla deficient
Fig. 3 Effects of Ang II (lmglkg, i.p., hatched column) or vehicle (200~1 saline, open column) on plasma corticosterone levels in wild-type mice (n= 8) and homozygous AT la deficient mice with (n=4) and without (n=4) pretreatment with nonselective ATlaIATl b antagonist CV-11974. Values are expressed as mean +SEM. *p < 0.05 vs. vehicle; +p < 0.05 vs. vehicle in wild type
Discussion The localization of ATla expression in the zona glomerulosa and zona fasciculata, and ATlb expression in the zona glomerulosa of the mouse adrenal cortex were clearly demonstrated by in situ hybridization. These results are in agreement with the studies in rats (10). The differential expression of ATla and ATlb suggests that they may have different biological roles in adrenocortical function. As the zona glomerulosa is the site of Aldo. production and the zona fasciculata is the site of corticosterone production, it is possible that ATla is involved both in Aldo. and corticosterone secretion, while ATlb is involved only in Aldo. secretion. This hypothesis was investigated by the combined usage of ATla deficient mice and pharmacological antagonists of the Ang II receptor. Basal plasma Aldo. levels were significantly decreased in ATla deficient mice compared to wild-type mice, suggesting that ATla is involved in Aldo. secretion. However, administration of Ang II produced a significant increase in plasma
Vol. 63, No. 18, 1998
AT1 Receptor Subtypes in Adrenals
1597
Aldo. levels in AT la deficient mice, suggesting that Ang II can stimulate Aldo. secretion through non-ATla receptors. When AT1 b function was blocked in ATla deficient mice using the nonselective ATla/ATlb antagonist CV-11974, plasma Aldo. levels were low both before and after Ang II administration. Thus it appears Ang II-dependent Aldo. secretion is also mediated by ATlb. Although compensatory enhancement of ATlb gene expression in ATla deficient mice cannot be completely excluded, parallel elevation of both ATla and ATlb gene expression during sodium depletion (20) supports involvement of ATlb as well as ATla in Ang II-induced Aldo. secretion. As Ang II significantly increased Aldo. levels even in the absence of AT la and AT 1b function, this suggests non-ATla/ATlb receptor(s) contribute to Aldo. secretion. However, the specific AT2 agonist did not increase Aldo. secretion in wild-type mice or mice with blocked ATla and ATlb function. Therefore our results suggest the existence of functionally significant nonATlIAT2 receptor(s) for Ang II. Whether the AT3 (21) and AT4 receptors (22) implied in neuronal cells play a part in Ang II-induced Aldo. secretion remains to be elucidated. Plasma corticosterone levels were also significantly decreased in ATla deficient mice compared to wild-type mice. However, in contrast to Aldo., Ang II did not show any increase in plasma corticosterone level in ATla deficient mice, indicating that corticosterone secretion is mediated mainly by the AT la receptor. The distinct functions of AT la and AT 1b in Aldo. secretion from the zona glomerulosa and corticosterone secretion from the zona fasciculata corresponds to the localization of the two subtypes of Ang II receptor in adrenal cortex. In conclusion, our results suggest that Ang II stimulates Aldo. secretion mainly through the ATla/AT lb receptors in the zona glomerulosa and corticosterone secretion through the AT la receptor in the zona fasciculata. Significant increase in Aldo. secretion by Ang II even without functional ATla/ATlb and lack of effects of AT2 agonist provide evidence for the existence of non-ATl/AT2 receptor(s). Acknowledgments We wish to thank Takeda Chemical Industries, Ltd. (Osaka, Japan) for providing CV-11974. This work was supported in part by research grants from the Yayoi Yoshioka Research Award of Tokyo Women’s Medical University for Akiyo Tanabe, the Japanese Ministry of Education, Science, and Culture, and the Japanese Ministry of Health and Welfare “Disorders of Adrenal Hormones” Research Committee. References 1. SE. WHITEBREAD, M. MELE, B. KAMBER AND M. DE GASPARO, B&hem. Biophys. Res. Commun. 163 284-291 (1989). 2. A.T. CHIU, W.F. HERBLIN, D.E. MCCALL, R.J. ARDECKY, D.J. CARINI, J.V. DUNCIA, L.J. PEASE, P.C. WONG, R.R. WEXLER, A.L. JOHNSON AND P.B.TIMMERMANS, B&hem. Biophys. Res. Commun. 165 196-203 (1989). 3. K. SASAKI, Y. YAMANO, S. BARDHAN, N. IWAI, J.J. MURRAY, M. HASEGAWA, Y. MATSUDA AND T. INAGAMI, Nature 351230-236 (1991). 4. S. TSUZUKI, T. ICHIKI, H. NAKAKUBO, Y. KITAMI, D-F. GUO, H. SHIRAI AND T. INAGAMI, Biochem. Biophys. Res. Commun. 200 1449-1454 (1994). 5. G. KOIKE, M. HORIUCHI, T. YAMADA, C. SZPIRER, H.J. JACOB AND V.J. DZAU, Biochem. Biophys. Res. Commun. 203 1842-1850 (1994). 6. G. AGUILERA, Mol. Cell. Endocrinol. 90 53-60 (1992). 7. A.M. KATZ, J. Mol. Cell. Cardiol. 22 739-747 (1990). 8. J.F. ACETO AND K.M. BAKER, Am. J. Physiol. 258 H806-H813 (1990). 9. T. ICHIKI, P.A. LABOSKY, C. SHIOTA, S. OKUYAMA, Y. IMAGAWA, A. FOGO, F. NIIMURA, I. ICHIKAWA, B.L. HOGAN AND T. INAGAMI, Nature 377 748-750 (1995). 10. J.M. GASC, S. SHANMUGAM, M. SIBONY AND P. CORVOL, Hypertension 24 531537 (1994). 11. N. IWAI AND T. INAGAMI, FEBS Letters 298 257-260 (1992).
1598
AT1 Receptor Subtypes in Adrenals
Vol. 63, No. 18, 1998
12. J.M. BURSON, G. AGUIERA. K.W. GROSS AND C.D. SIGMUND, Am. J. Physiol. 267 E260-E267 (1994). 13. H. KONISHI, S. KURODA, Y. INADA AND Y. FUJISAWA, Bicchem. Biophys. Res. Commun. 199 467-474 (1994). 14. H. SASAMURA, L. HEIN, J.E. KRIEGER, R.E. PRATT, B.K. KOBILKA AND V.J. DZAU, Biochem. Biophys. Res. Commun. 185 253-259 (1992). 15. C. LLORENS-CORTES, B. GREENBERG, H. HUANG AND P. CORVOL, Hypertension 24 x538-548 (1994). 16. T. SUGAYA, S. NISHIMATSU, K. TANIMOTO, E. TAKIMOTO, T. YAMAGISHI. K. IMAMURA, S. GOTO, K. IMAIZUMI, Y. HISADA AND A. OTSUKA, J. Biol. Chem. 270 18719-18722 (1995). 17. K. IMAIZUMI, M. TSUDA, A. WANAKA, M. TOHYAMA AND T. TAKAGI, Mol. Brain Res. 26 189-l% (1994). 18. Y. SHIBOUTA, Y. INADA. M. OJIMA, T. WADA, M. NODA, T. SANADA. K. KUBO, Y. KOHARA, T. NAKA AND K. NISHIKAWA, J. Pharmacol. Exp. Ther. 266 114-120 (1993). 19. B. BUISSON, S.P. BOTTARY, M. DE GASPARO, M. GALLO-PAYET AND M. PAYEI’, FEBS Lett. 309 161-164 (1992). 20. Y. DU, D.F. GUO, T. INAGAMI, R.C. SPETH AND D.H. WANG, Am. J. Physiol. 271 H440-H446 (19%). 21. S.S. CHAKI AND T. INAGAMI, Eur. J. Pharomacol. 225 355-356 (1992). 22. M.DE GASPARO, A. HUSAIN, W. ALEXANDER, K.J. CATT, A.T. CHIU, M. DREW, T. GOODFRIEND, J.W. HARDING, T. INAGAMI AND P.B. TIMMERMANS, Hypertension 25 924-927 (1995).