Regulatory Roles for APJ, a Seven-transmembrane Receptor Related to Angiotensin-type 1 Receptor in Blood Pressure in Vivo

Abstract

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II. APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II. A family of G protein-coupled receptors bind a large variety of ligands and plays an essential role for physiological functions in vivo including the maintenance of homeostasis in the cardiovascular system. APJ (a putative receptor protein related to the angiotensin-type 1 receptor (AT1)) 1The abbreviations used are: AT1, angiotensin-type 1 receptor; WKY, Wistar-Kyoto; SHR, spontaneously hypertensive rat; DMEM, Dulbecco's modified Eagle's medium; NO, nitric oxide; eNOS, endothelial NO synthase; l-NAME, NG-nitro-l-arginine methyl ester. is a G protein-coupled receptor that was isolated from human genomic DNA using the polymerase chain reaction (1O'Dowd B.F. Heiber M. Chan A. Heng H.H. Tsui L.C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. Gene (Amst.). 1993; 136: 355-360Crossref PubMed Scopus (687) Google Scholar). The APJ has a 31% amino acid sequence homology with the AT1, but APJ does not display specific binding for angiotensin II, which is the ligand of AT1 and exerts a pressor action in the blood pressure regulation (1O'Dowd B.F. Heiber M. Chan A. Heng H.H. Tsui L.C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. Gene (Amst.). 1993; 136: 355-360Crossref PubMed Scopus (687) Google Scholar). Recently, the endogenous ligand of APJ was identified from bovine stomach, and this peptide was named apelin (for APJ endogenous ligand) (2Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1349) Google Scholar). APJ and apelin are expressed in several tissues including the cardiovascular and the central nervous systems (3Hosoya M. Kawamata Y. Fukusumi S. Fujii R. Habata Y. Hinuma S. Kitada C. Honda S. Kurokawa T. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 21061-21067Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 4Kawamata Y. Habata Y. Fukusumi S. Hosoya M. Fujii R. Hinuma S. Nishizawa N. Kitada C. Onda H. Nishimura O. Fujino M. Biochim. Biophys. Acta. 2001; 1538: 162-171Crossref PubMed Scopus (435) Google Scholar, 5Reaux A. De Mota N. Skultetyova I. Lenkei Z. El Messari S. Gallatz K. Corvol P. Palkovits M. Llorens-Cortes C. J. Neurochem. 2001; 77: 1085-1096Crossref PubMed Scopus (319) Google Scholar, 6Medhurst A.D. Jennings C.A. Robbins M.J. Davis R.P. Ellis C. Winborn K.Y. Lawrie K.W. Hervieu G. Riley G. Bolaky J.E. Herrity N.C. Murdock P. Darker J.G. J. Neurochem. 2003; 84: 1162-1172Crossref PubMed Scopus (355) Google Scholar), and the structure of APJ and apelin is highly conserved among species, suggesting its important physiological roles. Intravenous administration of apelin suggested a hypotensive effect in rat (5Reaux A. De Mota N. Skultetyova I. Lenkei Z. El Messari S. Gallatz K. Corvol P. Palkovits M. Llorens-Cortes C. J. Neurochem. 2001; 77: 1085-1096Crossref PubMed Scopus (319) Google Scholar, 7Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (577) Google Scholar, 8Tatemoto K. Takayama K. Zou M.X. Kumaki I. Zhang W. Kumano K. Fujimiya M. Regul. Pept. 2001; 99: 87-92Crossref PubMed Scopus (565) Google Scholar, 9Cheng X. Cheng X.S. Pang C.C. Eur. J. Pharmacol. 2003; 470: 171-175Crossref PubMed Scopus (170) Google Scholar). On the other hand, apelin potently contracts human saphenous vein smooth muscle cells in vitro (10Katugampola S.D. Maguire J.J. Matthewson S.R. Davenport A.P. Br. J. Pharmacol. 2001; 132: 1255-1260Crossref PubMed Scopus (187) Google Scholar), indicating that apelin is a potent vasoconstrictor. Thus, at this moment, the action of apelin in blood pressure regulation is controversial, and it is still unclear whether these actions of apelin are really through APJ because of the absence of specific receptor blocker to clarify the in vivo functions of APJ. Therefore, in this study, by using animal models such as APJ-deficient mice, APJ/AT1a double knock-out mice, and spontaneously hypertensive rat and by using endothelial cells from mice, we evaluated the functional importance of apelin-APJ signaling in the blood pressure regulation in vitro and in vivo. Gene Targeting and Generation of Mutant Mice—The genomic DNA containing the APJ locus were isolated from a phage library from C57BL/6 mice (11Sugaya T. Nishimatsu S. Tanimoto K. Takimoto E. Yamagishi T. Imamura K. Goto S. Imaizumi K. Hisada Y. Otsuka A. Uchida H. Sugiura M. Fukuta K. Fukamizu A. Murakami K. J. Biol. Chem. 1995; 270: 18719-18722Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar) with the human AT1 cDNA as a probe. To construct a targeting vector for the APJ gene, the 156-bp fragment of the mouse APJ gene between the NcoI site including the translation initiation codon of the gene and the Csp45I site was replaced with the nuclear localization signal-lacZ cassette. The neomycin phosphotransferase (neo) gene cassette derived from pMC1neoPolyA (Stratagene) was placed downstream of the nuclear localization signal-lacZ gene. The 6.3-kb XhoI/NcoI fragment and the 1.6-kb Csp45I/Sau3AI fragment of the APJ gene were included upstream and downstream of these cassettes, respectively (Fig. 1A). Details of the negative selection with the diphtheria toxin-A cassette are described elsewhere (12Yagi T. Nada S. Watanabe N. Tamemoto H. Kohmura N. Ikawa Y. Aizawa S. Anal. Biochem. 1993; 214: 77-86Crossref PubMed Scopus (209) Google Scholar). The TT2 ES cells were grown on embryonic fibroblast feeder cells as described previously (13Yagi T. Tokunaga T. Furuta Y. Nada S. Yoshida M. Tsukada T. Saga Y. Takeda N. Ikawa Y. Aizawa S. Anal. Biochem. 1993; 214: 70-76Crossref PubMed Scopus (425) Google Scholar). Homologous recombination in TT2 ES cells was detected by Southern blotting using probe a (687-bp BanIII-Sau3AI fragment). Chimeric mice were generated by injecting the ES cells into ICR 8-cell embryos (13Yagi T. Tokunaga T. Furuta Y. Nada S. Yoshida M. Tsukada T. Saga Y. Takeda N. Ikawa Y. Aizawa S. Anal. Biochem. 1993; 214: 70-76Crossref PubMed Scopus (425) Google Scholar, 14Goto Y. Sugiyama F. Tanimoto K. Ishida J. Syoji M. Takahashi A. Sugiyama Y. Murakami K. Fukamizu A. Yagami K. Lab. Anim. Sci. 1995; 45: 601-603PubMed Google Scholar). AT1a-deficient mice were generated as described previously (11Sugaya T. Nishimatsu S. Tanimoto K. Takimoto E. Yamagishi T. Imamura K. Goto S. Imaizumi K. Hisada Y. Otsuka A. Uchida H. Sugiura M. Fukuta K. Fukamizu A. Murakami K. J. Biol. Chem. 1995; 270: 18719-18722Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Double knock-out mice for APJ and AT1a used in this study were generated from heterozygous mice after the crossing of single APJ-deficient and AT1a-deficient mice. RNA Preparation and Northern Blot Analysis—Total RNA was isolated from the heart and lung of four independent age-matched mice using ISOGEN (NipponGene) (15Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63196) Google Scholar). Fifteen micrograms of RNA were denatured with glyoxal, separated by electrophoresis, and transferred to a nylon membrane. The 728-bp NcoI/NaeI fragment that corresponds to the coding regions of APJ was used as the APJ receptor-specific probe (probe b). Probes for mouse glyceraldehyde-3-phosphate dehydrogenase were described previously (16Miyagishi M. Nakajima T. Fukamizu A. Int. J. Mol. Med. 2000; 5: 27-31PubMed Google Scholar). Measurement of Blood Pressure—The heart rate and systolic, mean, and diastolic blood pressures were measured by a programmable sphygmomanometer (BP-200, Softron, Japan) using the tail cuff method as described previously (17Fukamizu A. Sugimura K. Takimoto E. Sugiyama F. Seo M.S. Takahashi S. Hatae T. Kajiwara N. Yagami K. Murakami K. J. Biol. Chem. 1993; 268: 11617-11621Abstract Full Text PDF PubMed Google Scholar). Unanesthetized mice were introduced into a holder mounted in a thermostatically controlled warning plate and maintained at 37 °C during measurement. Intraperitoneal Injection of Apelin—Experiments were performed using 4-month-old male mice under the conscious and unrestrained conditions. [Pyr1]Apelin-13 (Peptide Institute 4361-v) was suspended in saline (0.9% NaCl in distilled water). After the measurement of the basal systolic blood pressure, [Pyr1]apelin-13 was administered by intraperitoneal injection at 285 μg/kg body weight and the systolic blood pressure was measured continuously. The data were calculated at 5-min intervals for 20 min after the administration of apelin. Intravenous Injection of Apelin in Wistar-Kyoto (WKY) Rat and SHR—SHR and WKY rats at 12 weeks of age were anesthetized with sodium pentobarbital (35 mg/kg were into the for blood pressure and into the for the administration of The was to a pressure and blood pressure was measured continuously. The was maintained by injection of mg/kg pentobarbital [Pyr1]Apelin-13 in of saline was administered through the vein and with and Intraperitoneal Injection of Apelin in and APJ-deficient were performed using 4-month-old male mice under the conscious and unrestrained conditions. was suspended in blood pressure was measured and after intraperitoneal injection of mg/kg body min after the administration of l-NAME, the administration of [Pyr1]apelin-13 μg/kg body saline was performed by intraperitoneal injection and the systolic blood pressure was measured continuously. The of systolic blood pressure to of apelin were calculated in a of apelin. Preparation of from and APJ-deficient Mice—The lung of wild-type and APJ-deficient male mice at weeks of age was with and in into and in 20 of in containing at 37 °C for min with The was through a nylon and in 20 of DMEM, and the was in of of were into the containing 12 of and at for of the with the endothelial cells were to which of were and the was The endothelial cells were in of containing bovine and endothelial from bovine for smooth muscle cells were as cells were at a of after and under the conditions. the of endothelial cells by blotting with not of NO endothelial cells from wild-type and APJ-deficient mice were in and by [Pyr1]apelin-13 bovine for and the reaction was by The were to and transferred to After with a containing the was with a and was detected by using to the phosphorylation of After with a of the and the was to blotting with a to the of as an with and Intraperitoneal Injection of blood pressure was measured in conscious and unrestrained mice at of age as The basal systolic blood pressure and the pressure against intraperitoneal injection of angiotensin and were measured to the administration of After the administration of in for 1 to the endogenous of angiotensin II, the pressure to angiotensin from were for min after the administration of angiotensin II. data were by for was are expressed as the Generation of APJ-deficient a at the mouse APJ gene we a targeting vector that a of the APJ coding with the gene 1A). After of TT2 cells with the targeting recombination was by Southern blotting (Fig. independent of cells recombination at the mouse APJ were into ICR 8-cell embryos to mice, and to by with mice. The heterozygous mice were to homozygous and the at APJ was detected by Southern of tail DNA (Fig. the were for the and were indicating the embryonic of the homozygous mutant mice. The of and not in between wild-type and heterozygous homozygous mutant mice not In the study, to the equivalent of other gene with the of for the APJ gene, we used these of heterozygous mice for physiological RNA whether APJ was in homozygous we performed Northern of heart and lung heart and lung highly the APJ gene in (3Hosoya M. Kawamata Y. Fukusumi S. Fujii R. Habata Y. Hinuma S. Kitada C. Honda S. Kurokawa T. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 21061-21067Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar), homozygous mutant mice APJ (Fig. A was with a glyceraldehyde-3-phosphate dehydrogenase probe to that the RNA was These results that APJ were from homozygous mutant mice Measurement of Blood and of whether in the regulation of the cardiovascular system, we measured the systolic blood pressure and heart rate under the steady state. in APJ-deficient mice and wild-type mice not in the base-line systolic blood pressure and and the heart rate and suggesting that APJ is not essential for the maintenance of base-line blood pressure. it is that apelin is in the regulation of (5Reaux A. De Mota N. Skultetyova I. Lenkei Z. El Messari S. Gallatz K. Corvol P. Palkovits M. Llorens-Cortes C. J. Neurochem. 2001; 77: 1085-1096Crossref PubMed Scopus (319) Google Scholar, 7Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (577) Google Scholar, S. K. M. E. Kennedy A. W. C. A. M. S. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar), but the of and the of of APJ-deficient mice are not from of wild-type is not has that blood pressure was decreased transiently by the administration of apelin, the endogenous ligand of APJ, in rat (5Reaux A. De Mota N. Skultetyova I. Lenkei Z. El Messari S. Gallatz K. Corvol P. Palkovits M. Llorens-Cortes C. J. Neurochem. 2001; 77: 1085-1096Crossref PubMed Scopus (319) Google Scholar, 7Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (577) Google Scholar, 8Tatemoto K. Takayama K. Zou M.X. Kumaki I. Zhang W. Kumano K. Fujimiya M. Regul. Pept. 2001; 99: 87-92Crossref PubMed Scopus (565) Google Scholar, 9Cheng X. Cheng X.S. Pang C.C. Eur. J. Pharmacol. 2003; 470: 171-175Crossref PubMed Scopus (170) Google Scholar). administered apelin to APJ-deficient mice to whether these actions of apelin are really through APJ. Apelin is derived from a acid and to several by the amino (3Hosoya M. Kawamata Y. Fukusumi S. Fujii R. Habata Y. Hinuma S. Kitada C. Honda S. Kurokawa T. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 21061-21067Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 4Kawamata Y. Habata Y. Fukusumi S. Hosoya M. Fujii R. Hinuma S. Nishizawa N. Kitada C. Onda H. Nishimura O. Fujino M. Biochim. Biophys. Acta. 2001; 1538: 162-171Crossref PubMed Scopus (435) Google Scholar). The of has to have the at the receptor in vitro (3Hosoya M. Kawamata Y. Fukusumi S. Fujii R. Habata Y. Hinuma S. Kitada C. Honda S. Kurokawa T. Onda H. Nishimura O. Fujino M. J. Biol. Chem. 2000; 275: 21061-21067Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). male mice were with The administration of apelin transiently and significantly decreased in the systolic blood pressure of wild-type mice (Fig. On the other hand, the apelin injection in systolic blood pressure of APJ-deficient mice (Fig. a in heart rate as as that of wild-type mice not These results demonstrate that the administration of apelin the blood pressure in wild-type but not in APJ-deficient mice and that APJ is really for this action of apelin on the blood pressure of Apelin to that the of the apelin-APJ signaling the blood pressure under the steady in mice (Fig. and rats (5Reaux A. De Mota N. Skultetyova I. Lenkei Z. El Messari S. Gallatz K. Corvol P. Palkovits M. Llorens-Cortes C. J. Neurochem. 2001; 77: 1085-1096Crossref PubMed Scopus (319) Google Scholar, 7Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (577) Google Scholar, 8Tatemoto K. Takayama K. Zou M.X. Kumaki I. Zhang W. Kumano K. Fujimiya M. Regul. Pept. 2001; 99: 87-92Crossref PubMed Scopus (565) Google Scholar, 9Cheng X. Cheng X.S. Pang C.C. Eur. J. Pharmacol. 2003; 470: 171-175Crossref PubMed Scopus (170) Google Scholar), is the hypotensive effect in the hypertensive To this we administered apelin to a hypertensive model animal, SHR, and measured the blood pressure. WKY rats were used as a the administration of apelin, the base-line blood pressure of WKY rats and was measured and apelin was into the WKY a and in blood pressure was (Fig. as previously (5Reaux A. De Mota N. Skultetyova I. Lenkei Z. El Messari S. Gallatz K. Corvol P. Palkovits M. Llorens-Cortes C. J. Neurochem. 2001; 77: 1085-1096Crossref PubMed Scopus (319) Google Scholar, 7Lee D.K. Cheng R. Nguyen T. Fan T. Kariyawasam A.P. Liu Y. Osmond D.H. George S.R. O'Dowd B.F. J. Neurochem. 2000; 74: 34-41Crossref PubMed Scopus (577) Google Scholar, 8Tatemoto K. Takayama K. Zou M.X. Kumaki I. Zhang W. Kumano K. Fujimiya M. Regul. Pept. 2001; 99: 87-92Crossref PubMed Scopus (565) Google Scholar, 9Cheng X. Cheng X.S. Pang C.C. Eur. J. Pharmacol. 2003; 470: 171-175Crossref PubMed Scopus (170) Google Scholar). The administration of apelin to was to significantly the blood pressure in a (Fig. Thus, the hypotensive effect by the administration of apelin was in hypertensive but the of was that of WKY The of apelin on blood pressure regulation in the hypertensive model have not previously to it has that apelin-APJ signaling were in the models in in the animal models of pressure and in with heart in vivo I. P. G. S. M. H. S. J. J. M. H. Res. PubMed Scopus Google Scholar, G. F. I. O. M. M. R. Z. R. H. M. Biochem. Biophys. Res. Commun. 2003; PubMed Scopus Google Scholar, A. A. R. A. E. M. Robbins R. T. H. Z. T. 2003; PubMed Scopus Google Scholar). In addition, the a is the angiotensin was identified as the for apelin C. P. J. J. K. T. E. F. S. M. A. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of apelin-induced hypotensive in compared with WKY rats to the in the of the and of apelin and in the of including receptor of a on the of Apelin K. Takayama K. Zou M.X. Kumaki I. Zhang W. Kumano K. Fujimiya M. Regul. Pept. 2001; 99: 87-92Crossref PubMed Scopus (565) Google Scholar) that apelin the of the nitric system. NO generated by has a central role in the regulation of Therefore, we the of a nitric-oxide synthase inhibitor, l-NAME, against the response of apelin-APJ signaling in wild-type mice. After a single intraperitoneal injection mg/kg body of l-NAME, the systolic blood pressure increased from to and from to in wild-type and APJ-deficient mice This in systolic blood pressure with the administration of was described previously in and the systolic blood pressure of a at min after administration not we the apelin peptide at min after The injection of apelin an and in systolic blood pressure in the wild-type mice. In the administration of the of apelin in systolic blood pressure in wild-type mice with (Fig. The systolic blood pressure in APJ-deficient mice was not by the apelin peptide injection of administration (Fig. suggesting that the action of against the hypotensive effect in wild-type mice by apelin injection under the K. Takayama K. Zou M.X. Kumaki I. Zhang W. Kumano K. Fujimiya M. Regul. Pept. 2001; 99: 87-92Crossref PubMed Scopus (565) Google Scholar) that the administration of apelin significantly increased the in the in the was not in the rats with These a that the action of apelin-APJ signaling is the of of the NO in from by has a role in the regulation of was that is by a variety of physiological and including and and by is by phosphorylation at the on the human sequence and is equivalent to bovine and mouse the phosphorylation that is by protein a downstream of and to the J.E. I. T. S. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Y. K. A. PubMed Scopus Google Scholar, S. I. C. R. PubMed Scopus Google Scholar). To whether apelin-APJ signaling eNOS, the endothelial cells derived from wild-type and APJ-deficient mice were with apelin and phosphorylation of at was by using a phosphorylation in in the endothelial cells from wild-type and APJ-deficient mice was significantly by compared with cells phosphorylation by apelin was in wild-type endothelial cells but not in APJ-deficient endothelial cells This is the that apelin in endothelial cells and that APJ plays a role in apelin-induced phosphorylation of eNOS, which the hypotensive effect on blood pressure of APJ with of blood pressure the and systems other to blood pressure. To whether APJ plays a role in blood pressure regulation as a counterregulatory against vasopressor we performed the angiotensin administration and measured the systolic blood pressure of wild-type and APJ-deficient mice. The intraperitoneal injection of of angiotensin and in a in systolic blood pressure in both (Fig. suggesting that the pressor response by AT1 in the steady was not by the of the APJ gene. used a for angiotensin administration to clarify the APJ in blood pressure 1995; PubMed Scopus Google Scholar) previously evaluated the role of angiotensin-type receptor in blood pressure regulation as a counterregulatory against vasopressor actions by to mice. mice with the of for 1 to the of endogenous angiotensin and and physiological of angiotensin II. The wild-type mice an increased systolic blood pressure by the intraperitoneal injection of angiotensin On the the APJ-deficient mice a increased to angiotensin and compared with wild-type (Fig. In to clarify a role of APJ in blood pressure we generated double knock-out mice for APJ and AT1a by crossing APJ-deficient and AT1a-deficient mice. in AT1a-deficient mice compared with that of wild-type mice as described previously (11Sugaya T. Nishimatsu S. Tanimoto K. Takimoto E. Yamagishi T. Imamura K. Goto S. Imaizumi K. Hisada Y. Otsuka A. Uchida H. Sugiura M. Fukuta K. Fukamizu A. Murakami K. J. Biol. Chem. 1995; 270: 18719-18722Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). the to the vasopressor actions on the AT1a-deficient the base-line blood pressure of double mutant mice homozygous for both APJ and AT1a was elevated significantly compared with that of AT1a-deficient mice and These data that APJ has a to the blood pressure. it was that mice the in to angiotensin vasopressor action 1995; PubMed Scopus Google Scholar, T. C. S. Y. A. F. I. T. 1995; PubMed Scopus Google Scholar). APJ as a of the in blood pressure regulation against the including angiotensin AT1 In the of the APJ gene in mice by gene targeting that this receptor is for the hypotensive effect of apelin in mice in vivo and plays a counterregulatory role against vasopressor In addition, by using endothelial cells derived from mice, it is suggested that the hypotensive effect by apelin-APJ signaling is through the by In the the of apelin-APJ the functions of APJ in the tissues including the central nervous and cardiovascular systems This animal model for the in vivo role of apelin-APJ signaling in and and for for the cardiovascular Nishimatsu and the Fukamizu for and