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Atrial and Brain Natriuretic Peptides Inhibit the Endothelin-1 Secretory Response to Angiotensin II in Porcine Aorta Masakazu Kohno, Koji Yokokawa, Takeshi Horio, Kenichi Yasunari, Koh-ichi Murakawa, and Tadanao Takeda We have recently shown that the porcine aorta releases immunoreactive endothelin-1 in a time-dependent way. Here, we examined the inhibition by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) of endothelin-1 secretion after stimulation with angiotensin II (Ang II) by using porcine aorta. Ang II dose-dependently stimulated immunoreactive endothelin-1 secretion. Porcine ANP-(1-28) and porcine BNP-26 both inhibited such secretion in a dose-dependent way. The addition of a cyclic guanosine 5'-monophosphate (cGMP) analogue, 8-bromo-cGMP, reduced the immunoreactive endothelin- 1 secretion after stimulation with Ang II. In cultured porcine endothelial cells the inhibition by porcine ANP-(1-28) and porcine BNP-26 of immunoreactive endothelin-1 secretion after stimulation with Ang II was paralleled by an increase in the cellular cGMP level. Rat ANP-(5-25) was weaker than porcine ANP-(1-28) in inhibiting immunoreactive endothelin-1 secretion and increasing cGMP in cultured cells. There was negative correlation between the percent decrease in immunoreactive endothelin-1 and the percent increase in cGMP. Neither porcine ANP-(1-28) nor BNP-26 affected the number or sensitivity of Ang II binding sites in cultured porcine endothelial cells. These results suggest that ANP and BNP inhibit endothelin-1 secretion after stimulation with Ang II, probably through a cGMP-dependent process. (Circulation Research 1992;70:241-247)
trial natriuretic peptide (ANP) is a diuretic, 4~ natriuretic, and vasodilatory peptide hor1k mone originally isolated from mammalian hearts.1 A novel natriuretic peptide, brain natriuretic peptide (BNP), has been identified in the porcine brain2 and later isolated from mammalian hearts.34 BNP has diuretic, natriuretic, and hypotensive effects and also relaxes the chick rectum, as ANP does.2 Both ANP and BNP may regulate vascular tonus and fluid homeostasis as cardiac hormones. Endothelin-1, a peptide of 21 amino acids that is produced by vascular endothelial cells, is a contractile agent.5,6 This peptide is present in human plasma and is at high levels in patients with acute myocardial infarction,7 uremia,8 or severe hypertension.9 This peptide is a potent secretagogue for ANP and BNP in atria.10-13 Cultured endothelial cells14 and aortic strips with an intact endothelium15,16 secrete immunoreactive endoFrom the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. Supported by a Grant-in-Aid for Scientific Research (6148210) from the Ministry of Education, Science, and Culture, Japan. Address for correspondence: Masakazu Kohno, MD, First Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan. Received October 11, 1990; accepted October 4, 1991.
thelin-1 in a time-dependent way. Angiotensin II (Ang II) stimulates endothelin-1 secretion from cultured bovine endothelial cells by a receptor-mediated process.14 On the other hand, ANP and BNP may act as physiological antagonists to the renin-angiotensin system in the regulation of vascular tonus and fluid homeostasis. So, this study was designed to elucidate whether the ANP-BNP system would interact with endothelin-1 secretion in the endothelium through the renin-angiotensin system. We examined the effects of porcine ANP-(1-28), rat ANP-(5-25), which is much weaker (2%) in vasorelaxant and natriuretic-diuretic potency than rat ANP-(1-28), and porcine BNP-26, the major secretory form of BNP in the porcine heart,'7 on the endothelin-1 secretory response to Ang II in porcine aorta. Furthermore, using cultured porcine endothelial cells, we examined the relation between the inhibition of immunoreactive endothelin-1 secretion by ANP and BNP and the level of cyclic guanosine 5'monophosphate (cGMP) in cells stimulated by Ang II. Materials and Methods Preparation of Porcine Aortic Strps The experiment was done with strips of adult porcine thoracic aortas as previously described.'5
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Aortas were obtained from 72 farm pigs killed at a nearby slaughterhouse (Nippon Meat Packers, Inc., Osaka, Japan) and were placed immediately in cold serum-free Dulbecco's modified Eagle's medium (DMEM) that contained penicillin (100 ,ug/ml) and streptomycin (50 ,ug/ml). All side branches and their connective tissue were then removed. Aortic segments were cut transversely (20 cm long) and vertically (4 cm wide). Each aortic segment was cut into 20 strips measuring 2 x 2 cm. Twenty strips from one animal were used in each experiment. Examination of the luminal surfaces of the aortas by light microscopy confirmed that the endothelium of the aorta was not damaged by this procedure. Endothelial Cell Culture Endothelial cells were isolated from five adult porcine thoracic aortas by a standard scraping technique and grown to confluence in DMEM containing 10% fetal calf serum (FCS), penicillin (100 ,ug/ml), and streptomycin (50 yg/ml). These cells were identified by their typical "cobblestone" appearance by phase-contrast microscopy and by their immunofluorescence when stained for factor VIII antigen. Contamination by cells with the morphological features of smooth muscle cells was not found. Cultures were maintained at 37°C with atmospheric air and 5% CO2, and subculturing was done after the treatment with Versene followed by trypsin. Materials and Supplies
Ang II, 8-bromo-cGMP, 3-isobutyl-1-methylxanthine (IBMX), and an endothelial cell growth supplement were purchased from Sigma Chemical Co., St. Louis, Mo. DMEM, trypsin, Versene, and FCS were purchased from GIBCO Laboratories, Grand Island, N.Y. Flasks and polypropylene tubes were purchased from Becton Dickinson & Co., Lincoln Park, N.J. Synthetic endothelin-1, endothelin-2, endothelin-3, big endothelin-1 (porcine, 1-39), somatostatin, /3-endorphin, human secretin, porcine ANP(1-28), rat ANP-(5-25), human BNP-32, and porcine BNP-26 were purchased from Peptide Institute, Inc., Osaka, Japan. Endothelin-1 antiserum was purchased from Peninsula Laboratories, Inc., Belmont, Calif. `25P-labeled endothelin-1 and `25P-labeled Ang II were purchased from Amersham Japan, Inc., Tokyo, Japan. The cGMP assay kit was purchased from Yamasa Shoyu Co., Ltd., Chiba, Japan.18 Pharnacological Treatment Protocol 1 (porcine aorta). Aortic strips were suspended in a 50-ml polypropylene tube filled with 30 ml serum-free DMEM that contained penicillin (100 ,ug/ml), streptomycin (50 ,ug/ml), and aprotinin (500 kallidinogenase inactivator units/ml). Ang II, ANP, and BNP were dissolved in distilled water, and then these agents were added to the medium (except for the control preparation) in a volume of less than 0.2% of the medium. Both control and treated media with aortic strips were maintained at
TABLE 1. Effect of Passage Numbers on Spontaneous and Angiotensin II-Stimulated Secretion of Immunoreactive Endothelin-1 in Cultured Porcine Endothelial Cells ir-Endothelin-1 level (pg/4 hours/5xl105 cells) after: Passage 6 Passage 3 Passage 4 19.2±3.1 20.7±3.3 19.7±2.8 Baseline 62.5 ±4.2* 60.4±4.8* 61.2±3.9* Ang II (108 M)
Values are mean+SEM of assays made of four cell cultures. ir, Immunoreactive; Ang II, angiotensin II. *Significant difference compared with the baseline level
(p
Atrial and Brain Natriuretic Peptides Inhibit the Endothelin-1 Secretory Response to Angiotensin II in Porcine Aorta Masakazu Kohno, Koji Yokokawa, Takeshi Horio, Kenichi Yasunari, Koh-ichi Murakawa, and Tadanao Takeda We have recently shown that the porcine aorta releases immunoreactive endothelin-1 in a time-dependent way. Here, we examined the inhibition by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) of endothelin-1 secretion after stimulation with angiotensin II (Ang II) by using porcine aorta. Ang II dose-dependently stimulated immunoreactive endothelin-1 secretion. Porcine ANP-(1-28) and porcine BNP-26 both inhibited such secretion in a dose-dependent way. The addition of a cyclic guanosine 5'-monophosphate (cGMP) analogue, 8-bromo-cGMP, reduced the immunoreactive endothelin- 1 secretion after stimulation with Ang II. In cultured porcine endothelial cells the inhibition by porcine ANP-(1-28) and porcine BNP-26 of immunoreactive endothelin-1 secretion after stimulation with Ang II was paralleled by an increase in the cellular cGMP level. Rat ANP-(5-25) was weaker than porcine ANP-(1-28) in inhibiting immunoreactive endothelin-1 secretion and increasing cGMP in cultured cells. There was negative correlation between the percent decrease in immunoreactive endothelin-1 and the percent increase in cGMP. Neither porcine ANP-(1-28) nor BNP-26 affected the number or sensitivity of Ang II binding sites in cultured porcine endothelial cells. These results suggest that ANP and BNP inhibit endothelin-1 secretion after stimulation with Ang II, probably through a cGMP-dependent process. (Circulation Research 1992;70:241-247)
trial natriuretic peptide (ANP) is a diuretic, 4~ natriuretic, and vasodilatory peptide hor1k mone originally isolated from mammalian hearts.1 A novel natriuretic peptide, brain natriuretic peptide (BNP), has been identified in the porcine brain2 and later isolated from mammalian hearts.34 BNP has diuretic, natriuretic, and hypotensive effects and also relaxes the chick rectum, as ANP does.2 Both ANP and BNP may regulate vascular tonus and fluid homeostasis as cardiac hormones. Endothelin-1, a peptide of 21 amino acids that is produced by vascular endothelial cells, is a contractile agent.5,6 This peptide is present in human plasma and is at high levels in patients with acute myocardial infarction,7 uremia,8 or severe hypertension.9 This peptide is a potent secretagogue for ANP and BNP in atria.10-13 Cultured endothelial cells14 and aortic strips with an intact endothelium15,16 secrete immunoreactive endoFrom the First Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. Supported by a Grant-in-Aid for Scientific Research (6148210) from the Ministry of Education, Science, and Culture, Japan. Address for correspondence: Masakazu Kohno, MD, First Department of Internal Medicine, Osaka City University Medical School, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545, Japan. Received October 11, 1990; accepted October 4, 1991.
thelin-1 in a time-dependent way. Angiotensin II (Ang II) stimulates endothelin-1 secretion from cultured bovine endothelial cells by a receptor-mediated process.14 On the other hand, ANP and BNP may act as physiological antagonists to the renin-angiotensin system in the regulation of vascular tonus and fluid homeostasis. So, this study was designed to elucidate whether the ANP-BNP system would interact with endothelin-1 secretion in the endothelium through the renin-angiotensin system. We examined the effects of porcine ANP-(1-28), rat ANP-(5-25), which is much weaker (2%) in vasorelaxant and natriuretic-diuretic potency than rat ANP-(1-28), and porcine BNP-26, the major secretory form of BNP in the porcine heart,'7 on the endothelin-1 secretory response to Ang II in porcine aorta. Furthermore, using cultured porcine endothelial cells, we examined the relation between the inhibition of immunoreactive endothelin-1 secretion by ANP and BNP and the level of cyclic guanosine 5'monophosphate (cGMP) in cells stimulated by Ang II. Materials and Methods Preparation of Porcine Aortic Strps The experiment was done with strips of adult porcine thoracic aortas as previously described.'5
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Circulation Research Vol 70, No 2 February 1992
Aortas were obtained from 72 farm pigs killed at a nearby slaughterhouse (Nippon Meat Packers, Inc., Osaka, Japan) and were placed immediately in cold serum-free Dulbecco's modified Eagle's medium (DMEM) that contained penicillin (100 ,ug/ml) and streptomycin (50 ,ug/ml). All side branches and their connective tissue were then removed. Aortic segments were cut transversely (20 cm long) and vertically (4 cm wide). Each aortic segment was cut into 20 strips measuring 2 x 2 cm. Twenty strips from one animal were used in each experiment. Examination of the luminal surfaces of the aortas by light microscopy confirmed that the endothelium of the aorta was not damaged by this procedure. Endothelial Cell Culture Endothelial cells were isolated from five adult porcine thoracic aortas by a standard scraping technique and grown to confluence in DMEM containing 10% fetal calf serum (FCS), penicillin (100 ,ug/ml), and streptomycin (50 yg/ml). These cells were identified by their typical "cobblestone" appearance by phase-contrast microscopy and by their immunofluorescence when stained for factor VIII antigen. Contamination by cells with the morphological features of smooth muscle cells was not found. Cultures were maintained at 37°C with atmospheric air and 5% CO2, and subculturing was done after the treatment with Versene followed by trypsin. Materials and Supplies
Ang II, 8-bromo-cGMP, 3-isobutyl-1-methylxanthine (IBMX), and an endothelial cell growth supplement were purchased from Sigma Chemical Co., St. Louis, Mo. DMEM, trypsin, Versene, and FCS were purchased from GIBCO Laboratories, Grand Island, N.Y. Flasks and polypropylene tubes were purchased from Becton Dickinson & Co., Lincoln Park, N.J. Synthetic endothelin-1, endothelin-2, endothelin-3, big endothelin-1 (porcine, 1-39), somatostatin, /3-endorphin, human secretin, porcine ANP(1-28), rat ANP-(5-25), human BNP-32, and porcine BNP-26 were purchased from Peptide Institute, Inc., Osaka, Japan. Endothelin-1 antiserum was purchased from Peninsula Laboratories, Inc., Belmont, Calif. `25P-labeled endothelin-1 and `25P-labeled Ang II were purchased from Amersham Japan, Inc., Tokyo, Japan. The cGMP assay kit was purchased from Yamasa Shoyu Co., Ltd., Chiba, Japan.18 Pharnacological Treatment Protocol 1 (porcine aorta). Aortic strips were suspended in a 50-ml polypropylene tube filled with 30 ml serum-free DMEM that contained penicillin (100 ,ug/ml), streptomycin (50 ,ug/ml), and aprotinin (500 kallidinogenase inactivator units/ml). Ang II, ANP, and BNP were dissolved in distilled water, and then these agents were added to the medium (except for the control preparation) in a volume of less than 0.2% of the medium. Both control and treated media with aortic strips were maintained at
TABLE 1. Effect of Passage Numbers on Spontaneous and Angiotensin II-Stimulated Secretion of Immunoreactive Endothelin-1 in Cultured Porcine Endothelial Cells ir-Endothelin-1 level (pg/4 hours/5xl105 cells) after: Passage 6 Passage 3 Passage 4 19.2±3.1 20.7±3.3 19.7±2.8 Baseline 62.5 ±4.2* 60.4±4.8* 61.2±3.9* Ang II (108 M)
Values are mean+SEM of assays made of four cell cultures. ir, Immunoreactive; Ang II, angiotensin II. *Significant difference compared with the baseline level
(p
