.J Mol
Cell Cardiol 22, 839-842
(1990)
RAPID Endothelin
Stimulates Cultured
COMMUNICATION
Angiotensin Pulmonary
I to Angiotensin Artery Endothelial
II Conversion Cells
(Received 12 December 1989, accepted in revisedform 12 Februay
in
1990)
We studied the effects of endothelin on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells. Endothelin had a novel effect on angiotensin I conversion. When endothelin was added to pulmonary artery endothelial cells, the conversion of angiotensin I to angiotensin II was enhanced about two-fold. The maximum stimulation was achieved at lo- o M of endothelin. This stimulatory effect was suppressed by angiotensin converting enzyme inhibitors such an enalapril. When the calcium antagonist, nifedipine, was incubated with lo- * M of endothelin, the conversion of angiotensin I to angiotensin II stimulated with endothelin was slightly suppressed by nifedipine. Enalapril (lo-* M) completely inhibited the conversion of angiotensin I to angiotensin II in the presence of endothelin. These results suggest that endothelin may play an important role in regulating vascular tone by modulating the conversion of angiotensin I to angiotensin II.
Endothelin1 (ET- 1) is a 2 1 amino acid peptide isolated from the culture medium of porcine aortic endothelial cells (Yanagisawa et al., 1988). ET- 1 possesses important biological and pharmacological properties: it induces a systemic hypertension in the rat (Yanagisawa et al., 1988) and bronchoconstriction in the guinea-pig (Braquet et al., 1989), produces a potent and long-lasting constriction in vascular and non-vascular isolated organs (Yanagisawa et al., 1988), and appears to have a specific reco.gnition site in smooth muscle cells (‘Hirata et al, 1988). On the other hand, angiotensin converting enzyme (ACE) exists in pulmonary vasculature (Ryan et al., 1970), kidney (Franklin et al., 1979), the mesenteric arcade (DiSalvo and Montefusco, 1957), and in isolated tissue preparations such as rabbit aortic strips (Ackerly et al., 1977). Since the quantity of angiotensin II produced across several intact vascular beds was greater than that attributable to plasma ACE activity, it was hypothesized that ACE was located on the luminal surface of the vascular endothelium. This hypothesis was confirmed (Ryan et al., 1976; Saye et al., 0022-2828/90/080839 + 04 $03.00/O
1984). Recently, we have demonstrated that ACE activity is stimulated by plateletactivating factor (Kawaguchi and Yasuda, 1984; Kawaguchi et al., 1989). On the other hand it is suppressed by atria1 natriuretic factor in cultured pulmonary artery endothelial cells (Kawaguchi et al., 1990). In this study we attempted to determine the effect of endothelin on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells and to clarify one of the mechanisms by which endothelin constricts vascular vessels. [rz51]Angiotensin I and [iz51]angiotensin II were obtained from Amersham International, UK; enalapril was a generous gift from Merck Sharp and Dohme, NJ, angiotensin I and angiotensin IT were purchased from Sigma, St Louis, MO; all other materials were of reagent grade. Aortic endothelial cells (bovine pulmonary artery; PEAC) were obtained from Flow Laboratories (Rockville, MD) and maintained in Minimum Eagles’ Medium (MEM) supplemented with lOo/0 fetal bovine serum (FBS) from Flow Laboratories. The cells were IC 1990 Acadrmic
Press Limitrd
Kapid
840
Communication:
incubated at 37°C in a humidified 5% CO2/95% air atmosphere (Kawaguchi and Yasuda, 1986a; Kawaguchi and Yasuda, 1988). Cells grown to confluence were subcultured at 2 x IO5 cells/dish in 3 ml of MEM containing 0.3% FBS in 35-mm dishes (Kawaguchi and Yasuda, 1986b,c). Cultures establishedin this manner were usedthroughout the courseof the study. Cellsfrom passages 20-50 were used in this seriesof experiments. The quality of this cell line was checked by measuring the conversion of angiotensin I to angiotensin II. It was constant during these passagesof the cell line. Angiotensin I conversion was done with confluent cultures (0.65 mg protein/dish) for all assays.Cells were washed with fresh MEM and then incubated with 0.3% FBS supplemented with MEM for 24 h at 37°C. After washing the cells again, [‘251]angiotensin I (0.1 &i/50 nmol) was added to the dishesand incubated for up to 60 min at 37°C with or without endothelin. [ 12-‘1] angiotensin I was separated from [ ‘251]angiotensin II on ODS column (Sep Pak, Waters Associated, Milford, MA), as described earlier. The column had been previously equilibrated with 10ml of Krebs-Henseleite buffer, followed by 20 ml of 0.1 M sodium phosphate buffer, pH 5.7. The sample was loaded onto the column and washed with 2 ml of 0.1 M sodium phosphate buffer. A solution (8.0 ml) of 80% phosphate
Kawaguchi
et al.
buffer, 20% acetonitrile (ACN, pH 5.7), was then passed through the column, and the fractions collected in tubes for measurementof radioactivity. This was followed by 5 ml of 0.1 M phosphate buffer (25%), 75% ACN (pH 5.7), after solution was collected in fractions and radioactivity was counted again. The columns were regenerated by washing with 5 ml of lOOo/oACN (Kawaguchi and Yasuda, 1984). Measurement of Ca2+ flux was carried out with cells prepared in 35-mm petri dishes as described above. For determination of Ca2+ influx, cells were washed with Hanks’s balanced salt solution with 1.26 mM calcium and incubated in 0.7 ml of Hanks’ balanced salt solution (containing 1.26 mM) in the presence of ET-l for 1 min at 37°C. Then 1 &i of 45Ca2+ was added for 1 min at 37°C. Thereafter, cells were scraped from dishes, filtered through a 0.45~ Millipore filter, washed twice with 3 ml of Hanks’ balanced salt solution and counted in lo-ml of Aquasol (Kawaguchi and Ysasuda, 1986c). For the cytoxicity assaycells were cultured in the samemanner as described for the assay of the angiotensin I converting study except that no [ ‘251]angiotensin I was used. Cell viability was assessed by the ability to exclude 0.23% trypan blue for 2 min at room temperature after 20 min incubation with endothelin, observed using an inverted microscope with
a)
, 0 12
II
, IO -Log
,
,
9
8
(ET-I)
M
,
,
7
6
0
IO
20 30 40 Time (min)
50
60
FIGURE 1. (a) Effect ofendothelin on angiotensin conversion. Endothelial cells were plated and assayed as described in the text. Points represent means & S.E. (b) Time course ofangiotensin conversion. Cells were cultured as described in the text. Angiotensin I was added and incubated for the indicated period with (0) or without (0) IO-* M ET-l. Points represent mean k S.E. Cell-free dishes without endothelin (A).
AngioteasiaConversioniaEndothelialCells
O-L,’
0
12
’ II
’ IO -Log
’ ’ ’ 9 8 7 (ET-0 M
’ 6
0 L,
841
,/I0
II
IO
L_1 ~ 1~ I 9 8 ,7 6
-LOCJ(ET-I)M
FIGURE 2. (a) Effect of endothelin on Ca’+-influx. Ca*+ -mflux was measured as described in the text. (b) Effect of angiotensin converting enzyme inhibitor and calcium antagonist on angiotensin conversion. Cells were, cultured as described in the text. _r tZsI]Angiotensin I and enalapril (0) or nifedipine (0) were added to dishes, then cells were _ incubated for 20 min. Points represent means + S.E.
phase optics (Kawaguchi and Yasuda, 1986c). In PAEC, 1.02 nmol/dish of angiotensin I was converted to angiotensin II for 20 min. When endothelin was added to dishes, the conversion of angiotensin I to angiotensin II was enhanced [Fig. l(a)]. The activation of ACE activity by endothelin was obvious at 10-t’ M, and it was dose-dependent. The stimulation persisted for up to 20 min [Fig. 1 (b)]. To clarify the mechanism of enhanced ACE activity stimulated by ET-l, Ca’+ was determined. ET-l stimulated Ca’+-influx in a dose-dependent manner [Fig. 2(a)]. As shown in Figure 2(b), ACE inhibitor completely inhibited endothelin-stimulated ACE activity. On the other hand, nifedipine ( 10e6 M) slightly suppressed its activity. Cytotoxicity was determined as described earlier. Cells were incubated with the concentrations of endothelin used for the ACE activity assay for 20 min, then trypan blue was added. The activation of ACE was seen from low9 to lo-’ M of endothelin. There was no cell damage at the concentrations which stimulated ACE activity. Endothelin stimulates Ca’+-influx into vascular smooth muscle cells and is insensitive to calcium antagonists (Chabrier et al., 1989). On the other hand, it is reported that calcium ionophore A23187 elevates ACE activity in cultured bovine endothelial cells (Dasarathy and Fanburg, 1990). We attempted to deterKEY WORDS:
Endothelin;
Angiotensin
converting
enzyme;
mine whether endothelin-induced Cazfinflux stimulates ACE activity in cultured pulmonary artery endothelial cells. In our experiments, endothelin stimulated the conversion of angiotensin I to angiotensin II. The maximal activity without cytotoxicity was seen at lo-’ M of endothelin. This stimulatory effect was inhibited by ACE inhibitor but not by a calcium antagonist. These results may suggest that ET-l stimulates ACE activity by elevating the intracellular calcium concentration in endothelial cells. Further investigation on the mechanism of the effect of elevated intracellular calcium induced by ET1 on ACE activity is under way. Acknowledgements This research was supported in part by Takeda Medical Research Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture 61132005 (Special Project Research “MetaResearches of Blood Vessels”), bolic and 62440040, 63480220, 63870037
63624502. HideakiKawaguchi,HirofumiSawaand HisakzauYasuda Department of Cardiovascular Medicine, Hokkaido University, School of Medicine, Sapporo 060, Japan Angiotensin
II
842
Rapid
Communication:
Kawaguchi
et al.
References MJ (1977) Role of converting enzyme in the response of atria, aorta and adrenal zona glomerulosa to des-Asp’ angiotensin I. Circ Res 41: 231-238. BRAQUET P, TOUVAY C, LACENTE V, VILAIN B, PONS F, HOSFORD D, CHABRIER P, MENCIA-HUERTA J (1989) Effect of endothelin-1 on blood pressure and bronchopulmonary system of the guinea pig. J Cardiovasc Pharmacol 13 [Suppl5]: S143-S146. CHABRIER PE, AUCUET M, ROUBERT P., LONCHAMPT MO, GILLARD-V, GUILLON JM, DEI.AFLOTTE S, BRAQUET P (1989) Vascular mechanism of action of endothelin-1: effect of Gas+ antagonist. J Cardiovasc Pharmacol 13 (Suppl 51: S32-S35. DASARATHY Y, FANBURG BL (1989) Calcium ionophore A23187 elevates-angiotensin-converting enzyme in cultured bovine endothelial cells. Biochim Biophys Acta 1010: 1619. DISALVO J, MONTEFUSCO CB (1957) Conversion of angiotensin I to angiotensin II in the canine mesenteric circulation. Am J Physiol221: 157661579. FRANKLIN WG, PEACH MJ, GILM~RE JP (1979) Evidence for the renal conversion of angiotensin 1 in the dog. Circ Res 27: 321-324. HIRATA Y, YOSHIMI M, TAKATA S (1988) Cellular mechanism ofaction by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commun 154: 868-875. KAWAGUCHI H, YASUDA H (1984) Platelet-activating factor stimulates phospholipase in quiescent Swiss mouse 31‘3 fibroblasts. FEBS Lett 176: 93-96. KAWAGUCHI H, YASUDA H (1986a) Effect of elastase on prostacyclin synthesis in aortic smooth muscle cells. Biochim Biophys Acta 878: 42-48. KAWAGUCHI H, YASUDA H (1986b) Platelet-activating factor stimulates prostaglandin synthesis in cultured cells. Hypertension 8: 192-197. KAWAGUCHI H, YASUDA H (1986c) Effect ofplatelet-activating factor on arachidonic acid metabolism in renal epithelial cells. Biochim Biophys Acta 875: 525-534. KAWAGUCHI H, YASUDA H (1988) Effect of elastase on phospholipase activity in aortic smooth muscle cells. Biochim Biophys Acta 958: 450-459. KAWAGUCHI H, SAWA H, YASUDA H ( 1989) Effect of atria1 natriuretic factor on angiotensin converting enzyme. J Mel Cell Cardiol 21: 959-961. KAWAGUCHI H, SAWA H, YASUDA H (1990) Platelet-activating factor stimulates angiotensin converting enzyme activity. J Hypertension 8: 1733177. RYAN JW, STEWART JM, LEARY WP, LEDINGHAM JG (1970) Metabolism ofangiotensin I in the pulmonary circulation. Biochem J 120: 221-223. RYAN US, RYAN JW, WnrrAkER C, CHIU A (1976) Localization ofangiotensin converting enzyme enzyme (kininase II) II. Immunocytochemistry and immunofluorescence. Tissue Cell 8: 125-145. SAYE JA, SINGER HA, PEACH MJ (1984) Role of endothelium in conversion of angiotensin I to angiotensin II in rabbit aorta. Hypertension 6: 216221. YANAGISAWA M, KURIHARA H, KIMURA S, TOMOBE Y, KOBAYASHI M, MITSUI Y, YAZAKI Y, GOTO K, MASAKI T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (Lond) 332: 411415. ACKERLY
JA,
TSAI BS, PEACH
Cell Cardiol 22, 839-842
(1990)
RAPID Endothelin
Stimulates Cultured
COMMUNICATION
Angiotensin Pulmonary
I to Angiotensin Artery Endothelial
II Conversion Cells
(Received 12 December 1989, accepted in revisedform 12 Februay
in
1990)
We studied the effects of endothelin on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells. Endothelin had a novel effect on angiotensin I conversion. When endothelin was added to pulmonary artery endothelial cells, the conversion of angiotensin I to angiotensin II was enhanced about two-fold. The maximum stimulation was achieved at lo- o M of endothelin. This stimulatory effect was suppressed by angiotensin converting enzyme inhibitors such an enalapril. When the calcium antagonist, nifedipine, was incubated with lo- * M of endothelin, the conversion of angiotensin I to angiotensin II stimulated with endothelin was slightly suppressed by nifedipine. Enalapril (lo-* M) completely inhibited the conversion of angiotensin I to angiotensin II in the presence of endothelin. These results suggest that endothelin may play an important role in regulating vascular tone by modulating the conversion of angiotensin I to angiotensin II.
Endothelin1 (ET- 1) is a 2 1 amino acid peptide isolated from the culture medium of porcine aortic endothelial cells (Yanagisawa et al., 1988). ET- 1 possesses important biological and pharmacological properties: it induces a systemic hypertension in the rat (Yanagisawa et al., 1988) and bronchoconstriction in the guinea-pig (Braquet et al., 1989), produces a potent and long-lasting constriction in vascular and non-vascular isolated organs (Yanagisawa et al., 1988), and appears to have a specific reco.gnition site in smooth muscle cells (‘Hirata et al, 1988). On the other hand, angiotensin converting enzyme (ACE) exists in pulmonary vasculature (Ryan et al., 1970), kidney (Franklin et al., 1979), the mesenteric arcade (DiSalvo and Montefusco, 1957), and in isolated tissue preparations such as rabbit aortic strips (Ackerly et al., 1977). Since the quantity of angiotensin II produced across several intact vascular beds was greater than that attributable to plasma ACE activity, it was hypothesized that ACE was located on the luminal surface of the vascular endothelium. This hypothesis was confirmed (Ryan et al., 1976; Saye et al., 0022-2828/90/080839 + 04 $03.00/O
1984). Recently, we have demonstrated that ACE activity is stimulated by plateletactivating factor (Kawaguchi and Yasuda, 1984; Kawaguchi et al., 1989). On the other hand it is suppressed by atria1 natriuretic factor in cultured pulmonary artery endothelial cells (Kawaguchi et al., 1990). In this study we attempted to determine the effect of endothelin on the conversion of angiotensin I to angiotensin II in pulmonary artery endothelial cells and to clarify one of the mechanisms by which endothelin constricts vascular vessels. [rz51]Angiotensin I and [iz51]angiotensin II were obtained from Amersham International, UK; enalapril was a generous gift from Merck Sharp and Dohme, NJ, angiotensin I and angiotensin IT were purchased from Sigma, St Louis, MO; all other materials were of reagent grade. Aortic endothelial cells (bovine pulmonary artery; PEAC) were obtained from Flow Laboratories (Rockville, MD) and maintained in Minimum Eagles’ Medium (MEM) supplemented with lOo/0 fetal bovine serum (FBS) from Flow Laboratories. The cells were IC 1990 Acadrmic
Press Limitrd
Kapid
840
Communication:
incubated at 37°C in a humidified 5% CO2/95% air atmosphere (Kawaguchi and Yasuda, 1986a; Kawaguchi and Yasuda, 1988). Cells grown to confluence were subcultured at 2 x IO5 cells/dish in 3 ml of MEM containing 0.3% FBS in 35-mm dishes (Kawaguchi and Yasuda, 1986b,c). Cultures establishedin this manner were usedthroughout the courseof the study. Cellsfrom passages 20-50 were used in this seriesof experiments. The quality of this cell line was checked by measuring the conversion of angiotensin I to angiotensin II. It was constant during these passagesof the cell line. Angiotensin I conversion was done with confluent cultures (0.65 mg protein/dish) for all assays.Cells were washed with fresh MEM and then incubated with 0.3% FBS supplemented with MEM for 24 h at 37°C. After washing the cells again, [‘251]angiotensin I (0.1 &i/50 nmol) was added to the dishesand incubated for up to 60 min at 37°C with or without endothelin. [ 12-‘1] angiotensin I was separated from [ ‘251]angiotensin II on ODS column (Sep Pak, Waters Associated, Milford, MA), as described earlier. The column had been previously equilibrated with 10ml of Krebs-Henseleite buffer, followed by 20 ml of 0.1 M sodium phosphate buffer, pH 5.7. The sample was loaded onto the column and washed with 2 ml of 0.1 M sodium phosphate buffer. A solution (8.0 ml) of 80% phosphate
Kawaguchi
et al.
buffer, 20% acetonitrile (ACN, pH 5.7), was then passed through the column, and the fractions collected in tubes for measurementof radioactivity. This was followed by 5 ml of 0.1 M phosphate buffer (25%), 75% ACN (pH 5.7), after solution was collected in fractions and radioactivity was counted again. The columns were regenerated by washing with 5 ml of lOOo/oACN (Kawaguchi and Yasuda, 1984). Measurement of Ca2+ flux was carried out with cells prepared in 35-mm petri dishes as described above. For determination of Ca2+ influx, cells were washed with Hanks’s balanced salt solution with 1.26 mM calcium and incubated in 0.7 ml of Hanks’ balanced salt solution (containing 1.26 mM) in the presence of ET-l for 1 min at 37°C. Then 1 &i of 45Ca2+ was added for 1 min at 37°C. Thereafter, cells were scraped from dishes, filtered through a 0.45~ Millipore filter, washed twice with 3 ml of Hanks’ balanced salt solution and counted in lo-ml of Aquasol (Kawaguchi and Ysasuda, 1986c). For the cytoxicity assaycells were cultured in the samemanner as described for the assay of the angiotensin I converting study except that no [ ‘251]angiotensin I was used. Cell viability was assessed by the ability to exclude 0.23% trypan blue for 2 min at room temperature after 20 min incubation with endothelin, observed using an inverted microscope with
a)
, 0 12
II
, IO -Log
,
,
9
8
(ET-I)
M
,
,
7
6
0
IO
20 30 40 Time (min)
50
60
FIGURE 1. (a) Effect ofendothelin on angiotensin conversion. Endothelial cells were plated and assayed as described in the text. Points represent means & S.E. (b) Time course ofangiotensin conversion. Cells were cultured as described in the text. Angiotensin I was added and incubated for the indicated period with (0) or without (0) IO-* M ET-l. Points represent mean k S.E. Cell-free dishes without endothelin (A).
AngioteasiaConversioniaEndothelialCells
O-L,’
0
12
’ II
’ IO -Log
’ ’ ’ 9 8 7 (ET-0 M
’ 6
0 L,
841
,/I0
II
IO
L_1 ~ 1~ I 9 8 ,7 6
-LOCJ(ET-I)M
FIGURE 2. (a) Effect of endothelin on Ca’+-influx. Ca*+ -mflux was measured as described in the text. (b) Effect of angiotensin converting enzyme inhibitor and calcium antagonist on angiotensin conversion. Cells were, cultured as described in the text. _r tZsI]Angiotensin I and enalapril (0) or nifedipine (0) were added to dishes, then cells were _ incubated for 20 min. Points represent means + S.E.
phase optics (Kawaguchi and Yasuda, 1986c). In PAEC, 1.02 nmol/dish of angiotensin I was converted to angiotensin II for 20 min. When endothelin was added to dishes, the conversion of angiotensin I to angiotensin II was enhanced [Fig. l(a)]. The activation of ACE activity by endothelin was obvious at 10-t’ M, and it was dose-dependent. The stimulation persisted for up to 20 min [Fig. 1 (b)]. To clarify the mechanism of enhanced ACE activity stimulated by ET-l, Ca’+ was determined. ET-l stimulated Ca’+-influx in a dose-dependent manner [Fig. 2(a)]. As shown in Figure 2(b), ACE inhibitor completely inhibited endothelin-stimulated ACE activity. On the other hand, nifedipine ( 10e6 M) slightly suppressed its activity. Cytotoxicity was determined as described earlier. Cells were incubated with the concentrations of endothelin used for the ACE activity assay for 20 min, then trypan blue was added. The activation of ACE was seen from low9 to lo-’ M of endothelin. There was no cell damage at the concentrations which stimulated ACE activity. Endothelin stimulates Ca’+-influx into vascular smooth muscle cells and is insensitive to calcium antagonists (Chabrier et al., 1989). On the other hand, it is reported that calcium ionophore A23187 elevates ACE activity in cultured bovine endothelial cells (Dasarathy and Fanburg, 1990). We attempted to deterKEY WORDS:
Endothelin;
Angiotensin
converting
enzyme;
mine whether endothelin-induced Cazfinflux stimulates ACE activity in cultured pulmonary artery endothelial cells. In our experiments, endothelin stimulated the conversion of angiotensin I to angiotensin II. The maximal activity without cytotoxicity was seen at lo-’ M of endothelin. This stimulatory effect was inhibited by ACE inhibitor but not by a calcium antagonist. These results may suggest that ET-l stimulates ACE activity by elevating the intracellular calcium concentration in endothelial cells. Further investigation on the mechanism of the effect of elevated intracellular calcium induced by ET1 on ACE activity is under way. Acknowledgements This research was supported in part by Takeda Medical Research Foundation and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture 61132005 (Special Project Research “MetaResearches of Blood Vessels”), bolic and 62440040, 63480220, 63870037
63624502. HideakiKawaguchi,HirofumiSawaand HisakzauYasuda Department of Cardiovascular Medicine, Hokkaido University, School of Medicine, Sapporo 060, Japan Angiotensin
II
842
Rapid
Communication:
Kawaguchi
et al.
References MJ (1977) Role of converting enzyme in the response of atria, aorta and adrenal zona glomerulosa to des-Asp’ angiotensin I. Circ Res 41: 231-238. BRAQUET P, TOUVAY C, LACENTE V, VILAIN B, PONS F, HOSFORD D, CHABRIER P, MENCIA-HUERTA J (1989) Effect of endothelin-1 on blood pressure and bronchopulmonary system of the guinea pig. J Cardiovasc Pharmacol 13 [Suppl5]: S143-S146. CHABRIER PE, AUCUET M, ROUBERT P., LONCHAMPT MO, GILLARD-V, GUILLON JM, DEI.AFLOTTE S, BRAQUET P (1989) Vascular mechanism of action of endothelin-1: effect of Gas+ antagonist. J Cardiovasc Pharmacol 13 (Suppl 51: S32-S35. DASARATHY Y, FANBURG BL (1989) Calcium ionophore A23187 elevates-angiotensin-converting enzyme in cultured bovine endothelial cells. Biochim Biophys Acta 1010: 1619. DISALVO J, MONTEFUSCO CB (1957) Conversion of angiotensin I to angiotensin II in the canine mesenteric circulation. Am J Physiol221: 157661579. FRANKLIN WG, PEACH MJ, GILM~RE JP (1979) Evidence for the renal conversion of angiotensin 1 in the dog. Circ Res 27: 321-324. HIRATA Y, YOSHIMI M, TAKATA S (1988) Cellular mechanism ofaction by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commun 154: 868-875. KAWAGUCHI H, YASUDA H (1984) Platelet-activating factor stimulates phospholipase in quiescent Swiss mouse 31‘3 fibroblasts. FEBS Lett 176: 93-96. KAWAGUCHI H, YASUDA H (1986a) Effect of elastase on prostacyclin synthesis in aortic smooth muscle cells. Biochim Biophys Acta 878: 42-48. KAWAGUCHI H, YASUDA H (1986b) Platelet-activating factor stimulates prostaglandin synthesis in cultured cells. Hypertension 8: 192-197. KAWAGUCHI H, YASUDA H (1986c) Effect ofplatelet-activating factor on arachidonic acid metabolism in renal epithelial cells. Biochim Biophys Acta 875: 525-534. KAWAGUCHI H, YASUDA H (1988) Effect of elastase on phospholipase activity in aortic smooth muscle cells. Biochim Biophys Acta 958: 450-459. KAWAGUCHI H, SAWA H, YASUDA H ( 1989) Effect of atria1 natriuretic factor on angiotensin converting enzyme. J Mel Cell Cardiol 21: 959-961. KAWAGUCHI H, SAWA H, YASUDA H (1990) Platelet-activating factor stimulates angiotensin converting enzyme activity. J Hypertension 8: 1733177. RYAN JW, STEWART JM, LEARY WP, LEDINGHAM JG (1970) Metabolism ofangiotensin I in the pulmonary circulation. Biochem J 120: 221-223. RYAN US, RYAN JW, WnrrAkER C, CHIU A (1976) Localization ofangiotensin converting enzyme enzyme (kininase II) II. Immunocytochemistry and immunofluorescence. Tissue Cell 8: 125-145. SAYE JA, SINGER HA, PEACH MJ (1984) Role of endothelium in conversion of angiotensin I to angiotensin II in rabbit aorta. Hypertension 6: 216221. YANAGISAWA M, KURIHARA H, KIMURA S, TOMOBE Y, KOBAYASHI M, MITSUI Y, YAZAKI Y, GOTO K, MASAKI T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (Lond) 332: 411415. ACKERLY
JA,
TSAI BS, PEACH
