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A Novel Inhibitory Effect on Prostacyclin Synthesis of Coupling Factor 6 Extracted from the Heart of Spontaneously Hypertensive Rats

1998; Elsevier BV; Volume: 273; Issue: 48 Linguagem: Inglês

10.1074/jbc.273.48.31778

1083-351X

Tomohiro Osanai, Takaatsu Kamada, Naoto Fujiwara, Takeshi Katoh, Koki Takahashi, Masao Kimura, Kiyohiko Satoh, Koji Magota, Shiho Kodama, Takaharu Tanaka, Ken Okumura,

Cancer, Hypoxia, and Metabolism

The possible presence of an unknown prostacyclin synthesis inhibitory substance has been reported in some strains of rats. We purified the inhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatography and two steps of reverse-phase high performance liquid chromatography. The amino acid composition and automated gas-phase sequencing of the full-length substance and fragments cleaved by AspN indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cleavable fusion protein strategy, attenuated base-line and bradykinin (10−6 m)-induced prostacyclin synthesis and [3H]arachidonic acid (AA) release in human umbilical vein endothelial cells in a dose-dependent manner (10−9–10−7 m). Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10−7 mrecombinant coupling factor 6. Base-line and bradykinin-induced [3H]AA release were suppressed by arachidonyltrifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 at 40 μm, and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine at 1 μmnor bromoenol lactone at 1 μm affected AA release. Preincubation (1 min) with 10−7 m recombinant coupling factor 6 had no influence on adenosine diphosphate- and collagen-induced platelet aggregations. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic phospholipase A2, although it is unclear whether coupling factor 6 functions in normal conditions or only in pathophysiological states. The possible presence of an unknown prostacyclin synthesis inhibitory substance has been reported in some strains of rats. We purified the inhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatography and two steps of reverse-phase high performance liquid chromatography. The amino acid composition and automated gas-phase sequencing of the full-length substance and fragments cleaved by AspN indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cleavable fusion protein strategy, attenuated base-line and bradykinin (10−6 m)-induced prostacyclin synthesis and [3H]arachidonic acid (AA) release in human umbilical vein endothelial cells in a dose-dependent manner (10−9–10−7 m). Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10−7 mrecombinant coupling factor 6. Base-line and bradykinin-induced [3H]AA release were suppressed by arachidonyltrifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 at 40 μm, and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine at 1 μmnor bromoenol lactone at 1 μm affected AA release. Preincubation (1 min) with 10−7 m recombinant coupling factor 6 had no influence on adenosine diphosphate- and collagen-induced platelet aggregations. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic phospholipase A2, although it is unclear whether coupling factor 6 functions in normal conditions or only in pathophysiological states. arachidonic acid phospholipase A2 human umbilical vein endothelial cells high performance liquid chromatography prostaglandin F arachidonyltrifluoromethyl ketone spontaneously hypertensive rats oleyloxyethyl phosphorylcholine bromoenol lactone Dulbecco's modified Eagle's medium. Prostacyclin, a potent vasodilator and the most potent endogenous inhibitor of platelet aggregation known, is synthesized from arachidonic acid (AA)1 by various stimuli in many types of cells, including vascular endothelial cells and smooth muscle cells. Of a number of stimuli, bradykinin (1Menke J.G. Borkowski J.A. Bierilo K.K. MacNeil T. Derrick A.W. Schneck K.A. Ransom R.W. Strader C.D. Linemeyer D.L. Hess J.F. J. Biol. Chem. 1994; 269: 21583-21586Abstract Full Text PDF PubMed Google Scholar,2Mceachern A.E. Shelton E.R. Bhakta S. Obernolte R. Bach C. Zuppan P. Fujisaki J. Aldrich R.W. Jarnagin K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7724-7728Crossref PubMed Scopus (402) Google Scholar) and arginine vasopressin (3Byron K.L. Circ. Res. 1996; 78: 813-820Crossref PubMed Scopus (42) Google Scholar), whose receptors are coupled to GTP-binding proteins, enhance AA release via Ca2+-dependent translocation of cytosolic phospholipase A2 (PLA2) (4Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265 (,): 5409-5413Abstract Full Text PDF PubMed Google Scholar) and the activation of PLA2 due to phosphorylation (5Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 6Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar). In contrast, growth factors (7Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 8Murakami M. Kudo I. Inoue K. J. Biol . Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar), whose receptors are coupled to tyrosine kinase, activate PLA2 by the additional mechanism of de novo protein synthesis. A novel substance, designated prostacyclin-stimulating factor (9Yamauchi T. Umeda F. Masakado M. Isaji M. Mizushima S. Nawata H. Biochem. J. 1994; 303: 591-598Crossref PubMed Scopus (84) Google Scholar), has recently been discovered to be a stimulus for AA release, and the physiological and pathophysiological roles of this new substance in health and disease are being investigated.The in vivo function of endogenous prostacyclin was unknown until quite recently. Mice lacking the gene for encoding the prostacyclin receptor (10Murata T. Ushikubi F. Matsuoka T. Hirata M. Yamasaki A. Sugimoto Y. Ichikawa A. Aze Y. Tanaka T. Yoshida N. Ueno A. Oh-ishi S. Narumiya S. Nature. 1997; 388: 678-682Crossref PubMed Scopus (680) Google Scholar), which was cloned from a human lung library (11Nakagawa O. Tanaka I. Usui T. Harada M. Sasaki Y. Itoh H. Yoshimasa T. Namba T. Narumiya S. Nakao K. Circulation. 1995; 90: 1643-1647Crossref Scopus (100) Google Scholar), demonstrated increased susceptibility to thrombosis, reduced pain perception, and decreased inflammatory response. These functions seem to be related to the amount of prostacyclin, which is regulated not only by stimuli that augment synthesis but also by the clearance from the circulating system and synthesis inhibitors. A prostaglandin (PG) transporter (12Kanai N. Lu R. Satria J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (346) Google Scholar), which was isolated from the protein encoded by the rat matrin complementary DNA, mediates the vascular clearance of classical PGs on passage through the pulmonary circulation but does not mediate the clearance of prostacyclin. Thus, it could not contribute to the regulation of prostacyclin effects. On the other hand, lipomodulin or macrocortin (13Flower R.J. Blackwell G.J. Nature. 1979; 278: 456-459Crossref PubMed Scopus (698) Google Scholar, 14Blackwell G.J. Carnuccio R. Di Rosa M. Flower R.J. Parente L. Persico P. Nature. 1980; 287: 147-149Crossref PubMed Scopus (691) Google Scholar, 15Hirata F. Carmine R.D. Nelson C.A. Axelrod J. Schiffmann E. Warabi A. De Blas A.L. Nirenberg M. Manganiello V. Vaughan M. Kumagai S. Green I. Decker J.L. Steinberg A.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3190-3194Crossref PubMed Scopus (110) Google Scholar), which is known to be an inhibitory protein of PLA2, inhibits the inflammatory effect of prostacyclin, but it is limited only when anti-inflammatory glucocorticoids were administered and only to the inside of the cell.In recent years, exposure to a high salt diet has been reported to increase the plasma concentration of an unknown substance that inhibits AA release from the plasma membrane in Dahl salt-sensitive rats (16Uehara Y. Tobian L. Iwai J. Hypertension. 1986; 8 (suppl.): 180-186Google Scholar) and Wistar rats (17Uehara Y. Ishii M. Shimitsu T.I. Sugimoto T. Hypertension. 1987; 9 (suppl.): 6-12Google Scholar). In addition, urinary excretion of 2,3-dinor-6-keto-PGF1α, an indicator for endogenous prostacyclin levels, was demonstrated to be similar in both spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats, despite the enhanced activity of prostacyclin synthesis in SHR-derived aortic strips (18Osanai T. Matsumura H. Kikuchi T. Minami O. Yokono Y. Akiba R. Eidou H. Konta A. Kanazawa T. Onodera K. Metoki H. Oike Y. Jpn. Circ. J. 1990; 54: 507-514Crossref PubMed Scopus (15) Google Scholar, 19Falardeau P. Robillard M. Martineau A. Prostaglandins. 1985; 29: 621-628Crossref PubMed Scopus (21) Google Scholar). Thus, these lines of accumulating evidence strongly suggest that there may be undiscovered endogenous prostacyclin synthesis inhibitor(s). Based on this reasoning, we tried to isolate and identify the prostacyclin synthesis inhibitory substance(s) by using SHR, which appear to be a suitable source for isolation, and to characterize the effects of the inhibitory substance synthesized by cleavable fusion protein strategy.DISCUSSIONIn the present study, a prostacyclin synthesis inhibitory substance was purified from the heart of SHR, and its structure was determined. It was a 76-amino acid peptide, and the sequence from the N terminus to the 40th amino acid was completely identical to the known peptide designated coupling factor 6. Further analysis of fragment peptides cleaved by AspN revealed that the amino acid sequences of the initial four fragments were identical to the 1–39th sequences and the other four to the 40–55th, the 56–64th, the 65–71st, and the 72–76th sequences of rat coupling factor 6. Thus, the purified peptide was completely identical to rat coupling factor 6. Then, we reconstructed rat coupling factor 6 using a cleavable fusion protein strategy and confirmed the effect of this recombinant peptide on prostacyclin synthesis. Recombinant coupling factor 6 manifested the same effect as the purified substance, and prostacyclin synthesis inhibitory substance was verified as identical to coupling factor 6.Coupling factor 6 (25Knowles A.F. Guillory R.J. Racker E. J. Biol. Chem. 1971; 246: 2672-2679Abstract Full Text PDF PubMed Google Scholar) is known to act inside the cell as an energy transducer in mitochondrial adenosine triphosphate synthase that consists of three domains, namely the extrinsic and intrinsic membrane domains, F1 and F0, respectively, joined by a stalk. F0 is the proton channel of the complex that spans the inner mitochondrial membrane, and the protons are conducted from F0 through the stalk to the catalytic sites in F1 composed of five kinds of polypeptides (26Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 27Walker J.E. Fearnley I.M. Gay N.J. Gibson B.W. Northrop F.D. Powell S.J. Runswick M.J. Saraste M. Tybulewicz V.L.J. J. Mol. Biol. 1985; 184: 677-701Crossref PubMed Scopus (380) Google Scholar). Four subunits of the stalk have been identified and designated as follows: the oligomycin sensitivity conferral protein, coupling factor 6, and subunit b and d (28Kagawa Y. Racker E. J. Biol. Chem. 1966; 241: 2475-2482Abstract Full Text PDF PubMed Google Scholar, 29Walker J.E. Runswick M.J. Poulter L. J. Mol. Biol. 1987; 197: 89-100Crossref PubMed Scopus (116) Google Scholar, 30Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar), of which coupling factor 6 is reported to be essentially required for energy transduction (25Knowles A.F. Guillory R.J. Racker E. J. Biol. Chem. 1971; 246: 2672-2679Abstract Full Text PDF PubMed Google Scholar). Coupling factor 6 is first synthesized as an immature form in the cytosol, then led to the mitochondria by an import signal peptide (32 amino acids) present in the upstream (31Higuti T. Tsurumi C. Kawamura Y. Tsujita H. Osaka F. Yoshihara Y. Tani I. Tanaka K. Ichikawa A. Biochem. Biophys. Res. Commun. 1991; 178: 793-799Crossref PubMed Scopus (21) Google Scholar), and then becomes an active form by the enzymatic deletion of the signal peptide. In the present study, we discovered a novel function of this peptide, an inhibitory effect on prostacyclin synthesis. This effect was demonstrated when the peptide was administered from the outside of the cell. Therefore, after organ damage such as myocardial infarction and myocarditis, this peptide may exert its inhibitory effect on prostacyclin synthesis. The questions whether coupling factor 6, a mitochondrial integral peptide, reaches an appropriate site of action in normal conditions and how coupling factor 6 contributes to the regulation of prostacyclin production in SHR remain to be elucidated.It is of interest to speculate the site of action of coupling factor 6. Because this peptide attenuated the use of endogenous AA in prostacyclin synthesis without affecting the uses of exogenous AA and PGH2, its effect appeared to be focused on PLA2independently of cyclooxygenase and prostacyclin synthase. To confirm this issue, we directly measured the release of [3H]AA from the pre-labeled cells. The result demonstrated that coupling factor 6 suppresses base-line [3H]AA release in HUVEC and blocks the bradykinin-induced increase in [3H]AA release, strongly suggesting that AA release from the plasma membrane is a target of this peptide. The complete blockade of the bradykinin-induced increase in AA release by coupling factor 6 may be overestimated by its suppressant effect on base-line AA release.Based on biochemical properties and structural features, the PLA2 superfamily, in which nine different PLA2groups have been identified to date (32Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (756) Google Scholar), can be subdivided into three main types, i.e. Ca2+-dependent secretory enzymes (sPLA2), Ca2+-dependent cytosolic enzymes (cPLA2), and Ca2+-independent cytosolic enzymes (iPLA2). Although PLA2 inhibitors currently available are not necessarily isoform-specific, the combined use of each inhibitor may allow us to examine the specific function of each PLA2 isoform in vitro. AACOCF3(cPLA2 inhibitor) exhibits 500-fold greater potency to cPLA2 than to sPLA2 (33Riendeau D. Guay J. Weech P.K. Laliberte J. Yergey L. Li C. Desmarais S. Perrier H. Liu S. Nicoll-Griffith D. Street I.P. J. Biol. Chem. 1994; 269: 15619-15624Abstract Full Text PDF PubMed Google Scholar, 34Street I.P. Lin H.K. Laliberte' F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weech P.K. Gelb M.H. Biochemistry. 1993; 32: 5935-5940Crossref PubMed Scopus (419) Google Scholar) but also inhibits macrophage iPLA2 with IC50 value of 15 μm (35Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). BEL (iPLA2 inhibitor) manifests greater than 1000-fold selectivity for iPLA2 than for sPLA2 and cPLA2 (36Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 37Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). However, BEL has been found to block Mg2+-dependent phosphatidate phosphohydrolase, a key enzyme in cellular phospholipid metabolism (38Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 31937-31941Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Base-line and bradykinin-induced AA releases were both suppressed by AACOCF3 but not by OPC (sPLA2 inhibitor) and BEL, suggesting that cPLA2 contributes to AA release under these conditions. Coupling factor 6 suppressed base-line and bradykinin-induced AA releases, and the simultaneous administration of AACOCF3 and coupling factor 6 had no additive effect to that by AACOCF3, suggesting that coupling factor 6 inhibits AA release from the plasma membrane via suppression of cPLA2 activity.A distinct difference in the responsiveness to coupling factor 6 was clearly observed between endothelial cells and platelets. We demonstrated that the acting site of coupling factor 6 was AA release from the plasma membrane and not prostacyclin synthase. Therefore, it is reasonable to presume that this peptide may inhibit thromboxane A2 synthesis in platelets and suppress aggregation of platelets. However, pretreatment of platelets with coupling factor 6 had no influence on the maximum value of platelet aggregation. This lack of effect suggests that coupling factor 6 may exert an AA release-inhibitory effect with cell specificity. Thus, the major PG suppressed by coupling factor 6 is prostacyclin. The mechanism for the cell specificity of the suppressant effect of coupling factor 6 is unknown. However, if coupling factor 6 indirectly inhibits cPLA2 via mediators such as receptors, one possible reason may be the presence or absence of its mediator(s) in the cell.Because prostacyclin receptor is expressed in many tissues such as aorta, lung, atrium, ventricle, and kidney (20Osanai T. Kanazawa T. Kamada T. Okuguchi T. Kosugi T. Mio Y. Imaoka Y. Metoki H. Oike Y. Onodera K. Hypertens. Res. 1994; 17: 227-232Crossref Scopus (3) Google Scholar), and because PG transporter (12Kanai N. Lu R. Satria J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (346) Google Scholar) does not mediate the vascular clearance of prostacyclin, the endogenous prostacyclin synthesis inhibitory peptide may have inhibitory effects against widespread biological actions of prostacyclin. This peptide also may counteract a biological action of AA such as inhibition of voltage-gated Ca2+ current, because a major acting site of coupling factor 6 is the inhibition of AA release from the plasma membrane. Further investigations, especially on the in vivo effects of exogenous coupling factor 6, will be required to establish the effect and role of this peptide. Prostacyclin, a potent vasodilator and the most potent endogenous inhibitor of platelet aggregation known, is synthesized from arachidonic acid (AA)1 by various stimuli in many types of cells, including vascular endothelial cells and smooth muscle cells. Of a number of stimuli, bradykinin (1Menke J.G. Borkowski J.A. Bierilo K.K. MacNeil T. Derrick A.W. Schneck K.A. Ransom R.W. Strader C.D. Linemeyer D.L. Hess J.F. J. Biol. Chem. 1994; 269: 21583-21586Abstract Full Text PDF PubMed Google Scholar,2Mceachern A.E. Shelton E.R. Bhakta S. Obernolte R. Bach C. Zuppan P. Fujisaki J. Aldrich R.W. Jarnagin K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7724-7728Crossref PubMed Scopus (402) Google Scholar) and arginine vasopressin (3Byron K.L. Circ. Res. 1996; 78: 813-820Crossref PubMed Scopus (42) Google Scholar), whose receptors are coupled to GTP-binding proteins, enhance AA release via Ca2+-dependent translocation of cytosolic phospholipase A2 (PLA2) (4Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265 (,): 5409-5413Abstract Full Text PDF PubMed Google Scholar) and the activation of PLA2 due to phosphorylation (5Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 6Hazen S.L. Gross R.W. J. Biol. Chem. 1993; 268: 9892-9900Abstract Full Text PDF PubMed Google Scholar). In contrast, growth factors (7Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 8Murakami M. Kudo I. Inoue K. J. Biol . Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar), whose receptors are coupled to tyrosine kinase, activate PLA2 by the additional mechanism of de novo protein synthesis. A novel substance, designated prostacyclin-stimulating factor (9Yamauchi T. Umeda F. Masakado M. Isaji M. Mizushima S. Nawata H. Biochem. J. 1994; 303: 591-598Crossref PubMed Scopus (84) Google Scholar), has recently been discovered to be a stimulus for AA release, and the physiological and pathophysiological roles of this new substance in health and disease are being investigated. The in vivo function of endogenous prostacyclin was unknown until quite recently. Mice lacking the gene for encoding the prostacyclin receptor (10Murata T. Ushikubi F. Matsuoka T. Hirata M. Yamasaki A. Sugimoto Y. Ichikawa A. Aze Y. Tanaka T. Yoshida N. Ueno A. Oh-ishi S. Narumiya S. Nature. 1997; 388: 678-682Crossref PubMed Scopus (680) Google Scholar), which was cloned from a human lung library (11Nakagawa O. Tanaka I. Usui T. Harada M. Sasaki Y. Itoh H. Yoshimasa T. Namba T. Narumiya S. Nakao K. Circulation. 1995; 90: 1643-1647Crossref Scopus (100) Google Scholar), demonstrated increased susceptibility to thrombosis, reduced pain perception, and decreased inflammatory response. These functions seem to be related to the amount of prostacyclin, which is regulated not only by stimuli that augment synthesis but also by the clearance from the circulating system and synthesis inhibitors. A prostaglandin (PG) transporter (12Kanai N. Lu R. Satria J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (346) Google Scholar), which was isolated from the protein encoded by the rat matrin complementary DNA, mediates the vascular clearance of classical PGs on passage through the pulmonary circulation but does not mediate the clearance of prostacyclin. Thus, it could not contribute to the regulation of prostacyclin effects. On the other hand, lipomodulin or macrocortin (13Flower R.J. Blackwell G.J. Nature. 1979; 278: 456-459Crossref PubMed Scopus (698) Google Scholar, 14Blackwell G.J. Carnuccio R. Di Rosa M. Flower R.J. Parente L. Persico P. Nature. 1980; 287: 147-149Crossref PubMed Scopus (691) Google Scholar, 15Hirata F. Carmine R.D. Nelson C.A. Axelrod J. Schiffmann E. Warabi A. De Blas A.L. Nirenberg M. Manganiello V. Vaughan M. Kumagai S. Green I. Decker J.L. Steinberg A.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3190-3194Crossref PubMed Scopus (110) Google Scholar), which is known to be an inhibitory protein of PLA2, inhibits the inflammatory effect of prostacyclin, but it is limited only when anti-inflammatory glucocorticoids were administered and only to the inside of the cell. In recent years, exposure to a high salt diet has been reported to increase the plasma concentration of an unknown substance that inhibits AA release from the plasma membrane in Dahl salt-sensitive rats (16Uehara Y. Tobian L. Iwai J. Hypertension. 1986; 8 (suppl.): 180-186Google Scholar) and Wistar rats (17Uehara Y. Ishii M. Shimitsu T.I. Sugimoto T. Hypertension. 1987; 9 (suppl.): 6-12Google Scholar). In addition, urinary excretion of 2,3-dinor-6-keto-PGF1α, an indicator for endogenous prostacyclin levels, was demonstrated to be similar in both spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats, despite the enhanced activity of prostacyclin synthesis in SHR-derived aortic strips (18Osanai T. Matsumura H. Kikuchi T. Minami O. Yokono Y. Akiba R. Eidou H. Konta A. Kanazawa T. Onodera K. Metoki H. Oike Y. Jpn. Circ. J. 1990; 54: 507-514Crossref PubMed Scopus (15) Google Scholar, 19Falardeau P. Robillard M. Martineau A. Prostaglandins. 1985; 29: 621-628Crossref PubMed Scopus (21) Google Scholar). Thus, these lines of accumulating evidence strongly suggest that there may be undiscovered endogenous prostacyclin synthesis inhibitor(s). Based on this reasoning, we tried to isolate and identify the prostacyclin synthesis inhibitory substance(s) by using SHR, which appear to be a suitable source for isolation, and to characterize the effects of the inhibitory substance synthesized by cleavable fusion protein strategy. DISCUSSIONIn the present study, a prostacyclin synthesis inhibitory substance was purified from the heart of SHR, and its structure was determined. It was a 76-amino acid peptide, and the sequence from the N terminus to the 40th amino acid was completely identical to the known peptide designated coupling factor 6. Further analysis of fragment peptides cleaved by AspN revealed that the amino acid sequences of the initial four fragments were identical to the 1–39th sequences and the other four to the 40–55th, the 56–64th, the 65–71st, and the 72–76th sequences of rat coupling factor 6. Thus, the purified peptide was completely identical to rat coupling factor 6. Then, we reconstructed rat coupling factor 6 using a cleavable fusion protein strategy and confirmed the effect of this recombinant peptide on prostacyclin synthesis. Recombinant coupling factor 6 manifested the same effect as the purified substance, and prostacyclin synthesis inhibitory substance was verified as identical to coupling factor 6.Coupling factor 6 (25Knowles A.F. Guillory R.J. Racker E. J. Biol. Chem. 1971; 246: 2672-2679Abstract Full Text PDF PubMed Google Scholar) is known to act inside the cell as an energy transducer in mitochondrial adenosine triphosphate synthase that consists of three domains, namely the extrinsic and intrinsic membrane domains, F1 and F0, respectively, joined by a stalk. F0 is the proton channel of the complex that spans the inner mitochondrial membrane, and the protons are conducted from F0 through the stalk to the catalytic sites in F1 composed of five kinds of polypeptides (26Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 27Walker J.E. Fearnley I.M. Gay N.J. Gibson B.W. Northrop F.D. Powell S.J. Runswick M.J. Saraste M. Tybulewicz V.L.J. J. Mol. Biol. 1985; 184: 677-701Crossref PubMed Scopus (380) Google Scholar). Four subunits of the stalk have been identified and designated as follows: the oligomycin sensitivity conferral protein, coupling factor 6, and subunit b and d (28Kagawa Y. Racker E. J. Biol. Chem. 1966; 241: 2475-2482Abstract Full Text PDF PubMed Google Scholar, 29Walker J.E. Runswick M.J. Poulter L. J. Mol. Biol. 1987; 197: 89-100Crossref PubMed Scopus (116) Google Scholar, 30Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar), of which coupling factor 6 is reported to be essentially required for energy transduction (25Knowles A.F. Guillory R.J. Racker E. J. Biol. Chem. 1971; 246: 2672-2679Abstract Full Text PDF PubMed Google Scholar). Coupling factor 6 is first synthesized as an immature form in the cytosol, then led to the mitochondria by an import signal peptide (32 amino acids) present in the upstream (31Higuti T. Tsurumi C. Kawamura Y. Tsujita H. Osaka F. Yoshihara Y. Tani I. Tanaka K. Ichikawa A. Biochem. Biophys. Res. Commun. 1991; 178: 793-799Crossref PubMed Scopus (21) Google Scholar), and then becomes an active form by the enzymatic deletion of the signal peptide. In the present study, we discovered a novel function of this peptide, an inhibitory effect on prostacyclin synthesis. This effect was demonstrated when the peptide was administered from the outside of the cell. Therefore, after organ damage such as myocardial infarction and myocarditis, this peptide may exert its inhibitory effect on prostacyclin synthesis. The questions whether coupling factor 6, a mitochondrial integral peptide, reaches an appropriate site of action in normal conditions and how coupling factor 6 contributes to the regulation of prostacyclin production in SHR remain to be elucidated.It is of interest to speculate the site of action of coupling factor 6. Because this peptide attenuated the use of endogenous AA in prostacyclin synthesis without affecting the uses of exogenous AA and PGH2, its effect appeared to be focused on PLA2independently of cyclooxygenase and prostacyclin synthase. To confirm this issue, we directly measured the release of [3H]AA from the pre-labeled cells. The result demonstrated that coupling factor 6 suppresses base-line [3H]AA release in HUVEC and blocks the bradykinin-induced increase in [3H]AA release, strongly suggesting that AA release from the plasma membrane is a target of this peptide. The complete blockade of the bradykinin-induced increase in AA release by coupling factor 6 may be overestimated by its suppressant effect on base-line AA release.Based on biochemical properties and structural features, the PLA2 superfamily, in which nine different PLA2groups have been identified to date (32Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (756) Google Scholar), can be subdivided into three main types, i.e. Ca2+-dependent secretory enzymes (sPLA2), Ca2+-dependent cytosolic enzymes (cPLA2), and Ca2+-independent cytosolic enzymes (iPLA2). Although PLA2 inhibitors currently available are not necessarily isoform-specific, the combined use of each inhibitor may allow us to examine the specific function of each PLA2 isoform in vitro. AACOCF3(cPLA2 inhibitor) exhibits 500-fold greater potency to cPLA2 than to sPLA2 (33Riendeau D. Guay J. Weech P.K. Laliberte J. Yergey L. Li C. Desmarais S. Perrier H. Liu S. Nicoll-Griffith D. Street I.P. J. Biol. Chem. 1994; 269: 15619-15624Abstract Full Text PDF PubMed Google Scholar, 34Street I.P. Lin H.K. Laliberte' F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weech P.K. Gelb M.H. Biochemistry. 1993; 32: 5935-5940Crossref PubMed Scopus (419) Google Scholar) but also inhibits macrophage iPLA2 with IC50 value of 15 μm (35Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). BEL (iPLA2 inhibitor) manifests greater than 1000-fold selectivity for iPLA2 than for sPLA2 and cPLA2 (36Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 37Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). However, BEL has been found to block Mg2+-dependent phosphatidate phosphohydrolase, a key enzyme in cellular phospholipid metabolism (38Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 31937-31941Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Base-line and bradykinin-induced AA releases were both suppressed by AACOCF3 but not by OPC (sPLA2 inhibitor) and BEL, suggesting that cPLA2 contributes to AA release under these conditions. Coupling factor 6 suppressed base-line and bradykinin-induced AA releases, and the simultaneous administration of AACOCF3 and coupling factor 6 had no additive effect to that by AACOCF3, suggesting that coupling factor 6 inhibits AA release from the plasma membrane via suppression of cPLA2 activity.A distinct difference in the responsiveness to coupling factor 6 was clearly observed between endothelial cells and platelets. We demonstrated that the acting site of coupling factor 6 was AA release from the plasma membrane and not prostacyclin synthase. Therefore, it is reasonable to presume that this peptide may inhibit thromboxane A2 synthesis in platelets and suppress aggregation of platelets. However, pretreatment of platelets with coupling factor 6 had no influence on the maximum value of platelet aggregation. This lack of effect suggests that coupling factor 6 may exert an AA release-inhibitory effect with cell specificity. Thus, the major PG suppressed by coupling factor 6 is prostacyclin. The mechanism for the cell specificity of the suppressant effect of coupling factor 6 is unknown. However, if coupling factor 6 indirectly inhibits cPLA2 via mediators such as receptors, one possible reason may be the presence or absence of its mediator(s) in the cell.Because prostacyclin receptor is expressed in many tissues such as aorta, lung, atrium, ventricle, and kidney (20Osanai T. Kanazawa T. Kamada T. Okuguchi T. Kosugi T. Mio Y. Imaoka Y. Metoki H. Oike Y. Onodera K. Hypertens. Res. 1994; 17: 227-232Crossref Scopus (3) Google Scholar), and because PG transporter (12Kanai N. Lu R. Satria J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (346) Google Scholar) does not mediate the vascular clearance of prostacyclin, the endogenous prostacyclin synthesis inhibitory peptide may have inhibitory effects against widespread biological actions of prostacyclin. This peptide also may counteract a biological action of AA such as inhibition of voltage-gated Ca2+ current, because a major acting site of coupling factor 6 is the inhibition of AA release from the plasma membrane. Further investigations, especially on the in vivo effects of exogenous coupling factor 6, will be required to establish the effect and role of this peptide. In the present study, a prostacyclin synthesis inhibitory substance was purified from the heart of SHR, and its structure was determined. It was a 76-amino acid peptide, and the sequence from the N terminus to the 40th amino acid was completely identical to the known peptide designated coupling factor 6. Further analysis of fragment peptides cleaved by AspN revealed that the amino acid sequences of the initial four fragments were identical to the 1–39th sequences and the other four to the 40–55th, the 56–64th, the 65–71st, and the 72–76th sequences of rat coupling factor 6. Thus, the purified peptide was completely identical to rat coupling factor 6. Then, we reconstructed rat coupling factor 6 using a cleavable fusion protein strategy and confirmed the effect of this recombinant peptide on prostacyclin synthesis. Recombinant coupling factor 6 manifested the same effect as the purified substance, and prostacyclin synthesis inhibitory substance was verified as identical to coupling factor 6. Coupling factor 6 (25Knowles A.F. Guillory R.J. Racker E. J. Biol. Chem. 1971; 246: 2672-2679Abstract Full Text PDF PubMed Google Scholar) is known to act inside the cell as an energy transducer in mitochondrial adenosine triphosphate synthase that consists of three domains, namely the extrinsic and intrinsic membrane domains, F1 and F0, respectively, joined by a stalk. F0 is the proton channel of the complex that spans the inner mitochondrial membrane, and the protons are conducted from F0 through the stalk to the catalytic sites in F1 composed of five kinds of polypeptides (26Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (913) Google Scholar, 27Walker J.E. Fearnley I.M. Gay N.J. Gibson B.W. Northrop F.D. Powell S.J. Runswick M.J. Saraste M. Tybulewicz V.L.J. J. Mol. Biol. 1985; 184: 677-701Crossref PubMed Scopus (380) Google Scholar). Four subunits of the stalk have been identified and designated as follows: the oligomycin sensitivity conferral protein, coupling factor 6, and subunit b and d (28Kagawa Y. Racker E. J. Biol. Chem. 1966; 241: 2475-2482Abstract Full Text PDF PubMed Google Scholar, 29Walker J.E. Runswick M.J. Poulter L. J. Mol. Biol. 1987; 197: 89-100Crossref PubMed Scopus (116) Google Scholar, 30Collinson I.R. van Raaij M.J. Runswick M.J. Fearnley I.M. Skehel J.M. Orriss G.L. Miroux B. Walker J.E. J. Mol. Biol. 1994; 242: 408-421PubMed Google Scholar), of which coupling factor 6 is reported to be essentially required for energy transduction (25Knowles A.F. Guillory R.J. Racker E. J. Biol. Chem. 1971; 246: 2672-2679Abstract Full Text PDF PubMed Google Scholar). Coupling factor 6 is first synthesized as an immature form in the cytosol, then led to the mitochondria by an import signal peptide (32 amino acids) present in the upstream (31Higuti T. Tsurumi C. Kawamura Y. Tsujita H. Osaka F. Yoshihara Y. Tani I. Tanaka K. Ichikawa A. Biochem. Biophys. Res. Commun. 1991; 178: 793-799Crossref PubMed Scopus (21) Google Scholar), and then becomes an active form by the enzymatic deletion of the signal peptide. In the present study, we discovered a novel function of this peptide, an inhibitory effect on prostacyclin synthesis. This effect was demonstrated when the peptide was administered from the outside of the cell. Therefore, after organ damage such as myocardial infarction and myocarditis, this peptide may exert its inhibitory effect on prostacyclin synthesis. The questions whether coupling factor 6, a mitochondrial integral peptide, reaches an appropriate site of action in normal conditions and how coupling factor 6 contributes to the regulation of prostacyclin production in SHR remain to be elucidated. It is of interest to speculate the site of action of coupling factor 6. Because this peptide attenuated the use of endogenous AA in prostacyclin synthesis without affecting the uses of exogenous AA and PGH2, its effect appeared to be focused on PLA2independently of cyclooxygenase and prostacyclin synthase. To confirm this issue, we directly measured the release of [3H]AA from the pre-labeled cells. The result demonstrated that coupling factor 6 suppresses base-line [3H]AA release in HUVEC and blocks the bradykinin-induced increase in [3H]AA release, strongly suggesting that AA release from the plasma membrane is a target of this peptide. The complete blockade of the bradykinin-induced increase in AA release by coupling factor 6 may be overestimated by its suppressant effect on base-line AA release. Based on biochemical properties and structural features, the PLA2 superfamily, in which nine different PLA2groups have been identified to date (32Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (756) Google Scholar), can be subdivided into three main types, i.e. Ca2+-dependent secretory enzymes (sPLA2), Ca2+-dependent cytosolic enzymes (cPLA2), and Ca2+-independent cytosolic enzymes (iPLA2). Although PLA2 inhibitors currently available are not necessarily isoform-specific, the combined use of each inhibitor may allow us to examine the specific function of each PLA2 isoform in vitro. AACOCF3(cPLA2 inhibitor) exhibits 500-fold greater potency to cPLA2 than to sPLA2 (33Riendeau D. Guay J. Weech P.K. Laliberte J. Yergey L. Li C. Desmarais S. Perrier H. Liu S. Nicoll-Griffith D. Street I.P. J. Biol. Chem. 1994; 269: 15619-15624Abstract Full Text PDF PubMed Google Scholar, 34Street I.P. Lin H.K. Laliberte' F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weech P.K. Gelb M.H. Biochemistry. 1993; 32: 5935-5940Crossref PubMed Scopus (419) Google Scholar) but also inhibits macrophage iPLA2 with IC50 value of 15 μm (35Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). BEL (iPLA2 inhibitor) manifests greater than 1000-fold selectivity for iPLA2 than for sPLA2 and cPLA2 (36Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar, 37Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). However, BEL has been found to block Mg2+-dependent phosphatidate phosphohydrolase, a key enzyme in cellular phospholipid metabolism (38Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 31937-31941Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Base-line and bradykinin-induced AA releases were both suppressed by AACOCF3 but not by OPC (sPLA2 inhibitor) and BEL, suggesting that cPLA2 contributes to AA release under these conditions. Coupling factor 6 suppressed base-line and bradykinin-induced AA releases, and the simultaneous administration of AACOCF3 and coupling factor 6 had no additive effect to that by AACOCF3, suggesting that coupling factor 6 inhibits AA release from the plasma membrane via suppression of cPLA2 activity. A distinct difference in the responsiveness to coupling factor 6 was clearly observed between endothelial cells and platelets. We demonstrated that the acting site of coupling factor 6 was AA release from the plasma membrane and not prostacyclin synthase. Therefore, it is reasonable to presume that this peptide may inhibit thromboxane A2 synthesis in platelets and suppress aggregation of platelets. However, pretreatment of platelets with coupling factor 6 had no influence on the maximum value of platelet aggregation. This lack of effect suggests that coupling factor 6 may exert an AA release-inhibitory effect with cell specificity. Thus, the major PG suppressed by coupling factor 6 is prostacyclin. The mechanism for the cell specificity of the suppressant effect of coupling factor 6 is unknown. However, if coupling factor 6 indirectly inhibits cPLA2 via mediators such as receptors, one possible reason may be the presence or absence of its mediator(s) in the cell. Because prostacyclin receptor is expressed in many tissues such as aorta, lung, atrium, ventricle, and kidney (20Osanai T. Kanazawa T. Kamada T. Okuguchi T. Kosugi T. Mio Y. Imaoka Y. Metoki H. Oike Y. Onodera K. Hypertens. Res. 1994; 17: 227-232Crossref Scopus (3) Google Scholar), and because PG transporter (12Kanai N. Lu R. Satria J.A. Bao Y. Wolkoff A.W. Schuster V.L. Science. 1995; 268: 866-869Crossref PubMed Scopus (346) Google Scholar) does not mediate the vascular clearance of prostacyclin, the endogenous prostacyclin synthesis inhibitory peptide may have inhibitory effects against widespread biological actions of prostacyclin. This peptide also may counteract a biological action of AA such as inhibition of voltage-gated Ca2+ current, because a major acting site of coupling factor 6 is the inhibition of AA release from the plasma membrane. Further investigations, especially on the in vivo effects of exogenous coupling factor 6, will be required to establish the effect and role of this peptide. We thank Drs. H. Matsue and T. Naraoka at Aomori Advanced Industrial Technology Center for peptide sequence analysis.