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Mitochondrial coupling factor 6 as an endogenous inhibitor of prostacyclin synthesis and an endogenous vasoconstrictor
International Congress Series 1244 (2002) 131 – 141
Mitochondrial coupling factor 6 as an endogenous inhibitor of prostacyclin synthesis and an endogenous vasoconstrictor Tomohiro Osanai a,*, Satoko Okada a, Masayuki Saitoh a, Hirotsugu Ono a, Koji Magota b, Shiho Kodama b, Ken Okumura a a
The Second Department of Internal Medicine, Hirosaki University School of Medicine, Zaifu-cho 5, Hirosaki 036-8562, Japan b Suntory Biomedical Research Limited, Osaka, Japan
Abstract 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 reverse-phase high performance liquid chromatography. The automated gas-phase sequencing 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 baseline and bradykinin (10 6 M)-induced prostacyclin synthesis and [3H] arachidonic acid (AA) release in human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10 7 M recombinant coupling factor 6. Baseline and bradykinin-induced [3H] AA release were suppressed by arachidonyl trifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 (PLA2), and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine nor bromoenol lactone affected AA release. Intravenous injection of recombinant coupling factor 6 increased arterial blood pressure in rats, whereas a specific antibody to coupling factor 6 decreased systemic blood pressure. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic PLA2 and functions as an endogenous vasoconstrictor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Prostacyclin; Mitochondria; Phospholipase A2; Vascular endothelial cell; Coupling factor 6
*
Corresponding author. Tel.: +81-172-39-5057; fax: +81-171-35-9190. E-mail address: [email protected] (T. Osanai).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 3 6 - 8
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1. Introduction Prostacyclin, a potent vasodilator and the most potent endogenous inhibitor of platelet aggregation known, is synthesized from arachidonic acid (AA) by various stimuli in many types of cells. Of a number of stimuli, bradykinin [1] and arginine vasopressin [2], whose receptors are coupled to G proteins, enhance AA release via Ca2+-dependent translocation of cytosolic phospholipase A2 (PLA2) [3]. In contrast, growth factors [4], 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 [5], has recently been discovered to be a stimulus for AA release. The in vivo function of endogenous prostacyclin seems 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 [6] 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 [7,8], 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 which inhibits AA release from the plasma membrane in Dahl salt-sensitive rats [9] and Wistar rats [10]. In addition, urinary excretion of 2,3-dinor-6-keto-PGF1, an indicator for endogenous prostacyclin levels, was demonstrated to be similar in both spontaneously hypertensive rats (SHR) and normotensive Wistar – Kyoto rats (WKY), despite the enhanced activity of prostacyclin synthesis in SHR-derived aortic strips [11,12]. 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 in vivo and in vitro effects of the inhibitory substance synthesized by cleavable fusion protein strategy.
2. Materials and methods 2.1. Materials SHR and WKY (16 – 20 weeks old) were obtained from Charles River, Japan. [3H] 6-keto-PGF1 and [3H] AA were purchased from New England Nuclear, Boston, MA, USA. Antibodies and standards for 6-keto-PGF1 were kindly provided by Ono Pharmaceutical, Japan. Arachidonyl trifluoromethyl ketone (AACOCF3; Ca2+-dependent cytosolic PLA2 inhibitor) was purchased from Biomol Research Lab., Campus, PA, USA. PGH2, Oleyloxyethyl phosphorylcholine (OPC; Ca2+-dependent secretory PLA2 inhibitor), and bromoenol lactone (BEL; Ca2+-independent cytosolic PLA2 inhibitor) were purchased from Cayman Chemical, Ann Arbor, MI, USA. Cell culture media were purchased from
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Gibco, Grand Island, NY, USA. All other reagents were of the finest grade available from Sigma, St. Louis, MO, USA. 2.2. Isolation and determination of prostacyclin-synthesis inhibitory substance Brain, heart, lung, kidney, liver, and intestine obtained from SHR were heat-treated in boiling water for 10 min and extracted with 1 M acetic acid (AcOH). After desalting through a Sepak C18 column, followed by acetone precipitation at a concentration of 66%, the resulting supernatant was then evaporated in vacuo to dryness. The inhibitory activity of each crude extract was determined by measuring the amount of 6-keto-PGF1a released from vascular smooth muscle cells exposed to the extract [13]. Next, the dry material of SHR hearts, extracted by the same procedure exactly, was dissolved in 1 M AcOH, subjected to a column of Sephadex G-25. The active fraction from the gel-filtration column chromatography was applied on reverse-phase high performance liquid chromatography (HPLC) using an Inertsil ODS-2 C18 column, and purified as a single peptide peak. The amino acid sequence of the purified full-length peptide was first analyzed by automated gas-phase peptide sequencing. Next, after the peptide was digested with aspN at 37 jC for 6 h, the amino acid sequence of the peptide fragments was determined by the same procedure. 2.3. Synthesis of recombinant coupling factor 6 Recombinant rat coupling factor 6, to which the prostacyclin-synthesis inhibitory peptide was found to be identical, was obtained from Escherichia coli using a cleavable fusion protein strategy [14,15]. The amino acid sequence and molecular mass of this recombinant peptide were checked by automated gas-phase peptide sequencing and mass spectrometry. 2.4. Synthesis of antibody (Ab) for rat coupling factor 6 Synthetic coupling factor 6 fragment (rat Cys-58-76 amino acid) solution was emulsified with an equal volume of Freund’s complete adjuvant and injected subcutaneously into New Zealand white rabbits at multiple sites in the interscapulovertebral region. The concentration of endotoxin in anti-coupling factor 6 Ab was less than 1 pg/ml. 2.5. Effects of recombinant coupling factor 6 on prostacyclin synthesis Dose-dependent effects of recombinant coupling factor 6 (10 9 – 10 7 M) on baseline and bradykinin (10 6 M)-induced prostacyclin production were assessed by measuring the amount of 6-keto-PGF1a released from subconfluent human umbilical vein endothelial cells (HUVEC) on 24-well plates for 30 min. The subconfluent cells were incubated with coupling factor 6 in the absence or presence of 10 6 M bradykinin in serum-free Dulbecco’s modified Eagle’s medium (DMEM) for 30 min at 37 jC. To determine the site of action of coupling factor 6, the effects of this recombinant peptide at 10 7 M on conversion of both exogenous AA (10 ng/ml) to prostacyclin and exogenous PGH2 (10 ng/ ml) to prostacyclin were assessed by the same method.
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2.6. Effects of recombinant coupling factor 6 on AA release HUVEC were labeled for 24 h with [3H] AA (0.25 ACi in complete medium/well). The cells were incubated with coupling factor 6 in both the absence of and presence of 10 6 M bradykinin in serum-free DMEM. In some experiments, the cells were incubated with AACOCF3 at 40 AM, OPC at 1 AM, or BEL at 1 AM in both the absence of and presence of 10 6 M bradykinin. After 30 min, the medium was collected and the [3H] level was counted with a liquid scintillation counter. In addition, the effect of co-administration of coupling factor 6 at 10 7 M and AACOCF3 was also assessed by the same method. 2.7. Blood pressure measurement Blood pressure was determined by direct arterial cannulation in rats anesthetized with 75 mg/kg of ketamine hydrochloride and 15 mg/kg of xylazine hydrochloride. Two polyethylene catheters filled with heparinized saline solution were inserted via the left carotid artery and left femoral vein, respectively. The carotid catheter was connected to a pressure transducer coupled to a polygraph. Rat recombinant coupling factor 6 and anticoupling factor 6 Ab were injected into the vena cava. The effect of either recombinant coupling factor 6 (0.1, 0.3, and 1.0 Ag/kg body weight) or anti-coupling factor 6 Ab (0.3, 1.0, and 3.0 Ag/kg body weight) on the arterial vascular tone was examined in SHR and WKY. The responses of blood pressure to recombinant coupling factor 6 and its Ab were also investigated 30 min after the intravenous injection of indomethacin at 10 mg/kg in SHR. 2.8. Statistics All data are shown as meanFone standard error. An unpaired t-test for comparison of two variables and analysis of variance (ANOVA) for repeated measures for multiple comparison were used for statistical analysis. The level of significance was <0.05.
3. Results 3.1. Purification and determination of prostacyclin-synthesis inhibitory peptide Six-keto-PGF1a production was most strongly suppressed by the heart extract, and its suppression was abolished by pretreatment with trypsin and chymotrypsin. Therefore, the prostacyclin-inhibitory peptide was purified from hearts of SHR by collecting active fractions after gel-filtration column chromatography. The active fraction was applied to reverse-phase HPLC and eventually purified as a single peak. Automated gas-phase peptide sequencing of the purified full-length peptide was interpretable up to the 40th amino acid from the N-terminus, and was completely identical to coupling factor 6. The sequences of four fragment peptides cleaved by aspN were DRELFKLKQMYGKGEM, DKFPTFNFE, DPKFEVL, and DKPQS, which were completely identical to the 40th – 55th, the 56th –64th, the 65th – 71st, and the 72th – 76th amino acid sequences of rat
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Fig. 1. Effect of recombinant coupling factor 6 at doses of 10 9 to 10 7 M on basal prostacyclin synthesis in human umbilical vein endothelial cells. *p<0.05, **p<0.02. Results are presented as meanFone standard error for five experiments.
coupling factor 6, respectively. These results indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. 3.2. Effects of recombinant coupling factor 6 on prostacyclin synthesis As shown in Fig. 1, recombinant coupling factor 6 suppressed prostacyclin synthesis at baseline in a dose-dependent manner (10 9 – 10 7 M). Treatment with bradykinin at 10 6 M elicited an increase in prostacyclin synthesis from 272F45 to 469F24 pg/well/30 min
Fig. 2. Effect of recombinant coupling factor 6 (CF6) at doses of 10 9 to 10 7 M on bradykinin (10 6 M)-induced prostacyclin synthesis in human umbilical vein endothelial cells. *p<0.05, **p<0.02. Results are presented as meanFone standard error for five experiments.
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Fig. 3. Effect of recombinant coupling factor 6 at doses of 10 9 to 10 7 M on basal arachidonic acid release in human umbilical vein endothelial cells. *p<0.05, **p<0.02. Results are presented as meanFone standard error for four experiments.
(Fig. 2). Simultaneous administration of coupling factor 6 suppressed bradykinin-induced prostacyclin production in a dose-dependent manner. In contrast, exogenous AA-induced increase in prostacyclin synthesis above the baseline was unchanged even after treatment with 10 7 M recombinant coupling factor 6. An increase in prostacyclin production induced by exogenous PGH2 also was not affected by coupling factor 6 at 10 7 M.
Fig. 4. Effect of phospholipase A2 inhibitors on basal arachidonic acid release in human umbilical vein endothelial cells. *p<0.05. Results are presented as meanFone standard error for four experiments. AACOCF3: arachidonyl trifluoromethyl ketone at 40 AM. OPC: oleyloxyethyl phosphorylcholine at 1 AM. BEL: bromoenol lactone at 1 AM. CF6: recombinant coupling factor 6 at 100 nM.
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Fig. 5. Effect of recombinant coupling factor 6 (CF6) at 10 9 to 10 7 M on bradykinin (10 6 M)-induced arachidonic acid release in human umbilical vein endothelial cells. *p<0.05, **p<0.01. Results are presented as meanFone standard error for eight experiments.
3.3. Effects of recombinant coupling factor 6 on AA release As shown in Fig. 3, recombinant coupling factor 6 suppressed baseline [3H] AA release in a dose-dependent manner. Baseline [3H] AA release was suppressed by 40 AM AACOCF3 but not by 1 AM OPC and 1 AM BEL (Fig. 4). The addition of 10 7 M
Fig. 6. Effect of phospholipase A2 inhibitors on bradykinin (10 6 M)-induced arachidonic acid release in human umbilical vein endothelial cells. *p<0.05. Results are presented as meanFone standard error for four experiments. AACOCF3: arachidonyl trifluoromethyl ketone at 40 AM. OPC: oleyloxyethyl phosphorylcholine at 1 AM. BEL: bromoenol lactone at 1 AM. CF6: recombinant coupling factor 6 at 100 nM.
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coupling factor 6 to 40 AM AACOCF3 showed no further suppressant effect on [3H] AA release. Administration of bradykinin at 10 6 M elicited an increase in [3H] AA release from 687F44 to 1112F36 cpm/well/30 min. As shown in Fig. 5, simultaneous administration of coupling factor 6 suppressed this bradykinin-induced [3H] AA release in a dose-dependent manner, as seen in prostacyclin synthesis. AACOCF3 at 40 AM sup-
Fig. 7. (a) Changes in the arterial blood pressure after intravenous injection of anti-coupling factor 6 Ab (CF6 Ab) in Wistar – Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). (b) Changes in the arterial blood pressure after intravenous injection of rat recombinant coupling factor 6 (CF6) at 1 Ag/kg in WKY and SHR.
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pressed [3H] AA release in the presence of 10 6 M bradykinin, while neither 1 AM OPC nor 1 AM BEL affected bradykinin-induced [3H] AA release (Fig. 6). The addition of coupling factor 6 at 10 7 M to AACOCF3 at 40 AM showed no further suppressant effect on the [3H] AA release induced by bradykinin. 3.4. Physiological effect of perturbing coupling factor 6 activity in vivo Fig. 7a illustrates tracings of changes in arterial blood pressure after an injection of anticoupling factor 6 Ab into the femoral vein. In 16-week-old WKY, the intravenous bolus injection of anti-coupling factor 6 Ab at 0.3, 1.0, and 3.0 Ag/kg body weight produced a transient and rapid reduction in systolic and diastolic blood pressures. In age-matched SHR, the reduction of blood pressure occurred in seconds, reached a maximal reduction within 30 s, and returned to the baseline after several minutes. It was noted that the reduction of blood pressure was greater in SHR than in WKY. The reduction of blood pressure was blocked after 30-min pretreatment with indomethacin at 10 mg/kg. In 16-week-old WKY, an intravenous bolus injection of rat recombinant coupling factor 6 at 1.0 Ag/kg body weight produced a transient and rapid increase in systolic and diastolic arterial blood pressures (Fig. 7b). In age-matched SHR, the peptide increased arterial blood pressure more markedly within seconds, reaching the maximal increase within 30 s and returning to baseline after several minutes.
4. Discussion The present study showed that a prostacyclin-synthesis inhibitory substance is identical to the known peptide designated coupling factor 6 and functions as an endogenous vasoconstrictor in rats. Coupling factor 6 [16] is known to act inside the cell as an energy transducer in mitochondrial adenosine triphosphate synthase. It is of interest to speculate the site of novel inhibitory 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 PLA2 independently of cyclooxygenase and prostacyclin synthase. To confirm this issue, we directly measured the release of AA from the pre-labeled cells. The result demonstrated that coupling factor 6 suppresses baseline AA release in HUVEC and blocks the bradykinin-induced increase in AA release, strongly suggesting that AA release from the plasma membrane is a target of this peptide. Based on biochemical properties and structural features, the PLA2 superfamily can be subdivided into three main types [17], i.e. Ca 2+-dependent secretory enzymes (sPLA2), Ca2+-dependent cytosolic enzymes (cPLA2) and Ca2+-independent cytosolic enzymes (iPLA2). Although PLA2 inhibitors currently available are not necessarily isoformspecific, the combined use of each inhibitor may allow us to examine the specific function of each PLA2 isoform in vitro. AACOCF3 (cPLA2 inhibitor) not only exhibits 500-fold greater potency to cPLA2 than to sPLA2 [18], but also inhibits macrophage iPLA2 with IC50 value of 15 AM [19]. BEL (iPLA2 inhibitor) manifests greater than 1000-fold selectivity for iPLA2 than for sPLA2 and cPLA2 [20]. However, BEL has been found to
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block Mg2+-dependent phosphatidate phosphohydrolase, a key enzyme in cellular phospholipid metabolism [21]. Baseline 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 baseline 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. Because prostacyclin receptor is expressed in many tissues such as aorta, lung, atrium, ventricle and kidney [13], and because PG transporter [6] 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. We evaluated whether endogenous coupling factor 6 affects arterial blood pressure in vivo by blocking the effect of endogenous coupling factor 6 with anti-coupling factor 6 Ab. The results clearly showed that intravenous administration of anti-coupling factor 6 Ab caused a transient and rapid reduction in arterial blood pressure. In addition to our work with the blocking antibody, we performed complementary studies by treating rats with exogenous recombinant coupling factor 6. Injected peptide induced an increase in arterial blood pressure, and its extent was similar to that expected from the finding of neutralization of coupling factor 6, suggesting that its vasoconstrictor effect is unaffected by a single pass through the pulmonary vascular bed. Taken together, these findings strongly suggest that endogenous coupling factor 6 has a direct vasoconstrictor effect in the fashion of a circulating hormone. In conclusion, this report shows that mitochondrial coupling factor 6 is an endogenous inhibitor of prostacyclin synthesis and a potent endogenous vasoconstrictor.
References [1] J.G. Menke, J.A. Borkowski, K.K. Bierilo, T. MacNeil, A.W. Derrick, K.A. Schneck, R.W. Ransom, C.D. Strader, D.L. Linemeyer, J.F. Hess, Expression cloning of a human B1 bradykinin receptor, J. Biol. Chem. 269 (1994) 21583 – 21586. [2] K.L. Byron, Vasopressin stimulates Ca2+ spiking activity in A7r5 vascular smooth muscle cells via activation of phospholipase A2, Circ. Res. 78 (1996) 813 – 820. [3] J.Y. Channon, C.C. Leslie, A calcium-dependent mechanism for associating a soluble arachidonyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7, J. Biol. Chem. 265 (1990) 5409 – 5413. [4] E.A. Dennis, Diversity of group types, regulation, and function of phospholipase A2, J. Biol. Chem. 269 (1994) 13057 – 13060. [5] T. Yamauchi, F. Umeda, M. Masakado, M. Isaji, S. Mizushima, H. Nawata, Purification and molecular cloning of prostacyclin-stimulating factor from serum-free conditioned medium of human diploid fibroblast cells, Biochem. J. 303 (1994) 591 – 598. [6] N. Kanai, R. Lu, J.A. Satria, Y. Bao, A.W. Wolkoff, V.L. Schuster, Identification and characterization of a prostaglandin transporter, Science 268 (1995) 866 – 869. [7] G.J. Blackwell, R. Carnuccio, M.D. Rosa, R.J. Flower, L. Parente, P. Persico, Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids, Nature 287 (1980) 147 – 149.
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Mitochondrial coupling factor 6 as an endogenous inhibitor of prostacyclin synthesis and an endogenous vasoconstrictor Tomohiro Osanai a,*, Satoko Okada a, Masayuki Saitoh a, Hirotsugu Ono a, Koji Magota b, Shiho Kodama b, Ken Okumura a a
The Second Department of Internal Medicine, Hirosaki University School of Medicine, Zaifu-cho 5, Hirosaki 036-8562, Japan b Suntory Biomedical Research Limited, Osaka, Japan
Abstract 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 reverse-phase high performance liquid chromatography. The automated gas-phase sequencing 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 baseline and bradykinin (10 6 M)-induced prostacyclin synthesis and [3H] arachidonic acid (AA) release in human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10 7 M recombinant coupling factor 6. Baseline and bradykinin-induced [3H] AA release were suppressed by arachidonyl trifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 (PLA2), and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine nor bromoenol lactone affected AA release. Intravenous injection of recombinant coupling factor 6 increased arterial blood pressure in rats, whereas a specific antibody to coupling factor 6 decreased systemic blood pressure. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic PLA2 and functions as an endogenous vasoconstrictor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Prostacyclin; Mitochondria; Phospholipase A2; Vascular endothelial cell; Coupling factor 6
*
Corresponding author. Tel.: +81-172-39-5057; fax: +81-171-35-9190. E-mail address: [email protected] (T. Osanai).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 5 3 6 - 8
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1. Introduction Prostacyclin, a potent vasodilator and the most potent endogenous inhibitor of platelet aggregation known, is synthesized from arachidonic acid (AA) by various stimuli in many types of cells. Of a number of stimuli, bradykinin [1] and arginine vasopressin [2], whose receptors are coupled to G proteins, enhance AA release via Ca2+-dependent translocation of cytosolic phospholipase A2 (PLA2) [3]. In contrast, growth factors [4], 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 [5], has recently been discovered to be a stimulus for AA release. The in vivo function of endogenous prostacyclin seems 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 [6] 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 [7,8], 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 which inhibits AA release from the plasma membrane in Dahl salt-sensitive rats [9] and Wistar rats [10]. In addition, urinary excretion of 2,3-dinor-6-keto-PGF1, an indicator for endogenous prostacyclin levels, was demonstrated to be similar in both spontaneously hypertensive rats (SHR) and normotensive Wistar – Kyoto rats (WKY), despite the enhanced activity of prostacyclin synthesis in SHR-derived aortic strips [11,12]. 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 in vivo and in vitro effects of the inhibitory substance synthesized by cleavable fusion protein strategy.
2. Materials and methods 2.1. Materials SHR and WKY (16 – 20 weeks old) were obtained from Charles River, Japan. [3H] 6-keto-PGF1 and [3H] AA were purchased from New England Nuclear, Boston, MA, USA. Antibodies and standards for 6-keto-PGF1 were kindly provided by Ono Pharmaceutical, Japan. Arachidonyl trifluoromethyl ketone (AACOCF3; Ca2+-dependent cytosolic PLA2 inhibitor) was purchased from Biomol Research Lab., Campus, PA, USA. PGH2, Oleyloxyethyl phosphorylcholine (OPC; Ca2+-dependent secretory PLA2 inhibitor), and bromoenol lactone (BEL; Ca2+-independent cytosolic PLA2 inhibitor) were purchased from Cayman Chemical, Ann Arbor, MI, USA. Cell culture media were purchased from
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Gibco, Grand Island, NY, USA. All other reagents were of the finest grade available from Sigma, St. Louis, MO, USA. 2.2. Isolation and determination of prostacyclin-synthesis inhibitory substance Brain, heart, lung, kidney, liver, and intestine obtained from SHR were heat-treated in boiling water for 10 min and extracted with 1 M acetic acid (AcOH). After desalting through a Sepak C18 column, followed by acetone precipitation at a concentration of 66%, the resulting supernatant was then evaporated in vacuo to dryness. The inhibitory activity of each crude extract was determined by measuring the amount of 6-keto-PGF1a released from vascular smooth muscle cells exposed to the extract [13]. Next, the dry material of SHR hearts, extracted by the same procedure exactly, was dissolved in 1 M AcOH, subjected to a column of Sephadex G-25. The active fraction from the gel-filtration column chromatography was applied on reverse-phase high performance liquid chromatography (HPLC) using an Inertsil ODS-2 C18 column, and purified as a single peptide peak. The amino acid sequence of the purified full-length peptide was first analyzed by automated gas-phase peptide sequencing. Next, after the peptide was digested with aspN at 37 jC for 6 h, the amino acid sequence of the peptide fragments was determined by the same procedure. 2.3. Synthesis of recombinant coupling factor 6 Recombinant rat coupling factor 6, to which the prostacyclin-synthesis inhibitory peptide was found to be identical, was obtained from Escherichia coli using a cleavable fusion protein strategy [14,15]. The amino acid sequence and molecular mass of this recombinant peptide were checked by automated gas-phase peptide sequencing and mass spectrometry. 2.4. Synthesis of antibody (Ab) for rat coupling factor 6 Synthetic coupling factor 6 fragment (rat Cys-58-76 amino acid) solution was emulsified with an equal volume of Freund’s complete adjuvant and injected subcutaneously into New Zealand white rabbits at multiple sites in the interscapulovertebral region. The concentration of endotoxin in anti-coupling factor 6 Ab was less than 1 pg/ml. 2.5. Effects of recombinant coupling factor 6 on prostacyclin synthesis Dose-dependent effects of recombinant coupling factor 6 (10 9 – 10 7 M) on baseline and bradykinin (10 6 M)-induced prostacyclin production were assessed by measuring the amount of 6-keto-PGF1a released from subconfluent human umbilical vein endothelial cells (HUVEC) on 24-well plates for 30 min. The subconfluent cells were incubated with coupling factor 6 in the absence or presence of 10 6 M bradykinin in serum-free Dulbecco’s modified Eagle’s medium (DMEM) for 30 min at 37 jC. To determine the site of action of coupling factor 6, the effects of this recombinant peptide at 10 7 M on conversion of both exogenous AA (10 ng/ml) to prostacyclin and exogenous PGH2 (10 ng/ ml) to prostacyclin were assessed by the same method.
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2.6. Effects of recombinant coupling factor 6 on AA release HUVEC were labeled for 24 h with [3H] AA (0.25 ACi in complete medium/well). The cells were incubated with coupling factor 6 in both the absence of and presence of 10 6 M bradykinin in serum-free DMEM. In some experiments, the cells were incubated with AACOCF3 at 40 AM, OPC at 1 AM, or BEL at 1 AM in both the absence of and presence of 10 6 M bradykinin. After 30 min, the medium was collected and the [3H] level was counted with a liquid scintillation counter. In addition, the effect of co-administration of coupling factor 6 at 10 7 M and AACOCF3 was also assessed by the same method. 2.7. Blood pressure measurement Blood pressure was determined by direct arterial cannulation in rats anesthetized with 75 mg/kg of ketamine hydrochloride and 15 mg/kg of xylazine hydrochloride. Two polyethylene catheters filled with heparinized saline solution were inserted via the left carotid artery and left femoral vein, respectively. The carotid catheter was connected to a pressure transducer coupled to a polygraph. Rat recombinant coupling factor 6 and anticoupling factor 6 Ab were injected into the vena cava. The effect of either recombinant coupling factor 6 (0.1, 0.3, and 1.0 Ag/kg body weight) or anti-coupling factor 6 Ab (0.3, 1.0, and 3.0 Ag/kg body weight) on the arterial vascular tone was examined in SHR and WKY. The responses of blood pressure to recombinant coupling factor 6 and its Ab were also investigated 30 min after the intravenous injection of indomethacin at 10 mg/kg in SHR. 2.8. Statistics All data are shown as meanFone standard error. An unpaired t-test for comparison of two variables and analysis of variance (ANOVA) for repeated measures for multiple comparison were used for statistical analysis. The level of significance was <0.05.
3. Results 3.1. Purification and determination of prostacyclin-synthesis inhibitory peptide Six-keto-PGF1a production was most strongly suppressed by the heart extract, and its suppression was abolished by pretreatment with trypsin and chymotrypsin. Therefore, the prostacyclin-inhibitory peptide was purified from hearts of SHR by collecting active fractions after gel-filtration column chromatography. The active fraction was applied to reverse-phase HPLC and eventually purified as a single peak. Automated gas-phase peptide sequencing of the purified full-length peptide was interpretable up to the 40th amino acid from the N-terminus, and was completely identical to coupling factor 6. The sequences of four fragment peptides cleaved by aspN were DRELFKLKQMYGKGEM, DKFPTFNFE, DPKFEVL, and DKPQS, which were completely identical to the 40th – 55th, the 56th –64th, the 65th – 71st, and the 72th – 76th amino acid sequences of rat
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Fig. 1. Effect of recombinant coupling factor 6 at doses of 10 9 to 10 7 M on basal prostacyclin synthesis in human umbilical vein endothelial cells. *p<0.05, **p<0.02. Results are presented as meanFone standard error for five experiments.
coupling factor 6, respectively. These results indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. 3.2. Effects of recombinant coupling factor 6 on prostacyclin synthesis As shown in Fig. 1, recombinant coupling factor 6 suppressed prostacyclin synthesis at baseline in a dose-dependent manner (10 9 – 10 7 M). Treatment with bradykinin at 10 6 M elicited an increase in prostacyclin synthesis from 272F45 to 469F24 pg/well/30 min
Fig. 2. Effect of recombinant coupling factor 6 (CF6) at doses of 10 9 to 10 7 M on bradykinin (10 6 M)-induced prostacyclin synthesis in human umbilical vein endothelial cells. *p<0.05, **p<0.02. Results are presented as meanFone standard error for five experiments.
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Fig. 3. Effect of recombinant coupling factor 6 at doses of 10 9 to 10 7 M on basal arachidonic acid release in human umbilical vein endothelial cells. *p<0.05, **p<0.02. Results are presented as meanFone standard error for four experiments.
(Fig. 2). Simultaneous administration of coupling factor 6 suppressed bradykinin-induced prostacyclin production in a dose-dependent manner. In contrast, exogenous AA-induced increase in prostacyclin synthesis above the baseline was unchanged even after treatment with 10 7 M recombinant coupling factor 6. An increase in prostacyclin production induced by exogenous PGH2 also was not affected by coupling factor 6 at 10 7 M.
Fig. 4. Effect of phospholipase A2 inhibitors on basal arachidonic acid release in human umbilical vein endothelial cells. *p<0.05. Results are presented as meanFone standard error for four experiments. AACOCF3: arachidonyl trifluoromethyl ketone at 40 AM. OPC: oleyloxyethyl phosphorylcholine at 1 AM. BEL: bromoenol lactone at 1 AM. CF6: recombinant coupling factor 6 at 100 nM.
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Fig. 5. Effect of recombinant coupling factor 6 (CF6) at 10 9 to 10 7 M on bradykinin (10 6 M)-induced arachidonic acid release in human umbilical vein endothelial cells. *p<0.05, **p<0.01. Results are presented as meanFone standard error for eight experiments.
3.3. Effects of recombinant coupling factor 6 on AA release As shown in Fig. 3, recombinant coupling factor 6 suppressed baseline [3H] AA release in a dose-dependent manner. Baseline [3H] AA release was suppressed by 40 AM AACOCF3 but not by 1 AM OPC and 1 AM BEL (Fig. 4). The addition of 10 7 M
Fig. 6. Effect of phospholipase A2 inhibitors on bradykinin (10 6 M)-induced arachidonic acid release in human umbilical vein endothelial cells. *p<0.05. Results are presented as meanFone standard error for four experiments. AACOCF3: arachidonyl trifluoromethyl ketone at 40 AM. OPC: oleyloxyethyl phosphorylcholine at 1 AM. BEL: bromoenol lactone at 1 AM. CF6: recombinant coupling factor 6 at 100 nM.
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coupling factor 6 to 40 AM AACOCF3 showed no further suppressant effect on [3H] AA release. Administration of bradykinin at 10 6 M elicited an increase in [3H] AA release from 687F44 to 1112F36 cpm/well/30 min. As shown in Fig. 5, simultaneous administration of coupling factor 6 suppressed this bradykinin-induced [3H] AA release in a dose-dependent manner, as seen in prostacyclin synthesis. AACOCF3 at 40 AM sup-
Fig. 7. (a) Changes in the arterial blood pressure after intravenous injection of anti-coupling factor 6 Ab (CF6 Ab) in Wistar – Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). (b) Changes in the arterial blood pressure after intravenous injection of rat recombinant coupling factor 6 (CF6) at 1 Ag/kg in WKY and SHR.
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pressed [3H] AA release in the presence of 10 6 M bradykinin, while neither 1 AM OPC nor 1 AM BEL affected bradykinin-induced [3H] AA release (Fig. 6). The addition of coupling factor 6 at 10 7 M to AACOCF3 at 40 AM showed no further suppressant effect on the [3H] AA release induced by bradykinin. 3.4. Physiological effect of perturbing coupling factor 6 activity in vivo Fig. 7a illustrates tracings of changes in arterial blood pressure after an injection of anticoupling factor 6 Ab into the femoral vein. In 16-week-old WKY, the intravenous bolus injection of anti-coupling factor 6 Ab at 0.3, 1.0, and 3.0 Ag/kg body weight produced a transient and rapid reduction in systolic and diastolic blood pressures. In age-matched SHR, the reduction of blood pressure occurred in seconds, reached a maximal reduction within 30 s, and returned to the baseline after several minutes. It was noted that the reduction of blood pressure was greater in SHR than in WKY. The reduction of blood pressure was blocked after 30-min pretreatment with indomethacin at 10 mg/kg. In 16-week-old WKY, an intravenous bolus injection of rat recombinant coupling factor 6 at 1.0 Ag/kg body weight produced a transient and rapid increase in systolic and diastolic arterial blood pressures (Fig. 7b). In age-matched SHR, the peptide increased arterial blood pressure more markedly within seconds, reaching the maximal increase within 30 s and returning to baseline after several minutes.
4. Discussion The present study showed that a prostacyclin-synthesis inhibitory substance is identical to the known peptide designated coupling factor 6 and functions as an endogenous vasoconstrictor in rats. Coupling factor 6 [16] is known to act inside the cell as an energy transducer in mitochondrial adenosine triphosphate synthase. It is of interest to speculate the site of novel inhibitory 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 PLA2 independently of cyclooxygenase and prostacyclin synthase. To confirm this issue, we directly measured the release of AA from the pre-labeled cells. The result demonstrated that coupling factor 6 suppresses baseline AA release in HUVEC and blocks the bradykinin-induced increase in AA release, strongly suggesting that AA release from the plasma membrane is a target of this peptide. Based on biochemical properties and structural features, the PLA2 superfamily can be subdivided into three main types [17], i.e. Ca 2+-dependent secretory enzymes (sPLA2), Ca2+-dependent cytosolic enzymes (cPLA2) and Ca2+-independent cytosolic enzymes (iPLA2). Although PLA2 inhibitors currently available are not necessarily isoformspecific, the combined use of each inhibitor may allow us to examine the specific function of each PLA2 isoform in vitro. AACOCF3 (cPLA2 inhibitor) not only exhibits 500-fold greater potency to cPLA2 than to sPLA2 [18], but also inhibits macrophage iPLA2 with IC50 value of 15 AM [19]. BEL (iPLA2 inhibitor) manifests greater than 1000-fold selectivity for iPLA2 than for sPLA2 and cPLA2 [20]. However, BEL has been found to
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block Mg2+-dependent phosphatidate phosphohydrolase, a key enzyme in cellular phospholipid metabolism [21]. Baseline 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 baseline 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. Because prostacyclin receptor is expressed in many tissues such as aorta, lung, atrium, ventricle and kidney [13], and because PG transporter [6] 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. We evaluated whether endogenous coupling factor 6 affects arterial blood pressure in vivo by blocking the effect of endogenous coupling factor 6 with anti-coupling factor 6 Ab. The results clearly showed that intravenous administration of anti-coupling factor 6 Ab caused a transient and rapid reduction in arterial blood pressure. In addition to our work with the blocking antibody, we performed complementary studies by treating rats with exogenous recombinant coupling factor 6. Injected peptide induced an increase in arterial blood pressure, and its extent was similar to that expected from the finding of neutralization of coupling factor 6, suggesting that its vasoconstrictor effect is unaffected by a single pass through the pulmonary vascular bed. Taken together, these findings strongly suggest that endogenous coupling factor 6 has a direct vasoconstrictor effect in the fashion of a circulating hormone. In conclusion, this report shows that mitochondrial coupling factor 6 is an endogenous inhibitor of prostacyclin synthesis and a potent endogenous vasoconstrictor.
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