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The effects of fatty acyl CoA esters on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes
Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65 & 2001 Harcourt Publishers Ltd doi:10.1054/plef.2000.0238, available online at http://www.idealibrary.com on
The effects of fatty acyl CoA esters on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes S. Sakuma,Y. Fujimoto,Y. Katoh, A. Kitao,T. Fujita Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, Osaka, Japan
Summary Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA).We have previously shown that palmitoyl-CoA (PA-CoA) shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla at a low concentration of AA (5 mM, close to the physiological concentration of substrate).In the present study, we investigated the effects of stearoyl (SA)-, oleoyl (OA)- and linoleoyl (LA)- CoAs on the formation of PG and AA-CoA from 5mM AA in rabbit kidney medulla microsomes.The kidney medulla microsomes were incubated with 5mM [14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2 and CoA). After incubation , PG (as total PGs), AA-CoA and residual AAwere separated by selective extraction using petroleum ether and ethyl acetate. SA- and OA-CoAs increased AA-CoA formation with a reduction of PG formation, as well as PA-CoA. On the other hand, LA-CoA decreased formation of both PG and AA-CoA.These results suggest that fatty acyl CoA esters can be regulators of PG and AA-CoA formation in kidney medulla under physiological conditions. & 2001Harcourt Publishers Ltd
INTRODUCTION Arachidonic acid (AA) released from membrane phospholipids by a variety of physiological stimuli is converted into eicosanoids by cyclooxygenase (COX) and the subsequent enzymes. On the other hand, acyl-CoA synthetase (ACS), which catalyzes the conversion of free AA into arachidonoyl-CoA (AA-CoA), is present in living cells. Fatty acyl CoA esters, including AA-CoA, have been shown to be regulators of various enzymes,1–5 and have therefore received much attention.
Received 1August 2000 Accepted 16 October 2000 Correspondence to: Satoru Sakuma, Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, 4^20^1 Nasahara,Takatsuki, Osaka 569^1094, Japan.Tel.: +81726 90 1055; Fax: +81726 90 1005; E-mail: [email protected]
& 2001Harcourt Publishers Ltd
The kidney medulla mainly synthesizes prostaglandins (PGs), which then regulate renal blood flow, glomerular excretion rate, and so on.6 COX and subsequent enzymes have been localized in microsomes from the medullary portion of the kidney. Also, ACS has been reported to be distributed in the same location.7–9 Furthermore, it has been reported that an ACS subtype that predominatly utilizes AA as the substrate is distributed in this region. As it is thought that, under normal physiological conditions, the free AA level is very low in kidney medulla as well as in polynuclear leukocytes, fibroblasts, platelets and cerebral cortex,10–14 COX and ACS distributed in the medullary portion competitively utilize this fatty acid to form PGs and AA-CoA. Recently, we have established in vitro assay conditions for simultaneously measuring COX and ACS activities (using 60 mM AA as substrate concentration), and for observing the PG and AA-CoA formation under conditions wherein COX and ACS competitively utilize AA Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
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(using 5 mM AA as substrate concentration), with one sample incubation using microsomes from rabbit kidney medulla.15 In this report, we also showed that Zn2+ and Cu2+ can be modulators of PG and AA-CoA formation by affecting the COX activity, ACS activity, and/or AA flow into the two enzymatic pathways.15 Furthermore, we have reported that palmitoyl-CoA (PA-CoA) shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla at a low concentration of free AA (5 mM).16 To further clarify the regulation of fatty acylCoA esters on the partitioning of AA to COX and ACS pathways, in the present study the effects of stearoyl (SA)-, oleoyl (OA)- and linoleoyl (LA)-CoAs on the formation of PG and AA-CoA from 60 or 5 mM AA by rabbit kidney medulla microsomes were examined. MATERIALS AND METHODS
Materials [1-14C] AA was obtained from Amersham International PLC (Buckinghamshire, UK) PA-, SA-, OA- and LA-CoAs, and ATP and CoA were purchased form Sigma Chemical Co. (St Louis, MO, USA) Reduced glutathione (GSH) and hydroquinone were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). All other reagents were of analytical grade.
Preparation of microsomes from rabbit kidney medulla Male rabbits (2–2.5 Kg) were used in the present study. Kidneys were removed from anaesthetized (sodium pentobarbitone, 30 mg/kg) rabbits, and the medullary portion of the kidney was homogenized in 2 vol of 0.1 M-Tris/HCl buffer (pH 7.5). The microsomal fraction was prepared according to the method of Tai et al.17 The medulla homogenate was centrifuged at 700 g for 10 min in a Hitachi model CR20E refrigerated centrifuge. The supernatant was then centrifuged at 8500 g for 10 min in the same centrifuge. The supernatant was further centrifuged at 105000 g for 60 min in a Beckman model TL-100 ultracentrifuge. The pellet resuspended in 0.1 MTris/HCl buffer (pH 7.5) was used as the microsomal fraction. The protein content of the microsomes was determined by the method of Lowry et al.18 using bovine serum albumin as standard.
Incubation conditions for simultaneous measurement of PG and AA-CoA formed from AA and determination of PG and AA-CoA The assay conditions for measurement of PG and AA-CoA formed from AA were as described in our previous paper.15 Briefly, the reaction mixture contained 60 or Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
5 mM [14C]-AA (1.5KBq/sample), 1mM GSH, 0.1mM hydroquinone, 6.7 mM ATP, 20mM MgCl2 and 67 mM CoA in 0.1 M-Tris/HCl buffer (pH 8.0) in a total volume of 0.5 ml. After preincubation for 5 min at 378C, the assay was started by the addition of the enzyme solution containing 0.5 mg of the microsomal protein. After a 5 min incubation at 378C, the reaction mixture was extracted twice with 6 vol of petroleum ether to remove residual AA. The aqueous phase was then acidified (to approximately pH 3) with 1N HCl and extracted twice with 6 vol of ethyl acetate. Total PG and AA-CoA formations were estimated by counting the radioactivities of the ethyl acetate phase, and the aqueous phase after ethyl acetate extraction, respectively. In our previous paper,15 validation of the assay methods was performed by selective synthesis inhibition using indomethacin (a COX inhibitor) and triacsin C (an ACS inhibitor19,20). Namely, when 60 mM AA was used as the substrate, indomethacin and triacsin C reduced only the radioactivity of the ethyl acetate phase and the aqueous phase, respectively. On the other hand, when 5 mM AA was used as the substrate, indomethacin and triacsin C came to increase significantly the radioactivity of the aqueous phase and the ethyl acetate phase, respectively. Thus, these experiments demonstrated that the radioactivities of the ethyl acetate and aqueous phases reflect the amounts of total PG and AA-CoA, respectively, and that incubation using 60 mM AA can simultaneously detect changes in the activities of COX and ACS caused by drugs, while incubation using 5 mM AA can detect changes in the products formation elicited by the resulting shunt of AA.
Statistical analysis The data shown are means + SE. Statistical analyses were performed using Student’s t-test and P50.05 was considered as significant. RESULTS Figure 1 shows the effect of LA-CoA on the PG and AA-CoA formation from exogenous AA (60 and 5 mM) by rabbit kidney medulla microsomes. When 60 and 5 mM AA were used as substrate concentrations, LA-CoA (10, 50 and 100 mM) decreased both PG and AA-CoA formations, and the effects were concentration-dependent. This means that under high and low concentrations of free AA, LA-CoA has the potential to reduce the PG and AA-CoA levels in the Kidney medulla by inhibiting COX and ACS activities. Figure 2 shows the effects of PA-, SA-, OA- and LA-CoA (100 mM) on the PG and AA-CoA formation from high and low concentrations of AA by rabbit kidney medulla microsomes. Different effects on PG and AA-CoA & 2001Harcourt Publishers Ltd
Fatty acyl CoA esters : effects on PG and AA-CoA formation in kidney medulla 63
Fig. 1 Effect of LA-CoA on the PG and AA-CoA formation by rabbit kidney medulla microsomes using 60 or 5 mM AA as the substrate.The microsomalfraction (0.5 mg of protein) wasincubatedwith 60 or 5 mM [14C]- AAfor 5 min at 378Cin 0.5 mlof 0.1 M Tris-HClbuffer (pH 8.0) containing GSH (1.0 mM), hydroquinone (0.1 mM), ATP (6.7 mM), MgCl2 (20 mM) and CoA (67 mM). Each point indicates the mean of 4 experiments; vertical lines show SE.*P50.05, **P50.01; significantly different from control. LA-CoA, linoleoyl-CoA; PG, prostaglandin; AA-CoA, arachidonoyl-CoA. *, PG ; ~, AA-CoA.
Fig. 2 Effects of various fatty acyl CoA esters on the PG and AA-CoA formation by rabbit kidney medulla microsomes using 60 or 5 mM AA as the substrate.The microsomal fraction (0.5 mg of protein) wasincubated with 60 or 5 mM [14C]- AA for 5min at 378C in 0.5 ml of 0.1 M Tris-HCl buffer (pH 8.0) containing GSH (1.0 mM), hydroquinone (0.1)mM), ATP (6.7 mM), MgCl2 (20 mM) and CoA (67 mM). Each point indicates the mean of 4^6 experiments; vertical lines show SE.*P50.05, **P50.01; significantly different from control. PA-CoA, palmitoyl-Coal; SA-CoA, stearoyl-CoA; OA-CoA, oleoyl-CoA; LA-CoA, linoleoyl-CoA; PG, prostaglandin; AA-CoA, arachidonoyl-CoA.
formation were observed with LA-CoA and with the other acyl CoA esters. Namely, when 60 mM AA was used as the substrate concentration, PA-, SA- and OA-CoA had little or no effect on the PG and AA-CoA formation. When 5 mM AA was used as the substrate concentration, these fatty acyl CoA esters markedly decreased the PG formation concomitantly with an increase in the AA-CoA formation. On the other hand, LA-CoA could inhibit the formation & 2001Harcourt Publishers Ltd
of both products under both high and low concentrations of AA. DISCUSSION The AA metabolites, PGs and AA-CoA, have a variety of functions in the body, and thus it is important to clarify the mechanisms by which the levels of these metabolites Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
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are controlled in living cells. There have been many reports concerning the factors regulating the activities of PG synthesizing enzymes. However, little information is available concerning the factors regulating ACS activity. Furthermore, under normal physiological conditions, as the level of free AA is very low, the COX and ACS competitively utilize the free AA to form PGs and AA-CoA. The competition between COX and ACS is thus an interesting and neglected area of research. We have recently established in vitro assay conditions for conversion of AA to PGs and AA-CoA by the COX and ACS in rabbit kidney medulla microsomes, using a simple extraction procedure to separate these two metabolites.15,16 When 60 mM AA was used as substrate, it was possible to simultaneously measure the COX and ACS activities, and to investigate the effects of drugs on these enzyme activities using one assay sample, resulting in a simple, rapid and low-cost method, compared with previous methods in which the COX and ACS activities were assayed separately. When 5 mM AA was used as the substrate, it was possible to observe the competitive formation of PGs and AA-CoA from AA, and to investigate the effects of drugs on the product formation elicited by the resulting shunt of AA. It is thought that the levels of free AA in gastric mucosa21 and kidney22 are about 26 and 5 mM, respectively. Thus, it seems likely that the results obtained using 5 mM AA reflect drug-induced changes in the PG and AA-CoA formation in the kidney medulla under normal physiological conditions. Furthermore, we have recently shown that PA-CoA shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla. Therefore, we investigated the effects of fatty acyl CoA esters on the PG and AA-CoA formation from high and low concentrations of AA by rabbit kidney medulla microsomes. In the present study, we demonstrated that SA- and OA-CoAs, in addition to PA- CoA, decreased the PG formation and increased the AA-CoA formation only at low concentration of AA. Furthermore, we found that LA-CoA decreased both PG and AA-CoA formation at both high and low concentrations of AA. LA intake has been reported to increase or decrease the PG levels in kidney. Lewis et al.23 and Adam and Wolfram24 have shown that LA intake causes a significant increase in the PG production in the kidney, and suggested that this effect is due, in part, to the resulting enrichment of the intracellular phosholipid substrate pools with AA. In contrast, Mahoney et al.25 have reported that dietary LA suppresses renal PG synthesis. Huang et al.26 have found that, during LA deprivation, PG levels in kidney increase in the first week, and fall to onethird of the control at 3 weeks. In vitro, the inhibitory effect of LA on PG biosynthesis has been repeatedly reported by many investigators, including us.27–31 The Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
previous observations, and our present finding that LACoA inhibits PG formation in rabbit kidney medulla microsomes may suggest that the reduction of PG formation by LA intake in the kidney can be ascribed to the inhibition of PG biosynthesis by LA and its CoA derivative, LA-CoA. Furthermore, the observation that LA-CoA can inhibit the PG formation even at a high concentration of AA may indicate that LA intake could prevent certain renal diseases32,33 partially caused by an excessive release of AA and the resulting increase in the PG levels. Under physiological conditions, a variety of fatty acyl CoAs exist in living cells. Previously, it has been shown that, in rat liver and heart, the total acyl-CoA content is 60–80 nmol/g wet weight.34,35 Among the fatty acyl CoAs, PA-, SA-, OA- and LA-CoAs accounts for 80–90% of the total acyl-CoA content in liver34,35 and kidney36. LA-CoA accounts for one-fifth of the four fatty acylCoA.34–36 In the present study, we showed that LA-CoA decreased the AA-CoA formation, whereas PA-, SA- and OA-CoAs increased it. Therefore, the balance of LA-CoA and other fatty acyl CoA esters could be critical for control of fatty acid metabolism in the kidney. Disturbance of this balance could have pathophysiological consequences. In conclusion, the present study clearly demonstrated that fatty acyl CoA esters can be regulators of formation of PG and AA-CoA in kidney medulla under normal physiological conditions.
ACKNOWLEDGMENT This work was supported by Scientific Grant (No. 11771457) from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. Tippett P. S., Neet K. E. Specific inhibition of glucokinase by long chain acyl coenzymes A below the critical micelle concentration. J Biol Chem 1982; 257: 12839–12845. 2. Kakar S. S., Huang W. -H., Askari A. Control of cardiac sodium pump by long-chain acyl coenzymes A. J Biol Chem 1987; 262: 42–45. 3. Bronfman M., Morales M. N., Orellanna A. Diacylglycerol activation of protein kinase C is modulated by long-chain acylCoA. Biochem Biophys Res Commun 1988; 152: 987–992. 4. Nieto M. L., Venable M. E., Bauldry S. A. et al. Evidence that hydrolysis of ethanolamine plasmalogens triggers synthesis of platelet-activating factor via a transacylation reaction. J Biol Chem 1991; 266: 18699–18706. 5. Hayashi M., Imai Y., Naraba H., Tomoda H., Omura S., Oh-ishi S. Enhanced production of platelet-activating factor in stimulated rat leukocytes pretreated with triacsin C, A novel acyl-CoA synthetase inhibitor. Biochem Biophys Res Commun 1992; 188: 1280–1285.
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6. Dunn M. J., Patrono C., Cinotti G. A. Prostaglandins and the kidney. New York: Plenum Press, 1983. 7. Tanaka T., Hosaka K. T., Hoshimaru M., Numa S. Purification and properties of long-chain acyl-coenzyme-A synthetase from rat liver. Eur J Biochem 1979; 98: 165–172. 8. Waku K. Origins and fates of fatty acyl-CoA esters. Biochim Biophys Acta 1992; 1124: 101–111. 9. Ramsammy L. S., Haynes B., Josepovitz C., Kaloyanides G. J. Mechanism of decreased arachidonic acid in the renal cortex of rats with diabetes mellitus. Lipids 1993; 28: 433–439. 10. Hunter S. A., Burstein S., Sedor C. Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate. Biochim Biophys Acta 1984; 793: 202–212. 11. Fuse I., Iwanaga T., Tai H. H. Phorbol ester, 1,2-diacylglycerol, and collagen induce inhibition of arachidonic acid incorporation into phospholipids in human platelets. J Biol Chem 1989; 264: 3890–3895. 12. Hornberger W., Patscheke H. Primary stimuli of icosanoid release inhibit arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. Mechanism of action of hydrogen peroxide and methyl mercury in platelets. Eur J Biochem 1990; 187: 175–181. 13. Reichman M., Nen W., Hokin L. E. Delta 9-tetrahydrocannabinol inhibits arachidonic acid acylation of phospholipids and triacylglycerols in guinea pig cerebral cortex slices. Mol pharmacol 1991; 40: 547–555. 14. Ohi-ishi S., Yamaki K., Abe M., Tomoda H., Omura S. The acyl-CoA synthetase inhibitor triacsin C enhanced eicosanoid release in leukocytes. Japan J Pharmacol 1992; 59: 417–418. 15. Sakuma S., Fujimoto Y., Kitao A., Sakamoto H., Nishida H., Fujita T. Simultaneous measurement of prostaglandin and arachidonoyl CoA formed from arachidonic acid in rabbit kidney medulla microsomes: the roles of Zn2+ and Cu2+ as modulators of formation of the two products. Prostaglandins Leukot Essent Fatty Acids 1999; 61: 105–112. 16. Sakuma S., Fujimoto Y., Katoh Y., Kitao A., Fujita T. The regulation of prostaglandin and arachidonoyl-CoA formation from arachidonic acid in rabbit kidney medulla microsomes by palmitoyl-CoA. Life Sci 2000; 66: 1147–1153. 17. Tai H. H., Tai C. L., Hollander C. S. Biosynthesis of prostaglandins in rabbit kidney medulla. Properties of prostaglandin synthase. Biochem J 1976; 154: 257–264. 18. Lowry O. H., Rosebrough N. J., Farr A., Randall R. J. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275. 19. Tomoda H., Igarashi K., Omura S. Inhibition of acyl-CoA synthetase by triacsins. Biochim Biophys Acta 1987; 921: 595–598. 20. Hartman E. J., Omura S., Laposata M. Triacsin C: a differential inhibitor of arachidonoyl-CoA synthetase and nonspecific long chain acyl-CoA synthetase. Prostaglandins 1989; 37: 655–671. 21. Ahlquist D. A., Madson T. H., Romero J. C., Dozois R. R, Malagelada J. -R. Factors influencing metabolism of arachidonic acid in guinea pig gastric mucosa. Am J Physiol 1983; 244: G131–G137.
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22. Erman A., Baer P. G., Nasijetti A. Augmentation of unesterified arachidonic acid in the renal inner medulla of dexamethasonetreated rats. Relationship to altered triglyceride metabolism. J Biol Chem 1985; 260: 4679–4683. 23. Lewis M. G., Kaduce T. L., Spector A. A. Effect of essential polyunsaturated fatty acid modifications on prostaglandin production by MDCK canine kidney cells. Prostaglandins 1981; 22: 747–760. 24. Adam O., Wolfram G. Effect of different linoleic acid intakes on prostaglandin biosynthesis and kidney function in man. Am J Clin Nutr 1984; 40: 763–770. 25. Mahoney D., Croft K., Beilin L. J. Influence of dietary polyunsaturated fatty acids on renal & aortic prostaglandin synthesis in 1 kidney 1 clip goldblatt hypertensive rats. Prostaglandins 1983; 26: 479–491. 26. Huang Y. S., Mitchell J., Jenkins K., Manku M. S., Horrobin D. F. Effect of dietary depletion and repletion of linoleic acid on renal fatty acid composition and urinary prostaglandin excretion. Prostaglandins Leukot Med 1984; 15: 223–228. 27. Pace-Asciak C., Wolfe L. S. Inhibition of prostaglandin synthesis by oleic, linoleic and linolenic acids. Biochim Biophys Acta 1968; 152: 784–787. 28. Wallach D. P., Daniels E. G. Properties of a novel preparation of prostaglandin synthetase from sheep seminal vesicles. Biochim Biophys Acta 1971; 231: 445–457. 29. Ziboh V. A. Biosynthesis of prostaglandin E2 in human skin: subcellular localization and inhibition by unsaturated fatty acids and anti-inflammatory drugs. J Lipid Res 1973; 14: 377–384. 30. Friedman Y., Lang M., Burke G. Further characterization of bovine thyroid prostaglandin synthase. Biochim Biophys Acta 1975; 397: 331–341. 31. Fujimoto Y., Nakajima T., Murakami Y. et al. Effects of fatty acyl-coenzyme A esters on prostaglandin synthesis in rabbit kidney medulla microsomes. Prostaglandins Leukot Essent Fatty Acids 1992; 47: 265–268. 32. Nakano J., Prancan A. V. Metabolic degradation of prostaglandin E1 in the lung and kidney of rats in endotoxin shock. Proc Soc Exp Biol Med 1973; 144: 506–508. 33. Pace-Asciak C. R. Decreased renal prostagladin catabolism precedes onset of hypertension in the developing spontaneously hypertensive rat. Nature 1976; 263: 510–511. 34. Prasad M. R. Sauter J., Lands W. E. M. Quantitative determination of acyl chain composition of subnanomole amounts of cellular long-chain acyl-coenzyme A esters. Anal Biochem 1987; 162: 202–212. 35. Tardi P. G., Mukherjee J. J., Choy P. C. The quantitation of long-chain acyl-CoA in mammalian tissue. Lipids 1992; 27: 65–67. 36. Mangino M. J., Zografakis J., Murphy M. K., Anderson C. B. Improved and simplified tissue extraction method for quantitating long-chain acyl-coenzyme A thioesters with picomolar detection using high-performance liquid chromatography. J Chromatogr 1992; 577: 157–162.
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The effects of fatty acyl CoA esters on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes S. Sakuma,Y. Fujimoto,Y. Katoh, A. Kitao,T. Fujita Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, Osaka, Japan
Summary Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA).We have previously shown that palmitoyl-CoA (PA-CoA) shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla at a low concentration of AA (5 mM, close to the physiological concentration of substrate).In the present study, we investigated the effects of stearoyl (SA)-, oleoyl (OA)- and linoleoyl (LA)- CoAs on the formation of PG and AA-CoA from 5mM AA in rabbit kidney medulla microsomes.The kidney medulla microsomes were incubated with 5mM [14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2 and CoA). After incubation , PG (as total PGs), AA-CoA and residual AAwere separated by selective extraction using petroleum ether and ethyl acetate. SA- and OA-CoAs increased AA-CoA formation with a reduction of PG formation, as well as PA-CoA. On the other hand, LA-CoA decreased formation of both PG and AA-CoA.These results suggest that fatty acyl CoA esters can be regulators of PG and AA-CoA formation in kidney medulla under physiological conditions. & 2001Harcourt Publishers Ltd
INTRODUCTION Arachidonic acid (AA) released from membrane phospholipids by a variety of physiological stimuli is converted into eicosanoids by cyclooxygenase (COX) and the subsequent enzymes. On the other hand, acyl-CoA synthetase (ACS), which catalyzes the conversion of free AA into arachidonoyl-CoA (AA-CoA), is present in living cells. Fatty acyl CoA esters, including AA-CoA, have been shown to be regulators of various enzymes,1–5 and have therefore received much attention.
Received 1August 2000 Accepted 16 October 2000 Correspondence to: Satoru Sakuma, Department of Hygienic Chemistry, Osaka University of Pharmaceutical Sciences, 4^20^1 Nasahara,Takatsuki, Osaka 569^1094, Japan.Tel.: +81726 90 1055; Fax: +81726 90 1005; E-mail: [email protected]
& 2001Harcourt Publishers Ltd
The kidney medulla mainly synthesizes prostaglandins (PGs), which then regulate renal blood flow, glomerular excretion rate, and so on.6 COX and subsequent enzymes have been localized in microsomes from the medullary portion of the kidney. Also, ACS has been reported to be distributed in the same location.7–9 Furthermore, it has been reported that an ACS subtype that predominatly utilizes AA as the substrate is distributed in this region. As it is thought that, under normal physiological conditions, the free AA level is very low in kidney medulla as well as in polynuclear leukocytes, fibroblasts, platelets and cerebral cortex,10–14 COX and ACS distributed in the medullary portion competitively utilize this fatty acid to form PGs and AA-CoA. Recently, we have established in vitro assay conditions for simultaneously measuring COX and ACS activities (using 60 mM AA as substrate concentration), and for observing the PG and AA-CoA formation under conditions wherein COX and ACS competitively utilize AA Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
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(using 5 mM AA as substrate concentration), with one sample incubation using microsomes from rabbit kidney medulla.15 In this report, we also showed that Zn2+ and Cu2+ can be modulators of PG and AA-CoA formation by affecting the COX activity, ACS activity, and/or AA flow into the two enzymatic pathways.15 Furthermore, we have reported that palmitoyl-CoA (PA-CoA) shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla at a low concentration of free AA (5 mM).16 To further clarify the regulation of fatty acylCoA esters on the partitioning of AA to COX and ACS pathways, in the present study the effects of stearoyl (SA)-, oleoyl (OA)- and linoleoyl (LA)-CoAs on the formation of PG and AA-CoA from 60 or 5 mM AA by rabbit kidney medulla microsomes were examined. MATERIALS AND METHODS
Materials [1-14C] AA was obtained from Amersham International PLC (Buckinghamshire, UK) PA-, SA-, OA- and LA-CoAs, and ATP and CoA were purchased form Sigma Chemical Co. (St Louis, MO, USA) Reduced glutathione (GSH) and hydroquinone were purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). All other reagents were of analytical grade.
Preparation of microsomes from rabbit kidney medulla Male rabbits (2–2.5 Kg) were used in the present study. Kidneys were removed from anaesthetized (sodium pentobarbitone, 30 mg/kg) rabbits, and the medullary portion of the kidney was homogenized in 2 vol of 0.1 M-Tris/HCl buffer (pH 7.5). The microsomal fraction was prepared according to the method of Tai et al.17 The medulla homogenate was centrifuged at 700 g for 10 min in a Hitachi model CR20E refrigerated centrifuge. The supernatant was then centrifuged at 8500 g for 10 min in the same centrifuge. The supernatant was further centrifuged at 105000 g for 60 min in a Beckman model TL-100 ultracentrifuge. The pellet resuspended in 0.1 MTris/HCl buffer (pH 7.5) was used as the microsomal fraction. The protein content of the microsomes was determined by the method of Lowry et al.18 using bovine serum albumin as standard.
Incubation conditions for simultaneous measurement of PG and AA-CoA formed from AA and determination of PG and AA-CoA The assay conditions for measurement of PG and AA-CoA formed from AA were as described in our previous paper.15 Briefly, the reaction mixture contained 60 or Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
5 mM [14C]-AA (1.5KBq/sample), 1mM GSH, 0.1mM hydroquinone, 6.7 mM ATP, 20mM MgCl2 and 67 mM CoA in 0.1 M-Tris/HCl buffer (pH 8.0) in a total volume of 0.5 ml. After preincubation for 5 min at 378C, the assay was started by the addition of the enzyme solution containing 0.5 mg of the microsomal protein. After a 5 min incubation at 378C, the reaction mixture was extracted twice with 6 vol of petroleum ether to remove residual AA. The aqueous phase was then acidified (to approximately pH 3) with 1N HCl and extracted twice with 6 vol of ethyl acetate. Total PG and AA-CoA formations were estimated by counting the radioactivities of the ethyl acetate phase, and the aqueous phase after ethyl acetate extraction, respectively. In our previous paper,15 validation of the assay methods was performed by selective synthesis inhibition using indomethacin (a COX inhibitor) and triacsin C (an ACS inhibitor19,20). Namely, when 60 mM AA was used as the substrate, indomethacin and triacsin C reduced only the radioactivity of the ethyl acetate phase and the aqueous phase, respectively. On the other hand, when 5 mM AA was used as the substrate, indomethacin and triacsin C came to increase significantly the radioactivity of the aqueous phase and the ethyl acetate phase, respectively. Thus, these experiments demonstrated that the radioactivities of the ethyl acetate and aqueous phases reflect the amounts of total PG and AA-CoA, respectively, and that incubation using 60 mM AA can simultaneously detect changes in the activities of COX and ACS caused by drugs, while incubation using 5 mM AA can detect changes in the products formation elicited by the resulting shunt of AA.
Statistical analysis The data shown are means + SE. Statistical analyses were performed using Student’s t-test and P50.05 was considered as significant. RESULTS Figure 1 shows the effect of LA-CoA on the PG and AA-CoA formation from exogenous AA (60 and 5 mM) by rabbit kidney medulla microsomes. When 60 and 5 mM AA were used as substrate concentrations, LA-CoA (10, 50 and 100 mM) decreased both PG and AA-CoA formations, and the effects were concentration-dependent. This means that under high and low concentrations of free AA, LA-CoA has the potential to reduce the PG and AA-CoA levels in the Kidney medulla by inhibiting COX and ACS activities. Figure 2 shows the effects of PA-, SA-, OA- and LA-CoA (100 mM) on the PG and AA-CoA formation from high and low concentrations of AA by rabbit kidney medulla microsomes. Different effects on PG and AA-CoA & 2001Harcourt Publishers Ltd
Fatty acyl CoA esters : effects on PG and AA-CoA formation in kidney medulla 63
Fig. 1 Effect of LA-CoA on the PG and AA-CoA formation by rabbit kidney medulla microsomes using 60 or 5 mM AA as the substrate.The microsomalfraction (0.5 mg of protein) wasincubatedwith 60 or 5 mM [14C]- AAfor 5 min at 378Cin 0.5 mlof 0.1 M Tris-HClbuffer (pH 8.0) containing GSH (1.0 mM), hydroquinone (0.1 mM), ATP (6.7 mM), MgCl2 (20 mM) and CoA (67 mM). Each point indicates the mean of 4 experiments; vertical lines show SE.*P50.05, **P50.01; significantly different from control. LA-CoA, linoleoyl-CoA; PG, prostaglandin; AA-CoA, arachidonoyl-CoA. *, PG ; ~, AA-CoA.
Fig. 2 Effects of various fatty acyl CoA esters on the PG and AA-CoA formation by rabbit kidney medulla microsomes using 60 or 5 mM AA as the substrate.The microsomal fraction (0.5 mg of protein) wasincubated with 60 or 5 mM [14C]- AA for 5min at 378C in 0.5 ml of 0.1 M Tris-HCl buffer (pH 8.0) containing GSH (1.0 mM), hydroquinone (0.1)mM), ATP (6.7 mM), MgCl2 (20 mM) and CoA (67 mM). Each point indicates the mean of 4^6 experiments; vertical lines show SE.*P50.05, **P50.01; significantly different from control. PA-CoA, palmitoyl-Coal; SA-CoA, stearoyl-CoA; OA-CoA, oleoyl-CoA; LA-CoA, linoleoyl-CoA; PG, prostaglandin; AA-CoA, arachidonoyl-CoA.
formation were observed with LA-CoA and with the other acyl CoA esters. Namely, when 60 mM AA was used as the substrate concentration, PA-, SA- and OA-CoA had little or no effect on the PG and AA-CoA formation. When 5 mM AA was used as the substrate concentration, these fatty acyl CoA esters markedly decreased the PG formation concomitantly with an increase in the AA-CoA formation. On the other hand, LA-CoA could inhibit the formation & 2001Harcourt Publishers Ltd
of both products under both high and low concentrations of AA. DISCUSSION The AA metabolites, PGs and AA-CoA, have a variety of functions in the body, and thus it is important to clarify the mechanisms by which the levels of these metabolites Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
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are controlled in living cells. There have been many reports concerning the factors regulating the activities of PG synthesizing enzymes. However, little information is available concerning the factors regulating ACS activity. Furthermore, under normal physiological conditions, as the level of free AA is very low, the COX and ACS competitively utilize the free AA to form PGs and AA-CoA. The competition between COX and ACS is thus an interesting and neglected area of research. We have recently established in vitro assay conditions for conversion of AA to PGs and AA-CoA by the COX and ACS in rabbit kidney medulla microsomes, using a simple extraction procedure to separate these two metabolites.15,16 When 60 mM AA was used as substrate, it was possible to simultaneously measure the COX and ACS activities, and to investigate the effects of drugs on these enzyme activities using one assay sample, resulting in a simple, rapid and low-cost method, compared with previous methods in which the COX and ACS activities were assayed separately. When 5 mM AA was used as the substrate, it was possible to observe the competitive formation of PGs and AA-CoA from AA, and to investigate the effects of drugs on the product formation elicited by the resulting shunt of AA. It is thought that the levels of free AA in gastric mucosa21 and kidney22 are about 26 and 5 mM, respectively. Thus, it seems likely that the results obtained using 5 mM AA reflect drug-induced changes in the PG and AA-CoA formation in the kidney medulla under normal physiological conditions. Furthermore, we have recently shown that PA-CoA shifts AA away from the COX pathway into the ACS pathway in rabbit kidney medulla. Therefore, we investigated the effects of fatty acyl CoA esters on the PG and AA-CoA formation from high and low concentrations of AA by rabbit kidney medulla microsomes. In the present study, we demonstrated that SA- and OA-CoAs, in addition to PA- CoA, decreased the PG formation and increased the AA-CoA formation only at low concentration of AA. Furthermore, we found that LA-CoA decreased both PG and AA-CoA formation at both high and low concentrations of AA. LA intake has been reported to increase or decrease the PG levels in kidney. Lewis et al.23 and Adam and Wolfram24 have shown that LA intake causes a significant increase in the PG production in the kidney, and suggested that this effect is due, in part, to the resulting enrichment of the intracellular phosholipid substrate pools with AA. In contrast, Mahoney et al.25 have reported that dietary LA suppresses renal PG synthesis. Huang et al.26 have found that, during LA deprivation, PG levels in kidney increase in the first week, and fall to onethird of the control at 3 weeks. In vitro, the inhibitory effect of LA on PG biosynthesis has been repeatedly reported by many investigators, including us.27–31 The Prostaglandins, Leukotrienes and Essential FattyAcids (2001) 64(1), 61^65
previous observations, and our present finding that LACoA inhibits PG formation in rabbit kidney medulla microsomes may suggest that the reduction of PG formation by LA intake in the kidney can be ascribed to the inhibition of PG biosynthesis by LA and its CoA derivative, LA-CoA. Furthermore, the observation that LA-CoA can inhibit the PG formation even at a high concentration of AA may indicate that LA intake could prevent certain renal diseases32,33 partially caused by an excessive release of AA and the resulting increase in the PG levels. Under physiological conditions, a variety of fatty acyl CoAs exist in living cells. Previously, it has been shown that, in rat liver and heart, the total acyl-CoA content is 60–80 nmol/g wet weight.34,35 Among the fatty acyl CoAs, PA-, SA-, OA- and LA-CoAs accounts for 80–90% of the total acyl-CoA content in liver34,35 and kidney36. LA-CoA accounts for one-fifth of the four fatty acylCoA.34–36 In the present study, we showed that LA-CoA decreased the AA-CoA formation, whereas PA-, SA- and OA-CoAs increased it. Therefore, the balance of LA-CoA and other fatty acyl CoA esters could be critical for control of fatty acid metabolism in the kidney. Disturbance of this balance could have pathophysiological consequences. In conclusion, the present study clearly demonstrated that fatty acyl CoA esters can be regulators of formation of PG and AA-CoA in kidney medulla under normal physiological conditions.
ACKNOWLEDGMENT This work was supported by Scientific Grant (No. 11771457) from the Ministry of Education, Science and Culture, Japan.
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