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Endogenous inhibitors of lipoxygenase
169
European Journal o f Pharmacology, 67 (1980) 169--170
© Elsevier/North-Holland Biomedical Press Rapid communication E N D O G E N O U S INHIBITORS OF LIPOXYGENASE SHEIKH A. SAEED, MARY DREW and HARRY O.J. COLLIER Research Department, Miles Laboratories Limited, Stoke Poges, Slough, Sl2 4L Y, U.K.
Received 19 August 1980, accepted 20 August 1980
Human plasma or serum inhibits the cyclooxygenation of arachidonic acid to prostaglandins E2 and F2~ by bovine vesicular gland prostaglandin (PG) synthase. This property has been attributed to the presence in plasma or serum of an endogenous inhibitor of PG synthase (EIPS) (Saeed et al., 1977; Collier et al., 1980). Since fatty acid precursors of PGs can also be oxygenated by a lipoxygenase that converts arachidonic acid to an h y d r o x y acid, 12-L-hydroxyeicosatetraenoic acid, 12-HETE (Hamberg and Samuelsson, 1974), we have investigated to what extent human plasma or serum would also inhibit lipoxygenase. For practical reasons, the experiments were carried o u t with soybean lipoxygenase, which we had found to be strongly inhibited by 3-amino-l-(m-(trifluoromethyl)-phenyl)-2pyrazoline (BW755C), a known inhibitor of horse platelet arachidonate lipoxygenase (Higgs et al., 1979) and of anaphylactic bronchoconstriction in the guinea pig (Nijkamp and Ramakers, 1980). The effect of plasma, serum and drugs on the lipoxygenase activity was determined polarographically with an oxygen electrode (Model 53, Yellow Springs Instrument Co., Yellow Springs, Ohio, U.S.A.). A standard assay procedure was developed by selecting the optimal condition for oxygen consumption during the oxidation of arachidonic acid by lipoxygenase. Incubations were conducted at 37°C in 50 mM sodium phosphate buffer, pH 7.4, containing arachidonic acid as its sodium salt (61 pM; final volume 4 ml). The mixture was stirred gently and, after 3 min,
20 pg of soybean lipoxygenase (Type II, Sigma) in phosphate buffer was added. Stirring was continued and the change in oxygen saturation of the mixture was recorded for 3 min. Each determination was carried o u t in triplicate with suitable controls containing all reagents except the enzyme. Table 1 shows that plasma or serum from several vertebrates inhibited the soybean arachidonate lipoxygenase activity. These effects were concentration-related (P ~ 0.005 to P < 0.001 for slopes of concentration-response lines). Human serum or plasma exhibited the strongest inhibitory effect, whereas foetal calf serum and foetal sheep plasma were about ten times less active. In our test system, c o m p o u n d BW755C also potently inhibited the enzyme activity with an IC50 value of 9.5 pM (table 1), compared with a value of 6.4 pM against horse platelet arachidonic lipoxygenase (Higgs et al., 1979). We conclude that mammalian and avian blood plasma or serum contain a factor that inhibits lipoxygenase. The presence of this factor in plasma indicates that it is n o t simply an artefact generated in serum during blood clotting. We, therefore, postulate that plasma and serum contain endogenous inhibitor(s) of lipoxygenase (EILO). The distribution of EILO activity among different sera and plasmas tested is different from that of EIPS activity (Saeed et al., 1977; Collier et al., 1980), indicating that EILO is a distinct entity. A preliminary biochemical analysis of human plasma EILO showed that it is of large
170 ~IABLE 1 Effect of plasma, serum and drugs on lipoxygenase activity. Plasma (P) or serum (S)
ICs0 with 95% limits 1
Slope + S.E.M.
Human (P) ltuman (S) Bovine (S) Normal calf (S) Foetal calf (S) Ram (P) Foetal sheep (P) Chicken (S) BW755C Indomethacin
0.32 0.18--0.48' 0.22 0.05--0.38 0.23 0 . 1 - - 0 . 3 9 ' 0.46 0.29--0.67' 2.04 1.31--4.48 0.50 0.31--0.77' 1.91 1.62--2.33' 1.12 0.90--1.40' 9.52 7 . 2 --13.2' Not active at 100
51.11-+ 6.214 92.38 _+ 17.82 4 53.65-+ 6.805 44.00-+ 3.725 4 9 . 5 9 + 7.784 49.27-+ 4.605 116.38-+ 7.97 s 8 4 . 8 2 + 6.305 3 3 . 3 0 + 2.555
1
2 2 2 2 2 2 2 3 /M
I IC50 concentration (% v/v or/aM) producing 50% inhibition of lipoxygenase calculated by least-squares regression analysis. 2 Concentration expressed as % v/v. 3 Concentration expressed as/aM. 4 For significance of slopes, P < 0.005. For significance of slopes, P < 0.001.
molecular size (molecular weight > 6 5 , 0 0 0 daltons) and fractionates into several fractions by gel filtration c h r o m a t o g r a p h y on Sephadex G-100. It was f o u n d t hat human plasma albumin and a-globulin fractions were rich in EILO activity. The EILO activity of human or adult bovine serum was non-dialyzable and heat-labile, b u t the EILO activity of foetal calf serum was heat-stable. This indicates that the nature of EILO activity in foetal calf serum is d if f er en t f r om t hat of adult bovine or hu man serum. More i nf or m at i on on the biochemical actions and nature o f plasma EILO is, however, needed before its pathophysiological significance and mechanism o f action can be established. F u r t h e r work on t h e purification o f EILO and its effect on human arachidonate lipoxygenase, as well as on the synthesis o f leukotrienes (SRS) is now in progress.
Acknowledgements We thank Wellcome Research Laboratories, England, for a gift of compound BW755C and Dr. M.D. Mitchell for specimens of ovine plasma.
References Collier, H.O.J., P.A. Denning-Kendall, W.J. McDonald-Gibson and S.A. Saeed, 1980, Plasma proteins that inhibit prostaglandin synthesis, in: Hemostasis, Prostaglandins and Renal Disease, eds. G. Remuzzi, G. Mecca and G. de Gaetano (Raven Press, New York) p. 257. Hamberg, M. and B. Samuelsson, 1974, Novel transformations of araehidonic acid in human platelets, Proc. Natl. Aead. Sci. USA 71, 3400. Higgs, G.A., R.J. Flower and J.R. Vane, 1979, A new approaeh to antiinflammatory drugs, Biochem. Pharmacol. 28, 1959. Nijkamp, F.P. and A.G.M. Ramakers, 1980, Prevention of anaphylactie bronchoconstriction by a lipoxygenase inhibitor, European J. Pharmacol. 62, 121. Saeed, S.A., W.J. McDonald-Gibson, J. Cuthbert, J.L. Copas, C. Schneider, P.J. Gardiner, N.M. Butt and H.O.J. Collier, 1977, Endogenous inhibitor of prostaglandin synthetase, Nature 270, 32.
European Journal o f Pharmacology, 67 (1980) 169--170
© Elsevier/North-Holland Biomedical Press Rapid communication E N D O G E N O U S INHIBITORS OF LIPOXYGENASE SHEIKH A. SAEED, MARY DREW and HARRY O.J. COLLIER Research Department, Miles Laboratories Limited, Stoke Poges, Slough, Sl2 4L Y, U.K.
Received 19 August 1980, accepted 20 August 1980
Human plasma or serum inhibits the cyclooxygenation of arachidonic acid to prostaglandins E2 and F2~ by bovine vesicular gland prostaglandin (PG) synthase. This property has been attributed to the presence in plasma or serum of an endogenous inhibitor of PG synthase (EIPS) (Saeed et al., 1977; Collier et al., 1980). Since fatty acid precursors of PGs can also be oxygenated by a lipoxygenase that converts arachidonic acid to an h y d r o x y acid, 12-L-hydroxyeicosatetraenoic acid, 12-HETE (Hamberg and Samuelsson, 1974), we have investigated to what extent human plasma or serum would also inhibit lipoxygenase. For practical reasons, the experiments were carried o u t with soybean lipoxygenase, which we had found to be strongly inhibited by 3-amino-l-(m-(trifluoromethyl)-phenyl)-2pyrazoline (BW755C), a known inhibitor of horse platelet arachidonate lipoxygenase (Higgs et al., 1979) and of anaphylactic bronchoconstriction in the guinea pig (Nijkamp and Ramakers, 1980). The effect of plasma, serum and drugs on the lipoxygenase activity was determined polarographically with an oxygen electrode (Model 53, Yellow Springs Instrument Co., Yellow Springs, Ohio, U.S.A.). A standard assay procedure was developed by selecting the optimal condition for oxygen consumption during the oxidation of arachidonic acid by lipoxygenase. Incubations were conducted at 37°C in 50 mM sodium phosphate buffer, pH 7.4, containing arachidonic acid as its sodium salt (61 pM; final volume 4 ml). The mixture was stirred gently and, after 3 min,
20 pg of soybean lipoxygenase (Type II, Sigma) in phosphate buffer was added. Stirring was continued and the change in oxygen saturation of the mixture was recorded for 3 min. Each determination was carried o u t in triplicate with suitable controls containing all reagents except the enzyme. Table 1 shows that plasma or serum from several vertebrates inhibited the soybean arachidonate lipoxygenase activity. These effects were concentration-related (P ~ 0.005 to P < 0.001 for slopes of concentration-response lines). Human serum or plasma exhibited the strongest inhibitory effect, whereas foetal calf serum and foetal sheep plasma were about ten times less active. In our test system, c o m p o u n d BW755C also potently inhibited the enzyme activity with an IC50 value of 9.5 pM (table 1), compared with a value of 6.4 pM against horse platelet arachidonic lipoxygenase (Higgs et al., 1979). We conclude that mammalian and avian blood plasma or serum contain a factor that inhibits lipoxygenase. The presence of this factor in plasma indicates that it is n o t simply an artefact generated in serum during blood clotting. We, therefore, postulate that plasma and serum contain endogenous inhibitor(s) of lipoxygenase (EILO). The distribution of EILO activity among different sera and plasmas tested is different from that of EIPS activity (Saeed et al., 1977; Collier et al., 1980), indicating that EILO is a distinct entity. A preliminary biochemical analysis of human plasma EILO showed that it is of large
170 ~IABLE 1 Effect of plasma, serum and drugs on lipoxygenase activity. Plasma (P) or serum (S)
ICs0 with 95% limits 1
Slope + S.E.M.
Human (P) ltuman (S) Bovine (S) Normal calf (S) Foetal calf (S) Ram (P) Foetal sheep (P) Chicken (S) BW755C Indomethacin
0.32 0.18--0.48' 0.22 0.05--0.38 0.23 0 . 1 - - 0 . 3 9 ' 0.46 0.29--0.67' 2.04 1.31--4.48 0.50 0.31--0.77' 1.91 1.62--2.33' 1.12 0.90--1.40' 9.52 7 . 2 --13.2' Not active at 100
51.11-+ 6.214 92.38 _+ 17.82 4 53.65-+ 6.805 44.00-+ 3.725 4 9 . 5 9 + 7.784 49.27-+ 4.605 116.38-+ 7.97 s 8 4 . 8 2 + 6.305 3 3 . 3 0 + 2.555
1
2 2 2 2 2 2 2 3 /M
I IC50 concentration (% v/v or/aM) producing 50% inhibition of lipoxygenase calculated by least-squares regression analysis. 2 Concentration expressed as % v/v. 3 Concentration expressed as/aM. 4 For significance of slopes, P < 0.005. For significance of slopes, P < 0.001.
molecular size (molecular weight > 6 5 , 0 0 0 daltons) and fractionates into several fractions by gel filtration c h r o m a t o g r a p h y on Sephadex G-100. It was f o u n d t hat human plasma albumin and a-globulin fractions were rich in EILO activity. The EILO activity of human or adult bovine serum was non-dialyzable and heat-labile, b u t the EILO activity of foetal calf serum was heat-stable. This indicates that the nature of EILO activity in foetal calf serum is d if f er en t f r om t hat of adult bovine or hu man serum. More i nf or m at i on on the biochemical actions and nature o f plasma EILO is, however, needed before its pathophysiological significance and mechanism o f action can be established. F u r t h e r work on t h e purification o f EILO and its effect on human arachidonate lipoxygenase, as well as on the synthesis o f leukotrienes (SRS) is now in progress.
Acknowledgements We thank Wellcome Research Laboratories, England, for a gift of compound BW755C and Dr. M.D. Mitchell for specimens of ovine plasma.
References Collier, H.O.J., P.A. Denning-Kendall, W.J. McDonald-Gibson and S.A. Saeed, 1980, Plasma proteins that inhibit prostaglandin synthesis, in: Hemostasis, Prostaglandins and Renal Disease, eds. G. Remuzzi, G. Mecca and G. de Gaetano (Raven Press, New York) p. 257. Hamberg, M. and B. Samuelsson, 1974, Novel transformations of araehidonic acid in human platelets, Proc. Natl. Aead. Sci. USA 71, 3400. Higgs, G.A., R.J. Flower and J.R. Vane, 1979, A new approaeh to antiinflammatory drugs, Biochem. Pharmacol. 28, 1959. Nijkamp, F.P. and A.G.M. Ramakers, 1980, Prevention of anaphylactie bronchoconstriction by a lipoxygenase inhibitor, European J. Pharmacol. 62, 121. Saeed, S.A., W.J. McDonald-Gibson, J. Cuthbert, J.L. Copas, C. Schneider, P.J. Gardiner, N.M. Butt and H.O.J. Collier, 1977, Endogenous inhibitor of prostaglandin synthetase, Nature 270, 32.