1 26s
Biochemical SocietyTransactions ( 1 992) 20
Production of HETEs in human neutrophils; Dependence of arachidonic acid.
GORANHANSSON AND BENGT EK
MATS JOHANSSON,
Biochemistry and Bioanalytical Chemistry, ASTRA HASSLE AB, S-43183 Mijlndal, Sweden Human lipoxygenases(LO) are known to induce oxidation of arachidonic acid (AA) a t the 5,12 or 15 position and thereby forming hydroperoxy- and hydroxy eicosatetraenoic acids (HPETEs and HETEs). The 5-LO is known to be activated by increased cytosollc Ca2+ and becomes a membrane associated enzyme, which then via the 5-LO activating protein (FLAP) induces the production of 5-HPETE and leukotrienes [l]. These products are inflammatory reaction mediators, potent activators of leucocyte and lymphocyte functions and a r e chemotactic to phagocytic cells. The activity of the other two pathways, 12- and 15-LO, is not related to increased levels of cytoplasmic Ca2+, and any activating protein is not yet defined. One of the major consequences of enzymic lipid hydroperoxide formation may be an altered membrane function, due to incorporation of the more hydrophilic hydroxy fatty acids in phospholipids. Incorporation of these lipids may induce remodeling and increased degradation of cellular membranes. In that respect it has been suggested that 15LO and its lipid hydroperoxide increased the differentiation of reticulocytes [21. These processes may also be the cause of the increased expression of 15-LO found in differentiated eosinophils and airway epithelial cells [3]. The biological role of 15-LO have recently been reviewed [4J]. Increase in 12- and 15-LO activity and formation of HPETEs may be related to an increased A A concentration. We have studied the production of 5-, 12- and 15-HETE in the presence of increased concentration of AA. To clarify the specificity of commonly used LO inhibitors we have studied the inhibition of HETE production with nordihydroguairetic acid (NDCA) and eicosatetraynoic acid (ETYA) in activated PMNs and t h e antioxidative effect in FG+/ascorbate activated liposomes. Human neutrophils (PMN) were purified by Percoll gradient centrifu tion and diluted to 4 6 x 1 0 6 celldml, in phosphate buffer with Ca9+, Mg2+ and 5.6mM glucose. The reaction mixture contained 2ml PMN suspension, lOpM A A and 1pM calcium ionophore (A23187). The reaction was stopped after 10 min with ethyl acetate, and free eicosanoids were extracted at pH 3. The 5-, 12- a n d 15-HETE were analyzed with reversed phase chromatography using a Nucleosil 120-3Cls column, with a mobile phase of methanol, water, acetic acid (7Yzs10.1, by vol). The peaks were identified by authentic eicosanoids from Cayman Chemicals, Ann Arbor, USA. The antioxidative potency of NDGA and ETYA was determined in ironlascorbate activated soybean liposomes [6]. The production of HETEs increased in the presence of increased concentration of AA (Fig 1).However, A23187 only stimulated the production of the 5-LO metabolite (Fig 1A) and no effect was seen on the 12- and 15-LO metabolites (Fig 1B).
..T
9 ..* ...
4 :c :
0.01 0.1
_* 1
-
MCONC (IrM)
.
LO
100
--w
Table 1.G o - v a l u e s of NDGA and ETYA. Each value represents mean f SD, n=2-3. 5-HETE NDCA ETYA
0.01 0.1
I
LO
la,
AA CONC (IrM)
Fig 1. Effect of AA on 5-HETE (A) and on 12- and 15-HETE (B) production in control and in A23187,lpM. activated PMN. Mean values + SD, n=4-6, P
Biochemical SocietyTransactions ( 1 992) 20
Production of HETEs in human neutrophils; Dependence of arachidonic acid.
GORANHANSSON AND BENGT EK
MATS JOHANSSON,
Biochemistry and Bioanalytical Chemistry, ASTRA HASSLE AB, S-43183 Mijlndal, Sweden Human lipoxygenases(LO) are known to induce oxidation of arachidonic acid (AA) a t the 5,12 or 15 position and thereby forming hydroperoxy- and hydroxy eicosatetraenoic acids (HPETEs and HETEs). The 5-LO is known to be activated by increased cytosollc Ca2+ and becomes a membrane associated enzyme, which then via the 5-LO activating protein (FLAP) induces the production of 5-HPETE and leukotrienes [l]. These products are inflammatory reaction mediators, potent activators of leucocyte and lymphocyte functions and a r e chemotactic to phagocytic cells. The activity of the other two pathways, 12- and 15-LO, is not related to increased levels of cytoplasmic Ca2+, and any activating protein is not yet defined. One of the major consequences of enzymic lipid hydroperoxide formation may be an altered membrane function, due to incorporation of the more hydrophilic hydroxy fatty acids in phospholipids. Incorporation of these lipids may induce remodeling and increased degradation of cellular membranes. In that respect it has been suggested that 15LO and its lipid hydroperoxide increased the differentiation of reticulocytes [21. These processes may also be the cause of the increased expression of 15-LO found in differentiated eosinophils and airway epithelial cells [3]. The biological role of 15-LO have recently been reviewed [4J]. Increase in 12- and 15-LO activity and formation of HPETEs may be related to an increased A A concentration. We have studied the production of 5-, 12- and 15-HETE in the presence of increased concentration of AA. To clarify the specificity of commonly used LO inhibitors we have studied the inhibition of HETE production with nordihydroguairetic acid (NDCA) and eicosatetraynoic acid (ETYA) in activated PMNs and t h e antioxidative effect in FG+/ascorbate activated liposomes. Human neutrophils (PMN) were purified by Percoll gradient centrifu tion and diluted to 4 6 x 1 0 6 celldml, in phosphate buffer with Ca9+, Mg2+ and 5.6mM glucose. The reaction mixture contained 2ml PMN suspension, lOpM A A and 1pM calcium ionophore (A23187). The reaction was stopped after 10 min with ethyl acetate, and free eicosanoids were extracted at pH 3. The 5-, 12- a n d 15-HETE were analyzed with reversed phase chromatography using a Nucleosil 120-3Cls column, with a mobile phase of methanol, water, acetic acid (7Yzs10.1, by vol). The peaks were identified by authentic eicosanoids from Cayman Chemicals, Ann Arbor, USA. The antioxidative potency of NDGA and ETYA was determined in ironlascorbate activated soybean liposomes [6]. The production of HETEs increased in the presence of increased concentration of AA (Fig 1).However, A23187 only stimulated the production of the 5-LO metabolite (Fig 1A) and no effect was seen on the 12- and 15-LO metabolites (Fig 1B).
..T
9 ..* ...
4 :c :
0.01 0.1
_* 1
-
MCONC (IrM)
.
LO
100
--w
Table 1.G o - v a l u e s of NDGA and ETYA. Each value represents mean f SD, n=2-3. 5-HETE NDCA ETYA
0.01 0.1
I
LO
la,
AA CONC (IrM)
Fig 1. Effect of AA on 5-HETE (A) and on 12- and 15-HETE (B) production in control and in A23187,lpM. activated PMN. Mean values + SD, n=4-6, P
