(Received for publication, November 20, 1995; and in revised form, December 15, 1995)
From the
Mammalian 15-lipoxygenases have been suggested to be involved in cell differentiation and atherogenesis because of their capability of oxygenating polyenoic fatty acids esterified to biomembranes and lipoproteins. We investigated the interaction of the lipid-peroxidizing 15-lipoxygenase and the hydroperoxy lipid-reducing phospholipid hydroperoxide glutathione peroxidase during their reaction with biomembranes and lipoproteins and obtained the following results. 1) Lipoxygenase treatment of submitochondrial membranes led to the formation of hydroperoxyphosphatidylethanolamine and hydroperoxyphosphatidylcholine as indicated by high performance liquid chromatography with chemiluminescence detection. In 15-lipoxygenase-treated low density lipoprotein cholesteryl hydroperoxylinoleate was the major oxygenation product. 2) Phospholipid hydroperoxide glutathione peroxidase was capable of reducing the hydroperoxy lipids formed by the 15-lipoxygenase to their corresponding alcohols. 3) Preincubation of low density lipoprotein and submitochondrial membranes with the phospholipid hydroperoxide glutathione peroxidase completely prevented the lipoxygenase reaction. However, addition of exogenous hydroperoxy lipids restored the oxygenase activity. 4) Short-term incubations of the complex substrates with the 15-lipoxygenase led to a specific pattern of oxidation products which was rendered more unspecific at long-term incubation or at high substrate concentrations. If the phosholipid hydroperoxide glutathione peroxidase was present during the reaction, the specific product pattern was preserved.
These data indicate that the phospholipid hydroperoxide glutathione peroxidase is capable of reducing hydroperoxy ester lipids formed by a 15-lipoxygenase, and that it may down-regulate the 15-lipoxygenase pathways in mammalian cells. The specificity of 15-lipoxygenase-derived hydroperoxy lipids depends on their immediate reduction to the corresponding alcohols preventing postcatalytic isomerization.
Enzymatic and nonenzymatic lipid peroxidation has been
implicated in a variety of physiological and pathophysiological
processes such as aging, cell differentiation, carcinogenesis,
inflammation, hypoxia, atherogenesis, and
others(1, 2, 3, 4, 5, 6, 7, 8) .
The formation of lipid hydroperoxides within the bilayer of
biomembranes alters the membrane structure (9, 10) and, thus, may lead to dysfunction of the
cellular metabolism. Among the lipid-peroxidizing enzymes in mammalian
cells, the 15-lipoxygenases (11, 12) and the closely
related leukocyte-type 12-lipoxygenases (13, 14) are
capable of oxidizing biomembranes and lipoproteins directly without the
preceding action of ester lipid-hydrolyzing enzymes. Because of this
property, 15-lipoxygenases may be considered as membrane-damaging
noxae, and, hence, the control of the cellular 15-lipoxygenase activity
appears to be of crucial cell-physiological relevance. While the
cellular regulation of the 5-lipoxygenase pathway has been studied in
detail(15, 16, 17, 18) , little is
known on the regulation of the 15-lipoxygenases in mammalian cells. One
general feature of the lipoxygenase reaction is the requirement for
small amounts of hydroperoxy lipids acting as essential activators of
the enzyme(19, 20) . For this reason, the cellular
``hydroperoxide tone'' constitutes a possible site of
regulation of lipoxygenase activity. In most mammalian cells, the
steady-state concentration of hydroperoxides is rather low. In fact,
even in cells exhibiting a high lipid-peroxidizing capacity, such as
rabbit reticulocytes, mainly hydroxy fatty acids have been detected in
the membrane lipids(21) . A preferential cleavage of
hydroperoxy lipids by synergistic action of phospholipases and
peroxidase appears unlikely(22) . Glutathione peroxidases are
known for their capability of reducing organic and inorganic
hydroperoxides in mammalian cells(23, 24) . So far, 4
different selenoperoxidases have been identified (25) as
translation products of 4 different genes(26) . Among these
enzymes, only the phospholipid hydroperoxide glutathione peroxidase
(PH-GPx) ()is capable of reducing hydroperoxy lipids
esterified to biomembranes (27) and lipoproteins (28, 29) . Recently, a regulatory role of the PH-GPx
has been reported for the 5-lipoxygenase in human
leukocytes(30) .
For this paper we investigated the concerted action of the 15-lipoxygenase and the PH-GPx during the oxygenation of biomembranes and lipoproteins in reconstituted model systems. The data presented suggest that the PH-GPx may play an important role as down-regulator of the cellular 15-lipoxygenase pathway and that the cell-protective action of this enzyme may in part be due to the suppression of the 15-lipoxygenase activity.
Figure 1: HPLC analysis of the hydroperoxy lipids formed during the interaction of the 15-lipoxygenase with submitochondrial particles and human LDL. Submitochondrial particles (A) and human LDL (B) were incubated with the pure rabbit reticulocyte 15-lipoxygenase (10 nanokatals/ml) for 15 min at 25 °C. Lipid extraction and HPLC analysis with chemiluminescence detection were carried out as described under ``Materials and Methods.'' Sensitivity settings for the uv and chemiluminescence detectors were the same for the paired comparisons. a, treatment with the native 15-lipoxygenase; b, treatment with the heat denatured enzyme. PEOOH, phosphatidylethanolamine hydroperoxide; PIOOH, hydroperoxyphosphatidylinositol; PCOOH, hydroperoxyphosphatidylcholine; CEOOH, cholesterol ester hydroperoxide. Hydroperoxides of free cholesterol which would chromatograph close to the solvent front under this conditions were not detected in significant amounts.
The hydroperoxy lipids formed during 15-lipoxygenase-catalyzed reaction are suitable substrates for the PH-GPx. As indicated in Fig. 2, the chemiluminescence signal which indicated the presence of the hydroperoxy group disappeared after incubation of the lipoxygenase-treated complex substrates with the PH-GPx. However, the absorbance at 235 nm was retained suggesting that the conjugated diene chromophore was still present. These data suggest that PH-GPx reduced the hydroperoxy lipids to the corresponding alcohols which are not any more detectable in the chemiluminescence assay. For direct evidence of the formation of the hydroxy fatty acids, the lipid extracts of submitochondrial particles treated with the 15-lipoxygenase and PH-GPx were hydrolyzed and analyzed by SP-HPLC. As indicated in Fig. 3, 13S-hydroxy-9Z,11E-octadecadienoic acid was detected as major oxygenated fatty acid, whereas the corresponding hydroperoxide was not found.
Figure 2: Reduction of the hydroperoxy lipids formed by the 15-lipoxygenase by PH-GPx. Submitochondrial particles (A) and human LDL (B) were incubated with the pure rabbit reticulocyte 15-lipoxygenase (10 nanokatals/ml) for 15 min at 25 °C in the absence (I) and presence (II) of PH-GPx (0.4 nanokatal/ml). Lipid extraction and HPLC analysis were carried out as described under ``Materials and Methods.'' The absorbance at 235 nm (a) and chemiluminescence (b) were recorded simultaneously. Sensitivity settings for the uv and chemiluminescence detectors were the same for the paired comparisons.
Figure 3: SP-HPLC of oxygenated polyenoic fatty acids formed during the reaction of the 15-lipoxygenase with submitochondrial particles in the presence of PH-GPx. Submitochondrial particles were treated with the 15-lipoxygenase (10 nanokatals/ml) in the presence of PH-GPx (0.4 nanokatal/ml). The lipids were extracted and hydrolyzed under alkaline conditions. The oxygenated free fatty acid derivatives were prepared by RP-HPLC and analyzed further by SP-HPLC as described under ``Materials and Methods.'' Inset, chiral phase-HPLC separation of the enantiomers of 13-HODE and 15-HETE.
Figure 4: Oxygraphic measurements of the 15-lipoxygenase reaction with submitochondrial particles in the absence and presence of PH-GPx. Submitochondrial particles (1 mg of protein/ml) were reacted in 0.1 M phosphate buffer, pH 7.4, with 15-lipoxygenase (10 nanokatals/ml) in the oxygraphic chamber at 25 °C in the absence (1) and presence (2) of PH-GPx (0.4 nanokatal/ml). For trace 3, the submitochondrial particles were preincubated with 0.4 nanokatal/ml PH-GPx in order to reduce all hydroperoxides present in the substrate. For trace 4, PH-GPx-treated submitochondrial particles were supplemented with 1 µM 13S-HPODE at the time point indicated by the asterisk. Inset, after the reaction (15 min), the hydroperoxides formed were reduced to the corresponding hydroxy derivatives by addition of sodium borohydride, the lipids were extracted, hydrolyzed under alkaline conditions and the resulting hydroxy fatty acids were analyzed by RP-HPLC. The numbers of the traces correspond to those in the main part of the figure.
Among the glutathione peroxidases described so far, the PH-GPx has the highest capability of reducing membrane-bound hydroperoxy lipids as compared with free organic and anorganic hydroperoxides(27) . Measurement of PH-GPx is usually carried out by monitoring the disappearance of NADH consumed during the glutathione reductase reaction which is coupled to the glutathione-dependent hydroperoxide reduction(32) . However, at high concentrations of biomembranes, this photometric assay system is not applicable because of the turbidity of the sample. In such cases, the HPLC analysis of the hydroperoxy phospholipids by chemiluminescence detection described here may be used to test for PH-GPx activity.
The 15-lipoxygenases constitute the major lipoxygenase subtype
capable of oxidizing membrane phospholipids without the preceding
action of lipid-cleaving enzymes(11, 38) . It may be
suggested that the concerted action of both 15-lipoxygenase and PH-GPx
regulate the hydroperoxide tone of the membranes. Usually, the
steady-state concentrations of hydroperoxy lipids in mammalian cells
and in particular in the biomembranes are rather low. We investigated
several mammalian cells (erythrocytes, liver cells, monocytes, adipose
tissue, U937 cells, several endothelial cell lines) for the occurrence
of oxygenated lipids in the membranes and found less than 0.01% of the
polyenoic fatty acids to be present as oxygenated derivatives. ()The actual hydroperoxide concentration should even be
lower and thus usually escapes detection by the currently available
analytical methods.
Our data on the concerted action of 15-lipoxygenase and PH-GPx indicate that the 15-lipoxygenase reaction is influenced by the PH-GPx in two different ways. (i) The 15-lipoxygenase activity is down-regulated probably by reducing the hydroperoxy lipids necessary as activator for the lipoxygenase reaction from the incubation mixture. In the case of preincubation of the substrate with PH-GPx, which may lead to a complete disappearance of the activator, the lipoxygenase activity is completely abolished. This effect may be of physiological relevance since the cellular hydroperoxide tone of the membranes appears to be crucial for the activity of cellular lipoxygenases(45) . In the absence of hydroperoxide activator, the lipoxygenase should remain enzymatically inactive even if the enzyme protein is expressed at relatively high levels. The in vitro activation of the soybean lipoxygenase by hydroperoxy linoleic acid has been studied in detail(41, 42, 43) . In contrast, only limited information is available on the mechanism of hydroperoxide activation of mammalian lipoxygenases during the oxygenation of biomembranes. Our data indicate that depletion of membranes from hydroperoxy lipids by reducing them to their corresponding hydroxy derivatives leads to a complete inhibition of the lipoxygenase activity. In cells expressing high levels of PH-GPx, the 15-lipoxygenase pathways should be inhibited simply by the fact that the concentration of hydroperoxide activator is too low. These cells may express the lipoxygenase protein at a high level but the enzyme remains catalytically silent. In fact, there are several reports in the literature indicating that cellular 15-lipoxygenase activity can only be measured after activation or disruption of the cells(46, 47) . Several authors suggest that the enzyme may be present in a cryptic form and becomes liberated upon cell activation. However, so far, no experimental evidence has been presented that the 15-lipoxygenase is sequestered in such a way. In light of our findings, the lack of hydroperoxide activation may also be considered as a reason for functional masking of the 15-lipoxygenase in cells. It is tempting to speculate that cell activation may lead to an increase in hydroperoxide-forming processes which in turn may act as 15-lipoxygenase activator. Work is in progress to address this issue on the cellular level. (ii) PH-GPx preserves the specificity of the product pattern of the lipoxygenase reaction which was shown to be rather unspecific under certain experimental conditions. The PH-GPx prevents hydroperoxide-induced secondary lipid peroxidation or peroxide rearrangement simply be reducing the specific hydroperoxy lipids formed by the lipoxygenase to their corresponding alcohols. These data suggest that the formation of unspecific oxygenation products during the lipoxygenase reaction appears to be a post-catalytic phenomenon (secondary reactions) rather than a syn-catalytic one (dissociation of free radicals from the enzyme during its catalytic cycle).
In atherosclerotic lesions, a functional 15-lipoxygenase is expressed (48, 49) . In the time course of plaque formation in cholesterol-fed rabbits, specific lipoxygenase products were detected after 12 weeks of feeding(50) . After longer feeding periods, these products were superimposed by large amounts of nonspecific oxygenation products originating from nonenzymatic lipid peroxidation. Our data suggest that the specificity of lipoxygenase products is preserved over long time periods only if the reducing capacity of the cells is high enough to quickly reduce the hydroperoxy lipids formed by the lipoxygenase. Such a situation may be found in early stages of plaque development. At later stages, the oxidizing processes may prevail, and hydroperoxides may accumulate and may undergo isomerization and/or provoke nonenzymatic lipid peroxidation leading to the more unspecific product pattern as detected advanced lesions(51, 52, 53) .
The data reported here support the assumption that the intracellular action of the PH-GPx may down-regulate the cellular 15-lipoxygenase pathways and that a balanced concerted interaction of both enzymes is a precondition for the formation of specific 15-lipoxygenase products in mammalian tissues.