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Effects of Oxygen Radicals Hydrogen Peroxide and Water-soluble Singlet Oxygen Carriers on 5- and 12-Lipoxygenase.

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819
Effects of Oxygen Radicals on Lipoxygenases
Effects of Oxygen Radicals, Hydrogen Peroxide and Water-soluble
Singlet Oxygen Carriers on 5- and 12-Lipoxygenase
Klaus Muller* and Klaus Ziereis
Institute of Pharmacy, University of Regensburg, P.O. Box 101042,D-8400 Regensburg, Germany
Received October 14, 1992
Wirkung von Sauerstoffradikalen, Wasserstoffperoxid und wasserloslichen Singulettsauerstoff-Tragernauf 5- und 12-Lipoxygenase
Inhibition of 5-lipoxygenase from bovine polymorphonuclear leukocytes
and 12-lipoxygenasefrom bovine platelets by active oxygen species has
been studied. Oxygen radicals and hydrogen peroxide markedly inhibited
12-lipoxygenase, whereas 5-lipoxygenase activity was only moderately
influenced. Singlet oxygen liberated from water-soluble naphthalene endoperoxides was without effect.
The products of 5-lipoxygenase (5-L0), particularly leukotriene B4
(LTB4), and 12-hydroxyeicosatetraenoicacid (12-HETE) of the 12-LO
pathway have been implicated in the development of inflammatory skin
diseases, such as psoriasis’). The etiology of psoriasis is still unknown’),
but a characteristic of lesional skin is the elevated levels of these oxygenation products of arachidonic acid.
The oxygenation reactions of LO involve free radical intermediates’) and
even singlet oxygen4). Moreover, the generation of hydroxyl or an
equivalent oxygen radical by conversion of various hydroperoxyeicosatetraenoic acids (HPETEs) to HETEs has been suggested5).
Accordingly, many LO inhibitors may be classified as antioxidants or
radical scavengers, whereas on the other hand 5-LO is prone to
inactivation by peroxides and even a possible inactivation by superoxide
(‘02-)
and hydroxyl radicals (’OH) has been proposed6). Consequently,
active oxygen species may play an ambiguous role in arachidonic acid
metabolism. They may function as activators of LO as well as suppressors
of eicosanoid biosynthesis in a self-catalyzedinactivation and may be able
to serve as “suicide” substrates’). These observations are in line with the
generation and/or scavenging of oxygen radicals or singlet oxygen by
antipsoriaticdrugss).For instance, benoxaprofen, which is a singlet oxygen
(lo2)sensitizer9),inhibits the LO and is beneficial in psoriasislO).Likewise
anthralin (dithranol), an effective antipsoriatic agent, has been shown to
inhibit both 5-LO”) and 12-LO”). In previous studies we have reported on
the formation of ‘0, and oxygen radicals by anthralin’3*’4z’5).
So there
might be a correlation between LO inhibition and the generation of active
oxygen species. The direct effect of active oxygen species on 5- and 12LO activity has not yet been established.
Die Hemmung der 5-Lipoxygenase von polymorphkernigen Rinder-Leukocyten und der 12-Lipoxygenase von Rinder-Thrombocyten durch aktive
Sauerstoffspezies wurde untersucht. Sauerstoffradikale und Wasserstoffperoxid zeigten eine deutliche Hemmung der 12-Lipoxygenase, wiihrend
sie die 5-Lipoxygenase-Aktivitatnur in geringem Ausmd beeintrachtigten. Aus wasserloslichen Naphthalin-Endoperoxiden freigesetzter Singulett
Sauerstoff war ohne Effekt.
nordihydroguaiaretic acid, xanthine, xanthine oxidase (EC 1.1.3.22) were
from Sigma; Munich, Germany; 5-HETE, I2-HETE, 5S,12S-diHETE, 12HHT, LTB4, PGB’ (Paesel GmbH FrankfurtM., Germany); FeC13 6H20,
FeS04 . H20, hydrogen peroxide (30%), nitro blue tetrazolium (NBT)
were obtained from Merck; Darmstadt, Germany; solvents for HPLC were
of HPLC quality (Roth; Karlsruhe, Germany); bovine blood was obtained
from the local slaughterhouse.
Active oxygen species generating systems
In order to produce a flux of ‘Oy a system of xanthine oxidase (XO, 0.02
U/mL) and xanthine (30 pM) was used’@. Generation of ’02-was
ascertained spectrophotometricallyby monitoring the reduction of NBT at
560 nrn. XO was added last to initiate the reaction. The phosphate buffer
solution was passed through a column of chelating resin to remove trace
levels of iron. DTPA (0.1 mM) was added to prevent hydroxyl radical
formation.
Hydroxyl radical was generated by a superoxide driven Fenton
reaction”) with the xanthine/XO system described above by addition of
0.1 mM FeS04 . HzO and 0.1 mM DTPA. Controls were performed with
the ferrous salt, H202, xanthine, and the chelators each alone. Additionally,
hydroxyl radicals were produced in a Fenton reaction with hydrogen
peroxide (17.6 pM) and Fe2+-DTPA(0.1 mM). H202 was added last in
three portions to initiate the reaction.
R’
In this study, we have examined the effects of active
oxygen species on the LTB, or 5-HETE production by
bovine polymorphonuclear leukocytes (PMNL) and 12HETE production by bovine platelets.
Materials and Methods
Chemicals
Arachidonic acid, calcium ionophore A23 187, chelating resin (sodium
form), diethylenetriaminepentaacetic acid (DTPA), histopaquea-1077,
Arch. Pharn. (Weinheim) 326,819-821 (1993)
Figure 1
0VCH VerlagsgesellschaftmbH, D-69451 Weinheim, 19930365-6233/93/1010-0819$5.00 + .25/0
Miiller and Ziereis
Singlet oxygen was produced by thermal decomposition of watersoluble endoperoxides of naphthalene derivatives (30 pM, Fig. 1) with different partition coefficients and half-lives between 25 and 52 min, as
described‘”’.
5-Lipmygenase assay
PMNL were prepared essentially as described”) from Na-EDTAanticoagulated bovine blood. Contaminating platelets were removed by
repeated centrifugations at 100 g for 20 min. The purified PMNL were
suspended at a concentration of 1 x lo7 cells/mL in phosphate buffered
saline (PBS) (composed of 8.00 g NaCl, 0.20 g KCI, 1.00 g Na,HPO,
2H,O, 0.15 g NaH2P0, H,O, 0.20 g KH,PO,, adjusted to pH 7.4 with 3
N NH, in a final volume of 1000 mL bidist. H20). Cells were counted with
a Sysmex microcellcounter CC-130 attached to an auto dilutor AD-241.
Preincubation was performed with 2.4 mL of the suspension and 10 pL
of active oxygen species generating systems at the desired concentrations
in PBS (endoperoxides in DMSO) or vehicle control (DMSO at final
concentration of 0.4%) for 30 rnin at 37°C in a shaking water bath. The
syntheses of LTB4 and 5-HETE were stimulated by the addition of CaC1,
and calcium ionophore A23187 (final concentrations 2 mM and 20 pM).
The incubation was allowed to proceed for 10 min at 37°C. Then the
incubation was terminated by the addition of 3.0 mL MeOH/CH3CN (1+1)
containing NDGA as a scavenger of active oxygen species (final
concentration 0.01 mM) and prostaglandin B2 as an internal standard (final
concentration 0.3 pM) and the incubation mixture was kept in an ice bath
for 20 min. After centrifugation at 4000 g for 5 min at 4°C the supernatant
was diluted with 5 mL of water and applied to a prewashed octadecylsilane
reversed phase cartridge (Baker), which had been washed with 10 mL of
MeOH and 5 mL of water. The eicosanoids were eluted with 3 mL of
MeOH, diluted with 3 mL of water and subjected to reverse phase HPLC
analysis.
HPLC was performed on a 250 x 4 mm column packed with Nucleosil
C i 8 (7 pm particles; Bischoff, Leonberg, Germany). The isocratic elution
conditions of LTB, were THF/MeOH/water (25+30+45, vol/vol), plus 0.1
vol % acetic acid, adjusted to pH 5.5 with 3N NH3, at a flow rate of 0.9
mL/min (Kontron 420 pump), monitored at 280 nm with a Kontron 735
LC UV detector, whereas 5-HETE was monitored at 232 nm using
MeOH/water (77+33, vol/vol), plus 0.1 vol % acetic acid, pH 5.5, flow
rate 0.9 mL/min. Integrated areas of the peaks were compared to the PGB,
internal standard and to external standards of authentic samples. Molar
absorption coefficients of Samuelsson et al.”) were used for calculations.
% Inhibition of the formation of LTB4 or 5-HETE by bovine PMNL was
calculated by the comparison of active oxygen generating system (N = 3,
SD < 10%) with control activity (N = 8, SD < 5%).
12-Lipoxygenase assay
The procedure was similar to the one described for 5-LO with the
following modification for cell preparation: The platelets were prepared
from Na-EDTA-anticoagulated bovine blood. After centrifugation at 100 g
for 20 min the platelet-rich plasma was removed by aspiration. The
platelets were collected by centrifugation at 1000 g for 15 min and the
platelet pellet was suspended in PBS at a volume equal to one third of the
original plasma volume. The suspension was centrifuged at 1000 g for 15
min and the washed platelets were resuspended at a concentration of 1 x
lo7 cells/mL in PBS. 12-HETE was detected at 232 nm.
Results and Discussion
Inhibition of 5- and 12-LO by active oxygen species has
been investigated with PMNL and platelets from bovine
blood, because it can be easily obtained in large quantities
and the production of LTB, by bovine PMNL’9,21)and 12HETE by bovine platelets22)has been well established.
Table 1 shows the influence of various active oxygen
species producing systems on the formation of 5-LO
products from bovine PMNL and 12-LO products from
bovine platelets.
Table 1 shows that 12-LO is markedly susceptible to
oxygen radicals and H202, whereas 5-LO is only moderately inhibited by these active oxygen species. On the other
hand, ‘02generated by the thermal decomposition of
naphthalene endoperoxides was completely ineffective. A
recent study showed that LO inhibition by phenidone and
BW755C only occurs after oxidative activation of the drugs
by the peroxidase-like activity, and reactive species formed
during this process, among others ‘ 0and
~ H202,have been
suggested for the ina~tivation~~).
The results from our studies indicate that in the case of
12-LO oxygen radicals and H202 are capable of enzyme
inactivation at concentrations that may be produced by
drugs acting as inhibitors of LO. Therefore, a mechanism
for enzyme inactivation which involves the participation of
active oxygen species appears likely for certain 12-LO
inhibitors, but not for 5-LO inhibitors, because 5-LO
activity is only slightly affected by active oxygen species.
In addition, the production of ‘02by LO inhibitors does not
contribute to their mechanism of inactivation of both enzymes.
Table 1: Inhibition of 5-LO and 12-LO by active oxygen species
Active oxygen species generatingsystem
’02- xanthine (30 )IM)/XO(0.02 U/mL)
‘OH
15
89
FeSOA-DTPA (0.1mMYxanthine (30 )IM)/XO (0.02U/mL)
15
87
FeS04-D-A
17
85
17
88
(0.1 mM)/H~O~
(17.6 PM)
H2Oz HzOz (17.6 W)
‘0,
5-LO
12-LO
(PMNL)
(Platelets)
% Inhibition % Inhibidon
naphthalene endoperoxides (30 NM, Fig. 1)
Inhibition was calculated by the comparison of the mean values of test system (n = 3)
with control (n = 6-8), range < 10%. Controls with FeSO,, DTPA or xanthine each alone
did not influence 5-LO and 12-LO activity.
Arch. Pharm. (Weinheim)326,819-821 (1993)
821
Effects of Oxygen Radicals on Lipoxygenases
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