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The effects of reduced glutathione and cysteine on prostaglandin synthesis in rabbit kidney medulla slices
0306.4492186 $3.00 + 0.00 80 1986Pergamon Press Ltd
Camp. Biochem. Phvsiol. Vol. 83C, No. I, pp. 29-31, 1986 Printed in Great Bhtain
THE EFFECTS OF REDUCED GLUTATHIONE AND CYSTEINE ON PROSTAGLANDTN SYNTHESIS IN RABBIT KIDNEY MEDULLA SLICES TADASHI
FUJITA, TAKU TOMOKO UENO
Department
of Hygienic
Chemistry,
Osaka
YAMAMOTO, YOHKO
and College
MOTOKO
TABATA,
FUJIMOTO
of Pharmacy,
Matsubara,
Osaka
580, Japan
(Receiued 31 MaJj 1985) Abstract-l. The effects of GSH and cysteine on the generation of medullary were examined. 2. The formation of prostaglandin E, was enhanced by GSH, but was dose-related manner. 3. GSH inhibited prostaglandin F,, production, while cysteine stimulated 4. These results suggest that GSH and cysteine have contrary effects on synthesis by affecting endoperoxide EZ isomerase or endoperoxide reductase.
INTRODUCTION
Measurement
AND METHODS
Male rabbits (222.5 kg) were used in the present study. The kidneys were removed from anaesthetized (sodium pentobarbital, 30mg/kg) rabbits and rapidly chilled in ice-cold 0.9% NaCl. Kidney medulla slices were prepared as described elsewhere (Fujimoto and Fujita, 1982). of medulla slices
In all experiments, rabbit kidney medulla slices (0.4g) were preincubated in 4.0 ml of 0.15 M KCl/0.02 M Tris-HCI buffer (pH 7.4) at 4°C for 5min. Following preincubation, the medium was discarded, the slices rinsed twice with Tris-HCI buffer and incubated with various concentrations of drugs at 37’C for 30 min. Determination
of prostaglandin
by cysteine
it. prostaglandins
in a
E, and F?,
of prostaglandins
E2 and F2z formation
We reported previously that the major prostaglandins produced in our incubation of medulla slices and recovered in the medium were prostaglandins E, and F,, (Fujimoto ef al., 1983). The medium was acidified (approx. pH 3) with 0.1 N HCI on an ice-cold water bath and prostaglandins were extracted with 15 ml of ethyl ether. The ether layer was washed with 5 ml of 0.01 N HCI and water. Prostaglandins E, and F,, in the extracted lipid were simultaneously determined by HPLC using 9-anthryldiazomethane (ADAM) (Nimura and Kinoshita, 1980). Since ADAM contains many impurities which interfere with the HPLC determination, the purification of prostaglandins esterified with ADAM (PGs-ADAM) was attempted using a reversedphase silica cartridge (Sep-pak C,,, Waters Associates). The cartridge was prepared by rinsing it with 5 ml of methanol followed by IO ml of benzene/ethyl acetate (60:40). The sample was passed through the cartridge. The cartridge was washed with benzene/ethyl acetate (60:40, 7 ml) and the PCs-ADAM was then quantitatively eluted with benzene/ ethyl acetate/methanol (60:40: 5, 7 ml). The HPLC separation and quantitative determination of prostaglandins E, and F,, esterified with ADAM have been described previously (Hatsumi et al., 1982). Briefly, prostaglandins E, and F,, esterified with ADAM are separated in reversed phase chromatography and simultaneously quantitated by employing a Shimadzu model RF-530 fluorescence spectrofluorometer (excitation 365 nm, emission 412 nm) with an 8 ~1 flow cell. In the present study, prostaglandins were separated with a YMC packed column (ODS A-303 type, 4.6 mm i.d. x 25 cm) eluted at 1.2 ml/mm (Hitachi model 633A pump) with methanol/water (78.5: 21.5). Peak heights were measured for the quantification of the PGs-ADAM relative to the standard derivatives prepared from authentic prostaglandins E, and F,,.
Tissues
Incubation
reduced
Ez and F,,
was measured after its base-catalysed conversion to prostaglandin B, (Jouvenaz et al., 1970). Peak heights were measured for the quantification of the extracted prostaglandin B, relative to a prostaglandin B, standard prepared from authentic prostaglandin E,. This method cannot be used to measure prostaglandin F,, in the medium.
Amid the literature concerning the protective role of reduced glutathione (GSH) and related thiols against lipid peroxidation (Little and O’Brien, 1968; Christophersen, 1968, 1969), there are reports that indicate thiols can also act as pro-oxidants (Misra, 1974; Rowley and Halliwell, 1982). We have reported that lipid peroxidation can modulate arachidonate turnover and prostaglandin synthesis in rabbit kidney medulla slices (Fujimoto and Fujita, 1982; Fujimoto et al., 1983) and that prostaglandin cycle-oxygenase activity can be enhanced or inhibited by antioxidants depending on their type and concentration (Fujita et al., 1982). This work was started to investigate the effects of GSH and cysteine on the biosynthesis of prostaglandin in kidney medulla slices. In this paper we describe our finding that GSH and cysteine have contrary effects on the in vitro production of prostaglandins E, and F,, in kidney medulla. MATERIALS
prostaglandins
E2 formation
After incubation the medium was assayed for prostaglandin E, content by a high-pressure liquid chromatographic (HPLC) method (Fujimoto ef al., 1983). Briefly, prostaglandin E, extracted with ethyl acetate (approx. pH 3)
Statistics The values are presented as means Ifr SE. significance was determined by Student’s t-test. 29
Statistical
30
TADASHI FUJITA et al.
J. 0
I
,
I
1
I
I
2
3
4
5 Time (min)
Concentration (mM) Fig. 1. Effects of GSH and cysteine on prostaglandin E, synthesis in rabbit kidney medulla slices. Slices were incu-
bated for 30 min at 37°C in 0.15 M KClj0.02 M Tris-HCI buffer in the presence of different concentrations of GSH (0) or cysteine (A). Each point indicates the mean of five experiments; vertical lines show SE. §P < 0.05 compared to corresponding value in the absence of GSH or cysteine. tP < 0.02 compared to corresponding value in the absence of GSH or cysteine. *P i 0.01 compared to corresponding value in the absence of GSH or cysteine.
RESULTS AND DISCUSSION
Figure 1 illustrates the effects of various concentrations of GSH or cysteine on prostaglandin E2 synthesis in rabbit kidney medulla slices. The generation of prostaglandin E, in medulla slices was stimulated
by GSH, but was inhibited by the addition of cysteine. The effects were concentration-dependent. The effects of GSH (1 mM) or cysteine (5 mM) were apparent within 10 min after addition to the incubation mixture, and they persisted for 30min (Fig. 2). These results show a clear difference between GSH and cysteine in the effects on prostaglandin Ez synthesis. Previous studies from our (Fujimoto et al., 1983) and other (Erman and Raz, 1979) laboratories have shown that the major prostagiandins produced in the incubation of medulla slices and recovered in the medium were prostaglandins E, and F,,. The conversion of arachidonate to prostaglandin E, or F,,
Fig. 2. Time course of prostaglandin E, release from rabbit kidney medulla slices. Incubations were for 30 min at 37C in 0.15 M KCl/O.OZM Tris-HCI buffer in the absence (Of and the presence of 1mM GSH (0) or 5 mM cysteine (A). Each point indicates the mean of five experiments; vertical lines show SE.
may be separated essentially
into two components. Firstly, prostaglandin endoperoxide synthetase (cycle-oxygenase) catalyses the oxygenation of ara~hidonate to prostaglandin Gz and the subsequent reduction of prostaglandin G, to prostaglandin H, (Nugteren and Hazelhof, 1973; Hamberg ef al., 1974; Miyamoto et al., 1976; Van der Ouderaa et al., 1977; Ogino et al., 1978). The oxygenase and peroxidase activities are, to date, inseparable and are thought to be part of the same protein (Roth et al., 1980; Van der Ouderaa et al., 1980). Secondly, an endoperoxide E, isomerase catalyses rearrangement of prostaglandin H, into prostaglandin E,, or an endoperoxide reductase catalyses reduction of prostaglandin H, into prostaglandin F,, (Hamberg and Samuelsson, 1973). To examine further the effects of Fe*+, GSH and cysteine on the ratio of prostaglandin F,, to prostaglandin E, formation, prostaglandins Ez and F,, were simultaneously determined by HPLC using ADAM. As shown in Table 1, medulla slice preparations produce prostaglan~n F,/E, in a ratio of 0.10. We have reported that lipid perox~dation induced by Fe’+ inhibits the formation of prostaglandin Ez and that this inhibitory effect may be mediated by lipid
Table I. Effects of Fe2+. GSH and cvsteine on the prostaalandin Prostaglandin Incubation conditions _...-..-..-._ None Fe’+ (0.4 mM) GSH (1 mM) Cysteine (5 mM)
E,
Prostaglandin
F,,
fag/g wet wt of tissue) 4.90 * 0.21 2.04 & 0.06*‘* 7.22 * 0.37*** 3.09&0.11**
0.49 0.20 0.36 0.65
* * i +
F,.JE, ratio
F, (nmoles/g wet wt of tissue) E, (nmoies,/g wet wt of tissue)
0.02 0.01*** 0.01* 0.02*
Slices were incubated for 30 min at 37°C in 0. I5 M KCljO.02 M Tris-HC1 buffer. Values are means _t SE (IV = 6). ‘P < 0.05 compared to corresponding value in the absence of Fe2+, GSH or cyst&e. +*P < 0.02 compared to corresponding value m the absence of Fe2+, GSH or cysteine. ***P < 0.01 compared to corresponding value in the absence of Fe2+, GSH or cyst&e.
0.10 0.10 0.05 0.21
Prostaglandin peroxides (Fujimoto
synthesis and thioi compounds
via the inhibition of cycle-oxygenase et al., 1983). In the present study, Fe’+
(0.4mM) reduced the production of basal prostaglandins E, and F2*, 58 and 59%, respectively. Fe2+ did not alter the prostaglandin F,,/E, ratio. This result supports the idea that the inhibition of prostaglandins EZ and F,, generation by Fe2+ occurs at the cycle-oxygenase reaction. The effects of GSH (1 mM) and cysteine (5 mM) on prostaglandin E, formation were consistent with Fig. 1. On the other hand, prostaglandin F,, production was reduced by GSW, but was enhanced by cysteine. Thus, GSH decreased the prostaglandin F,,/E, ratio (0.05), whereas cysteine increased it (0.21). These results indicate that the regulation of prostaglandins E, and F,, generation by GSH and cysteine occurs primarily at the endoperoxide E, isomerase or endoperoxide reductase step, which in turn suggests that they are specifically involved in the isomerase or reductase reaction besides their antioxidative or peroxidative action (Little and O’Brien, 1968; Christophersen, 1968, 1969; Misra, 1974; Rowley and Halliwell, 1982). This speculation is, at least partially, supported by the data using prostaglandin E isomerase from bovine vesicular gland microsomes, which showed that GSH was required for the isomerization of prostaglandin II, to prostaglandin E, (Miyamoto et al., 1974, 1976). However the role of GSH in the isomerase has not yet been elucidated. It is evident that further studies are needed to define the determinants of the difference in action between GSH and cysteine; however, the present study serves to emphasize that GSH and cysteine have contrary effects on prostagiandins Ez and F,, synthesis. A~~nortlledgemenr-This work was supported Scientific Grant (No. 59771726) from the Education, Science and Culture, Japan.
in part Ministry
by of
31
Fujimoto Y., Tanioka H., Keshi I. and Fujita T. (1983) The interaction between lipid peroxidation and prostaglandin svnthesis in rabbit kidnev medulla slices. Biochem. 1. 212. 167-171. Fujita T., Fujimoto Y. and Tanioka H. (1982) Antioxidant effects on prostaglandin synthesis in rabbit kidney medulla slices. Experientia 38, 1472. Hamberg M. and Samuelsson B. (1973) Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc. nam. Acad. Sci. U.S.A. 70,
899-903. Hamberg M., Svensson J., Wakabayashi T. and Samuelsson B. (1974) Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc. natn. Acad. Sci. U.S.A. 71, 345349. Hatsumi M., Kimata S. and Hirosawa K. (1982) 9-Anthryldiazomethane derivatives of prostaglandins for high-performance liquid chromatographic analysis. J.
Chromutogr. 253, 271-275. Jouvenaz G. H., Nugteren D. H., Beerthuis R. K. and Van Dorp D. A. (1970) A sensitive method for the determination of prostaglandins by gas chromatography with electron-capture detection. Biochim. biophys. Acta 202,
23 I-234. Little C. and O’Brien P. J. (1968) An intracellular GSHperoxidasc with a lipid peroxide substrate. Eiochem.
Biophys. Res. Common. 31, 145-150. Misra H. P. (1974) Generation of superoxide free radical during the autoxidation of thioIs. J. biol. Chem. 249, 2151-2155. Miyamoto T., Yamamoto S. and Hayaishi 0. (1974) Prostaglandin synthetase system-resolution into oxygenase and isomerase components. Proc. natn. Acad. Sci. U.S.A.
71, 3645-3648. Miyamoto T.. Ogino N., Yamamoto S. and Hayaishi 0. (1976) Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. biol.
Chem. 251, 2629-2636. Nimura N. and Kinoshita T. (1980) Fluorescent labeling of fatty acids with 9-anthryldiazomethane (ADAM) for high performance liquid chromatography. Anal. Left. 13,
191-202. Nugteren D. H. and Hazelhof E. (I9731 properties of intermediates in prostaglandin
Isolation and biosynthesis.
Biochir?z.biophys. Acta 326, 448-461.
Ogino N., Ohki S., Yamamoto S. and Hayaishi 0. (1978) Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. biol. Chem. 253, 5061-5068. REFERENCES Roth G. J., Siok C. J. and 0~01s J. (1980) Structural Christophersen B. 0. (1968) Formation of monohydroxycharacteristics of prostaglandin synthetase from sheep polyenic fatty acids from lipid peroxides by a glutathione vesicular eland. J. bioi. Chem. 255, 1301-1304. Rowley D.- A. and Halliwell B.’ (1982) Superoxideperoxidase. Biochim. biophys. Acta 164, 35-46. Christophersen B. 0. (1969) Reduction of linolenic acid dependent formation of hydroxyl radicals in thepresence hydroperoxide by a glutathione peroxidase. Biochim. of thiol comoounds. FEBS Lett. 138. 33-36. hiophys. Acta 176,463-470. Van der Ouderaa F. J., Buytenhek M., Nugteren D. H. and Erman A. and Raz A. (1979) Effects of bivalent cations on Van Dorp D. A. (1977) ~rification and characterization prostaglandin biosynthesis and phospholipase A, activaof prostaglandin endoperoxide synthetase from sheep tion in rabbit kidney medulla slices. Biochem. J. 182, vesicular glands. Biochim. biophys. Acta 487, 315-331, 821-825. Van der Ouderaa F. J., Buytenhek M., Nugteren D. H. and Fujimoto Y. and Fujita T. (1982) Effects of lipid perVan Dorp D. A. (1980) Acetylation of prostaglandin oxidation on prostaglandin synthesis in rabbit kidney endoperoxide synthetase with acetylsalicylic acid. Eur. J. medulla slices. Biochim. biophys. Acta 710, 82-86. Biochem. 109, l-8.
Camp. Biochem. Phvsiol. Vol. 83C, No. I, pp. 29-31, 1986 Printed in Great Bhtain
THE EFFECTS OF REDUCED GLUTATHIONE AND CYSTEINE ON PROSTAGLANDTN SYNTHESIS IN RABBIT KIDNEY MEDULLA SLICES TADASHI
FUJITA, TAKU TOMOKO UENO
Department
of Hygienic
Chemistry,
Osaka
YAMAMOTO, YOHKO
and College
MOTOKO
TABATA,
FUJIMOTO
of Pharmacy,
Matsubara,
Osaka
580, Japan
(Receiued 31 MaJj 1985) Abstract-l. The effects of GSH and cysteine on the generation of medullary were examined. 2. The formation of prostaglandin E, was enhanced by GSH, but was dose-related manner. 3. GSH inhibited prostaglandin F,, production, while cysteine stimulated 4. These results suggest that GSH and cysteine have contrary effects on synthesis by affecting endoperoxide EZ isomerase or endoperoxide reductase.
INTRODUCTION
Measurement
AND METHODS
Male rabbits (222.5 kg) were used in the present study. The kidneys were removed from anaesthetized (sodium pentobarbital, 30mg/kg) rabbits and rapidly chilled in ice-cold 0.9% NaCl. Kidney medulla slices were prepared as described elsewhere (Fujimoto and Fujita, 1982). of medulla slices
In all experiments, rabbit kidney medulla slices (0.4g) were preincubated in 4.0 ml of 0.15 M KCl/0.02 M Tris-HCI buffer (pH 7.4) at 4°C for 5min. Following preincubation, the medium was discarded, the slices rinsed twice with Tris-HCI buffer and incubated with various concentrations of drugs at 37’C for 30 min. Determination
of prostaglandin
by cysteine
it. prostaglandins
in a
E, and F?,
of prostaglandins
E2 and F2z formation
We reported previously that the major prostaglandins produced in our incubation of medulla slices and recovered in the medium were prostaglandins E, and F,, (Fujimoto ef al., 1983). The medium was acidified (approx. pH 3) with 0.1 N HCI on an ice-cold water bath and prostaglandins were extracted with 15 ml of ethyl ether. The ether layer was washed with 5 ml of 0.01 N HCI and water. Prostaglandins E, and F,, in the extracted lipid were simultaneously determined by HPLC using 9-anthryldiazomethane (ADAM) (Nimura and Kinoshita, 1980). Since ADAM contains many impurities which interfere with the HPLC determination, the purification of prostaglandins esterified with ADAM (PGs-ADAM) was attempted using a reversedphase silica cartridge (Sep-pak C,,, Waters Associates). The cartridge was prepared by rinsing it with 5 ml of methanol followed by IO ml of benzene/ethyl acetate (60:40). The sample was passed through the cartridge. The cartridge was washed with benzene/ethyl acetate (60:40, 7 ml) and the PCs-ADAM was then quantitatively eluted with benzene/ ethyl acetate/methanol (60:40: 5, 7 ml). The HPLC separation and quantitative determination of prostaglandins E, and F,, esterified with ADAM have been described previously (Hatsumi et al., 1982). Briefly, prostaglandins E, and F,, esterified with ADAM are separated in reversed phase chromatography and simultaneously quantitated by employing a Shimadzu model RF-530 fluorescence spectrofluorometer (excitation 365 nm, emission 412 nm) with an 8 ~1 flow cell. In the present study, prostaglandins were separated with a YMC packed column (ODS A-303 type, 4.6 mm i.d. x 25 cm) eluted at 1.2 ml/mm (Hitachi model 633A pump) with methanol/water (78.5: 21.5). Peak heights were measured for the quantification of the PGs-ADAM relative to the standard derivatives prepared from authentic prostaglandins E, and F,,.
Tissues
Incubation
reduced
Ez and F,,
was measured after its base-catalysed conversion to prostaglandin B, (Jouvenaz et al., 1970). Peak heights were measured for the quantification of the extracted prostaglandin B, relative to a prostaglandin B, standard prepared from authentic prostaglandin E,. This method cannot be used to measure prostaglandin F,, in the medium.
Amid the literature concerning the protective role of reduced glutathione (GSH) and related thiols against lipid peroxidation (Little and O’Brien, 1968; Christophersen, 1968, 1969), there are reports that indicate thiols can also act as pro-oxidants (Misra, 1974; Rowley and Halliwell, 1982). We have reported that lipid peroxidation can modulate arachidonate turnover and prostaglandin synthesis in rabbit kidney medulla slices (Fujimoto and Fujita, 1982; Fujimoto et al., 1983) and that prostaglandin cycle-oxygenase activity can be enhanced or inhibited by antioxidants depending on their type and concentration (Fujita et al., 1982). This work was started to investigate the effects of GSH and cysteine on the biosynthesis of prostaglandin in kidney medulla slices. In this paper we describe our finding that GSH and cysteine have contrary effects on the in vitro production of prostaglandins E, and F,, in kidney medulla. MATERIALS
prostaglandins
E2 formation
After incubation the medium was assayed for prostaglandin E, content by a high-pressure liquid chromatographic (HPLC) method (Fujimoto ef al., 1983). Briefly, prostaglandin E, extracted with ethyl acetate (approx. pH 3)
Statistics The values are presented as means Ifr SE. significance was determined by Student’s t-test. 29
Statistical
30
TADASHI FUJITA et al.
J. 0
I
,
I
1
I
I
2
3
4
5 Time (min)
Concentration (mM) Fig. 1. Effects of GSH and cysteine on prostaglandin E, synthesis in rabbit kidney medulla slices. Slices were incu-
bated for 30 min at 37°C in 0.15 M KClj0.02 M Tris-HCI buffer in the presence of different concentrations of GSH (0) or cysteine (A). Each point indicates the mean of five experiments; vertical lines show SE. §P < 0.05 compared to corresponding value in the absence of GSH or cysteine. tP < 0.02 compared to corresponding value in the absence of GSH or cysteine. *P i 0.01 compared to corresponding value in the absence of GSH or cysteine.
RESULTS AND DISCUSSION
Figure 1 illustrates the effects of various concentrations of GSH or cysteine on prostaglandin E2 synthesis in rabbit kidney medulla slices. The generation of prostaglandin E, in medulla slices was stimulated
by GSH, but was inhibited by the addition of cysteine. The effects were concentration-dependent. The effects of GSH (1 mM) or cysteine (5 mM) were apparent within 10 min after addition to the incubation mixture, and they persisted for 30min (Fig. 2). These results show a clear difference between GSH and cysteine in the effects on prostaglandin Ez synthesis. Previous studies from our (Fujimoto et al., 1983) and other (Erman and Raz, 1979) laboratories have shown that the major prostagiandins produced in the incubation of medulla slices and recovered in the medium were prostaglandins E, and F,,. The conversion of arachidonate to prostaglandin E, or F,,
Fig. 2. Time course of prostaglandin E, release from rabbit kidney medulla slices. Incubations were for 30 min at 37C in 0.15 M KCl/O.OZM Tris-HCI buffer in the absence (Of and the presence of 1mM GSH (0) or 5 mM cysteine (A). Each point indicates the mean of five experiments; vertical lines show SE.
may be separated essentially
into two components. Firstly, prostaglandin endoperoxide synthetase (cycle-oxygenase) catalyses the oxygenation of ara~hidonate to prostaglandin Gz and the subsequent reduction of prostaglandin G, to prostaglandin H, (Nugteren and Hazelhof, 1973; Hamberg ef al., 1974; Miyamoto et al., 1976; Van der Ouderaa et al., 1977; Ogino et al., 1978). The oxygenase and peroxidase activities are, to date, inseparable and are thought to be part of the same protein (Roth et al., 1980; Van der Ouderaa et al., 1980). Secondly, an endoperoxide E, isomerase catalyses rearrangement of prostaglandin H, into prostaglandin E,, or an endoperoxide reductase catalyses reduction of prostaglandin H, into prostaglandin F,, (Hamberg and Samuelsson, 1973). To examine further the effects of Fe*+, GSH and cysteine on the ratio of prostaglandin F,, to prostaglandin E, formation, prostaglandins Ez and F,, were simultaneously determined by HPLC using ADAM. As shown in Table 1, medulla slice preparations produce prostaglan~n F,/E, in a ratio of 0.10. We have reported that lipid perox~dation induced by Fe’+ inhibits the formation of prostaglandin Ez and that this inhibitory effect may be mediated by lipid
Table I. Effects of Fe2+. GSH and cvsteine on the prostaalandin Prostaglandin Incubation conditions _...-..-..-._ None Fe’+ (0.4 mM) GSH (1 mM) Cysteine (5 mM)
E,
Prostaglandin
F,,
fag/g wet wt of tissue) 4.90 * 0.21 2.04 & 0.06*‘* 7.22 * 0.37*** 3.09&0.11**
0.49 0.20 0.36 0.65
* * i +
F,.JE, ratio
F, (nmoles/g wet wt of tissue) E, (nmoies,/g wet wt of tissue)
0.02 0.01*** 0.01* 0.02*
Slices were incubated for 30 min at 37°C in 0. I5 M KCljO.02 M Tris-HC1 buffer. Values are means _t SE (IV = 6). ‘P < 0.05 compared to corresponding value in the absence of Fe2+, GSH or cyst&e. +*P < 0.02 compared to corresponding value m the absence of Fe2+, GSH or cysteine. ***P < 0.01 compared to corresponding value in the absence of Fe2+, GSH or cyst&e.
0.10 0.10 0.05 0.21
Prostaglandin peroxides (Fujimoto
synthesis and thioi compounds
via the inhibition of cycle-oxygenase et al., 1983). In the present study, Fe’+
(0.4mM) reduced the production of basal prostaglandins E, and F2*, 58 and 59%, respectively. Fe2+ did not alter the prostaglandin F,,/E, ratio. This result supports the idea that the inhibition of prostaglandins EZ and F,, generation by Fe2+ occurs at the cycle-oxygenase reaction. The effects of GSH (1 mM) and cysteine (5 mM) on prostaglandin E, formation were consistent with Fig. 1. On the other hand, prostaglandin F,, production was reduced by GSW, but was enhanced by cysteine. Thus, GSH decreased the prostaglandin F,,/E, ratio (0.05), whereas cysteine increased it (0.21). These results indicate that the regulation of prostaglandins E, and F,, generation by GSH and cysteine occurs primarily at the endoperoxide E, isomerase or endoperoxide reductase step, which in turn suggests that they are specifically involved in the isomerase or reductase reaction besides their antioxidative or peroxidative action (Little and O’Brien, 1968; Christophersen, 1968, 1969; Misra, 1974; Rowley and Halliwell, 1982). This speculation is, at least partially, supported by the data using prostaglandin E isomerase from bovine vesicular gland microsomes, which showed that GSH was required for the isomerization of prostaglandin II, to prostaglandin E, (Miyamoto et al., 1974, 1976). However the role of GSH in the isomerase has not yet been elucidated. It is evident that further studies are needed to define the determinants of the difference in action between GSH and cysteine; however, the present study serves to emphasize that GSH and cysteine have contrary effects on prostagiandins Ez and F,, synthesis. A~~nortlledgemenr-This work was supported Scientific Grant (No. 59771726) from the Education, Science and Culture, Japan.
in part Ministry
by of
31
Fujimoto Y., Tanioka H., Keshi I. and Fujita T. (1983) The interaction between lipid peroxidation and prostaglandin svnthesis in rabbit kidnev medulla slices. Biochem. 1. 212. 167-171. Fujita T., Fujimoto Y. and Tanioka H. (1982) Antioxidant effects on prostaglandin synthesis in rabbit kidney medulla slices. Experientia 38, 1472. Hamberg M. and Samuelsson B. (1973) Detection and isolation of an endoperoxide intermediate in prostaglandin biosynthesis. Proc. nam. Acad. Sci. U.S.A. 70,
899-903. Hamberg M., Svensson J., Wakabayashi T. and Samuelsson B. (1974) Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc. natn. Acad. Sci. U.S.A. 71, 345349. Hatsumi M., Kimata S. and Hirosawa K. (1982) 9-Anthryldiazomethane derivatives of prostaglandins for high-performance liquid chromatographic analysis. J.
Chromutogr. 253, 271-275. Jouvenaz G. H., Nugteren D. H., Beerthuis R. K. and Van Dorp D. A. (1970) A sensitive method for the determination of prostaglandins by gas chromatography with electron-capture detection. Biochim. biophys. Acta 202,
23 I-234. Little C. and O’Brien P. J. (1968) An intracellular GSHperoxidasc with a lipid peroxide substrate. Eiochem.
Biophys. Res. Common. 31, 145-150. Misra H. P. (1974) Generation of superoxide free radical during the autoxidation of thioIs. J. biol. Chem. 249, 2151-2155. Miyamoto T., Yamamoto S. and Hayaishi 0. (1974) Prostaglandin synthetase system-resolution into oxygenase and isomerase components. Proc. natn. Acad. Sci. U.S.A.
71, 3645-3648. Miyamoto T.. Ogino N., Yamamoto S. and Hayaishi 0. (1976) Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. biol.
Chem. 251, 2629-2636. Nimura N. and Kinoshita T. (1980) Fluorescent labeling of fatty acids with 9-anthryldiazomethane (ADAM) for high performance liquid chromatography. Anal. Left. 13,
191-202. Nugteren D. H. and Hazelhof E. (I9731 properties of intermediates in prostaglandin
Isolation and biosynthesis.
Biochir?z.biophys. Acta 326, 448-461.
Ogino N., Ohki S., Yamamoto S. and Hayaishi 0. (1978) Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. biol. Chem. 253, 5061-5068. REFERENCES Roth G. J., Siok C. J. and 0~01s J. (1980) Structural Christophersen B. 0. (1968) Formation of monohydroxycharacteristics of prostaglandin synthetase from sheep polyenic fatty acids from lipid peroxides by a glutathione vesicular eland. J. bioi. Chem. 255, 1301-1304. Rowley D.- A. and Halliwell B.’ (1982) Superoxideperoxidase. Biochim. biophys. Acta 164, 35-46. Christophersen B. 0. (1969) Reduction of linolenic acid dependent formation of hydroxyl radicals in thepresence hydroperoxide by a glutathione peroxidase. Biochim. of thiol comoounds. FEBS Lett. 138. 33-36. hiophys. Acta 176,463-470. Van der Ouderaa F. J., Buytenhek M., Nugteren D. H. and Erman A. and Raz A. (1979) Effects of bivalent cations on Van Dorp D. A. (1977) ~rification and characterization prostaglandin biosynthesis and phospholipase A, activaof prostaglandin endoperoxide synthetase from sheep tion in rabbit kidney medulla slices. Biochem. J. 182, vesicular glands. Biochim. biophys. Acta 487, 315-331, 821-825. Van der Ouderaa F. J., Buytenhek M., Nugteren D. H. and Fujimoto Y. and Fujita T. (1982) Effects of lipid perVan Dorp D. A. (1980) Acetylation of prostaglandin oxidation on prostaglandin synthesis in rabbit kidney endoperoxide synthetase with acetylsalicylic acid. Eur. J. medulla slices. Biochim. biophys. Acta 710, 82-86. Biochem. 109, l-8.