PROSTAGLANDINS
-WORK IN PROGRESSSYNTHESIS OF PROSTAGLANDIN F2a IN RAT LIVER
Ikuo Morita, Wen-Chang Chang and Sei-itsu Murota Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo-173, Japan
Prostaglandins are a group of biologically active fatty acids widelv distributed in animal tissues (1). In sumort of this fact. prostaglandin synthetase has been found.along with its substrate . fatty acids in virtually all mammalian tissues investigated to date (2). However only a small number of reports on prostaglandin synthesis in the liver are available, the smallest number concerning the synthesis of PGFza. Cohen and Jaffe have demonstrated negligible activity for PGF2, synthesis in rat liver homogenate as well as hepatoma cells (3). MacManus and Braceland have reported the appearance of PGF2, synthetic activity in regenerating rat liver, though the activity was absent in normal livers (4). This paper reported here describes our results that normal rat liver microsome can convert arachidonic acid into prostaglandin, and that the prostaglandin was identified with PGF2,. Livers from male rats, Sprague-Dawley strain weighing about 200 g, were homogenized with 0.05 M phosphate buffer, pH 7.6, and the homogenate was centrifuged 9000 x g for 30 min, and then the supernatant fraction was centrifuged again at 105000 x g for 60 min. The microsomal pellet was resuspended with the same buffer and it was used as an enzyme source. The reaction mixture contained 4 ml of the enzyme solution (20 mg protein/ ml) and O.ZuCi of [lJ'+C]arachidonic acid (specific activity 58 mCi/ mnol, purchased from the Radiochemical Centre, Amersham, Enqland) and 5.Oug of unlabeled arachidonic acid, was incubated in air at 37'C for 30 min with shaking. After termination of the reaction, radioactive products were extracted with ethyl acetate. The resulting organic phase was evaporated, and the residue was dissolved in a small amount of ethanol and applied to thin layer chromatography plate. The plate was developed with the AI solvent system (benzene/dioxane/aceticacid, 20:20:1 v/v) described by Green and Samuelsson (5). The radioactive products were detected by a radiochromato-scanneras well as by autoradiography with X-ray film. Details of all the procedures mentioned above are described in our previous paper (6). The obtained autoradiogram together with a scanning profile of the thin layer chromatogram are shown in Fig.1. Besides a large peak owing to unchanged substrate, 2 main peaks due to the radioactive products were detected. The Rf values of the 2 products were 0.38
AUGUST 1977 VOL. 14 NO. 2
403
PROSTAGLANDINS
and 0.51, which are completely similar to those of authentic PGFz and PGE2, respectively. The conversion rate from arachidonic aci% to the more polar product was approximately 2% and that to the less polar product was about 1.5% when 20 mg protein (equivalent to the quantity of microsome obtained from 1 g wet weight liver) per tube was used. Examination of cofactors on the conversion rate showed that reduced glutathione and hydroquinone, which have often been used in many tissues (7,8,g) to increase the conversion rate, had no effect in the case of liver microsome. The radioactive zone corresponding to authentic PGF,,,was scrapped off and the radioactivity was extracted with a mixture of chroloform/methanol (1:l v/v). NaBH, reduction for an aliquot of the extract showed that this radioactive product was inert to this reaction, suggesting it cannot have a keto-group in its molecule. Identification for the radioactive product was carried out first by the double isotope method described in our previous paper (6). Another aliquot of the extracted radioactive product was re-purified by thin layer chromatography using the AI solvent system. The purified [14C]labeled product was mixed with an appropriate quantity of authentic [WjPGFzcr and the mixture was successively chromatographed on thin layers with 3 kinds of solvent systems. The sequence was (A) the AI solvent system, (B) chroloform/ethyl acetate/ methanol (1:l:l v/v) and (C) the AI1 solvent system (ethyl acetate/ acetic acid/methanol/2,2,4-trimethylpentane/water, 110:30:35:10:100 v/v). Plate impregnated with AgNOs was used in the AI1 solvent system, which, owing to its high resolving power and sensitivity, enable the separation of all the known prostaglandins including their subfamilies (5). The method developed by Kunze and Bohn (10) was used for the extraction of the radioactivity from the silver impregnated plate. The radiochromatogram in each of the 3 ste s showed a single peak. A part of the radioactivity due to p'+C!Iand C3Hl in each zone were separately counted in the double isotope setting by a liquid scintillation counter. As shown in Table I, the ratio of 1'+C/3H remained constant throughout the successive chromatography with 3 different kinds of solvent systems. This result strongly suggests that the more polar radioactive product shown in Fig.1 was identical with PGFza. To confirm this, the product was further subjected to gas chromatography-mass spectrometry analysis. Details of the method for collection of the sample and preparation of methyl estermethoxime-trimethylsilyl ether, methyl ester-trimethylsilyl ether, methoxime-trimethylsilyl ester-trimethylsilyl ether and trimethylsilyl ester-trimethylsilyl ether derivatives of the sample for gas chromatography-mass spectrometry analysis have been described in our previous papers (11,12,13). The gas chromatogram and mass spectra of these derivatives were exactly identical to those of authentic PGFz,. From all the results mentioned above, the more polar product in Fig.1 was identified with PGFe,. The identification of the less polar product and an investigation of the properties of prostaglandin synthetase in rat liver are currently under way in our labolatory.
AUGUST 1977 VOL. 14 NO. 2
PROSTAGLANDINS
Fig. 1
Autoradiogram
and radiochromatogram scan showing the prostaglandin synthesizing activity by the microsomal fraction of rat liver.
Table I
Identification
of the more polar-radioactive
product
by the double isotope method.
solvent system AI
(original)
Rf value
14 C (cpm)
3H (cm)
14C/3H
0.38
11420
32300
0.35
AI
0.38
5256
15460
0.34
B
0.42
3350
9530
0.35
AI1
0.40
2022
6039
0.34
AUGUST 1977 VOL. 14 NO. 2
405
PROSTAGLANDINS
REFERENCES 1.
Bergstrom, S., Prostaglandins : Members of a new hormonal system. Science E,‘382 (1967)
2.
Silver, M.J. and Smith, J.B., Prostaglandins as intracellular messengers. Life Sciences 16, 1635 (1975)
3.
Cohen,,F. and Jaffe, B.M., Production of prostaglandins by cells in vitro : Radioimmunoassaymeasurement of the conver.sionof sachidonic acid to PGE, and PGF2a. Biochem. Biophys. Res. Commun. 55_,724 (1973)
4.
MacManus, J.P. and Braceland, B.M., A connection between the production of prostaglandins during liver regeneration and the DNA synthetic response. Prostaglandins11, 609 (1976)
5.
Green, K. and Samuelsson, B., Prostaglandins and related factors : XIX. Thin-layer chromatography of prostaglandins.J.Lipid Res. 5, 117 (1964)
6.
Chang, W.C., Murota, S. and Tsurufuji, S., Role of prostaglandin E in carrageenin induced inflammation in rat. Biochem. Pharmac. 3, 2045 (1976)
7.
Takeguchi, C., Kohno, E. and Chales, J.S., Mechanism of prostaglandin biosynthesis. I. Characterizationand assay of bovine prostaglandin synthetase. Biochemistry l& 2372 (1971)
8.
Blackwell, G.J., Flower, R.J. and Vane, J.R., Some characteristics of the prostaglandin synthesizing system in rabbit kidney microsomes. Biochemica Biophysics Acta 398, 178 (1975)
9.
Kingston, W.P. and Greaves, M.W., Factors affecting prostaglandin synthesis by rat skin microsomes. Prostaglandins12, 51 (1976)
10.
Kunze, H. and Bohn, E., Extraction of prostaglandin from AgNOsimpregnated silica gel layers from biological assay. NaunynSchmiedoberg's Arch. Pharmak. 267, 380 (1970)
11.
Chang, W.C., Murota, S..,Matsuo, M. and Tsurufuji, S., A new prostaglandin transformed from arachidonic acid in carrageenin induced granuloma. Biochem. Biophys. Res. Commun. 72_,1259 (1976)
12.
Chang, W.C. and Murota, S., Identificationof 6-keto prostaglandin F1, formed from arachidonic acid in bovine seminal vesicles. Biochemica Biophysics Acta 486, 136 (1977)
13.
Chang, W.C., Murota, S. and Tsurufuji, S., Thromboxane 82 transformed from arachidonic acid in carrageenin induced granuloma. Prostaglandins13, 17 (1977)
Received
406
3/24/77
- Approved
5/25/77
AUGUST 1977 VOL. 14 NO. 2
-WORK IN PROGRESSSYNTHESIS OF PROSTAGLANDIN F2a IN RAT LIVER
Ikuo Morita, Wen-Chang Chang and Sei-itsu Murota Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo-173, Japan
Prostaglandins are a group of biologically active fatty acids widelv distributed in animal tissues (1). In sumort of this fact. prostaglandin synthetase has been found.along with its substrate . fatty acids in virtually all mammalian tissues investigated to date (2). However only a small number of reports on prostaglandin synthesis in the liver are available, the smallest number concerning the synthesis of PGFza. Cohen and Jaffe have demonstrated negligible activity for PGF2, synthesis in rat liver homogenate as well as hepatoma cells (3). MacManus and Braceland have reported the appearance of PGF2, synthetic activity in regenerating rat liver, though the activity was absent in normal livers (4). This paper reported here describes our results that normal rat liver microsome can convert arachidonic acid into prostaglandin, and that the prostaglandin was identified with PGF2,. Livers from male rats, Sprague-Dawley strain weighing about 200 g, were homogenized with 0.05 M phosphate buffer, pH 7.6, and the homogenate was centrifuged 9000 x g for 30 min, and then the supernatant fraction was centrifuged again at 105000 x g for 60 min. The microsomal pellet was resuspended with the same buffer and it was used as an enzyme source. The reaction mixture contained 4 ml of the enzyme solution (20 mg protein/ ml) and O.ZuCi of [lJ'+C]arachidonic acid (specific activity 58 mCi/ mnol, purchased from the Radiochemical Centre, Amersham, Enqland) and 5.Oug of unlabeled arachidonic acid, was incubated in air at 37'C for 30 min with shaking. After termination of the reaction, radioactive products were extracted with ethyl acetate. The resulting organic phase was evaporated, and the residue was dissolved in a small amount of ethanol and applied to thin layer chromatography plate. The plate was developed with the AI solvent system (benzene/dioxane/aceticacid, 20:20:1 v/v) described by Green and Samuelsson (5). The radioactive products were detected by a radiochromato-scanneras well as by autoradiography with X-ray film. Details of all the procedures mentioned above are described in our previous paper (6). The obtained autoradiogram together with a scanning profile of the thin layer chromatogram are shown in Fig.1. Besides a large peak owing to unchanged substrate, 2 main peaks due to the radioactive products were detected. The Rf values of the 2 products were 0.38
AUGUST 1977 VOL. 14 NO. 2
403
PROSTAGLANDINS
and 0.51, which are completely similar to those of authentic PGFz and PGE2, respectively. The conversion rate from arachidonic aci% to the more polar product was approximately 2% and that to the less polar product was about 1.5% when 20 mg protein (equivalent to the quantity of microsome obtained from 1 g wet weight liver) per tube was used. Examination of cofactors on the conversion rate showed that reduced glutathione and hydroquinone, which have often been used in many tissues (7,8,g) to increase the conversion rate, had no effect in the case of liver microsome. The radioactive zone corresponding to authentic PGF,,,was scrapped off and the radioactivity was extracted with a mixture of chroloform/methanol (1:l v/v). NaBH, reduction for an aliquot of the extract showed that this radioactive product was inert to this reaction, suggesting it cannot have a keto-group in its molecule. Identification for the radioactive product was carried out first by the double isotope method described in our previous paper (6). Another aliquot of the extracted radioactive product was re-purified by thin layer chromatography using the AI solvent system. The purified [14C]labeled product was mixed with an appropriate quantity of authentic [WjPGFzcr and the mixture was successively chromatographed on thin layers with 3 kinds of solvent systems. The sequence was (A) the AI solvent system, (B) chroloform/ethyl acetate/ methanol (1:l:l v/v) and (C) the AI1 solvent system (ethyl acetate/ acetic acid/methanol/2,2,4-trimethylpentane/water, 110:30:35:10:100 v/v). Plate impregnated with AgNOs was used in the AI1 solvent system, which, owing to its high resolving power and sensitivity, enable the separation of all the known prostaglandins including their subfamilies (5). The method developed by Kunze and Bohn (10) was used for the extraction of the radioactivity from the silver impregnated plate. The radiochromatogram in each of the 3 ste s showed a single peak. A part of the radioactivity due to p'+C!Iand C3Hl in each zone were separately counted in the double isotope setting by a liquid scintillation counter. As shown in Table I, the ratio of 1'+C/3H remained constant throughout the successive chromatography with 3 different kinds of solvent systems. This result strongly suggests that the more polar radioactive product shown in Fig.1 was identical with PGFza. To confirm this, the product was further subjected to gas chromatography-mass spectrometry analysis. Details of the method for collection of the sample and preparation of methyl estermethoxime-trimethylsilyl ether, methyl ester-trimethylsilyl ether, methoxime-trimethylsilyl ester-trimethylsilyl ether and trimethylsilyl ester-trimethylsilyl ether derivatives of the sample for gas chromatography-mass spectrometry analysis have been described in our previous papers (11,12,13). The gas chromatogram and mass spectra of these derivatives were exactly identical to those of authentic PGFz,. From all the results mentioned above, the more polar product in Fig.1 was identified with PGFe,. The identification of the less polar product and an investigation of the properties of prostaglandin synthetase in rat liver are currently under way in our labolatory.
AUGUST 1977 VOL. 14 NO. 2
PROSTAGLANDINS
Fig. 1
Autoradiogram
and radiochromatogram scan showing the prostaglandin synthesizing activity by the microsomal fraction of rat liver.
Table I
Identification
of the more polar-radioactive
product
by the double isotope method.
solvent system AI
(original)
Rf value
14 C (cpm)
3H (cm)
14C/3H
0.38
11420
32300
0.35
AI
0.38
5256
15460
0.34
B
0.42
3350
9530
0.35
AI1
0.40
2022
6039
0.34
AUGUST 1977 VOL. 14 NO. 2
405
PROSTAGLANDINS
REFERENCES 1.
Bergstrom, S., Prostaglandins : Members of a new hormonal system. Science E,‘382 (1967)
2.
Silver, M.J. and Smith, J.B., Prostaglandins as intracellular messengers. Life Sciences 16, 1635 (1975)
3.
Cohen,,F. and Jaffe, B.M., Production of prostaglandins by cells in vitro : Radioimmunoassaymeasurement of the conver.sionof sachidonic acid to PGE, and PGF2a. Biochem. Biophys. Res. Commun. 55_,724 (1973)
4.
MacManus, J.P. and Braceland, B.M., A connection between the production of prostaglandins during liver regeneration and the DNA synthetic response. Prostaglandins11, 609 (1976)
5.
Green, K. and Samuelsson, B., Prostaglandins and related factors : XIX. Thin-layer chromatography of prostaglandins.J.Lipid Res. 5, 117 (1964)
6.
Chang, W.C., Murota, S. and Tsurufuji, S., Role of prostaglandin E in carrageenin induced inflammation in rat. Biochem. Pharmac. 3, 2045 (1976)
7.
Takeguchi, C., Kohno, E. and Chales, J.S., Mechanism of prostaglandin biosynthesis. I. Characterizationand assay of bovine prostaglandin synthetase. Biochemistry l& 2372 (1971)
8.
Blackwell, G.J., Flower, R.J. and Vane, J.R., Some characteristics of the prostaglandin synthesizing system in rabbit kidney microsomes. Biochemica Biophysics Acta 398, 178 (1975)
9.
Kingston, W.P. and Greaves, M.W., Factors affecting prostaglandin synthesis by rat skin microsomes. Prostaglandins12, 51 (1976)
10.
Kunze, H. and Bohn, E., Extraction of prostaglandin from AgNOsimpregnated silica gel layers from biological assay. NaunynSchmiedoberg's Arch. Pharmak. 267, 380 (1970)
11.
Chang, W.C., Murota, S..,Matsuo, M. and Tsurufuji, S., A new prostaglandin transformed from arachidonic acid in carrageenin induced granuloma. Biochem. Biophys. Res. Commun. 72_,1259 (1976)
12.
Chang, W.C. and Murota, S., Identificationof 6-keto prostaglandin F1, formed from arachidonic acid in bovine seminal vesicles. Biochemica Biophysics Acta 486, 136 (1977)
13.
Chang, W.C., Murota, S. and Tsurufuji, S., Thromboxane 82 transformed from arachidonic acid in carrageenin induced granuloma. Prostaglandins13, 17 (1977)
Received
406
3/24/77
- Approved
5/25/77
AUGUST 1977 VOL. 14 NO. 2
