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New aspects of arachidonic acid metabolism in human uterine cervix
European Journal of Pharmacology, 81 (1982) 515-516
515
Elsevier Biomedical Press
Rapid communication NEW A S P E C T S O F ARACHIDONIC ACID M E T A B O L I S M IN H U M A N UTERINE CERVIX S H E I K H A. SAEED and MURRAY D. MITCHELL *
The Cecil 1-1. and Ida Green Center for Reproductive Biology Sciences and the Departments of Biochemistry and Obstetrics and Gynecology, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA Received 2 June 1982, accepted 3 June 1982
Treatment of the human cervix by local application of prostaglandins (PGs) is used to ripen and dilate the unfavorable cervix prior to the induction of labour and abortion (MacKenzie, 1981). Data are also available that are supportive of the view that PGs are important factors in the natural softening, effacement and dilation of the human cervix during late pregnancy and labour. For example, human cervical tissue synthesizes PGs from arachidonic acid (AA) and increased rates of conversion and rates of PG formation from fatty acid substrate by tissue obtained after the onset of labour compared to those in tissues obtained before labour have been described (Ellwood et al., 1980; Tanaka et al., 1981). However AA also is oxygenated by lipoxygenases that catalyze the formation of hydroxyeicosatetraenoic acids (HETEs) from AA. HETEs are potent chemotactic factors for polymorphonuclear leukocytes and may be involved in the cellular component of the inflammatory response (Goetzl and Sun, 1979). Recently, Liggins (1981) proposed that cervical ripening may be likened to an inflammatory process, a process that involves leukocyte infiltration into the tissue. Hence we elected to evaluate the possibility that human uterine cervix may metabolize AA by way of the lipoxygenase pathway. In this study, we found that human cervical tissue, when incubated with [14C]AA, generates several lipoxygenase products of which two have been tentatively identified as 12-HETE and 5-HETE. Human uterine cervical tissue was obtained at hysterectomy from a non-pregnant woman of
* To whom correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
child-bearing age. The tissue was frozen immediately. Pieces of cervical tissue (2-3 g) were thawed and washed twice in an ice-cold solution of 0.15 M NaC1. The tissue was minced and placed in 3 vol of phosphate buffer (50 mM, p H 7.4) and homogenized for 20 sec with a Polytron PT10 homogenizer. The homogenate was centrifuged at 1000 x g for 20 min in a Beckman refrigerated centrifuge (Model J-6B) and thereafter 200 #1 of the supernatant fraction (containing 1.3 mg of protein) was incubated with 0.24/~Ci of [14C]AA (spec. "act. 58.4 mCi/mmol, Amersham) in phosphate buffer in a final volume of '1.5 ml. After 15 min with gentle shaking in air at 37°C, the radioactive metabolites and unchanged substrate were extracted with 9 vol of ethyl acetate. The organic phase was evaporated to dryness under a stream of nitrogen. Residues were dissolved in 40 #1 ethanol and applied quantitatively to silica gel G TLC plates (Analtech, New Delaware) that were developed in ether/petroleum ether (boiling range 2040°C)/acetic acid (50: 50 : 1, by vol) to a distance of 17 cm. By use of this solvent system the various HETEs are separated whilst PGs and thromboxane B2 remain at the origin. Zones of radioactivity on the chromatograms were located by use of a Berthold TLC Scanner (Wildbad, Germany) and the radioactivity in appropriate zones was assayed after scraping the silica gel into scintillation vials in a Packard Liquid Scintillation Spectrometer. Incubation of [14C]AA with the 1000 × g supernatant fraction of the homogenate of uterine cervical tissue resulted in the formation of several products. A typical radiochromatographic scan obtained after thin-layer chromatography of the products of incubation is shown in fig. 1. The
516
conversion of AA to lipoxygenase products by human cervix. The mean (+S.E.M.) values (/~M) for inhibition of the production of Pl-4 by 50% were PI, 7.8 ± 0.4; P2, 14.0 ± 0.6; P3, 5.2 ± 0.8 and P4, 90.0 ± 1.2. Indomethacin, an inhibitor of fatty acid cyclo-oxygenase activity caused a decrease in the production of P1 and P2 (3-10% inhibition) but stimulated 7-27% the production of P3 and P4. These results are indicative of the existence of lipoxygenase enzymes that can act on AA in human uterine cervix. Since lipoxygenase products are potent chemotactic and vasoactive agents, we suggest that lipoxygenase activities and H E T E formation in cervix may serve a significant role in regulating the processes that, together, are regarded as cervical ripening.
AA
PG
~B'
'
,u
Acknowledgements Origin
,1
Solvent Front
Fig. 1. Radiochromatograms of the products formed when [14C]AA was incubated with the 1000× g supernatant fraction of human uterine cervical tissue: (A) control; (B) control + 7 # M indomethacin, (C) control+ 13 #M nordihydroguaiaretic acid (NDGA). Solvent System: diethyl ether/petroleum ether (boiling range 20-40°C)/acetic acid (50 : 50 : 1, by vol). PG, prostaglandin; AA, arachidonic acid.
lipoxygenase products were identified by comigration with authentic H E T E standards. A characteristic pattern of two major and two minor peaks (P) can be seen (fig. 1). Of the two more polar compounds, P2, co-migrated with the standard 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5HETE) and the chromatographic mobilities of P3 and P4 were identical to those of the '5-1actone' form of 5-HETE and to 12-HETE, respectively. The identity of Pl is unknown. Addition of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase activity, to the incubation mixture caused a concentration-dependent inhibition of the
We thank Mrs. Lydia Morris for expert editorial assistance. This work was supported, in part, by USPHS Grant No. 5-P50-HDI 1149.
References Ellwood, D.A., A.B.IV/. Anderson, M.D. Mitchell and A.C. Turnbull, 1980, The in in vitro production of prostanoids by the human cervix during pregnancy: preliminary observations, Br. J. Obstet. Gynaecol. 87, 210. Goetzl, E.J. and F.F. Sun, 1979, Generation i f unique monohydroxy-eicosatetraenoic acids from arachidonic acid by human neutrophils, J. Exp. Med. 150, 406. Liggins, G.C., 1981, Cervical ripening as an inflammatory reaction, in: The Cervix in Pregnancy and Labour, eds. D.A. Ellwood and A.B.M. Anderson (Churchill Livingstone, London) p. 1. MacKenzie, I.Z., 1981, Clinical studies on cervical ripening, in: The Cervix in Pregnancy and Labour, eds. D.A. Ellwood and A.B.M. Anderson (Churchill Livingstone, London) p. 163. Tanaka, M., I. Morita, S. Hirakawa and Sei-itsu Murota, 1981, Increased prostacyclin synthesizing activity in human ripening uterine cervix, Prostaglandins 21, 83.
515
Elsevier Biomedical Press
Rapid communication NEW A S P E C T S O F ARACHIDONIC ACID M E T A B O L I S M IN H U M A N UTERINE CERVIX S H E I K H A. SAEED and MURRAY D. MITCHELL *
The Cecil 1-1. and Ida Green Center for Reproductive Biology Sciences and the Departments of Biochemistry and Obstetrics and Gynecology, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA Received 2 June 1982, accepted 3 June 1982
Treatment of the human cervix by local application of prostaglandins (PGs) is used to ripen and dilate the unfavorable cervix prior to the induction of labour and abortion (MacKenzie, 1981). Data are also available that are supportive of the view that PGs are important factors in the natural softening, effacement and dilation of the human cervix during late pregnancy and labour. For example, human cervical tissue synthesizes PGs from arachidonic acid (AA) and increased rates of conversion and rates of PG formation from fatty acid substrate by tissue obtained after the onset of labour compared to those in tissues obtained before labour have been described (Ellwood et al., 1980; Tanaka et al., 1981). However AA also is oxygenated by lipoxygenases that catalyze the formation of hydroxyeicosatetraenoic acids (HETEs) from AA. HETEs are potent chemotactic factors for polymorphonuclear leukocytes and may be involved in the cellular component of the inflammatory response (Goetzl and Sun, 1979). Recently, Liggins (1981) proposed that cervical ripening may be likened to an inflammatory process, a process that involves leukocyte infiltration into the tissue. Hence we elected to evaluate the possibility that human uterine cervix may metabolize AA by way of the lipoxygenase pathway. In this study, we found that human cervical tissue, when incubated with [14C]AA, generates several lipoxygenase products of which two have been tentatively identified as 12-HETE and 5-HETE. Human uterine cervical tissue was obtained at hysterectomy from a non-pregnant woman of
* To whom correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
child-bearing age. The tissue was frozen immediately. Pieces of cervical tissue (2-3 g) were thawed and washed twice in an ice-cold solution of 0.15 M NaC1. The tissue was minced and placed in 3 vol of phosphate buffer (50 mM, p H 7.4) and homogenized for 20 sec with a Polytron PT10 homogenizer. The homogenate was centrifuged at 1000 x g for 20 min in a Beckman refrigerated centrifuge (Model J-6B) and thereafter 200 #1 of the supernatant fraction (containing 1.3 mg of protein) was incubated with 0.24/~Ci of [14C]AA (spec. "act. 58.4 mCi/mmol, Amersham) in phosphate buffer in a final volume of '1.5 ml. After 15 min with gentle shaking in air at 37°C, the radioactive metabolites and unchanged substrate were extracted with 9 vol of ethyl acetate. The organic phase was evaporated to dryness under a stream of nitrogen. Residues were dissolved in 40 #1 ethanol and applied quantitatively to silica gel G TLC plates (Analtech, New Delaware) that were developed in ether/petroleum ether (boiling range 2040°C)/acetic acid (50: 50 : 1, by vol) to a distance of 17 cm. By use of this solvent system the various HETEs are separated whilst PGs and thromboxane B2 remain at the origin. Zones of radioactivity on the chromatograms were located by use of a Berthold TLC Scanner (Wildbad, Germany) and the radioactivity in appropriate zones was assayed after scraping the silica gel into scintillation vials in a Packard Liquid Scintillation Spectrometer. Incubation of [14C]AA with the 1000 × g supernatant fraction of the homogenate of uterine cervical tissue resulted in the formation of several products. A typical radiochromatographic scan obtained after thin-layer chromatography of the products of incubation is shown in fig. 1. The
516
conversion of AA to lipoxygenase products by human cervix. The mean (+S.E.M.) values (/~M) for inhibition of the production of Pl-4 by 50% were PI, 7.8 ± 0.4; P2, 14.0 ± 0.6; P3, 5.2 ± 0.8 and P4, 90.0 ± 1.2. Indomethacin, an inhibitor of fatty acid cyclo-oxygenase activity caused a decrease in the production of P1 and P2 (3-10% inhibition) but stimulated 7-27% the production of P3 and P4. These results are indicative of the existence of lipoxygenase enzymes that can act on AA in human uterine cervix. Since lipoxygenase products are potent chemotactic and vasoactive agents, we suggest that lipoxygenase activities and H E T E formation in cervix may serve a significant role in regulating the processes that, together, are regarded as cervical ripening.
AA
PG
~B'
'
,u
Acknowledgements Origin
,1
Solvent Front
Fig. 1. Radiochromatograms of the products formed when [14C]AA was incubated with the 1000× g supernatant fraction of human uterine cervical tissue: (A) control; (B) control + 7 # M indomethacin, (C) control+ 13 #M nordihydroguaiaretic acid (NDGA). Solvent System: diethyl ether/petroleum ether (boiling range 20-40°C)/acetic acid (50 : 50 : 1, by vol). PG, prostaglandin; AA, arachidonic acid.
lipoxygenase products were identified by comigration with authentic H E T E standards. A characteristic pattern of two major and two minor peaks (P) can be seen (fig. 1). Of the two more polar compounds, P2, co-migrated with the standard 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5HETE) and the chromatographic mobilities of P3 and P4 were identical to those of the '5-1actone' form of 5-HETE and to 12-HETE, respectively. The identity of Pl is unknown. Addition of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase activity, to the incubation mixture caused a concentration-dependent inhibition of the
We thank Mrs. Lydia Morris for expert editorial assistance. This work was supported, in part, by USPHS Grant No. 5-P50-HDI 1149.
References Ellwood, D.A., A.B.IV/. Anderson, M.D. Mitchell and A.C. Turnbull, 1980, The in in vitro production of prostanoids by the human cervix during pregnancy: preliminary observations, Br. J. Obstet. Gynaecol. 87, 210. Goetzl, E.J. and F.F. Sun, 1979, Generation i f unique monohydroxy-eicosatetraenoic acids from arachidonic acid by human neutrophils, J. Exp. Med. 150, 406. Liggins, G.C., 1981, Cervical ripening as an inflammatory reaction, in: The Cervix in Pregnancy and Labour, eds. D.A. Ellwood and A.B.M. Anderson (Churchill Livingstone, London) p. 1. MacKenzie, I.Z., 1981, Clinical studies on cervical ripening, in: The Cervix in Pregnancy and Labour, eds. D.A. Ellwood and A.B.M. Anderson (Churchill Livingstone, London) p. 163. Tanaka, M., I. Morita, S. Hirakawa and Sei-itsu Murota, 1981, Increased prostacyclin synthesizing activity in human ripening uterine cervix, Prostaglandins 21, 83.