Diacylglycerol Metabolism and Arachidonic Acid Release in Human Fetal Membranes and Decidua Vera*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 14, Issue of July 25, pp , 1981 Printed in U.S.A. Diacylglycerol Metabolism and Arachidonic Acid Release in Human Fetal Membranes and Decidua Vera* Takeshi Okazaki, Norimasa SagawaS, Janice R. Okitag, John E. Bleasdale, Paul C. MacDonald, and John M. Johnston From the Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Biochemistry and Obstetrics-Gynecology, The University of Texas Southwestern Medical School, Dallas, Texas Diacylglycerol lipase activity has been demonstrated in human fetal membranes and decidua vera tissues. The specific activity of the enzyme is highest in the microsomal fraction of decidua vera tissue. The acylester bond at the sn-1 position of 1,2-diacyl-sn-glycerol is hydrolyzed followed by release of the fatty acid at the sn-2 position. The diacylglycerol lipase activity present in the microsomal fraction of decidua vera tissue hydrolyzes preferentially a diacylglycerol con- taininganarachidonoylgroup in thesn-2position. Monoacylglycerol lipase activity was also demonstrated in these tissues. The specific activity of monoacylglycerol lipase was significantly greater than that were tested as substrates for the phospholipase A2, a preferof diacylglycerol lipase and catalyzed preferentially the ential hydrolysis of phosphatidylethanolamine containing arhydrolysis of monoacylglycerols containing an arachi- achidonic acid at the sn-2 position was observed (11). We donoyl group in the sn-2 position. Based on the subcellular distribution and the differential effects of various inhibitors, we suggest that the monoacylglycerol lipase and diacylglycerol lipase in decidua vera tissue are 2 distinct enzymes. Diacylglycerol kinase specific activity was examined also and was found to be 4-5 times greater in amnion than in either chorion laeve or decidua vera. The importance of diacylglycerol metabo- lism in the mechanism of arachidonic acid release and prostaglandin biosynthesis is discussed. Prostaglandins or related compounds play a central role in the metabolic events that lead to the initiation of parturition and maintenance of human labor (1). The prostaglandins of the 2 series are synthesized from free arachidonic acid [5, 8, 11, 14-eicosatetraenoic acid] (2, 3). The rate of release of arachidonic acid from glycerophospholipids is thought to be a major determinant of the rate of prostaglandin biosynthesis. During the course of human parturition, the level of free arachidonic acid in human amniotic fluid rises strikingly and disproportionately compared to the rise in the levels of other free fatty acids (5). The tissues of origin of the free arachidonic acid found in human amniotic fluid are believed to be the fetal membranes (amnion and chorion laeve) and decidua vera (1). The arachidonic acid composition of amnion is high (18 mol % of all fatty acids) before labor and is decreased in this tissue during labor (6). We (7) and others (8, 9) have reported that * This investigation was supported in part by United States Public Health Service Grant 5-P50-HD The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of a Chilton Foundation Postdoctoral Fellowship. 5 Recipient of a Predoctoral Fellowship Award from The Robert A. Welch Foundation fetal membranes and decidua vera tissue can synthesize prostaglandins. We have demonstrated that in amnion and chorion laeve tissues phosphatidylinositol and phosphatidylethanolamine are enriched with arachidonic acid and that there is a decrease in the arachidonic acid content of these glycerophospholipids in amnion tissue following labor (10). We have reported previously that fetal membranes contain a phospholipase A2 activity which has a substrate preference for phosphatidylethanolamine (11). Furthermore, when specific phosphatidylethanolamines containing various fatty acids in the sn-2 position have demonstrated also the presence of phosphatidylinositolspecific phospholipase C activity in human amnion, chorion laeve, and decidua vera tissues (12). Optimal in uitro specific activity of phospholipase C was much greater in amnion than in either chorion laeve or decidua vera tissues and was approximately 100 times greater than phospholipase Az specific activity in amnion tissue (12). We found also that the specific activities of phospholipases C and A2 in amnion tissue increased as pregnancy advanced (13). These several observations are consistent with the view that stimulation of hydrolysis of phosphatidylethanolamine and phosphatidylinositol could bring about the mobilization of arachidonic acid for prostaglandin production during the initiation and maintenance of human labor. In view of the high activity of phosphatidylinositol-specific phospholipase C in human amnion and the increase in the specific activity of this enzyme as pregnancy advances, we sought to characterize the mechanism by which arachidonic acid is released from phosphatidylinositol in amnion, chorion laeve, and decidua vera tissues. The products of the reaction catalyzed by phosphatidylinositol-specific phospholipase C are myoinositol-1-phosphate, myoinositol-1,2-cyclic phosphate, and diacylglycerols (12). The question then arises as to the sequence of events which leads to the release of arachidonic acid from the diacylglycerols. Several groups of investigators have demonstrated the presence of diacylglycerol lipase activity in human platelets (14, 15). In one of these studies, substrate specificity of the diacylglycerol lipase for diacylglycerols containing either arachidonic acid or oleic acid could not be demonstrated (14). It was suggested that arachidonic acid was released following direct hydrolysis at the sn-2 position of diacylglycerols (14). We have demonstrated the presence of diacylglycerol lipase activity in human amnion, chorion laeve, and decidua vera tissues (12). Diacylglycerol lipase activity is associated with the microsomal fraction of each of these tissues and the specific activity of this enzyme was greatest in the decidua vera tissue (13).

2 The purpose of the present investigation was to characterize the reaction sequence that leads to the release of arachidonic acid from the diacylglycerols produced by the action of phospholipase C on phosphatidylinositol in human amnion, chorion laeve, and uterine decidua vera tissues. Arachidonic Acid Release from Diacylglycerol 7317 EXPERIMENTAL PROCEDURES Materials-Human fetal membranes and decidua vera tissues were incubating the corresponding diacylglycerols (containing a radiolaremoved from placentae delivered per vagina and were separated beled fatty acid in the sn-2 position and palmitate in the sn-1 position) from each other as described previously (12).[l-14C]Arachidonic acid with purified pancreatic lipase. The diacylglycerols were suspended (55 mci/mmol) was purchased from Amersham, Arlington Heights, in 1.1 ml of Tris-HC1 buffer (0.18 M, ph 6.5) containing bovine serum IL. [9,10-3H]Oleic acid (5 Ci/mmol), 1-[1-'4C]palmitoyl-sn-glycero-3- albumin (2.27 mg/ml), CaCh (18 mm), and Triton X-100 (0.145 mg/ phosphocholine (lysophosphatidylcholine), and [Y-~~PIATP were pur- ml). The mixture was treated by sonication in a Bransonic 220 bath chased from New England Nuclear, Boston, MA. Nonradiolabeled sonicator (Bransonic Cleaning Co., Shelton, CT) for 4 min at 4 "C. arachidonic acid was obtained from Nu Chek Prep, Elysian, MN; Following sonication, 0.9 ml of pancreatic lipase (4 mg/ml) was added sodium oleate was purchased from Applied Science, State College, to the sonicated mixture and the sample was incubated for 1 h at 37 PA. l-palmitoyl-sn-glycero-3-phosphocholine, l-palmitoyl-sn-glycero-3-phosphoethanolamine, and 1,2-dioleoyl-sn-glycerol were obtained from Serdary Research Laboratories, London, Ontario, Canada. ~-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was purchased from Supelco, Inc., Bellefonte, PA. Phospholipase A2 obtained from Vipera russelli and porcine pancreatic lipase were purchased from Sigma Chemical Co., St. Louis, MO. ATP and phospholipase C (Bacillus cereus) were purchased from Boehringer Mannheim Biochemicals, Indianapolis, IN. Preparation of Subcellular Fractions-Fetal membranes and decidua vera tissues were homogenized at a concentration of 2.5 g wet weight of tissue/7.5 ml of ice-cold sucrose (0.32 M) solution using a Polytron PT 10 homogenizer (Brinkmann Instruments, Westbury, NY) at a setting of 6 for 30 s. The crude homogenate was centrifuged at 750 X g for 10 min at 4 "C, and the resulting supernatant fluid was filtered through 4 layers of cheesecloth. The filtered 750 X g supernatant fraction was centrifuged at 12,000 X g for 10 min at 4 "C. The resulting pellet was resuspended in sucrose (0.32 M) solution. The 12,000 X g supernatant fraction was removed and centrifuged at 105,000 X g for 1 h at 4 "C. The resulting microsomal pellet was resuspended in sucrose (0.32 M) solution. Rat liver microsomes were employed for the preparation of several of the substrates utilized in this investigation. Liver tissue (7-8 g) was obtained from adult male Sprague-Dawley rats ( g) and homogenized in 70 ml of sucrose (0.25 M) solution using a Potter- Elvehjem homogenizer. After centrifugation at 750 X g for 10 min at 4 "C, the resulting supernatant fraction was centrifuged at 12,000 X g for 20 min at 4 "C. The microsomal fraction was obtained by centrifugation of the 12,000 X g supernatant fraction at 105,000 x g for 60 rnin at 4 "C. The microsomal pellet was resuspended in 5 ml of sucrose (0.25 M) solution using a Potter-Elvehjem homogenizer. Preparation of Substrates-Substrates were synthesized employing rat liver microsomes as the enzyme source by a modification (11) of the procedure of Waite and van Deenen (16). l-palmitoyl-2- [3H]oleoyl-sn-glycero-3-phosphocholine was synthesized using l-palmitoyl-sn-glycero-3-phosphocholine and [9,10-3H]oleic acid. 1-Palmitoyl-2-['4C]arachidonoyl-sn-glycero-3-phosphoethanol~ine was synthesized employing 1-palmitoyl-sn-glycero-3-phosphoethanola- diacylglycerol lipase activity in these tissues had a broad ph optimum ( ). Reduced glutathione was included in the incubation mixture mine and [l-14c]arachidonic acid. l-['4c]palmitoyl-2-oleoyl-sn-glyc- since in preliminary experiments we demonstrated that the diacylero-3-phosphocholine was synthesized using l-[l-'4c]palmitoyl-sn- glycerol lipase was inhibited by sulfhydryl reagents such as Hg2+. glycero-3-phosphocholine and oleic acid. The reactions were termi- Ca2+ was included in the incubation mixture since it had been reported nated by addition of 3 volumes of methanolic HCl(O.1 M). Six volumes previously that the diacylglycerol lipase present in platelets required of Chloroform and 1.5 volumes of KC1 (2 M) were added to extract the Ca2+ for activity (14). In subsequent investigations, we could not phospholipids (12). Total lipid extracts were evaporated under N2 and demonstrate a Ca2+ requirement or an effect of EDTA on the activity purified by silicic acid column chromatography (17). of diacylglycerol lipase in fetal membranes and decidua vera.' The The purity of each of the glycerophospholipids was determined by reaction was terminated by addition of 1.5 ml of a mixture of chlorothin layer chromatography in a solvent system of chloroform/meth- form, methanol, and heptane (1.25:1.41:1.0, by volume), followed by anol/acetic acid/water (5015:4:2 by volume). Each radiolabeled 0.5 ml of a K2C03 (50 mm) and borate (50 mm) buffer, ph 10.0 (21). glycerophospholipid co-chromatographed with an authentic standard The mixture was centrifuged at 600 X g for 10 min. An aliquot (0.75 of that glycerophospholipid. The individual glycerophospholipids ml) of the resulting upper aqueous layer containing the free fatty acid were visualized by exposure to iodine vapor. The fatty acid composi- was evaporated in a counting vial and the radioactivity was assayed tion of the glycerophospholipid substrates was confirmed by gas in a Triton X-100 scintillation mixture (22). The lower organic layer chromatography (12). The distribution of the fatty acids between the was removed and evaporated under Nz. To the residue in the tube, 50 sn-1 and sn-2 positions of the glycerophospholipids synthesized was jd of a mixture of chloroform and methanol (2:1, v/v) was added and ascertained by incubation with phospholipase A2 obtained from V. 25 p1 of the mixture was applied on a thin layer plate (HETLC russelli as described (18). More than 97%of the radioactivity was Uniplates, Analtech, Inc., Newark, DE). The solvent system used for found to be associated with the free fatty acid when l-palmitoyl-2- the separation was diethyl ether/benzene/ethanol/acetic acid (2025 [3H]oleoyl-sn-glycero-3-phosphocholine or l-palmit0yl-2-['~c]arachi- 1:0.1, by volume). Lipids on the chromatograms were visualized by donoyl-sn-glycero-3-phosphoethanolamine was incubated with the phospholipase Az obtained from venom, whereas more than 91% of the radioactivity present in l-['4c]palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine was recovered as l-['4c]palmitoyl-sn-glycero-3-phosphocholine after snake venom treatment. The diacylglycerol lipase substrates were prepared immediately before use by incubating the radiolabeled glycerophospholipids described above with phospholipase C obtained from B. cereus employing the procedure described previously (19). The monoacylglycerol lipase substrates containing either ["C]arachidonate or r3h]oleate in the sn-2 position were prepared by "C. The reaction was terminated by the addition of 8.0 ml of chloroform/methanol (2:1, v/v) and 0.2 ml of HCl (5 M). The lower (chloroform) layer was removed and the aqueous upper layer washed twice with 5.3 ml of chloroform. The chloroform extracts were combined, taken to dryness rapidly under N2, and resuspended in 3 ml of petroleum ether (b.p "C). The monoacylglycerols were purified further by chromatography on 10% borate Unisil silicic acid (3 g) suspended in 30 ml of petroleum ether and packed into a column (1 X 30 cm). The column was washed with 40 ml of petroleum ether followed by 40 ml of diethyl ether/petroieum ether (1:3, v/v), followed by40 ml of diethyl ether/petroleum ether (91, v/v). The diethyl ether/petroleum ether (9:1, v/v) fraction which contained the monoacylglycerols was taken to dryness rapidly under N2 and resuspended in n-hexane. Radioactivity in an aliquot of the hexane solution was measured to determine the total monoacylglycerol recovery and another aliquot was subjected to thin layer chromatography to separate the sn-1 and 2 monoacylglycerols (20). Assay of Diacylglycerol Lipase Actiuity-Diacylglycerol lipase was assayed by a procedure similar to those described previously for the assay of this enzyme (14, 15). However, it should be emphasized that when the diacylglycerol substrate utilized for the assay is radiolabeled only in the sn-2 position, the assay does not distinguish between a diacylglycerol lipase and the combined action of a diacylglycerol lipase and a monoacylglycerol lipase. Diacylglycerol (approximately 500 nmol) in petroleum ether was transferred to a test tube and the petroleum ether removed by evaporation under Nz. The diacylglycerol was suspended in a substrate mixture consisting of 4-(2-hydroxy- ethyl)-i-piperazineethanesulfonic acid (Hepes)/NaOH buffer (50 mm, ph 7.0), NaCl (100 nm), and Triton X-100 (0.005%). The mixture was treated by sonication (Bransonic 220) for 2 min. The substrate mixture was translucent after sonication and contained 50 nmol/0.05 ml. Each assay mixture contained 0.05 ml of the enzyme suspended in sucrose (0.32 M), ml of the reaction mixture (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes)/NaOH (150 m, ph 7.0), NaCl (400 m), CaCL (25 nm), and reduced glutathione (62.5 mm)), and 0.05 ml of the substrate (50 nmol) in a final volume of ml. The ' T. Okazaki, N. Sagawa, and J. M. Johnston, unpublished observations.

3 7318 Arachidonic Acid Release from Diacylglycerol exposure to IZ vapor and the areas containing monoacylglycerol were identified by co-chromatography with authentic monooleoylglycerol. Following removal of 11, these areas were scraped and the radioactivity was assayed in Bray s scintillation solution (23). The rate of reaction was shown to be linear for 10 min with protein concentrations between 5 and 20 pg of protein/incubation vessel. Assay of Monoacylglycerol Lipase Activity-Monoacylglycerol lipase was assayed under conditions similar to those described for diacylglycerol lipase with the exception that the substrate 2-[l- C]arachidonoyl-sn-glycerol or 2-[9,10-3H]oleoyl-sn-glycerol was suspended in a mixture containing deoxycholate (0.004%) rather than Triton X-100. The concentration of monoacylglycerol used for the assay was 0.16 mm. We observed a broad ph optimum ( ) of monoacylglycerol lipase in these tissues. The enzymatic activity was inhibited by sulthydryl reagents, and, therefore, reduced glutathione was included in the assay of this enzyme. As was the case for diacylglycerol lipase, no Ca2+ requirement could be demonstrated for the activity of monoacylglycerol lipase. Assay of Diacyglycerol Kinase Actiuity-Diacylglycerol kinase activity was assayed according to the procedure described by Rittenhouse-Simmons (15). RESULTS Diacylglycerol Lipase Activity in Amnion, Chorion Laeve, and Decidua-The greatest specific activity of diacylglycerol lipase was found in the microsome-enriched fraction in each of the 3 tissues (Fig. 1). Of the total activity in the homogenate, the greatest amount of activity was found in the cytosolic fraction of amnion and chorion laeve tissues. However, we also found considerable activity of the microsomal marker enzyme NADPH cytochrome c reductase (EC ) in the cytosolic fraction of amnion tissue presumably because of the harsh homogenization procedure necessary to disrupt this tissue (12). The diacylglycerol lipase activity has been found to be associated primarily with a particulate fraction of human platelets (14). When l-palmitoyl-2-[3h]oleoyl-sn-glycerol was employed as the substrate, the greatest specific activity of diacylglycerol lipase among the subcellular fractions of the 3 tissues was found in the microsome-enriched fraction of decidua vera (Fig. 1). Characteristics of the Reaction Sequence for the Release of Fatty Acids from Diacylglycerols in Decidua-When l- [ 4C]palmitoyl-2-[3H]oleoyl-sn-glycerol was employed as the substrate, the rates of release of [%]pahnitic acid and [3H]- oleic acid as well as the rates of formation of 2-[3H]oleoyl-snglycerol and l-[ 4C]palmitoyl-sn-glycerol were measured. When the microsome-enriched fraction of decidua tissue was employed as the enzyme source, it was found that the release 100 f e Crude Extmct Mitochondrial Microsomal Cytosolic n Amnion Chorion Iawe M!tochandrial Microsomal - Cytosolic FIG. 1. Specific (A) and total (B) activities of diacylglycerol lipase in the various subcellular fractions of amnion, chorion laeve, and decidua vera tissues. t [ %]Palmltx Acid- /+ 2-[ H]Oleoyl-sn-glycerol lncuboticm Tome (men) FIG. 2. The rate of hydrolysis of l-[ 4C]palmitoyl-2-[3H]- oleoyl-sn-glycerol employing the microsomal fraction of decidua vera tissue. TABLE I Substrate specificity of the diacylglycerol lipase of human fetal membranes and decidua Vera tissues at term The rates of release of fatty acid from the sn-2 position of diacylglycerols were determined using l-pahnitoyl-2-[9,10-3h]oleoyl-snglycerol (PO-DG) and 1-palmitoy1-2-[1- C]arachidonoy1-sn-g1ycero1 (PA-DG) as substrates. Microsome-enriched fractions were used as the enzyme source. Substrate Fatty acid released AlNliOll Chorion laeve Decidua vera nmol x K x mg- protein PO-DG 27.6 f 9.2 " f 13.3 PA-DG f z!z 79.8 P p < 0.3 p i 0.2 p < 0.005~ a Mean f SE. Numbers in parentheses represent the number of experiments. b Only in the decidua vera tissue was there a statistically significant difference between the hydrolysis of PO-DG and PA-DG (p c 0.005). With both substrates, the specific activities of diacylglycerol lipase were statistically higher in decidua vera than in amnion or chorion (p < 0.005; decidua versus chorion with PA-DG, p < for other comparisons (paired t test)). of [ %]palmitic acid was greater than that of [3H]oleic acid at all incubation times analyzed (Fig. 2). It was found also that the rate of formation of 2j3H]oleoyl-sn-glycerol was considerably greater than that of l-[ 4C]palmitoyl-sn-glycerol which was barely detectable at all time periods. These findings are consistent with a reaction sequence in which the fatty acid at the sn-l position of diacylglycerol is released first followed by hydrolysis of the fatty acid at the sn-2 position. Substrate Specificity of the Diacylglycerol Lipase of Amnion, Chorion Laeue, and Decidua-When 1-palmitoyl-2- [3H]oleoyl-sn-glycerol and l-palmitoyl-2-[ 4C]arachidonoylsn-glycerol were employed as the substrates, the substrate specificity of the diacylglycerol lipase activity in these tissues was investigated. When the microsomal fraction obtained from any of the 3 tissues was employed as the enzyme source, the release of [i4c]arachidonic acid from l-pahnitoyl-2-[li4c]arachidonoyl-sn-glycerol was always greater than the release of [3H]oleic acid from l-palmitoyl-2-[3h]oleoyl-sn-glycerol (Table I). However, only when the microsomal fraction obtained from decidua vera tissue was employed was there a statistically significant difference between the hydrolysis of the 2 substrates (p < 0.005). Since the highest specific activity of diacylglycerol lipase was found in the microsomal fraction obtained from decidua vera tissue and since sufficient quantities of this tissue were available, this fraction was employed for the further characterization of diacylglycerol lipase activity.

4 When the release of [14C]arachidonic acid or [3H]oleic acid as well as the accumulation of the corresponding radiolabeled monoacylglycerols was studied as a function of incubation time, it was found that at all time points the amount of free [14C]arachidonic acid was greater than the amount of free [3H]oleic acid (Fig. 3A). A small amount of radioactivity was found in the corresponding monoacylglycerols. When the 2 substrates in petroleum ether were mixed, taken to dryness under NB, emulsified together, and incubated together with decidua vera microsomes, it again was found that the rate of release of arachidonic acid was greater than that of oleic acid at all incubation times analyzed (Fig. 3B). In this series of experiments, the accumulation of 2-['4C]arachidonoyl-sn-glycerol and that of 2-[3H]oleoyl-sn-glycerol was again monitored. The 2-[3H]oleoyl-sn-glycerol accumulated to a considerably greater extent than did 2-['4C]arachidonoyl-sn-glycerol. Again, the accumulation of 2-acyl-sn-glycerols is consistent with the postulate that the release of the fatty acids from the sn-2 position of diacylglycerols involves a 2-step reaction sequence. The results presented in Table I and Fig. 3 are consistent with the view that diacylglycerols (or monoacylglycerols) containing arachidonic acid are hydrolyzed preferentially by the diacylglycerol lipase (or monoacylglycerol lipase). When l-palmitoyl-2-['4c]arachidonoyl-sn-glycerol and l-palmitoyl-2-[3h]oleoyl-sn-glycerol were employed as substrates, the formation of ['4C]arachidonic acid plus 2-["C]- arachidonoyl-sn-glycerol exceeded the formation of [3H]oleic acid plus 2-[3H]oleoyl-sn-glycerol at all time points (Fig. 3). Based on these results, we suggest that diacylglycerol lipase hydrolyzes preferentially diacylglycerols containing arachidonic acid in the sn-2 position. Monoacylglycerol Lipase Activity in Fetal Membranes and Decidua-When the activity of monoacylglycerol lipase was measured in amnion, chorion laeve, and decidua vera tissues, it was found that the specific activity of this enzyme in the 750 x g supernatant fraction was approximately 7 times higher in decidua vera tissue than in amnion and chorion laeve tissue (Table 11). Furthermore, it should be noted that the specific activity of monoacylglycerol lipase in the 750 X g supernatant fraction of these 3 tissues was 5-18 times higher than that of the diacylglycerol lipase specific activities in these tissues. For this analysis, 2-[3H]oleoyl-sn-glycerol was used as the sub- Release Arachidonic Acid from Diacylglycerol Acyl-sn-glycerol 48.2 f f C 173.1h strate. In 2 experiments, the specific activity of monoacyl- " glycerol lipase in the microsomal and 105,000 X g supernatant 1,2-Diacyl-sn-glycerol 9.6 f C f 4.5' fractions of decidua tissue was measured with either 2-[14C]- arachidonoyl-sn-glycerol or 2-[3H]oleoyl-sn-glycerol as sub- Mean f S.E. Numbers in parentheses represent the number of experiments. strate. The rates of hydrolysis for 2-[14C]arachidonoyl-sn-glyc- Specific activities of both monoacylglycerol lipase and diacylglycerol and 2-[3H]oleoyl-sn-glycerol were 616 and 367 nmol X h" erol lipase in decidua vera tissue were significantly higher than those X mg" protein, respectively, employing microsomes as the of amnion and chorion laeve (p < 0.05 (paired t test)). r I [I4C] AracTdonlc Acid 0 A enzyme source. A similar increased rate of hydrolysis was observed for the 2-['4C]arachidonoyl-sn-glycerol substrate when the 105,000 X g supernatant fraction was employed as the enzyme source (701 uersus 456 nmol X h-' X mg" protein). Because of the limited availability of the 2-['4C]arachidonoyl- sn-glycerol, experiments in which 2-[~H]oleoyl-sn-glycerol and 2-['4C]arachidonoyl-sn-glycerol were mixed prior to the incubation were not conducted. Monoacylglycerol and diacylglycerol lipase activities in the various subcellular fractions obtained from decidua vera tissue were examined and the results obtained are given in Table 111. The specific activity of monoacylglycerol lipase was greatest in the 105,000 X g supernatant fraction, whereas the specific activity of diacylglycerol lipase was greatest in the microsomal fraction. In addition, more than 82% of the total monoacylglycerol lipase activity was recovered in the 105,000 x g supernatant fraction, whereas most of the diacylglycerol lipase activity was associated with the microsomal fraction (55%). In addition, we attempted to distinguish further between diacylglycerol and monoacylglycerol lipase activities by the use of various inhibitors. When 2-[3H]oleoyl-sn-glycerol was employed as the substrate for monoacylglycerol lipase activity and l-palmitoyl-2-[3h]oleoyl-sn-glycerol as substrate for diacylglycerol lipase, we compared the effect of Triton X- 100, F-, and the esterase inhibitor phenylmethylsulfonyl fluoride (24) on enzymatic activities. The results of these experiments are given in Table IV. With Triton X-100 (0.64 mg/ ml), the hydrolysis of diacylglycerols was inhibited by some TABLE I1 Specific activities of monoacylglycerol lipase and diacylglycerol lipase in human fetal membranes and decidua vera tissues at term Monoacylglycerol lipase and diacylglycerol lipase activities were assayed as described ("Experimental Procedures") using saturating quantities of either 2-[9,10-3H]oleoyl-sn-glycerol (0.16 m ~ or ) l-palmitoyl-2-[9,10-3h]oleoyl-sn-glycerol (0.40 mm) as substrates, respectively. The supernatant fraction obtained following centrifugation at 750 X g for 10 min was used as the enzyme source. Fatty acid released n Substrate Amnion Chorion laeve Decidua vera nmol x h" x mg" protein B 0.2 =-glycerol Incubation Time (min I FIG. 3. The rate of hydrolysis of l-palmitoyl-2-['4c]arachidonoyl-sn-glycerol and l-palmitoyl-2-[3h]oleoyl-sn-glycerol incubated separately (A) and together (B) with the microsomal fraction prepared from decidua vera tissue. when

5 7320 Arachidonic Acid Release from Diacylglycerol TABLE 111 Subcellular distribution of monoacylglycerol lipase and diacylglycerol lipase activities in human decidua vera tissue at term Monoacylglycerol lipase and diacylglycerol lipase activities were assayed as described ( Experimental Procedures ) using various subcellular fractions obtained from decidua vera tissue as the enzyme source. The substrates for monoacylglycerol lipase and diacylglycerol lipase were 2-[3H]oleoyl-sn-glycerol and I-palmit0yl-2-[~H]oleoyl-sn- glycerol, respectively. Specific activities are shown in nanomoles X h X mg protein (mean f S.E.). Distribution of total activities is shown in per cent (mean f S.E.). Numbers in parentheses represent number of experiments. 750xg supernatant 12,000 x g pellet 105,000 X g pellet 105,000 X g sudernant Monoacylglycerol lipase la f f f f f f f 2.8 Diacylglycerol lipase la 46.6 t (3) 56.5 f f 3.3 (3) (3) f f 10.8 (3) (3) 17.1 t f 10.7 (3) (3) TABLE IV Effects of Triton X-100, sodium fluoride, and phenylmethylsulfonyl fluoride on monoacylglycerol lipase and diacylglycerol lipase specific activities Enzymatic activities were assayed as described ( Experimental Procedures ) employing the microsome-enriched fraction obtained from decidua vera tissue. Values are the average of 2 experiments and are exaressed as aer cent of control. Inhibitor Monoacylglycerol li- Diacylglycerol lipase pase specific activity specific activity % of control Triton 10.9 X-100 (0.64 mg/ 99.5 ml) F- ( mm) 39.9 Phenylmethylsulfonyl fluoride (IO m) TABLE V Specific activities of diacylglycerol kinase in human fetal membranes and decidua vera tissues Diacylglycerol kinase activity was assayed employing the 750 X g supernatant fraction as enzyme source. The specific activity in the amnion was significantly higher than that in chorion laeve and decidua vera withp values less than 0.05 and 0.02, respectively (paired t test). Phosphatidate formation Chorion Amnion laeve Decidua vera nmol X h X mg protein 12.7 f 2.4 (5) (5) 2.5 f 0.4 (5) D 0.05b P < 0.02b Mean f S.E. Numbers in parentheses represent number of periments. p values are expressed as a comparison to amnion. 90% but no significant decrease in the hydrolysis of monoacylglycerol was observed. When F- (128 m ~ was ) present in incubations, diacylglycerol hydrolysis was inhibited by approximately 85% and monoacylglycerol hydrolysis was inhibited by 60%. When phenylmethylsulfonyl fluoride (10 mm) was used, the hydrolysis of diacylglycerol and monoacylglycerol was inhibited 45 and 78%, respectively. Although the results with use of inhibitors and the subcellular distribution of the diacylglycerol lipase and monoacylglycerol lipase activ- ex- ities are suggestive that 2 distinct enzymes are involved in the hydrolysis of these substrates, definitive proof awaits purification of the enzymes. Diacylglycerol Kinase Activity in Fetal Membranes and Decidua-The diacylglycerols formed in fetal membranes and uterine decidua by the action of phosphatidylinositol-specific phospholipase C could possibly be metabolized to phospha- tidic acid in a reaction catalyzed by diacylglycerol kinase. Therefore, we examined the activity of this enzyme in the 750 x g supernatant fraction of amnion, chorion laeve, and uterine decidua (Table V). In amnion tissue the specific activity of this enzyme is 4 to 5 times that in chorion laeve and decidua vera tissue. It is interesting to note that the enzymes that compete for diacylglycerols as substrate are present in the decidua vera in relative amounts greatly different than those in the amnion. The decidua contains a much greater lipase activity than kinase activity, whereas in amnion, the maximal in vitro activities of these 2 enzymes are similar. DISCUSSION In the present investigation, we found that microsomes obtained from human amnion, chorion laeve, and decidua vera tissues contained diacylglycerol lipase that effected the hydrolysis of fatty acids from diacylglycerols containing radiolabeled fatty acid esterified in the sn-2 position. The highest specific activity of diacylglycerol lipase was found in the microsomal fraction prepared from decidua vera tissue. Therefore, we characterized the hydrolysis of diacylglycerols in this tissue to define the reaction sequence for the stepwise hydrolysis of the fatty acids in the sn-1 and -2 positions. It was demonstrated that 1-palmitoyl-2-arachidonoyl-sn-glycerol was a better substrate than was 1-palmitoyl-2-oleoyl-sn-glycerol (Fig. 3). To our knowledge, this is the first demonstration of a diacylglycerol lipase with preference for the hydrolysis of diacylglycerols containing arachidonic acid. Acyl group specificity for the diacylglycerol lipase present in human platelets could not be demonstrated (14). When l-[14c]palmitoyl-2- [3H]oleoyl-sn-glycerol was employed as the substrate in incubation mixtures in which the microsome-enriched fraction obtained from decidua vera tissue was employed as the source of enzyme, it was demonstrated that l-[ 4C]palmitoyl-sn-glycerol was not formed however, the synthesis of 2-[3H]oleoylsn-glycerol was demonstrated (Fig. 2). Moreover, the release of [14C]palmitic acid was greater than the release of [3H]oleic acid at all incubation times. These results are consistent with the proposition that in decidua the reaction sequence leading to the release of the fatty acid from the sn-2 position of 1,2- diacyl-sn-glycerols is as follows. The fatty acid in the sn-1 position is hydrolyzed first in a reaction catalyzed by diacylglycerol lipase, followed by hydrolytic cleavage of the fatty acid from the sn-2 position in a reaction catalyzed by monoacylglycerol lipase. When the specific activities ofdiacylglycerol lipase and monoacylglycerol lipase in whole homogenates prepared from amnion, chorion laeve, and decidua vera tissues were compared, the specific activities of these enzymes in decidua tissue were significantly greater than those of amnion and chorion laeve tissues. Moreover, the monoacylglycerol lipase specific activity was 18 times the specific activity of diacylglycerol lipase in the 750 X g supernatant fraction from decidua vera tissue. The greater specific activity of monoacylglycerolipase no doubt provides the explanation as to why monoacylglycerols do not accumulate when diacylglycerols are utilized as substrates for measuring diacylglycerol lipase activity. We found that the greatest specific activity and most of the total activity of monoacylglycerol lipase was in the 105,000 X g supernatant fraction prepared from decidua vera tissue. In

6 contrast, the highest specific and total activities of diacylglycerol lipase were associated with the microsomal fraction (Table 111). We also found that various inhibitors affected the hydrolysis of di- and mono-acylglycerols to different degrees (Table IV). Based on the subcellular distribution of the 2 lipase activities and the differential effect of inhibitors, we suggest that 2 separate enzymes catalyze the hydrolysis of diacylglycerols and monoacylglycerols in this tissue. However, some reservations must be placed on such a conclusion since comparisons of the activities are based on assays that are optimized to in vitro conditions and the 2 substrates are not suspended in an identical manner. Future investigations employing purified enzyme preparations and the use of specific inhibitors wil be necessary in order to discern whether 1 or 2 enzymes are responsible for the hydrolysis of the 2 substrates. We compared the rate of hydrolysis of 2-acyl-sn-glycerols containing either oleate or arachidonate. The rate of arachidonic acid release was 50-70s greater than the rate of oleic acid release when the 105,000 X g supernatant and microsomal fractions prepared from decidua tissue were employed as the enzyme source. From these results, we suggest that the monoacylglycerol lipase has a substrate preference for monoacylglycerols containing arachidonic acid. The diacylglycerols produced from phosphatidylinositol as a result of phospholipase C action can also be phosphorylated by ATP to form phosphatidic acid in a reaction catalyzed by diacylglycerol kinase. The specific activity of diacylglycerol kinase was significantly greater in amnion tissue than in chorion laeve or decidua vera tissues. Presently we are investigating the mechanism(s) whereby the 2 pathways for diacylglycerol metabolism in amnion tissue are regulated. It was observed that the specific activities of the diacylglycerol kinase and diacylglycerol lipase in crude homogenates of amnion (12.7 and 9.6 nmol X h" X mg" protein, respectively) are similar. Therefore, the amount of diacylglycerol that was metabolized by the lipase pathway (destined for prostaglandin biosynthesis) may be regulated in part by the activity of the diacylglycerol kinase. It has been shown that the diacylglycerol lipase of platelets is activated by Ca2+ (14) and the diacylglycerol kinase of cerebral cortex is inhibited by Ca2+ (25). Presently, we are investigating the role of other agonists and antagonists on diacylglycerol lipase activity and diacylglycerol kinase activity in amnion. We suggest that competition for diacylglycerol may not be as important in decidua vera tissue since the specific activity of diacylglycerol lipase is times the specific activity of diacylglycerol kinase. In a previous study, it was established that there is a phosphatidylinositol-specific phospholipase C in amnion, chorion laeve, and decidua vera tissues (12). This finding, together with the findings of the present investigation that these tissues contain a diacylglycerol lipase with specificity for diacylglycerols containing arachidonic acid, provides a mechanism for the specific release 0C arachidonic acid from phosphatidylinositol. We have demonstrated also that amnion and chorion laeve tissues contain the enzyme phospholipase AP that catalyzes preferentially the release of arachidonic acid from phosphatidylethanolamine (11). We have shown also that there is preferential loss of arachidonic acid from phosphatidylethanolamine and phosphatidylinositol in amnion and chorion laeve during human parturition (10). Such an interlocking set of metabolic events, i.e. enzyme substrate specificity and substrate enrichment, has not been demonstrated in any other mammalian tissues involved in prostaglandin biosynthesis. It is of great importance to define the stimulus that brings about Release Arachidonic Acid from Diacylglycerol 7321 the activation of phospholipase C and phospholipase AP and, thus, the release of arachidonic acid that leads to the cascade of events that initiate parturition. We have demonstrated previously that the specific activities of phospholipases AP and C increase in amnion tissue as gestation advances (13). The activity of phospholipase C in fetal membranes and decidua that were obtained at term but prior to the onset of labor, when assayed under optimal in vitro conditions, are similar to those found in these tissues that were obtained after labor. This finding is suggestive of the possibility that an additional mechanism(s), other than the enzyme concentration, may be important in the regulation of phospholipase C activity as well as diacylglycerol lipase and phospholipase Az activity and in turn prostaglandin formation. Regulation of the enzymatic release of arachidonic acid for prostaglandin formation may involve changes in the ea2+ concentration at the sites of these lipases. Supportive of this postulate are the observations of Ca2+ requirements for phospholipase A2 (11) and for phospholipase C (12) in these tissues. Acknowledgments-We would like to thank Susan Cutrer and Sue MacDonald for obtaining the tissue samples and Dolly Tutton for editorial assistance in preparing this manuscript. REFERENCES 1. MacDonald, P. C., Porter, J. C., Schwarz, B. E., and Johnston, J. M. (1978) Sem. Perinatol. 2, Bergstrom, S., Danielsson, H., and Samuelsson, B. (1964) Biochim. Biophys. Acta 90, Van Dorp, D. A., Beerthuis, R. K., Nugteren, D. H., and Vonkeman, H. (1964) Biochim. Biophys. Acta 90, Lands, W. E. M., and Samuelsson, B. (1968) Biochim. Biophys. Acta 164, MacDonald, P. C., Schultz, F. M., Duenhoelter, J. H., Gant, N. F., Jimenez, J. M., Pritchard, J. A., Porter, J. C., and Johnston, J. M. (1974) Obstet. Gynecol. 44, Schwarz, B. E., Schultz, F. M., MacDonald, P. C., and Johnston, J. M. (1975) Obstet. Gynecol. 46, Okazaki, T., Casey, M. L., Okita, J. R., MacDonald, P. C., and Johnston, J. M. (1981) Am. J. Obstet. Gynecol. 139, Kinoshita, K., Satoh, K., and Sakamoto, S. (1977) Endocrinol. Jpn. 24, Mitchell, M. D., Bibby, J., Hicks, B. R., and Turnbull, A. C. (1978) Prostaglandins 15, IO. Okita, J. R. (1981) Ph.D. dissertation, University of Texas Health Science Center, Dallas, Texas 11. Okazaki, T., Okita, J. R., MacDonald, P. C., and Johnston, J. M. (1978) Am. J. Obstet. Gynecol. 130, Di Renzo, G. C., Johnston, J. M., Okazaki, T., Okita, J. R., MacDonald, P. C., and Bleasdale, J. E. (1981) J. Clin. Invest. 67, Okazaki, T., Sagawa, N., Bleasdale, J. E., Okita, J. R., MacDonald, P. C., and Johnston, J. M. (1981) Biol. Reprod., in press 14. Bell, R. L., Kennerly, D. A., Stanford, N., andmajerus, P. W. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, Rittenhouse-Simmons, S. (1980) J. Bwl. Chem. 255, Waite, M., and van Deenen, L. L. M. (1967) Biochim. Biophys. Acta 137, Sweeley, C. C. (1969) Methods Enzymoll4, Okuyama, H., and Nojima, S. (1965) J. Biochem. 57, Little, C., Aurebekk, B., and Otnaess, A. B. (1975) FEBSLett. 52, Thomas, A. E., 111, Scharoun, J. E., and Ralston, H. (1965) J. Am. Oil Chem. SOC. 42, Belfrage, P., and Vaughn, M. (1969) J. Lipid Res. 10, Bleasdale, J. E., Wallis, P., MacDonald, P. C., and Johnston, J. M. (1979) Biochim. Biophys. Acta 575, Bray, G. A. (1960) Anal. Biochem. 1, Fahrney, D. E., and Gold, A. M. (1963) J. Am. Chem. SOC. 85, Lapetina, E. G., and Hawthorne, J. N. (1971) Biochem. J. 122,