Phosphatidylinositol-specific C in Fetal Membranes and Uterine Decidua

Gian Carlo Di Renzo, … , Paul C. MacDonald, John E. Bleasdale

J Clin Invest. 1981;67(3):847-856. https://doi.org/10.1172/JCI110102.

Research Article

An assay procedure was developed in which phosphatidyl[2-3H]inositol was employed as substrate for the measurement of phosphatidylinositol-specific activity. Employing this assay, phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua was identified and characterized. The specific activity of this in amnion (4.4 μmol × mg−1 protein × h−1) was three times that in uterine decidua and more than five times that in chorion laeve. No difference was found between the specific activity of phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The products of phosphatidylinositol hydrolysis in short-term incubations were stoichiometric amounts of diacylglycerol and inositol-1,2-cyclic-phosphate plus inositol-1- phosphate. After longer periods of incubation, monoacylglycerol also was detected. Diacylglycerol activity also was demonstrated in these tissues. More than 90% of phosphatidylinositol-specific phospholipase C activity of amnion tissue was recovered in the 105,000-g supernatant fraction, and optimal enzymatic activity in vitro was observed at pH 6.5-7.5 in the presence of Ca2+ (8 mM) and mercaptoethanol (4 mM). Phosphatidylinositol-specific phospholipase C activity was stimulated by fatty acids in low concentrations, but was inhibited by lysophosphatidylcholine and a variety of detergents. No effect of labor on the specific activity of phosphatidylinositol-specific phospholipase C in either fetal membranes or uterine decidua could be detected. The finding of an active phosphatidylinositol-specific phospholipase C activity in human fetal […]

Find the latest version: https://jci.me/110102/pdf Phosphatidylinositol-specific Phospholipase C in Fetal Membranes and Uterine Decidua

GIAN CARLo Di RENZO, JOHN M. JOHNSTON, TAKESHI OKAZAKI, JANICE R. OKITA, PAUL C. MACDONALD, and JOHN E. BLEASDALE, 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 75235

A B S T R A C T An assay procedure was developed in specific phospholipase C in either fetal membranes or which phosphatidyl[2-3H]inositol was employed as uterine decidua could be detected. The finding of substrate for the measurement of phosphatidylinositol- an active phosphatidylinositol-specific phospholipase specific phospholipase C activity. Employing this C activity in human fetal membranes and uterine assay, phosphatidylinositol-specific phospholipase C decidua is complementary to our previous finding of a activity in human fetal membranes and uterine decidua selective loss of from phosphati- was identified and characterized. The specific activity dylinositol of human fetal membranes during labor. of this enzyme in amnion (4.4 umol x mg-' protein The action of phosphatidylinositol-specific phospho- x h-1) was three times that in uterine decidua and lipase C, coupled to diacylglycerol lipase action, could more than five times that in chorion laeve. No dif- provide a mechanism for the release of arachidonic ference was found between the specific activity of acid for prostaglandin biosynthesis during parturition. phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The INTRODUCTION products of phosphatidylinositol hydrolysis in short- term incubations were stoichiometric amounts of Prostaglandins or prostaglandin-related compounds diacylglycerol and inositol-1,2-cyclic-phosphate plus appear to play an important role in the initiation of inositol-l-phosphate. After longer periods of incuba- human parturition (1, 2). The obligate precursor of tion, monoacylglycerol also was detected. Diacyl- prostaglandins of the 2-series is free arachidonic acid glycerol lipase activity also was demonstrated in these (3). However, in mammalian tissues arachidonic acid is tissues. More than 90% of phosphatidylinositol-specific present predominantly in an esterified form; thus, the phospholipase C activity of amnion tissue was re- enzymatic release of free arachidonic acid is of signal covered in the 105,000-g supernatant fraction, and importance in the regulation of prostaglandin bio- optimal enzymatic activity in vitro was observed at pH synthesis (4). Human fetal membranes and uterine 6.5-7.5 in the presence of Ca2+ (8 mM) and mercapto- decidua contain an unusually high content of esterified ethanol (4 mM). Phosphatidylinositol-specific phos- arachidonic acid (5, 6); and -66% of the arachidonic pholipase C activity was stimulated by fatty acids in acid of human fetal membranes is present in the low concentrations, but was inhibited by lysophos- glycerophospholipids of these tissues (7). Greater phatidylcholine and a variety of detergents. No effect amounts of free fatty acids are found in amniotic fluid of labor on the specific activity of phosphatidylinositol- obtained during labor than in amniotic fluid obtained before labor, and the concentration of arachidonic acid in amniotic fluid during labor is disproportionately Dr. Di Renzo was a visiting fellow under the auspices of increased with the Fulbright-Hays Exchange Program. His present address is compared that of other fatty acids (6, 8). Clinica Obstetrica e Ginecologica, Policlinico Universitario, In a previous study we found that phospholipase 41100 Modena, Italy. Dr. Okita is the recipient of a predoctoral A2 of human fetal membranes exhibited substrate fellowship from the Robert A. Welch Foundation, Houston, specificity for the sn-2 arachidonoyl esters of phosphati- Tex. Address correspondence to Dr. John E. Bleasdale, dylethanolamine (9). Other evidence that phosphati- Department of Biochemistry, The Universtiy of Texas South- western Medical School, Dallas, Tex. 75235. dylethanolamine may be a storage form of arachidonic Received for publication 25 July 1980 and in revised form acid for prostaglandin biosynthesis was the finding 31 October 1980. that, during labor, there is a marked reduction in the

J. Clin. Invest. X The American Society for Clinical Investigation, Inc. * 0021-9738/81/03/0847/10 $1.00 847 Volume 67 March 1981 847-856 arachidonic acid content of phosphatidylethanol- at the time of spontaneous vaginal delivery or at the time of amine in amnion the arachidonic acid elective cesarean section performed before the onset of labor. (10). However, The membranes were washed several times with ice-cold content of phosphatidylinositol in amnion also de- NaCl (0.15 M). The amnion tissue was peeled away from creases during labor (10). The possibility that phos- chorion laeve, and for some experiments the amnion was phatidylinositol also may supply arachidonic acid for divided into placental and reflected parts. The uterine prostaglandin biosynthesis during labor is attractive decidua vera was removed from the chorion laeve by sharp dissection. Employing this technique, it was found by histo- since, in many tissues, I-stearoyl-2-arachidonoyl- logic examination that the chorion laeve after dissection glycerophosphoinositol is the predominant molecular was not contaminated by decidual tissue. The separated species of this phospholipid (11). However, a phos- fetal membranes and the uterine decidua were washed with phatidylinositol-specific has not been ice-cold sucrose solution (0.32 M), blotted, and weighed. A described. it was that portion (2 g) of each tissue was cut into small pieces, placed Recently, shown when platelets in 5 ml of ice-cold sucrose (0.32 M), and homogenized for were stimulated with thrombin, phosphatidylinositol 30 s, employing a Polytron homogenizer (Brinkmann Instru- was hydrolyzed rapidly to diacylglycerol (12-15), ments, Inc., Westbury, N. Y.) with the small blade and a dial which in turn was hydrolyzed by a diacylglycerol setting of 6. The whole homogenate was centrifuged at 750 g lipase to produce free arachidonic acid (13, 16). The for 10-min (Sorvall-Superspeed RC2B centrifuge, type SS-34 rotor; DuPont Instruments-Sorvall Biomedical Div., New- hydrolysis of phosphatidylinositol is catalyzed by a town, Conn.), and the resulting supernatant fluid was re- specific phospholipase C (phosphatidylinositol phos- moved and passed through four layers of cheesecloth. In phodiesterase, EC 3.1.4.10) in a reaction leading to some experiments, an aliquot of this extract ("homogenate") the formation of diacylglycerol, myo-inositol- 1- was employed as the enzyme source. The homogenate was and centrifuged at 10,000 g for 10 min. The 10,000-g pellet phosphate myo-inositol-1,2-cyclic phosphate. (mitochondria-enriched fraction) was resuspended in sucrose Phosphatidylinositol-specific phospholipase C activity (0.32 M) and the 10,000-g supematant fraction was centri- has been demonstrated in several tissues (17-26). fuged at 105,000 g for 60 min (Beckman L2-65 centrifuge, The purpose ofthe present investigation was to identify type TiSO rotor; Beckman Instruments, Inc., Spinco Div., and to characterize, if present, phosphatidylinositol- Palo Alto, Calif.). The 105,000-g pellet (microsome-enriched fraction) was resuspended in sucrose (0.32 M) and centri- specific phospholipase C activity in human fetal fuged at 105,000 g for 60 min. The resulting pellet was resus- membranes and uterine decidua. This research was pended in sucrose (0.32 M) by hand homogenization with a part of a continuing investigation designed to deter- Potter-Elvehjem teflon-glass homogenizer. Each of the sub- mine the mechanisms that give rise to the selective cellular fractions was assayed immediately after preparation, release of arachidonic acid during human parturition. since loss of phospholipase C activity was observed when these preparations were stored at -70°C for >24 h. After 48 h at -70°C, -25% of the activity was lost. METHODS Preparation of substrates. Phosphatidyl[2-3H]inositol Materials. All chemicals and solvents used in this study was prepared by a modification of the method of Bleasdale were reagent and/or analytical grade. Myo-[2-3H]inositol et al. (29). Microsomes (-9 mg of protein) were isolated from (12.5 Ci/mmol) and [1-_4C]oleic acid (55 mCi/mmol) were lung tissue of female New Zealand white rabbits (29) and purchased from New England Nuclear, Boston, Mass. Pig were incubated in Tris-HCl buffer (175 mM, pH 8.5) con- liver phosphatidylinositol, CDP-diacylglycerol (prepared taining [2-3H]myo-inositol (0.35 mM, 100 ,uCi,umol), CDP- from egg lecithin), phosphatidylserine (from beef brain), diacylglycerol from egg lecithin (0.5 mM), AMP (5 mM), phosphatidylethanolamine (from egg), phosphatidylglycerol mercaptoethanol (5 mM), and MnCl2 (3.8 mM) in a total (from egg lecithin), phosphatidylcholine (dioleoyl), 1-oleoyl- volume of 10 ml. The incubation was conducted at 370C in a sn-glycero-3-phosphocholine (oleoyl lysophosphatidyl- shaking water bath. After 20 min, and again after 40 min, choline), 1-palmitoyl-sn-glycero-3-phosphocholine and 2 ml of CDP-diacylglycerol (2.5 mM) was added to the in- 1-oleoylglycerol were obtained from Serdary Research cubation mixture. After 60 min, the reaction was stopped by Laboratories, Ontario, Canada. Phosphatidylinositol was the addition of 42 ml of methanolic HCl (0.1 M) followed by analyzed by two-dimensional thin-layer chromatography 84 ml of chloroform. After mixing, 28 ml of KCI (1.5 M) con- (27), and on the basis of phosphate determination (28) was taining myo-inositol (0.2 M) was added, and the mixture was found to be >94% pure. Rac-1,2-dioleoylglycerol and 1,3- centrifuged at 750 g for 10 min. The lower layer was removed dioleoylglycerol were gifts from Dr. F. H. Mattson of Proctor and retained; the upper layer was extracted twice more with and Gamble, Cincinnati, Ohio. Oleic and arachidonic acids 42 ml of chloroform. The pooled lower layers (three) were were obtained from Nu Check Prep., Elysian, Minn. Hepta- washed twice with 30 ml of methanol/KCl (1.5 M) containing decanoic acid was obtained from Analabs, Inc., North Haven, myo-inositol (0.2 M) (1:1, vol/vol). The washed extract was Conn. Triton X-100 (scintillar grade) was obtained from taken to dryness in vacuo, and the residue was dissolved in Mallinckrodt, Inc., St. Louis, Mo. Miranol H2M was obtained 5 ml of chloroform and stored at -20°C. The radiochemical from Miranol Chemical Co., Inc., Irvington, N. J. Cetyl- homogeneity of the substrate was evaluated by thin-layer pyridinium chloride, adenosine monophosphate, bovine chromatography as described (29). More than 97% of the serum albumin, myo-inositol and myo-inositol-2-phosphate incorporated radioactivity cochromatographed with authentic were purchased from Sigma Chemical Co., St. Louis, Mo. phosphatidylinositol. This extract (hereafter called "radio- Precoated thin-layer silica gel G and high resolution plates labeled total lipid extract") was used, without further were obtained from Analtech Inc., Newark, Del. purification, in the assay procedure described below. Preparation of fetal membranes. Human fetal mem- The diacylglycerol substrate (1-palmitoyl-2-oleoyl-sn- branes, which were not contaminated with meconium or glycerol) used for the assay of diacylglycerol lipase activity antiseptics, were removed from term gestation placentae was prepared immediately before use from 1-palmitoyl-2-

848 Di Renzo, Johnston, Okazaki, Okita, MacDonald, and Bleasdale [1-_4C]oleoyl-sn-glycero-3-phosphocholine as described by of the assay procedure were unchanged. Aliquots (0.02 ml) of Bell et al. (13). The 1-palmitoyl-2-[1-_4C]oleoyl-sn-3-phospho- the upper phases were treated by descending paper chroma- choline was prepared from 1-palmitoyl-sn-glycero-3-phos- tography on Whatman No. 1 paper (Whatman Inc., Clifton, phocholine and [1-14C]oleic acid employing rat liver micro- N. J.) developed in ethanol/ammonia (13.5 M) (3:2, vol/vol) somes by the procedure of Waite and van Deenen (30). as described by Dawson and Clarke (19). This chromato- Assay of phospholipase C activity. For the standard graphic procedure does not separate completely inositol from assay of phospholipase C activity, a stock solution of phos- inositol-1,2-cyclic phosphate. To ensure that the radioactive phatidyl[2-3H]inositol (5.0 mM) was prepared as follows. product recovered in inositol-1,2-cyclic phosphate was not An aliquot (2.5 ml, containing 24 ,umol) of phosphatidylinosi- contaminated by [3H]inositol, samples of the upper phases tol from pig liver in chloroform (10 mg/ml) was mixed with were subjected to acid hydrolysis (1 M HCI, 90°C for 3 min) 0.2 ml of radiolabeled total lipid extract (equivalent to -0.3 before chromatography. Employing this procedure, es- ,umol of total lipid phosphorus and 0.02 ,umol of phosphati- sentially all inositol-1,2-cyclic phosphate is converted to dylinositol) and taken to dryness under nitrogen. To the inositol-l-phosphate (31), which can be separated readily from dried lipid residue, 5.0 ml of Tris-HCl buffer (20 mM, pH inositol by this chromatographic procedure. Glycerophospho- 7.0) was added, followed by sonication for 60 s at 125 W in a inositol, although not a product of the enzymatic reaction Bransonic 220 bath sonicator (Bransonic Cleaning Company, measured here, was shown to be separated from inositol- Shelton, Conn.). The standard assay mixture consisted of Tris- 1,2-cyclic phosphate by this chromatographic procedure. HCI (20 mM, pH 7.0), phosphatidyl[2-3H]inositol (2 mM, The inositol-1,2-cyclic phosphate used as a chromatographic 0.05 ,ACi/4mol), mercaptoethanol (4 mM), CaCl2 (4 mM), and standard was prepared as described by Pizer and Ballou (31). enzyme (-80 itg protein), in a total volume of 0.25 ml. When Assay of diacylglycerol lipase activity. Diacylglycerol other components were added to the incubation mixture, lipase activity was assayed employing the procedure de- these were dissolved in Tris-HCl buffer. The incubations were scribed by Rittenhouse-Simmons (16). conducted at 37°C in a shaking water bath. After incubation Other methods. Protein concentration was measured by no change in pH was detected. After 30 min, the reaction the method of Lowry et al. (32), using bovine serum albumin was terminated by the addition of 1.0 ml of chloroform/ as the reference standard. methanol (1:2, vol/vol). After mixing, 0.3 ml of chloroform and 0.3 ml of KCI (2 M) were added and mixed, and phases RESULTS were separated by centrifugation at 750 g for 10 min. An aliquot (0.75 ml) of the upper phase (1.2 ml total) was dried For the characterization of phospholipase C activity in in a scintillation vial and assayed for radioactivity employing human amnion, the 105,000-g supematant fraction of a Triton X-100 scintillation mixture (29). All determinations were made in duplicate and were corrected for blank values, this tissue was employed. However, essentially the i.e., the amount of water-soluble radiolabeled products re- same characteristics were observed when the corre- covered from incubation mixtures in which the enzyme was sponding subcellular fraction from chorion, decidua, or added after the addition of 1.0 ml of chloroform/methanol the homogenate fraction from all three tissues was (1:2, vol/vol). Blank values were never >30% of experi- are either the results of mentally determined values of enzymatic activity. used. The data reported here Analysis of reaction products. The lipid-soluble products typical experiments or are the mean values from the were isolated by thin-layer chromatography on silica gel G number of experiments indicated in the figure legends. plates with hexane/diethyl ether/acetic acid (105:45:4.5, by Identification of phospholipase C activity. Upon vol) as the developing solvent for separation of mono- and incubation of phosphatidyl[2-3H]inositol with sub- diacylglycerols and chloroform/methanol/acetic acid/H20 or decidua, (100:50:14:6, by vol) as the developing solvent for separation cellular fractions of either amnion, chorion, of phospholipids. When lipid-soluble products were analyzed water-soluble tritium-labeled products were released for composition, the mono- and diacylglycerols and were analyzed by liquid scintillation spectrometry. were separated by thin-layer chromatography on silica gel After 30 min of incubation when the water-soluble high resolution plates with hexane/diethyl ether/methanol/ products were analyzed as above, these were found to acetic acid (80:21:3:2) as the developing solvent. After chromatography, areas of the chromatogram containing be inositol-1,2-cyclic phosphate (55%), inositol-l-phos- diacylglycerol were removed separately and added to 0.5 ml phate (36%), and inositol (9%). The lipid-soluble of spectrograde benzene, and 2.0 ml of boron trifluoride products from phosphatidylinositol breakdown cata- in methanol was then added. The mixture, in an atmosphere lyzed by all sources of enzyme used (i.e., amnion, of N2, was heated in a boiling water bath tor 30 min. After chorion, and decidua homogenates, the 105,000-g cooling, 20 ml of distilled H2O was added, and the sample was extracted three times with hexane (2.5 ml). The upper pellets, or the 105,000-g supernatant fractions) also layers were pooled and evaporated to dryness under nitrogen, were analyzed by thin-layer chromatography (Figs. 1A and the residue was redissolved in carbon disulfide. The and 1B). As the incubation time increased, there was fatty acid methyl esters in this solution were separated by a greater loss of substrate, and after 3 h of incubation gas chromatography, using a Hewlett-Packard model 5830 A was -95% com- gas chromatograph (Hewlett-Packard Co., Palo Alto, Calif.) the hydrolysis of phosphatidylinositol using columns of 10% Sp-2330 on 100/200 chromosorb W plete (Fig. 1A). The nonpolar lipids released during (Supelco Inc., Bellefonte, Pa.) at 200°C. Using heptadecanoic the incubation period were largely 1,2-diacylglycerols acid as internal standard, the amounts of the eluted fatty acid (Fig. 1B), with small amounts of 1,3-diacylglycerols methyl esters were computed. (probably derived from 1,2-diacylglycerols by isom- In experiments where water-soluble products were isolated and traces of un- and identified, the assay procedure was modified to accom- erization), monoacylglycerols, modate a total volume of 0.1 ml, and in the extraction pro- esterified fatty acids. The diacylglycerols were quanti- cedure KCI (2 M) was replaced by water. All other conditions fied by gas chromatography of their fatty acid sub-

Phospholipase C Activity in Fetal Membranes and Uterine Decidua 849 A phosphatidyl[2-3H]inositol hydrolysis after 6 min was 11.8% when assessed by measuring total radiolabeled water-soluble products, and 12.2% when assessed by measuring total diacylglycerol production. Further- more, after 6 min of incubation the fatty acid composi- tion of the diacylglycerol produced was not signifi- cantly different from that ofthe substrate, phosphatidyl- [2-3H]inositol. Expressed as mole percent, the fatty acid compositions of phosphatidyl[2-3H]inositol and diacyl- glycerol were, respectively, palmitate 5.2 and 4.5%, stearate 49.5 and 50.4%, oleate 5.4 and 5.4%, linoleate 5.3 and 3.3%, arachidonate 34.6 and 36.3%. After 30 12 3 4 { min of incubation, the molar stoichiometry between diacylglycerol recovered and total radiolabeled water- B soluble products was <1:1, since by this time appre- ciable quantities of monoacylglycerols had begun to accumulate (Fig. 1B). Specificity of phospholipase C. When either phos- phatidylcholine, phosphatidylethanolamine, phosphati- dylserine or phosphatidylglycerol was substituted for phosphatidylinositol, no diacylglycerol formation was detectable under the standard assay conditions (Fig. 2). A 'B Kinetics of phospholipase C activity. The rate of hydrolysis of phosphatidylinositol was constant for 30 min using the assay procedure as described, and also

SF * * Y 1 2 3 4 5 6 7 FIGURE 1 Time-course of phosphatidylinositol degradation and lipid product accumulation. Phospholipase C activity was measured in the 105,000-g supematant fraction pre- pared from amnion tissue. The assay mixture was composed of Tris-HCl buffer (20 mM, pH 7.0), mercaptoethanol (4 mM), CaCl2 (4 mM), phosphatidyl[2-3H]inositol (2 mM, 0.05 ACi/ ,tmol) and the 105,000-g supernatant fraction from amnion (80 ,ug protein) in a total volume of 0.25 ml. After incubation at 37°C for various times, reactions were terminated by the addition of 1.0 ml of chloroform/methanol (1:2, vol/vol), and lipids were extracted (Methods). Lipid extracts were analyzed A by two different thin-layer chromatographic procedures: (A) B For isolation of phosphatidyl[2-3H]inositol remaining after in- cubation, lipid extracts were chromatographed on silica gel G plates developed in chloroform/methanol/acetic acid/H20 C (100:50:14:6, by vol); PI, phosphatidylinositol. (B) For iso- a b c lation of the lipid products of phospholipase C action, lipid d e extracts were chromatographed on silica gel G plates de- FIGum 2 Specificity of phospholipase C activity of am- veloped in hexane/diethyl ether/acetic acid (105:45:4.5, by nion. Aliquots (80 ug protein) of the 105,000-g supematant vol); A, 1,3-diacylglycerol; B, 1,2-diacylglycerol; C, mono- fraction of amnion were incubated for 30 min as described in acylglycerol. Lipid spots on chromatograms were developed the legend of Fig. 1, except that phosphatidyl[2-3H]inositol by exposure to iodine vapor. Lane 1, no incubation; 2, 5 (2 mM) was replaced by (a) nonradiolabeled phosphatidylino- min; 3, 10 min; 4, 30 min; 5, 60 min; 6, 120 min, and 7, sitol (1 mM); (b) phosphatidylcholine (dioleoyl, 1 mM); 180 min. (c) phosphatidylglycerol (1 mM); (d) phosphatidylethanol- amine (1 mM); or (e) phosphatidylserine (1 mM). Following incubation, lipids were extracted from the reaction mixture stituents, and it was found that there was a 1:1 molar and separated by thin-layer chromatography on silica gel G stoichiometry between the amount of diacylglycerols plates developed in hexane/diethyl ether/acetic acid (105: (1,2- plus 1,3-) formed and the quantity of radio- 45:4.5, by vol). The nondesignated lane of the chromatogram labeled water-soluble products released at early time contained a mixture of known lipids: A, 1,3-diacylglycerol; B, 1,2-diacylglycerol; C, monoacylglycerol. Lipid spots were periods. For instance, when the 105,000-g super- rendered by exposure to iodine vapor. It was estimated that natant fraction of amnion (168 ,tg protein) was in- hydrolysis of as little as 2.5 nmol of added lipid could be cubated under standard assay conditions, the extent of detected by this procedure.

850 Di Renzo, Johnston, Okazaki, Okita, MacDonald, and Bleasdale A c Q.E

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30 60 90 120 150 180 0 50 1i0 150 200 INCUBATION TIME (min) PROTEIN (pIg)

FIGURE 3 Dependence of phosphatidylinositol-specific phospholipase C activity on time and protein concentration. Phosphatidylinositol-specific phospholipase C activity in the 105,000-g supematant fraction of amnion was measured as described in the legend of Fig. 1, except that radiolabeled water-soluble products were measured (Methods). (A) Time-dependence ofphospho- lipase C activity. After 180 min of incubation, hydrolysis of phosphatidylinositol was -95% com- plete. (Average of the results of two experiments). (B) Dependence of phospholipase C activity on protein concentration. was proportional to enzyme concentration up to 200 Effect of pH and various buffers on phospholipase ,ug protein in an assay volume of 0.25 ml (Figs. 3A C activity. The optimum pH for phosphatidylino- and 3B). sitol-specific phospholipase C activity in the presence The effect of substrate concentration on the rate of of Tris-HCl buffer (20 mM) was 7.0 (Fig. 5). Using Tris- product formation is depicted in Fig. 4. The rate of maleate buffer (20 mM), the phosphatidylinositol ap- product formation was independent of substrate con- peared to be better dispersed (less turbid suspension), centration between 0.5 and 4 mM phosphatidylinositol. but enzymatic activity decreased at pH values >5.0. Maleate might be expected to inhibit enzymatic activity 6 _

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0 1 2 3 4 5 6 7 8 [PHOSPHATIDYLINOSITOL] (mM) 4.0 5.0 6D 7.0 8.0 9.0 10.0 pH FIGuRE 4 Dependence of phosphatidylinositol-specific phos- pholipase C activity on phosphatidylinositol concentration. FIGuRE S The pH dependence of phosphatidylinositol- Aliquots (80 ,ug protein) of the 105,000-g supernatant fraction specific phospholipase C activity. Aliquots (80 ,ug protein) of amnion were incubated as described in the legend of of the 105,000-g supematant fraction from amnion tissue were Fig. 1 at 37°C for 30 min with phosphatidyl[2-3H]inositol at incubated under standard assay conditions (Methods) except different concentrations. Suspensions of phosphatidyl[2-3H]- that Tris-HCl (20 mM, pH 7.0) was replaced by (-) Tris- inositol were prepared (Methods) in Tris-HCl buffer (20 maleate (20 mM, pH 4.1-9.0); (0) sodium acetate-acetic acid mM, pH 7.0) at 2.5 times the desired final concentration in (100 mM, pH 4.1-6.0); (A) Clark and Lubs buffer (KH2P04 the incubation mixture. An identical dependence of phos- NaOH, 35 mM, pH 5.5-8.0); (0) Tris-HCl (20 mM, pH 5.5- pholipase C activity on phosphatidyl[2-3H]inositol concen- 9.0); or (A) 2-amino-2-methyl-propan-1-ol-HCl (50 mM, pH tration was observed when incubations were conducted at 7.5-10.0). Incubations were terminated after 30 min at 37°C either pH 5.5 (Tris-maleate, 130 mM) or pH 8.5 (Tris- and water-soluble products were measured as described in maleate, 130 mM). Methods. Mean of the results of five experiments.

Phospholipase C Activity in Fetal Membranes and Uterine Decidua 851 TABLE I Effect of Cations on Phosphatidylinositol-specific Phospholipase C Activity ox 4 F Phospholipase C specific activity Cation E / added Concentration With CaCi, (4 mM) Without CaCI, mM % control a. oxE/2K0 None - 100.0 15.0

0 4 8 12 16 32 Mg2+ 1.0 75.0 15.0 [CaCo2] (mM) 4.0 91.0 12.0 FIGURE 6 The effect of Ca2+ ions on phosphatidylinositol- Mn2+ 1.0 69.0 11.0 specific phospholipase C activity. Standard assays of phos- 4.0 68.0 7.0 pholipase C activity (Methods) were conducted employing the 105,000-g supematant fraction of amnion tissue (80 sg Fe2+ 1.0 53.0 4.0 protein) in the presence of CaCl2 at various concentrations 4.0 5.0 0.0 (average of the results of two experiments). A similar de- Co2+ 1.0 53.0 5.0 pendence of phosphatidylinositol-specific phospholipase C 4.0 24.0 5.0 on Ca2+ ions was observed when assays were conducted at pH 5.5 (Tris-maleate, 130 mM). Cd2+ 1.0 44.0 ND 4.0 0.0 ND by removing Ca2+ in the form of the poorly dissociated Na+ 50.0 52.0 20.0 calcium salt, or else by reacting with essential thiol 100.0 64.0 17.0 groups in the phospholipase C (18). Similarly, enzy- K+ 50.0 52.0 15.0 matic activity in the presence of phosphate buffer (pH 100.0 64.0 12.0 7.0) was less than that in Tris-HCl buffer (pH 7.0), pre- sumably due to formation of insoluble calcium phos- Cu2+ 0.1 15.0 10.0 phate. The other two buffer systems used (sodium 1.0 9.0 9.0 acetate-acetic acid and 2-amino-2-methyl-propan-1-ol- Hg2+ 0.1 9.0 11.0 HCI) have little or no buffering capacity in the pH 1.0 3.0 0.0 range that is optimal for enzymatic activity. Effect of cations on phospholipase C activity. In the Standard assays were conducted employing the 105,000-g absence of added Ca2+, enzymatic activity was only supernatant fraction of amnion tissue (80 Ag protein) as de- scribed (Methods), except that mercaptoethanol was omitted 15% of that observed in the presence of CaC12 (4 mM). from incubations. Various cations (as their chlorides) were Furthermore, the small amount of enzymatic activity added to incubation mixtures in the presence or absence of detected in the absence of added Ca2+ could be in- CaCl2 (4 mM). The values given are the phospholipase C hibited completely by EDTA (1.0 mM). Maximum specific activities as percentages of the specific activity phospholipase C activity was observed in the presence measured in the presence of CaCl2 (4 mM) with no other of Ca2+ (8 mM) (Fig. 6). When other cations were cation additions. ND, not determined. tested alone, none could substitute for Ca2+ ions, and in the presence of CaCl2 (4 mM), all inhibited enzy- in this study, has been found to stimulate phos- matic activity (Table I). It would appear that the phos- phatidylinositol-specific phospholipase C activity in rat phatidylinositol-specific phospholipase C of amnion cerebral cortex (20). Free oleic and arachidonic acids, tissue has an absolute requirement for Ca2 . at low concentrations, caused a slight increase in phos- Effect of detergents on phospholipase C activity. pholipase C activity of amnion tissue. At concentrations In an attempt to optimize in vitro conditions for the between 20 and 40 ,uM, free arachidonic acid in- assay of phospholipase C activity, the effects of five crease enzymatic activity by -30-40% (Table II). detergents on phospholipase C activity were investi- Other effectors of phospholipase C activity. Mer- gated. These were sodium deoxycholate (anionic), captoethanol (4 mM) increased phospholipase C ac- cetylpyridinium chloride (cationic), Triton X-100 tivity -25%. On the other hand, Hg2+ ions (Table I) (nonionic), Miranol H2M (zwitterionic), and lysophos- reduced drastically enzymatic activity, a finding that phatidylcholine, at various concentrations ranging from supports the importance of a sulfhydryl group for ac- 0.01 to 8 mg/ml. In the presence of Ca2+ (4 mM), all tivity of this enzyme, as reported by others (18). The detergents except cetylpyridinium chloride (at low inclusion of bovine serum albumin (5 mg/ml) in the concentrations) were inhibitory (Table II). Deoxy- standard assay mixture increased enzymatic activity cholate, at concentrations within the range employed by -50%.

852 Di Renzo, Johnston, Okazaki, Okita, MacDonald, and Bleasdale TABLE II phatidylinositol-specific phospholipase C activity of Effect of Surfactants on Phosphatidylinositol-specific 4.0±0.5 ,umol x mg-' protein x h-1 (mean+SE, n=4). Phospholipase C Activity Amnion tissue of an equivalent gestational age, but obtained at the time of vaginal delivery, ex- Phospholipase C Surfactant Concentration specific activity hibited a phosphatidylinositol-specific phospholipase C activity of 3.8±0.4 ,umol x mg-' protein x h-' mglml % control (mean±SE, n=4). None 100 When subcellular fractions were prepared from amnion, >90% of the phospholipase C activity was Deoxycholate 0.5 40 2.0 39 recovered in the 105,000-g supernatant fraction (Table 4.0 34 III). Presumably, because of the harsh procedure 8.0 15 necessary to homogenize amnion, the 105,000-g super- natant fraction contained activities of marker Triton X-100 0.3 80 for different subcellular organelles. In particular, 65% 3.0 54 of acid activity of the homogenate was re- Cetylpyridinium chloride 0.01 130 covered in the 105,000-g supernatant fraction, a finding 0.1 95 that indicates lysosome disruption. However, because 1.0 2 of the neutral pH optimum of the phospholipase C Miranol 0.1 96 activity, it seems unlikely that the enzyme in the 1.0 60 105,000-g supernatant fraction originated from lyso- somes. 1-Oleoyl-glycero- Diacylglycerol lipase activity in fetal membranes phosphocholine 0.05 100 and uterine decidua. Two observations indicated that 0.5 9 the amnion, chorion laeve, and decidua vera tissues Arachidonic acid 0.0012 (4 ,uM) 110 contained diacylglycerol lipase activity. First, when 0.006 140 phosphatidylinositol was incubated with these tissues 0.012 130 for longer time periods, the formation of monoacyl- 0.049 120 glycerol was observed. Second, the 1:1 stoichiometry Oleic acid 0.011 (40 ,uM) 105 between inositol phosphate and diacylglycerol forma- 0.044 115 tion was found only during the first 15 min of in- cubation; after this time the rate of accumulation Portions (80 ,ag protein) of the 105,000-g supernatant fraction of amnion tissue were incubated under standard assay con- ditions in the presence of different surfactants. The values TABLE III given are the phospholipase C specific activities as percent- Subcellular Distribution of Phosphatidylinositol-specific ages of specific activity measured in the absence of added Phospholipase C Activity of Amnion Tissue surfactant. Fatty acids and deoxycholate were added as the sodium salts. Phospholipase C activity Total Subcellular fraction protein Total activity Specific activity

Tissue and subcellular distribution of phospho- Imol x mg-' lipase C. The specific activity of phospholipase C in mg Amol x h-' protein x h-1 homogenates of amnion (4.4±0.7 ,umol x mg-' protein Homogenate 73.2 267.2 3.65 x h-l [mean+ SE, n = 11]) was three times that found in 10,000-g pellet 0.9 1.3 1.41 decidua (1.5+0.2 Amol x mg-' protein x h-l [mean 105,000-g pellet 3.1 1.3 0.42 +SE, n=4]) and more than five times that found in 105,000-g supernatant 58.8 245.8 4.18 chorion laeve (0.8±0.2 /jmol x mg-' protein x h-l [mean±SE, n=4]). No difference in phospholipase C To measure the subcellular distribution of phospholipase C activity was detected between placental and reflected activity, aliquots (-80 ,ug protein) of different subcellular frac- amnion. Furthermore, the phospholipase C activities tions of amnion tissue were incubated under standard assay in fetal membranes and uterine decidua obtained conditions (Methods). The small amount of enzymatic activity found in the 10,000-g pellet and in the 105,000-g pellet during or after labor were not significantly different exhibited a pH dependence and a Ca2+ dependence similar from the activities in the corresponding tissues ob- to those of the large amount of enzymatic activity present in tained before labor (but at the equivalent time in the 105,000-g supernatant fraction. Recoveries of protein and gestation). For example, the homogenate fraction of enzymatic activity throughout the subcellular fractionation amnion tissue obtained by elective cesarean section procedure were 86 and 93%, respectively (average of the before the onset of labor was found to have a phos- results of two experiments).

Phospholipase C Activity in Fetal Membranes and Uterine Decidua 853 of diacylglycerol was less than that of inositol-l- brain was stimulated markedly by oleic and arachi- phosphate plus inositol-1,2-cyclic phosphate. There- donic acids. Although stimulation of the same enzyme fore, we assayed diacylglycerol lipase activity in each in fetal membranes by free arachidonic acid is an of these tissues using 1-palmitoyl-2-[1-'4C]oleoyl-sn- attractive hypothesis, we were unable to demonstrate glycerol as the substrate. The rate of formation of a substantial increase in activity of phospholipase C in radiolabeled free fatty acid was determined. The amnion when arachidonic acid was added to the assay specific activities of diacylglycerol lipase (nmol x mg-' mixture. A slight increase (30-40%) in enzyme activity protein x h-1) observed in the 750-g supernatant was observed when the concentration of arachidonic fractions of amnion, chorion laeve, and uterine decidua acid was in the range of 20 to 40 ,uM. We observed were, respectively, 9.6+1.9 (mean+SE, n=4), 12.1 that deoxycholate, at all concentrations tested, in- ±4.4 (n=4) and 38.4±4.5 (n=4). We also observed hibited phospholipase C activity; of the other deter- that the subcellular distribution of diacylglycerol gents tested, only cetylpyridinium chloride, a cationic lipase activity in these tissues was similar to that of detergent, stimulated phospholipase C activity. NADPH cyctochrome c reductase activity, a finding Phosphatidylinositol of human fetal membranes, as which suggests a microsomal location of diacylglycerol in other tissues, is known to be rich in arachidonic acid lipase (Okazaki et al., manuscript in preparation). (7). This enrichment with arachidonic acid, together Although the substrate used to measure diacylglycerol with the presence in fetal membranes of phosphati- lipase activity was radiolabeled in the fatty acid in the dylinositol-specific phospholipase C, may lead to the sn -2 position, the assay procedure does not allow a formation of a diacylglycerol enriched in arachidonic distinction between a diacylglycerol lipase with sn -2 acid, from which arachidonic acid may be released positional specificity and a diacylglycerol lipase acting during parturition. Such a process would involve a in conjunction with a . diacylglycerol lipase. In the present study, using 1-palmitoyl-2-[1_14C]oleoyl-sn-glyceroI as substrate, DISCUSSION we demonstrated the presence of diacylglycerol lipase activity in fetal membranes and uterine decidua. In In the present study, phosphatidylinositol-specific addition, even though the present assay conditions phospholipase C activity was demonstrated in sub- were optimized for measurement of phospholipase C cellular fractions prepared from human amnion, activity, monoacylglycerol formation was detected chorion laeve, and uterine decidua. This enzymatic when the 105,000-g supernatant fraction of amnion activity was characterized partially in the 105,000-g tissue was incubated with phosphatidylinositol. This supernatant fraction of amnion tissue. The specific finding also suggests the presence of a diacylglycerol activity of the enzyme in amnion was much greater lipase in amnion tissue and contrasts with the failure than that in either chorion laeve or uterine decidua, to observe an accumulation of monoacylglycerol as a and also was greater than the specific activity of phos- result of diacylglycerol lipase activity in platelets (13). pholipase C of human platelets, which has been re- When phosphatidylinositol-specific phospholipase ported by others (14, 15, 33). Most of the phos- C activity was measured under the conditions de- phatidylinositol-specific phospholipase C enzymes scribed above, we found no difference in the specific studied in other mammalian tissues are either cytosolic activity of this enzyme in fetal membranes (or uterine (18, 21, 33-35) or membrane bound (20), and require decidua) obtained after labor compared with that in Ca2+ for activity. A second group of phosphatidylino- the corresponding tissues obtained before labor but at sitol-specific phospholipase C enzymes of lysosomal the equivalent time in gestation. Although no effect origin has a more acidic pH optimum and is not in- of labor on in vitro phospholipase C activity could be hibited by EDTA (21, 25, 26). From the results of detected, this finding does not preclude the possi- the present study, it is evident that the phospholipase bility that phospholipase C activity is regulated in vivo C activity of human fetal membranes and uterine during labor. Although a regulatory function for diacyl- decidua belongs to the former group. glycerol lipase cannot be discounted (13, 16), it is Some of the phosphatidylinositol-specific phos- possible that, in vivo, phosphatidylinositol-specific pholipase C enzymes are activated by the detergent phospholipase C is regulated by Ca2 , fatty acids, deoxycholate (20, 36, 37). It has been postulated that or neurohormones. There is precedent for suggesting deoxycholate might mimic some endogenous anionic that phosphatidylinositol-specific phospholipase C is amphiphile which regulates the activity of the enzyme a regulatory enzyme. In many tissues, a response to in vivo. Irvine et al. (37) presented evidence that extracellular stimuli is observed that involves specifi- supported the view that this amphiphilic regulator cally an enhanced turnover of phosphatidylinositol. It may be a free fatty acid, since the hydrolysis of has been proposed that in such responses, phos- membrane-bound phosphatidylinositol in rat liver phatidylinositol-specific phospholipase C activity is microsomes by the soluble phospholipase C from rat greatly increased (38). When considering phosphati-

854 Di Renzo, Johnston, Okazaki, Okita, MacDonald, and Bleasdale dylinositol as the source of arachidonic acid for prosta- Mobilization of arachidonic acid from glycerophospho- glandin biosynthesis, it is recognized that the con- lipids of human fetal membranes during parturition. Proc. Soc. Gynecol. Intvest. 27: 110. centration of this glycerophospholipid in human fetal 11. White, D. A. 1973. The phospholipid composition of membranes is low compared with that of phosphati- mammalian tissues. In Form and Function of Phospho- dylcholine and phosphatidylethanolamine. However, lipids. G. B. Ansell, J. N. Hawthorne, and R. M. C. in many tissues the turnover of phosphatidylinositol Dawson, editors. Elsevier, Amsterdam, The Netherlands. 441-482. is great compared with that of other glycerophospho- 12. Mauco, G., H. Chap, M. F. Simon, and L. Douste- lipids. Moreover, the optimal specific activity of the Blazy. 1978. Phosphatidic and lysophosphatidic acid phosphatidylethanolamine-specific phospholipase A2 production in phospholipase C- and thrombin-treated in human fetal membranes under optimal in vitro platelets, possible involvement of a platelet lipase. Bio- is with that of phos- chimie (Paris) 60: 653-661. conditions (9) very low compared 13. Bell, R. L., D. A. Kennerly, N. Stanford, and P. W. phatidylinositol-specific phospholipase C activity in Majerus. 1979. lipase: a pathway for arachi- these tissues. For these reasons, phospholipase C of donate release from human platelets. Proc. Natl. Acad. fetal membranes, as already suggested for that ofhuman Sci. U. S. A. 76: 3238-3241. platelets (13), may play a key role in the regulation 14. Billah, M. M., E. G. Lapetina, and P. Cuatrecasas. 1979. Phosphatidylinositol-specific phospholipase-C of plate- of the selective release of arachidonic acid for con- lets: association with 1, 2-diacylglycerol-kinase and in- version to prostaglandins in the initiation and/or hibition by cyclic-AMP. Biochem. Biophys. Res. Com- maintenance of human parturition. mun. 90: 92-98. 15. Rittenhouse-Simmons, S. 1979. Production of diglyceride ACKNOWLEDGMENT from phosphatidylinositol in activated human platelets. J. Clin. Invest. 63: 580-587. This work was supported in part by U. S. Public Health 16. Rittenhouse-Simmons, S. 1980. Indomethacin-induced Service grant 5-P50-HD11149. accumulation of diglyceride in activated human platelets. J. Biol. Chem. 255: 2259-2262. 17. Thompson, W. 1967. The hydrolysis of monophospho- REFERENCES inositide by extracts of brain. Can. J. Biochem. 45: 1. MacDonald P. C., J. C. Porter, B. E. Schwarz, and J. M. 853-861. Johnston. 1978. Initiation of parturition in the human fe- 18. Atherton, R. S., and J. N. 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856 Di Renzo, Johnston, Okazaki, Okita, MacDonald, and Bleasdale