Relationship between Androstenedione-lnduced Myometrial ...

BIOLOGY OF REPRODUCTION 56, 247-252 (1997)

Relationship between Androstenedione-lnduced Myometrial Contractions and Platelet-Activating Factor Acetylhydrolase in Late Gestation in Pregnant Rhesus Monkeys' Dino A. Giussani,2,3 John E. Parks,4 Shelley R. Hough,4 Susan L. Jenkins,3 James A. Winter, 3 and Peter W. Nathanielsz 3 Laboratory for Pregnancy & Newborn Research,3 Department of Physiology, College of Veterinary Medicine and Department of Animal Science, 4 Cornell University, Ithaca, New York 14853-6401 ABSTRACT An association between platelet-activating factor (PAF) and myometrial contractions has been established. Estrogens regulate PAF activity via reduction in the activity of plasma PAF acetylhydrolase (PAF-AH), the enzyme that catalyzes PAF inactivation. Administration of androstenedione to pregnant monkeys leads to sustained increases in maternal plasma estradiol (E2), with persistent nocturnal myometrial contractions. The present study tested the hypothesis that androstenedione-induced contractions are associated with a fall in maternal plasma PAF-AH activity in monkeys. Eight monkeys (132-136 days gestation, dGA) were instrumented under halothane anesthesia with maternal vascular catheters and uterine electromyogram electrodes. At 138-142 dGA, two baseline maternal arterial samples were taken for E2 and PAF-AH measurements. The following day a continuous i.v. androstenedione infusion was started in 4 monkeys while 4 control monkeys received i.v. infusions of vehicle alone. Arterial blood sampling was repeated 1 and 3 days after the start of either infusion. Despite an increase in maternal E2 to term levels and established myometrial contractions, no change in maternal plasma PAF-AH activity occurred after androstenedione treatment. Maternal plasma E2, PAF-AH activity, and contractions remained unchanged from baseline in control monkeys. In conclusion, androstenedione-induced increases in maternal plasma E2 and myometrial contractions are not associated with a fall in maternal plasma PAF-AH specific activity. INTRODUCTION Since its discovery, PAF (1-O-alkyl-2-acetyl-sn-glycero3-phosphocholine) has been implicated in playing important roles in numerous physiological processes including the initiation and maintenance of primate parturition (for review see [1]). An association between PAF and labor in primates is suggested since PAF concentrations increase in amniotic fluid of women at term and in labor [2, 3] and in amniotic fluid of women threatened with preterm delivery, namely patients with premature rupture of the membranes and/or preterm labor [4]. It has been suggested that amniotic PAF originates from fetal tissues, in particular the lung and the kidney, and several fetal tissues have an increased PAF biosynthetic capacity and content in the latter stages of gestation in the rabbit [5] and in the human [6]. In contrast, the activity of the major tissue intracellular degradative enzyme, PAF-acetylhydrolase (PAF-AH), is not changed during gestation in the fetal lung and kidney [7]. Accepted September 10, 1996. Received April 11, 1996. 'Supported by National Institutes of Health HD 21350. 2 Correspondence: Peter W. Nathanielsz, Laboratory for Pregnancy & Newborn Research, Dept. of Physiology, T9 016 VRT, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. FAX: (607) 253 3455; e-mail: pwnl @cornell.edu

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In addition, concentrations of PAF-AH in maternal plasma fall dramatically in late pregnancy in the rabbit [8] and at 32 wk of gestation in the pregnant woman [1]. A specific role for PAF in regulating the increase in myometrial contractility in labor is suggested by the observation that physiological concentrations of PAF stimulate in vitro smooth muscle contractions in myometrium taken from the guinea pig [9], from the rat [10], and from the pregnant woman [11]. In addition, high-affinity binding sites for PAF have been reported in myometrium from pregnant rabbits [12] and pregnant women [13], and administration of a PAF receptor antagonist prolongs parturition in the rat [14]. Evidence exists for a stimulatory influence of estrogens in the endocrine regulation of PAF since estradiol (E2 ) treatment of pregnant and nonpregnant rats led to a decrease in peripheral plasma PAF-AH concentrations [7]. We have previously reported that intravenous infusion of androstenedione to preparturient monkeys leads to sustained increases in maternal plasma E 2 to term concentrations, with persistent myometrial contractions [15]. In the present study, we have used this nonhuman primate model of labor associated with increased maternal plasma estrogen to test the hypothesis that androstenedione-induced increases in maternal plasma E 2 and myometrial contractions are accompanied by reduced maternal plasma PAF-AH specific activity in the pregnant rhesus monkey. MATERIALS AND METHODS Use of Animals Eight pregnant rhesus monkeys (6.9 0.6 kg; mean SEM) of known gestational ages were obtained from the California Regional Primate Research Center (Davis, CA) and acclimated to laboratory conditions and the tether restraint system as previously described [15-18]. In brief, after a full physical examination, the animals were housed in individual cages, in rooms with controlled light:dark cycles (14L:10OD), and placed in quarantine. During quarantine, the animals were jacketed and familiarized with the tether through which vascular catheters and electrode wires were to be connected after surgical instrumentation. One week later, the tether was fixed to a box on the back of the jacket. The animals were fed daily (Purina 5045 High Protein Monkey Chow [Purina, St. Louis, MO] and fresh fruits) and water was continuously available. Experimental procedures were approved by the Cornell University Institutional Animal Care and Use Committee. All facilities were accredited by the American Association of Laboratory Animal Care.

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TABLE 1. Animals used during the experimental protocol. Four monkeys (Group I) were infused with intralipid and four monkeys (Group II) were treated with androstenedione. Treatment group Group I

Group II

Rhl Rh2 Rh3 Rh4 Mean + SEM Rh5 Rh6 Rh7 Rh8 Mean + SEM

Weight (kg)

dGA surgery

dGA start infusion

dGA end experiment

5.9 7.3 5.9 6.8 6.5 ± 0.3 5.9 6.8 10.9 5.6 7.3 1.2

118 132 132 136 129 4 121 118 136 132 127 + 4

145 138 138 142 141 2 139 139 142 140 140 1

156 158 159 174 162 + 4 143 143 146 143 144 1

Surgical Instrumentation and Post-Surgical Management The monkeys were instrumented between 118 and 136 days gestation (dGA; term is ca. 162 days, see Table 1) using techniques previously described [15-18]. After withdrawal of food for 24 h, polyvinyl catheters (i.d. = 0.04 in; o.d = 0.07 in; Norton Performance Plastics Corp., Akron, OH) were placed in the dorsal aorta and inferior vena cava via insertion into the left femoral artery and vein, respectively, under general anesthesia (15 mg/kg ketamine for induction; 1-2% halothane in 02 for maintenance). In addition, following hysterotomy, three pairs of multi-stranded electrodes (AS 632; Cooner Wire Co., Chatsworth, CA) were sewn on different sites of the anterior surface of the uterine body to monitor myometrial electromyogram (EMG) activity. Catheters were filled with heparinized saline (25 IU/ml) and tunnelled subcutaneously with the electrode leads to exit between the shoulder blades. After surgery, the monkeys were rejacketed and maintained on the tether and swivel system, which allowed the animals to be free-ranging and permitted passage of vascular catheters and EMG leads to the top of the cage [15-18]. At least 5 days were allowed before commencement of all experiments, during which time antibiotics (Oxacillin; Marsam Pharmaceuticals Inc., Cherry Hill, NJ; i.v. infusion of 100 mg/kg per day) and analgesia (Buprenorphine; Reckitt-Colman Products, Richmond, VA; i.a. infusion of 15 ILg/kg per day for two days) were administered to the mother. Treatment Groups and Blood Sampling Catheters were maintained patent by continuous infusion of heparinized saline (25 IU/ml at 0.5 ml/h) from the time of surgery until the commencement of one of two treatments at 138-145 dGA (Table 1). In four monkeys (group I), the saline administration was switched to a continuous infusion of vehicle alone (intralipid 10%; Kabi Vitrum, Inc., Alameda, CA) set at the same rate, and in the other four monkeys (group II), androstenedione (4-androstenedione-3,17-dione; Sigma, St. Louis, MO) dissolved in intralipid was infused continuously at 0.3 mg/kg per h at 0.5 ml/h. In all animals, 2 x 4-ml maternal arterial blood samples were taken for measurement of plasma E 2 and calculation of PAF-AH specific activity during a baseline period at lights off and 4.5 h after lights-off in the animal's environment. In addition, 0.3 ml of arterial blood was taken for measurement of pH and of arterial pressure (Pa) of CO 2 and 02, measured in mm Hg. These sampling procedures were then repeated 1 (24-27 h) and 3 (72-75 h) days after the start of intralipid infusion in group I monkeys or androstenedione treatment in group II monkeys.

Androstenedione (mg/kg per h) 0.0 0.0 0.0 0.0 0.0 + 0 0.30 0.30 0.30 0.30 0.30 _+0

All androstenedione-treated monkeys (group II) underwent Cesarian section after 3.0 + 0.4 (mean SEM) induced nocturnal switches in myometrial activity patterns from contractures to labor-type contractions. All intralipidinfused (group I) animals were maintained until spontaneous onset of term labor-type contractions. At term in three of the controls, two further arterial samples (1 ml each) were taken for calculations of term PAF-AH specific activity at lights-off and 4.5 h after lights-off the last night before Cesarian section or live delivery (162 + 4 dGA, mean + SEM, n = 4; three animals underwent Cesarian section and one animal delivered). To validate an increase in maternal plasma androstenedione concentration following maternal androstenedione treatment, 1 ml of arterial blood was taken from group II monkeys during baseline and after 32-34 h of androgen infusion. All arterial blood samples for hormone analyses were collected using aseptic techniques and transferred into chilled polypropylene collection tubes. These were centrifuged at 4°C at 1200 X g for 5 min. Plasma was removed, aliquoted, flash-frozen, and stored at -20°C until assayed. Remaining red blood cells from the centrifuged tubes were resuspended in heparinized saline (25 IU/ml) and returned into the animal's arterial circulation. All hormone assays were performed within 2 mo of blood collection. Hormone Analyses E2. The assay for measurement of E2 has been previously validated for use in the rhesus monkey, and the assay procedure has been described in detail [18]. Maternal plasma E2 was measured in duplicate 200-1l plasma aliquots. The lower limit of detection of the assay (90% B/Bo ) was 12 pg/ml. The intraassay and interassay coefficients of variation were 8.5% and 7%, respectively. Androstenedione. A commercial assay kit (#TKAS2; Diagnostic Products Corporation, Los Angeles, CA) for human plasma androstenedione was used for analysis of rhesus plasma. Androstenedione recovery was determined by mixing rhesus plasma pool 1:1 with androstenedione of known concentration in human plasma (0.1, 0.2, 0.5, 1.0, 3.0, and 6.0 ng/ml). All androstenedione-spiked samples were diluted 1:1 with kit zero calibrator to ensure measurements from the linear region of the standard curve. Recovery was 98.4 +_7.6%. Parallelism was demonstrated by serial dilution of rhesus plasma in kit zero calibrator. Intraassay coefficient of variation was 3.5% for a rhesus quality control sample of 4.5 ng/ml (n = 3), and all samples of any particular animal were run within the same assay. Assay lower limit of detection (90% B/B) was 0.15 ng/tube,

PAF ACETYLHYDROLASE AND MYOMETRIAL CONTRACTIONS IN MONKEYS which represents 0.18 ng/ml (n = 4) when 1 ml of plasma was extracted. PAF-AH. Acetylhydrolase activity was determined by measuring the release of [3 H]acetate from PAF according to the method of Stafforini et al. [19]. Substrate was prepared in 0.1 M HEPES, pH 7.2, such that 100 M PAF (Sigma P9525) was made 1.125 1jCi/ml with 1-O-hexadecyl-2-acetyl-sn-glyceryo-3-phosphorylcholine, 1-O-acetyl[3H]N (NET-910, 10.0 Ci/mmol; New England Nuclear, Boston, MA). This working solution contained 10000 counts/min per 10 l and was stored at -20 0 C for up to 1 wk. The specificity of acetylhydrolase activity for the 2-acetyl moiety was determined by substituting PAF (1-0[3 H]alkyl (90 Ci/mmol, TRK.741; Amersham Corp., Arlington Heights, IL) for acetate-labeled PAF in the substrate preparation. The assay is based on the specific hydrolysis of acetate from PAF The acetate is recovered chromatographically. The tritiated 1-O-alkyl PAF was used as a control to confirm that the radioactivity recovered in the routine procedure was due to acetate and not to unhydrolyzed PAF (see [20]). Assays included a 10-xl sample volume containing a maximum of 40 Vig protein to which 40 l of substrate solution was added. To maintain zero-order kinetics, samples were diluted if necessary with HEPES buffer to insure that - 7.5% of total substrate was hydrolyzed during incubation [19]. Reactions were incubated at 37°C and stopped after 15 or 30 min by adding 50 l of 10 M acetic acid followed by 1.5 ml of 0.1 M sodium acetate. Each reaction mixture was applied to an octadecyl (C1 8) silica gel cartridge (Baker Chemical Co., Philipsburg, NJ), and the effluent was collected. Columns were washed twice with 1.5-ml aliquots of 0.1 M sodium acetate, and the combined effluent and washes were analyzed by liquid scintillation spectrometry. Unincubated sample preparations were also applied to columns to confirm that sample protein did not interfere with retention of unhydrolyzed substrate. Reactions in these preparations were stopped immediately after the addition of substrate to sample. Analysis of Myometrial Activity Recording of the myometrial EMG was performed with a computer-based data acquisition system. The signal was sampled at 32 Hz, integrated, and the average over 8 sec was digitized and stored as one data point reading with a time signal [21]. A switch in myometrial EMG activity to contractions was determined visually. A switch was defined if at least six contractions, each lasting ca. 1 min, occurred sequentially and the bout of contraction activity lasted at least 30 min. Individual contractions were counted visually for a 7-h period starting 2 h before the onset of darkness in the animal's environment. We have previously reported that a switch in myometrial activity patterns from contractures to contractions occurs around the onset of darkness [22, 23] and that maximal contraction activity occurs 2-4 h after the onset of darkness [23]. No myometrial EMG data were analyzed before 5 days postinstrumentation. Data and Statistical Analyses Arterial blood gases and pH, maternal plasma E2, plasma PAF-AH specific activity, and number of myometrial contractions are expressed as the means + SEM of each of the experimental periods: baseline, Day 1, and Day 3 for monkeys in groups I and II. There was no variation in maternal

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plasma concentrations of E2 or maternal plasma PAF-AH specific activity between the two blood samples taken at lights-off and 4.5 h after lights-off in each animal for each experimental period. Therefore, in each animal for each experimental period, the mean value obtained from these two samples was taken. Similarly, maternal plasma PAF-AH specific activity calculated from samples taken from group I animals after spontaneous onset of term labor-type contractions is exSEM. pressed as the mean Data for statistical analyses that were not normally distributed were log-transformed. Repeated-measures ANOVA with Dunnett's multiple comparison test was used to compare all measured variables between baseline and Day 1 or Day 3 for both groups of animals. Significance was accepted when p < 0.05. RESULTS Arterial Blood Gases and pH Mean arterial blood gases and pH values (mean + SEM) were similar for intralipid-(pH = 7.44 + 0.02; Pa CO 2 = 2.5) and androstenedione34.7 + 0.8; Pa 02 = 111.5 treated (pH = 7.44 ± 0.02; Pa CO 2 = 35.7 1.4; Pa 02 3.4) monkeys during the baseline period. Fur= 99.7 thermore, there were no differences in any of these variables measured after 1 day of intralipid infusion (pH = 7.45 ± 0.01; Pa CO2 = 32.7 + 0.9; Pa 02 = 113.1 + 3.3) and 3 days of intralipid infusion (pH = 7.43 0.01; Pa CO 2 = 34.6 0.8; Pa 02 = 110.2 ± 1.2), or after 1 day of androstenedione treatment (pH = 7.46 ± 0.01; Pa CO 2 = 35.9 + 1.4; Pa 02 = 107.4 ± 3.1) and 3 days of androstenedione treatment (pH = 7.44 + 0; Pa CO2 = 34.4 + 2.3; Pa 02 = 108.1 7.2). Endocrine Measurements Baseline maternal plasma androstenedione concentrations in group II monkeys were below the detection limit of the assay. However, after 32-34 h of treatment, maternal plasma androstenedione increased to 2.9 ± 0.8 ng/ml. This circulating concentration of maternal plasma androstenedione is similar to that previously reported for pregnant, untreated rhesus monkeys at term [24], and for late-gestation women [25]. In group I monkeys, maternal plasma E2, plasma PAFAH specific activity, and number of myometrial contractions following 1 and 3 days of intralipid infusion were unchanged from baseline (Fig. 1). In contrast, an increase in maternal plasma E2 accompanied by an increase in the number of myometrial contractions was measured by 3 days of androstenedione treatment. This increase in maternal plasma E2 in group II monkeys is similar to that measured in spontaneous term labor in the monkey [26]. However, despite increased maternal plasma E2 concentrations and established myometrial contractions, maternal plasma PAF-AH specific activity remained unchanged after 1 and 3 days of androstenedione treatment (Fig. 1). To address the possibility that androstenedione-induced myometrial contractions may be mediated via different mechanisms than those mediating myometrial contractions occurring at term, additional plasma samples were taken from group I monkeys after spontaneous onset of term labor contractions for calculation of plasma PAF-AH specific activity. The mean maternal plasma PAF-AH specific activity calculated in these samples was not different from baseline

GIUSSANI ET AL.

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=

>.0 0.24 -

A I

0.12

a.

I

-

o- II 180U.U

180

B

m

BL

D1

ii

M 900 90

0 120 0

0

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0

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FIG. 1. Effect of intralipid (open bars, n = 4) and androstenedione (closed bars, n = 4) on maternal plasma PAF-AH specific activity (A), maternal plasma concentrations of E, (B), and number of myometrial contractions (C) during baseline (BL), Day 1 (D), and Day 3 (D3) of the experimental protocol. * p < 0.05 vs. BL, ANOVA.

(0.17 + 0.04 nmol PAF hydrolyzed/min per mg protein [mean + SEM, n = 3]). DISCUSSION We have previously hypothesized that the increase in placental estrogen production occurring at term in the pregnant sheep [27], pregnant nonhuman primate [26, 28], and pregnant women [29, 30] plays a pivotal role in regulating the normal physiological preparturient, nocturnal switch that occurs in myometrial activity patterns from contractures to contractions which leads to delivery [16]. In primate pregnancy, androgens serve as the major precursors for placental estrogen synthesis [31]. According to the above hypothesis, estrogens act as the central link between androgen production and the switching of myometrial activity patterns. In support of this hypothesis, 48-h i.v. infusion of physiological doses of androstenedione to pregnant monkeys precipitates increases in maternal plasma E2 concentrations and switches myometrial activity patterns from contractures to contractions [15]. The increase in maternal plasma E2 following androstenedione treatment is similar to that measured in pregnant monkeys undergoing spontaneous term labor [26]. In addition, sustained administration of androstenedione to pregnant monkeys promotes sustained elevations in maternal plasma E2 concentrations, persistent myometrial contractions, fetal membrane rupture, cervical dilatation, and live, unassisted delivery of the monkey fetus [31]. The mechanism by which androstenedione promotes myometrial contractions has not been fully delineated. Current evidence strongly indicates that in the primate at term, estrogen-stimulated maternal oxytocin production and oxytocin receptor activity contribute significantly to the mechanism(s) mediating the spontaneous switch in myometrial

activity to contractions. Increased estrogen will promote production of maternal hypothalamic oxytocin [32] and uterine oxytocin receptors [33], as well as oxytocin production by intrauterine tissues [34, 35]. We have previously demonstrated that androstenedione will elevate maternal plasma oxytocin to term concentrations in the pregnant rhesus monkey [31, 36]. In addition, a close temporal association exists between the nocturnal transition to myometrial contractions and the nocturnal increase in maternal plasma oxytocin concentrations both in pregnant monkeys after androstenedione-induced labor [36] and in pregnant monkeys undergoing spontaneous term labor [18, 37]. Further, administration of oxytocin antagonists will prevent androstenedione-induced myometrial contractions [38] and abolish spontaneous term myometrial contractions in the pregnant monkey [18, 37]. Despite strong evidence for the involvement of oxytocin in mediating both androstenedione-induced myometrial contractions and spontaneous contractions occurring at term, alternative hypotheses have arisen since the discovery of PAF in the early 1970s and the subsequent numerous associations of PAF with the initiation and maintenance of primate parturition (see [] for review). One hypothesis is based on findings suggesting that increased PAF activity leads to stimulation of arachidonic acid and its prostaglandin metabolites via activation of phospholipase A (see [39]). This hypothesis states that during early pregnancy, high plasma PAF-AH activity present in the decidua plays a protective role by preventing PAF from reaching the myometrium and stimulating its contraction (see [8]). However, during the latter stage of gestation when biosynthesis of PAF is dramatically increased in the fetus [5, 6], the capacity of maternal plasma to inactivate PAF is significantly reduced because of a concomitant decrease in acetylhydrolase activity (see [8]). In support of this hypothesis, it is well established that large amounts of prostaglandins are produced by uterine and intrauterine tissues during primate parturition (for review, see [40]) and that prostaglandins are potent uterotonins [41]. It has been demonstrated that PAF produces marked stimulation of prostaglandin E, formation by intact fetal membranes [42]. E, treatment of adult female rats and pregnant rats produces a fall, but in contrast dexamethasone treatment produces an increase, in peripheral PAF-AH concentrations [7]. This evidence suggests a facilitatory role of estrogens and an inhibitory role of glucocorticoids in the regulation of PAF-AH. It is of further interest that administration of androgen precursors for placental estrogen synthesis led to premature labor and delivery in the pregnant monkey [15, 31, 36, 38] and that dexamethasone treatment of the pregnant monkey prolonged gestation [43, 44]. Therefore, it is reasonable to hypothesize that myometrial contractions induced by androstenedione may occur as a result of increased maternal plasma E2, estrogen-induced falls in maternal plasma PAF-AH specific activity, and thus increased PAF activity. Our results do not support this alternative hypothesis since maternal plasma PAF-AH specific activity remained unchanged after androstenedione treatment, despite physiological increases in maternal plasma androstenedione and E2 to term concentrations and the presence of established myometrial contractions. To our knowledge, this is the first study reporting maternal plasma PAF-AH specific activity in the pregnant monkey. It is also the first study during pregnancy in any species to relate PAF-AH measurements to precisely recorded myometrial activity. It could be argued that androstenedione-induced myo-

PAF ACETYLHYDROLASE AND MYOMETRIAL CONTRACTIONS IN MONKEYS

metrial contractions and spontaneous contractions occurring at term are mediated via different mechanisms. According to this view, PAF may still have a role in promoting spontaneous term contractions in the monkey. However, our data cannot be used to support this statement since maternal plasma PAF-AH specific activity also remained unchanged from baseline in monkeys undergoing spontaneous contractions at term. Narahara and colleagues [45] have isolated human decidual macrophages in rather pure form and have shown that they secrete PAF-AH of the plasma type. The presence of cells that secrete PAF-AH activity in the primate decidua may suggest that a local regulatory mechanism could also contribute to the regulation of PAF accumulation. Therefore, it could also be argued that, in addition to inactivation of PAF by plasma PAF-AH in the blood supply to this tissue, PAF-AH secreted by decidual macrophages may also play a role in the local metabolism of PAF within this tissue. Thus, the effect of androstenedione-induced myometrial contractions as mediated via estrogens may be localized within the decidua. Finally, it is possible that androstenedione administration may have confounded the resulting maternal plasma PAFAH specific activity in the pregnant monkey via its androgenic, in addition to its estrogenic, effects. However, this is unlikely since administration of testosterone to nonpregnant rats was without effect on PAF-AH measurements [7]. In conclusion, our results do not support the hypothesis that premature myometrial contractions induced by intravenous androstenedione treatment of the pregnant monkey are accompanied by a fall in maternal plasma PAF-AH specific activity. These results do not dismiss the possibility that androstenedione may induce changes in decidual PAFAH activity or that PAF may have a role in mediating premature labor under pathological conditions, such as that resulting from infection. The involvement of endotoxins and cytokines in premature rupture of the membranes and premature delivery has recently been stressed [46-48], and their interactions with PAF and PAF-AH have been recognized in a number of pathophysiological conditions (see [49] for review). ACKNOWLEDGMENT

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We are grateful to Karen Moore for her assistance with this manuscript. 21.

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