Estrogen effects on platelet-activating factor and platelet-activating factor acetylhydrolase activity in rat uterus during the late stages of pregnancy.

The platelet-activating factor (PAF) concentration of the uterus spontaneously increased during pregnancy. When 17alpha-ethynylestradiol (0.25 mg/kg) was administered subcutaneously to pregnant rats for 3 days starting on Day 17 of pregnancy, some rats delivered prematurely on Day 20. However, none of the vehicle-treated (80% dimethylsulfoxide and 20% ethanol) pregnant rats delivered prematurely. The PAF concentration of the uterus in pregnant rats treated with 17alpha-ethynylestradiol was significantly higher than in those treated with vehicle on Days 19 and 20. On the other hand, the specific activity of uterine PAF-acetylhydrolase (PAF-AH) in pregnant rats treated with 17alpha-ethynylestradiol was significantly lower than in those treated with vehicle on Days 19 and 20, and the plasma PAF-AH activity in pregnant rats treated with estrogen was also significantly lower than in treated with vehicle on Days 18, 19, and 20. These findings indicate that estrogen increases PAF concentrations in the rat uterus, and this was correlated with a decrease in PAF-AH in the uterus and plasma. The increase in PAF concentrations in the uterus may be related to premature delivery and labor caused by PAF's known effect on myometrial contraction.     

Effect of trifluoperazine, a calmodulin antagonist, on prostaglandin output from the guinea-pig uterus

Trifluoperazine, a calmodulin antagonist, inhibited the A23187-induced increase in outputs of prostaglandin (PG) F-2 alpha and 6-oxo-PGF-1 alpha from the Day 7 and Day 15 guinea-pig uterus superfused in vitro. The basal outputs of, and the arachidonic acid-induced increase in outputs of PGF-2 alpha, PGE-2 and 6-oxo-PGF-1 alpha from the guinea-pig uterus were not inhibited by trifluoperazine. In contrast, indomethacin inhibited A23187-stimulated, arachidonic acid-stimulated and the basal outputs of PGs from the guinea-pig uterus, indicating that trifluoperazine was not inhibiting cyclo-oxygenase. Since the action of A23187 is dependent upon extracellular Ca2+, the present findings provide evidence that calmodulin is involved in Ca2+-induced increases in uterine PG output from the guinea-pig uterus. Trifluoperazine, but not indomethacin, inhibited A23187-induced contraction of the guinea-pig uterus, which is consistent with calmodulin being involved in smooth muscle contraction. Arachidonic acid treatment did not contract the guinea-pig uterus. These findings indicate that PGs are not involved in the contraction induced by A23187. Other findings of interest were (i) trifluoperazine caused a small, sometimes significant (P less than 0.05), increase in uterine PG output, (ii) exogenous arachidonic acid failed to increase PGF-2 alpha output from the Day 15 uterus in contrast to the stimulant action of A23187, and (iii) exogenous arachidonic acid caused a fairly large increase in uterine PGE-2 output in contrast to the small effect with A23187.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1-100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC50 was 1 nM. The response to oxytocin (0.01-10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desensitization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1–100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC₅₀ was 1 nM. The response to oxytocin (0.01–10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desentization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

Mechanism of action of platelet-activating factor on guinea-pig lung parenchyma strips

The contribution of thromboxane A2 to platelet-activating factor (PAF)induced contraction of guinea-pig lung parenchyma strips (GPLPS) was investigated using an experimental design that allowed us to record the contractions of the tissues in parallel with the determination of thromboxane B2 (TXB2) levels in the organ baths by enzyme immunoassay. It was found that the first injection of PAF induced the contraction of GPLPS and the release of TXB2. Following subsequent additions of PAF to the same tissue, the contractile response was abolished but TXB2 levels were not significantly reduced. Pretreatment of the tissue with the thromboxane synthetase inhibitor OKY-046 (3.5, 170, and 350 microM) strongly inhibited the release of TXB2 but had no effect on the contraction of the tissues induced by PAF. The mechanism of PAF-induced contraction of GPLPS was further investigated using several drugs that interfere with arachidonic acid metabolism. It was found that pretreatment of the tissues with the cyclooxygenase and thromboxane synthetase inhibitors indomethacin (2.8, 28, and 56 microM) and OKY-046 (170 microM) or with the thromboxane antagonist SKF-88046 (1.25 and 12.5 microM) had no significant effect on the contractile response to PAF. The compound L-655,240 (2.5, 25, and 50 microM), which acts simultaneously as an antagonist of thromboxane and inhibitor of lipoxygenase, significantly reduced GPLPS contractions induced by PAF. Another lipoxygenase inhibitor, nordihydroguaiaretic acid (33 microM), and the inhibitor of both pathways of arachidonic acid metabolism, BW775c (110 microM), both reduced PAF-induced contractions of GPLPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.     

Effects of hydrocortisone, oestradiol and progesterone on A23187-stimulated prostaglandin output from the guinea-pig uterus superfused in vitro

Hydrocortisone (10 μg/ml) had no effect on the basal outputs and A23187-stimulated outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α from the Day 15 guinea-pig uterus superfused . These findings indicate that the high output of PGF_2α from the guinea-pig uterus during the last one-third of the oestrous cycle is not modulated by the adrenal glucocorticoid hormones. Progesterone (10 gmg/ml) had no effect on the A23187-induced increases in PG output from the Day 15 guinea-pig uterus. However, oestradiol (10 gmg/ml but not 1 μg/ml) significantly reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by A23187 from the Day 15 guinea-pig uterus, without affecting basal PG outputs. The increase in uterine tone induced by A23187 in the Day 15 guinea-pig uterus was reduced by 20–50% by oestradiol (10 μg/ml). The addition of oestradiol (10 μg/ml) and progesterone together (10 gmg/ml) produced the same effects on the Day 15 guinea-pig uterus as oestradiol alone. Oestradiol (10 μg/ml) also reduced the A23187-induced increases in PG output from the Day 7 guinea-pig uterus, but did not reduce the increase in uterine tone. Oestradiol (10 gmg/ml) reduced the increases in outputs of PGF_2α, PGE₂ and 6-keto-PGF_1α induced by exogenous arachidonic acid from the Day 7 and Day 15 guinea-pig uterus. Previous studies have shown that oestradiol is not a cyclo-oxygenase inhibitor. The present findings suggest that oestradiol, at a relatively high concentration, may interfere with the access of arachidonic acid to the cyclo-oxygenase enzyme. This action of oestradiol may explain its anti-luteolytic action when administered to guinea-pigs in large doses after Day 9 of the cycle.     

Effects of hydrocortisone, oestradiol and progesterone on A23187-stimulated prostaglandin output from the guinea-pig uterus superfused in vitro

Hydrocortisone (10 micrograms/ml) had no effect on the basal outputs and A23187-stimulated outputs of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha from the Day 15 guinea-pig uterus superfused in vitro. These findings indicate that the high output of PGF2 alpha from the guinea-pig uterus during the last one-third of the oestrous cycle is not modulated by the adrenal glucocorticoid hormones. Progesterone (10 micrograms/ml) had no effect on the A23187-induced increases in PG output from the Day 15 guinea-pig uterus. However, oestradiol (10 micrograms/ml but not 1 microgram/ml) significantly reduced the increases in outputs of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha induced by A23187 from the Day 15 guinea-pig uterus, without affecting basal PG outputs. The increase in uterine tone induced by A23187 in the Day 15 guinea-pig uterus was reduced by 20-50% by oestradiol (10 micrograms/ml). The addition of oestradiol (10 micrograms/ml) and progesterone together (10 micrograms/ml) produced the same effects on the Day 15 guinea-pig uterus as oestradiol alone. Oestradiol (10 micrograms/ml) also reduced the A23187-induced increases in PG output from the Day 7 guinea-pig uterus, but did not reduce the increase in uterine tone. Oestradiol (10 micrograms/ml) reduced the increases in outputs of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha induced by exogenous arachidonic acid from the Day 7 and Day 15 guinea-pig uterus. Previous studies have shown that oestradiol is not a cyclo-oxygenase inhibitor. The present findings suggest that oestradiol, at a relatively high concentration, may interfere with the access of arachidonic acid to the cyclo-oxygenase enzyme. This action of oestradiol may explain its anti-luteolytic action when administered to guinea-pigs in large doses after Day 9 of the cycle.     

Production and effects of platelet-activating factor in the rat brain

The synthesis of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in rat brain was evaluated. Extracted PAF was characterized using standard HPLC and TLC techniques, and by correlation of its bioactivity with the acetylation state of the 2-position of the molecule. PAF was quantified by bioassay, its ability to cause [3H]serotonin release from washed rabbit platelets. The low basal level of PAF (0.25 +/- 0.15 pmol/g wet wt., mean +/- S.E.) in the brain of the intact rat was greatly increased by intraperitoneal injection of the chemoconvulsant drugs picrotoxin or bicuculline, to levels of 10.68 +/- 2.18 and 4.97 +/- 0.75 pmol/g wet wt., respectively. Electroconvulsion also increased brain PAF, to 1.76 +/- 0.30 pmol/g wet wt. Equivalent experiments using bicuculline in the isolated perfused rat brain yielded qualitatively similar results, indicating that the production of PAF in the brain is independent of systemic metabolism. When a 32P-labeled nerve-ending (synaptosome) preparation from rat brain was challenged with synthetic PAF (denoted AGEPC) at 0.1 nM concentration, responses were observed consistent with accelerated turnover of polyphosphoinositides. AGEPC also caused an increase in the Na+-Ca2+ exchange of synaptic membrane vesicles. Furthermore, AGEPC infused into the vasculature of the isolated perfused rat brain caused changes consistent with an increase in blood-brain barrier permeability, although AGEPC did not itself significantly penetrate the blood-brain barrier. It is concluded from these studies that PAF is synthesized within the rat brain in response to convulsant stimuli and that one of its effects is to accelerate synaptic polyphosphoinositide turnover. In addition, circulating PAF can influence blood-brain barrier permeability without itself penetrating the blood-brain barrier.     

The effects of platelet-activating factor and platelet-derived compounds on bovine luteal cell progesterone production

This study was conducted to characterize bovine platelets with respect to serotonin (5-HT) concentration and platelet-activating factor (PAF)-activation and to examine the in vitro effects of PAF and platelet-derived compounds on bovine luteal progesterone (P4) production. The concentration of 5-HT in platelets, as determined by high-performance liquid chromatography, was 538.8 +/- 40.83 ng/1 x 10(8) platelets. Based on a circulating platelet concentration range of 2.3 x 10(8) 5.8 x 10(8) platelets/ml, the circulating concentration of 5-HT would be approximately 1239-3125 ng/ml of blood. Bovine platelets were found to aggregate in response to PAF (1-40 ng/0.5 ml), with maximal aggregation occurring at 20-40 ng/0.5 ml. Coincubation of luteal cells with platelets (1 x 10(7)-4 x 10(8] enhanced luteal P4 production (p less than 0.05). Addition of the 5-HT receptor antagonist mianserin blocked the platelet-induced increases in P4 (p less than 0.05). Preincubation of platelets with indomethacin did not alter the production of P4 (p greater than 0.05), nor did the addition of propranolol (p greater than 0.05). Platelet-derived growth factor at 8 and 16 ng/ml enhanced basal P4 production (p less than 0.05) but had no effect on the responsiveness of luteal cells to luteinizing hormone (LH) (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of endotoxin on platelet-activating factor synthesis in cultured rat glomerular mesangial cells

Platelet-activating factor (PAF), a potent vasoactive phospholipid, may contribute to acute renal failure and septic shock accompanying endotoxemia. Rat glomerular mesangial cells in culture synthesize PAF and contract after the addition of PAF. We thus investigated the potential of mesangial cells to respond to Escherichia coli lipopolysaccharide endotoxin with enhanced PAF synthesis in vitro. The mesangial cells were incubated with [3H]acetate, substrate for lyso-PAF: acetyl-CoA acetyltransferase, and endotoxin at different concentrations for various periods of time at 37 degrees C. Lipids were extracted and PAF was isolated by thin-layer chromatography. Endotoxin stimulated PAF generation in a time- and dose-related manner. Whereas most of the PAF was associated with the cells, endotoxin more than doubled the amount of PAF released into the extracellular medium as compared to control. Furthermore, the PAF-like material obtained from endotoxin-stimulated mesangial cells irreversibly aggregated washed rabbit platelets. This effect was lost after alkaline methanolysis and was totally blocked by L-652,731, a specific PAF-receptor antagonist. Finally, the PAF-like material exerted a hypotensive effect, which was abolished by L-652,731, when infused intravenously into healthy rats. These data indicate that rat glomerular mesangial cells have the ability to synthesize PAF in response to endotoxin. This suggests that PAF, so generated within the glomerulus, may contribute to acute decrements of glomerular filtration rate in endotoxemia.     

Effect of using calcium-free Krebs' solution on basal and A23187-stimulated prostaglandin output from the day 15 guinea-pig uterus superfused in vitro

The basal outputs of prostaglandin (PG) F_2α and PGE₂ from the Day 15 guinea-pig uterus superfused in vitro were unaffected by omitting Ca²⁺ from the Krebs' solution. In contrast, this omission of Ca²⁺ reduced the basal output of 6-oxo-PGF_1α (which reflects PGI₂ production) from the uterus by an average of 50%. Spontaneous and A23187-stimulated contractions of, and the stimulation by A23187 of PGF_2α, PGE₂ and 6-oxoPGF_1α outputs from the Day 15 guinea-pig uterus were all abolished by superfusing the tissue with Krebs' solution lacking Ca²⁺. It is concluded that the basal output of 6-oxo-PGF_1α, the occurrence of spontaneous contractions, and the effects of A23187 on PG output and contractility of the Day 15 guinea-pig uterus are dependent on extracellular Ca²⁺. However, the increase in PGF_2α output from the guinea-pig uterus on Day 15 compared to days much earlier in the cycle is apparently not dependent upon extracellular Cat+. The implications of these findings regarding the biochemical mechanisms involved in the increased synthesis of PGF_2α (the uterine luteolytic hormone) by the guinea-pig endometrium during the last one-third of the cycle are discussed.     

Alkyl chain homologs of platelet-activating factor and their effects on the mammalian heart

Platelet-activating factor (PAF; AGEPC) is a potent negative inotropic and coronary-vasoconstricting agent. Minor structural alterations in the PAF molecule are known to greatly affect its biological activity; thus, we have investigated the effects of selected synthetic saturated and unsaturated alkyl chain PAF homologs on the isolated guinea pig heart. The rank order of potency for the negative inotropic effect was C16:0- greater than C18:1- greater than beef-heart AGEPC greater than C15:0- greater than C18:0- greater than C14:0-AGEPC; the rank order for the coronary-vasoconstricting effect was C16:0- approximately C18:1- approximately beef-heart AGEPC greater than C15:0- greater than C18:0- approximately C14:0-AGEPC. With the exception of C16:0- and C18:1-AGEPC, the relative potencies for the cardiac and coronary effects of the alkyl chain AGEPC homologs did not correlate well with their relative potencies in stimulating rabbit platelets and human neutrophils. The differences in the rank order of potency for these AGEPC homologs suggest the presence of species and/or target cell PAF receptor heterogeneity.     

Paracrine interactions between platelet-activating factor and prostaglandins in hormonally-treated human luteal phase endometrium in vitro

Stromal cells and epithelial glands were separated after enzymic digestion of specimens obtained from 27 women at hysterectomy or endometrial biopsy during the luteal phase, and then cultured to confluence in vitro. PGE release into the culture medium (mean +/- s.e.m.: ng/mg protein/24 h) from gland cell cultures was not changed by oestradiol (17.6 +/- 1.3 for control and 25.5 +/- 2.8 for oestradiol, respectively). However, in the presence of oestradiol, PAF (5 ng/ml) significantly elevated PGE release to 44.2 +/- 5.8. No stimulation was observed in the presence of progesterone. Stromal cell medium had no effect on PGE release in gland cell cultures. PGE release was always much lower in stromal cell cultures than in glands (control: 4.7 +/- 0.6). PAF stimulated PGE release in the presence of oestradiol in these cells also; gland cell medium was without effect. In co-cultures of glandular and stromal cells, PGE release was more similar to that seen in gland cell cultures, with PAF being stimulatory under the influence of oestradiol. PGF release into the medium from the same gland cell cultures was significantly elevated by hormonal treatment, being greatest (62.0 +/- 11.3) with oestradiol alone, and was strongly inhibited in all wells by addition of PAF and stromal cell medium. In stromal cell cultures without hormonal addition, PGF levels (15.0 +/- 2.4) were similar to those seen in glands (18.1 +/- 3.1), and no stimulation was achieved by oestradiol (29.6 +/- 5.9). PAF was inhibitory on PGF release, while gland cell medium was without effect. Co-cultures gave PGF values generally similar to those of stromal cells; oestradiol was again stimulatory (55.0 +/- 9.3). PAF was significantly inhibitory in the presence of oestradiol. PAF (mean +/- s.e.m.: pmol/mg protein/24 h using a platelet serotonin release assay) in stromal cells was significantly increased from control [M199 alone] (0.31 +/- 0.12) by progesterone (1.00 +/- 0.17). Addition of PGE-2 (7.5 ng/ml) to progesterone-treated wells further increased PAF concentration (5.34 +/- 0.09), but was without effect in wells receiving oestradiol alone. Wells exposed to both hormones exhibited an intermediate response. Similar results were obtained with addition of gland cell culture medium, presumably due to its endogenous PGE content. In co-cultures, PAF concentrations were significantly elevated by progesterone alone (4.78 +/- 0.78) or when combined with oestradiol (2.38 +/- 0.51), but not by oestradiol alone. Treatment with PGE-2 caused no additional stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential production of prostaglandins within the human uterus

The ability of broken cell preparations of human endometrium, myometrium and a mixture of endometrium and myometrium to convert 14C arachidonic acid to prostaglandins (PG's) was compared. Endometrium metabolished arachidonic acid predominantly to a mixture of PGF2 alpha and PGE2. A similar weight of myometrium showed relatively little activity, the major product identified was 6 oxo PGF1 alpha. However, a combination of endometrium and myometrium showed an enhanced conversion of arachidonic acid to 6 oxo PGF1 alpha associated with a decreased production of PGF2 alpha and PGE2. This suggests that human endometrium and myometrium differ in their ability to metabolize arachidonic acid and in their ability to convert the endoperoxides formed, to PG's.     

Protein tyrosine sulfation in guinea-pig uterus: estradiol and progesterone effects

Tyrosine sulfation was studied in guinea-pig uterus by in vitro labelling with [35S]sulfate, after estradiol-17 beta (E2) and E2 plus progesterone (P) treatment. [35S]Sulfated tyrosine was identified in tissue and secreted proteins, ranged from 9.3 to 21.0% of total protein sulfation and was higher in secreted proteins than in tissue proteins. Sulfate incorporation into tyrosine increased with hormone treatments. The highest level was found in secreted proteins under the combined effect of E2 plus P. The effect of P may be related to both the increase of cellular uptake of sulfate and the increase of tyrosine sulfation of secreted proteins. These results are consistent with the effect of P on endometrium secretions.     

Existence of endogenous inhibitors of platelet-activating factor (PAF) with PAF in rat uterus

Two kinds of phospholipids in normal rat uterus were found to inhibit the aggregation of washed rabbit platelets induced by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and were named Inhibitor I and Inhibitor II and identified by mass spectrometry. Inhibitor I was a mixture of 1-acyl (16:0, 18:0, 18:1, 18:2, and 20:4)-2-lyso-sn-glycero-3-phosphocholine (acyllyso-GPC) and 1-alkyl (16:0, 18:0, and 18:1)-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC). 16:0 acyllyso-GPC was the most inhibitory, followed by 18:1, 18:2, 20:4, and 18:0 acyllyso-GPCs and 16:0 alkyllyso-GPC. Their IC50 values were in the range of 1-4 X 10(-5) M against the platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC, indicating that they were about 100 times weaker inhibitors than CV-3988. Inhibitor II was a mixture of N-acyl sphing-4-enyl phosphocholine (18:1/18:0, 18:1/20:0, 18:1/24:0, and 18:1/24:2). The most inhibitory of these components were 18:1/20:0 and 18:1/24:0, followed by 18:1/24:2 and 18:1/18:0, and their IC50 values were in the range of 4-5 X 10(-5) M against platelet aggregation induced by the alkylacetyl-GPC. Quantitatively, about 10(5) times higher concentrations of these inhibitors should be necessary to inhibit platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC. In fact, the contents of Inhibitors I and II, respectively, were approximately 10(5) times (4.7 X 10(-2) and 7.1 X 10(-2) mol/mol lipid-phosphorus of the original uterine phospholipids) than that of 16:0 alkylacetyl-GPC (1.4 X 10(-6) mol/mol lipid-phosphorus). The role of alkylacetyl-GPC in normal rat uterus is uncertain, but it coexists in situ with two kinds of endogenous inhibitors, choline containing lysoglycerophospholipids and sphingophospholipids.     

The release of platelet-activating factor from human endothelial cells in culture

The release of platelet-activating factor (PAF) from stimulated human endothelial cells (HEC) cultured from normal term, umbilical cord veins is described. HEC in primary cultures released PAF after challenge with A23187, rabbit anti-human factor VIII (RaHu/FVIII), angiotensin II, and vasopressin. HEC subcultures maintained the ability to release PAF in the presence of A23187 and RaHu/FVIII, whereas the release of PAF in response to angiotensin II and vasopressin was not constant and was reduced. Control cultured, smooth muscle cells derived from umbilical cord veins, previously depleted of endothelial cells, did not release PAF under the above-mentioned stimulation. Plastic-adherent or cultured monocytes released PAF with A23187, but not with RaHu/FVIII, angiotensin II, and vasopressin. The release of PAF from HEC in primary cultures required the presence of extracellular cations and the activation of membrane phospholipase A2. PAF release induced by A23187, RaHu/FVIII, angiotensin II, and vasopressin was unaffected by indomethacin, an inhibitor of cyclooxygenase, which, however, favored the release of PAF from HEC stimulated with thrombin, a stimulus that did not affect HEC in the absence of indomethacin. PGI2 inhibited PAF release from stimulated HEC. The relevance of an acetylation process in the biosynthesis of PAF and HEC was supported by the following evidence: 1) the increase in PAF yield in the presence of sodium acetate and, particularly, of acetyl-CoA; 2) the incorporation of [14C]acetate into PAF molecules; 3) the loss of radioactivity and of biologic activity after treatment with phospholipase A2. These results indicate that HEC in culture are able to release PAF and that metabolic pathways similar to those described for leukocytes are involved.     

Release of acetylhydrolase from platelets on aggregation with platelet-activating factor

Acetylhydrolase, the enzyme which inactivates platelet-activating factor (PAF, 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine), was selectively released from bovine platelets by aggregation with physiological concentrations (0.1-10 nM) of PAF with no cell lysis. The release of the acetylhydrolase paralleled that of serotonin. The acetylhydrolase released was active over a broad pH range (pH 5.4-8.6) and was not affected by Ca2+ (1-4 mM) or EDTA (1-8 mM). The Km value of the enzyme was 4.6 microM. Net specific acetylhydrolase activity recovered in the 130,000 x g supernatant after stimulation with PAF could be determined in the presence of EDTA without the activity of Ca2+-dependent phospholipase A2 which was also released from the cells at the same concentration of PAF. The acetylhydrolase was inhibited competitively by specific PAF antagonists, rac-3-(N-n-octadecylcarbamoyloxy)-2-methyoxypropyl-2-thiazolioe thyl phosphate (CV-3988) and (2RS)-1-O-hexadecyl-2-O-ethyl-3-O-(7-thiazolinoheptyl)-glycerol methanesulfonate (ONO-6040). Their Ki values for the enzyme were 1.17 microM and 0.84 microM, respectively. The release of the enzyme could also be detected when the platelets were aggregated with ADP (2.3 microM) or thrombin (0.5 unit). These results suggest that the enzyme released from the aggregated platelets to the blood plasma may also have a physiological function cooperating with the plasma acetylhydrolase.