In isolated human trophoblastic cells, dopamine (DA) significantly inhibited the angiotensin-II (AII)-stimulated inositol phosphate (IP) accumulation by 44 +/- 8% (EC50, 0.5 +/- 0.2 microM) and human placental lactogen (hPL) release by 85 +/- 5% (EC50, 1.0 +/- 0.8 microM). These effects were blocked by sulpiride, a specific D2 antagonist. On the contrary, scherring 23390 (a specific D1 antagonist) and propranolol (a specific beta-adrenergic antagonist) were ineffective, suggesting that these DA effects are mediated through a DA receptor of the D2 subtype. The mechanism by which DA inhibited AII-stimulated inositol phosphate production implicates a GTP-binding protein sensitive to the islet-activating-protein (IAP), since DA's effects on IP accumulation and hPL release were blocked by this toxin. To further characterize this GTP-binding protein, particulate fractions of placental cells were incubated with [alpha-32P]NAD and IAP. Solubilized extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins of 40 and 41 kDa mol wt were specifically ADP ribosylated. They were probably involved in the DA inhibitory processes, since IAP treatment, known to suppress the effects of DA, also reduced the labeling of these two molecules by around 40%. The effects of AII and DA on hPL release appear to be insensitive to the external calcium concentration, since the results were not significantly different in normal (1.8 mM Ca2+) and low Ca2+ (10(-5) M Ca2+) concentrations. On the other hand, increasing the intracellular concentration of cAMP by adding forskolin did not modify the effect of DA on either IP accumulation or hPL release, suggesting that cAMP is not implicated in hPL release from freshly isolated human trophoblastic cells.

Four labelled ligands, [3H]arginine vasopressin ([3H]AVP), [3H]oxytocin ([3H]OT), [3H]d(CH2)5[Tyr(Me)2]AVP ([3H]VPA), and [125I)d(CH2)5[Tyr(Me)2-Thr4-Orn8-Tyr(NH2)9]OT([125I]OTA)) and nine unlabelled analogues exhibiting enhanced selectivity for rat oxytocin (OT) and vasopressin (VP) receptors were used to characterize OT and VP receptors on myometrial membranes from non-pregnant and pregnant human uteri. On membranes from non-pregnant uteri, [3H]AVP, [3H]VPA, and [125I]OTA labelled with high affinity (Kd values: 3.2, 2 and 0.8 nM, respectively) a major and apparently homogeneous population of sites, the ligand selectivity of which resembled that of rat V1a VP receptors. On membranes from pregnant and non-pregnant uteri, [3H]OT labelled a single population of high-affinity sites that could be distinguished from VP receptors on the basis of ligand selectivity. Several analogues (in particular [125I]OTA) that are highly selective for rat OT receptors exhibited a much less pronounced selectivity for human OT receptors. Experiments with [3H]VPA allowed detection of VP receptors on myometrical membranes from pregnant uteri and confirmed that only OT but not VP receptors increase during pregnancy in humans.

Specific angiotensin II (AII) binding sites were identified and characterized in membranes from human term placenta. The binding of iodinated [125I](Sar1)AII was time-dependent and saturable; it could be totally reversed on addition of unlabelled (Sar1)AII and GTP + NaCl. Scatchard plot analysis of dose-dependent [125I](Sar1)AII binding indicated the presence of a single class of binding sites with an equilibrium dissociation constant of 0.27 +/- 0.06 nM and a maximum binding capacity of 38.4 +/- 4.3 fmol/mg protein. The affinity of five AII analogues for the placental receptor was determined in competitive binding assays; the order of inhibitory potency was: (Sar1)AII greater than (Sar1, Ile8)AII approximately (Sar1, Ala8)AII approximately AII greater than angiotensin I greater than (Des-Phe8)AII. (Sar1)AII did not cause any significant change in the basal or stimulated adenylate cyclase activity. In order to investigate the subunit molecular structure of the placenta AII receptor, membranes were covalently labelled with the photoaffinity ligand [125I](Sar1, (4N3Phe)8)AII. Sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by autoradiography showed that the labelling was specifically incorporated into a protein of Mr 92,000 in the presence or absence of dithiothreitol. It therefore appears that the AII receptor from human placenta has the same binding and pharmacological properties as other well-known AII receptors; by contrast, it is characterized by a significantly higher molecular weight, pointing out that structural differences in AII receptors may exist between species.

We studied the functional significance of the binding of angiotensin-II (AII) to human placentas. Human trophoblastic cell suspensions were prepared by trypsin digestion of minced tissue. Cell incubations with increasing doses of [125I](SAR1)AII, ranging from 0.01-2.5 nmol/L, were carried out for 20 min at 37 C. The results indicated the presence of specific low capacity [4300 +/- 1300 (+/- SE) sites/cell], high affinity (Kd = 0.38 +/- 0.06 nmol/L) binding sites for [125I](Sar1)AII. This binding was specific for AII analogs. When placental cells were preloaded with 40 microCi/mL [3H]myoinositol for 2 h at 37 C, AII stimulation resulted in a dose-dependent increase in inositol phosphate (InsP) production [EC50 = 1.4 +/- 0.4 (+/- SE) nmol/L], as measured by ion exchange chromatography. (Sar1)AII also stimulated InsP production, with an EC50 of 0.3 +/- 0.2 nmol/L. AII-stimulated production of InsP was completely blocked by the antagonist (Sar1,Ala8)AII. AII also stimulated human placental lactogen release from trophoblastic cells in a dose-dependent fashion. The EC50 was 18 +/- 9 pmol/L, and the stimulation was blocked by (Sar1,Ala8)AII, as found for AII-stimulated InsP production. These results suggest that stimulation of human placental lactogen release by AII may be mediated by activation of phospholipase-C, which, in turn, produces phosphoinositide breakdown. The results, therefore, provide evidence of a physiological role for the renin-angiotensin system within the human placenta.

Chemical ionization and fast atom bombardment mass spectra of a series of phospholipids related to PAF-acether are reported. The usefulness of these methods towards their structural determination is discussed. The mass spectra of sphingomyelin are also examined.

Platelet-activating factor (PAF-acether), a mediator of anaphylaxis and inflammation, is a 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine. Some analogs of PAF-acether were prepared by total synthesis or by partial synthesis from commercially available phospholipids and tested for platelet aggregating activity. This study indicated that the ether linkage at the position 1 of sn-glycerol and the short acyl chain at position 2 are structural features required for biological activity.

Platelet-activating factor (PAF) is a phospholipid mediator, released by basophils, macrophages and neutrophils under immunological and non immunological stimuli. It aggregates platelets and liberates their vasoactive contents. We studied the "spontaneous" release of PAF from hog blood leukocytes : optimal conditions were 22 degrees C, pH 9.5 in BSA and Ca2+-containing Tyrode's. This release was inhibited by the Ca2+-chelating agent, EDTA, and by the phospholipase A2 inhibitor, bromophenacyl bromide. Disruption of the cells did not yield PAF, indicating that it is not a "preformed" mediator. A preparative procedure for the extraction and purification of bulk quantities of PAF was developed. Purification was performed by silicic acid columns followed by high pressure liquid chromatography. The active fraction was eluted between sphingomyelin and lysophosphatidylcholine. The PAF purest fractions were still contaminated with these phospholipids as shown by thin layer chromatography and chemical ionization mass spectrometry. PAF activity was not affected by treatment with diazomethane, acetylation or hydrogenation. Our results combined with those obtained from our previous studies of the PAF structure using specific phospholipases indicate that PAF is a glycero-phospholipid devoid of ester function at position 1. This allowed us to establish precise criteria to distinguish PAF from other aggregating agents.

PLATELET aggregation is mediated by at least three distinct mechanisms1,2. The first involves the release of ADP and is inhibited by its conversion to ATP by the combination of creatine phosphate and creatine phosphokinase (CP/CPK). The second is mediated by metabolites of arachidonic acid, particularly thromboxane A2 (TXA2), and is blocked by aspirin or indomethacin, inhibitors of the arachidonate cyclo-oxygenase pathway3. It has been postulated that a third mechanism must exist, as neither CP/CPK nor aspirin, alone or combined, can inhibit aggregation induced by high concentrations of thrombin or the calcium ionophore A23187 (refs 1, 2, 4). Antigenic challenge of IgE-sensitised basophils releases a platelet-activating, factor (PAF), probably a 1-lysophosphatidylcholine5. PAF has a potent action on rabbit6,7 and human8,9 platelet aggregation and release which is independent of the cyclo-oxygenase arachidonate pathways6,10,11. We have also obtained PAF from A23187-stimulated rat peritoneal12 and alveolar (J.B., B. Arnoux and D. Duval, in preparation) macrophages. Moreover, thrombin and ionophore-induced platelet aggregation and the accompanying stimulation of phospholipase A2 (refs 13, 14) are inhibited by the phospholipase inhibitor bromophenacyl bromide (ref. 15 and B.B.V., F. Fouque and M.C., in preparation), suggesting that the third mechanism of platelet aggregation might involve a lipid mediator. These findings prompted us to investigate whether platelets can form and release PAF in experimental conditions in which the ADP and TXA2 pathways are fully blocked. We report here that this is indeed the case, and suggest PAF as a likely candidate for mediating the ‘third pathway’ of platelet aggregation.

Platelet aggregation induced by low concentrations of ionophore A23187(I) or thrombin (T) is due to ADP and to metabolites of arachidonic acid(AA) as shown by its inhibition by aspirin and by ADP scavangers. High concentrations of I or T surmount inhibition, thus involving other mediator(s) Platelet-activating factor (PAF)is a 1-lysophospholipid released from macrophages among other cells, in the presence of I. We now show that PAF is released from rabbit platelets during aggregation by I, T and collagen but not by AA nor by PAF itself. Formation and release of PAF by platelets is unaffected by cyclo-oxygenase blockers or by ADP scavengers, but is suppressed by inhibitors of phospholipase A2 activity (dibutyrylcyclic AMP and bromophenacylbromide). Platelet PAF exhibits similar absorption characteristics on silicic acid thin layer and hight pressure chromatography, and sensitivity to N. naja phospholipase A2 as compared to PAF from leukocytes. PAF may be like ADP and thromboxane A2, a Final effector for platelet aggregation and be responsible for the aspirin-resistant third pathway of platelet aggregation.

PLATELET-activating factor (PAF) is a newly described mediator of anaphylaxis which has been implicated in the deposition of immune complexes in acute serum sickness in rabbits1–4. It is released by leukocytes and tissues, most probably by the basophils or mastocytes from various mammalian species, under the influence of various substances known to activate these cells5,6. The study of the chemical nature of the compound responsible for the PAF activity led us to the preparation of large amounts of PAF from hog leukocytes5. Several fractionation steps of the crude material yielded a highly active chloroform-soluble fraction. We usually started these procedures with 1001 of hog blood which yielded 1001 of PAF, the biological activity of which could be detected at the 1-μ1 level. Nevertheless, we have not yet obtained sufficient homogeneous material to apply the current methods for structural analysis (mass spectrometry, magnetic nuclear resonance, hydrolysis reactions and so on). We therefore used different lipases to get some insight into the structure of PAF. We present here the results of these experiments which suggest that PAF is a 1-lyso-glycerophospholipid.

Evidence is presented for the simultaneous release of platelet-activating factor (PAF-acether) and of its deacetylated derivative (lyso-PAF-acether) from hog leukocytes. On the basis of spectroscopy and chemical reactions, the structure of 0-deacetyl-PAF is shown to be 1-aalkylglyceryl 3-phosphorylcholine, an alkyl ether analog of lyso-phosphatidylcholine. Acetylation of lyso-PAF yields a compound with biological activity and chromatographical behavior indistinguishable from those of native PAF. Lyso-PAF may be considered to be either the precursor or the enzymatic degradation product of PAF. The usefulness of chemical ionization mass for structural determination of phospholipids is Platelet-activating factor (PAF) is. a mediator of anaphylaxis and inflammation discovered in the early 1970s (1, 2). It aggregates rabbit, rat, guinea pig, and human platelets and liberates their vasoactive amines. PAF is released by blood leukocytes from various mammalian species and by macrophages, under immunological and nonimmunological stimuli (1-7). PAF was also shown to originate from platelets themselves during aggregation provoked by the ionophore A 23187 (8). Structural analysis of purified PAF preparations led, in 1977, to postulation of a phospholipidic structure for PAF, unique among the mediators of anaphylaxis (9). The low availability of PAF precluded its structural study by the currently used methods. Some insight into the structure of PAF nevertheless was gained by using different lipases and specific chemical treatments. The results obtained indicated that PAF was a 2O-acylglyceryl phosphorylcholine not having an ester group at position 1 (9, 10). The absence of a hydroxyl group at position 1 was evidenced by lack of effect of attempted acetylation of highly purified PAF preparations on either the activity or the chromatographical behavior of PAF (10). During these experiments it was discovered that acetylation of partially purified PAF gave rise to a significant increase in PAF activity and that the increased activity was maintained after thorough purification. This observation suggested that a "precursor" of PAF was present in this preparation and that the ester function present in PAF might be an acetate group. This led to the partial synthesis of a highly active platelet-activating component possessing the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 7019 physicochemical and biological properties of PAF. Because this was obtained by successive methylation, hydrogenation, and acetylation of the commercially available lyso-ethanolamine plasmalogen, the structure 1-O-alkyl-2-O-acetyl-sn-glyceryl 3-phosphorylcholine and the name PAF-acether were proposed (11). The same result has been reached by Hanahan's group (12) for PAF and also by Snyder's group (13) for antihypertensive polar renomedillary lipid (APRL) using choline plasmalogen as starting material. Recently, 1-O-octadecyl-2-O-acetyl-sn glyceryl 3-phosphorylcholine was synthesized and it showed biological properties identical with those of PAF-acether (unpublished data). We herein report the isolation and structural elucidation of the PAF-acether precursor, 1-O-alkylglyceryl 3-phosphorylcholine, which is released along with PAF by hog leukocytes. Acetylation of this glyceryl ether phosphorylcholine, named lyso-PAF-acether, produced a compound with biological activity and chromatogrAphical behavior indistinguishable from those of the native PAF-acether. We also demonstrate here the potential of chemical ionization (CI) mass spectrometry for structural determination of phospholipids. MATERIALS AND METHODS Chemicals. 1-O-Hexadecyl-rac-glycerol 3-phosphorylcholine (I; n = 14) was purchased from Medmark (Grfinwald, Munich). RED-Al [NaAlH2(OCH2-CH2-O-CH3)2, 70% in benzene] was supplied by Aldrich (Beerse, Belgium). 1-0Octadecyl-2-OAcglyceryl 3-phosphorylcholine (II; n = 16), 1-0-octadecyl-rac-glycerol (III; n = 16), and its isopropylidene derivative (IV; n = 16) were gifts of J. J. Godfroid (Universit6 Paris VII). Release of PAF-Acether and Lyso-PAF-Acether. Purification of hog blood leukocytes and extraction of the released lipids were detailed elsewhere (2, 9, 10). Briefly, leukocytes were purified by differential centrifugations and washings and then incubated for 18 hr at pH 9.5. The chloroform-soluble lipids extracted from leukocyte supernatants were then fractionated by silicic acid column chromatography and by highpressure liquid chromatography (HPLC) (9, 10). Abbreviations: PAF, platelet-activating factor; lyso-, devoid of an acyl group; HPLC, high-pressure liquid chromatography; TLC, thin-layer chromatography; CI, chemical ionization; EI, electron impact; amu, atomic mass units; PtdCho, phosphatidylcholine. 7020 Biochemistry: Polonsky et al. Bioassay. Assay for platelet aggregation was performed on washed rabbit platelets as described (9, 10). PAF activity was expressed in arbitrary units; 1 unit is the amount, in Al, of medium necessary for 50% of the maximum aggregation induced by thrombin at 0.1 unit/ml. Chromatographic, Spectroscopic, and Chemical Procedures. Thin-layer chromatography (TLC) of phospholipids was performed on silica gel 60 F 254 plates (Merck, Darmstadt, Federal Republic of Germany) in chloroform/methanol/water, 70:35:7 (vol/vol); TLC of other products was performed on silica gel F 1500 LS 254 plates (Schleicher & Schuell, Dassel, Federal Republic of Germany) which first were washed with ethanol, acetone, and diethyl ether and heated for 1 hr at 600C. Detection was by use of iodine vapor, Dittmer or Dragendorff reagents, or spraying with 50% sulfuric acid followed by heating. HPLC was conducted on a Varian liquid chromatograph, model 8500, equipped with a differential refractometer; Micropak Si-5 columns (Varian) 25 cm X 12.7 mm (8 mm inside diameter) were used and elution was performed with chloroform/methanol/water, 66:50:5 (vol/vol), at a flow rate of 200 ml/hr [at 100 bars (107 Pa)]. Infrared spectra were measured in chloroform solutions with a Perkin-Elmer 297 spectrophotometer. 1H NMR spectra were obtained with a Cameca spectrometer, and chemical shifts are given in ppm with respect to internal Me4Si. Electron impact (EI) mass spectra were recorded on an AEI MS50 instrument. CI mass spectra were measured at 200-260'C and a gas (isobutane) pressure of 0.5-0.6 torr (66.6-80.0 Pa) in an AEI MS9 spectrometer equipped with a CI source (14). Reduction with RED-Al: The reagent (0.5 ml) was added to 5 mg of the natural phospholipid (I) dissolved in 1.5 ml of benzene. The solution was heated at 370C for 1 hr; products were recovered by extraction with diethyl ether after addition