Effects of platelet-activating factor and platelet-activating factor: acetylhydrolase on in vitro post-thaw boar sperm parameters

Cryopreservation of boar sperm compromises fertility after thawing by reducing sperm longevity and inducing acrosome reaction-like changes. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using a modified Westendorf method in which the medium was supplemented with either platelet-activating factor (PAF) or a recombinant platelet-activating factor:acetylhydrolase (PAF:AH; Pafase) before or after freezing. Platelet-activating factor is a phospholipid that is present in boar semen and PAF:AH is the naturally occurring enzyme that converts PAF to biologically inactive Lyso-PAF. Addition of PAF to the cryopreservation medium improved post-thaw motility immediately after thawing and after 3h incubation at 37 degrees C (60.0+/-0.0% and 25.0+/-2.9%; mean+/-S.E.M.) compared to the control sperm (41.7+/-1.7% and 10.0+/-2.9%; P<0.05). Acrosome integrity was higher immediately after thawing and after 3 and 6h incubation at 37 degrees C when sperm were frozen in the presence of Pafase (55.7+/-3.2%, 45.7+/-3.7% and 23.0+/-3.1%), compared to the control sperm (42.7+/-1.5%, 25.7+/-5.7% and 12.3+/-2.7%) and sperm frozen in the presence of PAF (33.0+/-3.7%, 26.3+/-2.2% and 11.7+/-0.3%; P<0.05). Addition of PAF to sperm after thawing improved motility immediately post-thaw (41.6+/-2.6%), compared with addition of Pafase (23.3+/-2.2%) or the control sperm with no supplementation of the medium (26.7+/-2.2%; P<0.05). However, this beneficial effect was lost by 3h post-thaw. Supplementation of boar semen cryopreservation medium with PAF and Pafase appeared to have beneficial effects on the in vitro quality of the sperm post-thaw.     

Allergy-important advances in clinical medicine: platelet-activating factor

The Scientific Board of the California Medical Association presents the following inventory of items of progress in allergy. Each item, in the judgment of a panel of knowledgeable physicians, has recently become reasonably firmly established, both as to scientific fact and important clinical significance. The items are presented in simple epitome and an authoritative reference, both to the item itself and to the subject as a whole, is generally given for those who may be unfamiliar with a particular item. The purpose is to assist busy practitioners, students, research workers or scholars to stay abreast of these items of progress in allergy that have recently achieved a substantial degree of authoritative acceptance, whether in their own field of special interest or another.The items of progress listed below were selected by the Advisory Panel to the Section on Allergy of the California Medical Association and the summaries were prepared under its direction.     

Regulation of platelet-activating factor receptor and platelet-activating factor receptor-mediated biological responses by cAMP in rat Kupffer cells

Treatment of cultured Kupffer cells with the beta-adrenergic agonist isoproterenol (10 microM) for a short period of time (30 min) attenuated the subsequent platelet-activating factor (PAF)-induced arachidonic acid release and cyclooxygenase-derived eicosanoid (e.g. thromboxane B2 and prostaglandin E2) production. This effect of isoproterenol was highly specific since the alpha-adrenergic agonist phenylephrine and the beta-adrenergic antagonist propranolol had no effect on the stimulatory effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC). The inhibitory effect of isoproterenol on the AGEPC-induced arachidonic acid release was demonstrated through the use of a specific beta-adrenergic subtype agonist and antagonist to be mediated by beta 2-adrenergic receptors on Kupffer cells. These inhibitory effects of isoproterenol can be mimicked by dibutyryl cAMP but not by dibutyryl cGMP, suggesting that a cAMP-dependent mechanism is likely involved in the regulatory action of isoproterenol. Ligand binding studies indicated that short term (i.e. 30 min) treatment of the cultured Kupffer cells with either isoproterenol or dibutyryl cAMP had no effect on the specific [3H]PAF binding. However, long term incubation (9-24 h) with dibutyryl cAMP caused down-regulation of the PAF receptors in rat Kupffer cells. Forskolin (0.1 mM), an adenylyl cyclase activator, down-regulated the surface expression of the AGEPC receptors more rapidly, decreasing the specific [3H]AGEPC binding by approximately 40% within 2 h. The receptor regulatory effect of dibutyryl cAMP and forskolin was time- and concentration-dependent. These observations suggest that a cAMP-dependent mechanism coupled with beta 2-adrenergic receptors may have important regulatory effects on the PAF receptor and post-receptor signal transducing mechanisms for PAF in hepatic Kupffer cells.     

血小板活化因子受体研究进展

血汴板活化因子是通过靶细胞膜上的受体而发挥其作用的,该受体属Ｇ联的受体家族,含３４２个氨基酸,有７个疏水的跨膜片段。其作用机制是通过激活磷酯酰肌醇、钙信使系统及相关蛋白激酶,使某些蛋白质发生磷酸化并产生相应的生物学效应。     

Human macrophages secret platelet-activating factor acetylhydrolase

When monocytes mature to macrophages, their ability to accumulate the pro-inflammatory lipid autacoid, platelet-activating factor (PAF), is markedly decreased (Elstad, M. R. Stafforini, D. M., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1989) J. Biol. Chem. 264, 8467-8470) in conjunction with a 260-fold increase in the activity of intracellular PAF acetylhydrolase (PAF-AH). We now demonstrate that macrophages also secrete PAF-AH and that the secreted enzyme is biochemically and immunologically identical to the human plasma PAF-AH. It is sensitive to the same active-site-directed inhibitors, has the same electrophoretic mobility, is associated with lipoprotein particles, and transfers between low density lipoprotein and high density lipoprotein in a pH-dependent manner like the plasma PAF-AH. In addition, both activities hydrolyze oxidatively fragmented phospholipids and PAF. These data indicate that macrophages are a cellular source of the plasma PAF-AH. Thus, macrophages secrete an enzyme that inactivates lipid mediators at sites of inflammation and in plasma. These changes during the maturation of monocytes to macrophages may serve to limit the acute inflammatory response.     

Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor

Platelet-activating factor (PAF) is a bioactive phospholipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) synthesized by a variety of mammalian cell types. PAF induces hypotension, and activates neutrophils and platelets, among other actions. Removal of the acetyl moiety abolishes biological activity, so this reaction may regulate the concentration of PAF and its physiological effects. We have studied the significance of this reaction, which is catalyzed in vitro by an acetylhydrolase present in mammalian plasma, blood cells, and tissues. We have shown that the plasma PAF-acetylhydrolase is responsible for the degradation of PAF in whole human blood and that alternate pathways for PAF degradation in plasma or blood cells are negligible. Human plasma PAF-acetylhydrolase is associated with low and high density lipoproteins (LDL and HDL with apoE). We have confirmed that the activity is a stable component of these particles by density gradient ultracentrifugation, chromatography on heparin-agarose, and immunoprecipitation. The LDL-associated activity accounts for most or all of the PAF degradation that occurs in plasma ex vivo, while the HDL-associated activity contributes little to this process. However, the two activities likely are due to a single protein since the HDL- and LDL-associated PAF-acetylhydrolase activities can transfer from one lipoprotein to the other. These transfer processes are pH-dependent and specific, since they only occur from LDL to a well characterized subclass of HDL (apoE-containing HDL) and vice versa. We discuss the equilibrium between the two particles and the role that this process may have in vivo.     

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.     

A superfusion bioassay for platelet-activating factor

A superfusion bioassay for platelet-activating factor is described using various types of tissues. By washing the tissue with 0.1-0.5% bovine serum albumin for 2-3 min after each addition of platelet-activating factor, desensitization did not develop in most tissues studied. Because of the ability to apply a sample directly onto an assay tissue with negligible dilution, this bioassay can detect smaller amounts of platelet-activating factor than those previously reported in which an organ bath was utilized. The ascending colon of the rat and dog appeared to be the most sensitive of the tissues tested, with a limited of detectability in the range of 100-500 fg. Repeated additions of platelet-activating factor could be made for up to 4 h without desensitization. Release of platelet-activating factor from samples of rat stomach was measured using the superfusion bioassay and a platelet aggregation bioassay. There was a significant correlation (r = 0.96; p less than 0.01) between the values obtained using the two assay systems. Thus, the sensitivity, the reproducibility, and the inexpensive nature of this bioassay make it an attractive alternative to existing bioassays for platelet-activating factor.     

Lung endothelial and epithelial permeability after platelet-activating factor

We investigated whether platelet-activating factor (PAF) increased epithelial or endothelial permeability in isolated-perfused rabbit lungs. PAF was either injected into the pulmonary artery or instilled into the airway of lungs perfused with Tyrode's solution containing 1% bovine serum albumin. The effect of adding neutrophils or platelets to the perfusate was also tested. Perfusion was maintained 20-40 min after adding PAF and then a fluid filtration coefficient (Kf) was determined to assess vascular permeability. At the end of each experiment, one lung was lavaged, and the lavagate protein concentration (BALP) was determined. Wet weight-to-dry weight ratios (W/D) were determined on the other lung. PAF added to the vascular space increased peak pulmonary arterial pressure (Ppa) from 13.5 +/- 3.1 (mean +/- SE) to 24.2 +/- 3.3 cmH2O (P less than 0.05). The effect was amplified by platelets [Ppa to 70.8 +/- 8.0 cmH2O (P less than 0.05)] but not by neutrophils [Ppa to 22.0 +/- 1.4 cmH2O (P less than 0.05)]. Minimal changes in Ppa were observed after instilling PAF into the airway. The Kf, W/D, and BALP of untreated lungs were not increased by injecting PAF into the vasculature or into the air space. The effect of PAF on Kf, W/D, and BALP was unaltered by adding platelets or neutrophils to the perfusate. PAF increases intravascular pressure (at a constant rate of perfusion) but does not increase epithelial or endothelial permeability in isolated-perfused rabbit lungs.     

Endogenous platelet-activating factor and anti-platelet-activating factor in patients with renovascular hypertension

Renovascular hypertension is relieved by percutaneous transluminal renal angioplasty. In four patients with renovascular hypertension, platelet-activating factor (PAF) was found to be released into the ipsilateral renal venous blood after percutaneous transluminal renal angioplasty, but was not found in the contralateral renal venous blood following this procedure. Anti-platelet-activating factor with a lipid-like property was also found, and its polarity was slightly lower than that of PAF judging by its behavior on thin layer chromatography. Anti-platelet-activating factor completely blocked the aggregation of rabbit platelets induced by PAF, ADP or arachidonic acid. These results indicate that PAF and anti-platelet-activating factor are released into renal venous blood following percutaneous transluminal renal angioplasty in patients with renovascular hypertension.     

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.     

Occurrence of platelet-activating factor in rabbit spermatozoa

Spermatozoa obtained from rabbit ejaculate were analyzed for the presence of platelet-activating factor [PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)] by using standard HPLC and TLC procedures. Fractions corresponding to synthetic PAF (AGEPC) revealed PAF-like activity amounting to 0.35 +/- 0.06 pmol/10(8) cells (mean +/- SE) as determined by bioassays based on the release of [3H]serotonin from washed rabbit platelets. This activity was lost upon base-catalyzed methanolysis, but was restored to the original level after reacetylation. Analysis of the phosphatidylcholine (PC) fraction by GC-MS subsequent to base-catalyzed methanolysis showed that 1-O-alkyl-2-acylphosphocholine comprises about 12% of the PC fraction with alkyl chain lengths of 16:0 (88%) and 18:0 (12%).     

Fetal pulmonary vasodilation after exogenous platelet-activating factor

To determine the fetal pulmonary vascular response to platelet-activating factor (PAF), we studied the hemodynamic effects of the infusion of PAF directly into the left pulmonary artery in 21 chronically catheterized fetal lambs. Left pulmonary arterial blood flow (Q) was measured with electromagnetic flow transducers. Ten-minute infusions of low-dose PAF (10-100 ng/min) produced increases in Q from a baseline of 71 +/- 5 to 207 +/- 20 ml/min (P less than 0.001) without changes in pulmonary arterial pressure. Pulmonary vasodilation with PAF was further confirmed through increases in Q with brief (15-s) infusions and increases in the slope of the pressure-flow relationship as assessed by rapid incremental compressions of the ductus arteriosus during PAF infusion. Infusion of Lyso-PAF had no effect on Q or pulmonary arterial pressure. Treatment with CV-3988, a selective PAF receptor antagonist, but not with meclofenamate, atropine, or diphenhydramine and cimetidine blocked the response to PAF infusion and did not affect baseline tone. Systemic infusion of high-dose PAF (300 ng/min) through the fetal inferior vena cava increased pulmonary arterial pressure (46.5 +/- 1.0 to 54.8 +/- 1.9 mmHg, P less than 0.01) and aorta pressure (44.3 +/- 1.0 to 52.7 +/- 2.2 mmHg, P less than 0.01) while also increasing Q. Neither PAF nor CV-3988 changed the gradient between pulmonary arterial and aorta pressures, suggesting that PAF does not affect ductal tone. We conclude that PAF is a potent fetal pulmonary vasodilator and that the effects are not mediated through cyclooxygenase products or by cholinergic or histaminergic effects.     

Polymorphic phase behavior of platelet-activating factor.

Vibrational Raman and 31P NMR spectroscopic experiments have been performed as a function of temperature on aqueous dispersions of 1-0-octadecyl-2-acetoyl-sn-glycero-3-phosphocholine, a chemically synthesized platelet-activating factor. In the temperature range of -7 to 30 degrees C, the C(18)/PAF-H2O system is shown, upon heating, to undergo two thermal phase transitions centered at 9.2 degrees and 18.4 degrees C. The low temperature transition, attributed to the interdigitated lamellar gel (II)----gel (I) phase transition, is characterized by the breakdown of large lamellar organizations into small, but aggregated, bilayer vesicles. The high-temperature transition corresponds to the interdigitated lamellar gel (I)----micellar transition. The molecular ordering and packing structure of C(18)/PAF in the two lamellar phases and phase transition regions are described. It appears that the interdigitated lamellar gel (I) phase is unique for C(18)/PAF dispersions when compared with the behavior of other chemically closely related phospholipids in excess water.     

Role of spermatozoal platelet-activating factor in fertilization

Platelet-activating factor (PAF), a potent lipid mediator of inflammation, has been shown to play a role in both the implantation and viability of mammalian embryos. We examined whether human and mouse spermatozoa release PAF during in vitro incubation and assessed the effect of exogenous PAF and the PAF receptor antagonist WEB 2086, a thieno-triazolodiazepine, on mouse in vitro fertilization (IVF) rate. PAF biological activity was detected in 11 samples of leukocyte-free, purified human spermatozoa (28 pg PAF/10(6) cells/24 hr) and 5 samples of epididymal mouse spermatozoa (7.8 pg PAF/10(6) cells/3 hr). Exogenous PAF (10(-8) and 10(-6) M) increased (p less than 0.01) the fertilization rate 2- and 3-fold, respectively of mouse oocytes by mouse epididymal spermatozoa. 10(-4) M PAF, however, reduced sperm motility and decreased (p less than 0.05) the fertilization rate. 10(-6) M WEB 2086, decreased IVF to approximately 50% of the control fertilization rate (42% vs. 89%). WEB 2086 treatment also promoted the attachment of supernumerary spermatozoa to both fertilized and unfertilized oocytes. The fertilization rate in the presence of WEB 2086 returned to control levels when zona-pellucida-free oocytes were employed, indicating that WEB 2086 did not interfere with the spermatozoal acrosome reaction. These data suggest that PAF, of spermatozoal origin, may be important in mammalian fertilization.     

Production of platelet-activating factor by chick retina

In the present study it is demonstrated that platelet-activating factor (PAF) was produced by chick retinas, upon stimulation with neurotransmitters such as acetylcholine (ACh), dopamine, or with calcium ionophore A23187, but not upon stimulation with gamma-amino-n-butyric acid, L-glycine, L-glutamate, epinephrine, or histamine. PAF produced in response to ACh, dopamine, or A23187 was not released into supernatants but was extractable from retinas. The amounts of extractable PAF increased after sonication of stimulated retinas. While no PAF activity could be recovered from unstimulated retinas, small amounts of this lipid can be detected following sonication of the tissue. The amount of extractable PAF from ACh-, dopamine-, or A23187-stimulated retinas was dependent upon the incubation time and concentration of the agonists. PAF was identified on the basis of chemical and lipase treatments, biological activity with washed rabbit platelets, behavior on thin layer chromatography, and high pressure liquid chromatography. Control cell preparations (leukocytes, erythrocytes, and embryogenic fibroblasts) did not produce PAF upon neurotransmitter stimulation. ACh and dopamine promoted PAF production by increasing dithiothreitol-insensitive cholinephosphotransferase activity, without affecting the acetyltransferase activity. In contrast, the A23187 ionophore stimulated the acetyltransferase activity but did not affect the dithiothreitol-insensitive cholinephosphotransferase.