Characterization of eosinophil cell activation by peptides. Differential effects of substance P, melittin, and FMET-Leu-Phe.

The ability of three different peptides, substance P (SP), FMLP and melittin, to activate eosinophils purified from the peritoneal cavity of human serum-treated guinea pigs was investigated. Degranulation (eosinophil peroxidase, EPO), oxidative burst (O2-), [Ca2+]i mobilization, and arachidonic acid metabolism (thromboxane B2, TXB2) were used as indices of eosinophil activation. SP (100 nM to 100 microM), FMLP (1 to 100 microM) and melittin (10 nM to 100 microM) induced EPO release but only FMLP (1 to 100 microM) led to an elevation of [Ca2+]i. The melittin- and SP-induced EPO secretion occurred at both cytotoxic and noncytotoxic concentrations as assessed by lactate dehydrogenase release. In addition, the effect of SP was not inhibited by the SP analogue (D-Pro4, D-Trp7,9,10)SP(4-11) and SP failed to promote the generation and subsequent release of TXA2. In contrast, FMLP (10 to 100 microM) stimulated the release of TXB2 from guinea pig eosinophils that was selectively inhibited by pretreatment of the cells with BOC-FMLP. On an equimolar basis (1 microM), melittin was approximately fivefold more active at promoting TXB2 release than FMLP. The results indicate that eosinophils respond to the three peptides, SP, melittin, and FMLP in differential fashion. We conclude that activation of guinea pig eosinophils by FMLP is likely to be receptor-mediated whereas the actions of SP and melittin may act through nonspecific peptide-membrane phospholipid interactions.     

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.     

Effect of dipyridamole on prostanoid production in rat isolated atria.

The effect of 0.01 microM dipyridamole on prostanoid production was studied in atria from normal, acute diabetic and insulin-treated diabetic rats. Diabetes was induced by i.v. administration of 65 mg/kg of streptozotocin (STZ) and the rats were killed 5 days later. Atria were incubated during 60 min in Krebs solution. The prostanoids 6-keto-prostaglandin (PG) F1alpha (6-keto-PGF1alpha) and thromboxane (TX) B2, stable metabolites of prostacyclin and TXA2, respectively, as well as PGE2 were measured by reversed phase high-performance liquid chromatography-UV. In diabetic atria, 6-keto-PGF1alpha production was reduced by 50% whereas TXB2 release was increased two-fold compared to the controls, with a significant decrease in the 6-keto-PGF1alpha/TXB2 ratio. The preincubation with 0.01 microM dipyridamole for 30 min increased 6-keto-PGF1alpha production in control, diabetic and insulin-treated diabetic atria whereas TXB2 release was not modified. This effects provoked an significant increase in the 6-keto-PGF1alpha/TXB2 ratio. In conclusion, STZ diabetes reduces the 6-keto-PGF1alpha/TXB2 ratio impairing the functional status of the atria. Dipyridamole increased this ratio in atria from diabetic and insulin-treated diabetic rats, thus opposing the effects of STZ diabetes. This fact suggests the possibility of a participation of the drug in pathologies characterized by an imbalance in the production of vasodilator and vasoconstrictor prostanoids.     

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)     

A common binding site for primary prostanoids in vascular smooth muscles: a definitive discrimination of the binding for thromboxane A2/prostaglandin H2 receptor agonist from its antagonist

Differences in binding characteristics between agonists and antagonists for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were examined in rat cultured vascular smooth muscle cells (VSMC). Scatchard analysis indicated the existence of two binding sites for the TXA2/PGH2 agonist, whereas a single class of recognition sites for the receptor antagonists were observed with approximately the same maximum binding capacity (Bmax) as a high-affinity binding site of the agonist. Weak binding inhibition by approx. 100 nM of primary prostanoids (PGE1, PGF2 alpha and PGD2) was detected only with the TXA2/PGH2 agonist, and not with the antagonist. Primary prostanoids as well as TXA2/PGH2 agonists (U46619 and STA2) suppressed the [3H]PGF2 alpha and [3H]PGE1 binding with almost the same potency, whereas TXA2/PGH2 antagonists (S-145, SQ29,548 and ONO3708) did not. The Bmax value of the binding sites was roughly identical in PGF2 alpha, PGE1 and a low-affinity binding site of U46619. These results suggest the existence of two binding sites for TXA2/PGH2 in VSMC, i.e., a high-affinity binding site corresponding to that of the TXA2/PGH2 antagonists and a low-affinity binding site in common with primary prostanoids.     

磷脂酶A2活力与急性缺氧性肺动脉高压关系的探讨

通过动物实验探讨磷脂酶Ａ２及相关炎性介质在急性缺氧性肺动脉高压形成中的作用。３０只ＳＤ大鼠随机分为三组;正常对照组,缺氧组及缺氧加地塞米松组。均测定肺动脉压;缺氧３０ｍｉｎ后,取静脉血肺组织测定ＰＬＡ２活力,血小板活化因子,前列腺素Ｅ２,血栓素Ｂ２。     

Prostaglandin and thromboxane synthesis by tissue slices from human aortic aneurysms

Sliced portions of the walls of human aortic aneurysms were incubated with extracts of human plasma and serum to determine the profile of prostanoid production. 6-Oxo-prostaglandin (PG) F1 alpha, PGE2, PGF2 alpha, and thromboxane (TX) B2 were measured by gas chromatography/electron capture mass spectrometry. 6-Oxo-PGF1 alpha, the stable hydrolysis product of PGI2, was the major cyclooxygenase product but substantial amounts of TXB2 (the hydrolysis product of TXA2), with smaller amounts of PGE2 and PGF2 alpha were also synthesised. These prostanoids could contribute to the response of the vascular wall to injury, thereby influencing the disease process. Serum extracts stimulated PGI2 and TXA2 synthesis, probably as a result of their Ca2+ content.     

Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells

In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids.     

Effects of experimental diabetes on spontaneous contractions, on the output of prostaglandins and on the metabolism of labelled arachidonic acid, in uteri isolated from ovariectomized rats. Influences of estradiol

Spontaneous changes in isometric developed tension (IDT) as a function of time after isolation (contractile constancy) in uteri from control-castrated and castrated chronic streptozotocin-diabetic rats, were explored. The effects of injecting 17-beta estradiol (Eo) were also studied. No differences in the minor changes of contractile constancy, between control and diabetic preparations, during a period of 60 min, were detected, whereas uteri from non-diabetic Eo injected animals (0.5 + 1.0 ug, prior to sacrifice), exhibited a profound reduction of IDT, significantly greater than in tissues obtained from Eo injected-diabetic rats. Moreover, basal generation and outputs into the suspending solution of prostaglandins (PGs) E1, E2 and F2 alpha, were explored in the same groups, at 60 min following tissue isolation. The basal outputs of these three PGs were similar in castrated control rats, but preparations from castrated-diabetics released significantly more PGE1. The administration of Eo to castrated-diabetics, failed to alter the releases of the three PGs explored. In addition, the metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF1, PGF2, PGE2 and thromboxane B2-TXB2), was also investigated. The non-diabetic spayed rat uterus converted AA into these four prostanoids, the transformation into 6-keto-PGF1 alpha (as an index of PGI2 formation) being the most prominent. In preparations from diabetic rats the formation) being the most prominent. In preparations from diabetic rats the formation of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, was significantly smaller than in controls, whereas a greater % of TXB2 formation (as an index of TXA2), was detected. On the other hand uterine preparations from non-diabetic spayed rats injected with Eo formed less 6-keto-PGF1 alpha and PGE2 and similar amounts of PGF2 alpha or of TXB2 from AA, than Eo injected controls, whereas uteri from castrated diabetic animals injected with Eo, formed a similar % of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from AA, than tissue preparations from non-estrogenized controls. However, the enhanced transformation of the labelled fatty acid precursor (AA) into TXB2 in the diabetic group, was significantly reduced by the steroid. The role of the augmented generation and release of PGE1 in uteri from diabetic rats is discussed in terms of precedents indicating the relevance of PGs type E supporting rat uterine motility. In addition the influence of Eo is attractive, because its reducing effect on TX production, in diabetes, a disease known to be accompanied by enhanced synthesis of vasoconstrictor and platelet aggregation TXA2, and by frequent obstructive circulat     

Influence of some prostaglandins and prostaglandin analogues on PAF-induced shock in mice.

Prostaglandins and Prostaglandin-analogues were investigated for their ability to protect mice from platelet-activating factor (PAF) induced shock. 75% mortality in female NMRI mice was induced by i.v. injection of 75 micrograms/kg PAF. Nileprost and PGE1, the most potent substances, produced a dose dependent protection against PAF. Iloprost and PGI2 were less effective. PGE2, nalador, flunoprost and U 46619 were neither protective nor deleterious. The strong difference in the effectiveness between the two prostaglandins of the E-series and the poor effect of PGI2 and the PGI2 analogue is remarkable. Flunoprost and U 46619 that increased the TXB2 synthesis or release in two experimental models did not enhance the PAF mortality; TXA2 seems to be only a secondary mediator of the acute PAF-induced death.     

Pharmacological evaluation of both enantiomers of (R,S)-BM-591 as thromboxane A2 receptor antagonists and thromboxane synthase inhibitors

The aim of this work is to evaluate the anti-thromboxane activity of two pure enantiomers of (R,S)-BM-591, a nitrobenzene sulfonylurea chemically related to torasemide, a loop diuretic. The drug affinity for thromboxane A2 receptor (TP) of human washed platelets has been determined. In these experiments, (R)-BM-591 (IC50 = 2.4+/-0.1 nM) exhibited a significant higher affinity than (S)-BM-591 (IC50 = 4.2+/-0.15 nM) for human washed platelets TP receptors. Both enantiomers were stronger ligands than SQ-29548 (IC50 = 21.0+/-1.0 nM) and sulotroban (IC50 = 930+/-42 nM), two reference TXA2 receptor antagonists. Pharmacological characterisations of (S)-BM-591 and (R)-BM-591 were compared in several models. Thus, (R)-BM-591 strongly prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) and U-46619 (1 microM) while (S)-BM-591 showed a lower activity. On isolated tissues pre-contracted by U-46619, a stable TXA2 agonist, (S)-BM-591 was more potent in relaxing guinea-pig trachea (EC50 = 0.272+/-0.054 microM) and rat aorta (EC50 = 0.190+/-0.002 microM) than (R)-BM-591 (EC50 of 9.60+/-0.63 microM and 0.390+/-0.052 microM, respectively). Moreover, at 1 microM, (R)-BM-591 totally inhibited TXA2 synthase activity, expressed as TXB2 production from human platelets, while at the same concentration, (S)-BM-591 poorly reduced the TXB2 synthesis (22%). Finally, in rats, both enantiomers lost the diuretic activity of torasemide. In conclusion, (R)-BM-591 exhibits a higher affinity and antagonism on human platelet TP receptors than (S)-BM-591 as well as a better thromboxane synthase inhibitory potency. In contrast, (S)-BM-591 is more active than the (R)-enantiomer in relaxing smooth muscle contraction of rat aorta and trachea guinea pig. Consequently, (R)-BM-591 represents the best candidate for further development in the field of thrombosis disorders.     

Evidence for two platelet activating factor receptors on eosinophils: dissociation between PAF-induced intracellular calcium mobilization degranulation and superoxides anion generation in eosinophils

We have compared platelet activating factor (PAF)-induced eosinophil peroxidase (EPO) release and intracellular calcium mobilization with superoxide anion (.O2-) generation from guinea pig eosinophils. EPO release and Ca2+ mobilization occurred at lower concentrations of PAF (EC50 values of 1.3 nM and 11.5 nM, respectively) while .O2- production was observed at higher concentrations (EC50 of 31.7 microM). Receptor characterization with the competitive PAF antagonist, WEB 2086, gave pA2 values of 8.5 and 8.3 for EPO enzyme release and rise in [Ca2+]i, respectively, and 5.8 for the .O2- production. In addition, PAF-induced degranulation and elevation of [Ca2+]i were dependent on extracellular Ca2+ whereas PAF-stimulated .O2- generation was dependent on the presence of extracellular Mg2+ ions. These results suggest the existence either of two subtypes of the PAF receptor or a single receptor that can exist in one of two affinity states on guinea pig eosinophils.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.     

Release of tumor necrosis factor-alpha and prostanoids in whole blood cultures after in vivo exposure to low-dose aspirin

BACKGROUND: The preventive effect of low-dose aspirin in cardiovascular disease is generally attributed to its antiplatelet action caused by differential inhibition of platelet cyclooxygenase-1. However, there is evidence that aspirin also affects release of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha). It is not known whether this is caused by direct action on the cytokine pathway or indirectly through cyclooxygenase inhibition and altered prostanoid synthesis, or both. METHODS: We assessed the capacity of lipopolysaccharide-activated leukocytes in whole blood cultures of eight healthy subjects following a single oral dose of 80 mg aspirin to release TNF-alpha, prostanoid E2 (PGE2) and prostanoid I2 (PGI2), and thromboxane A2 (TXA2). TNF-alpha and prostanoids were determined by enzyme-linked immunoassays. RESULTS: In seven subjects, TNF-alpha release in blood cultures decreased 24h after intake of aspirin. The effect of aspirin on prostanoid release was assessed in three individuals: PGE2 increased in all subjects, PGI2 increased in two and remained unchanged in one, and TXA2 was reduced in two and unchanged in one individual The presence of DFU, a specific inhibitor of cyclooxygenase 2, did not affect the reduction of TNF-alpha release by aspirin, but abolished prostanoid production in all three individuals. Conclusion: The capacity of activated leukocytes to release TNF-alpha is reduced by ingestion of low-dose aspirin, independent of changes in prostanoid biosynthesis.     

Alpha-bulnesene, a novel PAF receptor antagonist isolated from Pogostemon cablin

Alpha-bulnesene is a sesquiterpenoid isolated from the water extract of Pogostemon cablin. It showed a potent and concentration-dependent inhibitory effect on platelet-activating factor (PAF) and arachidonic acid (AA) induced rabbit platelet aggregation. In a radioligand binding assay for the PAF receptor, alpha-bulnesene competitively inhibited [(3)H]PAF binding to the PAF receptor with an IC(50) value of 17.62+/-5.68microM. alpha-Bulnesene also dose-dependently inhibited PAF-induced intracellular Ca(2+) increase in fluo-3/AM-loaded platelets (IC(50) values of 19.62+/-1.32microM). Furthermore, alpha-bulnesene inhibited AA-induced thromboxane B(2) (TXB(2)) formation and prostaglandin E(2) (PGE(2)) formation. These results indicate that the inhibitory effect of alpha-bulnesene on platelet aggregation was due to a dual activity; specifically the chemical blocked PAF-induced intracellular signal transduction and interfered with cyclooxygenase activity, which resulted in a decrease in thromboxane formation. This study is the first to demonstrate that alpha-bulnesene is a PAF receptor antagonist as well as an anti-platelet aggregation agent.     

Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes

Lung cells have been isolated by enzymatic digestion of guinea pig lungs and mechanical dispersion to obtain a suspension of viable cells (approximately 500 X 10(6) cells). Type II pneumocytes have been purified to approximately 92% by centrifugal elutriation (2000 rpm, 15 ml/min) followed by a plating in plastic dishes coated with guinea pig IgG (500 micrograms/ml). We have investigated the arachidonic acid metabolism through the cyclooxygenase pathway in this freshly isolated type II cells (2 x 10(6) cells/ml). Purified type II pneumocytes produced thromboxane B2 (TxB2) predominantly and to a smaller extent the 6-keto prostaglandin PGF1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) after incubation with 10 microM arachidonic acid. The stimulation of pneumocytes with 2 microM calcium ionophore A23187 released less eicosanoids than were produced when cells were incubated with 10 microM arachidonic acid. There was no additive effect when the cells were treated with both arachidonic acid and the ionophore A23187. Guinea pig type II pneumocytes failed to release significant amounts of TxB2, 6-keto-PGF1 alpha and PGE2 after stimulation with 10 nM leukotriene B4, 10 nM leukotriene D4, 10 nM platelet-activating factor, 5 microM formyl-methionyl-leucyl-phenylalanine, 0.2 microM bradykinin and 10 nM phorbol myristate acetate. Our findings indicate that guinea pig type II pneunomocytes possess the enzymatic machinery necessary to convert arachidonic acid to specific cyclooxygenase products, which may suggest a role for these cells in lung inflammatory processes.     

Relationship between PAF-acether and thromboxane A2 biosynthesis in endotoxin-induced intestinal damage in the rat

PAF-receptor antagonists are known to inhibit gastrointestinal damage induced by endotoxin. In the present study, the interaction between the biosynthesis of PAF and thromboxane (TX) A2, as putative mediators of the acute intestinal damage induced by endotoxin, has been investigated in the anaesthetised rat. Bolus intravenous administration of lipopolysaccharide from E. coli (5-50 mg/kg) induced dose-related jejunal damage, assessed using both macroscopic and histological techniques. This damage was accompanied by significant increases in the jejunal formation of PAF determined by bioassay, and of TXB2, determined by radioimmunoassay. Pretreatment with the structurally-unrelated thromboxane synthase inhibitors, 1-benzyl imidazole (10-50 mg/kg) or OKY 1581 (25 mg/kg) substantially reduced both jejunal damage and TXB2 formation, but did not inhibit PAF formation. Likewise, pretreatment with indomethacin (5 mg/kg) or BW 755C (50 mg/kg) reduced jejunal damage and TXB2 formation but did not affect PAF formation. Pretreatment (2h) with dexamethasone (4 mg/kg) reduced jejunal damage and the formation of both TXB2 and PAF. Intravenous infusion of PAF (100 ng/kg/min for 10 min) induced jejunal damage and significantly increased the formation of TXB2, whereas non-specific jejunal damage induced by oral administration of ethanol did not augment PAF formation. The present findings that inhibition of jejunal thromboxane formation is associated with a substantial reduction in jejunal damage, with no corresponding inhibition in PAF formation, therefore suggests a complex interaction or sequential release of these tissue destructive mediators underlying the intestinal damage induced by endotoxin.     

Induction of prostanoid synthesis in human platelets by the late complement components C5b-9 and channel forming antibiotic nystatin: inhibition of the reacylation of liberated arachidonic acid

Treatment of human platelets by the purified late complement components C5b-9 results in a dose- and time-dependent release of prostaglandin E (PGE) and thromboxane B2 (TXB2). To study the mechanism underlying the complement-induced prostanoid synthesis, we examined whether C5b-9 affected the enzyme acyl-coA:lysolecithin acyltransferase (E.C.2.3.1.2.3) that catalyzes the reinsertion of liberated arachidonic acid, the precursor molecule of the prostanoids. With C5b-9 doses sufficient to induce prostanoid synthesis, the activity of lysolecithin acyltransferase, measured as conversion of lysophosphatidyl choline to phosphatidyl choline, was inhibited. For comparison, another channel-forming substance, nystatin, was studied. Nystatin had an effect similar to C5b-9: PGE and TXB2 release was stimulated, whereas acyltransferase activity was inhibited. These finding support the concept that inhibition of lysolecithin acyltransferase might be the prerequisite for prostanoid production.     

Zonal heterogeneity of prostaglandin and thromboxane release in the dog kidney

The release of prostaglandin(PG) and thromboxane(TX) was examined in the six different areas of the normal dog kidney, i.e., renal arterial and venous strips(RA and RV), superficial and deep cortical slices (SC and DC) and outer and inner medullary slices(Om and IM). These tissues were incubated in Krebs-bicarbonate buffer(pH 7.4, 37°C), and the released PGE2, PGF2α, 6-keto-PGF1α and TXB2(as stable metabolites of PG12 and TXA2, respectively) were determined by radioimmunoassay. In RA, RV, SC and DC, 6-keto-PGF1α was predominant, however, there were no quantitative differences between RA and RV, or SC and DC. The release of 6-keto-PGF1α reached a maximum in IM, similar to findings on the release of PGE2 and PGF2α. The release of TXB was uniform in OM and IM. The amount of PGE2, PGF2α, 6-keto-PGF1α and TXB2 released from IM was 2800, 400, 60 and 50 times higher, respectively, than the extent of the release from the cortical slices.These results suggest that PG12 as well as PGE2 and PGF2α, may be involved in renal PG, and that TXA2 is biosynthesized in the normal dog kidney.     

Effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced arachidonate release and 5-hydroxytryptamine secretion in human platelets. Dependence of effects on agonist type and time of incubation with PMA

We have previously demonstrated synergistic potentiation of secretion by phorbol 12-myristate 13-acetate (PMA) and platelet agonists such as thrombin and the thromboxane mimetic, U46619, with short (less than 2 min) pre-incubations of PMA, despite inhibition of agonist-induced [Ca2+]i mobilization and arachidonate/thromboxane release. In this study, the effect of PMA on 5-hydroxytryptamine secretion in relation to arachidonate/thromboxane B2 release induced by collagen as well as the 'weak agonists', ADP, adrenaline and platelet-activating factor (PAF), was investigated using human platelet-rich plasma. Short incubations (10-30 s) with PMA (400 nM) before agonist addition caused an inhibition (60-100%) of 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation in response to maximally effective doses of ADP (10 microM), adrenaline (10 microM) and PAF (0.5 microM) but potentiated collagen-induced 5-hydroxy[14C]tryptamine secretion and [3H]arachidonate/thromboxane release. However, a longer pre-incubation with PMA (5 min) caused a significant reduction (20-50%) in the extent of collagen-induced 5-hydroxy[14C]tryptamine secretion and thromboxane B2 formation as seen earlier with thrombin, although collagen-induced [3]arachidonate release was still unaffected. Pretreatment of platelets with the cyclo-oxygenase inhibitor, indomethacin (10 microM), abolished 5-hydroxy[14C]tryptamine secretion in response to the weak agonists and reduced collagen (2.5-10 micrograms/ml) -induced secretion by 50-90%, depending on the collagen concentration. Addition of PMA (400 nM) 10 s before these agonists in indomethacin-treated platelets resulted in synergistic interactions between agonist and PMA leading to enhanced 5-hydroxy[14C]tryptamine secretion, although this was notably less than the synergism observed previously between thrombin and PMA or U46619 and PMA. The results suggest that the effect of short incubations with PMA on 5-hydroxytryptamine secretion induced by 'thromboxane-dependent' agonists, such as those examined in this study, is determined by the effect on agonist-induced thromboxane synthesis. However, when endogenous thromboxane synthesis is blocked, weak agonists as well as collagen can synergize with PMA at potentiating 5-hydroxytryptamine secretion, albeit to a weaker extent than thrombin or U46619. The results also suggest that PMA has differential effects on arachidonate release induced by collagen and thrombin.