Developmental changes in endothelium-derived vasorelaxant factors in cerebral circulation

Endothelium-derived prostanoids are predominant vasorelaxant factors in the cerebral circulation of newborn pigs in vivo, whereas in older pigs nitric oxide (NO)-mediated responses also contribute to the regulation of cerebral vascular tone. We compared the expression and activities of NO synthase and cyclooxygenase in the cerebral microcirculation of newborn and adult pigs. In adult animals, expression and activity of endothelial NO synthase in cerebral microvessels and in cultured cerebral endothelial cells is two- to threefold higher than in newborn pigs; acetylcholine and bradykinin cause a greater increase in NO production in adult pigs. Expression and activity of cyclooxygenase in cerebral microvascular endothelial cells is similar in newborn and adult pigs; acetylcholine and bradykinin stimulated dilator prostanoid production to the same degree in both age groups. Endothelial prostanoid synthesis in cerebral microvessels and cultured endothelial cells was inhibited 30-70% by NS-398, reflecting a large contribution of COX-2 in both newborn and adult animals. These data indicate that in the cerebral circulation of pigs, NO synthase is age-dependently upregulated, whereas endothelial cyclooxygenase is not altered during postnatal development.     

Isomer-specific contractile effects of a series of synthetic f2-isoprostanes on retinal and cerebral microvasculature

F2-isoprostanes (F2-IsoP's) are biologically active prostanoids formed by free radical-mediated peroxidation of arachidonic acid. Four different F2-IsoP regioisomers (5-, 8-, 12-, and 15-series), each comprising eight racemic diastereomers, total 64 compounds. Information regarding the biological activity of IsoP's is largely limited to 15-F2t-IsoP (8-iso-PGF2alpha). We recently demonstrated that 15-F2t-IsoP and its metabolite, 2,3-dinor-5,6-dihydro-15-F2t-IsoP, evoked vasoconstriction and TXA2 generation in retina and brain microvasculature. We have now examined and compared the biological activities of a series of recently synthesized new 5-, 12-, and 15-series F2-IsoP isomers in pig retinal and brain microvasculature. We hereby show that other 15-series F2-IsoP isomers, 15-epi-15-F2t-IsoP, ent-15-F2t-IsoP, and ent-15-epi-15-F2t-IsoP, are also potent vasoconstrictors. The 12-series isomers tested, 12-F2t-IsoP and 12-epi-12-F2t-IsoP, also caused marked vasoconstriction. Of the 5-series isomers tested, 5-F2t-IsoP and 5-epi-5-F2t-IsoP possessed no vasomotor properties, whereas ent-5-F2t-IsoP caused modest vasoconstriction. The vasoconstriction of ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP was abolished by removal of the endothelium, by TXA2 synthase and receptor inhibitor (CGS12970, L670,596), and by receptor-mediated Ca2+ channel blockade (SK & F96365); correspondingly, these isomers increased TXB2 formation by activating Ca2+ influx (detected with fura 2-AM) through non-voltage-dependent receptor-mediated Ca2+ entry (SK & F96365 sensitive) in endothelial cells. In conclusion, as seen with 15-F2t-IsoP, ent-5-F2t-IsoP, 12-F2t-IsoP, and 12-epi-12-F2t-IsoP constricted both retinal and brain microvessels by inducing endothelium-dependent TXA2 synthesis. These new findings broaden the scope of our understanding regarding the potential involvement of F2-IsoP's as mediators of oxidant injury.     

Mechanisms of platelet activating factor-induced aggregation and secretion in human platelets

The role of TXA2 in PAF-induced aggregation and secretion of human platelets is unclear. We have studied the relationship between aggregation, synthesis of TXA2 and release of 5-HT during the time course of aggregation induced by PAF and collagen. For PAF-induced aggregation there was strong aggregation and secretion with minimal production of TXA2 in contrast to collagen in which a surge in TXA2 synthesis preceded both aggregation and secretion. To determine the role of calcium flux in PAF-induced aggregation we have similarly studied the temporal relationships between aggregation, secretion and TXA2 synthesis for calcium ionophore A23187 induced aggregation but found these to be distinctly different from those determined for PAF. A method for measuring absolute amounts of 5HT released from platelets in small volumes of plasma is described. We conclude that TXA2 is not important in the mechanism of PAF induced aggregation and that an increase in the level of intraplatelet calcium per se is not sufficient to explain the mediation of PAF-induced aggregation.     

Platelet-activating factor increases endothelial [Ca2+]i and NO production in individually perfused intact microvessels

We have demonstrated that inhibition of NO synthase (NOS) in endothelial cells by either the NOS inhibitor N(omega)-monomethyl-l-arginine (l-NMMA) or the internalization of caveolin-1 scaffolding domain attenuated platelet-activating factor (PAF)-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 286: H195-H201, 2004) indicating the involvement of an NO-dependent signaling pathway. To investigate whether an increase in endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is the initiating event and Ca(2+)-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca(2+)](i) and NO production were measured in individually perfused rat mesenteric venular microvessels via fluorescence microscopy. When venular microvessels were exposed to PAF, endothelial [Ca(2+)](i) increased from 69 +/- 8 nM to a peak value of 374 +/- 26 nM within 3 min and then declined to a sustained level at 190 +/- 12 nM after 15 min. Inhibition of NOS did not modify PAF-induced increases in endothelial [Ca(2+)](i). PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increased fluorescence intensity (FI), which is an indication of increased NO production, occurred in 75 +/- 7% of endothelial cells in each vessel. The mean maximum FI increase was 140 +/- 7% of baseline value. This increased FI was abolished by pretreatment of the vessel with l-NMMA and attenuated in the absence of extracellular Ca(2+). These results provide direct evidence from intact microvessels that increased endothelial [Ca(2+)](i) is the initial signal that activates endothelial NOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.     

Mechanisms of hypotension produced by platelet-activating factor

Platelet-activating factor (PAF) is a phospholipid mediator that induces cardiovascular collapse and release of the secondary mediator thromboxane A2 (TxA2). To clarify mechanisms involved in this collapse and, specifically, the relative contribution of left ventricular and right ventricular dysfunction, we studied 12 open-chest pigs. PAF infusion (0.04-0.28 nmol.kg-1.min-1) induced a 5- to 120-fold increase in pulmonary vascular resistance, a 75-98% fall in cardiac output, and systemic arterial hypotension. Right ventricular failure was indicated by chamber enlargement, decreased shortening, and increased right atrial pressures. In contrast, left ventricular dysfunction was accompanied by decreases in chamber dimensions and filling pressures that were unresponsive to volume expansion. U 46619 (a stable TxA2 analogue) and mechanical pulmonary artery constriction induced changes similar to PAF. In 11 additional closed-chest pigs, TxA2 blockade with indomethacin attenuated the PAF-induced rise in pulmonary vascular resistance, right ventricular dysfunction, and systemic hypotension. A specific TxA2 synthase inhibitor, OKY-046, also diminished hemodynamic effects of PAF in six other pigs. Tachyphylaxis was not observed in five pigs repeatedly given PAF. We conclude that acute right ventricular failure as the result of severe increase in pulmonary vascular resistance is the primary mechanism early in the course of PAF-induced shock in the pig. PAF-induced release of TxA2 may contribute significantly to these events.     

Effects of thromboxane synthase and cyclooxygenase inhibition on PAF-induced changes in lung function and arachidonic acid metabolism.

PAF was administered as an intravenous bolus (0.1 micrograms/kg) to eight chronically instrumented awake sheep. The effects of pretreatment with an inhibitor of cyclooxygenase (meclofenamate) on PAF-induced changes in lung function were compared to those observed with a specific inhibitor of thromboxane synthase (DP1904). Each animal was studied four times in varied order: PAF alone, PAF + DP1904, PAF + meclofenamate, and DP1904 alone. Saline alone (control), DP1904 alone, and meclofenamate alone did not cause changes in any of the measured variables. DP1904 and meclofenamate significantly attenuated the PAF-induced fall in lung compliance, elevation in peak pulmonary artery pressure, and increased lung lymph flow. Both drugs abolished the PAF-induced increases in lung lymph thromboxane B2 concentrations. Meclofenamate, but not DP1904, blocked the rise in lymph 6-keto-PGF1 alpha. Although meclofenamate blocked the rise in lymph PGE2, DP1904 resulted in levels 2.7 times higher than PAF alone. We conclude that: (1) inhibition of thromboxane synthase is as effective as inhibition of cyclooxygenase in attenuating PAF-induced changes in lung function, and (2) thromboxane synthase inhibition results in augmented production of PGE2 following PAF administration in vivo.     

Platelet-activating factor stimulates ovine foetal pulmonary vascular smooth muscle cell proliferation: role of nuclear factor-kappa B and cyclin-dependent kinases

Abstract.   Objective : Platelet-activating factor (PAF) is implicated in pathogenesis of persistent pulmonary hypertension of the neonate (PPHN); PAF is a mitogen for lung fibroblasts. PAF's role in pulmonary vascular smooth muscle cell (PVSMC) proliferation and in hypoxia-induced pulmonary vein (PV) remodelling has not been established and mechanisms for PAF's cell-proliferative effects are not well understood. We investigated involvement of PAF and PAF receptors in PVSMC proliferation. Materials and methods : Cells from pulmonary arteries (SMC-PA) and veins (SMC-PV) were serum starved for 72 h in 5% CO₂ in air (normoxia). They were cultured for 24 h more in normoxia or 2% O₂ (hypoxia) in 0.1% or 10% foetal bovine serum with 5 µCi/well of [³H]-thymidine, with and without 10 n m PAF. Nuclear factor-kappa B (NF-κB), CDK2 and CDK4 protein expression, and their roles in cell proliferation control were studied. Results : PAF and hypoxia increased SMC-PA and SMC-PV proliferation. WEB2170 inhibited PAF-induced cell proliferation while lyso-PAF had no effect. SMC-PV proliferated more than SMC-PA and PAF plus hypoxia augmented NF-κB protein expression. NF-κB inhibitory peptide attenuated PAF-induced cell proliferation by 50% and PAF increased CDK2 and CDK4 protein expression. The data show that hypoxia and PAF up-regulate PVSMC proliferation via PAF receptor-specific pathway involving NF-κB, CDK2 and CDK4 activations. Conclusion : They suggest that in vivo , in foetal lung low-oxygen environment, where PAF level is high, proliferation of PVSMC will occur readily to modulate PV development and that failure of down-regulation of PAF effects postnatally may result in PPHN.     

L-NAME augments PAF-induced venoconstriction in isolated perfused livers of rat and guinea pig, but not mouse

Platelet-activating factor (PAF), one of vasoconstrictive lipid mediators, is involved in systemic anaphylaxis. On the other hand, nitric oxide (NO) is known to attenuate anaphylactic venoconstriction of the pre-sinusoids in isolated guinea pig and rat livers. However, it is not known whether NO attenuates PAF-induced hepatic venoconstriction. We therefore determined the effects of L-NAME, a NO synthase inhibitor, on PAF-induced venoconstriction in blood- and constant flow-perfused isolated livers of mice, rats and guinea pigs. The sinusoidal pressure was measured by the double occlusion pressure (Pdo), and was used to determine the pre- (Rpre) and post-sinusoidal (Rpost) resistances. PAF (0.01-1 microM) concentration-dependently caused predominant pre-sinusoidal constriction in all livers of three species studied. The guinea pig livers were the most sensitive to PAF, while the mouse livers were the weakest in responsiveness. L-NAME pretreatment selectively increased the basal Rpre in all of three species. L-NAME also significantly augmented the PAF-induced increases in Rpre, but not in Rpost, in rat and guinea pig livers. This augmentation was stronger in rat livers than in guinea pig livers at the high concentration of 0.1 microM PAF. However, L-NAME did not augment PAF-induced venoconstriction in mouse livers. In conclusion, in rat and guinea pig livers, NO may be released selectively from the pre-sinusoids in response to PAF, and then attenuate the PAF-induced pre-sinusoidal constriction. In mouse liver, PAF-induced venoconstriction is weak and not modulated by NO.     

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.     

Endothelial dysfunction in aged humans is related with oxidative stress and vascular inflammation

Vascular endothelial dysfunction occurs during the human aging process, and it is considered as a crucial event in the development of many vasculopathies. We investigated the underlying mechanisms of this process, particularly those related with oxidative stress and inflammation, in the vasculature of subjects aged 18–91 years without cardiovascular disease or risk factors. In isolated mesenteric microvessels from these subjects, an age‐dependent impairment of the endothelium‐dependent relaxations to bradykinin was observed. Similar results were observed by plethysmography in the forearm blood flow in response to acetylcholine. In microvessels from subjects aged less than 60 years, most of the bradykinin‐induced relaxation was due to nitric oxide release while the rest was sensitive to cyclooxygenase (COX) blockade. In microvessels from subjects older than 60 years, this COX‐derived vasodilatation was lost but a COX‐derived vasoconstriction occurred. Evidence for age‐related vascular oxidant and inflammatory environment was observed, which could be related to the development of endothelial dysfunction. Indeed, aged microvessels showed superoxide anions (O₂^?) and peroxynitrite (ONOO^?) formation, enhancement of NADPH oxidase and inducible NO synthase expression. Pharmacological interference of COX, thromboxane A₂/prostaglandin H₂ receptor, O₂^?, ONOO^?, inducible NO synthase, and NADPH oxidase improved the age‐related endothelial dysfunction. In situ vascular nuclear factor‐κB activation was enhanced with age, which correlated with endothelial dysfunction. We conclude that the age‐dependent endothelial dysfunction in human vessels is due to the combined effect of oxidative stress and vascular wall inflammation.     

Three-dimensional localization and quantification of PAF-induced gap formation in intact venular microvessels

Combining single-vessel perfusion technique with confocal microscopy, this study presents a new approach that allows three-dimensional visualization and quantification of endothelial gaps under experimental conditions identical to those used to measure permeability coefficients, endothelial calcium concentration, and nitric oxide production in individually perfused intact microvessels. This approach provides an efficient means for defining the transport pathways and cellular mechanisms of increased microvascular permeability during inflammation. Platelet-activating factor (PAF) was used to increase the permeability of individually perfused rat mesenteric venules. Fluorescent microspheres (FMs, 100 nm) were used as leakage markers, and confocal images were acquired at successive focal planes through the perfused microvessel. Perfusion of FMs under control conditions produced a thin, uniform layer of FMs in the vessel lumen, but in PAF-stimulated microvessels significant amounts of FMs accumulated at endothelial junctions. Reconstructed confocal images three-dimensionally delineated the temporal and spatial development of endothelial gaps in PAF-stimulated microvessels. The FM accumulation, quantified as the total fluorescence intensity per square micrometer of vessel wall, was 8.4 +/- 1.8 times the control value within 10 min of PAF perfusion and declined to 5.0 +/- 0.6 and 1.4 +/- 0.2 times the control value when FMs were applied 30 and 60 min after PAF perfusion. The changes in the magnitude of FM accumulation closely correlated with the time course of PAF-induced increases in hydraulic conductivity (L(p)), indicating that the opening and closing of endothelial gaps contributed to the transient increase in L(p) in PAF-stimulated microvessels. Electron microscopic evaluations confirmed PAF-induced gap formation and FM accumulation at endothelial clefts.     

Pulmonary vascular reactivity: effect of PAF and PAF antagonists.

We investigated the effects of two different platelet-activating factor (PAF) antagonists, SRI 63-441 and WEB 2086, on PAF-, angiotensin II-, and hypoxia-induced vasoconstrictions in isolated rat lungs perfused with a physiological salt solution. Bolus injection of PAF (0.5 micrograms) increased pulmonary arterial and microvascular pressures and caused lung edema. Both SRI 63-441, a PAF-analogue antagonist, and WEB 2086, a thienotriazolodiazepine structurally unrelated to PAF, completely blocked PAF-induced vasoconstriction and lung edema at 10(-5) M. At a lower concentration (10(-6) M), WEB 2086 was more effective than SRI 63-441. WEB 2086 also blocked the pulmonary vasodilation induced by low-dose PAF (15 ng) in blood-perfused lungs preconstricted with hypoxia. SRI 63-441 and CV 3988 (another PAF analogue antagonist), but not WEB 2086, caused acute pulmonary vasoconstriction at 10(-5) M and severe lung edema at a higher concentration (10(-4) M). PAF-induced but not SRI- or CV-induced pulmonary vasoconstriction and edema were inhibited by WEB 2086. In addition, SRI 63-441 potentiated angiotensin II- and hypoxia-induced vasoconstrictions. This effect of SRI 63-441 is not due to PAF receptor blockade because 1) addition of PAF (1.6 nM) to the perfusate likewise potentiated angiotensin II-induced vasoconstriction and 2) WEB 2086 did not cause a similar response. We conclude that both SRI 63-441 and WEB 2086 are effective inhibitors of PAF actions in the rat pulmonary circulation. However, antagonists with structures analogous to PAF (SRI 63-441 and CV 3988) can have significant pulmonary vasoactive side effects.     

Effects of platelet activating factor antagonists on arachidonic acid-induced platelet aggregation and TXA2 formation

The effects of the PAF receptor antagonists WEB 2086, WEB 2170, BN 50739 and BN 52021 on AA-induced platelet aggregation (PA) and TXA2 formation were investigated in comparison with the TXA2 synthetase inhibitor HOE 944 and the TXA2 receptor antagonist BM 13.177. All PAF antagonists tested were weak inhibitors of AA-induced PA and TXA2 formation (IC50 values between 80 and 2,737 mumol/l). HOE 944 was effective in concentrations 2-3 orders of magnitude lower than PAF antagonists in inhibiting TXA2 generation. These results imply that the inhibition of TXA2 formation is of minor relevance for the actions of the investigated PAF antagonists in AA-induced PA.     

Internalization of caveolin-1 scaffolding domain facilitated by Antennapedia homeodomain attenuates PAF-induced increase in microvessel permeability

We demonstrated previously that inhibition of endothelial nitric oxide synthase (NOS), using pharmacological inhibitors, attenuated the ionomycin- and ATP-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 272: H176-H185, 1997). Recently, the scaffolding domain of caveolin-1 (CAV) has been implicated as a negative regulator of endothelial NOS (eNOS). To examine the role of CAV-eNOS interaction in regulation of permeability in intact microvessels, the effect of internalized CAV on the platelet-activating factor (PAF)-induced permeability increase was investigated in rat mesenteric venular microvessels. Internalization of CAV was achieved by perfusion of individual vessels using a fusion peptide of CAV with Antennapedia homeodomain (AP-CAV) and visualized by fluorescence imaging and electron microscopy. Changes in microvessel permeability were evaluated by measuring hydraulic conductivity (Lp) in individually perfused microvessels. We found that the PAF (10 nM)-induced Lp increase was significantly attenuated from 6.0 +/- 0.9 (n = 7) to 2.0 +/- 0.3 (n = 5) times control after microvessels were perfused with 10 microM AP-CAV for 2 h. The magnitude of this reduction is comparable with that of the inhibitory effect of Nomega-monomethyl-l-arginine on the PAF-induced Lp increase. In contrast, perfusion with 10 microM AP alone for 2 h modified neither basal Lp nor the vessel response to PAF. These results indicate that CAV plays an important role in regulation of microvessel permeability. The inhibitory action of CAV on permeability increase might be attributed to its direct inactivation of eNOS. In addition, this study established a method for studying protein-protein interaction-induced functional changes in intact microvessels and demonstrated AP as an efficient vector for translocation of peptide across the cell membrane in vivo.     

Oxygen-dependent PAF receptor binding and intracellular signaling in ovine fetal pulmonary vascular smooth muscle

Circulating levels of platelet-activating factor (PAF) are high in the fetus, and PAF is active in maintaining high PVR in fetal hypoxia (Ibe BO, Hibler S, Raj J. J Appl Physiol 85: 1079-1085, 1998). PAF synthesis by fetal pulmonary vascular smooth muscle cells (PVSMC) is high in hypoxia, but how oxygen tension affects PAF receptor (PAF-r) binding in PVSMC is not known. We studied the effect of oxygen tension on PAF-r binding and signaling in fetal PVSMC. PAF binding was saturable. PAF-r density (B(max): fmol/10(6) cells; means +/- SE, n = 6), 25.2 +/- 0.77 during hypoxia (Po(2) <40 Torr), was higher than 13.9 +/- 0.44 during normoxia (Po(2) approximately 100 Torr). K(d) was twofold lower in hypoxia than normoxia. PAF-r protein expression, 35-40% greater in hypoxia, was inhibited by cycloheximide, a protein synthesis inhibitor, suggesting translational regulation. IP(3) release, an index of PAF-r-mediated cell signaling, was greater in hypoxia (EC(50): hypoxia, 2.94 +/- 0.61; normoxia, 5.85 +/- 0.51 nM). Exogenous PAF induced 50-90% greater intracellular calcium flux in cells during hypoxia, indicating hypoxia augments PAF-r-mediated cell signaling. PAF-r phosphorylation, with or without 5 nM PAF, was 40% greater in hypoxia. These data show 1) hypoxia upregulates PAF-r binding, PAF-r phosphorylation, and PAF-r-mediated intracellular signaling, evidenced by augmented IP(3) production and intracellular Ca(2+) flux; and 2) hypoxia-induced PAF-r phosphorylation results in activation of PAF-r-mediated signal transduction. The data suggest the fetal hypoxic environment facilitates PAF-r binding and signaling, thereby promoting PAF-mediated pulmonary vasoconstriction and maintenance of high PVR in utero.     

Effect of long-term high-altitude hypoxia on fetal pulmonary vascular contractility.

Hypoxia in the fetus and/or newborn is associated with an increased risk of pulmonary hypertension. The present study tested the hypothesis that long-term high-altitude hypoxemia differentially regulates contractility of fetal pulmonary arteries (PA) and veins (PV) mediated by differences in endothelial NO synthase (eNOS). PA and PV were isolated from near-term fetuses of pregnant ewes maintained at sea level (300 m) or high altitude of 3,801 m for 110 days (arterial Po(2) of 60 Torr). Hypoxia had no effect on the medial wall thickness of pulmonary vessels and did not alter KCl-induced contractions. In PA, hypoxia significantly increased norepinephrine (NE)-induced contractions, which were not affected by eNOS inhibitor N(G)-nitro-l-arginine (l-NNA). In PV, hypoxia had no effect on NE-induced contractions in the absence of l-NNA. l-NNA significantly increased NE-induced contractions in both control and hypoxic PV. In the presence of l-NNA, NE-induced contractions of PV were significantly decreased in hypoxic lambs compared with normoxic animals. Acetylcholine caused relaxations of PV but not PA, and hypoxia significantly decreased both pD(2) and the maximal response of acetylcholine-induced relaxation in PV. Additionally, hypoxia significantly decreased the maximal response of sodium nitroprusside-induced relaxations of both PA and PV. eNOS was detected in the endothelium of both PA and PV, and eNOS protein levels were significantly higher in PV than in PA in normoxic lambs. Hypoxia had no significant effect on eNOS levels in either PA or PV. The results demonstrate heterogeneity of fetal pulmonary arteries and veins in response to long-term high-altitude hypoxia and suggest a likely common mechanism downstream of NO in fetal pulmonary vessel response to chronic hypoxia in utero.     

Endothelin-induced mobilization of Ca2+ and the possible involvement of platelet activating factor and thromboxane A2

Endothelin (ET) caused transient and sustained elevations of cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat and rabbit vascular smooth muscle cells (VSMC). Specific platelet activating factor (PAF) antagonists (CV-6209 and WEB-2086) and arachidonic acid (AA) cascade blockers (chlorpromazine, indomethacin, CV-4151 and AA-2414) potently inhibited the ET-induced increase in [Ca2+]i. Additionally, these compounds inhibited the PAF-induced increase in [Ca2+]i. These results suggest that PAF and thromboxane A2 (TXA2) may be involved in the mechanism of ET-induced mobilization of Ca2+ in cultured rat and rabbit VSMC.     

Role of leukotriene C4 in pulmonary hypertension: platelet-activating factor vs. hypoxia

The aim of this study was to determine whether leukotriene C4 (LTC4) is a mediator of hypoxic pulmonary vasoconstriction. We hypothesized that similar increases in LTC4, detected in the lung parenchyma and pulmonary vascular compartment during cyclooxygenase blockade with indomethacin (INDO), would be observed during an equal increase in pulmonary arterial pressure caused by acute alveolar hypoxia (HYP, 100% N2) or platelet-activating factor (PAF, 10 micrograms into the pulmonary artery). Rat lungs were perfused at constant flow in vitro with an albumin-Krebs-Henseleit solution. Mean pulmonary arterial pressure (n = 6 per group) increased from a base line of 10.9 +/- 1.2 to 15.8 +/- 2.1 (HYP + INDO) and 15.5 +/- 1.9 (SE) Torr (PAF + INDO). LTC4 levels increased only in response to PAF + INDO; perfusate levels increased from 0.4 +/- 0.07 to 5.3 +/- 1.1 ng/40 ml, and lung parenchymal levels increased from 1.9 +/- 0.07 to 22.8 +/- 5.3 ng/lung. Diethylcarbamazine (lipoxygenase inhibitor) reduced PAF-induced lung parenchymal levels of LTC4 by 68% and pulmonary hypertension by 63%. We conclude that 1) LTC4 is not a mediator of hypoxic pulmonary vasoconstriction and 2) intravascular PAF is a potent stimulus for LTC4 production in the lung parenchyma.     

PAF antagonists: different effects on platelets, neutrophils, guinea pig ileum and PAF-induced vasolidation in isolated rat lung

The effects of structurally different PAF receptor blockers were investigated in platelets, neutrophils, guinea pig ileum, rat isolated lung and rat isolated pulmonary artery. PAF caused serotonin release from platelets and a characteristic shape change and adhesion of neutrophils. The antagonists (CV 3988, alprazolam, 48740 RP and Merck-Sharp and Dohme L-652, 731) inhibited platelet serotonin release but not neutrophil shape change adhesion or lysosomal enzyme release. The antagonists in high concentrations (10⁻⁵ −10⁻⁴M) inhibited nonspecifically the PAF-induced (10⁻⁸M) guinea pig ileum contraction, but were ineffective at concentrations which inhibited platelet responses. In the rat lung the compounds, in high concentrations, partially inhibited the low dose PAF-induced pulmonary vasodilation and the high dose PAF induced pulmonary vasoconstriction and edema. Our data indicate that some platelet PAF antagonists may be ineffective in blocking the action of PAF on neutrophils and smooth muscle preparations and suggest either PAF-receptor independent actions of PAF or different classes of PAF receptors.     

PAF antagonists: different effects on platelets, neutrophils, guinea pig ileum and PAF-induced vasodilation in isolated rat lung

The effects of structurally different PAF receptor blockers were investigated in platelets, neutrophils, guinea pig ileum, rat isolated lung and rat isolated pulmonary artery. PAF caused serotonin release from platelets and a characteristic shape change and adhesion of neutrophils. The antagonists (CV 3988, alprazolam, 48740 RP and Merck-Sharp and Dohme L-652, 731) inhibited platelet serotonin release but not neutrophil shape change adhesion or lysosomal enzyme release. The antagonists in high concentrations (10(-5)-10(-4)M) inhibited nonspecifically the PAF-induced (10(-8)M) guinea pig ileum contraction, but were ineffective at concentrations which inhibited platelet responses. In the rat lung the compounds, in high concentrations, partially inhibited the low dose PAF-induced pulmonary vasodilation and the high dose PAF induced pulmonary vasoconstriction and edema. Our data indicate that some platelet PAF antagonists may be ineffective in blocking the action of PAF on neutrophils and smooth muscle preparations and suggest either PAF-receptor independent actions of PAF or different classes of PAF receptors.