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V Evangelista

Publications and source records attributed to V Evangelista.

At least 19 recordsLinked to original sources
PubMed2005

Oxidative stress and cardiovascular risk: the role of vascular NAD(P)H oxidase and its genetic variants.

Several risk factors for coronary artery disease (CAD) induce atherosclerosis through endothelial activation and dysfunction, and ample evidence now suggests that the balance between production and removal of reactive oxygen species (ROS) - a condition termed oxidative stress - is implicated in such processes. A main source of ROS in vascular cells is the reduced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase system. This is a membrane-associated enzyme, composed of five subunits, catalyzing the one-electron reduction of oxygen, using NADH or NADPH as the electron donor. One of the system subunits, termed p22-phox, has a polymorphic site on exon 4, associated with variable enzyme activity. This polymorphism is generated by a point mutation (C(242)T) producing a substitution of histidine with tyrosine at position 72, which affects one of the heme binding sites essential for the NAD(P)H enzyme activity. The consequent decrease of superoxide production thus characterizes a phenotype candidate for conferring to the carrier a reduced susceptibility to CAD. At present, however, the body of evidence from current literature is not yet sufficient to confirm or exclude the hypothesis that the C(242)T polymorphism protects from CAD. The functional effects of this polymorphism and the potential and its pathophysiological consequences also need further investigation.

Coronary Artery Disease↗
PubMed2003

TxA2-mediated myocardial ischemia as a consequence of an acute lung inflammatory reaction in the rabbit.

Epidemiological studies link acute infection of the respiratory tract to a transient increased risk of acute myocardial infarction. The underlying mechanisms remain unknown. We hypothesized that vasoactive mediators produced by inflammatory cells in the lungs and drained in the coronary circulation may trigger acute myocardial ischemia. To test this hypothesis we used an experimental model in the rabbit. Injection of the bacterial-derived peptide N-formyl-Met-Leu-Phe (or N-formyl-Methionyl-Leucyl-Phenylalanine)(fMLP) in the jugular vein induced massive recruitment of both polymorphonuclear leukocytes (PMN) and platelets in the microcirculation of the lungs, accompanied by rapid and marked increase of leukotriene B4, cysteinyl leukotrienes and thromboxane (Tx) A2 in the aortic blood. In all animals, fMLP evoked ischemic electrocardiographic changes: within the first minute of infusion a profound depression of the ST segment and inversion of the T wave were observed. Mean aortic pressure and heart rate fell to 64.0 +/- 6.9 and 83.5 +/- 3.1% of the basal levels at 3 and 10 min, respectively. All these alterations were transient. Aspirin, prevented electrocardiographic ischemic changes, reverted bradycardia and hypotension but did not significantly modify either PMN or platelet recruitment nor leukotriene synthesis. Ridogrel, a Tx-synthase and receptor inhibitor, prevented ECG alterations and bradycardia, but did not prevent and even worsened hypotension; it blocked platelet, but not PMN, sequestration. Pretreatment of animals with intravenous high dose of aspirin prevented ridogrel-dependent hypotension and platelet inhibition, suggesting that PGI2 contributes to the effects of Tx-synthase and receptor inhibitor. In hypercholesterolemic rabbits, ECG alterations persisted longer than in normal controls. In summary, our results indicate that acute activation of PMN and platelets in the lungs provokes transient myocardial ischemia, in normal animals that is exacerbated in hypercholesterolemic rabbits. TxA2 appears to be the major mediator of this phenomenon. Moreover the data suggest that a balance between TxA2 and PGI2 plays a pivotal role in platelet activation and recruitment in our model.

Acute Disease↗
PubMed2003

Role of P-selectin, beta2-integrins, and Src tyrosine kinases in mouse neutrophil-platelet adhesion.

BACKGROUND: The initial interaction of human polymorphonuclear leukocytes (PMN) with activated human platelets is mediated by P-selectin and its leukocyte ligand PSGL-1; subsequently the interaction is strengthened by activation of alphaMbeta2 via protein tyrosine phosphorylation mediated by Src kinases and binding of activated alphaMbeta2 to its platelet counterreceptor(s). OBJECTIVES: Because mouse models are being used to define the role of PMN-platelet interactions in thrombosis and the response to vascular injury, we investigated the molecular determinants responsible for the interaction of murine PMNs with activated murine platelets. METHODS: Mouse platelets were labeled with the green fluorescent dye BCECF and then activated with thrombin and fixed with 1% paraformaldehyde. Mouse PMNs were labeled with the red fluorescent dye hydroethidine and then stirred with the fixed platelets. After stopping the reaction with paraformaldehyde, formation of mixed cell conjugates was analyzed by flow cytometry. RESULTS: In time course experiments, 90 +/- 1.9% of PMNs formed mixed conjugates with platelets after 2 min and the mean (+/- SEM) number of platelets per positive PMN was 8.4 +/- 1.5. A monoclonal antibody to P-selectin reduced the percentage of PMNs with attached platelets to 16 +/- 2.4% (P = 0.001), and only 8 +/- 5% of PMNs interacted with platelets from P-selectin-/- mice. In contrast, monoclonal antibodies to PSGL-1, beta2-integrin, and alphaIIbbeta3 had much less or no effect on the production of mixed cell aggregates. To better identify a secondary contribution of beta2-integrins, P-selectin interactions were disrupted by briefly adding 5 mm EGTA to already-formed mixed cell aggregates. Brief EGTA treatment alone reduced the percentage of PMNs with attached platelets to 70 +/- 3.5% (P = 0.004 vs. no treatment), but did not modify the number of platelets per positive PMN (9.5 +/- 1.7). The combination of brief EGTA treatment and a monoclonal antibody to beta2-integrin lowered the percentage of PMN with attached platelets to 50 +/- 7% and reduced the number of platelets attached per positive PMN to 3.6 +/- 0.7 (P = 0.03 vs. brief EGTA treatment only). Brief EGTA treatment did not modify the effect of the other antibodies. When the incubation was stopped with EGTA the Src inhibitors PP1 and PP2 reduced PMN-platelet adhesion, while the inactive analog PP3 was ineffective. CONCLUSIONS: These results confirm that P-selectin plays a prominent role in mediating the initial interactions between mouse PMN and platelets, and provide support for additional contributions from beta2-integrins and Src family kinases.

Animals↗
PubMed2001

Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin.

Adhesion of polymorphonuclear leukocytes (PMNLs) to activated platelets requires a P-selectin-triggered, tyrosine kinase-dependent adhesiveness of Mac-1 and is accompanied by tyrosine phosphorylation of a 110-kd protein (P-110) in PMNLs. Inhibitors of SRC tyrosine kinases were found to inhibit PMNL adhesion to activated platelets or to P-selectin expressing Chinese hamster ovary (CHO-P) cells and the tyrosine phosphorylation of P-110. Adhesion of PMNLs to activated platelets or to CHO-P cells stimulated activity of LYN and HCK. Monoclonal antibody blockade of P-selectin or beta2-integrins reduced the activation of both kinases. In PMNLs either adherent to platelets or aggregated by P-selectin-IgG chimera, Mac-1 was rapidly redistributed to the Triton X-100-insoluble cytoskeletal fraction, and large clusters of Mac-1 colocalized with patches of F-actin at the sites of cell-cell contact. In PMNLs stimulated by P-selectin-IgG chimera, SRC kinase inhibition impaired Mac-1 clustering, F-actin accumulation, and CD18 redistribution to the cytoskeleton. Disruption of the actin filament network by cytochalasin D prevented PMNL-platelet adhesion and P-selectin-induced PMNL aggregation and impaired the clustering of Mac-1. In agreement with the requirement for the beta2-integrin in the functional up-regulation of LYN and HCK, integrin blockade by monoclonal antibodies resulted in a complete inhibition of P-selectin-induced Mac-1 clustering and F-actin accumulation. Taken together, the results indicate that, after an initial P-selectin-triggered beta2-integrin interaction with the ligand, SRC kinases are activated and allow the remodeling of cytoskeleton-integrin linkages and integrin clustering that finally strengthen cell-cell adhesion. This model highlights a new role for SRC kinases in a regulatory loop by which the Mac-1 promotes its own adhesive function.

Actins↗
PubMed2000

Polymorphonuclear leukocyte activation and hemostasis in patients with essential thrombocythemia and polycythemia vera.

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation. (Blood. 2000;96:4261-4266)

Adult↗
PubMed2000

Polymorphonuclear leukocyte apoptosis is inhibited by platelet-released mediators, role of TGFbeta-1.

Platelets regulate several polymorphonuclear leukocyte (PMN) functions. We have found that thrombin-stimulated platelets potently inhibited PMN apoptosis. Cell-free supernatant from increasing concentrations of stimulated platelets inhibited PMN apoptosis in a dose-dependent manner, with an effect similar to that of corresponding concentrations of platelets. At the plateau, platelet supernatant inhibited PMN apoptosis by 54.6 +/- 6.8%, the anti-apoptotic activity being higher than that of GM-CSF and comparable to that of LPS. Neither IL-1ra nor a combination of anti-IL1alpha + betamAb affected the activity of platelet supernatant. In contrast a mAb recognizing the active form of TGF-beta1 significantly decreased this activity. Moreover, exogenous TGF-beta1 inhibited PMN apoptosis in a dose-dependent manner. The active form of this cytokine was indeed present in the supernatant of stimulated platelets at a concentration able to elicit an anti-apoptotic effect. The p38 MAPK inhibitor SB203580 prevented the anti-apoptotic effect of TGF-beta1 in a dose-dependent manner. Interestingly, it also prevented the anti-apoptotic effect of IL-1alpha, but not that of GM-CSF, LPS and dexamethasone. In conclusion, we report for the first time that PMN apoptosis is potently inhibited by platelet-released mediators, that TGF-beta1 mediates an important part of this effect, and that p38 MAPK is involved in the TGF-beta1 signaling leading to its anti-apoptotic effect. These results provide novel evidence to support the central role of platelets in inflammation.

Apoptosis↗
PubMed1999

Neutrophil activation and hemostatic changes in healthy donors receiving granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) enhances neutrophil functions in vitro and in vivo. It is known that neutrophil-derived products can alter the hemostatic balance. To understand whether polymorphonuclear leukocyte (PMN) activation, measured as PMN degranulation and phenotypical change, may be associated to hemostatic alterations in vivo, we have studied the effect of recombinant human G-CSF (rHuG-CSF) administration on leukocyte parameters and hemostatic variables in healthy donors of hematopoietic progenitor cells (HPCs). Twenty-six consecutive healthy donors receiving 10 micrograms/kg/d rHuG-CSF subcutaneously for 5 to 7 days to mobilize HPCs for allogeneic transplants were included in the study. All of them responded to rHuG-CSF with a significant white blood cell count increase. Blood samples were drawn before therapy on days 2 and 5 and 1 week after stopping rHuG-CSF treatment. The following parameters were evaluated: (1) PMN activation parameters, ie, surface CD11b/CD18 antigen expression, plasma elastase antigen levels and cellular elastase activity; (2) plasma markers of endothelium activation, ie, thrombomodulin (TM) and von Willebrand factor (vWF) antigens; (3) plasma markers of blood coagulation activation, ie, F1+2, TAT complex, D-dimer; and (4) mononuclear cell (MNC) procoagulant activity (PCA) expression. The results show that, after starting rHuG-CSF, an in vivo PMN activation occurred, as demonstrated by the significant increment of surface CD11b/CD18 and plasma elastase antigen levels. Moreover, PMN cellular elastase activity, which was significantly increased at 1 day of treatment, returned to baseline at day 5 to 6, in correspondence with the elastase antigen peak in the circulation. This change was accompanied by a parallel significant increase in plasma levels of the two endothelial and the three coagulation markers. The PCA generated in vitro by unstimulated MNC isolated from rHuG-CSF-treated subjects was not different from that of control cells from untreated subjects. However, endotoxin-stimulated MNC isolated from on-treatment individuals produced significantly more PCA compared with both baseline and control samples. All of the parameters were decreased or normal 1 week after stopping treatment. These data show that rHuG-CSF induces PMN activation and transiently affects some hemostatic variables in healthy HPC donor subjects. The clinical significance of these findings remains to be established.

Adolescent↗
PubMed1999

Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule.

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated beta2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P approximately 110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P approximately 110. The inhibition of P approximately 110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin-transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin-IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin-IgG-triggered PMN/PMN aggregation as well as P approximately 110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered beta2-integrin-dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the beta2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P approximately 110.

Adult↗
PubMed1999

Recent advances in platelet-polymorphonuclear leukocyte interaction.

Epidemiological evidence suggests a positive correlation between the number of PMN and the risk of ischemic vascular disease. The observation that activated PMN induce platelet activation my provide some biological plausibility to the role of PMN in thrombogenesis. Between other PMN products, cathepsin G, a protease released during PMN activation, is a potent platelet agonist. However, the antiproteinases present in plasma could virtually abolish its activity. Indeed it was shown that, when PMN were stimulated after interaction with platelets in mixed cell population, P-selectin-mediated platelet-PMN adhesion may result in the formation of a sequestered microenvironment in which cathepsin G activity is protected by antiproteases. P-selectin-mediated adhesion was also shown to facilitate the transcellular metabolism of arachidonic acid, resulting in increased production of both thromboxane B2 and leukotriene C4. PMN adhesion to activated platelets in mixed cell suspensions subjected to high shear rate can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step followed by an adhesion-strengthening interaction mediated by the beta(2)-integrin Mac-1. Moreover, an intermediate tyrosine-kinase-dependent signal regulating beta(2)-integrin adhesiveness is required. Indeeed activated platelets express not only P-selectin but also different beta(2)-integrin ligands including fibrinogen and ICAM-2. Some of the functional responses elicited by P-selectin on PMN could be prevented by specific antibody to the P-selectin glycoprotein ligand-1, indicating that this adhesive receptor is able to transduce an 'outside-in' signal when engaged by the ligand. By using activated platelets, P-selectin-expressing CHO cells and soluble recombinant P-selectin, P-selectin was shown to trigger protein tyrosine phosphorylation in PMN and the tyrosine kinase-dependent function of Mac-1. In conclusion, adherence of activated platelets to PMN may be a key event in the sequence of thrombus formation. The recognition of the essential contribution of PMN beta(2)-integrins in addition to P-selectin in platelet-PMN adhesion provides an additional evidence to the broad range of function and mechanisms in which PMN integrins are involved and may be potential targets for pharmacological intervention.

Animals↗
PubMed1999

Platelet contribution to leukotriene production in inflammation: in vivo evidence in the rabbit.

The contribution of platelets to arachidonic acid transcellular metabolism may represent an important pathway of leukotriene (LT) production. The aim of this study was to investigate the role of platelets on LT production in an acute inflammatory model in the rabbit. Preliminary experiments showed that rabbit whole blood (5 ml) stimulated in vitro with the calcium ionophore A23187 produced LTB4 (52.7+/-13.9 ng) and the mixed 5,12-DiHETE (7.25+/-0.75 ng). In A23187-stimulated thrombocytopenic blood, LTB4 was significantly reduced to 19.5+/-8.6 ng and 5,12-DiHETE was undetectable. Peptido-LTs were undetectable in both conditions. In experiments using washed cells, addition of thrombin-activated platelets to fMLP-activated PMN resulted in the appearance of 5,12-DiHETE and in more than twofold increase of LTB4 synthesis. When 3H-arachidonic acid-labelled platelets were mixed with unlabelled PMN and challenged with fMLP and thrombin, radioactive LTB4 and 5,12-DiHETE were produced, indicating that platelet-derived arachidonic acid was utilized by PMN 5-lipoxygenase. Intravenous infusion of fMLP (2.5 nmol/kg/min) in the rabbit induced marked granulocytopenia, thrombocytopenia and increased TxB2 plasma concentrations within 3 min. Electron microscopy of lungs showed morphologically activated and aggregated platelets occluding the capillary lumen. Activation and recruitment of circulating cells was accompanied by the production of LTB4 (peak levels at 1 min: 30.0+/-9.5 ng/ml) and LTE4 (peak levels at 10 minutes: 77.8+/-11.6 ng/ml). The areas under the blood concentration-time curve (AUC, ng min/ml) corresponded to 812+/-182 and 3692+/-658 for LTB4 and LTE4, respectively. In immunologically thrombocytopenic rabbits, the AUC for LTB4 (86.0+/-23.0) and LTE4 (1165+/-542) were both significantly different from controls while in rabbits treated with an anti-leukocyte antiserum, both LTB4 and LTE4 were similar to controls. This experimental model provides in vivo evidence that platelets, involved in an acute inflammatory event contribute to the transcellular production of LTs.

Animals↗
PubMed1999

COX-2 is not involved in thromboxane biosynthesis by activated human platelets.

The occurrence of aspirin resistance has been inferred by the assessment of platelet aggregation ex vivo in patients with ischemic vascular syndromes taking aspirin. Since aspirin is a weak inhibitor of the inducible isoform of prostaglandin H synthase (COX-2), it was suggested that COX-2 may play a role in aspirin resistance. However the cellular source(s) of COX-2 possibly responsible for aspirin resistance remains unknown. Recently, the expression of the inducible isoform of COX-2 in circulating human platelets was reported. To investigate the possible contribution of COX-2 expression in platelet thromboxane (TX) biosynthesis, we have compared the inhibitory effects of NS-398 and aspirin, selective inhibitors of COX-2 and COX-1, respectively, on prostanoid biosynthesis by thrombin-stimulated platelets vs lipopolysaccharide (LPS)stimulated monocytes (expressing high levels of COX-2) isolated from whole blood of healthy subjects. NS-398 was 180-fold more potent in inhibiting monocyte COX-2 activity than platelet TXB2 production. In contrast, aspirin (55 micromol/L) largely suppressed platelet TXB2 production without affecting monocyte COX-2 activity. By using specific Western blot techniques, we failed to detect COX-2 in platelets while COX-1 was readily detectable. Our results argue against the involvement of COX-2 in TX biosynthesis by activated platelets and consequently dispute platelet COX-2 expression as an important mechanism of aspirin resistance.

Adult↗
PubMed1998

Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function.

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM, mean+/-s.e.mean), as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM, IC50 18.4+/-1.8 and 31+/-1.8 microM), and C5a (0.1 microM, IC50 41.6+/-3.5 and 42+/-8.3 microM), and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4), 6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.

Blood Platelets↗
PubMed1998

Extracellular signal-regulated kinases modulate capacitation of human spermatozoa.

Recent evidence indicates the presence of p21 Ras and of a protein with characteristics similar to mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), in mammalian spermatozoa, suggesting the occurrence of the Ras/ERK cascade in these cells. In the present study we investigated the subcellular localization of ERKs and their biological functions in human spermatozoa. Immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy demonstrated localization of ERKs in the postacrosomal region of spermatozoa. After stimulation of acrosome reaction with the calcium ionophore A23187 and progesterone, ERKs were mostly localized at the level of the equatorial region, indicating redistribution of these proteins in acrosome-reacted spermatozoa. Two proteins of 42 and 44 kDa that are tyrosine phosphorylated in a time-dependent manner during in vitro capacitation were identified as p42 (ERK-2) and p44 (ERK-1) by means of specific antibodies. The increase in tyrosine phosphorylation of these proteins during capacitation was accompanied by increased kinase activity, as determined by the ability of ERK-1 and ERK-2 to phosphorylate the substrate myelin basic protein. The role of this activity in the occurrence of sperm capacitation was also investigated by using PD098059, an inhibitor of the MAPK cascade. The presence of this compound during in vitro capacitation inhibits ERK activation and significantly reduces the ability of spermatozoa to undergo the acrosome reaction in response to progesterone. Since only capacitated spermatozoa are able to respond to progesterone, these data strongly indicate that ERKs are involved in the regulation of capacitation. In summary, our data demonstrate the presence of functional ERKs in human spermatozoa and indicate that these enzymes are involved in activation of these cells during capacitation, providing new insight in clarifying the molecular mechanisms and the signal transduction pathways of this process.

Acrosome↗
PubMed1997

Kinetics of endogenous leukotriene B4 and E4 production following injection of the chemotactic peptide FMLP in the rabbit.

The kinetic profiles of leukotriene B4 (LTB4) and E4 (LTE4) after intravenous administration (30 nmol/kg) of the inflammatory peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) were evaluated in male rabbits. LTB4 and LTE4 reached the maximal concentration of 84.2 +/- 60.0 and 162.2 +/- 51.4 nmol/L (mean +/- s.d.), at 2 and 5 min, respectively. The first elimination phase for LTB4 and LTE4, after FMLP administration, showed an apparent half-life of 24.6 +/- 6.7 and 36.9 +/- 13.0 min, respectively. The area under the blood concentration-time curve (AUC, nmol min/L) of LTB4 and LTE4 was 2178 +/- 1591 and 7627 +/- 3052, respectively. LTE4 and N-ac-LTE4 were the major components excreted in the urine, mostly in the first time interval (0-12 h) of urinary collection after FMLP treatment; 11-trans-LTE4 was recovered in the second interval (12-24 h). Two other more polar compounds, potential metabolites, were recovered in the first interval of urine collection. Knowledge of the kinetic characteristics of endogenously produced leukotrienes may be useful in understanding the role of these eicosanoids in inflammatory and thrombotic disease, as well as in evaluating the efficacy of drugs designed to modulate their production and effect.

Animals↗