Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro.
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1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP. (+info)
Oxytocin-induced Ca2+ responses in human myometrial cells.
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Complex spatiotemporal changes in intracellular Ca2+ were monitored in an immortalized human myometrial cell line (PHM1-41) and first-passage human myometrial cells after oxytocin stimulation (1. 0-1000 nM). Laser cytometry revealed intracellular Ca2+ oscillations in both culture systems starting at 1.0 nM, which were followed by repetitive Ca2+ transients by 10-15 min that lasted for at least 90 min. The amplitude of the initial Ca2+ spike was dose dependent, while the frequency of Ca2+ oscillations identified by Fast Fourier Transform (FFT) tended to increase with dose. Removal of oxytocin resulted in termination of oscillations. Analysis of the sources of the Ca2+ involved in oscillations indicated that the major contribution to oscillation frequencies of +info)
Gestational regulation of granulocyte-colony stimulating factor receptor expression in the human placenta.
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A number of cytokines and their receptors are abundantly expressed at the materno-fetal interface and are thought to have a function in the regulation of placentation. Granulocyte-colony stimulating factor (G-CSF) is expressed by stromal cells in both placental tissue and maternal decidua throughout placentation. In this study, we examined the expression of placental G-CSF receptor (G-CSFR) mRNA and protein throughout gestation by ribonuclease protection assays, Western blotting, and immunohistochemistry. The major placental form of G-CSFR mRNA, corresponding to a membrane-bound form of the protein, was present in first-trimester placental tissues; levels decreased in second- and were highest in third-trimester placental tissues. Two placental G-CSFR molecules, 120 kDa and 150 kDa, were detected in first- and third-, but not second-, trimester tissues. The level of the 150-kDa G-CSFR was greater in the third- than in first-trimester samples. These differences were irrespective of whether or not the patients had received prostaglandin E1 analogues, prostaglandin E1 analogues and oxytocin, oxytocin alone, or mifepristone before labor. We demonstrated by immunohistochemistry that interstitial cytotrophoblast in first- and second-trimester decidual tissue and cytotrophoblast in term fetal membranes express G-CSFR. These data demonstrate that the expression of specific forms of placental G-CSFR is strictly cell type- and developmental stage-specific, and they suggest that G-CSFR may be important in decidual invasion of cytotrophoblast and in trophoblast function during placentation. (+info)
Transduction mechanisms of porcine chondrocyte inorganic pyrophosphate elaboration.
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OBJECTIVE: To investigate cellular signaling mechanisms that influence chondrocyte production of inorganic pyrophosphate (PPi), which promotes calcium pyrophosphate dihydrate (CPPD) crystal deposition. METHODS: Articular chondrocyte and cartilage cultures were stimulated with protein kinase C (PKC) activator and adenyl cyclase activator. Generation of extracellular PPi was measured. RESULTS: Adenyl cyclase activation resulted in diminished pyrophosphate generation. PKC activation stimulated pyrophosphate elaboration. CONCLUSION: Two signaling pathways, cAMP and PKC, modulate generation of extracellular pyrophosphate by cartilage and chondrocytes. They are novel targets for potentially diminishing extracellular pyrophosphate elaboration that leads to CPPD crystal deposition. (+info)
Effects of the prostanoid EP3-receptor agonists M&B 28767 and GR 63799X on infarct size caused by regional myocardial ischaemia in the anaesthetized rat.
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1. This study investigates the effects of two agonists of the prostanoid EP3-receptor (M&B 28767 and GR 63799X) on the infarct size caused by regional myocardial ischaemia and reperfusion in the anaesthetized rat. 2. One hundred and sixty-seven, male Wistar rats were anaesthetized (thiopentone, 120 mg kg(-1) i.p.), ventilated (8-10 ml kg(-1), 70 strokes min(-1), inspiratory oxygen concentration: 30%; PEEP: 1-2 mmHg) and subjected to occlusion of the left anterior descending coronary artery (LAD, for 7.5, 15, 25, 35, 45 or 60 min) followed by reperfusion (2 h). Infarct size was determined by staining of viable myocardium with a tetrazolium stain (NBT), histological evaluation by light and electron microscopy and determination of the plasma levels of cardiac troponin T. 3. M&B 28767 (0.5 microg kg(-1) min(-1), i.v., n=7) or GR 63799X (3 microg kg(-1) min(-1), i.v., n=7) caused significant reductions in infarct size from 60+/-3% (25 min ischaemia and 2 h reperfusion; saline-control, n=8) to 39+/-6 and 38+/-4% of the area at risk, without causing a significant fall in blood pressure. Pretreatment of rats with 5-hydroxydecanoate (5-HD), an inhibitor of ATP-sensitive potassium channels, attenuated the cardioprotective effects of both EP3-receptor agonists. The reduction in infarct size afforded by M&B 28767 was also abolished by glibenclamide and the protein kinase C (PKC) inhibitors staurosporine and chelerythrine. 4. Thus, M&B 28767 and GR 63799X reduce myocardial infarct size in the rat by a mechanism(s) which involves the activation of PKC and the opening of ATP-sensitive potassium channels. (+info)
Transcranial Doppler pattern after intracarotid papaverine and prostaglandin E1 incorporated in lipid microsphere in patients with vasospasm.
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We studied the effects of intracarotid papaverine and prostaglandin E1 incorporated in lipid microsphere (Lipo-PGE1) in relation with transcranial Doppler parameters such as mean flow velocity (MFV) and pulsatile index (PI) of the proximal segment of the middle cerebral artery. Eighty patients with subarachnoid hemorrhage (SAH) were included in this study. In the case of angiographic vasospasm, papaverine at 7 mg/min with total dose below 300 mg per artery and 10-20 micrograms of Lipo-PGE1 were injected in the supraclinoid portion of the internal carotid artery. Vasospasm was improved in 24 patients (63%), however, it was unchanged in 14 patients (37%). The former patients had more favorable outcomes than the latter patients (p < 0.005). After intracarotid injection therapy, the correlation between MFV and PI was classified into three types: type 1, both MFV and PI decreased; type 2, MFV decreased but PI increased; and type 3, both MFV and PI fluctuated. The Glasgow Outcome Scale 3 months after SAH was as follows: type 1 (n = 15), good in 14 (93%) and moderate disability in one (7%); type 2 (n = 9), good in eight (89%) and vegetative state in one (11%); and type 3 (n = 14), moderate disability in five (36%), severe disability in seven (50%), and death in two (14%). Chi-square analysis showed significant differences between type 1 and type 3 (p < 0.005), and type 2 and type 3 (p < 0.005). In conclusion, intracarotid papaverine combined with Lipo-PGE1 was effective for vasospasm but type 3 patients require a different treatment protocol. (+info)
Platelet-stimulated thrombin and PDGF are normalized by insulin and Ca2+ channel blockers.
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Coronary artery disease is accelerated in chronic spinal cord injury (SCI). Because prostacyclin (PGI2) may retard atherogenesis through its inhibitory effects on platelet function, the role of PGI2 on SCI platelets was determined. The SCI platelets were neither hypersensitive to aggregating agonists nor resistant to the inhibitory effect of PGI2, but PGI2 failed to inhibit platelet-stimulated thrombin generation and the release of platelet-derived growth factor (PDGF) in SCI. Because thrombin and PDGF are atherogenic mitogens, the generation of these mitogens was investigated. Both the release of PDGF and thrombin generation in SCI platelets were higher when compared with control (n = 12). Treatment of non-SCI platelets with 100 nM PGE1 (a stable probe of PGI2) inhibited the release of the mitogens by 90% (P < 0.001), with no effect on SCI platelets. Scatchard analysis of prostaglandin E1 (PGE1) binding showed a 70% decrease of PGI2 receptors on the SCI platelet surface. Treatment of SCI platelets with insulin or Ca2+ channel blockers restored the PGI2-receptor number and "normalized" the inhibition of PDGF release and thrombin generation by PGI2. (+info)
Seminal plasma components stimulate interleukin-8 and interleukin-10 release.
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Human seminal plasma has potent anti-inflammatory properties which are thought to confer a survival advantage to the spermatozoa within the hostile female genital tract. In contrast, a profound pro-inflammatory leukocytosis has been observed post-coitus in animals and humans. Whether components of seminal plasma are involved in initiating this leukocytic reaction is not known. This study investigated the effect of human seminal plasma, a seminal plasma fraction and its principal constituent prostaglandins, prostaglandin E2 (PGE2) and 19-hydroxy PGE, on the release of the pro-inflammatory neutrophil chemotactic factor interleukin-8 (IL-8) and the anti-inflammatory cytokines interleukin-10 (IL-10) and secretory leukocyte protease inhibitor (SLPI). The tissues studied were non-pregnant cervical explants, peripheral blood and the monocyte cell line U937. Seminal plasma fraction (SPF) significantly (P < 0.05) stimulated release of IL-8 and inhibited release of SLPI from non-pregnant cervical explants. SPF, PGE2 and 19-hydroxy PGE significantly (P< 0.005) stimulated IL-8 release from peripheral blood and U937 cells. 19-hydroxy PGE was significantly (P< 0.005) more effective than PGE2 in stimulating IL-8 release. Seminal plasma, SPF and PGE2 significantly (P < 0.05) stimulated IL-10 release from U937 cells. 19-hydroxy PGE stimulated IL-10 release from U937 cells but this failed to reach significance. Release of IL-10 by cervical explants and SLPI by peripheral blood and U937 cells were below the detection limit of the assays employed. We suggest that the anti- and pro-inflammatory immune responses which seminal plasma induces might act in combination initially to promote sperm survival and then to facilitate their removal from the female genital tract. (+info)