Morphine preconditioning attenuates neutrophil activation in rat models of myocardial infarction.
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Previous results from our laboratory have suggested that morphine can attenuate neutrophil activation in patients with acute myocardial infarction. To elucidate if morphine preconditioning (PC) has the same effects via activation of neutrophil endopeptidase 24.11 (NEP), we measured serum levels of intercellular adhesion molecule-1 (ICAM-1), gp100MEL14 and NEP in adult Wistar rats subjected to ten different protocols (n = 10 for each) at baseline, immediately after and 2 h after morphine PC. All groups were subjected to 30 min of occlusion and 2 h of reperfusion. Similarly, morphine-induced PC was elicited by 3-min drug infusions (100 micrograms/kg) interspersed with 5-min drug-free periods before the prolonged 30-min occlusion. Infarct size (IS), as a percentage of the area at risk (AAR), was determined by triphenyltetrazolium staining. Pretreatment with morphine increased NEP activities (9.86 +/- 1.98 vs. 5.12 +/- 1.10 nmol/mg protein in control group; p < 0.001). Naloxone (mu-opioid receptor antagonist) (4.82 +/- 1.02 nmol/mg protein) and phosphoramidon (NEP inhibitor) (4.66 +/- 1.00 nmol/mg protein) inhibited morphine-activated NEP, whereas glibenclamide (ATP-sensitive potassium channel antagonist) and chelerythrine (protein kinase C inhibitor) had no effects. The ICAM-1 and gp100MEL14 of the third sampling were lowest for those with morphine PC (280 +/- 30 ng/ml and 2.2 +/- 0.7 micrograms/ml; p < 0.001), but naloxone (372 +/- 38 ng/ml and 3.8 +/- 0.9 micrograms/ml) and phosphoramidon (382 +/- 40 ng/ml and 4.2 +/- 1.1 micrograms/ml) abolished the above phenomenon. IS/AAR were definitely lowest for those with morphine PC (24 +/- 7%; p < 0.05). Morphine preconditioning increases NEP activities to attenuate shedding of gp100MEL14 and to ICAM-1 and, thus, provides myocardial protection. (+info)
Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells.
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Signals transduced through the B cell Ag receptor (BCR) drive B cell development. However, BCR-induced responses are developmentally regulated; immature B cells are tolerized following antigenic exposure while mature B cells are triggered to proliferate and differentiate. This differential responsiveness allows for the negative selection of self-reactive immature B cells while simultaneously allowing for clonal expansion of mature B cells in response to foreign Ags. Intrinsic differences in BCR-induced signal transduction at various stages of development may account for this functional dichotomy. We had previously demonstrated that the BCR-induced proliferation of mature B cells is accompanied by an increase in intracellular calcium levels and polyphosphoinositide bis phosphate (PIP2) hydrolysis. In contrast, immature B cells that undergo BCR-induced apoptosis increase intracellular calcium in the relative absence of PIP2 hydrolysis. Since PIP2 hydrolysis leads to the generation of diacylglycerol, a cofactor for protein kinase C (PKC) activation, these data suggested that an "imbalance" in BCR-induced signal transduction resulting from a relative inability to activate PKC may play a role in the susceptibility of immature B cells to BCR-induced apoptosis. In support of this hypothesis, we demonstrate that PKC activation can rescue immature B cells from BCR-induced apoptosis. Furthermore, the susceptibility of immature B cells to BCR-induced apoptosis is recapitulated in mature B cells that are either PKC depleted or are stimulated in the presence of PKC inhibitors, suggesting that an uncoupling of PKC activation from BCR-induced signaling is responsible for the apoptotic response of immature B cells. (+info)
A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages.
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This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC zeta, and this activation was inhibited by D609. LPS-activated PKC zeta phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS. (+info)
(-)-Stepholidine enhances K+ depolarization-induced activation of synaptosomal tyrosine 3-monooxygenase from rat striatum.
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AIM: To study the mechanism of K+ depolarization-induced activation of synaptosomal tyrosine 3-monooxygenase (TM) in rat striatum and the effect of (-)-stepholidine (SPD) on this activation. METHODS: The TM was assayed for DOPA by HPLC-ECD; the activities of Ca2+/calmodulin (CaM)-dependent protein kinase (PK II) and Ca2+/phosphoinositide-dependent protein kinase (PKC) were assayed using histidine as substrate. RESULTS: The incubation of striatal synaptosomes in K(+)-riched (60 mmol.L-1) medium resulted in a marked activation of TM. PKC inhibitor polymyxin B (PMB) completely blocked the activation of K+ 60 mmol.L-1 on TM. Selective D2 receptor agonist quinpirole (QP), Ca2+ removal from incubation medium and CaM antagonist W7 failed to affect the activation. However, SPD enhanced the activation of K+ 60 mmol.L-1 on TM. Meanwhile, the incubation in K+ 60 mmol.L-1 also activated PKC. Neither QP nor SPD affected K+ depolarization-induced activation of PKC. CONCLUSION: The activation of K+ depolarization on synaptosomal TM is enhanced by SPD and this activation is mediated by PKC rather than by PK II. (+info)
Effects of Ro 31-8220 on lipopolysaccharides-induced hepatotoxicity and release of tumor necrosis factor from rat Kupffer cells.
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AIM: To investigate protein kinase C (PKC) functions on lipopolysaccharide (LPS)-induced hepatotoxicity, a new potent PKC inhibitor Ro 31-8220 (Ro) was used to detect its effect on LPS-induced hepatotoxicity in rat hepatocytes and tumor necrosis factor (TNF) release from rat Kupffer cells (KC). METHODS: Hepatocytes (containing KC) were incubated with LPS (10 mg.L-1) and Ro (0.1-10 mumol.L-1) for 24 h, alanine aminotransferase (AlaA) leakage in the culture as indication of hepatotoxicity. The TNF activity in the supernatant of rat KC culture with LPS in the presence of Ro (0.1-10 mumol.L-1) was monitored by the L929 target cell lytic assay. RESULTS: Ro (0.1-10 mumol.L-1) reduced AlaA leakage in the hepatocyte culture. Ro inhibited dose-dependently the LPS-induced TNF production from rat KC. CONCLUSION: PKC inhibitor Ro protects the hepatocytes from LPS-induced cytotoxicity and inhibits the LPS-induced TNF production from rat KC. (+info)
Attenuation of myocardial injury due to oxygen free radicals (OFR) by pretreatment with OFR or calcitonin gene-related peptide.
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AIM: To study the cardioprotective effects of oxygen free radicals (OFR) and calcitonin gene-related peptide (OGRP) pretreatment on myocardial damages due to OFR in isolated perfused rat heart. METHODS: The hearts were perfused in a Langendorff mode. OFR were generated by electrolysis of Krebs-Henseleit (K-H) solution. RESULTS: OFR pretreatment reduced the impairment of cardiac contractile function, the decrease of coronary flow and the increase of creatinine kinase (CK) release due to OFR, and the effect exhibited period dependence and cycle-dependence. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, abolished the protection of OFR pretreatment (CK release = 110 +/- 7, 215 +/- 23, 169 +/- 14, 240 +/- 30, and 113 +/- 19 kU.L-1 for control, OFR, OFR pretreatment, OFR pretreatment plus H-7, and H-7, respectively). CGRP pretreatment also protected the myocardium damages elicited by OFR in isolated perfused rat heart. CONCLUSIONS: OFR or CGRP pretreatment protected myocardium against injury elicited by OFR, and the effect of OFR pretreatment was related to the activation of PKC. (+info)
Effect of quercetin on activities of protein kinase C and tyrosine protein kinase from HL-60 cells.
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AIM: To study the effect of quercetin (Que) on the activities of cytosol and membrane protein kinase C (PKC) and tyrosine protein kinase (TPK) from HL-60 cells in vitro. METHODS: The number of viable cells was counted by a trypan blue dye exclusion test. PKC activity was assayed by incubating PKC with histone III S and [gamma-32P]ATP. TPK activity was assayed by incubating TPK with poly glutamate.tyrosine (4:1). RESULTS: Que inhibited the proliferation of HL-60 cells in a concentration-dependent manner, its IC50 was 29 (22-37) mumol.L-1 after 48-h treatment; Que strongly inhibited the activity of cytosol PKC and membrane TPK with IC50 31 (20-48) mumol.L-1, 24 (13-45) mumol.L-1, respectively, but did not affect membrane PKC and cytosol TPK from HL-60 cells in vitro. CONCLUSION: The inhibitory effect of Que on the growth of tumor cells is related to its inhibitory effects on PKC and/or TPK. (+info)
Estrogens induce apoptosis in mouse peritoneal macrophages.
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AIM: To study whether estrogen might induce apoptosis in mouse peritoneal macrophages (MPM). METHOD: The MPM were isolated and incubated in culture medium containing 17-beta-estradiol, estrone, or equal volume of 100% ethanol as control. DNA fragmentation was visualized by agarose gel electrophoresis. RESULTS: 17-beta-Estradiol 0.01-1 mumol.L-1 or estrone 10-20 mumol.L-1 elicited typical morphological apoptosis and DNA fragmentation in a concentration-dependent manner in MPM. Staurosporine (Sta) 0.01 mumol.L-1, cycloheximide (Cyc) 1 mg.L-1, and tamoxifen (Tam) 10 mumol.L-1 inhibited the DNA fragmentation induced by 17-beta-estradiol 1 mumol.L-1 or estrone 20 mumol.L-1. CONCLUSION: Estradiol and estrone induced apoptosis in MPM. (+info)