共查询到20条相似文献,搜索用时 15 毫秒
1.
Many intracellular proteins and signaling cascades contribute to the ethanol sensitivity of native N-methyl-d-aspartate receptors (NMDARs). One putative protein is the serine/threonine kinase, protein kinase C (PKC). The purpose of this study was to assess if PKC modulates the ethanol sensitivity of native NMDARs expressed in primary cultured cerebellar granule cells (CGCs). With the whole-cell patch-clamp technique, we assessed if ethanol inhibition of NMDA-induced currents (INMDA) (100 μM NMDA plus 10 μM glycine) were altered in CGCs in which the novel and classical PKC isoforms were activated by phorbol-12-myristate-13-acetate (PMA). Percent inhibition by 10, 50, or 100 mM ethanol of NMDA-induced steady-state current amplitudes (ISS) or peak current amplitudes (IPk) of NMDARs expressed in CGCs in which PKC was activated by a 12.5 min, 100 nM PMA exposure at 37°C did not differ from currents obtained from receptors contained in control cells. However, PMA-mediated augmentation of IPk in the absence of ethanol was abolished after brief applications of 10 or 1 mM ethanol coapplied with agonists, and this suppression of enhanced receptor function was observed for up to 8 min post-ethanol exposure. Because we had previously shown that PMA-mediated augmentation of INMDA of NMDARs expressed in these cells is by activation of PKCα, we assessed the effect of ethanol (1, 10, 50, and 100 mM) on PKCα activity. Ethanol decreased PKCα activity by 18% for 1 mM ethanol and activity decreased with increasing ethanol concentrations with a 50% inhibition observed with 100 mM ethanol. The data suggest that ethanol disruption of PMA-mediated augmentation of INMDA may be due to a decrease in PKCα activity by ethanol. However, given the incomplete blockade of PKCα activity and the low concentration of ethanol at which this phenomenon is observed, other ethanol-sensitive signaling cascades must also be involved. 相似文献
2.
Mononuclear phagocytes from healthy human donors were incubatedwith or without ethanol (1255 mM, initial concentration)in non-sealed wells in an atmosphere of 5% CO2 in air for 6or 24 hr on day 1 or day 7 in culture. The actual ethanol concentrationwas assayed in the media at the beginning and at the end ofeach incubation period. The ethanol content was reduced to about70% of the initial concentration after 6 hr incubation, andto below 20% after 24 hr incubation. Binding properties of theFc-receptors, and their associated phagocytic activity, weretested after ethanol exposure of the cells. An initial concentrationof 12 or 22 mM ethanol caused no differences from controls atany time in culture. Mononuclear phagocytes assayed on day 1(=monocytes) showed reduced binding (60% of control) as wellas internationalization (70% of control) of particles via theFc-receptors after addition of 55 mM ethanol and incubationfor 6 or 24 hr. Incubation in corresponding ethanol concentrationfor 6 hr had no effect on cells cultured for 7 days (=macrophages),whereas 24 hr incubation depressed the Fc-receptor functionin these cells also. There were no changes in viability, morphologyor spreading ability after ethanol treatment. 相似文献
3.
We investigated the effects of iron deficiency in mice on protein kinase C (PKC) activation, an enzyme required for optimal lymphocyte proliferation. C57BL/6 mice were fed either an iron-deficient diet (ID; 10 mg Fe/kg diet), a control diet (C; 50 mg/kg diet), or were pair fed (PF) to ID mice for 34 d. PKC activity was studied in spleen cells by histone phosphorylation. Iron deficiency significantly reduced cytosol activity in unstimulated cells and membrane-bound activity in cells stimulated by concanavalin A (Con A) or phorbol-12-myristate-13-acetate (PMA), and the ratio of membrane-bound over cytosol activity in mitogen-stimulated cells. In PF mice the ratio of membrane-bound activity to cytosol activity was greater than normal in Con A-treated cells and only slightly decreased in PMA-treated cells. PKC activity positively correlated with iron status. We conclude that reduced PKC activity and poor translocation results in aberrant signal transduction, which in turn might be responsible for the impaired lymphocyte proliferation associated with iron deficiency. 相似文献
4.
Annayya R. Aroor 《Alcohol》2009,43(4):315-322
Hepato-subcellular effect of angiotensin II (Ang II) and ethanol on the p42/44 mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK 1/2) was investigated in the nucleus of rat hepatocytes. Hepatocytes were treated with ethanol (100 mM) for 24 h and stimulated with Ang II (100 nM, 5 min). The levels of p42/44 MAPK and MEK 1/2 were monitored in the nuclear fraction using antibodies. Ang II itself caused significant accumulation of phosphorylated p42/44 MAPK (phospho-p42/44 MAPK) in the nucleus without any significant translocation of p42/44 MAPK protein thereby suggesting activation of p42/44 MAPK in the nucleus. Ang II caused marked accumulation of phosphorylated MEK 1/2 (phospho-MEK 1/2) in the nucleus without any significant accumulation of MEK 1/2 protein. Ratio of phospho-MEK 1/2 to MEK 1/2 protein in the nucleus after Ang II treatment was 2.4 times greater than control suggesting phosphorylation of MEK 1/2 inside the nucleus. Ethanol had no effect on the protein level or the activation of p42/44 MAPK in the nucleus. Ethanol treatment potentiated nuclear activation of p42/44 MAPK by Ang II but not translocation of p42/44 MAPK protein. This was accompanied by potentiation of Ang II-stimulated accumulation of phospho-MEK 1/2 in the nucleus by ethanol. MEK 1/2 inhibitor, U-0126 inhibited Ang II response and its potentiation by ethanol. These results suggest that Ang II-mediated accumulation of phospho-p42/44 MAPK in the hepatocyte nucleus involves MEK 1/2-dependent activation and this effect is potentiated by ethanol. 相似文献
5.
《Alcohol》1993,10(4):259-262
We examined the effect of 60 days of ethanol treatment on protein kinase C (PKC) in membrane and cytosolic fractions of the rat cerebral cortex. Membranal and cytosolic PKC were determined by binding technique using [3H]-phorbol 12,13 dibutyrate (PDBU) as radioligand and phorbol 12-myristate 13-acetate (PMA) as displacer. Chronic ethanol consumption resulted in a decrease in the maximum number of binding sites (Brmmax) of [3H]-PDBU binding to membranal PKC without significant change in the apparent dissociation constant (KrmD) in the rat cortex. We also observed that chronic ethanol consumption had no significant effect on Brmmax or KrmD of [3H]-PDBU binding to cytosolic PKC in the rat cerebral cortex. These results suggest that chronic ethanol consumption leads to the down-regulation of brain PKC associated with membrane but not with cytosol. 相似文献
6.
Ahluwalia B Ahmad S Adeyiga O Wesley B Rajguru S 《Alcohol and alcoholism (Oxford, Oxfordshire)》2000,35(5):452-457
Phosphorylation and dephosphorylation of proteins associated with microtubules (MAPs) modulate the functional properties of microtubules (MT). A study was designed to test the hypothesis that ethanol at pharmacologically relevant levels affects phosphorylation of MAPs. Low (6, 12, 24, and 48 mM) and high (96, 384, and 768 mM) levels of ethanol were used in the study. MT prepared from rat brain by successive cycles of assembly-disassembly were found to contain two high molecular weight proteins (MAP2 and MAP1), tubulin, and 70-kDa neurofilament. The kinase activity was determined using [gamma(32)P]ATP as a phosphate donor. The results showed that ethanol primarily stimulated MAP2 phosphorylation. Low levels of ethanol stimulated, whereas high levels decreased, the kinase activity. MAP1 was phosphorylated to a lesser extent. 70-kDa neurofilament and tubulin were phosphorylated, however, the dose-dependent biphasic effect of ethanol on phosphorylation was not found in these cytoskeleton proteins. To determine whether the ethanol-induced kinase activity was cAMP-dependent, the catalytic subunit of cAMP-dependent protein kinase was isolated, purified, and kinase activity was determined with and without ethanol. The results showed that cAMP was not involved in ethanol-induced kinase activity. We conclude that ethanol predominantly stimulates phosphorylation of MAP2 in a dose-dependent manner. 相似文献
7.
Ethanol alone had no effect on neuronal nitric oxide synthase (nNOS) expression in PC12 cells. However, in the presence of nerve growth factor (NGF), nNOS expression was amplified (threefold, P < 0.05), compared to NGF alone. This increase was eliminated with pretreatment of PC12 cells with staurosporine, suggesting that the effects of ethanol on nNOS expression are mediated by a protein kinase C-dependent pathway. 相似文献
8.
《Alcohol》1988,5(6):499-503
Acute administration of ethanol reportedly decreases total sialic acid in brain. Here, we tested the hypothesis in brain and liver that the decrement is due to increased hydrolysis of sialoglycoconjugates. Mouse tissue slices were pulse-labeled with N-[3H]acetyl-D-mannosamine, the precursor of sialic acid. Incorporation was linear for up to 4 hr of incubation. When the labeled slices were incubated with three concentrations of ethanol (0.1, 0.5, and 1 M) for 5 hr, labeled liver sialoconjugates were significantly affected only at 0.5 and 1 M ethanol, whereas labeled brain sialoconjugates were markedly decreased even at 100 mM ethanol. Sialidase activity decreased steadily with increasing concentration of ethanol, indicating that the increased hydrolysis was not attributable to an enhanced sialidase activity. n-Propanol and t-butanol had the same degradative effect as ethanol on sialocompounds; and 3 mM pyrazole, an inhibitor of alcohol dehydrogenase (ADH), had no effect on ethanol-induced degradation of sialocompounds. The protein/DNA ratio in liver showed a steady decrease with increasing ethanol. The data thus confirm the in vivo reports of ethanol-enhanced cleavage and rule out any increase in sialidase activity as a major cause. 相似文献
9.
Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that gamma-irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following gamma-irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation. 相似文献
10.
11.
目的探讨蛋白酶体在乙醇对神经细胞产生毒性中的作用。方法分别用50、10、150、200mmol/L的乙醇对PC12细胞进行48h染毒后,用四甲基偶氮唑盐微量酶反应比色(MTT)法检测乙醇对PC12细胞的细胞毒性,用荧光底物法测定细胞蛋白酶体的活力,并与对照组比较。结果100mmol/L以上浓度的乙醇染毒48h对PC12细胞产生明显的毒性作用,且呈现良好的剂量-反应关系;蛋白酶体活力的测定表明,乙醇显著抑制蛋白酶体的活力,有剂量和时间依赖关系。结论乙醇可能通过抑制蛋白酶体的活力,干扰了某些蛋白质的正常代谢,从而产生了细胞毒性。 相似文献
12.
Ciereszko R Opalka M Kaminska B Górska T Dusza L 《Reproduction, fertility, and development》2003,15(1-2):27-35
The hypothesis that protein kinase C (PKC) and tyrosine kinases, as well as serine-threonine and tyrosine phosphatases, are involved in prolactin (PRL) signalling in theca cells harvested from porcine follicles was tested. Theca cells were incubated with PRL for 24 h to stimulate progesterone (P4) production. In addition, treatments included inhibitors of PKC and tyrosine kinases, as well as serine-threonine phosphatase inhibitor and tyrosine phosphatase inhibitor. Prolactin significantly stimulated P4 production by theca cells and all inhibitors suppressed the PRL-stimulated P4 production. After incubation with PRL for 2, 5, 10 or 20 min, theca cells were homogenized and cytosolic and membrane fractions were obtained. This was followed by determination of PKC activity in partially purified subcellular fractions by measuring the transfer of 32P from [gamma-32P] adenosine triphosphatase (ATP) to histone III-S. In unstimulated porcine theca cells the major proportion of PKC activity was present in the cytosol. Incubation of cells with PRL resulted in a rapid, time-dependent increase in the amount of PKC activity in the membrane fraction. Protein kinase C activity in the membrane fraction was maximal after 10 min of cells' exposure to PRL. Protein kinase C activation was assessed also by measuring the specific association of 3H-phorbol dibutyrate (3H-PDBu) with theca cells after treatment with PRL. Prolactin significantly increased 3H-PDBu-specific binding in theca cells. In contrast to PKC, total inositol phosphate accumulation was not affected by PRL in the current study. In summary, PRL stimulated P4 production by porcine theca cells derived from large follicles. The results of the study were consistent with the hypothesis that PKC is one of the intracellular mediators of PRL action in porcine theca cells. Protein kinase C activation does not appear to occur through the action of phosphatidylinositol-dependent phospholipase C. Moreover, the involvement of tyrosine kinases, as well as tyrosine and serine-threonine phosphatases, in PRL signalling in the examined cells is suggested. 相似文献
13.
Intestinal hyperpermeability is a causal factor for the development of alcoholic endotoxemia and steatohepatitis. However, the mechanisms governing this link remain unknown. The purpose of this study was to determine whether toll-like receptor 4 (TLR4) is involved in ethanol's deleterious effects on the intestinal barrier. Caco-2 cells were incubated in vitro with 1–10% ethanol. The results indicated that ethanol had a dose-dependent effect in increasing TLR4 expression and intercellular permeability. Then the effects of TLR4 on protein kinase C (PKC) and the intercellular junction protein occludin were assessed with and without pretreatment with a TLR4 inhibitor. The results indicated that TLR4 increased nonspecific PKC activity and reduced the expression of phosphorylated occludin in the membrane, which increased intercellular permeability. These effects were prevented by pretreatment with TLR4 mAb. 相似文献
14.
Hung MC Shibasaki K Yoshida R Sato M Imaizumi K 《The British journal of nutrition》2001,86(2):163-171
Our objective was to determine whether dietary supplementation with phosphatidylcholine (PC) plus vitamin B12 could afford beneficial effects on biochemical and biophysical events in the brain of senescence-accelerated mouse (SAM) substrain SAMP8. We measured learning behaviour, hippocampal protein kinase C (PKC) activity, cerebral antioxidant status, phospholipid composition and fatty acid composition in 6-month-old SAMP8 and in age-matched controls (SAM substrain SAMR1). In comparison with SAMR1, SAMP8 showed a significant elevation in total grading score of senescence and a significant decline in acquisition SAMP8 had a lower hippocampal PKC activity and cerebral PKC-beta mRNA abundance than SAMR1. SAMP8 had increased cerebral lipid peroxide levels and proportion of sphingomyelin, and a lower proportion of 20 : 4n-6 and 22 : 6n-3 in cerebral phosphtidylethanolamine than SAMR1. SAMP8 fed the PC combined with vitamin B12 diet had an increased PKC activity and a higher proportion of 22 : 6n-3 than SAMP8 fed the control diet. These results indicate the potential benefit of PC combined with vitamin B12 as a dietary supplement. 相似文献
15.
Ethanol increases phosphodiesterase 4 activity in bovine bronchial epithelial cells. 总被引:1,自引:0,他引:1
Ethanol exposure in airway epithelium increases cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. Activation of PKA and cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) has been shown to increase ciliary beat frequency (CBF) in bovine bronchial epithelial cells (BBECs). We have shown that biologically relevant concentrations of ethanol stimulate increases in CBF in a nitric oxide-dependent manner, mediated through elevated cAMP levels and subsequent PKA activation. This ethanol-driven rapid and transient increase in CBF occurs 15 to 30 min after exposure to 100 mM ethanol. However, after prolonged exposure to 100 mM ethanol (>/=6 h), CBF and the catalytic activity of PKA return to baseline levels. We hypothesize that cyclic nucleotide-dependent phosphodiesterase (PDE) activity attenuates the duration of ethanol-stimulated ciliary motility. The effect of ethanol on the PDE activity in BBECs was determined through direct assay of catalytic activity. When BBECs were incubated with 100 mM ethanol, significant increases in cAMP levels occurred within 1 h, with corresponding increases in PKA activity. Treatment of BBECs with 100 mM ethanol increased cAMP-PDE activity significantly by 4 h. 3-Isobutyl-1-methylxanthine, Ro 20-1724, and rolipram inhibited ethanol-stimulated cAMP-PDE activity. These agents inhibited ethanol-stimulated cAMP-PDE activity and increased the magnitude of ethanol-stimulated PKA activity observed under the same conditions. These findings support the idea that acute exposure (<6 h) to ethanol increases cAMP levels, and the associated increase in PKA activation is regulated by cAMP-dependent PDE, specifically PDE4. Other compensatory mechanisms however, may be responsible for the down-regulation of PKA, which occurs after chronic epithelial exposure (>/=6 h) to ethanol. 相似文献
16.
电磁辐射对大鼠海马PKC转位和激活的影响 总被引:1,自引:0,他引:1
目的 探讨电磁辐射对PKC转位和激活的影响。方法 以峰值功率密度为5 W/cm2的微波持续辐照大鼠1,3,5,10,15 d,用改良的Takai法检测海马PKC活性,用Western-blot法检测海马AMPA受体磷酸化程度。结果 微波辐照后,大鼠海马中的PKC被激活,时间为3~5 d,随着辐照时间增加,这种激活形式一直延续至辐照15 d;对照组和辐照组各个时相点的AMPA Glu2-Ser880位点的磷酸化程度差异无显著性。结论 峰值功率密度为5 W/cm2的微波辐照对中枢神经系统的损伤可能不涉及PKC-AMPA途径的功能变化。 相似文献
17.
The effects of ethanol on [3H]dopamine release were investigated in cultured PC12 cells using two methods to stimulate dopamine release: exposure to depolarizing concentrations of extracellular K+ and incubation with the highly active secretagogue, bradykinin. Both K+ and bradykinin dose-dependently increased [3H]dopamine release. The mean +/- S.E.M. EC50 for K+ was 35.8 +/- 1.2 mM; for bradykinin it was 1.07 +/- 0.23 x 10(-7) M. The characteristics of the bradykinin-stimulated dopamine release showed it to be dependent on extracellular Ca2+ and was attenuated by 1 mM Co2+ or 1 mM Ni2+. However, release was unaffected by either the voltage-dependent Ca2+ channel antagonist, verapamil, or the dihydropyridine (DHP) Ca2+ channel agonist, BAY K 8644. In contrast, 1 mM Co2+ completely blocked, verapamil inhibited and BAY K 8644 augmented K+-stimulated [3H]dopamine release. PC12 cells acutely exposed to ethanol (100 and 200 mM) showed diminished K+-stimulated [3H]dopamine release but an unaltered bradykinin-stimulated response. Cells exposed to 200 mM ethanol for 6 days showed significantly enhanced [3H]dopamine release in response to high concentrations of K+ but no changes were observed in their response to bradykinin. These data provide evidence that ethanol, within the same cell, can differentially affect neurotransmitter release, dependent upon the secretagogue used. 相似文献
18.
The role of dietary fibres in colon carcinogenesis is controversial. To elucidate the mechanisms by which different dietary fibre sources may affect colonic tumour development, we studied the effects of diets enriched with cereal brans or inulin on protein kinase C (PKC) activity and isozyme expression in rat colon. Male Wistar rats (twelve per group) were fed one of the following AIN-93G-based diets (Reeves et al. 1993) for 4 weeks: a non-fibre high-fat diet or one of the four high-fat diets supplemented with either rye, oat or wheat bran or inulin at 100 g/kg diet. The fat concentration (20 g/100 g) and fatty acid composition of the non-fibre high-fat diet was designed to approximate that in a typical Western-type diet. In the proximal colon, rats fed the inulin diet had a significantly higher membrane PKC activity and a higher membrane PKC delta level than rats fed the non-fibre diet In the distal colon, rats fed the inulin and oat bran diets had a higher total PKC activity and a higher membrane PKC beta 2 level than rats fed the wheat-bran diet. Rats in the non-fibre and wheat-bran groups had the lowest concentrations of luminal diacylglycerol. In conclusion, feeding of wheat bran resulted in low distal PKC activity and expression of PKC beta 2, a PKC isozyme related to colonic cell proliferation and increased susceptibility for colon carcinogenesis, which may explain in part the protective effect of wheat bran against tumour development in a number of experimental colon cancer studies. The increase in PKC activity and PKC beta 2 expression by feeding inulin may be a drawback of inulin as a functional food. 相似文献
19.
《Systems biology in reproductive medicine》2013,59(2-3):225-230
Soluble Mn2+ -dependent adenylate cyclase (MnAC) activity was found in testicular cytosol from blue foxes castrated during the breeding season. The rate of MnAC activity was approximately constant for 30 min at 35°C and for 2 hr after storage at 25°C. Activity was directly proportional to cytosol protein concentration and was optimal in the physiological pH range. Enzyme activity declined in the presence of an alkylating agent (N-ethyl maleimide, NEM) and was eliminated at a concentration of 1 mM NEM. Low concentrations (0.1—10 mM) of a reducing agent (β-mercapto ethanol, βME) did not increase MnAC activity, whereas a high concentration (100 mM) led to a significant reduction (p< 0.01) in activity. Substitution of Mn2- in the assay medium with Mg2- led to a total loss of enzyme activity, which could not be regained by adding hormones or by preincubation of cytosol for 60 min. The Km for Mn2+ was estimated to be 3.5 mM. The affinity of the enzyme for Mn2+ was not altered by varying the concentration of ATP. In contrast, increasing concentrations of Mn2+ appeared to increase the affinity of the enzyme for MnATP2-. The Km for MnATP2- thus varied from 6 to 18 mM. 相似文献
20.
Solanum nigrum L. (SNL) has been used in folk medicine for its anti-inflammatory activity. We isolated only the SNL glycoprotein from SNL and found that it was cytotoxic at low concentration. With respect to cytotoxicity, we investigated whether purified SNL glycoprotein is able to regulate protein kinase C (PKC) alpha activation and nuclear factor (NF)- kappaB activities in 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced tumor promotion, and whether it has an apoptosis-inducing effect in MCF-7 cells using western blot analysis. In addition, to elucidate the relationship between PKCalpha and NF-kappaB, inhibitory studies were performed with staurosporine (an inhibitor of phospholipid/calcium-dependent protein kinase) and pyrrolidine dithiocarbamate (an inhibitor of NF-kappaB activation). To verify induction of apoptosis by the SNL glycoprotein, we performed DNA fragmentation and nuclear staining assays using ethidium bromide and bisbenzamide H33342. The results in this study indicated that SNL glycoprotein induces apoptosis through modulation of PKCalpha and NF-kappaB activity in MCF-7 cells. In fact, SNL glycoprotein interfered with PKCalpha membrane translocation and inhibited NF-kappaB (p50) protein activity in MCF-7 cells stimulated with TPA (61.68 ng/mL, 100 nM) dose-dependently. Regarding the apoptotic-inducing effect, nucleosomal DNA fragmentation and nuclear staining by SNL glycoprotein in MCF-7 cells were shown. Collectively, the data demonstrate that SNL glycoprotein is a potential natural anticancer agent because of its ability to induce apoptosis in MCF-7 cells. 相似文献