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PubMed2006

Embryo development of prepubertal goat oocytes fertilised by intracytoplasmic sperm injection (ICSI) according to oocyte diameter.

The aim of this study was to evaluate embryo development of prepubertal goat oocytes fertilised by ICSI according to their diameter. Three experiments were carried out to achieve this objective. In all experiments, oocytes were matured in TCM199 supplemented with hormones, cysteamine and serum for 27 h at 38.5 degrees C. In Experiment 1, we studied the nuclear stage of goat zygotes produced by conventional ICSI and IVF using 20 nM ionomycin plus 10 microM heparin as sperm treatment. A group of Sham-injected oocytes was used as control. Results showed differences in the percentage of 2 PN (zygotes with male and female pronuclei) between ICSI, IVF and Sham (40.9, 26.6 and 3.0%, respectively; P<0.05). In Experiment 2, we evaluated the embryo development of prepubertal goat oocytes produced by ICSI and IVF after 192 h of culture in SOF medium. The percentage of morulae plus blastocysts obtained was higher in the ICSI than in the IVF group (13.4 and 5.1%, respectively; P<0.05). In Experiment 3, IVM-oocytes were classified in four groups depending on their diameter (Group A: <110 microm; Group B: 110-125 microm; Group C: 125-135 microm; Group D: >135 microm), fertilised by ICSI and cultured for 192 h. Results showed a positive correlation between oocyte diameter and embryo development (morulae+blastocysts: Group A: 0%; Group B: 6.2%; Group C: 46.4% and Group D: 33.3%). In conclusion, sperm treatment with ionomycin plus heparin using the conventional ICSI protocol improved fertilisation rates in comparison to IVF. Oocytes smaller than 125 microm were unable to develop up to blastocyst stage.

Animals↗
PubMed2004

Carbon monoxide inhibits capacitative calcium entry in human platelets.

The cytosolic calcium concentration in human platelets is elevated by several agonists via receptor-operated mechanisms involving both Ca(2+) release from intracellular stores and Ca(2+) entry. In order to get a mechanistic insight in the effect of carbon monoxide (CO)-containing solutions, this work examines the changes in [Ca(2+)](i) induced by 100 microM adenosine 5'diphosphate (ADP), 0.1 IU/ ml thrombin, 0.5 microM thapsigargin or 0.5 microM ionomycin in human platelets. In a saline solution bubbled with CO, the increase of [Ca(2+)](i) produced by thrombin was 72+/-4% of the response evoked in the control solution (CO-free) and the response elicited by ADP was 64+/-8% of the control. When a mixture of 5% CO/95% N(2) was used, the responses were 70+/-7% of control for thrombin and 79+/-6% of control for ADP. The mobilization of stored calcium produced by thrombin in a calcium-free solution and the increase of [Ca(2+)](i) produced by subsequent introduction of 1 mM extracellular calcium were both reduced in the presence of CO (82+/-6% and 78+/-5% of control, respectively). Similar reductions in the presence of CO were found when platelets were stimulated by ADP (62+/-8% and 60+/-8% for mobilization in calcium-free media and calcium entry, respectively). Although the change in [Ca(2+)](i) induced by ionomycin in the presence of extracellular calcium was almost the same in the absence or presence of CO (97+/-5% of control), the entry induced by depletion of reservoirs with the ionophore undergoes a significant reduction in a solution bubbled with CO (84+/-5% of control). In agreement with the concept that CO has a direct inhibitory effect on capacitative calcium entry, a reduction to 47+/-6% of control was obtained when sarco/endoplasmic reticulum ATPase was blocked by thapsigargin. Diverse mechanisms could be responsible for the effect of CO on calcium entry. On the one hand, a decrease in the calcium release from intracellular stores or an increase in the rate of its back-sequestration could occur, being the reduction of capacitative calcium entry an indirect consequence of a diminished emptying of reservoirs. On the other hand, CO could have a direct inhibitory effect on the pathway that produces the calcium entry. The decrease in the Ca(2+) signal in the presence of CO evoked by receptor-independent emptying of reservoirs indicates that a direct effect of CO on capacitative calcium entry participates in the antiaggregatory properties of CO. The proposal that CO inhibits directly store-operated calcium influx widens the potential mechanisms by which heme oxygenase regulates cell functions.

Adenosine Diphosphate↗
PubMed2006

Propionate induces pH(i) changes through calcium flux, ERK1/2, p38, and PKC in bovine neutrophils.

Propionate is a short-chain fatty acid produced under normal physiological conditions in the rumen of cattle. It is also involved in the inflammatory process and neutrophil function via calcium release, reactive oxygen species and intracellular pH (pH(i)) changes. This study examined the effect of propionate on the pH(i) of bovine neutrophils; specifically if pH(i) changes are controlled by calcium flux, and the mitogen-activated protein kinase (MAPK) pathway. Propionate caused rapid intracellular acidification and sustained alkalinization in bovine neutrophils loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a fluorescent indicator of pH(i). The acidification phase seems to be controlled by intracellular calcium release and p38 MAPK pathway. The pH recovery phenomenon was mediated by an amiloride-sensitive Na+/H+ exchanger and H+ channel, and was inhibited by UO126 (an ERK1/2 MAPK phosphorylation inhibitor), Gö6850 (a PKC inhibitor) and calcium chelating. Ionomycin, a calcium ionophore, induced intracellular acidification and sustained alkalinization. The intracellular acidification was strongly inhibited by BAPTA-AM (an intracellular calcium chelator) and SB203580 (a p38 MAPK inhibitor). In addition, the intracellular alkalinization was reduced by EGTA (a calcium chelator), UO126, LY294002 (a PI3K inhibitor) and Gö6850. Propionate did not increase superoxide production, however it reduced the superoxide production induced by platelet-activating factor (PAF), and increased the release of superoxide induced by ionomycin. Our results suggest that propionate-induced intracellular acidification is mediated by intracellular calcium release and p38 MAPK activation, and that pH recovery is controlled via ERK1/2 MAPK, PKC and calcium entry in bovine neutrophils.

Animals↗
PubMed1999

Modulation of Ca2+-dependent anion secretion by protein kinase C in normal and cystic fibrosis pancreatic duct cells.

The study investigated the role of protein kinase C (PKC) in the modulation of agonist-induced Ca2+-dependent anion secretion by pancreatic duct cells. The short-circuit current (ISC) technique was used to examine the effect of PKC activation and inhibition on subsequent ATP, angiotensin II and ionomycin-activated anion secretion by normal (CAPAN-1) and cystic fibrosis (CFPAC-1) pancreatic duct cells. The ISC responses induced by the Ca2+-mobilizing agents, which had been previously shown to be attributed to anion secretion, were enhanced in both CAPAN-1 and CFPAC-1 cells by PKC inhibitors, staurosporine, calphostin C or chelerythrine. On the contrary, a PKC activator, phorbol 12-myristate 13-acetate (PMA), was found to suppress the agonist-induced ISC in CFPAC-1 cells and the ionomycin-induced ISC in CAPAN-1 cells. An inactive form of PMA, 4alphad-phorbol 12, 13-didecanote (4alphaD), was found to exert insignificant effect on the agonist-induced ISC, indicating a specific effect of PMA. Our data suggest a role of PKC in modulating agonist-induced Ca2+-dependent anion secretion by pancreatic duct cells. Therapeutic strategy to augment Ca2+-activated anion secretion by cystic fibrosis pancreatic duct cells may be achieved by inhibition or down-regulation of PKC.

Adenosine Triphosphate↗
PubMed1988

5-Azacytidine induced inhibition of mitogenic response by agents that activate or augment activation of human peripheral blood T lymphocytes.

Previous work from our laboratory demonstrated the mitogenic response of human peripheral blood T lymphocytes by phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and ionomycin, or interleukin 2 (IL-2). Increasing levels of incorporated 5-azacytosine inhibited the action of the methyltransferase suggesting that incorporation of 5-azacytosine into DNA could be responsible for the inhibiting effect of 5-azacytidine (5-aza-CR) on DNA methylation. In this study, we first demonstrated the inhibition of mitogenic response by agents, such as PHA, PMA and ionomycin, or IL-2, that activate or augment activation of human peripheral blood T cells by treatment of the analog 5-azacytidine. Over 1 microM of 5-azacytidine, we detected significant inhibition of proliferative response and over 5 microM of 5-azacytidine toxic effect of cell viability. We found no significant change of T cell subsets after treatment of 5-azacytidine.

Adult↗
PubMed2002

Signaling pathways involved in Ca2+- and Pb2+-induced vesicular catecholamine release from rat PC12 cells.

Since Pb(2+) substitutes for Ca(2+) in essential steps leading to exocytosis, we have investigated whether Ca(2+) and Pb(2+) induce exocytosis through similar pathways. Vesicular catecholamine release was measured from dexamethasone-differentiated PC12 cells using carbon fiber microelectrode amperometry. Effects of drugs known to modulate PKC (PMA, staurosporine), calcineurin (cyclosporin A), calmodulin (W7), and CaM kinase II (KN-62) activity were investigated in intact and in ionomycin-permeabilized PC12 cells. Activation of PKC and inhibition of calmodulin decrease the frequency of exocytotic events evoked by high K(+) stimulation in intact cells. In addition, inhibition of calmodulin enhances the frequency of basal exocytosis from intact cells. Activation of PKC and inhibition of calcineurin enhance the frequency of basal exocytosis in intact as well as in ionomycin-permeabilized cells. Inhibition of PKC and of CaM kinase II cause no significant effects. None of the treatments has a significant effect on vesicle contents. The combined results indicate that PKC and calcineurin enhance and inhibit exocytosis through direct effects on the exocytotic machinery, whereas calmodulin and CaM kinase II exert indirect effects only. Conversely, Pb(2+)-evoked exocytosis in permeabilized cells is strongly reduced by inhibition of CaM kinase II, but is not sensitive to modulation of PKC and calcineurin activity. Inhibition of calmodulin only reduces the delay to onset of Pb(2+)-evoked exocytosis. Synaptotagmin I- and II-deficient PC12-F7 cells exhibit vesicular catecholamine release following depolarization or superfusion with Pb(2+). However, the frequency of exocytosis and the contents of vesicles released are strongly reduced as compared to PC12 cells. It is concluded that Ca(2+)-evoked exocytosis is modulated mainly by PKC and calcineurin, whereas Pb(2+)-evoked exocytosis is mainly modulated by CaM kinase II.

Animals↗
PubMed2003

Estrogen attenuates oxidative stress-induced apoptosis in C6 glial cells.

We examined the mechanism of 17beta-estradiol (estrogen)-mediated inhibition of apoptosis in C6 (rat glioma) cells following exposure to hydrogen peroxide (H(2)O(2)). Cells were preincubated with 4 microM estrogen for 2 h and then exposed to 100 microM H(2)O(2) for 24 h. Exposure to H(2)O(2) caused significant increases in intracellular calcium (Ca(2+)), as determined by fura-2, which was attenuated by preincubation with estrogen. H(2)O(2) and ionomycin caused cell death in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Preincubation with estrogen restored viability in cells exposed to H(2)O(2) but not in cells exposed to ionomycin. Western blot analysis showed an increase in Bax/Bcl-2 ratio, calpain activity, and caspase-3 activity following treatment with H(2)O(2), and estrogen pretreatment decreased levels of all three. Cell morphology, as evaluated by Wright staining, indicated apoptosis in cells treated with H(2)O(2), and pretreatment with estrogen reduced apoptosis. Results from MTT and Wright staining were further supported by the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) assay. These results indicate a role for estrogen in preventing apoptosis in C6 glial cells exposed to H(2)O(2). Our results suggest that estrogen may have a protective role in minimizing glial cell apoptosis in neurological diseases such as demyelinating disease or central nervous system trauma.

Animals↗
PubMed1997

BDNF prevents NO mediated glutamate cytotoxicity in cultured cortical neurons.

The effects of brain-derived neurotrophic factor (BDNF) on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. BDNF induced TrkB tyrosine phosphorylation in rat cultured cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to glutamate and incubated with normal medium for 24 h. Glutamate cytotoxicity was prevented by MK-801, which is a non-competitive blocker of N-methyl-D-aspartate and N(omega)-nitro-L-arginine, which is a blocker of nitric oxide synthetase. Delayed neurotoxicity was also induced by ionomycin, a calcium ionophore, and nitric oxide (NO) donors such as S-nitrosocysteine (SNOC) and 3-morpholinosydnonimine (SIN-1). Incubating cultures with BDNF for 10 min to 24 h protected cortical neurons against glutamate neurotoxicity. The protective effects of BDNF against glutamate cytotoxicity were dependent on both its concentrations and incubation time. BDNF also prevented the ionomycin-, SNOC-, and SIN-1 induced cytotoxicity. These results indicate that BDNF protects cultured cortical neurons from NMDA receptor-mediated glutamate neurotoxicity by reducing cytotoxic action of NO.

Animals↗
PubMed1997

Nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha7-neuronal receptors and neuronal CNS receptors.

We examined the effects of nicotine on glutamate-induced cytotoxicity using primary cultures of rat cortical neurons. The cell viability decreased significantly when cultures were exposed to glutamate for 10 min and then incubated with glutamate-free medium for 1 h. The exposure of cultures to nicotine (10 microM) for 8-24 h prior to glutamate application ameliorated the glutamate-induced cytotoxicity, with no significant effect of nicotine alone on the cell viability. Neuroprotection by nicotine was dependent on the incubation period. alpha-bungarotoxin (alpha-BTX) and methyllycaconitine (MLA), both of which are alpha7-neuronal receptor antagonists, and dihydro-beta-erythroidine (DHbetaE), a neuronal central nervous system (CNS) receptor antagonist, each significantly antagonized the protection by nicotine against glutamate-induced cytotoxicity. Ionomycin, a calcium ionophore, and S-nitrosocysteine (SNOC), a nitric oxide (NO) donor, also induced cytotoxicity in a manner similar to glutamate. Nicotine protected cultures against ionomycin-induced cytotoxicity, but not against SNOC-induced cytotoxicity. These results suggest that nicotine protects cultured cortical neurons against glutamate-induced cytotoxicity via alpha7-neuronal receptors and neuronal CNS receptors by reducing NO-formation triggered by Ca2+ influx.

Aconitine↗
PubMed2000

Enhancement of Ca2+-induced noradrenaline release by vanadate in PC12 cells: possible involvement of tyrosine phosphorylation.

Tyrosine phosphorylation has been shown to participate in the signal cascade after receptor stimulation with neurotransmitters and neurotrophins. However, the role of tyrosine phosphorylation in the process(es) of neurotransmitter release has not been well established. The effects of orthovanadate (Na3VO4), an inhibitor of protein-tyrosine phosphatases, on cytosolic free Ca2+ concentrations ([Ca2+]i), phosphotyrosine accumulation and noradrenaline (NA) release in neurosecretory PC12 cells were investigated. Addition of Na3VO4 enhanced ionomycin-stimulated [3H]NA release in a concentration-dependent manner, although Na3VO4 alone had no effect. Na3VO4 also enhanced [3H]NA release induced by P2 receptor stimulation with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or by depolarization with 50 mM KCl, which stimulated a [Ca2+]i increase. A cell permeable inhibitor of protein-tyrosine phosphatases, L-p-bromotetramisole oxalate, at 0.3 mM enhanced ionomycin-stimulated [3H]NA release, although pervanadate had no effect. Addition of 5 mM Na3VO4 stimulated phosphotyrosine accumulation in several protein bands such as p130cas, but did not increase [Ca2+]i in PC12 cells. These findings suggest that the tyrosine phosphorylation pathway regulates Ca2+-stimulated NA release without changes of [Ca2+]i in PC12 cells.

Animals↗
PubMed2001

Thapsigargin inhibits angiogenesis in the rat isolated aorta: studies on the role of intracellular calcium pools.

OBJECTIVE: Since the role of Ca2+ in angiogenesis is not fully understood, we investigated the effect of thapsigargin (TG: depletes intracellular Ca2+ pools) and other Ca2+ modulators [ionomycin, calcium ionophore A23187 and dibutyrylhydroquinone (DBHQ)] on in vitro angiogenesis by rat aortic rings. METHODS: Aortae from Sprague-Dawley rats were cut into 2-mm rings, embedded in a fibrin clot and cultured for 15 days in serum-free medium containing drugs and the microvessels counted. Rings were also pre-treated with TG and Ca2+ modulators for 1 h prior to embedding and culture. Viability was examined by the measurement of lactic acid dehydrogenase release. Rings were also treated with hydrocortisone and lavendustin A (a tyrosine kinase inhibitor), as positive controls. The effect of TG on the proliferation and migration of human umbilical artery endothelial cells (HUVECs) was studied in parallel. RESULTS: TG significantly inhibited microvessel formation and HUVEC proliferation and migration in a dose-dependent manner, all at <10 nmol/l, without affecting viability. In contrast, ionomycin, A23187 and DBHQ were cytotoxic at inhibitory concentrations. Continual exposure to hydrocortisone and lavendusin A also inhibited angiogenesis without affecting viability. CONCLUSION: Since low concentrations of TG deplete intracellular Ca2+ stores, it is concluded that these pools play a central role in mediating angiogenesis.

Analysis of Variance↗
PubMed2002

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles, a novel class of immunomodulators.

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.

Adjuvants, Immunologic↗
PubMed2003

Oscillatory CaMKII activity in mouse egg activation.

Fertilization-induced intracellular calcium (Ca(2+)) oscillations stimulate the onset of mammalian development, and little is known about the biochemical mechanism by which these Ca(2+) signals are transduced into the events of egg activation. This study addresses the hypothesis that transient increases in Ca(2+) similar to those at fertilization stimulate oscillatory Ca(2+)/calmodulin-dependent kinase II (CaMKII) enzyme activity, incrementally driving the events of egg activation. Since groups of fertilized eggs normally oscillate asynchronously, synchronous oscillatory Ca(2+) signaling with a frequency similar to fertilization was experimentally induced in unfertilized mouse eggs by using ionomycin and manipulating extracellular calcium. Coanalysis of intracellular Ca(2+) levels and CaMKII activity in the same population of eggs demonstrated a rapid and transient enzyme response to each increase in Ca(2+). Enzyme activity increased 370% during the first Ca(2+) rise, representing about 60% of maximal activity, and had decreased to basal levels within 5 min from the time Ca(2+) reached its peak value. Single fertilized eggs monitored for Ca(2+) had a mean increase in CaMKII activity of 185%. One and two ionomycin-induced Ca(2+) transients resulted in 39 and 49% mean cortical granule (CG) loss, respectively, while CG exocytosis and resumption of meiosis were inhibited by a CaMKII antagonist. These studies demonstrate that changes in the level of Ca(2+) and in CaMKII activity can be studied in the same cell and that CaMKII activity is exquisitely sensitive to experimentally induced oscillations of Ca(2+) in vivo. The data support the hypothesis that CaMKII activity oscillates for a period of time after normal fertilization and temporally regulates many events of egg activation.

Animals↗
PubMed2001

Enhancement of arachidonic acid release and prostaglandin F(2alpha) formation by Na3VO4 in PC12 cells and GH3 cells.

Both activation of phospholipase A2 causing arachidonic acid release and tyrosine phosphorylation have been proposed to be involved in neuronal functions. Previously, we reported that orthovanadate (Na3VO4), an inhibitor of tyrosine phosphatases, stimulated tyrosine phosphorylation in proteins and enhanced Ca2+-induced noradrenaline release in rat pheochromocytoma PC12 cells. However, the role of tyrosine phosphorylation on phospholipase A2 activity and/or arachidonic acid release in neuronal cells has not been well established. The effects of Na3VO4 on arachidonic acid release and prostaglandin F(2alpha) formation were investigated in two types of neuronal cell lines. In PC12 cells, addition of Na3VO4 stimulated [3H]arachidonic acid release and prostaglandin F(2alpha) formation in a concentration-dependent manner. Co-addition of 5 mM Na3VO4 enhanced ionomycin-stimulated [3H]arachidonic acid release. Na3VO4 also enhanced ionomycin-stimulated [3H]arachidonic acid release from GH3 cells, a clonal strain from rat anterior pituitary. These findings suggest that the tyrosine phosphorylation pathway regulates arachidonic acid release by phospholipase A2 and prostaglandin F(2alpha) formation in neuronal cells.

Animals↗
PubMed1997

Calcium influx inhibits MT1-MMP processing and blocks MMP-2 activation.

We have previously reported that concanavalin A (ConA)-induced MMP-2 activation involves both transcriptional and non-transcriptional mechanisms. Here we examined the effects of calcium influx on MT1-MMP expression and MMP-2 activation in MDA-MB-231 cells. The calcium ionophore ionomycin caused a dose-dependent inhibition of ConA-induced MMP-2 activation, but had no effect on MT1-MMP mRNA levels. However, Western analysis revealed an accumulation of pro-MT1-MMP (63 kDa), indicating that ionomycin blocked the conversion of pro-MT1-MMP protein to the active 60 kDa form. This suggests that increased calcium levels inhibit the processing of MT1-MMP. This finding may help to elucidate the mechanism(s) which regulates MT1-MMP activation.

Blotting, Northern↗
PubMed2000

Flow cytometric analysis of intracellular IFN-gamma, IL-4 and IL-10 in CD3(+)4(+) T-cells from rat spleen.

The application of multi-parameter flow cytometry for the assessment of T-cell and cytokine functioning has been used by several groups for studying human and mouse samples, although little has been reported for the rat. Here we report the optimisation of immunofluorescent staining for cell surface and intracellular antigens using three-colour flow cytometric analysis to measure the frequency of rat CD3(+)4(+) T-cells that produce IFN-gamma, IL-4 and IL-10. In vitro stimulation of IFN-gamma production required incubation of splenocytes with PMA and ionomycin in the presence of the protein transport inhibitor brefeldin A for 6 h. Three stimulation protocols for IL-4 and IL-10 production were evaluated. In vitro priming of splenic T-cells with antibodies against CD3 and CD28 and recombinant cytokines (IL-2 and IL-4) for 5 days followed by restimulation with PMA and ionomycin was required to stimulate cells to produce either IL-4 or IL-10. Brefeldin A was found to be a more suitable protein transport inhibitor than monensin. This method will be useful for analysing the nature of individual rat cytokine-producing cells in a variety of experimental model systems.

Animals↗
PubMed1999

Expression and kinetics of cytokines determined by intracellular staining using flow cytometry.

Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.

Cytokines↗
PubMed2002

Non-specific effects of the MEK inhibitors PD098,059 and U0126 on glutamate release from hippocampal synaptosomes.

In order to investigate a role for the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) on hippocampal neurotransmitter release, we studied the effect of commonly used MEK (mitogen-activated protein kinase [MAPK]/ERK kinase) inhibitors, PD098,059 and U0126, on depolarization-induced glutamate release. PD098,059 inhibited glutamate release from hippocampal synaptosomes stimulated with 15 mM KCl in a concentration-dependent manner. At the same range of concentrations, PD098,059 inhibited basal and KCl-stimulated ERK1/2 phosphorylation. U0126, however, did not significantly affect KCl-evoked glutamate release at concentrations shown to inhibit ERK activity. Nonetheless, U0126 unspecifically potentiated depolarization-induced Ca2+-independent glutamate release, which masked a small dose-dependent inhibitory effect on the Ca2+-dependent release. PD098,059 reduced the [Ca2+]i response to KCl by partially inhibiting Ca2+ entry through N- and P-/Q-type voltage-gated Ca2+ channels, whereas U0126 did not affect depolarization-induced Ca2+ influx. To overcome the unspecific effect of PD098,059 on Ca2+ entry, we studied the effect of both MEK inhibitors on glutamate release stimulated by a Ca2+ ionophore. PD098,029 and U0126 showed a small dose-dependent inhibitory effect on ionomycin-induced glutamate release, at concentrations shown to inhibit ionomycin-stimulated ERK phosphorylation. These findings uncover new unspecific actions for both MEK inhibitors and suggest a minor role for ERK in modulating glutamate release in the hippocampus.

Animals↗