Effects of restraint stress on the daily rhythm of hydrolysis of adenine nucleotides in rat serum

<p>Abstract</p> <p>Background</p> <p>Adenosine 5-triphosphate (ATP) and its breakdown products ADP and adenosine can act as extracellular messengers in a range of biological processes. Extracellular adenine nucleotides are metabolized by a number of enzymes including NTPDases and 5'-nucleotidase, which are considered to be the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATPase and ADPase activities in rat serum exhibit a 24-h temporal pattern, with higher enzyme activity during the dark (activity) phase. It was found that stress can cause disruptions in biological circadian rhythms and in the cardiovascular system. Therefore, the aim of the present study was to examine the influence of acute stress exposure upon temporal patterns of NTPDase and 5-nucleotidase enzyme activities in rat blood serum.</p> <p>Methods</p> <p>Adult male Wistar rats were divided into 4 groups: ZT0, ZT6, ZT12 and ZT18. Each group was subdivided in 4 groups: control, immediately, 6 h and 24 h after one hour of restraint stress. ATP, ADP and AMP hydrolysis were assayed in the serum.</p> <p>Results</p> <p>All stressed groups showed significant decreases in all enzyme activities at ZT 12 and ZT 18 when compared with control.</p> <p>Conclusion</p> <p>Acute stress provokes a decrease in nucleotidase activities dependent on the time that this stress occurs and this effect appears to persist for at least 24 hours. Stress can change levels of nucleotides, related to increased frequency of cardiovascular events during the activity phase. Altered levels of nucleotides in serum may be involved in cardiovascular events more frequent during the activity phase in mammals, and with their etiology linked to stress.</p

In vivo glioblastoma growth is reduced by apyrase activity in a rat glioma model

BACKGROUND: ATP is an important signalling molecule in the peripheral and central nervous system. Both glioma growth and tumor resection induces cell death, thus liberating nucleotides to the extracellular medium. Nucleotides are hydrolyzed very slowly by gliomas when compared with astrocytes and induce neuronal cell death and glioma proliferation. The objective of the present study was to test the involvement of extracellular ATP in glioblastoma growth in a rat glioma model. METHODS: To deplete the extracellular ATP, the enzyme apyrase was tested on the treatment of gliomas implanted in the rats CNS. One million glioma C6 cells in 3 microliters of DMEM/FCS were injected in the right striata of male Wistar rats, 250–270 g. After 20 days, the rats were decapitated and the brain sectioning and stained with hematoxylin and eosine. We performed immunohistochemical experiments with Ki67, CD31 and VEGF. Total RNA was isolated from cultured glioma C6 cells and the cDNA was analyzed by Real Time-PCR with primers for the NTPDase family. RESULTS: C6 glioma cells effectively have a low expression of all NTPDases investigated, in comparison with normal astrocytes. The implanted glioma co-injected with apyrase had a significant reduction in the tumor size (p < 0.05) when compared with the rats injected only with gliomas or with gliomas plus inactivated apyrase. According to the pathological analysis, the malignant gliomas induced by C6 injection and co-injected with apyrase presented a significant reduction in the mitotic index and other histological characteristics that indicate a less invasive/proliferative tumor. Reduction of proliferation induced by apyrase co-injection was confirmed by counting the percentage of Ki67 positive glioma cell nuclei. According to counts with CD31, vessel density and neoformation was higher in the C6 group 20 days after implantation. Confirming this observation, rats treated with apyrase presented less VEGF staining in comparison to the control group. CONCLUSION: These results indicate that the participation of extracellular ATP and the ecto-nucleotidases may be associated with the development of this type of brain tumor in an in vivo glioma model

Activity of novel quinoxaline-derived chalcones on in vitro glioma cell proliferation

Gliomas are the most common and devastating tumors of the central nervous system (CNS). Many pieces of evidence point out the relevance of natural compounds for cancer therapy and prevention, including chalcones. In the present study, eight synthetic quinoxaline-derived chalcones, structurally based on the selective PI3K gamma inhibitor AS605240, were evaluated for anti-proliferative activity and viability inhibition using glioma cell lines from human and rat origin (U-138 MG and C6, respectively), at different time-periods of incubation and concentrations. The results revealed that four chalcones (compounds 1, 6, 7 and 8), which present a methoxy group at A-ring, displayed higher efficacies and potencies, being able to inhibit either cell proliferation or viability, in a time- and concentration-dependent manner, with an efficacy that was greater than that seen for the positive control compound AS605240. Flow cytometry analysis demonstrated that incubation of C6 cells with compound 6 led to G1 phase arrest, likely indicating an interference with apoptosis. Furthermore, compound 6 was able to visibly inhibit AKT activation, allied to the stimulation of ERK MAP-kinase. The chalcones tested herein, especially those displaying a methoxy radical, might well represent promising molecules for the adjuvant treatment of glioma progression

Induction of M2 surface marker CD206 by DnaK treatment.

<p>Representative dot plots of surface (A) CD80 and (B) CD206 expression in BMMs treated with 30 ng/mL of LPS, 40 ng/mL of IL-4, 30 µg/mL or 60 µg/mL of DnaK, or left unstimulated for 24 h. The percentage values of (C) F4/80⁺CD80⁺ and (D) F4/80+CD206+ cells represent means ± SEM from triplicates. (E) and (F) show respective values for MFI analyses. (*) p<0.05, (**) p<0.01 and (***) p<0.001 indicate significant difference treated groups in relation to medium group. All data were analyzed by one-way ANOVA with Tukey post hoc test. Data are representative of three independent experiments.</p

DnaK-treated macrophages enhance tumor growth in murine allogeneic melanoma model.

<p>(A) The murine melanoma cell line B16F10 was co-injected with macrophages exposed to 30 µg/mL DnaK, 30 ng/ml of LPS, untreated macrophages, or no other cells for 24 h as illustrated in experimental design. (B) Cells were subcutaneously injected into BALB/c mice (4 mice per group) and the tumor volume was measured 8 days later as indicated. The values represent means ± SEM. (*) p<0.05 and (***) p<0.001 indicate significant difference between the macrophages exposed to DnaK in relation to B16 group. The data were analyzed by one-way ANOVA with Tuckey post hoc test in each time point. (C) Macroscopically view of tumor size. (D) Tumor weight on day 16 after tumor injection. The values represent means ± SEM. All data representative of three independent experiments.</p

Extracellular DnaK induces the expression of CD206 in peritoneal macrophages.

<p>Peritoneal macrophages were isolated from B6 mice and treated with 30 ng/mL of LPS, 40 ng/mL of IL-4, 30 µg/mL or 60 µg/mL of DnaK, or left unstimulated for 24 h. After that, cells were analyzed by flow cytometry and data presented as representative dot plots of (A) CD80, (B) CD206 expression. Data representative of three independent experiments.</p

Extracellular DnaK induces the expression of M2 markers in bone marrow-derived macrophages.

<p>BMMs were treated with LPS (30 ng/ml), IL-4 (40 ng/mL), DnaK (30 µg/mL or 60 µg/mL), or left unstimulated for 24 h. (A) iNOS activity was determinated by nitrite (NO²⁻) accumulation in the supernatant of macrophages. Data are the mean ± S.D. from triplicates. Data representative of three independent experiments. (***) p<0.001 indicates difference between LPS and other treatment groups. (B) Arginase activity was assessed by measuring the formation of urea from arginine. Data are the mean ± S.D. from triplicates. (**) p<0.01 and (***) p<0.001 indicate difference between treated groups and the medium group. Effect of DnaK on Ym1 (C) and FIZZ1 (D) expression in macrophages were quantified by real time PCR. The total amount of Ym1 and FIZZ1 mRNA were normalized to β-microglobulin signals and expressed as 2^−Δ/ΔCT. The values represent means ± SEM from triplicates. Data representative of three independent experiments. All data were analyzed by one-way ANOVA with Tukey post hoc test.</p