Data regarding M1 muscarinic receptor-mediated modulation of hepatic catalase activity in response to oxidative stress

We recently demonstrated the role of M1 muscarinic receptors (M1R) in modulating oxidative stress in liver and hepatocytes (Urrunaga et al., 2015) [1]. Here we provide data regarding the effect of a novel M1R agonist, VU0357017 (Lebois et al., 2010) [2], on H2O2-mediated hepatocyte injury, the effect of an M1R antagonist VU0255035 (Sheffler et al., 2009) [3] on catalase and super oxide dismutase (SOD) activities in H2O2–treated hepatocytes in vitro, and finally, the effect of M1R ablation on hepatic catalase activity in acetaminophen (APAP)-treated mice.


a b s t r a c t
We recently demonstrated the role of M1 muscarinic receptors (M1R) in modulating oxidative stress in liver and hepatocytes (Urrunaga et al., 2015) [1]. Here we provide data regarding the effect of a novel M1R agonist, VU0357017 (Lebois et al., 2010) [2], on H 2 O 2 -mediated hepatocyte injury, the effect of an M1R antagonist VU0255035 (Sheffler et al., 2009) [3] on catalase and super oxide dismutase (SOD) activities in H 2 O 2 -treated hepatocytes in vitro, and finally, the effect of M1R ablation on hepatic catalase activity in acetaminophen (APAP)-treated mice.
& In contrast to M1R inhibition that reduces hepatocyte injury [1], M1R activation has no effect. In addition to enhancing GSH recovery [1], M1R inhibition enhances hepatic catalase activity. However, M1R inhibition has no effect on hepatocyte SOD activity.

Experimental animals
Animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the United States National Academy of Sciences (National Institutes of Health), approved by the Institutional Animal Care and Use Committee, and described in detail previously [1]. The stored liver tissue from the Chrm1 À / À and WT mice were used to assess catalase activity. Fig. 2. Effects of a highly selective M1R antagonist VU0255035, on catalase and SOD activities were assessed in AML12 hepatocytes treated with 1 mM H 2 O 2 for 120 min. Compared to cells treated with vehicle (DMSO) alone, catalase and SOD activities were significantly reduced in H 2 O 2 -treated cells. Co-treatment with VU0255035 improved catalase activity (A), but had no effect on SOD activity (B). (C) VU0255035 alone had no effect on cell viability. Results are mean7 S.E.M. * P o0.05, ** Po 0.01. Fig. 3. Effect of M1R ablation on APAP-induced changes in hepatic catalase activity. WT and Chrm1 À / À mice were fasted overnight and treated with APAP 200 mg/kg intraperitoneally in the morning. Two and 4 h after APAP injection, mice were euthanized and their livers harvested and analyzed as described previously [1]. By 2 h, hepatic catalase activity reduced significantly in all APAP-treated mice. However, by 4 h, catalase activity was significantly higher in the livers of Chrm1 À / À mice when compared to WT mice. Results are mean7 S.E.M. * Po 0.05.

Preparation of cell extracts
AML12 cells (1 Â 10 5 cells/well) were maintained in six-well plates in the presence of 1 mM H 2 O 2 and vehicle or 1 mM VU0255035 (PubChem CID: 24768606). After 2 h, cells were lysed using a polytron homogenizer using isolation buffer (250 mM sucrose, 10 mM Tris HCL pH 7.4, and 0.1 mM EGTA).

Superoxide dismutase activity assay
SOD activity was estimated as described previously [4]. Reaction mixture contained 0.1 ml phenazine methosulphate (186 μM), 1.2 ml sodium pyrophosphate buffer (0.052 mM, pH 7.0) and 0.3 ml cell supernatant. Enzyme reaction was initiated by adding 0.2 ml NADH (780 μM) and terminated after 1 min by adding 1 ml glacial acetic acid. The chromogen formed was assessed by measuring absorbance at 560 nm on BioMate 3S Spectrophotometer (Thermo Scientific, USA). Results are expressed in units/mg protein.

Catalase activity assay
Catalase activity was assessed in cellular and tissue fractions as described earlier [5]. Briefly, the activity was determined by measuring the decrease in absorbance at 240 nm of a reaction mixture consisting H 2 O 2 in phosphate buffer, pH 7.0, and cellular/tissue extracts. The molar extinction coefficient of 43.6 M cm À 1 was used to determine catalase activity and expressed as units/mg protein.

Statistical analysis
All data are expressed as mean 7S.E.M. Normality was determined using the Shapiro-Wilk test. Student's t-test (normally distributed data) or the Mann-Whitney U test (nonparametric data) was used to determine significance. Analysis was performed using SigmaPlot (version 12.0; Systat Software, Inc. San Jose, CA). Significance was defined as Po0.05.