ABSTRACT
Title
Angiotensin-II modulates skeletal muscle chloride channel conductance via AT-1 receptor and activation of protein kinase C and reactive oxygen species signaling pathways: insight into strategies to contrast inflammation-related alterations in dystrophic myopathies
Authors
A. Cozzoli1, B. Nico2, V. Sblendorio1, R.F. Capogrosso1, V. Longo2, S. Gagliardi3, M. Montagnani3, A. De Luca1
1Unit of Pharmacology, Department of Pharmaco-biology, Faculty of Pharmacy, University of Bari, Italy; 2Department of Human Anatomy and Histology, and 3Department of Biomedical Sciences and Human Oncology, Faculty of Medicine, University of Bari, Italy.
1Unit of Pharmacology, Department of Pharmaco-biology, Faculty of Pharmacy, University of Bari, Italy; 2Department of Human Anatomy and Histology, and 3Department of Biomedical Sciences and Human Oncology, Faculty of Medicine, University of Bari, Italy.
Abstract
In fast-twitch muscle fibers, a large macroscopic chloride conductance (gCl), sustained by the expression/activity of ClC-1 channel, is important to ensure the electrical stability of sarcolemma. A decrease in gCl characterizes myofibers of dystrophic mdx mouse in relation to both spontaneous degeneration, as in diaphragm, or exercise-induced damage as in fast-twitch EDL muscle (De Luca et al. 2003). The molecular mechanism underlying gCl impairment might involve change in ClC-1 channel expression/turnover and/or function. We have shown that pro-inflammatorycytokine TNF-α can acutely reduce gCl through pathways involving protein kinase C (PKC) (Pierno et al. 2007), corroborating the view that ClC-1 channel is a marker of inflammatory states.
Angiotensin II (Ang II) is known to exert pro-inflammatory and pro-oxidative actions and recent evidence showed the presence of several components of RAS (Renin-Angiotensin System) in myofibers, corroborating an autocrine production of Ang II (Johnston et al. 2010). We then investigated if Ang II may have ClC-1 channel as a target, through phosphorylating/dephosphorylating pathways. Two microelectrodes current clamp recordings were used to measure resting gCl in EDL muscle fibers from adult wild-type mice (wt). Ang-II in vitro (10nM-30µM) produced a concentration-dependent decrease of gCl in EDL muscle, with a 40% decrease at 300 nM and a maximal block of 50% up to 30 µM. Then the calculated IC50 value was about 60 nM. Ang-II acted on gCl through the activation of the AT-1 receptor as its effects were fully inhibited by prior incubation of 300 nM losartan; accordingly no inhibition was observed with the pre-incubation of PD 123,319 (300nM), a specific AT-2 receptor antagonist. Furthermore, the effects of Ang II on gCl were antagonized by the PKC inhibitor chelerythrine (1 µM), suggesting the involvement of a PKC-dependent AT1-transduction signal. In line with the evidence that AT-1 receptor may also signal via NADPH-oxidase, the incubation of the anti-oxidant N-Acetyl Cysteine (NAC) (5mM) and the NADPH oxidase inhibitor apocynin (10µM) contrasted the effect of Ang-II in EDL muscle fibers. In line with an increased oxidative stress, Ang II significantly enhanced in a time-dependent manner, the creatin kinase released in vitro by isolated biceps muscles.
In vitro application of 300nM Ang-II significantly shifted by 4 mV the voltage threshold for mechanical activation of EDL muscle fibers toward more negative values, suggesting that Ang II increased intracellular Ca2+. This effect was inhibited by prior incubation of losartan but not of PD 123,319.
In agreement with these results, we tested the involvement of Ang-II in inflammation occurring musculardystrophy, by an in vivo treatment of dystrophic mdx mice with 1-5 mg/kg enalapril, an inhibitor of Angiotensin Converting Enzyme (6 days/week i.p. for 4-8 weeks). Enalapril significantly contrasted in a dose-dependent manner the reduction of gCl in mdx EDL muscle. In parallel the percentage of both NF-kB and dihydroethidium positive fibres weremarkedly reduced in limb muscles, supporting a drug-induced decrease of inflammation and oxidative stress.
Our data shows for the first time the ability of Ang-II to directly modulate muscle chloride channel function and support its interest as pharmacological target for inflammation-related muscle dysfunction (Supported by Telethon Italy).
De Luca et al. (2003).J Pharmacol Exp Ther. 304, 453-63
Pierno et al. (2007).Neuropathology and Applied Neurobiology33, 344–359
Johnston et al. (2010). J Renin Angiotensin Aldosterone Syst [Epub ahead of print]
Angiotensin II (Ang II) is known to exert pro-inflammatory and pro-oxidative actions and recent evidence showed the presence of several components of RAS (Renin-Angiotensin System) in myofibers, corroborating an autocrine production of Ang II (Johnston et al. 2010). We then investigated if Ang II may have ClC-1 channel as a target, through phosphorylating/dephosphorylating pathways. Two microelectrodes current clamp recordings were used to measure resting gCl in EDL muscle fibers from adult wild-type mice (wt). Ang-II in vitro (10nM-30µM) produced a concentration-dependent decrease of gCl in EDL muscle, with a 40% decrease at 300 nM and a maximal block of 50% up to 30 µM. Then the calculated IC50 value was about 60 nM. Ang-II acted on gCl through the activation of the AT-1 receptor as its effects were fully inhibited by prior incubation of 300 nM losartan; accordingly no inhibition was observed with the pre-incubation of PD 123,319 (300nM), a specific AT-2 receptor antagonist. Furthermore, the effects of Ang II on gCl were antagonized by the PKC inhibitor chelerythrine (1 µM), suggesting the involvement of a PKC-dependent AT1-transduction signal. In line with the evidence that AT-1 receptor may also signal via NADPH-oxidase, the incubation of the anti-oxidant N-Acetyl Cysteine (NAC) (5mM) and the NADPH oxidase inhibitor apocynin (10µM) contrasted the effect of Ang-II in EDL muscle fibers. In line with an increased oxidative stress, Ang II significantly enhanced in a time-dependent manner, the creatin kinase released in vitro by isolated biceps muscles.
In vitro application of 300nM Ang-II significantly shifted by 4 mV the voltage threshold for mechanical activation of EDL muscle fibers toward more negative values, suggesting that Ang II increased intracellular Ca2+. This effect was inhibited by prior incubation of losartan but not of PD 123,319.
In agreement with these results, we tested the involvement of Ang-II in inflammation occurring musculardystrophy, by an in vivo treatment of dystrophic mdx mice with 1-5 mg/kg enalapril, an inhibitor of Angiotensin Converting Enzyme (6 days/week i.p. for 4-8 weeks). Enalapril significantly contrasted in a dose-dependent manner the reduction of gCl in mdx EDL muscle. In parallel the percentage of both NF-kB and dihydroethidium positive fibres weremarkedly reduced in limb muscles, supporting a drug-induced decrease of inflammation and oxidative stress.
Our data shows for the first time the ability of Ang-II to directly modulate muscle chloride channel function and support its interest as pharmacological target for inflammation-related muscle dysfunction (Supported by Telethon Italy).
De Luca et al. (2003).J Pharmacol Exp Ther. 304, 453-63
Pierno et al. (2007).Neuropathology and Applied Neurobiology33, 344–359
Johnston et al. (2010). J Renin Angiotensin Aldosterone Syst [Epub ahead of print]