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Levosimendan inhibits peroxidation in hepatocytes by modulating apoptosis/autophagy interplay.

Grossini E, Bellofatto K, Farruggio S, Sigaudo L, Marotta P, Raina G, De Giuli V, Mary D, Pollesello P, Minisini R, Pirisi M, Vacca G - PLoS ONE (2015)

Bottom Line: In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased.Finally, all protective effects were prevented by both mitoKATP channels inhibition and NOS blocking.Such effects would involve mitoKATP channels opening and the modulation of NO release by the different NOS isoforms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physiology and Experimental Surgery, Department of Translational Medicine, University Eastern Piedmont "Amedeo Avogadro", Via Solaroli 17, Azienda Ospedaliera Universitaria Maggiore della Carità, corso Mazzini 36, Novara, Italy.

ABSTRACT

Background: Levosimendan protects rat liver against peroxidative injuries through mechanisms related to nitric oxide (NO) production and mitochondrial ATP-dependent K (mitoKATP) channels opening. However, whether levosimendan could modulate the cross-talk between apoptosis and autophagy in the liver is still a matter of debate. Thus, the aim of this study was to examine the role of levosimendan as a modulator of the apoptosis/autophagy interplay in liver cells subjected to peroxidation and the related involvement of NO and mitoKATP.

Methods and findings: In primary rat hepatocytes that have been subjected to oxidative stress, Western blot was performed to examine endothelial and inducible NO synthase isoforms (eNOS, iNOS) activation, apoptosis/autophagy and survival signalling detection in response to levosimendan. In addition, NO release, cell viability, mitochondrial membrane potential and mitochondrial permeability transition pore opening (MPTP) were examined through specific dyes. Some of those evaluations were also performed in human hepatic stellate cells (HSC). Pre-treatment of hepatocytes with levosimendan dose-dependently counteracted the injuries caused by oxidative stress and reduced NO release by modulating eNOS/iNOS activation. In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased. Finally, all protective effects were prevented by both mitoKATP channels inhibition and NOS blocking. In HSC, levosimendan was able to modulate the oxidative balance and inhibit autophagy without improving cell viability and apoptosis.

Conclusions: Levosimendan protects hepatocytes against oxidative injuries by autophagic-dependent inhibition of apoptosis and the activation of survival signalling. Such effects would involve mitoKATP channels opening and the modulation of NO release by the different NOS isoforms. In HSC, levosimendan would also play a role in cell activation and possible evolution toward fibrosis. These findings highlight the potential of levosimendan as a therapeutic agent for the treatment or prevention of liver ischemia/reperfusion injuries.

No MeSH data available.


Related in: MedlinePlus

Effects of levosimendan on eNOS (A) and iNOS activation (B) in hepatocytes subjected to peroxidation.In A and B, densitometric analysis of p-eNOS and iNOS and an example of lanes taken in one of 5 different experiments performed for each experimental protocol. eNOS = endothelial nitric oxide isoform; iNOS = inducible nitric oxide isoform; L = levosimendan; NM = L-NAME. The other abbreviations are as in Figs 1–4. In A, b, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e p<0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).
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pone.0124742.g005: Effects of levosimendan on eNOS (A) and iNOS activation (B) in hepatocytes subjected to peroxidation.In A and B, densitometric analysis of p-eNOS and iNOS and an example of lanes taken in one of 5 different experiments performed for each experimental protocol. eNOS = endothelial nitric oxide isoform; iNOS = inducible nitric oxide isoform; L = levosimendan; NM = L-NAME. The other abbreviations are as in Figs 1–4. In A, b, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e p<0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).

Mentions: As shown in Fig 4A, in physiologic condition levosimendan dose-dependently increased NO release in hepatocytes, which confirmed previous findings about the involvement of NO in the effects of levosimendan [23]. Moreover, pre-treatment of hepatocytes with 200 μM H2O2 strongly increased NO release (Fig 4B), an effect that was accompanied by p-eNOS inhibition and iNOS activation (Fig 5). As shown in Fig 4B, in peroxidative conditions levosimendan was able to counteract NO release by restoring eNOS/iNOS ratio in hepatocytes (Fig 5).


Levosimendan inhibits peroxidation in hepatocytes by modulating apoptosis/autophagy interplay.

Grossini E, Bellofatto K, Farruggio S, Sigaudo L, Marotta P, Raina G, De Giuli V, Mary D, Pollesello P, Minisini R, Pirisi M, Vacca G - PLoS ONE (2015)

Effects of levosimendan on eNOS (A) and iNOS activation (B) in hepatocytes subjected to peroxidation.In A and B, densitometric analysis of p-eNOS and iNOS and an example of lanes taken in one of 5 different experiments performed for each experimental protocol. eNOS = endothelial nitric oxide isoform; iNOS = inducible nitric oxide isoform; L = levosimendan; NM = L-NAME. The other abbreviations are as in Figs 1–4. In A, b, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e p<0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400069&req=5

pone.0124742.g005: Effects of levosimendan on eNOS (A) and iNOS activation (B) in hepatocytes subjected to peroxidation.In A and B, densitometric analysis of p-eNOS and iNOS and an example of lanes taken in one of 5 different experiments performed for each experimental protocol. eNOS = endothelial nitric oxide isoform; iNOS = inducible nitric oxide isoform; L = levosimendan; NM = L-NAME. The other abbreviations are as in Figs 1–4. In A, b, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e p<0.05 vs c; d, f, g P <0.05 vs e. In B, b, c, d, e, f, g P <0.05 vs a; c, d, e, f, g P <0.05 vs b; d, e P <0.05 vs c; d, f, g P <0.05 vs e. The results of densitometric analysis are expressed as means of 5 independent experiments (%) ± SD (indicated by the bars).
Mentions: As shown in Fig 4A, in physiologic condition levosimendan dose-dependently increased NO release in hepatocytes, which confirmed previous findings about the involvement of NO in the effects of levosimendan [23]. Moreover, pre-treatment of hepatocytes with 200 μM H2O2 strongly increased NO release (Fig 4B), an effect that was accompanied by p-eNOS inhibition and iNOS activation (Fig 5). As shown in Fig 4B, in peroxidative conditions levosimendan was able to counteract NO release by restoring eNOS/iNOS ratio in hepatocytes (Fig 5).

Bottom Line: In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased.Finally, all protective effects were prevented by both mitoKATP channels inhibition and NOS blocking.Such effects would involve mitoKATP channels opening and the modulation of NO release by the different NOS isoforms.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Physiology and Experimental Surgery, Department of Translational Medicine, University Eastern Piedmont "Amedeo Avogadro", Via Solaroli 17, Azienda Ospedaliera Universitaria Maggiore della Carità, corso Mazzini 36, Novara, Italy.

ABSTRACT

Background: Levosimendan protects rat liver against peroxidative injuries through mechanisms related to nitric oxide (NO) production and mitochondrial ATP-dependent K (mitoKATP) channels opening. However, whether levosimendan could modulate the cross-talk between apoptosis and autophagy in the liver is still a matter of debate. Thus, the aim of this study was to examine the role of levosimendan as a modulator of the apoptosis/autophagy interplay in liver cells subjected to peroxidation and the related involvement of NO and mitoKATP.

Methods and findings: In primary rat hepatocytes that have been subjected to oxidative stress, Western blot was performed to examine endothelial and inducible NO synthase isoforms (eNOS, iNOS) activation, apoptosis/autophagy and survival signalling detection in response to levosimendan. In addition, NO release, cell viability, mitochondrial membrane potential and mitochondrial permeability transition pore opening (MPTP) were examined through specific dyes. Some of those evaluations were also performed in human hepatic stellate cells (HSC). Pre-treatment of hepatocytes with levosimendan dose-dependently counteracted the injuries caused by oxidative stress and reduced NO release by modulating eNOS/iNOS activation. In hepatocytes, while the autophagic inhibition reduced the effects of levosimendan, after the pan-caspases inhibition, cell survival and autophagy in response to levosimendan were increased. Finally, all protective effects were prevented by both mitoKATP channels inhibition and NOS blocking. In HSC, levosimendan was able to modulate the oxidative balance and inhibit autophagy without improving cell viability and apoptosis.

Conclusions: Levosimendan protects hepatocytes against oxidative injuries by autophagic-dependent inhibition of apoptosis and the activation of survival signalling. Such effects would involve mitoKATP channels opening and the modulation of NO release by the different NOS isoforms. In HSC, levosimendan would also play a role in cell activation and possible evolution toward fibrosis. These findings highlight the potential of levosimendan as a therapeutic agent for the treatment or prevention of liver ischemia/reperfusion injuries.

No MeSH data available.


Related in: MedlinePlus