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Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.

Madhani M, Hall AR, Cuello F, Charles RL, Burgoyne JR, Fuller W, Hobbs AJ, Shattock MJ, Eaton P - Am. J. Physiol. Heart Circ. Physiol. (2010)

Bottom Line: PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated.The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive.Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.

ABSTRACT
The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na(+)/K(+)-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 muM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na(+)/K(+)-ATPase activation during reperfusion would attenuate Na(+) overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na(+)/K(+)-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

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The effect of sildenafil (Sild) at reperfusion in the absence or presence of KT-5823 on PLM Ser69 phosphorylation and total PLM. The PKG inhibitor KT-5823 reduced basal Ser69 phosphorylation and also blocked the sildenafil-dependant increase. *P < 0.05; **P < 0.01; ***P < 0.001.
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Figure 5: The effect of sildenafil (Sild) at reperfusion in the absence or presence of KT-5823 on PLM Ser69 phosphorylation and total PLM. The PKG inhibitor KT-5823 reduced basal Ser69 phosphorylation and also blocked the sildenafil-dependant increase. *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: To determine whether increased phosphorylation at the Ser69 site following sildenafil treatment was PKG dependent, we tested whether the PKG inhibitor KT-5823 reduced phosphorylation at this site (see protocol in Fig. 1D). Figure 5 reaffirms that sildenafil significantly increases PLM Ser69 phosphorylation. PKG inhibition with KT-5823 significantly reduced basal, as well as sildenafil-induced, PLM Ser69 phosphorylation. Thus KT5823 treatment at reperfusion blocks both sildenafil-induced PLM Ser69 phosphorylation and sildenafil-mediated cardioprotection.


Phospholemman Ser69 phosphorylation contributes to sildenafil-induced cardioprotection against reperfusion injury.

Madhani M, Hall AR, Cuello F, Charles RL, Burgoyne JR, Fuller W, Hobbs AJ, Shattock MJ, Eaton P - Am. J. Physiol. Heart Circ. Physiol. (2010)

The effect of sildenafil (Sild) at reperfusion in the absence or presence of KT-5823 on PLM Ser69 phosphorylation and total PLM. The PKG inhibitor KT-5823 reduced basal Ser69 phosphorylation and also blocked the sildenafil-dependant increase. *P < 0.05; **P < 0.01; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944484&req=5

Figure 5: The effect of sildenafil (Sild) at reperfusion in the absence or presence of KT-5823 on PLM Ser69 phosphorylation and total PLM. The PKG inhibitor KT-5823 reduced basal Ser69 phosphorylation and also blocked the sildenafil-dependant increase. *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: To determine whether increased phosphorylation at the Ser69 site following sildenafil treatment was PKG dependent, we tested whether the PKG inhibitor KT-5823 reduced phosphorylation at this site (see protocol in Fig. 1D). Figure 5 reaffirms that sildenafil significantly increases PLM Ser69 phosphorylation. PKG inhibition with KT-5823 significantly reduced basal, as well as sildenafil-induced, PLM Ser69 phosphorylation. Thus KT5823 treatment at reperfusion blocks both sildenafil-induced PLM Ser69 phosphorylation and sildenafil-mediated cardioprotection.

Bottom Line: PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated.The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive.Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, School of Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom.

ABSTRACT
The phosphodiesterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the Na(+)/K(+)-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 muM sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Ser68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the Na(+)/K(+)-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. Na(+)/K(+)-ATPase activation during reperfusion would attenuate Na(+) overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced Na(+)/K(+)-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion.

Show MeSH
Related in: MedlinePlus