Levosimendan induces NO production through p38 MAPK, ERK and Akt in porcine coronary endothelial cells: role for mitochondrial K(ATP) channel.
OBJECTIVE
Levosimendan acts as a vasodilator through the opening of ATP-sensitive K(+) channels (K(ATP)) channels. Moreover, the coronary vasodilatation caused by levosimendan in anaesthetized pigs has recently been found to be abolished by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester, indicating that nitric oxide (NO) has a role in the vascular effects of levosimendan. However, the intracellular pathway leading to NO production caused by levosimendan has not yet been investigated. Thus, the purpose of the present study was to examine the effects of levosimendan on NO production and to evaluate the intracellular signalling pathway involved.


METHODS
In porcine coronary endothelial cells (CEC), the release of NO in response to levosimendan was examined in the presence and absence of N(omega)-nitro-L-arginine methyl ester, an adenylyl cyclase inhibitor, K(ATP) channel agonists and antagonists, and inhibitors of intracellular protein kinases. In addition, the role of Akt, ERK, p38 and eNOS was investigated through Western blot analysis.


RESULTS
Levosimendan caused a concentration-dependent and K(+)-related increase of NO production. This effect was amplified by the mitochondrial K(ATP) channel agonist, but not by the selective plasma membrane K(ATP) channel agonist. The response of CEC to levosimendan was prevented by the K(ATP) channel blockers, the adenylyl cyclase inhibitor and the Akt, ERK, p38 inhibitors. Western blot analysis showed that phosphorylation of the above kinases lead to eNOS activation.


CONCLUSIONS
In CEC levosimendan induced eNOS-dependent NO production through Akt, ERK and p38. This intracellular pathway is associated with the opening of mitochondrial K(ATP) channels and involves cAMP.
Grossini
E
E
Laboratorio di Fisiologia, Dipartimento di Medicina Clinica e Sperimentale, Facoltà di Medicina e Chirurgia, Università del Piemonte Orientale A. Avogadro, via Solaroli 17, Novara, Italy. grossini@med.unipmn.it
Molinari
C
C
Caimmi
P P
PP
Uberti
F
F
Vacca
G
G
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D006835:Hydrazones / Q000494:pharmacology*; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D054086:KATP Channels / Q000819:agonists; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D015221:Potassium Channels / Q000819:agonists / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000502:physiology*; D011724:Pyridazines / Q000494:pharmacology*; D000077464:Simendan; D013552:Swine; D014665:Vasodilator Agents / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology*
D000067956:Adenylyl Cyclase Inhibitors; D006835:Hydrazones; D054086:KATP Channels; D015221:Potassium Channels; D011724:Pyridazines; D014665:Vasodilator Agents; C117780:mitochondrial K(ATP) channel; D009569:Nitric Oxide; D000077464:Simendan; D000242:Cyclic AMP; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019331:NG-Nitroarginine Methyl Ester