Erk1/2 and Akt kinases are involved in the protective effect of aniracetam in astrocytes subjected to simulated ischemia in vitro.
The present study focused on the mechanism of cytoprotective effect of aniracetam on the primary rat astrocyte cultures exposed to simulated ischemia conditions in vitro. To study these mechanisms, the aniracetam-mediated modulation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/Akt kinase pathways was determined. Simulated in vitro ischemia caused death of approximately 35% of astrocytes via apoptosis and decreased cell viability about 50% at 8 h. Exposure to aniracetam at concentrations of 0.1-10 microM in these conditions significantly decreased the number of apoptotic cells. Moreover, the intensification of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion and the decrease of lactate dehydrogenase (LDH) release after 1 and 10 microM aniracetam treatment were observed indicating a significant increase in cell viability. When cultured astrocytes were incubated during 8 h simulated ischemia with [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] (U0126), an extracellular regulated kinase 1 and 2 (Erk1/2) inhibitor or wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase)/Akt inhibitor, the cell apoptosis was accelerated. These effects of used kinase inhibitors (both U0126 and wortmannin) were antagonized by adding 1 and 10 microM aniracetam to the culture medium. In addition, aniracetam significantly stimulated of phospho-Erk1/2 kinase and phospho-Akt expression. Maximum levels of Erk1/2 and Akt activation were observed as a result of treatment with 10 microM aniracetam. U0126 and wortmannin markedly attenuated the effects of aniracetam on expression of activated kinases. Results of the present study indicate that both Erk1/2 and PI 3-K/Akt kinase pathways are vital for cytoprotective effect of aniracetam.
Gabryel
Bozena
B
Department of Pharmacology, Silesian Medical University, 18 Medyków Street, PL 40752 Katowice, Poland. bgabryel@interia.pl
Pudelko
Anna
A
Malecki
Andrzej
A
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000187:drug effects* / Q000473:pathology*; D001562:Benzimidazoles; D015153:Blotting, Western; D002545:Brain Ischemia / Q000473:pathology*; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009457:Neuroglia / Q000187:drug effects; D018696:Neuroprotective Agents; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013778:Tetrazolium Salts; D013844:Thiazoles
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D018696:Neuroprotective Agents; D011518:Proto-Oncogene Proteins; D011760:Pyrrolidinones; D013778:Tetrazolium Salts; D013844:Thiazoles; C036466:aniracetam; D007770:L-Lactate Dehydrogenase; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; C022616:thiazolyl blue; C017807:bisbenzimide ethoxide trihydrochloride