Apoptosis induced in neuronal cultures by either the phosphatase inhibitor okadaic acid or the kinase inhibitor staurosporine is attenuated by isoquinolinesulfonamides H-7, H-8, and H-9.
Protein phosphorylation is kept in balance by an orchestrated action of kinases and phosphatases; when this balance is lost, neuronal apoptosis may occur. Okadaic acid (OKA), a marine toxin that inhibits specifically protein phosphatases 1 and 2A (EC 3.1.3.16), and staurosporine, an inhibitor of protein kinase C (PKC; EC 2.7.1.37), induced apoptosis in primary cultures of rat cerebellar granule neurons. We assayed apoptosis by the DNA gel electrophoresis, by the in situ TUNEL assay, and by morphological appearance following propidium iodide staining. Cell viability was assessed by the Trypan blue assay. Both OKA- and staurosporine-induced neuronal apoptosis were prevented by a macromolecular synthesis inhibitor actinomycin D and by a group of isoquinolinesulfonamide kinase inhibitors (H-7, 1-[5-isoquinolinesulfonyl]-2-methylpiperazine; H-8, N-¿2-[methylamino]ethyl¿-5-isoquinolinesulfonamide; H-9, N-(2-aminoethyl)-5-isoquinolinesulfonamide, but not by inhibitors of PKC, cyclic-GMP- and cyclic-AMP-dependent kinases, calcium/calmodulin-dependent kinases, tyrosine kinases, or by antioxidants. We postulate that a common mechanism, possibly an increased protein phosphorylation, is responsible for apoptosis triggered by an inhibition of phosphatases 1 and 2A and PKC. Elucidating the isoquinolinesulfonamide-sensitive mechanism may help us find new therapies for neurodegenerative diseases that involve apoptosis.
Cagnoli
C M
CM
Allegheny-Singer Research Institute, Medical College of Pennsylvania, Pittsburgh, USA.
Kharlamov
E
E
Atabay
C
C
Uz
T
T
Manev
H
H
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects; D002531:Cerebellum / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D019319:Okadaic Acid / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine / Q000494:pharmacology*; D013449:Sulfonamides
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D019319:Okadaic Acid; C044388:N-(2-aminoethyl)-5-isoquinolinesulfonamide; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D019311:Staurosporine