Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells.
A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.
Chijiwa
T
T
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Mishima
A
A
Hagiwara
M
M
Sano
M
M
Hayashi
K
K
Inoue
T
T
Naito
K
K
Toshioka
T
T
Hidaka
H
H
D000310:Adrenal Gland Neoplasms; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology*; D003994:Bucladesine / Q000494:pharmacology; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology*; D007546:Isoquinolines / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D009363:Neoplasm Proteins / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000494:pharmacology; D010673:Pheochromocytoma; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides
D007546:Isoquinolines; D009363:Neoplasm Proteins; D009414:Nerve Growth Factors; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D005576:Colforsin; D003994:Bucladesine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide