Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells

Abstract

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na +, K +-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5–6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na +, K +-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethazone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na +, K +-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na +, K +-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na +, K +-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na +, K +-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na +, K +-ATPase activity via directly increasing cAMP and NO production in nerves.