PC12 cells are a rat pheochromocytoma cell line that synthesizes dopamine endogenously and are a commonly employed cellular model for investigating the neurotrophic effects of PACAP [
38]. PC12 cells share a similar mechanism of exocytosis with neurons [
30], and the mechanisms underlying the release of dopamine from PC12 cells, including membrane depolarization and increase in intracellular Ca
2+, have been extensively investigated through reconstitution of PAC1 receptors for PACAP in PC12 cells [
39]. As shown in this study, PACAP stimulated dopamine release from PC12 cells in a concentration-dependent manner with an EC
50 of 2.42 nM (Figure
2A). Dopamine was rapidly released within 1 min after stimulation with PACAP, and reuptake of it into the cells started by 10 min (Figure
2B). Although SNAP attenuated the evoked release at any time from 0.5 to 15 min, the extent of the inhibitory effect by SNAP was reduced during a longer incubation in the presence of imipramine (Figure
2C). Therefore, to simplify the elucidation of the action mechanisms of SNAP, we examined the effect of SNAP on the release for the initial 5 min. NO has long been considered to act largely through cGMP formed by activation of soluble guanylyl cyclase and subsequent cGK activation in the nervous system [
5]. It was previously shown that an ATP-sensitive K
+ channel is a target of cGK and that glibenclamide directly blocks both acute and persistent hypernociception via opening of an ATP-sensitive K
+ channel [
32]. The inhibition by SNAP was not attenuated by ODQ, a soluble guanylyl cyclase inhibitor; KT5823, a cGK inhibitor; or glibenclamide, an ATP-sensitive K
+ channel blocker (Figure
3A). Conversely, neither 8-Br-cGMP nor 8-Br-cAMP itself affected the basal release (data not shown) or PACAP-enhanced dopamine release (Figure
3B). Consistent with the distribution of cGKIα in the cerebellum and DRG [
40], it was detected in the homogenate of the cerebellum and DRG, but not in PC12 cells (Figure
3C). These results demonstrate that the inhibitory effect by SNAP was not mediated by the NO/cGMP/cGK pathway.