Oxidative stress, resulting from accumulation of reactive oxygen species, plays a critical role in neuronal cell death associated with neurodegenerative diseases and stroke. In the present study, we have investigated the potential neuroprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on oxidative stress-induced apoptosis. Incubation of cerebellar granule cells with PACAP inhibited hydrogen peroxide-evoked cell death in a concentration-dependent manner. The effect of PACAP on granule cell survival was not mimicked by vasoactive intestinal polypeptide and was blocked by the antagonist PACAP6-38. The protective action of PACAP upon hydrogen peroxide-induced neuronal cell death was abolished by the MAP-kinase kinase (MEK) inhibitor U0126 and mimicked by the caspase-3 inhibitor Z-DEVD-FMK. PACAP markedly inhibited hydrogen peroxide-evoked caspase-3 activation and DNA fragmentation. Taken together, these data indicate that PACAP, acting through PACAP receptor type 1, exerts a potent protective effect against neuronal degeneration induced by hydrogen peroxide. The anti-apoptotic effect of PACAP is mediated through the MAP-kinase pathway and can be accounted for by inhibition of caspase-3 activation resulting from oxidative stress.

Activation of potassium (K(+)) currents plays a critical role in the control of programmed cell death. Because pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to inhibit the apoptotic cascade in the cerebellar cortex during development, we have investigated the effect of PACAP on K(+) currents in cultured cerebellar granule cells using the patch-clamp technique in the whole-cell configuration. Two types of outward K(+) currents, a transient K(+) current (I(A)) and a delayed rectifier K(+) current (I(K)) were characterized using two different voltage protocols and specific inhibitors of K(+) channels. Application of PACAP induced a reversible reduction of the I(K) amplitude, but did not affect I(A), while the PACAP-related peptide vasoactive intestinal polypeptide had no effect on either types of K(+) currents. Repeated applications of PACAP induced gradual attenuation of the electrophysiological response. In the presence of guanosine 5'-[gammathio]triphosphate (GTPgammaS), PACAP provoked a marked and irreversible I(K) depression, whereas cell dialysis with guanosine 5'-[betathio]diphosphate GDPbetaS totally abolished the effect of PACAP. Pre-treatment of the cells with pertussis toxin did not modify the effect of PACAP on I(K). In contrast, cholera toxin suppressed the PACAP-induced inhibition of I(K). Exposure of granule cells to dibutyryl cyclic adenosine monophosphate (dbcAMP) mimicked the inhibitory effect of PACAP on I(K). Addition of the specific protein kinase A inhibitor H89 in the patch pipette solution prevented the reduction of I(K) induced by both PACAP and dbcAMP. PACAP provoked a sustained increase of the resting membrane potential in cerebellar granule cells cultured either in high or low KCl-containing medium, and this long-term depolarizing effect of PACAP was mimicked by the I(K) specific blocker tetraethylammonium chloride (TEA). In addition, pre-incubation of granule cells with TEA suppressed the effect of PACAP on resting membrane potential. TEA mimicked the neuroprotective effect of PACAP against ethanol-induced apoptotic cell death, and the increase of caspase-3 activity observed after exposure of granule cells to ethanol was also significantly inhibited by TEA. Taken together, the present results demonstrate that, in rat cerebellar granule cells, PACAP reduces the delayed outward rectifier K(+) current by activating a type 1 PACAP (PAC1) receptor coupled to the adenylyl cyclase/protein kinase A pathway through a cholera toxin-sensitive Gs protein. Our data also show that PACAP and TEA induce long-term depolarization of the resting membrane potential, promote cell survival and inhibit caspase-3 activity, suggesting that PACAP-evoked inhibition of I(K) contributes to the anti-apoptotic effect of the peptide on cerebellar granule cells.

Oxidative stress, induced by various neurodegenerative diseases, initiates a cascade of events leading to apoptosis, and thus plays a critical role in neuronal injury. In this study, we have investigated the potential neuroprotective effect of the octadecaneuropeptide (ODN) on 6-hydroxydopamine (6-OHDA)-induced oxidative stress and apoptosis in cerebellar granule neurons (CGN). ODN, which is produced by astrocytes, is an endogenous ligand for both central-type benzodiazepine receptors (CBR) and a metabotropic receptor. | 2013 | 125 | 620-633 doi: 10.1111/jnc.12140 these data demonstrate for the first time that ODN is a neuroprotective agent that prevents 6-OHDA-induced oxidative stress and apoptotic cell death.