PACAP inhibits delayed rectifier potassium current via a cAMP/PKA transduction pathway: evidence for the involvement of I_K in the anti‐apoptotic action of PACAP

Abstract

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.

Endogenous PACAP acts as a stress response peptide to protect cerebellar neurons from ethanol or oxidative insult

“…Concerning the mechanisms involved, it has been shown that ethanol and PACAP exert an opposite effect on the delayed outward rectifier potassium current which in turn modulate caspase-3 activity that finally contributes to the control of granule cell survival [15]. The antiapoptotic effect of PACAP after oxidative stress may also play a major role in the protection of the brain during several forms of neuronal insult due to a decrease of blood flow and/or oxidative damage [18].…”

Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 Years after the Discovery

“…11). The inhibitory effect of PACAP on potassium channels also contributes to the control of cell death (Zerr and Feltz, 1994;Mei et al, 2004;Castel et al, 2006). It has been proposed that activation by PACAP of the phosphatidylinositol 3Ј-OH kinase (PI3-K) neuroprotective pathway may synergize with the PKA cascade to promote cell survival (Fig.…”

Delineating the Factors and Cellular Mechanisms Involved in the Survival of Cerebellar Granule Neurons

“…41,42 In addition, it has been described that PACAP mediates its neuroprotective effect, in part, by inhibition of delayed rectifier K(+) current (I(K)) via cAMP/PKA transduction pathway. 43,44 Moreover, the survival effect of PACAP could be mediated by others pathways. It has been demonstrated that PACAP promotes the increase of intracellular calcium from intracellular stores and calcium influx through calcium channels.…”