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.
Mei
Y A
YA
Department of Physiology, School of Life Science, Fudan University, Shanghai 200433, China.
Vaudry
D
D
Basille
M
M
Castel
H
H
Fournier
A
A
Vaudry
H
H
Gonzalez
B J
BJ
D015761:4-Aminopyridine / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000494:pharmacology; D002531:Cerebellum / Q000166:cytology; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism* / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism* / Q000502:physiology; D051668:Delayed Rectifier Potassium Channels; D004347:Drug Interactions; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D008959:Models, Neurological; D009433:Neural Inhibition / Q000187:drug effects; D009474:Neurons / Q000187:drug effects; D009479:Neuropeptides / Q000494:pharmacology*; D018408:Patch-Clamp Techniques / Q000379:methods; D010446:Peptide Fragments / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects*; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects*; D013449:Sulfonamides; D019789:Tetraethylammonium / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
C495403:Adcyap1 protein, rat; D002492:Central Nervous System Depressants; D051668:Delayed Rectifier Potassium Channels; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D009479:Neuropeptides; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D013449:Sulfonamides; D013873:Thionucleotides; C108046:pituitary adenylate cyclase-activating-peptide (1-38), pig; D006153:Guanosine Diphosphate; D014660:Vasoactive Intestinal Peptide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000431:Ethanol; D013779:Tetrodotoxin; D019789:Tetraethylammonium; C023427:guanosine 5'-O-(2-thiodiphosphate); D002772:Cholera Toxin; D015761:4-Aminopyridine; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide