Mei Y A, Vaudry D, Basille M, Castel H, Fournier A, Vaudry H, Gonzalez B J
Department of Physiology, School of Life Science, Fudan University, Shanghai 200433, China.
Eur J Neurosci. 2004 Mar;19(6):1446-58. doi: 10.1111/j.1460-9568.2004.03227.x.
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.
摘要 钾离子（K⁺）电流的激活在程序性细胞死亡的调控中起着关键作用。由于垂体腺苷酸环化酶激活多肽（PACAP）已被证明在发育过程中可抑制小脑皮质的凋亡级联反应，我们使用全细胞膜片钳技术研究了PACAP对培养的小脑颗粒细胞中K⁺电流的影响。使用两种不同的电压方案和K⁺通道特异性抑制剂对两种外向K⁺电流，即瞬时K⁺电流（I(A)）和延迟整流K⁺电流（I(K)）进行了表征。应用PACAP可导致I(K)幅度可逆性降低，但不影响I(A)，而与PACAP相关的肽血管活性肠肽对两种类型的K⁺电流均无影响。重复应用PACAP可导致电生理反应逐渐减弱。在存在鸟苷5'-[γ硫代]三磷酸（GTPγS）的情况下，PACAP引起明显且不可逆的I(K)抑制，而用鸟苷5'-[β硫代]二磷酸（GDPβS）进行细胞透析则完全消除了PACAP的作用。用百日咳毒素预处理细胞并未改变PACAP对I(K)的作用。相反，霍乱毒素抑制了PACAP诱导的I(K)抑制。将颗粒细胞暴露于二丁酰环磷酸腺苷（dbcAMP）可模拟PACAP对I(K)的抑制作用。在膜片钳微管溶液中添加特异性蛋白激酶A抑制剂H89可防止PACAP和dbcAMP诱导的I(K)降低。PACAP可使在含高或低钾离子的培养基中培养的小脑颗粒细胞的静息膜电位持续升高，而PACAP的这种长期去极化作用可被I(K)特异性阻滞剂氯化四乙铵（TEA）模拟。此外，用TEA预孵育颗粒细胞可抑制PACAP对静息膜电位的作用。TEA模拟了PACAP对乙醇诱导的凋亡细胞死亡的神经保护作用，并且颗粒细胞暴露于乙醇后观察到的半胱天冬酶-3活性增加也被TEA显著抑制。综上所述，目前的结果表明，在大鼠小脑颗粒细胞中，PACAP通过激活与腺苷酸环化酶/蛋白激酶A途径偶联的1型PACAP（PAC1）受体，经霍乱毒素敏感的Gs蛋白降低延迟外向整流K⁺电流。我们的数据还表明，PACAP和TEA诱导静息膜电位的长期去极化，促进细胞存活并抑制半胱天冬酶-3活性，提示PACAP诱发的I(K)抑制有助于该肽对小脑颗粒细胞的抗凋亡作用。
