Zhuang Shou-Yuan, Bridges Daniel, Grigorenko Elena, McCloud Stephen, Boon Andrew, Hampson Robert E, Deadwyler Sam A
Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA.
Neuropharmacology. 2005 Jun;48(8):1086-96. doi: 10.1016/j.neuropharm.2005.01.005.
Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 microM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration (Ca2+) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in Ca2+ occurred with similar concentrations (25-100 nM) and in the same time-dependent manner (pre-exposure 1-15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in Ca2+ was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing Ca2+ via a cAMP/PKA-dependent process during the neurotoxic event.
外源性给予的大麻素在几种不同的细胞和动物模型中具有神经保护作用。在当前研究中，两种大麻素CB1受体配体（WIN 55,212-2、CP 55,940）以时间依赖性方式显著减少了在高水平NMDA（15微摩尔）作用下培养神经元中的海马细胞死亡。WIN 55,212-2还被证明能抑制NMDA诱导的细胞内钙浓度（Ca2+）升高，这是通过同一培养神经元中FURA-2荧光成像显示的。Ca2+的变化与CB1受体介导的神经保护作用具有相似的浓度（25 - 100纳摩尔）和相同的时间依赖性方式（预暴露1 - 15分钟）。这两种作用均被CB1受体拮抗剂SR141716A阻断。一个潜在机制表现为：（1）ryanodine抑制了NMDA诱导的Ca2+升高，这表明存在一种与ryanodine受体（RyR）偶联的细胞内钙通道；（2）大麻素的影响涉及降低cAMP依赖性蛋白激酶（PKA）对调节通道的相同RyR水平的磷酸化作用。此外，CB1受体介导的PKA磷酸化抑制的时间进程与大麻素神经保护的有效预暴露间隔直接相关。对照研究排除了肌醇三磷酸（IP3）途径、增强的钙再摄取和电压敏感性钙通道参与神经保护过程。结果表明，大麻素通过启动对腺苷酸环化酶的时间和剂量依赖性抑制来预防细胞死亡，这种抑制作用在CB1受体的直接作用之后持续存在，并且能够在神经毒性事件期间通过cAMP/PKA依赖性过程降低Ca2+。
