蛋白激酶C 参与内源性大麻素花生四烯乙醇胺对L型钙电流的抑制作用

摘要

Objective To investigate whether anandamide, one of endocannabinoids, can inhibit L-type calcium current by rebulating protein kinase C ( PKC ) activity, and to explore the possible signal pathway that may change PKC activity.Methods L-type calcium current of single cardiomyocyte was recorded by whole cell patch clamp technique;the PKC activity was detected by PepTag non radioactive protein kinase C detection system ( Promega);DAG content within cell was determined by ELISA kit;the expressions of PLCβand p-PLCβwere detected by Western Blot.Results Anandamide significantly inhibited L-type calcium current after perfusion of myocytes ( P <0.05),and the effect could be blocked by pretreatment of CB1 receptor antagonist AM251 or PKC nonspecific agonist PMA,however,CB2 recepor antagonist AM630 had no the effect.Anandamide could inhibit significantly PKC activity ( P <0.05),similarly,pretreatment of AM251 or PMA could block the effect,however,AM630 had no the effect.Anandamide had no effect on DAG content as well as expressions of PLCβand p-PLCβ.Conclusion The experimental results demonstrate that endocannabinoid-anandamide can inhibit PKC activity by activating CB1 receptor to inhibit L-type calcium current,however,PLCβ-DAG signal pathway is not involved in the process.%目的：研究内源性大麻素物质花生四烯乙醇胺是否通过改变蛋白激酶C（ PKC）活性从而抑制心肌L型钙电流，并进一步探讨可能改变PKC活性的信号途径。方法应用全细胞膜片钳技术记录单个心肌细胞的L型钙电流（P ＜0 n．05）；应用PepTag非放射性蛋白激酶C检测系统（ Promega）检测PKC活性；Elisa试剂盒测定细胞中二脂酰甘油（ DAG）的含量；western blot 技术测定磷脂酶Cβ（ PLCβ）和磷酸化磷脂酶C β（ p-PLCβ）表达。结果应用花生四烯乙醇胺灌流心肌细胞后显著抑制心肌L型钙电流（ P ＜0．05），预先应用大麻素1型受体（ CB1）阻断剂AM251或PKC非特异性激动剂佛波醇酯（PMA）可以完全阻断此抑制效应，而大麻素2型受体（CB2）阻断剂AM630没有阻断花生四烯乙醇胺抑制L型钙电流的作用。检测心肌细胞PKC活性发现，花生四烯乙醇胺明显抑制PKC活性（ P ＜0．05），同样预先应用AM251或PMA完全阻断花生四烯乙醇胺对PKC活性的抑制效应，而AM630无此效应。应用花生四烯乙醇胺没有影响心肌细胞DAG含量和PLCβ的磷酸化。结论本实验首次证明内源性大麻素花生四烯乙醇胺激活心肌细胞CB1受体后抑制细胞PKC的活性，从而抑制L型钙电流，此过程没有PLCβ-DAG途径参与。