中文摘要：

目的研究激活心肌内源性大麻素1型（CB1）受体是否通过改变蛋白激酶C（PKC）活性从而抑制L型钙电流,探讨激活CB1受体导致PKC活性变化的信号途径。方法利用酶解法制备大鼠心室肌细胞,分别单独应用CB1受体特异性激动剂2-氯乙胺花生四烯酸（ACEA）100 nmol·L-1、CB1受体阻断剂AM251 100 nmol·L-1或PKC非特异性激动剂十四烷酸乙酸大戟二萜醇酯（PMA）500 nmol·L-1孵育细胞10 min;此外提前应用AM251或PMA孵育细胞5 min后,再用ACEA孵育细胞10 min。应用全细胞膜片钳技术记录单个心肌细胞的L型钙电流;应用Pep Tag非放射性蛋白激酶C检测系统检测细胞PKC活性;ELISA试剂盒测定细胞中二酰甘油（DAG）的含量;Western蛋白印迹法测定磷脂酶Cβ（PLCβ）和磷酸化磷脂酶Cβ（p-PLCβ）表达。结果给予ACEA激动大鼠心肌细胞的CB1受体,可显著抑制L型钙电流,最大峰值电流密度由11.4±0.8降至（6.4±1.5）p A·p F-1;预先给予AM251或PMA可以完全阻断此抑制效应。检测心肌细胞PKC活性发现,与正常对照组（1.59±0.50）μmol·min-1·L-1相比,ACEA组心肌细胞PKC活性为（0.69±0.48）μmol·min-1·L-1,明显降低;同样预先给予AM251或PMA完全阻断ACEA对PKC活性的抑制效应。而给予ACEA没有影响心肌细胞DAG含量和PLCβ的磷酸化。结论激活心肌细胞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。

英文摘要：

OBJECTIVE To investigate whether activation of cardiomyocytes＇ endocannabinoid type 1receptor（ CB1 receptor） inhibits L-type calcium channel via protein kinase C（ PKC）,and the underlying mechanism. METHODS Rat ventricular myocytes were prepared by enzymatic hydrolysis before the cardiomyocytes were incubated by CB1 receptor agonist arachidonyl-2-chloroethylamide（ ACEA）100 nmol·L- 1,CB1 receptor antagonist AM251 100 nmol·L- 1or PKC nonspecific agonist phorbol myristate acetate（ PMA） 100 nmol·L- 1for 10 min,respectively. After application of AM251 or PMA for 5min,cardiomyocytes were again incubated by ACEA for 10 min. L-Type calcium current of single cardiomyocyte was recorded via whole cell patch clamp technique; Pep Tag non radioactive PKC detection system was used to determine PKC activity,diacylglycerol（ DAG） content was determined by ELISA kit,and the expression of phospholipase Cβ（ PLCβ） and phosphorylation phospholipase Cβ（ p-PLCβ） by Western blot technology. RESULTS ACEA significantly inhibited L-type calcium current. The maximum peak current density was decreased from 11. 4 ± 0. 8 to（ 6. 4 ± 1. 5） p A·p F- 1. However pretreatment of AM251 or PMA could completely block this effect. Similarly,ACEA significantly inhibited PKC activity.Compared to control group（ 1. 59 ± 0. 50） μmol·min- 1·L- 1,the PKC activity in ACEA group was dereased to（ 0. 69 ± 0. 48） μmol·min- 1·L- 1. But pretreatment of AM251 or PMA blocked the inhibitory effect of ACEA. ACEA had no effect on DAG content,PLCβ and p-PLCβ expression. CONCLUSION Our results,for the first time,demonstrate that activation of CB1 receptor inhibits L-type calcium channel via inhibition of PKC activity,but PLCβ-DAG signal pathway is not involved.