氯胺酮对大鼠心肌细胞内游离钙的影响

Effects of ketamine on free intracellular calcium in rat cardiomyocytes

目的 探讨氯胺酮对大鼠心肌细胞内游离钙的影响及其作用机制。方法 原代培养的SD大鼠心室肌细胞 ,用钙敏荧光探针Fluo 3/AM负载染色后 ,根据实验中加入氯胺酮终浓度不同分为六组 ,采用激光扫描共聚焦显微镜 ,测定心肌细胞内钙荧光强度的基础值及加入氯胺酮后 5min以及后续加入 4 0mM氯化钾 (KCl)或 1 0mM咖啡因后细胞内钙荧光强度值。结果 (1 )≤ 1 0 0 μM的氯胺酮对静息心肌细胞内钙荧光强度无明显影响 ,而 30 0 μM的氯胺酮降低钙荧光强度 (P <0 0 1 ) ;(2 )KCl和咖啡因使细胞内钙荧光强度较基础值均明显升高 (P <0 0 1 ) ;氯胺酮剂量依赖性地抑制KCl诱发胞内钙荧光强度升高的幅度 ,尤以 1 0 0和 30 0 μM氯胺酮组明显 ;而各浓度的氯胺酮不抑制咖啡因诱发胞内钙荧光强度的升高 (P >0 0 5 )。结论 1、1 0和 6 0 μM的氯胺酮可能不抑制整体状态下的心肌收缩 ;而 1 0 0和 30 0 μM的氯胺酮对心肌有剂量依赖性的负性肌力效应 ,其机制可能是氯胺酮抑制了胞外Ca2 + 经膜电压依赖性钙通道内流 ,胞内游离钙浓度降低所致 ,但不影响肌浆网释钙功能。

Objective To study the effects and mechanism of different doses of ketamine on free intracellular calcium concentration([Ca 2+ ]i)in rat cardiomyocytes.Methods Primary cultured ventricular myocytes obtained from neonatal SD rats,loaded with Fluo 3/AM 20μmol/L,were randomly divided into six groups.Equal volume of Hanks solution was given as control.Different doses of ketamine (1?10?60?100 and 300μM)were administered in the other five groups.KCl or caffeine was added subsequently as the stimulus ( n= 6).[Ca 2+ ] i was measured with fluorescent intensity(FI)by laser scanning confocal microscope in cardiomyocytes.After basal FI values of [Ca 2+ ] i were measured,ketamine was added in different doses.FI values were measured five minutes later.Then KCl 40mM or caffeine 10mM was applied to the myocytes and FI values were measured oneminute later. Results (1)There were no significant influences of ketamine at 1,10,60 and 100μM on FI values of [Ca 2+ ] i in resting cardiomyocytes,while ketamine 300μM decreased FI values significantly ( P< 0 01);(2)FI values elevated obviously when cardiomyocytes mobilized by KCl or caffeine in all groups( P< 0 01);(3)compared with control group,no significant inhibition was observed on KCl induced FI elevation in 1,10 and 60 of ketamine groups.However,ketamine 100 and 300μM decreased KCl induced FI elevation by 7%( P< 0 05)and 16%( P< 0 01);(4)compared with control group,there was no significant difference in caffeine induced FI increase in all ketamine groups( P> 0 05). Conclusions (1)Ketamine at 1μM,10μM and 60μM does not depress myocardial contractility;(2)ketamine 100μM and 300μM has dose dependent negative inotropic effect on the myocardium.One of the mechanisms seems to be that ketamine reduces Ca 2+ influx via the L type voltage dependent Ca 2+ channels,but without reducing Ca 2+ release from the sarcoplasmic reticulum. [