Effects Of Different Local Anesthetics On The Intracellular Ca～(2+) In Rat Ventricular Myocytes

Objective To compare the effects of racemic bupivacaine, levobupivacaine and ropivacaine at different concentrations on intracellular calcium concentration ([Ca2+]j) in rat ventricular myocytes induced by KCI using laser scanning confocal microscope (LSCM), and to investigate the mechanism of cardiotoxicity of local anesthetics. Methods 1. The hearts of newborn SD rats were cut into small pieces of 1mm3, digested with 0.08% collagenase and made into cell suspension by 10 % fetal bovine serum (FBS). The cell suspension was reached for about 90% after repeated purification, adjusted into 1 105/m1 and cultivated at 37 C in 5% C02 incubator. 2. The cultured myocytes were dyed by single-cloned a -actin antibody in order to identify if they are cardiomyocytes. 3. The cultured ventricular myocytes of newborn rats were divided randomly into 10 groups: the control group(group C), the group of racemic bupivacaine at 10 M (group B1), the group of racemic bupivacaine at 50 M (group B2), the group of racemic bupivacaine at 100 M (group B3), the group of levobupivacaine at 10 M (group L1), the group of levobupivacaine at 50 M (group L2), the group of levobupivacaine at 100 M (group L3), the group of ropivacaine at 10 M (group R1), the group of ropivacaine at 50 u M (group R2) and the group of ropivacaine at 100 M (group R3). 4. The cultured cardiomyocytes were rinsed twice with DMEM and loaded in Fluo 3/AM 20 M working solution containing 2% Pluronic F-127 at 37 C in 5% C02 incubator for 30 min, and washed again with DMEM to remove the extracellular Fluo 3/AM. 5. The fluorescent intensity (Fl) of intracellular Ca2+ in single culturedcardiomyocytes of newborn rats loaded with Fluo 3 was observed by LSCM in order to compare the effects of racemic bupivacaine, levobupivacaine and ropivacaine in different concentration on [Ca2+]i induced by KCI.Result 1. The cultured ventricular myocytes of newborn rats were growing with irregular starlike shape, adhereing to the wall of the culture dish and beating independently and rhythmically. They were proved to be myocytes by immunohistochemistry of single-cloned a -actin antibody showing brown in cytoplasm. 2.There were no differences in the changes of intracellular Ca2+ fluorescent intensity in rat ventricular myocytes induced by KCI in group B1.L1 and R1 as compared with group C (P>0.05) . Intracellular Ca2+ fluorescent intensity in rat ventricular myocytes induced by KCI was inhibited significantly in group B2 and B3, group L2 and L3, group R2 and R3 as compared with group C(P<0.05).3. Compared the same drug in different concentrations , the sequence of the degree of the inhibition of Intracellular Ca2+ fluorescent intensity in rat ventricular myocytes induced by KCI was: group B3>group B2>group B1, group L3>group L2>group L1, group R3 >group R2 >group R1 (P<0.05) . 4. Compared different drugs in the same concentration, the degree of the inhibition of Intracellular Ca2* fluorescent intensity in rat ventricular myocytes induced by KCI was: group B2 higher than that of group L2 and group R2 (P<0.05), no difference between group L2 and group R2 (P>.05); group B3 higher than that of group L3 and group R3(P<0.05), no difference between group L3 and group R3 (P>.05) . Conclusions 1 There were no significant effects of racemic bupivacaine, levobupivacaine and ropivacaine at low concentration on intracellular Ca2+ in rat ventricular myocytes induced by KCI. 2. Racemic bupivacaine, levobupivacaine and ropivacaine at high concentraton could inhibit significantly the effects on [Ca2+]j in rat ventricular myocytes induced byKCL. 3.The higher the concentration of racemic bupivacaine, levobupivacaine and ropivacaine, the more significant of inhibition of Intracellular Ca2+ fluorescent intensity in rat ventricular myocytes induced by KCI. 4. Racemic bupivacaine could inhibit the effect on [Ca2+]i in rat ventricular myocytes induced by KCI more significantly than levobupivacaine and ropivacaine did at the same concentration, and there was no significant difference between the latter two...