AIM:To investigate the
intracellular concentration of free calcium in the myocardial cells of guinea pig under the conditions
of normal oxygen and hypoxia,study the effects of intervention of puerarin injection, and compare the effects with those of nifedipine.METHODS:The in vitro guinea pig hearts were perfused with Langendorff method,myocardial cells were isolated with collagenase(Type I),and the number of cells was adjusted to 2× 109 L- 1.Under the extracellular
concentration of calcium was 2 mmol/L,the
intracellular content of calcium in myocardial cells was determined with F 2000 type spectrofluorometer,and intracellular concentration of free calcium ion was calculated.The myocardial cell suspension was divided into 6 groups:control group,puerarin injection group,nifedipine group under normal oxygen and hypoxia respectively.Under normal oxygen(partial pressure of oxygen=15.9 kPa) after observation for 100 s,10 μ L nutrient solution, 10 μ L puerarin injection(terminal concentration:0.1 mmol/L) and 10 μ L nifedipine(terminal concentration:20 mmol/L) were given to 3 groups with random selection and observed for 100,200,300,400 and 500 s.The other 3 groups were treated with the above 3 drugs of the same concentrations respectively under hypoxia(nitrogen gas:0.1 cm3/s),and were observed at the above 5 time points.The changes of the intracellular concentration of free calcium ion in myocardial cells in each group under different conditions were dynamically measured with spectrofluorometer.RESULTS:① Under normal oxygen(partial pressure of oxygen=15.9 kPa) at rest(0 to 100 s) and the extracellular concentration of free calcium ion was controlled at 2 mmol/L(physiological condition),there were no significant differences in the intracellular concentration of free calcium ion in myocardial cells among the 6 groups(P >0.05),it was known that the concordance among the 6 groups was good, and they were comparable.② The intracellular concentration of free calcium ion in myocardial cells 100,200,300,400 and 500 s after administration were obviously lower in the puerarin injection group and nifedipine group(P< 0.05 to 0.01),but it was decreased more slowly in the puerarin injection group than in the nifedipine group(P< 0.05 to 0.01).③ The intracellular concentration of free calcium ion in myocardial cells:Under hypoxia for 100,200,300,400 and 500 s,it was increased more slowly in the puerarin injection group than in the control group<(163.7± 19.7),(224.2± 20.7),(309.7± 15.1),(424.7± 14.0),(477.5± 21.5)nmol/L;(208.8± 28.9),(328.5± 14.4), (401.8 ± 21.2),(534.3± 20.8),(751.4± 36.0) nmol/L,P< 0.01>;The increases 100,200,300,400 and 500 s after hypoxia were fewer in the nifedipine group than in the control group(P< 0.01),and the decreasing speed was higher in the nifedipine group than in the puerarin injection group
.CONCLUSION:Under normal oxygen,puerarin injection can obviously decrease the intracellular concentration of free calcium ion in myocardial cells;Under hypoxia,puerarin injection can delay the increased concentration caused by hypoxia,so as to prevent the calcium overload in myocardial cells,and plays a role in protecting myocardial cells.Puerarin injection has the similar effect as nifedipine,which is a classic blocking agent of calcium passage;Compared with nifedipine,puerarin injection has the characteristics that it can inhibit relatively slowly the increase of the intracellular concentration of free calcium ion in myocardial cells after hypoxia.