三羟异黄酮对豚鼠心室肌细胞内游离钙浓度的影响.pdf

Acta Physiologica Sinica, April 25, 2004, 56(2): 204-209204Received 2003-06-19 Accepted 2003-07-30*Corresponding author. Tel: +86-311-6062490; Fax: 86-311-6062490; E-mail: syho@Research PaperEffects of genistein on intracellular free-calcium concentration in guinea pig ventricular myocytesJI En-Sheng1, WANG Chuan2, HE Rui-Rong 1,*Departments of 1Physiology and 2Pharmacology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, ChinaAbstract: The effects of genistein (GST) on intracellular calcium concentration ([Ca2+]i ) were investigated in guinea pig ventricularmyocytes. [Ca2+]i was detected by confocal microscopy and represented by relative fluorescent intensity (FI-F0) /FI0, %). The results showed that GST (10~40 µmol/L) reduced [Ca2+]i in normal Tyrode’s solution, Ca2+-free Tyrode’s solution and normal Tyrode’s solutioncontaining 3 mmol/L EGTA in a concentration-dependent manner. The effects of GST on [Ca2+]i in normal Tyrode’s solution were partially inhibited by pretreatment with sodium orthovanadate, a potent inhibitor of protein tyrosine phosphatase, or L-type Ca2+channel agonist Bay K8644. GST also markedly inhibited the ryanodine-induced [Ca2+]i responses in Ca2+-free Tyrode’s solution. When Ca2+ waves were produced by increasing extracellular Ca2+ concentration from 1 to 10 mmol/L, GST (40 µmol/L) could block thepropagating waves of elevated [Ca2+]i, and reduce the velocity and duration of propagating waves. These results suggest that GST may reduce the [Ca2+]i in isolated guinea pig ventricular myocytes. The inhibition of voltage-dependent Ca2+ channel , tyrosine kinase inhibition,and alleviation of Ca2+ release from SR are possibly involved in the GST effect.Key words: genistein; fluorescence intensity; myocytes; intracellular calcium; Ca2+ channel; intracellular Ca2+ release; confocal microscopy121,*12, 050017: (genistein, GST) ([Ca2+]i )(FI-F0/FI0, %) , 3 mmol/L EGTA, GST (10~40 µmol/L) (sodium orthovanadate) L-Ca2+Bay K8644GST1 mmol/L10 mmol/L , , GST (40 µmol/L) , , GST, Ca2+: ; ; ; ; ; ; : Q463Phytoestrogens are plant-derived dipheonolic com- pounds , which are structurally and functionally similar to estradiol. A growing number of reports have docu- mented that phytoestrogens may exert protective ac- tions on the cardiovascular system[1~3]. Genistein (GST), one of the well-known phytoestrogens, is an isoflavonethat is also proved to be a specific inhibitor of protein tyrosine kinase (PTK)[4]. Our previous work demonstrated that GST reduced the amplitude of action potential (APD), maximal rate of depolarization (Vmax), over shoot (OS), velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF) in sinoatrial node pace-205JI En-Sheng et al: Effects of Genistein on Intracellular Calciummaker cells of rabbits and human artrial fibers[5,6]. Moreover, GST inhibited early afterdepolarization (EDA) and triggered activity (TA) induced by ouabain in guinea pig papillary muscles[7]. These cardioprotective actions may be produced by reducing calcium influx. Our recent study indicated that GST had protective effects against myocardial ischemia- reperfusion injury in rabbits[8]. Thus, GST possesses po- tential clinical value in preventing and treating myocardial ischemia-reperfusion injury. Its cardioprotective effects may be related to the reduction of free intracellular cal- cium concentration ([Ca2+]i ). The present study was un- dertaken to observe the effects of GST on [Ca2+]i level and define its mechanism in guinea pig ventricular myocytes using confocal microscopy.1 MATERIALS AND METHODS1.1 Isolation of guinea-pig ventricular myocytes. Guinea- pig ventricular myocytes were obtained by modified en- zymatic dissociation technique[9]. Male guinea pigs weigh- ing 280~340 g, provided by Experimental Animal Center of Hebei Province (grade , Certificate No. 04064), were stunned by heavy blow on the head. The heart, with the aorta of 2~3 mm length, was then removed and placed in oxygenated ice-cold Ca2+-free Tyrode’s solu- tion . A Langendorff retrograde perfusion was performed through the aorta at a rate of 9 ml/min with Ca2+-free Tyrode’s solution for 5 min, and then with the same solu- tion containing 34 µmol/L CaCl2 and 360 mg/L collagenase (Sigma, type) for 9 min at 37ºC. The ventricles were incubated in Ca2+-free Tyrode’s solution at room tempera- ture for about 30 min. Afterwards, a piece of ventricle was cut out and teased into smaller pieces in KB solution. Myocytes were harvested after filtration through a 200 µm nylon mesh and stored in KB solution for at least 1 h before the experiment. The concentration of Ca2+ in Tyrode’s solution was gradually increased to 1.0 mmol/L. All experiments were performed within 12 h after isolation.1.2 Fluo3-AM loading. Isolated ventricular myocytes were incubated with Fluo3-AM working solution contain- ing 0.03% Pluronic F-127 (the final concentration of Fl。