GDP在体外对大鼠脑线粒体脱耦联蛋白活性和表达的影响

本研究通过GDP体外处理大鼠脑组织块,观察GDP对脑线粒体脱耦联蛋白(uncoupling proteins,UCPs)活性、UCP4和UCP5表达的影响,以探讨嘌呤核苷酸对大鼠脑UCPs的调节作用.取Sprague-Dawley大鼠双侧大脑半球,将脑组织切成约8-10 mm3的脑组织块,与含1 mmol/L GDP的孵育介质共孵育30 min后,匀浆并差速离心分离提取大鼠脑组织线粒体,采用3H]-GTP结合法测定UCPs活性,并以Scatehard作图法计算两者结合的解离常数(Kd)和最大结合量(Bmax);RT-PCR和Western blot分别检测UCP4和UCP5的mRNA和蛋白表达.结果显示,1 mmol/L GDP可降低体外大鼠脑组织线粒体中UCPs与3H]-GTP结合的Bmax,提高Kd,但对脑纰织中UCP4和UCP5 mRNA和蛋白表达量的改变无统计学意义.上述结果提示,GDP可直接抑制体外大鼠脑组织中UCPs的活性,但并不影响UCP4和UCP5的表达.

Effects of GDP on the activity and expression of mitochondrial uncoupling proteins in rat brain in vitro

To investigate the regulatory role of purine nucleotide on uncoupling proteins (UCPs), the activity of UCPs and the expressions of UCP4 and UCP5 in mitochondria of rat brain tissues incubated with GDP were observed in vitro. The cerebral hemispheres of adult male Sprague-Dawley rats were removed and clipped into 8-10 mm(3) tissue mass which incubated with 1 mmol/L GDP (GDP group), or only incubation medium (control group), for 30 min in vitro. The mitochondria from incubated tissue mass of rat brain were isolated by centrifugation. The activity of UCPs was detected by the method of (3)H]-GTP binding with UCPs specifically. The maximal binding content (B(max)) and the dissociation constant (K(d)) were determined from Scatchard plot. The mRNA and protein expressions of UCP4 and UCP5 were measured by RT-PCR and Western blot, respectively. The results showed that B(max) was increased and K(d) was decreased in rat brain mitochondria in GDP group compared with that in control group. But the mRNA and protein expressions of UCP4 and UCP5 exhibited no statistically significant changes. It is thus suggested that GDP inhibits the activity of mitochondrial UCPs in rat brain in vitro, but exhibits no effect on the expressions of UCP4 and UCP5.