Svoboda P, Unelius L, Dicker A, Cannon B, Milligan G, Nedergaard J
The Wenner-Gren Institute, University of Stockholm, Sweden.
Biochem J. 1996 Mar 15;314 ( Pt 3)(Pt 3):761-8. doi: 10.1042/bj3140761.
The significance of Gi proteins for the physiological desensitization phenomena observed in brown-fat cells from cold-acclimated hamsters was investigated. For this purpose, pertussis toxin (the inhibitor of Gi function) was injected into control and cold-acclimated hamsters. After 3 days the thermogenic response to noradrenaline injection was monitored in the intact animals. It was found that the pertussis-toxin pretreatment did not affect the thermogenic response to noradrenaline. Nonetheless, the pertussis toxin pretreatment had a dramatic effect on the noradrenaline-sensitivity of isolated brown-fat cells (measured the following day as the respiratory response): a 250-fold-increased sensitivity to noradrenaline was observed in cells from control animals that had been pertussis-toxin pretreated. However, only a 20-fold increase was observed in cells from cold-acclimated hamsters, implying a lower complement of the Gi system in these cells. Therefore the content of Gi proteins was determined by quantitative immunoblotting of purified plasma-membrane proteins. Cold acclimation resulted in a nearly 50% reduction in the content of Gi 1 alpha and Gi 2 alpha, as well as of the beta-subunit, both when expressed on a protein basis and when related to the content of forskolin-stimulated adenylyl cyclase; when expressed per unit of [3H]ouabain-binding (NA+/K+-ATPase), the reduction was even higher. In view of the magnitude of the pertussis-toxin effect, it was concluded that Gi proteins must play a substantial role in the regulation of the response of brown-fat cells to noradrenaline. As the capacity of the Gi pathway is reduced rather than augmented during cold acclimation, Gi activity cannot be responsible for the desensitization to noradrenaline observed in cells from cold-acclimated animals. However, the reduced Gi content may explain the earlier observed desensitization to adenosine that occurs after acclimation to cold.
研究了Gi蛋白对冷适应仓鼠棕色脂肪细胞中观察到的生理脱敏现象的意义。为此，将百日咳毒素（Gi功能抑制剂）注射到对照和冷适应仓鼠体内。3天后，在完整动物中监测对去甲肾上腺素注射的产热反应。发现百日咳毒素预处理不影响对去甲肾上腺素的产热反应。然而，百日咳毒素预处理对分离的棕色脂肪细胞的去甲肾上腺素敏感性有显著影响（第二天测量为呼吸反应）：在经过百日咳毒素预处理的对照动物的细胞中，观察到对去甲肾上腺素的敏感性增加了250倍。然而，在冷适应仓鼠的细胞中仅观察到20倍的增加，这意味着这些细胞中Gi系统的含量较低。因此，通过对纯化的质膜蛋白进行定量免疫印迹来测定Gi蛋白的含量。冷适应导致Gi 1α和Gi 2α以及β亚基的含量几乎降低50%，无论是以蛋白质为基础表达还是与福斯可林刺激的腺苷酸环化酶含量相关时；当以每单位[3H]哇巴因结合（Na+/K+-ATP酶）表示时，降低甚至更高。鉴于百日咳毒素效应的程度，得出结论：Gi蛋白在调节棕色脂肪细胞对去甲肾上腺素的反应中必须发挥重要作用。由于在冷适应过程中Gi途径的能力降低而不是增强，Gi活性不能解释在冷适应动物细胞中观察到的对去甲肾上腺素的脱敏。然而，Gi含量的降低可能解释了早期观察到的冷适应后对腺苷的脱敏。
