Quinlan P T, Thomas A P, Armston A E, Halestrap A P
Biochem J. 1983 Aug 15;214(2):395-404. doi: 10.1042/bj2140395.
Methods have been developed to measure the lysophospholipid content and matrix volume of liver cell mitochondria in situ in order to test the hypothesis that these parameters may be important in the hormonal control of mitochondrial function [Armston, Halestrap & Scott (1982) Biochim. Biophys. Acta 681, 429-439]. No change in the labelling of mitochondrial lysophospholipids with [32P]Pi was detected after treatment of liver cells with glucagon, phenylephrine or vasopressin. Incorporation of [32P]Pi into mitochondrial phosphatidylinositol was enhanced by phenylephrine and vasopressin. Mitochondrial volumes were measured using rapid disruption of cells by sonication into 3H2O and [14C]sucrose or without cell disruption using 3H2O and [14C]mannitol. In control cells the two methods gave values of 1.09 and 0.40 microliters/mg of mitochondrial protein respectively, which represent 19 and 7% respectively of the total cell volume measured with 3H2O and inulin [14C]carboxylic acid. Both methods showed that glucagon, phenylephrine and 1 nm-valinomycin produced significant increases (13% and 26% using sucrose and mannitol respectively) in mitochondrial volume. The increase was coincident with the stimulation of gluconeogenesis from L-lactate and pyruvate and of mitochondrial respiratory chain activity. The effects of glucagon and phenylephrine were additive on both mitochondrial volume and respiratory chain activity, but not on gluconeogenesis. Liver cells exposed to gluconeogenic hormones or low concentrations of valinomycin showed a decrease in light scattering at 520 nM correlating with the change in mitochondrial volume but without a change in whole-cell volume. The time course and hormone sensitivity of this response were similar to those for the hormonal stimulation of gluconeogenesis. The light-scattering response to glucagon, phenylephrine and vasopressin, but not to valinomycin, were greatly reduced or abolished in Ca2+-free media.
为了验证这些参数可能在激素对线粒体功能的调控中起重要作用这一假说，已开发出原位测量肝细胞线粒体溶血磷脂含量和基质体积的方法[阿姆斯特朗、哈利斯特雷普和斯科特（1982年）《生物化学与生物物理学报》681卷，429 - 439页]。用胰高血糖素、去氧肾上腺素或血管加压素处理肝细胞后，未检测到线粒体溶血磷脂与[³²P]Pi的标记有变化。去氧肾上腺素和血管加压素增强了[³²P]Pi掺入线粒体磷脂酰肌醇的过程。线粒体体积通过将细胞超声快速裂解到³H₂O和[¹⁴C]蔗糖中来测量，或者不进行细胞裂解，使用³H₂O和[¹⁴C]甘露醇来测量。在对照细胞中，这两种方法分别给出的值为1.09和0.40微升/毫克线粒体蛋白，分别占用³H₂O和菊粉[¹⁴C]羧酸测量的总细胞体积的19%和7%。两种方法均表明，胰高血糖素、去氧肾上腺素和1纳米缬氨霉素使线粒体体积显著增加（使用蔗糖和甘露醇分别增加13%和26%）。这种增加与L - 乳酸和丙酮酸的糖异生以及线粒体呼吸链活性的刺激同时发生。胰高血糖素和去氧肾上腺素对线粒体体积和呼吸链活性的影响是相加的，但对糖异生不是。暴露于糖异生激素或低浓度缬氨霉素的肝细胞在520纳米处的光散射减少，这与线粒体体积的变化相关，但全细胞体积没有变化。这种反应的时间进程和激素敏感性与激素对糖异生的刺激相似。在无钙培养基中，对胰高血糖素、去氧肾上腺素和血管加压素的光散射反应（但对缬氨霉素无反应）大大降低或消除。
