Metabolic organization in vascular smooth muscle: distribution and localization of caveolin-1 and phosphofructokinase.
Metabolic organization in vascular smooth muscle: distribution and localization of caveolin-1 and phosphofructokinase.
复制标题
血管平滑肌的代谢组织：caveolin-1 和磷酸果糖激酶的分布和定位。
DOI：
10.1152/ajpcell.00483.2002
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Hardin,ChristopherD
中科院分区：
文献类型：
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作者：
Vallejo,Johana;Hardin,ChristopherD
我们已经证明糖酵解和糖异生的区隔存在于血管平滑肌（VSM）中，并且完整的质膜对于区隔是必不可少的。先前，我们观察到小泡的破坏抑制糖酵解，但刺激糖异生，这表明小泡与糖酵解之间存在联系。我们假设糖酵解酶特异性地定位于小泡。我们用共聚焦显微镜检测了新分离的VSM细胞和培养的A7r5细胞中CAV-1和磷酸果糖激酶（PFK）的定位。新分离的细胞显示CAV-1的外周（膜）定位与PFK重叠85.3%。然而，只有59.9%的PFK定位于CAV-1，表明PFK比CAV-1分布更广。A7r5细胞表现出糖酵解和糖异生的区隔，并表现出两种明显的表型（纺锤形和卵形）。在两种表型中，CAV-1荧光与PFK荧光重叠（分别为83.1%和81.5%）。然而，在卵状细胞中，PFK与CAV-1的重叠率（35.9%）低于梭形细胞（53.7%）。在共定位模式上也有一个渐进的转变，从纺锤形细胞（包括新分离的和培养的细胞）的主要膜到卵形细胞的主要细胞质。总的来说，PFK与CAV-1的细胞共定位在所有细胞类型中都是显著的（0.68≥R2≤0.77）。PFK与CAV-1的共免疫沉淀进一步验证了蛋白之间可能的相互作用。我们得出结论，与CAV-1相似的PFK池分布有助于糖异生糖酵解的区室化。
We have shown that a compartmentation of glycolysis and gluconeogenesis exists in vascular smooth muscle (VSM) and that an intact plasma membrane is essential for compartmentation. Previously, we observed that disruption of the caveolae inhibited glycolysis but stimulated gluconeogenesis, suggesting a link between caveolae and glycolysis. We hypothesized that glycolytic enzymes specifically localize to caveolae. We used confocal microscopy to determine the localization of caveolin-1 (CAV-1) and phosphofructokinase (PFK) in freshly isolated VSM cells and cultured A7r5 cells. Freshly isolated cells exhibited a peripheral (membrane) localization of CAV-1 with 85.3% overlap with PFK. However, only 59.9% of PFK was localized with CAV-1, indicating a wider distribution of PFK than CAV-1. A7r5 cells exhibited compartmentation of glycolysis and gluconeogenesis and displayed two apparent phenotypes distinguishable by shape (spindle and ovoid shaped). In both phenotypes, CAV-1 fluorescence overlapped with PFK fluorescence (83.1 and 81.5%, respectively). However, the overlap of PFK with CAV-1 was lower in the ovoid-shaped (35.9%) than the spindle-shaped cells (53.7%). There was also a progressive shift in pattern of colocalization from primarily the membrane in spindle-shaped cells (both freshly isolated and cultured cells) to primarily the cytoplasm in ovoid-shaped cells. Overall, cellular colocalization of PFK with CAV-1 was significant in all cell types (0.68 ≥R2≤ 0.77). Coimmunoprecipitation of PFK with CAV-1 further validated the possible interaction between the proteins. We conclude that a similar distribution of one pool of PFK with CAV-1 contributes to the compartmentation of glycolysis from gluconeogenesis.