Frost S C, Lane M D, Gibbs E M
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
J Cell Physiol. 1989 Dec;141(3):467-74. doi: 10.1002/jcp.1041410304.
We have shown previously that insulin stimulates fluid phase endocytosis in 3T3-L1 adipocytes (Gibbs et al., 1986). Using [14C]sucrose as an endocytotic marker, we show here that phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols, blocked not only insulin-stimulated fluid phase endocytosis, but basal endocytosis as well. The Ki for this process was 6 microM in the presence or absence of insulin and the time required for inhibition was less than 2.5 min, the limit of detection in our assay system. These results can be compared with the inhibitory effect of phenylarsine oxide on insulin-stimulated glucose transport. Although the Ki for insulin-stimulated transport (7 microM) was similar to that for inhibition of endocytosis, basal glucose transport was not affected by the inhibitor. Further, when cells were prestimulated with insulin causing maximal stimulation of the glucose transport rate, phenylarsine oxide induced a time-dependent reduction to the basal rate (t 1/2 of 10 min), despite the fact that endocytosis was blocked immediately. This observation suggests that if the transporter is recycled by an exocytotic/endocytotic mechanism, it is distinct from fluid-phase endocytosis/exocytosis, which is a vesicle-mediated process, and provides further evidence that the transporter may undergo intrinsic activation/inactivation which does not require vesicle movement.
我们之前已经表明，胰岛素可刺激3T3-L1脂肪细胞中的液相内吞作用（吉布斯等人，1986年）。使用[14C]蔗糖作为内吞标记物，我们在此表明，苯胂化氧，一种能结合相邻二硫醇的三价砷化合物，不仅阻断了胰岛素刺激的液相内吞作用，也阻断了基础内吞作用。在有或没有胰岛素存在的情况下，该过程的抑制常数（Ki）为6微摩尔，抑制所需时间小于2.5分钟，这是我们检测系统的检测极限。这些结果可与苯胂化氧对胰岛素刺激的葡萄糖转运的抑制作用相比较。虽然胰岛素刺激转运的抑制常数（7微摩尔）与抑制内吞作用的抑制常数相似，但基础葡萄糖转运不受该抑制剂影响。此外，当细胞用胰岛素预刺激导致葡萄糖转运速率达到最大刺激时，尽管内吞作用立即被阻断，但苯胂化氧仍会导致葡萄糖转运速率随时间依赖性降低至基础速率（半衰期为10分钟）。这一观察结果表明，如果转运体通过胞吐/内吞机制进行再循环，那么它与液相内吞/胞吐作用不同，后者是一个囊泡介导的过程，并进一步证明转运体可能经历不需要囊泡移动的内在激活/失活。
