Martini Claudia N, Vaena de Avalos Silvia G, del Carmen Vila María
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, 1428, Buenos Aires, Argentina.
Mol Cell Biochem. 2004 Mar;258(1-2):191-9. doi: 10.1023/b:mcbi.0000012855.94291.dd.
We have previously reported that ACTH activates a phospholipase C that hydrolyzes glycosylphosphatidylinositol (GPI), which would release inositolphosphoglycan (IPG) to the extracellular medium, and that an IPG purified from Trypanosoma cruzi is able to inhibit ACTH-mediated steroid production in adrenocortical cells. In the present paper, it was found that anti-inositolphosphoglycan antibodies (anti-CRD) increased ACTH-mediated corticosterone production, which indicates that an endogenous IPG is a physiological inhibitor of ACTH response. On the other hand, we investigated the release to the extracellular medium of the GPI-anchored enzyme, alkaline phosphatase, by ACTH. We found that: (a) the released enzyme appeared in the aqueous phase after Triton X-114 partitioning, consistent with loss of the GPI, (b) the phospholipase C inhibitor, U73122, impaired the release of the enzyme by the hormone and (c) two inhibitors of IPG uptake, inositol 2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium. These results suggest that ACTH releases alkaline phosphatase by activation of a phospholipase C. Dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) was able to increase the release of alkaline phosphatase from adrenocortical cells and this effect was inhibited by U73122, suggesting that cAMP is involved in the activation of phospholipase C. In addition, it was found that a pertussis-toxin sensitive G-protein is required for ACTH- and db-cAMP-mediated release of alkaline phosphatase and that incorporation of anti-Gi antibodies in adrenocortical cells inhibited the release of alkaline phosphatase by ACTH. Our results suggest that ACTH increases the release of alkaline phosphatase by activation of a phospholipase C through cAMP and Gi which would contribute to produce IPG It was also found that the two inhibitors of IPG uptake, inositol-2-monophosphate and 2 M NaCl, increased the amount of alkaline phosphatase in the extracellular medium of ACTH-treated cells more than in control cells, indicating that ACTH also stimulates the uptake of IPG These data support a role of GPI and the involvement of Gi in ACTH action.
我们之前曾报道，促肾上腺皮质激素（ACTH）可激活一种磷脂酶C，该酶能水解糖基磷脂酰肌醇（GPI），从而将肌醇磷酸聚糖（IPG）释放到细胞外介质中，并且从克氏锥虫中纯化得到的一种IPG能够抑制肾上腺皮质细胞中ACTH介导的类固醇生成。在本文中，发现抗肌醇磷酸聚糖抗体（抗CRD）可增加ACTH介导的皮质酮生成，这表明内源性IPG是ACTH反应的生理抑制剂。另一方面，我们研究了ACTH促使GPI锚定酶碱性磷酸酶释放到细胞外介质的情况。我们发现：（a）经Triton X - 114分配后，释放的酶出现在水相中，这与GPI的丢失一致；（b）磷脂酶C抑制剂U73122可削弱激素对该酶的释放作用；（c）两种IPG摄取抑制剂，即肌醇2 - 单磷酸酯和2 M NaCl，可增加细胞外介质中碱性磷酸酶的量。这些结果表明，ACTH通过激活磷脂酶C来释放碱性磷酸酶。二丁酰腺苷 - 3',5'-环磷酸单酯（db - cAMP）能够增加肾上腺皮质细胞碱性磷酸酶的释放，且这种作用被U73122抑制，这表明cAMP参与了磷脂酶C的激活。此外，还发现ACTH和db - cAMP介导的碱性磷酸酶释放需要一种百日咳毒素敏感的G蛋白，并且在肾上腺皮质细胞中加入抗Gi抗体可抑制ACTH对碱性磷酸酶的释放。我们的结果表明，ACTH通过cAMP和Gi激活磷脂酶C来增加碱性磷酸酶的释放，这可能有助于产生IPG。还发现，两种IPG摄取抑制剂，即肌醇 - 2 - 单磷酸酯和2 M NaCl，在ACTH处理的细胞外介质中增加碱性磷酸酶的量比在对照细胞中更多，这表明ACTH也刺激IPG的摄取。这些数据支持了GPI的作用以及Gi参与ACTH作用。
