Sivaramakrishnan Venketesh, Fountain Samuel J
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom
Eukaryot Cell. 2015 Aug;14(8):775-82. doi: 10.1128/EC.00066-15. Epub 2015 Jun 5.
ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd(3+) caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd(3+). Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd(3+), while NO donors rescued apyrase- and Gd(3+)-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd(3+)-sensitive receptor that is coupled with intracellular NO production.
ATP在生物学中无处不在，在哺乳动物中作为一种细胞外信号分子发挥作用。目前缺乏关于单细胞真核生物中细胞外ATP信号功能的信息。在这里，我们探讨了细胞外ATP在变形虫盘基网柄菌渗透肿胀过程中细胞体积恢复中的作用。在低渗刺激的细胞肿胀和调节性细胞体积减小（RVD）阶段，可以检测到微摩尔浓度的ATP释放。用腺苷三磷酸双磷酸酶清除ATP会导致细胞严重肿胀和RVD丧失。100μMβγ-亚氨基ATP可以挽救腺苷三磷酸双磷酸酶诱导的肿胀。囊泡胞吐作用的抑制剂N-乙基马来酰亚胺（NEM）会导致细胞肿胀加剧、RVD丧失以及ATP释放受到抑制。收缩泡（CV）融合受损的变形虫（排水蛋白基因敲除[KO]细胞）表现出肿胀增加，但ATP释放完整。100μM的Gd(3+)会导致细胞肿胀，同时阻止βγ-亚氨基ATP的任何恢复作用。在Gd(3+)存在的情况下，ATP释放增加了4倍。细胞肿胀与细胞内一氧化氮（NO）的增加有关，NO清除剂会导致细胞肿胀。肿胀诱导的NO产生受到腺苷三磷酸双磷酸酶和Gd(3+)的抑制，而NO供体可以挽救腺苷三磷酸双磷酸酶和Gd(3+)诱导的肿胀。这些数据表明，细胞肿胀期间释放的细胞外ATP是引发RVD的重要信号。尽管盘基网柄菌中ATP的细胞表面受体仍然难以捉摸，但我们认为ATP通过一种对Gd(3+)敏感的受体发挥作用，该受体与细胞内NO的产生相关联。
