Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

Abstract

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 M ␤␥-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 ␤␥-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.T he ability to control cell volume is an essential function for cell survival in the face of osmotic challenge. Perturbations in cell volume evoke wide-ranging signaling events leading to acute protective responses (e.g., rearrangement of the cytoskeleton) and longer-term adaptive responses (e.g., alterations in osmolyte transport and gene expression) (1). During acute swelling, cells can respond by a process of regulatory cell volume decrease (RVD). Under normal physiological conditions, mammalian cells are exposed to extracellular fluid osmolarity of approximately 285 mosmol, which is kept constant by normal body fluid homeostasis. Cell swelling often occurs as a consequence of changes to the intracellular composition of osmolytes, which results in intracellular hypotonicity and the influx of water. Compositional changes may occur during increased cellular transport or accumulation of nutrients or metabolic waste. Osmotically swollen mammalian cells release K ϩ , Cl Ϫ , and nonessential organic osmolytes in an effort to reverse the flow of water by osmosis. In contrast to mammalian cells, free-living single eukaryotic cells can be subjected to rapid and harsh changes in the osmolarity of the extracellular environment. As a consequence, the majority of single-celled organisms have evolved a specialized organelle called the contractile vacuole, a bladder-like structure that plays a major role in extruding water from the cytoplasm and expelling it into the ex...

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“…6A. Considering the finding that ATP is released by bone cells via vesicular exocytosis, 148 the experiments were performed in the presence of N-ethylmaleimide and brefeldin A, 149,150 inhibitors which did not change the results significantly. In the absence of A(5 0 )P5(5 0 )A, polyP caused a strong upregulation of the ATP pool; 76 Fig.…”

Polyphosphate as a donor of high-energy phosphate for the synthesis of ADP and ATP

“…It is established that ATP is released by bone cells via vesicular exocytosis (Orriss et al, 2013). This process can be inhibited by N-ethylmaleimide (NEM) (Akopova et al, 2012;Sivaramakrishnan and Fountain, 2015) and brefeldin A (Yewdell and Bennink, 1989;Brandao-Burch et al, 2012). In turn, we applied these two inhibitors and determined that, even in their presence, the ATP level in the medium increased.…”

ATP regulation in bioproduction

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