title: 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 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.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
authors.forename: Venketesh
authors.initials: V
authors.lastname: Sivaramakrishnan
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom s.j.fountain@uea.ac.uk.
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Fountain
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D004023:Dictyostelium / Q000187:drug effects / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D005682:Gadolinium / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects / Q000502:physiology*
chemical_list: D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D005682:Gadolinium