title: Glutamate release from platelets: exocytosis versus glutamate transporter reversal.
abstract: Platelets express neuronal and glial glutamate transporters EAAT 1-3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in the membrane of secretory granules. This study is focused on the assessment of non-exocytotic glutamate release, that is, the unstimulated release, heteroexchange and glutamate transporter reversal in platelets. Using the glutamate dehydrogenase assay, the absence of unstimulated release of endogenous glutamate from platelets was demonstrated, even after inhibition of glutamate transporters and cytoplasmic enzyme glutamine synthetase by dl-threo-β-benzyloxyaspartate and methionine sulfoximine, respectively. Depolarization of the plasma membrane by exposure to elevated [K(+)] did not induce the release of glutamate from platelets that was shown using the glutamate dehydrogenase assay and radiolabeled l-[(14)C]glutamate. Glutamate efflux by means of heteroexchange with transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate (dl-THA) was not observed. Furthermore, the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and inhibitor of V-type H(+)-ATPase bafilomycin A1 also failed to stimulate the release of glutamate from platelets. However, exocytotic release of glutamate from secretory granules in response to thrombin stimulation was not prevented by elevated [K(+)], dl-THA, FCCP and bafilomycin A1. In contrast to nerve terminals, platelets cannot release glutamate in a non-exocytotic manner. Heteroexchange, transporter-mediated and unstimulated release of glutamate are not inherent to platelets. Therefore, platelets may be used as a peripheral marker/model for the analysis of glutamate uptake by brain nerve terminals only (direct function of transporters), whereas the mechanisms of glutamate release are different in platelets and nerve terminals. Glutamate is released by platelets exclusively by means of exocytosis. Also, reverse function of vesicular glutamate transporters of platelets is rather ambiguous.
authors.affiliation: Department of Neurochemistry, Palladin Institute of Biochemistry, NAS of Ukraine, 9 Leontovicha Street, Kyiv 01601, Ukraine. Electronic address: ludmilka.kasatkina@gmail.com.
authors.forename: Ludmila A
authors.initials: LA
authors.lastname: Kasatkina
authors.forename: Tatiana A
authors.initials: TA
authors.lastname: Borisova
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D000818:Animals; D001792:Blood Platelets / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004072:Digitonin / Q000494:pharmacology; D057075:Enzyme Assays; D005089:Exocytosis / Q000187:drug effects; D005372:Filipin / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D011188:Potassium / Q000494:pharmacology; D011522:Protons; D011817:Rabbits; D051381:Rats; D012235:Rhodamines / Q000378:metabolism; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism; D012964:Sodium / Q000378:metabolism
chemical_list: D027322:Amino Acid Transport System X-AG; D005456:Fluorescent Dyes; D011522:Protons; D012235:Rhodamines; C026188:rhodamine 6G; D018698:Glutamic Acid; D005372:Filipin; D012964:Sodium; D005969:Glutamate Dehydrogenase; D005974:Glutamate-Ammonia Ligase; D004072:Digitonin; D011188:Potassium
keywords: 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid; AMPA; EAAT; GABA; GDH; Glutamate; Human/rabbit/rat blood platelets; N-methyl-d-aspartate; NMDA; Platelet secretory granules; Rat brain nerve terminals; Reversal of Na(+)-dependent glutamate transporters; SBFI-AM; VGLUT; dl-TBOA; dl-THA; dl-threo-β-benzyloxyaspartate; dl-threo-β-hydroxyaspartate; excitatory amino-acid transporter; gamma-aminobutyric acid; glutamate dehydrogenase; sodium-binding benzofuran isophthalate acetoxymethyl ester; vesicular glutamate transporter