title: Ketamine Inhibits ATP-Evoked Exocytotic Release of Brain-Derived Neurotrophic Factor from Vesicles in Cultured Rat Astrocytes.
abstract: In the brain, astrocytes signal to neighboring cells via regulated exocytotic release of gliosignaling molecules, such as brain-derived neurotrophic factor (BDNF). Recent studies uncovered a role of ketamine, an anesthetic and antidepressant, in the regulation of BDNF expression and in the disruption of astrocytic Ca2+signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca2+signaling and exocytotic release of BDNF at the single-vesicle level in cultured rat astrocytes. Cells were transfected with a plasmid encoding preproBDNF tagged with the pH-sensitive fluorescent protein superecliptic pHluorin, (BDNF-pHse) to load vesicles and measure the release of BDNF-pHse when the exocytotic fusion pore opens and alkalinizes the luminal pH. In addition, cell-attached membrane capacitance changes were recorded to monitor unitary vesicle interaction with the plasma membrane. Intracellular Ca2+activity was monitored with Fluo-4 and confocal microscopy, which was also used to immunocytochemically characterize BDNF-pHse-laden vesicles. As revealed by double-fluorescent micrographs, BDNF-pHse localized to vesicles positive for the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins, vesicle-associated membrane protein 2 (VAMP2), VAMP3, and synaptotagmin IV. Ketamine treatment decreased the number of ATP-evoked BDNF-pHse fusion/secretion events (P < 0.05), the frequency of ATP-evoked transient (P < 0.001) and full-fusion exocytotic (P < 0.05) events, along with a reduction in the ATP-evoked increase in intracellular Ca2+activity in astrocytes by ~70 % (P < 0.001). The results show that ketamine treatment suppresses ATP-triggered vesicle fusion and BDNF secretion by increasing the probability of a narrow fusion pore open state and/or by reducing astrocytic Ca2+excitability.
authors.affiliation: Celica Biomedical, Tehnološki park 24, 1000, Ljubljana, Slovenia.|Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000, Ljubljana, Slovenia.
authors.forename: Matjaž
authors.initials: M
authors.lastname: Stenovec
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000, Ljubljana, Slovenia.
authors.forename: Eva
authors.initials: E
authors.lastname: Lasič
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000, Ljubljana, Slovenia.
authors.forename: Mićo
authors.initials: M
authors.lastname: Božić
authors.affiliation: Celica Biomedical, Tehnološki park 24, 1000, Ljubljana, Slovenia.|Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000, Ljubljana, Slovenia.
authors.forename: Saša Trkov
authors.initials: ST
authors.lastname: Bobnar
authors.affiliation: Department of Neurobiology, Civitan International Research Center and Center for Glial Biology in Medicine, Evelyn F. McKnight Brain Institute, Atomic Force Microscopy and Nanotechnology Laboratories, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 429, Birmingham, AL, 35294, USA.|The Dominick P. Purpura Department of Neuroscience, Rose F. Kennedy Center, Albert Einstein College of Medicine, Bronx, NY, 10461, USA.
authors.forename: Randy F
authors.initials: RF
authors.lastname: Stout
authors.affiliation: Department of Neurobiology, Civitan International Research Center and Center for Glial Biology in Medicine, Evelyn F. McKnight Brain Institute, Atomic Force Microscopy and Nanotechnology Laboratories, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 429, Birmingham, AL, 35294, USA.|Department of Physiology, Neuroscience Program, Michigan State University, East Lansing, MI, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Grubišić
authors.affiliation: Department of Neurobiology, Civitan International Research Center and Center for Glial Biology in Medicine, Evelyn F. McKnight Brain Institute, Atomic Force Microscopy and Nanotechnology Laboratories, University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 429, Birmingham, AL, 35294, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Parpura
authors.affiliation: Celica Biomedical, Tehnološki park 24, 1000, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si.|Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška 4, 1000, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si.
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D007649:Ketamine / Q000494:pharmacology*; D008561:Membrane Fusion / Q000187:drug effects; D011687:Purines / Q000378:metabolism; D017208:Rats, Wistar; D050600:SNARE Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D015415:Biomarkers; D019208:Brain-Derived Neurotrophic Factor; D011687:Purines; D050600:SNARE Proteins; D007649:Ketamine; D000255:Adenosine Triphosphate; D002118:Calcium
keywords: Astroglia; Brain-derived neurotrophic factor; Ca2+ signaling; Fusion pore; Ketamine; Regulated exocytosis