PMID- 26660497 OWN - NLM STAT- MEDLINE DCOM- 20171222 LR - 20181113 IS - 1559-1182 (Electronic) IS - 0893-7648 (Linking) VI - 53 IP - 10 DP - 2016 Dec TI - Ketamine Inhibits ATP-Evoked Exocytotic Release of Brain-Derived Neurotrophic Factor from Vesicles in Cultured Rat Astrocytes. PG - 6882-6896 AB - 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 Ca(2+) signaling, but it is unclear whether it affects astroglial BDNF release. We investigated whether ketamine affects ATP-evoked Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) excitability. FAU - Stenovec, Matjaz AU - Stenovec M AD - Celica Biomedical, Tehnoloski park 24, 1000, Ljubljana, Slovenia. AD - Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia. FAU - Lasic, Eva AU - Lasic E AD - Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia. FAU - Bozic, Mico AU - Bozic M AD - Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia. FAU - Bobnar, Sasa Trkov AU - Bobnar ST AD - Celica Biomedical, Tehnoloski park 24, 1000, Ljubljana, Slovenia. AD - Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia. FAU - Stout, Randy F Jr AU - Stout RF Jr AD - 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. AD - The Dominick P. Purpura Department of Neuroscience, Rose F. Kennedy Center, Albert Einstein College of Medicine, Bronx, NY, 10461, USA. FAU - Grubisic, Vladimir AU - Grubisic V AD - 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. AD - Department of Physiology, Neuroscience Program, Michigan State University, East Lansing, MI, USA. FAU - Parpura, Vladimir AU - Parpura V AD - 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. FAU - Zorec, Robert AU - Zorec R AD - Celica Biomedical, Tehnoloski park 24, 1000, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si. AD - Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si. LA - eng GR - R21 HD078678/HD/NICHD NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't DEP - 20151212 PL - United States TA - Mol Neurobiol JT - Molecular neurobiology JID - 8900963 RN - 0 (Biomarkers) RN - 0 (Brain-Derived Neurotrophic Factor) RN - 0 (Purines) RN - 0 (SNARE Proteins) RN - 690G0D6V8H (Ketamine) RN - 8L70Q75FXE (Adenosine Triphosphate) RN - SY7Q814VUP (Calcium) SB - IM MH - Adenosine Triphosphate/*pharmacology MH - Animals MH - Astrocytes/drug effects/*metabolism MH - Biomarkers/metabolism MH - Brain-Derived Neurotrophic Factor/*metabolism MH - Calcium/metabolism MH - Calcium Signaling/drug effects MH - Cells, Cultured MH - Cytoplasmic Vesicles/*metabolism MH - Exocytosis/*drug effects MH - Hydrogen-Ion Concentration MH - Ketamine/*pharmacology MH - Membrane Fusion/drug effects MH - Purines/metabolism MH - Rats, Wistar MH - SNARE Proteins/metabolism MH - Subcellular Fractions/metabolism OTO - NOTNLM OT - Astroglia OT - Brain-derived neurotrophic factor OT - Ca2+ signaling OT - Fusion pore OT - Ketamine OT - Regulated exocytosis EDAT- 2015/12/15 06:00 MHDA- 2017/12/23 06:00 CRDT- 2015/12/15 06:00 PHST- 2015/07/30 00:00 [received] PHST- 2015/11/29 00:00 [accepted] PHST- 2015/12/15 06:00 [pubmed] PHST- 2017/12/23 06:00 [medline] PHST- 2015/12/15 06:00 [entrez] AID - 10.1007/s12035-015-9562-y [pii] AID - 10.1007/s12035-015-9562-y [doi] PST - ppublish SO - Mol Neurobiol. 2016 Dec;53(10):6882-6896. doi: 10.1007/s12035-015-9562-y. Epub 2015 Dec 12.