Astrocytic CB1R enhances GLAST activity in a Ca2+ dependent manner
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1
Laboratório de Farmacologia Clínica e Terapêutica, Faculdade de Medicina da Universidade de Lisboa, Portugal
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2
Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal
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3
School of Biosciences, College of Biomedical and Life Sciences, Cardiff University, United Kingdom
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4
Instituto Superior de Engenharia de Lisboa, Portugal
Astrocytes perform many functions at the synapse level, including glutamate uptake, which is mainly mediated by specific glutamate transporters, GLT-1 and GLAST transporter (Perego et al., 2000) that are responsible for preventing excitotoxicity and for modulating synaptic function. Astrocytes can communicate with surrounding astrocytes and neurons by changes in the intracellular Ca2+ (Perea and Araque, 2005), being this achieved through the expression of several types of receptors in their membranes such as the cannabinoid type one receptor (CB1R) whose activation leads to Ca2+ mobilization from internal cellular stores (Navarrete and Araque, 2008). Thus, the aim of this work was to study the role of astrocytic CB1R activation-mediated Ca2+ signaling upon GLAST in rat primary astrocytic cultures.
Primary cortical astrocytic cultures were prepared from Sprague Dawley pups (0-2 days old). Variations in [Ca2+]i were detected by Ca2+ imaging technique using fura 2-acetoxymethyl ester (Fura2AM, 5 M). The transport was initiated by addition of [3H]glutamate and the specific transport mediated by GLAST was accessed in presence of UCPH-101. Ca2+ signalling and [3H]-glutamate uptake experiments were performed with 18-23 DIC astrocytes.
It was observed that astrocytic CB1R activation with ACEA, a CB1R specific agonist, led to Ca2+ transients, whose amplitude and frequency was dose-dependent for the two tested ACEA concentrations (0.5 and 1.0 µM, n = 3-7 independent cultures). The occurrence of these transients were impaired in the presence of a PLC antagonist (U73122, 3 µM) and a intracellular Ca2+ stores depletory (CPA, 10 µM). Low extracellular Ca2+ concentration decreased both frequency and amplitude of the transients. Regarding glutamate uptake, astrocytic CB1R activation increased significantly the maximum velocity of transport (Vmax) of GLAST transporter without significant changes in affinity (Km). This enhancement was lost in the presence of U73122 and a Ca2+ chelant (BAPTA, 20 µM), but not with a PKC antagonist (GF 10109203X, 1 µM).
In conclusion, in astrocytes, CB1R activation triggers Ca2+ transients by PLC activation, in a dose-dependent manner enhancing GLAST activity probably by increasing the number of functional transporters at the astrocytic membrane.
Acknowledgements
This project was funded by Fundação para a Ciência e a Tecnologia (PTDC/BTM-SAL/32147/2017).
Keywords:
Astrocytes,
CB1R,
Glutamate Uptake,
Calcium,
Cannabinoids
Conference:
XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019.
Presentation Type:
Poster presentation
Topic:
Glia / Neuroinflammation
Citation:
Gonçalves-Ribeiro
J,
Morais
TP,
Savchak
O,
Meneses
C,
Sebastião
AM and
Vaz
SH
(2019). Astrocytic CB1R enhances GLAST activity in a Ca2+ dependent manner.
Front. Cell. Neurosci.
Conference Abstract:
XVI Meeting of the Portuguese Society for Neuroscience (SPN2019).
doi: 10.3389/conf.fncel.2019.01.00039
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Received:
16 Apr 2019;
Published Online:
27 Sep 2019.
*
Correspondence:
Dr. Sandra H Vaz, Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Portugal, svaz@medicina.ulisboa.pt