title: A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes.
abstract: Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.
authors.affiliation: Unidad de Neurobiología Dinámica, Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía, México City, México.
authors.forename: Pavel
authors.initials: P
authors.lastname: Montes de Oca Balderas
authors.affiliation: Laboratorio de Patología Vascular Cerebral, Instituto Nacional de Neurología y Neurocirugía, México City, México.
authors.forename: Penélope
authors.initials: P
authors.lastname: Aguilera
mesh_terms: D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D001562:Benzimidazoles / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002232:Carbocyanines / Q000737:chemistry; D002478:Cells, Cultured; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D007477:Ions / Q000737:chemistry; D007736:Kynurenic Acid / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D016202:N-Methylaspartate / Q000494:pharmacology; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D001562:Benzimidazoles; D002121:Calcium Channel Blockers; D002232:Carbocyanines; C409648:Fluo 4; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007477:Ions; D021122:Protein Subunits; D034741:RNA, Small Interfering; D016194:Receptors, N-Methyl-D-Aspartate; D014966:Xanthenes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; D015763:2-Amino-5-phosphonovalerate; D007736:Kynurenic Acid; D002118:Calcium