The gliotransmitter D-serine is released upon (S)-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid͞kainate and metabotropic glutamate receptor stimulation, but the mechanisms involved are unknown. Here, by using a highly sensitive bioassay to continuously monitor extracellular D-serine levels, we have investigated the pathways used in its release. We reveal that D-serine release is inhibited by removal of extracellular calcium and augmented by increasing extracellular calcium or after treatment with the Ca 2؉ ionophore A23187. Furthermore, release of the amino acid is considerably reduced after depletion of thapsigargin-sensitive intracellular Ca 2؉ stores or chelation of intracellular Ca 2؉ with 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetate-acetoxymethyl ester. Interestingly, D-serine release also was markedly reduced by concanamycin A, a vacuolar-type H ؉ -ATPase inhibitor, indicating a role for the vesicular proton gradient in the transmitter storage͞release. In addition, agonist-evoked D-serine release was sensitive to tetanus neurotoxin. Finally, immunocytochemical and sucrose density gradient analysis revealed that a large fraction of D-serine colocalized with synaptobrevin͞VAMP2, suggesting that it is stored in VAMP2-bearing vesicles. In summary, our study reveals the cellular mechanisms subserving D-serine release and highlights the importance of the glial cell exocytotic pathway in influencing CNS levels of extracellular D-serine.glia ͉ synaptobrevin ͉ D-amino acid ͉ vesicles ͉ tetanus neurotoxin A strocytes play pivotal roles in synaptic transmission by controlling transmitter diffusion and concentration in the extracellular space (1) and also by back-signaling to neurons directly through the release of neuroactive substances (2). Although glutamate and ATP are the most recognized chemical transmitters that mediate astrocyte-neuron signaling (2), other cell-cell mediators also are involved in this pathway. D-Serine has recently been identified as a major gliotransmitter in the central nervous system that serves as an endogenous ligand for the glycine site of NMDA receptors (3-6).However, many aspects regarding D-serine release still need to be addressed. Although it has been suggested that astrocytes may release D-serine, through the reverse operation of a sodiumdependent transporter (7), the precise molecular mechanisms underlying D-serine release are currently unknown. To unravel the functional consequences of D-serine-mediated astrocyte-toneuron signaling, it is essential to shed light on the mechanisms controlling the gliotransmitter storage and release pathways.For this purpose, we have devised a previously undescribed bioassay to continuously monitor the release of D-serine from cultured glial cells. In this work, we show that astrocytes and C6 glioma cells synthesize and contain a large amount of D-serine that can be released upon glutamate receptor (GluR) stimulation. Moreover, our observations demonstrate that D-serine release, evoked by glutamatergic agonists, is linked to a [Ca 2ϩ...

Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo-and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.Membrane proteins such as tyrosine kinase, cytokine, or transforming growth factor receptors have been known for many years to form oligomers, and the link between their oligomerization and activity states has been well established (1). In contrast, G protein-coupled receptor (GPCR) 1 oligomerization has been documented only recently (2), and the relation between receptor activation and oligomerization is still poorly understood. Even the proportion between monomeric and oligomeric receptor species and the exact oligomerization state (dimer, trimer, tetramer, etc.) remains a matter of controversy. Currently, two models are proposed. In the first model GPCR are monomeric in their inactive state, and agonist activation induces the formation of receptor oligomers. This model is based on low basal and strong agonist-induced energy transfer signals observed in fluorescence and bioluminescence resonance energy transfer (FRET, BRET) experiments for the gonadotropin-releasing hormone (3), the somatostatin SSTR5 (4), and the SSTR5/dopamin D2R receptor oligomers (5). The second model proposes that GPCR are constitutively oligomerized and is supported by studies reporting high basal BRET or FRET signals for ␣-mating factor (6), ␤2-adrenergic (␤2AR) (7), tyrothropin-releasing hormone (8), ␦-opioid (9), type A cholecystokinin (10), and dopamine D2 receptors (11). Agonist-promoted increases in signals were observed in some of thes...

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca 2؉ stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca 2؉ increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals.