1 Previous studies have demonstrated the functional expression by osteoblasts of glutamate (Glu) signaling machineries responsible for the stimulation of cell proliferation and differentiation in bone, while there is no information available on the expression of the Glu signaling system by cartilage to date. 2 In cultured mouse embryonic metatarsals isolated before vascularization, chondral mineralization was almost completely inhibited in the presence of the group III metabotropic Glu receptor (mGluR) agonist L-(1)-2-amino-4-phosphonobutyrate (L-AP4) in a manner sensitive to an antagonist, with the total length being unchanged. 3 A group II mGluR agonist was similarly more effective in inhibiting the mineralization than a group I mGluR agonist, while none of ionotropic GluR agonists drastically affected the mineralization. 4 Both histological and in situ hybridization analyses revealed that L-AP4 specifically inhibited chondral mineralization, without apoptotic cell death, in cultured metatarsals. 5 In addition to the constitutive expression of mRNA for particular mGluRs in both cultured mouse metatarsals and rat costal chondrocytes, L-AP4 significantly inhibited the accumulation of cyclic AMP by forskolin and parathyroid hormone in a manner sensitive to a group III mGluR antagonist in cultured chondrocytes. 6 Moreover, L-AP4 drastically inhibited the expression of osteopontin mRNA in both cultured metatarsals and chondrocytes. 7 These results suggest that Glu may at least in part play a role as a signal mediator in mechanisms associated with chondral mineralization through the group III mGluR subtype functionally expressed by chondrocytes in rodent cartilage.

Although we have previously demonstrated the functional significance of excitatory amino acid transporters as well as glutamate (Glu) receptors (GluRs) expressed by chondrocytes, little attention has been paid to the possible expression of the cystine/ Glu antiporter responsible for the bi-directional transmembrane transport of Glu in chondrocytes to date. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, the chondral mineralization was significantly decreased in the presence of Glu at a high concentration. Apoptotic cells were detected within the late proliferating and prehypertrophic chondrocytic layers in metatarsals cultured in the presence of Glu. A group III metabotropic GluR (mGluR) antagonist partially, but significantly, prevented the inhibition of mineralization by Glu in metatarsals without affecting the number of apoptotic cells. Both decreased mineralization and apoptosis by Glu were significantly prevented by the addition of the cystine/Glu antiporter inhibitor homocysteic acid, as well as reduced glutathione (GSH) and cystine. Expression of mRNA for xCT and 4F2hc subunits, which are components of the cystine/Glu antiporter, was seen in both cultured mouse metatarsals and rat costal chondrocytes. In chondrocytes cultured with Glu, a significant decrease was seen in intracellular GSH levels, together with increases in the number of apoptotic cells and the level of intracellular reactive oxygen species. These results suggest that Glu could regulate chondrogenic differentiation toward mineralization through a mechanism associated with apoptosis mediated by the depletion of intracellular GSH after the retrograde operation of the cystine/Glu antiporter, in addition to the activation of group III mGluR, in chondrocytes.In the vertebrate central nervous system, glutamate (Glu) is one of the most abundant free amino acids with a neurotransmitter role involving signaling machineries that include Glu receptors (GluRs) 3 and Glu transporters (1, 2). In the glutamatergic synapses, Glu is condensed into synaptic vesicles through vesicular Glu transporters for subsequent exocytotic release into synaptic clefts upon stimulation. Glutamate is supposed to mediate the excitatory neurotransmission through GluRs categorized into two major groups. One is ionotropic Glu-gated ion channels (iGluRs) that are further classified into DL-␣-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), kainite, and N-methyl-D-aspartate subtypes, whereas the other is G-protein-coupled metabotropic receptors (mGluRs) classified into the three different subtypes, group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3), and group III (mGluR4, mGluR6, mGluR7, and mGluR8) (1, 2). The group I subtype stimulates the hydrolysis of membrane phospholipids in association with G q/11 protein, whereas both the group II and III subtypes inhibit the formation of cAMP with the aid of G i/o protein.However, several independent lines of evidence indicate that Glu may act as a "cytokine" rather than a "neurotransmi...

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L-Glutamate (Glu) is one of the most abundant free amino acids with a major excitatory neurotransmitter role in the vertebrate central nervous system, while recent trends are toward a role in neuronal differentiation, migration and survival in the developing brain.2-4) The actions of extracellular Glu are mediated by membranous receptors, which can be divided into two major groups. 4) One is ionotropic Glu-gated ion channels (iGluRs) that are further classified into DL-aamino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), kainite (KA) and N-methyl-D-aspartate (NMDA) subtypes according to sequential similarities as well as responsiveness to different agonists and antagonists, 5,6) whereas the other is G-protein-coupled metabotropic receptors (mGluRs) that are a member of the class 3 G protein-coupled receptor family. 7,8) In addition to GluRs, vesicular Glu transporters (VGLUTs) are essential for signal output through the condensation of Glu into vesicular constituents for subsequent exocytotic release. Within the central nervous system (CNS), both VGLUT-1 9) and VGLUT-2 10) isoforms are supposed to suffice for the definition of an excitatory neuronal phenotype, while VGLUT3 is expressed in a number of cells shown to release Glu through exocytosis including dopaminergic, GABAergic and serotonergic neurons as well as astrocytes. 11)Recently, evidence that glutamatergic signaling is also functional in non-neuronal tissues, such as bone, pancreas and skin, is accumulating in the literature.12,13) Glu may act as a more widespread "cytokine" rather than a "neurotransmitter" to influence a variety of cellular activities in different tissue types. 12,13) Recent studies have raised the possibility that Glu may be one of the endogenous paracrine (autocrine) factors used for intercellular communications in bone. 14,15) The addition of an NMDA receptor antagonist inhibits cell differentiation in cultured osteoclasts, 16) for example, while Glu induces elevation of intracellular free Ca 2ϩ in a manner sensitive to antagonism by the NMDA receptor antagonist dizocilpine in osteoblasts. 17) We have also recently demonstrated the exacerbation of osteoblastic differentiation by different NMDA receptor antagonists.18) In addition to NMDA receptors, osteoblasts constitutively express mRNA for non-NMDA receptors such as GluR3 subunit of AMPA receptors (AMPAR) as well as KA1 and KA2 subunits of KA receptors, 19) while AMPAR modulate the exocytotic release of Glu from cultured osteoblasts. 20) By contrast, no much attention has been paid to the possible functional expression by cartilage of particular glutamatergic signaling molecules required for the neurotransmission in the brain to date. Therefore, we have attempted to mimic our previous studies on cultured rat osteoblasts for the release of endogenous Glu using cultured rat chondrocytes. In the present study, functional expression was for the first time shown with a particular subtype of iGluRs in cartilage. MATERIALS AND METHODSMaterials Leica CM 3050s cryostat and a fluorescen...

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