Release of Endogenous Glutamate by AMPA Receptors Expressed in Cultured Rat Costal Chondrocytes

Abstract

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...

Differential regulation of cellular maturation in chondrocytes and osteoblasts by glycine

“…On the other hand, we have previously shown that the activation of intrinsic AMPA receptors leads to marked facilitation of the exocytic release of endogenous Glu from cultured rat calvarial osteoblasts (Hinoi et al 2002b) and costal chondrocytes (Wang et al 2005b). These findings raise the possibility that endogenous Gly might be also used as a local paracrine and/or autocrine signal mediator in association with Glu for intercellular communications between neighboring chondrocytes and osteoblasts Hinoi et al 2002b).…”

Poly(γ-Glutamic Acid) as an Exogenous Promoter of Chondrogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

“…It is expected that g-PGA might be consumed/degraded, thus generating a glutamate pool able to activate the glutamate signaling pathway. 96 In fact, glutamate signaling has been described in cartilage, namely in human articular chondrocytes, 28 rat costal and articular chondrocytes, [30][31][32] and also in mouse and human MSCs. [97][98][99] L-glutamate, but not D-glutamate, suppressed mouse MSC proliferation and differentiation in 2D without inducing cell death.…”

“…[27][28][29] In cartilage, glutamate signaling was shown to tune rat and human chondrocyte behavior and enhance matrix production. [30][31][32] g-PGA-blended scaffolds were reported to support rat chondrocyte cultures. 33 A patent describing the injection of g-PGA for the treatment of degenerative joint diseases has also been filed.…”

Glutamate Inhibits Chondral Mineralization through Apoptotic Cell Death Mediated by Retrograde Operation of the Cystine/Glutamate Antiporter

“…For example, recent studies have shown that Glu may be one of the endogenous paracrine (autocrine) factors used for intercellular communications through particular GluRs in cartilage (5-7) and bone (8,9). In cultured rat costal chondrocytes where constitutive expression is seen with mRNA for the GluR3 subunit of the iGluR subtype AMPA receptors, the addition of AMPA markedly stimulates the release of endogenous Glu in a Ca 2ϩ -dependent manner (5). Moreover, in cultured mouse embryonic metatarsals isolated before vascularization, chondral mineralization was almost completely abolished in the presence of the group III mGluR agonist L-(1)-2-amino-4-phosphonobutyrate (L-AP4) in a manner sensitive to an antagonist, without inducing any apoptotic cell death (6).…”