Brain Research 943 (2002) 112–116 www.elsevier.com / locate / bres Short communication Demonstration of expression of mRNA for particular AMPA and kainate receptor subunits in immature and mature cultured rat calvarial osteoblasts Eiichi Hinoi, Sayumi Fujimori, Akihiro Takemori, Hiroaki Kurabayashi, Yoichi Nakamura, * Yukio Yoneda Department of Molecular Pharmacology, Kanazawa University Faculty of Pharmaceutical Sciences, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan Accepted 21 March 2002 Abstract Reverse transcription polymerase chain reaction revealed expression of mRNA for particular subunits of ionotropic glutamate receptors (iGluR) in primary cultures of rat calvarial osteoblastic cells under immature to mature states. These included GluR3, KA1 and KA2 subunits, in addition to NR1 and NR2D subunits. These results suggest that glutamate may play an unidentified role in mechanisms associated with cellular development through particular subunits of iGluR in rat calvarial osteoblasts.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: Excitatory amino acids: structure, function and expression Keywords: Glutamate; AMPA receptor; GluR3; KA1; KA2; Osteoblast; RT-PCR L-Glutamic acid (Glu) is believed to be a neurotrans- the endogenous factors used for intercellular communica- mitter in the mammalian central nervous system (CNS). tions as a paracrine and / or autocrine substance via GluR Glutamate receptors (GluR) are categorized into two major [11] and Glu transporters [10] in bone. In this study, we subclasses, such as ionotropic (iGluR) and metabotropic have analyzed possible expression of mRNA for particular (mGluR) receptors, according to their differential intracel- iGluR subtypes by using reverse transcription polymerase lular signal transduction mechanisms. The former subclass chain reaction (RT-PCR) in primary cultures of rat osteob- is divided into three distinct subtypes, including N-methyl- lastic cells that are shown to contain particular NMDA D-aspartic (NMDA) (NR1 and NR2A–D), D,L-a-amino-3- receptor [2,8,11] and mGluR [8] subunits. Of NMDA hydroxy-5-methylisoxasole-4-propionic (GluR1–4) and receptor subunits tested, mRNA for NR1, NR2A-C and kainic (KA) (GluR5–7 and KA1-2) acids, which are all NR2D is all detected in the brain, while cultured osteob- associated with ion channels permeable to particular lasts contain mRNA for NR1 and NR2D subunits without cations [19]. The latter subclass is categorized into three expressing that for NR2A-C subunits irrespective of the different subtypes, including groups I, II and III, according maturity of cultured cells [8]. to exogenous agonists and intracellular second messengers Osteoblasts were prepared from calvaria of 1-day-old [17]. In agreement with our previous studies on localiza- Wistar rats by a sequential enzymatic digestion method 3 tion of [ H]Glu binding sites in particular peripheral previously reported [3] with some modifications [8]. In tissues such as adrenal [20] and pituitary [21], recent brief, calvaria were gently incubated at 37 8C for 10 min studies have raised the possibility that Glu may be one of with 0.1% collagenase and 0.25% trypsin in calcium and magnesium-free phosphate-buffered saline (PBS). This incubation was consecutively repeated five times. Cells *Corresponding author. Tel. / fax: 181-76-234-4471. E-mail address: yyoneda@anet.ne.jp (Y. Yoneda). obtained during the last four digestions were altogether 0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993(02)02726-9