Group III Metabotropic Glutamate Receptors in Rat Cultured Calvarial Osteoblasts Eiichi Hinoi, Sayumi Fujimori, Yoichi Nakamura, and Yukio Yoneda 1 Department of Molecular Pharmacology, Kanazawa University Faculty of Pharmaceutical Sciences, Kanazawa, Ishikawa 920-0934, Japan Received January 17, 2001 Reverse transcription polymerase chain reaction re- vealed expression of mRNAs for particular receptors for the central neurotransmitter L-glutamic acid (Glu) in primary cultures of rat calvarial osteoblastic cells under premature to mature states according to the duration of days in vitro. These included metabotropic Glu receptors (mGluR) such as mGluR4 and mGluR8, in addition to several ionotropic Glu receptor sub- units including NR1 and NR2D. Expression of mRNAs was not detected with other mGluR and NR2A-C sub- units irrespective of the maturity of cultured cells. The agonist for group III mGluR L-()-2-amino-4- phosphonobutyric acid significantly inhibited the forskolin-induced accumulation of cAMP in prema- ture osteoblasts, which occurred in a manner sensitive to prevention by the group III mGluR antagonist (RS)- -cyclopropyl-4-phosphonophenylglycine. These re- sults suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation and/or differentiation through group III mGluR func- tionally expressed in rat calvarial osteoblastic cells. © 2001 Academic Press Key Words: glutamate; group III mGluR; mGluR4; mGluR8; osteoblasts; RT-PCR; cAMP accumulation; NR1; NR2D. Osteoporosis is believed to be due to the imbalance of bone metabolism between formation by osteoblasts and resorption by osteoclasts (1, 2). The balancing mecha- nism involves maintenance of both the integrity and function by hormones such as estrogen, calcitonin, parathyroid hormone (PTH) and vitamin D as well as by cytokines including interleukin-6 (IL-6), IL-11 and leukemia inhibitory factor (1, 2). Recent studies have raised the possibility that L-glutamic acid (Glu) may be one of the endogenous factors used for intercellular communications as a paracrine and/or autocrine sub- stance via Glu receptors (GluR) (3) and Glu transport- ers (4) in bone, in addition to playing a neurotransmit- ter role in the central nervous system (CNS). GluR are categorized into two major subclasses, such as ionotropic (iGluR) and metabotropic (mGluR) recep- tors, according to their differential intracellular signal transduction mechanisms. The former subclass is di- vided into three distinct subtypes, including N-methyl- D-aspartic (NMDA) (NR1 and NR2A-D), DL--amino-3- hydroxy-5-methylisoxasole-4-propionic (GluR1-4) and kainic (KA) (GluR5-7 and KA1-2) acids, which are all associated with ion channels permeable to particular cations (5). The latter is sub-classified into three dif- ferent subtypes, including groups I, II, and III, accord- ing to exogenous agonists and intracellular second messengers (6, 7). Group I mGluR (mGluR1 and mGluR5) subtype is coupled to phospholipase C to stimulate hydrolysis of membrane phospholipids, while both group II (mGluR2 and mGluR3) and group III (mGluR4 and mGluR6-8) subtypes are linked to adenylate cyclase to inhibit formation of cAMP. In this study, therefore, we have analyzed possible expression of mRNAs for mGluR by using reverse tran- scription polymerase chain reaction (RT-PCR) in pri- mary cultured rat osteoblastic cells that are shown to contain particular iGluR subunits (3, 8). MATERIALS AND METHODS Materials. Quickprep Micro mRNA Purification Kit, First-strand cDNA Synthesis kit, DYEnamic ET Terminator Cycle Sequencing kit, and cAMP enzyme immunoassay system were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Taq DNA Abbreviations used: -MEM, -modified minimum essential me- dium; CNS, central nervous system; CPPG, (RS)--cyclopropyl-4- phosphonophenylglycine; FBS, fetal bovine serum; Glu, glutamic acid; GluR, glutamate receptors; IBMX, 3-isobutyl-1-methylxan- thine; iGluR, ionotropic glutamate receptors; IL, interleukin; KA, kainic acid; L-AP4, L-(+)-2-amino-4-phosphonobutyric acid; mGluR, metabotropic glutamate receptors; NMDA, N-methyl-D-aspartic acid; PBS, phosphate-buffered saline; PTH, parathyroid hormone; RT-PCR, reverse transcription polymerase chain reaction. 1 To whom correspondence should be addressed at Department of Molecular Pharmacology, Kanazawa University Faculty of Pharma- ceutical Sciences, 13-1 Takara-machi, Kanazawa, Ishikawa 920- 0934, Japan. Fax: 81-(0)76-234-4471. E-mail: yyoneda@anet.ne.jp. Biochemical and Biophysical Research Communications 281, 341–346 (2001) doi:10.1006/bbrc.2001.4355, available online at http://www.idealibrary.com on 341 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.