Glutamate receptors and the regulation of steroidogenesis in the human adrenal gland: The metabotropic pathway Saulo J.A. Felizola a , Yasuhiro Nakamura a,⇑ , Fumitoshi Satoh b , Ryo Morimoto b , Kumi Kikuchi b , Tomohiro Nakamura c , Atsushi Hozawa c , Lin Wang d , Yoshiaki Onodera a , Kazue Ise a , Keely M. McNamara a , Sanae Midorikawa e , Shinichi Suzuki f , Hironobu Sasano a a Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan b Division of Nephrology and Hypertension, Tohoku University Hospital, Sendai, Japan c Department of Preventive Medicine and Epidemiology, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan d Department of Physiology, Harbin Medical University Daqing Branch, Daqing, China e Department of Radiation Health Management, Fukushima Medical University, Fukushima, Japan f Department of Organ Regulatory Surgery, Fukushima Medical University, Fukushima, Japan article info Article history: Received 1 March 2013 Received in revised form 18 September 2013 Accepted 20 September 2013 Available online 27 September 2013 Keywords: Adrenal cortex Zona glomerulosa Adrenocortical adenoma Glutamate receptor cAMP Aldosterone abstract Background: L-glutamate is a major excitatory neurotransmitter in the mammalian brain. Glutamate receptors have been reported in the rat adrenal cortex and in human aldosterone-producing adenomas (APA). However, details regarding the expression levels and functions of these receptors in human adre- nocortical tissues remain unknown. Methods: The mRNA levels of glutamate receptors were evaluated by qPCR in: 12 normal adrenal cortex (NAC), 11 APA, and 12 cortisol-producing adenoma (CPA) tissues. Protein localization was evaluated by immunohistochemistry for 15 NAC, 5 idiopathic hyperaldosteronism cases, 15 APA and 15 CPA. H295R cells were treated with angiotensin-II or forskolin alone or combined with the GRM2/3 agonist LY354740. Results: The level of GRM3 mRNA was higher in APA than in CPA (P = 0.0086) or NAC (P = 0.0022). GRM1, IGLUR2, and IGLUR3 were also detected in adrenocortical tissues. When added to angiotensin-II/forskolin treatments, LY354740 decreased aldosterone and cortisol production in H295R cells. Conclusions: GRM3 is considered to regulate steroidogenesis in adrenocortical tissues. Ó 2013 Published by Elsevier Ireland Ltd. 1. Introduction L-glutamate (glutamate) is the major excitatory amino-acid neurotransmitter in the mammalian central nervous system (CNS) (Robinson and Coyle, 1987; Conn, 2003; Julio-Pieper et al., 2011). The glutamate concentrations in peripheral blood are stea- dy, ranging from 20 to 50 lM in healthy subjects (Hediger and Welbourne, 1999; Zlotnik et al., 2010; Julio-Pieper et al., 2011). However, a number of diseases have recently been reported to be associated with either increased (i.e., cancer and HIV) or decreased blood glutamate levels (i.e., sepsis with acute liver dysfunction and closed head injury) (Zlotnik et al., 2010; Julio-Pieper et al., 2011), and the presence of stress was also reported to decrease blood glutamate levels following the activation of peripheral corticotro- phin releasing factor receptors (Zlotnik et al., 2010). There are two known types of glutamate receptors, the metab- otropic (GRM) and the ionotropic receptors (IGLUR), both of which are responsible for glutamate signaling in the human CNS and peripheral tissues (Julio-Pieper et al., 2011). GRMs are G-protein coupled receptors (GPCRs) located at the cellular membrane. Three subgroups of GRM have been reported according to their function and sequence homology (Conn, 2003). Group I (GRM1 and GRM5) stimulates inositol phosphatase metabolism and the mobilization of intracellular Ca 2+ ; group II (GRM2 and GRM3) and group III (GRM4, GRM6, GRM7 and GRM8) receptors are negatively coupled to adenylyl cyclase and are known to decrease the intracellular lev- els of cAMP upon activation (Coutinho and Knopfel, 2002; Conn, 2003; Gill and Pulido, 2005). In contrast, the IGLUR receptors are ion gated cell membrane chan- nels. Three major subtypes of IGLUR have been reported thus far, clas- sified according to their sequence similarities, electrophysiological properties and affinity to selective agonists: N-methyl-D-aspartate (NMDA1 and NMDA2A-D), alpha-amino-3-hydroxy-5-methyl-4-sox- 0303-7207/$ - see front matter Ó 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.mce.2013.09.025 Abbreviations: NAC, normal adrenal cortex; APA, aldosterone producing ade- noma; CPA, cortisol producing adenoma; ZG, zona glomerulosa; ZF, zona fascicu- lata; ZR, zona reticularis; qPCR, quantitative RT-PCR. ⇑ Corresponding author. Address: Tohoku University Graduate School of Medi- cine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Tel.: +81 (22)717 8050; fax: +81 (22)717 8051. E-mail address: yasu-naka@patholo2.med.tohoku.ac.jp (Y. Nakamura). Molecular and Cellular Endocrinology 382 (2014) 170–177 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce