Brain Research 962 (2003) 122–128 www.elsevier.com / locate / brainres Research report Involvement of protein kinase C in glutamate release from cultured microglia * Yoichi Nakamura , Miho Ohmaki, Koji Murakami, Yukio Yoneda Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan Accepted 16 October 2002 Abstract Glutamate release from microglial cells may cause neuronal damage. To elucidate the mechanism of glutamate release, we examined the possible regulation by nitric oxide and protein kinase C. Cultured microglia prepared from the whole brains of newborn rats released glutamate by the stimulation with lipopolysaccharide (LPS) dose dependently. The time course study revealed that glutamate release showed a long lag time about 6 h after LPS stimulation, whereas about 3 h lag time was observed in LPS-induced NO production. An G inhibitor for NO synthase, N -nitro-L-arginine, could effectively inhibit the glutamate release. Glutamate release induced by LPS was enhanced by 1 nM phorbol myristate acetate (PMA). Furthermore, high concentrations of PMA (.10 nM) induced glutamate release even without LPS stimulation. Glutamate release stimulated either by 100 ng / ml LPS or 100 nM PMA was inhibited by staurosporine, and also by a-aminoadipate. These results provide insight into the pathways regulating microglial pathological activation by protein kinase C and may be a base for the protection against microglia-evoked neurotoxicity.  2002 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Neuroglia and myelin Keywords: Microglia; Glutamate; Nitric oxide; Lipopolysaccharide; Protein kinase C; Phorbol myristate acetate 1. Introduction microglial activation is a common phenomenon in many CNS diseases, the factors that trigger or regulate microglial Microglia, residential macrophages in the CNS, can activation are remained largely unknown. release a variety of trophic factors and cytokines to There have been recently a numerous papers published regulate the communication among neuronal and other on the regulation of microglial activation in vitro system types of glial cells [31,4,7]. Microglia also play immuno- using cultured microglia. Lipopolysaccharides (LPS), bac- logical roles to phagocytose invading microorganism and terial endotoxins, are most generally used for a stimulating to remove dead cells and / or being damaged cells [13]. agent. The addition of LPS stimulates cultured microglia to When microglia are hyper-activated due to a certain induce the release of a series of proinflammatory cyto- pathological imbalance, the functions of microglia may kines; tumor necrosis factor (TNF) a and interleukin cause neuronal degeneration. Pathological activation of (IL)-1b, then IL-6, and after a latent period of 3–4 h, nitric microglia has been reported in a wide range of injuries oxide (NO) is started to be released [22]. In addition to the such as cerebral ischemia, Alzheimer’s disease, multiple substances mentioned above, LPS-stimulated microglia sclerosis, and AIDS dementia [21,16,24,32]. Although release glutamate [14,25]. Glutamate is a major excitatory amino acid and the excess amount of glutamate causes the degeneration of *Corresponding author. Current address: Laboratory of Integrative neuronal cells in various CNS diseases. In vitro experi- Physiology in Veterinary Sciences, Osaka Prefecture University, 1-1 ments, cultured neurons are easily damaged by the expo- Gakuen-cho, Sakai, Osaka, 599-8531, Japan. Tel.: 181-72-254-9477; fax: sure to the excitatory amino acids, such as glutamate and 181-72-254-9478. E-mail address: yoichi@vet.osakafu-u.ac.jp (Y. Nakamura). aspartate [6,17]. Brain is the most vulnerable organ against 0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993(02)03979-3