The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Ca in C6 glioma cells Norimitsu Morioka a,b, *, Joynal Md. Abdin a , Katsuya Morita a , Tomoya Kitayama a , Yoshihiro Nakata b , Toshihiro Dohi a,c a Department of Dental Pharmacology, Division of Integrated Medical Science, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan b Department of Pharmacology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan c Department of Pharmaceutical Services, Yamaguchi Orthopedic Hospital, 2-13-20 Gion, Asaminami-ku, Hiroshima 731-0138, Japan 1. Introduction Glycine has an important neurotransmitter function at both inhibitory and excitatory synapses in the vertebrate central nerve system. The inhibitory action of glycine is initiated by the stimulation of strychnine-sensitive glycine receptors mainly located in the spinal cord, brainstem and cerebellum (Legendre, 2001). Additionally, glycine acts as an essential co-agonist of glutamate at NMDA receptors, and its binding to these receptors triggers both the opening of ion channels and the internalization of receptors (Johnson and Ascher, 1987; Nong et al., 2003; Sershen et al., 2008). The neurotransmission mediated by glycine in the synaptic cleft is terminated by the Na + –Cl À dependent reuptake system. Thus far, two glycine transporters GLYT1 and GLYT2 have been cloned in the brain or spinal cord (Guastella et al., 1992; Liu et al., 1993). In general, GLYT1 is expressed in glial cells that exist close to the glycinergic nerve terminals, and plays an essential role in the clearance of glycine at synapses (Adams et al., 1995; Arago ´n and Lo ´ pez-Corcuera, 2003). GLYT2 is present in axons and presynaptic terminals of inhibitory glycinergic neurons, and supplies glycine to the presynaptic cytosol for vesicular release (Liu et al., 1993; Zafra et al., 1995). In contrast, there are a report described the opposite location of GLYT1 or GLYT2 which are existed in neurons or astrocytes, respectively (Raiteri et al., 2008). It has been demonstrated that changes of GLYT1 activity caused by various factors directly affect the homeostasis of the glycinergic system, which is involved in both the excitatory neurotransmis- sion through NMDA receptors and the inhibitory neurotransmis- sion through glycine receptors, at synapses. For example, NMDA receptor-triggered currents were increased by the inhibition of Neurochemistry International 53 (2008) 248–254 ARTICLE INFO Article history: Received 12 March 2008 Received in revised form 8 August 2008 Accepted 8 August 2008 Available online 19 August 2008 Keywords: Protein kinase C Glycine transporter C6 glioma cells Uptake RNA interference ABSTRACT Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCa, PKCd, and PKCe of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca 2+ . Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Go ¨ 6983, which are broad inhibitors of PKC, or Go ¨ 6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCd and PKCe, did not affect the transport of glycine. Silencing of PKCd expression by using RNA interference or pretreatment with the inhibitor peptide for PKCe had no effect on the PMA- suppressed uptake of glycine. Together, these results suggest PKCa to be a crucial factor in the regulation of glycine transport in C6 cells. ß 2008 Elsevier Ltd. All rights reserved. * Corresponding author at: Department of Pharmacology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734- 8553, Japan. Tel.: +81 82 257 5312; fax: +81 82 257 5314. E-mail address: mnori@hiroshima-u.ac.jp (N. Morioka). Contents lists available at ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/neuint 0197-0186/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2008.08.002