Sulphur-containing amino acids are agonists for group 1 metabotropic receptors expressed in clonal RGT cell lines
Introduction
Glutamate is established as the major neurotransmitter in the mammalian brain that mediates fast synaptic transmission and induces neuronal plasticity (Schoepp and Conn, 1993, Hollman and Heinenmann, 1994, Pin and Duvoisin, 1995, Conn and Pin, 1997). Glutamate receptor subtypes are classified into two main types: ionotropic and metabotropic. The ionotropic receptors are homomeric or heteromeric cation specific ligand gated ion channels and are divided into three subtypes: N-methyl-d-aspartate (NMDA), α- amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors. The metabotropic glutamate receptors (mGluRs) were first identified by their coupling to signal transduction pathways via G proteins (Sladeczek et al., 1985, Nicoletti et al., 1986, Sugiyama et al., 1987) and later classified more specifically by expression cloning of brain cDNA libraries in oocytes (Houamed et al., 1991, Masu et al., 1991, Abe et al., 1992, Pin et al., 1992, Tanabe et al., 1992, Minakami et al., 1993, Nakajima et al., 1993, Okamoto et al., 1994, Saugstad et al., 1994, Duvoisin et al., 1995). Eight different subtypes of mGluRs have been cloned and are divided into three groups defined on the basis of their amino acid sequence homologies, ligand pharmacology and the type of signal transduction pathway to which they couple. Group 1 mGluRs comprise mGluR1 and mGluR5, each with splice variants, and these are coupled to the phosphoinositide (PI) hydrolysis/[Ca2+]i mobilisation signal transduction pathway. Both group 2 (mGluR2 and 3) and group 3 (mGluR4, 6, 7 and 8) receptors are negatively linked to adenyl cyclase and reduce cyclic AMP formation.
In addition to glutamate and aspartate, evidence has also accumulated to support a role for sulphur amino acids (S-amino acids) as endogenous excitatory amino acid neurotransmitter molecules (for reviews see Griffiths, 1990, Do et al., 1992). l-cysteine sulphinic acid, (l-CSA), l-homocysteine sulphinic acid (l-HCSA), l-homocysteic acid (l-HCA) and l-cysteic acid (l-CA) have been shown to exert excitatory effects on spinal cord neurones (Curtis and Watkins, 1960, Curtis and Watkins, 1963, Mewett et al., 1983); to be released from rat brain slices after depolarisation (Iwata et al., 1982, Recasens et al., 1984, Do et al., 1986, Kilpatrick and Mozley, 1986) to bind to synaptic membranes (Iwata et al., 1982, Recasens et al., 1983) and to elicit biochemical responses in neuronal cells (Sladeczek et al., 1985, Baba, 1987, Pullan et al., 1987, Porter and Roberts, 1993). However, cellular uptake of sulphur amino acids has been demonstrated to be mediated by a sodium dependent, high affinity mechanism that also transports l-glutamate and l-aspartate (Iwata et al., 1982, Griffiths et al., 1989, Arizza et al., 1994, Klockner et al., 1994). It is therefore conceivable that some of the observed effects of the sulphur amino acids may be due to their competitive heteroexchange effects on glutamate transport processes resulting in an elevation of extracellular glutamate concentration.
The derivation of mGluR clonal lines has greatly facilitated the definition of agonist pharmacology first described in native brain tissues. Studies on rat group 1 mGluR expressing cell lines have shown that mGluRs 1 and 5 are most potently stimulated by quisqualate>ibotenate>glutamate>ACPD (Abe et al., 1992, Aramori and Nakanishi, 1992, Thomsen et al., 1993) but these agonists also stimulate, with a different rank order of potency, group two and three receptors (Nakanishi, 1992, Tanabe et al., 1992). More recently, (S) 3,5-dihydroxyphenylglycine and (RS)-amino (3,5-dihydroxyphenyl) methylphosphinic acid (DHPMP) have been identified as the most selective of the group 1 agonists to date (Schoepp et al., 1994, Boyd et al., 1996).
In this study, the pharmacological effects of a range of l- and d-type sulphur containing amino acids have also been investigated on PI hydrolysis responses of mGluR1α and mGluR5α that were expressed in clonal cell lines deirved using identical cell systems. Further experimental consideration of the mechanism of effect of the sulphur containing (S-) amino acids have shown that their actions are not dependent on glutamate and that their stimulatory effects result from a direct interaction with mGluR1α and mGluR5α.
Section snippets
Materials
l-Quisqualate, 1S,3R-aminocyclopentane dicarboxylic acid (ACPD), (S)-4-carboxy-3-hydroxy-phenylglycine (4C3HPG), l- and d-cysteine sulphinic acids (CSA); l- and d-homocysteine sulphinic acids (HCSA), l- and d-cysteic acids (CA), l- and d-homocysteic acids (HCA), l-serine-O-sulphate (SOS), l-sulphocysteine (S-CYS), l-threo-β-hydroxy-aspartate (THA), dihydrokainate (DHK) and trans-pyrrolidine dicarboxylic acid (PDC) were purchased from Tocris Cookson (Bristol, UK). l-Glutamate was purchased from
Effects of S-amino acids on group 1 hmGluRs
Of the sulphur containing amino acids tested, the l-isomers of HCSA, CSA, CA and SOS produced stimulatory effects on PI hydrolysis in hmGluR1α and hmGluR5a cells (Fig. 1Fig. 2). The d-isomer of CSA showed agonist effects on hmGluR5a but not hmGluR1α and l- and d-forms of HCA were inactive on both the group 1 mGluR clones (Table 1). On hmGluR1α, l-HCSA and l-CSA had potencies similar to those of glutamate whereas the potencies of l-CA and l-SOS were comparable to 1S,3R-ACPD (Table 1). All the
Discussion
Characterisation and comparison of agonists effects on mGluR1α and mGluR5a included the investigation of S-amino acids as potential ligands for group 1 mGluRs. A number of previous studies have shown that S-amino acids fulfil many of the functional characteristics that define a neuro-transmitter molecule (Curtis and Watkins, 1960Curtis and Watkins, 1963Iwata et al., 1982Recasens et al., 1984Do et al., 1986Kilpatrick and Mozley, 1986Baba, 1987Pullan et al., 1987). More specifically, with respect
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