Hervé Schaffhauser

Characterization of [3H]-(2S,2′R,3′R)-2-(2′,3′-dicarboxy- cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes

The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]‐(2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine ([3H]‐DCG IV), was characterized in rat mGlu2 receptor‐transfected CHO cell membranes. [3H]‐DCG IV binding was pH‐dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg−1 protein. Binding was not sensitive to Na+‐dependent glutamate uptake blockers or Cl−‐dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N‐methyl‐D‐aspartic acid (NMDA), (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]‐DCG IV binding. Of the compounds observed to inhibit [3H]‐DCG IV binding, the most potent were the recently described selective group II agonist, (+)‐2‐aminobicyclo‐[3.1.0]hexane‐2,6‐dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2‐amino‐2‐(2‐carboxycyclopropan‐1‐yl)‐3‐(dibenzopyran‐4‐yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G‐protein‐coupled receptor, guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) inhibited [3H]‐DCG IV binding in a concentration‐dependent manner, with an IC50 value of 12 nM. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]‐DCG IV binding and potencies obtained for agonist activity in a GTPγ35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)‐2‐amino‐3‐phosphonopropionic acid (L‐AP3), L(+)‐2‐amino‐5‐phosphonopentanoic acid (L‐AP5), 3‐((RS)‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (R‐CPP), N‐acetyl‐L‐aspartyl‐L‐glutamic acid (NAAG) and (RS)‐α‐methylserine‐O‐phosphate (MSOP).

Distribution and abundance of metabotropic glutamate receptor subtype 2 in rat brain revealed by [3H]LY354740 binding in vitro and quantitative radioautography: Correlation with the sites of synthesis, expression, and agonist stimulation of [35S]GTPγs binding

Until recently, there was a lack of selective radioligands for the subtypes of metabotropic glutamate (mGlu) receptors. [3H]LY354740 ((+)‐2‐aminobicyclo[3,1,0]hexane‐2,6‐dicarboxylic acid), a selective agonist for group II receptors (mGlu2 and ‐3, which are negatively coupled to cAMP production), has now been used to map their brain distribution and abundance by in vitro binding and quantitative radioautography. The selective cation dependence of its binding allowed the discrimination between mGlu2 and mGlu3 receptor labeling. Thus, in the presence of Ca2+ and Mg2+ ions, the agonist bound selectively to mGlu2 receptors as evidenced by: 1) the correlative distribution and abundance of binding sites (highest in the lacunosum moleculare of the hippocampus and lowest in white matter) with mGlu2 receptor mRNA and protein revealed by in situ hybridization histochemistry and immunohistochemistry, respectively; 2) its selective pharmacology; and 3) the distribution of LY354740‐stimulated [35S]GTPγS binding (25–97% above basal, according to the brain region), revealing G protein‐coupled receptor coupling to Gi proteins. Nonspecific binding (in the presence of 10 μM DCG‐IV, a group II‐selective, mGlu2‐preferring, receptor agonist) was <10% of total. In adjacent sections, the distribution of binding sites for [3H]DCG‐IV was very similar. This extensive study paves the way for investigations of the regional expression and regulation of mGlu2 receptors in human CNS diseases, such as Alzheimers disease, which may reveal their functional roles and identify potential therapeutic drug targets. Indeed, it has recently been demonstrated (Higgins et al. [2004] Neuropharmacology 46:907–917) that pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent. J. Comp. Neurol. 487:15–27, 2005.

Multiple pathways for regulation of the KCl-induced [3H]-GABA release by metabotropic glutamate receptors, in primary rat cortical cultures

In rat cortical primary cultures, group II- and III-metabotropic glutamate receptor-selective agonists concentration-dependently reduced KCl-induced [3H]GABA release, with IC50 values of 11 nM for LY354740, 80 nM for L(+)-2-amino-4-phosphonobutyric acid (L-AP4), 180 nM for DCG-IV, and 330 nM for L-SOP. The group II antagonists, LY341495 and EGLU, reversed the effect of LY354740, and the group III antagonist MTPG reversed the effect of L-AP4. In the presence of omega-conotoxin GVIA, LY354740 inhibited the remaining [3H]GABA release, whereas L-AP4 was inactive. In contrast, in the presence of nifedipine, L-AP4 inhibited the remaining [3H]GABA release, but LY354740 was no longer active. The PKA inhibitor, H89, blocked the effects of both L-AP4 and LY354740, whereas the PKC inhibitor Ro 31-8220 blocked only the effect of LY354740. Both Ro 31-8220 and H89 reduced the [3H]GABA release to 60% of control. In whole-cell, voltage-clamp experiments, LY354740 and L-AP4 inhibited voltage-gated calcium channel currents with IC50 values of 28 nM and 22 microM, respectively. The results suggest that, in these cells, KCl-induced [3H]GABA release is modulated by two different mechanisms, one involving group II receptors and a direct control of the Ca2+ channel activity, and the other mediated by group III receptors and possibly involving a regulation located downstream of the Ca2+ channel activation.

mGluR-evoked augmentation of receptor-mediated cyclic AMP formation in neonatal and adult rat striatum

The effects of selective agonists at group I, II and III metabotropic glutamate receptors (mGluRs) on adenosine A2 receptor‐mediated cyclic AMP formation were compared in cross‐chopped slices of adult and neonatal (8 days old) rat striatum, in the presence of 1 u ml−1 adenosine deaminase. The group II selective agonist, (2S,1R,2R,3R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV), elicited a potentiation of 5′‐N‐ethylcarboxamidoadenosine (NECA)‐stimulated cyclic AMP production with similar potencies in adult (EC50 value 122±35 nM) and neonatal (EC50 value 285±6 nM) brain. In contrast, the group I selective agonist (S)‐dihydroxyphenylglycine ((S)‐DHPG) augmented the NECA cyclic AMP response in neonatal striatum (EC50 value 9±1 μM), but at a concentration of 100 μM, (S)‐DHPG failed to affect the NECA response in adult striatal slices. The potentiation evoked by (S)‐DHPG was specific for group I mGluRs as (2S,3S,4S,)‐2‐methyl‐2‐(carboxycyclopropyl)glycine (MCCG), a group II antagonist, was ineffective on the (S)‐DHPG (100 μM) response at a concentration (500 μM) which reversed a similar augmentation elicited by DCG‐IV (300 nM). Furthermore, a protein kinase C inhibitor (Ro 31‐8220, 10 μM) markedly reversed the effect of (S)‐DHPG without affecting the response to DCG‐IV. The mGluR agonist (2S,3S,4S,)‐α‐(carboxycyclopropyl)glycine (L‐CCG‐I), elicited a greater potentiation of NECA‐stimulated cyclic AMP production in neonatal striatum in comparison with that observed in adult rat brain. Moreover, EC50 values obtained from adult and neonatal striatum were 2±1 μM and 9±1 μM, respectively. These differences in potency might reflect co‐activation of both group I and group II mGluRs by L‐CCG‐I in neonatal striatum. Distinct patterns of mGluR expression in various brain areas might account for previous conflicting data on the nature of the mGluR able to evoke such potentiated responses.