PHARMACOLOGICAL CHARACTERIZATION OF METABOTROPIC GLUTAMATE RECEPTORS COUPLED TO PHOSPHOLIPASE-D IN THE RAT HIPPOCAMPUS

1 Phospholipase D (PLD) is the key enzyme in a signal transduction pat hway leading to the formation of the second messengers phosphatidic ac id and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR ag onists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2 The mGluR-selective agonist ( 1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induce d a concentration-dependent (10-300 mu M) activation of PLD in the hip pocampus,neocortex, and striatum, but not in the cerebellum. The effec t was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3 The rank order of potencies for ago nists stimulating the PLD response was: arboxycyclopropyl)-glycine>(1S ,3R)-ACPD>L-cysteine sulphinic acid>L-aspartate>L-glutamate. L-(+)-2-A mino-4-phosphonobutyric acid and the ionotropic glutamate receptor ago nists N-methyl-D-aspartate, lpha-amino-3-hydroxy-5-methyl-4-isoxazolep ropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyp henylglycine (100-300 mu M), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 mu M (1S ,3R)-ACPD. 4 (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a com petitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 mu M (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13- dibutyrate (PDBu), activators of protein kinase C, at 1 mu M had a sti mulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-ind uced phosphoinositide hydrolysis. The protein kinase C inhibitor, stau rosporine (1 and 10 mu M) reduced PLD activation induced by 1 mu M PDB u but not by 100 mu M (1S,3R)-ACPD. 6 Our results suggest that PLD-lin ked mGluRs in rat hippocampus may be distinct from any known mGluR sub type coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.