L-glutamate uptake inhibitors may stimulate phosphoinositide hydrolysis in baby hamster kidney cells expressing mGluR1a via heteroexchange with L-glutamate without direct activation of mGluR1a.
The functional efficacies of inhibitors of L-glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. L-Serine-O-sulfate was an agonist at mGluR1a (EC50 = 70 microM), mGluR2 (EC50 = 25 microM), and mGluR4 (EC50 = 324 microM). L-Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, L-cysteine, and DL-threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC50 values of 43, 64, 463, and 488 microM, respectively, and displaced L-[3H]glutamate binding from membranes prepared from these cells with respective IC50 values of 48, 44, 79, and 139 microM. However, D-aspartate, L-trans-pyrrolidine-2,4-dicarboxylate, L-threo-3-hydroxyaspartate, and L-aspartate-beta-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC50 values of 73, 54, 57, and 430 microM) but did not displace L-[3H]glutamate binding. These compounds inhibited Na(+)-dependent L-glutamate uptake into baby hamster kidney cells with IC50 values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this L-glutamate transporter, was significantly attenuated in the presence of L-glutamate decarboxylase (EC 4.1.1.15) or L-alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mM L-trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 +/- 0.2 microM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous L-glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.
Department of Receptor Neurochemistry, Novo Nordisk A/S, Måløv, Denmark.
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Suzdak
D000410:Alanine Transaminase / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000031:analogs & derivatives / Q000494:pharmacology; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003515:Cycloleucine / Q000031:analogs & derivatives / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D003998:Dicarboxylic Acids / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology*; D005968:Glutamate Decarboxylase / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney; D016202:N-Methylaspartate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*
D003998:Dicarboxylic Acids; D018691:Excitatory Amino Acid Antagonists; D010716:Phosphatidylinositols; D011759:Pyrrolidines; D018094:Receptors, Metabotropic Glutamate; D003515:Cycloleucine; C036361:1-amino-1,3-dicarboxycyclopentane; C011274:3-hydroxyaspartic acid; C024848:beta-aspartylhydroxamic acid; D001224:Aspartic Acid; D018698:Glutamic Acid; D016202:N-Methylaspartate; D001216:Asparagine; C083486:pyrrolidine-2,4-dicarboxylic acid; D000410:Alanine Transaminase; D005968:Glutamate Decarboxylase; D003545:Cysteine