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 (EC 50 = 70 µ M ), mGluR2 (EC 50 = 25 µ M ), and mGluR4 (EC 50 = 324 µ M ). l ‐Cysteine sulfinate, 1‐aminocyclobutane‐ trans ‐1,3‐dicarboxylate, l ‐cysteine, and dl ‐ threo ‐3‐methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC 50 values of 43, 64, 463, and 488 µ M , respectively, and displaced l ‐[ 3 H]glutamate binding from membranes prepared from these cells with respective IC 50 values of 48, 44, 79, and 139 µ M . However, d ‐aspartate, l ‐ trans ‐pyrrolidine‐2,4‐dicarboxylate, l ‐ threo ‐3‐hydroxyaspartate, and l ‐aspartate‐β‐hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC 50 values of 73, 54, 57, and 430 µ M ) but did not displace l ‐[ 3 H]glutamate binding. These compounds inhibited Na + ‐dependent l ‐glutamate uptake into baby hamster kidney cells with IC 50 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 m M l ‐ trans ‐pyrrolidine‐2,4‐dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µ M ) 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.