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MOLECULAR PHARMACOLOGY 52:406-414 (1997).

Enhanced Type 1 Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

Alan M. Carruthers, R. A. John Challiss, Rajendra Mistry, Ruth Saunders, Christian Thomsen, and Stefan R. Nahorski

Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, UK (A.M.C., R.A.J.C., R.M., R.S., S.R.N.), and Novo Nordisk A/S, Health Care Discovery, 2760 Måløv, Denmark (C.T.)

The regulation of phosphoinositide hydrolysis by the type 1 metabotropic glutamate receptor (mGluR1) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S,3R-dicarboxylic acid resulted in a marked accumulation of [³H]inositol monophosphate (InsP₁) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P₃] mass in a time- and concentration-dependent manner. Pretreatment of BHK-mGluR1 cells with pertussis toxin [ 100 ng/ml, 24 hr] led to a dramatic 12-16-fold increase in the accumulation of [³H]InsP₁ and a 2-fold increase in Ins(1,4,5)P₃ in the absence of added agonist. Although only very low levels (1 µM) of L-glutamate could be detected in medium taken from control and PTX-treated cell monolayers, the PTX-elicited effect on basal [³H]InsP₁ was fully reversed by preincubation of cells in the presence of glutamic-pyruvic transaminase and pyruvate, suggesting that an increased sensitivity to endogenous glutamate was responsible for the apparent agonist-independent activation of phosphoinositidase C (PIC) after PTX treatment. Consistent with this hypothesis, in the presence of glutamic-pyruvic transaminase/pyruvate, the maximal [³H]InsP₁ response to quisqualate was increased by 75%, and the EC₅₀ shifted leftward by 65-fold [log EC₅₀ values (molar), 7.26 ± 0.23 versus 5.45 ± 0.07; n = 4) in PTX-treated compared with control cells. In contrast, antagonist effects on agonist-stimulated [³H]InsP₁ responses were similar in control and PTX-treated BHK-mGluR1 cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (G_i/o) and PTX-insensitive stimulatory (G_q/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.