Abstract
The regulation of phosphoinositide hydrolysis by the type 1α metabotropic glutamate receptor (mGluR1α) 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-mGluR1α 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-mGluR1α 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 (Gi/o) and PTX-insensitive stimulatory (Gq/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1α-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.
Footnotes
- Received April 11, 1997.
- Accepted May 7, 1997.
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Send reprint requests to: Dr. R. A. J. Challiss, Department of Cell Physiology & Pharmacology, University of Leicester, P.O. Box 138, Maurice Shock Medical Sciences Building, University Road, Leicester LE1 9HN, United Kingdom. E-mail:jc36{at}le.ac.uk
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This work was supported by the Wellcome Trust of Great Britain. A.M.C. is the recipient of a Wellcome Trust Toxicology Initiative Studentship, and R.S. holds a Medical Research Council Postgraduate Studentship.
- The American Society for Pharmacology and Experimental Therapeutics
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