Supersensitivity of Human Metabotropic Glutamate 1a Receptor Signaling in L929sA Cells

Abstract

The effect of antagonist pretreatment on the signaling properties of the human metabotropic glutamate 1a (hmGlu1a) receptor was examined in stably transfected L929sA cells. Pre-exposure of hmGlu1a receptor-expressing cells to the mGlu1 receptor antagonists (S)-4-carboxy-3-hydroxyphenylglycine and 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester dramatically enhanced subsequent glutamate-induced phosphoinositide hydrolysis and intracellular [Ca 2ϩ ] rise. We found clear indications that the antagonist-mediated enhancement of glutamate-evoked mGlu1a receptor signaling is caused by the development of mGlu1a receptor supersensitivity: the potency of glutamate was increased by 3-fold after 24 h antagonist pretreatment and the potency of the antagonists was significantly decreased in antagonist-pretreated cells. The kinetic profile of the antagonist-mediated enhancement showed that the maximal increase in intracellular [Ca 2ϩ ] was already reached after 30-min pretreatment, suggesting that de novo receptor synthesis is not involved in the process of mGlu1a receptor supersensitization. Glutamate-mediated phosphoinositide hydrolysis increased up to 24 h after antagonist treatment. Although it seemed likely that the hmGlu1a receptor could desensitize after activation by endogenously present glutamate, removal of glutamate from the extracellular medium with GPT resulted in a much smaller enhancement of glutamate responsiveness. Moreover, the magnitude of antagonist-mediated receptor supersensitivity was much larger than the magnitude of agonist-induced receptor desensitization. These results suggest that antagonist-evoked mGlu1 receptor supersensitivity is not merely the result of a blockade of agonist-induced desensitization. Finally, we found that antagonist pretreatment doubled the amount of receptors at the cell surface. Our findings are the first lines of evidence that prolonged antagonist treatment can supersensitize the hmGlu1a receptor. In view of the potential therapeutic application of mGlu1 receptor antagonists, it will be important to know whether these phenomena occur in vivo.So far, eight subtypes of the family of mGlu receptors have been cloned (mGlu1 to mGlu8 receptors). These receptors can be further subdivided into three groups based on their amino acid sequence homology, signal transduction mechanisms, and pharmacological profiles. Group I mGlu receptors consist of subtypes 1 and 5. Splice variants have been found for both the mGlu1 receptor (mGlu1a to mGlu1f receptor) and mGlu5 receptor (mGlu5a, mGlu5b, and mGlu5d receptors). Stimulation of group I mGlu receptors activates phosphoinositide hydrolysis, giving rise to the generation of the intracellular messengers diacylglycerol and inositol trisphosphate. This coupling in turn results in protein kinase C activation and Ca 2ϩ mobilization from the endoplasmic reticulum (Aramori and Nakanishi, 1992). Group II (mGlu2 and mGlu3 receptor) and group III mGlu receptors (mGlu4, -6, -7, and -8 receptors) both inhibit adenylate cyclase...

Metabotropic Glutamate 1 Receptor: Current Concepts and Perspectives

“…Antagonist pretreatment of cells expressing the recombinant human mGlu1␣ receptor causes a marked increase in both potency and efficacy of glutamate stimulation (Lavreysen et al,2002). This particular form of "supersensitivity," which is induced by both noncompetitive and competitive mGlu1 receptor antagonists, cannot be simply described by the inhibition of receptor desensitization mediated by the endogenous glutamate (Lavreysen et al, 2002).…”

“…This particular form of "supersensitivity," which is induced by both noncompetitive and competitive mGlu1 receptor antagonists, cannot be simply described by the inhibition of receptor desensitization mediated by the endogenous glutamate (Lavreysen et al, 2002). Antagonist pretreatment increases cell surface expression of mGlu1␣ receptors, suggesting recruitment or stabilization of receptors at the plasma membrane (Lavreysen et al, 2002).…”

[3H]R214127: A Novel High-Affinity Radioligand for the mGlu1 Receptor Reveals a Common Binding Site Shared by Multiple Allosteric Antagonists

“…CHO-dhfr Ϫ cells stably expressing the rat mGlu1a, -2, -3, -4, -5, and -6 receptor were a kind gift from S. Nakanishi (Tokyo University, Tokyo, Japan) and were grown in Dulbecco's modified Eagle's medium with GlutaMAX I with 10% heat-inactivated dialyzed fetal calf serum, 0.4 mM L-proline, 0. (Lavreysen et al, 2002). The same procedure was followed for CHO-dhfr Ϫ cells expressing the rat mGlu1a receptor.…”

“…IP accumulation was measured as described previously (Lavreysen et al, 2002). Briefly, cells were seeded at 30,000 cells/well in 24-well plates and were labeled with 2.5 Ci/ml [myo-2-3 H (N)]inositol overnight.…”

“…L929sA cells stably expressing the human mGlu1a receptor were obtained as described previously (Lavreysen et al, 2002) and were cultured in GlutaMAX I medium supplemented with 10% heat-inactivated dialyzed fetal calf serum, 0.1 mg/ml streptomycin sulfate, and 100 units/ml penicillin. CHO-dhfr Ϫ cells stably expressing the rat mGlu1a, -2, -3, -4, -5, and -6 receptor were a kind gift from S. Nakanishi (Tokyo University, Tokyo, Japan) and were grown in Dulbecco's modified Eagle's medium with GlutaMAX I with 10% heat-inactivated dialyzed fetal calf serum, 0.4 mM L-proline, 0.…”