Animal models of chronic pain: Overexpression of group I metabotropic glutamate receptors attenuates receptor signaling

Abstract

Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system acting through interactions with ionotropic glutamate receptor channels and metabotropic glutamate (mGlu) G protein-coupled receptors (GPCRs). The 8 different mGlu receptors modulate neuronal excitability both pre- and postsynaptic, and at the level of glial cells. Substantial evidence has been accumulating supporting the role of group I mGlu receptors (mGlu1 and mGlu5) in nociceptive transmission. Group I receptor antagonism, by means of pharmacophores, receptor specific antibodies or antisense probes, overcomes hyperalgesia and allodynia in animal models of inflammatory and neuropathic pain suggesting that mGlu1/5 antagonists may represent an important target for chronic pain therapy. To develop in vitro tests for the human mGlu1 and mGlu5 receptors, HEK293, CHO, COS7, NIH3T3, SK-N-MC, HeLa, and L929 cells were transiently transfected with expression constructs of both receptors. The expression was analyzed by immunoblotting. Even though westernblot analysis demonstrated that the receptors were highly expressed in several cell types, hardly any calcium release could be measured in response to glutamate in a FLIPR assay for most cells. To further characterize the hmGlu1a expressing cells we performed receptor-binding assays using [3H]R214127 as described previously (Lavreysen et al., 2003). For the hmGlu5 expressing cells we used [3H]MPEP. These receptor-binding assays performed on membranes prepared from the transiently transfected cells conformed the expression and integrity of both receptors. Comparing the results of the immunoblot, receptor binding assay, and functional assay obtained with the seven different transiently transfected cells we found a novel principle for group I mGlu receptor signaling. We are currently studying the underlying mechanism of this process and evaluate whether this is also true for other classes of GPCRs. Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system acting through interactions with ionotropic glutamate receptor channels and metabotropic glutamate (mGlu) G protein-coupled receptors (GPCRs). The 8 different mGlu receptors modulate neuronal excitability both pre- and postsynaptic, and at the level of glial cells. Substantial evidence has been accumulating supporting the role of group I mGlu receptors (mGlu1 and mGlu5) in nociceptive transmission. Group I receptor antagonism, by means of pharmacophores, receptor specific antibodies or antisense probes, overcomes hyperalgesia and allodynia in animal models of inflammatory and neuropathic pain suggesting that mGlu1/5 antagonists may represent an important target for chronic pain therapy. To develop in vitro tests for the human mGlu1 and mGlu5 receptors, HEK293, CHO, COS7, NIH3T3, SK-N-MC, HeLa, and L929 cells were transiently transfected with expression constructs of both receptors. The expression was analyzed by immunoblotting. Even though westernblot analysis demonstrated that the receptors were highly expressed in several cell types, hardly any calcium release could be measured in response to glutamate in a FLIPR assay for most cells. To further characterize the hmGlu1a expressing cells we performed receptor-binding assays using [3H]R214127 as described previously (Lavreysen et al., 2003). For the hmGlu5 expressing cells we used [3H]MPEP. These receptor-binding assays performed on membranes prepared from the transiently transfected cells conformed the expression and integrity of both receptors. Comparing the results of the immunoblot, receptor binding assay, and functional assay obtained with the seven different transiently transfected cells we found a novel principle for group I mGlu receptor signaling. We are currently studying the underlying mechanism of this process and evaluate whether this is also true for other classes of GPCRs.