bay36-7620 and 7-(hydroxyimino)cyclopropan(b)chromen-1a-carbxoylic-acid-ethyl-ester

Homeostatic intrinsic plasticity is a cellular mechanism for maintaining a stable neuronal activity level in response to developmental or activity-dependent changes. Type 1 metabotropic glutamate receptor (mGlu1 receptor) has been widely known to monitor neuronal activity, which plays a role as a modulator of intrinsic and synaptic plasticity of neurons. Whether mGlu1 receptor contributes to the compensatory adjustment of Purkinje cells (PCs), the sole output of the cerebellar cortex, in response to chronic changes in excitability remains unclear. Here, we demonstrate that the mGlu1 receptor is involved in homeostatic intrinsic plasticity through the upregulation of the hyperpolarization-activated current (Ih) in cerebellar PCs. This plasticity was prevented by inhibiting the mGlu1 receptor with Bay 36-7620, an mGlu1 receptor inverse agonist, but not with CPCCOEt, a neutral antagonist. Chronic inactivation with tetrodotoxin (TTX) increased the components of Ih in the PCs, and ZD 7288, a hyperpolarization-activated cyclic nucleotide-gated channel selective inhibitor, fully restored reduction of firing rates in the deprived neurons. The homeostatic elevation of Ih was also prevented by BAY 36-7620, but not CPCCOEt. Furthermore, KT 5720, a blocker of protein kinase A (PKA), prevented the effect of TTX reducing the evoked firing rates, indicating the reduction in excitability of PCs due to PKA activation. Our study shows that both the mGlu1 receptor and the PKA pathway are involved in the homeostatic intrinsic plasticity of PCs after chronic blockade of the network activity, which provides a novel understanding on how cerebellar PCs can preserve the homeostatic state under activity-deprived conditions.

Topics: Action Potentials; Animals; Carbazoles; Chromones; Cyclic AMP-Dependent Protein Kinases; Homeostasis; Naphthalenes; Neuronal Plasticity; Purkinje Cells; Pyrroles; Rats; Receptors, Metabotropic Glutamate; Tetrodotoxin

Recently, new technologies based on biosensors and called label free have been developed. These technologies eliminate the need for using markers and dyes. The authors applied one of these technologies, based on measurement of cell impedance variation, to study the pharmacological profiles of ligands for the cannabinoid receptor 2 (CB2), a Gi-coupled receptor, and for the metabopotropic glutamate receptor 1 (mGluR1), a Gq-coupled receptor. Reference agonists and antagonists/inverse agonists for the 2 receptors were applied to recombinant cell lines and impedance monitored over time. Agonists (JWH133 and CP55940 for CB2; quisqualate, glutamate, 1S-3R-ACPD, and S-3,5-DHPG for mGluR1) triggered a variation of impedance consistent in both potency and efficacy with data obtained using classical assays measuring cAMP or Ca(2+) levels. This effect was not present in the parental nontransfected cell line, confirming specific receptor-mediated response. Application of antagonists (AM630 for CB2; YM298198, SCH1014222, J&J16259685, and CPCCOEt for mGluR1) reduced agonist-induced impedance changes. The only exception was the mGluR1 antagonist BAY367620 that, while active in the Ca(2+) assay, was inactive in the impedance assay. Overall, these results confirm the possibility of using cell impedance-based technology to study the pharmacological profile of ligands acting at G-protein-coupled receptors coupled to different downstream signaling pathways.

Topics: Analgesics; Animals; Benzimidazoles; Biological Assay; Calcium; Cannabinoids; CHO Cells; Chromones; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Cycloleucine; Electric Impedance; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Indoles; Naphthalenes; Neuroprotective Agents; Quinolines; Quisqualic Acid; Receptor, Cannabinoid, CB2; Receptors, Metabotropic Glutamate; Resorcinols; Signal Transduction; Thiazoles

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.

Topics: Animals; Arrestin; Benzoates; Cell Line; Cell Membrane; Chromones; Clathrin; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Humans; Inositol Phosphates; Kidney; Naphthalenes; Protein Isoforms; Protein Transport; Rats; Receptors, Metabotropic Glutamate

R214127 was shown to be a potent and noncompetitive metabotropic glutamate 1 (mGlu1) receptor-selective antagonist. The kinetics and pharmacology of [(3)H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127) binding to rat mGlu1a receptor Chinese hamster ovary (CHO)-dhfr(-) membranes was investigated, as well as the distribution of [(3)H]R214127 binding in rat brain tissue and sections. Specific binding to rat mGlu1a receptor CHO-dhfr(-) membranes was approximately 92% of total and was optimal at 4 degrees C. Full association was reached within 5 min, and [(3)H]R214127 bound to a single binding site with an apparent K(D) of 0.90 +/- 0.14 nM and a B(max) of 6512 +/- 1501 fmol/mg of protein. Inhibition experiments showed that [(3)H]R214127 binding was completely blocked by 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390), (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan-1-on (BAY 36-7620), and 7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), but was not displaced by competitive mGlu1 receptor ligands such as glutamate and quisqualate, suggesting that R214127, NPS 2390, BAY 36-7620, and CPCCOEt bind to the same site or mutually exclusive sites. Experiments using rat cortex, striatum, hippocampus and cerebellum revealed that [(3)H]R214127 labeled a single high-affinity binding site (K(D) approximately 1 nM). B(max) values were highest in the cerebellum (4302 +/- 2042 fmol/mg of protein) and were 741 +/- 48, 688 +/- 125, and 471 +/- 68 fmol/mg of protein in the striatum, hippocampus, and cortex, respectively. The distribution of [(3)H]R214127 binding in rat brain was investigated in more detail by radioligand autoradiography. A high density of binding sites was detected in the molecular layer of the cerebellum. Moderate labeling was seen in the CA3 and dentate gyrus of the hippocampus, thalamus, olfactory tubercle, amygdala, and substantia nigra reticulata. The cerebral cortex, caudate putamen, ventral pallidum, and nucleus accumbens showed lower labeling. The high affinity and selectivity of [(3)H]R214127 for mGlu1 receptors renders this compound the ligand of choice to study the native mGlu1 receptor in brain.

Topics: Adamantane; Allosteric Regulation; Animals; Binding Sites; Binding, Competitive; Brain; Cell Line; Cell Membrane; CHO Cells; Chromones; Cricetinae; Humans; Naphthalenes; Pyrans; Quinolines; Quinoxalines; Quisqualic Acid; Radioligand Assay; Rats; Receptors, Metabotropic Glutamate; Transfection; Tritium