title: Effects of positive allosteric modulators on single-cell oscillatory Ca2+ signaling initiated by the type 5 metabotropic glutamate receptor.
abstract: Agonist stimulation of the type 5 metabotropic glutamate (mGlu5) receptor initiates robust oscillatory changes in cytosolic Ca2+ concentration ([Ca2+]i) in single cells by rapid, repeated cycles of phosphorylation/dephosphorylation of the mGlu5 receptor, involving protein kinase C and as-yet-unspecified protein phosphatase activities. An emergent property of this type of Ca2+ oscillation-generating mechanism (termed "dynamic uncoupling") is that once a threshold concentration has been reached to initiate the Ca2+ oscillation, its frequency is largely insensitive to further increases in orthosteric agonist concentration. Here, we report the effects of positive allosteric modulators (PAMs) on the patterns of single-cell Ca2+ signaling in recombinant and native mGlu5 receptor-expressing systems. In a Chinese hamster ovary cell-line (CHO-lac-mGlu5a), none of the mGlu5 receptor PAMs studied [3,3'-difluorobenzaldazine (DFB), N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl]phenyl}-2-hydroxy-benzamide (CPPHA), 3-cyano-N-(1, 3-diphenyl-1H-prazol-5-yl)benzamide (CDPPB), S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone (ADX47273)], stimulated a Ca2+ response when applied alone, but each PAM concentration-dependently increased the frequency, without affecting the amplitude, of Ca2+ oscillations induced by glutamate or quisqualate. Therefore, PAMs can cause graded increases (and negative allosteric modulator-graded decreases) in the Ca2+ oscillation frequency stimulated by orthosteric agonist. Initial data in rat cerebrocortical astrocytes demonstrated that similar effects of PAMs could be observed in a native cell background, although at high orthosteric agonist concentrations, PAM addition could much more often be seen to drive rapid Ca2+ oscillations into peak-plateau responses. These data demonstrate that allosteric modulators can "tune" the Ca2+ oscillation frequency initiated by mGlu5 receptor activation, and this might allow pharmacological modification of the downstream processes (e.g., transcriptional regulation) that is unachievable through orthosteric ligand interactions.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.
authors.forename: Sophie J
authors.initials: SJ
authors.lastname: Bradley
authors.forename: Jeannette M
authors.initials: JM
authors.lastname: Watson
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D016466:CHO Cells; D017610:Calcium Compounds / Q000032:analysis; D020013:Calcium Signaling / Q000187:drug effects*; D002540:Cerebral Cortex / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D018698:Glutamic Acid / Q000494:pharmacology; D006834:Hydrazines / Q000494:pharmacology; D010069:Oxadiazoles / Q000494:pharmacology; D010087:Oxides / Q000032:analysis; D010797:Phthalimides / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000502:physiology
chemical_list: C481447:3,3'-difluorobenzaldazine; C494553:3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide; D001549:Benzamides; D017610:Calcium Compounds; D006834:Hydrazines; C481448:N-(4-chloro-2-((1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl)phenyl)-2-hydroxybenzamide; D010069:Oxadiazoles; D010087:Oxides; D010797:Phthalimides; D010880:Piperidines; D011720:Pyrazoles; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; C533862:S-(4-fluorophenyl)-(3-(3-(4-fluorophenyl)-(1,2,4)-oxadiazol-5-yl)piperidin-1-yl)methanone; D018698:Glutamic Acid; C016538:lime