Regulation and pharmacological manipulation of the type 5 metabotropic glutamate (mGlu5) receptor

Compelling evidence has emerged to indicate that astrocytes play crucial roles in neurotransmitter signalling, in addition to CNS homeostatic functions. Astrocytes can communicate with each other and with neurons, giving rise to the concept of the ‘tripartite synapse’. Expression of type 5 metabotropic glutamate (mGlu5) receptors in astrocytes is now well-established. Agonist stimulation of mGlu5 receptors initiates robust oscillatory changes in cytosolic Ca2+ concentration in single cells by rapid, repeated cycles of phosphorylation/dephosphorylation of the mGlu5 receptor, involving PKC and unidentified protein phosphatase activities in recombinant and native mGlu5 receptor-expressing systems. Each of the mGlu5 receptor positive allosteric modulators (PAMs) studied (DFB, CPPHA, CDPPB and ADX47273), failed to stimulate a Ca2+ response when applied alone, but increased the frequency of Ca2+ oscillations induced by glutamate or other orthosteric agonists. PAMs and negative allosteric modulators (NAMs) caused respectively graded increases and decreases in the Ca2+ oscillation frequency stimulated by orthosteric agonist. 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) not achievable through orthosteric ligand interactions. Further investigation into the mechanism of action of PAMs revealed marked differences with respect to their modulation of orthosteric agonist affinity and efficacy. Thus, while DFB and CDPPB primarily exert their allosteric modulatory effects through modifying orthosteric agonist affinity, effects of ADX47273 are primarily mediated through an efficacy-driven modulation. Investigating the mechanisms underlying mGlu5 receptor-mediated Ca2+ oscillations in astrocytes has provided further evidence for the involvement of PKC(s) and protein phosphatase(s) in the rapid phosphorylation/dephosphorylation cycles occurring at the C-terminal of mGlu5 receptors, a process termed ‘dynamic uncoupling’. This work has been extended using specific siRNA knockdown to elucidate the potential PKC isoenzyme(s) that determine mGlu5 receptor regulation of Ca2+ oscillation frequency in astrocytes.EThOS - Electronic Theses Online ServiceGBUnited Kingdo