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.
Footnotes
This work was supported by the Biotechnology and Biological Sciences Research Council by a CASE PhD studentship (to S.J.B.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.059170
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ABBREVIATIONS:
- mGlu
- metabotropic glutamate
- NAM
- negative allosteric modulator
- MPEP
- 2-methyl-6-(phenylethynyl)-pyridine
- PAM
- positive allosteric modulator
- DFB
- 3,3′-difluorobenzaldazine
- CPPHA
- N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide
- CDPPB
- 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide
- ADX47273
- S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone
- 5MPEP
- 5-methyl-2-(phenylethynyl)pyridine
- M-5MPEP
- 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine
- CHO
- Chinese hamster ovary
- DMEM
- Dulbecco's modified Eagle's medium
- FBS
- fetal bovine serum
- EBSS
- Earle's balanced salt solution
- DIV
- day(s) in vitro
- ANOVA
- analysis of variance
- FLIPR
- fluorometric imaging plate reader
- NR
- nonresponder
- SP
- single peak
- OS
- oscillatory
- PP
- peak-and-plateau.
- Received July 6, 2009.
- Accepted August 25, 2009.
- © 2009 The American Society for Pharmacology and Experimental Therapeutics
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