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.

Fig. 1.

Effects of the PAMs DFB, CPPHA, CDPPB, and ADX47273 on the frequency of Ca²⁺ oscillations in CHO-lac-mGlu5a cells. Representative traces showing the response of single CHO-lac-mGlu5a cells to perfusion with l-glutamate (100 μM; A, C, E, and G) or quisqualate (30 μM; B, D, F, and H) for 5 min followed immediately by the same concentration of agonist plus DFB (100 μM; A and B), CPPHA (3 μM; C and D), CDPPB (10 μM; E and F), or ADX47273 (10 μM; G and H) perfused for a further 5 min. Data are representative of at least 50 individual cells recorded over at least 3 separate days.

Fig. 2.

Concentration-dependent effects of CDPPB and ADX47273 on the frequency of Ca²⁺ oscillations stimulated by l-glutamate or quisqualate in CHO-lac-mGlu5a cells. Representative traces showing the effects of increasing concentrations of CDPPB (0.01, 0.1, 1, 10 μM; A and C) or ADX47273 (0.01, 0.1, 1, 10 μM; E and G) on Ca²⁺ oscillations elicited by glutamate (100 μM; A and E) or quisqualate (30 μM; C and G). Concentration-response curves showing the mean number of oscillations per minute when cells were stimulated with glutamate (100 μM; B and F) or quisqualate (30 μM; D and H) plus increasing concentrations of CDPPB (B and D) or ADX47273 (F and H). Data are shown as means ± S.E.M. from 25 individual cells recorded over 4 separate days. Mean pEC₅₀ (M) values for facilitation of glutamate and quisqualate responses were 6.46 ± 0.26 and 6.95 ± 0.27 for CDPPB and 6.32 ± 0.26 and 6.71 ± 0.39 for ADX47273, respectively.

Fig. 3.

Effects of DFB on the threshold for glutamate evoked on Ca²⁺ oscillations in CHO-lac-mGlu5a cells. A, representative trace showing the effect of stimulating cells with increasing concentrations of glutamate (each concentration applied for 3 min). B, a representative trace showing responses to increasing glutamate concentrations in the presence of DFB (30 μM). C, mean data showing the changes in oscillation frequency that occur when cells were stimulated with increasing concentrations of glutamate in the absence or presence of DFB (30 μM). Data are shown as means ± S.E.M. for at least 25 individual cells over at least three experiments.

Fig. 4.

Effects of the positive allosteric modulator, ADX47273, on orthosteric agonist-stimulated Ca²⁺ responses in CHO-lac-mGlu5a cells. Data show the percentage of the total number of cells analyzed that gave an NR, SP, OS, or PP response when stimulated with l-glutamate (100 μM; A) or quisqualate (30 μM; B) in the absence and presence of ADX47273 (10 μM). Data are shown as means ± S.E.M. from at least 50 individual cells recorded over 4 separate days. Criteria for classification of cell responses into NR, SP, OS, and PP subgroups were identical to those defined by Atkinson et al. (2006).

Fig. 5.

The positive allosteric modulators DFB, CPPHA, CDPPB, and ADX47273 possess no intrinsic agonist activity in the absence of orthosteric stimulation. Maximal concentrations of DFB (100 μM; A), CPPHA (3 μM; B), CDPPB (10 μM; C), and ADX47273 (10 μM; D) were perfused on to CHO-lac-mGlu5a cells for 5 min, followed by simultaneous perfusion of quisqualate (30 μM) plus each respective modulator. Traces are representative from at least 20 individual cells recorded over 3 separate days.

Fig. 6.

Comparison of effects of MPEP and 5MPEP on Ca²⁺ responses in single and populations of CHO-lac-mGlu5a cells. Representative traces showing the effects of MPEP (100 nM; A) or 5MPEP (30 μM; B) on Ca²⁺ oscillations elicited by glutamate (100 μM) in CHO-lac-mGlu5a cells. Traces shown are representative of at least 50 cells recorded over 3 separate days. FLIPR cell population responses for glutamate-stimulated Ca²⁺ concentration-response curves performed in the absence or presence of 10, 30, or 100 nM MPEP (C) or 0.1, 1, or 10 μM 5MPEP (D). MPEP and 5MPEP were added 30 min before challenge with glutamate at the concentrations indicated. Data are shown as means ± S.E.M. for three separate experiments performed in duplicate.

Fig. 7.

5MPEP abolishes the positive modulatory effects of ADX47273, DFB, or CDPPB on orthosteric agonist-stimulated Ca²⁺ oscillation frequency in CHO-lac-mGlu5a cells. Cells were perfused with glutamate (100 μM) for 5 min, followed by glutamate (100 μM) plus PAM for 5 min, and then glutamate (100 μM), PAM and 5MPEP (30 μM) (A). Perfusion periods with glutamate ± PAM ± 5MPEP followed on from each other without any washout between additions. A representative trace showing the effects of 5MPEP on the Ca²⁺ oscillation frequency elicited by glutamate (100 μM) plus ADX47273 (10 μM) is shown (A). Mean data for each PAM are also shown: ADX47273 (10 μM; B), DFB (100 μM; C), and CDPPB (10 μM; D). Histograms show means ± S.E.M. for 20 individual cells recorded over 4 separate days, with statistically significant differences (∗∗∗, P < 0.001) determined by one-way ANOVA.

Fig. 8.

5MPEP does not block the positive modulatory effect of CPPHA on orthosteric agonist-stimulated Ca²⁺ oscillation frequency in CHO-lac-mGlu5a cells. Cells were perfused with glutamate (100 μM) for 5 min, followed by glutamate (100 μM) plus CPPHA (3 μM) for 5 min, and then glutamate (100 μM), CPPHA (3 μM), and 5MPEP (30 μM) (A). Perfusion periods with glutamate ± CPPHA ± 5MPEP followed on from each other without any washout between additions. A representative trace showing the effects of 5MPEP on the Ca²⁺ oscillation frequency elicited by glutamate plus CPPHA is shown (A), whereas B presents mean data. Data are shown as means ± S.E.M. for 35 individual cells recorded over 7 separate days, with statistically significant differences (∗∗∗, P < 0.001) determined by one-way ANOVA.

Fig. 9.

Allosteric modulator site pharmacology at the mGlu5 receptor. Concentration-dependent effects of ADX47273, MPEP, 5MPEP, or M-5MPEP on glutamate (100 μM) evoked Ca²⁺ oscillations in CHO-lac-mGlu5a cells are summarized [pEC₅₀/IC₅₀ (M) values: ADX47273, 6.33 ± 0.13; MPEP, 7.69 ± 0.14 M; M-5MPEP, 6.26 ± 0.21]. Data are shown are means ± S.E.M. for at least 25 cells recorded over at least 3 separate days. Note that ordinate values shown are normalized to the oscillation frequency evoked by stimulation with glutamate (100 μM) alone.

Fig. 10.

Modulatory effects of MPEP and 5MPEP on l-glutamate-stimulated Ca²⁺ oscillations in rat cerebrocortical astrocytes. Representative trace (A) showing the pattern of Ca²⁺ oscillations evoked by glutamate (100 μM) and its attenuation by coaddition of increasing concentrations of MPEP (0.01–0.3 μM). Summary data are shown (B) comparing the effects of MPEP on glutamate-stimulated Ca²⁺ oscillation frequency in astrocytes and CHO-lac-mGlu5a cells. Data are shown as means ± S.E.M. for at least 25 individual cells over at least three separate experiments. The lack of effect of 5MPEP (10 μM) on glutamate-stimulated Ca²⁺ oscillations in astrocytes is also illustrated by a representative trace (C).

Fig. 11.

Effects of the mGlu5 receptor PAM CDPPB on glutamate-stimulated Ca²⁺ responses in rat cerebrocortical astrocytes. Under the experimental conditions used here [rapidly perfused (5 ml/min) cells on coverslips], addition of CDPPB (10 μM) alone did not evoke a Ca²⁺ response, in contrast to addition of glutamate (100 μM) (A). Representative traces are also shown illustrating the effect of CDPPB (10 μM) on Ca²⁺ responses evoked by a range of glutamate concentrations (C, 0.3 μM; E, 1 μM; G, 3 μM; I, 10 μM; K, 100 μM). Mean data are shown as the percentage of the total number of cells analyzed that gave NR, SP, OS, or PP response for each condition. These latter data are determined from analysis of least 50 individual astrocytes over at least three separate experiments. Note that CDPPB reduced the threshold for glutamate-stimulated Ca²⁺ oscillations (C), and although this PAM increased oscillation frequency at low orthosteric agonist concentrations (E, F and G, H), it transformed the response from oscillatory to peak-plateau at high agonist concentrations (I, J and K, L).