Quantitative analysis reveals multiple mechanisms of allosteric modulation of the mGlu5 receptor in rat astroglia

Abstract

Positive and negative allosteric modulators (PAMs and NAMs, respectively) of the type 5 metabotropic glutamate (mGlu5) receptor have demonstrable therapeutic potential in an array of neurological and psychiatric disorders. Here, we have used rat cortical astrocytes to investigate how PAMs and NAMs mediate their activity and reveal marked differences between PAMs with respect to their modulation of orthosteric agonist affinity and efficacy. Affinity cooperativity factors (α) were assessed using [(3)H]2-methyl-6-(phenylethynyl)-pyridine (MPEP)-PAM competition binding in the absence and presence of orthosteric agonist, whereas efficacy cooperativity factors (β) were calculated from net affinity/efficacy cooperativity parameters (αβ) obtained from analyses of the abilities of PAMs to potentiate [(3)H]inositol phosphate accumulation in astrocytes stimulated with a submaximal (EC(20)) concentration of orthosteric agonist. We report that whereas 3,3'-difluorobenzaldazine (DFB) and 3-cyano-N-(1,3-diphenyl-1H-prazol-5-yl)benzamide (CDPPB) primarily exert their allosteric modulatory effects through modifying the apparent orthosteric agonist affinity at the astrocyte mGlu5 receptor, the effects of S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone (ADX47273) are mediated primarily via efficacy-driven modulation. In [(3)H]MPEP-NAM competition binding assays, both MPEP and 2-(2-(3-methoxyphenyl)ethynyl)-5-methylpyridine (M-5MPEP) defined similar specific binding components, with affinities that were unaltered in the presence of orthosteric agonist, indicating wholly negative efficacy-driven modulations. It is noteworthy that whereas M-5MPEP only partially inhibited orthosteric agonist-stimulated [(3)H]inositol phosphate accumulation in astrocytes, it could completely suppress Ca(2+) oscillations stimulated by quisqualate or (S)-3,5-dihydroxyphenylglycine. In contrast, MPEP was fully inhibitory with respect to both functional responses. The finding that M-5MPEP has different functional effects depending on the endpoint measured is discussed as a possible example of permissive allosteric antagonism.

Fig. 1.

Saturation binding of [³H]MPEP to membranes prepared from rat cortical astrocytes (A) or adult rat cerebral cortex (B). To characterize the MPEP binding site, membranes were incubated with different concentrations of [³H]MPEP (0.1–40 nM) in the absence (Total) or presence (NSB) of 1 μM MPEP (see Materials and Methods). Single representative experiments are shown, with similar data being obtained on at least two additional occasions.

Fig. 2.

Effects of orthosteric binding site occupancy on the specific binding of [³H]MPEP in the presence of DFB, CPPHA, CDPPB, or ADX47273 in membranes prepared from rat cortical astrocytes (left) or rat cortex (right). The abilities of DFB (A), CPPHA (B), CDPPB (C), or ADX47273 (D) to affect specific [³H]MPEP binding was assessed in the absence and presence of quisqualate (30 μM). Data are presented as means ± S.E.M. of three to seven separate experiments performed in duplicate. Quantitative and statistical analyses of these data are presented in Table 1.

Fig. 3.

Concentration-dependent effects of DFB (A), CPPHA (B), CDPPB (C), and ADX47273 (D) on quisqualate-stimulated [³H]IP_x accumulation in rat cortical astrocytes. Astrocytes were preincubated (10 min) with increasing concentrations of mGlu5 receptor PAMs followed by stimulation with an approximate EC₂₀ quisqualate concentration (50 nM). Concentration-dependent increases in [³H]IP_x accumulation caused by mGlu5 receptor PAMs are shown in each panel compared with concentration-dependent [³H]IP_x accumulations stimulated by quisqualate alone. Data are shown as means ± S.E.M. for four to five separate experiments, each performed in duplicate.

Fig. 4.

Displacement of specific [³H]MPEP binding by mGlu5 receptor NAMs, MPEP, and M-5MPEP in membranes prepared from rat cortical astrocytes (A) or rat cortex (B). The indicated concentrations of NAMs were added to membranes immediately before the addition of [³H]MPEP (10 nM final concentration; see Materials and Methods). Effects of orthosteric binding site occupancy on the displacement of specific [³H]MPEP binding by MPEP (C) and M-5MPEP (D) was also investigated in rat cortex membranes. [³H]MPEP binding was performed in the absence and presence of quisqualate (30 μM). Data are shown as means ± S.E.M. of three to six separate experiments performed in duplicate. Quantitative and statistical analyses of these data are presented in Table 3.

Fig. 5.

Concentration-dependent effects of MPEP on Ca²⁺ oscillations stimulated by glutamate, quisqualate, or DHPG in rat cerebrocortical astrocytes. Representative traces showing the effects of incrementally increasing concentrations of MPEP (0.01–0.3 μM) on Ca²⁺ oscillations elicited by the continuous presence of glutamate (100 μM; A), quisqualate (10 μM; B), or DHPG (10 μM; C). Summary data are also shown for the concentration-dependent suppression of Ca²⁺ oscillations by MPEP when cells were stimulated by glutamate, quisqualate, or DHPG (D). Mean pIC₅₀ (M) values for inhibition of glutamate-, quisqualate- or DHPG-stimulated Ca²⁺ oscillation frequency by MPEP were 7.90 ± 0.06, 7.71 ± 0.07 and 7.82 ± 0.06, respectively. Data are shown as means ± S.E.M. from at least 50 individual cells recorded over at least three separate experiments.

Fig. 6.

Concentration-dependent effects of M-5MPEP on Ca²⁺ oscillation frequency stimulated by glutamate, quisqualate, or DHPG in rat cerebrocortical astrocytes. Representative traces showing the effects of incrementally increasing concentrations of M-5MPEP (0.01–10 μM) on Ca²⁺ oscillations elicited by the continuous presence of glutamate (100 μM; A), quisqualate (10 μM; B), or DHPG (10 μM; C). Summary data are also shown for the concentration-dependent suppression of Ca²⁺ oscillations by M-5MPEP when cells were stimulated by glutamate, quisqualate, or DHPG (D). Mean pIC₅₀ (M) values for inhibition of glutamate-, quisqualate-, or DHPG-stimulated Ca²⁺ oscillation frequency by M-5MPEP were 6.61 ± 0.08, 6.77 ± 0.13, and 6.38 ± 0.15, respectively. Data are shown as means ± S.E.M. from at least 50 individual cells recorded over at least 3 separate experiments.

Fig. 7.

Effects of MPEP and M-5MPEP on agonist-stimulated [³H]IP_x accumulation in rat cortical astrocytes. Increasing concentrations of mGlu5 receptor NAMs were preincubated for 10 min before orthosteric agonist addition. Data are expressed as the percentage of maximal [³H]IP_x accumulation on stimulation with quisqualate (10 μM; A) or DHPG (10 μM; B). Data shown are means ± S.E.M. for four to seven separate experiments performed in duplicate. Also shown are quisqualate-stimulated concentration-response curves performed in the absence or presence of 0.03, 0.1, 0.3, or 1 μM MPEP (C) or 1, 3, 10, or 30 μM M-5MPEP (D). Either MPEP or M-5MPEP was added 10 min before the addition of quisqualate at the concentrations indicated. Data are shown as means ± S.E.M. for three separate experiments performed in duplicate.