Data shown were performed in triplicate; Mean SEM. activity induced by small molecule positive allosteric modulators of mGlu4 is assessed, the potentiated signaling of mGlu4 is further biased by histamine toward calcium-dependent pathways. These results suggest that Gi/o-coupled mGlus may induce substantial, and potentially unexpected, calcium-mediated signaling NPB events if stimulation occurs concomitantly with activation of Gq receptors. Additionally, our results suggest that signaling induced by small molecule positive allosteric modulators may be substantially biased when Gq receptors are co-activated. This article is part of a Special Issue entitled mGluR = 0.029; unpaired = 0.017; unpaired = 0.76; unpaired = 0.65; unpaired = 0.10; unpaired = 0.0048, unpaired = 0.99; One-way ANOVA). C, The effect of 30 nM (), 100 nM () and 300 nM () histamine in potentiating calcium responses mediated by glutamate in mGluR4/H1/CHO-K1 cells is shown. Maximal responses in vehicle, 30 nM, 100 nM or 300 nM histamine-treated cells were 1548 230, 3390 636, 10099 819, 21261 1356 relative fluorescence units, respectively (*< 0.0001; One-way ANOVA). Data shown were performed in triplicate; Mean SEM. Statistical analysis was performed using GraphPad Prism (La Jolla, CA). 3.3. The potentiated calcium signal can be generalized to other receptor combinations According to our findings, the potentiated calcium response that we observed was mediated by concomitant activation of the Gq-coupled H1 receptor and Gi/o-coupled mGlu4 receptor. We NPB speculated that, if this potentiation was due to a signaling convergence, the phenomenon would extend to other Gq and Gi/o-coupled receptor pairs. To test this hypothesis, we co-expressed mGlu4 with the muscarinic acetylcholine M1 receptor, another Gq-coupled receptor which is also extensively expressed in the CNS. We observed that activation of the M1 receptor via acetylcholine in this mGlu4-co-expressing cell line induced similar glutamate-dependent calcium mobilization compared to cells co-expressing H1 and mGlu4 (Fig. 5A). We also hypothesized that such signaling crosstalk might be generalizable to other Gi/o-coupled mGlu receptors. As carried out for mGlu4, we constructed two mGlu2 cell lines in a CHO-K1 background, one of which expressed mGlu2 alone and the other in combination with H1 receptor. As shown in Fig. 5B, cells expressing mGlu2 alone did not respond to histamine; in contrast, cells CBP co-expressing H1 and mGlu2 exhibited robust potentiation of calcium responses after co-application of histamine and glutamate (Fig. 5C). As shown previously (Rives et al., 2009), signaling of Gi/o and Gq receptors converges on the PLC pathway. To determine if this was also the mechanism of potentiated calcium responses for the receptors examined here, phosphoinositide hydrolysis assays were performed in cells co-expressing mGlu2 and H1 receptors. Consistent with our observations in calcium mobilization assays, histamine dramatically potentiated mGlu2-induced phosphoinositide hydrolysis (Fig. 5D). Open in a separate window Fig. 5 Phospholipase C pathway potentiation extends to additional Gq and Gi/o pairs. A, Acetylcholine (Ach) potentiates calcium responses induced by mGlu4 activation in mGlu4/M1/CHO-K1 cells. 3 nM Ach () or vehicle () control was added to cells in the first add, while increasing concentrations of glutamate were applied 150 s later in the second add and calcium mobilization was measured. Maximal responses in the absence or presence of 3 nM Ach were: 2889 878 NPB vs. 6175 280 relative fluorescence units (*= 0.024; unpaired = 0.86; unpaired = 0.010; unpaired = 0.0005; unpaired potency and efficacy at mGlu4; additionally, VU0155041 displays allosteric agonist activity in some assays (Niswender et al., 2008) and has been proposed to bind to a different site on the mGlu4 receptor compared to PHCCC and 4PAM-2 (Drolet et al., 2011). In these experiments, we added increasing concentrations of each PAM either alone or in combination with histamine in the first addition. As shown in Fig. 7, addition of each PAM alone (white traces, Compound/Histamine Add) resulted in no calcium mobilization, even after glutamate addition (Glutamate Add). Addition of 300 nM histamine alone induced a relatively strong calcium response (dark gray traces); no potentiation of glutamate (second addition) was observed in this case due to the low concentration of glutamate added in these experiments. In contrast, addition of histamine + PHCCC, 4PAM-2, or “type”:”entrez-protein”,”attrs”:”text”:”ADX88178″,”term_id”:”323512724″,”term_text”:”ADX88178″ADX88178 (Fig. 7A, B, and C) resulted in a prolonged calcium transient after the first addition and a very strong potentiation of the glutamate addition. Consistent with its potential to display allosteric agonist activity in some assays, VU0155041 behaved differently from.