Results
β-alanine produced a concentration-dependent, rapid and transient increase in the DMR response when added to CHO-MRGPRD cells, reaching a maximum at approximately 4 minutes (figure 1A). To control for day-to-day variability the response at 4 minutes was used as the report point for calculating % effect relative to the responses observed with 20 mM β-alanine (100% effect) and unstimulated (0%) controls (figure 1B), with a pEC50 of 3.0 ± 0.018 (n = 5). To confirm the β-alanine response was specific to interaction with MRGPRD receptors, the experiment was repeated in untransfected CHO cells, which did not produce a fast transient response, although there was a slow increase in the DMR response at the highest concentration of β-alanine (20 mM) only (figure 1C). A similar protocol was used in a FLIPR Ca2+ assay. In order to control for plate-to-plate and day-to-day variability, data were normalised to the maximal concentration of 20mM β-alanine (100%) and vehicle only (0%) internal plate controls (Figure 1D) with pEC50 value of 4.3 ± 0.02 (n = 6). No response to β-alanine was observed in the control untransfected CHO cells (data not shown).
The β-alanine DMR response was partially, yet significantly inhibited in cells pretreated with PTX (Figure 2A). The remainder of the β-alanine response was inhibited by co-administration with the GαqG protein inhibitor YM-254890 (Xiong et al , 2016) (figure 2B). Addition of YM-254890 in the absence of PTX resulted in a modest, inhibition of the β-alanine DMR response (figure 2C). A different profile of coupling was observed in the FLIPR assay, in which the addition of YM-254890 completely abolished the β-alanine response whereas PTX reduced this by only 50% (Figure 2D). All pEC50 values and maximum effects are reported in table 1.
GABA, Glycine and alamandine were investigated for their agonist activity in both DMR and FLIPR assays. Similar to β-alanine, GABA produced a concentration-dependent, rapid and transient increase in the DMR signal with the peak response between 2 and 4 minutes (figure 3A) however this was considerably less potent than that of β-alanine. There was a modest inhibition in the magnitude of the GABA response following incubation with YM-254890 but no significant change in the pEC50 was observed (figure 3B, figure 3E), while the response was abolished following incubation with PTX (figure 3C, figure 3E). As with β-alanine, the Ca2+ response to GABA was abolished by YM-254890 while with PTX it was reduced by approximately 50% (Figure 3F). Glycine also produced a concentration-dependent, rapid and transient increase in the DMR response with a peak at between 2 to 4 minutes (figure 4A), which was of reduced potency and magnitude than both β-alanine and GABA. The response to glycine in the DMR assay was unaffected by pre-incubation with YM-254890 (figure 4B, figure 4E) but was inhibited by pre-incubation with PTX (figure 4C, figure 4E). In a similar manner to both β-alanine and GABA, the Ca2+response to glycine was abolished by the addition of YM-254890 while PTX reduced this by approximately 50% (figure 4F). All pEC50 values and maximum response data to GABA and glycine are described in table 1. No DMR response was observed by the addition of Alamandine, and there was also very little response in the FLIPR Ca2+ assay; see supplementary data.
Thioridazine hydrochloride, PD123,319 and MU-6840 were investigated for their ability to antagonise MRGPRD activity in both assay formats. Thioridazine hydrochloride inhibited the response to 1mM β-alanine in the FLIPR assay at the highest concentrations used (100µM and 50µM), while little effect was observed in the DMR assay except for inducing a very large negative response at the highest concentration tested (100µM) (figure 5A). This negative response was unaffected by PTX treatment (figure 5B) and was also observed in untransfected CHO cells (figure 5C). Thioridazine hydrochloride was also investigated in a CYQUANT cell viability assay, where at concentrations of 3.16 µM and above it appeared to induce cell death (figure 5E). PD123,319 showed no inhibition of β-alanine, GABA or glycine responses in either assay format (figure 6) in contrast to MU-6840, which inhibited all three agonists (figure 7). All pEC50 values and maximum effects ± SD are presented in table 2. Inhibition of the β-alanine response by MU-6840 was surmountable indicating a competitive mechanism of action, however the slope functions between the two assays were different; a Schild slope not significantly different from 1 was calculated from DMR data (n = 5), pA2 of 6.25 (95% CL, 6.630 - 6.044), whereas the slope in the FLIPR assay was 1.84 ± 0.08, pA2, 6.1 (95% CL, 6.0 – 6.3), n = 6 (Figure 8).