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).