In the patch clamp experiments, the DRG cells initially rested in the
whole-cell voltage clamp mode at a constant -80 mV holding potential.
When depolarized to +10 mV (100 ms), VDCCs opened to allow
Ca2+ ions to flow into the cell inducing an inward
current. NFEPP treatment dose-dependently decreased VDCC currents at
both pH values (6.5 and 7.4) but to different degrees. At pH 6.5, the
most effective NFEPP dose (100 µM) attenuated Ca2+currents by 41.8 %. This effect was significantly stronger than the
effect of NFEPP at pH 7.4 (28.8%) (Figure 2a). In contrast to NFEPP,
the dose-response curves upon fentanyl treatment were not different and
the most effective fentanyl dose (100 µM) attenuated +10 mV-induced
Ca2+ currents to the same degree
(~39%) at both pH 7.4 and pH 6.5 (Figure 2b). These
findings show that NFEPP is more effective at low pH, whereas the
conventional opioid agonist fentanyl is equally effective at both acidic
and physiological pH values. Naloxone dose-dependently recovered the +10
mV-induced VDCC currents during NFEPP treatment. The effects of NFEPP
were completely reversed by the highest naloxone dose (100 µM) at both
pH values (Figure 3a). This dose of naloxone also abolished fentanyl
effects at both pH values (Figure 3b).
To investigate the role of G-protein subunits in detail, we initially
used PTX which prevents the replacement of GDP by GTP at the
Gαi subunits and precludes subsequent
Gαβγ dissociation (Lu and Ikeda, 2016). PTX blocked
NFEPP’s effect in a dose-dependent manner (Figure 4a), fully reversing
the +10 mV-induced VDCC currents at the highest PTX dose (100 ng/ml).
The effect of fentanyl was also blocked in a dose-dependent manner after
PTX incubation (Figure 4b). Because PTX does not allow to distinguish
between the involvement of Gα versus Gβγsubunits (Lu and Ikeda, 2016), we sought to selectively block
Gβγ subunits by gallein (Lu and Ikeda, 2016). Gallein
dose-dependently decreased NFEPP-induced effects and fully recovered +10
mV-induced Ca2+ currents at the highest dose at both
pH values (Figure 5a). Fentanyl’s effects were also decreased and fully
blocked by the most effective dose of gallein at both pH values (Figure
5b). Together, these results indicate that the effects of both opioid
agonists on VDCCs are mediated via activation and subsequent
dissociation of heterotrimeric G-protein complexes at both pH values.
We then performed Ca2+ imaging experiments to
corroborate our electrophysiological results. Under our recording
conditions, both NFEPP and fentanyl decreased the Fura ∆ ratio 340/380,
i.e. the Ca2+ signals were reduced. Naloxone (100 µM)
completely reversed the effect of fentanyl (Figure 6b, c). However, in
NFEPP-treated neurons, the Ca2+ signal was
significantly lower at pH 6.5 when compared to pH 7.4 (Figure 6a, c). We
also examined whether the extracellular pH by itself had a role in the
decrease of Ca2+ signals. During short (60 s) (Figure
7a) or long (20 min) (Figure 7b) term pH changes, there was no
difference in the Fura ∆ ratio 340/380 at either pH 6, 6.5, 7 or 7.4.
This indicates that the pH difference by itself does not influence
Ca2+ signals in the cells and that NFEPP and fentanyl
were solely responsible for the decrease. The control experiments using
high potassium indicated that the signals indeed derived from the influx
of extracellular Ca2+ into neurons (Figure 7).
Finally, we examined the effects of NFEPP on MOR phosphorylation. MOR
expressing HEK293 cells were treated with either NFEPP or fentanyl at pH
6.0 - 8 (Figure 8a). The strongest NFEPP-mediated phosphorylation signal
was detected at pH 6.0. This signal diminished with increasing pH values
(Figure 8b). In contrast, fentanyl-induced phosphorylation was similar
at all pH values (Figure 8c).