Discussion
Developing new analgesics with less adverse effects is challenging
(reviewed in Yekkirala et al., 2017; Machelska and Celik, 2018).
Activation of peripheral opioid receptors by site-restricted opioid
administration was shown to produce strong analgesia in both acute and
chronic pain models as well as in patients with arthritis and
postoperative pain (reviewed in Machelska and Celik, 2018; Martínez and
Abalo, 2020). Additionally, animal studies using conditional opioid
receptor knockout in primary afferent Nav1.8 nociceptors clearly
demonstrated the essential role of peripheral opioid receptors
(Gaveriaux-Ruff et al., 2011; Weibel et al., 2013). Accordingly, the
selective activation of peripheral MOR on DRG neurons innervating
injured (inflamed) tissues appears to be a promising strategy to inhibit
pain while avoiding central side effects. NFEPP is an opioid with a
lower dissociation constant (pKa) that was shown to bind
and activate MORs more effectively at low pH values. Therefore, even
though NFEPP is presumably distributed throughout the body (including
the CNS) after systemic (intravenous) application, it only activates
MORs in peripheral inflamed tissues where the pH is more acidic
(reviewed in Stein, 2018).
In this study, we expanded our understanding of NFEPP’s antinociceptive
effects by investigating MOR signaling in transfected HEK cells and
sensory neurons. Opioid receptors exert their intracellular actions
through G-proteins, which interact with various cellular effector
systems. First, we found that NFEPP produced more efficient G-protein
activation at low pH, while fentanyl induced comparable G-protein
activation at both physiological and low pH values, as shown by the
[35S]-GTPγS binding experiments. Opioid receptor
activation can result in blockade of membrane VDCCs, which are essential
for the excitation of sensory neurons (reviewed in Machelska and Celik,
2018; Weiss and Zamponi, 2021). To verify that NFEPP acts through the
same mechanisms, we examined NFEPP-induced inhibition of inward calcium
currents at both physiological (7.4) and acidic (6.5) pH values. Our
experiments revealed that the electrical excitation of depolarized
sensory neurons was more efficiently reduced following NFEPP treatment
at acidic pH (in contrast to pH 7.4). However, fentanyl, a conventional
opioid, attenuated Ca2+ currents to similar degrees at
both pH values. By using naloxone, we confirmed that the effects of
NFEPP and fentanyl were mediated by opioid receptors. Together, these
findings support our previous studies where fentanyl, possibly due to
its higher pKa value (8.44) and comparable protonation,
was equally effective at both pH values (Spahn et al., 2017, 2018). In
contrast, NFEPP (pKa = 6.82) showed increased G-protein
activation and VDCC inhibition at acidic conditions, where an increase
in protonation due to lower pH improves NFEPP-MOR interaction (Spahn et
al. 2017). To examine whether the dissociation of G-protein subunits
differs between pH values following NFEPP or fentanyl treatment, we
blocked the GDP-GTP exchange at Gαi/o subunits using
PTX, and we inhibited Gβγ subunits using gallein in our
patch clamp analysis. Gβγ subunits were previously shown
to directly deactivate VDCCs following MOR activation (reviewed in Proft
and Weiss, 2015). Here, we have shown that NFEPP-induced inhibition of
inward calcium currents were dependent on Gβγ subunits.
Consistent with its indistinguishable effect on Gαversus Gβγ subunits (Lu and Ikeda, 2016), PTX also
blocked Ca2+ currents, indicating that, by blocking
GDP-GTP exchange, consequent Gαiβγ dissociation is also
impaired. Apparently, the extracellular pH changes did not significantly
affect intracellular activation of G-proteins since the dose ranges of
inhibitors (PTX, gallein) were similar for both opioid agonists and both
pH values.
Further validation of our electrophysiological data was performed using
Ca2+ imaging experiments. Ca2+signals were only reduced in NFEPP-treated DRG neurons at pH 6.5.
Fentanyl, however, significantly reduced Ca2+ signals
at both pH values, indicating no difference of its efficacy between
inflamed and non-inflamed environments. Control experiments using
naloxone and high potassium confirmed that the signals were modulated by
opioid receptors and derived from influx of Ca2+ into
neurons, similar to previous studies (Hochstrate et al., 1995). To
examine direct effects of low pH (without drug treatment), we
investigated VDCC activity at pH 6.5. Both calcium imaging and patch
clamp experiments (data not shown) demonstrated that pH alone did not
affect Ca2+ signals/ currents at low pH values.
Additionally, the ability of NFEPP to phosphorylate MOR under different
pH conditions was examined. Our western blot analysis showed that NFEPP
induced the strongest MOR phosphorylation at pH 6.0. The signal was most
pronounced at MOR residues T370 and S375 and decreased with increasing
pH levels. In comparison, fentanyl caused similar phosphorylation
signals of MOR regardless of increasing pH values. Notably, the
antibodies used in our experiments only detect intracellular
phosphorylation sites at the C-terminal of MOR (Mann et al., 2015).
Assuming that nonspecific pH effects would produce similar
phosphorylation signals for all pH values and both agonists, our
findings strongly suggest that the pH-dependent signals produced by
NFEPP were due to extracellular pH changes. Importantly, fentanyl
apparently stimulates MOR independently from extracellular pH, while
NFEPP activates MOR preferentially at low pH values.
In summary, our results show that during both NFEPP and fentanyl
treatment, VDCCs are directly modulated by Gβγ subunits
dissociated from Gαi/o. However, MOR signaling induced
by NFEPP is more effective at low pH. Given the known decrease of
extracellular pH in injured tissues (Stein, 2018) and the co-expression
of MOR and VDCC in sensory neurons (Proft and Weiss, 2015), our study
uncovers the neural mechanisms underlying the antinociceptive effects of
NFEPP in injured tissues, and strongly suggests that the enhanced
efficacy of NFEPP in acidic milieu is dependent on extra- rather than
intracellular effects on MOR function.