Introduction
Opioids are the strongest painkillers to date. Clinically used opioid
drugs (e.g. morphine, fentanyl) mainly target the mu opioid receptor
(MOR), a G-protein coupled receptor (GPCR). The activation of MORs in
the peripheral (PNS) and central nervous system (CNS) produces strong
pain relief, but their activation in the CNS also results in serious
side effects including addiction and respiratory depression (Imam et
al., 2018). Recently, our group designed a new MOR agonist,
(±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide
(NFEPP), with a lower acid dissociation constant (pKa)
(Spahn et al., 2017; Rosas et al., 2019; Lešnik et al., 2020; Ray et
al., 2020). NFEPP selectively activated peripheral MORs in inflamed
tissues at low pH values (common in most painful injuries) whereas a
conventional opioid (fentanyl) was equally active at both low and normal
pH values (Spahn et al., 2017; Rodriguez-Gaztelumendi et al., 2018;
Stein, 2018; Del Vecchio et al., 2019; Jiménez-Vargas et al., 2022).
Targeting opioid receptors in the PNS at the site of injury avoids CNS
side effects, while still contributing to strong analgesia, as
previously demonstrated in preclinical and clinical studies: (i) locally
applied opioids relieve severe clinical pain and a large proportion of
the analgesic effects of systemically administered opioids is mediated
by opioid receptors in the PNS (Jagla et al., 2014; Machelska and Celik,
2018; Martínez and Abalo, 2020); (ii) input from peripheral sensory
neurons is essential in numerous pain syndromes (Berta et al., 2017;
Sexton et al., 2018); (iii) upon injury, MOR expression is upregulated
in peripheral sensory neurons and the perineurial barrier is disrupted,
eventually increasing accessibility of the drugs’ target (reviewed in
Machelska and Celik, 2018; Jeske, 2019).
So far, the mechanisms underlying NFEPP-induced antinociception were not
examined in sufficient detail. At the level of sensory neurons, voltage
dependent Ca2+ channels (VDCC) play a major role in
the generation and inhibition of pain. These channels regulate the
excitation of neurons by allowing Ca2+ influx into the
neuron (Lu and Ikeda, 2016; Weiss and Zamponi, 2021). Following the
activation of MOR and dissociation of the heterotrimeric G-protein
complex, Gβγ subunits block VDCC function, leading to
decreased excitability of the sensory neuron (Proft and Weiss, 2015; Lu
and Ikeda, 2016). Finally, MOR is phosphorylated and internalized
(Machelska and Celik, 2018; Jeske, 2019; Mann et al., 2015).
In this study, we investigated the effects of NFEPP at acidic and
physiological pH in comparison to the conventional agonist fentanyl on
G-protein activation, on the activity of endogenous VDCCs and on MOR
phosphorylation.