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.