Discussion
In our study, we used morphine as a control to investigate whether β-arrestin2 is involved in fentanyl-induced respiratory depression by inhibiting the upstream signaling molecule GRK2 and knocking out β-arrestin2. From the aforementioned results, inhibition of GRK2 and β-arrestin2 knockout significantly alleviated morphine-induced respiratory depression in mice, which is consistent with the results reported in the literature (Raehal, Walker & Bohn, 2005). However, fentanyl-induced respiratory depression was not affected by GRK2 inhibition and β-arrestin2 knockout, suggesting that the mechanism of respiratory depression induced by fentanyl and morphine is different.
However, Kliewer et al. concluded from measurements of the respiratory rate that morphine, similarly as fentanyl, induced significant respiratory depression in βarr2−/− mice (Kliewer et al., 2020). We obtained similar results for the respiratory rate, but morphine and fentanyl had different effects on the inspiratory time and expiratory time after GRK2 was inhibited. The findings suggest that β-arrestin2, a key signaling protein, is involved in the regulation of respiratory depression induced by morphine but not by fentanyl.
Studies illustrated that morphine and fentanyl differ in their μ-opioid receptor (MOR) binding interactions and that different ligand–receptor interactions directly lead to differences in side chain conformational changes, which induce different activation modes (Ricarte, Dalton & Giraldo, 2021; Vo, Mahinthichaichan, Shen & Ellis, 2021). In addition, the effect of opioid binding to MORs also appears to depend on the location of opioids and MORs. Studies found that blocking presynaptic Kv+ channels with α-dendrotoxin or the postsynaptic GIRK channels with tertiapin-Q, morphine and fentanyl combined with MOR produced significant differences (Morgan, Tran, Wescom & Bobeck, 2020). This difference was sufficient to cause the drugs to activate different intracellular signaling pathways.
In addition, as opioid agonists, morphine and fentanyl broadly activate the same intracellular signaling pathways, but there may be subtle differences. Some studies found that PDE4 inhibitors can reverse the respiratory depressant effects of morphine by increasing intracellular cAMP levels in neuronal cells (Kimura, Ohi & Haji, 2015; Mosca, Ciechanski, Roy, Scheibli, Ballanyi & Wilson, 2014; Ruangkittisakul & Ballanyi, 2010); however, other studies revealed that PDE4 inhibitors do not reverse respiratory depression induced by fentanyl (Liang, Yong & Su, 2018). There are obvious differences in signal transduction, and the biological mechanism of respiratory depression might be different. In addition, in the β-arrestin signaling pathway, β-arrestin binds to Src, thereby increasing GRK degradation and inhibiting the initial termination of G protein-coupled receptor signaling (Laporte et al., 1999). In addition, Src complexes with β-arrestin to promote non-G protein-dependent signaling pathways, such as the activation of ERK and initiation of premitotic transcription processes (Luttrell & Lefkowitz, 2002). β-arrestin also independently mediates the activation of ERK (Daaka, 2004). The location at which activated ERKs are ultimately distributed within cells is related to the pathways that mediate ERK activation. The location of activated p-ERK in turn affects the activation of transcription factors involved in its downstream signaling, making it important for the entire cell. The difference in the p-ERK distribution induced by morphine and fentanyl might be one cause of their different mechanisms of respiratory depression.
The concept of “biased agonism” is important for the development of new drugs with greater efficacy and fewer side effects, and the study by Raehal et al. was central to the early development of new opioid drugs using this concept. However, experimental results increasingly confirm that “G protein-biased” opioids induce respiratory depression (Hertz, 2018; Hill et al., 2018), the concept of “biasing agitation” has also been called into question (Gregory, 2020). Our study suggests that whether opioid-induced respiratory depression is associated with the β-arrestin signaling pathway may depend on the type of opioid, and that other pathways or proteins are involved in this effect remain to be investigated.
In conclusion, our study confirmed that, unlike morphine, fentanyl does not induce β-arrestin2–biased respiratory depression, and the mechanisms of respiratory depression induced by the two drugs might differ. At the same time, this study highlighted the need to explore more precise bias signaling pathways and new ideas for the development of rescue drugs for respiratory depression caused by different opioids.