Rapid tolerance to morphine in the myenteric neurons of the small intestine is independent of β-arrestin-2 and mediated by PKC

Background and PurposeG-protein biased μ-opioid agonists against β-arrestin-2 activation are being investigated to reduce adverse effects. While opioid tolerance is strongly linked to the development of dependence, there is a dissociation between the two phenomena in the gut as tolerance does not develop to opioid-induced constipation, but diarrhea still manifests upon withdrawal. Here, we investigated the mechanism by which morphine tolerance in the small intestine develops.Experimental ApproachMechanism of morphine tolerance in the small intestine was evaluated in vivo and at the neuronal level. Whole-cell patch clamp electrophysiology was used to investigate tolerance in individual ileum myenteric neurons. Rate of morphine tolerance development in the small intestine was assessed against peripheral antinociception and whole gut transit.Key ResultsTolerance develops to inhibition of small intestinal motility after one day of morphine exposure, and is more rapid compared to peripheral antinociception and constipation in chronic morphine-treated mice. Morphine tolerance was reversed by the PKC inhibitor, Tamoxifen, but not by β-arrestin-2 deletion. Similarly, β-arrestin-2 deletion did not prevent morphine tolerance to inhibition of neuronal excitability in ileum myenteric neurons. However, neuronal tolerance was attenuated by inhibiting PKC.Conclusions and ImplicationsUnlike antinociceptive tolerance, rapid morphine tolerance in the small intestine is independent of β-arrestin-2 but is PKC-mediated. These findings reveal a potential mechanism for differences in the rates of tolerances to opioids, implicate myenteric neurons of the ileum as the primary cause for opioid-induced withdrawal effects and suggest that undesired gastrointestinal effects will persist with biased opioid agonist use.SummaryWhat is already known:Tolerance does not develop to chronic-opioid-induced constipation but diarrhea is produced upon withdrawalNovel G-protein biased agonists that preclude β-arrestin-2 activation at the μ-opioid receptor are in developmentWhat this study adds:Morphine tolerance in the ileum develops systemically and in individual myenteric neurons independent of β-arrestin-2Morphine tolerance in the small intestine develops before antinociception and is reversed by PKC inhibitionClinical significance:Clinical use of G-protein biased opioid agonists will not prevent tolerance development in the ileumTolerance in ileum myenteric neurons might be the basis of opioid-induced withdrawal in the gut