Selective up-regulation of functional mu-opioid receptor splice variants by chronic opioids

We recently reported (Verzillo, et al. J. Neurochem: 130, 790-796, 2014) that chronic systemic morphine selectively up-regulates mRNA encoding two C-terminal μ-opioid receptor (MOR) splice variants, MOR-1C1 and MOR-1B2 (MOR-1B2/-1C1). Given the known disconnects between changes in levels of mRNA and corresponding protein, it is essential to directly demonstrate that chronic opioid treatment elevates functional MOR-1B2/-1C1 protein prior to inferring relevance of these MOR variants to spinal opioid tolerance mechanisms. Accordingly, we investigated the ability of chronic opioid exposure to up-regulate MOR protein in Chinese hamster ovary cells stably transfected with rat MOR variants MOR-1B2, MOR-1C1, or MOR-1 (considered to be the predominant MOR). Findings revealed that chronic treatment with the clinically relevant opioids morphine, oxycodone and hydrocodone substantially up-regulated membrane MOR-1B2/-1C1 protein. This up-regulation was abolished by the protein synthesis inhibitor cycloheximide, eliminating contributions from receptor redistribution. The increment in MOR-1B2/-1C1 protein was paralleled by a significant increment in opioid agonist-stimulated GTPγS-binding (reflective of increased aggregate MOR G protein coupling) indicating that up-regulated MOR-1B2/-1C1 represented functional receptors. Strikingly, these tolerance-associated adaptations of MOR-1B2/-1C1 differed considerably from those of MOR-1. Antithetical regulation of MOR-1B2/-1C1 and MOR-1 by chronic opioids has significant implications for the design of new therapeutic agents to counteract opioid analgesic tolerance and accompanying addiction. Since chronic opioids induce MOR-1B2/-1C1 up-regulation in spinal cord of males, but not females, elucidating cellular compartments and intracellular pathways mediating MOR-1B2/-1C1 up-regulation and defining their unique signaling attributes would enable a precision medicinal approach to pain management and addiction therapy. In the spinal cord of males, but not females, chronic morphine up-regulates mRNA encoding two mu-opioid receptor (MOR) variants, MOR-1B2 and MOR-1C1 (MOR-1B2/-1C1). We now demonstrate that chronic treatment with the clinically relevant opioids morphine, hydrocodone or oxycodone up-regulates MOR-1B2/-1C1 functional protein, which is dependent on de novo protein synthesis. Findings underscore the importance of unique signaling attributes of MOR variants to sexually dimorphic tolerance mechanisms.