Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids.

In this exciting paper, the authors identify a molecular pathway by which opioids trigger an itch response as observed in patients treated with morphine. The authors show that this involves a novel splice isoform of the mu-opioid receptor that appears to form a dimer with the gastrin-releasing peptide receptor. Morphine administration results in the internalization of the receptor heterodimer, leading to activation of phospholipase C and ultimately itch.

The authors thus show that morphine-induced itch occurs at the same time as morphine-induced analgesia, but through distinct intracellular pathways. This then may yield novel strategies to selectively interfere with opioid-induced itch while sparing its analgesic properties. One additional neat aspect of the work is that morphine has been traditionally thought to be unable to trigger mu-opioid receptor internalization. The authors' work shows that this is not true for the mu-opioid receptor 1D splice variant.

This article addresses molecular differences between opioid-induced itch and analgesia and concludes they reside at the level of splice variants of the mu-opioid receptor MOR-1. In addition to providing insights into these two opioid-induced actions, this article also establishes the importance and relevance of MOR-1 alternative splicing and the importance of opioid receptor heterodimerization to generate alternative, pharmacologically distinct targets.

One of the problematic side effects of opioid use is pruritus, despite the fact that it often does not get the attention it deserves. The question addressed in the current article is whether or not the receptor targets mediating morphine-induced scratching (MIS; an indication of pruritus) and morphine-induced analgesia (MIA) can be differentiated. In a series of elegant studies, the authors present a very strong case that MIS is mediated through a splice variant (MOR-1D) of MOR-1. They carried this forward to demonstrate that the role of MOR-1D likely involves heterodimerization with the gastrin-releasing peptide receptor (GRPR) using a variety of biochemical approaches. The role of these receptors in behavior was demonstrated through exon mapping, documenting the role of exon 4 in MIA, and not MIS, and exon 9 in MIS, and not MIA, and knockout models.

In addition, the paper has great importance with regards to the relevance of alternative splicing of MOR-1. Although differences at the anatomical and biochemical levels have been reported previously for these splice variants, the current article presents compelling evidence for different phenotypic roles of the different splice variants. At the same time, it also emphasizes the importance of heterodimerization in extending the number of potential pharmacological targets. The authors also demonstrate that the dimerization likely involves only a small number of C-terminal amino acids -- the structural difference between MOR-1 and MOR-1D. This is intriguing in that there are a number of additional MOR-1 splice variants that also differ at the C-terminus, with wide variations in the length of the different sequences. It will be interesting to see whether these sequences also impact function through heterodimerization.

Pruritus is a very common complication of intrathecal morphine administration. This paper demonstrates that the mu-opioid receptor (MOR) isoform MOR1D plays a crucial role in intrathecal morphine-induced itch. Once activated by intrathecal narcotics, MOR1D forms heterodimers with gastrin-releasing peptide receptor (GRPR) in the neurons in the superficial dorsal horn to activate the downstream phospholipase C (PLC) pathway, which subsequently enhances the intracellular calcium level. Interestingly, Liu et al. have demonstrated that blocking of the MOR1D-GRPR association does not diminish the analgesic effect of morphine administration.

Although itch is a well-known and often observed side effect of intrathecally applied opioids, the underlying mechanisms are not well understood. This paper by Liu and colleagues provides a potential neuronal mechanism by which intrathecal application of opioids can lead to the sensation of itch in humans.

Previous work by this group on gastrin-releasing peptide and its receptor (GRPR) showed that GRPR-expressing dorsal horn neurons play a crucial role in the sensation of itch. In the present paper, the authors investigate the role of mu-opioid receptor (MOR) and GRPR in opioid-induced itch. They provide several lines of experimental evidence suggesting that MOR1D, a splice variant of MOR, can form heterodimers with GRPR and that opioids can activate this complex. Importantly, downregulation of MOR1D, knockout of GRPR, blocking the downstream signaling molecules of GRPR (PLCbeta1/3 and IP3R3) or preventing the interaction between MOR1D and GRPR inhibited opioid-induced itch but not opioid analgesia.

These and previous findings from the same group {1,2} suggest that the sensations of pain and itch are likely mediated through distinct afferent spinal pathways. This concept is consistent with recent findings regarding the role of VGlut2 in itch and pain {3,4} and the crucial role of inhibitory interneurons on itch sensation {5}. However, direct electrophysiological recordings from GRPR-expressing cells in the superficial dorsal horn will be necessary to determine if these cells are selective or specific for itch-producing stimuli.

This remarkable study used diverse methodologies to crack a clinical problem that has both vexed and perplexed a generation of anesthesiologists, pain physicians and their patients. Opioid-induced itch (pruritus) may seem little more than a trivial (dare I say ticklish) problem but it is distressing and affects the vast majority of patients receiving spinal or epidural opioids (e.g. morphine and fentanyl). The clinical application of these drugs includes labor analgesia, intraoperative and postoperative analgesia and chronic pain therapy. While not a dangerous complication of spinal opioids, it is certainly the commonest. Tens of millions of people have been literally scratching away at this irritating problem every year.

The authors, from the intriguingly named "Center for the Study of Itch" at Washington University School of Medicine Pain Center, St. Louis, MO, noted previous studies that demonstrated that mice lacking neurons expressing gastrin releasing peptide receptor (GRPR) in the spinal cord had almost complete absence of the scratch response to known pruritic stimuli. As opioid-induced pruritus is most prominent when administered spinally, they hypothesized that spinal opioids induce pruritus by activating spinal GRPR signaling pathways.

They first demonstrated that morphine-induced scratching (MIS) occurs independently of morphine-induced analgesia (MIA). They showed that MIA and MIS have different dose-response curves in mice and markedly different time courses of action. MIA was impaired by both acute and chronic morphine pre-exposure, while MIS was not.

Next, they used exon-specific siRNA knockdown studies in mouse spinal cord and observed the effects on MIS and MIA. Knockdown of either exon 1 (contained in most of the isoforms of the mu-opioid receptor [MOR] including MOR-1) or exon 9 (contained in isoforms 1C, 1D and 1E) markedly reduced MIS. However, siRNA knockdown of exon 4 (contained in MOR-1) or exon 7 (contained in 1C and 1E) failed to significantly reduce MIS. The knockdown of the MOR-1D protein in spinal cord by exon 9 siRNA was verified by Western blot. These results indicate that exon 9 is critical for MIS but not for MIA. Using identical methodology, they also showed that exon 4 is critical for MIA but not for MIS. From this step, they were able to establish that spinal MOR-1D is a MIS-specific isoform, while MOR-1 is specific for MIA.

Next they used double immunostaining to demonstrate co-expression of MOR-1D and GRPR in the dorsal horn spinal cord. This co-expression did not occur in double immunostaining for either MOR-1 and MOR-1D or for MOR-1 and GRPR. They then demonstrated that MIS was almost completely abolished in GRPR knockout mice, and that MIA was unaffected. Intrathecal injection of a GRPR antagonist inhibited MIS but not MIA. This dichotomy between drug-induced itch and drug-induced antinociception in GRPR knockout mice was also demonstrated for other opioid agonists.

Using co-immunoprecipitation techniques, both in vivo and in vitro, they demonstrated that GRPR and MOR-1D physically interact by a process of receptor heterodimerization, observed in other G protein-coupled receptors. Using receptor internalization assays, they demonstrated that morphine induced the internalization of GRPR in cells co-expressing both MOR-1D and GRPR. Interestingly, GRP was not able to induce MOR-1D internalization in these cells; this is seemingly a non-reciprocal relationship. Morphine was not able to induce the internalization of GRPR in cells expressing GRPR alone or in cells co-expressing MOR-1 and GRPR. The internalization of MOR-1D or GRPR was inhibited by naloxone in a dose-dependent manner.

They went on to assess calcium signals in cells co-expressing MOR-1D and GRPR; both morphine and GRP induced calcium spikes in these cells, but not in cells co-expressing MOR-1 and GRPR or in cells expressing MOR-1D alone. The morphine-induced calcium spike was blocked by a GRPR antagonist and to a lesser degree by naloxone. Furthermore, molecular and pharmacologic inhibition of PLCbeta3 and IP3R3, downstream effectors of GRPR, specifically blocked MIS but not MIA.

To further investigate PLC and IP3R mechanisms in MIS, they specifically ablated GRPR neurons with bombesin-saporin. They observed a complete loss of PLCbeta3 expression and a significant reduction in IP3R3 and MOR-1D mRNA, suggesting co-expression of GRPR with these proteins. Going back to the spinal siRNA knockdown technique, knockdown of PLCbeta3 and IP3R3 also inhibited MIS but not MIA.

Finally, they identified a likely pharmacological target. The seven amino acid tail of MOR-1D differentiates it structurally from MOR-1. They synthesized a fusion peptide targeting this amino acid sequence, hypothesizing that it would induce competitive antagonism for the GRPR interaction. As hypothesized, they observed that, when this fusion peptide was co-administered intrathecally together with morphine, it induced a specific inhibition of MIS with no impact on either MIA or GRP-induced scratch. The authors termed this last result 'remarkable'. I think that this description should be used for this entire elegant portfolio, which is a case of taking a clinical problem to the lab, using molecular, cellular, biochemical and behavioral assessments and then producing a clear therapeutic target.

They end by speculating whether MOR-1D may be responsible for other opioid-mediated side effects. They argue convincingly that the successful uncoupling of the desired analgesic effect and an unwanted side effect in this study highlights the need to further explore the function of individual isoforms of MOR. By implication, it may be possible to uncouple other, potentially more serious, complications of opioid analgesia. Their "Center for the Study of Itch" may yet become a "Center for the Study of Respiratory Depression". Apart from being a conceptual and technological masterpiece and a major advance in our understanding of opioid action, this contribution is likely to transform the analgesic experience for millions of patients.