Bovine Adrenal Medulla Peptide Research Articles

Key domains and residues of the receptor MRGPRX1 recognizing the peptide ligand BAM8-22

Mas-related G protein-coupled receptors (Mrgprs) are a newly discovered class of G protein-coupled receptors consisting of more than 50 members in recent years. MRGPRX1 can be activated by bovine adrenal medulla peptide 8–22 (BAM8–22), triggering Ca2+ influx and then causing pain and itch. It is very important for the discovery of analgesic and antipruritic drugs to elucidate the molecular mechanism of MRGPRX1 recognizing BAM8–22. Here, we identified the functional domains and residues of the receptor MRGPRX1 activating BAM8–22 through molecular model, mutation and living cell calcium imaging. The molecular docking predicted that BAM8–22 interacted with N-terminal, TM4, TM5, TM6 and ECL3 of MRGPRX1. Both ECL3 and TM6 domains were further revealed to play a critical role in the BAM8–22-induced MRGPRX1 activation, whereas TM3 region performed a secondary function. Moreover, the mutation F237A of MRGPRX1 completely lost the activation ability of BAM8–22. These results were consistent with the cryogenic electron microscopy (cryo-EM) structure of MRGPRX1-Gαq in complex with BAM8–22 reported most recently. Taken together, our work shows insights into the molecular mechanism of the interaction between the receptor MRGPRX1 and the peptide agonist BAM8–22, and will also provide some valuable clues for the design of analgesic and antipruritic drugs targeting MRGPRX1.

MMrgprA3/mMrgprC11/hMrgprX1: Potential therapeutic targets for allergic contact dermatitis-induced pruritus in mice and humans.

Although the Mas-related G-protein-coupled receptors (Mrgprs) play essential roles in itch detection, their contribution to allergic contact dermatitis (ACD)-associated itch remains unclear. To investigate whether Mrgprs are involved in ACD and whether Mrgprs can be identified as potential therapeutic targets. Mrgpr-clusterΔ-/- mice and human MrgprX1 (hMrgprX1) transgenic mice were used to evaluate the function of Mrgprs in oxazolone-induced ACD. Utilizing an ACD model, we found that Mrgpr-clusterΔ-/- mice display significantly reduced pruritus. Among 12 Mrgprs deleted in Mrgpr-clusterΔ-/- mice, the expression of MrgprC11 and MrgprA3 was significantly increased in the ACD model, which also innervated the skin and spinal cord at higher-than-normal densities. The proportions of dorsal root ganglia neurons responding to bovine adrenal medulla peptide 8-22 and chloroquine were also remarkably increased in the ACD model, resulting in enhanced itch behaviour. To study the function of human Mrgprs in ACD-induced itch, we used hMrgprX1 transgenic mice, which rescued the severe itch defect of Mrgpr-clusterΔ-/- mice in the ACD model. Remarkably, pharmacological blockade of hMrgprX1 significantly attenuates ACD itch in hMrgprX1 transgenic mouse. Our study provides the first evidence that Mrgprs are involved in ACD-induced chronic itch, which provides new avenues for itch management in ACD.

Microinjection of pruritogens in NGF-sensitized human skin

Single intradermal injections of nerve growth factor (NGF) evoke prolonged but temporally distinct sensitization patterns to somatosensory stimuli. Focal administration of the non-histaminergic pruritogen cowhage but not histamine resulted in elevated itch at day 21 after NGF administration. Here, we injected bovine adrenal medulla peptide 8–22 (BAM8–22), β-alanine (β-ALA) and endothelin-1 (ET-1) into NGF-treated skin of 11 healthy volunteers and investigated the corresponding itch/pain and flare reactions. β-ALA was the weakest pruritogen, while BAM8–22 and ET-1 were equally potent as histamine. NGF did not sensitize itch or flare reactions induced by any compound, but injection and evoked pain were increased at day 21 and 49. The involvement of histamine H1 receptors in itch was explored in eight subjects after oral cetirizine. ET-1-induced itch and flare were significantly reduced. BAM8–22 and β-ALA itch were not affected, but flare responses after BAM8–22 reduced by 50%. The results indicate that a single NGF injection does not sensitize for experimentally induced itch but increases pain upon pruritogen injection. In healthy humans, pruritic and algetic processing appear differentially regulated by NGF. However, in patients suffering chronic itch, prolonged elevation of NGF-levels under inflammatory conditions may contribute to elevated itch.

Pruriception and neuronal coding in nociceptor subtypes in human and nonhuman primates.

In humans, intradermal administration of β-alanine (ALA) and bovine adrenal medulla peptide 8-22 (BAM8-22) evokes the sensation of itch. Currently, it is unknown which human dorsal root ganglion (DRG) neurons express the receptors of these pruritogens, MRGPRD and MRGPRX1, respectively, and which cutaneous afferents these pruritogens activate in primate. In situ hybridization studies revealed that MRGPRD and MRGPRX1 are co-expressed in a subpopulation of TRPV1+ human DRG neurons. In electrophysiological recordings in nonhuman primates (Macaca nemestrina), subtypes of polymodal C-fiber nociceptors are preferentially activated by ALA and BAM8-22, with significant overlap. When pruritogens ALA, BAM8-22, and histamine, which activate different subclasses of C-fiber afferents, are administered in combination, human volunteers report itch and nociceptive sensations similar to those induced by a single pruritogen. Our results provide evidence for differences in pruriceptive processing between primates and rodents, and do not support the spatial contrast theory of coding of itch and pain.

Calcium imaging of primary canine sensory neurons: Small-diameter neurons responsive to pruritogens and algogens.

IntroductionRodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations.MethodsThis study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single‐cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease‐activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5‐hydroxytryptamine receptor agonist (5‐HT), bovine adrenal medulla peptide (BAM8‐22), substance P, allyl isothiocyanate (AITC), and capsaicin.ResultsThis study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens.ConclusionRatiometric calcium imaging demonstrated that small‐diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.

TRPA1 Channel is Involved in SLIGRL-Evoked Thermal and Mechanical Hyperalgesia in Mice.

Persistent itch (pruritus) accompanying dermatologic and systemic diseases can significantly impair the quality of life. It is well known that itch is broadly categorized as histaminergic (sensitive to antihistamine medications) or non-histaminergic. Sensory neurons expressing Mas-related G-protein-coupled receptors (Mrgprs) mediate histamine-independent itch. These receptors have been shown to bind selective pruritogens in the periphery and mediate non-histaminergic itch. For example, mouse MrgprA3 responds to chloroquine (an anti-malarial drug), and are responsible for relaying chloroquine-induced scratching in mice. Mouse MrgprC11 responds to a different subset of pruritogens including bovine adrenal medulla peptide (BAM8–22) and the peptide Ser-Leu-Ile-Gly-Arg-Leu (SLIGRL). On the other hand, the possibility that itch mediators also influence pain is supported by recent findings that most non-histaminergic itch mediators require the transient receptor potential ankyrin 1 (TRPA1) channel. We have recently found a significant increase of thermal and mechanical hyperalgesia induced by non-histaminergic pruritogens chloroquine and BAM8–22, injected into mice hindpaw, for the first 30–45 min. Pretreatment with TRPA1 channel antagonist HC-030031 did significantly reduce the magnitude of this hyperalgesia, as well as significantly shortened the time-course of hyperalgesia induced by chloroquine and BAM8–22. Here, we report that MrgprC11-mediated itch by their agonist SLIGRL is accompanied by heat and mechanical hyperalgesia via the TRPA1 channel. We measured nociceptive thermal paw withdrawal latencies and mechanical thresholds bilaterally in mice at various time points following intra-plantar injection of SLIGRL producing hyperalgesia. When pretreated with the TRPA1 antagonist HC-030031, we found a significant reduction of thermal and mechanical hyperalgesia.

Novel Probes Establish Mas-Related G Protein-Coupled Receptor X1 Variants as Receptors with Loss or Gain of Function.

The Mas-related G protein-coupled receptor X1 (MrgprX1) is a human seven transmembrane-domain protein with a putative role in nociception and pruritus. This receptor is expressed in dorsal root ganglion neurons and is activated by a variety of endogenous peptides, including bovine adrenal medulla peptide (BAM) and γ2-melanocyte-stimulating hormone (γ2-MSH). In the present work, we study how naturally occurring missense mutations alter the activity of MrgprX1. To characterize selected receptor variants, we initially used the endogenous peptide ligand BAM8-22. In addition, we generated and characterized a panel of novel recombinant and synthetic peptide ligands. Our studies identified a mutation in the second intracellular loop of MrgprX1, R131S, that causes a decrease in both ligand-mediated and constitutive signaling. Another mutation in this region, H133R, results in a gain of function phenotype reflected by an increase in ligand-mediated signaling. Using epitope-tagged variants, we determined that the alterations in basal and ligand-mediated signaling were not explained by changes in receptor expression levels. Our results demonstrate that naturally occurring mutations can alter the pharmacology of MrgprX1. This study provides a theoretical basis for exploring whether MrgprX1 variability underlies differences in somatosensation within human populations.

Role of spinal neuronal Mas-related gene receptor C in maintenance of bone cancer pain in mice

Objective To evaluate the role of spinal neuronal Mas-related gene receptor C (MrgC) in the maintenance of bone cancer pain (BCP) in mice. Methods A total of 132 SPF male C3H/HeJ mice, aged 8-10 weeks, weighing 18-22 g, were randomly divided into 4 groups (n=33 each) using a random number table: sham operation group (S group), BCP group, bovine adrenal medulla peptide 8-22 (BAM8-22, a highly selective MrgC agonist) group (group BAM), and MrgC antibody group (group MA). BCP was produced by injecting α-MEM 20 μl containing 2×105 NCTC2472 cells into the distal medullary cavity of right femur bone.While α-MEM 20 μl was injected only in group S. The artificial cerebrospinal fluid 5 μl was injected intrathecally in S and BCP groups, and BAM 8-22 8 nmol/5 μl and MrgC antibody 5 μl were injected intrathecally in BAM and MA groups, respectively, once a day for 7 consecutive days starting from the day 14 after inoculation of the tumor cells.At 1 day before inoculation (T0), before administration (T1), and at 14, 16 19 and 21 days after inoculation (T2-5, at 0.5 h before the initial administration and 2 h after each administration), the number of spontaneous flinches (NSF) and mechanical paw withdrawal threshold (MWT) were measured.Five animals selected from each group at each time point were sacrificed, and the lumbar enlargement segments of the spinal cord were removed for determination of MrgC expression in the spinal neurons (by immunofluorescence). Results Compared with group S, NSF was significantly increased, MWT was decreased, and the expression of MrgC was up-regulated at T1-5 in BCP, BAM and MA groups.Compared with group BCP, NSF was significantly decreased, MWT was increased, and the expression of MrgC was up-regulated at T2-5 in group BAM, and NSF was significantly increased, MWT was decreased, and the expression of MrgC was down-regulated at T2-5 in group MA. Conclusion Spinal neuronal MrgC is involved in the maintenance of BCP in mice. Key words: Receptors, G-protein-coupled; Bone neoplasms; Pain; Neurons; Spinal cord

Effect of Mas‐related gene (Mrg) receptors on hyperalgesia in rats with CFA‐induced inflammation via direct and indirect mechanisms

Mas oncogene-related gene (Mrg) receptors are exclusively distributed in small-sized neurons in trigeminal and dorsal root ganglia (DRG). We investigated the effects of MrgC receptor activation on inflammatory hyperalgesia and its mechanisms. A selective MrgC receptor agonist, bovine adrenal medulla peptide 8-22 (BAM8-22) or melanocyte-stimulating hormone (MSH) or the μ-opioid receptor (MOR) antagonist CTAP was administered intrathecally (i.t.) in rats injected with complete Freund's adjuvant (CFA) in one hindpaw. Thermal and mechanical nociceptive responses were assessed. Neurochemicals were measured by immunocytochemistry, Western blot, ELISA and RT-PCR. CFA injection increased mRNA for MrgC receptors in lumbar DRG. BAM8-22 or MSH, given i.t., generated instant short and delayed long-lasting attenuations of CFA-induced thermal hyperalgesia, but not mechanical allodynia. These effects were associated with decreased up-regulation of neuronal NOS (nNOS), CGRP and c-Fos expression in the spinal dorsal horn and/or DRG. However, i.t. administration of CTAP blocked the induction by BAM8-22 of delayed anti-hyperalgesia and inhibition of nNOS and CGRP expression in DRG. BAM8-22 also increased mRNA for MORs and pro-opiomelanocortin, along with β-endorphin content in the lumbar spinal cord and/or DRG. MrgC receptors and nNOS were co-localized in DRG neurons. Activation of MrgC receptors suppressed up-regulation of pronociceptive mediators and consequently inhibited inflammatory pain, because of the activation of up-regulated MrgC receptors and subsequent endogenous activity at MORs. The uniquely distributed MrgC receptors could be a novel target for relieving inflammatory pain.

Roles for substance P and gastrin-releasing peptide as neurotransmitters released by primary afferent pruriceptors

Recent studies support roles for neurokinin-1 (NK-1) and gastrin-releasing peptide (GRP) receptor-expressing spinal neurons in itch. We presently investigated expression of substance P (SP) and GRP in pruritogen-responsive primary sensory neurons and roles for these neuropeptides in itch signaling. Responses of dorsal root ganglion (DRG) cells to various pruritogens were observed by calcium imaging. DRG cells were then processed for SP, GRP, and isolectin B-4 (IB4; a marker for nonpeptidergic neurons) immunofluorescence. Of pruritogen-responsive DRG cells, 11.8-26.8%, 21.8-40.0%, and 21.4-26.8% were immunopositive for SP, GRP, and IB4, respectively. In behavioral studies, both systemic and intrathecal administration of a NK-1 receptor antagonist significantly attenuated scratching evoked by chloroquine and a protease-activated receptor 2 agonist, SLIGRL, but not histamine, bovine adrenal medulla peptide 8-22 (BAM8-22), or serotonin. Systemic or intrathecal administration of a GRP receptor antagonist attenuated scratching evoked by chloroquine and SLIGRL but not BAM8-22 or histamine. The GRP receptor antagonist enhanced scratching evoked by serotonin. These results indicate that SP and GRP expressed in primary sensory neurons are partially involved as neurotransmitters in histamine-independent itch signaling from the skin to the spinal cord.

Mas-related G-protein–coupled receptors inhibit pathological pain in mice

An important objective of pain research is to identify novel drug targets for the treatment of pathological persistent pain states, such as inflammatory and neuropathic pain. Mas-related G-protein-coupled receptors (Mrgprs) represent a large family of orphan receptors specifically expressed in small-diameter nociceptive primary sensory neurons. To determine the roles of Mrgprs in persistent pathological pain states, we exploited a mouse line in which a chromosomal locus spanning 12 Mrgpr genes was deleted (KO). Initial studies indicated that these KO mice show prolonged mechanical- and thermal-pain hypersensitivity after hind-paw inflammation compared with wild-type littermates. Here, we show that this mutation also enhances the windup response of dorsal-horn wide dynamic-range neurons, an electrophysiological model for the triggering of central pain sensitization. Deletion of the Mrgpr cluster also blocked the analgesic effect of intrathecally applied bovine adrenal medulla peptide 8-22 (BAM 8-22), an MrgprC11 agonist, on both inflammatory heat hyperalgesia and neuropathic mechanical allodynia. Spinal application of bovine adrenal medulla peptide 8-22 also significantly attenuated windup in wild-type mice, an effect eliminated in KO mice. These data suggest that members of the Mrgpr family, in particular MrgprC11, may constitute an endogenous inhibitory mechanism for regulating persistent pain in mice. Agonists for these receptors may, therefore, represent a class of antihyperalgesics for treating persistent pain with minimal side effects because of the highly specific expression of their targets.

Sensitisation of TRPV1 in rat sensory neurones by activation of SNSRs

The novel sensory neurone specific receptor (SNSR) family of G-protein coupled receptors are activated by non-opiate fragments of opioid precursor peptides. SNSRs are expressed in nociceptors, and SNSR agonists have been found to cause sensitisation to painful stimuli in vivo. We explored the basis of sensitisation caused by SNSR agonists in sensory neurones by investigating the effect of the SNSR-selective agonist bovine adrenal medulla peptide 8-22 (BAM (8-22)) on gating of the heat and capsaicin-sensitive ion channel TRPV1. Using calcium imaging we found that BAM (8-22) caused sensitisation of the TRPV1 response in approximately 13% of DRG neurones. Sensitisation of TRPV1 in a similar proportion of neurones was observed using whole-cell patch clamping. The PKC-specific inhibitor Ro-31-8220 reduced but did not completely abolish sensitisation, while the protein kinase A inhibitor H-89 was without significant effect. No translocation of the PKC delta, epsilon and zeta isoforms to the cell membrane was observed in response to BAM (8-22). These observations implicate PKC in the sensitisation of TRPV1, but suggest that other pathways are also involved.

Simultaneous Activation of the δ Opioid Receptor (δOR)/Sensory Neuron-Specific Receptor-4 (SNSR-4) Hetero-Oligomer by the Mixed Bivalent Agonist Bovine Adrenal Medulla Peptide 22 Activates SNSR-4 but Inhibits δOR Signaling

Hetero-oligomerization among G protein-coupled receptors has been proposed to contribute to signal integration. Because sensory neuron-specific receptors (SNSRs) and the opioid receptors (OR) share a common ligand, the bovine adrenal medulla peptide (BAM) 22, and have opposite effects on pain modulation, we investigated the possible consequences of deltaOR/SNSR-4 hetero-oligomerization on the signaling properties of both receptor subtypes. Bioluminescence resonance energy transfer revealed that the human deltaOR has similar propensity to homo-oligomerize and to form hetero-oligomers with human SNSR-4 when coexpressed in human embryonic kidney 293 cells. The hetero-oligomerization leads to a receptor form displaying unique functional properties. Individual activation of either deltaOR or SNSR-4 in cells coexpressing the two receptors led to the modulation of their respective signaling pathways; inhibition of adenylyl cyclase and activation of phospholipase C, respectively. In contrast, the deltaOR/SNSR-4 bivalent agonist BAM22, which could activate each receptor expressed individually, fully activated the SNSR-4-dependent phospholipase C but did not promote deltaOR-mediated inhibition of adenylyl cyclase in deltaOR/SNSR-4-coexpressing cells. Likewise, concomitant activation of the deltaOR/SNSR-4 hetero-oligomer by selective deltaOR and SNSR-4 agonists promoted SNSR-4 but not deltaOR signaling, revealing an agonist-dependent dominant-negative effect of SNSR-4 on deltaOR signaling. Furthermore, the deltaOR selective antagonist naltrexone trans-inhibited the SNSR-4-promoted phospholipase C activation mediated by BAM22 but not by the SNSR-4-selective agonists, suggesting a bivalent binding mode of BAM22 to the deltaOR/SNSR-4 hetero-oligomer. The observation that BAM22 inhibited the Leu-enkephalin-promoted cAMP inhibition in rat dorsal root ganglia neurons supports the potential physiological implication of such regulatory mechanism.

Modulation of ion channels and synaptic transmission by a human sensory neuron-specific G-protein-coupled receptor, SNSR4/mrgX1, heterologously expressed in cultured rat neurons.

Human sensory neuron-specific G-protein-coupled receptors (SNSRs) are expressed solely in small diameter primary sensory neurons. This restricted expression pattern is of considerable therapeutic interest because small nociceptors transmit chronic pain messages. The neuronal function of human SNSRs is difficult to assess because rodent orthologs have yet to be clearly defined, and individual isoforms are found only in a small subset of primary sensory neurons. To circumvent this problem, we expressed human SNSR4 (hSNSR4; also known as Hs.mrgX1) in rat superior cervical ganglion (SCG), dorsal root ganglion (DRG), and hippocampal neurons using nuclear injection or recombinant adenoviruses and examined modulation of ion channels and neurotransmission using whole-cell patch-clamp techniques. BAM8-22 (a 15 amino acid C-terminal fragment of bovine adrenal medulla peptide 22), a peptide agonist derived from proenkephalin, inhibited high (but not low) voltage-activated Ca2+ current in both DRG and SCG neurons expressing hSNSR4, whereas no response was detected in control neurons. The Ca2+ current inhibition was concentration dependent and partially sensitive to Pertussis toxin (PTX) treatment. Additionally, the peptide was highly effective in modulating current arising from M-type K+ channels in SCG neurons expressing hSNSR4. In hippocampal neurons expressing hSNSR4, BAM8-22 induced presynaptic inhibition of transmission that was abolished after PTX treatment. Our data indicate that hSNSR4, when heterologously expressed in rat neurons, can be activated by an opioid-related peptide, couples to G(q/11)-proteins as well as PTX-sensitive G(i/o)-proteins, and modulates neuronal Ca2+ channels, K+ channels, and synaptic transmission.

Proenkephalin A gene products activate a new family of sensory neuron--specific GPCRs.

Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.

Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity

A thiolprotease from rat brain membranes was shown to convert synthetic dynorphin B-29 (Dyn B-29, “leumorphin”) to the tridecapeptide dynorphin B (Dyn B, “rimorphin”). This represents a “single-arginine cleavage” between threonine-13 and arginine-14 of the substrate. The dynorphin converting activity displayed typical Michaelis-Menten kinetics with an apparent K m for the substrate of 0.58 μM. Surprisingly, a synthetic peptide, Dyn B-29-(9–22), which contains the cleavage site, did not inhibit the activity. Dyn A inhibited the activity competitively with an apparent K i of 3.7 μM. The converting activity was also inhibited by Dyn A-(6–17) but not by Dyn A-(8–17), suggesting a role of Arg 6-Arg 7 in the inhibition of converting activity. Bovine adrenal medulla Peptide E inhibited the converting activity substantially whereas metorphamide did not, suggesting the importance of COOH-terminal residues in recognition. β-Endorphin was an effective inhibitor of converting activity, and [α-N-acetyl]β-endorphin was not, indicating a crucial role of the free NH 2-terminus in recognition by the enzyme. ACTH inhibited the activity competitively with an apparent K i of 39 nM. The converting activity was also inhibited substantially by ACTH-(1–13) but not by α-MSH, again indicating a requirement of the free NH 2-terminus for recognition. The above results suggest that the converting enzyme recognizes peptides of the three known opioid gene families.