Bovine Adrenal Medulla 8-22 Research Articles

Effects of intravesical administration of sensory neuron-specific receptor agonist on voiding function in rats with cyclophosphamide-induced cystitis

IntroductionThe aim of this study was to investigate the urodynamic effects of intravesical administration of bovine adrenal medulla 8-22 (BAM8-22), a selective rat sensory neuron-specific receptor 1 agonist, on the micturition reflex in normal rats and rats with cyclophosphamide-induced bladder overactivity.Material and methodsContinuous cystometrograms (0.04 ml/min) were performed in urethane-anesthetized rats. After stable micturition cycles were established, vehicle (saline) or BAM8-22 was instilled intravesically and changes in bladder activity were monitored. The experiments using BAM8-22 were also performed in capsaicin-pretreated rats. In another experiment, vehicle (saline) or BAM8-22 was instilled intravesically and changes in bladder activity were monitored in cyclophosphamide-treated rats. Continuous cystometrograms were performed 48 hours after cyclophosphamide injection. Cystometric parameters were recorded and compared before and after intravesical drug administration.ResultsIntravesical administration of BAM8-22 significantly increased the intercontraction interval and threshold pressure in urethane-anesthetized rats, but did not affect the basal pressure or maximum pressure at any doses tested. The inhibitory effects of intravesical administration of BAM8-22 were not inhibited by capsaicin pretreatment. Intravesical administration of BAM8-22 also significantly increased intercontraction interval in the cyclophosphamide-treated rats.ConclusionsThe current results indicate that intravesical administration of a selective rat sensory neuron-specific receptor 1 agonist can inhibit the micturition reflex and can ameliorate cyclophosphamide-induced bladder overactivity in rats.

Effect of intrathecal injection of bovineadrenalmedulla 8-22 on pain behavior in a mouse model of bone cancer pain

Objective To investigate the effect of intrathecal injection bovineadrenalmedulla 8-22 (BAM8-22) on pain behavior in a mouse model of bone cancer pain. Methods 50 male 8-10 week old C3H/HeJ mice weighing 18-22 g were divided randomly into 5 groups (n=8): sham group (S group), BCP group: artificial cerebrospinal fluid treat group(B0 group) and BAM8-22 treat groups (B1-B3 group). Group C and B were induced mouse models of bone cancer pain by intra-right-femur inoculations of osteolytic NCTC2472 cells while group S were injected only α-MEM. On the 14 d after inoculations, group S and BCP received intrathecal injection of artificial cerebrospinal fluid 5 μl, while group B1-B3 received intrathecal injection of BAM8-22 0.8, 2.4, 8.0 nmol, which dissolved in 5 μl artificial cerebrospinal fluid. They were intrathecally injected, respectively, once a day for 7 consecutive days starting from day 14 after inoculation. Mice received pain behavior tests including quantification of spontaneous flinches (NSF), paw withdrawal mechanical threshold (PWMT) 0.5 h before and at 2 h, after administration. Results Compared with S group [(1.80±0.20) times, (1.87±0.22) g], NSF [(14.67±2.65) times] was significantly increased (P=0.000) and MWT [(0.42±0.22) g] was significantly decreased (P=0.002) on 7-21 days after inoculation in BCP group. Compared with B0 group [(12.38±1.12) times, (0.48±0.22) g] and the basal level on 14th day before administration [(12.65±1.10) times, (0.46±0.19) g, P=0.012, 0.011, 0.001, 0.001], PWMT was significantly decreased, and NSF was increased at 2 h after administration in B2 group [(7.28±0.82) times, (1.01±0.13) g] and B3 groups [(4.18±1.02) times, (1.38±0.12) g, P=0.027, 0.025, 0.013, 0.009]. The analgesic effect of group B3 was more conspicuous compared with group B2 (P=0.007, 0.031). No obvious change of pain ethology indexes wag observed in group B1 [(12.03±1.22) times, (0.51±0.16) g] (P=0.096, 0.074, 0.527, 0.474). Conclusion Intrathecal BAM8-22 injection can effectively attenuated bone cancer pain with increased doses in the mouse model of bone cancer pain. Key words: Bone cancer pain; bovineadrenalmedulla 8-22; Behavior; Mouse

Involvement of MrgprC in Electroacupuncture Analgesia for Attenuating CFA-Induced Thermal Hyperalgesia by Suppressing the TRPV1 Pathway.

Mas-related G-protein-coupled receptor C (MrgprC) plays an important role in modulating chronic inflammatory pain. Electroacupuncture (EA) has a satisfactory analgesic effect on chronic pain. This study aimed to investigate the involvement of MrgprC and its transient receptor potential vanilloid 1 (TRPV1) pathway in EA analgesia in chronic inflammatory pain. Chronic inflammatory pain was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into the left hind paw. EA (2/100 Hz) stimulation was administered. MrgprC siRNAs were intrathecally administered to inhibit MrgprC expression, and bovine adrenal medulla 8-22 (BAM8-22) was used to activate MrgprC. The mechanical allodynia was decreased by EA significantly since day 3. The piled analgesic effect of EA was partially blocked by 6 intrathecal administrations of MrgprC siRNA. Both EA and BAM8-22 could downregulate the expression of TRPV1 and PKC in both the DRG and the SCDH. Both EA and BAM8-22 could also decrease the TRPV1 translocation and p-TRPV1 level in both the DRG and the SCDH. The effects of EA on PKCε, TRPV1 translocation, and p-TRPV1 in both the DRG and the SCDH were reversed by MrgprC siRNA. The results indicated that MrgprC played crucial roles in chronic pain modulation and was involved in EA analgesia partially through the regulation of TRPV1 function at the DRG and SCDH levels.

Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1 This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8-22 (BAM8-22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8-22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8-22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain.

Activation of Mas Oncogene-Related G Protein-Coupled Receptors Inhibits Neurochemical Alterations in the Spinal Dorsal Horn and Dorsal Root Ganglia Associated with Inflammatory Pain in Rats.

Mas oncogene-related G protein-coupled receptor C (MrgC) is unequally expressed in sensory ganglia and has been shown to modulate pathologic pain. This study investigated the mechanism underlying the effect of MrgC receptors on inflammatory pain. Intrathecal administration of the selective MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22) (30 nmol) inhibited complete Freund's adjuvant-evoked hyperalgesia. This was associated with the inhibition of protein kinase C-γ and phosphorylated extracellular signal-regulated protein kinase in the spinal cord and/or dorsal root ganglia (DRG). The complete Freund's adjuvant injection in the hindpaw induced an increase in Gq, but not Gi and Gs, protein in the spinal dorsal horn. This increase was inhibited by the intrathecal administration of BAM8-22. The exposure of DRG cultures to bradykinin (10 μM) and prostaglandin E2 (1 μM) increased the expression of calcitonin gene-related peptide (CGRP) and neuronal nitric oxide synthase in small- and medium-sized neurons as well as the levels of CGRP, aspartate, and glutamate in the cultured medium. The bradykinin/prostaglandin E2-induced alterations were absent in the presence of BAM8-22 (10 nM). These results suggest that the activation of MrgC receptors can modulate the increase in the expression of CGRP and neuronal nitric oxide synthase as well as the release of CGRP and excitatory amino acids in DRG associated with inflammatory pain. This modulation results in the inhibition of pain hypersensitivity by suppressing the expression of Gq protein and protein kinase C-γ and extracellular signal-regulated protein kinase signaling pathways in the spinal cord and/or DRG. The present study suggests that MrgC receptors may be a novel target for relieving inflammatory pain.

Upregulation of pronociceptive mediators and downregulation of opioid peptide by adrenomedullin following chronic exposure to morphine in rats

Adrenomedullin (AM) belongs to a calcitonin gene-related peptide (CGRP) family and has been demonstrated to recruit CGRP following chronic use of morphine and neuronal nitric oxide synthase (nNOS) in inflammation. The present study investigated the possibility that AM initiates the changes of other molecules contributing to the development of morphine tolerance in its chronic use. Intrathecal (i.t.) co-administration of the AM receptor antagonist AM22–52 (35.8μg) inhibited tolerance to morphine-induced analgesia while a daily injection of the AM receptor agonist AM1–50 (8μg, i.t., bolus) for 9days induced a decrease in the potency of morphine analgesia and thermal hyperalgesia. Persistent exposure of cultured dorsal root ganglion (DRG) explants to morphine (3.3μM) for 4days resulted in an increase in AM and CGRP mRNA levels. However, morphine failed to produce these effects in the presence of AM22–52 (2μM). The i.t. administration of morphine for 6days increased the expression of nNOS in the spinal dorsal horn and DRG neurons but decreased expression of the endogenous opioid peptide bovine adrenal medulla 22 (BAM22) in small- and medium-sized neurons in DRG. Particularly, the co-administration of AM22–52 (35.8μg) inhibited the morphine-induced alterations in nNOS and BAM22. These results indicated that the increase in nNOS and CGRP expressions and the decrease in BAM22 were attributed to the increased AM receptor signaling induced by chronic morphine. The present study supports the hypothesis that the enhancement of AM bioactivity triggered upregulation of pronociceptive mediators and downregulation of pain-inhibiting molecule in a cascade contributing to the development of morphine tolerance.

Understanding the switch from pain-to-itch in dermatitis

evere chronic itch is debilitating symptom that results from a large umber of pathological conditions including skin disorders, organ ailure, and even some types of cancers [1]. In particular, conditions hat are associated with inflammation of the skin—such as atopic ermatitis and contact dermatitis—commonly result in itch that is ifficult to treat. Moreover, excessive scratching causes skin damge and release of inflammatory mediators that exacerbates itch, esulting in a pathological itch-scratch cycle. However, currently here are no effective drugs for the treatment of itch in dermatiis. While antihistamines reduce itch caused by hives, they have imited efficacy for most other types of itch, including atopic and ontact dermatitis. Recently, there has been a surge of interest in the field of itch, nd a large number of new pruritogens (itch-inducing agents) and heir receptors have been identified. Of these, histamine is the prootypical pruritogen, which is released from dermal mast cells and ctivates mechano-insensitive C-fibers to produce itch [2]. In addiion to histamine, there are numerous other pruritogens such as owhage, chloroquine, bovine adrenal medulla 8-22 (BAM8-22), nd serotonin, that likely cause itch via the activation of a disinct population (or populations) of primary afferents [3–8]. These gents cause acute itch in humans and/or trigger vigorous scratchng behavior when injected into the skin of mice. However, we still o not understand which factors are responsible for itch in disease tates like dermatitis, highlighting the pressing need for a better nderstanding of mechanisms underlying chronic itch. Just as people who suffer from chronic pain often experince hyperalgesia and allodynia, people suffering from chronic tch often experience hyperknesis and alloknesis. This ‘itchy-skin’ s commonly seen in dermatitis, where numerous innocuus stimuli—light touch, gentle brushing or contact with wool ber—cause extraneous itch [9,10]. Moreover, substances such as radykinin, which are normally experienced as painful, inappropritely cause itch rather than pain for people with atopic dermatitis 11].

Effects of sensory neuron-specific receptor agonist on bladder function in a rat model of cystitis induced by cyclophosphamide.

To investigate the effects of activation of sensory neuron-specific receptors (SNSRs) on cyclophosphamide (CYP) bladder overactivity in rats. Female Sprague-Dawley rats (235-258 g) were used. Rats were injected with either CYP (200 mg/kg, intraperitoneally) or saline (control). Continuous cystometrograms (0.04 ml/min) were recorded 48 h after CYP or saline injection under urethane anesthesia. After stable micturition cycles were established, a selective rat SNSR1 agonist, bovine adrenal medulla 8-22 (BAM8-22), was administered intravenously or intrathecally. Cyclophosphamide treatment-induced higher baseline pressure and shorter intercontraction intervals compared with the control group. Intravenous administration of BAM8-22 at 10, 30 and 100 μg/kg significantly increased intercontraction intervals in the CYP-treated group. Intrathecal administration of BAM8-22 at 0.03, 0.1 and 0.3 μg also significantly increased intercontraction intervals in the CYP-treated group. Intravenous or intrathecal administration of BAM8-22 did not change baseline pressure or maximum voiding pressure in the CYP-treated group. These findings indicate that activation of SNSRs can suppress CYP-induced bladder overactivity, probably due to suppression of bladder afferent activity.

Role of bovine adrenal medulla 22 (BAM22) in the pathogenesis of neuropathic pain in rats with spinal nerve ligation

The opioid peptide bovine adrenal medulla 22 (BAM22) is a cleavage product of proenkephalin and has been shown to be involved in inflammatory pain and morphine tolerance. This study was designed to investigate a role of BAM22 in neuropathic pain. L5 spinal nerve ligation (SNL) significantly reduced BAM22-immunoreactivity in small-sized neurons and depleted IB4 binding in injured L5 dorsal root ganglia (DRG) compared to sham rats. Double labeling study showed that the expression of BAM22-immunoreactivity was decreased mainly in IB4 neurons in the neighboring intact L4 and L6 DRGs following SNL. The nerve injury dramatically increased sensitivity of hindpaw to mechanical stimulation. Intrathecal (i.t.) administration of BAM22 on day 10 post-SNL attenuated mechanical allodynia in a dose-dependent manner (3–30nmol) and the effect lasted for up to 90min. Similar treatment with morphine at a dose of 30nmol produced a mild and brief inhibition on pain hypersensitivity. Furthermore, i.t. administration of 30nmol of BAM22 suppressed SNL-induced upregulation of interleukin-1β (IL-1β) in the spinal dorsal horn. The present study suggests that the reduction of BAM22 expression in small-sized neurons in both injured and the adjacent DRGs may contribute to pain hypersensitivity in peripheral nerve injury as a result of loss of inhibition of IL-1β upregulation in the spinal dorsal horn. Our results support the hypothesis that a reduction of antinociceptive activity loses the counteraction against activity of pronociceptive mediators, enhancing pain hypersensitivity following peripheral nerve injury.

Dual modulation effects of Mas-related gene (Mrg) receptors on pain sensitivity in rats

Mas-related gene G protein-coupled (Mrg) receptors have been identified to be uniquely distributed in subpopulations of small-diameter neurons in dorsal root and trigeminal ganglia in rodents and humans. It has been demonstrated that the activation of MrgC receptors in rodents inhibits pathological pain but does not alter pain sensitivity under normal conditions. In vitro studies have suggested that the activation of MrgX1 or MrgD receptors is associated with dual G proteins. The current study was designed to determine whether rat MrgC receptors are coupled to Gi/o and Gq proteins and what pain behavior response activation of these proteins could mediate. Intrathecal (i.t.) administration of bovine adrenal medulla 8-22 (BAM8-22), a specific MrgC receptor agonist, dose-dependently decreased tail flick latency (TFL) in rats pretreated with pertussis toxin, but not with saline. On the other hand, i.t. injected BAM8-22 increased TFL in the presence of U73122 but produced no changes in TFL following pretreatment with saline. The results in the present study provide in vivo evidence that both Gi/o and Gq proteins are involved in MrgC receptor signaling.

Sensory neurone‐specific receptor‐mediated regulation of micturition reflex in urethane‐anaesthetized rats

• To investigate the effect of sensory neurone-specific receptors (SNSRs) activation on the micturition reflex in rats. • Continuous cystometrograms (CMGs, 0.04 mL/min) were performed in female Sprague-Dawley rats under urethane anaesthesia. • After stable micturition cycles were established, a selective rat SNSR1 agonist, bovine adrenal medulla 8-22 (BAM8-22), was administered intravenously (i.v.) or intrathecally (i.t.) in normal rats or rats pretreated with capsaicin 4 days before the experiments. • Micturition variables were recorded and compared before and after drug administration. • Administration (i.v.) of BAM8-22 (3-100 µg/kg) significantly increased intercontraction intervals in a dose-dependent fashion, but did not affect residual urine or baseline pressure at any doses tested. • Administration (i.t.) of BAM8-22 (0.01-0.3 µg) also increased intercontraction intervals in a dose-dependent fashion, but did not affect residual urine or baseline pressure at any doses tested. • These inhibitory effects of i.v. (30 µg/kg) or i.t. (0.3 µg) administration of BAM8-22 still occurred after capsaicin pretreatment. • These results indicate that in urethane-anaesthetized rats activation of SNSRs can inhibit the micturition reflex via pathways independent of capsaicin-sensitive C-fibres. • Thus SNSRs could be a potential target for the treatment of bladder dysfunction, e.g. overactive bladder.

Blockade of adrenomedullin receptors reverses morphine tolerance and its neurochemical mechanisms

Adrenomedullin (AM) has been demonstrated to be involved in the development of opioid tolerance. The present study further investigated the role of AM in the maintenance of morphine tolerance, morphine-associated hyperalgesia and its cellular mechanisms. Intrathecal (i.t.) injection of morphine for 6 days induced a decline of its analgesic effect and hyperalgesia. Acute administration of the AM receptor antagonist AM 22–52 resumed the potency of morphine in a dose-dependent manner (12, 35.8 and 71.5 μg, i.t.). The AM 22–52 treatment also suppressed morphine tolerance-associated hyperalgesia. Furthermore, i.t. administration of AM 22–52 at a dose of 35.8 μg reversed the morphine induced-enhancement of nNOS (neuronal nitric oxide synthase) and CGRP immunoreactivity in the spinal dorsal horn and/or dorsal root ganglia (DRG). Interestingly, chronic administration of morphine reduced the expression of the endogenous opioid peptide bovine adrenal medulla 22 (BAM22) in small- and medium-sized neurons in DRG and this reduction was partially reversed by the administration of AM 22–52 (35.8 μg). These results suggest that the activation of AM receptors was involved in the maintenance of morphine tolerance mediating by not only upregulation of the pronociceptive mediators, nNOS and CGRP but also the down-regulation of pain-inhibiting molecule BAM22. Our data support the hypothesis that the level of both pronociceptive mediators and endogenous pain-inhibiting molecules has an impact on the potency of morphine analgesia. Targeting AM receptors is a promising approach to maintain the potency of morphine analgesia during chronic use of this drug.

The involvement of spinal bovine adrenal medulla 22‐like peptide, the proenkephalin derivative, in modulation of nociceptive processing

Bovine adrenal medulla 22 (BAM22), one of the cleavage products of proenkephalin A, possesses high affinity for opioid receptors and sensory neuron-specific receptor (SNSR). The present study was designed to examine the expression of BAM22 in the spinal cord and dorsal root ganglion (DRG) of naive rats as well as in a model of inflammation. BAM22-like immunoreactivity (BAM22-IR) was expressed in fibers in the spinal cord, with high density seen in lamina I in naïve rats. The expression of BAM22-IR in the superficial laminae was greatly reduced following dorsal rhizotomy. BAM22-IR was also located in 19% of DRG cells, mainly in the small- and medium-sized subpopulations. Following injection of complete Freund's adjuvant (CFA) in the hindpaw, the expression of BAM22-IR in the superficial laminae of the spinal cord and small-sized DRG neurons on the ipsilateral side was markedly increased. Double labeling showed that the Fos-positive nucleus was surrounded by BAM22-IR cytoplasm in the spinal dorsal horn neurons or closely associated with BAM22-IR fibers in the superficial laminae. Furthermore, CFA-induced mechanical allodynia in the inflamed paw was potentiated by intrathecal administration of anti-BAM22 antibody. Together, these results demonstrate for the first time that BAM22-like peptide is mainly located in the superficial laminae of the spinal cord and mostly originates from nociceptive DRG neurons. BAM22 could thus act as a ligand for presynaptic opioid receptors and SNSR. Our study also provides evidence suggesting that BAM22 plays a role in the modulation of nociceptive processing at the spinal level under normal and inflammatory conditions.

Effects of intrathecal BAM22 on noxious stimulus-evoked c- fos expression in the rat spinal dorsal horn

The effects of bovine adrenal medulla 22 (BAM22), a cleaved product of proenkephalin A, were investigated on the noxious stimulus-evoked expressions of spinal c- fos-like immunoreactivity (FLI). Heat (51 °C) applied to the tail evoked FLI predominantly in laminae I–II of the sacral spinal cord. Intrathecal (i.t.) BAM22 at a dose of 7 nmol decreased the expressions of the heat-evoked FLI by 68%, 64% and 56% in laminae I–II, III–IV and V–VI, respectively, and the decrease pattern was comparable to that induced by i.t. morphine (10 μg). Naloxone (1 mg/kg, i.p.) significantly enhanced the heat-evoked FLI in laminae III–VI, prevented the morphine-induced inhibition, and decreased the potencies of BAM22 in laminae I–II and V–VI by 23–40%. Higher dose of naloxone (10 mg/kg, i.p.) also partially reduced the BAM22-induced suppression. Following intraplantar injection of formalin (2.5%), FLI neurons were preferentially distributed not only in laminae I–II but also in laminae III–IV and V–VI of segments L4–L5. Pretreatment with BAM22 (7 nmol, i.t.) reduced the formalin-evoked FLI neurons by 72%, 61% and 58%, in laminae I–II, III–IV and V–VI, respectively. Naloxone (1 mg/kg. i.p.) enhanced the formalin-evoked expressions of FLI in laminae III–VI and decreased the potencies of BAM22 by 22–38% in laminae I–II and V–VI. The present study provided evidence at a cellular level showing that opioid and non-opioid effects of BAM22 on nociceptive processing in acute and persistent pain models were associated with modulation of noxious stimulus-evoked activity of the spinal dorsal horn neurons.