Martin N. Perkins

Bradykinin and inflammatory pain

There is compelling evidence linking bradykinin (BK) with the pathophysiological processes that accompany tissue damage and inflammation, especially the production of pain and hyperalgesia. Several mechanisms have been proposed to account for hyperalgesia including the direct activation of nociceptors as well as sensitization of nociceptors through the production of prostanoids or the release of other mediators. In keeping with this, antagonists of the BK B2 receptor are efficacious analgesic and anti-inflammatory agents in acute inflammatory pain. More recently it has been suggested that when inflammation is prolonged, BK B1 receptors, which are not expressed in healthy tissues to a significant degree, also play an important role in the maintenance of hyperalgesia. This may be one of a number of adaptive mechanisms that occur peripherally and centrally following the prolonged activation of nociceptors during inflammation or injury.

Antinociceptive activity of the bradykinin B1 and B2 receptor antagonists, des-Arg9, [Leu8]-BK and HOE 140, in two models of persistent hyperalgesia in the rat

&NA; There has been recent evidence linking bradykinin (BK) receptors with inflammation. This study has investigated the involvement of BK receptors in two models of persistent inflammatory hyperalgesia in rats. In a Freunds adjuvant‐induced hyperalgesia model and an ultraviolet (UV)‐induced hyperalgesia model in rats the specific B2 antagonist, D‐Arg[Hyp3, Thi5, D‐Tic7, Oic8]‐BK (HOE 140), was either ineffective or weakly active in reversing hyperalgesia. The specific B1 antagonist, des‐Arg9, [Leu8]‐BK, was effective in reversing or preventing the development of hyperalgesia in both Freunds adjuvant‐induced hyperalgesia and UV‐induced hyperalgesia. The B1 agonist, des‐Arg9‐BK, produced a small exacerbation of hyperalgesia in both models. Data suggest that in persistent inflammatory conditions in the rat bradykinin B1 receptors are involved in the accompanying hyperalgesia.

Kinins and kinin receptors in the nervous system

Kinins, including bradykinin and kallidin, are peptides that are produced and act at the site of tissue injury or inflammation. They induce a variety of effects via the activation of specific B1 or B2 receptors that are coupled to a number of biochemical transduction mechanisms. In the periphery the actions of kinins include vasodilatation, increased vascular permeability and the stimulation of immune cells and peptide-containing sensory neurones to induce pain and a number of neuropeptide-induced reflexes. Mechanisms for kinin synthesis are also present in the CNS where kinins are likely to initiate a similar cascade of events, including an increase in blood flow and plasma leakage. Kinins are potent stimulators of neural and neuroglial tissues to induce the synthesis and release of other pro-inflammatory mediators such as prostanoids and cytotoxins (cytokines, free radicals, nitric oxide). These events lead to neural tissue damage as well as long lasting disturbances in blood-brain barrier function. Animal models for CNS trauma and ischaemia show that increases in kinin activity can be reversed either by kinin receptor antagonists or by the inhibition of kinin production. A number of other central actions have been attributed to kinins including an effect on pain signalling, both within the brain (which may be related to vascular headache) and within the spinal dorsal horn where primary afferent nociceptors can be stimulated. Kinins also appear to play a role in cardiovascular regulation especially during chronic spontaneous hypertension. Presently, however, direct evidence is lacking for the release of kinins in pathophysiological conditions of the CNS and it is not known whether spinal or central neurones, other than afferent nerve terminals, are sensitive to kinins. A more detailed examination of the effects of kinins and their central pharmacology is necessary. It is also important to determine whether the inhibition of kinin activity will alleviate CNS inflammation and whether kinin receptor antagonists are useful in pathological conditions of the CNS.

Antihyperalgesic effects of δ opioid agonists in a rat model of chronic inflammation

Opioid receptors in the brain activate descending pain pathways to inhibit the nociceptive response to acute noxious stimuli. The aim of the present study was to clarify the role of supraspinal opioid receptors in modulating the nociceptive response to persistent inflammation in rats. Subcutaneous administration of 50 μl of complete Freunds Adjuvant (CFA) into the plantar surface of the hindpaw induced a significant decrease in paw withdrawal latency to thermal stimuli (P<0.01) at 24 h post‐injection. Intracerebroventricular (i.c.v.) administration of the μ opioid receptor agonists, DAMGO and morphine, and the δ opioid receptor agonists, deltorphin II and SNC80, significantly reversed the hyperalgesic response associated with peripheral inflammation in a dose‐dependent manner (P<0.0001). The μ and δ agonists also significantly attenuated the antinociceptive response to acute thermal stimulation in rats (P<0.001). However, deltorphin II and SNC80 were less potent, and in the case of SNC80 less efficacious, in modulating the response to acute thermal nociception in comparison to hyperalgesia associated with persistent inflammation. These results indicate that μ and δ opioid receptors in the brain modulate descending pain pathways to attenuate the nociceptive response to acute thermal stimuli in both normal and inflamed tissues. The heightened response to δ agonists in the hyperalgesia model suggests that δ opioid receptors in the brain are promising targets for the treatment of pain arising from chronic inflammation.

A peripherally restricted cannabinoid receptor agonist produces robust anti-nociceptive effects in rodent models of inflammatory and neuropathic pain.

&NA; Cannabinoids are analgesic in man, but their use is limited by their psychoactive properties. One way to avoid cannabinoid receptor subtype 1 (CB1R)‐mediated central side‐effects is to develop CB1R agonists with limited CNS penetration. Activation of peripheral CB1Rs has been proposed to be analgesic, but the relative contribution of peripheral CB1Rs to the analgesic effects of systemic cannabinoids remains unclear. Here we addressed this by exploring the analgesic properties and site of action of AZ11713908, a peripherally restricted CB1R agonist, in rodent pain models. Systemic administration of AZ11713908 produced robust efficacy in rat pain models, comparable to that produced by WIN 55, 212–2, a CNS‐penetrant, mixed CB1R and CB2R agonist, but AZ11713908 generated fewer CNS side‐effects than WIN 55, 212‐in a rat Irwin test. Since AZ11713908 is also a CB2R inverse agonist in rat and a partial CB2R agonist in mouse, we tested the specificity of the effects in CB1R and CB2R knock‐out (KO) mice. Analgesic effects produced by AZ11713908 in wild‐type mice with Freunds complete adjuvant‐induced inflammation of the tail were completely absent in CB1R KO mice, but fully preserved in CB2R KO mice. An in vivo electrophysiological assay showed that the major site of action of AZ11713908 was peripheral. Similarly, intraplantar AZ11713908 was also sufficient to induce robust analgesia. These results demonstrate that systemic administration of AZ11713908, produced robust analgesia in rodent pain models via peripheral CB1R. Peripherally restricted CB1R agonists provide an interesting novel approach to analgesic therapy for chronic pain.

Blocking spinal CCR2 with AZ889 reversed hyperalgesia in a model of neuropathic pain

BackgroundThe CCR2/CCL2 system has been identified as a regulator in the pathogenesis of neuropathy-induced pain. However, CCR2 target validation in analgesia and the mechanism underlying antinociception produced by CCR2 antagonists remains poorly understood. In this study, in vitro and in vivo pharmacological approaches using a novel CCR2 antagonist, AZ889, strengthened the hypothesis of a CCR2 contribution to neuropathic pain and provided confidence over the possibilities to treat neuropathic pain with CCR2 antagonists.ResultsWe provided evidence that dorsal root ganglia (DRG) cells harvested from CCI animals responded to stimulation by CCL2 with a concentration-dependent calcium rise involving PLC-dependent internal stores. This response was associated with an increase in evoked neuronal action potentials suggesting these cells were sensitive to CCR2 signalling. Importantly, treatment with AZ889 abolished CCL2-evoked excitation confirming that this activity is CCR2-mediated. Neuronal and non-neuronal cells in the spinal cord were also excited by CCL2 applications indicating an important role of spinal CCR2 in neuropathic pain. We next showed that in vivo spinal intrathecal injection of AZ889 produced dose-dependent analgesia in CCI rats. Additionally, application of AZ889 to the exposed spinal cord inhibited evoked neuronal activity and confirmed that CCR2-mediated analgesia involved predominantly the spinal cord. Furthermore, AZ889 abolished NMDA-dependent wind-up of spinal withdrawal reflex pathway in neuropathic animals giving insight into the spinal mechanism underlying the analgesic properties of AZ889.ConclusionsOverall, this study strengthens the important role of CCR2 in neuropathic pain and highlights feasibility that interfering on this mechanism at the spinal level with a selective antagonist can provide new analgesia opportunities.

Pro-nociceptive effects of neuromedin U in rat.

The neuropeptide neuromedin U (NMU) has been shown to have significant effects on cardiovascular, gastrointestinal and CNS functions. The peptide was first isolated from the porcine spinal cord and later shown to be present in spinal cords of other species. Little is known about the distribution of neuromedin U receptors (NMURs) in the spinal cord and the spinal action of the peptide. Here we report on the expression of NMURs and a potential role in nociception in the rat spinal cord using a combination of behavioral and electrophysiological studies. Receptor autoradiography showed that NMU-23 binding was restricted to the superficial layers of spinal cord, a region known to be involved in the control of nociception. In situ hybridization analysis indicated the mRNA of NMUR2 was located in the same region (laminae I and IIo) as NMU-23 binding, while the mRNA for NMU receptor 1 was observed in a subpopulation of small diameter neurons of dorsal root ganglia. Intrathecal (i.t.) administration of neuromedin U-23 (0.4-4.0 nmol/10 microl) dose-dependently decreased both the mechanical threshold to von Frey hair stimulation and the withdrawal latency to a noxious thermal stimulus. Mechanical allodynia was observed between 10 and 120 min, peaking at 30 min and heat hyperalgesia was observed 10-30 min after i.t. administration of NMU-23. A similar mechanical allodynia was also observed following i.t. administration of NMU-8 (0.4-4 nmol/10 microl). A significant enhancement of the excitability of flexor reflex was induced by intrathecal administration of NMU-23 (4 nmol/10 microl). Evoked responses to touch and pinch stimuli were increased by 439+/-94% and 188+/-36% (P<0.01, n=6) respectively. The behavioral and electrophysiological data demonstrate, for the first time, a pro-nociceptive action of NMU. The restricted distribution of NMU receptors to a region of the spinal cord involved in nociception suggests that this peptide receptor system may play a role in nociception.

A pro-nociceptive role of neuromedin U in adult mice.

Although the neuropeptide neuromedin U (NMU) was first isolated from the spinal cord, its actions in this site are unknown. The recent identification of the NMU receptor subtype 2 (NMU2R) in the spinal cord has increased the interest in investigating the role of NMU in this part of the central nervous system. Here, we report a novel function for NMU in spinal nociception in the mouse. Systemic perfusion of NMU (rat, NMU‐23) dose‐dependently (0.2, 0.5, 1, and 2.5 &mgr;M) potentiated both the background activity and noxious pinch‐evoked response of nociceptive or wide dynamic range, but not non‐nociceptive, dorsal horn neurons. At 2.5 &mgr;M, NMU‐23 increased the total background activity from 154±34 to 1374±260 spikes/160 s (P<0.005, n=28) and increased the evoked nociceptive response by 185±50% (P<0.01, n=13). Intrathecal administration of NMU‐23 (0.4, 1.1, and 3.8 nmol/10 &mgr;l) dose‐dependently decreased thermal withdrawal latencies and produced a morphine‐sensitive behavioral response. These electrophysiological and behavioral results suggest that NMU may be a novel physiological regulator in spinal nociceptive transmission and processing.

Differential expression and pharmacology of native P2X receptors in rat and primate sensory neurons.

Evidence suggesting the involvement of P2X2 and P2X3 in chronic pain has been obtained mostly from rodent models. Here we show that rodents may be poor predictors of P2X3 pharmacology in human. We demonstrate that monkey and human dorsal root ganglion (DRG) neurons do not express appreciable levels of P2X2 subunit, contrary to rat sensory neurons. Additionally, we report functional P2X3 activity in monkey DRG neurons and confirm the absence of functional P2X2/3 receptors. Interestingly, native P2X3 receptors in rat and monkey DRGs show similar agonist potency, but different antagonist potencies for TNP-ATP [2-O-(2,4,6-trinitrophenyl)-ATP] and RO51. This unexpected difference in antagonist potency was confirmed by comparing rat and human P2X3 receptors in HEK293 cells. Mutagenesis studies reveal that two extracellular residues, A197 and T202, are synergistically responsible for the potency drop in primate P2X3 receptors. These results uncover species-specific P2X3 pharmacology and identify key mechanisms impacting the translatability of potential analgesics targeting P2X3 receptors.

Activity of deep dorsal horn neurons in the anaesthetized rat during hyperalgesia of the hindpaw induced by ultraviolet irradiation

Thermal hyperalgesia was induced by UV irradiation of the glabrous skin of the hindpaw of adult female Sprague-Dawley rats. We have recorded single cell activity and studied excitability changes in wide dynamic range neurons in the lumbar spinal segments during the early phase (days 1-3) and late phase (days 5-7) of thermal hyperalgesia in animals under urethane anaesthesia. The proportion of spontaneously active wide dynamic range cells was increased following UV irradiation and the degree of spontaneous activity was enhanced during the course of hyperalgesia. In addition there was a significant increase in the total number of spikes evoked by standardized mechanical and noxious heat stimuli when tested at days 1-3 and days 5-7. The duration of the evoked responses was also significantly prolonged in both UV-treated groups. The noxious temperature threshold to radiant heat stimulation was significantly decreased on the UV-treated but not on the contralateral hindpaw. The average size of the receptive fields on the UV-treated paws was expanded in comparison to control. To differentiate between possible central and peripheral components of the hyperactivity of wide dynamic range cells we performed in situ dorsal rhizotomy during the recording. Cutting the dorsal roots (L2-5) evoked a significantly larger and more prolonged discharge in wide dynamic range cells in both UV-treated groups in comparison to control. Spontaneous activity in spinal wide dynamic range neurons was reduced after rhizotomy in each group. However, the decrease was only significant at days 1-3 (P < 0.05) but not at days 5-7.(ABSTRACT TRUNCATED AT 250 WORDS)