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Whether G protein-coupled receptors signal from endosomes to control important pathophysiological processes and are therapeutic targets is uncertain. We report that opioids from the inflamed colon activate δ-opioid receptors (DOPr) in endosomes of nociceptors. Biopsy samples of inflamed colonic mucosa from patients and mice with colitis released opioids that activated DOPr on nociceptors to cause a sustained decrease in excitability. DOPr agonists inhibited mechanically sensitive colonic nociceptors. DOPr endocytosis and endosomal signaling by protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways mediated the sustained inhibitory actions of endogenous opioids and DOPr agonists. DOPr agonists stimulated the recruitment of Gα and β-arrestin1/2 to endosomes. Analysis of compartmentalized signaling revealed a requirement of DOPr endocytosis for activation of PKC at the plasma membrane and in the cytosol and ERK in the nucleus. We explored a nanoparticle delivery strategy to evaluate whether endosomal DOPr might be a therapeutic target for pain. The DOPr agonist DADLE was coupled to a liposome shell for targeting DOPr-positive nociceptors and incorporated into a mesoporous silica core for release in the acidic and reducing endosomal environment. Nanoparticles activated DOPr at the plasma membrane, were preferentially endocytosed by DOPr-expressing cells, and were delivered to DOPr-positive early endosomes. Nanoparticles caused a long-lasting activation of DOPr in endosomes, which provided sustained inhibition of nociceptor excitability and relief from inflammatory pain. Conversely, nanoparticles containing a DOPr antagonist abolished the sustained inhibitory effects of DADLE. Thus, DOPr in endosomes is an endogenous mechanism and a therapeutic target for relief from chronic inflammatory pain.
Learn More >Children diagnosed with Christianson Syndrome (CS), a rare X-linked neurodevelopmental disorder characterized by intellectual disability, epilepsy, ataxia and mutism, also suffer from hyposensitivity to pain. This places them at risk of sustaining serious injuries that often go unattended. CS is caused by mutations in the alkali cation/proton exchanger SLC9A6/NHE6 that regulates recycling endosomal pH homeostasis and trafficking. Yet it remains unclear how defects in this transporter lead to altered somatosensory functions. In this study, we validated a Nhe6 knockout (KO) mouse as a model of CS and used it to identify the cellular mechanisms underlying the elevated pain tolerance observed in CS patients. Within the central nervous system, NHE6 immunolabelling is detected in a small percentage of cortical neurons involved in pain processing, including those within the primary somatosensory and the anterior cingulate cortices as well as the periaqueductal grey. Interestingly, it is expressed in a larger percentage of nociceptors. Behaviourally, Nhe6 KO mice have decreased nocifensive responses to acute noxious thermal, mechanical and chemical (i.e., capsaicin) stimuli. The reduced capsaicin-sensitivity in the KO mice correlates with a decreased expression of the transient receptor potential channel TRPV1 at the plasma membrane and capsaicin-induced Ca influx in primary cultures of nociceptors. These data indicate that NHE6 is a significant determinant of nociceptor function and pain behaviours, vital sensory processes that are impaired in CS.
Learn More >Endothelin-1 (EDN1) can evoke histamine-independent pruritus in mammals and is upregulated in the lesional epidermis of atopic dermatitis (AD). EDN1 increases the production of interleukin 25 (IL-25) from keratinocytes to accelerate T helper type 2 immune deviation. Plasma EDN1 levels are positively correlated with the clinical severity and itch intensity of AD. Therefore, we hypothesized that the inhibition of EDN1 might be useful for treating atopic inflammation and itch and investigated the effects of the topical application of the EDN1 receptor antagonist bosentan on the skin inflammation and itch in a murine AD model.
Learn More >Serotonin (5-hydroxytryptamine, 5-HT) released by platelets, mast cells, and immunocytes is a potent inflammatory mediator which modulates pain and itch sensing in the peripheral nervous system. The serotonergic receptors expressed by primary afferent neurons involved in these sensory functions are not fully identified and appear to be to a large extent species dependent. Moreover, the mechanisms through which 5-HT receptor activation is coupled to changes in neuronal excitability have not been completely revealed. Using a combination of in vitro (calcium and voltage imaging and patch-clamp) and in vivo behavioral methods, we used both male and female Wistar rats to provide evidence for the involvement of two 5-HT receptor subtypes, 5-HT1A and 5-HT3, in mediating the sustained and transient effects, respectively, of 5-HT on rat primary afferent neurons involved in pain and itch processing. In addition, our results are consistent with a model in which sustained serotonergic responses triggered via the 5-HT1A receptor are due to closure of background potassium channels, followed by membrane depolarization and action potentials, during which the activation of voltage-gated calcium channels leads to calcium entry. Our results may provide a better understanding of mammalian serotonergic itch signaling.
Learn More >At least one third of HIV-1-afflicted individuals experience peripheral neuropathy. Although the underlying mechanisms are not known, they may involve neurotoxic HIV-1 proteins. We assessed the influence of the neurotoxic HIV-1 regulatory protein, Tat, on inflammatory and neuropathic nociceptive behaviors using transgenic male and female transgenic mice that conditionally expressed (or did not express) HIV-1 Tat in glial fibrillary acidic protein-expressing glia in the central and peripheral nervous systems. Tat induction significantly attenuated the time spent paw-licking following formalin injection (2.5%, i.pl.) in both male and female mice. However, significant sex differences were observed in the onset and magnitude of inflammation and sensory sensitivity following complete Freund's adjuvant (CFA) injection (10%, i.pl.) after Tat activation. Unlike female mice, males showed a significant attenuation of paw swelling and an absence of mechanical/thermal hypersensitivity in response to CFA after Tat induction. Male Tat(+) mice also showed accelerated recovery from chronic constrictive nerve injury (CCI)-induced neuropathic mechanical and thermal hypersensitivity compared to female Tat(+) mice. Morphine (3.2 mg/kg) fully reversed CCI-induced mechanical hypersensitivity in female Tat(-) mice, but not in Tat(+) females. The ability of Tat to decrease edema, paw swelling, and limit allodynia suggest a sequela of events in which Tat-induced functional deficits precede the onset of mechanical hypersensitivity. Moreover, HIV-1 Tat attenuated responses to inflammatory and neuropathic insults in a sex-dependent manner. HIV-1 Tat appears to directly contribute to HIV sensory neuropathy and reveals sex differences in HIV responsiveness and/or the underlying peripheral neuroinflammatory and nociceptive mechanisms.
Learn More >The present series of studies examine the impact of systemically administered therapeutics on peripheral nerve injury (males; unilateral sciatic chronic constriction injury [CCI])-induced suppression of voluntary wheel running, across weeks after dosing cessation. Following CCI, active phase running distance and speed are suppressed throughout the 7-week observation period. A brief course of morphine, however, increased active phase running distance and speed throughout this same period, an effect apparent only in sham rats. For CCI rats, systemic co-administration of morphine with antagonists of either P2X7 (A438079) or TLR4 ((+)-naloxone) (receptors critical to the activation of NLRP3 inflammasomes and consequent inflammatory cascades) returned running behavior of CCI rats to that of shams through 5+ weeks after dosing ceased. This is a striking difference in effect compared to our prior CCI allodynia results using systemic morphine plus intrathecal delivery of these same antagonists, wherein a sustained albeit partial suppression of neuropathic pain was observed. This may point to actions of the systemic drugs at multiple sites along the neuraxis, modulating injury-induced, inflammasome-mediated effects at the injured sciatic nerve and/or dorsal root ganglia, spinal cord, and potentially higher levels. Given that our data to date point to morphine amplifying neuroinflammatory processes put into motion by nerve injury, it is intriguing to speculate that co-administration of TLR4 and/or P2X7 antagonists can intervene in these inflammatory processes in a beneficial way. That is, that systemic administration of such compounds may suppress inflammatory damage at multiple sites, rapidly and persistently returning neuropathic animals to sham levels of response.
Learn More >Temporomandibular Disorder (TMD) patients report amplification of pain in the masticatory muscles after psychological trauma or stressful conditions. The mechanisms underlying this phenomenon are yet to be elucidated. This study combined immunohistochemistry with single cell in vivo electrophysiology recordings of masticatory muscle afferent fibers to investigate the role of α-adrenergic receptors in muscle nociception. It was found that a subset of trigeminal afferent fibers which innervate the masseter and temporal muscles expressed α, α and α receptors, including a smaller number of putative nociceptors which co-expressed TrpV receptors. Local injection of the selective α adrenergic receptor agonist phenylephrine into masticatory muscle decreased and increased the mechanical activation threshold of slow and fast conducting afferent fibers, respectively. This effect was reversed by co-administration of the α selective antagonist terazosin. To rule out the possibility that local ischemia was responsible for the observed effect of phenylephrine on masticatory muscle afferent fibers, additional experiments were conducted where blood flow to the masticatory muscle was reduced by common carotid artery occlusion. This investigation found that muscle blood flow occlusion increased the mechanical activation threshold of the majority of masticatory muscle afferent fibers unrelated to conduction velocity. These findings suggest that under conditions of increased sympathetic tone, such as those related to stress, noradrenaline may sensitize masticatory muscle nociceptors to increase pain and desensitize muscle proprioceptors to alter muscle tone, through activation of α receptors.
Learn More >Pharmacological evidence indicated a functional interaction between neuropeptide FF (NPFF) and cannabinoid systems, and the cannabinoids combined with the NPFF receptor agonist neuropeptide VF (NPVF) produced antinociception without tolerance. In the present study, VF-13, a chimeric peptide containing the pharmacophores of the endogenous cannabinoid peptide VD-hemopressin(α) (VD-Hpα) and NPVF, was synthesized and pharmacologically evaluated. In vitro, VF-13 significantly upregulated the phosphorylated level of extracellular signal-regulated kinase 1/2 (ERK1/2) in CHO cells stably expressing CB1 receptors and inhibited forskolin-induced cAMP accumulation in HEK293 cells stably expressing NPFF or NPFF receptors. Moreover, VF-13 induced neurite outgrowth in Neuro 2A cells via CB1 and NPFF receptors. These results suggest that VF-13 exhibits multifunctional agonism at CB1, NPFF and NPFF receptors in vitro. Interestingly, intracerebroventricular VF-13 produced dose-dependent antinociception in mouse models of tail-flick and carrageenan-induced inflammatory pain via the TRPV1 receptor. In contrast, the reference compound (m)VD-Hpα-NH induced CB1 receptor-mediated supraspinal antinociception. Additionally, subcutaneous injection of (m)VD-Hpα-NH and VF-13 produced significant antinociception in carrageenan-induced inflammatory pain model. In the tetrad assay, our data demonstrated that VF-13 elicited hypothermia, but not catalepsy and hypoactivity after intracerebroventricular injection. Notably, VF-13 produced non-tolerance forming antinociception over 6 days treatment in both acute and inflammatory pain models. Furthermore, VF-13 had no apparent effects on gastrointestinal transit, pentobarbitone-induced sedation, food intake, and motor coordination at the supraspinal level. In summary, VF-13, a novel chimeric peptide of VD-Hpα and NPVF, produced non-tolerance forming antinociception in preclinical pain models with reduced cannabinoid-related side effects.
Learn More >In the peripheral nerve, mechanosensitive axons are insulated by myelin, a multilamellar membrane formed by Schwann cells. Here, we offer first evidence that a myelin degradation product induces mechanical hypersensitivity and global transcriptomics changes in a sex-specific manner. Focusing on downstream signaling events of the functionally active 84-104 myelin basic protein (MBP84-104) fragment released after nerve injury, we demonstrate that exposing the sciatic nerve to MBP84-104 via endoneurial injection produces robust mechanical hypersensitivity in female, but not in male, mice. RNA-Seq and systems biology analyses revealed a striking sexual dimorphism in molecular signatures of the dorsal root ganglia (DRG) and spinal cord response, not observed at the nerve injection site. Mechanistically, intra-sciatic MBP84-104 induced phospholipase C (PLC)-driven (females) and phosphoinositide 3-kinase-driven (males) phospholipid metabolism (tier 1). PLC/inositol trisphosphate receptor (IP3R) and estrogen receptor co-regulation in spinal cord yielded Ca2+-dependent nociceptive signaling induction in females that was suppressed in males (tier 2). IP3R inactivation by intrathecal xestospongin C attenuated the female-specific hypersensitivity induced by MBP84-104. According to sustained sensitization in tiers 1-2, T cell-related signaling spreads to the DRG and spinal cord in females, but remains localized to the sciatic nerve in males (tier 3). These results are consistent with our previous finding that MBP84-104-induced pain is T cell-dependent. In summary, an autoantigenic peptide endogenously released in nerve injury triggers multi-site, sex-specific transcriptome changes, leading to neuropathic pain only in female mice. MBP84-104 acts through sustained co-activation of metabolic, estrogen receptor-mediated nociceptive and autoimmune signaling programs.
Learn More >Unpleasant somatosensory stimuli such as pain and itch can interrupt normal behavior. But survival can depend on resuming normal behavior before these challenges are fully resolved. The neural mechanisms that prioritize behavior when individuals are challenged with unpleasant somatosensory sensations, however, are not fully understood. Recently, we identified a neural circuit activated by hunger that can inhibit pain, prioritizing food seeking over tending to an injury. Here, we examine the ability of hunger, and neurons activated by hunger, to inhibit behavioral responses to another unpleasant somatosensory sensation – itch. We demonstrate that food deprivation inhibits scratching induced by 3 different pruritogenic stimuli: histamine, serotonin, and chloroquine. The inhibition of scratching correlates with the level of food deprivation, suggesting a cross-competition of alarm systems in the brain whereby more energy need more efficiently inhibits competing drives. Finally, we show that activity in hunger-sensitive, hypothalamic agouti-related protein (AgRP)-expressing neurons is sufficient to inhibit itch. Taken together, we showed that hunger or AgRP neuron activity inhibits itch, demonstrating that organisms have neural systems to filter and process ascending spinal signals activated by unpleasant somatosensory stimuli to prioritize salient needs.
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