Substance P

Involvement of substance P/NK1 receptor system in central sensitization in chronic pain.

Juan Chen, Yimin Lai, Wei Li

Chronic pain has become a serious health issue, affecting more than 30% of people worldwide. One of the main mechanisms of chronic pain is central sensitization. It is well known that substance P (SP) and its receptor, Neurokinin 1 receptor (NK1R), play an important role in transmission of nociceptive signals. However, whether SP/NK1R system is involved in central sensitization in chronic pain remains controversial. In the present study, we adopted spared nerve injury (SNI) mouse model to induce neuropathic pain and assessed the role of SP/NK1R system in the development of hyperalgesia and central sensitization. We observed that hyperalgesia occurred in non-injured body part of SNI mice in tail withdrawal test. We also demonstrated hyperexcitability of S1 apical dendrites in SNI mice. Notably, the hyperalgesia behavior and hyperactivity of S1 apical dendrites were alleviated by NK1R antagonist L-703606. These results indicate that SP/NK1R system is involved in central sensitization in chronic pain.

Hemokinin-1 is a mediator of chronic restraint stress-induced pain.

Éva Borbély, Angéla Kecskés, József Kun +7 more

The Tac4 gene-derived hemokinin-1 (HK-1) binds to the NK1 receptor, similarly to Substance P, and plays a role in acute stress reactions and pain transmission in mice. Here we investigated Tac4 mRNA expression in stress and pain-related regions and its involvement in chronic restraint stress-evoked behavioral changes and pain using Tac4 gene-deleted (Tac4-/-) mice compared to C57Bl/6 wildtypes (WT). Tac4 mRNA was detected by in situ hybridization RNAscope technique. Touch sensitivity was assessed by esthesiometry, cold tolerance by paw withdrawal latency from 0°C water. Anxiety was evaluated in the light-dark box (LDB) and open field test (OFT), depression-like behavior in the tail suspension test (TST). Adrenal and thymus weights were measured at the end of the experiment. We found abundant Tac4 expression in the hypothalamic-pituitary-adrenal axis, but Tac4 mRNA was also detected in the hippocampus, amygdala, somatosensory and piriform cortices in mice, and in the frontal regions and the amygdala in humans. In Tac4-/- mice of both sexes, stress-induced mechanical, but not cold hyperalgesia was significantly decreased compared to WTs. Stress-induced behavioral alterations were mild or absent in male WT animals, while significant changes of these parameters could be detected in females. Thymus weight decrease can be observed in both sexes. Higher baseline anxiety and depression-like behaviors were detected in male but not in female HK-1-deficient mice, highlighting the importance of investigating both sexes in preclinical studies. We provided the first evidence for the potent nociceptive and stress regulating effects of HK-1 in chronic restraint stress paradigm. Identification of its targets might open new perspectives for therapy of stress-induced pain.

Effects of Electroacupuncture on Gastrointestinal Motility Function, Pain, and Inflammation via Transient Receptor Potential Vanilloid 1 in a Rat Model after Colonic Anastomoses.

Xuelai Zhong, Zhaodi Zhang, Jiaying Li +3 more

Complications after colon surgery are a major obstacle to postoperative recovery. The purpose of this study was to investigate the effect of electroacupuncture (EA) at Zusanli (ST36) on gastrointestinal motility in rats after colonic anastomosis and the mechanism of transient receptor potential vanillin 1 (TRPV1) channel in regulating gastrointestinal motility, pain, and inflammation.

Intrathecally administered substance P activated the spinal defecation center and enhanced colorectal motility in anesthetized rats.

Kiyotada Naitou, Honoka Iwashita, Hiromi H Ueda +6 more

Noxious stimuli on the colorectum cause colorectal contractions through activation of descending monoaminergic pathways projecting from the supraspinal defecation center to the spinal defecation center. Since it is known that substance P is involved in the response to peripheral noxious stimuli in the spinal cord, we investigated the effects of intrathecally administered substance P at L6-S1 levels on colorectal motility in rats that were anesthetized with α-chloralose and ketamine. Intrathecally administered substance P enhanced colorectal motility, even after transection of the thoracic spinal cord at the T4 level. Severing the pelvic nerves, but not the colonic nerves, abolished substance P enhanced colorectal motility. In the spinal cord at L6-S1 levels, expression of mRNA coding neurokinin (NK) 1-3 receptors was detected by RT-PCR. Immunohistological experiments revealed that preganglionic neurons of the pelvic nerves express NK1 receptors, whereas expression of NK2 receptors was not found. In addition, substance P-containing fibers densely innervated around the preganglionic neurons expressing NK1 receptors. An intrathecally administered NK1 receptor antagonist (spantide) attenuated capsaicin-induced colorectal contractions. These results suggest that the colokinetic action of substance P is mediated by the NK1 receptor in the spinal defecation center. Our findings indicate that substance P may function as a neurotransmitter in the spinal defecation center.NEW & NOTEWORTHY We found that intrathecally administered substance P enhanced colorectal motility in anesthetized rats. Neurokinin (NK) 1 receptors, but not NK2 receptors, were detected in preganglionic neurons of the pelvic nerves. Blockade of NK1 receptors in the spinal cord attenuated the enhanced colorectal motility in response to intracolonic noxious stimuli. The findings indicate that substance P may function as a neurotransmitter in the spinal reflex pathway controlling defecation.

Neuropeptide-induced modulation of carcinogenesis in a metastatic breast cancer cell line (MDA-MB-231LUC+).

Silvia Gutierrez, M Danilo Boada

Metastatic cancer to bone is well-known to produce extreme pain. It has been suggested that the magnitude of this perceived pain is associated with disease progression and poor prognosis. These data suggest a potential cross-talk between cancer cells and nociceptors that contribute not only to pain, but also to cancer aggressiveness although the underlying mechanisms are yet to be stablished.

The two-faced effects of nerves and neuropeptides in corneal diseases.

Romina Mayra Lasagni Vitar, Paolo Rama, Giulio Ferrari

Corneal nerves are instrumental to maintain cornea integrity through regulation of key physiological functions such as tear secretion, blink reflex, and neuropeptide turnover. Corneal nerve injury/stimulation can follow many insults including mechanical/chemical trauma, infections and surgeries. Nerve disruption initiates a process named neurogenic inflammation which leads to edema, pain, and recruitment and activation of leukocytes. Interestingly, leukocyte influx in the cornea can further damage nerves by releasing inflammatory mediators-including neuropeptides. The clinical outcome of neuroinflammation can be beneficial or detrimental to corneal integrity. On one side, it ensures prompt wound healing and prevents infections. On the other, prolonged and/or deranged neuroinflammation can permanently disrupt corneal integrity and impair vision. The cornea is an ideal site to study peripheral neuroinflammation and neurogenic inflammation since it receives the highest density of sensory nerves of the entire body. We will review the corneal nerve anatomy and neurochemistry, discuss the beneficial and detrimental effects of neurogenic inflammation in corneal wound healing, inflammatory processes, and pain. We will also examine the emerging remote impact of corneal nerve disruption on the trigeminal ganglion and the brain, highlighting the key role of neuropeptide Substance P. Finally, we will discuss the clinical relevance of such neuroinflammatory network in the context of severe and highly prevalent ocular diseases, including potential treatments.

Harnessing the Anti-Nociceptive Potential of NK2 and NK3 Ligands in the Design of New Multifunctional μ/δ-Opioid Agonist-Neurokinin Antagonist Peptidomimetics.

Charlène Gadais, Justyna Piekielna-Ciesielska, Jolien De Neve +3 more

Opioid agonists are well-established analgesics, widely prescribed for acute but also chronic pain. However, their efficiency comes with the price of drastically impacting side effects that are inherently linked to their prolonged use. To answer these liabilities, designed multiple ligands (DMLs) offer a promising strategy by co-targeting opioid and non-opioid signaling pathways involved in nociception. Despite being intimately linked to the Substance P (SP)/neurokinin 1 (NK1) system, which is broadly examined for pain treatment, the neurokinin receptors NK2 and NK3 have so far been neglected in such DMLs. Herein, a series of newly designed opioid agonist-NK2 or -NK3 antagonists is reported. A selection of reported peptidic, pseudo-peptidic, and non-peptide neurokinin NK2 and NK3 ligands were covalently linked to the peptidic μ-opioid selective pharmacophore Dmt-DALDA (H-Dmt-d-Arg-Phe-Lys-NH2) and the dual μ/δ opioid agonist H-Dmt-d-Arg-Aba-βAla-NH2 (KGOP01). Opioid binding assays unequivocally demonstrated that only hybrids SBL-OPNK-5, SBL-OPNK-7 and SBL-OPNK-9, bearing the KGOP01 scaffold, conserved nanomolar range μ-opioid receptor (MOR) affinity, and slightly reduced affinity for the δ-opioid receptor (DOR). Moreover, NK binding experiments proved that compounds SBL-OPNK-5, SBL-OPNK-7, and SBL-OPNK-9 exhibited (sub)nanomolar binding affinity for NK2 and NK3, opening promising opportunities for the design of next-generation opioid hybrids.

Continuous infusion of substance P into rat striatum relieves mechanical hypersensitivity caused by a partial sciatic nerve ligation via activation of striatal muscarinic receptors.

Yoki Nakamura, Ryo Fukushige, Kohei Watanabe +4 more

Previous studies have demonstrated that continuous substance P (SP) infusion into the rat striatum attenuated hind paw formalin-induced nociceptive behaviors and mechanical hypersensitivity via a neurokinin-1 (NK1) receptor dependent mechanism. However, whether there is a role of striatal infusion of SP on chronic, neuropathic pain has yet to be demonstrated. The present study investigated the effect of continuous SP infusion into the rat striatum using a reverse microdialysis method is antinociceptive in a rat model of chronic, mononeuropathic pain. Two weeks after partial sciatic nerve injury, the ipsilateral hind paw demonstrated mechanical hypersensitivity. Infusion of SP (0.2, 0.4, or 0.8 μg/mL, 1 μL/min) for 120 min into the contralateral striatum dose-dependently relieved mechanical hypersensitivity. The antinociceptive effect of SP infusion was inhibited by co-infusion with the NK1 receptor antagonist CP96345 (10 μM). Neither ipsilateral continuous infusion nor acute microinjection of SP (10 ng) into the contralateral striatum was antinociceptive. A role of striatal muscarinic cholinergic neurons is suggested since co-infusion of SP with atropine (10 μM), but not the nicotinic receptor mecamylamine (10 μM), blocked antinociception. The current study suggests that activation of striatal muscarinic receptors through NK1 receptors could be a novel approach to managing chronic pain.

The Challenge of Basic Itch Research.

Earl Carstens, Taylor Follansbee, Mirela Iodi Carstens

Basic mechanisms and pathways of itch signaling are reviewed, with an emphasis on the progress to date as well as remaining challenges in translating current knowledge to the clinical treatment of chronic itch. Recent studies reveal 3 subsets of pruriceptive sensory neurons highly expressing itch-related genes. Their fibers project into the spinal cord to activate neurons expressing gastrin releasing peptide (GRP) and its receptor (GRPR), which connect to neurons that express the substance P (NK-1) receptor and project to the parabrachial nucleus and thalamus. Spinal inhibitory interneurons release GABA, glycine and dynorphin to modulate segmental itch transmission. However, near-ly all pruriceptive neurons also respond to algogens such as capsaicin. Alternative theories of itch-pain discrimination, such as intensity or spatial contrast, are based on the observation that focal stimulation of nociceptive nerve endings elicits itch while more wide-spread stimulation elicits pain. These findings cloud the issue of a labeled line for itch- a long-debated but currently unresolved challenge. In higher primates there is a dichotomy of histaminergic and non-histaminergic itch-signaling pathways which is less demarcated in rodents, suggesting species differences. A cardinal symptom of chronic itch is alloknesis, i.e., mechanical or touch-evoked itch. Recent evidence indicates that low-threshold mechanosensory afferents can access the spinal itch pathway, but are normally kept in check by inhibitory interneurons expressing neuropeptide Y (NPY). In chronic itch, NPY-mediated inhibition is reduced, allowing touch to excite itch-signaling pathways. These recent advances provide novel targets for development of therapeutic strategies to relieve chronic itch.

Synthesis and Pharmacological Evaluation of Hybrids Targeting Opioid and Neurokinin Receptors.

Karol Wtorek, Anna Adamska-Bartłomiejczyk, Justyna Piekielna-Ciesielska +6 more

Morphine, which acts through opioid receptors, is one of the most efficient analgesics for the alleviation of severe pain. However, its usefulness is limited by serious side effects, including analgesic tolerance, constipation, and dependence liability. The growing awareness that multifunctional ligands which simultaneously activate two or more targets may produce a more desirable drug profile than selectively targeted compounds has created an opportunity for a new approach to developing more effective medications. Here, in order to better understand the role of the neurokinin system in opioid-induced antinociception, we report the synthesis, structure-activity relationship, and pharmacological characterization of a series of hybrids combining opioid pharmacophores with either substance P (SP) fragments or neurokinin receptor (NK1) antagonist fragments. On the bases of the in vitro biological activities of the hybrids, two analogs, opioid agonist/NK1 antagonist Tyr-[d-Lys-Phe-Phe-Asp]-Asn-d-Trp-Phe-d-Trp-Leu-Nle-NH2 (2) and opioid agonist/NK1 agonist Tyr-[d-Lys-Phe-Phe-Asp]-Gln-Phe-Phe-Gly-Leu-Met-NH2 (4), were selected for in vivo tests. In the writhing test, both hybrids showed significant an antinociceptive effect in mice, while neither of them triggered the development of tolerance, nor did they produce constipation. No statistically significant differences in in vivo activity profiles were observed between opioid/NK1 agonist and opioid/NK1 antagonist hybrids.

Hemokinin-1 is an important mediator of pain in mouse models of neuropathic and inflammatory mechanisms.

Ágnes Hunyady, Zsófia Hajna, Tímea Gubányi +5 more

The Tac4 gene-derived hemokinin-1 (HK-1) is present in pain-related regions and activates the tachykinin NK1 receptor, but with binding site and signaling pathways different from Substance P (SP). NK1 receptor is involved in nociception, but our earlier data showed that it has no role in chronic neuropathic hyperalgesia, similarly to SP. Furthermore, NK1 antagonists failed in clinical trials as analgesics due to still unknown reasons. Therefore, we investigated the role of HK-1 in pain conditions of distinct mechanisms using genetically modified mice. Chronic neuropathic mechanical and cold hyperalgesia after partial sciatic nerve ligation (PSL) were determined by dynamic plantar aesthesiometry and withdrawal latency from icy water, motor coordination on the accelerating Rotarod. Peripheral nerve growth factor (NGF) production was measured by ELISA, neuronal and glia cell activation by immunohistochemistry in pain-related regions. Acute somatic and visceral chemonocifensive behaviors were assessed after intraplantar formalin or intraperitoneal acetic-acid injection, respectively. Resiniferatoxin-induced inflammatory mechanical and thermal hyperalgesia by aesthesiometry and increasing temperature hot plate. Chronic neuropathic mechanical and cold hypersensitivity were significantly decreased in HK-1 deficient mice. NGF level in the paw homogenates of intact mice were significantly lower in case of HK-1 deletion. However, it significantly increased under neuropathic condition in contrast to wildtype mice, where the higher basal concentration did not show any changes. Microglia, but not astrocyte activation was observed 14 days after PSL in the ipsilateral spinal dorsal horn of wildtype, but not HK-1-deficient mice. However, under neuropathic conditions, the number of GFAP-positive astrocytes was significantly smaller in case of HK-1 deletion. Acute visceral, but not somatic nocifensive behavior, as well as neurogenic inflammatory mechanical and thermal hypersensitivity were significantly reduced by HK-1 deficiency similarly to NK1, but not to SP deletion. We provide evidence for pro-nociceptive role of HK-1, via NK1 receptor activation in acute inflammation models, but differently from SP-mediated actions. Identification of its targets and signaling can open new directions in pain research.

Genetic and pharmacological antagonism of NK1 receptor prevents opiate abuse potential.

A J Sandweiss, M I McIntosh, A Moutal +8 more

Development of an efficacious, non-addicting analgesic has been challenging. Discovery of novel mechanisms underlying addiction may present a solution. Here we target the neurokinin system, which is involved in both pain and addiction. Morphine exerts its rewarding actions, at least in part, by inhibiting GABAergic input onto substance P (SP) neurons in the ventral tegmental area (VTA), subsequently increasing SP release onto dopaminergic neurons. Genome editing of the neurokinin 1 receptor (NK1R) in the VTA renders morphine non-rewarding. Complementing our genetic approach, we demonstrate utility of a bivalent pharmacophore with dual activity as a μ/δ opioid agonist and NK1R antagonist in inhibiting nociception in an animal model of acute pain while lacking any positive reinforcement. These data indicate that dual targeting of the dopaminergic reward circuitry and pain pathways with a multifunctional opioid agonist-NK1R antagonist may be an efficacious strategy in developing future analgesics that lack abuse potential.

From the Anti-Nociceptive Substance P Metabolite Substance P (1-7) to Small Peptidomimetics.

Mathias Hallberg, Anja Sandstrom

Substance P (SP) is associated with pain and inflammatory processes and is released from terminals of specific sensory nerves. This undecapeptide that mediates its effects through the neurokinin type 1 (NK1) receptor, is rapidly degraded in vivo to smaller fragments. The heptapeptide SP(1-7) with a hitherto unknown receptor, is a major bioactive fragment and displays often opposite actions to those induced by SP. Hence, SP(1-7) elicits anti-nociceptive and anti-hyperalgesic effects. These observations have attracted a substantial interest and in this mini-review the efforts to transform the heptapeptide SP(1-7) into more drug-like small-molecule SP(1-7) peptidomimetics as a potential new class of analgesics are summarized. Structure-activity relationship studies and subsequent amidation of the C-terminal and truncations from the N-terminal of the heptapeptide delivered the bioactive dipeptide amide Gln- Phe-NH2 showing a high affinity at the SP(1-7) binding site. Similarly, endomorphin-2, an endogenous opioid ligand containing a C-terminal carboxamide group, demonstrated a high affinity at the SP(1-7) binding site. Endomorphin-2 subjected to truncations yielded the potent dipeptide amide Phe-Phe-NH2. Structural optimization of the latter furnished more drug-like high affinity ligands and among those a constrained cis-3-phenylpyrrolidine derivative that after peripheral administration produced a significant anti-allodynic effect in a mouse SNI model of neuropathic pain. This SP(1-7) peptidomimetic was as effective as SP(1-7) in alleviating mechanical allodynia in mice. Although, additional structural modifications are needed to achieve compounds exhibiting high/fair bioavailability after oral administration, the examples presented herein demonstrate that the bioactive peptides SP(1-7) and endomorphin-2 can be converted into low molecular weight compounds that are able to mimic the in vivo actions of the heptapeptide SP(1-7).

Involvement of the cystathionine-γ-lyase/Cav3.2 pathway in substance P-induced bladder pain in the mouse, a model for nonulcerative bladder pain syndrome.

Maho Tsubota, Yasumasa Okawa, Yuhei Irie +5 more

Hydrogen sulfide (H2S) formed by cystathionine-γ-lyase (CSE) enhances the activity of Cav3.2 T-type Ca2+ channels, contributing to the bladder pain accompanying hemorrhagic cystitis caused by systemic administration of cyclophosphamide (CPA) in mice. Given clinical and fundamental evidence for the involvement of the substance P/NK1 receptor systems in bladder pain syndrome (BPS)/interstitial cystitis (IC), we created an intravesical substance P-induced bladder pain model in mice and analyzed the possible involvement of the CSE/Cav3.2 pathway. Bladder pain/cystitis was induced by i.p. CPA or intravesical substance P in female mice. Bladder pain was evaluated by counting nociceptive behavior and by detecting referred hyperalgesia in the lower abdomen and hindpaw. The isolated bladder tissue was weighed to estimate bladder swelling and subjected to histological observation and Western blotting. Intravesical substance P caused profound referred hyperalgesia accompanied by little bladder swelling or edema 6-24 h after the administration, in contrast to i.p. CPA-induced nociceptive behavior/referred hyperalgesia with remarkable bladder swelling/edema and urothelial damage. The bladder pain and/or cystitis symptoms caused by substance P or CPA were prevented by the NK1 receptor antagonist. CSE in the bladder was upregulated by substance P or CPA, and the NK1 antagonist prevented the CPA-induced CSE upregulation. A CSE inhibitor, a T-type Ca2+ channel blocker and gene silencing of Cav3.2 abolished the intravesical substance P-induced referred hyperalgesia. The intravesical substance P-induced pain in mice is useful as a model for nonulcerative BPS, and involves the activation of the NK1 receptor/CSE/H2S/Cav3.2 cascade.

Analgesia by Deletion of Spinal Neurokinin 1 Receptor Expressing Neurons Using a Bioengineered Substance P-Pseudomonas Exotoxin Conjugate.

Michael J Iadarola, Matthew R Sapio, Xunde Wang +5 more

Cell deletion approaches to pain directed at either the primary nociceptive afferents or second-order neurons are highly effective analgesic manipulations. Second-order spinal neurons expressing the neurokinin 1 (NK1) receptor are required for the perception of many types of pain. To delete NK1+ neurons for the purpose of pain control, we generated a toxin–peptide conjugate using DTNB-derivatized (Cys0) substance P (SP) and a N-terminally truncated Pseudomonas exotoxin (PE35) that retains the endosome-release and ADP-ribosylation enzymatic domains but with only one free sulfhydryl side chain for conjugation. This allowed generation of a one-to-one product linked by a disulfide bond (SP-PE35). In vitro, Chinese hamster ovary cells stably transfected with the NK1 receptor exhibited specific cytotoxicity when exposed to SP-PE35 (IC50 = 5 × 10−11 M), whereas the conjugate was nontoxic to NK2 and NK3 receptor-bearing cell lines. In vivo studies showed that, after infusion into the spinal subarachnoid space, the toxin was extremely effective in deleting NK1 receptor-expressing cells from the dorsal horn of the spinal cord. The specific cell deletion robustly attenuated thermal and mechanical pain sensations and inflammatory hyperalgesia but did not affect motoric capabilities. NK1 receptor cell deletion and antinociception occurred without obvious lesion of non–receptor-expressing cells or apparent reorganization of primary afferent innervation. These data demonstrate the extraordinary selectivity and broad-spectrum antinociceptive efficacy of this ligand-directed protein therapeutic acting via receptor-mediated endocytosis. The loss of multiple pain modalities including heat and mechanical pinch, transduced by different populations of primary afferents, shows that spinal NK1 receptor-expressing neurons are critical points of convergence in the nociceptive transmission circuit. We further suggest that therapeutic end points can be effectively and safely achieved when SP-PE35 is locally infused, thereby producing a regionally defined analgesia.

Fosaprepitant for the Management of Refractory Pain in a Patient with Cancer-Related Dermatomyositis.

Jennifer D Dulin, Patrick J Coyne, Nicole M Bohm +1 more

Optimal pain management often requires multiple pharmacological interventions with the goal of disrupting the pain-signaling pathway and targeting the underlying pathophysiology. Off label use of nonpain medications may have a role in treating refractory pain syndromes.

Impact of N-methylation of the substance P 1-7 amide on anti-allodynic effect in mice after peripheral administration.

Anna Skogh, Anna Lesniak, Fabienne Z Gaugaz +6 more

Substance P 1-7 (SP1-7, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic degradation of the tachykinin substance P (SP). This heptapeptide often opposes the effects of the mother peptide. Hence, SP1-7 is having anti-inflammatory, anti-nociceptive and anti-hyperalgesic effects in experimental models. Despite all encouraging properties of SP1-7 its exact mode of action has not yet been elucidated which has hampered further development of this heptapeptide in drug discovery. Contrary to SP that mediates its biological activity via the NK-1 receptor, the N-terminal fragment SP1-7 acts through an unknown target that is distinct from all known opioid and tachykinin receptors. The SP1-7 amide 1 (Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) was previously shown to be superior to the endogenous SP1-7 in all experimental pain models where the two compounds were compared. Herein, we report that N-methylation scan of the backbone of the SP1-7 amide (1) results in peptides that are significantly less prone to undergo proteolysis in plasma from both mouse and human. However, with the two exceptions of the [MeLys3]SP1-7 amide (3) and the [MeGln5]SP1-7 amide (4), the peptides with a methyl group attached to the backbone are devoid of significant anti-allodynic effects after peripheral administration in the spared nerve injury (SNI) mouse model of neuropathic pain. It is suggested that the N-methylation does not allow these peptides to form the accurate bioactive conformations or interactions required for efficient binding to the macromolecular target. The importance of intact N-terminal Arg1 and C-terminal Phe7, anticipated to serve as address and message residues, respectively, for achieving the anti-allodynic effect is emphasized. Notably, the three heptapeptides: the SP1-7 amide (1), the [MeLys3]SP1-7 amide (3) amide and the [MeGln5]SP1-7 amide (4) are all considerably more effective in the SNI mouse model than gabapentin that is widely used in the clinic for treatment of neuropathic pain.

Hyperalgesia and sensitization of dorsal horn neurons following activation of NK-1 receptors in the rostral ventromedial medulla.

Sergey G Khasabov, Patrick Malecha, Joseph Noack +3 more

Neurons in the rostral ventromedial medulla (RVM) project to the spinal cord and are involved in descending modulation of pain. Several studies have shown that activation of neurokinin-1 (NK-1) receptors in the RVM produces hyperalgesia, although the underlying mechanisms are not clear. In parallel studies, we compared behavioral measures of hyperalgesia to electrophysiological responses of nociceptive dorsal horn neurons produced by activation of NK-1 receptors in the RVM. Injection of the selective NK-1 receptor agonist Sar9,Met(O2)11-substance P (SSP) into the RVM produced dose-dependent mechanical and heat hyperalgesia that was blocked by coadministration of the selective NK-1 receptor antagonist L-733,060. In electrophysiological studies, responses evoked by mechanical and heat stimuli were obtained from identified high-threshold (HT) and wide dynamic range (WDR) neurons. Injection of SSP into the RVM enhanced responses of WDR neurons, including identified neurons that project to the parabrachial area, to mechanical and heat stimuli. Since intraplantar injection of capsaicin produces robust hyperalgesia and sensitization of nociceptive spinal neurons, we examined whether this sensitization was dependent on NK-1 receptors in the RVM. Pretreatment with L-733,060 into the RVM blocked the sensitization of dorsal horn neurons produced by capsaicin. c-Fos labeling was used to determine the spatial distribution of dorsal horn neurons that were sensitized by NK-1 receptor activation in the RVM. Consistent with our electrophysiological results, administration of SSP into the RVM increased pinch-evoked c-Fos expression in the dorsal horn. It is suggested that targeting this descending pathway may be effective in reducing persistent pain.NEW & NOTEWORTHY It is known that activation of neurokinin-1 (NK-1) receptors in the rostral ventromedial medulla (RVM), a main output area for descending modulation of pain, produces hyperalgesia. Here we show that activation of NK-1 receptors produces hyperalgesia by sensitizing nociceptive dorsal horn neurons. Targeting this pathway at its origin or in the spinal cord may be an effective approach for pain management.

Redox-Dependent Modulation of T-Type Ca(2+) Channels in Sensory Neurons Contributes to Acute Anti-Nociceptive Effect of Substance P.

Dongyang Huang, Sha Huang, Haixia Gao +10 more

Neuropeptide substance P (SP) is produced and released by a subset of peripheral sensory neurons that respond to tissue damage (nociceptors). SP exerts excitatory effects in the central nervous system, but peripheral SP actions are still poorly understood; therefore, here, we aimed at investigating these peripheral mechanisms.

TRPA1, substance P, histamine and 5-hydroxytryptamine interact in an interdependent way to induce nociception.

Luana Fischer, Maria Isabel Lavoranti, Mariana de Oliveira Borges +5 more

Although TRPA1, SP, histamine and 5-hydroxytryptamine (5-HT) have recognized contribution to nociceptive mechanisms, little is known about how they interact with each other to mediate inflammatory pain in vivo. In this study we evaluated whether TRPA1, SP, histamine and 5-HT interact, in an interdependent way, to induce nociception in vivo.

Resolvin E1 Inhibits Substance P-Induced Potentiation of TRPV1 in Primary Sensory Neurons.

Youn Yi Jo, Ji Yeon Lee, Chul-Kyu Park

The neuropeptide substance P (SP) is expressed in primary sensory neurons and is commonly regarded as a "pain" neurotransmitter. Upon peripheral inflammation, SP activates the neurokinin-1 (NK-1) receptor and potentiates activity of transient receptor potential vanilloid subtype 1 (TRPV1), which is coexpressed by nociceptive neurons. Therefore, SP functions as an important neurotransmitter involved in the hypersensitization of inflammatory pain. Resolvin E1 (RvE1), derived from omega-3 polyunsaturated fatty acids, inhibits TRPV1 activity via activation of the chemerin 23 receptor (ChemR23)-an RvE1 receptor located in dorsal root ganglion neurons-and therefore exerts an inhibitory effect on inflammatory pain. We demonstrate here that RvE1 regulates the SP-induced potentiation of TRPV1 via G-protein coupled receptor (GPCR) on peripheral nociceptive neurons. SP-induced potentiation of TRPV1 inhibited by RvE1 was blocked by the Gαi-coupled GPCR inhibitor pertussis toxin and the G-protein inhibitor GDPβ-S. These results indicate that a low concentration of RvE1 strongly inhibits the potentiation of TRPV1, induced by the SP-mediated activation of NK-1, via a GPCR signaling pathway activated by ChemR23 in nociceptive neurons. RvE1 might represent a new therapeutic target for the treatment of inflammatory pain as a prospective endogenous inhibitor that strongly inhibits TRPV1 activity associated with peripheral inflammation.

Role of capsaicin-sensitive nerves and tachykinins in mast cell tryptase-induced inflammation of murine knees.

Éva Borbély, Katalin Sándor, Adrienn Markovics +6 more

Mast cell tryptase (MCT) is elevated in arthritic joints, but its direct effects are not known. Here, we investigated MCT-evoked acute inflammatory and nociceptive mechanisms with behavioural, in vivo imaging and immunological techniques.

Acute versus chronic phase mechanisms in a rat model of CRPS.

Tzuping Wei, Tian-Zhi Guo, Wen-Wu Li +2 more

Tibia fracture followed by cast immobilization in rats evokes nociceptive, vascular, epidermal, and bone changes resembling complex regional pain syndrome (CRPS). In most cases, CRPS has three stages. Over time, this acute picture, allodynia, warmth, and edema observed at 4 weeks, gives way to a cold, dystrophic but still painful limb. In the acute phase (at 4 weeks post fracture), cutaneous immunological and NK1-receptor signaling mechanisms underlying CRPS have been discovered; however, the mechanisms responsible for the chronic phase are still unknown. The purpose of this study is to understand the mechanisms responsible for the chronic phases of CRPS (at 16 weeks post fracture) at both the peripheral and central levels.

Environmental cold exposure increases blood flow and affects pain sensitivity in the knee joints of CFA-induced arthritic mice in a TRPA1-dependent manner.

Elizabeth S Fernandes, Fiona A Russell, Khadija M Alawi +12 more

The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor.

Involvement of spinal glutamate in nociceptive behavior induced by intrathecal administration of hemokinin-1 in mice.

Chizuko Watanabe, Hirokazu Mizoguchi, Giacinto Bagetta +1 more

The most recently identified tachykinin, hemokinin-1, was cloned from mouse bone marrow. While several studies indicated that hemokinin-1 is involved in pain and inflammation, the physiological functions of hemokinin-1 are not fully understood. Our previous research demonstrated that the intrathecal (i.t.) administration of hemokinin-1 (0.00625-1.6 nmol) dose-dependently induced nociceptive behaviors, consisting of scratching, biting and licking in mice, which are very similar with the nociceptive behaviors induced by the i.t. administration of substance P. Low-dose (0.0125 nmol) hemokinin-1-induced nociceptive behavior was inhibited by a specific NK1 receptor antagonist; however, high-dose (0.1 nmol) hemokinin-1-induced nociceptive behavior was not affected. In the present study, we found that the nociceptive behaviors induced by hemokinin-1 (0.1 nmol) were inhibited by the i.t. co-administration of MK-801 or D-APV, which are NMDA receptor antagonists. Moreover, we measured glutamate in the extracellular fluid of the mouse spinal cord using microdialysis. The i.t. administration of hemokinin-1 produced a significant increase in glutamate in the spinal cord, which was significantly reduced by co-administration with NMDA receptor antagonists. These results suggest that hemokinin-1-induced nociceptive behaviors may be mediated by the NMDA receptor in the spinal cord.

Selective innervation of NK1 receptor-lacking lamina I spinoparabrachial neurons by presumed nonpeptidergic Aδ nociceptors in the rat.

Najma Baseer, Abdullah S Al-Baloushi, Masahiko Watanabe +2 more

Fine myelinated (Aδ) nociceptors are responsible for fast, well-localised pain, but relatively little is known about their postsynaptic targets in the spinal cord, and therefore about their roles in the neuronal circuits that process nociceptive information. Here we show that transganglionically transported cholera toxin B subunit (CTb) labels a distinct set of afferents in lamina I that are likely to correspond to Aδ nociceptors, and that most of these lack neuropeptides. The vast majority of lamina I projection neurons can be retrogradely labelled from the lateral parabrachial area, and these can be divided into 2 major groups based on expression of the neurokinin 1 receptor (NK1r). We show that CTb-labelled afferents form contacts on 43% of the spinoparabrachial lamina I neurons that lack the NK1r, but on a significantly smaller proportion (26%) of those that express the receptor. We also confirm with electron microscopy that these contacts are associated with synapses. Among the spinoparabrachial neurons that received contacts from CTb-labelled axons, contact density was considerably higher on NK1r-lacking cells than on those with the NK1r. By comparing the density of CTb contacts with those from other types of glutamatergic bouton, we estimate that nonpeptidergic Aδ nociceptors may provide over half of the excitatory synapses on some NK1r-lacking spinoparabrachial cells. These results provide further evidence that synaptic inputs to dorsal horn projection neurons are organised in a specific way. Taken together with previous studies, they suggest that both NK1r(+) and NK1r-lacking lamina I projection neurons are directly innervated by Aδ nociceptive afferents.

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal-regulated kinase 1/2.

Yoki Nakamura, Hiroki Izumi, Ryo Fukushige +6 more

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an 'acute' phase (0-15 min) and 'tonic' phase (16-120 min), which is accompanied by significant phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post-formalin injection. To uncover a possible relationship between the slow-onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 μg/mL in microdialysis fiber, 1 μL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of 'tonic' phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre-treatment with NK1 receptor antagonist CP96345 (10 μM) blocked the effect of SP infusion. However, formalin-induced 'tonic' phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the 'tonic' phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP-NK1 receptor-ERK1/2 system. Noxious stimulation induces a slow-onset substance P (SP) release as a volume transmitter, activating extra-synaptic NK1 receptors, and evokes phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The SP-NK1-ERK1/2 system in the striatum decreases tonic nociception.

Ablating spinal NK1-bearing neurons eliminates the development of pain and reduces spinal neuronal hyperexcitability and inflammation from mechanical joint injury in the rat.

Christine L Weisshaar, Beth A Winkelstein

The facet joint is a common source of pain, especially from mechanical injury. Although chronic pain is associated with altered spinal glial and neuronal responses, the contribution of specific spinal cells to joint pain is not understood. This study used the neurotoxin [Sar(9),Met(O2)(11)]-substance P-saporin (SSP-SAP) to selectively eliminate spinal cells expressing neurokinin-1 receptor (NK1R) in a rat model of painful facet joint injury to determine the role of those spinal neurons in pain from facet injury. Following spinal administration of SSP-SAP or its control (blank-SAP), a cervical facet injury was imposed and behavioral sensitivity was assessed. Spinal extracellular recordings were made on day 7 to classify neurons and quantify evoked firing. Spinal glial activation and interleukin 1αα (IL1α) expression also were evaluated. SSP-SAP prevented the development of mechanical hyperalgesia that is induced by joint injury and reduced NK1R expression and mechanically evoked neuronal firing in the dorsal horn. SSP-SAP also prevented a shift toward wide dynamic range neurons that is seen after injury. Spinal astrocytic activation and interleukin 1α (IL1α) expression were reduced to sham levels with SSP-SAP treatment. These results suggest that spinal NK1R-bearing cells are critical in initiating spinal nociception and inflammation associated with a painful mechanical joint injury.

Botulinum toxin B in the sensory afferent: transmitter release, spinal activation, and pain behavior.

Marc J Marino, Tetsuji Terashima, Joanne J Steinauer +3 more

We addressed the hypothesis that intraplantar botulinum toxin B (rimabotulinumtoxin B: BoNT-B) has an early local effect upon peripheral afferent terminal releasing function and, over time, will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release, and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT-B (1 U) given unilaterally into the hind paw had no effect upon survival or motor function, but ipsilaterally decreased: (1) intraplantar formalin-evoked flinching; (2) intraplantar capsaicin-evoked plasma extravasation in the hind paw measured by Evans blue in the paw; (3) intraplantar formalin-evoked dorsal horn substance P (SP) release (neurokinin 1 [NK1] receptor internalization); (4) intraplantar formalin-evoked dorsal horn neuronal activation (c-fos); (5) ipsilateral dorsal root ganglion (DRG) vesicle-associated membrane protein (VAMP); (6) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; (7) ipsilateral activation of c-fos otherwise evoked bilaterally by intrathecal SP. These results indicate that BoNT-B, after unilateral intraplantar delivery, is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP, and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT-B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.

[The role of NMDA and NK1 receptors in cardiac nociceptive information transmissions in the spinal cords of rats].

Ling-Heng Kong, Na Sun, Juan-Xia Zhu +3 more

To determine the effects of NMDA and NK1 receptor agonist and antagonist on the EMG and the synaptic mechanism of nociceptive information transmissions in the spinal cords.