Calcitonin Gene-related Peptide In Mice Research Articles

Trimethoxyflavanone relieves Paclitaxel-induced neuropathic pain via inhibiting expression and activation of P2X7 and production of CGRP in mice

Paclitaxel (PTX) is an anticancer drug used to treat solid tumors, but one of its common adverse effects is chemotherapy-induced peripheral neuropathy (CIPN). Currently, there is limited understanding of neuropathic pain associated with CIPN and effective treatment strategies are inadequate. Previous studies report the analgesic actions of Naringenin, a dihydroflavonoid compound, in pain. Here we observed that the anti-nociceptive action of a Naringenin derivative, Trimethoxyflavanone (Y3), was superior to Naringenin in PTX-induced pain (PIP). An intrathecal injection of Y3 (1 μg) reversed the mechanical and thermal thresholds of PIP and suppressed the PTX-induced hyper-excitability of dorsal root ganglion (DRG) neurons. PTX enhanced the expression of ionotropic purinergic receptor P2X7 (P2X7) in satellite glial cells (SGCs) and neurons in DRGs. The molecular docking simulation predicts possible interactions between Y3 and P2X7. Y3 reduced the PTX-enhanced P2X7 expression in DRGs. Electrophysiological recordings revealed that Y3 directly inhibited P2X7-mediated currents in DRG neurons of PTX-treated mice, suggesting that Y3 suppressed both expression and function of P2X7 in DRGs post-PTX administration. Y3 also reduced the production of calcitonin gene-related peptide (CGRP) in DRGs and at the spinal dorsal horn. Additionally, Y3 suppressed the PTX-enhanced infiltration of Iba1-positive macrophage-like cells in DRGs and overactivation of spinal astrocytes and microglia. Therefore, our results indicate that Y3 attenuates PIP via inhibiting P2X7 function, CGRP production, DRG neuron sensitization, and abnormal spinal glial activation. Our study implies that Y3 could be a promising drug candidate against CIPN-associated pain and neurotoxicity.

Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice

Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

Protective Effect of TRPM8 against Indomethacin-Induced Small Intestinal Injury via the Release of Calcitonin Gene-Related Peptide in Mice.

Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by mild cooling and chemical agents including menthol. Nonsteroidal anti-inflammatory drugs have antipyretic, analgesic effects, and they can cause stomach and small intestinal injury. The current study investigated the role of TRPM8 in the pathogenesis of indomethacin-induced small intestinal injury. In male TRPM8-deficient (TRPM8KO) and wild-type (WT) mice, intestinal injury was induced via the subcutaneous administration of indomethacin. In addition, the effect of WS-12, a specific TRPM8 agonist, was examined in TRPM8KO and WT mice with indomethacin-induced intestinal injury. TRPM8KO mice had a significantly higher intestinal ulcerogenic response to indomethacin than WT mice. The repeated administration of WS-12 significantly attenuated the severity of intestinal injury in WT mice. However, this response was abrogated in TRPM8KO mice. Furthermore, in TRPM8-enhanced green fluorescent protein (EGFP) transgenic mice, which express EGFP under the direction of TRPM8 promoter, the EGFP signals in the indomethacin-treated intestinal mucosa were upregulated. Further, the EGFP signals were commonly found in calcitonin gene-related peptide (CGRP)-positive sensory afferent neurons and partly colocalized with substance P (SP)-positive neurons in the small intestine. The intestinal CGRP-positive neurons were significantly upregulated after the administration of indomethacin in WT mice. Nevertheless, this response was abrogated in TRPM8KO mice. In contrast, indomethacin increased the expression of intestinal SP-positive neurons in not only WT mice but also TRPM8KO mice. Thus, TRPM8 has a protective effect against indomethacin-induced small intestinal injury. This response may be mediated by the upregulation of CGRP, rather than SP.

CGRPergic Nerve TRPA1 Channels Contribute to Epigallocatechin Gallate-Induced Neurogenic Vasodilation.

Green tea polyphenol epigallocatechin gallate (EGCG), promotes vasodilation and reduces blood pressure, mechanisms of which are not fully resolved. Recent reports suggested that EGCG can activate heterologously expressed mouse and zebrafish TRPA1 channels. Activation of TRPA1 in sensory neurons triggers the release of calcitonin gene-related peptide (CGRP), a potent vasodilator. Whether CGRP-containing (CGRPergic) sensory nerves contribute to EGCG-induced reduction in vascular resistance remains unclear. In this study, we demonstrate that intravenous infusion of EGCG elevated the plasma level of CGRP in mice, an effect that was attenuated by TRPA1 channel blocker A-967079. EGCG-induced increase in mesenteric artery blood flow and reduction in mean arterial pressure were reversed by A-967079, CGRP receptor antagonist CGRP8-37, and CGRP depletion in perivascular nerves. Moreover, EGCG stimulated TRPA1-dependent intracellular Ca2+ elevation and CGRP release in a differentiated rat embryonic dorsal root ganglion/mouse neuroblastoma hybrid cell line. Together, these data suggest that EGCG-induced activation of TRPA1 channels in perivascular CGRPergic nerves decreases vascular resistance via Ca2+-dependent exocytosis of CGRP.

RAMP1 suppresses mucosal injury from dextran sodium sulfate-induced colitis in mice.

Calcitonin gene-related peptide (CGRP) is thought to be involved in the modulation of intestinal motility. CGRP receptor is composed of receptor activity-modifying protein (RAMP) 1 combined with calcitonin receptor-like receptor (CRLR) for CGRP. The study investigated the role of CGRP in mice with experimentally induced colitis. The study used dextran sodium sulfate (DSS) to induce colitis in mice. The study compared the severity of colitis in wild-type (WT) mice, mice treated with a CGRP receptor antagonist (CGRP8-37 ), and RAMP1 knockout (-/- ) mice. Pathological changes in the mucosa were assessed, and inflammatory cells and cytokine levels were measured. The severity of inflammation in DSS-induced colitis increased markedly in CGRP8-37 -treated mice and RAMP1-/- mice compared with WT mice. RAMP1-/- mice showed more severe damage compared with CGRP8-37 -treated mice. The number of periodic acid-Schiff-positive cells decreased in CGRP8-37 -treated mice compared with WT mice and was even further decreased in RAMP1-/- mice. RAMP1 was expressed by macrophages, mast cells, and T-cells. RAMP1-/- mice exhibited excessive accumulation of macrophages and mast cells into the colonic tissue with increased levels of tumor necrosis factor-α and interleukin-1β as compared with WT mice. Infiltration of T-cells into the colonic mucosa, which was associated with the expression of T helper (Th) cytokines including Th1 (interferon gamma) and Th17 (IL-17), was augmented in RAMP1-/- mice. The findings of this study suggest that RAMP1 exerted mucosal protection in DSS-induced colitis via attenuation of recruitment of inflammatory cells and of pro-inflammatory cytokines.

Activation of NPFFR2 leads to hyperalgesia through the spinal inflammatory mediator CGRP in mice

Neuropeptide FF (NPFF) is recognized as an opioid modulating peptide that regulates morphine-induced analgesia. The aim of this study was to delineate the role of NPFFR2 in pain transmission. We found the expression levels of NPFF and NPFFR2 were increased in the lumbar dorsal horn of animals with CFA- and carrageenan-induced inflammation and both NPFFR2 over-expressing transgenic (NPFFR2-Tg) and NPFFR2 agonist-treated mice displayed hyperalgesia. BOLD signals from functional MRI showed that NPFFR2-Tg mice exhibited increased activation of pain-related brain regions after painful stimulation when compared to WT mice. Inflammatory mediators within the spinal cord, calcitonin gene-related peptide (CGRP) and substance P (SP), were up-regulated in NPFFR2-Tg and chronic NPFFR2 agonist-treated mice. In DRG cultures, treatment with an NPFFR2 agonist induced the expression and release of CGRP, an action which was blocked by NPFFR2 siRNA. Furthermore, treatment with a CGRP antagonist ameliorated the pain hyperalgesia in NPFFR2-Tg mice, returning the pain threshold to a control level. However, treatment with a SP antagonist reduced the pain responses in both WT and NPFFR2-Tg mice and did not suppress pain hypersensitivity in NPFFR2-Tg mice. Together, these results demonstrate that NPFFR2 activation modulates pain transmission by up-regulating the pain mediator CGRP, leading to hyperalgesia.

Brain-derived neurotrophic factor enhances the contraction of intestinal muscle strips induced by SP and CGRP in mice

Background and aimsBrain-derived neurotrophic factor (BDNF) has been found in the intestinal tract of a variety of species. Its effects on visceral hyperalgesia have been examined to some degree, but limited studies have focused on gut motility. The aim of the present study was to investigate the effects of BDNF on gut motility of mice. MethodsLongitudinal muscle (LM) strips were prepared from mice ileum and distal colon. The motility of gut was evaluated by the contraction of LM strips, which was recorded by a polyphisograph in vitro. Firstly, the roles of substance P (SP), calcitonin gene-related peptide (CGRP), and acetylcholine (ACh) on the contraction of LM strips were clarified. Then the exogenous BDNF was administered, and the alterations of SP/CGRP/ACh-induced contractions of the muscle strips were explored. Finally, heterozygous BDNF+/− mice and antibody of TrkB were introduced to investigate the role of endogenous BDNF on the SP/CGRP/ACh-induced gut motility. Key resultsSP (10−8–10−6molL−1), CGRP (10−8–10−7molL−1) and ACh (10−8–10−6molL−1) dose-dependently caused the contraction of LM strips from ileum and distal colon, while the excitatory effect of CGRP was preceded by a transient inhibition. But 10−6molL−1 CGRP inhibited the contraction of LM strips. Pretreatment with exogenous BDNF (10−8molL−1) remarkably enhanced the contraction of LM strips induced by SP (10−9–10−7molL−1) and CGRP (10−8–10−9molL−1). However, exogenous BDNF couldn't affect the contraction induced by ACh (10−9–10−7molL−1). The excitatory effects of SP (10−8–10−6molL−1) and CGRP (10−8–10−7molL−1) on the contractions of LM strips from ileum and distal colon were significantly attenuated in BDNF+/− mice compared with those in BDNF+/+ mice, while no difference of the effects of ACh (10−8–10−6molL−1) on LM strips was observed between BDNF+/− mice and BDNF+/+ mice. The monoclonal antibody of TrkB (TrkB-Ab) dramatically attenuated the excitatory effects of SP and CGRP on the contractions of LM strips, without affecting the excitatory effects of ACh. Conclusions and inferencesThese data clarified the excitatory effects of SP, ACh and bilateral effects of CGRP on gut motility of mice and confirmed an essential role of BDNF on accelerating gut motility by enhancing the excitatory effects of SP/CGRP.

Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes

To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells. The full-length of CGRP gene cDNA was acquired by RT-PCR and cloned into pShuttle-CMV. After linearization with Pme I, the recombinant plasmid (pShuttle-CMV-CGRP) was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-pShuttle-CGRP. The recombinant adenovirus plasmids were transformed into E.coli XL10-Gold cells to be amplified. Then the recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. PCR technique was used to detect target gene. The recombinant adenovirus particles were purified by CsC1 density gradient. The purified recombinant adenovirus was transfected to neonatal rat cardiomyocytes,and the recombinant adenovirus production was observed by fluorescent microscope. Expression of CGRP in hearts 7 days after intravenous delivery of adenoviral vectors AV-CGRP was determined by radioimmunoassay. The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC(57) by sequencing. The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pShuttle-CMV. The recombinant adenovirus plasmid AdEasy-pShuttle-CGRP was digested by Pac I endonuclease to form the typical DNA segments, whose length was about 3 kb and 30 kb. PCR analysis and fluorescent microscope observation confirmed that the CGRP gene was inserted into the adenovirus vector with very strong power of transfection. The recombinant adenovirus particles infected neonatal rat cardiomyocytes successfully. Radioimmunoassay showed that delivery of AV-CGRP significantly increased the expression of CGRP in mice hearts. The recombinant adenovirus vector of CGRP gene has been constructed,and it can infect neonatal rat cardiomyocytes successfully. Somatic delivery of CGRP gene can significantly increase the expression of CGRP in mice hearts. The results may provide a sound foundation for further study on the value of CGRP as the target for gene therapy in both laboratory and clinical trials.

Role of Calcitonin Gene-Related Peptide in Light-Aversive Behavior: Implications for Migraine

Migraine is a chronic neurological disorder characterized by recurrent episodes of severe unilateral throbbing head pain and associated symptoms, such as photophobia. Our current understanding of the mechanisms underlying migraine has been hampered by limitations in ascertaining migraine symptoms in animal models. Clinical studies have established the neuropeptide calcitonin gene-related peptide (CGRP) as a key player in migraine. Here, we establish a genetic model of photophobia by engineering increased sensitivity to CGRP in mice. These transgenic mice (nestin/hRAMP1) display light-aversive behavior that is greatly enhanced by intracerebroventricular injection of CGRP and blocked by coadministration of the CGRP receptor antagonist olcegepant. This behavior appears to be an indicator of photophobia and cannot be fully explained by gross abnormality of ocular anatomy or differences in general anxiety or motor activity. Our findings demonstrate that a single gene, receptor activity-modifying protein 1 (RAMP1), can be a modifier of photophobia and, by extension, suggest that genetic or epigenetic modulation of RAMP1 levels may contribute to migraine susceptibility. Moreover, they validate CGRP hypersensitive mice as a tool for exploring the neurobiology and novel therapies for migraine and other disorders involving photophobia.

ENOS-dependent vascular interaction between nitric oxide and calcitonin gene-related peptide in mice: gender selectivity and effects on blood aggregation

The present study was performed to explore a possible vascular interplay between nitric oxide (NO) and calcitonin gene-related peptide (CGRP). We examined factors affecting CGRP release by the NO donor, nitroglycerin (NTG) and the potential involvement of endothelial NO synthase (eNOS) using eNOS knockout (−/−) vs. wild-type (+/+) mice. In the female eNOS (+/+) mice, but not in males, in vitro NTG (0.73 mM) induced significant increases in the release of CGRP-like immunoreactivity (CGRP-LI) from the aorta and the heart but not from the small intestine. In eNOS (−/−) mice, NTG incubation did not induce any CGRP-LI changes in either gender. These results suggest that NTG-induced CGRP release is eNOS-dependent and tissue- and gender-selective. The functional implication of this NO–CGRP interaction was further examined by testing the anti-aggregatory action of acetylcholine (Ach). Ach-induced platelet inhibition was significantly enhanced by the addition of aorta segments of either gender. However, the female aorta segments exhibited a greater platelet inhibitory effect, which could be reversed by the blockade of either CGRP or eNOS. Our study revealed a novel eNOS-dependent interaction between NO and CGRP, and the possible participation of regulatory peptides in affecting platelet function and possibly cardiovascular protection in females.

Nociceptive behavior after intrathecal injections of substance P, neurokinin A and calcitonin gene-related peptide in mice

Intrathecal injections of substance P (SP) or neurokinin A (NKA) in the mouse caused dose-dependent reciprocal hindlimb scratching, licking and biting responses directed to the caudal part of the body. NKA decreased the latency in the tail flick assay but like SP, did not alter the reactions in the hot plate test or the hypertonic saline assay. Although immunoreactivity of calcitonin gene-related peptide (CGRP) was detected in mouse spinal cord, CGRP caused no behavioral reactions, nor did it significantly affect thermo- or chemonociception or the scratching induced by SP. Since NKA-like immunoreactivity was found to be present in sensory neurons, NKA as well as SP are likely transmitters of nociceptive primary afferent neurons.