Calcitonin Gene-related Peptide Protein Levels Research Articles

Antagonism of CGRP Receptor: Central and Peripheral Mechanisms and Mediators in an Animal Model of Chronic Migraine.

Calcitonin-gene-related peptide (CGRP) plays a key role in migraine pathophysiology and more specifically in the mechanisms underlying peripheral and central sensitization. Here, we explored the interaction of CGRP with other pain mediators relevant for neuronal sensitization in an animal model of chronic migraine. Male Sprague-Dawley rats were exposed to nitroglycerin (NTG, 5 mg/kg, i.p.) or vehicle co-administered with the CGRP receptor antagonist olcegepant (2 mg/kg i.p.), or its vehicle, every other day over a 9-day period. Twenty-four hours after the last injection of NTG (or vehicle), behavioral test and ex vivo analysis were performed. Olcegepant attenuated NTG-induced trigeminal hyperalgesia in the second phase of the orofacial formalin test. Interestingly, it also reduced gene expression and protein levels of CGRP, pro-inflammatory cytokines, inflammatory-associated miRNAs (miR-155-5p, miR-382-5p, and miR-34a-5p), and transient receptor potential ankyrin channels in the medulla–pons area, cervical spinal cord, and trigeminal ganglia. Similarly, olcegepant reduced the NTG-induced increase in CGRP and inflammatory cytokines in serum. The findings show that the activation of the CGRP pathway in a migraine animal model was associated to the persistent activation of inflammatory pathways, which was paralleled by a condition of hyperalgesia. These molecular events are relevant for informing us about the mechanisms underlying chronic migraine.

Transient Receptor Potential Channel 4 Small-Molecule Inhibition Alleviates Migraine-Like Behavior in Mice.

Migraine is a common neurological disorder with few available treatment options. Recently, we have demonstrated the role of transient receptor potential cation channel subfamily C member 4 (TRPC4) in itch and the modulation of the calcitonin gene-related peptide (CGRP), a biomarker and emerging therapeutic target for migraine. In this study, we characterized the role of TRPC4 in pain and evaluated its inhibition as anti-migraine pain therapy in preclinical mouse models. First, we found that TRPC4 is highly expressed in trigeminal ganglia and its activation not only mediates itch but also pain. Second, we demonstrated that the small-molecule inhibitor ML204, a specific TRPC4 antagonist, significantly reduced episodic and chronic migraine-like behaviors in male and female mice after injection of nitroglycerin (NTG), a well-known migraine inducer in rodents and humans. Third, we found a significant decrease in CGRP protein levels in the plasma of both male and female mice treated with ML-204, which largely prevented the development of chronic migraine-like behavior. Using sensory neuron cultures, we confirmed that activation of TRPC4 elicited release of CGRP, which was significantly diminished by ML-204. Collectively, our findings identify TRPC4 in peripheral sensory neurons as a mediator of CGRP release and NTG-evoked migraine. Since a TRPC4 antagonist is already in clinical trials, we expect that this study will rapidly lead to novel and effective clinical treatments for migraineurs.

P2X4-receptor participates in EAAT3 regulation via BDNF-TrkB signaling in a model of trigeminal allodynia

ObjectivePrevious studies of neuropathic pain have suggested that the P2X4 purinoceptor (P2X4R) in spinal microglia is essential for maintaining allodynia following nerve injury. However, little is known about its role in inflammatory soup-induced trigeminal allodynia, which closely mimics chronic migraine status. Here, we determined the contributions of P2X4R and related signaling pathways in an inflammatory soup-induced trigeminal allodynia model.MethodsP2X4R gene and protein levels in the trigeminal nucleus caudalis were analyzed following repeated dural inflammatory soup infusions. p38, brain-derived neurotrophic factor, excitatory amino acid transporter 3, c-Fos, and calcitonin gene-related peptide protein levels in the trigeminal nucleus caudalis, as well as trigeminal sensitivity, were assessed among the different groups. Immunofluorescence staining was used to detect protein localization and expression in the trigeminal nucleus caudalis.ResultsRepeated inflammatory dural stimulation induced trigeminal hyperalgesia and the upregulation of P2X4R. Immunofluorescence revealed that P2X4R was expressed in trigeminal nucleus caudalis microglial cells. Blockage of P2X4R produced an anti-nociceptive effect, which was associated with an inhibition of inflammatory soup-induced increases in p38, brain-derived neurotrophic factor, excitatory amino acid transporter 3, c-Fos, and calcitonin gene-related peptide protein levels. The tyrosine receptor kinase B antagonist ANA-12 reversed trigeminal allodynia and the upregulation of excitatory amino acid transporter 3, c-Fos, and calcitonin gene-related peptide, whereas the agonist 7,8-dihydroxyflavone exacerbated these effects. Double immunostaining indicated that p38 and brain-derived neurotrophic factor were mainly expressed in microglial cells, whereas excitatory amino acid transporter 3 was primarily expressed in trigeminal nucleus caudalis neurons.ConclusionsThese data indicate that microglial P2X4R is involved in the regulation of excitatory amino acid transporter 3 via brain-derived neurotrophic factor-tyrosine receptor kinase B signaling following repeated inflammatory dural stimulation. Microglial P2X4R activation and microglia–neuron interactions in the trigeminal nucleus caudalis may play a role in the pathogenesis of migraine chronicity, and the modulation of P2X4R activation might be a potential therapeutic strategy.

The effect of 17β-estradiol on gene expression of calcitonin gene-related peptide and some pro-inflammatory mediators in peripheral blood mononuclear cells from patients with pure menstrual migraine.

The neuropeptide calcitonin gene-related peptide (CGRP) has long been postulated to play an integral role in the pathophysiology of migraine. Earlier studies showed that CGRP can stimulate the synthesis and release of nitric oxide (NO) and cytokines from trigeminal ganglion glial cells. The purpose of this study was to determine the effect of 17β-estradiol in regulation of CGRP expression, inducible nitric oxide synthase (iNOS) activity, and NO and interleukin-1beta (IL-1β) release in cultured peripheral blood mononuclear cells (PBMCs) from patients with pure menstrual migraine and healthy individuals. This study was performed on twelve patients with pure menstrual migraine and twelve age-and sex-matched healthy individuals. PBMCs treated with 17β-estradiol for 24 hr at physiological and pharmacological doses. Gene expression was evaluated by real time-PCR. CGRP and IL-1β proteins in culture supernatant were determined by ELISA method. Activity of iNOS in PBMCs and total nitrite in the culture supernatant were measured by colorimetric assays. Treatment with 17β-estradiol had a biphasic effect on expression of CGRP. We found that 17β-estradiol treatment at pharmacological dose significantly increases mRNA expression of CGRP in both groups (P<0.001), whereas at physiological dose it could significantly decrease CGRP mRNA expression (P<0.001), CGRP protein levels, IL-1β release, NO production and iNOS activity only in patient groups (P<0.05). Collectively, it appears that 17β-estradiol can exert protective effect on decrease of inflammation in migraine via decrease in levels of CGRP, IL-1β and iNOS activity; however, more studies are necessary in this regard.

Neuregulin-1β Regulates the migration of Different Neurochemical Phenotypic Neurons from Organotypically Cultured Dorsal Root Ganglion Explants.

Neuregulin-1β (NRG-1β) has multiple roles in the development and function in the nervous system and exhibits potent neuroprotective properties. In the present study, organotypically cultured dorsal root ganglion (DRG) explants were used to evaluate the effects of NRG-1β on migration of two major phenotypic classes of DRG neurons. The signaling pathways involved in these effects were also determined. Organotypically cultured DRG explants were exposed to NRG-1β (20 nmol/L), the phosphatidylinositol 3-kinase inhibitor LY294002 (10 μmol/L) plus NRG-1β (20 nmol/L), the extracellular signal-regulated protein kinase (ERK1/2) inhibitor PD98059 (10 μmol/L) plus NRG-1β (20 nmol/L), and LY294002 (10 μmol/L) plus PD98059 (10 μmol/L) plus NRG-1β (20 nmol/L), respectively, for 3 days. The DRG explants were continuously exposed to culture media as a control. After that, all above cultures were processed for detecting the mRNA levels of calcitonin gene-related peptide (CGRP) and neurofilament-200 (NF-200) by real-time PCR analysis. CGRP and NF-200 expression in situ was determined by fluorescent labeling technique. The results showed that NRG-1β elevated the mRNA and protein levels of CGRP and NF-200. NRG-1β also increased the number and the percentage of CGRP-immunoreactive (IR) migrating neurons and NF-200-IR migrating neurons. Inhibitors (LY294002, PD98059) either alone or in combination blocked the effects of NRG-1β. The contribution of NRG-1β on modulating distinct neurochemical phenotypic plasticity of DRG neurons suggested that NRG-1β signaling system might play an important role on the biological effects of primary sensory neurons.

Effects of rizatriptan on the expression of calcitonin gene-related peptide and cholecystokinin in the periaqueductal gray of a rat migraine model

Triptans are serotonin 5-hydroxytryptamine receptor 1B/D agonists that are highly effective in the treatment of migraine. We previously found that rizatriptan can reduce the expression of proenkephalin and P substance in the rat midbrain, suggesting that rizatriptan may exert its analgesic effects by influencing the endogenous pain modulatory system. Calcitonin gene-related peptide (CGRP) and cholecystokinin (CCK) are mainly responsible for antagonizing the analgesic effects of opioid peptides in the endogenous pain modulatory system. In this study, we investigated the effects of rizatriptan on the expression of CGRP and CCK in the periaqueductal gray (PAG), a key structure of the endogenous pain modulatory system, in a rat migraine model induced by nitroglycerin. We found that the mRNA and protein levels of CGRP and CCK in the PAG of migraine rats were significantly increased compared to those in control rats, and these levels were significantly reduced upon treatment with rizatriptan in migraine rats (P<0.05). Our results suggest that the expression of CGRP and CCK in the endogenous pain modulatory system may be increased during migraine attacks, which further antagonizes the analgesic effects of endogenous opioid peptides and induces sustained migraine. Rizatriptan, however, significantly reduces the levels of CGRP and CCK to enhance the inhibition of pain signals via the endogenous pain modulatory system, resulting in effective treatment of migraine.

Reactive Oxygen Species Induce Procalcitonin Expression in Trigeminal Ganglia Glia

To examine calcitonin gene-related peptide (CGRP) gene expression under inflammatory conditions using trigeminal ganglia organ cultures as an experimental system. These cultures have increased proinflammatory signaling that may mimic neurogenic inflammation in the migraine state. The trigeminal nerve sends peripheral pain signals to the central nervous system during migraine. Understanding the dynamic processes that occur within the trigeminal nerve and ganglion may provide insights into events that contribute to migraine pain. A neuropeptide of particular interest is CGRP, which can be elevated and play a causal role in migraine. However, most studies have overlooked a second splice product of the Calca gene that encodes calcitonin (CT), a peptide hormone involved in calcium homeostasis. Importantly, a precursor form of CT called procalcitonin (proCT) can act as a partial agonist at the CGRP receptor and elevated proCT has recently been reported during migraine. We used a trigeminal ganglion whole organ explant model, which has previously been demonstrated to induce pro-inflammatory agents in vitro. Quantitative polymerase chain reaction and immunohistochemistry were used to evaluate changes in messenger ribonucleic acid (mRNA) and protein levels of CGRP and proCT. Whole mouse trigeminal ganglia cultured for 24 hours showed a 10-fold increase in CT mRNA, with no change in CGRP mRNA. A similar effect was observed in ganglia from adult rats. ProCT immunoreactivity was localized in glial cells. Cutting the tissue blunted the increase in CT, suggesting that induction required the close environment of the intact ganglia. Consistent with this prediction, there were increased reactive oxygen species in the ganglia, and the elevated CT mRNA was reduced by antioxidant treatment. Surprisingly, reactive oxygen species were increased in neurons, not glia. These results demonstrate that reactive oxygen species can activate proCT expression from the CGRP gene in trigeminal glia by a paracrine regulatory mechanism. We propose that this glial recruitment pathway may occur following cortical spreading depression and neurogenic inflammation to increase CGRP nociceptive actions in migraine.

Transcutaneous Electrical Nerve Stimulation (TENS) Improves the Diabetic Cytopathy (DCP) via Up-Regulation of CGRP and cAMP

The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n = 15), DM group (n = 15) and control group (n = 15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG.

Calcitonin gene–related peptide regulates the early phase of liver regeneration

ObjectiveTo investigate the expression of calcitonin gene–related peptide (CGRP) and its role in the liver regeneration process after 70% hepatectomy (Hx). Materials and methodsWistar rats were divided into eight groups based on time after Hx. Remnant liver samples were collected serially 0 h, 1 h, 6 h, 12 h, 1 d, 2 d, 7 d, and 14 d after Hx (n = 6 for each time point). The expression level of the calcitonin/CGRP gene in the remnant liver was measured. Western bolts and immunohistochemistry were performed to determine the levels of CGRP in the regenerating liver. Furthermore, CGRP8-37 (a CGRP receptor antagonist) was used to examine the role of CGRP during liver regeneration. ResultsA marked upregulation of the calcitonin/CGRP gene was observed immediately after Hx, and the protein levels of CGRP in the liver, which were measured by western blot and immunohistochemistry, also rapidly increased after Hx. The liver regeneration rate was significantly attenuated by an administration of CGRP8-37 2 d after Hx. The mitotic index was evaluated by histologic examination 1 and 2 d after Hx and was also significantly lower in the CGRP8-37 group. In addition, CGRP8-37 treatment inhibited the phosphorylation of extracellular-signal regulated kinase 1/2. The levels of early response genes, such as c-fos, c-jun, and c-myc, were also downregulated by CGRP8-37. ConclusionThe calcitonin/CGRP gene may have an important role in the early phase of liver regeneration after Hx.