Induces Calcitonin Gene-related Peptide Research Articles

The effects of certain TRP channels and voltage-gated KCNQ/Kv7 channel opener retigabine on calcitonin gene-related peptide release in the trigeminovascular system.

Calcitonin gene-related peptide release in trigeminovascular system is a pivotal component of neurogenic inflammation underlying migraine pathophysiology. Transient receptor potential channels and voltage-gated KCNQ/Kv7 potassium channels expressed throughout trigeminovascular system are important targets for modulation of calcitonin gene-related peptide release. We investigated the effects of certain transient receptor potential (TRP) channels the vanilloid 1 and 4 (TRPV1 and TRPV4), the ankyrin 1 (TRPA1), and metastatin type 8 (TRPM8), and voltage-gated potassium channel (Kv7) opener retigabine on calcitonin gene-related peptide release from peripheral (dura mater and trigeminal ganglion) and central (trigeminal nucleus caudalis) trigeminal components of rats. The experiments were carried out using well-established in-vitro preparations (hemiskull, trigeminal ganglion and trigeminal nucleus caudalis) from male Wistar rats. Agonists and antagonists of TRPV1, TRPV4, TRPA1 and TRPM8 channels, and also retigabine were tested on the in-vitro release of calcitonin gene-related peptide. Calcitonin gene-related peptide concentrations were measured using enzyme-linked immunosorbent assay. Agonists of these transient receptor potential channels induced calcitonin gene-related peptide release from hemiskull, trigeminal ganglion and trigeminal nucleus caudalis, respectively. The transient receptor potential channels-induced calcitonin gene-related peptide releases were blocked by their specific antagonists and reduced by retigabine. Retigabine also decreased basal calcitonin gene-related peptide releases in all preparations. Our findings suggest that favorable antagonists of these transient receptor potential channels, or Kv7 channel opener retigabine may be effective in migraine therapy by inhibiting neurogenic inflammation that requires calcitonin gene-related peptide release.

Nociceptors Boost the Resolution of Fungal Osteoinflammation via the TRP Channel-CGRP-Jdp2 Axis

Candida albicans can enter skeletal tissue through askin wound in an immunocompromised host or by contamination during orthopedic surgery. Such Candida osteomyelitis is accompanied by severe pain and bone destruction. It is established that nociceptor innervation occurs in skin and bone, but the mechanisms of nociceptive modulation in fungal inflammation remain unclear. In this study, we show that C.albicans stimulates Nav1.8-positive nociceptors via the β-glucan receptor Dectin-1 to induce calcitonin gene-related peptide (CGRP). This induction of CGRP is independent of Bcl-10 or Malt-1 butdependent on transient receptor potential cation channel subfamily V member 1 (TRPV1)/transient receptor potential cation channel subfamily A member 1 (TRPA1) ion channels. Hindpaw β-glucan injectionafter Nav1.8-positive nociceptor ablation or inTRPV1/TRPA1 deficiency showed dramatically increased osteoinflammation accompanied by impaired CGRP production. Strikingly, CGRP suppressed β-glucan-induced inflammation and osteoclast multinucleation via direct suppression of nuclear factor-κB (NF-κB) p65 by the transcriptional repressor Jdp2 and inhibition of actin polymerization, respectively. These findings clearly suggest a role for Dectin-1-mediated sensocrine pathways in the resolution of fungal osteoinflammation.

Methylprednisolone blocks interleukin 1 beta induced calcitonin gene related peptide release in trigeminal ganglia cells

BackgroundMethylprednisolone (MPD) is a rapid acting highly effective cluster headache preventive and also suppresses the recurrence of migraine attacks. Previously, we could demonstrate that elevated CGRP plasma levels in a cluster headache bout are normalized after a course of high dose corticosteroids. Here we assess whether MPD suppresses interleukin-1β (IL-1β)- and prostaglandin E2 (PGE2)-induced CGRP release in a cell culture model of trigeminal ganglia cells, which could account for the preventive effect in migraine and cluster headache. Metoprolol(MTP), a migraine preventive with a slow onset of action, was used for comparison.MethodsPrimary cultures of rat trigeminal ganglia were stimulated for 24 h with 10 ng/ml IL-1β or for 4 h with 10 μM PGE2 following the exposure to 10 or 100 μM MPD or 100 nM or 10 µM MTP for 45 min or 24 h. CGRP was determined by using a commercial enzyme immunoassay.ResultsMPD but not MTP blocked IL-1β-induced CGRP release from cultured trigeminal cells. PGE2-stimulated CGRP release from trigeminal ganglia cell culture was not affected by pre-stimulation whether with MPD or MTP.ConclusionMPD but not MTP suppresses cytokine (IL-1β)-induced CGRP release from trigeminal ganglia cells. We propose that blockade of cytokine mediated trigeminal activation may represent a potential mechanism of action that mediates the preventive effect of MTP on cluster headache and recurrent migraine attacks.

Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons.

Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of allyl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knockdown of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by allyl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.

Acid-induced CGRP release from the stomach does not depend on TRPV1 or ASIC3

Acid-sensing and regulating reactions are vitally important in the upper gastrointestinal tract and disturbances are common. Sensory neurons in the mucosa detect the intrusion of hydrogen ions and, by their release of vasoactive neuropeptides, seem to play a predominantly protective role in these tissues. The model to investigate sensory transduction of proton stimuli in the isolated everted mouse stomach was to measure the induced calcitonin gene-related peptide (CGRP) release as an index of neuronal activation. Proton concentrations in the range of pH 2.5-0.5 stimulated the release of CGRP and substance P and profoundly decreased the prostaglandin E2 formation in outbred CD mice. A similar linearly pH-dependent CGRP release was observed in inbred C57BL/6 mice, fully dependent on extracellular calcium at pH 2, partially at pH 1. Both transient receptor potential vanilloid type 1 (TRPV1) and acid-sensing ion channel type 3 (ASIC3) are expressed in the sensory neurons innervating the stomach walls and are responsible for the transduction of acidic stimuli in other visceral organs. However, the proton-induced gastric CGRP release in mice lacking the TRPV1 or the ASIC3 receptor-channels was the same as in corresponding wild-type mice. Nonetheless, the pharmacological blockers N-(4-tertiarybutylphenyl)-4-(3-chlorophyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide and amiloride, respectively, inhibited the acid-stimulated CGRP release, although to the same extend in wild types as TRPV1 and ASIC3 knockout mice. Adequate proton concentrations inhibit prostaglandin and stimulate CGRP release from the stomach wall, however, the transduction mechanism in the gastric sensory neurons remains unclear.

Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells

The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a α 2-adrenoceptor blocker) and l-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of α 2-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (α- and β-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. l-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of α 2-adrenoceptor, which is related to the NO pathway.

Colitis induces calcitonin gene-related peptide expression and Akt activation in rat primary afferent pathways

Previous study has shown that colitis-induced increases in calcitonin gene-related peptide (CGRP) immunoreactivity in bladder afferent neurons result in sensory cross-sensitization. To further determine the effects of colitis on CGRP expression in neurons other than bladder afferents, we examined and compared the levels of CGRP mRNA and immunoreactivity in the lumbosacral dorsal root ganglia (DRG) and spinal cord before and during colitis in rats. We also examined the changes in CGRP immunoreactivity in colonic afferent neurons during colitis. Results showed increases in CGRP mRNA levels in L1 (2.5-fold, p < 0.05) and S1 DRG (1.9–2.4-fold, p < 0.05). However, there were no changes in CGRP mRNA levels in L1 and S1 spinal cord during colitis. CGRP protein was significantly increased in L1 (2.5-fold increase, p < 0.05) but decreased in S1 (50% decrease, p < 0.05) colonic afferent neurons, which may reflect CGRP release from these neurons during colitis. In L1 spinal cord, colitis caused increases in the number of CGRP nerve fibers in the deep lamina region extending to the gray commissure where the number of phospho-Akt neurons was also increased. In S1 spinal cord, colitis caused the increases in the intensity of CGRP fibers in the regions of dorso-lateral tract, and caused the increases in the level of phospho-Akt in the superficial dorsal horn of the spinal cord. In spinal cord slice culture, exogenous CGRP increased the phosphorylation level of Akt but not the phosphorylation level of extracellular-signal regulated kinase ERK1/2 even though our previous studies showed that colitis increased the phosphorylation level of ERK1/2 in L1 and S1 spinal cord. These results suggest that CGRP is synthesized in the DRG and may transport to the spinal cord where it initiates signal transduction during colitis.

M1742 Colitis Induces Calcitonin Gene-Related Peptide (CGRP) Expression and AKT Activation in Rat Primary Afferent Pathways

M1742 Colitis Induces Calcitonin Gene-Related Peptide (CGRP) Expression and AKT Activation in Rat Primary Afferent Pathways

A ketolide antibiotic, telithromycin, inhibits vascular adrenergic neurotransmission in the rat mesenteric vascular bed

A ketolide antibiotic, telithromycin, has side effects including temporary loss of consciousness in clinical use, but the underlying mechanisms remain unclear. This study investigated the effects of telithromycin on perivascular nerve function in rat mesenteric arteries, in comparison with those of macrolide (erythromycin and clarithromycin) and new quinolone antibiotics (levofloxacin and gatifloxacin). In vitro, vascular responses and release of noradrenaline induced by periarterial nerve stimulation (PNS) of rat perfused mesenteric vascular beds were measured in the presence of each antibiotic. In vivo blood pressure measurement was performed in Wistar rats. In mesenteric preparations with resting tone, telithromycin (10 nM-10 microM) markedly inhibited PNS (4-12 Hz)-induced adrenergic nerve- and exogenous noradrenaline-mediated vasoconstriction, whereas the other antibiotics slightly inhibited PNS-induced responses without affecting noradrenaline-induced responses. Telithromycin significantly reduced PNS (12 Hz)-evoked noradrenaline release in the perfusate. In pre-constricted preparations with or without endothelium, telithromycin (0.1 nM-10 microM) caused a concentration-dependent vasodilation. Telithromycin (10 nM) inhibited calcium-induced vasoconstriction in high KCl and calcium-free medium. None of the antibiotics used affected PNS (0.5-2 Hz)-induced calcitonin gene-related peptide (CGRP) nerve- and exogenous CGRP-mediated vasodilation. Intravenous injection of telithromycin significantly lowered blood pressure in anaesthetized rats. These results suggest that telithromycin causes not only strong inhibition of perivascular adrenergic neurotransmission but also a vasodilator action in mesenteric vascular beds and hypotension. It is thus possible that telithromycin increases visceral blood flow, consequently reducing cerebral blood flow and resulting in a temporary loss of consciousness.

The effects of iron-containing superoxide dismutases on haemodynamic parameters in spontaneously hypertensive rats

The product of FeSOD activity is hydrogen peroxide (H2O2). Furthermore, FeSOD can modify the chemical versatility of NO into its redox-active forms: nitrosonium cation (NO+) and nitroxyl anion (NO-). All of these low molecular weight species are vasoactive and, in particular, NO- induces calcitonin gene-related peptide (CGRP) synthesis (known to be the most potent relaxation-promoting peptide). In this study the effects of bolus infusions of iron-containing superoxide dismutase (FeSOD) and of superoxide dismutase containing both iron and manganese (FeMnSOD) on the arterial blood pressure (MAP), the arterial blood pressure (CO) and the total vascular resistance (TVR) in spontaneously hypertensive (SH) rats were determined. Bolus infusion of FeSOD induced a biphasic response in the MAP (an initial increase was followed by a significant decrease). At the end of the experiment the MAP returned to its basal value. FeMnSOD (the enzymatically inactive form of FeSOD) had no effect on the MAP in these experiments. Bolus infusions of FeSOD and of FeMnSOD had no effect either on the both the CO or on the TVR in SH rats. Our results indicate that arterial relaxation changes mediated by NO- may be important for regulation of blood pressure in SH rats.

Flurbiprofen inhibits capsaicin induced calcitonin gene related peptide release from rat spinal cord via an endocannabinoid dependent mechanism

Calcitonin gene related peptide (CGRP) is involved in nociceptive transmission and modulation at the spinal level. In the spinal superperfusion model, Δ 9 tetrahydrocannabinol inhibited capsaicin induced CGRP release in a concentration dependent manner. Similarly, flurbiprofen (3 μM) inhibited spinal CGRP release. This inhibition was reversed by the CB 1 antagonist AM-251 (1 μM), but not by co-administration of prostaglandin E 2 (PGE 2; 285 nM). AM-251 had no modulatory effect on flurbiprofen-induced cyclooxygenase (COX) inhibiting capacity as shown by PGE 2 levels. Furthermore, the phospholipase A 2 inhibitor palmityl trifluromethyl ketone (15 μM) reversed flurbiprofen's inhibitory effect. In conclusion the present work provides evidence on the shift of arachidonic acid metabolism towards endocannabinoids formation in response to COX inhibition as a mechanism for flurbiprofen inhibitory effect on spinal CGRP release.

Morphine treatment induced calcitonin gene-related peptide and substance P increases in cultured dorsal root ganglion neurons

The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342–2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 μM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 μM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5 mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments. These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.

Activin and Bone Morphogenetic Proteins Induce Calcitonin Gene-Related Peptide in Embryonic Sensory Neurons in Vitro

The neuropeptide calcitonin gene-related peptide (CGRP) expressed by one-third of rat dorsal root ganglion (DRG) neurons mediates pain sensation and vasodilation. The developmental regulation of CGRP is poorly understood, but may involve target-derived factors from skin or viscera. Few embryonic DRG neurons in defined culture express CGRP, indicating inductive signals are required. Follistatin blocked CGRP expression induced by serum or skin-conditioned medium, implicating transforming growth factor β (TGFβ) family members. Activin or bone morphogenetic proteins (BMPs) 2, 4, or 6 stimulated CGRP expression in 60% of DRG neurons. Brief BMP4 application supported maximal CGRP induction, suggesting that BMP4 is a “switch” rather than a continuous modulator of neuropeptide phenotype. DRG expressed corresponding receptor subunits and exhibited Smad1 transcription factor nuclear translocation following BMP stimulation. BMP mRNAs were present in embryonic targets innervated by CGRP-expressing neurons. Thus, specific TGFβ family members are candidate regulators of CGRP expression in sensory neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) protects dorsal root ganglion neurons from death and induces calcitonin gene-related peptide (CGRP) immunoreactivity in vitro.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a recently discovered neuropeptide which is present both in the central and peripheral nervous system of adult rats. Here we show that PACAP is also expressed by dorsal root ganglion sensory neurons of embryonic and newborn rats. To characterize the effects of PACAP on dorsal root ganglion (DRG) neurons, dissociated cultures were established and incubated in the absence or presence of this neuropeptide. The results show that PACAP increases the survival of cultured DRG neurons, and the effect was comparable to that of nerve growth factor (NGF). In DRG explants, PACAP induces the immunoreactivity for the neuropeptide calcitonin gene-related peptide (CGRP). PACAP also promoted the outgrowth of neurites in the DRG cultures. The present results show that PACAP acts as a trophic factor for DRG neurons and that it is able to modulate the expression of another neuropeptide in the ganglia. The presence of PACAP in normal DRG and after nerve lesions suggests that PACAP acts in a autocrine/paracrine manner possibly in conjunction with other neurotrophic factors such as nerve growth factor.