Release of calcitonin gene-related peptide (CGRP) from capsaicin-sensitive vasodilator nerves in the rat mesenteric artery

The effect of perivascular nerve stimulation (PNS) and capsaicin treatment on the release of calcitonin gene-related peptide (CGRP) was examined by radioimmunoassay (RIA) in isolated, perfused rat mesenteric arteries. In the preparation precontracted by methoxamine and treated with guanethidine, an adrenergic neuron blocker, PNS and capsaicin induced vasodilator responses and increase of CGRP-like immunoreactivity (CGRP-LI) in the perfusate in a frequency-dependent manner. The CGRP-LI released by capsaicin was identified as CGRP and its oxidized form by high-performance liquid chromatography coupled with RIA. After the tissue was treated with capsaicin, PNS didn't cause both the vasodilator response and the increase of CGRP-LI in the perfusate. These findings suggest that CGRP plays a neurotransmitter role in capsaicin-sensitive vasodilator nerves in rat mesenteric arteries.     

The role of calcitonin gene-related peptide in gastric mucosal protection in the rat

The presence of circulating antibodies to calcitonin gene-related peptide (CGRP) enhanced the damaging effect of ethanol on the rat gastric mucosa. Taken together with previous experimental and morphological data the results suggest that CGRP released from the peripheral terminals of visceral afferent fibres plays a role in mediating gastric mucosal defence mechanisms.     

Immunization with calcitonin gene-related peptide reduces the inflammatory response to adjuvant arthritis in the rat

The influence of circulating antibodies to calcitonin gene-related peptide on the inflammatory response was examined in rats with adjuvant-induced arthritis. Rats were immunized with alpha calcitonin gene-related peptide conjugated to thyroglobulin, and circulating antibodies were identified by their capacity to bind radiolabelled rat alpha or human calcitonin gene-related peptide. In unimmunized rats and rats immunized with thyroglobulin alone, the secondary lesions (characterized as paw swelling, nodules on ears and tail, and inflamed nose) produced after adjuvant-induced arthritis were similar. However, at 21 days, when these lesions were maximal, the animals immunized with calcitonin gene-related peptide showed decreased numbers of lesions. An additional marker of disease activity, namely alpha 1 glycoprotein levels in plasma, was also measured. Again, plasma alpha 1 glycoprotein levels were similar in rats that were unimmunized or received thyroglobulin alone, but at 21 days were significantly reduced in animals immunized with calcitonin gene-related peptide. In contrast, the initial foot swelling seen in the first few days after injection of adjuvant was not significantly different in the various groups. The results suggest that antibodies to calcitonin gene-related peptide are able to reduce the severity of the adjuvant arthritis syndrome, and that this peptide contributes to the inflammatory response seen in the later stages of the disease model.     

Depletion of calcitonin gene-related peptide from the caudal trigeminal nucleus of the rat after electrical stimulation of the Gasserian ganglion

Electrical stimulation of the Gasserian ganglion resulted in partial depletion of calcitonin gene-related peptide (CGRP) from ipsilateral central terminals of pseudounipolar primary sensory ganglion cells. Affected terminals exhibit decreased CGRP immunoreactivity as shown by cytophotometric densitomery of the caudal trigeminal nucleus. The decrease in CGRP immunoreactivity is statistically significant only in the medial one-third of the caudal trigeminal nucleus. Since earlier studies have shown that electrical stimulation of the Gasserian ganglion induces first accumulation then depletion of CGRP from perivascular sensory terminals in the dura mater, the present experiments suggest that CGRP is depleted also from central terminals of primary sensory trigeminal neurons, which might be of importance in the pathogenesis of migraine headache. Received: 18 December 1996 / Accepted: 26 June 1997     

The neurotransmitter role of calcitonin gene-related peptide in the rat and guinea-pig ureter: effect of a calcitonin gene-related peptide antagonist and species-related differences in the action of omega conotoxin on calcitonin gene-related peptide release from primary afferents.

In the rat and guinea-pig isolated ureter electrical field stimulation of intrinsic nerves (10 Hz for 10 s) produces transient inhibition of evoked (20 mM KCl or 0.1-1 microM neurokinin A) rhythmic contractions by releasing transmitter(s) from peripheral endings of capsaicin-sensitive primary afferents. The C-terminal fragment of human calcitonin gene-related peptide (8-37) blocked the inhibitory effect of electrical field stimulation as well as that produced by exogenous calcitonin gene-related peptide, while leaving unaffected the inhibitory response to isoprenaline. Human calcitonin gene-related peptide (8-37) was devoid of any inhibitory activity of its own but enhanced the amplitude and frequency of KCl-evoked rhythmic contractions in the rat ureter, probably by antagonizing the inhibitory effect of endogenous calcitonin gene-related peptide released by KCl. Omega conotoxin fraction GVIA, a peptide which possesses a potent blocking activity of N-type voltage-sensitive calcium channels, prevented the inhibitory response to electrical stimulation in the guinea-pig ureter, while leaving the response unaffected in the rat ureter. Conotoxin had no effect toward the inhibition produced by exogenous calcitonin gene-related peptide indicating its prejunctional site of action, demonstrated previously in the guinea-pig ureter [Maggi et al. (1990) Neurosci, Lett. 114, 203-206]. Dermorphin, an amphibian peptide with potent agonist activity on mu-type opioid receptors, inhibited the response to electrical stimulation in the guinea-pig ureter but had no effect in the rat ureter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Case reports: Electroacupuncture decreases the climacteric symptoms by calcitonin gene-related peptide modulation

ObjectiveThe purpose of the current study was to evaluate the modulation of Calcitonin gene-related peptide (CGRP) associated to the efficacy of Electroacupuncture (EA) in the reduction of climacteric symptoms.MethodsNine women between 51 and 59 years old with climacteric syndrome in menopause or perimenopause were included. Patients with hormone replacement therapy, psychiatric treatment with antidepressants, or acupuncture treatment in the last 3 months were excluded. A 4 Hz EA treatment was performed at acupoints Shenshu (BL-23), Pishu (BL-20), Guanyuan (REN-4), Taixi (KID-3), Fuliu (KID-7), Sanyinjiao (SP-6) and Neiguan (P-6) points. Women were treated two times a week for five consecutive weeks for a total treatment of 10 sessions. The menopause rating scale (MRS) was used to evaluate symptoms reduction and CGRP gene expression was measured before and after 10 EA session.ResultsThe results shown that climacteric symptoms diminish significantly after EA therapy. CGRP gene expression was down-regulated, evidencing a decrease of 5-fold after EA therapy respect to the initial condition.ConclusionEA treatment was associated with improvement in patients with climacteric syndrome and may be related to modulation of CGRP levels.     

蛛网膜下腔出血后鼠脑底动脉降钙素基因相关肽能神经纤维的变化

本文旨在探讨大鼠蛛网膜下腔出血(SAH)诱发脑血管痉挛时脑底动脉降钙素基因相关肽(CGRP)能神经纤维的变化。大鼠随机分为正常组、蛛网膜下腔出血组。SAH组大鼠取股动脉自体血注入蛛网膜下腔,正常组动物不做任何处理。3d后灌注固定后,应用免疫组织化学ABC法,对两组大鼠脑底动脉CGRP能神经纤维变化进行观察。结果显示:正常组大鼠脑底动脉可见棕褐色,细线状CGRP能免疫反应阳性纤维;而SAH后3d组大鼠脑底动脉与正常组大鼠脑底动脉相比CGRP能神经纤维密度明显减少,具有统计学意义。以上结果提示脑底动脉CGRP能神经纤维在蛛网膜下腔出血诱发脑血管痉挛的病理生理过程中可能发挥重要的作用。     

Origin and course of nerves immunoreactive for calcitonin gene-related peptide surrounding the femoral artery in rat

Appreciation of anatomic relationships between perivascular nerve fibers and blood vessels is essential in reconstructive surgery. We examined the origin and neural connections of perivascular nerve fibers containing calcitonin gene-related peptide surrounding the femoral artery that regulate vascular tone. We used immunohistochemistry, denervation, and retrograde labeling methods. Peptide-immunoreactive fibers surrounding the femoral artery formed a complex network, with numerous small fibers extending from nerve fiber bundles located in the perivascular connective tissue. In middle and distal arterial segments, these fibers originated from the femoral nerve, the arterys main accompanying nerve. More proximally, fibers arose from the genitofemoral nerve and sympathetic nerves. Nerve branches terminating in various arterial segments had origins corresponding to those of somatic sensory nerve fibers, although pathways innervating the femoral artery took different courses.     

Release of substance P, calcitonin gene-related peptide and prostaglandin E2 from rat dura mater encephali following electrical and chemical stimulation in vitro

Neurogenic inflammation of the dura, expressed in plasma extravasation and vasodilatation, putatively contributes to different types of headache. A novel in vitro preparation of the fluid-filled skull cavities was developed to measure mediator release from dura mater encephali upon antidromic electrical stimulation of the trigeminal ganglion and after application of a mixture of inflammatory mediators (serotonin, histamine and bradykinin, 10(-5) M each, pH 6.1) to the arachnoid side of rat dura. The release of calcitonin gene-related peptide, substance P and prostaglandin E2 from dura mater was measured in 5-min samples of superfusates using enzyme immunoassays. Orthodromic chemical and antidromic electrical stimulation of dural afferents caused significant release of calcitonin gene-related peptide (2.8- and 4.5-fold of baseline). The neuropeptide was found to be increased during the 5-min stimulation period and returned to baseline (20.9 +/- 12 pg/ml) in the sampling period after stimulation. In contrast, release of substance P remained at baseline levels (19.3 +/- 11 pg/ml) throughout the experiment. Prostaglandin E2 release was elevated during chemical and significantly also after antidromic electrical stimulation (6- and 4.2-fold of baseline, which was 305 +/- 250 pg/ml). Prostaglandin E2 release outlasted the stimulation period for at least another 5 min. The data support the hypothesis of neurogenic inflammation being involved in headaches and provide new evidence for prostaglandin E2 possibly facilitating meningeal nociceptor excitation and, hence, pain.     

Activation of renal pelvic chemoreceptors in rats: role of calcitonin gene-related peptide receptors

Substance P and calcitonin gene-related peptide (CGRP) increase afferent renal nerve activity (ARNA). A substance P receptor antagonist but not a CGRP receptor antagonist, h-CGRP (8–37), blocks the ARNA response to renal mechanoreceptor (MR) stimulation. We have examined whether calcitonin gene-related peptide activates renal pelvic sensory receptors and whether such activation contributes to renal chemoreceptor stimulation. The calcitonin gene-related peptide receptor antagonist, h-CGRP (8–37) [0.01–10 μmol L^?1] dose-dependently decreased (29 ± 4–86 ± 13%, P < 0.01) the ipsilateral afferent renal nerve activity in response to the renal pelvic administration of calcitonin gene-related peptide (0.26 μmol L^?1). Renal pelvic perfusion with 900 m M NaCl also increased ipsilateral ARNA (23 ± 3% increase, P < 0.02) and contralateral urinary sodium excretion (13 ± 4% increase, P < 0.05). However, these responses to hypertonic NaCl were unaltered by h-CGRP (8–37). Renal pelvic perfusion with 1 or 10 μM h-CGRP (8–37) also failed to alter the ARNA responses to KCl (31.25, 62.5 and 125 m M ). These results indicate that there are sensory receptors in the renal pelvic area that are responsive to calcitonin gene-related peptide. The activation of these receptors elicits a contralateral natriuretic response. In contrast, the activation of renal calcitonin gene-related peptide receptors does not contribute to renal chemoreceptor activation.     

Topographic localization of calcitonin gene-related peptide in the rat brain: an immunohistochemical analysis

The distribution of immunoreactive calcitonin gene-related peptide in the rat brain was investigated by means of an indirect immunofluorescence method. In addition to previously reported calcitonin gene-related peptide-like immunoreactive structure-containing sites such as the nucleus ambiguus, nucleus originis nervi facialis, nucleus originis nervi hypoglossi, nucleus peripeduncularis and nucleus parabrachialis, the present study demonstrated a far wider distribution of calcitonin gene-related peptide-like immunoreactive structure-containing cells in the rat brain, i.e. the nucleus hypothalamicus lateralis, nucleus ventromedialis thalami, colliculus superior, lemniscus lateralis, gyrus dentatus, nucleus olivaris superior, nucleus tractus solitarii, nucleus cuneiformis, nucleus parabigeminalis and a proportion of the Purkinje cells. We have also demonstrated a more extensive network of calcitonin gene-related peptide-like immunoreactive fibers distributed in various areas throughout the rat brain than has been reported previously such as the colliculus inferior, nucleus olivaris superior, nucleus vestibularis lateralis and inferioris, and nucleus cochlearis dorsalis and ventralis, etc.     

The presence of a calcitonin gene-related peptide in the olivocochlear bundle in rat

Summary The origins of calcitonin gene-related peptide immunoreactive (CGRPI) fibers in the cochlea were examined in rats. Parasagittal transection of the brain just medial to the principal sensory trigeminal nucleus resulted in the ipsilateral disappearance of CGRPI fibers in the cochlea, indicating that the origins of these fibers lie in the central nervous system. Next, we used a highly sensitive method combining retrograde tracing and immunohistochemistry to identify the origins of the CGRPI fibers in the cochlea. After injection of biotin-wheat germ agglutinin (b-WGA) into the cochlea, CGRPI neurons in the ipsilateral lateral superior olivary nucleus also contained b-WGA granules. These findings indicated tht CGRPI efferent fibers are major components of the olivocochlear bundle.     

Peripheral amplification of sweating – a role for calcitonin gene-related peptide

Neuropeptides are the mediators of neurogenic inflammation. Some pain disorders, e.g. complex regional pain syndromes, are characterized by increased neurogenic inflammation and by exaggerated sudomotor function. The aim of this study was to explore whether neuropeptides have a peripheral effect on human sweating. We investigated the effects of different concentrations of calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and substance P (SP) on acetylcholine-induced axon reflex sweating in healthy subjects (total n = 18). All substances were applied via dermal microdialysis. The experiments were done in a parallel setting: ACh alone and ACh combined with CGRP, VIP or SP in various concentrations were applied. Acetylcholine (10⁻² m ) always elicited a sweating response, neuropeptides alone did not. However, CGRP significantly enhanced ACh-induced sweating ( P < 0.01). Post hoc tests revealed that CGRP in physiological concentrations of 10⁻⁷–10⁻⁹ m was most effective. VIP at any concentration had no significant effect on axon reflex sweating. The duration of the sweating response ( P < 0.01), but not the amount of sweat, was reduced by SP. ACh-induced skin blood flow was significantly increased by CGRP ( P < 0.01), but unaltered by VIP and SP. The results indicate that CGRP amplifies axon reflex sweating in human skin.     

Activation of renal pelvic chemoreceptors in rats: role of calcitonin gene-related peptide receptors.

Substance P and calcitonin gene-related peptide (CGRP) increase afferent renal nerve activity (ARNA). A substance P receptor antagonist but not a CGRP receptor antagonist, h-CGRP (8-37), blocks the ARNA response to renal mechanoreceptor (MR) stimulation. We have examined whether calcitonin gene-related peptide activates renal pelvic sensory receptors and whether such activation contributes to renal chemoreceptor stimulation. The calcitonin gene-related peptide receptor antagonist, h-CGRP (8-37) [0.01-10 micromol L-1] dose-dependently decreased (29 +/- 4-86 +/- 13%, P < 0.01) the ipsilateral afferent renal nerve activity in response to the renal pelvic administration of calcitonin gene-related peptide (0.26 micromol L-1). Renal pelvic perfusion with 900 mM NaCl also increased ipsilateral ARNA (23 +/- 3% increase, P < 0.02) and contralateral urinary sodium excretion (13 +/- 4% increase, P < 0. 05). However, these responses to hypertonic NaCl were unaltered by h-CGRP (8-37). Renal pelvic perfusion with 1 or 10 microM h-CGRP (8-37) also failed to alter the ARNA responses to KCl (31.25, 62.5 and 125 mM). These results indicate that there are sensory receptors in the renal pelvic area that are responsive to calcitonin gene-related peptide. The activation of these receptors elicits a contralateral natriuretic response. In contrast, the activation of renal calcitonin gene-related peptide receptors does not contribute to renal chemoreceptor activation.     

Immunohistochemical analysis of calcitonin and calcitonin gene-related peptide in human lung

Calcitonin and calcitonin gene-related peptide (CGRP) have been localized immunohistochemically in neuroendocrine cells of normal and diseased human lungs. Cells immunoreactive for calcitonin and CGRP first appeared in immature bronchi in the 27th gestational week. Thereafter, both peptides were found in the same bronchial neuroendocrine cells throughout fetal and neonatal life. In adult lungs with or without neuroendocrine cell hyperplasia, only calcitonin was present. In 17 of 18 (94%) pulmonary tumorlets, variable numbers of calcitonin-positive cells were identified. A few CGRP immunoreactive cells, as a subset of calcitonin-containing cells, were found in only three (17%) lesions. Of 37 bronchial carcinoids, calcitonin was detected in 14 and CGRP was detected in 16 (38% and 43%, respectively), and both peptides were predominantly localized in the same cells. Ten of 45 (22%) small cell lung carcinomas were calcitonin-immunoreactive. CGRP was noted in only one (2%) of these tumors, and both peptides coexisted in single cells. These findings indicate that the patterns of calcitonin/CGRP expression in hyperplastic bronchial neuroendocrine cells, pulmonary tumorlets, and, to some extent, small cell lung carcinomas are similar to those of normal adult lungs. On the other hand, calcitonin/CGRP expression in bronchial carcinoids is similar to that of late fetal and neonatal lungs.     

Dietary calcium modulates spinal cord content of calcitonin gene-related peptide in the rat

This study was undertaken to determine if calcium status modulates calcitonin gene-related peptide (CGRP) neuronal content. In two separate experiments, young, growing rats and mature rats were placed on low, normal, and high calcium diets for four weeks. CGRP immunostaining was localized immunocytochemically in laminae I and II of the upper thoracic spinal cord in young rats and in the upper thoracic and lumbar spinal cord in mature rats. Low calcium intake decreased dorsal horn CGRP content in young, growing rats, while high calcium diet significantly increased CGRP content, as determined by computer-assisted image processing, in both young and adult rats. A significant positive correlation was found between the serum ionized calcium and CGRP content in laminae I and II. Thus, calcium balance appears to modulate neuronal CGRP content.     

The role of substance P and calcitonin gene-related peptide in neurogenic plasma extravasation and vasodilatation in the rat

Immunohistochemistry combined with retrograde tracing has been used to show that of the afferent neurons supplying the dorsomedial surface of the hind paw, approximately 30% contain substance P and 50% calcitonin gene-related peptide immunoreactivity. Stimulation of the saphenous nerve causes plasma extravasation and antidromic vasodilatation in this area of skin. The roles of calcitonin gene-related peptide and substance P released from peripheral afferent endings in mediating these effects were examined using immunoneutralization. In pilot experiments, the binding of radiolabelled peptide to the immunoglobulin fraction of calcitonin gene-related peptide and substance P antisera was characterized in quasi-physiological conditions. Systemic administration of either substance P or calcitonin gene-related peptide antibodies caused a significant decrease (P less than 0.05) in plasma extravasation measured by the Evans Blue method in response to topical application of mustard oil (0.5%) to the skin, or of capsaicin (5 microM) to the saphenous nerve. Topical application of mustard oil also produced a 52.9 +/- 5.1% increase in skin red cell flux. This increase was significantly decreased by both substance P and calcitonin gene-related peptide antibodies. The results suggest that both peptides are involved in mediating neurogenic inflammatory responses.