Regulation of calcitonin gene-related peptide release from rat trigeminal nucleus caudalis slices in vitro

Calcitonin gene-related peptide (CGRP) released from trigeminal primary afferents has been implicated in the pathophysiology of migraine. Here, we have used an in vitro slice preparation to investigate its release from nerve terminals in the rat trigeminal nucleus caudalis. Extracellular-calcium dependent CGRP release was stimulated by both capsaicin and neuronal depolarization with KCl. The capsaicin (1 microM)-evoked CGRP release was blocked by capsazepine and was also attenuated in the presence of the cyclooxygenase inhibitor, indomethacin, an effect that was reversed when slices were stimulated with capsaicin in the presence of the cyclooxygenase metabolite, prostaglandin E(2). Taken together, these data further highlight the importance of prostaglandins as enhancers of neuropeptide release and suggest that CGRP released from the central terminals of trigeminal neurones has the potential to be involved in the transmission of nociceptive information of relevance to migraine headache.     

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.     

Capsaicin-induced release of calcitonin gene-related peptide from dorsal horn slices is enhanced in adjuvant arthritic rats

The capsaicin (3 microM)-evoked release of the immunoreactive calcitonin gene-related peptide (iCGRP) from dorsal-half slices of the rat lumbar spinal cord was substantially reduced in slices from rats which had undergone dorsal rhizotomy. This suggests that the release is elicited mainly from the nerve terminals of primary afferents. The release was significantly increased in slices from adjuvant arthritic rats compared with those from control rats. This suggests that the amount of iCGRP in a capsaicin-sensitive pool of the nerve terminals of primary afferents is elevated by adjuvant arthritis. Since adjuvant arthritis is known to cause an enhancement of biosynthesis and axonal flow of iCGRP in primary sensory neurons, the present results imply an increased release of iCGRP from the central terminals of the primary afferents in the adjuvant arthritic rat.     

Coexistence of substance P and calcitonin gene-related peptide in the frog spinal cord

The localization of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the untreated spinal cord of the frog using single or double immunohistochemical stainings. SP and CGRP appear to coexist in the primary afferent fibers and in the marginal and submarginal dorsal horn zones, as well as in the dorsolateral zone. In other parts of the spinal cord CGRP immunoreactivity was scanty while diffuse SP systems were seen, suggesting that the coexistence of the two peptides is restricted to primary afferent fibers.     

降钙素基因相关肽合成与释放调节

降钙素基因相关肽（calcitonin gene-related peptide, CGRP）是迄今发现的最强的舒血管物质,具有多种重要的生物学作用。作为感觉神经主要递质,CGRP主要由背根神经节合成,某些非神经细胞(如淋巴细胞和内皮细胞)也有表达。CGRP的合成与释放受多种因素调节,除辣椒素受体外,肾上腺素受体、血管紧张素受体、神经生长因子受体和一氧化氮等也参与了CGRP合成与释放的调节。     

Nerve growth factor treatment enhances release of immunoreactive calcitonin gene-related peptide but not substance P from spinal dorsal horn slices in rats

The effect of systemic nerve growth factor (NGF) on neuropeptide content and capsaicin-evoked release of neuropeptide from in vitro spinal cord dorsal horn slices was examined. Rats were injected subcutaneously every other day with murine NGF (mNGF) 1 mg/kg or saline for 7 days, or mNGF 0.1/kg, mNGF 1 mg/kg or saline for 13 days. Lumbar dorsal horn slices of the rat spinal cord from all groups showed a significant increase in immunoreactive calcitonin gene-related peptide (CGRP) release upon exposure to capsaicin. This release was enhanced in rats pretreated with mNGF 1 mg/kg for 7 days, but not after 13 days. No enhancement was seen after 7 or 13 days in any treatment group for immunoreactive substance P release. Upon examination of neuropeptide content in dorsal horn, no significant differences were noted between treatment groups. The increased iCGRP release from dorsal horn slices suggests a preferential release of CGRP and provides further evidence that NGF indirectly plays a role in the modulation of inflammation through the regulation of neuropeptide release.     

Inflammatory mediators release calcitonin gene-related peptide from dorsal root ganglion neurons of the rat

The interactions between the inflammatory mediators bradykinin, serotonin, prostaglandin E(2) and acid pH were studied in rat dorsal root ganglion neurons in culture. For this purpose, the cultures were stimulated by inflammatory mediators (bradykinin, serotonin, prostaglandin E(2), 10(-5)M each) or acid solution (pH 6.1) for 5 min and the content of calcitonin gene-related peptide was determined in the supernatant before, during and after stimulation, using an enzyme immunoassay. Acid solution resulted in a threefold increase of the basal calcitonin gene-related peptide release which was entirely dependent on the presence of extracellular calcium. The release could not be blocked by the addition of the capsaicin antagonist capsazepine (10(-5)M). Bradykinin (10(-5)M) caused a 50% increase of the basal calcitonin gene-related peptide release which was again dependent on the presence of extracellular calcium, whereas serotonin and prostaglandin E(2) were each ineffective at 10(-5)M concentration. The combination of bradykinin, serotonin and prostaglandin E(2) led to a fivefold increase of the calcitonin gene-related peptide release which could not be further enhanced by acidification. The competitive capsaicin receptor antagonist capsazepine (10(-5)M) significantly reduced the release induced by the combination of bradykinin, serotonin and prostaglandin E(2).It is suggested that the inflammatory mediators co-operate and together may act as endogenous agonists at the capsaicin receptor to cause calcium influx and consecutive neuropeptide release.     

Adenosine inhibits action potential-dependent release of calcitonin gene-related peptide- and substance P-like immunoreactivities from primary afferents in rat spinal cord.

Electrical field stimulation (5 Hz) evoked a prompt outflow of calcitonin gene-related peptide- and substance P-like immunoreactivities (CGRP-LI and SP-LI, respectively) from superfused slices of the dorsal but not ventral half of the rat spinal cord. The evoked outflow was abolished by tetrodotoxin, calcium-free medium or previous exposure to capsaicin, indicating that it is produced through action potentials invading the central terminals of capsaicin-sensitive primary afferents. Adenosine as well as gamma-aminobutyric acid (GABA) or the GABAB receptor agonist (-)-baclofen produced a concentration-dependent inhibition of the evoked CGRP-LI outflow. Adenosine also inhibited the evoked SP-LI outflow. These findings demonstrate that inhibition of transmitter release from primary afferent neurons should be considered as a possible mechanism of the antinociceptive action of adenosine and adenosine analogs.     

Visceral and somatic afferent origin of calcitonin gene-related peptide immunoreactivity in the lower thoracic spinal cord of the rat

The origin of calcitonin gene-related peptide in the thoracic spinal cord of the rat was investigated by radioimmunoassay and immunohistochemistry. In transverse sections from normal animals there was a dense staining of calcitonin gene-related peptide-immunoreactivity in laminae I, II and V of the dorsal horn. In parasagittal sections this was found to consist of rostrocaudally orientated fibres in laminae I and II and longitudinal bundles of fibres interspersed with a plexus of immunoreactivity in lamina V. After sectioning the thoracic spinal nerves there was a significant reduction in immunoreactivity in the dorsal horn of the spinal cord which was seen as a marked reduction of staining in lamina II and in the bundles of fibres in lamina V. Section of the splanchnic nerve slightly reduced staining in lamina I and virtually abolished the plexuses of immunoreactivity in lamina V. However, measurement of calcitonin gene-related peptide in samples from coeliac-ganglionized rats revealed an increase in immunoreactivity in regions of the spinal cord containing lamina V. These results provide evidence of a visceral and somatic afferent origin of calcitonin gene-related peptide in the thoracic spinal cord of the rat.     

Leg uptake of calcitonin gene-related peptide during exercise in spinal cord injured humans.

Exercise-induced increases in cardiac output (CO) and oxygen uptake (VO2) are tightly coupled, as also in absence of central motor activity and neural feedback from skeletal muscle. Neuromodulators of vascular tone and cardiac function - such as calcitonin gene related peptide (CGRP) - may be of importance. Spinal cord injured individuals (six tetraplegic and four paraplegic) performed electrically induced cycling (FES) with their paralyzed lower limbs for 29 +/- 2 min to fatigue. Voluntary cycling performed both at VO2 similar to FES and at maximal exercise in six healthy subjects served as control. In healthy subjects, CGRP in plasma increased only during maximal exercise (33.8 +/- 3.1 pmol l(-1) (rest) to 39.5 +/- 4.3 (14%, P<0.05)) with a mean extraction over the working leg of 10% (P<0.05). Spinal cord injured individuals had more pronounced increase in plasma CGRP (33.2 +/- 3.8 to 46.9 +/- 3.6 pmol l-1, P<0.05), and paraplegic and tetraplegic individuals increased in average by 23% and 52%, respectively, with a 10% leg extraction in both groups (P<0.05). The exercise induced increase in leg blood flow was 10-12 fold in both spinal cord injured and controls at similar VO2 (P<0.05), whereas CO increased more in the controls than in spinal man. Heart rate (HR) increased more in paraplegic subjects (67 +/- 7 to 132 +/- 15 bpm) compared with controls and tetraplegics (P<0.05). Mean arterial pressure (MAP) was unchanged during submaximal exercise and increased during maximal exercise in healthy subjects, but decreased during the last 15 min of exercise in the tetraplegics. It is concluded that plasma CGRP increases during exercise, and that it is taken up by contracting skeletal muscle. The study did not allow for a demonstration of the origin of the CGRP, but its release does not require activation of motor centres. Finally, the more marked increase in plasma CGRP and the decrease in blood pressure during exercise in tetraplegic humans may indicate a role of CGRP in regulation of vascular tone during exercise.     

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, Delta(9) tetrahydrocannabinol inhibited capsaicin induced CGRP release in a concentration dependent manner. Similarly, flurbiprofen (3 microM) inhibited spinal CGRP release. This inhibition was reversed by the CB(1) antagonist AM-251 (1 microM), 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 microM) 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.     

Receptor for calcitonin gene-related peptide: localization in the dorsal and ventral spinal cord.

Although the distribution of calcitonin gene-related peptide has been extensively studied in the spinal cord, little is known about the precise subcellular localization of receptors for calcitonin gene-related peptide. The present study was undertaken to localize calcitonin gene-related peptide receptors in both the dorsal and ventral horns of the rat spinal cord. Immunocytochemical localization with specific monoclonal antibodies was performed at the light and electron microscopic levels. Calcitonin gene-related peptide receptor was expressed in neuronal but not glial elements. Discrete postsynaptic localization of receptor for the calcitonin gene-related peptide was evident in the cells and dendrites of the superficial dorsal horn. Some of the terminal endings apposing the stained synapses formed the central terminals of glomerular complexes. The endings were scallop shaped (Type I), typical of primary afferent terminations. Other dorsal horn structures with postsynaptic labeling were contacted by dome-shaped or elongated axonal endings. Presynaptic localization on some dorsal horn terminations may serve an autoreceptor function. Motoneurons, on the other hand, were contacted by axonal terminals with presynaptic calcitonin gene-related peptide receptors. These data suggest that (i) dorsal horn neurons are capable of direct primary afferent, calcitonin gene-related peptide receptor-mediated interactions and (ii) neuronal terminals contacting motor horn cells can be influenced through presynaptic paracrine-like calcitonin gene-related peptide receptor-mediated interactions. Thus, calcitonin gene-related peptide can have multiple modulatory effects on spinal cord neurons through site-specific receptors.     

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)     

Immunohistochemical mapping of neurophysins and calcitonin gene-related peptide in the human brainstem and cervical spinal cord

Our study investigates the distribution of neurophysins (Nph), proteins that are part of the precursors for vasopressin and oxytocin, and calcitonin gene-related peptide (CGRP) in the human brainstem by immunohistochemistry. Both peptides were found in discrete regions of the human hindbrain. Nph could be demonstrated exclusively in fibers and punctate perineural varicosities that were travelling within the mesencephalic central gray, substantia nigra, as well as locus coeruleus, medial longitudinal fascicle, raphe, nucleus of the solitary tract, lateral reticular nucleus and area postrema. A few varicosities were seen in the substantia gelatinosa of the spinal trigeminal tract and its continuation into the dorsal horn of the cervical spinal cord. In contrast to these observations. CGRP-immunoreactive fibers were found to be densest in the spinal tract of the trigeminal nerve and the dorsal horn of the spinal cord. In addition, fibers and varicosities could be demonstrated in numerous distinct brain regions, such as locus coeruleus and subcoeruleus, solitary tract, cuneate nucleus, raphe and periaqueductal gray. CGRP-immunoreactivity was also present in perikarya in the ventral horn of the spinal cord, as well as motor nuclei of cranial nerves, i.e., hypoglossal nucleus, ambiguous nucleus. Our results suggest that Nph-immunoreactivity in the human brainstem may be present predominantly within long fiber projections from hypothalamic neurosecretory nuclei, in analogy to data obtained from rodents, whereas CGRP may play a role in the branchiomotor system as well as in intrinsic or extrinsic projections involved in autonomic regulation and integration of sensory information.     

Immunoreactive tachykinins, calcitonin gene-related peptide and neuropeptide Y in human synovial fluid from inflamed knee joints

We have analysed the concentrations of substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) in the synovial fluid from 5 patients suffering from arthritis with inflamed knee joints as well as from 5 healthy control subjects with an earlier traumatic meniscal or cruciate ligament injury. Competitive radioimmunoassay was done using antisera SP2 (SP), K12 (NKA), R8 (CGRP), and NPY1 (NPY). No SP-like immunoreactivity (-LI) was detected in any patient NKA-LI was found in all control patients but in none of the arthritis patients. CGRP-LI was seen in all arthritis patients as well as in control patients, a non-significant difference. NPY-LI was found in a significantly higher concentration in the arthritis group vs the control patients. The results support an involvement of neuropeptides in human joint inflammation.     

Release of sensory neuropeptides in the spinal cord: studies with calcitonin gene-related peptide and galanin

In anaesthetized cats, antibody microprobes were used to investigate the release of immunoreactive calcitonin gene-related peptide and galanin in the lower lumbar spinal cord. In the absence of applied stimulation, a basal release of both peptides was detected at the level of the substantia gelatinosa. This release of calcitonin gene-related peptide was not altered by innocuous thermal cutaneous stimulation nor by electrical stimulation of low-threshold myelinated primary afferent fibres, but was increased by noxious thermal or noxious mechanical cutaneous stimuli and by electrical stimulation of unmyelinated primary afferents. A simultaneous release of both calcitonin gene-related peptide and substance P was detected in the substantia gelatinosa region by the use of pairs of microprobes. In contrast, none of the peripheral stimulation procedures increased intraspinal galanin release. The results suggest that the spinal transmission of nociceptive information may involve the simultaneous release and action of several neuropeptides within the superficial layers of the dorsal horn.     

Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat

Using immunohistochemistry evidence was obtained for the coexistence of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivity in spinal sensory neurons. Analysis of caudally directed biting and scratching (CBS) behavior was carried out after intrathecal administration of CGRP and SP alone or in combination. Thus, SP (up to 20 micrograms) alone caused CBS only for a few minutes after injection, whereas SP (10 micrograms) plus CGRP (20 micrograms) caused a response with a duration up to 40 min. CGRP (20 micrograms) alone had no effects in this model. These findings provide support for a possible interaction of the two peptides at synapses in the dorsal horn of the spinal cord.     

降钙素基因相关肽对内皮素释放的影响

本实验在大鼠的整体及离体血管条上观察了降钙素基因相关肽（ＣＧＲＰ）对内皮素（ＥＴ）释放的影响。结果表明：ＣＧＲＰ静脉注射（５μｇ／ｋｇ）能明显降低大鼠内毒素血症时血浆ＥＴ含量，ＣＧＲＰ孵育（１０＾－８ｍｏｌ／Ｌ）离体的大鼠主动脉血管条能有效地抑制凝血酶（ｔｈｒｏｍｂｉｎ）引起的ＥＴ释放，但是ＣＧＲＰ静注及孵育不影响血浆ＥＴ的基础含量及离体血管条ＥＴ的基础释放。结果提示ＣＧＲＰ能抑制病理条件下ＥＴ的     

Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig

In situ hybridization histochemistry was used to localize calcitonin gene-related peptide mRNAs in spinal cord, brain stem and dorsal root ganglion neurons of the rat and guinea-pig. A 32P-labeled 23-base-long (23mer) oligodeoxyribonucleotide (oligomer) complementary to calcitonin gene-related peptide mRNA sequences encoding residues 23-30 of calcitonin gene-related peptide was used primarily as a probe (CGRP I probe). A 32mer complementary to mRNA sequences for residues 10-20 of calcitonin gene-related peptide (CGRP II probe) was also used as a positive control for specificity of the 23mer for calcitonin gene-related peptide mRNA. In both the guinea-pig and rat calcitonin gene-related peptide mRNA was localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. Differences in the distribution of calcitonin gene-related peptide mRNA between the rat and guinea-pig included a higher proportion of rat dorsal root ganglion neurons containing calcitonin gene-related peptide mRNA and the localization of calcitonin gene-related peptide mRNA to motoneurons of the ambiguus motor nucleus, parabrachial and peripeduncular nucleus of the rat but not the guinea-pig. In the guinea-pig, in contrast, calcitonin gene-related peptide mRNA was localized also to motoneurons of the abducens, trigeminal, trochlear and oculomotor nerves. The neuronal groups in the intact rat found here to contain calcitonin gene-related mRNA have also been shown previously to contain calcitonin gene-related peptide immunoreactivity in colchicine-treated rats. Colchicine-treated rats, however, have been found to contain additional groups of calcitonin gene-related peptide immunoreactive neurons which, in the intact rats used in the present study, showed no detectable hybridization with the calcitonin gene-related peptide probe.