Tachykinins and calcitonin gene-related peptide as co-transmitters in local motor responses produced by sensory nerve activation in the guinea-pig isolated renal pelvis.

Electrical field stimulation of circular muscle strips from the guinea-pig isolated renal pelvis produces a frequency-dependent positive inotropic effect of the spontaneous contractions which is unaffected by atropine and guanethidine and abolished by tetrodotoxin or in vitro capsaicin desensitization. Omega conotoxin fraction GVIA markedly inhibited the response to low frequencies of stimulation but had only a partial or minor inhibitory effect at higher frequencies. Tachykinins produce a concentration-dependent positive inotropic effect, neurokinin A being more potent than substance P. On the other hand, rat alpha calcitonin gene-related peptide (CGRP) inhibited spontaneous contractions of the renal pelvis. MEN 10,376 a neurokinin A (4-10) analog, antagonized the positive inotropism produced by neurokinin A, without affecting the response to KCl, and suppressed the positive inotropic response produced by electrical field stimulation. In the presence of MEN 10,376, a negative inotropic response was produced by electrical field stimulation which was antagonized by the C-terminal fragment (8-37) of human alpha calcitonin gene-related peptide (hCGRP). hCGRP (8-37) antagonized the negative inotropic effect of exogenously administered CGRP without affecting inhibition by isoprenaline. Application of capsaicin (10 microM) produced a marked increase in the outflow of substance P-, neurokinin A- and CGRP-like immunoreactivities from the superfused guinea-pig renal pelvis. Substance P-, neurokinin A- and CGRP-like immunoreactivities were also detected in tissue extracts of the renal pelvis by radioimmunoassay. These experiments indicate that peptide release from peripheral endings of capsaicin-sensitive primary afferents represents the major type of nerve-mediated response affecting motility of the guinea-pig isolated renal pelvis. Tachykinins and CGRP act as physiological antagonists and the excitatory action of tachykinins prevails over the inhibitory action of CGRP. Local modulation of renal pelvis motility by sensory nerves could facilitate removal of irritants present in the urine, protecting the kidney during obstruction and ureteral antiperistalsis.     

Difference in the actions of calcitonin gene-related peptide on pig detrusor and vesical arterial smooth muscle

Calcitonin gene-related peptide has been demonstrated in urinary bladder nerves, and suggested to play a role in local control of bladder motility. In isolated strips of pig detrusor muscle, calcitonin gene-related peptide did not affect spontaneous contractile activity, or contractions induced by high K+, carbachol, substance P, and electrical field stimulation. In contrast, calcitonin gene-related peptide elicited a concentration-dependent and pronounced (78-99%) relaxation of vesical arteries precontracted with endothelin-1, noradrenaline or prostaglandin F2 alpha. As a vasodilator, CGRP was approximately 50 times more potent than acetylcholine. Removal of the endothelium abolished acetylcholine-induced relaxation, but did not affect the relaxation produced by calcitonin gene-related peptide. Pretreatment with methylene blue, glibenclamide or indomethacin had no influence on CGRP's ability to relax the vessels. The inhibitor of NO-synthesis, NG-nitro-L-arginine, had no effect on the maximum vascular relaxation induced by calcitonin gene-relate peptide. It is concluded that in the pig, calcitonin gene-related peptide has no functionally important mechanical effects on isolated detrusor muscle strips, but is a potent dilator of vesical arteries. The vascular effects of the peptide are endothelium-independent, and seem to be exerted directly on the vascular smooth muscle.     

Bradykinin-induced release of calcitonin gene-related peptide from capsaicin-sensitive nerves in guinea-pig atria: mechanism of action and calcium requirements

The mechanism of neuropeptide secretion induced by bradykinin from capsaicin-sensitive afferents was studied in guinea-pig atria. Both the inotropic response induced by bradykinin (0.1 microM) in the electrically driven isolated guinea-pig left atria and the bradykinin (10 microM)-induced release of calcitonin gene-related peptide calcitonin gene-related peptide-like immunoreactivity from slices of guinea-pig atria were abolished in vitro by capsaicin pretreatment or in the presence of indomethacin. Bradykinin-induced calcitonin gene-related peptide-like immunoreactive release was unaffected by tetrodotoxin (0.3 microM), the protein kinase C inhibitor, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (30 microM), nefedipine (1 microM) or Ruthenium Red (10 microM). It was significantly reduced by 79% in a Ca2(+)-free medium and by 52% in the presence of 0.1 microM omega-conotoxin (fraction GVIA). It is proposed that bradykinin releases calcitonin gene-related peptide from capsaicin-sensitive afferents in guinea-pig atria, via prostanoid generation. This mode of activation of the "efferent" function of capsaicin-sensitive nerves appears to be distinct from those produced by capsaicin or electrical field stimulation as they have been characterized in previous works. In fact, the bradykinin activation of capsaicin-sensitive afferents is not affected by tetrodotoxin and Ruthenium Red, but is partially sensitive to the selective blocker of N-type Ca2(+)-channels, omega-conotoxin.     

Antagonism of the effects of calcitonin gene-related peptide and of capsaicin on the guinea-pig isolated ileum by human alpha-calcitonin gene-related peptide(8-37)

The possible mediating role of calcitonin gene-related peptide (CGRP) in the effects of capsaicin in the guinea-pig ileum has been investigated by means of the CGRP antagonist hCGRP(8-37). Submaximal longitudinal muscle relaxation of the histamine-precontracted ileum evoked by rat CGRP (3 nM) or capsaicin (1 microM) was reversed by hCGRP(8-37) (1.5 microM), while that due to adrenaline or neurotensin was not affected. Inhibition of spontaneous circular muscle movements by capsaicin (100 nM) was also reversed by hCGRP(8-37). The CGRP antagonist had no effect on electrical stimulation-evoked ileal contractions or on the longitudinal or circular muscle tone. It is concluded that (a) hCGRP(8-37) is a specific CGRP antagonist in the ileum, apparently devoid of intrinsic activity or any effect not related to CGRP; (b) the inhibitory actions of capsaicin on the longitudinal and circular muscles of the ileum are mediated, at least in part, by CGRP.     

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.     

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.     

Dual capsaicin effects on ureteric motility: low dose inhibition mediated by calcitonin gene-related peptide and high dose stimulation by tachykinins?

The effects of capsaicin, in relation to substance P (SP), neurokinin A (NKA), neuropeptide K (NPK) and calcitonin gene-related peptide (CGRP) which coexist in local sensory nerves, on the motility of the guinea-pig ureter were studied in vivo and in vitro. Capsaicin in a low dose (10 nmol kg-1) given i.v. inhibited spontaneous, peristaltic contractions, as revealed by perfusion-pressure changes of the constantly perfused ureter in vivo. This action was independent of autonomic reflexes and prostaglandin formation. Capsaicin stimulated ureteric motility at higher doses (100 and 500 nmol kg-1). The dual effects of capsaicin on the ureteric contractility were absent 2 weeks after systemic capsaicin treatment, which depletes sensory neuropeptides. Both NKA and NPK initiated, as well as increased, the magnitude of the peristaltic contractions of the ureter, while SP only caused a minor excitatory effect. The CGRP inhibited spontaneous, as well as NKA- and NPK-induced ureteric peristaltic contractions. In vitro experiments on the ureter revealed that capsaicin (10(-6) M) induced phasic circular muscle contractions in 60% of the experiments. Neurokinin A, NPK and SP consistently increased the contractile activity. The NKA tachyphylaxis inhibited the contractile response to other tachykinins and capsaicin. The SP analogue Spantide (/D-Arg1, D-Trp7,9, Leu11/-SP) inhibited the contractile responses to SP, NKA and NPK. The CGRP also inhibited the NKA- and NPK-induced contractions of the ureter in vitro. In conclusion, capsaicin, which induces the release of mediators from sensory nerves within the ureter, has either stimulatory or inhibitory effects on ureteric smooth muscle, depending on the in vivo dose administered. The inhibitory response at a low capsaicin dose is similar to the effect of CGRP, while the contractile effects at higher doses resemble the response to tachykinins.     

Co-localization of tachykinins and calcitonin gene-related peptide in capsaicin-sensitive afferents in relation to motility effects on the human ureter in vitro

Tachykinin- and calcitonin gene-related peptide (CGRP) immunoreactivities were localized by immunohistochemistry in the same nerves of the kidney, renal pelvis and ureter as well as in spinal ganglion cells of both the guinea-pig and man. The tachykinin and CGRP-immunoreactive nerves in the ureter were present within the smooth muscle layers, around blood vessels, close to and within the lining epithelium. The levels of neurokinin A-, substance P- and CGRP-like immunoreactivity per tissue weight, as determined by radioimmunoassay, were about 30-100-fold higher in the guinea-pig than in the human ureter, which was in good agreement with the relative density of immunoreactive nerve fibres, as seen by immunohistochemistry. Capsaicin treatment caused an almost total disappearance of both neurokinin A-, substance P- and CGRP-immunoreactive nerve fibres in the guinea-pig ureter and a 90% depletion of neurokinin A, substance P- and CGRP-like immunoreactivity, further supporting a sensory origin of these nerves. Reversed-phase high performance liquid chromatography of water extracts of the human ureter revealed the presence of neurokinin A- and eledoisin-like material using antiserum K12, which does not cross-react with substance P. Most of the CGRP-like immunoreactivity in human ureter extracts co-eluted with synthetic human CGRP. Capsaicin both caused inhibition of spontaneous motility of the human ureter in vitro and initiated contractions in some preparations. Neurokinin A and neuropeptide K potently initiated phasic contractions of the ureter, while substance P had only minor contractile effects. CGRP inhibited both spontaneous and neurokinin A-induced ureteric contractions. In conclusion, peptides with potent opposite motility effects are present in the same, presumably sensory nerves of the ureter in both the guinea-pig and man. It will be of importance to determine whether local release of neuropeptides can account for ureteric motility changes accompanying sensory nerve activation upon ureteral obstruction, by e.g. renal calculi.     

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.     

Calcitonin gene-related peptide and prostaglandin E2 but not substance P release induced by antidromic nerve stimulation from rat skin in vitro

The liberation of calcitonin gene-related peptide from rat skin in vitro induced by antidromic electrical stimulation of unmyelinated units is demonstrated. Prostaglandin E2 was released concomitantly during C-fiber stimulation. A dose-dependent increase in prostaglandin E2 content of the eluate was also observed in response to stimulation with substance P (10(-7) to 10(-5) M) and calcitonin gene-related peptide (10(-6) and 10(-5) M). In contrast, prostaglandin E2 did not induce measurable release of neuropeptides. The amount of calcitonin gene-related peptide released during suprathreshold electrical stimulation increased with pulse frequency. Calcitonin gene-related peptide and prostaglandin release were completely inhibited in the presence of EMD 61753, a selective kappa-opioid receptor agonist. No significant release of substance P was observed. The data demonstrate a primary release of calcitonin gene-related peptide from unmyelinated but not myelinated primary afferents in the rat skin, which is accompanied by a secondary liberation of prostaglandin E2, connecting neurogenic inflammation to general mechanisms of 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.     

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.     

In vivo release of calcitonin gene-related peptide-like material from the cervicotrigeminal area in the rat. Effects of electrical and noxious stimulations of the muzzle.

The continuous perfusion with an artificial cerebrospinal fluid of the cervicotrigeminal area of the spinal cord in halothane-anaesthetized rats allowed the collection of calcitonin gene-related peptide-like material with the same immunological and chromatographic characteristics as authentic rat alpha-calcitonin gene-related peptide. The spinal release of calcitonin gene-related peptide-like material could be significantly increased by the local application of 60 mM K+ (approximately +100%), high-intensity percutaneous electrical stimulation (approximately +200%) and noxious heat (by immersion in water at 52 degrees C; approximately +150%) applied to the muzzle. By contrast, noxious mechanical (pinches) and chemical (subcutaneous formalin injection) stimulations and deep cooling (by immersion in water at 0 degrees C) of the muzzle did not alter the spinal release of calcitonin gene-related peptide-like material. In addition, low-intensity electrical stimulation, recruiting only the A alpha/beta primary afferent fibres, significantly reduced (approximately -30%) the release of calcitonin gene-related peptide-like material from the cervicotrigeminal area. These data suggest that among the various types of natural noxious stimuli, noxious heat may selectively excite calcitonin gene-related peptide-containing A delta and C primary afferent fibres projecting within the dorsal horn of the spinal cord, and that activation of A alpha/beta fibres reduces spontaneous calcitonin gene-related peptide-like material release possibly through an inhibitory presynaptic control of calcitonin gene-related peptide-containing A delta/C fibres.     

Inhibitory effects of calcitonin gene-related peptide on long-term potentiation induced in hippocampal slices of rats

It is well known that hippocampus plays important roles in learning and memory. Both calcitonin gene-related peptide (CGRP) and CGRP receptors are found in hippocampus. In the present study we explored the influence of CGRP on long term potentiation (LTP) in hippocampal Schaffer collateral-CA1. Our results demonstrated that CGRP inhibited the LTP induced by high frequency stimulation (HFS) in hippocampal CA1 neurons in rat brain slices. The inhibitory effect was blocked by CGRP receptor-1 antagonist CGRP 8-37. The results indicate that both CGRP and CGRP receptor 1 are involved in the modulation process of LTP in hippocampus of rats.     

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of 1.16 +/- 0.23 X 10(10) M-1 and a site concentration of 43.4 +/- 3.4 fmol/mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of 3.9 +/- 0.7 X 10(9) M-1 whereas salmon calcitonin was very weak with a binding affinity constant of only 6.8 +/- 4.0 X 10(5) M-1. CGRP binding localized by in vitro autoradiography, using [125I]rat calcitonin gene-related peptide, had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex, the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.     

Substance P and calcitonin gene-related peptide in the ureter of chicken and guinea-pig: distribution, binding sites and possible functions.

To elucidate the possible functional significance of sensory neuropeptides in visceral organs of mammals and birds the distribution, binding sites and the effects on ureteric peristalsis of substance P and calcitonin gene-related peptide (CGRP) were investigated in the ureter of guinea-pigs and chickens. In the guinea-pig numerous substance P and CGRP-immunoreactive fibres were located in the adventitia, smooth muscle layer, submucosa and occasionally in the epithelium. Varicose peptidergic fibres were often found on blood vessels. Binding sites for substance P were associated with blood vessels and epithelium in the following density order: venules greater than epithelium greater than arterioles. The highest density of CGRP binding sites was detected on the smooth muscle; venules and arterioles expressed moderate binding. The peristalsis frequency of the isolated ureter of the guinea-pig was increased by neurokinin A and substance P, whereas CGRP inhibited ureteric motility. In the chicken the immunoreactivity to substance P and CGRP was less pronounced. Immunoreactive fibres were found in the submucosa close to the epithelium and around ureteric ganglion cells. Correspondingly, substance P binding sites were located in the epithelium and in ureteric ganglia; however, specific CGRP binding was restricted to large blood vessels. In the chicken none of the sensory neuropeptides affected ureteric motility. Only high doses of the sensory neurotoxin capsaicin (greater than 10 microM) repeatedly produced a non-specific inhibitory effect, similar to that found in a capsaicin-desensitized guinea-pig ureter preparation. The data suggest that in the guinea-pig ureter sensory neuropeptides play a modulatory role in the regulation of ureteric motility and might have vascular and epithelial functions. In the chicken, substance P might be involved in the regulation of epithelial function and modulation of ganglionic transmission. The physiological or pathophysiological role of sensory neuropeptides and the efferent functions of afferent fibres appears to be much better developed in the guinea-pig than in the chicken.     

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.     

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.     

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.     

Acetylcholine receptor expression in primary cultures of embryonic chick myotubes--I. Discoordinate regulation of alpha-, gamma- and delta-subunit gene expression by calcitonin gene-related peptide and by muscle electrical activity

The neuropeptide calcitonin gene-related peptide calcitonin gene-related peptide, and muscle electrical activity regulate in opposite directions the content of nicotinic acetylcholine receptor alpha-subunit mRNA in primary cultures of chick embryonic myotubes. Indeed, treating the cells with calcitonin gene-related peptide or blocking the spontaneous activity of muscle cells by tetrodotoxin (an inhibitor of sodium channels) increases, although to different levels, the content of acetylcholine receptor alpha-subunit mRNA [Fontaine B., Klarsfeld A. and Changeux J. P. (1987) J. Cell Biol. 105, 1337-1342; Klarsfeld A. and Changeux J. P. (1985) Proc. natn. Acad. Sci. U.S.A. 82, 4558-4562]. In this paper, we demonstrate that, under these in vitro culture conditions, calcitonin gene-related peptide (0.1 microM) and tetrodotoxin (0.5 microM) regulate to a smaller extent (no more than 2.5-fold above control) the levels of acetylcholine receptor gamma- and delta-subunit mRNAs. No effect of either compound on acetylcholine receptor biosynthesis was observed during the initial three days of culture. The response to calcitonin gene-related peptide was already maximal when the cells were treated between days three and four after plating (about 3-fold increase of the alpha-subunit mRNA level). The effect of tetrodotoxin resulted in a six-fold increase of the acetylcholine receptor alpha-subunit mRNA level in cells treated between days three and four, and still increased when the cells were exposed to tetrodotoxin through days six and eight (up to a maximum of 20-fold).(ABSTRACT TRUNCATED AT 250 WORDS)