Α-calcitonin Gene-related Peptide Research Articles

Effect of Pilocarpine on Levels of Substance P and α-Calcitonin Gene-related Peptide in Human Saliva

The effects of pilocarpine on salivary secretion, and substance P immunoreactive substance (SP-IS) and α-calcitonin gene-related peptide immunoreactive substance (α-CGRP-IS) levels in human saliva were studied. After a single oral administration of 5 mg pilocarpine, or placebo (lactose), the peptide levels in saliva were measured using a sensitive enzyme immunoassay. Pilocarpine caused a significant increase in saliva SP-IS levels over the range 2.6±0.3 to 7.8±2.6 pg mL−1, 20–180 min after administration, compared with the placebo group. α-CGRP-IS also increased over the range of 14.9±2.7 to 19.2±3.4 pg mL−1, 40–120 min after administration. Pilocarpine significantly raised the saliva volume 20–120 min after administration and significantly increased the total area under the saliva peptide concentration-time curve of SP-IS (1028.3±386.9 min pg mL−1) and α-CGRP-IS (3401.7±852.4 min pg mL−1) compared with the placebo group (348.2±39.2 and 2508.7±106.6 min pg mL−1, respectively). Pilocarpine may have local effects on SP- and α-CGRP-nerves in salivary glands, and the elevated SP and α-CGRP may stimulate the production of saliva.

Effect of Pilocarpine on Levels of Substance P and α-Calcitonin Gene-related Peptide in Human Saliva

Effect of Pilocarpine on Levels of Substance P and α-Calcitonin Gene-related Peptide in Human Saliva

Mice Lacking α-Calcitonin Gene-Related Peptide Exhibit Normal Cardiovascular Regulation and Neuromuscular Development

α-Calcitonin gene-related peptide (αCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration, nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated αCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking αCGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in αCGRP-deficient animals. These results suggest that αCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

Expression of the genes for α-type and β-type calcitonin gene-related peptide during rat embryogenesis

Throughout rat embryogenesis we analysed the expression patterns of the three mature transcripts generated from the two calcitonin gene-related peptide genes: calcitonin, α-calcitonin gene-related peptide, and β-calcitonin gene-related peptide messenger RNAs. In addition, we examined in parallel the distribution of calcitonin gene-related peptide and calcitonin immunoreactivity. Of the three transcripts, β-calcitonin gene-related peptide messenger RNA was first detected in sensory ganglia on embryonic day 14, and by embryonic day 15 was seen to a lesser degree in motor neurons and autonomic ganglia. Starting at embryonic day 16, however, the highest levels of β-calcitonin gene-related peptide messenger RNA were found in motor neurons rather than sensory ganglia. α-calcitonin gene-related peptide messenger RNA was first detected on embryonic day 16 in both sensory ganglia and motor neurons, but at lower levels than β-calcitonin gene-related peptide, particularly in the motor neurons of the spinal cord. By embryonic day 20, transcripts for α- and β-calcitonin gene-related peptide were expressed in distinct brain regions. High levels of α-calcitonin gene-related peptide messenger RNA were detected in hypoglossal, facial, and parabrachial nuclei, and moderate levels in the trigeminal motor and ambiguus nuclei. By contrast, β-calcitonin gene-related peptide messenger RNA was detected at low levels in hypoglossal, ambiguus, facial, and parabrachial nuclei, and at high levels in the trigeminal nucleus. In the oculomotor-trochlear nucleus, β-calcitonin gene-related peptide messenger RNA was the sole isotype expressed. Low levels of messenger RNA for both calcitonin gene-related peptide transcripts were appreciated in the inferior olive. Outside the nervous system, α-calcitonin gene-related peptide messenger RNA was weakly expressed in the thyroid gland and β-calcitonin gene-related peptide messenger RNA in the thymus. Throughout embryogenesis, calcitonin gene-related peptide immunoreactivity usually followed the expression of either α- or β-calcitonin gene-related peptide messenger RNA. Calcitonin messenger RNA and protein were detected only in the thyroid gland from embryonic day 18 onward. This work shows that of the three mature transcripts produced by the two calcitonin gene-related peptide genes, β-calcitonin gene-related peptide messenger RNA is the predominant transcript produced early in rat embryogenesis. However, by perinatal stages α-calcitonin gene-related peptide shows the highest expression in the brain and spinal cord. In autonomic ganglia, β-calcitonin gene-related peptide is either the sole or the predominant transcript. Unlike the chick embryo in which calcitonin messenger RNA is expressed early in the CNS, in rat it was only expressed outside the nervous system in the thyroid gland during the last days of embryogenesis.

Localization of central cannabinoid CB1 receptor messenger RNA in neuronal subpopulations of rat dorsal root ganglia: a double-label in situ hybridization study

In situ hybridization histochemistry was used to show the distribution of messenger RNA for central cannabinoid CB1 receptors in dorsal root ganglia of the rat. CB1 messenger RNA was highly expressed in neuronal subpopulations of rat dorsal root ganglia. The phenotypes of neurons that express messenger RNA for CB1 were subsequently examined by combining a 35S-labeled ribonucleotide probe for CB1 messenger RNA with digoxigenin-labeled riboprobes for preprotachykinin A (substance P precursor), α-calcitonin gene-related peptide and preprosomatostatin (somatostatin precursor) messenger RNAs. Qualitative examination revealed expression of CB1 messenger RNA predominantly in medium- and large-sized cells distributed throughout the dorsal root ganglia. The majority of neurons expressing substance P messenger RNA were CB1 messenger RNA negative and smaller in size than the CB1 messenger RNA-positive cells. Only 13% of substance P messenger RNA-positive cells expressed CB1 messenger RNA. A similar degree of co-localization was observed with α-calcitonin gene-related peptide: 10% of cells expressing messenger RNA for this neuropeptide were CB1 messenger RNA positive. Co-localization of CB1 and somatostatin messenger RNAs was observed in less than 0.5% of somatostatin messenger RNA-positive cells. The data suggest that subpopulations of neurons in rat dorsal root ganglia are capable of synthesizing cannabinoid receptors and inserting them on terminals in the superficial dorsal horn. These findings provide anatomical evidence for cannabinoid modulation of primary afferent transmission. Although an anatomical basis for cannabinoid-mediated suppression of release of neurogenic peptides from nociceptive primary afferents is provided, our results demonstrate that the majority of CB1 messenger RNA-positive neurons in the dorsal root ganglia contain transmitters and/or neuromodulators other than the neuropeptides examined herein.

Calcitonin gene-related peptide increase in the rat spinal dorsal horn and dorsal column nucleus following peripheral nerve injury: Up-regulation in a subpopulation of primary afferent sensory neurons

Calcitonin gene-related peptide in sensory primary afferent neurons has an excitatory effect on postsynaptic neurons and potentiates the effect of substance P in the rat spinal dorsal horn. It has been established that calcitonin gene-related peptide expression in dorsal root ganglion neurons is depressed, and the effect of calcitonin gene-related peptide on dorsal horn neurons is attenuated, following peripheral nerve injury. We report here that a subpopulation of injured dorsal root ganglion neurons show increased expression of calcitonin gene-related peptide. Using in situ hybridization and the retrograde tracer, FluoroGold, we detected an increased number of medium- to large-sized rat dorsal root ganglion neurons projecting to the gracile nucleus that expressed α-calcitonin gene-related peptide messenger RNA following spinal nerve transection. Immunohistochemistry revealed a significant increase in calcitonin gene-related peptide immunoreactivity in the gracile nucleus and in laminae III–IV of the spinal dorsal horn.These results indicate that a subpopulation of dorsal root ganglion neurons express α-calcitonin gene-related peptide messenger RNA in response to peripheral nerve injury, and transport this peptide to the gracile nucleus and to laminae III–IV of the spinal dorsal horn. The increase of the excitatory neuropeptide, calcitonin gene-related peptide, in sites of primary afferent termination may affect the excitability of postsynaptic neurons, and have a role in neuronal plasticity following peripheral nerve injury.

Calcitonin gene-related peptide receptor in human placental syncytiotrophoblast brush-border and basal plasma membranes

Minerals, such as calcium and potassium, are essential for fetal development, but their transplacental transport, and in particular, the effect of hormones on this process has not been extensively studied. Human α-calcitonin gene-related peptide (hαCGRP), a hormone constituted of 37 amino acids, is obtained by the alternative splicing of the mRNA from the calcitonin gene, and could be implicated in placental ion transport. In order to study the presence of this receptor, brush-border and basal plasma membranes were purified, and membrane binding studies were conducted using [ 125I]hαCGRP. The initiation of binding of [ 125I]hαCGRP to both membranes was rapid and reached maximal value after 10 min of incubation at 37°C. Scratchard analysis revealed single-affinity binding sites for hαCGRP with K d equal to 4412.45 ± 604.81 p m and 2673.24 ± 552.51 p m for brush-border and basal plasma membranes, respectively, which were significantly different. Moreover, the maximal number of receptors was significantly different ( P<0.001) in both membranes, with B max of 627.94 ± 31.40 fmol/mg protein for brush-border membranes and 343.70 ± 43.52 fmol/mg protein in basal-plasma membranes. Competitive displacement of [ 125I]hαCGRP with other ligands showed the following potencies; hαCGRP ≈ hβCGRP ≈ Cys (acm) 2,7 CGRP>CGRP (8–37), but no competition was observed with human and salmon calcitonin. Half-maximal displacement for human α- and βCGRP was reached at approximately 10 −10 m for brush-border and basal-plasma membranes. α- and βCGRP, and their fragment and analogue, stimulated cyclic AMP production in placental homogenate ranging from 143–163 per cent. Thus, our results show the presence of CGRP-specific receptors in both the syncytiotrophoblast membranes of human placenta. The role(s) of this related peptide in placenta remains to be investigated

Calcitonin gene-related peptide is a potent intestinal, but not gastric, vasodilator in conscious dogs

The effects of human α-calcitonin gene-related peptide (α-CGRP), β-CGRP, and vasoactive intestinal polypeptide (VIP) on left gastric (LGA) and superior mesenteric arterial (SMA) blood flow, heart rate and systemic arterial blood pressure were investigated in 6 conscious beagle dogs. Both intravenous injections of α-CGRP and β-CGRP (5–200 pmol/kg) and infusion of α-CGRP (25–100 pmol/kg per h) induced a dose-related increase in SMA flow and a dose-related decrease in its resistance. At lower doses, α-CGRP was more potent than β-CGRP, but their maximal responses were the same. α-CGRP and β-CGRP had little effect on LGA flow. However, α-CGRP at 200 pmol/kg, but not β-CGRP, stimulated gastroduodenal contractions that were associated with a phasic increase of LGA flow. Atropine inhibited gastric, but not duodenal, motor and circulatory responses to α-CGRP. Tachycardia and hypotension induced by β-CGRP were significantly less than those by α-CGRP. VIP, on the other hand, increased mainly LGA flow. These results suggest that blood vessels of the small intestine of dogs are more sensitive to CGRP than those of the stomach, while the sensitivity to VIP is reversed.

Calcitonin gene-related peptide is a potent intestinal, but not gastric, vasodilator in conscious dogs

The effects of human α-calcitonin gene-related peptide (α-CGRP), β-CGRP, and vasoactive intestinal polypeptide (VIP) on left gastric (LGA) and superior mesenteric arterial (SMA) blood flow, heart rate and systemic arterial blood pressure were investigated in 6 conscious beagle dogs. Both intravenous injections of α-CGRP and β-CGRP (5–200 pmol/kg) and infusion of α-CGRP (25–100 pmol/kg per h) induced a dose-related increase in SMA flow and a dose-related decrease in its resistance. At lower doses, α-CGRP was more potent than β-CGRP, but their maximal responses were the same. α-CGRP and β-CGRP had little effect on LGA flow. However, α-CGRP at 200 pmol/kg, but not β-CGRP, stimulated gastroduodenal contractions that were associated with a phasic increase of LGA flow. Atropine inhibited gastric, but not duodenal, motor and circulatory responses to α-CGRP. Tachycardia and hypotension induced by β-CGRP were significantly less than those by α-CGRP. VIP, on the other hand, increased mainly LGA flow. These results suggest that blood vessels of the small intestine of dogs are more sensitive to CGRP than those of the stomach, while the sensitivity to VIP is reversed.

Effect of parasympathetic and sensory transmitters on human epicardial coronary arteries and veins.

Vasomotor effects of various agonists were tested on isolated human epicardial coronary arteries and veins at resting tension and after precontraction with U46619. Acetylcholine relaxed all arteries with intact endothelium but only some endothelium-denuded arteries. Most veins did not relax to acetylcholine. Higher concentrations of acetylcholine induced powerful contractions of all arteries and veins. Preincubation with atropine significantly lowered the pD(2) values but not E(max) values for contractile responses to acetylcholine in arteries and veins (pA(2) value for atropine 9.1 arteries and 9.6 veins). Vasoactive intestinal peptide, human alpha-calcitonin gene-related peptide and substance P potently relaxed all arteries with intact endothelium and all veins. Removal of the arterial endothelium abolished relaxation to substance P in most arteries whereas responses to vasoactive intestinal peptide were unaffected, and for alpha-calcitonin gene-related peptide the pD(2) value but not the E(max) value was significantly lowered. In both arteries and veins, the antagonists alpha-calcitonin gene-related peptide (8-37) and spantide lowered significantly the potency for alpha-calcitonin gene-related peptide and substance P, respectively, without significant changes in E(max) values (pA(2) value for alpha-calcitonin gene-related peptide (8-37) 7.9 arteries and 7.9 veins, for spantide 7.6 arteries and 8.1 veins).

Coexpression of preprotachykinin-A, alpha-calcitonin gene-related peptide, somatostatin, and neurotrophin receptor family messenger RNAs in rat dorsal root ganglion neurons.

Syntheses of substance P, somatostatin, and calcitonin gene-related peptide in sensory neurons have been suggested to be regulated by neurotrophic factors retrogradely transported from target tissues. In this study, we re-examined this idea by investigating the coexpression of neurotrophin receptor (trk family proto-oncogene) messenger RNAs, and preprotachykinin-A (a precursor peptide of substance P), alpha-calcitonin gene-related peptide and somatostatin messenger RNAs in lumbar dorsal root ganglion neurons by means of in situ hybridization histochemistry in rats. Approximately 35-40%, 5% and 15-20% of sensory neurons displayed signals for trkA, trkB, and trkC messenger RNAs, respectively. Approximately 28% of dorsal root ganglion neurons were positive for preprotachykinin-A messenger RNA, and were divided into two groups; those labeled strongly and those labeled weakly by in situ hybridization. All the strongly-labeled neurons (78% of preprotachykinin-A-positive cells) expressed trkA messenger RNA at the same time, while the weakly-labeled neurons did not. Thirty-seven per cent of dorsal root ganglion neurons expressed alpha-calcitonin gene-related peptide messenger RNA, and most of these neurons (84%) also expressed trkA messenger RNA. No or few preprotachykinin-A messenger RNA- and/or alpha-calcitonin gene-related peptide messenger RNA-expressing neurons were also positive for trkB or trkC messenger RNAs. Nine per cent of dorsal root ganglion neurons expressed somatostatin messenger RNA, and these neurons lacked all three trk messenger RNAs. Furthermore, most of these neurons (about 90%) showed positive, albeit weak, signals for preprotachykinin-A and alpha-calcitonin gene-related peptide messenger RNAs. The results suggest that expression of preprotachykinin-A and alpha-calcitonin gene-related peptide messenger RNAs is mediated by nerve growth factor via trkA receptor but not by brain-derived neurotrophic factor or neurotrophin-3, and that somatostatin gene transcription is not regulated by any member of the neurotrophin family in rat sensory neurons.

Pituitary adenylate cyclase-activating peptide stimulates neurite growth in PC12 cells.

The ability of PACAP-38 to stimulate morphological development was studied using rat pheochromocytoma PC12 cells. PACAP-38 produced concentration-dependent increases in percentage of cells exhibiting neurite extension. Similar increases were produced by forskolin (28 +/- 2% at 96 h) and 8-bromo cAMP (30 +/- 2%). Vasoactive intestinal peptide and alpha-calcitonin gene-related peptide were without effect. PACAP-38 produced significant increases in PC12 cell cAMP content and inositol phosphate turnover. Intracellular [Ca2+] increased from 169 +/- 14 nM to 560 +/- 58 nM in response to 1 microM PACAP-38. PACAP-stimulated neurite outgrowth was abolished by RpcAMPS, an inhibitor of cAMP-dependent kinases but was unaffected by the protein kinase C antagonist H7.

Skeletal muscle microcirculatory response to rat α-calcitonin gene-related peptide

We used in vivo video microscopy to determine the effect of increasing doses of rat α-calcitonin gene-related peptide (rCGRP) on rat cremaster muscle arterioles in the presence or absence of the nitric oxide synthase inhibitor N-ω-nitro-L-arginine (L-NNA). Male Sprague-Dawley rats (118–148 g) were anaesthetized with pentobarbital, and neurovascularly intact cremaster muscles were imaged. Changes in the diameter, erythrocyte velocity and volume flow in second- (A2), third- (A3), and fourth- (A4) order arterioles were determined. To produce uniform arteriolar tone, the cremaster preparation was challenged with norepinephrine (NE: 10 −7 M). L-NNA (10 −4 M), which was shown to inhibit acetylcholine- (ACh: 10 −6 M) induced arteriolar dilations, was added to 16 of the preparations. Preparations were then challenged by adding cumulative log concentrations of rCGRP (10 −12 – 10 −7 M; n = 16) or an equivalent volume of vehicle (n = 19) to the bath. Following rCGRP challenge, arterioles were maximally dilated with 10 −5 M nitroprusside (NP). rCGRP caused significant dose-dependent increases in erythrocyte velocity and volume flow in A2 arterioles, and in diameter, velocity, and volume flow in A3 and A4 arterioles, by 10 −8 M, when compared with vehicle-treated controls. L-NNA had no significant effect on rCGRP-induced responses. These data indicate that rCGRP causes dose-dependent dilation of skeletal muscle resistance arterioles at a concentration similar to that observed in larger vessels. This dilation does not appear to be dependent on the vascular production of nitric oxide from L-arginine.

In vitro expression of calcitonin gene-related peptide in human endothelial cells transfected with plasmid and retroviral vectors

Introduction of cDNA encoding human α-calcitonin gene-related peptide (CGRP), a potent vasodilator, into cultured human umbilical vein endothelial cells (HUVEC) was undertaken by using plasmids and retroviral vectors. In order to improve expression, modification of context coding sequence for the initiation of CGRP translation and deletion of nontranslated regions of CGRP cDNA in the transfection vectors were tested. Stable transfer of neo in the HUVEC was achieved with both plasmid and retroviral vectors. Integration rates obtained by using retrovirus (∼1%), where higher than those achieved with plasmid-mediated transfection ( < 1 1000 ). CGRP expressed in the transfected HUVEC was secreted into culture medium when a leading sequence was included in the expression vectors. CGRP was detected by enzyme-linked immunosorbent assay (ELISA) in the supernatants of both transiently transfected and stably transfected/infected HUVEC. Higher levels of expression were achieved by using plasmid (giving a maximum CGRP concentration of 6.5 ± 0.5 pM in the supernatant) than retroviruses. Lipofectin-mediated transfer of CGRP cDNA also resulted in transient expression of CGRP in the HUVEC.

Magnesium Sulphate Reverses the Carotid Vasoconstriction Caused by Endothelin-1, Angiotensin II and Neuropeptide-Y, but Not That Caused by NG-nitro-l-Arginine Methyl Ester, in Conscious Rats

1. Magnesium sulphate (MgSO4) has been used for many years in the prevention of eclamptic seizures, but its mechanism of action has never been elucidated. Recent studies suggest that cerebral vasospasm is an important feature of eclampsia and we have developed and tested the hypothesis that MgSO4 can reverse cerebral vasoconstriction. 2. Studies were performed in conscious, male Long Evans rats with pulsed Doppler probes sutured around both common carotid arteries after the external carotid artery had been ligated on the left, thus allowing simultaneous measurement of changes in common and internal carotid blood flow. Intravascular catheters were placed in the abdominal aorta for measurement of systemic blood pressure and in the right jugular vein for administration of drugs. Mean arterial blood pressure and mean Doppler shift signals were used to calculate percentage changes in common and internal carotid vascular conductance. 3. After a period of recovery the animals were infused with endothelin-1, angiotensin II, neuropeptide-Y or NG-nitro-L-arginine methyl ester alone or in combination, and MgSO4 in low or high dose was infused when the effects of the vasoconstrictors had become established. 4. MgSO4 itself, at the low dose, had no effect on carotid vascular conductance. Endothelin-1, angiotensin II and neuropeptide-Y all reduced common and internal carotid vascular conductance and this effect was significantly attenuated by low dose MgSO4. The carotid vasoconstrictor action of endothelin-1 was completely abolished by high dose MgSO4.(ABSTRACT TRUNCATED AT 250 WORDS)

CGRP 8-37 blocks the inhibition of gastric emptying induced by intravenous injection of α-CGRP in rats

The receptor subtype mediating rat α-calcitonin gene-related peptide (α-CGRP)-induced inhibition of gastric emptying of a non nutrient solution was tested in conscious rats using the CGRP 1 receptor antagonist, CGRP 8-37, and the CCK antagonist, MK-329. Intravenous injection of α-CGRP (0.5 μg) decreased gastric emptying to 26.5±5.8% from 46.4±3.9% in vehicle- treated group. Intravenous injection of CGRP 8-37 (15 μg) did not influence gastric emptying but completely prevented α-CGRP inhibitory effect whereas the 47% delay in gastric emptying induced by intravenous cholecystokinin-8 (CCK, 0.25 μg) was not modified. The CCK antagonist, MK-329 (1 mg) reversed CCK- but not α-CGRP-induced delay in gastric emptying. These results demonstrate that CGRP 8-37 is a specific tool to block α-CGRP inhibitory action on gastric motor function and suggest that gastric stasis elicited by peripheral injection of α-CGRP may involve an interaction with a CGRP 1 receptor subtype.

Microinjection of α-Calcitonin Gene-Related Peptide into the Hypothalamus Activates Sympathetic Outflow in Rats

Using isolated arteries, we demonstrated a marked difference in the angiotensin II-forming systems between human and rodent vessels. In human arteries, only 30-40% of the conversion of angiotensin I to angiotensin II depended on the angiotensin-converting enzyme (ACE), and the rest of the angiotensin II formation was ascribed to chymostatin-sensitive angiotensin II-generating enzyme (CAGE). On the contrary, angiotensin II formation in rodent arteries totally depended upon ACE, without any sign of CAGE involvement. Such a marked species-difference can be relevant to the reported difference between humans and rodents in the ACE inhibitor effects on the myointimal hyperplasia after intimal balloon injury.

Axonal flow blockade induces α-CGRP mRNA expression in rat motoneurons

Axonal flow of motoneurones was blocked in the sciatic nerve by the application of vinblastine at a low concentration (100 μM) not considered likely to cause axonal degeneration. The number of motoneurons expressing α-calcitonin gene-related peptide (CGRP) mRNA was significantly increased by vinblastine application, while the expression of ß-CGRP mRNA was not influenced. These findings suggest that the synthesis of these two peptides is differentially regulated and that α-CGRP mRNA is possibly down-regulated by unknown factor(s) which normally undergo retrograde transport from the periphery.

Differential regulation of α-calcitonin gene-related peptide and preprocholecystokinin messenger rna expression in α-motoneurons: Effects of testosterone and inactivity induced factors

α-Calcitonin gene-related peptide expression in α-motoneurons is regulated by spinal cord transection, axotomy and testosterone, but to date there are no studies which examine the regulation of cholecystokinin expression in motoneurons. In the present study, we compared the regulation of preprocholecystokinin and α-calcitonin gene-related peptide messenger RNA levels in motoneurons of the spinal nucleus of the bulbocavernosus. Previously, we demonstrated that manipulations which decrease activity in target muscles of the spinal nucleus of the bulbocavernosus motoneurons increase α-calcitonin gene-related peptide message and peptide levels in spinal nucleus of the bulbocavernosus motoneurons. This muscle-nerve interaction is mediated by a soluble factor which is increased by castration. We now report that decreasing plasma testosterone levels decreased preprocholecystokinin messenger RNA levels. Testosterone replacement at the time of castration restored preprocholecystokinin messenger RNA levels to intact values. Injections of crude extracts prepared from denervated bulbocavernosus/ levator ani into the homologous muscles of gonadally intact rats increased the levels of α-calcitonin gene-related peptide messenger RNA in spinal nucleus of the bulbocavernosus motoneurons. The levels of preprocholecystokinin messenger RNA did not differ in rats injected with denervated bulbocavernosus/ levator ani extract or buffer, both of which were significantly higher than in intact, untreated rats. The results of the present experiments imply that levels of preprocholecystokinin and α-calcitonin gene-related peptide messenger ribonucleic acid are differentially regulated in spinal nucleus of the bulbocavernosus motoneurons.

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 α-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 + (∼ + 100%), high-intensity percutaneous electrical stimulation (∼ + 200%) and noxious heat (by immersion in water at 52°C; ∼ + 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°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 Aα/β primary afferent fibres, significantly reduced (∼ − 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 Aδ and C primary afferent fibres projecting within the dorsal horn of the spinal cord, and that activation of aα/β fibres reduces spontaneous calcitonin gene-related peptide-like material release possibly through an inhibitory presynaptic control of calcitonin gene-related peptide-containing Aδ/C fibres.