Containing Calcitonin Gene-related Peptide Research Articles

Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers

Superoxide anion (O 2 −•) production was previously reported to be increased in celiac ganglia (CG) during DOCA–salt hypertension, possibly via activation of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. This suggested a role for neuronal NADPH oxidase in autonomic neurovascular control. However, the expression and localization of NADPH oxidase in the peripheral neurons are not fully known. The purpose of this study was to examine the subcellular localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. In rat CG, p22 phox and neuropeptide Y (NPY) were colocalized in all neurons. P22 phox was also localized to dorsal root ganglia (DRG) neurons that contain calcitonin gene related peptide (CGRP). In mesenteric arteries, p22 phox and p47 phox were colocalized with NPY or CGRP in perivascular nerve terminals. A similar pattern of nerve terminal staining of p22 phox and p47 phox was also found in cultured CG neurons and nerve growth factor (NGF)-differentiated PC12 cells. These data demonstrate a previously uncharacterized localization of NADPH oxidase in perivascular nerve fibers. The presence of a O 2 −•-generating enzyme in close vicinity to the sites of neurotransmitter handling in the nerve fibers suggests the possibility of novel redox-mediated mechanisms in peripheral neurovascular control.

Calcitonin Gene-Related Peptide Selectively Relaxes Contractile Responses to Endothelin-1 in Rat Mesenteric Resistance Arteries

We tested the hypothesis that endothelin-1 (ET-1) modulates sensory-motor nervous arterial relaxation by prejunctional and postjunctional mechanisms. Isolated rat mesenteric resistance arteries were investigated with immunohistochemistry, wire-myography, and pharmacological tools. ET(A)- and ET(B)-receptors could be visualized on the endothelium and smooth muscle and on periarterial fibers containing calcitonin gene-related peptide (CGRP). Arterial contractile responses to ET-1 (0.25-16 nM) were not modified by blockade of ET(B)-receptors, NO-synthase, and cyclooxygenase or desensitization of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) with capsaicin. ET-1 reversed relaxing responses to CGRP in depolarized arteries. This effect was inhibited by ET(A)-antagonists. It was not selective because ET-1 also reversed relaxing responses to Na-nitroprusside (SNP) and because phenylephrine (PHE; 0.25-16 microM) similarly reversed relaxing responses to CGRP or SNP. Conversely, contractile responses to ET-1 were, compared with PHE, hypersensitive to the relaxing effects of the TRPV1-agonist capsaicin and to exogenous CGRP, but not to acetylcholine, forskolin, pinacidil, or SNP. In conclusion, ET-1 does not stimulate sensory-motor nervous arterial relaxation, but ET(A)-mediated arterial contractions are selectively sensitive to relaxation by the sensory neurotransmitter CGRP. This does not involve NO, cAMP, or ATP-sensitive K(+) channels.

Activation of iGluR5 kainate receptors inhibits neurogenic dural vasodilatation in an animal model of trigeminovascular activation

Migraine is a disabling neurological disorder involving activation, or the perception of activation, of trigeminovascular afferents containing calcitonin gene-related peptide (CGRP). Released CGRP from peripheral trigeminal afferents causes dilatation of dural blood vessel, and this is used to measure trigeminal nerve activation. Kainate receptors with the GluR5 subunit (iGluR5, ionotropic glutamate receptor) are present in the trigeminal ganglion and may be involved in nociception. We investigated the possible involvement of prejunctional iGluR5 kainate receptors on CGRP release from trigeminal afferents. We used neurogenic dural vasodilatation, which involves reproducible vasodilatation in response to CGRP release after electrical stimulation of the dura mater surrounding the middle meningeal artery. The effects of the specific iGluR5 receptor antagonist UBP 302 and agonist (S)-(-)-5-iodowillardiine were investigated on neurogenic and CGRP-induced dural vasodilatation in rats, by using intravital microscopy. Administration of 10 and 20 mg.kg(-1) of iodowillardiine inhibited electrically induced dural vessel dilatation, an effect blocked by pretreatment with 50 mg.kg(-1) UBP 302. Administration of the iGluR5 receptor antagonist UBP 302 alone had no significant effect. CGRP (1 mg.kg(-1))-induced dural vasodilatation was not inhibited by the iGluR5 receptor agonist iodowillardiine. This study demonstrates that activation of the iGluR5 kainate receptors with the selective agonist iodowillardiine is able to inhibit neurogenic dural vasodilatation probably by inhibition of prejunctional release of CGRP from trigeminal afferents. Taken together with recent clinical studies the data reinforce CGRP mechanisms in primary headaches and demonstrate a novel role for kainate receptor modulation of trigeminovascular activation.

Characterisation of calcitonin gene-related peptide-immunoreactive neurons in the myenteric plexus of rat colon

A mechanical or chemical stimulus applied to the intestinal mucosa induces motility reflexes in the rat colon. Enteric neurons containing calcitonin gene-related peptide (CGRP) have been suggested as intrinsic primary afferent neurons responsible for mediating such reflexes. In the present study, immunohistochemistry was performed on whole-mount stretch preparations to investigate chemical profiles, morphological characteristics and projections of CGRP-containing neurons in the myenteric plexus of the rat colon. CGRP-positive neuronal cell bodies were detected in preparations incubated with colchicine-containing medium, whereas CGRP-positive nerve fibres were found in colchicine-untreated preparations. These neurons had large oval or round cell bodies that were also immunoreactive for the calcium-binding protein calretinin and neurofilament 200. Myenteric neurons positive for both calretinin and neurofilament 200 had several long processes that emerged from the cell body, consistent with Dogiel type II morphology. Application of the neural tracer DiI to the intestinal mucosa revealed that DiI-labelled myenteric neurons each had an oval or round cell body immunoreactive for calretinin. Thus, CGRP-containing myenteric neurons are Dogiel type II neurons and are immunoreactive for calretinin and neurofilament 200 in the rat colon. These neurons probably project to the intestinal mucosa.

Perivascular neuropeptides (NPY, VIP, CGRP and SP) in human brain vessels after subarachnoid haemorrhage.

INTRODUCTION--Cerebral blood vessels are innervated by sympathetic nerve fibres storing neuropeptide Y (NPY), parasympathetic nerves storing acetylcholine, vasoactive intestinal peptide (VIP) and sensory afferent fibres containing calcitonin gene-related peptide (CGRP), substance P (SP) and neurokinin A. In experimental studies on subarachnoid haemorrhage (SAH) there are indications that perivascular peptides are involved. In the present study we have in man measured the levels of NPY, VIP, SP and CGRP in brain vessels of patients that have suffered a fatal SAH and compared this with the levels encountered in subjects that died of an extracerebral cause. MATERIAL AND METHODS--Vessels from patients who have died from SAH or nonSAH were obtained during autopsy performed within 24 hrs after death. The peptides were extracted and fractionated with reversed phase liquid chromatography (HPLC). The levels of NPY, VIP, SP, and CGRP were measured with radioimmunoassay. Vasomotor responses of human cerebral arteries were performed using a sensitive in vitro system. RESULTS--Human cerebral vessels contained NPY, VIP, CGRP and SP which eluted at the same positions as the authentic peptides. The level of CGRP was significantly lower (p < 0.01) in arteries removed from SAH patients as compared to control subjects. The level of SP was not changed, if anything it tended to be increased after SAH. The levels of NPY and VIP were not significantly altered after SAH. In isolated brain vessels alpha-CGRP was a potent vasodilator of arteries precontracted with whole blood, prostaglandin F2 alpha or endothelin. It had a poor effect on vessels precontracted with 60 mM potassium. CONCLUSION--The evidence suggest that the trigemino-cerebrovascular system, storing CGRP and SP, is to a differential degree involved in the pathophysiology of SAH in man and supports the hypothesis of an exhaustion of CGRP as one important factor in the development of late spasm occurring after SAH.

L1 cell adhesion molecule is essential for the maintenance of hyperalgesia after spinal cord injury

Spinal cord injury (SCI) results in a loss of normal motor and sensory function, leading to severe disability and reduced quality of life. A large proportion of individuals with SCI also suffer from neuropathic pain symptoms. The causes of abnormal pain sensations are not well understood, but can include aberrant sprouting and reorganization of injured or spared sensory afferent fibers. L1 is a cell adhesion molecule that contributes to axonal outgrowth, guidance and fasciculation in development as well as synapse formation and plasticity throughout life. In the present study, we used L1 knockout (KO) mice to determine whether this adhesion molecule contributes to sensory dysfunction after SCI. Both wild-type (WT) and KO mice developed heat hyperalgesia following contusion injury, but the KO mice recovered normal response latencies beginning at 4 weeks post-injury. Histological analyses confirmed increased sprouting of sensory fibers containing calcitonin-gene related peptide (CGRP) in the deep dorsal horn of the lumbar spinal cord and increased numbers of interneurons expressing protein kinase C gamma (PKCγ) in WT mice 6 weeks after injury. In contrast, L1 KO mice had less CGRP + fiber sprouting, but even greater numbers of PKCγ + interneurons at the 6 week time point. These data demonstrate that L1 plays a role in maintenance of thermal hyperalgesia after SCI in mice, and implicate CGRP + fiber sprouting and the upregulation of PKCγ expression as potential contributors to this response.

Activity and expression of the vanilloid receptor 1 (TRPV1) is altered by long‐term diabetes in epineurial arterioles of the rat sciatic nerve

Epineurial arterioles of the sciatic nerve are innervated by sensory nerves containing calcitonin gene-related peptide (CGRP). We postulated that treating these resistance vessels with capsaicin would cause the release of endogenous CGRP and vascular relaxation. Videomicroscopy was used to examine the effect of capsaicin and neuropeptides on vascular reactivity of epineurial arterioles from control and streptozotocin-induced diabetic rats. Using immunohistochemistry, we examined expression of neuropeptide Y (NPY) and vanilloid receptor 1 in epineurial arterioles. Instead of relaxation, capsaicin was found to cause a concentration-dependent vasoconstriction in epineurial arterioles. The effect of capsaicin was transient, refractory, blocked by capsazepine and duplicated by resiniferatoxin. When examining potential candidates for the mediation of capsaicin-induced constriction, we found that vasopressin (VP), NPY, serotonin (5HT) and endothelin (ET), but not neurokinin A or substance P, caused a concentration-dependent vasoconstriction of epineurial arterioles. Epineurial arterioles express NPY and receptor antagonists to NPY significantly decreased capsaicin-induced vasoconstriction. In long-term diabetic rats, vasoconstriction to capsaicin was significantly attenuated. However, long-term diabetes did not impair vasoconstriction of epineurial arterioles to exogenous VP, NPY, 5HT or ET. Examining the expression of vanilloid receptor 1 in epineurial arterioles from control and long-term diabetic rats, we found that immunoreactivity for vanilloid receptor 1 was decreased by diabetes. These studies suggest that long-term diabetes causes vascular dysfunction in epineurial arterioles of the sciatic nerve that includes a decrease in capsaicin-induced vasoconstriction that is likely due to a decrease in the expression of vanilloid receptor 1.

Selective sensory denervation by capsaicin aggravates adriamycin-induced cardiomyopathy in rats

Capsaicin-sensitive sensory nerves that contain calcitonin gene-related peptide (CGRP) contribute significantly to cardioprotective mechanisms. In this study, the possible role of capsaicin-sensitive afferent nerves in the development of congestive heart failure was examined in an established model of adriamycin-induced experimental cardiomyopathy in rats. Systemic treatment with capsaicin was utilized to deplete sensory neuropeptides from cardiac afferent nerves. Echocardiography was applied to assess the cardiac function in adriamycin-treated rats pretreated with capsaicin or its vehicle. In control rats, adriamycin treatment produced a reduction in the fractional shortening of the left ventricle and an increase in the ratio of the left atrial diameter and the aortic diameter, indicative of a decreased myocardial contractility and heart failure only at 3-4 weeks post-treatment. In contrast, in capsaicin-pretreated rats, a deterioration of the cardiac function was already evident 1 week after the cessation of adriamycin administration, while the clinical signs associated with cardiomyopathy were more severe and displayed a significantly more rapid progression. Immunohistochemistry revealed a complete depletion of calcitonin gene-related peptide from cardiac sensory nerves after systemic capsaicin treatment. This study has demonstrated that elimination of capsaicin-sensitive afferent nerves promotes the development and progression of adriamycin-induced myocardial dysfunction. The results suggest that interfering with capsaicin/vanilloid receptor function and/or perturbation of the myocardial CGRP metabolism may open up new perspectives concerning prevention and/or alleviation of the pathological changes that follow adriamycin treatment.

INTRAVESICAL BOTULINUM TOXIN A ADMINISTRATION PRODUCES ANALGESIA AGAINST ACETIC ACID INDUCED BLADDER PAIN RESPONSES IN RATS

There is evidence that botulinum toxin A (BTX-A) might have analgesic properties. However, the mechanisms by which BTX-A alters pain remain largely unexplored. In the bladder afferent nerve fibers contain calcitonin gene-related peptide (CGRP). In this study we investigated the effect of intravesical BTX-A administration on CGRP immunoreactivity and bladder hyperactivity in an acetic acid induced bladder pain model in rats. Experimental and control animals were catheterized and intravesically exposed to protamine sulfate (1 ml, 10 mg/ml), followed by BTX-A (1 ml, 25 U/ml) or saline, respectively. Three or 7 days after intravesical therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemistry was used to detect CGRP. The intercontraction interval (ICI) was decreased after acetic acid instillation (50.2% and 65.0%) in the control group at days 3 and 7, respectively. However, rats that received BTX-A showed a significantly decreased response (28.6% ICI decrease) to acetic acid instillation at day 7. This effect was not observed at day 3 (62.2% ICI decrease). Increased CGRP immunoreactivity was detected in the BTX treated group at day 7, which was not detected at day 3. Intravesical BTX administration blocked acetic acid induced bladder pain responses and inhibited CGRP release from afferent nerve terminals. These results support the clinical application of BTX-A for the treatment of interstitial cystitis and other types of visceral pain.

Afferent connections of the parvocellular subparafascicular thalamic nucleus in the rat: evidence for functional subdivisions.

The parvocellular subparafascicular nucleus of the thalamus (SPFp) consists of separate subdivisions, i.e., a medial portion containing galanin-immunoreactive (-IR) axons and a lateral portion containing calcitonin gene related peptide (CGRP)-IR neurons and fibers. These subdivisions appear to have distinct functional roles. In particular, ejaculation-induced Fos expression is expressed in the medial SPFp. Hence, it was hypothesized that medial SPFp is involved in relay of copulation-related information. In contrast, lateral SPFp is involved in the processing of auditory and visual signals involved in fear-conditioned responses. Here we tested the hypothesis that medial and lateral subdivisions of SPFp receive different sets of afferents and that these differences contribute to the separate functional roles of the two subdivisions. Inputs to medial and lateral SPFp were identified following injections of FG restricted to either division in male rats. The medial SPFp receives unique inputs from lumbar spinothalamic cells and brain regions involved in processing of visceral stimuli, supporting the hypothesis that the medial SPFp is involved in the relay of genitosensory information critical for the expression of male sexual behavior. The afferents of the lateral SPFp include brain regions involved in processing of visual and auditory signals and support a role for this subdivision in relay of visual and auditory information. Thus, the two subdivisions of SPFp are anatomically and functionally distinctive.

Galanin‐Immunoreactive Cells and Their Relation to Calcitonin Gene‐Related Peptide‐, Substance P‐ and Somatostatin‐Immunoreactive Cells in Rat Lumbar Dorsal Root Ganglia

We report upon the distribution of galanin-immunoreactive (GAL-IR) cells in the lumbar dorsal root ganglia (DRG) of the rat, and upon the distribution of GAL-IR cells, which also contain calcitonin gene-related peptide (CGRP)-, substance P (SP)- and somatostatin (SOM)-immunoreactivity. Neuropeptide-immunoreactive lumbar DRG cells were 55.8% for CGRP, 12.7% for SP, and 6.5% for GAL in lumbar DRG cells. There was no significant difference between the right and left DRGs (L1-L6) for any neuropeptide-immunoreactive cell (P < 0.01). In terms of size distribution, CGRP-immunoreactive cells were identified below 1500 microm2, and SP-, and GAL-IR cells below 600 microm2. Neuropeptide immunoreactive cells showed various immunoreactivities in the cytoplasm according to each neuropeptide. CGRP and SP immunoreactive cells were colocalized with GAL immunoreactive cells in the serial sections about 83.3 and 60% respectively, but SOM colocalizing with GAL-IR cells were not in evidence. The current results confirm and extend previous results, and show that neuropeptides can coexist in single sensory neurones of the rat DRG. In addition, our results demonstrate that the normal distribution of some neurotransmitters modulating sensory action in Wistar Kyoto rat, make this model more prone to develop neuropathic pain than Sprague-Dawley rat.

Activation of Delta Opioid Receptors Induces Receptor Insertion and Neuropeptide Secretion

Here we describe a novel mechanism for plasma membrane insertion of the delta opioid receptor (DOR). In small dorsal root ganglion neurons, only low levels of DORs are present on the cell surface, in contrast to high levels of intracellular DORs mainly associated with vesicles containing calcitonin gene-related peptide (CGRP). Activation of surface DORs caused Ca 2+ release from IP 3-sensitive stores and Ca 2+ entry, resulting in a slow and long-lasting exocytosis, DOR insertion, and CGRP release. In contrast, membrane depolarization or activation of vanilloid and P2Y 1 receptors induced a rapid DOR insertion. Thus, DOR activation induces a Ca 2+-dependent insertion of DORs that is coupled to a release of excitatory neuropeptides, suggesting that treatment of inflammatory pain should include blockade of DORs.

Distribution and changes with age of calcitonin gene-related peptide- and substance P-immunoreactive nerves of the rat urinary bladder and lumbosacral sensory neurons.

In the distal parts of the urinary tract, nerves containing calcitonin gene-related peptide (CGRP) or substance P (SP) are sensory with their cell bodies located in lumbosacral dorsal root ganglia. These two neuropeptides are recognised as being present in pelvic sensory nerves, and may be involved in the mediation of pain, stretch and/or vasodilatation. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive (-ir) for CGRP and SP in the urinary bladder and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made for CGRP-ir and SP-ir fibres innervating the dome, body and base of the urinary bladder. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for CGRP and SP. There were very few immunoreactive axons in the dome and the overall density of innervation increased progressively towards the base of the bladder. The density of innervation in the aged rats revealed a slight reduction in CGRP and SP innervation of the detrusor muscle but was otherwise comparable to the young group. However, immunostaining of the lumbosacral dorsal root ganglia revealed that the percentage of CGRP- and SP-ir neuronal profiles showed a significant (P < 0.05) reduction from (mean +/- S.D) 44.5 +/- 2; 23.3 +/- 2 in young adult to 25.0 +/- 2.9; 14.8 +/- 1.6 in aged rats, respectively. These findings suggest that the involvement of CGRP and SP in urinary bladder innervation is relatively unchanged in old age, but their expression in dorsal root ganglion neurons is affected by age. The afferent micturition pathway from the pelvic region via these lumbosacral ganglia may be perturbed as a result.

Do sensory calcitonin gene-related peptide nerve fibres in the rat pelvic plexus supply autonomic neurons projecting to the uterus and cervix?

Sensory nerve fibres containing calcitonin gene-related peptide (CGRP) innervate neurons of the paracervical ganglion (PCG) in the female rat pelvic plexus. We have combined retrograde tracing with immunocytochemistry to investigate whether CGRP-immunoreactive (-IR) fibres supply neurons targeting the genital tract. Of the total neurons projecting to either the uterine horns or the cervix, 38 and 41% received CGRP-IR innervation, respectively. All these neurons displayed choline acetyltransferase-IR, thus are cholinergic. They were found throughout the PCG and other pelvic plexus ganglia, namely accessory ganglia (AG) and hypogastric plexus (HP). Pelvic nerve section showed that afferent fibres in these nerves provided most of the CGRP-IR fibres supplying uterine- or cervical-related neurons in the PCG/AG, none in HP. It is suggested that such sensory-motor network may provide a local pathway for reflex control of genital tract activity, acting through cholinergic nerve projections.

Neuropeptide expression in the human trigeminal nucleus caudalis and in the cervical spinal cord C1 and C2.

In migraine and other primary headaches there is a strong vascular component. Besides the trigeminovascular components some of the associated symptoms point to the involvement of brain stem regions. The central limb of the trigeminal vascular pathway is its projection to the trigeminal nucleus caudalis (TNC) and to the C1-C2 levels of the spinal cord. The aim of the present study was to demonstrate the occurrence of some neurotransmitters in these regions in man. In both the TNC and in the Rexed's laminae I and II of the dorsal horns at the C1 and C2 levels there were numerous substance P immunoreactive fibres. Fibres containing calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP) were moderately dense in number. Fibres containing vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS) were not seen in the TNC or at the C1 and C2 levels of the spinal cord.

Origins of Dorsal Root Ganglion Cells with CGRP innervating Quadriceps Femoris Tendon of Rat

Purpose : This study was undertaken to determine the origins of dorsal root ganglion (DRG) cells containing calcitonin gene-related peptide (CGRP) which innervate the quadriceps femoris tendon in the rat. Materials and Methods : DRG cells containing CGRP, which innervate the quadriceps femoris tendon, from 25 rats (Sprague-Dawley, 200-250 g) were examined using the retrograde tracing technique (neural tracers: horseradish peroxidase and fluorogold) combined with immunohistochemistry. Results : Injection of horseradish peroxidase (HRP) or fluoro-gold (FG) into the quadriceps femoris tendon resulted in the ipsilaterally labelling of cells between L1 and L6 DRGs. However, a large number of the labelled cells innervating the quadriceps femoris tendon were found in the L3 and L4 DRGs. Many DRG cells were immunostained with CGRP antibody in the L1-6 DRGs. The number of CGRP immunoreactive cells in the lumbar DRGs was larger than in the sacral DRG. FG labelled cells containing CGRP immunoreactivity (FG+CGRP cells) were found in the lumbosacral DRGs. Many FG+CGRP cells innervating the quadriceps femoris tendon were located in the L3 and L4 DRGs. Conclusion : These results show that the main DRG origin for the sensory innervation of the quadriceps femoris tendon is L3 or L4. The neurogenic pain of the quadriceps femoris tendon may originate from this region, and suggests that this may be important for the release of neurogenic pain.

Origin and Co-localization of nitric oxide synthase, CGRP, PACAP, and VIP in the cerebral circulation of the rat.

The origin of perivascular nerve fibres storing nitric oxide synthase (NOS) and co-localisation with perivascular neuropeptides were examined in the rat middle cerebral artery (MCA) by retrograde tracing with True Blue (TB) in combination with immunocytochemistry. Application of TB to the proximal part of the middle cerebral artery labelled nerve cell bodies ipsilaterally in the trigeminal, sphenopalatine, otic, and superior cervical ganglia. A few labelled cell bodies were seen contralaterally, suggesting bilateral innervation. In the parasympathetic sphenopalatine and otic ganglia, numerous TB-labelled cell bodies contained neuronal NOS (C- and N-terminal), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase activating peptide (PACAP). In the trigeminal ganglion, almost all TB-labelled cell bodies contained calcitonin gene-related peptide (CGRP) but only a few cells contained NOS. In the superior cervical ganglion, the majority of the TB-labelled nerve cells contained neuropeptide Y (NPY) but none of them contained NOS. Removal of the ipsilateral sphenopalatine ganglion caused a slight reduction in the number of perivascular VIP-, PACAP-, and NOS-containing fibres after 3 days in the MCA while there was no difference at 2 and 4 weeks after the denervation as compared to control. This indicates that the parasympathetic VIP-, PACAP-, and NOS-immunoreactive nerve fibres in the rat MCA originate from several sources.

Expression of vasoactive intestinal peptide, calcitonin gene-related peptide, substance P, and intermediate neurofilaments in nasal mucosal nerve fibers of horses without nasal disease.

To determine the distribution of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP), and intermediate neurofilaments in nasal mucosa of horses. 6 horses without evidence of nasal disease. Full-thickness nasal tissue specimens were obtained from the rostral portion of the nasal septum at necropsy, and fluorescence immunohistochemistry was performed to assess mucosal distribution of nerve fibers. Nerve fibers with CGRP-like immunoreactivity (CGRP-Li) formed a dense subepithelial network, and a large number of fibers were found coursing between epithelial cells. Fibers with CGRP-Li were also associated with blood vessels and mucous glands. Fibers with SP-like immunoreactivity (SP-Li) had a similar distribution and density. In contrast, there were few fibers with VIP-like immunoreactivity. Fibers containing intermediate neurofilaments were prominent and appeared as large nerve fiber bundles mainly adjacent to the nasal septum but also close to mucous glands and within the lamina propria. Intermediate neurofilaments were also identified in single nerve fibers at all sites, but the density of fibers with intermediate neurofilaments did not match that of fibers with CGRP- or SP-Li. The density and distribution of nerve fibers containing SP- or CGRP-Li in nasal mucosa of horses was similar to that reported for other species. However, expression of VIP in nerve fibers was low. Antibodies against intermediate neurofilaments identified many nerve fibers in nasal mucosa of horses but did not appear to identify small diameter fibers expressing SP or VIP.

Increased Blood Pressure in α-Calcitonin Gene–Related Peptide/Calcitonin Gene Knockout Mice

Nerves that contain calcitonin gene-related peptide (CGRP) are components of the sensory nervous system. Although these afferent nerves have traditionally been thought to sense stimuli in the periphery and transmit the information centrally, they also have an efferent vasodilator function. Acute administration of a CGRP receptor antagonist increases the blood pressure (BP) in several models of hypertension, which indicates that this potent vasodilator plays a counterregulatory role to attenuate the BP increase in these settings. To determine the role of this peptide in the long-term regulation of cardiovascular function, including hypertension, we obtained mice that have a deletion of the alpha-calcitonin gene-related peptide (alpha-CGRP) gene. Although the beta-calcitonin gene-related peptide (beta-CGRP) gene is intact in these mice, alpha-CGRP is by far the predominant species of CGRP produced in dorsal root ganglia (DRG) sensory neurons. Initially, we examined the effect of deletion of the alpha-CGRP on baseline BP and beta-CGRP and substance P mRNA expression. Systolic BP was significantly higher in the knockout mice (n=7) compared with wild-type in both male (160+/-6.1 vs 125+/-4.8 mm Hg) and female (163+/-4.8 vs 135+/-33 mm Hg) mice. Next, groups (n=7) of knockout and wild-type mice had catheters surgically placed in the right carotid artery for mean arterial pressure recording. With the animals fully awake and unrestrained, the knockout mice displayed an elevated mean arterial pressure compared with wild-type in both male (139+/-4.9 vs 118+/-4.9 mm Hg) and female (121+/-3.4 vs 107+/-3.1 mm Hg) mice. Northern blot analysis of DRG RNA samples confirmed the absence of alpha-CGRP mRNA in the knockout mice. Substance P mRNA content in DRG was unchanged between the 2 groups; however, beta-CGRP mRNA levels were reduced 2-fold in the knockout mice. These results indicate for the first time that alpha-CGRP may be involved in the long-term regulation of resting BP and suggest that these mice are particularly sensitive to challenges to BP homeostasis because of the loss of a compensatory vasodilator mechanism.

Pulpal exposure alters neuropeptide levels in inflamed dental pulp and trigeminal ganglia: Evaluation of axonal transport

Dental pulp is richly innervated with neurons containing calcitonin gene-related peptide (CGRP) or substance P (SP). Prior studies have demonstrated that inflammation alters these pulpal neuropeptides. In this study, we used a radioimmunoassay to evaluate the specificity of this response and the contribution of axonal transport. Rat mandibular molars were exposed and immunoreactive CGRP (iCGRP) and immunoreactive SP were measured. At 7 to 14 days after exposure, both pulpal iCGRP (73%) and immunoreactive SP (135%) displayed peak increases above control levels. This response was somatotopically restricted, with no changes observed in contralateral (intact) molars, or in ipsilateral mandibular molars after exposure of maxillary molars. Transection of the inferior alveolar nerve on day 13 significantly reduced pulpal levels of iCGRP on day 14. Collectively, these studies indicate that pulpal inflammation evokes a selective alteration in neuropeptide levels, due at least in part to alterations in transport or synthesis of neuropeptides in the trigeminal ganglion.