Presence Of Calcitonin Gene-related Peptide Research Articles

CGRP Inhibitor Use In Migraine Treatments

Several possible remedies for migraines have been discovered to date, the most prominent ones include various antiepileptic drugs such as divalproex sodium and topiramate, as well as beta (Amiri et al., 2021)blockers such as propranolol and timolol. However many of these treatments are not entirely effective treatments for patients who face chronic migraines due to their somewhat prosaic success rates. In 1985, researchers noticed the presence of Calcitonin Gene-Related Peptide (CGRP) in the plasma increased drastically in its levels during the presence of a migraine attack (Deen et al. 2). CGRP is a neuropeptide that is involved in the dilation of both dural and cerebral blood vessels, and this interaction is believed to be the main cause of migraines. Generally, when migraines are treated with triptans (a common symptom relief medication), CGRP levels in the blood generally reduce. It was further found that certain CGRP inhibitors reduced neurogenic inflammation and lead to an increased reduction of pain during the migraine (Deen et al. 4). When faced with a multitude of treatment options for chronic migraines, one must consider inhibitors of the CGRP pathway as a possible alternative. CGRP inhibitors introduce a possible better treatment option than previous medicinal drugs. A few previously used CGRP inhibitors include erenumab, galcanezumab, and fremanezumab, which have recently been used to treat patients of chronic migraines. In this paper we will consider the various CGRP inhibitors and their advantages and disadvantages in the face of conventional chronic migraine treatment options.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Interplay of Oral, Mandibular, and Facial Disorders and Migraine

Migraine and other primary headache disorders can be localized in the face resembling facial or dental pain, indicating the influence of the trigeminovascular system in the structures innervated by the maxillary (V2) and mandibulary (V3) branches of the trigeminal nerve. Disorders of oral and craniofacial structures may influence primary headache disorders. In the current article, we review the potential links of this interplay. This interplay may be related to anatomy, with the trigeminal pathway and the involvement of both peripheral and central mechanisms, and the presence of calcitonin gene-related peptide (CGRP), a key mediator in migraine pathophysiology. CGRP is also involved in the pathophysiology of temporomandibular disorders (TMD) and their comorbidity with migraine and is also implicated in dental and periodontal pathology. Inflammatory and pathological processes of these structures and their trigeminal nociceptive pathways may influence the trigeminovascular system and consequently may exacerbate or even potentially trigger migraine.

Nonsurgical mouse model of endometriosis-associated pain that responds to clinically active drugs.

Endometriosis is an estrogen-dependent inflammatory disease that affects approximately 10% of women. Debilitating pelvic or abdominal pain is one of its major clinical features. Current animal models of endometriosis-associated pain require surgery either to implant tissue or to remove the ovaries. Moreover, existing models do not induce spontaneous pain, which is the primary symptom of patients with chronic pain, including endometriosis. A lack of models that accurately recapitulate the disease phenotype must contribute to the high failure rate of clinical trials for analgesic drugs directed at chronic pain, including those for endometriosis. We set out to establish a murine model of endometriosis-associated pain. Endometriosis was induced nonsurgically by injecting a dissociated uterine horn into a recipient mouse. The induced lesions exhibited histological features that resemble human lesions along with an increase in proinflammatory cytokines and recruitment of immune cells. We also observed the presence of calcitonin gene-related peptide-, TRPA1-, and TRPV1-expressing nerve fibers in the lesions. This model induced mechanical allodynia, spontaneous abdominal pain, and changes in thermal selection behavior that indicate discomfort. These behavioral changes were reduced by drugs used clinically for endometriosis, specifically letrozole (aromatase inhibitor) and danazol (androgen). Endometriosis also induced neuronal changes as evidenced by activation of the NF-κB signaling pathway in TRPA1- and TRPV1-expressing dorsal root ganglion neurons. In conclusion, we have established a model of endometriosis-associated pain that responds to clinically active drugs and can, therefore, be used to identify novel therapies.

Fluorescently-labeled fremanezumab is distributed to sensory and autonomic ganglia and the dura but not to the brain of rats with uncompromised blood brain barrier.

BackgroundThe presence of calcitonin gene-related peptide and its receptors in multiple brain areas and peripheral tissues previously implicated in migraine initiation and its many associated symptoms raises the possibility that humanized monoclonal anti-calcitonin gene-related peptide antibodies (CGRP-mAbs) can prevent migraine by modulating neuronal behavior inside and outside the brain. Critical to our ability to conduct a fair discussion over the mechanisms of action of CGRP-mAbs in migraine prevention is data generation that determines which of the many possible peripheral and central sites are accessible to these antibodies – a question raised frequently due to their large size.Material and methodsRats with uncompromised and compromised blood-brain barrier (BBB) were injected with Alexa Fluor 594-conjugated fremanezumab (Frema594), sacrificed 4 h or 7 d later, and relevant tissues were examined for the presence of Frema594.ResultsIn rats with uncompromised BBB, Frema594 was similarly observed at 4 h and 7 d in the dura, dural blood vessels, trigeminal ganglion, C2 dorsal root ganglion, the parasympathetic sphenopalatine ganglion and the sympathetic superior cervical ganglion but not in the spinal trigeminal nucleus, thalamus, hypothalamus or cortex. In rats with compromised BBB, Frema594 was detected in the cortex (100 µm surrounding the compromised BBB site) 4 h but not 7 d after injections.DiscussionOur inability to detect fluorescent (CGRP-mAbs) in the brain supports the conclusion that CGRP-mAbs prevent the headache phase of migraine by acting mostly, if not exclusively, outside the brain as the amount of CGRP-mAbs that enters the brain (if any) is too small to be physiologically meaningful.

The Presence of Calcitonin Gene-Related Peptide and Its Receptors in Rat, Pig and Human Brain: Species Differences in Calcitonin Gene-Related Peptide Pharmacology

Aim: The aim of present study is to investigate the binding characteristics of non-peptide calcitonin gene-related peptide (CGRP) receptor antagonists (i.e., gepants) in the brain membranes of rat, pig and human. Methods: The interaction of available gepants with the CGRP receptor was studied in the brain membranes of 3 different species using a radioligand competitive binding assay. In addition, the distribution of CGRP and its receptor component receptor activity modifying protein 1 (RAMP1) in rat cerebellum and cortex was explored using immunohistochemistry. Results: All gepants, except SB268262, displaced 100% of the radioligand specific binding in the brain tissue of all 3 species and showed highest affinity for CGRP receptors in human brain as compared to rat and pig brain membranes. Furthermore, radioligand binding studies revealed the presence of higher CGRP receptor density in human cerebellum compared to human cortex. The morphology, size and density of CGRP immunoreactive cells suggest that all cerebral cortical neurons were positive for CGRP. Slender receptor immunoreactive fibres were found spanning through the entire cortex. CGRP immunoreactivity was displayed in the cell soma of cerebellar Purkinje cells and in large neurons in the medial cerebellar nucleus. RAMP1 was found on the surface of the Purkinje cells and in parallel fibres, indicating presence in the granule cell axons. Conclusion: Cerebellum and cerebral cortex are rich in CGRP and CGRP receptors, which can be antagonized by gepants. However, all gepants display higher affinity for human CGRP receptors as compared to rat and pig CGRP receptors. Furthermore, human cerebellum seems to express higher density of CGRP receptors.

Role of calcitonin gene-related peptide in energy metabolism.

Calcitonin gene-related peptide (CGRP) is a neuropeptide produced by alternative tissue-specific splicing of the primary transcript of the CALC genes. CGRP is widely distributed in the central and peripheral nervous system, as well as in several organs and tissues. The presence of CGRP in the liver and brown and white adipose tissue suggests an effect of this neuropeptide on regulation of energy homeostasis. In this review, we summarize the current knowledge of the effect of CGRP on the control of energy metabolism, primarily focusing on food intake, thermoregulation and lipid metabolism in adipose tissue, liver and muscle. CGRP induces anorexia, stimulating anorexigenic neuropeptide and/or inhibiting orexigenic neuropeptide expression, through cAMP/PKA pathway activation. CGRP also induces energy expenditure, increasing the skin temperature and brown adipose tissue thermogenesis. It has been also suggested that information related to peripheral lipid stores may be conveyed to the brain via CGRP-sensory innervation from adipose tissue. More recently, it was demonstrated that mice lacking αCGRP are protected from obesity induced by high-fat diet and that CGRP regulates the content of lipid in liver, muscle and adipose tissue. It is unclear the receptor responsible by CGRP effects, as well as whether this neuropeptide acts directly or indirectly in liver, muscle and adipose tissue.

Distribution of CGRP and CGRP receptor components in the rat brain.

Calcitonin gene-related peptide and its receptor, consisting of receptor activity-modifying protein 1 and calcitonin receptor-like receptor, are of considerable interest because of the role they play in migraine and recently developed migraine therapies. To better understand the function of this neuropeptide, we used immunohistochemistry to determine a detailed distribution of calcitonin gene-related peptide, receptor activity-modifying protein 1 and calcitonin receptor-like receptor in the rat brain in a region of 0.5-1.5 mm lateral to the midline. We found calcitonin gene-related peptide immunoreactivity in most of the neurons of the cerebral cortex, hippocampus, cerebellum, thalamic nuclei, hypothalamic nuclei and brainstem nuclei. In contrast, receptor activity-modifying protein 1 and calcitonin receptor-like receptor immunoreactivity were found almost exclusively in the neuronal processes in the investigated regions. Overall, the degree of expression of calcitonin gene-related peptide and calcitonin gene-related peptide receptor components in the central nervous system is astonishingly complex and suggestive of many different brain functions, including a possible role in migraine. However, currently, the presence of calcitonin gene-related peptide and the nature of its receptors throughout the brain is an enigma yet to be solved.

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood–brain barrier

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood–brain barrier (BBB). Autoradiography was performed with [3H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

Calcitonin gene-related peptide inhibits human immunodeficiency type 1 transmission by Langerhans cells via an autocrine/paracrine feedback mechanism.

Peripheral neurones innervating mucosal epithelia are in direct contact with resident immune cells, including Langerhans cells (LCs). Such neurones secrete the neuropeptide calcitonin gene-related peptide (CGRP) that modulates LCs function. We recently found that CGRP strongly inhibits human immunodeficiency virus type 1 (HIV-1) transmission, by interfering with multiple steps of mucosal LC-mediated HIV-1 transfer, including increased expression of the LC-specific lectin langerin. Herein, we investigated the anti-HIV-1 mechanism of CGRP. In the presence of CGRP, HIV-1 transfer from LCs to CD4+ T cells was tested with viral clones using either the HIV-1 co-receptor CCR5 (R5) or CXCR4 (X4). Surface expression of CCR5, CXCR4 and langerin was evaluated by flow cytometry. CGRP secretion by LCs was measured with an enzyme immunoassay. Expression of the multimeric CGRP receptor was examined by quantitative real-time RT-PCR and immuno-fluorescent microscopy. Calcitonin gene-related peptide decreased transfer of HIV-1 R5, but increased that of X4. These opposing effects correlated with decreased CCR5 vs. increased CXCR4 surface expression in LCs. Inhibition of HIV-1 R5 transfer by CGRP involved signal transducer and activator of transcription 4 (STAT4) activation. Both αCGRP and βCGRP were similarly efficient in decreasing HIV-1 R5 transfer and increasing langerin expression. LCs secreted low basal levels of endogenous CGRP, which increased markedly following CGRP treatment. CGRP also increased expression of its cognate receptor in LCs. CGRP engages a positive feedback mechanism that would further enhance its anti-HIV-1 activity. This information might be relevant for the therapeutic use of CGRP as a prophylactic agent against HIV-1.

Genome-Wide Analysis of Pain-, Nerve- and Neurotrophin -Related Gene Expression in the Degenerating Human Annulus

BackgroundIn spite of its high clinical relevance, the relationship between disc degeneration and low back pain is still not well understood. Recent studies have shown that genome-wide gene expression studies utilizing ontology searches provide an efficient and valuable methodology for identification of clinically relevant genes. Here we use this approach in analysis of pain-, nerve-, and neurotrophin-related gene expression patterns in specimens of human disc tissue. Control, non-herniated clinical, and herniated clinical specimens of human annulus tissue were studied following Institutional Review Board approval.ResultsAnalyses were performed on more generated (Thompson grade IV and V) discs vs. less degenerated discs (grades I-III), on surgically operated discs vs. control discs, and on herniated vs. control discs. Analyses of more degenerated vs. less degenerated discs identified significant upregulation of well-recognized pain-related genes (bradykinin receptor B1, calcitonin gene-related peptide and catechol-0-methyltransferase). Nerve growth factor was significantly upregulated in surgical vs. control and in herniated vs. control discs. All three analyses also found significant changes in numerous proinflammatory cytokine- and chemokine-related genes. Nerve, neurotrophin and pain-ontology searches identified many matrix, signaling and functional genes which have known importance in the disc. Immunohistochemistry was utilized to confirm the presence of calcitonin gene-related peptide, catechol-0-methyltransferase and bradykinin receptor B1 at the protein level in the human annulus.ConclusionsFindings point to the utility of microarray analyses in identification of pain-, neurotrophin and nerve-related genes in the disc, and point to the importance of future work exploring functional interactions between nerve and disc cells in vitro and in vivo. Nerve, pain and neurotrophin ontology searches identified numerous changes in proinflammatory cytokines and chemokines which also have significant relevance to disc biology. Since the degenerating human disc is primarily an avascular tissue site into which disc cells have contributed high levels of proinflammatory cytokines, these substances are not cleared from the tissue and remain there over time. We hypothesize that as nerves grow into the human annulus, they encounter a proinflammatory cytokine-rich milieu which may sensitize nociceptors and exacerbate pain production.

Differential roles of calcitonin family peptides in the dendrite formation and spinogenesis of the cerebral cortex in vitro

We examined roles of calcitonin family peptides in the initial stages of dendrite formation and the maturation of dendritic spines in the rat cerebral cortex in vitro. Embryonic day 18 cortical neurons were dissociated and cultured for 2-3days in the presence of calcitonin gene-related peptide (CGRP), calcitonin, amylin or adrenomedullin. The treatment of cortical neurons with CGRP promoted the formation of primary dendrites of non-GABAergic neurons. In contrast, the treatment with amylin and adrenomedullin for 3days inhibited the dendritic elongation of non-GABAergic neurons. Calcitonin had no effect on the initial dendrite formation. Next, we examined roles of the peptides in the spine formation. Embryonic day 16 cortical neurons were cultured for 14days and then treated acutely with CGRP, amylin or adrenomedullin for 24h. The density of filopodia, puncta/stubby spines and spines were increased by the CGRP treatment, whereas decreased by amylin. Therefore, CGRP and amylin showed opposite effects on the formation of dendritic filopodia, puncta and spines. Adrenomedullin had no effects on the spine formation. In conclusion, the present study showed that calcitonin family peptides have differential effects both in the dendrite formation during the initial stages and the spine formation of cortical neurons in vitro.

Morphological and biochemical characterisation of sensory neurons infected in vitro with rabies virus

This work was aimed at the morphological and biochemical characterisation of the most susceptible neuronal subpopulation to rabies virus (RABV) infection. Adult mouse DRG cultures were infected with RABV and double-processed for viral antigen detection and neuropeptides: calcitonin gene-related peptide (CGRP), galanin (GAL), substance P (SP), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP). It was found that 56% of the neurons in culture were small (diameter < 20 microm) but, in spite of this, 69% of the infected neurons had intermediate and large diameters (> or = 20 microm). More than 50% of infected neurons expressed NPY, VIP or SP, whereas no association was found between infected neurons and the presence of CGRP or GAL. Despite SP having been shown to be a small neuron marker, it was found that RABV infects medium and large-sized SP positive cells. RABV preference for larger neurons could explain part of the neuropathogenesis since it can be suggested that, following a rabid accident, the virus uses large neurons (mainly innervating muscle and joints) in vivo to be transported later on to the central nervous system.

Survival and neurotransmitter plasticity in cultured rat colonic myenteric neurons

The enteric nervous system is of great importance for maintenance and proper function of the gastrointestinal tract. The aim of this study was to quantify myenteric neuronal subpopulations expressing calcitonin gene-related peptide (CGRP), galanin, neuropeptide Y (NPY), somatostatin, vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in rat colon in vivo and after culturing. Further we investigated if culturing in the presence of CGRP, galanin, VIP, S-nitroso- N-acetyl- d, l-penicillamine (SNAP, a NO donor) or N-nitro- l-arginine methyl ester ( l-NAME, a NOS inhibitor) affect neuronal survival. After 4 days of culturing the proportions of neurons expressing CGRP, NPY, somatostatin or VIP increased as compared to in vivo, while the proportions of neurons expressing galanin or NOS did not change. Neuronal survival was unaffected after culturing in media enriched with CGRP, galanin, VIP, SNAP or l-NAME. Neither did addition of CGRP, galanin nor VIP to the cultures affect the relative numbers of neurons expressing CGRP, galanin or VIP respectively. Addition of SNAP or l-NAME did not change the percentage of neurons expressing NOS. In conclusion, cultured rat colonic myenteric neurons increase their expression of CGRP, NPY, somatostatin and VIP, suggesting that these neuropeptides are of importance for neuronal survival.

The Presence of CGRP in Forelimb Tendons in a Model of Upper Extremity Work-related Musculoskeletal Disorder

Purpose of Study: Increased presence of calcitonin gene–related peptide (CGRP) has been observed in patients with chronic tennis elbow. CGRP mediates pain within the dorsal horn of the spinal cord. The purpose of this study was 1) to examine for CGRP in forelimb flexor tendons of rats that performed a high repetition–high force (HRHF) or low repetition–low force (LRLF) reaching and grasping task and 2) to compare CGRP response to the immunoreactivity (IR) of Substance P and N-methyl-d-aspartate receptor 1 (NMDAr1) observed in this model.

Immunohistochemical localization of calcitonin gene-related peptide and substance P in the rat knee cartilage at birth.

Substance P (SP) and calcitonin gene-related peptide (CGRP) have been found in the perichondrium and within the cartilage canals. It is still unknown whether they exert a direct effect on chondrocytes during joint development. We processed 28 knees of newborn Wistar rats in 7 different fashions to perform histology and immunohistochemistry studies. Positive immunoreactivity against CGRP and SP was found in the inner aspect of the perichondrium in a close contact with chondrocytes. The presence of CGRP and SP indicates the presence of nerves fibers, and precedes the development of cartilage canals. Nerve fibers may play a role in the development of synovial joints before and during the presence of cartilage canals. The presence of CGRP and SP in the cartilage at birth may be involved in the early postnatal maturation of synovial joints. It remains to be determined whether autonomic innervation is later involved in age-related degenerative joint disease.

Effect of Neuropeptides (SP and CGRP) on Antigen Presentation by Macrophages

There is increasing evidence that neuropeptides regulate various functions of immune cells, including macrophages, which have functional receptors for neuropeptides. We have studied the effects of substance P (SP) and calcitonin gene-related peptide (CGRP) on the presentation of herpes simplex virus type-one (HSV-1) antigens by mouse peritoneal macrophages. Macrophages were treated with live or heat-killed virus in the presence or absence of one or both neuropeptides, then fixed. In vitro proliferation of lymphocytes, derived from intranasally immunized mice, was used to assess antigen presentation by the macrophages. Lymphocytes derived from cervical lymph nodes exhibited a greater proliferative response to heat-killed viral antigens than did lymphocytes derived from spleen. Macrophages treated with live virus did not induce lymphocyte proliferation in the immunized mice, but those treated with heat-killed viral particles did. When macrophages were treated with heat-killed virus in the presence of CGRP, the lymphocyte proliferative response was decreased; however, the effects of SP and SP + CGRP were not statistically significant. The results of interleukin-2 production were in accordance with proliferation assays. These findings suggest that neuropeptides regulate immune responses partly through their effects on macrophage function, including antigen presentation.

Regulation of pulpal microcirculation by calcitonin gene-related peptide

The purpose of this study was to investigate the function of calcitonin gene-related peptide (CGRP) in regulatory mechanism of pulpal microcirculation with the aim of elucidating neurogenic inflammation. Experiments were performed on twelve cats under general anesthesia. CGRP was administered through the femoral vein to see the systemic influence and through the external carotid artery to see the local effect. Sympathetic nerve to the dental pulp was stimulated electrically and pulpal blood flow (PBF) was measured with a laser Doppler flowmeter on the canine teeth to the drug administration. The paired variables of control and experimental data were compared by paired t-test and differences with p < 0.05 were considered statistically significant. Systemic administration of CGRP (0.3 ㎍/㎏) exerted decreases in systemic blood pressure and caused changes in PBF with an initial increase followed by decrease and a more marked second increase and decrease. Close intra-arterial (i.a.) injection of CGRP (0.03 ㎍/㎏) resulted in slight PBF increase. The effect of CGRP resulted in no significant increase in PBF in the presence of CGRP 8-37. The electrical stimulation of the sympathetic nerve alone resulted in PBF decreases. The i.a. administration of CGRP following the electrical stimulation of the sympathetic nerve compensated the decreased PBF. Therefore, CGRP effectively blocked the sympathetic nerve stimulation-induced PBF decrease. Results of the present study have provided evidences that even though the local vasodilatory function of CGRP are weak, CGRP is effectively involved in blocking the vasoconstriction caused by sympathetic nerve

Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits.

Serotonin 5-HT(3) receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT(3) receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT(3)A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT(3)A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT(3)A receptor subunits do not originate from the subtypes of primary afferent fibres that bind IB4 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres.