Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations

The occurrence and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in the rat respiratory tract were investigated by means of immunocytochemistry and radioimmunoassay using antibodies raised in rabbits to synthetic rat CGRP. Substantial amounts of CGRP immunoreactivity (range 5-37 pmol/g) were detected in all parts of the respiratory tract, the highest being in the stem bronchus. Gel filtration chromatography of extractable CGRP immunoreactivity revealed one single peak, eluting at the position of synthetic rat CGRP. CGRP immunoreactivity was localized both in mucosal endocrine cells and nerve fibres from the larynx down to the peripheral lung. CGRP-immunoreactive endocrine cells were found singly in trachea and stem bronchi and in groups in intrapulmonary airways. They appeared at a late stage of gestation (17 days), reached a maximum number near term and decreased after birth to maintain a population similar to that of the adult animals by postnatal day 21. Similarly, CGRP-immunoreactive nerve fibres were first identified by day 18 of the gestation period and reached the adult distribution by postnatal day 21. CGRP-immunoreactive nerve fibres were localized among smooth muscle, seromucous glands, beneath and within the epithelium of the airways and around blood vessels. CGRP was also found in sensory ganglia and in motor end plates of the larynx musculature. Neonatal pretreatment with capsaicin caused a marked reduction in CGRP immunoreactivity of nerve fibres in the respiratory tracts as well as a less marked decrease in the population of CGRP-containing endocrine cells of the lung. No change was seen in motor end plates immunostaining. Vagal ligation experiments revealed that CGRP-immunoreactive nerve fibres travelling in the vagus originate mainly from neurons located in the jugular ganglion. Infranodosal right vagal ligation induced a marked loss in CGRP-immunoreactive nerves of the trachea, and of the ipsilateral stem bronchus, but no changes were observed in peripheral lung. By contrast infranodosal left side vagal ligation caused a decrease in CGRP-immunoreactive nerves of the ipsilateral lung and bronchus without affecting the peptide content in the trachea. Left vagal ligation also induced a marked increase in both the intensity of staining and number of CGRP-immunoreactive endocrine cells in the lung. We conclude that CGRP immunoreactivity is localized in both nerve fibres and endocrine cells and is associated principally with the afferent (sensory) innervation of the respiratory tract.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in nerves and neuropeptides in skin from 100 leprosy patients investigated by immunocytochemistry

The cutaneous innervation is now known to contain neuropeptides including substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory nerves, and vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), principally in autonomic nerves. Skin biopsies from 100 leprosy patients and equivalent areas from 50 non-leprosy controls were fixed in p-benzoquinone solution for immunofluorescence staining and in Bouin's fluid for classification of leprosy type. Antisera to the neural markers, neurofilaments, and protein gene product 9.5 (PGP 9.5), and to neuropeptides were used. Cutaneous nerves and nerve endings immunoreactive for neuropeptides, neurofilaments, and PGP 9.5 were seen in all non-leprous control cases. In leprosy, PGP 9.5- and neurofilament-immunoreactive nerve fibres were seen in all 14 cases of the indeterminate (early) type and in the majority (33/43) of lepromatous cases, but in a smaller proportion (15/43) of tuberculoid cases. Neuropeptide immunoreactivity was seen in only 2/14 of the indeterminate leprosy specimens and was completely absent in other types. This early disappearance may be of diagnostic significance. Thus, cutaneous sensory and autonomic dysfunctions in leprosy are well reflected by changes in nerve fibres and neuropeptides.     

Capsaicin suppresses substance P-induced joint inflammation in the rat

Intra-articular injection of 20 micrograms substance P in rat knee joints results in a pronounced inflammatory response. However, prior intra-articular injection of 1% capsaicin solution (1-5 weeks previously) virtually abolishes this response. This is not a neurotoxic effect of capsaicin on nerve fibres as denervation of the knee produces no alteration of the response to injected substance P. The potent effect of capsaicin on substance P-mediated inflammation cannot be attributed to depletion of mast cells by this treatment as the mast cell degranulator compound 48/80 injected into capsaicin pre-treated knees still gives rise to a marked inflammatory response. Compound 48/80 does not activate nerve fibres to cause release of substance P as it is equally effective in eliciting an inflammatory response in the presence of 100 micrograms of the substance P antagonist D-Pro4, D-Trp7,9,10 SP(4-11) in the synovial cavity. The results suggest that capsaicin may act by depleting substance P receptors in joint tissue.     

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuronspecific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY- immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.     

Upregulation of the axonal growth and the expression of substance P and its NK1 receptor in human allergic contact dermatitis

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.     

Upregulation of the Axonal Growth and the Expression of Substance P and its NK1 Receptor in Human Allergic Contact Dermatitis

Nerve fibers and sensory neuropeptides substance P and calcitonin gene-related peptide (CGRP) have been reported to be involved in allergic contact dermatitis (ACD). In the present study, we investigated the general innervation (using antibody against protein gene product 9.5, PGP 9.5), axonal growth (using antibody against growth associated protein, GAP-43), CGRP, and substance P with its receptor neurokinin 1 (NK1), in positive epicutaneous reactions to nickel sulphate from nickel-allergic patients, at the peak of inflammation, 72 hr after challenge with the antigen. There was an increased (p < 0.01) number of GAP-43 positive fibers in the eczematous compared with control skin, indicating an increased axonal growth already at 72 hr postchallenge. Double staining revealed a coexpression of CGRP and GAP-43 on dermal nerve fibers. There was no difference in the number of substance P and CGRP positive nerve fibers between eczematous and control skin. However, semiquantification analyses showed an increased expression of substance P positive inflammatory cells, being CD3, CD4, or CD8 positive, and NK1R positive inflammatory cells, being tryptase or CD3 positive. These results indicate a contribution of regenerating nerve fibers and substance P to the contact allergic reaction.     

Nerve fibres in peritoneal endometriosis

BACKGROUND: Endometriosis is a gynaecological disease that can be associated with severe pelvic pain; however, the mechanisms by which pain is generated remain unknown. METHODS: Peritoneal endometriotic lesions and normal peritoneum were prepared from women with and without endometriosis (n = 40 and 36, respectively). Specimens were also prepared from endosalpingiosis lesions (n = 9). These sections were stained immunohistochemically with antibodies against protein gene product 9.5, neurofilament (NF), nerve growth factor (NGF), NGF receptor p75 (NGFRp75), substance P (SP), calcitonin gene-related peptide (CGRP), acetylcholine (ACh) and tyrosine hydroxylase (TH) to demonstrate myelinated, unmyelinated, sensory, cholinergic and adrenergic nerve fibres. RESULTS: There were significantly more nerve fibres identified in peritoneal endometriotic lesions than in normal peritoneum (P < 0.001) or endosalpingiosis lesions (P < 0.001). These nerve fibres were SP, CGRP, ACh or TH immunoreactive. Many of these markers were co-localized. There was an intense NGF immunoreactivity near endometriotic glands, and NGFRp75 immunoreactive nerve fibres were present near endometriotic glands and blood vessels in the peritoneal endometriotic lesions. CONCLUSIONS: Peritoneal endometriotic lesions were innervated by sensory Adelta, sensory C, cholinergic and adrenergic nerve fibres. These nerve fibres may play an important role in the mechanisms of pain generation in this condition.     

Distribution and coexistence of neuropeptides in nerve fibres in the temporomandibular joint of late gestation fetal sheep

The density and distribution of nerve fibres immunoreactive to antisera for PGP 9.5 (general neuronal marker), calcitonin gene related peptide (CGRP) and substance P (SP) (markers for sensory neurons), as well as neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) (markers for autonomic fibres), were examined in the temporomandibular joint (TMJ) of late gestation fetal sheep. This work formed part of a project investigating the influence of age and osteoarthritis on the innervation of the TMJ, and was undertaken to determine whether the innervation of the joint at 140 d gestation (17 d before birth) differed from that in the mature adult. Immunofluorescence microscopy was applied to serial sections of the capsule, disc and synovial membrane of 10 joints from 5 fetuses and image analysis was used for the quantitative assessment. The capsule, synovial membrane and the disc contained fibres immunoreactive (IR) to antisera for PGP 9.5, SP and CGRP. NPY-IR fibres were only visible in the loose connective tissue of the capsule. No VIP- or TH-IR nerve fibres were detected in the fetal TMJ. There was no statistically detectable difference between the density of nerve fibres immunoreactive to CGRP or PGP9.5 antisera in the capsule or disc. Substance P-immunoreactivity (IR) was relatively weak in all samples examined. Scattered branches of CGRP-IR fibres were found deep in the disc proper. The lack of receptor endings, other than free nerve endings in the TMJ of the late fetal sheep, might be a reflection of the functional and anatomical immaturity of the TMJ, as reflected in the immature, gross and microscopic appearance of the disc, the inferior joint compartment and articular surface of the condyle at this stage. These results demonstrate that the capsule, synovial membrane and disc in the TMJ of fetal sheep at 140 d gestation age are innervated with sensory fibres, while autonomic fibres are located in the capsule only. The findings also support the view that the disc is innervated at an early stage of life but at a later stage the density of innervation in the central part of the disc regresses and the innervation remains only peripherally in the adult TMJ disc. Further work is required to determine (1) at what stage sympathetic fibres innervate the disc and the synovium, and (2) when the mechanoreceptive nerve endings develop.     

Ultrastructural evidence for the coexistence of calcitonin gene-related peptide and substance P in secretory vesicles of peripheral nerves in the guinea pig

Summary Using double immunogold staining procedures, calcitonin gene-related peptide (CGRP)-like and substance P (SP)-like immunoreactivities were localized at the ultrastructural level to guinea pig trigeminal ganglia, dorsal root ganglia and peripheral nerve fibres associated with the vascular system. CGRP-like and SP-like immunoreactivities were found consistently in large granular secretory vesicles (70–100 nm in diameter), and both peptide immunoreactivities were co-localized to the same vesicle in both sensory ganglion cells and within axons and their terminals in the adventitia and adventitial-medial border of the superior mesenteric artery. These results suggest that CGRP and SP are co-stored and may be released together from peripheral axons in the guinea pig.     

Depletion by capsaicin of substance P-immunoreactivity and acetylcholinesterase activity from nerve fibres in the guinea-pig heart

The sensitivity to capsaicin of substance P-immunoreactive nerve fibres in the hearts of guinea-pigs was examined. Capsaicin decreased considerably the substance P-immunoreactive material in nerve fibres of the parietal pericardium, atria, bicuspid and tricuspid valves. Pericardial and valvar nerve fibres localized by acetylcholinesterase (AChE) histochemistry had a distribution and density pattern similar to that of the substance P immunoreactive fibres. Capsaicin treatment also decreased the number of visible AChE-positive nerve fibres. It is known that capsaicin has a selective action on those substance P-immunoreactive fibres that are of sensory origin; thus, these results imply that substance P-immunoreactive fibres in the heart are sensory. Moreover, the results suggest that some of the nerve fibres localized in the heart by AChE histochemistry are substance P-containing sensory fibres.     

Effects of capsaicin-induced sensory denervation on osteoclastic resorption in adult rats

Many recent findings suggest that the nervous system has efferent effects on bone. A putative role of the sensory innervation has been assessed by using a synchronised rat model of bone resorption after treating adult animals with the neurotoxin capsaicin. Fourteen days after capsaicin treatment (50 mg kg-1) the right maxillary molars were extracted to activate a wave of resorption along the mandibular cortex. The rats were killed 4 days later (i.e. at the peak of resorption in this model), and their right mandibles were processed for histometric evaluation of resorption along the cortex and of calcitonin gene-related peptide (CGRP)- and substance P (SP)-immunoreactive (IR) fibres in the dental pulp. CGRP-IR and SP-IR fibres were significantly reduced in numbers by the capsaicin treatment (by 58 and 49%, respectively), confirming the success of sensory denervation. The resorption surface was significantly reduced (P < 0.005) versus the sham-treated animals. Although the size of the osteoclast population recruited in the site was not modified, the number of actively resorbing osteoclasts was significantly reduced (P < 0.03). However, the activity of the resorbing cells was not modified. Non-specific esterase-positive osteoclast precursors were also significantly few after capsaicin treatment. These data show that the sensory nervous system is involved in the control of bone resorption at two different levels: (1) in the recruitment of osteoclast precursors, and (2) in regulating the access of recruited cells to the bone surface.     

Development of sensory innervation in rat tibia: co-localization of CGRP and substance P with growth-associated protein 43 (GAP-43)

The development of sensory innervation in long bones was investigated in rat tibia in fetuses on gestational days (GD) 16-21 and in neonates and juvenile individuals on postnatal days (PD) 1-28. A double immunostaining method was applied to study the co-localization of the neuronal growth marker growth-associated protein 43 (GAP-43) and the pan-neuronal marker protein gene product 9.5 (PGP 9.5) as well as that of two sensory fibre-associated neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP). The earliest, not yet chemically coded, nerve fibres were observed on GD17 in the perichondrium of the proximal epiphysis. Further development of the innervation was characterized by the successive appearance of nerve fibres in the perichondrium/periosteum of the shaft (GD19), the bone marrow cavity and intercondylar eminence (GD21), the metaphyses (PD1), the cartilage canals penetrating into the epiphyses (PD7), and finally in the secondary ossification centres (PD10) and epiphyseal bone marrow (PD14). Maturation of the fibres, manifested by their immunoreactivity for CGRP and SP, was visible on GD21 in the epiphyseal perichondrium, the periosteum of the shaft and the bone marrow, on PD1 in the intercondylar eminence and the metaphyses, on PD7 in the cartilage canals, on PD10 in the secondary ossification centres and on PD14 in the epiphyseal bone marrow. The temporal and topographic pattern of nerve fibre appearance corresponds with the development of regions characterized by active mineralization and bone remodelling, suggesting a possible involvement of the sensory innervation in these processes.     

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

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

Time course of effect of capsaicin on ultrastructure and histochemistry of substance P-immunoreactive nerves associated with the cardiovascular system of the guinea-pig

Capsaicin, a neurotoxin which depletes substance P from primary afferent nerve fibres, was injected systematically into adult guinea pigs. The effects of capsaicin were studied by immunohistochemistry, electron microscopy and radioimmunoassay at times from 5 min to 1 year. Within 5 min after a single injection of capsaicin (50 mg/kg) substance P immunofluorescence appeared less intense and less homogeneous than normal (i.e. it appeared granular). Large nerve trunks remained evident, but there were fewer fine single nerve fibres. With increasing time there was a progressive decrease in the number of immunoreactive fibres; by 4 h there was a marked reduction in the number of fibres and by 24 h only an occasional fibre was evident. In animals sacrificed 2 or more hours after treatment large brightly fluorescent swellings were seen in many nerves. Depletion of substance P-immunoreactivity persisted for as long as 365 days after treatment. Electron microscopy revealed alterations in capsaicin-sensitive nerve fibres within 5 min after treatment. Many fibres appeared swollen and there was disruption of their internal morphology, e.g. loss of microtubules and filaments and presence of an amorphous flocculent material in the axons. With increasing time after treatment, electron-dense profiles, indicative of degenerating nerve fibres, were commonly seen associated with Schwann cells. These findings demonstrate that the effects of systemic administration of capsaicin to adult guinea pigs occur rapidly in capsaicin-sensitive nerve fibres. The long lasting depletion of substance P-containing fibres is due to their degeneration.     

Calcitonin gene-related peptide in rat salivary glands: neuronal localization, depletion upon nerve stimulation, and effects on salivation in relation to substance P

Calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres occurred predominantly around blood vessels and large ducts and, to a minor extent, around acini and small ducts in the parotid, sublingual and submaxillary glands of the rat. Double immunostaining showed most of the CGRP-containing nerve fibres to contain substance P. However, the vast majority of substance P-immunoreactive periacinar nerve fibres in the parotid and submandibular glands lacked CGRP. After parasympathetic denervation of the parotid gland by section of the auriculotemporal nerve these periacinar substance P-immunoreactive nerve fibres disappeared almost completely, whereas the number of substance P/CGRP-immunoreactive nerve fibres seemed unchanged. After this operation the total amount of substance P in the parotid gland was reduced by about 90% as judged by radioimmunoassay; in denervation experiments the facial nerve was found to contribute to the residual substance P content. In contrast, the contribution of the auriculotemporal nerve to the CGRP content of the gland was small; the reduction in CGRP after section of the nerve was 20%. The facial nerve and the dorsal root nerves (C3 and C4) contributed to the CGRP content with about 50%. The source of the remaining 30% of the parotid gland CGRP is unknown. It is not the sympathetic nerve: sympathetic denervation resulted in a marked increase in CGRP, regardless of whether the auriculotemporal nerve was intact or not. Upon long-lasting electrical stimulation of the auriculotemporal nerve at a high frequency the parotid gland content of CGRP was gradually reduced, indicating depletion of this peptide in response to nerve stimulation. Intravenous injections of CGRP evoked no salivary flow; however, a release of amylase was revealed. Also, when CGRP was tested on isolated parotid gland lobules amylase was released into the medium. When, in vivo, CGRP was injected in combination with substance P, the substance P-evoked flow of parotid and submaxillary saliva was markedly enhanced. In addition, CGRP enhanced the in vivo secretory response to parasympathomimetics and to vasoactive intestinal peptide. The localization of CGRP-containing nerve fibres suggests that CGRP is involved in the regulation of secretion and blood flow of salivary glands. CGRP may interact positively with acetylcholine and certain nonclassical transmitters, and it may be involved (together with other neuropeptides) in the atropine-resistant parasympathetic secretion occurring in the glands under study.     

Temperature- and capsaicin-sensitive nerve fibers in brown adipose tissue attenuate thermogenesis in the rat

 We examined the function of putative sensory fibers that are contained in intercostal nerves and innervate interscapular brown adipose tissue (IBAT) in urethane-anesthetized rats. Warming the IBAT to 40–44°C with two small heaters placed bilaterally on the skin above it attenuated the subsequent noradrenaline-induced thermogenesis (NIT) of the IBAT. In this range of warming, higher IBAT temperatures resulted in more attenuation. Denervation of IBAT blocked the effect of thermal stimulation on the NIT. Thus, activation of nerve fibers in IBAT that are sensitive to warmth or to the nociceptive effects of heat probably attenuated the NIT. Since the putative sensory fibers in the IBAT contain calcitonin gene-related peptide (CGRP) and substance P, which are thought to act in peripheral tissues, we tested the effects of injection of these neuropeptides into the IBAT. Administration of 5.2 nmol CGRP but not substance P or vehicle saline mimicked the effect of thermal stimulation of IBAT. As the neuropeptide-containing primary sensory neurons are characterized by their sensitivity to capsaicin, we also tested its effects (1 mg/kg, s.c.) and found that it also attenuated the NIT. Denervation of the IBAT or pretreatment with capsazepine, a capsaicin receptor antagonist, blocked the effect of capsaicin. We propose that temperature- and capsaicin-sensitive nerve fibers release CGRP to attenuate the NIT of brown adipocytes. Received: 14 May 1998 / Received after revision: 6 August 1998 / Accepted: 11 August 1998     

Local application of capsaicin to one sciatic nerve of the adult rat induces a marked depletion in the peptide content of the lumbar dorsal horn

In an earlier study we have shown that local application of capsaicin directly to one sciatic nerve induces a decrease of substance P and cholecystokinin octapeptide (CCK8)-like peptide from the dorsal spinal cord using immunocytochemical analysis.¹ Here the effect of locally applied capsaicin on seven peptides known to be present in the L4 segment was assessed by radioimmunoassay and immunocytochemistry. The peptides investigated were substance P, somatostatin and CCK8-like peptide (which are present in small diameter primary afferent fibres), neurotensin, enkephalin (which are intrinsic to the spinal cord), neurophysin (of supraspinal origin) and bombesin (whose origin is unknown). Fourteen days after a single application of 49 mM solution of capsaicin a significant depletion of substance P and somatostatin was detected. These results were confirmed by parallel immunocytochemical analysis which localised the dramatic decreases of substance P and somatostatin to lamina 1 and lamina 2. In addition a depletion of CCK8-like immunoreactivity was observed by immunocytochemistry in this area, but quantitative radioimmunoassay of CCK8-like peptide did not detect this depletion. The capsaicin-induced changes were dose-dependent and reversible. Small decreases were noted with concentrations of capsaicin as low as 0.1 mM. The changes were apparent from day 9 onwards, maximal depletion seen by day 14. By 200 days post-operatively, a recovery to normal peptide levels in the ipsilateral dorsal horn was observed. In addition, a significant depletion of cutaneous substance P was noted in the area of the skin innervated by the capsaicin-treated nerve. These changes were accompanied by a significant increase in noxious thermal response (hind paw immersion test, T = 49°C, ipsilateral leg 9.11 ± 1.3 seconds, contralateral leg: 5.1 ± 1.3 seconds, P = < 0.005). The peptides neurotensin, enkephalin, neurophysin and bombesin were not affected by capsaicin treatment.These findings suggest that local application of capsaicin induces an indiscriminate depletion of peptide-containing primary sensory afferent fibres which is dose-dependent, long-lasting, but reversible.     

Calcitonin gene-related peptide (CGRP): Responsible for the increased blood flow induced by the stimulation of sensory nerves

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide contained in sensory nerves. We have examined the relative contribution of CGRP and substance P-like peptides (with which CGRP is commonly colocalized) to the increase in blood flow induced by the stimulation of sensory nerves. The sensory nerve stimulant capsaicin increased blood flow in rabbit and rat skin and this effect was substantially inhibited by the CGRP antagonist CGRP_8–37. Further, electrical stimulation of the rat saphenous nerve led to an increase in blood flow which was significantly inhibited by CGRP_8–37. CGRP_8–37 also had a partial inhibitory effect on oedema formation, an effect which is suggested to be a consequence of the inhibition of blood flow.

     

Distribution of PGP 9.5, TH, NPY, SP and CGRP immunoreactive nerves in the rat and guinea pig atrioventricular valves and chordae tendineae

The distribution of nerves immunoreactive to protein gene product 9.5 (PGP 9.5), tyrosine hydroxylase (TH), neuropeptide Y (NPY), substance P (SP) and calcitonin gene related peptide (CGRP) antisera was investigated in the atrioventricular valves of the Sprague–Dawley rat and the Dunkin–Hartley guinea pig using confocal and epifluorescence microscopy. No major differences were noted between the innervation of the mitral and tricuspid valves in either species. For all antisera the staining was more extensive in the guinea pig valves. Two distinct nerve plexuses separated by a ‘nearly nerve free’ zone were identified in both species with each antiserum tested. This was most apparent on the anterior cusp of the mitral valve. The major nerve plexus extends from the atrioventricular ring through the basal, intermediate and distal zones of the valves towards the free edge of the valve cusp. These nerve bundles, arranged as primary, secondary and tertiary components, ramify to the free edge of the valve and extend to the attachment of the chordae. They do not contribute to the innervation of the chordae tendineae. The second, minor chordal plexus, runs from the papillary muscles through the chordae tendineae and passes parallel to the free edge of the cusp. The nerves of this minor plexus are interchordal, branching to terminate mainly in the distal zone, free edge of the valve cusp and adjacent chordae tendineae. Some interchordal nerve fibres loop from a papillary muscle up through a chorda, along the free edge and pass down an adjacent chorda into another papillary muscle. The nerve fibres of the major and minor plexuses intermingle although no evidence was found for interconnectivity between them. In the distal zone between the major plexus which extends from the base of the valve and the minor chordal plexus there is a zone completely free of nerves staining with antisera to TH and NPY. Occasional nerves which stained positive for PGP 9.5, SP and CGRP immunoreactivities crossed this ‘nearly nerve free zone’ passing either from the chordal/free edge nerves to the intermediate and basal zones or vice versa. An additional small nerve plexus which displayed immunoreactivity to CGRP antiserum extended from the atrioventricular ring into the basal zone of the valve cusp. Not all chordae tendineae displayed immunoreactive nerve fibres. It is concluded that the innervation patterns of the sensory and sympathetic neurotransmitters and neuropeptides examined in the atrioventricular valves of the rat and guinea pig are ubiquitous in nature. The complexity of the terminal innervation network of the mammalian atrioventricular valves and chordae tendineae may contribute to the complex functioning of these valves in the cardiac cycle.     

Calcitonin gene-related peptide (CGRP): Responsible for the increased blood flow induced by the stimulation of sensory nerves

Calcitonin gene-related peptide (CGRP) is a potent vasodilator neuropeptide contained in sensory nerves. We have examined the relative contribution of CGRP and substance P-like peptides (with which CGRP is commonly colocalized) to the increase in blood flow induced by the stimulation of sensory nerves. The sensory nerve stimulant capsaicin increased blood flow in rabbit and rat skin and this effect was substantially inhibited by the CGRP antagonist CGRP_8–37. Further, electrical stimulation of the rat saphenous nerve led to an increase in blood flow which was significantly inhibited by CGRP_8–37. CGRP_8–37 also had a partial inhibitory effect on oedema formation, an effect which is suggested to be a consequence of the inhibition of blood flow.