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

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

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

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

Peptide containing nerves in the ureter of the guinea-pig and cat

We report the presence of two regulatory peptides, substance P and vasoactive intestinal polypeptide (VIP), in the ureter and their localisation by both light- and electron-microscopy to autonomic nerves. VIP- and substance P-like immunoreactive nerves showed, in general, a similar anatomical distribution in the various layers of the ureter. Immunoreactive nerves were observed running along the smooth muscle coat, parallel to muscle bundles, around blood vessels and in the submucosa, particularly beneath the epithelium. In addition, scattered VIP-like immunoreactive ganglion cells and nerve fibres were seen in adventitial ganglia around the most distal part of the ureter and ureter-bladder junction in the cat. The guinea-pig ureter contained principally substance P-like immunoreactivity, whereas the cat ureter possessed mainly VIP-like material.The distribution of adrenergic and cholinergic nerves was compared with those containing peptides. Peptide-containing nerves had a more extensive distribution than adrenergic ones, which were mainly associated with blood vessels; however, cholinergic nerves were often localised in the same areas as those possessing peptides. Conventional electron microscopy revealed that separate p-type (peptidergic) and cholinergic nerve terminals were frequently present in the same nerve bundles, although in the cat ureter some 50% of the p-type profiles contained a mixed population of vesicles, characteristic of both cholinergic and p-type nerves. VIP- and substance P-like immunoreactivity were also localised at the ultrastructural level by means of a gold-labelled goat-antirabbit serum.     

Autoradiographic localization of substance P binding sites in rat footpad skin

The autoradiographic distribution of binding sites for 60 pM [125I]Bolton-Hunter substance P (BHSP) was investigated in slide-mounted sections of rat footpad skin. BHSP binding sites were found over dermal papillae, postcapillary venules, sweat glands and arterioles. No binding was seen over mast cells, sebaceous glands or pacinian corpuscles. Specific BHSP binding was inhibited by substance P greater than neurokinin A greater than neurokinin B, indicating binding to an NK1 (substance P-preferring) site. The NK1 binding sites over postcapillary venules and arterioles probably represent receptors mediating plasma extravasation and vasodilatation, respectively. There was no apparent change in distribution or number of BHSP binding sites after neonatal capsaicin pretreatment, indicating a probable absence of NK1 receptors on sensory terminals. No binding sites were observed for 100 pM [125I]iodohistidyl neurokinin A (INKA).     

Substance P and neurokinin A in human nasal mucosa.

The tachykinins substance P (SP) and neurokinin A (NKA) were studied in human inferior turbinate nasal mucosa by radioimmunoassay, immunohistochemistry, and autoradiography and for their effect upon mucus release in an in vitro culture system in order to infer their potential functions in the upper respiratory tract. Similar amounts of SP (1.03 +/- 0.12 pmol/g wet weight; mean +/- SEM; n = 26) and NKA (0.76 +/- 0.23; n = 7) were found. NKA and SP immunoreactive nerve fibers were found in the walls of arterioles, venules, and sinusoids and as individual fibers in gland acini, near the basement membrane, and in the epithelium. [125I]SP bound to arterioles, venules, and glands. [125I]NKA bound only to arterioles. In short-term explant culture of fragments of human nasal mucosa, both 1 microM SP and 1 microM NKA stimulated release of [3H]glucosamine-labeled respiratory glycoconjugates. These results indicate that SP and NKA have similar distributions in nociceptive sensory nerves in human nasal mucosa. The distribution of [125I]SP binding sites is consistent with a role for SP as a vasodilator and mucous secretagogue. The presence of [125I] NKA binding sites on vessels suggests a primary role for NKA in regulating vasomotor tone.     

Afferent fibres from the guinea-pig ureter: size and peptide content of the dorsal root ganglion cells of origin.

A study was undertaken to determine the segmental organization of the dorsal root ganglion cells which give rise to ureteric primary afferent fibres in the guinea-pig. The size-distribution and peptide content of these dorsal root ganglion cells were examined and compared with a sample of all dorsal root ganglion cells from the same ganglia. Afferent fibres to the guinea-pig ureter were found to arise mainly from dorsal root ganglia L2-L3 and S1-S2. A large contralateral component of the afferent innervation of the ureter was found when either the right or the left ureter was injected with tracer. This amounted to approximately 40% of the total labelled cells. The cross-sectional areas of the dorsal root ganglion cells of ureteric afferents were found to be at the smaller end of the size-range for the whole ganglion. Most (90%) of the cells innervating the ureter were immunoreactive for one of the peptides studies, substance P or calcitonin gene-related peptide, and a large proportion (65%) were immunoreactive for both. This was very different for the ganglia as a whole, where only about 50% of the cells were immunoreactive for either of the peptides and only 14% were immunoreactive for both peptides. These results show a bilateral afferent innervation of the ureter by nerve fibres which, in the vast majority, contain substance P and/or calcitonin gene-related peptide.     

Receptor binding sites for substance P and substance K in the canine gastrointestinal tract and their possible role in inflammatory bowel disease

The mammalian tachykinins, substance P, substance K (neurokinin A) and neuromedin K (neurokinin B), are putative peptide neurotransmitters in both the brain and peripheral tissues. We used quantitative receptor autoradiography to localize and quantify the distribution of binding sites for radiolabeled substance P, substance K and neuromedin K in the canine gastrointestinal tract. Substance P binding sites were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, neurons in the myenteric plexus, mucosal epithelial cells, exocrine cells and lymph nodules. Substance K binding sites were distributed in a pattern distinct from substance P binding sites and were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, and neurons of the myenteric plexus. Neuromedin K binding sites were not observed in any area of the canine gastrointestinal tract although they were localized with high specific/non-specific binding ratios in the canine spinal cord. These results indicate that there are at least two distinct types of tachykinin receptor binding sites in the canine gastrointestinal tract, one of which probably recognizes substance P and the other substance K as endogenous ligands. In correlation with previous physiological data, these substance P and substance K receptor binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including gastric motility, mucosal ion transport, hemodynamics, digestive enzyme secretion and neuronal excitability. In addition these results demonstrate that receptor binding sites for substance P and substance K are expressed by cells involved in mediating inflammatory and immune responses. These data, together with our studies on surgical specimens from patients with inflammatory bowel disease, suggest that in a pathophysiological state tachykinins and their receptors may play a role in inflammatory bowel disease and should permit a rational approach to designing neuropeptide antagonists which may prove effective in treating inflammatory diseases.     

Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system

Electrophysiological studies within the lung have documented the presence of heterogenous groups of afferent fibers composed of Adelta and C-fibers and studies of somatosensory nerves within the skin reveal a complex pattern of distribution of sensory neuropeptides and transient receptor potential vanilloid (TRPV)1 positive nerves. However, the anatomical location of these different subpopulations of nerves within the lung has not been extensively studied. In the present study we have demonstrated that TRPV1 axons represented only a small proportion of the total number of PGP9.5 staining nerves within guinea-pig tracheal epithelium and only half the number of TRPV1 axons was immunopositive for substance P. In contrast, most TRPV1 positive neurones found within guinea-pig intrapulmonary airways were found to co-localize with sensory neuropeptides substance P and calcitonin gene-related peptide within and beneath the epithelium, around blood vessels, within airway smooth muscle and alveoli, indicative of heterogeneity of TRPV1 positive axons throughout the airways. However, in the smooth muscle layer of the trachea there was evidence of substance P and calcitonin gene-related peptide containing nerves that did not stain for TRPV1. We also demonstrated a complete loss of TRVP1 positive axons in the trachea and intrapulmonary airways and associated loss of bronchoconstriction induced by capsaicin, in animals chronically treated with capsaicin. However, some neuropeptide immunoreactive axons remained in the smooth muscle layer of capsaicin-treated animals which could represent the small subset of neuropeptide containing fibers which do not co-localize with TRPV1. We have provided evidence of heterogeneity of TRPV1 positive nerve fibers, including fibers characterized by lack of co-localization with neuropeptides in various regions of the airways and the existence of neuropeptide containing fibers that were not TRPV1 positive in guinea-pigs.     

Immunocytochemical investigation of neurovascular relationships in human tooth pulp

This study sought to explore the anatomical relationships between peptidergic nerves and blood vessels within human primary and permanent teeth. Extracted primary and permanent molars (n = 120) were split longitudinally, placed in Zamboni's fixative and the coronal pulps were processed for indirect immunofluorescence. Ten-micrometre-thick serial frozen pulp sections were triple-labelled using combinations of the following antisera: (1) protein gene-product 9.5 (PGP 9.5), a general neuronal marker; (2) one of the neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP) or neuropeptide Y (NPY); and (iii) the lectin Ulex europeus, a label for vascular endothelium. The mid-coronal pulp region was examined, using fluorescence microscopy, to determine the proportion of blood vessels showing a positive innervation (recorded when PGP 9.5-labelled nerves appeared to intersect the vessel wall). In addition, the percentage of these vascular-related nerves expressing each of the above neuropeptides was recorded. Overall, 20% of pulpal blood vessels appeared to have a positive innervation. In the main these were thick-walled arterioles. Capillaries, venules and lymphatics were mostly devoid of an associated innervation. Ninety-two per cent of vascular-related nerves expressed CGRP, 87% expressed SP, 15% expressed VIP and 80% expressed NPY. There were no significant differences in overall innervation or peptide-related innervation between primary and permanent teeth (P < 0.05, ANOVA), indicating that pulpal blood flow is likely to be subject to similar neurological control mechanisms in both dentitions.     

Regional differences in the motor response to capsaicin in the guinea-pig urinary bladder: relative role of pre- and postjunctional factors related to neuropeptide-containing sensory nerves

Capsaicin induced a contraction of isolated strips from the guinea-pig urinary bladder which was more evident in the dome than in the neck and inhibited contractions induced by field stimulation, particularly in the neck. Both responses exhibited prompt desensitization and were tetrodotoxin-resistant, suggesting a specific action on transmitter release from sensory nerve terminals. Indeed, the contractile response in the dome was prevented by a substance P antagonist while the inhibitory response in the neck was prevented by immunoblockade with anticalcitonin gene-related peptide (CGRP) serum. Substance P produced a contraction of the guinea-pig bladder, being about 5 times more potent in the dome than in the neck, while CGRP inhibited the evoked contractions, being about 8 times more potent in the neck than in the dome. Further, the maximal effect of CGRP in the neck was almost double that in the dome. Substance P- and CGRP-like immunoreactivity were detected in both the dome and the neck with no regional differences for each peptide. CGRP-like immunoreactivity was 6.3 and 7.9 times higher than substance P-like immunoreactivity in the dome and the neck, respectively. Exposure to capsaicin evoked release of both substance P- and CGRP-like immunoreactivity from the dome and the neck. Peak CGRP-like immunoreactivity released by capsaicin was 12.3 and 8 times greater than substance P-like immunoreactivity in the dome and the neck, respectively. For each peptide, no difference was found in peak release in the dome vs neck. Total substance P-like immunoreactivity released from the neck was 25% lower than that released from the dome. The ability of CGRP to stimulate accumulation of 3',5' cyclic adenosine monophosphate in membranes prepared from the bladder muscle was greater in preparations from the neck than from the dome. These findings indicate that postjunctional mechanisms (type and number of receptors for sensory neuropeptides, coupling with second messengers) are a major determinant of the type of motor responses consequent of the release of sensory neuropeptides from capsaicin-sensitive nerves.     

Reciprocal age-related changes in GAP-43/B-50, substance P and calcitonin gene-related peptide (CGRP) expression in rat primary sensory neurones and their terminals in the dorsal horn of the spinal cord and subintima of the knee synovium

Age-related changes in the expression of the growth associated protein GAP-43/B-50, and the neuropeptides substance P and calcitonin gene-related peptide (CGRP) were investigated in the sensory neurones of rat dorsal root ganglia, dorsal horns of the spinal cord and subintimal knee synovium. The two time-points studied were 2 months (young adults) and 14-month (aged)-old Sprague Dawley rats. Dorsal root ganglia: In young adults, 40 and 35% of the L4-L5 dorsal root ganglion neurones were positive for GAP-43/B-50 with a 1.5 fold increase in frequency in aged rats at the L5 ganglion. GAP-43/B-50 was strongly expressed by the non-neuronal satellite cells of some medium and many large sized neurones in aged rats. There were marked reciprocal shifts between small and medium sized sensory neurones in respect to their substance P and CGRP expression profiles. Dorsal horn of the spinal cord: there was a 1.3 fold decrease of substance P at L5 level and a 1.3 and 1.5 fold decrease of CGRP at L4-L5 levels in aged rats, respectively. Synovial membrane: There was a 2.3 fold increase in GAP-43/B-50 and a 2.5 fold decrease of CGRP with no changes in substance P expression. These results indicate that (i) primary sensory neurones undergo age-related changes already in early stages of aging, (ii) aging may result in a reduction of substance P and CGRP axonal transport, and (iii) reduced numbers of CGRP containing synovial perivascular fibres may imply a deficient regulation of the synovial microvasculature and therefore metabolic homeostasis of the joint in aged subjects.     

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.     

扬子鳄大肠和输尿管的神经肽分布

目的:观察扬子鳄大肠的神经肽Y（neuropeptide Y,NPY）、血管活性肠肽（vasoactive intestinal peptide,VIP）和输尿管的P物质（substance P,SP）、血管活性肠肽分布。方法:免疫荧光方法。结果:扬子鳄大肠的NPY和VIP免疫反应（NPY-and VIP-immunoreactive,NPY和VIP-IR）阳性神经纤维皆呈弯曲的细线状,主要位于粘膜下层,其次为浅部肌层,且在粘膜下层NPY-IR神经纤维交织成密集的神经丛;VIP-IR神经纡维在粘膜下层构成稀疏的网络状。输尿管的SP免疫反应（SP-immunoreactive,SP-IR）和VIP-IR神经纤维均呈点线状;其中,SP-IR神经纤维位于外膜层,较稀疏,VIP-IR神经纤维位于肌层,较密集,纤维间可见两个近似圆形的VIP-IR阳性神经细胞体。结论:扬子鳄大肠和输尿管分别存在有NPY、VIP和SP、VIP神经分布。     

Histochemical localization of nitric oxide-synthesizing neurons and vascular sites in the guinea-pig intestine.

Laminar preparations of fixed segments of the guinea-pig intestine were examined for nitric oxide synthase activity using reduced nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium salt as substrates. Under conditions specific for detecting nitric oxide synthase-related diaphorase activity, a subpopulation of neural elements in the myenteric plexus, deep muscular plexus and submucosa were intensely stained. Intensely stained nerve fibres were distributed throughout the meshworks of the myenteric plexus and its innervation of the circular muscle, and in the submucosa within Henle's plexus. Intensely stained nerve cells and their processes were evident in most myenteric ganglia but were rare in ganglia of Henle's plexus. Stained ganglion cells comprised types I, II and VI of the morphologically defined enteric nerve cells. Stained neural elements were increasingly prevalent within successively more caudal segments of the intestine. In addition to neuronal staining, arterioles of the submucosal vascular network displayed distinct, punctate patches of staining distributed over their surface. Perivascular nerve fibre staining was absent. These results show nitric oxide synthase activity to be present within neurons and fibres of the major enteric nerve layers and within submucosal blood vessels throughout the guinea-pig small and large intestine.     

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.     

Neuropeptide distributions in the colon,cecum, and jejunum of the horse

The pelvic flexure portion of the equine large colon is the proposed location of a pacemaker mechanism. This study was conducted to ascertain whether the distribution of certain putative neurotransmitters differs at the pelvic flexure compared to other sampling sites. Tissue samples were collected from the intestinal tracts of six horses. Serial sections from these samples were reacted with primary antisera specific for substance P, vasoactive intestinal polypeptide (VIP), methionine-Enkephalin, and calcitonin gene-related peptide (CGRP). The regional distribution of immunoreactive neuronal elements was uniform for each of the neuropeptides except VIP. Although neurons exhibiting VIP-like immunoreactivity were abundant throughout the colon, they were somewhat more plentiful near the apex of the pelvic flexure and the left dorsal colon. These neurons may participate in the initiation and propagation of the propulsive/retropulsive contraction waves, which emanate from this location and are believed to lend a sphincter-like capacity to the pelvic flexure. The submucosal plexus was replete with neurons with intense substance P and VIP-like reactivity. Reactive fibers left submucosal ganglia to project to the intestinal mucosa, reflecting a possible secretogogic role for these neurons. This role may be especially important for the horse as a hindgut fermenter. There were abundant methionine-Enkephalin and substance P-like reactive varicosities throughout the myenteric plexus, many of which established a pericellular plexus of varicose fibers. The abundance of these varicosities, which may correlate with a high degree of neuronal integration, did not vary regionally. These data may enhance our understanding of both normal colonic peristalsis and motility disorders caused by a depletion of these neuropeptides. © 1993 Wiley-Liss, Inc.     

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.     

Co-localization of calcitonin gene-related peptide- and substance P-like immunoreactivity in mucosal intra-epithelial nerves in the toad colon.

Mucosal intra-epithelial nerve terminals have been found in the colon of the toad, Bufo marinus. Co-localized in the fibres are calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and substance P (SP)-LI. The intraepithelial nerves are likely to be terminals of primary afferent sensory neurones. Fibres with vasoactive intestinal peptide (VIP)-LI or neuropeptide Y (NPY)-LI also supply the mucosa but do not penetrate the epithelium.     

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

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

Substance P-, calcitonin gene-related peptide- and C-flanking peptide of neuropeptide Y-immunoreactive fibres are present in normal synovium but depleted in patients with rheumatoid arthritis.

By means of antisera to cytoplasmic components of nerve fibres and neuropeptides which are known to be present in sensory or sympathetic nerves we have examined the distribution of both total and different types of nerve fibres in normal and inflamed human synovial tissue. Samples of synovia were obtained at surgery from five normal and five rheumatoid patients (age range 10-77 years). In order to map the overall neural innervation of the synovium, antiserum to the general neuronal marker protein gene product 9.5 was employed. Substance P and calcitonin gene-related peptide antisera were employed to identify sensory fibres and antisera to the C-flanking peptide of neuropeptide Y to distinguish sympathetic nerves. In normal synovium protein gene product 9.5-immunoreactive fibres were numerous, in particular, the vasculature was densely innervated. Free protein gene product 9.5-immunoreactive fibres were less numerous but were present in all synovia examined, and in many cases these extended to the intimal layer. Neuropeptide immunostaining was predominantly found in perivascular networks. Fibres immunoreactive for the C-flanking peptide of neuropeptide Y were exclusively located around blood vessels whereas free fibres were immunoreactive for substance P or calcitonin gene-related peptide. As with free protein gene product 9.5-immunoreactive fibres, fibres expressing substance P or calcitonin gene-related peptide immunoreactivity were often seen in the intimal cell layer. In rheumatoid arthritis a similar innervation to that seen in normal synovium was apparent in the deep tissue but fibres immunoreactive for protein gene product 9.5, the C-flanking peptide of neuropeptide Y, substance P or calcitonin gene-related peptide were not visible in the more superficial tissues or the intimal cell layer. In addition, immunostaining of neuropeptides in the deep tissue was weaker in the diseased tissues than in normal controls. The data unequivocally demonstrate that synovial tissues are richly innervated and confirm the presence of both sensory and sympathetic nerves. The absence of nerves which innervate the superficial synovium in rheumatoid arthritis might suggest that there is increased release of substance P, calcitonin gene-related peptide and the C-flanking peptide of neuropeptide Y, reducing the stores in the nerves to levels below that detectable by immunocytochemistry. However, since protein gene product 9.5-immunoreactive nerves were not seen in the inflamed tissue it is probable that synovial growth outflanks neural growth and consequently as the disease progresses neural structures become restricted to deeper tissues.(ABSTRACT TRUNCATED AT 400 WORDS)