Calcitonin gene-related peptide and the lung: neuronal coexistence with substance P, release by capsaicin and vasodilatory effect

The occurrence and distribution of calcitonin gene-related peptide (CGRP) in the lower airways was studied by means of immunohistochemistry and radioimmunoassay (RIA) in combination with high performance liquid chromatography (HPLC). CGRP-like immunoreactivity (-LI) was observed in nerves from the epiglottis down to peripheral bronchi in rat, cat and guinea pig and also in human bronchi. Double staining revealed colocalization of CGRP-LI and substance P (SP)-LI in cell bodies of nodose and jugular ganglia as well as in axons and nerve terminals of the airways. Systemic capsaicin pretreatment induced a marked loss of the CGRP- and SP-immunoreactive (-IR) nerves in the lower airways. CGRP-IR was also present in epithelial endocrine cells and neuroepithelial bodies. The content of CGRP-LI as measured with RIA in guinea pig bronchi was significantly lower after capsaicin pretreatment. Analysis of human bronchial extracts revealed that CGRP-LI coeluted with synthetic human CGRP on HPLC. In the isolated perfused guinea pig lung capsaicin exposure caused overflow of CGRP-LI suggesting release from peripheral branches of sensory nerves. Both in vivo experiments in the guinea pig measuring insufflation pressure as well as in vitro studies on isolated guinea pig and human bronchi showed that whereas tachykinins contracted bronchial smooth muscle no contractile or relaxing effect was elicited by human or rat CGRP. However, CGRP caused relaxation of serotonin precontracted guinea pig and human pulmonary arteries. In conclusion, the presence and release of CGRP-LI from capsaicin sensitive nerves in the lower airways adds another possible mediator, in addition to tachykinins, of vascular reactions upon sensory nerve irritation.     

Effect of neuropeptides released from sensory nerves on blood flow in the rat airway microcirculation.

Stimulation of sensory nerves in the airway mucosa causes local release of the neuropeptides substance P and calcitonin gene-related peptide (CGRP). In this study we used a modification of the reference-sample microsphere technique to measure changes in regional blood flow and cardiac output distribution produced in the rat by substance P, CGRP, and capsaicin (a drug that releases endogenous neuropeptides from sensory nerves). Three sets of microspheres labeled with different radionuclides were injected into the left ventricle of anesthetized F344 rats before, immediately after, and 5 min after left ventricular injections of capsaicin, substance P, or CGRP. The reference blood sample was withdrawn from the abdominal aorta and was simultaneously replaced with 0.9% NaCl at 37 degrees C. We found that stimulation of sensory nerves with a low dose of capsaicin causes a large and selective increase in microvascular blood flow in the extrapulmonary airways. The effect of capsaicin is mimicked by systemic injection of substance P but not by CGRP, suggesting that substance P is the main agent of neurogenic vasodilation in rat airways.     

Association between histamine-containing mast cells and sensory nerves in the skin and airways of control and capsaicin-treated pigs

Summary The association between mast cells (visualized by routine staining and immunohistochemistry for histamine) and capsaicin-sensitive nerves (containing calcitonin gene-related peptide (CGRP) and substance P (SP)) was studied in the pig. In the 1-ethyl-3(3-diethylaminopropyl)carbodiimide (EDCDI)-fixed skin tissue, histamine-containing mast cells and CGRP/SP-positive nerves were found in close association around blood vessels. In the EDCDI-fixed airway mucosa, only single histamine-containing mast cells were detected. However, many alcian blue-positive mast cells were found, sometimes close to the airway epithelium where CGRP/SP-containing nerve fibres were absent 2 days after systemic capsaicin pretreatment, but no changes in the number and distribution of tissue mast cells, granulocytes or lymphocytes, or the number of blood leukocytes were detected. Local injection of allergen, histamine and capsaicin into the skin of pigs actively sensitized with ascaris antigen caused a rapid light red-flare (vasodilation) reaction. Allergen and histamine, but not capsaicin, also produced plasma protein extravasation. In contrast to the absent flare, the protein extravasation response still occurred in capsaicin-treated pigs. The sensitivity to ascaris antigen was mediated by an IgE-like antibody. We conclude that a functional and morphological relationship exists between histamine-containing mast cells and capsaicin-sensitive sensory nerves in the pig skin. Mast cells and sensory nerves are also found in the airway mucosa and appear to be closely associated with the epithelium.     

Sensory and Sympathetic Nerve Sprouting in the Rat Cornea Following Neonatal Administration of Capsaicin

Corneal sensory and sympathetic nerves exert opposing actions on corneal mitogenesis and wound healing. The mechanisms by which these nerves exert their actions are unknown; however, the release of axonally transported neuropeptides has been postulated. In the present study, we investigated changes in innervation densities of calcitonin gene-related peptide (CGRP-) and tyrosine hydroxylase (TH-)immunoreactive (IR) nerves of the rat cornea following neonatal capsaicin administration, and the relationships between these changes and the development of neuroparalytic keratitis. Newborn rats were injected with capsaicin on each of the first 3 days of life. Forty-eight hours after the last injection, corneal CGRP immunostaining had totally disappeared from the cornea, whereas TH immunostaining was relatively unaffected. Over the next several weeks, a dramatic reinnervation of the cornea took place. By 6–8 weeks both the CGRP-and TH-IR corneal innervation density in the capsaicin-treated animals exceeded that of age-matched control or normal animals; that is, the corneas had become “hyper-reinnervated”. The pattern of innervation that returned was grossly abnormal and was characterized by the presence of a bizarre subepithelial plexus of fine stromal sprouts; an abundance of myelinated axons; and complex, atypical, epithelial leash morphologies. Retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) from the central cornea in control and capsaicin-treated adult animals labeled an average of 143 and 47 trigeminal ganglion cells, respectively (with mean diameters of 25.7 × 0.49 μm and 34.3 × 0.72 μm), suggesting a 67% decrease in corneal afferent neurons in the capsaicin-treated animals. Transection of the ophthalmomaxillary nerve in adult capsaicin-treated animals completely eliminated corneal CGRP-IR staining, and extirpation of the superior cervical ganglion resulted in the loss of 70–80% of corneal TH-IR nerves, thus demonstrating the sensory and predominantly sympathetic origins, respectively, of these fiber populations. Chronic keratitis and neovascularization developed in the capsaicin-treated animals by approximately 3 weeks of age, achieved a maximum intensity between 4 and 6 weeks, and showed some gradual improvement thereafter. However, the keratitis never completely disappeared, even after 13 months. In conclusion, these data show that corneal sensory (CGRP-IR) and sympathetic (TH-IR) nerve fibers undergo extensive sprouting following partial corneal sensory denervation with the neurotoxin capsaicin. However, the resultant “hyper-reinnervation” is morphologically abnormal and, for reasons unknown, functionally incapable of preventing or totally reversing the keratitis.     

The influence of sensory nerves and CGRP on the pancreatic regeneration after repeated episodes of acute pancreatitis in rats.

Stimulation of capsaicin sensitive nerves or administration of calcitonin gene-related peptide (CGRP) before induction of acute pancreatitis (AP) attenuates pancreatic damage, whereas CGRP administration after development of AP aggravates lesion of pancreatic tissue. The aim of this study was to determine the effect of prolonged activity of sensory nerves or CGRP administration on the pancreatic repair after repeated episodes of AP. Five episodes of acute caerulein-induced pancreatitis (10 microg/kg/h for 5 h s.c.) were performed at weekly intervals in rats receiving either vehicle or capsaicin at the sensory nerve stimulatory dose (0.5 mg/kg, 3 times daily), or CGRP (10 microg/kg, 3 times daily). Two weeks after the last induction of AP morphological signs of pancreatic damage, pancreatic blood flow (PBF), serum and pancreatic amylase activity, fecal chymotrypsin activity, pancreatic weight, pancreatic RNA and DNA content, as well as, serum interleukin-1beta (Il-1beta ) were assessed. Pancreata of animals receiving vehicle alone showed almost full recovery within two weeks after last episode of pancreatitis induction. In capsaicin-treated group of rats, we observed the increase in PBF by 44% and in serum Il-1beta concentration by 91%. The pancreatic amylase activity, fecal activity of chymotrypsin, pancreatic nucleic acids content and DNA synthesis were decreased. In rats treated with CGRP the alterations in PBF, serum Il-1beta concentration, as well as, in pancreatic and fecal activity of enzymes were similar to capsaicin treated group but less pronounced. We conclude that prolonged activity of capsaicin-sensitive sensory nerves and the presence of their main mediator-CGRP during pancreatic regeneration after AP leads to pancreatic functional insufficiency typical for chronic pancreatitis.     

Immunohistochemical studies on peptide- and amine-containing endocrine cells and nerves in the gut and the rectal gland of the ratfish Chimaera monstrosa

Summary The occurrence and distribution of endocrine cells and nerves were immunohistochemically demonstrated in the gut and rectal gland of the ratfish Chimaera monstrosa (Holocephala). The epithelium of the gut mucosa revealed open-type endocrine cells exhibiting immunoreactivity for serotonin (5HT), gastrin/cholecystokinin (CCK), pancreatic polypeptide (PP)/FMRFamide, somatostatin, glucagon, substance P or gastrin-releasing peptide (GRP). The rectum contained a large number of closed-type endocrine cells in the basal layer of its stratified epithelium; the majority contained 5HT- and GRP-like immunoreactivity in the same cytoplasm, whereas others were immunoreactive for substance P. The rectal gland revealed closed-type endocrine cells located in the collecting duct epithelium. Most of these contained substance P-like immunoreactivity, although some reacted either to antibody against somatostatin or against 5HT. Four types of nerves were identified in the gut and the rectal gland. The nerve cells and fibers that were immunoreactive for vasoactive intestinal peptide (VIP) and GRP formed dense plexuses in the lamina propria, submucosa and muscular layer of the gut and rectal gland. A sparse network of gastrin- and 5HT-immunoreactive nerve fibers was found in the mucosa and the muscular layer of the gut. The present study demonstrated for the first time the occurrence of the closed-type endocrine cells in the mucosa of the rectum and rectal gland of the ratfish. These abundant cells presumably secrete 5HT and/or peptides in response to mechanical stimuli in the gut and the rectal gland. The peptide-containing nerves may be involved in the regulation of secretion by the rectal gland.     

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, neuron-specific 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.     

Neurohormonal peptides, serotonin, and nitric oxide synthase in the enteric nervous system and endocrine cells of the gastrointestinal tract of neotenic and thyroid hormone-treated axolotls (Ambystoma mexicanum)

Immunoreactivity against vasoactive intestinal polypeptide (VIP), neurotensin (NT), substance P (SP), calcitonin gene-related peptide (CGRP), gastrin/cholecystokinin (GAS/CCK), somatostatin (SOM), serotonin (SER), and nitric oxide synthase (NOS) was investigated in the gastrointestinal tract of the urodele Ambystoma mexicanum, the axolotl, by the use of immunohistochemical techniques. The study also compares the distribution patterns and frequencies of the neurohormones, and NOS in neotenic and thyroxine-treated (metamorphosed) individuals. GAS/CCK, SP, NT, SOM, and SER immunoreactivities occurred in endocrine mucosal cells and VIP, SP, CGRP, NTSER, SER, and NOS immunoreactivities in the enteric nervous system. The GAS/CCK-immunoreactive (-IR) cells were restricted to the upper small intestine. NT-IR and SP-IR endocrine cells were found in the entire gastrointestinal tract and were most prominent in the distal large intestine. The density of the SOM-IR cells decreased from the stomach toward the large intestine. SER-IR endocrine cells were found throughout the gastrointestinal tract, with particularly high densities in the stomach and distal large intestine. The VIP-IR enteric nerve fibers were the most prominent ones, present in all layers of the entire gastrointestinal tract, and supplied the smooth muscle and the vasculature. The SER-IR fibers exhibited similar distribution patterns but were less numerous. Very few NT-IR but many SP-IR fibers were found in the muscle and submucosal layers. The NT-IR fibers mainly supplied blood vessels, while the SP-IR fibers were also in contact with the smooth muscle. In the muscle and submucosal layers, CGRP-IR fibers were associated to the vasculature; CGRP immunoreactivity occurred also in a minority of SP-IR fibers. NOS-IR nerve fibers were in contact with submucosal arteries but were the least frequent . After metamorphosis provoked by exogenous thyroxine, the number of SOM-IR endocrine cells in the stomach mucosa was increased as well as the density of VIP-IR, SER-IR, and SP-IR nerve fibers in the gastrointestinal tract. It is proposed that the observed increases may reflect refinements of the neurohormonal system after metamorphosis.     

Evidence that calcitonin gene-related peptide contributes to the capsaicin-induced relaxation of guinea pig cerebral arteries.

Pretreatment with capsaicin caused a depletion of substance P (SP)-, neurokinin A (NKA)- and calcitonin gene-related peptide (CGRP)-like immunoreactivity (-LI) from the trigeminal ganglion, dura mater and cerebral arteries. The effect of capsaicin on isolated basilar arteries of guinea pig resulted in a biphasic relaxant response of histamine precontracted vessels. The first phase of the capsaicin-induced relaxation was absent in capsaicin-treated guinea pigs. Furthermore, repeated administration of capsaicin decreased the first but not the second phase of relaxation, supporting the view that a stored agent was released, while the second phase probably was due to a direct effect of capsaicin per se. The biphasic effect was not modified by the SP antagonist Spantide in a concentration that blocks tachykinin response (3.10(-6) M), nor by removal of the endothelium. There was no significant difference in pD2 values (-log concentration eliciting half maximum relaxation) between relaxations induced by SP, NKA, neurokinin B, neuropeptide K or CGRP in capsaicin pretreated as compared to vehicle-treated animals. These results are in support of the assumption that CGRP is involved in the capsaicin-induced relaxation caused by release of vasoactive agents from sensory afferent nerves.     

Involvement of capsaicin-sensitive sensory neurons in gastrointestinal function

There is both morphological and functional evidence that capsaicin-sensitive sensory neurons innervate the digestive tract. The possible function of these neurons in gastric ulceration and gastrointestinal motility was investigated in rats which had been systemically pretreated with capsaicin (50-125 mg/kg). It was found that capsaicin-sensitive afferent neurons do not participate in the physiologic control of gastrointestinal propulsion. However, the inhibition of gastrointestinal transit due to surgical trauma or peritoneal irritation with iodine was reduced in capsaicin-treated rats. It was concluded that capsaicin-sensitive sensory neurons may be involved in sympathetic reflex inhibition of gastrointestinal propulsion. Gastric ulceration induced by the intraperitoneal injection of indomethacin or intragastric administration of ethanol was greatly aggravated in capsaicin-treated rats. Since an involvement of the autonomic nervous system as well as of histamine and prostaglandins in this effect of capsaicin treatment could be ruled out, further support was lent to the previously proposed hypothesis that sensory nerve endings can protect the gastric mucosa against ulceration by the local release of vasodilator substances.     

Anterograde transneuronal viral tract tracing reveals central sensory circuits from brown fat and sensory denervation alters its thermogenic responses

Brown adipose tissue (BAT) thermogenic activity and growth are controlled by its sympathetic nervous system (SNS) innervation, but nerve fibers containing sensory-associated neuropeptides [substance P, calcitonin gene-related peptide (CGRP)] also suggest sensory innervation. The central nervous system (CNS) projections of BAT afferents are unknown. Therefore, we used the H129 strain of the herpes simplex virus-1 (HSV-1), an anterograde transneuronal viral tract tracer used to delineate sensory nerve circuits, to define these projections. HSV-1 was injected into interscapular BAT (IBAT) of Siberian hamsters and HSV-1 immunoreactivity (ir) was assessed 24, 48, 72, 96, and 114 h postinjection. The 96- and 114-h groups had the most HSV-1-ir neurons with marked infections in the hypothalamic paraventricular nucleus, periaqueductal gray, olivary areas, parabrachial nuclei, raphe nuclei, and reticular areas. These sites also are involved in sympathetic outflow to BAT suggesting possible BAT sensory-SNS thermogenesis feedback circuits. We tested the functional contribution of IBAT sensory innervation on thermogenic responses to an acute (24 h) cold exposure test by injecting the specific sensory nerve toxin capsaicin directly into IBAT pads and then measuring core (T(c)) and IBAT (T(IBAT)) temperature responses. CGRP content was significantly decreased in capsaicin-treated IBAT demonstrating successful sensory nerve destruction. T(IBAT) and T(c) were significantly decreased in capsaicin-treated hamsters compared with the saline controls at 2 h of cold exposure. Thus the central sensory circuits from IBAT have been delineated for the first time, and impairment of sensory feedback from BAT appears necessary for the appropriate, initial thermogenic response to acute cold exposure.     

Mechanisms of depletion of substance P by capsaicin

Capsaicin is a neurotoxin that can deplete sensory nerves of their content of substance P and interfere with certain sensory functions, such as responses of animals to noxious heat stimuli. In adult guinea pigs, a species that is susceptible to the effects of capsaicin on both substance P content and sensory function, capsaicin induces selective depletion of substance P from dorsal root ganglia and the dorsal spinal cord, sites of the cell bodies and central terminals of primary afferent neurons, respectively. As the onset of thermal analgesia in guinea pigs precedes depletion of substance P, direct neural actions of capsaicin probably account for its effects on sensory function. Capsaicin interferes with the retrograde transport of nerve growth factor (NGF) to the cell bodies of sensory nerves. Decreased availability of NGF at the site of neural protein synthesis leads to decreased synthesis of substance P. After failure of synthesis of substance P, the content of the peptide in sensory nerves gradually decreases until depletion occurs.     

Gastroprotective action of orexin-A against stress-induced gastric damage is mediated by endogenous prostaglandins, sensory afferent neuropeptides and nitric oxide

Orexin-A, identified in the neurons and endocrine cells in the gut, has been implicated in control of food intake and sleep behavior but little is known about its influence on gastric secretion and mucosal integrity. The effects of orexin-A on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and restraint stress (WRS) or 75% ethanol were determined. Orexin-A (5-80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by WRS and this was accompanied by the significant rise in plasma orexin-A, CGRP and gastrin levels, the gastric mucosal blood flow (GBF), luminal NO concentration and an increase in mRNA for CGRP and overexpression of COX-2 protein and the generation of PGE(2) in the gastric mucosa. Orexin-A-induced protection was abolished by selective OX-1 receptor antagonist, vagotomy and attenuated by suppression of COX-1 and COX-2, deactivation of afferent nerves with neurotoxic dose of capsaicin, pretreatment with CCK(2)/gastrin antagonist, CGRP(8-37) or capsazepine and by inhibition of NOS with L-NNA. This study shows for the first time that orexin-A exerts a potent protective action on the stomach of rats exposed to non-topical ulcerogens such as WRS or topical noxious agents such as ethanol and these effects depend upon hyperemia mediated by COX-PG and NOS-NO systems, activation of vagal nerves and sensory neuropeptides such as CGRP released from sensory nerves probably triggered by an increase in gastric acid secretion induced by this peptide.     

Histamine is involved in gastric vasodilation during acid back diffusion via activation of sensory neurons

Protective vasodilation during acid back diffusion into the rat gastric mucosa depends on activation of sensory neurons and mast cell degranulation with histamine release. We hypothesized that these two mediator systems interact and that histamine partly exerts its effect via sensory nerves. Gastric blood flow (GBF) and luminal histamine were measured in chambered stomachs, and mast cell numbers were assessed by morphometry. Ablation of sensory neurons and depletion of mast cells were produced by pretreatment with capsaicin or dexamethasone, respectively. Mucosal exposure to 1.5 M NaCl and then to pH 1.0 saline in ablated and control rats caused increased luminal histamine and reduced numbers of mast cells. Enterochromaffin-like cell marker pancreastatin remained unchanged. Only control rats responded with an increase in GBF. Capsaicin stimulation (640 microM) of the undamaged mucosa induced identical increase in GBF and unchanged mast cell mass in normal and dexamethasone-treated rats. Increase in GBF after topical exposure to histamine (30 mM) in rats pretreated with capsaicin or a calcitonin gene-related peptide (CGRP)(1) antagonist human CGRP(8-37) or exposed to the calcium pore blocker ruthenium red was less than one-half of that in control rats. These data suggest that mast cell-derived histamine is involved in gastric vasodilatation during acid back diffusion partly via sensory neurons.     

Peptidergic innervation of rat lymphoid tissue and lung: Relation to mast cells and sensitivity to capsaicin and immunization

Summary The peptidergic innervation of lymphoid tissue and the lung in relation to mast cells was studied in rat. The sensitivity of neuropeptide-containing nerves to capsaicin treatment and immunization was also examined. Measurements of the content of neurokinin A and calcitonin gene-related peptide revealed that the lung contained the highest content of both neuropeptides; lymph nodes had intermediate levels, whereas the spleen had the lowest content. Immuhohistochemistry showed that the calcitonin gene-related peptide- and neurokinin A-immunoreactive nerves in lymph nodes were mainly found around blood vessels, whereas in the lung the nerves were present within the lining respiratory epithelium, bronchial smooth muscle, around blood vessels and close to lymphoid aggregates. Combined immunohistochemistry for serotonin (5-hydroxytryptamine), as a marker for mast cells, and tachykinins or calcitonin gene-related peptide revealed that a close association was often present between the nerves and 5-hydroxytryptamine-positive cells in the bronchi of the lung, while 5-hydroxytryptamine-positive cells were not observed in lymph nodes. The neurokinin A and calcitonin gene-related peptide content in lymph nodes, spleen and lung, but not the content of neuropeptide Y, was markedly decreased by capsaicin treatment, suggesting a sensory origin for the two former peptides. Aerosol immunization increased the levels of calcitonin gene-related peptide in the lung, whereas the content in mediastinal lymph nodes was not affected. These data demonstrate a peptidergic innervation mainly of blood vessels in lymphoid tissue and a close relation between sensory nerves and mast cells as well as lymphoid aggregates in the bronchi of the lung. This further suggests that the sensory innervation of lymph nodes is mainly related to regulation of vascular tone and lymph flow. Furthermore, at the site of immunization, i.e., in the airway mucosa, sensory nerve mediators may interact both with mast cells and lymphoid cells.     

Calcitonin gene-related peptide (CGRP)-immunoreactive sensory and motor nerves of the mammalian palate

The distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres in the palate of rat, cat and monkey was studied using immunocytochemistry and radioimmunoassay. CGRP-containing nerve fibres were found, in all species studied, to form a rich plexus in the subepithelial and submucous layers, around excretory ducts and blood vessels. A small number of CGRP-containing nerve fibres penetrated the epithelium of the hard and soft palate, and terminated as free endings. Some CGRP-containing nerve fibres were found in the vicinity of the mucous glands. CGRP-immunoreactive motor end plates were seen in the striated muscle (tensor veli palatini) of the soft palate. Following capsaicin treatment a small depletion in CGRP-immunoreactive nerve fibres in the rat palate epithelium was noted. In contrast, CGRP immunoreactive fibres forming rich plexuses in other layers of the palate, including motor end plates, were not affected. The extractable CGRP showed no significant depletion (normal animals [n = 10] 21.7 +/- 2.4 pmol/g compared with capsaicin-treated animals [n = 10] 17.5 +/- 1.8 pmol CGRP/g wet weight). The reduction in the number of visible immunoreactive nerves following capsaicin application tends to confirm the sensory character of the CGRP-containing nerve fibres terminating in the epithelium of the hard and soft palate. The capsaicin insensitive CGRP-immunoreactive nerve fibres may thus have a predominantly motor function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of neurotrophin receptors and transmitters in the developing Xenopus gut

The ontogeny of gut innervation in the anuran amphibian Xenopus laevis was studied using immunohistochemistry on sections of whole larvae from NF stages 38-52. Immunoreactivity to acetylated tubulin confirmed the presence of nerve fibres as early as stages 38-39. Actin immunoreactivity was found at stage 41, indicating the presence of smooth muscle cells. Trk-like neurotrophin receptors were occasionally found in nerve fibres as soon as stages 38-39. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) immunoreactivities coexisted in nerves innervating the gut wall from stages 40-41, and nitric oxide synthase (NOS) from stage 42. Substance P/neurokinin A (SP/NKA) occurred at stage 42. In all these cases, the first fibres were observed in the oesophagus. Calcitonin gene-related peptide (CGRP) was first observed in nerves at stage 48. In general, VIP/PACAP and NOS innervation was denser than the tachykinin innervation. In conclusion, the development of nerve fibres in the Xenopus gut is probably dependent on neurotrophins that may act via Trk-like receptors and occur before the gut wall is fully organised morphologically. Feeding in Xenopus larvae starts at NF stage 45. The study demonstrates that several of the transmitters investigated are expressed in the gut innervation (and in endocrine cells) prior to this stage.     

Sensory or sympathetic white adipose tissue denervation differentially affects depot growth and cellularity

Functional and histological evidence for the sympathetic nervous system (SNS) innervation of white adipose tissue (WAT) exists for several species; however, its sensory innervation has only been shown in laboratory rats, and its function is unclear. We tested the effects of sensory and SNS innervation of Siberian hamster epididymal and inguinal WAT (EWAT and IWAT) by assessing calcitonin gene-related peptide (CGRP)- and tyrosine hydroxylase-immunoreactivity (ir), respectively. Next, we tested the role of the sensory innervation of WAT on growth and cellularity because WAT surgical denervation increases pad mass via selective increases in fat cell number, an effect ascribed to SNS denervation but that could be due to the accompanying surgical disruption of WAT sensory innervation. Sensory denervation was accomplished via multiple local microinjections of capsaicin into WAT, and its effects were compared with those of surgical denervation. Surgically denervated IWAT and EWAT showed significantly decreased tyrosine hydroxylase-ir and CGRP-ir, whereas capsaicin-treated WAT had only significantly decreased CGRP-ir. Surgically denervated pad masses were significantly increased; this was accompanied by increased total fat cell number in IWAT, with no change in fat cell size. EWAT only showed a significant increase in the number of small- to medium-sized adipocytes (75-125 mum diameter). By contrast, sensory-denervated pad masses were unchanged, but IWAT showed significantly increased average fat cell size. Collectively, these data provide immunohistochemical evidence for sensory and SNS innervation of WAT in Siberian hamsters and differential control of WAT cellularity by these innervations, as well as the ability of locally applied capsaicin to selectively reduce WAT sensory innervation.     

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.