Expression of neuron-specific enolase, Leu-7, and neuropeptides in human fetal salivary gland epithelium

The immunoreactivity of anti-neuron-specific enolase (NSE) and anti-Leu-7 on formalin-fixed sections of human fetal salivary gland epithelium was determined by the avidin-biotin-peroxidase complex (ABC) method. In addition, expression of some neuropeptides such as vasoactive intestinal polypeptide (VIP), somatostatin (SRIF), and substance P in the human salivary gland epithelium during the gestational period was observed, whereas the other polypeptides examined, including glucagon, cholecystokinin (CCK), Leu-enkephalin, and calcitonin were absent. NSE and Leu-7 immunoreactivity in the fetal salivary gland epithelium was observed solitarily or in groups commonly restricted to the developing duct epithelium. Positive immunoreactivity was observed in 46 cases with NSE (73%) and 44 cases with Leu-7 (70%) in 63 fetal salivary glands examined. In contrast, the incidence of positive cases stained with neuropeptides was lower than those of NSE and Leu-7 immunoreactivity in the human fetal salivary gland epithelium. These findings indicate that certain neuropolypeptides, as well as VIP, SRIF, and substance P present in the human fetal salivary gland epithelium may play a significant role in the development of the gland.     

Distribution of immunoreactive neuropeptides in the pancreas of the bullfrog,Rana catesbeiana,demonstrated by immunofluorescence

Indirect double immunofluorescence labelling for eight neuropeptides in the pancreas of the bullfrog, Rana catesbeiana, demonstrated the occurrence, distribution, and coexistence of certain neuropeptides in the exocrine and endocrine pancreas. Immunoreactivity of substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), FMRFamide (FMRF), and galanin (GAL) was localized in nerve fibers distributed between the acini and around the duct system and vasculature of the exocrine pancreas. In these regions, CGRP-immunoreactive fibers were more numerous than those containing the other five peptides. Almost all SP fibers showed coexistence of SP with CGRP, and about one third of fibers also showed coexistence of SP with VIP, NPY, FMRF, and GAL. In the endocrine pancreas, SP, CGRP, VIP, and GAL were recognized in the nerve fibers around and within the islets of Langerhans, and VIP and GAL fibers were more numerous than SP and CGRP fibers. All CGRP fibers, and about half of the VIP and GAL fibers were immunoreactive for SP. NPY- and FMRF-immunoreactive cells were found at the periphery of the islets. These findings suggest that the exocrine and endocrine pancreatic functions of the bullfrog are under the control of peptidergic innervation.     

Growth-inhibitory properties of vasoactive intestinal polypeptide

It has recently been demonstrated that several neuropeptides can affect cell growth. The mammalian tachykinins substance P and neurokinin A, which are present in peripheral sensory neurons, stimulate growth of cultured connective tissue cells. Substance P-like immunoreactivity has been demonstrated in neuroblastoma cell lines. Neuroblastoma cells also produce other neuropeptides, among them vasoactive intestinal polypeptide (VIP). We report here that VIP is a potent inhibitor of serum-induced DNA synthesis in cultured smooth muscle cells (SMC), whereas no growth-inhibition was seen in SMC exposed to neurokinin A, calcitonin-gene related peptide, neuropeptide Y, somatostatin, or cholecystokinin. The growth-inhibitory effect of VIP was closely related to its ability to induce formation of cyclic AMP. Our results raise the possibility that peptides released by neurons, endocrine cells, as well as by transformed cells, may not only function as mitogens but also as inhibitory modulators of cell growth.     

Distribution and ontogeny of SP, CGRP, SOM, and VIP in chick sensory and sympathetic ganglia

The distribution and ontogeny of four neuropeptides in developing chick lumbosacral sensory and sympathetic ganglia were studied using immunohistochemical techniques. Antibodies to two of these peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), stained small neurons in the medial part of the dorsal root ganglia from embryonic Day 5 and Day 10, respectively, whereas neurons in the lateral part of the ganglia were negative; this distribution persisted throughout development. Both sets of neurons apparently send fibers to the dorsal horn of the spinal cord: SP to laminae I and II, and CGRP to lamina I, suggesting that the SP- and CGRP-positive sensory neurons are nociceptive or thermoreceptive. This correlation between the presence of SP or CGRP in a neuron and a particular functional modality thus provides evidence for a functional distinction between the mediodorsal and ventrolateral zones that are apparent during the development of chick dorsal root ganglia. Moreover, this study suggests that the type of neuron that develops within the dorsal root ganglion correlates with its position within the ganglion. In contrast to SP and CGRP, somatostatin (SOM) and vasoactive intestinal polypeptide (VIP) immunoreactivities were not seen in the lumbosacral sensory ganglia at any stage during development. However, both were present in sympathetic ganglia: SOM from embryonic Day 4.5 and VIP from embryonic Day 10. VIP immunoreactivity persisted throughout development in a large number of sympathetic neurons, but the number of cells with SOM immunoreactivity decreased from embryonic Day 10 onward. SOM therefore appears to be present only transiently in most chick lumbosacral sympathetic cells.     

Neuropeptides regulate the cardiac activity of a prosobranch mollusc,Rapana thomasiana

Summary Involvement of neuropeptides in the regulation of cardiac activity in a prosobranch mollusc, Rapana thomasiana, was studied physiologically as well as immunohistochemically. A catch-relaxing peptide (CARP) showed strong inhibitory effects on the heart with a lower threshold than acetylcholine. The action of CARP was in contrast to that of another neuropeptide, FMRFamide, which has previously been shown to enhance the heart beat. Benzoquinonium blocked the effects of acetylcholine and stimulation of right cardiac nerves 1 and 3b, but not those of CARP, suggesting that the effects of nerve stimulation are mainly due to the release of acetylcholine. Immunohistochemical examinations demonstrated that FMRFamide-like and CARP-like immunoreactive neurons are distributed in the visceral ganglia. Although a neuron appeared to show weak immunoreactivity to both antisera, evidence for the coexistence of peptides in a single neuron was not exhibited. Positive immunoreactivity to FMRFamide and CARP antisera also appeared in right cardiac nerves 1 and 3. In the heart, FMRFamide- and CARP-like immunoreactive fibers were restricted to the atrium and the aortic end of the ventricle, consistent with the morphological observation of innervation. The present results suggest that FMRFamide- and CARP-like peptides are involved in regulating the heart beat.     

Diversity in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and regulatory mechanisms

The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.     

The distribution of putative neurotransmitters in the nervous system of the dipteran Chironomus tentans insect larva: An immunohistochemical study using antisera to 5-hydroxytryptamine,tyrosine hydroxylase,methionine-enkephalin,proctolin and bombesin

Using the indirect immunofluorescence technique, the localization and distribution of transmitters, transmitter-related enzymes and neuropeptides was studied in the larvae of the dipteran species Chironomus tentans. Immunoreactivity could be seen for 5-hydroxytryptamine, tyrosine hydroxylase (the rate-limiting enzyme in the catecholamine synthesis), and the neuropeptides methionine-enkephalin (met-enk), proctolin and bombesin. The immunoreactivity was confined both to cell bodies as well as to nerve fibers within ganglia and along the alimentary canal. Furthermore, tyrosine hydroxylase immunoreactivity could also be seen in epithelial cells locally distributed along a short, middle part of the alimentary tract. These latter cells were regarded as endocrine-like cells. No immunoreactivity could be found with certainty for the enzyme phenylethanolamine-N-methyltransferase (PNMT) nor for the peptides vasoactive intestinal polypeptide (VIP), dynorphin, substance P, somatostatin, thyrotropin releasing hormone (TRH), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), neurotensin, galanin and cholecystokinin (CCK).     

Retinal neuropeptides in the skates,Raja clavata,R. radiata,R. oscellata (Elasmobranchii)

Summary The distribution of immunoreactive neuropeptides was investigated in the retina of three species of skates (Raja clavata, R. radiata, R. oscellata), elasmobranch fish often used in electrophysiological work on the retina. Enkephalins, neuropeptide Y (NPY), substance P and glucagon were found in different types of amacrine cells.All four peptides appeared in cell bodies in the innermost part of the inner nuclear layer. Processes from the cells containing enkephalins were numerous and ramified throughout the inner plexiform layer. Processes from the cells containing glucagon were thick and rare, and were found throughout the inner plexiform layer, at times with a predominance in sublaminae 1 and 4. NPY-immunoreactive fibres appeared mainly in sublamina 1 but also in 2 or 3, and substance-P-immunoreactive fibres in sublaminae 1,4 and 5.Antisera against somatostatin, VIP or neurotensin did not show any immunoreactivity in the skate retina.     

Neuropeptides modulate the transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia

The effects of four neuropeptides (AKH, FMRFamide, proctolin, VIP) on the alterations in glycogen levels induced by other transmitters were studied in isolated leech segmental ganglia. With the exception of FMRFamide, which produced a small increase (14%) in glycogen, the peptides were by themselves without effect on the glycogen levels. Proctolin abolished the glycogenolytic effects of 5-HT, dopamine and octopamine but not histamine. AKH in combination with ACh produced glycogenolysis although each by themselves were ineffective. AKH modified the effects of other transmitters in different ways i.e. by reduction or reversal of effect. VIP and noradrenaline produced an increase in glycogen (cf. noradrenaline alone which decreased glycogen), but did not modify the effects of the other transmitters. FMRFamide produced a complex variety of modulatory effects on the other transmitters. It is concluded that the glycogen stores in leech ganglia, which are localized principally in the glial cells, may be controlled in complex ways by the different combinations of monoamines, amino acids and neuropeptides.     

Presence and distribution of immunoreactive and bioactive FMRFamide-like peptides in the nervous system of the horseshoe crab, Limulus polyphemus

FMRFamide immunoreactivity was detected in all regions of the Limulus nervous system, including the brain (6.5 +/- 0.6 pg FMRFamide/mg), cardiac ganglion (2.06 +/- 0.67 pg FMRFamide/mg), and ventral nerve cord (5.8 +/- 0.7 pg FMRFamide/mg). The distribution of immunoreactive FMRFamide (irFMRFamide) was mapped by immunofluorescence and the distribution corresponded to regional RIA data. A good proportion of the CNS and cardiac ganglion neuropile contained irFMRFamide, and fluorescent cell bodies were observed in several areas. High performance liquid chromatography (HPLC) was employed to separate and characterize the FMRFamide-like peptides from extracts of Limulus brains. HPLC fractions were analyzed using coincidental radioimmunoassay and bioassay (the radula protractor muscle of Busycon contrarium). There appear to be at least three FMRFamide-like peptides in the Limulus brain, including one similar to clam FMRFamide. FMRFamide acts on Limulus heart in a biphasic manner at relatively high concentrations (10(-5)M), but has no effect on the activity of the isolated ventral nerve cord. These data suggest that in Limulus FMRFamide-like peptides are acting as neurotransmitters, or neuromodulators.     

Distribution and function of biogenic amines in the heart of <Emphasis Type="Italic">Nautilus pompilius</Emphasis> L. (Cephalopoda,Tetrabranchiata)

Summary Biogenic amines (serotonin and catecholamines), play an important role in the control of the blood flow not only in vertebrates, but also in invertebrates such as cephalopods. In contrast to the well investigated hearts of the ȁ8modern,ȁ9 coleoid cephalopods, the innervation of the heart of the archaic Nautilus pompilius L. has not been studied in detail. In this study the distribution and effects of biogenic amines in the Nautilus heart were investigated. Serotonin and catecholamines were visualised by the glyxoylic acid induced fluorescence. High performance liquid chromatotography analysis was performed to discriminate between the catecholamines, which showed a high content of noradrenaline in the 4 auricles, the aorta and the ventricle, whereas the ventricle showed a high dopamine content. Adrenaline was found at a very low concentration in the ventricle. Serotonin and dopamine were also immunohistochemically localised to larger nerves and throughout the heart, respectively. In organ bath experiments, the auricles showed little spontaneous activity. After adding serotonin, they displayed rhythmical contractions, which were accelerated dose-dependently by noradrenaline. In summary, these data suggest an important role for biogenic amines in the control of the heart of Nautilus pompilius L., with serotonin possibly stimulating excitatory nerve fibres, whereas noradrenaline is likely to influence the muscle contraction itself.     

Putative neurohemal areas in the peripheral nervous system of an insect,Gryllus bimaculatus,revealed by immunocytochemistry

The morphology and position of putative neurohemal areas in the peripheral nervous system (ventral nerve cord and retrocerebral complex) of the cricket Gryllus bimaculatus are described. By using antisera to the amines dopamine, histamine, octopamine, and serotonin, and the neuropeptides crustacean cardioactive peptide, FMRFamide, leucokinin 1, and proctolin, an extensive system of varicose fibers has been detected throughout the nerves of all neuromeres, except for nerve 2 of the prothoracic ganglion. Immunoreactive varicose fibers occur mainly in a superficial position at the neurilemma, indicating neurosecretory storage and release of neuroactive compounds. The varicose fibers are projections from central or peripheral neurons that may extend over more than one segment. The peripheral fiber varicosities show segment-specific arrangements for each of the substances investigated. Immunoreactivity to histamine and octopamine is mainly found in the nerves of abdominal segments, whereas serotonin immunoreactivity is concentrated in subesophageal and terminal ganglion nerves. Immunoreactivity to FMRFamide and crustacean cardioactive peptide is widespread throughout all segments. Structures immunoreactive to leucokinin 1 are present in abdominal nerves, and proctolin immunostaining is found in the terminal ganglion and thoracic nerves. Codistribution of peripheral varicose fiber plexuses is regularly seen for amines and peptides, whereas the colocalization of substances in neurons has not been detected for any of the neuroactive compounds investigated. The varicose fiber system is regarded as complementary to the classical neurohemal organs.     

Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae

The insect myosuppressins (X1DVX2HX3FLRFamide) are neuropeptides that generally block insect muscle activities. We have used the genomic sequence information from the malaria mosquito Anopheles gambiae Genome Project to clone a G protein-coupled receptor that was closely related to the two previously cloned and characterized myosuppressin receptors from Drosophila [Proc. Natl. Acad. Sci. USA 100 (2003) 9808]. The mosquito receptor cDNA was expressed in Chinese hamster ovary cells and was found to be activated by low concentrations of Anopheles myosuppressin (TDVDHVFLRFamide; EC50, 1.6 x 10(-8)M). The receptor was not activated by a library of 35 other insect neuropeptides and monoamines, including neuropeptides that resembled myosuppressin in their C-terminal moiety, such as PDRNFLRFamide (Anopheles FMRFamide-3), other Anopheles FMRFamide peptides, or neuropeptide F-like peptides, showing that the receptor was quite selective for myosuppressin. These results also showed that the myosuppressin receptor needs a much larger portion than the C-terminal FLRFamide sequence for its activation. The insect myosuppressins are often grouped together with the insect FMRFamides under the name FaRPs (FMRFamide-related peptides). However, this is not justified anymore, because the insect myosuppressin receptor/ligand couple is both functionally and evolutionarily fully unrelated to the insect FMRFamide receptor/ligand couple. To our knowledge, this is the first report on the molecular identification of a mosquito neuropeptide receptor.     

Peptide immunocytochemistry of neurons projecting to the retrocerebral complex in the blow fly, <Emphasis Type="Italic">Protophormia terraenovae</Emphasis>

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to characterize neurons with somata in the pars intercerebralis (PI), pars lateralis (PL), and subesophageal ganglion (SEG), designated as PI neurons, PL neurons, and SEG neurons, respectively, all of which project to the retrocerebral complex in the blow fly, Protophormia terraenovae. Immunocytochemistry combined with backfills through the cardiac-recurrent nerve revealed that at least two pairs of PI and SEG neurons for each were FMRFamide-immunoreactive. Immunoreactivity against [Arg7]-corazonin, beta-pigment-dispersing hormone (beta-PDH), cholecystokinin8, or FMRFamide was observed in PL neurons. Immunoreactive colocalization of [Arg7]-corazonin with beta-PDH, [Arg7]-corazonin with cholecystokinin8, or beta-PDH with FMRFamide was found in two to three somata in the PL of a hemisphere. Based on their anatomical and immunocytochemical characteristics, PI neurons were classified into two types, PL neurons into six types, and SEG neurons into two types. Fibers in the retrocerebral complex showed [Arg7]-corazonin, beta-PDH, cholecystokinin8, and FMRFamide immunoreactivity. Cholecystokinin8 immunoreactivity was also detected in intrinsic cells of the corpus cardiacum. The corpus allatum was densely innervated by FMRFamide-immunoreactive varicose fibers. These results suggest that PI, PL, and SEG neurons release [Arg7]-corazonin, beta-PDH, cholecystokinin8, or FMRFamide-like peptides from the corpus cardiacum or corpus allatum into the hemolymph, and that some PL neurons may simultaneously release several neuropeptides.     

Immunocytochemical demonstration of neuropeptides in the fish-gill parasite, Diclidophora merlangi (Monogenoidea)

Using the indirect immunofluorescence technique, immunoreactivity (IR) to three mammalian and one invertebrate regulatory peptide has been demonstrated in the nervous system of the monogenean gill parasite Diclidophora merlangi. IR to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY) and FMRFamide was evident throughout central and peripheral nervous tissues, whereas vasoactive intestinal polypeptide (VIP)-IR was confined to a portion of the longitudinal ventral nerve cords. Staining patterns revealed the orthogonal arrangement of the nervous system consisting of paired cerebral ganglia, connecting post-pharyngeal commissure, three pairs of longitudinal nerve cords and associated neurones. PP-IR, PYY-IR and FMRFamide-IR were intense throughout the central nervous system of the worm. A small plexus of nerve fibres and somata in each peduncle was immunoreactive for FMRFamide and provided innervation to each of the eight posterior clamps. In the peripheral nervous system, PP-IR, PYY-IR and FMRFamide-IR occurred in an extensive nerve-net with fine, possibly sensory nerve endings in the tegument. PP-IR was also present in nerve fibres in the walls of the ootype, seminal vesicle and uterus. PYY- and FMRFamide-IRs, while evident in nerve fibres of the ootype wall, were also present in a distinct population of cells that encircles the ootype, and which are linked to it by fine cytoplasmic connectives. The majority of these somata were bipolar or multipolar. PYY-IR and FMRFamide-IR were also associated with nerve fibres and bipolar cells in the wall of the vitelline reservoir. Regulatory peptides would appear to play an integral role in neuronal functioning and egg development in D. merlangi.     

Tachykinin receptors and the airways.

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.     

Mapping, quantitative distribution and origin of substance p- and VIP-containing nerves in the uvea of guinea pig eye

VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3 +/- 4.8 pmol/g, mean +/- SEM; substance P:11.1 +/- 1.8 pmol/g) in the uveal portion of the guinea pig eye. Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p less than 0.0005) than those immunoreactive for substance P (95 +/- 7 nm and 82 +/- 9 nm respectively; mean +/- SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1 +/- 1.5 pmol/g, mean +/- SEM, control; 5.3 +/- 1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.     

Quantitative comparison of growth-associated protein-43 and substance P in ulcerative colitis.

The aim of this study was to compare immunoreactivities for substance P with other enteric neuropeptides and GAP-43, a general marker for enteric nerves, in normal human colon and in different stages of ulcerative colitis. Tissue samples from normal colon and regions of ulcerative colitis colon were obtained at surgery and immunostained for substance P, vasoactive intestinal polypeptide (VIP), somatostatin, calcitonin gene-related peptide (CGRP), enkephalin, galanin, GAP-43, and neuron-specific enolase (NSE). Visual examination and semiquantitative analysis revealed a clear increase in the immunoreactivity for substance P in ulcerative colitis, whereas no differences were observed in the distribution of the other peptides. Therefore, quantitative analysis was performed only for substance P immunoreactivity in the lamina propria, circular muscle layer, and myenteric ganglia. In the lamina propria, the score of total intensity of substance P immunoreactivity was 0.55 +/- 0.15 (mean +/- SEM) in normal colon, 1.30 +/- 0.35 (p = 0.087) in least affected colon, and 2.22 +/- 0.28 (p < 0.001) in moderately affected colon, whereas no significant differences were observed in immunoreactivities for GAP-43. Similar results were obtained for the mean substance P- or GAP-43-immunoreactive area. In the circular muscle layer, the number, density, total intensity, and perimeter of substance P- and GAP-43-immunoreactive fibers were essentially similar in normal colon, and in mild or moderately affected colon. We conclude that ulcerative colitis does not change the density of gut innervation as a whole. However, the density of substance P-containing nerves is specifically increased, probably due to increased peptide synthesis leading to better visibility of the fibers.     

Distribution of substance P receptor (neurokinin-1 receptor) in normal ovine lung and during the progression of bronchopneumonia in sheep.

Substance P contributes to the physiological homeostasis of pulmonary airways and vasculature. During pneumonia, alterations in substance P production and receptor expression can influence bronchoconstriction and vascular perfusion. The distribution of substance P receptor [neurokinin-1 receptor (NK-1R)] in lungs of normal sheep and sheep with acute (1 day), subacute (15 days), and chronic (45 days) bronchopneumonia caused by Mannheimia haemolytica was determined by immunohistochemistry (IHC). Three rabbit polyclonal antibodies generated to the same cytosolic C-terminal portion of NK-1R (residues 393-407) were tested. NK-1R immunoreactivity was traced in digital images and quantified with IPLAB software. There were no significant differences in NK-1R protein density between normal and infected lambs. Antibody 1 had the broadest distribution and intensity, and stained alveolar septae, smooth muscle cells of airways and vessels, epithelial cells of airways and alveoli, and submucosal glands. When all animals from the study were included, there was a trend towards decreased NK-1R immunoreactivity over time. The work suggests that (a) the density of NK-1R does not change during progression of bacterial (M. haemolytica) bronchopneumonia, (b) NK-1R is widely distributed in ovine lung and decreases with age, and (c) antibodies to the same NK-1R cytosolic region can vary in specificity and affinity.     

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.