Association of substance-P-immunoreactive nerves with the murine olfactory mucosa

Location and distribution of nerve fibers immunoreactive to substance P were studied in the mouse olfactory mucosa. A moderately dense plexus of fibers is present at the interface of the olfactory epithelium and the connective tissue of the lamina propria. In addition, many immunoreactive nerve fibers are noted in close association with Bowman's glands and blood vessels in the lamina propria. However, such fibers were not observed in olfactory epithelium proper nor in the fila olfactoria. Substance-P-immunoreactivity is almost totally abolished by treatment of animals with capsaicin, an agent known to deplete substance P from primary sensory neurons. It is suggested that the substance-P-immunoreactive fibers are of sensory origin, with their perikarya most likely located in the trigeminal ganglia. Functionally, they might influence local blood flow and/or the secretion of Bowman's glands.     

Peptidergic regulation of secretory activity in amphibian olfactory mucosa: Immunohistochemistry,neural stimulation,and pharmacology

Summary The role of substance P in the regulation of secretion from sustentacular cells, Bowman's glands and deep glands in the amphibian olfactory mucosa was investigated using immunohistochemical, electrophysiological, and pharmacological methods. Substance P-like immunoreactive varicose fibers extended through the olfactory epithelium, terminating at or near the surface. In addition, immunoreactive varicose fibers innervated Bowman's glands, deep glands, and blood vessels in the lamina propria. Innervation of Bowman's gland was sparse, with fibers terminating on basal acinar cell membranes; deep gland innervation was abundant, with fibers often extending between acinar cells almost to the lumen. Stimulation of the ophthalmic branch of the trigeminal nerve resulted in slow potentials recorded at the surface of the olfactory epithelium. When the olfactory mucosae from trigeminal-stimulated animals were examined histologically, morphological signs of secretory activity were observed, suggesting that substance P was released from the trigeminal nerve terminals. Topical application of 10^-5 to 10^-3 mol substance P resulted in morphological signs of secretion that were very similar to those seen as a result of trigeminal stimulation. Thus, substance P released from trigeminal fibers may modulate secretory activity within the olfactory mucosa.     

Neurokinin A-like immunoreactivity in feline dental pulp: its distribution,origin and coexistence with substance P-like immunoreactivity

Summary The distribution and origin of neurokinin A (NKA)-like immunoreactivity were investigated in feline dental pulp by an indirect immunofluorescence method. NKA-containing nerve fibres with varicosities, which entered the dental pulp via apical foramen, were distributed throughout this tissue. Many NKA-containing nerve fibres were localized around blood vessels, but some were observed apart therefrom. At the odontoblastic layer, thin NKA-containing nerve fibres were observed running straight toward the pulp-predentinal border between odontoblasts. After inferior alveolar nerve section, all NKA-containing nerve fibres disappeared in the dental pulp, while the removal of the superior cervial ganglion resulted in no change in the distribution of these fibres. The correlation of NKA-like immunoreactivity and substance P (SP)-like immunoreactivity was also investigated by double-immunofluorescence technique. The distribution of NKA-containing nerve fibres was very similar to that of SP-containing nerve fibres; it appeared that all NKA-containing nerve fibres contained SP.     

Innervation of lower airways and neuropeptide effects on bronchial and vascular tone in the pig

Summary The occurrence and distribution of peptide-containing nerve fibres [substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), neuropeptide Y (NPY)] and noradrenergic nerve fibres [tyrosine hydroxylase (TH)- and dopamine beta hydroxylase (DBH)-positive] in the airways of the pig were studied by means of immunohistochemistry. SP- and CGRP-immunoreactive (-IR) nerve fibres were present close to and within the lining respiratory epithelium, around blood vessels, within the tracheobronchial smooth muscle layer and around local tracheobronchial ganglion cells. The content of CGRP- and neurokinin A (NKA)-like immunoreactivity (-LI) measured by radioimmunoassay (RIA) was twice as high in the trachea compared to that in the peripheral bronchi. SP was a more potent constrictor agent than NKA on pig bronchi in vitro. CGRP had a relaxant effect on precontracted pig bronchi. On blood vessels CGRP exerted a relaxant effect that was more pronounced on pulmonary arteries than on bronchial arteries. VIP/PHI-IR fibres were seen in association with exocrine glands and in the tracheobronchial smooth muscle layer. VIP-positive nerve fibres were abundant around blood vessels in the trachea but sparse or absent around blood vessels in the peripheral bronchi. This histological finding was supported by RIA; it was shown that the content of peptides displaying VIP-like immunoreactivity (-LI) was 18 times higher in the trachea compared to peripheral bronchi. VIP was equally potent as CGRP in relaxing precontracted pig bronchi in vitro. Both bronchial and pulmonary arteries were relaxed by VIP. NPY was colocalized with VIP in tracheal periglandular nerve fibres and in nerve fibres within the tracheobronchial smooth muscle layer. NPY was also present in noradrenergic (DBH-positive) vascular nerve fibres. The content of NPY was much higher (15-fold) in the trachea compared to small bronchi. NPY caused a contraction of both pulmonary and bronchial arteries. The bronchial smooth muscle contraction to field stimulation in vitro was purely cholinergic. A non-cholinergic relaxatory effect following field stimulation was observed after bronchial precontraction. Capsaicin had no effect on pig bronchi in vitro.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

Target cells for methylsulphonyl-2,6-dichlorobenzene in the olfactory mucosa in mice

Previously we reported that methylsulphonyl-2,6-dichlorobenzene, 2, 6-(diCl-MeSO(2)-B), was irreversibly bound to the olfactory mucosa of mice and induced necrosis of the Bowman's glands with subsequent neuroepithelial degeneration and detachment. In this study, autoradiography and histopathology were used to determine tissue-localization and toxicity of 2,6-(diCl-MeSO(2)-B) in the olfactory mucosa of control mice and animals pretreated with cytochrome P450 (CYP) and glutathione (GSH) modulators. The Bowman's glands of the olfactory mucosa were the major target sites of non-extractable binding of 2,6-(diCl-(14)C-MeSO(2)-B), whereas the olfactory neuroepithelium and nerve bundles showed only background levels of silver grains. Metyrapone pretreatment slightly decreased binding in the Bowman's glands and markedly decreased toxicity in the olfactory mucosa after 2,6-(diCl-MeSO(2)-B) administration. These results support that a CYP-mediated activation of 2, 6-(diCl-MeSO(2)-B) takes place in the Bowman's glands giving rise to toxic reactive intermediates. In mice pretreated with the GSH-depleting agent phorone, a marked increase of irreversible binding of 2,6-(diCl-(14)C-MeSO(2)-B) in the Bowman's glands was observed. Tape-section autoradiograms also revealed a significant increase of uptake of radioactivity in the olfactory bulb. As determined by histopathology, GSH-depletion increased both the extent and severity of the lesion in the mucosa. These results imply that 2,6-(diCl-MeSO(2)-B)-reactive intermediates are conjugated with GSH. The amount of irreversible binding and toxicity in the olfactory mucosa seems to be associated with the level of 2, 6-(diCl-MeSO(2)-B)-reactive intermediates.     

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.     

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in salivary glands.

VIP-like immunoreactivity was found in rat, cat and human salivary glands at a concentration of 17.0 – 29.3 pmol/g of tissue. The immunoreactivity was localised by immunocytochemistry in beaded nerve fibres which occurred in association with secretory acini, ducts, blood vessels and larger nerves. In salivary glands VIP may act as a neurotransmitter or neuromodulator involved in the regulation of blood flow and in the composition and volume of saliva.     

Glutathione and γ-glutamyl transpeptidase are differentially distributed in the olfactory mucosa of rats

Summary Components of the -glutamyl cycle, including thiols, glutathione (GSH) and -glutamyl transpeptidase (-GT), were localized in the nasal mucosae of rats using histochemical and immunohistochemical methods. In olfactory mucosa, thiols were widely distributed, with intense staining in the mucociliary complex (MC), basal cells, acinar cells of Bowman's glands (BG), and olfactory nerve bundles, and with moderate staining in olfactory receptor neurons (ORNs). GSH was localized in MC, BG acinar cells, nerve bundles and, to a lesser extent, in ORNs. -GT immunoreactivity was restricted to the MC and to basolateral and apical membranes of BG acinar and duct cells. The basolateral membrane of BG acinar cells, located in close association with blood vessels and connective tissue, showed granule-like immunoreactivity. Inrespiratory mucosa, all three compounds were localized in the MC and acinar cells of respiratory glands (RG). In the MC, -GT immunoreactivity was associated primarily with brush borders of ciliated cells. Granular immunoreactivity was also apparent in the supranuclear region of RG acinar cells. These results demonstrate that components of the -glutamyl cycle are localized in olfactory and respiratory glands, and that they are secreted into the mucus, where they may mediate perireceptor events such as detoxification and/or solubilization of air-borne xenobiotics, toxicants and odorants.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Substance P and neurokinin A immunoreactive nerve fibres in the developing salivary glands of the rat

The time of appearance and distribution of substance P (SP) and neurokinin A (NKA) immunoreactive nerve fibres in developing salivary glands of the rat were studied by the use of indirect immunohistochemical methods. The glands were examined at daily intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th and 30th postnatal day. The findings were compared to samples from adult. The first SP- and NKA-immunoreactive (IR) nerve fibres appeared on the 19th day i.u. in the parotid and submandibular glands and were abundantly distributed around developing ductal branches. In the mesenchyme around the developing ductal branches of the parotid gland the fibres appeared on the 20th day i.u. In the submandibular gland NKA-IR fibres appeared in the mesenchyme surrounding the developing ductal branches on the 19th day i.u. and SP-IR fibres on the 21st day i.u. Around blood vessels of both glands, SP- and NKA-IR fibres made their appearance only much later, on the second postnatal day. The number of SP- and NKA-IR nerve fibres in the developing salivary glands was already high on the 19th day i.u. when they were first detected. From this point up to the 16th postnatal day the glands were richly innervated by the fibres, but later the numbers slowly decreased to adult levels. The abundance of SP- and NKA-IR nerve fibres especially around the ductal branches and secretory structures in the developing salivary glands suggests a role in the functional maturation of salivary glands.     

Substance P and neurokinin A immunoreactive nerve fibres in the developing salivary glands of the rat.

The time of appearance and distribution of substance P (SP) and neurokinin A (NKA) immunoreactive nerve fibres in developing salivary glands of the rat were studied by the use of indirect immunohistochemical methods. The glands were examined at daily intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th and 30th postnatal day. The findings were compared to samples from adult. The first SP- and NKA-immunoreactive (IR) nerve fibres appeared on the 19th day i.u. in the parotid and submandibular glands and were abundantly distributed around developing ductal branches. In the mesenchyme around the developing ductal branches of the parotid gland the fibres appeared on the 20th day i.u. In the submandibular gland NKA-IR fibres appeared in the mesenchyme surrounding the developing ductal branches on the 19th day i.u. and SP-IR fibres on the 21st day i.u. Around blood vessels of both glands, SP- and NKA-IR fibres made their appearance only much later, on the second postnatal day. The number of SP- and NKA-IR nerve fibres in the developing salivary glands was already high on the 19th day i.u. when they were first detected. From this point up to the 16th postnatal day the glands were richly innervated by the fibres, but later the numbers slowly decreased to adult levels. The abundance of SP- and NKA-IR nerve fibres especially around the ductal branches and secretory structures in the developing salivary glands suggests a role in the functional maturation of salivary glands.     

异型一氧化氮合酶在爪蛙鼻粘膜中的分布

用还原型辅酶Ⅱ黄递酶组织化学和一氧化氮合酶（ＮＯＳ）免疫细胞化学技术研究了成年爪蛙（Ｘｅｎｏｐｕｓｌａｅｖｉｓ）鼻粘膜ＮＯＳ的阳性结构。嗅上皮中嗅感觉神经元和支持细胞,以及固有层中的神经束、血管和粘膜下腺均呈还原型辅酶Ⅱ黄递酶阳性染色。在嗅上皮中,未见Ⅰ型或Ⅱ型ＮＯＳ抗体免疫反应阳性结构,但鼻内侧窦和内侧窦口顶嗅上皮中的嗅感觉神经元见有Ⅲ型ＮＯＳ强免疫反应。在固有层中,Ⅰ型或Ⅲ型ＮＯＳ免疫反应性存在于神经束和血管中,未见于粘膜下腺的腺泡中。结果表明,不同异型的ＮＯＳ存在于爪蛙鼻粘膜中,提示一氧化氮可能参与爪蛙的化学感觉活动。     

Vasoactive intestinal peptide and substance P in salivary glands of the rat following denervation or duct ligation

Immunoreactive vasoactive intestinal peptide (VIP) and substance P (SP) were studied in parotid, submaxillary and sublingual glands of the rat. The concentration of VIP was highest in the submaxillary gland and lowest in the parotid gland. The concentration of SP was highest in the parotid gland; it was at, or below the limit of detection in the sublingual gland. In the parotid gland the total amounts of VIP and SP were reduced by 95% after parasympathetic denervation (section of the auriculo-temporal nerve). In the submaxillary gland the total amounts of the peptides were unchanged after parasympathetic decentralization (section of the chorda-lingual nerve). In this gland the total amount of SP was reduced by 92% and that of VIP by 50%, when the chorda tympani nerve fibres were cut deep into the hilum. Cutting the nerve fibres at the hilum left the total amounts of the peptides unchanged in the submaxillary gland, whereas in the sublingual gland the total amount of VIP was reduced by 70%. Sympathetic denervation did not reduce the total amounts of the peptides. Duct ligation caused gland atrophy. In the parotid gland the total amounts of VIP and SP were reduced by 40%. In the submaxillary gland the same percentage reduction occurred with regard to SP; however, the total amount of VIP was reduced by 99%. The VIP- and SP-containing nerve fibres reach the salivary glands by the parasympathetic nerves. In both submaxillary and sublingual glands a certain fraction of VIP originates within the glands.     

Aquaporin pathways and mucin secretion of Bowman's glands might protect the olfactory mucosa

The sense of smell is conveyed by the olfactory sensory neurons of the olfactory mucosa. Uniquely for sensory systems, the olfactory neurons directly face the external environment and are thus vulnerable to infections and changes in the airway surface liquid, but the surface liquid production and maintenance is not well understood. Here we show in rats and mice that Bowman's glands secrete the mucin MUC5AC. Aquaporin-5 was present at the apical face of the olfactory epithelium, completing a water transport pathway to the surface of the epithelium. Immunogold electron microscopy analysis revealed an intricate network of fine Aquaporin-1-positive fibroblast processes that surround Bowman's glands, whereas deeper blood vessels were unlabeled for Aquaporin-1. Our results show how the olfactory mucosa might be protected against infections and dehydration generally and how neuronal function is protected against ion concentration changes in the airway surface liquid by rapid replacement of water losses through the aquaporin pathways.     

Immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa

Previously, we described two olfactory-specific cytochromes P-450: rat cytochrome P-450olf1 (IIG1), identified by cDNA cloning, and bovine cytochrome P-450olf2 (IIA), identified by peptide microsequencing of a transmembranal polypeptide (p52). Here we describe the preparation of polyclonal antisera against peptide sequences of these proteins and their use in the immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa. Immunoreactivities related to both enzymes are found in the subepithelial Bowman's glands of olfactory mucosa. Practically no immunoreactivity was found in other rat tissues, including liver, lung, kidney and respiratory mucosa. In addition, double-labeling experiments demonstrated that cytochromes P-450olf1 and P-450olf2 are present in the same population of Bowman's glands. The olfactory-specific localization of cytochromes P-450olf1 and P-450olf2 is consistent with a role for these enzymes in the modification or clearance of odorants from the chemosensory tissue.     

The distribution of substance P-, CGRP-, galanin-and ANP-like immunoreactive nerves in human sweat glands

Summary The distribution in immunoreactivities towards atrial natriuretic peptide, calcitonin gene-related peptide, galanin and substance P were demonstrated in human skin at the light and electron microscopic levels. Nerves immunoreactive to the first three of these peptides were found around eccrine sweat glands, whereas only a few positively-labelled nerve fibres could be seen around apocrine glands. At the ultrastructural level, immunoreactivity to the neuropeptides was localized in the large dense-cored vesicles of the nerve terminals. No immunoreactivity to substance P could be detected around sweat glands. In addition to these findings, the four types of immunoreactivity were seen in the thick preterminal nerve bundles.     

Calcitonin gene-related peptide (CGRP) in the nipple of the rat mammary gland

The distribution of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) was investigated by immunohistochemistry in nipples and mammary glands from lactating and non-lactating rats and compared to the immunoreactivity of other neuropeptides including substance P (SP), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and somatostatin (SOM). The study revealed an extensive innervation of the mammary nipples, in which CGRP-immunoreactive (IR) nerve fibres were abundantly present in the epidermis, dermal connective tissue and intralobular connective tissue of the mammary gland parenchyma. Several of the dermal CGRP-IR fibres seemed to follow blood vessels, or formed ringlet-like structures. The latter were mostly observed in the dermal connective tissue of the nipple from the lactating rat and may have a mechanoreceptive function, e.g. for the suckling stimuli. The location of SP-IR appeared to be comparable to CGRP-IR, but in fewer fibres. Dense NPY-IR networks of nerve fibres were closely associated with the fascicles of smooth musculature in the core of the nipple base. In contrast, VIP-IR fibres were only sparsely present, and SOM-IR was not detected in the mammary nipples. The immunoreactive content of CGRP and SP was determined by radioimmunoassays. The total amount of immunoreactive CGRP was significantly higher in the nipples from the pregnant and the lactating rats when compared to SP. The maximum concentration of CGRP (65.9±4.0 pmol/g) measured in the nipples of the pregnant (day 10) rats exceeded almost ninefold the maximum concentration of SP (7.7±2.0 pmol/g). Thus, the immunoreactive content of CGRP in the nipples confirmed the immunohistochemical observations, and the present results strongly suggest that CGRP is a major neuropeptide in the rat nipple.