VIP like immunoreactive nerves in human respiratory tract. Light and electron microscopic study

The present study provides light and electron microscopical evidence of Vasoactive Intestinal Peptide - (VIP) like immunoreactive nerves in human lower respiratory tract. Peroxidase antiperoxidase (PAP) technique was used to localize VIP-like immunoreactivity light microscopically and ultrastructurally. Under light microscopy, VIP-like immunoreactive nerves were observed in the smooth muscle layer of secondary bronchi to small bronchioli, and in bronchial glands. In addition, positive immunoreactive nervous network to VIP was found around nerve cell bodies in small microganglia. The bronchial epithelium of airway tract did not receive any VIP positive nerve fibers. Ultrastructurally VIP-like positive immunoreaction was localized in large granular vesicles ranging from 90 to 210 nm. Usually VIP-like positive immunoreactive nerve profiles contained several immunoreactive large vesicles (100-210). However, nerve profiles containing only a few positive large vesicles (80-150) were also observed. Under electron microscopy VIP-positive nerve profiles corresponded ultrastructurally to nerve profiles containing large granular vesicles observed in conventional electronmicroscopy. The present study provides new information about the innervation of human lower airway tract and widens the concept of their functional regulation on the anatomical basis reported here.     

Catecholamine- and acetylcholinesterase-containing nerves in human lower respiratory tract

Summary The innervation of human lower respiratory tract was studied with special emphasis on airways with sodium-potassium glyoxylic acid (SPG) and acetylcholinesterase (AChE) methods to demonstrate catecholamine-containing and acetylcholinesterase-containing nerve fibers. AChE-method revealed a rich network of cholinesterase positive nerves both inside the bronchial glands where they run around and between the acini, and the airway smooth muscle from secondary bronchi to terminal bronchioli. No AChE-positive fibers were found in connection with the blood vessels or within the epithelium of bronchi or bonchioli. The AChE-positive nerve fibers in bronchial smooth muscle greatly outnumbered those containing catecholamine. The SPG-method revealed the presence of adrenergic nerves from the level of secondary bronchi to that of terminal bronchioli. These nerve fibers were most abundant in bronchial glands, where their amount was equal and distribution similar to those of AChE-containing nerve fibers. Outside the glands adrenergic fibers were constantly seen in connection with the bronchial blood vessels in connective tissues surrounding bronchi. A few nerve fibers were also present in airway smooth muscle from the secondary bronchi to terminal bronchioli.     

Innervation of the pancreas by vasoactive intestinal polypeptide (VIP) immunoreactive nerves

The vasoactive intestinal polypeptide (VIP) has been shown to exert effects on endocrine and exocrine pancreatic secretion. Immunocytochemistry reveals that VIP immunoreactive nerves occur in the porcine, canine, feline and avian pancreas. In the pancreas of pig and cat VIP nerves are abundant around non-immunoreactive nerve cell bodies of the intrapancreatic ganglia but scarce in the islets and in the exocrine parenchyma. In the dog pancreas, however, the intrapancreatic ganglia contain strongly immunoreactive VIP nerve cell bodies which give off axons that seem to heavily innervate vessels as well as endocrine and exocrine cells. We suggest that in the pig and cat the pancreatic VIP nerves mainly affect the activity of a second type of intrapancreatic neuron, whose transmitter is unknown, whereas in the dog pancreas VIP nerves directly contact their putative effector structures.     

Distribution and characterisation of immunoreactive somatostatin in human gastrointestinal tract

Immunoreactive somatostatin (IRS) was measured in acid extracts of human gastrointestinal tissue. The highest levels were found in the duodenum, pancreas, jejunum and stomach with lower levels in the ileum and colon. In the antrum, pylorus, duodenum and pancreas the main peak of IRS (1.6K IRS) coeluted with synthetic somatostatin-14 on both gel filtration chromatography and HPLC. In the body of stomach, jejunum, ileum and colon, a large peak coeluting with synthetic somatostatin-28 (3.5K IRS) on both chromatographic systems was also identified, while minor peaks of IRS assigned molecular weights of 6000 (6K) and greater than 15 000 (15K) were seen in some extracts. The total IRS content and pattern of molecular forms were similar in tissues obtained from adults at surgery or rapid post mortem, and in tissue taken from human fetuses after prostaglandin termination of pregnancy. When tissues were divided into mucosal and muscle layers, greater than 90% of the IRS was in the mucosa with less than 10% in the muscle layer. In the muscle layer the IRS was almost entirely the 1.6K form in all tissues. Immunohistochemical studies showed the IRS in the mucosa to be localised in endocrine-type cells, while in the muscle layer the IRS is present in nerve fibres and neurones of the myenteric plexus. It is suggested that (1) different mechanisms may control the biosynthesis of somatostatin-14 and somatostatin-28 in mucosal cells in different parts of the gut, (2) different biosynthetic controls may operate in endocrine-like and neuronal cells in the same region of the gut.     

Occurrence and distribution of neuropeptide-Y-immunoreactive nerves in the respiratory tract and middle ear

Summary Nerve fibres displaying neuropeptide-Y (NPY) immunoreactivity are abundantly distributed in the respiratory tract of cats, guinea-pigs, rats and mice. Fine beaded NPY fibres were seen in whole-mount spreads of the middle-ear mucosa. In the nasal mucosa and in the wall of the Eustachian tube NPY fibres were numerous around arteries and arterioles but sparse in the vicinity of veins; single fibres were found close to the acini of seromucous glands. In the tracheobronchial wall NPY fibres occurred in the proximity of blood vessels, in the subepithelial layer and in the smooth muscle. Surgical and chemical (6-hydroxydopamine treatment) sympathectomy resulted in disappearance of adrenergic and NPY-containing nerve fibres in the nasal mucosa. Sequential staining with antibodies against dopamine--hydroxylase (DBH) and NPY revealed that DBH and NPY occur in the same perivascular nerve fibres in the nasal mucosa. The distribution of NPY fibres in the respiratory tract suggests multiple functions of NPY, such as regulation of local blood flow, glandular secretion and smooth muscle activity.     

Ontogeny of immunoreactive glicentin in the human gastrointestinal tract and endocrine pancreas

The gestational time of appearance and distribution of immunoreactive glicentin was compared to that of immunoreactive glucagon in the gastrointestinal tract and endocrine pancreas of human fetuses, aged between 5 and 24 weeks, by an indirect immunoperoxidase method. With the glicentin antiserum No. R 64, the first immunoreactive cells were detected at the 10th week of gestation in the oxyntic mucosa and proximal small intestine, at the 8th week in the ileum and at the 12th week in the colon. In the endocrine pancreas, the first immunoreactive cells were observed as early as 8 weeks within the walls of the primitive pancreatic ductules. At a more advanced stage of development (12 weeks), they were found interspersed among the islet cell clusters and still later (16 weeks) inside the recognizable islets of Langerhans. With the glucagon antiserum No. GB 5667, no immunoreactive cells were demonstrated in the gastrointestinal tract whatever the age of the fetuses. In the endocrine pancreas, the first immunoreactive cells were observed at the 8th week of gestation in the pancreatic parenchyma. The distribution of glucagon-containing cells in the pancreas was similar to that of glicentin immunoreactivity throughout ontogenesis. In the pancreatic islets of one 18-week-old human fetus, the study of consecutive semithin sections treated by both antisera showed that the same cells were labelled. The significance of these findings concerning the role of glicentin as a glucagon precursor is discussed.     

Flow simulation in the human upper respiratory tract

Computer simulations of airflow patterns within the human upper respiratory tract (URT) are presented. The URT model includes airways of the head (nasal and oral), throat (pharyngeal and laryngeal), and lungs (trachea and main bronchi). The head and throat morphology was based on a cast of a medical school teaching model; tracheobronchial airways were defined mathematically. A body-fitted three-dimensional curvilinear grid system and a multiblock method were employed to graphically represent the surface geometries of the respective airways and to generate the corresponding mesh for computational fluid dynamics simulations. Our results suggest that for a prescribed phase of breath (i.e., inspiration or expiration), convective respiratory airflow patterns are highly dependent on flow rate values. Moreover, velocity profiles were quite different during inhalation and exhalation, both in terms of the sizes, strengths, and locations of localized features such as recirculation zones and air jets. Pressure losses during inhalation were 30-35% higher than for exhalation and were proportional to the square of the flow rate. Because particles are entrained and transported within airstreams, these results may have important applications to the targeted delivery of inhaled drugs.     

Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man

Summary The occurrence and origin of substance P (SP)-immunoreactive (IR) nerves in the lower respiratory tract was studied by means of immunohistochemistry in the guinea-pig, rat, cat and man. In addition, biopsies from human material were also analysed by radioimmunoassay. SP-IR nerves were seen in four principal locations: 1) under or within the lining epithelium, 2) around blood vessels, 3) within the bronchial smooth muscle layer, and 4) around local tracheobronchial ganglion cells. Ligation experiments combined with capsaicin pretreatments indicated that all SP-IR nerves in the respiratory tract are sensory. The trachea seems to be mainly supplied by the vagal nerves, while intrapulmonary bronchi and blood vessels receive SP-IR nerves of both vagal and non-vagal (spinal) origin. SP-IR nerves were also found in the human bronchi with principally similar location as in the guinea-pig. The levels of SP-IR in the trachea and peripheral bronchi of man were about 3–4 pmol/g, which is in the same range as the content of corresponding tissues from the guinea-pig.In conclusion, the present experimental findings of SP-IR nerves in the lower respiratory tract in both experimental animals and man support the functional evidence for the importance of SP in the vagal and non-vagal (spinal) control of bronchial smooth muscle tone and vascular permeability.     

Ontogeny of immunoreactive CCK and VIP in pig brain and gut

The concentrations and hormonal forms of CCK and VIP have been determined in extracts of the brain and duodenum of the developing and adult pig. In methanol extracts of the brain cortex, the single hormone form, CCK8, increased from 130 +/- 20 (Mean +/- SEM) pmol/g at birth to an adult level of 300 +/- 50 pmol/g. In acid extracts of brain, the predominant immunoreactive form had N-terminal immunoreactivity and increased from 240 +/- 20 pmol/g at birth to an adult level 490 +/- 30 pmol/g; the C-terminal immunoreactivity was about 10-fold lower. The concentrations and hormonal forms of immunoreactive CCK in duodenal extracts did not appear to be age-related. C-terminal immunoreactivity in methanol extracts averaged 140 +/- 20 pmol/g and in acid extracts 240 +/- 60 pmol/g. The concentration of N-terminal immunoreactivity in acid extracts averaged 490 +/- 70 pmol/g. The VIP concentrations in acid extracts of the brain cortex was 13.5 +/- 2 pmol/g at birth and rose gradually to 30 +/- 9 pmol/g in the adult; in duodenal extracts it was 240 +/- 18 pmol/g at birth and 195 +/- 38 pmol/g in the adult. These results are in marked contrast with the ontogeny of these hormones in the rat in which brain concentrations of CCK and VIP in the neonate are less than 10% of adult levels and in which there are age-related changes in the content of these hormones in the duodenum as well.     

Arginine-vasopressin immunoreactive material in the gastrointestinal tract

Summary Like many other neuropeptides, vasopressin is not confined to the hypothalamic neurohypophysial system. Furthermore, vasopressin was found to be a potent vasoconstrictor in the rat jejunum, reducing myenteric artery flow. These associations were the basis of this investigation on the presence of vasopressin in the gastrointestinal (GI) tract by both RIA and immunohistochemistry.Portions of the gastrointestinal tract and pancreatic islets of the rat were extracted with 0.1N HCl for RIA measurements of AVP content. Similar portions from the male cat GI tract were used for immunohistochemistry studies.Acid extracts of the GI tract were found to contain immunoreactive AVP with the highest concentration (pg/mg protein) in the fundus portion of the stomach (15.0±1.6) and slightly lower values down along the antrum-pylorus portion (6.7±0.6), proximal jejunum (8.6±0.2), distal ileum (9.7±0.3) and colon (11.9±0.5). In the pancreatic islets the concentration was much higher (72.0 pg/mg protein). The extract inhibition curves showed parallelism with the appropriate standard preparation of AVP in the specific RIA.Immunohistochemical localization showed IR-AVP in the nerve fibers around the myenteric plexus of the second portion of the duodenum. It was also found in fibers starting from where the myenteric plexus goes through the layer of muscle fibers, penetrating the submucosa and duodenal mucosa, ending near the capillaries situated along the basal side of the villous epithelium cells. Similar IR-AVP activity was found in cells located in the mucosal epithelium of the duodenum, jejunum, ileum, colon and rectum.These results show that the gastrointestinal tract of different species and pancreatic islets of the rat are a rich source of immunoreactive neurohypophysial AVP. Because of its distribution, this peptide might have some physiological significance in intestinal circulatory regulation.     

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/ VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22–79, PHM, prepro-VIP 111–122, VIP and prepro-VIP 156–170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single-or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.     

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.     

Arginine-vasopressin immunoreactive material in the gastrointestinal tract

Like many other neuropeptides, vasopressin is not confined to the hypothalamic neurohypophysial system. Furthermore, vasopressin was found to be a potent vasoconstrictor in the rat jejunum, reducing myenteric artery flow. These associations were the basis of this investigation on the presence of vasopressin in the gastrointestinal (GI) tract by both RIA and immunohistochemistry. Portions of the gastrointestinal tract and pancreatic islets of the rat were extracted with 0.1 N HCl for RIA measurements of AVP content. Similar portions from the male cat GI tract were used for immunohistochemistry studies. Acid extracts of the GI tract were found to contain immunoreactive AVP with the highest concentration (pg/mg protein) in the fundus portion of the stomach (15.0 +/- 1.6) and slightly lower values down along the antrum-pylorus portion (6.7 +/- 0.6), proximal jejunum (8.6 +/- 0.2), distal ileum (9.7 +/- 0.3) and colon (11.9 +/- 0.5). In the pancreatic islets the concentration was much higher (72.0 pg/mg protein). The extract inhibition curves showed parallelism with the appropriate standard preparation of AVP in the specific RIA. Immunohistochemical localization showed IR-AVP in the nerve fibers around the myenteric plexus of the second portion of the duodenum. It was also found in fibers starting from where the myenteric plexus goes through the layer of muscle fibers, penetrating the submucosa and duodenal mucosa, ending near the capillaries situated along the basal side of the villous epithelium cells. Similar IR-AVP activity was found in cells located in the mucosal epithelium of the duodenum, jejunum, ileum, colon and rectum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive-intestinal-polypeptide (VIP)-like immunoreactive cells in the skull base of rats

Summary We investigated the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactive cell bodies in relation to the major cerebral and internal carotid arteries at the skull base in rats. Acetylcholinesterase (AChE) histochemistry was also applied to investigate the localization of this enzyme. VIP staining revealed a few positive cell bodies in nerves close to the internal carotid artery at the base of the skull as well as in the cerebral arterial wall. Ganglion-like cell bodies were detectable within the greater superficial petrosal (GSP) nerve. AChE activity was observed in VIP-like immunoreactive cell bodies along the whole of the GSP nerve. These cell bodies in the GSP nerve may give rise to at least some of the perivascular VIP-and AChE-containing nerves of the internal carotid arteries at the base of the skull.     

VIP release from enteric nerves is independent of extracellular calcium

The release of endogenous vasoactive intestinal polypeptide (VIP) from enteric nerves of isolated rat ileum and the role of extracellular calcium on the release mechanism have been investigated. Evaluation of simultaneous release of endogenous acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) from enteric nerves was used to establish the reliability of the technique. Electrical field stimulation of the ileal preparation induced an increase in the release of endogenous ACh, ATP and VIP. The evoked, but not the basal, release of these substances was blocked by tetrodotoxin (TTX), indicating that the release was a result of nerve stimulation. However, while increase in release of ACh and ATP during nerve stimulation was suppressed in Ca2+-free medium and by the addition of the Ca2+ channel blocker cadmium, nerve-mediated VIP release was unaffected. Further, while K+-depolarization induced release of ACh and ATP from the ileal preparations, it did not lead to an increase in the release of VIP. These results demonstrate that, unlike ACh and ATP release, release of endogenous VIP from enteric nerves is independent of extracellular calcium. The implications of these results in terms of the mechanism of transmitter release in the gastrointestinal tract are discussed.     

Cyclooxygenase metabolites are compartmentalized in the human lower respiratory tract

Cyclooxygenase metabolites of arachidonic acid are thought to play an important role in the regulation of diverse physiological functions in the lung. Although the concentration of these metabolites required to have effects is several orders of magnitude greater than the concentration of these mediators in the blood, it has been postulated that local concentrations within tissues are much higher. In a direct test of this hypothesis, the concentrations of the cyclooxygenase products of arachidonic acid including prostaglandin (PG) E, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane B2, were measured in a specialized tissue compartment, the epithelial surface of the lower respiratory tract. The concentration of these mediators within this compartment was 50- to 80-fold greater than concurrent blood levels and are sufficient to likely have physiological effects. Thus the epithelial surface of the lower respiratory tract represents a specialized compartment with high local levels of cyclooxygenase products of arachidonic acid.     

Dopamine beta-hydroxylase like immunoreactive cells in the frog taste disc

We immunohistochemically examined the existence of dopamine beta-hydroxylase (DBH), a noradrenalin (NA)-synthesizing enzyme from dopamine, in the taste disc of frog, Rana catesbeiana. DBH-like immunoreactive cells were located in the intermediate layer in the taste disc; the cells showed an apical process reaching the surface of the disc and one or several basal processes. Cells with a thick apical process and those with a thin apical process were both immunoreactive: these cells corresponded to type II and III receptor cells of the frog taste disc. Immunoreactive granules were observed in the cytoplasm of those cells. In the frog taste disc, only type III cells are reported to have afferent synapses with the nerve via basal processes but those basal processes have not been reported in type II cells. In the present study, we found that type II-like cells possessed a long basal process extending toward the basal lamina. Mucous (type Ia) cells, wing (type Ib) cells, and glia-like sustentacular (type Ic) cells were all immunohistochemically unreactive. The present observations support the argument that NA (or adrenalin) may work as a chemical transmitter in the frog taste organ.     

Growth of respiratory syncytial virus in primary epithelial cells from the human respiratory tract

Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in infants and children. To study RSV replication, we have developed an in vitro model of human nasopharyngeal mucosa, human airway epithelium (HAE). RSV grows to moderate titers in HAE, though they are significantly lower than those in a continuous epithelial cell line, HEp-2. In HAE, RSV spreads over time to form focal collections of infected cells causing minimal cytopathic effect. Unlike HEp-2 cells, in which wild-type and live-attenuated vaccine candidate viruses grow equally well, the vaccine candidates exhibit growth in HAE that parallels their level of attenuation in children.