A. Luts

Pituitary adenylate cyclase activating peptide is a sensory neuropeptide: Immunocytochemical and immunochemical evidence

Pituitary adenylate cyclase activating peptide (PACAP) is a vasoactive intestinal peptide-like hypothalamic peptide occurring as two variants, PACAP-27 and the C-terminally extended PACAP-38. Immunoreactive PACAP has also been demonstrated in the enteric nervous system and in the innervation of the respiratory tract. We have examined the possibility that PACAP occurs in the sensory nervous system of the rat. Immunocytochemistry revealed PACAP in numerous nerve fibres in the superficial layer of the dorsal horns of the spinal cord, in nerve cell bodies (most of them of small size) of the spinal ganglia and trigeminal ganglia and in nerve fibres running close to and within the surface epithelium in the skin of the nose, the tongue, the larynx-trachea, and the urinary bladder as well as around the ducts of the submandibular gland. In all locations, PACAP co-existed with calcitonin gene-related peptide and substance P, the PACAP-immunoreactive fibres and cell bodies constituting a subpopulation of those storing substance P and/or calcitonin gene-related peptide. Additional PACAP-immunoreactive fibres not associated with epithelia seemed to lack calcitonin gene-related peptide and substance P. Capsaicin treatment reduced the density of PACAP- and calcitonin gene-related peptide/substance P-immunoreactive fibres in the tissues examined. On the whole, the immunocytochemical results agreed with those obtained by radioimmunoassay for PACAP and CGRP. The data favour a role for PACAP in primary sensory neurons.

Plasma exudation as a first line respiratory mucosal defence

A great variety of provocations of the airway mucosa produce extravasation of plasma from the abundant subepithelial microvessels. A plasma exudate has important actions through its volume, its specific and unspecific binding proteins, its enzyme systems, and its potent peptides (of kinin. complement, coagulation, fibrinolysis and other systems). If allowed to operate on the surface of an intact mucosa the plasma exudale would have important roles in normal airway defence. Recent observations in guinea‐pig tracheo‐bronchial airways and in human nasal airways suggest that the mucosal exudation of plasma into the airway lumen is a non‐injurious fully reversible process. Threshold exudative responses thus resulted in the appearance of an ‘unfiltered’ plasma exudate not only in the lamina propria but also on the surface of an undisrupted mucosa. Even after extensive luminal entry of exudate the epithelial lining was intact, as judged by light, fluorescence and electron microscopy. Hence, the epithelial barrier was reversibly permeable when approached from beneath by the plasma exudate. This was a distinct increase in outward permeability, because even during the exudation of plasma the mucosa remained a barrier to luminal solutes. It is possible that the exudate itself, by a slight compressive action on the basolateral aspect of epithelial cells, creates intercellular pathways for its entry into the lumen. Contrary to current beliefs, we propose that plasma exudation should be considered a first line respiratory defence mechanism operating together with other systems of the mucosal surface.

Pituitary adenylate cyclase-activating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract

SummaryPituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PACAP-immunoreactive fibers was seen in the nasal mucosa of guinea pigs. Few to moderate numbers of PACAP-containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the immunoreactive fibers were observed close to small bronchioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guinea pigs. Simultaneous double immunostaining of the respiratory tract of sheep and ferrets revealed that all PACAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.

Occurrence and distribution of calcitonin gene-related peptide in the mammalian respiratory tract and middle ear

SummaryNerve fibres displaying immunoreactivity to calcitonin gene-related peptide (CGRP) are abundantly distributed in the respiratory tract of man, dog, cat, guineapig, rat and mouse. Numerous fine, beaded CGRP fibres were seen in the middle ear mucosa, and a moderate supply was found in the ear drum. In the nasal mucosa and in the wall of the Eustachian tube CGRP fibres occurred around blood vessels, arteries in particular. A conspiciously rich supply of CGRP fibres was seen beneath and within the epithelium. In addition, a few fibres were seen in smooth muscle bundles and close to sero-mucous glands. In the tracheo-bronchial wall CGRP fibres were distributed beneath and within the epithelium, in vascular and non-vascular smooth muscle and sometimes close to small glands. A few CGRP-immunoreactive endocrine-like cells were, in addition, distributed in the tracheal epithelium of cat, rat and mouse. The trigeminal, spinal and nodose ganglia, studied in rats and guinea-pigs, harboured numerous CGRP-immunoreactive nerve cell bodies. The cervical sympathetic ganglia were devoid of immunoreactive neuronal perikarya. Surgical and chemical (6-hydroxydopamine treatment) sympathectomy did not affect the number and distribution of CGRP fibres. The distribution of CGRP fibres in the respiratory tract suggests that CGRP may take part in sensory transmission. In addition, CGRP may affect the regulation of local blood flow, smooth muscle tone and glandular secretion.

Peptide-Containing Nerve Fibers in Human Airways: Distribution and Coexistence Pattern

The occurrence, distribution and coexistence pattern of an array of neuropeptides and tyrosine hydroxylase in the human larynx, trachea, bronchi and lungs were studied by immunocytochemistry. A rich supply of nerve fibers containing vasoactive intestinal peptide (VIP) was seen close to blood vessels, glands and nonvascular smooth muscle. Pituitary adenylate cyclase-activating peptide (PACAP)-containing fibers were numerous among bundles of smooth muscle. Moderate numbers of helospectin-containing nerve fibers were seen in the nonvascular smooth muscle. The majority of neuropeptide Y (NPY)-containing fibers were located in the nonvascular smooth muscle; some fibers also occurred around blood vessels and glands. Substance P (SP) and calcitonin gene-related peptide (CGRP)-containing fibers were generally few and distributed beneath the epithelium, among bundles of smooth muscle, around blood vessels and glands. A conspicuous finding was the lack of SP- and CGRP-containing fibers within the respiratory epithelium. Galanin-containing nerve fibers were moderate in number among bundles of smooth muscle. Tyrosine hydroxylase-containing fibers were numerous around blood vessels and glands. The majority of the VIP-containing nerve fibers present in nonvascular smooth muscle also stored PACAP and helospectin. A subpopulation of VIP-containing fibers in both vascular and nonvascular smooth muscle and around glands stored NPY. Additionally, galanin was found to occur in many VIP-containing fibers located among bundles of smooth muscle.

Pituitary adenylate cyclase activating peptide (PACAP) in guinea-pig lung: distribution and dilatory effects.

The lower airways of guinea-pigs were analyzed for pituitary adenylate cyclase activating peptide (PACAP) using immunocytochemistry. In the trachea a moderate supply of PACAP-immunoreactive nerve fibers occurred around smooth muscle bundles, glands and small blood vessels. In the lung, PACAP-immunoreactive nerve fibers were distributed around small glands and bronchi. A rich supply of PACAP immunoreactive nerve fibers was found around blood vessels in the lungs. PACAP-suppressed smooth muscle responses were analysed using isolated circular segments of trachea, pulmonary arteries and aorta of guinea-pigs. In both airways and arteries PACAP caused a concentration-dependent relaxation of precontracted segments. The maximal relaxation effects were more pronounced in the airways than in the arteries while the order of potency was aorta greater than pulmonary artery greater than trachea. The effect of PACAP was compared to those of acetylcholine (ACh) and vasoactive intestinal peptide (VIP). In the pulmonary artery the vasomotor responses expressed as maximal dilatation had the order: ACh greater than VIP = PACAP while the order of potency was PACAP = VIP greater than ACh. In the trachea, PACAP was slightly more potent than VIP. The relaxatory responses to PACAP in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine, cimetidine and Spantide. Removal of the endothelium abolished PACAP-induced vascular relaxation. Conceivably, PACAP-containing nerve fibers play a role in the regulation of airway resistance and local blood flow.

Peptide-containing nerve fibers in the respiratory tract of the ferret

SummaryThe ferret is widely used in functional and neuromorphological studies on the respiratory tract. We have examined the occurrence and distribution of peptide-containing and adrenergic nerve fibers (using dopamine-β-hydroxylase as a marker). Adrenergic nerve fibers and fibers storing vasoactive intestinal peptide have a widespread distribution along the entire respiratory tract. Adrenergic nerve fibers were found in the lamina propria, as well as around blood vessels and glands and in smooth muscle. Nerve fibers storing vasoactive intestinal peptide occurred in the epithelium, the lamina propria, around blood vessels and glands, and among muscle bundles. Substance P-, neurokinin A- and calcitonin gene-related peptide-containing nerve fibers predominated beneath and within the epithelium along the entire respiratory tract. Neuropeptide Y-containing nerve fibers were prominent among smooth muscle bundles and around glands. The blood vessels in the wall of the airways were richly supplied with peptidecontaining nerve fibers and adrenergic fibers. Ganglia located over the outer or dorsal surface of the tracheal wall harbored vasoactive intestinal peptide-containing nerve cell bodies. Substance P and neurokinin A invariably coexisted in the same nerve fibers. Further, coexistence of substance P/neurokinin A and calcitonin gene-related peptide was observed in the nerve fibers associated with the epithelium. Vasoactive intestinal peptide, neuropeptide Y and occasionally also substance P coexisted in the population of nerve fibers associated with blood vessels and smooth muscle. Many adrenergic nerve fibers contained neuropeptide Y.

PACAP, a VIP-like peptide, in neurons of the esophagus.

The lower esophagus of guinea-pig, cat, sheep and man was analyzed for pituitary adenylate cyclase activating peptide (PACAP), a novel vasoactive intestinal peptide (VIP)-like peptide, using immunocytochemistry and radioimmunoassay. PACAP-immunoreactive nerve fibers were numerous in the longitudinal and circular muscle layers of sheep and man, moderate in numbers in cat, while being few in the esophagus of guinea-pig. A few PACAP-immunoreactive nerve cell bodies and numerous nerve fibers were seen in the myenteric ganglia of the esophagus of cat, sheep and man. In the lower esophagus of cat, sheep and man all PACAP-containing nerve cell bodies and nerve fibers stored VIP. The results of radioimmunoassay of PACAP in extracts of specimens from man were in good agreement with the immunocytochemical findings. High performance liquid chromatography revealed one major peak of PACAP-like immunoreactivity in extracts of human esophagus. We suggest that neuronal PACAP may serve to modulate motor activity and secretion in the lower esophageal sphincter region.

Subepithelial Hydrostatic Pressure May Regulate Plasma Exudation across the Mucosa

This study demonstrated in guinea pig tracheal tubes in vitro that small increases in serosal hydrostatic pressure caused significant mucosal crossing of serosal macromolecules. Reversibility and repeatability of this passage agree with inflammatory stimulus-induced appearance of exuded plasma in airway lumen in vivo. Bradykinin, histamine, and terbutaline, which induce and inhibit, respectively, plasma exudation in vivo, were without effect on the present in vitro permeability. Carbachol, similar to histamine, contracted the trachea, and did not increase, but rather decreased the pressure-induced luminal entry of serosal macromolecules. It is proposed that a plasma-exudation-induced hydrostatic pressure load transiently separates epithelial cells, providing a direction-selective and non-injurious intercellular pathway for passage of bulk plasma exudate into the airway lumen. This mechanism would allow potent plasma protein systems to operate on mucosal surfaces at sites of insults without compromising the mucosa as a barrier to luminal solutes.

Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides

SummaryThe epithelium of the airways is rich in endocrine cells containing serotonin and/or a wide variety of regulatory peptides. These cells usually occur in clusters in the lungs but are also found scattered in the larynx and trachea. In the present study, endocrine cells in the airways of mouse, rat, hamster, guinea pig, pig, sheep and squirrel monkey were examined for the presence of serotonin, helodermin-like peptides and other regulatory peptides using immunocytochemistry and radioimmunoassay. In addition, we looked for the protein gene product 9.5 (PGP), which occurs in many peptide hormone-producing endocrine cells in the body. Both clustered and scattered endocrine cells in the airways were found to display coexistence of serotonin and peptides, such as a helodermin-like peptide, calcitonin and calcitonin gene-related peptide (CGRP). The PGP-immunoreactive cells were numerous and included elements containing serotonin and/or regulatory peptides. An additional PGP-immunoreactive endocrine cell population lacked serotonin and regulatory peptides. Helodermin-immunoreactive material was demonstrated in endocrine cells of the airways in the mouse and hamster but not in any of the other species studied. Serotonin was an endocrine cell constituent in all the species studied. Calcitonin and CGRP could be demonstrated by immunocytochemistry in the mouse, rat, and hamster, but not in the guinea pig, sheep, pig and monkey. In the hamster airways double immunostaining indicated that the helodermin-like peptide occurred in a subpopulation of the CGRP- and serotonin-containing cells. Most of the CGRP-containing cells stored serotonin; some of them also contained calcitonin. The chemical coding of these cells resembled that of the thyroid C cells.