Immunohistochemical localization of substance P, vasoactive intestinal polypeptide and gastrin-releasing peptide in vas deferens and seminal vesicle, and the effect of these and eight other neuropeptides on resting tension and neurally evoked contractile activity

Immunohistochemical studies of the vas deferens and seminal vesicle of mouse, guinea-pig, and rabbit showed the presence of nerve fibres containing vasoactive intestinal polypeptide (VIP), substance P (SP), and gastrin-releasing peptide (GRP) supplying the smooth muscle layers as well as blood vessels. The nerve supply was better developed in the seminal vesicle than in the vas deferens. The motor activity of the vas deferens and seminal vesicle of the guinea-pig was studied in vitro. The vas deferens responded to transmural electrical stimulation with a twitch followed by a slow contraction. The twitch was blocked by guanethidine and tetrodotoxin, but not by atropine, propranolol, phenoxybenzamine, or fluphenazine. The slow contraction exhibited features of an alpha-receptor-mediated response. SP, physalaemin and eledoisin contracted the smooth muscle and also potentiated the twitch response to electrical nerve stimulation in a concentration-dependent manner. The SP blocking agent, (D-Pro2,D-Trp7,9)-SP, affected neither the resting tension nor the response to electrical stimulation. It is therefore suggested that the SP fibres act mainly prejunctionally. VIP, Leu-enkephalin, cholecystokinin octapeptide (CCK-8), angiotensin II, vasopressin, neurotensin, bombesin, and GRP had no effect on either the resting tension or the response to electrical nerve stimulation. The seminal vesicle responded to electrical stimulation with a contraction which was unimpaired by atropine, propranolol, phenoxybenzamine, and guanethidine, but abolished by tetrodotoxin. Hence, this contraction is mediated by a non-adrenergic, non-cholinergic neurotransmitter. Bombesin, GRP, SP, physalaemin and eledoisin contracted the smooth muscle and potentiated the response to electrical stimulation. VIP, Leu-enkephalin, CCK-8, angiotensin II, vasopressin, and neurotensin had no effect on the resting tension or on the response to transmural electrical stimulation. The SP antagonist abolished the contraction elicited by SP but did not influence the response to nerve stimulation. The results suggest that the SP and GRP nerves may have prejunctional and facilitating postjunctional effects in the seminal vesicle.     

Peptide histidine methionine and vasoactive intestinal peptide: occurrence and relaxant effect in the human female reproductive tract

The occurrence of the neuropeptides peptide histidine methionine (PHM) and vasoactive intestinal peptide (VIP) in the human female genital tract was studied by means of immunochemistry and radioimmunoassay in combination with gel chromatography. In addition, the effect of PHM and VIP on smooth muscle activity was investigated in vitro. The regional distribution of PHM as determined by radioimmunoassay correlated with that of VIP. This finding agreed with the immunohistochemical data, which, in addition, provided evidence for colocalization of the two peptides in nerve fibers. These fibers were most abundant in the vagina and the uterine cervix, where they seemed to innervate blood vessels, smooth muscle, and epithelial cells. The concentrations of immunoreactive PHM and VIP were found to be similar in all areas except in the vagina, where the PHM concentration was fourfold that of VIP. Gel chromatography of vaginal extract revealed a high concentration of a C-terminally extended form of PHM, suggesting differential processing pathways of the VIP precursor. Both PHM and VIP inhibited, in a dose-dependent manner, the smooth muscle activity in strips from the Fallopian tube and the myometrium. Administered in combination, PHM and VIP had an additive effect and displayed the same efficacy as VIP alone, indicating that the peptides act via a common receptor.     

Non-amidated forms of VIP (glycine-extended VIP and VIP-free acid) have full bioactivity on smooth muscle

The aim of the present study was to elucidate the importance of the C-terminal amide group for the biological activity of vasoactive intestinal peptide (VIP). Two synthetic peptides lacking the amide group: VIP having a carboxyl group at the C-terminus and the intermediate biosynthetic precursor, glycine-extended VIP were compared with VIP itself regarding the ability to inhibit spontaneous activity in smooth muscle strips from rat stomach and human Fallopian tube. Both the glycine-extended VIP and VIP having a carboxyl group at the C-terminus caused a significant and dose-dependent inhibition of smooth muscle activity and displayed dose-response curves similar to VIP. The potencies of the VIP variants did not differ significantly from that of VIP. Thus, alpha-carboxyamidation of VIP is not a prerequisite for biological activity.     

Peptide-containing neurons intrinsic to the gut wall

Summary Nerve fibers containing substance P, VIP, enkephalin or somatostatin are numerous in the porcine gut wall. They are particularly numerous in the submucosal and myenteric plexuses where peptide-containing cell bodies are also observed. Peptide-containing nerve fibers occur also in the vagus nerves, suggesting that the gut receives an extrinsic supply of peptidergic nerves. The extrinsic contribution to the peptide-containing nerve supply of the gut wall has not yet been quantitatively assessed. In an attempt to clarify this question pigs were subjected to bilateral subdiaphragmatic vagotomy. Another group of animals was subjected to complete extrinsic denervation by autotransplantation of a jejunal segment. The pigs were killed at various time intervals after the operations; the longest time interval studied was four months. Following vagotomy the innervation pattern of the jejunum appeared completely unaffected. Following complete extrinsic denervation the adrenergic nerve fibers disappeared, while peptide-containing and acetylcholinesterase-positive nerve fibers remained apparently unaltered. This was confirmed chemically in the case of substance P.The motor activity of smooth muscle from the jejunum was studied in vitro. At low stimulation frequencies the smooth muscle from control jejunum responded by relaxation; upon cessation of stimulation a contraction occurred. With increasing stimulation frequencies the duration of the relaxation decreased; at high frequency stimulation only a contraction was recorded. In the autotransplant low frequency stimulation induced no or only a weak relaxation; high frequency stimulation induced contraction. After cholinergic and adrenergic blockade, the muscle responded with relaxation at all frequencies; the response was similar in innervated and denervated specimens. On the whole, the effects of extrinsic denervation on the motor activity of smooth muscle from porcine jejunum were minor, possibly reflecting the high degree of autonomy of the gut.     

Effects of VIP and NO on the motor activity of vascularly perfused rat proximal colon

The effects of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) on the motor activity of the rat proximal colon were examined in an ex vivo model of vascularly perfused rat proximal colon. VIP reduced motor activity and this inhibitory effect was not altered by either atropine, hexamethonium, tetrodotoxin (TTX) nor TTX plus acetylcholine (ACh), but was completely antagonized by NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) and by VIP receptor antagonist, VIP(10-28). These results suggest that VIP may exert a direct inhibitory effect on the motor activity of the rat proximal colon via a VIP receptor located on the smooth muscle and this effect is mediated by NO but not by cholinergic pathways. Atropine and hexamethonium reduced but ACh stimulated motor activity and the effect of ACh was not changed by TTX, suggesting that the cholinergic pathway may exert a direct stimulatory effect on motor activity. Single injection of TTX, VIP(10-28) or L-NA induced a marked increase in motor activity, suggesting that the motor activity of rat proximal colon is tonically suppressed by VIP and NO generating pathways, and elimination of inhibitory neurotransmission by TTX may induce an abnormal increase of the motor activity. The interaction between VIP and NO in regulation of motor activity was further examined by a measurement of NO release from vascularly perfused rat proximal colon. Results showed that NO release was significantly increased during infusion of VIP and this response was reversed by L-NA. These results suggest that VIP generating neurons may inhibit colonic motility by stimulating endogenous NO production in either smooth muscle cells or nerve terminals.     

Nerves containing substance P,vasoactive intestinal polypeptide,enkephalin or somatostatin in the guinea-pig taenia coli

Summary The guinea-pig taenia coli is rich in peptide-containing nerves. Nerve fibres containing substance P (SP), vasoactive intestinal peptide (VIP), or enkephalin, were numerous in the smooth muscle while somatostatin fibres were very few. Nerve fibres displaying SP or VIP immunoreactivity were numerous in the myenteric plexus. Enkephalin nerve fibres were fairly numerous in the plexus while somatostatin nerve fibres were sparse. Nerve cell bodies containing immunoreactive SP or VIP were regularly seen in the plexus. Delicate varicose elements of the different types of nerve fibres were found to ramify around nerve cell bodies in a manner suggestive of innervation.In the electron microscope the various peptide-storing nerve fibres (i.e., elements containing SP, VIP or enkephalin) were found to contain a varying number of fairly large, electron-opaque vesicles in the varicose swellings. These vesicles represent the storage site of the neuropeptides.The isolated taenia coli responded to electrical nerve stimulation with a contraction. After cholinergic and adrenergic blockade the contractile response was replaced by a relaxation followed by a contraction upon cessation of stimulation. SP contracted the taenia while VIP caused a relaxation. The enkephalins raised the resting tension slightly while somatostatin had no effect. These observations are compatible with a role for SP as an excitatory neurotransmitter and for VIP as an inhibitory one, and with the view that both SP neurones and VIP neurones act as motor neurones. In preparations contracted by SP the electrically induced contractions were reduced in amplitude while the electrically induced relaxations seen after adrenergic and cholinergic blockade were enhanced in amplitude. In preparations relaxed by VIP there was an increased contractile response to electrical stimulation, while in the atropine + guanethidine-treated preparation the electrically induce relaxations were reduced in amplitude. The enkephalins reduced the contractile response to electrical stimulation, while somatostatin induced a very small reduction in the amplitude of such responses. These observations suggest that SP neurones and VIP neurones may play additional roles as interneurones. Somatostatin neurones probably act as interneurones. Enkephalin-containing fibres may serve to modify the release of transmitter from other nerves in the smooth muscle, perhaps through axo-axonal arrangements. Alternatively, the enkephalin nerve fibres in the smooth muscle are afferent elements involved in mediating sensory impulses to the myenteric plexus.     

Interstitial Cajal-like cells of human Fallopian tube express estrogen and progesterone receptors

Cells of the female reproductive tract are subject to hormonal control via sex steroid genomic receptors expressed at nuclear level. We previously showed that interstitial Cajal-like cells (ICLC) of human myometrium expressed estrogen and progesterone receptors (ER/PR). Our aim, based on these results, was to see if ER and/or PR could be found also in tubal ICLC. Indeed, we present here immunohistochemical evidence that ICLC of human Fallopian tube (isthmic region) have such receptors. Stromal ICLC, as well as ICLC among smooth muscle layers, were identified in tissue sections by their morphological features (e.g. several very long, moniliform, prolongations of cell body) as well as by c-kit positivity, vital staining with methylene blue or silver impregnation. Additional evidence was provided by sequential staining for c-kit and for PR on the same cell, by ‘sandwich method’. In vitro, the 4th passage cell cultures from Fallopian tube muscularis exhibiting ICLC morphology showed the presence of ER-alpha and/or PR-A by immunofluorescence. In conclusion, our data suggest that ICLC could function as steroid sensors, and might be implicated in Fallopian tube motility (via gap junctions or juxta- and/or paracrine mechanisms).     

Vasoactive intestinal polypeptide (VIP) inhibits neurally evoked smooth muscle activity of rat uterine cervix in vitro

VIP inhibits the spontaneous motor activity (including tone) in isolated preparations of uterine cervix from oophorectomized rats, but has no direct effect on preparations from estrogen-treated animals. Electrical field stimulation of nerves in the tissue evokes a contractile response that is inhibited by VIP in a concentration-dependent manner. The neuronal link probably involves a cholinergic mechanism, since the contraction is blocked by atropine. The results suggest the presence of VIP receptors both in cholinergic nerves and in smooth muscle cells of the rat uterine cervix.     

Mechanism of noncholinergic excitation of canine ileal circular muscle by motilin.

In the isolated perfused canine ileal segment, exogenous motilin infused for 9 min, at concentrations from 10(-10) M and 10(-8) M, increased circular muscle motility concomitant with inhibiting tonic VIP release, maximum at 10(-8) M. Both effects increased with increasing motilin concentrations. Atropine 10(-7) M pretreatment did not alter these responses. Naloxone 10(-7) M pretreatment eliminated both the increase in motor activity and the inhibition of VIP levels. Thus the nonmuscarinic neural pathway responsible for motor activation by motilin probably involves the stimulation of release of opiates, which in turn inhibit the release of VIP. Reduction of tonic inhibition of the muscle by continuous VIP release may in part account for increases in motor activity induced by motilin.     

Sex hormone binding globulin expression and colocalization with estrogen receptor in the human Fallopian tube.

The detection of sex hormone binding globulin (SHBG) or SHBG mRNA in several sex steroid target tissues, has raised the possibility that SHBG modulates the action of sex steroids outside the vascular compartment. The presence of SHBG mRNA was investigated by RT-PCR in the poly (A+) RNA fraction of the human Fallopian tube. Human and rat liver were used as positive and negative control tissues, respectively. The electrophoretic analysis of the amplified PCR products showed bands at 219 bp, corresponding to the expected size of the SHBG cDNA, in the Fallopian tube and human liver but not in rat liver, indicating that SHBG might be synthesized by the Fallopian tube. The cellular localization of SHBG and of estrogen receptor (ER) was examined by immunohistochemistry in consecutive sections of Fallopian tube tissues for individual staining or double immunostaining in the same section. Specific immunostaining of SHBG was present in the epithelial, vascular and muscle cells of the ampullary and isthmic region. In epithelial cells, immunoreactive SHBG was present in the apical end with the highest concentration close to the luminal membrane. The ER was localized in the nuclei of epithelial, stromal and muscle cells of the ampulla and isthmus. Double immunostaining showed that SHBG and ER are colocalized principally in epithelial cells of the ampulla and in muscle cells of the isthmus. In conclusion, the detection of SHBG and SHBG mRNA and the localization of SHBG in estrogen target cells was shown. These findings support the hypothesis that SHBG might regulate sex steroid action at the tissue level.     

Maturation of respiratory reflex responses in the piglet

Stimulation of chemo-, irritant, and pulmonary C-fiber receptors reflexly constricts airway smooth muscle and alters ventilation in mature animals. These reflex responses of airway smooth muscle have, however, not been clearly characterized during early development. In this study we compared the maturation of reflex pathways regulating airway smooth muscle tone and ventilation in anesthetized, paralyzed, and artificially ventilated 2- to 3- and 10-wk-old piglets. Tracheal smooth muscle tension was measured from an open tracheal segment by use of a force transducer, and phrenic nerve activity was measured from a proximal cut end of the phrenic nerve. Inhalation of 7% CO2 caused a transient increase in tracheal tension in both age groups, whereas hypoxia caused no airway smooth muscle response in either group. The phrenic responses to 7% CO2 and 12% O2 were comparable in both age groups. Lung deflation and capsaicin (20 micrograms/kg iv) administration did not alter tracheal tension in the younger piglets but caused tracheal tension to increase by 87 +/- 28 and 31 +/- 10%, respectively, in the older animals (both P less than 0.05). In contrast, phrenic response to both stimuli was comparable between ages: deflation increased phrenic activity while capsaicin induced neural apnea. Laryngeal stimulation did not increase tracheal tension but induced neural apnea in both age groups. These data demonstrate that between 2 and 10 wk of life, piglets exhibit developmental changes in the reflex responses of airway smooth muscle situated in the larger airways in response to irritant and C-fiber but not chemoreceptor stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

VIP and PHM and their role in nonadrenergic inhibitory responses in isolated human airways

There is increasing evidence in many species that vasoactive intestinal peptide (VIP) may be a neurotransmitter in nonadrenergic inhibitory nerves. We have studied the effect of electrical field stimulation (EFS), exogenous VIP, and isoproterenol (Iso) on human airways in vitro. We have also studied a related peptide, peptide histidine methionine (PHM), which coexists with VIP in human airway nerves, and in separate experiments studied fragments of the VIP amino acid sequence (VIP1-10 and VIP16-28) for agonist and antagonist activity. Human airways were obtained at thoracotomy and studied in an organ bath. In bronchi EFS gave an inhibitory response that was unaltered by 10(-6) M propranolol but was blocked by tetrodotoxin, whereas in bronchioles there was little or no nonadrenergic inhibitory response. VIP, PHM, and Iso all caused dose-dependent relaxation of bronchi, VIP and PHM being approximately 50-fold more potent than Iso. VIP, but not Iso, mimicked the time course of nonadrenergic inhibitory nerve stimulation. In contrast bronchioles relaxed to Iso but not to VIP or PHM. Neither propranolol nor indomethacin altered the relaxant effects of VIP or PHM, suggesting a direct effect of these peptides on airway smooth muscle. Neither of the VIP fragments showed either agonist or antagonist activity. We conclude that VIP and PHM are more potent bronchodilators of human bronchi than Iso and that the association between the relaxant effects of these peptides and nonadrenergic inhibitory responses suggests that they may be possible neurotransmitters of nonadrenergic inhibitory nerves in human airways.     

Existence and coexistence of peptides in nerves of the mammalian ovary and oviduct demonstrated by immunocytochemistry

The immunocytochemical distribution of substance P (SP), gastrin releasing peptide (GRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), and neuropeptide Y (NPY) was studied in the ovary and the Fallopian tube (oviduct) of rats, guinea-pigs, cows, pigs and humans. Generally, the nerve supply was better developed in the oviduct than in the ovary. GRP fibers were most scarce in all tissues. Nerves containing SP were particularly numerous in the oviduct of rat and guinea-pig, supplying the muscular wall and blood vessels. VIP and PHI coexisted in dense plexuses of nerves, not only around blood vessels but also in the follicular wall and the interstitial gland of the ovary, as well as within the smooth muscle layers and subepithelially in the oviduct. The general distribution of NPY was similar, but these immunoreactive nerves were even more numerous. Sequential staining for dopamine-beta-hydroxylase and NPY together with results of chemical sympathectomy with 6-hydroxydopamine suggested that NPY was stored in the noradrenergic sympathetic nerves.     

Effect of pharmacological agents on the activity of the circular and longitudinal smooth muscle layers of human fallopian tube ampullar segments

The in vitro determination of the effects of some pharmacological agents on the outer diameter and axial tension of human Fallopian tube ampullar segments showed that some drugs may act on the circular and longitudinal smooth muscle layers relatively separately. PGF2 alpha elicited an excitatory response in both muscle layers while norepinephrine and isoproterenol decreased the activity. Oxytocin and indomethacin had relatively limited effects on the spontaneous motility of the ampullar segments. The calcium antagonist verapamil inhibited the spontaneous periodic activity.     

Phenotypic analyses of a medaka mutant reveal the importance of bilaterally synchronized expression of isthmic fgf8 for bilaterally symmetric formation of the optic tectum

Developing neural tubes are bilaterally symmetric in all vertebrate embryos, irrespective of the presence of gene networks that generate left-right asymmetry. To explore the mechanisms that underlie the bilaterally symmetric formation of the neural tube, we examined a medaka (Oryzias latipes) dominant mutant, Oot, the neural tube of which transiently lacks normal symmetry in the optic tectum. We found that spatial changes in isthmic fgf8 expression do not occur on one side of the mutant, resulting in a transient desynchronized expression that correlates with tectal asymmetry. The application of exogenous FGF8 on one side of a wild-type embryo mimics the Oot phenotype, indicating that the bilaterally equivalent expression of isthmic fgf8 is crucial for the bilaterally symmetric development of the tectum. These results suggest that tectal symmetry is not a "default" state, but rather is maintained actively by a bilaterally coupled and synchronized regulation of isthmic fgf8 expression.     

Inspiratory rhythm in airway smooth muscle tone

In anesthetized paralyzed open-chested cats ventilated with low tidal volumes at high frequency, we recorded phrenic nerve activity, transpulmonary pressure (TPP), and either the tension in an upper tracheal segment or the impulse activity in a pulmonary branch of the vagus nerve. The TPP and upper tracheal segment tension fluctuated with respiration, with peak pressure and tension paralleling phrenic nerve activity. Increased end-tidal CO2 or stimulation of the carotid chemoreceptors with sodium cyanide increased both TPP and tracheal segment tension during the increased activity of the phrenic nerve. Lowering end-tidal CO2 or hyperinflating the lungs to achieve neural apnea (lack of phrenic activity) caused a decrease in TPP and tracheal segment tension and abolished the inspiratory fluctuations. During neural apnea produced by lowering end-tidal CO2, lung inflation caused no further decrease in tracheal segment tension and TPP. Likewise, stimulation of the cervical sympathetics, which caused a reduction in TPP and tracheal segment tension during normal breathing, caused no further reduction in these parameters when the stimulation occurred during neural apnea. During neural apnea the tracheal segment tension and TPP were the same as those following the transection of the vagi or the administration of atropine (0.5 mg/kg). Numerous fibers in the pulmonary branch of the vagus nerve fired in synchrony with the phrenic nerve. Only these fibers had activity which paralleled changes in TPP and tracheal tension. We propose that the major excitatory input to airway smooth muscle arises from cholinergic nerves that fire during inspiration, which have preganglionic cell bodies in the ventral respiratory group in the region of the nucleus ambiguus and are driven by the same pattern generators that drive the phrenic and inspiratory intercostal motoneurons.     

Neuroplasticity in the smooth muscle of the myenterically and extrinsically denervated rat jejunum

The objective of this study was to examine the effects of two different denervation procedures on the distribution of nerve fibers and neurotransmitter levels in the rat jejunum. Extrinsic nerves were eliminated by crushing the mesenteric pedicle to a segment of jejunum. The myenteric plexus and extrinsic nerves were eliminated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). The effects of these two denervation procedures were evaluated at 15 and 45 days. The level of norepinephrine in whole segments of jejunum was initially reduced by more than 76% after both denervation procedures, but by 45 days the level of norepinephrine was the same as in control tissue. Tyrosine hydroxylase (nor-adrenergic nerve marker) immunostaining was absent at 15 days, but returned by 45 days. However, the pattern of noradrenergic innervating axons was altered in the segment deprived of myenteric neurons. Immunohistochemical studies showed protein gene product 9.5 (PGP 9.5)-immunoreactive fibers in whole-mount preparations of the circular smooth muscle in the absence of the myenteric plexus and extrinsic nerves. At 45 days, the number of nerve fibers in the circular smooth muscle increased. Vasoactive intestinal polypeptide (VIP)-immunoreactive fibers, a subset of the PGP 9.5 nerve fibers, were present in the circular smooth muscle at both time points examined. Choline acetyltransferase (CAT) activity and VIP and leucine enkephalin levels were measured in separated smooth muscle and submucosa-musosal layers of the denervated jejunum. VIP and leucine-enkephalin levels were no different from control in tissue that was extrinsically denervated alone. However, the levels of these peptides were elevated two-fold in the smooth muscle 15 and 45 days after myenteric and extrinsic denervation. In the submucosa-mucosa, VIP and leucine enkephalin levels also were elevated two-fold at 15 days, but comparable to control at 45 days. CAT activity was equal to control in the smooth muscle but elevated two-fold in the submucosa-mucosa at both times. These results provide evidence for innervation of the circular smooth muscle by the submucosal plexus. Moreover, these nerve fibers originating from the submucosal plexus proliferate in the absence of the myenteric plexus. Furthermore, the myenteric neurons appear to be essential for normal innervation of the smooth muscle by the sympathetic nerve fibers. It is speculated that the sprouting of the submucosal plexus induced by myenteric plexus ablation is mediated by increased production of trophic factors in the hyperplastic smooth muscle.     

Functional anatomy of the vagal innervation of the cervical trachea of the dog.

The canine cervical trachea has been used for numerous studies regarding the neural control of tracheal smooth muscle. The purpose of the present study was to determine whether there is lateral dominance by either the left or right vagal innervation of the canine cervical trachea. In anesthetized dogs, pressure in the cuff of the endotracheal tube was used as an index of smooth muscle tone in the trachea. After establishment of tracheal tone, as indicated by increased cuff pressure, either the right or left vagus nerve was sectioned followed by section of the contralateral vagus. Sectioning the right vagus first resulted in total loss of tone in the cervical trachea, whereas sectioning the left vagus first produced either a partial or no decrease in tracheal tone. After bilateral section of the vagi, cuff pressure was recorded during electrical stimulation of the rostral end of the right or left vagus. At the maximum current strength used, stimulation of the left vagus produced tracheal constriction that averaged 28.5% of the response to stimulation of the right vagus (9.0 +/- 1.8 and 31.6 +/- 2.5 mmHg, respectively). In conclusion, the musculature of cervical trachea in the dog appears to be predominantly controlled by vagal efferents in the right vagus nerve.     

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.     

Inhibitory innervation to the guinea pig trachealis muscle

The inhibitory innervation of the cervical trachea was studied in situ in anesthetized male guinea pigs. We measured effects of electrical stimulation of vagal motor and sympathetic trunk nerve fibers, during atropine, on trachealis muscle tension. Effects of direct transmural stimulation of trachealis muscle were also determined. We confirmed the dual nature of the inhibitory innervation to this muscle. Vagal motor inhibitory nerves are shown to be preganglionic. Neural transmission at the level of the ganglia is characterized by filtering of high frequency action potentials. The neurotransmitter at the myoneural junction is unidentified but is not norepinephrine. Maximal relaxation accounts for about 20-40% of maximal relaxations seen with transmural stimulation of trachealis muscle in the presence of atropine. Sympathetic trunk nerve fibers are also preganglionic. Neurotransmission at the level of the ganglia is apparently 1:1 at high-action potential frequencies. Norepinephrine released presynaptically has access to smooth muscle beta- but not alpha-receptors. Maximal adrenergic relaxations account for 60-80% of total transmural stimulation relaxations. Transmural stimulation relaxations appear to be accounted for by release of neurotransmitter from sympathetic adrenergic plus vagal nonadrenergic postganglionic nerve fibers.