Osmotic stimuli induce epithelial-dependent relaxation in the guinea pig trachea

Epithelium in airways, like endothelium in blood vessels, may regulate responses of adjacent smooth muscle. To study the intact trachea from guinea pigs we developed an in vitro preparation that permits independent stimulation from either the inner epithelial surface or the outer serosal surface. The whole guinea pig trachea was excised, cannulated, and perfused at a constant flow with Krebs-Henseleit (KH) solution that was in direct contact with the inner epithelial-lined surface. The outer serosal surface of the trachea was immersed in a separate system (bath) containing KH solution. Tracheal responses were assessed by measuring the pressure drop between the tracheal inlet and the outlet under conditions of constant flow. When the trachea was precontracted with carbachol or KCl, hyperosmolar stimuli (KCl, mannitol, urea, or NaCl) produced concentration-dependent relaxation when applied to the inner epithelial surface. Relaxation was not produced when the hyperosmolar stimulus was applied to the serosal surface and was markedly reduced or abolished when the epithelial surface had been physically damaged or removed. These results indicate that hyperosmotic stimuli induce epithelial-dependent relaxation of trachea. A defect in this mechanism may be partially responsible for the bronchoconstriction seen in asthmatic subjects after exercise.     

Role of the epithelium in airway smooth muscle responses to relaxant agonists.

We studied the role of the guinea pig tracheal epithelium in modulating tracheal smooth muscle responses to the relaxant agonists albuterol, sodium nitroprusside, and theophylline. We used an in vitro preparation that allowed separation of the fluids bathing the luminal (internal) and serosal (external) surfaces of the trachea, and bronchodilators were administered to either surface of carbachol-contracted tracheae. All three drugs produced dose-dependent relaxation. However, albuterol and nitroprusside were less potent (concentration that produced half-maximal effect increased by 100- and 32-fold, respectively) when given to the epithelial side with the epithelium intact compared with the epithelium denuded or compared with serosal administration with the epithelium intact. These differences were not observed for theophylline, where smooth muscle responses were independent of either the side of stimulation or of the presence or absence of the epithelium. Direct measurements of the diffusion of theophylline across the tracheal wall in the presence or absence of epithelium showed that after 5 h of incubation with a fixed luminal concentration of theophylline, only 1.7% had diffused across the tracheal wall with the epithelium intact. This increased to only approximately 3.3% when the epithelium was denuded. These results suggest that the epithelial is a relatively weak barrier for lipophilic agents but has a major role as a diffusion barrier to hydrophilic substances.     

The effect of verapamil is reduced in isolated airway smooth muscle preparations lacking the epithelium

The effect of epithelium removal on the reactivity of rabbit airway smooth muscle to bronchoactive agents and on the effect of verapamil was studied in vitro using preparations from several levels within the respiratory tree, i.e., trachea, primary (10) and secondary (20) bronchus. Methacholine contracted tissues from all three levels of airway. Histamine contracted strips from 20 bronchus, had an inconsistent action in strips from 10 bronchus and was without effect in tracheal preparations. K+ contracted tissues from the trachea and 10 bronchus, and had a mixed action in 20 bronchial strips. Removal of the epithelial cell layer variably affected the reactivity of the smooth muscle to the three agents studied. In 20 bronchus, epithelium removal potentiated responses to histamine and methacholine. In 10 bronchus, only responses to methacholine were consistently augmented. In tracheal preparations epithelium removal did not alter the reactivity of the tissue to any agent examined. Verapamil (1 microM) attenuated responses to all agents and increased in its potency from tracheal through 10 to 20 bronchial preparations. Following epithelium removal, verapamil was substantially less effective in 20 bronchi, yet its effects were unchanged in the trachea. The results indicate that the epithelial cell layer modulates airway smooth muscle reactivity; this phenomenon is apparently widespread in mammals, the modulatory effect is more prominent in the smaller airways, and the magnitude of the effect of verapamil on airway smooth muscle is, in part, related to the presence of the epithelium.     

Murine nasal septa for respiratory epithelial air-liquid interface cultures

Air-liquid interface models using murine tracheal respiratory epithelium have revolutionized the in vitro study of pulmonary diseases. This model is often impractical because of the small number of respiratory epithelial cells that can be isolated from the mouse trachea. We describe a simple technique to harvest the murine nasal septum and grow the epithelial cells in an air-liquid interface. The degree of ciliation of mouse trachea, nasal septum, and their respective cultured epithelium at an air-liquid interface were compared by scanning electron microscopy (SEM). Immunocytochemistry for type IV beta-tubulin and zona occludens-1 (Zo-1) are performed to determine differentiation and confluence, respectively. To rule out contamination with olfactory epithelium (OE), immunocytochemistry for olfactory marker protein (OMP) was performed. Transepithelial resistance and potential measurements were determined using a modified vertical Ussing chamber SEM reveals approximately 90% ciliated respiratory epithelium in the nasal septum as compared with 35% in the mouse trachea. The septal air-liquid interface culture demonstrates comparable ciliated respiratory epithelium to the nasal septum. Immunocytochemistry demonstrates an intact monolayer and diffuse differentiated ciliated epithelium. These cultures exhibit a transepithelial resistance and potential confirming a confluent monolayer with electrically active airway epitheliumn containing both a sodium-absorptive pathway and a chloride-secretory pathway. To increase the yield of respiratory epithelial cells harvested from mice, we have found the nasal septum is a superior source when compared with the trachea. The nasal septum increases the yield of respiratory epithelial cells up to 8-fold.     

Epithelial modulation of allergen and drug effects in guinea pig airways.

The effect of egg albumin (EA) challenge on tracheal tube preparations from sensitized guinea pigs was studied with regard to EA permeability, histamine release and penetrability, and the contractile response of the preparation. We used a plethysmographic method that allowed simultaneous measurement of changes in smooth muscle tension and collection of samples for determination of mediators. Our results clearly show that epithelial damage potentiates the contractile response to histamine, potassium ions, and acetylcholine. Epithelial damage did not alter the maximal contractile response in preparations challenged with high antigen concentrations (EA, 1 mg/ml), but histamine release measured in the perfusate increased substantially. The permeability of the preparations to EA was greater when the epithelium was damaged. No increase in the permeability in response to the EA challenge was observed. The present study has demonstrated that guinea pig airway epithelium constitutes a barrier for both antigen and drugs. We also present a method for recording contractile responses from intact whole tracheal preparations, in which the epithelium can still act as a barrier, as is the case in vivo.     

Influence of ventrolateral surface of medulla on reflex tracheal constriction

To assess the role of structures located superficially near the ventrolateral surface of the medulla on the reflex constriction of tracheal smooth muscle that occurs when airway and pulmonary receptors are stimulated mechanically or chemically, experiments were conducted in alpha-chloralose-anesthetized, paralyzed, and artificially ventilated cats. Pressure changes within a bypassed segment of the trachea were used as an index of alterations smooth muscle tone. The effects of focal cooling of the intermediate areas or topically applied lidocaine on the ventral surface of the medulla on the response of the trachea to mechanical and chemical stimulation of airway receptors were examined. Atropine abolished tracheal constriction induced by mechanical stimulation of the carina or aerosolized histamine, showing that the responses were mediated over vagal pathways. Moderate cooling of the intermediate area (20 degrees C) or local application of lidocaine significantly decreased the tracheal constrictive response to mechanical activation of airway receptors. Furthermore, when the trachea was constricted by histamine, cooling of the intermediate area significantly diminished the increased tracheal tone, whereas rewarming restored tracheal tone to the previous level. These findings suggest that under the conditions of the experiments the ventral surface of the medulla plays an important role in constriction of the trachea by inputs from intrapulmonary receptors and in the modulation of parasympathetic outflow to airway smooth muscle.     

In vivo restitution of airway epithelium

Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-m wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [³H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2–3 m/min) over the denuded basement membrane. Within 8–15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.     

Epithelial modulation of tracheal smooth muscle response to antigenic stimulation

The role of the epithelium has been studied in the contractile responses of rat trachea. The different modulations observed are discussed in respect to vagal components of the epithelial layer. Responses of rat trachea to immunologic stimulation are shown to be dependent on the presence of the epithelium, which prolongs the relaxation stage without affecting the contractions. This prolongation is abolished by neonatal capsaicin pretreatment, whereas substance P induces a significantly greater relaxation of serotonin-precontracted intact than deepithelialized trachea. Serotonin concentration-response curves are shifted to the right in intact preparations, which is partly reversed by neonatal capsaicin pretreatment, but a hyporeactivity of the tissue exists. A relaxing factor released by the epithelium is hypothesized, possibly dependent on substance P-ergic innervation. Muscarinic cholinergic innervation slightly modulates the contractions but not the relaxations in antigen-induced responses, independently on the presence of the epithelial layer. 4-Aminopyridine induces epithelium-dependent potentiations of contractions to antigen and to serotonin, which involves acetylcholine at one step of the reaction cascade. Epithelial-dependent contracting and relaxing factors are thus suggested in rat trachea.     

Ultrastructural modifications of frog urinary bladder epithelium under the influence of hypertonic media

Summary The frog urinary bladder undergoes a marked increase in its water permeability when incubated in hypertonic media. Many similarities are found between this effect and the hydrosmotic action of antidiuretic hormone. The ultrastructural modifications of the epithelium observed under the influence of serosal hypertonicity (the intercellular spaces are dilated while the tight junctions remain closed) lead us to assume that the pathways of water movement across the epithelium could be the same in this case and in hydrosmotic response to the hormone. In contrast, when the mucosal medium is made hypertonic, the ultrastructure is differently altered: the intercellular spaces are closed, the tight junctions show small vesicles and numerous large vacuoles appearing in the cytoplasm.     

Action of Antidiuretic Hormone on the Equivalent Pore Radius at both Surfaces of the Epithelium of the Isolated Toad Skin

A previously described method (1) allows the observation of swelling and shrinking of the epithelial cells of the isolated toad skin, when the solution bathing either the outer or inner side of the skin is modified. Thus, the concentration of probing molecules of graded size, isotonic to the epithelial cells, across each face of the isolated toad skin can be determined. These concentrations have been used for the estimation of the equivalent pore radius at the outer and inner face of the skin epithelium, following the approach of Goldstein and Solomon for red cells (3). An equivalent pore radius of 4.5 A for the outer surface, and one of 7 A for the inner surface have been obtained. Antidiuretic hormone had an effect only when added to the inner side. This effect was only at the outer surface and is interpreted as widening of the 4.5 A pores to about 6.5 A. A model membrane, formed by narrow and wide pores in series, may explain some of the apparent inconsistencies previously observed.     

Epithelium modulates the reactivity of ovalbumin-sensitized guinea-pig airway smooth muscle

Mechanical removal of the airway epithelium alters the in vitro reactivity of airway smooth muscle. The modulation of reactivity may involve the release of inhibitory and excitatory factors from epithelial cells. Guinea pigs sensitized with ovalbumin have been used as an animal model of airway hyperreactivity. We evaluated the influence of the epithelium on the reactivity of in vitro tracheal smooth muscle from control and ovalbumin-sensitized guinea pigs, and the extent to which the presence of the epithelium affects the contractile response to in vitro challenge with ovalbumin. In both control and ovalbumin-sensitized tissues, epithelium removal increased the sensitivity of the preparations to histamine, methacholine and isoproterenol to a similar extent, i.e., 2- to 2.5-fold. Epithelium removal resulted in an 8.1-fold increase in sensitivity to ovalbumin in sensitized tissues. The epithelium appears not only to modulate the reactivity of the tissues to bronchoactive agents, but it also influences the magnitude of the contractile response following antigen challenge.     

Differential expression of a WD protein during squamous differentiation of tracheal epithelial cells

The lining of the trachea consists of a pseudostratified, mucociliary epithelium that under a variety of conditions, such as vitamin A deficiency, toxic and mechanical injury, becomes a stratified squamous epithelium. Several in vitro cell culture models have been established to study the process of differentiation of airway epithelium. Such studies have indicated that mucosecretory differentiation of tracheal epithelial cells can be modulated by substratum. This study was undertaken to understand molecular mechanisms of squamous differentiation in tracheal epithelia. Primary cultured tracheal cells grown on uncoated filters were differentiated to single layer of squamous cells, whereas cells were grown as stratified columnar cells on collagen-I coated filters. The responses to secretagogues were altered according to culture conditions. DD-PCR revealed that FAK and a WD protein expression was increased in squamous tracheal epithelia. Expression of a WD protein was changed by the treatment of retinoic acid in various epithelial cells. These results indicated that squamous differentiation of tracheal cells changes the expression of a variety of genes, and that the experimental model for this study can be employed to study molecular mechanisms of squamous differentiation in airway epithelial cells.     

支气管上皮细胞在气道高反应中的作用

气道高反应的发病机制目前仍然不清楚,但人多数人认同是气道的一种慢性炎症。近十年来,上皮缺陷学说逐渐成为解释气道高反应机制的主流观点。气道上皮不再被仅仅看作为单纯的机械屏障,而是机体内环境与外部环境相互作用的界面。气道上皮具有广泛的生理作用,包括抗氧化、内分泌和外分泌、黏液运输、生物代谢、结构性黏附、损伤修复、应激或炎症信号传递、抗原递呈作用等。借助这些生理作用,支气管上皮细胞在气道局部微环境稳态维持中发挥重要作用。有理由相信,气道上皮的结构完整性缺陷或功能紊乱是哮喘和慢性阻塞性肺疾病等气道高反应性疾病的启动环节。     

Release of epithelium-derived relaxing factor after ozone inhalation in dogs

Airway epithelium has been reported to release epithelium-derived relaxing factor (EpDRF), which inhibits contraction of airway smooth muscle. This study tested the hypothesis that airway hyperresponsiveness after inhalation of ozone in dogs results from an inability to produce EpDRF. Two groups of five dogs each were studied; one group inhaled ozone, the other dry room air. Ozone-treated dogs developed airway hyperresponsiveness, whereas the control group did not. The acetylcholine provocative concentration decreased from 4.17 (%SE 1.35) to 0.56 mg/ml (%SE 1.24) (P = 0.0006) in the ozone-treated dogs and was 18.76 (%SE 2.04) and 29.77 mg/ml (%SE 2.07) in the air-treated dogs (P = 0.47). In vitro the presence of airway epithelium reduced the constrictor responses to acetylcholine, histamine, serotonin, and KCl in trachealis strips from the control dogs. This effect of epithelium was still present in trachealis strips from dogs with airway hyperresponsiveness. These results demonstrate that EpDRF is released from canine tracheal epithelium, that this function is not impaired in dogs with airway hyperresponsiveness after inhaled ozone, and that loss of EpDRF is not responsible for the development of airway hyperresponsiveness after inhaled ozone in dogs.     

The effect of osmotically induced water flows on the permeability and ultrastructure of the rabbit gallbladder

Summary The purpose of these experiments was to determine the effect of osmotic gradients on the permeability of the rabbit gallbladder. Increasing the tonicity of the mucosal solution reduced the permeability of the gallbladder to both ions and nonelectrolytes, whereas there was no significant effect when the serosal solution was made hypertonic. These results cannot be explained by solvent/solute interactions in either the epithelial membranes or the unstirred layers. Associated with the changes in permeability was the appearance of the transport number effect and current induced resistance changes. Morphological studies of the gallbladder under these conditions showed that the extracellular spaces of the epithelium and the rest of the wall dilate in the presence of osmotic flow to the serosa, but that the spaces collapse when the flow is in the opposite direction. Reconstruction of the permeability changes from the dimensions of the tissue show that all the physiological phenomena are accounted for by the changes in morphology, the dominant effect being in the lateral intercellular spaces. These results suggest that the lateral spaces are a common pathway shared by all solutes crossing the epithelium, and that diffusion along these spaces becomes rate limiting as the spaces collapse.     

Mechanisms underlying the relaxation induced by isokaempferide from Amburana cearensis in the guinea-pig isolated trachea

The present study examines possible mechanisms by which the flavonoid isokaempferide (IKPF; 5,7,4'-trihydroxy-3-methoxyflavone) from Amburana cearensis, a Brazilian medicinal plant popularly used as bronchodilator, induces relaxation of guinea-pig isolated trachea. In the trachea (with intact epithelium) contracted by carbachol, IKPF (1-1000 microM) caused a graded relaxation, and the epithelium removal increased the sensitivity of the airway smooth muscle to IKPF (EC50, in intact tissue: 77.4 [54.8-109.2] microM; in denuded epithelium: 15.0 [11.3-20.1] microM). The IKPF-induced relaxation was inhibited in 41% by the nitric oxide (NO) synthase inhibitor L-NAME (100 microM); in 31% and 50% by the soluble guanylate cyclase (sGC) inhibitor ODQ (3 and 33 microM); by propranolol (31%) and also by capsaicin (37%). In the trachea pre-contracted by 40 mM KCl the pre-incubation with glibenclamide (33 microM) or iberiotoxin (IbTX, 0.1 microM), selective K(+) channel inhibitors, inhibited the IKPF-induced relaxation by 39% and 38%, respectively. On the other hand, 4-aminopyridine (100 microM), a nonselective K(+) channel antagonist, did not significantly influence the effect of IKPF, while IbTX induced a rightward displacement of the IKPF concentration-response curve. However, in muscle pre-contracted with 120 mM KCl the relaxant effect of IKPF was significantly reduced and not affected by glibenclamide. In conclusion, these results indicate a direct and epithelium-independent relaxant effect of IKPF on smooth muscle fibers. Although this IKPF relaxant action seems to be multi-mediated, it occurs via both Ca(2+) and ATP-sensitive K(+) channels, but some other possible mechanisms unrelated to K(+) channels cannot be excluded.     

Bronchial mucosal permeability.

The tracheobronchial epithelium has well-developed tight junctions which on a morphologic basis should be markedly resistant to penetration by protein molecules. Despite this, antigen inhalation in monkeys allergic to Ascaris suum results in the rapid onset of pulmonary physiologic changes. Recent studies in man and animals have shown that a substantial number of mast cells exist in the bronchial lumen and epithelium. We suggest that antigen-antibody interaction initially occurs on these superficial mast cells leading to mediator release and the stimulation of airway irritant receptors. Antigen challenge also results in increased epithelial permeability to protein in the Ascaris-allergic monkey, and from studies on guinea pigs we suggest that this is due to alterations in the tight junctions. Antigen challenge in the monkey also produces increased permeability to labeled histamine and hyperresponsiveness to low concentrations of histamine. We suggest that the apparent airway hyperreactivity to inhaled histamine seen after inhalation of ozone, and NO2, or after upper respiratory infections could be due to damage to epithelial tight junctions. The resultant increase in mucosal permeability would result in an increased amount of histamine reaching airway smooth muscle for a given inhaled concentration.     

Pharmacological analysis of the peristalsis block induced by magnesium applied at the serosal and mucosal surfaces of the isolated guinea pig ileum]

Acetylcholine, metacholine, eserine and neostigmine, acting from the serosal surface antagonised the peristaltic block produced by mucosal application of magnesium, as well as when choline esters and anticholinesterases were injected intraluminally and magnesium acted from the serosal side of the guinea-pig isolated ileum. On the other hand, 5-hydroxytryptamine and histamine did not remove the peristaltic block produced by magnesium. It is concluded that cholinoceptive sites within the myenteric plexus which, when stimulated by cholinergic substanced, produce regular peristaltic waves, are accessible from either sides of the guinea-pig isolated ileum.     

Repair and regeneration of the airway epithelium

Despite an efficient defence system, the airway surface epithelium, in permanent contact with the external milieu, is frequently injured by inhaled pollutants, microorganisms and viruses. The response of the airway surface epithelium to an acute injury includes a succession of cellular events varying from the loss of the surface epithelium integrity to partial shedding of the epithelium or even to complete denudation of the basement membrane. The epithelium has then to repair and regenerate to restore its functions, through several mechanisms including basal cell spreading and migration, followed by proliferation and differentiation of epithelial cells. The cellular and molecular factors involved in wound repair and epithelial regeneration are closely interacting and imply extracellular matrix proteins, matrix metalloproteinases (MMPs) and their inhibitors as well as cytokines and growth factors secreted by airway epithelial and mesenchymal cells. The development of in vitro and in vivo models of airway epithelium wound repair allowed the study of the spatio-temporal modulation of these factors during the different steps of epithelial repair and regeneration. In this context, several studies have demonstrated that the matrix and secretory environment are markedly involved in these mechanisms and that their dysregulation may induce remodelling of the airway mucosa. A better knowledge of the mechanisms involved in airway epithelium regeneration may pave the way to regenerative therapeutics allowing the reconstitution of a functional airway epithelium in numerous respiratory diseases such as asthma, chronic obstructive pulmonary diseases, cystic fibrosis and bronchiolitis.     

Absorption of intact beta-casomorphins (beta-CM) in rabbit ileum in vitro

The functional significance of the presence of opioid peptides in enzymatic digestion of bovine milk beta-casein remains unclear. Opiates modify intestinal electrolyte transport by acting on receptors located on the serosal side of the intestine. The aim of the present study is to determine under which conditions beta-casomorphins could act from the luminal side of the intestine. The effect of natural morphiceptin (beta-CM4-NH2) and the non metabolized analogue beta-[DAla2,4, Try5]-CM5-NH2 were studied on isolated rabbit ileum mounted in Ussing chambers. Both peptides caused a naloxone-reversible reduction in short-circuit current (lsc) and stimulated Na and Cl absorption after addition to the serosal side of the tissue. After mucosal addition, only the analogue (10(-3) M) crossed the epithelium intact (Jm-s = 3.5 +/- 1.2 nmol.h-1.cm-2) and reduced lsc. Morphiceptin, under the same conditions, was degraded by the intestinal mucosa without opiate action on electrolyte transport. Pretreatment of the ileum by 10(-3)M diisopropylfluorophosphate that inhibited brush-border dipeptidylpeptidase IV, prevented mucosal degradation of morphiceptin. Under these conditions, the peptide (10(-3)M) crossed the epithelium intact (Jm-s = 1.8 +/- 0.16 nmol.h-1.cm-2) and stimulated electrolyte absorption by means of an opioid mechanism. These results show that both natural morphiceptin and the protected analogue have an opiate activity on intestinal electrolyte transport. Their action from the lumen depends on their transfer intact to the serosal side of the intestine where opiate receptors are located. The limiting step in this transfer is at the brush-border membrane where dipeptidylpeptidase IV in particular seems to play a major role.