Protective role of epithelium in the guinea pig airway

We developed an in vitro system to assess the role of the epithelium in regulating airway tone using the intact guinea pig trachea (J. Appl. Physiol. 64: 466-471, 1988). This method allows us to study the response of the airway when its inner epithelial surface or its outer serosal surface is stimulated independently. Using this system we evaluated how the presence of intact epithelium can affect pharmacological responsiveness. We first examined responses of tracheae with intact epithelium to histamine, acetylcholine, and hypertonic KCl when stimulated from the epithelial or serosal side. We then examined the effect of epithelial denudation on the responses to these agonists. With an intact epithelium, stimulation of the inner epithelial side always caused significantly smaller changes in diameter than stimulation of the outer serosal side. After mechanical denudation of the epithelium, these differences were almost completely abolished. In the absence of intact epithelium, the trachea was 35-fold more sensitive to histamine and 115-fold more sensitive to acetylcholine when these agents were applied to the inner epithelial side. In addition, the presence of an intact epithelium almost completely inhibited any response to epithelial side challenge with hypertonic KCl. These results indicate that the airway epithelial layer has a potent protective role in airway responses to luminal side stimuli, leading us to speculate that changes in airway reactivity measured in various conditions including asthma may result in part from changes in epithelial function.     

Quantitative measurement of smooth muscle shortening in isolated pig trachea

Matched porcine tracheal rings were exposed to theophylline and increasing doses of carbachol in Krebs solution. Histological sections of each ring were traced and each of the following dimensions measured: the external perimeter (Pe) and external area (Ae) defined by the outer border of smooth muscle and inner surface of cartilage, and the internal perimeter (Pi) and internal area (Ai) defined by the luminal surface of the epithelium and the muscle length (L) along its outer border. Absolute wall area (WA = Ae - Ai) and relative wall area (PW = WA/Ae) were calculated. Carbachol-treated tracheal ring dimensions were compared with those of their matched theophylline-treated rings. In tracheal rings with intact cartilage, maximal smooth muscle shortening of 44% was achieved with 10(-2) M carbachol. In tracheal rings in which anterior and posterior segments of cartilage were excised, the trachealis muscle passively shortened by 20% and maximal shortening (10(-3) M carbachol) was 57%. Although Ai decreased with maximal smooth muscle shortening, there were no changes in the length of Pi or in WA. These data show that the cartilage in the porcine trachea exerts both a preload that passively stretches the trachealis muscle and an afterload that limits maximal smooth muscle shortening.     

Respiratory epithelium inhibits bronchial smooth muscle tone

The aim of the present study was to determine whether or not the respiratory epithelium can modulate the responsiveness of bronchial smooth muscle. Paired rings of canine bronchi (4-6 mm OD), in some of which the epithelium had been removed mechanically (by rubbing the luminal surface), were mounted in physiological saline solution, gassed with 95% O2-5% CO2, and maintained at 37 degrees C. The presence or absence of the epithelium was confirmed by histological examination. Removal of the epithelium increased the contractile responses evoked by acetylcholine, histamine, and 5-hydroxytryptamine. Transmural nerve stimulation evoked similar peak responses in the presence and absence of epithelium. In unrubbed preparations, the peak response was followed by a gradual decrease when the stimulation was continued. This decrease, which persisted in the presence of propranolol, was not observed in epithelium-denuded preparations. In bronchial rings contracted with acetylcholine, isoproterenol produced concentration-dependent relaxations which were significantly greater in rings with epithelium compared with denuded rings. These results suggest that respiratory epithelial cells may generate an inhibitory signal to decrease the responsiveness of bronchial smooth muscle to contractile agonists and augment the effectiveness of inhibitory stimuli.     

Bronchial responses to substance P after antigen challenge in the guinea-pig: in vivo and in vitro studies

The effect of antigen challenge on the airway responses to substance P and on the epithelial neutral endopeptidase (NEP) activity was investigated in aerosol sensitized guinea-pigs. In vivo, bronchial responses to aerosolized substance P were similar to the responses observed in antigen-challenged guinea-pigs and in the control groups. In contrast, when the guinea-pigs were pretreated with the NEP inhibitor, phosphoramidon, a significant increase in the airway responses to substance P was observed after antigen challenge in vivo. However, in vitro, the contractile responses of the tracheal smooth muscle to substance P were similar between groups of guinea-pigs, in respect to the presence or absence of the epithelium and/or phosphoramidon. Histological studies showed an accumulation of eosinophils in the tracheal submucosa after antigen challenge and intact epithelial cells. These results show that in vivo bronchial hyperresponsiveness to substance P after antigen challenge in the guinea-pig is not associated with increased responses of the smooth muscle to exogenous SP in vitro. In addition, the results with phosphoramidon suggest that loss of NEP activity cannot account for the in vivo bronchial hyperresponsiveness to substance P presently observed.     

Embryonic chicken gizzard: smooth muscle and non-muscle myosin isoforms

Antibodies to smooth muscle and non-muscle myosin allow the development of smooth muscle and its capillary system in the embryonic chicken gizzard to be followed by immunofluorescent techniques. Although smooth muscle development proceeds in a serosal to luminal direction, angiogenetic cell clusters develop independently at the luminal side close to the epithelial layer, and the presumptive capillaries invade the developing muscle in a luminal to serosal direction. The smooth muscle and non-muscle myosin heavy chains in this avian system cannot be separated by SDS polyacrylamide gel electrophoresis and do not show isoform specificity in immunoblotting, unlike the system found in mammals. Only two myosin heavy chains with M_r of 200 and 196 kDa were separable and considerable immunological cross-reactivity was found between the denatured myosin isoform heavy chains.     

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.     

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.     

Functional identification of epithelial and smooth muscle histamine-dependent relaxing mechanisms in the bovine trachea,but not in bronchi

Theoretically, the overall effect of histamine on respiratory smooth muscle is the result of a subtle balance of contraction and relaxation. The aim of the study was to identify histamine type 2 (H2) and 3 (H3) receptor-dependent relaxing mechanisms in the contractile elements of the bovine tracheobronchial tree. In bronchial preparations, histamine induced very weak contractions, which were not exacerbated with the H2-antagonist cimetidine. Moreover, precontracted bronchial rings never relaxed in response to cumulative doses of histamine or amthamine (H2-agonist). In intact tracheal preparations, histamine induced strong contractions that were exacerbated by cimetidine (E(max): +17.2+/-6.6%) but not by thioperamide (H3-antagonist). Precontracted tracheal bundles did not relax in response to cumulative doses of the H3-agonist R-alpha-methylhistamine. The tracheal contractile response was higher in denuded compared to intact preparations (11.0+/-1.2 vs. 6.0+/-1.7 g). Cimetidine effect was dramatically potentiated in denuded tracheal strips (+40.0+/-11.7%). It is concluded that the weak response of bovine bronchi to histamine is due to a relative scarcity of H1 receptors on bronchial smooth muscle rather than to H2- or H3-dependent relaxation. In the bovine trachea, the smooth muscle possesses relaxing H2 but no H3 receptors. The epithelium exercises a relaxation, which is independent from H2 and H3 receptors.     

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.     

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.     

Epithelial modulation of trachealis muscle tension is calcium and temperature dependent

The tracheobronchial epithelium produces inhibitory substance(s) that alter the tracheal smooth muscle tension. This study examined the effect of changes in extracellular Ca2+ and temperature in vitro on the tension response of rabbit trachealis muscle to mechanical removal of the epithelium. Tension during acetylcholine- and KCl-induced contractions was examined at 0, 0.75, 1.5, 2.5, and 5 mM bath Ca2+ concentrations and at 37, 30, 23, and 41 degrees C bath temperature. At most extracellular Ca2+ concentrations (i.e., 0.75, 1.5, 2.5, and 5 mM), epithelial removal shifted the acetylcholine concentration response approximately one-half log to the left (P less than 0.001 for each condition) but had no effect on the responses to KCl (P = NS). Reductions in bath Ca2+ to 0 mM eliminated the epithelial inhibitory effect on the acetylcholine response. In contrast to the effects of reductions in Ca2+, cooling the airway to 30 and 23 degrees C progressively diminished the magnitude of the epithelial inhibitory effect. Our results indicate that the influence of the tracheal epithelium on tracheal smooth muscle responses to constrictor agonists is substance specific and can be diminished by reductions in tracheal temperature and extracellular Ca2+ concentration.     

Serosal zinc attenuate serotonin and vasoactive intestinal peptide induced secretion in piglet small intestinal epithelium in vitro.

This study addressed the mechanisms by which dietary zinc affects diarrhoea and aimed to study possible interactions between zinc status and the presence of zinc in vitro on secretagogue-induced secretion from piglet intestinal epithelium in Ussing chambers. In addition, it was studied from which side of the epithelium zinc would perform an effect and if copper caused similar effects. Twenty-four piglets (28 days of age) were weaned and fed diets containing 100 or 2500 mg zinc/kg (as ZnO) for 5 or 6 days (12 piglets per group). Intestinal epithelium underwent the following 5 treatments: zinc at the mucosal side (M(Zn)), zinc at the serosal side (S(Zn)), zinc at both sides (MS(Zn)), copper at both sides (MS(Cu)) or water at both sides (control). Provoked secretion in terms of short circuit responses to serotonin (5-HT) and vasoactive intestinal peptide (VIP) were measured. Zinc at the serosal or both sides of the epithelium reduced the 5-HT induced secretion (P<0.001); however, due to interactions (P=0.05) the effect of zinc in vitro was only present in the ZnO(100) group. The secretion caused by VIP was not affected by the diet (P=0.33), but zinc at the serosal side or both sides reduced the response to VIP (P<0.001). Copper reduced the 5-HT and VIP induced secretion to a larger extent than zinc. However, copper also disturbed intestinal barrier function as demonstrated by increased transepithelial conductance and increased short circuit current, which was unaffected by zinc. In conclusion, zinc at the serosal side of piglet small intestinal epithelium attenuated 5-HT and VIP induced secretion in vitro. These in vitro studies indicate that in vivo there will be no positive acute effect of increasing luminal Zn concentration on secretagogue-induced chloride secretion and that zinc status at the serosal side of the epithelium has to be increased to reduce secretagogue-induced chloride secretion and thereby diarrhoea.     

Serosal zinc attenuate serotonin and vasoactive intestinal peptide induced secretion in piglet small intestinal epithelium in vitro

This study addressed the mechanisms by which dietary zinc affects diarrhoea and aimed to study possible interactions between zinc status and the presence of zinc in vitro on secretagogue-induced secretion from piglet intestinal epithelium in Ussing chambers. In addition, it was studied from which side of the epithelium zinc would perform an effect and if copper caused similar effects. Twenty-four piglets (28 days of age) were weaned and fed diets containing 100 or 2500 mg zinc/kg (as ZnO) for 5 or 6 days (12 piglets per group). Intestinal epithelium underwent the following 5 treatments: zinc at the mucosal side (M(Zn)), zinc at the serosal side (S(Zn)), zinc at both sides (MS(Zn)), copper at both sides (MS(Cu)) or water at both sides (control). Provoked secretion in terms of short circuit responses to serotonin (5-HT) and vasoactive intestinal peptide (VIP) were measured. Zinc at the serosal or both sides of the epithelium reduced the 5-HT induced secretion (P<0.001); however, due to interactions (P=0.05) the effect of zinc in vitro was only present in the ZnO(100) group. The secretion caused by VIP was not affected by the diet (P=0.33), but zinc at the serosal side or both sides reduced the response to VIP (P<0.001). Copper reduced the 5-HT and VIP induced secretion to a larger extent than zinc. However, copper also disturbed intestinal barrier function as demonstrated by increased transepithelial conductance and increased short circuit current, which was unaffected by zinc. In conclusion, zinc at the serosal side of piglet small intestinal epithelium attenuated 5-HT and VIP induced secretion in vitro. These in vitro studies indicate that in vivo there will be no positive acute effect of increasing luminal Zn concentration on secretagogue-induced chloride secretion and that zinc status at the serosal side of the epithelium has to be increased to reduce secretagogue-induced chloride secretion and thereby diarrhoea.     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD4, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE2, PGF2 alpha and PGI2 (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD4, methacholine and histamine. LTD4 (3-100 nM), methacholine (0.1-10 microM) or histamine (0.3-30 microM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE2 and PGF2 alpha, but not PGI2, was decreased in tissues lacking epithelium. Indomethacin (1 microM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI2 and is unlikely to be PGF2 alpha or PGE2. However, the possibility remains that the basal release of PGE2 and/or PGF2 alpha derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE2 and PGF2 alpha which may be involved in the maintenance of baseline tone.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

STRUCTURE OF THE TOAD'S URINARY BLADDER AS RELATED TO ITS PHYSIOLOGY

The structure of the urinary bladder of the toad Bufo marinus was studied by light and electron microscopy. The epithelium covering the mucosal surface of the bladder is 3 to 10 microns thick and consists of squamous epithelial cells, goblet cells, and a third class of cells containing many mitochondria and possibly representing goblet cells in early stages of their secretory cycle. This epithelium is supported on a lamina propria 30 to several hundred microns thick and containing collagen fibrils, bundles of smooth muscle fibers, and blood vessels. The serosal surface of the bladder is covered by an incomplete mesothelium. The cytoplasm of the squamous epithelial cells, which greatly outnumber the other types of cells, is organized in a way characteristic of epithelial secretory cells. Mitochondria, smooth and rough surfaced endoplasmic reticulum, a Golgi apparatus, "multivesicular bodies," and isolated particles and vesicles are present. Secretion granules are found immediately under the plasma membranes of the free surfaces of the epithelial cells and are seen to fuse with these membranes and release their contents to contribute to a fibrous surface coating found only on the free mucosal surfaces of the cells. Beneath the plasma membranes on these surfaces is an additional, finely granular component. Lateral and basal plasma membranes are heavily plicated and appear ordinary in fine structure. The cells of the epithelium are tightly held together by a terminal bar apparatus and sealed together, with an intervening space of only 0.02 mµ near the bladder lumen, in such a way as to prevent water leakage between the cells. It is demonstrated in in vitro experiments that water traversing the bladder wall passes through the cytoplasm of the epithelial cells and that a vesicle transport mechanism is not involved. In vitro experiments also show that the basal (serosal) surfaces of the epithelial cells are freely permeable to water, while the free (mucosal) surfaces are normally relatively impermeable but become permeable when the serosal surface of the bladder is treated with neurohypophyseal hormones. The permeability barrier found at the mucosal surface may be represented, structurally, either by the filamentous layer lying external to the plasma membrane, by the intracellular, granular component found just under the plasma membrane, or by both of these components of the mucosal surface complex. The polarity of the epithelial sheet is emphasized and related to the physiological role of the urinary bladder in amphibian water balance mechanisms.     

Physiology of the tracheal epithelium

The mucosa that lines the airways is covered with a fluid film forming a hypophase between mucus and cell surface. To study the function of this epithelium aims at describing the mechanisms by which fluid is normally produced. Another goal to be pursued consists in looking for the origin of pathological situations, such as cystic fibrosis, in which the functioning of epithelial cell is altered. The elucidation of transport mechanisms present in the apical and in the basolateral membrane results in a conceptual model that illustrates the asymmetrical functioning of epithelial cells. Recent discoveries enlarge our understanding of membrane transport processes; in particular, a concerted, reciprocal regulation of the activity of both membranes was shown to be exerted via the intracellular composition. The tracheal epithelium absorbs Na+ and secretes Cl-. These two transports are active and electrogenic; their sum corresponds approximately to the short-circuit current measured in vitro. Na+ absorption is sensitive to amiloride from the luminal side and also to ouabain added to the serosal compartment. The process is a primary active transport, analogous to that found in amphibian epithelia or in mammalian colon. Cl- secretion is abolished by furosemide (or bumetanide), by ouabain or by Na+ suppression in the serosal incubation solution. The mechanism is a secondary active transport: Cl- influx across the basolateral membrane is coupled to Na+ (probably through Na+, K+, Cl- symport); energy is dissipated by the Na+-K+-ATPase localised in the basolateral membrane. Thus, Na+ is recirculated across that membrane by the pump activity, which maintains a favorable gradient for influx via the symport. Cl- efflux takes place by diffusion through the luminal membrane. This model applies to other epithelia in which Na+-coupled Cl- secretion was shown to take place. It is confirmed by isotopic fluxes measurements and by electrophysiologic properties of the apical and the basolateral membrane. Various agents are known to influence ion transports. In particular Cl- secretion is stimulated by substances that increase the intracellular concentration of cyclic AMP. At the membrane level, the number of active Cl- channels in the apical membrane is primarily controlled, then the basolateral membrane K+ permeability. Yet, species differences are worth to note: the trachea of the cow is barely sensitive to agents that exert a marked action on dog trachea. The tracheal epithelium is used as an experimental model for studying cystic fibrosis, a disease in which the apical membrane is almost devoid of functional Cl- channels, so that Cl- permeability is quite low.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacological differentiation of epithelium-derived relaxing factor from nitric oxide

We examined the possibility that nitric oxide is one of the epithelium-derived relaxing factors in guinea pig airways. First we studied whether nitric oxide could relax isolated tracheal strips, and then we examined the effects of known inhibitors of endothelium-dependent relaxation (EDR) in the vascular system [hemoglobin, methylene blue, and NG-monomethyl-L-arginine (L-NMMA)] on epithelium-dependent relaxation (EpDR) induced by hyperosmotic stimuli in perfused whole tracheal preparations. Mannitol (160 mM in Krebs-Henseleit solution) applied to the epithelial surface was used as an osmotic stimulus to induce EpDR after carbachol-induced contraction (2 microM, serosal side). Nitric oxide produced concentration-dependent and complete relaxation of epithelium-denuded tracheal strips. Preincubation of the whole trachea with hemoglobin significantly inhibited osmotic-induced EpDR (P less than 0.05), but preincubation with methylene blue and L-NMMA did not. Hemoglobin introduced into the epithelial side after EpDR induced by hyperosmotic stimuli reversed relaxation, but methylene blue and L-NMMA did not. These results suggest that, although EpDR and vascular EDR have some pharmacological similarities and nitric oxide can relax airway smooth muscle, nitric oxide is not responsible for osmotic-induced EpDR.     

Physiological and morphological effects of alendronate on rabbit esophageal epithelium

Alendronate, an aminobisphosphonate, produces as a side effect a topical (pill induced) esophagitis. To gain insight into this phenomenon, we assessed the effects of luminal alendronate on both esophageal epithelial structure and function. Sections of rabbit esophageal epithelium were exposed to luminal alendronate at neutral or acidic pH while mounted in Ussing chambers to monitor transmural electrical potential difference (PD), short-circuit current (I(sc)), and resistance (R). Morphological changes were sought by light microscopy in hematoxylin and eosin-stained sections. Impedance analysis was used for localization of alendronate-induced effects on ion transport. Luminal, but not serosal, alendronate (pH 6.9-7.2), increased PD and I(sc) in a dose- and time-dependent manner, with little change in R and mild edema of surface cell layers. The changes in I(sc) (and PD) were reversible with drug washout and could be prevented either by inhibition of Na,K-ATPase activity with serosal ouabain or by inhibition of apical Na channels with luminal acidification to pH 2.0 with HCl. An effect on apical Na channel activity was also supported by impedance analysis. Luminal alendronate at acidic pH was more damaging than either alendronate at neutral pH or acidic pH alone. These data suggest that alendronate stimulates net ion (Na) transport in esophageal epithelium by increasing apical membrane sodium channel activity and that this occurs with limited morphological change and no alteration in barrier function. Also alendronate is far more damaging at acidic than at neutral pH, suggesting its association with esophagitis requires gastric acid for expression. This expression may occur either by potentiation between the damaging effects of (refluxed) gastric acid and drug or by acid-induced conversion of the drug to a more toxic form.     

Epithelial augmentation of trachealis contraction caused by major basic protein of eosinophils

We studied the effect of epithelial removal and intraepithelial administration of human eosinophil granule major basic protein (MBP) on the contraction of underlying canine tracheal smooth muscle in 23 dogs in vivo. A dual in situ tracheal preparation was utilized that allowed sharp excision of epithelium. The response to intra-arterial acetylcholine (ACh) was augmented substantially in five dogs receiving 200 micrograms MBP by intraepithelial instillation. Active tension elicited by 10(-8) mol intra-arterial ACh was 34.0 +/- 2.2 g/cm before and 46.1 +/- 2.6 g/cm 30 min after MBP (P less than 0.002). There was no change in active tension in the control segment in the same dogs after intraepithelial instillation of vehicle only (34.7 +/- 3.2 vs. 34.4 +/- 2.3 g/cm; P = NS). Instillation of MBP directly into the subepithelial tracheal smooth muscle did not alter contraction. To assess whether this augmentation was caused by inhibition of an epithelial-derived relaxant factor, additional studies were performed in nine other dogs in which the epithelium was excised discretely from one of the two tracheal segments. No significant differences in contractile response to ACh or relaxation response to isoproterenol were observed at 2, 15, 30, or 60 min after epithelial excision. We demonstrate that intraepithelial administration of MBP augments the contraction of underlying canine tracheal smooth muscle elicited by ACh. This augmentation is a direct effect of MBP and does not require antagonism of epithelial inhibition.