Epithelium-derived Relaxing Factor Research Articles

Epithelial expression and role of secreted STC1 on asthma airway hyperresponsiveness through calcium channel modulation

Asthma is characterized by airway hyperresponsiveness (AHR), inflammation, and airway remodeling. Airway hyperresponsiveness results from enhanced airway smooth muscle (ASM) contraction potentially under the control of an epithelium-derived relaxing factor (EpDRF). However, relatively rare is known about EpDRF. We aimed to elucidate the role of epithelium-derived stanniocalcin-1 (STC1) on AHR and ASM contraction. Stanniocalcin-1 levels in the serum of asthmatic patients and healthy volunteers and in bronchoalveolar lavage fluid (BALF) from ovalbumin (OVA)-challenged mice were measured by ELISA. The effects of exogenous STC1 on AHR and on inflammation were examined in mice. IL-13 modulation of STC1 mRNA and protein levels was studied in human bronchial epithelial cell lines (16HBE). The function of STC1 on Ca2+ influx and ASM contraction was examined ex vivo. Serum STC1 was decreased in asthma (n=93) compared with healthy volunteers (1071±30.4 vs 1414±75.1pg/ml, p<0.0001, n=23) and correlated with asthma control (p=0.0270), lung function (FEV1, p=0.0130), and serum IL-13 levels (p=0.0009). Treatment of ten asthmatic subjects with inhaled corticosteroids/long-acting beta2-agonists (ICS/LABA) for 1year enhanced STC1 expression which correlated with improved asthma control (p=0.022). STC1 was mainly expressed in bronchial epithelium and intranasal administration of recombinant human STC1 (rhSTC1) reduced AHR and inflammation in mice. IL-13 suppressed STC1 release from 16HBE, whereas rhSTC1 blocked store-operated Ca2+ entry (SOCE) by suppressing stromal interaction molecule 1 (STIM1) and further inhibited ASM cell contractility by suppressing Ca2+ -dependent myosin light chain (MLC) phosphorylation. Our data indicate that STC1 deficiency in asthmatic airways promotes STIM1 hyperactivity, enhanced ASM contraction, and AHR. STC1 may be a candidate EpDRF.

Airway epithelium-derived relaxing factor: myth, reality, or naivety?

The presence of a healthy epithelium can moderate the contraction of the underlying airway smooth muscle. This is, in part, because epithelial cells generate inhibitory messages, whether diffusible substances, electrophysiological signals, or both. The epithelium-dependent inhibitory effect can be tonic (basal), synergistic, or evoked. Rather than a unique epithelium-derived relaxing factor (EpDRF), several known endogenous bronchoactive mediators, including nitric oxide and prostaglandin E2, contribute. The early concept that EpDRF diffuses all the way through the subepithelial layers to directly relax the airway smooth muscle appears unlikely. It is more plausible that the epithelial cells release true messenger molecules, which alter the production of endogenous substances (nitric oxide and/or metabolites of arachidonic acid) by the subepithelial layers. These substances then diffuse to the airway smooth muscle cells, conveying epithelium dependency.

Tracheal epithelium cell volume responses to hyperosmolar, isosmolar and hypoosmolar solutions: relation to epithelium-derived relaxing factor (EpDRF) effects.

In asthmatic patients, inhalation of hyperosmolar saline or D-mannitol (D-M) elicits bronchoconstriction, but in healthy subjects exercise causes bronchodilation. Hyperventilation causes drying of airway surface liquid (ASL) and increases its osmolarity. Hyperosmolar challenge of airway epithelium releases epithelium-derived relaxing factor (EpDRF), which relaxes the airway smooth muscle. This pathway could be involved in exercise-induced bronchodilation. Little is known of ASL hyperosmolarity effects on epithelial function. We investigated the effects of osmolar challenge maneuvers on dispersed and adherent guinea-pig tracheal epithelial cells to examine the hypothesis that EpDRF-mediated relaxation is associated with epithelial cell shrinkage. Enzymatically-dispersed cells shrank when challenged with ≥10 mOsM added D-M, urea or NaCl with a concentration-dependence that mimics relaxation of the of isolated perfused tracheas (IPT). Cells shrank when incubated in isosmolar N-methyl-D-glucamine (NMDG) chloride, Na gluconate (Glu), NMDG-Glu, K-Glu and K2SO4, and swelled in isosmolar KBr and KCl. However, isosmolar challenge is not a strong stimulus of relaxation in IPTs. In previous studies amiloride and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) inhibited relaxation of IPT to hyperosmolar challenge, but had little effect on shrinkage of dispersed cells. Confocal microscopy in tracheal segments showed that adherent epithelium is refractory to low hyperosmolar concentrations that induce dispersed cell shrinkage and relaxation of IPT. Except for gadolinium and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), actin and microtubule inhibitors and membrane permeabilizing agents did not affect on ion transport by adherent epithelium or shrinkage responses of dispersed cells. Our studies dissociate relaxation of IPT from cell shrinkage after hyperosmolar challenge of airway epithelium.

Effects of cytokines on mechanical and epithelial bioelectric responses to methacholine and hyperosmolarity in guinea-pig airways: An in vitro study

We observed previously that lipopolysaccharide (LPS) 18 h after i.p. injection of guinea pigs increased transepithelial potential difference ( V t), hyperpolarization responses to methacholine, and hyperosmolarity-induced, epithelium-derived relaxing factor (EpDRF)-mediated relaxation responses, in excised and perfused tracheal segments. To investigate their roles in these changes, the effects of cytokines on in vitro epithelial bioelectric and smooth muscle mechanical responses were investigated using the isolated, perfused trachea preparation. Tracheas were incubated (6 h) with LPS or IL-1β, IL-4, IL-13, IFN-γ, TNF-α, singly or in combination. Incubation with LPS and cytomix (IL-1β + IFN-γ + TNF-α together) had no effect on muscle reactivity to methacholine, but potentiated D-mannitol-induced relaxation. Individually, IL-1β and IFN-γ inhibited methacholine-induced contractions and potentiated D-mannitol-induced relaxation responses. TNF-α increased contractions to methacholine but had no effect on relaxation responses to D-mannitol. Methacholine elicited hyperpolarization in low concentrations and depolarization in high concentrations. The individual cytokines decreased the hyperpolarization response to low methacholine concentrations and increased the depolarization response to high methacholine concentrations but had no effect on V t responses to D-mannitol. Cytomix did not affect V t responses to methacholine, but potentiated both the hyperpolarization and depolarization responses to D-mannitol. In Ussing chambers all agents except IL-1β and IFN-γ increased V t; IL-1β decreased slightly but none of the other agents affected transepithelial resistance ( R t). The results indicate that cytokines and LPS alter smooth muscle reactivity to methacholine, potentiate EpDRF-mediated relaxation responses and, thereby, mimic the effects of LPS treatment in vivo, but do not recapitulate LPS' effects on V t responses.

Hyperosmolarity-Induced Dilation and Epithelial Bioelectric Responses of Guinea Pig Trachea in Vitro: Role of Kinase Signaling

Exercise-induced airway obstruction is thought to involve evaporative water loss and hyperosmolarity of the airway surface liquid. Hyperosmolar challenge of the epithelium of isolated, perfused guinea pig trachea rapidly alters transepithelial potential difference (V(t)), and it elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). In many cell types, protein kinases mediate responses to hyperosmolarity and regulatory volume increase. In this study, inhibitors were used to investigate the involvement of kinases and phosphatases in bioelectric responses of epithelium to hyperosmolarity and their possible relationship to EpDRF-mediated relaxation. After contraction of the perfused trachea with extraluminal methacholine, D-mannitol applied intraluminally (< or = 80 mosM) increased V(t) and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased V(t). In tracheas exposed to 30 mosM D-mannitol (approximately EC(50)), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) and SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole] (p38 inhibitors) potentiated the dilation, whereas SP 600125 [anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone] and dicumarol [c-Jun NH(2)-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO(2) (mitogen-activated protein kinase stress inducer) and Na(3)VO(4) (protein tyrosine phosphatase inhibitor) inhibited the hyperpolarization. Large increases in the phosphorylation of p38 and JNK occurred at concentrations higher than those needed to elicit functional responses. The phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and Na(3)VO(4) did not affect the V(t) responses, but they inhibited methacholine-induced constriction; SP 600125 and dicumarol potentiated, and chelerythrine inhibited, methacholine-induced epithelial hyperpolarization. These results suggest that JNK, PKC, and phosphatase(s) are involved in hyperosmolarity-induced hyperpolarization of the tracheal epithelium but that p38 is involved in EpDRF-mediated relaxation.

Altered ion transport and responsiveness to methacholine and hyperosmolarity in air interface-cultured guinea-pig tracheal epithelium

Introduction: Challenge of guinea-pig tracheal epithelium with hyperosmolar solution alters ion transport and evokes the release of epithelium-derived relaxing factor (EpDRF). Cultured tracheal epithelial cells (CE) offer the potential to examine biochemical pathways related to EpDRF release, but whether the bioelectric properties and responses of fresh, adherent epithelial cells (FE) are modeled by CE has not been established. Methods: Tracheal epithelial cells grown in air-interface culture and fresh tracheal segments were mounted in Ussing chambers to determine short circuit current ( I sc) and transepithelial resistance ( R t) and to compare responses to transport inhibitors, methacholine and hyperosmolarity. Results: Significant differences in basal I sc and R t between FE and CE were observed ( I sc, 41.3 ± 3.5 and 8.5 ± 0.8 μA/cm 2, P < 0.05; R t, 106 ± 7 and 422 ± 4 Ω cm 2, P < 0.05; respectively); basal spontaneous potential difference values were not different (4.2 ± 0.3 and 3.4 ± 0.3 mV, respectively). Amiloride (mucosal, 3 × 10 − 5 M), bumetanide (basolateral, 10 − 5 M) and ouabain (basolateral, 10 − 5 M) reduced I sc equally in FE and CE. In contrast, NPPB (10 − 5 M) in the presence of amiloride had a differential effect, decreasing I sc by 11% in FE and 71% in CE ( P < 0.05). Iberiotoxin (basolateral, 10 − 7 M) was without effect in either preparation. In FE, serosal methacholine (3 × 10 − 5 M) elicited an NPPB-insensitive monotonic increase in I sc, but in CE caused a large, transient, NPPB-inhibitable increase which was followed by an NPPB-resistant plateau. Addition of apical d-mannitol (0.3–267 mosM) to increase osmolarity decreased I sc in FE, whereas in CE d-mannitol initially increased (0.3–84.3 mosM) and then decreased (84.3–267 mosM) I sc. Discussion: Cell culture causes substantial changes in the bioelectric and pharmacological properties of respiratory epithelium. Caution should be exercised when using CE as a substitute for FE in studies of ion transport- and cell volume-dependent processes.

Popcorn worker's lung: In vitro exposure to diacetyl, an ingredient in microwave popcorn butter flavoring, increases reactivity to methacholine☆

Workers who inhale microwave popcorn butter flavorings experience decrements in lung function and can develop clinical bronchiolitis obliterans, i.e., "popcorn worker's lung" (Kreiss, K., Gomaa, A., Kullman, G., Fedan, K., Simoes, E.J., Enright, P.L., 2002. Clinical bronchiolitis obliterans in workers at a microwave-popcorn plant. N. Engl. J. Med. 347, 330-338.). In a rat inhalation model, vapors of an artificial butter flavoring damaged the epithelium of the upper and lower airways (Hubbs, A.F., Battelli, L.A., Goldsmith, W.T., Porter, D.W., Frazer, D., Friend, S., Schwegler-Berry, D., Mercer, R.R., Reynolds, J.S., Grote, A., Castranova, V., Kullman, G., Fedan, J.S., Dowdy, J., Jones, W.G., 2002. Necrosis of nasal and airway epithelium in rats inhaling vapors of artificial butter flavoring. Toxicol. Appl. Pharmacol. 185, 128-135.). Diacetyl, a butter flavoring component, is a major volatile ketone in the popcorn-processing workplace. We investigated the effects of diacetyl on epithelium of guinea pig isolated airway preparations and the effects of diacetyl in vitro on reactivity to bronchoactive agents. In the isolated, perfused trachea preparation, diacetyl added to the intraluminal (mucosal) bath elicited responses that began with contraction (threshold ca. 3 mM) and ended with relaxation. After a 4-h incubation with intraluminal diacetyl (3 mM), contractions to extraluminal (serosal) methacholine (MCh) were slightly increased; however, sensitivity to intraluminally (mucosally) applied MCh was increased by 10-fold. Relaxation responses of MCh (3 x 10(-7) M)-contracted tracheas to extraluminally applied terbutaline and intraluminally applied 120 mM KCl, to evoke epithelium-derived relaxing factor release, were unaffected by diacetyl. Exposure of the tracheal epithelium in Ussing chambers to diacetyl decreased transepithelial potential difference and resistance. These findings suggest that diacetyl exposure compromised epithelial barrier function, leading to hyperreactivity to mucosally applied MCh. The respiratory epithelium appears to serve as an initial target for the toxic effects of diacetyl in the airways.

Effects of Na + and Cl − Channel Blockers on Guinea‐pig (GP) Isolated Tracheal epithelial cell (EC) shrinkage in response to hyperosmolar challenge

In GP trachea that are precontracted with methacholine (MCh; 3H10−7 M), hyperosmolar D-mannitol (D-M) induces changes in epithelial Na+ and ClΓ transport and smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). We examined the effects of sequentially-added ion channel blockers, MCh and D-M (30 mosM) on EC cell volume (CV) changes in ECs isolated enzymatically (2% protease, 1 h) from GP-trachea and suspended in Krebs-Henseleit solution, under conditions in which these agents are used in the isolated, perfused trachea preparation to examine D-M-induced EpDRF release. CV was measured with a cell sizer. D-M elicited shrinkage of EC (~15 %) under conditions in which it caused EpDRF-mediated relaxation. The Na+ channel blocker, amiloride (A; 10−4 M), did not affect EC CV. MCh had no effect on CV (±A). Shrinkage stimulated by D-M was unaffected by A. On the other hand, incubation with the ClΓ blocker, DIDS (10−4 M), resulted in an immediate and prolonged decrease in CV (~9%). Again, MCh had no effect on CV (±DIDS). However, in the presence of DIDS the CV decrease in response to D-M was diminished. Thus, the inhibitory effect of A on EpDRF release is not due to an effect on EC shrinkage, whereas the inhibitory effect of DIDS on EpDRF release may be linked to the decrease in the CV response. The findings and conclusions in this abstract have not been formally disseminated by the National Institute for Occupational Safety and Health and should not be construed to represent any agency determination of policy. Funded by NIOSH.

Guinea‐pig Tracheal Epithelial Cell (EC) Shrinkage Induced by Hyperosmolar Challenge

Hyperventilation during exercise is thought to increase the osmolarity of airway surface liquid. Hyperosmolar challenge of EC with mucosal D-mannitol (D-M) in methacholine (MCh)-contracted guinea-pig isolated, perfused trachea elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). To understand the relationship between EpDRF release and hyperosmolarity-induced changes in cell volume (CV), we examined CV changes in response to challenge of EC with 120 mosM D-M. EC were isolated from tracheas by protease digestion (2%; 1 h) and suspended in Krebs-Henseleit solution. Suspended EC remained viable for more than 5 h as indicated by active beating of cilia and exclusion of trypan blue (90 to 95%). CV was measured using a cell sizer. D-M induced a rapid decrease in CV (~9% shrinkage) during the first minute. The peak CV decrease occurred between 3 to 5 min (~22% shrinkage). No increase in CV was observed during the next 5 h. The results indicate that hyperosmolar conditions which cause EpDRF-mediated relaxation also evoke EC shrinkage. The rapid onset of the relaxation and CV responses could suggest that signaling pathways are shared. The findings and conclusions in this abstract have not been formally disseminated by the National Institute for Occupational Safety and Health and should not be construed to represent any agency determination of policy. Funded by NIOSH.

Bronchial epithelium-associated pulmonary arterial muscle relaxation in the rat is absent in the fetus and suppressed by postnatal hypoxia

We recently reported the existence of a bronchial epithelium-derived relaxing factor (BrEpRF) capable of reducing pulmonary arterial smooth muscle force generation in the newborn rat. We reasoned in this study that BrEpRF has physiological significance in the control of pulmonary vascular tone. We hypothesized that the release and/or activity of this factor can be stimulated and is suppressed prenatally or under hypoxic conditions postnatally. Therefore, we evaluated the pathways stimulated by the BrEpRF in fetal and newborn rat intrapulmonary arteries mounted with their adjacent bronchi in a wire myograph under both normoxic and hypoxic conditions. Under normoxic conditions, BrEpRF release/activation was observed in newborn vessels following methacholine stimulation of M(2) muscarinic receptors, which was mediated via a nitric oxide (NO)-dependent mechanism involving the phosphatidylinositol 3-kinase pathway. Hypoxia suppressed the BrEpRF-dependent modulation of basal and methacholine-induced pulmonary arterial muscle tone in newborn vessels without altering endothelium-dependent or -independent NO-mediated relaxation. In fetal pulmonary arteries studied under normoxic conditions, BrEpRF neither was active under basal conditions nor could it be stimulated with methacholine. We conclude that release/activation of the BrEpRF occurs by an oxygen-dependent mechanism in the newborn and is suppressed during late fetal life. These results suggest that the BrEpRF may be involved in postnatal adaptation of the pulmonary circulation and that its suppression may contribute to hypoxic pulmonary vasoconstriction.

Effects of lipopolysaccharide on epithelium-dependent relaxation in coaxial bioassay

This study investigated the effects of airway inflammation elicited by intraperitoneal and intratracheal lipopolysaccharide administration to guinea pigs on the activity of tracheal epithelium-derived relaxant factor (EpDRF). Acetylcholine induced epithelium-dependent relaxation in precontracted rat anococygeus muscle placed in guinea pig trachea (coaxial bioassay). Indomethacin, N-nitro- l-arginine methyl ester, aminoguanidine and l-canavanine did not alter this relaxation excluding the role of prostaglandins and nitric oxide (NO). Intraperitoneal lipopolysaccharide potentiated the acetylcholine response, which was reversed by aminoguanidine and l-canavanine while intratracheal lipopolysaccharide inhibited acetylcholine-induced relaxation. Lipopolysaccharide pretreatments did not cause epithelial damage but induced inflammatory cell infiltration. These results suggested that systemic lipopolysaccharide administration did not alter the EpDRF response but resulted in NO synthase induction, thus NO participated in relaxation to acetylcholine in coaxial bioassay system. On the other hand, airway inflammation induced by intratracheal lipopolysaccharide attenuated the synthesis/release of EpDRF without altering the epithelium morphology.

Hyperosmolar solution effects in guinea pig airways. IV. Lipopolysaccharide-induced alterations in airway reactivity and epithelial bioelectric responses to methacholine and hyperosmolarity.

We investigated the in vivo and in vitro effects of lipopolysaccharide (LPS) treatment (4 mg/kg i.p.) on guinea pig airway smooth muscle reactivity and epithelial bioelectric responses to methacholine (MCh) and hyperosmolarity. Hyperosmolar challenge of the epithelium releases epithelium-derived relaxing factor (EpDRF). Using a two-chamber, whole body plethysmograph 18 h post-treatment, animals treated with LPS were hyporeactive to inhaled MCh aerosol. This could involve an increase in the release and/or actions of EpDRF, because LPS treatment enhanced EpDRF-induced smooth muscle relaxation in vitro in the isolated perfused trachea apparatus. In isolated perfused tracheas the basal transepithelial potential difference (Vt) was increased after LPS treatment. The increase in Vt was inhibited by amiloride and indomethacin. Concentration-response curves for changes in Vt in response to serosally and mucosally applied MCh were biphasic (hyperpolarization, <3 x 10(-7)M; depolarization, >3 x 10(-7)M); MCh was more potent when applied serosally. The hyperpolarization response to MCh, but not the depolarization response, was potentiated after LPS treatment. In both treatment groups, mucosally applied hyperosmolar solution (using added NaCl) depolarized the epithelium; this response was greater in tracheas from LPS-treated animals. The results of this study indicate that airway hyporeactivity in vivo after LPS treatment is accompanied by an increase in the release and/or actions of EpDRF in vitro. These changes may involve LPS-induced bioelectric alterations in the epithelium.

Hyperosmolar solution effects in guinea pig airways. III. Studies on the identity of epithelium-derived relaxing factor in isolated perfused trachea using pharmacological agents.

Hyperosmolar challenge of airway epithelium stimulates the release of epithelium-derived relaxing factor (EpDRF), but the identity of EpDRF is not known. We examined the effects of pharmacological agents on relaxant responses of methacholine (3 x 10(-7) M)-contracted guinea pig perfused trachea to mucosal hyperosmolar challenge using D-mannitol. Responses were inhibited by gossypol (5 x 10(-6) M), an agent with diverse actions, by the carbon monoxide (CO) scavenger hemoglobin (10(-6) M), and by the heme oxygenase (HO) inhibitor zinc (II) protoporphyrin IX (10(-4) M). The HO inhibitor chromium (III) mesoporphyrin IX (10(-4) M) was not inhibitory, and the HO activator heme-L-lysinate (3 x 10(-4) M) did not evoke relaxant responses. The CO donor tricarbonyldichlororuthenium (II) dimer (2.2 x 10(-4) M) elicited small relaxation responses. Other agents without an effect on responses included: apyrase, adenosine, 6-anilino-5,8-quinolinequinone (LY83583), proadifen, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK 571), diphenhydramine, glibenclamide, HgCl2, tetrodotoxin, nystatin, alpha-hemolysin, 8-bromoguanosine 3',5'-cyclic monophosphothioate, Rp-isomer, 12-O-tetradecanoylphorbol-13-acetate, cholera toxin, pertussis toxin, thapsigargin, nifedipine, Ca(2+)-free mucosal solution, hydrocortisone, and epidermal growth factor. Cytoskeleton inhibitors, includingerythro-9-(2-hydroxyl-3-nonyl)adenine, colchicine, nocodazole, latrunculin B, and cytochalasins B and D, had no effect on relaxation responses. The results suggest provisionally that a portion of EpDRF activity may be due to CO and that the release of EpDRF does not involve cytoskeletal reorganization.

Hyperosmolar solution effects in guinea pig airways. I. Mechanical responses to relative changes in osmolarity.

In the guinea pig isolated perfused trachea contracted with serosal methacholine (MCh), increasing the osmolarity of the mucosal bathing solution elicits relaxation of smooth muscle mediated by epithelium-derived relaxing factor (EpDRF). The present study was undertaken to determine whether a specific modality of the hyperosmolar stimulus induced the relaxation response. Mucosal hyperosmolar challenge with D-mannitol, N-methyl-D-glucamine (NMDG)-chloride, NMDG-gluconate (NMDG-Glu), or urea elicited relaxation with equal potency. In contrast, hyperosmolar solutions at the serosal surface induced diverse, osmolyte-specific responses. In tracheae contracted with MCh, abrupt replacement of the mucosal modified Krebs-Henseleit solution (MKHS) with isosmolar osmolyte solutions to stimulate cell shrinkage elicited five discrete response patterns related to the membrane permeance of the solute, but increasing the osmolarity of the isosmolar solution via the further addition of the same solute always induced relaxation. Similarly, perfusion of the lumen with water induced a transient contraction, but subsequent addition of MKHS, or isosmolar D-mannitol, urea, NMDG-Glu, NaCl, or KCl induced relaxation. Subsequent hyperosmolar addition of the same osmolyte-evoked relaxation. Compatible osmolytes had no effect on smooth muscle tone and did not affect responses to hyperosmolar challenge. The results suggest that the airway epithelium acts as an osmolarity sensor, which communicates with airway smooth muscle through EpDRF. The mechanical responses of the smooth muscle resulting from changes in the osmotic environment are associated with discrete modalities of the osmolar stimulus, including membrane reflection of the particles, incremental change in osmolarity and directionality, but not cell shrinkage.

Effects of Various Reactive Oxygen Species on the Guinea Pig Trachea and Its Epithelium

Reactive oxygen species (ROS) are key factors playing important roles in tissue damage of airways under different pathological conditions. Effects of ROS (superoxide anion, H2O2 and hydroxyl radical) were recorded on isometric tension of intact and epithelium denuded, not precontracted guinea pig trachea. Superoxide anion was produced by xanthine/xanthine oxidase and hydroxyl radical either by FeSO4/H2O2 or FeSO4/ascorbic acid. In intact preparations, the muscle tension was unaffected by superoxide anion, while H2O2 and hydroxyl radical produced a biphasic response, contraction followed by relaxation. Both the amplitude and duration of contractions evoked by H2O2 were larger than those caused by hydroxyl radical producing systems. On denuded tracheal strips, superoxide anion elicited also a biphasic response, and the H2O2 and hydroxyl radical produced contractions were of higher amplitude and of longer duration than in intact tissues. Indomethacin pretreatment enhanced or slightly reduced the amplitude of contractions evoked by both H2O2 and hydroxyl radical on the intact and denuded preparations, respectively. Moreover, the duration of contractions of the trachea induced by oxidative systems was prolonged. Indomethacin did not affect the action of superoxide anion on the intact tissues and reduced the amplitude of the biphasic response on denuded ones. Nordihydroguaiaretic acid pretreatment did not alter the responses elicited by ROS in intact preparations and reduced their action on the denuded ones. Our results suggest that a) various ROS contract tracheal smooth muscle with simultaneous release of epithelium derived relaxing factors, b) epithelium possesses superoxide anion scavenging capacity which is high enough to protect smooth muscle from its actions, and c) cyclooxygenase products participate in relaxation and lipoxygenase products in contraction caused by ROS in the guinea pig trachea.

Osmotic regulation of epithelium-derived relaxing factor (EPDRF) release in airway epithelium

Osmotic regulation of epithelium-derived relaxing factor (EPDRF) release in airway epithelium

Role of endogenous nitric oxide in asthma.

Endogenous nitric oxide (NO) is an ubiquitous signaling molecule with important regulatory functions such as regulation of blood pressure, neurotransmission, and host and immune defense. In the respiratory tract, NO is formed and released by various sources including endothelial and epithelial cells, nerves, airway smooth muscle, and inflammatory cells. Recent evidence suggests that endogenous NO is the neurotransmitter of the nonadrenergic noncholinergic inhibitory (iNANC) system, the only bronchorelaxant neural pathway of human airways. A number of studies also suggest that in some species epithelium-derived NO accounts for the functional bronchoprotective role of the so-called epithelium-derived relaxing factor. In human airways, endogenous NO counteracts the bronchoconstriction induced by pharmacologic stimuli such as bradykinin, histamine, and methacholine. On the basis of these and other observations, it is suggested that a reduced synthesis and/or activity of endogenous NO may contribute to the pathogenesis of airway hyperresponsiveness that characterizes asthma and other respiratory disorders. This short paper summarizes the activities of endogenous NO in the airways of experimental animals and man, and discusses the evidence supporting the view that NO confers bronchoprotection.

Epithelium-dependent bronchodilatory activity is preserved in pig bronchioles after normothermic cardiopulmonary bypass.

Analogous to vascular endothelium, bronchial epithelium modulates bronchomotor activity by releasing epithelium-derived relaxing factors. Cardiopulmonary bypass (CPB) is associated with endothelial dysfunction. We examined whether CPB may be associated with bronchiolar epithelial dysfunction in pigs. Pigs were exposed to normothermic CPB for 1.5 h and then separated from CPB. Lung tissues were biopsied before and 30 min after CPB. For time control, lung tissues were biopsied at baseline and after 2 hr of anesthesia. Bronchioles measuring about 100 microm were dissected, and the epithelium was either left intact or denuded. Each bronchiolar segment was preconstricted with 10 microM 5-hydroxytryptamine and relaxation responses to nitroprusside 10(-9)-10(-4) M, isoproterenol 10(-9)-10(-4) M, or the inhaled anesthetics halothane or isoflurane 0-2.5 minimum alveolar anesthetic concentration were examined in vitro by videomicroscopy. Bronchiolar segments demonstrated concentration-dependent relaxation responses to each of the dilators examined. Epithelial denudation reduced bronchodilation to isoproterenol, isoflurane, and halothane, but not to nitroprusside. Bronchodilation was not significantly affected by CPB. We conclude that, unlike vascular endothelial function, porcine bronchiolar epithelium-modulated bronchomotor activity is not significantly affected by normothermic CPB. Normothermic cardiopulmonary bypass does not result in epithelial dysfunction in pigs. Epithelium-dependent and epithelium-independent bronchodilators may be equally effective before and after cardiopulmonary bypass.

Impairment of epithelium-dependent relaxation in coaxial bioassay by reactive oxygen species

The purpose of the present study was to investigate the effects of reactive oxygen species on the activity of epithelium-derived relaxant factor (EpDRF) released by guinea-pig tracheal epithelium. Reactive oxygen species were generated by the electrolysis of the physiological buffer in which the tissues were bathed. Epithelium-dependent relaxation induced by acetylcholine in precontracted rat anococcygeus muscle that was placed in epithelium-intact guinea-pig trachea (coaxial bioassay system) was significantly attenuated when the tissues were exposed to electrolysis. Impairment of the acetylcholine response was prevented by incubation with free radical scavengers prior to electrolysis. In isolated rings of guinea-pig trachea, the contractile responses elicited by acetylcholine, histamine and 5-hydroxytryptamine were not altered after electrolysis of the bathing solution. The results of the present study suggested that exposure to reactive oxygen species impaired EpDRF release from guinea-pig trachea epithelium but did not alter the contractility of tracheal smooth muscle.

Folkerts and Nijkamp reply.

Airway epithelium: more than just a source for epithelium-derived relaxing factors!