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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-1beta, IL-4, IL-13, IFN-gamma, TNF-alpha, singly or in combination. Incubation with LPS and cytomix (IL-1beta+IFN-gamma+TNF-alpha together) had no effect on muscle reactivity to methacholine, but potentiated D-mannitol-induced relaxation. Individually, IL-1beta and IFN-gamma inhibited methacholine-induced contractions and potentiated D-mannitol-induced relaxation responses. TNF-alpha 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-1beta and IFN-gamma increased V(t); IL-1beta 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.

The isolated, perfused trachea preparation has been used to compare reactivity of the intact airway in response to differential exposure of the mucosal (intraluminal) and serosal (extraluminal) surfaces to contractile and relaxant agonists and other agents, and to gain insight into the modulatory role of the epithelium and the pathways involved. The apparatus has also been configured for simultaneous measurement of transepithelial potential difference and changes in tracheal diameter, thereby providing parallel observations of epithelial and smooth muscle function and reactivity to drugs. The transepithelial potential difference is a product of transepithelial resistance and short circuit current, and the present study describes a novel isolated, perfused tracheal apparatus which allows simultaneous measurement of transepithelial potential difference, transepithelial resistance and mechanical responses of the smooth muscle. The apparatus was validated using well-known ion transport inhibitors [intraluminal amiloride and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB), extraluminal ouabain and bumetanide], bronchoactive agonists (extraluminal methacholine, histamine and terbutaline), and osmolytes (intraluminal d-mannitol and NaCl) to induce epithelium-derived relaxing factor-mediated relaxations. This apparatus will facilitate investigation of interactions between the epithelium and smooth muscle in airways that retain their in situ structure, and signaling mechanisms potentially involved in the regulation of airway smooth muscle tone.

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.

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 microA/cm(2), P<0.05; R(t), 106+/-7 and 422+/-4 Omega 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 x 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 (3x10(-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.

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 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.

Osmotic challenge of airways alters the bioelectric properties of the airway epithelium and induces the release of factors that modulate smooth muscle tone. Recent studies in our laboratory suggested that methacholine-contracted airways relax in response to incremental increases in osmolarity, rather than from cell shrinkage or absolute solute concentration. In the present study, guinea pig tracheae were mounted in Ussing chambers to elucidate the bioelectric effects of challenge of the epithelium with hyperosmolar and isosmolar solutions. Transepithelial short-circuit current (Isc) across tracheae stimulated with basolateral methacholine was inhibited by apical amiloride, apical 5-nitro-2-(3-phenylpropylamino)benzoic acid, basolateral bumetanide, basolateral ouabain, and Cl(-)-free solution, but not by basolateral iberiotoxin. Apical hyperosmolar challenge with NaCl variably decreased or increased Isc, but D-mannitol (D-M) always inhibited Isc; bumetanide attenuated decreases in Isc. The effects of the transport blockers depended upon whether Isc was initially decreased or increased. Unique concentration-dependent changes in Isc and transepithelial resistance (Rt) were observed when ionic (NaCl and KCl), nonionic impermeant (D-M and sucrose), and nonionic permeant (urea) osmolytes were added to the apical and basolateral baths. At concentrations that doubled the osmolarity of the apical bath, D-M, urea, and N-methyl-D-glucamine-gluconate (NMDG-Glu) decreased Isc. Apical isosmolar NMDG-Glu solution decreased Isc, and additional NMDG-Glu caused a further decrease in Isc. Inclusion of one permeant ion, either Na+,K+, or Cl-, reversed the response to apical isosmolar and hyperosmolar solutions. Thus, bioelectric responses of the airway epithelium to hyperosmolar solution are induced by incremental increases 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.

The relationship between epithelial bioelectric events and epithelium-dependent relaxant and contractile responses of airway smooth muscle in response to hyperosmolar and hypo-osmolar solutions was investigated in guinea pig isolated trachea. Tracheae were perfused with normal or nonisosmotic modified Krebs-Henseleit solution while simultaneously monitoring transepithelial potential difference (VT) and contractile and relaxant responses of the muscle. Baseline VT was -10.1 to -13.3 mV (distal and proximal ends, respectively). Intraluminal amiloride (10(-4) M) induced a 3.7-mV depolarization, verifying that the VT was of epithelial origin. Extraluminal methacholine (3 x 10(-7) M; EC50) caused hyperpolarization and smooth muscle contraction; intraluminal methacholine had very little effect. Increasing intraluminal bath osmolarity via addition of 240 mOsM NaCl or KCl caused an immediate and prolonged depolarization and epithelium-dependent relaxation. Increasing intraluminal bath osmolarity with sucrose evoked similar responses, except that an immediate, transient hyperpolarization and contraction preceded the depolarization and relaxation. Increasing extraluminal bath osmolarity with 240 mOsM NaCl induced depolarization and a longer lasting epithelium-dependent relaxation, whereas extraluminally added 240 mOsM KCl induced a complex smooth muscle response (i.e., transient relaxation followed by contraction), which was accompanied by prolonged depolarization. Intraluminal hypo-osmolarity produced a transient hyperpolarization followed by depolarization along with contraction of the smooth muscle. Bioelectric responses always preceded smooth muscle responses. These results suggest that bioelectric events in the epithelium triggered by nonisosmotic solutions are associated with epithelium-dependent responses in tracheal smooth muscle.

The characteristics of endothelin (ET) release from guinea-pig tracheal epithelium were investigated, including examination of the effects of several pro-inflammatory mediators. In confluent cultured guinea-pig tracheal epithelial cells (GPTECs) there was a time-dependent basal release of immunoreactive ET (ir-ET) from 4-48 h. Basal ir-ET release from GPTECs was unaffected by the peptidase inhibitors, thiorphan (10 microM), benzamidine (1 mM), pepstatin-A (30 microM), aprotinin (1 microgram/ml), bacitracin (20 micrograms/ml) or leupetin (50 microM), but was inhibited by phosphoramidon, the neutral metalloprotease inhibitor (IC50 = 16.8 microM), or the calcium chelator, EGTA (10 mM). There was little ir-ET release 1 day after placing GPTECs in culture, although appreciable release (> 10-fold higher) was detected on days 5 and 7. No significant release of ir-ET was demonstrated from intact guinea-pig trachea. Human thrombin (0.1-10 U/ml), LPS (0.3-10 ng/ml) and the phorbol ester, phorbol 12-myristate-13-acetate (0.1 nM-1 microM), significantly increased ir-ET release, whereas TNF-alpha (0.1-10 ng/ml), RANTES (0.1-100 nM), IL-1 (0.01-10 ng/ml), bradykinin (1 nM-10 microM), CGRP (0.01 nM-1 microM), PDGF (0.1-3 ng/ml), Sar9, Met(O2)11-Sub P, Nle10-NKA 4-10 and senktide (selective NK-1, NK-2 and NK-3 receptor agonists, respectively; 1 nM-10 microM), LTD4 (1 nM-10 microM) or major basic protein (10 nM-1 microM) were without stimulatory effect. The results indicate that the enzyme responsible for the basal release of ET from cultured GPTECs is a Ca(2+)-dependent, phosphoramidon-sensitive, neutral metalloprotease. Furthermore, normally there is minimal ET release from guinea-pig airway epithelium but this can be increased markedly by culturing the cells to confluence, and by select pro-inflammatory mediators.

Effects of cationic proteins on electrolyte transport depend on the specific channels and electrochemical driving forces expressed by epithelia. The bioelectric responses of canine tracheal and bronchial epithelia (CTE and CBE, respectively) to a polycationic protein, protamine, were therefore compared. CTE exhibited a brief transient inhibition of shortcircuit current (I(SC)) followed by a prolonged increase of 18 microA/cm2. The apical membrane transiently hyperpolarized and then depolarized by 11 mV. The increase in I(SC) was inhibited by bumetanide. Adenosine 3',5'-cyclic monophosphate, ionomycin, and thapsigargin attenuated the response whereas indomethacin or hypotonic solution had no effect, indicating that latent cystic fibrosis transmembrane regulator Cl- channels were activated. CBE preparations exhibited a 4-microA/cm2 decrease in I(SC), 2 mV hyperpolarization of the apical membrane, and an increase in fractional resistance of the apical membrane on exposure to protamine. These results were consistent with inhibition of the Na+ conductance in the apical membrane of CBE and confirmed that polycationic proteins exert differential effects on Cl- secretory and Na+ absorptive airways.

The utility of a transgenic murine model of cystic fibrosis (CF) lung disease will likely depend on whether the mouse's proximal airway epithelium is characterized by Na(+)- and Cl(-)-conductive pathways comparable to those found in human airways. Therefore, the electrophysiological properties of primary cultures of mouse tracheal epithelium (MTE) were investigated using double-barreled, Cl(-)-selective microelectrodes. Epithelial cells isolated from freshly excised mouse tracheae formed confluent polarized monolayers on permeable collagen supports and developed significant transepithelial potential differences (approximately -10 mV) within 5-6 days postseeding. Under basal conditions, the MTE monolayers had an equivalent short-circuit current (Ieq) of -21.1 +/- 2.1 microA/cm2 and a transepithelial resistance of 424 +/- 49 omega.cm2. Intracellular measurements indicated that the apical (Va) and basolateral (Vb) membrane potential differences were -16.9 +/- 1.5 and -25.4 +/- 1.5 mV, respectively; apical membrane fractional resistance was 0.36 +/- 0.03; and intracellular Cl- activity was 56.1 +/- 2.3 mM. The presence of an apical Na+ conductance was demonstrated by luminal amiloride application (10(-4)M), which decreased Ieq, hyperpolarized Va, and increased the fractional resistance of the apical membrane. The presence of an apical Cl- conductance was demonstrated by substitution of Cl- with gluconate in the luminal bath, which decreased intracellular Cl- activity and increased the fractional resistance of the apical membrane. Luminal application of ATP (10(-4) M was also found to increase the rate of Cl- secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Disaggregated airway epithelial cells replicate in serum-free media containing supraphysiologic concentrations of insulin. To examine the hypothesis that the type 1 insulin-like growth factor (IGF) receptor mediates the mitogenic action of insulin on these cells, we studied the mitogenic effects of IGF-I and insulin, and the expression of type 1 IGF receptors in primary cultures of adult canine tracheal epithelial cells. Isolated tracheal epithelial cells were grown in varying concentrations of IGF-I or insulin in Ham's F12 medium supplemented with transferrin, cholera toxin, and endothelial cell growth supplement. Both IGF-I and insulin increased DNA synthesis (measured as [3H]thymidine incorporation into DNA) and cell number in a concentration-dependent fashion, but IGF-I was at least 20 to 60 times more potent than insulin in its mitogenic effects. No additive or synergistic effect was observed with the simultaneous addition of IGF-I and insulin in maximally effective doses. A monoclonal antibody directed against the type 1 IGF receptor (alpha IR3) blocked the mitogenic activity of both IGF-I and insulin. Affinity labeling of type 1 IGF receptors by covalent cross-linking with disuccinimidyl suberate demonstrated the tracheal epithelial cell IGF-I binding moiety to have a relative molecular weight of 130,000 D. Binding of [125I]IGF-I to this protein was inhibited by low concentrations of IGF-I, relative to insulin, and by alpha IR3. An 11-kb transcript characteristic of mRNA for the type 1 IGF receptor was recognized in poly(A+) RNA derived from cultured canine tracheal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin (INS), endothelial cell growth supplement (ECGS), transferrin (TF), cholera toxin (CT), hydrocortisone (HC), triiodothyronine (T3), and epidermal growth factor (EGF) were systematically examined for their effects on proliferation, ion transport activity, and morphological differentiation of canine tracheal epithelial (CTE) cells in culture. INS, ECGS, TF, and CT increase proliferation of CTE cells cultured on plastic Petri dishes but exhibit no acute effect on the bioelectric activity of freshly excised CTE. CT increases amiloride-insensitive transepithelial ion transport across both freshly excised canine trachea and CTE cultures. EGF has no effect on proliferation of CTE cells on plastic but induces hyperplasia of CTE cells on collagen matrices. EGF does not alter basal transepithelial ion transport across cultures but increases cellular responsiveness to beta-adrenergic stimulation. HC and T3 have no effect on proliferation or transepithelial bioelectric properties of CTE cells but improve morphological differentiation yielding cultures of complex epithelia containing cuboidal ciliated and nonciliated cells. These results demonstrate that growth and function of cultured CTE cells are affected by specific growth factors.