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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Hyperosmolar Solution Effects in Guinea Pig Airways. I. Mechanical Responses to Relative Changes in Osmolarity

Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia (J.S.F., J.A.D., R.A.J.); Department of Pharmacology and Toxicology, Robert C. Byrd Health Sciences Center of West Virginia University, Morgantown, West Virginia (J.S.F., R.A.J.); and Department of Physiology, The Brody School of Medicine at East Carolina University, Greenville, North Carolina (M.R.V.S.)

Received March 14, 2003; accepted October 8, 2003.

	Abstract

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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 effects of hyperosmolar solutions on airways have been studied in vitro to gain insight into the mechanisms of the effect of exercise and hyperosmolar aerosols in normal and asthmatic individuals. To date, the alteration that occurs in asthmatic patients has not been defined in in vitro studies or in animal models, but several in vitro studies have described the effects of hyperosmolarity on the airway epithelium and smooth muscle. Application of hyperosmolar solution to airway epithelium leads to the release of epithelium-derived relaxing factor (EpDRF) and relaxation of airway smooth muscle (Munakata et al., 1988; Fedan et al., 1990, 1999), bioelectric changes reflective of altered ion transport in the epithelium (Willumsen et al., 1994; Dortch-Carnes et al., 1999; Fedan et al., 2003), and shrinkage of epithelial cells (Willumsen et al., 1994; Hjoberg, 1999). In guinea pig tracheal epithelium, EpDRF is released by hyperosmolar challenge of the apical or basolateral membranes (Dortch-Carnes et al., 1999; Fedan et al., 1999). EpDRF is neither nitric oxide nor a prostanoid (Munakata et al., 1990; Spina and Page, 1991; Fedan et al., 1999; Johnston et al., 2003) but has attributes that resemble, in part, but not completely, carbon monoxide (Fedan et al., 2003).

Most cells placed in a hyperosmolar environment shrink initially and then undergo a regulatory volume increase, the latter involving early electrolyte accumulation and delayed uptake of compatible osmolytes, e.g., betaine, myo-inositol, sorbitol, and taurine (Lang et al., 1998). The response of the airway epithelium to hyperosmolarity in some species is dependent upon the epithelial surface that is exposed to hyperosmolar solution. Hyperosmolar challenge of the mucosal surface of human cultured nasal epithelium caused cell shrinkage and epithelial depolarization, whereas serosal exposure to hyperosmolar solution did not (Willumsen et al., 1994). Dog tracheal epithelial cells depolarized in response to mucosally but not serosally applied hyperosmolar solution; in contrast, cell shrinkage occurred only when the epithelium was challenged with hyperosmolar solution on the basolateral surface (Man et al., 1984). In guinea pig trachea, mucosally applied hyperosmolarity elicited shrinkage of the epithelium (Hjoberg, 1999).

The present study had several aims. The first was to determine whether mechanical responses of the perfused trachea to hyperosmolar challenge are polarized across the epithelium, and whether they show a dependence on the specific solute used to raise osmolarity. A second aim was to address the question of whether hyperosmolarity-induced EpDRF release occurs in response to epithelial cell shrinkage, absolute osmolarity, or a step-increase in osmolarity ("osmolar jump"). In studies (Krump et al., 1997; Szászi et al., 1997) of the relationship between cell shrinkage and protein phosphorylation, shrinkage was triggered both by elevating osmolarity with sucrose or by incubating the cells in an inorganic ion-free isosmolar solution; both conditions stimulated tyrosine phosphorylation, suggesting a cell volume dependence, not an osmolarity dependence, of the phosphorylating event. Therefore, to cause efflux of ions out of the epithelium and cell shrinkage, the trachea was perfused with isosmolar solutions of diverse permeant and nonpermeant solutes. Mechanical responses to these solutions were compared with those triggered by hyperosmolar solutions and osmolar jump. Third, we evaluated the possible role of compatible organic osmolytes in hyperosmolar solution-induced relaxation.

	Materials and Methods

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Animals. These studies were conducted in facilities accredited fully by the Association for the Assessment and Accreditation of Laboratory Animal Care International and were approved by the institutional Animal Care and Use Committee. Male guinea pigs (400-600 g), HsdPoc:DH from Harlan (Indianapolis, IN), monitored free of endogenous viral pathogens, parasites, and bacteria were used in all experiments. The animals were acclimated before use and were housed in filtered ventilated cages on -Dri virgin cellulose chips and hardwood -chips as bedding, provided HEPA-filtered air, Teklad 7006 diet, and tap water ad libitum, under controlled light cycle (12-h light) and temperature (22-25°C) conditions. The animals were anesthetized with sodium pentobarbital (65 mg/kg i.p.) and sacrificed by thoracotomy and bleeding before removing the trachea.

Isolated Perfused Trachea Preparation. The isolated perfused trachea preparation (Munakata et al., 1988) was used to measure responses of the smooth muscle elicited by the application of hyperosmolar solutions separately to the mucosal or serosal surfaces of the trachea. The smooth muscle lies in a band on the serosal surface of the trachea. To reach the muscle, agents applied to the mucosal surface must first diffuse across the epithelium.

As described previously (Fedan and Frazer, 1992), a 4-cm segment of trachea was removed, cleaned, and mounted onto a perfusion holder that contained indwelling side-hole catheters that were connected to the positive (inlet) and negative (outlet) sides of a differential pressure transducer. The holder was placed into a bath containing modified Krebs-Henseleit solution (MKHS; 37°C). This bath is referred to hereafter as the extraluminal (serosal) bath. The trachea was perfused (34 ml/min) with recirculating MKHS (37°C) from a separate, 30-ml bath, referred to as the intraluminal (mucosal) bath. Transmural pressure was adjusted to zero. Responses were measured as changes in the inlet minus outlet pressure difference (P; centimeters of H₂O) and recorded. A 1.5-h equilibration period was allowed before the experiment, during which the MKHS in both baths was changed at 15-min intervals.

Epithelium Removal. When appropriate, the trachea was denuded of epithelium before mounting by inserting a 5- to 6-cm piece of trimmed pipe cleaner brush into the lumen and withdrawing while rotating slowly (Fedan and Frazer, 1992).

Tracheal Strip Preparation. The trachea was removed, cleaned, and cut into transverse strips two cartilage-rings wide. Each strip was attached to a holder and placed in organ chambers containing MKHS for the measurement of isometric responses. An optimum resting force of 0.5 g was applied. The preparations were equilibrated for 1 h before any intervention, during which the MKHS was changed at 15-min intervals.

Concentration-Response Curves for Mechanical Responses of Perfused Trachea to Intraluminal and Extraluminal Hyperosmolar Solutions. After the equilibration period, the trachea was contracted with extraluminally added methacholine (MCh; 3 x 10^-7 M; approximate extraluminal EC₅₀ value; Fedan and Frazer, 1992). At the plateau of the response, the osmolyte of interest was added to the MKHS in the intraluminal or extraluminal baths in cumulative amounts to elevate osmolarity. At the conclusion of the additions, the intraluminal and extraluminal baths were washed at 15-min intervals for 1.5 h with fresh MKHS. The trachea was contracted again with extraluminally applied MCh, and, at the plateau of the response, the same osmolyte was added in cumulative amounts to the abluminal bath. In the results, the order of obtaining the two curves, i.e., intraluminal before extraluminal and extraluminal before intraluminal, is indicated with the following convention: IL-1 refers to the intraluminal curve obtained first, EL-2 to the extraluminal curve obtained second, EL-1 to the extraluminal curve obtained first, and IL-2 to the intraluminal curve obtained second. The responses to the osmolytes were quantified and expressed as a percentage of the MCh-induced contraction.

Mechanical Responses to Intraluminal, Isosmolar Non-MKHSs in Perfused Trachea. After the equilibration period ended, the tracheae were contracted with extraluminally added MCh (3 x 10^-7 M). At the plateau of the response, the solution perfusing the lumen was rapidly switched from MKHS to an isosmolar solution (oxygenated, 37°C) containing osmolyte dissolved in water. The osmolarity of the isosmolar solution was adjusted to the same osmolarity as the MKHS (Osmette A automatic osmometer; Precision Systems, Inc., Sudbury, MA). The pHs of the isosmolar solutions were D-M, 5.34; NaCl, 6.26; KCl, 6.26; urea, 8.41; NMDG-gluconcate (NMDG-Glu), 6.54; Na-Glu, 6.92; and NMDG-Cl, 7.4. Experiments were done using untitrated isosmolar solutions, and in separate experiments, solutions were titrated to pH 7.4 with NaOH or HCl.

Effect of Indomethacin on Mechanical Responses of Perfused Trachea to Intraluminal Isosmolar, Non-MKHSs. In separate experiments, after the control response to isosmolar intraluminal solution in extraluminal MCh (3 x 10^-7 M)-contracted preparations became stable, the intraluminal and extraluminal baths were washed at 15-min intervals with fresh MKHS for 1.5 h. Indomethacin (3 x 10^-6 M) was added to both baths and 30 min later the MCh-contracted preparations were perfused again with isosmolar solution of the same solute.

"Osmolar Jump" Experiments in Perfused Trachea. Two types of experiments were performed on MCh-contracted perfused trachea to examine the effects of rapid increase in osmolarity (osmolar jump) from two starting points. In the first, after perfusing the lumen with isosmolar osmolyte, the perfusing solution was made hyperosmolar by administering additional solute to the perfusate. In the second, the lumen of the trachea was abruptly perfused with distilled water. A contraction ensued. After stabilization of this response, the perfusion fluid was rapidly changed to MKHS (control) or one of several isosmolar solutions to increase osmolarity, i.e., an osmolar jump to normosmolarity. After stabilization of this response, the perfusion solution was made hyperosmolar through the addition of the same osmolyte to the perfusion solution, i.e., a hyperosmolar jump from isosmolar solution. Experiments were performed with epithelium-containing and -denuded tracheae.

Effect of Compatible Osmolytes on Airway Smooth Muscle. Tracheal strips were used to assess the pharmacological activity of compatible osmolytes. After the equilibration period, the strips were either left at basal tone or contracted with MCh (3 x 10^-7 M). An osmolyte (10^-4 M), either taurine, sorbitol, myo-inositol, or betaine, was added at the plateau of the response to MCh.

Effect of Compatible Osmolytes on Responses to Hyperosmolar Solution. The perfused trachea was used to examine whether responses to hyperosmolar solution are affected in the presence of compatible osmolytes. Hyperosmolar D-M was added to serosal MCh-contracted perfused trachea to obtain a control relaxation response. After changing the intraluminal and extraluminal MKHS every 15 min for 1 h, the trachea was incubated for 30 min with a mixture of taurine, sorbitol, myo-inositol, and betaine (each 10^-4 M) added to the intraluminal and extraluminal baths before the tracheae were contracted a second time with MCh and challenged with hyperosmolar D-M.

Drugs and Reagents. All drugs and reagents were from Sigma-Aldrich (St. Louis, MO).

MKHS. MKHS contained 113.0 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 25.0 mM NaHCO₃, and 5.7 mM glucose, pH 7.4 (37°C); gassed with 95% O₂, 5% CO₂. The osmolarity of MKHS was 281 ± 5 mosM.

Analysis of Results. Geometric mean EC₅₀ values were derived from least-squares analysis of a four-parameter logit curve fit. Statistical comparisons of EC₅₀ values were done using normally distributed -logEC₅₀ values. The results are expressed as mean ± S.E.; n is the number of separate experiments. Differences in the shapes of concentration-response curves for extraluminal and intraluminal D-M were evaluated using a general linear model for full factorial analysis of repeated measures optimized for polynomial contrast of doses (SPSS Science, Chicago, IL); F value was corrected for the correlation structure using the Greenhouse-Geisser epsilon factor (Holbert et al., 1990). Other results were analyzed for differences using Student's t test for paired or nonpaired samples, or analysis of variance, as appropriate. p < 0.05 was considered significant.

	Results

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Effect of Hyperosmolar MKHS in Perfused Trachea. Figure 1 illustrates that perfusion of the MCh-contracted trachea with increased-strength MKHSs containing all ingredients in 1.05-, 1.1-, 1.2-, and 2 times their normal concentrations (osmolarity elevated by 11.0, 21.5, 51.5, and 250 mosM, respectively), resulted in osmolar concentration-dependent relaxation responses of the smooth muscle. These responses occurred over a range of hyperosmolarity that is thought to occur during exercise, i.e., 40 to 60 mosM. A series of impermeant and permeant osmolytes were used subsequently to determine whether the chemical and physical properties of the osmotic particles affected the relaxation response (Fig. 2). Increasing the osmolarity of the intraluminal, or mucosal, bath induced relaxation, regardless of the osmolyte that was used. In contrast, the responses obtained after additions to the extraluminal, or serosal, bath were osmolyte-specific.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Effect of increased-strength MKHS on MCh (3 x 10-7 M MCh)-contracted perfused trachea. Top tracings, the trachea was contracted with MCh, whereas the lumen was perfused with normal MKHS. At the plateau of the response, the perfusion MKHS was rapidly changed to a MKHS in which the concentrations of all ingredients were elevated. Shown here are typical responses to 1.1-, 1.2-, and 2-fold-concentrated MKH solutions. Bottom, summary of osmolar concentration-dependent relaxation responses to increased-strength MKHSs of 1.05-, 1.1-, 1.2-, and 2-times its normal strength (1.05x, 1.1x, 1.2x, and 2x). The increments in the osmolarity of the MKH solutions were 11.0, 21.5, 51.5, and 250 mosM, respectively. n = 3, 4, 5, and 5 for 1.05-, 1.1-, 1.2-, and 2 times MKHS strength, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Concentration-response curves for responses to elevation of osmolarity with cumulative additions of NMDG-Cl (A and B), D-M (C and D), urea (E and F), and NMDG-Glu (G) to the IL or EL baths. The left panels (A, C, E, and G) show curves obtained when the intraluminal curve (IL-1) was obtained before the extraluminal curve (EL-2); the right panels (B, D, and F) depict the results when the extraluminal curve (EL-1) was obtained before the intraluminal curve (IL-2). Positive values on the ordinate signify relaxation and negative values signify contraction. When the extraluminal D-M curve was obtained after the intraluminal D-M curve the earliest responses to extraluminal D-M were contraction (C); contraction was less evident when the extraluminal curve was obtained first (D). Contraction responses to extraluminal urea were evident irrespective of the order of the curves; the responses plotted here are the net stable responses at each concentration, irrespective of whether a relaxation preceded a contraction (Fig. 3). The transient relaxations observed with extraluminal urea (Fig. 3) are not plotted here, but they occurred in concentrations higher than those that gave rise to contraction. It is noted that the concentrations of urea which initiated contraction and relaxation are similar (i.e., 2.67 mosM), whereas extraluminal urea was less potent than intraluminal urea at initiating relaxation. Responses to intraluminal and extraluminal NMDG-Glu contained transient contractions; the results plotted here summarize the net response after stabilization. *IL versus EL, p < 0.05. n = 6 separate experiments for IL before EL and EL before IL for each osmolyte.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Representative tracings showing the effects of increasing osmolarity in the intraluminal (top tracing) and extraluminal (bottom tracing) baths with D-M. The preparations were contracted with extraluminal 3 x 10-7 M MCh as indicated. In this figure, the intraluminal curve was obtained before the extraluminal curve; both were obtained from the same trachea. The numbers under the traces refer to the cumulative concentrations of D-M (milliosmolar), as follows: 1, 0.84; 2, 2.67; 3, 8.43; 4, 26.68; 5, 84.34; and 6, 266.80. Note that intraluminal D-M caused only relaxation; extraluminal D-M first caused contractions, and then transient contractions were followed by relaxations. Horizontal bar, 5 min; vertical bar, 1 cm of H2O.

Intraluminally applied D-M evoked only relaxation responses (Figs. 2 and 3). When the extraluminal D-M concentration-response curve (EL-2) followed the intraluminal curve (IL-1), extraluminal D-M evoked contraction initially, and at the highest concentrations a transient contraction preceded the relaxation. When the extraluminal concentration-response curve (EL-1) was obtained before the intraluminal curve (IL-2), at higher concentrations the preparations responded with a transient contraction followed by net relaxation. The reason for this transformation in the shape of the responses is not known. EL-1 responses were significantly smaller than IL-2 responses, indicating a greater reactivity of the preparations to intraluminal D-M than to extraluminal D-M. The unusual shape of the extraluminal curve in the IL-1 before EL-2 experiment, and the lack of an achievable maximum response for the extraluminal curve in the IL after EL experiment due to limits of solubility, required general linear modeling analysis to compare the extraluminal and intraluminal curves for differences; likewise, EC₅₀ values for extraluminally added D-M could not be calculated. The analysis indicated that the shapes of extraluminal and intraluminal curves in the two experimental protocols were significantly different. The -logEC₅₀ (osM) values for intraluminal D-M were unaffected by curve order (IL before EL, 1.75 ± 0.11; IL after EL, 1.92 ± 0.40; p > 0.05). As was observed with NMDG-Cl, the response to MCh before the extraluminal curve (EL-2; 3.26 ± 1.30 cm of H₂O) was significantly reduced compared with the response preceding the intraluminal curve (IL-1; 5.43 ± 1.55 cm of H₂O); no effect on responses to MCh was seen in the EL-1, IL-2 experiments.

In intact tracheae, intraluminally applied urea caused relaxation (Figs. 2 and 4), whereas extraluminal urea caused contraction at all concentrations (Fig. 4). At higher concentrations, the contraction was preceded by a transient relaxation. Intraluminally applied urea was significantly more potent than extraluminally applied urea (Table 1). In contrast to NMDG-Cl and D-M, the responses to MCh before the first and second curves were not different (data not shown). Contractile and relaxant responses to extraluminally and intraluminally added urea were abolished in the absence of the epithelium, indicating that they did not result from a direct effect on smooth muscle (n = 6 for IL-1, EL-2, and EL-1, IL-2 experiments; data not shown).

View larger version (8K): [in this window] [in a new window]   Fig. 4. Representative tracings showing the effects of increasing osmolarity in the intraluminal (top tracing) or extraluminal (bottom tracing) baths with urea (U). The preparations were contracted with extraluminal 3 x 10-7 M MCh as indicated. In this figure the intraluminal curve was obtained before the extraluminal curve; both were obtained from the same trachea. The numbers under the traces refer to the cumulative concentrations of urea (milliosmolar), as follows: 1, 0.84; 2, 2.67; 3, 8.43; 4, 26.68; 5, 84.34; 6, 266.80; and 7, 843.4. Note that intraluminal urea caused only relaxation; extraluminal urea first caused transient relaxations, and then relaxations were followed by contractions. Relaxations to urea occurred in lower concentrations in the intraluminal bath than in the extraluminal bath. Horizontal bar, 5 min; vertical bar, 1 cm of H2O.

Intraluminal and extraluminal NMDG-Glu elicited relaxation responses (Fig. 2) that were preceded, at the three highest concentrations, with a transient contraction. In five of six preparations, the highest intraluminal concentration of the solute (266.8 mosM) elicited a sustained contraction after the initial transient phase, thereby giving rise to an upward inflection of the curve. The osmolyte was more potent when applied intraluminally than extraluminally (Table 1).

Comparison of Osmolyte Potencies as Relaxants. There were no differences in the EC₅₀ values for the IL-1 curves and the IL-2 curves obtained with NMDG-Cl, D-M, urea, and NMDG-Glu (one-way analysis of variance; Table 1). The intraluminal potencies of these osmolytes were identical (except for NMDG-Cl, which was slightly but significantly more potent than urea and NMDG-Glu), and they also were similar to those for NaCl and KCl (Fedan et al., 1999). Thus, the stimulus to relaxation in response to mucosal hyperosmolarity seems to be independent of the solute.

Responses to Perfusion with Isosmolar Osmolytes. To evaluate whether the release of EpDRF in response to hyperosmolar solutions was linked to cell shrinkage or to detection of the hyperosmolar environment per se, the trachea was perfused with isosmolar solutions of osmolytes to initiate cell shrinkage (Szászi et al., 1997; Fig. 5). Not every osmolyte used in this study has been demonstrated previously to elicit shrinkage when epithelial cells are exposed to an isosmolar solution of the solute. However, the loss of isotonic conditions should stimulate shrinkage through efflux of K⁺, Cl^-, and/or Na⁺ and water.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Mechanical responses of the trachea to perfusion with isosmolar solutions. Five classes of response were obtained; a representative example of each class is shown at the top of the figure. The occurrence and frequency of each type of response is shown for each osmolyte as a ratio of the incidence divided by the number of experiments of that type (n), and as percentage in parentheses. The preparations were contracted with MCh (3 x 10-7 M; dot labeled A) while the trachea was perfused with normal MKHS. Where indicated (dot labeled B), the lumen was rapidly perfused with isosmolar solution of the solute while MCh remained present. For clarity, calibration bars for time and P have not been included with the response traces. The asterisks (*) denote the most prevalent response classes.

Abrupt perfusion with isosmolar solutions of permeant and impermeant osmolytes gave rise to five classes of response. Perfusion with NaCl induced sustained relaxation in 60% of the preparations and contraction in 20% of the tracheae; the remaining tracheae did not respond.

Isosmolar D-M, an impermeant nonionic organic osmolyte, induced a transient contraction in 94% of the preparations, and NMDG-Glu, a nonpermeant organic salt, induced a contraction in all the preparations. Thus, a strong association existed between impermeant osmolytes and contractile responses. In 81% of the preparations, the transient contraction in response to D-M was followed by a prolonged relaxation; in 13% of the preparations, only the transient contraction was observed.

Thirty-eight percent of the preparations exhibited only relaxation during perfusion with Na-Glu, and 62.5% exhibited only transient contraction. No preparations exhibited a mixture of the two responses. In contrast, relaxation responses to NMDG-Cl were observed in 70% of the tracheae, whereas only 7% of the tracheae exhibited relaxation when perfused with isosmolar NMDG-Glu. Thus, an association between the solution anion and the mechanical response begins to emerge. When Cl^- was present in the solution, 60 to 70% of the tracheae exhibited relaxation; when gluconate was substituted for Cl^- (NMDG-Glu), 60 to 90% of the tracheae exhibited contraction. Hence, variation in responses to isosmolar solutions of impermeant solutes was linked to differential responsiveness of tracheal preparations to substitution of impermeant ions for Cl^-.

Isosmolar KCl stimulated only large monotonic contractile responses in five of six preparations; in one preparation there was no response.

Sixty-seven percent of the tracheae exhibited only sustained contraction to isosmolar urea, a permeant solute (Fig. 6). Twenty-five percent exhibited sustained relaxation, but the response was not strong.

View larger version (8K): [in this window] [in a new window]   Fig. 6. Four classes of responses to perfusion of trachea with isosmolar urea (U). See Fig. 5 legend for details. This figure shows representative tracings of the responses obtained in n = 12 separate experiments. Most tracheae contracted in response to isosmolar urea. One trachea did not respond at all, whereas others responded with relaxation and contraction. Vertical bars, 2, 2, 5, and 5 cm of H2O for A, B, C, and D, respectively; horizontal bar, 5 min.

The pHs of the isosmolar solutions varied, but identical results were obtained in separate follow-up experiments with isosmolar solutions, which were adjusted to pH 7.4 (n = 3 for NaCl; n = 4 for D-M, urea, KCl, and Na-Glu).

The shape of the responses to the isosmolar osmolytes varied in tracheae from different animals, but the responses produced by a trachea from a given animal was, except for urea (see below), consistent and reproducible. The variability was not attributable to technique, protocol, or the individual performing the experiment, but reflected phenotypic differences between animals.

Effect of Indomethacin on Responses to Perfusion with Isosmolar Solutions. Indomethacin (3 x 10^-6 M) had no effect on the responses to isosmolar D-M, NaCl, KCl, and NMDG-Glu solutions (data not shown; respective n values for control and indomethacin experiments were D-M, 6 and 10; NaCl, 11 and 6; KCl, 3 and 4; and NMDG-Cl, 4 and 4). The effect of indomethacin on isosmolar urea-induced responses could not be determined because the shape of the second control responses often changed. The results indicate that the complex shapes of isosmolar solution-induced responses are not attributable to prostanoids.

Effects of Incremental Increase in Osmolarity (Osmolar Jump) in Perfused Trachea. We hypothesized that if perfusion of tracheae with isosmolar solution causes cell shrinkage, creation of hyperosmolar conditions with the same solute should have little additional effect. Furthermore, responses to hyperosmolar jump under these conditions would reflect release of EpDRF related to the osmolarity of the intraluminal solution. Thus, a series of experiments was performed in which the isosmolar perfusion solution was abruptly made hyperosmolar (Fig. 7). Irrespective of the shape of the responses to the isosmolar solution, hyperosmolar jump led to rapid and large relaxations. Thus, relaxation always occurs in response to hyperosmolarity, whether or not cell shrinkage has been caused by isosmolar solution.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Osmolar jump from isosmolar (IO) perfusion solution. The perfused tracheae were contracted with MCh (3 x 10-7 M) while the trachea was perfused with normal MKHS. Where indicated the lumen was perfused with an isosmolar solution of osmolyte in distilled water. After stabilization of the response, the osmolarity of the perfusion solution was increased (hyperosmolar, HO) through the addition of 120 mosM of the same osmolyte. Perfusion with isosmolar solutions caused modest changes in P; however, hyperosmolar jump from isosmolarity resulted in a marked relaxation response to the osmolytes. n = 6, 4, 4, 4, and 4 for D-M, urea, NMDG-Glu, NaCl, and KCl, respectively. Vertical bar, 5 cm of H2O; horizontal bar, 5 min.

We then asked the question of whether EpDRF release and relaxation induced by hyperosmolar jump is caused by the concentration of osmolyte or by the change in osmolarity, regardless of the starting point, by examining whether relaxation responses could be elicited by an incremental jump from hyposmolar conditions to isosmolar conditions (Fig. 8). In these experiments, the lumen of MCh-contracted tracheae was first perfused with water to establish extreme hypoosmolarity (and cell swelling). Both in the presence (Fig. 8) and absence (n = 4; data not shown) of the epithelium water elicited an abrupt contraction, which subsided to the prestimulation level. Subsequent replacement of water with MKHS or isosmolar D-M elicited an abrupt relaxation response. The relaxation response was transient, in contrast to the response to hyperosmolarity (see above), and the MCh-induced level of contraction was reestablished. The relaxation response caused by jump to isosmolar D-M occurred both in intact and epithelial-denuded (n = 4; data not shown) tracheae, raising the possibility that the epithelium did not mediate the response. This possibility was tested by examining the effect of epithelium removal on responses to perfusion with isosmolar D-M. These preparations were contracted with MCh but were not challenged with intraluminal water. Whereas every preparation (n = 4; data not shown) lacking the epithelium relaxed in response to isosmolar D-M, no preparation containing the epithelium responded in this manner. Thus, the presence of the epithelium in intact tracheae would serve as a barrier against access of D-M to the smooth muscle. The effect of osmolar jump was further investigated by perfusing the preparations with MKHS or isosmolar D-M and then adding D-M to effect an additional 281 mosM jump in osmolarity. Again, the osmolar jump induced a long-lasting relaxation. After the osmolar jump, changing from isosmolar D-M to MKHS, i.e., osmolar-equivalent solutions, did not elicit relaxation. Identical results were obtained using urea, NMDG-Glu, NaCl, and KCl (Fig. 9) and a combination of isosmolar NMDG-Glu first and hyperosmolar D-M second (n = 4; data not shown). It is important to note that responses to urea are epithelium-dependent (see above). Isosmolar KCl (Fig. 9) differed from the other solutes in that a contraction stronger than that elicited with MCh was consistently obtained; this may have reflected depolarization of the smooth muscle.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Osmolar jump from distilled water and from isosmolar perfusion solution using MKHS and D-M as the jump osmolytes. A, perfused tracheae were contracted with MCh (3 x 10-7 M) while the trachea was perfused with MKHS. Where indicated the lumen was perfused with distilled water. After stabilization of the response, the osmolarity of the perfusion solution was increased abruptly by perfusing the tracheal lumen with MKHS. After the large, transient relaxation became stabilized the perfusion solution was made hyperosmolar (HO) through the addition of 120 mosM D-M. B, same as A, but osmolar jump from water was accomplished by perfusion with isosmolar (IO) D-M, and a transient relaxation ensued. After stabilization of the response, 120 mosM D-M was added to the perfusion solution to achieve hyperosmolarity, and a sustained relaxation ensued. C, experiment shown here involved a protocol similar to that shown in B, except that the isosmolar D-M perfusion solution was not made hyperosmolar with additional D-M but was instead replaced rapidly with MKHS. A relaxation response typical of that triggered by hyperosmolarity was not evoked. The tracings shown here are typical of several experiments (n = 6, 7, and 6 for A, B, and C, respectively).

View larger version (11K): [in this window] [in a new window]   Fig. 9. Typical tracings showing the effect of osmolar jump from distilled water and from isosmolar solution using urea (A), NMDG-Glu (abbreviated as N-G) (B), NaCl (C), and KCl (D) as the jump agents. These experiments followed the protocol described in Fig. 8. The tracings shown here are typical of several experiments (n = 5, 6, 4, and 4 for A, B, C, and D, respectively).

These findings suggest that epithelial-dependent relaxation is initiated by an increase in osmolarity rather than by the concentration or species of osmolyte.

Examination of the Effects of Compatible Osmolytes in Tracheal Strips and Perfused Trachea. Cell shrinkage in response to hyperosmolar challenge results in the uptake of compatible osmolytes after up-regulation of membrane osmolyte transporters (Lang et al., 1998). Inasmuch as the compatible osmolytes are involved in cell volume regulation, one or more of them could have EpDRF-like activity. Therefore, the pharmacological activity of these compounds was examined. Tracheal strips under basal force or contracted with MCh neither contracted nor relaxed in response to sorbitol, taurine, betaine or myo-inositol (10^-4 M; n = 4; data not shown). As such, these substances do not mimic EpDRF.

If compensatory changes in epithelium are initiated during hyperosmolar challenge to restore cell volume (Lang et al., 1998), the compatible osmolytes could be involved, and osmolyte uptake or loss could affect the response. However, sorbitol, taurine, betaine, and myo-inositol together (each 10^-4 M) had no effect on relaxation to hyperosmolar D-M (n = 6; data not shown).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study was performed to gain insight into the mechanisms involved in bronchodilation during exercise in normal individuals, exercise-induced obstruction and obstruction after inhalation of hypertonic solutions in asthmatic patients, and EpDRF release. The results provide evidence that the airway epithelium acts as an osmolarity sensor, which communicates with the airway smooth muscle, through EpDRF. The mechanical responses of the smooth muscle resulting from changes in the osmolar environment are associated with discrete modalities of the osmolar stimulus, including membrane reflection of the solute particles, the incremental change in osmolarity, and directionality.

All osmolytes examined here elicited relaxation responses with similar potency after application to the mucosal bath to increase osmolarity. Neither the chemical nature nor the cellular permeance of the solute was a determinant of the response. In contrast, the osmolytes produced solute-specific effects when applied to the serosal bath to increase osmolarity. Whereas extraluminal NMDG-Cl caused only relaxation, the responses to extraluminal D-M and urea contained contractile and relaxant components. To a lesser degree, NMDGGlu also elicited contractions, albeit transient ones. Under hyperosmolar conditions, the appearance of contraction is associated with nonionic osmolytes. Two phases in the responses indicates that two signals were triggered by these solutes. Nevertheless, the osmolytes were more potent when applied intraluminally than extraluminally, suggesting that the apical epithelial membrane is more sensitive to hyperosmolarity than the basolateral membrane. It is possible that the direct effects of the osmolytes on the smooth muscle may have given rise to the complex responses to the agents when added to the extraluminal bath. However, inhibition of both contractile and relaxant responses to urea in epitheliumdenuded preparations argues against this possibility. Generation of contractile responses could suggest the production of an epithelium-derived contractile substance(s) (Lamport and Fedan, 1990) in response to hyperosmolar challenge with D-M and urea.

Exposure of ovary cells and neutrophils to hyperosmolar solutions (added sucrose, NaCl, or KCl), or isosmolar solution (NMDG-Glu) initiated cell shrinkage and protein phosphorylation; phosphorylation did not occur if shrinkage was prevented by various means (Krump et al., 1997; Szászi et al., 1997). Thus, cell shrinkage, not an increase in intracellular osmolarity or ion concentrations, initiated protein phosphorylation in these cells. Human nasal (Willumsen et al., 1994) and guinea pig tracheal epithelia shrink when exposed to hyperosmolar medium (added NaCl; Hjoberg, 1999). In preliminary experiments using confocal microscopy, we have obtained evidence that guinea pig tracheal epithelial cells shrink in response to challenge with mucosal hyperosmolarity (D-M). We sought to ascertain whether epithelial cells release EpDRF in response to hyperosmolar solution because they shrink, sense osmolarity in the extracellular milieu, and/or respond to an incremental increase in osmolarity.

Hyperosmolarity, whether achieved by increasing the osmolarity of MKHS or isosmolar perfusing solution, always caused relaxation of the perfused trachea. In contrast, only a small percentage of the preparations relaxed in response to isosmolar solution. Thus, challenge with isosmolar solution did not mimic the effects of hyperosmolar solution. In contrast to the unimodal responses to hyperosmolar solutions, the complex responses to isosmolar solutions indicated that both relaxant and contractile pathways were involved. Prostanoids do not mediate this complexity. The issue of whether EpDRF is released by isosmolar solutions has not been resolved unequivocally by our experiments. It is clear, however, that challenge of the epithelium with hyperosmolar solution is a greater stimulus to EpDRF release than isosmolar solution and occurs in every trachea and that it can be achieved irrespective of whether the solution is rendered hyperosmolar from a starting point that is MHKS or isosmolar solution of osmolyte.

The weight of the evidence suggests that EpDRF is released in response to incremental increase in osmolarity rather than in response to cell shrinkage or sensing of the absolute osmolarity per se. This evidence is as follows: 1) Hyperosmolar addition of osmolyte to intraluminal MKHS relaxed the trachea, irrespective of the osmolyte used (Munakata et al., 1988; Dortch-Carnes et al., 1999; Fedan et al., 1999; this study); 2) hyperosmolar addition of every solute to isosmolar perfusing solution elicited relaxation; 3) administration of isosmolar osmolyte, or MKHS itself, to restore normosmolarity to preparations that had been perfused with water led to a relaxation response; 4) while already perfusing with an isosmolar solution of a given solute, administration of additional osmolyte to achieve hyperosmolarity elicited relaxation, irrespective of the osmolyte used; and 5) cytoskeleton disruptors did not inhibit relaxation stimulated with hyperosmolar solution (Fedan et al., 2003). The responses to incremental increase in osmolarity under the two conditions were not the same, however; if the jump was made from water the relaxation response was transient, whereas the response was sustained if the jump was made from isosmolar solution. The reason for this difference is not understood.

In conclusion, the tracheal epithelium releases EpDRF in response to an incremental increase in osmolarity sensed by a putative osmosensor. Cell shrinkage such as that stimulated by isosmolar solution perfusion may also cause EpDRF release, but this pathway may be secondary in its intensity or activate the release of opposing contractile mediators.

	Acknowledgements

 
We thank Deborah C. Sbarra and Nicole Diotte for technical assistance.

	Footnotes

 
This work was supported, in part, by National Institutes of Health Grant 5-T32-GM07039 (to R.A.J.). Mention of brand name does not constitute product endorsement. This article is the first one of a series of four companion articles that report the effects of hyperosmolar solutions in guinea pig airways (Fedan et al., 2003; Johnston et al., 2003; Wu et al., 2003).

DOI: 10.1124/jpet.103.051607.

ABBREVIATIONS: EpDRF, epithelium-derived relaxing factor; MKHS, modified Krebs-Henseleit solution; MCh, methacholine; IL, intraluminal; EL, extraluminal; NMDG, N-methyl-D-glucamine; NMDG-Cl, N-methyl-D-glucamine-chloride; NMDG-Glu, N-methyl-D-glucamine-gluconate; Na-Glu, Na gluconate; D-M, D-mannitol.

¹ Hypertonic solutions are those that cause cell shrinkage. Hyperosmolar solutions have osmolarity greater than that of the physiological extracellular solution. For simplicity, in this report we do not draw distinctions between the two terms when describing general phenomena.

Address correspondence to: Dr. Jeffrey S. Fedan, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Rd., Morgantown, WV 26505-2888. E-mail: jsf2{at}cdc.gov

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