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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia (D.X.-Y.W., R.A.J., A.R., J.S.F.); 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 for publication
March 14, 2003
Accepted
October 8, 2003.
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; Godfrey, 1997). The increased minute ventilation during exercise causes evaporative water loss. The ensuing elevation in the osmolarity of the airway surface liquid is thought to stimulate bronchoconstriction (Anderson et al., 1982; Freed and Davis, 1999; Anderson and Daviskas, 2000) resulting from the effects of released mediators, including leukotrienes (Umeno et al., 1990; Makhdum and Pearce, 1993; Freed, 1995; Israel and Drazen, 1999). In addition to reacting to exercise, asthmatic patients are hyperreactive to inhaled hypertonic saline solution (Makker et al., 1994).
Several in vitro studies have suggested that challenge of the epithelium with hyperosmolar solution stimulates bioelectric events. Application of hyperosmolar solution1 to ferret tracheal epithelium resulted in ion transport, the result of which was to restore isoosmolarity to the solution (Price et al., 1993). In cultured human nasal epithelium (Willumsen et al., 1994), hyperosmolar challenge of the apical (but not the basolateral) surface of the cells decreased Na+ absorption and deactivated basolateral membrane K+ and apical membrane Cl- conductance. In dog tracheal epithelium (Yankaskas et al., 1987), hyperosmolar NaCl but not L-mannitol (L-M) increased paracellular permeability. Hyperosmolar solution caused cell shrinkage of human nasal (Willumsen et al., 1994) and guinea pig tracheal epithelial cells (Hjoberg et al., 1999). In dog trachea, hyperosmolar solution caused depolarization of epithelium upon challenge of the apical but not the basolateral membrane; however, the cells were shrunken by basolateral hyperosmolarity, not apical hyperosmolarity (Man et al., 1984).
In the guinea pig isolated perfused trachea preparation, hyperosmolar solutions applied to the mucosal or basolateral surfaces caused relaxation of the smooth muscle (Munakata et al., 1988; Fedan et al., 1990, 1999), which is mediated via the release of an epithelium-derived relaxing factor (EpDRF). EpDRF is released in association with transepithelial depolarization (Dortch-Carnes et al., 1999) and is associated with Na+ and Cl- transport, as judged by the inhibitory effects of Na+ and Cl- channel blockers (Fedan et al., 1999). EpDRF is neither nitric oxide nor a prostanoid (Munakata et al., 1990; Spina and Page, 1991; Fedan et al., 1999, 2003a; Johnston et al., 2003).
In the guinea pig isolated perfused trachea, permeant and impermeant osmolytes were equieffective in their ability to elicit relaxation, i.e., release EpDRF, when used to raise the osmolarity of the physiological salt solution (Fedan et al., 2003a). Osmolytes differed in their activity as contractile and relaxant agents when applied to the apical2 and basolateral surfaces of the trachea to raise osmolarity. Exposure of the apical surface of the epithelium to isosmolar solutions, a procedure that stimulates cell shrinkage in neutrophils and ovary cells (Krump et al., 1997; Szászi et al., 1997), caused a diversity of mechanical responses that rarely mimicked the relaxation caused by hyperosmolar solution. Experiments in which the osmolarity of the perfusion solution was increased from isosmolar to hyperosmolar, or from hypoosmolar to isosmolar ("osmolar jump"), suggested that increment in osmolarity, rather than the absolute osmolar concentration or cell shrinkage, is the stimulus to the release of EpDRF (Fedan et al., 2003a).
Here we examined bioelectric responses of the guinea pig tracheal epithelium to challenge with hyperosmolar and isosmolar osmolyte solutions to compare these with mechanical responses (Fedan et al., 2003a). We characterized the effects of ion transport blockers and the effects of different classes of osmolytes, examined polarity of the responses, and investigated the bioelectric responses to osmolar jump.
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).
Bioelectric Measurements in Tracheal Segments. The Ussing chamber (WPI, Sarasota, FL) was used to measure changes in transepithelial short-circuit current (Isc) and transepithelial resistance (Rt) in response to various solutions and agents. After sacrifice of the animal, a 4-cm segment of trachea was removed, placed in modified Krebs-Henseleit solution (MKHS), cleaned, and slit along its length through the smooth muscle band. The segment was stretched to its original length, reflected open, and bisected so that the proximal end of the trachea was anchored across an aperture of 0.125 cm2, thereby separating the two hemi-chambers of the apparatus. Both hemi-chambers were perfused separately with recirculating MKHS (37°C). Two silver/silver chloride-agar bridge voltage electrodes containing 0.9% NaCl, and two silver/silver chloride-agar bridge current electrodes containing 0.9% NaCl, were placed to monitor transepithelial potential difference and to deliver current, respectively. Isotonic NaCl-containing bridge electrodes were used in place of 3 M KCl-containing bridges to prevent possible changes in osmolarity arising from KCl diffusion from the electrodes, inasmuch as the epithelium responds to slight elevations in osmolarity (Johnston et al., 2003). The apical and basolateral baths contained 5 ml of recirculating MKHS (37°C) in each reservoir. The preparations were allowed to equilibrate and the MKHS was changed at 15- to 30-min intervals. Isc was measured with an automatic voltage/current clamp amplifier (DVC 1000 or EVC 4000; WPI). The preparation was continuously short-circuited. In experiments involving an examination of the effects of ion channel blockers and the development of osmolyte concentration-response curves, for 0.5 s every 30 s a constant voltage pulse (dV = 10 mV) was applied to yield a current response (dI). In the experiments in which the effects of hyperosmolar and isosmolar solutions and osmolar jump were being studied, a 1-mV, 5-s pulse was delivered at 50-s intervals. In both cases, Rt was determined from the relation dV/dI. The spontaneous potential difference (SPD) was determined from Ohm's law, i.e., Rt x Isc.
Effect of Ion Transport Blockers on Epithelial Bioelectric Responses to Apical Hyperosmolarity. In these experiments, conditions were used that mimicked those in studies of the relaxant effects of hyperosmolarity (Fedan et al., 2003a). While recording Isc and delivering voltage pulses, the preparations were equilibrated for 3 h in the Ussing chamber. Methacholine (MCh; 3 x 10-7 M) was then added to the basolateral bath. At the plateau of the response, either L-M (120 mosM) or NaCl (120 mosM) was administered to the apical bath to obtain a control response. Volume equivalents of MKHS were added simultaneously to the basolateral bath to equalize hydrostatic pressure. After stabilization of the response, both chambers were washed with fresh MKHS at 15-min intervals over a 90-min period. An ion transport blocker was then added to the apical or basolateral bath, as appropriate, and incubated for 30 min. MCh was again added to the basolateral bath, and the same osmolyte was added to the apical bath to raise osmolarity. Vehicle control preparations (inhibitor not present) were run separately.
Concentration Dependence of Osmolyte-Induced Bioelectric Responses. After equilibration an osmolyte was then added cumulatively to the MKHS of the apical bath to obtain an osmolar concentration-response curve. A volume equivalent of MKHS was added simultaneously to the basolateral bath. At the conclusion of this procedure the preparation was washed bilaterally with fresh MKHS and, after 1 h, the same osmolyte was added in cumulative concentrations to the basolateral bath. Each preparation was used to study one osmolyte. In preliminary experiments, the preparations became unstable after the basolateral concentration-response curve; therefore, the apical curves were obtained before the basolateral curves.
Bioelectric Responses of Tracheal Epithelium to Apical Hyperosmolar and Isosmolar Non-MKHSs, and Incremental Osmolar Jump. After equilibration, the preparations were challenged with apical hyperosmolar solution, in an amount needed to double the osmolarity of the MKHS, which was measured in every experiment (Osmette A automatic osmometer; Precision Systems, Inc., Sudbury, MA). In experiments in which the effects of apical isosmolar solutions and incremental osmolar jump were also investigated, at the conclusion of the response both hemi-chambers were washed with MKHS at 15-min intervals for 1 h. The solution in the apical hemi-chamber was then rapidly changed to one containing isosmolar osmolyte dissolved in water. [The pH of these solutions were not adjusted to 7.4 to avoid introduction of transportable ions, and because responses to mechanical responses to isosmolar solutions adjusted to pH 7.4 were not different from those using untitrated solutions (Fedan et al., 2003a).] Upon stabilization of this response, the apical solution was made hyperosmolar by administering additional amounts of the same osmolyte to the isosmolar solution. The osmolytes chosen for study of the bioelectric effects of challenge with isosmolar solution were necessarily ionic. These experiments were conducted both in the absence, or in separate experiments, in the presence of basolateral MCh.
Solution Resistance. Solution resistances of the various solutions were measured in Ussing chambers under current clamp conditions. These solutions were MKHS, MKHS containing hyperosmolar osmolyte, isosmolar solution in water, and twice isosmolar solution in water, for the osmolytes NaCl, KCl, N-methyl-L-glucamine gluconate (NMDG-Glu), N-methyl-L-glucamine chloride (NMDG-Cl), and Na gluconate (Na-Glu). The resistances (0.0002-0.003 
· cm2) were negligible compared with those recorded when the tracheal segment was present (see Results).
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 the MKHS was 281 ± 5 mosM.
Analysis of Results. The results are expressed as mean ± S.E.; n is the number of separate experiments. The 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.
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) but differs from those obtained from cable analysis of intact trachea (91 µA/cm2; Croxton, 1993) and primary culture of epithelial cells (14 µA/cm2; Robison and Kim, 1994). Apical amiloride (Na+ channel blocker; 3 x 10-5 M) decreased basal Isc by 35.6%, whereas apical 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; Cl- channel blocker; 10-4 M) decreased Isc by 83.3 ± 4.9%, indicating that the guinea pig tracheal epithelium is a predominantly Cl--secretory rather than a Na+-absorptive epithelium. Basolateral bumetanide (Na+,K+,2Cl--cotransport inhibitor; 10-5 M) decreased Isc by 8.9 ± 1.6%, but iberiotoxin (Ca2+-activated K+-channel inhibitor; 10-7 M) in both the apical and basolateral baths had no effect on basal Isc. Basolateral ouabain (Na+,K+-pump inhibitor; 10-5 M) decreased Isc by 94.3 ± 3.3%.
Basolateral MCh increased Isc. This Isc response was inhibited substantially by apical NPPB and basolateral bumetanide, modestly by basolateral ouabain, but was not inhibited by apical amiloride or basolateral iberiotoxin (Table 1). These findings suggest that the bioelectric response to MCh involved activation of Cl- secretion. In the presence of MCh, apical hyperosmolar L-M (120 mosM) decreased Isc (Figs. 1, 10, and others).
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). This variability was never observed with L-M, and preparations that responded to apical hyperosmolar NaCl with an increase in Isc responded with a decrease in Isc when challenged with apical L-M. The basis for differences in passive flux behavior between animals is not known. In preparations in which apical hyperosmolar NaCl decreased Isc, the prolonged decrease in response to NaCl and L-M was smaller in the presence of apical amiloride and basolateral bumetanide (Fig. 2); apical NPPB and basolateral ouabain reversed the polarity of the responses to both solutes; and iberiotoxin had no effect. These effects were not seen in control preparations (blockers omitted) nor were they due to vehicle. The decreases in basal Isc caused by amiloride, NPPB, and bumetanide could have affected the subsequent responses to hyperosmolar challenge. Analysis of the results in terms of the percentage change of Isc caused by the osmolyte from the value in the presence of the inhibitors revealed that only bumetanide inhibited the Isc responses (Fig. 2). These findings suggest that Na+,K+,2Cl--cotransport was activated during hyperosmolar challenge.
In preparations in which apical hyperosmolar NaCl increased Isc, the blockers did not inhibit the Isc response (
Isc or %Isc) to NaCl but, in most cases, potentiated the responses (Fig. 3). The different pharmacological effects of the blockers suggest that passive ion fluxes were preponderant in preparations in which hyperosmolar NaCl increased Isc.
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Effect of Cl--Free MKHS on the Bioelectric Response to L-M. In the absence of Cl- (gluconate was substituted for Cl- in the apical and basolateral baths), the reduction in Isc in response to apical 266.8 mosM L-M was attenuated. (Fig. 4). Thus, the decrease in Isc in response to L-M reflects an active response of the epithelium to hyperosmolar challenge.
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Comparison of Bioelectric Responses to Apically and Basolaterally Applied Osmolytes: Cumulative Concentration-Response Curves. In these experiments, basal Isc was 45.3 ± 2.4 µA/cm2. Figures 5, 6, 7, 8 illustrate the bioelectric effects of cumulative additions of NaCl, KCl, urea, L-M, and sucrose to the MKHS in the apical and basolateral baths. MCh was not present in these experiments. It should be noted that asymmetrical ion concentrations across the epithelium were induced by addition of NaCl and KCl but not urea, L-M, or sucrose. There was a clear delineation in the type of responses obtained that depended on the osmolyte used and the bath to which it was added. The agents could be grouped into one of three categories of response profile. Apically applied NaCl and KCl increased Isc (Figs. 5 and 6). The increases in Isc in lower concentrations of the salts were small, but beginning at 502 mosM large increases occurred. Rt was decreased, as has been reported previously (Yankaskas et al., 1987), and the changes in Rt were observed at lower concentrations than those that altered Isc appreciably. The large changes in Isc observed at higher osmolar concentrations could be attributed to induction of passive nonspecific cation absorption, but changes in Rt in the absence of changes in Isc was evidence of an active compensation of the epithelium to apical challenge. Basolaterally-applied NaCl and KCl decreased Isc and Rt. The epithelium was more reactive to basolateral than apical hyperosmolarity. Given the known differences in channel distribution between the apical and basolateral membranes in airway epithelium, it was interesting that apical and basolateral addition of NaCl and KCl resulted in identical osmolarity-response curves.
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Urea decreased Isc after addition to the apical and basolateral chambers (Fig. 7); the responses in both baths were essentially the same, although two points on the curves exhibited small, significant differences. Basolateral urea decreased Rt, but apical urea increased Rt. The difference in Rt responses could not be an artifact of voltage clamping because the SPD also showed a separation of the two curves that was consistent with the differences in Rt.
The third Isc response profile, exhibited by the impermeant osmolytes L-M and sucrose, was characterized by a greater decrease in Isc in the apical bath than in the basolateral bath (Fig. 8). L-M and sucrose had minimal effects on Rt. Thus, all five osmolytes decreased Isc when applied to the basolateral bath and decreased SPD in both baths. Only the salts increased Isc when applied apically, but the increase was not linear or as large as would be expected for a pure Nernstian relationship. The remaining solutes decreased Isc after apical bath application. The results demonstrate polarity in the reactivity of the epithelium to increases in osmolarity. The bioelectric responses to increases in osmolarity due to permeant ions reflect both passive and active responses, and the latter can be studied using nonionic, impermeant solutes.
Bioelectric Effects of Hyperosmolar MKHS. Exposure of the apical membrane to twice-strength MKHS, which relaxed the perfused trachea (Fedan et al., 2003a), elicited an increase in Isc (Fig. 9); Rt was slightly but significantly decreased. These changes emulated those caused by the higher osmolar concentrations of apically applied NaCl and KCl (Fig. 5).
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Bioelectric Effects of Hyperosmolar L-M and Urea. Addition of L-M to the apical chamber to double the osmolarity decreased Isc without changing Rt (Fig. 10). In the same preparations, hyperosmolar urea decreased Isc in most experiments, but in some preparations complex responses were obtained; in one experiment urea increased Isc. Identical results were obtained in the absence or presence (n = 4; data not shown) of basolateral MCh.
Bioelectric Effects of Apical Hyperosmolar and Isosmolar Solutions and Incremental Osmolar Jump. Permeant and nonpermeant solutes have distinct effects on relaxation responses of the guinea pig perfused trachea (Fedan et al., 2003a). Initially, the osmolarity of the apical bath was elevated by adding NMDG-Glu to the MKHS, thereby maintaining the concentrations of the ions in MKHS. This manipulation decreased Isc, in the manner of L-M, and decreased Rt (Fig. 11). In contrast, elevation of apical bath osmolarity with NaCl (Fig. 12) increased Isc and decreased Rt, as before (Fig. 5). Hyperosmolar KCl increased Isc but did not affect Rt (Fig. 13).
Isosmolar NMDG-Glu solution decreased Isc and increased Rt (Fig. 11); in fact, the polarity of the epithelium was reversed. These results were consistent with termination of transepithelial Na+ absorption and induction of transepithelial cation secretion. Subsequent doubling of the osmolarity of the apical bathing solution by addition of NMDG-Glu resulted in a further decrease in Isc.
To elucidate further the roles of permeant and nonpermeant ions in the bioelectric responses, Cl- and Na+ were substituted for NMDG and gluconate. Elevation of apical osmolarity by addition of NMDG-Cl to MKHS increased Isc and decreased Rt (Fig. 14). In one-half of the preparations, the increase in Isc was sustained; the other one-half exhibited a transient increase in Isc followed by a sustained decrease. The responses were not random, but were consistent for a preparation, as evidenced by subsequent exposure to isosmolar NMDG-Cl and rechallenge with hyperosmolar NMDG-Cl (Fig. 14). Isosmolar NMDG-Cl caused a large decrease in Isc in all preparations, resulting in a reversal of polarity; Rt was increased. It should be noted that under these conditions, there was minimal difference between apical and basolateral concentrations of Cl-, yet Isc was greatly affected in all preparations, and reversal of the Isc was observed in 50% of the preparations. Hence, induction of passive anion absorption is unable to explain the effects of isosmolar NMDG-Cl. In contrast to NMDG-Glu, addition of hyperosmolar NMDG-Cl increased Isc back toward the original baseline and decreased Rt (Fig. 14). Thus, the presence of Cl- in the apical bath is linked to the active increase in Isc observed in response to hyperosmolar solutions. It should also be noted that the preparations that had exhibited biphasic responses to addition of NMDG-Glu to MKHS also exhibited biphasic responses to addition of NMDG-Cl to isosmolar NMDG-Cl, and preparations that exhibited monotonic responses to addition of NMDG-Cl to MKHS exhibited monotonic responses to addition of NMDG-Cl to isosmolar NMDG-Cl. A possible explanation for these findings is that the response to hyperosmolar NMDG-Cl has several potential components: absorption of Cl- due to an elevated concentration in the apical bath, secretion of Na+ and 
due to asymmetrical concentrations across the epithelium, and an active, electrogenic cellular response involving anion secretion. The balance between these processes within a given preparation could determine the response pattern for that preparation under a variety of conditions. It is also possible that a paracellular conductance of NMDG leads to an electrodiffusive NMDG absorption. This is unlikely, however, because the transepithelial driving force for NMDG is infinite under both isosmolar and hyperosmolar conditions.
Elevation of osmolarity of the apical bath via addition of Na-Glu to MKHS increased Isc (Fig. 15). In 18 of the 20 preparations, the increase in Isc was sustained above baseline, and in the remaining two preparations Isc eventually decreased to below baseline. The increase in Isc was consistent with induction of a passive absorption of Na+ due to asymmetrical concentrations. Hyperosmolar Na-Glu decreased Rt. Isosmolar Na-Glu induced a large biphasic increase in Isc consistent with induction of passive Cl- secretion. Subsequent addition of Na-Glu to isosmolar Na-Glu decreased Rt, but had little additional effect on Isc compared with isosmolar Na-Glu. This observation provides further evidence that the increase in Isc in response to isosmolar Na-Glu was due to transepithelial secretion of anions, because the chemical driving force would be increased by isosmolar Na-Glu but would not be increased further by increasing the concentration of Na-Glu.
This set of experiments was concluded by repeating the protocol with NaCl, KCl, and K-gluconate (K-Glu). As predicted from the osmolarity-response curves, increasing the osmolarity of the apical bathing solution by addition of NaCl or KCl induced similar increases in Isc (Figs. 12 and 13). Isosmolar NaCl (Fig. 12) and KCl (Fig. 13) caused small decreases in Isc, and addition of NaCl or KCl to the respective isosmolar solutions yielded increases in Isc consistent with the induction of driving forces for passive transepithelial cation absorption. Consistency in responses to Na+ and K+ was also seen in response to hyperosmolar K-Glu (Fig. 16).
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When applied to preparations already incubated in apical isosmolar solution, the administration of the same osmolyte to cause hyperosmolarity elicited Isc and Rt responses that were generally similar to those obtained when the osmolyte was added to MKHS to create hyperosmolar conditions (Figs. 11, 12, 13, 14, 15, 16). Together, these experiments indicate that responses to incremental increases in osmolarity (i.e., osmolar jump) were similar, whether by elevation of osmolarity in MKHS or in isosmolar solution of osmolytes, regardless of the direction of the Isc response. Solutes containing Na+, K+, and Cl- increased Isc when added in hyperosmolar amounts to MKHS or isosmolar solution (osmolar jump). The impermeant osmolytes, in contrast, decreased Isc under both conditions. Curiously, hyperosmolar urea also decreased Isc, and the reason why this permeant substance should do so is not understood.
Bioelectric Effects of Apical Hyperosmolar and Isosmolar Solutions and Hyperosmolar Jump in the Presence of MCh. To examine whether MCh could affect the bioelectric responses to apical hyperosmolar and isosmolar solutions and incremental osmolar jump, the experiments with ionic osmolytes were repeated using the same protocols but in the presence of basolateral 3 x 10-7 M MCh. MCh had no appreciable effect on bioelectric responses to isosmolar and incremental osmolar jump (NMDG-Glu, NMDG-Cl, NaCl, KCl, Na-Glu, and K-Glu; n = 4 separate experiments for each osmolyte; data not shown).
Solution Resistance. Changes in Rt caused by non-MKHS osmolyte solutions could have involved a change in the fluid resistance of the apical solution. Therefore, the electrical resistance of the various solutions was measured (see Materials and Methods), and it was observed that the solution resistances were negligible compared to these developed by the epithelium. These findings indicate that the fluid resistance of the solutions cannot explain the Isc and Rt responses, both their direction and magnitude. In addition, the effects of changed fluid resistance would have been instantaneous, whereas the responses measured throughout this study were slow-developing.
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). The bioelectric responses to hyperosmolarity induced with Na+-, K+-, and/or Cl--containing osmolytes seems to involve passive and active responses in the epithelium, giving rise to increases or decreases, respectively, in Isc. The preponderance of these two processes varied apparently between animals when these ions were used as solutes. However, in all preparations hyperosmolar L-M decreased Isc; this response involves Cl- transport.
The effects of the ion transport blockers on bioelectric responses to hyperosmolarity using L-M and NaCl (in those preparations in which the active, cellular response prevailed) were similar to the effects of the same or similar blockers on relaxation responses (Fedan et al., 1999, 2003b). Both amiloride and NPPB inhibited relaxation and attenuated the decrease in Isc; Cl--free MKHS also blunted bioelectric responses to hyperosmolarity. Likewise, K+-channel blockade did not affect relaxation (glibenclamide) and had no effect on Isc responses (iberiotoxin). Such correlations buttress the hypothesis that apical membrane Na+ and Cl- transport and associated bioelectric events are linked to and may initiate the release of EpDRF (Dortch-Carnes et al., 1999; Fedan et al., 1999). This parallelism diverged in the case of bumetanide and ouabain: both of these agents were silent in relaxation studies but inhibited the decrease in Isc in response to L-M. Analysis of the effects of the blockers on Isc with their initial baseline actions in mind revealed that only bumetanide antagonized bioelectric responses to hyperosmolar challenge. It is, therefore, difficult at present to model the ion transport changes that occur in the epithelium during hyperosmolar challenge in the context of EpDRF release and airway smooth muscle relaxation. Moreover, the effects of amiloride and NPPB are not absolutely restricted to Na+ and Cl- channels, and it is conceivable that the agents could interfere with Na+,H+- and/or Cl-, exchange, respectively, to alter ion transport secondarily.
In those preparations in which hyperosmolar NaCl elicited an increase in Isc, none of the ion transport blockers inhibited the responses. This evidence suggests that passive ion absorption stimulated by the asymmetrical distribution of the salt was responsible for the bioelectric events in these tracheal segments. In addition, the absence of any effect of the blockers on these responses would suggest that passive ion absorption is not associated with EpDRF release.
As in the mechanical studies (Fedan et al., 2003a), there was substantial diversity in the bioelectric responses when the epithelium was challenged with hyperosmolar and isosmolar solutions. So long as an osmolyte was impermeant (L-M, sucrose and NMDG-Glu) the bioelectric response to elevation of osmolarity in MKHS was a decrease in Isc without a change in Rt. We interpret these results as indicative of the active response of the cells to hyperosmolarity unmitigated by passive diffusion of the osmolyte as a component of the response, such as would be the case for the other ionic osmolytes. Urea also decreased Isc when applied cumulatively (and caused multiphasic responses in some tissues when added in a single concentration). Thus, with respect to their ability to relax the perfused trachea and elicit bioelectric responses, L-M, NMDG-Glu, and urea were pharmacologically equivalent when they were used to elevate osmolarity. Inasmuch as urea would not induce long-term shrinkage of the epithelial cells, these findings, by themselves, could suggest that the stimulus to the relaxant and bioelectric responses to these four osmolytes is the osmolar concentration of the perfusing MKHS rather than shrinkage of the cells. However, the hyperosmolar jump experiments indicated that increment in osmolarity rather than absolute osmolarity is a far greater stimulus of the cells.
The results indicate that osmolytes containing one permeant ion, when added to increase the osmolarity of MKHS, stimulated a transient or sustained increase in Isc. Interestingly, the charge of the permeant ion is irrelevant, because NaCl, KCl, NMDG-Cl, Na-Glu, and K-Glu exhibit approximately the same effect, i.e., to increase Isc (left tracings of Figs. 12, 13, 14, 15, 16). This is in contrast to the decrease in Isc caused by L-M, NMDG-Glu, sucrose, and urea. Yet, all of the osmolytes elicited relaxation when added to normosmolar solutions (Fedan et al., 2003a).
In conclusion, guinea pig tracheal epithelium is sensitive to alterations in extracellular osmolarity at the apical and basolateral membranes, although bioelectric responses to osmolytes differ among permeant and nonpermeant solutes, and the responses are polarized. As with the mechanical responses, bioelectric responses to hyperosmolarity seem to be stimulated by an incremental increase in osmolarity rather than by solute concentration or cell shrinkage per se. However, a direct link between the mechanical and bioelectric responses to osmolar challenge is not evident.
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,b; Johnston et al., 2003). ABBREVIATIONS:L-M,L-mannitol; EpDRF, epithelium-derived relaxing factor; MKHS, modified Krebs-Henseleit solution;Isc, short-circuit current; Rt, transepithelial resistance; SPD, spontaneous potential difference; MCh, methacholine; NMDG,N-methyl-L-glucamine; NMDG-Cl, N-methyl-L-glucamine-chloride; NMDG-Glu,N-methyl-L-glucamine-gluconate; Na-Glu, Na-gluconate; NPPB, 5-nitro-2-(3-phenylpropylamino) benzoic acid; K-Glu, K-gluconate.
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. 
2 Apical and mucosal are terms that correspond to the intraluminal bath in perfused trachea experiments and the "air side" of the trachea. Basolateral and serosal are terms that correspond to the extraluminal bath in perfused trachea experiments and the "blood side" of the trachea.
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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