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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Biochemistry and Molecular Pharmacology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia (Y.J., J.S.F.); Department of Physiology, The Brody School of Medicine, East Carolina University, Greenville, North Carolina (M.R.V.S.); and Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia (J.A.D., J.S.F.)
Received December 21, 2007; accepted April 14, 2008.
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Abstract |
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80 mosM) increased Vt and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased Vt. In tracheas exposed to 30 mosM D-mannitol (
EC50), 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 NH2-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO2 (mitogen-activated protein kinase stress inducer) and Na3VO4 (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 Na3VO4 did not affect the Vt 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.
; Hamilton et al., 2001), and it regulates the depth and composition of the airway surface liquid (ASL) (Widdicombe, 2002). Under physiological conditions, ASL osmolarity is approximately isosmolar with that of the interstitium (Boucher, 1999; Jayaraman et al., 2001; Tarran, 2004). During exercise and hyperventilation, evaporative water loss is thought to cause an increase in the osmolarity of the ASL (Anderson and Daviskas, 2000). In dogs, Freed and Davis (1999) observed increases in osmolarity of approximately 130 mOsM during hyperventilation with dry air, which was reduced to an 40 mOsM increase after the end of hyperventilation. In nonasthmatic subjects, exercise causes a decrease in respiratory resistance due to bronchial dilation (Kagawa and Kerr, 1970; Silverman et al., 2005). In asthmatic patients, however, exercise causes airway obstruction (Anderson and Daviskas, 2000), which is thought to be triggered by a 40 to 60 mOsM increase in the osmolarity of the ASL (Freed and Davis, 1999; Anderson and Kippelen, 2005; Hallstrand et al., 2005a,b; Anderson, 2006). Hyperosmolar stimulation of the airways, such as that induced with inhalation of the nonpermeant osmolyte D-mannitol (D-M), also evokes bronchoconstriction in asthmatic subjects (Porsbjerg et al., 2007). Studies using the guinea pig isolated, perfused trachea demonstrated that the airway epithelium is an osmometer that responds to very small increases in lumen osmolarity, within the range occurring during exercise, by releasing epithelium-derived relaxing factor (EpDRF) and relaxing the airway smooth muscle (Munakata et al., 1988; Fedan et al., 1990). EpDRF has not been isolated. Dilation responses of the perfused trachea triggered by challenge of the epithelium with hyperosmolar solution are not mediated by nitric oxide or prostanoids (Munakata et al., 1990; Johnston et al., 2004). The responses are partially inhibited by hemoglobin and zinc II protoporphyrin(IX) (Fedan et al., 2004b), which suggests that carbon monoxide contributes to the activity known as EpDRF. We have hypothesized that EpDRF participates in bronchodilation in normal individuals during exercise and that reduced secretion of EpDRF may contribute to obstruction in asthmatic subjects.
The relationship between bioelectric responses of the epithelium in response to hyperosmolar challenge and the ensuing relaxation has been under investigation. In fresh guinea pig tracheas mounted in Ussing chambers, elevation of osmolarity by 120 mOsM depolarized the epithelium and decreased transepithelial short-circuit current (Wu et al., 2004). In guinea pig isolated, perfused trachea, the depolarization preceded dilation (Dortch-Carnes et al., 1999). Blockade of epithelial Na+ and Cl– channels inhibited EpDRF-mediated airway relaxation, whereas blockade of Na+,K+,2Cl– cotransport had no effect (Fedan et al., 1999). The timing of the two responses and the effects of Na+ and Cl– channel blockade have suggested that the bioelectric and mechanical responses are linked.
Extracellular hyperosmolarity causes shrinkage in epithelium and other cells (Lang et al., 1998). The EpDRF-mediated relaxation is not linked to cell shrinkage (Fedan et al., 2004a, 2007a,b). Many cells, but not airway epithelial cells, undergo a compensatory regulatory volume increase (RVI) in the presence of raised osmolarity, resulting from activation of the Na+,K+,2Cl– cotransporter and other ion transport systems, causing an uptake of Na+,Cl–, and water to restore cell volume. The preponderance of studies of the effects of hyperosmolarity have been focused on pathways involved in RVI and long-term regulation of cell volume, have used supraphysiological osmolarities and long incubation periods, and have limited relevance for understanding the rapid, early events in EpDRF release in response to small increments in osmolarity. These studies, however, have revealed that several signaling pathways are involved in RVI, including mitogen-activated protein (MAP) kinases, p38 [c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)], protein kinase C (PKC), calmodulin kinase-II (CaM-K-II), myosin light chain kinase (MLCK), and phosphatidylinositol 3-kinase (PI-3-K) (Shrode et al., 1998; Puddicombe and Davies, 2000; Sheikh-Hamad and Gustin, 2004; Zhao et al., 2004). p38 and JNK are also involved in the release of inflammatory mediators from epithelium after hyperosmolar challenge (Hashimoto et al., 1999a,b; Furuichi et al., 2002).
We hypothesized that one or more of these kinases may play a role in EpDRF release and bioelectric responses of the epithelium to hyperosmolar challenge. The guinea pig perfused trachea preparation was used to compare the osmolar concentration dependencies of epithelial bioelectric responses and EpDRF release. We found that only p38 modulates EpDRF-mediated relaxant responses, whereas JNK, PKC, and phosphatase(s) participate in epithelial bioelectric responses to methacholine (MCh) and hyperosmolarity. Small increases in osmolarity that are relevant to exercise and that maximally release EpDRF do not elevate phosphorylation to the degree caused by supraphysiological hyperosmolar challenges.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-Dri virgin cellulose chips and hardwood β-chips as bedding. They were provided HEPA-filtered air, and Teklad 7006 diet and tap water ad libitum, under controlled light cycle (12 h of light) and temperature (22–25°C) conditions. The animals were anesthetized with sodium pentobarbital (65 mg/kg i.p.), and then they were sacrificed by thoracotomy and bleeding before removing the trachea. Materials. Modified Krebs-Henseleit (MKH) solution, pH 7.4, at 37°C contained 113 mM NaCl, 4.8 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, and 5.7 mM glucose, and it was saturated with 95% O2 and 5% CO2. The osmolarity of MKH was 281 ± 5 mOsM. Acetyl-β-methylcholine chloride (MCh) and D-M (Sigma-Aldrich, St. Louis, MO) solutions were prepared in saline. Kinase inhibitors (SB 203580, SKF 86002, SP 600125, dicumarol, PD 98059, U 0126, chelerythrine, LY 294002, KN-62, and ML-7) were from Calbiochem-Novabiochem (La Jolla, CA), and they were dissolved in dimethyl sulfoxide (DMSO). NaAsO2 (Mallinckrodt Laboratory Chemicals, Phillipsburg, NJ) and Na3VO4 (Sigma-Aldrich) were prepared in MKH solution. Radioimmunoprecipitation assay cell lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) contained 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 0.004% sodium azide in Tris-buffered saline, to which phenylmethanesulfonyl fluoride, sodium orthovanadate, and protease inhibitors were added according to the manufacturer's directions. Laemmli protein sample buffer and 10% SDS-polyacrylamide gels were from Bio-Rad (Hercules, CA). Phosphorylated and nonphosphorylated p38 and JNK control proteins and their primary antibodies, horseradish peroxidase-linked anti-rabbit secondary antibody, and β-actin antibody were from Cell Signaling Technology Inc. (Beverly, MA). Bicinchoninic acid protein assay reagents and Western blot stripping buffer were from Pierce Chemical (Rockford, IL). Enhanced chemiluminescence solutions were from GE Healthcare (Piscataway, NJ).
Perfused Trachea Preparation. The guinea pig isolated, perfused trachea preparation, which allows agents to be applied separately to the mucosal (intraluminal) or serosal (extraluminal) surfaces of the trachea (Munakata et al., 1988, 1990; Fedan et al., 1990), was used to measure simultaneously epithelial bioelectric and smooth muscle mechanical responses induced by various agents (Dortch-Carnes et al., 1999). With this preparation, agents can be added to either bath to target epithelium or smooth muscle. A 4.2-cm segment of guinea pig trachea was removed, and it was mounted at its in situ length onto a plastic perfusion holder that allows indwelling cannulae with side-holes to be inserted into the lumen from either end of the trachea. The cannulae were made of stainless steel, and they were coated with nail polish to provide electrical insulation. MKH solution in the intraluminal and extraluminal baths was kept at 37°C and saturated with 95% O2 and 5% CO2. The holder was placed into an extraluminal bath filled with MKH solution. The tracheal lumen was perfused with MKH solution from the intraluminal bath at a constant rate (20 ml/min) using a pump; the transmural pressure was adjusted to zero. The inlet minus outlet perfusion pressure difference (
P), a fifth power function of tracheal diameter (Munakata et al., 1988, 1990), was measured through the side-holes in the indwelling cannulae that were connected to a differential pressure transducer. Contraction of the smooth muscle increases P (constriction), whereas relaxation of the smooth muscle decreases P (dilation). Voltage electrodes (silver/AgCl) were in continuity via a bridge (filled with 4% agar/saline) with the intraluminal and extraluminal baths to record transepithelial potential difference (Vt). The inner voltage electrode consisted of the side-holes of the indwelling cannula that had been inserted into the proximal end of the trachea; the apertures were 6 mm from the point of entry of MKH into the lumen of the trachea. The outer voltage electrode consisted of an MKH solution-filled glass tube placed close (1 cm) to the tracheal wall that was in continuity via a 4% agar/saline bridge with a silver/AgCl electrode. Both voltage and current electrodes were connected to a voltage/current clamp amplifier (DVC-1000; WPI, Sarasota, FL). Thus, the preparation allowed simultaneous monitoring of both Vt and P in the same trachea.
Concentration-Response Curves for Hyperosmolar D-M-Induced Vt and P Responses. The tracheal preparation was equilibrated by perfusing MKH solution for 2.5 to 3 h to allow Vt and P to become stable. To prepare the trachea for dilation responses, the smooth muscle was contracted by adding MCh (3 x 10–7 M, EC50) to the extraluminal bath. MCh elicited epithelial hyperpolarization. When the MCh-induced Vt and P responses reached their plateaus, the osmolarity of the perfused solution was elevated cumulatively with stepwise additions of D-M to the intraluminal bath.
Effects of Kinase and Phosphatase Inhibitors on Vt and P. The guinea pig genome has not been sequenced, and genetic gain or loss of function models are not available for this species. Therefore, a pharmacological approach was taken to investigate the involvement of kinases and phosphatases in hyperosmolarity-induced responses.
To avoid variability between animals and shipments, control responses and the effects of the inhibitors were compared using a paired design protocol wherein responses were measured in both the absence and presence of an agent in each trachea. After the equilibration period, the tracheas were incubated with vehicle for 30 min in the intraluminal bath. MCh was then added to the extraluminal bath. When the constriction and hyperpolarization reached their plateaus, the osmolarity of intraluminal perfused MKH solution was elevated by adding 30 mOsM D-M; this concentration approximates the EC50 value for hyperosmolarity-induced relaxation responses (Fedan et al., 2004a). After the D-M-induced Vt and P responses became stable, the preparation was washed with fresh MKH solution to remove all agents. Ninety minutes after the baselines were reestablished, the trachea was incubated with a test agent in the intraluminal bath for 30 min, after which the trachea was challenged again with MCh and D-M in the presence of the agent. The possible effects of DMSO on the two sets of responses to MCh and D-M were evaluated in a separate group of control preparations.
The choice of the inhibitors and the concentrations to be used was decided after a search of the literature for investigations describing the kinetic properties of the agents (Table 1). When available, two structurally different agents were used to lessen the likelihood of interpreting findings caused by nonspecific agent effects. Concentrations were chosen that were ca. 1 order of magnitude above the reported EC50 values for enzyme inhibition for the reason that the concentrations used in intact epithelial cells would need to be higher than those used in broken cell preparations. Chelerythrine was used as a broad-spectrum inhibitor of the family of PKCs in these experiments to gauge the potential scope of PKC involvement.
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Western Blots. In these experiments, tracheal segments were challenged with D-M in the absence of MCh. Two animals were required for each experiment. Tracheas were removed, placed in MKH solution, cleaned, and cut longitudinally into three strips. In a paired design, the strips from each animal were randomly divided into three experimental groups to give a total of two strips for each condition, one from each animal: 1) controls, incubated in MKH solution; 2) D-M-exposed (20, 40, 80, 160, 240, and 320 mOsM was added to MKH solution; each strip was exposed to a single concentration); and 3) NaAsO2-exposed (0.5 mM in MKH solution; the concentration of NaAsO2 was increased in these experiments), as a positive control. Each condition was investigated at least four times. After a 2-h equilibration period in MKH (37°C), the strips were exposed to MKH, D-M, or NaAsO2 for 20 min at 37°C. At the end of this period, the strips were transferred into ice-cold MKH solution. At 0°C, the epithelium was scraped off with a scalpel blade, collected and centrifuged (600g) for 3 min at 4°C; the pellets were sonicated in 50 µl of radioimmunoprecipitation assay buffer. Protein concentrations were measured with the bicinchoninic acid assay.
Samples (20 µg of protein per well) were loaded on a 10% SDS-polyacrylamide gel together with phosphorylated and nonphosphorylated p38 or JNK standards, and then they were subjected to electrophoresis. The proteins were then transferred to a nitrocellulose membrane. After blocking nonspecific protein binding by incubating the membrane in phosphate-buffered saline with 0.1% Triton X-100 and 5% nonfat milk at room temperature for 1 h, the membrane was incubated overnight at 4°C with phosphorylated p38 or JNK primary antibodies, followed by incubation with the horseradish peroxidase-linked anti-rabbit secondary antibody at room temperature for 1 h. The membrane was washed in phosphate-buffered saline, and the blots were incubated with enhanced chemiluminescence solution for 1 min and exposed to Kodak BioMax XAR film. To determine the total amount of p38 and JNK proteins on the blots, the membrane was stripped and reprobed with nonphosphorylated p38 or JNK antibodies. β-Actin was included to assess the accuracy of sample loading.
Data Analysis. The epithelial bioelectric responses were quantified as Vt in millivolts. Responses to MCh were quantified as the increase in P, in cm H2O. D-M-induced dilation responses were normalized as a percentage of the MCh response. The EC50 values for Vt and P responses to D-M were derived from concentration-response curves using least-squares analysis of four-parameter logit curve fits (SigmaPlot version 9; Systat Software, Inc., Point Richmond, CA), and they are given along with 95% confidence intervals. Other results are presented as means ± S.E. Differences were analyzed statistically using Student's paired t test (SigmaStat version 3.1; Systat Software, Inc.). p < 0.05 was considered significant. Bands on Western blots were scanned with a General Electric Molecular Dynamics scanner, and pixel intensity was quantified (ImageQuant 5.2 software; Molecular Dynamics). The effects of agents on the phosphorylated and total amounts of p38 and JNK were plotted as -fold increases above the levels observed in paired, unstimulated control samples.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Concentration-response curves for Vt and P responses induced by raising intraluminal osmolarity with D-M were obtained from extraluminal MCh-contracted preparations (Fig. 1). Hyperpolarization and dilation responses were obtained at very low D-M concentrations, i.e., the threshold was 1–3 mOsM (Fedan et al., 2004b), attesting to the sensitivity of the epithelium to increases in osmolarity. Elevations in D-M up to 80 mOsM caused hyperpolarization. The maximum dilation response was essentially reached at 80 mOsM. The hyperpolarizing and relaxant responses exhibited the same concentration dependence; the EC50 values for hyperpolarization [16.6 (13.0–21.2) mOsM] and dilation [24.4 (19.2–31.3) mOsM] were not significantly different. The congruence of the two concentration-response curves suggests that the bioelectric and dilation responses to hyperosmolarity were closely, if not causally, linked. In addition, the EC50 values were in a range that is pertinent to the increase in ASL osmolarity that accompanies exercise. At concentrations greater than 80 mOsM, a transition occurred and the bioelectric response of the epithelium became converted from hyperpolarization to depolarization, whereas the dilation response was not increased appreciably. This reversal in the bioelectric response was not an artifact of the concentration-response determination, because elevations in osmolarity above 80 mOsM with a single addition evoke depolarization (data not shown; Dortch-Carnes et al., 1999; Wu et al., 2004). On the basis of these results, a D-M concentration of 30 mOsM, which produced consistent dilation and hyperpolarization responses, was used to investigate the effect of the inhibitors.
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P. The purpose of these experiments was to ascertain the roles of kinases in EpDRF-mediated relaxation and bioelectric responses to hyperosmolarity.
Experiments were first conducted to establish a no-effect concentration for DMSO. DMSO had no effect on P, but it increased Vt by 0.6 ± 0.1 mV at concentrations as low as 0.1%. After investigating progressively lower concentrations, it was found that DMSO at a concentration of 0.04% had no effect.
Next, the effects of the intraluminally added kinase and phosphatase inhibitors on P and Vt were investigated. Of the six MAP kinase inhibitors examined (SB 203580, SKF 86002, SP 600125, dicumarol, PD 98059, and U 0126), only SB 203580 affected Vt, eliciting a small depolarization (0.8 ± 0.3 mV). This small response not only could reflect modulation of ion transport by p38 but also it could represent a nonspecific effect, because SKF 86002, the other inhibitor of p38, had no effect. SB 203580, dicumarol, and PD 98059 decreased P slightly (0.06 ± 0.02, 0.46 ± 0.13, and 0.16 ± 0.04 cm H2O, respectively) but, compared with responses to MCh, these effects were negligible. The other three MAP kinase inhibitors had no effect on P. Chelerythrine, LY 294002, KN-62, and ML-7, decreased Vt by 1.5 ± 0.3, 3.9 ± 0.6, 0.6 ± 0.1, and 0.6 ± 0.5 mV, respectively. The magnitude of these effects suggests that PKC and PI-3-K are involved in the regulation of ion transport, whereas the roles of CaM-K-II and MLCK are minor.
In contrast, the protein tyrosine phosphatase inhibitor Na3VO4 and the kinase activator NaAsO2 increased Vt by 0.7 ± 0.2 and 1.7 ± 0.7 mV, respectively. The nonselective PKC inhibitor chelerythrine increased Pby0.2 ± 0.1 cm H2O, a negligible effect; the other kinase inhibitors had no effect on P. These findings indicate together that Vt is regulated by some phosphorylation pathways but that this regulation is of little consequence to smooth muscle tone.
Effects of Kinase and Phosphatase Inhibitors on MCh-Induced Epithelial Hyperpolarization and Dilation Responses. Because some kinase and phosphatase inhibitors affected Vt, we investigated whether bioelectric responses of the epithelium to MCh are also influenced by these agents. Extraluminally applied MCh (3 x 10–7 M) caused constriction and hyperpolarized the epithelium. None of the MAP kinase inhibitors (SB 203580, SKF 86002, SP 600125, dicumarol, PD 98059, and U 0126) affected the contractile response to MCh (n = 7, 6, 10, 6, 6, and 6, respectively; data not shown). Intraluminally applied LY 294002 (Fig. 2A) and Na3VO4 (Fig. 2B) inhibited contractile responses to MCh. Apparent effects of chelerythrine and NaAsO2 did not reach significance; KN-62 and ML-7 had no effect. Because they were added to the intraluminal bath and the epithelium constitutes a 400 to 800-fold diffusion barrier for agents (Fedan and Frazer, 1992), these findings indicate that the inhibitory effects of LY 294002 and Na3VO4 on smooth muscle responses were indirect and mediated by the epithelium.
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SP 600125 and dicumarol potentiated the hyperpolarization to MCh (Fig. 3A), whereas chelerythrine inhibited this response (Fig. 3B). Hyperpolarization in response to MCh is thought to involve primarily activation of Cl– secretion (Wu et al., 2004; Fedan et al., 2007b), a possible downstream target of these pathways. The remaining kinase inhibitors, as well as NaAsO2 and Na3VO4 (n = 9 and 6, respectively; data not shown), had no effect on bioelectric responses to MCh.
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Using human cultured bronchial epithelial cell to explore the role of p38 in hyperosmolarity-induced release of cytokines, Hashimoto et al. (1999b) observed little, if any, phospho-p38 in unstimulated cells. In contrast, at the outset of this study, we observed high levels of phospho-p38 when the epithelium was scraped off and centrifuged at room temperature, and little further increase occurred in response to D-M. This outcome was not encountered to the same degree with JNK. The warmer temperatures had been used to minimize disruption of ion gradients which, itself, might alter catalytic activity. We determined after performing extensive preliminary experiments that the phospho-p38 background was reduced greatly but not eliminated by immersing the tracheal strips into ice-cold MKH solution at the end of the challenge with D-M, and preparing cell extracts at 4°C. Nevertheless, basal phospho-p38 was not reduced to the low levels reported by Hashimoto et al. (1999b).
D-M stimulated a concentration-dependent increase in the phosphorylation of p38 (Fig. 7) and JNK II (Fig. 8). For p38, the increase in phospho-p38 ranged from 1.4 to 1.7-fold at D-M concentrations 80 mOsM; these changes were not significant. Significant increases (up to 4-fold) in phospho-p38 were observed at the two highest D-M concentrations. Similar results were seen with JNK, with significant increases in phosphorylation occurring at the three highest concentrations. The -fold increases in phospho-JNK were appreciably higher than obtained for p38. The amounts of p38 and JNK proteins were unaffected by D-M (Figs. 7 and 8). To investigate the extent of phosphorylation of these two enzymes stimulated by D-M challenge against the levels that the cells might be potentially capable of producing in response to a nonspecific stress stimulus, the effects of NaAsO2 (0.5 mM) were examined as a positive control. A stress-inducing agent, NaAsO2 has multiple sites of action on protein kinases and phosphatases (Ludwig et al., 1998). NaAsO2 stimulated strongly the phosphorylation of p38 and JNK II (Figs. 7 and 8). Interestingly, NaAsO2 also caused an increase in immunostaining of total p38 protein (Fig. 7), which suggests that de novo protein synthesis had occurred. However, inasmuch as the epithelium was incubated with NaAsO2 for only 20 min, this increase in immunostaining is unexplained. NaAsO2 also caused an increase in JNK II phosphorylation. Unlike p38, the total amount of JNK protein was not markedly changed (Fig. 8).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The epithelium is very sensitive to small increases in luminal osmolarity. The concentration-response curves for hyperpolarization and dilation at concentrations of D-M lower than 80 mOsM were coincident, suggesting that a link exists between the two types of responses over an osmolar concentration range that is relevant to changes in the ASL during exercise. However, above 80 mOsM D-M, depolarization ensued even though dilation continued to occur. Earlier, a single challenge with 240 mOsM sucrose was reported to depolarize the epithelium (Dortch-Carnes et al., 1999).
The transformation in the bioelectric responses to D-M is not understood; a graded continuum of bioelectric response of one type would be expected. At D-M concentrations 80 mOsM, the hyperpolarization is likely to result from rapid (within seconds) apical Cl– and basolateral K+ secretion as the cells shrink (Russell, 2000). Upon shrinkage, most cells readjust their volume, principally through activation of Na+,K+,2Cl– cotransport (O'Neill, 1999). However, airway epithelial cells challenged with hyperosmolarity do not undergo RVI (Willumsen et al., 1994; Fedan et al., 2007a,b). At D-M concentrations 
80 mOsM, apical D-M (120 mOsM) rapidly decreased short-circuit current (Wu et al., 2004), suggesting that the driving force for transport, the Na+,K+-pump, was inhibited. However, depolarization after inhibition of the Na+,K+-pump with ouabain is slow in onset and completion compared with the rapid depolarization triggered by high concentrations of D-M. Another explanation is epithelial Na+ absorption was decreased by large elevations in osmolarity. This notion is supported by the finding that high levels of osmolarity (using NaCl) decreased Vt of human nasal epithelium as a result of a decrease in Na+ conductance (Hebestreit et al., 2007). The phenomenon is also supported by findings in other cell types. For example, in rat hepatocytes, Na+ channel conductance was increased in response to 80 mOsM and higher concentrations of sucrose (Wehner et al., 2000). The depolarization at D-M concentrations 80 mOsM could result from a decrease in transepithelial resistance (Rt). However, in the Ussing chamber (Wu et al., 2004) and the perfused trachea apparatus (Y. Jing and J. S. Fedan, unpublished observations), D-M did not decrease Rt. Further studies will be necessary to establish the mechanisms involved in the bioelectric responses to low and high concentrations of D-M.
We investigated the possible roles of kinases and phosphatases in epithelial signaling involved in the regulation of Vt and P and responses to MCh and D-M. Of the six MAP kinase inhibitors, only SB 203580 decreased basal Vt; because SKF 86002 did not produce the same effect, the effect of SB 203580 might not involve p38. Chelerythrine, LY 294002, KN-62, and ML-7 decreased Vt. In contrast, NaAsO2 and Na3VO4 increased basal Vt. By increasing protein phosphorylation, the effects of NaAsO2 and Na3VO4 on ion transport should be opposite those of the kinase inhibitors. Thus, these results suggest that ion transport of unstimulated epithelial cells is regulated by PKC, PI-3-K, CaM-K-II, and MLCK.
Administered intraluminally, the MAP kinase inhibitors were generally without effect on the constriction responses to MCh. However, evidence was obtained that PI-3-K, phosphatases, and, possibly, PKC, may regulate reactivity of the muscle to MCh indirectly at the level of the epithelial cells.
MCh-induced epithelial hyperpolarization in dog (Phillips et al., 2002) and guinea pig tracheal epithelium (Wu et al., 2004) results primarily from Cl– secretion and activation of the Na+,K+,2Cl– cotransporter (Wu et al., 2004). MCh activated JNK in Chinese hamster ovary cells, and the activation of JNK was negatively regulated by PKC (Wylie et al., 1999). This suggests that PKC and JNK could have differential regulatory effects on MCh-induced responses. These findings help to explain the diverse effects of SP 600125, dicumarol, and chelerythrine on the MCh-induced hyperpolarization. PKC down-regulation of the activity of JNK was also observed in Rat-1 fibroblasts (Cadwallader et al., 1997). Because the activity of the Na+,K+,2Cl– cotransporter is modulated by phosphorylation, PKC and JNK may regulate responses to MCh at this locus (Flatman, 2002). The recent findings that Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) may be the primary regulatory kinases of the Na+,K+,2Cl– cotransporter (Gagnon et al., 2006) could suggest that PKC may be involved in a secondary but related capacity.
SP 600125, dicumarol, and chelerythrine inhibited D-M-induced hyperpolarization. The mechanism may also have involved inhibition of the Na+,K+,2Cl– cotransporter and secondary secretion of Cl– and K+, because hyperosmolar sucrose induced activation of the Na+,K+,2Cl– cotransporter through the regulation of JNK and PKC in endothelial and epithelial cells, respectively (Klein et al., 1999; Liedtke and Cole, 2002). Inhibition of hyperpolarization by SP 600125 and dicumarol occurs not only through inhibition of transcellular ion transport but also involves decreases in Rt (Y. Jing and J. S. Fedan, unpublished observations).
The effects of NaAsO2 and Na3VO4 may have resulted from their interactions with multiple kinase pathways involved in the regulation of the Na+,K+,2Cl– cotransporter (Zagórska et al., 2007). Inhibition of the D-M-induced hyperpolarization by NaAsO2 involves decreases in Rt (Y. Jing and J. S. Fedan, unpublished observations). Although it inhibits the Na+,K+pump (Dafnis and Sabatini, 1994), which would lead to depolarization, Na3VO4 increased Vt. Na3VO4 had no effect on Rt (Y. Jing and J. S. Fedan, unpublished observations); its inhibition of hyperpolarization could result from decreased transcellular ion transport.
p38 inhibition led to potentiation of EpDRF-mediated dilation responses. Immunoblotting experiments were performed to obtain biochemical evidence to support the pharmacological evidence for an involvement of p38 in EpDRF release. An important observation was the high levels of phosphorylated p38 in unstimulated epithelium, compared with what is observed in epithelial cell culture (Hashimoto et al., 1999b). A question that we are unable to answer is whether this represents the constitutive level of phosphorylation or an artifact of tissue handling. Nevertheless, the immunoblotting results suggested that there are small increases in phospho-p38 over the concentration range in which D-M initiates hyperpolarization and dilation. It is unequivocal that large increases in phosphorylation occurred at supraphysiological concentrations associated with depolarization. Is it possible that only small increases in phospho-p38 are required to modulate EpDRF release over osmolar concentrations that are pertinent to exercise? If the high basal levels of phospho-p38 do reflect the influence of experimental manipulations, the -fold increases in phosphorylation at the lower D-M concentrations would have been greater.
In conclusion, our results indicate that physiologically relevant increases in luminal osmolarity elicit epithelial hyperpolarization and smooth muscle dilation; the two responses share the same osmolarity concentration dependence. p38 plays a modulatory, inhibitory role in the EpDRF-mediated dilation. As such, p38 inhibition could be a useful therapeutic approach for preventing obstruction during exercise without affecting epithelial ion transport. Hyperosmolarity-induced hyperpolarization may be regulated by JNK, PKC, and phosphatase, but these pathways seem not to be involved in the regulation of EpDRF release.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ASL, airway surface liquid; D-M, D-mannitol; EpDRF, epithelium-derived relaxing factor; RVI, regulatory volume increase; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; CaM-K-II, calmodulin kinase-II, MLCK, myosin light chain kinase; PI-3-K, phosphatidylinositol 3-kinase; MCh, methacholine; MKH, modified Krebs-Henseleit; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PD 98059, 2'-amino-3'-methoxyflavone; U 0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; LY 294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; DMSO, dimethyl sulfoxide; Vt, transepithelial potential difference; P, inlet minus outlet perfusion pressure difference; Rt, transepithelial resistance; SKF 86002, 6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole; SP 600125, anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone; ML-7, 1-(5-iodonaphthalene-1-sulfonyl)homopiperazine.
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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Anderson SD (2006) How does exercise cause asthma attacks? Curr Opin Allergy Clin Immunol 6: 37–42.[Medline]
Anderson SD and Daviskas E (2000) The mechanism of exercise-induced asthma is... J Allergy Clin Immunol 106: 453–459.[CrossRef][Medline]
Anderson SD and Kippelen P (2005) Exercise-induced bronchoconstriction: pathogenesis. Curr Allergy Asthma Rep 5: 116–122.[Medline]
Boucher RC (1999) Molecular insights into the physiology of the "thin film" of airway surface liquid. J Physiol 516: 631–638.
Cadwallader K, Beltman J, McCormick F and Cook S (1997) Differential regulation of extracellular signal-regulated protein kinase 1 and Jun N-terminal kinase 1 by Ca2+ and protein kinase C in endothelin-stimulated Rat-1 cells. Biochem J 321: 795–804.[Medline]
Cross JV, Deak JC, Rich EA, Qian Y, Lewis M, Parrott LA, Mochida K, Gustafson D, Vande Pol S, and Templeton DJ (1999) Quinone reductase inhibitors block SAPK/JNK and NF
B pathways and potentiate apoptosis. J Biol Chem 274: 31150–31154.
Dafnis E and Sabatini S (1994) Biochemistry and pathophysiology of vanadium. Nephron 67: 133–143.[Medline]
Dortch-Carnes J, Van Scott MR, and Fedan JS (1999) Changes in smooth muscle tone during osmotic challenge in relation to epithelial bioelectric events in guineapig isolated trachea. J Pharmacol Exp Ther 289: 911–917.
English JM and Cobb MH (2002) Pharmacological inhibitors of MAPK pathways. Trends Pharmacol Sci 23: 40–45.[CrossRef][Medline]
Fedan JS and Frazer DG (1992) Influence of epithelium on the reactivity of guineapig isolated, perfused trachea to bronchoactive drugs. J Pharmacol Exp Ther 262: 741–750.
Fedan JS, Dowdy J, and Van Scott MR (2007a) Comparison of the effects of isosmolar (IM) and hyperosmolar (HM) solutions on cell volume (CV) of guinea-pig fresh tracheal epithelial cell (EC) suspensions and epithelium-derived relaxing factor (EpDRF) release (Abstract). Am J Respir Crit Care Med 175: A887.
Fedan JS, Dowdy JA, Johnston RA, and Van Scott MR (2004a) Hyperosmolar solution effects in guinea pig airways. I. Mechanical responses to relative changes in osmolarity. J Pharmacol Exp Ther 308: 10–18.
Fedan JS, Dowdy JA, Van Scott MR, Wu DX, and Johnston RA (2004b) Hyperosmolar solution effects in guinea pig airways. III. Studies on the identity of epithelium-derived relaxing factor in isolated perfused trachea using pharmacological agents. J Pharmacol Exp Ther 308: 30–36.
Fedan JS, Ismailoglu UB, Dowdy J, and Roberts JR (2007b) Cell volume (CV) responses of adherent cells and cell suspensions of guinea-pig fresh tracheal epithelial cells (EC) to hyperosmolar challenge (Abstract). FASEB J 21: A1170.
Fedan JS, Nutt ME, and Frazer DG (1990) Reactivity of guinea-pig isolated trachea to methacholine, histamine and isoproterenol applied serosally vs. mucosally. Eur J Pharmacol 190: 337–345.[CrossRef][Medline]
Fedan JS, Yuan LX, Chang VC, Viola JO, Cutler D, and Pettit LL (1999) Osmotic regulation of airway reactivity by epithelium. J Pharmacol Exp Ther 289: 901–910.
Flatman PW (2002) Regulation of Na-K-2Cl cotransport by phosphorylation and protein-protein interactions. Biochim Biophys Acta 1566: 140–151.[Medline]
Flatman PW and Creanor J (1999) Stimulation of Na+-K+-2Cl– cotransport by arsenite in ferret erythrocytes. J Physiol 519: 143–152.
Freed AN and Davis MS (1999) Hyperventilation with dry air increases airway surface fluid osmolality in canine peripheral airways. Am J Respir Crit Care Med 159: 1101–1107.
Furuichi S, Hashimoto S, Gon Y, Matsumoto K, and Horie T (2002) p38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase regulate interleukin-8 and RANTES production in hyperosmolarity stimulated human bronchial epithelial cells. Respirology 7: 193–200.[CrossRef][Medline]
Gagnon KB, England R, and Delpire E (2006) Characterization of SPAK and OSR1, regulatory kinases of the Na-K-2Cl cotransporter. Mol Cell Biol 26: 689–698.
Göggel R and Uhlig S (2005) The inositol trisphosphate pathway mediates platelet-activating-factor-induced pulmonary oedema. Eur Respir J 25: 849–857.
Gordon JA (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 201: 477–482.[Medline]
Hallstrand TS, Moody MW, Aitken ML and Henderson WR Jr (2005a) Airway immunopathology of asthma with exercise-induced bronchoconstriction. J Allergy Clin Immunol 116: 586–593.[CrossRef][Medline]
Hallstrand TS, Moody MW, Wurfel MM, Schwartz LB, Henderson WR Jr, and Aitken ML (2005b) Inflammatory basis of exercise-induced bronchoconstriction. Am J Respir Crit Care Med 172: 679–686.
Hamilton LM, Davies DE, Wilson SJ, Kimber I, Dearman RJ, and Holgate ST (2001) The bronchial epithelium in asthma-much more than a passive barrier. Monaldi Arch Chest Dis 56: 48–54.[Medline]
Hashimoto S, Gon Y, Asai Y, Asai Y, Machino T, Jibiki I, Takeshita I, Anazawa H, and Horie T (1999a) p38 MAP kinase regulates RANTES production by TNF--stimulated human pulmonary vascular endothelial cells. Allergy 54: 1168–1172.[CrossRef][Medline]
Hashimoto S, Matsumoto K, Gon Y, Maruoka S, Kujime K, Hayashi S, Takeshita I, and Horie T (2000) p38 MAP kinase regulates TNF -, IL-1 - and PAF-induced RANTES and GM-CSF production by human bronchial epithelial cells. Clin Exp Allergy 30: 48–55.[CrossRef][Medline]
Hashimoto S, Matsumoto K, Gon Y, Nakayama T, Takeshita I, and Horie T (1999b) Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase. Am J Respir Crit Care Med 159: 634–640.
Hebestreit A, Kersting U, and Hebestreit H (2007) Hypertonic saline inhibits luminal sodium channels in respiratory epithelium. Eur J Appl Physiol 100: 177–183.[CrossRef][Medline]
Herbert JM, Augereau JM, Gleye J, and Maffrand JP (1990) Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun 172: 993–999.[CrossRef][Medline]
Hirst SJ, Hallsworth MP, Peng Q, and Lee TH (2002) Selective induction of eotaxin release by interleukin-13 or interleukin-4 in human airway smooth muscle cells is synergistic with interleukin-1β and is mediated by the interleukin-4 receptor -chain. Am J Respir Crit Care Med 165: 1161–1171.
Jayaraman S, Song Y, and Verkman AS (2001) Airway surface liquid osmolality measured using fluorophore-encapsulated liposomes. J Gen Physiol 117: 423–430.
Johnston RA, Van Scott MR, Kommineni C, Millecchia LL, Dortch-Carnes J, and Fedan JS (2004) Hyperosmolar solution effects in guinea pig airways. IV. Lipopolysaccharide-induced alterations in airway reactivity and epithelial bioelectric responses to methacholine and hyperosmolarity. J Pharmacol Exp Ther 308: 37–46.
Kagawa J and Kerr HD (1970) Effects of brief graded exercise on specific airway conductance in normal subjects. J Appl Physiol 28: 138–144.
Klein JD, Lamitina ST, and O'Neill WC (1999) JNK is a volume-sensitive kinase that phosphorylates the Na-K-2Cl cotransporter in vitro. Am J Physiol 277: C425–C431.[Medline]
Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, and Haussinger D (1998) Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247–306.
Liedtke CM and Cole TS (2002) Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-
and ERK. Biochim Biophys Acta 1589: 77–88.[Medline]
Ludwig S, Hoffmeyer A, Goebeler M, Kilian K, Hafner H, Neufeld B, Han J, and Rapp UR (1998) The stress inducer arsenite activates mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 via a MAPK kinase 6/p38-dependent pathway. J Biol Chem 273: 1917–1922.
Massé T and Kelly PT (1997) Overexpression of Ca2+/calmodulin-dependent protein kinase II in PC12 cells alters cell growth, morphology, and nerve growth factor-induced differentiation. J Neurosci 17: 924–931.
Munakata M, Huang I, Mitzner W, and Menkes H (1989) Protective role of epithelium in the guinea pig airway. J Appl Physiol 66: 1547–1552.
Munakata M, Masaki Y, Sakuma I, Ukita H, Otsuka Y, Homma Y, and Kawakami Y (1990) Pharmacological differentiation of epithelium-derived relaxing factor from nitric oxide. J Appl Physiol 69: 665–670.
Munakata M, Mitzner W, and Menkes H (1988) Osmotic stimuli induce epithelial-dependent relaxation in the guinea pig trachea. J Appl Physiol 64: 466–471.
O'Neill WC (1999) Physiological significance of volume-regulatory transporters. Am J Physiol 276: C995–C1011.[Medline]
Pelaia G, Cuda G, Vatrella A, Gallelli L, Caraglia M, Marra M, Abbruzzese A, Caputi M, Maselli R, Costanzo FS, et al. (2005) Mitogen-activated protein kinases and asthma. J Cell Physiol 202: 642–653.[CrossRef][Medline]
Phillips JE, Hey JA, and Corboz MR (2002) Effects of ion transport inhibitors on MCh-mediated secretion from porcine airway submucosal glands. J Appl Physiol 93: 873–881.
Porsbjerg C, Rasmussen L, Thomsen SF, Brannan JD, Anderson SD, and Backer V (2007) Response to mannitol in asymptomatic subjects with airway hyperresponsiveness to methacholine. Clin Exp Allergy 37: 22–28.[CrossRef][Medline]
Prabhakar U, Lipshutz D, and Truneh A (1993) Inhibition of CD44, CD45 and LFA-3 mediated cytokine release from human monocytes by SK&F 86002 and pentoxifylline. Int J Immunopharmacol 15: 205–209.[CrossRef][Medline]
Puddicombe SM and Davies DE (2000) The role of MAP kinases in intracellular signal transduction in bronchial epithelium. Clin Exp Allergy 30: 7–11.[CrossRef][Medline]
Pugazhenthi S, Tanha F, Dahl B, and Khandelwal RL (1996) Inhibition of a Src homology 2 domain containing protein tyrosine phosphatase by vanadate in the primary culture of hepatocytes. Arch Biochem Biophys 335: 273–282.[CrossRef][Medline]
Russell JM (2000) Sodium-potassium-chloride cotransport. Physiol Rev 80: 211–276.
Sheikh-Hamad D and Gustin MC (2004) MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals. Am J Physiol Renal Physiol 287: F1102–F1110.
Shen MR, Furla P, Chou CY, and Ellory JC (2002) Myosin light chain kinase modulates hypotonicity-induced Ca2+ entry and Cl– channel activity in human cervical cancer cells. Pflugers Arch 444: 276–285.[CrossRef][Medline]
Shrode L, Krump E, and Grinstein S (1998) Activation of protein kinases upon volume changes: role in cellular homeostasis, in Cell Volume Regulation (Lang F ed) pp 79–93, Karger, Basel, Switzerland.
Silverman NK, Johnson AT, Scott WH, and Koh FC (2005) Exercise-induced respiratory resistance changes as measured with the airflow perturbation device. Physiol Meas 26: 29–38.[CrossRef][Medline]
Tarran R (2004) Regulation of airway surface liquid volume and mucus transport by active ion transport. Proc Am Thorac Soc 1: 42–46.
Tokumitsu H, Chijiwa T, Hagiwara M, Mizutani A, Terasawa M, and Hidaka H (1990) KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 265: 4315–4320.
Vlahos CJ, Matter WF, Hui KY, and Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269: 5241–5248.
Wehner F, Bohmer C, Heinzinger H, van den Boom F, and Tinel H (2000) The hypertonicity-induced Na+ conductance of rat hepatocytes: physiological significance and molecular correlate. Cell Physiol Biochem 10: 335–340.[Medline]
Widdicombe JH (2002) Regulation of the depth and composition of airway surface liquid. J Anat 201: 313–318.[CrossRef][Medline]
Willumsen NJ, Davis CW, and Boucher RC (1994) Selective response of human airway epithelia to luminal but not serosal solution hypertonicity. Possible role for proximal airway epithelia as an osmolality transducer. J Clin Invest 94: 779–787.[Medline]
Woo CH, Lim JH, and Kim JH (2005) VCAM-1 upregulation via PKC-p38 kinase-linked cascade mediates the TNF--induced leukocyte adhesion and emigration in the lung airway epithelium. Am J Physiol Lung Cell Mol Physiol 288: L307–L316.
Wu DX, Johnston RA, Rengasamy A, Van Scott MR, and Fedan JS (2004) Hyperosmolar solution effects in guinea pig airways. II. Epithelial bioelectric responses to relative changes in osmolarity. J Pharmacol Exp Ther 308: 19–29.
Wylie PG, Challiss RA, and Blank JL (1999) Regulation of extracellular-signal regulated kinase and c-Jun N-terminal kinase by G-protein-linked muscarinic acetylcholine receptors. Biochem J 338: 619–628.[CrossRef][Medline]
Zagórska A, Pozo-Guisado E, Boudeau J, Vitari AC, Rafiqi FH, Thastrup J, Deak M, Campbell DG, Morrice NA, Prescott AR, et al. (2007) Regulation of activity and localization of the WNK1 protein kinase by hyperosmotic stress. J Cell Biol 176: 89–100.
Zhao H, Hyde R, and Hundal HS (2004) Signalling mechanisms underlying the rapid and additive stimulation of NKCC activity by insulin and hypertonicity in rat L6 skeletal muscle cells. J Physiol 560: 123–136.
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