1Department of Pediatric Otolaryngology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; and Departments of 2Otolaryngology and 3Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
Submitted 26 November 2002 ; accepted in final form 7 May 2003
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ABSTRACT |
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ion channels; sodium; chloride; hypoxia
The primary determinant of baseline transepithelial potential difference in cultured gerbil middle ear epithelial cells is apical sodium (Na+) absorption through amiloride-sensitive Na+ channels (4, 9, 10). Compared with gerbil middle ear epithelial cells exposed to 21% O2, a significant, progressive decrease in apical Na+ absorption has been shown with up to 18 h of exposure to 0%, but not 5 and 10% O2 concentrations (14).
At baseline, a slight amount of apical Cl secretion is present in gerbil middle ear epithelial cells (7), and it is Cl secretion that is believed to account for residual current after middle ear epithelium apical Na+ channels are blocked with amiloride (4, 10). Stimulation of Cl secretion can be achieved by applying UTP to either the apical or the basolateral surfaces of middle ear epithelial cells, possibly due to involvement of Ca2+-activated Cl channels [Cl(Ca)] (5). The effect of changes in ambient O2 concentration on middle ear epithelial Cl secretion is not known.
The purpose of the present study was to compare the effect of 24 h of exposure to 7% O2 (normal middle ear physiological conditions) vs. 21% O2 (middle ear conditions after ventilation tube placement) on transepithelial Na+ absorption and Cl secretion in cultured gerbil middle ear epithelial cell monolayers.
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MATERIALS AND METHODS |
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Reagents. Amiloride (105 M) was obtained from Sigma and was made as a 1,000-fold stock solution in water. UTP (100 µM) was obtained from Calbiochem (La Jolla, CA) and was also made as a 1,000-fold stock solution in water. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) (100 µM) was obtained from Sigma and was made as a 1,000-fold stock solution in dimethyl sulfoxide. All chemicals were of analytical grade.
Hypoxic and hyperoxic exposure. To achieve hypoxic exposure, 47 days after plating, culture dishes were placed in a humidified airtight incubation chamber with inflow and outflow valves (Billups-Rothenberg, Del Mar, CA). The desired gas mixture (either 7% O2-5% CO2-88% N2 or 14% O2-5% CO2-81% N2) was delivered at a constant flow rate (1 l/min) for about 30 min until equilibrated. The closed chamber was placed in a tissue culture incubator at 37°C for 24 h. Control normoxic cells (21% O2-5% CO2-74% N2) were placed in a humidified chamber with the valves left open for the same time period. Before culture plates were removed from the chambers, the O2 concentration was confirmed by using an oximeter (MiniOxI oxygen analyzer, Catalyst Research, Owings Mills, MD). The corresponding O2 partial pressures for O2 concentrations of 7, 14, and 21% were 53, 106, and 159 mmHg, respectively.
Other cultures were similarly exposed to hyperoxic conditions of 50, 70, or 95% O2, plus 5% CO2, for 24 h. Under all experimental conditions of O2, 5% CO2 was used as part of the pH buffering of the cell culture medium.
Bioelectric measurements. Transwell cell culture inserts containing the cultured cells were mounted into an Ussing chamber (Jim's Instruments, Dept. of Bioengineering, Univ. of Iowa, Iowa City, IA). The monolayers were continuously short-circuited via an automatic voltage clamp (Harvard Apparatus, Holliston, MA). Transepithelial short-circuit current (Isc) was continuously monitored with a 4-s, 5-mV pulse applied every 56 s to determine the resistance (RT), which was calculated using Ohm's law (R = V/I). The bath solution contained 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose. The pH of this solution was 7.4 when gassed with a mixture of 95% O2-5%CO2 at 37°C. A minimum of 10 min was allowed to elapse before determining baseline Isc and RT measurements, after which pharmacological agents were added to the apical and/or the basal bath, with Isc and RT measured immediately and at 10 min after drug addition.
Statistical analysis. Results are expressed as means ± SE.
Statistical analysis was performed using Student's t-test. A
P value of 0.05 was considered statistically significant. The
value n refers to the number of replicate experiments performed.
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RESULTS |
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Figure 2 demonstrates the effect of mucosal amiloride (10 µM) and UTP (100 µM) on Isc in MESV cell monolayer exposed to 24 h of 21% O2 in a representative tracing.
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Application of the Na+ channel blocker amiloride (1 x 105 M) resulted in a drop in baseline Isc of 89, 89, and 88% in cells exposed to 7% O2 (2.7 ± 0.3 µA/cm2), 14% O2 (3.0 ± 0.2 µA/cm2), and 21% O2 (2.9 ± 0.1 µA/cm2), respectively (see Figs. 3 and 4). There were no significant differences in postamiloride Isc among cells exposed to the three O2 concentrations 7, 14, or 21%. After amiloride application, RT was similar between cells exposed to all three oxygen concentrations. In cells exposed to 0% O2, application of amiloride resulted in a drop in baseline Isc of 71% (2.0 µA/cm2), suggesting that even in the setting of low baseline Isc in the presence of 0% O2, the majority of transepithelial ion activity was still due to Na+ channel activity.
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With the cell monolayers in a hyperpolarized state following blockade of Na+ absorption with amiloride, isolated Cl secretion in response to UTP was able to be studied (see Fig. 3). Application of 100 µM UTP to the apical surface of the cells resulted in an initial dramatic increase in Isc that was greatest in cells exposed to 21% O2 (31.3 ± 1.9 µA/cm2) and was significantly lower in cells exposed to 14% O2 (20.5 ± 1.4 µA/cm2) and 7% O2 (20.1 ± 1.6 µA/cm2). The initial increase in Isc after UTP application was similar between cells exposed to 7% O2 (20.1 ± 1.6 µA/cm2) and 14% O2 (20.5 ± 1.4 µA/cm2). Initially after UTP application, RT was similar among cells exposed to all three O2 concentrations.
After the initial increase in Isc following apical UTP application in the setting of amiloride blockade, Isc decreased to a sustained level, lower than baseline Isc (before any pharmacological intervention), but higher than the Isc following amiloride application only (see Fig. 3). The sustained Isc following apical UTP application was highest in cells that had been exposed to 21% O2 (10.8 ± 1.0 µA/cm2) and was noticeably, but not significantly, lower in cells that had been exposed to both 14% O2 (8.4 ± 0.5 µA/cm2) and 7% O2 (8.5 ± 0.7 µA/cm2).
In another set of experiments, cell monolayers were hyperpolarized with amiloride and 100 µM UTP was then applied to the basolateral surface of the monolayers, yielding changes in Isc that were similar to those observed after apical UTP application (see Fig. 4). The initial increase in Isc following basolateral UTP administration was greatest in cells exposed to 21% O2 (24.1 ± 1.9 µA/cm2) and was significantly lower in cells exposed to 14% O2 (17.1 ± 1.7 µA/cm2) and 7% O2 (14.8 ± 1.2 µA/cm2). RT was similar among cells exposed to all three O2 concentrations.
After the initial increase in Isc following basolateral UTP application in the setting of amiloride blockade, Isc decreased to a sustained level, lower than baseline Isc (before any pharmacological intervention), but higher than the Isc following amiloride application (see Fig. 4). This pattern was similar to that seen after apical UTP application. The sustained Isc following basolateral UTP application was highest in cells that had been exposed to 21% O2 (9.7 ± 0.6 µA/cm2), lower in cells that had been exposed to 14% O2 (8.2 ± 1.0 µA/cm2), and least in cells that had been exposed to 7% O2 (7.3 ± 0.8 µA/cm2). The difference in sustained Isc between cells exposed to 7% vs. 21% O2 was statistically significant; the differences between cells exposed to 7 vs. 14%, and 14 vs. 21%, were not significant.
To determine whether the enhanced UTP-induced stimulation of Cl secretion in the setting of 21% O2 exposure involved Ca2+-activated Cl (Cl(Ca)) channels, cell monolayers exposed to 21% O2 were hyperpolarized with amiloride and then 100 µM DIDS (a Cl(Ca) channel inhibitor) was applied to the apical surface (see Fig. 5). In the presence of DIDS, basal application of UTP generated an initial increase in Isc (12.2 µA/cm2) that was of significantly less magnitude compared with the initial spike in Isc seen with UTP application in the absence of DIDS under otherwise similar conditions (24.1 µA/cm2). However, as Isc decreased to a new sustained level after the initial response to UTP, the difference in sustained Isc was similar between cells that were (10.9 µA/cm2) and were not (9.7 µA/cm2) exposed to DIDS in the setting of 21% O2 exposure.
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DISCUSSION |
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The effect, if any, of relative middle ear hyperoxia after ventilation tube placement on mucosal ion transport and thus on mucosal absorptive or secretory properties is not known. Given the paucity and inaccessibility of human middle ear mucosal specimens, most studies of middle ear mucosa have utilized transformed cell cultures derived from the Mongolian gerbil (Meriones unguiculatus) (9), because in this species the distribution of middle ear epithelial cells is similar to that of the human (3).
Amiloride-sensitive apical Na+ channels have been demonstrated to be the major determinant of baseline electrogenic ion transport in Mongolian gerbil middle ear epithelium, accounting for 62 (4) to 90% (9) of baseline Isc.
At baseline, a slight amount of transepithelial Cl secretion is present in gerbil middle ear cells (7). Residual current after blocking apical Na+ channels with amiloride is believed to be due to passive transepithelial Cl secretion (4, 10). Stimulation of Cl secretion can be achieved by applying UTP to either the apical or the basolateral surfaces of middle ear cells, possibly due to activation of Cl(Ca) channels (5). UTP is known to activate a Cl(Ca) channel, resulting in stimulation of Cl secretion (12). In the present study, the initial marked increase in Isc seen in response to UTP at 21% O2 concentration was significantly less robust in the presence of DIDS, a Cl(Ca) channel blocker, suggesting that Cl(Ca) channels may play an important role in fluid secretion in the middle ear.
In transformed cultured Mongolian gerbil middle ear epithelium, it has been shown that 618 h of exposure to 0% O2 concentrations results in a progressive decrease in apical Na+ absorption compared with cells exposed to 21% O2 (14). Similarly, in cultured rat type II alveolar epithelial cells exposed to 328 h of 0% O2 concentration, a time-dependent decrease in apical Na+ channel activity was noted (13). Exposure to 3% O2 also resulted in a decrease in Na+ channel activity, whereas exposure to 5% O2 did not. In another study, fetal distal lung epithelial cell monolayers grown for 3 days in 20% O2 demonstrated significantly higher levels of total and amiloride-sensitive Isc compared with cells exposed to 3 days of 2.5 to 5% O2 (16).
In the present study, exposure of transformed cultured Mongolian gerbil middle ear epithelial monolayers to 24 h of 0% O2 also resulted in decreased amiloride-sensitive Isc compared with similar monolayers exposed to higher O2 concentrations, similar to other studies. However, a concentration of 0% O2 is experimental and does not reflect the O2 concentration that is found in either the normal middle ear or the ear with a serous middle ear effusion (7%, PO2 = 43 mmHg) in humans (6). In the present study, exposure of cultured gerbil middle ear epithelial monolayers to 7% O2 resulted in no change in Na+ channel activity compared with similar cells exposed to 21% O2 concentration. Therefore, our results suggest that changes in Na+ transport cannot account for alterations in fluid clearance from the middle ear after tympanostomy tube placement.
In the present study, transepithelial Cl secretion was isolated by blocking apical Na+ channels with amiloride. Then, Cl secretion was stimulated using UTP, applied to either the apical or basolateral cell monolayer surface. The typical response of Isc after UTP application (an initial marked increase, or spike, in Isc followed by a sharp decrease and new sustained level of Isc) occurred in cells exposed to both 7 and 21% O2. However, in cells that had been exposed to 21% O2, the initial increase in Cl secretion was significantly greater than that seen in cells that had been exposed to 7% O2. The subsequent drop and final sustained level of Isc, which represented ongoing Cl secretion at a level above that seen before UTP application, was still greater in cells exposed to 21% O2 compared with cells exposed to 7% O2, although the difference was only significant with basolateral UTP application. Another study has shown, using a cultured canine kidney cell line, that exposure to relative hyperoxia (20%) results in an increase in forskolin-stimulated Cl secretion compared with exposure to lower O2 tensions (as low as 2.5%) (2).
We theorize that the increase in UTP-stimulated transepithelial
Cl secretion seen in middle ear mucosal cells exposed to 21
vs. 7% O2 will result in a relatively small amount of apical water
secretion via osmosis. Because Cl secretion constitutes a
relatively small amount of Isc (10%), the amount of
water that is secreted into the middle ear space may not result in the
development of an effusion but, rather, may contribute to the middle ear space
in a more subtle manner.
Ciliated pathways leading to the Eustachian tube have been identified in both human (8) and Mongolian gerbil (3) middle ear epithelium. Secretions propelled by ciliated respiratory epithelium consist of two layers: a viscous mucus (gel) layer above the cilia that is propelled via intermittent contact with the tips of the cilia, and a serous periciliary (sol) layer within which the cilia beat (1, 15). The origin of the periciliary fluid is not known but has been proposed to originate via transepithelial osmosis as governed by mucosal ion transport mechanisms (15).
If the periciliary fluid layer is reduced, the cilia may become entangled in the mucus and thus beat less efficiently (1, 15). A decrease in periciliary fluid has been demonstrated in middle ear mucosal specimens from humans with otitis media with effusion (11). The pathophysiology of this decrease in periciliary fluid is unknown and is unlikely to be due to changes in middle ear O2 concentration, because O2 concentration is similar in nontubulated ears with and without a serous effusion (6).
Irrespective of the cause of decreased middle ear periciliary fluid in patients with otitis media with effusion, we theorize that this periciliary fluid deficiency may be overcome by enhancement of apical Cl secretion after ventilation tube placement due to a resultant increase in middle ear O2 concentration from 7 to 21%. Although the middle ear cells in our study are not ciliated, periciliary fluid has been found to be present on the surface of both ciliated and nonciliated human middle ear cells (11), suggesting that even nonciliated middle ear mucosal cells may regulate sol layer fluid production via their microvilli.
Transepithelial chloride secretion requires the coordinate action of both apical chloride channels and basolateral potassium channels (5). Future work is planned to further elucidate the effect upon these channels of an increase in ambient O2 concentration from 7 to 21%.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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