Hydrostatic pressure-regulated ion transport in bladder uroepithelium

Edward C. Y. Wang,1,2 Jey-Myung Lee,1 John P. Johnson,1 Thomas R. Kleyman,1,2 Robert Bridges,2 and Gerard Apodaca1,2

1Renal-Electrolyte Division, Department of Medicine, Laboratory of Epithelial Cell Biology, and 2Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Submitted 13 November 2002 ; accepted in final form 21 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The effect of hydrostatic pressure on ion transport in the bladder uroepithelium was investigated. Isolated rabbit uroepithelium was mounted in modified Ussing chambers and mechanically stimulated by applying hydrostatic pressure across the mucosa. Increased hydrostatic pressure led to increased mucosal-to-serosal Na+ absorption across the uroepithelium via the amiloride-sensitive epithelial Na+ channel. In addition to this previously characterized pathway for Na+ absorption, hydrostatic pressure also induced the secretion of Cl and K+ into the mucosal bathing solution under short-circuit conditions, which was confirmed by a net serosal-to-mucosal flux of 36Cl and 86Rb+. K+ secretion was likely via a stretch-activated nonselective cation channel sensitive to 100 µM amiloride, 10 mM tetraethylammonium, 3 mM Ba2+, and 1 mM Gd3+. Hydrostatic pressure-induced ion transport in the uroepithelium may play important roles in electrolyte homeostasis, volume regulation, and mechanosensory transduction.

mechanical force; epithelial sodium channel; nonselective cation channel


CELLS ARE EXPOSED TO AN ARRAY of physical forces, including compression, shear stress, and hydrostatic pressure, which occurs when bodily fluids push against the epithelium that lines the tubes and sacs that form many of the body's organs (3). Changes in these forces can result in alterations in cellular structure, function, gene expression, and membrane traffic (2, 9, 18, 55). A common response of cells exposed to mechanical stimuli is activation of stretch-activated ion channels. The first such channel described in detail is the stretch-activated nonselective cation channel of chick embryonic skeletal muscle (42), which is cation selective but discriminates poorly between Na+ and K+. Since these initial studies, stretch-activated K+, Cl, and nonselective cation channels have been identified in many cell types, including erythrocytes, oocytes, fibroblasts, aortic endothelium, heart cells, kidney cells, muscle cells, and epithelial cells (18).

In the urinary tract, where cells experience shear stress and hydrostatic pressure, mechanosensitive ion channels may play an important role in volume regulation and ion homeostasis. For example, a stretch-activated cation-selective channel is found in the apical membrane of proximal tubule cells. This channel is permeable to Ca2+, K+, and Na+ and is not gated by membrane potential or cytosolic Ca2+ (14), but its activity is dependent on changes in extracellular osmolarity. Mechanical stimulation of this channel (e.g., by cell swelling) could lead to cell volume regulation. In renal cortical collecting ducts, changes in hydrostatic pressure may increase the open probability, or number, of stretch-sensitive epithelial Na+ channels (ENaC), enhancing the rate of Na+ reabsorption (44). Moreover, the rate of K+ secretion by large-conductance maxi-K channels in cortical collecting ducts is also affected by variations in intraluminal flow rates (56).

The lower urinary tract is also subject to mechanical stimuli; hydrostatic pressure increases as the bladder fills and decreases during bladder emptying. The cell type most directly affected by these changes is the umbrella cell. These large polygonal cells constitute the innermost layer of the uroepithelium, and the combination of an impermeable apical membrane and high-resistance tight junctions forms an effective barrier to solute and ion permeability. Umbrella cells respond to mechanical stimuli in a number of ways, including an increase in surface area as well as ion transport (12, 36, 37, 52). Membrane "punching," performed by rapidly increasing and then decreasing hydrostatic pressure across the mucosal surface of the bladder uroepithelium, leads to increased ion conductance after 10–30 min (36, 37). The observed rise in ion conductance is contributed by the amiloride-sensitive ENaC as well as by an amiloride-insensitive nonselective cation channel (29, 35). Alternatively, ENaC activity can be acutely stimulated by removing fluid from the serosal side of bladder tissue mounted in an Ussing chamber, thereby increasing hydrostatic pressure across the mucosal surface of the bladder. In addition to Na+ absorption, a K+ secretory pathway, sensitive to the K+ channel blocker tetraethylammonium (TEA), has been identified in resting bladder tissue (13), but the nature of this secretory pathway and whether it is stimulated by hydrostatic pressure have not been defined.

The goal of this study was to examine the possibility that, like in the upper urinary tract, hydrostatic pressure stimulates multiple ion transport pathways in bladder uroepithelium. Rabbit uroepithelium was mounted in modified Ussing chambers and was then subjected to cycles of increased hydrostatic pressure while changes in short-circuit current (Isc) and ion conductance were monitored. We found that in addition to hydrostatic pressure-induced Na+ absorption across the uroepithelium, there was also under short-circuit conditions hydrostatic pressure-induced electroneutral Cl and K+ secretion into the mucosal chamber. Na+ absorption was via the previously described amiloride-sensitive ENaC, whereas K+ secretion was probably via a nonselective cation channel sensitive to 100 µM amiloride, TEA, Ba2+, and Gd3+. These results provide additional evidence that besides retaining urine, the mammalian bladder epithelium might play an important role in electrolyte homeostasis.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Unless otherwise specified, all chemicals were of reagent quality or better and were obtained from Sigma (St. Louis, MO). Stocks of the following inhibitors were prepared as follows: amiloride was dissolved in water at 10 mM, or when used at 500 µM it was dissolved directly into buffer solution; apamin was dissolved in water at 100 µM; BaCl2 was dissolved in Na+, Cl-free buffer at 1.5 M; GdCl3 was dissolved in Na+, Cl-free buffer at 100 mM; glibenclamide was dissolved in DMSO at 50 mM; ouabain was dissolved in water at 13 mM; and TEA was dissolved in Na+, Cl-free buffer at 1 M. The following K+ channel blockers were obtained from Alomone Laboratories (Jerusalem, Israel), and stocks were prepared as follows: charybdotoxin (CTX) was dissolved in water at 100 µM; iberiotoxin was dissolved in water at 1 µM; and margatoxin was dissolved in water at 10 µM. All inhibitors were freshly prepared before use.

Solutions. Control Krebs solution was prepared by mixing the following (in mM): 110 NaCl, 5.8 KCl, 25 NaHCO3, 1.2 KH2PO4, 2.0 CaCl2, 1.2 MgSO4, and 11.1 glucose. Na+-free Krebs had the following composition (in mM): 120 tetramethylammonium (TMA) Cl or N-methyl-D-glucamine (NMDG)-Cl, 3.33 KH2PO4, 0.83 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, 28 choline-HCO3, 0.01 atropine, and 2.7 glucose. Na+- and Cl-free Krebs had the following composition (in mM): 120 NMDG-gluconate buffer (prepared as a stock by adjusting 240 mM NMDG to pH 7.4 with gluconic acid), 3.33 KH2PO4, 0.83 K2HPO4, 4 Ca +-gluconate, 4 Mg2+-gluconate, 28 choline-HCO3, 0.01 atropine, and 2.7 glucose. For Cs+ transport experiments, the NMDG-gluconate solution had the following composition (in mM): 135 NMDG, 4 hemi-Ca2+-gluconate, and 10 HEPES. The pH was adjusted to 7.4 with D-gluconic acid. The Cs+-gluconate solution had the following composition (in mM): 135 CsOH, 4 hemi-Ca2+-gluconate, and 10 HEPES. The pH of the Cs+-gluconate buffer was adjusted to 7.4 with D-gluconic acid. All the solutions had an osmolarity of 280–300 mosM. Solutions that contained bicarbonate were aerated with a 95% O2-5% CO2 mixture and maintained at a pH of 7.4 at 37°C.

Preparation and mounting of uroepithelium and system for increasing hydrostatic pressure across the mucosal surface of the tissue. Animal experiments were performed in accordance with the Animal Use and Care Committee of the University of Pittsburgh (Pittsburgh, PA). Urinary bladders were obtained from female New Zealand White rabbits (3–4 kg; Myrtle's Rabbitry, Thompson Station, TN). Rabbits were euthanized with 300 mg of pentobarbital sodium, the bladder was excised, and after careful dissection of the muscle layers the mucosa was placed on tissue rings that nominally exposed 2 cm2 of tissue. The tissue rings were then mounted between two halves of a custom Ussing chamber as described previously (see Fig. 1 in Ref. 52). The serosal side of this Ussing chamber was open, whereas the mucosal chamber was enclosed and, after being filled, could be closed off. Once the tissue was mounted, each hemichamber (mucosal and serosal) was filled with 12.5 ml of Krebs solution, the serosal hemichamber was bubbled with 95% air-5% CO2 gas (when solutions contained bicarbonate), and the tissue was equilibrated for 30–60 min. Normally, each bladder yielded three rings of mounted tissue. Voltage-sensing and current-passing Ag/AgCl wires were placed in the chambers, the electrodes in the serosal chamber served as the reference electrodes, and tissue capacitance and transepithelial resistance (TER) were monitored throughout the equilibration period using the MacLab system described below. Only preparations that exhibited a starting capacitance of 1.8–2.1 µF and a TER of >5,000 {Omega} · cm2 were used.

To increase hydrostatic pressure across the mucosal surface of the tissue, additional Krebs solution was added to the mucosal hemichamber to a volume of 14 ml (hemichamber capacity), and an additional 0.5 ml of Krebs solution was injected with a syringe to increase the back pressure in the mucosal hemichamber to 8 cmH2O, as measured by a force transducer (AD Instruments, Mountain View, CA). This pressure is similar to that observed during the extended filling stage of the rabbit bladder (25). The chamber was then closed off for the times specified. To relieve the pressure, an identical amount of Krebs solution was removed from the mucosal hemichamber. When necessary, the mucosal and/or serosal Krebs solutions were isovolumetrically replaced by injecting 70 ml of modified Krebs solution toward the base of the hemichamber via a syringe needle while the solution was simultaneously withdrawn by vacuum suction connected to another syringe needle directed toward the top of the hemichamber. With the use of this technique, the volume of hemichamber solution was kept constant (12.5 ml) during the isovolumetric wash.

Measure of tissue capacitance. Capacitance (where 1 µF ~ 1cm2 of actual membrane area) was measured by monitoring the voltage response to a square-current pulse as described previously (52). The time constant, {tau}, of the resulting voltage response was determined by calculating the length of time required to reach 63% of the steady-state voltage by using a data transformation routine that included curve fitting the voltage response to a single exponential. The R values for curve fitting were >0.99 under all conditions. The capacitance was determined using the formula C = {tau}/R, where C is capacitance, and R is resistance. Resistance was determined by dividing the amplitude of the steady-state voltage response by the amplitude of the square-current pulse. Because the area of the tissue mounted on the ring is 2 cm2, a capacitance of ~2 µF ensured that the tissue was smooth and free of large folds. This was confirmed previously by transmission electron microscopy and scanning electron microscopy (52). As previously described, changes in capacitance primarily reflect changes in the apical surface area of the umbrella cells (8, 30).

Isc and conductance measurements. Bladder tissue was mounted as described above and equilibrated with a VCC MC6 current/voltage clamp (Physiological Instruments, San Diego, CA) set to open-circuit mode. Voltage asymmetry between the voltage-sensing electrodes and fluid resistance was corrected by positioning the voltage-sensing electrodes in the Ussing chambers before the tissue was mounted and the potential difference was adjusted to zero. The reference electrode was in the serosal hemichamber. The voltage clamp was switched from the open-circuit mode to the short-circuit mode, and the Isc values were recorded with a frequency of 10 Hz, digitized by a MacLab 8s A/D converter (AD Instruments, Mountain View, CA), and then displayed and captured using the Chart program (AD Instruments). Isc data were normalized to the exposed surface area of the tissue ring (2 cm2). To measure TER, a square-voltage pulse generated by the MacLab 8s A/D converter was applied with a frequency of 0.1 Hz across the tissue for 500 ms to a new clamp potential of 50 mV. TER was calculated from the change in voltage divided by the change in Isc, where TER = ({Delta}V/{Delta}Isc) · exposed surface area of tissue ring (2 cm2). Data are expressed as conductance, which is defined as the reciprocal of the TER value. The Isc and conductance values during the 5-min periods of increased hydrostatic pressure were averaged, and the SE was calculated from up to six separate experiments. Significant changes in Isc were assessed by t-test.

Unidirectional 36Cl, 22Na+, and 86Rb+ flux measurements. 36Cl, 22Na+, and 86Rb+ flux measurements were performed as described previously (5). One set of Ussing chamber setups was used for the mucosal-to-serosal flux (JMS) measurements, and one set of Ussing chamber setups was used to measure serosal-to-mucosal flux (JSM). Tissue was mounted in standard Krebs solution, and when the Isc became stable, 36Cl (specific activity = 3.66 mCi/mg 36Cl, concentration = 20 mCi/ml, PerkinElmer, Boston, MA); 22Na+ (specific activity = 100 mCi/mg 22Na+, concentration = 2 mCi/ml, Amersham, Piscataway, NJ); or 86Rb+ (specific activity = 1.53 mCi/mg 86Rb+, concentration = 20 mCi/ml, NEN Life Sciences, Boston, MA) was added to the serosal or mucosal side of the chamber at a final concentration of 1 µCi/ml, and sampling was performed as previously described (5). Unidirectional fluxes (Jnet) were calculated using standard equations, and all fluxes were corrected for the exposed surface area of the tissue ring (5). Isc values were averaged and converted to µeq · cm2 · h1 using the following formula: 1 µA/cm2 = 3.736 x 102 µeq · cm2 · h1 (5).


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Increasing hydrostatic pressure across the mucosal surface of the uroepithelium is accompanied by increases in ion transport. Because of its rapid effect, reversibility, and physiological relevance, we used an adaptation of the hydrostatic pressure method to mimic bladder filling and emptying (12, 37). Rabbit uroepithelium was mounted in a modified Ussing chamber that allowed hydrostatic pressure to be increased and then decreased across the mucosal surface of the tissue. Under control conditions, the uroepithelium exhibited a small basal Isc of 0.94 ± 0.03 µA/cm2 and a conductance of ~0.1 mS/cm2 (~10,000 {Omega} · cm2) (Fig. 1A). These are well within the range of previously reported values for Isc and conductance in the bladder uroepithelium (26, 3133). Increasing hydrostatic pressure across the mucosal surface of the tissue resulted in an almost instantaneous nearly fivefold increase in Isc from 0.94 ± 0.03 to 4.20 ± 0.21 µA/cm2, indicating that active ion transport accompanied the mechanical stimulus. Hydrostatic pressure was also accompanied by a significant increase in conductance to ~0.2–0.3 mS/cm2 (arrows in Fig. 1A). The increased values for Isc and conductance remained relatively constant during the 5-min period of increased hydrostatic pressure, and this was true when the pressure remained elevated for up to 60 min (data not shown). The changes in Isc and conductance were rapidly reversible and returned to near control levels when the pressure was decreased (arrowheads in Fig. 1A).



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Fig. 1. Hydrostatic pressure-induced changes in short-circuit current (Isc), conductance (G), and membrane capacitance. A: uroepithelium was mounted in modified Ussing chambers and equilibrated for 30–60 min. After the equilibration period, the tissue was subjected to increased hydrostatic pressure for 5 min (arrows) followed by release for 10 min (arrowheads). This experimental treatment was repeated 9 times. A representative tracing is shown with Isc in top trace (grey) and conductance in bottom trace (black). Values are means ± SE of 6 separate experiments. The grey bar above the graph indicates the bathing solution used during the experimental protocol (Krebs). B: membrane capacitance was measured in Krebs solution. The start of the period of a 5-min increase in hydrostatic pressure is marked by arrows, whereas the start of the 10-min period of recovery is marked by arrowheads. Shown is a representative tracing from 3 separate experiments.

 

The changes in Isc and conductance were highly reproducible and were seen when tissue was subjected to multiple cycles of raising and then lowering of the hydrostatic pressure head (Fig. 1A). The change in Isc at each pressure cycle was relatively constant, as was the basal Isc value when the hydrostatic pressure was released. In contrast, the conductance values decreased during periods of increased hydrostatic pressure and during the intervening periods after the release of hydrostatic pressure, leading to a 50% reduction in conductance by the end of the experiment. The cyclical increase in hydrostatic pressure caused no apparent damage to the tissue, as the conductance values returned to baseline levels immediately on release of hydrostatic pressure and, as noted, decreased as the experiment progressed. Furthermore, we have previously shown that epithelium exposed to hydrostatic pressure remains intact when examined by transmission electron microscopy and urea permeability remains low and unchanged (52).

Hydrostatic pressure induces increases in the apical, but not basolateral, surface area of umbrella cells (52). Because large increases in surface area would result in an overestimation of changes in Isc and conductance, we measured hydrostatic pressure-induced changes in membrane capacitance, which are proportional to changes in surface area (where 1 µF ~ 1 cm2 of surface area) (34). However, the cyclical increases in hydrostatic pressure were accompanied by only a small (~10%) increase in capacitance (Fig. 1B), indicating that the Isc (µA/cm2) and conductance values (mS/cm2) obtained for tissue exposed to hydrostatic pressure (shown in Fig. 1A and subsequently as described below) are overestimated by ~10%. Unfortunately, the system we routinely use for monitoring Isc does not allow for the simultaneous measurement of Isc, conductance, and capacitance. Similar changes in capacitance were obtained using impedance analysis that employed sinusoidal current waveforms (data not shown).

Hydrostatic pressure increases Na+ transport across the uroepithelium. The mammalian bladder expresses ENaC, which transports Na+ across the mucosal surface of the bladder (26, 32), and changes in hydrostatic pressure alter the Na+-transporting properties of the uroepithelium in a highly specific and reproducible manner (12). To determine whether the hydrostatic pressure-induced changes in Isc were the result of increased Na+ conductance by ENaC, we performed the following experiment. Ussing chamber-mounted tissue was first subjected to three cycles of increased and then decreased hydrostatic pressure, which resulted in the expected changes in Isc and conductance (Fig. 2A). Next, tissue was exposed to three cycles in the presence of the diuretic amiloride, which, when used at 1–10 µM, acts as a specific blocker of ENaC (23, 24). When 10 µM amiloride was added to the mucosal hemichamber, hydrostatic pressure-induced Isc was reduced from 4.75 ± 0.50 to 1.66 ± 0.08 µA/cm2, an inhibition of ~65% (Fig. 2A). Similar levels of inhibition were observed when amiloride was used at 1 µM (data not shown). When amiloride was isovolumetrically washed from the mucosal chamber with Krebs solution, followed by three cycles of increased and then decreased hydrostatic pressure, the inhibitory effect of amiloride was reversed (Fig. 2A).



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Fig. 2. Characterization of uroepithelial Na+ transport in the presence of amiloride and in Na+-free Krebs solution. A: isolated uroepithelium was mounted in Ussing chambers. Tissue was subjected to 3 cycles of raising (arrows) and then lowering (arrowheads) of hydrostatic pressure in control Krebs solution (grey bar above the graph), followed by 3 cycles in the presence of 10 µM amiloride added to mucosal (M) Krebs solution (horizontal double arrow). Amiloride was removed by isovolumetric replacement with Krebs solution, and the tissue was subjected to an additional 3 cycles of increased and then decreased hydrostatic pressure. B: isolated uroepithelium was subjected to 3 cycles of increased and then decreased hydrostatic pressure, and the Krebs solution was then isovolumetrically replaced in the serosal and mucosal chambers with Na+-free Krebs solution (light grey bar above the graph). After 3 more cycles of increased and then decreased hydrostatic pressure, the Na+-free Krebs solution was replaced with Krebs solution and then subjected to 3 additional cycles of increased and then decreased hydrostatic pressure. In each panel, a representative tracing is shown. Average values for Isc and conductance during the 5-min periods of increased hydrostatic pressure are shown above the graph and are means ± SE of 6 separate experiments.

 

To further confirm that increased Na+ transport accompanied increases in hydrostatic pressure, Krebs solution in both the mucosal and serosal chambers was isovolumetrically replaced with Krebs solution in which Na+ ions were substituted with the nontransportable cation TMA, generating nominally Na+-free Krebs solution. The actual concentration of Na+ under these conditions was <10 mM, as measured by an Na+-sensitive ion probe. Under these conditions, the Isc was inhibited by ~58% (Fig. 2B), which was reversed when the modified Krebs solution was replaced with the regular Krebs solution (Fig. 2B). Similar results were obtained when Na+ ions were replaced with NMDG; however, the level of Isc inhibition was ~87% (3.61 ± 0.01 µA/cm2 in Krebs solution and 0.48 ± 0.01 µA/cm2 in NMDG-Cl-containing Krebs solution).

The Isc observed in the presence of hydrostatic pressure was consistent with Na+ absorption. To confirm this observation, unidirectional 22Na+ flux studies were performed in the mucosal-to-serosal and serosal-to-mucosal directions both before and during application of hydrostatic pressure. JMS was 0.01 ± 0.01 µeq · cm2 · h1 before pressure was increased and 0.33 ± 0.02 µeq · cm2 · h1 after pressure was increased. In contrast, JSM was 0.0 ± 0.01 µeq · cm2 · h1 before and 0.15 ± 0.01 µeq · cm2 · h1 after pressure was increased. In all experiments, there was a net JMS of Na+ in the presence of hydrostatic pressure equal to 0.15 ± 0.01 µeq · cm2 · h1 (Fig. 3A). This value was not significantly different (P > 0.05) from Isc measured in the presence of hydrostatic pressure, which when converted to chemical equivalent units was equal to 0.17 ± 0.02 µeq · cm2 · h1.



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Fig. 3. Characterization of 22Na+ flux and Na+ transport in the presence of ouabain. A: net 22Na+ flux (grey bars) and Isc (filled bars) were measured before (–) and after (+) increased hydrostatic pressure. Values for Isc were converted to µeq · cm2 · h1. There was no significant difference between net 22Na+ flux and converted Isc ± pressure (P > 0.05). B: uroepithelium was exposed to 3 cycles of increased and then decreased hydrostatic pressure in Krebs solution, ouabain was added to the serosal (S) chamber (double arrow), and after 30 min the tissue was subjected to 3 additional cycles of increased and then decreased hydrostatic pressure. A representative tracing is shown. Average values for Isc and conductance during the 5-min periods of increased hydrostatic pressure are shown above the graph and are means ± SE of 6 separate experiments. JMS, mucosal-to-serosal flux; JSM, serosal-to-mucosal flux.

 

The driving force for Na+ conductance across the bladder uroepithelium is the electrochemical gradient generated by the Na+-K+-ATPase (10, 19), which favors the net absorption of Na+ across the uroepithelium. The importance of this driving force in hydrostatic pressure-induced ion transport was assessed using ouabain, a selective inhibitor of the Na+-K+-ATPase. When hydrostatic pressure was increased in the presence of 1 mM ouabain (added to the serosal hemichamber), the values for Isc were reduced from 4.48 ± 0.22 to 0.77 ± 0.37 µA/cm2 (Fig. 3B), and conductance concomitantly decreased from 0.25 ± 0.19 to 0.06 ± 0.01 mS/cm2.

K+ is secreted into the mucosal hemichamber in response to hydrostatic pressure. Previously, Ferguson and colleagues (13) presented preliminary data that a K+ secretory pathway may exist in the umbrella cell. To determine whether K+ transport was elevated by hydrostatic pressure, the uroepithelium was exposed to hydrostatic pressure in Krebs solution in which Na+ and Cl were substituted with nontransportable ions to form nominally Na+, Cl-free Krebs solution. In the modified Krebs solution, a reversal in Isc was observed when the tissue was exposed to hydrostatic pressure (Fig. 4A).



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Fig. 4. Effects of Na+, Cl-free Krebs solution, Ba2+, and ouabain on hydrostatic pressure-activated ion secretion. A: isolated uroepithelium was subjected to 3 cycles of increased (arrow) and then decreased (arrowhead) hydrostatic pressure in Krebs solution, 3 cycles in Na+, Cl-free Krebs solution, and 3 cycles in Krebs solution. B: isolated uroepithelium was subjected to 3 cycles of increased and then decreased hydrostatic pressure in Krebs solution, 3 cycles in Na+, Cl-free Krebs solution, 3 cycles after addition of 3 mM Ba2+ to the mucosal chamber, 3 cycles in Na+, Cl-free solution, 3 cycles after addition of Ba2+ to the serosal chamber, and 3 cycles in Krebs solution. C: isolated uroepithelium was subjected to 3 cycles of increased and then decreased hydrostatic pressure in Krebs solution, 3 cycles in Na+, Cl-free Krebs solution, and 3 cycles after addition of ouabain to the serosal chamber. A representative tracing is shown for each panel. Average values for Isc and conductance during the 5-min periods of increased hydrostatic pressure are shown above the graph and are means ± SE of 6 separate experiments.

 

To test whether K+ was secreted in response to hydrostatic pressure, Ba2+ was added to either the mucosal or serosal Na+,Cl-free Krebs solution during application of hydrostatic pressure (Fig. 4B). Ba2+ is a commonly used blocker of K+ channels; however, it can block some nonselective cation channels as well (11, 15, 17, 59). After three cycles of increased and then decreased hydrostatic pressure in Krebs solution and three in Na+, Cl-free Krebs solution, 3 mM Ba2+ was added to the mucosal hemichamber. In the presence of mucosal Ba2+, hydrostatic pressure-induced Isc was reduced from –9.63 ± 0.11 to 1.02 ± 0.83 µA/cm2 during increased pressure (Fig. 4B). These inhibitory effects were reversed after isovolumetric washout of Ba2+ with Na+, Cl-free Krebs solution (Fig. 4B). When Ba2+ was added to the serosal medium, hydrostatic pressure led to an increase in Isc from –9.61 ± 0.12 to –16.54 ± 0.88 µA/cm2 (Fig. 4B). These effects were reversed when the cells were returned to normal Krebs solution. The mucosal and serosal effects of another K+ channel blocker, 10 mM TEA, were also evaluated, and we observed essentially identical effects to those observed with Ba2+ (Table 1).


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Table 1. Effects of channel inhibitors on K+ conductance

 



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Fig. 5. Effects of charybdotoxin (CTX), Gd3+, and amiloride on hydrostatic pressure-activated K+ secretion. A: isolated uroepithelium was subjected to 3 cycles of increased (arrows) and then decreased (arrowheads) hydrostatic pressure in Krebs solution, 3 cycles in Na+, Cl-free Krebs solution, 3 cycles after addition of 100 nM CTX to the mucosal chamber, 3 cycles in Na+, Cl-free solution, 3 cycles after addition of 100 nM CTX to the serosal chamber, and 3 cycles in Krebs solution. B: isolated uroepithelium was subjected to 3 cycles of increased and then decreased hydrostatic pressure in Krebs solution, 3 cycles in Na+, Cl-free Krebs solution, 3 cycles after addition of 1 mM Gd3+ to the mucosal chamber, 3 cycles in Na+, Cl-free solution, 3 cycles after addition of 1 mM Gd3+ to the serosal chamber, and 3 cycles in Krebs solution. C: isolated uroepithelium was subjected to 3 cycles of increased and then decreased hydrostatic pressure in Krebs solution, 3 cycles in Na+, Cl-free Krebs solution, 3 cycles after addition of 100 µM amiloride to the mucosal chamber, 3 cycles in Na+, Cl-free solution, and 3 cycles in Krebs solution. A representative tracing is shown for each panel. Average values for Isc and conductance during the 5-min periods of increased hydrostatic pressure are shown above the graph and are means ± SE of 6 separate experiments.

 
As additional evidence that hydrostatic pressure stimulated K+ transport under short-circuit conditions, we measured, in standard Krebs buffer, the unidirectional flux of 86Rb+, which is transported by both K+ channels and nonselective cation channels (4, 6, 39, 48). Consistent with the results presented in Fig. 4, there was a net JSM of 86Rb+ in the presence of hydrostatic pressure equal to 0.37 ± 0.03 µeq · cm2 · h1 (Table 2). Essentially identical results were observed when 86Rb+ fluxes were performed in Na+, Cl-free Krebs solution (data not shown), indicating that the magnitude and direction of K+ transport were similar regardless of the bathing solution.


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Table 2. Effects of hydrostatic pressure on 86Rb+ flux

 

As described above, the ion gradients generated by the Na+-K+-ATPase provide the driving force for transport of Na+ and K+ across the uroepithelium. Isovolumetric washing with Na+, Cl-free Krebs solution is likely to leave some Na+ both outside and within the cell, and this may have been sufficient to allow K+ entry via the Na+-K+-ATPase. Alternatively, the K+ chemical gradient, generated by the high intracellular K+ pool coupled with a low extracellular K+ pool, may have been sufficient to drive the hydrostatic pressure-induced K+ exit via mucosal K+ channels. To determine whether stretch-induced K+ secretion was dependent on the activity of the Na+-K+-ATPase, uroepithelium was hydrostatically stimulated in the presence of 1 mM ouabain added to the Na+, Cl-free Krebs solution bathing the serosal hemichamber (Fig. 4C). Ouabain effectively blocked the putative hydrostatic pressure-induced K+ secretion, the inhibition being greater during each subsequent pressure cycle (Fig. 4C). This likely represents the depletion of intracellular K+ stores, which could not be replenished in the presence of ouabain. Treatment with ouabain also decreased the magnitude of the hydrostatic pressure-induced increase in conductance, consistent with a block in ion transport. These data indicated that K+ secretion was in fact dependent on basolateral K+ import by the Na+-K+-ATPase.

To characterize further the putative K+ conductance pathway, we used commercially available toxins and venoms that are selective for different classes of K+ channels. CTX, a scorpion venom that is selective for high and intermediate conductance Ca2+-activated and some voltage-gated K+ channels, had little effect on hydrostatic pressure-induced Isc when added to the mucosal hemichamber (–9.45 ± 0.58 µA/cm2 in Na+, Cl-free Krebs solution vs. –9.42 ± 0.61 µA/cm2 in Na+, Cl-free Krebs solution containing 100 nM CTX) (Fig. 5A). When CTX was subsequently added to the serosal chamber, the level of K+ transport in the presence of hydrostatic pressure was augmented (–14.92 ± 0.58 µA/cm2) compared with secretion in Na+, Cl-free Krebs solution (–9.43 ± 0.57 µA/cm2) (Fig. 5A). Other K+ channel blockers were tried as well. The addition of apamin, an inhibitor of the small-conductance Ca2+-activated K+ channels, to the mucosal hemichamber caused Isc to increase from –9.89 ± 0.27 to –12.78 ± 2.24 µA/cm2 after the application of hydrostatic pressure (Table 1). The addition of iberiotoxin, an inhibitor of the high-conductance Ca2+-activated K+ channel, and margatoxin, an inhibitor of some voltage-gated K+ channels, also had no inhibitory effect on hydrostatic pressure-induced K+ secretion (Table 1). Finally, glibenclamide, which blocks plasma membrane ATP-sensitive K+ channels (1), did not demonstrate any inhibitory effects but did marginally stimulate Isc (Table 1). These results indicated that hydrostatic pressure-induced K+ secretion from the mucosal surface of the tissue was not dependent on CTX-, iberiotoxin-, apamin-, margatoxin-, or glibenclamide-sensitive transport pathways.

K+ secretion is via a nonselective cation channel. Despite numerous trials, we were unable to find a selective K+ channel blocker that could inhibit the pressure-activated K+ secretion described above. Next, we determined whether the trivalent cation Gd3+ had any effect, as this is a general inhibitor of mechanosensitive ion channels (43). After three cycles of hydrostatic pressure/relaxation in control Krebs solution and three in Na+, Cl-free Krebs solution, 1 mM Gd3+ was added to the mucosal Na+, Cl-free Krebs solution (Fig. 5B). In the presence of Gd3+, hydrostatic pressure-activated Isc was almost completely blocked (–9.66 ± 0.05 µA/cm2 in Na+, Cl-free Krebs solution vs. 0.34 ± 0.25 µA/cm2 in the presence of Gd3+) (Fig. 5B). The addition of Gd3+ to the serosal hemichamber caused an increase in Isc from –9.65 ± 0.06 µA/cm2 in Na+, Cl-free Krebs solution to –14.68 ± 0.71 µA/cm2 in Na+, Cl-free Krebs solution containing Gd3+ (Fig. 5B). These effects were reversible when the tissue was returned to control Krebs solution (Fig. 5B). When used at high concentrations (>=100 µM), amiloride inhibits several types of mechanically activated ion channels (16, 21, 41). In the presence of 100 µM mucosal amiloride, hydrostatic pressure-activated Isc was blocked ~40% compared with stretch-activated Isc in Na+,Cl-free Krebs solution (Fig. 5C). Similar levels of inhibition were observed with 500 µM amiloride (data not shown). Addition of amiloride at 100 µM did not potentiate the effects of Gd3+ or Ba2+ (data not shown).

The sensitivity of the K+ secretion pathway to Gd3+ and high concentrations of amiloride prompted us to explore the possibility that the transport pathway we were exploring was a mechanosensitive nonselective cation channel. One characteristic of nonselective cation channels is their ability to conduct Cs+. Classic K+ channels do not conduct this ion (20). To test whether Cs+ could be transported across the mucosal membrane in a hydrostatic pressure-dependent manner, we designed an experiment in which regular Krebs solution was replaced with NMDG-gluconate buffer in the mucosal chamber and Cs+-gluconate buffer in the serosal chamber, generating a Cs+ chemical gradient from the serosal to mucosal hemichamber. To move down this chemical gradient, Cs+ needs to cross the basolateral membranes of the umbrella cells and subsequently exit the apical membranes. Because there is no known pathway for Cs+ to enter the basolateral membrane of the umbrella cells, nystatin, an ionophore, was added to the serosal hemichamber to allow transport of Cs+ across the basolateral membrane of the umbrella cells. The exit of Cs+ from the umbrella cell apical membrane via nonselective cation channels was then monitored by Isc measurements.

After a period of equilibration, control Krebs solution was isovolumetrically replaced with mucosal NMDG-gluconate buffer and serosal Cs+-gluconate buffer. Other than Cs+, there were no transportable ions in these buffers. In the absence of nystatin, hydrostatic pressure had no effect on Isc, indicating that Cs+ could not cross the basolateral membrane of the umbrella cells (Fig. 6). In addition, there was no change in conductance, further indicating that changes in conductance were dependent in part on ion transport. However, after the addition of nystatin to the serosal hemichamber, a significant change in Isc was observed after application of hydrostatic pressure (–10.59 ± 0.02 µA/cm2) that was similar in magnitude to the hydrostatic pressure-induced Isc observed in Na+, Cl-free Krebs solution (about –9.5 to –9.11 µA/cm2) (Figs. 4 and 5). Additionally, the transport of Cs+ was accompanied by a significant, reversible increase in conductance, suggesting opening and closing of ion channels. One characteristic of the K+ conductance we have described is its sensitivity to Gd3+. As predicted, the hydrostatic pressure-activated Isc was blocked when Gd3+ was added to the mucosal hemichamber (Fig. 6). These results indicated that hydrostatic pressure activated a nonselective cation channel in the apical membrane of the umbrella cells that was capable of conducting K+.



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Fig. 6. Hydrostatic pressure-induced transport of Cs+ in nystatin-permeabilized uroepithelium. Isolated uroepithelium was equilibrated in Krebs solutions, and the Krebs solution was replaced by N-methyl-D-glucamine (NMDG)-gluconate buffer in the mucosal chamber and Cs+-gluconate buffer in the serosal chamber. Hydrostatic pressure was increased (arrows) and then decreased (arrowheads) 3 times. Nystatin (2 µM) was added to the serosal chamber, and the tissue was again subjected to 3 cycles of increased and then decreased hydrostatic pressure. Finally, 1 mM Gd3+ was added to the mucosal chamber, and 3 more cycles of increased and then decreased hydrostatic pressure were performed. A representative tracing is shown. Average values for Isc and conductance during the 5-min periods of increased hydrostatic pressure are shown above the graph and are means ± SE of 6 separate experiments.

 

Hydrostatic pressure induces Cl secretion into the mucosal hemichamber. In Krebs solution, the net hydrostatic pressure-induced change in Isc was ~4 µA/cm2 (equivalent to ~0.15 µeq · cm2 · h1) (Fig. 1A), which was not significantly different from the net 22Na+ flux we measured (~0.15 µeq · cm2 · h1) (Fig. 3A). However, the 86Rb+ flux experiments in Krebs solution (Table 2) and the experiments in Na+, Cl-free Krebs solution indicated that hydrostatic pressure also induced an ~10 µA/cm2 (equal to a flux of ~0.4 µeq · cm2 · h1) secretory K+ current in the opposite direction. This prompted us to explore whether pressure stimulated transport of an additional ion species that was of a similar magnitude but opposite charge to the K+ current, thus masking the contribution of the K+ current to the net Isc. Previous studies indicated that a pathway for Cl entry exists on the basolateral membrane of bladder umbrella cells (35). To determine whether a pressure-sensitive pathway for Cl transport exists in these cells, the uroepithelium was exposed to hydrostatic pressure and unidirectional flux measurements were performed. These experiments confirmed that in standard Krebs solution and under short-circuit conditions, there was a net JSM of 36Cl in the presence of hydrostatic pressure equal to 0.41 ± 0.03 µeq · cm2 · h1 (Table 3). This value was not significantly different (P > 0.05) from the net 86Rb+ flux in the presence of hydrostatic pressure, indicating that hydrostatic pressure induced electroneutral K+ and Cl secretion under short-circuit conditions.


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Table 3. Effects of hydrostatic pressure on 36Cl- flux

 


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The bladder is not simply a vessel for storing urine but instead may play an active part in regulating the total Na+, Cl, and K+ content of the organism, a function mediated by ion transport across the apical surface of the umbrella cell (26). We observed that raising and then lowering hydrostatic pressure, which mimics bladder filling and voiding, was accompanied by increased and then decreased ion transport. Like previous reports, which employed membrane punching and small changes in hydrostatic pressure (12, 37), we observed that mechanical stimulation was accompanied by increased Na+ absorption across the apical membrane of the umbrella cells (12, 13, 26, 27, 3133, 37). This was confirmed by demonstrating a net JSM of 22Na+ of ~0.15 µeq · cm2 · h1. Furthermore, we observed a significant decrease in Isc when tissue was exposed to pressure in Na+-free Krebs solution, or when pressure was increased in the presence of ouabain, indicating that the ion gradients generated by the Na+-K+-ATPase provided the driving force for Na+ entry across the apical membrane of the umbrella cell (Fig. 7). The sensitivity of this transport pathway to low concentrations of amiloride (1–10 µM) and previous localization of ENaC to the apical surface of the umbrella cell (46) were consistent with an absorptive Na+ transport pathway mediated by ENaC (Fig. 7).



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Fig. 7. Model for hydrostatic pressure-induced ion transport in bladder umbrella cells. Increased hydrostatic pressure causes increased conductance of Na+, K+, and Cl. Na+ enters the cell via the amiloride-sensitive epithelial Na+ channel (ENaC), driven by the favorable electrochemical gradient (low-cellular Na+ concentration and cellular negative voltage), and exits the cell, in exchange for K+, via the ouabain-sensitive Na+-K+-ATPase located at the basolateral surface of the cell. K+ can exit the cell via basolaterally localized K+ leak channels [which are inhibited by Ba2+, CTX, Gd3+, and tetraethylammonium (TEA)] or under short-circuit conditions through an apically localized nonselective cation channel, which is inhibited by amiloride (>=100 µM), Ba2+, Gd3+, and TEA. The former may also conduct Ca2+ and Na+, but this has not been directly demonstrated. Apical K+ conductance is balanced by an electroneutral Cl conductance, which may represent transport of Cl through basolateral and then apical Cl channels. A basolaterally localized Cl conductance has been described (35).

 

In addition to this Na+ transport pathway, we also characterized a pressure-sensitive K+ secretion pathway, which was revealed under short-circuit conditions when tissue was exposed to hydrostatic pressure in Na+, Cl-free Krebs solution. This pathway was confirmed by measuring a net JSM of 86Rb+ in standard Krebs solution. Mechanically stimulated K+ secretory pathways have been described in several cell types, including marginal cells of the stria vascularis, gall bladder epithelial cells, epithelial cells of Reissner's membrane, apical membrane of rabbit cortical collecting tubule, and vestibular dark cells (50, 51, 54, 56, 58). In each of these cell types, K+ is secreted via a large-conductance K+ channel that is blocked by TEA and Ba2+. The K+ conductance in the umbrella cells is also sensitive to these channel blockers. However, we found that CTX and iberiotoxin (both inhibitors of large-conductance K+ channels), apamin, or margatoxin had either no effect or a small effect on K+ secretion when added to the mucosal surface of the uroepithelium. In contrast, Ba2+, TEA, Gd3+, and CTX stimulated secretion when added to the serosal surface of the tissue. This likely reflects inhibition of basolateral K+ channels, which would enhance apical release of K+ when pressure was increased. The driving force for K+ secretion was apparently the ion gradients generated by the Na+-K+-ATPase, as ouabain inhibited K+ secretion (Fig. 7).

The observation that Ba2+ and TEA can inhibit the activity of nonselective cation channels (17) prompted us to explore whether the K+ secretory pathway we observed was possibly via a mechanosensitive nonselective cation channel. Several observations indicated that this was the case. First, K+ secretion in the uroepithelium was inhibited by mucosal addition of Gd3+, a blocker of both mechanosensitive ion channels and nonselective cation channels (7, 18, 40, 43). Second, we observed that high concentrations of amiloride, which block mechanosensitive nonselective cation channels (7), partially inhibited the K+ secretory pathway. Third, the Gd3+-sensitive K+ secretory pathway also conducted Cs+, a hallmark of a nonselective cation conductance pathway (20, 57). Based on these observations, we propose that hydrostatic pressure may increase the activity of a mechanosensitive nonselective cation channel in the apical membrane of the umbrella cell (Fig. 7).

A nonselective cation channel has previously been described in bladder uroepithelium (28, 60). This so-called "leak" channel is localized to the apical membrane of the umbrella cells and is thought to be a degradation product of ENaC (29). Zweifech and Lewis (60) showed that the conductivity for K+ by the leak channel was greater than that of Na+ or Cl. Despite the similarity in ion conductivity and localization between the leak channel and the putative nonselective cation channel that we described here, there are notable differences between the two channels. First, the leak channel is unaffected by 10–100 µM amiloride (28, 36, 60), whereas the nonselective cation channel we have characterized is partially inhibited by 100 µM amiloride. Second, the amiloride-sensitive K+ conductance that we observed is relatively stable (>10 min) during increased hydrostatic pressure and can be activated during multiple cycles of increased pressure and relaxation. This contrasts with the short residency time of the leak channel (~2 min) in the apical membrane of the umbrella cell (60). Third, the pressure-activated nonselective cation channel described in this communication produced a secretory K+ current, whereas Zweifech and Lewis (60) described an absorptive leak current. In fact, in the presence of Ba2+, TEA, or Gd3+ we observe a small residual absorptive current that may represent leak channel activity.

In addition to Na+ and K+ transport, increased hydrostatic pressure led to increased Cl secretion under short-circuit conditions, which was of a similar magnitude to the K+ secretory pathway we described (Fig. 7). Mechanically sensitive Cl channels have been described in several other tissues and cell types, including Reissner's membrane, the stria vascularis, and cortical collecting duct (45, 49, 58); however, none have been described in bladder epithelium. Known pathways for Cl entry across the basolateral membrane of umbrella cells include Cl channels and exchangers (26, 35). An apical pathway for Cl conductance has not been described in bladder epithelium, and we found that the Cl secretory pathway was insensitive to mucosal addition of DIDS, serosal addition of bumetanide, or mucosal addition of niflumic acid (unpublished observations), all inhibitors of other known Cl conductance pathways (22, 38, 53). The nature of the Cl secretory pathway requires additional experimentation.

Finally, the relatively stable change in Isc during increased pressure (see Fig. 1) but the tendency for the conductance to fall during increased pressure and during the period between cycles of elevated pressure indicates that paracellular conductance and/or the magnitude of the different ion channel activities may be affected by pressure. These changes may also explain why mucosal addition of Ba2+ and Gd+ inhibited pressure-induced changes in the Isc contributed by K+ secretion but had less of an effect on changes in conductance (Figs. 4B and 5B).

In summary, our findings further increase our understanding of the mechanically sensitive ion transport processes in the mammalian urinary bladder. Our data indicate that in addition to the previously characterized Na+ absorption pathway, hydrostatic pressure also stimulates Cl and K+ secretion under short-circuit conditions. Although the bladder is generally thought to store urine and maintain the ion composition of the urine generated by the kidney, the ion transport pathways we and others have described indicate that the uroepithelium could alter the water and ion composition of the urine. Furthermore, absorption of ions from the urine (e.g., Na+) or secretion of ions into the urine (e.g., K+) may provide a means of regulating the electrolyte composition of the extracellular fluid by modifying kidney-generated ion gradients. Finally, transport of Cl, K+, and Na+ during the bladder-filling process may also play an important role in sensory transduction and may couple changes in hydrostatic pressure to release of agonists such as ATP that would modulate other cellular functions such as neural-epithelial signaling (12, 13, 47).


    DISCLOSURES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants T32-DK-61296 (to E. C. Y. Wang), RO1-DK-54425 (to G. Apodaca), 1 P50-DK-56490 (to R. Bridges), RO1-DK-47874 (to J. P. Johnson), and DK-51391 (to T. R. Kleyman).


    ACKNOWLEDGMENTS
 
We thank Steven Truschel for assistance during the preparation of this manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. Apodaca, Renal Div., Univ. of Pittsburgh, 982 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261 (E-mail: gla6{at}pitt.edu).

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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