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Hydrostatic pressure-regulated ion transport in bladder uroepithelium [复制链接]

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发表于 2009-4-21 13:46 |只看该作者 |倒序浏览 |打印
作者:Edward C. Y. Wang, Jey-Myung Lee, John P. Johnson, Thomas R. Kleyman, Robert Bridges,  Gerard Apodaca,作者单位:1 Renal-Electrolyte Division, Department ofMedicine, Laboratory of Epithelial Cell Biology, and Department of Cell Biology and Physiology, Universityof Pittsburgh, Pittsburgh, Pennsylvania 15261 7 ]6 P# I+ D4 n& W" _  [% u3 W
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5 V, i+ D) W! Y- D+ j1 i          【摘要】
2 i  W8 c  d& v# g) i, `      The effect of hydrostatic pressure on ion transport in the bladder uroepithelium was investigated. Isolated rabbit uroepithelium was mounted inmodified Ussing chambers and mechanically stimulated by applying hydrostaticpressure across the mucosa. Increased hydrostatic pressure led to increasedmucosal-to-serosal Na   absorption across the uroepithelium via theamiloride-sensitive epithelial Na   channel. In addition to thispreviously characterized pathway for Na   absorption, hydrostaticpressure also induced the secretion of Cl - and K   into the mucosal bathing solution under short-circuit conditions, which wasconfirmed by a net serosal-to-mucosal flux of 36 Cl - and 86 Rb  . K   secretion was likely via a stretch-activated nonselective cationchannel sensitive to 100 µM amiloride, 10 mM tetraethylammonium, 3 mMBa 2  , and 1 mM Gd 3  . Hydrostaticpressure-induced ion transport in the uroepithelium may play important rolesin electrolyte homeostasis, volume regulation, and mechanosensory transduction. * c: {3 Y% Z% D; n
          【关键词】 mechanical force epithelial sodium channel nonselective cation channel; K' }3 v& R7 X; }8 \8 R6 v0 W$ i
                  CELLS ARE EXPOSED TO AN ARRAY of physical forces, including compression, shear stress, and hydrostatic pressure, which occurs when bodilyfluids push against the epithelium that lines the tubes and sacs that formmany 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 ofcells exposed to mechanical stimuli is activation of stretch-activated ionchannels. The first such channel described in detail is the stretch-activatednonselective cation channel of chick embryonic skeletal muscle( 42 ), which is cationselective but discriminates poorly between Na   and K  .Since these initial studies, stretch-activated K  ,Cl -, and nonselective cation channels have been identified inmany cell types, including erythrocytes, oocytes, fibroblasts, aortic endothelium, heart cells, kidney cells, muscle cells, and epithelial cells( 18 ).! @, I, B/ L' h6 a! A5 c( z
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In the urinary tract, where cells experience shear stress and hydrostaticpressure, mechanosensitive ion channels may play an important role in volumeregulation and ion homeostasis. For example, a stretch-activatedcation-selective channel is found in the apical membrane of proximal tubulecells. This channel is permeable to Ca 2  , K  ,and Na   and is not gated by membrane potential or cytosolicCa 2   ( 14 ), but its activity isdependent on changes in extracellular osmolarity. Mechanical stimulation ofthis channel (e.g., by cell swelling) could lead to cell volume regulation. Inrenal cortical collecting ducts, changes in hydrostatic pressure may increasethe open probability, or number, of stretch-sensitive epithelialNa   channels (ENaC), enhancing the rate of Na   reabsorption ( 44 ). Moreover,the rate of K   secretion by large-conductance maxi-K channels incortical collecting ducts is also affected by variations in intraluminal flowrates ( 56 ).
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The lower urinary tract is also subject to mechanical stimuli; hydrostaticpressure 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 theuroepithelium, and the combination of an impermeable apical membrane andhigh-resistance tight junctions forms an effective barrier to solute and ion permeability. Umbrella cells respond to mechanical stimuli in a number ofways, including an increase in surface area as well as ion transport( 12, 36, 37, 52 ). Membrane"punching," performed by rapidly increasing and then decreasinghydrostatic pressure across the mucosal surface of the bladder uroepithelium, leads to increased ion conductance after 10-30 min( 36, 37 ). The observed rise in ionconductance is contributed by the amiloride-sensitive ENaC as well as by anamiloride-insensitive nonselective cation channel( 29, 35 ). Alternatively, ENaC activity can be acutely stimulated by removing fluid from the serosal side ofbladder tissue mounted in an Ussing chamber, thereby increasing hydrostaticpressure across the mucosal surface of the bladder. In addition toNa   absorption, a K   secretory pathway, sensitive to theK   channel blocker tetraethylammonium (TEA), has been identified inresting bladder tissue ( 13 ), but the nature of this secretory pathway and whether it is stimulated byhydrostatic pressure have not been defined.
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& m) f: v* v* Z) M3 G$ o/ v: F: wThe goal of this study was to examine the possibility that, like in theupper urinary tract, hydrostatic pressure stimulates multiple ion transportpathways in bladder uroepithelium. Rabbit uroepithelium was mounted inmodified Ussing chambers and was then subjected to cycles of increasedhydrostatic pressure while changes in short-circuit current( I sc ) and ion conductance were monitored. We found that inaddition to hydrostatic pressure-induced Na   absorption across theuroepithelium, there was also under short-circuit conditions hydrostaticpressure-induced electroneutral Cl - and K   secretion into the mucosal chamber. Na   absorption was via thepreviously described amiloride-sensitive ENaC, whereas K   secretionwas probably via a nonselective cation channel sensitive to 100 µMamiloride, TEA, Ba 2  , and Gd 3  .These results provide additional evidence that besides retaining urine, themammalian bladder epithelium might play an important role in electrolytehomeostasis.* Y' |" P4 ?6 m2 ?- @" q; m
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EXPERIMENTAL PROCEDURES% d! Y& @: C- X/ R  o# \0 q/ q

; O1 G: }6 H+ \/ CMaterials. Unless otherwise specified, all chemicals were of reagent quality or better and were obtained from Sigma (St. Louis, MO). Stocksof the following inhibitors were prepared as follows: amiloride was dissolvedin water at 10 mM, or when used at 500 µM it was dissolved directly intobuffer solution; apamin was dissolved in water at 100 µM; BaCl 2 was dissolved in Na  , Cl - -free buffer at 1.5 M;GdCl 3 was dissolved in Na  , Cl - -freebuffer at 100 mM; glibenclamide was dissolved in DMSO at 50 mM; ouabain wasdissolved in water at 13 mM; and TEA was dissolved in Na  ,Cl - -free buffer at 1 M. The following K   channelblockers were obtained from Alomone Laboratories (Jerusalem, Israel), andstocks were prepared as follows: charybdotoxin (CTX) was dissolved in water at100 µM; iberiotoxin was dissolved in water at 1 µM; and margatoxin wasdissolved in water at 10 µM. All inhibitors were freshly prepared beforeuse.
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! o* s8 l; ?/ l3 D1 S- NSolutions. Control Krebs solution was prepared by mixing the following (in mM): 110 NaCl, 5.8 KCl, 25 NaHCO 3, 1.2KH 2 PO 4, 2.0 CaCl 2, 1.2 MgSO 4, and11.1 glucose. Na   -free Krebs had the following composition (in mM):120 tetramethylammonium (TMA) Cl or N -methyl- D -glucamine(NMDG)-Cl, 3.33 KH 2 PO 4, 0.83 K 2 HPO 4, 1.2 CaCl 2, 1.2 MgCl 2, 28choline-HCO 3, 0.01 atropine, and 2.7 glucose. Na   - andCl - -free Krebs had the following composition (in mM): 120NMDG-gluconate buffer (prepared as a stock by adjusting 240 mM NMDG to pH 7.4with gluconic acid), 3.33 KH 2 PO 4, 0.83K 2 HPO 4, 4 Ca   -gluconate, 4Mg 2   -gluconate, 28 choline-HCO 3, 0.01atropine, and 2.7 glucose. For Cs   transport experiments, theNMDG-gluconate solution had the following composition (in mM): 135 NMDG, 4hemi-Ca 2   -gluconate, and 10 HEPES. The pH was adjustedto 7.4 with D -gluconic acid. The Cs   -gluconate solutionhad the following composition (in mM): 135 CsOH, 4hemi-Ca 2   -gluconate, and 10 HEPES. The pH of the Cs   -gluconate buffer was adjusted to 7.4 with D -gluconicacid. All the solutions had an osmolarity of 280-300 mosM. Solutions that contained bicarbonate were aerated with a 95% O 2 -5%CO 2 mixture and maintained at a pH of 7.4 at 37°C.3 \2 b. @% ?4 r; D5 W% v* [
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Preparation and mounting of uroepithelium and system for increasing hydrostatic pressure across the mucosal surface of the tissue. Animalexperiments were performed in accordance with the Animal Use and CareCommittee of the University of Pittsburgh (Pittsburgh, PA). Urinary bladderswere obtained from female New Zealand White rabbits (3-4 kg; Myrtle'sRabbitry, Thompson Station, TN). Rabbits were euthanized with 300 mg ofpentobarbital sodium, the bladder was excised, and after careful dissection ofthe muscle layers the mucosa was placed on tissue rings that nominally exposed2 cm 2 of tissue. The tissue rings were then mounted between twohalves 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, afterbeing filled, could be closed off. Once the tissue was mounted, eachhemichamber (mucosal and serosal) was filled with 12.5 ml of Krebs solution,the serosal hemichamber was bubbled with 95% air-5% CO 2 gas (whensolutions contained bicarbonate), and the tissue was equilibrated for30-60 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, andtissue capacitance and transepithelial resistance (TER) were monitoredthroughout the equilibration period using the MacLab system described below.Only preparations that exhibited a starting capacitance of 1.8-2.1 µFand a TER 5,000 · cm 2 were used.
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To increase hydrostatic pressure across the mucosal surface of the tissue,additional Krebs solution was added to the mucosal hemichamber to a volume of14 ml (hemichamber capacity), and an additional 0.5 ml of Krebs solution wasinjected with a syringe to increase the back pressure in the mucosalhemichamber to 8 cmH 2 O, as measured by a force transducer (ADInstruments, Mountain View, CA). This pressure is similar to that observed during the extended filling stage of the rabbit bladder( 25 ). The chamber was thenclosed off for the times specified. To relieve the pressure, an identicalamount of Krebs solution was removed from the mucosal hemichamber. Whennecessary, the mucosal and/or serosal Krebs solutions were isovolumetrically replaced by injecting 70 ml of modified Krebs solution toward the base of thehemichamber via a syringe needle while the solution was simultaneouslywithdrawn by vacuum suction connected to another syringe needle directedtoward the top of the hemichamber. With the use of this technique, the volumeof hemichamber solution was kept constant (12.5 ml) during the isovolumetricwash.
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Measure of tissue capacitance. Capacitance (where 1 µF 1cm 2 of actual membrane area) was measured by monitoring the voltage response to a square-current pulse as described previously ( 52 ). The time constant,, of the resulting voltage response was determined by calculating thelength of time required to reach 63% of the steady-state voltage by using adata transformation routine that included curve fitting the voltage responseto a single exponential. The R 0.99 under all conditions. The capacitance was determined using the formula C = / 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 cm 2, acapacitance of 2 µF ensured that the tissue was smooth and free oflarge folds. This was confirmed previously by transmission electron microscopyand scanning electron microscopy ( 52 ). As previously described,changes in capacitance primarily reflect changes in the apical surface area ofthe umbrella cells ( 8, 30 ).
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I sc and conductance measurements. Bladder tissue wasmounted 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 resistancewas corrected by positioning the voltage-sensing electrodes in the Ussingchambers before the tissue was mounted and the potential difference wasadjusted to zero. The reference electrode was in the serosal hemichamber. Thevoltage clamp was switched from the open-circuit mode to the short-circuit mode, and the I sc 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). I sc data were normalized to the exposed surface area ofthe tissue ring (2 cm 2 ). To measure TER, a square-voltage pulsegenerated by the MacLab 8s A/D converter was applied with a frequency of 0.1Hz 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 I sc, where TER =( V / I sc ) · exposed surfacearea of tissue ring (2 cm 2 ). Data are expressed as conductance, which is defined as the reciprocal of the TER value. The I sc and conductance values during the 5-min periods ofincreased hydrostatic pressure were averaged, and the SE was calculated fromup to six separate experiments. Significant changes in I sc were assessed by t -test.1 B4 v8 @+ P' j, J& B  f- }9 g! u

/ @0 d2 J7 p1 JUnidirectional 36 Cl -, 22 Na  , and 86 Rb   flux measurements. 36 Cl -, 22 Na  , and 86 Rb   flux measurementswere performed as described previously( 5 ). One set of Ussing chambersetups was used for the mucosal-to-serosal flux ( J MS )measurements, and one set of Ussing chamber setups was used to measureserosal-to-mucosal flux ( J SM ). Tissue was mounted instandard Krebs solution, and when the I sc became stable, 36 Cl - (specific activity = 3.66 mCi/mg 36 Cl -, concentration = 20 mCi/ml, PerkinElmer, Boston, MA); 22 Na   (specific activity = 100 mCi/mg 22 Na  , concentration = 2 mCi/ml, Amersham, Piscataway,NJ); or 86 Rb   (specific activity = 1.53 mCi/mg 86 Rb  , concentration = 20 mCi/ml, NEN Life Sciences, Boston, MA) was added to the serosal or mucosal side of the chamber at a finalconcentration of 1 µCi/ml, and sampling was performed as previouslydescribed ( 5 ). Unidirectional fluxes ( J net ) were calculated using standard equations,and all fluxes were corrected for the exposed surface area of the tissue ring( 5 ). I sc values were averaged and converted to µeq ·cm - 2 · h - 1 using the following formula: 1 µA/cm 2 = 3.736 x 10 - 2 µeq ·cm - 2 · h - 1 ( 5 ).
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  |! v/ {+ M' x, `7 ?3 \) ^Increasing hydrostatic pressure across the mucosal surface of theuroepithelium is accompanied by increases in ion transport. Because ofits rapid effect, reversibility, and physiological relevance, we used anadaptation of the hydrostatic pressure method to mimic bladder filling andemptying ( 12, 37 ). Rabbit uroepithelium wasmounted in a modified Ussing chamber that allowed hydrostatic pressure to beincreased and then decreased across the mucosal surface of the tissue. Undercontrol conditions, the uroepithelium exhibited a small basal I sc of 0.94 ± 0.03 µA/cm 2 and aconductance of 0.1 mS/cm 2 ( 10,000 ·cm 2 ) ( Fig.1 A ). These are well within the range of previouslyreported values for I sc and conductance in the bladder uroepithelium ( 26, 31 - 33 ).Increasing hydrostatic pressure across the mucosal surface of the tissueresulted in an almost instantaneous nearly fivefold increase in I sc from 0.94 ± 0.03 to 4.20 ± 0.21µA/cm 2, indicating that active ion transport accompanied themechanical stimulus. Hydrostatic pressure was also accompanied by asignificant increase in conductance to 0.2-0.3 mS/cm 2 (arrows in Fig. 1 A ).The increased values for I sc and conductance remainedrelatively constant during the 5-min period of increased hydrostatic pressure, and this was true when the pressure remained elevated for up to 60 min (datanot shown). The changes in I sc and conductance wererapidly reversible and returned to near control levels when the pressure wasdecreased (arrowheads in Fig.1 A ).
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6 u7 \2 ~4 X/ i9 YFig. 1. Hydrostatic pressure-induced changes in short-circuit current( I sc ), conductance ( G ), and membrane capacitance. A : uroepithelium was mounted in modified Ussing chambers andequilibrated for 30-60 min. After the equilibration period, the tissuewas subjected to increased hydrostatic pressure for 5 min (arrows) followed byrelease for 10 min (arrowheads). This experimental treatment was repeated 9times. A representative tracing is shown with I sc 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 theexperimental protocol (Krebs). B : membrane capacitance was measuredin Krebs solution. The start of the period of a 5-min increase in hydrostaticpressure is marked by arrows, whereas the start of the 10-min period ofrecovery is marked by arrowheads. Shown is a representative tracing from 3separate experiments.
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The changes in I sc and conductance were highlyreproducible and were seen when tissue was subjected to multiple cycles of raising and then lowering of the hydrostatic pressure head ( Fig. 1 A ). The changein I sc at each pressure cycle was relatively constant, aswas the basal I sc value when the hydrostatic pressure wasreleased. In contrast, the conductance values decreased during periods ofincreased hydrostatic pressure and during the intervening periods after therelease of hydrostatic pressure, leading to a 50% reduction in conductance bythe end of the experiment. The cyclical increase in hydrostatic pressurecaused no apparent damage to the tissue, as the conductance values returned tobaseline levels immediately on release of hydrostatic pressure and, as noted,decreased as the experiment progressed. Furthermore, we have previously shownthat epithelium exposed to hydrostatic pressure remains intact when examinedby transmission electron microscopy and urea permeability remains low andunchanged ( 52 ).
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2 ]0 J. ~( p: a' s6 nHydrostatic pressure induces increases in the apical, but not basolateral,surface area of umbrella cells( 52 ). Because large increasesin surface area would result in an overestimation of changes in I sc and conductance, we measured hydrostatic pressure-induced changes in membrane capacitance, which are proportional tochanges in surface area (where 1 µF 1 cm 2 of surface area)( 34 ). However, the cyclicalincreases in hydrostatic pressure were accompanied by only a small ( 10%) increase in capacitance ( Fig.1 B ), indicating that the I sc (µA/cm 2 ) and conductance values (mS/cm 2 ) obtained for tissue exposed to hydrostatic pressure (shown in Fig. 1 A and subsequently as described below) are overestimated by 10%. Unfortunately, the system we routinely use for monitoring I sc does notallow for the simultaneous measurement of I sc,conductance, and capacitance. Similar changes in capacitance were obtainedusing impedance analysis that employed sinusoidal current waveforms (data not shown).
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Hydrostatic pressure increases Na   transport acrossthe uroepithelium. The mammalian bladder expresses ENaC, which transportsNa   across the mucosal surface of the bladder( 26, 32 ), and changes inhydrostatic pressure alter the Na   -transporting properties of theuroepithelium in a highly specific and reproducible manner( 12 ). To determine whether thehydrostatic pressure-induced changes in I sc were theresult of increased Na   conductance by ENaC, we performed thefollowing experiment. Ussing chamber-mounted tissue was first subjected tothree cycles of increased and then decreased hydrostatic pressure, whichresulted in the expected changes in I sc and conductance( Fig. 2 A ). 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 I sc was reduced from 4.75 ± 0.50 to 1.66 ±0.08 µA/cm 2, an inhibition of 65%( Fig. 2 A ). Similarlevels of inhibition were observed when amiloride was used at 1 µM (datanot shown). When amiloride was isovolumetrically washed from the mucosalchamber with Krebs solution, followed by three cycles of increased and thendecreased hydrostatic pressure, the inhibitory effect of amiloride wasreversed ( Fig.2 A ).
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Fig. 2. Characterization of uroepithelial Na   transport in the presenceof amiloride and in Na   -free Krebs solution. A : isolateduroepithelium was mounted in Ussing chambers. Tissue was subjected to 3 cyclesof raising (arrows) and then lowering (arrowheads) of hydrostatic pressure incontrol Krebs solution (grey bar above the graph), followed by 3cycles in the presence of 10 µM amiloride added to mucosal (M) Krebssolution (horizontal double arrow). Amiloride was removed by isovolumetricreplacement with Krebs solution, and the tissue was subjected to an additional3 cycles of increased and then decreased hydrostatic pressure. B :isolated uroepithelium was subjected to 3 cycles of increased and thendecreased hydrostatic pressure, and the Krebs solution was thenisovolumetrically replaced in the serosal and mucosal chambers withNa   -free Krebs solution (light grey bar above the graph).After 3 more cycles of increased and then decreased hydrostatic pressure, theNa   -free Krebs solution was replaced with Krebs solution and thensubjected to 3 additional cycles of increased and then decreased hydrostaticpressure. In each panel, a representative tracing is shown. Average values for I sc and conductance during the 5-min periods of increasedhydrostatic pressure are shown above the graph and are means ± SE of 6separate experiments.
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+ P+ W' z1 j6 U2 {8 `. J+ J4 STo further confirm that increased Na   transport accompanied increases in hydrostatic pressure, Krebs solution in both the mucosal andserosal chambers was isovolumetrically replaced with Krebs solution in whichNa   ions were substituted with the nontransportable cation TMA,generating nominally Na   -free Krebs solution. The actualconcentration of Na   under these conditions was   -sensitive ion probe. Under these conditions, the I sc was inhibited by 58% ( Fig. 2 B ), which wasreversed when the modified Krebs solution was replaced with the regular Krebssolution ( Fig. 2 B ).Similar results were obtained when Na   ions were replaced withNMDG; however, the level of I sc inhibition was 87%(3.61 ± 0.01 µA/cm 2 in Krebs solution and 0.48 ±0.01 µA/cm 2 in NMDG-Cl-containing Krebs solution).
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The I sc observed in the presence of hydrostaticpressure was consistent with Na   absorption. To confirm thisobservation, unidirectional 22 Na   flux studies wereperformed in the mucosal-to-serosal and serosal-to-mucosal directions bothbefore and during application of hydrostatic pressure. J MS was 0.01 ± 0.01 µeq · cm - 2 · h - 1 before pressure was increased and0.33 ± 0.02 µeq · cm - 2 ·h - 1 after pressure was increased. In contrast, J SM was 0.0 ± 0.01 µeq ·cm - 2 · h - 1 before and 0.15 ± 0.01 µeq · cm - 2 · h - 1 after pressure was increased. In allexperiments, there was a net J MS of Na   in thepresence of hydrostatic pressure equal to 0.15 ± 0.01 µeq ·cm - 2 · h - 1 ( Fig. 3 A ). This value was not significantly different ( P 0.05) from I sc measured in the presence of hydrostatic pressure,which when converted to chemical equivalent units was equal to 0.17 ±0.02 µeq · cm - 2 ·h - 1.
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Fig. 3. Characterization of 22 Na   flux and Na   transport in the presence of ouabain. A : net 22 Na   flux (grey bars) and I sc (filled bars) were measured before (-) and after ( ) increasedhydrostatic pressure. Values for I sc were converted toµeq · cm - 2 ·h - 1. There was no significant difference betweennet 22 Na   flux and converted I sc ± pressure ( P 0.05). B : uroepithelium wasexposed to 3 cycles of increased and then decreased hydrostatic pressure inKrebs solution, ouabain was added to the serosal (S) chamber (double arrow),and after 30 min the tissue was subjected to 3 additional cycles of increasedand then decreased hydrostatic pressure. A representative tracing is shown.Average values for I sc and conductance during the 5-minperiods of increased hydrostatic pressure are shown above the graphand are means ± SE of 6 separate experiments. J MS,mucosal-to-serosal flux; J SM, serosal-to-mucosal flux.
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2 j, G, N- j$ O5 X7 QThe driving force for Na   conductance across the bladderuroepithelium is the electrochemical gradient generated by theNa   -K   -ATPase ( 10, 19 ), which favors the netabsorption of Na   across the uroepithelium. The importance of thisdriving force in hydrostatic pressure-induced ion transport was assessed usingouabain, 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 I sc werereduced from 4.48 ± 0.22 to 0.77 ± 0.37 µA/cm 2 ( Fig. 3 B ), andconductance concomitantly decreased from 0.25 ± 0.19 to 0.06 ±0.01 mS/cm 2.
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K   is secreted into the mucosal hemichamber in response to hydrostatic pressure. Previously, Ferguson and colleagues( 13 ) presented preliminarydata that a K   secretory pathway may exist in the umbrella cell. Todetermine whether K   transport was elevated by hydrostaticpressure, the uroepithelium was exposed to hydrostatic pressure in Krebssolution in which Na   and Cl - were substitutedwith nontransportable ions to form nominally Na  ,Cl - -free Krebs solution. In the modified Krebs solution, areversal in I sc was observed when the tissue was exposedto hydrostatic pressure ( Fig.4 A ).7 W+ d  J- a8 m# }, Z9 Z+ U" B, F* K

' D. B" O( C: L) }; `% |3 wFig. 4. Effects of Na  , Cl - -free Krebs solution,Ba 2  , and ouabain on hydrostatic pressure-activated ionsecretion. A : isolated uroepithelium was subjected to 3 cycles ofincreased (arrow) and then decreased (arrowhead) hydrostatic pressure in Krebssolution, 3 cycles in Na  , Cl - -free Krebssolution, and 3 cycles in Krebs solution. B : isolated uroepitheliumwas subjected to 3 cycles of increased and then decreased hydrostatic pressurein Krebs solution, 3 cycles in Na  , Cl - -free Krebssolution, 3 cycles after addition of 3 mM Ba 2   to themucosal chamber, 3 cycles in Na  , Cl - -freesolution, 3 cycles after addition of Ba 2   to the serosalchamber, and 3 cycles in Krebs solution. C : isolated uroepitheliumwas subjected to 3 cycles of increased and then decreased hydrostatic pressurein Krebs solution, 3 cycles in Na  , Cl - -free Krebssolution, and 3 cycles after addition of ouabain to the serosal chamber. Arepresentative tracing is shown for each panel. Average values for I sc and conductance during the 5-min periods of increasedhydrostatic pressure are shown above the graph and are means ± SE of 6separate experiments.
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To test whether K   was secreted in response to hydrostatic pressure, Ba 2   was added to either the mucosal orserosal Na  ,Cl - -free Krebs solution duringapplication of hydrostatic pressure ( Fig. 4 B ). Ba 2   is a commonly usedblocker of K   channels; however, it can block some nonselectivecation channels as well ( 11, 15, 17, 59 ). After three cycles ofincreased and then decreased hydrostatic pressure in Krebs solution and threein Na  , Cl - -free Krebs solution, 3 mMBa 2   was added to the mucosal hemichamber. In thepresence of mucosal Ba 2  , hydrostatic pressure-induced I sc was reduced from -9.63 ± 0.11 to 1.02± 0.83 µA/cm 2 during increased pressure( Fig. 4 B ). These inhibitory effects were reversed after isovolumetric washout ofBa 2   with Na  , Cl - -freeKrebs solution ( Fig.4 B ). When Ba 2   was added to theserosal medium, hydrostatic pressure led to an increase in I sc from -9.61 ± 0.12 to -16.54± 0.88 µA/cm 2 ( Fig.4 B ). These effects were reversed when the cells werereturned to normal Krebs solution. The mucosal and serosal effects of anotherK   channel blocker, 10 mM TEA, were also evaluated, and we observedessentially identical effects to those observed withBa 2   ( Table1 ).1 W- R  Z/ c& z; l* T, ^' A+ R) ^

3 c8 }8 t# s( n1 v1 X3 {Table 1. Effects of channel inhibitors on K   conductance
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" V) D: m! K; V$ q0 ]& f5 N9 FFig. 5. Effects of charybdotoxin (CTX), Gd 3  , and amilorideon hydrostatic pressure-activated K   secretion. A :isolated uroepithelium was subjected to 3 cycles of increased (arrows) andthen decreased (arrowheads) hydrostatic pressure in Krebs solution, 3 cyclesin Na  , Cl - -free Krebs solution, 3 cycles afteraddition of 100 nM CTX to the mucosal chamber, 3 cycles in Na  ,Cl - -free solution, 3 cycles after addition of 100 nM CTX tothe serosal chamber, and 3 cycles in Krebs solution. B : isolateduroepithelium was subjected to 3 cycles of increased and then decreasedhydrostatic pressure in Krebs solution, 3 cycles in Na  ,Cl - -free Krebs solution, 3 cycles after addition of 1 mMGd 3   to the mucosal chamber, 3 cycles in Na  ,Cl - -free solution, 3 cycles after addition of 1 mMGd 3   to the serosal chamber, and 3 cycles in Krebssolution. C : isolated uroepithelium was subjected to 3 cycles ofincreased and then decreased hydrostatic pressure in Krebs solution, 3 cyclesin Na  , Cl - -free Krebs solution, 3 cycles afteraddition of 100 µM amiloride to the mucosal chamber, 3 cycles inNa  , Cl - -free solution, and 3 cycles in Krebssolution. A representative tracing is shown for each panel. Average values for I sc and conductance during the 5-min periods of increasedhydrostatic pressure are shown above the graph and are means ±SE of 6 separate experiments.$ v# w4 ^: ?. ~1 f+ w2 x. y. m
) X: t. j( W! Z& A" h
As additional evidence that hydrostatic pressure stimulated K   transport under short-circuit conditions, we measured, in standard Krebsbuffer, the unidirectional flux of 86 Rb  , which istransported by both K   channels and nonselective cation channels( 4, 6, 39, 48 ). Consistent with theresults presented in Fig. 4,there was a net J SM of 86 Rb   in thepresence of hydrostatic pressure equal to 0.37 ± 0.03 µeq · cm - 2 · h - 1 ( Table 2 ). Essentiallyidentical results were observed when 86 Rb   fluxes wereperformed in Na  , Cl - -free Krebs solution (datanot shown), indicating that the magnitude and direction of K   transport were similar regardless of the bathing solution.; V3 H+ {. i; c# m6 Q- g

3 w* A4 B) }! O7 xTable 2. Effects of hydrostatic pressure on 86 Rb   flux
; G! {. @4 B+ M0 s1 V& K
8 j0 U5 F/ q1 u* F% T6 c3 CAs described above, the ion gradients generated by theNa   -K   -ATPase provide the driving force for transport ofNa   and K   across the uroepithelium. Isovolumetricwashing with Na  , Cl - -free Krebs solution islikely to leave some Na   both outside and within the cell, and thismay have been sufficient to allow K   entry via theNa   -K   -ATPase. Alternatively, the K   chemical gradient, generated by the high intracellular K   pool coupled witha low extracellular K   pool, may have been sufficient to drive thehydrostatic pressure-induced K   exit via mucosal K   channels. To determine whether stretch-induced K   secretion wasdependent 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 bathingthe serosal hemichamber ( Fig.4 C ). Ouabain effectively blocked the putative hydrostaticpressure-induced K   secretion, the inhibition being greater duringeach subsequent pressure cycle ( Fig.4 C ). This likely represents the depletion ofintracellular K   stores, which could not be replenished in thepresence of ouabain. Treatment with ouabain also decreased the magnitude ofthe hydrostatic pressure-induced increase in conductance, consistent with ablock in ion transport. These data indicated that K   secretion wasin fact dependent on basolateral K   import by theNa   -K   -ATPase.5 I: c$ B$ q! z+ H8 g* k
" ~) a) S$ ]& d- ^, G- R' B
To characterize further the putative K   conductance pathway, weused commercially available toxins and venoms that are selective for differentclasses of K   channels. CTX, a scorpion venom that is selective forhigh and intermediate conductance Ca 2   -activated andsome voltage-gated K   channels, had little effect on hydrostatic pressure-induced I sc when added to the mucosal hemichamber (-9.45 ± 0.58 µA/cm 2 in Na  ,Cl - -free Krebs solution vs. -9.42 ± 0.61µA/cm 2 in Na  , Cl - -free Krebssolution containing 100 nM CTX) ( Fig. 5 A ). When CTX was subsequently added to the serosalchamber, the level of K   transport in the presence of hydrostaticpressure was augmented (-14.92 ± 0.58 µA/cm 2 )compared with secretion in Na  , Cl - -free Krebssolution (-9.43 ± 0.57 µA/cm 2 )( Fig. 5 A ). OtherK   channel blockers were tried as well. The addition of apamin, aninhibitor of the small-conductance Ca 2   -activatedK   channels, to the mucosal hemichamber caused I sc to increase from -9.89 ± 0.27 to-12.78 ± 2.24 µA/cm 2 after the application ofhydrostatic pressure ( Table 1 ).The addition of iberiotoxin, an inhibitor of the high-conductanceCa 2   -activated K   channel, and margatoxin, aninhibitor of some voltage-gated K   channels, also had no inhibitoryeffect on hydrostatic pressure-induced K   secretion( Table 1 ). Finally,glibenclamide, which blocks plasma membrane ATP-sensitive K   channels ( 1 ), did notdemonstrate any inhibitory effects but did marginally stimulate I sc ( Table1 ). These results indicated that hydrostatic pressure-induced K   secretion from the mucosal surface of the tissue was not dependent on CTX-, iberiotoxin-, apamin-, margatoxin-, orglibenclamide-sensitive transport pathways.
- e4 ~2 [" S( q. P3 T& O3 C' }+ C  K/ N; M4 ~
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 Gd 3   had any effect, as this is a general inhibitor ofmechanosensitive ion channels( 43 ). After three cycles ofhydrostatic pressure/relaxation in control Krebs solution and three inNa  , Cl - -free Krebs solution, 1 mMGd 3   was added to the mucosal Na  ,Cl - -free Krebs solution( Fig. 5 B ). In thepresence of Gd 3  , hydrostatic pressure-activated I sc was almost completely blocked (-9.66 ±0.05 µA/cm 2 in Na  , Cl - -free Krebssolution vs. 0.34 ± 0.25 µA/cm 2 in the presence ofGd 3   ) ( Fig.5 B ). The addition of Gd 3   to theserosal hemichamber caused an increase in I sc from-9.65 ± 0.06 µA/cm 2 in Na  ,Cl - -free Krebs solution to -14.68 ± 0.71µA/cm 2 in Na  , Cl - -free Krebssolution containing Gd 3   ( Fig. 5 B ). Theseeffects were reversible when the tissue was returned to control Krebs solution( Fig. 5 B ). When used at high concentrations ( 100 µM), amiloride inhibits several types ofmechanically activated ion channels ( 16, 21, 41 ). In the presence of 100µM mucosal amiloride, hydrostatic pressure-activated I sc was blocked 40% compared with stretch-activated I sc in Na  ,Cl - -free Krebssolution ( Fig. 5 C ).Similar levels of inhibition were observed with 500 µM amiloride (data notshown). Addition of amiloride at 100 µM did not potentiate the effects ofGd 3   or Ba 2   (data not shown).6 K  [; u+ u: _( C1 Q

$ `' f6 c# ?) R# G: pThe sensitivity of the K   secretion pathway toGd 3   and high concentrations of amiloride prompted us toexplore the possibility that the transport pathway we were exploring was amechanosensitive nonselective cation channel. One characteristic ofnonselective cation channels is their ability to conduct Cs  .Classic K   channels do not conduct this ion( 20 ). To test whetherCs   could be transported across the mucosal membrane in ahydrostatic pressure-dependent manner, we designed an experiment in which regular Krebs solution was replaced with NMDG-gluconate buffer in the mucosalchamber and Cs   -gluconate buffer in the serosal chamber, generatinga Cs   chemical gradient from the serosal to mucosal hemichamber. Tomove down this chemical gradient, Cs   needs to cross thebasolateral membranes of the umbrella cells and subsequently exit the apicalmembranes. Because there is no known pathway for Cs   to enter thebasolateral membrane of the umbrella cells, nystatin, an ionophore, was addedto the serosal hemichamber to allow transport of Cs   across the basolateral membrane of the umbrella cells. The exit of Cs   fromthe umbrella cell apical membrane via nonselective cation channels was thenmonitored by I sc measurements.
, T% l2 K$ c0 [, l% D; e
2 `. e; X% L& F3 z2 k8 NAfter a period of equilibration, control Krebs solution was isovolumetrically replaced with mucosal NMDG-gluconate buffer and serosalCs   -gluconate buffer. Other than Cs  , there were notransportable ions in these buffers. In the absence of nystatin, hydrostaticpressure had no effect on I sc, indicating that Cs   could not cross the basolateral membrane of the umbrella cells( Fig. 6 ). In addition, therewas no change in conductance, further indicating that changes in conductancewere dependent in part on ion transport. However, after the addition ofnystatin to the serosal hemichamber, a significant change in I sc was observed after application of hydrostatic pressure(-10.59 ± 0.02 µA/cm 2 ) that was similar inmagnitude to the hydrostatic pressure-induced I sc observedin Na  , Cl - -free Krebs solution (about -9.5to -9.11 µA/cm 2 ) (Figs. 4 and 5 ). Additionally, the transportof Cs   was accompanied by a significant, reversible increase inconductance, suggesting opening and closing of ion channels. Onecharacteristic of the K   conductance we have described is itssensitivity to Gd 3  . As predicted, the hydrostaticpressure-activated I sc was blocked whenGd 3   was added to the mucosal hemichamber ( Fig. 6 ). These resultsindicated that hydrostatic pressure activated a nonselective cation channel inthe apical membrane of the umbrella cells that was capable of conductingK  .1 O+ q0 G+ F7 }6 U

$ h5 c4 Y+ Y" T6 H& OFig. 6. Hydrostatic pressure-induced transport of Cs   innystatin-permeabilized uroepithelium. Isolated uroepithelium was equilibratedin Krebs solutions, and the Krebs solution was replaced by N -methyl- D -glucamine (NMDG)-gluconate buffer in themucosal chamber and Cs   -gluconate buffer in the serosal chamber.Hydrostatic pressure was increased (arrows) and then decreased (arrowheads) 3times. Nystatin (2 µM) was added to the serosal chamber, and the tissue wasagain subjected to 3 cycles of increased and then decreased hydrostaticpressure. Finally, 1 mM Gd 3   was added to the mucosalchamber, and 3 more cycles of increased and then decreased hydrostaticpressure were performed. A representative tracing is shown. Average values for I sc and conductance during the 5-min periods of increasedhydrostatic pressure are shown above the graph and are means ±SE of 6 separate experiments.
4 n% ~; s  o5 W
+ g6 m( T. X! z/ D, D' {& a/ _Hydrostatic pressure induces Cl - secretioninto the mucosal hemichamber. In Krebs solution, the net hydrostaticpressure-induced change in I sc was 4µA/cm 2 (equivalent to 0.15 µeq ·cm - 2 · h - 1 )( Fig. 1 A ), which was not significantly different from the net 22 Na   flux wemeasured ( 0.15 µeq · cm - 2 ·h - 1 ) ( Fig. 3 A ). However, the 86 Rb   fluxexperiments in Krebs solution ( Table2 ) and the experiments in Na  ,Cl - -free Krebs solution indicated that hydrostatic pressurealso induced an 10 µA/cm 2 (equal to a flux of 0.4µeq · cm - 2 ·h - 1 ) secretory K   current in the opposite direction. This prompted us to explore whether pressure stimulatedtransport of an additional ion species that was of a similar magnitude butopposite charge to the K   current, thus masking the contribution ofthe K   current to the net I sc. Previous studiesindicated that a pathway for Cl - entry exists on thebasolateral membrane of bladder umbrella cells( 35 ). To determine whether apressure-sensitive pathway for Cl - transport exists in thesecells, the uroepithelium was exposed to hydrostatic pressure andunidirectional flux measurements were performed. These experiments confirmedthat in standard Krebs solution and under short-circuit conditions, there wasa net J SM of 36 Cl - in thepresence of hydrostatic pressure equal to 0.41 ± 0.03 µeq ·cm - 2 · h - 1 ( Table 3 ). This value was notsignificantly different ( P 0.05) from the net 86 Rb   flux in the presence of hydrostatic pressure,indicating that hydrostatic pressure induced electroneutral K   andCl - secretion under short-circuit conditions." G3 B- I/ l, A; g

/ b# e! x) i- G. bTable 3. Effects of hydrostatic pressure on 36 Cl - flux
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DISCUSSION
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The bladder is not simply a vessel for storing urine but instead may playan 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 raisingand then lowering hydrostatic pressure, which mimics bladder filling andvoiding, was accompanied by increased and then decreased ion transport. Likeprevious reports, which employed membrane punching and small changes inhydrostatic pressure ( 12, 37 ), we observed thatmechanical stimulation was accompanied by increased Na   absorptionacross the apical membrane of the umbrella cells( 12, 13, 26, 27, 31 - 33, 37 ). This was confirmed bydemonstrating a net J SM of 22 Na   of 0.15 µeq · cm - 2 ·h - 1. Furthermore, we observed a significantdecrease in I sc when tissue was exposed to pressure inNa   -free Krebs solution, or when pressure was increased in thepresence of ouabain, indicating that the ion gradients generated by theNa   -K   -ATPase provided the driving force forNa   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 anabsorptive Na   transport pathway mediated by ENaC( Fig. 7 ).! B: [3 Y& n, D/ t% h* a
* O# ~( O- o& F9 h* ~
Fig. 7. Model for hydrostatic pressure-induced ion transport in bladder umbrellacells. Increased hydrostatic pressure causes increased conductance ofNa  , K  , and Cl -. Na   entersthe 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 exchangefor K  , via the ouabain-sensitiveNa   -K   -ATPase located at the basolateral surface of thecell. K   can exit the cell via basolaterally localizedK   leak channels [which are inhibited byBa 2  , CTX, Gd 3  , andtetraethylammonium (TEA)] or under short-circuit conditions through anapically localized nonselective cation channel, which is inhibited byamiloride ( 100 µM), Ba 2  ,Gd 3  , and TEA. The former may also conductCa 2   and Na  , but this has not been directlydemonstrated. Apical K   conductance is balanced by anelectroneutral Cl - conductance, which may represent transportof Cl - through basolateral and then apicalCl - channels. A basolaterally localized Cl - conductance has been described( 35 ).
; m' y2 T0 G; y& _: v
3 X9 u- C' }; ^- W6 CIn addition to this Na   transport pathway, we also characterized a pressure-sensitive K   secretion pathway, which was revealed undershort-circuit conditions when tissue was exposed to hydrostatic pressure inNa  , Cl - -free Krebs solution. This pathway wasconfirmed by measuring a net J SM of 86 Rb   in standard Krebs solution. Mechanicallystimulated K   secretory pathways have been described in severalcell types, including marginal cells of the stria vascularis, gall bladderepithelial 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 celltypes, K   is secreted via a large-conductance K   channelthat is blocked by TEA and Ba 2  . The K   conductance in the umbrella cells is also sensitive to these channel blockers.However, we found that CTX and iberiotoxin (both inhibitors oflarge-conductance K   channels), apamin, or margatoxin had either noeffect or a small effect on K   secretion when added to the mucosalsurface of the uroepithelium. In contrast, Ba 2  , TEA,Gd 3  , and CTX stimulated secretion when added to theserosal surface of the tissue. This likely reflects inhibition of basolateralK   channels, which would enhance apical release of K   when pressure was increased. The driving force for K   secretion wasapparently the ion gradients generated by theNa   -K   -ATPase, as ouabain inhibited K   secretion ( Fig. 7 ).
, h) t5 U: r7 Z) M- f: g; C# r& n# I; b+ l  p( q; U
The observation that Ba 2   and TEA can inhibit theactivity of nonselective cation channels( 17 ) prompted us to explore whether the K   secretory pathway we observed was possibly via amechanosensitive nonselective cation channel. Several observations indicatedthat this was the case. First, K   secretion in the uroepitheliumwas inhibited by mucosal addition of Gd 3  , a blocker ofboth mechanosensitive ion channels and nonselective cation channels( 7, 18, 40, 43 ). Second, we observed that high concentrations of amiloride, which block mechanosensitive nonselectivecation channels ( 7 ), partiallyinhibited the K   secretory pathway. Third, theGd 3   -sensitive K   secretory pathway alsoconducted Cs  , a hallmark of a nonselective cation conductancepathway ( 20, 57 ). Based on theseobservations, we propose that hydrostatic pressure may increase the activity of a mechanosensitive nonselective cation channel in the apical membrane ofthe umbrella cell ( Fig. 7 ).
0 d3 M( }' [% f  m' f6 K! G% t, W3 h5 r
A nonselective cation channel has previously been described in bladderuroepithelium ( 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 theconductivity for K   by the leak channel was greater than that ofNa   or Cl -. Despite the similarity in ionconductivity and localization between the leak channel and the putativenonselective cation channel that we described here, there are notabledifferences between the two channels. First, the leak channel is unaffected by10-100 µM amiloride( 28, 36, 60 ), whereas the nonselectivecation channel we have characterized is partially inhibited by 100 µMamiloride. Second, the amiloride-sensitive K   10 min) during increased hydrostaticpressure and can be activated during multiple cycles of increased pressure andrelaxation. This contrasts with the short residency time of the leak channel ( 2 min) in the apical membrane of the umbrella cell( 60 ). Third, thepressure-activated nonselective cation channel described in this communicationproduced a secretory K   current, whereas Zweifech and Lewis( 60 ) described an absorptiveleak current. In fact, in the presence of Ba 2  , TEA, orGd 3   we observe a small residual absorptive current thatmay represent leak channel activity.
/ k. U5 e* i. D! T7 `. N6 _2 Q
% q% g" f# u/ }1 K$ k+ f& xIn addition to Na   and K   transport, increasedhydrostatic pressure led to increased Cl - secretion undershort-circuit conditions, which was of a similar magnitude to theK   secretory pathway we described( Fig. 7 ). Mechanicallysensitive Cl - channels have been described in several othertissues and cell types, including Reissner's membrane, the stria vascularis, and cortical collecting duct( 45, 49, 58 ); however, none have beendescribed in bladder epithelium. Known pathways for Cl - entryacross the basolateral membrane of umbrella cells include Cl - channels and exchangers ( 26, 35 ). An apical pathway forCl - conductance has not been described in bladder epithelium,and we found that the Cl - secretory pathway was insensitiveto mucosal addition of DIDS, serosal addition of bumetanide, or mucosal addition of niflumic acid (unpublished observations), all inhibitors of otherknown Cl - conductance pathways( 22, 38, 53 ). The nature of theCl - secretory pathway requires additional experimentation.2 L0 A! o% m) O2 C. n/ T% G/ N) Y

7 C1 Q; ~$ C* ]$ _/ H/ @7 XFinally, the relatively stable change in I sc duringincreased pressure (see Fig. 1 )but the tendency for the conductance to fall during increased pressure andduring the period between cycles of elevated pressure indicates thatparacellular conductance and/or the magnitude of the different ion channelactivities may be affected by pressure. These changes may also explain whymucosal addition of Ba 2   and Gd   inhibitedpressure-induced changes in the I sc contributed byK   secretion but had less of an effect on changes in conductance(Figs. 4 B and 5 B ).
- X! @7 e" C$ \# F- c
  I0 ?* d8 E6 |, f" R' ?In summary, our findings further increase our understanding of themechanically sensitive ion transport processes in the mammalian urinarybladder. Our data indicate that in addition to the previously characterizedNa   absorption pathway, hydrostatic pressure also stimulatesCl - and K   secretion under short-circuit conditions. Although the bladder is generally thought to store urine andmaintain the ion composition of the urine generated by the kidney, the iontransport pathways we and others have described indicate that theuroepithelium could alter the water and ion composition of the urine.Furthermore, absorption of ions from the urine (e.g., Na   ) orsecretion of ions into the urine (e.g., K   ) may provide a means ofregulating the electrolyte composition of the extracellular fluid by modifyingkidney-generated ion gradients. Finally, transport of Cl -,K  , and Na   during the bladder-filling process may alsoplay an important role in sensory transduction and may couple changes inhydrostatic pressure to release of agonists such as ATP that would modulate other cellular functions such as neural-epithelial signaling ( 12, 13, 47 ).
3 K$ t% s2 m# r- F7 I, Z- |3 ~
% F+ y# W+ b+ m5 n. {DISCLOSURES# k" `9 b. ?0 j! O2 Y

8 x$ N, D# d  y: H/ ~This work was supported by National Institute of Diabetes and Digestive andKidney 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), andDK-51391 (to T. R. Kleyman).: z8 h3 t0 H! b  p) B
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ACKNOWLEDGMENTS
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We thank Steven Truschel for assistance during the preparation of thismanuscript.
& o7 M2 x  _" I4 m          【参考文献】
' v4 ~9 i1 m# V% |5 S+ n: c Alves D andDuarte I. Involvement of ATP-sensitive K   channels in theperipheral antinociceptive effect induced by dipyrone. Eur JPharmacol 24:47-52, 2002.9 l. t* W* w$ c$ b# `8 A: M+ R; f0 N

+ B8 N7 E* Q! m  T+ [  @' a; `- P# Z% S

/ ?! K+ x" j; QApodaca G. Modulation of membrane traffic by mechanical stimuli. Am J PhysiolRenal Physiol 282:F179-F190, 2002.9 V6 H* v6 _1 K$ v0 M
! c" \3 R( j! g, b% I" B
' C0 @0 d' y, I, s3 Q* P

) {  Q: ^8 l  r. h3 X) h  yBanes AJ,Tsuzaki M, and Yamamoto J. Mechanoreception at the cellular level: thedetection, interpretation, and diversity of responses to mechanical signals. Biochem Cell Biol 73:349-365, 1995.
8 D0 Y3 [  I# o
8 x. f7 g. [$ A+ _' u1 }1 X6 A6 w- k  `5 O, v1 {! e
/ \5 P  w1 n- j. z+ y* Z) `
Blokkebak-Poulsen M, Sheikh I, and Jacobsen C. Nonselective cationchannels in basolateral-membrane vesicles from pars recta of rabbit kidneyproximal tubule. Biochem J 272:839-842, 1990.( M7 X* v: R2 c% W, I  ?

, B, Z1 }, I2 ]
8 Y8 c" G7 _, f# B8 q
8 R$ h9 s5 w! ?/ g, VBridges RJ. Transepithelial measurements of bicarbonate secretion in calu-3 cells. Methods Mol Med 70:111-128, 2002.8 L1 l2 t* C* e8 m- j

1 P( ?. O; ~* M9 p3 o7 e8 N8 w5 H+ Z* O3 U- \( f, C- E, b

+ m4 J/ \& z) Q9 OChepilko S,Zhou H, Sackin H, and Palmer L. Permeation and gating properties of acloned renal K   channel. Am J Physiol CellPhysiol 268:C389-C401, 1995.% a3 f. o0 z" f. y! Z  ^) i. c2 z
: \; o! K8 B  d! Z  D/ q

+ J: h# M  S/ z* P# f+ [6 }% m: J$ y% q2 ]
Chiba T andMarcus D. Nonselective cation and BK channels in apical membrane of outersulcus epithelial cells. J Membr Biol 174: 167-179,2000.
9 p( a/ I, P0 h  C4 G, y* d) D( V  L" B" L; F  V4 O! T+ {

' D. b' v) O7 z. @& M2 O& W0 f$ R' B) G5 n
Clausen C,Lewis SA, and Diamond JM. Impedance analysis of a tight epithelium using adistributed resistance model. Biophys J 26: 291-318,1979.3 Y* N* y. ]$ Y: z/ i& \1 ]

+ r4 ^0 X! A: X. S6 J  g* Z) ]- v# i8 L# o% h3 U& d

% s; T& g. F# E) [( x, yDavies PF andTripathi SC. Mechanical stress mechanisms and the cell. CircRes 72:239-245, 1993.+ }8 ?7 e0 Q/ m( W; h7 }0 D" a
* z: h0 g& f8 V6 G$ S1 q+ S% w
: p( _& F+ p. w, [' s
8 A5 B  l& i' p+ M) M" K. L# u
Eaton DC,Hamilton KL, and Johnson KE. Intracellular acidosis blocks the basolateralNa-K pump in rabbit urinary bladder. Am J Physiol Renal FluidElectrolyte Physiol 247:F946-F954, 1984., i; z4 K9 ~8 T/ e; w6 u) P

4 j7 z7 i, R1 W. t. x8 p3 M" S! ?! K$ x+ |+ o7 ~

; }/ ^! y4 ~- xEgee S, LapaixF, Cossins AR, and Thomas S. The role of anion and cation channels involume regulatory responses in trout red blood cells. Bioelectrochemistry 52:133-149, 2000.
. x/ ]' _! N( t6 Z) L& Z& I( u* O% A* D' r1 M6 {" t6 M0 F

8 r/ R6 x  g0 a+ L, \- Q
/ q" O4 j  n0 m1 @/ NFerguson DR. Urothelial function. Br J UrolInt 84:235-242, 1999.
0 `! I& Q# O5 u8 G. b& ^
0 n* i+ P: H' R9 |/ ~3 x' B% I
( O' e8 @4 M, P& w  p# S: ~
3 M  B; m* s2 J8 y" |Ferguson DR,Kennedy I, and Burton TJ. ATP is released from rabbit urinary bladderepithelial cells by hydrostatic pressure changes-a possible sensorymechanism. J Physiol 505:503-511, 1997./ f* F% X* Y2 X3 n
( _3 i7 Z: o. P* O, }, v; P
( ]" g5 E9 \# R; x9 W. y

( v% z9 m$ w3 KFilipovic D andSackin H. Stretch- and volume-activated channels in isolated proximaltubule cells. Am J Physiol Renal Fluid ElectrolytePhysiol 262:F857-F870, 1992.
' B7 s7 s. j: s, N8 `' K0 `  W1 k. P4 l; M8 S1 ]+ Y  f" j/ m

, U, _8 x1 \" t, ~; k! G1 I1 W5 H" _, H( {) N7 X
Gargus JF,Frace AM, and Jung F. The role of a PDGF-activated nonselective cationchannel in the proliferative response. EXS 66: 289-295,1993.' Q& ~2 S' s* u' \% v: n# h. s
* e5 ]8 k$ [$ j5 W

2 b- g# r9 K8 _7 f  [# f# l* G; K& [/ t7 w2 h. A, b
Gonzalez-Perrett S, Kim K, Ibarra C, Damiano AE, Zotta E, Batelli M,Harris PC, Reisin IL, Arnaout MA, and Cantiello HF. Polycystin-2, theprotein mutated in autosomal dominant polycystic kidney disease (ADPKD), is aCa 2   -permeable nonselective cation channel. Proc Natl Acad Sci USA 98:1182-1187, 2001.$ R/ ^$ W1 G; O  \' B* N
' F! Y0 H5 L3 O- `+ r
3 u4 k+ X5 S0 K! Q% r1 T

8 c' ^3 Q8 t  h# O* [Guerineau NC,Bossu JL, Gahwiler BH, and Gerber U. Activation of a nonselective cationicconductance by metabotropic glutamatergic and muscarinic agonists in CA3pyramidal neurons of the rat hippocampus. J Neurosci 15: 4395-4407,1995.0 V" R5 k+ i! z/ L! O: X4 B

5 G9 [) p" Q1 Q# M$ W0 D! u" X, V6 n0 G" L
* A$ d+ k! e1 ?& y; q4 @- r1 \
Hamill OP andMartinac B. Molecular basis of mechanotransduction in living cells. Physiol Rev 81:685-740, 2001.
2 a0 k$ G6 c. l
$ S  a/ \2 }  S1 o+ V* u3 F' D. _: @) S

" U; D( B9 y" X3 _* ^  V# c+ rHill AE andHill BS. Transcellular sodium fluxes and pump activity in Necturus gall-bladder epithelial cells. JPhysiol 382:35-49, 1987.
( p: W% z' t$ a
: x: |  [6 Y4 k- `  N( \, Z2 R! b% q1 v! v# f) L5 F& C

7 L2 i- o, O3 E; T! V' dHille B. Ion Channels of Excitable Membranes (3rd ed.).Sunderland, MA: Sinauer, 2001.( |3 Q% ^  H/ h* q0 g

) L; b5 _( i5 Z2 V- [5 J- K& C
# f/ @- S8 u. ^  D& b6 Y2 S# @/ z
Hoger U,Torkkeli PH, Seyfarth E, and French AS. Ionic selectivity of mechanicallyactivated channels in spider mechanoreceptor neurons. JNeurophysiol 78:2079-2085, 1997.& y* \, y9 |! c* _, ]6 v
/ ~3 h3 ?/ l1 n! H- l

9 J! d3 B/ w9 X4 k4 y' V9 G" }7 O, g, T/ u2 K4 z: r+ `
Kitano I, DoiK, Mori N, and Matsunaga T. Involvement of Cl - transportin forskolin-induced elevation of endocochlear potential. HearRes 71:23-27, 1993.
0 E6 T* c$ P+ i5 N; v% m
) r5 ?. [" O/ a9 j
! L5 J8 X5 {' c+ N- @
5 [  s& ^$ @% t- N3 p' ?4 h$ B9 o7 BKleyman TR andCragoe EJJ. Amiloride and its analogs as tools in the study of iontransport. J Membr Biol 105:1-21, 1988.
$ U; V  c' \4 r6 |; z1 {1 r, _* g  J. b5 v7 j* P+ I

1 i+ q4 L' ], t; t1 k
3 M1 `- x! [! ]+ _, v' u1 F; G, E" [Kleyman TR andCragoe EJJ. Cation transport probes: the amiloride series. Methods Enzymol 191:739-755, 1990.
$ I& d" A. Z0 A- C6 c4 Q: F; V
* _4 ^# e$ ]# Y; Q( {( J
3 v& ]% `0 T+ g' R( z
( {8 y3 N0 D, F/ f0 n7 l! ]Levin R andWein A. Response of the in vitro whole bladder (rabbit) preparation toautonomic agonists. J Urol 128:1087-1090, 1982.
' l7 Z9 P" s. t& s' Q: w& ^
% ^8 a' R6 w) d" t# f4 U: B
/ c6 `( }/ c* P
0 W6 c$ x% o2 A% n7 gLewis SA. Everything you wanted to know about the bladder epithelium but were afraid toask. Am J Physiol Renal Physiol 278: F867-F874,2000.- J) c( [$ a5 M" K$ X

5 ^0 k5 l8 q$ g4 r' v. T- g( e2 s/ Y

1 Y  B$ G5 X$ I- n$ b2 J* @Lewis SA. The mammalian urinary bladder: it's more than accommodating. NewsPhysiol Sci 1:61-65, 1986.
0 H$ m& `6 L' P9 C
( i! e7 H& D/ Z- ^# R1 g, B+ F
  K; C* H# R5 o
2 ~+ `; F3 m) D( |9 rLewis SA andAlles WP. Urinary kallikrein: a physiological regulator of epithelialNa   absorption. Proc Natl Acad Sci USA 83: 5345-5348,1986.1 P* F5 d5 {+ W

4 \6 v$ g' w1 g4 X/ Z* V+ D( Z/ C$ x; P2 T

' z  Z$ J! ]; q) |1 K$ P" ^4 aLewis SA andClausen C. Urinary proteases degrade epithelial sodium channels. J Membr Biol 122:77-88, 1991.% ]( O  I9 t8 ~; _2 }' d) a

6 a9 U) W7 v$ k: `% K  {0 ~
1 ~5 {* g' m% p0 @; @# I! [' f6 G& s* d' f; q9 P9 y, W& k8 {$ k
Lewis SA and deMoura JL. Apical membrane area of rabbit urinary bladder increases byfusion of intracellular vesicles: an electrophysiological study. JMembr Biol 82:123-136, 1984.
+ V( r$ c6 [3 Z7 r- M/ x+ z3 j; c- k5 S$ i, G; Q. _6 \% n

# [; a$ Q2 M) _8 e, W6 l
! U( J5 g: M! m) iLewis SA andDiamond JM. Active sodium transport by mammalian urinary bladder. Nature 253:747-748, 1975.
4 S# `; K4 g) e" Y5 J" F! V& D. w" y( D: X( w$ t2 {$ T7 P* z0 _

2 |- Z6 F! ?. ~, T, I9 R6 F& _: S/ {; u9 e" M. P9 M2 m! T4 j2 R0 j5 i  X
Lewis SA andDiamond JM. Na   transport by rabbit urinary bladder, a tightepithelium. J Membr Biol 28:35-40, 1976.
/ I- X+ {6 ?$ \* L; l4 R' N! k/ N! B9 p2 L
) w# E; K5 _' Y+ s  e
( I- G( }0 t4 y
Lewis SA, EatonDC, and Damiano AE. The mechanism of Na   transport by rabbiturinary bladder. J Membr Biol 28: 41-70,1976.+ u1 S/ i6 m, f2 n  s: V

. C7 j" f& D' ?6 N" o# t: @1 {
0 E" A& }! g; g6 \5 O- j5 l
; a$ F( x! L" O: ULewis SA andHanrahan J. Physiological approaches for studying mammalian urinarybladder epithelium. Methods Enzymol 192: 632-650,1990.8 ], X; R( e/ P, H

) X6 v+ c7 D" Q9 T0 Z9 d
- z0 S: w- W9 q' a' m! M2 |; J' }/ Y/ a  w7 Z, O0 J/ E2 b% E
Lewis SA andHanrahan JW. Apical and basolateral membrane ionic channels in rabbiturinary bladder epithelium. Pflügers Arch 405: S83-S88,1985.. R  ?0 L5 f: d, a3 K# S" i7 \: `
8 |4 o* Z* w* U5 @6 x
8 E& G' U* }2 g9 @) [
) ^1 }3 e/ ^2 l. v
Lewis SA,Ifshin MS, Loo DDF, and Diamond JM. Studies of sodium channels in rabbiturinary bladder by noise analysis. J Membr Biol 80: 135-151,1984.
$ O! f; w# ~* C& ]. F0 \/ D$ \1 F+ M: r5 o6 R9 |+ O- X: e- h

2 c% O  g) \3 ?8 N/ i1 T: L
- @* r- e( q5 }+ y- H, p' wLewis SA and deMoura JLC. Incorporation of cytoplasmic vesicles into the apical membraneof rabbit urinary bladder epithelium. Nature 297: 685-688,1982.
" A$ g. d0 m& \( J/ ?2 X: t' ?7 i$ G  q9 y, |+ u' ^1 j
. z1 ~$ B/ d- ?7 s/ K  p/ z
6 ^" W: q: M9 E6 O
Light DB,Schwiebert EM, Fejes-Toth G, Naray-Fejes-Toth A, Karlson KH, McCann FV, andStanton BA. Chloride channels in the apical membrane of corticalcollecting duct cells. Am J Physiol Renal Fluid ElectrolytePhysiol 258:F273-F280, 1990.& E2 I) h$ Z: b
6 o5 [) V7 H3 ?& K6 [- G

! Q% S6 F" A* I8 n% i7 ^/ K: n2 u$ h1 x6 a! e* `
Lindinger MI,Hawke TJ, Vickery L, Bradford L, and Lipskie SL. An integrative, in situapproach to examining K   flux in resting skeletal muscle. Can J Physiol Pharmacol 79:996-1006, 2001.
) K. j9 k9 `8 W; r# a0 ?% F, w: s+ U& E% b6 A0 ^' K
8 |3 i6 ]7 f+ m* N" N: e
: f1 B  z% L. c9 r% F
Morris CE. Mechanosensitive ion channels. J Membr Biol 113: 93-107,1990.2 t+ E% h5 q5 {7 w

& O  s/ M& }  e1 B0 W& G! ]" K7 w! B6 h; s) V" x' _& |* W* H
, M/ N4 R3 O8 ^6 L. W. W; D
Rusch A, KrosCJ, and Richardson GP. Block by amiloride and its derivatives ofmechano-electrical transduction in outer hair cells of mouse cochlearcultures. J Physiol 474:75-86, 1994.% t; W4 b7 `! {3 R9 r
  A$ m# l  W# k, j% J( n
! ~& }: j! C/ d, i' u% A
; K) a6 j. O: R+ o$ v. e7 Y
Sach F. Baroreceptor mechanisms at the cellular level. FederationProc 46:12-16, 1987." }+ I. L9 |1 P5 h  F0 r8 `4 n
2 }/ W' R; h* i- O$ F7 w$ ?
3 `' N! r# h8 x: b
/ ]- Q6 h' G3 P
Sackin H. Stretch-activated ion channels. Kidney Int 48: 1134-1147,1995.
: z* U" w8 u! i
1 O# ?1 }% v( B% Y$ m+ ~' c
4 W& ]) E/ R0 m! U
* s2 S5 h( `3 i% S! r1 X) p* K+ J& dSatlin LM,Sheng S, Woda CB, and Kleyman TR. Epithelial Na   channels areregulated by flow. Am J Physiol Renal Physiol 280: F1010-F1018,2001.5 E. X3 ?, t* Y& I0 X
: o4 m1 K6 U& k: U, L
/ Q* |+ ^5 h: C" ^; W6 ?5 D
9 O; T0 g+ y! I6 U
Schweibert E,Mills JW, and Stanton BA. Actin-based cytoskeleton regulates a chloridechannel and cell volume in a renal cortical collecting duct cell line. J Biol Chem 269:7081-7089, 1994.
) Y6 T4 x, ?3 p+ A9 {2 @9 X2 h" k; k

1 l, z0 G7 }  l1 m( F- a9 |+ H# j
8 a$ R9 N0 `. W  @0 |Smith PR,Mackler SA, Weiser PC, Brooker DR, Ahn YJ, Harte BJ, McNulty KA, and KleymanTR. Expression and localization of epithelial sodium channel in mammalianurinary bladder. Am J Physiol Renal Physiol 274: F91-F96,1998.; u- b, l' B) _5 j3 ]& X7 I  l
, ]/ ~2 O# y5 t( q# b( M* S
7 I: ?! Y' D; Z0 `. L

5 |1 L9 N3 ~9 ^# R( g3 \, v6 USun Y, Keay S,De Deyne P, and Chai TC. Augmented stretch activated adenosinetriphosphate release from bladder uroepithelial cells in patients withinterstitial cystitis. J Urol 166: 1951-1956,2001.( n6 D5 y2 \. k" ^% g
( ~! ^% ]$ e/ x  }, t% g. q$ i2 m
- E5 ?8 @1 Q8 S8 g$ {" H2 N: f2 c
9 z1 C5 q/ Q, n0 K( L- f
Takeuchi S,Ando M, Kozakura K, Saito H, and Irimajiri A. Ion channels in basolateralmembrane of marginal cells dissociated from gerbil stria vascularis. Hear Res 83:89-100, 1995.
* h( S' |! g4 q3 ]; _& r$ k, S% W( z7 B$ ~) Q" t3 }# W5 P
) u8 g6 ~/ E5 ]  o' J7 {! ~/ k/ B

4 h$ |+ I3 d- Q& FTakeuchi S,Marcus D, and Wangemann P. Ca 2   activatednonselective cation, maxi K   and Cl - channels inapical membrane of marginal cells of stria vascularis. HearRes 61:86-96, 1992.
# n! l# _& S) M( W( s" @: d" L# m/ O: A3 g# ]. l9 E

/ B4 ~2 m# M( O, X7 A( Y6 B. g) @& m! y' x
Takeuchi S,Marcus D, and Wangemann P. Maxi-K   channel in apical membraneof vestibular dark cells. Am J Physiol Cell Physiol 262: C1430-C1436,1992.. b( H! b" J5 O  v

8 |+ Z* G. K- c, m/ m: u5 l; @: `7 i4 n4 K; _  ?# H

& ]5 ^/ W3 y+ a$ E3 G4 @9 UTaniguchi J andImai M. Flow-dependent activation of maxi-K   channels in apicalmembrane of rabbit connecting tubule. J Membr Biol 164: 35-45,1998.
5 z( G7 X$ }$ Q; g5 W; ~2 `  `' V$ G) e  e  d9 c. @6 l

: u% ^/ D: T  K# T( [* H# u
3 |, ?' U; m! k& y- W9 J. ]2 ZTruschel S,Wang EC, Ruiz W, Leung S, Rojas R, Lavelle J, Stoffer D, and Apodaca G. Stretch-regulated exocytosis/endocytosis in bladder umbrella cells. Mol Biol Cell 13:830-846, 2002.
/ n& I) p+ s; v- o4 s0 c6 N
( S- ]0 p0 b( A* b( J# h! A6 I' Q
8 `2 f( I4 l. ~( |. ~0 ]0 Z4 E2 Y3 a1 T: z
Vaca L andKunze DL. Anion and cation permeability of a large conductance anionchannel in the T84 human colonic cell line. J MembrBiol 130:241-249, 1992.
0 p! D4 P  u( Z6 i9 ^$ V2 l$ w
1 ?- n, G7 R) q8 f& Z8 p1 H: B6 B+ \0 e0 ]) L1 M
: {$ I9 T/ M& X
Vanoye CG andReuss L. Stretch-activated single K   channels account forwhole-cell currents elicited by swelling. Proc Natl Acad SciUSA 96:6511-6516, 1999.
; o1 n" o) a: E8 M1 }9 b9 w& H
. W8 L4 k/ \3 t5 c9 y) f
4 D9 }7 b4 n$ N' I" i6 D4 _$ N; }
Watson PA. Function follows form: generation of intracellular signals by celldeformation. FASEB J 5:2013-2019, 1991.
0 e! q3 Q, U; ~2 D4 a8 C9 [% y* m. c
- X2 w0 p! N* E

: V0 T$ I4 z& `( M5 h2 r- ~+ |- pWoda CB, BraginA, Kleyman TR, and Satlin LM. Flow-dependent K   secretion inthe cortical collecting duct is mediated by a maxi-K channel. Am JPhysiol Renal Physiol 280:F786-F793, 2001.6 f, E5 v9 ^5 U& U9 g& T

3 b; E/ {; Y+ Z5 B. n+ P% H( D- K' Z& N( r, I2 }2 j5 s

0 F) H; r% O5 k0 ]9 U% U% @Yang X and SachF. Mechanically sensitive, nonselective cation channels. In: Nonselective Cation Channels: Pharmacology, Physiology andBiophysics, edited by Siemen D and Hescheler J. Boston, MA:Birkhauser, 1993, p.79-92.# l1 @; L' q* i
1 Q6 e5 D0 |0 N3 D2 o

* P, F( P' b6 m+ N1 w
5 F' k, s; I: m% u& H  c: xYeh T, Tsai M,Lee S, Hsu M, and Huy P. Stretch-activated nonselective cation,Cl - and K   channels in apical membrane ofepithelial cells of Reissner's membrane. Hear Res 109: 1-10,1997.
, \5 p, O$ I- w% j3 \4 c- n# L0 ?$ B/ f7 n$ t

2 z7 u. z! J4 ?7 u2 |# [
' i, l8 V5 ~$ Y, T( YZhang WH,Skerrett M, Walker NA, Patrick JW, and Tyerman SD. Nonselective currentsand channels in plasma membranes of protoplasts from coats of developing seedsof bean. Plant Physiol 128:388-399, 2002.3 E4 Q: a. n: u, U  y: B

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9 v' d, h2 ^( u' P& d8 D+ i( A  k& c  d% A3 j' ~7 X% a
Zweifach A andLewis SA. Characterization of a partially degraded Na   channelfrom urinary tract epithelium. J Membr Biol 101: 49-56,1988.

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发表于 2015-6-6 09:01 |只看该作者
很有吸引力  

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发表于 2015-6-9 15:35 |只看该作者
初来乍到,请多多关照。。。嘿嘿,回个贴表明我来过。  

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发表于 2015-6-13 09:43 |只看该作者
干细胞之家微信公众号
回帖是种美德.  

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发表于 2015-6-18 09:53 |只看该作者
努力,努力,再努力!!!!!!!!!!!  

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地板
发表于 2015-7-8 18:08 |只看该作者
我卷了~~~~~~~  

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发表于 2015-7-20 15:40 |只看该作者
回帖是种美德.  

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真的有么  

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发表于 2015-8-19 22:02 |只看该作者
勤奋真能造就财富吗?  

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肌源性干细胞
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