Ammonium effects on colonic Cl- secretion: anomalous mole fraction behavior

Roger T. Worrell,1,2 Jennifer Oghene,1 and Jeffrey B. Matthews1,2

1Epithelial Pathobiology Group, Department of Surgery and 2Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45219

Submitted 29 April 2003 ; accepted in final form 20 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A significant amount of ammonium () is absorbed by the colon. The nature of effects on transport and transport itself in colonic epithelium is poorly understood. The goal of this study was to elucidate the effects of on cAMP-stimulated Cl- secretion in the colonic cell line T84. In HEPES-buffered solutions, application of basolateral resulted in a reduced level of Cl- secretory current. The effect of appears to occur by at least three mechanisms: 1) basolateral membrane depolarization, 2) a competitive effect with K+, and 3) a long-term (>20 min) increase in transepithelial resistance (TER). The competitive effect with K+ exhibits anomalous mole fraction behavior. Transepithelial current relative to that in 10 mM basolateral K+ was inhibited 15% by 10 mM alone and by 30% with a mixture of 2 mM K+ and 8 mM . A mole fraction mix of 2 mM K+:8 mM produced a greater inhibition of basolateral membrane K+ current than pure K+ or alone. Similar anomalous behavior was also observed for inhibition of bumetanide-sensitive 36Cl- uptake, e.g., Na+-K+-2Cl--cotransporter (NKCC-1). No anomalous effect was observed on Na+-K+-ATPase current. Both NKCC-1 and Na+-K+-ATPase activity were elevated in 10 mM with respect to 10 mM K+. The effect on TER did not exhibit anomalous mole fraction behavior. The overall effect of basolateral on cAMP-stimulated transport is dependent on the ratio at the basolateral membrane, where o is outside of the cell.

transepithelial resistance; Na+-K+-2Cl--cotransporter


COLONIC LUMEN CONCENTRATION can range from 15 up to 100 mM (28). Normal arterial plasma levels of are relatively low at 45 µM (5, 28). The fact that portal vein levels are elevated with respect to arterial plasma (~350 µM) indicates a net absorption of across the colonic epithelium (28). The level at or near the base of the colonic epithelium is unknown but is likely to be significantly higher, ~3–10 mM than that in the portal vein due to the >10-fold dilution of "colonic crypt" blood by the time it reaches the portal vein. Despite this, little is known about ammonium () transport or effects on colonic ion transport. Excess in systemic levels can lead to hyperammonemia-associated encephalopathy that can be life threatening. Although the kidney is responsible for carrying out the bulk of body homeostasis, the possibility exists that some level of body control can be accomplished via changes in colonic function and/or in the level of production within the lumen of the colon.

Previous studies in rat and human colon as well as cultured epithelial cell lines have demonstrated that can inhibit both sodium absorption (1, 2) as well as Cl- secretion (2, 12, 23, 24, 26). With the use of the T84 secretory colonic cell line, it was shown that can affect cAMP- and cGMP-dependent Cl- secretion but not carbachol-induced Ca2+-dependent secretion (23). Although did not inhibit the secretory response to carbachol or thapsigargin, pretreatment with was found to blunt the secretory response of T84 cells to Ca2+ ionophore-mediated secretion. Posttreatment with did not affect the Ca2+ ionophore-mediated secretion (23).

is a weak acid with an acid-base ionization constant (pKa) = 9.2 and with some notable exceptions (14, 30) can diffuse across the plasma membrane. However, at physiological pH, ~98% of NH3 exists in the protonated form of . The / prepulse method has been used extensively as a means of altering intracellular pH (pHi). Although, in previous studies (12) in which did alter pHi in T84 cells, the changes in pHi did not correlate with the changes in Cl- secretion. In addition, there was a sidedness to the effect on cAMP-stimulated Cl- secretion, with application to the basolateral side having an inhibitory constant (Ki) of 5 mM and apical application of Ki = 50 mM, which suggested that the effect on Cl- secretory rate occurred by affecting the basolateral membrane transport processes. Indeed, in the report of Hrnjez et al. (12), was found to not alter the apical Cl- conductance (CFTR) but was found to affect the basolateral K+ conductance.

The minimalist model for electrogenic Cl- secretion in T84 cells includes an apical Cl- conductance in series with basolateral Na+-K+-2Cl- cotransporter, K+ conductance, and Na+-K+-ATPase. has been shown to interact with a number of K+ transport proteins, including K+ channels (3, 4, 9, 40), Na+-K+-2Cl- and KCl cotransporters (15, 35), and Na+-K+-ATPase (17, 33). The purpose of this study was to address the effects of interactions on the basolateral transport processes in T84 cells that are of key importance in the Cl--secretory model (29, 36).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. T84 cells (8) obtained from the American Type Culture Collection and K. Barrett (Univ. of California, San Diego) were grown to confluence at pH 7.4 in 162-cm2 flasks with DMEM/Ham's F-12, 1:1 mix supplemented with 6% FBS, 15 mM HEPES, 14 mM NaHCO3, 170 µM penicillin G, and 69 µM streptomycin sulfate. Amphotericin B was not included in the medium to avoid potential complications due to its ionophoretic activity. Cells were maintained in culture with weekly passage by trypsinization in Ca2+- and Mg2+-free phosphate-buffered saline at a split ratio of 1:2. Cells for experimentation were plated on uncoated 12- or 24-mm Costar Transwell (3-µm pore size) inserts at a seeding density of 4–5 x 105 cells/cm2 and cultured 8–14 days with feeding in the above medium 3 times per week. Cell monolayers were determined to be of acceptable use when the transepithelial resistance (TER) reached >1,200 {Omega}·cm2 as measured by epithelial volt-ohm meter (EVOM) (see below). All experiments were performed at 37°C.

Measurement of transepithelial current. The quality of high resistance monolayer formation was monitored by using an EVOM (World Precision Instruments) as described previously (38). This instrument consists of a pair of Ag/AgCl electrodes mounted in "chopstick" fashion attached to a customized voltohmmeter. Both transepithelial potential (mV) and TER (K{Omega}) were measured with this instrument. Transepithial current (ITR) was calculated by Ohm's law and expressed as µA/cm2 and is referred to as open-circuit current in the text to clearly distinguish it from the short-circuit current technique. Although the open-circuit current technique tends to underestimate the amount of current that would be obtained in short-circuit current measurements, EVOM measurements proved consistent and reliable (Fig. 1, error bars, as an example) for detecting changes in transepithelial voltage, resistance, and current. Moreover, measurements in the open-circuit mode represent the normal condition (e.g., not short circuited) of native epithelia.



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Fig. 1. Inhibition of PGE2-stimulated Cl- secretion by basolateral ammonium (). Cl- secretion was accessed by measurement of open-circuit current (Ioc) in confluent monolayers of T84 cells grown on permeable supports. Cells were stimulated with 10 nM basolateral PGE2 at time 0, with the exception of the control ({bullet}). Application of NH4Cl ({triangledown}, 0 mM; {square}, 1 mM; {diamondsuit}, 10 mM; {blacktriangleup}, 20 mM) was at the 5-min point. Inhibition was significantly different from control for each dose and time point. Representative data shown from 4 experiments for each data point.

 

Transepithelial measurements were carried out in the following base solution (in mM): 140 NaCl, 5 KCl 0.5 CaCl2, 2 MgCl2, 10 Na-HEPES at a pH of 7.4. "K+-like" cations were added as indicated in the text to a K+-free base solution, and the solution was pH adjusted to pH 7.4 as necessary. cAMP-dependent Cl- secretion was stimulated either by basolateral 10 nM PGE2 or 10 µM forskolin. This was initially done to rule out possible effects of due to very high cAMP levels vs. those achieved with an endogenous agonists. Results were similar with each method, as indicated in Fig. 3.



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Fig. 3. inhibition of PGE2-stimulated Ioc at 5-min exposure to displays anomalous mole fraction behavior. A: current relative to that in 10 mM K+ alone is plotted vs. a varying mix of 10 mM total + K+. Maximal current inhibition is observed at a ratio of ~2 mM K+ to 8 mM . Data represent at least an n of 4 experiments for each condition. *Significant vs. <7:3 and >9:1, #significant vs. 10 mM K+ and vs. 7.5:2.5 and 8:2 mix. Cl- salts were used for cation substitutions. B: inhibition of forskolin-stimulated Ioc at 5-min exposure to displays anomalous mole fraction behavior. Current in the presence of 2 mM K+ or 8 mM alone are depicted. Current at 2 mM K+ + 8 mM was 61% relative to 10 K+ alone. Current at 10 mM was ~90% relative to 10 mM K+ alone. Current is shown at 5-min exposure to the given concentration mix of K+ and/or . Representative data shown from 4 experiments for each data set. *Significant vs. K+ or alone. Gluconate salts were used for cation substitutions.

 

Basolateral potassium currents. The basolateral K+ channel current was isolated by using nystatin permeabilization of the apical membrane (18) in the following manner. The apical solution was switched to one containing (in mM): 135 K-gluconate, 0.5 Ca-gluconate, 2 Mg-gluconate, 10 K-HEPES at pH 7.4 containing 200 U/ml nystatin. The basolateral solution was: 130 Na-gluconate, 10 K or 2 K + 8 NH4 or 10 NH4-gluconate, 0.5 Ca-gluconate, 2 Mg-gluconate, 10 Na-HEPES at pH 7.4 containing 10 µM forskolin and 100 µM ouabain. Nernst potentials were equilibrium potential for K+ (EK) ~60 mV, equilibrium potential for Na+ (ENa) >125 mV, Enonselective ~0 mV with 10 mM basolateral K+ and apical nystatin. Effectiveness of apical permeabilization was gauged by the transepithelial potential (ETR). Experiments in which ETR approached EK with 10 mM basolateral K+ were used for analysis.

Measurement of pHi. pHi was determined flurometrically, as previously described (12). Briefly, confluent T84 cell monolayers were grown on Anocell inserts (0.33 cm2). Cells were loaded with BCECF-AM for 1 h at room temperature and mounted in a customized quartz cuvette that allowed for isolation between the apical and basolateral solutions. was added to the basolateral side of the monolayer while monitoring the fluorescence output. Dye calibration with pHi was accomplished by running pH standards at the end of each experiment.

Na+-K+-2Cl--cotransporter activity. Na+-K+-2Cl--cotransporter (NKCC-1) activity was determined by bumetanide-sensitive basolateral 86Rb or 36Cl uptake. Cells grown on 24-mm Costar inserts were stimulated with 10 µM forskolin and transepithelial currents measured at 5 min. Individual inserts were then placed in wells containing 1 µCi/ml 86Rb or 1 µCi/ml 36Cl with or without 50 µM bumetanide for 1 min. Inserts were washed by sequentially dunking into two reservoirs of >500 ml ice-cold 0.1 M MgCl2 and 10 mM Tris-Cl pH 7.4. Cells were lysed with 1% Triton X-100, scraped, and collected into liquid scintillation vials. Correcting for protein content by protein measurements on individual inserts did not lead to a significant decrease in variability and were thus omitted to simplify the assay.

Pump current. Current due to Na+-K+-ATPase was assessed by measuring the ouabain-sensitive transepithelial current under the following conditions: apical: 120 Na-gluconate, 5 K-gluconate, 0.5 Ca-gluconate, 2 Mg-gluconate, 10 Na-HEPES at pH 7.4 containing 200 U/ml nystatin; and basolateral: 120 Na-gluconate, 5, 10, or 2 K + 8 NH4, or 10 NH4-gluconate, 0.5 Ca-gluconate, 2 Mg-gluconate, 10 Na-HEPES at pH 7.4 containing 10 µM forskolin and ±100 µM ouabain. Nernst equalibrium potential were ENa ~0 mV and EK ~0 mV with 5 mM basolateral K+ for the given solutions in the presence of apical nystatin.

Reagents. Forskolin was obtained from Calbiochem, PGE2 was from BioMol, 86Rb (0.5–10 mCi/mg) and 36Cl (3 mCi/mg) were from Amersham, FBS and PenStrep were from GIBCO-BRL, all other chemicals were of the highest grade obtainable from Sigma.

Significance tests. Data are presented as means ± SE. Significance was estimated by using Student's t-test and two-way ANOVA when indicated, with a P value of <0.05 indicating a significant difference.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
effects on transepithelial transport. Figure 1 depicts PGE2 stimulated cAMP-dependent Cl- secretion in T84 monolayers. Open-circuit current increased significantly from 1.6 ± 0.1 to 50 ± 0.4 µA/cm2 within 5-min treatment with basolateral 10 nM PGE2. Current was relatively stable for 30 min, with only a slight decline in current magnitude observed. The addition of increasing amounts of basolateral on a background of 5 mM basolateral K+ produced an increasing amount of current inhibition. At 5-min exposure, current (in µA/cm2) was -51 ± 0.5 for control and -42 ± 1, -23 ± 3, and -17 ± 2 for 1, 10, and 20 mM for added , respectively. This corresponds to current inhibition of 18, 54, and 66% by 1, 10, and 20 mM , respectively when added in the presence of 5 mM K+. These results are consistent with the effect previously reported (26) for forskolin stimulation of current.

Because is of similar size to K+, it is possible that conduction through basolateral K+ channels is similar to that of K+. It is well appreciated that increasing basolateral K+ will result in an intracellular depolarization and thus a decrease in the driving force for Cl- secretion (36, 37). Thus we chose to measure open-circuit current under conditions in which basolateral K+ was replaced, under zero basolateral [K+], by various congeners of K+. Because 10 mM produced a substantial inhibitory effect on Cl- secretion (Fig. 1), each congener including K+ itself was used at a 10 mM basolateral concentration.

Figure 2 shows PGE2-stimulated open-circuit current relative to that with 5 mM basolateral K+. Basolateral solutions were changed to those indicated at the 5-min time point. Current decreased slightly but was relatively stable with replacement of the basolateral control solution (5 mM KCl). Interestingly, shifting the basolateral solution to one containing 10 mM K+ produced a similar inhibitory effect to that of 10 mM with 7-min exposure (41 ± 1.2 and 37 ± 1.0%, respectively). This is consistent with producing a basolateral membrane depolarization by conduction through basolateral K+ channels. However, inhibition by 10 mM was greater than that of 10 mM K+ at 15-min exposure (40 ± 1.2 and 50 ± 1.4%, respectively). Both 10 mM Rb+ and 10 mM Tl+, ions known to permeate some K+ channels, produced current inhibition greater than that of 10 mM K+. Rb+ inhibited current by 54 ± 1.8% and Tl+ inhibited current by 64 ± 2.5% with 7-min exposure. Whereas inhibition produced by Rb+ was stable between 7 and 15 min, that of Tl+ continued to increase. All basolateral solutions were subsequently replaced to control (5 mM K+) at the 20-min time point. On washout, current inhibition by 10 mM K+ and 10 mM Rb+ was fully reversible. Compared with a control value of 80 ± 1% initial current, current with washout of K+ and Rb+ were 94 ± 1 and 87 ± 1% of initial values. The observation that both are relatively flat between the 12- and 20-min time points is consistent with a pure membrane depolarization effect. Unlike, Rb+ and K+, current inhibition by 10 mM was not fully reversible; current magnitude was 67 ± 1.5% of the initial value on removal of (significantly different from control). This, taken together with the observation of continued inhibition after 7-min exposure, suggests that may exert effects other than can be explained by simple basolateral membrane depolarization. Current inhibition by 10 mM Tl+ was irreversible as has been seen by others in frog skin (41).



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Fig. 2. Effect of basolateral cations on PGE2-stimulated Ioc. Current relative to the PGE2-stimulated level (at 5 mM K+) is depicted. Basolateral medium was replaced with: {bullet}, 5 mM KCl; {blacktriangleup}, 10 mM KCl; {blacksquare}, 10 mM NH4Cl; {diamondsuit}, 10 mM RbCl; {blacktriangledown}, TlCl at the 5-min point. Tl+, Rb+, K+, and at 10 mM significantly reduced current compared with control. At the 20-min time point, basolateral solution was changed back to 5 mM K+. Current inhibition by K+ or Rb+ was fully reversible, whereas inhibition by Tl+ or was not fully reversible. Representative data shown from 4 experiments for each data point.

 

The degree of current inhibition by each cation after 5-min exposure relative to that seen with basolateral 10 mM K+ was as follows. NMDG-Cl was similar to K+ 1.08 ± 0.02 for NMDG and 1.00 ± 0.05 for 10 mM K+.1 Currents were significantly reduced by 16 ± 2% by Cs+, 21 ± 2% by Rb+, 27 ± 2% by , and 65 ± 5% by Tl+ compared with 10 mM K+. The lack of difference between and K+ inhibition at 10 mM compared with 5 mM K+ in Fig. 2 and that above likely relates to variability in timing of the effect.

Anomalous mole fraction effect of on transepithelial current. Given that 10 mM K+ and 10 mM both produced current inhibition relative to 5 mM K+ at 5 min and that this likely relates to conductance through basolateral K+ channels, it was reasonable to determine whether and K+ mixes behaved similarly to either pure or pure K+. Figure 3A shows the current relative to that of 10 mM K+ with various mixes of K+ and in which the sum of K+ and is held at a constant 10 mM. As shown, the inhibition of current with 2 mM K+ plus 8 mM is greater than with 10 mM alone. Current relative to that in 10 mM K+ was 85 ± 6% with 10 mM , 70 ± 8% for a 2 mM K+/8 mM mix, and 70 ± 4% for a 2.5 mM K+/7.5 mix. Current with a 3 mM K+/7 mM mix was 86 ± 6% relative to that in 10 mM K+. Transepithelial current was >90% of that seen with 10 mM K+ alone for mole fractions of <0.6 []/[]+[]. Figure 3B depicts an experiment in which current was stimulated with basolateral 10 µM forskolin. Shown are open-circuit current for 2, 5, and 10 mM K+, a mix of 2 mM K+/8 mM , and 8 and 10 mM . Current (in µA/cm2) was 35 ± 1.5, 23 ± 1.2, and 18 ± 1.7 for 2, 5, and 10 mM K+. Current with basolateral alone was 17 ± 2.9 and 16 ± 2.6 µA/cm2 for 8 and 10 mM . A mixture of 2 mM K+ and 8 mM produced a current level of 11 ± 0.3 µA/cm2. Current with 10 mM was 90% that of current in 10 mM K+, and current in the mix of 2 mM K+/8 mM was 61% that of current in 10 mM K+. Current with 2 mM K+ or with 8 mM alone cannot account for the lower currents (anomalous mole fraction effect) observed with the mixture of K+ and .

The anomalous mole fraction effect observed with / mixtures compared with either cation alone was further characterized in experiments designed to isolate each of the basolateral membrane K+ transport pathways.

effect on basolateral K+ conductance. Figure 4 shows open-circuit current that is due to the basolateral K+ conductance (monolayers treated in Cl--free solutions with apical nystatin and basolateral ouabain). As predicted from the data in Fig. 4, a mix of basolateral 2 mM K+/8 mM produces a greater inhibition of current through K+ channels than does 10 mM . Current (in µA/cm2) was 23 ± 2, 11 ± 1, and 16 ± 2 for 10 mM K+, a mix of 2 mM K+/8 mM , and 10 mM , respectively. Relative to current in 10 mM K+, this corresponds to a current level of 72% for 10 mM and 47% for the K+, mix. Such anomalous mole fraction behavior has been associated with a number of ion channels (11). These data also support the notion that the basolateral K+ conductance activated with cAMP-stimulated Cl- secretion has a measurable permeability and conductance.



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Fig. 4. Forskolin-stimulated basolateral K+ channel current is inhibited by in an anomalous mole fraction-dependent manner. Ioc was measured in apical nystatin-permeabilized monolayers in the presence of basolateral 200 µM ouabain. Current at 2 mM K+ + 8 mM was 47% relative to 10 K+ alone. Current at 10 mM was 72% relative to 10 K+ alone. Representative data from 4 experiments shown. *Significant vs. K+ and alone, #significant vs. K+ and 2:8 mix. Gluconate salts were used for cation substitutions.

 

effect on NKCC-1. has been reported to be a substrate for NKCC-1 (15, 35). One means to determine the relative rates of NH3 and entry into cells is to monitor pHi changes with the addition or removal of external . Figure 5A shows the change in pHi with the basolateral addition of 10 mM . Initially, there is an alkalinization of pHi consistent with net NH3 entry. However, this is followed by an intracellular acidification phase consistent with net entry. On removal of basolateral , pHi further acidifies, indicating net NH3 exit. Figure 5B shows a representative monolayer that has been pretreated with bumetanide to block basolateral NKCC-1. Basolateral addition of with bumetanide pretreatment results in a pronounced intracellular alkalinization that is not followed by acidification, indicating net NH3 entry throughout the 20-min time course. This is consistent with the idea that net acidification with external seen in Fig. 5A is due to entry via NKCC-1. Intracellular acidification on washout either in the presence or absence of bumetanide, suggests that the exit pathway, once has entered the cell, is limited. That is, the exit pathway represents net NH3 exit independent of NKCC-1. entry via NKCC-1 is further supported by the data in Fig. 6.



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Fig. 5. entry through NKCC-1 in forkolin-stimulated T84 cells as indicated by change in intracellular pH ({Delta}pHi). A: addition of basolateral 10 mM NH4Cl causes pHi to alkalinize followed by acidification, indicating the net movement of NH3 followed by net entry into the cell. On washout of basolateral , pHi further acidifies indicating net NH3 exit. B: pretreatment of cells with bumetanide, to block NKCC-1, prevents net intracellular acidification by basolateral . Note that the subsequent wash still produces a net acidification. Representative data from 4 experiments shown.

 


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Fig. 6. Effect of bumetanide-sensitive 36Cl- uptake in forskolin-stimulated T84 cells. Both increased basolateral K+ and basolateral decreased bumetanide-sensitive 36Cl- uptake. Inhibition of 36Cl- uptake exhibited anomalous mole fraction behavior with a mixture of 2 mM K+ and 8 mM producing a greater inhibition than pure K+ or . Uptake time was for 1 min in monolayers stimulated with 10 µM forskolin. Gluconate salts were used for cation substitutions. Representative data from 4 experiments shown. *Significant vs. K+ and , #significant vs. K+ and mix.

 

Direct assessment of NKCC-1 activity was accomplished by using 36Cl uptake. Figure 6 shows the bumetanide-sensitive 36Cl uptake in forskolin-stimulated cells. Differing basolateral conditions have little effect on bumetanide-insensitive uptake. Uptake was 886 ± 82, 1,140 ± 166, 924 ± 209, 952 ± 72 cpm x 103/4.7 cm2 with 5 mM K+, 10 mM K+, a mix of 2 mM K+/8 mM , and 10 mM , respectively. However, there is a marked difference observed in the bumetanide-sensitive uptake. NKCC-1 activity was suppressed 73% by an increase in basolateral K+ from 5 to 10 mM, uptakes of 1,573 ± 347 and 565 ± 174 counts/min (cpm) x 103/4.7 cm2, respectively. More interestingly though, a mix of 2 mM K+/8 mM produced a greater inhibition of uptake than did 10 mM K+ or 10 mM alone. Uptake was 247 ± 159 cpm x 103/4.7 cm2 for the mix of K+ and and was 1,013 ± 111 cpm x 103/4.7 cm2 for 10 mM . This corresponds to a 56 and 76% reduction in uptake with the K+, mix vs. uptake in 10 mM K+ and 10 mM , respectively. Uptake in the presence of 10 mM was 79% greater than that in 10 mM K+. Interestingly, bumetanide-sensitive 86Rb uptake did not display an anomalous mole fraction effect. Each condition produced a similar inhibition of bumetanide-sensitive 86Rb uptake compared with 5 mM K+ (P < 0.05 for each condition with respect to 5 mM K+). Bumetanide-sensitive 86Rb uptake was 20 ± 0.5, 20 ± 1, 17 ± 2 cpm x 103/4.7 cm2 for 10 mM K+, a mix of 2 mM K+/8 mM , and 10 mM , respectively. Compared with 5 mM K+ (uptake of 37 ± 5 cpm x 103/4.7 cm2), this suggests that K+ and are similarly effective competing with Rb+ for the K+ site on NKCC-1. The implications of the difference between 36Cl and 86Rb uptake measurements are contained in DISCUSSION.

effect on Na+-K+-ATPase. Pump activity was assessed by measuring ouabain-sensitive pump currents in apical nystatin-permeabilized monolayers. Changing basolateral K+ from 5 to 10 mM had a slight effect on pump current (14 ± 2 and 11 ± 0.6 µA/cm2, respectively). More importantly, no anomalous mole fraction behavior was observed between K+ and . A slight increase in pump activity occurred with 10 mM K+, 10 mM , or a mix of 2 mM K+/8 mM ; current in µA/cm2 was 11 ± 0.6, 13 ± 0.9, and 14 ± 2 for each respective condition. Pump current was 15% greater with a mix of 2 mM K+/8 mM and 25% greater with 10 mM than with 10 mM K+. The possibility that the pump may slightly favor over K+ is supported by the bumetanide-insensitive 86Rb uptake (predominately due to pump activity) that is enhanced by compared with 10 mM K+. Uptake was 4.2 ± 0.1, 8.49 ± 0.4*, and 9.5 ± 0.77* cpm x 103/4.7 cm2 for 10 mM K+, a mix of 2 mM K+ + 8 mM , and 10 mM , respectively (*P < 0.05 vs. 10 mM K+).

effect on TER. Lack of full reversibility with inhibition suggests possible effects in addition to interactions with ion binding sites on K+ channels, NKCC-1, or Na+-K+-ATPase. Figure 7 depicts an additional effect of that is most prominent with longer-term exposure to . Exposure of monolayers to over a longer term results in a significant increase in the TER. TER is >1,500 {Omega}·cm2 under control conditions for the data presented. On stimulation of Cl- secretion with PGE2, TER drops to 523 ± 23, 490 ± 4, and 483 ± 3 {Omega}·cm2 for 5, 10 mM K+, and 10 mM , respectively primarily due to a drop in the apical membrane resistive element (37). Over time with basolateral , TER significantly increases to levels near control. TER was 1,400 ± 40 {Omega}·cm2 after 115 min in compared with 743 ± 34 and 690 ± 13 {Omega}·cm2 for 5 and 10 mM K+. An increase in TER is not observed with basolateral 10 mM K+. On replacement of basolateral with control (5 mM K+) TER further increases significantly instead of reversing, indicating a possible relationship with intracellular acidification (refer to DISCUSSION). With washout, TER increased from 1,400 ± 41 to 1,696 ± 51 {Omega}·cm2 for -treated monolayers and increased to 792 ± 40 and 730 ± 12 {Omega}·cm2 for 5 and 10 mM K+-treated monolayers.



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Fig. 7. Long-term exposure to basolateral increases transepithelial resistance (TER). Basolateral PGE2 (10 nM) was added at time 0. At 5 min, basolateral solutions were changed to 5 mM KCl ({bullet}), 10 mM KCl ({blacktriangledown}), or 10 mM NH4Cl ({blacksquare}) with PGE2. The effect was not reversible on return to basolateral 5 mM K+ (last data points). Representative data from 4 experiments shown for each data set.

 

Of the "K+-like" ions tested, only produces a substantial increase in TER. Figure 8A shows the TER relative to that in 10 mM K+ with 35-min exposure to the indicated ions. TER was 81 ± 1% that of K+ for Tl+ and 97 ± 1% for Rb+. TER was increased 7 ± 1% by Cs+, 13 ± 1% by NMDG, and 52 ± 3% by with respect to K+. Figure 8B depicts the relative current for the same data set. Current relative to that in 10 mM K+ (in %) was 71 ± 2 for NMDG, 63 ± 4 for Rb+, 60 ± 1 for , 23 ± 1 for Cs+ and 8 ± 1 for Tl+. Note that the change in TER does not correlate with the changes in current (e.g., the series are not the same). This argues against the -induced increase in TER being due to a reduction in the basolateral membrane K+ conductance.



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Fig. 8. Other ions that inhibit current do not produce an increase in TER. A: of the K+ congeners tested, only produced a large increase in TER. TER is depicted relative to that in 10 mM basolateral K+. B: relative current for the resistance data shown in A. Note the difference in the rank order between A and B, as well as A with Fig. 3. Cl salts were used for cation substitutions. Representative data shown from at least 4 experiments for each data set.

 

Further evidence that the effect on TER is distinct from that on the basolateral transport processes is provided in that the effect on TER does not display anomalous mole fraction behavior but rather increases as a function of the concentration. TER was increased 9 ± 1, 14 ± 2, 23 ± 7, and 55 ± 5% by 2.5, 5, 7.5, and 10 mM , respectively.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Data presented demonstrate that , when present at the basolateral membrane of a model cell line for electrogenic Cl- secretion, can impact Cl- secretion through multiple mechanisms. Effects of were consistent with: 1) exerting a depolarization effect similar to that of K+, thereby reducing the driving force for Cl- secretion; 2) having a competitive interaction with K+ for K+ binding sites on the K+ channel NKCC-1 as well as Na+-K+-ATPase; and 3) inducing a long-term increase in TER. was found to influence each of the basolateral membrane transport processes involved in Cl- secretion.

effects on transepithelial transport. The fact that PGE2 (cAMP)-stimulated Cl- secretion in T84 cells is inhibited by the addition of to the transporting monolayer is consistent with earlier findings. We have previously reported that both forskolin (cAMP)- and heat-stable enterotoxin (cGMP)-stimulated secretion is inhibited by basolateral and that this effect, in part, relates to an inhibition of the cAMP-activated basolateral K+ conductance in these cells (12, 26).

Electrogenic Cl- secretion is dependent on the driving force for Cl- exit across the apical membrane, which is principally due to a negative intracellular potential maintained by the basolateral transport processes, namely the combined function of the Na+-K+-ATPase and a "K+ leak" pathway. Increasing the K+ leak pathway will increase the driving force for Cl- exit at the apical membrane. On the other hand, decreasing the K+ leak pathway will decrease the driving force for apical Cl- exit (32, 36, 37).

and K+ are of similar size; thus can substitute for, and/or compete with, K+ in many K+ channels. We have shown here (Fig. 1) that under conditions in which basolateral is added in the presence of basolateral K+, has a significant inhibitory effect on transepithelial current. Previously reported experiments were performed in this manner (12, 26). However, when is applied to the external side under nominally K+-free conditions (Fig. 2), the inhibitory effect compared with equal molar K+ is less pronounced. Other congeners of K+ produce a current inhibition (Fig. 2), thus suggesting that, in part, is able to slow K+ exit in a manner similar to externally applied K+ and other K+ congeners. Assuming that and K+ permeability on the external side of the basolateral K+ channel are similar, one would predict that an increase in basolateral could exert a basolateral membrane depolarization effect and thereby decrease the rate of Cl- secretion. The prediction of this model is that once the basolateral concentration of the K+ congener is reduced, the basolateral membrane depolarization effect would be reduced and transepithelial current would increase to previous levels. This appears to be the case as basolateral K+ or Rb+ concentrations are changed but only partially for and not at all for Tl+ (Fig. 2). Lack of reversibility with Tl+ is not unexpected, because others (40) have shown that Tl+ produces an irreversible deterioration of transport. Two lines of evidence point to additional effects of beyond simple basolateral membrane depolarization. First, as the data in Fig. 2 show, current inhibition by does not plateau as does inhibition of current with equal molar K+ or Rb+. Second, inhibition of current by is only partially reversible after 20-min exposure.

Magnitude of transepithelial current inhibition in mixed / conditions displays a maximal inhibitory point at a ratio of ~2 mM K+:8 mM as shown in Fig. 3A, a so called anomalous mole fraction effect. Zeiske and Van Driessche (40) with the use of short-circuit current and noise fluctuation analysis have reported Tl+ and K+ interaction resulting in anomalous mole fraction behavior in frog skin. They also reported , Rb+, and Tl+ permeation and conductance through the apical K+ channels. Observation of an anomalous effect is consistent with the notion previously reported that -induced inhibition of current is not attributable to -induced changes in pHi, but rather is due to effects on the basolateral transport processes. The notion that can substitute for K+ is supported by the observation that current values with mole fractions <0.6 []/[]+[] are roughly equal (>90%) to those seen with 10 mM K+ alone, despite the observation that lower basolateral [K+] in the absence of produce more current (refer to Fig. 3).

effect on basolateral K+ conductance. Previous data indicated that inhibition of Cl- secretion in T84 was due to block of the cAMP-stimulated basolateral K+ channel (12). Data presented herein are consistent with and amplify that finding. Because anomalous mole fraction behavior is considered to occur with ion channels, experiments to determine basolateral K+ conductance inhibition were carried out. As is shown in Fig. 4, a K+, mix at 2:8 reduces basolateral K+ current to a greater extent than an equal molar amount of . Anomalous mole fraction behavior has been demonstrated for a number of ion channel types and is most often attributed to an ion channel with multiple ion binding sites and single file flow (9). A number of basolateral K+ channels have been reported to occur in colonic crypts (16, 19, 34) and in T84 cells (6, 7, 13). Although the most widely accepted cAMP-activated channel is KvLQT1(KCNQ1), the presence of other cAMP-responsive K+ channels may indicate a less simplistic model for basolateral K+ exit. Yang and Sigworth (39) have reported a permeability sequence for KvLQT1 but did not include or mole fraction experiments in their study. To our knowledge, possible anomalous mole fraction effects of and K+ in KvLQT1 or other K+ channels identified in these secretory cells have not been reported.

effect on NKCC-1. has been shown to be a substrate for NKCC-1 (35) and NKCC-2 (15) in kidney. Thus we wanted to determine whether NKCC-1 activity in the T84 cells was affected by . The observation that intracellular alkalinization on the addition of is bumetanide sensitive (Fig. 5) suggests that can be transported by NKCC-1 in these cells. To assess whether NKCC-1 activity was affected by , we examined bumetanide-sensitive 86Rb and 36Cl uptake in the presence of . Experiments with 86Rb uptake indicated that K+ and acted in a similar manner to inhibit apparent NKCC-1 activity, likely by competition with 86Rb thereby reducing 86Rb uptake. This is supported by a study involving NKCC-2 from rabbit kidney thick ascending limb in which bumetanide-sensitive 86Rb uptake was inhibited by , whereas 22Na uptake was not (15). Thus to accurately address Cl- entry across the basolateral membrane via NKCC-1, bumetanide-sensitive 36Cl uptake was used.

Our experiments demonstrate (Fig. 6) the novel finding that exhibits anomalous mole fraction behavior on NKCC-1. Although anomalous mole fraction effects have been most strongly associated with multipore single-file ion channels (9), they may also occur due to the electrostatic consequences of localized ion specific binding (25). The anomalous behavior may be a reflection of the anomalous behavior associated with inhibition of Cl- secretion. That is a change in intracellular Cl- and or K+ at or near the cytoplasmic side of NKCC-1. Increases in intracellular Cl- or in extracellular K+ will reduce NKCC-1 activity (21). In addition, Gillen and Forbush (10) showed a steep relationship between intracellular Cl- concentration ([Cl-]i) and NKCC-1 activity within the physiological range of [Cl-]i. Reduction in 36Cl uptake seen on increasing extracellular K+ from 5 to 10 mM is consistent with a [K+]out-induced reduction in NKCC-1 activity. Interestingly, uptake in 10 mM is higher than in equal molar K+, which is not the case for the effect on basolateral K+ current (Fig. 6 vs. Fig. 4), thus suggesting more than a simple indirect effect of / mix on NKCC-1. Moreover, if this were the case, one would expect that the bumetanide-sensitive 86Rb uptake would match that of the 36Cl uptake, which is not the case. One interesting possibility is that the / interaction might favor the exchange mode of NKCC-1. NKCC-1 is known to exhibit K+/K+ exchange activity under which no Cl- flux occurs (22), thus one could get a 86Rb uptake which exceeded that of 36Cl uptake by 86Rb/K exchange. Indeed, for the mixed condition of 2 mM K+ and 8 mM , 86Rb uptake was reduced 47%, whereas 36Cl uptake was reduced 84% with respect to 5 mM K+.

effect on Na+-K+-ATPase. No anomalous mole fraction effect was observed with an , K+ mix on Na+-K+-ATPase activity as measured by ouabain-sensitive current. However, there does appear to be a slight increase in pump current (~25%) with pure vs. pure K+, which implies that the Na+-K+-ATPase in these cells may prefer . substitution for K+, with equal affinity, on Na+-K+-ATPase from crab gill membrane vesicles has been demonstrated (33). Similarly, was shown to substitute for K+ on Na+-K+-ATPase from rat proximal tubules (17). Early studies of Na+-K+-ATPase activity, however, have demonstrated enhanced activity with (27, 31). It is reasonable to assume on the basis of the slight increase in pump current with and the ouabain-insensitive 86Rb uptake (predominately due to the pump), that under conditions in which the extracellular [] approaches that of [K+], a significant amount of would be pumped into the cell by Na+-K+-ATPase.

effect on TER. Longer-term exposure (refer to Fig. 7) leads to an increase in TER. Although, on first thought, this might suggest an increase in basolateral membrane resistance due to block of basolateral K+ channels, a number of observations suggest otherwise. K+-like ions that produce a greater inhibition than do not substantially affect TER (Fig. 8), and the long-term effect of is not readily reversible. Indeed, on washout of , TER increased further (Fig. 7). Because does not affect the apical Cl- conductance (26), an -induced increase in the apical component of TER is unlikely. The -induced increase in TER is thus likely to involve an increase in the paracellular component of TER. Interestingly, pHi acidifies with long-term as well as on washout of , thus the increase in TER might involve pHi changes. effect on TER is distinct from that of the effect on transepithelial current in that the effect on TER does not show anomalous mole fraction behavior.

effect on transepithelial transport, implications. effects on both K+ conductance and NKCC-1 shows anomalous mole fraction behavior, but no such behavior was observed on Na+-K+-ATPase activity or for the effect on TER. Figure 9 provides a qualitative summary of these effects relative to 10 mM basolateral K+ for comparison. inhibition of transepithelial current is most closely correlated with the effects of on the basolateral K+ conductance. In the mix of K+ with , inhibition of NKCC-1 activity correlated well with the inhibition of Cl- secretion. However, under conditions of pure , NKCC-1 activity is elevated, whereas total transepithelial current is slightly inhibited. Na+-K+-ATPase activity is also elevated relative to that seen with 10 mM basolateral K+.



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Fig. 9. Relative anomalous mole fraction effect on transepithelial current compared with individual basolateral transport processes. Data are extracted from Figs. 3, 4, 8, and 9.

 

It is noteworthy that the maximal inhibition relative to 10 mM basolateral K+ seen on any one component of Cl- secretion is 44%. Thus Cl- secretion can be supported by basolateral ; that is, NKCC-1 can function in a -- mode and Na+-K+-ATPase in an --ATPase mode in these cells. Indeed, the data indicate that under conditions of nominally free basolateral K+ with , both NKCC-1 and Na+-K+-ATPase activities are elevated, which would further support uptake of . Given an apical exit pathway for , we hypothesize that secretion is likely to occur in colonic crypts. Unfortunately, T84 cells do not have the ability to secrete K+ presumably due to the lack of a functional apical K+ channel and thus are limited to Cl- secretory studies. However, in native crypts, under conditions in which cAMP is only slightly elevated, K+ secretion occurs and predominates. This is due to the preferential activation of apical vs. basolateral K+ channels (19) and basolateral vs. apical Cl- channels (20). The notion that secretion can be driven by NKCC and Na+-K+-ATPase with an opposing exit pathway is supported by work involving renal cells (15, 35). In fact, Kinne et al. (15) estimated that it is energetically possible for NKCC and Na+-K+-ATPase under physiological conditions to generate a / ratio of ~2,000!

Mayol et al. (24) reported a less significant effect of basolateral on transepithelial short-circuit current in native rat and human colon. However, K+ secretion was not isolated from Cl- secretion. Some argument was made indicating that Cl- was the predominant current carrier. Indeed, the concentrations of forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate used would result in the predominant stimulation of Cl- secretion. However, also noted was an active apical K+ conductance. Because the relative contribution of K+ and Cl- to transepithelial current was not determined, it is a possibility that basolateral is affecting the balance between secretory modes (K+ vs. Cl- secretion) without having a significant effect on the overall current magnitude.

As reported by Mayol et al. (24), apical also inhibited cAMP-stimulated Cl- secretion via a process thought to involve apical K+ channels. In light of the high luminal concentration of in the colon, this raises an interesting question as to whether apical or basolateral has a more significant impact on Cl- secretion in vivo. Lack of an apical K+ conductance in T84 cells limits their use in this regard. Although apical does not appreciably inhibit Cl- secretion in T84 cells under HEPES-buffered conditions, under -buffered conditions, inhibition of secretion is observed despite the lack of an apical K+ conductance (unpublished observation). The aim of the present study was to address the mechanisms of the basolateral effects of in T84. We chose to use basolateral concentrations that might be expected to occur in vivo. The range over which serosal levels near the crypt basolateral membrane may vary in normal and pathological states is currently unknown. Clearly, additional studies are warranted to determine the overall impact of on Cl- secretion as well as K+ (and possibly ) secretion in the native colon.

The anomalous mole fraction effect in a / mix in the present findings suggests a relevance of physiological importance and not merely a biophysical phenomena. Thus the colon may be self regulating regarding net Cl- and () secretion on the basis of the [K+]o-to- ratio at the basolateral membrane of the crypt cells. It is most likely that the basolateral level of is most important, but as indicated in this report, cannot be viewed independently of [K+]. Data presented support the idea that T84 cells are sensitive not only to the basolateral level of but also to the ratio of to K+.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-051630 (to J. B. Matthews) and by the Epithelial Pathobiology Group.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. T. Worrell, Epithelial Pathobiology Group, Dept. of Surgery and Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati, Cincinnati, OH 45219 (E-mail: Roger.Worrell{at}uc.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.

1 Lack of inhibition with NMDG at 5-min exposure may relate to its larger size and access restriction to the basolateral membrane or an inability to interact with the K+ channel. Thus any residual K+ could support transport. At later time points, current with 10 mM NMDG is reduced related to that in K+. Back


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