The Basolateral NHE1 Na (cid:1) /H (cid:1) Exchanger Regulates Transepithelial HCO 3 (cid:2) Absorption through Actin Cytoskeleton Remodeling in Renal Thick Ascending Limb*

In the renal medullary thick ascending limb (MTAL), inhibiting the basolateral NHE1 Na (cid:1) /H (cid:1) exchanger with amiloride or nerve growth factor (NGF) results secondarily in inhibition of the apical NHE3 Na (cid:1) /H (cid:1) exchanger, thereby decreasing transepithelial HCO 3 (cid:2) absorption. MTALs from rats were studied by in vitro microperfusion to identify the mechanism underlying cross-talk between the two exchangers. The basolateral addition of 10 (cid:3) M amiloride or 0.7 n M NGF decreased HCO 3 (cid:2) absorption by 27–32%. Jasplakinolide, which stabilizes F-actin, or latrunculin B, which disrupts F-actin, decreased basal HCO 3 (cid:2) absorption by 30% and prevented the inhibition by amiloride or NGF. Jasplakinolide had no effect on HCO 3 (cid:2) absorption in tubules bathed with amiloride or a Na (cid:1) -free bath to inhibit NHE1. Jasplakinolide and latrunculin B did not prevent inhibition of HCO 3 (cid:2) absorption by vasopressin or stimulation by hyposmolality, factors that regulate HCO 3 (cid:2) absorption through primary effects on apical Na (cid:1) /H (cid:1) exchange. These that 45 The supernatants were then collected, and the pellets were resus- pended in a volume equal to that of the supernatant. Proteins in the Triton X-100-soluble (supernatant) and -insoluble (pellet) fractions were treated with Laemmli sample buffer, separated by SDS-PAGE on 12% gels, and transferred to polyvinylidene difluoride membranes (34). The membranes were blocked with TBS, 0.1% Tween 20, and 1% bovine serum albumin and exposed to monoclonal anti-actin antibody (AC-40; 1:1000; Sigma) for 2 h atroom temperature. Immunoreactive bands were detected by ECL (Amersham Biosciences) after application of horseradish peroxidase-conjugated goat anti-mouse secondary antibody. Relative band intensities were quantified by densitometry (34), and the results are expressed as the percentage of total cell actin present in the detergent-soluble and -insoluble fractions. Equal amounts of total cellular actin in different experimental conditions were verified by immunoblotting unextracted tissue lysates. Statistical Analysis— Differences between means were evaluated using Student’s t test for paired data or analysis of variance with New- man-Keul’s multiple range test, as appropriate. p (cid:6) 0.05 was considered statistically significant. absorption by AVP and hyposmolality is preserved in the presence of the F-actin modifier. These results that the effect of jasplakinolide to block 3 (cid:2) transport regulation is specific for factors (bath amiloride and NGF) that act via the basolateral NHE1 Na (cid:1) /H (cid:1) exchanger.

Na ϩ /H ϩ exchangers (NHEs) 1 are transmembrane proteins that mediate the electroneutral exchange of Na ϩ for H ϩ (1,2). At least eight NHE isoforms (NHE1 to -8) have been identified in mammalian cells (1)(2)(3)(4)(5). These differ in their tissue distribution, membrane localization, inhibitor sensitivity, and physiological functions. NHE1 is expressed ubiquitously in the plasma membrane of nonpolarized cells and in the basolateral membrane of epithelial cells, where it mediates housekeeping functions such as regulation of intracellular pH and cell volume (1)(2)(3)6). Activation of NHE1 also is involved in other important cellular processes, including growth, migration, survival, and adhesion (2,6). In contrast, other exchangers (NHE2 to -5) exhibit a more restricted tissue distribution (1,2,7). In particular, NHE3 is localized selectively to the apical membrane of epithelial cells in the kidney and gastrointestinal tract, where it mediates the transepithelial absorption of NaCl and/or NaHCO 3 (1,3,7,(8)(9)(10)(11)(12). Regulation of NHE3 activity in the kidney is important for the maintenance of acid-base balance, Na ϩ balance and extracellular fluid volume, and blood pressure.
The medullary thick ascending limb (MTAL) of the mammalian kidney participates in acid-base homeostasis by reabsorbing most of the filtered HCO 3 Ϫ not reabsorbed by the proximal tubule (12,13). HCO 3 Ϫ absorption in the MTAL depends on luminal H ϩ secretion mediated by the apical membrane NHE3 Na ϩ /H ϩ exchanger (8,9,(13)(14)(15). The MTAL also expresses basolateral NHE1 (16 -18), and we have recently identified a novel role for this exchanger in transepithelial HCO 3 Ϫ absorption. Inhibition of basolateral Na ϩ /H ϩ exchange with either amiloride or nerve growth factor (NGF) results secondarily in inhibition of apical Na ϩ /H ϩ exchange, thereby decreasing HCO 3 Ϫ absorption (19,20). NHE1 was identified conclusively as the basolateral exchanger responsible for this regulation based on the observations that basal HCO 3 Ϫ absorption is reduced, and inhibition of HCO 3 Ϫ absorption by basolateral amiloride and NGF is eliminated, in MTALs from NHE1 null mutant mice (18). The control of HCO 3 Ϫ absorption in the MTAL thus involves cross-talk between the basolateral and apical membrane Na ϩ /H ϩ exchangers, whereby basolateral NHE1 enhances the activity of apical NHE3 (18 -20). The effect of NHE1 to increase apical NHE3 activity cannot be explained by a change in the net driving force for the apical exchanger and thus appears to be mediated through a signal transduction pathway (19,20). However, the identity of this signaling pathway has remained undefined.
The cytoskeleton plays a role in the targeting, anchoring, and regulation of a variety of ion transport proteins (21). Recent work has identified important regulatory interactions between the actin cytoskeleton and NHEs. NHE1 binds to actin filaments and anchors the actin cytoskeleton to the plasma membrane (6,21). NHE1 is also a component of signaling pathways that regulate several cytoskeleton-dependent processes, including cell adhesion and motility, and selective inhibition of NHE1 has been shown to impair actin filament assembly in cell lines (6,(22)(23)(24). Thus, NHE1 is involved in regulating the assembly and organization of the actin cytoskeletal network. The epithelial NHE3 Na ϩ /H ϩ exchanger is linked indirectly to the cytoskeleton via actin-binding adapter proteins such as ezrin (7,25,26), and cytoskeletal interactions play a role in the regulation of NHE3 by factors such as cAMP and endothelin (26 -29). In an antiporter-deficient fibroblast cell line transfected with NHE3, either stabilizing or depolymerizing actin filaments markedly inhibited NHE3 by decreasing its intrinsic activity (25). When considered in the context of epithelial cells such as the MTAL that express NHE1 in the basolateral membrane and NHE3 in the apical membrane, the above findings raise the possibility that the cytoskeleton could mediate crosstalk between the two exchangers, whereby NHE1 could induce changes in the actin network that in turn modulate NHE3.
The purpose of the present experiments was to investigate the role of the actin cytoskeleton in transepithelial HCO 3 Ϫ absorption in the MTAL and to determine whether the cytoskeleton mediates the regulatory interaction between the basolateral NHE1 and apical NHE3 Na ϩ /H ϩ exchangers. The results demonstrate that NHE1 regulates the organization of the actin cytoskeleton in the MTAL and that actin remodeling is involved in mediating NHE1-induced regulation of apical NHE3 and HCO 3 Ϫ absorption.

Tubule Perfusion and Measurement of Net HCO 3
Ϫ Absorption-MTALs from male Sprague-Dawley rats (50 -100-g body weight; Taconic, Germantown, NY) were isolated and perfused in vitro as described (19,30). Tubules were dissected from the inner stripe of the outer medulla at 10°C in control bath solution (see below), transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipettes for perfusion at 37°C. In most experiments, the tubules were perfused and bathed in control solution that contained 146 mM Na ϩ , 4 mM K ϩ , 122 mM Cl Ϫ , 25 mM HCO 3 Ϫ , 2.0 mM Ca 2ϩ , 1.5 mM Mg 2ϩ , 2.0 mM phosphate, 1.2 mM SO 4 2Ϫ , 1.0 mM citrate, 2.0 mM lactate, and 5.5 mM glucose (osmolality ϭ 295 mosmol/kg H 2 O). In one series of experiments (Fig. 4B), Na ϩ in the bath solution was replaced completely with N-methyl-D-glucammonium (19,20). Hyposmotic solutions (245 mosmol/kg H 2 O) (Figs. 3B and 6B) were produced by removing 25 mM NaCl from the control solution or by removing 50 mM mannitol from control solution in which 50 mM mannitol replaced 25 mM NaCl (15). Bath solutions contained 0.2 g/100 ml fatty acid-free bovine albumin. All solutions were equilibrated with 95% O 2 , 5% CO 2 and were pH 7.45 at 37°C. Experimental agents were added to the bath solutions as described under "Results." Jasplakinolide and latrunculin B were prepared as stock solutions in dimethyl sulfoxide and diluted into bath solutions to final concentrations given under "Results." Equal concentrations of vehicle were added to control solutions. Solutions containing other experimental agents were prepared as described (15,19,20).
The protocol for study of transepithelial HCO 3 Ϫ absorption was as described (15,19,30). In most experiments, tubules were equilibrated for 20 -30 min at 37°C in the initial perfusion and bath solutions, and the luminal flow rate (normalized per unit of tubule length) was adjusted to 1.6 -2.0 nl/min/mm. 1-5 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 5-10 min after an experimental agent was added to or removed from the bath solution. In some experiments, longer treatment periods were used, as described under "Results." The absolute rate of HCO 3 Ϫ absorption (JHCO 3 Ϫ , pmol/min/mm) was calculated from the luminal flow rate and the difference between total CO 2 concentrations measured in perfused and collected fluids (30). An average HCO 3 Ϫ absorption rate was calculated for each period studied in a given tubule. When repeat measurements were made at the beginning and end of an experiment (initial and recovery periods), the values were averaged. Single tubule values are presented in the figures. Means Ϯ S.E. (n ϭ number of tubules) are presented under "Results".
Phalloidin Staining and Fluorescence Microscopy-MTALs were microdissected and mounted on Cell-Tak-coated coverslips at 4°C. The tubules were then incubated in a flowing bath at 37°C using the same solutions and protocols as in HCO 3 Ϫ transport experiments (see "Results"). Following incubation, the tubules were washed with phosphatebuffered saline (PBS) and fixed for 15 min at room temperature with 4% paraformaldehyde in PBS. After washing in PBS, the tubules were permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. The tubules were then blocked with PBS plus 1% bovine serum albumin for 20 min at room temperature, washed, and incubated with Alexa 488-conjugated phalloidin (Molecular Probes, Inc., Eugene, OR) in blocking solution (1:200) for 1 h at room temperature to label F-actin. After staining, the tubules were washed and placed in fresh PBS for fluorescence imaging.
Fluorescence staining was examined using a Zeiss confocal microscope (LSM 510 META) equipped with a ϫ63 oil immersion objective. The tubules were imaged longitudinally, and optical sections were obtained with a step displacement of Ͻ0.4 m. Total fluorescence intensity of phalloidin staining normalized for tubule volume was quantified after background subtraction by integrating pixel intensities of tubule images over the total number of optical sections using Metamorph software (Universal Imaging). Three-dimensional images were constructed using Metamorph. For individual experiments, two to four tubules from the same kidney were imaged for each experimental condition, and the results were pooled to obtain an average value for each condition. To compare different experimental conditions, tubules in individual experiments were fixed and stained identically and imaged in a single session under identical conditions of illumination, gain, and exposure time. The fluorescence intensity for treatment groups was expressed as a percentage of the control value measured in the same experiment. Mean values for repeat experiments were used for statistical analysis.
Actin Detergent Solubility-The relative amounts of actin in Triton X-100-soluble and -insoluble fractions were determined using established protocols (25,31,32) and a previously described inner stripe tissue preparation (33,34). In brief, thin strips of tissue were dissected at 4°C from the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs. The strips were divided into 2-4 samples of equal amount and then incubated in vitro at 37°C in the same solutions used for HCO 3 Ϫ transport experiments (33,34). The specific protocols for incubation are given under "Results." After incubation, the tissue samples were homogenized in ice-cold buffer (150 mM NaCl, 20 mM HEPES, pH 7.4) and then incubated for 3 h at 4°C after the addition of an equal volume of Triton extraction buffer (final composition 5 mM imidazole, 37.5 mM KCl, 2.5 mM MgCl 2 , 0.5 mM EGTA, 1% Triton X-100, and protease inhibitor mixture (Sigma), titrated to pH 7.2) (32). The lysates were centrifuged at 100,000 ϫ g at 4°C for 45 min. The supernatants were then collected, and the pellets were resuspended in a volume equal to that of the supernatant. Proteins in the Triton X-100-soluble (supernatant) and -insoluble (pellet) fractions were treated with Laemmli sample buffer, separated by SDS-PAGE on 12% gels, and transferred to polyvinylidene difluoride membranes (34). The membranes were blocked with TBS, 0.1% Tween 20, and 1% bovine serum albumin and exposed to monoclonal anti-actin antibody (AC-40; 1:1000; Sigma) for 2 h at room temperature. Immunoreactive bands were detected by ECL (Amersham Biosciences) after application of horseradish peroxidase-conjugated goat anti-mouse secondary antibody. Relative band intensities were quantified by densitometry (34), and the results are expressed as the percentage of total cell actin present in the detergent-soluble and -insoluble fractions. Equal amounts of total cellular actin in different experimental conditions were verified by immunoblotting unextracted tissue lysates.
Statistical Analysis-Differences between means were evaluated using Student's t test for paired data or analysis of variance with Newman-Keul's multiple range test, as appropriate. p Ͻ 0.05 was considered statistically significant.
Effects of Jasplakinolide on HCO 3 Ϫ Absorption-To investigate the role of the cytoskeleton in HCO 3 Ϫ absorption, we examined the effects of jasplakinolide, a membrane-permeant cyclic peptide that binds and stabilizes actin filaments and promotes actin polymerization (35). Adding 0.05 M jasplakinolide to the bath decreased HCO 3 Ϫ absorption by 32%, from 14.8 Ϯ 0.3 to 10.0 Ϯ 0.4 pmol/min/mm (p Ͻ 0.001) ( Fig. 2A). This inhibition was observed within 15-20 min after the addi-tion of jasplakinolide and was reversible. In MTAL bathed with 0.05 M jasplakinolide for 25-30 min, the addition of either 10 M amiloride or 0.7 nM NGF to the bath had no effect on HCO 3 Ϫ absorption (Fig. 2, B and C). These results suggest that the basal rate of HCO 3 Ϫ absorption and the inhibition of HCO 3 Ϫ absorption by bath amiloride and NGF depend on the state of the actin cytoskeleton.
To assess the specificity of jasplakinolide's actions on the regulation of HCO 3 Ϫ absorption, we examined the effects of vasopressin (AVP) and hyposmolality. These factors were studied because AVP inhibits and hyposmolality stimulates HCO 3 Ϫ absorption in the MTAL through primary effects on the apical Na ϩ /H ϩ exchanger (13,15,30,36), contrary to bath amiloride and NGF, which act primarily on the basolateral Na ϩ /H ϩ exchanger. In MTAL bathed with 0.05 M jasplakinolide for 25-50 min, adding 10 Ϫ10 M AVP to the bath decreased HCO 3 Ϫ absorption by 43%, from 11.4 Ϯ 0.5 to 6.5 Ϯ 0.9 pmol/min/mm (p Ͻ 0.001; Fig. 3A), and hyposmolality increased HCO 3 Ϫ absorption from 10.5 Ϯ 0.7 to 16.0 Ϯ 1.4 pmol/min/mm (p Ͻ 0.025; Fig. 3B). These effects are similar to those observed in the absence of jasplakinolide (15,30). Thus, the regulation of HCO 3 Ϫ absorption by AVP and hyposmolality is preserved in the presence of the F-actin modifier. These results demonstrate that the effect of jasplakinolide to block HCO 3 Ϫ transport regulation is specific for factors (bath amiloride and NGF) that act via the basolateral NHE1 Na ϩ /H ϩ exchanger.
The preceding results suggest that jasplakinolide and factors that inhibit NHE1 may decrease HCO 3 Ϫ absorption through a common mechanism, namely modification of the actin cytoskeleton. If this hypothesis is correct, then the effect of jasplakinolide to inhibit HCO 3 Ϫ absorption should be diminished under conditions in which basolateral Na ϩ /H ϩ exchange is inhibited. To test this, we examined the effect of jasplakinolide in the presence of bath amiloride or in the absence of bath Na ϩ , two conditions that inhibit basolateral Na ϩ /H ϩ exchange (19,20). In MTAL bathed with 10 M amiloride, adding 0.05 M jasplakinolide to the bath decreased HCO 3 Ϫ absorption only by 11%, from 11.0 Ϯ 0.6 to 9.7 Ϯ 0.8 pmol/min/mm (p Ͻ 0.005) (Fig. 4A). When compared with the effect of jasplakinolide in control experiments ( Fig. 2A), 10 M bath amiloride reduced the inhibition by jasplakinolide by 70% (p Ͻ 0.05). In tubules studied in a Na ϩ -free bath, adding 0.05 M jasplakinolide to the bath had no effect on HCO 3 Ϫ absorption (14.4 Ϯ 0.8 pmol/min/ mm, 0 Na ϩ bath versus 14.3 Ϯ 1.0 pmol/min/mm, 0 Na ϩ bath ϩ jasplakinolide; p ϭ not significant) (Fig. 4B). Thus, the effect of jasplakinolide to inhibit HCO 3 Ϫ absorption was virtually eliminated under two different conditions in which basolateral Na ϩ /H ϩ exchange was inhibited. These results support the view that jasplakinolide and NHE1 regulate HCO 3 Ϫ absorption through a common mechanism involving modification of the actin cytoskeleton.
Effects of Latrunculin B on HCO 3 Ϫ Absorption-To investigate further the role of the actin cytoskeleton in HCO 3 Ϫ absorption, we examined the effects of latrunculin B. This compound sequesters monomeric actin and induces F-actin depolymerization (37), actions opposite to the F-actin-stabilizing effects of jasplakinolide. Adding 1 M latrunculin B to the bath decreased HCO 3 Ϫ absorption by 31%, from 14.9 Ϯ 0.8 to 10.3 Ϯ 0.9 pmol/min/mm (p Ͻ 0.005) (Fig. 5A). The inhibition by latrunculin B occurred after a time delay of 70 -80 min. This time course parallels the effect of latrunculin B on the actin cytoskeleton (see below). The HCO 3 Ϫ absorption rate returned to its initial control value within 30 -40 min after the removal of latrunculin B from the bath solution. In MTALs bathed with 1 M latrunculin B for 80 -100 min, adding 10 M amiloride or 0.7 nM NGF to the bath had no effect on HCO 3 Ϫ absorption (Fig. 5, B and C). Thus, either disrupting F-actin with latrunculin B or stabilizing F-actin with jasplakinolide prevents inhibition of HCO 3 Ϫ absorption by bath amiloride and NGF (see "Discussion"). These results support further the hypothesis that regulation of HCO 3 Ϫ absorption by bath amiloride and NGF involves the actin cytoskeleton.

Effects of Colchicine on HCO 3
Ϫ Absorption-To determine whether the regulation of HCO 3 Ϫ absorption via basolateral Na ϩ /H ϩ exchange involves cytoskeletal structures in addition to F-actin, we examined the effects of colchicine, an inhibitor of microtubule assembly. In MTAL bathed with 50 M colchicine for 70 -80 min, the addition of 10 M amiloride to the bath decreased HCO 3 Ϫ absorption by 38%, from 12.4 Ϯ 1.0 to 7.7 Ϯ 0.6 pmol/min/mm (p Ͻ 0.005) (Fig. 7). A similar or less severe colchicine treatment has been shown to block other microtubuledependent processes in renal cells (38 -40). Thus, these results suggest that the effect of bath amiloride to inhibit HCO 3 Ϫ absorption via basolateral Na ϩ /H ϩ exchange is not dependent on microtubules.
Effects of Inhibitors of NHE1 on the Actin Cytoskeleton-To examine more directly possible interactions between NHE1 and the cytoskeleton, F-actin was studied in microdissected MTALs by Alexa 448-phalloidin staining and confocal fluorescence microscopy (Fig. 8). Tubules were optically sectioned, three-dimensional images were constructed, and the fluorescence intensity of phalloidin staining normalized for tubule volume was quantified. MTALs exhibit F-actin staining along the inner surface of the plasma membranes (cortical actin), a diffuse F-actin network throughout the cytoplasm, and a dense annular bundle of actin filaments surrounding the apical cell pole (actin belt of the zonula adherens) (Fig. 8, A and C). Treatment with either 10 M amiloride or 0.7 nM NGF for 15 min decreased the intensity of fluorescence staining by 30% (p Ͻ 0.05; Fig. 8, A and B). This decrease is attributable to a decrease in cellular F-actin content rather than to a change in cell volume based on the following observations: 1) treatment with amiloride or NGF has no effect on cell volume (Metamorph analysis) (19,20); 2) amiloride and NGF cause a similar decrease in fluorescence intensity when normalized per mm of tubule length (indicating decreased F-actin per cell); and 3) decreased cellular F-actin is confirmed independently by measurement of actin detergent solubility (see below). At the cellular level, amiloride or NGF treatment decreased F-actin lining the basal cell surface, reduced cortical actin underlying the cell membranes, and decreased the cytoplasmic F-actin network (Fig. 8C). In contrast, treatment for 15 min with 10 Ϫ10 M AVP, which inhibits HCO 3 Ϫ absorption via a primary effect on the apical Na ϩ /H ϩ exchanger, had no effect on F-actin fluorescence intensity (Fig. 8, A and B). Thus, cytoskeletal remodeling was induced selectively by factors (amiloride and NGF) that inhibit HCO 3 Ϫ absorption through primary inhibition of basolateral NHE1. These results demonstrate that inhibitors of NHE1 induce reorganization of the cytoskeleton that involves a decrease in cellular F-actin.
Treatment with 1 M latrunculin B for 60 min decreased fluorescence intensity (Fig. 8, A and B), consistent with its action to depolymerize F-actin. Latrunculin B disrupted the cortical and cytoplasmic F-actin networks, with relative preservation of the annular F-actin bundle (Fig. 8A). Latrunculin B had no effect on fluorescence intensity at 15 min (not shown). Thus, the time course for latrunculin B-induced disruption of actin filaments parallels its effects on HCO 3 Ϫ absorption. Effects of Inhibitors of NHE1 on Actin Solubility-To examine further the interaction of NHE1 with the cytoskeleton, the relative amounts of actin in Triton X-100-soluble and -insoluble fractions were determined. Because actin filaments are resistant to mild detergent extraction, quantification of the fraction of Triton X-100-insoluble actin provides a measure of cellular F-actin content and the extent of actin polymerization (25,31,32). Inner stripe tissue was incubated in vitro using the same solutions and treatments as in HCO 3 Ϫ transport and fluores-  (15). JHCO 3 Ϫ , data points, lines, and p values are as in Fig. 1. Mean values are given under "Results."

FIG. 4. Conditions that inhibit basolateral Na ؉ /H ؉ exchange prevent inhibition of HCO 3
؊ absorption by jasplakinolide. MTALs were studied with 10 M amiloride in the bath (A) or in a Na ϩ -free bath (B), and then 0.05 M jasplakinolide was added to the bath. B, Na ϩ in the bath solution was replaced completely with Nmethyl-D-glucammonium; the lumen was perfused with control solution containing 146 mM Na ϩ (19, 20) (see "Experimental Procedures"). JHCO 3 Ϫ , data points, lines, and p values are as in Fig. 1. NS, not significant. Mean values are given under "Results." cence imaging experiments. In tissue incubated in control solution, 40% of cellular actin is in the soluble form, and 60% is in the insoluble (F-actin) form (Fig. 9, A and B). This distribution did not differ for control incubations of 15 or 60 min. Treatment with either 10 M amiloride or 0.7 nM NGF for 15 min reversed the distribution, increasing soluble actin from 40 to 60% and decreasing insoluble actin from 60 to 40% (p Ͻ 0.05; Fig. 9). As expected, treatment with 1 M latrunculin B for 60 min converted a large fraction of the actin to its soluble form. There was no difference in total cellular actin determined by immunoblotting between the different experimental conditions (not shown). These results agree closely with the results of the phalloidin labeling experiments and support further the conclusion that inhibitors of NHE1 induce reorganization of the cytoskeleton that involves a decrease in F-actin.
Further experiments examining actin detergent solubility were carried out to verify that the effect of jasplakinolide to block NHE1-mediated regulation of HCO 3 Ϫ absorption was related to an effect on the cytoskeleton. This approach was used because effects of jasplakinolide on F-actin content cannot be quantified by phalloidin labeling due to the fact that jasplakinolide binds to F-actin competitively with phalloidin (35).
Inner stripe tissue was incubated in control solution in the absence and presence of 0.05 M jasplakinolide for 15 min. The tissue was then either maintained in these solutions or treated with 10 M amiloride for an additional 15 min. Treatment with jasplakinolide alone did not alter actin detergent solubility. 2 However, jasplakinolide blocked completely the effects of amiloride to increase the Triton X-100-soluble fraction and to decrease the Triton X-100-insoluble fraction of cellular actin (Fig. 10, A and B). Thus, in the MTAL, jasplakinolide blocks both the effects of basolateral amiloride to induce F-actin remodeling and to inhibit HCO 3 Ϫ absorption.

DISCUSSION
Previously, we identified an important role for the basolateral NHE1 Na ϩ /H ϩ exchanger in regulation of transepithelial HCO 3 Ϫ absorption in the MTAL. This involves a novel and paradoxical mechanism whereby inhibition of basolateral NHE1 results secondarily in the inhibition of apical NHE3, thereby decreasing HCO 3 Ϫ absorption (18 -20). These results provided the first evidence of a regulatory role for NHE1 in transepithelial transport in the kidney. In the present study, we examined the mechanism of cross-talk between the two Na ϩ /H ϩ exchangers. The results show that NHE1 regulates NHE3 and HCO 3 Ϫ absorption by controlling the organization of the actin cytoskeleton. Our data support a model (Fig. 11) in which inhibition of basolateral NHE1 induces a decrease in polymerized actin that inhibits the apical NHE3 Na ϩ /H ϩ exchanger.
The conclusion that NHE1 regulates NHE3 via the cytoskeleton is supported by functional and biochemical evidence. This includes the following: 1) the specific actin modifiers jasplakinolide and latrunculin B decrease basal HCO 3 Ϫ absorption by an amount similar to that observed with inhibition of NHE1; 2) jasplakinolide or latrunculin B eliminates completely the inhibition of HCO 3 Ϫ absorption by bath amiloride or NGF, con-2 Jasplakinolide has been shown to increase the fraction of Triton X-100-insoluble (polymerized) actin in some systems (25,31,32). However, these studies used much higher jasplakinolide concentrations (1-10 M) and longer exposure times (45-120 min) than those used in our experiments (0.05 M for 15-30 min). We used a relatively low dose of jasplakinolide that is above the IC 50 for F-actin binding (35), induced a rapid, stable, and reversible inhibition of HCO 3 Ϫ absorption, and blocked amiloride-induced F-actin rearrangement. The stabilizing effect of jasplakinolide on F-actin in our experiments did not result in a measurable change in actin Triton X solubility. Ϫ , data points, lines, and p value are as in Fig. 1. Mean values are given under "Results." sistent with actin modifiers and NHE1 inhibitors acting via a common mechanism; 3) jasplakinolide has no effect on HCO 3 Ϫ absorption under conditions in which NHE1 is inhibited; 4) the actin modifiers block selectively the inhibition of HCO 3 Ϫ absorption by factors that act via NHE1 (amiloride and NGF) but have no effect on regulation by factors that act primarily on NHE3 (AVP and hyposmolality); 5) inhibitors of NHE1 induce cytoskeletal remodeling that involves a decrease in polymerized actin, as verified using two independent methods of measurement (fluorescence microscopy and detergent solubility); 6) similar cytoskeletal changes are induced by two different NHE1 inhibitors, which argues against amiloride and NGF having nonspecific cytoskeletal effects; 7) AVP, which inhibits HCO 3 Ϫ absorption by an amount similar to that observed with NGF and amiloride but does not act through NHE1, had no effect on cellular F-actin content; and 8) jasplakinolide prevents amiloride-induced transport inhibition by blocking cytoskeletal remodeling.
Taken together, these findings indicate that reorganization of the cytoskeleton is necessary for NHE1-induced regulation of NHE3 and HCO 3 Ϫ absorption. It is noteworthy that basal HCO 3 Ϫ absorption is decreased, and NHE1-induced regulation is prevented, by agents that either disrupt or stabilize actin filaments. This suggests that the NHE1-induced regulation of NHE3 depends on dynamic actin rearrangement and not on a static actin network. These findings are consistent with previous results in AP-1 cells demonstrating that either disruption of F-actin with latrunculin B or stabilization of F-actin with jasplakinolide inhibits NHE3 activity (25). Our results show that inhibiting NHE1 in the MTAL shifts the steady state for actin assembly toward F-actin depolymerization, which results in inhibition of NHE3.
NHE1 was identified unambiguously as the basolateral exchanger that regulates apical NHE3 and HCO 3 Ϫ absorption through studies of MTALs from wild-type and NHE1 knockout mice (18). Of significance, genetic ablation of NHE1 produces regulatory changes in HCO 3 Ϫ absorption that are strikingly similar to those observed with cytoskeletal modifiers. Both cause a decrease in the basal rate of HCO 3 Ϫ absorption and eliminate inhibition of HCO 3 Ϫ absorption by basolateral amiloride or NGF but do not prevent inhibition by AVP or stimulation by hyposmolality (18) (Figs. 2-6). The fact that NHE1 knockout and actin-modifying agents have virtually identical effects on HCO 3 Ϫ absorption in five independent conditions (basal, amiloride, NGF, AVP, and hyposmolality) provides further strong support for the conclusion that NHE1 and the cytoskeleton are components of a common regulatory mechanism.
Several lines of evidence indicate that the effects of jasplakinolide and latrunculin B on HCO 3 Ϫ absorption are attributable to their effects on the actin cytoskeleton and not to nonspecific metabolic or cytotoxic effects. First, the inhibition of basal HCO 3 Ϫ absorption by both agents is stable and reversible and is similar in magnitude to that induced by amiloride and NGF. Second, jasplakinolide and latrunculin B inhibit HCO 3 Ϫ absorption with different time courses, but for each agent the time course for inhibition of HCO 3 Ϫ absorption parallels its effect on the cytoskeleton. The different time courses argue against an effect of these agents to bind and directly inhibit NHE1, consistent with previous results (25). Third, jasplakinolide has no effect on HCO 3 Ϫ absorption under two different conditions in which basolateral NHE1 is inhibited, arguing against nonspecific effects on ion transport pathways. Fourth, disruption of microtubules does not prevent NHE1induced transport regulation, indicating that there is specificity in the cytoskeletal signaling pathway for actin filaments. Fifth, the regulation of HCO 3 Ϫ absorption by AVP and hyposmolality is unaffected by jasplakinolide or latrunculin B, indicating that these agents act selectively to block regulation mediated through NHE1. In the MTAL, AVP decreases HCO 3 Ϫ absorption by inhibiting apical NHE3 via cAMP (13,30,36); hyposmolality increases HCO 3 Ϫ absorption by stimulating apical NHE3 via phosphatidylinositol 3-kinase (33). Our results suggest that these regulatory pathways are intact in MTAL cells in which the actin cytoskeleton is modified by latrunculin B or jasplakinolide or by amiloride or NGF (Figs. 3 and 6) (19,20). The normal response to AVP under these conditions is somewhat surprising in view of previous studies indicating that cAMP/protein kinase A-dependent inhibition of NHE3 involves cAMP-induced cytoskeletal reorganization (28) and a NHERF-ezrin signaling complex that links NHE3 to actin filaments (7,26). It is possible that inhibition of NHE3 by cAMP/ protein kinase A in the MTAL occurs independently of cytoskeletal interactions or that the cystoskeletal changes that underlie NHE1-induced regulation of NHE3 do not impair cytoskeletal interactions necessary for inhibition by cAMP. Further work on how cAMP inhibits NHE3 in the MTAL will be required to address this issue.
There is precedent for NHE1-induced control of the actin cytoskeleton in nonpolarized cells. Barber and co-workers have demonstrated in fibroblasts that NHE1 functions as a cytoskeleton anchoring protein and regulates a number of cytoskeletondependent processes, including adhesion, motility, and actin filament organization and assembly (6,(21)(22)(23)(24). This regulation is mediated in part through ERM proteins, which bind NHE1 and form a structural link between NHE1 and actin filaments (24). An unanswered question involves the relationship between the ion transport and F-actin-anchoring functions of NHE1. Pharmacological inhibition of NHE1 was found to impair actin filament assembly and cell adhesion in response to various stimuli in fibroblasts (21)(22)(23). In the MTAL, we found that inhibiting NHE1 activity by two different methods (amiloride and NGF) resulted in cytoskeletal changes involving decreased F-actin. These results suggest that NHE1-induced cytoskeletal remodeling may be related to or dependent upon changes in NHE1 transport activity. On the other hand, NHE1dependent formation of actin stress fibers and focal adhesion assembly were preserved in fibroblasts expressing an NHE1 mutant that bound F-actin but was devoid of transport activity (24). Also, disruption of the cytoskeleton with cytochalasin B had no effect on NHE1 activity in AP-1 cells (25), further supporting a dissociation between NHE1 transport activity and cytoskeletal organization. It is possible, therefore, that conformational changes in NHE1 related to inhibition of transport rather than changes in ion translocation itself are important for mediating NHE1-induced cytoskeletal changes (21)(22)(23)(24). Alternatively, the cytoskeletal anchoring and ion transport functions of NHE1 may act cooperatively to regulate actin-dependent cell processes (42). Further studies to examine the relationship between the cytoskeleton and NHE1 transport activity and to identify accessory proteins that may mediate NHE1-cytoskeleton interactions are needed to address these questions in the MTAL.
NHE1-induced reorganization of the cytoskeleton regulates apical NHE3 activity in the MTAL. A role for the actin cytoskeleton in modulating NHE3 activity has been identified previously. In NHE3-transfected AP-1 cells, either latrunculin B or jasplakinolide decreased NHE3 activity (25), consistent with results of the present study showing that these agents inhibited basal HCO 3 Ϫ absorption in the MTAL. The cytoskeletal regulation of NHE3 involves a region of its cytoplasmic tail that may interact with NHERF and ezrin (25,27). However, the mechanism by which the cytoskeleton regulates NHE3 activity remains unclear. In AP-1 cells, cytoskeletal changes inhibit NHE3 through effects on its intrinsic activity with no effect on the number of cell surface transporters, arguing against an effect on NHE3 trafficking (25,27). In contrast, a role for the cytoskeleton in mediating endothelin-induced exocytotic insertion of NHE3 was suggested in OKP cells (29), and cytochalasin D was found to alter the amount of surface biotinylated NHE3 in cultured mouse kidney cells (43). Thus, the mechanisms involved in cytoskeleton-induced regulation of NHE3 are likely to be complex and may depend on the experimental conditions, physiological stimulus, or cell type. Studies examining NHE3 activity, kinetics, and subcellular localization will be required to understand how NHE1-induced cytoskeletal changes regulate NHE3 in the MTAL. Although inhibition of NHE3 is the primary mechanism responsible for the NHE1-induced decrease in HCO 3 Ϫ absorption in the MTAL (19,20), our results do not rule out the possibility that cytoskeleton remodeling also may influence basolateral HCO 3 Ϫ efflux pathways. In addition to NHE3, other ion transporters critical for epithelial function are regulated through their interactions with the cytoskeleton. These include the epithelial Na ϩ channel ENaC, the ROMK K ϩ channel, the Na ϩ -K ϩ -2Cl Ϫ cotransporter NKCC2, the cystic fibrosis transmembrane conductance regulator Cl Ϫ channel, and the AE1 Cl Ϫ /HCO 3 Ϫ exchanger (21, 39, 44 -48). NHE1 is expressed along with these transporters in a variety of epithelial tissues, including the renal proximal tubule, thick ascending limb, and collecting duct; the gastrointestinal tract; pancreatic ascinar cells; bile duct; and salivary gland. Our results thus raise the possibility that NHE1 acting via the cytoskeleton could influence a broad range of membrane transporters and epithelial functions, as it does NHE3 and HCO 3 Ϫ absorption in the MTAL. A loss of cross-talk between basolateral NHE1 and apical Na ϩ /H ϩ exchange has been suggested recently as a possible explanation for decreased NaCl Ϫ absorption in the MTAL is mediated by H ϩ secretion via apical NHE3 and efflux of HCO 3 Ϫ across the basolateral membrane via mechanisms not yet established. Inhibiting NHE1 with amiloride or NGF induces reorganization of F-actin that decreases NHE3 activity and HCO 3 Ϫ absorption. The cell model is modified from Ref. 18. ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. absorption by parotid gland cells from NHE1 knockout mice (49). An effect of NHE1 to control NHE3 or other transporters also may be relevant to pathophysiological conditions in which NHE1 expression and activity are altered. For example, increased NHE1 activity contributes to ischemic-reperfusion injury of myocardial cells, a process that involves regulatory interactions of NHE1 with other transporters such as Na ϩ / Ca 2ϩ exchange (50,51). Also, NHE1 activity is increased in a variety of cells and tissues in patients with essential hypertension and in hypertensive animal models (52,53), and transgenic mice overexpressing NHE1 exhibit salt-sensitive hypertension in association with renal Na ϩ retention (54). Our results suggest that an increase in basolateral NHE1 activity leading to secondary stimulation of apical NHE3 could promote renal Na ϩ retention and contribute to the pathophysiology of salt-sensitive hypertension.
In summary, our findings indicate that basolateral NHE1 controls the activity of apical NHE3 in the MTAL through actin cytoskeleton remodeling. Inhibition of NHE1 induces a decrease in polymerized actin that decreases apical NHE3 activity and transepithelial HCO 3 Ϫ absorption. Our results suggest that NHE1 could influence a broad range of ion transporters and epithelial transport functions by controlling the organization of the actin cytoskeleton.