Aldosterone inhibits apical NHE3 and HCO 3 - absorption via a nongenomic ERK-dep - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:34 |只看该作者 |倒序浏览 |打印

作者：Bruns A. Watts, III, Thampi George, and David W. Good作者单位：Departments of Medicine and Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas : F9 Z$ P6 j+ X; b' a

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      【摘要】
   Although aldosterone influences a variety of cellular processes through nongenomic mechanisms, the significance of nongenomic pathways for aldosterone-induced regulation of epithelial function is not understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO 3 - absorption in the medullary thick ascending limb (MTAL) through a nongenomic pathway. This inhibition is mediated through a direct cellular action of aldosterone to inhibit the apical membrane NHE3 Na /H exchanger. The present study was designed to identify the intracellular signaling pathway(s) responsible for this aldosterone-induced transport regulation. In rat MTALs perfused in vitro, addition of 1 nM aldosterone to the bath decreased HCO 3 - absorption by 30%. This inhibition was not mediated by cAMP/PKA and was not prevented by inhibitors of PKC or PI3-K, pertussis toxin, or rapamycin. The inhibition of HCO 3 - absorption by aldosterone was largely eliminated by the MEK/ERK inhibitors U-0126 and PD-98059. Aldosterone increased ERK activity 1.8-fold in microdissected MTALs. This ERK activation is rapid ( 5 min) and is blocked by U-0126 or PD-98059 but is unaffected by spironolactone or actinomycin D. Pretreatment with U-0126 to block ERK activation prevented the effect of aldosterone to inhibit apical NHE3. These data demonstrate that aldosterone inhibits NHE3 and HCO 3 - absorption in the MTAL through rapid activation of the ERK signaling pathway. The results identify NHE3 as a target for nongenomic regulation by aldosterone and establish a role for ERK in the acute regulation of NHE3 and its epithelial absorptive functions. 8 Z0 r  {4 G" Y6 \1 x' j! r
      【关键词】 Na/H exchange ERK/ sodium absorption acid secretion kidney
   NA / H EXCHANGERS (NHE) are a family of transmembrane proteins that mediate the electroneutral exchange of Na for H  . These exchangers are present in virtually all mammalian tissues and participate in a variety of vital cell functions, including intracellular pH and cell volume regulation, epithelial Na absorption, and cell growth. At least eight mammalian NHE isoforms have been identified (NHE1-NHE8) that differ in their tissue expression and membrane localization, regulation, and physiological functions ( 42, 44, 54, 64 ). NHE3 is expressed selectively in the apical membrane of epithelial cells in the kidney and gastrointestinal tract, where it plays a major role in mediating absorption of NaCl, NaHCO 3, and fluid ( 2, 3, 6, 16, 25, 35, 39, 42, 48, 54, 55, 60, 64 ). The regulation of NHE3 is crucial for the normal maintenance of Na balance and extracellular fluid volume, blood pressure, and acid-base homeostasis. Factors involved in the fine control of NHE3 include ANG II, endothelin, catecholamines, dopamine, and changes in osmolality ( 2, 39, 42, 60, 64 ).
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Aldosterone plays a major role in regulating systemic Na  , K  , and acid-base balance through its effects on renal electrolyte excretion. This regulation is mediated primarily through classical actions of aldosterone to stimulate Na absorption, K secretion, and H secretion by segments of the collecting duct through changes in gene expression and synthesis of new proteins ( 2, 34, 46 ). More recently, aldosterone has been found to induce rapid cellular effects that are not dependent on transcription or translation ( 9 ). These nongenomic effects are observed in a variety of nonpolarized and epithelial cells, and include the activation of signal transduction pathways and membrane transporters such as the epithelial sodium channel (ENaC), K channels, and the vacuolar H -ATPase ( 9, 26, 36, 63, 65 ). Aldosterone also induces rapid activation of Na /H exchange in multiple cell types, an effect attributed to stimulation of the NHE1 isoform expressed ubiquitously in the plasma membrane of nonpolarized cells and the basolateral membrane of epithelial cells ( 9, 11, 12, 26, 27, 36, 62 ). At present, the physiological significance of nongenomic pathways for aldosterone-induced regulation of epithelial transport functions is not understood.
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Recently, we demonstrated that aldosterone regulates the epithelial NHE3 Na /H exchanger through a nongenomic pathway. In the renal medullary thick ascending limb (MTAL), aldosterone rapidly inhibits apical NHE3, resulting in a decrease in transepithelial HCO 3 - absorption ( 21, 23 ). This inhibition is not dependent on transcription or translation and is not mediated through the classical mineralocorticoid receptor ( 21, 23 ). These studies identify NHE3 as a target for nongenomic regulation by aldosterone and demonstrate that aldosterone can modulate epithelial absorptive functions important for volume and acid-base homeostasis through direct regulation of this exchanger. At present, the intracellular signaling pathways through which aldosterone regulates NHE3 are unknown. However, aldosterone has been shown in nonpolarized cells and epithelial cell lines to induce rapid activation of several pathways known to influence NHE3, including cAMP and protein kinase A (PKA), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3-kinase), and G proteins ( 9, 11, 26, 33, 37, 62 ).4 {! ^, m* z5 `3 l0 M+ U

The purpose of the present study was to identify the signal transduction mechanism(s) involved in nongenomic regulation by aldosterone in the MTAL. The results demonstrate that aldosterone inhibits NHE3 and HCO 3 - absorption through rapid activation of the extracellular signal-regulated kinase (ERK) signaling pathway. These studies identify a role for ERK in the acute regulation of epithelial NHE3 activity.

METHODS5 u& G2 g) y/ J. c' l$ D0 U+ p2 a
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Tubule Perfusion and Measurement of Net HCO 3 - Absorption" _. u" @9 Y, E

MTALs from male Sprague-Dawley rats (50-100 g body wt; Taconic, Germantown, NY) were isolated and perfused in vitro as described ( 15, 21 ). Tubules were dissected from the inner stripe of the outer medulla at 10°C in control bath solution, transferred to a bath chamber on the stage of an inverted microscope, and mounted on concentric glass pipets for perfusion at 37°C. For HCO 3 - transport experiments, the tubules were perfused and bathed in control solution that contained (in mM): 146 Na  , 4 K  , 122 Cl -, 25 HCO 3 -, 2.0 Ca 2 , 1.5 Mg 2 , 2.0 phosphate, 1.2 SO 4 2-, 1.0 citrate, 2.0 lactate, and 5.5 glucose (equilibrated with 95% O 2 -5% CO 2, pH 7.45 at 37°C). Bath solutions also contained 0.2% fatty acid-free bovine albumin. Experimental agents were added to the bath solution as described in RESULTS. Solutions containing aldosterone and other experimental agents were prepared as described ( 15, 17, 19, 21, 61 ). Equal concentrations of vehicle were added to control solutions in all protocols.
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The protocol for study of transepithelial HCO 3 - absorption was as described ( 15, 21 ). 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 tubule length) was adjusted to 1.5-1.9 nl·min -1 ·mm -1. One to three 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 aldosterone was added to or removed from the bath solution. In one series (see Fig. 2 B ), a longer treatment period was used, as described in RESULTS. The absolute rate of HCO 3 - absorption ( J HCO 3 -, pmol·min -1 ·mm -1 ) was calculated from the luminal flow rate and the difference between total CO 2 concentrations measured in perfused and collected fluids ( 15, 21 ). An average HCO 3 - 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. Mean values ± SE ( n = number of tubules) are presented in the text.
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Fig. 2. Protein kinase C inhibitors and pertussis toxin (PTX) do not prevent inhibition of HCO 3 - absorption by aldosterone. MTALs were bathed with 10 -7 M chelerythrine Cl or 10 -7 M staurosporine ( A ), or with 5 x 10 -9 M PTX ( B ), and then 1 nM aldosterone was added to and removed from the bath solution. Tubules were exposed to PTX for 150 min before aldosterone addition ( 17 ). J HCO 3 -, data points, lines, and P values are as in Fig. 1. Mean values are given in RESULTS., L4 |6 O( y2 ~' S# }! t

Measurement of Intracellular pH and Apical Na /H Exchange Activity

pH i was measured in isolated, perfused MTALs by use of the pH-sensitive dye BCECF and a computer-controlled spectrofluorometer (CM-X, SPEX Industries) coupled to the perfusion apparatus ( 59, 60 ). The tubules were perfused in the same manner used for HCO 3 - transport experiments except that the lumen and bath solutions were delivered via rapid flow systems that permit complete exchange of the solutions in less than 2 s. Apical membrane Na /H exchange activity was determined as described ( 57, 59, 60 ). Tubules were perfused and bathed in Na -free, HEPES-buffered solution that contained (in mM): 145 NMDG  , 4 K  , 147 Cl -, 2.0 Ca 2 , 1.5 Mg 2 , 1.0 phosphate, 1.0 SO 4 2-, 1.0 citrate, 2.0 lactate, 5.5 glucose, and 5 HEPES (equilibrated with 100% O 2; titrated to pH 7.4). The lumen solution also contained furosemide to block Na -K -2Cl - cotransport activity and the bath contained ethylisopropyl amiloride (EIPA) to eliminate any contribution of basolateral Na /H exchange to changes in pH i. Apical Na /H exchange activity was determined by measurement of the initial rate of pH i increase after addition of 145 mM Na to the lumen solution (Na replaced NMDG ) ( 59 ). Interruption of pH i recovery at various points along the recovery curve permits determination of the apical Na /H exchange rate over a broad range of pH i values (6.4 to 7.7), with appropriate corrections for a variable background acid-loading rate ( 59 ). The Na -dependent pH i recovery was inhibited 90% by lumen EIPA (50 µM) under all experimental conditions. Apical Na /H exchange rates ( J Na /H  , pmol·min -1 ·mm -1 ) were calculated as (dpH i /d t ) x i x V, where dpH i /d t (pH U/min) is the initial slope of the record of pH i vs time, i is the intrinsic intracellular buffering power (mM·pH), and V is cell volume per unit tubule length (nl/mm), measured as previously described ( 59, 60 ). Experimental agents were added to the bath solution as described in RESULTS.
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Determination of ERK Activity

ERK activity was studied using two previously described preparations ( 4, 56, 61 ): 1 ) thin strips of tissue dissected from the inner stripe of the outer medulla (the region of the kidney highly enriched in MTALs) and 2 ) microdissected MTALs. Following dissection at 4°C, the tissue strips or MTALs were divided into two to four samples of equal amount and then incubated in vitro at 37°C in the same solutions used for HCO 3 - transport experiments ( 4, 56, 61 ). The specific protocols used for incubations are given in RESULTS. Following incubation, the tissue was lysed and ERK1/2 activity was measured in an immune complex kinase assay using myelin basic protein as substrate as previously described ( 56, 61 ). Phosphorylated substrate was isolated by SDS-PAGE, visualized by autoradiography, and quantified by densitometry. Equal amounts of ERK in immunoprecipitates under different experimental conditions were verified in parallel samples by immunoblotting ( 4, 56 ). We demonstrated previously that changes in protein kinase activities measured in the inner stripe accurately reproduce changes in the MTAL ( 4, 19, 56, 61 ).4 k$ d4 Z; Y% B+ O

Analysis
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Results are presented as means ± SE. Differences between means were evaluated using Student's t -test for paired data or ANOVA with the Newman-Keuls multiple range test, as appropriate. P 2 q( [+ h$ x, ]- S% V- f: Y

RESULTS! W3 Y1 Y8 d' M5 n' o( Y  K: b

Aldosterone Inhibits HCO 3 - Absorption( v( g2 `  f3 z* U

Adding 1 nM aldosterone to the bath decreased HCO 3 - absorption by 29%, from 15.3 ± 0.5 to 10.9 ± 0.4 pmol·min -1 ·mm -1 ( P
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Fig. 1. Aldosterone (Aldo) inhibits HCO 3 - absorption in the medullary thick ascending limb (MTAL). Rat MTALs were isolated and perfused in vitro. The absolute rate of HCO 3 - absorption ( J HCO 3 - ) was measured in control solution, and then 1 nM aldosterone was added to and removed from the bath solution. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. P value is for paired t -test. Mean values are given in RESULTS.

Role of PKC and Pertussis Toxin-Sensitive G Proteins

Aldosterone induces rapid activation of PKC in several cell types ( 9, 12, 26, 27, 31, 36, 38, 62, 63 ), and PKC is a regulator of Na /H exchange activity ( 2, 12, 36, 39, 54, 62 ). The role of PKC in mediating aldosterone-induced inhibition of HCO 3 - absorption was examined using chelerythrine Cl and staurosporine, inhibitors that selectively abolish PKC-dependent regulation of HCO 3 - absorption in the MTAL ( 4, 17 - 20 ). In tubules bathed with 10 -7 M chelerythrine Cl or 10 -7 M staurosporine, addition of 1 nM aldosterone to the bath decreased HCO 3 - absorption by 30%, from 13.4 ± 0.4 to 9.4 ± 0.5 pmol·min -1 ·mm -1 ( P . x4 F/ j) e# K' \* O, C

Aldosterone-induced stimulation of Na /H exchange in certain colonic and renal epithelial cells depends on pertussis toxin-sensitive G proteins ( 12, 53, 62 ). We therefore examined the effect of aldosterone in MTALs bathed with 5 x 10 -9 M pertussis toxin for 150 min, a treatment that blocks G protein-mediated regulation of HCO 3 - absorption in the MTAL ( 17 ). As shown in Fig. 2 B, aldosterone decreased HCO 3 - absorption by 30% (from 14.2 ± 0.8 to 10.0 ± 0.6 pmol·min -1 ·mm -1; P
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Role of cAMP

Aldosterone has been shown to induce a rapid increase in intracellular cAMP in several cell systems ( 9, 28, 30, 49, 53 ), and the cAMP/protein kinase A pathway inhibits apical Na /H exchange and HCO 3 - absorption in the MTAL ( 7, 15, 16, 20 ). To determine whether cAMP is involved in mediating inhibition by aldosterone, MTALs were bathed with 10 -4 M 8-BrcAMP or 10 -6 M forskolin, agents that induce maximal cAMP-mediated inhibition of HCO 3 - absorption ( 15, 20 ). In the presence of 8-BrcAMP or forskolin, aldosterone decreased HCO 3 - absorption by 48%, from 9.6 ± 0.6 to 5.0 ± 0.3 pmol·min -1 ·mm -1 ( P
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Fig. 3. Inhibition of HCO 3 - absorption by aldosterone is not mediated by cAMP. MTALs were bathed with 10 -4 M 8-BrcAMP or 10 -6 M forskolin, and then 1 nM aldosterone was added to and removed from the bath solution. J HCO 3 -, data points, lines, and P value are as in Fig. 1. Mean values are given in RESULTS.* e. S; O8 {1 [, F/ g1 O/ O

Role of PI3-Kinase and p70 S6 Kinase; x/ M  X/ z4 C; a5 t( ]# x* P1 N
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Aldosterone has been reported in vascular tissue to induce rapid activation of PI3-kinase and its downstream effector p70 S6 kinase ( 33, 37 ), pathways that regulate HCO 3 - absorption in the MTAL ( 19, 24 ). To examine the role of PI3-kinase in inhibition by aldosterone, MTALs were bathed with 20 µM LY-294002 or 100 nM wortmannin, inhibitors that selectively block PI3-kinase-mediated regulation of HCO 3 - absorption in the MTAL ( 19, 20, 24 ). In the presence of LY-294002 or wortmannin, aldosterone decreased HCO 3 - absorption by 34%, from 13.8 ± 0.6 to 9.1 ± 0.6 pmol·min -1 ·mm -1 ( P

Fig. 4. PI3-kinase inhibitors and rapamycin do not prevent inhibition of HCO 3 - absorption by aldosterone. MTALs were bathed with 20 µM LY-294002 or 100 nM wortmannin ( A ), or with 30 nM rapamycin ( B ), and then 1 nM aldosterone was added to and removed from the bath solution. J HCO 3 -, data points, lines, and P values are as in Fig. 1. Mean values are given in RESULTS.
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Further experiments examined the effect of rapamycin, an immunosuppressive agent that can block aldosterone-induced signaling by inhibiting the activation of p70 S6 kinase and/or by binding to heat shock proteins and altering the stability of an HSP-aldosterone receptor complex ( 8, 52 ). Tubules were bathed with 30 nM rapamycin, which blocks p70 S6 kinase-mediated regulation of HCO 3 - absorption in the MTAL ( 24 ). As shown in Fig. 4 B, aldosterone decreased HCO 3 - absorption from 13.0 ± 1.0 to 7.2 ± 0.5 pmol·min -1 ·mm -1 in the presence of rapamycin ( P $ k& V  X  h3 H2 L0 a

Role of ERK
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Inhibitors of ERK activation reduce inhibition of HCO 3 - absorption by aldosterone. Aldosterone induces rapid activation of ERK in several cell types, including renal epithelial cell lines ( 11, 33, 47 ). To determine whether the ERK pathway is involved in inhibition of HCO 3 - absorption by aldosterone, we examined the effects of U-0126 and PD-98059. These compounds selectively inhibit the mitogen-activated protein kinase kinase MEK1/2, the direct upstream activator of ERK1/2 ( 1, 10 ), and block MEK/ERK signaling in the MTAL ( 24, 56, 61 ). As shown in Fig. 5, the inhibition of HCO 3 - absorption by aldosterone was markedly reduced in tubules bathed with either 15 µM U-0126 ( Fig. 5 B ) or 15 µM PD-98059 ( Fig. 5 C ). In the presence of the inhibitors, aldosterone decreased HCO 3 - absorption only by 11%, from 14.4 ± 0.4 to 12.8 ± 0.4 pmol·min -1 ·mm -1 ( n = 7; P

Fig. 5. Inhibitors of ERK activation reduce inhibition of HCO 3 - absorption by aldosterone. MTALs were studied in control solution ( A ), or bathed with 15 µM U-0126 ( B ) or 15 µM PD-98059 ( C ), and then 1 nM aldosterone was added to and removed from the bath solution. Control data ( A ) are repeated from Fig. 1 to facilitate comparison. J HCO 3 -, data points, lines, and P values are as in Fig. 1. Mean values are given in RESULTS.

Aldosterone increases ERK activity. The effect of aldosterone on ERK activity was examined directly in the MTAL. Microdissected MTALs were incubated in vitro in the absence and presence of 1 nM aldosterone for 15 min and then ERK activity was measured by immune complex assay as described ( 56, 61 ). As shown in Fig. 6 A, aldosterone increased ERK activity 1.8-fold. These studies establish directly that ERK is activated by aldosterone in the MTAL. Previously, we demonstrated that aldosterone increases ERK activity by a similar amount in inner stripe tissue ( 22 ). Thus changes observed in the inner stripe reflect changes in the MTAL.
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Fig. 6. Aldosterone increases ERK activity in the MTAL. A : microdissected MTALs were incubated in vitro at 37°C in the absence and presence of 1 nM aldosterone for 15 min and then ERK activity was measured by immune complex assay that used myelin basic protein (MBP) as substrate ( 56, 61 ). Phosphorylated MBP was analyzed by SDS-PAGE, autoradiography, and densitometry. Aldosterone increased ERK activity 1.8-fold. One of two similar experiments is shown. B : time course of ERK activation. Inner stripe tissue was incubated in the absence and presence of 1 nM Aldo for the indicated times and ERK activity was assayed as in A. Autoradiograph is representative of 4 independent experiments. Relative ERK activities (determined by densitometry) are given in RESULTS. C : MEK inhibitors block ERK activation. Inner stripe tissue was incubated for 30 min in the absence (Cont) and presence of 15 µM U-0126 or 15 µM PD-98059 and then treated with 1 nM aldosterone for 15 min in the absence (Aldo) or continued presence (Inhibitor Aldo) of the inhibitors. ERK activity was assayed as in A and is presented as a percentage of control activity measured in the same experiment. Autoradiographs are of representative experiments. Bars are means ± SE for 7 independent experiments (3 with U-0126 and 4 with PD-98059). * P 4 r4 z- v" ~- G; t+ V

Further experiments were carried out using inner stripe tissue to determine the time course of ERK activation. ERK activity increased within 5 min of exposure to aldosterone and remained elevated for at least 30 min ( Fig. 6 B ). ERK activity was increased 1.6 ± 0.1-fold at 5 min ( n = 4; P 1 f2 i6 p' ]: ^/ ^8 B9 k
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Inhibiting ERK activation prevents inhibition of NHE3 by aldosterone. Recently, we demonstrated that aldosterone inhibits HCO 3 - absorption in the MTAL through primary inhibition of the apical NHE3 Na /H exchanger ( 21, 23 ). To determine whether the inhibition of NHE3 is dependent on ERK activation, we examined the effects of U-0126. MTALs were studied with and without 1 nM aldosterone in the bath for 15-20 min in the absence and presence of 15 µM U-0126. Apical Na /H exchange activity was determined by measurement of the initial rate of pH i increase in response to lumen Na addition (see METHODS ). Under basal conditions (Control, Fig. 7 A ), the exchanger exhibits a sigmoidal dependence on pH i, as demonstrated previously ( 59, 60 ). U-0126 alone has no effect on exchanger activity ( Fig. 7, A and C ). Consistent with previous results ( 23 ), aldosterone decreased apical Na /H exchange activity over the pH i range studied, due to a 28% decrease in V max ( Fig. 7, B and C ). The effect of aldosterone to decrease apical Na /H exchange activity was prevented in tubules bathed with U-0126 ( Fig. 7, B and C ). These results demonstrate that aldosterone inhibits apical NHE3 in the MTAL through activation of the ERK signaling pathway.
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Fig. 7. Inhibitors of ERK activation block inhibition of apical Na /H exchange by aldosterone. MTALs were studied under control conditions and with either 15 µM U-0126, 1 nM aldosterone, or 15 µM U-0126 1 nM aldosterone in the bath for 15-20 min. Apical Na /H exchange rates ( J Na /H ) were determined at various pH i values from initial rates of pH i increase measured after addition of Na to the tubule lumen (see METHODS ). A : U-0126 alone has no effect on apical Na /H exchange activity. Data points are from 12 control tubules and 6 tubules with U-0126. Line is from a least-squares fit of the combined data to the Hill equation ( 59, 60 ). B : inhibition of apical Na /H exchange by aldosterone is prevented by U-0126. Data are from 9 tubules with aldosterone (dashed line) and 10 tubules with U-0126 aldosterone (dotted line). Solid line for combined Control/U-0126 data is repeated from A for comparison. Lines, maximal velocity ( V max, pmol·min -1 ·mm -1 ), and apparent affinity for intracellular H ( K ' app, pH) are from least-squares fits to the Hill equation. * P 4 c! ]) G0 C2 P$ S* ^; Z
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DISCUSSION

Aldosterone plays a major role in Na  , volume, and acid-base homeostasis through its regulatory actions on electrolyte transport in the kidney and gastrointestinal tract. Although rapid, nongenomic effects of aldosterone have been demonstrated in many cell types, their physiological significance for aldosterone-induced regulation of epithelial function remains undefined. Recently, we demonstrated that aldosterone inhibits transepithelial HCO 3 - absorption in the MTAL through a nongenomic pathway ( 21 ). This inhibition is the result of a direct cellular action of aldosterone to decrease the activity of the apical membrane NHE3 Na /H exchanger ( 23 ). The present study demonstrates that aldosterone inhibits NHE3 and HCO 3 - absorption through activation of the ERK signaling pathway. These results identify NHE3 as a target for nongenomic regulation by aldosterone and establish a physiological role for ERK in the acute regulation of NHE3 activity.

The conclusion that aldosterone inhibits NHE3 through activation of the MEK/ERK pathway is supported by several observations: 1 ) the inhibition of apical Na /H exchange by aldosterone is blocked by U-0126 and PD-98059, two chemically unrelated MEK1/2 inhibitors with different mechanisms of action ( 1, 10 ), 2 ) aldosterone activates ERK in the MTAL in physiological solutions used for HCO 3 - transport experiments, 3 ) U-0126 and PD-98059 block this ERK activation, 4 ) both the rapidity ( 5 min) and duration ( 30 min) of ERK activation correlate temporally with aldosterone-induced inhibition of NHE3 and HCO 3 - absorption ( 21, 23 ), 5 ) the aldosterone-induced transport inhibition is not prevented by inhibitors of several other signaling pathways, indicating a high degree of selectively for inhibitors of the MEK/ERK pathway, 6 ) U-0126 and PD-98059 do not prevent inhibition of HCO 3 - absorption in the MTAL by several other stimuli ( 20, 22, 24, 56 ), and U-0126 alone has no effect on apical Na /H exchange activity ( Fig. 7, A and C ), indicating that these agents do not prevent aldosterone-induced inhibition through nonspecific cytotoxic or metabolic effects or an effect on NHE3 that prevents its physiological regulation, 7 ) both activation of ERK and inhibition of NHE3 by aldosterone occur independently of gene transcription (are not blocked by actinomycin D) and are unaffected by the mineralocorticoid receptor antagonist spironolactone ( Fig. 6 ) ( 21, 22 ). Activation of ERK has been shown previously to play a role in the long-term stimulation of NHE3 by acid media in renal epithelial (OKP) cells ( 51 ) and in the control of NHE3 by -adrenergic agents in cultured proximal tubule cells ( 32 ). The present study provides evidence for direct coupling of the ERK pathway to the acute regulation of NHE3 and its physiological functions to mediate epithelial Na absorption and H secretion./ J- H8 i) B( e: t' M" W
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Previously, we identified a role for ERK in mediating inhibition of HCO 3 - absorption by nerve growth factor (NGF) in the MTAL. This regulation occurs through a novel mechanism in which activation of ERK is coupled to inhibition of the basolateral NHE1 Na /H exchanger ( 57, 61 ). The inhibition of NHE1 induces actin cytoskeleton remodeling that secondarily inhibits apical NHE3 and HCO 3 - absorption ( 57, 58, 61 ). A relevant component of this mechanism is that the activation of ERK by NGF has no direct effect on NHE3 but instead inhibits NHE3 only indirectly through inhibition of NHE1 ( 57, 61 ). These findings are in sharp contrast to the regulatory effects induced by aldosterone in the MTAL: activation of ERK by aldosterone is coupled directly to inhibition of NHE3 ( Fig. 7 ), and NHE1 plays no role in aldosterone-induced inhibition of HCO 3 - absorption ( 23 ). Thus NGF and aldosterone both inhibit MTAL HCO 3 - absorption through activation of ERK, but this inhibition occurs through entirely different transport mechanisms: with aldosterone, the ERK pathway is coupled primarily to inhibition of NHE3; with NGF, the ERK pathway is coupled primarily to inhibition of NHE1, with no direct coupling to NHE3. Thus, in the MTAL, ERK-dependent signals are targeted to regulate different Na /H exchangers in different membrane domains, depending on the physiological stimulus. An important goal for future work will be to understand how this signal targeting occurs, and how it enables different stimuli to act through a common signaling pathway to induce distinct physiological responses in the MTAL.2 e( s( L3 m# |4 T$ X% o0 ?9 Q

Aldosterone has been shown to induce rapid increases in ERK activity in other cell types ( 11, 33, 47 ), and ERK activation has been linked to aldosterone-induced stimulation of the NHE1 Na /H exchanger in Madin-Darby canine kidney cells ( 11 ). The latter finding is consistent with numerous studies demonstrating that the ERK pathway plays an important role in mediating activation of NHE1 by growth factors and other mitogenic stimuli ( 5, 42, 44, 50 ). These factors stimulate NHE1 by increasing its apparent affinity for intracellular H ( 5, 11, 42, 44, 54 ). In contrast, we found that aldosterone acts via ERK to inhibit NHE3 by decreasing its maximal velocity, without altering the intracellular H affinity ( Fig. 7 ) ( 23 ). Thus aldosterone-induced ERK activation leads to a decrease in the turnover number of individual NHE3 transporters, a decrease in the number of functional transporters in the apical membrane, or both. Regulation of NHE3 in other cell systems involves trafficking between the plasma membrane and intracellular vesicles ( 39, 42, 64 ), and a role for ERK in regulating intracellular trafficking of membrane proteins has been described ( 13, 29 ). Whether NHE3 undergoes trafficking in the MTAL, and whether this process is influenced by ERK, remains to be determined. The regulation of NHE1 by ERK involves multiple molecular mechanisms that include direct phosphorylation of the exchanger, phosphorylation of NHE1 through the downstream effector p90 ribosomal S6 kinase, and ERK-dependent phosphorylation of accessory regulatory proteins that interact with NHE1 ( 5, 40, 42, 44, 50, 54 ). The possible role of these mechanisms in mediating ERK-induced regulation of NHE3 is unknown. The cytoplasmic terminus of rat NHE3 ( 43 ) contains at least two potential ERK concensus sequences (P-X-S/T-P; 14), raising the possibility that ERK could phosphorylate NHE3 directly. NHE3 also is regulated through its interactions with multiple ancillary/scaffolding proteins such as NHERF1 and NHERF2, the actin-binding protein ezrin, and the Ca 2  -binding protein CHP ( 39, 42, 64 ), which provide potential targets for ERK-induced regulation.: ~, Z$ R8 ^4 W- W. I

Although ERK plays the predominant role in mediating inhibition by aldosterone in the MTAL, a significant decrease in HCO 3 - absorption persists when ERK activation is prevented ( Fig. 5 ). This suggests that an as yet unidentified signaling pathway may be activated by aldosterone and functions in parallel with the ERK pathway to mediate inhibition of HCO 3 - absorption. The present study found no evidence of a role for cAMP/PKA, PKC, PI3-kinase, p70 S6 kinase, or pertussis toxin-sensitive G proteins. The aldosterone-induced regulation also is unlikely to involve a cytochrome P -450 pathway based on the finding that the inhibition of HCO 3 - absorption by aldosterone is additive to inhibition by ANG II ( 21 ). Our results also show that, in the presence of ERK inhibitors, aldosterone had no effect on apical Na /H exchange activity but induced a small decrease in HCO 3 - absorption ( Figs. 5 and 7 ). This suggests that a small portion of the inhibition by aldosterone may be mediated through a transporter other than NHE3. However, it is also possible that the small ( 10%) decrease in apical Na /H exchange activity needed to account for the decrease in HCO 3 - absorption in the presence of ERK inhibitors may have gone undetected in our experiments (e.g., technical limitations preclude measuring apical Na /H exchange activity in the presence and absence of aldosterone with ERK inhibitors in the same tubule). Nevertheless, the results of our current and previous ( 23 ) studies show that most, if not all, of the inhibition of HCO 3 - absorption by aldosterone is mediated through inhibition of NHE3.

NHE3 is the primary absorptive exchanger in the kidney and gastrointestinal tract. In addition to mediating NaHCO 3 absorption by the MTAL ( 3, 6, 16, 25, 60 ), apical NHE3 is responsible for most of NaCl, fluid, and NaHCO 3 absorption by the renal proximal tubule and the intestine ( 35, 39, 42, 48, 55, 64 ). Our results demonstrate that aldosterone acts directly via ERK to regulate NHE3 in the MTAL. Thus, in addition to its actions on classic genomic targets such as ENaC and the Na -K -ATPase, aldosterone may influence epithelial absorptive processes important for volume and acid-base balance through nongenomic regulation of NHE3. As discussed previously ( 21, 23 ), the effect of aldosterone to inhibit NHE3 and HCO 3 - absorption in the MTAL may play an important role in enabling the kidneys to maintain acid-base balance during changes in Na and volume balance. In addition, aldosterone-induced inhibition of NHE3 could play a role in pathophysiological processes such as aldosterone escape or the effect of changes in systemic K balance to alter renal HCO 3 - absorptive capacity and acid excretion ( 2, 21, 23 ). Whether aldosterone regulates NHE3 directly in other epithelia remains to be determined. However, in a recent study infusion of aldosterone into rats induced a rapid increase in renal Na excretion ( 45 ), an effect consistent with aldosterone-induced inhibition of NHE3. Whether ERK plays a physiological role in regulating NHE3 by other stimuli and in other cell types also requires further investigation. In a recent study of LLC-PK 1 -F cells, which exhibit functional properties of proximal tubule cells, the insulin-sensitizing agent troglitazone inhibited Na /H exchange through ERK activation, resulting in an intracellular acidosis that increased ammoniagenesis and decreased DNA synthesis ( 41 ). Based on results of the present study, these effects may be mediated by ERK-induced inhibition of NHE3. Our results raise the possibility that a wide variety of important stimuli in addition to aldosterone, including peptide hormones and growth factors acting through receptor tyrosine kinases and G protein-coupled receptors, could influence NHE3 and its biological functions through the ERK pathway.
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In summary, the present study demonstrates that aldosterone inhibits apical NHE3 in the MTAL through rapid activation of the ERK signaling pathway. This regulation is nongenomic and results in a decrease in transepithelial HCO 3 - absorption. These results identify NHE3 as a direct target for aldosterone-induced regulation of epithelial Na absorption and acid secretion and establish a role for ERK in the acute regulation of NHE3 and its physiological functions.$ S' c$ T5 t% M# k+ n0 I

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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38217.
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