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(Hypertension. 2000;35:1160.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Activation of MAPKs in Proximal Tubule Cells From Spontaneously Hypertensive and Control Wistar-Kyoto Rats

Astrid Parenti; Xiao-Lan Cui; Ulrich Hopfer; Marina Ziche; Janice G. Douglas

From the Division of Hypertension (A.P., X.-L.C., J.G.D.), Department of Medicine, and the Department of Physiology and Biophysics (U.H.), School of Medicine, Case Western Reserve University and University Hospitals, Cleveland, Ohio; and the Department of Pharmacology (A.P.) and CIMMBA (M.Z.), University of Florence, Florence, Italy.

Correspondence to Dr Janice G. Douglas, Division of Hypertension, Department of Medicine W165, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106-4982. E-mail jgd3{at}po.cwru.edu

	Abstract

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Abstract—The aim of this study was to test the hypothesis that differences exist in the activity and/or expression of mitogen-activated protein kinases (MAPKs) between spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) and that these differences may account for the enhanced activity of the Na⁺/H⁺ exchanger (NHE) previously observed in the renal proximal tubule of SHR. Therefore, the activities of c-jun N-terminal kinase₁ (JNK₁), extracellular signal-regulated kinase_1/2 (ERK_1/2), and p38 were investigated. A reduced amount of ERK₁ and JNK₁ protein was found in renal cortex specimens of SHR as compared with WKY; however, their activities were the same. To study the cellular basis of this difference, immortalized proximal tubule cell lines were grown on Millicell-CM filter inserts where the cell lines organize as polarized monolayers with separate access to apical and basolateral compartments. Although basal JNK₁ and ERK_1/2 activities were not significantly different between WKY and SHR cells, anisomycin stimulated JNK₁ activity in WKY cells more than in SHR cells (eg, at 15 minutes 300% versus 30%, respectively). Similarly, angiotensin II increased JNK₁ and ERK_1/2 activity in a time- and concentration-dependent manner in WKY cells but not in SHR cells. Western blot analyses showed a deficit in JNK₁ and ERK₁ protein in SHR (0.25 and 0.5, respectively, of the levels in WKY cells), although ERK₂ and p38 protein levels were the same. These observations suggest that, although angiotensin II activates MAPKs and MAPKs have been shown to regulate NHE, this regulatory pathway is unlikely to account for the increased activity of NHE in the proximal tubular epithelium of SHR.

Key Words: epithelial cells • protein kinases • hypertension, essential • angiotensin II

	Introduction

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The genetic basis for essential hypertension in humans and animal models, such as the spontaneously hypertensive rat (SHR), has not been elucidated. In the SHR model, the pathogenesis of hypertension appears to be related to changes in the renal set point for Na⁺ reabsorption or cellular Na⁺ homeostasis, in general. The long-term control of Na⁺ homeostasis appears to depend on pressure natriuresis in the kidney (ie, the renal set point for Na⁺ reabsorption is set such that hypertension is necessary to achieve a normal extracellular fluid volume).¹ Evidence for the crucial role of the kidneys comes from transplantation experiments of SHR kidneys into normotensive recipient strains: SHR kidneys exhibit an altered pressure-natriuresis relationship that is maintained after transplantation,² and the transplanted SHR kidneys confer hypertension to animals from normotensive strains.³ The proximal tubule is the major site for Na⁺ reabsorption⁴ and thus is potentially a site for changes in the set point for Na⁺ reabsorption. Considerable evidence points toward intrinsic differences in tubule function between SHR and the normotensive Wistar-Kyoto (WKY) control strain. Of particular importance to the pathogenesis of hypertension appears to be abnormalities of the 2 most important regulatory agents of Na⁺ transport: dopamine and angiotensin II (Ang II). D1 dopaminergic receptor signaling is impaired resulting in enhanced Na⁺ reabsorption.⁵ Alterations in responses to Ang II are more complex and age dependent, but they generally also result in enhanced proximal tubule Na⁺ reabsorption.^{6 7} In young, prehypertensive SHR, type 1 Ang II receptor numbers are increased, although their sensitivity is decreased. In contrast, in 12-week old rats, the sensitivity to Ang II is increased. With cell lines from SHR and WKY proximal tubular epithelial cells, it has been possible to confirm that there is enhanced Ang II–dependent activation of the Na⁺/H⁺ exchanger (NHE).⁸ Moreover, isolated brush border membranes⁹ and cultured proximal tubule cells¹⁰ from SHR exhibit higher NHE activity even during the prehypertensive state, which is consistent with enhanced proximal tubular Na⁺ reabsorption as a cause of hypertension in SHR. These observations suggest that there may be an underlying defect in NHE activity (basal and Ang II–stimulated) that may be responsible for enhanced Na⁺ reabsorption in SHR. Interestingly, mitogen-activated protein kinases (MAPKs) have been linked to Na⁺ homeostasis, although the nature of this connection has not been completely unraveled. For example, extracellular signal-regulated kinase (ERK) activation is a major mediator of growth factor–induced activation of NHE-1.¹¹ Moreover, cells that overexpress a dominant negative mutant of MAPK have a 50% reduction in NHE activity.¹²

Because MAPKs have been linked to regulation of NHE activity and epithelial cell Ang II signaling,^{11 13 14} the present studies were designed to test the hypothesis that enhanced basal and/or Ang II–stimulated activity of MAPKs in proximal tubular epithelium may be associated with SHR cells. Basal levels as well as stress- and Ang II–dependent activation of endogenous ERK_1/2, c-jun N-terminal kinase (JNK₁), and p38 were assessed in renal cortex specimens, and well-differentiated, proximal tubule cell lines were derived from SHR and WKY.

	Methods

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Renal Cortex Specimens
Male 4- to 8-week-old WKY and SHR (Charles River Laboratories, Wilmington, Mass) were anesthetized with pentothal (40 mg/kg body weight), and their kidneys were perfused through the renal artery with DMEM/F12 (1:1 vol/vol) medium supplemented with 5% fetal bovine serum, 5 µg/mL transferrin, 5 µg/mL insulin, 10 ng/mL epithelial growth factor, 4 µg/mL dexamethasone, and 2 mmol/L L-glutamine (rat–renal tubular epithelial [RTE] medium). After complete washout of blood, small pieces of outer renal cortex were excised and promptly frozen in liquid N₂ until use. To measure protein levels and activities of MAPK, the frozen specimens were homogenized on ice in lysis buffer followed by centrifugation at 14 000xG for 10 minutes at 4°C.^{15 16} Aliquots of 100 µg of protein from the harvested supernatants were used for immunoprecipitation of ERK₁ and JNK₁ and for Western blot analyses of JNK₁, p38, and phosphorylated p38. Fifty micrograms of lysate protein was used for Western blot analysis of ERK.

Protein Determination
Protein concentrations were measured by the BCA method (Pierce Chemical Co).

Cell Culture
Immortalized epithelial cell lines derived from renal proximal tubules of normotensive WKY 1292 (clone 8) and SHR 0193 (clone 2) rats were grown to confluence on Ethicon collagen-coated 30-mm Millicell-CM culture plate inserts.⁸ The culture medium was rat-RTE.

Monolayer Resistance
Confluence of monolayers was assessed quantitatively by measuring the electrical resistance with a Millicell ERS probe as previously described.¹⁷ The electrical resistance of the monolayers for the reported experiments was 510±18 cm² and 390±17 cm² for the SHR and WKY cell lines, respectively, which is in accordance with the values reported previously.⁸

[³⁵S]-Labeling
Cells were uniformly labeled by growing them for 8 to 12 hours in methionine-free rat-RTE medium in the presence of 100 µCi/mL of [³⁵S]-L-methionine (1000 Ci/mmol, New England Nuclear [NEN]).

MAPK Activity
MAPK protein levels and activities were measured in confluent, polarized monolayers. Before each experiment, serum and EGF were omitted from the medium for 1 night. Stimuli were added to either the inside or the outside of the insert to differentially stimulate the apical or basolateral side of the polarized monolayers, respectively. JNK₁ and ERK₁ activities were measured by immunoprecipitation, with polyclonal antibodies against JNK₁ and ERK₁, and immune-complex kinase assays as previously described.^{15 16} The anti-JNK₁ antibody recognized all JNK isoforms, and the anti-ERK₁ antibody recognized both ERK₁ and ERK₂, but preferentially bound to ERK₁. The activated form of p38 was measured by immunoblotting with anti-phospho p38 antibody.

Western Blot Analysis
Cell lysates containing 50 to 100 µg proteins were subjected to 8% SDS-polyacrylamide gel electrophoresis (PAGE) and proteins were then transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting. The blots were treated with rabbit polyclonal antibodies against JNK₁, ERK₁, or p38 overnight at 4°C. Immunoreactive proteins were detected by enhanced chemiluminescence. The intensities of the bands corresponding to MAPKs were quantified by densitometric analysis (scanned on UMAX MagicScan with Adobe Photoshop and analyzed with the software package IMAGE, United States Biochemical).

Statistical Analysis
Results are expressed as mean±SEM for (n) experiments with duplicate measurements. Differences between groups were tested for significance by Student’s t test for unpaired data, and a P value <0.05 was considered significant.

Materials
Purified rabbit IgG, anti-rabbit IgG coupled to agarose beads, myelin basic protein, PMSF, dithiothreitol, R24571, okadaic acid, and anisomycin were purchased from Sigma Chemical Co. [³²P]ATP and [³⁵S]-L-methionine were from NEN. Rabbit polyclonal antibodies against JNK₁, ERK₁, and p38 as well as the recombinant activating factor 2 (ATF-2) protein were purchased from Santa Cruz Biotechnology, Inc. Anti-phospho p38 polyclonal antibody was from New England Biolabs. Aprotinin, leupeptin, fetal bovine serum, and trypsin-EDTA were purchased from Boehringer Mannheim. Collagen dispersion was from Ethicon, Inc. Millicell-CM culture plate inserts (diameter 30 mm) were from Millipore. Acrylamide, TEMED, ammonium persulfate, and Coomassie brilliant blue were from Bio-Rad Laboratories. [Sar]-Ang II was from Upstate Biotechnology.

	Results

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MAPK Expression and Activity in Renal Cortex of WKY and SHR
A comparison of the relative concentrations and activities of different MAPKs was performed in freshly isolated renal cortex from 5-week-old WKY and SHR. ERK_1/2 and JNK₁ activities were measured by immunocomplex kinase assays, whereas p38 activity was quantified as a phosphorylated isoform of the enzyme on Western blots. The activities were normalized to lysate protein. The results are summarized in Table 1. No differences were found in the activities of ERK_1/2, JNK₁, and p38 between SHR and WKY. However, when MAPK protein levels were measured by Western blot analysis and normalized to actin levels, smaller amounts of JNK₁ and ERK₁ were found in SHR than in WKY, whereas ERK₂ and p38 protein expression levels appeared to be identical in both strains (Table 1).

View this table: [in this window] [in a new window]   Table 1. MAPK Activity and Protein Levels in Renal Cortex of WKY and SHR

Taken at face value, the observation of similar levels of MAPK activities in SHR and WKY renal cortex seems inconsistent with the hypothesis that these enzymes are responsible for elevated Na⁺ transport in the proximal tubules in SHR. However, the finding that the fraction of activated to total ERK₁ and JNK₁ was higher in SHR than in WKY suggests intrinsic strain differences that complicate the interpretation of the results. To get insight into the cellular basis of the differences between SHR and WKY strains and to study MAPKs under more controlled conditions than is possible in vivo, further experiments were performed with proximal tubule cell lines derived from these 2 rat strains.⁸

MAPK Activities in WKY and SHR Cell Lines
Basal Activities
Similar to renal cortex specimens, baseline activity of ERK_1/2 and JNK₁ were the same in both cell lines (ERK₁ 1026±200 cpm and 830±95 cpm with n=5; JNK₁ 128±9 cpm and 122±15 cpm with n=9, for WKY and SHR, respectively).

Stimulated JNK₁ Activity
JNK₁ is activated by a number of different stresses, including application of certain drugs, such as anisomycin.¹⁸ First, we evaluated whether differences existed between apical and basolateral stimulation in response to anisomycin because epithelial monolayers often exhibit different properties when stimulated from different sides.^{19 20} Activation of JNK₁ by apical application was significantly greater than by basolateral application, as shown for WKY monolayers in Figure 1A. Furthermore, 100 nmol/L anisomycin stimulated JNK₁ more in WKY cells than in SHR cells, although basal levels of JNK₁ activity were the same. Figure 1B illustrates the time-dependence of activation in WKY cells whereby the maximal activity was observed at 15 minutes with stimulation of 300% over basal levels. In contrast, anisomycin did not significantly stimulate JNK₁ in the SHR cells (Figure 1B) as the increase was 30%. At a higher concentration of 500 nmol/L, anisomycin was able to increase JNK₁ activity in SHR cells, although this activation was still considerably less than in WKY cells (179±45% and 444±80% increase over basal activity at 15 minutes for SHR and WKY, respectively, n=3).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of anisomycin on JNK1 activity in WKY and SHR cells. The activity is measured as 32P phosphorylation of ATF-2. A, Representative autoradiograph of WKY cells stimulated with 100 nmol/L anisomycin (second and third lane) added to the apical or basolateral side. First lane=unstimulated cells. B, Time dependence of JNK1 activation. WKY and SHR cells were grown on Millicell inserts until they reached confluence. Anisomycin (100 nmol/L) was added to the apical side, and JNK1 activity was measured by an immune-complex kinase assay with ATF-2 as the substrate. Results are expressed as radioactivity (cpm) recovered in the ATF-2 gel band. Mean±SEM, n= 4. **P<0.01, ***P<0.001 vs unstimulated cells.

We next compared the response to Ang II, which represents a physiological stimulus involved in the regulation of salt reabsorption. Experiments throughout this study were performed with [Sar]-Ang II, although the plain term Ang II is used. At 0.1 µmol/L, Ang II significantly stimulated JNK₁ activity in the WKY cell line (Figure 2a). Interestingly, the time course of activation was different with apical and basolateral stimulation. With basolateral stimulation, JNK₁ was maximally activated within 5 minutes, whereas with apical stimulation, activation increased more slowly and became stable at 30 minutes. Moreover, the Ang II stimulation of JNK₁ activity in WKY cells was concentration dependent with maximal activation at 1 µmol/L [Sar]-Ang II (50% over basal levels, Figure 2b). Interestingly, Ang II did not change JNK₁ activity in SHR cells (Figure 2c), even at concentrations as high as 10 µmol/L (data not shown). To test whether the differences can be explained on the basis of regulation of MAPKs by phosphatases,²¹ JNK₁ activity was measured in the presence of phosphatase inhibitors. The addition of R24571 (10 µmol/L) and okadaic acid (100 nmol/L), which inhibit phosphatase 2B and phosphatases 1 and 2A, respectively, did not augment JNK₁ activation by Ang II, suggesting that phosphatases are not responsible for the weaker JNK₁ activation in SHR cells (Table 2).

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of Ang II stimulation on JNK1 activity in polarized WKY and SHR cells. a, Time dependence of JNK1 activity in WKY cells in response to 0.1 µmol/L [Sar]-Ang II added apically or basolaterally to the monolayer. JNK1 activity is expressed as percent increase relative to unstimulated cells. b, Concentration dependence of stimulation of JNK1 in WKY cell line by apical or basolateral stimulation with [Sar]-Ang II (15-minute time point). Mean±SEM, n=3. *P<0.05, **P<0.01 vs basal activity. c, Effect of Ang II on JNK1 activity in SHR cells. Apical and basolateral stimulation with 0.1 µmol/L [Sar]-Ang II from 5 to 30 minutes. Mean±SEM, n=4.

View this table: [in this window] [in a new window]   Table 2. Effect of Protein Phosphatase Inhibitors on JNK1 Activity in SHR Cell Line

ERK₁ Activity in WKY and SHR Proximal Tubule Cells
Because Ang II has been reported to stimulate ERK in rabbit proximal tubular epithelial¹⁴ and opossum kidney epithelial cells¹³ and has been suggested to be an important signaling modulator of ion transport,¹¹ the ability of Ang II to stimulate ERK₁ in proximal tubule cell lines was assessed. Ang II stimulated ERK₁ activity in a time- and concentration-dependent manner in confluent WKY epithelial monolayers. Significant stimulation of ERK₁ activity was already seen by 5 minutes when Ang II (0.1 µmol/L) was added to the basolateral side, an effect that persisted for at least 15 minutes (data not shown). Dose-response relationships differed between apical and basolateral application of Ang II; for example, 0.1 µmol/L Ang II was required for maximal stimulation on the basolateral side, whereas 1 µmol/L Ang II was required for maximal stimulation on the apical side (Figure 3a). As with JNK₁, Ang II did not significantly stimulate ERK₁ in the SHR cell line (Figure 3b).

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of Ang II on ERK1 activity in WKY and SHR cells. ERK1 activity was measured by an immune-complex kinase assay with MBP as substrate. a, Concentration dependence of apical and basolateral Ang II stimulation in WKY cells (5-minute time point). *P<0.05 vs basal activity. b, Time dependence (5 to 30 minutes) of ERK1 stimulation with separate apical or basolateral addition of 0.1 µmol/L [Sar]-Ang II in SHR cells. Mean±SEM, n=3.

MAPKs Levels in SHR and WKY Proximal Tubule Cell Lines
One reason JNK₁ and ERKs were not activated in SHR cells by either stress or Ang II might be that the protein levels of these particular MAPKs were reduced in SHR; thus, the fractional activation under basal conditions was already close to the maximum. This explanation is supported by the findings in cortex specimens (ie, the greater fractional activation in SHR reported above). Therefore, the protein levels of MAPKs were also determined in the cell lines by immunoblot analysis.

Interestingly, SHR cells contained lower levels of both JNK₁ isoforms (p46 and p54), than WKY cells. Figure 4a illustrates representative data for the p46 JNK₁ isoform. Densitometric analysis of all immunoblots performed showed that, despite using the same amount of protein from cell lysates, JNK₁ expression in SHR cells was approximately one fourth of that observed in WKY cells (SHR/WKY= 0.25±0.03 densitometric units, P<0.01, n=3). A second approach to measure JNK₁ expression was to label cells for 8 to 12 hours with [³⁵S]-methionine, immunoprecipitate JNK₁, and then visualize it by means of autoradiography. Again, [³⁵S]-methionine–labeled JNK₁ was less in SHR than in WKY cells (Figure 4b).

View larger version (46K): [in this window] [in a new window]   Figure 4. ERK1/2 and JNK1 expression in WKY and SHR cells. Immunoblot of cell lysates (a, c) and immunoprecipitated ERK1/2 and JNK1 from [35S]met-labeled cells (b, d). Immunoblot: WKY (lanes 1 and 2) and SHR (lanes 3 and 4) cells (untreated, lanes 1 and 3, or stimulated, lanes 2 and 4) were lysed and 50 µg of total protein was fractionated in 8% SDS-PAGE. a, Cells stimulated with [Sar]-Ang II for 15 minutes (lanes 2 and 4) and immunoblotted with anti-JNK1 antibody. c, Cells stimulated for 5 minutes with 1 µmol/L [Sar]-Ang II (lanes 2 and 4) and immunoblotted with anti-ERK1 antibody. [35S]met-labeling: Cells were grown for 8 to 12 hours in methionine-free rat-RTE medium in the presence of 100 µCi/mL of [35S]-L-met (1000 Ci/mmol, NEN). JNK1 (b) and ERK1 (d) were immunoprecipitated, separated on 10% SDS-PAGE, and visualized by autoradiography.

The same experimental approach was used to measure ERK₁ expression. As shown in Figure 4c, Western blot analysis revealed a lower amount of ERK₁ in SHR compared with WKY cells. Densitometric analysis produced a ratio of 0.58±0.15 for ERK₁ expression in SHR relative to WKY cells (P<0.05, n=4). The anti-ERK₁ antibody also recognized ERK₂; however, the expression of ERK₂ was the same in both cell lines. Similar results were obtained with [³⁵S]-methionine labeling (Figure 4d).

p38 MAPK Activity and Expression
The levels of activated p38 were determined by immunoblotting with anti-phospho p38 antibody. In contrast to what was found for JNK₁ and ERK₁, p38 activity appeared the same in WKY and SHR cells stimulated for 20 minutes with 100 nmol/L anisomycin (Figure 5a). Moreover, immunoblot analysis did not reveal any difference in the amount of this member of the MAPK superfamily (Figure 5b). The ratio of protein levels of ERK₁ in SHR/WKY was 1.00±0.05 (n=3).

View larger version (60K): [in this window] [in a new window]   Figure 5. Activities and protein levels of p38 in WKY and SHR cells. WKY and SHR cells were left untreated (ctr) or were stimulated with 100 nmol/L anisomycin (anis) for 20 minutes. Cells were lysed, and aliquots of 50 µg of total protein were resolved by 10% SDS-PAGE. a, Immunoblot with anti-phospho p38 antibody for estimating the amount of activated form of p38. b, Immunoblot with anti-p38 antibody for estimating protein levels. c, Immunoblot with anti–ß-actin antibody. Representative results from 3 separate experiments.

	Discussion

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The present studies were aimed at testing the hypothesis that proximal tubule cells from SHR possess enhanced basal or stimulated activity of some members of the MAPK superfamily as compared with WKY. Such enhanced activity could be an explanation for enhanced Na⁺ reabsorption. Previous studies from this laboratory had documented enhanced Ang II–stimulated activity of NHE and Na⁺ reabsorption in SHR as compared with WKY epithelial cell lines.⁸ Others have documented that NHE is regulated by members of the MAPK superfamily.¹¹ This report represents an assessment of activity and expression levels of many members of the MAPK superfamily comparing renal cortex specimens and proximal tubule cells from SHR and WKY. With the same cell lines and culture conditions (growth on filters, same medium) as used for ion transport earlier,⁸ we observed that the basal activities of ERK_{1/2, p38,} and JNK₁ were similar in these cells. However, stimulation by Ang II or anisomycin was reduced in SHR. This lower capacity for activating both ERK₁ and JNK₁ may be a result of the lower amount of enzymes expressed. Greater fractional activation of ERK₁ and JNK₁ in SHR was seen in both experimental preparations used (cell lines, cortex) under baseline conditions (no stimuli), suggesting that the cell lines reflect in vivo behavior and are useful model systems.

Activation of MAPKs had been associated with higher NHE activity.^{9 10 11 12 13 14} Therefore, the results also suggest that neither ERK₁ or JNK₁ can be responsible for enhanced activation of NHE by Ang II in SHR epithelial cells. This conclusion differs strikingly from vascular smooth muscle cells, wherein increased activity of ERKs or a difference in the time-dependency of activation was observed in SHR as compared with WKY. Moreover, the differences in VSMC were due to altered regulation of MAPK phosphorylation and dephosphorylation rather than a difference in the relative abundance of the enzyme.^{22 23} Of interest is the observation that p38 and ERK₂ levels were the same in both the cell lines and the tissue samples, which demonstrated that deficit protein expression does not involve all members of the MAPK superfamily.

The pathogenesis and pathophysiology of essential hypertension are complex and are influenced by many interrelated factors.²⁴ The kidney is central to the pathogenesis of high blood pressure in salt-sensitive individuals, because its dominant role in the regulation of Na⁺ homeostasis. Studies on SHR suggest proximal tubular abnormalities in several signal transduction pathways, apart from MAPKs, that can affect NHE-3 activity and Na⁺ absorption.^{5 6 7} Our data document comparable basal levels of JNK₁ and ERK₁ activities and deficient activation in SHR cells and therefore suggest that the MAPK superfamily is unlikely to be a crucial regulatory factor for enhanced activity of proximal tubular NHE-3. This conclusion leaves other altered signaling pathways as possible explanations, such as defective receptor/G-protein coupling. Evidence for an abnormal G-protein coupling in SHR is emerging in the case of D₁ agonist inhibition of NHE-3.^{5 25}

The present study demonstrates different time-course and dose-response relationships when Ang II was added to the apical versus basolateral side of the polarized monolayer. This is not surprising given that the Ang II receptor subtypes differ in the 2 compartments.^{18 26 27} The apical angiotensin type 2 (AT₂) receptor is linked to phospholipase A₂ and arachidonic acid and inhibits Na⁺ reabsorption. Arachidonic acid is also critical for MAPK activation.^{14 15} By contrast, the basolateral angiotensin type 1 (AT₁) receptor has been linked to enhanced Na⁺ reabsorption, decrements in cAMP, and activation of MAPK as well.^{28 29 30} The mechanism of AT₁-mediated MAPK activation has not been determined. Several reports have documented that the AT₁ receptor increases activity of NHE,^{31 32} whereas the AT₂ receptor decreases activity of the NHE-3.³³ However, the importance of MAPKs as modulators of ion transport under physiological circumstances has not been resolved. This question remains of significant potential interest despite the exclusion of MAPKs as crucial factors for increasing proximal tubule Na⁺ transport in SHR.

	Acknowledgments

 
This work was supported by the National Institutes of Health (NIH) grants HL-44618 and HL-07714 to J.G.D., and a grant from the Ministry of University and Scientific and Technological Research (MURST 40%) to M.Z. Astrid Parenti was recipient of a fellowship from the Italian Pharmacology Society and by the National Heart, Lung, and Blood Institute (NHLBI). Xiao-Lan Cui was supported by NIH Training Grant HL-07714. We wish to thank Dr Philip G. Woost for helpful comments during the course of this work.

Received September 7, 1999; first decision September 28, 1999; accepted December 28, 1999.

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