Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Nov 3.
Published in final edited form as: Hypertension. 2008 Apr 7;51(5):1332–1338. doi: 10.1161/HYPERTENSIONAHA.107.102434

H2O2 Stimulation of the Cl−/HCO3− Exchanger by Angiotensin II and Angiotensin II Type 1 Receptor Distribution in Membrane Microdomains

Rui Pedrosa 1, Van Anthony M Villar 1, Annabelle M Pascua 1, Sónia Simão 1, Ulrich Hopfer 1, Pedro A Jose 1, Patrício Soares-da-Silva 1
PMCID: PMC2772095  NIHMSID: NIHMS151192  PMID: 18391104

Abstract

The present study tested the hypothesis that angiotensin II (Ang II)–induced oxidative stress and Ang II–stimulated Cl−/HCO3− exchanger are increased and related to the differential membrane Ang II type 1 (AT1) receptor and reduced nicotinamide-adenine dinucleotide phosphate oxidase expression in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) relative to its normotensive control (Wistar Kyoto rat [WKY]). The exposure of cells to Ang II increased Cl−/HCO3− exchanger activity with EC50s of 0.10 and 12.2 nmol/L in SHR and WKY PTE cells, respectively. SHR PTE cells were found to overexpress nicotinamide-adenine dinucleotide phosphate oxidase 2 and 4 and were endowed with an enhanced ability to generate H2O2. The reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor apocynin reduced the production of H2O2 in SHR PTE cells and abolished their hypersensitivity to Ang II. The expression of the glycosylated form of the AT1 receptor in both lipid and nonlipid rafts were higher in SHR cells than in WKY PTE cells. Pretreatment with apocynin reduced the abundance of AT1 receptors in both microdomains, mainly the glycosylated form of the AT1 receptor in lipid rafts, in SHR cells but not in WKY PTE cells. In conclusion, differences between WKY and SHR PTE cells in their sensitivity to Ang II correlate with the higher H2O2 generation that provokes an enhanced expression of glycosylated and nonglycosylated AT1 receptor forms in lipid rafts.

Keywords: Cl−/HCO3− exchanger, AT1R, H2O2, lipid rafts, hypertension, spontaneously hypertensive rats, Wistar Kyoto rats

An abnormal relationship between Na+/H+ exchanger (NHE) activity and natriuretic and antinatriuretic agents, such as dopamine or angiotensin II (Ang II), respectively, has been implicated in hypertension, but the link is less established for the anion exchangers. We have shown recently that D1-like dopamine receptor stimulation inhibits the activity of the Cl−/HCO3− exchanger in immortalized renal proximal tubular epithelial (PTE) cells from normotensive Wistar Kyoto rats (WKYs) but not in PTE cells from the spontaneously hypertensive rat (SHR).1 Most likely SHR cells have a defective transduction of the D1 receptor signal downstream to the Cl−/HCO3− exchanger, as shown for the NHE3 and Na+-K+-ATPase.2–4 On the other hand, preliminary studies showed that immortalized WKY and SHR PTE cells responded to Ang II with stimulation of Cl−/HCO3− exchanger activity through AT1 activation.5 However, SHR PTE cells were endowed with an enhanced sensitivity to Ang II–induced stimulation of the Cl−/HCO3− exchanger activity.5

Caveolae/lipid rafts are specialized membrane microdomains that coordinate and regulate a variety of signaling processes. A crosstalk among Ang II type 1 (AT1) receptors, lipid rafts, and reactive oxygen species derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase was postulated in vascular smooth muscle cells. Indeed, Ushio-Fukai et al6 have shown that transactivation of the epidermal growth factor receptor induced by Ang II is mediated through cSrc, a proximal target of reactive oxygen species derived from NADPH oxidase, and depends on AT1 receptor trafficking through lipid rafts. In addition, renal H2O2 that influences the development of hypertension and renal function7,8 is an indirect product of the NADPH oxidase activity, the expression of which is increased in the SHR.9

The study hypothesizes that PTE cells from SHRs have an enhanced oxidative stress condition coupled with NADPH oxidase expression that provokes changes in the function and membrane distribution of AT1 receptors. In line with this view, this study was designed to understand the relationships among the enhanced sensitivity of Ang II–induced stimulation of the Cl−/HCO3− exchanger, oxidative stress, and distribution of AT1 receptors in membrane lipid rafts in cultured, immortalized PTE cells from SHRs.

Methods

Cell Culture

Immortalized renal PTE cells from 4- to 8-week–old WKY rats and SHRs10 were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C (for details please see the data supplement available online at http://hyper.ahajournals.org).

Intracellular pH Measurements

In intracellular pH (pHi) measurement experiments, WKY and SHR PTE cells were grown in 96-well plates. pHi was measured as described previously.3

Cl−/HCO3− Exchanger Activity

The Na+-independent HCO3− transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal), in the absence of Na+, as described previously1 (for details please see the online data supplement).

AT1 Receptor and NADPH Oxidase Immunoblotting

WKY and SHR PTE cells cultured to 90% confluence were washed twice with PBS, and total cell protein was extracted for AT1 receptor NADPH oxidase (NOX) NOX2 and NOX4 detection (for protein extraction details please see the online data supplement). Proteins were subjected to SDS-PAGE (10% SDS polyacrylamide gel) and electrotransferred onto nitrocellulose membranes. The transblot sheets were blocked with 5% nonfat dry milk in Tris.HCl 25 mmol/L (pH 7.5), NaCl 150 mmol/L, and 0.1% Tween 20, overnight at 4°C. Then the membranes were incubated overnight at 4°C with polyclonal antibody rabbit anti-AT1 receptor (1:1000; Santa Cruz Biotechnology), goat polyclonal NOX2 (1:1000; Santa Cruz Biotechnology), goat polyclonal NOX4 (1:1000; Santa Cruz Biotechnology), and mouse monoclonal β-actin primary antibody (1:20 000; Laboratory Vision Corporation).

Membranes were incubated with appropriate fluorescently labeled secondary antibodies for 60 minutes at room temperature and protected from light, and an Odyssey Infrared Imaging System was used for detection. The immunoblots against the AT1 receptor were subsequently washed and incubated with fluorescent-labeled goat antirabbit (1:10 000; IRDye 800, Rockland). The immunoblots against NOX2 and NOX4 were subsequently washed and incubated with fluorescent-labeled donkey antigoat (1:20 000; IRDye 800, Rockland). The immunoblots against β-actin were subsequently washed and incubated with fluorescent-labeled goat antimouse secondary antibody (1:20 000; AlexaFluor 680, Molecular Probes). All of the membranes were washed and imaged by scanning at both 700 and 800 nm, with an Odyssey Infrared Imaging System (LI-COR Biosciences).

AT1 Receptor Expression in Lipid and Nonlipid Rafts

To prepare lipid raft and nonlipid raft fractions, the cell pellets were subjected to sucrose gradient centrifugation using a detergent-free protocol, with slight modifications.11 The cell pellets were lysed in 1.5 mL of sodium carbonate (500 mmol/L; pH 11) in clear ultracentrifuge tubes, homogenized with 15 strokes of Dounce homogenizer, and sonicated using three 30-second bursts of the tip sonicator. One mL of the homogenate was diluted in 2 volumes of 80% sucrose, overlaid with 6 mL of 35% sucrose and 3 mL of 5% sucrose, and spun at 160 000g in a Beckman SW40 rotor at 4°C for 18 hours. The sucrose solutions were prepared in MBS (25 mmol/L of 2-[N-morpholino] ethanesulfonic acid; pH 6.7; 150 mmol/L NaCl). After centrifugation, twelve 1-mL fractions were collected and labeled as fractions 1 to 12 from top to bottom, representing the lipid raft (1 to 6) and nonlipid raft (6 to 12) fractions. Aliquots of each fraction were mixed with Laemmli buffer, boiled for 5 minutes, and subjected to immunoblotting using a rabbit polyclonal anti-AT1R antibody (Santa Cruz, Biotechnology). Immunoblotting for flottilin-1, a specific lipid raft marker shown previously to be expressed in rat proximal tubule epithelial cells, was also performed to confirm the lipid and nonlipid raft fractions and for annexin 2, annexin 4, and annexin 5 (markers for intracellular membranes) to determine the presence of intracellular membrane proteins.

Measurement of H2O2

H2O2 was measured fluorometrically using the Amplex Red Hydrogen Peroxide Assay kit (Molecular Probes, Inc), as described previously.12

Data Analysis

Arithmetic means are given with SEM or geometric means with 95% confidence values. Statistical significance was determined using 1-way ANOVA, followed by the Student t test or the Newman-Keuls test for multiple comparisons. A P<0.05 was assumed to denote a significant difference.

Drugs

Apocynin, chelerythrine chloride, phorbol-12,13-dibutyrate, and PD 123319 were purchased from Sigma Chemical Co. Angiotensin II, PD 98059, SB 203580, U0126, and anisomycin were obtained from Research Biochemicals International. Losartan was obtained from Merck Portuguesa. Acetoxymethyl ester of 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein nigericin and the Amplex Red Hydrogen Peroxide Assay kit were obtained from Molecular Probes. Okadaic acid was obtained from Tocris Cookson. Anti–β-actin antibody was purchased from Laboratory Vision Corp. Anti-AT1 antibody was purchased from Santa Cruz Biotechnology.

Results

The activity of the Cl−/HCO3− exchanger was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in the absence of sodium to avoid the contribution of other transporters, such as the NHE3, as reported previously. 1 The addition of Ang II before (25 minutes) and during (10 minutes) the HCO3−-dependent recovery of pHi increased the Cl−/HCO3− exchanger activity in a concentration-dependent manner in WKY and SHR PTE cells (Figure S1). However, as observed in Figure S1, the sensitivity of Ang II–dependent stimulation of Cl−/HCO3− exchange activity was significantly higher in SHR than in WKY PTE cells. This is also evidenced by the 10-fold difference in EC50 values for the Ang II–induced stimulation of Cl−/HCO3− activity between SHR (0.10 nmol/L [0.09 to 0.12 nmol/L]) and WKY (12.2 nmol/L [11.3 to 13.2 nmol/L]) PTE cells. To investigate the type of Ang II receptor and the signal transduction pathway involved in the stimulation of the Cl−/HCO3− exchanger after exposure to Ang II, it was decided to use 2 different concentrations of Ang II that elicit similar increases in Cl−/HCO3− exchange activity in WKY and SHR PTE cells, ie, 30 and 1 nmol/L in WKY and SHR PTE cells, respectively. As shown in Figure S2, treatment of WKY PTE cells with 30 nmol/L Ang II increased Cl−/HCO3− exchange activity to an extent similar to that observed with 1 nmol/L Ang II in SHR PTE cells. As depicted in Figure S3, the effect of Ang II (30 nmol/L in WKY PTE cells and 1 nmol/L in SHR PTE cells) on the activity of the Cl−/HCO3− exchanger was completely blocked by the AT1 receptor antagonist losartan (100 nmol/L) in both SHR and WKY PTE cells. By contrast, the effect of Ang II was unaffected by the selective AT2 receptor antagonist PD 123 319 (100 nmol/L; Figure S3).

To evaluate the contribution of protein kinase C and mitogen-activated protein kinase (MAPK), which are normally related with signal transduction coupled to Ang II receptors and also linked to Ang II–induced stimulation of sodium reabsorption in kidney and vascular tissues,13,14 we used specific inhibitors for protein kinase C, MAPK/extracellular signal–regulated kinase kinase (MEK) kinases, and p38 MAPK. Pretreatment of cells for 30 minutes with the MAPK inhibitors PD 098059 (MEK 1 inhibitor) and U0126 (MEK 1/2 inhibitor) abolished the Ang II–induced stimulation of Cl−/HCO3− exchanger activity in both SHR and WKY PTE cells (Figure S4). A similar effect was obtained with the p38 MAPK inhibitor SB 203580, which completely abolished the Ang II–induced stimulation of Cl−/HCO3− exchanger activity (Figure S4). However, chelerythrine (1 µmol/L) did not affect the effect of Ang II in both WKY and SHR PTE cells (Figure S4).

Because recent studies have demonstrated a role for renal H2O2 in the development of hypertension and renal dysfunction, 8,15 particularly in the SHR model,9 it was felt worthwhile to measure H2O2 generation and evaluate the involvement of H2O2 in the regulation of Cl−/HCO3− exchanger activity by Ang II in WKY and SHR PTE cells. The SHR PTE cells had an increased rate of H2O2 production (50.7±0.4 versus 11.3±0.1 nmol/L per minute) when compared with WKY PTE cells (Figure 1A). Treatment of cells with apocynin (100 µmol/L), an inhibitor of the NADPH oxidase complex, for 4 days after seeding reduced the extracellular levels of H2O2 in a concentration-dependent manner in SHR PTE cells but not in WKY cells (Figure 1B).

Figure 1.

Figure 1

Open in a new tab

A, Rate of H2O2 (nmol/L per minute) released from WKY or SHR cell culture when cells reached confluence (4 days after of seeding). The inset shows the time course in real time scale. B, Levels of extracellular H2O2 (nmol/L) in WKY and SHR PTE cells in control cell culture conditions and in the presence of apocynin (100 µmol/L; 4 days after seeding). Each column represents the mean of 6 to 16 experiments per group, and vertical lines show SEM. Data are significantly different from corresponding values for WKY (*P<0.05) or the corresponding control values (#P<0.05) using the Newman-Keuls test.

NOXs are transmembrane proteins that transfer electrons across biological membranes to reduce oxygen to superoxide. To date, 7 members of the NOX family have been discovered. The NOX enzyme family has been identified as a major source of reactive oxygen species that include superoxide anion, hydrogen peroxide, and hydroxyl radical.16 As shown in Figure 2A, the level of expression of NOX2 was significantly higher in SHR than in WKY PTE cells. As shown in Figure 2B, the level of expression of the NOX4 was also significantly higher in SHR than in WKY PTE cells.

Figure 2.

Figure 2

Open in a new tab

Expression of NOX2 (A) and NOX4 (B) in WKY and SHR PTE cells. Representative immunoblots are depicted on top of the bar graphs. Columns represent mean of 4 independent immunoblots; vertical lines show SEM. NOX2: ≈70 kDa; NOX4: ≈70 kDa; β-actin: ≈40 kDa. Data show significantly different from values for WKY (*P<0.05) using the Student t test.

Because enhanced H2O2 generation in SHR cells maybe related to Ang II receptor activity, eventually resulting from the presence of angiotensinogen in the serum or the constitutively activity of the AT1 receptors, we treated WKY and SHR PTE cells for 4 days after seeding with losartan (100 nmol/L) or PD 123 319 (100 nmol/L); the antagonists did not change the levels of H2O2 generation in both types of cells (data not shown). The treatment with apocynin (100 µmol/L) for 4 days after seeding also did not change the basal Cl−/HCO3− exchanger activity in both WKY (0.084±0.004 versus 0.085±0.006 pH units per minute) and SHR (0.239±0.008 versus 0.240±0.020 pH units per minute) PTE cells. However, treatment of SHR PTE cells with apocynin (100 µmol/L) completely blocked the ability of Ang II to stimulate Cl−/HCO3− exchanger activity with no changes in WKY PTE cells (Figure 3A and 3B). To confirm the relationship among the NADPH oxidase, oxidative stress, and enhanced response to angiotensin II–mediated stimulation of Cl−/HCO3− exchanger activity in SHR cells, we decided to test a lower concentration of apocynin. As shown in Figure 3B, treatment of SHR PTE cells with apocynin (10 µmol/L) during 4 days after seeding reduced the sensitivity of these cells to Ang II–mediated stimulation of Cl−/HCO3− exchanger activity, to an extent identical to that observed in WKY PTE cells.

Figure 3.

Figure 3

Open in a new tab

Effect of apocynin (10 and 100 µmol/L; during 4 days after seeding) and Ang II (1 and 30 nmol/L for 25 minutes) on Cl−/HCO3− exchanger activity in WKY and SHR PTE cells. Each column represents the mean of 6 to 15 experiments per group; vertical lines indicate SEM. Data are significantly different from corresponding values for vehicle (*P<0.05) and corresponding values for control (#P<0.05) using the Newman-Keuls test.

The distribution and abundance of AT1 receptors in membrane microdomains were evaluated in WKY and SHR PTE cells using sucrose gradient fractionation followed by Western blotting using uniform amounts of proteins in corresponding fractions. Flotillin-1, a specific lipid raft protein marker17,18 shown previously to be expressed in rat membrane microdomains,11 was used to distinguish lipid from nonlipid raft fractions. In both cell lines, 2 distinct bands corresponding with the native, nonglycosylated (≈45 kDa) and glycosylated (≈60 kDa; preliminary data) forms of AT1 receptors were observed in both lipid (fractions 1 to 6) and nonlipid rafts (fractions 7 to 12; Figure 4). Apart from the difference in the distribution of the AT1 receptor in lipid and nonlipid rafts, the most significant difference was the greater abundance of the glycosylated form of the AT1 receptor in both lipid and nonlipid rafts in SHR PTE cells compared with WKY PTE cells. Pretreatment with apocynin (10 µmol/L) drastically reduced the AT1 receptor abundance in both microdomains in SHR PTE cells but not in WKY cells PTE (Figure 4). This effect was more marked in lipid rafts, particularly with the glycosylated form of the AT1 receptor. Ang II (1 nmol/L) slightly increased the amount of glycosylated AT1 receptor in lipid rafts in SHR PTE cells but not in WKY PTE cells. Pretreatment with apocynin (10 µmol/L) abolished the Ang II–induced increased abundance of the glycosylated AT1 receptor form in lipid rafts in SHR PTE cells (Figure 4).

Figure 4.

Figure 4

Open in a new tab

Distribution of the native nonglycosylated (≈45 kDa) and glycosylated (≈60 kDa) forms of AT1 receptors in rat membrane microdomains (lipid raft: fractions 1 to 6; nonlipid rafts: fractions 7 to 12) before and after Ang II (1 nmol/L) stimulation in the presence or in the absence of apocynin (Apo; 10 µmol/L) pretreatment (4 days after seeding) in WKY and SHR PTE cells. The distribution of flotillin-1, a marker of lipid rafts, is also shown. These experiments were performed thrice with similar results.

To determine the inclusion of intracellular membranes in the various fractions obtained after ultracentrifugation, the fractions were also probed for annexin 2, annexin 4, and annexin 5, which all belong to a family of calcium ion (Ca2+)–dependent, phospholipid-binding proteins involved in vesicle trafficking.19 Annexins are generally regarded as cytosolic proteins19 and, as such, are commonly used as markers for intracellular membranes. No bands were observed after probing for annexin 2 and annexin 4; however, bands corresponding with annexin 5 were observed mostly in nonlipid rafts (Figure S5). These data suggest that the signal bands observed in the lipid raft fractions are mainly surface membrane lipid raft proteins and are unadulterated by proteins found in intracellular membranes.

The AT1 receptor transcripts and AT1 receptor expression were also evaluated in total cell extract from immortalized WKY and SHR PTE cells by RT-PCR and by immunoblot analysis, respectively. As shown in Figure S6 and Figure S7, both the total expression of transcripts and protein of AT1 receptors in WKY PTE cells were similar to those in SHR PTE cells.

Discussion

The data reported here show that SHR PTE cells were 10 times more sensitive to Ang II than WKY PTE cells in stimulating Cl−/HCO3− exchanger activity. Despite differences in sensitivity to Ang II between SHR and WKY PTE cells, the results obtained indicated that Ang II–induced stimulation of Cl−/HCO3− exchanger activity was mediated through the activation of losartan-sensitive AT1 receptors coupled to MEK and MAPK pathways in both WKY and SHR PTE cells. The enhanced sensitivity to Ang II–induced stimulation of the Cl−/HCO3− exchanger activity through the AT1 receptor in SHR PTE cells was associated with the higher H2O2 generation that may have provoked an enhanced expression of glycosylated and nonglycosylated AT1 receptor forms in lipid rafts.

To elucidate the mechanism involved in the enhanced sensitivity to Ang II–mediated stimulation on the Cl−/HCO3− exchanger, we considered 3 possibilities. First, differences in signal transduction pathways between WKY and SHR PTE cells might explain the enhanced sensitivity to Ang II. Second, oxidative stress might influence the responses to Ang II. Third, differences between WKY and SHR PTE cells were because of differences in the expression of AT1 receptors.

To evaluate the contribution of MAPK in the signal transduction pathway coupled to Ang II–induced stimulation of Cl−/HCO3− exchanger activity, specific MEK and p38 MAPK inhibitors were used. The MEK1/MEK2 inhibitor U0126 and the specific MEK1 inhibitor PD98059 blunted the stimulation of Cl−/HCO3− exchanger activity induced by Ang II in both WKY and SHR PTE cells. Similarly, the p38 MAPK inhibitor SB 203580 blocked the stimulation of Cl−/HCO3− exchanger activity by Ang II in both WKY and SHR PTE cells. The results presented here clearly establish a connection between MEK and p38 MAPK in the signal transduction pathways coupled to Ang II–induced stimulation of Cl−/HCO3− exchanger activity in both WKY and SHR PTE cells. In conclusion, the signal transduction pathway coupled to Ang II–induced stimulation of Cl−/HCO3− exchanger activity in SHR PTE cells appears to be similar to that in WKY PTE cells.

Because of the finding that the signal transduction pathway associated with Ang II–induced stimulation of Cl−/HCO3− exchanger activity was similar in WKY and SHR PTE cells, it was hypothesized that oxidative stress, which has been clearly implicated in hypertension,7–9 could be involved in such differences in the response to Ang II in WKY and SHR PTE cells. To test this possibility, the generation of H2O2, a marker of oxidative stress, was measured in WKY and SHR PTE cells. SHR PTE cells were found to be endowed with an increased rate of H2O2 production, which was 5-fold that of WKY cells. As a result of this enhanced ability to generate H2O2, SHR PTE cells accumulated greater amounts of H2O2 in the extracellular medium. One of the mechanisms involved in the enhanced generation of H2O2 in SHR PTE cells could be the overexpression of NADPH oxidase, as has been found in the SHR.9 In fact, the abundance of NOX2 and NOX4 was found markedly increased in SHR PTE cells. The finding that immortalized renal PTE cells from the SHR overexpress NOX2 and NOX4 is in line with the finding that increased NOX2 and NOX4 mRNA and protein were found in the kidney of SHRs.9,20,21 The view that apocynin, an inhibitor of NADPH oxidase subunit assembly, clearly reduced the extracellular levels of H2O2 in SHR PTE cells but not in WKY PTE cells implicates this enzyme in the enhanced oxidative stress in SHR PTE cells. In SHR PTE cells, treatment with apocynin apart from decreasing the levels of H2O2 in the extracellular medium also abolished the enhanced sensitivity in Ang II–mediated stimulation of Cl−/HCO3− exchanger activity. It should be emphasized that, after treatment with apocynin, WKY PTE cells were endowed with identical sensitivity to Ang II in terms of the AT1 receptor–mediated stimulation of Cl−/HCO3− exchanger activity. This would agree with the view that the enhanced sensitivity to Ang II–mediated stimulation of Cl−/HCO3− exchanger activity in SHR cells is a consequence of the oxidative stress condition, resulting from increases in the generation of H2O2.

Next we evaluated whether the enhanced sensitivity to Ang II in SHR PTE cells was linked to differences in the expression of AT1 receptors. The distribution of AT1 receptors in lipid and nonlipid rafts was clearly distinct in WKY and SHR PTE cells. SHR PTE cells were found to be endowed with more AT1 receptors in both microdomains than WKY PTE cells, with particular emphasis in lipid rafts. Moreover, differences between WKY and SHR PTE cells were more pronounced in the heavy bands that correspond with the glycosylated form of the AT1 receptor, which correspond with the active form of the receptor (data not shown). These differences are in agreement with the view that SHR cells have an enhanced sensitivity to the Ang II–induced stimulation of Cl−/HCO3− exchanger activity.

Although differences in the distribution of the glycosylated and the nonglycosylated forms of the AT1 receptor in lipid and nonlipid rafts, per se, may explain the enhanced response of SHR PTE cells to Ang II–induced stimulation of Cl−/HCO3− exchanger activity, the relationship between that response and the higher H2O2 generation is not linear. To evaluate the relationship among oxidative stress, the enhanced response to Ang II, and differences in the distribution of the glycosylated and the nonglycosylated forms of the AT1 receptor in lipid and nonlipid rafts, we felt that it was worthwhile to evaluate the effect of apocynin on the distribution of AT1 receptors. In SHR but not in WKY PTE cells, a drastic reduction in the expression of AT1 receptors in both microdomains, especially in the glycosylated forms of the receptor in lipid rafts, was observed when cells were grown in the presence of apocynin. This result is in agreement with the view that apocynin changed neither the H2O2 generation nor the Ang II–induced stimulation of Cl−/HCO3− exchanger activity in WKY PTE cells. Moreover, 1 nmol/L Ang II, a concentration that only produced stimulation of Cl−/HCO3− exchanger activity in SHR PTE cells, slightly increased the amount of glycosylated AT1R in lipid rafts from SHR but not WKY PTE cells. Pretreatment with apocynin abolished the Ang II–induced increases in the glycosylated form of the AT1 receptor in lipid rafts in SHR PTE cells. All together these results demonstrate that the aberrant distribution of the AT1 receptor in lipid and nonlipid rafts in SHR PTE cells is a consequence of the enhanced H2O2 generation that leads to increases in the sensitivity to Ang II on the stimulation of the Cl−/HCO3− exchanger.

It is concluded that SHR PTE cells were 10 times more sensitive to Ang II than WKY PTE cells in stimulating Cl−/HCO3− exchanger activity. In both cell lines, the Ang II–mediated stimulation of Cl−/HCO3− exchanger activity proceeds through a molecular pathway that involves MEK and p38 MAPK downstream AT1 receptor activation. Differences between WKY and SHR PTE cells in their sensitivity to Ang II correlate with the higher H2O2 generation that provokes an enhanced expression of the glycosylated and nonglycosylated AT1 receptor form in lipid rafts.

Perspectives

Woost et al,10 in a clone of immortalized cells used in the current studies, demonstrated that SHR cells have an enhanced Ang II– dependent activation of NHE. However, a sustained activation of the NHE is only possible in the presence of a parallel increase of an acidifying pathway, such as the Cl−/HCO3− exchanger, as demonstrated in the current report. This is also in agreement with the view that Ang II leads to enhanced proximal tubular sodium reabsorption in proximal tubular cells of SHRs.10,22–24

Our study also confirms the view that H2O2 influences renal ion transport.7,8 The enhanced sensitivity to Ang II–mediated stimulation of Cl−/HCO3− exchanger activity that is associated with increased oxidative stress results from the enhanced generation of H2O2 by NADPH oxidase in SHR cells. Indeed, H2O2 causes the uncoupling of dopamine D1–like receptors from G proteins in renal proximal tubules of Sprague-Dawley rats.7 This is particularly relevant in renal proximal tubules of the SHRs, which have a defective transduction of the D1 receptor signal downstream to the Cl−/HCO3− exchanger, NHE3, and Na+-K+-ATPase.2–4 All together it is likely that oxidative stress, NADPH oxidase, Ang II, and the uncoupling of D1 receptor are involved in the development of hypertension in SHRs. This hypothesis needs to be confirmed by future studies “in vivo.”

Supplementary Material

01

Acknowledgments

Sources of Funding

This work was supported by Fundação para a Ciência e a Tecnologia, POCI, FEDER, and Programa Comunitário de Apoio (POCI/SAUFCF/ 59207/2004), HL074940, and HL68686.

Footnotes

Reprints: Information about reprints can be found online at http://www.lww.com/reprints

Disclosures

None.

References

  • 1.Pedrosa R, Jose PA, Soares-da-Silva P. Defective D1-like receptor-mediated inhibition of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells. Am J Physiol Renal Physiol. 2004;286:F1120–F1126. doi: 10.1152/ajprenal.00433.2003. [DOI] [PubMed] [Google Scholar]
  • 2.Hussain T, Lokhandwala MF. Altered arachidonic acid metabolism contributes to the failure of dopamine to inhibit Na+,K+-ATPase in kidney of spontaneously hypertensive rats. Clin Exp Hypertens. 1996;18:963–974. doi: 10.3109/10641969609097911. [DOI] [PubMed] [Google Scholar]
  • 3.Pedrosa R, Gomes P, Zeng C, Hopfer U, Jose PA, Soares-da-Silva P. Dopamine D3 receptor-mediated inhibition of Na+/H+ exchanger activity in normotensive and spontaneously hypertensive rat proximal tubular epithelial cells. Br J Pharmacol. 2004;142:1343–1353. doi: 10.1038/sj.bjp.0705893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Xu J, Li XX, Albrecht FE, Hopfer U, Carey RM, Jose PA. Dopamine1 receptor, Gsalpha, and Na+-H+ exchanger interactions in the kidney in hypertension. Hypertension. 2000;36:395–399. doi: 10.1161/01.hyp.36.3.395. [DOI] [PubMed] [Google Scholar]
  • 5.Pedrosa R, Soares-da-Silva P. AT1 receptors mediate the enhanced sensitivity to angiotensin II and upon overexpressed Cl−/HCO3−exchanger (SLC26A6) in hypertension. Nephrol Dial Transplant. 2006;21 suppl 4:iv24. [Google Scholar]
  • 6.Ushio-Fukai M, Zuo L, Ikeda S, Tojo T, Patrushev NA, Alexander RW. cAbl tyrosine kinase mediates reactive oxygen species- and caveolin-dependent AT1 receptor signaling in vascular smooth muscle: role in vascular hypertrophy. Circ Res. 2005;97:829–836. doi: 10.1161/01.RES.0000185322.46009.F5. [DOI] [PubMed] [Google Scholar]
  • 7.Asghar M, Banday AA, Fardoun RZ, Lokhandwala MF. Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2. Free Radic Biol Med. 2006;40:13–20. doi: 10.1016/j.freeradbiomed.2005.08.018. [DOI] [PubMed] [Google Scholar]
  • 8.Makino A, Skelton MM, Zou AP, Cowley AW., Jr Increased renal medullary H2O2 leads to hypertension. Hypertension. 2003;42:25–30. doi: 10.1161/01.HYP.0000074903.96928.91. [DOI] [PubMed] [Google Scholar]
  • 9.Adler S, Huang H. Oxidant stress in kidneys of spontaneously hypertensive rats involves both oxidase overexpression and loss of extracellular superoxide dismutase. Am J Physiol Renal Physiol. 2004;287:F907–F913. doi: 10.1152/ajprenal.00060.2004. [DOI] [PubMed] [Google Scholar]
  • 10.Woost PG, Orosz DE, Jin W, Frisa PS, Jacobberger JW, Douglas JG, Hopfer U. Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int. 1996;50:125–134. doi: 10.1038/ki.1996.295. [DOI] [PubMed] [Google Scholar]
  • 11.Yu P, Yang Z, Jones JE, Wang Z, Owens SA, Mueller SC, Felder RA, Jose PA. D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells. Kidney Int. 2004;66:2167–2180. doi: 10.1111/j.1523-1755.2004.66007.x. [DOI] [PubMed] [Google Scholar]
  • 12.Pedrosa R, Soares-da-Silva P. Oxidative and non-oxidative mechanisms of neuronal cell death and apoptosis by L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine. Br J Pharmacol. 2002;137:1305–1313. doi: 10.1038/sj.bjp.0704982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Du Z, Ferguson W, Wang T. Role of PKC and calcium in modulation of effects of angiotensin II on sodium transport in proximal tubule. Am J Physiol Renal Physiol. 2003;284:F688–F692. doi: 10.1152/ajprenal.00261.2002. [DOI] [PubMed] [Google Scholar]
  • 14.Bianchini L, L’Allemain G, Pouyssegur J. The p42/p44 mitogen-activated protein kinase cascade is determinant in mediating activation of the Na+/H+ exchanger (NHE1 isoform) in response to growth factors. J Biol Chem. 1997;272:271–279. doi: 10.1074/jbc.272.1.271. [DOI] [PubMed] [Google Scholar]
  • 15.Minuz P, Patrignani P, Gaino S, Degan M, Menapace L, Tommasoli R, Seta F, Capone ML, Tacconelli S, Palatresi S, Bencini C, Del Vecchio C, Mansueto G, Arosio E, Santonastaso CL, Lechi A, Morganti A, Patrono C. Increased oxidative stress and platelet activation in patients with hypertension and renovascular disease. Circulation. 2002;106:2800–2805. doi: 10.1161/01.cir.0000039528.49161.e9. [DOI] [PubMed] [Google Scholar]
  • 16.Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev. 2007;87:245–313. doi: 10.1152/physrev.00044.2005. [DOI] [PubMed] [Google Scholar]
  • 17.Dermine JF, Duclos S, Garin J, St-Louis F, Rea S, Parton RG, Desjardins M. Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes. J Biol Chem. 2001;276:18507–18512. doi: 10.1074/jbc.M101113200. [DOI] [PubMed] [Google Scholar]
  • 18.Foster LJ, De Hoog CL, Mann M. Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci U S A. 2003;100:5813–5818. doi: 10.1073/pnas.0631608100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Moss SE, Morgan RO. The annexins. Genome Biol. 2004;5:219. doi: 10.1186/gb-2004-5-4-219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chabrashvili T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita T, Welch WJ, Wilcox CS. Expression and cellular localization of classic NADPH oxidase subunits in the spontaneously hypertensive rat kidney. Hypertension. 2002;39:269–274. doi: 10.1161/hy0202.103264. [DOI] [PubMed] [Google Scholar]
  • 21.Zhan CD, Sindhu RK, Vaziri ND. Up-regulation of kidney NAD(P)H oxidase and calcineurin in SHR: reversal by lifelong antioxidant supplementation. Kidney Int. 2004;65:219–227. doi: 10.1111/j.1523-1755.2004.00372.x. [DOI] [PubMed] [Google Scholar]
  • 22.Cheng HF, Wang JL, Vinson GP, Harris RC. Young SHR express increased type 1 angiotensin II receptors in renal proximal tubule. Am J Physiol. 1998;274:F10–F17. doi: 10.1152/ajprenal.1998.274.1.F10. [DOI] [PubMed] [Google Scholar]
  • 23.Kost CK, Jr, Jackson EK. Enhanced renal angiotensin II subtype 1 receptor responses in the spontaneously hypertensive rat. Hypertension. 1993;21:420–431. doi: 10.1161/01.hyp.21.4.420. [DOI] [PubMed] [Google Scholar]
  • 24.Thomas D, Harris PJ, Morgan TO. Altered responsiveness of proximal tubule fluid reabsorption of peritubular angiotensin II in spontaneously hypertensive rats. J Hypertens. 1990;8:407–410. doi: 10.1097/00004872-199005000-00002. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS151192-supplement-01.pdf (180.6KB, pdf)

RESOURCES