Published online before print April 17, 2006, doi: 10.1161/01.HYP.0000220112.91724.fc
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(Hypertension. 2006;47:1117.)
© 2006 American Heart Association, Inc.
Original Articles |
Angiotensin II Type 2 Receptor Agonist Directly Inhibits Proximal Tubule Sodium Pump Activity in Obese But Not in Lean Zucker Rats
From the Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, Tex.
Correspondence to Tahir Hussain, Department of Pharmacological and Pharmaceutical Sciences, Science and Research Bldg 2, University of Houston, 4800 Calhoun, Houston, TX 77204-5037. E-mail thussain2{at}uh.edu
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Abstract |
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We have reported recently that the renal angiotensin II type 2 (AT2) receptors are upregulated and involved in promoting natriuresis/diuresis in obese but not in lean Zucker rats. In the present study, we tested the hypothesis that there is an enhanced AT2 receptor signaling via NO/cGMP pathway leading to greater inhibition of the Na+, K+-ATPase (NKA) activity in the proximal tubules (PT) of obese rather than lean Zucker rats. The AT2 agonist CGP42112 (0.1 to 100 nmol/L) inhibited (33% at 100 nmol/L) the NKA activity in the PTs of obese but not in lean Zucker rats. The AT2 antagonist PD123319 (1 µmol/L), not the angiotensin II type 1 antagonist losartan (1 µmol/L), significantly diminished the CGP42112-induced inhibition of the NKA activity in obese rats. The AT2 agonist (10 nmol/L)–induced NKA inhibition was abolished by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 µmol/L), the NO synthase inhibitor NG-nitro-L-arginine methyl ester (100 µmol/L), and the protein kinase G inhibitor K1388 (2 µmole/L). CGP42112 (10 nmol/L) caused an increase in serine phosphorylation of NKA
1-subunit in PT of obese rats. Measurement of cGMP and NO revealed that CGP42112 (0.1 to 100 nmol/L) increased cGMP and NO accumulation in the PTs of obese but not lean rats. The CGP42112-induced stimulation of NO and cGMP was blocked by PD123319 (1 µmol/L), NG-nitro-L-arginine methyl ester (100 µmol/L), and 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10 µmol/L) but not by losartan (1 µmol/L). The data suggest that the AT2 receptor activation via stimulation of the NO/cGMP/protein kinase G pathway directly inhibits the tubular NKA activity that provides as a mechanism responsible for the AT2 receptor–mediated natriuresis in obese but not in lean Zucker rats.
Key Words: diabetes mellitus • hypertension, sodium-dependent • kidney • sodium • epithelium
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Introduction |
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The renin–angiotensin system (RAS) is a major hormonal regulator of sodium homeostasis. The RAS mediates most of its effects via the octapeptide angiotensin II (Ang II). Ang II signals are mediated via 2 major receptor subtypes, Ang II type 1 (AT1) and Ang II type 2 (AT2),1 belonging to the G protein–coupled receptor superfamily. The AT1 receptors are ubiquitously expressed and are involved in mediating the Ang II–associated vasoconstriction, cell growth, and sodium retention.2 The AT2 receptors are expressed in various tissues3,4 and are associated with vasodilatation, apoptosis, antiproliferation, and natriuresis2,3,5 and, therefore, can be considered as functional antagonists to the AT1 receptors.
We have shown recently that the AT2 receptors are upregulated in the proximal tubular membranes of obese Zucker rats.3 Selective activation of the AT2 receptors by CGP42112 promotes natriuresis/diuresis to a greater degree in obese compared with lean Zucker rats.3 The obese Zucker rat is a genetic model of insulin resistance that develops mild hypertension.3,6 It has been shown that the RAS is overactive in obesity7 and that obesity-associated hypertension is linked to increased sodium reabsorption.8 The proximal tubules (PT) are involved in reabsorbing &75% of the filtered sodium.9 Ang II, via the activation of the AT1 receptors expressed on the PT membranes, activates the Na+, K+-ATPase10 (NKA), Na+, H+ exchanger,11 and Na+, HCO3– cotransporter12 and thereby contributes to sodium retention.
The NKA is a major active transporter involved in pumping sodium from the cytoplasm to the interstitium against its density gradient.9 We have reported recently that AT2 receptor activation causes NKA inhibition via the NO/cGMP pathway in the PTs of Sprague Dawley rats.13 Because AT2 receptors are upregulated in PTs, and the selective activation of the AT2 receptors causes greater natriuresis in obese Zucker rats,3 we hypothesized on an enhanced AT2 receptor–mediated NKA inhibition because of enhanced NO/cGMP signaling in PTs of obese Zucker rats. We found that selective activation of the AT2 receptors mediates an inhibitory effect on the NKA activity associated with enhanced serine phosphorylation of the NKA in the PT isolated from obese Zucker rats but not in lean rats. We also found that this inhibition was mediated via an NO/cGMP/protein kinase G (PKG) pathway, and the AT2 receptor activation stimulates NO/cGMP production in the PTs of obese but not lean rats.
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Methods |
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Animals
Male obese and lean Zucker rats (10 weeks of age) were purchased from Charles River Laboratories (Wilmington, MA). The animals were housed in the University of Houston animal care facility and had free access to standard rat chow and tap water. The Institutional Animal Use and Care Committee approved animal experimental protocols.
Preparation of Renal Proximal Tubular Suspensions
Renal proximal tubular suspensions were prepared as described previously.14,15 Rats were anesthetized with sodium pentobarbital (50 mg/kg, IP). After a midline incision, selective perfusion of the kidneys was performed with modified Krebs–Hensleit buffer containing collagenase type IV (230 U/mL) and hyaluronidase type III (250 U/mL). Kidneys were excised, and the outer cortex was removed, minced into fine pieces, and digested with collagenase–hyaluronidase solution under 95% O2:5% CO2 atmosphere until uniformly dispersed. Enrichment of PTs was carried out using 20% Ficoll density gradient. Trypan blue exclusion test was used to determine tubule cell viability. More than 95% of the tubules excluded trypan blue, indicating viable tubular preparation. Protein in the proximal tubular suspension was assayed using a kit (Pierce Products).
NKA Activity
The PT suspensions (1 mg/mL) were incubated without (basal) and with AT2 receptor agonist CGP42112 (0.1 to 100 nmol/L) in the presence and absence of the AT2 receptor antagonist PD123319 (1 µmol/L). The AT1 receptor antagonist losartan (1 µmol/L), the NO synthase inhibitor Nw-nitro-L-arginine methyl ester ([L-NAME] 100 µmol/L),16 and the NO-dependent soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one ([ODQ] 10 µmol/L)17 were added with the AT2 agonist CGP42112 (10 nmol/L) in the PT suspension. In a second set of experiments, the PT suspensions (1 mg/mL) were incubated without (basal) and with AT2 receptor agonist CGP42112 (0.1 to 100 nmol/L) in the presence and absence of the cAMP-dependent protein kinase A (PKA) and the cGMP-dependent PKG inhibitors K3761 (500 nmol/L) and K1388 (2 µmol/L), respectively. To test the effect of intracellular sodium on the CGP42112-mediated effect of NKA, PTs were incubated with CGP42112 (10 nmol/L) in the presence and absence of gramicidin (1 µmol/L), an antibiotic that forms pores in the plasma membrane and allows only monovalent ions to pass. These various inhibitors were added to the tubule suspension 10 minutes before the agonist was added. After incubation at 37°C for 30 minutes, the PTs were permeabilized by rapid freezing on a dry ice/acetone mixture and thawed and were used for the NKA activity assay. The ouabain (1 mmol/L)-sensitive NKA activity was determined as described previously.18,19 Briefly, the samples (100 µL each) were suspended in 1 mL of reaction mixture A (mmol/L: NaCl 70, KCl 5, MgCl2 5, NaN3 6, imidazole 37.5, NaEGTA 1, Tris-HCl 75; pH 7.4) for total ATPase activity and reaction mixture B (mmol/L: MgCl2 5, NaN3 6, imidazole 37.5, Na EGTA 1, Tris-HCl 150; pH 7.4) with ouabain 1 mmol/L for ouabain-insensitive ATPase activity. The reaction was initiated by the addition of 4 mmol/L ATP and incubated for 15 minutes at 37°C. The reaction was terminated by the addition of 50 µL of ice-cold trichloroacetic acid solution (50%). The tubes were transferred onto ice and kept for few minutes. Coloring reagent (1 mL; 5% FeSO4 in 1% ammonium molybdate in 1 N H2SO4) was added. The liberated inorganic phosphate (Pi) was determined by measuring the absorbance at 740 nm. The NKA activity was measured as a function of liberated Pi. The total ATPase activity minus the ATPase activity in presence of ouabain (nonspecific) represents the specific NKA activity.
cGMP and cAMP Measurement
The PTs (1 mg/mL) were incubated without (basal) and with CGP42112 (0.1 to 100 nmol/L) at 37°C for 30 minutes in a shaking water bath. Various inhibitors, PD123319 (1 µmol/L), L-NAME (100 µmol/L), and ODQ (10 µmol/L) were added to the tubular suspension 10 minutes before CGP 42112 (10 nmol/L). After incubation, samples were boiled for 5 minutes to stop the reaction. The samples were acidified by adding 10 µL of 0.1 N HCl and were centrifuged for 10 minutes at 600g. The supernatant was aliquoted and stored at –20°C for cGMP and cAMP determination using ELISA kits (R&D Systems). A set of standards for cAMP and for cGMP was assayed in duplicate along with the samples. Nonspecific binding and the background were subtracted from each reading, and the average optical density was calculated. Values were presented as picomole of cGMP or cAMP per milligram of protein.
Nitrite/Nitrate Measurement
Total nitrites/nitrates were determined as a measure of NO production using an enzymatic kit (R&D Systems). The PTs (1 mg/mL) were incubated without (basal) and with CGP42112 (0.1 to 100 nmol/L) for 30 minutes at 37°C in a shaking water bath. When applicable, the PTs were incubated with PD123319 (1 µmol/L) and L-NAME (100 µmol/L) 10 minutes before CGP42112 (10 nmol/L) was added. After incubation, samples were boiled for 5 minutes to stop the reaction. After boiling, the samples were filtered by centrifuging at 5000g for 1 hour in 10 000-molecular weight protein cutoff centrifuge tubes (Millipore). The filtrate was aliquoted and stored at –20°C for nitrite/nitrate measurement. A set of standards (3.25 to 100 pmol/L) was assayed in duplicate along with the samples. The background was subtracted from each reading, and the average optical density was calculated. The values are represented as picomole of nitrite/nitrate per milligram of protein.
Immunoprecipitation of NKA
Proximal tubular suspensions isolated from lean and obese Zucker rats were incubated in the absence and presence of CGP42112 (10 nmol/L) for 30 minutes as described above. After incubation, the samples were frozen on acetone and dry ice and stored at –70°C until use. The immunoprecipitation was carried out as described previously.20 The samples were thawed on ice, then homogenized in immunoprecipitation buffer containing (mmol/L) Tris-HCl 20, NaCl 150, NaF 10, Na2P4O7 10, EDTA 2, PMSF 0.1, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor mixture. Homogenates were centrifuged at 8000g for 15 minutes, and the supernatant was precleared with protein A/G plus agarose for 30 minutes. The precleared supernatant was incubated with mouse monoclonal antibody for the 1-subunit of NKA. The antigen–antibody complex was precipitated using protein A/G with agarose. The complex was then washed with immunoprecipitation buffer and once with 50 mmol/L of Tris-HCl. All of the steps were carried out at 4°C. The antigen–antibody complex was dissociated with 50 µL of 2x Laemmli buffer (125 mmol/L Tris-HCl, 4% SDS, 5% ß-mercaptoethanol, and 20% glycerol).
Western Blotting
The immunoprecipitated samples (3 µL each) were resolved by SDS-PAGE (10%). The proteins were electrophoretically transblotted on PVDF membranes (blot). The blots were incubated with mouse monoclonal phosphoserine antibody or mouse monoclonal 1-subunit NKA antibody for 1 hour. Anti-mouse IgG-horseradish peroxidase conjugate and chemiluminescent substrate were used to detect the signal that was recorded on x-ray film. The values are presented as the ratio of the phosphoband to the total NKA band in respective lanes. The specification of the phosphoserine and NKA antibodies has been confirmed by us earlier.21
Materials
CGP42112, PD123319, L-NAME, ODQ, K3761, K1388, and all of the other chemicals were purchased from Sigma Aldrich. Losartan was a generous gift from Merck Sharp & Dohme. Antibody for NKA 1-subunit was purchased from Research Diagnostics. Phosphoserine antibody was purchased from Calbiochem.
Statistical Analysis
Data are presented as mean±SEM. One-way ANOVA with post-hoc tests (Neumann–Keul) was used to analyze variation within the group. Student t test was used to compare variation between groups. We used a total of 9 lean and 11 obese rats. All of the statistical analyses were done using Graph Pad Prism, version 3.02 (GraphPad Software). A value of P<0.05 was considered statistically significant.
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Results |
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Effect of AT2 Receptor Agonist on NKA Activity
The AT2 receptor agonist CGP42112, in a dose-dependent manner (0.1 to 100 nmol/L), inhibited the NKA activity (Figure 1) in PTs isolated from obese but not lean Zucker rats. The minimal inhibitory effect of &6% was observed at 100 pmol/L, and the maximal inhibitory effect of &33% was observed at 100 nmol/L of the agonist. The ouabain-insensitive (Mg+-ATPase) activity was not affected by the CGP42112 treatment (data not shown). The presence of the AT2 receptor antagonist (PD123319, 1 µmol/L) attenuated the CGP42112-mediated inhibition at all of the concentrations in obese rats while not affecting the NKA activity in lean rats. This suggested that the inhibition of NKA in PTs isolated from obese rats was AT2 receptor mediated (Figure 1). PD123319 by itself did not affect the basal NKA activity in obese (basal, 158±5 versus PD123319, 163±6 nmol Pi/mg protein per minute) or in lean (basal, 167±15 versus PD123319, 170±13 nmol Pi/mg protein per minute) rats. We tested various inhibitors to investigate the potential pathway involved in mediating the CGP42112 (100 nmol/L)-induced inhibition of the NKA activity in obese rats. The AT1 receptor antagonist losartan (1 µmol/L) did not alter the inhibitory effect of CGP42112 (10 nmol/L) on the NKA activity (Figure 2A) in obese rats. The NO synthase inhibitor L-NAME (100 µmol/L) and the soluble guanylate cyclase inhibitor ODQ (10 µmol/L) abolished the AT2 receptor agonist–induced inhibition of NKA activity (Figure 2A) in obese rats. These inhibitors on their own had no effect on the basal NKA activity in obese rats (basal, 158±5 versus L-NAME, 154±6 and ODQ, 155±4 nmol Pi/mg protein per minute) suggesting that the AT2 receptor–mediated inhibition of the NKA activity is NO and cGMP dependent. Treating the PT isolated from lean rats with CGP42112 (10 nmol/L), L-NAME (100 µmol/L), or ODQ (10 µmol/L) did not affect the NKA activity (Figure 2A). The CGP42112-induced inhibition of the NKA activity in obese rats was abolished in the presence of the PKG inhibitor K1388 (2 µmol/L) and not the PKA inhibitor K3761 (500 nmol/L; Figure 2B). To investigate the role of intracellular sodium concentration on the CGP42112-induced NKA inhibition in obese rats, we used gramicidin (1 µmol/L) to create holes in the plasma membranes of PT cells and, therefore, diminish the sodium gradient in these cells. Gramicidin did not affect the CGP42112-induced NKA inhibition, suggesting that this AT2 effect is not dependent on the changes in intracellular sodium (basal, 160±8 versus CGP42112, 127±7 versus CGP42112+ gramicidin, 125±10; n=3).
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Effect of AT2 Receptor Agonist on cGMP Accumulation
The AT2 receptor agonist (CGP42112) in a dose-dependent manner (0.1 to 100 nmol/L) increased cGMP accumulation in the PTs (Figure 3A) isolated from obese but not lean rats. In the PT isolated from obese rats, the maximal stimulatory effect of &5-fold was observed with 100 nmol/L of the agonist. Preincubating the PTs with the AT2 receptor antagonist PD123319 (1 µmol/L) abolished the CGP42112 (10 nmol/L)-induced cGMP accumulation (Figure 3B) in obese but not in lean rats, suggesting the involvement of the AT2 receptors. PD123319 by itself did not significantly alter the basal cGMP levels in obese (basal, 0.4±0.07 versus PD123319, 0.5±0.06 pmol/mg protein) or in lean (basal, 0.5±0.09 versus PD123319, 0.4±0.07 pmol/mg protein) rats. The NO synthase inhibitor L-NAME (100 µmol/L) and the NO-dependent soluble guanylate cyclase inhibitor ODQ (10 µmol/L) both abolished the CGP42112-induced cGMP accumulation (Figure 3B) in obese but not lean rats, suggesting that the cGMP accumulation observed in PT isolated from obese rats is NO synthase dependent and soluble guanylate cyclase mediated. Both inhibitors alone did not significantly alter the basal cGMP levels in obese rats (basal, 0.4±0.07 versus ODQ, 0.33±0.09 and L-NAME, 0.44±0.07 pmol/mg protein). PT isolated from lean rats did not show cGMP accumulation when treated with CGP42112 (0.1 to 100 nmol/L) and, therefore, the various inhibitors used in the study did not have any effect (Figure 3A and 3B).
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Effect of AT2 Receptor Agonist on Nitrite/Nitrate Formation
Total nitrite/nitrate levels, a measure of NO production, were stimulated in the PTs isolated from lean and obese Zucker rats by the AT2 receptor agonist CGP42112 (100 pmol/L to 100 nmol/L) in a dose-dependent manner (Figure 4). The maximal stimulatory effect of &3.4-fold was observed at 100 nmol/L in PT isolated from obese rats. PT isolated from lean rats, when treated with CGP42112, did not have a significant change in nitrite/nitrate production (Figure 4A). Preincubating the PTs with the AT2 receptor antagonist PD123319 (1 µmol/L) abolished CGP42112 (100 nmol/L)-induced nitrite/nitrate formation in obese rats, suggesting the involvement of the AT2 receptors. The NO synthase inhibitor L-NAME (100 µmol/L) also abolished the AT2 receptor–mediated stimulation of nitrite/nitrate in obese rats, suggesting that it is NO synthase mediated. Because the PT isolated from lean rats did not show any change in nitrite/nitrate production, the treatment with the various inhibitors did not have any effect (Figure 4B). PD123319 or L-NAME alone did not affect the basal nitrite/nitrate levels in obese (basal, 2.2±0.3 versus PD123319, 2.0±0.3 and L-NAME, 1.8±0.2 nmol/mg protein) or in lean (basal, 2.4±0.2 versus PD123319, 2.0±0.3 and L-NAME, 1.9±0.2 nmol/mg protein) rats.
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Effect of AT2 Receptors on cAMP Levels
The AT2 receptor agonist (CGP42112) in a dose-dependent manner (0.1 to 100 nmol/L) inhibited cAMP accumulation to a similar extent in the PTs (Figure 5A) isolated from lean and obese rats. Preincubating the PTs with the AT2 receptor antagonist PD123319 (1 µmol/L) abolished the CGP42112 (10 nmol/L)-induced cAMP inhibition (Figure 5B), whereas the AT1 antagonist losartan (1 µmol/L) did not affect the inhibition in obese and lean rats, suggesting the involvement of the AT2 and not AT1 receptors. Neither the NO synthase inhibitor L-NAME (100 µmol/L) nor the NO-dependent soluble guanylate cyclase inhibitor ODQ (10 µmol/L) affected the CGP42112-induced cAMP inhibition (Figure 5B) in lean and obese rats, suggesting that the cAMP inhibition observed is not NO synthase dependent and soluble guanylate cyclase mediated as observed with the NKA inhibition. Both of the inhibitors alone did not significantly alter the basal cAMP levels in obese rats (basal, 0.045±0.005 versus ODQ, 0.04±0.005 and L-NAME, 0.046±0.007 pmol/mg protein).
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Effect of AT2 Receptor Agonist on NKA Phosphorylation
The monoclonal antibody against the NKA 1-subunit detected a single band (&100 kDa) in the immunoprecipitated samples (Figure 6A, bottom). The phosphoserine antibody also detected a major band at &100 kDa (Figure 6A, top). The effect of the AT2 agonist CGP42112 on NKA phosphorylation was presented as the ratio of the density of phosphorylated NKA to the total NKA protein (Figure 6B). CGP42112 (10 nmol/L) enhanced the level by 50% of phosphorylated NKA in PT isolated from obese but not lean rats (Figure 6B). The enhanced level of phosphorylation of the NKA suggests a direct effect of the AT2 receptor activation on the NKA activity.
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Discussion |
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Obesity is a major risk factor for the development of hypertension.22 Although the mechanism responsible for obesity related hypertension is not understood, numerous reports suggest that a balance between antinatriuretic and natriuretic hormone function on renal sodium metabolism is impaired, which contributes to the development of hypertension in obesity.6 The obese Zucker rat is a genetic model of insulin resistance and exhibits mildly elevated blood pressure.3,6 This animal model has close resemblance to the human metabolic syndrome. We have shown that, on one hand, the renal dopamine D1 receptor, a natriuretic hormone receptor, function is defective,18 and on the other hand, Ang II function with an antinatriuretic effect is hyperactive in obese Zucker rats.11 However, we reported recently that AT2 receptors are upregulated in the cortical membranes of obese Zucker rats, and on selective activation, the AT2 receptors mediate greater natriuresis in these animals.3 Obese Zucker rats respond with greater natriuresis/diuresis when infused with candesartan, an AT1 receptor antagonist,3,23 which was initially believed to be because of upregulation of the AT1 receptor function. However, the AT2 receptor antagonist PD 123319 infusion abolished candesartan-induced natriuresis in obese rats. Also, selective stimulation of the AT2 receptors by CGP42112 produced, without affecting glomerular filtration rate or blood pressure, greater diuresis/natriuresis in obese rats compared with lean rats.3
In an attempt to investigate the signaling mechanism of AT2 receptor function, we have reported that AT2 receptor activation via the NO/cGMP pathway inhibits NKA activity in the PTs of Sprague Dawley rats,13 where we have also shown that AT2 receptors mediate natriuresis.13 In the present study, we tested the hypothesis that AT2 receptor activation produces greater inhibition in the NKA activity in the isolated PTs of obese compared with lean Zucker rats. Interestingly, we found that AT2 receptor agonist CGP42112 produced concentration-dependent inhibition of the NKA activity in the PT suspension prepared only from obese but not lean Zucker rats. Because L-NAME and ODQ abolished the CGP42112-induced NKA inhibition in obese Zucker rats, it suggested the role of NO/cGMP pathway in the AT2 receptor-mediated NKA inhibition. Earlier, NO was suggested as the primary second messenger leading to the physiological effects mediated by the AT2 receptor activation.24 The AT2 receptor activation increases NO and cGMP production in the kidney, including in the PTs.25 The cGMP is known to mediate a vast array of signaling pathways,25 including stimulation of cGMP-dependent protein kinase (PKG) and modulation of phosphodiesterase activity.25 The PKG is a potential enzyme that may affect NKA activity directly,26 and phosphodiesterase causes changes in the cAMP metabolism, hence affecting the activity of PKA, an enzyme shown to regulate NKA activity.9 Here we found that PKG inhibitor, not PKA inhibitor, completely abolished the CGP42112-induced NKA inhibition in obese Zucker rats, suggesting the role of PKG in AT2 receptor–mediated NKA inhibition.
The NKA inhibition by CGP42112 may explain the exaggerated sodium excretion reported previously in obese Zucker rats in response to AT2 receptor agonist.3 However, it is interesting to note that, whereas AT2 receptors also are expressed in cortical membrane of lean rats, the AT2 receptor agonist CGP42122 completely failed to affect NKA activity, as well as to increase NO/cGMP levels in the PTs of lean Zucker rats. Similarly, we have shown that the AT2 receptor agonist also failed to promote natriuresis in lean Zucker rats.3 This may not be attributed to the expression of the nonfunctional AT2 receptors in lean rats, because the AT2 receptor agonist CGP42112, independent of the NO/cGMP pathway, reduces cAMP accumulation in the PTs of both lean and obese Zucker rats equally. This also suggests that, whereas the AT2 receptor activation inhibits cAMP generation, it does not seem to participate in the AT2 receptor–mediated NKA inhibition. The AT2 receptors are also expressed in distal nephron segments27; therefore, it is likely that the activation of the AT2 receptor will also lead to the NKA inhibition distal tubules in obese rats. Also, it is expected that, similar to the PTs, distal tubule NKA in lean Zucker rats may not be sensitive to the AT2 receptor activation.
The selective activation of the AT2 receptor signaling via NO/cGMP in obese Zucker rats seems to be proximal to the NO/cGMP and is not because of higher activity of the NO/cGMP pathway, per se, in the PTs of obese rats. This notion is supported by the reports that cortical NO synthase activity was lower28 and sodium nitroprusside, an NO donor that directly activates soluble guanylate cyclase, produced a similar degree of NKA inhibition in obese and lean Zucker rats.29 To test whether AT2 receptor-mediated NKA inhibition was a direct effect or whether changes in the sodium entry from apical side were responsible for this inhibition, we performed 2 sets of experiments. In 1 set, gramicidin, which disrupts the sodium gradient between the intracellular and extracellular spaces, did not affect the AT2-mediated inhibition of the NKA activity in obese rats. In the second experiment, we found that CGP42112 induced a significant increase in serine phosphorylation of the NKA 1-subunit in the PTs of obese and not in lean rats. The gramicidin and phosphorylation data suggest that AT2 receptor–mediated inhibition of the NKA activity is direct and not dependent on apical sodium entry. Other studies have shown that NO, which is a mediator of NKA inhibition in the present study, inhibits the NKA activity independent of Na,H-exchange inhibition or apical sodium entry into the cell.26,30 Serine phosphorylation of the NKA 1-subunit has been suggested as a mechanism of inhibition induced by dopamine and parathyroid hormone.21,31 Because the signaling (NO/cGMP/PKG) involved in AT2 receptor–mediated NKA inhibition is different than dopamine D1 and parathyroid hormone receptors9 (cAMP and protein kinase C), the intermediary mechanisms of NKA phosphorylation and inhibition by AT2 receptor may be different. The NKA protein expression, per se, may not contribute to the enhanced NKA inhibition by AT2 receptor, because the basal NKA activity observed in the present study and reported earlier,18,32 as well as the NKA protein expression,18,32 are similar in the PTs of lean and obese Zucker rats.
It is believed that hyperactivity of antinatriuretic hormones and defective natriuretic hormones contribute to the increased renal sodium absorption and the development of hypertension in obese Zucker rats.6 In numerous studies, we have shown that dopamine D1 receptor, which is known to increase tubular sodium excretion, is defective and unable to inhibit NKA activity in the PTs18 and promote natriuresis in response to D1 receptor agonist in obese rats.33 Although D1 receptor agonist was reported recently to promote AT2 receptor translocation to the membrane and thereby enhance the AT2 receptor function to promote natriuresis,34 it is unlikely that defective D1 receptor18 contributed to the enhanced AT2 receptor function as a compensatory mechanism in obese Zucker rats.18 Similarly, an upregulation of the renal AT2 receptor function on tubular sodium metabolism was found in streptozotocin-induced diabetic rats,35 where dopamine D1 receptors also are shown to be defective.36 In addition to the defective dopamine D1 receptor, we3,11 and others23 have reported enhanced Ang II function on tubular sodium transport and hyperantinatriuretic activity in obese compared with lean Zucker rats. Compared with the AT2 receptors, in general, the AT1 receptors are the predominant receptor subtype; therefore, the net effect of Ang II is believed to be mediated via the activation of the AT1 receptors. It is likely that the enhanced AT1 receptor function, along with defective dopamine D1 receptor function, lead to increased renal sodium retention in obese rats. It is important to note that because of likely dominance of the AT1 receptors, the effect of AT2 receptors is evident only by selectively activating with AT2 agonist or by blocking the AT1 receptor with AT1 antagonist.3,18 The AT2 receptor antagonist PD123319 alone did not affect the basal sodium excretion in obese Zucker rats.3 Although selective activation of the AT2 receptor inhibits sodium transport and increases its urinary excretion, whether AT2 receptors participate and provide a compensatory mechanism is yet to be determined. Long-term treatment of obese Zucker rats with selective AT2 receptor antagonist may shed light as to whether AT2 receptors are protective against enhanced renal sodium absorption and the development of sever hypertension. It should be noted that obese Zucker rats develop only mild hypertension.3,6
Perspectives
Recent focus on the understanding of the AT2 receptor function in renal sodium metabolism provides novel aspects of the RAS in the regulation of renal function under normal and pathophysiological conditions. Whereas the AT1 receptor is implicated in the stimulation of the tubular sodium transport, the AT2 receptor not only antagonizes the AT1 receptor function, but it also seems to produce direct effect on sodium excretion by inhibiting tubular NKA activity. These findings support the notion that selective activation of the AT2 receptors, in addition to selective inhibition of the AT1 receptors, may provide a better therapeutic approach to treat salt-sensitive hypertension, although it remains to be a subject of further investigation.
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Acknowledgments |
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This work is supported by the National Institutes of Health grant R01-DK61578. Losartan was a generous gift from Merck Sharp & Dohme (Australia).
Received February 23, 2006; first decision March 13, 2006; accepted March 22, 2006.
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References |
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