The Dopamine D₁ Receptor and Angiotensin Type-2 Receptor are Required for Inhibition of Sodium Transport Through a Protein Phosphatase 2A Pathway

Abstract

Activation of the renal dopamine D₁-like receptor (D₁R) or angiotensin II type-2 receptor (AT₂R), individually or both, simultaneously, is necessary in the normal regulation of renal sodium (Na⁺) transport and blood pressure. However, little is known regarding the precise mechanism of this interaction. Pharmacologic stimulation, membrane biotinylation, and cell surface immunofluorescence were used to study the effect of the D₁R/AT₂R interaction in human renal proximal tubule cells (RPTCs). D₁R activation of Gα_S stimulates adenylyl cyclase and induces apical plasma membrane recruitment of AT₂Rs. We now show for the first time the reciprocal reaction, AT₂R stimulation with angiotensin III leads to the apical plasma membrane recruitment of the D₁R. The cell-permeable second messenger analogs of cAMP (8-Br-cAMP) or cGMP (8-Br-cGMP) induce translocation of both D₁R and AT₂R to the plasma membrane. Inhibition of protein kinase A (PKA) with Rp-cAMPS and protein kinase G (PKG) with Rp-8-CPT-cGMPS blocks D₁R and AT₂R recruitment, respectively, indicating that both PKA and PKG are necessary for D₁R and AT₂R trafficking. Both 8-Br-cAMP and 8-Br-cGMP activate protein phosphatase 2A (PP2A) which is necessary for both plasma membrane recruitment of D₁R and AT₂R and the inhibition of NHE3-dependent Na⁺ transport. These studies provide insights into the D₁R/AT₂R transregulation mechanisms that play a crucial role in maintaining Na⁺ and ultimately blood pressure homeostasis.

Summary

This study extends previous observations about the D₁R and AT₂R role in regulating Na⁺ transport by demonstrating in human RPTCs that there is an absolute co-dependency between D₁R and AT₂R to inhibit Na⁺ transport despite their differences in second messenger linkage.

INTRODUCTION

The renal dopaminergic and renin-angiotensin systems are major regulators of sodium (Na⁺) homeostasis^1–4. Both dopamine D_1-like receptors (D₁R) and angiotensin II type-2 receptors (AT₂R) are natriuretic receptors, as stimulation by either dopamine or angiotensin III (Ang III), respectively, inhibits renal Na⁺ reabsorption, resulting in natriuresis^4,5. Upon agonist stimulation, D₁Rs are recruited by microtubules from the cytoplasm to the apical cell surface^6,7. Renal tubular plasma membrane D₁Rs are further stimulated by dopamine synthesized in the renal proximal tubule cell (RPTC) that is released in a paracrine manner, increasing G protein coupling and a subsequent activation of adenylyl cyclase (AC) and protein kinase A (PKA)^6,8. Renal angiotensin II (Ang II) is converted to Ang III by aminopeptidase A, which stimulates AT₂Rs to decrease Na⁺ transport through an increase in soluble guanylyl cyclase (GC) production of cGMP which stimulates protein kinase G (PKG)⁹. (Figure 6).

Figure 6: Suggested Model of D₁R and AT₂R Recruitment to the Apical Plasma Membrane of Human RPTCs.

Fenoldopam (FEN), adenylyl cyclase (AC), protein kinase A (PKA), angiotensin III (Ang III), guanylyl cyclase (GC), protein kinase G (PKG), and okadaic acid (OA).

Previous studies have not determined whether the D₁R or AT₂R acts independently or collectively to inhibit renal Na⁺ transport. In rodents, AT₂Rs are recruited to the RPTC apical plasma membrane upon D₁R stimulation and the AT₂R antagonist, PD-123319 (PD), blocks the D₁R-mediated natriuretic response, suggesting that AT₂R is necessary for D₁R activity¹⁰. In human RPTCs, we and others have reported that D₁R and AT₂R activation increases cAMP and cGMP, respectively, leading to a decrease in Na⁺ transport^11–14, but the interdependence of D₁R and AT₂R stimulation on receptor translocation to the apical plasma membrane is unknown. The goal of this study is to investigate further the relationship between the D₁R and AT₂R in human RPTCs. We have reported that the resensitization of the desensitized, phosphorylated D₁R is dependent on its dephosphorylation by protein phosphatase 2A (PP2A)^15–18. The current study tests the hypothesis that downstream activation of either D₁R or AT₂R converges at PP2A which is necessary for both receptors to be translocated to the apical cell surface to inhibit Na⁺ transport.

MATERIAL AND METHODS

The authors declare that the data that support the findings of this study are available from the corresponding author upon reasonable request.

Pharmacological agents

All pharmacological agents were purchased from Sigma-Aldrich. Fenoldopam, (FEN) is a D₁R agonist; it stimulates the two D₁-like receptors, D₁R and D₅R. 7-Methyl-6,7,8,9,14,15-hexahydro-5H-benz[d]indolo[2,3-g]azecine (LE300¹⁹) is a potent and selective D₁R antagonist²⁰. H-Arg-Val-Tyr-Ile-His-Pro-Phe-OH, Angiotensin III (Ang III) is the preferred endogenous AT₂R agonist²¹. 1-[[4-(Dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate, PD-123319 (PD) is a potent and selective non-peptide antagonist to the AT₂R²². 8-Bromoadenosine cyclic 3’,5’-monophosphate (8-Br-cAMP) is a cell-permeable cAMP analog. 8-Bromoguanosine cyclic 3’,5’-monophosphate (8-Br-cGMP) is a cell-permeable cGMP analog²³. Rp-adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS) is a PKA antagonist²⁴. 8-(4-chlorophenylthio) guanosine-3,5-cyclic monophosphorothioate, Rp-isomer triethylammonium salt (Rp-8-CPT-cGMPS) is a PKG antagonist²⁵. 9,10-Deepithio-9,10-didehydroacanthifolicin, Okadaic Acid (OA) is a PP2A antagonist¹⁴. Ouabain (OUB) is a Na⁺/K⁺ATPase alpha inhibitor. 3,3′-(4-Methyl-1,2-phenylene)bis[N-(aminoiminomethyl)-2-methyl-2-propenamide] dihydrochloride, S3226, is a potent and selective NHE3 inhibitor²⁶

Human RPTCs

Human kidney tissue and cell culture procedures were followed as reported previously^27,28. For more details, please see the Online Supplement.

Quantification of Cell Surface Recruitment of D₁R and AT₂R by Flow Cytometry

RPTCs were grown in iPT-G418 media in 100 mm plates for 2 days until 60–80% confluence at 37°C in full humidity with 5% CO₂. Vehicle (control) and agonists were added to cells and inhibitors were added 15 min prior to the addition of receptor agonists. Cells were stained in suspension and stained without permeabilization according to details found in the online supplement. Treated and stained cells were run on a benchtop flow cytometer with a robotic autoloader (Accuri C6Sampler, Accuri Cytometers).

Quantification of Cell Surface Recruitment of D₁R and AT₂R by 96-Well In-Cell Western

Procedures were followed as reported previously²⁹. For more details, please see the Online Supplement.

Biotinylation and Western Blot Analysis

Procedures were followed as reported previously^14,28. For more details, please see the Online Supplement.

PP2A Activity Assay

RPTCs at 80% confluence in 100 mm plates were incubated in serum-free DMEM-F12 media with or without agonists, washed, and lysed in phosphate-free buffer, according to kit instructions (Millipore, cat# 17–313). Briefly, 500 μg of protein were immunoprecipitated using a monoclonal antibody specific for the catalytic subunit of PP2A (clone 1D6). Phosphatase activity was measured by the release of phosphate from a phosphorylated peptide K-R-pT-I-R-R using malachite green by absorbance at 620 nm in a 96-well plate reader (BMG, Pherastar FSX) and compared to a standard curve made by diluting phosphate directly in malachite green.

NHE3-Mediated Na⁺ Accumulation Assay

Procedures were followed as reported previously²⁶. For more details, please see the Online Supplement.

Specific Protocols

1. Effects of D₁-Like Agonist FEN±D₁R Antagonist LE300 and the Effects of Endogenous AT₂R Agonist Ang III±AT₂R Antagonist PD on D₁R and AT₂R Recruitment Measured by Flow Cytometry.

Human RPTCs were treated as follows: (1) VEH (N=12); normal media, (2) FEN (N=6); 1 μmol/L for 30 min, (3) FEN+LE300 (N=6); pretreated with LE300 (1 μmol/L) for 15 min followed by 30 min co-incubation with FEN, (4) LE300 (N=6); 30 min, (5). Ang III (N=6); 10 nmol/L for 30 min, (6) Ang III+PD (N=6); pretreated with PD (1 μmol/L) for 15 min followed by 30 min co-incubation with Ang III, and (7) PD (N=6); 30 min.

2. Dose Response Curves for 8-Br-cAMP and 8-Br-cGMP to Measure Maximal D₁R and AT₂R Recruitment by Flow Cytometry.

Human RPTCs were treated as follows: 8-Br-cAMP and 8-Br-cGMP for 30 min (N=6 per dose): 10 mmol/L, 3 mmol/L, 1 mmol/L, 0.3 mmol/L, 0.1 mmol/L, 0.03 mmol/L, and 0.01 mmol/L.

3. Effects of 8-Br-cAMP and 8-Br-cGMP on D₁R and AT₂R Recruitment Measured by Flow Cytometry and Western Blot Analysis.

Human RPTCs were treated as follows: (1) VEH (N=15); normal media, (2) 8-Br-cAMP (N=15); 1 mmol/L for 30 min, (3) 8-Br-cGMP (N=15); 1 mmol/L for 30 min, and (4) 8-Br-cAMP+8-Br-cGMP (N=15); 30 min. In a separate set of experiments (N=3 for each condition), RPTCs were treated with either 8-Br-cAMP or 8-Br-cGMP to measure both D₁R and AT₂R recruitment by Western blot analysis.

4. Effects of 8-Br-cGMP±PKG Inhibitor Rp-8-CPT-cGMPS and the Effects of 8-Br-cAMP±PKA Inhibitor Rp-cAMPS on D₁R and AT₂R Recruitment Measured by Flow Cytometry.

Human RPTCs were treated as follows: (1) VEH (N=12), normal media, (2) 8-Br-cGMP (N=6); 1 mmol/L for 30 min, (3) 8-Br-cGMP+Rp-8-CPT-cGMPS (N=6); pretreated with Rp-8-CPT-cGMPS (100 μmol/L) for 15 min followed by 30 min co-incubation with 8-Br-cGMP, (4) Rp-8-CPT-cGMPS (N=6); 30 min, (5) 8-Br-cAMP (N=6); 1 mmol/L for 30 min, (6) 8-Br-cAMP+Rp-cAMPS (N=6); pretreated with Rp-cAMPS (100 μmol/L) for 15 min followed by 30 min co-incubation with 8-Br-cAMP, and (7) Rp-cAMPS (N=6); 30 min.

5. Effects of 8-Br-cAMP and 8-Br-cGMP on PP2A Activity.

Studies followed the same protocol stated in Protocol 3 and N=3 for each condition.

6. Effects of PP2A Inhibition with OA on 8-Br-cAMP- and 8-Br-cGMP-Induced D₁R and AT₂R Recruitment Measured by Flow Cytometry.

Human RPTCs were treated as follows: (1) VEH (N=6); normal media, (2) 8-Br-cAMP (N=6); 1 mmol/L for 30 min, (3) 8-Br-cGMP (N=6); 1 mmol/L for 30 min, (4) 8-Br-cAMP+OA (N=6); pretreated with OA (10 nmol/L) for 15 min followed by 30 min co-incubation with 8-Br-cAMP, (5) 8-Br-cGMP+OA (N=6); pretreated with OA for 15 min followed by 30 min co-incubation with 8-Br-cGMP, and (6) OA (N=6); 30 min.

7. Effects of PP2A Inhibition with OA on Na⁺ Transport.

Human RPTCs were treated as follows: (1) VEH (N=6); normal media, (2) 8-Br-cAMP (N=6); 1 mmol/L for 30 min, (3) 8-Br-cGMP (N=6); 1 mmol/L for 30 min, (4) 8-Br-cAMP+8-Br-cGMP (N=6); both for 30 min, (5) 8-Br-cAMP+OA (N=6); pretreated with OA (10 nmol/L) for 15 min followed by 30 min co-incubation with 8-Br-cAMP, (6) 8-Br-cGMP+OA (N=6); pretreated with OA for 15 min followed by 30 min co-incubation with 8-Br-cGMP, (7) 8-Br-cAMP+8-Br-cGMP+OA (N=6); pretreated with OA for 15 min followed by the co-incubation with 8-Br-cAMP+8-Br-cGMP, and (8) OA (N=6); 30 min.

Statistical Analysis

The data are presented as mean ± SEM. One-way ANOVA, followed by Tukey’s test was used to compare ≥3 groups. A two-tailed Student’s t-test was used to compare 2 groups. P<0.05 was considered statistically significant.

RESULTS

Flow Cytometry Analysis of the Effects of Exogenously Added FEN and Ang III on D₁R and AT₂R Recruitment.

Figure 1A. RPTCs stimulated with FEN (1 μmol/L, 30 min) increased the recruitment of the D₁R to the apical plasma membrane (1.56±0.07-fold, FEN vs. VEH, P<0.05, N=6). The addition of the D₁R antagonist LE300 (1 μmol/L, 30 min) + FEN significantly inhibited D₁R membrane recruitment (FEN vs. FEN+LE300, P<0.05, N=6 per group), while LE300, alone, had no effect compared to VEH. Figure 1B. RPTCs stimulated with FEN also increased the recruitment of the AT₂R to the apical plasma membrane (1.53±0.12-fold, FEN vs. VEH, P<0.05, N=6 per group), which was inhibited in the presence of LE300 (FEN vs. FEN+LE300, P<0.05, one-way ANOVA, N=6 per group). LE300 alone had no effect on AT₂R recruitment compared to VEH. Figure 1C. RPTCs stimulated with Ang III (10 nmol/L, 30 min) increased recruitment of the D₁R to the apical plasma membrane (1.39±0.06-fold, Ang III vs. VEH, P<0.05, N=6 per group). The addition of the AT₂R antagonist PD (1 μmol/L, 30 min) + Ang III significantly inhibited D₁R membrane recruitment (Ang III vs. AngIII+PD, P<0.05, N=6 per group), while PD alone had no effect compared to VEH. Figure 1D. RPTCs stimulated with Ang III increased the apical plasma membrane recruitment of the AT₂R (1.49±0.10-fold, Ang III vs. VEH, P<0.05, N=6 per group). The addition of the AT₂R antagonist PD+Ang III significantly inhibited AT₂R membrane recruitment (Ang III vs. AngIII+PD, P<0.05, N=6 per group), while PD alone, had no effect compared to VEH. This is the first time AT₂R stimulation with Ang III is shown to induce D₁R apical plasma membrane recruitment.

Figure 1: Flow Cytometry Analysis of the Effects of Exogenous FEN and Ang III Treatment on D₁R and AT₂R Recruitment.

FEN (1 μmol/L, 30 min) significantly increases cell surface D₁R (A) and AT₂R (B) in human RPTCs. *P<0.05 vs. VEH and #P<0.05 vs. FEN. Ang III (10 nmol/L, 30 min) significantly increases cell surface D₁R (C) and AT₂R (D), respectively. *P<0.05 vs. VEH and #P<0.05 vs. Ang III.

Dose-Response Analysis of 8-Br-cAMP and 8-Br-cGMP

Dopamine binding to the D₁R leads to AC activation and accumulation of cAMP³⁰. Similarly, Ang III binding to the AT₂R leads to GC activation and accumulation of cGMP³¹. Using cell permeable analogs of cAMP and cGMP, 8-Br-cAMP and 8-Br-cGMP, respectively, allows us to study downstream signaling leading to D₁R and AT₂R membrane trafficking. Concentration-response curves were measured for both 8-Br-cAMP and 8-Br-cGMP stimulation of D₁R and AT₂R apical plasma membrane recruitment, using a variant of an In-Cell Western blotting technique, where the directly labeled antibodies are added to cells without cell permeabilization. Semi-log dilutions from 10 mmol/L to 10 nmol/L for both 8-Br-cAMP and 8-Br-cGMP were added to RPTCs for 30 min and both D₁R and AT₂R plasma membrane recruitment were analyzed. A concentration-response curve for 8-Br-cAMP and 8-Br-cGMP showed maximal recruitment of the D₁R and AT₂R at 1 mmol/L for both 8-Br-cAMP Figure S1A and S1B and 8-Br-cGMP Figure S1C and S1D. For the D₁R, the EC₅₀ of 8-Br-cAMP was 296.1 μmol/L (Log EC₅₀ was −3.529) and the EC₅₀ of 8-Br-cGMP was 144.3 μmol/L (Log EC₅₀ was −3.841). For the AT₂R the EC₅₀ of 8-Br-cAMP was 206.4 μmol/L (Log EC₅₀ was −3.685) and the EC₅₀ of 8-Br-cGMP was 165.6 μmol/L (Log EC₅₀ was −3.781).

Flow Cytometry Analysis of 8-Br-cAMP, 8-Br-cGMP, or 8-Br-cAMP+8-Br-cGMP Stimulation on D₁R and AT₂R Recruitment

The Emax of 1 mmol/L, determined from Figure 2 for both 8-Br-cAMP and 8-Br-cGMP, measured by In-Cell Western experiments, was confirmed using flow cytometry (Figure 2A and 2B). 8-Br-cAMP, 8-Br-cGMP, or 8-Br-cAMP+8-Br-cGMP significantly increased the plasma membrane recruitment of D₁R (1.50±0.08-, 1.51±0.08-, and 1.56±0.0-fold, respectively, P<0.05, N=15 per group). 8-Br-cAMP, 8-Br-cGMP, or 8-Br-cAMP+8-Br-cGMP also significantly increased the plasma membrane recruitment of AT₂R (1.58±0.07-, 1.61±0.06-, and 1.58±0.08-fold respectively, P<0.05, N=15 per group). Interestingly, the simultaneous addition of both 8-Br-cAMP and 8-Br-cGMP did not cause an additional increase in apical plasma membrane recruitment, relative to either agonist alone.

Figure 2: Flow Cytometry and Western Blot Analysis of the Effects of Exogenously 8-Br-cAMP and 8-Br-cGMP Treatment on D₁R and AT₂R Recruitment.

8-Br-cAMP (1 mmol/L, 30 min) or 8-Br-cGMP (1 mmol/L, 30 min) significantly increases cell surface D₁R (A) and AT₂R (B) in human RPTCs measured by flow cytometry. *P<0.05 vs. VEH. 8-Br-cAMP or 8-Br-cGMP also significantly increases cell surface D₁R (C) and AT₂R (D) measured by Western blot analysis. All blots are normalized to β-actin and then to VEH. *P<0.05 vs. VEH.

Biotinylation and Western Blot Analysis of 8-Br-cAMP and 8-Br-cGMP Stimulation on D₁R and AT₂R Recruitment

Biotinylation and Western blot analysis were performed to verify the results generated from flow cytometry and In-Cell Western experiments. We measured D₁R (Figure 2C) and AT₂R (Figure 2D) recruitment when the cells were stimulated with 8-Br-cAMP and 8-Br-cGMP (both at 1 mmol/L) for 30 min. RPTCs stimulated with 8-Br-cAMP and 8-Br-cGMP induced an increase in apical plasma membrane-localized D₁R expression by 2.71±0.21-fold (P<0.05) and 1.98±0.31-fold (P<0.05), N=3 per group, respectively. 8-Br-cAMP and 8-Br-cGMP also induced an increase in apical plasma membrane-localized AT₂R expression by 2.11±0.42-fold (P<0.05) and 1.64±0.10-fold (P<0.05), N=3 per group respectively. These results are consistent with the flow cytometry results, indicating that the flow cytometry technique is a reliable method for measuring receptor recruitment.

Flow Cytometry Analysis of the Effects of PKG and PKA Inhibitors on Cell Surface D₁R and AT₂R Recruitment.

In order to determine, more precisely, the signaling downstream of cAMP and cGMP, we next examined the effect of PKA and PKG inhibition on D₁R and AT₂R recruitment in RPTCs. PKA and PKG are inhibited by Rp-cAMPS and Rp-8-CPT-cGMPS, respectively for 15 min prior to 8-Br-cAMP and 8-Br-cGMP stimulation. Exogenous 8-Br-cGMP (1 mmol/L, 30 min) increased the apical plasma membrane recruitment of D₁R (1.56±0.05-fold vs. VEH, *P<0.05, N=6), which was inhibited in the presence of the PKG inhibitor Rp-8-CPT-cGMPs, (100 μmol/L, #P<0.05, 8-Br-cGMP+Rp-c8 vs. 8-Br-cGMP, N=6). Rp-8-CPT-cGMPS, alone, had no effect (Figure 3A). Exogenous 8-Br-cAMP (1 mmol/L, 30 min) recruited AT₂Rs to the cell surface (1.58±0.04-fold vs. VEH, *P<0.05, N=6), which was inhibited in the presence of the PKA inhibitor Rp-cAMPS (100 μmol/L, #P<0.05, 8-Br-cAMP+Rp-cA vs. 8-Br-cAMP, N=6). Rp-cAMPS alone had no effect (Figure 3B). These results demonstrate that in the absence of PKA or PKG, neither D₁R nor AT₂R can be recruited to the cell surface, suggesting important roles for PKA and PKG in D₁R and AT₂R recruitment.

Figure 3. Flow Cytometry Analysis of the Effects of PKG and PKA Inhibition on D₁R and AT₂R Recruitment.

(A). In human RPTCs, 8-Br-cGMP (1 mmol/L, 30 min) recruits D₁R to the cell surface and the PKG inhibitor Rp-8-CPT-cGMPS (Rp-c8; 100 μmol/L) blocks this response. *P<0.05 vs. VEH and #P<0.05 vs. 8-Br-cGMP. (B). 8-Br-cAMP (1 mmol/L, 30 min) recruits AT₂R to the cell surface and the PKA inhibitor Rp-cAMPS (Rp-cA; 100 μmol/L) blocks this response. *P<0.05 vs. VEH and #P<0.05 vs. 8-Br-cAMP.

The Importance of PP2A in D₁R and AT₂R Recruitment

We have previously shown that the addition of the D₁R agonist FEN to RPTCs leads to the activation of PP2A^15–18 and AT₂R cell surface recruitment, which are inhibited in the presence of OA. However, we have not tested whether cAMP or cGMP is capable of directly activating PP2A and whether OA is capable of blocking 8-Br-cAMP- and 8-Br-cGMP-dependent D₁R or AT₂R apical plasma membrane recruitment. Figure 4A depicts the amount of phosphate released from the phosphorylated PP2A target peptide. PP2A activity increased by 1.78±0.12-fold (*P<0.05 vs. VEH, N=3) following the addition of 8-Br-cAMP. The addition of 8-Br-cGMP also induced a 1.77±0.14-fold increase in PP2A activity (*P<0.05, N=3). Either 8-Br-cAMP or 8-Br-cGMP stimulated the recruitment of D₁Rs to the cell surface (1.50±0.08-fold and 1.51±0.09-fold, respectively, *P<0.05, N=15) (Figure 4B). Either 8-Br-cAMP or 8-Br-cGMP also stimulated the recruitment of AT₂Rs to the cell surface (1.76±0.05-fold and 1.79±0.08-fold, respectively, *P<0.05, N=15) (Figure 4C). Recruitment of either D₁R or AT₂R using cell permeable downstream second messenger agonists 8-Br-cAMP or 8-Br-cGMP was completely blocked by OA (10 nmol/L), a specific PP2A inhibitor.

Figure 4: The Importance of PP2A on D₁R and AT₂R Recruitment.

(A). 8-Br-cAMP (1 mmol/L, 30 min) or 8-Br-cGMP (1 mmol/L, 30 min) increases PP2A activity in human RPTCs. *P<0.05 vs VEH. 8-Br-cAMP or 8-Br-cGMP significantly increases cell surface D₁R (B) and AT₂R (C) recruitment that is blocked with concomitant okadaic acid (OA, a specific PP2A inhibitor; 10 nmol/L) treatment. *P<0.05 vs. VEH and #P<0.05 vs. 8-Br-cAMP or 8-Br-cGMP.

Na⁺ Transport–Na⁺ Influx Assay

Both the D₁R and the AT₂R, as well as their respective endogenous agonists, dopamine and Ang III, respectively, have been implicated in inhibition of proximal tubule Na⁺ reabsorption that results in natriuresis. Using this cell culture model, we tested whether direct cytoplasmic stimulation with 8-Br-cAMP or 8-Br-cGMP inhibits Na⁺ transport and whether PP2A activity is necessary for this inhibition (Figure 5). 8-Br-cAMP (1 mmol/L, 30 min), 8-Br-cGMP (1 mmol/L, 30 min), or both 8-Br-cAMP+8-Br-cGMP significantly inhibited NHE3-mediated Na⁺ influx, (34.37±1.48, 27.74±1.58, 31.38±1.24 % reduction vs. VEH, respectively, *P<0.05, N=6) with no increase in the effect when combined. The PP2A inhibitor OA (10 nmol/L, 30 min) completely blocked the inhibition of NHE3-mediated Na⁺ influx, caused by 8-Br-cAMP, 8-Br-cGMP, or both 8-Br-cAMP+8-Br-cAMP (N=6/group).

Figure 5: Sodium (Na⁺) Transport – Na⁺ Influx Assay.

8-Br-cAMP (1 mmol/L, 30 min), 8-Br-cGMP (1 mmol/L, 30 min), or both 8-Br-cAMP+8-Br-cGMP significantly inhibits NHE3-mediated Na⁺ influx. *P<0.05 vs. VEH. The PP2A inhibitor okadaic acid (OA, 10 nmol/L) blocks the inhibition of NHE3-mediated Na⁺ influx caused by 8-Br-cAMP, 8-Br-cGMP, or 8-Br-cAMP+8-Br-cAMP. #P<0.05 vs. 8-Br-cAMP, 8-Br-cGMP, or 8-Br-cAMP+8-Br-cAMP.

Model Depicting Molecular Interactions

The model in Figure 6 summarizes our results. A slice of plasma membrane is depicted with attached microvilli. D₁R and AT₂R previously recruited to the apical plasma membrane appear on the membrane surface. Stimulation by D₁R agonist FEN and the endogenous AT₂R agonist Ang III [inhibited by the D₁R and AT₂R antagonists LE 300 (LE) and PD, respectively] causes an increase in D₁R and AT₂R recruitment to the apical plasma membrane (solid arrows) through a common serine/threonine PP2A-mediated mechanism. PP2A is stimulated via the D₁R stimulation of cAMP production via AC and PKA activation, as well as AT₂R stimulation of cGMP production via GC and PKG activation.

DISCUSSION

Cell surface membrane recruitment of G protein-coupled receptors from intracellular compartments is crucial to the renewal of response to autocrine and paracrine agonist stimulation. We have presented evidence that the D₁R- and AT₂R-mediated inhibition of RPTC Na⁺ transport are linked in order to assure redundancy in Na⁺ transport regulation. The cellular messengers associated with these receptors, PKA and PKG, are essential for D₁R and AT₂R recruitment through downstream activation via cAMP and cGMP.

Previous studies demonstrated that the D₁R inhibition of renal Na⁺ transport depends on activation of the AT₂R^10,21, Simultaneous stimulation of D₁R and AT₂R induces an increase in cGMP³². D₁R stimulation can also induce the recruitment of not only D₁R, but also AT₂R to the apical brush border membrane of human RPTCs¹⁴. However, cAMP, cGMP, or AT₂R stimulated D₁R translocation has not been reported. In the current study cell permeant cAMP and cGMP were able to induce both D₁R and AT₂R recruitment. The use of both compounds simultaneously does not synergistically or additively increase D₁R and AT₂R recruitment to the plasma membrane. The dose chosen for both 8-Br-cAMP and 8-Br-cGMP is the minimum dose that shows maximal translocation of these receptors and may have depleted the limited pool of internalized receptors. Alternatively the doses used could have maximally stimulated a common saturable shared pathway like PP2A. The selectivity of each cyclic nucleotide is quite high because of the relative affinities to their respective kinases, and the dose responses curves show that a dose of one tenth the chosen dose does not significantly activate recruitment of either receptor, suggesting that direct cross-activation is unlikely. The interdependence of these pathways is supported by the fact that Ang III stimulation of the AT₂R is also uncoupled to cGMP in the SHR³³. The D₁R is also uncoupled from cAMP, in the SHR and Dahl salt-sensitive rat^34,35. Future studies will focus on the role of dietary Na⁺ on the synthesis and release of dopamine relative to that of conversion of Ang II to Ang III.

The shared molecular mechanism how PKA or PKG activation of PP2A leads to translocation of D₁R and AT₂R is unknown. G Protein-Coupled Receptor Kinase 4 (GRK4) is known to phosphorylate and inactivate the D₁R and may be involved in a particular inherited form of human hypertension⁵. β-Arrestin2 is involved with internalization and inactivation of the D₁R³⁶. We hypothesize that PP2A dephosphorylates the D₁R, releasing the receptor from engagement with this well studied internalization mechanism. We have previously shown that the D₁R and AT₂R are found together by protein-protein interaction¹⁴, and perhaps are translocated together when released from internal pools.

Renal interstitial cGMP inhibits renal tubule Na⁺ reabsorption via PKG, which regulates Na⁺ excretion^37,38. In this study, the inhibition of either PKA or PKG prevents AT₂R and D₁R translocation respectively to the cell surface. This novel finding further extends the receptor-cAMP/cGMP model to include PKA and PKG which are also closely linked and that both PKA and PKG need to be present in order for D₁R and AT₂R to be recruited to the apical plasma membranes. We also blocked PP2A activity to determine if this is where these two pathways converge and become integrated into one signaling mechanism; our previous studies demonstrated the role of PP2A in D₁R signaling^15–18,39. PKA has been reported to activate PP2A by phosphorylating its regulatory subunit, B56δ⁴⁰. In the renal proximal tubule B56δ associates with and dephosphorylates NHE3 to facilitate the inhibitory effect of dopamine on NHE3 activity and expression⁴¹. PP2A has also been reported to be involved in the D₁R-mediated recruitment of Na⁺/K⁺ATPases from cytoplasmic pools into the basolateral membrane and its subsequent inhibition in human adenocarcinoma cells³⁹. We now report the convergence of D₁R- and AT₂R-mediated inhibition of luminal Na⁺ transport in human RPTCs at PP2A. This effect of PP2A may be a general phenomenon because we have also reported that the D₂R-mediated inhibition of inflammation is also mediated by PP2A⁴².

Perspective:

Previous studies have not determined whether the D₁R or AT₂R acts independently or collectively to inhibit up to 75% of renal Na⁺ transport. The phenomenon of linked G protein coupled receptors has not been readily appreciated until recently. Studies by others have demonstrated elaborate interactions such as allosteric interactions between monomer receptors either as pairs, oligomers, and receptor mosaic assemblies. These assemblies can inhibit or enhance each other’s activities, or have an absolute dependence on each other to exert their downstream activities, which is the case for the D₁R/AT₂R interaction. Future studies will focus on determining which other proteins are involved in the Na⁺ transport inhibition, as we found for PP2A, and whether there is symmetric or asymmetric allosteric modulation involved in their regulation of Na⁺ transport.

NOVELTY AND RELEVANCE.

What is new?

We have demonstrated that there is an absolute co-dependency between D₁R and AT₂R to inhibit Na⁺ transport despite their differences in second messenger linkage. This paper is the first to show that direct stimulation of PKA and PKG can induce both D₁R and AT₂R recruitment to the cell surface. Inhibition of either PKA or PKG prevents both D₁R and AT₂R recruitment, highlighting the co-dependency of third messengers in these mechanisms. Furthermore, these two interdependent pathways converge and increase the activity of PP2A which has been shown to increase both D₁R and AT₂R receptor activity.

What is relevant?

This study addresses the controversy over whether the D₁R or the AT₂R dominates over the regulation of Na⁺ excretion in the human RPTC. These studies pave the way to determine why codependent systems which regulate an important aspect of electrolyte balance are located in the same renal tubule segment.