Role of Gα₁₂- and Gα₁₃-protein subunit linkage of D₃ dopamine receptors in the natriuretic effect of D₃ dopamine receptor in kidney

Abstract

The D₃ dopamine receptor is the major D₂-like receptor that regulates sodium transport in the renal proximal tubule (RPT) and helps maintain blood pressure in the normal range. In Wistar–Kyoto (WKY) rats chronically fed high-salt diet, the intrarenal arterial infusion of a D₃ receptor agonist, PD128907, increased absolute and fractional sodium excretion. We have reported that Gα₁₂ and Gα₁₃, which participate in the signal transduction of the D₅ receptor, are expressed in RPTs. As the D₃ receptor is also expressed in RPTs, we hypothesized that it may also interact with Gα₁₂/Gα₁₃ in RPTs from WKY rats. There were co-localization and co-immunoprecipitation of D₃ receptor and Gα₁₂/Gα₁₃ in renal brush border membranes (BBMs) and RPT cells. The intrarenal infusion of PD128907 (1 μg kg⁻¹ min⁻¹) that increased sodium excretion also increased the co-immunoprecipitations of D₃/Gα₁₂ and D₃/Gα₁₃ in renal BBMs; their co-immunoprecipitation was confirmed in RPT cells. As Gα₁₂ and Gα₁₃ increase sodium pump and transporter activity (for example, Na⁺–K⁺–ATPase, NHE3), an increased association of D₃ receptors with Gα₁₂/Gα₁₃ receptors after D₃ receptor activation may be a mechanism to prevent Gα₁₂/Gα₁₃-mediated stimulation of sodium transport (and thus enhance natriuresis). We conclude that a D₃ receptor interaction with Gα₁₂/Gα₁₃ that increases sodium excretion may have a role in the regulation of blood pressure.

Introduction

Dopamine produced in neural and non-neural tissues is now recognized to serve an important role in the regulation of blood pressure and sodium balance by direct actions on renal and intestinal epithelial ion transport, by interaction with other receptors, by modulation of the secretion of hormonal/humoral agents, such as aldosterone, catecholamines, renin and vasopressin, and by actions on brain appetite centers.^{1, 2, 3} Dopamine receptors are classified into D₁- (D₁, D₅) and D₂-like (D₂, D₃ and D₄) subtypes based on their structure and pharmacology. Under euvolemic conditions or volume expansion, dopamine, via D₁-like and D₃ receptors, acts to increase sodium excretion and decrease blood pressure.^{1, 2, 3}

The effects of dopamine are exerted by cell surface receptors that belong to the rhodopsin-like or class A family of membrane receptors. These receptors, characterized by seven membrane-spanning domains, are called G protein-coupled receptors because of their interaction with heterotrimeric G proteins, composed of α, β and γ subunits.^{1, 2, 3, 4, 5} There are more than 20 Gα-subunits, grouped into four subfamilies (Gα_S, Gαi, Gαq and Gα12). In mammals, the two D₁-like dopamine receptors, D₁ and D₅, are coupled to the stimulatory Gα subunit (Gα_S) and Gαq,⁶ whereas the three D₂-like receptors, D₂, D₃ and D₄, are coupled to the inhibitory Gα subunit, Gα_i. Gα_S is stimulatory, whereas Gαi is inhibitory of adenylyl cyclase activity.^{1, 2, 3} However, D₃ receptor linkage to Gα_i is not robust, in contrast to that observed for the D₂ and D₄ receptors.⁷ In some instances, the D₃ can be linked to Gα_S, Gα_o and β/γ from Gα_i. Our previous study showed that Gα₁₂ and Gα₁₃, members of the fourth family of G protein subunits, are not linked to D₁ receptors, but are linked to D₅ receptors.⁸ As with the D₃ receptor,^{9, 10, 11} Gα₁₂ and Gα₁₃ are expressed in the kidney, especially in the renal proximal tubules (RPTs).⁸ However, it is not known whether or not Gα₁₂ and/or Gα₁₃ are also involved in the mechanisms by which the D₃ receptor promotes sodium excretion. Therefore, we studied the effect of the D₃ receptor on sodium excretion in normotensive Wistar–Kyoto (WKY) rats, and investigated the effect of the D₃ receptor on the linkage between D₃ receptor and the members of the fourth family of G protein subunits (Gα₁₂ and Gα₁₃) in kidney and RPT cells from WKY rats.

Methods

Blood pressure and renal function studies in rats

Nine- to 16-week-old WKY rats (n=6) (Taconic Farms, Germantown, NY, USA) were maintained on rat chow (6% NaCl) until one day before the experiment; water was given ad libitum. The rats were anesthetized with pentobarbital (50 mg per kg body wt i.p.), placed on a heated board to maintain their body temperature at about 37 °C and then tracheotomized. Anesthesia was maintained by infusion of pentobarbital at 0.8 mg per 100 g body wt per hour. Catheters (PE-50) were placed into the external jugular and femoral veins and femoral artery. Systemic arterial pressure was monitored electronically (Cardiomax II, Columbus Instruments, Columbus, OH, USA). Laparotomy was performed and both the right and left ureters were catheterized (PE-10). The right renal artery was exposed; the right suprarenal artery, which originates from the right renal artery, was catheterized (PE-10 heat stretched to 180 μm); and the vehicle (saline) or PD128907 (1 μg kg⁻¹ min⁻¹)¹² was infused at a rate of 40 μl h⁻¹.¹³ The duration of the surgical procedures was about 60 min. Fluid losses during surgery were replaced with 5% albumin at 1% body weight over 30 min. Glomerular filtration rate was determined by the clearance of [¹⁴C]-inulin (NEN, Boston, MA, USA) in normal saline infused at 5 ml per 100 g body wt for 30 min, followed by a rate of 0.8 ml per 100 g body wt per h until the end of the experiment, as previously reported.¹⁰ After an equilibration period of 120 min, urine was collected every 40 min for clearance measurements.¹³

Preparation of renal brush border membranes (BBMs)

Kidneys were obtained from WKY rats. Renal BBMs were prepared by MnCl₂ precipitation and differential centrifugation and studied under approved protocols with institutional guidelines.¹⁰ The BBMs have no immunoblottable sodium-hydrogen exchanger 1 (NHE1) and Na⁺–K⁺–ATPase (markers for basolateral membranes), but express immunoreactive NHE3, γ-glutamyl transpeptidase and alkaline phosphatase (markers for BBMs), indicating minimal contamination with basolateral membranes.^{14, 15} Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).

Cell culture

Immortalized RPT cells from 4- to 8-week-old WKY rats were cultured at 37 °C in 95% air/5% CO₂ atmosphere in DMEM/F-12.^{13, 16} The cells (80% confluence) were extracted in ice-cold lysis buffer, sonicated, kept on ice for 1 h and centrifuged at 16 000 g for 30 min. All samples were stored at −70 °C until use.

Co-localization of D₃ receptor and Gα₁₂, Gα₁₃ in RPT cells

RPT cells grown on coverslips were treated with PD128907 (10 nM) for 15 min, fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 in PBS and double immunostained as follows: D₃ receptor was probed using a polyclonal rabbit anti-D₃ receptor antibody (1:200; Abcam, Cambridgeshire, UK) followed by Alexa Fluor 488-donkey anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA), while the Gα₁₂ or Gα₁₃ was visualized using an IgG affinity-purified goat anti-Gα₁₂ or anti-Gα₁₃ antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by Alexa Fluor 555-donkey anti-goat IgG antibody (Molecular Probes). The cover slips were mounted on microscope slides using Fluoro-Gel mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA). Confocal and differential interference contrast images were obtained using Carl Zeiss LSM 510 META with an × 63/1.4 NA oil immersion objective and processed using Zeiss 510 META with Physiology Software ver. 3.5 and Multiple Time Series Software ver. 3.5 (Carl Zeiss International, Dublin, CA, USA).

Immunoprecipitation studies

BBMs or RPT cells were lysed with lysis buffer (50 mM Tris-Cl, pH, 7.4, 1% NP-40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 10 μg ml⁻¹ aprotinin and 10 μg ml⁻¹ leupeptin) on ice for about 1 h, and centrifuged at 16 000 g for 30 min. The lysates (supernatant, 300 μg protein ml⁻¹) were then incubated with affinity-purified anti-D₃ receptor antibodies (1 μg ml⁻¹) at 4 °C for 1 h and protein-G agarose at 4 °C for 2 h. The immunoprecipitates were pelleted and washed four times with lysis buffer. After the sample buffer was added, the samples were boiled for 10 min and subjected to immunoblotting with the Gα₁₂ or Gα₁₃ antibody. To determine the specificity of the bands found on the immunoblots, IgG (negative control) and Gα₁₂ or Gα₁₃ antibodies (positive control) were used as the immunoprecipitants instead of the D₃ receptor antibodies (data not shown).^{8, 13}

Na⁺–K⁺–ATPase activity assay

Rat RPT cells were treated with vehicle (dH₂O), or a D₃ receptor agonist (PD128907, Sigma, St Louis, MO, USA), at the indicated concentrations and durations of incubation. Na⁺–K⁺–ATPase activity was determined as the rate of inorganic phosphate released in the presence or absence of ouabain.¹⁷ To prepare membranes for Na⁺–K⁺–ATPase activity assay, RPT cells cultured in 21 cm² plastic culture dishes were collected and centrifuged at 3000 g for 10 min. The cells were then placed on ice and lysed in 2 ml of lysis buffer (1 mM NaHCO₃, 2 mM CaCl₂ and 5 mM MgCl₂). Cellular lysates were centrifuged at 3000 g for 2 min to remove intact cells, debris and nuclei. The resulting supernatant was suspended in an equal volume of 1 M sodium iodide, and the mixture was centrifuged at 48 000 g for 25 min. The pellet (membrane fraction) was washed twice and then suspended in 10 mM Tris containing 1 mM EDTA (pH 7.4). Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories) and adjusted to 1 mg ml⁻¹. The membranes were stored at −70 °C until further use. To measure Na⁺–K⁺–ATPase activity, 100 μl aliquots of membrane fraction were added to an 800 μl reaction mixture (75 mM NaCl, 5 mM KCl, 5 mM MgCl₂, 6 mM sodium azide, 1 mM Na₄EGTA, 37.5 mM imidazole, 75 mM Tris-HCl and 30 mM histidine; pH 7.4) with or without 1 mM ouabain (final volume=1 ml) and pre-incubated for 5 min in a water bath at 37 °C. Reactions were initiated by adding Tris-ATP (4 mM) and terminated after 15 min of incubation at 37 °C by adding 50 μl of 50% trichloroacetate. For determination of ouabain-insensitive ATPase activity, NaCl and KCl were omitted from the reaction mixtures containing ouabain. To quantify the amount of phosphate produced, 1 ml of coloring reagent (10% ammonium molybdate in 10 N sulfuric acid + ferrous sulfate) was added to the reaction mixture. The mixture was then mixed thoroughly and centrifuged at 3000 g for 10 min. Formation of phosphomolybdate was determined spectrophotometrically at 740 nm, against a standard curve prepared from K₂HPO₄. Na⁺–K⁺–ATPase activity was estimated as the difference between total and ouabain-insensitive ATPase activity and expressed as percent change of control.

To eliminate the effect of proteases and phosphatases, protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 μg ml⁻¹ each leupeptin and aprotinin) and a phosphatase inhibitor (50 μM sodium orthovanadate) were added in all solutions used after drug/vehicle incubations.¹⁸

Statistical analysis

The data are expressed as mean±s.e.m. Comparison within groups was made by repeated-measures analysis of variance, and comparison among groups was made by factorial analysis of variance and Duncan's test; t-test was used when only two groups were compared. A value of P<0.05 was considered significant.

Results

Stimulation of renal D₃ receptors increases sodium excretion in WKY rats

To determine the effect of D₃ receptors on sodium excretion, the D₃ receptor agonist PD128907 (1.0 μg kg⁻¹ min⁻¹ for four periods, each period lasting 40 min) was infused into the right renal artery in WKY rats (n=6) maintained on high sodium diet (6% NaCl), using a protocol reported previously.¹³ The intrarenal arterial infusion of the vehicle into the right kidney had no effect on blood pressure, urine flow (V), fractional sodium excretion (FE_Na), absolute sodium excretion (U_NaV), potassium excretion (U_KV) or glomerular filtration rate (data not shown). PD128907 had no effect on blood pressure (Figure 1a), but increased glomerular filtration rate, V, U_NaV and FE_Na in WKY rats (Figures 1b–e).

Figure 1

Effect of the intrarenal infusion of a D₃ receptor agonist (PD128907) on blood pressure and renal function in WKY rats. The D₃ receptor agonist, PD128907 (1.0 μg kg⁻¹ min⁻¹), was infused into the right renal artery of WKY rats. Blood pressure (a), glomerular filtration rate (b), urine volume (c), absolute sodium excretion (d) and fractional sodium excretion (e) were measured (n=6). During the control (C1–C3) and recovery periods, only the vehicle was infused; during periods 3–6 (P1–P4, respectively), PD128907 was infused. Each period lasted for 40 min. ^*P<0.05 vs. control, ANOVA, Duncan's test.

Stimulation of D₃ receptors inhibits Na⁺–K⁺–ATPase activity in RPT cells

To determine whether or not the natriuretic effect of D₃ receptor is related to inhibition of Na⁺–K⁺–ATPase activity, the effect of PD128907, on Na⁺–K⁺–ATPase activity was measured in RPT cells. We found that PD128907 inhibited Na⁺–K⁺–ATPase activity in a concentration-dependent manner. The inhibitory effect was evident at 10⁻⁸ M (Figure 2).

Figure 2

Effect of a D₃ receptor agonist (PD128907) on Na⁺–K⁺–ATPase activity in RPT cells. Concentration–response of Na⁺–K⁺–ATPase activity in RPT cells incubated with the D₃ receptor agonist, PD128907, for 15 min. Results are shown as % of control (n=6, ^*P<0.01 vs. control, ANOVA, Duncan's test).

D₃ receptor colocalizes with Gα₁₂ or Gα₁₃ in RPT cells

We next evaluated the colocalization of these proteins via laser-scanning confocal microscopy to determine the capacity of receptor and Gα subunits to interact in RPT cells. Under basal condition, both the D₃ receptor and Gα₁₂ are localized at the plasma membrane and the cytoplasm, where they partially colocalize. D₃ receptor stimulation with PD128907 promoted the endocytosis of both the receptor and Gα₁₂ and enhanced the extent of colocalization at the perinuclear area (Figure 3a). Similarly, D₃ receptor and Gα₁₃ are basally distributed and colocalized at the plasma membrane and cytoplasm. Receptor activation promoted the internalization of both proteins and markedly increased the colocalization between D₃ receptor and Gα₁₃ (Figure 3b).

Figure 3

Co-localization of Gα₁₂, Gα₁₃ and D₃ receptors in RPT cells. The cells grown on coverslips were serum-starved before being treated with PD128907 (10⁻⁸ M/15 min). The cells were then fixed and double-immunostained for D₃ receptor and Gα₁₂ or Gα₁₃, as described in Methods. Colocalization was evaluated via laser-scanning confocal microscopy and appears as yellow punctate areas in merge images between D₃R and Gα₁₂ (a) or Gα₁₃ (b). Differential interference contrast (DIC) images were obtained to indicate the cellular confines of the cells. Magnification × 600, scale bar=10 μm.

Immunoprecipitation of Gα₁₂, Gα₁₃ and D₃ receptors in RPT cells

To confirm the apparent interaction between Gα₁₂ or Gα₁₃ with D₃ receptors noted in the laser confocal microscopic studies, we determined whether or not Gα₁₂ or Gα₁₃ co-immunoprecipitated with D₃ receptors in renal BBMs and RPT cells. Gα₁₂ and Gα₁₃ co-immunoprecipitated with D₃ receptors in BBMs (Figures 4a and b) and this co-immunoprecipitation was increased following D₃ receptor agonist stimulation (PD128907, 1 μg kg⁻¹ min⁻¹) in WKY rats. There was negligible co-immunoprecipitation when the immunoprecipitant was IgG instead of anti-D₃ receptor antibodies (data not shown).

Figure 4

Co-immunoprecipitation of Gα₁₂ or Gα₁₃ and D₃ receptors in kidney and RPT cells. (a, b) Co-immunoprecipitation of Gα₁₂ or Gα₁₃ and D₃ receptors in rat kidney. BBMs were prepared from kidneys of WKY rats treated with vehicle or PD128907 (1.0 μg kg⁻¹ min⁻¹). The same amounts of protein were then immunoprecipitated with D₃ receptor antibody and immunoblotted for Gα₁₂ (a) or Gα₁₃ (b). For positive controls, anti-Gα₁₂ and Gα₁₃ antibodies (1 μg ml⁻¹) were used as the immunoprecipitants; for a negative control, normal rabbit IgG (1 μg ml⁻¹) was used as the immunoprecipitant instead of the anti-D₃ antibodies and immunoblotted with anti-Gα₁₂ or Gα₁₃ antibodies as above. ^*P<0.05 vs. control (n=6), ANOVA, Duncan's test, DU=density units. (c, d) Co-immunoprecipitation of Gα₁₂ or Gα₁₃ and D₃ receptors in RPT cells. RPT cells from WKY rats were treated with vehicle or PD128907 (10⁻⁸ M every 30 min). The same amounts of protein were then immunoprecipitated with D₃ receptor antibody and immunoblotted for Gα₁₂ (c) or Gα₁₃ (d). For positive controls, anti-Gα₁₂ and Gα₁₃ antibodies (1 μg ml⁻¹) were used as the immunoprecipitants; for a negative control, normal rabbit IgG (1 μg ml⁻¹) was used as the immunoprecipitant instead of the anti-D₃ antibodies and immunoblotted with anti-Gα₁₂ or Gα₁₃ antibodies as above. ^*P<0.05 vs. control (n=7), ANOVA, Duncan's test, DU=density units.

Consistent with the results of the in-vivo study, we found that in RPT cells the co-immunoprecipitation between D₃ receptor and Gα₁₂ or Gα₁₃ was increased by PD128907 (10⁻⁸ M/30 min) (Figures 4c and d).

Discussion

In the current report, we confirmed that stimulation of D₃ receptors with the D₃ receptor selective agonist, PD128907, increases sodium excretion in WKY rats. The natriuretic effect may be, in part, via Na⁺–K⁺–ATPase, as activation of the D₃ receptor inhibits Na⁺–K⁺–ATPase activity in RPT cells. We now report that there is a linkage between D₃ receptors and Gα₁₂, and D₃ receptors and Gα₁₃ in RPTs in native kidneys. We also report that the dose of PD128907 that increases sodium excretion in WKY rats increases the co-immunoprecipitation of D₃ receptors with Gα₁₂ or Gα₁₃ in renal BBMs, which was confirmed in RPT cells from WKY rats.

Dopamine receptors are classified into D₁- and D₂-like subtypes based on their structure and pharmacology.^{1, 2, 3} Under euvolemic conditions or volume expansion, dopamine receptors act to increase sodium excretion and normalize blood pressure.^{1, 2, 3, 19} Most in vivo studies have shown that the natriuretic effect of dopamine is exerted via D₁-like receptors.^{15, 20} The effect of D₂-like receptors, independent of D_l-like receptors, on sodium excretion has ranged from anti-natriuresis, to no effect, to natriuresis. It is possible that these discrepant effects are related to the use of drugs that have poor selectivity to the D₂-like receptor subtypes. Thus, bromocriptine, a drug that has a similar affinity to the D₂ receptor and D₃ receptor, stimulates sodium transport.^{21, 22} In contrast, 7-OH-DPAT and PD128907, which are D₃ receptor agonists with preferential selectivity for D₃ over D₂ and D₄ receptors, decrease sodium transport and increase sodium excretion.^{13, 23} Acute intravenous administration of 7-OH-DPAT in Dahl salt-resistant rats increases glomerular filtration rate and sodium and water excretion without affecting blood pressure.²³ That D₃ receptors can mediate natriuresis is supported by the decreased ability of D₃ receptor-deficient mice to excrete an acute saline load.²⁴ Consistent with our previous study,¹³ the intrarenal infusion of PD128907, a D₃ receptor agonist, increases sodium excretion in salt-loaded WKY rats.

We now report a linkage between the D₃ receptor and Gα₁₂ and the D₃ receptor and Gα₁₃. The D₃ receptor negatively regulates renin secretion.²⁴ Gα₁₂ and Gα₁₃ can increase intracellular calcium, and calcium can decrease renin secretion.^{25, 26} It is tempting to speculate that Gα₁₂ or Gα₁₃ may be important in the D₃ receptor-mediated negative regulation of renin secretion, as well as sodium transport in normotensive rats. Gα₁₂, Gα₁₃ and D₃ receptor influence sodium transport by regulating the activities of the Na⁺–K⁺–ATPase and sodium–hydrogen exchanger 1. Gα₁₂ and Gα₁₃ stimulate sodium–hydrogen exchanger 1,²⁷ whereas D₃ receptor inhibits Na⁺–K⁺-ATPase and NHE3 activities^{9, 28, 29} in adult WKY rats. The fact that a D₃ receptor agonist increases the interaction between the D₃ receptor with either Gα₁₂ or Gα₁₃ in renal BBMs and RPT cells from WKY rats suggests that the D₃ receptor may participate in the regulation of sodium transport/pump activity by hampering Gα₁₂ or Gα₁₃ action, similar to that suggested for the D₅ receptor.⁸ It is possible that the increase in co-immunoprecipitation following agonist stimulation between the D₃ receptor, Gα₁₂ and Gα₁₃, and therefore, reduction of ‘free’ Gα₁₂ and Gα₁₃, may explain the inhibitory effect of D₃ receptor on other sodium transporters (for example, Na/Pi, Na⁺/HCO₃⁻, Cl⁻/HCO₃⁻), which needs to be confirmed in the future. However, the negative regulation of renin secretion by the D₃ receptor is probably independent of Gα₁₂ as it is not expressed in juxtaglomerular cells⁸ or Gα₁₃, but by its ability to decrease cAMP production.^{26, 30}

In summary, we found that stimulation of the D₃ receptor increases sodium excretion in WKY rats and the effect is, in part, via inhibition of Na⁺–K⁺–ATPase activity. There is linkage between D₃ receptors, Gα₁₂ and Gα₁₃ in RPTs, which is increased by stimulation of D₃ receptor in WKY rats, suggesting that the D₃ receptor may participate in the regulation of sodium transport by hampering Gα₁₂ and/or Gα₁₃ action.