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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Free Radic Biol Med. 2010 Dec 28;50(7):832–840. doi: 10.1016/j.freeradbiomed.2010.12.027

Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A-protein kinase C cross-talk

Peiying Yu 1, Weixing Han 2, Van Anthony M Villar 1, Hewang Li 1, Francis B Arnaldo 3, Gisela P Concepcion 3, Robin A Felder 4, Mark T Quinn 5, Pedro A Jose 1
PMCID: PMC3066436  NIHMSID: NIHMS261494  PMID: 21193028

Abstract

Dopamine cellular signaling, via the D1 receptor (D1R), involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D1R-mediated inhibition of NADPH oxidase activity involves cross-talk between PKA and specific PKC isoform(s). In HEK-293 cells heterologously expressing human D1R (HEK-hD1), fenoldopam, a D1R agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D1R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two different PKA inhibitors, H89 (10 µmol/L) (88±8.1%) and Rp-cAMP (10 µmol/L) (97.7±6.7%), and two different PKC inhibitors, bisindolylmaleimide I (1 µmol/L) (94±6%) and staurosporine (10 nmol/L) (93±8%), which by themselves, had no effect (n=4–8/group). The inhibitory effect of PMA (1 µmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%) (n=5–6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθS676, an effect that was also blocked by H89. Fenoldopam (1 µmol/L) also increased PKCθS676 in HEK-hD1 and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross-talk between PKA and PKCθ plays an important role in the D1R-mediated negative regulation of NADPH oxidase activity in human kidney cells.

Keywords: NADPH oxidase, protein kinase A, protein kinase C, D1 dopamin receptor

Introduction

Dopamine is an important regulator of blood pressure, sodium balance, and renal and adrenal function through the peripheral dopaminergic system (1, 2). Dopamine receptors are grouped into two subfamilies: D1-like receptors (D1R and D5R) stimulate adenylyl cyclases, while D2-like receptors (D2R, D3R, and D4R) inhibit adenylyl cyclases. D1-like receptors also stimulate phospholipase C (PLC) and activate protein kinase C (PKC) in many cells, including neural and renal proximal tubule (RPT) cells (3–8). D2-like receptor signaling may also involve protein kinase A (PKA) and PKC pathways (9–12).

The interaction between PKA and PKC pathways is important in the regulation of G protein-coupled receptor (GPCR) signaling (3, 6, 13–16). Phorbol-12-myristate-13-acetate (PMA), a direct activator of PKC, stimulates adenylyl cyclase activity and enhances agonist-induced cAMP production (15). In addition, activation of PKC with PMA stimulates intracellular cAMP production in human eosinophils, in the absence of any agonist (17). Dopamine, via D1R, decreases renal sodium transport and increases sodium excretion, in part, by activation of PKA- and PKC-dependent and independent mechanisms (1–6). Norepinephrine, via the α1-adrenergic receptor (α1AR), and angiotensin II, via the angiotensin type I receptor (AT1R), also increase PKC activity in many cells, including RPT cells, but increase, rather than decrease, renal sodium transport (18, 19). The differential effect of dopamine and α1AR or AT1R on sodium transport may occur because α1AR (18, 20), AT1R (19, 21) and dopamine D1-like receptors activate different PKC isoforms (4, 14, 16).

PKC isoforms are classified into four subgroups based on their requirements for Ca2+ and phospholipids for activation: classical PKCs (cPKCs: α, β and γ), novel PKCs (nPKCs: ε, δ, θ and η), atypical PKCs (aPKCs: ζ, ι and λ), and PKCμ, also called PKD which consists of PKD1 and PKD2/3 (22, 23). AT1R-mediated stimulation of renal sodium transport is associated with an increase in the levels of PKCα, PKCε, PKCδ, and PKCζ (19, 21, 24) in the plasma membrane, while α1AR-mediated stimulation of renal sodium transport is associated with increased levels of PKCδ and PKCζ (25, 26). In contrast, dopamine receptor-mediated inhibition of renal sodium transport is associated with an increase in membrane levels of PKCα, PKCε (14), and PKCθ (4), but a decrease in renal membrane of PKCδ (4). Although D1-like receptors have been reported to increase membrane PKCζ levels in RPT cells from hypertensive rats (4) and in an opossum kidney cell line (16), D1-like receptor stimulation actually decreases PKCζ in RPT cells from normotensive rats (4). Thus, the PKC isoforms that are differentially regulated by AT1R and α1AR on the one hand and D1-like receptors on the other are PKCδ and PKCθ, respectively.

Overproduction of reactive oxygen species (ROS) plays an important role in the pathogenesis of cardiovascular diseases, including hypertension, heart failure, and atherosclerosis (27). NADPH oxidase enzymes are major sources of ROS in the cardiovascular system and the kidney (27). Several lines of evidence have shown that PKC isoforms are involved in the regulation of NADPH oxidase activity (28–32). PKCδ is involved in the phosphorylation and translocation of p47phox from cytosol to membrane, which is required for the subsequent activation of several NADPH oxidase isoforms (28). PKCα- and δ-dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury of RPT cells (29). Angiotensin II has been reported to increase superoxide production, in part, by stimulation of PKC/PLD2 activity (30). EXP3179, a metabolite of the AT1R antagonist losartan, inhibits the activity of NADPH oxidase and PKC, specifically, PKCα and PKCβ (31). However, PKA has also been reported to inhibit NADPH oxidase activity (32).

We and others have reported that both D1R and D5R inhibit NADPH oxidase activity in vascular smooth muscle and RPT cells (33–36). Vascular smooth muscle cells and renal RPT cells express all subtypes of dopamine receptors but only the D1-like receptors (D1R and D5R in human or D1a and D1b in rodents) have been reported to have antioxidant property (27, 33). We have previously reported that in HEK-293 cells heterologously expressing the human D5R, stimulation of D5R inhibits NADPH oxidase activity, which is PKA-independent, partly PLD dependent, and partly due to interference with the distribution and assembly of NADPH oxidase components (34). Jackson et al. have also reported that PMA increases D1R but decreases D5R signaling (15). Therefore, it is necessary to use cell lines that heterologously and exclusively express D1R or D5R to distinguish the mechanisms of involved in D1R or D5R activation because there are no commercially available D1-like receptor agonists or antagonists that can distinguish D1R from D5R. We tested the hypothesis that a specific PKC isoform may be involved in the D1R-mediated inhibition of NADPH oxidase activity. We hypothesized further that phosphorylated PKCθ may be involved because this PKC isoform is activated by D1-like receptors, but not by AT1R and α1AR. Because PKC can augment D1R function but can decrease D5R function (15), studies were performed in HEK-293 cells heterologously expressing human D1R (HEK-hD1) or human D5R (HEK-hD5). We also studied the effect of PKC activation in human RPT cells which express all the subtypes of dopamine receptors in order to determine if the results obtained in an expression system (HEK-293) are relevant in cells that endogenously express these receptors. We now report that D1R but not D5R exerts its negative effect on NADPH oxidase activity by increasing the phosphorylation of PKCθS676, which is PKA-dependent.

For the materials used in this report, please see the supplementary file

Methods

Cell Treatment

D1R and D5R density and cAMP response to D1-like receptor stimulation, in stably transfected HEK-hD1 and HEK-hD5 cells, were reported previously (34, 35). To demonstrate that the D1R inhibits NADPH oxidase activity, HEK-hD1 cells, pre-starved in serum-free MEM for 1 hour, were incubated for 20 min with vehicle, the D1-like receptor agonist, fenoldopam (1.0 μmol/L/20 min), or the D1-like receptor antagonist, SCH 23390 (5.0 μmol/L, added to the cells 5 min prior to the addition of fenoldopam). To determine if PKA or PKC mediates the inhibition of NADPH oxidase activity by D1R, pre-starved HEK-hD1 cells were pre-treated for 40 min with vehicle, two different PKA inhibitors (H89 [10 μmol/L] or Rp-cAMP [50 µmol/L]), or two different PKC inhibitors, (bisindolylmaleimide I [1 μmol/L] or staurosporine [10 nmol/L]). In HEK- D5 studies, fenoldopam and H89 were used. The pre-treated cells were then incubated for an additional 20 min with vehicle, the PKA activator Sp-cAMP (10 μmol/L), PMA (1.0 μmol/L), fenoldopam (1 µmol/L) alone, or in combination with the PKA or PKC inhibitors. In the concentration-dependent experiments, the cells were treated with fenoldopam or PMA (10−9, 10−8, 10−7, 10−6, and 10−5 mol/L) for 20 min. In the time-course experiments, the cells were treated with fenoldopam (1 µmol/L) or PMA (1 µmol/L) for 0, 20, 40, 60 and 120 min. The temperature of all incubations was set at 37°C. After treatment, the cells were rinsed, and the cell membranes were isolated (36).

Measurement of NADPH oxidase activity by lucigenin chemiluminescence

NADPH oxidase activity was measured using lucigenin chemiluminescence in the presence of NADPH (36). Whole cell membranes were prepared as described in “Methods” in the supplementary file. Equal amounts of cell membranes were incubated with lucigenin (5 µmol/L) for 10 min at 37°C in a final volume of 0.9 ml assay buffer. Dynamic tracings were recorded for 180 sec (Autolumet Plus LB953, EG&G Berthhold, Germany) following the injection of 0.1 ml of NADPH (final concentration, 100 µmol/L) (36). The activity was expressed as arbitrary light units per 50 µg protein and converted to percent of control. In some experiments NADPH oxidase activity was measured using a luminometer (MicroWin 2000). The activity was expressed as relative light units per mg protein and converted to percent of control.

siRNA knockdown of PKCθ in human RPT cells

Human RPT cells were seeded in 6-well plates (for immunoblotting) or in 60 mm dishes (for oxidase assay) at a density of 2×105/well or dish at day 1 and then transfected with vehicle, scrambled RNA, or PKCθ-specific siRNA at day 2, as described previously (36). Cells were treated with or without drug and harvested at day 4 and assayed for NADPH oxidase activity, as described above. Protein levels were quantified by immunoblotting.

Statistical analysis

Data are expressed as mean ± standard error. Significant difference between two groups was determined by Student’s t-test and factorial ANOVA followed by Newman-Keuls test for groups >2. P<0.05 was considered significant.

Results

D1R inhibits NADPH oxidase activity in HEK-hD1 cells

Since our studies utilized HEK-293 cells, we confirmed that the various Nox isoforms (Nox1, Nox2, Nox3, Nox4 and Nox5) were expressed endogenously in these cells (Figure S1). To test the specificity of the inhibitory effect of D1R on NADPH oxidase activity, non-transfected HEK-293 cells, HEK-293 cells transfected with the empty vector (HEK-EV), and HEK-293 cells heterologously expressing hD1R (HEK-hD1) were treated with vehicle or the D1-like receptor agonist, fenoldopam (1 µmol/L/20min) and NADPH oxidase activity was measured. As shown in Figure 1A, fenoldopam inhibited NADPH oxidase activity in HEK-hD1 cells (33±8.2%) but not in HEK-293 (10.5±3.6%) or HEK-EV cells (7±7.3%) (compared to vehicle). In addition, the inhibitory effect of fenoldopam on NADPH oxidase activity in HEK-hD1 cells was concentration- and time-dependent (supplementary Figure S2 and supplementary Table S1). Furthermore, the inhibitory effect of fenoldopam on NADPH oxidase activity (37±5.0% inhibition) was blocked by the D1-like receptor antagonist, Sch23390 (5 µM) (16±6.2%), which by itself had no effect (2±5.7%) (Figure 1B).

Figure 1. Effect of D1-like receptor agonist and antagonist on NADPH oxidase activity.

Figure 1

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A. Cells [HEK-293, HEK-293 expressing human D1R (HEK-hD1) and empty vector (HEK-EV)] were treated for 20 min at 37°C with vehicle (Con), or the D1-like receptor agonist, fenoldopam (Fen, 1 µmol/L). Membranes were prepared and oxidase activity was measured in the presence of 5 µmol/L lucigenin and 100 µmol/L NADPH as described in “Methods”. Values are mean ± SEM (n = 6/group). *P<0.001 vs. control, t-test

B. HEK-hD1 cells were treated for 20 min at 37°C with vehicle (Con), fenoldopam (Fen, 1 µmol/L/20min), the D1-like receptor antagonist, Sch23990 (Sch, 5 µmol/L), alone or in combination (S+F), as described in “Methods”. Membranes were assayed for NADPH oxidase activity (Arbitrary Light Units per 50 µg protein). The left panel shows the dynamic tracings of oxidase-dependent chemiluminescence recorded over a period of 180 seconds; right panel shows the oxidase activity expressed as % of control. Values are the mean ± SEM (n=5/group). *P<0.01 vs. others, ANOVA, Newman-Keuls test

D1R-mediated inhibition of NADPH oxidase activity involves both PKA and PKC in HEK-hD1 cells

Dopamine has been reported to inhibit NADPH oxidase activity through PKA and PKC pathways in rat vascular smooth muscle cells (33, 34) that endogenously express both D1R and D5R (D1A and D1B in rodents) (1–3). In the current study, the inhibitory effect of D1R, independent of D5R, on NADPH oxidase activity (Fen=68.1±3.6% of control, set at 100%) was almost completely prevented by two different PKA inhibitors, H89 (10 µmol/L) (Figure 2A), and Rp-cAMP (50 µmol/L) (supplementary Figure S3), which by themselves had no effect on oxidase activity (H89=88±8.1% and Rp-cAMP=97.7±6.7% of control) in HEK-hD1 cells. The inhibitory effect of D1R on NADPH oxidase activity was also reversed by two different PKC inhibitors, bisindolylmaleimide I and staurosporine, which by themselves, also had no effect (bisindolylmaleimide I = 89.04±3.4%, and staurosporine= 98±4.5 % of control) (Figure 2B and supplementary Table S2).

Figure 2. Effect of PKA and PKC inhibitors on NADPH oxidase activity in HEK-hD1 cells.

Figure 2

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HEK-hD1 cells were pre-incubated for 40 min at 37°C with vehicle, PKA inhibitor H89 (10 µmol/L), two different PKC inhibitors, bisindolylmaleimide I (Bis, 1 μmol/L), or staurosporine (St, 10nmol/L), and then were treated for 20 min at 37°C with vehicle (Con), fenoldopam (Fen, 1 µmol/L), or inhibitor combinations (H89+Fen) (A) and (Bis+Fen, St+Fen) (B), as described in “Methods”. Membranes were assayed for oxidase activity (Arbitrary Light Units per 50 µg protein). The left panel (2A) and the upper panels (2B) show dynamic tracings of oxidase-dependent chemiluminescence recorded over a period of 180 seconds; the right panel (2A) and the lower panel (2B) show oxidase activity expressed as % of control. Values are mean ± SEM (n=4–8/group). *P<0.01 vs. others, ANOVA, Newman-Keuls test

Activation of PKC inhibits NADPH oxidase activity in HEK-hD1 cells

To evaluate the effect of PKC activation on NADPH oxidase activity, HEK-EV and HEK-hD1 cells were treated with PMA (1 µM/20 min), and NADPH oxidase activity was measured. PMA significantly increased NADPH oxidase activity in HEK-EV cells (121.6±6.2% vs. control=100±3.0%). In contrast, PMA significantly decreased NADPH oxidase activity in HEK-hD1 cells (80.5±2.8% vs. 100±4.9%) (Figure 3A), indicating that direct activation of PKC inhibits oxidase activity in HEK-hD1 but not HEK-EV cells. We next determined the concentration- and time-dependent effect of PMA on NADPH oxidase in HEK-hD1 cells. The cells were treated with PMA at various concentrations for 20 min and PMA (1 µmol/L) at various durations, as indicated. As shown in Figure 3B and supplementary Table S3, NADPH oxidase activity was decreased by PMA in a concentration- and time-dependent manner. To confirm that the effect of PMA on oxidase activity was mediated by PKC, the PKC inhibitor bisindolylmaleimide I (1 µM) was used. Indeed, the suppressive effect of PMA on NADPH oxidase activity (77.3±3.4 % of control) was prevented by bisindolylmaleimide I (97.3±8.9 % of control) (Figure 3C).

Figure 3. Effect of PKC activator and inhibitor on NADPH oxidase activity in HEK-hD1 cells.

Figure 3

Figure 3

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A. Effect of PMA on NADPH oxidase activity in HEK-EV and HEK-hD1 cells. HEK-EV and HEK-hD1 cells were treated with the PKC activator PMA (1 µmol/L) for 20 min at 37°C, as described in “Methods”. Membranes were prepared and assayed for oxidase activity (Arbitrary Light Units per 50 µg protein). The left panel shows the dynamic tracings of oxidase-dependent chemiluminescence recorded over a period of 180 seconds; the right panel shows oxidase activity expressed as % of control. Values are mean ± SEM (n=8/group). *P=0.007 vs. control in HEK-EV and **P=0.004 vs. control in HEK-hD1, t-test

B. Concentration- and time-dependent effect of PMA on membrane NADPH oxidase activity. In the concentration-dependent experiments, HEK-hD1 cells were treated for 20 min at 37°C at the indicated concentrations of PMA. In the time-dependent experiments, cells were treated at 37°C with PMA (1 µmol/L) for the indicated times. Membranes were prepared and assayed for oxidase activity (Arbitrary Light Units per 50 µg protein). The upper panels show the dynamic tracings of oxidase-dependent chemiluminescence recorded over a period of 180 seconds; the lower panels show oxidase activity expressed as % of control. Values represent the mean ± SEM (n=6/group). *P<0.01 vs. all concentrations except 10−9M, #P<0.05, vs. others (time), ANOVA, Newman-Keuls test

C. Effect of PKC inhibitor on PMA-induced increase in NADPH oxidase activity. HEK-hD1 cells pre-incubated for 40 min at 37°C with vehicle, or a PKC inhibitor bisindolylmaleimide I (Bis, 1 μmol/L) were incubated for an additional 20 min at 37°C with vehicle (Con), PMA (1 μmol/L), or drug combination (Bis+PMA) as described in “Methods”. Membranes were prepared and assayed for oxidase activity which is expressed as % of control. Values represent the mean ± SEM (n=6/group). *P<0.05 vs. others, ANOVA, Newman-Keuls test

Activation of PKC decreases NADPH oxidase activity through cross-talk with PKA in HEK-hD1 cells

To determine if the effect of PMA on NADPH oxidase activity involves PKA signaling, cells were treated with a PKA inhibitor, H89. The inhibitory effect of PMA on oxidase activity (73±3.2% of control) was prevented by the addition of H89 (100±7.8 % of control), which by itself had no effect on NADPH oxidase activity (104±7.4 % of control) in HEK-hD1 cells (Figure 4A and supplementary Table S4).

Figure 4. Effect of PKA inhibitor on PMA-induced decrease in NADPH oxidase activity and phosphorylated PKC isoforms.

Figure 4

Figure 4

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A. Effect of PKA inhibitor on PMA-induced decrease in NADPH oxidase activity. HEK-hD1 cells pre-incubated for 40 min at 37°C with vehicle or a PKA inhibitor H89 (10 µmol/L) were incubated for an additional 20 min at 37°C with vehicle (Con) or PMA (1 µmol/L), as described in “Methods”. Membranes were prepared and assayed for oxidase activity using a luminometer. Oxidase activity is expressed as % of control. Values are mean ± SEM (n=5–6/group). *P<0.01 vs. others, ANOVA, Newman-Keuls test

B. Effect of PMA on the phosphorylation of PKC. HEK-hD1 cells pre-incubated for 40 min at 37°C with vehicle or PKA inhibitor H89 (10 µmol/L) were incubated for an additional 20 min at 37°C with vehicle, PMA (1 µmol/L) or drug combination (H+P). The cells were then fractionated into cytosol and membrane, as described in “Methods”. Protein loading was verified by Ponceau-S staining in Figure S4, supplementary file. The proteins were probed with specific anti-phospho-PKC isoforms, as indicated. N=3/group.

C–D. Graphic representation of the effect of PMA on PKCθS676 (Figure 4C) and PKCδS645 (Figure 4D) proteins in the cells studied in Figure 4B. In the some cases, cell lysates were used for immunoblotting because PKCθS676 protein was increased in both cytosol and membrane fractions. The immunoblots of PKCθS676 and PKCδS645 (upper band) from Figure 4B were semi-quantified, as described in “Methods”. Values are mean ± SEM (n= 4–6/group). *P<0.05 vs. others, ANOVA, Newman-Keuls test

To determine if activation of PKC by PMA is isoform-specific, we studied the abundance of phosphorylated PKC isoform in HEK-hD1 cells treated with PMA (1 µmol/L for 20 min). PMA decreased the level of cytosolic PKC, but increased the membrane abundance of serine-phosphorylated PKCα, PKCε, and PKCη and threonine-phosphorylated PKCθ in HEK-hD1 cells (Figure 4B). A similar pattern has been reported for the PMA-mediated translocation of PKC isoforms without taking into account their phosphorylation status (37). In contrast, the serine-phosphorylated PKCθS676 was increased by PMA in both cytosol and membrane, whereas PKCδS645 was increased in the membrane but not in the cytosol fraction. Additionally, the PMA-mediated increase in phosphorylated PKCθS676 and PKCδS645 was prevented by the PKA inhibitor H89 (Figure 4C) and (Figure 4D).

To determine the PKC isoforms involved in D1R signaling, HEK-hD1 cells were treated with fenoldopam (D1R agonist), Sch23390 (D1R antagonist), the PKA inhibitor Rp-cAMP, the PKC inhibitor bisindolylmaleimide I, or inhibitor combinations. Whole cell lysates were used to determine PKCθS676 protein because it was increased in both cytosol and membrane fractions (Figure 4B) of the cells. PKCθS676 was increased by fenoldopam (153±11.3 % of control) in whole cell lysates, an effect that was abolished by the D1R antagonist Sch23390 (97.1 ±9.2 % of control) (Figure 5A). The increase in PKCθS676 induced by fenoldopam was prevented by the PKA inhibitor Rp-cAMP (112±7.7 % of control) (Figure 5B, left panel) and the PKC inhibitor bisindolylmaleimide I (112±5.4% of control) (Figure 5B, right panel), which by themselves had no effect.

Figure 5. Effect of D1R stimulation on PKCθS676 protein.

Figure 5

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Cell lysates were prepared and used for immunoblotting following drug treatment. Protein loading was verified by Ponceau-S staining, Figure S5, supplementary file.

A. Effect of fenoldopam on PKCθS676. HEK-hD1 cells were treated for 20 min at 37°C with vehicle (Con), D1-like receptor agonist fenoldopam (Fen, 1 µmol/L), or D1-like receptor antagonist Sch23990 (Sch, 5 µmol/L) added to cells 5 min prior to the addition of fenoldopam) or drug combination (Sch+Fen). The cell lysates were probed with anti-PKCθS676, and the immunoreactive bands semi-quantified, as described in “Methods”. Values are mean ± SEM (n=4–6/group). *P< 0.05 vs. others, ANOVA, Newman-Keuls test

B. Effect of fenoldopam and PKA and PKC inhibitors on PKCθS676. HEK-hD1 cells pre-incubated for 40 min at 37°C with vehicle, PKA inhibitor Rp-cAMP and (50 µmol/L) (left panel) or PKC inhibitor bisindolylmaleimide I (Bis, 1 μmol/L) (right panel) were incubated for an additional 20 min at 37°C with vehicle (Con), fenoldopam (1 µmol/L), or drug combinations (Rp+Fen, Bis+Fen), as described in “Methods”. The cell lysates were probed with anti-PKCθS676, and the immunoreactive bands were semi-quantified as described in “Methods”. Values are mean ± SEM (n=4–6/group). *P< 0.05 vs. others, ANOVA, Newman-Keuls test

To determine if D5R can also increase PKCθS676, we assessed the effect of a D1-like receptor agonist, fenoldopam, and a PKA inhibitor, H89, on the protein level of PKCθS676 in HEK-293 cells heterologously expressing D5R (HEK-D5) (Figure S7, supplementary file). Stimulation of D5R with fenoldopam did not significantly increase PKCθS676 (P>0.05, ANOVA, Duncan’s test) (Fenoldopam=108.2±2.7 vs. control=100±1.5 % change). However, the PKA inhibitor H89, by itself or with fenoldopam significantly decreased PKCθS676 (H89=82.4±6.8 and H89+fenoldopam =77.8±6.8 % change, P<0.05, control vs. H89 and H89+fen, n=3/group, ANOVA, Duncan’s test).

To determine the relevance of our findings to cells that endogenously express functional D1R and D5R, we studied the effect of D1-like receptor stimulation in human RPT cells. In these cells, we found that stimulation of D1-like receptors increased the phosphorylated PKCθS676 (Supplementary Figure S6), but not the phosphorylated PKCδS645 (data not shown). To determine which of the D1-like receptor (D1R or D5R) is responsible for the fenoldopam-mediated increase in PKCθS676 abundance in human RPT cells, we knocked down of D5R protein expression using siRNA specific for D5R in human RPT cells (Figure S8–S9, supplementary file), which decreased D5R protein levels by 45% (45±6.7 % vs. control siRNA=100±0.73 %, P<0.05, n=3, t-test) (Figure S8). Knockdown of D5R did not prevent the fenoldopam-mediated stimulation of PKCθS676 (P<0.05, Fen vs. control, H89 and H89+Fen, ANOVA, Duncan’s test) (Figure S9 and Table S6 in the supplementary file).

The involvement of PKCθS676 in D1R signaling in HEK-hD1 cells was also studied by confocal microscopy. Fenoldopam (1 µmol/L/20 min) increased the colocalization of PKCθ and PKCθS676, an effect that was attenuated by the PKA inhibitor H89, which by itself had no effect (supplementary Figure S10).

The D1-like receptor-mediated inhibition of NADPH oxidase activity is abrogated by the knockdown of PKCθ

To demonstrate further that PKCθ plays an important role in D1-like receptor-mediated negative regulation of NADPH oxidase activity, the expression of PKCθ was knocked-down by PKCθ-specific siRNA in human RPT cells. Knockdown of PKCθ, which decreased PKCθ protein by 70% (Figure 6A), prevented the inhibitory effect of fenoldopam (1 µmol/L/20 min) on NADPH oxidase activity (Figure 6B and supplementary Table S6).

Figure 6. Knockdown of PKCθ with PKCθ-specific siRNA in human RPT cells.

Figure 6

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A. Effect of PKCθ-specific siRNA on PKCθ in human RPT cells. Human RPT cells were transfected with control siRNA (siCon) or specific PKCθ siRNA (si PKCθ) for 48 hr as described in “Methods”. Cell lysates were immunoblotted with anti-PKCθ monoclonal antibody. The immunoreactive bands were semi-quantified (siCon=209.94±4.5, siPKCθ=51±8.9 density units). Values are mean ± SEM (n=3/group). *P< 0.05, t-test

B. Effect of PKCθ knockdown on D1-like receptor-mediated inhibition of NADPH oxidase activity in human RPT cells. Human RPT cells were transfected with vehicle (Con), siCon, or siPKCθ as in Figure 6A. Membranes were prepared and assayed for oxidase activity using a luminometer. Oxidase activity is expressed as % of control. Values are mean ± SEM (n=3–6/group). *P<0.01 vs. respective controls, ANOVA, Newman-Keuls test

Discussion

The current studies provide firm evidence that D1R inhibits NADPH oxidase activity in HEK-hD1 cells heterologously expressing hD1R but not hD5R. These studies are consistent with previous reports using rat vascular smooth muscle and human RPT cells, which endogenously express both D1-like receptors, D1R and D5R (33, 36). Although the inhibitory effect of D1-like receptors on NADPH oxidase activity has been reported to be mediated by PKA- and PKC-dependent pathways (33, 34, 36), the role of cross-talk between PKA and various PKC isoforms in D1R-mediated oxidase inhibition has not been determined. We now report that D1R inhibits NADPH oxidase activity by increasing the PKA-dependent phosphorylated PKCθS676. The current studies show that direct activation of PKC with PMA decreases NADPH oxidase activity in a time- and concentration-dependent manner in HEK-hD1 cells. PMA, a direct activator of PKC, induces the translocation of conventional and novel PKC isoforms from cytosol to membranes (37). In the current studies, PMA also induced the translocation of serine-phosphorylated PKCα, PKCδ, PKCθ, PKCη, and PKCε and threonine-phosphorylated PKCθ from cytosol to membranes in HEK-hD1 cells. In addition, the PMA-mediated increase in membranes of PKCθS676 and PKCδS645, but not the other phosphorylated PKC isoforms, was attenuated by the PKA inhibitor H89 in HEK-hD1 cells. PKCθ, a Ca2+-independent novel PKC isoform (37), has been implicated in T cell receptor signal transduction (38). PKCθ is the only PKC isoform involved in the formation of a membrane-signal complex when the T cell comes into contact with a stimulator cell (39). In addition, PKCθ is constitutively phosphorylated at the activation-loop (threonine 538) and turn-motif (serine 676) sites in T cells (40). PKCθ activity is also involved in aromatic hydrocarbon receptor signal transduction and nicotinic acetylcholine receptor (nAChR) cluster formation (41, 42). The inhibitory effect of PKC and PKA on NADPH oxidase activity in our studies is similar to that recently reported for Nox 1 in HEK-293 cells (43). We now report, for the first time, that D1R-mediates the inhibition of NADPH oxidase activity via a cross-talk between PKA/PKC that is linked to PKCθ. In contrast, the increase in ROS production in the renal medullary thick ascending limb of diabetic rats and adipocytes of obese, insulin-resistant mice has been reported to be PKC-dependent but involves PKCδ rather than PKCθ (44, 45). PKCα and PKCζ may also be involved in the PKC-mediated activation of NADPH oxidase in rat mesangial cells (46, 47). PKCα and PKCδ contribute to NADPH oxidase activation in a pig kidney cell line (LLC-PK1) (48). Thus, the PKC-mediated inhibition or activation of NADPH oxidase is PKC isoform-dependent and tissue- and cell-specific. Activation of PKC with phorbol ester generally results in increase NADPH-oxidase activity (44–48). In HEK-293 cells heterologously expressing human D1R, we found that PMA decreases NADPH oxidase activity. This indicates that PMA, in the presence of D1R, exerts a strong antioxidant effect PMA has been reported to stimulate D1R but inhibit D5R signaling in HEK-293 cells heterologously expressing human D1R or D5R (15). PKC can also potentiate forskolin- and dopamine-induced cAMP formation in a concentration-dependent manner in vitro (49). Therefore, the activation of D1R by PMA along with elevated cAMP concentration (and PKA activity) can synergistically activate PKC (3, 6, 15). An activated D1R can also directly stimulate PLA2 (50). The activation of PLA2 increases the release of arachidonic acid that is converted to 20-hydroxy-eicosatetraenoic acid (20-HETE) and stimulates PKC activity (50). Therefore, the PKC-mediated decrease in NADPH oxidase activity in HEK-hD1 cells may be due to activation of D1R, resulting in a positive feedback.

In RPT cells, the activation of D1-like receptors (D1R and D5R in human and D1A and D1B in rodents) stimulates PKCθ and PKCζ and inhibits PKCδ. In addition, D1-like receptor stimulation induces the translocation of specific PKC isoforms from cytosol to membrane (α, β, and ε) and membrane to cytosol (δ) (3, 4, 14, 17). We and others have reported previously that D1R-mediated stimulation of PLC can be a result of PKA and PKC activation in epithelial cells (3–6, 14, 15). We have also reported that intrarenal arterial infusion of two different D1-like receptor agonists, SKF38393 and fenoldopam, for 10 min increases PKCθ protein in rat RPT membranes (4). In the present study, activation of D1R (fenoldopam, 1 µmol/20 min) did not significantly increase PKCθ protein in HEK-hD1 cells. The difference between our previous in vivo study and the current in vitro study may lie in the inability of D1-like receptor agonists to distinguish between the two D1-like receptors, D1R and D5R. In the current in vitro study in which the D1R but not the D5R is expressed, D1R stimulation increases membrane PKCθS676. This effect is exerted at the D1R because it is prevented by a D1R antagonist. In contrast, D5R stimulation with the D1-like receptor agonist fenoldopam does not increase PKCθS676 in HEK-D5 cells. Additionally, knockdown of D5R in RPT cells does not prevent the fenoldopam-mediated increase PKCθS676 protein level in human RPT cells. The D1R-mediated increase in the membrane PKCθS676 is mediated via PKA, because the effect is prevented by pre-treatment with the PKA inhibitor H89. These results suggest that the D1R-mediated increase in PKCθS676 abundance is PKA-dependent. PKCθS676 has been reported to have a limited effect on kinase activity but may negatively regulate other aspects of PKCθ function, at least as related to NF-kB activation in Jurkat T-cells (40). Nevertheless, the fact that knockdown of PKCθ with PKCθ-specific siRNA prevented the inhibitory effect of fenoldopam on oxidase activity in HEK-hD1 cells confirms the important role of PKCθ in D1R-mediated inhibition of NADPH oxidase activity.

In summary, our studies demonstrate that the D1R-mediated inhibition of NADPH oxidase activity is via the phosphorylated PKC isoform, PKCθS676 in HEK-hD1 and human RPT cells and that the D1R-mediated increase in the phosphorylated PKCθS676 involves the PKA pathway. In contrast, the D5R-mediated inhibition of NADPH oxidase activity is independent of PKA and PKC and is rather due to interference with the distribution and assembly of NADPH oxidase components and stimulation of PLD (34).

Supplementary Material

01

Acknowledgments

These studies were supported in part by grants from the National Institutes of Health (HL023081, HL074940, DK039308, HL068686, HL092196, and RR020185).

Footnotes

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