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Abstract
The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Because this interaction may be perturbed in genetic hypertension, we studied D1 dopamine and AT1 angiotensin receptors in immortalized renal proximal tubule (RPT) and A10 aortic vascular smooth muscle cells. In normotensive Wistar-Kyoto (WKY) rats, the D1-like agonist fenoldopam increased D1 receptors but decreased AT1 receptors. These effects were blocked by the D1-like antagonist SCH 23390 (10−7 mol/L per 24 hours). In spontaneously hypertensive rat (SHR) RPT cells, fenoldopam also decreased AT1 receptors but no longer stimulated D1 receptor expression. Basal levels of AT1/D1 receptor coimmunoprecipitation were greater in WKY RPT cells (29±2 density units, DU) than in SHR RPT cells (21±2 DU, n=7 per group, P<0.05). The coimmunoprecipitation of D1 and AT1 receptors was increased by fenoldopam (10−7 mol/L per 24 hours) in WKY RPT cells but decreased in SHR RPT cells. The effects of fenoldopam in RPT cells from WKY rats were similar in aortic vascular smooth muscle cells from normotensive BD IX rats, that is, fenoldopam decreased AT1 receptors and increased D1 receptors. Our studies show differential regulation of the expression of D1 and AT1 receptors in RPT cells from WKY and SHR. This regulation and D1/AT1 receptor interaction are different in RPT cells of WKY and SHR. An altered interaction of D1 and AT1 receptors may play a role in the impaired sodium excretion and enhanced vasoconstriction in hypertension.
Dopamine, a neurotransmitter in neural tissue, acts as an autocrine/paracrine substance in nonneural tissues. Dopamine produced in neural and nonneural 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 Dopamine receptors are classified into D1-like (D1, D5) and D2-like (D2, D3 and D4) subtypes, based on their structure and pharmacology. Whereas the D1-like receptors couple to the stimulatory G protein, GαS and thus, activate adenylyl cyclases, the receptors of the D2-like subfamily couple to the inhibitory G protein, Gαi/GαO and inhibit adenylyl cyclases and calcium channels and modulate potassium channels.1–3 Under euvolemic conditions and magnified during moderate volume expansion, dopamine, through D1-like and D3 receptors, acts to increase sodium excretion and decrease blood pressure.1,4,5
The renin-angiotensin system is another important regulator of sodium transport and blood pressure.6 Like dopamine, angiotensin exerts its action through several receptor subtypes. AT1 receptors are linked to Gq/11 and Gαi/GαO, whereas AT2 receptors are linked to Gαi2/32,7; the G protein linkage of AT4 receptors is not clear.8 Although angiotensin, through AT2 and AT4 receptors, can decrease blood pressure by vasodilation and increase in sodium excretion, the major effect of angiotensin II is increasing sodium reabsorption and blood pressure, counteracting the effect of dopamine.6–10
Dopamine and angiotensin receptors counteract each other in the paracrine regulation of renal sodium transport. Thus, the natriuretic effect of D1-like receptors is enhanced when angiotensin II production is decreased or when AT1 receptors are blocked.11 Dopamine, through D1-like and D2-like receptors, by themselves or in concert, antagonizes the stimulatory actions of angiotensin II on proximal tubular luminal sodium transport. Dopamine, through D1-like receptors, also decreases AT1 receptor expression and angiotensin II binding sites in renal proximal tubules.12–15 D2-like receptors,14 and more specifically, the D3 receptor, also decrease AT1 receptor expression in immortalized renal proximal tubule (RPT) cells from normotensive Wistar-Kyoto (WKY) rats.16 Immortalized RPT cells have characteristics similar to freshly obtained RPTs and renal brush border membranes.17–20
Similar to the counterregulatory effects of these systems on renal sodium transport, the renal vasoconstrictor effect of angiotensin II is counteracted by the vasodilator action of dopamine, through D1-like receptors, in normotensive WKY rats.21,22 In spontaneously hypertensive rats (SHR), the renal vasodilatory effect of D1-like receptors is decreased and there is a lack of D1-like receptor antagonism of the vasoconstrictor effect of angiotensin II.21,22 Our previous study showed that angiotensin II increases D1 receptor expression in RPT cells from WKY rats but not from SHR.23 We hypothesize that there is a differential interaction of D1 and AT1 receptors not only in RPT cells but also in vascular smooth muscle cells of WKY and SHR. We now report that fenoldopam, an agonist for D1-like receptors (D1 and D5), increases expression of D1 receptors and decreases expression of AT1 receptors in RPT cells from WKY rats and A10 cells,24,25 a primary culture of vascular smooth muscle cells from the embryonic thoracic aorta of normotensive BD IX rats. The actions of fenoldopam are exerted through D1-like receptors because the effects are blocked by SCH23390, an antagonist for D1-like receptors (D1 and D5). Although D1/AT1 receptors physically interact in both cell lines, fenoldopam increases D1/AT1 receptor coimmunoprecipitation in RPT cells from WKY rats but decreases it in SHR. In SHR, fenoldopam no longer affects D1 receptor expression but its ability to decrease AT1 receptors is preserved; the effect of fenoldopam on AT1 receptor expression may be exerted through another D1-like receptor.1–3
Methods
Cell Culture
Immortalized RPT cells from WKY and SHR and primary cultures of embryonic thoracic aortic smooth muscle cells (A10) (passage 10 to 20) from normotensive Berlin-Druckrey IX (BD IX) rats were cultured at 37°C in 95% air/5% CO2 atmosphere in DMEM/F-12 or DMEM, respectively.17,24,25 The cells (80% confluence) were extracted in ice-cold lysis buffer,17 sonicated, kept on ice for 1 hour, and centrifuged at 16 000g for 30 minutes. The supernatants were stored at −70°C until use.
Immunoblotting
The antibodies are polyclonal, purified antipeptides. The human AT1 receptor immunogenic peptide is QDDCPKAGRHC, amino acids 15 to 24 of the AT1 receptor. The rat D1 receptor antibody corresponds to positions 299 to 307 (GSEETQPFC) of the D1 receptor. The specificities of these antibodies have been reported.17–19,26–30 The cells were treated with vehicle (dH2O), fenoldopam, or a D1-like receptor antagonist (SCH23390) at the indicated concentrations and times. Immunoblotting was performed as reported17–20,26 except that the transblots were probed with the D1 (1:800) or the AT1 receptor antibody (1:400). Equality of total amount of protein per sample (50 μg) transferred onto the membranes was ascertained by Ponceau-S staining and by immunoblotting for α-actin.
Immunoprecipitation
RPT cells were incubated with vehicle or fenoldopam (10−7 mol/L) for 24 hours, as described above. The cells were lysed with ice-cold lysis buffer for 1 hour and centrifuged at 16 000g for 30 minutes. Supernatant (500 μg protein/mL) was incubated with affinity-purified rabbit anti-rat D1 receptor antibody (2 μL/mL) for 1 hour and protein-G agarose at 4°C for 12 hours. The immunoprecipitates were pelleted and washed 4 times with lysis buffer. The pellets were suspended in sample buffer, boiled for 10 minutes, and subjected to immunoblotting with the AT1 receptor antibody. To determine the specificity of the bands, preimmune serum of D1 receptor antibody (negative control) and AT1 receptor antibody (positive control) were used as immunoprecipitants instead of the D1 receptor antibody. The bands were quantified by densitometry.17–20,26
Materials
Rabbit anti-human AT1 receptor antibodies were purchased from Santa Cruz Biotechnology, Inc; D1 receptor antibodies were custom-produced (Research Genetics). Fenoldopam and SCH23390 were from Sigma. A10 cells were from ATCC. Other reagents were of the highest purity available (Sigma or Gibco).
Statistical Analysis
The data are expressed as mean±SEM. Comparisons within and among groups were made by repeated-measures and factorial ANOVA, respectively, with the Duncan test. A value of P<0.05 was considered significant.
Results
Fenoldopam Decreases AT1 Receptor Expression in RPT Cells From WKY and SHR
Fenoldopam decreased AT1 receptor expression (≈45 kDa) in a concentration- and time-dependent manner in WKY RPT cells. The inhibitory effect was evident at 10−9mol/L, with a 50% decrease at 1.4×10−9mol/L (Figure 1A). The inhibitory effect of fenoldopam (10−7 mol/L) was noted as early as 8 hours and maintained for at least 30 hours (t1/2=15.5 hours) (Figure 1B). In SHR RPT cells, fenoldopam also decreased AT1 receptor expression (WKY: control=31±1, fenoldopam (10−7mol/L)=21±1 density units, [DU]; SHR: control=27±2, fenoldopam (10−7 mol/L) =20±2 DU; n=8/group), (Figure 1C).
Figure 1. Effect of D1-like agonist fenoldopam on AT1 receptor expression in RPT cells from WKY and SHR. A and B, Concentration-response (24 hours) and time course (10−7 mol/L) of AT1 receptor expression (≈45 kDa), determined by immunoblotting, in RPT cells from WKY rats treated with fenoldopam. All immunoblotting results are expressed as relative density units (DU) (n=7 to 10, *P<0.05 vs control [C], ANOVA, Duncan test). One immunoblot is depicted in the inset. C, Effects of fenoldopam (10−7 mol/L per 24 hours) on AT1 receptor expression in RPT cells from WKY and SHR. Cells were incubated at indicated times and concentrations (n=8, *P<0.05 vs control, ANOVA, Duncan test). One immunoblot is depicted in the inset. D, Effects of fenoldopam (Fen) and a D1-like receptor antagonist (SCH23390, SCH) on AT1 receptor expression in RPT cells from WKY rats. Cells were incubated with the indicated reagents (fenoldopam, 10−7 mol/L; SCH23390, 10−7 mol/L) for 24 hours (n=9, *P<0.05 vs others, ANOVA, Duncan test). One immunoblot is depicted in the inset. The apparent molecular size of the AT1 receptor (≈45 kDa) is in agreement with our previous report.17
The specificity of fenoldopam as a D1-like receptor agonist was determined by studying the effect of the D1-like receptor antagonist SCH23390. In WKY RPT cells, consistent with the study shown in Figures 1A and 1B, fenoldopam (10−7mol/L per 24 hours) decreased AT1 receptor expression (control=26±2 DU, fenoldopam=19±2 DU; n=9, P<0.05). SCH23390 (10−7 mol/L) by itself had no effect (28±2 DU) but reversed the inhibitory effect of fenoldopam on AT1 receptor expression (28±3 DU) (Figure 1D). In SHR RPT cells, SCH23390 also blocked the inhibitory effect of fenoldopam on AT1 receptor expression (control=29±3 DU, fenoldopam=16±3 DU, SCH23390=28±4, SCH23390+ fenoldopam=29±3; n=12, P<0.05).
Fenoldopam Increases D1 Receptor Expression in RPT Cells From WKY Rats But Not From SHR
To investigate the effect of fenoldopam on the D1 receptor, RPT cells were incubated with fenoldopam (10−7 mol/L) for 24 hours. Fenoldopam increased D1 receptor expression (≈80 kDa) in RPT cells from WKY rats (control=25±3 DU, fenoldopam=37±3 DU; P<0.05, n=8) but had no significant effect in cells from SHR (control=22±2 DU, fenoldopam=17±4 DU; n=8/group) (Figure 2A).
Figure 2. A, Different effect of fenoldopam on D1 receptor expression in RPT cells from WKY and SHR. Cells were incubated with indicated reagents (fenoldopam, 10−7 mol/L) for 24 hours (n=8, *P<0.05 vs others, ANOVA, Duncan test). One immunoblot is depicted in the inset. The major D1 receptor had a molecular size of ≈80 kDa.18,19 B, Effects of fenoldopam (Fen) and a D1-like receptor antagonist (SCH23390, SCH) on D1 receptor expression in RPT cells from WKY rats. Cells were incubated with indicated reagents (fenoldopam, 10−7 mol/L; SCH23390, 10−7 mol/L) for 24 hours (n=11, *P<0.05 vs others, ANOVA, Duncan test). One immunoblot is depicted in the inset.
The specificity of fenoldopam as a D1-like receptor agonist also was determined by studying the effect of the D1-like receptor antagonist SCH23390 on D1 receptor protein expression. In WKY RPT cells, consistent with the study shown in Figure 2A, fenoldopam (10−7 mol/L per 24 hours) increased D1 receptor expression (control=22±1 DU, fenoldopam=30±2 DU; n=11, P<0.05). SCH23390 (10−7 mol/L) by itself had no effect (24±2 DU) but reversed the stimulatory effect of fenoldopam on D1 receptor expression (24±3 DU) (Figure 2B).
Fenoldopam Increases D1 Receptors and Decreases AT1 Receptors in A10 Cells
To test our hypothesis that the effects of fenoldopam on AT1 and D1 receptors happens not only in RPT cells but also in vascular smooth muscle cells, A10 cells were treated with fenoldopam at the indicated concentrations and times. In A10 cells, as in RPT cells, fenoldopam decreased AT1 receptors and increased D1 receptors (D1: EC50=1.0×10−9mol/L, t1/2=16.0 hours; AT1: EC50=5.1×10−10 mol/L, t1/2=16.1 hours) in a time-dependent (2 to 30 hours) and concentration-dependent (10−11 to 10−5 mol/L) manner (D1 receptor: Figures 3A and 3B; AT1 receptor: data not shown). In A10 cells, as in RPT cells, fenoldopam exerted its effects on D1-like receptors; SCH23390 completely blocked the effect of fenoldopam on AT1 receptor expression (Figure 4).
Figure 3. Effect of fenoldopam on D1 receptor expression in A10 cells. A and B, Concentration response (24 hours) and time course (10−7 mol/L) of D1 receptor expression, determined by immunoblotting, in A10 cells treated with fenoldopam. All immunoblotting results are expressed as relative density units (DU) (n=6 to 7, *P<0.05 vs control [C], ANOVA, Duncan test). One immunoblot is depicted in the inset. Major D1 receptor had a molecular size of ≈80 kDa.18,19
Figure 4. Effect of fenoldopam (Fen) and SCH23390 (SCH) on AT1 receptor expression in A10 cells. Cells were incubated with the indicated reagents (fenoldopam, 10−7 mol/L; SCH23390, 10−7 mol/L) for 24 hours (n=13, *P<0.05 vs others, ANOVA, Duncan test). One immunoblot is depicted in the inset.
AT1 Receptor Coimmunoprecipitates With the D1 Receptor in Rat RPT Cells
To determine whether there is a physical interaction between the D1 and the AT1 receptor, additional experiments were performed. D1 receptors were first immunoprecipitated with anti-D1 receptor antibodies and then probed with anti-AT1 receptor antibodies. As shown in Figure 5A, basal coimmunoprecipitation was greater in RPT cells from WKY rats than those in SHR (45-kDa bands represented the coimmunoprecipitated D1 and AT1 receptors). The 24-hour treatment of fenoldopam (10−7 mol/L) increased D1/AT1 coimmunoprecipitation in RPT cells from WKY rats but decreased it in SHR (WKY: control=29±2 DU, fenoldopam=39±2 DU; n=7, P<0.05; SHR: control=21±2 DU, fenoldopam=11±1 DU; n=7, P<0.05). The changes in D1/AT1 coimmunoprecipitation caused by fenoldopam were blocked by 10−7 mol/L SCH23390 (n=2) (Figure 5B).
Figure 5. A, Effect of fenoldopam on the coimmunoprecipitation of D1 and AT1 receptors in rat RPT cells. Cells were incubated with fenoldopam (10−7 mol/L) for 24 hours. Thereafter, samples were immunoprecipitated with anti–D1 receptor antibodies and immunoblotted with anti–AT1 antibodies (#P<0.05 vs control, *P<0.05, vs WKY, n=7, ANOVA, Duncan test). One immunoblot (≈45 kDa) is depicted in the inset: (lane 1=positive control, lane 2=negative control, lane 3=vehicle-treated RPT cell of WKY rats, lane 4=fenoldopam-treated RPT cell of WKY rats, lane 5=vehicle-treated RPT cell of SHR, lane 6=fenoldopam-treated RPT cell of SHR). For positive control, anti–AT1 antibodies (1 μg/mL) were used as immunoprecipitant; for negative control, preimmune serum of D1 receptor (2 μL/mL) was used as immunoprecipitant instead of anti–D1 receptor antibodies and immunoblotted with anti–AT1 receptor antibodies as above. B, Effect of fenoldopam (Fen) and SCH23390 (SCH) on D1/AT1 receptor coimmunoprecipitation in rat RPT cells from SHR and WKY rats. Cells were incubated with the indicated reagents (fenoldopam, 10−7 mol/L; SCH23390, 10−7 mol/L) for 24 hours (n=2). One immunoblot is shown.
Discussion
There are several novel observations in our study. First, we show that the D1-like receptor agonist fenoldopam increases D1 receptor expression but decreases AT1 receptor expression in RPT cells from WKY rats. This effect is exerted at the D1 receptor because a D1-like receptor antagonist, SCH23390, completely blocks the effect of fenoldopam. Second, in SHR, although fenoldopam also decreases AT1 receptor expression, it has no effect on D1 receptor expression. Third, AT1 receptors coimmunoprecipitate with D1 receptors in rat RPT cells. The basal level of AT1/D1 coimmunoprecipitation is greater in WKY than in SHR. Moreover, fenoldopam increases the physical interaction between AT1 and D1 receptors in rat RPT cells from WKY rats but decreases it in SHR. Fourth, the stimulatory effect of fenoldopam on D1 receptor expression and inhibitory effect on AT1 receptor in RPT cells from WKY rats also occur in A10 cells.
Both dopamine and renin-angiotensin systems exist in the RPT.1,3,6,13 The components of the renin-angiotensin system, including angiotensinogen mRNA, renin, and ACE, have been localized the proximal tubule.6,13,31,32 The proximal tubule is also the site of local dopamine production.1,33 Urinary dopamine and angiotensin II concentrations exceed circulating levels, suggesting that both systems may modulate RPT function in an autocrine or paracrine function.6,13,31,33–35 Dopamine and angiotensin II are two important regulators of sodium and water absorption in the kidney serving counteracting functions in the proximal tubule. Low concentrations of angiotensin II stimulate net reabsorption through activation of Na+/H+ exchanger-3, Na+-HCO3 cotransporter, and Na+-K+ATPase,6,12–14,36,37 whereas dopamine exerts the opposite effect.1,3,14,19,37–41 Salt depletion increases angiotensin II production and AT1 receptor expression in renal proximal tubules in normotensive rats, resulting in increased reabsorption of sodium and water.6,13 Conversely, sodium loading increases the production of dopamine, which by activation of D1- and D2-like receptors, promotes renal sodium and water excretion.1,3,33,34
The current study shows that long-term D1-like receptor stimulation (hours) increases D1 receptors and decreases AT1 receptors both in RPT from WKY rats17 and in A10 vascular smooth muscle cells from normotensive BD IX rats.24 This may be a mechanism by which D1-like receptors continue to exert their effects long after D1 receptor desensitization should have occurred.42–44 The inhibitory effect of the D1-like receptor on sodium transport has been consistent,1,3,14,19,37–41 whereas its effect on resistance vessels has not been so.1 Thus, although most studies have reported a vasorelaxant effect of D1-like receptors,1,3,42 a vasoconstriction effect has been reported in the rat tail artery.45 In our study, we found that fenoldopam increased D1 receptor expression in A10 cells but decreased AT1 receptor expression. These effects together with a D1-like receptor-induced increase in cAMP levels should lead to vasodilation in resistance vessels, at least in the normotensive state.
We found a direct interaction between AT1 and D1 receptors by immunoprecipitation study. The basal level of AT1/D1 coimmunoprecipitation is much higher in WKY rats as compared with SHR. D1 receptor stimulation with fenoldopam results in an increase in the coimmunoprecipitation of AT1 and D1 receptors in WKY rats, whereas a decrease occurs in SHR. The increase in AT1/D1 coimmunoprecipitation in WKY rats could have been caused by the increase in D1 receptor expression per se or an increased physical interaction between AT1 and D1 receptors. We favor the latter because D1 receptor expression in SHR RPT cells is not affected by fenoldopam, yet the AT1/D1 receptor coimmunoprecipitation is still decreased. It is possible that the D1 receptors antagonize AT1 receptor function in WKY rats by sequestering AT1 receptors. In SHR, the defective D1 receptor is unable to perform this function, allowing more AT1 receptors to respond to angiotensin II. Whether this differential D1/AT1 interaction in WKY and SHR has functional consequences remains to be determined. Further studies are also needed to determine whether the increased interaction between these two receptors is a direct or an indirect mechanism, possibly by the alteration of an adaptor gene or adaptor proteins. Others have shown that several G protein–coupled receptors are known to interact directly with each other by homo- or hetero-oligomerization.46,47 Thus, an adaptor protein for D2 and D3 receptors (protein 4.1N) has been recently identified that is important in their localization in plasma membranes.48
It is known that the D1-like receptor function is impaired in the renal proximal tubule and medullary thick ascending limb of genetically hypertensive rats, such as the SHR.1,3 The impaired D1-like receptor function is not caused by abnormalities in G proteins or effectors proteins but rather the D1-like receptor is uncoupled from its G protein/effector complex because of increased activity of the G protein–coupled receptor kinase type 4.1,49 Therefore, it is not unexpected that the ability of the D1-like receptor agonist to increase D1 receptor expression in WKY rats is no longer present in SHR. However, the persistence of the ability of D1-like receptor stimulation to inhibit AT1 receptor expression in RPT cells of SHR is unexpected. Because there are two D1-like receptors expressed in RPT cells,1,3 we hypothesize that the effect of D1-like agonist stimulation may have been exerted at the other D1-like receptor, the D5 receptor, which remains functional in the SHR. Our preliminary data show that in D5 knockout mice, AT1 receptor expression is higher in the renal cortical membrane as compared with its wild-type control animals (C. Zeng, J.E. Jones, L.D. Asico, and P.A. Jose, unpublished data, 2003). Because the increase in cAMP levels after D1-like receptor stimulation is due mainly to D1 receptor rather than D5 receptor effect,50 we presume that the inhibitory effect of D5 receptors on AT1 receptor expression may not have a significant impact on overall D1-like receptor function mediated by cAMP in the kidney of normotensive animals.
In mesangial, renal proximal tubular, vascular smooth muscle, and adrenocortical cells, increasing intracellular cAMP levels decreases steady state AT1 receptor expression.11,13,51–53 Since the 5′ promoter region of rat AT1 receptor expression has a cAMP response element, further studies will be required to determine whether cAMP decreases receptor expression by transcriptional and/or posttranscriptional mechanisms. We have reported that D1-like receptors can inhibit NHE3 activity independent of cAMP, through GαS.19 The linkage between D1 and D5 receptors to G protein subunits are different. Thus, the D5 receptor (but not the D1 receptor) is linked to Gα12 and Gα13.28 Gβ1 and Gγ7 transduce D1 (but not D5) receptor function.54 It is therefore possible that the regulation of AT1 receptors by D1-like receptors can occur by cAMP-independent and G protein subunit–dependent mechanisms.
In summary, we have demonstrated that the D1-like agonist fenoldopam positively regulates the expression of D1 receptors and negatively regulates AT1 receptors in vascular smooth muscle and RPT cells from normotensive rats. Whereas fenoldopam does not stimulate D1 receptor expression in SHR RPT cells, the inhibitory effect on AT1 receptor expression is seen. AT1 and D1 receptors physically interact with each other; fenoldopam increases the interaction between these two G protein–coupled receptors in WKY RPT cells but decreases it in SHR RPT cells.
Perspectives
The dopaminergic and renin-angiotensin systems are two important systems that regulate blood pressure. Dopamine causes natriuresis and vasorelaxation, whereas angiotensin II leads to opposite effects.1,3–6,12–14,19,36–41 The major D1-like receptor subtype mediating the increase in sodium excretion is probably the D1 receptor,1,3,50 whereas the major angiotensin II receptor mediating the decrease in renal sodium excretion is the AT1 receptor.6–10 In SHR, renal proximal tubular D1 receptor function is impaired.4 It is possible that the increased renal vascular resistance and increased renal sodium transport in SHR6,37,55 are caused by a defective interaction between D1 and AT1 receptors. However, which is primary and which is secondary remains to be determined.
Acknowledgments
These studies were supported in part by grants from the National Institutes of Health, HL-23081, DK-39308, HL-68686, DK-52612, HL-62211, and HL-41618.
- Received May 12, 2003.
- Revision received May 27, 2003.
- Accepted June 26, 2003.
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- Perturbation of D1 Dopamine and AT1 Receptor Interaction in Spontaneously Hypertensive Rats
