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Am J Physiol Heart Circ Physiol 288: H55-H61, 2005; doi:10.1152/ajpheart.00627.2004
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2004 CARDIOVASCULAR AND KIDNEY INVESTIGATORS MEETING

D5 dopamine receptor regulation of phospholipase D

Zhiwei Yang,1,2 Laureano D. Asico,2 Peiying Yu,2 Zheng Wang,2 John E. Jones,2 Ren-kui Bai,3 David R. Sibley,4 Robin A. Felder,5 and Pedro A. Jose1,2

1Department of Physiology and Biophysics, 2Department of Pediatrics, 3Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, District of Columbia; 4National Institute Neurological Disorders and Stroke, Bethesda, Maryland; and 5Department of Pathology, The University of Virginia Center for the Health Sciences, Charlottesville, Virginia

Submitted 24 June 2004 ; accepted in final form 18 August 2004


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
D1-like receptors have been reported to decrease oxidative stress in vascular smooth muscle cells by decreasing phospholipase D (PLD) activity. However, the PLD isoform regulated by D1-like receptors (D1 or D5) and whether abnormal regulation of PLD by D1-like receptors plays a role in the pathogenesis of hypertension are unknown. The hypothesis that the D5 receptor is the D1-like receptor that inhibits PLD activity and serves to regulate blood pressure was tested using D5 receptor mutant mice (D5–/–). We found that in the mouse kidney, PLD2, like the D5 receptor, is mainly expressed in renal brush-border membranes, whereas PLD1 is mainly expressed in renal vessels with faint staining in brush-border membranes and collecting ducts. Total renal PLD activity is increased in D5–/– mice relative to congenic D5 wild-type (D5+/+) mice. PLD2, but not PLD1, expression is greater in D5–/– than in D5+/+ mice. The D5 receptor agonist fenoldopam decreases PLD2, but not PLD1, expression and activity in human embryonic kidney-293 cells heterologously expressing the human D5 receptor, effects that are blocked by the D5 receptor antagonist SCH-23390. These studies show that the D5 receptor regulates PLD2 activity and expression. The hypertension in the D5–/– mice is associated with increased PLD expression and activity. Impaired D5 receptor regulation of PLD2 may play a role in the pathogenesis of hypertension.

hypertension

DOPAMINE has been shown to be an important regulator of blood pressure, sodium balance, and renal and adrenal function through an independent peripheral dopaminergic system. The effects of dopamine are exerted by two subfamilies of cell surface receptors D1-like and D2-like receptors, which belong to the rhodopsin-like family (family A) of G protein-coupled receptors. D1-like receptors (D1 and D5) are coupled to the stimulatory G proteins G{alpha}S and Golf and stimulate adenylyl cyclases, whereas D2-like receptors (D2, D3, D4) are coupled to the inhibitory G protein G{alpha}i and Go and inhibit adenylyl cyclases (14, 17, 30). There are no ligands that can distinguish the D5 from the D1 receptor, but the D5 receptor has a higher affinity for dopamine than the D1 receptor and exhibits some constitutive activity (33, 34).

Phospholipase D (PLD) is a ubiquitous enzyme stimulated by many cell surface receptors that hydrolyze phospholipids, such as phosphatidylcholine (PC), to form phosphatidic acid (PA) and the free polar head group of the phospholipid substrate (5, 8, 23). PA can be cleaved by PA phosphohydrolase to produce diacylglycerol, both of which are important second messengers in the "late" response of cells to certain stimuli (8). The two mammalian isoforms of PLD (PLD1 and PLD2) have ~50% identity and are distributed widely in mammalian tissues and cells. They are believed to play an important role in the regulation of cell function and cell fate by a variety of extracellular signals (5, 8, 23). PLD1 is localized to perinuclear regions (endoplasmic reticulum, Golgi apparatus, and late endosomes), whereas PLD2 is localized primarily to the plasma membrane (5, 23). PLD1 has low basal activity and can be activated by ADP-ribosylation factor (ARF), Rho, phosphatidylinositol 4,5-bisphosphate, and protein kinase C (PKC) (5, 9, 22). PLD2 is activated by oleate, other unsaturated fatty acids, and PKC (5, 28). PLD2 is constitutively active and regulated primarily by inhibitory mechanisms (5, 32).

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension (14, 17). Hollon et al. (12) have reported that a deficiency of the D5 receptor in mice produces arterial hypertension. Because oxidative stress is thought to be important in the pathogenesis of hypertension (41), it is of interest that dopamine, via a D1-like receptor, has been reported to have antioxidant properties by inhibition of PLD activity that apparently involves both D1 and D5 receptors (43). Both protein kinase A (PKA) and PKC were also suggested to be involved. However, although both D1 and D5 receptors increase PKA activity, D1 receptors stimulate, whereas D5 receptors inhibit phospholipase C (PLC) activity (6, 40).

Human embryonic kidney (HEK)-293 cells endogenously express both PLD1 and PLD2 (25) but express neither D1 nor D5 receptors (39). Therefore, studies were conducted to determine the effects of D5 receptor on the activity and expression of PLD and isoforms in HEK-293 cells heterologously expressing the D5 receptor, as well as catalytically inactive variants of PLD1 and PLD2. We also studied PLD isoform expression and activity in congenic D5 receptor mutant (D5–/–) mice on a C57BL/6 Taconic background and an F2 generation of mixed B129 and C57BL/6 background.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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D5 Receptor Mutant Mice Blood Pressure

D5–/– and D5 wild-type (D5+/+) mice were generated by injecting C57BL/6 mouse blastocysts with 129/SV embryonic stem cells containing the targeting construct generated by ligating the neomycin resistance gene in reverse orientation at the unique SfiI site in the second intracellular loop of the D5 receptor. Disruption of the D5 receptor gene is not associated with any alteration of the expression of other dopamine receptors, including D1, D2, D3, and D4 receptors (12, 13). The second generation (F2) of D5–/– mice is in a mixed background of 129/SV and C57BL/6. Backcrossing to the sixth generation (F6) results in a C57BL/6 (Taconic) genetic background that is >98% congenic (11, 42). We studied 6-mo-old gender-matched F2 generation and >F6 generation mice as indicated. D5+/+ littermates were used as controls. The statistical analyses were performed separately for the F2 and >F6 mice; i.e., the >F6 D5–/– mice were compared with their >F6 D5+/+ controls and the F2 D5–/– mice were compared with their F2 D5+/+ controls. Mice were fed a 0.4% sodium diet. Arterial blood pressures were first directly measured via the femoral artery in pentobarbital-anesthetized (50 mg/kg ip) mice. The kidneys were then removed and the mice euthanized with an overdose of pentobarbital (150 mg/kg iv).

Cell Cultures and Transfections

HEK-293 cells were cultured in Dulbecco's modified Eagle's medium (GIBCO-BRL) containing 4.5 g/l glucose, 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. The full-length human (h) D5 receptor cDNA subcloned into pcDNA6/V5-His vector (Invitrogen) between EcoRI and XbaI or an empty vector control was used for transfection (48). Wild-type hPLD1, mouse (m) PLD2, and their catalytically inactive variants K898R-PLD1 and K758R-PLD2 were fused to Aequorea victoria green fluorescent protein by subcloning into pEGFP-C1 (generously provided by Dr. Guillermo G. Romero) (29). hD5 receptor and K898R-PLD1 or K758R-PLD2 were cotransfected in HEK-293 cells. Stably transfected single colonies were selected with 10 µg/ml blasticidin for the D5 receptor and 600 µg/ml neomycin (G418) for the PLDs. The successful transfections of D5 receptor cDNA were verified by immunoblotting for V5/D5 expression and measuring cAMP production for hD5 receptor function. Transfection efficiency of PLDs was assessed by conventional epifluorescence microscopy before experimentation.

Immunohistochemistry

Four-micrometer sections were cut from a Bouin's solution-fixed, paraffin-embedded mouse kidney. The tissue sections were deparaffinized and rehydrated by successive incubations in xylene and decreasing concentrations of ethanol (100%, 95%, and 75%) and PBS. The tissue sections were then briefly treated with 0.1% Triton X-100, 10 mM sodium citrate (pH 6.0), and 3% H2O2 and then blocked sequentially with 10% normal serum in phosphate-buffered saline overnight at 4°C. The tissue sections were incubated with anti-PLD1/2 (Biosource International) and D5 receptor (Santa Cruz) antibodies at a dilution of 1:100 at room temperature for 1 h. After being stained with a VIP substrate kit (Vector), the slides were permanently mounted. Primary antibodies previously incubated with the immunizing peptides were used in control experiments.

Tissue and Subcellular Fractions

After measurement of blood pressure, the kidneys were immediately removed, cut into small pieces, and mixed with 5 vol of buffer A [in mM: 50 HEPES-NaOH (pH 7.4), 1 MgCl2, 1 EGTA, and 0.25 sucrose] containing 10 µM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 20 µg/ml aprotinin. The tissue was homogenized with a Potter-Elvehjem Teflon-glass homogenizer and sonically disrupted. The homogenate was centrifuged for 10 min at 1,000 g to remove nuclei and unbroken tissues. The postnuclear fraction was used for PLD activity assay and lysed with 1% Triton X-100 for immunoblotting.

The cells were starved in serum-free medium for 2 h and then treated with vehicle or fenoldopam (D1-like agonist) and/or SCH-23390 (D1-like antagonist) at the indicated time and concentration. The cells were then washed twice with cold PBS and scraped after adding lysis buffer (buffer A with 1% Triton X-100). The cell lysates were centrifuged at 12,000 g for 30 min. Protein concentration was determined by the Bradford method.

Immunoblotting

Mouse kidney tissue and cellular samples were subjected to SDS-PAGE and immunoblotted as previously reported (46) but using specific human PLD1/2 antibodies. Uniformity of protein loading for immunoblotting was determined by staining the membranes with 0.1% Ponceau S and immunoblotting them against {beta}-actin.

Real-Time Quantitative PCR

Total mRNA was extracted from HEK-293 cells heterologously expressing the hD5 receptor (HEK-hD5R) with or without fenoldopam treatment using RNAzol B RNA isolation kit. Reverse transcription of total RNA was performed using Moloney murine leukemia virus RT with 1 µg total RNA in 20 µl of the reaction mixture. PLD2 primers were based on unique sequences: 5'-AGA GAC TTC CTA CAG CTG CAC-3' (sense primer, nucleotide positions 1030–1050) and 5'-TGC CAC AGC AGC AAA GTA ACC-3' (antisense primer, nucleotide positions 1134–1114) of the human PLD2 mRNA sequence (accession no. NW-002663) separated by a 3,465-bp intron (accession no. NT-010718). {beta}2-Microglobulin cDNA ({beta}2M) primers were based on unique sequences: 5'-TTC TGG CCT GGA GGC TAT CC-3' (sense primer, nucleotide positions 45–64) and 5'-ACA TAG CAA TTC AGG AAA TTT GAC-3' (antisense primer, nucleotide positions 142–117) of the human full open reading frame cDNA for gene {beta}2M (accession no. CR457066) separated by a 3,810-bp intron (accession no. NT-030828). Real-time quantitative PCR (RT-qPCR) assays were performed in triplicate, using SYBR Green qPCR SuperMix UDG kit (Invitrogen). Real-time PCR conditions were 2 min at 50°C and 10 min at 95°C, followed by 45 cycles of denaturation (15 s/cycle) at 95°C and annealing/extension for 30 s at 60°C. The fluorescent signals were recorded and analyzed during PCR in an ABI Prism 7700 sequence detector system (Applied Biosystems) using SDS software (Version 1.91). Dissociation curves of the amplifications were generated after each run to make sure that the increased fluorescent intensities were not due to nonspecific signal (primer dimer). The increase in fluorescent signal is associated with an exponential growth of PCR products during the linear log phase. The threshold cycle or the Ct value is the cycle at which a significant increase in the reaction product is first detected. The higher the initial amount of cDNA, the sooner the accumulated product is detected in the PCR process, and the lower the Ct value. The expression of PLD2 gene was normalized to the expression of {beta}2M gene and presented as the cDNA ratio of the target PLD2 to {beta}2M gene calculated by 2, where {Delta}Ct = C– C.

PLD Activity Assay

PLD activity was performed by using the transphosphatidylation method, as previously described (27, 29). PLD activity in kidney tissue was initiated by adding 100 µl of a solution containing 2 µmol HEPES-NaOH (pH 7.4), 2 nmol [14C]PC (13,750 dpm/nmol) (NEN), 34.2 µmol ethanol, 10 nmol GTP[{gamma}S], and 160 µmol ammonium sulfate neutralized with ammonium hydroxide to 100 µg postnuclear fraction. After incubation for 20 min at 37°C, the reaction was stopped by the addition of 1 ml of ice-cold chloroform-methanol-HCl (1:1:0.006 vol/vol/vol) and mixed well. The lipid phase was extracted and developed by thin-layer chromatography on silica gel 60 plates using the upper phase of a mixture of ethyl acetate-2,2,4-trimethylpentane-acetic acid-H2O (13:2:3:10 vol/vol/vol) as a solvent. The position of major phospholipids was determined using true standards, phosphatidylethanol, and autoradiography. The total amount of radioactivity associated with each lipid species was determined by liquid scintillation counting (27).

The PLD activity measurement in HEK-293 cells was initiated after cells, subcultured into 60-mm dishes, reached 80% confluence. After an overnight labeling with [3H]palmitate (5 µCi/ml) (NEN) in quiescent medium, the cells were treated with fenoldopam and SCH-23390 at the doses and times specified in the RESULTS, followed by the addition of 1% ethanol (final concentration). The reaction was stopped by the addition of chloroform-methanol (1:1 vol/vol). The lipid phase was extracted and developed using ethyl acetate-trimethylpentane-acetic acid (9:5:2 vol/vol/vol) as the solvent. The same procedure as the described above was used to determine phosphatidylethanol formation (29).

Statistical Analysis

Data are expressed as means ± SE. Comparison within groups was made by ANOVA for repeated measurements and Scheffé's (multiple comparisons) or Dunnett's (versus control) test, and a comparison between two groups utilized the t-test. A value of P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vivo Studies

Blood pressure. The F2 generation D5–/– mice exhibited significantly elevated systolic, diastolic, and mean arterial blood pressures under pentobarbital anesthesia. This finding is consistent with our previous report in anesthetized and conscious D5–/– mice (12). The >F6 generation of D5–/– mice also had elevated blood pressure (Table 1). As described above, the F2 generation mice are in mixed 129/SV and C57BL/6 genetic background, and >F6 generation mice are in a C57BL/6 genetic background that is >98% congenic. The 129/SV mouse is more susceptible than the C57BL/6 mouse to the development of DOCA-induced hypertension and renal damage (10). However, we did not find a significant difference in the blood pressure between F2 generation and >F6 generation D5+/+ or D5–/– mice (Table 1).


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  		Table 1. Arterial blood pressure in pentobarbital-anesthetized D5+/+ and D5–/– mice

 
PLD distribution in mouse kidney. Both D5 and PLD2 were mainly expressed in the brush-border membrane of the renal proximal tubule (Fig. 1, A,a and B,a). PLD1 was mainly expressed in renal vessels, with faint staining in brush-border membranes (Fig. 1C) and collecting ducts (not shown). There was no staining when the primary antibodies were blocked by their respective immunizing peptides (Fig. 1, A,b; B,b; and C,b).



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  		Fig. 1. Mouse kidney immunohistochemistry with anti-human phospholipase D (hPLD1), anti-mouse PLD (mPLD2), and D5 receptor antibodies. A,a: D5 receptor antibody; A,b: D5 receptor antibody blocked by its immunizing peptide. B,a: mPLD2 antibody; B,b: PLD2 antibody blocked by its immunizing peptide. C,a: hPLD1 antibody; C,b: PLD1 antibody blocked by its immunizing peptide (magnification = x10, Bars = 0.1 mm). The studies were performed at least three times in each group.

 
PLD protein expression. PLD2 (110 kDa) expression was increased in F2 generation D5–/– relative to D5+/+ mice (t-test, P < 0.05, n = 6 mice/group). There was no difference in PLD1 expression between the two strains (Fig. 2). The anti-human PLD1 antibody recognized a 120- and 110-kDa species in the brain and mouse kidney, respectively. No bands were seen when the PLD1 and PLD2 antibodies were preadsorbed with the immunizing peptide (not shown). Similar results were found in the >F6 mice (not shown).



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  		Fig. 2. PLD expression in mouse kidney. Tissue postnuclear particulate protein (100 µg) was subjected to immunoblotting with polyclonal rabbit hPLD1 and mPLD2 antibodies. Both hPLD1 and mPLD2 antibodies recognize a 110-kDa protein in mouse kidney. All immunoblotting results are expressed as relative density units (*P < 0.05 vs. D5+/+, t-test, n = 6/group). Inset, immunoblot with anti-PLD1 and anti-PLD2 and {beta}-actin.

 
PLD activity. PLD activity was increased in F2 or >F6 generation D5–/– relative to D5+/+ mice (t-test, P < 0.05, n = 3 mice/group) (Fig. 3).



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  		Fig. 3. PLD activity in mouse kidney. PLD activity was assayed by measuring the formation of radioactive phosphatidylethanol by phosphatidyl transfer reaction of [14C]-[3H]PC to ethanol. F2 and F6, second and sixth generation mice, respectively. Results are expressed as means ± SE (*P < 0.05 vs. D5+/+, t-test, n = 6/group).

 
In Vitro Studies

The mechanism by which the D5 receptor regulates PLD expression and activity was studied further in HEK-hD5R cells. In HEK-hD5R cells, a 30-min incubation with the D1-like agonist fenoldopam (10–7 mol/l) stimulated cAMP accumulation. No stimulation was found in empty vector-transfected cells. Radioligand binding assay ([3H]SCH-23390 with specific binding defined by 1 µM d-butaclamol) in this cell line showed a maximum receptor density of 2,400 fmol/mg protein and a dissociation constant of 0.80 nmol/l (n = 3).

PLD protein expression. In HEK-hD5R, the D1-like agonist fenoldopam caused a time-dependent (Fig. 4A) and a concentration-dependent (Fig. 4B) decrease in PLD2 but not in PLD1 (not shown) expression. The effect of fenoldopam was exerted at a D1-like receptor because its inhibitory effect was blocked by the D1-like receptor antagonist SCH-23390 (Fig. 4C). The fenoldopam-mediated inhibition of PLD2 expression was restored 12 h after removal of fenoldopam (Fig. 4D). PLD2 expression was unchanged for up to 48 h of incubation in cells not exposed to fenoldopam (not shown). There were no effects in vector-transfected cells (not shown).



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  		Fig. 4. PLD expression in human embryonic kidney (HEK)-293 cells heterologously expressing the hD5 receptor (HEK-hD5R) cells. Protein (100 µg) from HEK-hD5R cells was subjected to immunoblotting with polyclonal rabbit mPLD2 antibody, which recognizes a 110-kDa protein in this cell line. A and B: dose response (24 h) and time course (1 µmol/l) of PLD2 expression in HEK-hD5R cells treated with the D1-like receptor agonist fenoldopam (Fen). C: PLD2 expression in HEK-hD5R cells treated with the D1-like agonist Fen and/or the D1-like antagonist SCH-23390 (SCH, 1 µmol/l per 24 h). All immunoblotting results (A–C) are expressed as relative density units. Insets, immunoblots of PLD2 and {beta}-actin (A–C). [*P < 0.05 vs. controls (C), ANOVA Dunnett's test, n = 4/group]. D: HEK-hD5R cells were serum starved for 2 h and then treated with Fen (1 µmol/l) for 12 h. Thereafter, Fen was removed from the culture media, and the cells were reincubated with fresh serum-free medium without Fen for 0 (control), 6, 12, 24, or 36 h. This experiment was performed in triplicate. Protein samples were then subjected to immunoblotting with polyclonal mPLD2 antibody. Results are expressed as percentage of recovery of PLD2 expression. *P < 0.05 vs. control, ANOVA Dunnett's test, n = 3/group.

 
PLD2 mRNA levels. Fenoldopam had no effect on PLD2 mRNA expression, indicating that the D5 receptor does not regulate the transcription or postranscription of PLD2 in this cell line (cDNA ratio of the target PLD2 to {beta}2M gene, control = 0.040 ± 0.002, fenoldopam treatment = 0.043 ± 0.002, n = 3).

PLD activity. In preliminary studies (44), we found that the D1-like agonist fenoldopam inhibited PLD activity in a time- and a concentration-dependent manner in Chinese hamster ovary cells transfected with hD5 cDNA. We used the same conditions to test PLD activity in HEK-hD5R cells. Fenoldopam inhibited PLD activity in HEK-hD5R cells, expressing the catalytically inactive PLD1 K898R-PLD1 (Fig. 5). However, fenoldopam did not inhibit basal PLD or phorbol myristate acetate (PMA)-stimulated (5, 31) activity in HEK-hD5R cells expressing the catalytically inactive PLD2 K758R-PLD2 (Fig. 5), suggesting that the PLD2 isoform is the target of the D5 receptor.



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  		Fig. 5. PLD activity in HEK-hD5R cells. PLD activities in HEK-hD5R cells expressing a catalytically inactive variant of PLD1 (K898R-PLD1), and thus with PLD2 activity, and expressing a catalytically inactive variant of PLD2 (K758R-PLD2), and thus with PLD1 activity, treated with Fen, and/or SCH (1 µmol/l, 30 min), and/or phorbol myristate acetate (PMA, 0.1 µmol/l, 20 min). Results are expressed as means ± SE. PtdEtOH, phosphatidylethanol. *P < 0.05 vs. K898R-PLD1 control; #P < 0.05 vs. K758R-PLD2 control, ANOVA Dunnett's test, n = 3–7/group.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The data presented in this study suggest that the D5 receptor inhibits PLD2 expression and activity. In the absence of the D5 receptor, as is the case in D5–/– mice, PLD activity and PLD2 but not PLD1 expression were increased compared with wild-type controls. The negative regulatory effect of the D5 receptor on PLD2 activity and expression was confirmed by in vitro studies in which the D1-like receptor agonist fenoldopam inhibited PLD2, but not PLD1, expression in HEK-hD5R cells and PLD activity in the cell line coexpressing the catalytically inactive K898R-PLD1 but not the catalytically inactive K758R-PLD2. More interestingly, we found that PLD2 and the D5 receptor were mainly expressed in the brush-border membrane of proximal tubules. Because both of them are cell-membrane proteins and constitutively active, it is possible that the D5 receptor is important in the regulation of PLD2 in vivo.

In this study, we found that the D1-like agonist fenoldopam decreased PLD activity in HEK-hD5R cells coexpressing K898R-PLD1 even without prior PLD stimulation, in agreement with reports that PLD2 is constitutively active (32). However, fenoldopam does not show any evidence of inhibiting PLD activity in HEK-hD5R cells coexpressing K758R-PLD2. Because the low basal of PLD1 (8, 22) may have precluded the observation of an inhibitory effect of fenoldopam, we stimulated PLD activity with the potent PLD stimulator PMA (5, 31) in the cells cotransfected with the dominant negative PLD2. Despite increasing PLD (presumably PLD1) activity in these cells, fenoldopam still failed to inhibit PLD activity. Therefore, the D5 receptor specifically regulates PLD2 and not PLD1 activity.

PLD has been shown to be regulated by a number of G protein-coupled and tyrosine kinase receptors (5). As mentioned above, unlike PLD1, which is quiescent in vitro and in vivo until stimulated by PKC, ARF, and Rho, PLD2 is constitutively active and regulated primarily by specific inhibitory proteins in cells (5, 32). The reported PLD inhibitors include a heat-stable 18-kDa protein, adolase, ceramide, actinin, {alpha}- and {beta}-synucleins, and a vesicle exocytosis-related protein Munc-18–1, most of which inhibit PLD2 activity by protein-protein interaction via a PLD-PX binding domain (3, 16, 18, 20, 21, 31). PKA has been reported to inhibit PLD activity in cell-free systems of human neutrophils (19) and in vascular smooth muscle cells (43). It has also been suggested that PLD1, not PLD2, is phosphorylated by PKA (15) and that PKA stimulates PLD activity in tracheal smooth muscle strips (24). However, we found that forskolin, an activator of all adenylyl cyclase isoforms, except isoform 9, did not affect PLD activity in both Chinese hamster ovary and HEK-293 cells (unpublished observations). Both PLD2 and D5 receptors are membrane proteins and are found in similar locations in the brush-border membrane of renal proximal tubules. Therefore, a possible mechanism for the inhibition of PLD2 by the D5 receptor is a direct interaction between the two membrane proteins. We also found that the D5 receptor decreased PLD2 protein expression but not PLD2 mRNA levels in HEK-hD5R cells. Therefore, the decrease in PLD2 protein expression caused by the D5 receptor may be via translational or posttranslational (degradation) mechanisms.

PLD has been suggested to be involved in the pathophysiology of hypertension. PLD activity and protein levels of PLD1 were increased in the brain and liver of the spontaneously hypertensive rats (SHR) compared with their normotensive controls Wistar-Kyoto (WKY) rats (26). PLD, stimulated by angiotensin II, has been implicated in vascular contractility and remodeling, cardiac hypertrophy, and vascular smooth muscle cell proliferation (4, 8, 35). Angiotensin II, via the AT1 receptor, increased PLD activity to a greater degree in SHR than in WKY rats (1). The magnitude of the increase in PLD activity and the rate of activation in response to angiotensin II were greater in aortic vascular smooth muscle cells in SHR than in WKY rats (37). Angiotensin II-stimulated PLD activity and DNA and protein synthesis were also greater in vascular smooth muscle cells from the aorta of hypertensive than normotensive rats (7). Dopamine, acting via D1-like receptors, attenuates angiotensin responses by downregulating AT1 receptors. The D1-like agonist fenoldopam decreased AT1 mRNA and protein expression in renal proximal tubule cells (2, 47). We also found that AT1 expression is increased in D5–/– mice compared with D5+/+ mice (unpublished observations). The ability of PLD to increase NADH/NADPH oxidase activity and generation of reactive oxygen species has been implicated in vascular smooth muscle hypertrophy and remodeling in hypertension (8, 36). Yang et al. (45) reported preliminary evidence that the D5 receptor decreases oxidative stress by inhibiting NADPH oxidase expression and activity. Therefore, the increase in AT1 receptors (which stimulate PLD expression and activity) associated with the absence of D5 receptors (which inhibit PLD2 expression and activity) may both contribute to the increase in PLD expression and activity in hypertension.

We conclude that dopamine inhibits PLD2 expression and activity via the D5 receptor. We suggest that by inhibiting PLD and NADPH oxidase expression and activity and by downregulating AT1 expression, the constitutively active D5 receptor plays an important role in preventing the development of hypertension.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants DK-39308 and HL-68686.


   ACKNOWLEDGMENTS
 
We thank Dr. Peter M. Andrews for assistance in immunohistochemistry studies, Drs. Rhian M. Touyz and Guillermo G. Romero for help in the assay of PLD activity, and Dr. Lee-Jun C. Wong for help in real-time quantitative PCR.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. Jose, Pediatrics and Physiology and Biophysics, Georgetown Univ. Medical Center, 3800 Reservoir Rd. NW, Washington, DC 20057 (E-mail: pjose01{at}georgetown.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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