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(Hypertension. 1997;29:962-968.)
© 1997 American Heart Association, Inc.

Articles

Dopamine D_1A Receptors and Renin Release in Rat Juxtaglomerular Cells

Ikuyo Yamaguchi; Lynne Yao; Hironobu Sanada; Ryoji Ozono; M. Maral Mouradian; Pedro A. Jose; Robert M. Carey; ; Robin A. Felder

From the Department of Pathology (I.Y., H.S., R.A.F.) and Department of Medicine (R.O., R.M.C.), The University of Virginia Health Sciences Center, Charlottesville; Department of Pediatrics, Georgetown University Children's Medical Center, Washington, DC (L.Y., P.A.J.); and Genetic Pharmacology Unit, National Institute of Neurological Disorders and Stroke, The National Institutes of Health, Bethesda, Md (M.M.M.).

Correspondence to Robin A. Felder, PhD, Department of Pathology, Box 168, MSB6171 J, The University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail raf7k{at}virginia.edu

	Abstract

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Abstract Two dopamine D₁-like receptors have been cloned from mammals, the D₁ and D₅ receptors, also known as D_1A and D_1B receptors, respectively, in rodents. Although D₁-like receptors are known to stimulate renin release, the receptor subtype mediating this action has not been determined. We investigated D₁ receptor subtype expression in rat juxtaglomerular cells obtained after enzymatic dispersion of kidney cortex and differential centrifugation. Juxtaglomerular cells in primary culture were immunocytochemically 85% to 95% renin positive. These cells expressed the D_1A but not the D_1B receptor (mRNA and protein). D₁-like receptor function was demonstrated by a concentration-dependent stimulation of cAMP production by dopamine (n=5-9 per group). Fenoldopam, a D₁-like receptor agonist, also caused a concentration-dependent increase in cAMP production and renin secretion that was blocked by the selective D₁-like receptor antagonist SCH23390 (n=4-13 per group). Although the D₁ ligands do not distinguish between the cloned D₁-like receptors, the actions of fenoldopam were due to occupancy of the D_1A receptor: (1) the D_1B receptor, the only other mammalian D₁-like receptor, is not expressed in juxtaglomerular cells; (2) antisense but not sense D_1A oligonucleotides completely blocked the stimulatory effect of fenoldopam on cAMP production and renin secretion. We conclude that there is selective dopamine receptor gene expression in juxtaglomerular cells; the dopamine receptor subtype linked to the stimulation of cAMP and renin secretion in juxtaglomerular cells is the D_1A subtype.

Key Words: renin • receptors, dopamine • cyclic AMP • immunohistochemistry • oligonucleotides, antisense

	Introduction

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The renin-angiotensin system is a major hormonal system involved in the regulation of electrolyte and fluid balance and blood pressure. Renin secretion is regulated by numerous mechanisms, including the renal afferent arteriolar baroreceptor, macula densa, and sympathetic nervous system as well as humoral factors and certain ions.^{1 2} Since the kidney produces dopamine and dopaminergic nerves terminate directly on JG cells, it has been suggested that dopamine may directly stimulate renin release.³ However, dopamine activates not only dopamine receptors but also - and ß-adrenergic receptors. Moreover, the effects of dopamine on blood pressure, cardiac output, and regional blood flow distribution may indirectly influence renin activity. Indeed, dopamine has been reported to increase, decrease, or not affect renin secretion in vivo.^{4 5 6 7 8 9 10 11 12 13} In vitro studies in rats have shown that pharmacological stimulation of renal dopamine D₁-like receptors with dopamine or fenoldopam, a D₁ agonist, increases renin secretion.^{14 15} However, D₂-like receptors may produce the opposite effect.¹⁶

The determination of the dopamine receptor subtype involved in these actions is complicated by the fact that several D₁-like and D₂-like receptors have been cloned.^{17 18 19 20 21 22 23 24 25 26} The D₁-like receptors, which include the D_1A, D_1B, D_1C, and D_1D receptors (D_1A and D_1B are also known as D₁ and D₅ in humans), are linked to stimulation of adenylyl cyclase; the D₂-like receptors, which include the D₂, D₃, and D₄ receptors, are linked to inhibition of adenylyl cyclase.^{17 18 19 20 21 22 23 24 25 26} D_1A, D_1B, D_2L, and D₃ receptors have been identified in the kidney.^{18 27 28 29 30 31 32} However, the role of dopamine receptor subtypes in renal physiology is not well understood.

Studies of the control of renin release in vivo are complicated by the multiplicity of factors that directly or indirectly interact with the JG cell.^{1 2 3} To determine which dopamine receptor subtype is involved in stimulating renin secretion, we studied dopamine receptor gene expression in rat JG cells grown in primary culture. This method enabled us to study renin release independently of confounding factors in vivo, such as changes in systemic and renal hemodynamics, renal nerve activity, and ionic composition of the fluid bathing the JG cell and macula densa.

	Methods

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JG Cell Preparation
All studies were approved by the University of Virginia Animal Welfare Committee. Renal cortical cells were harvested with a modification of a method previously described by our laboratory³³ from male Sprague-Dawley rats (Hilltop Lab Animals, Inc, Scottdale, Pa) weighing 50 to 60 g. The use of young rats facilitated the harvesting and growth of JG cells in primary culture (unpublished observations, 1996). For each cell preparation, one rat was killed by decapitation, the descending aorta was cannulated, and both kidneys were perfused with RPMI-1640 medium (GIBCO) containing an enzyme solution consisting of 0.07% collagenase type A (Boehringer Mannheim Biochemicals), 0.12 mg/mL elastase (type III), 0.25 mg/mL soybean trypsin inhibitor, 0.16 mg/mL deoxyribonuclease I, 0.3% crystallized BSA (Sigma Chemical Co), 100 U/mL penicillin, and 100 µg/mL streptomycin (Pen/Strep, GIBCO-BRL).

Both kidneys were excised, decapsulated, hemisected, and demedullated. The cortices were minced and transferred to a Spinner flask (Bellco), brought to 25 mL with enzyme solution, and incubated at 37°C with spinning for three consecutive 30-minute periods. Between periods, the cells were triturated and gassed with 95% O₂/5% CO₂. After 90 minutes, the enzymatic dispersion was stopped by addition of 5 mL fetal calf serum and further dilution of the solution threefold to fourfold in RPMI-1640 medium containing 0.1% BSA and Pen/Strep (RPMI-BSA-P/S). The cells were collected by centrifugation at 200g for 10 minutes and resuspended in 100 mL RPMI-BSA-P/S. They then were centrifuged in a Percoll density gradient; an isotonic 40% Percoll solution (Sigma) was prepared and centrifuged at 31 000g for 20 minutes. The cells that sedimented at a density of 1.067 g/mL were carefully aspirated from the gradient and washed free of Percoll by centrifugation (200g) in 5 vol RPMI-BSA-P/S.

The cells were resuspended in culture medium (RPMI-1640, 25 mmol/L HEPES, 0.66 U/mL insulin, 2% fetal calf serum, and Pen/Strep)³⁴ and diluted to a concentration of 1.25x10⁵ to 2x10⁵ cells per milliliter. The cells were then seeded at 2x10³ to 4x10³ cells per centimeter squared in plastic tissue culture dishes. The cells were incubated at 37°C in 21% O₂/5% CO₂ for 24 to 72 hours.

Immunoperoxidase Immunocytochemistry
Immunocytochemistry of D_1A receptors was performed as previously described.³² Rabbit polyclonal antibody was raised against a synthetic peptide sequence corresponding to the third extracellular portion of the rat D_1A receptor (²⁹⁹GSEETQPFC³⁰⁷). The specificity of the polyclonal antisera was verified by its ability to recognize the native receptor that had been stably transfected and expressed in a murine fibroblast LTK^- cell line.³² The transfected cells, grown on poly-L-lysine–coated glass well slides, were positively stained with the antisera using the immunoperoxidase technique, but no staining was observed (1) when antisera were preincubated with the immunogen peptide (preadsorption), (2) when antisera were replaced with preimmune rabbit serum, or (3) when the transfected cells were replaced with wild-type or D_1B-transfected LTK^- cells.

JG cells were grown on poly-L-lysine–coated glass chamber slides. On day 3, the cells were fixed for 45 to 60 minutes in 4% paraformaldehyde in PBS at room temperature. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol (30 minutes). The cells were incubated with antisera diluted 1:4000 to 1:1000 in PBS containing 1.5% normal goat serum and 0.5% nonfat dry milk with 0.01% saponin (to enhance permeabilization) overnight at 4°C. Thereafter, positive staining was detected with the avidin-biotin immunoperoxidase reaction (Vectastain ABC Elite Kit, Vector Laboratories, Inc) using diaminobenzidine (DAB Fast Tablets, Sigma) as a substrate. Before the incubation with antisera, the cells were treated with blocking serum (PBS with 3% normal goat serum and 1% nonfat dry milk, 0.01% saponin) for 30 minutes to reduce the nonspecific binding of secondary goat biotinylated antibody to JG cells. Negative controls included substitution of antisera with preimmune sera and preabsorption of antisera. For preadsorption, antisera were incubated overnight at 4°C with the antigen peptide in 10- to 30-fold molar excess before application to JG cells.

Reverse Transcription–Polymerase Chain Reaction
Total cellular RNA was prepared from cultured cells with Tri-Reagent LS (Molecular Research Center, Inc). Reverse transcription (reaction volume, 20 µL) was performed in the presence of 20 pmol antisense primer (bases 1010-1029) for the D_1A receptor or oligo(dT) for the D_1B receptor (1 mmol/L deoxynucleotides, 5 mmol/L Tris-HCl [pH 8.3], 25 mmol/L KCl, 5 mmol/L MgCl₂, 1 U RNase inhibitor, and 2.5 U Moloney murine leukemia virus reverse transcriptase).²⁷ The reaction tubes were incubated in a Twin Block system (ERICOMP) at 42°C for 30 minutes, 99°C for 5 minutes, and 5°C for 5 minutes (GeneAmp RNA PCR Kit, Perkin-Elmer Cetus). A control group in which all the reactants were added except for reverse transcriptase was tested in parallel with each sample.

PCR was performed with the GeneAmp RNA PCR Kit. Specific D_1A primers, 20 to 21 nucleotides in length, were designed with 50% to 60% GC composition.²⁷ The primers for the D_1A receptor flanked the portion of the cDNA that corresponded to the third cytoplasmic loop of the receptor (sense primer: identical to bases 783-803, 5'-TGCCCAGAAGCAAATCCGGCG-3'; antisense primer: complementary to bases 1010-1029, 5'-CTCCTCAGAGCCACAGAAGG-3').²⁷ The specific amplification product was 247 bp in length. The sense primer for the D_1B receptor was identical to bases 651-669 (5'-AACCTATGCCATCTCCTCG-3') and the antisense primer was complementary to bases 1131-1151 (5'-ATGTTTACCGTCTGCACTGGG-3').²⁰ PCR was carried out (total volume of 100 µL) in the presence of 20 pmol of each primer, 0.20 mmol/L of each deoxynucleotide, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.0 mmol/L MgCl₂, and 2.5 U Taq DNA polymerase. The reaction cycles were set at 94°C for 3 minutes (initial melt), followed by 40 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute, with maintenance at 4°C until recovery for analysis.²⁷

Western Blotting
JG cells were seeded on 150-cm² plates at approximately 25 000 cells per centimeter squared and were washed after 1 day of growth and used after 2 days. The cells were detached from the plates by scraping in buffer A (20 mmol/L Tris-HCl [pH 7.4], 2 mmol/L EDTA, 2 mmol/L phenylmethylsulfonyl fluoride [PMSF], 10 mmol/L Na₃VO₄, 100 mmol/L NaCl, 10% glycerol, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). The cell fragments were then centrifuged at 30 000g for 30 minutes at 4°C. The supernatant was discarded and the pellet resuspended in a minimal volume of buffer B (buffer A plus 1% NP-40, Sigma) and stirred on ice for 2 hours to solubilize the receptor. The suspension was recentrifuged and the resulting supernatant aliquoted and loaded onto an 8% sodium dodecyl sulfate–polyacrylamide gel. The gels were run on an electrophoresis unit (Mighty Small II Slab Gel Electrophoresis Unit, Hoefer Scientific Instruments) for approximately 5 hours (8 V/cm through the stacking gel and 15 V/cm through the resolving gel) to optimize resolution in the desired size range. The gels were blotted onto nitrocellulose (Schleicher & Schuell) with a Trans-Blot SD semi-Dry Electrophoretic Transfer Cell (Bio-Rad).^{35 36}

The blot was protected by treatment with a 5% milk solution and then probed with an antibody to D_1A for 1 hour. After 1 hour of washing, the gels were reincubated with secondary antibody (rabbit IgG, horseradish peroxidase–linked) for 1 hour. The sites of antibody binding were illuminated bychemiluminescence with the enhanced chemiluminescence system (ECL, Amersham International). A visible protein ladder was run as an indicator of protein size.

cAMP Accumulation
The culture media were aspirated and the cells washed twice with Dulbecco's phosphate-buffered saline (D-PBS). After the second wash, 400 mL D-PBS containing 1 mmol/L 3-isobutyl-1-methylxanthine was added to each well. The cells were incubated at 37°C for 30 minutes with or without drugs. Since currently available drugs do not distinguish the D₁-like receptor subtypes (eg, D_1A from D_1B receptor), the linkage of the D₁-induced cAMP accumulation to the D_1A receptor was determined with antisense phosphorothioate oligonucleotides purified by high-performance liquid chromatography (GENSET SA).³⁷ Thus, before drug treatment in some experiments, JG cells were incubated with sense (5'-ATGGCTCCTAACACTTCTACC-3') (5 µmol/L) or antisense (5'-GGTAGAAGTGTTAGGAGCCAT-3') (5 µmol/L) oligonucleotides for 6 to 12 hours. Then, the media were aspirated and the cells washed with D-PBS twice and frozen at -80°C for 30 minutes. The cells were lysed with 0.1N HCl, and cAMP was measured by radioimmunoassay as we reported previously.³⁸

Renin Activity Assay
To quantify renin secretion from JG cells, we treated the cells similarly as in the cAMP accumulation studies. After a 30-minute incubation with or without added drugs, the media were removed from the culture wells and centrifuged at 400g for 10 minutes to pellet any residual nonadherent cells. The supernatant was collected and assayed for renin activity. Cell lysates were prepared by washing the adherent cells twice in PBS and lysing them in a buffer containing 150 mmol/L NaCl, 0.5 mmol/L EDTA, 25 mmol/L HEPES, 1% Triton X, 0.5% deoxycholic acid, and 0.5 mmol/L PMSF. The cells were scraped off of the culture wells and sonicated for 15 seconds with an ultrasonic processor (Heat Systems). Total intracellular renin content was measured by assaying for renin activity with an angiotensin I generation assay and angiotensin I radioimmunoassay. Cell supernatants or lysates were incubated with an excess of rat renin substrate (plasma from 48-hour bilaterally nephrectomized rats) for 60 minutes at 37°C. The angiotensin I generation was carried out in 50 mmol/L PO₄ buffer (pH 6.2) containing 4 mmol/L EDTA and 1.4 mmol/L PMSF. The reaction was stopped by immersion of the tubes in ice, and the amount of angiotensin I generated was determined by radioimmunoassay.^{39 40} Protein concentration was measured in the cell lysates with the BCA protein assay kit (Pierce Corp).

Data Analysis
Data are expressed as mean±SE. Significant differences were determined by ANOVA for repeated measures and Scheffé's test.

	Results

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Morphological and Immunological Characterization of JG Cell Culture
Experiments were performed on JG cells after 48 hours of primary culture. At this time, the adherent layer was 50% to 60% confluent and composed of round patches of cells with prominent cytoplasmic granules. Immunoperoxidase stains of the 48-hour cultures demonstrated that greater than 85% of the adherent cells contained renin, as demonstrated by brown cytoplasmic stain (data not shown). No immunostaining was present in the control preparation in which the renin antiserum was preadsorbed with a 10-fold molar excess of purified rat renin or when normal rabbit serum was used. Immunocytochemical staining of 48-hour cultured cells was negative for other kidney cell–specific markers except for -smooth muscle actin, which was used to verify their smooth muscle origin (data not shown).

Immunocytochemistry for the D_1A Receptor
Fig 1A demonstrates that in the cells treated with D_1A receptor antisera, D_1A receptor immunoreactivity was detected in cytoplasm and on cell membranes. The intensity of the staining was highest with the 1:1000 dilution of antisera, and staining was progressively reduced as the dilution was increased to 1:4000. Substitution of preimmune sera for antisera (Fig 1B) resulted in elimination of positive staining in the cytoplasm. Minimal nonspecific staining was still observed in the nucleus. In cells exposed to preadsorbed antisera, no positive staining was observed in the cytoplasm (data not shown).

View larger version (149K): [in this window] [in a new window]   Figure 1. Light photomicrographs of cultured rat JG cells immunostained for D1A receptors. A, JG cells demonstrating positive staining with antisera against the third extracellular epitope of the D1A receptor (1:1000 dilution). B, Preimmune control sera demonstrating only light nuclear staining. Original magnification x500.

Western Blotting for D_1A and D_1B Receptors
Western blotting revealed a band of approximately 74 kD, consistent with the presence of the D_1A receptor in rat JG cells (Fig 2). LTK^- cells transfected with rat D_1A receptor cDNA were used as positive controls, and nontransfected LTK^- cells were used as negative controls. Similarly, Chinese hamster ovary (CHO) cells transfected with rat D_1B receptor cDNA demonstrated no bands in the immunoblots (data not shown). Western blots revealed the expected presence of D_1B receptors in renal proximal tubules but not in JG cells (data not shown).

View larger version (105K): [in this window] [in a new window]   Figure 2. Western blotting of D1A receptor in rat JG cells. JG cells demonstrated a band at the appropriate molecular size of 74 kD (third lane). LTK- cells transfected with the D1A receptor cDNA served as positive control (middle lane), and untransfected LTK- cells (first lane) and Chinese hamster ovary cells transfected with rat D1B receptor cDNA (not shown) served as negative controls.

RT-PCR for Dopamine Receptor Subtypes
The top panel of Fig 3 shows size analysis of the PCR products in 2% agarose gel stained with ethidium bromide. Amplification products were of the predicted size (247 bp for D_1A and 355 bp for ß-actin). The bottom panel of Fig 3 shows that the D_1B receptor mRNA is absent in rat JG cells (lane 1) but present in the medullary thick ascending limb of Henle (lane 3, 501 bp). No products were noted in the absence of reverse transcriptase, indicating that the amplified products were from cDNA and not from genomic DNA (lanes 2 and 4). The specificity of the D_1B reaction products was shown by restriction enzyme digestion with Pst I (lane 7).

View larger version (76K): [in this window] [in a new window]   Figure 3. Top, Analysis of D1A receptor PCR products in 2% agarose gel stained with ethidium bromide (n=3, one representative experiment is shown). An amplification product of the predicted size (247 bp) was evident in RT-PCR reactions using RNA extracted from rat JG cells (100 ng). There were no amplified products when PCR was performed in the absence of reverse transcriptase (D1A PCR). ß-Actin was used as a positive RT-PCR control. Bottom, Analysis of D1B receptor PCR products in 2% agarose gel stained with ethidium bromide (n=3, one representative experiment is shown). No amplification products were seen using RNA (1000 ng) from rat JG cells in the presence or absence of reverse transcriptase (lanes 1 and 2). However, an amplification product of the correct size (501 bp) was present using RNA (1000 ng) from rat medullary thick ascending limbs (lane 3 with reverse transcriptase) compared with the amplified product using D1B cDNA clone (lane 5). There were no amplification products when PCR was performed without reverse transcriptase (lane 4). The amplified PCR products from the medullary thick ascending limb corresponded with the D1B receptor since the digestion pattern with Pst I was consistent with the predicted sequence; there was a minor nonspecific band of 250 bp. X174 RF DNA/Haemophilus aegyptius III ladder is shown in lane 6.

Effect of D₁-Like Receptors on cAMP Accumulation
To assess the functional association between the D₁-like receptor and adenylyl cyclase in JG cells, we examined the effect of dopamine and a selective D₁-like receptor agonist, fenoldopam, on cAMP production. Both dopamine and fenoldopam significantly stimulated cAMP production in a concentration-dependent manner (Fig 4, top). The greater efficacy of fenoldopam compared with dopamine in stimulating cAMP accumulation may be due to the opposing effect of the D₃ receptor expressed in rat JG cells (data not shown). The stimulatory effects of dopamine and fenoldopam were completely blocked by the D₁-selective antagonist SCH23390 (1 µmol/L) (Fig 4, bottom). SCH23390 alone had no effect (data not shown). These results indicate that the D₁-like receptor in rat JG cells is coupled to stimulation of adenylyl cyclase activity.

View larger version (31K): [in this window] [in a new window]   Figure 4. Effect of dopamine and dopaminergic drugs on cAMP accumulation in cultured JG cells. Both dopamine (DA) and fenoldopam (Fen), a D1-like receptor, increased cAMP accumulation in JG cells in a concentration-dependent manner (top). The stimulatory effect of dopamine and fenoldopam was blocked by the D1-like receptor antagonist SCH23390 (SCH) (bottom). SCH23390 by itself was without effect (data not shown). Values represent mean±SE of five to nine experiments performed in duplicate. *P<.05 vs basal activity; **P<.05 vs dopamine or fenoldopam alone; ANOVA for repeated measures, Scheffé's test.

Effect of D₁-Like Receptors on Renin Secretion
Fig 5 shows that the D₁-selective agonist fenoldopam significantly stimulated renin secretion in a concentration-dependent manner, with a maximum increase of 215% at 10 µmol/L. This stimulatory effect of fenoldopam on renin secretion was completely blocked by the D₁-selective antagonist SCH23390 (1 µmol/L). SCH23390 alone had no effect (data not shown). Forskolin (1 µmol/L), which directly stimulates adenylyl cyclase activity, also stimulated renin secretion in cultured JG cells.

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of dopaminergic drugs and forskolin on renin secretion in cultured JG cells. Fenoldopam (Fen) increased angiotensin I (AGI) generation in a concentration-dependent manner. The stimulatory effect of fenoldopam on angiotensin I generation was blocked by the D1-like receptor antagonist SCH23390 (SCH). Forskolin (FSK) by itself also increased angiotensin I generation. SCH23390 by itself was without effect (data not shown). Data are mean±SE of 4 to 13 experiments performed in quadruplicate. *P<.05 vs basal activity or Fen+SCH; ANOVA for repeated measures, Scheffé's test.

Effect of D_1A Oligonucleotides on Fenoldopam-Stimulated cAMP Accumulation and Renin Release
The above studies suggest that the D₁-like receptor involved in the stimulation of cAMP production and renin release in rat JG cells is the D_1A receptor (the D_1A but not the D_1B receptor is expressed in these cells). However, other as yet uncloned D₁-like receptors have been suggested to be expressed in the kidney and other organs.⁴¹ We therefore determined the consequences of inhibition of translation of the D_1A receptor protein using an antisense phosphorothioate oligonucleotide corresponding to the ATG start codon and the subsequent 18 bases of the coding sequence of the D_1A receptor.^{37 42} We have previously demonstrated that the D_1A antisense but not sense phosphorothioate oligonucleotide used in the present study blocked translation of the D_1A receptor and prevented the ability of the D₁-selective agonist fenoldopam to stimulate phospholipase C protein expression in LTK^- cells transfected with the rat D_1A receptor cDNA.³⁷ Moreover, the antisense and sense oligonucleotides by themselves did not have any effect. In the current studies, we found that treatment of JG cells with antisense D_1A oligonucleotide (5 µmol/L) for 6 to 12 hours prevented the ability of fenoldopam to stimulate cAMP accumulation (Fig 6, top) and renin secretion (Fig 6, bottom). This effect was not due to toxicity of the phosphorothioate oligonucleotides, because the sense oligonucleotide (10 µmol/L) had no such effect. As expected, forskolin stimulated both cAMP accumulation and renin secretion.

View larger version (45K): [in this window] [in a new window]   Figure 6. Effect of D1A receptor sense and antisense phosphorothioate oligonucleotides on cAMP accumulation and renin secretion in cultured JG cells. Antisense but not sense oligonucleotide prevented the ability of fenoldopam to stimulate cAMP accumulation (top) and renin secretion (bottom). Forskolin, which directly stimulates adenylyl cyclase, increased cAMP accumulation and renin secretion. *P<.05 vs other groups; ANOVA for repeated measures, Scheffé's test. n=4 per group.

	Discussion

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Previous studies have demonstrated that dopamine stimulates renin secretion via D₁-like receptors.^{3 14 15} D₁-like receptors in rat JG cells have been demonstrated by autoradiographic techniques⁴³ ; this receptor is linked to stimulation of adenylyl cyclase activity and renin secretion.^{19 20 21 22 23 25 26 41} The findings in the current report confirm these observations. In our studies, dopamine and fenoldopam, a D₁-like receptor agonist, stimulated cAMP production and renin secretion in cultured rat JG cells. The actions of these drugs in JG cells were blocked by the D₁-like receptor antagonist SCH23390, indicating involvement of a D₁-like receptor.

A limitation of previous studies was the inability to determine the D₁-like receptor subtype in rat JG cells linked to stimulation of adenylyl cyclase and renin release. Two D₁-like receptors, D_1A and D_1B, have been reported to be expressed in mammalian kidneys.^{27 29 30 31 32 41} These receptors are expressed in low abundance, and their mRNA has been demonstrated by ribonuclease protection and RT-PCR in rat proximal tubules.^{27 30 44} We have recently reported the immunohistochemical demonstration of the D_1A receptors in renal proximal and distal convoluted tubules, cortical collecting duct, renal vasculature, and JG apparatus in the rat.³² Using the same D_1A receptor antibodies and primers for the rat D_1A cDNA, we now report the presence of the D_1A receptor in cultured rat JG cells. The D₁-like receptor in JG cells linked to stimulation of cAMP production and renin release is probably the D_1A type because neither the D_1B mRNA nor protein was found in cultured rat JG cells. More importantly, antisense D_1A oligonucleotides prevented the stimulatory effect of a D₁-like receptor agonist on cAMP accumulation and renin secretion. The complete abrogation by the D_1A antisense oligonucleotide of the stimulatory effect of the D₁-like receptor agonist fenoldopam on both cAMP accumulation and renin secretion suggests that any potential (as yet to be cloned) D₁-like receptor is unlikely to mediate the dopaminergic stimulation of renin secretion in primary cultures of JG cells. We cannot infer from these studies the involvement of D_1B receptors or any other D₁-like receptors in the regulation of renin secretion in vivo since Nash et al²⁹ have reported that D_1B receptors, which are present in opossum kidneys, are not expressed in an opossum kidney cell line. Selection of cells that do not express certain functional receptors may occur in clonal cell lines, but this is unlikely to happen in cells in primary culture.

Although the stimulatory effects of dopamine and D₁-like receptor agonists are consistently demonstrable in studies in vitro,^{3 14 15} the effect of dopamine on renin secretion in vivo is far from resolved. Several studies in conscious and anesthetized dogs have shown that intrarenal dopamine infusion did not affect PRA.^{4 5} In contrast, Imbs et al⁷ showed in anesthetized dogs that haloperidol, a D₁- and D₂-like receptor antagonist, but not propranolol, a ß-adrenergic receptor antagonist, was effective in antagonizing the dopamine-induced increase in renin release. Similarly, Mizoguchi et al⁶ reported that intrarenal arterial infusion of dopamine in conscious dogs resulted in an increase in renin secretion that was not inhibited by propranolol but was inhibited by two mixed D₁ and D₂ antagonists, sulpiride and haloperidol. A D₁-like receptor was probably involved in the stimulatory effect of dopamine because its effect was mimicked by the D₁-like receptor agonist fenoldopam.^{3 14 15} Moreover, SCH23390, a D₁-like receptor antagonist, blocked the stimulatory effect of fenoldopam.⁸

As in the studies in dogs, the effects of intravenous infusion of dopamine on renin secretion have also been inconsistent in humans. Murphy et al¹² found that intravenous infusion of fenoldopam in individuals with essential hypertension led to a small increase in PRA and suggested that this effect was probably mediated through enhanced sympathetic activity caused by baroreflex activation. Similarly, Hughes et al¹³ reported no changes in PRA in normotensive subjects during intravenous infusion of fenoldopam. In contrast, Harvey et al^{9 10} reported a marked increase in renin levels 1 hour after oral administration of fenoldopam in hypertensive individuals. Francis et al¹¹ also found a tendency toward higher PRA levels in individuals with congestive heart failure receiving fenoldopam. However, gludopa, a dopamine prodrug, suppressed PRA.^{16 45} Metoclopramide, a mixed dopamine antagonist, has also been reported to increase renin activity, apparently mediated by cyclooxygenase products.⁴⁶

The apparent contradictory effects of dopamine on renin secretion in vivo may be related to differences in experimental design. For example, the ability of dopamine to stimulate renin secretion is enhanced by a low sodium diet and blunted by a high sodium diet.⁴⁷ An additional confounding variable in the in vivo studies is the ability of dopamine in certain species to decrease PRA via central nervous system mechanisms.⁴⁸ Since a dopamine-mediated increase in renin secretion occurs via D₁-like receptors,^{3 14 15} the use of mixed dopamine antagonists may also confound the results. For example, D₂-like receptors can inhibit renin release directly¹⁶ or indirectly by decreasing norepinephrine release from nerve terminals.⁴⁸ We have preliminary evidence that rat JG cells and afferent arterioles express at least two D₂-like receptors, the D₃ and D₄ receptors.⁴⁹

The cellular mechanism or mechanisms by which dopamine induces its stimulatory effect on renin remain to be established. Renin secretion is under the control of both calcium and cAMP messenger systems acting in concert.^{2 50 51 52} We have demonstrated that stimulation of the D_1A receptor in the rat JG cell increased cAMP accumulation. Forskolin, which directly stimulates adenylyl cyclase, also increased renin secretion. However, the D_1A receptor is linked not only to adenylyl cyclase but also to phospholipase C stimulation.⁴¹ This is of particular interest because phospholipase C activation generally leads to a decrease in renin secretion.⁵² The intracellular events elicited by activation of D_1A, D₃, and D₄ receptors leading to the regulation of renin release in JG cells are currently being evaluated.

In summary, we have demonstrated the presence of D_1A but not D_1B receptors in cultured rat JG cells. Stimulation of the D_1A receptor in cultured rat JG cells increases cAMP accumulation and renin secretion. However, the role of cAMP or other second messengers in the D_1A receptor–induced increase in renin secretion remains to be determined. We suggest that dopamine produced by the renal proximal tubules should be included as one of the paracrine regulators of renin secretion in the kidney.

	Selected Abbreviations and Acronyms

 

BSA = bovine serum albumin JG = juxtaglomerular PBS = phosphate-buffered saline PRA = plasma renin activity RT-PCR = reverse transcription–polymerase chain reaction

	Acknowledgments

 
This research was supported in part by grants from the National Institutes of Health (DK-42185, DK-39308, and HL-23081) and Zambon Group (Milano, Italy). We thank the technical assistance of Hakan A. Dagli, Helen McGrath, Scott Walk, and Dr Pei-Ying Yu.

Received August 23, 1996; first decision September 26, 1996; accepted October 14, 1996.

	References

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