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(Hypertension. 1996;27:693-703.)
© 1996 American Heart Association, Inc.

Articles

Expression of the Subtype 1A Dopamine Receptor in the Rat Heart

Ryoji Ozono; Damian P. O'Connell; Carl Vaughan; Suzanne J. Botkin; Scott F. Walk; Robin A. Felder; Robert M. Carey

From the Departments of Medicine and Pathology, University of Virginia Health Sciences Center, Charlottesville (R.O., S.J.B., S.F.W., R.A.F., R.M.C.), and Department of Pharmacology and Therapeutics, University College Cork, Cork, Ireland (D.P.O., C.V.).

	Abstract

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Abstract The subtype 1A dopamine receptor (D_1A) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D_1A receptor in Sprague-Dawley and Wistar-Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D_1A receptor expressed in stably transfected LTK^- cells. D_1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription–polymerase chain reaction. D_1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D_1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D_1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D_1A bands in transfected LTK^- cells and the atrium but not in nontransfected LTK^- cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390. These results demonstrate that the D_1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.

Key Words: dopamine • SKF38393 • rat heart • RNA • in situ amplification • immunohistochemistry • adenyl cyclase

	Introduction

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Dopamine exerts its physiological functions through specific dopamine receptors. Recent advances in molecular biology have afforded the identification of at least five subtypes of dopamine receptors,^{1 2 3 4 5} which have been classified pharmacologically into D1-like (D_1A and D_1B or D₅) or D2-like (D_2L, D_2S, D₃, D₄) subtypes on the basis of their ability to stimulate or inhibit adenylyl cyclase, respectively.⁶ Dopamine is an established neurotransmitter in the central nervous system. Dopamine also has been characterized as an important modulator of peripheral organ function, as evidenced by the ability of local tissues to synthesize dopamine,⁷ the peripheral localization of dopamine receptors, and significant physiological effects of dopaminergic agents on renal,^{8 9 10} adrenal,¹¹ and cardiovascular¹² functions. Our research group has recently demonstrated that both mRNA¹³ and protein¹⁴ of D_1A receptor are expressed in the rat kidney, and intrarenal administration of a selective D1-like receptor antagonist caused a large increase in tubular sodium reabsorption.⁸ Other peripheral tissues in which dopamine receptors have been identified include the renal¹⁵ and mesenteric¹⁶ vasculatures, adrenal gland,¹⁷ and gastrointestinal tract.¹⁸

The heart, as well as the kidney, is a potentially important target of dopamine administered exogenously as a therapeutic agent in patients with compromised hemodynamic function. A recent genetic study suggested that the D_1A receptor gene may be associated with increased ventricular weight in spontaneously hypertensive rats.¹⁹ Dopamine has been detected in the heart,^{11 20} so there may be a dopaminergic system in the heart; however, it is still uncertain whether dopamine receptors are present in the heart.

The present study, using a combination of light and electron microscopic immunohistochemistry, Western blot analysis, RT-PCR, and a novel in situ mRNA amplification system, demonstrates the D_1A receptor in rat coronary vessels and myocardium, to our knowledge for the first time.

	Methods

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All experiments were conducted with the approval of the Animal Research Committee of the University of Virginia School of Medicine.

Light Microscopic Immunohistochemistry
Immunohistochemistry was performed as previously described.¹⁴ Polyclonal antibodies were raised against two synthetic peptide sequences derived from the predicted D_1A receptor. The specific sequences of the peptides were (1) CQTTAGNGNPVE, amino acids 241-252 located on the third intracellular domain; and (2) GSEETQPFC, amino acids 299-307 located on the third extracellular domain. The antibodies were IgG affinity purified by the method of Lindmark et al.²¹ Selectivity of the antibodies was validated by recognition of D_1A receptor expressed in stably transfected murine fibroblast LTK^- cells, as previously described.^{14 22} A full-length rat D_1A receptor cDNA was subcloned in the expression vector pRc/CMV (Invitrogen) at the Xba I site and transfected into the LTK^- cell line. Cell cultures grown on poly-L-lysine–coated glass well slides were fixed in 4% paraformaldehyde in PBS at room temperature. The cells were treated with the antisera diluted at 1:1000 overnight at 4°C, and the staining was visualized with an avidin-biotin immunoperoxidase reaction (Vectastain ABC Kit, Vector Laboratories).

For light microscopic immunohistochemistry, male young (<4 weeks old, 70 to 100 g) and adult (200 to 250 g) Sprague-Dawley rats were purchased from Hilltop Inc (Scottsdale, Pa). After perfusion fix with 1% paraformaldehyde, or without perfusion in other cases, the heart was excised and fixed with 1% paraformaldehyde in PBS for 1 hour. The tissue was cryoprotected overnight at 4°C in 30% sucrose in PBS, and frozen sections (12 µm) were cut. For detection of the D_1A receptor, endogenous peroxidase was blocked with 0.3% H₂O₂ in methanol for 30 minutes. The sections were washed in PBS and then blocked for 30 minutes with 3% normal goat serum and 1% nonfat dry milk in PBS and were incubated overnight at 4°C with one of the following, diluted in 1.5% normal goat serum and 0.5% nonfat dry milk in PBS: (1) D_1A receptor primary antiserum, (2) preimmune serum, or (3) D_1A receptor primary antiserum preabsorbed against its antigen. For preadsorption, antiserum was incubated overnight at 4°C with a 10-fold molar excess of peptide. After washes in PBS, immunostaining was detected with an avidin-biotin immunoperoxidase reaction (Vectastain ABC Kit) and visualized by diaminobenzidine (Fast DAB tablets, Sigma Chemical Co). Tissue sections were lightly stained with hematoxylin, dehydrated, and placed under coverslips.

Electron Microscopic Immunocytochemistry
For electron microscopic immunocytochemistry, male Sprague-Dawley rats weighing 200 to 250 g were used. Rats were anesthetized with pentobarbital sodium (50 mg/L) and perfused with 4% paraformaldehyde fixative in 0.05 mol/L sodium cacodylate buffer (pH 7.3). The left ventricle was removed, minced into 1-mm³ pieces, and postfixed for an additional hour. Tissue was processed and embedded into Lowicryl K4M resin (Polysciences Inc), as previously described.¹⁴

Immunolabeling was performed on ultrathin sections mounted on nickel grids, as previously described.¹⁴ For the primary label, the sections were incubated overnight at 4°C on either primary antibody (D_1A), diluted 1:100, or Tris buffer. Protein A gold, diluted 1:10, was used for the secondary label. After counterstaining with uranyl acetate and lead acetate, sections were examined in a transmission electron microscope (10 CA, Carl Zeiss) at 60 kV.

RT-PCR
RT-PCR was performed as previously described.¹³ RNA was extracted from rat ventricles with TRI-REAGENT LS (Molecular Research Center Inc) according to the method of Chomczynski and Sacchi.²³ The protocol includes homogenization of adult male Sprague-Dawley rat ventricles (100 mg) in 1 mL TRI-REAGENT, phase separation by addition of 0.2 mL chloroform, RNA precipitation (-80°C overnight) from the aqueous phase by mixing with 0.5 mL isopropanol, RNA wash in 75% ethanol, and resuspension in diethyl pyrocarbonate–treated water. RT-PCR was performed with a Gene Amp RNA PCR kit (Perkin-Elmer Corp) as previously described.¹³ The RT reaction was performed in a reaction mixture (20 µL total) containing 2.5 IU/µL murine leukemia virus reverse transcriptase, 1 µL RNA sample, 2.5 µmol/L oligo(dT), 1 mmol/L of each dNTP, 5 mmol/L Tris-HCl (pH 8.3), 25 mmol/L KCl, 5 mmol/L MgCl₂, and 1 IU/µL RNase inhibitor. The tubes were incubated in a thermal cycler (Twin Block System, Ericomp Inc) at 42°C for 15 minutes, 99°C for 5 minutes, and 5°C for 5 minutes sequentially. Subsequent PCR was carried out (100 µL total volume) in the presence of 0.25 µmol/L sense primer (identical to oligonucleotides 783-803, 5'-TGCCCAGAAGCAAATCCGGCG-3'), 0.25 µmol/L antisense primer (complementary to oligonucleotides 1010-1029, 5'-CTCCTCAGAGCCACAGAAGG-3'), 0.025 IU/µL Taq DNA polymerase, 0.20 mmol/L of each dNTP, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, and 2 mmol/L MgCl_2. The reaction mixture was incubated at 94°C for 3 minutes (initial melt), followed by 30 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 3 minutes. PCR was completed by a final extension at 72°C for 3 minutes. PCR products were size-fractionated in 2% agarose gels stained with ethidium bromide. Controls included omission of the RT step, amplification of oligonucleotides specific to ß-actin mRNA by RT-PCR, and amplification of control RNA (pAW109 RNA) with interleukin-1 primers as provided by the manufacturer.

In Situ mRNA Amplification
The procedure of in situ amplification involves continuous cycling of RT of D_1A receptor gene mRNA, conversion of the transcripts to cDNA containing promoter sequences for bacteriophage T7 RNA polymerase, and RNA synthesis by the RNA polymerase,²⁴ which was performed with a 3SR system kit (Bartels Diagnostic Division, Baxter Healthcare). We amplified a sense product, oligonucleotides 270 bp in length located on the 5' flanking region of the mRNA encoding the N-terminus of the D_1A receptor, using the sense primer 5'-GTAGATTTATTTGTCTGG-3', spanning nucleotides 49-66, and the antisense primer, 5'-CTAAAGAGATGACAAAGA-3', spanning nucleotides 302-319. The sense primer contained a promoter sequence for T7 RNA polymerase (5'-AATTTAATACGACTCACTATAGGGA-3') at the 5' end.

Male Wistar-Kyoto rats (Harlan Sprague Dawley, Inc, Indianapolis, Ind) were sequentially perfused with 0.9% saline, 1% sucrose in 0.12 mol/L sodium phosphate, and 4% paraformaldehyde in 0.12 mol/L phosphate. The heart was removed and postfixed in the 4% paraformaldehyde solution for 1 hour at 4°C and cryoprotected overnight at 4°C in 30% sucrose and 0.12 mol/L phosphate. Frozen sections were cut at between 7 and 12 µm in a cryostat at -26°C, mounted on poly-L-lysine–coated slides, and stored at -70°C. Tissues were incubated with antisense primer at 1 ng/25 µL (7 nmol/L) in 50% formamide 4x SSC solution overnight at room temperature. Subsequently, tissue was washed twice, 15 minutes each at room temperature, in 2x SSC, followed by two other washes in 0.5x SSC, 30 minutes each at 40°C. Then, transcriptional amplification was initiated by addition of 60 IU of avian myeloblastosis virus reverse transcriptase, 200 IU of T7 RNA polymerase, 6 IU of RNAse H, sense and antisense primers both at 20 and 50 nmol/L, respectively, dNTP mix at 5 mmol/L, and nucleotide 5'-triphosphates (rNTP) mix at 1.5 mmol/L in reaction buffer (40 mmol/L Tris-HCl [pH 8.1], 30 mmol/L MgCl₂, 20 mmol/L KCl, 10 mmol/L dithiothreitol, and 4 mmol/L spermidine). The reaction lasted 2 hours at 42°C, and then sections were washed with two changes in 2x SSC for 15 minutes at room temperature. Subsequent in situ hybridization was conducted with 24-mer high-performance liquid chromatography–purified antisense oligonucleotide probes directed to the nucleotides 163-187 (5'-AAAGGAGAAATCCCTCTCCGCTGG-3') lying within the amplified 270-bp product of a D_1A mRNA. The probe was labeled by tailing the 3' end with digoxigenin-11-dUTP with terminal transferase from a commercially available kit (Genius 5 Oligonucleotide 3'-End Labeling Kit, Boehringer Mannheim). Amplified and nonamplified tissue sections were prehybridized by incubation at room temperature for 1 hour with the hybridization solution (50% deionized formamide, 4x SSC, 1x Denhardt's bovine serum albumin, 0.5 mg/mL denatured sheared herring sperm DNA, 0.25 mg/mL yeast transfer RNA, and 10% dextran sulfate). The slides were briefly submerged in 2x SSC and then incubated overnight at 37°C with digoxigenin-labeled sense or antisense probes diluted in the hybridization solution. After hybridization, the sections were washed in decreasing concentrations of SSC as follows: 2x SSC for 1 hour at room temperature, 1x SSC for 1 hour at room temperature, 0.5x SSC for 0.5 hour at 37°C, and 0.5x SSC for 0.5 hour at room temperature. Immunoreactive hybridized digoxigenin-labeled probe was detected with an alkaline phosphatase–NBT/BCIP technique with the Genius Nonradioactive Nucleic Acid Detection Kit (Boehringer Mannheim). After the posthybridization washes, the sections were immersed first for 5 minutes in buffer A (0.1 mol/L Tris [pH 7.5], 1 mol/L NaCl, and 2 mmol/L MgCl₂) containing 2% normal goat serum and 0.1% Triton X-100 and second in buffer A containing alkaline phosphatase–labeled anti-digoxigenin F(ab) fragment (1:5000) and were incubated overnight at 4°C. The sections then were washed in buffer A (three times for 10 minutes), in buffer B (0.1 mol/L Tris [pH 9.5], 1 mol/L NaCl, and 2 mmol/L MgCl₂) (5 minutes), and in buffer C (0.1 mol/L Tris [pH 9.5], 0.1 mol/L NaCl, and 5 mmol/L MgCl₂) (5 minutes). Tissue-bound alkaline phosphatase activity was detected by incubation of the tissue sections with 92 mmol/L NBT salt in 70% (vol/vol) dimethylformamide and 115 mmol/L BCIP (X-phosphast) toluidine salt in 100% dimethylformamide diluted in buffer C in the absence of light for 2 to 24 hours. The enzymatic reaction was stopped by rinsing the section in PBS. Finally, sections were stained with neutral red, delipidated with xylene, and cover-slipped in DPX (Electron Microscopy Science) mounting media.

Western Blot Analysis of D_1A Receptor Protein Expression
The atria and ventricles of adult Sprague-Dawley rats were dissected, minced, and homogenized with Tissuemizer (Tekmar Corp) in buffer A (10% glycerol, 20 mmol/L Tris-HCl [pH 7.3], 100 mmol/L NaCl, 2 mmol/L phenylmethylsulfonyl fluoride, 2 mmol/L EDTA, 2 mmol/L EGTA, 10 mmol/L sodium orthovanadate, 10 µg/mL [approximately 20 µmol/L] leupeptin, and 10 µg/mL [approximately 1.5 µmol/L] aprotinin). The homogenate was centrifuged at 30 000g for 30 minutes at 4°C. The pellet was resuspended in buffer B (buffer A with 1% NP-40 [Sigma]), stirred for 1 hour at 4°C, and centrifuged again at 30 000g for 30 minutes at 4°C. The supernatant was used for the analysis. For control samples, D_1A receptor–transfected or nontransfected LTK^- cells were processed in the same way. The sample was analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (5% acrylamide stacking gel and 8% running gel) in a standard protocol as described.²⁵ The resolved proteins were transferred by electroblotting (15 V for 20 minutes, Trans Blot SD DNA, Bio-Rad) onto a nitrocellulose sheet (BA-S 83, Schleicher & Schull). The nitrocellulose sheet was then soaked in 5% nonfat dry milk in Tween 20 solution (0.05% Tween 20, 10 mmol/L Tris-HCl [pH 7.2], 250 mmol/L NaCl) for 1 hour, incubated with the antisera against the third extracellular epitope of the rat D_1A receptor (1:5000 dilution in Tween 20 solution) for 1 hour, and reacted with a peroxidase-labeled secondary antibody (1:10 000 dilution) for 1 hour. Specific bands were visualized with chemiluminescence (ECL Western Blotting Detecting Kit, Amersham).

Assay for Adenylyl Cyclase Activity
cAMP was generated and measured by radioimmunoassay as previously described.^{26 27} The rat heart was surgically removed and placed in Tris buffer (10 mmol/L Tris-HCl [pH 7.4] at 4°C, 20% glycerol, 100 µmol/L ascorbic acid, 10 µmol/L vitamin E [Trorox, Aldrich], 10 µg/mL [approximately 20 µmol/L] leupeptin, 10 µg/mL [approximately 0.5 µmol/L] trypsin inhibitor). Ventricles were dissected, minced, and homogenized in 10 vol ice-cold Tris buffer with a Tissuemizer (3x10 seconds at medium setting). The homogenate was centrifuged at 30 000g for 15 minutes at 4°C. The plasma membrane pellet was then gently swirled off of the dark brown lower pellet and resuspended in Tris buffer at 1.0 to 2.0 mg/mL protein concentration. In preliminary experiments, we confirmed that the adenylyl cyclase activity and responses to agonist were not different among the crude homogenate, the membrane described above, and a membrane prepared by alternative methods in which the homogenate was centrifuged at low speed (2000g and/or 10 000g) for 15 minutes, the supernatant was centrifuged at 30 000g, and the pellet was obtained as described above. cAMP was generated by incubation of 20 µL membrane suspension or crude homogenate (1.5 to 2.5 mg/mL protein) for 30 to 45 minutes at 37°C in a reaction mixture (100 µL total) with or without drugs to be studied. Dopamine (Sigma) at 10 µmol/L and a D1-like agonist, SKF38393 (RBI), at concentrations of 0.1, 1.0, and 100 µmol/L were used. Dopamine was dissolved in distilled water containing 100 µmol/L ascorbic acid. The selective D1-like receptor antagonist SCH23390 (RBI) at 10 µmol/L was used to block agonist-stimulated activity. To reduce cAMP generation by binding of endogenous catecholamines to ß-adrenergic receptor, 0.1 µmol/L propranolol was added to all samples. The reaction mixture contained 50 mmol/L Tris-HCl, 10 mmol/L MgCl₂, 0.4 mmol/L EGTA, 0.1 mmol/L GTP, 0.25 mmol/L ATP, 1 mmol/L 1-methyl-3-isobutylxanthine, 20 mmol/L phosphocreatinine, 100 IU/mL creatinine phosphokinase, and 0.06% bovine serum albumin (final concentrations). The reaction was terminated by placing samples on ice and adding 10 µL of 0.5 mol/L EDTA and then 400 µL of ice-cold sodium acetate buffer (50 mmol/L, pH 4.75). The cAMP concentration in the sample was determined by radioimmunoassay with ¹²⁵I-cAMP (10 000 cpm/50 µL) as previously described.^{26 27} Each 500-µL sample was acetylated with 6 µL triethylamine and 4 µL acetic anhydride before analysis. Protein concentrations were determined by the method of Lowry et al.²⁸

Statistical Analysis
Results are expressed as mean±SE. Comparison between the cAMP responses to drugs was made with two-tailed paired Student's t test. A value of P<.05 was accepted as statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Light Microscopic Immunohistochemistry
The D_1A receptor–transfected LTK^- cells were positively stained with both antibodies against the third extracellular and intracellular domains, whereas the following controls were all negative: (1) substitution of preimmune serum, (2) omission of primary antiserum, and (3) incubation of antiserum with nontransfected LTK^- cells (Fig 1).

View larger version (0K): [in this window] [in a new window]   Figure 1. Light photomicrograph of D1A-transfected LTK- cells. a, Cells demonstrating positive staining with antisera against the third extracellular epitope of D1A receptor (1:1000 dilution); b, preimmune control. Magnification x1750.

Light microscopic immunohistochemical staining for the D_1A receptor was obtained in all of the rat hearts studied. The staining was localized to the myocardium and homogeneously distributed throughout the atria and ventricles (Fig 2). A greater overall density was observed in the atrium (Fig 2). In the young rats, blood vessels, regardless of the size, were more intensely stained compared with the myocardium (Fig 2e and 2f), whereas vascular staining was less prominent in adult rats (Fig 3). Both antisera against the third extracellular and intracellular epitopes of the native receptor showed a similar staining pattern (Fig 3). Consecutive sections processed with either the antibody preabsorbed against its peptide antigen (Fig 2) or preimmune serum (Fig 3) at the same dilution as the anti-D_1A receptor serum did not produce significant staining.

View larger version (0K): [in this window] [in a new window]   Figure 2. Light photomicrograph of frozen section of young (1-month-old) rat heart demonstrating positive immunostaining for D1A receptor. Panels a, c, e, Sections treated with antiserum (1:1500 dilution) directed to the third extracellular epitope; Panels b, d, f, sections treated with preabsorbed antisera against the peptide antigen. a and b, Consecutive sections of atrium (magnification x875); c and d, high-power magnification of left ventricular myocardium (magnification x1750); e and f, coronary artery (magnification x875).

View larger version (0K): [in this window] [in a new window]   Figure 3. Light micrograph of frozen section of adult (2-month-old) rat heart demonstrating positive immunostaining for D1A receptor. a and c, Left ventricular myocardium and intracardiac vessels treated with antisera (1:1000 dilution) directed to the third extracellular (a) and third intracellular (c) epitopes; b and d, consecutive sections treated with preimmune sera. Magnification x875.

Electron Microscopic Immunocytochemistry
Figs 4 through 6 are electron micrographs of rat ventricle immunostained with D_1A extracellular antiserum (diluted 1:100). Electron microscopic immunocytochemistry further defined the intracellular sites of cardiac D_1A receptor immunoreactivity. Both vasculature and myocardium showed plasma membrane–bound staining (Figs 4 and 6). Figs 4a and 5a show the protein A gold staining pattern in cardiac muscle cells with D_1A antiserum along the M-line and on the thick filament of the striated tissue. Fig 6a demonstrates the vascular staining on both vascular smooth muscle cells and endothelial cells. The D_1A labeling is located on the membrane within small vesicles and also intracellularly. Control sections (Figs 4b, 5b, and 6b), incubated with the absence of primary antibody, showed no specific staining.

View larger version (116K): [in this window] [in a new window]   Figure 4. Electron micrographs of ventricular cardiomyocyte and neighboring capillary. a, D1A plasma membrane staining of myocyte (arrows) and M-line (open arrow). The neighboring capillary (C) is stained on the basal membrane of the endothelial cell (arrowheads). b, Control section shows no staining on the membrane of the myocyte or on the M-lines (M, within brackets) or thick filaments. L indicates capillary lumen. Magnifications x62 500 (a) and x78 750 (b).

View larger version (116K): [in this window] [in a new window]   Figure 5. Electron micrographs of cardiac striated muscle. a, D1A staining of M-lines (M, arrows) and thick filaments of striated muscle (arrowheads); b, control section shows no staining of myosin containing tissue. Magnifications x57 500 (a) and x72 450.

View larger version (123K): [in this window] [in a new window]   Figure 6. Electron micrographs of a cardiac vessel within the ventricle. a, D1A staining in vascular smooth muscle (VSM) cell in vesicles along plasma membrane (arrows) and in cytoplasm. The endothelial cell (E) also has staining in vesicles along the basal membrane (arrowhead) and within the cytoplasm. b, Control section for a. Magnification x72 450.

RT-PCR
Fig 7 shows the size analysis of the PCR products in 2% agarose gel stained with ethidium bromide. An amplification product of the predicted size (247 bp) for the D_1A receptor was evident in the RT-PCR reactions using extracted rat ventricular RNA, but no band was observed when PCR was performed without RT. Bands for ß-actin and control RNA (pAW109) served as positive controls for the RNA preparation and RT-PCR system, respectively.

View larger version (105K): [in this window] [in a new window]   Figure 7. Analysis of PCR products in 2% agarose gel stained with ethidium bromide. Amplification product of the predicted size (247 bp) for the D1A receptor is observed in the RT-PCR reaction using extracted rat ventricular RNA but not when PCR is performed in the absence of reverse transcriptase (-). There are also products for ß-actin (ß-A) in the RT-PCR reaction using rat ventricular RNA.

In Situ Amplification of D_1A Receptor mRNA
A positive hybridization signal to the antisense oligonucleotide probes for D_1A mRNA was observed in the rat heart sections processed for in situ amplification (Fig 8). The signals were localized to both the atria and ventricles and in coronary and intracardiac vessels, regardless of their size. Consecutive heart sections probed with sense oligonucleotide, absence of anti-digoxigenin, or nonamplified sections probed with antisense oligonucleotide failed to show any hybridization signal. Background labeling was minimal in all sections examined.

View larger version (0K): [in this window] [in a new window]   Figure 8. Light photomicrographs of rat left ventricular tissue processed for in situ amplification (see "Methods") and probed with digoxigenin-dUTP–labeled antisense or sense oligonucleotides. A positive hybridization signal was visualized with an alkaline phosphatase–conjugated anti-digoxigenin antibody and alkaline phosphatase-NBT/BCIP reaction yielding a purple substrate reaction. a and c, Sections probed with antisense oligonucleotide. Positive hybridization signals were noted in both the blood vessel wall (a) and myocardium (c). b and d, Control sections of a and c, respectively, probed with sense oligonucleotide. Magnifications x1600 (a, b, c) and x1000 (d).

Western Blot Analysis
Fig 9 shows Western blot analysis of D_1A receptor in the D_1A-transfected or nontransfected murine LTK^- cell line and in rat atria and ventricles. Bands of the predicted size (approximately 70 kD) for D_1A receptor were detected in D_1A-transfected murine LTK^- cells but not in nontransfected cells. In the atria, a specific band was observed, but not in the ventricles. The molecular weight of the receptor is predicted as approximately 70 kD because of glycosylation and phosphorylation. The molecular weight of the D_1A receptor itself is approximately 50 kD.²⁶

View larger version (21K): [in this window] [in a new window]   Figure 9. Western blot analysis of D1A receptor in D1A-transfected and nontransfected (LTK) murine LTK- cell lines and rat atria (A) and ventricles (V). Bands of the predicted size (approximately 70 kD) for D1A receptor were detected in D1A and A but not in LTK and V.

Adenylyl Cyclase Activity
The time course of cAMP generation was studied with the use of adult rat ventricular crude homogenate. cAMP generation was linear until 30 to 45 minutes (Fig 10). In addition, adenylyl cyclase activity at 30 minutes was significantly increased in the presence of 10 µmol/L dopamine compared with the control (n=10, P<.05) (Fig 10). In the plasma membrane of adult rat hearts, basal adenylyl cyclase activity was 77.7±6.2 pmol/mg protein per minute (n=12). SKF38393 stimulated cAMP generation in a dose-related fashion (Fig 11). At the highest dose of SKF38393 (100 µmol/L), the cAMP level was increased by 13.9±5.3% (P<.05 versus baseline, n=12) compared with the basal value, whereas forskolin (Sigma) at 1 µmol/L stimulated adenylyl cyclase by 128.4±13.4% (P<.01 versus baseline, n=7). This effect of SKF38393 was not observed when 10 µmol/L SCH23390 was simultaneously present with the SKF38393. The difference between cAMP levels at 100 µmol/L SKF38393 with and without SCH23390 was statistically significant (P<.05).

View larger version (29K): [in this window] [in a new window]   Figure 10. Time course of cAMP generation. Crude homogenates of rat ventricles were incubated in the absence (control) or presence of 10 µmol/L dopamine (DOP). Data are mean±SEM (n=10). *P<.05, DOP vs control at 30 minutes.

View larger version (19K): [in this window] [in a new window]   Figure 11. Effect of D1-like receptor agonist SKF38393 (SKF) and antagonist SCH23390 (SCH) on cAMP generation in rat heart plasma membranes. Plasma membranes were incubated with or without drugs for 45 minutes. Percent increase over baseline was plotted. Baseline cAMP value in the absence of SKF and SCH was 77.7±6.2 pmol/mg protein per minute. Data are mean±SEM (n=12). *P<.05 vs baseline; P<.05 vs SKF at 100 µmol/L.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we demonstrate that the gene and protein of the D_1A receptor are expressed in the coronary arteries, atria, and ventricles of rat heart. We also demonstrate pharmacological evidence of a D1-like receptor in the heart by showing the ability of a D1-selective agonist to stimulate adenylyl cyclase in a dose-dependent fashion and blockade of this response with a specific D1-like receptor antagonist.

Our immunohistochemical findings provide strong evidence for the presence of D_1A receptor protein in the heart. Our antibodies were directed toward both intracellular and extracellular epitopes that are unique to the D_1A receptor subtype, and positive immunohistochemical signals were obtained with both of these antibodies. Our antigen peptides have no homology to known amino acid sequences, including other dopamine receptor subtypes. Controls included substitution of preimmune serum as well as the preadsorption of the antibody with the peptide antigen. Selectivity of the antisera was established with an LTK^- cell line stably expressing the D_1A receptor as previously described.¹⁴

In addition, our electron microscopic study confirmed a positive immunocytochemical signal along plasma membranes of myocardium and vascular smooth muscle cells, where a functioning membrane-bound receptor should be located. It is of interest that we also detected receptors in cytosol, cytosolic vesicles attached to the membrane, and myosin filaments as well as in the M-line. These vesicles are consistent with previously described plasma membrane invaginations termed caveolae.²⁹ The M-line is the locus of specific proteins that link adjacent myosins to each other. A substantial body of recent evidence supports a hypothesis that G protein–coupled receptors are internalized by "potocytosis" and transported toward a specific intracellular site of action (see References 29 and 30 for review). Our immunocytochemical observations appear to reflect such an intracellular mechanism of receptor–G protein signaling. The localization of the D_1A receptor on the M-line may suggest the regulatory role of this receptor in myocardial contraction. Cytoskeletal proteins including actin and myosin are postulated to be important for potocytosis³⁰ and a dynamic interplay between G proteins and receptors.³¹ In fact, we have detected an amino acid sequence of ß-actin in a purification product of immunoprecipitated D_1A receptor protein (unpublished data, 1995) despite the fact that the antibody does not cross-react immunologically with ß-actin. However, these ideas are speculative, and further study will be necessary to confirm the specificity and significance of D_1A receptor localization in the M-line and myosin thick filaments and to clarify the intracellular signal transduction pathway or pathways involving D_1A receptor and associated G proteins.

Significant advances in technology have allowed amplification of specific sequences of nucleic acids by a transcription reaction.^{24 32} The in situ amplification technique used in the present study was a modification of the method described by Guatelli et al.²⁴ These authors described the self-sustained sequence replication system (3SR) involving the collective activities of avian myeloblastosis virus reverse transcriptase, Escherichia coli RNase H, and T7 RNA polymerase for isolated RNA. We carried out this reaction on tissue sections. The major advantage of the 3SR reaction is the use of RNase H, which eliminates the need for any thermal denaturation steps. The 3SR reaction is more appropriate for whole tissue sections than PCR or other nucleotide amplification systems³² because it avoids nonspecific amplification of heat-denatured genomic DNA as well as heat-related damage to the tissue. Furthermore, compared with in situ PCR, the enzyme reactions have rapid kinetics and the inherent ability to distinguish between RNA and DNA targets. As a result, our novel in situ technique allows for improved sensitivity and selectivity in detecting a low copy signal with delineation of the specific cell types compared with other amplification techniques.

Previously, several attempts have been made to detect cardiac dopamine receptors. Sandrini et al³³ have shown specific binding of [³H]dopamine in the guinea pig heart, but the ligand is not specific enough to differentiate the subtypes of dopamine or other receptors (eg, ß-adrenergic receptors). Amenta et al³⁴ failed to detect D1-specific binding sites ([³H]SCH23390 binding) in the human heart. On the other hand, these authors and Ganguly et al³⁵ have reported the presence of D2 binding sites ([³H]spiperone binding) in the human and rat heart, respectively. In physiological studies, several authors have reported D1-like receptor–specific coronary vasodilation³⁶ and negative³⁷ and positive^{38 39} inotropism after a specific D1 agonist. However, Van Woerkens et al¹² have reported that intracoronary infusion of fenoldopam, a specific D1-like receptor agonist, provided no evidence to support a major role of D1-like receptor in myocardium and pig coronary artery. Using newly obtained molecular information, O'Malley et al⁴⁰ reported that the D₄ receptor gene was expressed abundantly in the rat heart. Other than this latter study, dopamine receptor subtypes have not been characterized by means of molecular biological techniques.

The fact that mRNA of the D_1A receptor was detectable only after amplification indicates that the expression of the receptor subtype in the heart is in low abundance. Consistently, we failed to detect sufficient specific binding of D1-specific radioligand (¹²⁵I-SCH23982) to plasma membranes of the rat heart to analyze the binding kinetics (data not shown). Northern blot analysis also failed to detect D_1A receptor gene expression.² Our Western blot analysis detected D_1A receptor protein only in the atria and not in the ventricles. This observation, consistent with our immunohistochemical findings, suggests that the receptor is relatively abundant in the atria compared with the ventricles. However, such a low-copy receptor could play a significant physiological role, as is the case with D_1A receptors in the kidney,¹³ particularly when the receptors function by means of a local cell-to-cell communication (paracrine) system.

It is noteworthy, however, that the activation of adenylyl cyclase through D1-like receptor stimulation was only by 14% compared with the baseline value. Such a small response may be explained by low abundance of the receptor or by the possibility that this receptor is coupled to another cell signaling system, such as the phospholipase C pathway.⁴¹

A major question that needs to be addressed is the physiological role of the cardiac D_1A receptor. Our data indicate that the myocardial D1-like receptor stimulates adenylyl cyclase, which should produce a positive inotropism. It is likely that the D_1A receptor in the coronary artery contributes to the inotropism by dilation and increased coronary blood flow. However, the observed change in cAMP, which mainly reflects the myocardial adenylyl cyclase, was relatively small, and previous physiological studies do not support the hypothesis that cardiac D_1A receptor is involved as a major inotropic mechanism in the normal physiological state.^{12 39}

Evidence is beginning to accumulate that the D_1A receptor is important in left ventricular hypertrophy. Chromosomal mapping studies in spontaneously hypertensive rats have indicated that left ventricular weight, and not blood pressure, was most tightly correlated with a marker for the D_1A receptor locus.¹⁹ Our preliminary data (not shown) have suggested that the density of D_1A receptors in the rat heart is increased with the development of left ventricular hypertrophy due to aortic banding. In the same rat model, left ventricular hypertrophy was enhanced and attenuated by fenoldopam and SCH23390, respectively.⁴² In this regard, it is of interest that Hackman et al³⁸ have reported a large increase in cardiac function after fenoldopam infusion in patients with severe arterial hypertension. These observations suggest that the D_1A receptor may play a role in compensatory hypertrophic responses to increased ventricular afterload.

In summary, we have identified the D_1A receptor gene and protein in the rat heart. The physiological and pathophysiological roles and the predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.

	Selected Abbreviations and Acronyms

 

BCIP = 5-bromo-4-chloro-3-indolyl phosphate D1A = dopamine subtype 1A NBT = nitro blue tetrazolium PBS = phosphate-buffered saline PCR = polymerase chain reaction RT = reverse transcription SSC = standard sodium citrate

	Acknowledgments

 
This project was supported in part by National Institutes of Health grant RO1-HL-49575 to Dr Carey.

	Footnotes

 
Reprint requests to Robert M. Carey, MD, Box 395, University of Virginia Health Sciences Center, Charlottesville, VA 22908.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Bunzow JR, Van Tol HHM, Grandy DK, Albert P, Salon J, Christe M, Machida CA, Neve KA, Civelli O. Cloning and expression of a rat D2 dopamine receptor cDNA. Nature. 1988;336:783-787. [Medline] [Order article via Infotrieve]

2. Zhou QY, Grandy DK, Thambi L, Kushner JA, Van Tol HHM, Cone R, Pribnow D, Salon J, Bunzow JR, Civelli O. Cloning and expression of human and rat D1 dopamine receptors. Nature. 1990;347:76-80. [Medline] [Order article via Infotrieve]

3. Socoloff P, Giros B, Martres MP, Bouthenet ML, Schwaltz JC. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature. 1990;347:146-150. [Medline] [Order article via Infotrieve]

4. Weinshank R, Adham N, Macchi M, Olsen MA, Branchek TA, Hartig PR. Molecular cloning and characterization of a high affinity dopamine receptor (D1ß) and its pseudogene. J Biol Chem. 1991;266:22427-22435. [Abstract/Free Full Text]

5. Van Tol HHM, Bunzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB, Civelli O. Cloning for gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature. 1991;350:610-614. [Medline] [Order article via Infotrieve]

6. Goldberg LI, Kohli JD. Peripheral pre- and postsynaptic dopamine receptors: are they different from dopamine receptors in the central nervous system? Commun Psychopharmacol. 1979;3:447-456. [Medline] [Order article via Infotrieve]

7. Aperia A, Bertorello A, Seri I. Dopamine causes inhibition of Na-K-ATPase activity in proximal tubular segments. Am J Physiol. 1987;252:F32-F45. [Abstract/Free Full Text]

8. Siragy HM, Felder RA, Howell NL, Chevalier RL, Peach MJ, Carey RM. Evidence that intrarenal dopamine acts as a paracrine substance at the renal tubule. Am J Physiol. 1989;257:F469-F477. [Abstract/Free Full Text]

9. Siragy HM, Felder RA, Howell NL, Chevalier RL, Peach MJ, Carey RM. Evidence that dopamine-2 mechanisms control renal function. Am J Physiol. 1990;259:F793-F800. [Abstract/Free Full Text]

10. Siragy HM, Felder RA, Peach MJ, Carey RM. Intrarenal DA₂ dopamine receptor stimulation in the conscious dog. Am J Physiol. 1992;262:F932-F938. [Abstract/Free Full Text]

11. Kujacic M, Hansson LO, Carlsson A. Acute dopaminergic influence on plasma adrenaline levels in the rat. Eur J Pharmacol. 1995;273:247-257. [Medline] [Order article via Infotrieve]

12. Van Woerkens LJ, Duncker DJ, Den Boer MO, McFalls EO, Sassen LMA, Saxena PR, Verdouw PD. Evidence against a role for dopamine D1 receptors in the myocardium of the pig. Br J Pharmacol. 1991;104:246-250. [Medline] [Order article via Infotrieve]

13. Yamaguchi I, Jose PA, Mouradian M, Canessa LM, Monsma FJ, Sibley DR, Takeyasu K, Felder RA. Expression of dopamine D_1A receptor gene in proximal tubule of rat kidneys. Am J Physiol. 1993;264:F280-F285. [Abstract/Free Full Text]

14. O'Connell DP, Botkin SJ, Ramos SI, Sibley DR, Ariano MA, Felder RA, Carey RM. Localization of dopamine D_1A receptor protein in rat kidneys. Am J Physiol. 1995;268:F1185-F1197. [Abstract/Free Full Text]

15. Felder RA, Blecher M, Eisner GM, Jose PA. Cortical tubular and glomerular dopamine receptors in the rat kidney. Am J Physiol. 1984;246:F557-F568.

16. Missale C, Castelletti L, Memo M, Carruba MO, Spano PF. Identification and characterization of postsynaptic D₁- and D₂-dopamine receptors in the cardiovascular system. J Cardiovasc Pharmacol. 1988;11:643-650. [Medline] [Order article via Infotrieve]

17. Pupilli C, Lanzillotti R, Fiorelli G, Selli C, Gomez RA, Carey RM, Serio M, Mannelli M. Dopamine D2 receptor gene expression and binding sites in adrenal medulla and pheochromocytoma. J Clin Endocrinol Metab. 1994;79:56-61. [Abstract]

18. Marmon LM, Albrecht F, Canessa LM, Hoy GR, Jose PA. Identification of dopamine D_1A receptors in the rat small intestine. J Surg Res. 1993;54:616-620. [Medline] [Order article via Infotrieve]

19. Pravenec M, Gauguier D, Schott JJ, Buard G, Kren V, Bila V, Szpirer C, Szpirer J, Wang JM, Huang H, St. Lezin M, Spence MA, Flodman P, Prinz M, Lathrop GM, Vergnaud G, Kurtz TW. Mapping of quantitative trait loci for blood pressure and cardiac mass in the rat by genome scanning of recombinant inbred strains. J Clin Invest. 1995;96:1973-1978.

20. Elayan H, Kennedy B, Ziegler MG. Propranolol reduces rat dopamine-ß-hydroxylase activity and catecholamine levels. Eur J Pharmacol. 1992;212:259-262. [Medline] [Order article via Infotrieve]

21. Lindmark R, Thoren-Tolling K, Sjoquist J. Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera. J Immunol Methods. 1983;62:1-13. [Medline] [Order article via Infotrieve]

22. Horiuchi A, Takeyasu K, Mouradian MM, Jose PA, Felder RA. D_1A dopamine receptor stimulation inhibits Na⁺/K⁺-ATPase activity through protein kinase A. Mol Pharmacol. 1993;43:281-285.[Abstract]

23. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

24. Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richman DD, Gingeras TR. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc Natl Acad Sci U S A. 1990;87:1874-1878. [Abstract/Free Full Text]

25. Shambrock J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989:18.47-18.75.

26. Kinoshita S, Sidhu A, Felder RA. Defective dopamine-1 receptor adenylyl cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat. J Clin Invest. 1989;84:1849-1856.

27. Ohbu K, Hendley ED, Yamaguchi I, Felder RA. Renal dopamine-1 receptors in hypertensive inbred rat strains with and without hyperactivity. Hypertension. 1993;21:485-490. [Abstract/Free Full Text]

28. Lowry OH, Rosebrough NS, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]

29. Chang W-J, Ying Y-S, Rothberg KG, Hooper NM, Turner AJ, Gambliel HA, DeGunzburg JD, Mumby SM, Gilman AG, Anderson RGW. Purification and characterization of smooth muscle cell caveolae. J Cell Biol. 1994;126:127-138. [Abstract/Free Full Text]

30. Lisanti MP, Scherer PE, Tang ZL, Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Biol. 1994;4:231-235. [Medline] [Order article via Infotrieve]

31. Jahageer S, Rodbell M. The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated. Proc Natl Acad Sci U S A. 1993;90:8782-8786. [Abstract/Free Full Text]

32. Kwoh DY, Davis GR, Whitfield KM, Chappelle HL, DiMichele LJ, Gingeras TR. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad Sci U S A. 1988;86:1173-1177.

33. Sandrini M, Benelli A, Baraldi M. Dopamine receptors in the guinea-pig heart: a binding study. Life Sci. 1984;35:1839-1849. [Medline] [Order article via Infotrieve]

34. Amenta F, Gallo P, Rossodivita A, Ricci A. Radioligand binding and autoradiographic analysis of dopamine receptors in the human heart. Naunyn Schmiedebergs Arch Pharmacol. 1993;347:147-154. [Medline] [Order article via Infotrieve]

35. Ganguly PK, Mukherjee K, Chen Y. Altered renal dopamine receptors during development of cardiac hypertrophy. Am J Physiol. 1992;262:E569-E573. [Abstract/Free Full Text]

36. Kopia GA, Valocik RE. Demonstration of specific dopamine-1 receptor-mediated coronary vasodilation in the anesthetized dog. J Pharmacol Exp Ther. 1989;248:215-221. [Abstract/Free Full Text]

37. Hieble JP, Owen DAA, Harvey CA, Blumberg AL, Valocik RE, Demarinis RM. Hemodynamic effects of selective receptor agonists in the rat and dog. Clin Exp Hypertens. 1987;9:889-912.

38. Hackman BB, Griffin B, Mills M, Ramanathan KB. Comparative effects of fenoldopam mesylate and nitroprusside on left ventricular performance in severe systemic hypertension. Am J Cardiol. 1992;69:918-922. [Medline] [Order article via Infotrieve]

39. Zhao RR, Fennell WH, Abel FL. Effects of dopamine D₁ and dopamine D₂ receptor agonists on coronary and peripheral hemodynamics. Eur J Pharmacol. 1990;190:193-202. [Medline] [Order article via Infotrieve]

40. O'Malley KL, Harmon S, Tang L, Todd RD. The rat dopamine D₄ receptor: sequence, gene structure, and demonstration of expression in the cardiovascular system. New Biol. 1992;4:137-146. [Medline] [Order article via Infotrieve]

41. Felder CC, Blecher M, Jose PA. Dopamine-1-mediated stimulation of phospholipase C activity in rat renal cortical membranes. J Biol Chem. 1989;264:8739-8745. [Abstract/Free Full Text]

42. Ganguly PK, Mukherjee K, Sahai A. Renal dopamine receptors are involved in the development of cardiac hypertrophy. Mol Cell Biochem. 1995;144:81-84.[Medline] [Order article via Infotrieve]

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