Expression and Distribution of Dopamine Transporter in Cardiac Tissues of the Guinea Pig
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- Reynoso Palomar, A., Navarrete Larios, B., Chagoya De Sánchez, V. et al. Neurochem Res (2011) 36: 399. doi:10.1007/s11064-010-0344-7
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Dopamine transporter (DAT) is a membrane protein that it is a marker for dopaminergic neurons. In the present work, throught Western blot and autoradiographic studies with a selective ligand for DAT ([3H] WIN-35428) and noradrenaline transporter (NET) ([3H] Nisoxetine), we search the expression and distribution of DAT in comparison with NET, in cardiac tissue of guinea pig in order to support the presence of dopaminergic nerve cells into the heart. Expression of DAT, and NET were evidenced by a bands of 75 and 54 kDa, respectively in the heart. Binding for DAT and NET were found in the four cardiac chambers. However, DAT show heterogeneous distribution with binding in right atria and in both ventricles, whereas NET show homogenous distribution in the four cardiac chambers. The results show the expression of DAT in cardiac tissues with a different distribution compared with NET, being an evidence for the presence of dopaminergic nerve cells into the heart.
KeywordsDopamine transporter Noradrenaline transporter Western blot Autoradiographic studies Guinea pig heart
Dopamine transporter (DAT) is a membrane transporter protein involved in the reuptake of dopamine in the nerve terminals, and is an important mechanism to maintain dopamine homeostasis in the synapses [1, 2, 3]. Functional dopamine neurotransmission imply a balance between dopamine synthesis, release and reuptake. Particularly for dopamine neurotransmission, DAT is the main mechanism for dopamine reuptake, an then clearing extracellular dopamine, since enzymatic hydrolysis has poor participation [4, 5, 6]. DAT is considered a marker to distinguishes dopaminergic neurons from others catecholaminergic neurons [7, 8]. Regional and cellular localization of DAT in different tissues, have been performed by uptake, binding, hybridization assays, and in the last years throughout Western blot and light microscopic immunocytochemistry studies, using highly specific antibodies directed against unique region of DAT [9, 10, 11, 12, 13, 14].
The mammal heart is an organ with a complex intrinsic nervous system that is organized in ganglion and extents network of nerve fibers [15, 16, 17, 18, 19, 20, 21]. Numerous studies have demonstrated the presence of different components of cholinergic and sympathetic neurotransmission markers as choline and noradrenaline transporters that support the presence of cholinergic and sympathetic nerve cells into the heart [22, 23, 24, 25, 26]. Also it has been reported the presence of different neural markers, that suggest the presence of histaminergic, nitrergic, peptidergic, and serotonergic neurotransmission in the same organ [27, 28, 29]. However, there is a little information about the expression of proteins substrates for dopamine neurotransmission in cardiac tissues, in spite of different reports that suggest the presence of dopaminergic nerve cell into the mammal heart [30, 31, 32].
The first purpose of this study was to evaluate the expression of dopamine transporter using Western blot and autoradiographic studies in the cardiac tissues of guinea pig heart in order to support the presence of dopaminergic nerve cells into the heart. Secondly, to analyze the distribution of the expression of the dopamine transporter in comparison with the well studied noradrenaline transporter in the guinea pig heart.
All experiments were conducted in male guinea pigs (weighing 250–300 g) bred in our facilities. Animals were maintained under constant room temperature (23°C) and a 12:12 h light/dark cycle; with food and water ad libitum. All the procedures described in this study were approved by the BUAP Animal Care Committee and the governmental guidelines (Mexican Council for Animal Care, Norma Oficial Mexicana NOM-062-ZOO-1999) and in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Guinea-pigs were killed by decapitation (n = 16), and after a thoracotomy the hearts were removed quickly and washed during 5 min with Krebs-Henseleit solution in a Langendorff system, then the hearts were rapidly frozen in a 2-methylbutane/dry ice mixture (−40°C) and then stored at −80°C until use.
Frozen guinea pig hearts (n = 4, for each protocol) were first sectioned transversely, at the atrioventricular groove, to separate the rigth atria. After sagital sections throughout the long longitudinal axes, in the ventricles plus the left atria and in the isolated right atria were made. Tissues were sectioned at 15 μm thickness using a cryostat (Leica Germany), and sections were mounted on precleaned, gelatin-coated microscope slides, (three sections/slide), thaw-mounted, desiccated under vacuum at 4°C overnight and then stored at −80°C until the day of the experiment.
Autoradiography for Dopamine Transporter
DAT binding autoradiography was assayed in accord with previous protocols . Cardiac tissue sections, were preincubated for 20 min at 4°C in 50 mM Tris–HCl, containing 120 mM NaCl, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, 4 mM de MgCl2, pH de 7.4. After sections were incubated in the same buffer during 2 h at 4°C containing 10 nM [3H]WIN-35428 (specific activity: 84 Ci/mmol) from New England Nuclear (Boston, MA, USA). Non-specific binding was determined in other cardiac tissue sections adding 1 μM GBR-12909 in the same buffer. Incubations were terminated by washing the cardiac tissue sections twice for 1 min each in ice-cold 50 Tris–HCl, pH 7.4, 4°C. After a brief dipping in ice-cold distilled water, sections were rapidly dried an apposed to [3H]-Hyperfilm for 6 weeks, along side calibrated tritium standards. GBR-12909, desipramine and most other reagents were purchased from Sigma–Aldrich (St. Louis, MO). [3H]-Hyperfilm was purchased from Amersham Canada (Toronto, Ontario).
Autoradiography for Noradrenaline Transporter
NET binding autoradiography was carried out as described previously [34, 35]. Cardiac tissue sections were preincubated for 20 min at 4°C in 50 mM Tris–HCl, containing 300 mM NaCl, 1 mM EDTA, 5 mM KCl, 1.5 mM CaCl2, 4 mM de MgCl2, pH de 7.4, a room temperature during 10 min. After sections were incubated in the same buffer during 2 h at 4°C containing 3 nM [3H] Nisoxetine hydrochloride (specific activity: 82 Ci/mmol) from New England Nuclear (Boston, MA, USA). Non-specific binding was determined in other cardiac tissue sections adding 100 μM desipramine in the same buffer. Incubations were terminated by washing the cardiac tissue sections twice for 1 min each in ice-cold 50 Tris–HCl, pH 7.4, 4°C. After a brief dipping in ice-cold distilled water, sections were rapidly dried an apposed to [3H]-Hyperfilm for 4 weeks, along side calibrated tritium standards.
Heart tissues from 8 male guinea pigs, four for each protocol were minced in separated tubes (right and left atria, and right and left ventricle) with 10 ml of cold buffer containing 20 mM Tris–HCL (pH 7.4), 1 mM EDTA, leupeptin (5 μg/ml), soybean trypsin inhibitor (5 μg/ml), and benzamidine (10 μg/ml). Tissues were then lyzed with a polytron homogenizer (three 10 s bursts at maximum speed) and the lysates were centrifuged at 1,000g for 10 min at 4°C. The lysate was filtered through 4 layers of cheesecloth in a corex tube and centrifuged at 45,000g (18,500 rpm) for 20 min at 4°C. The pellets were resuspended in 250 μl of solubilization buffer containing triton X-100, Nonidet (NP-40), 2 M NaCl, Tris–HCL 1 M (pH 7.4), EDTA 250 mM (pH 7.4). The tubes were inverted during 2 h at 4°C, and centrifuged at 45,000g (18,500 rpm) for 20 min at 4°C. The supernatants were kept for protein assay and Western blot . Protein content was determined according to Bradford’s method using a Bio-Rad kit.
Equal amounts of solubilized membrane proteins (50 μl/well) were fractionated on 10% SDS polyacrylamide gels. The separated proteins were transferred to 0.2 μm nitrocellulose membranes and the intensity of the bands was verified with red ponceau. Membranes were blocked using 5% nonfat dry milk in TBST (Tris–HCl 1 M, NaCl 2 M; pH 7.4, Tween-20 250 μM) for 1 h at room temperature. The membranes were then incubated with polyclonal antibodies for DAT and NET, respectively (AB1591P and AB5066P from Chemicon-Millipore, USA) at a dilution of 1:400 for at 4°C overnight. After three 10 min washes with TBST, the membranes were incubated with peroxidase-conjugated secondary antibody (anti-goat IgG and anti-rabbit IgG, diluted at 1:2,500, from Santa Cruz Biotech, USA) for 45 min and washed 5 times, 10 min, with TBST. Antibody detection was performed by chemiluminescent detection system (Renaissance reagent, Mandel). Membranes were exposed to a Kodak film and developed right away.
Autoradiograms were analyzed using a computerized image analysis system (MCID-4, Imaging Research, St Catherine’s, Ontario, Canada). The film optical densities were converted to fmol/mg tissue using a standard curve generated by the [3H] microescale. Binding data were analyzed in the each cardiac chamber and expressed in fmol/mg wet tissue weight, as mean ± SEM. Statistical analysis was done with one-way analysis of variance with post-ANOVA (Bonferroni). A P < 0.05 was considered significant.
Western blot results were analyzed by qualitative method with the presence of band corresponding with molecular weight in kDa of each protein analyzed. Films were scanned and images were processed with Adobe Photoshop 7.0.
[3H] WIN-35428 Binding
Quantitative analysis and regional distribution of specific binding of [3H] WIN-35428 and [3H] Nisoxetine, to dopamine and norepinephrine uptake sites in cardiac tissues slides from guinea pig hearts
Radioligand for selective transporter
Dopamine transporter [3H] WIN-35428
21.3 ± 3.4
15.3 ± 3.5*
24.1 ± 3.4
24.2 ± 3.4
12.6 ± 3.6*
Noradrenaline transporter [3H] Nisoxetine
44.9 ± 1.2
44.4 ± 1.5
36.5 ± 3.3
40.4 ± 3.7
56.9 ± 2.8*
[3H] Nisoxetine Binding
Figures 1 and 2 shows Western blot detection of DAT and NET in cardiac membranes of the guinea pig heart. Bands at 54 and 75 kDa for DAT and NET, respectively were detected in tissues from the four cardiac chambers. The rat anti-dopamine transporter used, is polyclonal antibody in which the immunogen is an 18 amino acid peptide sequence near the NH2-terminus of rat brain dopamine transporter, and by Western blot the antibody recognizes a broad 80 kDa band of extracts from rat striatum, whereas the anti-norepinephrine transporter is a polyclonal antibody with 22 aminoacid peptide sequence mapping to the 1st extracellular domain of rat NET, and it recognize a diffuse band at approximately 54 and 80 KDa. It has been reported that NET can exist at least in two forms, a highly glycosylated 80 kDa form, which is the most common in brain, and a less glycosylated 54 kDa forms also seen in some regions and tissues. We found the less glycosylated 54 kDa forms in the cardiac tissues.
In the present study we provide evidence to support the presence of dopamine transporter protein in the guinea pig heart. In addition, we found different distribution of DAT in comparison with NET in the four cardiac chambers of the guinea pig heart, suggesting the presence of dopaminergic nerve cells into the heart.
Dopamine transporter (DAT) is an exchanger protein, involved in the reuptake of dopamine from the synapses and is distributed in the plasma membrane of the soma, the neural extensions, and varicosities along these extensions in midbrain dopaminergic neurons [1, 2, 3]. DAT is considered the main mechanism to terminate the dopamine action for clearing extracellular dopamine and then regulate the intensity and duration of dopaminergic neurotransmission. Since DAT is essential for dopamine homeostasis in the synapses is considered a specific marker to identify dopaminergic neurons [4, 5, 6].
Although dopamine transporter display highest amino acid homology with other amino transporters, particularly with the norepinephrine transporter (67%), it has been possible discriminate the presence of dopaminergic and noradrenergic neurons in the same region, using high selectively ligands for both transporters . In the present study, was used a highly selective ligand for the DAT, [3H]WIN 35,428 ([3H]2-β-carbomethoxy-3β-(4-fluoro-phenyl) tropane) that recognizes two sites binding for DAT (high and low affinity components Ki = 1.33 to 5.55 nM and Ki = 164 to 404 nM, respectively), in membranes and in intact sections from rat and rodent brains, to investigate differences in binding densities in the four cardiac chambers. It has been reported that [3H]WIN 35,428 has excellent qualities for autoradiographic studies and it has a good selectivity for DAT over other monoamine transporters [8, 10, 11]. The results show that through [3H]WIN 35,428 there is an expression of DAT, particularly the high affinity component, which could represent the functional state of the DAT in the guinea pig heart, and the results show positive [3H]WIN 35,428 binding in the tissues of the four cardiac chambers with a different distribution, being predominantly in the right atria and in both ventricles. This different distribution could be related to predominantly location of cardiac nerve cells in some region into the heart, than can synthesize dopamine.
Through autoradiographic studies with [3H]-Nisoxetine as a ligand we identify the presence of NET in the cardiac tissues of guinea pig heart. Previous studies have proven that [3H]-Nisoxetine is useful for measuring uptake sites for NE in homogenates and in tissues slides for quantitative autoradiography, in different tissues, since is a potent and selective inhibitor of the uptake of norepinephrine, being approximately 1000-fold more potent in blocking the uptake of NE than the serotonine and 400-fold more potent in blocking the uptake of NE than dopamine [23, 32, 36]. In spite that [3H]-Nisoxetine has been used as a ligand to identify NET in different tissues, there is s little information about their distribution in the heart. In the present work besides to identify the presence of NET in the four cardiac chambers we analyzed their distribution in comparison with the DAT distribution, and the result show that both transporters are present in the four cardiac chambers, however, DAT has heterogeneous distribution in the chambers and is predominantly in right atria and in both ventricles whereas NET has a homogenous distribution in the four cardiac chambers, in addition the hight [3H]-Nisoxetine binding was found in proximal region from aorta and pulmonary artery. This could represent that cardiac nerve cells with capacity to synthesize norepinephrine are present in the four cardiac chambers of the guinea pig heart particularly in great vessels.
Western blot analysis also provided strong evidence for the presence of DAT protein in the four cardiac chambers of the guinea pig heart. The antibodies were directed toward the N-terminus region of the structure of DAT, which no cross reactivity to the closely related norepinephrine transporter [12, 13]. In fact two different bands were clearly indentified in the homogenates from the four cardiac chambers in accord with the molecular weight corresponding to the DAT (75 kDa) and NET (54 kDa), respectively. Our resulted are in accord with previous reports in cardiac tissue particularly for NET, where it has been reported the less glycosylated 54 kDa form [23, 36].
A role of neuromodulation of dopamine into the mammal heart has been demonstrated from different studies where activation of prejuntional dopamine D-2 Family receptors decreases the norepinephrine releases from heart nerve terminals [37, 38, 39]. Then dopamine has predominantly role in the control of cardiac function since it is regulating the sympathetic tone throughout regulating the release of norepinephrine from terminal sympathetic nerves. In addition others studies have been demonstrated the expression of dopamine receptors in mammal hearts [40, 41, 42]. Also mRNA levels for D1, and D4 receptors have been reported in mammals heart including human heart [43, 44]. In addition administration of dopamine receptor agonists, generate changes in parameters of cardiac functions [42, 45]. Throughout high performance liquid chromatography, analysis of catecholamine content in heart has shown that dopamine is a normal component in the heart. Different report shows that dopamine concentration is significantly less than norepinephrine concentration in the heart tissues (dopamine 4.67 ng/g and norepinephrine 0.28 μg/g) [32, 46, 47]. It is considered that dopamine only serve as a precursor of norepinephrine, however, dopamine concentrations in heart tissues could be much greater than can be ascribed to their role as precursors in the biosynthesis of norepinephrine and it have been suggested that could be related to the presence of dopaminergic cell nerve into the heart that can synthesize dopamine . In fact it has been reported that dopamine is the higher catecholamine in the atrias from invertebrate heart .
The intrinsic cardiac nerve cells displayed immunoreactivity for different active substances than can be colocalized, and it is a representation of their capacity to synthesize and release different neurotransmitters as acetylcholine and norepinephrine. Different reports have been demonstrated inmunorreactivity for cholinergic, noradrenergic, histaminergic, nitrergic, peptidergic, and serotonergic neurotransmitters in cardiac tissues [15, 19, 20, 24, 27, 28, 29]. Also it has been reported, different distributions of immunoreactivity for tyrosine hidroxilase and dopamine beta hydroxilase a specific markers for adrenergic neurons in mammal hearts [30, 31]. Neurons that exhibit a positive labeled for tyrosine hydroxylase (TH) and negative labeled for dopamine-beta-hydroxylase (DBH) are more probably dopaminergic neurons. Neurons with positive labeled for both markers are noradrenergic neurons [7, 8]. Tyrosine hydroxilase immunoreactivity has been reported particularly in thick connecting nerve trunks and in fine varicose nerve fibers in the myocardium and in several ganglia from the right atria myocardium, whereas dopamine-beta-hydroxylase immunoreactivity, it has been reported in ganglion cell bodies from the left atria suggesting the presence of different cardiac nerve cells than can synthesize dopamine and norepinephrine in an independent form [30, 31].
The information reported in this study that support the presence of dopamine transporter protein in the guinea pig heart that is considered a marker for dopaminergic neurons, taken together with other information as quantitation of dopamine concentration in the heart, the expression of dopamine receptors in cardiac tissues, changes in parameter of cardiac function induced with dopamine agonist receptors, and the different distributions of immunoreactivity for tyrosine hidroxilase and dopamine beta hydroxilase in heart tissues, support the hypothesis that dopaminergic nerve cells could exist in the mammal heart, which could release dopamine in the junctional space between nerve cell and the myocyte. The interaction of dopamine with dopamine receptors could be explain some effects of dopamine agonist drugs in the heart [42, 47]. Then the intrinsic cardiac nerve cell has a complex neurochemical anatomy that is not clear until now. The functional repercussion of the multiple neurotransmitters released in the heart make more complex their understanding role in the control of the cardiac function in normal and pathological states of the heart and constitutes a wide field to research.
Our findings demonstrate the presence of DAT in the guinea pig heart, distributed in the four cardiac chambers, predominantly in the right atria and both ventricles, and show different distribution in comparison with NET. Since DAT is considered a marker of dopaminergic neurons, the result suggest the presence of dopaminergic nerve cell into the guinea pig heart, and in consequences functionally dopaminergic neurotransmission.
This study was supported by grants from VIEP-BUAP (GOVM-SAL08-I). We are grateful to Ruth Giles for their technical assistance, and we thank Dr. Ellis Glazier for editing this English-language text.