Abstract
To investigate the effects of the dopamine D4 receptor agonist A-412997 and the D4 antagonist sonepiprazole in human isolated umbilical artery (HUA) and vein (HUV) and the expression of the D4 receptor by immunohistochemistry in these vessels. A segment of the umbilical cord (10–20 cm) from the insertion point in the placenta and 5 cm from the umbilicus was removed by the obstetrician and placed in a container with Krebs–Henseleit solution (KHS). The Wharton’s jelly was removed, and HUA and HUV rings (3 mm) were suspended in 10 mL organ baths containing oxygenated (95% O2:5% CO2) and heated (37 °C) KHS. For immunohistochemistry, the vessels were fixed in 10% formalin, embedded in paraffin wax and sectioned (4 µm). A-412997 did not induce contractions in the HUA rings. In HUA pre-contracted rings, A-412997 induced concentration-dependent relaxations, which were reduced when the HUA rings were pre-incubated with L-NAME. A-412997 caused concentration-dependent contractions of HUV rings, which were potentiated by pre-treatment with L-NAME and reduced by pre-incubation with 6-nitrodopamine. In HUV pre-contracted rings, A-412997 failed to induce relaxations. Sonepiprazole antagonized A-412997-induced contractions in HUV rings and provoked concentration-dependent relaxations in pre-contracted HUA and HUV rings. Dopamine D4 receptor was positive in both HUA and HUV, especially in the endothelium, and detected only in HUV smooth muscle cells. Activation of HUA D4 receptor is associated with relaxation, whereas in HUV, it leads to contraction. Differential expression of D4 receptors may modulate umbilical-placental blood flow.
Introduction
The umbilical cord is constituted of two arteries and one vein (1, 2), and the umbilical vein is the vessel that carries oxygenated, nutrient-rich blood from the placenta to the fetus, i.e., it behaves as an artery. The umbilical-placental circulation is characterized by a network of blood vessels without innervation (3, 4), so that the umbilical-placental blood flow is mainly modulated by perfusion pressure, fetal heart rate and umbilical-placental resistance (5). Infusion of the nitric oxide (NO) synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME) in the left umbilical arterial hypogastric branch caused increased umbilical-placental vascular resistance and arterial pressures, accompanied by a fall in umbilical blood flow (6), indicating basal release of NO modulates the resting umbilical-placental vascular resistance in normal fetal lambs (7).
Human umbilical cord vessels (HUCVs) present basal release of both dopamine (8) and 6-nitrodopamine (6-ND) (9). In endothelium-intact HUCV rings pre-contracted with the endoperoxide analogue U-46619 (10 nM), 6-ND caused concentration-dependent relaxations, which were not affected by pre-incubation of the rings with L-NAME, indicating that endothelium-derived 6-ND is a novel mechanism by which NO may modulate vascular reactivity in the HUCV (9).
Dopamine receptors are classified into D1-like and D2-like subtypes based on their structure and pharmacology. D1-like receptors are composed of D1 and D5 receptors, while D2-like receptors are composed of D2, D3 and D4 receptors (10). In vitro receptor binding using the dopamine D1-like receptor antagonist [3H]-SCH-23390 revealed binding sites in human umbilical arteries (HUAs) (11), suggesting that endogenous dopamine could modulate HUA tonus. Interestingly, in HUA obtained from pre-eclamptic patients, the density of dopamine D1-like receptors was decreased, but the affinity using [3H]-SCH-23390 as a ligand was unaffected (12). However, the role of dopamine D1-like receptors is controversial since, in U-46619 (10 nM) pre-contracted HUA, the dopamine D1 receptor agonist fenoldopam (13), caused either contraction (14) or relaxation (9).
The relaxations induced by 6-ND in both HUA and human umbilical vein (HUV) pre-contracted with U-46619 are dependent on the integrity of the endothelium and were similar to those induced by the dopamine D2-like receptor antagonist haloperidol (9), indicating that 6-ND could be acting as a D2-like receptor antagonist (15). Indeed, the relaxations induced by 6-ND were not affected by pre-incubation of the HUCV rings with the dopamine D1-like receptor antagonist SCH-23390 (8).
Differential expression of placental D2-like receptors is observed during normal pregnancy, indicating a potential role of dopamine in placental physiology (16). Although the mean antepartum plasma concentration of dopamine was not different from the nonpregnant value, it was significantly less than the corresponding vaginal and cesarean section values (17). Urine dopamine levels were also increased in women with pre-eclampsia compared with matched control subjects or compared with a group of multigravidae with hypertension in pregnancy (18). Dopamine arterial and venous plasma concentrations were also higher in pre-eclamptic patients compared to normotensive pregnant women (19), indicating a potential role of this catecholamine in this pathology.
Although D1, D2, D3, D4 and D5 dopamine receptors are present in the peripheral vasculature (20, 21, 22), there is no evidence of the presence of D2-like receptors in either HUA or HUV. In here, it was investigated the actions of both the selective dopamine D4 receptor agonist A-412997 (23) and the selective dopamine D4 receptor antagonist sonepiprazole (24, 25) in these tissues, and the expression of the D4 receptor in both HUA and HUV by immunohistochemistry and by RNAscope®. RNAscope® is a highly sensitive and specific method employed for testing gene expression levels (26). It has a high concordance rate (CR) with current ‘gold standard’ methods such as qPCR and qRT-PCR (26), and it allows for the precise cellular localization of the messenger RNA (mRNA) of interest, thus providing insightful information based on morphological/topographical correlations. RNAscope was used in the present study as a means to confirm and cross-validate immunohistochemistry results.
Methods
Study participants
Normotensive pregnant women (18–40 years; n = 34) who did not have preeclampsia or gestational hypertension (27) or pregestational or gestational diabetes mellitus (28) participated in the study. They underwent either natural or cesarean delivery (Campinas Maternity Hospital), and none of them were on regular medication. The protocol was approved by the local institutional review board, and the volunteers signed the informed consent form.
The investigation conformed to the principles outlined in the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Institute of Biomedical Sciences of the University of São Paulo – ICB/USP (protocol number 3.165.417).
Preparation of HUAs and HUV for the in vitro vascular reactivity assays
A segment of the umbilical cord (10–20 cm) from the insertion point in the placenta and 5 cm from the umbilicus was removed by the obstetrician and placed in a container with Krebs–Henseleit solution (KHS). The Wharton’s jelly was removed, and HUA and HUV were dissected (29). Briefly, HUA and HUV rings (3 mm) were vertically hung between two metal hooks in 10 mL organ baths containing KHS, aerated with carbogen (95% O2:5% CO2; pH 7.4) at 37 °C. Tissues were allowed to equilibrate for 90 min under a resting tension (10 mN), and the isometric tension was recorded using a PowerLab system (ADInstruments, Austrália).
Effect of the dopamine D4 receptor agonist A-412997 on HUA and HUV rings
To evaluate the integrity of the endothelium, both HUA and HUV were pre-contracted with 5-HT (1 μM), and ATP (10 μM) was used as the endothelium-dependent relaxing agent. For the other experiments where pre-contracted HUA and HUV were used, the contracting agent employed was U-46619 (10 nM). The effect of A-412997 (1 nM to 1 μM) was assessed in endothelium-preserved HUA in the absence and presence of the NOS inhibitor L-NAME (100 μM, 30 min) or the selective, irreversible, heme-site inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 100 μM, 30 min). Progressive concentration–response curves to A-412997 (10 pM to 1 μM) were also performed in HUV rings in the absence and in the presence of L-NAME (100 μM, 30 min). In addition, in HUV pretreated with L-NAME (100 μM, 30 min), the effects of A-412997 (1 nM to 10 μM) were assessed in the presence and absence of the selective dopamine D4 antagonist sonepiprazole (0.01–0.1 μM) and 6-ND (0.03–0.1 μM). The effect of A-412997 (0.01–300 nM) was also evaluated in endothelium-preserved HUA and HUV rings pre-contracted with U-46619 (10 nM).
Effect of the dopamine D4 receptor antagonist sonepiprazole on HUA and HUV rings
The evaluation of the effect of the dopamine D4 receptor antagonist sonepiprazole was carried out in HUA and HUV vessels pretreated with L-NAME (100 μM, 30 min). Progressive concentration–response curves to dopamine (10 nM to 1 mM) were conducted with and without the selective DDR4 antagonist sonepiprazole (0.1–1 μM, 30 min). The relaxant effects of sonepiprazole (0.1–1 μM) were evaluated in endothelium-intact HUA and HUV rings pre-contracted with U-46619 (10 nM).
Immunohistochemistry for DDR4
Umbilical cord samples (n = 8) from normal placentae were fixed in 10% neutral-buffered formalin for 24 h at 25 °C, dehydrated, embedded in paraffin wax, and sectioned at 4 µm. Subsequently, these sections were deparaffinized in xylene and rehydrated in a series of ethanol baths of decreasing concentration and eventually incubated with a rabbit polyclonal anti-DDR4 antibody (catalog code: PA5-89196; dilution: 1:50 in PBS; Invitrogen, USA). Negative controls consisted of the omission of the primary antibody and incubation with the primary antibody diluent (one negative control per section) to identify any nonspecific background staining. Furthermore, formalin-fixed, paraffin-embedded human adrenal parenchyma (medulla, cortex and periadrenal adipose tissue) and human central nervous system (CNS – cerebellum) tissue sections were used as positive controls for the presence of the dopamine D4 receptor in development and main assays. The purpose of using these positive control tissues was to ascertain that the primary antibody was properly working (i.e., identifying the target protein in tissues known to contain it), especially if the tissues of interest (umbilical cord sections) were found to be negative. Furthermore methodological details are described as supplemental information (see section on Supplementary materials given at the end of the article).
In situ hybridization (RNAscope®) – DDR4 probe
For the RNAscope assay, the tissue sections were incubated with RNAscope® hydrogen peroxidase (Ref. 322335, ACD, USA) (Pretreat 1) for 10 min at RT, and then, they were rinsed in dH2O. The sections were incubated with RNAscope® Target Retrieval (Ref. 322000, ACD, USA) 1× Pretreatment Solution in dH2O (Pretreat 2) for 15 min at approximately 99 °C. The methodological details are described as supplemental information.
Data analysis
Data are expressed as mean ± S.E.M of the pharmacological experiments, and the number of experiments is expressed as x/y, where x represents the number of umbilical vessels and y the number of rings employed in the experiment. Emax values are presented in mN, and the data were analyzed by an unpaired t-test.
Results
Effect of the dopamine D4 receptor agonist A-412997 on HUA and HUV rings
A-412997 (1 nM to 100 μM) did not induce contractions in the HUA rings (n = 5/10; data not shown), even when the rings were pre-incubated (30 min) with either L-NAME (100 μM; n = 5/10; data not shown) or ODQ (100 μM; n = 5/10; data not shown). A-412997 (1 nM to 1 μM) caused concentration-dependent contractions of the HUV rings (Fig. 1A), which were significantly increased when the HUV rings were pre-incubated (30 min) with L-NAME (100 μM; Fig. 1A; Table 1). In L-NAME (100 μM) pre-treated (30 min) HUV rings, A-412997 induced concentration-dependent contractions (Fig. 1B; Table 2), which were significantly reduced when the rings were pre-incubated with 6-ND (Fig. 1B). The pEC50 and Emax values are described in Table 2. In HUA rings pre-contracted with U-46619 (10 nM), A-412997 induced concentration-dependent (0.01–100 nM) relaxations, which were significantly reduced when the HUA rings were pre-incubated (30 min) with L-NAME (100 μM; Fig. 1C; Table 3). In contrast, in HUV rings pre-contracted with U-46619 (10 nM), A-412997 failed to induce relaxations (Fig. 1D).
Effect of the dopamine D4 receptor agonist A-412997 on human umbilical artery (HUA) and vein (HUV) rings. A-412997 (0.001–10 μM) caused concentration-dependent HUV contractions, which were potentiated by pre-incubation (30 min) with L-NAME (100 μM; panel A). In L-NAME-pretreated HUV rings (panel B), the A-412997-induced contractions were significantly reduced by incubation with 6-nitrodopamine (6-ND). In HUA rings precontracted with U-46619 (10 nM), A-412997 induced concentration-dependent relaxations, which are significantly reduced by L-NAME pretreatment (panel C). A-412997 caused a small relaxation in HUV rings precontracted with U-46619 (panel D). The results of the statistical analysis are reported in Tables 1, 2, 3. The number of experiments (n) is reported as x/y, where x represents the number of parturients and y the number of rings employed.
Citation: Vascular Biology 7, 1; 10.1530/VB-24-0010
The potency (pEC50) and the maximum response (Emax) of A-412997 on the HUV in the absence (control) and presence of the NO inhibitor L-NAME (100 μM).
| A-412997 | pEC50 (log(M)) | P value | Emax (mN) | P value | n |
|---|---|---|---|---|---|
| Control | 7.27 ± 0.29 | 4.79 ± 1.30 | 4/4 | ||
| L-NAME | 7.52 ± 0.28 | 0.5612 | 25.65 ± 11.05 | 0.0202 | 4/6 |
NO, nitric oxide; HUV, human umbilical vein.
The potency (pEC50) and the maximum response (Emax) of dopamine- and/or A-412997-induced contractions on the HUA and HUV rings in the absence (control) and presence of 6-ND or sonepiprazole.
| pEC50 (log(M)) | P value | Emax (mN) | P value | n | |
|---|---|---|---|---|---|
| HUV | |||||
| L-NAME (100 μM) | 7.30 ± 0.26 | 26.6 ± 3.25 | 5/11 | ||
| L-NAME+6-ND 30 nM | 6.24 ± 0.10 | 0.0029 | 22.36 ± 3.35 | 0.6200 | 4/4 |
| L-NAME+6-ND 100 nM | 6.04 ± 0.36 | 0.0089 | 16.46 ± 3.22 | 0.0402 | 5/5 |
| HUV | |||||
| Dopamine | |||||
| L-NAME (100 μM) | 4.67 ± 0.21 | 16.30 ± 1.87 | 5/10 | ||
| L-NAME+Sonepiprazole 10 nM | 4.23 ± 0.15 | 0.0311 | 16.43 ± 2.40 | 0.4770 | 5/10 |
| L-NAME+Sonepiprazole 100 nM | 4.10 ± 0.16 | 0.0380 | 9.10 ± 1.85 | 0.0094 | 5/7 |
| A-412997 | |||||
| L-NAME (100 μM) | 7.30 ± 0.26 | 26.6 ± 3.25 | 5/11 | ||
| L-NAME+Sonepiprazole 10 nM | 6.75 ± 0.42 | 0.1574 | 18.63 ± 2.24 | 0.0287 | 5/6 |
| L-NAME+Sonepiprazole 100 nM | 6.50 ± 0.13 | 0.4296 | 10.46 ± 0.28 | 0.0008 | 4/4 |
| HUA | |||||
| Dopamine | |||||
| L-NAME (100 μM) | 3.75 ± 0.16 | 6.83 ± 2.15 | 3/5 | ||
| L-NAME+Sonepiprazole 10 nM | 3.66 ± 0.17 | 0.7158 | 7.15 ± 1.10 | 0.8959 | 3/5 |
| L-NAME+Sonepiprazole 100 nM | 3.80 ± 0.25 | 0.8786 | 5.73 ± 0.40 | 0.6433 | 3/5 |
6-ND, 6-nitrodopamine; HUA, human umbilical artery; HUV, human umbilical vein.
The potency (pEC50) and the maximum response (Emax) of A-412997- and/or sonepiprazole-induced relaxation on the HUA and HUV rings in the absence (control) and presence of L-NAME.
| pEC50 (log(M)) | P value | Emax (mN) | P value | n | |
|---|---|---|---|---|---|
| HUA | |||||
| A-412997 | |||||
| Control | 9.03 ± 0.10 | 53.65 ± 2.86 | 4/7 | ||
| L-NAME (100 μM) | 6.04 ± 0.36 | 0.0417 | 33.10 ± 6.62 | 0.0114 | 4/6 |
| HUA | |||||
| Sonepiprazole | |||||
| Control | 7.69 ± 0.27 | 109.39 ± 7.79 | 3/5 | ||
| L-NAME (100 μM) | 7.70 ± 0.28 | 0.4960 | 80.92 ± 8.41 | 0.0189 | 3/5 |
| HUV | |||||
| Sonepiprazole | |||||
| Control | 8.19 ± 0.12 | 96.23 ± 8.04 | 4/8 | ||
| L-NAME (100 μM) | 8.06 ± 0.10 | 0.2268 | 94.74 ± 18.88 | 0.4715 | 4/8 |
HUA, human umbilical artery, HUV, human umbilical vein.
Effect of the dopamine D4 receptor antagonist sonepiprazole on HUCV rings
Dopamine (1 μM to 3 mM) induced concentration-dependent contractions of L-NAME (100 μM) pre-treated (30 min) of HUV rings, which were antagonized when the HUV rings were pre-incubated with sonepiprazole (0.1–1 μM; Fig. 2A). The concentration-dependent contractions induced by A-412997 in the HUV rings were also antagonized by pre-incubation with sonepiprazole (0.01–0.1 μM; Fig. 2B). The pEC50 and Emax values are described in Table 2. In contrast to HUV rings, pre-incubation of the HUA rings with sonepiprazole (0.1–1 μM) did not affect the contractions induced by dopamine (Table 2).
Effect of the dopamine D4 receptor antagonist sonepiprazole on human isolated artery (HUA) and vein (HUV) rings. Panels A and B illustrate the experiments performed in HUV pretreated with L-NAME (100 μM). Dopamine induces concentration-dependent contractions of the HUV (panel A). Pre-incubation (30 min) of the HUV with the D4 receptor antagonist sonepiprazole caused concentration-dependent inhibition of the contractions induced by dopamine (panel A). The dopamine D4 agonist A-412997 induces concentration-dependent contractions of the HUV (panel B) and pre-incubation (30 min) with sonepiprazole caused concentration-dependent inhibition of the contractions induced by this agonist (panel B). In U-46619 (10 nM) precontracted HUA (panel C) and HUV (panel D) rings, sonepiprazole caused concentration-dependent relaxations of the vascular tissues, which were insensitive to pretreatment with L-NAME. The results of the statistical analysis are reported in Tables 2 and 3. The number of experiments (n) is reported as x/y, where x represents the number of parturients and y the number of rings employed.
Citation: Vascular Biology 7, 1; 10.1530/VB-24-0010
Sonepiprazole caused concentration-dependent (100 pM to 1 μM) relaxations of both pre-contracted (U-46619, 10 nM) HUA rings (Fig. 2C; Table 3) and HUV rings (Fig. 2D; Table 3). In HUA rings, pre-incubation with L-NAME 100 μM minimally reduced the Emax (Fig. 2C), whereas in HUV rings, the relaxations induced by sonepiprazole were not affected by pre-incubation with L-NAME (Fig. 2D).
Immunohistochemistry
The results regarding the immunodetection of DDR4 are summarized in Tables 4, 5, 6 and illustrated in Figs. 3 and 4. Briefly, DDR4 was strongly positive in the endothelium of both HUA (Fig. 3A, B, C; Table 4) and HUV (Fig. 3B, C, D; Table 4). In all HUA cases, DDR4 was either completely absent or very faint in tunica media smooth muscle cells and it was thus considered as negative in this vascular compartment (Fig. 3A, B, C, Table 4). By contrast, in all HUVs, DDR4 was consistently positive in tunica media smooth muscle cells (Fig. 3B, C, D, Table 4). As expected, positive control tissue sections (cerebellum) showed diffuse positivity in molecular, granular and Purkinje cell layers (Fig. 4A, Table 5). In addition, cerebellum small/medium-sized vessels were also positive for DDR4 (both in endothelial and smooth muscle cells) (Fig. 4B, Table 5). DDR4 was again detected in the human adrenal cortex and medulla (both in parenchymal cells and vascular structures; Fig. 4C; Table 6), not to mention in the adjacent adipose tissue cells and vessels (Fig. 4D; Table 6).
Dopamine receptor D4 (DDR4) detection by immunohistochemistry in HUAs and HUVs (n = 8 samples).
| Vessels | DDR4 | ||
|---|---|---|---|
| Histologic compartment | Frequency of positive cases | Intensity range | |
| Arteries | Tunica intima (endothelium) | 8/8 | +++ |
| Arteries | Tunica media (smooth muscle layer) | 0/8 | |
| Veins | Tunica intima (endothelium) | 8/8 | +++ |
| Veins | Tunica media (smooth muscle layer) | 8/8 | + to +++ |
HUAs, human umbilical arteries; HUV, human umbilical vein; DDR4, D4 dopamine receptor; anti-DDR4 Ab dilution = 1:50 (established using human adrenal tissue); intensity range: 0/+ = absent or very faint intensity; + = weak positivity; ++ = moderate positivity; +++ = strong positivity.
Dopamine receptor D4 (DDR4) detection by immunohistochemistry in human CNS tissue and vessels (n = 1).
| CNS | DDR4 | |||
|---|---|---|---|---|
| Histologic compartment | Frequency of positive cases | Distribution | Intensity range | |
| Brain parenchyma | Neuropil | 1/1 | Diffuse | ++ to +++ |
| Brain parenchyma | Neuron cell bodies | 1/1 | <10% of Purkinje cells | +++ |
| Vessels* | Tunica intima (endothelium) | 1/1 | Most vessels | +++ |
| Vessels* | Tunica media (smooth muscle layer) | 1/1 | Most vessels | +++ |
CNS, central nervous system, Anti-DDR4 Ab dilution = 1:50 (established using human adrenal tissue); intensity range: 0/+ = absent to very faint intensity; + = weak positivity; ++ = moderate positivity; +++ = strong positivity.
Small arteries and veins.
Dopamine receptor D4 (DDR4) detection by immunohistochemistry in human adrenal parenchyma (medulla and cortex) and periadrenal adipose tissue vessels (n = 1).
| Adrenal | DDR4 | |||
|---|---|---|---|---|
| Histologic compartment | Frequency of positive cases | Distribution | Intensity range | |
| Adrenal parenchyma | Medulla | 1/1 | Diffuse | +++ |
| Adrenal parenchyma | Cortex | 1/1 | Diffuse | + |
| Adrenal and periadrenal ATV* | Tunica intima (endothelium) | 1/1 | Most vessels | +++ |
| Adrenal and periadrenal ATV* | Tunica media (smooth muscle layer) | 1/1 | Most vessels | +++ |
Anti-DDR4 Ab dilution = 1:50; intensity range: 0/+ = absent to very faint intensity; + = weak positivity; ++ = moderate positivity; +++ = strong positivity. ATV, adipose tissue vessels.
Mostly arterioles.
Dopamine receptor D4 (DDR4) detection in HUCVs by immunohistochemistry. (A) Human umbilical artery (HUA): in this example, DDR4 was positive only in the endothelium (in all HUA cases, DDR4 was either completely absent or very faint in smooth muscle cells and it was thus considered as negative in this vascular compartment); (B) HUV: in all HUV cases such as this one, DDR4 was present both in the endothelium and in the smooth muscle layer; (C) HUA, detail showing DDR4 positivity restricted to the endothelium (E) tunica media (TM) smooth muscle cells are negative for this marker; (D) HUV, detail showing both endothelium and TM smooth muscle cells are positive for DDR4; (E) HUA, negative control (omission of the primary antibody); (F) HUV, negative control (omission of the primary antibody). (A and B) Immunoperoxidase/Hematoxylin, 400× (original magnification); (C, D, E and F) Immunoperoxidase/Hematoxylin, 200× (original magnification). L, lumen; TM, tunica media; E, endothelium.
Citation: Vascular Biology 7, 1; 10.1530/VB-24-0010
Dopamine receptor D4 (DDR4) detection in the human CNS and in adrenal gland. (A) Cerebellum, diffuse positivity (in molecular, granular and Purkinje cell layers); (B) cerebellum small/medium-size vessels, positivity in endothelial and smooth muscle cells; (C) adrenal (cortex and medulla); (D) small vessels in periadrenal adipose tissue. Immunoperoxidase, 200× (original magnification). M, molecular layer; P, Purkinje cells; G, granular layer; TI, tunica intima; TM, tunica media; V, vessel.
Citation: Vascular Biology 7, 1; 10.1530/VB-24-0010
RNAscope in situ hybridization
To confirm the presence of DDR4 in HUCVs and to cross-validate the immunohistochemistry technique, we attempted to detect DDR4 messenger RNA (mRNA) using the RNAscope in situ hybridization technique. However, the RNAscope technique failed to demonstrate the presence of any DDR4 mRNA in the positive control tissue (human basal ganglia) (Fig. 5A) and in HUCV (tissue of interest) (Fig. 5B).
Dopamine receptor D4 (DDR4) detection in the human brain (basal ganglia, external positive control tissue), HUCVs and HeLa human cells (external positive control cells) by in situ hybridization (RNAscope® technique): (A) absence of positivity with the RNAscope Hs-DDR4 probe in the basal ganglia; (B) absence of positivity with the RNAscope Hs-DDR4 probe in HUCVs; (C) basal ganglia, negative control probe (for DAPB – ACD); (D) basal ganglia, positive control probe (for HSPPIB – panels ACD); (E) HeLa cells, negative control probe (for bacterial transcript); (F) HeLa cells, PPIB positive control probe; 400× (A, B, C, D), 200× (E and F); original magnification.
Citation: Vascular Biology 7, 1; 10.1530/VB-24-0010
Discussion
The results clearly demonstrated that both HUA and HUV express dopamine D4 receptors, as evaluated by both the selective dopamine receptor agonist A-412997 (30) and by the selective dopamine receptor antagonist sonepiprazole (25), and by immunohistochemistry. However, the pharmacological effects resulting from the activation were distinct depending on the nature of the vessel (artery or vein).
Activation of the D4 receptor by A-412997 caused contractions only in the HUV. The contraction induced by A-412997 was due to D4 receptor activation, since sonepiprazole induced concentration-dependent rightward shifts of the A-412997 concentration–response curve. Indeed, in contrast to the HUA, immunoreactivity for the D4 receptor subtype was found only in HUV smooth muscle cells. Dopamine D4 receptors are expressed in a smooth muscle cell line originating from rat thoracic aorta (31), and in the human placenta (32). Although D4 receptor expression was found in the HUV endothelium and the contractions induced by A-412997 were potentiated by NOS inhibition, it is unlikely that the D4 receptor in the HUV endothelium plays an important role in HUV, since its activation by A-412997 in pre-contracted HUV did not lead to relaxation. Thus, the potentiation observed by NOS inhibition is certainly due to the inhibition of basal release of NO/6-ND. Indeed, the relaxation induced by sonepiprazole in the HUV was insensitive to NO inhibition, confirming that endothelium-derived dopamine plays a major role as a contractile agent in this tissue. In contrast to the HUV, the D4 receptor was expressed only in the endothelium of the HUA, and its activation provoked only relaxation, which was mildly sensitive to NO synthase inhibition. Cross-validation of the immunohistochemistry technique using RNAscope failed to confirm the presence of D4 receptor mRNA in the tested samples. Currently, it is not clear if the DDR4 probe is not technically suitable to detect the human DDR4 mRNA in these tissues or if the quantities of such mRNA are actually too small to be detected by this technique. For instance, one could speculate that DDR4 mRNA may have a very short half-life in the examined tissues. We are currently running some assays with a different probe in other to further investigate this hypothesis. So far, we have ruled out problems involving pre-analytical variables such as mRNA preservation in the FFPE tissue sections used and the quality/stability of the RNAscope detection system (notice that all positive and negative control probes worked fine, as expected), in both human basal ganglia tissue sections (Fig. 5C and D) and HeLa cells (Fig. 5E and F).
It is known that the section of the HUV entering the abdomen has an important role in regulating umbilical venous blood flow. Fetuses can regulate blood blow by changing the diameter of the HUV at the umbilical ring (33). The pEC50 of A-412997 as a contractile agent was similar to that reported for adrenaline (34) and 500 times more potent than dopamine, indicating D4 receptor expression may play a major role in modulating HUV contractility. Dopamine D4 receptors are expressed in the adventitia and adventitia-media border of human pulmonary (35) and coronary arteries (36). It is interesting that, in a prospective analysis of 50 families (patient and both parents), a statistically significant correlation was found between DDR4 gene polymorphism and the risk of pre-eclampsia development (16). It would be important to investigate whether the expression and reactivity of dopamine D4 receptors would vary in HUCVs from pathological pregnancies, such as hypertension, pre-eclampsia or eclampsia.
It is interesting that in the HUA, both the dopamine D4 receptor agonist A-412997 and the D4 receptor antagonist sonepiprazole caused relaxation. The pEC50 for the relaxation of the HUA induced by A-412997 was 9.03 ± 0.10, which is very close to the Ki for the human D4 receptor 5.7 ± 0.3 (30). The D4 receptor in the HUA is located only in the endothelium, and the relaxation is sensitive to L-NAME, indicating that it is due to the release of either NO and/or 6-ND. The D4 receptor antagonist sonepiprazole has a Ki for the human D4 receptor of 10 nM, whereas for D1, D2 and D3 receptors, it is over 2 μM (24). As mentioned above, the D4 receptor was found only in the endothelium, yet the pEC50 for the relaxation of the HUA induced by sonepiprazole was 7.62 ± 0.10. This pharmacological paradox can be explained by the heterodimerization of the dopamine D4 receptor. The contractions induced by dopamine in the HUA are mainly due to activation of D2 receptors (9), and in HEK293T cells transiently co-expressing D2–D4 heterodimers, the D4 receptor agonist caused a leftward shift of the D2R agonist concentration–response curve of ERK1/2 phosphorylation (37). D2R–D4R heteromers have been described in localized in the perisomatic region of frontal cortical pyramidal neurons and their striatal terminals (38). Since endothelium-derived dopamine is considered as a major modulator of the cardiovascular system (15), this pharmacological evidence of expression of dopamine D4 heteromers in the vasculature may have important therapeutic implications.
The strengths of this study rely on the use of both, a selective D4 receptor agonist and antagonist, the use of both HUA and HUV rings, and the supporting immunohistochemical evaluation. The only major technical limitation facing this study is the fact that the immunohistochemistry results could not be confirmed/cross-validated by the RNAscope® technique. This was not entirely unexpected given the fact that the CR between IHC and RNAscope® is reported to be lower (58.7–95.3%) than that between other methods for gene expression investigation (e.g. qPCR and qRT-PCR) and RNAscope® (26). This discrepancy could be due to the different products that each technique measures (protein vs RNA).
Conclusions
The specific distribution of dopamine D4 receptors in systemic arteries indicates that they may have a different role in regulating blood flow through the umbilical-placental circulation.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/VB-24-0010.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was funded by Sao Paulo Research Foundation (FAPESP) grants 2021/14414-8 (JBJ); 2021/13726-6 (VBS); 2021/13593-6 (ATL); 2017/15175-1 (EA) and 2019/16805-4 (GDN). GDN is also in receipt of the National Council for Scientific and Technological Development (CNPq) grant 303839/2019-8.
Acknowledgments
JBJ and VBA thank FAPESP for postdoctoral fellowships (2021/14414-8 and 2021/13726-6). ATL thanks FAPESP for a PhD fellowship (2021/13593-6). EA thanks FAPESP (2017/15175-1). GDN thanks FAPESP (2019/16805-4) and CNPq (303839/2019-8).
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