Endothelium‐derived dopamine modulates EFS‐induced contractions of human umbilical vessels

Abstract Electrical field stimulation (EFS) induces contractions of both snake aorta and human umbilical cord vessels (HUCV) which were dependent on the presence of the endothelium. This study aimed to establish the nature of the mediator(s) responsible for EFS‐induced contractions in HUCV. Rings with or without endothelium from human umbilical artery (HUA) or vein (HUV) were mounted in organ bath chambers containing oxygenated, heated Krebs‐Henseleit's solution. Basal release of dopamine (DA), noradrenaline, and adrenaline was measured by LC‐MS‐MS. Cumulative concentration‐response curves were performed with dopamine in the absence and in the presence of L‐NAME or of dopamine antagonists. EFS studies were performed in the presence and absence of L‐NAME, the α‐adrenergic blockers prazosin and idazoxan, and the dopamine antagonists SCH‐23390 and haloperidol. Tyrosine hydroxylase (TH) and dopa‐decarboxylase (DDC) were studied by immunohistochemistry and fluorescence in situ hybridizations. Basal release of dopamine requires an intact endothelium in both HUA and HUV. TH and DDC are present only in the endothelium of both HUA and HUV as determined by immunohistochemistry. Dopamine induced contractions in HUA only in the presence of L‐NAME. Dopamine‐induced contractions in HUV were strongly potentiated by L‐NAME. The EFS‐induced contractions in both HUA and HUV were potentiated by L‐NAME and inhibited by the D2‐like receptor antagonist haloperidol. The α‐adrenergic antagonists prazosin and idazoxan and the D1‐like receptor antagonist SCH‐23390 had no effect on the EFS‐induced contractions of HUA and HUV. Endothelium‐derived dopamine is a major modulator of HUCV reactivity in vitro.


| INTRODUC TI ON
Electrical field stimulation (EFS) is a technique in which an electrical stimulus is applied uniformly to an isolated tissue in short pulse width waves. 1,2 It may cause tissue contraction or relaxation depending on the mediators released. 1,3,4 The proposed mechanism for EFS in isolated tissues is stimulation of intramural nerve endings. 5 The sodium channel blocker tetrodotoxin is classically used to block neural stimulation. 6 The EFS-induced contractions of the aortae of the snakes Crotalus durissus terrificus and Bothrops jararaca, 7 Panterophis guttatus, 8 and of the tortoise Chelonoidis carbonaria 9 are endothelium dependent and tetrodotoxin insensitive. The EFS-induced contractions of human umbilical cord vessels (HUCV) are also dependent on the presence of the endothelium and are not affected by tetrodotoxin, 10 the latter indicating lack of involvement of nerve terminals. Indeed, the umbilical cord has no innervation since no cholinergic or adrenergic nerve fibers have been identified by fluorescence. 11 The nonselective alpha-blocker phentolamine caused a significant inhibition of EFS-induced HUCV contractions. However, this inhibition was observed only at high concentrations, indicating that it may be acting on a different population of receptors. 10 In this study, the nature of the mediator was identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS), followed by a pharmacological characterization of the EFS-induced contractions in both HUA and HUV in vitro.

| Study participants
Participants over the age of 18, undergoing the natural or cesarean delivery from Santa Casa de Vinhedo (Vinhedo-SP) and Campinas Maternity Hospital (Campinas-SP), were invited to take part in the study. The women were normotensive, and did not have preeclampsia, pregestational, or gestational diabetes mellitus and none were on regular medication. Written consent was obtained from those who agreed to participate. Umbilical cords from 67 volunteers aged

| LC-MS-MS analysis
This study was carried out on human umbilical cord specimens The dopamine, noradrenaline, and adrenaline concentrations in the Krebs-Henseleit's solution were determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The extraction procedure was similar to that described for extracting methyldopa from plasma. 12 Briefly, 100 µL of the internal standards (dopamine-d3, noradrenaline-d6, and adrenaline-d6 at 100 ng/mL) were added to the Krebs' solution (2 mL) followed by 1.5 mL of  (Table 1).

| Immunohistochemistry
This assay was carried out on human umbilical cord specimens ob- Tissue sections were deparaffinized and rehydrated in graded alcohols to distilled water, and then they were incubated in 3% hydrogen peroxide for 10 minutes to block the endogenous peroxidase. Antigen retrieval was performed by heating slides in citrate buffer (10 mm, pH 6.0) at 95°C for 20 minutes (in a steamer set).
Subsequently, they were left to cool down at room temperature and rinsed with PBS. Each slide was then incubated for 2 hours at Negative controls consisted of the omission of the primary antibody and incubation with the primary antibody diluents (as well as with the secondary/tertiary antibodies, where applicable, and the detection system) and were performed in all immunohistochemistry assays (one negative control per section) to identify any background staining. All solutions (including primary, secondary, and tertiary antibody stocks) were prepared for a single use on the same day of the immunohistochemistry assay, and kept at 4ºC until use.
All slides were examined and photomicrographed using a trinocular Eclipse 50i microscope (Nikon) coupled to a 10MP CMOS digital camera (AmScope, EUA). Positivity was assessed by an experienced MD, PhD pathologist (AAS), who was blind to the presence/absence of the primary antibody on the sample under examination (the observer did not know whether a test sample or an omission control was being assessed). Blinding was achieved by covering the slide labels with a removable occluding sticker.
For FISH analysis, sections from 5 randomly selected from the 8 human umbilical cords used for immunohistochemistry were deparaffinized with xylene and rehydrated in graded alcohols for

| Pharmacological experiments
This study was carried out on human umbilical cord specimens ob- The effect of the antagonists was evaluated always in the presence of L-NAME (100 μM). Student's t test was used and a P-value of < .05 was considered as significant.

| Data analysis
Nonlinear regression analysis to determine the pEC 50 was car-

MS-MS
Dopamine, noradrenaline, and adrenaline calibration curves were linear for concentrations of 0.1-10.0 ng/mL, with a correlation coefficient higher than 0.99. The limit of quantification was 0.1 ng/ mL. The method was fully validated, and the results reported elsewhere. 13 Only dopamine concentrations were above the limit of quantification, and were only observed in endothelium-intact HUA and HUV ( Figure 1A and 1B).

| Umbilical cord vessels. Immunohistochemistry and fluorescence in situ hybridization (FISH) analysis
Tyrosine hydroxylase was detected by immunohistochemistry only in endothelial cells, in all samples of both HUA (n = 8; Figure 2A) and HUV (n = 8; Figure 2B). Negative controls were obtained by the omission of the primary antibody, as illustrated for HUA (n = 8; Figure 2C) and HUV (n = 8, Figure 2D).  Figure 4A) and HUV (n = 8; Figure 4B). Negative controls were obtained by the omission of the primary antibody, as illustrated for HUA (n = 8; Figure 4C) and HUV (n = 8; Figure 4D). FISH could not be used to detect dopa decarboxylase mRNA because there were no commercially available probes at the time.
Using immunohistochemistry, we attempted to identify calretinin  Figure 5A) or HUV (n = 8; Figure 5B), which indicates lack of neural tissue within the vessels walls (thus, ruling out a neural origin for the vessel-derived catecholamines detected in the pharmacological assays).
There was no significant difference in the Emax, but the pEC 50 2. and HUV ( Figure 7B).

| Effect of alpha-adrenergic receptor antagonists
Incubation with prazosin (100 µM), a selective α 1 -adrenoceptor antagonist, had no effect in the EFS-induced contraction of the HUA

| Effect of dopamine receptor antagonists
In L-NAME-treated vessels, the dopamine D1-like receptor antago-

| D ISCUSS I ON
The endothelium of umbilical cord vessels is capable of releasing mediators capable of modulating the contractile activity induced by EFS. 9 The results presented here clearly demonstrate, for the first time in human vessels, that HUA and HUV display a basal endothelium-derived dopamine release, as identified by tandem mass spectrometry. Furthermore, the enzymes involved in dopamine synthesis, tyrosine hydroxylase, which is the enzyme responsible for the conversion of tyrosine into L-dihydroxy-phenylalanine (L-DOPA) 14 and dopa-decarboxylase, also responsible for the conversion of L-DOPA into dopamine, 15  In previous studies in the field of primary care, schizophrenic patients and nonschizophrenic patients treated with antipsychotics were strongly associated, after adjusted analysis, with a lesser F I G U R E 6 Dopamine concentrationresponse curves in the absence and presence of L-NAME in HUA rings (Panel A [control n = 6/6 and L-NAME 100 µM n = 6/12]) and in HUV rings (Panel B [control n = 5/5 and L-NAME 100 µM n = 5/10]). Effect of the D1-like receptor antagonist SCH-23390 on the dopamine concentration-response curves in HUA rings (Panel C [L-NAME 100 µM n = 5/10 and L-NAME + SCH-23390 10 μM n = 5/10]) and in HUV rings (Panel D [L-NAME 100 µM n = 5/10 and L-NAME + SCH-23390 10 μM n = 5/10]). Effect of the D2like receptor antagonist haloperidol on the dopamine concentration-response curves in HUA rings (Panel E [L-NAME 100 µM n = 5/10 and L-NAME + haloperidol 10 μM n = 5/10]) and in HUV rings (Panel F [L-NAME 100 µM n = 5/10 and L-NAME + haloperidol 10 μM n = 5/10]). In the six panels, there was a significant difference in the Emax (P < .05) presence of hypertension. 33 This was particularly unexpected since patients affected by schizophrenia have an increased cardiovascular morbidity and mortality. 34 The alpha 1 -adrenergic receptor antagonist prazosin 35 and the alpha 2 -adrenergic receptor antagonist idazoxan 36 had no effect on EFS-induced contraction of HUCV, confirming that dopamine is the major catecholamine responsible for this phenomenon. The inhibition observed with phentolamine at higher concentration is possibly due to binding of phentolamine in D2-like receptors, as previously suggested. 10 Indeed, phentolamine at higher concentrations (>2 µM) displaces 3 H-haloperidol binding to dopamine receptors in calf brain membranes. 37 The interaction between dopamine and NO should not be restricted to their pharmacological actions. Nitro-catecholamines such as nitro-dopamine, nitro-noradrenaline, and nitro-adrenaline have been found in rat brain. 38 Thus, it is possible that endothelium-de- What is the possible physiological role of endothelial-derived dopamine? Although cardiac output is defined by the product of heart rate and systolic volume, it is known that the pumping function of the heart has a permissive role in the determination of cardiac output. 40 Indeed, the cardiac output was largely unaffected by heart rate when subjects were electrically paced. 41 The characteristics of the peripheral circulation such as capacitance and conductance/resistance play a major role F I G U R E 7 EFS caused a contraction in both HUA (panel A [control n = 5/5 and L-NAME 100 µM n = 5/5]) and HUV rings (panel B [control n = 6/6 and L-NAME 100 µM n = 6/6]). The response was significantly potentiated in both HUA and HUV by previous treatment with L-NAME. The incubation with SCH-23390 had no effect on the EFS-induced contractions in either HUA (panel C [L-NAME 100 µM n = 5/5 and L-NAME + SCH-23390 10 μM n = 5/5]) and HUV rings (panel D [L-NAME 100 µM n = 5/8 and L-NAME + SCH-23390 10 μM n = 5/8]). The treatment with haloperidol caused significant reduction in EFS-induced contractions in both HUA (panel E [L-NAME 100 µM n = 5/8 and L-NAME + haloperidol 10 μM n = 5/8]) and HUV rings (panel F [L-NAME 100 µM n = 5/9 and L-NAME + haloperidol 10 μM n = 5/9]). Data are expressed as mean ± SEM *P < .05. Vs control

CO N FLI C T O F I NTE R E S T
The authors of this manuscript declare that they have no conflicts of interest.
Research data are not shared.