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¹ Centro de Regulación Celular y Patología J.V. Luco, Instituto Milenio de Biología Fundamental y Aplicada MIFAB, Departamento de Fisiología, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Santiago, Chile

	Abstract

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The expression of purinergic P2Y receptors (P2YRs) along the cord, superficial chorionic vessels and cotyledons of the human placenta was analysed and functional assays were performed to determine their vasomotor activity. Immunoblots for the P2Y₁R and P2Y₂R revealed a 6- to 8-fold increase in receptor expression from the cord to the chorionic or cotyledon vessels. In the cord and chorionic vessels the receptor distribution was mainly in the smooth muscle, whereas in the cotyledon vessels these receptors were equally distributed between the endothelium and smooth muscle cells. An exception was the P2Y₂R at the umbilical artery, which was distributed as in the cotyledon. mRNA coding for the P2Y₁R and P2Y₂R were detected by RT-PCR and the mRNA coding for the P2Y₄R, P2Y₆R and P2Y₁₁R was also identified. Application of 2-MeSADP and uridine triphosphate (UTP), preferential P2Y₁R and P2Y₂R ligands, respectively, resulted in contraction of isolated rings from umbilical and chorionic vessels. The vasoconstriction was blocked in a concentration-dependent manner by 10–100 nM indomethacin or 10 nM GR32191, suggesting the involvement of thromboxane receptors. MRS 2179, a selective P2Y₁R antagonist, reduced the 2-MeSADP- but not the UTP-evoked contractions. Perfusion of cotyledons with 2-MeSADP or UTP evoked concentration-dependent reductions in perfusion pressure mediated by the NO–cGMP pathway. Blockade of NO synthase abolished the vasodilatation and the rise in luminal NO elicited by either agonist. MRS 2179 antagonized the dilatation and rise in luminal NO evoked by 2-MeSADP but not by UTP. In summary, P2Y₁R and P2Y₂R are unevenly distributed along the human placental vascular tree; both receptors are coupled to different signalling pathways in the cord/chorionic vessels versus the cotyledon leading to opposing vasomotor responses.

(Received 22 January 2006; accepted after revision 15 March 2006; first published online 16 March 2006)
Corresponding author J. Pablo Huidobro-Toro: Dept. of physiology, Faculty of Biological Science, P. Catholic University of Chile, PO Box 114-D, Santiago 1, Chile. Email: jphuid{at}bio.puc.cl

	Introduction

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The importance of signalling by extracellular nucleotides has rapidly gained recognition. The identification and characterization of membrane receptors, which are widely expressed in a variety of tissues, was essential for understanding their physiological role. Two families of nucleotide receptors, P2XR and P2YR, have been described in the nervous system, smooth muscles, endothelium, platelets and epithelial cells. A set of seven clones constitute the P2X family of ATP-gated ionic channels. Their amino acid sequences differ notoriously from other ligand-gated ionic channels (North & Surprenant, 2000; Khakh et al. 2001). In addition, cloning has identified eight members of the P2Y receptor family, each coupled to heteromeric G-proteins (Ralevic & Burnstock, 1998; Abbracchio et al. 2003; Fumagalli et al. 2004).

The recent finding that perivascular sympathetic nerves corelease ATP and noradrenaline, as well as ectonucleotidases (Mihaylova-Todorova et al. 2001; Westfall et al. 2002), poses new challenges and highlights the role of nucleotide signalling in sympathetic reflexes. Boarder & Hourani (1998) have summarized the role of extracellular nucleotides and adenosine in the control of blood pressure regulation. The pioneering study by Drury & Szent-Gyorgyi (1929) first demonstrated that a mixture of heart-derived nucleosides and nucleotides lowered systemic blood pressure. Approximately 60 years past before a more detailed knowledge was gained of the mechanisms of action of purines in the vascular system (Burnstock, 1990, 2006). A number of important studies were performed in the rat isolated arterial mesenteric bed, which is a suitable model to study resistance vessels. In this model, nucleotides were shown to act both in endothelial and vascular smooth muscle cells, activating selective receptors. Ralevic & Burnstock (1988, 1991 and 1996), proposed that ATP mediates both relaxant and contractile mechanisms, which are now attributed to P2YR and P2XR, respectively. The finding that ATP, ADP, uridine triphosphate (UTP) and adenosine are released from endothelial cells and platelets (Kunapuli & Daniel, 1998), and that blood pressure is lowered as a result of the relaxation of conductance blood vessels and the microcirculation (Burnstock, 1990; Lewis et al. 2000), further argued for the role of nucleotides in the control of vascular tone. The subsequent cloning and development of selective tools to activate these receptors (Sak & Webb, 2002) allowed the functional identification of P2Y₁R and P2Y₂R in the endothelial cells of the rat mesenteric bed and provided evidence to suggest that they are coupled to the nitric oxide (NO)–cGMP pathway (Buvinic et al. 2002). In addition to endothelial P2Y receptors, P2XR subtypes have been identified in vascular smooth muscles of several blood vessels, including the human saphenous vein and placental vessels (Cario-Toumaniantz et al. 1998; Valdecantos et al. 2003).

Notwithstanding the proposed role of ATP and related nucleotides in vascular beds, little is known about the physiology of purines in human vascular territories, and even less in the human placenta. However, Ralevic et al. (1997) advanced the notion that nucleotides have a dual role in human placental motor tone. Nucleotides are released locally from a variety of vascular beds under several conditions and may reach micromolar and even millimolar concentrations during hypoxia or tissue damage (Forrester & Lin, 1969; Harkness et al. 1984; Lazarowski et al. 2003). Therefore, extracellular nucleotides and their receptors could serve as autocrine and paracrine signals, especially in the placenta, which lacks significant innervation (Walker & McLean, 1971). Previous studies from our laboratory described the presence and pharmacological identification of multiple P2X receptors along the human vascular bed of the placenta (Valdecantos et al. 2003). In addition, we recently described the vasocontractile role of adenosine in human placental vessels using a combination of functional bioassays and molecular biology strategies to identify and define the physiological significance of the adenosine A_2B receptor in placental blood vessels (Donoso et al. 2005).

The aim of this investigation was to expand our current understanding of the role of purinergic signalling in human placental blood vessels by assessing the localization and distribution of the P2Y₁R and P2Y₂R along the human placental vessels and determine their functional role in the placental vasculature. Immunoblots for the P2Y₁R and P2Y₂R show that the expression of these receptors increased 6- to 8-fold from the cord to the superficial chorionic vessels and the vessels in the internal placental cotyledons. We demonstrated that the distribution of these receptors varied as the placental tree descends with, in particular, a shift from smooth muscle to endothelium at the cotyledon, and that this is reflected by vasoconstriction in response to selective receptor agonists in rings from the cord and chorionic vessels and relaxation of the cotyledon vessels. Furthermore, whereas the isolated ring contraction involved the activation of the arachidonic acid cascade with the probable release of thromboxane as the ultimate mediator, in the cotyledons the activation of the P2Y₁R and P2Y₂R was coupled to the NO–cGMP pathway leading to relaxation. The differential expression of the P2Y₁R and P2Y₂R along the human placental vasculature plus the shift in their distribution from smooth muscle to the endothelium as the vascular tree descend to the placental cotyledon, are novel findings that highlight the role of extracellular nucleotides in the control of the human placental vascular tone. Some of these results were presented to the Physiological Society in the joint meeting of the Physiological Society and the Chilean Physiological Society held at Kings College, London, December 2004 (Huidobro-Toro et al. 2005).

	Methods

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Human placentae

Full term placentae from normal pregnancies delivered by vaginal or Caesarean sections were obtained from the maternity unit associated with the Department of Obstetrics and Gynaecology of the School of Medicine at the Catholic University Clinical Hospital, Santiago. The placentae were transported to the laboratory within 5–15 min of childbirth. Immediately thereafter, the placentae were either prepared to perfuse the cotyledons or 2–4 cm sections of umbilical cord or superficial chorionic arteries and veins were dissected to prepare rings for vascular reactivity assessment. For the latter protocols, vessel rings were dissected. Some conserved an intact endothelium, whereas others were manually denuded from the endothelial layer. The Ethics committee from the School of Medicine and Biology approved the protocols and the guidelines for the handling of human material were strictly adhered to. Consent was obtained as requested by the School of Medicine.

P2Y₁R and P2Y₂R immunoblots

Antibodies for the P2Y₁R were generated against the C-terminus of the receptor, as described by Moore et al. (2000). We injected pre-immunized rabbits with the synthetic peptide NH₂-CPEFKQNGDTSL-COOH and the antibodies were characterized by immunoblot using extracts of either wild-type astrocytoma cells or astrocytoma cells stably transfected with the cDNA for the human P2Y₁ receptor. The P2Y₂R was detected using a commercial antibody (Santa Cruz Biotechnology, CA, USA).

Segments (length, 3–4 cm) of second- or third-order chorionic artery and vein were carefully dissected from surrounding tissues, removing all visible trophoblast. Separate vessels were likewise prepared but the endothelial layer was gently removed manually as reported by Valdecantos et al. (2003). Cotyledons were perfused for about 1 h with regular Kiebs-Ringa buffer and next dissected from the whole placenta to isolate the vascular tree from the placental stroma and surrounding trophoblast. The interstitial tissue and trophoblast from a section of the cotyledon were carefully peeled off with tweezers. A 55-s perfusion with 0.1% saponin allowed the removal of the endothelial layer from the dissected cotyledon. The vessels were homogenized with ultraturrax in 0.5 ml lysis buffer plus 2 mg ml^–1 antipain, 1 mg ml^–1 pepstatin, 35 mg ml^–1 phenylmethanesulfonyl fluoride (PMSF) and 2 mg ml^–1 leupeptin. After centrifugation for 20 min at 2500 g, supernatants were separated and stored at –80°C. Protein (50 µg) from each sample was denatured with SDS and ß-mercaptoethanol and separated by SDS-PAGE (8%). Electrophoresed proteins were blotted onto nitrocellulose membranes and were blocked overnight with 10% non-fat dry milk in PBS. Membranes were incubated with rabbit anti-P2Y₁ (1: 500), rabbit anti-P2Y₂ (1: 500) or the mouse anti-actin (1: 5000, AbCam, Cambridge, UK) antibodies. Immunoreactive bands were visualized by incubation with the horseradish peroxidase-conjugated anti-rabbit (1: 10000) or anti-mouse (1: 1000) IgG and detected by enhanced chemiluminescence (ECL). Reactive bands were quantified using the NIH Image 1.62 software (National Institute of Health, USA). Relative amounts of P2Y₁ or P2Y₂ receptors detected in the immunoblot were normalized with respect to actin. For comparisons between the levels of these receptors along the placental vascular tree, we expressed the values as a percentage of the levels obtained for the umbilical vein. For comparisons between the levels of these receptors in each vessel with and without the endothelium, we standardized the values with respect to each vessel with the intact endothelial cell layer.

Placental P2YR mRNA determinations by RT-PCR

We prepared samples as described for immunoblot assays, but tissues were placed in RNA stabilizing solution (RNA Latter, Ambion Inc, Austin, TX, USA) instead of lysis buffer. Total RNA from placental vessels was extracted using the standard procedure as described by Chomczynski & Sacchi (1987). The RT-PCR reaction was performed as described by Valdecantos et al. (2003). The amplification primers for each receptor were: P2Y₁ forward 5'-CCG CCG CCT AAG TCG AG and reverse 5'-GGC ATT TCT ACT TCT AT (Ayyanathan et al. 1996); P2Y₂ forward 5'-CTC TAC TTT GTC ACC ACC AG and reverse 5'-TTC TGC TCC TAC AGC CGA AT (Adrian et al. 2000); P2Y₄ forward 5'-CGT CTT CTC GCC TCC GCT CTC T and reverse 5'-GCC CTG CAC TCA TCC CCT TTT CT (Maier et al. 1997); P2Y₆ forward 5'-CGC TTC GTC TTC TAT GCC AA and reverse 5'-CCA TCC TGG CGG CAC AGG CG (Adrian et al. 2000); P2Y₁₁ forward 5'-CAG CGT CAT CTT CAT CACC and reverse 5'-GCT ATA CGC TCT GTA GGC (Adrian et al. 2000); P2Y₁₂ forward 5'-CCA GAA TCA ACA GTT ATC A and reverse 5'-GTC TGC CTC ATC TCG AAG GAA GG (Hollopeter et al. 2001). The annealing temperatures were: P2Y₁, 50°C; P2Y₂, 56°C; P2Y₄, 58°C; P2Y₆, 58°C; P2Y₁₁, 52°C; and P2Y₁₂, 57°C.

As a control of the endothelium-removal technique, additional protocols involved the detection of selective endothelium and smooth muscle markers. For this purpose we chose human CD31 and human myosin alkaline light chain (MALC) isoform 6, respectively, using the RT-PCR protocol and primers described by Buvinic et al. (2002).

Vascular reactivity assays

To examine the functional activity of the P2Y₁R and P2Y₂R, vascular reactivity tests were performed in isolated ring segments of umbilical and chorionic arteries and veins. Intact segments of arteries and veins from umbilical cord and chorionic vessels were dissected; rings 0.3–0.5 cm in width were carefully prepared as detailed by Racchi et al. (1999) and Valdecantos et al. (2003). Parallel studies were also conducted in endothelium-denuded vessels prepared by gently rubbing the lumen of these vessels with a cotton swab, a procedure that successfully removes the endothelial layer without damaging the adjacent smooth muscles (Valdecantos et al. 2003; Donoso et al. 2005). Rings placed in buffer were maintained at 37°C within a double-jacketed organ bath gassed with 95% O₂–5% CO₂. The composition of the Krebs–Ringer solution used was the same as the buffer described by Buvinic et al. (2002). Isometric muscular tension from the circular layer of the rings was measured by means of a force–displacement transducer connected to a Grass multichannel polygraph.

The general procedures for the performance of concentration–response curve protocols to determine potency and the maximal effect (E_max) of agonists were described by Valdecantos et al. (2003). Briefly, the rings were first challenged with 70 mM KCl, which elicited a sustained contraction within 2–3 min. This nearly maximal muscle contraction was used as a standard to normalize all vasomotor responses. Next, tissues were washed with buffer for 30 min. To test the vasomotor activity of nucleotides, varying concentrations of nucleotides were added randomly to the organ bath to record vasomotor responses and to perform concentration–response studies. These studies were performed in rings precontracted with 75 nM serotonin (5-HT) to rule out the possibility of nucleotide-induced vasodilatation. Comparative studies were performed in non-precontracted tissues. Samples were washed in drug-free buffer for 30 min after each stimulus. Care was taken to test each ring with one application of 0.3 mM ATP; tissues that responded to this challenge with less than 50% of the magnitude of the KCl-induced contracture were discarded (14/235).

Separate protocols were used to examine the mechanism of vasoconstriction evoked by the nucleotides. In the first set of experiments, our aim was to establish the relative potency of ATP and structurally related analogues, including 2-methylthioATP (2-MeSATP), 2-methylthioADP (2-MeSADP), UTP and ,ß-methyleneATP (,ß-meATP). For this purpose, randomised concentration–response protocols were performed in rings from umbilical cord and chorionic vessels precontracted with 75 nM 5-HT. Each ring was used to examine only one nucleotide concentration–response curve. The nucleotide responses were reproducible within 4 h of preparation; thereafter, vasomotor responses declined and the experiment was stopped. Parallel sets of protocols were performed using rings manually denuded of the endothelial layer as described above.

Use of MRS 2179 and the involvement of an arachidonic acid metabolite in the nucleotide-induced vasoconstriction

We next assessed whether 100 nM 2'-deoxy-N⁶-methyl adenosine 3',5'-diphosphate (MRS 2179), a potent and competitive P2Y₁ receptor antagonist (Boyer et al. 1998; Nandanan et al. 2000; Buvinic et al. 2002), blocked the 2-MeSADP or UTP-evoked vasoconstrictions.

In addition, we investigated possible mechanisms and the intracellular pathways associated with the vasomotor response elicited by P2Y₁ and P2Y₂ receptor agonists. To assess whether prostanoids are involved in the vasomotor response of ATP and analogues, we used two complementary pharmacological protocols. In the first set of experiments, we assessed the influence of the non-selective cyclooxygenase1/2 inhibitor, indomethacin (10–100 nM) (Donoso et al. 2005), on the vasoconstrictions elicited by either 2-MeSADP or UTP. For this purpose, rings were challenged with either 10 nM 2-MeSADP or 20 nM UTP following 30 min of tissue incubation with 10–100 nM indomethacin, which was present during the challenge with nucleotide. In a separate set of experiments, we studied whether 10 nM [1R-[1(Z),2ß,3ß,5]]-(+)-7-[5-([1,1'-biphenyl]-4-ylmethoxy)-3-hydroxy-2-(1piperidinyl)cyclopentyl]-4-heptonic acid (GR32191) (Lumley et al. 1989; Hornby et al. 1989), a potent thromboxane receptor antagonist, reduced the vasocontractile responses elicited by either 10 nM 2-MeSADP or 20 nM UTP. The aim of this latter protocol was to evaluate the participation of an arachidonate metabolite in the vasomotor response of ATP and structural analogues. Control protocols also assessed the influence of indomethacin and GR32191 on basal tone.

Quantification of the vasocontractile responses

The contractions elicited by the nucleotides were normalized with respect to the contraction evoked by KCl in each vessel ring studied at the beginning of each experiment; the response was plotted on a concentration–response graph using Graph Pad software (Graph Pad Inc., San Diego, CA, USA). The negative logarithm of the agonist concentration required to elicit the median effective response (EC₅₀) was determined by interpolation from every concentration–response curve used for potency assays.

Perfusion of human placental cotyledons

Immediately upon arrival of the placentae to the laboratory, the tissue was placed in a hard polystyrene dish. The protocol described by Ralevic et al. (1997) and Donoso et al. (2005) was followed. In brief, one of the superficial chorionic arteries was cannulated with P100 tubing and perfused with Krebs solution gassed with 95% O₂–5% CO₂ at 37°C and a flow rate of 4 ml min^–1. Next, the vein of each cotyledon was also cannulated with P150 tubing and the perfusate was collected. Care was taken with each perfused cotyledon to ensure that the outflow of the venular buffer was within at least 90% of the inflow, (i.e. the outflow was not less than 3.6 ml min^–1) otherwise, the preparation was discarded and a separate cotyledon was cannulated. A pressure transducer placed at the entrance of the artery recorded continuously the pressure of the cotyledon; the transducer was coupled to a Grass multichannel polygraph. The cotyledons were routinely perfused for 1 h with drug-free buffer to washout residual blood. Two cotyledons from a same placenta were simultaneously assessed for better use of the human tissue. In some protocols, one cotyledon served as a control for the other, particularly when using receptor antagonists or NO synthase inhibitors.

As a control for the results obtained using the single-sided cotyledon perfusion (Ralevic et al. 1997), a separate set of experiments was compared with those obtained using the dual-perfusion method. In the latter case, we perfused simultaneously the uterine and the chorionic arteries. A set of five placentae was used to assess the vasodilatation and NO outflow evoked by varying concentrations of 2-MeSADP. These results demonstrated 2-MeSADP-induced vasodilatation for the one sided and the dual-perfusion (n = 3) method with EC₅₀ values of 6.3 and 5.0 nM, respectively. The EC₅₀ for the 2-MeSADP-evoked NO production in the dual-perfusion assay was also similar to that of the single-sided perfusion (n = 2). In view of the similar results with the two methods, and considering that the one-sided perfusion technique is much simpler to handle, the rest of the studies were performed using the single-sided procedure as described by Ralevic et al. (1997) and Donoso et al. (2005).

Quantification of nucleotide-induced vasodilatation: concentration–response protocols

To assess the vasodilatation induced by ATP, 2-MeSATP, 2-MeSADP, UTP or UDP, placental cotyledons were precontracted with 0.3 µM 5-HT. When the perfusion pressure reached stable values, the nucleotides were perfused for 4 min with the 5-HT remaining in the buffer. The return to basal pressure was achieved by perfusing buffer without nucleotides or 5-HT for 15 min. Each cotyledon was used to perform a concentration–response protocol for a single nucleotide. The vasodilatation was expressed as a percentage of the contraction obtained with 5-HT, which was generally 50–70 mmHg, following the protocol of Buvinic et al. (2002). Prism GraphPad software was used to calculate by interpolation the nucleotide EC₅₀ values.

Samples for luminal NO and cGMP determinations

To quantify the luminally accessible NO released by varying applications of 1–30 000 nM ATP, 2-MeSATP, 2-MeSADP, UTP and UDP, the nucleotides were perfused for 1 min; tissues were not precontracted in order to avoid NO release by vasoconstriction, as noted by Figueroa et al. (2001). The cotyledon perfusate was collected every minute in 5-ml plastic tubes, from 3 min before and up to 8 min after the perfusion with each P2Y receptor agonist. Each cotyledon was used for a separate and individual nucleotide test. Luminal NO was quantified in the superfusate by chemiluminescence using a Sievers 280 analyser as described by Figueroa et al. (2001) and Buvinic et al. (2002). Results are expressed as the integrated area of the NO peak produced by the nucleotides above basal values (NO in pmol).

Parallel studies were conducted in separate placentae to determine the tissue cGMP production elicited by single concentrations of each agonist. After a 1-min perfusion with 10 µM ATP, 0.1 µM 2-MeSATP, 0.1 µM 2-MeSADP, 10 µM UTP or 1 µM UDP (n = 6–12 for each), the cotyledons were transferred to an ice-cold plate; the cotyledon stroma was gently peeled off with tweezers dissecting the blood vessel, tissues were homogenized in 10% trichloroacetic acid and centrifuged at 4°C for 30 min at 1900 g. The aqueous phase was extracted four times with four volumes of ethyl ether each run. The samples were dried using a speed vac and stored at –20°C for less than 2 weeks, until the radioimmunoassay for cGMP was performed, as described by Figueroa et al. (2001) and Buvinic et al. (2002). To increase the detection of cGMP, all protocols were performed perfusing the placentae with 0.5 mM 3-isobutylmethyl xanthine (IBMX), a non-selective phosphodiesterase inhibitor, from 20 min before and during agonist application. Results are expressed as the increase in cGMP production induced by agonists above the basal values (cGMP in pmol (g tissue)^–1) produced in the cotyledons perfused with 0.5 mM IBMX alone.

Influence of endothelial NO synthase and MRS 2179 on the nucleotide-induced vasodilatation and NO production by placental cotyledons

To assess the influence of the endothelial NO synthase on the production of NO elicited by nucleotides, 2-MeSADP- or UTP-induced vasodilatation concentration–response protocols were performed in 5-HT precontracted tissues, before and after NOS inhibition with 100 µM N-nitro-L-arginine (L-NNA) for 45-min (the L-NNA remained in the buffer during nucleotide application). In addition, the same protocol was used to test the vasomotor effect of 0.1 µM 2-MeSATP (n = 4), 100 µM ATP (n = 4) or 1 µM UTP (n = 4) before and after treatment with L-NNA. After L-NNA treatment, the concentration of 5-HT used to raise the placental vascular tone was reduced from 0.3 µM to 0.1 µM, because tissues become hypersensitive to the vasomotor effect of this vasoconstrictor (Buvinic & Huidobro-Toro, 2001; Buvinic et al. 2002). Parallel experiments assessed the luminal release of NO elicited by either 0.1 µM 2-MeSADP or 10 µM UTP in cotyledons either with or without the NOS inhibitor (n = 4 per assay). In another assay, we further examined whether 100 nM indomethacin influenced the contractile effect of 2-MeSADP in the L-NNA-treated placentae. Results are expressed in mmHg, as the increase in perfusion pressure above the 5-HT-evoked precontraction.

In a separate series of protocols, we assessed the selectivity of the nucleotide-induced vasorelaxation and NO production by perfusing the tissues with 100 nM MRS 2179 for 5 min before and during the application of the nucleotides.

Drugs

ATP trisodium salt, 2-MeSATP tetrasodium salt, 2-MeSADP disodium salt, UTP trisodium salt, UDP disodium salt, ,ß-meATP lithium salt, MRS 2179 diammonium salt, L-NNA, indomethacin, leupeptin, pepstatin, PMSF and antipain were purchased from Sigma, St Louis, MO, USA. Glaxo Smith Kline kindly provided a sample of GR32191. Analytical grade reagents for preparation of buffers were obtained from Merck (Darmstadt, Germany). All molecular biology reagents and buffers were obtained from Gibco (BRL Life Tech., CA, USA).

Statistical analysis

Multiple statistical tests were utilized as appropriate. ANOVA, Wilcoxon's paired test or Student's t test was used to compare the effect of endothelium denudation, simple drug treatment effects or changes in the EC₅₀ values. P < 0.05 was set to establish significance. Dunnett's tables were used as appropriate to compare multiple observations against a common control.

	Results

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Immunodetection of P2Y₁R and P2Y₂R

Blots for the P2Y₁R and P2Y₂R provide evidence for the expression of these proteins in umbilical, chorionic and cotyledon vessel extracts. The density of the P2Y₁R and the P2Y₂R significantly increased 7- to 10-fold from the cord to the superficial chorionic vessels and the cotyledon (Fig. 1), establishing a non-uniform pattern of expression along this vascular territory (ANOVA: F_4,49 = 7.6, P < 0.00015, Fig. 1A; F_4,49 = 9.9, P < 0.000016, Fig. 1B).

View larger version (25K): [in this window] [in a new window]   Figure 1.  The expression of the P2Y1R and the P2Y2R varies along the placental vascular tree Immunoblots show the P2Y1R (A) and P2Y2R (B) in extracts from segments of the umbilical cord artery (UA) or vein (UV), chorionic artery (ChA) and vein (ChV) and cotyledons (Cot) from the same human placenta. The relative expression of these receptors was normalized with respect to actin and expressed in arbitrary units as a percentage of the UV value (n = 9, for each tissue). Representative blots for the receptors and actin were derived from a same placenta and are shown in the top part of the figure. Columns represent the mean values and bars show the S.E.M. *P < 0.05; **P < 0.01 (Dunnett's test).

Removal of the endothelial cell layer did not elicit a significant variation in the levels of the P2Y₁R and P2Y₂R in segments of the umbilical vein, or either the superficial chorionic arteries or veins (Fig. 2, n = 9), suggesting that the P2Y₁R and P2Y₂R are mainly expressed in the vascular smooth muscles rather than in the vessel endothelium. However, endothelial denudation of the umbilical artery resulted in a significant reduction only of the P2Y₂R (Fig. 2). In the vessels of the cotyledon, we consistently observed that removal of the endothelium halved the density of the P2Y₁R and the P2Y₂R in each tissue extract (Fig. 2, n = 9), suggesting that these receptors are expressed in apparently equal proportions in the smooth muscle and in the endothelial cells. The differential receptor localization between smooth muscle and the endothelium, suggests functional differences related to their particular anatomical distribution.

View larger version (25K): [in this window] [in a new window]   Figure 2.  P2Y1R and the P2Y2R immunoblots from vessels with and without endothelium Immunoblots for P2Y1R (A) and P2Y2R (B) were derived from extracts of segments of the same vessels either intact (filled columns) or manually denuded from their endothelial cell layer (open columns). From each placenta, segments of the umbilical cord artery (UA) or vein (UV), chorionic artery (ChA) and vein (ChV) and cotyledons (Cot) were dissected (n = 9). For comparative purposes, the expression of these receptors was normalized against actin and standardized with respect to each vessel with an intact endothelial layer. Columns represent the mean values and bars show the S.E.M. *P < 0.05; **P < 0.01 and ***P < 0.001 (Wilcoxon's paired test).

mRNA for multiple P2YR subtypes detected by PCR

Consistent with the expression of the P2Y₁R and P2Y₂R, PCR determinations confirmed the presence of mRNA coding for these receptors. In addition, we identified the mRNA for the P2Y₄, P2Y₆ and P2Y₁₁ receptors in the superficial chorionic vein and the cotyledon vessels (Fig. 3); similar results were observed in chorionic artery (data not shown). No evidence for the expression of the mRNA coding for the P2Y₁₂ receptor was found.

View larger version (55K): [in this window] [in a new window]   Figure 3.  RT-PCR identification of P2YR mRNA in human chorionic veins and cotyledons Gels show PCR products of the estimated molecular weights corresponding to P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11R. Identical results were obtained in three experiments from separate placentae.

In view of the non-homogenous distribution of P2YR between the smooth muscle and the endothelial cells, we anticipated differences in vascular reactivity. Therefore, bioassays were used to examine the functional responses mediated by P2Y₁R and P2Y₂R activation. 2-MeSADP and UTP, used as preferential ligands for the P2Y₁R and P2Y₂R, respectively, contracted isolated rings from umbilical and chorionic vessels whether precontracted or not, whereas the same nucleotides reduced the perfusion pressure of the cotyledon, supporting the interpretation that these agonists relaxed the cotyledon vasculature. Representative tracings of the 2-MeSADP-evoked vasomotor effects are shown in Fig. 4; similar results were obtained with UTP (data not shown).

View larger version (8K): [in this window] [in a new window]   Figure 4.  Bioassays show that 2-MeSADP contracted cord and chorionic vessel rings and reduced the perfusion pressure of cotyledons Functional bioassays were performed in rings from segments of umbilical artery (UA) and vein (UV) and the chorionic artery (ChA) or vein (ChV) that were precontracted with 75 nM 5-HT (). Representative polygraphic tracings show that 100 nM 2-MeSADP contracted these isolated vessels. In contrast, perfusion with 2-MeSADP reduced the perfusion pressure in the cotyledon, an indication of the nucleotide-evoked relaxation.

Potency studies.  2-MeSADP was slightly less potent than UTP whereas both nucleotides resulted in the same force of contraction in chorionic vein rings (Fig. 5A); their EC₅₀ values were 4.5 ± 1 and 1.3 ± 0.4 nM, respectively (Table 1). The EC₅₀ of ATP was almost 150 µM, whereas 2-MeSATP, a structural analogue less prone to enzymatic degradation, was 10-fold more potent. The E_max for ATP was at least 4-fold larger than that exhibited by 2-MeSADP or UTP (Table 1). The E_max for ,ß-meATP, a preferential P2X receptor ligand, was close to that of ATP, a finding consistent with the interpretation that ATP may also interact with P2X receptors. Very similar results were obtained with several nucleotides in isolated chorionic arteries and umbilical vessels; see data summarized in Table 1.

View larger version (19K): [in this window] [in a new window]   Figure 5.  Characterization of the mechanism of the nucleotide-evoked contraction of chorionic veins A, concentration–response curves for contraction induced by 2-MeSADP and UTP. B, illustrates that while 100 nM MRS 2179 significantly reduced the 10 nM 2-MeSADP-evoked contraction of chorionic veins (n = 5) it did not modify the 20 nM UTP-evoked contractions (n = 6). In addition, 100 nM indomethacin (n = 5) or 10 nM GR32191 (n = 4), significantly reduced the contractions evoked by the two agonists. Symbols and columns are mean values and bars show the S.E.M. *P < 0.05, **P < 0.01 (Dunnett's test).

Influence of the endothelium.  Consistent with a major smooth muscle localization of the nucleotide receptors in the cord and chorionic vessels, the removal of the endothelial layer did not result in an obliteration of the nucleotide-evoked contractions (Table 1). Although we observed tissue-to-tissue variability in these responses, the changes in E_max after endothelium removal may be interpreted to indicate that a fraction of the nucleotide receptors is localized in the endothelial cells.

The contractile responses evoked by 5-HT, used as a control for the nucleotide effects, were not altered by endothelium denudation (data not shown).

Receptor selectivity of the nucleotide-evoked contractions.  In chorionic vein rings, 100 nM MRS 2179 antagonized exclusively the motor responses elicited by 2-MeSADP without affecting the UTP-mediated vasoconstriction (Fig. 5B).

Involvement of the arachidonic acid cascade.  Blockade of cyclooxygenase with 100 nM indomethacin reduced by more than 70% the vasomotor response elicited by either 2-MeSADP or UTP (Fig. 5B). The effect of indomethacin was concentration dependent: 10 nM was inactive but 30 nM caused on average 45% inhibition of the vasomotor response induced by either nucleotide (data not shown). Moreover, 10 nM GR32191, a thromboxane receptor antagonist, inhibited by 70–90% the nucleotide-evoked contraction (Fig. 5B), suggesting that thromboxane A2 is probably the common downstream mediator of the nucleotide-induced vasoconstriction. As controls, neither 100 nM indomethacin nor 10 nM GR32191 significantly reduced the KCl-evoked contractions (data not shown).

Nucleotide-evoked decrease in perfusion pressure of cotyledons

Vasodilator potency of selective P2Y₁R and P2Y₂R agonists.  All nucleotides examined reduced the perfusion pressure of the cotyledons, probably as a result of dilatation of its vasculature. 2-MeSADP was 12-fold more potent than UTP (Fig. 6A and Table 2). ATP was 40-fold less potent than 2-MeSADP, but 2-MeSATP was about as potent as 2-MeSADP, suggesting that the tissue metabolizes a large fraction of the nucleotides. UDP was inactive (Table 2). The E_max for each active nucleotide was comparable and ranged from 55 to 75%. Control experiments demonstrated that two applications of these nucleotides spaced 20 min apart caused comparable vasodilatations (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 6.  Pharmacodynamics of the vasorelaxation and increase in luminal outflow of NO evoked by 2-MeSADP or UTP in the cotyledon vascular bed Concentration-dependent vasodilatation (A) and increase in luminal NO outflow (C) elicited by 2-MeSADP and UTP which were used as P2Y1R and P2Y2R agonists, respectively. 100 nM MRS 2179 only reduced the vasodilatation and the increase in NO outflow evoked by 0.1 µM 2-MeSADP (B and D), whereas blockade of NO synthase with L-NNA abolished the vasodilatation and significantly reduced the outflow of luminal NO evoked by both 0.1 µM 2-MeSADP and 10 µM UTP. Symbols and columns are mean values and bars show the S.E.M. *P < 0.05; **P < 0.01 as compared to the control (Dunnett's test).

Nucleotide-evoked relaxations involve activation of the NO–cGMP pathway.  NO overflow and nucleotide potency. The nucleotide-evoked relaxations were associated with a concentration-dependent rise in luminally accessible NO (Fig. 6C). Consistent with the greater vasodilator potency of 2-MeSADP over UTP, its ability to increase the outflow of luminal NO was correspondingly larger. The maximal NO production elicited by 2-MeSADP was almost double that evoked by UTP (Fig. 6C and Table 2). UDP was almost inactive as a vasorelaxant and NO producer (Tables 1 and 2).

MRS 2179 antagonism of nucleotide-evoked NO overflow.  MRS 2179 selectively reduced the 2-MeSADP-evoked release of luminal NO without affecting the rise in NO production mediated by UTP (Fig. 6D). MRS 2179 alone failed to change basal release of NO.

Blockade of nitric oxide synthase and luminal NO outflow.  Blockade of NO synthase obliterated the vasorelaxant action of the nucleotides as well as the outflow of NO elicited by 2-MeSADP or UTP (Fig. 6B and D), indicating that the rise in the luminal NO outflow is linked to enzymatic activity. L-NNA treatment also blunted the rise in NO elicited by 100 nM 2-MeSATP (from 124.5 ± 29.7 to 16.8 ± 8.6, n = 5, P < 0.001).

cGMP determinations in cotyledons.  In separate assays we determined that 2-MeSATP, UTP and related nucleotides augmented by 2- to 3-fold the tissue production of cGMP (Table 3). UDP consistently elicited a significant rise in tissue levels of cGMP, whereas we were unable to detect a significant output of NO (Tables 2 and 3). Basal levels of tissue cGMP averaged in these vessel samples 237.5 ± 22.7 pmol (g tissue)^–1 (n = 10).

View larger version (16K): [in this window] [in a new window]   Figure 7.  2-MeSADP or ATP, but not UTP, contract the cotyledon vasculature following blockade of NO synthase A, representative set of recordings shows that while 0.1 µM 2-MeSADP relaxed the cotyledon vasculature (left-hand traces), it contracted the cotyledon vessels following pretreatment with 100 µM L-NNA (middle traces). Addition of 0.1 µM indomethacin plus L-NNA (right-hand traces) abolished the 2-MeSADP-evoked contractions. All the recordings were obtained from the same placenta; three separate identical protocols were conducted. The concentration of 5-HT used for precontractions was changed from 0.3 () to 0.1 µM (•) in the presence of L-NNA, because tissues become hypersensitive to the vasomotor effect of this vasoconstrictor. B, the relaxation evoked by 100 µM ATP became a sustained contractile response following perfusion with L-NNA. C, the UTP-evoked relaxation was abolished by L-NNA treatment, but in contrast to in the presence of ATP, it did not evoke contractions.

	Discussion

Top Abstract Introduction Methods Results Discussion References

View larger version (30K): [in this window] [in a new window]   Figure 8.  Schematic model shows the gradient of P2Y1R and P2Y2R expression along the placental blood vessels and its uneven distribution between smooth muscle and endothelial cells The expression of P2Y1R and P2Y2R increase from the cord to the cotyledon. In addition, the distribution of these receptors between the endothelial cell (EC) and the vascular smooth muscle cell (VSMC) is not uniform, but changes gradually along the vasculature. The cord and superficial chorionic vessels express these receptors primarily in the vascular smooth muscle cells, with a lesser proportion distributed in the endothelial cell layer. In contrast, towards the cotyledon the endothelial cells are preferentially enriched in these receptors with a lower receptor density localized in the vascular smooth muscle layer. In addition to a differential localization of these receptors, we also illustrate that the large-sized vessels elicit a contractile response that seems to be due to the synthesis of an arachidonic acid (AA) metabolite, probably thromboxane A2, which activates the thromboxane receptor. In contrast, in the placental cotyledon vasculature, the P2Y1R and P2Y2R are coupled to the NO–cGMP pathway to promote vasodilatation. The VSMCs are represented by a dotted rectangle; contraction or dilatation is depicted by continuous rectangles of smaller and larger size, respectively.

Methodological intricacies are compounded by the lack of selective tools to explore in detail the pharmacology of the P2Y₁R and P2Y₂R. Notwithstanding, we relied on the use of 2-MeSADP and UTP, agonists with a preferential although not absolute affinity for the P2Y₁R and P2Y₂R, respectively. We routinely used MRS 2179 as a selective P2Y₁R antagonist, to confirm the specificity of 2-MeSADP. Although UTP might also activate the P2Y₄R, the finding that UDP, a preferential P2Y₄R/P2Y₆R ligand was almost inactive as a vasodilator, argues against the notion that the P2Y₄ receptor is involved in the UTP-evoked vasodilatation. Moreover, in isolated chorionic vein rings, UDP evoked an almost negligible contraction, reaching less than a third of the maximal effect elicited by UTP or 2-MeSADP, a finding that might be interpreted in favour of the notion that the UTP-mediated contractions are not due to P2Y₄ receptor activation, although P2Y receptor classification by selective agonists remains controversial (von Kugelgen, 2005). We cannot rule out the possibility that some of the nucleotides examined may interact also with P2X receptors, because both ATP and 2-MeSATP elicited contractions that reached maximal effects four to five times larger than those of 2-MeSADP or UTP, even though their potencies were three to five orders of magnitude lower. The latter finding might be interpreted to indicate that at these large concentrations, nucleotides may activate smooth muscle P2X receptors, whose affinities are within three order of magnitude less than required for P2YR activation (Ralevic & Burnstock, 1998). The finding that ,ß-meATP, a potent although not selective P2X₁ receptor ligand, is 20- to 50-fold more potent that ATP (Table 1 and Valdecantos et al. 2003), supports the notion that placental vascular smooth muscle also expresses P2XRs that lead to vasoconstriction. This finding highlights the notion that both the P2YR and the P2XR may play a fundamental role in the regulation of placental blood flow, and that ATP has a dual vasomotor action in this vascular bed, which makes the nucleotides particularly suited to regulate blood flow distribution. As proof of this dual role, in specific protocols we contracted the placental vasculature with ,ß-meATP, and relaxed the cotyledon vessels with either 2-MeSADP or UTP. The notion that nucleotides play a dual role in placental vessels, dilating via endothelial P2YRs and contracting via smooth muscle P2X receptors, is common to other vascular beds, as recently reviewed by Burnstock (2006).

The differential expression of the P2Y₁R and P2Y₂R along the human placenta vessels, reveals that the cord and superficial chorionic vessels express significantly less receptors than the cotyledon vessels. In addition, the distribution of these receptors between muscle and endothelial cells shows a shift towards the endothelium as the placental tree descends; these two concepts are illustrated schematically in Fig. 8. It is interesting that the physiological activity of nucleotides changes along this vascular tree, contracting umbilical and chorionic vessels but dilating cotyledons. To systematically rule out the possibility that 2-MeSADP or UTP relax the cord or chorionic vessels, the bioassays were always performed in rings precontracted with 5-HT, a strategy that favours smooth muscle relaxation. Nevertheless, we repeatedly observed that 2-MeSADP and UTP did not relax, but contracted human placental vessel rings. In addition, the vasocontractile potencies of 2-MeSADP and UTP range between 1 and 5 nM (Table 1), values that are within the affinity of these ligands for P2Y receptors allowing us to rule out major participation of P2XR in this vasomotor response, as the affinity of ATP for the P2XR is in the micromolar range (Khakh et al. 2001). In addition, both the 2-MeSADP-induced vasoconstriction and cotyledon vasorelaxation were sensitive to MRS 2179, which is a pharmacological demonstration that 2-MeSADP activates a common receptor independent of the different transduction mechanisms involved.

The metabolism of arachidonic acid and the role of eicosanoids in the regulation of the human placental blood flow have been addressed by several research groups. The role of the arachidonic acid cascade in the generation of potent and efficacious vasoactive metabolites is of paramount importance for human placental vasomotor tone (Ylikorkala & Makila, 1985; Walters & Boura, 1991; Donoso et al. 2005). We recently demonstrated that prostaglandin E₂ or F₂ (Valdecantos et al. 2003) and even adenosine contracts main conductance placental vessel rings, putatively mediated by the release of thromboxane (Donoso et al. 2005), reiterating the importance of arachidonate metabolites in the regulation of placental vasomotor tone. Consistent with these findings, the blockade of the 2-MeSADP- and UTP-evoked contractions with indomethacin suggested the involvement of cyclooxygenase in the purinergic vasomotor response. Moreover, the vasoconstriction was also abolished using GR32191, a preferential thromboxane receptor antagonist. These results suggest that the activation of P2Y₁R and P2Y₂R, localized in the cord and chorionic vessels, generate prostanoids, which probably act as the ultimate effector at thromboxane receptors (Fig. 8). In contrast, 2-MeSADP or UTP vasodilatate the cotyledons through the activation of the NO–cGMP pathway; nanomolar concentrations elicit a concentration-dependent rise of luminal NO, an effect that is paralleled by an increase in tissue cGMP levels. MRS 2179 blocked the rise in tissue NO elicited by 2-MeSADP but not by UTP, an indication of the selectivity of action of 2-MeSADP on the P2Y₁R.

Blockade of NO synthase in the perfused cotyledon abolished the vasodilatation elicited by either 2-MeSADP or UTP. However, in the case of 2-MeSADP we consistently observed vasoconstriction, an a priori indication that the remnant muscular P2Y₁R becomes unmasked favouring vasoconstriction. It is interesting that the vasoconstriction induced by 2-MeSADP was abolished when the tissue was pretreated with both L-NNA and indomethacin, which is consistent with the notion that muscular P2Y₁R elicited contractions through an arachidonic acid metabolite. Consisitent with this interpretation, no vasoconstriction was evoked by 2-MeSADP following P2Y₁R blockade with MRS 2179, as this antagonist blocked both the endothelial and the muscular P2Y₁R. Similarly, we consistently observed that the ATP-evoked cotyledon vasorelaxation shifted to contraction only after NO synthase blockade. Likewise, 2-MeSATP, an analogue that is 1000-fold more potent than ATP, also elicited dual vasorelaxation/contractile responses after NO synthase blockade, highlighting once again the dual vasomotor action of ATP. In contrast, UTP did not elicit a vasocontractile effect upon blockade of NO synthase. Although we lack an explanation for this finding, for some reason the remnant P2Y₂R in the vascular smooth muscle is not able to promote contractions. Altogether, these findings allow us to conclude that in addition to a gradient of receptor expression along the human placental vasculature and a shift of receptor distribution between smooth muscle and endothelial cells, both the P2Y₁R and P2Y₂R are functionally coupled to different signalling pathways along the placental vascular tree (Fig. 8).

Whereas the endothelium does not seem to play a relevant role in nucleotide-induced vasoconstrictions, these cells are crucial for the nucleotide-evoked cotyledon vasorelaxations. The reduction in the maximal nucleotide-evoked contractions in some cord and chorionic preparations upon endothelial cell removal (Table 1) might reflect the fact that a small fraction of these receptors must be localized on the endothelial cells, as depicted in Fig. 8. Although removal of the endothelial layer in the cotyledon for the perfusion assays was not possible due to a violent rise in the perfusion pressure of all the tissues examined, we relied on blockade of endothelial NO synthase to demonstrate the influence of the NO–cGMP pathway in the cotyledon-evoked vasorelaxation. Regarding the endothelium, although vascular reactivity bioassays gave a negative result with acetylcholine due to the lack of muscarinic receptor expression in this model, we relied on the expression of CD31 mRNA as an endothelial marker. In endothelium-denuded vessels, PCR studies consistently demonstrated the absence of this marker, whereas the signal for the smooth muscle marker MALC was preserved. 5-HT reactivity was essentially unaltered by endothelium removal, indicating that in this model, these receptors must be mainly located in smooth muscle, a finding supported by other authors (Cruz et al. 1997, 1998).

With regard to the physiology of purinergic mechanisms, the dual vasoactive mechanisms of nucleotides in the human placenta offer novel regulatory mechanisms to modulate the vascular tone. It has not escaped our attention that the dual actions of nucleotides are reminiscent of the physiology of several ligands that have dual actions in other vascular beds, such as adrenaline, endothelin and/or angiotensin II. Whether this dichotomy reflects differential receptor densities and signalling pathways in endothelium compared with smooth muscle remains a challenging new possibility. In the same way, adrenaline has a dual vascular action ensuring the contraction of blood vessels via ₁ and ₂ receptors activation and vasorelaxation as a result of ß₂-adrenoceptor activation, part of which may be of endothelial localization (Guimaraes & Moura, 2001). Different vascular beds are known to express different proportions of these adrenoceptors both in the smooth muscle cells (Wier & Morgan, 2003; Briones et al. 2005) and in the endothelial cell layer (Boric et al. 1999). The same pattern of receptor distribution might hold true for the endothelin ET_A and ET_B (Loesch, 2005) receptor subtypes and the angiotensin II AT₁ and AT₂ receptors (Jackson, 2001). As with adrenaline, the presence of P2Yrs in both vascular smooth muscles and in endothelial cells plus their differential intracellular signalling leading to opposing physiological effects highlight their role in regulation of vascular tone. In addition, we need to emphasize the role of several P2XRs in human placental vascular smooth muscles, first demonstrated by Valdecantos et al. (2003). These receptors contract intact rings of cord and chorionic vessels as well as perfused cotyledons.

As placental vessels lack perivascular innervation and sympathetic nerves (Walker & McLean, 1971), the ATP in these vessels must come from the endothelium, muscle cells or adjacent tissues, including the trophoblast or blood cells such as platelets and erythrocytes. Several mechanisms have been proposed to account for the release of ATP, including exocytosis, transporters or simply leakage following cell damage. Furthermore, it is well characterized that ATP and related nucleotides are continuously released from cell cultures as well as from the endothelium by shear stress. Additionally, pathophysiological mechanisms such as ischaemia can exacerbate the release of nucleotides allowing the nucleotides in blood vessels to reach millimolar concentrations (Lazarowski et al. 2003), finding lacking evidences in the human placenta.

After years of uncertainty, it is now widely recognized that ATP is an active extracellular signal. The cloning and pharmacological characterization of at least 15 nucleotide receptor subtypes has allowed the development of tools to study physiology of ATP in multiple in vivo and in vitro systems. Nowadays, ATP is regarded as a novel and widespread communication signal that encompasses physiological processes as varied as development, transmission or co-transmission role, platelet aggregation and blood clotting, and epithelial transport. Clinical opportunities for purinergic drugs are emerging: P2Y₁₂R antagonists are widely used as antiplatelet drugs (Hollopeter et al. 2001; Storey, 2001; Bauer, 2003), while novel P2Y₂/P2Y₄R agonists are in different phases of clinical trials for the treatment of cystic fibrosis (Yerxa et al. 2002; Deterding et al. 2005). The present investigation focused on the human placenta as a model vascular bed to study the physiology of ATP; however, we hope that future studies will address the role of ATP and its receptors in other human vascular beds and address their involvement in vascular disorders. Likewise, the role of ATP in the regulation of placental vascular tone in eclampsia and related vascular diseases during pregnancy and parturition remains a challenge.

In summary, the gradient of P2Y₁R and P2Y₂R distribution in addition to their differential transduction mechanisms along the placental vasculature highlights the role of extracellular nucleotides in the regulation of the human placental vascular tone. Although the physiological determinants of the gradual change in receptor expression between endothelium and vascular smooth muscle along the placental vasculature remain unknown, multiple mechanisms may be responsible for this pattern of distribution. For example, it may be due to ligand availability, receptor stability and half-life in the different cells of the vessel wall, or the influence of shear stress and partial oxygen tension along the placental vascular tree. A final result of the activation of these multiple receptors is the diverse physiological responses that vary in accordance to their expression and coupling to signalling pathways in the different cell types of blood vessels. Altogether the present results show the intricacies of purinergic signalling in the placental vascular tree, the functional significance of these receptors in the maternal–fetal blood flow distribution remains to be elucidated. In this regard, the vascular role of extracellular nucleotides shows physiological redundancy with other, better-explored systems involved in vascular control, such as adrenaline or angiotensin II, which are implicated in the haemodynamics of rest and exercise in health and disease. In view of the fact that nucleotides are ubiquitous signals that are released from a variety of cell types, they may play a primordial role as local regulators of the vascular tone.

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