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Am J Physiol Heart Circ Physiol 288: H2439-H2449, 2005. First published January 6, 2005; doi:10.1152/ajpheart.00548.2004
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A2B adenosine receptor mediates human chorionic vasoconstriction and signals through arachidonic acid cascade

M. Verónica Donoso, Rodrigo López, Ramiro Miranda, René Briones, and J. Pablo Huidobro-Toro

Centro de Regulación Celular y Patología Prof. J. V. Luco, Instituto Milenio de Biología Fundamental y Aplicada, Departamento de Fisiología, Unidad de Regulación Neurohumoral, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

Submitted 10 June 2004 ; accepted in final form 4 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Because adenosine is a vascular tone modulator, we examined the effect of adenosine and congeners in the vascular reactivity of isolated human placental vessels and in perfused cotyledons. We characterized its vasomotor action and tentatively identified the receptor subtypes and their intracellular signaling mechanisms. We recorded isometric tension from the circular layer of chorionic vessel rings maintained under 1.5 g of basal tension or precontracted with KCl. The relative order of potency of adenosine and structural analogs is consistent with the expression of A2B receptors, 5'-(N-ethylcarboxamido)adenosine (NECA) being the most potent. The maximal contraction ranged from 45% to 60% of the KCl standard response, except for an A2A receptor agonist that did not exceed 15%. Consistently, NECA was 100-fold more potent than adenosine to raise the perfusion pressure of ex vivo perfused cotyledons. In contrast, a selective A3 receptor agonist relaxed precontracted rings of chorionic vessels. Whereas a selective A3 receptor antagonist was ineffective to antagonize adenosine-induced contraction, A2 or A1 receptor antagonists reduced adenosine-induced vasoconstriction concentration-dependently. Denudation of the endothelial layer reduced adenosine- and NECA-induced contractions by 50–70%. Furthermore, indomethacin reduced adenosine- or NECA-induced contractions concentration-dependently in intact and endothelium-denuded rings. A thromboxane receptor antagonist blocked adenosine- and NECA-induced contractions in intact and endothelium-denuded rings, suggesting the involvement of an arachidonic acid metabolite as the mediator of the vasoconstriction. We propose that adenosine A2B receptors mediate the adenosine-induced contraction vasomotor effect in human chorionic vessels and that this involves synthesis of a thromboxane receptor activator or a related prostanoid.

adenosine-induced vasoconstriction; human chorionic vessels; arachidonate metabolite; thromboxane receptor antagonism; placental vasculature

THE ADENOSINE RECEPTOR FAMILY comprises four gene products, identified by pharmacological, biochemical, and molecular biology studies (45). Whereas A1 and A3 receptors are coupled to adenylate cyclase through the Gi/Go proteins and therefore decrease intracellular cAMP, the A2A and A2B receptors are coupled through Gs; the human A3 receptor has also been observed to be coupled to Gq and regulates phospholipase C (8, 9, 11, 51, 52). The diverse physiological effects mediated by the different adenosine receptors present in the central nervous, cardiovascular, and immune systems have been confirmed by studies with transgenic mice lacking these receptors (30). Specific adenosine receptor ligands have emerged (23, 26) and proven critical in tentatively identifying functional receptor subtypes, suggesting their involvement in pathophysiological processes (31). The selectivity of these agonists/antagonists is only relative, however, because the selectivity is lost at large concentrations.

In blood vessels, adenosine derives in large part from nucleotides released from platelets, endothelial cells, and perivascular nerve endings or tissue damage. On tissue ischemia, adenosine may protect the myocardium and blood vessels from reperfusion injury, playing a relevant pathophysiological role (39). Adenosine vasodilatates vessels by acting predominantly on adenosine A2A receptors on vascular smooth muscle cells (19). In mammals, including humans, coronary vessels are dilated by adenosine, which increases heart perfusion, an effect due to the activation of adenosine A2B and A3 receptors (18, 39). In contrast, A1 receptors located in the kidneys mediate a vasocontractile response that ensures a transient and local rise in blood pressure (36). In addition to controlling vascular tone, A2B receptors have been implicated in the regulation of mast cell secretion (14, 34), gene expression (15), intestinal function, and neurosecretion (18, 37). In the ovine and human placental vasculature, arachidonic acid metabolites play a critical role in the regulation of fetal-placental circulation in health and disease (41, 55); although debatable, prostaglandin E2 (46) and even prostacyclin were once considered potent vasoconstrictors (40, 55). The role of adenosine in the placental vessels remains poorly investigated. Reid et al. (47) reported that adenosine has a biphasic response in the ovine fetal placental vasculature, an observation that may indicate the expression of a mixed adenosine receptor population along the placental vasculature or signaling mechanisms at variance. On the other hand, our group recently reported (21, 54) that ATP contracts superficial chorionic vessels from the human placenta. Considering that ATP is rapidly inactivated by releasable ectonucleotidases (56), the possibility exists that even though the placenta lacks sympathetic perivascular nerves, the ATP released to the fetal-placental circulation will be rapidly degraded to adenosine and modulate the ATP vasomotor response.

Because adenosine has been proposed to play a modulator role in the regulation of human vascular tone, we aimed at examining the vascular reactivity of adenosine along the human placental vasculature by ascertaining its vasomotor action and typifying the putative adenosine receptor subtypes that might mediate these effects in isolated human chorionic vessels and intact perfused cotyledons. We further aimed at characterizing the intracellular signaling pathways that may govern the adenosine response in a tissue-specific manner. In view of the paramount role of prostanoids in placental vasculature, we ascertained whether eicosanoids might act as the mediators of the adenosine-induced vasomotor action in human chorionic vessels.

The present vascular reactivity assays and ex vivo cotyledon perfusion protocols show that both endothelial cells and vascular smooth muscles from human chorionic vessels and cotyledons have predominantly A2B receptors, which are coupled to the arachidonic acid cascade and cause a vasoconstriction, mediated apparently by the release of a thromboxane or related prostanoid. RT-PCR studies confirmed the expression of A2B receptors in both endothelial and smooth muscles of these vessels. The finding that placental vessels contract to applications of adenosine, in contrast to most other arteries or veins in which adenosine is known to cause vasorelaxation, illustrates the concept that the vascular reactivity of adenosine varies in humans in a tissue-specific manner.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adenosine Receptor Ligands and Source of These Compounds

Adenosine and related structural analogs such as 5'-(N-ethylcarboxamido)adenosine (NECA), 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine HCl (CGS-21680), N6-cyclohexyladenosine (CHA), 9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)-[1,2,4]triazolo[1,5-c]quinazoline (MRS-1220), serotonin hydrochloride (5-HT), 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid (indomethacin), prostaglandin E2, and prostaglandin F2{alpha} were purchased from Sigma-Aldrich (St. Louis, MO). 1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-{beta}-D-ribofuranuronamide (2-Cl-IB-MECA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) were purchased from Tocris Cookson (Ellisville, MO). ([1R-[1{alpha}(Z),2{beta},3{beta},5{alpha}]]-(+)-7-[5-[[1,1'-biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl)-cyclopentyl]-4-heptenoic acid (GR32191) was provided by Glaxo Wellcome. Analytical grade reagents for buffer preparation were purchased from Merck (Darmstadt, Germany).

Source of Human Placentas

Over 160 full-term placentas from normal pregnancies delivered by vaginal or cesarean section were transported from the delivery room to the laboratory within <20 min after childbirth. The Committee on Ethics of the Medical Research Department approved these protocols and consent forms, and ethical regulations were strictly followed.

Dissection of Placentas to Obtain Rings from Superficial Chorionic Vessels

Immediately after placentas arrived in the laboratory, a 2- to 4-cm segment of second- and third-order superficial chorionic artery and vein vessels were routinely dissected to obtain 0.5-cm rings and mounted on a bath chamber used for vascular reactivity studies as detailed by Valdecantos et al. (54). Most of the protocols were repeated at least four times, using vessels dissected from different placentas each time. Studies on artery or vein rings were performed with intact (E+) or manually endothelium-denuded (E–) vessels (2, 54). Vessel rings were placed in Krebs-Ringer buffer maintained at 37°C within a double-jacketed organ bath chamber bubbled with a 95% O2-5% CO2 mixture. Isometric muscular tension from the circular layer was recorded with a Grass force displacement transducer connected to a Grass 7 multichannel polygraph. Vessel rings were manually adjusted to an artificial tension of 1.5 g, which, as described by Valdecantos et al. (54), is an optimal value obtained from length-tension curves; this tension was maintained throughout the experiment. After an equilibration period of 60 min, with buffer washouts every 15 min, the rings were challenged with three successive applications of 70 mM KCl to evoke the maximal smooth muscle contraction, used to quantify and standardize responses within vessels. This concentration of KCl proved optimal for this caliber of vessels [see Valdecantos et al. (54) and Racchi et al. (44) for human saphenous vein biopsies]. This long-lasting protocol was routinely followed with each vessel ring preparation; it allowed a thorough washout of hormones or drugs that could have been present in the tissues due to parturition or cesarean section surgery.

Vascular Reactivity Bioassays in Intact and Endothelium-Denuded Vessels

Determination of agonist potency. The contractile potency of adenosine, the endogenous ligand for all four adenosine receptor subtypes, and agonists with preferential selectivity for each of the alleged receptor subtypes, CHA (a preferential A1 receptor ligand), CGS-21680 (a rather selective A2A receptor agonist), NECA (a nonselective A2 receptor agonist), and 2-Cl-IB-MECA (recognized as a relatively selective A3 ligand), was assessed by means of noncumulative concentration-response protocols. Varying adenosine concentrations were added directly to the tissue bath chambers in random order within two or three orders of magnitude concentration range. The adenosine applications were spaced every 45 min, a time lag required to avoid desensitization of the adenosine-induced contraction. Parallel control experiments were conducted to examine whether the adenosine vasomotor response decayed over time during the 4- to 5-h course of the experiment. In the particular case of NECA, CHA, or CGS-21680, because the second application caused a much diminished response, a single concentration was assessed per bioassay, i.e., in the concentration-response curve protocols, each data point is derived from separate vessel rings, each obtained from an individual placenta. In every case, at least three to five rings were used to quantify the contractions elicited by NECA, CHA, or CGS-21680. In addition, we also examined whether, in precontracted vessels, adenosine and analogs also elicited contractions. For this purpose, a single concentration of 19 µM adenosine, 57 µM CHA, 37 µM CGS-21680, or 1 µM NECA was also applied in 20 mM KCl-precontracted rings.

In every single bioassay protocol, rings were challenged with a 70 mM KCl standard at the beginning and the end of the experiment, a concentration previously demonstrated to cause the maximal contracture of these vessels (54). This procedure allowed the standardization of the concentration-response protocols described. Furthermore, adenosine concentration-response studies were performed in E+ and E– vessels. Likewise, in the case of NECA, a single concentration was assayed in tissues with and without the endothelial cell layer.

Studies with adenosine receptor antagonists. Intact vessel rings were challenged repeatedly with adenosine at 19 µM, a value close to its EC50, which did not evidence desensitization after successive applications. Each antagonist was examined in a wide range of concentrations (6–1,500 nM); the antagonists were preapplied 10 min before the standard 19 µM adenosine challenge. The results are plots of the percentage of 70 mM KCl values of the vasomotor action of the adenosine challenge vs. the antagonist concentration. Independent protocols, performed in separate vessels, examined the potency of DPCPX, ZM-241385, or MRS-1220 to block the standard adenosine-evoked contractions. These antagonists have relative selectivity for the A1, A2A, and A3 receptor subtypes, respectively. The concentration of these antagonists that halved the adenosine-induced contraction was interpolated from the respective concentration-response curves and expressed as the mean ± SE for each receptor blocker. To assess whether more than one adenosine receptor mediates the adenosine response, additional studies were performed applying 200 nM ZM-241385 and 200 nM DPCPX together.

Blockade of tissue cyclooxygenase and thromboxane receptor. Because eicosanoids often act as mediators of the vasomotor actions elicited by serotonin and other vasoconstrictors in umbilical vessels (3, 7, 13), we assessed the involvement of the arachidonic acid cascade in adenosine-induced vasoconstrictions. For this purpose, tissue cyclooxygenases were blocked with indomethacin (42), a nonselective COX-1/COX-2 inhibitor. The indomethacin concentration necessary to reduce the 19 µM adenosine-induced vasoconstriction was determined in a first set of experiments. Next, chorionic rings were incubated with 100 nM indomethacin for 40 min before the performance of a complete adenosine concentration-response protocol. As control, tissues were maintained in the bath chamber during the same 40 min but without drug treatments, except for the challenge with 19 µM adenosine. Tissue preincubation with 100 nM indomethacin was also used to assess the blockade of the vasoconstriction elicited by 1 µM NECA. Reversibility was examined by challenging the tissues several times until the original contractile response was recovered. Analogous protocols were performed in endothelium-denuded vessels.

In the next series of protocols we assessed whether the thromboxane receptor blocker GR32191 (33) antagonizes the adenosine-induced contraction. Adenosine concentration-response protocols were performed in the absence of GR32191 and then after a 5-min exposure to 10, 70, or 210 nM GR32191. Additional experiments assessed whether 140 nM GR32191 also reduced the vasomotor response elicited by 1 µM NECA in E+ and E– vessels. Separate experiments addressed the specificity of the GR32191-induced blockade of the adenosine-evoked contractions by challenging vessels with 70 mM KCl before and after a 5-min incubation with 140 nM GR32191.

RT-PCR Amplification Studies

Four- to five-centimeter segments of chorionic arteries or veins with and without the endothelial cell layer were placed in RNA stabilizing solution for RNA extraction and PCR studies. Total RNA from each chorionic vessel was extracted by the standard Chomczynski and Sacchi procedure (5). The oligonucleotide amplification primers for each human (h) adenosine receptor subtype and the length of the expected PCR products (in parentheses) were A1: sense 5'-CTCTAGAGATGCCGCCCTCC-3', antisense 5'-CGGAATTCCCAGGGCCAGGA-3' (311 bp) (35); A2A: sense 5'-AGATGGAGAGCCAGCCTCTG-3', antisense 5'-GCTAAGGAGCTCCACGTCTG-3' (427 bp) (20); A2B: sense 5'-GAGCTGATGGAGCACTCGAG-3', antisense 5'-ACACCGAGAGCAGGCTGTAC-3' (342 bp) (22); and A3: sense 5'-ACCCCCATGTTTGGCTG-3', antisense 5'-GCACAAGCTGTGGTACCTCA-3' (361 bp) (29).

The PCR reactions were performed in 25-µl final volumes, containing each primer at 0.5 µM, 2 µl of cDNA, 200 µM dNTPs, 1 U of Taq DNA polymerase, and 1x Taq DNA polymerase reaction buffer. The PCR thermal profile for hA1, hA2A, and hA2B were 3 min at 94°C and 30 cycles of 1 min at 94°C, 1 min at 60°C, and 3 min at 72°C. A final extension at 72°C for 7 min was completed. PCR conditions for hA3 were 2 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C and a last step of 10 min at 72°C. A sample without cDNA was subjected to this protocol as a negative control. The PCR products were separated by electrophoresis in agarose gel and visualized with ethidium bromide staining. The specificity of product bands was confirmed by analysis of the nucleotide sequence with an ABI 3100 Sequencing Automatic Analyzer.

Additional protocols were followed to evaluate the presence of human CD31 and human myosin alkaline light chain (MALC) isoform 6, selective endothelium and vascular smooth muscle markers, respectively. The corresponding primers were obtained from the GenBank database as detailed by Buvinic et al. (4). For this purpose, RNA extracts from chorionic vessels with or without the endothelial layer were utilized. All molecular biology reagents and buffers were obtained from GIBCO-BRL Life Technologies, Promega, and Ambion RNA.

Perfusion of Placental Cotyledons and Assessment of Vascular Resistance

Individual cotyledons were perfused with Krebs-Ringer buffer gassed with 95% O2-5% CO2 through plastic tubing cannulas inserted into the corresponding placental artery. After a 40-min equilibration period, 300 µM adenosine or 10 µM NECA dissolved in buffer was perfused through the arterial cannula for 1 min. Perfusion pressure was monitored continually on a Grass polygraph; the changes in perfusion pressure were recorded through a strain gauge connected to the artery. Generally, for adenosine we tested seven concentrations in four different placentas, whereas for NECA four concentrations were tested; a single agonist concentration was examined per cotyledon, and each concentration was examined in at least four separate placentas. This procedure was determined to be optimal for concentration-response determinations, because we noted in a preliminary series of experiments that a second perfusion of NECA, but not adenosine, performed 25 min later, evoked a markedly reduced contraction. In a further set of three protocols, the vasomotor action elicited by 300 µM adenosine was assessed in cotyledons precontracted with 0.3 µM 5-HT, a procedure that allowed us to discard the idea that adenosine might act as a vasorelaxant in precontracted tissues. Results are plotted as the mean changes in perfusion pressure elicited by both adenosine and NECA.

Data Quantification and Statistical Analysis

The median effective contractile concentration (EC50) was interpolated from each agonist concentration-response curve (at least 4 points defined a curve, and each point was repeated in individual vessel rings from at least 3–8 separate placentas) and served to establish the relative order of potency of adenosine and structurally related analogs; EC50 values are expressed as micromolar. The contraction elicited by each agonist concentration was recorded in grams of tension, normalized according to the KCl standard, allowing comparisons within multiple vessel rings, and plotted in a standard concentration-response curve format. With regard to the relaxant effect induced by 2-Cl-IB-MECA, this effect was quantified by normalizing the percent relaxation of the 20 mM KCl-induced tension; the median agonist concentration required to elicit relaxation was interpolated from each concentration-response curve protocol. In the case of the receptor subtype antagonist studies, the concentration required to halve the contractile response to the standard adenosine challenge (IC50) was interpolated for each antagonist experiment (n = 4).

GraphPad software (GraphPad, San Diego, CA) was used to fit concentration-response curves. Analysis of variance established the statistical significance of several treatments on the concentration-response curves. When necessary, the Student’s t-test tables (paired or nonpaired) were used; Dunnett's tables for multiple comparisons with a single control were likewise used when appropriate. Significance was established by a P value <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Vasomotor Action of Adenosine and Structurally Related Analogs in Intact Vessels

Adenosine contracted isolated chorionic arteries or veins concentration-dependently, reaching slightly more than 40% of the KCl standard contracture. The adenosine EC50 was almost identical in artery and vein and averaged 37 µM (Table 1 and Fig. 1). NECA, a highly potent but nonselective adenosine receptor agonist, was almost 100-fold more potent than adenosine and reached 60% of the KCl contracture, and CHA, a classic A1 receptor ligand, was 2-fold more potent than adenosine and maintained the maximal response; that is, they both reached 40% of the KCl standard contracture, whereas the potency of CGS-21680, a prototype A2A receptor agonist, was indistinguishable from that of CHA, but its maximal contracture did not exceed 15% (Table 1). Furthermore, adenosine and congeners also raised the tension of precontracted vessels; the magnitude of the contraction was similar to that obtained in nonprecontracted tissues (see tracings in Fig. 1), although only 2-Cl-IB-MECA, an alleged A3 receptor agonist, which per se was inactive as a vasoconstrictor, concentration-dependently relaxed KCl-precontracted chorionic vessels (Fig. 2). The potency of 2-Cl-IB-MECA to induce relaxations was 27.5 ± 7 µM (n = 4) in vein rings; a similar value was found in arteries (18.5 ± 8.5 µM n = 3). The maximal relaxation achieved 58.7 ± 7% of the KCl-induced tension (n = 4).


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  		Table 1 Potency and maximal response evoked by NECA, adenosine, and structurally related analogs to contract human superficial chorionic vessels

 


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  		Fig. 1. Potency determinations; concentration-response studies in isolated chorionic artery (ChA) and vein (ChV) rings. Top: adenosine (Ado) contracts a human ChV ring to about the same extent with or without a 20 mM KCl precontracture (horizontal bars), indicating that this agonist does not cause vasorelaxation. Similarly, N6-cyclohexyladenosine (CHA), 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine HCl (CGS-21680, CGS), and 5'-(N-ethylcarboxamido)adenosine (NECA) contract precontracted vein rings; these recordings were obtained from the same ChV ring. Bottom: adenosine and congener concentration-response curves performed in nonprecontracted ChA and ChV ring vessels. Each symbol denotes the mean average tension elicited by each agonist concentration ([agonist]) normalized against the 70 mM KCl standard; error bars indicate SE. Full adenosine concentration-response protocols were performed in 8 separate ChA and ChV vessels; each ring was used to run a full-curve protocol. Concentration-response curves for CHA (n = 5), NECA (n = 5), CGS-21680 (n = 3), and 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-{beta}-D-ribofuranuronamide (2-Cl-IB-MECA; n = 4) were performed in separate ChA and ChV rings. Because the vasomotor responses to these adenosine congeners desensitize, each vessel ring was used to test a separate agonist concentration.

 


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  		Fig. 2. Vasorelaxation evoked by 2-Cl-IB-MECA in precontracted chorionic veins with or without endothelium. Top: representative polygraph tracings show that 36 µM 2-Cl-IB-MECA relaxed a 20 mM KCl-precontracted human ChV (horizontal bars). The relaxation was halved by tissue preincubation with 0.1 µM indomethacin, whereas the cyclooxygenase inhibitor at 1 µM abolished the 2-Cl-IB-MECA-evoked relaxation. This protocol was repeated in 3 different ChV rings obtained from separate placentas. Bottom: 2-Cl-IB-MECA concentration-dependent relaxations in intact (+) and endothelium-denuded (–) chorionic veins (n = 4 for each condition). Symbols indicate the mean values; error bars indicate SE.

 
Successive CHA, CGS-21680, or NECA applications in the range of their EC50 values resulted in a gradual and substantial loss of vasomotor activity (Table 2). In contrast, three or more sequential adenosine applications elicited contractions of similar magnitude (Table 2).


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  		Table 2 Loss of vasomotor responses elicited by successive applications of adenosine analogs, but not adenosine, in rings prepared from superficial human chorionic vessels

 
The KCl standard challenge elicited a significantly larger contraction in chorionic vein than in artery rings [1.54 ± 013 g (n = 29) vs. 0.88 ± 0.08 g (n = 29), P < 0.001]; however, we did not observe significant differences between the KCl contracture in vessels derived from cesarean section placentas vs. vaginal birth placentas, ruling out the influence of drugs used during the surgical and/or anesthetic procedures.

Adenosine-Induced Vasoconstriction Raises Perfusion Pressure of Isolated Cotyledons

Consistent with the finding that NECA is much more potent than adenosine to contract isolated chorionic ring vessels, NECA was at least 100-fold more potent than adenosine to raise the perfusion pressure of human placenta cotyledons (Fig. 3). Consonant with the vascular reactivity observations in the isolated rings from the chorionic vessels, a second NECA application did not cause a rise in the perfusion pressure, in contrast to adenosine, which showed essentially no desensitization (see recordings in Fig. 3). Moreover, paralleling the observations in isolated vessel rings, adenosine caused a further rise in perfusion pressure in precontracted cotyledons (Fig. 3). Interestingly, the magnitude of the rise in perfusion elicited by 300 µM adenosine in noncontracted and precontracted tissues was the same (28.3 ± 4 mmHg; n = 4).



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  		Fig. 3. Adenosine and NECA cause a rise in the perfusion pressure of isolated perfused cotyledons; a concentration dependence study. Top: representative tracings from prototype protocols used to assess the rise in perfusion pressure elicited by cotyledon perfusion with either 300 µM adenosine or 10 µM NECA. Two agonist applications, spaced ~25 min apart, demonstrate NECA- but not Ado-induced vasocontractile desensitization. Inset: representative tracing showing that 300 µM adenosine evoked vasoconstriction even in a cotyledon precontracted with 0.3 µM serotonin (5-HT), providing evidence that adenosine does not relax this preparation. Bottom: adenosine and NECA concentration-response curves. The adenosine concentration-response curves were performed with 4 separate placentas; however, in the experiments with NECA, because of desensitization, each placenta was used to obtain a single data point (n = 4 per agonist concentration). Symbols refer to the mean average increase in perfusion pressure ({Delta}mmHg); bars indicate SE.

 
Additional experiments demonstrated that prostaglandin F2{alpha} elicited a concentration-dependent rise in the perfusion pressure (its estimated EC50 was 3 ± 0.43 µM), whereas the application of 5 µM prostaglandin F2{alpha} evoked a rise in the perfusion pressure of 137.5 ± 12.5 mmHg (n = 3) and that of 0.5 µM 5-HT caused a 91 ± 15 mmHg rise in perfusion pressure (n = 3). Therefore, prostaglandin F2{alpha} is at least 100-fold more potent than adenosine as a human cotyledon vasoconstrictor.

Adenosine Receptor Classification Based on Use of Receptor Antagonists

DPCPX and to a much lesser extent, ZM-241385, concentration-dependently blocked the challenge adenosine-induced contractions. The estimated potency of DPCPX and ZM-241385 to halve the adenosine contraction standard in chorionic arteries was 15 ± 3.7 (n = 6) and 207 ± 42 (n = 3) nM, respectively (Fig. 4). Likewise, in veins, these values were 22 ± 3.8 (n = 6) and 633 ± 84 (n = 3) nM, respectively. MRS-1220 did not significantly block the adenosine-induced contractions in either chorionic arteries or veins [Fig. 4; linear regression r = 0.29 and 0.014 (not significant) in chorionic artery and vein, respectively]. Successive adenosine applications caused a similar vasoconstriction, as exemplified in Table 2, ruling out the idea that the antagonist-evoked reduction of the adenosine vasoconstriction is due to muscular damage or nonspecific metabolic alterations.



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  		Fig. 4. Antagonism of adenosine-induced vasoconstriction by prototype selective receptor subtype antagonists. Adenosine challenges were applied before and 10 min after incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a high-affinity A1 receptor antagonist (n = 6), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385), an alleged A2 adenosine receptor antagonist (n = 3), or 9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)-[1,2,4]triazolo[1,5-c]quinazoline (MRS-1220), a prototype A3 adenosine receptor antagonist (n = 3) in concentrations that ranged from 6 to 3,000 nM. Complete antagonist concentration-response curves were performed in both ChA and ChV rings. Symbols denote the mean average adenosine contraction normalized as % of the respective 70 mM KCl-induced contracture; bars indicate SE. *P < 0.05 compared with nontreated controls, by multiple comparisons against a common control (Dunnett's t-test).

 
In addition, 200 nM ZM-241385 reduced the magnitude of the contractions induced by the 19 µM adenosine standard in artery rings by 47% [from 18.7 ± 2.2% of the KCl contracture (n = 21) to 8.7 ± 1.6% of the maximal KCl response (n = 7); P < 0.05], 200 nM DPCPX caused a 69% reduction of the response (from the control value to 5.8 ± 1.4% of the maximal KCl response; n = 12; P < 0.01). However, the coapplication of both antagonists only reduced the adenosine response by 77% (4.3 ± 0.7%; n = 4; P < 0.05), a value that is not significantly different from that attained by the separate application of each antagonist. Similar findings were also observed in chorionic vein rings (data not shown).

RT-PCR Products Identify Adenosine Receptor mRNAs

RT-PCR further identified the mRNAs coding for multiple adenosine receptors. In E+ and E– vessels, RT-PCR products identified the mRNAs coding for the human adenosine A2A, A2B, and A3 receptors in chorionic arteries or veins (Fig. 5). In addition, RT-PCR products for the A1 receptor were detected only in E+ veins (Fig. 5). The removal of the endothelial cell layer was confirmed by the loss of the CD31 marker, whereas in the presence of the MALC, a smooth muscle tracer was preserved in the absence of endothelium (Fig. 5). This experiment was repeated with mRNA extracted from three separate placentas.



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  		Fig. 5. Identification of the mRNAs coding for the adenosine receptor subtypes by RT-PCR analysis. Gels show RT-PCR products of the expected sizes corresponding to the 2 A2 receptors (A2A = 427 bp and A2B = 342 bp) in chorionic artery and vein vessels with (E+) and without (E–) endothelium, whereas the A1 receptor mRNA (311 bp) was detected only in veins with an intact endothelial cell layer; the product for the A3 receptor (361 bp) was detected in intact and endothelium-denuded vessels. As controls, smooth muscle RT-PCR products for myosin alkali light chain (MALC) were observed in vessels with and without endothelium. In contrast, CD31, the endothelium cell marker, was not evidenced on manual endothelial denudation of the rings. Similar results were obtained in 3 gels prepared with total RNA extracts obtained from 3 different placentas. MW, molecular weight.

 
Role of Endothelium

Removal of the endothelial cell layer reduced by about two-thirds the adenosine- and NECA-induced contractions. Endothelial cell removal caused a flattening and downward shift of the adenosine concentration-response curve in both artery and vein rings (P < 0.01, n = 7; Fig. 6). The NECA-induced contraction was likewise more than halved after endothelium removal (P < 0.05; Fig. 6). Acetylcholine, a widely accepted endothelium-dependent vasodilator, is used to functionally evaluate the role of the endothelium in vascular reactivity assays. However, it did not relax the intact isolated chorionic vessels; in fact, 1–10 µM acetylcholine contracted one-third of chorionic rings. Likewise, 10–100 µM acetylcholine failed to relax precontracted perfused human placental cotyledons. Therefore, because the acetylcholine challenge was not useful as a functional test of endothelium denudation, the absence of CD31, revealed in the RT-PCR studies, confirmed in this case the efficacy of the endothelium removal procedure.



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  		Fig. 6. Endothelium influences adenosine- and NECA-evoked vasoconstrictions. Top: representative polygraph tracings of the contractions evoked by 6.4 and 19 µM adenosine in a ChA ring with endothelium (left) and an adjacent segment ring that was endothelium denuded (right). Middle: adenosine concentration-response curves in ChA (left) and ChV (right) rings with and without endothelium. Symbols denote the mean average normalized tension; bars indicate SE. Each complete concentration-response curve protocol was repeated in 7 intact endothelium rings and in 7 rings without endothelium obtained from separate ChA and ChV, each derived from individual placentas. Bottom: 1 µM NECA-evoked contraction in ChA and ChV is also partially sensitive to endothelium denudation (*P < 0.05). Each experiment was replicated in 9 vessel rings with endothelium and 4 rings without endothelium from both ChA and ChV vessels; each preparation was derived from a separate placenta. Columns represent the mean average tension; bars indicate SE.

 
Additionally, the 2-Cl-IB-MECA-induced relaxation was independent of the endothelium (Fig. 2). Removal of the endothelial cell layer did not significantly modify the potency or the maximal relaxation evoked by this agonist in isolated chorionic veins [15.4 ± 2.4 (n = 4) vs. 27.5 ± 7 µM (n = 4)].

Involvement of Arachidonic Cascade in Adenosine- and NECA-Evoked Contractions

In view of the fact that arachidonate metabolites have a preponderant role in vascular tone regulation, we assessed the putative role of eicosanoids as mediators of adenosine-evoked contractions.

Cyclooxygenase inhibition. Indomethacin concentration-dependently inhibited adenosine-evoked contraction in both chorionic artery and vein rings (Fig. 7). Indomethacin blockade was reduced by 30–60% after a 120-min washout period (Fig. 7). Moreover, 100 nM indomethacin obliterated and flattened the adenosine concentration-response curve in both artery and vein rings (Fig. 7). Likewise, 100 nM indomethacin reduced by 92 ± 4% (P < 0.01) the 1 µM NECA-induced contraction in either vessel (Fig. 7, inset). Moreover, 100 nM indomethacin also reduced by 80% the 1 µM NECA-evoked vasoconstriction in vessel rings denuded of the endothelial layer (P < 0.01; n = 3). Furthermore, 2-Cl-IB-MECA-evoked relaxation was also blocked in a concentration-dependent manner by 0.1–1 µM indomethacin (see recordings in Fig. 2).



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  		Fig. 7. Indomethacin blocks adenosine-induced vasoconstriction, involving the participation of the arachidonic acid cascade. Top: tissue preincubation with indomethacin, a cyclooxygenase inhibitor, for 40 min concentration-dependently reduced the standard 19 µM adenosine-induced contractions in intact ChA (n = 4, left) and ChV (right); 100 nM indomethacin obliterated the adenosine response (*P < 0.05, Dunnett's tables). The indomethacin-induced blockade is partially reversible after 120 min of drug washout (open columns) (*P < 0.05 compared with the 100 nM indomethacin). Inset: 1 µM NECA-induced contraction was also significantly reduced by 100 nM indomethacin (Indo) in either ChA or ChV vessel rings (**P < 0.01; n = 3). Columns indicate the mean average of the normalized tension; bars indicate SE. Bottom: 100 nM indomethacin flattened the adenosine concentration-response curve, which was performed with 13 control rings and 3 separate indomethacin-treated rings. Symbols indicate the mean average normalized tension; bars indicate SE.

 
Thromboxane receptor blockade. The thromboxane inhibitor GR32191, which per se did not alter the basal tone of the rings, concentration-dependently reduced the vasoconstriction evoked by adenosine (P < 0.05 in arteries and veins), whereas 70 nM GR32191 halved the vasoconstrictions and increasing the concentration to 210 nM obliterated the adenosine concentration-response curve (Fig. 8).In separate experiments, 140 nM GR32191 reduced by 70–90% the 1 µM NECA-induced vasoconstriction (P < 0.05) in artery or vein chorionic rings. Likewise, GR32191 abolished the 19 µM adenosine- and 1 µM NECA-induced vasoconstriction in endothelium-denuded vessels (data not shown). In separate control protocols, 140 nM GR32191 reduced only by 8.7% the 70 mM KCl-evoked contractions (n = 4), evidencing antagonist selectivity. Previous studies from our laboratory (54) showed that the EC50 for prostaglandin F2{alpha} is 0.09 ± 0.004 µM, a value 400-fold more potent than adenosine, with a maximal effect that doubled that elicited by the KCl standard challenge. The present results show that prostaglandin E2 is also a constrictor of isolated chorionic vein segments; although its EC50 is 170-fold less than prostaglandin F2{alpha} (15.5 ± 2.1 µM; n = 4), it is still 2.4-fold more potent than adenosine.



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  		Fig. 8. Involvement of thromboxane receptors in adenosine and NECA-induced vasoconstriction. Top: adenosine concentration-response curves were performed in ChA and ChV rings in the absence (n = 10) or the presence of 10, 70, or 210 nM ([1R-[1{alpha}(Z),2{beta},3{beta},5{alpha}]]-(+)-7-[5-[[1,1'-biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl)-cyclopentyl]-4-heptenoic acid (GR32191; n = 3 per antagonist concentration). Symbols indicate the mean average contraction normalized against the 70 mM KCl standard; bars indicate SE. Bottom: ring preincubation with 140 nM GR32191 significantly (*P < 0.05) reduced the 1 µM NECA-evoked contractions. Left: data from ChA rings. Right: data from ChV rings. These protocols were repeated 4 times in vessels obtained from separate placentas. Columns indicate mean average tension; bars indicate SE.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present results support the hypothesis that the adenosine-induced vasoconstriction of human chorionic vessels is mediated by the activation of A2B receptors, an effect that appears to be related to the synthesis of an arachidonate metabolite that ultimately activates a thromboxane receptor in the vascular smooth muscles. Figure 9 summarizes and schematizes this proposal, indicating that adenosine contracts chorionic vessels via the activation of a dominating population of adenosine A2B receptors located in both endothelial and vascular smooth muscle cells. These receptors are coupled to the synthesis of an arachidonate metabolite, likely thromboxane A2, which might activate a thromboxane TP{tau} receptor, as the final effector of the adenosine contractile response. At present we cannot exclude the possible participation of related prostanoids. The mechanism that links A2B receptor signaling to phospholipase activity, and thereafter activation of tissue cyclooxygenase, remains to be further described. The present results highlight for the first time a role for adenosine as an extracellular signal involved in blood flow regulation in the human placental vasculature and links its transduction mechanism to an arachidonic acid cascade metabolite.



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  		Fig. 9. Heuristic model illustrates the localization and signal transduction cascade of the adenosine receptors in human placental vessels that cause vasoconstriction. Adenosine A2B receptors are localized in both the endothelial and vascular smooth muscle cells of human chorionic blood vessels. In both cell types these receptors appear to be coupled through a G protein (G) to the activation of phospholipase A2 (PLA2), releasing arachidonic acid, which gives rise, via the activation of the cyclooxygenase pathway (COX), to the synthesis of thromboxane A2 (TxA2), which activates the thromboxane receptor (TP{tau}). We propose that a thromboxane is ultimately responsible for the adenosine- or NECA-induced contractions in human chorionic vessels and placental cotyledons with and without endothelium, although we cannot discard the idea that a related prostanoid may act as a mediator.

 
Adenosine contracted isolated chorionic arteries and veins and, moreover, increased the perfusion pressure of cotyledons, a finding that does not imply, but suggests, commonalities in receptor distribution along the placental vasculature. We are aware that vasoconstrictors such as angiotensin II or norepinephrine do not have the same responses along the whole fetal-placental circulation, at least in the sheep, a common animal model used to study cardiovascular development. Almost 20 years ago, Adamson et al. (1) showed that angiotensin II increased umbilical artery resistance in the sheep, whereas no change was detected in cotyledon or venous resistances. Likewise, norepinephrine had no effect on cotyledon vascular resistance but constricted downstream vessels (1). Radiolabeling binding studies further confirmed the data of Adamson et al. (1) and established that only umbilical vessels expressed type 1 angiotensin II receptor (6, 50). In addition, the density of angiotensin II receptors in placental primary/secondary stem vascular tissue depends on whether the delivery is vaginal or by cesarean section (25), a finding that has pathophysiological consequences because it depends on the circulating levels of angiotensin II, which change with type of delivery. Furthermore, 2-Cl-IB-MECA, an alleged adenosine A3 receptor ligand, relaxed human chorionic vessels, an effect that is only detectable in precontracted rings and that also appears to involve the activation of the arachidonate cascade. Reviriego et al. (49) and Huidobro-Toro and Valdecantos (21) previously reported that 500 µM adenosine is required to vasodilatate precontracted human chorionic vessels, an indication of a dual mechanism of adenosine on human chorionic vessels. We infer that in the human superficial chorionic vessels, although adenosine might vasodilatate, this effect is hindered by the more potent adenosine A2B receptor-mediated vasoconstriction, revealing that multiple adenosine receptors might regulate the placental vascular tone, a conclusion consonant with the findings of Reid et al. (47), who first observed the biphasic action of adenosine while studying the physiology of ovine placental vessels.

For a comparison, we have not overlooked the fact that adenosine contracts the human bronchia, an effect that is also indirect because adenosine activates mast cell adenosine A2B receptors (53), triggering the release of a variety of chemical mediators, including prostanoids of the leukotriene family. In addition, Nicholls et al. (38) reported that adenosine and congeners contract the rat duodenum muscularis mucosae, in contrast to the rat duodenum longitudinal muscle that is relaxed by adenosine. In common with the present findings, Nicholls et al. (38) inferred that this adenosine-induced contraction is mediated through the preponderant activation of A2B receptors.

Several arguments substantiate our hypothesis that the adenosine-induced vasoconstriction in human placental vessels is mediated predominantly by activation of the A2B receptor. The finding that CGS-21680, a prototype A2A receptor agonist, was inactive at 1 µM suggests that the A2A receptor may be ruled out (28). Furthermore, the potency of NECA is within the range of the potency described for this agonist as an A2B receptor agonist (17). Likewise, and by the same argument, we may also discard the influence of A3 receptors, because 2-Cl-IB-MECA, a high-affinity A3 receptor agonist (27), was inactive as a vasoconstrictor and instead induced relaxations. With regard to the role of A1 receptors, we conclude that the large CHA concentrations required to contract chorionic vessels may be mediated by a nonspecific interaction with the A2B receptor (15). Furthermore, the use of receptor antagonists is consonant with our proposal. In the absence of a selective A2B receptor antagonist, we examined the blockade of the adenosine-induced vasoconstriction with DPCPX, ZM-241385, and MRS-1220. The potency of DPCPX is four- to sixfold lower than what is described for the human A1 receptor, but closer to its potency as an A2B receptor antagonist (28). ZM-241385, an alleged selective and potent A2A receptor antagonist (43), was found to be even less potent than DPCPX, indicating that the nature of the ZM-241385-induced antagonism could be related to a nonspecific blockade of the alleged A2B receptor (32). Confirming the existence of a single adenosine receptor population, and that both antagonists act at a common site, the joint application of ZM-241385 and DPCPX did not result in an increased antagonism. Furthermore, MRS-1220, a specific A3 receptor antagonist (24), did not block the adenosine-induced contraction.

Consistent with this interpretation, RT-PCR studies identified the A2B receptor subtype mRNA in these vessels. A1 receptor mRNA was found only in intact veins, suggesting that the mRNA might be further restricted to endothelial cells. We are aware that the mRNAs for the A1 and A2A receptors were detected by this procedure; however, at present we have no indication for a functional role of these receptors in the placental vasculature, except for the mRNA coding for the A3 receptor, which might be linked to vasodilatation.

Regarding the intracellular signaling mechanisms of adenosine in human chorionic vessels, the present results might be interpreted to indicate that arachidonate metabolites are in all likelihood the mediators of the adenosine effects in the placental vasculature. Furthermore, we infer that the thromboxane TP{tau} receptor (3, 10, 33) is the final effector involved in the adenosine-induced contraction. The present results highlight the preponderant role of arachidonate metabolites, such as prostaglandin E2, prostaglandin F2{alpha}, and likely thromboxane A2, in the control of the placental vascular tone (40, 41). Furthermore, the present data demonstrate the potent and efficacious contractions mediated by prostanoids in isolated vessels as in perfused cotyledons, a finding that further supports their role as mediators of the adenosine contractions. Why the adenosine-induced contractions do not desensitize, in contrast to the rapid loss of response observed with NECA, CHA, or CGS-21680, remains an open question. However, we recognize that the lack of desensitization of the adenosine responses allowed us to study its physiology in human chorionic vessels.

An issue that has gained increasing interest within the past few years, and that may have profound implications for this research and clinically oriented pathophysiological studies, refers to the discovery that tissue hypoxia upregulates the expression of the adenosine A2B receptor while decreasing the mRNA for the A2A receptor (12, 16) in both human umbilical vein endothelial cells and bronchial smooth muscle cells. Because the placentas used in this study were not oxygenated in the 20-min period that elapsed between child delivery and laboratory manipulation, we are aware that some degree of modulation might have occurred and may have a definitive influence on our vascular reactivity studies. The group of Biaggioni (16) demonstrated that 3 h of hypoxia sufficed to triple the mRNA for the adenosine A2B receptor in endothelial cells from the umbilical vein, while it reduced by almost 90% the mRNA coding for the A2A receptor. However, it is difficult at present to evaluate the sole influence of this regulatory mechanism in vascular reactivity.

Although the present research was not aimed at determining the origin of the local placental adenosine production, it is important to keep in mind that adenosine in blood vessels derives from platelet release, endothelial cells, nerve endings, tissue damage, and so forth. These processes may be exacerbated by a variety of stimuli, including, among others, ischemia, local hypoxia, and platelet aggregation. As to the physiological role that adenosine may play in fetal adaptation, the present results indicate the importance of the adenosine receptors in the regulation of fetal-placental circulation in health and disease states, including eclampsia and related diseases.

It remains a challenge to examine whether there is a common intracellular signaling mechanism that mediates the A2B receptor activation in different tissues, confirming the generality of our proposal. Adenosine is known to cause a transient rise in blood pressure, a finding that appears unique to the kidney microcirculation and involves A1 receptor activation (36). Although at present we cannot distinguish whether the indirect adenosine-evoked vasocontractile mechanism operates exclusively in human placental vessels, the vasomotor response developed by serotonin in placental vessels is also indirect in nature and dependent on the synthesis of an arachidonate metabolite (7, 13), suggesting that in the human placenta, which lacks sympathetic innervation (48), the arachidonic acid cascade has a major role in mediating the vasomotor regulation discussed for adenosine. The pathophysiological implications of the present findings for preeclampsia and related vascular diseases during pregnancy are open for future research.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded by Fondo Nacional de Desarollo Áreas Prioritarias-Biomedicine Grant 13980001; the Instituto Mileniode Biología Fundamental y Aplicada also contributed to the funding of this investigation. R. López did an extended summer rotation at the Department of Physiology as a medical student and was funded by local intramural grants.


   ACKNOWLEDGMENTS
 
We are indebted to Prof. Drs. A. Germain and J. Carvajal and the personnel of the Clinical Hospital Maternity Ward for help with the expeditious handling of the placentas. The collaboration of the personnel of the Obstetrics Department and the fellows of the maternity ward at the Medical School of the Pontifical Catholic University is much appreciated. The thromboxane receptor antagonist GR32191 was a kind donation from Dr. P. Humphrey, Glaxo-Wellcome UK. The editorial assistance of M. Brenda Watt and the review of the manuscript by Prof. E. Contreras are greatly appreciated.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. P. Huidobro-Toro, Dept. of Physiology, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago 1, Chile (E-mail: jphuid{at}genes.bio.puc.cl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adamson SL, Morrow RJ, Bull SB, and Langille BL. Vasomotor responses of the umbilical circulation in fetal sheep. Am J Physiol Regul Integr Comp Physiol 256: R1056–R1062, 1989.[Abstract/Free Full Text]
  2. Arens YH, Rosenfeld CR, and Kamm KE. Maturational differences between vascular and bladder smooth muscle during ovine development. Am J Physiol Regul Integr Comp Physiol 278: R1305–R1313, 2000.[Abstract/Free Full Text]
  3. Boersma JI, Janzen KM, Oliveira L, and Crankshaw DJ. Characterization of excitatory prostanoid receptors in the human umbilical artery in vitro. Br J Pharmacol 128: 1505–1512, 1999.[CrossRef][ISI][Medline]
  4. Buvinic S, Briones R, and Huidobro-Toro JP. P2Y1 and P2Y2 receptors are coupled to the NO/cGMP pathway to vasodilate the rat arterial mesenteric bed. Br J Pharmacol 136: 847–856, 2002.[CrossRef][ISI][Medline]
  5. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]
  6. Cox BE and Rosenfeld CR. Ontogeny of vascular angiotensin II receptor subtype expression in ovine development. Pediatr Res 45: 414–424, 1999.[ISI][Medline]
  7. Cruz MA, Gallardo V, Miguel P, Carrasco G, and Gonzalez C. Serotonin-induced vasoconstriction is mediated by thromboxane release and action in the human fetal-placental circulation. Placenta 18: 197–204, 1997.[CrossRef][ISI][Medline]
  8. Cunha RA. Adenosine as a neuromodulator and as a homeostatic regulator in the nervous system: different roles, different sources and different receptors. Neurochem Int 38: 107–125, 2001.[CrossRef][ISI][Medline]
  9. Cunha RA, Constantino MD, and Ribeiro JA. G protein coupling of CGS 21680 binding sites in the rat hippocampus and cortex is different from that of adenosine A1 and striatal A2A receptors. Naunyn Schmiedebergs Arch Pharmacol 359: 295–302, 1999.[CrossRef][ISI][Medline]
  10. Dogné JM, de Leval X, Delarge J, David JL, and Masereel B. New trends in thromboxane and prostacyclin modulators. Curr Med Chem 7: 609–628, 2000.[ISI][Medline]
  11. Dunwiddie TV and Masino SA. The role and regulation of adenosine in the central nervous system. Annu Rev Neurosci 24: 31–55, 2001.[CrossRef][ISI][Medline]
  12. Eltzschig HK, Ibla JC, Furuta GT, Leonard MO, Jacobson KA, Enjyoji K, Robson SC, and Colgan SP. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J Exp Med 198: 783–796, 2003.[Abstract/Free Full Text]
  13. Ezimokhai M, Aloamaka CP, Osman NA, Mensah-Brown EP, and Morrison J. The role of the vascular endothelium in the contractile responses of human chorionic plate artery in pre-eclampsia to prostaglandin F2{alpha}, 5-hydroxytryptamine, and potassium chloride. Res Exp Med (Berl) 195: 171–182, 1995.[Medline]
  14. Feoktistov I and Biaggioni I. Adenosine A2B receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma. J Clin Invest 96: 1979–1986, 1995.[ISI][Medline]
  15. Feoktistov I and Biaggioni I. Adenosine A2B receptors. Pharmacol Rev 49: 381–402, 1997.[Abstract/Free Full Text]
  16. Feoktistov I, Ryzhov S, Zhong H, Goldstein AE, Matafonov A, Zeng D, and Biaggioni I. Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype. Hypertension 44: 649–654, 2004.[Abstract/Free Full Text]
  17. Fozard JR, Baur F, and Wolber C. Antagonist pharmacology of adenosine A2B receptors from rat, guinea pig and dog. Eur J Pharmacol 475: 79–84, 2003.[CrossRef][ISI][Medline]
  18. Gharib A, Delton I, Lagarde M, and Sarda N. Evidence for adenosine A2b receptors in the rat pineal gland. Eur J Pharmacol 225: 359–360, 1992.[CrossRef][ISI][Medline]
  19. Grbovic L and Radenkovic M. Analysis of adenosine vascular effect in isolated rat aorta: possible role of Na+/K+-ATPase. Pharmacol Toxicol 92: 265–271, 2003.[CrossRef][ISI][Medline]
  20. Hinschen AK, Rose'Meyer RB, and Headrick JP. Adenosine receptor subtypes mediating coronary vasodilation in rat hearts. J Cardiovasc Pharmacol 41: 73–80, 2003.[CrossRef][ISI][Medline]
  21. Huidobro-Toro JP and Valdecantos P. Purinergic vasocontractile mechanisms in human chorionic vessels. J Physiol 523P: 102P–103P, 2000.
  22. Iwamoto T, Umemura S, Toya Y, Uchibori T, Kogi K, Takagi N, and Ishii M. Identification of adenosine A2 receptor-cAMP system in human aortic endothelial cells. Biochem Biophys Res Commun 199: 905–910, 1994.[CrossRef][ISI][Medline]
  23. Kie -Kononowicz K, Drabczynska A, Pe

    {ecedil}

    cala E, Michalak B, Müller CE, Schumacher N, Karolak-Wojciechowska J, Duddeck H, Rockitt S, and Wartchow R.     New developments in A1 and A2 adenosine receptor antagonists. Pure Appl Chem 73: 1411–1420, 2001.
  24. Kim YC, Ji XD, and Jacobson KA. Derivatives of the triazoloquinazoline adenosine antagonist (CGS15943) are selective for the human A3 receptor subtype. J Med Chem 39: 4142–4148, 1996.[CrossRef][ISI][Medline]
  25. Kingdom JC, McQueen J, Connell JM, and Whittle MJ. Fetal angiotensin II levels and vascular (type I) angiotensin receptors in pregnancies complicated by intrauterine growth retardation. Br J Obstet Gynaecol 100: 476–482, 1993.[ISI][Medline]
  26. Klotz K. Adenosine receptors and their ligands. Naunyn Schmiedebergs Arch Pharmacol 362: 382–391, 2000.[CrossRef][ISI][Medline]
  27. Klotz KN, Camaioni E, Volpini R, Kachler S, Vittori S, and Cristalli G. 2-Substituted N-ethylcarboxamidoadenosine derivatives as high-affinity agonists at human A3 adenosine receptors. Naunyn Schmiedebergs Arch Pharmacol 360: 103–108, 1999.[CrossRef][ISI][Medline]
  28. Klotz KN, Hessling J, Hegler J, Owman C, Kull B, Fredholm BB, and Lohse MJ. Comparative pharmacology of human adenosine receptor subtypes—characterization of stably transfected receptors in CHO cells. Naunyn Schmiedebergs Arch Pharmacol 357: 1–9, 1998.[ISI][Medline]
  29. Kohno Y, Sei Y, Koshiba M, Kim HO, and Jacobson KA. Induction of apoptosis in HL-60 human promyelocytic leukemia cells by adenosine A3 receptor agonists. Biochem Biophys Res Commun 219: 904–910, 1996.[CrossRef][ISI][Medline]
  30. Ledent C, Vaugeois JM, Schiffmann SN, Pedrazzini T, Yacoubi ME, Vanderhaeghen JJ, Costentin J, Heath JK, Vassart G, and Parmentier M. Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2A receptor. Nature 388: 674–678, 1997.[CrossRef][Medline]
  31. Linden J. Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection. Annu Rev Pharmacol Toxicol 41: 775–787, 2001.[CrossRef][ISI][Medline]
  32. Linden J, Thai T, Figler H, Jin X, and Robeva AS. Characterization of human A2B adenosine receptors: radioligand binding, Western blotting, and coupling to Gq in human embryonic kidney 293 cells and HMC-1 mast cells. Mol Pharmacol 56: 705–713, 1999.[Abstract/Free Full Text]
  33. Lumley P, White BP, and Humphrey PPA. GR32191, a highly potent and specific thromboxane A2 receptor blocking drug on platelets and vascular and airways smooth muscle in vitro. Br J Pharmacol 97: 783–794, 1989.[ISI][Medline]
  34. Marquardt DL, Walker LL, and Heinemann S. Cloning of two adenosine receptor subtypes from mouse bone marrow-derived mast cells. J Immunol 152: 4508–4515, 1994.[Abstract]
  35. McCoy DE, Schwiebert EM, Karlson KH, Spielman WS, and Stanton BA. Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells. Am J Physiol Cell Physiol 268: C1520–C1527, 1995.[Abstract/Free Full Text]
  36. Modlinger PS and Welch WJ. Adenosine A1 receptor antagonists and the kidney. Curr Opin Nephrol Hypertens 12: 497–502, 2003.[ISI][Medline]
  37. Murthy KS, McHenry L, Grider JR, and Makhlouf GM. Adenosine A1 and A2b receptors coupled to distinct interactive signaling pathways in intestinal muscle cells. J Pharmacol Exp Ther 274: 300–306, 1995.[Abstract/Free Full Text]
  38. Nicholls J, Brownhill VR, and Hourani SM. Characterization of P1-purinoceptors on rat isolated duodenum longitudinal muscle and muscularis mucosae. Br J Pharmacol 117: 170–174, 1996.[ISI][Medline]
  39. Obata T. Adenosine production and its interaction with protection of ischemic and reperfusion injury of the myocardium. Life Sci 71: 2083–2103, 2002.[CrossRef][ISI][Medline]
  40. Parisi VM and Rankin JH. The effect of prostacyclin on angiotensin II-induced placental vasoconstriction. Am J Obstet Gynecol 151:444–449, 1985.[ISI][Medline]
  41. Parisi VM and Walsh SW. Arachidonic acid metabolites and the regulation of placental and other vascular tone during pregnancy. Semin Perinatol 10:288–298, 1986.[ISI][Medline]
  42. Patrignani P, Panara MR, Greco A, Fusco O, Natoli C, Iacobelli S, Cipollone F, Ganci A, Creminon C, and Maclouf J. Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases. J Pharmacol Exp Ther 271: 1705–1712, 1994.[Abstract/Free Full Text]
  43. Poucher SM, Keddie JR, Singh P, Stoggall SM, Caulkett PW, Jones G, and Coll MG. The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2A selective adenosine receptor antagonist. Br J Pharmacol 115: 1096–1102, 1995.[ISI][Medline]
  44. Racchi H, Irarrazabal MJ, Howard M, Moran S, Zalaquett R, and Huidobro-Toro JP. Adenosine 5'-triphosphate and neuropeptide Y are co-transmitters in conjunction with noradrenaline in the human saphenous vein. Br J Pharmacol 126: 1175–1785, 1999.[CrossRef][ISI][Medline]
  45. Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50, 413–492, 1998.[Abstract/Free Full Text]
  46. Rankin JH and Phernetton TM. Alpha and angiotensin receptor tone in the near-term sheep fetus. Proc Soc Exp Biol Med 158: 166–169, 1978.[Medline]
  47. Reid DL, Davidson SR, Phernetton TM, and Rankin JH. Adenosine causes a biphasic response in the ovine fetal placental vasculature. J Dev Physiol 13: 237–240, 1990.[ISI][Medline]
  48. Reilly FD and Russel PT. Neurohistichemical evidence supporting an absence of adrenergic and cholinergic innervation in the human placenta and umbilical cord. Anat Rec 188: 277–286, 1977.[CrossRef][Medline]
  49. Reviriego J, Alonso MJ, Ibañez C, and Marin J. Action of adenosine and characterization of adenosine receptors in human placental vasculature. Gen Pharmacol 21: 227–233, 1990.[ISI][Medline]
  50. Rosenfeld CR, Cox BE, Magness RR, and Shaul PW. Ontogeny of angiotensin II vascular smooth muscle receptors in ovine fetal aorta and placental and uterine arteries. Am J Obstet Gynecol 168: 1562–1569, 1993.[ISI]