Functional Evidence for a Novel Suramin-Insensitive Pyrimidine Receptor in Rat Small Pulmonary Arteries

From the University of Oxford, Department of Pharmacology, Oxford, UK.

Correspondence to Roland Z. Kozlowski, University Department of Pharmacology, Mansfield Rd, Oxford, OX1 3QT, UK. E-mail roland.kozlowski{at}pharm.ox.ac.uk

	Abstract

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Abstract—Uridine nucleotides are known to cause constriction of pulmonary arterial smooth muscle. However, the P2 receptor subtypes underlying the contractile effects of these nucleotides in the pulmonary circulation have not been determined. We have used myography and the patch-clamp recording technique to compare the effects of UTP and UDP in isolated small pulmonary arteries (diameter 100 to 400 µm) and their constituent smooth muscle cells. In endothelium-denuded arteries, both UTP and UDP (0.01 to 3 mmol/L) induced concentration-dependent increases in tension that were independent of P2X receptor stimulation. The UDP-mediated increase in tension was significantly less sensitive to the nonselective P2 receptor blocker suramin than the UTP-mediated increase in tension. In single isolated arterial myocytes, voltage-clamped at -50 mV (close to the resting membrane potential of these cells), application of both UTP and UDP evoked periodic oscillations of inward current primarily because of a Ca²⁺-activated Cl^- current (I_Cl,Ca). Oscillations of I_Cl,Ca evoked by UTP were reversibly inhibited by suramin, although those evoked by UDP were insensitive to the antagonist. In addition to confirming the presence of classical P2Y₂ receptors, these results also provide functional evidence for the existence of a novel UDP receptor in pulmonary arterial myocytes, which may contribute to pyrimidine-evoked vasoconstriction. This notion is supported by molecular evidence that demonstrates the presence of P2Y₆ receptor transcripts in rat pulmonary arterial smooth muscle.

Key Words: artery, pulmonary • nucleotide, uridine • channel, Cl^-

	Introduction

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Adenine and uracil nucleotides such as ATP and UTP are potent modulators of cardiovascular function. These nucleotides can be released either intraluminally from perivascular nerves or extraluminally from a range of sources, such as aggregating platelets and endothelial cells.^{1 2 3} These nucleotides interact with various P2 receptors to produce a range of physiological responses. P2 receptors located on the endothelium mediate vasodilatation, and receptors located on the smooth muscle mediate vasoconstriction.⁴ Consequently, the effects of such nucleotides are dependent on their site of action. Direct evidence of the regulated release of uridine nucleotides is limited,⁵ but the vasomotor activity of UTP in human cerebral arteries⁶ in addition to activation of other cell functions,⁷ has led to the suggestion that extracellular uridine nucleotides are also important signaling molecules that mediate their effects through P2 receptor stimulation. These receptors have been divided into 2 broad groups: P2X receptors, which are intrinsic ion channels, and P2Y receptors, which are G protein–coupled receptors. The P2X receptor family comprises subtypes P2X_1–6. The P2Y family includes the receptors P2Y₁ and P2Y₂ (formerly known as P2Y and P2U, respectively), the P2T receptor (yet to be cloned), and 5 further cloned subtypes (P2Y_3–7).⁸

Since the initial observation in cerebral vessels, UTP has been shown to produce constriction of a number of arterial vessels including guinea pig coronary artery,⁹ rabbit ear artery,¹⁰ rat tail and femoral arteries,¹¹ and rat pulmonary artery.¹² The first evidence that a novel class of uridine nucleotide-responsive receptors may exist came with the observation that stimulation of G protein–coupled P_2U (P2Y₂) receptors (originally cloned by Lustig et al¹³ ) by either ATP or UTP promoted an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Evidence for selective "pyrimidinergic" receptors was provided by Lazarowski et al,¹⁴ who reported the existence of uridine nucleotide–specific receptors on C6-2B rat glioma cells. Since the identification of this receptor type, 2 further nucleotide sequences encoding 2 different G protein–coupled receptors that are selectively activated by uridine nucleotides have been described,^{15 16} indicating the possible existence of a family of pyrimidinergic receptors. Studies that use expression cloning¹⁷ indicate that these 3 receptors can be clearly delineated into (1) a UDP-preferring, uridine nucleotide–specific receptor (P2Y₆), (2) a UTP-preferring receptor (P2Y₄), and (3) a P2Y₂ receptor activated by both UTP and ATP. Although mRNA for both P2Y₂ receptors and P2Y₆ receptors is present in vascular smooth muscle cells,¹⁵ existing functional studies have demonstrated only the presence of P2Y₂ receptors. Studies in isolated pulmonary arterial smooth muscle cells of both the rat and the rabbit have shown the existence of P2Y₂ receptors blocked by the nonselective P2 receptor antagonist suramin.^{18 19} However, in the pulmonary vascular bed, both UTP- and UDP-induced vasoconstriction is thought to be insensitive to suramin, indicating the existence of a suramin-insensitive receptor activated by uridine nucleotides.¹² Consistent with these observations, the existence of a UTP-specific receptor has been shown in guinea pig endothelial cells²⁰ and canine coronary arteries.²¹ In the present study, we have investigated the actions of UTP and UDP in whole arteries and single-isolated myocytes of the rat small pulmonary artery. Our experiments have been designed to determine whether (1) a novel suramin-insensitive receptor activated by uridine nucleotides exists, and (2) whether it plays a functional role in mediating contraction.

	Materials and Methods

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Arterial Preparation and Mounting
Male albino Wistar rats (200 to 250 g body weight) were killed by an overdose of IP Euthatol (pentobarbitone sodium B.P.), and cervically dislocated. Small pulmonary arterial vessels, 100 to 400 µm in diameter, were removed from the lungs and placed in physiological salt solution (PSS) of the following composition (mmol/L): NaCl 150, KCl 5.4, MgCl₂ 1.2, CaCl₂ 1.2, HEPES 5, and glucose 10 (pH 7.4 with NaOH). These vessels, 1 to 2 mm in length, were denuded of endothelium by gentle rubbing with thin surgical thread and mounted in a Mulvaney myograph (volume 10 mL), which was perfused at a rate of 2 mL/min with solution A maintained at 37°C. Two tungsten wires (30 µm diameter) were inserted through the lumen, and mechanical activity was recorded isometrically by a force transducer connected to 1 of the 2 tungsten wires; the other wire was attached to a support carried by a micromanipulator. After subjecting the muscle to an initial tension of 25 mm Hg (3.3 Kpa), the muscle was equilibrated for 30 minutes before being challenged with 50 mmol/L KCl and until reproducible contractions were obtained. Tension (T) of vessels was normalized through the use of the following equation: T_N=(T-T_O)/(LxIC), in which T_N is the normalized force, T_O is the initial tension, L is the tissue wall length, and IC is the internal circumference (calculated after applying the initial tension). To verify effective endothelium removal, the sensitivity of vessels to acetylcholine after preconstriction with noradrenaline was determined. All vessels denuded of endothelium were not relaxed by acetylcholine (Figure 1). Concentration-response curves to UTP (0.01 to 3 mmol/L) and UDP (0.01 to 3 mmol/L) were constructed noncumulatively, which allowed the tension to return to baseline between additions.

View larger version (12K): [in this window] [in a new window]   Figure 1. Control response to phenylephrine (1 µmol/L) and acetylcholine (ACh, 10 µmol/L) in the absence (upper) and presence (lower) of the endothelium. Note that ACh is ineffective at causing relaxation of vessels preconstricted with phenylephrine in the absence of the endothelial cell layer.

Single Cell Isolation and Electrophysiological Experiments
Pulmonary arterial smooth muscle cells were isolated through the use of a dispersion procedure, previously described by Albarwani et al,²² and modified to include an incubation with collagenase (type VIII, 1.5 mg/mL), protease (type I, 0.1 mg/mL), and elastase (type II-A, 0.3 mg/mL) for 4 minutes at 37°C after pretreatment with papain. Cells were stored at 4°C and remained viable for 10 hours.

Membrane current from pulmonary arterial myocytes was recorded with the use of the perforated patch configuration of the patch-clamp recording technique²³ at room temperature (20°C to 25°C). The cells were bathed in PSS, and the pipette was filled with solution consisting of the following (mmol/L): 125 KCl, 4 MgCl₂, 10 HEPES, 0.02 EGTA, and pH 7.3 with KOH. The viability of each cell preparation was assessed by qualitatively visualizing the ability of isolated cells to contract in response to UTP and UDP. In general, cells isolated by the use of our methods could be maintained in the perforated patch configuration for up to 1 hour. However, the experiments presented here rarely lasted for >15 minutes. To examine the effects of the uridine nucleotides on membrane current, cells were voltage-clamped at -50 mV, unless otherwise stated. Electrodes (3 to 6 M) were pulled from borosilicate glass capillaries (Clark Electromedical) with a vertical puller (Narishige Ltd). Ionic currents were detected with the use of an Axopatch 200A amplifier (Axon Instruments). Series resistance and capacity compensation facilities were used when necessary. Data were filtered at 2 kHz with the use of a Digidata 1200 interface (Axon Instruments) and recorded either on-line with a personal computer or off-line on a modified DAT recorder (Sony DTC-100ES). Data were analyzed with the use of pClamp software (version 6.1; Axon Instruments Inc).

Molecular Studies
Total RNA was isolated from endothelium-denuded rat pulmonary arteries and aortas using TRIZOL reagent (Gibco Life Technologies) according to the recommendations of the manufacturer. cDNA was synthesized from 1 µg total RNA, in the presence of (100 ng) random hexamer primer (Perkin Elmer), 1 mmol/L dNTPs (Promega), 20 U of RNasin ribonuclease inhibitor (Promega), and 50 U of Moloney murine leukemia virus reverse transcriptase (Perkin Elmer) according to the recommendations of the manufacturer. Polymerase chain reaction (PCR) was conducted through the use of oligonucleotide primers designed from the published rat P2Y₆ sequence,¹⁵ which has been described previously by Webb et al²⁴ ; sense primer 5'-GGAGACCTTGCCTGCCGCCTGGTA-3', and antisense primer 5'-TACCACGACAGCCATACGGGCCGC-3'. PCR was performed in a buffer containing (in mmol/L) 50 KCl, 10 Tris-HCl (pH 8.3), 1.5 MgCl₂, and 0.01% (wt/vol) gelatin (Perkin Elmer), with the use of 10% of the cDNA synthesis reaction, 80 ng of each primer, 200 µmol/L of each dNTP, and 1.25 U of Taq DNA polymerase (Promega) in a total volume of 50 µL. The reaction conditions were as follows: 30 seconds at 94°C, 55°C, and 72°C, respectively, for 35 cycles followed by 1 cycle at 72°C for 10 minutes. Both negative control reactions (in which no cDNA was included in the PCR reaction mix) and positive control reactions (with primers designed to amplify GAPDH and with cDNA synthesized from total RNA isolated from rat aorta, which is known to express the P2Y₆ receptor) were performed. A mock cDNA synthesis reaction, in which no reverse transcriptase was added, was also performed as a control for genomic DNA contamination of the RNA sample and for DNA contamination in the PCR reaction.

PCR products were separated on a 1.2% (wt/vol) agarose gel by electrophoresis, and the bands excised. After extraction of the DNA using the QIAquick gel extraction kit (QIAGEN), the PCR product was cloned into the pCRII dual vector Invitrogen (INV) and introduced into INVF' competent cells. Plasmid DNA was prepared using QIAfilter plasmid midipreps (QIAGEN), and the double-stranded plasmid inserts were sequenced in both directions with the use of the Sequenase 2.0 kit (Amersham International) to confirm their identity.

Specialty Chemicals
Amphotericin B, collagenase (type VIII), dithiothreitol, elastase (type IIA), EGTA, HEPES, niflumic acid, papain (papaya latex), protease (type I), UDP (sodium salt), and UTP (sodium salt) were all purchased from Sigma (Poole). Suramin (hexasodium salt) was obtained from ICN Biochemicals Ltd.

Data Analysis
To compare the ability of a range of nucleotides to activate oscillatory inward currents, membrane current elicited over a 1-minute period was digitized at 100 Hz and integrated (pClamp software; version 6.2), as previously described,²⁵ giving a value in nA · ms. Contractions were normalized and expressed as N/mm². Data are presented throughout as mean±SEM. When presented graphically, the SEM is represented by the associated error bars. Statistical significance was assessed using a Student t test. P values 0.05 were considered significant.

	Results

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Contractile Effects of UTP and UDP
UTP (0.01 to 3 mmol/L) and UDP (0.1 to 3 mmol/L) produced reproducible concentration-dependent contractions of endothelium-denuded rat small pulmonary arteries. The endothelium was removed to ensure that effects of the nucleotides could be attributed to their actions on the smooth muscle cells and to obviate any relaxation mediated through P2Y₁ and P2Y₂ receptors present on vascular endothelial cells.²⁶ Contractions in response to UTP and UDP developed slowly and were maintained during administration of the nucleotide. Typical traces showing the changes in contraction produced by the addition of the nucleotides UTP (0.01 to 3 mmol/L) and UDP (0.01 to 3 mmol/L) are shown in Figure 2A and 2B. The concentration-response curves to UTP and UDP are shown in Figure 2C. In the presence of ,ß-methylene ATP (100 µmol/L), a selective P2X receptor agonist that causes desensitization of these receptors, the magnitude of the contractile response induced by either UTP or UDP was unchanged, which indicates that P2X receptor stimulation is not involved in the contractile responses to either uridine nucleotide (not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. Contractile effects of UTP and UDP on endothelium-denuded arteries. A and B, Representative isometric records showing the concentration-dependent contractile effect of UTP (0.01 to 3 mmol/L) and UDP (0.01 to 3 mmol/L), respectively, on isolated arteries. C, Concentration-response relationships for UTP- and UDP-induced contractions of isolated arteries (n=8 arteries for both nucleotides). The values are normalized to and expressed as mN/mm2.

Effects of Suramin on the Contractile Actions of UTP and UDP
In a number of vascular and neuronal preparations, suramin has been a nonselective antagonist at P2 receptors.²⁷ To investigate the action of suramin on UTP- and UDP-induced contractions, pulmonary arteries were preincubated with the antagonist for 30 minutes before the addition of the nucleotides (used at 1 mmol/L). The antagonist was present in the bath solution throughout the experiment. As illustrated in Figure 3A, suramin (100 µmol/L) significantly reduced the contractile response of UTP to 57±7% (n=8) of control, which was obtained in the absence of the antagonist. The UDP-induced contraction was only slightly (24±5%, n=8), but significantly, inhibited on application of 100 µmol/L suramin (Figure 3B). A significant difference was found between the inhibitory effects of suramin on the UTP- and UDP-induced contractions. The UDP-induced contraction was significantly less resistant to suramin than the UTP-induced contraction (Figure 3C). This difference indicated the possible existence of a novel UDP-activated receptor relatively insensitive to suramin. To verify this notion and to minimize the potential influence of nucleoside diphosphokinase or ectonucleotidase enzymes known to reside on the extrafacial surface of most cells,⁷ the effects of UTP and UDP were investigated with the use of single rat pulmonary arterial myocytes.

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of suramin on the UTP- and UDP-induced contractions of isolated arteries. A and B, Representative isometric records showing the inhibitory effect of 100 µmol/L suramin on the contractile responses of 1 mmol/L UTP and 1 mmol/L UDP, respectively. C, Bar graph showing the mean±SEM inhibition by 100 µmol/L suramin on the UTP- and UDP-induced contraction of isolated arteries. The number of arteries used for each nucleotide is shown in parentheses. *Significant (P0.05) difference between the suramin-sensitivity of the UTP- and UDP-induced contractions.

Electrophysiological Effects of UTP and UDP
We¹⁸ have shown that extracellular application of UTP (10 µmol/L) to myocytes isolated from rat small pulmonary arteries evokes periodic oscillations of a Ca²⁺-activated Cl^- current (I_Cl,Ca) via the activation of P2Y₂ receptors. We showed in an earlier study that myocytes isolated from rat small pulmonary arteries have a resting membrane potential between -44 mV and -53 mV (-49.3±1.1 mV, n=15). We have found consistently that the extracellular addition of UTP (10 µmol/L) to cells maintained in the perforated patch configuration at a holding potential of -50 mV (close to the resting membrane potential of these cells) evoked periodic oscillations of inward current in 16 of 21 cells. Under identical conditions, extracellular application of UDP (10 µmol/L) evoked periodic oscillations of inward current in 24 of 36 cells. These oscillations were reversibly inhibited (n=5) by niflumic acid (50 µmol/L, a blocker of Cl^- channels),²⁸ which confirms that they were caused by activation of I_Cl,Ca (data not shown). The magnitude of I_Cl,Ca measured over a 1-minute period, as discussed in Materials and Methods, was not significantly different between the 2 nucleotides: 420±113 nA · ms (n=11) for UTP and 412±64 nA · ms (n=20) for UDP. Higher concentrations of UDP (100 µmol/L and 500 µmol/L) evoked currents of similar magnitude: 429±55 nA · ms (in 4 of 4 cells) and 437±34 nA · ms (in 3 of 3 cells), respectively. This suggests activation of I_Cl,Ca by UTP and UDP is "all or nothing," and if activated, the current magnitude may be a maximal response. Because there is significant cross-contamination in commercially available UTP and UDP, we examined the effects of the 2 nucleotides at a concentration of 0.5 µmol/L. This concentration represents the maximum (5%) likely to be found as a contaminant within commercially available UTP or UDP when used at 10 µmol/L. Neither nucleotide had any effect at this concentration (data not shown), which obviated the possibility that the responses presented were the consequences of cross-contamination.

Effects of Antagonists on the Electrophysiological Actions of UTP and UDP
Application of suramin (100 µmol/L throughout, unless otherwise stated) totally inhibited oscillations of I_Cl,Ca evoked by 10 µmol/L UTP (Figure 4A, n=5). In contrast, application of suramin had no significant inhibitory effect on activation of I_Cl,Ca evoked by 10 µmol/L UDP (Figure 4B, n=6). Furthermore, pretreatment of cells with suramin for 3 minutes had no effect on the action of 10 µmol/L UDP (in the continued presence of suramin). The nucleotide still evoked oscillations of I_Cl,Ca of similar magnitude to those evoked in the absence of the antagonist (n=4, data not shown). Additional experiments using a range of concentrations of suramin (0.01 to 1 mmol/L) further highlighted the differential sensitivity of the UDP- and UTP-evoked oscillations of I_Cl,Ca to the antagonist. The results are expressed quantitatively in Figure 4C. Unlike suramin, application of the known P2Y receptor blocker Cibacron Blue (10 µmol/L), a concentration reported to be selective for P2Y receptors (Burnstock and Warland),²⁹ significantly inhibited oscillations induced by both UTP and UDP (Figure 5A, 5B, and 5C). This finding further supports that UTP and UDP are acting via P2Y receptors.

View larger version (31K): [in this window] [in a new window]   Figure 4. Effect of suramin on the UTP- and UDP-evoked periodic oscillations of ICl,Ca in single isolated arterial myocytes. A, Representative record demonstrating the inhibitory effect of extracellular suramin (100 µmol/L) on UTP-evoked oscillations of ICl,Ca. B, Representative record demonstrating the insensitivity of UDP-evoked oscillations of ICl,Ca to extracellular suramin (100 µmol/L). C, Bar graph showing the mean±SEM inhibition by suramin (0.01 1 mmol/L) of ICl,Ca activated by UTP and UDP (both 10 µmol/L). The number of cells is shown in parentheses. *Significant (P0.05) inhibition by suramin.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effect of Cibacron Blue on the UTP- and UDP-evoked periodic oscillations of ICl,Ca in single isolated arterial myocytes. A, Representative record demonstrating the inhibitory effect of extracellular Cibacron Blue (10 µmol/L) on UTP-evoked oscillations of ICl,Ca. B, Representative record demonstrating the insensitivity of UDP-evoked oscillations of ICl,Ca to extracellular Cibacron Blue (10 µmol/L). C, Bar graph showing the mean±SEM inhibition by Cibacron Blue of ICl,Ca activated by (1) UTP and (2) UDP (both 10 µmol/L). The number of cells is shown in parentheses. *Significant (P0.05) inhibition by Cibacron Blue.

Molecular Studies
To determine whether P2Y₆ receptor transcripts are expressed in rat pulmonary arteries, reverse transcription (RT)-PCR experiments were performed. In these experiments, rat aortas, which have already been shown to express P2Y₆ receptors,¹⁵ were used as a positive control. RT-PCR, which used oligonucleotide primers designed to amplify a 450-bp region of the P2Y₆ receptor, yielded a single PCR product in experiments through the use of total RNA isolated from rat pulmonary arteries and aortas (Figure 6). Cloning and sequencing of these products confirmed that they represented amplification of the selected region of the published P2Y₆ receptor sequence (Accession No. D63665).²⁴

View larger version (53K): [in this window] [in a new window]   Figure 6. Agarose gel electrophoresis of PCR amplification products from rat pulmonary artery and aorta using oligonucleotide primers for P2Y6 receptor and GAPDH. Both sets of primers yield a single band (450 and 561 bp, respectively) in reactions that contained RNA and reverse transcriptase. No bands were observed in any of the negative control lanes (2, 4, 5, 7, 9, and 10).

	Discussion

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The present study compares the contractile and electrophysiological effects of UTP and UDP in rat small pulmonary arterial muscle. Our report provides for the first time functional evidence of the existence of UDP receptors in arterial myocytes pharmacologically distinct from P2Y₂ receptors.

We agree with previous studies performed in the pulmonary vascular bed of the rat,¹² which indicated that the nucleotides UTP and UDP (0.01 to 3 mmol/L) caused concentration-dependent increases in the tension of isolated small pulmonary arteries of the rat. There are several reports indicating the presence of P2Y₂ receptors in pulmonary arterial smooth muscle cells that are equally sensitive to the purine ATP and the pyrimidine UTP.^{18 19} The existence of such receptors could explain the ability of UTP to cause contraction of pulmonary arteries independently of P2X receptor stimulation. However, because UDP has been shown unequivocally not to be an agonist at the P2Y₂ receptor,¹⁷ UDP could only be mediating its contractile effects in this tissue through either (1) the in situ conversion of UDP to UTP through the activity of an ectonucleoside diphosphokinase³⁰ and subsequent activation of P2Y₂ receptors, or (2) the activation of an additional pyrimidine receptor that is exquisitely sensitive to UDP. The increases in tension induced by both UTP and UDP were inhibited by suramin. However, the UDP-mediated contraction was significantly less sensitive to the antagonist than was the UTP-mediated contraction. This marked difference in the sensitivity to suramin obviates the possibility that the 2 nucleotides are acting via the same P2Y₂ receptor.

Our original observations were extended in this study when we showed that UDP, like UTP,¹⁸ can result in activation of I_Cl,Ca. However, in contrast to UTP, the oscillations in I_Cl,Ca evoked by UDP were totally insensitive to suramin (100 µmol/L) and were only significantly inhibited when suramin was used at concentrations >100 µmol/L: a concentration known to have nonspecific inhibitory effects on other ion channels and cell functions.³¹ Furthermore, as suramin selectively inhibited activation of I_Cl,Ca by UTP but not UDP, calcium release from intracellular stores is probably not affected. Thus, under conditions un which the influence of ectonucleotidases is negligible (isolated single cells⁷ ) and no significant conversion of UDP to UTP can occur, UDP can still elicit effects (activation of I_Cl,Ca) that are insensitive to suramin. Because of this pharmacological difference and the fact that UDP, unlike UTP, does not activate P2Y₂ receptors,¹⁷ a novel UDP selective receptor probably exists in pulmonary arterial smooth muscle.

Because UDP is known to be inactive at both the P2Y₂ and P2Y₄ receptors, the most likely receptor candidate for the effect of UDP in pulmonary arterial smooth muscle cells is the P2Y₆ receptor. When expressed in C6 rat glioma cells, the P2Y₆ receptor was relatively resistant to blockade by suramin, with receptor-mediated increases in IP₃ being reduced by only 20%.¹⁵ This lack of potency is consistent with our findings with suramin (1) in isolated vessels in which UDP-mediated increases in tension were only reduced by 23%, and (2) in single cells in which UDP-mediated activation of I_Cl,Ca was only reduced by 15%. P2Y₆ receptors (and their encoding mRNA) are expressed in the rat aortas in which they have been suggested to perform a potential physiological role in vascular function.¹⁵ The P2Y₆ receptor transcript is also found in cultured rat aortic smooth muscle cells and may be expressed in the smooth muscle layer of the vascular wall where it may be involved in the regulation of vascular tone. Our molecular data are consistent with these results through the expression of identical mRNA in pulmonary arterial smooth muscle. Like P2Y₂ and P2Y₄ receptors, P2Y₆ receptors are coupled to phospholipase C, and their stimulation mediates an increase in the [Ca²⁺]_i, which in vascular smooth muscle may elicit activation of Ca²⁺-sensitive membrane currents (such as I_Cl,Ca) and, therefore, induce contraction.

The existence of P2Y₆ receptors in pulmonary arterial smooth muscle explains the observed suramin insensitivity of the UDP-induced vasoconstriction of the pulmonary vascular bed of the rat.¹² The 2 chief reasons for this conclusion are as follows. First, the P2Y₆ receptor is reportedly UDP-selective but is weakly activated by UTP.¹⁷ Consequently, in multicellular tissue preparations that require higher concentrations of nucleotides to elicit a given response than single cells, vasoconstriction evoked by UTP may be mediated at least in part through stimulation of suramin-resistant P2Y₆ receptors. This could also explain the apparent insensitivity of UTP- and UDP-induced contraction of canine coronary artery.²¹ Second, a much greater ectonucleotidase activity exists in multicellular preparations that could potentially hydrolyze UTP into UDP, which would then act via P2Y₆ receptors to elicit vasoconstriction. Our finding that the UTP-mediated activation of I_Cl,Ca in single cells is significantly more sensitive to inhibition with suramin than the UTP-mediated contraction in intact vessels also supports the two explanations.

In summary, our report provides for the first time evidence of the existence of an additional suramin-insensitive UDP receptor, distinct from the classical P2Y₂ subtype, which mediates pyrimidine-evoked constriction of the rat small pulmonary artery. The results obtained through myography, electrophysiology, and molecular methods make the probable candidate for this receptor the UDP-selective P2Y₆ receptor. The functional implications of separate pyrimidine-sensitive receptors with a common signaling cascade in pulmonary arterial smooth muscle is unknown. However, a possibility remains that they may have different, as yet undiscovered, regulatory functions in controlling vascular tone. Only limited information is available concerning the storage and regulation of the release of uridine nucleotides. These nucleotides are known to be stored in platelets, and by analogy, with purine nucleotides may be released from cells under a variety of pathological conditions such as inflammation, trauma, and hypoxia.³² With the existence of constriction-mediating pyrimidine receptors on vascular smooth muscle, the release of uridine nucleotides under such conditions would be of extreme importance in the pulmonary circulation. In the pulmonary circulation, their release may play a role in hypoxic pulmonary vasoconstriction or in certain forms of pulmonary hypertension when there is loss or trauma to the endothelium.

	Acknowledgments

 
This work was supported by Glaxo Wellcome, the Medical Research Council (United Kingdom), and the British Heart Foundation, with which Roland Z. Kozlowski is a lecturer.

Received September 9, 1997; accepted July 30, 1998.

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