Functional Evidence for a Novel Suramin-Insensitive
Pyrimidine Receptor in Rat Small Pulmonary Arteries
S. Anthony Hartley, Kenichi Kato, Katie J. Salter, Roland Z. Kozlowski
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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 mm) 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 250 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 Ca21-activated Cl2 current (ICl,Ca). Oscillations of ICl,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. (Circ Res. 1998;83:940-946.)
Key Words: artery, pulmonary n nucleotide, uridine n channel, Cl2
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 P2U (P2Y2)
receptors (originally cloned by Lustig et al13) by either ATP
or UTP promoted an increase in the intracellular Ca21
concentration ([Ca21]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
A
denine 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.1–3 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 P2X1– 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 (P2Y3–7).8
Since the initial observation in cerebral vessels, UTP has
been shown to produce constriction of a number of arterial
Received September 9, 1997; accepted July 30, 1998.
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
[email protected]
© 1998 American Heart Association, Inc.
940
Hartley et al
November 2, 1998
941
Figure 1. Control response to phenylephrine
(1 mmol/L) and acetylcholine (ACh, 10 mmol/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.
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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 suramininsensitive receptor activated by uridine nucleotides.12 Consistent with these observations, the existence of a UTPspecific 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
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 mm 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 mm 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: TN5(T2TO)/(L3IC), in
which TN is the normalized force, TO 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.
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 250
mV, unless otherwise stated. Electrodes (3 to 6 MV) 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 mg 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;
942
UDP Receptor in Arterial Myocytes
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sense primer 59-GGAGACCTTGCCTGCCGCCTGGTA-39, and antisense primer 59-TACCACGACAGCCATACGGGCCGC-39. 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 mmol/L of each dNTP, and 1.25 U of Taq DNA
polymerase (Promega) in a total volume of 50 mL. 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 INVaF9 competent cells. Plasmid DNA was prepared using QIAfilter plasmid midipreps (QIAGEN), and the doublestranded 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 z ms. Contractions were normalized and expressed as N/mm2.
Data are presented throughout as mean6SEM. 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
Figure 2. Contractile effects of UTP and UDP on endotheliumdenuded 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 (n58
arteries for both nucleotides). The values are normalized to and
expressed as mN/mm2.
stimulation is not involved in the contractile responses to
either uridine nucleotide (not shown).
Contractile Effects of UTP and UDP
Effects of Suramin on the Contractile Actions 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 a,bmethylene ATP (100 mmol/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
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 mmol/L) significantly reduced the contractile response of
UTP to 5767% (n58) of control, which was obtained in the
absence of the antagonist. The UDP-induced contraction was
only slightly (2465%, n58), but significantly, inhibited on
application of 100 mmol/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
Hartley et al
November 2, 1998
943
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tion of ICl,Ca (data not shown). The magnitude of ICl,Ca
measured over a 1-minute period, as discussed in Materials
and Methods, was not significantly different between the 2
nucleotides: 4206113 nA z ms (n511) for UTP and 412664
nA z ms (n520) for UDP. Higher concentrations of UDP
(100 mmol/L and 500 mmol/L) evoked currents of similar
magnitude: 429655 nA z ms (in 4 of 4 cells) and 437634 nA z
ms (in 3 of 3 cells), respectively. This suggests activation of
ICl,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 mmol/L. This concentration represents the maximum (5%) likely to be found as a contaminant
within commercially available UTP or UDP when used at
10 mmol/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
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 mmol/L suramin on
the contractile responses of 1 mmol/L UTP and 1 mmol/L UDP,
respectively. C, Bar graph showing the mean6SEM inhibition by
100 mmol/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 (P#0.05) difference
between the suramin-sensitivity of the UTP- and UDP-induced
contractions.
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.
Electrophysiological Effects of UTP and UDP
We18 have shown that extracellular application of UTP
(10 mmol/L) to myocytes isolated from rat small pulmonary
arteries evokes periodic oscillations of a Ca21-activated Cl2
current (ICl,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 244 mV and 253 mV (249.361.1 mV, n515). We
have found consistently that the extracellular addition of UTP
(10 mmol/L) to cells maintained in the perforated patch
configuration at a holding potential of 250 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 mmol/L) evoked periodic oscillations of inward current in
24 of 36 cells. These oscillations were reversibly inhibited
(n55) by niflumic acid (50 mmol/L, a blocker of Cl2
channels),28 which confirms that they were caused by activa-
Application of suramin (100 mmol/L throughout, unless
otherwise stated) totally inhibited oscillations of ICl,Ca evoked
by 10 mmol/L UTP (Figure 4A, n55). In contrast, application
of suramin had no significant inhibitory effect on activation
of ICl,Ca evoked by 10 mmol/L UDP (Figure 4B, n56).
Furthermore, pretreatment of cells with suramin for 3 minutes
had no effect on the action of 10 mmol/L UDP (in the
continued presence of suramin). The nucleotide still evoked
oscillations of ICl,Ca of similar magnitude to those evoked in
the absence of the antagonist (n54, 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 ICl,Ca to the
antagonist. The results are expressed quantitatively in Figure
4C. Unlike suramin, application of the known P2Y receptor
blocker Cibacron Blue (10 mmol/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.
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
Discussion
The present study compares the contractile and electrophysiological effects of UTP and UDP in rat small pulmonary
944
UDP Receptor in Arterial Myocytes
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 mmol/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 mmol/L). C, Bar graph showing the
mean6SEM inhibition by suramin (0.01 1 mmol/L)
of ICl,Ca activated by UTP and UDP (both 10 mmol/
L). The number of cells is shown in parentheses.
*Significant (P#0.05) inhibition by suramin.
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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
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 mmol/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 mmol/L). C, Bar graph showing the mean6SEM
inhibition by Cibacron Blue of ICl,Ca activated by (1) UTP and (2)
UDP (both 10 mmol/L). The number of cells is shown in parentheses. *Significant (P#0.05) inhibition by Cibacron Blue.
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
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).
Hartley et al
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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
ICl,Ca. However, in contrast to UTP, the oscillations in ICl,Ca
evoked by UDP were totally insensitive to suramin
(100 mmol/L) and were only significantly inhibited when
suramin was used at concentrations .100 mmol/L: a concentration known to have nonspecific inhibitory effects on other
ion channels and cell functions.31 Furthermore, as suramin
selectively inhibited activation of ICl,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 ICl,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 ICl,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 [Ca21]i, which in vascular smooth
muscle may elicit activation of Ca21-sensitive membrane
currents (such as ICl,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
November 2, 1998
945
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 ICl,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 constrictionmediating 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.
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Functional Evidence for a Novel Suramin-Insensitive Pyrimidine Receptor in Rat Small
Pulmonary Arteries
S. Anthony Hartley, Kenichi Kato, Katie J. Salter and Roland Z. Kozlowski
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Circ Res. 1998;83:940-946
doi: 10.1161/01.RES.83.9.940
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