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Department of Neurosurgery, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of extraluminal UTP
were studied and compared with vascular responses to ATP and its
analogs in rat cerebral-penetrating arterioles. UTP, UDP,
2-methylthio-ATP, and 
,
-methylene-ATP dilated arterioles at the
lowest concentration and constricted them at high concentrations. Low
concentrations of ATP dilated the vessels; high concentrations caused a
biphasic response, with transient constriction followed by dilation.
Endothelial impairment inhibited ATP- and UTP-mediated dilation and
potentiated constriction to UTP but not to ATP. ATP- and
2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by
desensitization with 10
6 M ,-methylene-ATP or
3 × 106 M pyridoxal
phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). PPADS at
104 M abolished the UTP-mediated constriction and induced
vasodilation in a dose-dependent manner but did not affect the dilation
to ATP. These results suggest that in rat cerebral microvessels
1) ATP and 2-methylthio-ATP induce transient constriction
via smooth muscle P2X1 receptors in the cerebral arteriole,
2) UTP stimulates two different classes of P2Y
receptors, resulting in constriction (smooth muscle P2Y4)
and dilation (possibly endothelial P2Y2), and 3)
ATP and UTP produce dilation by stimulation of a single receptor
(P2Y2).
ATP; cerebral circulation; UTP; purine receptors; pyrimidine receptors
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PURINES SUCH AS ATP
and pyrimidines such as UTP are involved in many important regulatory
systems (6, 39, 53). In the cerebral circulation, ATP and
UTP are natural agonists with prominent vasoactivity and numerous
physiological sources (8, 19, 22, 30, 40, 41, 43, 44).
Although ATP was initially found to cause vasodilation and UTP was a
strong vasoconstrictor, further studies showed that ATP and UTP can
cause dilation, constriction, or both, depending on the species, vessel
type, location within the vascular tree, and/or route of administration
(14, 20, 25, 26, 38-41, 48, 50, 54, 60, 61). This
variability in responses can be explained by purines and pyrimidines
acting on a number of different receptors that can reside on the
vessel's smooth muscle and/or endothelial cells, with their
distribution also variable along the vascular tree (Table
1).
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P2 receptors have been divided into two broad groups: ionotropic P2X receptors, which are intrinsic ion channels, and metabotropic P2Y receptors, which are G protein coupled (3, 46) and may activate different phospholipases (11). In the vasculature, P2X1 receptors on smooth muscle mediate constriction via Ca2+ influx (Table 1) (46, 59). P2Y receptors were found on endothelial cells of several vessel types causing dilation via activation of P2Y1(P2Y) and P2Y2(P2U) receptors. On smooth muscle, P2Y2-, P2Y4-, and P2Y6-receptor activation causes constriction, while similar stimulation of the P2Y1 receptor induces vessel dilation (3, 46).
Only recently have studies appeared elucidating purinergic vasomotor
responses and receptor distribution in cerebral vessels (Table 1). In
dog cerebral arteries, ATP, coreleased from perivascular sympathetic
nerves, stimulates P2X1 receptors, causing transient constriction (39, 40). However, in rat cerebral arteries, ATP causes endothelium-dependent dilation through P2Y2
receptors, while 2-methylthio-ATP (2-MeSATP) induces dilation through
P2Y1 receptors (60). Constriction induced by
stimulation of P2X1 receptors by ,-methylene-ATP
(,-MetATP) was less effective than that induced by ATP
(60). In rat cerebral artery and bovine middle cerebral
artery, UTP acts on endothelial and smooth muscle P2Y2
receptors, with stimulation of the endothelial receptors directly
causing dilation (60) or opposing smooth muscle-induced vasoconstriction (38). In large cerebral vessels, ATP and
UTP appear to act predominantly through endothelial P2Y2
receptors (60), with the smooth muscle possibly
contracting via P2Y2 receptors in addition to
P2X1 receptors (Table 1).
In cerebral microvessels, purinergic receptor distribution has not been identified. However, several studies found that ATP elicits vasodilator and vasoconstrictor responses after intra- or extraluminal administration in vivo and in vitro (14, 25-27, 34, 49, 61). Tested only in mouse, UTP constricted pial arterioles in vivo independent of the endothelium (48, 50).
Since vessel reactivity to agonists changes along the cerebrovascular tree (61), extrapolation of the results obtained from the macrocirculation to microvessels could be problematic. Penetrating cerebral arterioles, which originate from pial vessels and supply the cerebral cortex, represent the last smooth muscle vessels before the blood is distributed into the capillary bed. These vessels may contribute as much as 23% of the total arterial cerebrovascular resistance (47), making them an important regulator of cerebral blood flow. Since little is known about the distribution of purinergic receptors on these important resistance vessels, we evaluated vasomotor responses to extraluminal UTP and compared them with those induced by ATP using isolated, pressurized cerebral-penetrating arterioles of the rat. We used P2-purinoceptor antagonists and agonists to elucidate pharmacologically the type of P2 purinoceptor involved in the purinergic transmission in these vessels (Table 1). To differentiate between smooth muscle- and endothelium-mediated responses, we impaired endothelial function using air embolization in some vessels.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation and cannulation. Studies were approved by the Washington University Animal Studies Committee. The procedure for preparation and cannulation has been described previously (13, 14). Briefly, male Sprague-Dawley rats (n = 49), weighing 383 ± 9 g, were anesthetized with pentobarbital sodium (65 mg/kg ip) and killed. The brain was removed and transferred to a dissection chamber filled with physiological salt solution (PSS; 4°C; see below) with 1% BSA. A penetrating arteriole was isolated from the middle cerebral artery and transferred to a temperature-controlled organ bath (2.5 ml volume) mounted on the stage of a Nikon or Zeiss inverted microscope. The arteriole was cannulated at one end with glass pipettes. The other end was occluded. All experiments were conducted without intraluminal flow. The transmural pressure (60 mmHg) was monitored continuously with a pressure transducer (model P23, Gould, Cleveland, OH) and recorded on a strip chart recorder (model 3200, Gould). The internal diameter of the vessel was observed with a high-resolution videocamera (CCD 72 with GenIIsis, Dage-MTI, Michigan City, IN) and displayed on a monitor. The chamber temperature was increased to 37.5°C, and the vessels were allowed to develop spontaneous tone for 45 min at pH 7.3. The chamber was superfused with PSS without BSA at a constant flow rate (0.5 ml/min) with a peristaltic pump (model 203, Scientific Industries, Bohemia, NY). The arterioles developed spontaneous tone, and we assessed their responsiveness and viability by changing the extraluminal pH from 7.3 to 6.8 and from 7.3 to 7.65. We discarded vessels with poor tone (<20% decrease from the maximum diameter) or poor pH response (<15% diameter change). In some vessels, we compared vessel tone and pH-induced responses at the beginning and end of experiments to confirm the stability of the preparation.
Diameter measurements. We used a calibrated video-dimensional analyzer (modified model 321, Colorado Video, Boulder, CO) (13, 14) and computerized diameter tracking system (video resolution 320 × 200 pixels with 256 shades of gray; Diamtrak, Montech) to measure the internal vessel diameter. The resolution of this tracking system was 0.5 µm/pixel with a sample rate of 9-10 Hz. The data were recorded on a strip chart recorder and stored digitally.
Experimental protocols.
In the first series of experiments, dose-response curves of UTP,
UDP, ,-MetATP, and 2- MeSATP ranging from 1012 to
104 M were obtained. UTP was used as
P2Y2(P2U)-, P2Y4-, and weaker P2Y6-subtype agonists (9, 42, 45, 46). UDP is
selective for the P2Y6 subtype (42), and
,-MetATP is selective for the P2X1 subtype (28,
59). 2-MeSATP is a P2Y1(P2Y)- and a
P2X1-subtype agonist (Table 1) (45, 59).
6 M propidium iodide
extraluminally before and after air embolism and counted stained
endothelial and smooth muscle cells in the field of view. Staining of
cell nuclei indicated cell damage, which we visualized using
epifluorescence with a standard TRITC filter set. Air embolization does
not necessarily remove the endothelium but has been shown to disrupt
the endothelium in microvessels (52). Only vessels that
regained spontaneous tone after air embolization were accepted for data
analysis. We used sodium nitroprusside (SNP) to ensure that vascular
smooth muscle dilation was not changed before and after air embolization.
In the third series of experiments, 106 M ,-MetATP
was used to desensitize P2X1 receptors (28,
46). Dose-response curves of UTP and 2-MeSATP and responses to
104 M ATP were conducted in the absence or presence of
,-MetATP. We also used 3 × 106 M pyridoxal
phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS) as another
P2X1 antagonist (59) to analyze responses to
104 M ATP, 104 M UTP, 104 M
2-MeSATP, and 106 M ,-MetATP.
In the fourth series of experiments, dose-response curves for UTP were
obtained in the absence and presence of 106 M suramin (a
P2-purinoceptor antagonist) or 104 M PPADS (a
P2X1- and P2Y-receptor antagonist) (9,
45, 46). We also tested 104 M ATP and compared its
response with that of UTP. The incubation periods were 
20 min for
each antagonist.
Drugs and solutions. The composition of the PSS (in mmol/l) was as follows: 144 NaCl, 3 KCl, 2.5 CaCl2, 1.4 MgSO4, 2.0 pyruvate, 5.0 glucose, 0.02 EDTA, 2.0 MOPS, and 1.21 NaH2PO4. Solutions used for dissection and cannulation contained 1% BSA.
All drugs were obtained from Sigma Chemical (St. Louis, MO).Statistical analysis. Only one vessel was studied from each rat brain. Experimental values represent means ± SE; n indicates the number of vessels used in the present study. For dose-response curves, the data are presented as absolute diameter. To demonstrate the effect of antagonists or endothelial impairment on the vessel responses, we expressed the magnitude of constriction and dilation as percent changes from resting diameter (control before administration of drug defined as 0%) and compared the diameter changes before and after the inhibition. Statistically significant differences (P < 0.05) were determined by repeated-measures ANOVA with Bonferroni's multiple comparisons test as posttest or paired Student's t-test as appropriate.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The passive maximum vessel diameter of 49 cerebral arterioles was
65.7 ± 1.4 µm. The vessels developed spontaneous tone,
constricting by 28.3 ± 0.9% to an average diameter of 47.3 ± 1.3 µm. They dilated by 25.9 ± 1.4% to pH 6.8 and
constricted by 
27.9 ± 1.0% to pH 7.65.
Stability of the preparation.
In six arterioles, the pH challenge was repeated at the end of
experiment. The duration of the experiments was 180 ± 22 min. We
found no significant difference in vessel tone (32.3 ± 2.9 and
33.4 ± 4.4% at the beginning and end, respectively),
acidosis-induced dilation (34 ± 1.9 vs. 26 ± 4.3%), and
alkalosis-induced constriction (28.2 ± 1.5 vs. 29.0 ± 3.3%).
Responses to P2-purinoceptor agonists.
The concentration-response curves to UTP, UDP, 2-MeSATP, and
,-MetATP are shown in Fig. 1,
A and B. Since the data for
the UTP dose-response curves used in the second and fourth series did
not differ statistically, we pooled these into the data shown in Fig.
1A. Representative traces of the vasomotor responses to these agonists are shown in Fig. 1C. UTP, UDP, 2-MeSATP, and
,-MetATP dilated arterioles at the lowest concentration
(1012 M) and constricted them at higher concentrations
(104 and 106 M, except for UDP at
106 M; Fig. 1, A and B).
Furthermore, responses of these agonists were monophasic; i.e., only
dilation or constriction was observed. The maximum dilations of UTP,
UDP, 2-MeSATP, and ,-MetATP were 10.7 ± 2.0%
(n = 23), 4.8 ± 2.3% (n = 6),
5.8 ± 1.3% (n = 10), and 7.6 ± 5.9%
(n = 5), respectively, at 1012 M
(P < 0.05 vs. control). The maximum constrictions to
all agonists except ,-MetATP were observed at 104
M. For ,-MetATP, 106 M induced maximum
constriction. The maximum constriction values for UTP, UDP, 2-MeSATP,
and ,-MetATP were 25.2 ± 1.8% (n = 23),
7.0 ± 1.1% (n = 6), 22.5 ± 4.3%
(n = 10), and 17.3 ± 2.0% (n = 5), respectively. UDP was ~3.5-fold less potent than UTP. UTP- and
UDP-induced constrictions were steady and sustained, while 2-MeSATP and
,-MetATP evoked only a temporary constriction (Fig.
1C). There is a marked difference in the time course of the
vasomotor effect of the pyrimidines (UTP and UDP) compared with the
purine analogs (2-MeSATP and ,-MetATP). We previously reported
that, at high concentrations, extraluminal ATP evoked a biphasic
response: initial temporary constriction (18%) followed by dilation
(17%) (14, 27) (Figs. 1C and
2B). None of the other
agonists used in this study showed such dual behavior. This may
indicate that ATP acts on different receptors/signal transduction pathways to elicit the various responses.
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Effects of air emboli on ATP- and UTP-induced responses.
Endothelial impairment by air embolization significantly constricted
arterioles from 48.1 ± 3.4 to 43.6 ± 2.9 µm (9.4% of control diameter before air embolization), indicating endothelial disruption. Under control conditions, neither smooth muscle cells nor
endothelial cells were stained by propidium iodide. After air
embolization, 12.2 ± 1.9 endothelial cells/250 µm and 0.7 ± 0.3 smooth muscle cells/250 µm vessel length were stained with propidium iodide, indicating that predominantly endothelial cell damage
had occurred (Fig. 2A). In five additional experiments, we
applied the endothelium-independent nitric oxide donor SNP. The
dilation in response to 107 and 105 M SNP
(11.3 ± 2.6 and 21.4 ± 2.0% before air embolization vs. 11.8 ± 2 and 19.5 ± 0.9% after air embolization) was not
altered by air embolization (n = 5). ATP-induced
initial constrictions were not altered by the endothelial impairment;
however, secondary dilations were significantly inhibited (Fig.
2B). Air embolization also attenuated UTP-mediated dilation
and, in contrast to ATP, potentiated vessel constriction (Fig.
2C). These results demonstrate that UTP and ATP dilate
arterioles via endothelial stimulation and that UTP- but not
ATP-mediated constriction is decreased by an intact endothelium.
Effects of P2X1-purinoceptor desensitization by
,-MetATP.
,-MetATP (106 M) itself constricted the vessel
transiently, with the arteriole subsequently returning to its control
diameter (Fig. 1C). ATP- and 2-MeSATP-induced constrictions
were attenuated in the presence of 106 M ,-MetATP,
which indicates that P2X1-receptor stimulation is involved
in the contractile response (Figs.
3A and
4). In contrast, 106 M ,-MetATP did not attenuate the dilation of
2-MeSATP or ATP (Figs. 3A and 4). The dose response of UTP
was not affected by ,-MetATP (Fig. 3B).
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Effects of PPADS on vasoconstrictor and vasodilator responses.
PPADS had no effect on the basal vessel diameter. PPADS at 3 × 106 M (P2X1-receptor antagonist at this
concentration) significantly inhibited constrictions of 2-MeSATP,
,-MetATP, and ATP, but not UTP (Figs.
5 and 6).
These results further support that ATP, 2-MeSATP, and
,-MetATP, but not UTP, constrict the vessel through P2X1-receptor stimulation. The ATP-induced dilation was
neither attenuated nor potentiated by 3 × 106 and
104 M PPADS (Fig. 6). However, in the presence of
104 M PPADS (P2X1- and
P2Y-receptor antagonists at this concentration) (45,
46), UTP-induced constrictions were reversed, with dilation occurring at high concentration (Fig. 7).
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Effects of suramin on ATP- and UTP-induced vasoconstriction and
vasodilation.
Suramin (106 M) had no effect on the UTP- and ATP-induced
responses (n = 4, data not shown). We could not use
higher concentrations of suramin (up to 104 M), as used
in other studies (36, 51), because suramin induced vasodilation (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The new findings in the present study are as follows:
1) The pyrimidines UTP and UDP and the ATP analogs 2-MeSATP
and ,-MetATP applied extraluminally dilate isolated, pressurized
rat cerebral-penetrating arterioles at the low concentration
(1012 M) and constrict the vessels at high concentrations
(>106 M). 2) Constrictions induced by the
pyrimidines UTP and UDP and the ATP analogs 2-MeSATP and ,-MetATP
were monophasic; no biphasic response with initial constriction
followed by a secondary dilation, as seen with ATP, occurred (Fig.
1C) (14, 27). 3) UDP was ~3.5-fold
less potent than UTP. 4) Using pharmacological tools and
endothelial impairment, we suggest that ATP and 2-MeSATP induce transient constriction via smooth muscle P2X1-receptor
stimulation. 5) UTP stimulates two P2Y-receptor
types, resulting in dilation via stimulation of endothelial
P2Y2 receptors and sustained constriction through smooth
muscle P2Y4 receptors. 6) ATP and UTP produce
dilation by the same receptor stimulation, probably endothelial
P2Y2.
Purine (ATP, 2-MeSATP, and ,-MetATP)-mediated
vasoconstriction in cerebral arterioles.
At high concentrations 2-MeSATP and ,-MetATP caused transient
vasoconstriction similar to ATP (Fig. 1C). However, 2-MeSATP and ,-MetATP did not cause a secondary dilation, as observed with
ATP stimulation (Figs. 1C and 2B) (14,
27). Constrictions induced by ATP, 2-MeSATP, and ,-MetATP
were diminished after desensitization of the P2X1 receptor
by ,-MetATP or pretreatment with 3 × 106 M
PPADS (Figs. 3A, 4, and 5). Our results are consistent with ATP and 2-MeSATP mediating transient constrictions via stimulation of
P2X1 receptors. In addition, ATP mediates a secondary
dilation that is not mimicked by 2-MeSATP or ,-MetATP, indicating
that ATP also acts on other P2 receptor(s). Our results are
similar to those reported after sympathetic stimulation in dog cerebral artery (41) but differ from those found in isolated rat
cerebral artery. In the latter studies, extraluminal ATP and 2-MeSATP
caused a sustained constriction, while stimulation with ,-MetATP
caused some constriction at 106 M but a vasodilation at
104 M (60). This would suggest that large
cerebral vessel vasoconstriction is the result of stimulation of
P2X1 and P2Y receptors, which differs from the
results observed in smaller cerebral vessels.
Purine-mediated vasodilation in cerebral arterioles. 2-MeSATP is a strong vasodilator of large cerebral arteries, possibly via endothelial P2Y1 receptors (61). In our study, extraluminal application of 2-MeSATP caused only a small vasodilation at lower concentration (Fig. 1B). You and co-workers (61) reported that the intraluminal application of 2-MeSATP did not significantly dilate third-order branches of the middle cerebral artery and large penetrating arterioles of the rat, while it did induce a pronounced dilation of the middle cerebral artery. They hypothesized that this heterogeneity of 2-MeSATP-induced responses might result from an altered distribution or function of P2Y1 receptors along the vascular tree.
Since 2-MeSATP can stimulate P2X1 and P2Y1 receptors, another possible explanation is that 2-MeSATP also stimulated P2X1 receptors, thus counteracting a vasodilator P2Y1 effect. In our experiments, 2-MeSATP-mediated dilation was not potentiated after desensitization of P2X1 receptors (Fig. 3A). In addition, the ATP-induced dilation was unaffected by the desensitization of P2X1 receptors (Fig. 4), and endothelial impairment did not potentiate ATP-induced constrictions (Fig. 2B). If stimulation of P2X1 receptors were to counteract a vasodilator effect of 2-MeSATP and ATP in our preparations, agonist-induced dilation should be potentiated by the desensitization of P2X1 receptors, and constriction should be enhanced after the endothelial impairment. Our results indicate that transient constrictions by a high concentration of ATP and 2-MeSATP did not result from the competition between constriction and dilation. Thus ATP and 2-MeSATP probably stimulated and then desensitized P2X1 receptors, causing the temporary constriction. Such desensitization has been previously reported in numerous tissues (39, 46). We suggest either that 2-MeSATP is not a potent agonist of P2Y1 receptors in rat cerebral arterioles or that there is only a limited distribution of P2Y1 receptors on the endothelium. In either case, ATP does not dilate the cerebral arteriole through the P2Y1 receptor in penetrating cerebral arterioles. The small dilation produced by,-MetATP at lower concentrations
has been occasionally observed in the rat middle cerebral artery
(60) and mouse pial arteriole (49). Although
the mechanism for this vasodilation is unclear, it is possible that low
concentration of ,-MetATP might also stimulate P2Y receptors.
Pyrimidine (UTP and UDP)-mediated vasomotor responses in cerebral
arterioles.
Recently, it has become apparent that initial tone and route of
administration are important factors determining the magnitude and
direction of vascular responses to UTP in vitro (20, 38, 59,
60). In the rat middle cerebral artery, UTP induces a dose-dependent dilation with intraluminal administration or
constriction with extraluminal administration (60). UTP
relaxed precontracted human pial artery strips at
107-105 M and constricted them at high
concentration (20). Endothelial removal potentiated the
UTP-induced constriction in human pial artery and bovine middle
cerebral artery strips (20, 38).
Receptors involved in pyrimidine-induced vasoconstriction and vasodilation. There are marked differences in purine- and pyrimidine-induced constrictions with respect to receptor subtype, desensitization, and the release and inhibition of the other neurotransmitters (53, 58). P2X1 receptors are desensitized by purines such as ATP, while P2Y receptors do not readily desensitize (46).
In the present study, UTP-evoked constrictions in rat cerebral arterioles were resistant to desensitization and blockade of P2X1 receptor by,-MetATP and 3 × 106 M PPADS. The endothelial impairment diminished
UTP-mediated dilations at low concentrations and potentiated the
constriction at high concentrations (Fig. 2C), with
UTP-induced constriction abolished by 104 M PPADS.
Furthermore, 104 M PPADS converted the UTP-induced
constriction to a strong dilation (Fig. 7). These results indicate that
1) UTP-evoked constriction is not due to the stimulation of
P2X1 receptors in the rat cerebral arteriole, 2)
vascular responses to UTP result from the competition of endothelial
and smooth muscle receptor stimulation, 3) 104
M PPADS is an antagonist of UTP-induced constriction, and 4) UTP-induced dilation is not affected by 104 M PPADS. Thus
UTP appears to stimulate two distinct P2Y-purinoceptor subtypes present simultaneously on the smooth muscle and endothelium.
Smooth muscle P2Y2-, P2Y4-, and
P2Y6-receptor subtypes, working through the phospholipase
C-inositol (1,4,5)-trisphosphate-Ca2+ cascade,
elicit pyrimidine nucleotide-induced constriction due to increase of
intracellular Ca2+ activity (3, 23, 46). UDP
is a strong P2Y6 agonist (42) and a partial
agonist at P2Y2 and P2Y4 receptors (4, 9, 42), while UTP is a potent agonist at P2Y2 and
P2Y4 receptors (9, 42). In the present study,
the rat cerebral-penetrating arterioles constricted less to UDP than to
UTP, suggesting that the UTP-evoked constriction is not caused by
stimulation of P2Y6 receptors. Thus UTP-induced responses
are probably more likely due to stimulation of P2Y2- and/or
P2Y4-receptor subtypes.
High concentrations of PPADS are considered to act as a general
P2Y-receptor inhibitor. However, in several studies, PPADS inhibited only P2Y1, P2Y4, and, to a lesser
degree, P2Y6 receptors, but not P2Y2 receptors
(9, 10, 45, 46). Our data would suggest that, in rat
cerebral arterioles, UTP may induce constriction via activation of
smooth muscle P2Y4 receptors and dilation via endothelial
P2Y2-receptor stimulation, respectively. We applied suramin, which may antagonize P2Y2 receptors and not
P2Y4 receptors (4, 9, 46). However,
106 M suramin did not inhibit UTP- or ATP-mediated
responses. These results suggest that suramin at this concentration
does not antagonize P2 purinoceptors in our preparation.
Higher concentrations of suramin (up to 104 M) (36,
51) and, similarly, reactive blue-2 (general P2Y antagonist) (46) induced a strong vasodilation and thus
could not be used in our experiments (data not shown). While the lack of a specific P2Y2 antagonist in our preparation does not
exclude an unknown mechanism involved in UTP-induced dilation, the
results obtained with PPADS support that the observed dilation may be due to P2Y2-receptor stimulation.
To further substantiate that UTP causes constriction via
P2Y4-receptor stimulation, we compared the vasoconstriction
induced by UTP with that produced by ATP. UTP and ATP are considered to be equipotent agonists for P2Y2 receptors (46)
located on the endothelium and smooth muscle (3, 33, 50).
Stimulation of endothelial and smooth muscle P2Y2 receptors
causes dilation and constriction, respectively (3, 38,
51). UTP- and ATP-induced constrictions via
P2Y2-receptor stimulation were reported in rat pulmonary
(51) and bovine middle cerebral (38)
arteries. In the present study, ATP caused constriction via
P2X1 but not P2Y2 receptors (Fig. 4 and 6).
This result supports the idea that UTP-induced constriction is mediated
via P2Y4 receptors, a finding similar to that reported in
the canine epicardial coronary circulation (33).
UTP- and ATP-mediated dilation in cerebral arterioles. P2Y1 and P2Y2 receptors are generally located on the endothelium, and their stimulation causes dilation (3, 46). These receptors are G protein coupled, with the most common pathway for P2Y-receptor signaling activation of phospholipase C leading to inositol (1,4,5)-trisphosphate release. This causes Ca2+ mobilization and activation of protein kinase C, phospholipase A2, or Ca2+-dependent K+ channels, leading to the formation of an endothelial nitric oxide- or prostanoid-based relaxation factor depending on species and P2y-receptor type studied (46). However, it is interesting that there is also evidence for relaxation via smooth muscle P2Y1 receptors in some blood vessels (12, 57).
At low concentrations, UTP induced dilation in rat cerebral arterioles, which dose dependently increases in the presence of 104 M
PPADS. ATP-induced dilation following constriction was not inhibited by
PPADS. Higher concentrations of PPADS
(105-104 M) have been used as
P2Y1-receptor antagonists (45, 59). These
results suggest that 1) the receptor subtype mediating the dilation induced by UTP is different from that mediating the
constriction induced by UTP and similar to that mediating the dilation
induced by ATP and 2) UTP and ATP probably mediate dilations
via endothelial P2Y2-receptor stimulation in the rat
penetrating arteriole. In the cerebral circulation, release of nitric
oxide and endothelium-derived hyperpolarizing factor, rather than
prostacyclin, appears to be involved in the dilation to endothelial
P2Y1- and P2Y2-receptor stimulation (25,
26, 38, 60, 61). In rat cerebral artery, endothelial
P2Y1 stimulation causes exclusively nitric oxide release, while P2Y2 stimulation released nitric oxide and another
not yet defined relaxation factor (60). In rat
cerebral-penetrating arterioles, scavenging of nitric oxide with
oxyhemoglobin reduced ATP-induced vasodilation at low but not at high
concentrations (27). These results indicate that, in these
vessels, ATP may initially release nitric oxide at low ATP
concentrations and another yet unidentified relaxation factor is
released at high ATP concentrations. Second messenger systems, which
may account for the observed difference in mediators released, have not
been studied in the cerebral microcirculation.
Sources for ATP and UTP and possible physiological relevance. Natural sources for ATP in the brain include corelease from perivascular sympathetic nerves to modulate pressor responses (40, 41), which could overflow into the vessel lumen to elicit vasomotor responses downstream (2). Release from cerebral nerves (30) may act on cerebral vessels directly via diffusion or indirectly through astrocyte stimulation (7, 21). ATP is also released from red blood cells during hypoxia and/or acidosis, causing vasodilation (1, 16), and may be a mechanism for sensing metabolic need in the tissue (1). Platelets release basal amounts of ATP (22, 44), which acts to inhibit platelet aggregation (56) via nitric oxide generation (37), while platelet activation is associated with the release of large amounts of ATP (17). Thus, in rat cerebral arterioles, intraluminal ATP may be mainly involved in maintaining blood flow by mediating hypoxic vasodilation and inhibiting platelet activation. Since ATP can diffuse from the vessel lumen to arteriolar smooth muscle cells (35), transient smooth muscle constriction may counteract an overdilation due to large amounts of intraluminal ATP, preventing vessel damage. However, massive ATP release from brain tissue after trauma or platelet activation may have only a short-term effect on arteriolar vessel constriction (due to P2X1-receptor desensitization), while the large cerebral arteries would develop vasospasm due to smooth muscle P2Y-receptor stimulation (32, 61).
Cerebral sources for UTP are less well studied, and its physiological function is less well described (31). UTP is likewise liberated from platelets (44) and has been shown to be released from neuronally derived tissue such as chromaffin cells (19). Furthermore, UTP and ATP were found in high concentrations in synaptosomes of cerebral tissue (8, 43), suggesting that ATP and UTP can be released into the tissue after trauma. While a physiological function for ATP in the cerebral circulation is apparent (18, 29), such a role for UTP is less clear. Our data show that UTP dilates penetrating arterioles at low concentrations. We speculate that UTP, similar to ATP, may be involved in maintaining local blood flow by preventing platelet aggregation and countering smooth muscle tone. Pathologically, a massive release of UTP from brain tissue and/or platelets could be responsible for the vasospasm observed after cerebral injury and subarachnoid hemorrhage. In conclusion, we analyzed the responses to UTP, ATP, and its analogs in the rat cerebral arteriole. ATP and 2-MeSATP induce temporary constriction via P2X1 receptors on the smooth muscle cell. ATP-mediated dilation is due to endothelial P2Y2-receptor stimulation. In contrast, UTP causes sustained constriction at high concentrations and dilation at low concentrations. UTP probably activates P2Y4 receptors in the smooth muscle cell, causing constriction, and P2Y2 receptors on the endothelial cell, causing dilation. P2Y4-receptor stimulation of UTP may counteract the dilatory response of P2Y2 receptors to UTP.| |
ACKNOWLEDGEMENTS |
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We are very grateful to Dr. Mary L. Ellsworth for invaluable suggestions and to Robyn L. Reese for excellent technical assistance.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grants HL-57540 and NS-30555.
Address for reprint requests and other correspondence: H. H. Dietrich, Dept. of Neurosurgery, Washington University School of Medicine, Box 8057, 660 South Euclid Ave., St. Louis, MO 63110 (E-mail: dietrich_h{at}kids.wustl.edu).
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.
Received 1 March 2000; accepted in final form 3 August 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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