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(Stroke. 2003;34:1473.)
© 2003 American Heart Association, Inc.

Original Contributions

Comparison of P₂ Receptor Subtypes Producing Dilation in Rat Intracerebral Arterioles

Tetsuyoshi Horiuchi, MD; Hans H. Dietrich, PhD; Kazuhiro Hongo, MD Ralph G. Dacey, Jr, MD

From the Department of Neurosurgery, Washington University School of Medicine, St Louis, Mo (T.H., H.H.D., R.G.D.), and Department of Neurosurgery, Shinshu University School of Medicine, Matsumoto, Japan (T.H., K.H.).

Correspondence to Hans H. Dietrich, PhD, Department of Neurosurgery, Washington University School of Medicine, Box 8057, 660 S Euclid Ave, St Louis, MO 63110. E-mail DietrichH{at}Nsurg.wustl.edu

	Abstract

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Background and Purpose— P₂ receptors are important regulators of cerebrovascular tone. However, there is functional heterogeneity of P_2Y receptors along the vascular tree, and the functionality of P_2Y receptors in small arterioles has not been studied in detail. We investigated the effects of activating P_2Y1 and P_2Y2 receptors and their underlying dilator mechanisms in rat intracerebral arterioles.

Methods— We used computer-aided videomicroscopy to measure diameter responses from isolated and pressurized rat penetrating arterioles (39.9±1.2 µm) to the natural P₂ receptor agonist ATP in addition to ADP-ß-S (P_2Y1-selective) and ATP--S (P_2Y2-selective) and inhibitors of signaling pathways.

Results— Extraluminal application of ATP--S and ADP-ß-S initiated a biphasic response (initial constriction followed by the secondary dilation) similar to ATP-induced responses. Pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (0.1 mmol/L; a P_2Y1 receptor antagonist) blocked ADP-ß-S- but not ATP--S-induced dilation and affected the ATP-mediated dilation at low concentrations. N-Monomethyl-L-arginine partially inhibited the dilation of ATP and ADP-ß-S but not ATP--S. High K⁺ saline suppressed the dilation of all agonists. Indomethacin had no effect.

Conclusions— Both P_2Y1 and P_2Y2 receptors are functionally present in cerebral arterioles. ATP stimulates P_2Y1 receptors at low concentrations, while high concentrations of ATP activate P_2Y2 in addition to P_2Y1 receptors. Nitric oxide is involved in P_2Y1 but not P_2Y2 receptor activation. Potassium channels play an important role in the regulation of P_2Y receptor-mediated dilation.

Key Words: adenosine • cerebral circulation • nitric oxide • potassium channels • receptors, purinergic P₂

	Introduction

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The importance of purinergic regulation has been recognized in the cerebral circulation. ATP is one of the purines and the natural agonist to control blood flow. Purines released from the parenchyma may be important in regulating microvascular blood flow.¹ Both vasoconstriction and vasodilation can be produced by ATP in the cerebral vasculature.^2,3 These vasomotor responses depend on distribution of P₂ receptor subtypes. Thus, species, location, and size of vessels greatly influence the vasomotor response to ATP.³

It has been demonstrated that both endothelial P_2Y1 and P_2Y2 receptors are present and their stimulation dilates rat middle cerebral artery.^4–6 In contrast, we² and others⁴ speculated that P_2Y1 receptor may not be present or its function may be silent in the cerebral microcirculation under physiological conditions because 2-methylthio-ATP (2-MeSATP; a P_2Y1-receptor agonist) did not dilate the cerebral arteriole effectively. However, P_2Y1 and P_2Y2 receptors have been detected in primary cultures of rat brain capillary endothelium.^1,7 In rat middle cerebral artery, P_2Y1 releases only nitric oxide (NO), while P_2Y2 stimulation liberates both NO and endothelium-derived hyperpolarizing factor (EDHF).⁵ It is of interest that the involvement of NO in response to ATP-induced dilation decreases along the cerebrovascular tree, whereas EDHF seems to be the major contributor to ATP-induced dilation in smaller vessels.⁴

It is not known whether P_2Y1 receptors are functionally present in penetrating arterioles, which are important regulators of cerebral microvascular blood flow. Furthermore, the mechanism of P_2Y1 receptor-induced dilation has not been studied. Therefore, in rat isolated cerebral arterioles, we evaluated the functional P_2Y receptor distribution using ADP-ß-S (a selective P_2Y1 receptor agonist) and ATP--S (a selective P_2Y2 receptor agonist) on arteriolar diameter. We compared the results with the natural agonist ATP and also investigated the relaxing factor(s) released by these agonists.

	Materials and Methods

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The Animal Studies Committee at Washington University approved the experimental protocol for this study. Fifty-six male Sprague-Dawley rats (weight, 350 to 450 g; Harlan, Indianapolis, Ind) were anesthetized with pentobarbital sodium (65 mg/kg IP) and decapitated. The brain was immediately removed and stored in cold physiological salt solution (PSS). The technique for isolation and cannulation of the intracerebral arteriole has been showed in previous studies.^8–10 In short, the cerebral penetrating arterioles were obtained from the proximal portion of middle cerebral artery. An arteriole without branches was transferred to a temperature-controlled organ bath and cannulated with glass pipettes. The luminal pressure was maintained at 60 mm Hg, and no luminal perfusion was given in all experiments. The vessel was magnified with an inverted microscope (Diaphot, Nikon) equipped with a video camera and monitor. The internal diameter of the vessel was measured with a computerized diameter tracking system (Diamtrak, Montech Pty Ltd; 320x200 pixels; 0.5 µm per pixel), and the measurements were stored digitally (WinDaq, DataQ Instruments) for further evaluation.

After the diameter was measured at 60 mm Hg (maximum diameter [D_max]), the organ bath temperature was brought to 37.5°C with circulating PSS (pH 7.3) at a rate of 0.5 mL/min. After an equilibration period, the arterioles developed spontaneous tone (D_tone), constricting by at least 20%. Subsequently the vessel viability was confirmed with diameter responses to acidosis (pH 6.8) and alkalosis (pH 7.65).

Only 1 arteriole was used from each brain. Vasomotor responses to ATP, ATP--S (selective P_2Y2 receptor agonist),³ and ADP-ß-S (selective P_2Y1 receptor agonist)³ were determined by changing the bath solution in a cumulative manner. Initially, we generated a concentration-response curve for each agonist as control response. Then we reapplied the agonist in the presence of the inhibitor. We applied only 1 agonist on each vessel and generated concentration-response curves maximally 3 times per vessel. In preliminary experiments, we found no difference among 3 concentration-response curves obtained in the same vessel.

We investigated effects of 0.1 mmol/L pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on ATP-, ATP--S-, and ADP-ß-S-induced responses. We² have previously shown that 0.1 mmol/L PPADS is an antagonist for P_2X1 and P_2Y1 but not P_2Y2 receptors in this preparation.

The effects of 10 µmol/L N-monomethyl-L-arginine (L-NMMA; a NO synthase inhibitor)⁹ or 10 µmol/L indomethacin (cyclooxygenase inhibitor) on ATP-, ATP--S-, and ADP-ß-S-mediated vessel responses were examined in response to the 3 agonists. We also used the high-K⁺ saline (30 and 80 mmol/L) to negate any effects of K⁺ channels.¹¹ Isotonic high-K⁺ PSS was made by substituting NaCl with an equimolar amount of KCl.

The composition of PSS was as follows (in mmol/L): 144 NaCl, 3 KCl, 2.5 CaCl₂, 1.4 MgSO₄, 2.0 pyruvate, 5.0 glucose, 0.02 EDTA, 2.0 3-(N-morpholino) propanesulfonic acid (MOPS), 1.21 NaH₂PO₄. ATP, ATP--S, ADP-ß-S, L-NMMA, indomethacin, and PPADS were obtained from Sigma Chemical.

All data are presented as mean±SEM; n represents the number of vessels.

Vessel tone is calculated as follows: %Tone=[(D_max-D_tone)/ D_max]x100.

For concentration-response curves, the results are presented as percentages of the maximal dilation of the vessel, calculated as follows: %Maximum Dilation=[(D_agonist-D_base)/(D_max-D_base)]x100, where D_max is the maximum diameter of the vessel at 60 mm Hg before the development of spontaneous tone, D_tone is the spontaneous tone, D_base is the baseline diameter of the vessel before the stimulation with agonist, and D_agonist is the diameter of the vessel after agonist stimulation. D_max is identical to the diameter produced by papaverine¹² and calcium-free buffer.¹³ With this method, the maximal dilation is represented as 100%, and baseline diameter is 0%. Dilation from baseline is presented as positive and constriction as negative relative values.

Comparisons were made with the use of paired Student’s t test or ANOVA with a post hoc Bonferroni test, as appropriate. The acceptable level of significant was defined as P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
All vessels (n=56) developed spontaneous tone and responded to pH challenge (Table). There were no significant differences in passive diameter, spontaneous tone, dilation to acidosis, and constriction to alkalosis between vessel groups (ATP, ATP--S, or ADP-ß-S) (Table).

View this table: [in this window] [in a new window]   Passive Diameter, Tone Diameter, and pH Response in Isolated Rat Intracerebral Arterioles

Previous studies in our laboratory showed that extraluminal ATP caused a biphasic response consisting of initial constriction followed by secondary dilation.^2,10 Extraluminal ATP--S and ADP-ß-S also produced dilation after constriction, as seen with ATP (Figure 1). We previously demonstrated that the initial constriction is caused by smooth muscle cell P_2X1 receptors.² We also showed that this constriction did not affect the subsequent dilation.² The following results, therefore, focus on the secondary dilation induced by the agonists. Group data for concentration responses of ATP, ATP--S, and ADP-ß-S are shown in Figure 1. Dilator responses to ATP--S and ADP-ß-S (10 and 100 µmol/L) were significantly more potent than those to ATP. ATP, ATP--S, and ADP-ß-S at 100 µmol/L produced 60.1±4.3%, 90.2±2.5%, and 90.2±2.5% of maximal dilation, respectively. Furthermore, ATP showed a right-shifted EC₅₀ value of 9.69 µmol/L compared with ATP--S (EC₅₀=6.40 µmol/L) and ADP-ß-S (EC₅₀=3.72 µmol/L). We compared the control dose-response curves for ATP, ATP--S, and ADP-ß-S in Figure 1 with the respective curves in the subsequent protocols and found them to be not statistically different (ANOVA).

View larger version (24K): [in this window] [in a new window]   Figure 1. Concentration-response curves comparing vasomotor responses induced by ATP (n=19), ATP--S (P2Y2-receptor agonist; n=17), and ADP-ß-S (P2Y1-receptor agonist; n=20). All agonists cause a biphasic response consisting of initial transient constriction followed by secondary dilation. *P<0.05, significantly different compared with ATP-induced responses.

Figure 2 illustrates the effects of 0.1 mmol/L PPADS (a P_2X1 plus P_2Y1 receptor antagonist at this concentration²) on ATP-, ATP--S-, and ADP-ß-S-induced dilation. PPADS had no effect on the baseline vessel diameter. PPADS significantly inhibited dilation to ADP-ß-S but not to ATP--S. Interestingly, ATP-mediated dilations at 10 nmol/L to 1 µmol/L were inhibited by PPADS, whereas PPADS did not affect 10 µmol/L ATP- and 0.1 mmol/L ATP-induced dilation. The initial constrictions of all agonists were abolished by 0.1 mmol/L PPADS (data not shown).² In preliminary experiments, 3 µmol/L PPADS inhibited the agonist-induced initial constriction. However, 3 µmol/L PPADS did not affect the secondary dilation.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effects of 0.1 mmol/L PPADS on vasomotor responses induced by ATP (n=5), ATP--S (n=4), and ADP-ß-S (n=5). *P<0.05, significantly different compared with the percentage of dilation in presence of PPADS. Note that ATP-mediated dilations at low concentration are inhibited by PPADS.

Figure 3 shows the effects of 10 µmol/L L-NMMA on ATP-, ATP--S-, and ADP-ß-S-induced dilation. The vessels constricted significantly to L-NMMA from 42.2±2.0 to 32.5±1.7 µm. L-NMMA attenuated the dilation of ATP and ADP-ß-S but not ATP--S. In preliminary experiments (2 vessels), 100 µmol/L L-NMMA did not produce further inhibition of the dilation.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effects of 10 µmol/L L-NMMA on vasomotor responses induced by ATP (n=5), ATP--S (n=4), and ADP-ß-S (n=6). *P<0.05, significantly different compared with percentage of dilation in presence of L-NMMA.

Figure 4 shows the effects of 10 µmol/L indomethacin on ATP-, ATP--S-, and ADP-ß-S-induced dilation. Indomethacin affected neither the control diameter nor dilations to the 3 agonists.

View larger version (16K): [in this window] [in a new window]   Figure 4. Effects of 10 µmol/L indomethacin on vasomotor responses induced by ATP (n=4), ATP--S (n=4), and ADP-ß-S (n=4).

Figure 5 demonstrates the effects of 30 and 80 mmol/L KCl on ATP-, ATP--S-, and ADP-ß-S-induced dilation. The high-K⁺ saline (30 and 80 mmol/L) decreased the baseline diameter of the vessels (41.6±2.5 µm [control diameter]; 35.0±2.3 µm [30 mmol/L]; 24.1±2.1 µm [80 mmol/L]) and attenuated the dilation of ATP, ADP-ß-S, and ATP--S in a dose-dependent manner. Papaverine (10 µmol/L) fully dilated 3 vessels in the presence of high-K⁺ saline, indicating that high-K⁺ saline did not cause vascular paralysis.

View larger version (19K): [in this window] [in a new window]   Figure 5. Effects of 30 and 80 mmol/L KCl on vasomotor responses induced by ATP (n=5), ATP--S (n=5), and ADP-ß-S (n=5). *P<0.05, significant difference vs control; P<0.05, significant difference vs 30 mmol/L.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The purpose of the present study was to clarify the functional contribution of P_2Y1 receptors in particular to purinergic stimulation with the use of specific agonists/antagonists and to elucidate the relaxing factors released to specific P_2Y receptor stimulation. We found that ADP-ß-S and ATP--S potently dilate rat intracerebral arterioles, indicating P_2Y1 and P_2Y2 receptor presence, respectively. With the use of the specific P_2Y1 inhibitor PPADS, low concentrations of ATP stimulated P_2Y1 receptors, whereas P_2Y2 receptors were additionally activated by higher concentrations of ATP. NO partially contributed to P_2Y1 receptor- but not P_2Y2 receptor-produced dilation. Cyclooxygenase products were not involved. Potassium channels may play a major role in dilation to both P_2Y1 and P_2Y2 receptor stimulation.

Distribution of P₂ Receptors in Cerebral Arterioles
Extraluminal application of ATP, ADP-ß-S, and ATP--S resulted in a biphasic response consisting of the initial constriction followed by the secondary dilation. The initial constriction was inhibited by PPADS (3 µmol/L and 0.1 mmol/L). PPADS is a P₂ receptor antagonist,³ and its effects depend on concentration, species, and vessel location. In our preparation, 3 µmol/L PPADS inhibits P_2X1 receptor but not P_2Y receptor.² A higher concentration of 0.1 mmol/L PPADS, however, blocks P_2X1 plus P_2Y1 receptors but not P_2Y2 receptors.² These results indicate that both ADP-ß-S and ATP--S activate P_2X1 receptors to induce transient arteriolar constriction. We² reported that biphasic vasomotor responses after extraluminal application of agonists not only resulted in the activation of smooth muscle cells (to cause transient constriction) but also activated the endothelium to cause vessel dilation. In cerebral macro- and micro-circulation, P_2Y2 receptor subtypes function as an important regulator of cerebral blood flow.^2,4–6 However, on the basis of previous studies,^2,4 P_2Y1 receptor subtypes seem to be less important in the regulation of the cerebral microcirculation because 2-MeSATP as a P_2Y1 agonist produced no vasodilation in third-order branches of the middle cerebral artery and large penetrating arterioles of the rat.⁴ These findings suggested that functional P_2Y1 receptors may be absent in the cerebral microcirculation. Recently, our study,² which also used 2-MeSATP, confirmed their results. However, we² also speculated that 2-MeSATP is not a potent P_2Y1 receptor agonist in the cerebral arteriole because 2-MeSATP can also stimulate P_2X1 receptors in our preparation.² However, we excluded that the P_2X1 stimulation has an inhibitory/subtractive effect on ATP-induced secondary dilation.² Another explanation for the apparent lack of potency of 2-MeSATP is based on differences in smooth muscle sensitivity to the relaxing factors along the vessel trees, ie, NO.⁴

In the present study ATP--S in addition to ADP-ß-S dilated the cerebral arterioles of the rat in a dose-dependent manner. PPADS (0.1 mmol/L) can clearly distinguish P_2Y1 from P_2Y2 receptor subtypes in our preparation and inhibited ADP-ß-S-induced dilation. Thus, P_2Y1 receptors are also present in the arteriole and can regulate the vascular tone in the cerebral microcirculation. Furthermore, the water-soluble purinergic agonists we used reached endothelial receptors on the abluminal side to cause endothelium-dependent dilation. Our results are consistent with studies in which P_2Y agonists are used intraluminally.^4–6 However, if endothelial P_2y1 and P_2y2 receptors are heterogeneously distributed on luminal versus abluminal surfaces, it is possible that the effects of the receptor specific agonists we used may differ when applied intraluminally.

ADP-ß-S and ATP--S strongly dilated the vessels compared with ATP. It is a possibility that ATP was metabolized by ectonucleotidases to decrease its efficacy. However, we found that ADP as a dephosphorylated ectonucleotidase product dilated penetrating arterioles in a manner similar to that of ATP.¹⁴ Thus, ATP degradation by ectonucleotidases to cause the reduced dilation seems less likely.

Our results show that high concentrations of ATP (10 to 100 µmol/L) activate P_2Y2 in addition to P_2Y1 receptors, resulting in arteriolar dilation. By contrast, low concentrations of ATP (10 nmol/L to 1 µmol/L) mainly stimulated P_2Y1 receptors. These results indicate that ATP acts as both a P_2Y1 and P_2Y2 receptor agonist, and its function depends on the agonist concentration. Sipos et al⁷ discussed the possible relevance of multiple purinergic receptors on the brain endothelium and concluded that in addition to liberating various dilators such as NO and EDHF, multiple receptors could also contribute to "cross-talk" between second messenger systems.

Mechanisms of P_2Y1 and P_2Y2 Purinoceptor-Induced Vasodilation
It is well known that the activation of endothelial P_2Y1 and P_2Y2 receptors in the cerebral circulation dilates the vessel via different mechanisms.^4,5,15 The stimulation of endothelial P₂ receptors dilates vessels through releasing NO, prostanoid, and/or EDHF in the vasculature.³ However, these relaxing factors may vary from species, size, and location of vessels, even though the P_2Y receptor subtype is the same.³ Prostacyclin seems to be a less important factor in response to endothelial P_2Y receptor stimulation in cerebral circulation.^3,4 These findings are consistent with the present study because inhibition of cyclooxygenase in the intracerebral arteriole had no effect in any agonist-induced dilations. Thus, a cyclooxygenase metabolite such as prostacyclin was not involved in P_2Y-induced dilation. However, we cannot exclude a possible contribution of cyclooxygenase metabolites to P_2Y receptor-induced dilation after NO inhibition because we did not examine the combined effects of L-NMMA and indomethacin.

Our laboratory reported that ATP hyperpolarized the smooth muscle cell, resulting in dilation of the cerebral arteriole,¹⁶ and oxyhemoglobin (a scavenger of NO) partially inhibited ATP-induced dilation.¹⁴ We also reported that an intact endothelium is necessary for the ATP-induced dilation.² These findings indicate that endothelial NO production and a hyperpolarization factor caused the dilation of the cerebral arteriole to ATP. In the rat middle cerebral artery,^4,15 P_2Y1 receptor activation produces vasodilation exclusively through NO release, whereas P_2Y2 receptor activation results in vasodilation via NO and EDHF. Interestingly, EDHF rather than NO prominently contributes to dilation of P_2Y receptor stimulation in the cerebral microcirculation compared with the macrocirculation.⁴

In the present study NO was partially involved in both ATP- and ADP-ß-S-induced (P_2Y1 receptor-induced) dilations. This contribution was observed at low but not high concentration of ADP-ß-S. By contrast, ATP--S (P_2Y2 receptor agonist) dilated the arteriole via a NO-independent pathway. High-K⁺ saline strongly diminished dilation in response to both P_2Y1 and P_2Y2 receptor stimulation, suggesting that potassium channels contribute to the vessel dilation. Potassium channels are one of the main contributors to the EDHF-induced dilation.^17,18 It is possible that NO dilates the vessel via potassium channel stimulation. However, our previous studies^12,19 demonstrated that NO/potassium channel interactions did not seem to be important in the cerebral arteriole. Thus, NO and potassium channel activation may act as the mediator of P_2Y1 receptor independently. On the basis of this finding, the relaxing factors released in response to P_2Y receptor stimulation depended on receptor subtypes and agonist concentrations in rat cerebral arterioles.

Intracellular Mechanisms and Mediators Released to Purinergic Stimulation Along the Cerebrovascular Tree
P_2Y1 and P_2Y2 receptors are G protein coupled.³ However, the subsequent signal transduction pathway linked to P_2Y receptors varies between the cell types even though receptor subtypes on the cells may be identical.³ Nevertheless, an increase in endothelial intracellular Ca²⁺ concentration [(Ca²⁺)_in] is seen as an essential intracellular response.^3,5 In rat middle cerebral artery, Marrelli⁵ reported that P_2Y2 receptor stimulation resulted in a significantly greater increase in (Ca²⁺)_in than P_2Y1 receptor stimulation. P_2Y1 stimulation produced a purely NO-dependent dilation, while P_2Y2 stimulation caused the release of both NO and EDHF. These results suggest that (Ca²⁺)_in thresholds regulate NO- and EDHF-dependent dilation. Our results, however, indicate that in cerebral arterioles P_2Y1 stimulation releases both NO and an indomethacin-independent factor, while P_2Y2 stimulation was independent of NO. Thus, our results differ from those seen in middle cerebral arteries but agree with studies in pial arterioles, in which P_2Y1 stimulation depended partly (approximately one half) on NO.²⁰ However, if P_2Y2 stimulation results in a higher calcium increase than P_2Y1, we would also expect a release of NO, which is calcium dependent. Since we did not find NO involvement after P_2Y2 stimulation, a possible explanation is that P_2Y2 receptors are directly coupled to phospholipase A₂, which could release EDHF.²¹ Further studies are needed to elucidate the intracellular coupling of P_2Y1 and P_2Y2 receptors in rat penetrating arterioles.

In summary, the results of the present study provide functional evidence that both P_2Y1 and P_2Y2 receptors are present and mediate dilation of rat cerebral arterioles via different mechanisms. The P_2Y1 receptor dilates via both NO and potassium channels (probably EDHF), whereas the P_2Y2 receptor dilates the cerebral arteriole via potassium channel activation but not NO. Cyclooxygenase products are not involved. At low concentrations, the natural agonist ATP stimulates predominantly P_2Y1 receptors with subsequent NO release. Higher ATP concentrations stimulate P_2Y2 in addition to P_2Y1 receptors, releasing a NO- and cyclooxygenase-independent but potassium channel-dependent factor(s). Our results confirm the functional P_2Y receptor heterogeneity along the cerebrovascular tree, where stimulation of similar P_2Y receptors results in the release of different dilators.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL57540 and NS30555.

Received September 12, 2002; revision received December 17, 2002; accepted December 18, 2002.

	References

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