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Am J Physiol Heart Circ Physiol 287: H157-H164, 2004. First published February 12, 2004; doi:10.1152/ajpheart.00617.2003
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P2Y purine receptor responses and expression in the pulmonary circulation of juvenile rabbits

Girija G. Konduri,1,3 Ivane Bakhutashvili,1,3 Recia Frenn,1,3 Indira Chandrasekhar,1,3 Elizabeth R. Jacobs,2,3 and Ashwani K. Khanna2,3

Departments of 1Pediatrics and 2Medicine, 3Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Submitted 30 June 2003 ; accepted in final form 9 February 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purine nucleotide ATP mediates pulmonary vasodilation at birth by stimulation of P2Y purine receptors in the pulmonary circulation. The specific P2Y receptors in the pulmonary circulation and the segmental distribution of their responses remain unknown. We investigated the effects of purine nucleotides, ATP, ADP, and AMP, and pyrimidine nucleotides, UTP, UDP, and UMP, in juvenile rabbit pulmonary arteries for functional characterization of P2Y receptors. We also studied the expression of P2Y receptor subtypes in pulmonary arteries and the role of nitric oxide (NO), prostaglandins, and cytochrome P-450 metabolites in the response to ATP. In conduit size arteries, ATP, ADP, and AMP caused greater relaxation responses than UTP, UDP, and UMP. In resistance vessels, ATP and UTP caused comparable vasodilation. The response to ATP was attenuated by the P2Y antagonist cibacron blue, the NO synthase antagonist N{omega}-nitro-L-arginine methyl ester (L-NAME), and the cytochrome P-450 inhibitor 17-octadecynoic acid but not by the P2X antagonist {alpha},{beta}-methylene ATP or the cyclooxygenase inhibitor indomethacin in conduit arteries. In the resistance vessels, L-NAME caused a more complete inhibition of the responses to ATP and UTP. Responses to AMP and UMP were NO and endothelium dependent, whereas responses to ADP and UDP were NO and endothelium independent in the conduit arteries. RT-PCR showed expression of P2Y1, P2Y2, and P2Y4 receptors, but not P2Y6 receptors, in lung parenchyma, pulmonary arteries, and pulmonary artery endothelial cells. These data suggest that distinct P2Y receptors mediate the vasodilator responses to purine and pyrimidine nucleotides in the juvenile rabbit pulmonary circulation. ATP appears to cause NO-mediated vasodilation predominantly through P2Y2 receptors on endothelium.

ATP; UTP; nitric oxide

THE PULMONARY VASCULAR RESISTANCE undergoes a rapid decrease at birth to facilitate initiation of gas exchange during postnatal life. Our previous studies demonstrated that the purine nucleotide ATP contributes to this birth-related pulmonary vasodilation (17–22). The pulmonary vascular effects of ATP in the fetus appear to be mediated by the activation of G protein-coupled P2Y receptors (17). Although four different G protein-coupled P2Y receptors, P2Y1, P2Y2, P2Y4, and P2Y6, have been identified (Fig. 1) on blood vessels (3) and may potentially mediate the effects of ATP (7), these receptors have not been characterized in the perinatal pulmonary circulation. In addition, the response of the pulmonary circulation to vasodilators shows segmental variation from conduit to resistance pulmonary arteries (1). The contributions of endothelium-derived nitric oxide (NO), vasodilator prostaglandins, and cytochrome P-450 metabolites to the vasodilator response to ATP also appear to vary by the vascular bed and the postnatal age (11, 13, 15). The specific objectives of this study were to 1) define the functional contribution of the P2Y receptor subtypes to the vasodilator responses by investigation of the agonist profiles for the purine and pyrimidine nucleotides (Fig. 1) in isolated pulmonary arteries; 2) determine the segmental variation in the responses by investigation of the effects of representative nucleotides, ATP and UTP, in both conduit and resistance arterioles; 3) determine the role of NO, prostaglandins, and cytochrome P-450 metabolites in the vasodilator response to ATP; and 4) determine the expression of P2Y receptor subtypes by RT-PCR in pulmonary arteries, pulmonary artery endothelial cells, and peripheral lung tissue. Characterization of these receptors allows us to investigate their role in the pulmonary vasodilation that occurs at birth.



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  		Fig. 1. Sensitivity of different P2Y purine receptor subtypes to purine and pyrimidine nucleotides. The functional classification of these receptors is based on this agonist profile (3, 7).

 

   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The Animal Investigation Committee of the Medical College of Wisconsin approved the study. Lungs were harvested from 7- to 14-day-old New Zealand White rabbits after euthanasia with an overdose of pentobarbital and exsanguination.

Isolation of pulmonary arteries and investigation of the response to ATP. Pulmonary arteries were dissected out of the lung parenchyma with care taken not to damage the endothelium. Second- and third-generation pulmonary arteries were cut into 1-mm rings with an internal diameter of 0.4–0.5 mm. The rings were suspended with stainless steel hooks in water-jacketed chambers and were connected to force displacement transducers (model FTO3, Grass Instruments). The tissue was bathed in 2 ml of physiological salt solution (PSS) kept at 37°C and aerated with a gas mixture of 95% O2-5% CO2 to maintain normal pH, PCO2, and adequate oxygenation of tissue. In some studies, 21% O2 was used instead of 95% O2 to verify whether oxygen tension had an independent effect on the response of vessel rings to ATP and UTP. Rings were allowed to equilibrate for 45 min, stretched to a passive tension of 0.4 g, and preconstricted with 10–8–10–7 M U-46619. The dose of U-46619 was adjusted to allow the rings to develop 50% of maximum tension reached with 100 mM KCl. The tension reached with U-46619 contraction for each ring was normalized to 100%, and the percent change from this tension for all the rings with each dose of the nucleotide was expressed as means ± SD. Relaxation responses to 10–8–10–3 M doses of each nucleotide were determined, and the responses were compared with rings treated with PSS alone. Separate rings were pretreated with 10–4 M N{omega}-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, 10–3 M indomethacin, a cyclooxygenase inhibitor, 10–5M cinnamyl-3,4-dihydroxy-{alpha}-cyanocinnamate (CDC), a lipooxygenase inhibitor, or 10–4 M 17-octadecynoic acid (17-ODYA), a cytochrome P-450 inhibitor. Selected rings were pretreated with 10–3 M {alpha},{beta}-methylene ATP, a P2X purine receptor antagonist, or 10–3 M cibacron blue, a P2Y receptor antagonist (3, 26). Relaxation responses to nucleotides were then studied after 15 min of incubation with the antagonist. The endothelium was denuded from some rings by passing a blunt needle through the lumen and gently rotating the ring around the needle. Loss of endothelium was verified by a lack of relaxation response to acetylcholine and by light microscopy after the study was completed (30). The responses to ADP, AMP, UDP, and UMP were evaluated in the endothelium-denuded vessel rings to investigate whether the vasodilator response observed with these nucleotides is mediated by endothelial cell receptors or by a direct effect on the vascular smooth muscle.

Fifth- to seventh-generation intrapulmonary arterioles with an internal diameter of 60–100 µm were connected to two glass pipettes tapering to an outside diameter of 40 µm and were tied in place. Arterioles were superfused with PSS (temperature of 37°C, equilibrated with 95% O2-5% CO2) and luminally pressurized to 15 mmHg. The pressure chosen reflects the transmural pressure for the resistance vessels in newborn lungs (1, 24, 27). Internal diameter of the arterioles was monitored using a stereomicroscope (Zeiss), charge-coupled device television system camera (9KP 130 AU, Hitachi), a monitor (CVM 1271, Sony), and a calibrated video measuring system (Colorado Video). After an equilibration period of 30 min, reactivity of the vessel was confirmed by constriction with 30 mM KCl. The KCl-containing buffer was then removed, and the arterioles were preconstricted with 10–7–10–6 M norepinephrine to achieve a 50% decrease in luminal diameter before dilator responses were tested. The presence of intact endothelium was confirmed by evaluation of a vasodilator response to acetylcholine (10–5 M). Treatment of vessels with L-NAME (10–4 M) was done with the superfusion stopped, and the vessels were allowed to incubate with the drug for 20 min. Responses to intraluminal application of 10–6–10–3 M doses of ATP and UTP were determined with or without pretreatment of vessels with L-NAME. Our pilot studies demonstrated no vasodilator response to ATP at 10–6 M and plateau of the vasodilator response at 10–3–10–2 M doses in control resistance pulmonary arteries. The doses we chose are therefore expected to reproduce the range of vasodilator responses of these arteries to ATP and UTP.

Drugs and reagents. ATP, ADP, AMP, UTP, UDP, UMP, {alpha},{beta}-methylene ATP, and cibacron blue were obtained from Sigma Chemical (St. Louis, MO) and were dissolved in PSS to obtain the necessary concentration. L-NAME (Sigma) was dissolved in PSS, and indomethacin (Sigma), 17-ODYA, and CDC (Sigma) were dissolved in ethyl alcohol initially and diluted with PSS to obtain the required concentration.

Endothelial cell isolation. The main pulmonary artery was separated from the right ventricle. The left and right pulmonary arteries were dissected out of the lung, and the branches were tied off. The pulmonary arterial tree was flushed clear of blood with PSS and was incubated with 0.1% collagenase type A (Roche) for 10–12 min. The endothelial cells were then flushed into a 25-mm flask and grown to confluence in growth media (endothelial SFM, Life Technologies). Cells were used at passage 2 for RNA isolation and RT-PCR. In some studies, freshly dispersed endothelial cells were used for RNA extraction to determine whether passaging of cells in culture alters the receptor expression.

RT-PCR for P2Y receptors. Peripheral lung tissue and third- to fifth-generation pulmonary arteries were flash frozen in liquid nitrogen. The tissue was subsequently homogenized, and RNA was extracted (SV total RNA isolation system, Promega; Madison, WI). RNA was isolated from pulmonary artery endothelial cells using TRIzol reagent (Life Technologies) following the manufacturer's instructions. Single-strand cDNA was synthesized from 1 µg of total RNA (Superscript II, Life Technologies). Target cDNA was then amplified by PCR using gene-specific primers for P2Y1, P2Y2, P2Y4, and P2Y6 receptors and for {beta}-actin control. The primer sequences for each receptor subtype are as follows: P2Y1 sense 5'-GCATCTCGGTGTACATGTTC-3' and antisense 5'-GCTGTTGAGACTTGCTAGACCT-3'; P2Y2 sense 5'-TACAGCTCTGTCATGCTGGG-3' and antisense 5'-GCCAGGAAGTAGAGCACAGG-3'; P2Y4 sense 5'-CTTTGCAAGTTTGTCCGCTTTC-3' and antisense 5'-CCGGGCCATGAGTCCATA-3'; and P2Y6 sense 5'-CTGTGTCATCGCCCAGATATGC-3' and antisense 5'-GGTTGCCGCCGGAACTTC-3'. The primer sequences for {beta}-actin are as follows: sense 5'-TGACGGGGTCACCCACACTGTGAACATCTA-3' and antisense 5'-CTTGAAGCATTTGCGGTGGACGATGGAGGG-3'. The expected PCR product sizes for different receptors are P2Y1 698 bp, P2Y2 348 bp, P2Y4 357 bp, and P2Y6 801 bp and {beta}-actin 650 bp. PCR amplification parameters are as follows: denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and primer extension at 72°C for 60 s. PCR was done for 30 cycles for {beta}-actin and 40 cycles for all P2Y receptor subtypes. Appropriate negative controls were done with the omission of primers or Taq polymerase. PCR products were resolved by agarose gel electrophoresis and were identified by molecular size standards. The identity of the PCR products was also confirmed by direct sequencing.

Statistical analysis. Data are shown as means ± SD. Comparison of baseline data with those obtained at different doses of each nucleotide was done by single-factor ANOVA for repeated measures. Comparison of control data with those obtained after pretreatment with an inhibitor was done by two-way ANOVA (31). The two factors affecting the outcome were assumed to be the dose of ATP and presence or absence of the inhibitor. When a significant difference (P < 0.05) was found, Duncan's multiple-range test was done to determine which means were different.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Response of conduit vessels to purine and pyrimidine nucleotides. The control rings suspended in PSS alone maintained their tension for the duration of the study (Fig. 2). The purine nucleotides ATP, ADP, and AMP caused significant relaxation of the conduit vessel rings with an agonist profile of ATP > ADP = AMP (Fig. 2A). The pyrimidine nucleotides UTP, UDP, and UMP also caused relaxation of conduit vessel rings (Fig. 2B). The relaxation responses to UTP, UDP, and UMP were less than the responses observed with the purine nucleotides ATP, ADP, and AMP. The response to ATP was attenuated by cibacron blue, a P2Y receptor antagonist, but not by {alpha},{beta}-methylene-ATP, a P2X receptor antagonist (Fig. 3A). The response to UTP was also inhibited by cibacron blue, whereas {alpha},{beta}-methylene ATP did not alter the response (Fig. 3B).



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  		Fig. 2. Effects of purine nucleotides ATP, ADP, and AMP (A) and pyrimidine nucleotides UTP, UDP, and UMP (B) on the tension of conduit pulmonary artery rings. The tension obtained with U-46619 was normalized to 100%, and the percent change from the U46619 [GenBank] contraction is expressed as means ± SD for 24 rings from 6 rabbits at each dose. Control data were obtained from rings treated with physiological salt solution (PSS) alone. * P < 0.05 from control and #P < 0.05 from ADP and AMP. Both purine and pyrimidine nucleotides caused relaxation of pulmonary artery rings. The responses to purines were greater than to pyrimidines. ATP caused a greater relaxation response than ADP and AMP.

 


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  		Fig. 3. Effects of the P2Y receptor antagonist cibacron blue (CB) and the P2X receptor antagonist {alpha},{beta}-methylene ATP (A-BATP) on the response of conduit size pulmonary artery rings to ATP (A) and UTP (B). The tension obtained with U-46619 was normalized to 100%, and the percent change from the U-46619 contraction is expressed as means ± SD for 20 rings from 5 rabbits at each dose. *P < 0.05 from ATP or UTP alone. Cibcacron blue but not {alpha},{beta}-methylene ATP significantly attenuated the responses to ATP and UTP.

 
Response of resistance pulmonary arterioles to purine and pyrimidine nucleotides. The purine nucleotide ATP and pyrimidine nucleotide UTP caused vasodilation of the resistance pulmonary arterioles (Fig. 4A). The maximum increase in luminal diameter was observed at the 10–3 M doses of ATP and UTP. Although the response to ATP was greater than to UTP in conduit size arteries (Fig. 4A), the responses to ATP and UTP were similar in the resistance pulmonary arterioles (Fig. 4B).



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  		Fig. 4. Effects of ATP and UTP on the tone of conduit size pulmonary artery rings (A) and on luminal diameter (LD) of resistance size pulmonary arterioles (B). For the conduit size rings, the tension observed with U-46619 was normalized to 100%, and the percent change from the U-46619 contraction was assessed with each dose of nucleotide for 24 rings from 6 animals. For the resistance arterioles, the luminal diameter observed after constriction with norepinephrine was normalized to 100%, and the percent increase in LD with each dose of nucleotide is shown as means ± SD for 6 vessels from 6 rabbits. *P < 0.05 from UTP and #P < 0.05 from 10–6 M doses of ATP and UTP. The vasodilator response to ATP was greater than that to UTP in conduit size vessels, but the responses were equal in resistance arterioles.

 
Role of NO in the relaxation response to ATP and UTP. In the conduit vessels, pretreatment with NOS antagonist L-NAME partially attenuated the relaxation response to ATP, whereas it abolished the response to UTP (Fig. 5). The use of 21% O2 in the bath did not change the relaxation responses to ATP and UTP or the NO-dependent relaxation responses to ATP and UTP (Fig. 6). In the resistance pulmonary arterioles, pretreatment with L-NAME caused a greater inhibition of the response to ATP and complete inhibition of the response to UTP (Fig. 7). These data suggest that NO plays an important role in the relaxation responses to ATP and UTP. However, there was a segmental variation in the role of NO in the vasodilator response with NO playing a greater role in the resistance arterioles compared with conduit arteries and in the response to UTP compared with ATP. Because ATP and UTP caused comparable vasodilation in resistance arteries, these data suggest the possibility that P2Y2 receptors mediate the response to ATP and UTP by an NO-dependent mechanism. L-NAME inhibited the response to AMP but not to ADP (Fig. 8). L-NAME also inhibited the response to UMP but had no effect on the response to UDP (Fig. 9). Endothelial denudation did not alter the relaxation responses to ADP and UDP but attenuated the responses to AMP and UMP (Figs. 8 and 9). Endothelium-derived NO appears to play a major role in the vasodilator response to AMP and UMP but not ADP and UDP. Thus the role of NO in the vasodilator response appears to vary for different purine and pyrimidine nucleotides. These data suggest that the purine receptors mediating the responses to ADP and UDP are distinct from that of AMP and UMP.



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  		Fig. 5. Effects of nitric oxide synthase (NOS) antagonist N{omega}-nitro-L-arginine methyl ester (L-NAME) on the responses of conduit pulmonary arteries to ATP (A) and UTP (B). Data are means ± SD for 24 rings from 6 animals at each dose of ATP or UTP. *P < 0.05 from L-NAME + ATP or L-NAME + UTP. L-NAME caused partial attenuation of the response to ATP but complete inhibition of the response to UTP in conduit size pulmonary arteries.

 


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  		Fig. 6. Effect of changing oxygen tension on the response of conduit pulmonary arteries to ATP (A) and UTP (B). Pulmonary artery rings were incubated in 21% O2-5% CO2-74% N2 in these studies. Data are means ± SD for 16 rings from 4 animals at each dose of ATP or UTP. *P < 0.05 from L-NAME + ATP or L-NAME + UTP. The relaxation responses to ATP and UTP and the effect of L-NAME, a NOS antagonist, on the responses to ATP and UTP were similar to what we observed when vessels were incubated in 95% O2-5% CO2, as shown in Fig. 5.

 


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  		Fig. 7. Effects of L-NAME on the vasodilator responses to ATP and UTP in resistance size pulmonary arterioles. Resistance arterioles were preconstricted with norepinephrine, and the percent increases in LD are shown for 6 vessels from 6 rabbits as means ± SD. *P < 0.05 from ATP or UTP treatment alone. L-NAME caused a more complete inhibition of vasodilator response to ATP in these vessels compared with conduit size arteries and complete inhibition of the response to UTP.

 


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  		Fig. 8. Effects of L-NAME and denudation of endothelium (denuded) on the responses of conduit size pulmonary artery rings to ADP (A) and AMP (B). The percent changes in vascular ring tension with each dose of nucleotide from U-46619 contraction are shown as means ± SD for 24 rings from 6 different animals. *P < 0.05 from L-NAME + AMP and denuded + AMP. L-NAME and endothelial denudation had no effect on the response to ADP and attenuated the response to AMP equally.

 


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  		Fig. 9. Effects of L-NAME and denudation of endothelium on the responses of conduit size pulmonary artery rings to UDP (A) and UMP (B). The percent changes in vascular ring tension with each dose of nucleotide from U46619 [GenBank] contraction are shown as means ± SD for 24 rings from 6 different animals. *P < 0.05 from L-NAME + UMP and denuded + UMP. L-NAME and endothelial denudation had no effect on the response to UDP and attenuated the response to UMP equally.

 
Mechanism of NOS-independent vasodilator response to ATP in conduit arteries. Pretreatment of the conduit artery segments with cyclooxygenase inhibitor indomethacin or lipooxygenase inhibitor CDC did not attenuate the response to ATP (Fig. 10). Pretreatment of the rings with the cytochrome P-450 inhibitor 17-ODYA caused partial attenuation of the response to ATP (Fig. 10). In conduit artery rings pretreated with L-NAME to inhibit NO release, 17-ODYA did not cause significant additional inhibition of the vasodilator response to ATP (Fig. 11), although the rings developed higher tension than with L-NAME alone. These data suggest that cytochrome P-450 metabolites contribute to the vasodilator response to ATP in conduit size pulmonary arteries.



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  		Fig. 10. Effects of cyclooxygenase, lipooxygenase, and cytochrome P-450 inhibition on the vasodilator response to ATP in conduit size pulmonary arteries. Indo, indomethacin; CDC, cinnamyl-3,4-dihydroxy-{alpha}-cyanocinnamate; 17-ODYA, 17-octadecynoic acid. Data are means ± SD for 20 vessels from 5 animals with each inhibitor. *P < 0.05 from other groups. Only 17-ODYA, a cytochrome P-450 inhibitor, caused attenuation of the vasodilator response to ATP.

 


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  		Fig. 11. Effects of pretreatment with the NOS inhibitor L-NAME and combined NOS and cytochrome P-450 inhibition with L-NAME + 17-ODYA on the response to ATP in conduit size arteries. Data are means ± SD for 20 vessels from 5 animals with each blocker. *P < 0.05 from the other two groups. 17-ODYA did not cause additional inhibition of vasodilator response to ATP in L-NAME-pretreated vessels.

 
Expression of P2Y receptors in the pulmonary circulation. The pulmonary arteries (Fig. 12A) and peripheral lung tissue (Fig. 12B) showed expression of P2Y1, P2Y2, and P2Y4 receptors. However, P2Y6 receptor transcript was not detected in the lung tissue or pulmonary arteries (Fig. 12). Pulmonary artery endothelial cells, whether freshly dispersed or passaged, also showed expression of P2Y1, P2Y2, and P2Y4 but not P2Y6 receptors (Fig. 13). These data suggest that several P2Y receptor subtypes that can potentially mediate the vasodilator responses to purine and pyrimidine nucleotides (Fig. 1) are expressed in the pulmonary circulation. The expression of these receptors in endothelial cells suggests that the NO-dependent pulmonary vasodilation caused by ATP and UTP may be mediated by these receptors.



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  		Fig. 12. Expression of P2Y purine receptor subtypes in pulmonary arteries (A) and in peripheral lung tissue (B) by RT-PCR with {beta}-actin mRNA included for comparison. The transcript for P2Y1, P2Y2, and P2Y4 but not P2Y6 receptors was detected in RNA from isolated pulmonary arteries and whole lung.

 


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  		Fig. 13. Expression of P2Y purine receptor subtypes in cultured early passage pulmonary artery endothelial cells (A) and in freshly dispersed pulmonary artery endothelial cells (B) by RT-PCR with {beta}-actin mRNA included for comparison. The transcript for P2Y1, P2Y2, and P2Y4 but not P2Y6 receptors was detected in RNA from both cultured and freshly dispersed pulmonary artery endothelial cells.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study demonstrated that the vasodilator responses to purine and pyrimidine nucleotides in the juvenile rabbit pulmonary circulation are mediated by distinct purine receptors. We also observed that the role of endothelium-derived NO in this vasodilation varies by the specific purine receptor and by the segment of the pulmonary circulation. We observed a correlation between the functional responses of these pulmonary arteries and the expression of the purine receptors. These studies will facilitate the investigation of the role of purine receptors in birth-related pulmonary vasodilation. To the best of our knowledge, this is the first report of the distribution of P2Y receptors in the perinatal pulmonary circulation.

Our previous studies demonstrated that release of ATP contributes to birth-related pulmonary vasodilation. The plasma levels of ATP increased during exposure of fetal lambs to oxygen, alone or accompanied by lung distension (18, 19, 21). Infusion of ATP and its metabolites into the fetal pulmonary artery reproduced the pulmonary vasodilation that occurs at birth in fetal lambs (22). Inhibition of ATP synthesis (19) or receptors for ATP and its metabolite, adenosine (18, 21), attenuated birth-related pulmonary vasodilation in fetal lambs. ATP causes vasodilation in fetal lambs, partly by a NO-dependent mechanism and partly by its direct effect on the vascular smooth muscle (20). Inhibition of P2Y purine receptors by cibacron blue attenuated the vasodilator response to ATP (17). However, the specific P2Y receptor subtypes that are expressed in the pulmonary circulation or the functional effects of their stimulation were previously unknown.

The P2Y receptor subtypes have been functionally characterized by the agonist profile for different purine and pyrimidine nucleotides for these subtypes (Fig. 1). The four G protein-coupled receptors in the blood vessels (P2Y1, P2Y2, P2Y4, and P2Y6) have distinct agonist profiles, with P2Y1 receptors showing greater sensitivity to ADP and ATP and insensitivity to pyrimidines (7). P2Y2 receptors show dual specificity for ATP and UTP, whereas P2Y4 receptor show greater sensitivity for UTP, UDP, and UMP than for purines (3). P2Y6 receptors are reported to be insensitive to purines (7). We observed that ADP and UDP cause NO-independent vasodilation in pulmonary arteries, suggesting the possibility that the receptors activated by these nucleotides are different from those activated by other nucleotides that cause NO-dependent vasodilation. On the basis of previously described functional responses and expression of these receptors by RT-PCR, the profile suggests that P2Y1 receptors on vascular smooth muscle mediate the NO-independent vasodilator response to ADP and P2Y4 receptors on smooth muscle mediate the NO-independent response to UDP. The nearly equal vasodilator response observed with ATP and UTP in the resistance vessels suggests a P2Y2-mediated response in these smaller arteries. Because the small intrapulmonary arterioles are the most important component of pulmonary vascular resistance (27), these data suggest that P2Y2 receptors on vascular endothelium may mediate the response to ATP in the rabbit pulmonary circulation. However, there is a limitation to the functional characterization of these receptors in pulmonary circulation using vascular tone as a marker. The responses of vascular ring tension or diameter of the vessel to the nucleotides represent a summation of the contributions from several receptor subtypes present on both endothelial and smooth muscle cells. Stimulation of these different P2Y and P2X receptor subtypes may have opposing effects, and the resulting changes in vessel diameter or tension may also be influenced by other factors, such as preexisting tone and the alterations induced by dissection of these vessels from the lung.

We observed that ATP causes both NO-mediated and NO-independent vasodilation in the rabbit pulmonary arteries. The contribution of NO to ATP-mediated vasodilation was greater in the resistance arterioles compared with conduit size pulmonary arteries. Previous studies with different vasodilators have reported similar heterogeneity in NO-dependent vasodilation in the pulmonary circulation (8, 9). Although several previous studies suggested that EDHF plays a larger role than NO in the small resistance arteries, our data are consistent with the report of Gao et al. (8) showing that in the newborn pulmonary circulation, NO is the primary endothelium-derived vasodilator. These data suggest developmental differences in the segmental distribution of pulmonary vasodilator responses to physiological agonists. We also observed that the vasodilator response to ATP is mediated in part by cytochrome P-450 metabolites but not by the products of cyclooxygenase or lipooxygenase pathways. In addition, a direct effect of ATP on pulmonary vascular smooth muscle may account for part of the NO-independent vasodilation caused by ATP. Our previous studies in isolated rabbit pulmonary arteries suggested that ATP causes endothelium-independent vasodilation by activation of voltage-gated K+ channels on vascular smooth muscle (28).

We used U-46619 to preconstrict the pulmonary artery rings and observed that the control rings treated with U-46619 maintained a stable contraction for the duration of the study. Previous studies demonstrated species-related differences in the effects of U-46619 with dog pulmonary arteries showing no response to this agent, whereas rat and human pulmonary artery rings showed a constrictor response to this agent (4, 16). We used norepinephrine to constrict the intrapulmonary resistance arteries based on our observation of a stable constriction of these vessels to norepinephrine for the duration of the study. In addition, the vessel chamber was superfused with 50 ml of PSS, and the addition of norepinephrine to maintain the constriction of vessel represented a fraction of the cost of U-46619. We used 95% O2 in the tissue bath because the lack of hemoglobin in PSS decreases the oxygen availability for the endothelium and smooth muscle, and we observed a more consistent contraction and relaxation of the vessel with 95% O2 in the gas mixture, as previously reported (30). We also observed that the use of 95% O2 did not change the contribution of NO to the vasodilator responses to ATP and UTP in the conduit artery rings compared with 21% O2.

McMillan et al. (25) have previously reported that ATP and UTP cause predominantly endothelium-independent vasodilation in U-46619-constricted, conduit size rings from newborn piglets. In contrast, our studies have shown a significant component of NO-dependent vasodilation in juvenile rabbit pulmonary arteries. Although species differences may account for these results, we believe that these studies support our concept of segmental differences in the contribution of NO to vasodilation. The studies of McMillan et al. (25) were done on the main conduit pulmonary artery in the lower lobe of the lung, whereas our studies were done with rings from smaller branches and resistance arterioles. Our studies therefore suggest that NO makes an increasing contribution to ATP-mediated vasodilation with progression from larger conduit arteries to smaller resistance arterioles. ATP and UTP were previously shown to increase NO release from cultured vascular endothelial cells (2). We previously reported that ATP causes NO-dependent vasodilation in intact fetal lambs (20). Because the small intrapulmonary resistance arterioles are the major component of pulmonary vascular resistance (27), these data support a role for NO in mediating the vasodilator response to ATP in the pulmonary circulation. Previous studies have also reported that purine and pyrimidine nucleotides may cause vasoconstrictor effects, particularly with extraluminal application in both pulmonary and cerebral vascular beds (15, 25). We applied ATP intraluminally in the resistance arteries in our studies. However, our studies in conduit pulmonary artery rings were done with the addition of the nucleotides to the tissue bath, resulting in the exposure of both endothelium and vascular smooth muscle to the nucleotides. We did not observe a vasoconstrictor effect of the nucleotides in these studies and in studies done with endothelium-denuded rings. The difference in these results may represent developmental maturation of the response of these vessels to purine and pyrimdine nucleotides, as reported by McMillan et al. (25) previously in porcine pulmonary arteries.

Previous studies of the role of NO in mediating purine- and pyrimidine-induced vasodilation in systemic vascular beds have given variable results (26). You et al. (32) reported that NO mediates the vasodilator response to ATP in larger but not smaller cerebral arteries of rats. However, Horiuchi et al. (15) reported that ATP and UTP cause NO-dependent vasodilation in small penetrating arteries of the rat cerebral circulation. Hammer et al. (13) reported a significant contribution of the prostaglandin system to the vasodilator response to ATP in the hamster cremaster muscle arterioles, whereas our studies suggest that prostaglandins do not contribute to the vasodilator response to ATP. These studies suggest diversity in the purine receptor distribution as well as the signaling mechanisms involved in different vascular beds and in different species.

Several studies have suggested a physiological role for ATP in the regulation of pulmonary vascular tone. Sprague and colleagues (6, 29) have shown that ATP derived from red blood cells plays a significant role in the modulation of tone in systemic and pulmonary vascular beds during hypoxia and shear stress. Shear stress has been shown to evoke ATP release and contributes to NO-mediated vasodilation in the rat lung (14). These studies are supported by our observation that ATP mediated vasodilation is present in both conduit and resistance size pulmonary arteries in rabbits. The purine receptors are expressed and functional early in the juvenile rabbit pulmonary circulation. Although a physiological role for ATP appears to be established, the role of UTP, UDP, and UMP in maintaining pulmonary vascular tone is less clear. UTP is released from platelets (10) and is colocalized with ATP in the chromaffin tissue and perivascular nerves (5). Extracellular fluid contains UTP in micromolar concentrations (23). Our studies have shown that the receptors for pyrimidine nucleotides are expressed in pulmonary arteries, and a significant vasodilator response is observed in response to these nucleotides. However, their physiological role in maintaining pulmonary vascular tone requires further study.

The limitations of our study are that we did not address the individual contribution of each receptor subtype to the vasodilation caused by ATP and UTP, because selective antagonists of P2Y receptor subtypes are currently not available. The isolated pulmonary arteries were less sensitive to the vasodilator effects of ATP than what we observed in our previous studies in animals with an intact pulmonary circulation (17, 22). The resistance pulmonary arteries only showed vasodilator response to millimolar concentrations of the nucleotides, which are 10- to 1,000-fold higher than the plasma concentrations we previously measured in the pulmonary circulation. Therefore, we cannot extrapolate a physiological role for these receptors from the vasodilator responses we observed in the resistance arteries. We also based our conclusions of receptor expression on the mRNA detected by RT-PCR and not by Western blot analysis for the receptor protein. The antibodies currently available for the receptor protein have given inconsistent results with the rabbit pulmonary arteries. Because the half-life of mRNA can be short, the absence of transcript for some P2Y receptors may not imply absence of expression.

In conclusion, our study provides evidence that three distinct P2Y purine receptors are expressed in the juvenile rabbit pulmonary circulation. The response of the pulmonary arteries to purine and pyrimidine nucleotides shows segmental variation for both agonist profile and the contribution of the endothelium-derived NO to the vasodilator response. The potential role of these receptors in birth-related pulmonary vasodilation and its alteration in pulmonary hypertension require further investigation.


   ACKNOWLEDGMENTS
 
This study was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-57268 (to G. G. Konduri).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. G. Konduri, Medical College of Wisconsin, CHW OB 213 A, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: gkonduri{at}mcw.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aschner JL, Smith TK, Kovacs N, Pinheiro JMB, and Fuloria M. Mechanisms of bradykinin-mediated dilation in newborn piglet pulmonary conducting, and resistance vessels. Am J Physiol Lung Cell Mol Physiol 283: L373–L382, 2002.[Abstract/Free Full Text]
  2. Brown CA, Patel V, Wilkinson G, and Boarder MR. P2 purinoceptor-stimulated conversion of arginine to citrulline in bovine endothelial cells is reduced by inhibition of protein kinase C. Biochem Pharmacol 52: 1849–1854, 1996.[CrossRef][ISI][Medline]
  3. Burnstock G. The past, present, and future of purine nucleotides as signaling molecules. Neuropharmacology 36: 1127–1139, 1997.[CrossRef][ISI][Medline]
  4. Chand N and Altura BM. Reactivity of isolated rat, and canine pulmonary blood vessels to synthetic prostaglandin endoperoxide (PGH2) analogs. Prostaglandins Med 5: 469–75, 1980.[CrossRef][ISI][Medline]
  5. Chapman AG, Westerberg E, and Siesjo BK. The metabolism of purine and pyrimidine nucleotides in rat cortex during insulin-induced hypoglycemia and recovery. J Neurochem 36: 179–189, 1981.[ISI][Medline]
  6. Dietrich HH, Ellsworth ML, Sprague RS, and Dacey RG Jr. Red blood cell regulation of microvascular tone through adenosine triphosphate. Am J Physiol Heart Circ Physiol 278: H1294–H1298, 2000.[Abstract/Free Full Text]
  7. Dubyak GR. Knock-out mice reveal tissue specific roles of P2Y receptor subtypes in different epithelia. Mol Pharmacol 63: 773–776, 2003.[Free Full Text]
  8. Eichinger MR and Walker BR. Segmental heterogeneity of NO-mediated pulmonary vasodilation in rats. Am J Physiol Heart Circ Physiol 267: H494–H499, 1994.[Abstract/Free Full Text]
  9. Gao Y, Tolsa JF, and Raj JU. Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs. Pediatr Res 44: 723–729, 1998.[ISI][Medline]
  10. Goetz U, Da Prada M, and Pletscher A. Adenine-, guanine-, and uridine-5'-phosphonucleotides in blood platelets and storage organelles of various species. J Pharmacol Exp Ther 178: 210–215, 1971.[Abstract/Free Full Text]
  11. Gorman MW, Ogimoto K, Savage MV, Jacobson K, and Feigl EO. Nucleotide coronary vasodilation in guinea pig hearts. Am J Physiol Heart Circ Physiol 285: H1040–H1047, 2003. First published May 22, 2003; 10.1152/ajpheart.00981.2002.[Abstract/Free Full Text]
  12. Hakim TS, Ferrario L, Freedman JC, Carlin RE, and Camporesi EM. Segmental pulmonary vascular responses to ATP in rat lungs: role of nitric oxide. J Appl Physiol 82: 852–858, 1997.[Abstract/Free Full Text]
  13. Hammer LW, Ligan AL, and Hester RL. ATP-mediated release of arachidonic acid metabolites from venular endothelium causes arteriolar dilation. Am J Physiol Heart Circ Physiol 280: H2616–H2622, 2001.[Abstract/Free Full Text]
  14. Hassessian H, Bodin P, and Burnstock G. Blockade by glibenclamide of the flow-evoked endothelial release of ATP that contributes to vasodilatation in the pulmonary vascular bed of the rat. Br J Pharmacol 109: 466–472, 1993.[ISI][Medline]
  15. Horiuchi T, Dietrich HH, Tsugane S, and Dacey RG. Analysis of purine, and pyrimidine-induced vascular responses in the isolated rat cerebral arteriole. Am J Physiol Heart Circ Physiol 280: H767–H776, 2001.[Abstract/Free Full Text]
  16. Joiner PD, Kadowitz PJ, Hughes JP, and Hyman AL. Actions of prostaglandins E1 and F2alpha on isolated intrapulmonary vascular smooth muscle. Proc Soc Exp Biol Med 150: 414–421, 1975.[Abstract]
  17. Konduri GG, Forman K, and Mital S. Characterization of purine receptors in fetal lamb pulmonary circulation. Pediatr Res 47: 114–120, 2000.[ISI][Medline]
  18. Konduri GG, Gervasio CT, and Theodorou AA. Role of adenosine triphosphate, and adenosine in oxygen induced pulmonary vasodilation in fetal lambs. Pediatr Res 33: 533–539, 1993.[ISI][Medline]
  19. Konduri GG and Mattei J. Role of oxidative phosphorylation and ATP release in mediating birth-related pulmonary vasodilation in fetal lambs. Am J Physiol Heart Circ Physiol 283: H1600–H1608, 2002. First published June 27, 2002; 10.1152/ajpheart.00245.2002.[Abstract/Free Full Text]
  20. Konduri GG and Mital S. Adenosine and ATP cause nitric oxide dependent pulmonary vasodilation in fetal lambs. Biol Neonate 78: 220–229, 2000.[CrossRef][ISI][Medline]
  21. Konduri GG, Mital S, Gervasio CT, Rotta AT, and Forman K. Purine nucleotides contribute to pulmonary vasodilation caused by birth-related stimuli in the ovine fetus. Am J Physiol Heart Circ Physiol 272: H2377–H2384, 1997.[Abstract/Free Full Text]
  22. Konduri GG, Theodorou AA, Mukhopadhyay A, and Deshmukh DR. Adenosine triphosphate and adenosine increase pulmonary blood flow to postnatal levels in fetal lambs. Pediatr Res 31: 451–457, 1992.[ISI][Medline]
  23. Lazarowski ER and Harden TK. Quantitation of extracellular UTP using a sensitive enzymatic assay. Br J Pharmacol 127:1272–1278, 1999.[CrossRef][ISI][Medline]
  24. Liu YA, Theis JGW, and Coceani F. Contractile and relaxing mechanisms in pulmonary resistance arteries of the preterm fetal lamb. Biol Neonate 77: 253–260, 2000.[CrossRef][ISI][Medline]
  25. McMillan MR, Burnstock G, and Haworth SG. Vasodilation of intrapulmonary arteries to P2-receptor nucleotides in normal, and pulmonary hypertensive newborn piglets. Br J Pharmacol 128: 543–548, 1999.[CrossRef][ISI]
  26. Pearson JD and Carter TD. Effects of extracellular ATP on the release of vasoactive mediators from endothelium. In: Biological Actions of Extracellular ATP, edited by Dubyak GR and Fedan JS. New York: N. Y. Acad. Sci., 1990, vol 603, p. 267–274.
  27. Raj JU and Chen P. Microvascular pressures measured by micropuncture in isolated perfused lamb lungs. J Appl Physiol 61: 2194–2201, 1986.[Abstract/Free Full Text]
  28. Shash T, Bakhutashvillli I, and Konduri GG. Role of vascular K+ channels in vasodilator response to ATP in isolated pulmonary arteries from juvenile rabbits (Abstract). Pediatr Res 51: 397A, 2002.
  29. Sprague RS, Ellsworth ML, Stephenson AH, and Lonigro AJ. ATP: the red blood cell link to NO and local control of the pulmonary circulation. Am J Physiol Heart Circ Physiol 271: H2717–H2722, 1996.[Abstract/Free Full Text]
  30. Steinhorn RH, Morin FC 3rd, Van Wylen DG, Gugino SF, Giese EC, and Russell JA. Endothelium-dependent relaxations to adenosine in juvenile rabbit pulmonary arteries and veins. Am J Physiol Heart Circ Physiol 266: H2001–H2006, 1994.[Abstract/Free Full Text]
  31. Winer BJ. Single-factor experiments having repeated measures on the same elements. In: Statistical Principles in Experimental Design, edited by Winer BJ. New York: McGraw-Hill, 1971, p. 261–308.
  32. You J, Johnson TD, Marrelli SP, and Bryan RM. Functional heterogeneity of endothelial P2 purinoceptors in the cerebrovascular tree of the rat. Am J Physiol Heart Circ Physiol 277: H893–H900, 1999.[Abstract/Free Full Text]



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