Ann Thorac Surg 2003;76:50-57
© 2003 The Society of Thoracic Surgeons
Original article: cardiovascular
Contractions in human coronary bypass vessels stimulated by extracellular nucleotides
Catharina Borna, MDa,
Lingwei Wang, MDa,
Tomas Gudbjartsson, MDb,
Lena Karlsson, MD, PhDc,
Sverker Jern, MD, PhDc,
Malin Malmsjö, MD, PhDa,
David Erlinge, MD, PhDa*
a department of Cardiology, University Hospital, Lund, Sweden,
b department of Cardiothoracic Surgery, University Hospital, Lund, Sweden
c Clinical Experimental Research Laboratory, Department of Medicine, Sahlgrenska University Hospital/Östra, Göteborg University, Göteborg, Sweden
Accepted for publication December 22, 2002.
* Address reprint requests to Dr Erlinge, Department of Cardiology, Lund University Hospital, S-221 85 Lund, Sweden.
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Abstract
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BACKGROUND: The present study was designed to evaluate the relative contribution of different contractile P2 receptors in human saphenous vein compared with internal mammary artery obtained during coronary artery surgery.
METHODS: The isometric tension in endothelium-denuded isolated vessel segments was recorded in vitro. The P2 receptor mRNA expression was quantified by real-time polymerase chain reaction.
RESULTS: The P2X1 receptor agonist, 
ß-MeATP (ß-methylene-adenosine triphosphate), was the most potent vasoconstrictor, with more efficacious contractions in the saphenous vein than in the internal mammary artery. The selective P2Y6 receptor agonist, UDPßS (uridine 5’-O-thiodiphosphate), stimulated more potent contractions in saphenous vein compared with internal mammary artery. Furthermore, UDPßS induced long-lasting contractions for more than 2 hours, explained by the low desensitization rate of the P2Y6 receptor. The ATP-induced vasoconstriction could not be abolished by desensitization of P2X1 receptors with ß-MeATP, or P2Y2/4 receptors with UTP
S (uridine 5’-O-3-thiotriphosphate), indicating the presence of yet another contractile ATP receptor. Based on quantification with real-time polymerase chain reaction, the P2Y11 receptor could be responsible for this ATP contraction.
CONCLUSIONS: The P2X1 and P2Y6 receptors elicit more prominent contractions in the saphenous vein as compared with the internal mammary artery. These results may present one explanation for the differences in the two conduits. It is possible that selective antagonists of P2X1 and P2Y6 receptors could be used to prevent vasospasm and restenosis in the saphenous vein during and after revascularization surgery.
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Introduction
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One of the treatment options for ischemic heart disease is coronary artery bypass graft (CABG) surgery, where a blood vessel graft is inserted to bypass the occlusion. There is a marked difference in long-term patency of the two main coronary bypass vessels. In the saphenous vein the occlusion rate amounts to 50% 10 years after CABG mainly due to atherosclerotic processes in the vein termed "venous graft disease" [1]. In contrast, the internal mammary artery has a good long-term patency [1]. The molecular mechanisms for these differences between the two vessels remain unknown but it is clear that both vasospasm and vascular smooth muscle cell proliferation may contribute.
Extracellular nucleotides (adenosine triphosphate [ATP], adenosine diphosphate [ADP], uridine triphosphate [UTP], and uridine diphosphate [UDP]) could have a potential role in this process. Their receptors—the P2 receptors—have been shown to constitute one of the largest receptor families with at least 14 known subtypes divided into P2X and P2Y receptors [2]. Extracellular nucleotides are released in high concentrations in close proximity to the vessel wall from endothelial cells during hypoxia and shear stress. They are also released from aggregating platelets, inflammatory cells, from vascular smooth muscle cells when damaged, and from the myocardium during ischemia [3].
Recently the cardiovascular effect of extracellular nucleotides have received increasing attention since the platelet inhibitory ADP receptor antagonist clopidogrel has been shown to prevent myocardial infarction and stroke (CAPRIE and CURE studies) [4, 5]. On endothelial cells P2Y receptors mediate vasodilatation by release of prostaglandin, nitric oxide [2] and endothelium-derived hyperpolarizing factor [6, 7] but P2X1 receptor agonists have no effect on endothelial cells. Instead, P2X1 and several P2Y receptors on vascular smooth muscle cells mediate vasoconstriction [2]. Furthermore extracellular nucleotides act as growth factors for vascular smooth muscle cells by activation of several P2Y receptors (Table 1)
[8].
The aim of this study was to compare contractile effects of different P2 receptor subtypes in the main coronary bypass vessels, saphenous vein and internal mammary artery. Selective P2 receptor agonists were used for the pharmacological evaluation of the contractile effects and the levels of mRNA expression was quantified with real time polymerase chain reaction (PCR).
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Material and methods
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Tissue preparation
Saphenous vein and internal mammary artery were obtained from 14 patients undergoing coronary bypass surgery (mean age, 71 years; 12 of 14 male, and 5 of 14 with noninsulin dependent diabetes mellitus). The internal mammary artery was harvested on a pedicle and the saphenous vein by open approach from the lower leg and the knee region. The distal 2 cm of the internal mammary artery and proximal 3 to 4 cm of the saphenous vein were cut off and kept in cold buffer solution (for composition, see below) and transported to the laboratory where they were dissected free of adhering tissue under a microscope. The internal mammary artery had a relaxed inner diameter of approximately 1 mm while the inner diameter of the saphenous vein was approximately 3 mm. The endothelium was removed by gently rubbing the intimal surface with a wooden stick. The vessels were then cut into 4 to 5 mm long cylindrical segments (4 to 8 segments from each vessel). Each cylindrical segment was mounted on two L-shaped metal prongs, one of which was connected to a force displacement transducer (FT03C) attached to a Grass Polygraph (Grass Instruments, MA), for continuous recording of the isometric tension. The position of the holder could be changed by means of a movable unit allowing fine adjustments of the vascular resting tension by varying the distance between the metal prongs. The mounted vessel segments were immersed in temperature controlled (37°C) tissue baths (2 mL), containing bicarbonate based buffer solution of the following composition (mM): NaCl 119, NaHCO3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5, and glucose 5.5. The solution was continuously gassed with 5% CO2 in O2 resulting in a pH of 7.4. The artery segments were allowed to stabilize at a resting tension of 4 mN and the vein segments at a resting tension of 5 mN for 1 hour before the experiments were started. The vessels were washed with fresh buffer solution every 15 minutes except for the experiments with were prolonged contractions were studied. The contractile capacity of each vessel segment was examined by exposure to a potassium-rich (60 mmol/L) buffer solution in which NaCl was exchanged for an equimolar concentration of KCl (for composition, see above). When two reproducible contractions had been achieved the vessels were used for further studies. The vessels were allowed to rest for 1.5 hours after K+ contractions. Except for the first ß-mATP contraction, each vessel segment was only exposed to one agonist. Endothelium removal was checked by monitoring responses to acetylcholine (ACh), in noradrenaline (NA) precontracted vessel segments. Abolished relaxation indicated a properly removed endothelium whereas unaffected K+-induced contractions indicated intact vascular smooth muscle cell function.
Drugs
Agonist selectivity and stability are potential problems when analyzing the pharmacologic profiles of natural nucleotides in intact tissue. Therefore more stable compounds were used. The UTPS and UDPßS were donated by Inspire Pharmaceuticals (Chapel Hill, NC); the ß-MeATP, ACh, 2-MeSADP (2-methylthio-adenosine diphosphate), and NA were purchased from Sigma. All drugs were solved in 0.9% saline. The drugs were stored in high concentrations at –20 degrees.
Vascular contraction
Eight ring segments were studied at the same time in separate tissue baths. Concentration-response curves to ß-MeATP were constructed in single concentrations. Each artery segment was then exposed to a single concentration of ß-MeATP and the resultant responses of several segments exposed to different concentrations were grouped together to form a concentration-response curve. This way the problem of desensitization of P2X receptors was avoided. To study the P2Y receptor stimulated contractions without interference of simultaneous P2X1 receptor induced responses, ATP, 2-MeSADP, UTP, UTPS, UDP, and UDPßS were added after desensitization of P2X1 receptors using 10 µM ß-MeATP [9]. As the P2Y receptors are only very slowly desensitized, these agonists could be added cumulatively to determine concentration-response relationships.
Rna extraction
Saphenous vein and internal mammary artery smooth muscle layers were obtained by dissecting free of adhering tissue, stripping off the adventitia and outer media, and removing the endothelial layer by gently rubbing the intimal surface with a wooden stick in sterile conditions. The media were cleansed with cold bicarbonate based buffer solution and snap frozen in liquid nitrogen. Then samples were put in a –70°C freezer for RNA extraction. TRIzol reagent (Invitrogen AB, Lidingö, Sweden) was used to isolate RNA according to the supplier’s instructions. The RNA samples were stored at –70°C until used.
Reverse transcription real-time PCR
Oligonucleotide primers, TaqMan probes (Perkin-Elmer Applied Biosystems, Foster City, CA) were designed and reverse transcription was performed as described elsewhere [10]. Relative quantification of mRNA was performed on an ABI PRISM7700 Sequence Detector (Perkin-Elmer Applied Biosystems) [10]. Constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was selected as endogenous control to correct for potential variation in RNA loading or efficiency of the amplification reaction. The threshold cycle (CT) is defined as the fractional cycle number at which the reporter fluorescence reaches a certain level (set to 10 times the SD of the base line). Target gene normalized to GAPDH was expressed as 
CT (CT of target gene minus CT of GAPDH). For amplicons designed and optimized according to the manufacturer’s guidelines, amplification efficiency is typically close to one, namely, product accumulation increases twofold until the plateau phase is reached. Thus the ratio of target gene/GAPDH could be calculated by 2-Ct. The relative value of target genes were shown as the percentages of GAPDH.
Polymerase chain reaction was carried out in a 50 µL reaction mixture that contains 1x TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems), 15 pmol of both forward and reverse primers, 5 pmol of probe, and 1 µL of the cDNA templates. Thermal cycling conditions included the following steps: 2 minutes at 52°C to activate ampUNG, then the reaction mixture was preheated for 10 minutes at 95°C to activate Taq polymerase. Then, a 50-cycle two-step PCR was performed consisting of 15 seconds at 95°C and 1 minute at 60°C. Samples were amplified simultaneously in triplicate in one-assay runs.
Ethics
The Ethics Committee of Lund University approved the project. All patients gave written consent for the study.
Calculation and statistics
All results are expressed as mean values ± SEM with n being the sample size. The experiments were repeated six to eight times for each substance. Gene expressions statistical analysis was used the normalized CT values (CT) that were performed with a one-way analysis of variance (ANOVA), followed by a multiple comparison posttest (Tukey’s test) using GraphPad and InStat version 3.00 (GraphPad Software). Other statistical analysis used Student’s t test (two-tailed). The significance was accepted when p was less than 0.05. The individual pEC50 values (calculated concentration of agonist that stimulates 50% contraction of the maximum possible contraction [Emax] of the agonist) were not calculated because the responses were still increasing at the highest available concentration of agonist. Instead, K25 was used, which is the concentration of the agonist that causes a vasoconstriction equal to 25% of 60 mmol/L K+. Because the maximum possible contraction of the agonists could not be reached owing to the limited supply of substance, the contraction at the highest agonist concentration (10 mmol/L for endogenous agonists, 0.1 mmol/L for stable agonists) calculated as percent of the contractile capacity of 60 mmol/L K+ was registered and called maximum contraction (Cmax).
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Results
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The contractile capacity of the vein and artery were examined by the addition of 60 mmol/L K+ (internal mammary artery, 2.9 ± 1.0 mN; saphenous vein, 11.1 ± 5.2 mN). The vascular contractions were studied after removal of the endothelium. Addition of 10 µM ACh in a NA precontracted vessel did not result in relaxation, indicating that the endothelium was properly removed.
Contractile responses to ß-MeATP
Single doses of ß-MeATP evoked concentration-dependent contractions in both vessels (Fig 1). The contractions to ß-MeATP were more prominent in the saphenous vein
(K25 = 4.96 ± 0.16, Cmax = 76 ± 10) as compared with the internal mammary artery (K25 = 4.18 ± 0.11, Cmax = 24 ± 13, p < 0.05; Table 2).
The K25-value was shifted to the left in saphenous vein compared with internal mammary artery. The weak contractions in internal mammary artery resulted in only three of the ß-MeATP stimulated vessels reaching K25 and this low n-value explains the lack of significance between the K25 values in saphenous vein and internal mammary artery for ß-MeATP.
P2x1 receptor desensitization
A transient contraction was induced by 10 µM ß-MeATP. When ß-MeATP was added a second time no contraction could be observed, indicating desensitized P2X1 receptors. To exclude the contribution of P2X receptors when studying P2Y receptor mediated contractions, these experiments were performed in the presence of 10 µM ß-MeATP.
P2y receptor mediated contractions
The P2Y1 receptors do not seem to mediate contraction in any of the vessels as 2-MeSADP had no effect with the exception of one subject who had a weak contraction to 2-MeSADP in the internal mammary artery.
The stable UDP analogue UDPßS was markedly more potent than UDP and induced strong contractions in the saphenous vein (K25 = 4.86 ± 0.25, Cmax = 80 ± 30). Less potent and less efficacious UDPßS responses could be observed in the internal mammary artery (K25 = 4.26 ± 0.11, p < 0.05, Cmax = 42 ± 14; Table 2). The UDPßS induced a sustained contraction that was maintained for more than 2 hours in the saphenous vein (Fig. 2). Such a prolonged contraction could not be observed for any of the other agonists used. The stable UTP analogue UTPS induced a stronger response in the internal mammary artery (K25 = 4.68 ± 0.23, Cmax = 149 ± 44) as compared with the saphenous vein (K25 = 4.29 ± 0.18, Cmax = 36 ± 12, p < 0.05). However if compared in mN this difference was not apparent. The ATP, UTP, and UDP induced vasoconstriction in a concentration-dependent manner although high concentrations compared with the stable agonists were needed in both the internal mammary artery and the saphenous vein owing to degrading ectonucleotides in the vessel wall. In general these unstable nucleotides were more potent in internal mammary artery because of more ectonucleotidases in the thicker vessel wall of the saphenous vein.
Contraction induced by ATP after desensitization with ß-MeATP and UTPS in saphenous vein
The contraction of ATP was not abolished after desensitization with ß-MeATP suggesting that P2X1 is not the only receptor mediating ATP induced contraction in the saphenous vein. To desensitize the P2Y2 receptors (that are activated by both UTP and ATP), the vessels were exposed to UTPS for 1 hour. After the vessel segments had been washed UTPS was added once more resulting in a transient contraction measuring about 10% of the initial contraction, indicating that the P2Y2 receptors were desensitized (Fig 3).
The ß-MeATP was then added to desensitize the P2X1 receptors. The UTPS and ß-MeATP were left in the vessel bath for further experiments with ATP. After this desensitization of P2X1 and P2Y2 receptors, ATP elicited contractions of approximately 65% of the initial contraction, indicating that other receptors than P2X1 and P2Y2 are involved in the ATP contraction (Fig 3).
Rna quantification in vascular smooth muscle cells from saphenous vein and internal mammary artery
In the saphenous vein and internal mammary artery, mRNA for all the target genes P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, and P2X7 could be detected. The ratios of GAPDH were shown as relative values. As shown in Figure 4,
the P2X1 receptor had the highest mRNA expression in saphenous vein (n = 5 different patients) and internal mammary artery (n = 5 different patients) compared with the other receptors (p < 0.05). The mRNA level order for the P2Y receptors was P2Y2 more than P2Y11 more than P2Y6 more than P2Y1 equal to P2Y4.
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Fig 4. The mRNA expression profiles of P2 receptors in saphenous vein (open bars) and internal mammary artery (solid bars). Data are shown as the ratios of targets/GAPDH (glyceraldehyde-3phosphate dehydrogenase; see methods) and the mean values are given over each column. *p less than 0.05 versus other target genes in each tissue.
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Comment
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The study demonstrated a clear difference in the distribution of contractile P2 receptors in the two main coronary bypass vessels, saphenous vein and internal mammary artery. The UDP-activated P2Y6 receptors and ATP-activated P2X1 receptors mediated more prominent contraction in the saphenous vein than in the internal mammary artery. Interestingly the UDP-activated P2Y6 receptors mediated longlasting contractions that could be important in the development of vasospasm.
Compared with arteriosclerosis in the arteries that begins in childhood the vein graft disease is a rapidly progressing condition. The major problems seem to be damage to the endothelium and ischemia in the vessel wall. The mechanisms behind these problems are not fully understood but a traumatic surgical technique together with a high pressure in a "low-pressure" system could be contributing factors. Differences in contractile P2 receptors may also be of importance. It is not surprising that differences in receptor expression exists as cells from the coronary vessels arise from the epicardial lining, the thoracic arteries develop from the mesectoderm of the neural crest, and peripheral blood vessels such as the saphenous vein arise from mesenchymal cells [11].
Recent cloning of P2 receptors has facilitated characterization of their vasocontractile responses but still the identification of P2 receptors expressed on vascular smooth muscle cells is made difficult particularly because of the absence of selective antagonists. Ligand instability is also a problem especially when investigations are performed in intact tissue, because nucleotides are metabolized by ectonucleotidases on the cells membranes. Even though there is a lack of selective antagonists, some stable and selective agonists are available (Table 1). The ß-MeATP is selective for P2X1 and P2X3 receptors and the 2-MeSADP for the P2Y1 receptor [2]. The vascular actions of the pyrimidine sensitive receptors have not been possible to evaluate until the synthesis and characterization of the stable analogues, UTPS and UDPßS, on selectively transfected P2Y receptor subtypes [12, 13]. Although UTPS is selective for P2Y2 and P2Y4 receptors it cannot discriminate between them [12]; UDPßS is selective for P2Y6 receptors with no effects on the other P2 receptors [13]. Using these selective agonists it was recently demonstrated that both P2Y2/4 and P2Y6 receptors mediate strong arterial contractions in rat mesenteric arteries [14]. However in human coronary arteries UDPßS did not mediate any contraction, indicating a lack of contractile P2Y6 receptors [15]. Selective agonists for the P2Y11 receptor are still not available.
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P2x receptor mediated contractions
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In our study, the selective P2X1 receptor agonist ß-MeATP induced transient contractions, which is typical for the rapidly desensitized P2X1 receptor. The P2X1 receptor has long been regarded as the dominant P2X receptor in blood vessels. Even though ß-MeATP also activates P2X3 receptors there is no evidence for P2X3 expression in vascular smooth muscle cells [16]. This together with the present results that demonstrate high levels of P2X1 receptor mRNA in the saphenous vein makes it probable that ß-MeATP induced contractions are mediated by P2X1 receptors. In the saphenous vein ß-MeATP induced a powerful contraction. The response in the internal mammary artery was significantly weaker, indicating that P2X1 induced contraction is more important in the saphenous vein than in the internal mammary artery.
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P2y2 receptor mediated contraction
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In the internal mammary artery UTPS induced a potent contraction similar to that seen in coronary arteries [15], indicating that P2Y2 or P2Y4 receptors are important contractile receptors in the internal mammary artery. In the saphenous vein UTPS also induced contractions but weaker than in internal mammary artery when expressed as percentage of K+-induced contraction. However because saphenous veins in absolute terms (mN) have much stronger contractions to the reference (K+), the contractions to UTPS in mN did not differ between saphenous vein and internal mammary artery. The results of the quantitative real-time PCR contribute to answering the frequently recurring question, if the vascular effects of UTP and UTPS are mediated by P2Y2 or P2Y4 receptors. In smooth muscle cells of the saphenous vein and internal mammary artery the P2Y4 receptor levels are less than half of the P2Y2 levels [10]. Thus the P2Y2 receptor seems to be the most important UTP sensitive receptor on vascular smooth muscle cells in the saphenous vein and the internal mammary artery.
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P2y6 receptor mediated contraction
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One of the most interesting findings was the strong contractile effect of UDPßS in the saphenous vein. The agonist UDPßS is a selective P2Y6 receptor agonist with no effect on any of the other cloned P2Y receptors [13]. Our findings indicate that the P2Y6 receptor is one of the most important contractile P2 receptors in the saphenous vein. The agonist UDPßS also induced contractions in the internal mammary artery but to a lesser extent. Furthermore, UDPßS does not mediate any contraction at all in coronary arteries [15]. Similarly contractile adrenergic -receptors are also absent in coronary arteries. Thus the coronary arteries are protected from the contractile effects of UDP owing to the lack of P2Y6 receptors. On the other hand in the saphenous vein the stable UDP analogue UDPßS was the strongest vasoconstrictor, indicating that UDP may be important as a vasoconstrictor in the vein. This could be beneficial when the vein acts as a capacitance vessel with the need for strong long-acting contractions to bring venous blood back to the heart. However it could be detrimental in coronary artery surgery by causing vasospasm.
The P2Y6 receptors have mitogenic effects and stimulate progression through the whole cell cycle per se, but also act in synergy with other growth factors such as platelet-derived growth factor [13]. This could be important because the smooth muscle cell proliferation in the venous graft disease contributes to the development of stenosis. Because the barrier of the endothelium in the saphenous vein is damaged during surgery, causing lack of endothelium-derived ectonucleotidases and increased levels of nucleotides, the proliferative and contractile properties of nucleotides could be more important in venous grafts compared with arteries. These processes could contribute to the development of venous graft disease and in restenosis after balloon angioplasty in the saphenous vein.
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P2y11 receptor mediated contraction
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There is strong evidence for ATP inducing contraction by stimulating P2X1 and P2Y2 receptors [17]. In our study half of the ATP-induced contraction in the saphenous vein remained even after desensitization of the P2X1 receptor. Our results indicate that there could be other receptors involved in the ATP contraction. Cario-Toumaniantz and coworkers [18] suggested that P2X7 could be involved in the contractility of the saphenous vein. However the mRNA expression of P2X7 receptors was markedly low in contrast to high P2X1 receptor levels in our results with quantitative real-time PCR.
Conversely the P2Y receptors are present to a large extent in vascular smooth muscle cells [10, 19]. The ATP contractions could be mediated by degradation of ATP to ADP with subsequent activation of P2Y1 or P2Y12 receptors but this is contradicted by the lack of contractile effects of the selective activator of these receptors, 2-MeSADP. The only P2Y receptors that are directly activated by ATP are P2Y2 and P2Y11. To discriminate between these, the P2Y2 receptor was desensitized by repeated UTPS stimulation, resulting in lost contractions to UTPS. Despite this ATP induced vasoconstriction suggesting that the ATP contraction was mediated by P2Y11 receptors. This was also supported by mRNA quantification with real-time PCR where P2Y11 together with P2Y2 were the second most expressed receptors. This is the first evidence that the P2Y11 receptor is expressed and could mediate contraction in vascular smooth muscle cells.
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Different duration of contractions induced by different p2 receptors
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It is not only the maximum contractile effect that mirrors a compounds importance as a vasoconstrictor. The duration of the contraction is of equal importance. To study this we compared the vasocontractile effect of single doses of ß-MeATP, UTPS, and UDPßS in the saphenous vein. The contractions were monitored until they spontaneously decreased (see Fig 4). The contraction induced by ß-MeATP was transient and lasted for about 5 minutes whereas the contraction mediated by UTPS was more prolonged, lasting for about 45 minutes. The UDPßS caused a slow onset and a long-lasting contraction that did not decrease even after 2 hours. In isolated cell systems UDP induces a slow and sustained accumulation of inositol triphosphate mediated by the P2Y6 receptor. The mechanism for this slow desensitization is that, in contrast to other P2Y receptors, the P2Y6 receptor lacks two serine residues in the cytoplasmic carboxyterminal that are crucial for agonist-dependent phosphorylation, desensitization, and loss of surface receptors [20]. Our results confirm that the P2Y6 receptor exhibits the same slow rate of desensitization resulting in a prolonged contraction and that could be important for the spasm induced in the saphenous vein. Spasm in the vein is a common problem both during harvesting and when the vein is connected to the heart. To deal with this problem both pharmacologic and mechanical methods have been used. It has become clear that both canalizing and distending the vein and the use of papaverin have a negative effect on the endothelial function [21]. For this purpose it would be interesting to use a selective and stable UDP receptor antagonist during coronary artery surgery and balloon angioplasty in venous grafts.
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Quantification of mRNA
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Using GAPDH as reference gene, high levels of the P2X1 receptor were found in the saphenous vein and internal mammary artery. The mRNA levels of P2Y receptor were generally lower but as they are coupled to different second messenger systems that does not mean that the functional significance has to be smaller. That is clearly shown by our pharmacologic experiments that demonstrate a stronger and more sustained contraction for P2Y than for P2X receptors. One explanation could be that the P2X1 receptor is an ion-channel (needing three to five subunits to form one channel) whereas the P2Y receptors are G-protein coupled. The P2Y2 and P2Y11 receptor expression were highest among the P2Y receptors. This finding is in agreement with the contractile effect of UTP and ATP. Somewhat surprising was that the P2Y6 receptor had a relatively low expression in the saphenous vein. It could be that fewer P2Y6 receptors are needed to give a strong contractile effect owing to its slow desensitization rates (see above).
Previously we analyzed the P2 receptor mRNA levels in internal mammary artery to compare with endothelial expression [10]. After finding pharmacologic differences in the contractile patterns we expected the mRNA levels to be different between internal mammary artery and saphenous vein. The powerful P2X1 and weaker P2Y2 receptor mediated contraction in the saphenous vein compared with the internal mammary artery was supported by finding a P2X1: P2Y2 mRNA ratio of 11:1 in the saphenous vein and 7:1 in the internal mammary artery (Fig 4) [10]. However the P2Y6 receptor mRNA expression pattern was similar in the two vessels, in contrast to the contraction experiments where UDPßS was most potent in saphenous vein. One possible explanation may be that the amount of receptors present on the cell surface is not dependent on regulation of the mRNA level or the results are affected by the different cell contents in the vessels. The saphenous vein has a much higher fibroblast content on the adventitial side, which may represent a larger part of the cells than the vascular smooth muscle cells. These fibroblasts may have "diluted" the P2 receptor mRNA and affected the results. We also examined the protein expression of these receptors (data not shown). However the band intensities for all the receptors were markedly lower in the saphenous vein than in the internal mammary artery. We believe that this nonspecific attenuation is caused by the high extracellular matrix content in the vein compared with the internal mammary artery that causes a relatively lower general receptor expression compared with the total protein content.
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Conclusion
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Both the selective P2X1 and P2Y6 receptor agonists, ß-MeATP and UDPßS, induced more prominent contractions in the saphenous vein as compared with the internal mammary artery. The agonist UDPßS induced stable long-lasting contractions, which is explained by the low desensitization rate of the P2Y6 receptor. The ATP-induced vasoconstriction could not be abolished by desensitization of P2X1 and P2Y2 receptors. In addition mRNA expression for the ATP sensitive P2Y11 receptor was high, indicating that P2Y11 may act as a contractile receptor, which has not been reported before. The UDP-activated P2Y6 receptors and the ATP-activated P2X1 and P2Y11 receptors play a primary role in mediating contraction of the saphenous vein. All together these results may present one explanation for the differences in saphenous vein and internal mammary artery graft properties and suggest that selective antagonists of P2X1, P2Y6, and P2Y11 receptors could be used to prevent vasospasm in the saphenous vein. Antagonists of P2Y6 could reduce vascular smooth muscle cell proliferation and restenosis in the saphenous vein.
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Acknowledgments
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The study has been supported by the Swedish Heart and Lung Foundation, the Franke and Margareta Bergqvist Foundation, the Wiberg Foundation, the Bergwall Foundation, the Zoegas Foundation, the Netpharma Foundation, the Tore Nilsson Foundation, and by Swedish Medical Research Council Grant 13130 (DE) and 9046 (SJ).
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Abstract
Introduction
