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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2013 Jan 3;33(3):629–636. doi: 10.1161/ATVBAHA.112.300600

Ecto-5’-nucleotidase, CD73, is an endothelium-derived hyperpolarizing factor synthase

Masanori Ohta 1,#, Kazuyoshi Toyama 1,#, David D Gutterman 1,2,3, William B Campbell 2, Vincent Lemaître 4, Ryutaro Teraoka 4, Hiroto Miura 1,4
PMCID: PMC3587694  NIHMSID: NIHMS436337  PMID: 23288168

Abstract

Objective

Adenosine dilates human coronary arteries by activating potassium channels in an endothelial cell-independent manner. Cell surface ecto-5’-nucleotidase (CD73) rapidly dephosphorylates extracellular adenosine 5’-monophosphate to adenosine. We tested the hypothesis that coronary vasodilation to adenine nucleotides is mediated by an endothelial CD73-dependent, extracellular production of adenosine that acts as an endothelium-derived hyperpolarizing factor (EDHF).

Methods and Results

Videomicroscopy showed that adenine nucleotides but not inosine potently dilated and hyperpolarized human coronary arteries independent of NO, prostacyclin and classical EDHFs, whereas endothelial denudation, adenosine receptor antagonism, adenosine deaminase or CD73 blockers reduced vasodilations. Liquid chromatography-electrospray ionization-mass spectrometry revealed adenosine accumulation in perfusates from arteries in the presence of adenosine 5’-diphosphate. CD73 was localized on the cell surface of endothelial cells but not of vascular smooth muscle cells, and its deficiency suppressed vasodilation of mouse coronary arteries to adenine nucleotides and augmented vasodilation to adenosine. Adenosine dose-dependently dilated and hyperpolarized human coronary arteries to a similar extent as adenosine 5’-diphosphate.

Conclusions

Coronary vasodilation to adenine nucleotides is associated with endothelial CD73-dependent production of extracellular adenosine that acts as an EDHF by relaxing and hyperpolarizing underlying vascular smooth muscle cells via activating adenosine receptors. Thus CD73 is a novel EDHF synthase in human and mouse coronary arteries.

Keywords: adenosine, endothelium-derived relaxant factor, vasodilation

Introduction

In response to physiological and pathophysiological stimuli, adenosine 5’-triphosphate (ATP) and -diphosphate (ADP), both cell membrane-impermeable adenine nucleotides, are released into the bloodstream via membrane transporters and/or leakage upon cell lysis1. Cardiovascular sources of ATP and ADP include erythrocytes, endothelial cells (ECs), platelets, inflammatory cells, and sympathetic nerves. Once released, ATP and ADP can influence vascular tone, platelet aggregation, and inflammatory responses in autocrine and paracrine fashions through the interaction with purinoceptors as they are rapidly and efficiently hydrolyzed1,2. In general, ATP and ADP elicit EC-dependent dilation in a variety of vasculatures in several species. Proposed mediators include nitric oxide (NO), prostacyclin (PGI2), endothelium-derived hyperpolarizing factor (EDHF)3,4, P2 purinoceptors5, and P1 (adenosine) receptors6. Thus it is likely that the mechanisms involved are largely dependent upon either receptor-mediated EC activation or the hydrolysis kinetics of adenine nucleotides in vasculatures. However, the precise mechanism is largely unknown in human coronary arteries (HCA).

Adenosine, a metabolite of adenine nucleotides, contributes to maintaining coronary blood flow during basal conditions in humans7 and swine8 but not in dogs9, while it plays an important role in the regulation of coronary circulation under pathophysiological conditions such as increased myocardial workload, hypoxia and ischemia in many species10. It is supposed that adenosine may possess the potential properties to act as an EDHF, since it dilates arteries via activating potassium channels11 and hyperpolarizes vascular smooth muscle cells (VSMCs)12. However, potassium channel-dependent hyperpolarization of VSMCs is not always associated with vasodilation13, and it has not been directly proven in intact arteries if adenosine induces vasodilation concomitantly with VSMC hyperpolarization, a common criteria of EDHF action in VSMCs. In addition, it is unknown whether ECs and/or VSMCs are capable of generating adenosine sufficient to produce vasodilation via VSMC hyperpolarization. Furthermore, if capable, the enzymatic source of adenosine production and its localization in vasculatures, and the mechanisms for adenosine-mediated vasodilations remain elusive.

Nucleotidases are major enzymes implicated in the intracellular and extracellular metabolism of adenine nucleotides to adenosine. Extracellular nucleotides are successively dephosphorylated to adenosine 5’-monophosphate (AMP) by cell surface ecto-ATPase (CD39), then to adenosine by ecto-5’-nucleotidase (CD73) which is bound onto the cell membrane via a glycosyl-phosphatidylinositol anchor, with the catalytic site facing the extracellular region1. Recent studies have demonstrated that global deficiency of CD73 reduces basal coronary perfusion, increases platelet activation, and accelerates leukocyte adhesion to ECs by suppressing adenosine formation, implying CD73 as an enzymatic source for local adenosine production in mouse hearts and peripheral circulation14,15. However, little is known about the precise role of endothelial CD73 in the regulation of coronary arterial tone. Furthermore, as we reported previously, adenosine induces an EC-independent vasodilation by activating potassium channels in VSMCs of HCA11. Endothelial CD73 may therefore generate extracellular adenosine that is transferred to underlying VSMCs, causing membrane hyperpolarization and relaxation - an EDHF phenomenon.

The aims of this study were to investigate whether adenine nucleotides cause HCA vasodilation concomitantly with VSMC hyperpolarization via extracellular formation of adenosine but not via receptor-mediated EC activation; whether vasodilations to adenine nucleotides are mediated by endothelial CD73, and finally whether adenosine-induced dilation of EC-denuded arteries occurs with VSMC hyperpolarization. Here, we provide novel evidence that CD73 is an EDHF synthase that generates extracellular adenosine as an EDHF.

Materials and Methods

HCA (n=108; passive diameter, 144±5 µm) were dissected from 106 right atrial appendages obtained at surgery with waiver of consent. Demographic data and diagnoses of patients were obtained from hospital records at the time of surgery, as summarized in Table I in the online supplement. Mouse left anterior descending coronary arteries (MCA; n=15 from male 12-week old CD73 knockout and 14 from the wild-type littermates, 154±6 µm) were isolated from mouse hearts. Vasodilator responses of HCA and MCA were examined by videomicroscopy. In separate experiments, we measured simultaneous changes in vessel diameter and membrane potential (Em) of VSMCs in response to ADP and adenosine in HCA. EC-denuded HCA were used for experiments with adenosine. Whole mount tissue immunostaining was performed to localize cell surface CD73 expression in non-permeabilized HCA, MCA, and mouse aortas. Levels of adenosine in the perfusates collected from perfused HCA were quantified using liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI-MS). A full description of the Materials and Methods is given in the online-only Data Supplement.

Results

EDHF-mediated vasodilation of HCA to ADP

The roles of NO, PGI2, and EDHF in HCA dilation to ADP was analyzed. As we have shown previously16, ADP (10−10 to 10−4 mol/L) produced a potent vasodilation that was abolished by EC denudation (max dilation: denudation 13±5, p<0.05 vs. control 89±2%, n=7–15), indicating that ADP is an EC-dependent dilator in HCA. Neither 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of soluble guanylyl cyclase; 5×10−5 mol/L) alone nor the combination of Nω-nitro-L-arginine methyl ester (LNAME, a NO synthase inhibitor; 10−4 mol/L) with indomethacin (INDO, a cyclooxygenase inhibitor; 10−5 mol/L) inhibited ADP-induced vasodilation (Fig. 1A, ODQ: max dilation: 87±7, p=ns vs. control 81±6%, n=5 and Fig.1B, LNAME+INDO: max dilation: 97±1, p=ns vs. control 94±1%, n=8), whereas KCl (50×10−3 mol/L) abolished the dilation (Table 1). Measurements of simultaneous changes in vascular tone and Em of VSMCs to ADP in EC-intact HCA showed that vasodilation occurred concomitantly with a significant hyperpolarization of VSMCs (Fig. 1C). EC-dependent dilation of HCA to ADP is therefore independent of NO and PGI2, but is associated with VSMC hyperpolarization due to potassium efflux, implying a possible involvement of EDHF.

Figure 1. EDHF-mediated dilation to ADP.

Figure 1

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(A) ADP produced a dose-dependent dilation in HCA. ODQ, a selective inhibitor of soluble guanylyl cyclase, had no effect on ADP-induced vasodilation. (B) The combination of LNAME, a NO synthase inhibitor, and INDO, a cyclooxygenase inhibitor, had no effect on ADP-induced vasodilation. (C) The vasodilation was associated with VSMC membrane hyperpolarization (n=5, #p<0.05 vs. preconstriction).

Table 1.

Role of proposed EDHFs in ADP-induced dilation of HCA.

ED50
(−log[mol/L])		 Max dilation (%)
Inhibitors Targets n Control Inhibitor Control Inhibitor
KCl K+ channels 5 6.3±0.3 6.2±0.5 83±4 26±16 *
PPOH Cytochrome P450 6 5.7±0.1 5.8±0.2 94±4 95±2
Catalase H2O2 5 6.2±0.1 6.1±0.1 97±1 94±2
Carbenoxolone Gap junctions 5 6.3±0.1 6.2±0.2 98±1 99±0
BaCl2 + Ouabain Kir and Na+/K+-ATPase 5 6.3±0.1 5.9±0.3 98±1 96±2
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Values are shown as mean±SE, with n indicating the number of HCA.

*

p<0.05 vs. control.

Classical EDHFs include epoxyeicosatrienoic acids (cytochrome P450 epoxygenase metabolites of arachidonic acid)17, H2O218,19, gap junction communication20, and potassium ions21. ADP-induced HCA dilation was not affected after inhibiting cytochrome P450 epoxygenase activity using 6-(2-propargyloxyphenyl)hexanoic acid (PPOH; 3×10−5 mol/L), scavenging H2O2 with catalase (1000 U/L), blocking gap junction communication by carbenoxolone (10−4 mol/L), nor inhibiting potassium ion-induced activation of inwardly-rectifying potassium channels (Kir) and Na+/K+-ATPase by BaCl2 (a Kir blocker, 30×10−6 mol/L) and ouabain (an inhibitor of Na+/K+-ATPase, 10−3 mol/L) (Table 1). Therefore, ADP-induced HCA dilation is likely mediated by a new and unidentified EDHF.

Role of adenosine in vasodilation of HCA to adenine nucleotides

The role of purinoceptors in HCA dilation to adenine nucleotides was examined. ATP (10−8 to 10−4 mol/L) elicited a dose-dependent HCA dilation that was insensitive to antagonizing P2 receptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS; 10−4 mol/L) (Fig. 2A; max dilation: 95±4, p=ns vs. control 91±6%). Vasodilation to ADP was also resistant to PPADS (Fig. 2B; dilation at 10−5 mol/L: 88±4, p=ns vs. control 80±16%, n=5). In addition, P2Y receptor stimulation with adenosine 5´-O-[2-thiodiphosphate] (ADPβS, a non-hydrolyzable ADP analog; 10−7 to 10−5 mol/L) mimicked vasodilation to ADP with a nearly equal potency (dilation at 10−5 mol/L: 67±12, p=ns vs. control 78±11%, n=7), indicating that P2Y receptors in HCA are functional. These results suggest that HCA dilation to adenine nucleotides is independent of their specific P2 receptors, likely involving a product of the high energy phosphate metabolism.

Figure 2. Role of extracellular adenosine and adenosine receptors.

Figure 2

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(A and B) HCA dilation to ATP and ADP was resistant to PPADS, a P2 receptor antagonist. (C and D) 8-SPT, a P1 receptor antagonist, reduced vasodilation to ATP and ADP. (E and F) ADA, which catalyzes the deamination of adenosine to inosine, decreased vasodilation to ATP and ADP. # p<0.05 vs. control.

To determine if HCA dilation is mediated by adenosine, a metabolite of adenine nucleotides, the effect of 8-(p-sulfophyenyl) theohpylline (8-SPT, a P1 [adenosine] receptor antagonist; 10−4 mol/L) was tested. As shown in Fig. 2C, 8-SPT significantly inhibited vasodilation to ATP (dilation at 10−6 mol/L: 18±1, p<0.05 vs. control 62±8%, n=6). A similar effect was found in ADP-induced vasodilation (Fig. 2D, dilation at 10−6 mol/L: 1±1, p<0.05 vs. control 37±9%, n=6). In contrast, 8-SPT had no effect on EDHF-dependent vasodilation to bradykinin (10−6 mol/L), which is mediated largely by H2O219 (dilation at 10−6 mol/L: 93±4, p=ns vs. control 94±4%, n=5). In addition, intraluminal administration of cell-impermeable adenosine deaminase (ADA; 6 U/ml) which catalyzes the deamination of adenosine to inosine significantly decreased dilations to ATP (Fig. 2E; dilation at 10−6 mol/L: 13±6, p<0.05 vs. control 63±17%) and ADP (Fig. 2F; dilation at 10−6 mol/L: 16±12, p<0.05 vs. control 58±20%) and abolished the dilation to AMP (10−9 to 10−4 mol/L, dilation at 10−6 mol/L: 5±3, p<0.05 vs. control 83±3%), but not to bradykinin (10−6 mol/L; data not shown). On the other hand, inosine (10−9 to 10−4 mol/L), the primary metabolite of adenosine, had no vasodilator effect (dilation at 10−6 mol/L: 1±3%, n=3). EC-independent vasodilation to papaverine (10−4 mol/L) was insensitive to 8-SPT and ADA (data not shown).

Isolated HCA were next perfused at physiological range of shear stress19 with PSS containing ADP or bradykinin, and adenosine concentration in the collected perfusate was quantified by LC/ESI-MS. A large amount of adenosine accumulated in the perfusate from vessels treated with ADP during a single passage and within seconds (10−5 mol/L ADP yielded 13 pg/µl ≈ 5×10−8 mol/L of adenosine, and 10−4 mol/L ADP yielded 494 pg/µl ≈ 2×10−6 mol/L of adenosine, n=3), whereas perfusion without ADP or with bradykinin (10−8 to 10−6 M) yielded little adenosine production (control; ~ 1 pg/µl). Therefore, adenosine is rapidly and efficiently produced from ADP, possibly at the intraluminal surface of HCA, whereas neither shear stress nor bradykinin produces a substantial amount of adenosine. These findings suggest that extracellular adenosine is a mediator of HCA dilation to adenine nucleotides.

Role of EC surface CD73 in vasodilation to adenine nucleotides

Whole mount tissue immunostaining of non-permeabilized HCA showed that the cell surface of ECs, but not of VSMCs, was strongly positive for CD73. Several cells in the tunica adventitia were also faintly stained (Fig. 3A). This positive staining for endothelial CD73 was not observed in EC-denuded vessels (Fig. 3B) and in negative controls (Fig. 3C). In addition, MCA and aortas also showed CD73-positive staining on the EC surface in wild-type mice (Fig. 3D and F), but not in CD73 knockout mice (Fig. 3E and G). These data confirm that, in the vasculatures, cell surface CD73 is present on ECs but not on VSMCs.

Figure 3. CD73 expression on the extracellular membrane of ECs.

Figure 3

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Whole mount immunostaining for CD73 showed an extracellular localization of CD73 on ECs in a non-permeabilized HCA (A, arrow). This signal was not detected in EC-denuded HCA (B) and in negative controls (C). MCA and ascending aortas from wild type mice also exhibited cell surface expression of CD73 on ECs (D and F, arrow), whereas no signal was detected in MCA and aortas from CD73 knockout mice (E and G). HCA were counterstained with hematoxylin. Positive staining appears in brown. Magnification ×20~100. L indicates the lumen, M the medial layer, and A the adventitia.

The effect of CD73 inhibition or deficiency on coronary dilation to adenine nucleotides was tested. α,β-methylene adenosine 5’-diphosphate (APCP, a selective and competitive inhibitor of CD73; 10−4 mol/L) significantly reduced vasodilation to ATP (Fig. 4A, dilation at 10−6 mol/L: 26±10, p<0.05 vs. control 78±4%, n=4) and ADP (Fig. 4B, dilation at 10−6 mol/L: 11±7, p<0.05 vs. control 53±12%, n=6), and abolished AMP-induced dilation (dilation at 10−6 mol/L: 6±6, p<0.05 vs. control 83±3%). No inhibitory effect was seen on the dilation to bradykinin (10−6 mol/L; data not shown). Intraluminal administration of the anti-CD73 antibody IE9 (10 µg/mL), which inhibits cell surface CD73 nucleotidase activity, also suppressed ATP-induced dilation (dilation at 10−6 mol/L: −3±3, p<0.05 vs. control 54±11%). Neither APCP nor the antibody IE9 had an effect on the dilation to papaverine (10−4 mol/L, data not shown). In addition, CD73 deficiency also caused a marked reduction in vasodilation to ATP and ADP compared to wild-type mice (ATP, Fig. 4C; dilation at 10−6 mol/L: −3±2, p<0.05 vs. control 32±9%, n=6–7 and ADP, Fig. 4D; dilation at 10−6 mol/L: 5±1, p<0.05 vs. control 20±2%, n=8), while it slightly enhanced vasodilation to adenosine (dilation at 10−5 mol/L: 56±7, p<0.05 vs. control 38±6%). CD73 disruption had no effect on EC-dependent or -independent vasodilation to acetylcholine (10−7 to 10−4 mol/L, dilation at 10−4 mol/L: 58±8, p=ns vs. control 54±10%) and to papaverine (10−4 mol/L, data not shown). These findings indicate that vasodilator responses to adenine nucleotides are largely dependent upon EC surface CD73 activity in HCA and MCA.

Figure 4. CD73 is required for adenine nucleotides-induced dilation.

Figure 4

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(A and B) APCP, a selective inhibitor of CD73, reduced vasodilations to ATP and ADP. (C and D) CD73 deficiency markedly reduced vasodilations to ATP and ADP in mice. # p<0.05 vs. control

Adenosine acts as an EDHF

We determined whether adenosine acts as an EDHF by measuring simultaneous changes in both Em and vascular diameter in response to adenosine in EC-denuded HCA. Adenosine caused a rapid drop in Em in a single VSMC of HCA in a dose-dependent fashion (Fig. 5A; upper panel). A representative measurement of steady-state Em and vascular tone in a single HCA by a series of VSMC impalements in the presence of increasing doses of adenosine is presented in Fig. 5A, lower panel. Exogenous application of adenosine to the superfusate simultaneously hyperpolarized VSMCs and dilated HCA in a dose-dependent manner. Fig. 5B summarizes the steady-state changes in Em and vascular tone of HCA to adenosine. Following preconstriction of HCA to acetylcholine with a modest depolarization of VSMCs, adenosine induced vasodilation, together with a significant hyperpolarization of VSMCs. These results indicate that EC-independent HCA dilation to adenosine depends largely upon membrane hyperpolarization of VSMCs, suggesting that adenosine acts as an EDHF.

Figure 5. Adenosine elicits VSMC relaxation and hyperpolarization.

Figure 5

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(A) Upper panel: adenosine hyperpolarized a single VSMC of isolated and pressurized HCA (continuous measurements) in time-dependent and dose-dependent fashions. Lower panel: Adenosine simultaneously decreased VSMC Em (measurements by a series of VSMC impalements) and increased vessel diameter of a HCA in a dose-dependent manner. (B) Summary of simultaneous changes in VSMC Em and vessel diameter to adenosine (n=5, # p<0.05 vs. preconstriction).

Discussion

Our study directly identifies endothelial CD73 as a novel mediator of coronary vasodilation to adenine nucleotides in humans and mice. The major new findings are six-fold: (1) EC-dependent vasodilation to adenine nucleotides is associated with VSMC hyperpolarization and is independent of NO and PGI2; (2) the dilation is mediated largely by the activation of adenosine receptors; (3) extracellular production of adenosine is necessary to elicit vasodilation; (4) CD73 is localized on the surface of ECs but not of VSMCs and its activity is required to mediate vasodilation; (5) CD73 deficiency abolishes coronary vasodilation, and (6) adenosine EC-independently induces vasodilation together with a significant hyperpolarization of VSMCs. These findings suggest that adenosine generated by CD73 on ECs plays an important role in adenine nucleotides-induced vasodilation of coronary arteries. EC surface CD73 is most likely a novel EDHF synthase, generating extracellular adenosine as an EDHF.

Adenine nucleotides and adenosine in the coronary circulation

Variable amounts of ATP and ADP are continually present in the coronary circulation, and the local concentration of extracellular ATP often exceeds micromolar levels due to its release into intraluminal spaces of the coronary circulation1. Stimuli that induce ATP release include increased blood flow22 hypoxia23,24, catecholamine25, vascular injury associated with platelet aggregation26, and myocardial workload27. Adenine nucleotides are quickly hydrolyzed during a single coronary passage28 or when incubated with cultured ECs29. This rapid metabolism correlates with vasodilation accompanied by an increase in venous adenosine concentration in the coronary circulation30. We also found that ADP is hydrolyzed to adenosine within seconds in the lumen of HCA. Moreover, scavenging extracellular adenosine with ADA significantly reduced HCA dilation to adenine nucleotides, consistent with the finding that ADA attenuates the increase in coronary conductance caused by both adenosine and ATP in guinea-pig hearts6. Adenosine contributes to maintaining coronary blood flow during increased myocardial workload, and hypoxic and ischemic conditions10. The contribution during basal conditions is varied among species10; the blockade of adenosine axis significantly lowers basal coronary blood perfusion in humans7 and swine8 but not in dogs9. These findings suggest that extracellular adenosine generated from adenine nucleotides play a significant role in the regulation of human coronary circulation under physiological and pathophysiological conditions.

Role of purinoceptors in coronary dilation to adenine nucleotides

Adenine nucleotides have been shown to cause coronary vasodilation through P2 receptor activation5. However, P2 receptor antagonism did not impair HCA dilation to adenine nucleotides, while P1 receptor antagonism reduced vasodilation. Furthermore, ADPβS, a P2Y1 receptor agonist, elicited a dose-dependent dilation similar to ADP, indicating that P2 receptors were functional. Thus it is unlikely that alteration in P2 receptors in function or expression caused the insusceptibility to P2 receptor antagonism in HCA. Consistent with our results, vasodilation to adenine nucleotides is dependent on adenosine receptor activation in guinea pig hearts6. Moreover, we previously reported that adenosine EC-independently induces HCA dilation by activating adenosine A2a receptors in VSMCs11. Consequently, coronary vasodilation to adenine nucleotides is likely mediated largely by adenosine, which activates adenosine A2a receptors in VSMCs.

Role of CD73 in adenosine production in the coronary circulation

CD73 is the predominant AMP-hydrolyzing enzyme on human ECs29 and in hearts of other species15. We found that CD73 is expressed primarily on the extracellular membrane of ECs, but not VSMCs, in HCA, MCA, and mouse aortas. A large amount of extracellular adenosine was produced from HCA in the presence of intraluminal ADP, and blockade or deficiency of CD73 activity significantly reduced coronary dilations to adenine nucleotides. These results support our hypothesis that ECs extracellularly convert adenine nucleotides to adenosine that is transferable to and relaxes adjacent VSMCs. CD73 deficiency slightly augmented MCA dilation to adenosine. Since peripheral and tissue levels of adenosine may be lower in CD73 knockout mice15, enhanced vasodilation to adenosine in these mice could be a compensatory mechanism for the loss of CD73-derived adenosine, similar to the enhanced vasorelaxation to NO donors in endothelial NO synthase knockout mice18. Our results are consistent with a study demonstrating a role for CD73-derived adenosine in the inhibition of endothelial permeability in response to adenine nucleotides31 and suggest that CD73 plays a crucial role in the regulation of coronary arterial tone via generating adenosine from ECs.

CD73 is a novel EDHF synthase

We show here that EC-dependent HCA dilation to ADP is accompanied by VSMC hyperpolarization. This dilation was inhibited by a high concentration of KCl but not by ODQ, by the combination of LNAME and INDO, or by inhibitors of classical EDHFs, indicating that these responses were mediated by an unidentified EDHF. We previously reported that adenosine induces an EC-independent HCA dilation by activating adenosine receptors and calcium-activated potassium channels in VSMCs11. Here, we demonstrate extracellular conversion of adenine nucleotides to adenosine and inhibition of vasodilations to adenine nucleotides by ADA, blockade and genetic disruption of CD73, and antagonism of adenosine receptors in HCA or MCA. Adenosine induced vasodilation concomitantly with VSMC hyperpolarization, which has also been observed in rabbit carotid arterial VSMCs12. The concept that adenosine may function as an EDHF is widely recognized, but there has been little evidence that adenosine plays a discernible role as an EDHF32 in intact arteries. In addition, the enzymatic source, its localization, and the precise mechanisms for induced vasodilations remained elusive. We therefore propose that endothelial CD73 is a novel EDHF synthase producing adenosine as an EDHF in coronary arteries in humans and mice.

Potential limitations of this study

An intrinsic limitation of this study is the lack of normal HCA, since fresh cardiac tissue cannot be obtained from a healthy person. However, this limitation allows us to study adenine nucleotides in the setting of chronic cardiovascular disease that cannot be adequately mimicked in animal models. In addition, it is not clear if endothelial CD73 plays a role in ventricular HCA. Atrial HCA exhibit vasodilator responses similar to those in ventricular HCA in response to a variety of endothelium-dependent vasodilators, although acetylcholine constricts atrial arteries but dilates ventricular arteries33. The MCA used in the present study were isolated from ventricles and similarly showed the importance of CD73 in the regulation of vasodilation to adenine nucleotides. We therefore propose that this mechanism of vasodilation commonly exists both in atrial and ventricular arteries.

Vasodilation to adenine nucleotides was not completely abolished by the inhibitors used in our study. CD73 inhibitors and a P1 receptor antagonist caused greater inhibition of vasodilation to ADP than that to ATP, while vasodilation to AMP was more sensitive to inhibitors than those to ATP and ADP. This leaves the possibility that P2 receptor subtypes insensitive to PPADS and/or other unknown vasodilator(s) mediate the remaining component of dilations to ATP. On the other hand, CD73 deficiency led to an almost complete suppression of vasodilation to ATP in MCA. Thus, this difference is most likely explained by an incomplete blockade of CD73 activity by competitive inhibitors or by the existence of divergent pathways between humans and mice.

In addition to ECs, cardiac myocytes also express CD73 and release adenine nucleotides. In isolated and perfused mouse hearts deficient in CD73, the loss of adenosine generated by cardiac myocytes could account for the lower basal coronary perfusion15. Further studies are needed to determine whether CD73 deficiency alters tissue perfusion, including coronary perfusion in the mouse, in which circulating erythrocytes also release a large amount of adenine nucleotides24. Moreover, the relative contribution of endothelial and myocardial CD73-generated adenosine to the regulation of coronary circulation also needs to be clarified.

Clinical implications

EDHF is a predominant vasodilator in the human coronary circulation, in particular in diseases where it compensates for the loss of NO19,34. Adenosine plays a role in the maintenance and metabolic regulation of coronary circulation in healthy subjects7 and during hypoxia and ischemia-reperfusion in animals10. CD73 deficiency reduced the basal level of coronary perfusion in Langendorff mouse hearts without altering ventricular size and functions, while it slightly increased systemic blood pressure15,35. Thus it may be of clinical benefit to further explore the precise role of endothelial CD73 as an EDHF synthase in the regulation of coronary circulation and systemic blood pressure in normal and diseased states.

Adenine nucleotides are generally recognized as pro-inflammatory and pro-thrombotic molecules, whereas adenosine has the opposite effects1. CD73 deficiency promotes endothelial inflammation leading to leukocyte adhesion, edema, and neointimal formation14,15,36. Furthermore, CD73 is cardio-protective in ischemic hearts37, and its expression and activity are decreased in certain disease conditions such as diabetes mellitus38, irradiation39, and atherosclerosis40. Administration of soluble CD73 to mice abolished ADP-induced platelet aggregation and increased tail bleeding time41. Thus, increasing CD73 expression and activity may constitute a novel therapeutic strategy for cardiovascular ailments.

Conclusions

Coronary artery dilation to adenine nucleotides is mediated by an endothelial CD73-dependent, extracellular production of adenosine that induces VSMC hyperpolarization and relaxation through activation of adenosine receptors and potassium channels. Therefore, CD73 may play an important role in the regulation of the coronary circulation by generating adenosine as an EDHF.

Supplementary Material

01

Acknowledgments

The authors wish to thank the Division of Cardiothoracic Surgery at the Medical College of Wisconsin, the Cardiovascular Surgery Associates of Milwaukee, the Cardiothoracic Surgery Group of Milwaukee, the Midwest Heart Surgery Institute, the Wisconsin Heart Group, and Reno Heart Surgeons at the Renown Regional Medical Center for providing surgical specimens.

Funding sources

This study was supported by American Heart Association Northland Affiliate Beginning Grant-in-Aid 0360035Z, Advancing a Healthier Wisconsin grant from Medical College of Wisconsin, and NIH grants R01 HL080173, HL080173-02S1, P20 RR018751, and P20 GM103513 (to H.M.); NIH grants P01 HL68769 and P50 HL65203 and Veterans Administration Merit Review grant (to D.D.G.); and R01 HL51055 and HL103673 (to W.B.C.).

Footnotes

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Disclosures

None.

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