Cardiovascular Ecto-5′-Nucleotidase

• adenosine
• CD 73
• cell adhesion molecules
• coronary flow
• ecto-phosphatases
• inflammation

Elsewhere in this issue, Koszalka et al¹ add another chapter to the long-running saga of the significance of ecto-5′-nucleotidase (CD-73) in cardiovascular function, showing that this enzyme catalyzes the formation of adenosine in amounts sufficient to have important physiological effects. A brief sketch of the tortuous path to our present understanding of the role of this enzyme puts this work in perspective. It also illustrates how science evolves stepwise, testing hypotheses based on available evidence and modifying them to fit the new evidence. In the early 1960s, Berne² and, independently, Gerlach et al³ showed that hypoxia stimulated adenosine production in the heart. Within a few years, histochemists showed phosphatase activity able to hydrolyze AMP on the surface of cardiac and skeletal muscle myocytes. At first this seemed to provide an explanation for the adenosine released from hypoxic heart muscle, but there was a serious problem. The enzyme was outside the cell, but its substrate, AMP, was in the cytoplasm, separated by a sarcolemma thought at the time to be impermeable to nucleotides. Theories advanced to resolve this dilemma included the idea that the ectoenzyme also had transport capacity, but this faltered for lack of direct evidence. There was little further progress for approximately a decade, in part because the discovery of nitric oxide-mediated vasodilation⁴ dispelled the notion that adenosine was “the” regulator of coronary vascular resistance. Then, Japanese and American workers isolated cytosolic nucleotidases, and Andrew Newby clinched the importance of the cytosolic enzyme by showing that leukocytes poisoned with 2-deoxyglucose still produced adenosine despite inactivation of the ectoenzyme by antibodies directed against it.⁵ Cloning the genes for the cytosolic enzyme in the late 1990s seemed the final step in establishing the cytosol as the site of adenosine production. By then, thinking about the function of CD73 had largely shifted to its role as a B lymphocyte adhesion molecule.⁶

However, there were still inconsistencies in the evidence supporting a solely cytosolic origin of cardiac adenosine. Inhibitors of the equilibrative nucleoside transporter, which should prevent adenosine export from the site of its formation, actually augmented the accumulation of adenosine in the extracellular space. Measurements of the transmembrane gradient of adenosine concentration⁷ showed that the concentration is higher in the extracellular space, reflecting in part the efficiency of the incorporation of adenosine into the adenylate pool by adenosine kinase, the cytosolic enzyme that converts adenosine to AMP. The simplest explanation for such findings is that adenosine indeed forms outside cells and, in well-oxygenated hearts, the cytosolic compartment acts as a sink rather than as the source of adenosine leaving the heart.

We now know that cells can release ATP and other adenine nucleotides through any of several kinds of channels or from within secretory granules such as those of platelets, whereupon several ecto-phosphatases⁸ degrade it to adenosine (Figure). The questions now become, “How important is the extracellular pathway of adenosine formation, and does that adenosine actually do anything?” Koszalka et al have now answered both. They found that although the ecto-enzyme accounts for only a minor fraction of cardiac 5′-nucleotidase activity, the adenosine it generates has a significant impact on basal vascular tone, hemostasis, and leukocyte adhesion. In other words, the enzyme not only regulates the function of cells in the walls of blood vessels but also regulates the function of the blood cells traversing them. Although the authors draw their conclusions very narrowly, CD73-catalyzed extracellular adenosine formation might be physiologically important in organs other than the heart and blood vessels. For example, a large body of evidence suggests that adenosine plays a key role in tubuloglomerular feedback.⁹ Although there is now persuasive evidence¹⁰ that ATP participates in this critical function, the blunting of the response by the CD73 inhibitor adenosine-α,β-methylene diphosphonate¹¹ and almost complete loss of autoregulatory capacity in mice lacking the A₁ adenosine receptor (A₁AR^−/−)^12,13 or CD73¹⁴ suggests the ATP might serve as a substrate supporting adenosine production in addition to whatever role it might play as a primary signaling molecule.

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Roles of cytosolic and ecto-5′-nucleotidases in adenosine formation. AK indicates adenosine kinase; AP, unspecific alkaline phosphatase; APCs, ATP-permeable channels; ATPases, ATP-hydrolyzing enzymes; c-5′NP and e-5′-NP, cytosolic and ecto-5′- nucleotidases; eNT, equilibrative nucleoside transporter; MK, myokinase; NPP, nucleotide (pyro)phosphatases. The “stalks” connecting NPP, AP, and e5′-NP to the plasma membrane represent the peptide (NPP) and glycophosphatidylinositol chains (AP, e5′-NP) anchoring these enzymes to the membrane. For simplicity, this model does not include extracellular adenosine production from cAMP catalyzed by ecto-phosphodiesterase or production in the cytosol through the S-adenosylhomocysteine hydrolase pathway, both of which contribute to net adenosine production.

Coronary resistance was slightly but significantly higher in the CD73^−/− mice, suggesting that the small amount of adenosine generated by that pathway might still participate in setting basal coronary tone. Such is probably not the case during hypoxia, when hyperemia owes to the exuberant outpouring of adenosine resulting from a rapid, high-degree inhibition of recycling through adenosine kinase.¹⁵

Recently, Colgan et al¹⁶ discovered another example of the involvement of CD73 in cardiovascular regulation. Activated neutrophils traversing the endothelial barrier release ATP, which undergoes sequential hydrolysis by CD39, a diphosphohydrolase, and CD73 to release adenosine, which then activates A_2B adenosine receptors on the endothelial cells to seal the endothelial gap and restore barrier integrity.

And there is more. Using immunohistochemistry, Koszalka et al showed that in addition to CD73, coronary microvessels contain low levels of unspecific alkaline phosphatase, another AMP-hydrolyzing ectoenzyme. The minor effect of the alkaline phosphatase inhibitor levamisole authoritatively established the primacy of CD73 in the extracellular formation of adenosine. Interestingly, the two enzymes occurred in different microvessels, adding to other evidence of the regional heterogeneity of vascular endothelial cells.

By activating adenosine A_2A receptors, adenosine suppresses the expression of cell adhesion molecules on both leukocytes and endothelial cells.¹⁷ This easily accounts for the increased monocyte adhesion the authors measured in CD73^−/− mice (apparently, the function of CD73 as a leukocyte adhesion molecule is limited to B lymphocytes⁶). The same receptors on platelets antagonize activation by agents such as ADP and thrombin, likewise accounting for the impaired coagulation they measured in these mice.

So, this elegant multidisciplinary (and multinational) study, which combined molecular biology, histochemistry, and coronary physiology, has shown that ecto-5′-nucleotidase is a very important player in cardiovascular physiology after all, although not in ways anyone imagined 40 years ago when Berne and Gerlach first floated the “adenosine hypothesis.”