Abstract 241: Role of CD73, Ecto-5′ Nucleotidases, in Human Coronary Arteriolar Dilation to Adenine Nucleotides
Adenosine triphosphate (ATP) has been proposed as a metabolic regulator of microcirculation. We previously reported in human coronary arterioles (HCA) that vasodilation to ATP or ADP is mediated by adenosine. Since ecto-nucleotidases or alkaline phosphatases rapidly and efficiently hydrolyze circulating ATP, adenosine diphosphate (ADP) and adenosine monophos-phate (AMP) to adenosine, we tested the hypothesis that vasodilation to adenine nucleotides is associated with CD73 activity in HCA.
Methods: Changes in internal diameter to ATP, ADP, AMP or inosine were recorded in HCA (156±12μm, n=24) dissected from right atrial appendage and in left anterior descending coronary arteries (mLAD) of CD73 knockout mice (165±7μm, n=7) with videomicroscopy. CD73 localization or adenosine production was examined by immunohistochemistry or by liquid chromatography-electrospray ionization-mass spectrometry (LC/ESI-MS).
Results: ATP, ADP and AMP but not inosine potently dilated HCA in a dose-dependent manner. α,β-methylene adenosine 5′-diphosphate (10−4 M, a competitive inhibitor of CD73) partially but significantly inhibited these vasodilations, whereas pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (10−4 M, a selective P2 receptor antagonist) had no effect. LC/ESI-MS showed that a large amount of adenosine accumulates in the perfusate from HCA treated with 10−4 M ADP (≅500 pg/μl), whereas perfusion without ADP yields little adenosine production (≅1 pg/μl). Immunohistochemistry revealed CD73 localization in HCA, similar to mLAD, showing abundant expression of CD73 in endothelial cells but no or little expression in the medial layer or adventitia. Intraluminal application of hCD73 antibody 1E9 (10μg/ml, an hCD73 blocker) abolished vasodilation to ATP (%Max dilation; 1±9% vs control 83±7 at 10−5 M). CD73 deficiency similarly abolished vasodilation of mLAD to ATP (1±9 vs control 83±8% at 10−5 M). None of the inhibitors affected dilation to papaverine.
In conclusion: Vasodilation to adenine nucleotides is dependent largely upon endothelial CD73 activity but not upon P2 receptor activation. Thus CD73 activity may play an important role in the regulation of human coronary microcirculation through adenine nucleotide hydrolyzation.