Identification and characterization of adenosine 5′-tetraphosphate in human myocardial tissue

Abstract

Endocrine functions of the human heart have been studied extensively. Only recently, nucleotidergic mechanisms have been studied in detail. Therefore, an isolation strategy was developed to isolate novel nucleotide compounds from human myocardium. The human myocardial tissue was fractionated by several chromatographic studies. A substance purified to homogeneity was identified as adenosine 5′-tetraphosphate (Ap4) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), post-source decay MALDI MS, and enzymatic cleavage analysis. Furthermore, Ap4 was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap4 elicited dose-dependent vasodilations. Vasodilator responses were abolished in the presence of the P2Y1 receptor antagonist MRS 2179 (1 μM) or the NO synthase inhibitor NG-nitro-L-arginine methyl ester (50 μM). After removal of the endothelium by Triton X-100, Ap4 induced dose-dependent vasoconstrictions. Inhibition of P2X receptors by pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (30 μM) or desensitization of P2X receptors by α,β-methylene ATP (α,β-meATP, 1 μM) diminished these vasoconstrictor responses completely. In the present study Ap4 has been isolated from human tissue. Ap4 was shown to exist in human myocardial tissue and was identified in ventricular specific granules. In coronary vasculature the nucleotide exerted vasodilation via endothelial P2Y1 receptors and vasoconstriction via P2X receptors on vascular smooth muscle cells. Ap4 acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion.