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 (Ap(4)) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), post-source decay MALDI MS, and enzymatic cleavage analysis. Furthermore, Ap(4) was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap(4) elicited dose-dependent vasodilations. Vasodilator responses were abolished in the presence of the P(2Y1) receptor antagonist MRS 2179 (1 microm) or the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (50 microm). After removal of the endothelium by Triton X-100, Ap(4) induced dose-dependent vasoconstrictions. Inhibition of P(2X) receptors by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (30 microm) or desensitization of P(2X) receptors by alpha,beta-methylene ATP (alpha,beta-meATP, 1 microm) diminished these vasoconstrictor responses completely. In the present study Ap(4) has been isolated from human tissue. Ap(4) was shown to exist in human myocardial tissue and was identified in ventricular specific granules. In coronary vasculature the nucleotide exerted vasodilation via endothelial P(2Y1) receptors and vasoconstriction via P(2X) receptors on vascular smooth muscle cells. Ap(4) acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion.
Highlights
The endocrine functions of myocardial tissue have been investigated widely in the past
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
An isolation procedure was constructed that was suitable to detect further novel nucleotides secreted by human myocardium
Summary
Human myocardial tissue was obtained from two graft recipients after heart transplantation. Purification of Adenosine 5Ј-Tetraphosphate from Human Myocardial Tissue—After excision from the heart transplant recipient, human myocardial tissue (10 g) was immediately placed in ice-cooled physiological saline solution and processed within 30 min. The tissue was lyophilized and powdered (step 1). The powder was suspended in 200 ml of 0.6 M ice-cold perchloric acid and homogenized at 25,000 rpm three times for 1 min. After centrifugation at 4,000 rpm for 10 min at 4 °C, the supernatant was titrated to pH 6.5 with HCl and centrifuged again as above (step 2). The supernatant with 40 mM TEAA added (final concentration) was pumped to the column, washed with 40 mM TEAA, and eluted with 40% acetonitrile in water at a flow rate of 1 ml/min. The 40% acetonitrile eluate was collected, frozen at Ϫ80 °C, and lyophilized
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