Identification and characterization of diadenosine 5′,5‴‐P¹,P²‐diphosphate and diadenosine 5′,5‴‐P¹,P³‐triphosphate in human myocardial tissue

Abstract

We examined whether human cardiac tissue contains diadenosine polyphosphates and investigated their physiological role. Extracts from human cardiac tissue from transplant recipients were fractionated by size exclusion-, affinity-, anion exchange- and reversed-phase chromatography. MALDI-MS analysis of two absorbing fractions revealed molecular masses of 676.2 Da and 756.0 Da. The UV spectra of both fractions were identical to that of adenosine. Postsource decay MALDI mass spectrometry indicated that the molecules with a mass of 676.2 Da and 757.0 Da contained AMP and ATP, respectively. As shown by enzymatic cleavage, both molecules consist of two adenosines interconnected by either two or three phosphates in 5'-positions of the riboses. Two substances can be identified as 5',5"'-P1,P2-diphosphate (Ap2A) and 5',5"'-P1, P3-triphosphate (Ap3A). Ap2A and Ap3A, together with ATP and ADP, are stored in myocardial-specific granules in biologically active concentrations. In the isolated perfused rat heart, Ap2A and Ap3A caused dose-dependent coronary vasodilations. In myocardial preparations, Ap2A and Ap3A attenuated the effect of isoproterenol, exerting a negative inotropic effect. The calcium current of guinea pig ventricular myocytes, stimulated by isoproterenol, was also attenuated by Ap2A and Ap3A. The presence of Ap2A and Ap3A in cardiac-specific granules and the actions of these substances on the myocardium and coronary vessels indicate a role for these substances as endogenous modulators of myocardial functions and coronary perfusion.

Cloning, expression and characterization of YSA1H, a human adenosine 5′-diphosphosugar pyrophosphatase possessing a MutT motif

“…Of the other potential substrates, Ap # A is the most recent of the diadenosine polyphosphates to be identified in i o. It is present in specific granules in human myocardium and can attain an intragranular concentration of 9 mM [29]. When released extracellularly, it acts as a coronary vasodilator.…”

Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases

“…Until now, the lowest K i values known were for phosphonate analogues of Ap 4 A. For example, AppCH 2 ppA inhibited the (asymmetrical) Ap 4 A hydrolases from lupin [31] and Artemia [32] with K i values of 0.25 µM and 0.4 µM respectively, and the (symmetrical) Ap 4 A hydrolase from E. coli with a K i of 7 µM [32]. Comparable effects were also observed with halomethylene analogues.…”

“…The Abbreviations used: Ap 4 A, diadenosine tetraphosphate; Np n N , dinucleoside 5 ,5 -P 1 ,P n -polyphosphate (where N and N are 5 -O-nucleosides and n is the number of phosphate residues in the polyphosphate chain that links the two 5 -esterified nucleosides). 1 To whom correspondence should be addressed (e-mail guranow@au.poznan.pl).…”

“…No or poor inhibition of the plant enzyme by many analogues that affect the corresponding human enzyme can be explained by the more restricted space in the substrate-binding cleft in the former enzyme. Because compound (4b) is the strongest inhibitor of both of these (asymmetrical) Ap 4 A hydrolases, it may most resemble the natural substrate, Ap 4 A.…”

“…Most predominant are the dinucleoside tri-and tetra-phosphates, but diadenosine penta- [2], hexa- [3] as well as di- [4] and heptaphosphates [5] have been found in some mammalian cells. The biological roles of Np n N s are still fairly obscure, with data suggesting a variety of possible functions in addition to toxic properties [6].…”