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

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'''-P-1,P-2-diphosphate (Ap(2)A) and 5',5'''-P-1,P-3-triphosphate (Ap(3)A). Ap(2)A and Ap(3)A, together with ATP and ADP, are stored in myocardial-specific granules in biologically active concentrations. In the isolated perfused rat heart, Ap(2)A and Ap(3)A caused dose-dependent coronary vasodilations. In myocardial preparations, Ap(2)A and Ap(3)A 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 Ap(2)A and Ap(3)A. The presence of Ap(2)A and Ap(3)A 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.