Abstract-Diadenosine polyphosphates have been identified as important regulators of vascular tone and blood pressure.In reference to the background of the well-known vasoconstriction induced by hemolysate, we questioned whether this action may be due in part to the presence of diadenosine polyphosphates in human erythrocytes. Therefore, erythrocytes were separated from other blood cells and deproteinated. To concentrate and purify nucleotides, the extract was chromatographed by anion exchange, affinity, and reversed-phase columns. In one of the purified fractions, diadenosine hexaphosphate (diadenosine 5Ј, 5Ј-P 1 , P 6 hexaphosphate [AP 6 A]) was identified by matrix-assisted laser desorption/ ionization mass spectrometry, ultraviolet spectroscopy, and enzymatic analysis. Hemolysate of erythrocytes injected into the isolated perfused rat kidney induced a dose-dependent vasoconstriction, which was partially inhibited by P 2 -purinoceptor antagonist. The presence of diadenosine phosphates in granules releasable into the circulation suggests an important role in the control of local perfusion or blood pressure. 1,5,6 Vasoactive action of the diadenosine polyphosphates was demonstrated in numerous vascular models, including most of the physiologically important elements of blood pressure regulation. Diadenosine polyphosphates have different actions on the vasculature, depending on the number of phosphate groups. Diadenosine 5Ј, 5Ј-P 1 , P 2 diphosphate (AP 2 A), diadenosine 5Ј, 5Ј-P 1 , P 3 triphosphate (AP 3 A), and diadenosine 5Ј, 5Ј-P 1 , P 4 tetraphosphate (AP 4 A) are potent vasodilators in the mesenteric and coronary vasculature, 3,5,6 whereas diadenosine 5Ј, 5Ј-P 1 , P 5 pentaphosphate (AP 5 A) and diadenosine 5Ј, 5Ј-P 1 , P 6 hexaphosphate (AP 6 A) appear to have vasoconstrictor properties. 1 The vasoconstrictive effect of hemolysate of dog erythrocytes on dog basilar artery in vitro is caused by ATP. 7 Furthermore, a hypertensive factor that induced a prolonged elevation of blood pressure in normotensive rats has been reported in the erythrocytes of spontaneously hypertensive rats. It appeared that the substance occurred in normotensive as well as hypertensive rats, albeit at a lower concentration in normal animals. 8 Diadenosine tetraphosphate was found to be synthesized within avian erythrocytes. 9 Therefore, in the present study we examined whether diadenosine polyphosphates also exist in human erythrocytes.
MethodsBlood from normotensive subjects (men aged 26Ϯ4 years) was collected in tubes containing sodium heparin (1%), and the erythrocyte number was counted. The blood was centrifuged at 2000g at 4°C for 10 minutes, and the plasma together with the buffy coat was discarded. The erythrocytes were washed twice with ice-cold saline. After centrifugation, 5 volumes of ice-cold high-performance liquid chromatography (HPLC)-grade water were added and placed in the refrigerator at 4°C for 20 minutes to hemolyze the erythrocytes. This hemolysate was centrifuged at 6000g for 10 minutes
Purification ProceduresT...