Catabolism of Ap4A and Ap3A in whole blood. The dinucleotides are long-lived signal molecules in the blood ending up as intracellular ATP in the erythrocytes

Adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A) are stored in large amounts in human platelets. After activation of the platelets both dinucleotides are released into the extracellular milieu where they play a role in the modulation of platelet aggregation and also in the regulation of the vasotone. It has recently been shown that the dinucleotides are degraded by enzymes present in the plasma [Lüthje, J. & Ogilvie, A. (1987) Eur. J. Biochem. 169, 385-388]. The further metabolism as well as the role of blood cells has not been established. The dinucleotides were first degraded by plasma phosphodiesterases yielding ATP (ADP) plus AMP as products which were then metabolized to adenosine and inosine. The nucleosides did not accumulate but were very rapidly salvaged by erythrocytes yielding intracellular ATP as the main product. Although lysates of platelets, leucocytes and red blood cells contained large amounts of Ap3A-degrading and Ap4A-degrading activities, these activities were not detectable in suspensions of intact cells suggesting the lack of dinucleotide-hydrolyzing ectoenzymes. Compared to ATP, which is rapidly degraded by ectoenzymes present on blood cells, the half-life of Ap4A was two to three times longer. Since the dinucleotides are secreted together with ADP and ATP from the platelets, we tested the influence of ATP on the rate of degradation of Ap4A. ATP at concentrations present during platelet aggregation strongly inhibited the degradation of Ap4A in whole blood. It is suggested that in vivo the dinucleotides are protected from degradation immediately after their release. They may thus survive for rather long times and may act as signals even at sites far away from the platelet aggregate.     

Cytochemical detection of carbohydrate and immunocytochemical detection of relaxin in the same secretory granule

We administered estradiol and progesterone to spayed guinea pigs, with resultant accumulation of secretory granules in endometrial gland cells. By initially employing protein A-colloidal gold immunolocalization of relaxin, followed by cytochemical staining of carbohydrate with the thiocarbohydrazide-silver proteinate method on the same section, we showed clearly that the secretory granules were composed of a central core containing relaxin and a cortex of carbohydrate-rich material. Use of normal rabbit serum rather than relaxin antiserum, and omission of periodic acid, demonstrated the specificity of the technique.