Adenine nucleotide storage and secretion in platelets as studied by 31P nuclear magnetic resonance.

Abstract

Suspensions of human and pig blood platelets have been studied by 31p NMR at 145.7 MHz and by chemical and radiochemical determination of nucleotide levels. In both types of platelets the cytoplasmic nucleotide pool, which was prelabeled by incubation with 1j4CJadenine, was selectively reduced by addition of H202/NaN3 or 2-deoxyglucose/antimycin A. After the reduction of cytoplasmic ATP in human platelets, the 31P NMR spectra showed an almost complete loss of the nucleoside di-and triphosphate resonances at temperatures examined (4-50'C), indicating that only the cytoplasmic nucleotides had been observed, with no detectable contributions from the granular ATP, ADP, and pyrophosphate. Slow tumbling of the granular nucleotides, possibly due to aggregation, is the probable explanation of their undetectability at 145.7 MHz. Similar experiments showed that, in pig platelets, granular ATP and ADP were not detected by 31P NMR at 4VC but were observed at higher temperatures, indicating that aggregation may be occurring at the lower temperatures. Upon thrombin stimulation of human platelets, the NMR spectra and the chemical and radioactivity analyses showed that the granular adenylates and pyrophosphate were secreted, and that cytoplasmic ATP levels were appreciably reduced.Blood platelets are free-floating anucleate cells that play a fundamental role in hemostasis and the maintenance of vascular integrity. These functions are mediated, in part, by adenine nucleotides, which are segregated into two distinct compartments (1). One of the two compartments is the membraneenclosed dense granules, in which as much as 65% of the total cellular ATP and ADP is sequestered together with serotonin, pyrophosphate, and Ca2+ in human platelets (2) and with serotonin and Mg2+ in pig (3) platelets. The cytoplasmic pool contains the remaining adenylates with an ATP/ADP ratio of 7-10; this pool participates in the energy metabolism of the cell and, unlike the granular pool, is affected by metabolic inhibitors (4, 5). During platelet secretion at 370C the granular pool is extruded to the extracellular volume, whereas the cytoplasmic ATP is retained intracellularly, and is partially converted into hypoxanthine (1). When platelets are incubated with radioactive nucleotide precursors such as l14C]adenine, the cytoplasmic pool is labeled rapidly (half time~40 sec); in contrast, the granules incorporate the label slowly (half time -18 hr) (6).High-resolution 31P nuclear magnetic resonance (NMR) provides a rapid, accurate, and noninvasive method for the study of phosphorylated metabolites in vivo (7-9). Furthermore, 31P NMR measurements possess the capability to distinguish among nucleoside phosphate resonances arising from different subcellular compartments when these compartments maintain differences in pH, divalent cation concentration, or both. With this in mind, we have examined the 31P NMR spectra of resting platelets, of cells challenged with thrombin to induce secretion, and of cells metabolically perturbed with H202 plus NaN3 or...

Compartmentation of Nucleotides in Corn Root Tips Studied by 31P-NMR and HPLC

“…4). Qualitatively similar results have been reported from NMR studies of human blood platelets (4,28), skeletal muscle (7), and rat kidney (6), where it was concluded that NMR-invisible nucleotides present in vivo were immobilized; immobilized species give broad signals that are undetectable in high resolution experiments. Hence, existence of an immobilized pool of NDP in corn root tips is one possible explanation for the result in Figure 4.…”

“…Any such distinctions between nucleotides in vivo is lost on extraction. Compartmentation of nucleotides between immobilized and freely mobile pools was suggested in NMR studies of human blood platelets (4,28), skeletal muscle (7), and rat kidney (6), from comparison of the amounts of nucleotides visible in vivo and in extracts. Such comparisons were made in this study, and our results indicate the existence of an NMR-invisible pool of NDP in vivo.…”

Trypanosoma cruzi Contains Major Pyrophosphate Stores, and Its Growth in Vitro and in Vivo Is Blocked by Pyrophosphate Analogs

“…The presence of pyrophosphate in "volutin" granules (vacuoles stained pink with basic blue dyes), which are also rich in calcium and magnesium, has been described in the free-living ciliate Tetrahymena pyriformis by direct chemical analysis (25,26) and 31 P NMR (27). The dense granules of blood platelets are also known to contain pyrophosphate together with ATP, ADP, and serotonin (28). Previous studies in trypanosomatids have demonstrated the presence of a high concentration of pyrophosphate in Leishmania major (11), although its subcellular localization was not established.…”

31P NMR studies of intact anchorage-dependent mouse embryo fibroblasts.

“…Some of the ADP in this extract may have come from hydrolysis of ATP during the extraction procedure. ADP bound to high molecular weight proteins, such as actin, is not detected in NMR spectra of intact cells (11). Therefore, some of the ADP recovered in the extract may fall in this category.…”