In adult non-replicating tissues such as heart, demand for dNTPs (deoxynucleoside triphosphates) is low but essential for mitochondrial DNA replication and nuclear DNA repair. dNTPs may be synthesized from salvage of deoxyribonucleosides or by reduction of ribonucleotides. We have hypothesized that the cardiac mitochondrial toxicity of the nucleoside analogue AZT (3'-azido-3'-deoxythymidine; known as zidovudine) is caused by inhibition of thymidine kinase 2 of the salvage pathway and subsequent TTP pool depletion. The extent to which this hypothesis has merit depends on how much the heart relies on thymidine phosphorylation for maintenance of the TTP pool. In the present study, we used isotopic tracing to demonstrate that both TTP and dCTP are solely synthesized by phosphorylation of thymidine and deoxycytidine respectively, with no evidence for synthesis from other precursors. We have also shown that UTP and CTP are synthesized by phosphorylation of uridine and cytidine respectively, with no detectable role for the de novo pyrimidine synthesis pathway. Lastly, we have demonstrated that AZT decreased the TTP pool by 50% in 30 min of perfusion, while having no effect on other dNTPs. In summary, the present study demonstrated that adult rat heart has a limited mechanism for dCTP and TTP synthesis and thus these pools may be more sensitive than replicating cells to drugs such as AZT that affect the salvage pathway.

Both zinc (Zn) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. To understand the cross talk between the two of them, here we studied Zn and ROS accumulation by employing fluorescent probes in HeLa cells to further the understanding of the cause and effect relationship of these two important cellular signaling systems during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn rises that were divided into four phases in the course of 30 min of OGD. The first Zn rise was a transient, which was followed by a latent phase during which Zn levels recovered; however, levels remained above a basal level in most cells. The final phase was the second Zn rise, which reached a sustained plateau called Zn overload. Zn rises were not observed when Zn was removed by TPEN (a Zn chelator) or thapsigargin (depleting Zn from intracellular stores) treatment, indicating that Zn was from intracellular storage. Damaging mitochondria with FCCP significantly reduced the second Zn rise, indicating that the mitochondrial Zn accumulation contributes to Zn overload. We also detected two OGD-induced ROS rises. Two Zn rises preceded two ROS rises. Removal of Zn reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn contributes to mitochondrial ROS generation. There was a Zn-induced increase in the functional component of NADPH oxidase, p47, thus suggesting that NADPH oxidase may mediate Zn-induced ROS accumulation. We suggest a new mechanism of cross talk between Zn and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn and ROS accumulations during the course of ischemic stress.

It is well known that zinc (Zn(2+)) is required for the process of insulin biosynthesis and the maturation of insulin secretory granules in pancreatic beta (β)-cells, and that changes in Zn(2+) levels in the pancreas have been found to be associated with diabetes. Glucose-stimulation causes a rapid co-secretion of Zn(2+) and insulin with similar kinetics. However, we do not know whether Zn(2+) regulates insulin availability and secretion. Here we investigated the effect of Zn(2+) on glucose-stimulated insulin secretion (GSIS) in isolated mouse pancreatic islets. Whereas Zn(2+) alone (control) had no effect on the basal secretion of insulin, it significantly inhibited GSIS. The application of CaEDTA, by removing the secreted Zn(2+) from the extracellular milieu of the islets, resulted in significantly increased GSIS, suggesting an overall inhibitory role of secreted Zn(2+) on GSIS. The inhibitory action of Zn(2+) was mostly mediated through the activities of KATP/Ca(2+) channels. Furthermore, during brief paired-pulse glucose-stimulated Zn(2+) secretion (GSZS), Zn(2+) secretion following the second pulse was significantly attenuated, probably by the secreted endogenous Zn(2+) after the first pulse. Such an inhibition on Zn(2+) secretion following the second pulse was completely reversed by Zn(2+) chelation, suggesting a negative feedback mechanism, in which the initial glucose-stimulated Zn(2+) release inhibits subsequent Zn(2+) secretion, subsequently inhibiting insulin co-secretion as well. Taken together, these data suggest a negative feedback mechanism on GSZS and GSIS by Zn(2+) secreted from β-cells, and the co-secreted Zn(2+) may act as an autocrine inhibitory modulator.

New Findings r What is the central question of this study?The main aim of the present study was to determine glucose-stimulated zinc secretion and the effect of zinc on glucose-stimulated insulin secretion in pancreatic β-cells. r What is the main finding and its importance?Using a newly developed approach, we demonstrated a robust glucose-stimulated zinc secretion. Importantly, the application of zinc inhibited glucose-stimulated insulin secretion. Our findings provide evidence that zinc, after being secreted, can regulate the insulin secretion of β-cells by a negative feedback mechanism.Diminished or inappropriate secretion of insulin is associated with type II diabetes. The cellular/molecular mechanism coupled with the regulation of insulin secretion is still under intense investigation. Divalent ion zinc (Zn 2+ ) is co-packaged and co-secreted with insulin and is intimately involved in the process of insulin biosynthesis and the maturation of insulin secretory granules. The study reported here investigated glucose-stimulated zinc secretion (GSZS) and the effect of zinc on glucose-stimulated insulin secretion (GSIS) in the HIT-T15 pancreatic β-cell line. Zinc secretion was measured using a newly developed fluorescent zinc imaging approach, and the insulin secretion was measured using an enzyme-linked immunosorbent assay. There was apparent granular-like zinc staining in β-cells. The application of glucose induced detectable zinc secretion or GSZS. Like GSIS, GSZS was dependent on the glucose concentration (5-20 mm) and the presence of extracellular calcium. The application of a zinc chelator enhanced GSZS. When brief paired-pulse glucose stimulations, which involve the initial glucose stimulation followed by a second round of glucose stimulation, were applied, zinc secretion or GSZS that followed the first pulse was inhibited. This inhibition was reversed by zinc chelation, suggesting a feedback mechanism on GSZS by zinc secreted from β-cells. Finally, the application of zinc (50 μm) strongly inhibited GSIS as measured by enzyme-linked immunosorbent assay. The present study suggests that insulin secretion is regulated by co-secreted zinc that may act as an autocrine inhibitory modulator.