Photolysis of cell-permeant caged inositol pyrophosphates controls oscillations of cytosolic calcium in a ß-cell line.

Among many cellular functions, inositol pyrophosphates (PP-InsPs) are metabolic messengers involved in the regulation of glucose uptake, insulin sensitivity, and weight gain. However, their mechanisms of action are still poorly understood. So far, the influence of PP-InsPs on cellular metabolism has been studied by overexpression or knockout/inhibition of relevant metabolizing kinases (IP6Ks, PPIP5Ks). These approaches are, inter alia, limited by time-resolution and potential compensation mechanisms. Here, we describe the synthesis of cell-permeant caged PP-InsPs as tools to rapidly modulate intracellular levels of defined isomers of PP-InsPs in a genetically non-perturbed cellular environment. We show that caged prometabolites readily enter live cells where they are enzymatically converted into still inactive, metabolically stable, photocaged PP-InsPs. Upon light-triggered release of 5-PP-InsP5, the major cellular inositol pyrophosphate, oscillations of intracellular Ca2+ levels in MIN6 cells were transiently reduced to spontaneously recover again. In contrast, uncaging of 1-PP-InsP5, a minor cellular isomer, was without effect. These results provide evidence that PP-InsPs play an active role in regulating [Ca2+]i oscillations, a key element in triggering exocytosis and secretion in ß-cells.

Synthesis of 2-diphospho-myo-inositol 1,3,4,5,6-pentakisphosphate and a photocaged analogue.

Diphosphoinositol polyphosphates (inositol pyrophosphates, X-InsP7) are a family of second messengers with important roles in eukaryotic biology. Their chemical synthesis and modification remains a challenging task due to the high density of phosphate groups arranged around the myo-inositol core. Here, a novel approach is presented that facilitates the incorporation of the diphosphate in the 2-position (2-InsP7) and that enables the introduction of a photocage subunit.

Effect of cigarette smoke on endothelial regeneration in vivo and nitric oxide levels.

BACKGROUND: Cigarette smoking accelerates atherosclerosis and restenosis after vascular reconstruction. The mechanisms by which smoking alters vessel structure after injury are unclear. This study examined the effects of cigarette smoking on endothelial regeneration, an important component of arterial remodeling. MATERIALS AND METHODS: Adult male rats were subjected to balloon injury of the thoracic aorta and exposed to mainstream cigarette smoke via a Griffith-type smoking machine for 2 weeks. Control groups included rats which were restrained in the machine but not smoked and a group not utilizing the machine. Aortic reendothelialization was determined using Evan's blue staining of the arterial surface. Serum levels of nitric oxide were measured to determine if smoke exposure altered this potential endothelial cell mitogen. RESULTS: Cigarette smoking increased aortic endothelial regeneration (78.4 +/- 4.6% vs 59.2 +/- 2.1%, P < 0.05) and was associated with an increase in serum nitric oxide level (59.9 +/- 7. 1 microM vs 28.5 +/- 1.8 microM, P < 0.05). Daily restraint alone in the smoking machine had no effect on endothelial regeneration. CONCLUSIONS: This is the first report on the effects of smoking on endothelial regeneration and demonstrates that smoking increases reendothelialization after large vessel injury and serum levels of nitric oxide, an EC mitogen.