Inorganic polyphosphate regulates functions of thymocytes via activation of P2X purinoreceptors.

Inorganic polyphosphate (polyP) is an ancient polymer, which was proven to be a signalling molecule in the mammalian brain, mediating the communication between astrocytes via activation of P2Y1 purinoreceptors and modulating the activity of neurons. There is very limited information regarding the ability of polyP to transmit the information as an agonist of purinoreceptors in other cells and tissues. Here, we show that application of polyP to the suspension of primary thymocytes increases the concentration of intracellular calcium. PolyP evoked calcium signal was dependent on the presence of P2X inhibitors but not P2Y1 inhibitor. PolyP dependent increase in intracellular calcium concentration caused mild mitochondrial depolarization, which was dependent on inhibitors of purinoreceptors, extracellular calcium and inhibitor of mitochondrial calcium uniporter but wasn't dependent on cyclosporin A. Application of polyP modulated cell volume regulation machinery of thymocytes in calcium dependent manner. Molecular docking experiments revealed that polyP can potentially bind to several types of P2X receptors with binding energy similar to ATP - natural agonist of P2X purinoreceptors. Further molecular dynamics simulations with P2X4 showed that binding of one molecule of polyP dramatically increases permeability of this receptor-channel for water molecules. Thus, in this research we for the first time showed that polyP can interact with P2X receptors in thymocytes and modulate physiological processes.

Ecdysterone prevents negative effect of acute immobilization stress on energy metabolism of rat liver mitochondria.

Ecdysterone is a naturally occurring steroid hormone, which presents in arthropods and in a number of plants as an insect defence tool. There are many studies showing that application of ecdysterone can alter mitochondrial functions of mammalian cells, however it is not clear whether its effects are direct or mediated by activation of other cellular processes. In our study, we have shown how ecdysterone acts at the mitochondrial level in normal conditions and in certain pathology. We have demonstrated that application of immobilization stress to male rats causes uncoupling of mitochondrial oxidative phosphorylation, the preliminary application of ecdysterone prevents negative effect of immobilization stress on mitochondria. In-vitro experiments with isolated mitochondria have shown that ecdysterone can increase mitochondrial coupling and hyperpolarise mitochondria but without a noticeable effect on ADP/O ratio. Molecular docking experiments revealed that ecdysterone has high binding energy with mitochondrial FOF1 ATP synthase, but further biochemical analysis have not revealed either stimulatory or inhibitory effect of ecdysterone on FOF1 ATPase activity of the enzyme. Thus, ecdysterone can directly affect mitochondrial bioenergetics, though we assume that its preventive effect on mitochondria during immobilization stress is also coupled with the activation of some other cellular processes.