Probiotics are an upcoming group of nutraceuticals claiming positive effects on athlete’s gut health, redox biology and immunity but there is lack of evidence to support these statements.

The members of the nitric oxide synthase (NOS) family, eNOS, nNOS and iNOS, are well-characterized enzymes. However, due to the lack of suitable direct NO sensors, little is known about the kinetic properties of cellular NO generation by the different nitric oxide synthase isoenzymes. Very recently, we developed a novel class of fluorescent protein-based NO-probes, the geNOps, which allow real-time measurement of cellular NO generation and fluctuation. By applying these genetic NO biosensors to nNOS-, eNOS- and iNOS-expressing HEK293 cells we were able to characterize the respective NO dynamics in single cells that exhibited identical Ca²⁺ signaling as comparable activator of nNOS and eNOS. Our data demonstrate that upon Ca²⁺ mobilization nNOS-derived NO signals occur instantly and strictly follow the Ca²⁺ elevation while NO release by eNOS occurs gradually and sustained. To detect high NO levels in cells expressing iNOS, a new ratiometric probe based on two fluorescent proteins was developed. This novel geNOp variant allows the measurement of the high NO levels in cells expressing iNOS. Moreover, we used this probe to study the L-arginine-dependency of NO generation by iNOS on the level of single cells. Our experiments highlight that the geNOps technology is suitable to detect obvious differences in the kinetics, amplitude and substrate-dependence of cellular NO signals-derived from all three nitric oxide synthase isoforms.

Nitric oxide (NO) homeostasis maintained by neuronal/endothelial nitric oxide (NO) synthase (n/eNOS) contributes to regulate cardiac function under physiological conditions. At the early stages of ischaemia, NO homeostasis is disturbed due to Ca²⁺-dependent e/nNOS activation. In endothelial cells, successive drop in NO concentration may occur due to both uncoupling of eNOS and/or successive inhibition of nNOS catalytic activity mediated by arachidonic acid-induced tyrosine phosphorylation of this enzyme. The reduced NO bioavailability triggers nuclear factor (NF)-κB activation followed by the induction of inducible NOS (iNOS) expression. In cardiomyocytes ischaemia also triggers the induction of iNOS expression during reperfusion. The massive amounts of NO which are subsequently produced following iNOS induction may exert on cardiomyocytes and the other cell types of cells of the heart, such as endothelial and smooth muscle cells, macrophages and neutrophils, opposing effects, either beneficial or toxic. The balance between these two double-faced actions may contribute to the final clinical outcomes, determining the degree of functional adaptation of the heart to ischaemia/reperfusion injury. In the light of this new vision on the critical role played by the cross-talk between n/eNOS and iNOS as well as the non enzymatic NO production by nitrite, we have reason to believe that new pharmacological measurements or experimental interventions, such as ischaemic preconditioning, aimed at counteracting the drop in NO levels beyond the normal range of NO homeostasis during early reperfusion can represent an efficient strategy to reduce the extent of functional impairment and cardiac damage in the heart exposed to ischaemia/reperfusion injury.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2–related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.