P2X7 receptor-mediated calcium dynamics in HEK293 cells: experimental characterization and modelling approach.

The P2X7 receptor (P2X7R) induces ionotropic Ca²âº signalling in different cell types. It plays an important role in the immune response and in the nervous system. Here, the mechanisms underlying intracellular Ca²âº variations evoked by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), a potent agonist of the P2X7R, in transfected HEK293 cells, are investigated both experimentally and theoretically. We propose a minimal model of P2X7R that is capable of reproducing, qualitatively and quantitatively, the experimental data. This approach was also adopted for the P2X7R variant, which lacks the entire C-terminus tail (trP2X7R). Then we introduce a biophysical model describing the Ca²âº dynamics in HEK293. Our model gives an account of the ionotropic Ca²âº influx evoked by BzATP on the basis of the kinetics model of P2X7R. To explain the complex Ca²âº responses evoked by BzATP, the model predicted that an impairment in Ca²âº extrusion flux through the plasma membrane is a key factor for Ca²âº homeostasis in HEK293 cells.

Regulation of innate immunity by the nucleotide pathway in children with idiopathic nephrotic syndrome.

Activation of the oxidative burst and failure of CD4(+) CD25(+) cell regulation have been implicated in idiopathic nephrotic syndrome (iNS). The intimate mechanism is, however, unknown and requires specifically focused studies. We investigated reactive oxygen species (ROS) generation [di-chlorofluorescein-diacetate (DCFDA)] fluorescence assay and the regulatory adenosine 5'-triphosphate (ATP) pathways in the blood of 41 children with iNS, utilizing several agonists and antagonists of nucleotide/nucleoside receptors, including the addition of soluble apyrase. The CD4(+) CD25(+) CD39(+) /CD73(+) expression was determined in vivo in parallel during disease activity. Overall, we found that the percentage of CD39(+) CD4(+) CD25(+) was reduced markedly in iNS by 80% (3·43±0·04% versus 13·14±0·07% of total lymphocytes, P<0·001). In these patients, reactive oxygen species (ROS) generation by polymorphonuclear neutrophils (PMN) at rest was a function of apyrase (CD39) expressed by CD4(+) CD25(+) , with higher rates in patients with very low CD39(+) CD4(+) CD25(+) levels (<7·5%). Addition of apyrase reduced ROS generation by 40% in both iNS and controls and was mainly effective in patients. The quota of ROS surviving ATP elimination was higher still in iNS. In vitro studies to limit ROS generation with adenosine analogues (2'-chloroadenosine and 5'-N-ethylcarboxamidoadenosine) produced minor effects. At variance, antagonizing ATP efflux with carbenoxolone or by antagonizing ATP effects (Brilliant Blue G, KN62 and A437089) reduced ROS generation comparable to apyrase. These results confirm a key role of ATP in the regulation of innate immunity and minimize the effect of adenosine. Decreased CD39(+) CD4(+) CD25(+) expression in iNS highlights an impairment of ATP degradation in this pathology. However, high ROS surviving ATP consumption implies a major role of other regulatory pathways.