Unveiling the agonistic properties of Preyssler-type Polyoxotungstates on purinergic P2 receptors

Abstract

The Preyssler-type polyoxotungstate ({P₅W₃₀}) belongs to the family of polyanionic metal-oxides formed by group V and VI metal ions, such as V, Mo and W, commonly known as polyoxometalates (POMs). POMs have demonstrated inhibitory effect on a significant number of ATP-binding proteins in vitro. Purinergic P2 receptors, widely expressed in eukaryotic cells, contain extracellularly oriented ATP-binding sites and play many biological roles with health implications. In this work, we use the immortalized mouse hippocampal neuronal HT-22 cells in culture to study the effects of {P₅W₃₀} on the cytosolic Ca²⁺ concentration. Changes in cytosolic Ca²⁺ concentration were monitored using fluorescence microscopy of HT-22 cells loaded with the fluorescent Ca²⁺ indicator Fluo3. ³¹P-Nuclear magnetic resonance measurements of {P₅W₃₀} indicate its stability in the medium used for cytosolic Ca²⁺ measurements for over 30 min. The findings reveal that addition of {P₅W₃₀} to the extracellular medium induces a sustained increase of the cytosolic Ca²⁺ concentration within minutes. This Ca²⁺ increase is triggered by extracellular Ca²⁺ entry into the cells and is dose-dependent, with a half-of-effect concentration of 0.25 ± 0.05 μM {P₅W₃₀}. In addition, after the {P₅W₃₀}-induced cytosolic Ca²⁺ increase, the transient Ca²⁺ peak induced by extracellular ATP is reduced up to 100% with an apparent half-of-effect concentration of 0.15 ± 0.05 μM {P₅W₃₀}. Activation of metabotropic purinergic P2 receptors affords about 80% contribution to the increase of Fluo3 fluorescence elicited by {P₅W₃₀} in HT-22 cells, whereas ionotropic receptors contribute, at most, with 20%. These results suggest that {P₅W₃₀} could serve as a novel agonist of purinergic P2 receptors.