Multi-Nucleation of P2X4 and P2X7 HEK Cells

Purinergic receptors, P2X4 and P2X7, function as ligand-gated channels stimulated by extracellular ATP.  P2X4 and P2X7 receptors form high calcium permeabilization and membrane pore expansion.  Previously, the purinergic P2X7 receptor has been shown to induce giant multinucleation morphology in Human Embryonic Kidney 293 (HEK) cells, while P2X4 is still under study.  The exact mechanism behind the signaling cascades of these receptors is unclear, and must be elaborated upon. Here in this study we are modifying the same HEK293 cells by over expressing both the purinergic P2X4 and P2X7 receptors to demonstrate multi-nucleation.  By utilizing cell tracker green (5-chloromethylfluorescein diacetate) and cell tracker red (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetra-methylrhodamine) at various time points with and without inhibition of multi-nucleation with the purinergic antagonist, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS) solution; multi-nucleation is examined using flow cytometry and fluorescence microscopy.  Our results show that the over expression of these receptors in HEK293 cells induces these cells to aggregate and fuse, causing multiple nuclei to bind and share a cytoplasm.  Purinergic receptors have been identified as stimulating the opening of large 900da pores that release a variety of cytokines by characterizing the receptors involvement in multi-nucleation we learn more about the importance of these purinergic receptors in general cell function.