Microfluorimetric analysis of a purinergic receptor (P2X7) in GH4C1 rat pituitary cells: effects of a bioactive substance produced by Pfiesteria piscicida.

Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP.