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Ana Clara Melo, Peter D.R. Moeller, Howard Glasgow, JoAnn M. Burkholder, and John S. Ramsdell

Abstract

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. Key words: GH₄C₁, Pfiesteria, purinergic receptors, P2X₇, toxin. -- Environ Health Perspect 109(suppl 5) :731-737 (2001) .

http://ehpnet1.niehs.nih.gov/docs/2001/suppl-5/731-737melo/abstract.html