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Karen L. Kimm-Brinson,¹ Peter D. R. Moeller,¹ Michéle Barbier,¹ Howard Glasgow, Jr.,² JoAnn M. Burkholder,² and John S. Ramsdell¹

¹Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, National Oceanic and Atmospheric Administration-National Ocean Service, Charleston, South Carolina, USA; ²Department of Botany, North Carolina State University, Raleigh, North Carolina, USA

Abstract

We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH₄C₁ rat pituitary cells. Adenosine-5´-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH₄C₁ rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx) . The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2´,4´-disulfonic acid (PPADS) . Further characterization of a P2X receptor on the GH₄C₁ cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist ,ß-methylene ATP (,ß-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2´,3´-(4-benzoyl) benzoyl ATP (BzATP) , which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH₄C₁ cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH₄C₁ cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin. Key words: c-fos, GH₄C₁, P2X7, Pfiesteria, pituitary, purinergic, toxin. Environ Health Perspect 109:457-462(2001) . [Online 1 May 2001]

http://ehpnet1.niehs.nih.gov/docs/2001/109p457-462kimm-brinson/ abstract.html

Address correspondence to J. S. Ramsdell, Chief, Coastal Research Branch Center for Coastal Environmental Health and Biomolecular Research, NOAA-National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412 USA. Telephone: (843) 762-8510. Fax: (843) 762-8700. E-mail: john.ramsdell@noaa.gov

This work was funded by the National Oceanic and Atmospheric Administration (NOAA-NOS) and by the North Carolina General Assembly, the Z. Smith Reynolds Foundation, and an anonymous foundation (grants to coauthors J.M. Burkholder and H. Glasgow) . The National Ocean Service (NOS) does not approve, recommend, or endorse any proprietary product or material mentioned in this publication. No reference shall be made to NOS, or to this publication furnished by NOS, in any advertising or sales promotion that would indicate or imply that NOS approves, recommends, or endorses any proprietary product or proprietary material mentioned herein or that has as its purpose any intent to cause directly or indirectly the advertised product to be used or purchased because of NOS publication.

Received 1 September 2000 ; accepted 14 November 2000.