Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: a potential target for a bioactive substance produced by Pfiesteria piscicida.
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 GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) 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 GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-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 GH(4)C(1) 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 GH(4)C(1) 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
Kimm-Brinson
K L
KL
Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, National Oceanic and Atmospheric Administration-National Ocean Service, Charleston, South Carolina, USA.
Moeller
P D
PD
Barbier
M
M
Glasgow
H
H
Burkholder
J M
JM
Ramsdell
J S
JS
D000255:Adenosine Triphosphate / Q000819:agonists / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D016762:Genes, fos; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008387:Marine Toxins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology*; D020045:Pfiesteria piscicida / Q000235:genetics / Q000378:metabolism*; D010902:Pituitary Gland / Q000166:cytology; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction
D008387:Marine Toxins; C546468:P2RX7 protein, human; C546470:P2rx7 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; D008156:Luciferases