16672692

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4

The plasmalemmal Na(+)-K(+)-ATPase (NKA) pump is the receptor for the potent marine toxin palytoxin (PTX). PTX binds to the NKA and converts the pump into a monovalent cation channel that exhibits a slight permeability to Ca(2+). However, the ability of PTX to directly increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) via Na(+) pump channels and to initiate Ca(2+) overload-induced oncotic cell death has not been examined. Thus the purpose of this study was to determine the effect of PTX on [Ca(2+)](i) and the downstream events associated with cell death in bovine aortic endothelial cells. PTX (3-100 nM) produced a graded increase in [Ca(2+)](i) that was dependent on extracellular Ca(2+). The increase in [Ca(2+)](i) initiated by 100 nM PTX was blocked by pretreatment with ouabain with an IC(50) < 1 microM. The elevation in [Ca(2+)](i) could be reversed by addition of ouabain at various times after PTX, but this required much higher concentrations of ouabain (0.5 mM). These results suggest that the PTX-induced rise in [Ca(2+)](i) occurs via the Na(+) pump. Subsequent to the rise in [Ca(2+)](i), PTX also caused a concentration-dependent increase in uptake of the vital dye ethidium bromide (EB) but not YO-PRO-1. EB uptake was also blocked by ouabain added either before or after PTX. Time-lapse video microscopy showed that PTX ultimately caused cell lysis as indicated by release of transiently expressed green fluorescent protein (molecular mass 27 kDa) and rapid uptake of propidium iodide. Cell lysis was 1) greatly delayed by removing extracellular Ca(2+) or by adding ouabain after PTX, 2) blocked by the cytoprotective amino acid glycine, and 3) accompanied by dramatic membrane blebbing. These results demonstrate that PTX initiates a cell death cascade characteristic of Ca(2+) overload.

eng

IM

MEDLINE

0363-6143

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NLM

C657-67

pubmed-meshheading:16672692-Acrylamides, pubmed-meshheading:16672692-Animals, pubmed-meshheading:16672692-Aorta, pubmed-meshheading:16672692-Calcium, pubmed-meshheading:16672692-Cattle, pubmed-meshheading:16672692-Cell Death, pubmed-meshheading:16672692-Cells, Cultured, pubmed-meshheading:16672692-Cnidarian Venoms, pubmed-meshheading:16672692-Computer Systems, pubmed-meshheading:16672692-Dose-Response Relationship, Drug, pubmed-meshheading:16672692-Drug Administration Schedule, pubmed-meshheading:16672692-Endothelial Cells, pubmed-meshheading:16672692-Ethidium, pubmed-meshheading:16672692-Fluorescent Dyes, pubmed-meshheading:16672692-Green Fluorescent Proteins, pubmed-meshheading:16672692-Intracellular Membranes, pubmed-meshheading:16672692-Microscopy, Video, pubmed-meshheading:16672692-Osmolar Concentration, pubmed-meshheading:16672692-Ouabain, pubmed-meshheading:16672692-Pertussis Toxin, pubmed-meshheading:16672692-Propidium

Palytoxin-induced cell death cascade in bovine aortic endothelial cells.

Journal Article, Research Support, N.I.H., Extramural