Mitochondrial K+ as modulator of Ca(2+)-dependent cytotoxicity in hepatocytes. Novel application of the K(+)-sensitive dye PBFI (K(+)-binding benzofuran isophthalate) to assess free mitochondrial K+ concentrations.
In isolated rat hepatocytes a sustained high increase in intracellular free Ca2+ ([Ca2+]i), induced by extracellular ATP, is associated with mitochondrial dysfunction and cell death. The Ca(2+)-induced effects are Pi-dependent and less severe when the intracellular K+ content is low. In this study, the involvement of mitochondrial K+ processing in Ca(2+)-induced loss of mitochondrial membrane potential (MMP) and viability was investigated. The recently introduced K(+)-sensitive dye PBFI (K(+)-binding benzofuran isophthalate) has been used in combination with video-microscopy to assess intramitochondrial free K+ concentration ([K+]mito) in rat liver mitochondria in situ. After rapid permeabilization of the plasma membrane to remove cytosolic PBFI, the remaining PBFI was localized in mitochondria, and a 'resting' [K+]mito of approx. 15 mM could be measured. Increased [K+]mito levels were measured after induction of a prolonged increase in [Ca2+]i by ATP. Much lower [K+]mito, more comparable with control levels, were observed when intracellular K+ was depleted by omission of extracellular K+. In permeabilized cells the Ca(2+)-induced, Pi-dependent, dissipation of the MMP was markedly delayed in the absence of K+. These observations suggest involvement of [K+]mito as modulating agent in Ca(2+)-induced cytotoxicity in hepatocytes.
Zoeteweij
J P
JP
Division of Toxicology, Leiden University, The Netherlands.
van de Water
B
B
de Bont
H J
HJ
Nagelkerke
J F
JF
D000818:Animals; D001572:Benzofurans / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D004988:Ethers, Cyclic / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats
D001572:Benzofurans; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D010710:Phosphates; C073886:potassium-binding benzofuran isophthalate; D011188:Potassium; D002118:Calcium