Intracellular calcium dynamics and cellular energetics in ischemic NG108-15 cells studied by concurrent 31P/19F and 23Na double-quantum filtered NMR spectroscopy.
The role of voltage-sensitive Ca2+ channels in mediating Ca2+ influx during ischemia was investigated in NG108-15 cells, a neuronal cell line that does not express glutamate-sensitive receptor-mediated Ca2+ channels. Concurrent 31P/19F and 23Na double-quantum filtered (DQF) NMR spectra were used to monitor cellular energy status, intracellular [Ca2+] ([Ca2+]i), and intracellular Na+ content in cells loaded with the calcium indicator 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) during ischemia and reperfusion. Cells loaded with 5FBAPTA were indistinguishable from unloaded cells except for small immediate decreases in levels of phosphocreatine (PCr) and ATP. Ischemia induced a steady decrease in intracellular pH and PCr and ATP levels, and a steady increase in intracellular Na+ content; however, a substantial increase in [Ca2+]i (about threefold) was seen only following marked impairment of cellular energy status, when PCr was undetectable and ATP content was reduced to 55% of control levels. A depolarization-induced increase in [Ca2+]i could be completely blocked by 1 microM nifedipine, whereas up to 20 microM nifedipine had no effect on the increase in [Ca2+]i seen during ischemia. These data demonstrate that voltage-gated Ca2+ channels do not mediate significant Ca2+ flux during ischemia in this cell line and suggest an important role for Ca2+i stores, the Na+/Ca2+ antiporter, or other processes linked to cellular energy status in the increase in cytosolic Ca2+ level during ischemia.
Montreal Neurological Institute, Quebec, Canada.
J S
JS
Tauskela
E A
EA
Shoubridge
D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002451:Cell Compartmentation; D015687:Cell Hypoxia; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects; D006801:Humans; D006822:Hybrid Cells / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000378:metabolism; D007511:Ischemia / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009420:Nervous System / Q000098:blood supply*; D009447:Neuroblastoma / Q000473:pathology*; D009457:Neuroglia / Q000473:pathology*; D009543:Nifedipine / Q000494:pharmacology; D010477:Perfusion; D011189:Potassium Chloride / Q000494:pharmacology; D015424:Reperfusion; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004533:Egtazic Acid; D011189:Potassium Chloride; C039877:5,5'-difluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D009543:Nifedipine; D002118:Calcium