Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals.
Using the membrane-permeant chelator of heavy metal ions, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), we demonstrate that in pancreatic acinar cells, hepatocytes, and a variety of mammalian cell lines, endogenous heavy metal ions bind to cytosolic fura-2 causing basal cytosolic free [Ca2+] ([Ca2+]i) to be overestimated. TPEN had most effect in cells lightly loaded with fura-2, suggesting the presence of a limited pool of heavy metal ions (> or = 12 microM in pancreatic acinar cells) that does not rapidly exchange across the plasma membrane. In fura-2-loaded hepatocytes, vasopressin failed to evoke a detectable change in fluorescence, but after preincubation of cells with TPEN, it caused fluorescence changes characteristic of an increase in [Ca2+]i. We conclude that in many mammalian cells, a slowly exchanging mixture of cytosolic heavy metal ions binds to fura-2 both to quench its fluorescence and to mimic the effects of Ca2+ binding, thereby causing basal [Ca2+]i to be overestimated. By chelating endogenous heavy metal ions, TPEN allows basal [Ca2+]i to be accurately measured and, by preventing competition between heavy metal ions and Ca2+ for binding to fura-2, unmasks the full effect of agonists in increasing [Ca2+]i.
Snitsarev
V A
VA
Department of Pharmacology, Cambridge, UK.
McNulty
T J
TJ
Taylor
C W
CW
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology*; D016257:Fura-2; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008322:Mammals; D051379:Mice; D008815:Mice, Inbred Strains; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*; D004369:Pentetic Acid / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods; D014667:Vasopressins / Q000494:pharmacology*
D002413:Cations, Divalent; D002614:Chelating Agents; D005029:Ethylenediamines; D014667:Vasopressins; D004369:Pentetic Acid; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; D002118:Calcium; D016257:Fura-2