Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals

Vladislav Snitsarev, Tracy J. McNulty, Colin W. Taylor

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Abstract

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 (≤12 μM 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).

Original languageEnglish
Pages (from-to)1048-1056
Number of pages9
JournalBiophysical Journal
Volume71
Issue number2
DOIs
StatePublished - 1 Jan 1996

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Heavy Ions
Fura-2
Heavy Metals
Hormones
Acinar Cells
Fluorescence
Hepatocytes
Diamines
Chelating Agents
Vasopressins
Cell Membrane
Cell Line
Membranes
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine

Cite this

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title = "Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals",
abstract = "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 (≤12 μM 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).",
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Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals. / Snitsarev, Vladislav; McNulty, Tracy J.; Taylor, Colin W.

In: Biophysical Journal, Vol. 71, No. 2, 01.01.1996, p. 1048-1056.

Research output: Contribution to journalArticleResearchpeer-review

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