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Electronic Letters to:
- Cellular:
Christian J. Stork and Yang V. Li- Intracellular Zinc Elevation Measured with a "Calcium-Specific" Indicator during Ischemia and Reperfusion in Rat Hippocampus: A Question on Calcium Overload
J. Neurosci. 2006; 26: 10430-10437[Abstract] [Full text] [PDF]
eLetters: Submit a response to this article
Electronic letters published:
Zinc has not been ignored
- Ian J Reynolds, Kirk E Dineley and Olga Vergun (13 November 2006)
- Yang V Li (28 January 2007)
Zinc has not been ignored 13 November 2006 
Ian J Reynolds,
Senior Director
Merck Research Laboratories, West Point, PA 19486,
Kirk E Dineley and Olga VergunSend letter to journal:
Re: Zinc has not been ignored
ian_reynolds{at}merck.com Ian J Reynolds, et al.
In this paper, Stork and Li describe intraneuronal zinc changes following oxygen glucose deprivation (OGD) in hippocampal slices. They make several claims, which include the suggestion that the detection of zinc by calcium sensitive dyes has been ignored, and that the role of calcium may be over-stated. These claims are exaggerated, and ignore a substantial body of literature that has both recognized and utilized the zinc sensitivity of dyes designed to detect calcium. A recent clear example is the paper of Devinney and colleagues (2005), who clearly delineated the zinc sensitivity of calcium dyes in the context of excitoxicity. Indeed, this paper has a section in the discussion entitled "How are calcium and zinc distinguished?". This and a number of papers cited therein belie the claim that the topic has largely been ignored by others. A second concern is that the key experiment, where TPEN decreases the fluorescence signal of the calcium sensitive dye, may be conceptually flawed. As noted by the authors, TPEN does bind calcium but with a much lower affinity than zinc. However, as discussed by Dineley and colleagues (2002), when chelators are present in excess over the free ion concentration, binding is driven by the chelator concentration rather than the affinity. At the high concentrations used here, TPEN could be having an important impact on the calcium concentration, and may not be specifically chelating zinc at all. The simple experiment of using a much lower concentration of TPEN was not reported. Although we certainly agree that zinc may make an under- appreciated contribution to excitotoxic neuronal injury, this paper does a disservice to the concept by failing to recognize the substantial body of work that has gone before, and by basing key conclusions on flawed experiments.
Dineley KE, Malayandi LM and Reynolds, IJ. (2002) A reevaluation of intraneuronal zinc measurements: artifacts associated with high intracellular dye concentrations. Mol. Pharmacol. 62:618-627. Devinney, MJ, Reynolds IJ and Dineley, KE (2005) Simultaneous detection of intracellular free calcium and zinc using fura2FF and fluozin3. Cell Calcium 37:225-232.
A selective calcium indicator is needed. Re: Zinc has not been ignored 28 January 2007 
Yang V Li,
Assistant Professor
Ohio University, Athens, OH 45701Send letter to journal:
Re: A selective calcium indicator is needed. Re: Zinc has not been ignored
li{at}oucom.ohiou.edu Yang V Li
Dr. Reynolds commented that we overlooked his works and suggested the zinc had not been ignored. The groundbreaking works of others are recognized in our paper. While using ‘calcium indicators’ to measure calcium, the fact is that few indicate the possibility that zinc might be the cause of their fluorescent signals. A recent paper by Martin and colleagues studied zinc sensitivity of Fura-2, Calcium Green-1, and fluo- 3. Do we not wish to know whether zinc accumulation contributes to the calcium transient measured with these widely used fluorophores? The paper by Reynolds and colleagues made an effort to separate zinc and calcium using two indicators, FluoZin-3 and Fura-2FF. We disagree with their assertion that there are high [Ca2+]i and low/small [Zn2+]i in neurotoxicity. In fact, Fura-2FF may be more sensitive to zinc than to calcium. A comprehensive metal-ion response screening for fluorophores can be found in vendor’s “The Handbook” by Molecular Probes (Figure 19.76. in page 915). Regarding the use of TPEN, we note that 50-100 µM TPEN has been widely used in the cultured cells and tissue preparation. Reynolds himself uses 50 µM TPEN. Evidence that ischemic zinc accumulation as detected by the low affinity Newport Green suggests there are substantial amounts of zinc. Because we don’t know concentration of [Ca2+]i, it is hard to conclude whether or how much TPEN will affect calcium homeostasis. Perhaps it underscores the need for a selective calcium indicator. Only when truly selective calcium probes are developed can investigators unambiguously define the role of calcium signaling in biological processes with confidence. Given the potential advances that are at the heart of this issue, a discussion, in a collegial environment, is beneficial in the advancement of our understanding of zinc biology.
Haugland RP. (2005) The Handbook — A Guide to Fluorescent Probes and Labeling Technologies. Molecular Probes, Invitrogen, Eugene, Oregon.
Martin JL, Stork CJ, Li YV (2006) Determining zinc with commonly used calcium and zinc fluorescent indicators, a question on calcium signals. Cell Calcium 40:393–402
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