The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/ L free zinc (pZn = 8.3, where pZn is defined as − log 10 [free Zn 2+ ]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

No abstract

No abstract

S U M M A R Y The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2-to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain. (J Histochem Cytochem 56:3-6, 2008) K E Y W O R D S mossy fibers ZNT3 glutamate zinc release hilus X-ray fluorescence knockout MORE THAN A DECADE AGO, Dr. Richard Palmiter discovered and cloned a gene (Slc30a3) the protein from which (ZNT3) he showed to be selectively located on the vesicles of zinc-secreting neurons, such as those comprising the hippocampal mossy-fiber pathway (Palmiter et al. 1996;Wenzel et al. 1997). Mice congenitally lacking the ZNT3 protein (ZNT3 knockout mice) were developed by Cole et al. (1999), and these mice proved to have no histochemically detectable zinc whatsoever in their mossy-fiber pathway, or in other zinc-secreting pathways (Cole et al. 1999); implying that the ZNT3 protein serves as a zinc transporter, responsible for the loading of zinc into those vesicles. The experiments performed by both Palmiter et al. (1996) and Cole et al. (1999) used immunocytochemistry to show that ZNT3 in the hippocampus is localized to the CA4/hilar region of the dentate gyrus, the stratum lucida of CA3, and the pyramidal cells of CA3 and CA1. Cole et al. (1999) also performed an elemental analysis of digested brain regions, demonstrating that total hippocampal zinc was reduced by 20%. The present work was undertaken to confirm that knocking out ZNT3 results in the loss of elemental zinc from the normally zincenriched regions of the hippocampus by using a novel non-destructive technique, microprobe synchrotron X-ray fluorescence (mSXRF).The specific question under examination in the present work was, Does the ZNT3 protein control the amount of zinc stored in vesicles of zinc-secreting neurons, or does ZNT3 merely control the amount of zinc that is detectable by histochemical methods, i.e., control the speciation, or distribution between bound and free (rapidly exchangeable) zinc in the vesicle? The answer we have obtained by quantitative imaging of total elemental zinc, using mSXRF, in the hippocampal regions studied by Cole et al. (1999), of ZNT3 knockout mice and wild-type mice is unambiguous: mice lacking the ZNT3 protein have no detectable enrichment of elemental zinc in their vesicles. Moreover,...

(CJC,SJL) S U M M A R Y We have used a new family of zinc-specific-responsive fluorescent dyes (ZPs) to study the sequestration and secretion of zinc from Paneth cells, which are located in the bases of the crypts of Lieberkü hn within the rat small intestine. Vivid ZP fluorescence zinc staining of Paneth cell secretory granules is seen in both cryostat sections and isolated crypts, providing firm evidence for a pool of labile (rapidly exchangeable) zinc within these cells. We further demonstrate that this ionic zinc pool is secreted under physiological conditions. In vivo stimulation of the small intestine by IP injection of the secretagogue pilocarpine results in discrete zinc staining within the lumens of subsequently isolated crypts, concomitant with a decrease in the zinc staining of Paneth cell granules located within the same crypts. In contrast, the secretion of zinc into the lumens of isolated crypts stimulated in vitro with either carbachol or LPS (lipopolysaccharide) is not observed. However, a distinct change in Paneth cell morphology, suggesting attempted secretion, is seen in response to the direct application of cholinergics but not LPS. These findings suggest that zinc is coreleased with other Paneth cell anti-microbials, and that the intact intestine is necessary for secretion into the crypt lumen. (J Histochem Cytochem 54:311-316, 2006)