calcimycin and zinc-chloride

Several chemicals have been widely used to evaluate the involvement of free Ca(2+) in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca(2+)-sensitive reagents in diverse cultured cells.. In rat astrocytic C6 glioma cells loaded with the fluorescent Ca(2+) dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca(2+) chelator EGTA irrespective of added CaCl2. The addition of the Ca(2+) ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn(2+) ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters.. Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca(2+) in diverse cells enriched of Zn(2+).

Topics: Aniline Compounds; Animals; Calcimycin; Calcium; Cell Line; Cell Line, Tumor; Cells, Cultured; Chelating Agents; Chlorides; Ethylamines; HEK293 Cells; Humans; Indicators and Reagents; Mice; Polycyclic Compounds; Pyridines; Rats; Tetrazolium Salts; Xanthenes; Zinc; Zinc Compounds

The Maf protein family consists of eight transcription factors containing a basic-leucine zipper (bZIP) domain. We have previously reported that the mRNA to one of these members, mafG/adapt66, is induced by oxidative stress in hamster HA-1 cells. It has subsequently been reported that mafG is induced bystress that activates the expression of genes under the control of the antioxidant/electrophile response element (ARE/EpRE), and that small Maf proteins are present in ARE/EpRE-protein complexes. Here we extend these studies to assess the effects of various types of stress on maf mRNA induction in human cells. The oxidative stressor cadmium, and the heavy metals cadmium, zinc, and arsenite induced mafG RNA levels within two hours, and maximally at five hours for cadmium and zinc. This induction was observed for multiple transcripts including two not normally associated with mafG, suggesting that these stress agents induced the expression of other related maf family RNAs. Modest induction of mafG mRNA was also observed with heat shock but not calcium elevation. These results suggest that mafG is a human stress-response gene induced by multiple stress, and that several maf (proto-)oncogene members play an important role in cellular stress response.

Topics: Animals; Arsenites; Cadmium Chloride; Calcimycin; Chlorides; Cricetinae; DNA-Binding Proteins; Gene Expression Regulation; HeLa Cells; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Kinetics; MafG Transcription Factor; Metals, Heavy; Oxidative Stress; Proto-Oncogene Mas; Repressor Proteins; RNA, Messenger; Sodium Compounds; Transcription, Genetic; Zinc Compounds