Intracellular Free Zinc Research Articles

Diverse sensitivity of cells representing various stages of colon carcinogenesis to increased extracellular zinc: Implications for zinc chemoprevention

The relationship between zinc intake and risk of colon cancer is widely recognized. Despite reported mechanisms of zinc-mediated effects in colonic cells no information is available on whether zinc is capable of inducing cell death of malignant colonocytes. The present study shows that increased external zinc concentrations inhibit cell growth of three different colon cancer cell lines representing different stages of colon cancer: HCT-116, HT-29 and SW620 cells and induce their death. Of the tested cell lines, SW620 cells proved to be the most sensitive to externally added zinc and this sensitivity was at least partly due to increased levels of intracellular free zinc and the inability to overexpress metallothionein. Further studies into the mechanisms of zinc-induced cell injury and cell death revealed oxidative stress as the most important underlying mechanism activating stress kinase-dependent signaling, perturbation of mitochondria and plasma membrane damage. In addition, observed cell death in individual cell populations was cell line-dependent and variable including cells displaying features of apoptosis, necrosis, autophagy and other mixed-types. In conclusion, presented results for the first time show variability of responses to zinc in colon cancer at different stages as modeled in vitro and suggest that zinc-induced cell death despite common underlying mechanism(s) might have a variable nature.

Zinc and Redox Signaling: Perturbations Associated with Cardiovascular Disease and Diabetes Mellitus

Cellular signal transduction pathways are influenced by the zinc and redox status of the cell. Numerous chronic diseases, including cardiovascular disease (CVD) and diabetes mellitus (DM), have been associated with impaired zinc utilization and increased oxidative stress. In humans, mutations in the MT-1A and ZnT8 genes, both of which are involved in the maintenance of zinc homeostasis, have been linked with DM development. Changes in levels of intracellular free zinc may exacerbate oxidative stress in CVD and DM by impacting glutathione homeostasis, nitric oxide signaling, and nuclear factor-kappa B-dependent cellular processes. Zinc ions have been shown to influence insulin and leptin signaling via the phosphoinositide 3′-kinase/Akt pathway, potentially linking an imbalance of zinc at the cellular level to insulin resistance and dyslipidemia. The oxidative modification of cysteine residues in zinc coordination sites in proteins has been implicated in cellular signaling and regulatory pathways. Despite the many interactions between zinc and cellular stress responses, studies investigating the potential therapeutic benefit of zinc supplementation in the prevention and treatment of oxidative stress-related chronic disease in humans are few and inconsistent. Further well-designed randomized controlled trials are needed to determine the effects of zinc supplementation in populations at various stages of CVD and DM progression.

Intracellular free zinc during cardiac excitation–contraction cycle: calcium and redox dependencies

Zinc exists in biological systems as bound and histochemically reactive free Zn(2+). It is an essential structural constituent of many proteins, including enzymes from cellular signalling pathways, in which it functions as a signalling molecule. In cardiomyocytes at rest, Zn(2+) concentration is in the nanomolar range. Very little is known about precise mechanisms controlling the intracellular distribution of Zn(2+) and its variations during cardiac function. Live-cell detection of intracellular Zn(2+) has become feasible through the recent development of Zn(2+)-sensitive and -selective fluorophores able to distinguish Zn(2+) from Ca(2+). Here, in freshly isolated rat cardiomyocytes, we investigated the rapid changes in Zn(2+) homeostasis using the Zn(2+)-specific fluorescent dye, FluoZin-3, in comparison to Ca(2+)-dependent fluo-3 fluorescence. Zn(2+) sparks and Zn(2+) transients, in quiescent and electrically stimulated cardiomyocytes, respectively, were visualized in a similar manner to known rapid Ca(2+) changes. Both Zn(2+) sparks and Zn(2+) transients required Ca(2+) entry. Inhibiting the sarcoplasmic reticulum Ca(2+) release or increasing the Ca(2+) load in a low-Na(+) solution suppressed or increased Zn(2+) movements, respectively. Mitochondrial inhibitors slightly reduced both Zn(2+) sparks and Zn(2+) transients. Oxidation by H₂O₂ facilitated and acidic pH inhibited the Ca(2+)-dependent Zn(2+) release. It is proposed that Zn(2+) release during the cardiac cycle results mostly from intracellular free Ca(2+) increase, triggering production of reactive oxygen species that induce changes in metal-binding properties of metallothioneins and other redox-active proteins, aside from ionic exchange on these proteins.

Endothelial metallothionein expression and intracellular free zinc levels are regulated by shear stress

We examined the effects of fluid shear stress on metallothionein (MT) gene and protein expression and intracellular free zinc in mouse aorta and in human umbilical vein endothelial cells (HUVECs). Immunostaining of the endothelial surface of mouse aorta revealed increased expression of MT protein in the lesser curvature of the aorta relative to the descending thoracic aorta. HUVECs were exposed to high steady shear stress (15 dyn/cm(2)), low steady shear stress (1 dyn/cm(2)), or reversing shear stress (mean of 1 dyn/cm(2), 1 Hz) for 24 h. Gene expression of three MT-1 isoforms, MT-2A, and zinc transporter-1 was upregulated by low steady shear stress and reversing shear stress. HUVECs exposed to 15 dyn/cm(2) had increased levels of free zinc compared with cells under other shear stress regimes and static conditions. The increase in free zinc was partially blocked with an inhibitor of nitric oxide synthesis, suggesting a role for shear stress-induced endothelial nitric oxide synthase activity. Cells subjected to reversing shear stress in zinc-supplemented media (50 μM ZnSO(4)) had increased intracellular free zinc, reduced surface intercellular adhesion molecule-1 expression, and reduced monocyte adhesion compared with cells exposed to reversing shear stress in normal media. The sensitivity of intracellular free zinc to differences in shear stress suggests that intracellular zinc levels are important in the regulation of the endothelium and in the progression of vascular disease.

Cytotoxicity and its mechanism of zinc oxide nanoparticles on human leukemic monocyte lymphoma cell line U937

To investigate the cytotoxicity and its mechanism of ZnO nanoparticles on human leukemic monocyte lymphoma cell line U937. Four different size ZnO (10, 30, 60, 500 nm) were carefully characterized. The survival rate and viability were measured by trypan blue assay and MTT assay for each size ZnO particles at different concentrations (12, 120, 240, 600, 1200 µmol/L). The zinc probe, Fluozin-3, was used to detect the intracellular free zinc. Transmission electron microscopy was adopted to observe the cellular ultrastructure and the uptake of ZnO. All four kinds of ZnO were rod shape, with a purity of > 99.9 wt%, and they were classified as zincite phase crystal and their surface areas were in accordance with the sizes. The viability (ZnO-n10: (97 ± 19)%, (91 ± 4)%, (24 ± 4)%, (15 ± 2)%; ZnO-n30: (111 ± 4)%, (81 ± 3)%, (24 ± 2)%, (27 ± 8)%; ZnO-n60: (105 ± 11)%, (73 ± 20)%, (43 ± 11)%, (28 ± 14)%; ZnO-µm: (88 ± 16)%, (62 ± 7)%, (22 ± 4)%, (13 ± 5)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r values were 0.965, 0.979, 0.998, 0.992, and the t values were 19.8, 25.3, 76.3, 40.9, respectively, P < 0.05). The liability (ZnO-n10: (98 ± 1)%, (67 ± 2)%, (59 ± 7)%, (13 ± 13)%, (5 ± 4)%; ZnO-n30: (98 ± 1)%, (97 ± 2)%, (50 ± 3)%, (20 ± 14)%, (7 ± 2)%; ZnO-n60: (97 ± 2)%, (88 ± 5)%, (48 ± 10)%, (12 ± 5)%, (4 ± 1)%; ZnO-µm: (96 ± 1)%, (76 ± 3)%, (58 ± 3)%, (19 ± 5)%, (20 ± 10)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r valued at 0.982, 0.956, 0.972, 0.980, and the t valued at 19.3, 12.1, 15.6, 18.5, respectively, P < 0.05). The increase of the zinc concentration showed by the zinc fluorescence probe was 121 ± 11, which was similar to the fluorescence of cells treated with ZnAc(2) (132 ± 14, F = 0.6, P > 0.05) at the Zn-equivalent concentration. There was no statistic difference for the percents of high zinc content cells in total cells exposed to ZnO-n30 (87.6 ± 2.6)% and these exposed to ZnAc(2) (86.9 ± 3.2)% (F = 1.5, P > 0.05). ZnO nanoparticles are highly cytotoxic to U937 cells and the solubilization of ZnO is the main toxicological mechanism.

Monochloramine-induced toxicity and dysregulation of intracellular Zn2+in parietal cells of rabbit gastric glands

Monochloramine (NH(2)Cl) is a potent, thiol-directed oxidant capable of oxidizing thiol (S-H) residues in a wide variety of proteins. Generated in the stomach by the interaction of bacterial and host products, monochloramine has been shown to dysregulate Ca(2+) homeostasis and disrupt mucosal integrity. In this report, we show that monochloramine also leads to disturbances in intracellular free zinc concentration ([Zn(2+)](i)) in the gastric gland of the rabbit and that the increased Zn(2+) within the cell causes an independent decrease in cell viability. Changes in [Zn(2+)](i) were measured by using the fluorescent reporter FluoZin-3, whereas cell viability was assessed by measuring the conversion of calcein-AM to fluorescent calcein, an assay that is not affected by intracellular oxidation state. Cell death was confirmed using propidium iodide and YO-PRO-1 dye uptake measurements. Our experiments demonstrate that [Zn(2+)](i) is increased in gastric glands exposed to NH(2)Cl and that elevated [Zn(2+)](i) decreases cell viability. Chelation of Zn(2+) with tetrakis-(2-pyridylmethyl) ethylenediamine decreases the toxicity of NH(2)Cl, but only when administered concurrently. These findings suggest that the toxic effect of thiol oxidants present during chronic gastritis is partially due to dysregulation of [Zn(2+)](i) early in the process and that zinc chelation can protect, but not rescue, gastric glands exposed to toxic doses of NH(2)Cl.

Zinc signals promote IL‐2‐dependent proliferation of T cells

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells.

Measuring Readily Exchangeable Zinc in Escherichia coli by Carbonic Anhydrase‐based Expressible Ratiometric Zinc Sensor

Zinc is essential in numerous cellular processes and its homeostasis is highly regulated in living organisms. Monitoring readily exchangeable zinc concentration in the cells under various conditions will provide insight into the regulation of zinc homeostasis. Here we engineered a series of genetically encoded fluorescence resonance energy transfer (FRET)‐based excitation ratiometric zinc sensors based on carbonic anhydrase (CA) that exhibit large signal increases in response to zinc. A fluorescent inhibitor of carbonic anhydrase, Dapoxyl sulfonamide (DPS), binds selectively to the active site zinc of CA, undergoing &gt;100 fold increase in quantum yield. A red fluorescent protein (RFP) fused to the C‐terminus of CA functions as the FRET acceptor to zinc‐dependent DPS emission. The FRET signal normalized by the fluorescence from RFP is then calibrated in a series of chelated zinc buffers. These CA_RFP sensors were expressed in the E. coli strain BL21(DE3) in minimal medium and the readily exchangeable zinc concentration was measured at 30 ± 10 pM. The zinc concentration is not dependent on the sensor concentration or the growth stage but varies significantly (from 5 pM to &gt; 300 pM) with changes in the zinc content in the medium. Furthermore, the cells’ ability to maintain low intracellular free zinc concentration is impaired during sugar starvation. This work was supported by National Institutes of Health (NIH) grant NIH/NIBIB RO1 EB003924.

The role of zinc deficiency in alcohol-induced intestinal barrier dysfunction

Disruption of the intestinal barrier is a causal factor in the development of alcoholic endotoxemia and hepatitis. This study was undertaken to determine whether zinc deficiency is related to the deleterious effects of alcohol on the intestinal barrier. Mice were pair fed an alcohol or isocaloric liquid diet for 4 wk, and hepatitis was detected in association with elevated blood endotoxin level. Alcohol exposure significantly increased the permeability of the ileum but did not affect the barrier function of the duodenum or jejunum. Reduction of tight-junction proteins at the ileal epithelium was detected in alcohol-fed mice although alcohol exposure did not cause apparent histopathological changes. Alcohol exposure significantly reduced the ileal zinc concentration in association with accumulation of reactive oxygen species. Caco-2 cell culture demonstrated that alcohol exposure increases the intracellular free zinc because of oxidative stress. Zinc deprivation caused epithelial barrier disruption in association with disassembling of tight junction proteins in the Caco-2 monolayer cells. Furthermore, minor zinc deprivation exaggerated the deleterious effect of alcohol on the epithelial barrier. In conclusion, epithelial barrier dysfunction in the distal small intestine plays an important role in alcohol-induced gut leakiness, and zinc deficiency attributable to oxidative stress may interfere with the intestinal barrier function by a direct action on tight junction proteins or by sensitizing to the effects of alcohol.

Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes

Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.

Nitric oxide synthase activation and oxidative stress, but not intracellular zinc dyshomeostasis, regulate ultraviolet B light-induced apoptosis

Aims To investigate the role of nitric oxide synthase (NOS) and intracellular free zinc ion (Zn 2+) in regulation of ultraviolet B light (UVB)-induced cell damage and apoptosis. Main methods Real-time confocal microscopy measurement was used to determine the changes of intracellular free zinc concentration under different conditions. Cell apoptotic death was determined using fluorescein isothiocyanate (FITC) conjugated-annexin V (ANX5)/PI labeling followed by flow cytometry. Western analysis was used to determine cell apoptosis and eNOS uncoupling. Key findings UVB induced an elevation of Zn 2+ within 2 min of exposure. The UVB-induced intracellular Zn 2+ elevation was dependent on the increase of constitutive nitric oxide synthase (cNOS) activity and production of superoxide. Removal of Zn 2+ with a lower concentration (< 25 μM) of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn 2+-specific chelator, did not induce cell death or prevent cells from UVB-induced apoptosis. However, a higher [TPEN] (> 50 μM) was cytotoxic to cells, but prevented cells from further UVB-induced apoptosis. The higher [TPEN] also induced cNOS uncoupling. Furthermore, treating the cells with a membrane permeable superoxide dismutase (PEG-SOD) inhibited Zn 2+ release and reduced apoptotic cell death after UVB treatment. The results demonstrated a complex and dynamic regulation of UVB-induced cell damage. Significance Our findings not only advance our understanding of the correlations between cNOS activation and Zn elevation, but also elucidated the role of cNOS in regulation of oxidative stress and apoptosis upon UVB-irradiation.

The critical role of intracellular zinc in adenosine A2 receptor activation induced cardioprotection against reperfusion injury

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A2 receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A1/A2 receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30min ischemia followed by 2h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl2 given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (ΔΨm) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl2 inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA.

Heat shock but not cold shock leads to disturbed intracellular zinc homeostasis

The heat shock response is a highly conserved process essential for surviving environmental stress, including extremes of temperature. To investigate whether heat shock has an impact on intracellular Zn(2+) homeostasis, cells were subjected to heat shock, and subsequently the intracellular free zinc concentration was investigated. Sublethal heat shock induced a temperature-dependent and transient intracellular Zn(2+) release that was repeatable after 24 h. The free zinc was localized in round-shaped nuclear bodies identified as nucleoli. Metallothionein, the main cellular zinc storing protein, was found to be not functionally essential for this heat-shock-induced effect. No significant oxidative stress within the cells was detected after heat shock. Cold shock and subsequent rewarming did not result in disturbed intracellular zinc homeostasis. These results show that heat shock and cold shock differ with respect to intracellular Zn(2+) release. A role for zinc as signaling ion during fever is conceivable.

Identification of LIV1, a Putative Zinc Transporter Gene Responsible for HDACi-Induced Apoptosis, Using a Functional Gene Screen Approach

Histone deacetylase inhibitors (HDACi) show promise as a novel class of antitumoral agents and have shown the ability to induce apoptosis of tumor cells. To gain a better understanding of the action of HDACi, we conducted a functional gene screen approach named suppression of mortality by antisense rescue technique to identify the key genes responsible for the tumor-selective killing trichostatin A. Over 20 genes associated with HDACi-induced mortality were identified. One of the confirmed positive hits is LIV1, a putative zinc transporter. LIV1 is significantly induced by treatment with HDACi in a number of tumor cells, but not in normal cells. Knockdown of LIV1 suppressed apoptosis induced by HDACi in tumor cells. Although HDACi induced a slight increase in the free intracellular zinc concentration, knockdown of LIV1 significantly enhanced the intracellular zinc level, which was associated with resistance to apoptosis. On the other hand, pretreatment of the cells with a specific zinc chelator TPEN reversed the apoptosis resistance conferred by knockdown of LIV1. However, the biological effects of TPEN were abolished by addition of physiologic concentrations of zinc. Taken together, the present study identifies LIV1 as a critical mediator responsible for HDACi-induced apoptosis. The effect of LIV1 is, at least in part, mediated by affecting intracellular zinc homeostasis, which may be related to alteration of the catalytic activity of the Caspase 3 and expression of some BCL-2 family genes. As such, these findings highlight a novel mechanism underlying the action of HDACi that could be potentially useful in the clinical setting.

Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator

Zinc is the second-most abundant transition metal within cells and an essential micronutrient. Although adequate zinc is essential for cellular function, intracellular free zinc (Zn(2+)) is tightly controlled, as sustained increases in free Zn(2+) levels can directly contribute to apoptotic endothelial cell death. Moreover, exposure of endothelial cells to acute nitrosative and/or oxidative stress induces a rapid rise of Zn(2+) with mitochondrial dysfunction and the initiation of apoptosis. This apoptotic induction can be mimicked through addition of exogenous ZnCl(2) and mitigated by zinc-chelation strategies, indicating Zn(2+)-dependent mechanisms in this process. However, the molecular mechanisms of Zn(2+)-mediated mitochondrial dysfunction are unknown. Here we report that free Zn(2+) disrupts cellular redox status through inhibition of glutathione reductase, and induces apoptosis by redox-mediated inhibition of the mitochondrial adenine nucleotide transporter (ANT). Inhibition of ANT causes increased mitochondrial oxidation, loss of ADP uptake, mitochondrial translocation of bax, and apoptosis. Interestingly, pre-incubation with glutathione ethyl ester protects endothelial cells from these observed effects. We conclude that key mechanisms of Zn(2+)-mediated apoptotic induction include disruption of cellular glutathione homeostasis leading to ANT inhibition and decreases in mitochondrial ATP synthesis. These pathways could represent novel therapeutic targets during acute oxidative or nitrosative stress in cells and tissues.

Effect of resveratrol and zinc on intracellular zinc status in normal human prostate epithelial cells

To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated with resveratrol (0, 0.5, 1, 2.5, 5, and 10 microM) and zinc [0, 4, 16, and 32 microM, representing zinc-deficient (ZD), zinc-normal (ZN), zinc-adequate (ZA), and zinc-supplemented (ZS) conditions, respectively]. A progressive reduction in cell growth was observed in cells treated with increasing amounts of resveratrol (2.5-10 microM). Resveratrol at 5 and 10 microM resulted in a dramatic increase in cellular total zinc concentration, especially in ZS cells. Flow cytometry indicated that 10 microM resveratrol induced arrest of the cell cycle at the G(2)/M phase in association with the observed cell growth inhibition. Data from an in vitro experiment using zinquin as an indicator of intracellular free Zn(II) status demonstrated complex interactions between resveratrol and Zn(II). Fluorescence spectrofluorometry and fluorescence microscopic analyses revealed that intracellular free labile zinc was progressively elevated from nearly twofold in ZS cells with no resveratrol to multifold in ZA and ZS cells with 10 microM resveratrol compared with the corresponding ZN cells. Furthermore, increases in cellular zinc status were associated with elevated levels of reactive oxygen species and senescence, as evidenced by morphological and histochemical changes in cells treated with 2.5 or 10 microM resveratrol, especially in ZA and ZS cells. Taken together, the interaction between resveratrol and zinc in NHPrE cells increases total cellular zinc and intracellular free labile zinc status and, subsequently, reactive oxygen species production and senescence.

Intracellular zinc inhibits KCC2 transporter activity

We found that K(+)/Cl(-) co-transporter 2 (KCC2) activity, monitored with wide-field fluorescence, was inhibited by intracellular Zn(2+), a major component of neuronal injury. Zn(2+)-mediated KCC2 inhibition produced a depolarizing shift of GABA(A) reversal potentials in rat cortical neurons. Moreover, oxygen-glucose deprivation attenuated KCC2 activity in a Zn(2+)-dependent manner. The link between Zn(2+) and KCC2 activity provides a previously unknown target for neuroprotection and may be important in activity-dependent regulation of inhibitory synaptic transmission.

Hypoxia and zinc induced changes in the contractile machinery of isolated rat pulmonary microvascular endothelial cells (RPMVECs)

Our recent data suggest that hypoxic induced increases in NO biosynthesis result in increases in intracellular free zinc that in turn contribute to vasoconstriction of small, non‐muscularized intra‐acinar arteries (&lt;50 um). In order to understand the cellular changes behind this contractile behavior, we examined actin‐myosin dynamics and focal adhesion changes in isolated rat pulmonary microvascular endothelial cells (RPMVEC) in response to 30 minutes of hypoxia or exogenously applied zinc. Immunocytochemistry and confocal analysis showed that contraction in response to either 2% oxygen or zinc, were accompanied by increases in actin‐myosin interactions; stress fiber formation; and recruitment of focal adhesion proteins to cell‐matrix contacts. Live cell imaging of GFP tagged proteins provided real‐time information about the strengthening and alignment of actin stress fibers while total internal reflection (TIRF) imaging showed recruitment of paxillin, talin and zyxin to focal adhesions. These data suggest that the reorganization of cytoskeletal and focal adhesion proteins in response to hypoxia or zinc may be responsible for the force induction and cell stability necessary to elicit an active contractile response in pulmonary endothelium.Funded by NIH HL 081421 (CMS)

THE EFFECT OF RESVERATROL AND ZINC ON INTRACELLULAR ZINC STATUS IN NHPrE CELLS

To evaluate the influence of resveratrol on cellular zinc status, normal human prostate epithelial (NHPrE) cells were treated in 6 levels of resveratrol (0, 0.5, 1, 2.5, 5 and 10 µM) and 4 levels of zinc [0, 4, 16 and 32 µM or zinc deficient (ZD), zinc normal (ZN), zinc adequate (ZA) and zinc supplemented (ZS), respectively]. Among each zinc treatment, a progressive reduction in cell growth or increase in cellular total zinc was observed with increases of resveratrol from 2.5, 5 and 10 µM or from 5 and 10 µM, respectively. In ZS cells, a much higher increase in cellular total zinc was observed as early as 1 µM resveratrol. A flow cytometry study revealed that the resveratrol (10 µM) induced G2/M arrest was responsible for the depressed cell growth. Data from an in vitro experiment using zinquin, as an indicator of intracellular free Zn(II) status, demonstrated complex formation between resveratrol and zinc ion. Zinquin ethyl ester fluorescence spectrofluorimetry and microscope imaging revealed that intracellular labile free zinc decreased in ZD and ZN NHPrE cells but increased in high zinc (ZA and ZS) cells. Furthermore, increases in cellular zinc status induced enhanced levels of reactive oxygen species (ROS) as well as senescence detected by morphological and histochemical changes in cells treated with 2.5 or 10 µM resveratrol, especially in ZA and ZS cells. Thus, increases in free labile zinc may induce ROS and senescence.Grant Funding SourceUNIV. MARYLAND

Carbon Monoxide-releasing Antibacterial Molecules Target Respiration and Global Transcriptional Regulators

Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO)(3)Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation.