Roles for endothelial zinc homeostasis in vascular physiology and coronary artery disease.

The discovery of the roles of nitric oxide (NO) in cardiovascular signaling has led to a revolution in the understanding of cardiovascular disease. A new perspective to this story involving zinc (Zn) is emerging. Zn and its associated Zn transporter proteins are important for the integrity and functions of both the large conduit vessels and the microvascular resistance vessels. The Zn and NO pathways are tightly coordinated. Zn ions are required for the dimerization of endothelial nitric oxide synthase and subsequent generation of NO while generation of NO leads to a rapid mobilization of endothelial Zn stores. Labile Zn may mediate important downstream actions of NO including vascular cytoprotection and vasodilation. Several vascular disease risk factors (including aging, smoking and diabetes) interfere with Zn homeostatic mechanisms and both hypozincaemia and Zn transporter protein abnormalities are linked to atherosclerosis and microvascular disease. Some vegetarian diets and long-term use of certain anti-hypertensives may also impact on Zn status. The available evidence supports the existence of a Zn regulatory pathway in the vascular wall that is coupled to the generation and actions of NO and which is compromised in Zn deficiency with consequent implications for the pathogenesis and therapy of vascular disease.

Apoptosis-regulatory factors as potential drug targets in the epithelium of normal and inflamed airways.

Airway epithelium (AE) lines the conducting airways of the respiratory system and functions to maintain airway integrity by providing both a physical barrier to inhaled noxious agents and a mechanism for their clearance via the mucociliary escalator. Normal AE cells are relatively refractory to a number of apoptotic stimuli and survival mechanisms are in place to maintain the integrity of the epithelial barrier that is exposed to agents such as reactive oxygen species (ROS) and death receptor ligands secreted by immune cells during inflammation. When damage to AE does occur, there is increased AE apoptosis, such as in the airway damage that occurs in the chronically inflamed airways in diseases like asthma where rates of AE apoptosis can be increased many-fold. The usual treatment for persistent asthma in humans involves a combination of bronchodilator and inhaled corticosteroid; there is however a need to develop strategies to better control other aspects of the disease, including minimizing the ongoing damage to AE and consequent airway remodeling. Targeting of the major apoptosis-regulatory factors in AE may be one such strategy. Here we review what is known about apoptosis and its regulatory factors in normal AE and abnormalities in these factors in the inflamed airways of mice and humans.

Zinc and its specific transporters as potential targets in airway disease.

The dietary group IIb metal zinc (Zn) plays essential housekeeping roles in cellular metabolism and gene expression. It regulates a number of cellular processes including mitosis, apoptosis, secretion and signal transduction as well as critical events in physiological processes as diverse as insulin release, T cell cytokine production, wound healing, vision and neurotransmission. Critical to these processes are the mechanisms that regulate Zn homeostasis in cells and tissues. The proteins that control Zn uptake and compartmentalization are rapidly being identified and characterized. Recently, the first images of sub-cellular pools of Zn in airway epithelium have been obtained. This review discusses what we currently know about Zn in the airways, both in the normal and inflamed states, and then considers how we might target Zn metabolism by developing strategies to monitor and manipulate airway Zn levels in airway disease.

Zinc in health and chronic disease.

Zinc is a trace element essential for the optimal function of a variety of biochemical and physiological processes. Its role in healthy aging is particularly important as it prevents neo plastic cell growth, is involved in mitotic cell division, DNA and RNA repair. Although zinc is widely available in food, the daily intake in many persons may be suboptimal. Other causes of low zinc concentrations may be due to small bowel conditions that cause mucosal damage and thus decrease absorption. Chronic diseases associated with alterations in zinc status are bronchial asthma, rheumatoid arthritis and Alzheimer disease. At present it is uncertain if therapy with zinc would assist in the management of these chronic diseases. In view of the important cellular functions of zinc in the human body, a diet with an adequate zinc content is beneficial in promoting healthy aging and maintaining good health.

New insights into the role of zinc in the respiratory epithelium.

Over the past 30 years, many researchers have demonstrated the critical role of zinc (Zn), a group IIb metal, in diverse physiological processes, such as growth and development, maintenance and priming of the immune system, and tissue repair. This review will discuss aspects of Zn physiology and its possible beneficial role in the respiratory epithelium. Here we have detailed the mechanisms by which Zn diversely acts as: (i) an anti-oxidant; (ii) an organelle stabilizer; (iii) an anti-apopototic agent; (iv) an important cofactor for DNA synthesis; (v) a vital component for wound healing; and (vi) an anti-inflammatory agent. This paper will also review studies from the authors' laboratory concerning the first attempts to map Zn in the respiratory epithelium and to elucidate its role in regulating caspase-3 activated apoptosis. We propose that Zn, being a major dietary anti-oxidant has a protective role for the airway epithelium against oxyradicals and other noxious agents. Zn may therefore have important implications for asthma and other inflammatory diseases where the physical barrier is vulnerable and compromised.

The role of zinc in caspase activation and apoptotic cell death.

In addition to its diverse role in many physiological systems, zinc (Zn) has now been shown to be an important regulator of apoptosis. The purpose of this review is to integrate previously published knowledge on Zn and apoptosis with current attempts to elucidate the mechanisms of action of this biometal. This paper begins with an introduction to apoptosis and then briefly reviews the evidence relating Zn to apoptosis. The major focus of this review is the mechanistic actions of Zn and its candidate intracellular targets. In particular, we examine the cytoprotective functions of Zn which suppress major pathways leading to apoptosis, as well as the more direct influence of Zn on the apoptotic regulators, especially the caspase family of enzymes. These two mechanisms are closely related since a decline in intracellular Zn below a critical threshold level may not only trigger pathways leading to caspase activation but may also facilitate the process by which the caspases are activated. Studies by our laboratory in airway epithelial cells show that Zn is co-localized with the precursor form of caspase-3, mitochondria and microtubules, suggesting this Zn is critically placed to control apoptosis. Further understanding the different pools of Zn and how they interact with apoptotic pathways should have importance in human disease.

Visualization of labile zinc and its role in apoptosis of primary airway epithelial cells and cell lines.

The respiratory epithelium is vulnerable to noxious substances, resulting in the shedding of cells and decreased protection. Zinc (Zn), an antioxidant and cytoprotectant, can suppress apoptosis in a variety of cells. Here we used the novel Zn-specific fluorophore Zinquin to visualize and quantify labile intracellular Zn in respiratory epithelial cells. Zinquin fluorescence in isolated ciliated tracheobronchial epithelial cells and intact epithelium from sheep and pigs revealed an intense fluorescence in the apical and mitochondria-rich cytoplasm below the cilia. Zinquin fluorescence was quenched by the Zn chelator N,N,N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and increased by the Zn ionophore pyrithione. We also assessed whether changes in intracellular labile Zn would influence susceptibility of these cells to apoptosis by hydrogen peroxide. Our results confirm that Zn deficiency enhanced hydrogen peroxide-induced caspase activation from 1.24 +/- 0.12 to 2.58 +/- 0.53 units. microg protein(-1). h(-1) (P

Intracellular zinc depletion induces caspase activation and p21 Waf1/Cip1 cleavage in human epithelial cell lines.

To better understand the mechanisms by which zinc deficiency induces epithelial cell death, studies were done of the effects of intracellular zinc depletion induced by the zinc chelator TPEN on apoptosis-related events in human malignant epithelial cell lines LIM1215 (colonic), NCI-H292 (bronchial), and A549 (alveolar type II). In TPEN-treated cells, depletion of zinc was followed by activation of caspase-3 (as demonstrated by enzymatic assay and Western blotting), DNA fragmentation, and morphologic changes. Increase in caspase-3 activity began 12 h after addition of TPEN, suggesting that zinc may suppress a step just before the activation of this caspase. Caspase-6, a mediator of caspase-3 processing, also increased, but later than caspase-3. Effects of TPEN on apoptosis were completely prevented by exogenous ZnSO4 and partially prevented by peptide caspase inhibitors. A critical substrate of caspase-3 may be the cell cycle regulator p21Waf1/Cip1, which was rapidly cleaved in TPEN-treated cells to a 15-kDa fragment before further degradation.

Cellular zinc fluxes and the regulation of apoptosis/gene-directed cell death.

The maintenance of discrete subcellular pools of zinc (Zn) is critical for the functional and structural integrity of cells. Among the important biological processes influenced by Zn is apoptosis, a process that is important in cellular homeostasis (an important cellular homeostatic process). It has also been identified as a major mechanism contributing to cell death in response to toxins and in disease, offering hope that novel therapies that target apoptotic pathways may be developed. Because Zn levels in the body can be increased in a relatively nontoxic manner, it may be possible to prevent or ameliorate degenerative disorders that are associated with high rates of apoptotic cell death. This review begins with brief introductions that address, first, the cellular biology of Zn, especially the critical labile Zn pools, and, second, the phenomenon of apoptosis. We then review the evidence relating Zn to apoptosis and address three major hypotheses: (1) that a specific pool or pools of intracellular labile Zn regulates apoptosis; (2) that systemic changes in Zn levels in the body, due to dietary factors, altered physiological states or disease, can influence cell susceptibility to apoptosis, and (3) that this altered susceptibility to apoptosis contributes to pathophysiological changes in the body. Other key issues are the identity of the molecular targets of Zn in the apoptotic cascade, the types of cells and tissues most susceptible to Zn-regulated apoptosis, the role of Zn as a coordinate regulator of mitosis and apoptosis and the apparent release of tightly bound intracellular pools of Zn during the later stages of apoptosis. This review concludes with a section highlighting areas of priority for future studies.

Involvement of intracellular labile zinc in suppression of DEVD-caspase activity in human neuroblastoma cells.

Age-related tissue Zn deficiency may contribute to neuronal and glial cell death by apoptosis in Alzheimer's dementia. To investigate this, we studied the effects of increasing or decreasing the levels of intracellular labile Zn on apoptosis of human neuroblastoma BE(2)-C cells in vitro. BE(2)-C cells were primed for 18 h with butyrate (1 mM) before addition of staurosporine (1 microM), an effector enzyme of apoptosis, for a further 3 h to induce DEVD-caspase activity. An increase in intracellular Zn using Zn ionophore pyrithione suppressed DEVD-caspase activity, while a decrease in intracellular Zn induced by Zn chelator TPEN mimicked staurosporine by activating DEVD-caspase in butyrate-primed cells. The distribution of intracellular Zn in the cells was demonstrated with the UV-excitable Zn-specific fluorophore Zinquin. Confocal images showed distinct cytoplasmic and cytoskeletal fluorescence. We propose that Zn decreases the level of apoptosis in neuronal cells exposed to toxins, possibly by stabilizing their cytoskeleton.

Involvement of p21(Waf1/Cip1) and its cleavage by DEVD-caspase during apoptosis of colorectal cancer cells induced by butyrate.

Butyrate, a short chain fatty acid produced in the colon, induces apoptosis in cancer cell lines by a sequential process involving inhibition of histone deacetylase, de novo protein synthesis and activation of DEVD-caspase, a major effector of apoptotic DNA fragmentation and membrane blebbing. We now show, in LIM 1215 colorectal cancer cells, that butyrate, in addition to activating DEVD-caspase and inducing apoptosis, also increases expression and cleavage of the universal cyclin-dependent kinase inhibitor p21(Waf1/Cip1) and leads to hypo-phosphorylation of retinoblastoma protein. Accompanying these molecular changes was a progressive loss of G(0)/G(1) and S phase cells. Expression of p21 had similar kinetics to that of the essential protein required for DEVD-caspase activation, indicating parallel effects of butyrate on anti-apoptotic and pro-apoptotic mechanisms. LIM 1215 cells, which were resistant to butyrate-induced apoptosis, were selected by three cycles of exposure to butyrate and removal of floating apoptotic cells. These cells showed markedly enhanced p21 expression and were in cell cycle arrest as determined by flow cytometry. On the other hand, subsequent culture of these cells for 2-3 days in the absence of butyrate resulted in down-regulation of p21 and restoration of sensitivity to apoptosis by butyrate. Western blots of butyrate-treated cells undergoing apoptosis consistently demonstrated a 15 kDa band (p15) that was not present in control cultures. This band became apparent immediately after the onset of DEVD-caspase activation, was enriched in the floating apoptotic cell population when compared with the adherent, non-apoptotic cells and was absent in butyrate-resistant cells lacking DEVD-caspase activity. Peptide caspase inhibitors partially blocked appearance of p15. Here we show, for the first time, that p21 is a target of effector caspases in colorectal cancer cells and that the resistance to butyrate-induced apoptosis is characterized by failure of p21 cleavage.

The activity of caspase-3-like proteases is elevated during the development of colorectal carcinoma.

Activated caspase-3-like proteases promote apoptotic cell death by cleaving cellular substrates. Caspase-3-like activity was measured in colonic carcinomas and in matched normal colonic mucosa from 31 patients and was significantly elevated in 25/ 31 colonic carcinomas and adenomas when compared to normal mucosa (P < 0.0001). Caspase-3-like activity was much higher in normal mucosa and tumours of female subjects than of males (P < 0.0001). No correlation was obtained between caspase-3-like activity and location of the tumour, tumour grade, stage, or patient age. The marked increase in caspase-3-like activity in colorectal carcinomas may reflect an increase in the proportion of cells undergoing spontaneous apoptosis.

Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: A review.

Non-toxic agents that target intracellular signalling pathways in apoptosis may have potential therapeutic use in many diseases. One such agent is the transition metal Zn, a dietary cytoprotectant and anti-oxidant, which stimulates cell proliferation and suppresses apoptosis. Zn is maintained in discrete subcellular pools that are critical for the functional and structural integrity of cells. The present review initially describes the current state of knowledge on the cellular biology of Zn, especially the critical free or loosely bound (labile) pools of Zn, which are thought to regulate apoptosis. We then review the evidence relating Zn to apoptosis, including studies from our laboratory showing potent synergy between intracellular Zn deficiency and the short chain fatty acid butyrate in induction of caspase activation and the downstream events of apoptosis. Our studies have also reported the suppressive effects of micromolar concentrations of Zn on caspase-3 activation in cell-free models. Other key issues that will be discussed include the identification of the putative molecular targets of Zn and the evidence that systemic changes in labile Zn levels are sufficient to alter susceptibility to apoptosis and lead to physiopathological changes in the human body. Finally, we propose that labile Zn may serve as a coordinate regulator of mitosis and apoptosis to regulate tissue growth.

Induction of caspase-3 protease activity and apoptosis by butyrate and trichostatin A (inhibitors of histone deacetylase): dependence on protein synthesis and synergy with a mitochondrial/cytochrome c-dependent pathway.

The induction of apoptosis of tumor cells by the colonic fermentation product butyrate is thought to be an important mechanism in protection against colorectal cancer. Because a major action of butyrate is to inhibit histone deacetylase (leading to chromatin relaxation and altered gene expression), butyrate may induce apoptosis by derepression of specific cell death genes. Here we show that butyrate and trichostatin A (a more selective inhibitor of histone deacetylase) induce the same program of apoptosis in Jurkat lymphoid and LIM 1215 colorectal cancer cell lines that is strictly dependent on new protein synthesis (within 10 h) and that leads to the conversion of the proenzyme form of caspase-3 to the catalytically active effector protease (within 16 h) and apoptotic death (within 24 h). Cells primed with a low concentration of butyrate that itself did not induce activation of caspase-3 or apoptosis were, nevertheless, rendered highly susceptible to induction of apoptosis by staurosporine (an agent that has recently been shown to act by causing mitochondrial release of cytochrome c). Synergy between butyrate and staurosporine was due to the presence of a factor in the cytosol of butyrate-primed cells which enhanced over 7-fold the activation of caspase-3 induced by the addition of cytochrome c and dATP to isolated cytosol. We propose that changes at the level of chromatin structure, induced by a physiological substance butyrate, lead to the expression of a protein that facilitates the pathway by which mitochondria activate caspase-3 and trigger apoptotic death of lymphoid and colorectal cancer cells.

Changes in distribution of labile zinc in mouse spermatozoa during maturation in the epididymis assessed by the fluorophore Zinquin.

The Zn(II)-specific fluorophore Zinquin was used to determine the regional distribution of free or loosely-bound Zn(II) in mouse spermatozoa. Spermatozoa from the testes exhibited bright fluorescence over the entire head; those from the caput epididymides generally fluoresced more brightly in the post-acrosomal region; and spermatozoa from the caudae epididymides fluoresced less brightly, with foci of fluorescence over the sperm head which were lost after extraction with Triton X-100 and hence appeared to be membrane-associated. Treatment of cauda sperm with sodium dodecyl sulfate resulted in a bright uniform Zinquin fluorescence in the heads, similar to that observed in caput sperm, indicating that the two types of sperm have similar amounts of head Zn(II) but that the availability of Zn(II) for binding Zinquin is different. By contrast, the intensity of tail fluorescence was similar in spermatozoa from different regions of the male reproductive tract and was largely unaffected by Triton X-100 extraction, consistent with an intracellular location. Similar differences were observed between caput sperm and cauda sperm in the rat. It is concluded that visualization and measurement of free or loosely-bound Zn(II) in subcellular compartments of spermatozoa should facilitate investigation of the role of this metal in the development and function of spermatozoa and abnormalities that might accompany infertility and Zn(II) deficiency.

Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship to metallothionein and intracellular zinc.

Zinquin [ethyl (2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetate], a new intracellular zinc fluorophore, was used to reveal and to measure Zn in cultured rat hepatocytes before and after metallothionein (MT) induction. Hepatocytes labelled with an intense extranuclear fluorescence. Culture with combinations of Zn, dexamethasone and interleukin-6, increased intracellular MT by 24-fold, Zn 3-fold, and Zinquin fluorescence by approx. 2-fold above control values. Zinquin fluorescence correlated in descending order with the total cellular Zn (r = 0.747), exchangeable Zn (r = 0.735), soluble cytosolic Zn (r = 0.669) and MT (r = 0.666). When Zinquin was incubated with a cytosolic fraction of liver proteins before Sephadex G-75 column chromatography, it fluoresced with free, MT-incorporated and protein-bound Zn. Although only a slight attenuation of fluorescence was seen with high-molecular-mass protein-bound Zn, MT was degraded by 60% in the presence of Zinquin. The undegraded Zn-MT fluoresced at about 20% of the expected intensity. Although Zinquin fluoresces with all cytosolic Zn, caution is required when comparisons are made between samples with different concentrations of MT. This limitation was demonstrated by staining liver slices from adjuvant-treated rats where MT was increased 24-fold, intracellular Zn by 77%, but Zinquin fluorescence by only 19% above controls. Nevertheless, Zinquin should prove to be a useful tool for studying the distribution of Zn in living cells.

Flux of intracellular labile zinc during apoptosis (gene-directed cell death) revealed by a specific chemical probe, Zinquin.

BACKGROUND: The transition metal Zn(II) is thought to regulate cell and tissue growth by enhancing mitosis (cell proliferation) and suppressing the counterbalancing process of apoptosis (gene-directed cell death). To investigate the role of Zn(II) further, we have used a UV-excitable Zn(II)-specific fluorophore, Zinquin. The ester group of Zinquin is hydrolyzed by living cells, ensuring its intracellular retention; this allows the visualization and measurement of free or loosely-bound (labile) intracellular Zn(II) by fluorescence video image analysis or fluorimetric spectroscopy. RESULTS: Here we show that in cells undergoing early events of apoptosis, induced spontaneously or by diverse agents, there is a substantial increase in their Zinquin-detectable Zn(II). This increase occurred in the absence of exogenous Zn(II) and before changes in membrane permeability, consistent with a release of Zn(II) from intracellular stores or metalloproteins rather than enhanced uptake from the medium. We propose that there is a major redistribution of Zn(II) during the induction of apoptosis, which may influence or precipitate some of the later biochemical and morphological changes. CONCLUSIONS: The phenomenon of Zn(II) mobilization, revealed by Zinquin, presents a new element in the process of apoptosis for investigation and may permit rapid and sensitive identification of apoptotic cells, particularly in those tissues where their frequency is low.

Video image analysis of labile zinc in viable pancreatic islet cells using a specific fluorescent probe for zinc.

We used an intracellular zinc-specific fluorophore, Zinquin, in conjunction with fluorescence video image analysis, to reveal labile zinc in pancreatic islet cells, which concentrate this metal for use in synthesis, storage, and secretion of insulin. Zinquin vividly demonstrated zinc in the islet cell secretory granules, which formed a brightly labeled crescent in the cytoplasm between one side of the nucleus and the plasma membrane. Lower but still appreciable amounts of zinc were detected in the remaining cytoplasm, but there was little labeling in the nucleus. Fluorescence intensity varied among islet cells, suggesting differences in zinc content. Their average fluorescence intensity greatly surpassed that of the surrounding pancreatic acinar cells in frozen sections of pancreas and in all other types of cell studied, including lymphocytes, neutrophils, fibroblasts, and erythrocytes. Less labile zinc was detected in cells of the mouse insulinoma cell line NIT-1, regardless of whether they were maintained in long-term culture in the presence or absence of exogenous extracellular zinc. Exposure of islet or insulinoma cells to a high concentration of glucose or other secretagogue decreased the content of labile zinc. Zinquin should be a useful probe for revealing changes in zinc homeostasis in islet B-cells that may be important in their dysfunction and death during diabetes.

Correlation of apoptosis with change in intracellular labile Zn(II) using zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic acid], a new specific fluorescent probe for Zn(II).

Zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)-acetic acid], a membrane-permeant fluorophore specific for Zn(II), was used with spectrofluorimetry and video image analysis to reveal and quantify labile intracellular Zn. Zinquin labelled human chronic-lymphocytic-leukaemia lymphocytes, rat splenocytes and thymocytes with a weak diffuse fluorescence that was quenched when intracellular Zn was chelated with NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) and was greatly intensified by pretreatment of cells with the Zn ionophore pyrithione and exogenous Zn. There was substantial heterogeneity of labile Zn among ionophore-treated cells, and fluorescence was largely extranuclear. The average contents of labile Zn in human leukaemic lymphocytes, rat splenocytes and rat thymocytes were approx. 20, 31 and 14 pmol/10(6) cells respectively. Morphological changes and internucleosomal DNA fragmentation indicated substantial apoptosis in these cells when the level of intracellular labile Zn was decreased by treatment with TPEN. Conversely, increasing labile Zn by pretreatment with Zn plus pyrithione suppressed both spontaneous DNA fragmentation and that induced by the potent apoptosis-induced agents colchicine and dexamethasone. These results suggest that prevention of apoptosis is a function of labile Zn, and that a reduction below a threshold concentration in this Zn pool induces apoptosis.

Induction of apoptosis in chronic lymphocytic leukemia cells and its prevention by phorbol ester.

Chronic lymphocytic leukemia lymphocytes were used to study mechanisms involved in apoptosis (programmed cell death). Apoptosis, which was determined by morphological changes including cell death and by internucleosomal DNA fragmentation, occurred during culture for 1 to 2 days in a portion of the cells from three of the four patients tested. Most of the cells underwent apoptosis and DNA fragmentation was greatly enhanced when cells were cultured in the presence of the microtubule inhibitor colchicine, the topoisomerase II inhibitor etoposide, or the glucocorticoid methylprednisolone. Tumor-promoting phorbol esters inhibited spontaneous DNA fragmentation and cell death including that induced by colchicine, etoposide, and methylprednisolone, indicating that they act on an event common to apoptosis caused by diverse stimuli. Phorbol esters probably act through protein phosphorylation, since they were effective at concentrations which modulated protein kinase C (PKC) and their action was prevented by H-7, which binds to and inactivates the catalytic site of PKC. In the absence of phorbol ester, H-7 itself induced some apoptosis. These findings implicate PKC in the suppression of apoptosis, but its precise role requires systematic investigation.