Metal ion chelation enhances tissue plasminogen activator (tPA)-induced thrombolysis: an in vitro and in vivo study.

Stroke is the third leading cause of death in the United States and the leading cause of adult disability. Despite enormous research efforts including many clinical trials, tissue plasminogen activator (tPA) remains the only FDA-approved treatment for acute ischemic stroke. Unfortunately, only 1-3% of stroke patients in the US receive this therapy because of the narrow time window and severe side effects for using tPA. The most deadly and damaging side effect is the risk of intracranial bleeding or hemorrhage. For that reason, the dose of tPA and its overall administration are under tight control, which may compromise the effect of thrombolysis. Studies have been focused on improving the effectiveness of tPA for higher rate of reperfusion, and the safety for less adverse bleeding episode. We studied how metal ions (zinc & iron) affect tPA-induced thrombolysis in vitro and in vivo, and proposed a method to improve the rate of thrombolysis. The amount of hemoglobin in the blood clot lysis was measured by a spectrophotometer. The tPA-induced thrombolysis was measured in vivo in femoral artery. Our results showed that Zn2+, Fe3+ and Fe2+ inhibited tPA-induced thrombolysis, with Zn2+ and Fe2+ being the most effective. Metal ion chelating agent EDTA when it was co-applied with tPA significantly enhanced the tPA-induced thrombolysis. The chelation alone did not have noticeable thrombolytic effect. In in vivo study of tPA-induced thrombosis following femoral artery thrombosis, the co-application of tPA and EDTA achieved significant higher rate of reperfusion than that by tPA treatment alone, suggesting that ion chelation facilitates tPA-induced thrombolysis and potentially improves the safety of tPA application by reducing the necessary dose of tPA application. Our results suggest that the co-application of a chelator and tPA improves the efficacy and, potentially, safety of tPA application, by reducing the necessary dose of tPA for thrombolysis.

Expression of SSEA-4 and Oct-4 from somatic cells in primary mouse gastric cell culture induced by brief strong acid.

Environmental changes can stress and alter biology at the molecular and cellular level. For example, metal-protein interaction is a classic physic and biological property of nature, which is fundamentally influenced by acidity. Here, we report a unique cellular reprogramming phenomenon in that a brief strong acid treatment induced the expression of pluripotent stem cell (PSC) markers. We used strong acid to briefly challenge mix-cultured gastric cells, and then subcultured survived cells in a normal cell culture medium. We found that survival acid-treated cells expressed PSC markers detected by commonly used pluripotent antibodies such as SSEA-4 and Oct4. In addition, we observed that the survived cells from the acid challenge grew faster during the second and third weeks of subculture and had a relative short doubling time (DT) than the controls. PSC marker-labeled 'older' cells also presented immature cell-like morphology with some having marker Oct4 in the nucleus. Finally, the expression of the markers appeared to be sensitive to metal ion chelation. Removal of the metals during a brief acid treatment reduced pluripotent marker-positive cells, suggesting the dissociation of metals from metal-binding proteins may be a factor involved in the induction of stem cell markers. Our findings reveal that somatic cells appear to possess a plasticity feature to express pluripotent marker proteins or to select cell subpopulations that express pluripotent marker proteins when cells are transiently exposed to strong acid. It opens new directions for understanding conserved regulatory mechanisms involved in cellular survival under stressful stimulation.

Zinc modulates synaptic transmission by differentially regulating synaptic glutamate homeostasis in hippocampus.

A subset of presynaptic glutamatergic vesicles in the brain co-releases zinc (Zn2+ ) with glutamate into the synapse. However, the role of synaptically released Zn2+ is still under investigation. Here, we studied the effect of Zn2+ on glutamate homeostasis by measuring the evoked extracellular glutamate level (EGL) and the probability of evoked action potential (PEAP ) at the Zn2+ -containing or zincergic mossy fiber-CA3 synapses of the rat hippocampus. We found that the application of Zn2+ (ZnCl2 ) exerted bidirectional effects on both EGL and PEAP : facilitatory at low concentration (~1 µM) while repressive at high concentration (~50 µM). To determine the action of endogenous Zn2+ , we also used extracellular Zn2+ chelator to remove the synaptically released Zn2+ . Zn2+ chelation reduced both EGL and PEAP , suggesting that endogenous Zn2+ has mainly a facilitative role in glutamate secretion on physiological condition. We revealed that calcium/calmodulin-dependent protein kinase II was integral to the mechanism by which Zn2+ facilitated the release of glutamate. Moreover, a glutamate transporter was the molecular entity for the action of Zn2+ on glutamate uptake by which Zn2+ decreases glutamate availability. Taken together, we show a novel action of Zn2+ , which is to biphasically regulate glutamate homeostasis via Zn2+ concentration-dependent synaptic facilitation and depression. Thus, co-released Zn2+ is physiologically important for enhancing weak stimulation, but potentially mitigates excessive stimulation to keep synaptic transmission within optimal physiological range.

Cross talk between increased intracellular zinc (Zn2+) and accumulation of reactive oxygen species in chemical ischemia.

Both zinc (Zn2+) and reactive oxygen species (ROS) have been shown to accumulate during hypoxic-ischemic stress and play important roles in pathological processes. To understand the cross talk between the two of them, here we studied Zn2+ and ROS accumulation by employing fluorescent probes in HeLa cells to further the understanding of the cause and effect relationship of these two important cellular signaling systems during chemical-ischemia, stimulated by oxygen and glucose deprivation (OGD). We observed two Zn2+ rises that were divided into four phases in the course of 30 min of OGD. The first Zn2+ rise was a transient, which was followed by a latent phase during which Zn2+ levels recovered; however, levels remained above a basal level in most cells. The final phase was the second Zn2+ rise, which reached a sustained plateau called Zn2+ overload. Zn2+ rises were not observed when Zn2+ was removed by TPEN (a Zn2+ chelator) or thapsigargin (depleting Zn2+ from intracellular stores) treatment, indicating that Zn2+ was from intracellular storage. Damaging mitochondria with FCCP significantly reduced the second Zn2+ rise, indicating that the mitochondrial Zn2+ accumulation contributes to Zn2+ overload. We also detected two OGD-induced ROS rises. Two Zn2+ rises preceded two ROS rises. Removal of Zn2+ reduced or delayed OGD- and FCCP-induced ROS generation, indicating that Zn2+ contributes to mitochondrial ROS generation. There was a Zn2+-induced increase in the functional component of NADPH oxidase, p47phox, thus suggesting that NADPH oxidase may mediate Zn2+-induced ROS accumulation. We suggest a new mechanism of cross talk between Zn2+ and mitochondrial ROS through positive feedback processes that eventually causes excessive free Zn2+ and ROS accumulations during the course of ischemic stress.

Zebrafish (Danio rerio) Developed as an Alternative Animal Model for Focal Ischemic Stroke.

Thrombotic cerebral ischemia is one of the leading causes of mortality and chronic disability. Animal models provide an essential tool for understanding the complex cellular and molecular pathophysiology of ischemia and for improving treatment and testing novel neuroprotective drugs in the preclinical setting. In this study, we tested zebrafish as a novel model for thrombotic ischemic brain damage. Zebrafish were intraperitoneally injected with Rose Bengal and light exposure was directed onto the optic tectum region of the brain to induce photothrombosis. After full recovery from anesthesia, zebrafish consistently exhibited abnormal swimming patterns, indicating brain injury from the procedure. The staining of 2,3,5-triphenyltetrazolium chloride (TTC) 24 h after the treatment showed lack of staining of the exposed area of the brain, which further confirmed the ischemic injury. Application of Activase®-tPA improved viability of the brain. The tPA treatment also reduced the occurrence of moving disability as well as the mortality rate, demonstrating that the zebrafish model not only showed focal ischemic injury but also responded well to tPA therapy. Our results suggest that the current photothrombotic method induced focal ischemia in zebrafish and produced consistent brain damage that can be measured by behavioral changes and quantified by histological staining.

Elevated Cytoplasmic Free Zinc and Increased Reactive Oxygen Species Generation in the Context of Brain Injury.

Intracellular zinc release and the generation of reactive oxygen species (ROS) have been reported to be common ingredients in numerous toxic signaling mechanisms in neurons. A key source for intracellular zinc release is its liberation from metallothionein-III (MT-III). MT-III binds and regulates intracellular zinc levels under physiological conditions, but the zinc-binding thiols readily react with certain ROS and reactive nitrogen species (RNS) to result in intracellular zinc liberation. Liberated zinc induces ROS and RNS generation by multiple mechanisms, including the induction of mitochondrial ROS production, and also promotes ROS formation outside the mitochondria by interaction with the enzymes NADPH oxidase and 12-lipoxygenase. Of particular relevance to neuronal injury in the context of ischemia and prolonged seizures, the positive feedback cycle between ROS/RNS generation and increasing zinc liberation will be examined.

Inhibitory effect of zinc on glucose-stimulated zinc/insulin secretion in an insulin-secreting ß-cell line.

Diminished or inappropriate secretion of insulin is associated with type II diabetes. The cellular/molecular mechanism coupled with the regulation of insulin secretion is still under intense investigation. Divalent ion zinc (Zn(2+)) is co-packaged and co-secreted with insulin and is intimately involved in the process of insulin biosynthesis and the maturation of insulin secretory granules. The study reported here investigated glucose-stimulated zinc secretion (GSZS) and the effect of zinc on glucose-stimulated insulin secretion (GSIS) in the HIT-T15 pancreatic ß-cell line. Zinc secretion was measured using a newly developed fluorescent zinc imaging approach, and the insulin secretion was measured using an enzyme-linked immunosorbent assay. There was apparent granular-like zinc staining in ß-cells. The application of glucose induced detectable zinc secretion or GSZS. Like GSIS, GSZS was dependent on the glucose concentration (5-20 mm) and the presence of extracellular calcium. The application of a zinc chelator enhanced GSZS. When brief paired-pulse glucose stimulations, which involve the initial glucose stimulation followed by a second round of glucose stimulation, were applied, zinc secretion or GSZS that followed the first pulse was inhibited. This inhibition was reversed by zinc chelation, suggesting a feedback mechanism on GSZS by zinc secreted from ß-cells. Finally, the application of zinc (50 µm) strongly inhibited GSIS as measured by enzyme-linked immunosorbent assay. The present study suggests that insulin secretion is regulated by co-secreted zinc that may act as an autocrine inhibitory modulator.

The change of intracellular zinc distribution after strong acid challenge.

Zinc (Zn2+) is stored in the nucleus, endoplasmic reticulum (ER), Golgi apparatus, mitochondria, lysosomes, and zinc-binding proteins. The acidity of the microenvironment affects the binding between zinc and proteins in which zinc become free or loosely bound. In this study, when cells were treated with an acidic medium, we started seeing free zinc 'hot spots' or zincosomes where we found bright zinc fluorescence. The rising free zinc quickly across whole cells with both intensity and distribution were pH-dependent. Interestingly, the nucleus was more sensitive to acidic treatment as the increase of nuclear zinc was faster and higher than the increase of cytosolic zinc. In addition, we re-cultured strong acid-challenged cells in a normal medium. Comparing to the control, these cells exhibited multiple zinc 'hot spots' beside the nucleus, suggesting that free zinc became more extensively distributed. To investigate further the function of zinc in cell shaping and morphological changes, we categorized strong acid-challenged cells into different shapes and found that the proportion of each cell shape had changed after the acid challenge. These acid-induced changes of the cell shape percentage were partially reversed by the reduction of zinc, suggesting that zinc participated in directing the cell shapes and morphologies during cell growth. Our findings reveal that acidic pH affects the dynamics of cellular zinc by making zinc more accessible to cellular compartments and zinc-binding proteins, which provided new insights into understanding the cellular behavior and the function of zinc in it.

Zinc cytotoxicity induces mitochondrial morphology changes in hela cell line.

Zinc (Zn2+) is important in cellular processes. In the cell, free zinc is tightly regulated and found in minuscule amounts. However, in an unhealthy cellular environment, such as hypoxia, zinc increases in the cell and zinc overload may occur. Studies have shown that zinc overload causes cellular and mitochondrial stress. Mitochondrial stress affects mitochondrial morphology. In normal cells, mitochondrial morphology resembles a long, tubular shape. In unhealthy cells, mitochondrial morphology resembles fragmented, circular shape. To address whether zinc overload contributes directly to the abnormal changes of mitochondrial morphology, we imaged and analyzed mitochondria that were treated with the application of exogenous zinc. In the first part of the study, exogenous zinc was applied to HeLa cells at 1 µM, 10 µM, 50 µM, 100 µM, or 200 µM zinc chloride along with 10 µM pyrithione. Mitochondrial morphology was analyzed with Mito-Morphology micro in ImageJ. Mitochondrial morphology changed from a healthy tubular shape to an unhealthy circular shape and fragmentation. Mitochondrial morphology changes were observed in a dose-dependent fashion. The second part of the study involved applying the metal ion chelator TPEN after applying 50 µM zinc chloride along with 10 µM pyrithione. TPEN reduced zinc-induced abnormal mitochondrial morphology after zinc treatment. This present study supports that zinc overload may cause morphology changes induced by mitochondrial stress that may lead to cell death.

Presynaptic evidence for zinc release at the mossy fiber synapse of rat hippocampus.

Vesicular zinc (Zn(2+)) is found in a subset of glutamatergic nerve terminals throughout the mammalian forebrain and is colocalized with glutamate. Despite well-documented neuromodulatory roles, exocytosis of endogenous Zn(2+) from presynaptic terminals has never been directly demonstrated, because existing studies have measured elevated Zn(2+) concentrations by examining the perfusate. Thus, the specific origin of synaptic Zn(2+) remains a controversial subject. Here, we describe synaptic Zn(2+) trafficking between cellular compartments at hippocampal mossy fiber synapses by using the fluorescent indicator Zinpyr-1 to label the hippocampal mossy fiber boutons. We determined endogenous Zn(2+) exocytosis by direct observation of vesicular Zn(2+) as decreasing fluorescence intensity from presynaptic axonal boutons in the stratum lucidum of CA3 during neural activities induced by the stimulation of membrane depolarization. This presynaptic fluorescence gradually returned to a level near baseline after the withdrawal of moderate stimulation, indicating an endogenous mechanism to replenish vesicular Zn(2+). The exocytosis of the synaptic Zn(2+) was also dependent on extracellular Ca(2+) and was sensitive to Zn(2+)-specific chelators. Vesicular Zn(2+) loading was sensitive to the vacuolar-type H(+)-ATPase inhibitor concanamycin A, and our experiments indicated that blockade of vesicular reloading with concanamycin A led to a depletion of that synaptic Zn(2+). Furthermore, synaptic Zn(2+) translocated to the postsynaptic cell body upon release to produce increases in the concentration of weakly bound Zn(2+) within the postsynaptic cytosol, demonstrating a feature unique to ionic substances released during neurotransmission. Our data provide important evidence for Zn(2+) as a substance that undergoes release in a manner similar to common neurotransmitters.

Intracellular zinc increase affects phosphorylation state and subcellular localization of protein kinase C delta (δ).

Protein kinase C delta (PKCδ) is a Ser/Thr-specific kinase involved in many fundamental cellular processes including growth, differentiation and apoptosis. PKCδ is expressed ubiquitously in all known cell types, and can be activated by diacylglycerol, phorbol esters and other kinases. Multiple lines of evidence have indicated that the mode of activation greatly influences the role PKCδ plays in cellular function. Divalent metal ions, such as zinc are released as a response to cellular stress and injury, often resulting in oxidative damage and cell death. In this study, we evaluate the effect increased concentrations of intracellular zinc has on the phosphorylation state and subcellular localization of PKCδ. More specifically, we demonstrate that intracellular zinc inhibits the phosphorylation of PKCδ at Thr505 in a concentration-dependent manner and facilitates the translocation of PKCδ from the cytosol to the Golgi complex. Analysis of a PKCδ structural model revealed a potential His-Cys3 zinc-binding domain adjacent to residue Thr505 and suggests that interaction with a Zn2+ ion may preclude phosphorylation at this site. This study establishes zinc as a potent modulator of PKCδ function and suggests a novel mechanism by which PKCδ is able to "sense" changes in the concentration of intracellular zinc. These findings illuminate a new paradigm of metal ion-protein interaction that may have significant implications on a broad spectrum of cellular processes.

Intracellular zinc elevation measured with a "calcium-specific" indicator during ischemia and reperfusion in rat hippocampus: a question on calcium overload.

Much of our current evidence concerning of the role of calcium (Ca2+) as a second messenger comes from its interaction with fluorescent probes; however, many Ca2+ probes also have a higher affinity for another divalent cation: zinc (Zn2+). In this study, using a selective Zn2+ probe (Newport Green), we investigated the accumulation of intracellular Zn2+ transients in acute rat hippocampal slices during ischemia, simulated by oxygen and glucose deprivation (OGD). Subsequent reperfusion with glucose-containing oxygenated medium resulted in an additional increase in intracellular Zn2+. Such observations compelled us to investigate the contribution of Zn2+ to the alleged intracellular Ca2+ overload occurring in ischemia and reperfusion. Using confocal fluorescent microscopy of Calcium Green-1, a widely used Ca2+ indicator, we detected increases in fluorescence intensity during OGD and reperfusion. However, application of a Zn2+ chelator, at the peak of the fluorescence elevation (interpreted as Ca2+ overload), resulted in a significant drop in intensity, suggesting that rising Zn2+ is the primary source of the increasing Calcium Green-1 fluorescence. Finally, staining with the cell viability indicator propidium iodide revealed that Zn2+ is responsible for the ischemic neuronal cell death, because Zn2+ chelation prevented cells from sustaining ischemic damage. Current cellular models of ischemic injury center on Ca2+-mediated excitotoxicity. Our results indicate that Zn2+ elevation contributes to conventionally recognized Ca2+ overload and also suggest that the role of Ca2+ in neurotoxicity described previously using Ca2+ probes may need to be re-examined to determine whether effect previously attributed to Ca2+ could, in part, be attributable to Zn2+.

Fluorescence imaging study of extracellular zinc at the hippocampal mossy fiber synapse.

Although synaptically released, vesicular Zn(2+) has been proposed to play a neuromodulatory or neuronal signaling role at the mossy fiber-CA3 synapse, Zn(2+) release remains controversial, especially when detected using fluorescent imaging. In the present study, we investigated synaptically released Zn(2+) at the mossy fiber (MF) synapse in rat hippocampal slices using three chemically distinct, fluorescent Zn(2+) indicators. The indicators employed for this study were cell membrane impermeable (or extracellular) Newport Green [K(DZn2+) approximatelly 1 microM] , Zinpyr-4 K(DZn2+) approximately 1 nM and FluoZin-3 K(DZn2+) approximately 15 nM, chosen, in part, for their distinct dissociation constants. Among the three indicators, FluoZin-3 was also sensitive to Ca(2+) K(DCa2+) approximately 200-300 microM which was present in the extracellular medium ([Ca(2+)](o)>2mM). Hippocampal slices loaded with either Newport Green or FluoZin-3 showed increases in fluorescence after electrical stimulation of the mossy fiber pathway. These results are consistent with previous studies suggesting the presence of synaptically released Zn(2+) in the extracellular space during neuronal activities; however, the rise in FluoZin-3 fluorescence observed was complicated by the data that the addition of exogenous Zn(2+) onto FluoZin-3 loaded slices gave little change in fluorescence. In the slices loaded with the high-affinity indicator Zinpyr-4, there was little change in fluorescence after mossy fiber activation by electrical stimulation. Further study revealed that the sensitivity of Zinpyr-4 was mitigated by saturation with Zn(2+) contamination from the slice. These data suggest that the sensitivity and selectivity of a probe may affect individual outcomes in a given experimental system.

Zinc chelation promotes streptokinase-induced thrombolysis in vitro.

Cardiovascular disorder occurs when a local blood clot obstructs an artery or a vein to its surround organs, causing related tissues to lose function and die. It is one of the leading causes of mortality and a major cause of disability. The effect of thrombolysis induced by injecting intravenous thrombolytic agents is critical for reducing tissue damages. Streptokinase (SK) is a widely used thrombolytic agent in the treatment of thromboembolism in the blood vessels. A high unit of streptokinase is used in thrombolytic therapies for thrombotic disorders and could improve tissue reperfusion. It is a potent plasminogen activator. However, safety concerns for the usage of a high unit of streptokinase have been raised for the hemorrhagic transformation. In the present study, we studied how zinc would affect streptokinase-induced thrombolysis in vitro, and proposed a strategy to improve streptokinase's effectiveness in promoting thrombolysis. The mice whole blood was used to form the blood clot in vitro by incubating with calcium at 37°C for 30 minutes. Streptokinase was used for inducing thrombolysis measured with the spectrophotometer. Zinc and its chelator, Ca-EDTA, were applied with streptokinase, respectively. Results showed that the co-application zinc inhibited the thrombolytic effect of streptokinase in a dose-dependent manner. Zinc chelator, Ca-EDTA, significantly increased the effect of streptokinase-induced thrombolysis. Our results suggest that zinc chelation improved the efficiency of streptokinase in thrombolysis. The results may have a significant clinical implication by potentially reducing the adverse effect of streptokinase application.

Determining zinc with commonly used calcium and zinc fluorescent indicators, a question on calcium signals.

Investigations into the roles of Ca(2+) and Zn(2+) in cell biology have been facilitated by the development of sensitive fluorometric probes that have enabled the measurement of Ca(2+) or Zn(2+) in both extracellular and intracellular environments. It is critical to be aware of the specificity and relative selectivity of a probe for the targeted ion. Here, we investigated metal-ion responses by screening nominally Zn(2+)- or Ca(2+)-selective fluorophores in solutions containing various concentrations of Ca(2+), as a potential interferent for Zn(2+), or Zn(2+), as a potential interferent for Ca(2+). The results suggested that Zn(2+)-sensitive dyes were more specific for their targeted ion than dyes that targeted Ca(2+). Ca(2+)-sensitive dyes such as Calcium Green-1, Fura-2, and Fluo-3 showed a wide range of interaction with Zn(2+), even responding to Zn(2+) in the presence of high concentrations of Ca(2+). We demonstrate that these Ca(2+) indicators can effectively measure dynamic changes of cytosolic Zn(2+). Our results appeal for a new generation of Ca(2+) fluorophores that are more specific for Ca(2+) over Zn(2+). One implication of these results is that data obtained using Ca(2+)-sensitive dyes may need to be re-examined to determine if results previously attributed to Ca(2+) could, in part, be due to Zn(2+).

Measuring cell viability with membrane impermeable zinc fluorescent indicator.

Recent findings suggest that the accumulation of cytoplasmic zinc [Zn2+]i is a ubiquitous component in the cell death cascade. Zn2+ can be liberated from intracellular stores following oxidative stress and contribute to cell death processes. Here we show that the membrane/cell impermeable Zn2+ fluorescent indicator Newport Green (NG), which is non-toxic and impermeable to the membranes of healthy cells, can label unhealthy cells in tissue slices in a manner comparable to the traditional viability indicator propidium iodide (PI). Using confocal microscopy, we detected PI labeled nuclei colocalized with NG fluorescence. Our results indicate that cells which absorbed PI into their nuclei also allowed cell-impermeable Zn2+ dye to penetrate their plasma membranes, subsequently exhibiting cytosolic and nuclear fluorescence. As in PI staining, we observed marked increases in NG fluorescence in damaged/dead cells of tissue slices. Two other cell impermeable fluorescent Zn2+ dyes, Fluozin-3 and Zinpyr-4, also stained cytosolic Zn2+ in PI labeled cells. Our data indicates that the application of a Zn2+ fluorescent indicator is a fast, simple, non-toxic and reliable method for visualizing cell viability within in vitro tissue preparations. Accordingly, we demonstrate that intracellular accumulation of Zn2+ correlates with neuronal death.

Zinc wave during the treatment of hypoxia is required for initial reactive oxygen species activation in mitochondria.

Mitochondrial reactive oxygen species (ROS) are known to accumulate during chemical hypoxia, causing adverse effects on cell function and survival. Recent studies show important role zinc accumulation plays in dysfunction associated with hypoxia. It is well known that ROS accumulation also plays a major role in cellular damage by hypoxia. In this study, fluorescent imaging and pharmacological methods were used in live HeLa cells to determine role of zinc in initial ROS accumulation in mitochondria during chemical hypoxia (oxygen glucose depravation with 4 mM sodium dithionite). Accumulation of both was observed as a very rapid phenomenon with initial rapid zinc increase (zinc wave) within 60 seconds of hypoxia onset and ROS increase within 4.5 minutes. Zinc chelation with TPEN removed the initial zinc wave which in turn abolished ROS accumulation. Influx of exogenous zinc induced rapid ROS accumulation. Inhibition of NADPH oxidase with apocynin, a NADPH oxidase inhibitor, showed significant and prolonged reduction in zinc induced ROS accumulation. We proposed a novel mechanism of intracellular zinc increase that activates NADPH oxidase which in turn triggers mitochondrial ROS production.

Intracellular zinc distribution in mitochondria, ER and the Golgi apparatus.

Zinc (Zn(2+)) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, and BODIPY TR ceramide respectively, suggesting the presence of free zinc in mitochondria, endoplasmic reticulum, and the Golgi apparatus. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. It is known that nucleus contains high amount of zinc binding proteins that have high zinc binding affinity. The absence of zinc fluorescence suggests that there is little free zinc in these two regions. It also indicates that the zinc fluorescence detected in mitochondria, ER and Golgi apparatus represents free chelatable zinc. Taken together, our results support that these organelles are potential zinc storing organelles during cellular zinc homeostasis.

Intranasal Delivery of Granulocyte Colony-Stimulating Factor Enhances Its Neuroprotective Effects Against Ischemic Brain Injury in Rats.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor with strong neuroprotective properties. However, it has limited capacity to cross the blood-brain barrier and thus potentially limiting its protective capacity. Recent studies demonstrated that intranasal drug administration is a promising way in delivering neuroprotective agents to the central nervous system. The current study therefore aimed at determining whether intranasal administration of G-CSF increases its delivery to the brain and its neuroprotective effect against ischemic brain injury. Transient focal cerebral ischemia in rat was induced with middle cerebral artery occlusion. Our resulted showed that intranasal administration is 8-12 times more effective than subcutaneous injection in delivering G-CSF to cerebrospinal fluid and brain parenchyma. Intranasal delivery enhanced the protective effects of G-CSF against ischemic injury in rats, indicated by decreased infarct volume and increased recovery of neurological function. The neuroprotective mechanisms of G-CSF involved enhanced upregulation of HO-1 and reduced calcium overload following ischemia. Intranasal G-CSF application also promoted angiogenesis and neurogenesis following brain ischemia. Taken together, G-CSF is a legitimate neuroprotective agent and intranasal administration of G-CSF is more effective in delivery and neuroprotection and could be a practical approach in clinic.

Do we need zinc to think?

Chelatable Zn(2+), which is found in the synaptic vesicles of certain glutamatergic neurons in several regions of the forebrain, is released during neuronal activity. Zn(2+) exhibits numerous effects on ligand-gated and voltage-dependent ion channels, and released Zn(2+) is therefore likely able to modulate synaptic transmission. The physiologically relevant actions of Zn(2+), however, have remained unclear. Recent research exploiting improved Zn(2+)-sensitive optical probes has suggested some intriguing effects for synaptically released Zn(2+), including heterosynaptic regulation of N-methyl-D-aspartate (NMDA) receptor function, and a novel role as a trans-synaptic second messenger that may enter postsynaptic neurons to modulate various signal transduction pathways.