Kirk E. Dineley

Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells

Edited by Louis J. Ignarro, University of California, Los Angeles School of Medicine, Los Angeles, CA, and approved August 27, 2001 (received for review May 16, 2001)

Induction of neuronal apoptosis by thiol oxidation: putative role of intracellular zinc release.

Abstract: The membrane‐permeant oxidizing agent 2,2′‐dithiodipyridine (DTDP) can induce Zn2+ release from metalloproteins in cell‐free systems. Here, we report that brief exposure to DTDP triggers apoptotic cell death in cultured neurons, detected by the presence of both DNA laddering and asymmetric chromatin formation. Neuronal death was blocked by increased extracellular potassium levels, by tetraethylammonium, and by the broad‐spectrum cysteine protease inhibitor butoxy‐carbonyl‐aspartate‐fluoromethylketone. N,N,N′,N′‐Tetrakis‐(2‐pyridylmethyl)ethylenediamine (TPEN) and other cell‐permeant metal chelators also effectively blocked DTDP‐induced toxicity in neurons. Cell death, however, was not abolished by the NMDA receptor blocker MK‐801, by the intracellular calcium release antagonist dantrolene, or by high concentrations of ryanodine. DTDP generated increases in fluorescence signals in cultured neurons loaded with the zinc‐selective dye Newport Green. The fluorescence signals following DTDP treatment also increased in fura‐2‐ and magfura‐2‐loaded neurons. These responses were completely reversed by TPEN, consistent with a DTDP‐mediated increase in intracellular free Zn2+ concentrations. Our studies suggest that under conditions of oxidative stress, Zn2+ released from intracellular stores may contribute to the initiation of neuronal apoptosis.

Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.

Direct visualization of mitochondrial zinc accumulation reveals uniporter-dependent and -independent transport mechanisms

Current evidence suggests that zinc kills neurons by disrupting energy production, specifically by inhibiting mitochondrial function. However it is unclear if the inhibitory effect requires zinc accumulation, and if so, precisely how zinc enters mitochondria. Here, using fluorescence microscopy to visualize individual rat brain mitochondria, we detected matrix zinc uptake using the fluorophore FluoZin‐3. Fluorescence increased rapidly in mitochondria treated with micromolar free zinc, and was quickly returned to baseline by membrane permeant chelation. Zinc uptake occurred through the calcium uniporter, because depolarization or uniporter blockade reduced fluorescence changes. However, increased fluorescence under these conditions suggests that zinc can enter through a uniporter‐independent pathway. Fluorescence steadily declined over time and was unaffected by acidification or phosphate depletion, suggesting that zinc precipitation is not a mechanism for reducing matrix zinc. Uniporter blockade with ruthenium red also did not change the rate of zinc loss. Instead, zinc appears to exit the matrix through a novel efflux pathway not yet identified. Interestingly, dye‐loaded mitochondria showed no fluorescence increase after treatment with strong oxidants, arguing against oxidant‐labile intra‐mitochondrial zinc pools. This study is the first to directly demonstrate zinc accumulation in individual mitochondria and provides insight about mechanisms mediating mitochondrial zinc uptake and efflux.

Divergent consequences arise from metallothionein overexpression in astrocytes: Zinc buffering and oxidant-induced zinc release

Excessive accumulation of the heavy metal zinc is cytotoxic. As a consequence, cellular vulnerability to zinc‐induced injury may be regulated by the abundance of proteins that maintain intracellular free zinc concentrations ([Zn2+]i). In this study, we overexpressed the zinc‐binding protein metallothionein‐II (MT) in astrocytes to assess its impact as (1) an acute zinc buffering mechanism, and (2) an oxidant‐releasable zinc pool. Overexpression of MT in primary astrocyte cultures was accomplished using an adenoviral vector. Using the zinc‐sensitive fluorescent indicator mag‐fura‐2, we monitored [Zn2+]i after stimulating zinc influx or oxidant treatment. With MT overexpression, we observed an acute buffering effect manifested as a dampening of stimulus‐induced increases in [Zn2+]i. In contrast, we also saw enhanced zinc release with application of the sulfhydryl oxidizing agent 2,2′‐dithiodipyridine. These results indicate that overexpression of a zinc‐binding protein can quickly diminish [Zn2+]i following zinc influx, but elevate [Zn2+]i under conditions of oxidative stress, providing protective yet potentially endangering effects.

The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green.

Zinc-sensitive fluorescent probes have become increasingly important in the investigation of the cellular roles of zinc. There is, however, little information on how the other transition metals in cells may influence the measurement of zinc. We have characterized in vitro the interaction of the nominal zinc indicators FluoZin-3 and Newport Green with all the cationic transition metals found within cells, Cr, Mn, Fe, Co, and Cu, as well as Ni and Cd, by measuring their dissociation constants. In addition, we have shown how FluoZin-3 can be used to quantify the concentration of copper in a cell-free assay and report that the fluorescence of Newport Green is boosted by both Cu(I) and Fe(II). Furthermore, we have introduced diagnostics for detecting the interference of metals other than zinc with its measurement within cells.

Glutamate mobilizes [Zn2+]i through Ca2+-dependent reactive oxygen species accumulation

Liberation of zinc from intracellular stores contributes to oxidant‐induced neuronal injury. However, little is known regarding how endogenous oxidant systems regulate intracellular free zinc ([Zn2+]i). Here we simultaneously imaged [Ca2+]i and [Zn2+]i to study acute [Zn2+]i changes in cultured rat forebrain neurons after glutamate receptor activation. Neurons were loaded with fura‐2FF and FluoZin‐3 to follow [Ca2+]i and [Zn2+]i, respectively. Neurons treated with glutamate (100 μM) for 10 min gave large Ca2+ responses that did not recover after termination of the glutamate stimulus. Glutamate also increased [Zn2+]i, however glutamate‐induced [Zn2+]i changes were completely dependent on Ca2+ entry, appeared to arise entirely from internal stores, and were substantially reduced by co‐application of the membrane‐permeant chelator TPEN during the glutamate treatment. Pharmacological maneuvers revealed that a number of endogenous oxidant producing systems, including nitric oxide synthase, phospholipase A2, and mitochondria all contributed to glutamate‐induced [Zn2+]i changes. We found no evidence that mitochondria buffered [Zn2+]i during acute glutamate receptor activation. We conclude that glutamate‐induced [Zn2+]i transients are caused in part by [Ca2+]i‐induced reactive oxygen species that arises from both cytosolic and mitochondrial sources.

A comparison of Zn2+- and Ca2+-triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition

Intracellular Zn(2+) toxicity is associated with mitochondrial dysfunction. Zn(2+) depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn(2+)-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn(2+)-induced depolarization with the effects of Ca(2+) in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca(2+) caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg(2+), ADP and cyclosporine A. Zn(2+) also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn(2+)-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca(2+) and Zn(2+) in a calcein-retention assay. Consistent with the well-documented ability of Ca(2+) to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn(2+)-treated mitochondria. Considered together, our results suggest that Ca(2+) and Zn(2+) depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn(2+)-induced depolarization, and that Zn(2+) is not a particularly potent mitochondrial inhibitor.

Drugs to Treat Head and Neck Cancers: Mechanisms of Action

This chapter presents the anticancer agents used for the treatment of head and neck squamous cell carcinomas (HNSCC), emphasizing the mechanisms of action of the various drug classes. Current therapies for HNSCC can be broadly divided into four categories: (1) DNA damaging agents, (2) Antimetabolites that interfere with DNA synthesis, (3) Antimitotic agents that interfere with cell division, (4) Agents that target pathways whose dysregulation are critical for tumorigenesis, including apoptosis and angiogenesis. Agents from the first three groups interfere with cell division and are therefore fundamentally non-selective. Most of their significant adverse effects result from the damage they inflict on normal cells that divide or remodel rapidly. Targeted therapies in contrast have greater potential to selectively inhibit transformed cells while sparing normal tissues. All HNSCC therapies are affected by resistance mechanisms that decrease drug efficacy. Typical mechanisms of tumor resistance include reduced drug uptake, increased drug efflux, rapid metabolism, and overexpression/mutation of target enzymes and receptors. Resistance can be pre-empted using combination chemotherapy regimens in which several anticancer agents are given simultaneously. These agents are also used in multimodal therapies, i.e. as a complement to surgery and/or radiation. Indeed, most HNSCC is treated with multimodal therapy and combination chemotherapy. Intravenous injection is the typical route of administration, however a few can be given orally. We also discuss several compounds in various stages of investigation.

M3-subtype muscarinic receptor activation stimulates intracellular calcium oscillations and aldosterone production in human adrenocortical HAC15 cells

A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.