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Zinc is an essential micronutrient and cytoprotectant involved in the host response to inflammatory stress. We tested whether zinc transporters, the critical regulators that maintain intracellular zinc concentrations, play a role in cell survival, particularly in lung epithelia, during inflammation. Initially, mRNA transcripts were quantitatively measured by RT-PCR for all known human zinc transporters, including 14 importers (SLC39[A.sub.1-14]) and 10 exporters (SLC30[A.sub.1-10]), in primary human lung epithelia obtained from multiple human donors and BEAS-2B cell cultures under baseline and TNF-[alpha]-stimulated conditions. While many zinc transporters were constitutively expressed, only SLC39A8 (Zip8) mRNA was strongly induced by TNF-[alpha]. Endogenous Zip8 protein was not routinely detected under baseline conditions. In sharp contrast, TNF-[alpha] induced the expression of a glycosylated protein that translocated to the plasma membrane and mitochondria. Increased Zip8 expression resulted in an increase in intracellular zinc content and coincided with cell survival in the presence of TNF-[alpha]. Inhibition of Zip8 expression using a short interfering RNA probe reduced cellular zinc content and impaired mitochondrial function in response to TNF-[alpha], resulting in loss of cell viability. These data are the first to characterize human Zip8 and remarkably demonstrate that upregulation of Zip8 is sufficient to protect lung epithelia against TNF-[alpha]-induced cytotoxicity. We conclude that Zip8 is unique, relative to other Zip proteins, by functioning as an essential zinc importer at the onset of inflammation, thereby facilitating cytoprotection within the lung. inflammation; tissue injury; cell death

Treatment of cancer patients with anthracyclin-based chemotherapeutic drugs induces congestive heart failure by a mechanism involving p53. However, it is not known how p53 aggravates doxorubicin (Dox)-induced toxicity in the heart. On the basis of in vitro acute toxicity assay using heat shock factor-1 (HSF-1) wild-type (HSF-[1.sup.+/+]) and HSF-1-knockout (HSF-[1.sup.-/-]) mouse embryonic fibroblasts and neonatal rat cardiomyocyte-derived H9c2 cells, we demonstrate a novel mechanism whereby heat shock protein 27 (HSP27) regulates transcriptional activity of p53 in Dox-treated cells. Inhibition of p53 by pifithrin-[alpha] (PFT-[alpha]) provided different levels of protection from Dox that correlate with HSP27 levels in these cells. In HSF-[1.sup.+/+] cells, PFT-[alpha] attenuated Dox-induced toxicity. However, in HSF-[1.sup.-/-] cells (which express a very low level of HSP27 compared with HSF-[1.sup.+/+] cells), there was no such attenuation, indicating an important role of HSP27 in p53-dependent cell death. On the other hand, immunoprecipitation of p53 was found to coimmunoprecipitate HSP27 and vice versa (confirmed by Western blotting and matrix-assisted laser desorption/ionization time of flight), demonstrating HSP27 binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-[1.sup.+/+] and H9c2 cells, indicating that HSP27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in [G.sub.2]/M phase cell cycle arrest in Doxtreated cells. Overall, HSP27 binding to p53 attenuated the cellular toxicity by upregulating p21 and prevented cell death. redox signaling; DNA repair; cell survival; oxidative stress

Our group (Patschan S, Chen J, Gealekman O, Krupincza K, Wang M, Shu L, Shayman JA, Goligorsky MS; Am J Physiol Renal Physiol 294: F100-F109, 2008) previously observed an accumulation of gangliosides coincident with development of cell senescence and demonstrated lysosomal permeabilization in human umbilical vein endothelial cells exposed to glycated collagen I (GC). Therefore, we investigated whether the lysosome-dependent, caspase-independent or type 2-programmed cell death (autophagy) is involved in development of premature senescence of endothelial cells. The cleaved microtubule-associated protein 1 light-chain 3 (LC3), a marker of autophagosome formation, was overexpressed within 24 h of GC treatment; however, by 4-5 days, it was nearly undetectable. Early induction of autophagosomes was associated with their fusion with lysosomes, a phenomenon that later became subverted. Autophagic cell death can be triggered by the products of damaged plasma membrane, sphingolipids, and ceramide. We observed a clustering of membrane rafts shortly after exposure to GC; later, after 24 h, we observed an internalization, accompanied by an increased acid sphingomyelinase activity and accumulation of ceramide. Pharmacological inhibition of autophagy prevented development of premature senescence but did lead to the enhanced rate of apoptosis in human umbilical vein endothelial cells exposed to GC. Pharmacological induction of autophagy resulted in reciprocal changes. These observations appear to represent a mechanistic molecular cascade whereby advanced glycation end products like GC induce sphingomyelinase activity, accumulation of ceramide, clustering, and later internalization of lipid rafts. advanced glycation end product; lysosomes; sphingomyelinase activity; lipid rafts

Skeletal muscle in congestive heart failure is responsible for increased fatigability and decreased exercise capacity. A specific myopathy with increased expression of fast-type myosins, myocyte atrophy, secondary to myocyte apoptosis triggered by high levels of circulating tumor necrosis factor-[alpha] (TNF-[alpha]) has been described. In an animal model of heart failure, the monocrotaline-treated rat, we have observed an increase of apoptotic skeletal muscle nuclei. Proapoptotic agents, caspase-3 and -9, were increased, as well as serum levels of TNF-[alpha] and its second messenger sphingosine. Treatment of rats with L-carnitine, known for its protective effect on muscle metabolism injuries, was found to inhibit caspases and to decrease the levels of TNF-[alpha] and sphingosine, as well as the number of apoptotic myonuclei. Staurosporine was used in in vitro experiments to induce apoptosis in skeletal muscle cells in culture. When L-carnitine was applied to skeletal muscle cells, before staurosporine treatment, we observed a reduction in apoptosis. These findings show that L-carnitine can prevent apoptosis of skeletal muscles cells and has a role in the treatment of congestive heart failure-associated myopathy. tumor necrosis factor; sphingosine; caspases; staurosporine

We have shown previously that depletion of polyamines delays apoptosis induced by camptothecin in rat intestinal epithelial cells (IEC-6). Mitochondria play an important role in the regulation of apoptosis in mammalian cells because apoptotic signals induce mitochondria to release cytochrome c. The latter interacts with Apaf-1 to activate caspase-9, which in turn activates downstream caspase-3. Bcl-2 family proteins are involved in the regulation of cytochrome c release from mitochondria. In this study, we examined the effects of polyamine depletion on the activation of the caspase cascade, release of cytochrome c from mitochondria, and expression and translocation of Bcl-2 family proteins. We inhibited ornithine decarboxylase, the first rate-limiting enzyme in polyamine synthesis, with [alpha]-difluoromethylornithine (DFMO) to deplete cells of polyamines. Depletion of polyamines prevented camptothecin-induced release of cytochrome c from mitochondria and decreased the activity of caspase-9 and caspase-3. The mitochondrial membrane potential was not disrupted when cytochrome c was released. Depletion of polyamines decreased translocation of Bax to mitochondria during apoptosis. The expression of antiapoptotic proteins Bcl-[x.sub.L] and Bcl-2 was increased in DFMO-treated cells. Caspase-8 activity and cleavage of Bid were decreased in cells depleted of polyamines. These results suggest that polyamine depletion prevents IEC-6 cells from apoptosis by preventing the translocation of Bax to mitochondria, thus preventing the release of cytochrome c. polyamines; [alpha]-difluoromethylornithine; mitochondria; Bcl-2 protein; caspase-9

The effects of insulin-like growth factor I (IGF-I) on the survival and regeneration of lymphocytes in the thymus and spleen were investigated in rats treated with dexamethasone and control subjects. Results show that IGF-I reduced lymphocyte apoptosis in the spleen but not in the thymus. In control animals, IGF-I increased the total number of lymphocytes. These results indicate that IGF-I modulates immune homeostasis by influencing the survival and generation of lymphocytes.

This review addresses the hypothesis that polyunsaturated fatty acids (PUFA), particularly those of the n-3 family, play pivotal roles as 'fuel partitioners' in that they direct fatty acids away from triglyceride storage and toward oxidation and they enhance glucose flux to glycogen. In doing this, PUFA may reduce the risk of enhanced cellular apoptosis associated with excessive cellular lipid accumulation. PUFA exert their beneficial effects by upregulating the expression of genes encoding proteins involved in fatty acid oxidation while simultaneously downregulating genes encoding proteins of lipid synthesis. PUFA govern oxidative gene expression by activating the transcription factor peroxisome proliferator-activated receptor-[Alpha]. PUFA suppress lipogenic gene expression by reducing the nuclear abundance and DNA binding affinity of transcription factors responsible for imparting insulin and carbohydrate control to lipogenic and glycolytic genes. In particular, PUFA suppress the nuclear abundance and expression of sterol regulatory element binding protein-1 and reduce the DNA binding activities of nuclear factor Y, stimulatory protein 1, and possibly hepatic nuclear factor-4. Collectively, the studies discussed suggest that the fuel 'repartitioning' and gene expression actions of PUFA should be considered among the criteria used in defining the dietary needs of n-6 and n-3 fatty acids and in establishing the dietary ratio of n-6 to n-3 fatty acids needed for optimum health benefit. sterol regulatory element binding protein; fatty livers; nuclear factor Y; transcription

Mitochondrial membrane potential ([Delta][[Psi].sub.m]) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive [K.sup.+] (mito[K.sub.ATP]) channel activation prevents the collapse of [Delta][[Psi].sub.m] in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mito Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed [Delta][[Psi].sub.m], as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome c immunostaining. Activation of mito[K.sub.ATP] channel with diazoxide (100 [micro]mol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized [Delta][[Psi].sub.m]. This protection was blocked by 5-hydroxydecanoate (500 [micro]mol/l), a mito[K.sub.ATP] channel-selective inhibitor, but not by HMR-1098 (30 [micro]mol/l), a putative sarcolemmal [K.sub.ATP] channel-selective inhibitor. Dissipation of [Delta][[Psi].sub.m] also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts [Delta][[Psi].sub.m] and induces apoptosis, which are prevented by the activation of the mito[K.sub.ATP] channel. This further emphasizes the therapeutic significance of mito[K.sub.ATP] channel agonists in the prevention of ischemia-reperfusion cell injury. apoptosis; myocytes; ATP; permeability transition pore; cytochrome c

Bcl-2 decreases voltage-gated [K.sup.+] channel activity and enhances survival in vascular smooth muscle cells. Am J Physiol Cell Physiol 281: C157-C165, 2001.--Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to [K.sup.+] efflux; blockade of plasmalemmal [K.sup.+] channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated [K.sup.+] (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents ([I.sub.Kv]). In contrast, the apoptosis inducer staurosporine (ST) enhanced [I.sub.Kv]. In bcl-2-infected cells, however, the ST-induced increase in [I.sub.Kv] was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in [I.sub.Kv] and apoptesis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of [I.sub.Kv] and downregulated the mRNA expression of the pore-forming Ky channel [Alpha]-subunits (Kv1.1, Kv1.5, and Ky2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells. apoptotic volume decrease; pulmonary artery smooth muscle cells; current density of voltage-gated potassium channels

Edwards, Yasmin S., Leanne M. Sutherland, and Andrew W. Murray. NO protects alveolar type II cells from stretch-induced apoptosis. A novel role for macrophages in the lung. Am J Physiol Lung Cell Mol Physiol 279: L1236-L1242, 2000.--We have previously shown that mechanical distortion or stretch of alveolar type II (ATII) cells induces both surfactant release and the induction of apoptosis. We hypothesize that nitric oxide (NO) secreted from alveolar macrophages (AMs) prevents cyclic stretch-induced apoptosis. We show that S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a chemical donor of NO, protects cells against nuclear condensation and DNA fragmentation induced by stretch (30% at 60 cycles/min) as well as by sorbitol. SNAP depleted of NO had no protective effect, and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide blocked the antiapoptotic effect of SNAP. We also show that AMs isolated from rat lung lavage fluid actively synthesize and secrete NO. Using a novel technique in which AMs were cocultured with ATII cells while adhered to floating membrane rafts, we found that NO released from AMs was effective in protecting ATII cells from undergoing apoptosis. We therefore propose that NO secreted by AMs may function as part of a physiological antiapoptotic mechanism that prevents ATII cells from undergoing stretch-induced cell death in the lung. apoptotic stimuli; antiapoptotic factors; mechanical distortion; S-nitroso-N-acetyl-D,L-penicillamine; sorbitol-induced osmotic shock.

Teshima, Shigetada, Hiromu Kutsumi, Tsukasa Kawahara, Kyoichi Kishi, and Kazuhito Rokutan. Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1. Am J Physiol Gastrointest Liver Physiol 279: G1169-G1176, 2000.--We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]. We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [[sup.3]H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-[CH.sub.2]F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-[Kappa]B, and inhibition of this activity by nuclear factor-[Kappa]B decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation. NADPH oxidase; superoxide anion; hydrogen peroxide; anti-apoptosis; cell growth

Howlett, Clare E., James S. Hutchison, John P. Veinot, Aaron Chiu, Pradeep Merchant, and Henry Fliss. Inhaled nitric oxide protects against hyperoxia-induced apoptosis in rat lungs. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L596-L605, 1999.--Inhaled nitric oxide (NO), frequently administered in combination with hyperoxic gas mixtures, was recently shown to protect against the injurious consequences of prolonged hyperoxia. We investigated the possibility that this protective effect is attributable to the ability of NO to block pulmonary apoptosis. We show that rats exposed to 100% [O.sub.2] for 60 h develop severe lung injury consisting of pronounced vascular leak and alveolar apoptosis as inferred from the presence of positive terminal deoxynucleo-tidyltransferase-mediated dUTP nick end labeling and DNA ladders in agarose gels and a decrease in constitutive procaspase-3 levels. However, the inclusion of NO (20 parts/ million) in the hyperoxic gas mixture significantly attenuated both the vascular leak and apoptosis. NO reversed the hyperoxia-associated changes in the activity of the redoxsensitive transcription factors nuclear factor-[Kappa]B, activator protein-1, and Sp1 after 24 h, lowered intercellular adhesion molecule-1 levels, and increased glutathione content. We therefore show, for the first time, that NO can protect against both hyperoxia-induced apoptosis and inflammation. The data suggest that this protection may occur at the transcriptional and caspase-activation levels. DNA fragmentation; nuclear factor-[Kappa]B; activator protein-1; Sp1; intercellular adhesion molecule-1; caspase-3