Role of zinc in pulmonary endothelial cell response to oxidative stress

Zi-Lue Tang, Karla Wasserloos, Claudette M. St. Croix, and Bruce R. Pitt

Department of Environmental and Occupational Health, The Graduate School of Public Health, University of Pittsburgh, and Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although zinc is a well-known inhibitor of apoptosis, it may contribute to oxidative stress-induced necrosis. We noted that N,N,N',N'- tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; >10 µM), a zinc chelator, quenched fluorescence of the zinc-specific fluorophore Zinquin and resulted in an increase in spontaneous apoptosis in cultured sheep pulmonary artery endothelial cells (SPAECs). Addition of exogenous zinc (in the presence of pyrithione, a zinc ionophore) to the medium of SPAECs caused an increase in Zinquin fluorescence and was associated with a concentration-dependent increase in necrotic cell death. Exposure of SPAECs to TPEN (10 µM) resulted in enhanced apoptosis after lipopolysaccharide or complete inhibition of t-butyl hydroperoxide (tBH)-induced necrosis. We further investigated the role of two zinc-dependent enzymes, poly(ADP-ribose) polymerase (PARP) and protein kinase (PK) C, in tBH toxicity. tBH toxicity was only affected by the PARP inhibitors 4-amino-1,8-naphthalimide or 3-aminobenzamide over a narrow range, whereas the PKC inhibitors bisindolylmaleimide and staurosporine significantly reduced tBH toxicity. tBH caused translocation of PKC to the plasma membrane of SPAECs that was partially inhibited by TPEN. Thus pulmonary endothelial cell zinc inhibits spontaneous and lipopolysaccharide-dependent apoptosis but contributes to tBH-induced necrosis, in part, via a PKC-dependent pathway.

endothelium; apoptosis; necrosis; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AFTER IRON, ZINC is the most abundant trace metal. Its intracellular levels are maintained by the dynamic process of zinc transport, intracellular vesicular storage, and binding to a large number of proteins (6). Although the transporters and binding sites have not been completely enumerated, they appear ubiquitous and account for the extraordinarily low level of free intracellular zinc (8). Zinc itself is redox inert but is associated with a large number of proteins (estimated between 1 and 10% of the human genome). As such, zinc is an integral component of numerous metalloenzymes, structural proteins, and transcription factors and contributes to physiological processes including neurotransmission, hormone secretion, and DNA synthesis and gene expression. In addition to these diverse physiological roles, it is apparent that zinc is an inhibitor of many forms of apoptosis (3), including a recent report (35) identifying such a role for zinc in airway epithelium. Alternatively, zinc may contribute to various forms of necrosis, especially those associated with excitotoxicity and other forms of neuronal injury (4).

As in most other cells studied, zinc appears to inhibit apoptosis in systemic endothelial cells. For example, in cultured aortic endothelial cells, zinc can block cadmium (32)-, cholesterol (24)-, linoleic acid-, and tumor necrosis factor-alpha (25)-induced apoptosis. Zinc appears to be anti-inflammatory for some endothelial cells by inhibiting oxidative stress (16) and tumor necrosis factor-alpha -induced changes in various transcription factors (7) and enhancing repair in a cultured endothelial cell wound model (12). Nonetheless, as in the central nervous system, high levels of zinc can be lethal to cultured cerebral endothelial cells (19). Nothing is known, however, about the role of zinc in the pulmonary endothelium.

We examined some of these issues for the first time in pulmonary endothelial cells. We utilized two simple models of oxidative stress that result in discrete forms of cell death. In particular, our laboratory has previously shown that lipopolysaccharide (LPS) is associated with oxidative stress in sheep pulmonary artery endothelial cells (SPAECs) (37) and causes cell death in large part via apoptosis (18), whereas t-butyl hydroperoxide (tBH) causes oxidative stress (31) that is associated with necrotic cell death. In the current study, we show that zinc inhibits LPS-induced apoptosis in SPAECs. In contrast, zinc appears to contribute to tBH-induced necrosis in these cells. The mechanism underlying this latter phenomenon is unclear, but poly(ADP-ribose) polymerase (PARP) activation is not critical, whereas protein kinase (PK) C activation may participate.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and Culture of SPAECs

SPAECs were isolated and cultured from collagenase-digested pulmonary arteries as described previously (18). Cells were subcultured in Opti-MEM (GIBCO BRL, Life Technologies, Grand Island, NY) with endothelial cell growth supplement (15 µg/ml; Collaborative Biomedical Products, Bedford, MA), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% sheep serum (Sigma, St. Louis, MO). The cells were grown in the above medium at 37°C in 95% air-5% CO2. They were routinely passaged 1:3 by detaching the cells with a balanced salt solution containing trypsin (0.05%) and EDTA (0.02%; GIBCO BRL) and were used between passages 6 and 12.

Microspectrofluorometric Determination of Labile Zinc

SPAECs were plated on polylysine-coated glass coverslips. The cells were washed with phosphate-buffered saline (PBS) and incubated with 15 µM Zinquin (Toronto Research Chemicals, Toronto, ON) in PBS for 30 min at 37°C. The coverslips were then washed with PBS and placed onto a Nikon Diaphot inverted microscope. All recordings were made at room temperature. Zinquin fluorescence was imaged with a DAPI dichroic mirror and collected with a Photometrics charge-coupled device camera with RatioTool software (29).

Endothelial Cell Toxicity

The viability of SPAECs was determined by quantifying reduction of a fluorogenic indicator Alamar blue (Alamar Biosciences, Sacramento, CA). SPAECs (5 × 104) were allowed to attach in 48-well tissue culture clusters and were exposed to the experimental conditions. Alamar blue was added to the medium, and 3 h later, fluorescence was determined with a cytofluorometer (Cytofluor II, PerSeptive Biosystems, Framingham, MA). It has previously been shown that oxidized Alamar blue is taken up by cells and is reduced by intracellular dehydrogenases, and the water-soluble changes in fluorescence emission (590 nm) are utilized as an index of viability (10, 28).

SPAECs were also examined for evidence of apoptosis as indicated by condensed, clumped, or segmented nuclei as detected with a fluorescence microscope. Both detached and attached cells (released with EDTA-trypsin) were collected, combined, and centrifuged. After resuspension in PBS containing 2% paraformaldehyde, the cells were stained with Hoechst 33342 (1 µg/ml; Molecular Probes, Eugene, OR). Three hundred cells per coverslip were counted. Those cells with clumped or condensed chromatin as detected with fluorescence microscopy were counted and are expressed as a fraction of the total cells counted (2).

PKC Activity

After the incubation period, the cells were immediately washed three times with precooled PBS, detached with a cell scraper, transferred to 10-ml Falcon tubes, and pelleted by centrifugation. The pellet was sonicated in 1 ml of extraction buffer (20 mM Tris · HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 330 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml each of aprotinin and leupeptin) and then ultracentrifuged at 100,000 g for 30 min. The resulting supernatant contained the soluble cytosolic fractions of PKC. The pellet was resolubilized in extraction buffer (without sucrose) with 1% Triton X-100 and sonicated. The membrane fraction of PKC was retained after ultracentrifugation at 100,000 g for 30 min. PKC from the membrane and cytosolic fractions was partially purified with a DEAE-Sephacel chromatography column (Sigma). PKC activity was measured with a commercial kit (Calbiochem, San Diego, CA) on the same day of extraction by its ability to transfer 32P from [gamma -32P]ATP into the specific biotinylated PKC pseudosubstrate. The results are expressed as the percent distribution of PKC activity between the cytosolic and membrane fractions.

Experimental Protocols

Detection of labile zinc in SPAECs. SPAECs were incubated with Zinquin, and labile zinc was detected with microspectrofluorometric techniques. ZnCl2 (100 µM) was added to the medium along with 10 µM pyrithione, and the relative fluorescence was quantified for an additional 2-5 min. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; 100 µM) was then added to the medium to assess the reversibility of ZnCl2-induced changes. In some instances, TPEN was added before exogenous ZnCl2 and pyrithione.

Effect of zinc on SPAECs. SPAECs were exposed to ZnCl2 with and without the zinc ionophore pyrithione (4 µM) in concentrations up to 100 µM for 5 h. Viability was determined with Alamar blue. A separate subculture of SPAECs was exposed to ZnCl2 and pyrithione with and without the zinc chelator TPEN (10 µM), and viability was quantified. In the next series of experiments, TPEN (1-50 µM) was added to the medium, and cell viability was determined with both Alamar blue and nuclear morphology after 5 h of incubation. This experiment was repeated by adding equimolar ZnCl2 (as in TPEN) to the medium.

Effect of TPEN on LPS-induced apoptosis in SPAECs. SPAECs were plated in 25-cm2 tissue culture flasks with and without TPEN (10 µM). The cells were then exposed to LPS (1 µg/ml for 4 h, Escherichia coli 0111:B4; Sigma) followed by phase-contrast microscopy, Hoechst 33342 staining, and fluorescence chromatin determination and quantification.

Effect of TPEN on tBH-induced necrosis in SPAECs. SPAECs were exposed to tBH (0-100 µM) for 5 h, and cell viability was determined with an Alamar blue assay. Additional subcultures of SPAECs were pretreated with TPEN (10 µM) with and without equimolar ZnCl2, and cytotoxicity to tBH was assessed.

Inhibition of PARP or PKC on tBH-induced necrosis in SPAECs. SPAECs were exposed to tBH in the presence of the PARP inhibitor 4-amino-1,8-naphthalimide (ANI; 20 µM) or 3-aminobenzamide (3-AB; 4 mM), and cell viability was determined via an Alamar blue assay. A separate group of SPAECs was exposed to tBH in the presence of the PKC inhibitors bisindolylmaleimide (GFX; 300 nM and 10 µM), staurosporine (STP; 200 nM), Gö-6976 (1-100 nM), calphostin C (10-1,000 nM), chelerythrine chloride (0.3-30 µM), and rottlerin (1-100 µM), and cell viability was determined via Alamar blue. In addition, a group of SPAECs was exposed to 0.1 µM phorbol 12-myristate 13-acetate for 24 h, and then the effect of tBH on cell viability was determined. Finally, a group of SPAECs was exposed to tBH (100 µM) with and without TPEN (10 µM), and PKC activity was determined in the cytosolic and membrane fractions 10, 30, and 60 min after exposure to tBH.

Statistical Analyses

All values are means ± SE. The effect of zinc and tBH on cell viability, TPEN on LPS-induced apoptosis, and TPEN, ANI, 3-AB, and PKC inhibitors on cell viability was determined by two-factor analysis of variance. Multiple means were compared by Newman-Keuls test, and significance was established at the P < 0.05 level (39).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Determination of Labile Zinc in SPAECs

As previously reported (29), labile zinc appears to partition in discrete vesicles of SPAECs (Fig. 1A). The relative intensity of Zinquin fluorescence in SPAECs can be greatly enhanced by acute exposure to ZnCl2 in the presence of the zinc ionophore pyrithione. This increase was completely reversible by addition of the membrane-permeant Zn chelator TPEN (Fig. 1B). Indeed, the addition of TPEN to resting SPAECs resulted in a decrease in constitutive levels of labile zinc (data not shown). The effects of exogenous zinc or TPEN were reversible and repetitive over a short time period (<1 h).


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Fig. 1.   Detection of labile zinc in cultured sheep pulmonary artery endothelial cells (SPAECs) that were incubated with 15 µM Zinquin. Zinquin appears to partition in discrete vesicles of SPAECs (A). The intensity of Zinquin fluorescence was increased by acute exposure to 100 µM ZnCl2 in the presence of 10 µM pyrithione (B). The increase in Zinquin fluorescence was reversed by exposure to 100 µM TPEN.

Effect of Zinc on SPAECs

SPAECs were not affected by exposure to exogenous zinc (0-100 µM) unless sodium pyrithione (4 µM) was present (Fig. 2). The concentration-dependent toxicity of zinc (in the presence of pyrithione) was due to necrosis as detected by the decrease in cell viability with Alamar blue (with no detectable DNA fragmentation or nuclear condensation; data not shown). The toxicity was a result of the abrupt elevation in labile zinc (Fig. 1B) because it was significantly inhibited by the addition of 10 µM TPEN (Fig. 2). Although TPEN (<10 µM) caused a decrease in labile zinc as detected by Zinquin quenching, exposure to this low level of TPEN was not significantly toxic to SPAECs (Fig. 3). TPEN > 10 µM caused a concentration-dependent toxicity, of which the greatest proportion was due to apoptosis (Fig. 3), suggesting that labile zinc inhibited spontaneous apoptosis.


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Fig. 2.   Zinc-induced necrosis in SPAECs. SPAECs were not affected by exposure to exogenous ZnCl2. In the presence of sodium pyrithione (4 µM), however, ZnCl2 induced concentration-dependent necrosis as determined with Alamar blue. This zinc-induced toxicity was attenuated in the presence of 10 µM TPEN.



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Fig. 3.   Zinc chelation by TPEN promotes spontaneous apoptosis in SPAECs. TPEN (>10 µM) induced a concentration-dependent decrease in viability, which was reversible with equimolar ZnCl2. The greatest proportion of this toxicity was due to apoptosis that was inhibited by the addition of equimolar ZnCl2.

Effect of Zinc on LPS-Induced Apoptosis in SPAECs

As previously reported (2, 18, 37), exposure of SPAECs to LPS (1 µg/ml for 4 h) resulted in a significant increase in apoptosis (Fig. 4). Although LPS by itself did not result in acute (<30 min) changes in labile zinc as determined by Zinquin fluorescence (data not shown), TPEN (10 µM) caused a significant further increase in LPS-induced apoptosis (Fig. 4). Collectively (Figs. 3 and 4), it appears that labile zinc (e.g., TPEN chelatable) inhibits spontaneous or LPS-induced apoptosis.


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Fig. 4.   Effect of zinc on lipopolysaccharide (LPS)-induced apoptosis in SPAECs. A low percentage of SPAECs showed spontaneous apoptosis that was not significantly increased by TPEN (10 µM). Treatment with LPS (1 µg/ml for 4 h) induced apoptosis in SPAECs that was enhanced with TPEN (10 µM). * Significantly different from control. ** Significantly different from TPEN and from LPS.

Effect of Zinc on tBH-Induced Necrosis in SPAECs

tBH caused a concentration-dependent toxicity in SPAECs that was primarily necrotic and sensitive to TPEN (Fig. 5). The inhibitory effect of TPEN was lost with coadministration of equimolar concentrations of zinc (Fig. 5), suggesting that tBH-induced increases in labile zinc contributed to its toxicity in SPAECs. Nonetheless, there were no acute changes in Zinquin fluorescence in response to toxic levels (100 µM) of tBH for short periods of time (<30 min).


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Fig. 5.   TPEN inhibits t-butyl hydroperoxide (tBH)-induced necrosis in SPAECs. tBH caused a concentration-dependent necrosis as measured with Alamar blue. The specific zinc chelator TPEN (10 µM) blocked tBH toxicity. Coadministration of ZnCl2 (10 µM) reversed the protection with TPEN.

Role of PARP and PKC in tBH Toxicity in SPAECs

Incubation of SPAECs with either 3-AB (4 mM) or ANI (20 µM) limited cell death over a very narrow concentration range, near the threshold for cell death (Fig. 6), suggesting that activation of PARP was not a significant component of such toxicity. In contrast, the PKC inhibitor STP (200 nM) significantly inhibited tBH-induced toxicity. Although high doses of GFX (10 µM) significantly attenuated tBH-induced toxicity, a lower concentration (<300 nM; IC50 for PKC activity = 10 nM) did not show significant protection. Gö-6976, a specific calcium-dependent PKC isozyme (conventional PKC) inhibitor did not affect tBH toxicity over a large concentration range (1-100 nM; Fig. 7). Pretreatment of SPAECs with phorbol 12-myristate 13-acetate (0.1 µM for 24 h), a condition usually associated with the downregulation of PKC, also reduced tBH toxicity (data not shown). Three other PKC inhibitors, calphostin C, chelerythrine chloride, and rottlerin, were directly toxic to SPAECs at concentrations near their IC50 for PK inhibition, and thus their effect on tBH toxicity was not pursued (data not shown). Acute (<1-h) exposure of SPAECs to 100 µM tBH resulted in translocation of PKC activity from the cytosol to the plasma membrane, and such translocation was significantly inhibited by TPEN (Fig. 8).


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Fig. 6.   Inhibition of poly(ADP-ribose) polymerase (PARP) does not affect tBH-induced necrosis. tBH induced concentration-dependent toxicity in SPAECs, which was reversible with TPEN (10 µM). Pretreatment of SPAECs with 1 of 2 PARP inhibitors, 20 µM 4-amino-1,8-naphthalimide (ANI) or 4 mM 3-aminobenzamide (3-AB), only affected tBH-induced necrosis near the threshold for cell death.



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Fig. 7.   Protein kinase (PK) C inhibitors reduce tBH-induced toxicity in SPAECs. Coadministration of 10 µM bisindolylmaleimide (GFX) or 200 nM staurosporine (STP) each significantly inhibited tBH-induced toxicity, whereas 100 nM Gö-6976 and a low concentration (300 nM) of GFX did not protect SPAECs against tBH.



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Fig. 8.   PKC activity in SPAECs. PKC activity is expressed as percent distribution between cytosolic and membrane fractions. PKC translocated from cytosolic fractions to membrane fractions after exposure to 100 µM tBH (left) and 10 µM TPEN (right) inhibited tBH-induced PKC activation.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Zinc is the second most abundant intracellular metal after iron. Perhaps secondary to its biophysical characteristics (promoting favorable aspects of protein folding while itself being redox inert), zinc appears to be involved in a myriad of biological processes including apoptosis (34) and/or necrosis (4, 20). A recent study (35) of the inhibitory role of labile zinc in oxidant-mediated apoptosis of epithelial cells is the first report of the role of zinc in apoptosis within the lung. The contribution of elevated intracellular zinc to necrotic cell death has largely been limited to cells of the central nervous system (19-21). Although a dual role for zinc in affecting cell death was recently reported in thymocytes (36), the contribution of zinc homeostasis to pulmonary endothelial cell injury has not been reported. In the present study, we show that like many other cell types including systemic endothelial cells (11, 25, 32-34), the chelation of zinc enhances LPS-induced apoptosis in SPAECs, likely via secondary effects on the functioning of zinc-containing enzymes or proteins. Alternatively, zinc can contribute to at least one model (e.g., tBH) of necrotic death in SPAECs, and this appears to be secondary to an association between elevated intracellular zinc and PKC activation.

Zinc Homeostasis in SPAECs

Although total intracellular zinc concentrations are in the millimolar range, concentrations of free zinc in most cells are several orders of magnitude less. Nonetheless, it is the labile pool of zinc that appears to be critical in affecting various cellular processes. Quantitation of such labile pools remains challenging, but some progress has been made with sulfonamidoquinoline-based ultraviolet-excitable zinc fluorophores (26, 38). Incubation of SPAECs with one such probe, Zinquin, resulted in an appearance of fluorescence consistent with vesicular concentrations of labile zinc (Fig. 1A). The sensitivity of this pool to elevations in intracellular zinc secondary to the addition of zinc and an ionophore, pyrithione, to the medium (Fig. 1B) are also suggestive that Zinquin fluorescence is reporting changes in labile zinc. The fact that TPEN, a strong chelator, can compete with this pool is further evidence that Zinquin is responding to changes in physiologically relevant levels of labile zinc (Fig. 1B). The currently available Zinquin derivatives are not suitable for ratiometric measurements, and thus quantitation of labile zinc is precluded. It is possible that the future generation of zinc fluorophores will become available, with isobestic features compatible with quantitation (26). As noted above, despite its current limitations (relatively low affinity for zinc and lack of isosbestic spectral properties), Zinquin has been useful to reveal a labile pool of zinc with a potentially important subcellular distribution in cultured airway epithelium (35) and in rat aortic endothelial cells (1).

Zinc Inhibits Apoptosis in SPAECs

Hennig and colleagues (15-17), Meerarani et al. (25), and others (32, 33) have shown that culturing systemic endothelial cells in zinc-depleted medium or exposing such cells to TPEN enhances their sensitivity to various proapoptotic stimuli. In the present study, TPEN (>10 µM) by itself produced a concentration-dependent increase in spontaneous apoptosis in SPAECs (Fig. 3). In subthreshold concentrations (e.g., 10 µM), TPEN increased the level of LPS-induced apoptosis. Accordingly, this is the first demonstration of an antiapoptotic effect of zinc in pulmonary endothelial cells. Nonetheless, because zinc was one of the first antiapoptotic molecules described, these results are somewhat predictable. Initially, it was theorized that zinc inhibited apoptosis by suppressing the Ca/Mg endonuclease responsible for the laddering pattern of DNA oftentimes associated with apoptosis (5). A more recent study (30) suggested that caspase-3 is inhibited by low concentrations of zinc. In this regard, it is noteworthy that Ceneviva et al. (2) initially identified the central role of caspase-3 in LPS-induced apoptosis in SPAECs. It is highly likely that there is another zinc-sensitive step in apoptosis in these cells. For instance, in other cells, the ratio of Bcl-2 to Bax-like proteins (11) is increased in response to zinc, thereby potentially reducing apoptosis in response to a variety of stimuli.

Zinc Contributes to tBH-Induced Necrosis in SPAECs

Zinc has been extensively studied as a pronecrotic molecule in the excitotoxicity of neurons (4, 9, 20) and necrotic death of cultured cerebral endothelial cells (19), but its role outside the central nervous system has received considerably less attention. In this regard, we note that exposing SPAECs to excess zinc with pyrithione results in an increase in labile zinc (Fig. 1B) that is associated with a TPEN-sensitive decrease in cell survival (Fig. 2). Although oxidative stress is a central feature of many forms of apoptosis, in SPAECs, the prooxidative agent tBH (2) results primarily in necrosis. tBH-induced toxicity was sensitive to TPEN (Fig. 5). This is in contrast to H2O2-induced apoptosis in cultured airway epithelium in which TPEN enhanced the apoptotic response (35). Thus zinc has a dual role in SPAECs much like that described in lymphocytes (36) in that it can inhibit apoptosis or contribute to necrosis in the same cell type depending on the injurious stimuli and the respective process of cell death that ensues.

PKC But Not PARP Activation Contributes to tBH-Induced Necrosis in SPAECs

A recent report (36) noted the association between nitrosative stress, necrotic cell death in lymphocytes, and poly(ADP-ribose) synthetase activation. PARP is a zinc-dependent enzyme that maintains genomic integrity and facilitates DNA repair. Activation of PARP has been shown to contribute to necrotic cell death in a number of experimental models, perhaps by depleting its substrate beta -nicotinamide adenine dinucleotide of ATP (14). Although TPEN inhibited PARP activation in competent lymphocytes and protected them against peroxynitrite-induced necrosis, such protection could only partially be ascribed to PARP inhibition because TPEN was also effective in lymphocytes from PARP-deficient mice (36). The limited inhibition of either 3-AB or ANI (Fig. 6) on tBH toxicity in SPAECs suggests that the protective effect of TPEN may be PARP independent. This possibility was raised regarding tBH toxicity in rat hepatocytes (23).

An alternative zinc-dependent family of enzymes that may be involved in oxidative stress and cellular toxicity is PKC (27). Oxidative stress is thought to activate PKC by modifying zinc thiolate clusters of the regulatory domain of the NH2 terminus, thereby relieving autoinhibition and facilitating cofactor independent PKC activation (13). This general prediction may underlie the translocation of PKC activity after tBH in SPAECs in our study (Fig. 8). It is less clear how TPEN inhibited such translocation (Fig. 8), although it is pertinent to the current study that oxidation of PKC results in zinc release from the enzyme (22). In this regard, the cytoprotection of TPEN against tBH-induced necrosis (Fig. 5) is mimicked to a certain degree by inhibitors of PKC (Fig. 7), suggesting that zinc binding to PKC may be another cytoprotective pathway toward inhibition of tBH-mediated necrosis in SPAECs.

There are more than 12 PKC isozymes that can be simply categorized into two classes: conventional PKCs (alpha , beta , and gamma ) that are calcium dependent and nonconventional PKCs that are calcium independent. Nonconventional PKCs include three subclasses: 1) novel PKCs (delta , epsilon , eta , and sigma ), 2) atypical PKCs (zeta , iota , and lambda ), and 3) PKC-µ. It is well known that STP is a broad PKC inhibitor, whereas Gö-6976 only selectively inhibits calcium-dependent PKC isozymes (conventional PKC). GFX is sensitive to conventional isozymes but also inhibits nonconventional PKCs at high concentrations. Our pharmacological studies suggest that tBH-induced cytotoxicity may be related to the activation of nonconventional PKCs.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-32154 and HL-65697 and National Institute of General Medical Sciences Grant GM-53789.


    FOOTNOTES

Z.-L. Tang and C. M. St. Croix are recipients of Postdoctoral Fellowships from the American Heart Association.

Address for reprint requests and other correspondence: B. R. Pitt, Dept. of Environmental and Occupational Health, The Graduate School of Public Health, Univ. of Pittsburgh, 260 Kappa Drive, Pittsburgh, PA 15238 (E-mail: Brucep{at}pitt.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 December 2000; accepted in final form 15 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 281(1):L243-L249
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