Improved spatial resolution of matrix-assisted laser desorption/ionization imaging of lipids in the brain by alkylated derivatives of 2,5-dihydroxybenzoic acid.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) is one of the major techniques for mass spectrometry imaging (MSI) of biological systems along with secondary-ion mass spectrometry (SIMS) and desorption electrospray mass spectrometry (DESI). The inherent variability of MALDI-MSI signals within intact tissues is related to the heterogeneity of both the sample surface and the matrix crystallization. To circumvent some of these limitations of MALDI-MSI, we have developed improved matrices for lipid analysis based on structural modification of the commonly used matrix 2,5-dihydroxybenzoic acid (DHB). METHODS: We have synthesized DHB containing -C6H13 and -C12H25 alkyl chains and applied these matrices to rat brain using a capillary sprayer. We utilized a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer to analyze lipid extracts and tissue sections, and examined these sections with polarized light microscopy and differential interference contrast microscopy. RESULTS: O-alkylation of DHB yields matrices, which, when applied to brain sections, follow a trend of phase transition from crystals to an oily layer in the sequence DHB → DHB-C6H13 → DHB-C12H25 . MALDI-MSI images acquired with DHB-C12H25 exhibited a considerably higher density of lipids than DHB. CONCLUSIONS: Comparative experiments with DHB and DHB-C12H25 are presented, which indicate that the latter matrix affords higher lateral resolution than the former.

Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein.

We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn(2+), plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn(2+) by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn(2+)-specific fluorophore Zinquin that were rapidly reversed by the Zn(2+) chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn(2+) in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn(2+) homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn(2+)-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn(2+) that were reversed by Zn(2+) supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn(2+) release from thiolate clusters. This effect was restored by Zn(2+) supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn(2+) and suggest that the ratio of MT to T can regulate Zn(2+) homeostasis in response to nitrosative stress.

Signal transduction pathways of IL-1beta-mediated iNOS in pulmonary vascular smooth muscle cells.

Interleukin (IL)-1beta is an important early mediator of inflammation in pulmonary artery smooth muscle cells. We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1beta-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction. In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1beta induction of iNOS. Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1beta activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways. The former two pathways were not associated with IL-1beta-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression. These data suggest that a broad intracellular signaling response to IL-1beta in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways.

Role of zinc in pulmonary endothelial cell response to oxidative stress.

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 microM), 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 microM) 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.

Identification of respiratory complexes I and III as mitochondrial sites of damage following exposure to ionizing radiation and nitric oxide.

In 32D cl 3 hematopoietic progenitor cells, the overexpression of manganese superoxide dismutase (MnSOD, SOD2), the enzyme normally found in mitochondria, protects against the damaging effects of ionizing radiation. In the presence of a nitric oxide donor, which exacerbates the damage, inhibition of mitochondrial function can be demonstrated to be associated with respiratory complexes I (NADH dehydrogenase) and III (cytochrome c reductase), but not II (succinate dehydrogenase), IV (cytochrome c oxidase), or V (ATP synthase). The same pattern of inhibition is observed in the case of isolated bovine heart mitochondria exposed to ionizing radiation and the nitric oxide donor. The addition of authentic peroxynitrite (ONO2(-)) to isolated mitochondria also results in damage to complexes I and III (but not II, IV, and V), as shown by assays of electron-transfer activities and electron paramagnetic resonance (EPR) spectroscopic measurements, suggesting ONO2(-) to be responsible for most of the observed radiation damage in both the cultured cell lines and isolated mitochondria. It is argued that, in general, production of ONO2(-) is an important contributor to radiation damage in biological systems and the implications of these findings in relation to possible mechanisms of oxidant-linked apoptosis are briefly considered.

Effect of sodium arsenite on iNOS expression and vascular hyporeactivity associated with cecal ligation and puncture in the rat.

Induction of the heat shock response protects animals from either endotoxemia or peritonitis. In endotoxemia, heat shock protein (HSP) induction is associated with reversal of vascular hyporeactivity and inhibition of iNOS expression. Recent studies suggest differences in the inflammatory mechanisms during endotoxemia and peritonitis animal models and their response to therapeutic interventions. We therefore studied the effect of the HSP inducer sodium arsenite (SA) on vascular reactivity and iNOS expression in rats undergoing cecal ligation and puncture (CLP). CLP resulted in suppression of the pressor effect of norepinephrine (NE) in vivo (measured by changes in blood pressure in response to NE boluses) and ex vivo (changes in contraction force in isolated mesenteric arteries in response to NE concentrations), and in the expression of iNOS protein. Pretreatment of the rats with SA resulted in reversal of CLP-induced vascular hyporeactivity in vivo and ex vivo, and inhibition of iNOS expression after 22 h. SA pretreatment improved 7-day survival after CLP from 18.2% to 70% (P < 0.005). Glucocorticoid receptor inhibition did not affect the effect of HSP induction on iNOS expression. The similarity of the effect of HSP on vascular reactivity and iNOS expression in two distinct sepsis models suggests that this effect may be clinically important and that a causative relationship between HSP induction, iNOS inhibition, and reversal of vascular reactivity is likely.

Nitric oxide-dependent pro-oxidant and pro-apoptotic effect of metallothioneins in HL-60 cells challenged with cupric nitrilotriacetate.

Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl(2)-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl(2)-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.

Antioxidant and antiapoptotic function of metallothioneins in HL-60 cells challenged with copper nitrilotriacetate.

Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with elevated concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for antioxidant function as effective copper chelators. In the study presented here, we tested this hypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigated antiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partial purification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showed that zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quench redox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress in membrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristic biochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation, and externalization of phosphatidylserine in plasma membranes). In separate experiments, we used lung fibroblasts derived from MT1, MT2 knockout mice (MT(-)(/)(-)) and MT wild-type (MT(+/+)) mice. ZnCl(2) pretreatment resulted in a more than 10-fold induction of MTs in MT(+/+) cells, whereas the MT content in MT(-)(/)(-) cells remained low, at levels approximately 100-fold lower than in their MT wild-type counterparts. MT(-)(/)(-) cells were very sensitive to Cu-NTA and, most importantly, showed no response to ZnCl(2) pretreatment. In contrast, MT(+/+) cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl(2) pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.

Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins.

Little is known about copper transfer from Cu-metallothionein (Cu-MT) to various target proteins, such as apo-SOD, and the potential role of redox mechanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD in a cell-free model system and found that Cu(5)-MT and Cu(10)-MT were able to reconstitute SOD activity only in the presence of a nitric oxide donor, (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (NOC-15). The percentage of reconstitution by Cu(5)-MT and Cu(10)-MT was 34 and 83%, respectively, compared with that reconstituted by free Cu alone. A Cu chelation assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced release of free Cu from Cu(10)-MT but not from Cu(5)-MT. The transfer of Cu from Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generation of ascorbate radicals or hydroxyl radicals as measured by EPR spectroscopy. We found a 70% decrease in the number of 2,2'-dithiodipyridine titratable SH groups on MT after incubation with NOC-15. Overall, our results suggest that Cu-MT could potentially function in a nitric oxide-dependent pathway for the delivery of Cu to apo-SOD in copper-challenged cells.

Lung redox homeostasis: emerging concepts.

This symposium was organized to present some aspects of current research pertaining to lung redox function. Focuses of the symposium were on roles of pulmonary endothelial NADPH oxidase, xanthine oxidase (XO)/xanthine dehydrogenase (XDH), heme oxygenase (HO), transplasma membrane electron transport (TPMET), and the zinc binding protein metallothionein (MT) in the propagation and/or protection of the lung or other organs from oxidative injury. The presentations were chosen to reflect the roles of both intracellular (metallothionein, XO/XDH, and HO) and plasma membrane (NADPH oxidase, XO/XDH, and unidentified TPMET) redox proteins in these processes. Although the lung endothelium was the predominant cell type under consideration, at least some of the proposed mechanisms operate in or affect other cell types and organs as well.

Actin-containing sera from patients with adult respiratory distress syndrome are toxic to sheep pulmonary endothelial cells.

Actin released from damaged cells after a variety of tissue injuries appears to be involved in multiple organ dysfunction syndrome. Under experimental conditions, when the quantity of actin present in plasma is made to exceed the protective capacity of the actin-scavenging mechanism, microembolism and pulmonary vascular angiopathy have been noted in rats. It remains to be determined whether this injury is a result of a direct toxic effect or occurs indirectly via platelet activation or fibrin interactions. We examined the effect of sera from patients with adult respiratory distress syndrome (ARDS), as well as G-actin added to normal serum, on the viability, morphology, and function of cultured sheep pulmonary artery endothelial cells (SPAEC). Both patient sera and normal sera to which actin was added were toxic in the cell culture model; this toxicity could be abrogated, at least partially, by preincubation with gelsolin, which is known to complex with actin. A significant portion of the toxicity of sera from patients with ARDS was sensitive to heat (56 degrees C), suggesting an important role of complement. Sera from patients with ARDS were shown to contain filaments of F-actin by immunoblot and rhodamine phalloidin staining after ultracentrifugation. Thus, saturation of the actin-scavenging system by addition of exogenous G-actin to plasma produces direct pulmonary endothelial cell injury. Furthermore, plasma from patients with ARDS secondary to bacterial pneumonia is toxic to SPAEC, and a small but significant contributory role of actin is apparent in these studies.

Induced nitric oxide inhibits IL-6-induced stat3 activation and type II acute phase mRNA expression.

Inducible nitric oxide synthase (iNOS) can be coexpressed with acute phase reactants in hepatocytes; however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOS-derived nitric oxide (NO) attenuates the acute phase response by inhibiting IL-6-enhanced Stat3 DNA-binding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS (AdiNOS), and Stat3 DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA). EMSAs demonstrated that AdiNOS inhibits IL-6-induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMA). The induction of beta-fibrinogen mRNA by IL-6, a Stat3 dependent process, is attenuated in AdiNOS-transduced cells and partially reversed by L-NMA. Thus, iNOS overexpression suppresses IL-6-induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO down-regulates the acute phase response.

Metallothionein, nitric oxide and zinc homeostasis in vascular endothelial cells.

Recent in vitro studies suggest that the oxidoreductive capacity of metal thiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothesis in intact endothelial cells, focusing on the contributory role of the important redox signaling molecule, nitric oxide. Microspectrofluorometry with Zinquin revealed that the exposure of cultured sheep pulmonary artery endothelial cells to S-nitrosocysteine resulted in the release of N, N,N',N'-tetrakis(2. pyridylmethyl)ethylendiamine (TPEN) chelatable zinc. Cultured sheep pulmonary artery endothelial cells were transfected with a plasmid expression vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. The exposure of cultured sheep pulmonary artery endothelial cells transfected with this chimera to nitric oxide donors or to agents that increased cytoplasmic Ca(2+) via endogenously generated nitric oxide decreased the efficiency of fluorescence resonance energy transfer in a manner consistent with the release of metal (Zn) from MT. A physiological role for this interaction in intact tissue was supported by the lack of myogenic reflex in resistance arteries of MT knockout mice unless endogenous nitric oxide synthesis was blocked. These data suggest an important role for metal thiolate clusters of MT in nitric oxide signaling in the vascular wall.

Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model.

Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P < 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself.

Pulmonary arterial smooth muscle cells modulate cytokine- and LPS-induced cytotoxicity in endothelial cells.

Cytokines and lipopolysaccharide (LPS) are known to be injurious to vascular endothelial cells (ECs), but the influence of adjacent vascular smooth muscle cells (SMCs) on this injury is unknown. Exposure of cultured rat (RPMECs) or human (HPMECs) pulmonary microvascular ECs on tissue culture plastic to a mixture of cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) and LPS (cytomix) resulted in a significant increase in (51)Cr release to 35-40%. When unstimulated RPMECs were cocultured with cytomix-pretreated rat pulmonary microvascular SMCs (RPMSMCs) there was an increase in (51)Cr release to 8.4%, which was nitric oxide dependent. However, when RPMECs or HPMECs were stimulated in direct contact with their respective SMCs, rather than a further increase in cytomix-induced injury (e.g., >35-40%), (51)Cr release decreased to <10%. This cytoprotection was fully reproduced with fixed RPMSMCs, and partially reproduced by plating HPMECs on gelatin. These data show that the direct toxicity of a cytokine and endotoxin mixture on cultured ECs can be reduced by contact with vascular smooth muscle.

Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody.

To achieve efficient systemic gene delivery to the lung with minimal toxicity, a vector was developed by chemically conjugating a cationic polymer, polyethylenimine (PEI), with anti-platelet endothelial cell adhesion molecule (PECAM) antibody (Ab). Transfection of mouse lung endothelial cells with a plasmid expression vector with cDNA to luciferase (pCMVL) complexed with anti-PECAM Ab-PEI conjugate was more efficient than that with PEI-pCMVL complexes. Furthermore, the anti-PECAM Ab-PEI conjugate mediated efficient transfection at lower charge plus-to-minus ratios. Conjugation of PEI with a control IgG (hamster IgG) did not enhance transfection of mouse lung endothelial cells, suggesting that the cellular uptake of anti-PECAM Ab-PEI-DNA complexes and subsequent gene expression were governed by a receptor-mediated process rather than by a nonspecific charge interaction. Conjugation of PEI with anti-PECAM Ab also led to significant improvement in lung gene transfer to intact mice after intravenous administration. The increase in lung transfection was associated with a decrease compared with PEI-pCMVL with respect to circulating proinflammatory cytokine (tumor necrosis factor-alpha) levels. These results indicate that targeted gene delivery to the lung endothelium is an effective strategy to enhance gene delivery to the pulmonary circulation while simultaneously reducing toxicity.

Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein.

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca(2+) act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT(-/-)) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.

The peroxynitrite reductase activity of cytochrome c oxidase involves a two-electron redox reaction at the heme a(3)-Cu(B) site.

Fully and partially reduced forms of isolated bovine cytochrome c oxidase undergo a two-electron oxidation-reduction process with added peroxynitrite, leading to catalytic oxidation of ferrocytochrome c to ferricytochrome c. The other major reaction product is nitrite ion, 86% of the added peroxynitrite being measurably converted to this species. The reaction is inhibited in the presence of cyanide, implicating the heme a(3)-Cu(B) binuclear pair as the active site. Moreover, provided peroxynitrite is not added to excess, the reductase activity of the enzyme toward this oxidant efficiently protects other protein and detergent molecules in vitro from nitration of tyrosine residues and oxidative damage. If the enzyme is exposed to approximately 10(2)-fold excesses of peroxynitrite, then significant irreversible loss of electron transfer activity results, and the heme a(3)-Cu(B) binuclear pair no longer undergo a characteristic carbon monoxide-driven reduction. The accompanying rather small changes in the observed electronic absorption spectrum are suggestive of a modification in the vicinity of one or both hemes but probably not to the cofactors themselves.