Cytoprotective effects of albumin, nitrosated or reduced, in cultured rat pulmonary vascular cells

Abstract

S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H₂S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to N^G-nitro-l-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H₂S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H₂S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an N^G-nitro-l-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H₂S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS.

s-nitrosoalbumin (sno-alb) was recently shown to be effective in mitigating LPS-induced acute lung injury (ALI) in intact rats (12) and hypoxia-regenerated ALI in sickle cell disease model in mice (6). Pretreatment of intact pigs with polynitrosated bovine albumin decreased LPS-induced cardiopulmonary dysfunction (9). These reports were a logical extension of initial observations demonstrating a cytoprotective role of SNO-Alb in ischemia-reperfusion injury of skeletal muscle (11) and heart (20) and hemorrhagic shock-induced changes in liver (1).

Although the above studies provide a compelling preclinical interest in the uniqueness and advantages of SNO-Alb over other nitric oxide (NO) donors, including low-molecular-weight S-nitrosothiols (RSNO) and authentic NO, itself, in sepsis or shock, most of the efforts 1) were directed toward cytoprotection of microcirculation in the context of platelet aggregation or leukocyte-endothelial cell adhesion (1); 2) generally discounted a contribution of reduced albumin (12); and 3) overall formed the basis of new questions with respect to cellular mechanisms by which SNO-Alb was protective. In the present study, we examined the potential of SNO-Alb to affect necrotic, as well as apoptotic, pathways in both pulmonary endothelium and smooth muscle. Our laboratory previously reported (31) that SNO-Alb was indeed capable of transnitrosating targets in intact pulmonary endothelium, and this effect was sensitive to inhibition of cell surface protein disulfide isomerase, as originally shown by Ramachandran et al. (19). This raises the possibility that (reduced) albumin formed by denitrosation of SNO-Alb can contribute to the overall effect under study (e.g., cytoprotection), perhaps secondary to activation of gp60 in pulmonary endothelial cells (25), accounting for albumin transcytosis, as well as activation of endothelial NO synthase (eNOS, NOSIII) (16). We now show that pulmonary smooth muscle cells also express gp60 [ and neuronal NO synthase (nNOS) ] and are also capable of transporting albumin. The N^G-nitro-l-arginine methyl ester (l-NAME)-sensitive component [ secondary to activation of NO synthase (NOS) via reduced albumin (SH-Alb) ] of SNO-Alb in pulmonary smooth muscle and endothelium underscores the contributory role of albumin-mediated NOS activation in such cytoprotection. Insight into potential cytoprotective mechanisms is provided in hydrogen sulfide (H₂S)-mediated apoptosis of pulmonary smooth muscle, where SNO-Alb interferes with ERK1/2 and p38 activation.

EXPERIMENTAL PROCEDURES

Materials.

All reagents were obtained from Sigma Chemical (St. Louis, MO), unless stated otherwise. alamarBlue was purchased from Invitrogen (Carlsbad, CA). Antibodies raised against phosphorylated-eNOS (Ser¹¹⁷⁹), caspase-3, ERK1/2, phosphorylated-ERK1/2, p38, and phosphorylated-p38 were purchased from Cell Signal Technology (Beverly, MA). eNOS, nNOS, Bcl-2, horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody to gp60 was a kind gift from Dr. Asrar Malik (University Illinois at Chicago). PD-98059 and SB-203580 were purchased from Calbiochem (San Diego, CA). Sodium hydrosulfide (NaHS) solution was freshly prepared on the day of every experiment by mixing the stock solutions of sodium sulfide and hydrochloric acid.

Cell culture.

Rat pulmonary microvascular endothelial cells (RPMEC) were cultured in MCDB-131 complete medium (VEC Technologies, Rensselaer, NY). Rat pulmonary artery smooth muscle cells (RPASMC) were isolated as previously described (18) and grown in DMEM (low glucose) with 10% FBS, 1,000 U/ml penicillin, and 1 mg/ml streptomycin. All cells were incubated at 37°C in a humidified atmosphere of air and 5% CO₂.

Preparation of SNO-Alb.

Human albumin [ human serum albumin (HSA) ] (50 mg/ml) was initially treated for 45–60 min at room temperature in the dark with 1 mM dithiothreitol in PBS supplemented with 640 μM diethylenetriaminepentaacetic acid to reduce the cysteine-34 thiol group (HSA-SH). After extensive washing with Chelex-treated PBS (Sigma, pH 7.4), the mixture was centrifuged (14,000 g, 25 min, 4°C) and fractionated with 30,000-Da cutoff filter (Millipore, MA). Albumin concentration was then determined by its absorbance at 279 nm (0.609 absorbance units per 1 mg/ml) and sulfhydryl groups at 412 nm (ε = 13.6 mmol·l⁻¹·cm⁻¹) by using the Ellman's reagent. Nitrosylated human albumin (SNO-Alb) was prepared using transnitrosylation reaction and S-nitroso-glutathione (GSNO) as a donor. GSNO was prepared by the reaction of acidified NaNO₂ with glutathione. Briefly, 100 mM glutathione was mixed with 100 mM NaNO₂ in 200 mM HCl at room temperature. pH was adjusted to 7.4 with NaOH, and the solution was used for S-transnitrosylation of HSA-SH. For S-transnitrosylation, reduced HSA (SH-Alb) was mixed with 50 times excess of GSNO, and the solution was incubated in the dark for 20 min at room temperature. To prevent S-glutathiolation of SH-Alb, the reaction was stopped with N-ethylmaleimide (4:1, mole N-ethylmaleimide to mole HSA-SH), and the samples were extensively washed, as described above, to remove low-molecular-weight components. The resultant SNO-Alb preparation was checked for the level of residual SH-Alb, as well as SNO-Alb using Ellman's reagent and 4,5-diaminofluorescein assays, respectively. The molar ratio of reduced sulfhydryls to total albumin was <0.01, and the molar ratio of SNO-Alb to total albumin was ∼0.55 (2).

Cell viability assay.

Cell viability was determined using alamarBlue assay, according to the manufacturer's instruction. The assay is based on the detection of metabolic activity in living cells using a redox indicator that changes from an oxidized (blue) to a reduced (red) form. The intensity of the red color is proportional to the viability of the cells. Data were obtained by reading fluorescence with excitation wavelength of 540–570 nm and emission wavelength of 580–610 nm.

Lactate dehydrogenase release assay and caspase activity assay.

Lactate dehydrogenase (LDH) release was measured using CytoTox-ONE homogeneous membrane integrity assay (Promega). Caspase activity was determined using Caspase-Glo-3/7 assay (Promega) and immunoblot with caspase-3 antibody to determine the cleavage of caspase-3.

Flow cytometry.

Cells were stained with annexin V-FITC and propidium iodide (PI) and then evaluated for necrosis or apoptosis by flow cytometry, according to the manufacturer's protocol (Biovision, Mountain View, CA). Briefly, cells were washed with PBS and trypsinized and stained with 5 μl of annexin V and 5 μl of PI (50 μg/ml) in 400 μl of binding buffer containing 10 mM HEPES (pH 7.4), 140 mM NaOH, and 2.5 mM CaCl₂ for 5 min in the dark before flow cytometry analysis. Ten thousand events were collected on a FACScan flow cytometer supplied with CellQuest software.

Immunoblot.

Cells were washed three times with ice-cold PBS and then lysed on ice in SDS sample buffer (62.5 mM Tris·HCl, pH 6.8, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% wt/vol bromophenol blue). The extracts were sonicated briefly, boiled for 5 min, then centrifuged at 14,000 rpm for 5 min. Equivalent amounts of protein were separated by electrophoresis using 10% SDS-PAGE gels. The proteins were then transferred to polyvinylidene difluoride membrane (Invitrogen, CA) and blocked with PBS containing 5% nonfat milk for 1 h at RT. The membranes were incubated with primary antibody overnight at 4°C; horseradish peroxidase-conjugated secondary antibody was used for visualization by chemiluminescence using enhanced chemiluminescence reagent (Perkin-Elmer Life Science). Blots were stripped with stripping buffer (Pierce) and reprobed. Densitometric analysis was performed using Image J software from the National Institutes of Health.

eNOS phosphorylation.

RPMEC were grown on six-well plates to 90% confluence, subsequently serum deprived for 2 h in serum-free MCDB-131 medium, and then treated with SNO-Alb (20 μM), SH-Alb (20 μM), and anti-gp60 polyclonal antibody (10 μg/ml) in serum-free HBSS (Invitrogen) for 30 min. Cell lysates were collected for immunoblot analysis.

Uptake of reduced albumin.

RPMEC and RPASMC were plated into Lab-Tek II four-well chamber slides (Nalge Nunc) and grown until 90% confluent. After being washed with HBSS, cells were incubated with HBSS-BSA (5 mg/ml) at 37°C for 10 min and then exposed to 50 μg/ml of Alexa 488-BSA (Invitrogen) for 20 min at 37°C. Cells were fixed with 4% paraformaldehyde and stained with 4,6-diamidino-2-phenylindole (Invitrogen) to reveal cell nuclei, and then slides were peeled off and sealed with Fluoromount-G (Southern Biotechnology Associates) under coverslips.

Data analysis.

Statistical analyses were performed with one-way ANOVA within groups, followed by pairwise multiple-comparison procedure (Holm-Sidak method). Data are presented as means ± SD. Statistical significance was defined as P < 0.05.

RESULTS

Tert-butyl hydroperoxide-induced necrosis rather than apoptosis in RPMEC.

Tert-butyl hydroperoxide (tBH) caused a concentration-dependent toxicity in RPMEC (Fig. 1A) as determined by alamarBlue. There was no apparent toxicity of tBH (0–100 μM) in RPASMC (data not shown). Toxicity to tBH in RPMEC was likely due to necrosis as there was 1) a concentration-dependent (Fig. 1B) increase in PI-positive cells by fluorescence-activated cell sorting (Fig. 1C); 2) a concentration-dependent increase in LDH release that approached maximum release from standard lysis protocol (Fig. 1D); and 3) no change in caspase-3/7 activity in an otherwise staurosporine-sensitive RPMEC (Fig. 1E) or immunostaining of M30 (data not shown). Accordingly, tBH was associated with necrotic, but not apoptotic, cell death in RPMEC.

Fig. 1.

Tert-butyl hydroperoxide (tBH) results in necrosis rather than apoptosis in rat pulmonary microvascular endothelial cells (RPMEC). A: tBH treatment (0–100 μM) in serum-free HBSS for 6 h induced concentration-dependent cytotoxicity in RPMEC. Cell viability was assessed by alamarBlue assay. B: RPMEC were treated with indicated concentrations of tBH for 6 h, then stained with annexin V/propidium iodide (PI), and flow cytometry was performed. Necrotic cells are PI positive, whereas apoptotic cells are annexin V positive. C: representative flow cytometry dot blots of necrosis induced by tBH in RPMEC are shown. The percentages in the top left quadrant (annexin V negative, PI positive) and the top right quadrant (annexin V and PI positive) correspond to necrotic cells. D: a significant increase in lactate dehydrogenase (LDH) release occurred after cells were exposed to tBH (0–80 μM) in serum-free HBSS for 6 h. Lysis buffer was used as a positive control to induce necrosis in RPMEC. E: no increase in caspase-3/7 activation occurred after cells were exposed to tBH (0–80 μM) in serum-free HBSS for 6 h, according to caspase-Glo-3/7 assay. Treatment of staurosporine (Stauro; 1 μM) for 4 h was used as a positive control to induce apoptosis in RPMEC. Values are means ± SD; 3 independent experiments were performed. *P < 0.05 compared with tBH (0 μM) treatment. RFU, relative fluorescence units; RLU, relative light units.

Effect of SNO-Alb on tBH-induced cytotoxicity in RPMEC.

We then examined the effect of 1-h pretreatment with SNO-Alb (0–20 μM) on tBH (40 μM)-induced toxicity in RPMEC and noticed a statistically significant inhibition of tBH cytotoxicity in a concentration-dependent manner, as ascertained by alamarBlue (Fig. 2A). SNO-Alb (0–20 μM), by itself, was without measureable effect (Fig. 2A). We then evaluated the contributing role of endogenous NO production in the cytoprotective effect of SNO-Alb and noted that a statistically significant component of SNO-Alb cytoprotection was l-NAME sensitive in RPMEC (Fig. 2B). A likely explanation is that denitrosation of SNO-Alb occurred, and that the reduced form (SH-Alb) activated eNOS (16). Such denitrosation was not likely to have occurred in the extracellular space in a readily detectable fashion under the conditions of our experiment, as there was no increase in nitrite in the medium of SNO-Alb-conditioned RPMEC (Fig. 2C). We noted, however, that SH-Alb, itself, was cytoprotective in an l-NAME-sensitive fashion in RPMEC (Fig. 2B), while rat IgG had no protective effect on tBH-induced cytotoxicity. We then noted that RPMEC expressed gp60 and eNOS (Fig. 3A) and were capable of transporting fluorescent-labeled (Alexa 488) reduced albumin (Fig. 3B). Furthermore, exposure of RPMEC to SNO-Alb or SH-Alb mimicked activation of eNOS observed via gp60 activation by gp60 antibody (Fig. 3C).

Fig. 2.

S-nitrosoalbumin (SNO-Alb) has a protective effect on tBH-induced cytotoxicity in RPMEC. A: SNO-Alb protected RPMEC against tBH-induced cytotoxicity in a concentration-dependent manner. RPMEC were pretreated with indicated concentrations of SNO-Alb (0–20 μM) 1 h before tBH treatment (40 μM) for 6 h in serum-free HBSS. Cell viability was assessed by alamarBlue assay. B: SNO-Alb and reduced albumin (SH-Alb) reduced sensitivity of RPMEC to tBH in an N^G-nitro-l-arginine methyl ester (l-NAME)-sensitive fashion. RPMEC were treated with 1 mM l-NAME 1 h before the addition of tBH (40 μM), SNO-Alb (20 μM), and SH-Alb (20 μM). Cell viability was determined by alamarBlue assay. C: time course of nitrite formation in culture medium after incubation of cells with SNO-Alb (20 μM) for 1 h. Nitrite quantitation was performed by the Griess reaction, and the concentration was calculated by standard curve method. Values are means ± SD; 3 independent experiments were performed. P < 0.05 compared with *control, #tBH treatment, †tBH and SNO-Alb treatment, and ‡tBH and SH-Alb treatment.

Fig. 3.

Endocytosis of SH-Alb and activation of endothelial nitric oxide synthase (eNOS) in RPMEC. A: expression of gp60 and eNOS in RPMEC. Three subcultures of RPMEC were tested for gp60 and eNOS expression by using Western blotting. B: fluorescently labeled (Alexa 488) reduced albumin uptake in RPMEC. Cells were incubated with HBSS-BSA (5 mg/ml) at 37°C for 10 min, and then 50 μg/ml of Alexa 488-BSA were added for 20 min at 37°C. Cells were fixed and stained with 4,6-diamidino-2-phenylindole. Representative images of stained cells are shown by fluorescent microscopy. C: eNOS phosphorylation induced by SNO-Alb (20 μM), SH-Alb (20 μM), and gp60 antibody (10 μg/ml) in RPMEC. RPMEC were serum deprived for 2 h and then treated with indicated concentration of SNO-Alb, SH-Alb, and gp60 antibody for 30 min. Results in the representative Western blot were samples, as labeled, that were cut from a single gel and rearranged for clarity. Values are means ± SD. Results shown are representative of 3 experiments. Densitometric analysis was performed using Image J software from the NIH. *P < 0.05 compared with control. P-eNOS, phosphorylated eNOS.

H₂S-induced apoptosis rather than necrosis in RPASMC.

In screening the toxicity of other conditions in RPASMC, we noted that these cells were sensitive to NaHS, whereas RPMEC were resistant over a comparable large concentration range (Fig. 4A). In investigating the pathway of cell death after exposing RPASMC to 0.5 mM NaHS, we noted activation of caspase-3 (Fig. 4D) at 4 h without significant release of LDH (Fig. 4B), as well as early and late apoptosis detection in flow cytometry (Fig. 4C). Accordingly, H₂S was associated with apoptotic, but not necrotic, cell death in RPASMC.

Fig. 4.

Sodium hydrosulfide (NaHS) results in apoptosis rather than necrosis in rat pulmonary artery smooth muscle cells (RPASMC). A: effects of NaHS on cell viability in RPMEC and RPASMC. Cells were serum starved for 3 h and then exposed to indicated concentrations of NaHS in serum-free HBSS for 12 h. Cell viability was measured by alamarBlue assay. NaHS caused a concentration-dependent cell cytotoxicity in RPASMC, but not in RPMEC. B: no significant increase in LDH release occurred after RPASMC were exposed to indicated concentrations of NaHS in serum-free HBSS for 12 h. Lysis buffer was used as a positive control to induce necrosis in cells. C: representative flow cytometry dot blots of apoptosis induced by NaHS (0.5 mM) in RPASMC are shown. The percentages in the bottom right quadrant (annexin V positive, PI negative) correspond to early apoptotic cells, and the top right quadrant (annexin V and PI positive) correspond to late apoptotic cells. Treatment of staurosporine (0.3 μM) for 12 h was used as a positive control to induce apoptosis in RPASMC. D: caspase-3 was activated and subsequently released as cleaved caspase-3, starting at 4 h after treatment with 0.5 mM NaHS in RPASMC. Values are means ± SD; 3 independent experiments were performed. *P < 0.05 compared with NaHS (0 mM) treatment.

Effect of SNO-Alb on H₂S-induced cytotoxicity in RPASMC.

Pretreatment of RPASMC with 20 μM SNO-Alb, which did not affect cell viability in itself, significantly reduced the toxicity of NaHS, as determined by alamarBlue (Fig. 5A). We confirmed this effect using Trypan blue exclusion by showing that 0.5 mM NaHS reduced cell viability to 56% of control, and this toxicity was completely inhibited by pretreatment with 20 μM SNO-Alb (data not shown). In further studies using alamarBlue and a high-dose (1.0 mM) NaHS, we noted that the protective effect of SNO-Alb (20 μM) was sensitive to l-NAME (1 mM) and could be mimicked by 20 μM SH-Alb, and this effect was also sensitive to l-NAME (Fig. 5B). Although lower doses of NaHS resulted in toxicity that was sensitive to SNO-Alb, there was no significant l-NAME-sensitive component. RPASMC, like endothelial cells, transported Alexa 488-labeled reduced albumin (Fig. 5C). In addition (Fig. 5D), they expressed gp60 and nNOS (but not eNOS) that may account for the l-NAME-sensitive component of SNO-Alb and SH-Alb cytoprotection. Furthermore, early (by 2 h) in the course of NaHS-induced apoptosis, we noted activation of ERK1/2 (Fig. 6A) and a decrease in Bcl-2 expression (Fig. 6B). We then showed that NaHS activated not only ERK1/2 (Fig. 6C), but also p38 (Fig. 6D), and that phosphorylation of both of these kinases was decreased in a concentration-dependent fashion with pretreatment of SNO-Alb (5–20 μM) 1 h before the addition of 0.5 mM NaHS. The protective effect of SNO-Alb (20 μM) could be mimicked by pretreatment with ERK inhibitor PD-98059 (20 μM), or p38 inhibitor SB-203580 (10 μM) (Fig. 6E), suggesting the importance of MAPK kinases in mediating the toxicity of NaHS in RPASMC and consistent with the likelihood that protective effect of SNO-Alb may reside in its impact in this complex signaling pathway.

Fig. 5.

SNO-Alb has a protective effect on NaHS-induced cytotoxicity in RPASMC. A: pretreatment with SNO-Alb (20 μM) 1 h before addition of NaHS (0.5 mM) attenuated its cytotoxicity, according to alamarBlue assay. B: SNO-Alb and SH-Alb reduced sensitivity of RPASMC to NaHS in an l-NAME-sensitive fashion. RPASMC were treated with 1 mM l-NAME 1 h before addition of NaHS (1 mM), SNO-Alb (20 μM), and SH-Alb (20 μM). Cell viability was determined by alamarBlue assay. C: fluorescently labeled (Alexa 488) SH-Alb uptake in RPASMC. Cells were incubated with HBSS-BSA (5 mg/ml) at 37°C for 10 min, then 50 μg/ml of Alexa 488-BSA were added for 20 min at 37°C. Cells were extensively washed and fixed. Cellular nucleoli were stained with 4,6-diamidino-2-phenylindole. Representative images of stained cells are shown by fluorescent microscopy. D: expression of gp60 and neuronal nitric oxide synthase (nNOS), but no eNOS expression in RPASMC. Three subcultures of RPASMC were tested for gp60, nNOS, and eNOS expression by using Western blotting. Values are means ± SD; 3 independent experiments were performed. P < 0.05 compared with *control, #NaHS treatment, †NaHS and SNO-Alb treatment, and ‡NaHS and SH-Alb treatment.

Fig. 6.

Mechanism of protection of SNO-Alb against NaHS-induced cytotoxicity in RPASMC. A: ERK1/2 was maximally activated at 90 min and 2 h after 0.5 mM NaHS treatment. B: reduction of Bcl-2 expression was noticed starting at 2 h after 0.5 mM NaHS treatment. β-Actin was used as control. Pretreatment with SNO-Alb (5, 10, 20 μM) inhibited ERK1/2 (C) and p38 (D) activation at 2 h after addition of NaHS (0.5 mM) in a concentration-dependent manner. Representative images of 3 independent experiments are shown. Densitometric analyses were performed using Image J software. E: pretreatment with PD-98059 (PD; 20 μM), specific inhibitor of ERK1/2, and SB-203580 (SB; 10 μM), specific inhibitor of p38, mimicked the protective effect of SNO-Alb (20 μM) in RPASMC exposed to NaHS (0.5 mM). Cell viability was assessed by alamarBlue assay. Values are means ± SD; 3 independent experiments were performed. P < 0.05 compared with *control and #NaHS treatment. P-ERK1/2, phosphorylated ERK1/2; P-p38, phosphorylated p38.

DISCUSSION

In the present study, we note that pretreatment of RPMEC and RPASMC with SNO-Alb (Figs. 2A and 5A) reduces their sensitivity to tBH-induced necrosis (Fig. 1) and NaHS-induced apoptosis (Fig. 4), respectively. The disposition of SNO-Alb in the extracellular space of endothelium (31) and vascular smooth muscle (15, 33) is complex and may involve 1) denitrosation in the extracellular space with S-transnitrosation of cysteines and subsequent transport of nitroso-cysteine (15, 33); or 2) denitrosation at plasma membrane via cis protein sulfide deisomerase (19, 31) with subsequent S-transnitrosation outside (19) and/or inside (19, 31) the cell. We report that pulmonary smooth muscle cells, like pulmonary endothelium (Fig. 3A), express gp60 (Fig. 5D) and can transport SH-Alb (Fig. 5C). Such transport appears coupled to activation of NOS in RPMEC and RPASMC, as the protective effect of SNO-Alb was sensitive to l-NAME and mimicked by SH-Alb (Figs. 2B and 5B, respectively). Although the mechanism underlying protection against tBH in pulmonary endothelium remains unclear, it is noteworthy that SNO-Alb appears to protect RPASMC from toxicity (apoptosis) of H₂S by inhibition of activation of MAP kinases (ERK1/2 and p38; Fig. 6).

Several preclinical studies support the therapeutic potential and uniqueness of SNO-Alb to protect and mitigate against microvascular injury, including experimental models of ALI. The original work by Hallstrom and colleagues (11) demonstrated that SNO-Alb could preserve endothelial cell integrity in heart and skeletal muscle in ischemia-reperfusion injury in intact animals and was not associated with a hypotensive effect. Subsequently, a number of investigators have revealed a protective role for SNO-Alb in intact animals in experimental models of ALI, including hypoxia-reoxygenation of sickle cell mice (6) and pretreatment of LPS-exposed pigs (9) and posttreatment of LPS-exposed rats (12). A common experience in this collective body of work is that SNO-Alb, in contrast to other NO donors, including low-molecular-weight thiols, has minimal untoward effects, such as systemic hypotension. SNO-Alb enhances microvascular function in these models by inhibiting platelet aggregation or neutrophil accumulation. An added feature in some studies is the contribution of albumin in the mixture (6) (see below) and an important therapeutic feature that mitigation (delivering SNO-Alb after onset of LPS exposure) is possible (12). SNO-Alb is more stable than other sources of NO and indeed, under various conditions, is considered as a major endogenous plasma reservoir of circulating NO (22, 26).

SNO-Alb and tBH toxicity.

Exposure of RPMEC to tBH, like our laboratory's previous report with cultured sheep pulmonary artery endothelial cells (24), resulted in necrosis without apoptosis (Fig. 1), as determined by alamarBlue (Fig. 1A), PI and fluorescence-activated cell sorting (Fig. 1, B and C), LDH release (Fig. 1D), and lack of change in caspase-3/7 activation (Fig. 1E). SNO-Alb had a concentration-dependent protective effect in RPMEC, as reflected in cell viability measured with alamarBlue (Fig. 2A) or Trypan blue exclusion (data not shown). tBH acts as a membrane-permeant peroxide donor, and a critical role for intracellular iron in mediating its toxicity is well known (8, 17). Although the mechanism by which SNO-Alb mediated protection is unclear, a likely pathway involves nitrosation of heme and nonheme iron, as our laboratory has previously shown for the protective effect of other NO donors (10) and inducible NOS-derived NO (29) in nonendothelial myeloid-like cells. Although we have not identified specific nitrosated targets in rat pulmonary endothelium, our laboratory previously reported the capabilities of SNO-Alb to nitrosate a surrogate fluorescence resonance energy transfer reporter for transnitrosation in rat pulmonary endothelial cells (31), suggesting that such biochemistry is apparent in SNO-Alb-treated RPMEC.

Maniatis et al. (16) described a unique phenomenon in which binding to gp60 (albumin receptor) in cultured pulmonary endothelial cells results in activation of eNOS secondary to caveolae-mediated endocytosis. In our study, it is noteworthy that SH-Alb produced partial protection of RPMEC to tBH in an l-NAME-sensitive fashion, and that SNO-Alb protection partially involved an l-NAME-sensitive pathway (Fig. 2B). The likely role of endogenous NO (e.g., eNOS-derived NO) in cytoprotective effects of SNO-Alb and reduced albumin is also apparent in the ability of SNO-Alb, SH-Alb, and gp60 to activate eNOS (Fig. 3C).

SNO-Alb and H₂S toxicity.

H₂S is synthesized in a number of mammalian tissues from l-cysteine by the action of cysathionine-β-synthase or cysathionine-γ-lyase and has emerged as an important newly recognized signal transduction molecule joining NO and carbon monoxide as so-called gaseous transmitters (27, 28). The physiological effects of H₂S are oftentimes confounding (anti- vs. pro-inflammatory; vasodilator vs. vasoconstrictor), and such diametrically opposed observations may be secondary to the concentration of H₂S (27). At a somewhat supraphysiological level, inhaled H₂S has attracted extraordinary attention by virtue of its ability to induce suspended animation (4). Investigations into its physiological or therapeutic role are always influenced and balanced by the long history of toxicity of H₂S [ including its telltale odor of rotten eggs and its well-known ability to inhibit cytochrome-c oxidase (14) ] . As such, most information of toxicity of H₂S in vascular tissue is derived from systemic smooth muscle (30), and little is known regarding toxicity of H₂S in pulmonary vascular tissue or the ability of NO donors to affect H₂S toxicity in the lung.

In the present study, we utilized NaHS as a source of exogenous H₂S. NaHS solution was freshly prepared on the day of every experiment by mixing the stock solution of sodium sulfide and hydrochloric acid. In normal physiological solution, one-third of NaHS exists as H₂S, and the other two-thirds exist as HS⁻ (3). Although this approach is common in cell biology and greatly simplifies use of H₂S, as with any chemical donor, it is only a surrogate for authentic H₂S. Nonetheless, the potential concentrations used in the present study most likely exceeded physiological concentrations (45–300 μM) of H₂S in plasma (32).

Our studies extend previous information on proapoptotic effects of H₂S in systemic vascular smooth muscle (7, 30). We report that exogenous H₂S released from NaHS had a proapoptotic effect on RPASMC (Fig. 4), but endothelial cells were resistant to comparable concentration range of H₂S (Fig. 4A). H₂S induced ERK1/2 and p38 (Fig. 6) phosphorylation, accompanied by downregulation of Bcl-2 and caspase-3 activation (Fig. 4D). The critical role of MAPK activation and H₂S-induced apoptosis was apparent in the sensitivity of this process (Fig. 6E) to PD-98059 (ERK1/2 inhibitor) and SB-203580 (p38 inhibitor). The cytoprotective effect of SNO-Alb against NaHS reinforced this concept and suggested a possible mechanism in which SNO-Alb protects RPASMC from NaHS, since SNO-Alb inhibited NaHS-induced activation of ERK1/2 and p38. Nonetheless, the precise molecular mechanism in which SNO-Alb affects these complex kinase pathways is unclear. In addition, the protective effect of SNO-Alb was sensitive to l-NAME, and the effect could be mimicked (at high doses of NaHS) with SH-Alb (Fig. 5B), which, in turn was sensitive to l-NAME. Apparently, activation of nNOS secondary to binding and uptake of SH-Alb (Fig. 5C) contributed to the cytoprotective effects of SNO-Alb. It remains to be determined whether the comparable components (gp60 and albumin transcytosis and activation of NOS) reported in endothelial cells (16) operate in a similar fashion in RPASMC.

The biochemical basis by which SNO-Alb produces resistant phenotypes to diverse stimuli in the present study remains uncertain. It quite likely involves trans-S-nitrosation of one or more intracellular targets, as 1) there is a large body of literature indicating that exposure of cells to low-molecular-mass protein RSNOs markedly affects their homeostasis; 2) our laboratory previously reported (31) that SNO-Alb trans-S-nitrosated a surrogate fluorescence resonance energy transfer chimera in intact rat pulmonary endothelium, and this effect was sensitive to inhibition of cell surface protein disulfide isomerase, as originally shown by Ramachandran et al. (19); and 3) plasma membrane components (e.g., ion channels, receptors, etc.) are not likely to be trans-S-nitrosated by bulky RSNOs, such as SNO-Alb. In contrast, as our laboratory has shown (5), the first site of action of low-molecular-weight RSNO, especially unhindered compounds, is likely to be the thiol-rich plasma membrane of cells. It could be further speculated that, in cells, SNO-Alb will participate in a complex equilibrium with glutathione, GSNO, and thioredoxin type 1, which is likely to be shifted in favor of GSNO (21, 23).

In conclusion, SNO-Alb appears to be a unique NO donor in that it is relatively stable, and its secondary reactions are primarily transnitrosation, perhaps limiting more oxidizing species of secondary nitrogen monoxides common to other NO donors, including authentic NO itself. Although it is unclear why RPMEC were resistant to NaHS (in comparison to RPASMC), it is noteworthy that SNO-Alb reduced the sensitivity of pulmonary vascular cells to either necrosis (tBH in RPMEC) or apoptosis (NaHS in RPASMC). Details of the latter protection are consistent with SNO-Alb interfering with H₂S-induced activation of MAPK. As such, a possible rational approach to combined therapies (SNO-Alb and H₂S) in the context of use of H₂S in suspended animation or other unusual pathophysiological states is apparent.

GRANTS

This work was supported by National Institutes of Health Grants K08 HL79456-01A1 to L.M. Zhang, NIH R37 HL65697 and P50 GM53789 to B. R. Pitt, and HL-70755 and HL-094488 to V. E. Kagan.

DISCLOSURES

No conflicts of interest, financial or otherwise are declared by the author(s).