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Am J Physiol Heart Circ Physiol 295: H1943-H1955, 2008. First published September 5, 2008; doi:10.1152/ajpheart.00642.2008
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Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole

Somshuvra Mukhopadhyay,1 Jason Lee,1 and Pravin B. Sehgal1,2

Departments of 1Cell Biology & Anatomy and 2Medicine, New York Medical College, Valhalla, New York

Submitted 18 June 2008 ; accepted in final form 1 September 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Investigations of regulated S-nitrosylation and denitrosylation of vasorelevant proteins are a newly emergent area in vascular biology. We previously showed that monocrotaline pyrrole (MCTP)-induced megalocytosis of pulmonary arterial endothelial cells (PAECs), which underlies the development of pulmonary arterial hypertension, was associated with a Golgi blockade characterized by the trapping of diverse vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) in the Golgi; reduced trafficking of caveolin-1 (cav-1) and endotheial nitric oxide (NO) synthase (eNOS) from the Golgi to the plasma membrane; and decreased caveolar NO. We have investigated whether NSF, the ATPase involved in all SNARE disassembly, might be the upstream target of MCTP and whether MCTP might regulate NSF by S-nitrosylation. Immunofluorescence microscopy and Golgi purification techniques revealed the discordant decrease of NSF by ~50% in Golgi membranes after MCTP despite increases in {alpha}-SNAP, cav-1, eNOS, and syntaxin-6. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide failed to affect the initiation or progression of MCTP megalocytosis despite a reduction of 4,5-diaminofluorescein diacetate fluorescence and inhibition of S-nitrosylation of eNOS as assayed using the biotin-switch method. Moreover, the latter assay not only revealed constitutive S-nitrosylation of NSF, eNOS, cav-1, and clathrin heavy chain (CHC) in PAECs but also a dramatic 70–95% decrease in the S-nitrosylation of NSF, eNOS, cav-1, and CHC after MCTP. These data point to depletion of NSF from Golgi membranes as a mechanism for Golgi blockade after MCTP and to denitrosylation of vasorelevant proteins as critical to the development of endothelial cell megalocytosis.

pulmonary arterial hypertension; subcellular trafficking; N-ethylmaleimide-sensitive factor; nitric oxide; monocrotaline pyrrole; adenosine 5'-triphosphatase

PULMONARY ARTERIAL HYPERTENSION (PAH) is an unrelenting disease process with high morbidity, mortality, and limited therapeutic options (42, 47, 48, 56). The pathological hallmark of PAH is the obliteration of the lumen of medium-sized pulmonary arteries by enlarged, vacuolated, and apoptosis-resistant (megalocytotic) pulmonary arterial endothelial cells (PAECs) and pulmonary arterial smooth muscle cells (PASMCs) (42, 47, 48, 56), which have characteristic increases in endoplasmic reticulum (ER) and Golgi stacks (47, 48). Since the initiating events that lead to the onset of megalocytosis in target PAECs and PASMCs in PAH in humans far precede the onset of clinical symptoms, experimental models such as monocrotaline (MCT)-induced PAH in rats and hypoxia-induced PAH in rats and mice have been extensively used to study the initiation of PAH (47, 48). Studies in humans and experimental models have led to the identification of multiple different and seemingly unrelated changes in vasoactive substances [such as increases in the vasoconstrictors thromboxane A2 (6) and endothelin-1 (14) and decreases in the vasodilators prostacyclin synthase (55) and nitric oxide (NO) (13)] as well as alterations in cell-surface receptors, caveolar proteins, growth factors, and signaling pathways in affected vascular cells in this disease (reviewed in Refs. 47 and 48). Although many of these alterations in cell surface and signaling proteins depend on the integrity of membrane-associated intracellular trafficking (Refs. 35, 49, 59, and citations therein), the contribution of disrupted intracellular trafficking to the pathobiology of PAH has rarely been discussed.

In a line of research beginning in 2003, our laboratory investigated the integrity of intracellular trafficking in MCT pyrrole (MCTP)-treated PAECs in culture and in the MCT-treated rat model (31, 35–38, 46, 50). In this model, which is used extensively in the PAH field today, exposure of PAECs to MCTP for as short a time as 2 min in culture (see Fig. 7 for an example) leads to the development of megalocytosis 12–18 h later. Pioneering studies by various groups over the past four decades have extensively characterized the phenotypic changes that underlie megalocytosis in PAECs in the MCT model both in the rat in vivo as well as in cell culture (reviewed in Refs. 47 and 48). These studies have demonstrated that in addition to dramatic increases in cell size and ER/Golgi, MCTP-induced megalocytosis in PAECs is associated with increased DNA synthesis, hyperploidy, and a premitotic cell cycle arrest (36, 50; reviewed in Refs. 47 and 48). We observed that megalocytotic PAECs exhibit caveolar/raft disruption with the loss of proteins such as caveolin-1 (cav-1), endothelial NO synthase (eNOS), and E-cadherin from the cell surface and the trapping of these proteins and vasorelevant receptor molecules such as bone morphogenetic protein receptor II (BMPRII) in the Golgi (Golgi blockade hypothesis) (31, 37, 38, 46, 50). The trapping of different cargo proteins in the Golgi in megalocytosis was explained by the fact that all vesicle tethers, soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs), and soluble NSF-attachment proteins (SNAPs) (the molecular machinery required to regulate intracellular membrane fusion events) investigated were also trapped in the Golgi in this condition (46). Moreover, in lungs of the MCT-treated rat with right ventricular hypertrophy, there was loss of cav-1 from PAECs together with enlargement and dispersal of the Golgi tethers Golgi matrix (GM)130, giantin, and golgin 84 (31, 46). That diverse and apparently unrelated tethers, SNAREs, and SNAPs were trapped in the Golgi suggested that some common mediator of intracellular trafficking was likely the upstream target in MCTP-induced megalocytosis. One such candidate upstream target at the level of the Golgi membranes is NSF, the cysteine-rich ATPase required for the disassembly of all cis-SNARE complexes (4, 19). In the present study, we have investigated this possibility and find a relative depletion of NSF from Golgi membranes after MCTP.

From a mechanistic standpoint, our laboratory had reported earlier that the trapping of eNOS in the ER/Golgi in PAECs in MCTP-induced megalocytosis led to a loss of plasma membrane/caveolar NO generation as assayed by 4,5-diaminofluorescein diacetate (DAF-2DA) fluorescence concomitant with an increase in intracellular NO (38). Since the SNARE disassembly function of NSF is known to be inhibited by S-nitrosylation (32) and Golgi-targeted eNOS mutants are known to S-nitrosylate NSF and subsequently inhibit trafficking of cargo proteins from the Golgi to the plasma membrane (21), we investigated whether the increased intracellular localization of NO in MCTP-induced megalocytosis might underlie the development of megalocytosis and be reflected in increased S-nitrosylation of NSF. However, the experimental observations were the opposite; NO scavenging not only did not affect the development of megalocytosis but enhanced the dispersal and fragmentation of the Golgi apparatus, and NSF and other trafficking proteins [cav-1 and clathrin heavy chain (CHC)] were hypo-S-nitrosylated after MCTP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Bovine PAECs in T-75 flasks, 10-cm Petri dishes, or 6-well plates were grown in DMEM and 15% fetal bovine serum as described previously by our laboratory (31, 35–38, 46, 50). MCTP was prepared by the method of Mattocks et al. (33) as described previously by our laboratory (31, 35–38, 46, 50). For all experiments, near-confluent cultures of PAECs received either 0.4% vol/vol dimethyl formamide vehicle (DMF; control cultures) or MCTP in DMF (equivalent to 200 µM MCT with 25–30% conversion to the active pyrrole; see Ref. 46 for details). Megalocytosis began to develop within 12–18 h and was fully evident by 24–36 h. Cultures were usually used 2 or 4 days after exposure to MCTP.

In experiments designed to study the initiation and progression stages of megalocytosis, PAECs in 6-well plates were treated with MCTP for 2, 5, or 20 min. The cultures were then washed with PBS and replenished with fresh serum-containing medium without MCTP, and megalocytosis was allowed to develop over the next 2 days.

Cell fractionation. Two different approaches were used: 1) fractionation based on the differential solubilization of cellular proteins in different detergents (differential detergent fractionation) and 2) mechanical cell breakage and isolation of Golgi membranes using flotation through sucrose gradients.

Differential detergent fractionation of PAECs was carried out as described previously (18, 36, 40, 45) using confluent PAEC cultures in 10-cm plates by sequential extraction with an isotonic 0.25 M sucrose buffer containing digitonin (50 µg/ml; this releases cytosolic proteins; the cytosol fraction), followed by buffer containing Brij-58 (0.5%; this solubilizes plasma membrane and some cytoplasmic membranes; the membrane 1 fraction) and then one containing Na-deoxycholate (DOC)-Tween 40 (0.5% DOC and 1% Tween 40; this solubilizes cytoplasmic membranes including the outer nuclear membrane but not the inner nuclear membrane; the membrane 2 fraction). The pellet left after the isotonic DOC-buffer wash represents the inner nuclear membrane and nuclear contents (the nuclear fraction) (18, 40, 45).

Mechanical breakage and isolation of Golgi membranes by flotation through a sucrose gradient was carried out as described earlier (38, 46) using the method of Xu and Shields (60). Briefly, PAECs grown in 10-cm plates were harvested directly in a 0.25 M sucrose-containing isotonic buffer containing 10 mM Tris-Cl (pH 7.4) and 1 mM MgCl2 (Tris-Mg buffer) (60). Cells were broken with a Tekmar Tissumiser for 15 s. A clarified cell homogenate was obtained by two sequential low-speed centrifugation spins (800 rpm for 5 min followed by 2,500 rpm for 5 min). In all experiments, total protein was estimated in the homogenate using the Bradford reagent (Bio-Rad). Protein-matched volumes of the cell homogenates derived from control or MCTP-treated cultures were adjusted to 1.4 M sucrose and floated up through a step gradient consisting of ~3 ml of cell lysate (adjusted to 1.4 M sucrose at the bottom) overlaid with ~6 ml of 1.2 M sucrose and ~2.5 ml of 0.8 M sucrose. The gradient was centrifuged in a Beckman SW41Ti rotor at 35,000 rpm for 4 h. Our laboratory and others have previously extensively characterized this gradient and shown that the band between 1.2 M and 0.8 M sucrose represents the Golgi membranes, whereas ER membranes reside at the bottom of the gradient (38, 46, 60). This fraction has been characterized to represent Golgi membranes by Shields and colleagues (60) by Western blotting with the Golgi marker trans-Golgi network protein 38 and the localization of the enzymes galactosyl transferase and sialyl transferase (Fig. 3 in Ref. 60). Moreover, we have previously characterized this fraction derived from PAECs in our hands to represent Golgi membranes using Western blots probed for several Golgi markers (GM130, p115, giantin, and golgin84) and the trans-Golgi SNARE syntaxin-6 (Fig. 3, A and B, in Ref. 46) and the enzymes {alpha}-D-mannosidase, β-D-mannosidase, {alpha}-D-glucosidase, and β-D-glucosidase (Fig. 8 in Ref. 46). Five hundred microliter fractions were collected sequentially from the top of the gradient. Proteins from each fraction were precipitated using 10% cold TCA for 2 h (250 µl/fraction for each TCA precipitate). Western blot analyses were then performed to analyze changes in the distribution of NSF and other proteins; as expected, the trans-Golgi marker syntaxin-6 peaked in the 0.8 M/1.2 M interface (Fig. 5B). In experiments based on matching for protein amounts constituting the Golgi membranes, the two to three fractions comprising the Golgi band from each gradient at the 0.8 M/1.2 M sucrose interface were pooled and assayed for total protein using the Bradford assay. Protein-matched amounts of control and MCTP-treated Golgi fractions were then used for TCA precipitation and Western blot analyses of different proteins. Alternatively, the pooled Golgi fractions (a total volume of 1–1.5 ml) were diluted with 0.25 M sucrose buffer, sedimented using an SW50.1 rotor (35,000 rpm for 20 min), and resuspended in 100 µl of sucrose-free Tris-Mg buffer.

Western blot analyses. These were carried out as described previously (31, 35–38, 46, 50). Quantitation was done using National Institutes of Health (NIH) ImageJ software.

Immunopanning using protein A magnetic beads. Immunopanning (IPN) using protein A magnetic beads (New England Biolabs, Beverly, MA) was performed essentially as described previously by our laboratory (38). Protein-matched aliquots of the Brij-58- and DOC-extracted cell fractions (membranes 1 and 2, respectively) derived from control and 4-day MCTP-treated culture samples were immunopanned using buffer containing 20 mM Tris-Cl, 100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 1 mM PMSF without and with the addition of 0.1% SDS and respective rabbit polyclonal antibodies (PAbs) (38).

Immunofluorescence microscopy. Immunofluorescence assays were performed essentially as described previously (31, 35–38, 46, 50). Briefly, PAECs in 6-well plates were appropriately exposed to vehicle (DMF), MCTP, and/or (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; see respective figure legends), fixed using 4% cold paraformaldehyde for 1 h, and permeabilized using 0.1% Triton X-100. Fixed cultures were then stained using various combinations of goat and rabbit PAbs and murine monoclonal antibodies (MAbs). AlexaFluor 488-, AlexaFluor 594- or AlexaFluor 647-tagged secondary antibodies were used in this study (Molecular Probes, Eugene, OR). Images were collected using a Bio-Rad MRC 1024 ES confocal imaging system with a black-and-white charge-coupled device (CCD) camera with AlexaFluor 488, AlexaFluor 594, or AlexaFluor 647 displayed in green, red, and blue pseudocolors, respectively. Alternatively, a Nikon Eclipse 50i epifluorescence microscopy system equipped with an RGB camera was used; this allowed the demarcation of nuclei in blue using 4',6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO) as in Fig. 7. Nuclear DAPI staining was not possible in the confocal microscopy system used in this study. Controls included the omission of the primary antibody, peptide competition assays, and the use of multiple different antibodies toward the same antigen (see Refs. 31, 35–38, 46, and 50, supplemental data in Ref. 49, and Fig. 1C). All data within each experiment were collected at identical imaging settings for the control and experimental groups, and images were subjected to iterative deconvolution using the NIH ImageJ software.


Figure 1
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  		Fig. 1. Confocal immunofluorescence imaging showing separation of N-ethylmaleimide-sensitive factor (NSF) from Golgi tethers and soluble NSF-attachment protein receptors (SNAREs) in monocrotaline pyrrole (MCTP)-induced megalocytosis of endothelial cells. Pulmonary arterial endothelial cells (PAECs) in 6-well plates were treated with MCTP, and megalocytosis was allowed to develop for 4 days. Control and MCTP-treated cultures were then fixed using the cold paraformaldehyde-Triton X-100 fixation protocol. Two-color immunofluorescence analyses were carried out using goat polyclonal antibodies (PAbs) to NSF in combination with either of murine monoclonal antibodies (MAbs) to Golgi matrix (GM)130, Golgi snare (GS)28, or syntaxin (Syn)-6 (A) or rabbit PAbs to giantin or p115 (A and B) as indicated. As a further control, a murine MAb to NSF was used in combination with the goat PAb to the NSF in C. A, arrowheads: cells with a diffuse NSF pattern after MCTP. *Cells with punctate NSF. Scale bars, 50 µm. eNOS, endothelial nitric oxide synthase.

 
Quantitative image analyses. These were performed using NIH ImageJ software (available as a free download from http://rsb.info.nih.gov/ij/). For quantitating fluorescence images, per-cell pixel intensities were measured by visually defining cell outlines and measuring the total integrated intensity (as per cell) and the average or the mean pixel intensities for the outlined area, which corrects for changes in cell size. For each image, the background pixel intensity measured over clear plastic was adjusted to zero (38).

Quantitative analyses of colocalization with statistical testing were performed using the just another colocalization plug-in (JACoP) plug-in of NIH ImageJ (3, 38). In addition to the Pearson's correlation coefficient, this plug-in provides Manders' M1 and M2 coefficients for evaluating colocalization between pairs of images in the red and green channels. Manders' coefficients are indicators of the level of colocalization between red and green channels (and vice versa) and are independent of the intensity of fluorescent signals (3). The values of Manders' coefficients range from 0 to +1, with 1 indicating complete colocalization and 0 indicating no colocalization (3). Manders' M1 is the ratio of the summed intensities of pixels from the green channel for which the intensity in the red channel is above zero to the total intensity in the green channel; Manders' M2 is the reverse (see Ref. 38 for a detailed description of various colocalization coefficients obtained using the JACoP and their respective properties).

Use of the NO scavenger c-PTIO and DAF-2DA fluorescence assay for NO. PAECs in 6-well plates were treated with c-PTIO (Biomol International, Plymouth Meeting, PA) at concentrations of either 1 mM (for short exposures of 30–40 min) or 50 or 100 µM (for longer exposures of 24–48 h) in various combinations with MCTP in different experiments. The ability of c-PTIO to scavenge intracellular NO was assayed in live-cell imaging experiments using the membrane-permeant NO-reporter DAF-2DA as described previously by our laboratory (38).

Biotin-switch method to detect protein S-nitrosylation. S-nitrosylation of proteins was detected using the biotin-switch method of Jaffrey et al. (22, 23) in which S-nitrosylated proteins are specifically derivatized with biotin using a reversible sulphydryl cross-linker, purified using an avidin column, and eluted using β-mercaptoethanol, and the originally S-nitrosylated proteins were detected by Western blotting of the eluate fractions. This procedure was applied using a S-nitrosylation protein detection assay kit purchased from Cayman Chemical (Ann Arbor, MI). Briefly, PAECs in 10-cm plates were treated with MCTP or DMF, and megalocytosis was allowed to develop for 4 days. For experiments with c-PTIO, cultures were exposed to 100 µM c-PTIO for 2 days. For the S-nitrosylation assay, cultures were washed four times with cold PBS and harvested in 1 ml of wash buffer (from the S-nitrosylation assay kit). The Bradford assay was performed on aliquots of extracts from control or MCTP or c-PTIO-treated cultures, and protein-matched aliquots of respective lysates were then processed through the blocking of free thiols, reduction of S-NO bonds, and labeling with biotin using the manufacturer's protocol. The final yield of the biotinylated proteins obtained was dissolved in 100 µl of wash buffer and separated from nonbiotinylated proteins using NeutrAvidin agarose resin (Thermo Scientific, Rockford, IL) packed in centrifuge minicolumns (100 µl resin with binding capacity of ~20 µg biotin per ml resin per spin column). The bound proteins were eluted using three to six sequential aliquots (100 µl each) of elution buffer containing (in mM) 20 HEPES (pH 7.7), 100 NaCl, 1 EDTA, and 100 mercaptoethanol, as per Jaffrey et al. (22, 23). Equal volumes of each elute fraction from the control and MCTP- or c-PTIO-treated groups were used for Western blot analysis. Respective protein-matched aliquots of the prebiotinylation cell lysates from control and respective experimental groups were also included in the Western blot assays.

Antibody reagents. Goat PAb to NSF and rabbit PAbs to eNOS, cav-1, and steroid receptor coactivator-1 (SRC-1) were obtained from Santa Cruz Biotechnology. Murine MAbs to NSF, {alpha}-SNAP, GM130, Golgi snare (GS)28, and syntaxin-6 were from BD Biosciences (Eugene, OR). Rabbit PAb to giantin was from Abcam (Cambridge, MA), and p115 was a gift from Dr. Dennis Shields (Albert Einstein College of Medicine, New York, NY).

Statistical analyses. Statistical analyses were performed using the two-tailed Student's t-test and Microsoft Excel software.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Since NSF is involved in the disassembly of all cis-SNARE complexes at the conclusion of the membrane fusion event in intracellular trafficking (4, 19, 24, 53), the relative depletion of NSF from Golgi membranes would account for the trapping of multiple different tethers, SNAREs, and SNAPs and of cargo such as eNOS, cav-1, and BMPRII in that organelle in MCTP-treated PAECs as previously reported by our laboratory (38, 46, 50). The effect of MCTP on the relative content of NSF in Golgi membranes in PAECs was investigated using two different methods: immunofluorescence microscopy coupled with quantitative colocalization analyses (Figs. 1–3) and cell fractionation approaches coupled with quantitative Western blot analyses (Figs. 4–6).

Relative depletion of NSF from Golgi membranes in MCTP-treated PAECs assayed using immunofluorescence methods and quantitative colocalization analyses. Figures 1 and 2 summarize data for the colocalization of NSF with three different Golgi tethers and scaffolding proteins, GM130, giantin, and p115, and with two Golgi SNAREs, GS28 and syntaxin-6, in control and 4-day MCTP-treated PAECs. In control PAECs, there was a diffuse immunostaining for NSF characteristic of a cytosolic protein, whereas the Golgi markers all showed a discrete single punctum (Fig. 1A, top). MCTP-treated megalocytotic cells showed a dramatic redistribution of Golgi tethers and SNAREs, indicative of Golgi enlargement and dispersal (Fig. 1A, middle and bottom), confirming earlier observations of our laboratory (36–38, 46). Importantly, NSF was aggregated into large cytoplasmic puncta in many of the megalocytotic PAECs in a manner clearly distinct from the Golgi elements (Fig. 1A, middle and bottom). A triple-label immunofluorescence analysis verified that even in cells in which NSF did not colocalize with Golgi markers, two different Golgi markers, as expected, did strongly colocalize with each other (GM130 and giantin in Fig. 1B, left, and GM130 and p115 in Fig. 1B, right). Furthermore, Fig. 1C shows that two different antibodies to NSF (one a polyclonal and another one a monoclonal) revealed identical patterns of NSF distribution in MCTP-treated PAECs. Additionally, the omission of the respective primary antibodies used in Fig. 1 led to no immunofluorescence (Refs. 31, 35–38, 46, and 50 and data not shown). However, there was heterogeneity in the pattern of subcellular distribution of NSF among different MCTP-treated PAECs, one phenotype (asterisks in Fig. 1A) in which there were large cytoplasmic punctae/aggregates of NSF clearly separable from the Golgi markers and a second phenotype (arrowheads in Fig. 1C) with diffuse cytoplasmic NSF. Overall, 44% of cells (n = 869) had the punctate NSF phenotype. We do not understand the basis for this cellular heterogeneity at the present time, other than the recognition that we are working with primary bovine PAEC cultures and not cloned cell lines.


Figure 2
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  		Fig. 2. Quantitative image analyses of changes in colocalization of NSF with Golgi tethers and SNAREs in MCTP-induced megalocytosis of endothelial cells. Multiple 246 µm x 246 µm frames (n = 10–15/culture) were collected from each of the experiments in Fig. 1, and quantitative colocalization analyses were carried out using the just another colocalization plug-in (JACoP) of National Institutes of Health ImageJ. Colocalization between NSF and each of the Golgi tethers or SNAREs investigated was assessed using the Manders' colocalization coefficients M1 and M2, which, respectively, represent the overlap between red pixels compared with green pixels as the denominator and vice versa. For each Golgi marker, n > 100 cells in both control and MCTP-treated groups were quantitated. A–E summarize data derived from these 2-way comparisons with M1 and M2 expressed as means ± SE. F diagrams that Manders' coefficients range from 0 to 1 with a higher numerical value indicating increasing colocalization. *P < 0.00001, using the Student's t-test.

 
To obtain a quantitative assessment of changes in the colocalization of NSF with Golgi markers in megalocytosis throughout a culture, we performed colocalization analyses using multiple 246 µm x 246 µm frames (n = 10–15 frames containing 10–15 cells each) collected in an unbiased manner irrespective of punctate or diffuse NSF phenotype considerations. We used Manders' coefficients to assess colocalization since these are independent of the intensity of fluorescent labeling. Figure 2 shows that there was a statistically significant decrease in the colocalization of each of the five Golgi markers investigated with NSF in pairwise comparisons (Manders' M1) and of NSF with each of the five Golgi markers (assayed using Manders' M2) in MCTP-treated PAECs compared with corresponding controls. Thus there was a relative depletion of NSF from the Golgi in megalocytosis as investigated using confocal immunofluorescence imaging assays.

Figure 3, A and B, summarizes data for the relationship between NSF and the cargo proteins eNOS and cav-1. Although both eNOS and cav-1 were increasingly trapped in a perinuclear compartment in MCTP-treated PAECs, the cytoplasmic NSF punctae in these cells did not colocalize with either eNOS (Fig. 3A) or cav-1 (Fig. 3B). Figure 3C shows an experiment using a different NSF antibody (different from that used in Fig. 3, A and B, but the same as that in Fig. 1C), which again confirms that the bulk of the NSF did not colocalize with the perinuclear eNOS in MCTP-treated PAECs. Figure 3D shows that although in MCTP-treated PAECs eNOS partially colocalized with the Golgi marker GM130, NSF was discretely separable from the Golgi and also largely from that portion of eNOS, which did not colocalize with the Golgi. Indeed, we have previously shown that this eNOS observed in the non-Golgi compartment was predominantly in a novel ionomycin-releasable ER compartment (38).


Figure 3
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  		Fig. 3. Confocal immunofluorescence imaging showing separation of NSF from Golgi cargo proteins eNOS and caveolin (cav)-1 in MCTP-induced megalocytosis of endothelial cells. PAECs in 6-well plates were treated with MCTP, and megalocytosis was allowed to develop for 4 days. Control and MCTP-treated cultures were then fixed using the cold paraformaldehyde-Triton X-100 fixation protocol. Two-color immunofluorescence analyses were carried out using goat PAb to NSF in combination with either of rabbit PAbs to eNOS (A) or cav-1 (B) or with a murine MAb to NSF along with a rabbit PAb to eNOS (C). In D, a murine MAb to the Golgi tether GM130 was used for immunofluorescence in combination with the goat PAb to NSF and the rabbit PAb to eNOS. Scale bars, 50 µm.

 
Taken together, the data in Figs. 1 and 2 revealed that NSF was largely depleted from the region of the Golgi organelle in MCTP-treated PAECs and those in Fig. 3 that this NSF was in a compartment predominantly distinct from the cytoplasmically trapped eNOS.

Cellular content of NSF in PAECs after MCTP and interaction with {alpha}-SNAP. The depletion of NSF from the Golgi in MCTP-treated PAECs, as indicated by the immunofluorescence data in Figs. 1–3, could simply be a consequence of a decrease in total cellular levels of NSF as such after MCTP on a per-unit cellular protein basis. Figure 4A shows Western blot data for NSF using protein-matched aliquots of whole-cell extracts derived from control or MCTP-treated PAECs. There was little change in the cellular content of NSF. Figure 4B shows that there was little change in the detergent extractability of NSF between control and MCTP-treated cells; the bulk (>95%) of the Western-blottable NSF in control and MCTP-treated cells was in the Brij-resistant DOC-soluble membrane 2 fraction together with {alpha}-SNAP, cav-1, and syntaxin-6. The detection of cav-1 and SNAREs in the nuclear pellet fraction shown in Fig. 4B was replicable and has been recently commented upon by other investigators (5, 43, 61).


Figure 4
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  		Fig. 4. Cellular levels of NSF and its capacity to associate with {alpha}-soluble NSF-attachment protein (SNAP) are maintained in MCTP-induced megalocytosis of endothelial cells. PAECs in 10-cm plates were treated with MCTP (8 cultures) or with vehicle alone (4 cultures), and megalocytosis was allowed to develop over 4 days. A: whole-cell extracts were prepared using the lysis buffer in the S-nitrosylation kit (Cayman Chemicals) and assayed for total protein using the Bradford assay, and protein-matched aliquots were Western blotted for respective proteins. Quantitation of the blot shown in A revealed that the total cellular content of NSF was 114% after MCTP compared with that in the control cells. B: cytoplasmic extracts were prepared by sequentially harvesting each group in isotonic buffer containing 50 µg/ml of digitonin (cytosol), 0.5% Brij-58 [membrane 1 (Memb1)], and 0.5% deoxycholate/1% Tween 40 [membrane 2 (Memb2)], leaving a pellet fraction (nucleus). A composite of the Western blot analyses performed using protein-matched aliquots of extracts derived from control and MCTP-treated cells is shown. Quantitation of the blot shown in B revealed that the total cellular content of NSF was 101% after MCTP compared with that in the control cells. In C, protein-matched aliquots of respective membrane fractions derived from control and MCTP-treated samples were immunopanned using PAbs to {alpha}-SNAP and steroid receptor coactivator (SRC)-1 in the presence of 0.1% SDS. Arrow indicates cross-immunopanning (IPN) of NSF by anti-{alpha}-SNAP PAb. CHC, clathrin heavy chain.

 
As the SNARE disassembly function of NSF is inherently dependent on its recruitment to cis-SNARE complexes by {alpha}-SNAP (4, 19, 24, 53, 62), we used a cross-IPN approach to investigate whether there were changes in the ability of NSF to form protein complexes with {alpha}-SNAP per se in megalocytotic PAECs compared with controls. To this end, we used protein-matched aliquots of the two cytoplasmic membrane fractions (membrane 1 and membrane 2) derived from control and MCTP-treated cultures and performed the cross-IPN assay using rabbit PAbs to {alpha}-SNAP and SRC-1 (which was used as a control irrelevant antibody). Figure 4C, lanes 1 and 2, confirms that on a per-unit protein basis the levels of NSF and {alpha}-SNAP did not differ between MCTP and control PAECs in the membrane 2 fraction. Figure 4C, bottom, next shows that although the rabbit PAb to {alpha}-SNAP successfully pulled out {alpha}-SNAP from both the cytoplasmic membrane fractions derived from both control and MCTP-treated cultures (lanes 3–6), that to SRC-1 did not (lanes 7–10). This confirmed the specificity of the IPN assay. Figure 4C, top, shows that a pool of NSF in the membrane 2 fractions of both control and MCTP-treated cultures formed complexes with {alpha}-SNAP (lanes 5 and 6). Thus there was no apparent defect in the ability of NSF to interact with its recruiter {alpha}-SNAP after MCTP.

Relative depletion of NSF from Golgi membranes in MCTP-treated PAECs assayed using Golgi membrane isolation and Western blotting methods. The association of NSF with the Golgi membranes per se was investigated further by cell fractionation and purification of Golgi membranes and quantitative Western blotting. In these analyses, the relative content of NSF, {alpha}-SNAP, eNOS, cav-1, and syntaxin-6 was evaluated in purified Golgi membrane fractions (Figs. 5 and 6). Golgi membranes were isolated from protein-matched aliquots of the cell homogenates derived from control and MCTP-treated PAECs by the method of Xu and Shields (60) as previously applied by our laboratory to endothelial cells in culture (46). To derive protein-matched aliquots of the respective cell homogenates, ~4 control cultures and 8 MCTP-treated cultures in 10-cm plates were harvested; the latter contained two- to threefold less total cells. In Fig. 5A, fractions 3, 4, and 5 represent the Golgi membrane band at the 0.8/1.2 M sucrose interface (see Refs. 46 and 60 and MATERIALS AND METHODS; in Fig. 5B, the trans-Golgi SNARE syntaxin-6 peaks in the G1 and G2 regions). There was a marked increase in the visible Golgi band at the 0.8/1.2 M sucrose interface in MCTP-treated PAECs compared with untreated controls despite loading aliquots of the respective cell homogenates containing matched amounts of total protein in each of the flotation gradients. The visible increase in the Golgi band was reflected in the approximately twofold increase in total protein in the Golgi fractions after MCTP (Fig. 5C). With respect to specific proteins, Fig. 5A, left, shows that in control cells, NSF was distributed in heterogeneous fractions throughout the gradient and displayed a peak in the Golgi fractions. Similarly, {alpha}-SNAP and the cargo proteins cav-1 and eNOS were also present in heterogeneous fractions through the gradient and also displayed a peak in the Golgi fractions. Megalocytotic PAECs 4 days after MCTP exposure showed dramatic increases in the association of {alpha}-SNAP, cav-1, and eNOS with the Golgi but not of NSF (Fig. 5A, right). Similar results were obtained when Golgi membranes were isolated from PAECs 2 days after MCTP exposure (Fig. 5B). A summary of the quantitation of the changes in the levels of association of NSF, {alpha}-SNAP, eNOS, and cav-1 with the isolated Golgi fractions is presented in Fig. 5C, which combines data from three independent experiments. Figure 5C confirms the increase in total protein in the Golgi membranes as such in MCTP-treated PAECs. Despite this increase in total protein in the Golgi, there was a 54% decrease in NSF, whereas the levels of {alpha}-SNAP and cav-1 increased approximately twofold (nearly the same level of increase as that of total Golgi protein itself) and those of eNOS increased six- to sevenfold (greater than the increase in total Golgi protein). The key observation was the discordant decrease of NSF levels in Golgi membranes compared with the other proteins investigated, which increased.


Figure 5
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  		Fig. 5. Selective depletion of NSF from purified Golgi membranes derived from endothelial cells with MCTP-induced megalocytosis. PAECs in 10-cm plates were treated with MCTP (8 cultures) or vehicle (4 cultures), and megalocytosis was allowed to develop for 4 (A) or 2 (B) days. Golgi membranes were purified from protein-matched aliquots of the respective clarified cell homogenates by the flotation up a discontinuous sucrose gradient using the method of Xu and Shields (Ref. 60). Five hundred microliter fractions were collected sequentially from the top, and 250 µl of each fraction were used for trichloroacetic acid (TCA) precipitation. Western blot analyses were carried out using the TCA-precipitated aliquots of each fraction and MAbs to NSF, {alpha}-SNAP, or Syn-6 or rabbit PAbs to eNOS or cav-1. C: quantitation of the change in association of total protein or various specific proteins with the Golgi fractions (fractions 3, 4, and 5 in each gradient) at the 0.8/1.2 M sucrose interface (pooled as G1 and G2) from 3 independent experiments expressed in terms of the values in controls (as percent change means ± SE relative to that in controls). *P < 0.01.

 

Figure 6
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  		Fig. 6. Selective depletion of NSF per-unit Golgi protein in MCTP-induced megalocytosis of endothelial cells. The respective Golgi fractions from the experiment depicted in Fig. 5B (fractions 3, 4, and 5 from both the control and MCTP gradients) were pooled into the G1 and G2 pools. Aliquots were used for determination of total protein in the respective G1 and G2 pools. Western blotting was carried out following TCA precipitation in 2 ways: either based upon protein-matched (Prt Mat) aliquots (250 µl for G1 and 88 µl for G2) or Golgi compartment volume-matched (Vol Mat) aliquots (250 µl each of G1 and G2) using respective antibodies. For comparison, protein-matched aliquots of the starting cell homogenate (Homog) were included in lanes 1 and 2. B: quantitation of the Western blot data shown in A.

 
Figure 6 summarizes a more detailed bookkeeping analysis of this issue. The Golgi fractions (fractions 3, 4, and 5) in each of the two gradients in Fig. 5B were pooled and evaluated for total protein, and protein-matched aliquots were Western blotted for different proteins. Figure 6A shows that although there was little change in the levels of NSF in the whole-cell homogenate between control and MCTP-treated PAECs (confirming the data in Fig. 4, A and B), there was a 79% decrease in the association of NSF with purified Golgi membranes when assayed on a per-unit protein basis (Fig. 6A, lanes 3 and 4, and Fig. 6B). In contrast {alpha}-SNAP, eNOS, and cav-1 were all increased when assayed on a Golgi compartment (volume) basis, with eNOS showing an increase even when Golgi membranes were assayed on a protein-matched basis. Again, the key observation was that NSF was relatively depleted in Golgi membranes, whereas the other proteins investigated were either unchanged or increased. Similar decreases in the association of NSF with Golgi membranes (on a per-unit Golgi protein basis) were obtained when the analyses were carried out using 4-day MCTP-treated PAEC cultures and corresponding controls and TCA precipitation to concentrate sample aliquots or when the Golgi membrane fractions were concentrated by washing with isotonic sucrose buffer and an additional round of sedimentation (data not shown). However, from a logistics standpoint, there was insufficient NSF in the purified Golgi membranes, even from control PAECs, to successfully carry out a cross-IPN assay for interaction between NSF and {alpha}-SNAP as was illustrated in Fig. 4C for detergent-extracted cellular membranes.

The NO scavenger c-PTIO blocks neither the initiation nor progression of MCTP-induced megalocytosis in PAECs. In addition to its subcellular localization per se, the ability of NSF to disassemble cis-SNARE complexes is critically regulated by S-nitrosylation (32). Lowestein and colleagues (32) have shown that a modification of specific cysteine residues of NSF by S-nitrosylation inhibits the ability of NSF to either associate with or disassemble cis-SNARE complexes. Figures 3, 5, and 6 show marked increases in eNOS in MCTP-treated PAECs in a perinuclear compartment partially colocalizing and fractionating with the Golgi. We thus asked whether the intracellular NO produced by the eNOS increasingly trapped in the ER/Golgi in megalocytotic PAECs could affect MCTP-induced megalocytosis and, more specifically, increase the level of S-nitrosylation of NSF and other vasorelevant proteins (eNOS itself, cav-1, and CHC). The hypothesis tested was that increased intracellular NO after MCTP would increase S-nitrosylation of NSF, which would inhibit trafficking through the Golgi and thus lead to megalocytosis in a feed-forward inhibitory mechanism. Thus we investigated the effect of a NO scavenger, c-PTIO, on the initiation and progression of MCTP-induced megalocytosis and on the state of S-nitrosylation of NSF and additional trafficking proteins in MCTP-treated cells.

Figure 7A focuses on the initiation phase of MCTP-induced megalocytosis in which cultures of PAECs were exposed to MCTP for 2, 5, or 20 min and then washed, and development of megalocytosis was assayed by phase-contrast microscopy 2 days later. Figure 7A, bottom, confirms this hit-and-run aspect of the initiation of megalocytosis by MCTP. Figure 7A, top, shows that a preexposure of cells to c-PTIO (1 mM) for 20 min with continuing treatment with c-PTIO together with MCTP for 20 min failed to affect the initiation phase of the effects of MCTP. In separate experiments we confirmed the ability of c-PTIO to scavenge NO under these conditions using the DAF-2DA fluorescence assay. Treatment of cultures of PAECs with c-PTIO (1 mM) for 40 min inhibited DAF-2DA fluorescence by 47% compared with untreated controls [mean DAF-2DA fluorescence per cell was 12.3 ± 0.4 in arbitrary units (means ± SE; n = 200 cells) for control and 6.5 ± 0.25 (n = 240) for the c-PTIO group; P < 0.0001]. Nevertheless, the initiation of MCTP-induced megalocytosis was not affected by c-PTIO (Fig. 7A).


Figure 7
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  		Fig. 7. (4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) blocked neither the initiation nor progression of MCTP-induced megalocytosis in endothelial cells. In A, PAECs in 6-well plates were left untreated; treated with MCTP for 2, 5, or 20 min and then replenished with MCTP-free medium; treated with 1 mM c-PTIO alone for 40 min and then replenished with c-PTIO-free medium; or treated with 1 mM c-PTIO for 20 min followed by MCTP and 1 mM c-PTIO for an additional 20 min and then replenished with MCTP and c-PTIO-free medium as indicated. Phase contrast pictures were taken 2 days after the beginning of the experiment. Scale bar, 50 µM. In B, PAECs in 6-well plates were first treated with MCTP for 20 min, washed, and then treated with 1 mM c-PTIO for 30 min and maintained in medium containing 50 or 100 µM c-PTIO for 2 days. Respective controls included cultures, which received MCTP alone or c-PTIO alone or neither as indicated. Phase contrast images were collected 2 days after the beginning of the experiment. Cultures were then fixed using the cold paraformaldehyde Triton X-100 protocol, and immunofluorescence analyses were carried out using a rabbit PAb to giantin and 4',6-diamidino-2-phenylindole (DAPI). Scale bars, 50 µm.

 
Figure 7B summarizes our experiments aimed at investigating whether c-PTIO affected the progression of MCTP-induced megalocytosis. In these experiments, PAECs were first either exposed to MCTP for 20 min or left untreated and then c-PTIO was added for 2 days. For the c-PTIO addition in these experiments, cells were loaded with c-PTIO at 1 mM for 30 min beginning 20 min after exposure to MCTP and washout of MCTP followed by a maintenance concentration of c-PTIO at 50 and 100 µM for 2 days. The phase contrast images in Fig. 7B(i), bottom left, shows that exposure to MCTP for 20 min led to the development of megalocytosis 2 days later [compare with Fig. 7B(i), top left]. Exposure to c-PTIO alone for 2 days led to a more enlarged, cuboidal morphology of the PAECs with increased numbers of rounded cells, which were in mitosis [Fig. 7B(i), top middle and top right. See Ref. 25 for the effects of c-PTIO on cell cycle progression; the rounded cells were confirmed to be annexin V-FITC negative and thus not in apoptosis and displayed mitotic figures by DAPI staining]. However, c-PTIO did not affect the development of megalocytosis in PAEC cultures that received MCTP for 20 min before being treated with c-PTIO [Fig. 7B(i), bottom middle and bottom right].

To investigate whether MCTP-induced enlargement and dispersion of the Golgi was also not affected by c-PTIO, we performed immunofluorescence analyses of cultures in Fig. 7B(i) using an antibody to the Golgi tether giantin together with DAPI to demarcate nuclei. Figure 7B(ii) shows that in control PAECs (without any drug treatment), the Golgi as demarcated using giantin was a discrete juxtanuclear punctum [Fig. 7B(ii), top left]. Exposure to MCTP for 20 min led to the development of megalocytosis with a change in the giantin staining pattern from juxtanuclear to perinuclear/circumnuclear [Fig. 7B(ii), bottom left]. If c-PTIO could block the progression of megalocytosis, we would anticipate that the structure of the Golgi would revert back to a more juxtanuclear pattern as in control cells. However, Fig. 7B(ii), bottom middle and bottom right, shows that in the PAECs treated with MCTP and then exposed to c-PTIO, the pattern of giantin staining was, in fact, further dispersed beyond that seen with MCTP alone. Thus not only did c-PTIO not block the progression of MCTP-induced megalocytosis but c-PTIO further enhanced the dispersal of the Golgi. This was the opposite of what we had expected to find.

MCTP-induced megalocytosis is characterized by hypo-S-nitrosylation of NSF and other vasorelevant proteins. To biochemically investigate whether the increased intracellular NO in megalocytotic PAECs might lead to an increase in the S-nitrosylation of NSF per se, we used the biotin-switch method devised by Jaffrey et al. (22, 23). These biotin-switch assays were performed using protein-matched aliquots from the cell lysates of control and 4-day MCTP-treated PAEC cultures; the input lanes in Fig. 8 confirm the equivalent presence of the indicated proteins in the respective whole-cell extracts. As a control for the biotin-switch assay, we confirmed the inhibitory effect of the NO scavenger c-PTIO on the S-nitrosylation of eNOS. Figure 8A, top, shows that although levels of eNOS in the prebiotinylated cell lysates were nearly identical in c-PTIO-treated and control PAECs when assayed on a per-unit protein basis (input lanes on the left), there was a 70% decrease in the levels of S-nitrosylation of eNOS after c-PTIO (summed over the 4 eluate fractions shown in Fig. 8A, right). Additionally, we identified the trafficking proteins cav-1 and CHC as two new proteins, which were modified by S-nitrosylation in control endothelial cells (Fig. 8A, see control eluate fractions 1–4). The levels of S-nitrosylation of cav-1 and CHC were also decreased after c-PTIO (by >95% and 87%, respectively). Thus c-PTIO effectively decreased the level of S-nitrosylation of eNOS, CHC, and cav-1 in PAECs, validating the biotin-switch assay in our hands. Moreover, in comparision with the amounts of these proteins in the input lanes, it is clear that only a fraction of these proteins are S-nitrosylated under the basal culture conditions used.


Figure 8
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  		Fig. 8. Constitutive S-nitrosylation of eNOS, NSF, CHC, and cav-1 in endothelial cells and hypo-S-nitrosylation after c-PTIO or MCTP. PAEC cultures in 10-cm plates were left untreated or treated with 100 µM c-PTIO for 2 days (4 cultures/group; A) or were treated with MCTP (8 cultures) or vehicle (4 cultures), and megalocytosis was allowed to develop for 4 days (B and C). S-nitrosylated proteins were detected using the biotin-switch assay (Refs. 22 and 23) in which S-nitrosylated proteins in protein-matched aliquots of the whole-cell extracts are first selectively biotinylated, then purified using a NeutrAvidin spin column, and the biotinylation reversed by elution in buffer containing 100 mM β-mercaptoethanol (Refs. 22 and 23) in sequential 100-µl elution steps. Western blot analyses of the respective eluate fractions were carried out using 50-µl aliquots in A, B, and C(i) and 35 µl in C(ii) and respective antibodies as indicated; input lanes containing protein-matched aliquots of the respective whole-cell extracts were included within the same blot [for comparison the input lanes in B (which are the same as in Fig. 4A) and in C are illustrated next to the eluate lanes as an uncut set of continuous lanes in the same enhanced chemiluminescence exposure]. Quantitation of S-nitrosylated pools of the respective proteins detected in the Western blots was carried out by summing across the eluted fractions. In A, c-PTIO reduced S-nitrosylation of eNOS by 70%, of cav-1 by 99%, and of CHC by 89%. In B, MCTP reduced S-nitrosylation of NSF by >95%. In C, MCTP reduced S-nitrosylation of NSF by 81%, of CHC by 97%, of eNOS by 95%, and of cav-1 by 94%. C, control; P, c-PTIO; M, MCTP.

 
Figure 8, B and C, summarizes two independent experiments investigating changes in protein S-nitrosylation in MCTP-treated PAECs compared with controls. Figure 8B, top, shows that the levels of NSF in the starting whole-cell lysates derived from MCTP-treated PAECs did not differ from that derived from control cultures on a per-unit protein basis (confirming data in Fig. 4 and Fig. 6A). However, contrary to what we had expected, although endogenous NSF was S-nitrosylated (see fractions 2 and 3 in Fig. 8B), there was a clear decrease in the S-nitrosylation of NSF after MCTP (>95% compared with control when fractions 2 and 3 are summed). Additionally, as a negative control, {alpha}-SNAP, a protein not known to be S-nitrosylated, was not detected in the eluate fractions of either the control or the MCTP-treated groups. Thus, contrary to what we would have expected, NSF was hypo-S-nitrosylated after MCTP and not hyper-S-nitrosylated.

Figure 8C illustrates an independent experiment in which the eluate fractions were subjected to two separate Western blot analyses (i and ii). Figure 8C(i) confirms that although the levels of NSF in the starting lysate did not change between control and MCTP-treated PAECs, there was an 80% decrease in S-nitrosylation of NSF after MCTP (when eluate fractions 2 and 3 are summed) and also reconfirms that {alpha}-SNAP was not modified by S-nitrosylation and not detected in any of the eluate fractions in the control or MCTP-treated groups. Moreover, Fig. 8C(i) shows a >90% decrease in the S-nitrosylation of CHC, and Fig. 8C(ii) shows >90% decrease in S-nitrosylation of eNOS and cav-1 after MCTP. Taken together, the data in Fig. 8 provide the first evidence for the S-nitrosylation of endogenous cav-1 and CHC in endothelial cells and for the hypo-S-nitrosylation of membrane trafficking (NSF, cav-1, and CHC) and vasorelevant (eNOS) proteins in MCTP-induced megalocytosis. The data in Figs. 7 and 8 taken together help us exclude a role for increased intracellular NO in the initiation and progression of MCTP-induced megalocytosis and point to hypo- and not hyper-S-nitrosylation of trafficking proteins after MCTP as the underlying mechanistic event(s).


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The present investigation revealed that the SNARE disassembly ATPase NSF was relatively depleted from Golgi membranes in MCTP-treated PAECs and that MCTP-induced endothelial-cell megalocytosis was associated with hypo-S-nitrosylation of membrane trafficking (NSF, cav-1, and CHC) and vasorelevant (eNOS) proteins. Furthermore, the present data identify two new proteins not previously known to be modified by S-nitrosylation: cav-1 and CHC. Since cav-1 and CHC affect the subcellular trafficking of a wide array of proteins, the modulation of the biological functions of these proteins by S-nitrosylation is clearly of broad significance in vascular biology. Although we had set out with the working hypothesis that increased accumulation of intracellular NO might enhance S-nitrosylation and thus inhibit NSF function, as indicated by the work of Lowestein and colleagues (32), scavenging NO did not affect MCTP-induced megalocytosis, and MCTP itself led to hypo- and not hyper-S-nitrosylation of NSF. Thus we reject our working hypothesis as it relates to the effects of increased intracellular NO. However, NSF was depleted from Golgi membranes and this, irrespective of nitrosylation status, provides a mechanistic explanation for the trapping of multiple vesicle tethers, SNAREs, SNAPs, and cargo proteins in the Golgi in megalocytotic endothelial cells. The biochemical basis for how MCTP leads to NSF depletion from the Golgi remains an open question.

The Golgi blockade hypothesis proposed by our laboratory (31, 50) posited that an initiating event in MCTP-induced megalocytosis of PAECs both in cell culture and in vivo was a block in intracellular trafficking of vasorelevant cell surface proteins and growth factor receptors from the Golgi to the plasma membrane. Since diverse cargo proteins were trapped in the Golgi in MCTP-induced megalocytosis and there was a change in the organization of the Golgi organelle itself with enlargement and circumnuclear dispersal in megalocytotic endothelial cells (36–38, 46), subsequent investigations focused on the effects of MCTP on vesicle tethers, SNAREs, and SNAPs, which mediate trafficking of different cargo proteins to/through the Golgi organelle. We observed that all tethers, SNAREs, and SNAPs examined were also trapped in the Golgi after MCTP (46).

Briefly, the process of membrane fusion at each subcellular site is mediated by a different combinatorial set of vesicle tethers, SNAREs, and SNAPs (4, 19, 24, 53). There are ~38 different SNARE proteins in mammalian cells, and the Golgi organelle itself has over 12 different tether proteins. However, the disassembly of cis-SNARE complexes is mediated by two proteins at all subcellular sites: {alpha}-SNAP and NSF (4, 19, 24, 53). NSF is a cysteine-rich ATPase that resides in the cytosol. It is recruited to cis-SNARE complexes by {alpha}-SNAP. NSF does not bind {alpha}-SNAP in the cytosol and cannot bind SNARE complexes without {alpha}-SNAP first associating with these complexes (62). The recruited NSF then uses its ATPase activity to pry apart the v- and t-SNAREs and thus helps recycle the individual SNAREs for additional rounds of membrane fusion (4, 19, 24, 53). Moreover, there is growing literature on the non-SNARE functions of NSF in cells (1, 27, 39, 62) and the existence of another AAA-family ATPase, p97, which overlaps with NSF in some of the non-SNARE functions of the latter (51, 58).

Our previous work showed that in addition to Golgi tethers and SNAREs, the plasma membrane SNAREs syntaxin-4 and SNAP-23 were also trapped in the Golgi in megalocytosis (46). This provided the clue that a step common to all SNARE-mediated membrane fusion might be affected in MCTP-induced megalocytosis. We therefore focused on investigating the subcellular localization of {alpha}-SNAP and NSF. The present data derived using both immunofluorescence and cell fractionation/Golgi purification methods revealed a relative depletion of NSF from the Golgi, whereas levels of {alpha}-SNAP increased. This depletion of NSF in Golgi membranes occurred despite the fact that the total cellular content of NSF did not decrease after MCTP and NSF retained the capacity of binding to {alpha}-SNAP.

Regulation of protein function by S-nitrosylation. Since the SNARE disassembly function of NSF depends on the integrity of its cysteine residues and is inhibited by S-nitrosylation (32) and there is an increase in intracellular eNOS and NO in MCTP-treated PAECs (38), we anticipated that there might be increased S-nitrosylation of NSF. However, our present data reveal the opposite: NSF was hypo-S-nitrosylated in MCTP-treated and megalocytotic PAECs. Moreover, eNOS, cav-1, and CHC were also hypo-S-nitrosylated in such cells. The hypo-S-nitrosylation of NSF was accompanied by a depletion of NSF from Golgi membranes. Clearly, there are complex changes in NSF biology in MCTP-induced megalocytosis.

Over the past few years, a wide array of proteins have been reported to be modified by S-nitrosylation, and changes in protein S-nitrosylation have been associated with different human diseases (2, 7–12, 15–17, 20, 26, 29, 30, 34, 41, 44, 52, 54, 57). Although considerable attention has been directed at increases in protein S-nitrosylation in pathological states with the inhibition of the function of a protein subsequent to S-nitrosylation, it has been recognized that 1) S-nitrosylation of proteins can occur under basal (unstimulated) conditions, 2) S-nitrosylation can lead to an enhancement of function of specific proteins and removal of S-NO groups or hyponitrosylation of specific proteins can lead to aberrant cellular functions or pathological states, and 3) regulated S-nitrosylation/denitrosylation can control the activation or inactivation of a signaling pathway or transcriptional responses (17, 52). Proteins in which activities are inhibited by S-nitrosylation include protein tyrosine phosphatase 1B, GAPDH, enzymes of arginine metabolism, NADPH oxidase, and the N-methyl-D-aspartate receptor (see supplemental Table S1 in Ref. 52 for a list of proteins modified by S-nitrosylation and the effect of the S-nitrosylation on their activity/function). On the other hand, proteins activated by S-nitrosylation include Src kinase and N-ras in tumor cells, the type 1 ryanodine receptor in skeletal muscles and type 2 ryanodine receptor in cardiac muscles, and cyclooxygenase-2 in neuronal cells (see Ref. 17 and citations therein for details and Refs. 8, 15, 20, and 54). Procaspase-3 and caspase-9 are kept in their inactive state by constitutive S-nitrosylation, and activation of caspases during the induction of apoptosis by Fas or TNF-{alpha} requires regulated denitrosylation of procaspase-3 and caspase-9 (2, 17). Additionally, S-nitrosylation can affect protein-protein interactions; S-nitrosylation of murine double minute-2 inhibits its ability to associate with p53 and leads to enhanced p53-mediated transcription (17), whereas S-nitrosylation of hypoxia inducible factor-1{alpha} (HIF-1{alpha}) enhances its binding to its transcriptional cofactor p300 and results in increased HIF-1{alpha}-mediated transcription (17). Thus S-nitrosylation is a dynamic process, and changes in S-nitrosylation can affect a myriad of subcellular processes. Indeed, MCTP is known to directly derivatize thioredoxin, a denitrosylase enzyme, but the effect of this derivatization on enzyme activity is not known (28). Similarly, the contribution of S-nitrosylation to the ability of NSF to be recruited to Golgi membranes in MCTP-induced megalocytosis requires further investigation. More generally, protein hypo-S-nitrosylation may be relevant to the pathobiology of human PAH in that decreased levels of S-nitrosohemoglobin have been reported in red blood cells of human patients with PAH (12, 34).

In the present study, we have identified two new trafficking proteins not previously known to be modified by S-nitrosylation: cav-1 and CHC. Indeed, in the context of membrane trafficking, it is known that the GTPase dynamin, which regulates vesicle budding in endocytosis, is activated by S-nitrosylation and that this modification increases the self-assembly of dynamin, promotes its GTPase activity, and facilitates endocytosis (26). The present data showing hypo-S-nitrosylation of NSF, cav-1, CHC, and eNOS in MCTP-treated PAECs raise the possibility that it is the hypo-S-nitrosylation of proteins that mediate trafficking and those that affect vascular biology that may represent the underlying biochemical defect.

To summarize, we suggest that a mechanistic explanation for the dysfunctional intracellular trafficking evident in MCTP-induced megalocytosis is likely to be the depletion of NSF from the Golgi. We further show that, contrary to our initial working hypothesis, MCTP-induced megalocytosis is associated with hypo-S-nitrosylation of NSF, cav-1, CHC, and eNOS. The biochemical bases for these effects of MCTP on endothelial cells remain to be elucidated.


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This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-077301 and R01-HL-087176 (to P. B. Sehgal) and a Predoctoral Research Fellowship from the American Heart Association, a Sigma Xi Research Grant, and a Research Grant from the American Foundation for Aging Research (all to S. Mukhopadhyay).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. B. Sehgal, Rm. 201 Basic Sciences Bldg., Dept. of Cell Biology & Anatomy, New York Medical College, Valhalla, NY 10595 (e-mail: pravin_sehgal{at}nymc.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.


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