Exogenous and Endogenous NO Inhibits Weibel-Palade Body Exocytosis
To explore the effect of NO upon granule exocytosis, we studied thrombin-induced exocytosis of Weibel-Palade bodies from human aortic endothelial cells (HAEC), which release von Willebrand’s Factor (vWF). We pre-treated HAEC with 10 mM NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NONOate) or with DEA as a control, stimulated the cells with thrombin 1 U/ml for 1 hr, and then measured the amount of vWF released into the media. Thrombin induces a rapid release of vWF from HAEC (). However, exogenous NO blocks the effects of thrombin (). Endogenous NO produced from endothelial cells also inhibits vWF release, since 1 mM L-nitroarginine-methyl-ester (L-NAME) inhibition of NOS for 16 hr increases the amount of vWF released after thrombin stimulation (). NO inhibition of vWF release is dose dependent. We pretreated HAEC with increasing amounts of various NO donors, including a rapidly releasing NO donor DEA-NONOate for 10 min, a slowly releasing NO donor DETA-NONOate for 16 hr, or a nitrosothiol S-nitroso-penicillamine (SNAP) for 6 hr; or we treated cells with NEM for 1 hr. Pretreatment with any one of these reagents inhibits thrombin induced vWF release ().
NO Inhibits vWF Release from Human Aortic Endothelial Cells
We then explored the role of endogenous NO in the regulation of Weibel-Palade body exocytosis. We pre-treated HAEC with L-NAME for 16 hr in order to inhibit endogenous NOS and then stimulated the cells with 1 U/ml thrombin for 1 hr and measured vWF release. L-NAME increases thrombin-stimulated vWF release in a dose-dependent manner (). (The iNOS inhibitor 1400W had no effect [data not shown]). We next pretreated HAEC with vascular endothelial growth factor (VEGF) in order to activate endogenous NOS3. Cells were incubated with 50 ng/ml VEGF or control for 2 hr, and then stimulated with thrombin. VEGF treatment decreases thrombin stimulated vWF release (). L-NAME 1 mM blocks the effects of VEGF treatment, implying that NO mediates VEGF inhibition of exocytosis (). Since L-NAME inhibition of NOS increases exocytosis and since VEGF activation of NOS decreases exocytosis, taken together, these data suggest that endogenous NO regulates endothelial cell exocytosis.
SNARE Molecules Regulate Weibel-Palade Body Exocytosis
The molecular machinery that regulates Weibel-Palade body exocytosis is unknown. However, since SNARE molecules regulate exocytosis of vesicles and granules from other cells, we reasoned that they might also regulate endothelial cell exocytosis of Weibel-Palade bodies as well. We first determined the expression of SNARE molecules in endothelial cells, using brain extracts as a control. HAEC and human brain extracts were immunoblotted with antibodies to SNARE molecules. HAEC express VAMP-3, syntaxin-2 and syntaxin-4, and SNAP-23 (). To explore the role of syntaxin-4 and VAMP-3 in the regulation of vWF release, we permeabilized HAEC and then incubated them with antibody to syntaxin-4, antibody to VAMP, or IgG as a control. Cells were then resealed, treated with thrombin, and the amount of vWF released into the media was measured. Antibody to syntaxin-4 inhibits vWF release by over 75% (). Antibody that reacts with VAMP isoforms 1, 2, and 3 inhibits vWF release by approximately 25%. These results suggest that SNARE molecules regulate Weibel-Palade body exocytosis.
NSF and SNARE Molecules Regulate Weibel-Palade Body Secretion
NSF Regulates Weibel-Palade Body Exocytosis
We next explored the role of NSF in regulating Weibel-Palade body exocytosis. We first determined that HAEC express NSF (). We then used two approaches to show that NSF regulates Weibel-Palade body exocytosis. Our first approach involved antibody inhibition of exocytosis. We permeabilized HAEC as above, incubated cells with antibody to NSF or antibody to α-SNAP, resealed the cells, and stimulated them with thrombin. Antibody to NSF inhibits vWF release by approximately 25% (). Antibody to α-SNAP also inhibits vWF release.
Our second approach to demonstrating NSF regulation of Weibel-Palade body exocytosis involved peptide inhibition of NSF. We designed a peptide inhibitor of NSF that crosses cell membranes. This peptide, designated TAT-NSF222, consists of the protein transduction domain of HIV TAT (residues 47–57) previously shown to transduce polypeptides into cells (Becker-Hapak et al., 2001
) fused to a fragment of NSF (residues 222–243) previously shown to inhibit NSF (Schweizer et al., 1998
). To show that this peptide enters HAEC, we incubated HAEC with 10 μM FITC-labeled TAT-NSF or 10 μM FITC-albumin for 20 min, added ethidium bromide to quench extracellular fluorescence, and analyzed fluorescence of the cells by FACS. Over 75% of cells treated with FITC-TAT-NSF222 contained FITC (). We then incubated HAEC for 20 min with TAT-NSF222 or with a control peptide TAT-NSF222scr that consisted of the intact TAT domain followed by the amino acid residues of NSF 222-243 in a scrambled order. HAEC were transduced with TAT-NSF peptides, treated with media or thrombin, and the amount of vWF released into the media was measured by ELISA. TAT-NSF222 inhibits vWF release in a dose-dependent manner ().
Taken together, the antibody and peptide inhibition data suggest that NSF regulates Weibel-Palade body exocytosis.
NO Does Not Inhibit NSF ATPase Activity
We next explored the effect of NO upon NSF activity since NO inhibits Weibel-Palade body exocytosis and since NSF regulates Weibel-Palade body exocytosis. We first examined the effect of NO upon the ATPase activity of NSF. DEA-NONOate was added to recombinant NSF, and the ATPase activity of NSF was measured. NO does not significantly inhibit NSF hydrolysis of ATP ().
NO Inhibits NSF Disassembly Activity
We next explored the effect of NO upon NSF disassembly activity. ATP is required for NSF to disassemble the SNARE complex; ATP-γS can lock NSF onto the SNARE complex. The SNARE complex alone sediments at 7S, and the NSF-α-SNAP-SNARE complex sediments at approximately 20S. Accordingly, we pretreated recombinant NSF with the NO donor DEA-NONOate 1 mM for 10 min. We then mixed pretreated NSF, α-SNAP, and detergent extracts of HAEC membranes in the presence of 0.5 mM ATP or ATP-γS. These reactions were then fractionated by sucrose density gradient ultracentrifugation and fractions were collected from the bottom of the tube and immunoblotted with antibody to syntaxin-4. The SNARE complex sediments at 7S, as expected in the presence of NSF and ATP (, top). The SNARE complex sediments at 20S, as expected in the presence of NSF and ATP-γS (, middle). However, the SNARE complex also sediments at 20S in the presence of ATP and NSF pretreated with DEA-NONOate (, bottom). These results suggest that NO inhibits the ability of NSF to disassemble a 20S SNARE complex derived from endothelial cell extracts.
We next examined the effect of NO upon NSF disassembly of purified, recombinant SNARE molecules. Recombinant (His)6-NSF was pretreated or not with 1 mM DEA-NONOate for 10 min and then mixed with (His)6-α-SNAP and recombinant SNARE fusion polypeptides identified in endothelial cells: GST-syntaxin-4, VAMP-3, and SNAP-23. ATP or ATP-γS was added, the mixture was precipitated with glutathione-sepharose beads, and precipitated proteins were fractionated by SDS-PAGE and immunoblotted with antibody to the NSF tag.
NSF with α-SNAP interacts with GST-syntaxin-4 (). ATP decreases the interaction of NSF with GST-syntaxin-4; ATP also decreases the coprecipitation of VAMP-3 and SNAP-23 along with GST-syntaxin-4, presumably by NSF disassembly of the SNARE complex. As expected, ATP-γS blocks NSF disassembly activity. However, NO blocks NSF disassembly of the SNARE complex, even in the presence of ATP (). Furthermore, DTT restores the disassembly activity of NSF pretreated with NO, suggesting that NO inhibition of NSF disassembly is reversible. Finally, NO inhibits NSF disassociation from GST-syntaxin-4 in a dose-dependent manner ().
(The low level of interaction between NSF and SNAREs in the absence of adding additional ATP to the reaction mixture may be due to residual ATP in the storage buffer used to prepare recombinant NSF. Small amounts of ATP present in the reaction buffer may permit an interaction between NSF and SNAREs, while larger amounts enable NSF to disassemble the SNARE/α-SNAP/NSF complex.)
TAT-NSF Peptides Inhibit NSF ATPase Activity and NSF Disassociation from SNAREs
NO and TAT-NSF peptides inhibit NSF by different mechanisms. NO inhibits NSF disassembly activity but does not affect NSF ATPase activity, as shown above. In contrast, the TAT-NSF222 peptide inhibits both NSF ATPase activity () and also NSF disassembly activity (). Thus, the mechanisms by which NO and TAT-NSF peptides inhibit NSF are different: both inhibit NSF disassembly activity, but only TAT-NSF peptides inhibit NSF ATPase activity as well. Since NO reversibly inhibits NSF disassembly activity without affecting its ATPase activity, we reasoned that NO targets NSF cysteine residues in regions of NSF that couple the energy of ATP hydrolysis to the mechanical energy of disassembling the SNARE complex.
NO Inhibits Exocytosis by Inhibiting NSF
To confirm that NSF is a primary physiological target of NO, we pretreated HAEC with 1 mM DEA-NONOate for 10 min to inhibit exocytosis, permeabilized the endothelial cells as above, and then added recombinant NSF 100 μg/ml. HAEC were then resealed, stimulated with thrombin, and the amount of vWF released into the media was measured. As before, NO inhibits exocytosis (). However, exogenous recombinant NSF restores the ability of NO treated HAEC to undergo exocytosis (). In contrast, recombinant NSF pre-treated with NO cannot restore the ability of NO treated HAEC to undergo exocytosis (). These data suggest that NSF is indeed a primary target of NO in cells.
Cysteine Residues Mediate NSF Activity
NSF contains 9 cysteine residues, 3 in the N-terminal domain, 3 in the D1 domain, and 3 in the D2 domain (). To determine the importance of individual cysteine residues in NSF functions, we made nine individual NSF mutants, each lacking one of the nine cysteine residues, and then compared the activity of wild-type NSF to mutant NSF.
Cysteine Residues Mediating NSF Activity
We first determined which cysteine residues mediate NSF ATPase activity. We measured the ATPase activity of wild-type NSF and of each NSF mutant. Mutation of cysteine residues 21, 91, 264, and 334 partially decrease NSF ATPase activity (, white bars).
We next determined which cysteine residues mediate NSF interactions with SNARE molecules. We repeated the pull-down assay with wild-type and mutant NSF. NSF and α-SNAP were incubated with GST-SNARE fusion polypeptides in the presence of ATP or ATP-γS, and the mixture was precipitated with glutathione-sepharose beads, and finally, immunoblotted with antibody to the NSF tag. Wild-type NSF interacts with GST-SNAREs in the presence of ATP-γS and disassembles GST-SNAREs in the presence of ATP (). Mutation of cysteine residues 250 and 599 have no effect on NSF interaction and disassembly activity. Mutation of cysteine residues 11, 21, 334, 568, and 582 block the ability of NSF to interact with GST-SNARE molecules. Mutation of cysteine residues 91 and 264 permit NSF to interact with the SNARE complex, but inhibit the ability of NSF to disassemble the GST-SNARE complex. These data suggest that one set of NSF cysteine residues regulate NSF interactions with SNARE complexes and another set of NSF cysteine residues regulate NSF disassembly of SNARE complexes. In particular, cysteine residues 91 and 264 appear to regulate NSF disassembly activity.
NSF Cysteine Residue Targets of NO
We next used these NSF cysteine mutants to explore which cysteine residues of NSF are targets of NO. We added NO to wild-type and mutant NSF and measured the ATPase activity. NO does not affect ATPase activity of wild-type NSF and does not affect ATPase activity of any of the NSF mutants (, black bars).
We then tested the effect of NO upon the disassembly activity of NSF mutants, using the pull-down assay. NO blocks the ability of wild-type NSF to disassemble the SNARE complex in the presence of ATP (). Mutation of cysteine residues 250 and 599 have no effect on the ability of NO to inhibit NSF disassembly activity. The effect of NO upon cysteine residues 11, 21, 334, 568, and 582 cannot be ascertained, since mutation of these residues abrogates NSF interaction with SNARE molecules. Mutation of cysteine residues 91 and 264 blocks the ability of NSF to disassemble the SNARE complex, and NO has no effect upon these mutants.
We next measured the number of cysteine residues that are nitrosylated by exogenous NO. We exposed recombinant NSF to increasing amounts of DEA-NONOate and then used the Saville reaction to measure the number of nitrosocysteine residues per molecule of NSF. NO nitrosylates NSF in a dose-dependent manner (). At 1 mM DEA-NONOate, approximately 3 cysteine residues are modified by NO per each molecule of NSF. (This dose of 1 mM DEA-NONOate also inhibits NSF disassembly activity ().)
We next identified the cysteine residues that are targets of NO by measuring the number of cysteines that are nitrosylated in wild-type and mutant NSF. DEA-NONOate was added to wild-type or mutant NSF that lack specific cysteine residues, and the Saville reaction was used to measure the number of nitrosocysteine residues per molecule of NSF. Mutation of cysteine residues 11, 250, 334, and 568 have no effect upon the nitrosocysteine content (). However, mutation of cysteine residues 21, 91, or 264 decreases the nitrosocysteine content per NSF molecule by one (). These data suggest that NO nitrosylates cysteine residues 21, 91, and 264 of NSF.
NO Nitrosylation of NSF Is Reversible
Since NSF plays a general role in vesicle trafficking, permanent inactivation of NSF would slow vesicular trafficking in general. However, local production of NO at the plasma membrane and reversible nitrosylation of NSF could provide spatial regulation of NSF. In order to measure how long NO inhibition of exocytosis lasts, we pretreated HAEC with the NO donor SNAP 100 μM for 4 hr, washed the cells to remove the NO donor, and then at various times afterwards stimulated the HAEC with thrombin and measured the amount of vWF released into the media. NO inhibits thrombin-induced release of vWF, and a single application of NO continues to inhibit exocytosis 1 hr after treatment (). However, NO inhibition of exocytosis gradually decreases after 1–2 hr; and 2–4 hr after NO treatment about 75% of Weibel-Palade body exocytosis is restored (). Thus, NO inhibition of exocytosis is reversible.
NO Inhibition of Exocytosis and Nitrosylation of NSF Is Reversible
In order to examine directly the reversibility of NSF nitrosylation, we pretreated endothelial cells with an NO donor as above and then harvested cell lysates at various times after NO treatment. Cell lysates were immunoprecipitated with antibody to nitrosocysteine, fractionated by SDS-PAGE, and immunoblotted with antibody to NSF. Treatment with NO increases the level of nitrosylated NSF (, top: − versus + at 0 hr). The level of nitrosylated NSF decreases 2 hr after treatment with NO, and 2–4 hr after treatment the level of nitrosylated NSF is similar to nontreated cells (, top). (In addition to the expected signal for NSF at 82 kDa, another band at approximately 70 kDa is observed.) Treatment with NO donors does not change the total amount of NSF for the first 4 hr, but total amounts of NSF increase 4–16 hr after NO treatment (, bottom).
Taken together, these data show that within 2–4 hr of exposure to NO, nitrosylation of NSF, and inhibition of endothelial cell exocytosis is reversible.
NSF Is Nitrosylated and Regulates Exocytosis In Vivo
In order to test whether or not NO modifies NSF in vivo, we prepared cell lysates from HAEC treated with media or the NOS inhibitor L-NAME. Polypeptides in this lysate containing nitrosothiols were biotinylated, precipitated with avidin-agarose, and immunoblotted with antibody to NSF (Jaffrey et al., 2001
). NSF is nitrosylated in endothelial cells, and treatment of cells with increasing amounts of NOS inhibitor decreases NSF nitrosylation (). We next searched for nitrosylated NSF in mice. Polypeptides were prepared from spleens of wild-type and eNOS null mice as above. NSF is nitrosylated in wild-type mice expressing eNOS (). In contrast, NSF is not nitrosylated in eNOS-deficient mice. These data show that NO nitrosylates NSF in vivo.
NSF Is Nitrosylated and Inhibits Exocytosis In Vivo
To explore the physiological relevance of NSF regulation of Weibel-Palade body exocytosis, we measured the effect of the NSF inhibitory peptide upon the bleeding time in mice. Anesthetized mice were injected intravenously with saline, TAT-NSF222, or the control peptide TAT-NSF222scr; after 45 min, the distal 5 mm of tail was amputated and the bleeding time measured (if the animals bled continuously for 20 min, the experiment was stopped, and the bleeding time was recorded as 20 min). Treatment with saline or the control peptide has no effect upon bleeding time (). In contrast, treatment with TAT-NSF222 dramatically prolongs the bleeding time (). In fact, three of the six mice treated with the TAT-NSF222 peptide had bleeding times in excess of 20 min. (The NSF inhibitory peptides did not enter platelets and did not affect platelet exocytosis [data not shown]. The NSF inhibitory peptides also did not affect vascular contractility of mouse aortas perfused in organ baths [data not shown]). These data show that NSF regulates Weibel-Palade body exocytosis in vivo and suggest that NSF is a novel target for treatment of thrombotic and cardiovascular diseases.
NO Regulates Exocytosis In Vivo
We explored the physiological relevance of NO regulation of Weibel-Palade body exocytosis in two murine models. We measured the bleeding time in wild-type and eNOS-deficient mice. Lack of eNOS decreases the bleeding time in mice, which would be predicted if a lack of NO decreased NSF inhibition and permitted an increase in exocytosis of vWF (). We also measured serum levels of vWF and soluble P-selectin in wild-type and eNOS-deficient mice. Serum levels of vWF are higher in eNOS-deficient mice compared to wild-type mice (20 ± 25 versus 11 ± 14 mU/ml, although these differences are not significant for n = 6). Serum levels of soluble P-selectin are also higher in eNOS-deficient mice compared to wild-type mice (81 ± 21 versus 73 ± 14 mU/ml, although these differences are not significant for n = 8). Conclusions from this physiological model of bleeding are limited because NO has effects upon platelets as well as endothelial cells. Despite the limitations of this in vivo model, increased levels of vWF and soluble P-selectin and decreased bleeding times would be expected in eNOS–deficient mice, if NO inhibits exocytosis in vivo.
To further examine the physiological relevance of NO regulation of Weibel-Palade body exocytosis, we measured the effect of endogenous NO upon platelet interactions with endothelial cells in vivo. Platelet rolling, or transient adherence of platelets to the walls of blood vessels, is mediated by vWF released by exocytosis (Andre et al., 2000
). We hypothesized that inhibition of endogenous NO synthesis would permit an increase in Weibel-Palade body exocytosis, an increase in released vWF, and an increase in platelet adherence to venule walls. We first showed that exogenous NO can inhibit histamine-induced exocytosis from HAEC (). We next employed intravital microscopy to explore the effects of endogenous NO upon platelet rolling in vivo. Anesthetized mice were pretreated or not with L-NAME and then transfused with calcein-AM labeled platelets. The mesentery was externalized, superfused with histamine, and intravital microscopy was used to record interactions of fluorescently labeled platelets with mesenteric venules. Platelets were classified as adherent if they were transiently captured by the endothelium and then translocated in a stop-and-go fashion for a minimum of 2 s. Histamine rapidly induces platelet adhesion to the venule wall without aggregation, starting within 30 s and peaking 3 min after histamine treatment (). Inhibition of endogenous NOS with L-NAME increases histamine-induced platelet interactions with the venule wall to a frequency more than double that observed in nontreated mice (). Furthermore, inhibition of endogenous NOS permits an increase in platelet-venule interactions, which finally peaks at 6 min after histamine treatment, indicating that the vWF release continues twice as long as in the control mice. This increase in transient platelet adherence to the venule would be predicted if less NO synthesis led to increased exocytosis of Weibel-Palade bodies and more release of vWF.
NO Inhibits Exocytosis In Vivo and Consequently Platelet Adhesion to Endothelium
The bleeding time and platelet rolling data taken together suggest that NO regulates hemostatically important granule secretion in vivo.