1Vascular Biology Center and Department of Pharmacology, Medical College of Georgia, Augusta, Georgia; and 2Department of Pharmacology and Molecular Cardiobiology Division, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut
Submitted 7 April 2005 ; accepted in final form 26 May 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
nitric oxide; Akt; Golgi
In endothelial cells, both in culture and in situ within isolated blood vessels, eNOS resides in two distinct subcellular locations, the perinuclear Golgi complex and the plasma membrane. Recently, we reported (8) that subcellular targeting of eNOS to either the cytoplasmic face of the Golgi or to the plasma membrane strongly influenced the ability of the enzyme to produce NO in response to both the calcium-mobilizing agent, thapsigargin, and to Akt-dependent phosphorylation. Plasma membrane-targeted eNOS was constitutively active and highly responsive to transmembrane calcium fluxes, whereas cis-Golgi-targeted eNOS was less responsive to calcium and fully activated by the protein kinase Akt. In contrast, eNOS targeted to the trans-Golgi or to the cytoplasm exhibited both reduced calcium and Akt-dependent activation. Furthermore, the translocation of eNOS from the plasma membrane to internal endomembranes, induced via binding the proteins eNOS interacting protein/NOS traffic inducer (NOSTRIN/NOSIP) or through exposure to oxidized low-density lipoprotein, is also associated with reduced capacity to make NO (4, 30, 42). The mechanisms underlying the reduced activity of eNOS within these intracellular locations remain to be identified.
The site-specific phosphorylation of eNOS modulates enzyme activity in response to a variety of stimuli (6). The extent of phosphorylation is strongly influenced by subcellular localization as shown by the intense serine 1179 (S1179) phosphorylation of plasma membrane-targeted eNOS and the reduced phosphorylation of cytoplasmic G2A eNOS (8, 15). To exclude the possibility that Akt and or other kinases are spatially restricted from activating eNOS in different compartments, we mutated S1179 to the phosphomimetic aspartic acid (S1179D) (8). However, the cytosolic and trans-Golgi targeted eNOS S1179D were not fully activated compared with the native enzyme. These data suggest that mechanisms other than phosphorylation constrain the activity of eNOS in various subcellular compartments.
The activation of endothelial cells by "classic" transmembrane receptor ligands such as bradykinin and acetylcholine and by shear stress is accompanied by the elevation of intracellular calcium and subsequent liberation of NO (27). The initial depletion of calcium from intracellular stores triggers the influx of extracellular calcium through a variety of different plasma membrane calcium channels (32). The location of signaling molecules within the cell and their proximity to the influx of calcium plays a major role in the efficiency of coupling enzyme activity to local calcium concentrations. In this regard, membrane associated eNOS is more sensitive to transmembrane calcium fluxes than a cytosolic eNOS mutant (21). However, because of the presence of eNOS in both locations it is not known whether the influx of calcium primarily activates the plasma membrane eNOS or eNOS bound to intracellular membranes such as the Golgi. The presence of eNOS or eNOS-like activity has also been reported in the mitochondria and the nucleus (1, 5, 13) and the contribution of calcium to the activity of eNOS in these discreet intracellular environments, i.e., cis- vs. trans-Golgi, mitochondria, and nuclei remains unknown.
Therefore, the goal of the present study is to determine the calcium dependency of eNOS in these organelles and at the Golgi and plasma membrane by using targeted fusion proteins of eNOS and iNOS. eNOS and iNOS are structurally similar enzymes and have virtually identical co-factor and substrate affinities with one major exception, calcium-calmodulin. These properties will enable us to investigate both the calcium and substrate/co-factor dependency of NOS activity within discrete subcellular locations.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The wild type (WT), 17 amino acid transmembrane sequence derived from syntaxin-3 (S17), 87 amino acid targeting sequence derived from Golgin-97 (GRIP), 81 amino acid transmembrane sequence of -1,4-galactosyltransferase (GAL), and 15 amino acid membrane targeting sequence from K-ras (CAAX) eNOS constructs were generated as previously described (8).
Nuclear-targeted eNOS. Nuclear-targeted eNOS (NLS) was achieved by fusion of a tripartite nuclear localization sequence (PKKKRKVD) derived from SV40 to the untargeted G2A-eNOS by using the following primer: 5'-TAT CTA GAT CTA GAC TAT ACC TTT CTC TTC TTT TTT GGA TCT ACC TTT CTC TTC TTT TTT GGA TCT ACC TTT CTC TTC TTT TTT GGA TCG GGG CCG GGG GTG TCT GGG CCG GG-3'.
Mitochondrial targeted eNOS. Mitochondrial (Mito) eNOS was constructed by fusion of the targeting sequence derived from human cytochrome-c oxidase subunit VIII (MSVLTPLLLRGLTGSARRLPVPRA KIHSL) to G2A-eNOS by using the following primers: 5'-GTT ACT CAA GCT TGC CAC CAT GTC CGT CCT GAC GCC G-3',5'-GCC AAG ATC CAT TCG TTG GCG GCC GCA TCG TAA-3'.
Plasma membrane-targeted eNOS. eNOS was targeted to the plasma membrane via fusion to the amino terminal neuromodulin targeting sequence (accession no. NM_002045) using the following primers: sense, 5'-AGC TTG CCA CCA TGC TGT GCT GTA TGA GAA GAA CCA AAC AGG TTG AAA AAA ATG ATG ACG ACC AAA AGA TTG C-3'; and antisense, 5'-GGC CGC AAT CTT TTG GTC GTC ATC ATT TTT TTC AAC CTG TTT GGT TCT TCT CAT ACA GCA CAG CAT GGT GGC A-3'.
Golgi lumen targeted eNOS. Targeting the lumen of the Golgi apparatus was achieved via fusion of a Golgi-targeting sequence from the glycosyltransferase FKRP (accession no. NM_024301) using the following primers: sense 5'-ATG CGG CTC ACC CGC TGC CAG GCT GCC CTG GCC GCC GCC ATC ACC CTC AAC CTT CTG C-3', and antisense 5'-GCG GCC GCC CGC ACC AGG ACG GTG ACA C-3'.
Generation of iNOS Targeting Proteins
Golgi-targeted iNOS constructs. The trans-Golgi GRIP domain, and cis-Golgi S17 were derived from the eNOS constructs described above and fused to the full-length mouse iNOS cDNA (accession no. M87039) via 3' XhoI and XbaI sites.
Golgi and plasma membrane-targeted iNOS. The first 75 amino acids of eNOS, required for targeting of eNOS to Golgi and plasma membrane [MGNLKSVGQEPGPPCGLGLGLGLGLCGKQGPA-SPAPEPSRAPAPATPHAPDHSPAPNSPTLTRPPEGPKFPRVKN (24)] were fused to the NH2 terminus of iNOS using the following primers: sense, 5'-AAG CTT GCC ACC ATG GGC AAC TTG AAG AGT G-3'; and antisense, 5'-GCG GCC GCG TTC TTC ACG CGA GGG AAC TTG-3'.
Plasma membrane-targeted iNOS constructs. The CAAX motif was derived from eNOS-CAAX as described above and fused to the COOH terminus of iNOS via XhoI-XbaI restriction sites. Neuromodulin (MLCCMRRTKQVEKNDDDQKI)-tagged iNOS was generated using the eNOS targeting sequence described above.
Nuclear and mitochondria-targeted iNOS constructs. Nuclear, mitochondria, and intra-Golgi lumen targeting sequences were obtained from the eNOS constructs described above and ligated to the XhoI-XbaI sites and HindIII-NotI sites, respectively, of iNOS.
Green fluorescent protein-tagged iNOS. Enhanced green fluorescent protein (EGFP) was amplified by PCR from the plasmid pEGFP-N1 (BD Bioscience Clontech) using the following primers and fused to the NH2- and COOH-terminus of iNOS via HindIII-NotI and XhoI-XbaI sites, respectively. Sense, 5'-AAG CTT GTC GCC ACC ATG GTG AGC AAG GGC G-3'; antisense, 5'-GCG GCC GCC TTG TAC AGC TCG TCC ATG CC-3'; sense, 5'-CTC GAG CAT GGT GAG CAA GGG CGA GGA GC-3'; and antisense, 5'-TCT AGA TTA CTT GTA CAG CTC GTC CAT GCC-3'.
Nuclear- and Golgi-targeted red fluorescent protein. Monomeric red fluorescent protein (accession no. (AF506027) was a generous gift from Roger Tsien (University of California, San Diego). Nuclear-targeted RFP was achieved via fusion to the nuclear targeting sequence described above, using the following primers: sense, 5'-AAG CTT GTC GCC ACC ATG GCC TCC TCC GAG GAC G-3'; and antisense, 5'-GGC CGC CAC TCC ACC GGC GCC GGC GGC AGC CTC GAG-3'.
Golgi targeting of RFP was achieved via fusion with -galactosyltransferase as described previously (8) using the following primers: sense, 5'-GCG GCC GCA GGC GGC AGC ATG GCC TCC TCC GAG GAC GTC-3'; and antisense, 5'-TCT AGA TTA GGC GCC GGT GGA GTG GCG-3'.
Cell Culture Conditions and Transfection
COS-7 cells were grown in Dulbeccos modified Eagles medium containing penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% (vol/vol) fetal calf serum (complete Dulbeccos modified Eagles medium). For transfection, COS-7 cells were seeded at a density of 3 x 106 cells/60-mm dish and transfected the next day with the cDNAs according to the manufacturers instructions (Lipofectamine 2000, Invitrogen).
Western Blot Analysis
Cells were washed twice with phosphate-buffered saline (PBS), lysed on ice in 50 mM Tris·HCl, pH 7.5, 1% Nonidet P-40 (vol/vol), 10 mM NaF, 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and lysates were transferred to an Eppendorf tube and rotated for 45 min at 4°C. Lysates were Dounce homogenized (50 strokes), insoluble material was removed by centrifugation at 12,000 g for 10 min at 4°C, size fractionated by SDS-PAGE, and were Western blotted as described previously (9).
Reagents. All buffer reagents and chemicals were acquired from Sigma-Aldrich.
Immunofluorescence
COS-7 cells were transfected as described and plated onto sterile coverslips. Cells were then fixed in acetone/methanol 1:1 for 3 min at 20°C and rinsed twice with PBS plus 0.1% bovine serum albumin (PBS/BSA) for 5 min at room temperature. The cells were incubated with 5% goat serum in PBS/BSA for 30 min at room temperature, followed by incubation for 2-h with primary antibody either (polyclonal or monoclonal) at room temperature. Anti-rabbit Texas Red-labeled (diluted 1:100) or anti-mouse FITC-labeled (1:100) secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) was incubated for 1 h at room temperature. The slides were mounted with Slowfade (Molecular Probes; Eugene, OR), and the cells were observed with an inverted Zeiss microscope fitted with a Bio-Rad MRC 600 confocal imaging system. Primary antibodies for eNOS were obtained from BD Transduction Labs (eNOS MAb) and GM130 from G. Warren (Yale University). Mitotracker and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen.
Live Cell Imaging
COS-7 cells were transfected with cDNAs encoding fusion proteins of EGFP and monomeric RFP as described above. From 24 to 48 h later, cells were replated onto glass-bottomed culture dishes (MatTek). All imaging was performed with the use of the confocal microscope (LSM 510 Meta 3.2, Zeiss). Magnification power was set at x40 with oil.
NO Release
Media (100 µl) containing nitrite and nitrate (primarily NO2) was ethanol precipitated to remove proteins and refluxed in sodium iodide/glacial acetic acid to convert NO2 to NO. NO was measured via specific chemiluminescence after reaction with ozone (Sievers). Net NO2 from cells transfected with eNOS cDNAs was calculated after subtracting NO2 levels from mock transfected cells (10). NO release from cells transfected with iNOS constructs was determined in a 120-min period immediately after the addition of fresh media (DMEM) containing 400 µM L-arginine.
Statistical Analysis
NO release data are expressed as means ± SE. All analyses were performed using InStat software (GraphPad) and were made using a two-tailed Students t-test or ANOVA with a post hoc test where appropriate. Differences were considered as significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The restricted expression of eNOS in various intracellular locations was obtained by inframe fusion of the cytosolic G2A mutant of eNOS to the targeting sequences of select organelle resident proteins. The strategy, as illustrated in Fig. 1 (also see Table 1), shows cis- (S17) and trans- (GRIP) Golgi, plasma membrane (CAAX, NEURO), nuclear (NLS), and mitochondria (Mito)-targeted eNOS. To demonstrate that these constructs do indeed target their intended domains, we used confocal microscopy to record their intracellular location. As shown in Fig. 2A, WT-eNOS is found at both the perinuclear/Golgi complex and the plasma membrane. eNOS targeted to the cis-Golgi with S17 is exclusively perinuclear (Fig. 2B), whereas trans-Golgi expression of eNOS co-localizes with the Golgi marker GM130 (Fig. 2E). In Fig. 2, C and D, targeting of eNOS to the plasma membrane is clearly evident by the presence of eNOS in the outer membrane. eNOS targeted to the nucleus (Fig. 2F) is present exclusively within the nucleus, as determined by comparison with DAPI staining (Fig. 2F, right). Restricted expression of eNOS within the mitochondria is shown by overlap with the mitochondria stain with Mitotracker (Fig. 2G, left and right).
|
|
|
To assess the functional significance of eNOS subcellular localization, COS-7 cells were transfected with the targeted eNOS constructs and NO release was determined via NO-specific chemiluminescence with the use of a NO analyzer (model 280, Sievers) (Fig. 3). In Fig. 3A, basal NO was measured in unstimulated COS-7 cells. Plasma membrane-targeted eNOS is constitutively active and releases large amounts of NO from unstimulated cells (Fig. 3A, right). The cis-Golgi-targeted S17 eNOS shows comparable activity to the WT enzyme, whereas the trans-Golgi-targeted (GRIP), mitochondrial (Mito), and nuclear (NLS) enzymes produce very little basal NO. As shown in the Western blot in Fig. 3A, bottom, equal amounts of eNOS enzyme were present in the lysates of cells transfected with the fusion proteins. To ensure that the capacity of the organelle-restricted enzymes to produce NO is not compromised by the targeting sequence, NOS activity assays were performed in cell free extracts. In detergent-soluble lysates, the enzymatic activity, as determined by arginine to citruline conversion, of NLS and Mito-eNOS were not significantly different from that of WT eNOS (81 ± 7, 79 ± 10, vs. 73 ± 5 pmol·min1·mg protein1, respectively). In Fig. 3B, stimulated NO release was determined from transfected COS-7 cells using the calcium mobilizing agent thapsigargin. Stimulated NO release was similar from WT and cis-Golgi targeted S17 eNOS and enhanced in plasma membrane targeted eNOS. In contrast, the trans-Golgi, mitochondria, and nucleus-targeted eNOS had greatly attenuated calcium-dependent NO synthesis. To further address the ability of localized calcium to activate targeted eNOS, we stimulated transfected COS cells with a G protein coupled receptor agonist, ATP and the calcium ionophore, ionomycin. As shown in Fig. 3, C and D, both ATP and ionomycin stimulated significantly less NO release from the NLS, Mito, and GRIP eNOS constructs. However, in response to ionomycin there was less difference between the activities of the WT and the Mito and GRIP eNOS (44% and 65% of WT for ATP and 70% and 80% of WT activity in response to ionomycin, respectively).
|
In the next set of experiments, our goal was to determine the calcium dependence of eNOS and iNOS in transfected COS-7 cells. As shown in Fig. 4, the calcium chelator EGTA, effectively eliminated NO release from eNOS, but not the calcium-independent iNOS. In contrast, the calcium ionophore, ionomycin, stimulated a large increase in NO release from eNOS transfected cells, but did not influence NO release from iNOS. Thus the activity of iNOS is completely independent from calcium with regard to both positive and negative regulation.
|
|
Targeting of iNOS constructs. The affinities of iNOS and eNOS toward their shared substrates and co-factors are virtually identical (Table 2) with the exception of calcium and calmodulin. Thus the calcium-independent activity of iNOS in transfected COS-7 cells presents a novel way of isolating this variable to determine whether the reduced activity of eNOS in discrete intracellular locations is due to reduced access to cofactors or substrate or calcium. iNOS was targeted to the cis and trans Golgi, mitochondria, nucleus, both Golgi and plasma membrane and the plasma membrane as outlined by the strategy in Fig. 1 and Table 1. As shown in Fig. 6A, WT murine iNOS is predominantly cytosolic, distributed throughout the cytosol, and excluded from the nucleus of transfected COS-7 cells. Attaching the first 75 amino acids of eNOS, which are responsible for both its membrane association and subcellular targeting (24), to iNOS resulted in the enzyme being transported to both the perinuclear Golgi and plasma membrane (Fig. 6B) as per eNOS. Targeting iNOS to the cis- and trans-Golgi with the S17 and GRIP motifs is confirmed by the predominant perinuclear expression and the absence of plasma membrane staining (Fig. 6, C and D, respectively). iNOS targeted to the mitochondria exhibits a classic mitochondrial staining pattern (Fig. 6E) and colocalizes with mitochondria targeted RFP and Mitotracker (data not shown). The nuclear targeting of iNOS is clearly shown in Fig. 6F and is distinct from the perinuclear, Golgi targeted RFP. The plasma membrane targeting of iNOS via fusion to the CAAX or NEURO motif is shown in Fig. 6, G and H. The iNOS constructs were fused in frame with either COOH or NH2 terminal GFP and cotransfected with either nuclear or Golgi-targeted RFP to aid in the identification of subcellular localization. The fusion of GFP to either the NH2 or COOH terminus of iNOS did not reduce the ability of the enzyme to produce NO (1135.7375 ± 82.5613 pmol/ml for WT-iNOS, 1228.6102 ± 83.5684 pmol/ml for GFP-iNOS, and 1417.0998 ± 227.3476 pmol/ml for iNOS-GFP, n = 4).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Calcium-calmodulin is an essential requirement for NO production from all NOS isoforms. The inducible iNOS isoform tightly binds calmodulin in an almost irreversible manner. In combination with the absence of calmodulin-sensitive autoinhibitory loops, the activity of iNOS within cells is essentially independent of calcium, a finding confirmed in transfected COS-7 cells. In contrast, eNOS and nNOS are calcium-dependent isoforms. Both genes have evolved a targeting strategy to efficiently couple enzyme activity to discrete changes in the intracellular calcium environment. eNOS is anchored to intra-cellular membranes via a combination of myristoylation, palmitoylation, and possibly polybasic domains (13, 16). The targeting of eNOS to these intracellular domains facilitates the efficient coupling of enzyme activity to intracellular second messengers as evidenced by the reduced NO release seen from cells expressing mistargeted acylation-deficient mutants (23, 37). Similarly, nNOS is concentrated at synaptic junctions and motor endplates via the interaction of its NH2-terminal PDZ domain to adapter proteins such as PSD93/95, CAPON, and -syntrophin (20, 41). The central dogma of eNOS targeting is that eNOS must target plasma membrane organelles such as caveolae or lipid rafts to be fully active. Consistent within this theme, plasma membrane-targeted eNOS is highly responsive to transmembrane calcium fluxes (8). The clustering of proteins that facilitate calcium entry, such as voltage independent-calcium channels and the Na/Ca exchanger within these organelles (38) and the detection of higher concentrations of calcium at the plasma membrane (19, 21, 33) strengthen this concept. When stimulated with ionomycin, which produces larger calcium transients than thapsigargin or the G protein-coupled receptor agonist, ATP, the difference between the WT enzyme and the targeted eNOS constructs was reduced, suggesting that calcium is the key variable influencing eNOS activity. In addition, the data showing that the activity of iNOS is invariant when targeted to the plasma membrane and peripheral aspects of the Golgi is also consistent with this concept and argues against the intracellular limitation of substrate or cofactors.
The reduced activity of eNOS within the nucleus suggests that a physiological role of eNOS within this location is unlikely, except perhaps as a mechanism to silence eNOS activity. Similarly, the inner leaflet of the mitochondria is a less efficient site of NO production and the possibility remains that mitochondrial production of NO is more effective on the peripheral membranes of this organelle (13). Despite their close proximity, it is not clear why substantial differences in eNOS activity occur between the trans- and cis-Golgi-targeted eNOS constructs. The local concentration of calcium within the microenvironment of the perinuclear/Golgi is not known; however, the ability of iNOS to produce equivalent amounts of NO at both locations suggests that there are significant differences.
In transfected COS cells, iNOS-GFP displays a predominantly cytosolic distribution, which is in agreement with the localization of iNOS reported in many other cell types (3, 35). iNOS immunoreactivity has also been reported in perinuclear organelles, peroxisomes, and the plasma membrane (3, 39). The significance of iNOS in these locations is poorly understood and it is not clear whether intracellular location facilitates NO synthesis or whether it has a functional or protective role. Evidence for the latter comes from studies demonstrating the colocalization of iNOS and catalase in peroxisomes, where the presence of antioxidant enzymes has been proposed to reduce iNOS-derived oxidative stress (3) and from our current findings, which to a large extent, demonstrates that iNOS functions independently of its intracellular location. The invariant activity of iNOS also suggests that the concentrations of NOS substrates and cofactors are sufficient throughout the cell to support NOS activity. However, within the lumen of the Golgi both eNOS and iNOS activities were significantly reduced suggesting that one and more factors essential for NOS activity is deficient.
In conclusion, we have shown that targeted expression of eNOS within the cell dictates the calcium-dependent activation of the enzyme. Restricted expression of eNOS at the plasma membrane produces an enzyme that is highly active in response to the elevation of intracellular calcium and subsequent transmembrane calcium fluxes. The activity of the cis-Golgi eNOS was comparable to the WT enzyme, whereas eNOS expressed in the trans-Golgi, mitochondria, and nucleus displayed significantly reduced capacity to produce NO. The reduced NO release seen with the trans-Golgi, mitochondrial, and nuclear-targeted eNOS cannot be due to insufficient co-factors or substrate as the calcium-independent iNOS, targeted to the same locations, produced equivalent levels of NO as the WT enzyme. Thus the proximity of eNOS to local pools of intracellular calcium is the major factor governing the synthesis of NO.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, and Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 340: 11111115, 1992.[CrossRef][ISI][Medline]
3. Collins JL, Vodovotz Y, Hierholzer C, Villavicencio RT, Liu S, Alber S, Gallo D, Stolz DB, Watkins SC, Godfrey A, Gooding W, Kelly E, Peitzman AB, and Billiar TR. Characterization of the expression of inducible nitric oxide synthase in rat and human liver during hemorrhagic shock. Shock 19: 117122, 2003.[CrossRef][ISI][Medline]
4. Dedio J, Konig P, Wohlfart P, Schroeder C, Kummer W, and Muller-Esterl W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 15: 7989, 2001.
5. Feng Y, Venema VJ, Venema RC, Tsai N, and Caldwell RB. VEGF induces nuclear translocation of Flk-1/KDR, endothelial nitric oxide synthase, and caveolin-1 in vascular endothelial cells. Biochem Biophys Res Commun 256: 192197, 1999.[CrossRef][ISI][Medline]
6. Fleming I and Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 284: R1R12, 2003.
7. Fossetta JD, Niu XD, Lunn CA, Zavodny PJ, Narula SK, and Lundell D. Expression of human inducible nitric oxide synthase in Escherichia coli. FEBS Lett 379: 135138, 1996.[CrossRef][ISI][Medline]
8. Fulton D, Babbitt R, Zoellner S, Fontana J, Acevedo L, McCabe TJ, Iwakiri Y, and Sessa WC. Targeting of endothelial nitric-oxide synthase to the cytoplasmic face of the Golgi complex or plasma membrane regulates Akt- versus calcium-dependent mechanisms for nitric oxide release. J Biol Chem 279: 3034930357, 2004.
9. Fulton D, Fontana J, Sowa G, Gratton JP, Lin M, Li KX, Michell B, Kemp BE, Rodman D, and Sessa WC. Localization of endothelial nitric-oxide synthase phosphorylated on serine 1179 and nitric oxide in Golgi and plasma membrane defines the existence of two pools of active enzyme. J Biol Chem 277: 42774284, 2002.
10. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, and Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399: 597601, 1999.[CrossRef][ISI][Medline]
11. Fulton D, Gratton JP, and Sessa WC. Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough? J Pharmacol Exp Ther 299: 818824, 2001.
12. Furfine ES, Harmon MF, Paith JE, Knowles RG, Salter M, Kiff RJ, Duffy C, Hazelwood R, Oplinger JA, and Garvey EP. Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline. J Biol Chem 269: 2667726683, 1994.
13. Gao S, Chen J, Brodsky SV, Huang H, Adler S, Lee JH, Dhadwal N, Cohen-Gould L, Gross SS, and Goligorsky MS. Docking of endothelial nitric oxide synthase (eNOS) to the mitochondrial outer membrane: a pentabasic amino acid sequence in the autoinhibitory domain of eNOS targets a proteinase K-cleavable peptide on the cytoplasmic face of mitochondria. J Biol Chem 279: 1596815974, 2004.
14. Ghosh S, Wolan D, Adak S, Crane BR, Kwon NS, Tainer JA, Getzoff ED, and Stuehr DJ. Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase. J Biol Chem 274: 2410024112, 1999.
15. Gonzalez E, Kou R, Lin AJ, Golan DE, and Michel T. Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric oxide synthase. J Biol Chem 277: 3955439560, 2002.
16. Govers R and Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol 280: F193F206, 2001.
17. Guan ZW, Kamatani D, Kimura S, and Iyanagi T. Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human neuronal nitric-oxide synthase and inducible nitric-oxide synthase flavin domains. J Biol Chem 278: 3085930868, 2003.
18. Hevel JM, White KA, and Marletta MA. Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. J Biol Chem 266: 2278922791, 1991.
19. Isshiki M, Mutoh A, and Fujita T. Subcortical Ca2+ waves sneaking under the plasma membrane in endothelial cells. Circ Res 95: 1121, 2004.[CrossRef]
20. Jaffrey SR, Benfenati F, Snowman AM, Czernik AJ, and Snyder SH. Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON. Proc Natl Acad Sci USA 99: 31993204, 2002.
21. Lin S, Fagan KA, Li KX, Shaul PW, Cooper DM, and Rodman DM. Sustained endothelial nitric-oxide synthase activation requires capacitative Ca2+ entry. J Biol Chem 275: 1797917985, 2000.
22. List BM, Klosch B, Volker C, Gorren AC, Sessa WC, Werner ER, Kukovetz WR, Schmidt K, Mayer B, Klatt P, Schmidt K, Lehner D, Glatter O, Bachinger HP, and Mayer B. Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization. Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer. Biochem J 323: 159165, 1997.[ISI][Medline]
23. Liu J, Garcia-Cardena G, and Sessa WC. Palmitoylation of endothelial nitric oxide synthase is necessary for optimal stimulated release of nitric oxide: implications for caveolae localization. Biochemistry 35: 1327713281, 1996.[CrossRef][ISI][Medline]
24. Liu J, Hughes TE, and Sessa WC. The first 35 amino acids and fatty acylation sites determine the molecular targeting of endothelial nitric oxide synthase into the Golgi region of cells: a green fluorescent protein study. J Cell Biol 137: 15251535, 1997.
25. Martasek P, Miller RT, Liu Q, Roman LJ, Salerno JC, Migita CT, Raman CS, Gross SS, Ikeda-Saito M, and Masters BS. The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding. J Biol Chem 273: 3479934805, 1998.
26. McCabe TJ, Fulton D, Roman LJ, and Sessa WC. Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. J Biol Chem 275: 61236128, 2000.
27. Muller JM, Davis MJ, Kuo L, and Chilian WM. Changes in coronary endothelial cell Ca2+ concentration during shear stress- and agonist-induced vasodilation. Am J Physiol Heart Circ Physiol 276: H1706H1714, 1999.
28. Mungrue IN, Bredt DS, Stewart DJ, and Husain M. From molecules to mammals: whats NOS got to do with it? Acta Physiol Scand 179: 123135, 2003.[CrossRef][ISI][Medline]
29. Nishimura JS, Narayanasami R, Miller RT, Roman LJ, Panda S, and Masters BS. The stimulatory effects of Hofmeister ions on the activities of neuronal nitric-oxide synthase. Apparent substrate inhibition by L-arginine is overcome in the presence of protein-destabilizing agents. J Biol Chem 274: 53995406, 1999.
30. Nuszkowski A, Grabner R, Marsche G, Unbehaun A, Malle E, and Heller R. Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase. J Biol Chem 276: 1421214221, 2001.
31. Panda K, Adak S, Konas D, Sharma M, and Stuehr DJ. A conserved aspartate (Asp-1393) regulates NADPH reduction of neuronal nitric-oxide synthase: implications for catalysis. J Biol Chem 279: 1832318333, 2004.
32. Plant TD and Schaefer M. TRPC4 and TRPC5: receptor-operated Ca2+-permeable nonselective cation channels. Cell Calcium 33: 441450, 2003.[CrossRef][ISI][Medline]
33. Rizzuto R, Brini M, Murgia M, and Pozzan T. Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science 262: 744747, 1993.[ISI][Medline]
34. Rodriguez-Crespo I and Ortiz de Montellano PR. Human endothelial nitric oxide synthase: expression in Escherichia coli, coexpression with calmodulin, and characterization. Arch Biochem Biophys 336: 151156, 1996.[CrossRef][ISI][Medline]
35. Roman V, Zhao H, Fourneau JM, Marconi A, Dugas N, Dugas B, Sigaux F, and Kolb JP. Expression of a functional inducible nitric oxide synthase in hairy cell leukaemia and ESKOL cell line. Leukemia 14: 696705, 2000.[CrossRef][ISI][Medline]
36. Ruan J, Xie Q, Hutchinson N, Cho H, Wolfe GC, and Nathan C. Inducible nitric oxide synthase requires both the canonical calmodulin-binding domain and additional sequences in order to bind calmodulin and produce nitric oxide in the absence of free Ca2+. J Biol Chem 271: 2267922686, 1996.
37. Sakoda T, Hirata K, Kuroda R, Miki N, Suematsu M, Kawashima S, and Yokoyama M. Myristoylation of endothelial cell nitric oxide synthase is important for extracellular release of nitric oxide. Mol Cell Biochem 152: 143148, 1995.[CrossRef][ISI][Medline]
38. Schneider JC, El Kebir D, Chereau C, Mercier JC, DallAva-Santucci J, and Dinh-Xuan AT. Involvement of Na+/Ca2+ exchanger in endothelial NO production and endothelium-dependent relaxation. Am J Physiol Heart Circ Physiol 283: H837H844, 2002.
39. Stolz DB, Zamora R, Vodovotz Y, Loughran PA, Billiar TR, Kim YM, Simmons RL, and Watkins SC. Peroxisomal localization of inducible nitric oxide synthase in hepatocytes. Hepatology 36: 8193, 2002.[CrossRef][ISI][Medline]
40. Stuehr DJ, Cho HJ, Kwon NS, Weise MF, and Nathan CF. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci USA 88: 77737777, 1991.
41. Thomas GD, Shaul PW, Yuhanna IS, Froehner SC, and Adams ME. Vasomodulation by skeletal muscle-derived nitric oxide requires alpha-syntrophin-mediated sarcolemmal localization of neuronal nitric oxide synthase. Circ Res 92: 554560, 2003.
42. Zimmermann K, Opitz N, Dedio J, Renne C, Muller-Esterl W, and Oess S. NOSTRIN: a protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 99: 1716717172, 2002.