©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Golgi Association of Endothelial Nitric Oxide Synthase Is Necessary for the Efficient Synthesis of Nitric Oxide(*)

(Received for publication, May 18, 1995; and in revised form, June 7, 1995)

William C. Sessa (§) Guillermo Garca-Cardea (¶) Jianwei Liu Agnes Keh (1) Jennifer S. Pollock (2) John Bradley (3) Sathia Thiru (3) Irwin M. Braverman (1) Kaushik M. Desai (**)

From the  (1)Departments of Pharmacology and Molecular Cardiobiology andDepartment of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06536, (2)Abbott Laboratories, Abbott Park, Illinois 60064, and (3)Departments of Medicine and Pathology, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Cambridge CBZ 2QQ, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The particulate enzyme, endothelial nitric oxide synthase (eNOS), produces nitric oxide to maintain normal vasodilator tone in blood vessels. In this study, we demonstrate that eNOS is a Golgi-associated protein in cultured endothelial cells and intact blood vessels. Using a heterologous expression system in HEK 293 cells, we show that wild-type myristoylated and palmitoylated eNOS, but not mutant, non-acylated eNOS targets to the Golgi. More importantly, HEK 293 cells expressing wild-type eNOS release substantially more NO than cells expressing the mutant, non-acylated enzyme. Thus, eNOS is a novel Golgi-associated protein, and Golgi compartmentalization is necessary for the enzyme to respond to intracellular signals and produce NO.


INTRODUCTION

Under physiological conditions, nitric oxide (NO)()produced by the vascular endothelium exerts a major inhibitory influence on the contractile properties of vascular smooth muscle and contributes to the non-thrombogenic surface of the endothelium. NO is produced via the metabolism of one of the chemically equivalent guanidino nitrogens of L-arginine by the enzyme endothelial nitric oxide synthase (eNOS)(1, 2) . eNOS is localized in membrane fractions (>90% of the total protein) of freshly isolated and serially passaged endothelial cells and of recombinant cells expressing the eNOS cDNA(3, 4) . eNOS is the only N-myristoylated protein in the NOS family, and N-myristoylation is necessary for the particulate localization of the enzyme as demonstrated by the inability of non-myristoylated mutants to efficiently bind to biological membranes(5, 6) . Moreover, N-myristoylation is necessary for palmitoylation of eNOS(7) ; however, the site(s) of palmitoylation and the contribution of this post-translational modification to the membrane association are not known. Since the precise biological membrane(s) where eNOS resides and the functional significance of such localization are not well described(8, 9) , we set out to examine the localization of eNOS in bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC).


MATERIALS AND METHODS

Cell Culture and Immunofluorescent, Light, and Electron Microscopy

Endothelial cells were isolated from BAEC or HUVEC and were isolated and cultured as described previously(5, 10) . Cells were grown in plastic tissue culture flasks and used at passages 2-3.

For immunofluorescence microscopy, BAEC or HUVEC were plated on Permanox 4-chamber slides. The cells were fixed in 1.5% formaldehyde, 0.1 M sodium phosphate (pH 7.4) for 10 min at 25 °C and permeabilized with 0.1% Triton X-100 in PBS/0.1% BSA for 5 min. Cells were incubated with the appropriate primary antibodies for 2 h (eNOS mAb, H32, IgG isotype, 56 µg/ml tissue culture supernatant, 1:500 dilution(8) , and mannosidase II polyclonal antisera, 1:500 dilution(11) ) followed by washing and incubation for 1 h with fluorescein isothiocyanate- and/or Texas Red-labeled second antibodies (Jackson Laboratories, 1:400 dilutions). After washing in PBS/BSA, slides were mounted with Slowfade (Molecular Probes) and observed with a Bio-Rad MRC 600 confocal inverted microscope. Controls using non-immune at equivalent dilutions as immune antibody or secondary antibody alone were negative. Identical perinuclear localization was detected with a commercially available eNOS mAb (1:300 dilution, Transduction Laboratories) and a rabbit polyclonal eNOS Ab directed against a glutathione S-transferase-eNOS fusion protein (amino acids 134-579 of bovine eNOS).

For light microscopy, frozen sections (10 µm) of lymph nodes were air-dried, acetone-fixed for 10 min, and incubated overnight with tissue culture supernatant mAb H32 (56 µg/ml, 1:300) in PBS/0.1% BSA. Sections were washed and incubated with peroxidase-conjugated rabbit anti-mouse antibody (Dako Corp.) for 45 min at room temperature and visualized with DAB reagent for 10 min. Controls with non-immune IgG and secondary antibody alone were negative.

For electron microscopy, BAEC were grown as confluent monolayers onto tissue culture slides with chambers (Tissue-Tek) and fixed in situ at room temperature for 15 min in 3% paraformaldehyde/0.1% glutaraldehyde, buffered to pH 7.4 in 0.1 M phosphate buffer. Cells were washed with 0.1% BSA/phosphate-buffered saline (PBS-BSA, 30 min) and permeabilized with 0.1% Triton X-100 in PBS (5 min) and then washed again with PBS-BSA. Cells were then incubated overnight at 4 °C with primary eNOS antibody (H32, protein A-purified ascites fluid, 910 µg/ml) at a dilution of 1:500. An irrelevant IgG (1 mg/ml), at the same dilution, served as a negative control. Sites of antibody binding were visualized by the avidin/biotin peroxidase technique (Vectastain ABC kit, Vector Laboratories). After the staining reaction with diaminobenzidine/hydrogen peroxide, the cells were refixed with half-strength Karnovsky's glutaraldehyde/paraformaldehyde fixative, followed by 1% OsO. Cells were dehydrated in increasing concentrations of ethanol and embedded in epoxy resin. Ultrathin serial sections, cut parallel to the original surface, were left unstained or were double stained with uranyl acetate and lead citrate and viewed in a Zeiss 109 transmission electron microscope, operated at an accelerating voltage of 80 kV.

Generation of Stable Cell Lines and Measurement of NO Release

Wild-type and G2A mutant (in which glycine-2 has been mutated to alanine) eNOS cDNAs were constructed (6) and subcloned into the mammalian expression vector, pcDNA3 (containing the neomycin-resistant gene). Human embryonic kidney cells (HEK293, ATCC) were transfected with wild-type or G2A eNOS cDNAs in pcDNA3 (15 µg of plasmid DNA/1 10 cells in 100-mm dishes) according to the standard calcium phosphate precipitation method. Transfected cells were selected for growth in Dulbecco's modified Eagle's medium (DMEM) containing penicillin (100 IU/ml), streptomycin (100 µg/ml), and 10% (v/v) fetal calf serum (complete DMEM) in the presence of G418 (800 µg/ml). After 14 days, G418-resistant colonies were isolated and maintained in DMEM containing G418 (250 µg/ml). Lines WT.HEK5 and G2A.HEK6 were used for this study. Western blot analysis was performed as described previously(5) .

For measurement of NO (NO and NO) release from stably transfected cells, HEK-293 cells were grown in 35-mm dishes. The medium was changed to Hanks' balanced salt solution supplemented with L-arginine (100 µM) and CaCl (1.3 mM), and cells were equilibrated for 30 min at 37 °C. In some experiments cells were treated with N-monomethyl-L-arginine (300 µM) in the absence or in the presence of additional L-arginine (2 mM). To stimulate NO release, ionomycin (3 µM) was added for 1 h, and the supernatant was collected for analysis of NO by chemiluminescence. Samples (100 µl) containing NO were refluxed in 0.1 M vanadium(III) chloride in 2 M HCl. Under these conditions, both NO and NO are quantitatively reduced to NO, which was quantified by a chemiluminescence detector after reaction with ozone. Cells were scraped and lysed with 1 N NaOH for total protein determination using the Lowry assay. Net NO per mg of protein was calculated after subtracting unstimulated basal release from the control wells.


RESULTS AND DISCUSSION

Localization of eNOS in BAEC and HUVEC by indirect immunofluorescence using an eNOS monoclonal antibody, H32, revealed specific, crescent-shaped, perinuclear staining and additional diffuse, peripheral staining of the antigen (Fig. 1, A and B). In both subconfluent and confluent EC monolayers, no significant plasma membrane staining was observed. Due to the difficulty in visualizing the spatial orientation of proteins in flat endothelial monolayers that line large blood vessels, we stained human lymph node microvessels from frozen biopsy samples that contain cuboidal shaped, high endothelial venules. As seen in Fig. 1, C and D, staining of lymphatic tissue demonstrated an intense, perinuclear peroxidase product in high endothelial venules, a pattern identical to that seen in cultured BAEC and HUVEC. More interestingly, the staining appeared to be polarized toward the vessel lumen. This polarized orientation suggests that activation of eNOS by shear forces and local factors (bradykinin, ATP) would favor NO to be released lumenally to influence lymphocyte trafficking or leukocyte-platelet adhesion to the vascular endothelium.


Figure 1: eNOS is localized in the perinuclear region of cultured endothelial cells and in intact human blood vessels. BAEC (A) and HUVEC (B) were fixed, permeabilized, and labeled with the H32 anti-eNOS mAb. Both endothelial cell lines showed intense perinuclear staining in a Golgi-like distribution. In C and D lymphatic tissues from biopsy specimens were stained with H32, followed by peroxidase labeling and counterstained with eosin (C) or hematoxylin and eosin (D). Notice the perinuclear lumenal (L) staining pattern and polarized localization of eNOS in high endothelial venules.



Next, we colocalized eNOS in BAEC with various markers for cellular organelles by confocal microscopy. eNOS did not colocalize with dihydrotetramethylrosamine, a redox-sensitive dye for mitochondria, or with calnexin, a resident glycoprotein chaperone of the endoplasmic reticulum. Additionally, eNOS did not colocalize with iron-loaded transferrin, suggesting an association of eNOS with structures other than those associated with recycling plasmalemma-derived endocytic vesicles.()However, a majority of the eNOS staining did colocalize with mannosidase II (1:500 dilution of rabbit polyclonal antibody(11) , a lumenal Golgi protein (Fig. 2A). Immunoperoxidase labeling/electron microscopy shows high concentrations of eNOS at the cytoplasmic face of Golgi stacks (Fig. 2B) and some staining in adjacent vesicles. There was no obvious reaction product in endoplasmic reticulum or plasma membrane of various cells in multiple experiments.


Figure 2: Colocalization of eNOS with mannosidase II, a Golgi marker, and electron microscopic localization of eNOS on Golgi membranes. In A, BAEC were double labeled by simultaneous incubations of cells with mannosidase II (ManII) and H32 eNOS mAb antibodies and processed as above. B, transmission electron micrograph of Golgi stacks in cultured BAEC after immunoperoxidase labeling with eNOS mAb. The electron-dense peroxidase reaction product is concentrated on Golgi cisternae. Bar = 0.5 µm; N = nucleus; PM = plasma membrane. Arrows show areas of intense peroxidase product on Golgi membranes.



To characterize if recombinantly expressed eNOS targets to the Golgi region as seen in endothelial cells, we generated stable cell lines that expressed either wild-type (WT) or mutant (glycine to alanine mutation, G2A) eNOS. G2A eNOS is not myristoylated or palmitoylated and is a soluble protein that behaves identically to membrane-associated, wild-type eNOS in direct measurements of NOS activity(6, 7) . Immunolocalization of eNOS reveals a majority of the WT enzyme in a perinuclear crescent that colocalizes with mannosidase II, whereas the G2A enzyme exhibits a diffuse cytoplasmic stain that does not overlap with the Golgi marker (Fig. 3, middle and lower panels). Non-transfected HEK 293 cells do not stain for eNOS but stain for mannosidase II (Fig. 3, top panel). Measurements of eNOS specific activities (assessed by nitro-L-arginine-inhibitable conversion of H-labeled L-arginine to H-labeled L-citrulline) were similar in lysates from both cell lines (89 and 92 pmol/mg/min in WT and G2A eNOS-transfected HEK 293 cells, respectively) as were the amounts of immunoreactive NOS protein (Fig. 4A). Pulse-chase analysis of S-labeled eNOS demonstrated identical turnovers for both WT and G2A eNOS (18-20 h). These data suggest that myristoylation and/or palmitoylation of eNOS are necessary for Golgi targeting of the protein in a heterologous expression system.


Figure 3: Wild-type eNOS colocalizes with mannosidase II in stably transfected HEK 293 cells, whereas G2A mutant eNOS does not. Non-transfected HEK 293 cells (NT) and WT and G2A eNOS stably transfected HEK 293 cells were labeled with mannosidase II (Man II) and eNOS antibodies and processed as described.




Figure 4: Ionomycin stimulates the release of more NO from wild-type eNOS-transfected HEK 293 cells than G2A mutant eNOS-transfected cells. In A, eNOS protein in total lysates (60 µg) or proportional amounts of membrane (m) or cytosolic (c) proteins from WT and G2A-transfected cells were Western blotted with eNOS mAb H32. NT, lysate from non-transfected HEK 293 cells. In B, transfected cells were stimulated with ionomycin (3 µM) as described, and NO release was measured by chemiluminescence. Bars represent means; cappedverticallines represent S.E. (n = 5). L-NMMA, N-monomethyl-L-arginine.



To examine the functional significance of eNOS localized to the Golgi, we then challenged both WT and G2A-transfected cells with the calcium-mobilizing ionophore, ionomycin, and measured NO release (the sum of all nitrogen oxides). Agonist-dependent increases in cytoplasmic calcium facilitate calcium/calmodulin-dependent activation of endothelial nitric oxide synthase and cause the subsequent release of NO(1, 3) . Ionomycin (3 µM) induced the release of NO from both WT and G2A-transfected HEK 293 cells; however, 3-4 times more NO was released from WT eNOS cells (Fig. 4B). The release of NO from both WT and G2A cells was L-arginine-dependent, attenuated by the NOS inhibitor, N-monomethyl-L-arginine and partially reversed by L-arginine (L-Arg). These data suggest that compartmentalization of WT eNOS on the Golgi is necessary for the enzyme to respond to intracellular signals (calcium/calmodulin) and to efficiently utilize the cellular cofactors (O, NADPH, FMN, FAD, and tetrahydrobiopterin) required for the stoichiometric conversion of L-arginine to NO.

The mechanism of how eNOS or other fatty acylated membrane proteins get to their final destination is a subject of intense investigation in many laboratories(12) . For myristoylated eNOS, there are two possible fates; either it can be targeted to the Golgi where it is palmitoylated by a Golgi membrane-associated palmitoyltransferase (13, 14) or it can be palmitoylated in the cytosol by a cytoplasmic palmitoyltransferase and then targeted to the Golgi. For plasma membrane proteins such as p21 or Src-related proteins, palmitoylation is necessary for membrane binding and localization(15, 16) . However, for the Golgi-associated enzyme, glutamic acid decarboxylase (GAD 65), palmitoylation is not required for membrane association or localization(17, 18) . Whether palmitoylation of eNOS is necessary for Golgi localization is not known and is presently being investigated.

The presence of eNOS on the Golgi is functionally important for the activation of eNOS and the release of NO as demonstrated by the ability of cells expressing Golgi-localized eNOS to release more NO than mutant cells. Presently, we cannot rule out the possibility that the diffuse cytoplasmic staining in endothelial cells represents eNOS in non-endosomal, post-Golgi vesicles destined for another cellular compartment. However, preliminary data demonstrate that stimulation of early passage BAEC with bradykinin or HUVECs with histamine does not influence the localization of eNOS, suggesting that the enzyme is a resident Golgi protein (data not shown). Quantitative imaging of intracellular calcium has shown high resting levels of calcium in the perinuclear and Golgi regions of cells(19, 20) , and upon activation of surface receptors, calcium accumulation occurs primarily in these same regions(21, 22) . In endothelial cells, laminar fluid shear stress, a stimulus for NO release, also increases perinuclear calcium accumulation(23) . The perinuclear, Golgi localization of eNOS would promote a productive interaction between calcium, calmodulin, and the enzyme. In addition to the optimal spatial orientation of the enzyme with its primary activator, calcium, the Golgi localization of eNOS suggests that NO may exert a more generalized role in cellular processes such as the secretion, nitration, and ADP-ribosylation of proteins.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants R29-HL51948 (to W. C. S.) and F32-HL09224 (to J. L.) and by grants from the Pharmaceutical Manufacturers' Association and the Patrick and Catherine Weldon Donaghue Medical Research Foundation. The Molecular Cardiobiology Program at Yale is supported by Lederle Pharmaceuticals. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Rm. 436D, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06536. Tel.: 203-737-2291; Fax: 203-737-2290.

Partially supported by a National Council of Science and Technology fellowship (Mexico).

**
Supported by Wellcome International Prize Travelling Research Fellowship 038282/Z/93 from the Wellcome Trust.

The abbreviations used are: NO, nitric oxide; eNOS, endothelial nitric oxide synthase; NOS, nitric oxide synthase; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; WT, wild-type.

G. Garcia-Cardena and W. C. Sessa, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Kelley Moreman for the generous supply of mannosidase II antibody, Dr. Ari Helenius for calnexin antibody, Drs. Louis Ignarro and David Harrison for vanadium reflux/NO chemiluminescence methodology, Dr. Mary Gerritsen for helpful suggestions during the early phases of this project, and Rong Zhang for expert technical assistance.


REFERENCES

  1. Moncada, S., Palmer, R. M. J., and Higgs, E. A.(1991)Pharmacol. Rev. 43, 109-142 [Medline] [Order article via Infotrieve]
  2. Nathan, C., and Xie, Q. (1994)J. Biol. Chem.269,13725-13728 [Free Full Text]
  3. Sessa, W. C.(1994) J. Vasc. Res.31,131-143 [Medline] [Order article via Infotrieve]
  4. Pollock, J. S., Forstermann, U., Mitchell, J. A., Warner, T. D., Schmidt, H. H. H. W., Nakane, M., and Murad, F.(1991)Proc. Natl. Acad. Sci. U. S. A. 88, 10480-10484 [Abstract]
  5. Liu, J., and Sessa, W. C. (1994)J. Biol. Chem.269,11691-11694 [Abstract/Free Full Text]
  6. Sessa, W. C., Barber, C. M., and Lynch, K. R.(1993)Circ. Res. 72, 921-924 [Abstract]
  7. Robinson, L. J., Busconi, L., and Michel, T.(1995)J. Biol. Chem. 270, 995-998 [Abstract/Free Full Text]
  8. Pollock, J. S., Nakane, M., Buttery, L. D. K., Martinez, A., Springall, D., Polak, J. M., Forstermann, U., and Murad, F.(1993)Am. J. Physiol. 265,C1379-C1387
  9. Hecker, M., Mulsch, A., Bassenge, E., Forstermann, U., and Busse, R.(1994) Biochem. J. 299, 247-252 [Medline] [Order article via Infotrieve]
  10. Gimbrone, M. A. (1976) in Progress in Hemostasis and Thrombosis (Spacet, T. H., ed) pp. 1-28, Grune & Stratton Inc., New York
  11. Moremen, K. W., and Touster, O.(1986)J. Biol. Chem. 261, 10945-10951 [Abstract/Free Full Text]
  12. Resh, M. D.(1994) Cell76,411-413 [Medline] [Order article via Infotrieve]
  13. Schmidt, J. W., and Catterall, W. A.(1987)J. Biol. Chem. 262, 13713-13723 [Abstract/Free Full Text]
  14. Gutierrez, L., and Magee, A. I.(1991)Biochim. Biophys. Acta 1078, 147-154 [Medline] [Order article via Infotrieve]
  15. Hancock, J. F., Paterson, H., and Marshall, C. J.(1990)Cell 63, 133-139 [Medline] [Order article via Infotrieve]
  16. Koegl, M., Zlatkine, P., Ley, S. C., Courtneidge, S. A., and Magee, A. I.(1994) Biochem. J. 303, 749-753 [Medline] [Order article via Infotrieve]
  17. Solimena, M., Aggujaro, D., Muntzel, C., Dirkx, R., Butler, M., DeCamilli, P. D., and Hayday, A.(1993)Proc. Natl. Acad. Sci. U. S. A. 90, 3073-3077 [Abstract]
  18. Shi, Y., Veit, B., and Baekkeskov, S.(1994)J. Cell Biol. 124, 927-934 [Abstract]
  19. Chandra, S., Gross, D., Ling, Y., and Morrison, G. H.(1989)Proc. Natl. Acad. Sci. U. S. A. 86, 1870-1874 [Abstract]
  20. Chandra, S., Kable, E. P., Morrison, G. H., and Webb, W. W.(1991)J. Cell Sci. 100, 747-752 [Abstract]
  21. Chandra, S., Fewtrell, C., Millard, P. J., Sandison, D. R., Webb, W. W., and Morrison, G. H. (1994)J. Biol. Chem. 269, 15186-15194 [Abstract/Free Full Text]
  22. Burnier, M., Centeno, G., Burki, E., and Brunner, H. R.(1994)Am. J. Physiol.266,C1118-C1127
  23. Geiger, R. V., Berk, B. C., Alexander, R. W., and Nerem, R. M.(1992)Am. J. Physiol.262,C1411-C1417

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.