Skip Navigation

Cardiovascular Research 2004 63(2):338-346; doi:10.1016/j.cardiores.2004.04.025
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Weis, M. T
Right arrow Articles by Stallone, J. N
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weis, M. T
Right arrow Articles by Stallone, J. N
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Inhibiting long chain fatty Acyl CoA synthetase increases basal and agonist-stimulated NO synthesis in endothelium

Margaret T Weis*,a, Jason L Crumleya, Lindon H Youngb and John N Stallonec

aDepartment of Pharmaceutical Sciences, Texas Tech University Health Science Center, 1300 Coulter, Amarillo, TX 79106, USA
bDepartment of Pathology, Philadelphia College of Osteopathic Medicine, Philadelphia, PA 19131, USA
cDepartment of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843, USA

* Corresponding author. Tel.: +1-806-356-4015x281; fax: +1-806-356-4034. Email address: margaret.weis{at}tuuhsc.edu

Received 12 January 2004; revised 7 April 2004; accepted 21 April 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objectives: Endothelial nitric oxide synthase (eNOS) activation/deactivation is associated with cyclic depalmitoylation/repalmitoylation of specific Cys residues. The mechanism of depalmitoylation has been identified recently, but repalmitoylation remains undefined. We hypothesized that long chain fatty acyl CoA synthetase (LCFACoAS) modulates endothelial nitric oxide synthase repalmitoylation by limiting palmitoyl CoA availability. Methods: Human coronary endothelial cells were treated with triacsin-C, an inhibitor of long chain fatty acyl CoA synthetase, for 24 h. Media nitrite accumulation, eNOS activity, and eNOS palmitoylation were measured. Methacholine-induced NO synthesis or vascular relaxation were measured in endothelium-intact rat aortae in the presence and absence of triacsin-C. Results: Triacsin-C significantly reduced incorporation of [3H] palmitate into immunoreactive endothelial nitric oxide synthase and over a concentration range of 0.1 to 10 µM, increased media nitrite accumulations 2- to 2.5-fold over baseline. Total in vitro catalytic activity of nitric oxide synthase in triacsin-C treated cells did not differ significantly from control. Triacsin-C significantly increased methacholine-induced NO synthesis in the isolated rat aorta, and significantly enhanced methacholine-induced relaxation of rat aortic rings. Conclusions: These data are consistent with the interpretation that inhibition of palmitoylation increases endothelial nitric oxide synthase activity without changing endothelial nitric oxide synthase expression, suggesting that inhibiting palmitoylation increases the catalytically active fraction of endothelial nitric oxide synthase.

KEYWORDS Coronary circulation; Endothelial factors; Endothelial function; Hypertension; Nitric oxide


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The role of endothelial nitric oxide synthase (eNOS) in regulating vascular smooth muscle tone is demonstrated in the eNOS knock-out mouse. These animals are invariably hypertensive, and exhibit the vessel wall changes characteristic of chronic hypertension [33]. The hypertension of advancing age is associated with reduced activity of eNOS [1,3,4], as well as reduced bioavailability of NO [4]. Blood pressure increases in direct proportion to plasma fatty acid concentration [5,8,9,38], independent of atherosclerosis [9], by a mechanism which appears to involve reduced synthesis or availability of nitric oxide (NO) [38]. Thus, elucidating the mechanisms that regulate eNOS activation is critical to understanding the regulation of vascular tone as well as the pathogenesis of hypertension.

Endothelial NOS is located predominantly in the caveolae [15], where the structural protein caveolin-1 (cav-1) is the primary regulator of eNOS activity. The two proteins form a heterodimer [19] through interaction at well-defined domains [14,26]. Displacement of cav-1 by Ca2+/calmodulin results in elevation of eNOS catalytic activity, suggesting reciprocal regulation by these proteins [25]. Under basal conditions in stationary cell cultures, more than 95% of eNOS is bound to cav-1 and sequestered in the caveolae [11].

Co-translational, irreversible N-myristoylation and post-translational palmitoylation target newly synthesized eNOS to the caveolae [11,16,31,32]. Agonist-induced activation of eNOS is accompanied by depalmitoylation mediated via a calcium/calmodulin dependent acyl hydrolase [44]. Re-formation of the cav-1/eNOS heterodimer is facilitated by re-palmitoylation [10], by a process that remains poorly understood.

The importance of palmitoylation in the regulation of eNOS activity in intact cells and whole tissues has remained largely unstudied. Both the rate limiting step for eNOS palmitoylation and the effect of inhibiting palmitoylation on NO generation in intact cells remain unknown, as do the functional consequences of inhibiting eNOS palmitoylation in intact blood vessels, whether measured by agonist-induced NO generation or by vascular smooth muscle relaxation.

Recently, a long-chain fatty acyl CoA synthetase that appears to be expressed only in the vascular endothelium (eLCFACoAS) was isolated, cloned and sequenced from rabbits [41]. It is hypothesized here that a function of an eLCFACoAS might be to provide fatty acyl CoA derivatives for use in modification (e.g. palmitoylation) of membrane proteins such as eNOS. If so, then inhibition of eLCFACoAS should increase the duration of cytoplasmic residence of activated eNOS in intact cells and blood vessels. The predicted consequence would be increased duration of eNOS catalytic activity, resulting in increased synthesis of NO. The experiments outlined here were undertaken to test this hypothesis.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1. Cell cultures
Human coronary endothelial cells (passage 3) were purchased from Clonetics (Bethesda, MD), passaged once and re-frozen. These cells (passage 4) were used for all experiments. Cells were grown in EGM-2MV medium (Bio-Whittaker), supplemented with 5% fetal bovine serum. Cells were fed daily until confluent (>95% of the growth surface covered, and <1 mitotic figure per low-power field). Experimental drugs were added directly to the medium.

2.2 [3H]fatty acid incorporation into lipids
Triacsin-C is an inhibitor of long chain fatty acyl CoA synthetase (LCFACoAS) [20,21,27,40]. Its specificity and efficacy in cultured human endothelial cells was tested by measuring the incorporation of tracer quantities of palmitic or arachidonic acid into lipid-extractable material. Confluent cultures in six-well tissue culture cluster plates were re-fed with EGM-2MV medium supplemented with [3H] palmitic (specific activity, 60 Ci/mmol) or [3H] arachidonic acid (specific activity 217 Ci/mmol), with or without 5 µM triacsin-C. The final concentrations of the fatty acids were 7 to 10 nM. The medium contained about 3.7 mg/ml protein (from fetal bovine serum), and no additional albumin was added. Twenty-four hours later, the monolayers were rinsed 3 x with ice-cold calcium–magnesium free phosphate buffered saline (CMFPBS, in mM, 140 NaCl, 2.7 KCl, 10 Na2HPO4, 1.76 KH2PO4, pH 7.4). Subsequently, the cells were scraped into 400 µl of ice cold imidazole buffer (10 mM EGTA, 10 mM imidazole, 100 mM KCl, pH 7.4) and transferred to 15 ml screw-top glass tubes. Lipids were extracted as described [28], and incorporated radioactivity was measured by liquid scintillation spectroscopy.

In one experiment, the lipid classes (phospholipids, monoacylglycerol, diacylglycerol, triacylglycerol, unesterified fatty acids and cholesteryl esters) were separated by thin layer chromatography as described [42]. Zones corresponding to authentic standards were identified with I2 vapor, and radioactivity in each zone was determined by liquid scintillation spectroscopy.

2.3. NOS assay
Total nitric oxide synthase activity was estimated by measuring the conversion of [14C]arginine to [14C] citrulline. Confluent monolayers were washed then scraped into CMFPBS. Cells were pelleted by centrifugation at 7000 x g for 10 min, and then homogenized in 500 µl CMFPBS containing 1 mM EDTA. Cytoplasmic and membrane-bound eNOS were not separated.

Activity was assayed by mixing 10 µl of cell homogenate with 40 µl of NOS assay cocktail (final concentration: 25 mM Tris HCl, pH 7.4, 0.83 µg/ml FAD, 0.94 µg/ml tetrahydrobiopterin, 0.585 µg/ml FMN, 1 mg/ml NADPH, 600 µmol/l CaCl2,1 µCi [14C] arginine (340 mCi/mmol)). After 60 min at 37 °C in a humidified atmosphere, the reaction was terminated by adding 400 mL of stop solution (50 mM HEPES, pH 5.5, 5 mM EDTA). Unreacted substrate was removed by Dowex AG8 resin (Na+ form) chromatography. Product formation was calculated as moles product/min/µg protein.

2.4. Media nitrite determination
Media nitrite was determined by the Greiss method. Briefly, 200 µl samples of tissue culture medium, 40 µl of 4 M HCl, and 30 µl of sulfanilic acid (2 g/l) were pipetted into 96-well microtiter plates and incubated at room temperature for 10 min. Thirty microliters of color reagent (1 mg/ml N-(1-naphthyl)ethylenediamine in distilled demineralized water) were added to each well. After 30 min at room temperature, absorbance at 570 nm was measured on a Tcam plate reader. Sodium nitrite standards (0 to 2 µM in H2O) were used to construct a standard curve. A medium blank was included with each assay.

2.5. eNOS palmitoylation
Confluent monolayers were incubated for 24 h with [3H] palmitic acid (5 mCi/T-25 flask, 60 Ci/mmol; final [3H] palmitate concentration was about 17 µM). The cells were rinsed 3 x with ice cold CMFPBS, scraped into 1 ml ice cold CMFPBS, pelleted by centrifugation (7000 x g for 10 min), then homogenized in 50 µl NOS homogenization buffer (vide supra). Immunoblots of proteins in 10 µl of homogenate were prepared, visualized with BCIP/NBT (immunoreactive protein), then exposed to the tritium screen (radioactivity) of a BioRad GS525 image analysis system. The radioactivity image and the immunoreactivity image were captured and analyzed by densitometry.

2.6. Methacholine induced NO synthesis
Male Sprague–Dawley rats (Ace Animals, Boyertown, PA, USA) were anesthetized with pentobarbital sodium (50 mg/kg, IP), and the thoracic aortas were isolated and immersed in 37 °C Krebs buffer (KHB, in mM: 10 dextrose, 119 NaCl, 12.5 NaHCO3, 2.5 CaCl2, 4.8 KCl, 1.2 KH2PO4, and 1.2 MgSO4, equilibrated with 95%O2/5% CO2). Vessels were cleaned of adherent fat and connective tissue and rings 6 to 7 mm in length (i.e. approximately 15 mg of tissue wet weight) were carefully cut, opened, and fixed (with the endothelial surface facing up) by small pins in 24-well culture dishes containing 1 ml of Krebs buffer. After equilibration at 37 °C, triacsin-C (5 µM) or vehicle (DMSO, 0.1%) was added and the incubation was continued for another 15 min. NO was measured using a calibrated NO meter (Iso-NO; World Precision Instruments, Sarasota FL, USA) connected to a polarographic, internally shielded NO electrode positioned within 1 mm of the endothelium as described previously [45,46]. An NO release blank was determined by placing the electrode in Krebs buffer. The probe was then placed in the wells containing aortic tissue; the difference between the two readings was used to calculate basal NO release. Methacholine (MeCh, 5 µM) was added to the tissue and NO release was measured for 5 min. After stimulation, 400 µM L-NAME was added to both the control and experimental wells; MeCh-induced NO release was reassessed 30 min later. Data were captured on the Iso-NO data acquisition system; peak height and area under the curve were determined using the Iso-NO software package.

2.7. Methacholine-induced vascular smooth muscle relaxation
Female Sprague–Dawley rats (Zivic Miller Laboratories, Zelienople, PA, USA) were sacrificed by rapid decapitation, and the thoracic aortae were removed and placed in chilled, Krebs–Henseleit–bicarbonate solution (KHB) (4 °C), gassed with 95%O2/5%CO2. After careful removal of fat and adventitia, the vessels were cut into 3 mm segments and mounted in tissue baths containing warmed gassed KHB (37 °C) for measurement of isometric tension using standard methods [35–37]. Passive force was adjusted to optimal levels, and a 90-min equilibration was allowed prior to experimental intervention. Following the equilibration period, the aortae were stabilized by a near-maximal contraction with phenylephrine (PE, 10–6 M final concentration). Once the aortas achieved a stable plateau force, the functional integrity of the endothelium was evaluated by determining the response to 0.1 µM acetylcholine (ACh). The baths were rinsed twice and the aortas were re-equilibrated in fresh KHB for 30 to 45 min before adding vehicle (0.1% DMSO), triacsin-C (5 µM), vehicle+L-NAME (100 or 250 µM) or vehicle+L-NAME+triacsin-C to the baths. Fifteen minutes later, the rings were pre-contracted with PE to 75% of maximal force, before adding MeCh (final concentration 10–11–10–5 M) to the tissue baths in a cumulative manner. At the end of the experiment, the aortas were dried to a constant weight; contractile force was normalized per mg aortic ring dry weight, as in previous studies [35–37].

2.8. Protein determination
Protein was determined by the method of Lowry. Samples containing NP-40 were treated with SDS prior to assay to prevent precipitation of detergent by the Lowry reagent.

2.9. Materials
Triacsin-C, L-NAME and sterile, tissue-culture grade DMSO were purchased from Sigma, St. Louis, MO. Triacsin-C was prepared as a 5 mM stock solution in DMSO and stored at –20 °C. L-NAME was prepared fresh daily as a 10 mM stock solution in CMF-PBS. All other chemicals were of reagent grade or better. Polyclonal eNOS antibody was purchased from Cayman Chemical, Ann Arbor, MI, USA (Cat #160880), and used as per manufacturer's instructions.

2.10. Statistical analysis of data
All data are presented as the mean±S.E.M. For the NO release experiments, the treated and untreated aortic segments were compared by Student's t-test for paired data. For the vascular relaxation experiments, data were analyzed by non-linear regression analysis (ALLFIT program) to determine maximal response and effective dose for 50% response (ED50 [6]). Data from cultured endothelial cell experiments were compared by Student's t-test for paired or unpaired data (as appropriate) or by one-way ANOVA. Differences were considered significant at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Incorporation of radioactivity
Triacsin-C (5 µM) significantly reduced the incorporation of [3H] palmitic acid, while the incorporation of [3H] arachidonic acid was unchanged (Fig. 1). Even at 30 min, the earliest time point tested, palmitate incorporation in the presence of triacsin-C was only 41.7±1.8% of control (p<0.05; n=cells from 3 different donors).


Figure 1
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 The effect of triacsin-C on incorporation of palmitic or arachidonic acid into extractable lipids. At both 30 min and 24 h, the incorporation of palmitate, but not arachidonate, was significantly reduced. Open bars, 0.1% DMSO vehicle control; shaded bars, 5 µM triacsin-C. *p<0.05 (Triacsin-C vs. Control); N.S.=not significant. Bars represent mean±S.E.M.; n=cells from each of three donors, each determined in triplicate.

 
The effect of triacsin-C on lipid class distribution of [3H] palmitic acid is shown in Table 1. In general, triacsin-C treatment reduced the absolute amount of radioactivity associated with a lipid class, but did not change the distribution of radioactivity among the lipid classes. Triacsin-C treated and control cells had equal [3H] palmitate associated with diacylglycerol (5.44±0.856 nCi treated vs. 7.48±3.8 nCi control; p=0.56); when expressed as a fraction of the total incorporated radioactivity, these correspond to 5.63%±0.89% for triacsin-C treated vs. 1.30±0.73% for control cultures.


View this table:
[in this window]
[in a new window]

  		Table 1 The effect of triacsin-C (5 µM) on the incorporation and percent distribution of radioactivity from cultured human coronary endothelial cells

 
3.2. Triacsin-C and basal nitrite accumulation
The effect of triacsin-C on basal nitrite accumulation is shown in Fig. 2. Palmitic acid (60 µM) did not change the accumulation of nitrite in the medium compared to control (P>0.50). When both palmitic acid and triacsin-C (5 µM) were included in the media, the 24-h nitrite accumulation increased approximately 2.5-fold (P<0.05). When palmitic acid, triacsin-C and L-NAME (100 µM) were all included, the 24-h nitrite accumulation did not differ from control (P>0.50). In a separate experiment without palmitic acid, basal nitrite concentrations were 518±78 nM in control, 451±150 nM in L-NAME treated cultures and 1060±151 in triacsin-C treated cultures (n=cells from 3 donors, 3 flasks per donor; P>0.25 L-NAME vs. control; P<0.05 triacsin-C vs. control).


Figure 2
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 The effect of 5 µM triacsin-C and 60 µM palmitic acid on 24-h medium nitrite accumulation. Palmitic acid alone had no effect on medium nitrite accumulation. Triacsin-C significantly increased 24-h medium nitrite accumulation, an effect that was prevented by 100 µM L-NAME. *p<0.05 (Triacsin-C+Palmitate vs. all other groups). n=4 flasks from a single donor.

 
The accumulation of nitrite was measured in the medium of cells cultured in the presence of 0 to 10 µM triacsin-C (Fig. 3). The 24-h accumulation of media nitrite increased in a concentration-dependent fashion, with the maximal effect observed at 5 to 10 µM triacsin-C.


Figure 3
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 The in vivo production of nitrite by cells cultured for 24 h in the presence of increasing concentrations of triacsin-C. Medium nitrite concentrations increased in proportion to the concentration of triacsin-C. 0F0194 and 0F1041 represent cells from two different donors. Bars represent mean±S.E.M.; n=3 culture flasks from each donor.

 
3.3. Effect of triacsin-C on NOS activity and expression
The effect of triacsin-C on nitrite accumulation could be explained either by an increase in total NOS activity or by an increase in the fraction of NOS that is catalytically active under basal conditions. These two possibilities were differentiated by measuring NOS activity in homogenates of cells treated with 5 µM triacsin-C or its vehicle (DMSO). Total NOS activity from cells cultured in the presence of 5 µM triacsin-C was 22.2±4.32 fmol/min/µg protein, not significantly different from the 22.2±0.23 fmol/min/µg protein observed in cells cultured in the presence of DMSO alone. As shown in Fig. 4, immunoreactive eNOS was not changed in cells treated with triacsin-C. In a separate study, neither palmitate (50 µM) nor triacsin-C (5 µM) had any effect on total immunoreactive eNOS (data not shown).


Figure 4
View larger version (55K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 A representative experiment showing the effect of 5 µM triacsin-C on incorporation of [3H] palmitic acid into immunoreactive eNOS. Cultured human coronary endothelial cells were incubated in the presence of [3H] palmitic acid (5 mCi/5 ml/flask, 60 mCi/mmol; chemical concentration=16.6 µM) for 24 h. Lanes 1 and 4, 5 µM triacsin-C. Lanes 2 and 5, vehicle-control (0.1% DMSO). Lane 3, authentic eNOS standard. Panel A, incorporation of [3H] palmitate. Panel B, immunoreactive eNOS. Panel C, results of densitometric scan of panel A ({Delta} optical density compared to background). Panel D, results of densitometric scan of lanes 1, 2, 4 and 5 of panel B ({Delta} optical density compared to authentic eNOS standard). Triacsin-C reduced the radioactivity associated with eNOS by about 65%, but had no effect on the amount of immunoreactive eNOS.

 
3.4 Effect of triacsin-C on [3H] palmitate incorporation into eNOS
The previous experiment indicated that triacsin-C does not increase total in vitro NOS activity in homogenates of cultured human coronary endothelial cells. However, triacsin-C could increase basal eNOS activity by inhibiting palmitoylation, delaying caveolar re-sequestration. To test this hypothesis, palmitoylation of eNOS was measured directly. Cells cultured in the presence of [3H] palmitic acid with or without 5 µM triacsin-C were immunoblotted for eNOS. Both immunoreactivity and radioactivity on the blots were visualized and quantified by densitometry. As shown in Fig. 4, triacsin-C reduced the radioactivity associated with eNOS, but had no effect on the amount of immunoreactive eNOS. Triacsin-C reduced incorporation of [3H] palmitate into immunoreactive eNOS from 37.5±2.56 relative units (ratio of tritium scan to immunoreactive scan) to 11.7±2.96 relative units (n=cells from three donors; P<0.05).

3.5. Effect of triacsin-C on methacholine-stimulated NO synthesis
The previous experiments examined the role of LCFACoAS in the basal synthesis of NO in cultured cells. The effect of inhibiting LCFACoAS on MeCh (5 µM)-stimulated eNOS activity was evaluated in endothelium-intact segments of rat aorta. As shown in Fig. 5 (upper panel), pre-treatment with triacsin-C did not alter peak NO synthesis, but did prolong the duration of the NO response. The area under the NO vs. time curve was nearly three times greater in the presence of 5 µM triacsin-C (Fig. 5, lower panel). MeCh-stimulated NO synthesis was completely abolished by L-NAME (400 µM) whether or not triacsin-C was present.


Figure 5
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 The effect of triacsin-C on MeCh-induced synthesis of nitric oxide in segments of rat aorta, measured with an internally shielded NO electrode. Upper panel: representative tracings of MeCh-induced NO release in control (left) and triacsin-C-treated (right) aortic segments from the same animal. Lower panel: the areas under the MeCh-stimulated NO concentration curves were computed. *p<0.05 by Student's t-test for paired data; n=8 animals. When 400 µM L-NAME was present, MeCh-stimulated NO synthesis was undetectable (data not shown).

 
3.6. Effect of triacsin-C on methacholine stimulated vascular smooth muscle relaxation
The previous experiment showed that MeCh stimulated NO generation is increased by 5 µM triacsin-C. The ability of that enhanced NO release to relax vascular smooth muscle was tested in a series of experiments on rat thoracic aortic rings pre-contracted with phenylephrine. The concentration–response curves for MeCh-induced relaxation in the presence or absence of triacsin-C (5 µM) and/or L-NAME (100 µM) are shown in Fig. 6. In the presence of triacsin-C, the EC50 for MeCh-induced relaxation was 39.9±9.2 nM for control rings, and 18.2±4.8 nM for triacsin-C-treated rings (n=6; p<0.05), indicating an increased sensitivity to MeCh, consistent with increased MeCh-induced NO synthesis. Pretreatment of aortic rings with L-NAME (100 µM) virtually abolished relaxation to MeCh in control aortae and significantly attenuated the MeCh-induced response in the presence of triacsin-C. When the L-NAME concentration was increased to 250 µM, the triacsin-C mediated enhancement of MeCh-induced relaxation was abolished.


Figure 6
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 The effect of triacsin-C on MeCh-induced vascular relaxation of isolated, endothelium intact rat aortic rings. In control tissues, 50% relaxation was achieved at 39.9±9.2 nM MeCh. In the presence of 5 µM triacsin-C, that concentration was significantly reduced to 18.2±4.8 nM (*p<0.01 by one-way ANOVA). 100 µM L-NAME abolished the MeCh-induced relaxation (#p<0.01 vs. control by one-way ANOVA) in the absence of triacsin-C, and significantly attenuated the MeCh-induced response in the presence of triacsin-C ({dagger}p<0.01 vs. triacsin-C by one-way ANOVA). 250 µM L-NAME completely abolished the effect of triacsin-C to enhance MeCh-induced relaxation.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The evidence for reciprocal regulation of eNOS by cav-1 and Ca2+/calmodulin is overwhelming. Overexpression of cav-1 attenuates eNOS activity [25]; further, cav-1 null mice exhibit enhanced endothelium-dependent responses [7,30]. Acylation and phosphorylation appear to play supporting roles in eNOS function, by directing the nascent protein to the caveolar membrane (myristoylation [2,16,32]), facilitating membrane/cytoplasmic cycling (palmitoylation [10]), and increasing or decreasing intrinsic activity (phosphorylation [13,16]).

A Ca2+/calmodulin dependent acyl hydrolase is co-activated with eNOS [44]. The re-formation of the eNOS/cav-1 heterodimer in palmitoylation-incompetent (palm–) eNOS mutants is delayed following stimulation with carbachol [10]. More than 75% of palm– eNOS is present in the cytoplasmic fraction in the unstimulated cell, and is catalytically active in an in vitro assay [11]. However, to our knowledge, the present experiments are the first to determine the functional consequences of inhibiting eNOS palmitoylation in intact endothelial cells.

Triacsin-C inhibition of LCFACoAS has been amply demonstrated by others [20,21,27,40]. Fatty acid uptake is diffusion mediated, and so is unlikely to be affected by triacsin-C or any other drug. In the present study, triacsin-C reduced the incorporation of [3H] palmitate but not [3H]arachidonate into extractable lipids, and, in general, the inhibition was uniform over the various lipid classes. This finding confirms that triacsin-C inhibits synthesis of palmitoyl CoA in human coronary endothelial cells, and is consistent with observations [23] that palmitate and arachidonate are activated by different CoA synthetases.

The absolute molar quantity of [3H] palmitate in diacylglycerol did not differ between experimental and control cells. This suggests that the flux of palmitate through diacylglycerol is not dependent upon the formation of the CoA derivative of palmitic acid, and may be mediated by a CoA-independent transacylase. While this is an interesting speculation, its resolution is beyond the scope of the present study.

Free fatty acids did not accumulate in triacsin-C treated cells. In the presence of triacsin-C, unesterified intracellular free fatty acid simply diffuses out [43]. As unesterified fatty acids are very low at all times, and since the lipid extraction process itself generates free fatty acids, it is expected that this fraction will be quite variable. As shown in Table 1, the variability of the free fatty acid fraction is very large for both the treated and control cells. In any case, even if triacsin-C were acting by any mechanism other than inhibition of fatty acyl CoA formation, the data still demonstrate that triacsin-C treatment reduces palmitoylation of eNOS and increases in vivo eNOS catalytic activity, both basal and agonist stimulated.

If inhibiting palmitoylation prolongs the eNOS activity cycle, then triacsin-C treatment would be expected to increase nitrite production without changing the total (cytoplasmic+membrane bound) eNOS present in cultured endothelial cells. There was no significant difference between control and triacsin-C treated cells for immunoreactive eNOS (Fig. 4, panels B and D) or total in vitro NOS catalytic activity, consistent with the interpretation that triacsin-C treatment does not change eNOS expression. Furthermore, the experiments measuring agonist induced NO generation in the presence and absence of triacsin-C (see Fig. 5) were performed after 15–30 min pre-treatment with triacsin-C. This time frame is sufficient for the inhibition of LCFACoAS (see Fig. 1), but is too brief to permit up-regulation at the transcriptional or translational level.

The effect of triacsin-C on iNOS expression was not measured in these studies. However, as triacsin-C inhibits transcription of iNOS mRNA in isolated pancreatic islets of Zucker obese diabetic rats [34], it is unlikely that iNOS expression accounts for the increased 24 h basal nitrite accumulation. Further, the iNOS promoter contains an HNF4{alpha} response element [17], a transcription factor whose endogenous ligand is palmitoyl CoA [18,29]. Thus, it would be expected that inhibition of palmitoyl CoA formation would either have no effect on iNOS transcription, or would decrease it.

When the effect of triacsin-C on the incorporation of [3H] palmitic acid into eNOS was measured, significantly less radioactivity was associated with immunoreactive eNOS in the triacsin-C-treated cells. These results are consistent with the notion that palmitoyl CoA availability is rate-limiting in eNOS palmitoylation and suggest that limiting eNOS re-palmitoylation increases the catalytically active fraction of eNOS.

Twenty-four-hour nitrite accumulation in the medium of cells treated with triacsin-C+60 µM palmitate was significantly greater than in the vehicle-control. Since L-NAME co-treatment prevented the increase in nitrite accumulation, it is inferred that triacsin-C treatment increases NOS activity (or NO release). The effect was concentration-dependent, and was observed even when palmitate was omitted from the medium (Fig. 3). That L-NAME treatment did not reduce NO synthesis below basal levels is consistent with the observation that L-NAA does not change basal nitrite accumulation in stationary (i.e. not subjected to shear stress) endothelial cell cultures [22].

It was somewhat surprising to discover that 60 µM palmitic acid did not significantly reduce basal nitrite accumulation. However, in unstimulated stationary endothelial cell cultures, more than 95% of eNOS is membrane-bound, and catalytically inactive [10]. Perhaps then, the basal levels of eNOS activity in vivo cannot be further depressed by providing palmitate substrate. Others [5] have noted that exogenous oleic and linoleic acids (10 to 100 µM) suppress eNOS activation in cultured bovine aortic endothelial cells. The current study differs substantively in that agonist-induced activity was measured over 15 min in the former studies, while in the present experiments basal activity was measured over 24 h.

If triacsin-C is indeed acting to delay repalmitoylation and re-sequestration of eNOS in the caveolar membrane, then it is necessary for eNOS to be deacylated in order to observe an effect. The effect of triacsin-C on cultured cells was to more than double basal nitrite accumulation over 24 h, suggesting that the basal deacylation/reacylation rate is slow. Thus, it is not surprising that an effect on basal activity was not observed in the endothelium-intact rat aorta during the 15 min pre-treatment before challenge with MeCh. Furthermore, the absence of a short-term effect of triacsin-C on basal NO synthesis suggests that the compound is not activating protein kinase(s), thereby directly activating eNOS (vide infra).

Triacsin-C treatment prolonged duration of MeCh stimulated NO synthesis, without changing peak NO output, suggesting that triacsin-C prolonged the active period of eNOS. The 55% reduction in MeCh EC50 in the presence of triacsin-C is consistent with prolonged eNOS activity consequent to delayed re-sequestration. As blood vessel resistance is inversely proportional to the 4th power of the radius, even small changes in vascular reactivity would have very large consequences for resistance and flow. The effect of triacsin-C treatment on vascular smooth muscle relaxation demonstrates that the increased NO generated is physiologically available and sufficient to decrease vessel tone.

The effect of triacsin-C is likely mediated through its inhibition of long chain fatty acyl CoA synthetase activity [20,21,27,40] rather than by modulating eNOS phosphorylation. In response to at least some agonists, eNOS is phosphorylated [12,13,16]. Protein kinase Akt mediates phosphorylation at S1179; however palm– eNOS is not phosphorylated by this pathway [12,13,16]. Triacsin-C reduces eNOS palmitoylation, thus making Akt phosphorylation less likely. eNOS catalytic activity is decreased by phosphorylation at T495 [13]. The membrane anchorage requirement for phosphorylation at this site is unknown. Furthermore, in vitro catalytic activity of NOS in whole homogenates of triacsin-C treated cells was not different from control cells. If triacsin-C affected eNOS phosphorylation, either directly or indirectly, one would expect greater in vitro catalytic activity in the homogenates of the treated cells.

In any case, the preponderance of studies show that the major regulator of eNOS activation/deactivation is cav-1, an event that is facilitated by, though not absolutely dependent upon, palmitoylation. Thus it is feasible that delayed eNOS/cav-1 interaction consequent upon reduced rates of re-palmitoylation could prolong eNOS catalytic activity.

More than 95% of eNOS is bound (in an inactive state) to the caveolar membrane in quiescent cultures [10]. When shear force is applied to cultures, the steady state partitioning of eNOS between the caveolar membrane and the cytoplasm shifts in direct proportion to the magnitude of the shear force [22]. The ability of eNOS activity to respond to changes in shear force generated by changes in blood flow is one of the primary mechanisms for autoregulation of blood flow in the resistance vessels. As shear force in a vessel increases, eNOS is activated and moves from the membrane to the cytoplasm. When the shear force decreases, eNOS shifts from the cytoplasm back to the membrane. This last step is facilitated by palmitoylation [10]. As plasma fatty acids (i.e. palmitic acid) increase, the high substrate availability would favor eNOS palmitoylation, facilitating re-sequestration and inactivation of eNOS in the caveolar membrane.

The clinical implications of the present findings may be significant. Hypertension in humans is associated with increased plasma fatty acid concentrations [8,24,39]. Although many of these studies have focused on the role of plasma fatty acids in obesity/hypertension/insulin resistance (syndrome X), there are data indicating that the effects of plasma fatty acids on blood pressure may be mediated, in part, by inhibition of eNOS [5,8,38]. To date, a mechanism directly linking elevated plasma fatty acids to reduced eNOS activity has not been proposed. The finding that endothelial cell long chain fatty acyl CoA synthetase activity may be directly linked to eNOS activity suggests a mechanism by which plasma fatty acids could affect vascular smooth muscle tone. The studies reported here are consistent with the notion that reducing endothelial LCFACoAS activity inhibits re-palmitoylation of eNOS, slowing the re-sequestration of eNOS and increasing NO generation, and demonstrate that inhibition of LCFACoAS enhances NO dependent vascular smooth muscle relaxation.


   Notes
 
{star} Supported by NIH HL48204, NIH AG022614 [GenBank] , and Women's Health Research Institute of Amarillo.

Time for Primary Review 25 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Andrawis N, Jones D, Abernathy D. Aging is associated with endothelial dysfunction in the human forearm vasculature. J. Am. Geriatr. Soc. (2000) 48:193–198.[Web of Science][Medline]
  2. Busconi L, Michel T. Endothelial nitric oxide synthase: N-terminal myristoylation determines subcellular localization. J. Biol. Chem. (1993) 268:8410–8413.[Abstract/Free Full Text]
  3. Cerñadas M, Sanchez de Miguel L, Garcia-Duran M, et al. Expression of constituitive and inducible nitric oxide synthases in the vascular wall of young and aging rats. Circ. Res. (1998) 83:279–286.[Abstract/Free Full Text]
  4. Csiszar A, Ungvari Z, Edwards J.G, et al. Aging-induced phenotypic changes and oxidative stress impair coronary arteriolar function. Circ. Res. (2002) 90:1159–1166.[Abstract/Free Full Text]
  5. Davda R.K, Stepniakowski K.T, Lu G, Ullian M.E, Goodfriend T.L, Egan B.M. Oleic acid inhibits endothelial nitric oxide synthase by a protein kinase C-independent mechanism. Hypertension (1995) 26:764–770.[Abstract/Free Full Text]
  6. DeLean A, Munson P.J, Rodbard D. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose–response curves. Am. J. Physiol. (1978) 235:E97–E102.[Web of Science][Medline]
  7. Drab M, Verkade P, Elger M, et al. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science (2001) 293:2449–2452.[Abstract/Free Full Text]
  8. Egan B.M, Greene E.L, Goodfriend T.L. Nonesterified fatty acids in blood pressure control and cardiovascular complications. Curr. Hypertens. Rep. (2001) 3:107–116.[Medline]
  9. Fagot-Campagna A, Balkau B, Simon D, et al. High free fatty acid concentration: an independent risk factor for hypertension in the Paris Prospective Study. Int. J. Epidemiol. (1998) 27:808–813.[Abstract/Free Full Text]
  10. Feron O, Michel J.B, Sase K, Michel T. Dynamic regulation of endothelial nitric oxide synthase: complementary roles of dual acylation and caveolin interactions. Biochemistry (1998) 37:193–200.[CrossRef][Web of Science][Medline]
  11. Feron O, Saldana F, Michel J.B, Michel T. The endothelial nitric-oxide synthase–caveolin regulatory cycle. J. Biol. Chem. (1998) 273:3125–3128.[Abstract/Free Full Text]
  12. Fulton D, Gratton J.P, McCabe T.J, et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature (1999) 399:597–601.[CrossRef][Medline]
  13. Fulton D, Gratton J.-P, Sessa W.C. Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough? J. Pharmacol. Exp. Ther. (2001) 299:818–824.[Abstract/Free Full Text]
  14. García-Cardeñas G, Martasek P, Masters B.S.S, et al. Dissecting the interaction between nitric oxide synthase and caveolin. J. Biol. Chem. (1997) 272:25437–25440.[Abstract/Free Full Text]
  15. Goligorsky M.S, Li H, Brodsky S, Chen J. Relationships between caveolae and eNOS: everything in proximity and the proximity of everything. Am. J. Physiol. (2002) 283:F1–F10.[Web of Science]
  16. Gonzalez E, Kou R, Lin A.J, et al. Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase. J. Biol. Chem. (2002) 277:39554–39560.[Abstract/Free Full Text]
  17. Guo H, Cai C.Q, Kuo P.C. Hepatocyte nuclear factor-4{alpha} mediates redox sensitivity of inducible nitric-oxide synthase gene transcription. J. Biol. Chem. (2002) 277:5054–5060.[Abstract/Free Full Text]
  18. Hertz R, Magenheim J, Berman I, Bar-Tana J. Fatty acyl Co-A thioesters are ligands of hepatic nuclear factor-4{alpha}. Nature (1998) 392:512–516.[CrossRef][Medline]
  19. Ju H, Zou R, Venema V.J, Venema R.C. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J. Biol. Chem. (1997) 272:18522–18525.[Abstract/Free Full Text]
  20. Knoll L.J, Schall O.F, Suzuki I, Gokel G.W, Gordon J.I. Comparison of the reactivity of tetradecenoic acids, a triacsin and unsaturated oximes with four purified Saccharomyces cerevisiae fatty acid activation proteins. J. Biol. Chem. (1995) 270:20090–20097.[Abstract/Free Full Text]
  21. Korchak H.M, Kane L.H, Rossi M.W, Corkey B.E. Long chain acyl coenzyme A signaling in neutrophils: an inhibitor of acyl coenzyme A synthetase, triacsin-C, inhibits superoxide anion generation and degranulation by human neutrophils. J. Biol. Chem. (1994) 269:30281–30287.[Abstract/Free Full Text]
  22. Kuchan M.J, Frangos J.A. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am. J. Physiol. (1994) 266:C628–C636.[Web of Science][Medline]
  23. Laposata M, Reich E.L, Majerus P.W. Arachidonoyl-CoA synthetase, separation from non-specific acyl-CoA synthetase and distribution in various cells and tissues. J. Biol. Chem. (1985) 260:11016–11020.[Abstract/Free Full Text]
  24. Lopes H.F, Stojiljkovic M.P, Zhang D, Goodfriend T.L, Egan B.M. The pressor response to acute hyperlipidemia is enhanced in lean normotensive offspring of hypertensive parents. Am. J. Hypertens. (2001) 14:1032–1037.[CrossRef][Web of Science][Medline]
  25. Michel J.B, Feron O, Sacks D, Michel T. Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J. Biol. Chem. (1997) 272:15583–15586.[Abstract/Free Full Text]
  26. Michel J.B, Feron O, Sase K, Prabhakar P, Michel T. Caveolin versus calmodulin—counterbalancing allosteric modulators of endothelial nitric oxide synthase. J. Biol. Chem. (1997) 272:25907–25912.[Abstract/Free Full Text]
  27. Noel R.J, Antinozzi P.A, McGarry D.J, Newgard C.B. Engineering of glycerol-stimulated insulin secretion in islet beta cells: differential metabolic fates of glucose and glycerol provide insight into mechanisms of stimulus-secretion coupling. J. Biol. Chem. (1997) 272:18621–18627.[Abstract/Free Full Text]
  28. Palazzo A.J, Malik K.U, Weis M.T. Vasopressin stimulates the mobilization and metabolism of triacylglycerol in perfused rabbit hearts. Am. J. Physiol. (1991) 260:H604–H612.[Web of Science][Medline]
  29. Petrescu A.D, Hertz R, Bar-Tana J, Schroeder F, Kier A.B. Ligand specificity and conformational dependence of the hepatic nuclear factor-4{alpha} (HNF-4{alpha}). J. Biol. Chem. (2002) 277:23988–23999.[Abstract/Free Full Text]
  30. Razani B, Engelman J.A, Wang X.B, et al. Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. J. Biol. Chem. (2001) 276:38121–38138.[Abstract/Free Full Text]
  31. Robinson L.J, Busconi L, Michel T. Agonist-modulated palmitoylation of endothelial nitric oxide synthase. J. Biol. Chem. (1995) 270:995–998.[Abstract/Free Full Text]
  32. Robinson L.J, Michel T. Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting. Proc. Natl. Acad. Sci. U. S. A. (1995) 92:11776–11780.[Abstract/Free Full Text]
  33. Shesely E.G, Maeda N, Kim H.-S, et al. Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc. Natl. Acad. Sci. U. S. A. (1996) 93:13176–13181.[Abstract/Free Full Text]
  34. Shimabukuo M, Zhou Y.-T, Levi M, Unger R.H. Fatty acid-induced β cell apoptosis: a link between obesity and diabetes. Proc. Natl. Acad. Sci. U. S. A. (1998) 95:2498–2502.[Abstract/Free Full Text]
  35. Stallone J.N. Role of endothelium in sexual dimorphism in vasopressin-induced contraction of rat aorta. Am. J. Physiol. (1993) 265:H2073–H2080.[Web of Science][Medline]
  36. Stallone J.N. Sex differences in nitric oxide-mediated attentuation of vascular reactivity to vasopressin are abolished by gonadectomy. Eur. J. Pharmacol. (1994) 259:273–283.[Web of Science][Medline]
  37. Stallone J.N, Crofton J.T, Share L. Sexual dimorphism in vasopressin-induced contraction of rat aorta. Am. J. Physiol. (1991) 260:H453–H458.[Web of Science][Medline]
  38. Steinberg H.O, Tarshoby M, Monestel R, et al. Elevated circulating free fatty acid levels impair endothelium-dependent vasodilation. J. Clin. Invest. (1997) 100:1230–1239.[Web of Science][Medline]
  39. Stojiljkovic M.P, Zhang D, Lopes H.F, Lee C.G, Goodfriend T.L, Egan B.M. Hemodynamic effects of lipids in humans. Am. J. Physiol. (2001) 280:R1674–R1679.[Web of Science]
  40. Tomoda H, Igarishi K, Cyong J.-C, Omura S. Evidence for an essential role of long chain acyl CoA synthetase in animal cell proliferation: inhibition of long chain acyl CoA synthetase by triacsins caused inhibitions of Raji cell proliferation. J. Biol. Chem. (1991) 266:4214–4219.[Abstract/Free Full Text]
  41. Uberti M.A, Pierce J.C, Weis M.T. Molecular characterization of a rabbit long-chain fatty acyl CoA synthetase that is highly expressed in the vascular endothelium. Biochim. Biophys. Acta (2003) 1645:193–204.[Medline]
  42. Weis M.T, Malik K.U. Uptake and hormonally induced hydrolysis of [3H]phosphatidyl choline in isolated rabbit heart. J. Pharmacol. Exp. Ther. (1989) 248:614–620.[Abstract/Free Full Text]
  43. Yaney G.C, Civelek V.N, Richard A.M, et al. Glucagon-like peptide stimulates lipolysis in clonal pancreatic beta cells (HIT). Diabetes (2001) 50:56–62.[Abstract/Free Full Text]
  44. Yeh D.C, Duncan J.A, Yamashita S, Michel T. Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca2+-calmodulin. J. Biol. Chem. (1999) 274:33148–33154.[Abstract/Free Full Text]
  45. Young L.H, Ikeda Y, Lefer A.M. Caveolin-1 peptide exerts cardioprotective effects in myocardial ischemia–reperfusion via nitric oxide mechanism. Am. J. Physiol. (2001) 280:H2489–H2495.[Web of Science]
  46. Young L.H, Ikeda Y, Scalia R, Lefer A.M. C-peptide exerts cardioprotective effects in myocardial ischemia–reperfusion. Am. J. Physiol. (2000) 279:H1453–H1459.[Web of Science]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
O. Feron and J.-L. Balligand
Caveolins and the regulation of endothelial nitric oxide synthase in the heart
Cardiovasc Res, March 1, 2006; 69(4): 788 - 797.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Weis, M. T
Right arrow Articles by Stallone, J. N
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Weis, M. T
Right arrow Articles by Stallone, J. N
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?