Endothelin-1- and endothelin-3-induced vasorelaxation via common generation of endothelium-derived nitric oxide.

The vasorelaxant effects by endothelin-1 (ET-1) and endothelin-3 (ET-3), and their mechanisms of action were studied in isolated porcine pulmonary arterial strips. ET-1 and ET-3 dose-dependently (10(-9) - 10(-8) M) relaxed vascular strips precontracted with norepinephrine only in the presence of endothelium. The maximal vasorelaxant effect by ET-1 was about 70% of that by ET-3. The ET-1- and ET-3- induced vasorelaxation was blocked by NG-nitro-L-arginine, an inhibitor of nitric oxide synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The present data suggest that vascular smooth muscle relaxation induced by ET-1 and ET-3 is mainly ascribed to synthesis and release of nitric oxide from L-arginine in endothelium.     

Insulin stimulates production of nitric oxide via ERK in osteoblast cells

We explored to determine if iNOS could be induced by insulin in osteoblast-like UMR-106 cells. Insulin (100 nM) stimulated nitric oxide production by twofold and significantly increased iNOS mRNA and protein levels. Insulin also increased collagen synthesis, but had little effect on alkaline phosphatase activity. In contrast, IGF-1 had little effect on NO production below 10 nM and it stimulated NO production by only 57% at 100 nM. IGF-1 had little effect on collagen levels, whereas it inhibited alkaline phosphatase activities in a dose-dependent manner. When an MEK inhibitor was preincubated, insulin failed to stimulate NO production, whereas insulin dramatically increased NO production in the ERK1 overexpressed cells. Taken together, it is proposed that insulin increases iNOS mRNA, iNOS protein, and NO production, possibly via activation of ERK. These may play an important role in osteoblast functions such as collagen synthesis.     

Pharmacology of endothelium-derived nitric oxide and nitrovasodilators.

Nitric oxide is the active chemical species responsible for the vasodilator action of nitroglycerin, nitroprusside, and related nitrovasodilators. The most potent vasodilator and inhibitor of platelet aggregation known, nitric oxide was recently discovered to occur endogenously as the endothelium-derived relaxing factor. The pharmacology of endothelium-derived nitric oxide is virtually identical to that of the clinically used nitrovasodilators. Although endothelium-derived relaxing factor or endothelium-derived nitric oxide seems to be important in animals, its significance in humans still needs to be shown. We review the recent discoveries in the identification, biosynthesis, metabolism, and biologic actions of endothelium-derived nitric oxide, its significance in humans, and its relation to the clinically used nitrovasodilators.     

Insulin stimulates nitric oxide production in rat adipocytes

In adipocytes, insulin regulates the activity of different protein kinases (PI3K/Akt, MAPK, PKC) and protein phosphatases (PP-1, PP-2A). Since these enzymes are implicated in the regulation of NOS activity which is present in adipose tissue, we tested the effects of insulin on white adipocyte NOS activity. Exposure of adipocytes to insulin resulted simultaneously in NOS activity stimulation and Akt activation with maximal effect observed at 1 nM. Higher concentrations of insulin induced a progressive decline of NOS activity. In the presence of wortmannin, a PI3K inhibitor, 1 nM insulin failed to stimulate NOS activity. Insulin (1 nM)-stimulated NOS activity was also abolished by U0126, an inhibitor of p42/p44 MAPK activation, and by 1 microM okadaic acid (OA), which inhibits both PP-1 and PP-2A but not by 1 nM OA which inhibits only PP-2A. Moreover, inhibition of cPKC allowed a high (1 microM) insulin concentration to stimulate NOS activity. These results (i) demonstrate that insulin activates NO production in adipocytes through both PI3K/Akt and MAPK/PP-1 activation and (ii) suggest that PP-1 activation protects NOS against the inhibitory effect of cPKC activation.     

Endothelin-1 stimulates phosphoinositide hydrolysis in the rat pineal gland

The presence of functional endothelin receptors and their signal transduction mechanism has not been determined so far in the pineal gland. We examined the effect of endothelin-1 (ET-1) on phosphoinositide turnover in whole pineal gland. Endothelin-1 increased monophosphate accumulation in a dose-dependent manner. The phosphoinositide (PI) response elicited by ET-1 was dependent on the presence of extracellular Ca (++) since its chelation resulted in a marked decrease in ET-1-stimulated InsP(1) accumulation. On the contrary, phosphoinositide hydrolysis was not changed by the calcium blocker amlodipine. ET-1 induced PI breakdown was inhibited by neomycin, an inhibitor of phospholipase C. However, mastoparan 7, a G protein activator via Gi/Go s timulation, did not alter ET-1-induced InsP(1) accumulation. Our data indicate that stimulation of PI turnover constitutes one of the signaling pathways of ET in rat pineal gland through the stimulation of a receptor-coupled phospholipase C. And they demonstrate, for the first time, the presence of functional binding sites for endothelin in the pineal gland.     

Endothelin-3 stimulates the hypothalamic-pituitary-adrenal axis.

Endothelin-3 (ET-3) is a member of the novel vasoconstrictive peptide family, identified in porcine central nervous system. Intravenous bolus injection of 1000 pmol/kg of ET-3 in freely moving rats caused significant increases in plasma ACTH and corticosterone levels, almost equivalent to those of 100 pmol/kg of rat corticotropin-releasing hormone (rCRH). The action of ET-3 was virtually abolished by pretreatment of CRH-antagonist, alpha-helical CRH. When ET-3 was added to cultured anterior pituitary cells, neither direct stimulation of ACTH release nor potentiation of rCRH action was noted. The results indicate that ET-3 may function as a neuropeptide and stimulation of the CRH-neurons, direct or inderect, is mainly responsible for activation of ACTH and corticosterone release.     

Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production

The mechanism of endothelin-1 (ET-1)-induced nitric oxide (NO) production, MMP-1 production and MMP-13 production was investigated in human osteoarthritis chondrocytes. The cells were isolated from human articular cartilage obtained at surgery and were cultured in the absence or presence of ET-1 with or without inhibitors of protein kinase or LY83583 (an inhibitor of soluble guanylate cyclase and of cGMP). MMP-1, MMP-13 and NO levels were then measured by ELISA and Griess reaction, respectively. Additionally, inducible nitric oxide synthase (iNOS) and phosphorylated forms of p38 mitogen-activated protein kinase, p44/42, stress-activated protein kinase/Jun-N-terminal kinase and serine-threonine Akt kinase were determined by western blot. Results show that ET-1 greatly increased MMP-1 and MMP-13 production, iNOS expression and NO release. LY83583 decreased the production of both metalloproteases below basal levels, whereas the inhibitor of p38 kinase, SB202190, suppressed ET-1-stimulated production only. Similarly, the ET-1-induced NO production was partially suppressed by the p38 kinase inhibitor and was completely suppressed by the protein kinase A kinase inhibitor KT5720 and by LY83583, suggesting the involvement of these enzymes in relevant ET-1 signalling pathways. In human osteoarthritis chondrocytes, ET-1 controls the production of MMP-1 and MMP-13. ET-1 also induces NO release via iNOS induction. ET-1 and NO should thus become important target molecules for future therapies aimed at stopping cartilage destruction.     

Angiotensin II stimulates nitric oxide production in pulmonary artery endothelium via the type 2 receptor

We previously reported that angiotensin II stimulates an increase in nitric oxide production in pulmonary artery endothelial cells. The aims of this study were to determine which receptor subtype mediates the angiotensin II-dependent increase in nitric oxide production and to investigate the roles of the angiotensin type 1 and type 2 receptors in modulating angiotensin II-dependent vasoconstriction in pulmonary arteries. Pulmonary artery endothelial cells express both angiotensin II type 1 and type 2 receptors as assessed by RT-PCR, Western blot analysis, and flow cytometry. Treatment of the endothelial cells with PD-123319, a type 2 receptor antagonist, prevented the angiotensin II-dependent increase in nitric oxide synthase mRNA, protein levels, and nitric oxide production. In contrast, the type 1 receptor antagonist losartan enhanced nitric oxide synthase mRNA levels, protein expression, and nitric oxide production. Pretreatment of the endothelial cells with either PD-123319 or an anti-angiotensin II antibody prevented this losartan enhancement of nitric oxide production. Angiotensin II-dependent enhanced hypoxic contractions in pulmonary arteries were blocked by the type 1 receptor antagonist candesartan; however, PD-123319 enhanced hypoxic contractions in angiotensin II-treated endothelium-intact vessels. These data demonstrate that angiotensin II stimulates an increase in nitric oxide synthase mRNA, protein expression, and nitric oxide production via the type 2 receptor, whereas signaling via the type 1 receptor negatively regulates nitric oxide production in the pulmonary endothelium. This endothelial, type 2 receptor-dependent increase in nitric oxide may serve to counterbalance the angiotensin II-dependent vasoconstriction in smooth muscle cells, ultimately regulating pulmonary vascular tone.     

Adiponectin stimulates production of nitric oxide in vascular endothelial cells

Adiponectin is secreted by adipose cells and mimics many metabolic actions of insulin. However, mechanisms by which adiponectin acts are poorly understood. The vascular action of insulin to stimulate endothelial production of nitric oxide (NO), leading to vasodilation and increased blood flow is an important component of insulin-stimulated whole body glucose utilization. Therefore, we hypothesized that adiponectin may also stimulate production of NO in endothelium. Bovine aortic endothelial cells in primary culture loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA) were treated with lysophosphatidic acid (LPA) (a calcium-releasing agonist) or adiponectin (10 microg/ml bacterially produced full-length adiponectin). LPA treatment increased production of NO by approximately 4-fold. Interestingly, adiponectin treatment significantly increased production of NO by approximately 3-fold. Preincubation of cells with wortmannin (phosphatidylinositol 3-kinase inhibitor) blocked only adiponectin- but not LPA-mediated production of NO. Using phospho-specific antibodies, we observed that either adiponectin or insulin treatment (but not LPA treatment) caused phosphorylation of both Akt at Ser473 and endothelial nitric-oxide synthase (eNOS) at Ser1179 that was inhibitable by wortmannin. We next transfected bovine aortic endothelial cells with dominant-inhibitory mutants of Akt (Akt-AAA) or AMP-activated protein kinase (AMPK) (AMPKK45R). Neither mutant affected production of NO in response to LPA treatment. Importantly, only AMPKK45R, but not Akt-AAA, caused a significant partial inhibition of NO production in response to adiponectin. Moreover, AMPK-K45R inhibited phosphorylation of eNOS at Ser1179 in response to adiponectin but not in response to insulin. We conclude that adiponectin has novel vascular actions to directly stimulate production of NO in endothelial cells using phosphatidylinositol 3-kinase-dependent pathways involving phosphorylation of eNOS at Ser1179 by AMPK. Thus, the effects of adiponectin to augment metabolic actions of insulin in vivo may be due, in part, to vasodilator actions of adiponectin.     

Endothelin-1 stimulates leptin production in adipocytes

Leptin is an adipocyte-derived hormone that regulates body fat stores and feeding behavior. In an effort to identify endogenous diffusible modulators of leptin production, we found that endothelin-1 (ET-1) up-regulates leptin expression in adipocytes. ET-1 is as potent and efficacious as insulin in stimulating leptin production in two different adipocyte cell lines. Endothelins stimulate leptin production via the endothelin-A receptor (ET(A)), as judged by a potency rank order of ET-1 ET-3. We detected expression of ET(A) but not ET(B) in both cell lines by Northern blot analysis. In addition, the ET(A)-selective antagonist FR139317 inhibited ET-1-induced leptin expression more potently than did the ET(B)-selective antagonist BQ788. ET-1 and insulin positively interact with each other in increasing leptin production in adipocytes. In primary mouse white fat cells, we detected expression of both ET(A) and ET(B) by Northern blot and in situ hybridization analyses. We conclude that ET-1 stimulates leptin production via the ET(A) receptor in cultured adipocytes.     

Maitotoxin stimulates phosphoinositide breakdown in neuroblastoma hybrid NCB-20 cells

1. Maitotoxin (MTX) was an extraordinarily potent stimulant of phosphoinositide breakdown in the neuroblastoma hybrid NCB-20 cells. 2. Maximal responses were obtained at 0.25-0.5 ng MTX/ml, and resulted in increased formation of [3H]inositol mono-, bis-, and trisphosphates. Increased formation of [3H]inositol bis- and trisphosphate was observed as early as 15 sec after the addition of MTX. 3. MTX-induced phosphoinositide breakdown in NCB-20 cells was not antagonized by organic (nifedipine, methoxyverapamil) or inorganic (Mn2+, Co2+, Cd2+) calcium channel blockers. However, the response on phosphoinositide breakdown was completely eliminated in the absence of extracellular calcium. 4. The results suggest that MTX either directly stimulates phosphoinositide breakdown in a calcium-dependent manner or acts indirectly through calcium channels insensitive to organic/inorganic calcium channel blockers.     

Interleukin-13 stimulates production of nitric oxide in cultured human nasal epithelium

The diversity and extent of signaling functions of nitric oxide (NO) in cell physiology as well as its presence and influence as a common component of ambient air pollution and tobacco smoke are gaining increasing research attention relative to both health and disease. While cellular NO production is typically associated with inflammatory cells and processes, the airway epithelium particularly of the paranasal sinuses, has been documented to be a rich source of excreted NO. Inasmuch as excreted NO derives from both mucosal and inflammatory cell sources, distinguishing the individual contribution of these compartments to total excreted cellular NO is potentially problematic. We simulated an inflammatory mucosal environment by stimulating human nasal epithelial cultures with interleukin-13 (IL-13), a mediator produced by eosinophils in asthma, allergic rhinitis, and sinusitis. While a consistent baseline of NO excretion in control cultures was documented, widely variable individual responses to IL-13 exposure were observed in companion cultures maintained under identical conditions and tested at the same time. These studies suggest that cellular NO excretion by the healthy epithelial mucosa is subject to considerable individual variability and may be significantly elevated among some individuals in the presence of IL-13 stimulation.     

Chicken cystatin stimulates nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages via cytokine synthesis.

Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon-gamma-activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077-28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon-gamma and then stimulated with interferon-gamma plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon-gamma. Interferon-gamma-activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon-gamma plus chicken cystatin produced increased amounts of both tumor necrosis factor alpha and interleukin 10. The addition of recombinant murine tumor necrosis factor alpha alone or in combination with recombinant murine interleukin-10 mimicked the effect of chicken cystatin. The addition of neutralizing anti-(tumor necrosis factor alpha) antibodies reduced sharply NO production by chicken cystatin/interferon-gamma-activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor alpha and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon-gamma-activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.     

Measurement of acetylcholine-induced endothelium-derived nitric oxide in aorta using a newly developed catheter-type nitric oxide sensor

Intra-aortic measurement of nitric oxide (NO) would provide valuable insights into NO bioavailability in systemic circulation and vascular endothelial function. In the present study, we thus developed a catheter-type NO sensor to measure intra-aortic NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter to measure intra-aortic plasma NO concentration in vivo in anesthetized dogs. Infusion of acetylcholine (10 microg/kg) increased base-to-peak plasma NO level in the aorta by 2.4+/-0.4 nM (n=7). After 20-min infusion of N(G)-methyl-L-arginine (NO synthase inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8+/-0.4 nM, P<0.003, n=7). In conclusion, the newly developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo and demonstrated applicability to direct evaluation of intravascular NO bioavailability.     

The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion

The ability of Salmonella enterica to invade and replicate within host cells depends on two type III secretion systems (TTSSs) encoded on pathogenicity islands 1 and 2 (SPI1 and SPI2). The current paradigm holds that these systems translocate two classes of effectors that operate sequentially and independently. In essence, the SPI1 TTSS mediates early events (i.e. invasion) whereas the SPI2 TTSS mediates post-invasion processes (i.e. replication, vacuole maturation). Contrary to this model, we have found in infected macrophages that a SPI1 effector, SopB/SigD, increased inducible nitric oxide synthase levels and nitric oxide production, host cell process previously known only to be a target of the SPI2 TTSS. Furthermore, SopB protein and message persist many hours after invasion. Our findings reveal an unanticipated potential for dialogue between the SPI1 and SPI2 TTSS and the host cell response.     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria     

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.     

Heat stress stimulates nitric oxide production in Symbiodinium microadriaticum: a possible linkage between nitric oxide and the coral bleaching phenomenon

Nitric oxide (NO) is a gas displaying multiple physiologicalfunctions in plants, animals and bacteria. The enzymes nitratereductase and NO synthase have been suggested to be involvedin the production of NO in plants and algae, but the implicationof those enzymes in NO production under physiological conditionsremains obscure. Symbiodinium microadriaticum, commonly referredto as zooxanthellae, is a marine microalga commonly found insymbiotic association with a cnidarian host including reef-buildingcorals. Here we demonstrate NO production in zooxanthellae uponsupplementation of either sodium nitrite or L-arginine as asubstrate. The nitrite-dependent NO production was detectedelectrochemically and confirmed by the application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(cPTIO), a specific NO scavenger. Cells stained with the diaminofluorescein,DAF-2 DA, an NO fluorescent probe, showed an increase in fluorescenceintensity upon supplementation of both sodium nitrite and L-arginine.Microscopic observations of DAF-stained cells verified thatNO was produced inside the cells. NO production in S. microadriaticumwas found to increase upon exposure of cells to an acute heatstress which also caused a decline in the photosynthetic efficiencyof PSII (F_v/F_m). This study provides substantial evidence toconfirm that zooxanthellae can synthesize NO even when theyare not in a symbiotic association with a coral host. The increasein NO production at high temperatures suggests that heat stressstimulates the microalgal NO production in a temperature-dependentmanner. The implications of these findings are discussed inthe light of the coral bleaching phenomenon which is associatedwith elevated sea surface temperature due to global warming.     

Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release

Endothelium-derived nitric oxide (NO) is synthesized in response to chemical and physical stimuli. Here, we investigated a possible role of the endothelial cell glycocalyx as a biomechanical sensor that triggers endothelial NO production by transmitting flow-related shear forces to the endothelial membrane. Isolated canine femoral arteries were perfused with a Krebs-Henseleit solution at a wide range of perfusion rates with and without pretreatment with hyaluronidase to degrade hyaluronic acid glycosaminoglycans within the glycocalyx layer. NO production rate was evaluated as the product of nitrite concentration in the perfusate and steady-state perfusion rate. The slope that correlates the linear relation between perfusion rate and NO production rate was taken as a measure for flow-induced NO production. Hyaluronidase treatment significantly decreased flow-induced NO production to 19 +/- 9% of control (mean +/- SD; P < 0.0001 vs. control; n = 11), whereas it did not affect acetylcholine-induced NO production (88 +/- 17% of pretreatment level, P = not significant; n = 10). We conclude that hyaluronic acid glycosaminoglycans within the glycocalyx play a pivotal role in detecting and amplifying the shear force of flowing blood that triggers endothelium-derived NO production in isolated canine femoral arteries.     

Rosiglitazone stimulates nitric oxide synthesis in human aortic endothelial cells via AMP-activated protein kinase

The thiazolidinedione anti-diabetic drugs increase activation of endothelial nitric-oxide (NO) synthase by phosphorylation at Ser-1177 and increase NO bioavailability, yet the molecular mechanisms that underlie this remain poorly characterized. Several protein kinases, including AMP-activated protein kinase, have been demonstrated to phosphorylate endothelial NO synthase at Ser-1177. In the current study we determined the role of AMP-activated protein kinase in rosiglitazone-stimulated NO synthesis. Stimulation of human aortic endothelial cells with rosiglitazone resulted in the time- and dose-dependent stimulation of AMP-activated protein kinase activity and NO production with concomitant phosphorylation of endothelial NO synthase at Ser-1177. Rosiglitazone stimulated an increase in the ADP/ATP ratio in endothelial cells, and LKB1 was essential for rosiglitazone-stimulated AMPK activity in HeLa cells. Infection of endothelial cells with a virus encoding a dominant negative AMP-activated protein kinase mutant abrogated rosiglitazone-stimulated Ser-1177 phosphorylation and NO production. Furthermore, the stimulation of AMP-activated protein kinase and NO synthesis by rosiglitazone was unaffected by the peroxisome proliferator-activated receptor-gamma inhibitor GW9662. These studies demonstrate that rosiglitazone is able to acutely stimulate NO synthesis in cultured endothelial cells by an AMP-activated protein kinase-dependent mechanism, likely to be mediated by LKB1.