No abstract

The endothelial nitric-oxide synthase (eNOS) is a key determinant of vascular homeostasis. Like all known nitric-oxide synthases, eNOS enzyme activity is dependent on Ca 2؉ -calmodulin. eNOS is dynamically targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae and interacts with caveolin, an integral membrane protein that comprises a key structural component of caveolae. We have previously reported that the association between eNOS and caveolin is quantitative and tissue-specific (Feron, O., Belhassen, L., Kobzick, L., Smith, T. W., Kelly, R. A., and Michel, T. (1996) J. Biol. Chem. 271, 22810 -22814). We now report that in endothelial cells the interaction between eNOS and caveolin is importantly regulated by Ca 2؉ -calmodulin. Addition of calmodulin disrupts the heteromeric complex formed between eNOS and caveolin in a Ca 2؉ -dependent fashion. In addition, overexpression of caveolin markedly attenuates eNOS enzyme activity, but this inhibition is reversed by purified calmodulin. Caveolin overexpression does not affect the activity of the other NOS isoforms, suggesting eNOSspecific inhibition of NO synthase by caveolin. We propose a model of reciprocal regulation of eNOS in endothelial cells wherein the inhibitory eNOS-caveolin complex is disrupted by binding of Ca 2؉

Opposite changes occur in beta(1)- and beta(3)-adrenoceptor abundance in the failing left ventricle, with an imbalance between their inotropic influences that may underlie the functional degradation of the human failing heart.

Nitric oxide production in the vascular endothelium is promoted by diverse agonists that transiently increase intracellular Ca 2؉ concentration and activate the endothelial nitric-oxide synthase (eNOS), a Ca 2؉ /calmodulindependent enzyme. eNOS is acylated by the fatty acids myristate and palmitate and is targeted thereby to plasmalemmal signal-transducing domains termed caveolae. eNOS enzyme activity is markedly attenuated by its interactions with caveolin, the structural scaffolding protein of caveolae. We have discovered that in living cells, the eNOS-caveolin heteromeric complex undergoes cycles of dissociation and re-association modulated by Ca 2؉ -mobilizing agonists. Calcium ionophore A23187 and the muscarinic cholinergic agonist carbachol both promote the dissociation of eNOS from caveolin in cultured cells, associated with translocation of eNOS from caveolae. As [Ca 2؉ ] i returns to basal levels, eNOS reassociates with caveolin, and the inhibited enzyme complex is then restored to caveolae, a process accelerated by palmitoylation of the enzyme. These data establish an eNOS-caveolin regulatory cycle, wherein enzyme activation is modulated by reversible protein-protein interactions controlled by Ca The endothelial isoform of nitric-oxide synthase (eNOS) 1 is robustly expressed in the vascular endothelium and in cardiac myocytes, and the cellular regulation of eNOS may represent an important determinant of cardiovascular homeostasis (reviewed in Ref. 1). In endothelial cells and in cardiac myocytes, eNOS is targeted to specialized invaginations of the plasmalemma termed caveolae (2). Plasmalemmal caveolae serve as sites for the sequestration of signaling proteins and are further characterized by the presence of caveolin, an intrinsic membrane protein that forms a structural "scaffold," organizing both proteins and lipids within this key membrane organelle (3, 4). Caveolin directly interacts with several structurally distinct signaling proteins in caveolae, including G proteins and cellular oncogenes (4) as well as eNOS (2, 5-9). The activity of purified eNOS, a Ca 2ϩ /calmodulin-dependent enzyme (10, 11), is markedly attenuated by its interaction with caveolin (5-9). We have also shown that purified Ca 2ϩ /calmodulin can overcome the inhibitory interaction between eNOS and caveolin in vitro (5, 7, 9), but the relevance of these observations to the dynamic regulation of eNOS in endothelial cells is less well understood. In vascular endothelial cells and in cardiac myocytes, the cycle of eNOS activation and deactivation is intimately coupled to the changes in intracellular Ca 2ϩ that are promoted by stimulation of diverse G protein-coupled receptors (12,13). In this report, we describe a series of experiments that have explored the relationships between intracellular Ca 2ϩ regulation and the dynamics of eNOS-caveolin interactions in living cells. We also document the role of eNOS palmitoylation in the reversible caveolar targeting of the eNOS-caveolin complex following muscarinic cholinergic stimulat...

We provide biochemical and functional evidence that atorvastatin promotes NO production by decreasing caveolin-1 expression in ECs, regardless of the level of extracellular LDL-Chol. These findings highlight the therapeutic potential of inhibiting cholesterol synthesis in peripheral cells to correct NO-dependent endothelial dysfunction associated with hypercholesterolemia and possibly other diseases.

Hypercholesterolemia is a central pathogenic factor of endothelial dysfunction caused in part by an impairment of endothelial nitric oxide (NO) production through mechanisms that remain poorly characterized. The activity of the endothelial isoform of NO synthase (eNOS) was recently shown to be modulated by its reciprocal interactions with the stimulatory Ca 2+ -calmodulin complex and the inhibitory protein caveolin. We examined whether hypercholesterolemia may reduce NO production through alteration of this regulatory equilibrium. Bovine aortic endothelial cells were cultured in the presence of serum obtained from normocholesterolemic (NC) or hypercholesterolemic (HC) human volunteers. Exposure of endothelial cells to the HC serum upregulated caveolin abundance without any measurable effect on eNOS protein levels. This effect of HC serum was associated with an impairment of basal NO release paralleled by an increase in inhibitory caveolin-eNOS complex formation. Similar treatment with HC serum significantly attenuated the NO production stimulated by the calcium ionophore A23187. Accordingly, higher calmodulin levels were required to disrupt the enhanced caveolin-eNOS heterocomplex from HC serum-treated cells. Finally, cell exposure to the low-density lipoprotein (LDL) fraction alone dose-dependently reproduced the inhibition of basal and stimulated NO release, as well as the upregulation of caveolin expression and its heterocomplex formation with eNOS, which were unaffected by cotreatment with antioxidants. Together, our data establish a new mechanism for the cholesterol-induced impairment of NO production through the modulation of caveolin abundance in endothelial cells, a mechanism that may participate in the pathogenesis of endothelial dysfunction and the proatherogenic effects of hypercholesterolemia.

Abstract-3-Hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors or statins exert direct beneficial effects on the endothelium in part through an increase in nitric oxide (NO) production. Here, we examined whether posttranslational modifications of the endothelial NO synthase (eNOS) could account for the proangiogenic effects of statins. We used endothelial cells (ECs) isolated from cardiac microvasculature, aorta, and umbilical veins, as well as dissected microvessels and aortic rings, that were cultured on reconstituted basement membrane matrix (Matrigel). Tube or precapillary formation was evaluated after statin treatment, in parallel with immunoblotting and immunoprecipitation experiments. Atorvastatin stimulated NO-dependent angiogenesis from both isolated and outgrowing (vessel-derived) ECs, independently of changes in eNOS expression. We found that in macro-but not microvascular ECs, atorvastatin stabilized tube formation through a decrease in caveolin abundance and its inhibitory interaction with eNOS. We also identified the chaperone protein hsp90 as a key target for the proangiogenic effects of statins. Using geldanamycin, an inhibitor of hsp90 function, and overexpression of recombinant hsp90, we documented that the statin-induced phosphorylation of eNOS on Ser1177 was directly dependent on the ability of hsp90 to recruit Akt in the eNOS complex. Finally, we showed that statin promoted the tyrosine phosphorylation of hsp90 and the direct interaction of hsp90 with Akt, which further potentiated the NO-dependent angiogenic processes. Our study provides new mechanistic insights into the NO-mediated angiogenic effects of statins and underscores the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis. ( Key Words: statin Ⅲ angiogenesis Ⅲ nitric oxide Ⅲ hsp90 Ⅲ caveolin I t is now established that statins, in addition to their ability to improve serum lipid profile, 1 exert beneficial effects on endothelial function through an increase in nitric oxide (NO) production and/or bioavailability. 2 A major area in which such effects of statins on peripheral cells would be valuable is therapeutic angiogenesis, eg, the development of neovascularization in ischemic tissues. NO has, indeed, been identified as a downstream mediator of various growth factors initiating the angiogenic signaling cascade in endothelial cells (ECs). 3 Recently, Kureishi et al 4 reported that simvastatin could promote angiogenesis in ischemic limbs of normocholesterolemic rabbits. These authors further documented that statins could acutely induce Akt-dependent endothelial NO synthase (eNOS) phosphorylation in cultured ECs and therefore proposed that the Akt activation of eNOS could account for the long-term beneficial effects of simvastatin observed in vivo. Although the reversal of this effect by mevalonate suggested an inhibitory effect of the statin on the isoprenoid synthesis and downstream isoprenylation/activatio...

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca 2؉ -dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca 2؉ /calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca 2؉ ] i , this late stage of eNOS activation is strictly conditional on the initial VEGFinduced Ca 2؉ -dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90. Nitric oxide (NO)1 contributes to the cardiovascular homeostasis through its profound effects on blood pressure, vascular remodeling, platelet aggregation, and angiogenesis (1). Under normal conditions, the endothelial isoform of NO synthase (eNOS) expressed both in endothelial cells (EC) and cardiac myocytes is the major source of NO in the cardiovascular system (2). eNOS was originally identified as a particulate enzyme, associated primarily with the plasma membrane and with intracellular organelles such as the Golgi complex (3). Recent findings suggest that trafficking of eNOS from plasmalemmal to intracellular structures is part of a physiological cycle highly sensitive to the state of cell activation (4 -8). The dynamic trafficking of eNOS between cell surface and intracellular compartments is supported in part by its close interaction with the scaffold protein caveolin (4). In addition to regulating eNOS subcellular localization, this stable interaction with caveolin leads to the inhibition of the enzyme activity in basal conditions. Upon increases in intracellular calcium ([Ca 2ϩ ] i ), Ca 2ϩ /calmodulin (CaM) displaces the inhibitory binding of caveolin to eNOS and allows enzyme activation (9). Similarly, increasing vascular flow and pressure in an in situ artery perfusion model rapidly leads to activation of caveolar eNOS with apparent eNOS ...