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Cardiovascular Research 2006 71(3):478-485; doi:10.1016/j.cardiores.2006.04.013
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Copyright © 2006, European Society of Cardiology

The calcium channel blocker amlodipine promotes the unclamping of eNOS from caveolin in endothelial cells

Suzan Batova, Julie DeWever, Théophile Godfraind, Jean-Luc Balligand, Chantal Dessy1 and Olivier Feron*,1

Unit of Pharmacology and Therapeutics, University of Louvain Medical School, UCL-FATH5349, 53 Avenue E. Mounier, Brussels B-1200, Belgium

* Correspondence author. Tel.: +32 2 764 5264; fax: +32 2 764 5461. Email address: feron{at}mint.ucl.ac.be

Received 22 November 2005; revised 30 March 2006; accepted 17 April 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
 References
 
Objectives Amlodipine is a calcium channel blocker (CCB) known to stimulate nitric oxide production from endothelial cells. Whether this ancillary property can be related to the capacity of amlodipine to concentrate and alter the structure of cholesterol-containing membrane bilayers is a matter of investigation. Here, we reasoned that since the endothelial nitric oxide synthase is, in part, expressed in cholesterol-rich plasmalemmal microdomains (e.g., caveolae and rafts), amlodipine could interfere with this specific locale of the enzyme and thereby modulate NO production in endothelial cells.

Methods and results Using a method combining lubrol-based extraction and subcellular fractionation on sucrose gradient, we found that amlodipine, but not verapamil or nifedipine, induced the segregation of endothelial NO synthase (eNOS) from caveolin-enriched low-density membranes (8±2% vs. 42±3% in untreated condition; P<0.01). We then performed co-immunoprecipitation experiments and found that amlodipine dose-dependently disrupted the caveolin/eNOS interaction contrary to other calcium channel blockers, and potentiated the stimulation of NO production by agonists such as bradykinin and vascular endothelial growth factor (VEGF) (+138±28% and +183±27% over values obtained with the agonist alone, respectively; P<0.01). Interestingly, we also documented that the dissociation of the caveolin/eNOS heterocomplex induced by amlodipine was not mediated by the traditional calcium-dependent calmodulin binding to eNOS and that recombinant caveolin expression could compete with the stimulatory effects of amlodipine on eNOS activity. Finally, we showed that the amlodipine-triggered, caveolin-dependent mechanism of eNOS activation was independent of other pleiotropic effects of the CCB such as superoxide anion scavenging and angiotensin-converting enzyme (ACE) inhibition.

Conclusions: This study unravels the modulatory effects of amlodipine on caveolar integrity and the capacity of caveolin to maintain eNOS in its vicinity in the absence of any detectable changes in intracellular calcium levels. The resulting increase in caveolin-free eNOS potentiates the NO production in response to agonists including VEGF and bradykinin. More generally, this work opens new avenues of treatment for drugs able to structurally alter signaling pathways concentrated in caveolae.

KEYWORDS Nitric oxide; Endothelial function; Calcium channel; Signal transduction; Caveolin

This article is referred to in the Editorial by B. Mayer (pages 411–413) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Material and methods
 3. Results
 4. Discussion
 References
 
Caveolae are plasmalemmal invaginations formed by the sequestration of cholesterol with self-associating molecules termed caveolins, resulting in a platform for the assembly of signaling complexes at the cell surface [1]. The lipid-modifications of proteins, including the endothelial nitric oxide synthase (eNOS) which is both myristoylated and palmitoylated, account for their high tropism towards caveolae [2] but also to the larger family of cholesterol-enriched microdomains termed rafts [3]. The relationship between membrane cholesterol and eNOS was further confirmed by different set of studies: (i) We documented that increasing cell cholesterol uptake by exposing endothelial cells to native LDL increases the abundance of caveolin (the structural protein of caveolae) and the strength of the caveolin/eNOS interaction,[4] a process reversed by the hypolipidemic drugs statins [5,6]. (ii) Blair and colleagues reported that oxidized LDL by depleting cholesterol from caveolae induced the intracellular translocation of eNOS away from the cell surface [7]. Moreover, the same authors documented that the ability of HDL to fuel caveolae with cholesterol esters could reverse the effects of oxLDL [8]. (iii) Drugs such as cyclodextrins, filipin or nystatin by binding/extracting cholesterol from membranes are known to alter the structure of numerous membrane microdomains, including rafts and caveolae [9]. Changes in eNOS subcellular location and/or activity were reported upon treatment with these drugs [10].

It may therefore be postulated that lipophilic drugs that avidly partition into cholesterol-rich membrane bilayers could also impact on the eNOS locale, and thereby on the activity of the enzyme. One such drug could be amlodipine, a long-lasting calcium channel blocker (CCB) with a marked ability to partition to the cell membrane [11]. Amlodipine is indeed several-fold more lipohilic than the first- and second-generation CCBs and was documented to modify the structure of cholesterol-containing membranes [12], and in particular to reduce the width of synthetic cholesterol-rich double leaflets or native smooth muscle cell (SMC) membranes isolated from hypercholesterolemic rabbits [13]. Amazingly, another independent group of studies has identified eNOS activation as a major source of atheroprotection by amlodipine [12]. Actually, although vasodilatory properties of CCB are traditionally related to their blocking effects on vascular SMC L-type calcium channels, amlodipine was shown to exert additional vasorelaxant effects through stimulation of NO release from endothelial cells [12,14,15]. This eNOS-agonist effect was attributed to one enantiomer of amlodipine that does not possess the ability to block L-type calcium channels [16]. Moreover, the absence of L-type calcium channels in macrovascular EC further pleads for an ancillary property independent of the channel blocking effects. Different mechanisms accounting for the activation of eNOS by amlodipine have been identified in a variety of in vitro and in vivo models [12,14]. They relate to one or several of the following effects: (i) inhibition of the angiotensin converting enzyme (ACE), thereby promoting the kinin-dependent activation of eNOS [15,17]; (ii) anti-oxidant effects increasing the half-life of NO by scavenging superoxide anion [18]; and (iii) inhibition of PKC and consecutive changes in the eNOS phosphorylation status [19].

Here, we reasoned that a pre-requisite to all of the documented amlodipine-mediated eNOS-activating processes was the segregation of eNOS from caveolae and the associated unclamping of eNOS from caveolin. This process could be mediated by the high tropism of amlodipine for membranes rich in cholesterol, a peculiarity of rafts and caveolae. The impact of amlodipine on eNOS subcellular location, caveolin interaction and catalytic activity was examined and confronted with the previously reported kinin-mediated effects of amlodipine and the traditional calcium–calmodulin-dependent activation of eNOS.


   2. Material and methods
Top
 Abstract
 1. Introduction
 2. Material and methods
3. Results
 4. Discussion
 References
 
2.1 Endothelial cells and drug treatments
Bovine aortic endothelial cells (BAEC) were cultured on gelatin-coated flasks using endothelial cell growth medium (EGM-MV, Clonetics). In some experiments, endothelial cells were transfected with 5 µg caveolin-1 cDNA (per 35-mm dish) using the Amaxa device and recommended reagents; a delay of 48 h before amlodipine/agonist treatments was respected to allow recombinant caveolin expression to occur. Endothelial cells were collected for immunoprecipitation experiments 60 min after calcium channel blocker treatment. In experiments requiring calcium chelation or blockade of the bradykinin B2 receptor, 20 µM BAPTA/AM or 1 µmol/L HOE140, respectively, was added 30 min before adding amlodipine. In experiments where both amlodipine (10 nmol/L) and agonists (VEGF or bradykinin) were combined, they were simultaneously added to endothelial cell cultures (with or without preincubation in 10% fetal calf serum for 4 h at 37 °C). All drugs were from Sigma.

2.2 Subcellular fractionation
Endothelial cells were lysed in ice-cold TNE buffer (25 mmol/L Tris, 150 mmol/L NaCl, and 1 mmol/L EGTA) containing a cocktail of protease inhibitors (Sigma). After 10 passages through 23 G and 26 G needles, postnuclear supernatants were obtained after centrifugation at 800 x g at 4 °C for 10 min. PNS was then incubated with 1% detergent (lubrol or triton) at 4 °C for 30 min under constant agitation. The solution was brought to 40% sucrose in TNE/detergent and, after homogenization, overlaid by a two-step sucrose gradient (30% to 10% sucrose in TNE/detergent). Gradients were centrifuged at 38000 rpm for 16 to 18 h in a SW41 rotor (Beckman Instruments Inc) at 4 °C. Eight fractions (1.5 mL) plus the pellet (resuspended in 1.5 mL TNE/lubrol) were harvested from the top. In several experiments, fractions 1 to 4 and 6 to 9 were pooled and named low-density (LD) and high-density (HD) membranes, respectively.

2.3 eNOS immunoblotting and NO determination
Immunoblotting was performed on caveolin-1 immunoprecipitates or eNOS immunoprecipitates (from the supernatant of caveolin immunoprecipitation), as previously described [20]; both caveolin and eNOS antibodies were from BD PharMingen. NO production was determined by the measurement of the accumulation of NO derivatives (NOx) in the cell-bathing medium for a fixed period of 8 h. The Nitric Oxide Colorimetric Assay (Roche Diagnostics, Mannheim, Germany) was used and only the L-NAME-sensitive component of NOx production was considered.

2.4 Statistical analyses
Data are normalized to control condition and are presented for convenience as mean±S.E.M. Two-way ANOVA was performed where appropriate.


   3. Results
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
4. Discussion
 References
 
Cholesterol-enriched microdomains, such as rafts and caveolae, are traditionally ascribed as detergent-insoluble membranes. Still, detergents do not lead to the same extent/nature of membrane extraction and differences may also arise from cell types. In a first set of experiments, we therefore compared the subcellular fractionation on a 10–20–30–40% discontinuous sucrose gradient of EC membranes insoluble in either triton or lubrol. Fig. 1A reveals a distinct pattern according to the detergent used. While no detectable amounts of caveolin could be identified in low-density fractions from triton-insoluble membranes, a significant amount of the scaffold protein was found in the low-density fractions of lubrol-insoluble membranes. Furthermore, since equi-volumes (and not equivalent protein concentrations) of the 9 fractions collected were loaded on polyacrylamide gels, the real enrichment in caveolin had therefore to be corrected by the amounts of proteins recovered in each fraction (see Fig. 1B). Accordingly, fractions 1–4 from lubrol-extracted membranes represented more than 70% of caveolin present in endothelial cells. We then searched to concentrate the caveolin fraction by modifying the sucrose concentration steps. Fig. 1C shows that caveolin was very efficiently recovered in fractions 3 and 4 of the two discontinuous sucrose gradients tested (i.e., 10–25–40% and 10–30–40%), the 10–30–40% formulation being however slightly more efficient in concentrating caveolin in low-density fractions.


Figure 1
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  		Fig. 1 Caveolin is enriched in lubrol-insoluble low-density endothelial cell membranes. BAECs were cultured to confluence before lysis and incubation with the indicated detergents. Subcellular fractionation was then performed on sucrose gradient and the different fractions (low to high densities) were loaded onto acrylamide gels. (A) Representative caveolin immunoblotting performed on 9 (equi-volume) fractions collected from a discontinuous 10–20–30–40% sucrose gradient. (B) Bar graph represents the abundance of caveolin in each fraction (as determined by densitometric analyses of immunoblots) but corrected for the amounts of recovered proteins. (C) Comparison of caveolin immunoblottings performed on fractions collected from discontinuous 10–25–40% and 10–30–40% sucrose gradients.

 
Using the above optimized sucrose gradient concentrations, we aimed at evaluating the extent of eNOS enrichment in the caveolin-containing low-density fraction. Fig. 2A (top panel) revealed that a large proportion of eNOS was found in low-density fractions 1–4 (LD). Correction for protein contents led to the finding that 42±3% of eNOS was found enriched in pooled LD fractions 1–4 (see also control bars in Fig. 2B). We then evaluated the effects of exposure to three different calcium channel blockers (CCB) to determine their impact on the distribution of eNOS between low- and high-density membranes. Fig. 2B (bottom panel) shows that only amlodipine led to a net shift from the low- to high-density membranes. Correction by protein content confirmed that verapamil and nifedipine did not significantly alter the proportion of eNOS in LD fraction (37±1% and 35±1%, respectively), whereas amlodipine exposure allowed to recover only 8±2% of total eNOS protein present in LD lubrol-insoluble membranes (Fig. 2B). Of note, the distribution of caveolin across the gradient fractions remained unaltered by the different CCB treatments (not shown).


Figure 2
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  		Fig. 2 Amlodipine, but not verapamil and nifedipine, induced eNOS translocation from caveolin-rich low-density membranes to high-density membranes. BAECs were cultured to confluence and exposed (or not) to a high dose of CCB (10 µM verapamil, 1 µM nifedipine, 10 nM amlodipine) before lysis. Subcellular fractionation of lubrol-resistant membranes was then performed on a discontinuous 10–30–40% sucrose gradient and the different fractions were loaded onto acrylamide gels. (A) Representative eNOS immunoblotting performed (top) on each collected fraction and (bottom) on pooled fractions 1–4 and 6–9, named low-density (LD) and high-density (HD) membranes, respectively. (B) Bar graph represents the abundance (% of total) of eNOS in LD and HD fractions isolated from extracts of EC treated as indicated (n=3).

 
To determine whether the segregation from caveolin-enriched membranes was associated with a disruption of the caveolin–eNOS interaction, we carried out immunoprecipitation experiments on EC membranes following exposure to the different CCB. We used caveolin antibodies to determine the proportion of eNOS that could be immunoprecipitated with caveolin. Fig. 3A shows that amlodipine treatment led to a dose-dependent disruption of the caveolin/eNOS interaction, whereas verapamil did not exert any treatment and only the highest nifedipine concentration led to a partial dissociation of eNOS from caveolin. Since the caveolin/eNOS dissociation is usually described as a calcium-dependent process requiring the binding of a calcium–calmodulin complex to eNOS, we used the calcium chelator BAPTA to determine the calcium dependence of the amlodipine effects. Fig. 3B shows that BAPTA pre-incubation failed to prevent the dissociation of eNOS from caveolin by amlodipine; as a control, we also showed that BAPTA alone did not alter the amounts of eNOS associated with caveolin (Fig. 3B, right).


Figure 3
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  		Fig. 3 Amlodipine-induced disruption of the caveolin/eNOS interaction in a calcium-independent manner. BAECs were cultured to confluence and exposed (or not) to the indicated CCB before lysis. In some experiments, the calcium chelator BAPTA was added 30 min before amlodipine exposure. Shown are eNOS immunoblots of caveolin immunoprecipitates from extracts of EC exposed (A) to increasing doses of CCB (verapamil, nifedipine, amlodipine) and (B) to BAPTA with or without 10 nM amlodipine. These experiments were repeated twice with similar results. The extent of the reduction in eNOS co-immunoprecipitated with caveolin is also given for the maximal dose of each CCB tested.

 
We then attempted to correlate the effects of amlodipine on eNOS translocation from caveolin-enriched membranes with changes in eNOS activity. Thus, we measured the accumulation of NOx derivatives in the medium of EC exposed for 8 h to 10 nM amlodipine in basal conditions as well as in the presence of agonists. Fig. 4A shows that in the limits of detection of our assay, amlodipine treatment alone did not lead to an increase in NOx production whereas bradykinin or VEGF when administered alone led to a significant increase in NOS activity (+96±8% and +108±40%, respectively. Interestingly, however, when both amlodipine and either agonist (bradykinin or VEGF) were co-administered, a synergistic effect was observed: NOx values reached 3.3- and 3.9-fold the amounts measured in the medium of EC treated with amlodipine alone. To attribute the effects of amlodipine to the unclamping of eNOS from caveolin (and the consecutive facilitation of agonist stimulation), we transfected EC with a caveolin plasmid in order to artificially increase the amounts of caveolin. We previously documented that this could reinforce the caveolin/eNOS interaction and prevent the disruption of the complex [21]. Fig. 4B shows that, indeed, in the presence of an excess caveolin, the NOx production was largely attenuated in EC treated with the combination of amlodipine with either bradykinin or VEGF. Note also that the basal NO production (in the presence of amlodipine or not) was slightly but significantly reduced by the caveolin overexpression (Fig. 4B).


Figure 4
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  		Fig. 4 Amlodipine potentiates the activation of eNOS by bradykinin and VEGF in a caveolin-dependent manner. (A) Bar graph represents the L-NAME-sensitive NOx production by EC in basal conditions (control) and on exposure to agonists (1 µmol/L bradykinin or 25 ng/mL VEGF) in the presence or the absence of 10 nM amlodipine; **P<0.01 vs. control; §§P<0.01 vs. corresponding agonist given alone (n=3). The absolute levels of NOx (primarily nitrites) in basal conditions amounted to 0.451±0.023 µM. (B) Bar graph represents the L-NAME-sensitive NOx production by EC transfected (or not) with a caveolin plasmid and exposed (or not) 48 h later to 10 nmol/L amlodipine and either 1 µmol/L bradykinin or 25 ng/mL VEGF; expression of recombinant caveolin was verified by immunoblotting. *P<0.05, **P<0.01 vs. corresponding untransfected conditions (n=3).

 
Finally, we examined whether the previously reported pleiotropic effects of amlodipine, namely the anti-oxidant effects and the ACE inhibition (see Introduction), could interplay with our own findings. To show the influence of the potential anti-oxidant effects of amlodipine in our experimental conditions, we measured the NOx production in response to VEGF in the presence of increasing concentrations of PEG–SOD. Fig. 5A shows that irrespective of the presence of amlodipine, PEG–SOD slightly increased the amounts of NOx produced upon VEGF stimulation. Moreover, the effect of a maximal dose of PEG–SOD led to an increase in NOx levels still lower than that triggered by amlodipine (+17±11% vs. +88±28%, respectively).


Figure 5
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  		Fig. 5 Evaluation of the anti-oxidant and ACE inhibitory effects of amlodipine vs. the caveolin-mediated regulation of eNOS. (A) Effects of increasing doses of PEG–SOD on the VEGF-induced (L-NAME-sensitive) NOx production in the absence ({square}) or the presence ({blacksquare}) of amlodipine. **P<0.01 vs. basal condition (no PEG–SOD, no amlodipine); §§P<0.01 vs. basal condition (no amlodipine) in the presence of a maximal dose of PEG–SOD. (B) Bar graph represents the L-NAME-sensitive NOx production by EC exposed (or not) to 10% serum. In some experiments, the serum was pre-incubated for 2 h at 37 °C with 10 nmol/L amlodipine or 10 µmol/L lisinopril and 10 µmol/L bradykinin or 100 ng/mL VEGF, or with each drug separately. **P<0.01 vs. control (serum alone); §§P<0.01 vs. bradykinin or VEGF alone; {dagger}P<0.05 vs. amlodipine+bradykinin; {ddagger}{ddagger}P<0.01 vs. amlodipine+VEGF (n=3).

 
To evaluate the potential impact of the inhibitory effects of amlodipine on ACE activity (e.g. kinin degradation) in our experimental conditions, we co-incubated amlodipine with bradykinin in bovine serum (as source of ACE) for 4 h at 37 °C, and then exposed EC to the drug/serum mixture. Fig. 5B shows that the native serum alone did increase NOx production by more than two-fold when compared with serum-free condition, and that the presence of amlodipine in the serum further increased NOx production by 40±8% (P<0.01). More importantly, we found that bradykinin-containing serum was unable to stimulate NOx production (above the serum condition) but that in the presence of amlodipine, bradykinin-containing serum did increase NOx production by 2.6-fold (Fig. 5B). The use of the specific ACE inhibitor lisinopril did recapitulate these effects but to a much lesser extent than amlodipine (Fig. 5B). Furthermore, the combination of amlodipine and VEGF in the same experimental condition (serum+drug pre-incubation) did reveal a similar potentiation of NO production as observed with bradykinin co-incubated with amlodipine (Fig. 5B).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Material and methods
 3. Results
 4. Discussion
References
 
The major findings of this manuscript are that, in endothelial cells, amlodipine can segregate eNOS from its preferential location in caveolae and that the associated unclamping of eNOS from caveolin favors a configuration where eNOS is more activable by agonists such as bradykinin and VEGF. Importantly, these effects appear to be related to the structure of amlodipine and its ability to interfere with cholesterol-rich membranes. Indeed, we showed that two other CCB failed to induce a similar dose-dependent caveolin/eNOS disruption and to stimulate NOS activity (although a small effect of nifedipine was observed at high dosages). Major anti-oxidant effects could be excluded in our experimental conditions since the effects of amlodipine were maintained in the presence of large concentrations of PEG–SOD (see Fig. 5A). Importantly, the traditional signaling pathway leading to caveolin/eNOS complex disruption that involves calcium (e.g., calcium–calmodulin binding to eNOS) was not involved since the calcium chelator BAPTA did not prevent the amlodipine-induced dissociation of eNOS from its inhibitory interaction with caveolin (see Fig. 3B). These data are reminiscent of previous reports on the capacity of amlodipine, and not other CCB, to alter the structure of cholesterol-rich membrane bilayers and influence the cell biology through a "steric" effect (more than through initiation of specific signaling cascades) [12]. For instance, Kahn and colleagues [13] recently reported that when amlodipine was added to native SMC membranes isolated from hypercholesterolemic rabbits, it reduced the swelling of membrane bilayer generally observed in these atherosclerotic animals [22].

Although further biophysical studies are warranted to dissect how amlodipine specifically alters caveolae and/or raft structures, this study illustrates the lability of the caveolin/eNOS interaction, independently of changes in intracellular calcium concentrations. This observation can therefore be related to the so-called calcium-independent activation of eNOS reported by several authors [23–27], where changes in the phosphorylation status of eNOS (e.g., phosphorylation on Ser1177 and dephosphorylation of Thr495) ensures the activation of the enzyme at low intracellular calcium concentration. Here, the effects of amlodipine on the dissociation of the caveolin/eNOS complex appears "truly" calcium-independent and should instead be related to the effects of oxidized LDL or cyclodextrins on caveolae, where alterations in the local cholesterol concentration were shown to induce the translocation of eNOS from the caveolae to an intracellular locale. In this latter situation, however, a decrease in NO production was observed both in basal and agonist-stimulated endothelial cells whereas with amlodipine, basal NO production was unchanged and agonist stimulation led to a dramatic increase in NO production (above the effects of the agonist alone). These apparent discrepancies can be explained by the new locales reached by eNOS after either oxLDL or amlodipine treatment. In the former case, uncoupling of eNOS from receptor activation was proposed to account for a reduced NO production [7] whereas in the latter, the enzyme was dissociated from caveolin but remained sensitive to agonists. Interestingly, bradykinin stimulation of BAEC was previously documented to increase the triton-insolubility of caveolin-associated membranes [28]. Together with our observations according to which in the same cells but in basal conditions, caveolin-enriched membranes are soluble in triton but not in lubrol (see Fig. 1), these data suggest that the dynamics of cholesterol-enriched microdomains within the cell may indeed favor the segregation of caveolar/raft constituents (such as caveolin or eNOS) in specific compartments. It is however still unknown whether these locales are artificially created by the ability of a given detergent to preferentially extract some proteins following post-translational alterations [29] or whether differential detergent extraction correspond to the existence of rafts subtypes, as recently proposed for some lubrol-insoluble and triton-soluble proteins [30,31]. It is also worth noting that the two agonists that we used, bradykinin and VEGF, have both receptors located in caveolae [32,33] and that translocation of these receptors out of caveolae have also been reported following agonist stimulation [34,35].

Another interesting observation of the current study is that the amlodipine potentiating effect on eNOS stimulation by bradykinin or VEGF could be prevented by an excess of recombinant caveolin (see Fig. 4B). The implications of this observation are twofold: (i) the amlodipine-triggered caveolin/eNOS disruption applied to any type of "eNOS agonists" (i.e. not only G protein-coupled receptor – but also tyrosine kinase receptor agonists) and independently of the potential other effects of amlodipine on the activity of these agonists (such as anti-oxidant effects or through ACE inhibition) (see Fig. 5A and B). The mechanism proposed by Lenasi and colleagues [19] (e.g., the activating eNOS Ser1177 phosphorylation on the de-phosphorylation of Thr495) also perfectly fits a model where the equilibrium between caveolin-bound and caveolin-free eNOS is displaced towards the latter, thereby facilitating the consecutive changes in the eNOS phosphorylation status (ensuring the calcium-independent activation of the enzyme); (ii) in situations where caveolin is pathologically increased, such as hypercholesterolemia [4] and other cardiovascular disease states [36], the NO-mediated vasodilatory effects of amlodipine could be less effective than expected. Interestingly, we have previously documented that statins could reduce caveolin abundance in endothelial cells and thereby favor the disruption of the caveolin/eNOS heterocomplex [5]. Altogether, these data support the hypothesis that statins and amlodipine could have synergistic effects, a property that could account for recent clinical data where combination of both drugs appeared particularly efficient in reducing arterial wall compliance (AVALON–AWC).

In summary, this work identifies for the first time how an established drug such as amlodipine may impact the caveolae integrity and consecutively influence signaling pathways concentrated in these specialized microdomains. This study, indeed, unravels the calcium-independent interfering effects of amlodipine with one of the physiological locale of eNOS and the enzyme interaction with caveolin. The resulting eNOS configuration appears more prone to agonist stimulation and to posttranslational modifications ensuring long-term potentiation of NO production by amlodipine, thereby establishing the therapeutic interest to combine this CCB with any "eNOS agonist", including statins.


   Notes
 
1 These two authors have equally supervised this work. Back

Time for primary review 21 days


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

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