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Cardiovascular Research 2003 59(4):844-853; doi:10.1016/S0008-6363(03)00505-4
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Copyright © 2003, European Society of Cardiology

Amlodipine activates the endothelial nitric oxide synthase by altering phosphorylation on Ser1177 and Thr495

Helena Lenasi, Karin Kohlstedt, Birgit Fichtlscherer, Alexander Mülsch, Rudi Busse and Ingrid Fleming*

Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany

fleming{at}em.uni-frankfurt.de

* Corresponding author. Tel.: +49-69-6301-6972; fax: +49-69-6301-7668.

Received 10 February 2003; revised 7 June 2003; accepted 1 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The Ca2+ antagonist amlodipine increases the generation of nitric oxide (NO) from native and cultured endothelial cells. The aim of this investigation was to determine whether or not the activation of the endothelial NO synthase (eNOS) by this Ca2+ antagonist is related to alterations in eNOS phosphorylation. Methods and results: In isolated, pre-contracted, endothelium-intact porcine coronary arteries, amlodipine elicited an NO-mediated relaxation and a leftward shift in the concentration-relaxation curve to bradykinin. Moreover, the Ca2+ antagonist increased the generation of NO from native endothelial cells, as detected by electron spin resonance spectroscopy and stimulated an 8-fold increase in cyclic GMP levels in cultured endothelial cells. In unstimulated endothelial cells, eNOS was not phosphorylated on Ser1177 but was phosphorylated on Thr495. Amlodipine elicited the phosphorylation of Ser1177 and attenuated Thr495 phosphorylation, with a time course similar to that of eNOS activation. The amlodipine-induced relaxation of porcine coronary arteries was attenuated by the B2 kinin receptor antagonist, icatibant, but this antagonist did not affect amlodipine-induced changes in eNOS phosphorylation in cultured endothelial cells. Moreover, amlodipine elicited the NO-mediated relaxation of rat aortic rings which do not express the B2 receptor. Amlodipine time-dependently attenuated the phosphorylation of protein kinase C (PKC) in endothelial cells, with a time course similar to the changes in eNOS phosphorylation, and prevented the phorbol-12-myristate-13-acetate-induced activation of PKC. The PKC inhibitor, Ro 31-8220, also elicited the phosphorylation of Ser1177 and the dephosphorylation of Thr495 in cultured cells and induced a leftward shift in the concentration–relaxation curve to bradykinin in rings of porcine coronary artery. Conclusion: The Ca2+ antagonist, amlodipine, enhances endothelial NO generation by inducing changes in the phosphorylation of eNOS. Although the activation of eNOS was related to the activation of the B2 kinin receptor in the porcine coronary artery, a B2 receptor-independent mechanism involving the inhibition of PKC appears to account for the effects observed in the rat aorta as well as in cultured endothelial cells.

KEYWORDS Endothelial factors; Signal transduction; Nitric oxide; Protein phosphorylation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Dihydropyridine Ca2+ antagonists, such as amlodipine, are reported to improve endothelial function in patients with essential hypertension [1] and to significantly slow down carotid atherosclerosis [2]. In cholesterol-fed rabbits amlodipine treatment reduces lesion formation in the thoracic aorta [3] and infarct size after coronary occlusion and reperfusion at the same time as improving acetylcholine-stimulated, NO-mediated relaxation of the aorta [4]. The molecular mechanisms responsible for these effects have not been fully elucidated but have been suggested to be related to the antioxidant properties of this compound [5], reduced uptake of iron into vascular cells [6] or an increase in the production of nitric oxide (NO) [7].

Macrovascular endothelial cells do not express L-type Ca2+ channels [8] and although substances such as nifedipine and amlodipine do not increase intracellular Ca2+ levels ([Ca2+]i) in cultured endothelial cells [9–11], both have been reported to increase the generation of NO [7,10]. It has been proposed that some of the effects of nifedipine may be attributed to an increase in the expression of the endothelial NO synthase (eNOS) [12], but this response has not been universally observed [11]. Moreover, an increase in eNOS expression cannot account for the acute vasodilator effects of Ca2+ antagonists in vivo [13] or in isolated vascular preparations [7]. It therefore appears that Ca2+ antagonists elicit the activation of eNOS via a signaling pathway that does not require a maintained increase in [Ca2+]i. Since a number of stimuli can activate eNOS by modulating the phosphorylation of the enzyme (for review see [14]), the aim of the present investigation was to determine whether or not the amlodipine-induced activation of eNOS and the generation of NO in native and cultured endothelial cells was associated with alterations in eNOS phosphorylation.


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

2.1 Vessel preparation
Porcine hearts were obtained from a local slaughterhouse, placed immediately into ice-cold modified Tyrode’s solution and transported to the laboratory. Male Wistar-Kyoto rats (WKY, 8 weeks) from Charles-River (Sulzfeld, Germany) were anesthetized with isofuren, killed by decapitation and the thoracic aorta was isolated. Once dissected, porcine coronary arteries and rat aortae were cleaned from adventitial adipose and connective tissue and cut into rings (3–4 mm) for the measurement of isometric force. In all cases, the period between isolation and the start of experiments was less than 6 h.

2.2 Vascular reactivity studies
Artery rings were mounted between force transducers and a rigid support for measurement of isometric force as described [15]. Artery rings were incubated in static organ baths containing Tyrode’s solution of the following composition (mmol/l): NaCl, 132; KCl, 4; CaCl2 1.6, MgCl2, 0.98; NaHCO3, 11.9; NaH2PO4, 0.36; glucose, 10; Ca-Titriplex, 0.05 and gassed with 20% O2, 5% CO2 and 75% N2 to give a pO2 of approximately 140 mmHg and pH 7.4 at 37°C. Passive tension was adjusted over a 120-min period to 5 g for porcine coronary arteries and 1 g for rat aorta; thereafter segments were repeatedly exposed to a modified Tyrode’s solution rich in KCl (80 mmol/l) until stable contractions were obtained. After washing, rings were exposed to U46619 [GenBank] (0.1–1 µmol/l) or the combination of U46619 [GenBank] and phorbol 12-myristate 13-acetate (PMA) to achieve a stable contraction which was approximately 80% of the maximal KCl (80 mmol/l)-induced contraction. Thereafter, the integrity of the endothelium was assessed by the application of either bradykinin (1 µmol/l, coronary arteries) or acetylcholine (1 µmol/l, rat aorta) and vessels exhibiting less than 80% relaxation were discarded.

2.3 Cell culture
Human umbilical vein endothelial cells and porcine coronary artery endothelial cells were isolated and cultured as described [16]. Due to the loss of several signalling pathways with time in culture, endothelial cells were used after only one passage. The intracellular concentration of Ca2+ ([Ca2+]i) was measured as described previously [17] and eNOS activity in cultured endothelial cells was assessed by determining the N{omega}-nitro-L-arginine (L-NA)-sensitive accumulation of cyclic GMP under resting conditions and following stimulation using a specific radioimmunoassay, as described [18].

2.4 Assay of NO production
In freshly isolated segments of porcine coronary artery, NO release from the endothelium was detected by electron spin resonance spectroscopy by measuring the rate of formation of paramagnetic mono-nitrosyl-iron complex, as described [19]. The nitrosyl complex exhibits an anisotropic triplet signal with axial symmetry at g{perp}=2.035, g||=2.02 in frozen state. The concentration was determined by comparison of its ESR spectrum with that of a standard (12 µmol/l NOFe(DETC)2 dissolved in DMSO) recorded under identical instrument settings. The ESR spectra were recorded at 77 K on a Bruker ESR 300E at a frequency of 9.47 GHz, modulation frequency 100 kHz, modulation amplitude 0.5 mT, microwave power 20 mW and time constant 0.1–1.3 s.

2.5 Immunoblotting
Cells were lysed in buffer containing Tris–HCl (pH 7.5; 50 mmol/l), NaCl (150 mmol/l), NaF (100 mmol/l), Na4P2O7 (15 mmol/l), Na3VO4 (2 mmol/l), leupeptin (2 µg/ml), pepstatin A (2 µg/ml), trypsin inhibitor (10 µg/ml), phenylmethylsulfonyl fluoride (PMSF; 44 µg/ml) and Triton X-100 (1%, v/v), left on ice for 10 min and centrifuged at 10 000xg for 10 min. Proteins in the cell supernatant or Triton-insoluble pellet were heated with SDS–PAGE sample buffer and separated by SDS–PAGE, as described [20]. Proteins were detected using their respective antibodies, and visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Germany). The eNOS polyclonal antibody as well as the phospho-specific antibodies recognising Thr495 eNOS and Ser1177 eNOS were from Upstate (Milton Keynes, UK) and the phospho-protein kinase C (PKC) antibody was from Cell Signaling Technology (Frankfurt am Main, Germany).

2.6 PKC activity assay
Endothelial cells were stimulated as described in the results section, washed once with Tyrode’s solution and frozen in liquid nitrogen. Cells were lysed by three cycles of freeze–thawing and the phosphorylation of acetylated myelin basic protein (Ac-MBP 4-14, Sigma, Deisenhofen, Germany) assessed in the absence and presence of the PKC inhibitor RO 31-8220 (300 nmol/l) as described previously [21]. Ac-MBP phosphorylation was quantified by scintillation counting, and the specific PKC activity of each sample was determined by subtracting the value obtained in the presence of the PKC inhibitor.

2.7 Materials
Bradykinin was from Bachem (Heidelberg, Germany), U46619 [GenBank] from Alexis (Grünberg, Germany), amlodipine from Pfizer (Sandwich, UK), and icatibant from Aventis (Frankfurt am Main, Germany). All other substances were obtained from Sigma.

2.8 Statistical analysis
Rmax represents the maximal relaxation recorded in response to the cumulative addition of a given agonist and pD2 (–log EC50) values were calculated by non-linear regression of the concentration-relaxation curves to bradykinin. Values presented are the mean±S.E.M and were compared by paired- and unpaired t-test or ANOVA for repeated measurements followed by the Newman–Keuls test, as appropriate. P values less than 0.05 was considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of amlodipine on the vascular tone in the porcine coronary artery
In isolated, endothelium-intact porcine coronary artery rings pre-contracted with U46619 [GenBank] , bradykinin induced a concentration-dependent relaxation. The concentration–response curve to bradykinin was unaffected by pre-incubation (30 min) with amlodipine at concentrations up to 100 nmol/l (pD2 values were 8.24±0.19 and 8.42±0.42 in the absence and presence of amlodipine, n=6, Fig. 1A). The concentration–response curve was however significantly shifted to the left in rings pre-incubated with 1 µmol/l amlodipine (30 min; pD2 values were 8.18±0.19 and 9.02±0.32 in the absence and presence of amlodipine, n=6, P=0.047, Fig. 1B).


Figure 1
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  		Fig. 1 Effect of amlodipine on vascular tone in precontracted rings of porcine coronary artery. (A,B) Concentration–response curves to bradykinin (0.1 nmol/l to 1 µmol/l) obtained the presence of either solvent or amlodipine (Aml, 100 nmol/l or 1 µmol/l, 30 min preincubation). (C,D) Time course of amlodipine-induced NO-mediated relaxation. Solvent or amlodipine (Aml) were added to precontracted coronary artery rings in the absence or presence of the NOS inhibitor N{omega}-nitro-L-arginine (L-NA, 300 µmol/l). Some experiments were performed using endothelium-denuded (-E) arterial rings. The results are presented as the mean±S.E.M. from six separate experiments; *P<0.05, **P<0.01 (ANOVA) compared with the respective control group.

 
When applied directly to U46619 [GenBank] -precontracted coronary artery rings, amlodipine (100 nmol/l and 1 µmol/l) induced a time-dependent relaxation which was markedly attenuated in the presence of the NOS inhibitor, L-NA (300 µmol/l; Fig. 1C,D). Amlodipine failed to elicit the relaxation of endothelium-denuded rings (Fig. 1D).

3.2 Effect of amlodipine on NO production and cyclic GMP levels and [Ca2+]i in endothelial cells
The production of NO, assessed using ESR spectroscopy, by freshly isolated segments of porcine coronary artery was significantly enhanced when segments were treated with amlodipine (1 µmol/l, 30 min, Fig. 2A). The average increase in NO production observed in segments treated with amlodipine was similar to that detected in segments treated with bradykinin. An increase in NO production was also detected in cells stimulated with nifedipine, [NOFe(DETC)2] was 51±14 nmol/l in solvent-treated coronary arteries versus 121±18 nmol/l in coronary arteries treated with nifedipine (3 µmol/l, 10 min; n=7).


Figure 2
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  		Fig. 2 Effect of amlodipine on NO production. (A) Original tracings showing characteristic signals of paramagnetic mono-nitrosyl-iron complex (MNIC) obtained from freshly isolated, endothelium-intact segments of porcine coronary artery incubated with Fe-citrate (100 µmol/l) and DETC (10 mmol/l) in the absence (control) and presence of bradykinin (1 µmol/l), amlodipine (1 µmol/l) or N{omega}-nitro-L-arginine (L-NA, 300 µmol/l). (B) Bar graphs showing the accumulation of cyclic GMP in cultured human umbilical vein endothelial cells under basal conditions (time 0) and in response to cell stimulation with amlodipine (1 µmol/l) for the time indicated. Results are expressed as mean±S.E.M. from six separate experiments; **P<0.01 compared with cyclic GMP levels under basal conditions.

 
In cultured human endothelial cells, amlodipine also induced a time-dependent increase in intracellular cyclic GMP levels (Fig. 2B). Nifedipine also increased cyclic GMP levels in cultured endothelial cells, albeit to a lesser extent than amlodipine (see Fig. 6C).


Figure 6
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  		Fig. 6 Pharmacological characterisation of the amlodipine-induced accumulation of cyclic GMP in cultured human umbilical vein endothelial cells. Graphs showing the accumulation of cyclic GMP in cultured human umbilical vein endothelial cells under basal conditions (CTL) and in response to cell stimulation with (A) amlodipine (1 µmol/l), (B) bradykinin (Bk, 0.1 µmol/l) and (C) nifedipine (Nif, 1 µmol/l). Experiments were performed in the absence (open symbols and columns) and presence (black symbols and columns) of icatibant (0.1 µmol/l). (D) Cyclic GMP levels in endothelial cells under basal conditions (CTL) and in response to cell stimulation with amlodipine in the absence (open columns) and presence of wortmannin (40 nmol/l, grey columns) and H 98 (5 µmol/l, shaded columns). Results are expressed as mean±S.E.M. from eight to 16 separate experiments; *P<0.05, **P<0.01, ***P<0.001 (ANOVA) compared with the appropriate control (CTL) signal.

 
Neither amlodipine (1 µmol/l, 30 min) nor nifedipine (1 µmol/l, 30 min) altered endothelial cell Ca2+; [Ca2+]i was 128±13 versus 122±20 and 134±19 nmol/l in the absence and presence of amlodipine and nifedipine, respectively (n=9).

3.3 Effect of amlodipine on the phosphorylation of eNOS
eNOS in unstimulated human endothelial cells was weakly phosphorylated on Ser1177 (Fig. 3A) but strongly phosphorylated on Thr495 (Fig. 3B). Stimulation of endothelial cells with amlodipine (1 µmol/l) elicited a slow increase in phosphorylation of Ser1177and the dephosphorylation of Thr495, both effects were maximal 20 to 30 min after the addition of amlodipine. Identical results were obtained using cultured porcine coronary artery endothelial cells (data not shown). Solvent (0.1% DMSO) did not affect eNOS phosphorylation but cell stimulation with bradykinin led to a rapid but transient increase in the phosphorylation of eNOS on Ser1177 and a decrease in Thr495 phosphorylation, as reported previously [20].


Figure 3
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  		Fig. 3 Time course of the amlodipine-induced phosphorylation of eNOS. Representative Western blots and summary data showing the effects of amlodipine (1 µmol/l) on eNOS Ser1177 and Thr495. Bradykinin (Bk, 0.1 µmol/l, 30 s)-stimulated cells were included as a positive control. Results are presented as the mean±S.E.M. of five to eight separate experiments, *P<0.05, **P<0.01 (ANOVA) compared with the control (CTL) signal.

 
3.4 Effect of icatibant on amlodipine-induced relaxation
Since some of the actions of amlodipine have been proposed to be mediated via the B2 kinin receptor, we determined the effects of a B2 kinin receptor antagonist/inverse agonist on the amlodipine-induced relaxation of rings of the porcine coronary artery and the rat aorta.

Although amlodipine induced an L-NA-sensitive relaxation of porcine coronary artery rings pre-contracted with U46619 [GenBank] (Fig. 4A), an amlodipine-induced, NO-mediated relaxation was not observed in rings pre-treated (30 min) with icatibant (1 µmol/l; Fig. 4A). Icatibant alone exerted no significant effect on vessel tone (Fig. 4A) and failed to affect the substance P-mediated relaxation of the coronary artery (pD2 values were 8.50±0.20 vs. 8.44±0.23, in the absence and presence of icatibant, respectively, n=6).


Figure 4
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  		Fig. 4 Effect of icatibant on amlodipine-induced relaxation. Time course of amlodipine (Aml, 1 µmol/l)-induced, NO-mediated vascular relaxation in U46619-pre-contracted rings of endothelium-intact porcine coronary artery (A) and rat aorta (B). Experiments were performed in the absence and presence of icatibant (0.1 µmol/l), and N{omega}-nitro-L-arginine (L-NA, 300 µmol/l). Results are expressed as mean±S.E.M. from six separate experiments; **P<0.01 (ANOVA) compared with the amlodipine+L-NA group.

 
In rings of rat aorta, amlodipine also induced a significant L-NA-sensitive decrease in tone. The maximal relaxation (Rmax) observed in rat aortic rings 30 min after the application of amlodipine (1 µmol/l) was slightly lower than that observed in coronary artery rings (Fig. 4) but was unaffected by icatibant (Fig. 4B).

3.5 Effect of icatibant on amlodipine-induced changes in eNOS phosphorylation and endothelial cyclic GMP levels
To determine whether the amlodipine-induced changes in eNOS phosphorylation were mediated via the B2 kinin receptor, we assessed the effects of icatibant on the phosphorylation of eNOS Ser1177 and Thr495 in human endothelial cells.

Icatibant (0.1 µmol/l) had little effect on the amlodipine-induced changes in eNOS phosphorylation in primary cultures of human umbilical vein endothelial cells (Fig. 5), thus suggesting that the Ca2+-antagonist-induced changes in eNOS phosphorylation are unrelated to B2 kinin receptor activation.


Figure 5
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  		Fig. 5 Effect of icatibant on the amlodipine-induced changes in eNOS phosphorylation in cultured human umbilical vein endothelial cells. Representative Western blots showing the effects of amlodipine (Aml, 1 µmol/l) on eNOS Ser1177 and Thr495 phosphorylation in the absence and presence of the B2 kinin receptor icatibant (0.1 µmol/l). Bradykinin (Bk, 0.1 µmol/l, 30 s)-stimulated cells were included as a positive control. Identical results were obtained in three additional experiments.

 
Amlodipine increased intracellular cyclic GMP in human endothelial cells and icatibant did not attenuate this response (Fig. 6A). Icatibant did however significantly attenuate the bradykinin-induced increase in cyclic GMP (Fig. 6B). The nifedipine-induced increase in intracellular cyclic GMP was also insensitive to icatibant (Fig. 6C).

We next determined the effects of inhibiting kinase activation on the amlodipine-induced increase in endothelial cyclic GMP. Neither wortmannin (40 nmol/l), which inhibits the phosphatidylinositol 3-kinase and prevents the activation of Akt, nor H98 (5 µmol/l), which is a potent inhibitor of PKA, affected the increase in cyclic GMP observed after the application of amlodipine (Fig. 6D).

3.6 Effect of amlodipine on the activation of protein kinase C
PKC phosphorylates Thr495, therefore, we assessed the effects of amlodipine on the phosphorylation of PKC in the Triton X-100 insoluble fraction of primary cultures of human umbilical vein endothelial cells which is thought to contain active PKC isoforms [22]. The phospho-PKC antibody used detects PKC{alpha}, βI, βII, {zeta}, {varepsilon} and {delta} isoforms only when phosphorylated at a carboxy terminal residue.

A basal PKC phosphorylation was detected in the Triton X-100-insoluble fraction of unstimulated endothelial cells. Amlodipine induced a slow and time-dependent decrease in the phospho-PKC signal, a response which was most prominent 10–20 min after the application of the Ca2+ channel antagonist (Fig. 7A). Moreover, preincubation of endothelial cells with amlodipine for 20 min prior to the addition of the PKC activator PMA (1 µmol/l), largely prevented PKC phosphorylation (Fig. 7B). Pre-treatment with amlodipine also attenuated the PMA-induced increase in PKC activity, assessed by determining the PKC inhibitor-sensitive phosphorylation of acetylated myelin basic protein (Fig. 7C).


Figure 7
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  		Fig. 7 Effect of amlodipine on the phosphorylation and activity of PKC. (A) Representative Western blot showing the effect of amlodipine (1 µmol/l) on PKC phosphorylation (p-PKC) in cultured human endothelial cells. (B) Effect of amlodipine (1 µmol/l; 20 min preincubation) on the PMA (1 µmol/l)-induced phosphorylation of PKC. The bar graph summarizes data obtained in four additional experiments. (C) Effect of amlodipine (1 µmol/l; 20 min preincubation) on the PMA (1 µmol/l, 20 min)-induced activation of PKC. The bar graph summarizes data obtained in three experiments; *P<0.05, **P<0.01, ***P<0.001 (ANOVA) compared with the appropriate control (CTL) signal.

 
3.7 Effect of the protein kinase C inhibitor Ro 31-8220 on eNOS phosphorylation and NO-mediated relaxation
To determine whether or not a PKC inhibitor could elicit a similar response to amlodipine we determined the effects of Ro 31-8220 on the phosphorylation of eNOS as well as on NO-mediated relaxation of the porcine coronary artery.

In cultured human endothelial cells, Ro 31-8220 (30 nmol/l) time-dependently increased the phosphorylation of eNOS on Ser1177 and attenuated the phosphorylation of Thr495 with a kinetic similar to that of amlodipine (Fig. 8A). Preincubation of porcine coronary artery rings with Ro 31-8220 for 30 min, significantly enhanced the bradykinin-induced, NO-mediated relaxation (Fig. 8B). pD2 values for bradykinin were 7.04±0.30 vs. 8.01±0.20, in the absence and presence of Ro 31-8220, respectively (n=14, P=0.012). Ro 31-8220 did not affect the relaxant response to the endothelium-independent vasodilator, sodium nitroprusside (data not shown).


Figure 8
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  		Fig. 8 Effect of Ro 31-8220 on the phosphorylation of eNOS and NO-dependent relaxation. (A) Representative Western blots showing the effects of Ro 31-8220 (30 nmol/l) on eNOS phosphorylation on Ser1177 and Thr495 in cultured human endothelial cells. Identical results were obtained in three additional experiments. (B) Concentration–response curves to bradykinin (0.1 to 1 µmol/l) in endothelium-intact rings porcine coronary artery. Experiments were performed in the presence of either solvent or Ro 31-8220 (30 nmol/l, 30 min preincubation). Results are expressed as mean±S.E.M. from 14 separate experiments.

 
Stimulating U46619 [GenBank] -pre-contracted coronary artery rings with PMA (1 µmol/l) to activate PKC prior to the addition of amlodipine, attenuated the amlodipine-induced NO-mediated relaxation of these arteries (Rmax 30 min after the addition of amlodipine was 56.1±8.0 vs. 36.4±6.3% in the absence and presence of PMA; n=8, P=0.03).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The results of the present investigation demonstrate that amlodipine activates eNOS by a mechanism involving the inhibition of PKC and subsequent changes in the phosphorylation of eNOS on Ser1177 and Thr495. In the porcine coronary artery this phenomenon seemed linked to the activation of B2 kinin receptors, inasmuch as an inverse agonist at the B2 receptor [23] prevented the amlodipine-induced, NO-mediated relaxation.

A number of dihydropyridine Ca2+ antagonists, including nifedipine [24] and amlodipine [7], are reported to enhance the production of NO from endothelial cells and to relax coronary arteries by an NO-mediated mechanism. In our study we used a concentration of amlodipine that was previously reported to enhance NO production by endothelial cells [7]. Although this concentration (1 µmol/l) is higher than the plasma concentration of the drug, these compounds are all lipophilic and accumulate intracellularly suggesting the effective concentration of the drug may be higher that that in plasma. Since neither nifedipine nor amlodipine increase [Ca2+]i in endothelial cells [9–11], the signalling mechanisms involved in the Ca2+ antagonist-induced activation of eNOS remain unclear. The relaxant effect of amlodipine in the canine coronary artery has been linked to the activation of the B2 kinin receptor, mainly because a B2 receptor inverse agonist abrogated the amlodipine-induced generation of NO from native endothelial cells [7,25]. However, a classical B2 receptor signalling to eNOS is unlikely to mediate the effects of amlodipine on NO production as the response to bradykinin is Ca2+-dependent and involves the activation of kinases such as the Ca2+/calmodulin-dependent kinase II (CaMKII) [20]. It is therefore difficult to determine the exact role of the B2 kinin receptor in the Ca2+ antagonist-induced generation of NO. Indeed, although the B2 receptor appears to be involved in the amlodipine-induced generation of NO in porcine coronary arteries, other B2 receptor-independent but PKC-related mechanisms dominate in native endothelial cells that lack the B2 receptor (i.e., rat aorta) as well as in cultured human endothelial cells which appear to possess a fully functional B2 receptor signalling pathway. It is, however, important to note that the only evidence presented in favour of the activation of the B2 receptor in the coronary artery was that icatibant prevented the amlodipine-induced increase in NO production. However, icatibant is an inverse agonist of the B2 kinin receptor [23], and the previously reported effects on endothelial function may be just as easily be explained by inhibition of the basal activity of the B2 receptor as by an angiotensin converting enzyme-inhibitor like action of the Ca2+ antagonist [13].

Over the last 5 years it has become clear that the activity of eNOS can be regulated by changes in its phosphorylation. Two phosphorylation sites have been particularly well studied. One is a serine residue (Ser1177) in the reductase domain which is not phosphorylated in unstimulated cells but is rapidly phosphorylated in response to the application of fluid shear stress [26], vascular endothelial growth factor (VEGF) [27] or receptor-dependent agonists such as bradykinin [20]. The second residue is a threonine (Thr495) in the calmodulin binding domain which is constitutively phosphorylated in all of the endothelial cells studied to date and which is rapidly dephosphorylated in response to agonist stimulation [20,28].

A number of kinases have been reported to phosphorylate Ser1177 and increase eNOS activity and NO production. Some of these kinases, e.g., CaMKII [20] and PKA [29], are activated by an increase in endothelial [Ca2+]i while kinases such as Akt [26] and the AMP-activated protein kinase (AMPK) [30], can be activated independently of an increase in [Ca2+]i, and are responsible for the phenomenon previously referred to as the Ca2+-independent activation of eNOS [18]. Although the stimulation of endothelial cells with bradykinin can result in the activation of CaMKII and PKA [20], the latter kinases do not appear to play a role in the amlodipine-induced phosphorylation of eNOS as we were unable to detect activation of these kinases by Western blotting and selective kinase inhibitors failed to influence the amlodipine-induced increase in endothelial cell cyclic GMP. We also found no evidence to indicate the involvement of Akt since inhibition of the phosphatidylinositol 3-kinase failed to affect the amlodipine-induced increase in cyclic GMP levels.

The dephosphorylation of Thr495 facilitates the binding of calmodulin to its binding domain and thus is a prerequisite for maximal NO production [20]. The constitutively active kinase that phosphorylates Thr495 is PKC [20,29], a finding that accounts for earlier reports that eNOS is a PKC substrate and that PKC-mediated phosphorylation inhibits eNOS activity [31–33]. Thus, the inhibition of PKC, either by a PKC inhibitor such as Ro 31-8220 or by amlodipine, would be expected to activate eNOS by decreasing the phosphorylation of Thr495 and alleviating the intrinsic PKC-mediated inhibition of enzyme activity. Since nifedipine as well as amlodipine have been reported to inhibit the activation of PKC in endothelial cells [10,34,35], we determined whether or not the activation of eNOS by the Ca2+ antagonist could be related to an effect on PKC. We used an antibody that recognizes several phosphorylated and thus activated isoforms of PKC as well as a PKC activity assay to determine the effects of the Ca2+ channel antagonists on the kinase and found that amlodipine attenuated both the basal activity of the kinase in unstimulated cells and largely prevented the activation of PKC by PMA. Therefore, it is feasible that the main effect of amlodipine on endothelial NO production can be attributed to the inhibition of PKC activity. Indeed, the amlodipine-induced NO-mediated relaxation of porcine coronary arteries was not observed in rings which were pre-incubated with PMA in order to activate PKC. The inhibition of PKC by amlodipine can also account for the changes in Ser1177 phosphorylation since PKC can physically associate with PP2A [36], the phosphatase that dephosphorylates Ser1177 [20,29]. Indeed, we and others [29] have observed that PKC inhibitors enhance the phosphorylation of eNOS on Ser1177 presumably by affecting the activity of the phosphatase.

Taken together our data indicate that although the activation of eNOS was apparently related to the activation of the B2 kinin receptor in the porcine coronary artery, a B2 receptor-independent mechanism involving the inhibition of PKC appears to account for the amlodipine-induced activation of eNOS in the rat aorta as well as in cultured endothelial cells.

Time for primary review 24 days.


   Acknowledgements
 
The authors are indebted to Isabel Winter and Tanja-Maria Mareczek for expert technical assistance. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 553, B5).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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B. Fisslthaler, A. E. Loot, A. Mohamed, R. Busse, and I. Fleming
Inhibition of Endothelial Nitric Oxide Synthase Activity by Proline-Rich Tyrosine Kinase 2 in Response to Fluid Shear Stress and Insulin
Circ. Res., June 20, 2008; 102(12): 1520 - 1528.
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