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RECEPTORS AND SIGNAL TRANSDUCTION

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca²⁺ in human atrial myocardium

¹Department of Molecular and Cellular Sport Medicine, German Sport University, Cologne; and ²Laboratory for Muscle Research and Molecular Cardiology, Department of Internal Medicine III and ³Department of Cardiothoracic Surgery, University of Cologne, Cologne; and ⁴Clinic II of Internal Medicine and Cardiology, Oberpfalz, Germany

Submitted 7 January 2005 ; accepted in final form 2 December 2005

	ABSTRACT

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In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser¹¹⁷⁷ (Ser¹¹⁷⁷ eNOS). We have recently shown that agonist-induced Ser¹¹⁷⁷ eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca²⁺ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser¹¹⁷⁷ eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca²⁺ chelator BAPTA before and after application of BRL 37344 (BRL), a ₃-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser¹¹⁷⁷ eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca²⁺ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser¹¹⁷⁷ eNOS phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not Ser¹¹⁷⁷ eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca²⁺ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation.

₃-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca²⁺ regulation

In this scheme, the endothelial NO synthase (eNOS) is regarded as the predominant generator of NO in not only vascular (23) but also myocardial (2) tissue. Intracellular signal mediation and mechanisms of eNOS activation have been well defined in the vascular system (14). Thus two different molecular mechanisms have been uncovered in endothelial cells that independently change eNOS from an inactive to an active state: 1) a dissociation of the enzyme from the cellular membrane into the cytosol (i.e., translocation) (5, 30) and 2) an Akt kinase-dependent serine phosphorylation of eNOS in position 1177 (i.e., Ser¹¹⁷⁷ eNOS) (12, 15, 16). Whereas eNOS translocation has been shown to be dependent on the binding of CaM, thus requiring elevation of cytosolic Ca²⁺ concentration (9, 23, 39), the phosphorylation of Ser¹¹⁷⁷ eNOS has been found to occur even in Ca²⁺-free endothelial cell medium (12).

Less is known about the molecular mechanisms of eNOS activation and their dependence on cytosolic Ca²⁺ in cardiac myocytes, in which a number of functional and structural differences from endothelial cells are known to exist, including drastic beat-to-beat alterations in myocardial cytosolic Ca²⁺ concentration. Recently, we reported that both eNOS translocation and Ser¹¹⁷⁷ phosphorylation also occur in human myocardium (10, 34). Because cytosolic Ca²⁺ concentration is transiently elevated with each heartbeat and is also known to be altered by -adrenergic stimulation and several pathological states of the heart (4), the degree of Ca²⁺ dependence of these mechanisms may have important physiological and pathophysiological implications for the role of NO in the heart.

In the present study, we therefore examined eNOS activation and NO release in human right atrial myocardium before and after chelating cytosolic Ca²⁺ with BAPTA. We used antibodies specifically directed against the translocated form of eNOS, phosphorylated Akt, and phosphorylated Ser¹¹⁷⁷ eNOS, as well as the NO-sensitive dye diaminofluorescein (DAF). To elicit agonist-induced eNOS activation, we applied the preferential ₃-adrenoceptor agonist BRL 37344 (BRL) (1, 18), which we recently demonstrated both translocates and Ser¹¹⁷⁷ phosphorylates eNOS (10, 34).

	MATERIALS AND METHODS

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Patients

Human right atrial myocardium tissue was obtained from patients with coronary disease who underwent aortocoronary bypass operations without clinical signs of heart failure (14 males, 8 females; mean age, 56.3 ± 5.8 yr). Immediately after explantation, the myocardial tissues were placed into ice-cold, aerated, modified Tyrode solution (see below) and then were delivered to the laboratory within 10 min. Our study protocol was approved by the local ethics committee and conformed to the requirements stated in the Declaration of Helsinki. All patients gave global consent for explantation of tissue.

Immunohistochemistry

Tissue pretreatment. Investigation of BRL- or forskolin-mediated changes in the phosphorylation and translocation state of eNOS and Akt using specific antibodies (5, 10, 35) made it necessary to conduct preincubation procedures with freshly obtained tissue. Therefore, two pieces of myocardial tissue obtained from one patient were suspended in separated organ baths containing carbogen gassed Tyrode solution at 37°C (composition in mM: 119.8 NaCl, 5.4 KCl, 1.05 MgCl₂, 1.8 CaCl₂, 22.6 NaHCO₃, 0.42 NaH₂PO₄, 5.5 glucose, 0.28 ascorbic acid, and 0.05 Na₂-EDTA; pH 7.4) for at least 45 min. One of the myocardial tissue sections was then incubated with BRL (10 µM) in the organ bath for 5 min. Both tissue pieces were then taken from the bath and placed immediately into 4% paraformaldehyde. When experiments were performed in the presence of Ca²⁺ chelation, BAPTA (20 µM) was added to the organ bath at least 20 min before experiments were conducted.

Fixation procedures and immunocytochemistry were conducted as described previously (10, 34). To prevent day-to-day variations in experimental conditions, all immunohistochemical preparations for a specific subset of experiments were obtained simultaneously in one procedure on the same day under identical conditions.

Identification of cell type. Vascular structures of all sizes were found frequently throughout the immunohistochemical preparations and were consistently observed to have higher-than-average staining for phospho-Akt, phosphorylated Ser¹¹⁷⁷ eNOS, and translocated eNOS (Fig. 1B, arrows). This finding is consistent with the relatively higher occurrence of eNOS in endothelial cells (29). Because this study was focused on myocardial tissue only, we strictly excluded all vascular and/or endothelial cells from our densitometric quantifications.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Negative and positive controls for immunohistochemical staining. A: lowest staining intensity was observed in immunohistochemical controls, i.e., when no specific antibody was used. B: maximum staining intensity was observed in endothelial cells of vascular structures, which were stained for Akt-dependent phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at Ser1177 (Ser1177 eNOS) in this image (arrows). Vascular structures are shown for positive control of staining intensity but were excluded from the study because only cardiomyocytes were investigated (see Figs. 2 and 3).

Densitometry. For analysis of immunostaining intensity in cardiomyocytes, we measured the gray values of 30 cardiomyocytes from three randomly selected areas of each slice. The immunostaining intensity is reported as the mean of measured cardiomyocyte gray value minus background gray value. The background gray value was measured in a cell-free area of the slice. For staining intensity detection, a Zeiss Axiophot microscope coupled to a three-chip charge-coupled device camera was used, and image analysis was performed using a VIA-Optima 6.01 system (Horiba Jobin Yvon, Munich, Germany).

Western Blot Analysis of Ser¹¹⁷⁷ eNOS Phosphorylation

Tissue pretreatment. For the determination of BRL-induced Ser¹¹⁷⁷ eNOS phosphorylation, the tissue was pretreated as described above. For the subsequent experiments, it was shock frozen in liquid nitrogen and then stored at –80°C.

Tissue preparation. For experiments, 0.1–1 g of myocardium was powdered in liquid nitrogen and thawed on ice in 3 volumes of chilled preparation buffer composed of (in mM) 300 sucrose, 1 PMSF, 20 PIPES, 10 EDTA, and 50 NaH₂PO₄, pH 7.4. Care was taken to dissect myocardial tissue from connective tissue, vessels, and epicardium. Samples were minced with three strokes of 30 s using an Ultra-Turrax homogenizer (IKA, Staufen, Germany) after homogenization with a Teflon potter under constant temperature (4°C). The resulting homogenate was further diluted with the same volumes of a storage buffer containing (in mM) 400 saccharose, 5 HEPES, 5 Tris, 10 EDTA, and 50 NaH₂PO₄, pH 7.2, frozen in liquid nitrogen, and stored at –80°C until use. For the experiments, myocardial homogenates were further diluted in the above storage buffer to a final protein concentration of 4,000 µg/ml, which was suitable for Western blot analysis, and verified using the Bradford assay method.

Western blot analysis. For the detection of phosphorylation of eNOS at Ser¹¹⁷⁷, we performed immunoblot analysis according to the method of Laemmli (25) using an antibody directed specifically against the phosphorylated form of Ser¹¹⁷⁷ eNOS (10). Therefore, crude homogenates were thawed on ice and suspended in buffer (0.5 mmol/l Tris·HCl, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue). Protein separation was performed in a continuous SDS-PAGE gel with 4% stacking gel and 12% separation gel under constant currents of 70 and 100 mA. Protein transfer onto PVDF membrane was performed at 4°C overnight with 70-mA constant current. Transfer efficiency was verified using total protein staining of the gels with Coomassie blue. Membranes were blocked in Tris-buffered saline containing 5% milk. Antibody binding to the PVDF membrane with the specific MAb was performed overnight at 4°C. After being washed thoroughly, the membrane was exposed to a secondary rabbit MAb for 2 h at room temperature. For detection of antibody binding, an ECL assay was performed and exposed to X-ray film according to the signal. Quantification of protein expression was performed after scanning the films into a personal computer file and analyzing the densitometric volume of bands using the personal densitometer and ImageQuant software (Amersham Biosciences, Little Chalfont, UK).

DAF Fluorometry

4-Amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) is converted via an NO-specific mechanism to an intensely fluorescent triazole derivative (24). We used DAF-FM diacetate to detect changes in NO level induced by BRL or by forskolin. Therefore, right atrial myocardial tissue was collected as described above. It was then shock frozen and stored at –80°C. For experiments, the tissue was equilibrated at –20°C for at least 1 h and sliced to 25-µm thickness. Slices were fixed to a plastic slide that had been coated with 15% gelatin. Incubation medium containing CaCl₂, MgCl₂, KCl, NaCl, NaH₂PO₄, glucose, NaHCO₃, and 1 mM L-arginine was added immediately, and DAF (10 µM) and BRL (10 µM) or forskolin (0.3 µM) were added for the relevant experiments. In the subsequent experiments, the intensity of DAF-FM fluorescence was measured every 10 s for 10 min. The intensity of DAF-FM in the absence of any of the agents listed above was set at 100% as a reference value for each time point. The effects of BRL and forskolin on NO level were then investigated in relation to DAF-FM fluorescence intensity. When experiments where performed in the presence of BAPTA or LY-292004, the compound was added 20 min before the experiment. For positive controls, we repeated the experiments using the NO donor (z)-1-{N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino}-diazen-1-ium-1,2-diolate (spermine NONOate; Alexis, Grünberg, Germany) (10 µM) or L-arginine (10 mM) instead of BRL. For negative controls, we used L-NAME (100 µM).

Materials

The preferential ₃-adrenoceptor agonist BRL was obtained from Tocris (Bristol, UK). The following primary antibodies were used for immunohistochemistry: 1) rabbit anti-eNOS antibody against the bovine eNOS peptide [amino acids (aa) 599–613] plus additional COOH-terminal Cys conjugated to KLH (PYNSSPREQHKSYKC; Biomol, Hamburg, Germany), an antibody previously shown to be specific in detecting the eNOS protein after dissociation from caveolin, i.e., after translocation (5, 35); 2) anti-phospho-Akt/PKB (Upstate Biotechnology, Lake Placid, NY), corresponding to aa 301–312 of mouse pAkt/PKB; and 3) anti-phospho-Ser¹¹⁷⁷ eNOS (Upstate Biotechnology), corresponding to aa 1,172–1,183 of human eNOS. As secondary antibodies, a biotinylated goat anti-rabbit or biotinylated goat anti-mouse antibody (Dako, Hamburg, Germany) was used for accentuation.

Further substances used were the cell-permeant Ca²⁺ chelator BAPTA-AM (Calbiochem, Schwalbach, Germany), DAF-FM (Molecular Probes, Eugene, OR), the NO inhibitor N-nitro-L-arginine (L-NAME) (Buchs, Switzerland), and the NO donor spermine NONOate.

All other chemicals were of analytical grade or the best grade commercially available. For studies with isolated myocardium and trabeculae, stock solutions were prepared and added to the organ bath. All compounds were dissolved twice in distilled water and did not change the pH of the medium.

Statistical Analysis

Means were compared between groups and across time using repeated-measures ANOVA. Pairwise mean comparisons were performed using the Tukey-Fisher criterion with repeated-measures ANOVA (JMP 5.01a; SAS Institute, Cary, NC).

	RESULTS

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eNOS Translocation

To assess eNOS activation by translocation of the enzyme in the absence and presence of BAPTA, we performed immunohistochemical eNOS staining in isolated, nonfailing right atrial trabeculae under control conditions (time 0) and after the application of preferential ₃-adrenoceptor agonist and eNOS activator BRL (10 µM for 5 min). We therefore used an eNOS antibody that has been shown to specifically detect the activated, translocated eNOS protein (5, 35). Figure 2 shows original immunostaining images. Staining intensity for activated and/or translocated eNOS was significantly raised after incubation with BRL (densitometric evaluation of 178 cells obtained from 6 patients; +BRL, +53.52 ± 15.21%) (Fig. 2, A and B).

View larger version (73K): [in this window] [in a new window]   Fig. 2. Immunohistochemical detection of translocated eNOS (5) in human right atrial myocardium in the absence (A and B) and presence (C and D) of BAPTA. A distinct increase in immunohistochemically detectable translocated eNOS was visible in right atrial cardiomyocytes after stimulation with BRL 37344 (BRL), a 3-adrenoceptor agonist (10 µM for 5 min) (B), compared with unstimulated cardiomyocytes (A). This experiment was repeated in the presence of BAPTA (20 µM for 20 min) (C and D). In this situation, BRL (10 µM for 5 min) (D) failed to elicit eNOS translocation. Images are representative of experiments with tissues from 6 patients in the absence of BAPTA and from 4 patients in the presence of BAPTA.

Ser¹¹⁷⁷ eNOS Phosphorylation

We recently showed that BRL induces Akt-dependent phosphorylation of Ser¹¹⁷⁷ eNOS in human myocardium (10). To test whether this effect is also influenced by Ca²⁺ chelation, the effects of BRL on Ser¹¹⁷⁷ eNOS and Akt phosphorylation were assessed in the presence of BAPTA. We therefore conducted immunohistochemical experiments, this time using antibodies specifically directed against phosphorylated Akt and phosphorylated Ser¹¹⁷⁷ eNOS (10, 12). Although BAPTA (20 µM for 20 min) was present, BRL led to a strong increase in both phosphorylated Ser¹¹⁷⁷ eNOS (Fig. 3, A and B) and phosphorylated Akt (Fig. 3, C and D) compared with basal conditions (Fig. 3, A and C). Densitometric quantification (Fig. 4) showed an increase in staining intensity of +148.1 ± 39.2% (P < 0.05) for phosphorylated eNOS (densitometric evaluation of 89 cells obtained from 3 patients) and phosphorylated Akt (densitometric evaluation of 114 cells obtained from 4 patients) upon BRL stimulation. Densitometric quantification of the immunohistochemical experiments is shown in Fig. 4.

View larger version (71K): [in this window] [in a new window]   Fig. 3. Influence of BRL on intensity of immunostaining for phosphorylated Ser1177 eNOS (A and B) and phosphorylated Akt (C and D) proteins in the presence of BAPTA (20 µM for 20 min) in human right atrial myocytes. Although eNOS translocation was abolished in the presence of BAPTA (Fig. 2), BRL (10 µM for 5 min) distinctly raised the level of both phosphorylated Ser1177 eNOS (B) and phosphorylated Akt (D) compared with unstimulated myocytes (A and C), even in the presence of BAPTA. Images represent experiments with tissue from 4 patients for phosphorylated Akt and 3 patients for phosphorylated Ser1177 eNOS.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Densitometric quantification of immunohistochemical experiments. Staining intensity was quantified by comparing the gray-shaded values of randomly selected cardiomyocytes before (basal) and after 5-min stimulation with BRL. Values are average percentages ± SE of fluorescence intensity under basal conditions. *P < 0.01 vs. translocation of eNOS in presence of BAPTA using 2-way ANOVA. Note that in the presence of BAPTA, BRL did not induce eNOS translocation (n = 122) but distinctly raised the phosphorylation of Akt (n = 114) and Ser1177 eNOS (n = 89).

View larger version (16K): [in this window] [in a new window]   Fig. 5. Western blot analysis of homogenates from human atrial myocardium. BRL (10 µM for 5 min) led to phosphorylation of Ser1177 eNOS in the absence and presence of BAPTA. A: original blots shown are representative of 3 patients in the absence of BAPTA (left) and 4 patients in the presence of BAPTA (right) (20 µM for 20 min). B: densitometric quantification of Western blots. Values are percentages ± SE of signal intensity under basal conditions. *P < 0.05 vs. basal condition.

The fluorescent NO-sensitive dye DAF (24) was used to measure agonist-induced NO release in the presence and absence of BAPTA. Figure 6 shows representative fluorescent images obtained after reaction times of 5 and 10 min in human right atrial myocardium. Under control conditions (Fig. 6, A–C), fluorescence modestly increased over time as DAF labeled NO produced under basal conditions (n = 4; +23.7 ± 5.9%). A strong increase in fluorescence was observed after reaction times of 5 min (Fig. 6E) and 10 min (Fig. 6F) when BRL (10 µM) was added at time 0 (+BRL, +90.7 ± 16.3%; n = 6) (P < 0.05 vs. control 2-way ANOVA) (Fig. 6, D–F). When this experiment was repeated in the presence of BAPTA (20 µM for 20 min) (Fig. 6, G–I), the effect of BRL on fluorescence was completely lost. To further asses the contribution of Ca²⁺-independent eNOS phosphorylation to atrial NO release, we repeated the experiment in the presence of LY-292004 (40 µM), a blocker of phosphatidylinositol 3-kinase, which has been demonstrated to have the potential to block myocardial Ser¹¹⁷⁷ eNOS phosphorylation (10). Other than the presence of BAPTA, LY-292004 did not affect BRL-induced NO release [+BRL + LY-292004, +96 ± 28%, n = 3, P < 0.05 vs. control; and P > 0.05 vs. BRL in absence of LY-292004 (2-way ANOVA)] (Fig. 6, J–L). These findings were quantified and are shown in Fig. 6B.

View larger version (64K): [in this window] [in a new window]   Fig. 6. NO imaging with diaminofluorescein (DAF). A: under control conditions (DAF only; A–C), fluorescence mildly increased as DAF accumulated in atrial myocytes during 5-min (B) and 10-min (C) periods. A strong increase in fluorescence intensity was observed after 5-min (E) and 10-min (F) incubation with BRL (10 µM) compared with basal conditions (D). In the presence of BAPTA (20 µM for 20 min) (G–I), only a weak increase in DAF fluorescence was induced upon BRL incubation (H and I) that did not exceed that observed under control conditions. When experiments were repeated in the absence of BAPTA but in the presence of LY-292004 (40 nM) to inhibit eNOS phosphorylation (J–L), BRL-induced NO release was undisturbed (J–L). Images are representative of 7 patients in the absence of BAPTA and of 3 patients in the presence of BAPTA, as well as of 3 patients in the presence of LY-292004. B: densitometric quantification of DAF NO imaging. Values are percentages of densitometric units at time 0, i.e., under basal conditions. *P < 0.05, **P < 0.01 vs. controls (2-way ANOVA).

Effects of Forskolin on eNOS Translocation and NO Release

Because -adrenergic stimulation is mediated via an adenylate cyclase-activated increase in cytosolic Ca²⁺ concentration, we asked whether this effect may directly influence myocardial eNOS activation. We therefore tested the effects of the adenylate cyclase activator forskolin (0.3 µM) on eNOS translocation and NO release in immunohistochemical and DAF experiments (Fig. 7). Forskolin neither affected eNOS translocation (Fig. 7, A and B) nor changed the NO level (Fig. 7, C and D).

View larger version (66K): [in this window] [in a new window]   Fig. 7. Effects of forskolin on eNOS translocation and NO release. A and B: immunohistochemical analysis showing that eNOS translocation did not change before (A) or after (B) stimulation with forskolin (0.3 µM for 5 min; n = 3 patients). B: NO imaging via DAF fluorescence: Forskolin (0.3 µM for 10 min) also did not have a significant influence on NO release (C and D); n = 3 patients.

	DISCUSSION

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Translocation and Phosphorylation of eNOS

The molecular mechanisms of eNOS activation are well defined in vascular tissue. Thus it is known that eNOS activation occurs via dissociation from caveolin and subsequent translocation into the cytosol, a process that requires the binding of CaM and thus increased cytosolic Ca²⁺ concentration (5, 29, 30). Dimmeler and Fulton (12, 16) were the first to recognize that eNOS phosphorylation poses an alternative mechanism of eNOS activation in endothelial cells that occurs independently of eNOS translocation and CaM binding and thus does not require cytosolic Ca²⁺. Recently, we were able to demonstrate that agonist-induced Ser¹¹⁷⁷ phosphorylation of eNOS occurs not only in endothelium but also in human myocardium (10).

In the present study, myocardial eNOS translocation was abolished after depletion of cytosolic Ca²⁺, a finding consistent with the theory of Ca²⁺/CaM-induced eNOS activation in endothelial cells. Because CaM cannot bind to eNOS in Ca²⁺-free medium, eNOS cannot dissociate from caveolin and thus cannot translocate into the cytosol. To the contrary, we were able to clearly detect agonist-induced phosphorylation of Ser¹¹⁷⁷ eNOS clearly, along with Akt phosphorylation, even after chelation of cytosolic Ca²⁺. This finding also is consistent with the observation of persistent shear stress-induced Akt phosphorylation in endothelial cells in the absence of Ca²⁺ (12) and allows the conclusion that not only phosphorylation of Akt but also subsequent Ser¹¹⁷⁷ eNOS phosphorylation are Ca²⁺ independent. To our knowledge, this study is the first to show that Ca²⁺-dependent and Ca²⁺-independent molecular mechanisms of eNOS activation occur not only in endothelium but also in atrial cardiomyocytes. Until now, Ca²⁺-independent eNOS phosphorylation has not been observed to occur as a consequence of agonist stimulation; it is furthermore most notable that in this study we were able to elicit it by stimulation with the ₃-adrenoceptor agonist BRL.

NO Release

In endothelial cells, a release of NO subsequent to phosphorylation of eNOS has been reported to occur even in the absence of Ca²⁺, although usually with delayed kinetics, i.e., with a maximum between 5 and 10 min (7, 14, 20, 32, 40). Although we chose similar conditions to lower cytosolic Ca²⁺ and we monitored the full first 10 min after stimulation with BRL, we were unable to detect an increase in NO level in the presence of BAPTA in atrial myocytes. When we suppressed Ser¹¹⁷⁷ eNOS phosphorylation using LY-294002, BRL-induced NO release persisted. On the basis of these observations, we conclude that other than in endothelium, eNOS phosphorylation at Ser¹¹⁷⁷ in atrial myocytes plays only a minor role in NO production compared with eNOS translocation. Yet, this may not account for all circumstances. In this study, we used BRL to elicit eNOS activation via ₃-adrenergic stimulation (17, 34) and thus we cannot exclude the possibility that non-receptor-mediated eNOS phosphorylation, for example, by shear stress (33), ceramides (20), or insulin (40), might have more impact on myocardial eNOS activity in the absence of Ca²⁺. Furthermore, recent studies have shown that additional phosphorylation sites, such as at Ser⁶³⁵, may be involved in Ca²⁺-independent regulation of eNOS in endothelial cells (3, 6, 13, 31). We cannot exclude the possibilities that BRL may fail to act via this phosphorylation site and that only the convergence of phosphorylation at Ser⁶³⁵ and Ser¹¹⁷⁷ ultimately may be decisive for Ca²⁺-independent eNOS activity in atrial cardiomyocytes.

Also, -adrenoceptor distribution and coupling have been reported to differ between atrium and ventricle (36–38). Because we have provided evidence that eNOS activation in ventricular myocytes occurs without eNOS translocation (10), Ser¹¹⁷⁷ eNOS phosphorylation may be of much greater significance for NO production in ventricular myocytes than in atrial myocardium.

Lack of Effects of Forskolin

₁-adrenergic stimulation is one of the most important mechanisms of cardiac regulation. In our study, the application of forskolin to mimic ₁-adrenergic effects did not affect eNOS translocation or NO release. This finding is consistent with those reported in previous studies that demonstrated that ₁-adrenergic stimulation cannot by itself induce cardiac NO release (18, 34). Thus our findings indicate that although Ca²⁺-dependent myocardial NO release does not occur as a direct, sole consequence of cytosolic Ca²⁺ increase by adrenergic stimulation. Yet, it is possible that alterations of cytosolic Ca²⁺ may have modulatory effects on agonist-induced myocardial NO release, i.e., that the effects of NO-releasing agonists such as acetylcholine, bradykinin, or ₃-adrenoceptor agonists are enhanced or diminished, depending on the cytosolic Ca²⁺ level of the cardiomyocyte.

Functional Implications

Our present results show that NO release in human atrium is Ca²⁺ dependent and that Ca²⁺-independent phosphorylation, although existent in human atrium, does not contribute substantially to NO production. This may be different in human ventricles, in which NO production seems to depend on eNOS phosphorylation and therefore is independent of cytosolic Ca²⁺ level (10). Thus eNOS activity in human atrium could be much more dependent on beat-to-beat changes in cytosolic Ca²⁺ levels compared with ventricular myocardium, which may result in differences in contractile regulation between atrium and ventricle. Also, longer-term changes in diastolic and systolic Ca²⁺ concentration as they occur during hypertrophy and heart failure (4) potentially could have greater effects on atrial than on ventricular NO metabolism. On the other hand, stretch-activated cardiac NO release, which is known to be mediated via Ca²⁺-independent eNOS phosphorylation (33), would thus have more pronounced effects in ventricle than in atrium. Finally, the fact that NO release in atrium is Ca²⁺ dependent could play an important role during ischemia-reperfusion injury, in which an increase in cytosolic Ca²⁺ mediates myocardial damage (21).

Summary

In conclusion, the present study shows that in human atrial myocardium, receptor-mediated eNOS translocation is dependent on intracellular Ca²⁺ concentration, whereas receptor-mediated Ser¹¹⁷⁷ eNOS phosphorylation is not. In this regard, our present findings are consistent with current understanding of eNOS activation in the endothelium and extend this knowledge to the atrial myocardium. Furthermore, we have demonstrated that in atrial myocardium, eNOS translocation and not Ser¹¹⁷⁷ eNOS phosphorylation generates bulk NO upon agonist stimulation. We also have provided evidence that Ca²⁺-dependent eNOS translocation and NO release do not occur directly upon ₁-adrenergic stimulation alone.

	GRANTS

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This work was supported by funds from the Deutsche Forschungsgemeinschaft (to C. Pott and R. H. G. Schwinger) and the Cologne Fortune Program, Faculty of Medicine, University of Cologne (to C. Pott and K. Brixius).

	ACKNOWLEDGMENTS

 
We are indebted to all colleagues of the Department of Cardiothoracic Surgery of the University of Cologne (Prof. Dr. R. E. de Vivie) for providing human myocardium tissue samples. We are thankful to K. Rösler and M. Ghilav for technical support.

Present address of C. Pott: Department of Physiology, University of California, Los Angeles, 675 Charles E. Young Dr. South, MRL Building, Room 3645, Los Angeles, CA 90095 (e-mail: cpott@mednet.ucla.edu).

	FOOTNOTES

 

Address for reprint requests and other correspondence: W. Bloch, Dept. of Molecular and Cellular Sport Medicine, German Sport Univ. Cologne, Institutsgebäude 1, 9.OG, Carl-Diem-Weg 6, Rm. 911, D-50933 Cologne, Germany (e-mail: w.bloch{at}dshs-koeln.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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