Na+/Ca2+ Exchange Facilitates Ca2+-dependent Activation of Endothelial Nitric-oxide Synthase*

Recent evidence suggests the expression of a Na+/Ca2+ exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca2+ signaling and Ca2+-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na+gradients and after loading of endothelial cells with Na+ions using the ionophore monensin. Monensin-induced Na+loading markedly reduced Ca2+ entry and, thus, steady-state levels of intracellular free Ca2+([Ca2+] i ) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca2+] i , Ca2+-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca2+ concentration response curve in monensin-treated cells. This facilitation of Ca2+-dependent activation of eNOS was strictly dependent on the presence of Na+ ions during treatment of the cells with monensin. Na+-induced facilitation of eNOS activation was not due to a direct effect of Na+ ions on the Ca2+ sensitivity of the enzyme. Moreover, the effect of Na+ was not related to Na+ entry-induced membrane depolarization or suppression of Ca2+ entry, since neither elevation of extracellular K+ nor the Ca2+ entry blocker 1-(β-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) mimicked the effects of Na+loading. The effects of monensin were completely blocked by 3′,4′-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na+/Ca2+ exchange, was ineffective. Consistent with a pivotal role of Na+/Ca2+ exchange in Ca2+-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na+ gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.

The endothelial isoform of nitric-oxide synthase (eNOS) 1 is constitutively expressed in endothelial cells and cardiac myocytes and dynamically regulated by Ca 2ϩ /calmodulin. The enzyme is unique among the three known NOS isoforms in being targeted to specialized cell surface signal-transducing domains termed plasmalemmal caveolae (1,2). This feature appears to allow for an efficient control of eNOS activity, as numerous signaling molecules such as G-protein-coupled receptors, the plasma membrane Ca 2ϩ pump, an inositol 1,4,5-trisphosphatesensitive Ca 2ϩ channel, and protein kinase C are enriched in caveolae (3,4). The targeting of eNOS to caveolae is promoted by direct interaction of the enzyme with the caveolae structural protein, caveolin-1. The interaction of eNOS with caveolin-1 renders the enzyme inactive, apparently due to a functional competition between caveolin-1 and Ca 2ϩ /calmodulin (5)(6)(7). In the presence of high concentrations of Ca 2ϩ , the NOS-caveolin complex dissociates, and a catalytically active NOS-Ca 2ϩ /calmodulin complex is formed.
Although it is well established that depletion of intracellular Ca 2ϩ stores and capacitative Ca 2ϩ entry across the endothelial plasma membrane are key events in endothelial Ca 2ϩ signaling (8 -10), little is known about the role of individual Ca 2ϩ transport systems in eNOS regulation. Recent studies provide evidence that significant increases in subplasmalemmal [Ca 2ϩ ] i , which may well be sufficient for enzyme activation, may occur even in the absence of detectable changes in perinuclear [Ca 2ϩ ] i , suggesting that focal elevations in subplasmalemmal [Ca 2ϩ ] i rather than increases in overall [Ca 2ϩ ] i trigger NO biosynthesis in endothelial cells (11,12). Thus, the subcellular Ca 2ϩ distribution and the subplasmalemmal Ca 2ϩ concentration at the caveolae may be of particular importance for modulation of eNOS activity. It has recently been postulated that endothelial subplasmalemmal [Ca 2ϩ ] i may be controlled for a large part by Na ϩ /Ca 2ϩ exchange (11). This Ca 2ϩ transport system was first identified in cardiac muscle (13) and transports Ca 2ϩ in exchange for Na ϩ in either direction, depending on the electrochemical gradients of Na ϩ and Ca 2ϩ (14,15). In the forward mode (Na ϩ entry/Ca 2ϩ extrusion), the exchanger represents the primary mechanism for Ca 2ϩ efflux in the myocardium and thus plays a prominent role in contractile function (15)(16)(17). During depolarization, the exchanger operates in reversed mode (Ca 2ϩ entry/Na ϩ extrusion) and triggers Ca 2ϩinduced Ca 2ϩ release during cardiac excitation (18,19).
In endothelial cells, the presence of a Na ϩ /Ca 2ϩ exchanger (NCX) has recently been demonstrated by immunoblotting and immunofluorescence microscopy (20), and these results have been verified by the detection of cDNA coding for NCX1 (21). However, the role of this protein in endothelial Ca 2ϩ homeostasis is still a matter of debate. Since inhibition of NCX diminished the endothelium-dependent relaxation of arteries (22,23), it has been suggested that reversed mode Na ϩ /Ca 2ϩ exchange may contribute to the plateau phase of [Ca 2ϩ ] i after agonist stimulation of endothelial cells (24). However, no change in overall [Ca 2ϩ ] i was observed when extracellular Na ϩ was replaced by N-methyl-D-glucamine or Li ϩ in resting or bradykinin-stimulated endothelial cells (25,26). Nonetheless, a significant increase in [Ca 2ϩ ] i was observed when extracellular Na ϩ was replaced by Li ϩ in endothelial cells that were Na ϩloaded by a preceding incubation with the Na ϩ ionophore monensin (26). Thus, changes in [Ca 2ϩ ] i due to Na ϩ /Ca 2ϩ exchange are clearly detectable when the intracellular Na ϩ concentration is elevated, supporting the hypothesis that Na ϩ /Ca 2ϩ exchange contributes to Ca 2ϩ homeostasis in endothelial cells.
The present study was designed to elucidate the functional relevance of Na ϩ /Ca 2ϩ exchange for Ca 2ϩ -dependent activation of eNOS in cultured porcine aortic endothelial cells. We provide evidence for facilitation of eNOS activation due to reversed mode Na ϩ /Ca 2ϩ exchange.
Determination of Intracellular Ca 2ϩ Levels in Store-depleted Endothelial Cells-[Ca 2ϩ ] i was determined using the Ca 2ϩ indicator FURA-2 as described previously (29,30). Briefly, endothelial cells were harvested, suspended in Dulbecco's modified Eagle's medium (ϳ10 6 cells/ ml), and incubated with 2 M FURA-AM at 37°C. After 45 min, cells were washed and resuspended in nominal Ca 2ϩ -free incubation buffer (50 mM HEPES buffer, pH 7.4, containing 100 mM NaCl, 5 mM KCl, 1 mM MgCl 2 and 0.1 mM EGTA). Where indicated, 100 mM NaCl was replaced by 100 mM choline chloride (nominal Na ϩ -free buffer) or 100 mM KCl ϩ 20 mM NaCl (high K ϩ buffer). Fluorescence measurements were carried out at 37°C at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Experiments were started by the addition of 0.1 M thapsigargin and, where indicated, 1 M monensin or 30 M SK&F 96365. After 15 min, aliquots of Ca 2ϩ stock solutions were added to obtain the indicated concentrations of extracellular Ca 2ϩ , which were determined in parallel experiments with a Ca 2ϩ -sensitive electrode. [Ca 2ϩ ] i was calculated using the ratio of fluorescence intensity at 340/380 nm.
Determination of eNOS Activity in Store-depleted Endothelial Cells-NOS activity in intact cells was determined by monitoring the conversion of L-[ 3 H]arginine into L-[ 3 H]citrulline as described previously (31,32). Briefly, endothelial cells grown in 6-well plates were washed with nominal Ca 2ϩ -free incubation buffer, nominal Na ϩ -free buffer, or high K ϩ buffer (see above) and preincubated for 15 min at 37°C in the presence of 100 nM thapsigargin and, where indicated, with 1 M monensin or 30 M SK&F 96365. Reactions were started by addition of L-[2,3,4,5- Isolation of Caveolin-rich Membrane Fractions-Caveolae membranes were isolated by a method adapted from Song et al. (33). Briefly, endothelial cells were harvested, washed twice with ice-cold phosphatebuffered saline (PBS), and resuspended in 2 ml of 0.5 M sodium carbonate, pH 11.0 (2 ϫ 10 7 cells/ml). Cells were homogenized by sonication, and the homogenate was adjusted to 45% sucrose by adding 2 ml of 90% sucrose prepared in MBS (25 mM MES, pH 6.5, 0.15 M NaCl). 1.8 ml of the 45% sucrose homogenate was placed at the bottom of a 4-ml ultracentrifuge tube overlaid by 1.8 ml of 35% and 0.4 ml of 5% sucrose (each prepared in MBS containing 0.25 M sodium carbonate) and centrifuged for 20 h at 134,000 ϫ g. From the top of the gradient, 0.4-ml fractions were collected and mixed with 0.1 ml of a solution of 50% trichloroacetic acid. After centrifugation, the precipitated proteins were either dissolved in 0.1 ml of 1 N NaOH for the determination of total protein or in 0.1 ml of Laemmli buffer for immunoblotting experiments.
Immunofluorescence Microscopy-Endothelial cells, cultured on glass coverslips, were washed twice with PBS and fixed in PBS containing 3.7% formaldehyde and 1.5% methanol for 10 min at room temperature (35). After two washes with 0.2% Triton X-100 in PBS, cells were preincubated at 37°C in PBS containing 0.3% Triton X-100, 1% bovine serum albumin, and 1% goat serum (blocking buffer). After 45 min, the antibody against eNOS (final dilution 1/10) or NCX (final dilution 1/500) was added, and cells were incubated for 90 min at 37°C. Cells were then washed three times with blocking buffer and incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG in blocking buffer (dilution 1/200) for 90 min at 37°C. After 2 washes with 0.2% Triton X-100 in PBS and 1 wash with PBS, cells were mounted in PBS containing 50% glycerol and 0.1% p-phenylendiamine, pH 8.0. Fluorescence microscopy was carried out on a Leica TCS 4D confocal microscope equipped with an Ar/Kr laser and set up with the appropriate filter set for fluorescein isothiocyanate detection (488 nm excitation, TK515 beam splitter, BP-fluorescein isothiocyanate emission filter). Fig. 1  To test for a contribution of NCX in Ca 2ϩ homeostasis, endothelial cells were loaded with Na ϩ by incubation with the Na ϩ ionophore monensin, which is known to promote Ca 2ϩ entry via reversed mode Na ϩ /Ca 2ϩ exchange (26,36). Alternatively, Na ϩ was omitted from the extracellular solution and replaced by choline chloride to avoid Na ϩ loading of the cells. As shown in Fig. 2A, replacement of Na ϩ by choline chloride in the extracellular solution did not change the correlation between the extracellular and the corresponding intracellular Ca 2ϩ levels under control conditions. Preincubation of endothelial cells with the Na ϩ -ionophore monensin (1 M) in Na ϩcontaining solution markedly reduced [Ca 2ϩ ] i from 663 Ϯ 23 nM to 278 Ϯ 27 nM (mean Ϯ S.E., n ϭ 8 -12) at 11.2 mM [Ca 2ϩ ] e , indicating that Na ϩ loading inhibits rather than augments Ca 2ϩ entry. Thus, an involvement of reversed mode Na ϩ /Ca 2ϩ exchange in homeostasis of overall [Ca 2ϩ ] i was not detectable in these experiments. Since a monensin-induced reduction of [Ca 2ϩ ] i was not observed in Na ϩ -free buffer ( Fig. 2A), the effect of monensin was presumably due to Na ϩ entry and consequent membrane depolarization, which is known to suppress storeoperated Ca 2ϩ entry.

RESULTS
The addition of CaCl 2 to store-depleted endothelial cells increased eNOS activity as expected from Ca 2ϩ entry into the cells (Fig. 2B, open circles). Interestingly, replacement of extracellular Na ϩ by choline chloride (filled circles) induced a rightward shift of the concentration response curve despite a lack of effect on Ca 2ϩ entry (cf. Fig. 2A). In the presence of monensin, the discrepancy between the effects of extracellular Ca 2ϩ on [Ca 2ϩ ] i and eNOS activity was even more pronounced. As shown in Fig. 2B (open squares), Na ϩ loading with the Na ϩ ionophore potentiated the effect of extracellular Ca 2ϩ on eNOS activity despite a marked reduction in overall [Ca 2ϩ ] i (cf. Fig.  2A). Similar to the monensin-induced reduction of [Ca 2ϩ ] i , the effect of the Na ϩ ionophore on eNOS activity was completely abolished when extracellular Na ϩ was replaced by choline chloride (Fig. 2B, filled squares). To evaluate the Ca 2ϩ dependence of eNOS activation under the various conditions, a correlation was drawn between L-citrulline formation and [Ca 2ϩ ] i (Fig.  2C). The data revealed that, under control conditions, halfmaximal activation of eNOS occurred at a [Ca 2ϩ ] i of ϳ310 nM, whereas the EC 50 was ϳ390 nM in nominal Na ϩ -free buffer. Pretreatment of the cells with monensin in the presence of Na ϩ resulted in a pronounced leftward shift of the concentration response curve (EC 50 ϳ 210 nM), but the Na ϩ -ionophore had no effect in the absence of extracellular Na ϩ (EC 50 ϳ 390 nM).
To test whether the monensin-induced facilitation of eNOS activation was due to a change in cellular Na ϩ gradients or rather a general phenomenon associated with membrane de-polarization and/or inhibition of Ca 2ϩ entry, we investigated the effects of K ϩ -induced membrane depolarization and inhibition of Ca 2ϩ entry by SK&F 96365. As evident from Fig. 3A (Fig. 3B). Consequently, the correlation between L-citrulline formation and [Ca 2ϩ ] i was neither affected by SK&F 96365 nor by KCl (Fig. 3C), demonstrating that the potentiation of Ca 2ϩ -dependent eNOS activation observed in Na ϩ -loaded endothelial cells was not related to diminished Ca 2ϩ entry and/or membrane depolarization.
Thus, Na ϩ -loading-induced facilitation of endothelial NO synthesis appears to be mediated either by a direct effect of Na ϩ ions on the enzyme or by a local elevation of free [Ca 2ϩ ] i due to reversed mode Na ϩ /Ca 2ϩ exchange, which is not detectable by measurement of overall Ca 2ϩ signals. Direct effects of Na ϩ on the Ca 2ϩ sensitivity of eNOS were studied by measuring Ca 2ϩ -dependent activation of the enzyme in homogenates of endothelial cells. The data revealed that Na ϩ ions up to 100 mM failed to modify the effects of Ca 2ϩ on enzyme activity (EC 50 for Ca 2ϩ ϭ 0.19 Ϯ 0.02 M and 0.20 Ϯ 0.03 M (mean Ϯ S.E., n ϭ 4 each) in the absence and presence of 100 mM Na ϩ , respectively). We, therefore, tested whether DCB, an inhibitor of Na ϩ /Ca 2ϩ exchange, influences the effects of Na ϩ loading. For comparison, amiloride, a structural analog of DCB that barely affects Na ϩ /Ca 2ϩ exchange but inhibits Na ϩ /H ϩ exchange and nonselective cation channels (37), was studied. Since both compounds interfered with the fluorometric detection of [Ca 2ϩ ] i , the respective experiments were confined to the measurements of L-citrulline formation. As shown in Fig. 4, treatment of endothelial cells with 30 M amiloride did not modify the effects of extracellular Ca 2ϩ on eNOS activity in Na ϩ -loaded cells (EC 50 ϳ 0.3 mM). However, in the presence of 30 M DCB, the Ca 2ϩ sensitivity of L-citrulline formation was markedly reduced, resulting in an EC 50 of ϳ1.5 mM, which is comparable to the value observed in Na ϩ -free buffer. These data strongly support the concept that Na ϩ -dependent facilita-tion of eNOS activation is due to Ca 2ϩ entry via reversed mode Na ϩ /Ca 2ϩ exchange.
Similar to the results obtained with store-depleted endothelial cells, Na ϩ loading also facilitated eNOS activation in resting and bradykinin-stimulated cells. As shown in Fig. 5, basal L-citrulline formation under control conditions (i.e. in the presence of 100 mM Na ϩ and 2.5 mM Ca 2ϩ ) was 2.4 Ϯ 0.5% and stimulated up to 17.2 Ϯ 2.1% upon addition of bradykinin (mean Ϯ S.E., n ϭ 4 each). Pretreatment of the cells with monensin (1 M) not only potentiated the effect of bradykinin but also enhanced eNOS activity ϳ3-fold in the absence of the agonist. No effects of the Na ϩ -ionophore were observed when experiments were performed in Na ϩ -free buffer.
Since the effect of Na ϩ loading was not associated with elevated overall [Ca 2ϩ ] i (cf. Fig. 2A), we speculated that the involved NCX is localized in close proximity of eNOS and, thus, preferentially regulates the Ca 2ϩ concentration in the local environment of the enzyme. This hypothesis would imply that the NCX protein is present in caveolae. We, therefore, fractionated endothelial cell homogenates on a discontinuous sucrose gradient and analyzed the fractions by immunoblotting (Fig. 6, upper panel). In accordance with other reports (33,38), the caveolae structural protein, caveolin-1, was found at the interface between the 5 and 35% sucrose layer (fraction 2). The caveolin-rich membrane fraction also contained the majority of eNOS, but minor portions of the enzyme were found in higher density fractions. In contrast to caveolin-1 and eNOS, the NCX protein was found in all fractions of the density gradient. To determine the proportion of NCX relative to the total protein (Fig. 6, lower panel), we quantified the NCX bands by densitometric analysis. The data revealed that NCX was substantially enriched in fractions 1 (ϳ25-fold) and 2 (ϳ5-fold), which represent plasma membrane vesicles of low density and caveolae, respectively.
To get further insights into the subcellular distribution of eNOS and NCX, the localization of both proteins was investigated by immunofluorescence microscopy. Consistent with the immunoblotting data, a substantial immunoreactivity against the NCX antibody was observed in regions of the plasma membrane, the Golgi, and the cytoskeleton (Fig. 7, upper image), whereas in most cells investigated, eNOS immunostaining was restricted to the plasma membrane (Fig. 7, lower image), and only a minority of cells showed an additional staining in the perinuclear region (data not shown). These data suggest that both eNOS and NCX are indeed present in caveolae and sup- port our concept that Na ϩ /Ca 2ϩ exchange may play a prominent role in the regulation of eNOS activity. DISCUSSION As several previous reports have indicated the contribution of Na ϩ /Ca 2ϩ exchange to Ca 2ϩ homeostasis in endothelial cells (11,26,36), it was of interest to study the role of NCX in cellular regulation of eNOS activity. Thereby, overall intracellular Ca 2ϩ levels and L-citrulline formation were determined in parallel. The readdition of extracellular Ca 2ϩ after store depletion with thapsigargin in nominally Ca 2ϩ -free incubation buffer allowed for reproducible and sustained elevation of [Ca 2ϩ ] i and for reliable determination of Ca 2ϩ concentration response curves. Two approaches were used to test for a contribution of NCX in the control of eNOS: (i) the cellular Na ϩ gradient was altered by use of monensin, and (ii) NCX was selectively blocked by DCB.
Na ϩ Loading Facilitates Ca 2ϩ -dependent Activation of eNOS-Na ϩ loading of endothelial cells is an intervention that was used previously to demonstrate NCX-mediated Ca 2ϩ signals in endothelial cells (26,36). In those studies, transient rises in overall [Ca 2ϩ ] i were observed when extracellular Na ϩ was removed after agonist stimulation of Na ϩ -loaded endothelial cells. However, we observed that Na ϩ loading reduced overall [Ca 2ϩ ] i of thapsigargin-stimulated cells. This result is probably explained by the fact that in the present study total steady-state levels of [Ca 2ϩ ] i were analyzed instead of the increments of [Ca 2ϩ ] i induced by Na ϩ removal. The total, steady-state level of [Ca 2ϩ ] i after stimulation with thapsigargin is the result of Ca 2ϩ entry via store-operated Ca 2ϩ channels and concomitant Ca 2ϩ transport via exchange mechanisms (NCX, Ca 2ϩ -ATPase). Na ϩ loading promotes Ca 2ϩ entry via reversed mode Na ϩ /Ca 2ϩ exchange (26, 36) but suppresses store-operated Ca 2ϩ entry via a reduction of the electrochemical gradient (25,39). In our experimental setup, inhibition of store-operated Ca 2ϩ entry was apparently the prominent effect of Na ϩ loading and masked the contribution of other Ca 2ϩ transport systems, e.g. Na ϩ /Ca 2ϩ exchange. Treatment of endothelial cells with monensin augmented NOS activity without detectable changes of [Ca 2ϩ ] i , resulting in a striking change in the correlation between overall [Ca 2ϩ ] i and eNOS activity. This FIG. 6. Distribution of caveolin-1, eNOS, NCX, and total protein in sucrose gradient fractions. Endothelial cells were homogenized and fractionated on a discontinuous sucrose gradient. Eight fractions were collected from the top of each tube, and proteins were separated by SDS-polyacrylamide gel electrophoresis (8% acrylamide for eNOS and NCX, 12% acrylamide for caveolin-1). Immunoblot analysis was carried out with antibodies against caveolin-1, eNOS, or NCX (upper panel). Note that in accordance with other reports, only the proteolytic fragment of NCX migrating at ϳ70 kDa was detected by the antibody (47,48). In the lower panel, the protein content of the sucrose gradient fractions is shown.

FIG. 7. Distribution of eNOS and NCX in endothelial cells.
Endothelial cells were incubated with rabbit polyclonal antibodies against NCX (upper image) and eNOS (lower image) and stained using a goat anti-rabbit IgG linked to fluorescein isothiocyanate. Fluorescence microscopy was carried out on a Leica TCS 4D confocal microscope. facilitation of eNOS stimulation was clearly dependent on the entry of Na ϩ ions, since preincubation of endothelial cells with monensin in the absence of Na ϩ failed to affect Ca 2ϩ -dependent enzyme activation. Thus, our results provide the first evidence for a role of Na ϩ ions in cellular regulation of eNOS.
Mechanism of Na ϩ -dependent Facilitation of eNOS Activation-Various mechanisms such as direct modulation of eNOS activity by Na ϩ ions, membrane depolarization, changes in intracellular pH, or Na ϩ /Ca 2ϩ exchange may explain the observed Na ϩ -dependent facilitation of eNOS activation. A direct modulatory effect of Na ϩ ions on Ca 2ϩ sensitivity of the enzyme was excluded in experiments with cell-free eNOS preparations. Moreover, the Na ϩ entry-induced membrane depolarization itself is apparently not involved in promotion of eNOS activation, since the effect of monensin was not mimicked by elevation of extracellular K ϩ . Similarly, a mechanism based on changes in intracellular pH can be excluded, since Na ϩ loading did not significantly affect intracellular pH of endothelial cells. 2 Moreover, amiloride, a potent inhibitor of Na ϩ /H ϩ exchange, did not prevent the effects of monensin. In contrast to amiloride, the structural analog DCB, which is a potent inhibitor of Na ϩ /Ca 2ϩ exchange, completely prevented Na ϩ -induced facilitation of eNOS activation. These results strongly support the view that the observed effects of Na ϩ loading were indeed due to reversed mode Na ϩ /Ca 2ϩ exchange. Since we did not detect an elevation of overall [Ca 2ϩ ] i in Na ϩ -loaded cells, the increase in [Ca 2ϩ ] i induced by Na ϩ /Ca 2ϩ exchange may be restricted to the subplasmalemmal region. It appears reasonable to speculate about a buildup of a subcellular Ca 2ϩ gradient, since such preferential elevation of subplasmalemmal [Ca 2ϩ ] i has been also observed upon activation of endothelial cells with bradykinin or sodium fluoride (11).
Presence of Endothelial NCX in Caveolae-The hypothesis that Na ϩ loading enhances eNOS activity via reversed mode Na ϩ /Ca 2ϩ exchange and focal rises in subplasmalemmal [Ca 2ϩ ] i requires the assumption that the involved NCX protein is located in close proximity of eNOS. Since regulation of eNOS is likely to take place in caveolae, it appeared of interest to test for the presence of NCX in these specialized plasma membrane domains. Separation of endothelial cell homogenates on a discontinuous sucrose gradient revealed the presence of both eNOS and NCX in the caveolin-1-positive fraction. In accordance with a previous report, eNOS was also found in higher density fractions, presumably representing Golgi-associated and cytosolic enzyme (38). Interestingly, the proportion of caveolae-associated eNOS reported by these authors was ϳ35% and, thus, considerably lower than observed in the present study (ϳ90%). This variation in the subcellular distribution may be explained by variability of eNOS localization due to species differences, type of endothelial cells, or culture conditions (1,40).
The predominant localization of eNOS in caveolae observed in our study was confirmed by immunofluorescence microscopy. Consistent with previous immunofluorescence staining of eNOS in bovine aortic endothelial cells (40), we observed that the majority of cells exhibited a substantial immunoreactivity in the plasma membrane region, whereas a perinuclear staining was only found in a minority of cells. In contrast to this heterogeneous pattern of eNOS staining, the immunoreactivity against the NCX antibody did not exhibit considerable cell-tocell variations; as in all cells investigated, a substantial staining in the regions of the plasma membrane, the Golgi, and the cytoskeleton was observed. Consistent with these immunofluorescence data, NCX was enriched in the plasma membrane and caveolae fractions of the sucrose gradient but was also present in higher density fractions. Albeit eNOS and NCX exhibit a clearly different pattern of subcellular localization, our results strongly suggest the presence of both proteins in caveolae. Since eNOS has been shown to interact with a variety of proteins present in caveolae (e.g. the structural protein caveolin-1 (5-7), the bradykinin receptor B2 (41), or the Larginine transporter CAT-1 (42)), it is reasonable to speculate that eNOS may also form a complex with NCX. However, we did not observe a substantial coimmunoprecipitation of eNOS and NCX from endothelial cell lysates, 3 indicating that both proteins apparently do not form a tight and stable complex.
Regulation of Endothelial Ca 2ϩ Homeostasis by Na ϩ Ions-Based on our data, we suggest that Ca 2ϩ concentrations at the cytoplasmic side of caveolae and, thus, eNOS activity is for a large part controlled by Na ϩ /Ca 2ϩ exchange and propose the following model (Fig. 8). Ca 2ϩ -dependent stimulation of eNOS in endothelial cells involves two distinct Ca 2ϩ transport systems in the plasma membrane. Ca 2ϩ channels, activated by depletion of intracellular Ca 2ϩ stores, provide a general pathway for Ca 2ϩ entry into activated endothelial cells. Albeit storeoperated Ca 2ϩ entry is the major determinant of the overall Ca 2ϩ signal observed in agonist-stimulated endothelial cells, essential subcellular Ca 2ϩ gradients are established by Na ϩ / Ca 2ϩ exchange. An NCX protein is present in caveolae and, thus, in close proximity of eNOS. Thereby, NCX essentially contributes to local Ca 2ϩ homeostasis and cellular control of eNOS activity. This interaction between eNOS and NCX allows for modulation of eNOS activity via the cellular Na ϩ gradient and, thus, via a variety of Na ϩ transport systems.
Physiological and Pathophysiological Implications-In the present study, Ca 2ϩ -dependent activation of eNOS was primarily investigated in store-depleted endothelial cells. This method is widely accepted as a physiological relevant model of endothelial cell activation, since direct depletion of intracellular Ca 2ϩ stores by inhibitors of endoplasmic Ca 2ϩ -ATPase such as thapsigargin mimics the stimulation of phospholipase C-coupled receptors in terms of Ca 2ϩ /NO signaling due to activation of the same Ca 2ϩ entry pathway (8,30). It is therefore conceivable to conclude that the effects of Na ϩ loading observed in thapsigargin-treated endothelial cells will take place similarly in cells activated by stimulation of phospholipase C-coupled receptors. Consistently, we observed that Na ϩ loading of endothelial cells also potentiates eNOS activation by bradykinin 2 K. Groschner, unpublished observations. 3 M. Teubl and K. Schmidt, unpublished observations.
FIG. 8. Proposed scheme for facilitation of Ca 2؉ -dependent activation of eNOS by Na ؉ . Na ϩ entry into endothelial cells mediated either by monensin or via Na ϩ -permeable ion channels results in reduction of the electrochemical gradient for Ca 2ϩ entry via store-operated Ca 2ϩ channels (SOCC) and in decreased overall [Ca 2ϩ ] i (a) and reversed mode Na ϩ /Ca 2ϩ exchange leading to a focal elevation of subplasmalemmal [Ca 2ϩ ] i (b). Since eNOS co-localizes with NCX in caveolae, the reversed mode Na ϩ /Ca 2ϩ -exchange enables efficient enzyme activation at low overall [Ca 2ϩ ] i . due to Ca 2ϩ entry via reversed mode Na ϩ /Ca 2ϩ exchange.
In the absence of Na ϩ loading, forward mode Na ϩ /Ca 2ϩ exchange is likely to contribute to Ca 2ϩ extrusion during cell activation (11). It appears reasonable to expect that even a moderate elevation of intracellular Na ϩ , in particular in association with membrane depolarization, may substantially suppress Ca 2ϩ extrusion and, thus, facilitates Ca 2ϩ -dependent activation of eNOS. The contribution of Na ϩ /Ca 2ϩ exchange is likely to vary, depending on the ability of a physiological stimulus to induce Na ϩ loading (36). The recent demonstration of an inositol 1,4,5-trisphosphate-dependent, endothelial Na ϩ permeability suggests that various hormones or neurotransmitters are able to affect endothelial Na ϩ gradients (43). In pathophysiological situations such as oxidative stress, excessive Na ϩ entry via redox-activated cation channels has been observed (44,45). The resulting profound Na ϩ loading is expected to shift Na ϩ /Ca 2ϩ exchange to reversed mode, thereby promoting the buildup of an even more pronounced subplasmalemmal Ca 2ϩ gradient. These Ca 2ϩ gradients are likely to control not only eNOS but also other Ca 2ϩ -dependent proteins that are present in caveolae (46). Therefore, we suggest Na ϩ / Ca 2ϩ exchange as a physiological and pathophysiological link between Na ϩ homeostasis and caveolae-associated cell signaling.