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1 Departments of Pharmacology and Toxicology and Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 2 The iCAPTUR4E Center, University of British Columbia, St. Paul's Hospital, Vancouver, British Columbia, Canada V6Z 1Y6
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We developed an in situ assay system to simultaneously monitor intracellular Ca2+ concentration ([Ca2+]i, fura 2 as indicator) and nitric oxide (NO) levels [4,5-diaminofluorescein as probe] in the intact endothelium of small bovine coronary arteries by using a fluorescent microscopic imaging technique with high-speed wavelength switching. Bradykinin (BK; 1 µM) stimulated a rapid increase in [Ca2+]i followed by an increase in NO production in the endothelial cells. The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO; 10 µM) induced a gradual, small increase in [Ca2+]i and a slow increase in intracellular NO levels. Removal of extracellular Ca2+ and depletion of Ca2+ stores completely blocked BK-induced increase in NO production but had no effect on PAO-induced NO production. However, a further reduction of [Ca2+]i by application of BAPTA-AM or EGTA with ionomycin abolished the PAO-induced NO increase. These results indicate that a simultaneous monitoring of [Ca2+]i and intracellular NO production in the intact endothelium is a powerful tool to study Ca2+-dependent regulation of endothelial nitric oxide synthase, which provides the first direct evidence for a permissive role of Ca2+ in tyrosine phosphorylation-induced NO production.
endothelium-derived relaxing factor; signal transduction; protein kinase; coronary circulation; heart
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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NITRIC OXIDE SYNTHASES (NOSs) comprise a family of three mammalian gene products that each possess an NH2-terminal heme-containing oxygenase domain and a COOH-terminal flavin-containing reductase domain, bridged by a canonical CaM-binding polypeptide (33). NOS isoforms are activated and regulated by different signaling mechanisms. Neuronal NOS (nNOS) and endothelial NOS (eNOS) are activated by agonist-induced elevation of intracellular Ca2+ concentration ([Ca2+]i) with subsequent binding of Ca2+/CaM to these enzymes (35). In contrast, inducible NOS (iNOS) binds to CaM with such high affinity that it is already maximally activated at the Ca2+ concentrations within resting cells (32). These different regulatory mechanisms determine the NOS activity in response to various physiological or pathological stimuli, thereby participating in the control of a variety of cell functions (1).
Endothelium-derived nitric oxide (NO) contributes to cardiovascular homeostasis through its profound effects on blood pressure, vascular remodeling, platelet aggregation, and angiogenesis (15). It is well known that for some agonists, such as acetylcholine (29) and bradykinin (BK) (36), a rise in intracellular Ca2+ is necessary for NO production. However, recent studies indicated that shear stress (9, 30), isometric contraction (10), tyrosine phosphatase inhibitors (9, 28, 30), IGF-I (34), estrogen (5), and insulin (25) activate eNOS in isolated vessel preparations or in cultured cells in the absence of a change in [Ca2+]i. These Ca2+-insensitive responses are blocked by tyrosine kinase inhibitors. It thus appears that eNOS activity is also regulated by a Ca2+-independent mechanism. However, purified eNOS and broken endothelial cell preparations containing eNOS are unable to produce NO in the absence of Ca2+ in the reaction mixtures (14, 23). The reason for these apparent conflicting findings remains unknown. It is possible that an alternative signaling pathway for eNOS activation exists that does not require elevation of [Ca2+]i but is dependent on a basal level of [Ca2+]i.
This hypothesis can be tested by simultaneous measurements of endothelial [Ca2+]i and NO production. Recently, a novel fluorescent NO indicator, 4,5-diaminofluorescein (DAF-2) diacetate (DAF-2 DA), was developed (18) and applied to in situ monitoring of NO production (2, 37). This dye can readily enter the cells and be hydrolyzed by cytosolic esterases to DAF-2 that is trapped inside the cells. The fluorescent chemical transformation of DAF-2 is based on the reactivity of the aromatic vicinal diamines with NO in the presence of dioxygen. This N-nitrosation of DAF-2 produces a highly green-fluorescent triazole form, DAF-2T. Because this nitrosation reaction is essentially irreversible (20), DAF-2 fluorescence reflects a sum total of NO production. Thus this dye can be used to monitor NO production in response to different stimuli. In the present study, we combined DAF-2 fluorescent microscopic imaging with fura 2 fluorescence measurements to monitor simultaneous changes in accumulating intracellular NO levels and [Ca2+]i in the intact endothelium of freshly dissected small bovine coronary arteries. This in situ simultaneous assay of [Ca2+]i and NO production in endothelial cells circumvents the problems encountered with cultured endothelial cells, in which the expression of cell surface receptors, ion channels, and intracellular signaling mechanism might be changed because of enzymatic, mechanical, or culturing treatments (19, 22). Using this assay system, we examined the simultaneous responses of [Ca2+]i and NO production to different agonists or stimuli to determine the [Ca2+]i dependence or -independence of eNOS and to explore the mechanisms regulating eNOS activity in the endothelium of coronary arteries.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated small coronary artery preparation and dye loading. Fresh bovine hearts were obtained from a local abattoir. Small coronary arteries (400- to 600-µm ID) were prepared as we described previously (37). In brief, the left ventricular wall was rapidly dissected and immersed in ice-cold modified Hanks' buffered saline solution containing (in mM) 137 NaCl, 5.4 KCl, 4.2 NaHCO3, 3 Na2HPO4, 0.4 KH2PO4, 1.5 CaCl2, 0.5 MgCl2, 0.8 MgSO4, 10 glucose, 10 HEPES, pH 7.4. This myocardial section was transported immediately to a dissection station, and small intramural coronary arteries from the left anterior descending artery were carefully dissected and placed in cold Hanks' buffer and then transferred to a 0.9-mm microdissecting dish filled with Sylgard (World Precision Instruments) immersed in ice-cold Hanks' buffer. The arterial segment was cut open along its longitudinal axis and pinned onto the Sylgard with the lumen side upward. Care was taken not to disrupt the endothelium. Four additional supportive pins were placed around the lumen-opened artery with the top ends 1.2 mm over the arterial endothelium. After the artery was fixed, the 0.9-mm dish was put into a recording chamber with the vessel lumen side downward facing the objective of an inverted microscope. A 1.2-mm space between the arterial endothelium and the glass bottom of the recording chamber was made by the supportive pins. This kept the artery endothelium from direct contact with glass bottom and allowed the solution a free passage. The recording chamber was filled with Hanks' buffer. After a 30-min equilibration, the bath solution was exchanged with 1 ml of Hanks' buffer containing DAF-2 DA (10 µM; Sigma), fura 2-AM (10 µM; Molecular Probes), Pluronic F-127 (0.025%; Molecular Probes), and BSA (0.05%), and the artery was incubated in this loading solution for 40 min at room temperature. The arteries were then rinsed three times with Hanks' buffer to remove extracellular fura 2-AM and DAF-2 DA, and an additional 30 min were allowed for intracellular deesterification of fura 2-AM to fura 2.
In situ simultaneous measurement of
[Ca2+]i and NO production
in intact endothelium of coronary arteries.
[Ca2+]i and NO fluorescent imaging analysis
was performed with a modification of the method used by Li and van
Breemen (21) for [Ca2+]i
measurement in intact endothelium of rabbit cardiac valve, with a
combination of DAF-2 and Ca2+ assay through the high-speed
excitation and emission wavelength switching systems. After dye
loading, the chamber solution was kept at 37°C by a temperature
control system (Warner Instruments, Hamden, CT). The chamber was
mounted on an inverted microscope with epifluorescence attachments
(Diaphot 200; Nikon, Tokyo, Japan) so that a ×20 phase/fluor objective
(Nikon Diaphot) was focused on the endothelium and individual
endothelial cells were visualized. The excitation light from a xenon
lamp was filtered to provide wavelengths of 340 ± 10, 380 ± 10 (for fura 2), and 480 ± 20 (for DAF-2) nm with a high-speed
wavelength switcher (Lambda DG-4; Sutter, Novato, CA). Emission light
from endothelial cells was passed through a dichroic mirror (500 nm)
and through an emission filter of 510 ± 20 nm for fura 2 or
535 ± 25 nm for DAF-2 with a high-speed rotating filter wheel
(Lambda 10-2; Sutter). The fluorescence images were recorded by a
digital camera (SPOT RT Monochrome; Diagnostic Instruments). Metafluor
imaging and analysis software (Universal Imaging) was used to acquire,
digitize, and store the images and data for off-line processing and
statistical analysis. The autofluorescence of the unloaded endothelium
was minimal, and the background images were obtained from a focal plane
1 mm away from the endothelium. The relative fluorescence intensities
for fura 2 (128 ± 38 for F380) and DAF-2 (147 ± 42) are quantitatively comparable. When the endothelium was only loaded with fura 2-AM, no signal at 480/535 nm (for NO imaging) was detected. In contrast, when the endothelium was only loaded with DAF-2 DA, no
detectable signal could be recorded at 340/510- and 380/510-nm wavelengths (for Ca2+ imaging) (data not shown). To reduce
photobleaching of these fluorescent dyes, images with excitation of 340 and 380 nm for Ca2+ were acquired at 2-s intervals, and
images at excitation of 480 nm for NO were acquired at 10-s intervals.
F340/F380, a fluorescence ratio of excitation
at 340 nm to that at 380 nm, was determined after background
subtraction, and [Ca2+]i was calculated by
using the following equation (13):
[Ca2+]i = Kd
[(R 
Rmin)/(Rmin R)], where
Kd for the fura 2-Ca2+ complex is
224 nM; R is the fluorescence ratio
(F340/F380); Rmax and
Rmin are the maximal and minimal fluorescence ratios
measured by addition of 10 µM of Ca2+ ionophore ionomycin
to Ca2+-replete (2.5 mM CaCl2) solution and
Ca2+-free (5 mM EGTA) solution, respectively; and is
the fluorescence ratio at 380-nm excitation determined at
Rmin and Rmax, respectively (13).
F/F0 × 1,000), where F is DAF-2 fluorescence
intensity obtained during experiments and F0 is its basal
fluorescence intensity.
Differential conversion of NO fluorescence intensity. Because NO does not dissociate from DAF-2 once this dye reacts with NO, the detected NO-sensitive fluorescence with DAF-2 primarily represents a cumulative amount of NO within the cells as described above. An obvious disadvantage of this method for continuous NO measurements is that the plateau area of the NO-DAF-2 fluorescence curve does not represent the actual NO concentration. In fact, the formation of this plateau mainly results from the termination of NO production within the cells. To more representatively show the features of NO-DAF-2 fluorescence and to accurately present the relationship between NO production and Ca2+ concentration in the cells, we performed a differential conversion of time-dependent NO-DAF-2 fluorescence curve to calculate df/dt, which represents NO production rate. We plotted the converted df/dt against the reaction time and presented this curve with [Ca2+] changes in parallel. We also calculated the area under the df/dt curve (AUC), which indicates the cumulative amount of NO in the cells.
We first performed regression analysis of the NO-DAF-2 fluorescence curve recorded during different stimuli to obtain a regression equation. A differential conversion was then undertaken based on the regression equation. For example, the NO-DAF-2 curve recorded during BK incubation was fitted with a sigmoidal four-parameter Gompertz growth model analyzed with SigmaPlot 5.0. An equation for BK-induced time-dependent change in fluorescence (f) was described as follows
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(1) |
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(2) |
Statistical analysis. Data are presented as means ± SE. Significant differences in mean values between and within multiple groups were examined by using ANOVA for repeated measures, followed by a Duncan multiple-range test. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Simultaneous monitoring of
[Ca2+]i and NO production
in intact endothelium of small bovine coronary arteries.
Figure 1 shows representative images and
recordings of simultaneously measured [Ca2+]i
and NO production in the intact endothelium of small coronary arteries.
Figure 1A shows that the
F340-to-F380 ratio dramatically increased from
0.8 (green color) to 1.3 (red color) in 2 min with addition of BK (1 µM) to the bath solution. The green DAF-2 fluorescence was also
simultaneously stimulated by BK. As shown in Fig. 1B, a
digitized on-line recording of [Ca2+]i and
DAF-2 fluorescence in this arterial endothelium preparation was also
monitored throughout the experiment. A plateau of DAF-2 fluorescence
was observed. As discussed above, this plateau was formed because of
the termination of NO production, which was not associated with a
constant association and dissociation of NO and DAF-2. To more
accurately present this feature of NO/DAF-2 fluorescence, a
differential conversion was performed, and data are presented in Fig.
1C; df/dt represents the production rate for NO.
From this plot, a peak response of [Ca2+]i to
BK was observed within 2 min and the rise of
[Ca2+]i decreased gradually by 50% and was
sustained for 20 min. However, NO production, as shown by
df/dt, reached maximum at 2 min of BK treatment and then
stopped 7 min after BK treatment. The sustained Ca2+
increase seems not to influence NO production induced by BK.
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Confirmation of location and specificity of detected
[Ca2+]i and NO level.
When the endothelium of coronary arteries was gently scraped after
loading, a dark image (<5% fluorescence intensity compared with
intact endothelium) was obtained, indicating that little fluorescence
signal was derived from the underlying smooth muscle layer in this
preparation. In contrast to the intact endothelium (Fig. 1), removal of
the endothelium abolished the BK-induced increase in
[Ca2+]i and DAF-2 fluorescence (Fig.
2).
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Ca2+ dependence of BK-induced NO
production.
BK is a typical agonist that activates eNOS by increasing
[Ca2+]i. As summarized in Fig.
4A, BK induced a rapid peak in
[Ca2+]i followed by a sustained plateau,
which may represent Ca2+ release (peak response) and
Ca2+ influx (plateau), respectively (16, 17).
DAF-2 fluorescence increased after a rise of
[Ca2+]i with a time delay of ~20 s. This NO
production terminated 7 min after BK treatment. As shown in Fig.
4B, removal of extracellular Ca2+ and depletion
of the intracellular sarcoplasmic reticulum Ca2+ store by
exchanging the bath solution with a Ca2+-free Hanks'
solution containing 1 mM EGTA and 1 µM thapsigargin (TSG) completely
blocked the BK-induced increase in both
[Ca2+]i and NO levels in the
endothelium.
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Effects of phenylarsine oxide on
[Ca2+]i and NO production
in coronary endothelium.
The protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO)
activates eNOS in the preparations of endothelial cells in a
Ca2+-independent manner (9, 30). Therefore, we
tested whether this compound also stimulates NO production without an
increase in [Ca2+]i in the intact endothelium
of coronary arteries. As shown in Fig.
5A, PAO (10 µM) induced a
gradual, slow increase in [Ca2+]i from
110 ± 17 nM to a maximum of 198 ± 31 nM 7-8 min after its addition, which was then sustained throughout the experimental period (n = 6). PAO also induced a gradual, slow
increase in NO production in the endothelial cells, which continued
with an increasing production rate (df/dt) as late as 30 min
after addition of PAO (Fig. 5A). When the endothelium was
gently removed, the PAO-induced increases in both
[Ca2+]i and [NO]i were
abolished (data not shown), indicating that these PAO responses occur
in the endothelium.
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Inhibition of PAO-induced NO production by L-NAME
and tyrosine kinase inhibitor.
To confirm that PAO-induced NO production is associated with protein
tyrosine phosphorylation, the effects of the tyrosine kinase inhibitor
erbstatin A were examined. As shown in Fig.
6, preincubation of coronary arteries
with erbstatin A substantially reduced PAO-induced NO production but
was without effect on the small increase in
[Ca2+]i in response to PAO. Similarly, the
NOS inhibitor L-NAME did not alter PAO-induced increase in
[Ca2+]i but significantly inhibited
PAO-induced NO production.
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Permissive role of Ca2+ in
PAO-induced NO production.
As shown in Fig. 5B, PAO still increased NO production in
coronary endothelium, in which Ca2+ stores were depleted by
1 µM TSG and Ca2+ influx was abolished by the use of
Ca2+-free solution containing 1 mM EGTA. This NO production
by enhancing tyrosine phosphorylation does not seem to require
Ca2+ as a trigger. However, when
[Ca2+]i was maximally decreased to almost 0 nM by preincubation of the arterial endothelium with
Ca2+-free buffer plus EGTA (1 mM) in combination with the
cell-permeant intracellular Ca2+ chelator BAPTA-AM (100 µM) for 30 min, PAO could no longer induce NO production (Fig.
7). Similarly, complete removal of
intracellular Ca2+ by incubation of the endothelium with
Ca2+-free solution containing EGTA (1 mM) and
Ca2+ ionophore ionomycin (10 µM) (to remove all
Ca2+ inside the cells) abolished the PAO-induced NO
production (8.9 ± 7.4 AUC of ionomycin + EGTA vs. 480 ± 69 AUC of PAO control in 30 min; P < 0.05, n = 6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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By simultaneously monitoring [Ca2+]i and NO levels in the intact endothelium of small coronary arteries, the present study provides direct evidence that eNOS can be activated by agonist-induced increases in [Ca2+]i and by enhanced phosphorylation of the enzyme associated with inhibition of tyrosine phosphatase or stimulation of tyrosine kinase. Although the activation of eNOS by phosphorylation does not require a large increase in [Ca2+]i, a prerequisite of basal [Ca2+]i for this phosphorylation-induced NOS activation has been demonstrated. Therefore, intracellular Ca2+ not only mediates the activation of eNOS in response to agonists such as BK but also plays a permissive role in eNOS activation by stimulation independent of Ca2+ increase.
Simultaneous measurements of [Ca2+]i and NO production in intact endothelium. Despite considerable evidence that eNOS is a Ca2+-dependent enzyme (1, 12), recent studies indicated that eNOS also produces NO independently of intracellular Ca2+ (5, 9, 10, 25, 28, 30, 34). The present study was designed to further determine the role of intracellular Ca2+ in the regulation of eNOS activity and to explore the mechanism by which Ca2+ activates or modulates NO production via eNOS. In these experiments, we developed an imaging analysis to simultaneously monitor alterations of [Ca2+]i with fura 2 and the total amount of intracellular NO with DAF-2 in the intact endothelium of freshly dissected small coronary arteries. This technique allows us to directly determine the relationship between [Ca2+]i and NO levels in the endothelium and to continuously observe the role of Ca2+ in the activation or modulation of NOS activity. By an analysis of the time course for the parallel changes in [Ca2+]i and NO levels in endothelial cells, the mechanism mediating the effect of Ca2+ on NOS activity can be explored. To validate this assay system, we performed a series of experiments to determine the possible influences of Ca2+ and NO signals on each other. First, by using excitation and emission wavelength switching systems, we successfully recorded Ca2+ and NO fluorescence images "simultaneously." These images from representative cells or areas were digitized and stored for off-line data analysis (Fig. 1). A response curve of both Ca2+ and NO clearly exhibited dynamic changes in [Ca2+]i and NO levels in response to stimuli such as BK and PAO.
By separately loading fura 2-AM or DAF-2 DA into the endothelial cells in coronary arteries, we demonstrated that there was no cross signal at the corresponding wavelength region for DAF-2 or fura 2 fluorescence, respectively. Because fura 2 has a broad emission signal that significantly overlaps the excitation region of DAF-2, there is a concern that the fura 2 emission may excite DAF-2 to produce fluorescence, thereby resulting in an artifact in Ca2+ measurement. In general, however, the ratio of emitted fluorescence intensity to excitation light intensity is between 1:10,000 and 1:1,000,000 (31); it is impossible for the fura 2 emission to excite DAF-2 to produce detectable fluorescence because the fura 2 emission intensity is not comparable to DAF-2 excitation light intensity. To further exclude the interaction of two fluorescence signals, we first tested the effects of the NO donor deta NONOate on [Ca2+]i production. It was found that deta NONOate increased the NO signal, but it had no effect on Ca2+ signal, suggesting that DAF-2 fluorescence is without influence on Ca2+ signal in this assay. On the other hand, the NOS inhibitor L-NAME inhibited the BK-induced increase in NO production by ~70% but did not alter BK-induced increase in [Ca2+]i. When a combination of both L-NAME and the intracellular NO scavenger carboxy-PTIO was used, the BK-induced increase in DAF-2 fluorescence was completely abolished but Ca2+ response to BK was not altered, suggesting that NO signal also has no influence on Ca2+ signal. In light of these results, we have confidence that these assays can simultaneously detect both [Ca2+]i and NO production in situ in endothelial cells without interference with each other and can reliably determine the role of Ca2+ in the regulation of NOS activity. This in situ measurement of [Ca2+]i and NO levels in the intact endothelium of freshly dissected coronary arteries excludes the possible influences of various experimental manipulations on NOS activity in isolated or cultured endothelial cells or purified enzymes. Therefore, the results from the intact endothelium obtained from the present experiments may more precisely represent the behavior of NOS and Ca2+ within the endothelium under physiological conditions.Role of Ca2+ in regulation of eNOS activity. BK is a classic Ca2+-dependent activator of eNOS (4, 36). In the present study, it produced a rapid and simultaneous increase in intracellular Ca2+ and intracellular NO. Although the rise in [Ca2+]i in response to BK was sustained after a peak increase, it seems that this sustained [Ca2+]i increase is not responsible for activation of NOS, because the increase in DAF-2 fluorescence stopped 5 min after BK treatment. Given that a rapid peak increase of [Ca2+]i is primarily determined by intracellular Ca2+ mobilization (16, 17), this BK-induced NOS activation may be largely dependent on Ca2+ release from the intracellular stores. These results are consistent with the view that eNOS activity is activated by intracellular Ca2+ mobilization and subsequent binding of Ca2+/CaM to NOS and displacement from caveolin-1 (7, 24).
The tyrosine phosphatase inhibitor PAO also activated NO production in the endothelium. The PAO-induced NOS activation was not associated with increase in [Ca2+]i, because PAO only produced a small increase in [Ca2+]i and blockade of this Ca2+ increase by removal of extracellular Ca2+ and depletion of Ca2+ stores had no effect on PAO-induced increase in NO levels in the endothelium. This suggests that inhibition of dephosphorylation of tyrosine by PAO directly activates eNOS in endothelial cells independently of a Ca2+ increase. Previous studies also demonstrated that PAO-induced NOS activation is associated with tyrosine phosphorylation but not with a Ca2+ increase in these cells (9, 30). Indeed, the tyrosine kinase inhibitor erbstatin A blocked PAO-induced increase in NO production in the endothelium in our experiments, further supporting the role of tyrosine phosphorylation in the activation of eNOS. However, this Ca2+-independent eNOS activation conflicts with published results that eNOS did not have Ca2+-independent activity in broken cell preparations or as a purified enzyme (14, 23). In this regard, PAO-induced NOS activation was even reported also to depend on Ca2+ increase in cultured endothelial cells (8). It appears that intracellular Ca2+ may also participate in the regulation of eNOS activity induced by PAO, even though it may not directly mediate eNOS activation. To test this hypothesis, we further examined the effects of [Ca2+]i on eNOS with the cell-permeant Ca2+ chelator BAPTA-AM. Interestingly, when basal [Ca2+]i was further reduced by addition of the intracellular Ca2+ chelator BAPTA-AM, PAO-induced NO production was completely blocked. When CaCl2 was added to restore [Ca2+]i to ~80 nM, PAO-induced NO production was substantially restored. These results suggest that [Ca2+]i may be a prerequisite of PAO-induced NO production, despite the fact that it may not directly trigger eNOS activation by PAO. This view is further supported by our findings that complete removal of [Ca2+]i with the calcium ionophore ionomycin and EGTA blocked PAO-induced NOS activation. As Brouet et al. (3) pointed out recently, the term "Ca2+-independent activation of eNOS" may be misleading and the Ca2+ dependence of eNOS is an important feature of the enzyme. However, the mechanisms mediating the actions of intracellular Ca2+ in NOS activation may be different depending on the stimuli. It may directly mediate the activation of eNOS in response to classic Ca2+-dependent stimulators such as BK, acetylcholine, and Ca2+ ionophore A-23198 (29, 36). It may also play a permissive role in the activation of eNOS induced by other stimuli such as shear stress, inhibition of tyrosine dephosphorylation, activation of tyrosine kinase, IGF-I, and insulin (9, 10, 25, 30, 34). In summary, a simultaneous assay system for [Ca2+]i and NO production was used to determine the role of Ca2+ in the activation of NOS in the intact endothelium from small coronary arteries. Two Ca2+-dependent mechanisms were found to regulate the NOS activity in this in situ preparation. An increase in intracellular Ca2+ mediates the activation of eNOS in response to BK, and basal [Ca2+]i is a prerequisite for activation of eNOS by tyrosine phosphorylation.| |
ACKNOWLEDGEMENTS |
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This study was supported by National Institutes of Health Grants HL-57244 (to P.-L. Li), HL-51055 (to W. B. Campbell), and DK-54927 (to A.-P. Zou) and American Heart Association Established Investigator Grant 9940167N (to P.-L. Li).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P.-L. Li, Dept. of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (E-mail: pli{at}mcw.edu).
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.
August 22, 2002;10.1152/ajpheart.00428.2002
Received 28 May 2002; accepted in final form 19 August 2002.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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