AJP - Heart Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 283: H1819-H1828, 2002. First published July 8, 2002; doi:10.1152/ajpheart.00214.2002
0363-6135/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/5/H1819    most recent
00214.2002v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boo, Y. C.
Right arrow Articles by Jo, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boo, Y. C.
Right arrow Articles by Jo, H.
Vol. 283, Issue 5, H1819-H1828, November 2002

Shear stress stimulates phosphorylation of eNOS at Ser635 by a protein kinase A-dependent mechanism

Yong Chool Boo1, Jinah Hwang1, Michelle Sykes1, Belinda J. Michell2, Bruce E. Kemp2, Hazel Lum3, and Hanjoong Jo1

1 Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, Georgia 30322; 2 St. Vincent's Institute of Medical Research, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia; and 3 Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at Ser1179 in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including Ser116, Thr497, and Ser635. Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at Ser635, which was consistently slower than that at Ser1179. Thr497 was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated Ser116. Whereas shear-stimulated Ser635 phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of Ser635 may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli.

endothelial cells; vascular endothelial growth factor; cAMP


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) produced from endothelial nitric oxide synthase (eNOS) plays critical roles in normal vascular biology and pathophysiology. In addition to its well-known vascular functions such as vessel relaxation and inhibition of platelet aggregation, NO also inhibits some of the key steps in atherogenesis, including cell death and monocyte adhesion induced by proatherogenic factors (14, 15, 24, 45). NO production from eNOS is stimulated by a variety of mechanical forces such as shear stress and stretching, and humoral factors ranging from growth factors and peptide hormones such as vascular endothelial growth factor (VEGF), estrogen, sphingosine-1-phosphate, acetylcholine, and bradykinin (13, 17, 18, 25, 36, 37).

The eNOS is known as a Ca2+/calmodulin (CaM)-dependent form of NOS (34). Indeed, most humoral ligands including bradykinin, acetylcholine, and ATP stimulate NO production from eNOS by raising the level of intracellular Ca2+, which forms Ca2+/CaM complex (34). On the other hand, mechanical forces such as fluid shear stress and stretching stimulate NO production from eNOS by Ca2+-independent mechanisms (1, 10). Moreover, eNOS has been shown to be regulated by interactions with other positive and negative protein modulators such as caveolin and heat shock protein 90 (20, 41). In the basal state, the majority of eNOS appears to be bound to caveolin-1 with its enzyme activity repressed in the caveolae (27, 33). This tonic inhibition of eNOS can be released by displacing caveolin-1 with Ca2+/CaM in response to Ca2+-mobilizing agonists (27).

In addition to those modulators, phosphorylation of eNOS at key regulatory sites plays an important role in regulation of enzyme activity in response to several physiological stimuli (3, 13, 17, 23, 35). It has been shown that phosphorylation of eNOS-Ser1179 (based on bovine eNOS sequence and equivalent to human eNOS-Ser1177) is associated with increased activity of the enzyme (19, 32). Phosphorylation of eNOS-Ser1179 is regulated by phophosinositide-3-kinase (PI3K)-dependent mechanisms (19). Akt, one of the major regulatory targets of PI3K, has been shown to directly phosphorylate eNOS at Ser1179 and activate the enzyme in response to vascular endothelial growth factor (VEGF), sphingosine-1-phosphate, and estrogen (13, 17, 25, 36, 37). However eNOS-Ser1179 can also be phosphorylated by AMP-activated protein kinase (8), or protein kinase A (PKA), and protein kinase G (PKG) (5). Exactly which protein kinase(s) phosphorylates eNOS-Ser1179 in intact cells appears to be regulated by the cellular context and given stimuli. For example, we have shown that shear stress phosphorylates eNOS-Ser1179 by a PI3K- and PKA-dependent manner without involving Akt, whereas VEGF phosphorylates eNOS-Ser1179 by a PI3K- and Akt-dependent manner (3, 21).

Phosphorylation of eNOS at Thr497 has also been shown to play an essential role in the regulation of enzyme activity (8, 23, 35, 36). For example, bradykinin has been shown to stimulate NO production by a mechanism involving dephosphorylation at Thr497 (23).

In addition to Ser1179 and Thr497, eNOS has several other potential phosphorylation sites, including Ser116, Ser635 (13, 17, 19, 35, 36), and some unknown sites (2, 19). Differential phosphorylation of eNOS at various sites could play an important role in the regulation of enzyme activity. However, little information is available regarding their phosphorylation in response to various stimuli. In the present study, we systematically compared phosphorylation of eNOS at Ser116, Thr497, Ser635, and Ser1179 in response to shear stress, VEGF, and cAMP. The current study, for the first time, demonstrates that phosphorylation eNOS-Ser635 was stimulated by shear stress, VEGF, and cAMP. More interestingly, we show evidence that shear-dependent phosphorylation of eNOS-Ser635 is regulated by a different mechanism from that of eNOS-Ser1179.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Bovine aortic endothelial cells (BAEC) harvested from descending thoracic aortas were maintained (37°C, 5% CO2) in a growth medium [Dulbecco's modified Eagle's medium (DMEM) containing 1 g/l glucose (GIBCO) and 20% fetal bovine serum (FBS, Atlanta Biologicals) without antibiotics] (26). BAEC used in this study were between passages 5 and 10. Unless specified otherwise, 2 million cells were seeded in 100-mm tissue culture dishes (Falcon) and grown to confluency in the growth medium before exposure to stimuli.

Adenoviral infections. For the infection of BAEC with recombinant adenovirus, 1 million cells were seeded in 100-mm tissue culture dishes (Falcon) 1 day before infection. Cells were infected with recombinant adenovirus at 100 multiplicity of infection in serum-free DMEM for 1 h and then incubated 48 h in a growth medium before the treatment. Construction of the recombinant adenovirus encoding PKA inhibitor (Ad-PKI) has been described previously (30). Recombinant adenovirus encoding green fluorescence protein (Ad-GFP) was used as a control.

Shear stress studies. A confluent monolayer of BAEC grown in a 100-mm dish was exposed to nonpulsatile, laminar shear stress in a shear medium (phenol red-free DMEM containing 0.5% FBS and 25 mM HEPES, pH 7.4) by rotating a Teflon cone (0.5° cone angle) as we described previously (21, 43). Cells were exposed to an arterial level of shear stress (15 dyn/cm2) (3, 21).

Preparation of cell lysates. After experimental treatments, cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed in 0.75 ml lysis buffer A (20 mM Tris · HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerophosphate, 1 mM sodium vanadate, 1 µg/ml leupeptin, 1 mM phenylenemethylsulfonyl fluoride, 1 µM microcystin, and 1% Triton X-100). Cell lysates were clarified by spinning at 14,000 g for 15 min at 4°C. Protein content of each sample was measured by using a Bio-Rad DC assay (26).

Immunoblotting. Aliquots of cell lysates (20 µg protein each) were resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore) (26). The membrane was incubated with a primary antibody overnight at 4°C and then with a secondary antibody conjugated with alkaline phosphatases (1 h at room temperature), which were detected by a chemiluminescence method (26). The intensities of immunoreactive bands in Western blots were analyzed by using the NIH Image program. The following primary antibodies were used: polyclonal antibodies for phosphorylated forms of Akt-Thr308, Akt-Ser473, and eNOS-Ser1179; a monoclonal antibody for phosphorylated form of cAMP response element binding (CREB) protein (CREB-Ser133); a polyclonal antibody for total CREB from Cell Signaling Technology; and a polyclonal antibody for total Akt from Santa Cruz biotechnology; polyclonal and monoclonal antibodies for total eNOS from Transduction Laboratories. Rabbit polyclonal antibodies specific for phosphorylated forms of eNOS-Ser116, eNOS-Thr497, and eNOS-Ser635, respectively, were raised and purified as previously described (8).

Statistical analysis. Statistical analysis was performed by Student's t-test. The P < 0.05 based on at least three or more independent experiments was considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Differential phosphorylation of eNOS at Ser116, Thr497, Ser635, and Ser1179 in response to shear stress, VEGF, and cAMP. To determine whether shear stress stimulates phosphorylation of eNOS at amino acid residues other than the well-characterized Ser1179, Western blot studies were carried out using antibodies specific to eNOS phosphorylated at Ser116, Thr497, Ser635, and Ser1179, respectively. As expected, exposure of BAEC to an arterial level of laminar shear stress (15 dyn/cm2) stimulated phosphorylation of eNOS-Ser1179 in a time-dependent manner as shown previously (3). Phosphorylation of eNOS-Ser1179 was apparent as early as 2 min after shear onset, reached maximum by 30 min, and remained elevated at least for 1 h (Fig. 1A).


View larger version (52K):
[in this window]
[in a new window]
  		Fig. 1.   Phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Thr497, Ser635, and Ser1179 in response to shear stress, vascular endothelial growth factor (VEGF), and cAMP. Confluent monolayers of bovine aortic endothelial cells (BAEC) were exposed to laminar shear stress (15 dyn/cm2) (A), 50 ng/ml VEGF (B), or 1 mM 8-bromo-cAMP (8-BRcAMP, C) for the time periods indicated. Cell lysates (20 µg/lane) were analyzed by Western blot with antibodies specific for phosphorylated forms of eNOS-Ser116, eNOS-Thr497, eNOS-Ser635, and eNOS-Ser1179. Membranes were reprobed with antibodies detecting total eNOS to monitor equal loading of samples. Blots shown are representatives of at least 3 independent studies. Intensity of each band of phosphorylated eNOS was quantified, and data are expressed as percentage of maximum stimulation (set as 100%). Line graphs represent means ± SE (n = 3-4 experiments).

We then examined the phosphorylation status of eNOS at Ser116, Thr497, and Ser635. Shear stress stimulated phosphorylation of Ser635 in a time-dependent manner (Fig. 1A). The shear-stimulated phosphorylation of Ser635 was significantly slower than that of Ser1179. At least 15 min of shear exposure was required to observe any significant phosphorylation of eNOS-Ser635. In contrast, shear stress had no effect on the phosphorylation status of eNOS-Ser116 and eNOS-Thr497 (Fig. 1A). In additional studies, we compared the phosphorylation of eNOS-Ser116 in subconfluent cells (~75% confluent) and confluent monolayers (1 day postconfluent) and found that it was decreased as cells became confluent (data not shown).

Next, we decided to determine whether eNOS phosphorylation at the four sites is also regulated by VEGF, another well-known activator of eNOS. As demonstrated previously (3, 17), VEGF rapidly stimulates phosphorylation of eNOS-Ser1179 in a transient manner reaching a maximum by 2 min and returning to a basal level by 15 min (Fig. 1B). Similar to shear stress, VEGF stimulated phosphorylation of eNOS at Ser635, whereas it did not affect phosphorylation of Ser116 and Thr497 (Fig. 1B). Similar to that found in response to shear stress, the time required for any significant phosphorylation of Ser635 was consistently slower than that of Ser1179 in each of our experiments. Whereas the Ser1179 was maximally phosphorylated within 2 min after VEGF stimulation, it required at least 5 min to observe a relatively small change and 30 min for a maximum stimulation of Ser635 phosphorylation in response to VEGF (Fig. 2B).


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 2.   Phosphorylation of Akt in response to shear stress, VEGF and cAMP. Confluent monolayers of BAEC were exposed to laminar shear stress (15 dyn/cm2) (B), 50 ng/ml VEGF (C), or 1 mM 8-BRcAMP (D) for the time periods indicated. Cell lysates (20 µg/lane) were analyzed by Western blot with antibodies specific for phosphorylated forms of Akt-Thr308 and Akt-Ser473. Membranes were reprobed with antibodies detecting total Akt to monitor equal loading of samples. Blots shown are representatives of at least 3 independent experiments. Phosphorylation of Akt-Thr308 was determined by densitometry, and data are represent means ± SE (n = 3 experiments) (A).

Recently, we showed that phosphorylation of eNOS-Ser1179 induced by shear stress is mediated by the PKA-dependent mechanisms (3). To examine whether direct stimulation of PKA can also induce similar phosphorylation patterns, BAEC were treated with 8-bromocAMP (BrcAMP), a cell-permeable cAMP analog. Treatment of cells with 8-BRcAMP stimulated phosphorylation of eNOS at Ser1179 and Ser635 in a time-dependent manner as shown in Fig. 1C. Treatment with 8-BRcAMP rapidly stimulated phosphorylation of Ser1179 reaching maximum within 2 to 5 min, which remained elevated at least for up to 1 h (Fig. 1C). Phosphorylation of eNOS-Ser635 was again significantly slower (requiring at least 15 min to observe a discernable change and 30 min for maximum stimulation) than that of Ser1179 (Fig. 1C).

Unlike shear stress and VEGF, 8-BRcAMP induced a rapid dephosphorylation of eNOS-Thr497 within 2 min and for as long as 1 h (Fig. 1C). This result is consistent with a previous report (35). On the other hand, Ser116 was not affected by 8-BRcAMP treatment (Fig. 1C).

All three experimental conditions used in this study, shear stress, VEGF, and 8-BRcAMP, had no effect on the total amount of eNOS as determined by using an antibody specific for eNOS protein. This result demonstrated that the changes observed with the phosphospecific antibodies were not due to changes in the eNOS protein expression level. Rather they represent changes in the phosphorylation status.

These results identify eNOS-Ser635 as a new amino acid residue that becomes phosphorylated in response to shear stress, VEGF, and cAMP. It also shows that phosphorylation of eNOS-Ser635 is consistently preceded by phosphorylation of Ser1179, suggesting a potential priming effect induced by Ser1179 phosphorylation. Another important finding is that shear stress and VEGF do not induce dephosphorylation of the Thr497 residue as reported in the case of bradykinin (23).

Differential time course of Akt phosphorylation in response to shear stress, VEGF, and cAMP. Recently, we showed that shear stress stimulates phosphorylation of eNOS-Ser1179 in an Akt-independent manner, whereas VEGF phosphorylates eNOS-Ser1179 in an Akt-dependent manner (3). Therefore, we compared the activation patterns of Akt by monitoring phosphorylation at two major regulatory sites, Thr308 and Ser473. As shown in Fig. 2B, shear stress stimulated Akt phosphorylation in a time-dependent manner, reaching maximum on 30-min shear exposure and remaining at maximum for at least 1 h. In contrast, VEGF stimulated Akt phosphorylation rapidly (maximum within 2-5 min) and in a transient manner (returning to a basal level by 15 min) (Fig. 2C). Unlike shear stress and VEGF, 8-BRcAMP is a relatively weak and slow activator of Akt (Fig. 2D). Any discernible changes in Akt phosphorylation by 8-BRcAMP required at least 15 min and remained stimulated by twofold for up to 1 h.

It is noteworthy that treatment of BAEC with 8-BRcAMP induced phosphorylation of eNOS-Ser1179 in >2 min, whereas Akt phosphorylation required more than 15 min of exposure (compare Fig. 1C and 2D). This result provides additional evidence that eNOS-Ser1179 can be phosphorylated in the absence of Akt activation if cells are activated by PKA-dependent mechanisms.

Shear stress regulates phosphorylation of eNOS-Ser635 in a PI3K-independent manner. We then determined whether phosphorylation of eNOS-Ser635 by shear stress is regulated in a PI3K-dependent manner. For this study, BAEC were pretreated for 30 min with two structurally distinct PI3K inhibitors, wortmannin and LY-294002, and then exposed to shear stress for 30 min. As expected, treatment of BAEC with either wortmannin or LY-294002 inhibited eNOS-Ser1179 phosphorylation as well as Akt phosphorylation (Fig. 3, A and B). However, the eNOS-Ser635 phosphorylation stimulated by shear stress was not affected by either inhibitor (Fig. 3, A and B). This result suggests that shear-dependent phosphorylation of eNOS-Ser635 is regulated by a PI3K-independent mechanism.


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 3.   Phosphoinositide-3-kinase (PI3K) inhibitors wortmannin and LY-294002 inhibit phosphorylation of eNOS at Ser1179 but not phosphorylation at Ser635 in response to shear stress. BAEC were pretreated with vehicle (DMSO) or PI3K inhibitors (100 nM wortmannin in A or 30 µM LY-294002 in B) for 30 min before exposure to shear stress (15 dyn/cm2) for 30 min (A and B). C: BAEC were pretreated with wortmannin as in A and followed by incubation with 1 mM 8-BRcAMP for 30 min. Cell lysates were analyzed by Western blot with antibodies specific for phosphorylated forms of eNOS-Ser1179, eNOS-Ser635, and Akt-Thr308, and total eNOS and total Akt. Blots shown are representatives of at least 3 independent studies.

Because eNOS-Ser635 can be phosphorylated by 8-BRcAMP as shown above (Fig. 1C), we examined further whether it is regulated by PI3K-dependent mechanisms by using wortmannin. Similar to that of shear stress, phosphorylation of Ser635 residue induced by 30 min treatment of 8-BRcAMP was not inhibited by wortmannin (Fig. 3C). On the other hand, wortmannin inhibited phosphorylation of eNOS-Ser1179 and Akt induced by 30 min treatment with 8-BRcAMP (Fig. 3C).

These results establish that Ser635 and Ser1179 residues are phosphorylated by different protein kinases in response to both shear stress and 8-BRcAMP.

Shear stress stimulates phosphorylation of eNOS-Ser635 in a PKA-dependent manner. The 8-BRcAMP effect shown above (Fig. 3C) suggested a role of PKA in the phosphorylation of eNOS-Ser635 in response to shear stress. To examine this further, PKA was inhibited either by treating cells with a pharmacological inhibitor H89 or by expressing PKI in cells by using an adenoviral construct Ad-PKI. Treatment of BAEC with H89 for 30 min inhibited shear-induced phosphorylation of eNOS-Ser635 (Fig. 4A). Under the same conditions, shear-dependent phosphorylation of eNOS-Ser1179 was also inhibited, but Akt phosphorylation was further enhanced by H89 (Fig. 4A) as shown previously (3).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4.   Protein kinase A (PKA) inhibitor H89 inhibits phosphorylation of eNOS at Ser1179 and Ser635 in response to shear stress. BAEC were pretreated with vehicle (DMSO) or a PKA inhibitor (10 µM H89) for 30 min and then exposed to shear stress (15 dyn/cm2) for 30 min (A) or 1 mM 8-BRcAMP for 30 min (B). Cell lysates were analyzed by Western blot with antibodies specific for phosphorylated forms of eNOS-Ser1179, eNOS-Ser635, and Akt-Thr308, and total eNOS and total Akt. Blots shown are representatives of at least 3 independent studies.

We then determined whether the phosphorylation of Ser635 induced by 8-BRcAMP could also be inhibited by H89. As shown in Fig. 4B, treatment with H89 completely blocked phosphorylation of eNOS-Ser635. In addition, as expected, H89 also inhibited Ser1179 phosphorylation induced by 8-BRcAMP (Fig. 4B). These results show that H89 prevents phosphorylation of Ser635 and Ser1179 induced by either shear stress or 8-BRcAMP.

Whereas H89 has been used widely as a highly selective PKA inhibitor, it may have other unexpected effects. To further confirm the role of PKA by using an independent approach in shear-dependent eNOS phosphorylation, BAEC were infected with recombinant adenovirus Ad-PKI before exposure to shear stress. As shown in Fig. 5A, infection of BAEC with Ad-PKI blocked shear stress-stimulated eNOS phosphorylation at Ser635 and Ser1179 without inhibiting Akt phosphorylation.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 5.   Adenovirus-mediated expression of protein kinase A inhibitor (Ad-PKI) inhibits phosphorylation of eNOS at Ser1179 and Ser635 in response to shear stress. Two days after BAEC were infected with Ad-PKI or Ad-GFP at 100 multiplicity of infection, cells were exposed to shear stress (15 dyn/cm2) for 30 min (A) or 10 µM forskolin for 30 min (B). Cell lysates were analyzed by Western blot with antibodies specific for phosphorylated forms of eNOS-Ser1179, eNOS-Ser635, and Akt-Thr308, CREB-Ser133 and total eNOS, total Akt, total CREB. Blots shown are representatives of at least 3 independent studies.

CREB is a well-known target of PKA, although several other protein kinases are also involved in CREB regulation (11). Treatment of BAEC with an adenylate cyclase activator forskolin (10 µM, 30 min) stimulated phosphorylation of CREB-Ser133, which was inhibited in cells infected with Ad-PKI (Fig. 5B). This result clearly demonstrates the effectiveness of PKI on a known PKA substrate CREB-Ser133, providing a positive control.

These results establish that PKA plays an essential role in shear-dependent phosphorylation of eNOS-Ser635 and Ser1179.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The most significant finding reported in the current study is that eNOS is phosphorylated at Ser635 in response to mechanical (shear stress) and humoral (VEGF and cAMP) stimuli. We also report evidence supporting that shear-dependent phosphorylation of eNOS-Ser635 is regulated in a PKA-dependent but PI3K-independent manner. In addition, we found that two other potential phosphorylation sites of eNOS at Thr497 and Ser116 were not regulated by shear stress or VEGF. However, cAMP induced a rapid dephosphorylation of Thr497.

It has been well documented that phosphorylation of eNOS-Ser1179 plays a key role in stimulation of eNOS activity in response to various physiological stimuli, which activate eNOS in a Ca2+-independent manner, including shear stress, VEGF, and estrogen (3, 12, 17, 21, 25). Other stimuli that activate eNOS by Ca2+-dependent mechanisms may not require phosphorylation. For example, although bradykinin stimulates phosphorylation of eNOS-Ser1179, it does not appear to have any regulatory role in eNOS activation (23).

Recent studies including our own work have shown that phosphorylation of eNOS-Ser1179 is mediated by a PI3K-dependent mechanism. However, it is not likely that PI3K directly phosphorylates eNOS-Ser1179. Instead it is highly likely that PI3K leads to activation of PDK1, and PDK1 in turn stimulates downstream target protein kinases, including Akt, PKA, PKG, and AMP kinase, which directly phosphorylate eNOS (3, 5, 13, 17, 35). PDK1 has been shown to activate, not only the well-known Akt, but also other protein kinases such as PKA, PKG, PKC, serum- and glucocorticoid-inducible kinase, and p70S6 kinase (9, 44). Whether the PI3K/PDK1 pathway indeed regulates these other protein kinases in response to a particular stimulus remains to be determined. At present it is not known whether AMP kinase can also be activated in a PI3K-dependent manner. Which protein kinase phosphorylates eNOS-Ser1179 appears to be determined by each given stimulus. For example, we showed that shear stress stimulates phosphorylation of eNOS-Ser1179 by PKA-dependent but Akt-independent mechanisms (3). An earlier study (13) suggested that shear stimulates eNOS-Ser1179 phosphorylation by the Akt-dependent mechanisms. This notion was based on indirect studies using mainly PI3K inhibitors and overexpression of constitutively active Akt mutants. However, the use of dominant negative mutants as performed more recently by Boo et al. (3) directly demonstrated that Akt is not the protein kinase-regulating eNOS-Ser1179 phosphorylation in response to shear stress. On the other hand, the same study confirmed that VEGF stimulates phosphorylation of eNOS-Ser1179 by Akt-dependent mechanisms (3).

Although it is clear that eNOS-Ser1179 is one of the key regulatory sites in response to shear stress and VEGF, eNOS contains many other potential phosphorylation sites, including Ser116, Thr497, and Ser635 (18). Therefore, we examined whether shear stress regulates phosphorylation of these potential sites by taking advantage of the phosphorylation site-specific eNOS antibodies.

Using phosphorylation of eNOS-Ser1179 in response to shear stress as a positive control and reference, we compared phosphorylation patterns of Ser116, Thr497, Ser635, and Ser1179 in response to shear stress, VEGF, and cAMP. First, eNOS-Ser1179 was phosphorylated by all three stimuli with unique time courses. Shear stress stimulated phosphorylation of eNOS-Ser1179 gradually and relatively slowly reaching maximum by 30-60 min exposure (Fig. 1A). On the other hand, both VEGF and cAMP rapidly stimulated phosphorylation of eNOS-Ser1179 reaching maximum levels within 2 min exposure (Fig. 1, B and C). Interestingly, VEGF-dependent phosphorylation of Ser1179 was transient returning to a basal level by 15 min exposure, whereas the maximal phosphorylation of Ser1179 induced by cAMP was maintained for as long as 1 h (Fig. 1C).

Currently, it is not known why VEGF-dependent phosphorylation of eNOS-Ser1179 is transient, whereas those induced by either shear stress or cAMP remain elevated for a longer period. One potential mechanism underlying different response patterns of eNOS-Ser1179 phosphorylation may be related to the upstream kinases Akt and PKA. Consistent with this possibility, the VEGF-dependent phosphorylation of Akt was indeed transient reaching maximum by 2-5 min and returning to a basal level by 15 min treatment (Fig. 2C). In comparison, the maximum phosphorylation of eNOS-Ser1179 induced by cAMP occurred (within 2 min) before there was any sign of Akt phosphorylation (requires at least 15 min) (Fig. 1C and 2D). In addition, phosphorylation of eNOS-Ser1179 induced by either shear stress or cAMP was prevented by PKA inhibitor H89 or Ad-PKI, further supporting the role of PKA, as shown previously (3).

We then found no evidence that the phosphorylation status of eNOS-Ser116 is regulated in response to shear stress, VEGF, or cAMP. However the Ser116 site was reported to be phosphorylated in response to shear stress in BAEC as determined by mass spectroscopy (19). Although the exact reasons are not clear, there are subtle differences in experimental conditions that could have contributed to the discrepancy between the two studies. The presence or absence of NaVO3 in the shear media and confluency of endothelial cells used for shear studies appear to be especially relevant. For example, in the study by Gallis et al. (19) study subconfluent (60-80% confluency) BAEC were labeled with [32P]orthophosphate in the presence of 200 µM NaVO3 in DMEM supplemented with 10% serum. In comparison, our studies were carried out using confluent BAEC monolayers in serum-deficient medium (DMEM containing 0.5% serum) in the absence of NaVO3.

The eNOS-Thr497 was rapidly dephosphorylated when BAEC were treated with 8-BRcAMP (Fig. 1A). In contrast, shear stress and VEGF did not have any effect on the phosphorylation status of eNOS-Thr497 (Fig. 1A). 8-BRcAMP-induced dephosphorylation of Thr497 was consistent with a previous finding by using an independent approach with isobutyl methylxanthine to increase the cellular cAMP level (35). Unlike in BAEC, however, VEGF stimulated dephosphorylation of eNOS-Thr497 in endothelial cells obtained from a different vascular bed, human umbilical vein endothelial cells (35). These differences in the behavior of endothelial cells depending on vascular origin are not unusual (28, 40). These results also provide an example that the response of cells to a physiological stimulus such as shear stress and VEGF are not necessarily identical compared with that induced by a bolus addition of 8-BRcAMP. It is possible that PKA activation induced by the physiological stimuli may be more restricted thus ensuring signaling specificity, whereas a bolus addition of 8-BRcAMP could activate many PKA isoforms regardless of their subcellular locations or cellular context. These results do not support a role for phosphorylation/dephosphorylation of eNOS-Thr497 in shear stress-dependent activation of eNOS.

We found in this study that shear stress, as well as VEGF and 8-BRcAMP, stimulate phosphorylation of eNOS-Ser635 (Fig. 1B). As far as we are aware, this is the first demonstration of eNOS-Ser635 phosphorylation under physiologically relevant stimuli. It is interesting to note that phosphorylation of eNOS-Ser635 is consistently slower than that of eNOS-Ser1179 in response to all three stimuli (Fig. 1). This raises the possibility that phosphorylation of Ser1179 may prime Ser635 for a subsequent phosphorylation. This possibility was further supported by the data showing that PKA inhibition by H89 or Ad-PKI blocked phosphorylation of both eNOS-Ser1179 and Ser635 (Fig. 4 and 5A). One simple interpretation of this result was that PKA might phosphorylate both sites at Ser1179 and Ser635 in a sequential manner. However, this speculation was not supported by subsequent experiments using PI3K inhibitors wortmannin and LY-294002 because both inhibitors blocked phosphorylation of Ser1179 but not of Ser635 (Fig. 3). If the Ser1179 phosphorylation was a prerequisite step, wortmannin or LY-294002 should have blocked phosphorylation of Ser635 as well. However, that was not the case. An alternative mechanism is that shear stress may stimulate two independent signaling pathways: one that activates a PI3K-dependent PKA pathway phosphorylating eNOS-Ser1179 and another that activates a PI3K-independent PKA pathway phosphorylating eNOS-Ser635 (Fig. 6). This scenario, however, would require the presence of at least two different pools of PKA.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 6.   Differential regulation of eNOS phosphorylation at Ser635 and Ser1179 in response to shear stress-speculated pathway. Shear stress stimulates PI3K, which in turn activates phosphoinositide-dependent protein kinase (PDK1). Shear stress may activate PKA through a PI3K-PDK1 pathway. Then PKA either directly or indirectly phosphorylates eNOS-Ser1179. On the other hand, we speculate that shear stress activates another type of PKA in a PI3K-independent manner (?), which is responsible for phosphorylation of eNOS-Ser635. PI3K inhibitors (wortmannin and LY-294002), PKA inhibitors (H89 and PKI), and PKA stimulator (cAMP) are shown. Also shown is the shear-stimulated activation of Akt in a PI3K-dependent manner.

One potential support for this speculation regarding two different pools of PKA is the preferential subcellular localization of different isoforms of PKA (4). PKA is a tetramer composed of two regulatory (R) and two catalytic (C) subunits. The mammalian PKA family includes four types of R subunits (RIalpha , RIbeta , RIIalpha , and RIIbeta ) and two C subunits (Calpha and Cbeta ) (4). Whereas RI types tend to be found in the cytosolic fraction of cells, RII subunits have been found to be associated with particulate fractions (4).

A typical activation of PKA occurs when cAMP binds to the R subunit thereby releasing the C subunit and leading to activation of the released C subunit. However, it has been previously shown that shear stress does not affect cAMP level in endothelial cells (31). Then, how would PKA be activated by shear stress without changing total cAMP levels?

Accumulating evidence suggest that PKA can be regulated in a cAMP-independent manner. One mechanism is phosphorylation of Thr197 in the activation loop of C subunit by PDK1 (6, 9). Other mechanisms include interactions of PKA with caveolin, A-kinase-anchoring protein (AKAP110), and inhibitory nuclear factor kappa B (Ikappa B) protein (16, 39, 42). It is especially interesting that the phosphorylation of the key regulatory site Thr197 is mediated by PDK1 (9) because shear stress has been shown to stimulate PI3K (22), which in turn activates PDK1. We speculate that shear stress activates PKA through a PI3K-PDK1 pathway and then PKA phosphorylates eNOS-Ser1179 either directly or indirectly (Fig. 6).

The sequence surrounding Thr197 matches the consensus PDK1 phosphorylation sequence and is conserved in both PKA catalytic subunits Calpha and Cbeta (46). Therefore, once PDK1 is activated, it is likely to activate both isoforms if they were in the close vicinity. However, PDK1 is believed to be localized in the plasma membrane, and it may not be able to phosphorylate those PKAs that are not colocalized. Because shear-dependent phosphorylation of eNOS-Ser635 was not prevented by PI3K inhibitors, it is unlikely that the PKA phosphorylating this site is a PI3K/PDK-dependent isoform. Therefore, we propose that PKA is activated in a compartmentalized and time-dependent manner in response to shear stress.

What is the physiological significance of phosphorylation of eNOS? Whereas the importance of eNOS-Ser1179 and Thr497 has been explored, the role of Ser635 is unknown at this point. The eNOS is composed of two identical monomers and each monomer contains the amino-terminal oxidase domain and carboxy-terminal reductase domain (18). To produce NO from substrates O2 and L-arginine, electron flux has to occur from the reductase domain of one monomer to the oxygenase domain of the other monomer. Binding of Ca2+/CaM plays a key role in coupling the two monomers so that electron can be fluxed and, in turn, NO can be produced. It has been proposed that the carboxy-terminal tail of eNOS, including Ser1179 is "wedged" in between the two monomers and act as autoinhibitory domain by blocking electron transfer between the two monomers (29). Phosphorylation of Ser1179 upon challenge with eNOS stimulators is proposed to induce the conformation change of the carboxy-terminal, which removes the wedge and lowers Ca2+ requirement leading to enzyme activation (29, 32). eNOS-Thr497 is adjacent to the CaM-binding region, and its phosphorylation has been proposed to hamper CaM binding and prevent enzyme activation (8).

It was previously shown that mutation of eNOS-Ser635 to Ala (S635A) did not induce any changes in phosphorylation and activation of eNOS stimulated by Akt-dependent mechanisms (13, 17). Taken together with our current studies, these results support the concept that Ser635 is phosphorylated by an Akt-independent but PKA-dependent mechanism. In addition, another eNOS mutant (S635D) in which eNOS-Ser635 was mutated to Asp to mimic a continuously phosphorylated state showed the identical enzyme activity as that of wild-type eNOS in an in vitro assay using a saturating concentration of Ca2+ (13). However, it remains to be determined whether phosphorylation of eNOS-Ser635 would alter enzyme activity in cells as well as in enzyme assays using subsaturating levels of Ca2+.

It has been recognized that a ~50-residue segment present in the flavin mononucleotide -binding domain of eNOS (residue 596-647 based on the bovine sequence) represents a putative "autoinhibitory element" that impedes CaM binding to eNOS and electron flux between the two monomers (7, 38). Deletion of this element from eNOS decreases Ca2+ and CaM requirement for enzyme activation and enhances maximal activity (7, 38). Because Ser635 is located in this "autoinhibitory element," it is a reasonable speculation that phosphorylation of Ser635 may affect CaM binding and electron transfer of the enzyme. However, this speculation awaits further study.

In conclusion, we found that mechanical shear stress stimulates phosphorylation of eNOS at Ser635. In addition, VEGF also stimulates phosphorylation at the same site indicating that eNOS-Ser635 may be an important regulatory site in response to both mechanical and humoral stimuli. We also showed evidence supporting that shear-dependent phosphorylation of eNOS-Ser635 is regulated by PI3K-independent but PKA-dependent mechanisms. The slow time course suggests that phosphorylation of Ser635 is likely to play a role in a chronic regulation of eNOS activity rather than in the acute NO production phase within minutes of the agonist stimulation. For example, phosphorylation of eNOS-Ser635 may determine its subcellular localization and interaction with other cofactors and regulators of eNOS. These questions are interesting subjects for future studies.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-60905, National Aeronautics and Space Administration Grant NAG2-1348, and a Whitaker Development fund (to H. Jo).


    FOOTNOTES

Address for reprint requests and other correspondence: H. Jo, Wallace H. Coulter Dept. of Biomedical Engineering at Georgia Tech and Emory Univ., 308D WMB, Atlanta, GA 30322 (E-mail: hanjoong.jo{at}bme.gatech.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.

July 8, 2002;10.1152/ajpheart.00214.2002

Received 13 March 2002; accepted in final form 21 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ayajiki, K, Kindermann M, Hecker M, Fleming I, and Busse R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ Res 78: 750-758, 1996[Abstract/Free Full Text].

2.   Bernier, SG, Haldar S, and Michel T. Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase. J Biol Chem 275: 30707-30715, 2000[Abstract/Free Full Text].

3.   Boo, YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, and Jo H. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 277: 3388-3396, 2002[Abstract/Free Full Text].

4.   Brandon, EP, Idzerda RL, and McKnight GS. PKA isoforms, neural pathways, and behaviour: making the connection. Curr Opin Neurobiol 7: 397-403, 1997[ISI][Medline].

5.   Butt, E, Bernhardt M, Smolenski A, Kotsonis P, Frohlich LG, Sickmann A, Meyer HE, Lohmann SM, and Schmidt HH. Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem 275: 5179-5187, 2000[Abstract/Free Full Text].

6.   Cauthron, RD, Carter KB, Liauw S, and Steinberg RA. Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase. Mol Cell Biol 18: 1416-1423, 1998[Abstract/Free Full Text].

7.   Chen, PF, and Wu KK. Characterization of the roles of the 594-645 region in human endothelial nitric-oxide synthase in regulating calmodulin binding and electron transfer. J Biol Chem 275: 13155-13163, 2000[Abstract/Free Full Text].

8.   Chen, ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, and Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285-289, 1999[ISI][Medline].

9.   Cheng, X, Ma Y, Moore M, Hemmings BA, and Taylor SS. Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase. Proc Natl Acad Sci USA 95: 9849-9854, 1998[Abstract/Free Full Text].

10.   Corson, MA, James NL, Latta SE, Nerem RM, Berk BC, and Harrison DG. Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res 79: 984-991, 1996[Abstract/Free Full Text].

11.   Daniel, PB, Walker WH, and Habener JF. Cyclic AMP signaling and gene regulation. Annu Rev Nutr 18: 353-383, 1998[ISI][Medline].

12.   Dimmeler, S, Assmus B, Hermann C, Haendeler J, and Zeiher AM. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res 83: 334-341, 1998[Abstract/Free Full Text].

13.   Dimmeler, S, Fleming I, Fisslthaler B, Hermann C, Busse R, and Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt- dependent phosphorylation. Nature 399: 601-605, 1999[Medline].

14.   Dimmeler, S, Haendeler J, Nehls M, and Zeiher AM. Suppression of apoptosis by nitric oxide via inhibition of interleukin- 1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)- 32-like proteases. J Exp Med 185: 601-607, 1997[Abstract/Free Full Text].

15.   Dimmeler, S, Hermann C, Galle J, and Zeiher AM. Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells. Arterioscler Thromb Vasc Biol 19: 656-664, 1999[Abstract/Free Full Text].

16.   Dulin, NO, Niu J, Browning DD, Ye RD, and Voyno-Yasenetskaya T. Cyclic AMP-independent activation of protein kinase A by vasoactive peptides. J Biol Chem 276: 20827-20830, 2001[Abstract/Free Full Text].

17.   Fulton, D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, and Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399: 597-601, 1999[Medline].

18.   Fulton, D, Gratton JP, and Sessa WC. Post-translational control of endothelial nitric oxide synthase: why isn't calcium/calmodulin enough? J Pharmacol Exp Ther 299: 818-824, 2001[Abstract/Free Full Text].

19.   Gallis, B, Corthals GL, Goodlett DR, Ueba H, Kim F, Presnell SR, Figeys D, Harrison DG, Berk BC, Aebersold R, and Corson MA. Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002. J Biol Chem 274: 30101-30108, 1999[Abstract/Free Full Text].

20.   Garcia-Cardena, G, Fan R, Shah V, Sorrentino R, Cirino G, Papapetropoulos A, and Sessa WC. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 392: 821-824, 1998[Medline].

21.   Go, YM, Boo YC, Park H, Maland MC, Patel R, Pritchard KA, Jr, Fujio Y, Walsh K, Darley-Usmar V, and Jo H. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress. J Appl Physiol 91: 1574-1581, 2001[Abstract/Free Full Text].

22.   Go, YM, Park H, Maland MC, Darley-Usmar VM, Stoyanov B, Wetzker R, and Jo H. Phosphatidylinositol 3-kinase gamma mediates shear stress-dependent activation of JNK in endothelial cells. Am J Physiol Heart Circ Physiol 275: H1898-H1904, 1998[Abstract/Free Full Text].

23.   Harris, MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, and Venema RC. Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem 276: 16587-16591, 2001[Abstract/Free Full Text].

24.   Hermann, C, Zeiher AM, and Dimmeler S. Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. Arterioscler Thromb Vasc Biol 17: 3588-3592, 1997[Abstract/Free Full Text].

25.   Hisamoto, K, Ohmichi M, Kurachi H, Hayakawa J, Kanda Y, Nishio Y, Adachi K, Tasaka K, Miyoshi E, Fujiwara N, Taniguchi N, and Murata Y. Estrogen induces the akt-dependent activation of endothelial nitric- oxide synthase in vascular endothelial cells. J Biol Chem 276: 3459-3467, 2001[Abstract/Free Full Text].

26.   Jo, H, Sipos K, Go YM, Law R, Rong J, and McDonald JM. Differential effect of shear stress on extracellular signal-regulated kinase and N-terminal Jun kinase in endothelial cells. Gi2- and Gbeta/gamma-dependent signaling pathways. J Biol Chem 272: 1395-1401, 1997[Abstract/Free Full Text].

27.   Ju, H, Zou R, Venema VJ, and Venema RC. Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272: 18522-18525, 1997[Abstract/Free Full Text].

28.   Kuchan, MJ, Jo H, and Frangos JA. Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells. Am J Physiol Cell Physiol 267: C753-C758, 1994[Abstract/Free Full Text].

29.   Lane, P, and Gross SS. Disabling a C-terminal autoinhibitory control element in endotelial NO synthase by phosphorylation provides a molecular explanation for activation of vascular NO synthesis by diverse physiological stimuli. J Biol Chem 277: 19087-19094, 2002[Abstract/Free Full Text].

30.   Lum, H, Jaffe HA, Schulz IT, Masood A, RayChaudhury A, and Green RD. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction. Am J Physiol Cell Physiol 277: C580-C588, 1999[Abstract/Free Full Text].

31.   Malek, AM, Greene AL, and Izumo S. Regulation of endothelin 1 gene by fluid shear stress is transcriptionally mediated and independent of protein kinase C and cAMP. Proc Natl Acad Sci USA 90: 5999-6003, 1993[Abstract/Free Full Text].

32.   McCabe, TJ, Fulton D, Roman LJ, and Sessa WC. Enhanced electron flux and reduced calmodulin dissociation may explain "calcium-independent" eNOS activation by phosphorylation. J Biol Chem 275: 6123-6128, 2000[Abstract/Free Full Text].

33.   Michel, JB, Feron O, Sacks D, and Michel T. Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J Biol Chem 272: 15583-15586, 1997[Abstract/Free Full Text].

34.   Michel, T, and Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest 100: 2146-2152, 1997[ISI][Medline].

35.   Michell, BJ, Chen Z, Tiganis T, Stapleton D, Katsis F, Power DA, Sim AT, and Kemp BE. Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem 276: 17625-17628, 2001[Abstract/Free Full Text].

36.   Michell, BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, de Montellano PR, Kemp BE, and Pearson RB. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol 9: 845-848, 1999[ISI][Medline].

37.   Morales-Ruiz, M, Lee MJ, Zollner S, Gratton JP, Scotland R, Shiojima I, Walsh K, Hla T, and Sessa WC. Sphingosine 1-phosphate activates akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells. J Biol Chem 276: 19672-19677, 2001[Abstract/Free Full Text].

38.   Nishida, CR, and Ortiz de Montellano PR. Autoinhibition of endothelial nitric-oxide synthase. Identification of an electron transfer control element. J Biol Chem 274: 14692-14698, 1999[Abstract/Free Full Text].

39.   Niu, J, Vaiskunaite R, Suzuki N, Kozasa T, Carr DW, Dulin N, and Voyno-Yasenetskaya TA. Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation. Curr Biol 11: 1686-1690, 2001[ISI][Medline].

40.   Ohno, M, Gibbons GH, Dzau VJ, and Cooke JP. Shear stress elevates endothelial cGMP. Role of a potassium channel and G protein coupling. Circulation 88: 193-197, 1993[Abstract/Free Full Text].

41.   Pritchard, KA, Jr, Ackerman AW, Gross ER, Stepp DW, Shi Y, Fontana JT, Baker JE, and Sessa WC. Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase. J Biol Chem 276: 17621-17624, 2001[Abstract/Free Full Text].

42.   Razani, B, Rubin CS, and Lisanti MP. Regulation of cAMP-mediated signal transduction via interaction of caveolins with the catalytic subunit of protein kinase A. J Biol Chem 274: 26353-26360, 1999[Abstract/Free Full Text].

43.   Rieder, MJ, Carmona R, Krieger JE, Pritchard KA, Jr, and Greene AS. Suppression of angiotensin-converting enzyme expression and activity by shear stress. Circ Res 80: 312-319, 1997[Abstract/Free Full Text].

44.   Toker, A, and Newton AC. Cellular signaling: pivoting around PDK-1. Cell 103: 185-188, 2000[ISI][Medline].

45.   Tsao, PS, Buitrago R, Chan JR, and Cooke JP. Fluid flow inhibits endothelial adhesiveness. Nitric oxide and transcriptional regulation of VCAM-1. Circulation 94: 1682-1689, 1996[Abstract/Free Full Text].

46.   Uhler, MD, Chrivia JC, and McKnight GS. Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem 261: 15360-15363, 1986[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 283(5):H1819-H1828
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Circ. Res.Home page
L. E. Goldfinger, E. Tzima, R. Stockton, W. B. Kiosses, K. Kinbara, E. Tkachenko, E. Gutierrez, A. Groisman, P. Nguyen, S. Chien, et al.
Localized {alpha}4 Integrin Phosphorylation Directs Shear Stress-Induced Endothelial Cell Alignment
Circ. Res., July 18, 2008; 103(2): 177 - 185.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
C. I. Jones III, Z. Han, T. Presley, S. Varadharaj, J. L. Zweier, G. Ilangovan, and B. R. Alevriadou
Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite
Am J Physiol Cell Physiol, July 1, 2008; 295(1): C180 - C191.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
O. Yalcin, P. Ulker, U. Yavuzer, H. J. Meiselman, and O. K. Baskurt
Nitric oxide generation by endothelial cells exposed to shear stress in glass tubes perfused with red blood cell suspensions: role of aggregation
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2098 - H2105.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
D. Fulton, L. Ruan, S. G. Sood, C. Li, Q. Zhang, and R. C. Venema
Agonist-Stimulated Endothelial Nitric Oxide Synthase Activation and Vascular Relaxation: Role of eNOS Phosphorylation at Tyr83
Circ. Res., February 29, 2008; 102(4): 497 - 504.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
E. Gkaliagkousi, J. Ritter, and A. Ferro
Platelet-Derived Nitric Oxide Signaling and Regulation
Circ. Res., September 28, 2007; 101(7): 654 - 662.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
D. M. Dudzinski and T. Michel
Life history of eNOS: Partners and pathways
Cardiovasc Res, July 15, 2007; 75(2): 247 - 260.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
A. Csiszar, N. Labinskyy, K. E. Smith, A. Rivera, E. N.T.P. Bakker, H. Jo, J. Gardner, Z. Orosz, and Z. Ungvari
Downregulation of Bone Morphogenetic Protein 4 Expression in Coronary Arterial Endothelial Cells: Role of Shear Stress and the cAMP/Protein Kinase A Pathway
Arterioscler. Thromb. Vasc. Biol., April 1, 2007; 27(4): 776 - 782.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
C. D. Mazer, F. Briet, K. R. Blight, D. J. Stewart, M. Robb, Z. Wang, A. M. Harrington, W. Mak, X. Li, and G. M.T. Hare
Increased cerebral and renal endothelial nitric oxide synthase gene expression after cardiopulmonary bypass in the rat
J. Thorac. Cardiovasc. Surg., January 1, 2007; 133(1): 13 - 20.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
W. Sun, T.-S. Lee, M. Zhu, C. Gu, Y. Wang, Y. Zhu, and J. Y-J. Shyy
Statins Activate AMP-Activated Protein Kinase In Vitro and In Vivo
Circulation, December 12, 2006; 114(24): 2655 - 2662.
[Abstract] [Full Text] [PDF]

Home page
 Integr. Comp. Biol.Home page
S. G. Lee and D. L. Mykles
Proteomics and signal transduction in the crustacean molting gland
Integr. Comp. Biol., December 1, 2006; 46(6): 965 - 977.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
K. Yayama, H. Hiyoshi, D. Imazu, and H. Okamoto
Angiotensin II Stimulates Endothelial NO Synthase Phosphorylation in Thoracic Aorta of Mice With Abdominal Aortic Banding Via Type 2 Receptor
Hypertension, November 1, 2006; 48(5): 958 - 964.
[Abstract] [Full Text] [PDF]

Home page