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Am J Physiol Heart Circ Physiol 290: H2376-H2384, 2006. First published January 20, 2006; doi:10.1152/ajpheart.00614.2005
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cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation

Xiao-Ping Zhang and Thomas H. Hintze

Department of Physiology, New York Medical College, Valhalla, New York

Submitted 8 June 2005 ; accepted in final form 9 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The objective of this study was to determine whether activation of protein kinase B (PKB) is involved in the production of nitric oxide (NO) induced by cAMP signal transduction. Mongrel dogs were used for this study. Coronary microvessels were isolated from the left ventricular free wall of these dog hearts. Forskolin (an activator of adenylyl cyclase that increases intracellular cAMP level) and 8-bromo-cAMP (a membrane-permeable cAMP analog) were used to stimulate NO release and activation of PKB and endothelial NO synthase (eNOS) in these blood vessels. We found that forskolin and 8-bromo-cAMP increased NO release (quantified by using the Griess reaction) from coronary microvessels by 80 ± 6 and 78 ± 11 pmol/mg (mean ± SE), respectively (P < 0.05 vs. control). Western blot analysis showed that forskolin elicited a significant increase in eNOS phosphorylation (59 ± 11%) at serine-1177 (a positively regulatory phosphorylation site for eNOS) and a significant increase in dephosphorylation (28 ± 6%) at threonine-495 (a negatively regulatory phosphorylation site of eNOS) (P < 0.05 vs. control). Interestingly, forskolin also increased the phosphorylation of PKB at serine-473 (by 49 ± 17%) and threonine-308 (by 53 ± 17%), respectively (P < 0.05 vs. control; phosphorylation of both sites is required for a full activation of PKB). N{omega}-nitro-L-arginine methyl ester (an NOS inhibitor) blocked NO formation, Rp diastereomer of cAMP (a PKA inhibitor), and LY-294002 [a PI3-kinase (an activator of PKB) inhibitor] prevented the production of NO, phosphorylation of PKB, and eNOS induced by forskolin. Our data clearly show an involvement of PKB activation in cAMP signal-induced NO production. We are reporting for the first time that cAMP signal transduction stimulates eNOS activation through a PKB-mediated mechanism.

nitric oxide; microvessels; protein kinases; adenylyl cyclase; protein phosphatases; adenosine 3',5'-cyclic monophosphate; protein kinase B; endothelial nitric oxide synthase

ENDOTHELIAL nitric oxide (NO) synthase (NOS) activity is regulated by multiple mechanisms, including regulation of multisite phosphorylation of endothelial NOS (eNOS) protein (17). Two phosphorylation sites on eNOS, Ser1177(human)/Ser1179(bovine) in the COOH-terminal tail of the enzyme and Thr495(human)/Thr497(bovine), located in the calmodulin (CaM)-binding sequence, have been studied intensively and are considered to be the most important. Phosphorylation of eNOS at Ser1177 reduces Ca2+ dependence of the enzyme (7), increases the rate of electron flux from the reductase domain to the oxygenase domain (24a), and increases the enzyme activity (11, 16, 18). In contrast, phosphorylation at Thr495 increases Ca2+-CaM dependence of the enzyme and decreases eNOS activity (5, 15, 25). Several laboratories have shown that multiple protein kinases, including PKB (11, 16, 18, 24, 25, 26), AMPK (7), PKA and PKG (5, 6, 25), and CaM II protein kinase (15), cause the phosphorylation of eNOS at Ser1177 and/or a coordinated dephosphorylation of eNOS at Thr495 and increase eNOS activity. However, the detailed molecular signaling pathways are not clear. Moreover, much of the current knowledge for these kinase-mediated eNOS activations is based on studies using a variety of transfected and tissue-derived endothelial cells in culture. Little is known about the presence and regulation for these pathways in intact vascular tissue. A recent study series in our laboratory (23, 40, 41) has shown that cAMP signal transduction induces eNOS activation and increases endothelial NO production from isolated canine and porcine coronary microvessels and that this is regulated by a PKA/PI3-kinase-mediated mechanism. However, whether this mechanism is involved in a kinase-regulated eNOS phosphorylation is unknown. Furthermore, PI3-kinase is an essential factor for activation of PKB; whether PKB activation is also involved in this PKA-mediated NO formation induced by cAMP signal transduction in these vessels is also not known. In cultured endothelial cells, both PKA and PKB have been reported to have an effect on eNOS phosphorylation and activation (5, 11, 16, 25). Therefore, we hypothesized that cAMP signal transduction stimulates endothelial NO generation by inducing phosphorylation of eNOS through both PKA and PKB activation. Thus the goals of this study were to determine 1) whether stimulation of cAMP signal transduction induces eNOS phosphorylation and 2) whether this increase in phosphorylation of eNOS is due to an increase in phosphorylation and activation of PKB. Because protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) have also been reported to regulate eNOS dephosphorylation, we also determined in this study 3) whether PP1 and PP2A also play a role in the regulation of eNOS activity mediated by cAMP signaling pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Preparation

All of the studies were approved by the Institutional Animal Care and Use Committee of New York Medical College and conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and the current "Guiding Principles in the Care and Use of Animals" of the American Physiological Society. Twenty-three adult mongrel dogs (body weight 21–29 kg) were anesthetized with pentobarbital sodium (50 mg/kg). The heart was excised immediately and kept in ice-cold phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin at pH 7.4.

Isolation of Coronary Microvessels

Isolation of myocardial microvessels from the left ventricular free wall of the dog hearts was performed using the method described in our previous studies (23, 33, 40, 41). Microvessels were obtained after separation from large arteries and veins, connective tissue, fat, and myocytes by a series of steps involving sequential dissection, homogenization, sieving, and glass bead purification. This preparation of microvessels (diameter range 20–70 µm) was virtually free of myocytes and consisted only of arterioles, venules, and capillaries. Approximately 2,000 mg of microvessels were collected per heart (215 ± 7 g; mean ± SE).

Incubation of Coronary Microvessels

Microvessels were placed in a small package of 80-µm nylon mesh, transferred into a tissue bath containing PBS, and oxygenated with 95% O2-5% CO2 for 30 min. Approximately 20 mg (wet weight) of tissue were placed in 5-ml plastic tubes that contained 500 µl of PBS as control or 450 µl of PBS and 50 µl of drugs dissolved in PBS used to stimulate (e.g., forskolin and 8-bromo-cAMP) or inhibit [e.g., N{omega}-nitro-L-arginine methyl ester (L-NAME) or Rp diastereomer of cAMP (Rp-cAMP)] PKB and eNOS phosphorylation and endothelial NO formation. All tissues were incubated with drugs for 20 min at 37°C. The inhibitors were added 20 min before incubation of tissue with the stimuli. In a parallel experiment, we also incubated microvessels with forskolin for 2, 5, 10, and 20 min, respectively, at 37°C to study the time course for the effect of cAMP signal transduction. At the end of the incubation time, the tubes were removed from the tissue bath. The supernatants were removed from these tubes to a matched empty tube for measurement of NO production, and the microvessels from each of these tubes were placed in liquid nitrogen immediately for the later measurement of PKB and eNOS phosphorylation.

Measurement of NO production from myocardial microvessels using Griess reaction. Sulfanilamide (450 µl of 1%) and N-(1-naphthyl)ethylene diamine (50 µl of 0.2%) were added to the supernatant of each tube described above for diazotization of sulfanilic acid by NO. After 5–10 min of incubation at room temperature for full color (pink) development, formation of NO was measured as nitrite with a spectrophotometer (Uvikon 930 Spectrophotometer, Kontron Instruments) as the increase in absorbance at 540 nm and compared with known concentrations of nitrite. Absorbance was measured and converted to a straight line by use of linear regression analysis (y = a + bx, r > 0.99). We have described these methods recently (23, 33, 40, 41).

Measurement for phosphorylation of PKB and eNOS protein from myocardial microvessels using Western blotting. Western blotting was used to quantify the phosphorylated PKB-Ser473 and PKB-Thr308 (59 kDa) and the phosphorylated eNOS-Ser1177 and phosphorylated eNOS-Thr495 (140 kDa) protein from the previously saved microvessels from normal dog hearts (n = 9). Briefly, total protein from these frozen (at –80°C) microvessels was extracted on liquid nitrogen, separated by using 10% SDS-PAGE gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia). The membrane was blocked for 1.5 h at room temperature in 5% dry nonfat milk blocking solution (DNMBS) in 1x PBS with 0.05% Tween 20 (PBS-T), followed by an overnight incubation at 4°C with the primary appropriate antibody diluted 1:1,000 in PBS-T-1% DNMBS. The membrane was washed four times in PBS-T-0.1% DNMBS, incubated 1.5 h with the secondary antibody 1:10,000, and finally washed four times in PBS-T-0.1% DNMBS before signal detection using chemiluminescence Super-Signal method (Pierce). PKB and the phosphorylated PKB-Ser473 and PKB-Thr308 (59 kDa) and eNOS and the phosphorylated eNOS-Ser1177 and eNOS-Thr495 protein (140 kDa) bands were detected by scanning densitometry of film (Kodak) autograms. The primary antibodies for PKB and phosphorylated PKB were rabbit polyclonal anti-human PKB, PKB-Ser473, or PKB-Thr308 peptide antibody (Cell Signaling). The primary antibodies for phosphorylated eNOS were mouse monoclonal anti-human eNOS-Ser1177 and mouse monoclonal anti-human eNOS-Thr495 peptide antibody (BD Biosciences), and the primary antibody for eNOS was rabbit polyclonal anti-human eNOS peptide antibody (Affinity Bioreagents). All these antibodies react with dog tissue. We have described these methods recently (40, 33).

Experimental Protocols

Time course of NO production, phosphorylation of PKB, and phosphorylation of eNOS in coronary microvessels induced by forskolin or okadaic acid. Forskolin (10–4 mol/l), okadaic acid (10–6 mol/l, a selective inhibitor for PP2A in the concentration range of 5 x 10–9 to 10–6 mol/l, 25), and combined forskolin (10–4 mol/l) with okadaic acid (10–7 mol/l, a subthreshold concentration that has no effect on NO production shown by a concentration-response study in these blood vessels) were incubated with coronary microvessels for 2, 5, 10, and 20 min individually. Nitrite release from one set of these microvessels and protein level of phosphorylated PKB and phosphorylated eNOS from another set of these microvessels were measured separately at each time point by using Griess reaction and Western blot analysis, respectively.

Effects of PP1 and PP2A on cAMP signal transduction-mediated NO production in coronary microvessels. Increasing concentrations of forskolin (10–10 to 10–4 mol/l) and 8-bromo-cAMP (10–8 to 10–2 mol/l) alone and in the presence of okadaic acid (10–7 mol/l) were incubated with tissue for 20 min. Nitrite release was measured. To determine the effect of PP1 on forskolin- and 8-bromo-cAMP-induced NO production, calyculin A (a potent inhibitor of PP1 at this concentration) (10–8 mol/l) was incubated with these microvessels before the addition of the highest concentration of forskolin and 8-bromo-cAMP. L-NAME (a competitive inhibitor of NOS) (10–4 mol/l), Rp-cAMP (a selective potent inhibitor of PKA) (10–3 mol/l), and LY-294002 (a specific PI3-kinase inhibitor) (3 x 10–5 mol/l), were also incubated with tissue before the addition of forskolin or 8-bromo-cAMP. Nitrite release from these differently treated coronary microvessels was measured by using Griess reaction.

Effects of cAMP signal transduction on regulation of eNOS and PKB phosphorylation in coronary microvessels and effect of PKA, PI3-kinase, and PP1 on this process. Forskolin (10–4 mol/l) was incubated with isolated canine coronary microvessels in the presence and absence of Rp-cAMP, LY-294002, and calyculin A. Protein levels of PKB and eNOS and phosphorylated PKB and eNOS in these microvessels under the conditions of control and these different treatments were measured with Western blotting. To avoid any undetectable protein level of phosphorylated eNOS or PKB under different conditions, all these experiments were performed in the presence of okadaic acid (10–7 M) to control the activity of PP2A at a certain level. Control experiments with microvessels in the presence and absence of okadaic acid (10–7M) for the basal level of eNOS and PKB, and phosphorylated eNOS and PKB, and the experiments for the effect of forskolin, PP1, PKA, and PI3-kinase inhibitors in the absence of okadaic acid on the basal level of eNOS and PKB, and phosphorylated eNOS and PKB in the microvessels, were also performed in this study.

Drugs and Chemicals

The PBS used in these studies consisted of 139 mM NaCl, 2.7 mM KCl, 8.1 mM NaHPO4, 1.5 mM KH2PO4, 0.68 mM CaCl2, 0.49 mM MgCl2, and 0.1% bovine serum albumen. Drugs and chemicals (8-bromo-cAMP, okadaic acid, calyculin A, L-NAME, LY-294002, nitrite, and bovine serum albumin) were purchased from Sigma Chemicals (St. Louis, MO). Forskolin was purchased from Calbiochem-Novabiochem (La Jolla, CA). Rp-cAMP was purchased from Research Biochemicals International (Natick, MA).

Statistical Analysis and Calculation

To construct a standard curve for nitrite, a stock solution of NaNO2 (10–5 mol/l) was prepared and diluted for each experiment. Sulfanilamide (450 µl of 1%) and N-(1-naphthyl)ethylene diamine (50 µl of 0.2%) were mixed with NaNO2 and allowed to stand at room temperature for 5–10 min for full color (pink) development. Absorbance of nitrite was measured at 540 nm and converted to a straight line using a regression analysis (y = ax + b, r > 0.99). Nitrite production was calculated using the linear regression formula. Data were expressed as means ± SE in picomoles per milligram wet weight tissue per 20-min incubation time (or per 2-, 5-, 10-, or 20-min incubation time for time course studies). The data in the figures that show nitrite releases are the changes in nitrite production in picomoles per milligram wet weight tissue per 20-min incubation time (or per 2-, 5-, 10-, or 20-min incubation, for time course studies) whereas the data in the text are the percentages of changes in nitrite and absolute values. Differences of nitrite production from control were determined by using a two-way analysis of variance. The differences between individual data points were determined using Tukey's test. P < 0.05 was considered statistically significant. The data in the figures that show expressions of PKB and phosphorylated PKB and eNOS and phosphorylated eNOS protein are the densities of the Western blotting bands for 150 µg of protein from each of these samples scanned by a spectrophotometer. The data in the text for differences of expression of protein from control were shown as the percentages of changes of density of these Western blotting bands compared with the control levels and were determined by using a two-way analysis of variance. The differences between individual data points were determined by using Tukey's test. P < 0.05 was considered statistically significant. Statistical analysis and graphs were produced on a Pentium III computer (Dell) using commercially available software (Lotus 1–2-3, Sigmastat, and Slide Write).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Time Course of NO Production From Coronary Microvessels Induced by Forskolin or Okadaic Acid

Forskolin (10–4 mol/l) increased nitrite release by 60 ± 16, 79 ± 20, 122 ± 12, and 145 ± 12 pmol/mg at 2-, 5-, 10-, and 20-min incubation time points, respectively (from control value: 53 ± 8 pmol/mg). Okadaic acid (10–6 mol/l) also increased nitrite release accumulatively. Okadaic acid (10–7 mol/l) significantly potentiated forskolin-induced nitrite release within 2-, 5-, 10-, and 20-min incubation time points, respectively (Fig. 1).


Figure 1
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  		Fig. 1. Time course for changes in nitrite formation in response to forskolin (10–4 mol/l; For, bullet), okadaic acid (10–6 mol/l; OK, {blacksquare}), and forskolin (10–4 mol/l) in the presence of okadaic acid (10–7 mol/l; For+OK, {blacktriangleup}) from coronary microvessels at 2-, 5-, 10-, and 20-min incubation. Values are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. forskolin or okadaic acid alone.

 
There was a rapid rate of NO production during 5- to 10-min incubation of microvessels with forskolin (8 ± 1 and 7 ± 0.8 pmol·mg–1·min–1, respectively; P < 0.05 vs. control = zero) or forskolin in the presence of okadaic acid (14 ± 3 and 11 ± 3 pmol·mg–1·min–1, respectively; P < 0.05 vs. control = zero). However, there was a marked drop of the rate of NO production at 20-min incubation (3 ± 0.4 and 3 ± 0.7 pmol·mg–1·min–1, respectively; P < 0.05 vs. either at 5- or 10-min incubation).

Time Course of Expression of Phosphorylated eNOS From Coronary Microvessels Induced by Forskolin or Okadaic Acid

Okadaic acid (10–6 mol/l) induced increase in eNOS phosphorylation at Ser1177 by –0.8 ± 4%, 16 ± 4%, 32 ± 18%, and 12 ± 6% (from control value: 0%), respectively, at 2-, 5-, 10-, and 20-min incubation time point. Forskolin (10–4 mol/l) in the presence of okadaic acid (10–7 mol/l) induced a marked increase in phosphorylation of eNOS at Ser1177 by 7 ± 3%, 57 ± 181%, 128 ± 48%, and 103 ± 52% (from control value: 0%) at 2-, 5-, 10-, and 20-min incubation time point, respectively. There was an immediately increased rate of eNOS phosphorylation during the first 10-min incubation of tissue with either okadaic acid or forskolin in the presence of okadaic acid. However, there was a dramatic drop of the rate of eNOS phosphorylation after 10-min incubation. The time course for protein level of phosphorylated eNOS and the rates of eNOS phosphorylation in coronary microvessels are shown in Fig. 2 (top; summary data).


Figure 2
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  		Fig. 2. Time course for rate of endothelial nitric oxide (NO) synthase (eNOS) phosphorylation at Ser1177 (top) and PKB phosphorylation at Ser473 (bottom) in response to forskolin (10–4 mol/l) in the presence of okadaic acid (10–7 mol/l; For+OK, hatched bar) and okadaic acid (10–6 mol/l; OK, striped bar) alone from coronary microvessels at 2-, 5-, 10-, and 20-min incubation. Values are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. okadaic acid alone.

 
Time Course of Expression of Phosphorylated PKB From Coronary Microvessels Induced by Forskolin or Okadaic Acid

Okadaic acid (10–6 mol/l) induced increase in PKB phosphorylation at Ser473 by 43 ± 19%, 8 ± 8%, 4 ± 4%, and 5 ± 3% (from control value: 0%) at 2-, 5-, 10-, and 20-min incubation time point, , respectively. Forskolin (10–4 mol/l) in the presence of okadaic acid (10–7 mol/l) induced a marked increase in phosphorylation of PKB at Ser473 by 156 ± 52%, 224 ± 61%, 166 ± 37%, and 129 ± 34% (from control value: 0%) at 2-, 5-, 10-, and 20-min incubation time point, respectively. There was an immediately increased rate of PKB phosphorylation in the first 2-min incubation of tissue with either okadaic acid or forskolin in the presence of okadaic acid. However, there was a dramatic drop of the rate of PKB phosphorylation after 2-min incubation. The time course for the protein level of phosphorylated PKB and the rates of PKB phosphorylation in coronary microvessels are shown in Fig. 2 (bottom; summary data).

Effects of PP1 and PP2A on cAMP Signal Transduction-Mediated NO Production in Coronary Microvessels

Forskolin (10–10 to 10–4 mol/l) and 8-bromo-cAMP (10–8 to 10–2 mol/l) concentration dependently increased nitrite release by 6 ± 7% and 16 ± 1% to 100 ± 10% and 99 ± 15% (from control value: 79 ± 1 pmol/mg), respectively. Preincubation with okadaic acid (10–7 mol/l) increased nitrite release induced by forskolin and 8-bromo-cAMP by 34 ± 9% to 177 ± 22% and by 28 ± 3% to 154 ± 37%, respectively. In a comparison of the effects of the highest concentration of forskolin and 8-bromo-cAMP, okadaic acid potentiated the change in nitrite production by 68% and 48% (P < 0.05), respectively. The effect of okadaic acid on nitrite production induced by forskolin and 8-bromo-cAMP is shown in Fig. 3 (top). In the presence of L-NAME, Rp-cAMP, LY-294002, and calyculin A, nitrite release induced by the highest concentration of forskolin was blocked by 39, 48, 53, and 35%, and of 8-bromo-cAMP was blocked by 47, 45, 44, and 48%, respectively (all P < 0.05). There was a detectable basal level of nitrite production. However, L-NAME, Rp-cAMP, LY-294002, or calyculin A, at the concentration used in this study, did not have any significant effect on basal level NO release from these coronary microvessels. The effects of L-NAME, Rp-cAMP, LY-294002, and calyculin A on nitrite production induced by forskolin and 8-bromo-cAMP are shown in Fig. 3 (bottom).


Figure 3
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  		Fig. 3. Top: change in formation of nitrite from coronary microvessels in response to increasing concentrations of forskolin (For, bullet) and 8-bromo-cAMP (8Br, {blacksquare}). Forskolin and 8-bromo-cAMP significantly increased nitrite production from coronary microvessels. Okadaic acid (10–7 mol/l; OK, {blacktriangleup}) markedly potentiated production of nitrite induced by forskolin (For+OK, {circ}) and 8-bromo-cAMP (8Br+OK, {square}). *P < 0.05 vs. control; #P < 0.05 vs. forskolin or 8-bromo-cAMP alone. Values are means ± SE. Bottom: changes in nitrite in response to highest concentration of forskolin (For, hatched bar) and 8-bromo-cAMP (8-Br, striped bar) were reduced by N{omega}-nitro-L-arginine methyl ester (L-NAME, 10–4 mol/l), Rp-diastereomer of cAMP (Rp-cAMP; 10–3 mol/l), LY-294002 (LY-294, 3 x 10–5 mol/l), and calyculin A (Caly, 10–8 mol/l). Values are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. highest concentration of forskolin or 8-Bromo-cAMP alone.

 
Effects of cAMP Signal Transduction on Regulation of eNOS Phosphorylation in Coronary Microvessels and Effect of PKA, PI3-Kinase, and PP1 on This Process

Forskolin (10–4 mol/l) induced a significant increase in eNOS phosphorylation at Ser1177 by 59 ± 11% and decrease in phosphorylation of eNOS at Thr495 by –28 ± 6%, respectively (P < 0.05 vs. control). These effects were blocked by Rp-cAMP, LY-294002, and calyculin A, respectively (all P < 0.05). There was a detectable basal level of eNOS protein and phosphorylated eNOS protein at either Ser1177 or Thr495. However, Rp-cAMP, LY-294002, or calyculin A, at the concentration used in this study, did not have any significant effect on basal level eNOS phosphorylation. The effects of forskolin on the phosphorylation of eNOS and the effects of Rp-cAMP, LY-294002, and calyculin A on forskolin-induced eNOS phosphorylation are shown in Fig. 4. (top, Western blotting; bottom, summary data).


Figure 4
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  		Fig. 4. Top: Western blotting shows protein level of phosphorylated eNOS (P-eNOS) at Ser1177 and Thr495 in response to forskolin (10–4 mol/l) in the presence of okadaic acid (10–7 mol/l; For) and in the presence of Rp-cAMP (10–3 mol/l; Rp), LY-294 (3 x 10–5 mol/l; LY), and calyculin (10–8 mol/l; Cal) in coronary microvessels after 20-min incubation (Con, control; PC, positive control). Phosphorylated eNOS-Ser1177, eNOS-Thr495, and eNOS (140 kDa) protein bands were detected by scanning densitometry of film (Kodak) autograms. Bottom: %changes in eNOS phosphorylation at Ser1177 (hatched bar) and Thr495 (striped bar) induced by forskolin (For, 10–4 mol/l) in the presence of okadaic acid (10–7 mol/l) and effects of Rp-cAMP (10–3 mol/l; Rp), LY-294 (3 x 10–5 mol/l), and calyculin A (10–8 mol/l; Caly) on eNOS phosphorylation at Ser1177 (hatched bar) and Thr495 (striped bar). Values are means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. highest concentration of forskolin alone.

 
Effects of cAMP Signal Transduction on Regulation of PKB Phosphorylation in Coronary Microvessels and Effect of PKA, PI3-Kinase, and PP1 on This Process

Forskolin (10–4 mol/l) induced a significant increase in PKB phosphorylation at Ser473 by 40 ± 17% and at Thr308 by 53 ± 17%, respectively (P < 0.05 vs. control). These effects were blocked by Rp-cAMP and LY-294002, respectively (all P < 0.05). Calyculin A decreased PKB phosphorylation at Ser473 by 31 ± 11% and at Thr308 by 33 ± 17%, respectively (all P < 0.05). There was a detectable basal level of PKB protein and phosphorylated PKB protein at either Ser473 or Thr308. However, Rp-cAMP, LY-294002, or calyculin A, at the concentration used in this study, did not have any significant effect on basal level of PKB phosphorylation. The effects of forskolin on PKB phosphorylation and the effect of Rp-cAMP, LY-294002, and calyculin A on PKB phosphorylation induced by forskolin are shown in Fig. 5 (top, Western blotting; bottom, summary data).


Figure 5
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  		Fig. 5. Top: Western blotting shows protein level of phosphorylated PKB (P-PKB) at Ser473 and Thr308 in response to forskolin (10–4 mol/l) in the presence of okadaic acid (10–7 mol/l) and in the presence of Rp-cAMP (10–3 mol/l), LY-294 (3 x 10–5 mol/l), and calyculin A (10–8 mol/l) in coronary microvessels after 20-min incubation. Phosphorylated PKB-Ser473, PKB-Thr308, and PKB (60 kDa) protein bands were detected by scanning densitometry of film (Kodak) autograms. Bottom: %changes in PKB phosphorylation at Ser473 (hatched bar) and Thr308 (striped bar) induced by forskolin (10–4 mol/l) in the presence of okadaic acid (10–7 mol/l) and the effects of Rp-cAMP (10–3 mol/l), LY-294 (3 x 10–5 mol/l), and calyculin A (10–8 mol/l) on PKB phosphorylation at Ser473 (hatched bar) and Thr308 (striped bar). Values are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. highest concentration of forskolin alone.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The significant findings in this study were twofold. First, stimulation of canine coronary microvessels with forskolin elicited a significant increase in eNOS phosphorylation at Ser1177 and dephosphorylation of eNOS at Thr495 and a similar time course for increase in endothelial NO production. These changes were associated with a slightly earlier but rapid increase in PKB phosphorylation at Ser473 and Thr308. Both PKA and PI3-kinase inhibitors substantially blocked PKB phosphorylation, as well as eNOS phosphorylation at Ser1177 and dephosphorylation at Thr495, and microvascular NO production induced by forskolin. These data suggest that cAMP signal transduction stimulates endothelial NO formation by modulating eNOS phosphorylation at Ser1177 and Thr495 through a PKA/PI3-kinase-mediated mechanism and that this process involves an increase in PKB phosphorylation and activation. PKB is a demonstrated activator of eNOS by phosphorylation of this enzyme at Ser1177; thus, to our knowledge, we have, for the first time, demonstrated a cAMP signal transduction-mediated, NO-forming pathway through a PKB-related mechanism. Second, incubation of microvessels with forskolin induced a rapid phosphorylation of eNOS at Ser1177 in the first 10 min, and this was associated with a rapid increase in NO formation. However, after 10 min of incubation, there was a reduction of eNOS phosphorylation at Ser1177 accompanied by a low rate of NO production, indicating a considerable effect of eNOS dephosphorylation at Ser1177 and the subsequent inactivation of this enzyme. Okadaic acid (a selective inhibitor for PP2A at 5 x 10–9 mol/l and up to 10–6) (25, 38) significantly potentiated nitrite production induced by forskolin and 8-bromo-cAMP, whereas, calyculin A (a potent inhibitor for PP1 at 10–8 mol/l) (25) essentially blocked nitrite release by these two agents. Okadaic acid also potentiated eNOS phosphorylation at Ser1177 induced by forskolin, whereas calyculin A blocked the dephosphorylation of eNOS at Thr495 stimulated by forskolin. These results suggest that protein phosphatases PP1 and PP2A both play an important role in maintaining eNOS activation even under a stimulated condition, and PP1 and PP2A may constitutively contribute to the regulation of eNOS activity in coronary blood vessels by controlling the equilibrium between phosphorylation and dephosphorylation of this enzyme.

Consistent with its classification as a Ca2+/CaM-dependent enzyme, the activity of eNOS is thought to be tightly controlled by an intramolecular autoinhibitory element that hinders CaM binding (38). Receptor-dependent and -independent agonists that increase intracellular Ca2+ or the association of the Ca2+/CaM complex with eNOS can increase its activity. Recent studies (9, 14, 38) have found that in addition to this Ca2+-dependent regulation, posttranslational mechanisms also play a very important role in the regulation of eNOS activity. These include the interaction of eNOS with other proteins (such as caveolin-1, heat shock protein 90, or membrane phospholipids), enzyme translocation, as well as eNOS phosphorylation. These posttranslational processes are actually calcium independent (14, 38) and can enhance the activity of this enzyme two to three times above the basal level (14). Increasing evidence shows that phosphorylation of eNOS is a critical regulatory mechanism for control of eNOS activity, and multiple protein kinases regulate eNOS activity through phosphorylation at multiple sites. Because several consensus sites for phosphorylation by protein kinases such as PKA, PKB (Akt), PKC, and CaM kinase II are found on eNOS, the key role of these kinases in the regulation of NOS through phosphorylation at specific site of this enzyme becomes of critical interest. To date, at least five specific phosphorylation sites on eNOS have been recognized. These are Ser1177, Thr495, Ser633, Ser615, and Ser114 (the amino acid numbers of eNOS used here are based on human sequence) (3). However, the functional importance of the phosphorylation on these sites and a detailed understanding regarding the mechanisms of these protein kinases regulated phosphorylation on each site are still controversial and unclear.

In a comparison of the phosphorylation of other sites, the effect of phosphorylation of eNOS at Ser1177 is relatively well described and plays an important role in stimulation of eNOS activity in response to various physiological stimuli, including shear stress, VEGF, bradykinin, estrogen, and hydrogen peroxide (3, 5, 10, 11, 16, 18, 21, 25, 26, 34, 37). Ser1177 on eNOS can be phosphorylated by a variety of protein kinases, including PKB (11, 16, 18, 24, 25, 26) and PKA (3, 5, 6, 27). In contrast, phosphorylation of eNOS at Thr495 has been reported to have an inhibitory effect on eNOS activity (7, 25), and it can be stimulated by AMPK (37) and PKC (15, 25). However, the molecular signaling pathways for the phosphorylation of even these two sites are still open questions at the current time. It is known that there are putative phosphorylation sites of PKB on eNOS (11, 16). Thus PKB may have a direct effect on phosphorylation of eNOS (at least at Ser1177) and, therefore, a subsequent activation of this enzyme. Indeed, numerous studies (11, 13, 18, 19) have shown that PKB increases NO release by promoting the phosphorylation and activation of eNOS in response to shear stress, whereas a study by Boo et al. (5) showed recently that in cultured bovine aortic endothelial cells (BAEC), shear stress and 8-bromo-cAMP both induced a PKA-dependent, but PKB-independent, phosphorylation of eNOS at Ser1177 and an increase in endothelial NO release. Bae et al. (1) also reported a PKA-, but not PKB-, dependent phosphorylation of eNOS at Ser1177 by bradykinin. Interestingly, in the present study, we found that incubation of canine coronary microvessels with forskolin induced a rapid (peak rate at 2 min) increase in phosphorylation of PKB at Ser473 and Thr308, a delayed (peak rate at 5 min) but associated increase in eNOS phosphorylation at Ser1177, a coordinated dephosphorylation of eNOS at Thr495, and an increase in endothelial NO production (peak rate similar to eNOS phosphorylation). Rp-cAMP and LY-294002 significantly attenuated the phosphorylation of PKB at Ser473 and Thr308, the phosphorylation of eNOS at Ser1177, the dephosphorylation of eNOS at Thr495, and the production of NO induced by forskolin, indicating that at least in our preparation, both PKA and PI3-kinase are important for the phosphorylation and activation of both PKB and eNOS stimulated by cAMP signaling. Because PKB is a demonstrated activator for eNOS by phosphorylating this enzyme at Ser1177, our data strongly suggest a PKB-dependent mechanism on the cAMP signal transduction-mediated, NO-forming pathway. These results are counter to the findings by Boo et al. (5) and Bae et al. (1), in which they described a PKA-mediated mechanism for eNOS phosphorylation at Ser1177 and activation of this enzyme independent of PKB. In isolated canine and porcine coronary microvessels, we demonstrated that cAMP signal transduction increases endothelial NO production through a PKA-dependent and PI3-kinase/PKB-mediated mechanism. The discrepancy between our results and the previous studies by Boo et al. (5) and Bae et al. (1) could be due to the different study conditions or to species difference. For instance, the experiments in both the Boo et al. (5) and Bae et al. (1) studies have been performed in cultured bovine aortic endothelial cells. The extracellular, perhaps even the intracellular, environments of these endothelial cells are completely different from those freshly isolated canine coronary microvessels used in the present study. Phosphorylation or dephosphorylation of eNOS occurs after intracellular molecular signaling cascades initiated by different stimuli from either intracellular or extracellular compartment after possibly complicated cross talk between intracellular-intracellular or intracellular-extracellular signal transduction pathways. Boo et al. (5) have found that to activate eNOS through PKA, vascular endothelial growth factor (VEGF) needs PKB to be involved, but shear stress does not. It has also been reported (20) that both eNOS and PKA catalytic subunits are restrictively located in the different cellular compartment in endothelial cells, suggesting that different PKA pools may be operational in the regulation of eNOS activity by different stimuli through different molecular signaling pathways. Furthermore, Michell et al. (25) also found that VEGF stimulation led to a transient increase in eNOS phosphorylation at Ser1177 and dephosphorylation at Thr495 in human umbilical vein endothelial cells but not in BAEC, suggesting a species difference for regulation of eNOS phosphorylation and its activity. All these reasons described above could significantly contribute to the discrepancy observed in these different studies.

Although our data strongly suggest a role of PKB in PKA-mediated phosphorylation and activation of eNOS, and Filippa et al. (14) have also previously reported that PKB can be phosphorylated and activated by cAMP dependent kinase, how and why PI3-kinase is involved in this regulation of PKB phosphorylation and activation mediated by PKA and whether PI3-kinase has an effect on either PKB or PKA translocation and therefore an effect on protein-protein interaction are unknown. In addition, we were not able to directly block the effect of PKB on phosphorylation and activation of eNOS induced by cAMP, because there are no specific PKB inhibitors currently available. Therefore, a further study for the function of eNOS by using a negative mutation of PKB after stimulation with cAMP is needed to confirm this mechanism.

Phosphorylation of eNOS at Ser635 promoted by PKA has also been reported (2, 4, 27) and is thought to be important for the chronic regulation of eNOS activity (2). In the current study with microvessel preparation, despite a detectable level of eNOS phosphorylation at Ser633 (corresponds to bovine Ser635) at basal condition (data not shown), we did not observe any significant change in phosphorylation of eNOS at this site after stimulation with forskolin in the presence or absence of either PKA or PI3-kinase inhibitors (data not shown). The discrepancy between our results and previous studies could be also due to the different tissue preparation and stimulation time period with different agonists. Michell et al. (27) reported that incubation of BAEC with isobutylmethylxanthine (a phosphodiesterase inhibitor), stimulated phosphorylation of Ser635 slightly from 5 min but reached a maximum after 60 min. Boo et al. (2, 4) also found that phosphorylation of Ser635 was significantly slower (requiring at least 15 min to observe a discernable change and 30 min for maximum stimulation) than that of Ser1179, whereas 8-bromo-cAMP rapidly stimulated phosphorylation of Ser1179 (reaching maximum within 2 to 5 min), which remained elevated at least for up to 60 min. However, in our present study, we did expose the microvessels to forskolin only for 20 min. Although the peak level of phosphorylation of eNOS at Ser1177 started from 5 min and was maintained up to 10 min, this effect was dramatically dropped after 10 min, indicating a slightly slower but markedly shorter-period phosphorylation of eNOS in our experiment than that in these previous studies. Therefore, it is conceivable to reason that there may also be a much slower (perhaps later than 20 min after stimulation with forskolin) phosphorylation of Ser633 in our experiments. However, further studies are needed to confirm this speculation.

The phosphorylation level of protein depends on the equilibrium between protein kinases and protein phophatases. Although the studies for eNOS phosphorylation have been done comprehensively, not enough attention has been paid to the protein phosphatase-mediated eNOS dephosphorylation and the associated activation or inactivation of this enzyme. It is known that both PP1 and PP2A are serine and threonine specific protein phosphatases (9). Recent studies by Michell et al. (25) and Fleming et al. (15) have found that PP1 preferentially dephosphorylates eNOS at Thr495, whereas PP2A is preferentially responsible for the dephosphorylation of eNOS at Ser1177. In cultured bovine aortic and human umbilical vein endothelial cells, PP1 dephosphorylated Thr497 by >80%, whereas PP2A caused <40% dephosphorylation. In contrast, PP1 dephosphorylated the Ser1179 by 30%, whereas PP2A caused >70% dephosphorylation at this site. There is a basal level of eNOS phosphorylation at both Ser1179 and Thr495 (5, 7). In the present study, we found that there was a detectable basal level of phosphorylated eNOS protein at Ser1177 and Thr495. There was a rapid reduction in PKB phosphorylation at Ser473 and eNOS phosphorylation at Ser1177 after 10 min of stimulation of tissue with forskolin. This phenomenon was associated with a rapid drop in the rate of coronary microvascular NO production, indicating the importance of dephosphorylation of the enzyme. Okadaic acid significantly potentiated nitrite production induced by forskolin and 8-bromo-cAMP, whereas calyculin A substantially blocked nitrite release by these two agents. Okadaic acid potentiated eNOS phosphorylation at Ser1177, and calyculin A blocked the dephosphorylation of eNOS at Thr495 induced by forskolin. These results suggest that protein phosphatases PP1 and PP2A both play an important role in the control of eNOS activation even during protein kinase stimulation. The data in the current study suggest that stimulation of cAMP signal transduction induces coronary vascular NO production perhaps by causing a coordinated phosphorylation of PKB at both Ser473 and Thr308, a subsequent eNOS phosphorylation at the stimulatory site, Ser1177, and a dephosphorylation of the inhibitory site, Thr495, through a PKA- and PI3-kinase mediated mechanism. On the other hand, PP1 and PP2A regulate eNOS activity by inducing dephosphorylation of this enzyme at their relevant sites of amino acid. Both protein kinases and phosphatases together regulate eNOS activity by maintaining the equilibrium of phosphorylation and dephosphorylation of this enzyme.

There are some unexpected findings in the current study. First, we found that calyculin A prevented the phosphorylation of eNOS at Ser1177 induced by forskolin. Because PP1 preferentially dephosphorylates eNOS at Thr495 and calyculin A is a potent inhibitor for PP1 at the concentration 10–8 M (15, 25), therefore, a preventive effect of calyculin A on dephosphorylation of Thr495 on eNOS induced by forskolin is presumable, whereas the prevention of phosphorylation of Ser1177 on eNOS by calyculin A is completely unexpected. The mechanism for this result is unknown. A reasonable explanation for it could be that during the inhibition of PP1, the effect of PP2A is relatively elevated to compensate the reduced effect of PP1 on the substrate of phosphorylated eNOS at Ser1177, because both enzymes have similar, but differently potent, effects on some identical substrates, such as phospho-eNOS-Ser1177. Because PP2A has an effect preferentially on dephosphorylation of Ser1177 on eNOS (15, 25), therefore, during the inhibition of PP1 by calyculin A, there is a prevention of dephosphorylation of Thr495 on eNOS; however, at the same time, there is also an increase in dephosphorylation of Ser1177 on eNOS due to a relatively elevated effect of PP2A after PP1 inhibition. This phenomenon may present a general mechanism for the regulation in activity of the enzymes that share the same substrates. However, this speculated mechanism awaits further study. Second, the data in our present study showed that calyculin A also has a significant effect on the prevention of PKB phosphorylation, and this may also, at least in part, contribute to the reduction of eNOS activation and endothelial NO production mediated by cAMP signaling. It is known that both PP1 and PP2A have effects on dephosphorylation and inactivation of PKB; however, PP2A serves as a major PKB phosphatase (28). Therefore, we infer that the unexpected effect of calyculin A on PKB dephosphorylation could be also due to a relatively increased effect of PP2A on PKB during PP1 inhibition. However, again, a further study is needed for this presumable explanation.

cAMP signal transduction is a novel pivotal mechanism for regulation of endothelial NO production and may play a crucial role in the control of cardiovascular function. For example, many studies (29, 22, 39) have shown that heart failure is associated with a depressed systemic and cardiac endothelial NO production, and defective endothelial NO formation has been recognized as an important mechanism contributing to the progressive deterioration of this disease. Thus any method that restores endothelial NO production may be beneficial for control of this disease. Recently, we (40) have found that although there is a selectively impaired bradykinin- or acetylcholine-induced endothelial NO formation in coronary microvasculature during heart failure, there is a significant elevation of NO production after stimulation of cAMP signal transduction, which may serve to compensate for the reduction of endothelial eNOS expression in this diseased state. NO produced by endothelial cells plays a crucial role for regulation of many biological functions such as vasodilatation, host defense, tissue respiration, and substrate utilization (30, 36). Many physiological factors, such as adrenomedullin, ATP, adenosine, or beta-adrenergic receptor agonist, activate adenylyl cyclase, increase intracellular cAMP, and initiate cAMP signal transduction. It has been reported that plasma adrenomedullin levels are elevated in a variety of disease states, including hypertension, congestive heart failure, and septic shock (8, 31), and plasma levels of adrenomedullin are positively correlated with left ventricular function in heart failure in humans and predict survival in septic shock (32). Therefore, better understanding of the underlying mechanism for activation of eNOS mediated by cAMP signal transduction in coronary microvessels and appropriate regulation of this pathway by modulating these physiological factors under different pathological conditions may have broad implications for control of the cardiovascular function and protection from these diseases.

In summary, our study demonstrates that cAMP signal transduction increases endothelial NO production in isolated canine coronary microvessels by increasing phosphorylation of eNOS at Ser1177 and dephosphorylation of eNOS at Thr495 through a PKA/PI3-kinase-dependent and PKB-mediated mechanism. Okadaic acid enhanced and calyculin A blocked eNOS phosphorylation at Ser1177 and dephosphorylation of eNOS at Thr495 and endothelial NO production induced by forskolin, indicating both PP1- and PP2A-mediated protein dephosphorylation plays an important role in the regulation of eNOS activation. Because PKA/PI3-kinase/PKB pathway and PP1 or PP2A all are important physiological factors or signaling systems, appropriate modulation of these pathways may have a critical impact on the regulation of endothelial NO production and control of biological function in normal and diseased states.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by a Scientist Development Award (0235493T) from American Heart Association in Northeast Affiliate (to X.-P. Zhang) and National Heart, Lung, and Blood Institute Grants PO-1-HL-43023, HL-50142, and HL-61290 (to T. H. Hintze).


   ACKNOWLEDGMENTS
 
We thank Dr. John G Edwards and Ziping Wang for technical support with Western blot analysis.


   FOOTNOTES
 

Address for reprint requests and other correspondence: X.-P. Zhang, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 (xpz789{at}hotmail.com)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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