doi: 10.1161/CIRCULATIONAHA.104.524454
(Circulation. 2005;112:I-190 – I-195.)
© 2005 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Bradykinin Preconditioning Improves the Profile of Cell Survival Proteins and Limits Apoptosis After Cardioplegic Arrest
From the Division of Cardiothoracic Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Mass.
Correspondence to Dr Frank W. Sellke, Division of Cardiothoracic Surgery, BIDMC, LMOB 2A, 110 Francis St, Boston, MA 02215. E-mail fsellke{at}caregroup.harvard.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background— We hypothesized that preconditioning the heart with bradykinin (BK) would improve the profile of antiapoptotic proteins and inhibit myocardial apoptosis.
Methods and Results— Eighteen rabbit hearts were retrogradely perfused with Krebs-Henseleit buffer (KHB). Six control hearts were perfused with KHB for 90 minutes without cardioplegia ischemia. Six hearts were arrested for 30 minutes (37°C) with crystalloid cardioplegia (CCP). Six BK preconditioning (BKPC) hearts received a 10-minute coronary infusion of 10–8 M BK-enriched KHB followed by a 5-minute recovery period and were then arrested for 30 minutes with CCP. The hearts were reperfused for 30 minutes with KHB. BKPC significantly improved the recovery of left ventricular pressure (73±5 versus 51±4 mm Hg; P<0.05) and reduced the percentage of myocardial apoptosis (3.4±0.3% versus 1.2±0.2%; P<0.05) as compared with CCP. There were no significant differences in total protein levels of caspase 3, Bcl-2, Bad, and Bax, among the groups. Both BKPC and CCP induced phosphorylation of Bad at Ser112, but the BKPC group had higher phosphorylated Bad than CCP (4.4±0.5 versus 2.0±0.3; P<0.05). Both BKPC and CCP alone increased caspase 3 cleavage and activity as compared with controls (P<0.05 and P<0.01, respectively), but BKPC caused less cleavage and activation of caspase 3 than CCP alone (P<0.05).
Conclusions— BKPC increased Bad phosphorylation, inhibited caspase 3 activation, and limited myocardial apoptosis, which were associated with improvement of left-ventricular performance. These results identify novel molecular mechanisms underlying the protective effects of BKPC during cardiac surgery.
Key Words: apoptosis • bradykinin • cardioplegia • ischemia • protein
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Ischemia preconditioning (IPC) is a phenomenon whereby exposure of the myocardium to a brief period of ischemia and reperfusion (I/R) markedly limits myocardial necrosis induced by subsequent prolonged I/R.1 Recent studies have suggested that IPC prevents myocardial apoptosis.2 Apoptosis and necrosis are two different types of cell death. In contrast to necrosis, apoptosis is a "programmed cell death," and it is associated with nuclear alterations, such as chromatin condensation and DNA fragmentation, and with the formation of apoptotic bodies.3 Apoptosis has been considered as one of the mechanisms of cell loss during I/R injury.3 Although the mechanism responsible for I/R-induced apoptosis is incompletely understood, it has been demonstrated that I/R-induced apoptosis is mainly regulated by the Bcl-2 family of proteins and cysteine protease family of caspases.3,4
Several cell survival factors have been shown to improve the profile of antiapoptotic proteins and prevent myocardial apoptosis.4 Bradykinin (BK) has been widely accepted as one of the endogenous protective factors to improve-left ventricular (LV) performance, attenuate myocardial necrosis, and reduce the incidence of arrhythmias after myocardial I/R.5,6 However, it is not known if BK also affects I/R-induced apoptosis-related proteins and myocardial apoptosis. Therefore, we hypothesized that BKPC would improve the profile of antiapoptotic proteins and prevent apoptosis after myocardial I/R.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Experimental Model
Animals were cared for in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center and Harvard Medical Area Standing Committee approved the protocols used in this study.
New Zealand white rabbits (1.5 to 2.5 kg) were used in this study (Millbrook Farm). Rabbits were anesthetized with ketamine (35 mg/kg) and xylazine (2.5 mg/kg IM) and anticoagulated with heparin (2000 U/kg IV), after which the heart was rapidly exposed. The aorta was cannulated, and the heart was retrogradely perfused in situ to avoid ischemia. The heart was then excised and mounted in an organ chamber on a Langendorff perfusion system. The heart was retrogradely perfused at 70 mm Hg with a modified Krebs-Henseleit buffer (KHB) with the following composition (mmol/L): 118 NaCl, 25 NaHCO3, 1.2 KHPO4, 4.7 KCl, 1.2 MgSO4, 1.8 CaCl2, and 11 glucose. The KHB was equilibrated with 95% O2 and 5% CO2, adjusted to a pH of 7.35 to 7.4 at 37°C, and filtered with a 5 µm filter (Gilman Scientific, Inc.). Right ventricular myocardial temperature was measured with a thermistor needle probe (Mallinckrodt, Inc.) and was maintained at 37°C throughout the experiment. Our Langendorff apparatus permits instantaneous change of the perfusion fluids from standard KHB to one containing different pharmacological substances or cardioplegia solution by adjusting an inlet valve to the aortic perfusion cannula.
Measurements
Isovolumetric measurement of LV performance was made using a compliant latex balloon connected to a pressure transducer inserted in the LV across the mitral valve. A calibrated syringe attached to the pressure transducer system was used to fill the balloon with a volume of saline needed to maintain a LV end diastolic pressure (LVEDP, mm Hg) of 5 mm Hg during the measurement of baseline LV performance. This same balloon volume was used for subsequent measurements of LV performance after reperfusion. LV performance was assessed by measurement of LV systolic pressure (LVSP, mm Hg) and LVEDP [mm Hg, LVSP – LVEDP = LV developed pressure (LVDP)]. Positive and negative first derivatives of LVSP (+dP/dt and –dP/dt, mm Hg/s) were calculated as indices of ventricular contractility and compliance, respectively. Analog pressure data from the LV balloon were amplified and converted to a digital signal for on-line data recording and computation (Gould-PONEMAN, Gould). Continuous pressure measurements were sampled at specific time points in each experiment. Coronary flow (CF; mL/min) was measured by timed collection of effluent from the right ventricle exiting the heart from the severed pulmonary artery. Hearts failing to generate a LVDP >80 mm Hg or a CF <25 mL/min during the stabilization phase of the experiment were excluded from additional study.
Experimental Protocols
After 30 minutes of equilibration, hearts were divided into the following 3 groups. The control group was additionally buffer-perfused for 60 minutes without cardioplegic ischemia (n =6). In the crystalloid cardioplegia (CCP) groups, the hearts were arrested for 30-minute ischemia with CCP (n =6). In the BKPC group, hearts received a 10-minute coronary perfusion of 10–8 M BK-enriched KHB, followed by a 5-minute recovery period, and were then arrested for 30 minutes with CCP (BKPC + CCP; n =6). The composition of the crystalloid cardioplegic solution was as follows (mmol/L): 121 NaCl, 25 KCl, 12 NaHCO3, 1.5 CaCl2, 16 MgCl2, and 11.1 glucose (pH 7.6). A single dose (50 mL) of cardioplegia solution (37°C) was infused at 60 mm Hg via a separate perfusion column. After a total of 30 minutes of cardioplegic arrest, the hearts from the CCP and BKPC groups were reperfused for 30 minutes with KHB. At the conclusion of reperfusion, the hearts were then excised, and the LV-free wall was cut into several pieces for apoptosis analysis and molecular comparisons.
Immunoblotting
Whole-cell lysates were isolated from the homogenized LV samples (100 mg) with a radioimmunoprecipitation assay solution (Biotech) and centrifuged at 12,000 x g for 10 minutes at 4°C to separate soluble from insoluble proteins. The supernatant protein concentration was measured spectrophotometrically at a 595-nm wavelength with a DC protein assay kit (Bio-Rad). Forty micrograms of total protein were fractionated on 10% SDS-polyacrylamide gel electrophresis and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation) using a semidry transfer apparatus (Millipore). All of the incubations were at room temperature. Membranes were then incubated with 5% nonfat dry milk in 50 mmol/L Tris-HCl (pH 8.0), 100 mmol/L NaCl, and 0.1% Tween 20 buffer (TBST) for 1 hour at room temperature to block nonspecific binding. Membranes were incubated with rabbit polyclonal anti-Bcl-2, Bad, and Bax (Cell Signaling) 1:1000 (v/v) dilution in 5% nonfat dry milk, mouse monoclonal anti-phospho-Bad (Cell Signaling Tech. Inc.) 1:2000 (v/v) dilution with 5% bovine serum albumin, rabbit polyclonal anti-caspase 3 (Santa Cruz Biotechnology, Inc.) 1:1000 (v/v) for 1 hour. After being washed with TBST, the membranes were incubated for 1 hour in 2.5% nonfat dry milk in TBST (Jackson Immunolabs), Peroxidase activity was visualized by means of an enhanced chemiluminescence substrate system and exposed to X-ray films (Amersham). NIH Image 1.6 was used for band intensities.
Caspase 3-Like Activity
Myocardial tissue (50 mg) was homogenized in lysis buffer [1% Triton X-100, 0.32 sucrose, NaCl, 1 mmol/L phenylmethanesulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 10 mmol/L Tris/Hcl (pH 8.0)] followed by centrifugation (16 000 x g, 4°C, 10 minutes). The supernatants (200 µg of protein) were incubated with 2X reaction buffer (containing 10 mmol/L dithiothreitol) and 50 µmol/L DEVD-7-amino-4-trifluromethyl coumarin (AFC) substrate at 37°C for 60 minutes (BioVision). Caspase 3-like activity was detected in a luminescence spectrometer (Perkin-Elmer, LS50B, EG&G) by measuring the proteolytic cleavage of the fluorogenic DEVD-AFC substrate and AFC as standard at an excitation wavelength of 400 nm versus an emission wavelength of 505 nm. Fold-increase in caspase 3 activity was determined by comparing these results with level of the uniduced controls.
TUNEL Staining
LV tissues were fixed in formalin for 24 hours, embedded in paraffin, and a 4-µm section obtained. The apoptotic cells were identified by TUNEL using an apoptosis detection kit according to the manufacturer’s protocol (Chemicon Inc). Ten photographs (magnification, x200) of each tissue section were taken. The nuclei were viewed and manually counted by 2 observers, who were blinded to the experimental conditions. The number of TUNEL-positive cardiomyocytes, indicating apoptosis, was expressed in mean number per microscopic field.
Drugs
BK was obtained from Sigma Chemical. BK was dissolved in KHB prepared on the day of the study. Other chemicals were obtained from Fisher Chemical.
Data Analysis
Data were presented as the mean ±SEM. Comparisons among the groups were assessed by ANOVA followed by Bonferroni’s post hoc test. The paired Student t test was used to compare hemodynamic variables after reperfusion among groups. Statistical significance was taken at P<0.05.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Effects of BKPC on the Recovery of LV Function
There were no significant differences in baseline LV performance before ischemia among any of the groups. Ten minutes of BK 10–8 M infusion induced a slight but not significant increase in CF (35.3±6 mL/min of BKPC+CCP versus 34±7 mL/min of baseline). LVDP and +/– dP/dtmax were not changed during BK coronary infusion. There were significant differences in the recoveries of LVDP (P<0.05), +dP/dtmax (P<0.05), and –dP/dtmax (P<0.01) as compared with CCP alone, respectively (Table).
|
Effects of BKPC on Bcl-2-Family Protein Expression and Bad Phosphorylation
The changes in expression of Bcl-2, Bad, and Bax proteins were analyzed by immunobloting (Figure 1a through 1c). Densitometrically, there were no significant differences in expression of the Bcl-2, Bad, and Bax proteins after I/R among the groups. CCP induced phosphorylation of Bad at Ser112 as compared with control, but BKPC caused greater phosphorylation of Bad (4.4±0.5 versus 2.0±0.3; P<0.05) (Figure 1d) than CCP.
|
P<0.05 vs CCP.
BKPC Inhibits Caspase 3 Cleavage and Activation
There were no alterations in total caspase 3 proteins among control, BKPC, and CCP (Figure 2a). Both BKPC and CCP induced caspase 3 cleavage, a prerequirement for caspase 3 activation, but CCP caused more caspase 3 cleavage than BKPC (6.2±0.4-fold versus 2.7±0.3-fold, respectively; P<0.05) (Figure 2b). Caspase 3-like activity in the myocardium was significantly increased after CCP-induced I/R compared with the control. BKPC significantly inhibited the CCP-induced activation of caspase 3 (Figure 2c).
|
BKPC Reduces Myocardium Apoptosis
Figure 3a through 3c illustrates the TUNEL-positive myocyte nuclei photographed at a magnification of x200. Figure 4 shows the percentage of TUNEL-positive nuclei in LV sections in the 3 groups. The percentage of TUNEL-positive myocyte nuclei was significantly increased in the CCP group compared with the control group (P<0.05). The number of TUNEL-positive myocyte nuclei was significantly reduced by BKPC as compared with CCP (P<0.05).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
BK has been considered as one of the mediators or triggers for early and delayed IPC.7 Pretreating the heart with BK before crystalloid cardioplegic ischemia significantly improves postischemic LV and microvascular function, suggesting that pharmacological BKPC may be an important addition to our current methods of myocardial and microvascular protection with hyperkalemic cardioplegia.8–10 Recent clinical studies have confirmed those animal studies and have shown that BKPC improves ischemia tolerance in patients undergoing angioplasty and coronary artery bypass surgery.11–12 Consistent with previous studies, the present study used a pharmacologic preconditioning regiment with 10 minutes of 10–8 M BK infusion and 5 minutes of KHB before crystalloid cardioplegic ischemia indicating that BK preconditioned the rabbit heart against postischemic LV dysfunction.
Recent studies have demonstrated that cardioplegia infusion and cardiopulmonary bypass trigger apoptosis signal pathways and induce cardiomyocyte apoptosis in animals and humans.13–15 These studies suggest that apoptosis may play an important role in myocardial stunning after open heart surgery.3,13–15 The present study investigated the effects of BKPC on I/R-induced myocardial apoptosis in rabbits. Our results show that cardioplegia I/R significantly induces myocardial apoptosis. In contrast, BKPC significantly inhibits cardioplegia I/R-related myocardial apoptosis. The antiapoptotic effects of BKPC may be associated with its improving postischemic LV performance.
The Bcl-2 family of proteins controls apoptosis by modifying mitochondrial permeability and the release of cytochrome c.3,16 I/R stress may induce the formation of proapoptotic Bcl-2 family protein complexes in mitochondria, which, in turn, leads to the release of cytochrome c and activation of death-effector caspases, such as caspase 3.3,16 Phosphorylation and inactivation of the proapoptotic protein Bad prevent the release of mitochondrial cytochrome c, thereby preventing apoptosis.4,17 Our studies demonstrated that cardioplegic I/R did not increase the total Bcl-2 family proteins, but induced phosphorylation of Bad. The discrepancies between the other studies and this one may be because of the different animal models used, such as a long period of I/R versus a short period of I/R. Moreover, BKPC did not change the expression of the Bcl-2 family proteins but caused greater phosphorylation of Bad at Ser112 than cardioplegia alone. The enhanced phosphorylation of Bad by BKPC may contribute to its antiapoptotic effects (Figure 5).
|
Signaling through tyrosine kinase (TK) has been demonstrated to confer protection against I/R injury through its activation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS).18 Several studies have shown that BKPC activates these prosurvival signaling kinase cascades.9,19,20 Blockades of TK and PI3K using genistein and wortamannin, respectively, have been found to abolish the protective effects of BKPC.9,19,20 Several investigators have reported that BK phosphorylated Akt in the myocardium,19,20 and the activation of the PI3K-Akt pathway has been shown to phosphorylate and subsequently inactivate proapoptotic protein Bad and inhibit apoptosis.4,17,21 Based on these observations, it would be interesting to speculate that BKPC-induced phosphorylation and inactivation of Bad in the present study may be through a PI3K-Akt-eNOS signal pathway.
Akt has been reported to phosphorylate and activate eNOS, producing nitric oxide (NO), which has been implicated in myocardial protection.19,22,23 NO, in turn, has been shown to inhibit opening of the mitochondrial permeability transition pore and caspase 3 activation.24 BK has been found to induce phosphorylation and activation of eNOS via Akt and subsequently enhance NO release. 8,9 19,20 The beneficial effects of BKPC have been shown to be abrogated in the presence of N-
-nitro-L-arginine methyl ester, a nonselective eNOS inhibitor.9 Thus, eNOS may also participate in the antiapoptotic effects of BKPC.
In the present study, we observed that terminal caspase 3 was cleaved into the active p17-subunit of caspase 3 after cardioplegic I/R. We additionally found that the cleaved caspase 3 was activated and increased after cardioplegic arrest and reperfusion. BKPC significantly inhibited the cleavage and activation of caspase 3. The BKPC-induced inactivation of terminal caspase 3 may lead to its inhibiting myocardial apoptosis after cardioplegic I/R. To the best of our knowledge, this is the first report showing that BK is capable of improving Bad phosphorylation, inhibiting caspase 3 activation, and limiting apoptosis after myocardial I/R.
Angiotensin converting enzyme (ACE) inhibitors have been widely used to limit I/R injury, which appears to be dependent on inhibition of BK degradation.25 The cardioprotective effects of ACE inhibitors could be abolished in the presence of HOE 140 (icatibant), a specific BK-B2 receptor antagonist.25 Thus, our data support the use of ACE inhibitors in the treatment of myocardial infarction, hypertrophy, and heart failure, because cell apoptosis often occurs during these pathophysiological processes.3,25
The isolated buffer-perfused heart has been widely used in the investigation of cardioplegic I/R. However, there are some advantages and disadvantages of this experimental preparation. For example, the advantage of isolated buffer-perfused heart is the elimination of extrinsic neural input and hormonal factors. The disadvantage of the isolated heart perfused with low viscosity media lacking red cells is the abnormally high-coronary flow rate as compared with the blood-perfused heart.
In conclusion, we demonstrate that BKPC improves LV performance and limits myocardial apoptosis with concomitant phosphorylation of Bad and inactivation of caspase 3. These results may provide novel molecular mechanisms responsible for the protective effects of BKPC and open new avenues for research into the clinical limitations of I/R injury after open-heart surgery.
|
Acknowledgments |
|---|
This research project was supported in part by the National Heart, Lung, and Blood Institute grants HL-69024 and HL-46716.
|
Footnotes |
|---|
Guest Editor for this article was Robert C. Robbins, MD, Stanford University School of Medicine, Stanford, Calif.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Murry C, Jennings R, Reimer K. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124–1136.
2. Nakamura M, Wang NP, Zhao ZQ, Wilcox JN, Thourani V, Guyton RA, Vinten-Johansen J. Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart. Cardiovasc Res. 2000; 45: 661–670.
3. Valen G. The basic biology of apoptosis and its implications for cardiac function and viability. Ann Thorac Surg. 2003; 75: S656–S660.
4. Kuwahara K, Saito Y, Kishimoto I, Miyamoto Y, Harada M, Ogawa E, Hamanaka I, Kajiyama N, Takahashi N, Izumi T, Kawakami R, Nakao K. Cardiotrophin-1 phosphorylates Akt and Bad, and prolongs cell survival via a PI3K-dependent pathway in cardiac myocytes. J Mol Cell Cardiol. 2000; 32: 1385–1394.[CrossRef][Medline] [Order article via Infotrieve]
5. Goto M, Liu Y, Yang XM, Ardell JL, Cohen MV, Downey JM. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res. 1995; 77: 611–621.
6. Feng J, Yamaguchi N, Foucart S, Chahine R, Lamontagne D, Nadeau R. Transient ischemia inhibits nonexocytotic release of norepinephrine in the rat heart: Is bradykinin involved? Can J Physiol Pharmacol. 1997; 75: 665–670.[CrossRef][Medline] [Order article via Infotrieve]
7. Baxter GF, Ebrahim Z. Role of bradykinin in preconditioning and protection of the ischemic myocardium. Br J Pharmacol. 2002; 135: 843–854.[CrossRef][Medline] [Order article via Infotrieve]
8. Feng J, Rosenkranz ER. Bradykinin pretreatment preconditions the heart by tyrosine kinase mediated pathways. Ann Thorac Surg. 1999; 68: 1567–1572.
9. Feng J, Li HL, Rosenkranz ER. Bradykinin protects the rabbit heart after cardioplegic ischemia via NO-dependent mechanisms. Ann Thorac Surg. 2000; 70: 2119–2124.
10. Feng J, Bianchi C, Li JY, Sellke FW. Bradykinin preconditioning preserves coronary microvascular reactivity during cardioplegia-reperfusion. Ann Thorac Surg. 2005; 79: 911–916.
11. Leesar MA, Stoddard MF, Manchikalapudi S, Bolli R. Bradykinin-induced preconditioning in patients undergoing coronary angioplasty. J Am Coll Cardiol. 1999; 34: 639–650.
12. Wei M, Wang X, Kuukasjarvi P, Laurikka J, Rinne T, Honkonen EL, Tarkka M. Bradykinin preconditioning in coronary artery bypass grafting. Ann Thorac Surg. 2004; 78: 492–497.
13. Schmitt JP, Schrader J, Schunkert H, Birnbaum DE, Aebert H. Role of apoptosis in myocardial stunning after open-heart surgery. Ann Thorac Surg. 2002; 73: 1229–1235.
14. Cooper WA, Duarte IG, Thourani, Nakamura M, Wang NP, Brown WM 3rd, Gott JP, Vinten-Johansen J, Guyton RA. Hypothemic circulatory arrest causes multisystem vascular endothelial dysfunction and apoptosis. Ann Thorac Surg. 2000; 69: 696–703.
15. Fischer UM, Klass O, Stock U, Easo J, Geissler HJ, Fischer JH, Bloch W, Mehlhorn U. Cardioplegia arrest induces apoptosis signal-pathway in myocardial endothelial cells and cardiac myocytes. Eur J Cardio Thorac Surg. 2003; 23: 984–990.
16. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997; 275: 1132–1136.
17. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME. Akt phosphorylation of Bad couples survival signals to the cell-intrinsic death machinery. Cell. 1997; 91: 231–241.[CrossRef][Medline] [Order article via Infotrieve]
18. Hausenloy DJ, Mocanu MM, Yellon DM. Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning. Cardiovsc Res. 2004; 305–312.
19. Bell RM, Yellon DM. Bradykinin limits infarction when administered as an adjunct to reperfusion in mouse heart: the role of PI3kinase, Akt and eNOS. J Mol Cell Cardiol. 2002; 35: 185–193.
20. Li YL, Sato T. Dual Signaling via protein kinase C and phosphatidylinositol 3'-kanse/Akt contributes to bradykinin B2 receptor-induced cardioprotection in guinea pig hearts. J Mol Cell Cardiol. 2001; 33: 2047–2053.[CrossRef][Medline] [Order article via Infotrieve]
21. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14–3-3 not Bcl-X(L). Cell. 1996: 619–628.
22. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605.[CrossRef][Medline] [Order article via Infotrieve]
23. Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, Ma XL. Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the role of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation. 2002; 105: 1497–1452.
24. Brookes PS, Salinas EP, Darley-Usmar K, Eiserich JP, Freeman BA, Darley-Usmar VM, Anderson PG. Concentration-dependent effects of nitric oxide on mitochondrial permeability transition and cytochrome C release. J Biol Chem. 2000; 275: 20474–20479.
25. Linz W, Weimer G, Gahle P, Unger T, Scholkens BA. Contribution of kinins to the cardiovascular action of angiotensin converting enzyme inhibition. Pharmacol Rev. 1995; 47: 25–44.[Abstract]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||