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Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning

Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Submitted 26 February 2004 ; accepted in final form 1 May 2004

	ABSTRACT

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The opening of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels has a significant role in delayed ischemic preconditioning, and nitric oxide (NO) is a well-known trigger for its activation. However, the source of NO remains unknown. Phosphorylation of endothelial NO synthase (eNOS) increases NO production and reduces apoptosis through the Akt signaling pathway. To elucidate the Akt signaling pathway involved in the opening and antiapoptotic effect of mitoK_ATP channel during delayed pharmacological preconditioning, the mitoK_ATP channel opener diazoxide (DE, 7 µg/kg ip) alone or DE plus N-nitro-L-arginine methyl ester (L-NAME, 30 µg/kg iv), an inhibitor of NOS, or wortmannin (WTN, 15 µg/kg iv), an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), was administered to wild-type (WT) or eNOS^–/– mice during DE treatment. Twenty-four hours later, hearts were isolated and subjected to 40 min ischemia and 30 min reperfusion (I/R). The effect of DE and other interventions on hemodynamic, terminal dUTP nick-end labeling staining and biochemical changes during I/R was assessed in mouse hearts. Treatment with DE resulted in a 2.2-fold increase in phosphorylation of Akt and a significant increase in eNOS and inducible NOS (iNOS) proteins. Akt is upstream of NOS and the mitoK_ATP channel as simultaneous pretreatment of WTN with DE abolished phosphorylation of Akt, which was not affected by L-NAME and 5-hydroxydecanoate. In hearts treated with DE, cardiac function was significantly improved after I/R, and apoptosis was also significantly decreased. WTN abolished the antiapoptotic effect of DE. Similarly, S-methylisothiourea, a specific iNOS inhibitor, when given to eNOS^–/– mice that were pretreated with DE completely abolished the beneficial effects of DE on reduction of apoptotic death. DE was partially effective in eNOS^–/– mice against the ischemic injury. It is concluded that DE activates Akt through the PI3 kinase signaling pathway and iNOS and eNOS is downstream of Akt.

diazoxide; apoptosis

Apoptosis plays a key role in ischemia-reperfusion (I/R) injury (3). Recently, it has been demonstrated that DE plays an important regulatory role in apoptotic cell death (20). NO is also a key trigger of mitoK_ATP channel activation (24) and is involved in the regulation of apoptosis (10). High concentrations of NO produced from iNOS induce apoptosis, whereas low concentrations of NO produced from eNOS or pharmacological concentrations of exogenous NO released by NO donors reduce apoptosis (16). Similarly, NO, which is generated upon ischemic stress, triggers the opening of mitoK_ATP channel leading to cardioprotection (24). A recent report showed that phosphatidylinositol 3'-kinase (PI3 kinase)-Akt played a role in eNOS phosphorylation and NO production (9), which can also be induced by DE (2). However, the relationship between the antiapoptotic effect of NO and DE-mediated stimulatory effects of NO are still unclear.

To study the contribution of endogenous NO to I/R damage without interference by pharmacological nonspecific inhibitors, eNOS knockout (eNOS^–/–) mice were used. The use of genetically manipulated mice is important due to nonspecific effects of pharmacological inhibitors currently in use, thus making less complex for the interpretation of data. The knockout animal model is therefore important to determine isoform-specific function of eNOS. To our knowledge, the present study is the first to utilize eNOS^–/– mice to investigate the effect of both DE and NO in regulating apoptosis and late cardiac protection.

	MATERIALS AND METHODS

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Adult male eNOS^–/– (B6.129P2-Nos3^tm1Unc) and wild-type (WT) mice weighing 25–30 g were obtained from Jackson Laboratory. The University of Cincinnati Animal Care and Use Committee approved the use of mice in these experiments.

Heart preparation. Mice weighing 25–30 g were anesthetized with pentobarbital sodium (40 mg/kg ip) and heparinized (5,000 U/kg) to protect the heart against microthrombi. The chest was opened at the sternum, and the heart was quickly removed and was then cannulated with a 20-gauge phalanged stainless steel cannula. Hearts were retrogradely perfused through the aorta in a noncirculating Langendorff apparatus with Krebs-Henseleit buffer, which consisted of (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.5 CaCl₂, 25 NaHCO₃, 0.5 Na-EDTA, and 11 glucose. The buffer was saturated with 95% O₂-5% CO₂ (pH 7.4, 37°C) for 50 min. Hearts were perfused at a constant pressure of 80 mmHg. A home-made water-filled balloon was inserted into the left ventricle and was adjusted to a left ventricular (LV) end-diastolic pressure (LVEDP) of 5–8 mmHg during initial equilibration. Thereafter, the balloon volume was not changed. The distal end of the catheter was connected to a Digi-Med Heart Performance Analyzer- (model 210, version 1.01, Micro-Med) via a pressure transducer (Case; Lakewood, CO). Hearts were paced at 350 beats/min except during ischemia. Pacing was reinitiated after 3 min of reperfusion in all groups. The index of myocardial function was determined as previously described (23). The current study conforms to the guidelines established by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985) Guide for the Care and Use of Laboratory Animals.

Experimental protocol. The study consisted of seven groups (Fig. 1). After a 25-min equilibration period, hearts were subjected to 40 min of no-flow normothermic global ischemia and 30 min of reperfusion.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Experimental groups and protocol. I/R, ischemia-reperfusion; DE, diazoxide; eNOS–/–, endothelial nitric oxide (NO) synthase knockout mice; L-NAME, N-nitro-L-arginine methyl ester; SMT, S-methylisothiourea; WTN, wortmannin; WT, wild-type mice.

Terminal dUTP nick end-labeling staining for the determination of myocardial apoptosis. Terminal dUTP nick-end labeling (TUNEL) assay was performed in deparaffinized 5-µm for thick sections using a MEBSTAIN Apoptosis Kit II (Medical and Biological Laboratories). Sections from the mid left ventricle were stained for TUNEL, as previously described (20). Sections were then stained with 4,6-diamidino-2-phenylindole to visualize nuclei and photographed with an Olympus BX41 microscope (Olympus America; Melville, NY) equipped with a digital camera.

Western blotting for iNOS and eNOS. Hearts were weighed and homogenized at 4°C with six bursts of 15 s each with a Polytron PT 20 in 1 ml RIPA buffer (1x PBS, 1% Nonidat P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µmol/l PMSF, 30 µl/ml aprotinin, and 100 µmol/l sodium orthovanadate). Subsequently, the samples were centrifuged at 14,000 rpm for 10 min. The pellet was discarded, and the protein in the supernatant was determined. Standard SDS gel electrophoresis was performed with 20 µg of protein loaded in each well of a 12% polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane for 2 h at 100 V. The membrane was blocked with 5% no-fat dry milk in 1x Tris-buffered saline containing 0.1% Tween 20 (0.1% TBST) for 1 h. The membrane was incubated with the primary iNOS and eNOS antibody (Santa Cruz, CA) at 1:1,000 dilution for 1 h at room temperature. After being washed with 0.1% TBST, the membrane was incubated with an anti-rabbit horseradish peroxidase-linked antibody (dilution, 1:500) for 1 h. The membranes were developed with enhanced chemiluminescence (Amersham) and exposed to X-ray film for the appropriate time.

Western blotting for Akt. Protein samples were mixed with equal volume of sample buffer to measure Akt kinase activity with the PhosphoPlus Akt [Ser-473] antibody kit (Cell Signaling Technology) as described previously (6). The kit included a phosphospecific antibody that recognizes Akt only when phosphorylated at Ser-473, a control Akt antibody (phosphorylation-state independent), protein controls, and protein markers for Western blot analyses, plus reagents and protocols for the rapid analysis of Akt phosphorylation.

Statistical analysis. All values are expressed as means ± SE. Group comparisons were analyzed by one-way ANOVA (Statview 4.0) followed by a Bonferroni-Dunn test. A value of P < 0.05 was considered to be statistically significant.

	RESULTS

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Preischemic cardiac hemodynamic parameters. Preischemic baseline values of LV function in various treatment groups are summarized in Table 1. The mean values of LV developed pressure (LVDP), LVEDP, and coronary flow (CF) were not significantly different among the groups. There was also no significant difference in the body and heart weights among the groups.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Effect of various interventions on left ventricular (LV) developed pressure (LVDP, A), LV end-diastolic pressure (LVEDP, B), coronary flow (CF, C), and lactate dehydrogenase (LDH, D). I/R CON, ischemic-reperfusion control. Values are means ± SE; n = 6 for each group. *P < 0.05 vs. ischemic control; P < 0.05 vs. DE group and CON.

Effect of DE on ischemic injury in eNOS^–/– mice. After pretreatment with DE, LVDP was significantly increased, whereas LVEDP was markedly reduced compared with ischemic control group. However, LVEDP did not rise significantly compared with the control ischemic group. No important changes in CF and LDH retention were observed in the eNOS gene-knockout mice compared with WT mice after I/R.

Pretreatment of eNOS^–/– mice with S-methylisothiourea (SMT), a selective iNOS inhibitor, completely abolished the protective effect of DE, as indicated by decreased LVDP and CF and increased LVEDP and LDH release.

Similarly, pretreatment with N-nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, or Wortmannin (WTM), an inhibitor of PI3-kinase, blocked the cardiac protective effect of DE, as indicated by decreased LVDP and CF and increased LVEDP and LDH release (Fig. 2D).

Effect of DE on eNOS and iNOS upregulation. To determine whether DE upregulates eNOS and iNOS, Western blot analysis was performed. As shown in Fig. 3, treatment with DE resulted in a time-dependent increase in eNOS and iNOS proteins reaching a peak at 12 h and leveling off after 24 h (Fig. 3, A and B). However, eNOS activity was inhibited when hearts were pretreated with a general inhibitor of L-NAME, and eNOS synthesis was also prevented in eNOS^–/– mice with SMT, a specific inhibitor of iNOS, but no effect on eNOS was observed in WT mice (Fig. 3C).

View larger version (35K): [in this window] [in a new window]   Fig. 3. Effect of DE on expression of inducible nitric oxide (NO) synthase (iNOS) and eNOS. Top: DE-treated mice demonstrate time-dependent enhanced expression of iNOS (A) or eNOS (B). Bottom: quantitative densitometric data (n = 6 for each group). *P < 0.05 vs. saline group. *P < 0.01 vs. 24-h groups. C: effect of DE on eNOS expression in various treatment groups. Top: representative Western blot analysis showing the 130-kDa eNOS protein band. Bottom: quantitative measurement of eNOS levels. Data are means ± SE. *P < 0.05 vs. control.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Effect of DE on phosphorylation and total Akt in various treatment groups. A, top: representative Western blots showing 60-kDa Akt protein bands in various treatment groups; bottom: quantitative measurement of phosphorylated Akt. Data are means ± SE. n = 6 for each group. *P < 0.05 vs. control. B, top: representative Western blots showing total Akt protein bands in various treatment groups; bottom: quantitative measurement of total Akt levels. Data are means ± SE. No significant differences were observed in all groups, P > 0.05 vs. control. C, top: time-dependent Akt activation by DE. Bottom: time-dependent phosphorylation of Akt. Data are means ± SE. n = 6 for each group. *P < 0.05 vs. control. D, top: representative Western blots showing 60-kDa Akt protein bands in various treatment groups; bottom: quantitative measurement of phosphorylated Akt. Data are means ± SE. n = 6 for each group. *P < 0.05 vs. control.

Antiapoptotic effect of DE through NO signaling pathway. Because NO is an important trigger for the opening of mito K_ATP channels in DE-induced preconditioning, the source of NO production is not clear from previous studies. To ascertain whether eNOS was involved in the PI3 kinase-Akt-NO signaling pathway in the reduction of apoptosis, adult eNOS^–/– mice were used to avoid further nonspecific effects of pharmacological inhibitors. In WT mice, an increased percentage of cells underwent apoptosis (Fig. 5), although some studies report that the severity of an apoptosis is dependent on the period of reperfusion (3). Administration of DE markedly reduced TUNEL-positive cells. However, the percentage of apoptotic cells increased significantly in both eNOS^–/– mice and mice treated with SMT, a specific inhibitor of iNOS, suggesting that eNOS and iNOS are the major source of NO during activation of Akt by DE. The antiapoptotic effect of DE was also abolished in the WT mice on pretreatment with L-NAME or WTN. These data provided further support to the concept that the PI3 kinase-Akt-NO pathway plays a critical role in the antiapoptotic effect of DE.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Representative photomicrographs of terminal dUTP nick-end labeling (TUNEL)-positive nuclei in various treatment groups. A: normal myocyte nuclei are shown with 4,6-diamidino-2-phenylindole (DAPI) (yellow arrow). B and C: apoptotic nuclei have been shown in yellow. D: green fluoresence shows TUNEL-positive nuclei (white arrow). The number of TUNEL-positive myocytes was siginificantly decreased by DE treatment. A: normal control; B: ischemic control; C: DE; D: DE + WTN with FITC filter; E: same section as in D except staining with DAPI; F: same section as in D and E except double stained for -sarcomeric actin. Apoptosis in DE + SMT + eNOS–/– and DE + L-NAME groups is similar to ischemic control except DE + SMT and DE + eNOS–/– groups show less apoptosis than ischemic control but more than DE group (data not shown). Bottom: percentage of TUNEL-positive nuclei in different treatment groups. *P < 0.05 vs. I/R control; *#P < 0.05 vs. DE.

	DISCUSSION

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Apoptosis. This study demonstrates that Akt phosphorylation by DE is upstream of NOS, and both NOS and Akt phosphorylation are important in preventing cell death in the ischemic myocardium. However, despite Akt phosphorylation in eNOS^–/– mice, partial protection by DE suggests that eNOS deletion is compensated by iNOS upregulation. In addition, administration of DE in eNOS^–/– with SMT, a selective iNOS inhibitor, significantly diminished postischemic cardiac functional recovery, but without SMT, only partial protection was observed, suggesting that iNOS may be involved in DE-induced late preconditioning through a different signaling pathway from eNOS. It is not unreasonable to conclude that iNOS has more impact on functional recovery, whereas eNOS mediates apoptosis reduction. It became more clear when SMT was given to DE + eNOS^–/– mice, functional recovery was further reduced accompanied by an increase in apoptosis. eNOS is responsible for maintaining systemic blood pressure, vascular remodeling, and angiogenesis and is phosphorylated in response to various cellular stimuli (18). It has recently been reported that eNOS is a novel substrate for Akt and that phosphorylation of eNOS by Akt results in calcium-independent NO production (4). NO could modulate mitoK_ATP channels and provide cardioprotection (11). Yang et al. (27) demonstrated that eNOS is expressed mainly in the coronary vasculature and endothelium, and it may function as a tissue protective factor against damage to the heart, which is consistent with our studies that DE upregulated eNOS proteins leading to antiapopototic effect through a PI3 kinase-Akt pathway. As mentioned above, treatment with DE significantly reduced myocardial apoptosis, as evidenced by reduced TUNEL-positive myocyte nuclei. Surprisingly, administration of DE to eNOS^–/– mice or to WT mice together with SMT significantly reduced apoptosis. However, L-NAME was totally ineffective in preventing apoptosis, despite Akt activation. This suggests that Akt is upstream of NOS (Fig. 6). Gao et al. reported (4) that treatment with L-NAME significantly reduced the antiapoptotic effect of insulin, whereas L-NAME alone only slightly increased the number of TUNEL-positive cells, suggesting that the basal production of NO during I/R is not sufficient to exert significant antiapoptotic effect in the myocardial tissue. Taken together, these results demonstrated that DE, an important agonist of mitoK_ATP channel opener, exerts its antiapoptotic effect through the PI3 kinase-Akt pathway and that the phosphorylation of eNOS with subsequent NO production is an important downstream effector that contributes significantly to the cardioprotective effect of DE against myocardial I/R injury (Fig. 6).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Proposed pathway of delayed cardioprotection by DE. PI3 kinase; phosphatidylinositol 3'-kinase; PKC, protein kinase C; NF-B, nuclear factor- B; mitoKATP, mitochondrial ATP-sensitive K+ channels; 5-HD, 5-hydroxydecanoate.

In summary, we have demonstrated that Akt and eNOS proteins are rapidly upregulated by DE and remain upregulated for at least 24 h. Thus NO and Akt could be the basis of protection in early and delayed preconditioning through PI3-kinase-dependent pathways.

	GRANTS

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This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-23597 and HL-70062 (to M. Ashraf).

	ACKNOWLEDGMENTS

 
The authors thank Boyu Wang for technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Ashraf, Dept. of Pathology and Laboratory Medicine, Univ. of Cincinnati Medical Center, 231 Albert Sabin Way, Cincinnati, OH 45267-0529 (E-mail: Muhammad.Ashraf{at}UC.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.

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This Article Abstract Full Text (PDF) All Versions of this Article: 287/3/H1125    most recent 00183.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (22) Citing Articles via Google Scholar Google Scholar Articles by Wang, Y. Articles by Ashraf, M. Search for Related Content PubMed PubMed Citation Articles by Wang, Y. Articles by Ashraf, M.