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Exogenous nitric oxide generates ROS and induces cardioprotection: involvement of PKG, mitochondrial K_ATP channels, and ERK

Department of Anesthesiology, University of North Carolina, Chapel Hill, North Carolina 27599

Submitted 16 September 2003 ; accepted in final form 2 December 2003

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
We examined whether cGMP-dependent protein kinase (PKG) and mitochondrial ATP-sensitive potassium (K_ATP) channels are involved in S-nitroso-N-acetyl penicillamine (SNAP)-induced reactive oxygen species (ROS) generation. SNAP significantly increased ROS generation in cardiomyocytes. This increase was suppressed by both 5-hydroxydecanoate (5-HD) and glibenclamide. Direct opening of mitochondrial K_ATP channels with diazoxide led to ROS generation. The increased ROS generation was reversed by N-(2-mercaptopropionyl)glycine (MPG), a scavenger of ROS. Myxothiazol partially suppressed the ROS generation. KT-5823, an inhibitor of PKG, prevented ROS generation, indicating that PKG is required for ROS generation. In addition, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), an activator of PKG, induced ROS generation. The effect of 8-BrcGMP was reversed by either 5-HD or MPG. YC-1, an activator of guanylyl cyclase, also increased ROS production, which was reversed by 5-HD. Neither LY-294002 nor wortmannin, the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), affected SNAP's action. In a whole heart study, SNAP significantly reduced infarct size. The anti-infarct effect of SNAP was abrogated by either MPG or 5-HD. This effect was also blocked by PD-98059, an ERK inhibitor, but not by LY-294002. A Western blotting study showed that SNAP significantly enhanced phosphorylation of ERK, which was reversed by MPG. These results suggest that SNAP-induced ROS generation is mediated by activation of PKG and mitochondrial K_ATP channels and that opening of mitochondrial K_ATP channels is the downstream event of PKG activation. ROS and mitochondrial K_ATP channels participate in the anti-infarct effect of SNAP. Moreover, phosphorylation of ERK is the downstream signaling event of ROS and plays a role in the cardioprotection of SNAP.

reactive oxygen species; guanylyl cyclase; cGMP-dependent protein kinase; mitochondrial ATP-sensitive potassium channel; extracellular signal-regulated kinase; phosphatidylinositol 3-kinase

As to the mechanism for the cardioprotective effect of NO, cGMP (13, 25) and reduced mitochondrial calcium uptake (26) have been suggested to be possible mechanisms of the protection. Recently, reactive oxygen species (ROS) have been reported to be essential in the cardioprotective effect of NO. In late preconditioning, NO induces production of ROS, which in turn activate PKC (3, 21, 29, 30). Similar to findings from the late preconditioning studies, the SNAP-induced anti-infarct effect was abrogated by N-(2-mercaptopropionyl)glycine (MPG), an inhibitor of ROS, suggesting an involvement of ROS in the protection in rabbit heart (14). However, the role of ROS in the protection of NO was based on indirect evidence that the protection was aborted by MPG, without showing direct evidence that comes from measurement of ROS. Moreover, it is intriguing to investigate the mechanism by which NO induces ROS generation, if ROS are indeed involved in NO's action.

Opening of mitochondrial ATP-sensitive potassium (K_ATP) channels has been demonstrated to trigger ischemic preconditioning by generation of ROS (7, 17, 19). In rabbit cardiomyocytes, acetylcholine causes generation of ROS by opening mitochondrial K_ATP channels (17). Interestingly, it was reported that exogenous NO activates mitochondrial K_ATP channels in rabbit cardiomyocytes (10, 27). Therefore, we hypothesized that opening of mitochondrial K_ATP channels by NO is an essential step to cause ROS generation. Furthermore, if mitochondrial K_ATP channel opening is crucial for NO-induced ROS production, it is intriguing to investigate the signaling pathway by which NO causes K_ATP channel opening. Recently, it was reported that exogenous NO and cGMP-dependent protein kinase (PKG) activate K_ATP channels in rabbit ventricular myocytes (10). Thus we determined whether the NO-cGMP-PKG pathway is involved in the mechanism of ROS generation.

In addition, very little information is available about the signal cascade beyond ROS in the protective effect of NO (or SNAP). ERK has been reported to be involved in ischemic preconditioning (8, 22, 28) and antiapoptotic pathways (34). ROS, especially H₂O₂, can induce ERK activation (1, 15). Therefore, if SNAP acts through ROS, it is important to test whether ERK is a component of the signal cascade for the protection of SNAP. Because phosphatidylinositol 3-kinase (PI3-kinase) has been implicated in the mechanism for ischemic preconditioning (31) and ERK activation by ROS (15), PI3-kinase may also contribute to the protection of SNAP.

In the present study, we first observed whether SNAP could induce ROS generation in isolated rat cardiomyocytes and whether PKG and mitochondrial K_ATP channels were involved in SNAP-induced ROS generation. We then observed the roles of ROS and mitochondrial K_ATP channels in the anti-infarct effect of SNAP. Finally, we examined whether ERK and PI3-kinase were involved in SNAP's protection.

	METHODS

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All procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill.

Isolation of adult rat cardiomyocytes. Male Wistar rats weighing 250–350 g were anesthetized with thiobutabarbital sodium (100 mg/kg). A central thoracotomy was performed, and the heart was removed and rapidly mounted on a Langendorff apparatus. The heart was perfused in a nonrecirculating mode with Krebs-Henseleit buffer (37°C) containing (in mM) 118 NaCl, 25 NaHCO₃, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.25 CaCl₂, and 10 glucose for 5 min to wash out blood. The buffer was bubbled with 95% O₂-5% CO₂. The heart was then perfused with calcium-free buffer that contained the above components except for CaCl₂. After 5 min of perfusion, collagenase (type II, Worthington Biochemical) was added to the buffer (0.1%) and the heart was perfused in a recirculating mode for 15 min. The heart was removed from the apparatus, and the ventricles were placed into a beaker containing the calcium-free buffer. The ventricles were agitated in a shaking bath (37°C) at a rate of 50 cycles/min until cells were released. The released cells were suspended in an incubation buffer containing all the components of the calcium-free buffer, 1% bovine serum albumin, 30 mM HEPES, 60 mM taurine, 20 mM creatine, and amino acid supplements at 37°C. Calcium was gradually added to the buffer containing the cells to a final concentration of 1.2 mM. The cells were filtered through nylon mesh and centrifuged briefly. Finally, the cells were suspended in culture medium 199 (no. 7653, Sigma) for 4 h before experiments.

Measurement of intracellular ROS. Cardiomyocytes cultured on a 24-well tissue culture plate were incubated with 20 µM dichlorodihydrofluorescein diacetate (H₂DCFDA) at 37°C for 20 min. After being washed with PBS, the cells were incubated in standard Tyrode solution including (in mM) 140 NaCl, 6 KCl, 1 MgCl₂, 5 HEPES, 1.8 CaCl₂, and 5.8 glucose (pH 7.4) for ROS measurement. Dichlorofluorescein (DCF) fluorescence was measured with a fluorescent plate reader (SpectraMAX GeminiXS, Molecular Probes). The fluorescence was excited at 480 nm and collected at 530 nm. The change in fluorescent intensity for each experimental group was expressed as a percentage of respective control value. Temperature was maintained at 37°C throughout the experiment.

Perfusion of isolated rat heart. Male Wistar rats (280–350 g) were anesthetized with thiobutabarbital sodium (100 mg/kg ip). The hearts were removed rapidly and mounted on a Langendorff apparatus. The hearts were perfused with Krebs-Henseleit buffer containing (in mM) 118.5 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.8 CaCl₂, 24.8 NaHCO₃, 1.2 KH₂PO₄, and 10 glucose, which was heated to 37°C and gassed with 95% O₂-5% CO₂. A latex balloon connected to a pressure transducer was inserted into the left ventricle through the left atrium. The left ventricular pressure and heart rate were continuously recorded with a PowerLab system (ADInstruments, Mountain View, CA). A 5-0 silk suture was placed around the left coronary artery. The ends of the suture were passed through a small piece of soft vinyl tubing to form a snare. All hearts were allowed to stabilize for at least 20 min. Ischemia was induced by pulling the snare and then fixing it by clamping the tubing with a small hemostat. Total coronary artery flow was measured by timed collection of the perfusate dripping from the heart into a graduated cylinder.

Measurement of infarct size. At the end of the experiments, the coronary artery was reoccluded and fluorescent polymer microspheres (2- to 9-µm diameter, Duke Scientific) were infused to demarcate the risk zone as the tissue without fluorescence. The hearts were weighed, frozen, and cut into 1-mm slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in sodium phosphate buffer at 37°C for 20 min. The slices were immersed in 10% formalin to enhance the contrast between stained (viable) and unstained (necrotic) tissue and then squeezed between glass plates spaced exactly 1 mm apart. The myocardium at risk was identified by illuminating the slices with UV light. The infarcted and risk zone regions were traced on a clear acetate sheet and quantified with ImageTool. The areas were converted into volumes by multiplying the areas by slice thickness. Infarct size is expressed as a percentage of the risk zone.

Western blotting analysis of phosphorylated ERK activity. After exposure to SNAP for 30 min, cells were homogenized in ice-cold lysis buffer. Equal amounts of protein were loaded and electrophoresed on 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was probed with the primary antibody that recognizes phospho-p42/p44 MAPK (phospho-ERK). To confirm equal loading, the membrane was stripped and reprobed with an antibody recognizing both phosphorylated and nonphosphorylated forms of p42/p44 MAPK. The primary antibody binding was detected with a secondary anti-rabbit antibody and visualized by the enhanced chemiluminescence method.

Experimental protocols. The agonists and antagonists used in the studies are described in Table 1. In the ROS measurement study, the cells were treated with 20 µM SNAP for 20 min. YC-1 and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) were given for 20 min. The blockers were given 10 min before SNAP and were present throughout the experiment. In the isolated, perfused heart study, all hearts were subjected to a 30-min sustained ischemia followed by 2 h of reperfusion. Infusion of SNAP (10 µM) was started 30 min before ischemia and lasted for 20 min. The blockers MPG (300 µM), 5-hydroxydecanoate (5-HD), PD-98059 (20 µM), and LY-294002 (10 µM) were administered for 30 min, starting 5 min before the infusion of SNAP. To measure ERK activity with Western blotting, myocytes were treated with SNAP (10 and 100 µM) for 30 min and then processed as described above. To observe the relationship between ROS and ERK in the action of SNAP, we performed another series of ERK studies in which MPG (0.5 mM) was given 10 min before the treatment with SNAP.

Statistics. All data are expressed as means ± SE. One-way ANOVA followed by Tukey's test was used to test for differences in baseline hemodynamics, infarct size, fluorescence values, and phospho-ERK activity among groups. A P value of <0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Measurement of ROS production. As shown in Fig. 1, 20 µM SNAP caused a 72.4 ± 7.1% increase in DCF fluorescence, indicating that NO can cause ROS generation in cardiomyocytes. To confirm that NO level is increased by SNAP, we measured NO concentration in cardiomyocytes with 4,5-diaminofluorescein diacetate (DAF-2DA), an indicator of NO. SNAP (20 µM) significantly increased (660% of baseline) DAF-2DA fluorescence, confirming that SNAP actually increased NO level in the cardiomyocytes. Because photooxidation of H₂DCFDA in the solution may affect fluorescence reading, we tested whether SNAP or H₂O₂ can alter fluorescence in the solution without the cells and found that neither SNAP nor H₂O₂ affected fluorescence. The enhanced ROS production induced by SNAP was partially but significantly suppressed by the selective mitochondrial K_ATP channel blocker 5-HD (500 µM; 45 ± 5.4% increase). The nonselective K_ATP channel blocker glibenclamide (50 µM) also significantly prevented the increased ROS generation induced by SNAP. Therefore, it is unlikely that a nonspecific effect of the blockers on the mitochondrial K_ATP channels contributed to the inhibition of ROS production. Here, neither 5-HD nor glibenclamide itself affected ROS generation. These results indicate that opening of mitochondrial K_ATP channels is responsible for SNAP-induced ROS generation. To test whether the opening of mitochondrial K_ATP channels leads to ROS production, we carried out another experiment in which direct opening of mitochondrial K_ATP channels by diazoxide (200 µM) significantly increased DCF fluorescence (148.3 ± 15.3% of control; Fig. 1). Figure 2 shows that the ROS scavenger MPG (1 mM) completely blocked SNAP-induced increase in DCF fluorescence and that MPG alone did not affect fluorescence intensity. Thus it is reasonable to conclude that the increased DCF signal induced by SNAP was related to ROS generation. Because neither 5-HD nor glibenclamide completely reversed SNAP's effect, we examined whether SNAP-induced ROS generation was partially due to the direct effect of NO on mitochondrial electron transport chain (ETC). Figure 3 shows that the inhibitor of mitochondrial electron transport myxothiazol partially suppressed SNAP-induced ROS production, implying that the direct inhibition of NO may partially contribute to ROS generation. To test whether SNAP-induced ROS generation was dependent on PKG activation, we applied the PKG inhibitor KT-5823. Figure 4 shows that KT-5823 (1 µM) blocked the increase in ROS generation induced by SNAP, suggesting that activation of PKG is required for ROS generation. The involvement of PKG in ROS generation was also confirmed by another experiment in which the PKG activator 8-BrcGMP (500 µM) significantly increased ROS generation (116.6 ± 2.9% of control; Fig. 5). 5-HD (500 µM) completely blocked-8-BrcGMP's action (Fig. 5), suggesting that PKG produced ROS via activation of mitochondrial K_ATP channels. MPG (1 mM) also prevented 8-BrcGMP-induced ROS generation-(98.2 ± 3.7% of control). Because cGMP is an upstream signal of PKG activation, we then examined whether activation of guanylyl cyclase (GC) with YC-1 could produce ROS. As shown in Fig. 6, YC-1 (30 µM) significantly increased ROS generation, which was reversed by 5-HD (500 µM). Finally, the increased ROS generation induced by SNAP was not affected by either LY-294002 (10 µM) or wortmannin (100nM), indicating that PI3-kinase is not involved in the action of SNAP on ROS generation (Fig. 7).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Effects of S-nitroso-N-acetyl penicillamine (SNAP) on reactive oxygen species (ROS) generation in isolated rat cardiomyocytes. SNAP (20 µM) induced marked ROS generation. SNAP-induced increased ROS generation was partially but significantly reduced by either 5-hydroxydecanoate (5-HD; 500 µM) or glibenclamide (Gli; 50 µM). Neither 5-HD nor glibenclamide itself had a significant effect on ROS generation. Diazoxide (200 µM) increased ROS generation. *P < 0.05 vs. SNAP.

View larger version (14K): [in this window] [in a new window]   Fig. 2. SNAP-induced ROS generation was blocked by the ROS scavenger N-(2-mercaptopropionyl)glycine (MPG; 1 mM). MPG itself did not affect ROS generation. *P < 0.05 vs. SNAP.

View larger version (13K): [in this window] [in a new window]   Fig. 3. SNAP-induced ROS generation was partially inhibited by the mitochondrial electron transport inhibitor myxothiazol (Myxo; 2 µM). *P < 0.05 vs. SNAP.

View larger version (14K): [in this window] [in a new window]   Fig. 4. The cGMP-dependent protein kinase (PKG) inhibitor KT-5823 (KT; 1 µM) blocked SNAP-induced ROS generation. KT-5823 itself did not affect ROS generation. *P < 0.05 vs. SNAP.

View larger version (16K): [in this window] [in a new window]   Fig. 5. The PKG activator 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP; 500 µM) induced ROS generation. This effect was completely reversed by either 5-HD (500 µM) or MPG (1 mM). *P < 0.05 vs. control.

View larger version (13K): [in this window] [in a new window]   Fig. 6. The guanylyl cyclase activator YC-1 (30 µM) significantly increased ROS generation. 5-HD (500 µM) abolished the YC-1-induced ROS generation. *P < 0.05 vs. control and YC-1 + 5-HD.

View larger version (17K): [in this window] [in a new window]   Fig. 7. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors LY-294002 (LY; 10 µM) and wortmannin (Wort; 100 nM) did not affect SNAP-induced ROS generation. LY-294002 itself has no influence on ROS production.

Infarct size study. Baseline heart rate, left ventricular developed pressure, and coronary flow were not different among any of the experimental groups (Table 2). There were no significant differences in body weight, heart weight, or the size of the risk zone among the groups (Table 3). Infarct size in the control hearts was 36.8 ± 3.6% of the risk zone (Fig. 8). Pretreatment with 10 µM SNAP significantly reduced infarct size to 14.8 ± 1.5% of the risk zone (P < 0.05). The anti-infarct effect of SNAP was assessed in hearts treated with MPG, a ROS scavenger. As shown in Fig. 8, the protective effect of SNAP was completely blocked by 300 µM MPG (35.1 ± 3.6% infarction of the risk zone), suggesting an involvement of ROS in the protection of SNAP in isolated rat hearts. MPG alone did not affect infarct size. The anti-infarct effect of SNAP was also abolished by closing mitochondrial K_ATP channels with 500 µM 5-HD (34.1 ± 2.0% infarction of the risk zone), indicating that opening of mitochondrial K_ATP channels is needed for cardioprotection by SNAP. The ERK inhibitor PD-98059 (20µM) was used to test whether the ERK pathway is involved in the protection. The anti-infarct effect of SNAP was totally reversed by 20 µM PD-98059 (31.7 ± 1.4% infarction of the risk zone), as shown in Fig. 9. PD-98059 alone did not affect infarct size (33.1 ± 3.0% infarction of the risk zone). The PI3-kinase inhibitor LY-294002 (10 µM) did not alter the protective effect of SNAP (13.9 ± 1.4% infarction of the risk zone). These results suggest that SNAP protects the hearts from ischemia-reperfusion injury through ROS and ERK pathways but not through PI3-kinase.

View larger version (11K): [in this window] [in a new window]   Fig. 8. Effect of SNAP on myocardial infarct size in isolated rat hearts. SNAP (10 µM) significantly reduced infarct size compared with control. The anti-infarct effect of SNAP was completely blocked by MPG (300 µM). MPG itself did not affect infarct size. The protective effect of SNAP was also reversed by the mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-HD (500 µM). , Individual infarct sizes; , group means. *P < 0.05 vs. control.

View larger version (11K): [in this window] [in a new window]   Fig. 9. Effects of SNAP, PD-98059, and LY-294002 on myocardial infarct size in isolated rat hearts. The protective effect of SNAP was blocked by PD-98059 (20 µM) but not by LY-294002 (10 µM). PD-98059 alone did not affect infarct size. , Individual infarct sizes; , group means. *P < 0.05 vs. control.

Western blotting analysis of ERK activity. The cardiomyocytes were incubated with SNAP (10 and 100 µM) for 30 min. As shown in Fig. 10, treatment with 10 µM SNAP resulted in a significant increase in activities in phosphorylated ERKs (134.2 ± 13.5% of the control in ERK1 and 134.9 ± 12.8% in ERK2; P < 0.05 vs. control). ERK1 and -2 activities were further increased by 100 µM SNAP to 182.3 ± 22.4% and 187.7 ± 23.1% of control, respectively. In another series of experiments, the increased phospho-ERK activities were reversed by 1 mM MPG, indicating that SNAP activated ERK through ROS generation (Fig. 11). These results further suggest that ERK is an important element of the signal pathway leading to cardioprotection by SNAP.

View larger version (36K): [in this window] [in a new window]   Fig. 10. Western blotting analysis of phospho-ERK (p-ERK) activity in rat cardiomyocytes. SNAP significantly increased both ERK1 and ERK2 activities. *P < 0.05 vs. control.

View larger version (33K): [in this window] [in a new window]   Fig. 11. Western blotting analysis of phospho-ERK activity in rat cardiomyocytes. Effect of SNAP (10 µM) on phospho-ERK activities was reversed by MPG (500 µM). *P < 0.05 vs. control.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Although the detailed mechanism for the protection of NO is not clearly elucidated, ROS have been proposed to be involved in the action. Takano et al. (30) reported that late preconditioning induced by NO donors was blocked by treatment with MPG, indicating an involvement of ROS in the phenomenon. Similarly, Nakano et al. (14) demonstrated that SNAP-induced early preconditioning was abrogated by MPG. In the present study, we have shown direct evidence that SNAP caused ROS generation in isolated rat cardiomyocytes. To exclude the possibility that the SNAP-induced increased DCF fluorescence signal was caused by factors other than ROS, we tested whether MPG, a scavenger of ROS, could affect the signal. The finding that MPG completely reversed SNAP-induced ROS generation would strongly indicate that the SNAP-induced increase in DCF signal was related to ROS generation. Our infarct size study also confirmed that MPG-sensitive ROS were implicated in the protective effect of SNAP in isolated rat hearts. As to the mechanism for generation of ROS by exogenous NO, Takano et al. (30) and Ping et al. (21) proposed that NO forms peroxynitrite (ONOO^–), which then decomposes to generate hydroxyl radical (·OH) or ·OH-like radicals. In the current study, both 5-HD and glibenclamide suppressed the increase in ROS generation by SNAP, indicating that SNAP caused ROS generation through opening mitochondrial K_ATP channels. The importance of mitochondrial K_ATP channel opening was further evidenced by the finding that the anti-infarct effect of SNAP was reversed by 5-HD. Mitochondria have been reported as the major source of ROS generation in hypoxic preconditioning (32). In ischemic preconditioning, opening of mitochondrial K_ATP channels causes ROS generation, which is a crucial step to trigger preconditioning (16). Recently, Lebuffe et al. (12) reported that exogenous NO was cardioprotective in chick myocytes and that the protection of NO was blocked by 5-HD, a K_ATP channel blocker, or MPG. Involvement of K_ATP channels in exogenous NO action was further demonstrated by Bell et al. (2). Therefore, it is reasonable to conclude that SNAP increases ROS generation via opening of K_ATP channels in cardiomyocytes and that ROS are crucial for the protective effect of SNAP. However, it should be mentioned that SNAP-induced ROS generation was partially but not completely suppressed by 5-HD or glibenclamide, suggesting that something other than mitochondrial K_ATP channel opening may contribute to ROS generation. NO has been reported to generate ROS by inhibiting mitochondrial ETC (23). In this study, we have found that the inhibitor of mitochondrial electron transport myxothiazol partially abolished SNAP-induced ROS generation. Thus it is likely that SNAP-induced ROS generation is partially attributable for the direct inhibitory effect of NO on mitochondrial ETC.

In the present study, we demonstrated that SNAP-induced ROS generation was blocked by the PKG inhibitor KT-5823, implying that PKG is needed for SNAP-induced ROS generation. To further confirm the role of PKG in ROS generation, we examined whether direct activation of PKG with 8-BrcGMP could result in ROS production. Our data clearly show that 8-BrcGMP significantly increases ROS generation, supporting the concept that SNAP induces ROS generation via activation of PKG. Furthermore, 8-BrcGMP-induced ROS generation was also reversed by 5-HD, confirming that PKG induces ROS generation through activation of mitochondrial K_ATP channels. Because NO activates PKG via a GC-cGMP pathway, we then tested whether activation of GC could duplicate PKG's effect. Similar to PKG, activation of GC with YC-1 significantly increased ROS generation and this effect was dependent on mitochondrial K_ATP channel opening. Thus our data would suggest a unique signal pathway leading to ROS generation: NO-GC-cGMP-PKG-mitochondrial K_ATP channel opening-ROS production. Similarly, Han et al. (10) reported that the cGMP-PKG pathway is involved in phosphorylation of K_ATP channels in rabbit cardiomyocytes.

The present investigation identifies ERK as a likely downstream target of ROS in SNAP-induced protection. ERK has been proposed to participate in preconditioning (8, 28) and antiapoptosis action (34). ROS are known to activate ERK (9, 15). Aikawa et al. (1) reported that H₂O₂ rapidly and transiently activated ERK in rat cardiomyocytes. In their study, 0.01 mM (10 µM) H₂O₂ for 10 min significantly increased ERK activity. The dose and duration of H₂O₂ treatment were quite similar to those of another study in which 15 µM H₂O₂ for 10 min mimicked the preconditioning effect in chick cardiomyocytes (32). In the current study, the anti-infarct effect of SNAP was blocked by the MEK inhibitor PD-98059 and ERK was phosphorylated to its active catalytic form by treatment with SNAP. Furthermore, the enhanced phosphorylation of ERK by SNAP was blocked by MPG, suggesting that ROS serve as the upstream signal of ERK in SNAP's action. Therefore, we conclude that the increased ERK activity induced by ROS is responsible for the cardioprotective effect of SNAP.

PI3-kinase is involved in cell survival pathways and in metabolic control. Tong et al. (31) reported that the preconditioning effect was blocked by PI3-kinase inhibitors and that PI3-kinase is the upstream signal of PKC in the mechanism of preconditioning. Recently, Oldenburg et al. (18) reported that PI3-kinase is involved in the effect of acetylcholine on ROS production and proposed that a PI3-kinase step exists between the surface receptor and mitochondrial K_ATP channels. If their hypothesis is correct, PI3-kinase should be an upstream trigger of ROS because ROS production is dependent on the opening of K_ATP channels. In the present study, both LY-294002 and wortmannin affected neither the ROS-generating nor anti-infarct effects of SNAP, suggesting that SNAP's protection does not require PI3-kinase activation.

Finally, we summarize the signal pathway of SNAP's action in Fig. 12. SNAP can cause ROS generation, and ROS play a crucial role in the protective effect of SNAP. SNAP-induced ROS generation is dependent on opening of mitochondrial K_ATP channels, activation of PKG, and the direct inhibition of mitochondrial ETC. Although PI3-kinase is not involved, ERK activation appears to be the downstream signal of ROS in SNAP's action.

View larger version (13K): [in this window] [in a new window]   Fig. 12. Signal transduction pathway leading to the cardioprotection effect of SNAP. NO, nitric oxide; mKATP, mitochondrial KATP; ETC, electron transport chain; +, activation; –, inhibition.

	ACKNOWLEDGMENTS

 
GRANTS

This work was partially supported by American Heart Association Beginning Grant-In-Aid 0365534U (to Z. Xu).

	FOOTNOTES

 

Address for reprint requests and other correspondence: Z. Xu, Dept. of Anesthesiology, CB#7010, Univ. of North Carolina, Chapel Hill, NC 27599-7010 (E-mail: zxu{at}aims.unc.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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