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In the rabbit heart, bradykinin and ACh trigger preconditioning by a mechanism involving ATP-sensitive potassium channel-dependent production of reactive oxygen species (ROS). Recent evidence indicates that the pathway by which bradykinin causes ROS generation includes nitric oxide synthase (NOS) and protein kinase G (PKG). On the other hand, Akt was shown to be essential for ACh to generate ROS. This study determines whether these two G-coupled receptor agonists indeed have similar signaling targets, i.e., whether Akt is involved in bradykinin's pathway and whether NOS is involved in ACh's pathway. Isolated adult rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 [micro]M) caused a 51.4 [+ or -] 14.8% and 39.8 [+ or -] 11.7% increase, respectively, in ROS production (P < 0.005). Coincubation of either agonist with Akt inhibitor (20 [micro]M) or infection of cells with an adenovirus containing dominant negative Akt abolished this increase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP, 1 [micro]M) also increased the ROS signal by 40.8 [+ or -] 15.7%, but this increase was unaffected by Akt inhibitor (39.0 [+ or -] 6.4%), implying that Akt is upstream of NOS. ACh-induced ROS production could be abolished by either of the NOS inhibitors [N.sup.[omega]]-monomethyl-L-arginine monoacetate (100 [micro]M) and L-[N.sup.5]-(1-iminoethyl)ornithine hydrochloride (L-NIO, 5 [micro]M). L-NIO also blocked the anti-infarct effect of ACh (550 [micro]M) in isolated rabbit hearts exposed to 30 min of regional ischemia. We conclude that both bradykinin and ACh trigger ROS generation by sequentially activating Akt and NOS. nitric oxide; [N.sup.[omega]]-monomethyl-L-arginine monoacetate; L-[N.sup.5]-(1-iminoethyl)omithine; S-nitroso-N-acetyl penicillamine

Exogenous nitric oxide (NO) triggers a preconditioning-like effect in heart via a pathway that is dependent on reactive oxygen species. This study examined the signaling pathway by which the NO donor S-nitroso-N-acetylpeniciilamine (SNAP, 2 [micro]M) triggers its anti-infarct effect. Isolated rabbit hearts experienced 30 min of regional ischemia and 120 min of subsequent reperfusion, infarct size was determined by triphenyltetrazolium chloride staining, infarct size was reduced from 30.5 [+ or -] 3.0% of the risk zone in control hearts to 10.2 [+ or -] 2.0% in SNAP-treated hearts. Bracketing the SNAP infusion with either the guanylyl cyclase blocker 1H-[1,2,4]oxadiazoie[4,3-a]quinoxalin-1-one (2 [micro]M) or the mitochondrial ATP-sensitive [K.sup.+] (mito[K.sub.ATP]) channel blocker 5-hydroxydecanoate (200 [micro]M) completely blocked the infarct-sparing effect of SNAP (34.3 [+ or -] 3.8 and 32.2 [+ or -] 1.6% infarction, respectively). Pretreatment of hearts with 8-(4-chlorophenylthio)-guanosine 3',5'cyclic monophosphate (10 [micro]M), which is a cell-permeable cGMP analog that activates protein kinase G, mimicked the preconditioning effect of SNAP by reducing infarct size to 7.5 [+ or -] 1.1% of the risk zone. This salutary effect was abolished by either the free radical scavenger N-(2-mercaptopropionyl)glyciue (1 mM) or 5-hydroxydecanoate (100 [micro]M; 28.9 [+ or -] 2.7 and 33.6 [+ or -] 5.0% infarction of the risk zone, respectively). To confirm these functional data and the effect of SNAP on the guanylyl cyclase-protein kinase G signaling pathway, cGMP levels were measured. SNAP increased the level from 0.18 [+ or -] 0.04 to 0.61 [+ or -] 0.14 pmol/mg of protein (P < 0.05). These data suggest that exogenous NO triggers the preconditioning effect by initiating a cascade of events including stimulation of guanylyl cyclase to make cGMP, activation of protein kinase G, opening of mito[K.sub.ATV] channels, and, finally, production of reactive oxygen species. mitochondrial ATP-sensitive potassium channel; protein kinase G; S-nitroso-N-acetylpenicillamine; guanylyl cyclase

Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 [+ or -] 16% increase in ROS generation (P < 0.001). Coincubation with either the BK [B.sub.2]-receptor blocker HOE-140 (5 [micro]M) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive [K.sup.+] (mito[K.sub.ATP]) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME, 200 [micro]M), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with l-H-[l,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 [micro]M) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mito[K.sub.ATP], channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 [micro]M) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 [micro]M) increased ROS generation (39 [+ or -] 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 [micro]M) increased ROS by 34 [+ or -] 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 [+ or -] 3.0 of the risk zone in control hearts to 11.9 [+ or -] 1.4% (P < 0.01). This protection was aborted by either 200 [micro]M L-NAME or 2 [micro]M ODQ (35.4 [+ or -] 5.7 and 30.4 [+ or -] 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mito[K.sub.ATP]. Subsequent release of ROS triggers cardioprotection. mitochondrial ATP-sensitive potassium channel; nitric oxide; preconditioning; reactive oxygen species; protein kinase G

Adenosine and acetylcholine (ACh) trigger preconditioning by different signaling pathways. The involvement of phosphatidylinositol 3-kinase (PI3-kinase), a protein tyrosine kinase, and Src family tyrosine kinase in preconditioning was evaluated in isolated rabbit hearts. Either wortmannin (PI3-kinase blocker), genistein (tyrosine kinase blocker), lavendustin A (tyrosine kinase blocker), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2; Src family tyrosine kinase blocker) was given for 15 min to bracket a 5-min infusion of either adenosine or ACh (trigger phase). The hearts then underwent 30 min of regional ischemia. Infarct size for ACh alone was 9.3 [+ or -] 3.5% of the risk zone versus 34.3 [+ or -] 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30rain ischemic period (mediator phase), ACh-induced protection was not affected (7.4 [+ or -] 2.1% and 9.7 [+ or -] 1.7% infarction, respectively). Adenosine-triggered protection was not blocked by any of the inhibitors. Therefore, PI3-kinase and at least one protein tyrosine kinase, probably Src kinase, are involved in the trigger phase of ACh-induced, but not adenosine-induced, preconditioning. Neither PI3-kinase nor Src kinase is a mediator of the protection of ACh. phosphatidylinositol 3-kinase; Src kinase

Objectives: Ischemic preconditioning (PC) reduces myocardial infarction by a mechanism that involves opening of mitochondrial ATP-dependent potassium channels (mKATP), reactive oxygen species (ROS), and possibly activation of p38 mitogen-activated protein kinase (p38 MAPK). The actual order of these steps, however, is a matter of current debate. This study examined whether protection afforded by menadione, which protects by causing mitochondria to produce ROS, requires mKATP opening. In addition, we tested whether protection from anisomycin, a p38 MAPK activator, is dependent on ROS production. Methods and Results: Isolated, buffer-perfused rat hearts were pretreated with menadione, and infarction was assessed after 30 min of regional ischemia and 120 min of reperfusion. Menadione reduced infarction in a dose-dependent manner with an EC50 of 270 nM. Menadione’s infarct-limiting effect was insensitive to 200 μM 5-hydroxydecanoate (5HD), an mKATP channel blocker, whereas protection by diazoxide and PC were blocked by 5HD. Anisomycin caused hearts to resist infarction and this protective effect was abrogated by SB203580, a p38 MAPK inhibitor, and 2-mercaptopropionylglycine (MPG), a free radical scavenger. Conclusions: These results indicate that mKATP opening occurs upstream of mitochondrial ROS generation in the protective pathway. Furthermore, protection afforded by anisomycin was p38 MAPK- and ROS-dependent. [Copyright &y& Elsevier]

Objective: Acetylcholine (ACh) mimics ischemic preconditioning (PC) and therefore protects the heart against lethal ischemia. Steps common to both ischemic and drug-induced PC are opening of mitochondrial KATP channels (mito KATP) and generation of reactive oxygen species (ROS). The aim of this study was to test whether ACh-induced ROS production could be seen in a vascular smooth muscle cell line, and, if so, to investigate the underlying signaling pathway. Methods: Mitochondrial ROS generation was quantified by measuring changes in fluorescence of ROS-sensitive intracellular markers in vascular smooth muscle cells (A7r5). Results: Fluorescence, and, therefore, ROS production, was increased to 197.5±8.5% of baseline after 45 min of exposure of cells to 2 mM ACh (P<0.001 vs. untreated controls). This effect was blocked by co-treatment with a muscarinic receptor antagonist (atropine 102.8±2.9%, 4-DAMP 92.6±7.4%) or by inhibition of Gi with pertussis toxin (PTX) (90.5±4.4%), implicating a receptor-mediated rather than non-specific effect of ACh. The increased fluorescence induced by ACh was also abrogated by the free radical scavenger N-(2-mercaptopropionyl) glycine (104.2±10.1%), documenting that ROS were indeed the cause of the enhanced fluorescence. Both diazoxide, a KATP channel opener, and valinomycin, a potassium ionophore, also significantly increased ROS production, and these effects were not blocked by PTX, while the KATP channel closer 5-hydroxydecanoate blocked ACh-induced ROS production (92.3±3.8%). These results suggest ROS production is directly influenced by KATP activity and K+ movements in the cell. The tyrosine kinase inhibitor genistein (102.8±6.6%) and the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (90.7±4.1%) also inhibited the ability of ACh to increase ROS production. Conclusion: The signaling pathway by which ACh leads to ROS generation in A7r5 cells involves a muscarinic surface receptor, a pertussis toxin-sensitive G protein, PI3-kinase, at least one tyrosine kinase, and a 5-hydroxydecanoate (5-HD)-dependent KATP (presumably that in mitochondria). [Copyright &y& Elsevier]

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 +/- 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 +/- 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G(i) protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.

Objectives: Ischemic preconditioning (PC) reduces myocardial infarction by a mechanism that involves opening of mitochondrial ATP-dependent potassium channels (mK(ATP)), reactive oxygen species (ROS), and possibly activation of p38 mitogen-activated protein kinase (p38 MAPK). The actual order of these steps, however, is a matter of current debate. This study examined whether protection afforded by menadione, which protects by causing mitochondria to produce ROS, requires mK(ATP) opening. In addition, we tested whether protection from anisomycin, a p38 MAPK activator, is dependent on ROS production., Methods and Results: Isolated, buffer-perfused rat hearts were pretreated with menadione, and infarction was assessed after 30 min of regional ischemia and 120 min of reperfusion. Menadione reduced infarction in a dose-dependent manner with an EC(50) of 270 nM. Menadione's infarct-limiting effect was insensitive to 200 microM 5-hydroxydecanoate (5HD), an mK(ATP) channel blocker, whereas protection by diazoxide and PC were blocked by 5HD. Anisomycin caused hearts to resist infarction and this protective effect was abrogated by SB203580, a p38 MAPK inhibitor, and 2-mercaptopropionylglycine (MPG), a free radical scavenger., Conclusions: These results indicate that mK(ATP) opening occurs upstream of mitochondrial ROS generation in the protective pathway. Furthermore, protection afforded by anisomycin was p38 MAPK- and ROS-dependent.

Objective: Acetylcholine (ACh) mimics ischemic preconditioning (PC) and therefore protects the heart against lethal ischemia. Steps common to both ischemic and drug-induced PC are opening of mitochondrial K(ATP) channels (mito K(ATP)) and generation of reactive oxygen species (ROS). The aim of this study was to test whether ACh-induced ROS production could be seen in a vascular smooth muscle cell line, and, if so, to investigate the underlying signaling pathway., Methods: Mitochondrial ROS generation was quantified by measuring changes in fluorescence of ROS-sensitive intracellular markers in vascular smooth muscle cells (A7r5)., Results: Fluorescence, and, therefore, ROS production, was increased to 197.5+/-8.5% of baseline after 45 min of exposure of cells to 2 mM ACh (P<0.001 vs. untreated controls). This effect was blocked by co-treatment with a muscarinic receptor antagonist (atropine 102.8+/-2.9%, 4-DAMP 92.6+/-7.4%) or by inhibition of G(i) with pertussis toxin (PTX) (90.5+/-4.4%), implicating a receptor-mediated rather than non-specific effect of ACh. The increased fluorescence induced by ACh was also abrogated by the free radical scavenger N-(2-mercaptopropionyl) glycine (104.2+/-10.1%), documenting that ROS were indeed the cause of the enhanced fluorescence. Both diazoxide, a K(ATP) channel opener, and valinomycin, a potassium ionophore, also significantly increased ROS production, and these effects were not blocked by PTX, while the K(ATP) channel closer 5-hydroxydecanoate blocked ACh-induced ROS production (92.3+/-3.8%). These results suggest ROS production is directly influenced by K(ATP) activity and K(+) movements in the cell. The tyrosine kinase inhibitor genistein (102.8+/-6.6%) and the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin (90.7+/-4.1%) also inhibited the ability of ACh to increase ROS production., Conclusion: The signaling pathway by which ACh leads to ROS generation in A7r5 cells involves a muscarinic surface receptor, a pertussis toxin-sensitive G protein, PI3-kinase, at least one tyrosine kinase, and a 5-hydroxydecanoate (5-HD)-dependent K(ATP) (presumably that in mitochondria).