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Michael V Cohen

Publications and source records attributed to Michael V Cohen.

At least 37 records · Page 2Linked to original sources
PubMed2004

Multiple, brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways.

OBJECTIVES: An in situ model was used to test whether and how multiple occlusions at reperfusion can protect rabbit myocardium. BACKGROUND: Recently it was demonstrated that postconditioning in dogs salvaged ischemic myocardium. METHODS: Control hearts underwent 30-min regional ischemia/3-h reperfusion, whereas in experimental hearts four postconditioning cycles of 30-s occlusion/30-s reperfusion starting 30 s after release of the index coronary occlusion were added in the presence or absence of various cell signaling antagonists. RESULTS: Postconditioning decreased infarction from 35.4 +/- 2.7% of the risk zone in control hearts to 19.8 +/- 1.8% (p < 0.05). Six cycles did not result in greater protection. If postconditioning cycles were begun after 10 min of reperfusion, protection was no longer evident. Either the non-selective K(ATP) channel closer glibenclamide or the putatively selective mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate administered 5 min before reperfusion blocked the protection afforded by postconditioning, indicating involvement of the mitochondrial K(ATP) channel. PD98059, a mitogen-activated protein/extracellular-signal regulated kinase (MEK) 1/2 and therefore extracellular-signal regulated kinase (ERK) inhibitor, and N(omega)-nitro-L-arginine methyl ester, an antagonist of nitric oxide synthase, infused shortly before reperfusion also aborted the protection afforded by postconditioning. Combined ischemic postconditioning and preconditioning resulted in significantly greater protection than either alone. CONCLUSIONS: Multiple, short, regional coronary occlusions immediately after prolonged myocardial ischemia are an effective cardioprotective intervention in the rabbit, and the mechanism of protection involves activation of ERK, production of nitric oxide, and opening of mitochondrial K(ATP) channels. These observations suggest that a similar approach could be applied in the cardiac catheterization laboratory to protect reperfused myocardium after primary angioplasty in patients with acute myocardial infarction.

Animals↗
PubMed2004

Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS.

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 microM) caused a 51.4 +/- 14.8% and 39.8 +/- 11.7% increase, respectively, in ROS production (P <0.005). Coincubation of either agonist with Akt inhibitor (20 microM) or infection of cells with an adenovirus containing dominant negative Akt abolished this increase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP, 1 microM) also increased the ROS signal by 40.8 +/- 15.7%, but this increase was unaffected by Akt inhibitor (39.0 +/- 6.4%), implying that Akt is upstream of NOS. ACh-induced ROS production could be abolished by either of the NOS inhibitors Nomega-monomethyl-L-arginine monoacetate (100 microM) and L-N5-(1-iminoethyl)ornithine hydrochloride (L-NIO, 5 microM). L-NIO also blocked the anti-infarct effect of ACh (550 microM) 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.

Acetylcholine↗
PubMed2004

Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism.

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-acetylpenicillamine (SNAP, 2 microM) 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 +/- 3.0% of the risk zone in control hearts to 10.2 +/- 2.0% in SNAP-treated hearts. Bracketing the SNAP infusion with either the guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (2 microM) or the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (200 microM) completely blocked the infarct-sparing effect of SNAP (34.3 +/- 3.8 and 32.2 +/- 1.6% infarction, respectively). Pretreatment of hearts with 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (10 microM), 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 +/- 1.1% of the risk zone. This salutary effect was abolished by either the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) or 5-hydroxydecanoate (100 microM; 28.9 +/- 2.7 and 33.6 +/- 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 +/- 0.04 to 0.61 +/- 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 mitoK(ATP) channels, and, finally, production of reactive oxygen species.

Animals↗
PubMed2004

Protective and anti-protective effects of acute ethanol exposure in myocardial ischemia/reperfusion.

Epidemiological data suggest that a cardioprotective effect of acute alcohol exposure exists in humans. Recent studies in animal models indicate that transient alcohol exposure can indeed reduce myocardial infarct size and exert a preconditioning-like protective effect akin to ischemic preconditioning (IPC). Moreover, the mechanism of alcohol-induced cardioprotection in part resembles the signaling cascade triggered by ischemic preconditioning, with adenosine receptors, PKC, protein tyrosine kinases, and mitochondrial K(ATP) channels all being implicated as key components. However, it was also seen, at least in the rabbit, that if alcohol were present during the ischemic insult, it could block its own protection. The primary protective effect of alcohol use is probably in the chronic setting where alcohol exposure causes a second window type of protection that persists for many days thereafter. Regular moderate drinking should keep the heart in a second window state indefinitely.

Journal Article↗
PubMed2004

NECA and bradykinin at reperfusion reduce infarction in rabbit hearts by signaling through PI3K, ERK, and NO.

The adenosine A1/A2 adenosine agonist 5'-(N-ethylcarboxamido) adenosine (NECA) and bradykinin both limit infarction when administered at reperfusion in rabbits. This study compares the signal transduction pathways responsible for their anti-infarct effect. Receptor agonists were administered to isolated rabbit hearts starting 25 min after the onset of a 30-min period of ischemia and continued into the 2-h reperfusion period. Infarct size was measured. Both NECA and bradykinin decreased infarction from 31.5 +/- 2.4% of the risk zone in untreated hearts to 11.8 +/- 2.0% and 15.4 +/- 2.4%, respectively (P<0.05). Protection from both agents was blocked by PD98059, wortmannin, and Nomega-nitro-L-arginine methyl ester (L-NAME), thus demonstrating dependence on activation of extracellular regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) and stimulation of nitric oxide synthase (NOS). Both wortmannin and PD98059 prevented phosphorylation of ERK 1/2 in NECA-treated hearts, whereas only wortmannin and not PD98059 blocked Akt phosphorylation. These data suggest Akt is upstream of ERK 1/2. In addition, 8-(3-chlorostyryl) caffeine blocked NECA's protection indicating that A2 adenosine receptors trigger NECA's anti-infarct effect. Of note, both bradykinin and acetylcholine (ACh) administered before ischemia to trigger preconditioning's cardioprotection use PI3K and NOS in their signaling pathway. Curiously, however, ACh, unlike bradykinin, was not protective when administered at reperfusion. Hence, both NECA and bradykinin administered at reperfusion protect through a common signaling pathway that includes PI3K, NO, and ERK.

Adenosine A2 Receptor Antagonists↗
PubMed2004

Mitochondrial ROS generation following acetylcholine-induced EGF receptor transactivation requires metalloproteinase cleavage of proHB-EGF.

Acetylcholine (ACh) mimics ischemic preconditioning by a mechanism dependent on phosphatidylinositol 3-kinase (PI3-kinase) and reactive oxygen species (ROS). In other tissues muscarinic receptors activate a metalloproteinase, which liberates surface-associated heparin-binding epidermal growth factor (HB-EGF) and causes transactivation of epidermal growth factor receptors (EGFRs) with activation of PI3-kinase. We tested whether this pathway is operative in myocardium. Adult rabbit cardiomyocytes were incubated in reduced MitoTracker Red, which fluoresces after ROS exposure. ACh caused a 36 +/- 6% increase in fluorescence (P<0.001) and metalloproteinase inhibitor III (MPI) abolished this increase. Both exogenous EGF as well as HB-EGF caused similar increases in the ROS signal (41 +/- 12%, P=0.005 and 40 +/- 7%, P<0.001, respectively). The ROS burst from HB-EGF was unaffected by MPI (37 +/- 6%, P=0.002), confirming that inhibition of metalloproteinase activity blocked ACh's effect at a site upstream of EGFR. CRM-197, which inhibits HB-EGF activity, also blocked ACh-induced ROS generation, again implicating release of HB-EGF as a necessary step for ROS generation. An HB-EGF-neutralizing antibody also prevented ACh-induced increase in ROS. In isolated, perfused rabbit hearts ACh increased phosphorylation of EGFR by 127.4 +/- 43.7%, and this increase was abolished by MPI. Finally, ACh decreased infarct size from 30.1 +/- 2.9% of the risk zone in control hearts to 13.7 +/- 3.0% (P=0.002), and this protection could be abolished by co-treatment with MPI (28.7 +/- 2.6%, P=n.s. vs. control). Stimulation of a second Gi-protein-coupled receptor by the delta-opioid agonist [D-Ala2, D-Leu5]-enkephalin acetate (DADLE) also protected the heart (9.1 +/- 2.0% infarction, P<0.005 vs. control), and this protection was similarly blocked by MPI (28.9 +/- 2.3% infarction). We conclude that ACh-induced ROS generation in myocytes is mediated via transactivation of EGFR through metalloproteinase-dependent release of HB-EGF, and that this pathway is also operative in the intact heart and is required for ACh's cardioprotection.

Acetylcholine↗
PubMed2003

Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection.

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 +/- 16% increase in ROS generation (P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 microM) 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+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nomega-nitro-m-arginine methyl ester (m-NAME, 200 microM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 microM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 microM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 microM) increased ROS generation (39 +/- 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 microM) increased ROS by 34 +/- 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 +/- 3.0 of the risk zone in control hearts to 11.9 +/- 1.4% (P < 0.01). This protection was aborted by either 200 microM m-NAME or 2 microM ODQ (35.4 +/- 5.7 and 30.4 +/- 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 mitoKATP. Subsequent release of ROS triggers cardioprotection.

Adenosine Triphosphate↗
PubMed2003

Activation of Akt is essential for acetylcholine to trigger generation of oxygen free radicals.

OBJECTIVES: Acetylcholine (ACh) receptor activation in the heart causes mitochondrial production of reactive oxygen species (ROS) that is dependent on mitochondrial K(ATP) channel opening. Recent data show that Akt (also known as protein kinase B) is phosphorylated at its activation site following exposure to ACh. However, since no reliable Akt inhibitor is available, it has not been possible to determine whether Akt activation is an actual step in the protective pathway. METHODS: Cultured rat vascular smooth muscle cells (A7r5) were transiently transfected with a dominant negative Akt (Akt-AAA), thus inhibiting the ability of ACh in these cells to phosphorylate Akt. Transfected cells were identified by co-transfection of enhanced green fluorescent protein (EGFP). ROS production was determined by incubating the cells for 15 min with 1 mM reduced MitoTracker Red which becomes fluorescent only after reacting with ROS. Cells were then triple-washed to remove any voltage-dependent pool of dye and single cell fluorescence was measured. RESULTS: ACh exposure (2 mM) led to a 1.64+/-0.15-fold increase in the average fluorescence over that seen in untreated cells (P=0.002). A similar increase in ROS production occurred after treatment with either the K(ATP) channel opener diazoxide (DIAZ) or the potassium ionophore valinomycin (VAL). Akt-AAA transfection abolished ACh-induced ROS production, but not increased ROS production after treatment with either DIAZ or VAL. CONCLUSION: Thus, at least in the smooth muscle cell model, Akt phosphorylation is an important step in the signal transduction pathway leading from ACh receptor activation to the generation of ROS. The experiments reveal that Akt is positioned between the receptor and the K(ATP) channel in this model.

Acetylcholine↗
PubMed2003

Acetylcholine-induced production of reactive oxygen species in adult rabbit ventricular myocytes is dependent on phosphatidylinositol 3- and Src-kinase activation and mitochondrial K(ATP) channel opening.

Acetylcholine (ACh), like ischemic preconditioning (PC), protects against infarction and is dependent on generation of reactive oxygen species (ROS). To investigate the mechanism by which ACh causes ROS production, isolated adult rabbit cardiomyocytes underwent a timed incubation in reduced MitoTracker Red, which is oxidized to a fluorescent form after exposure to ROS. The mitochondrial ATP-sensitive potassium (mK(ATP)) channel opener diazoxide (50 microM) increased fluorescence by 47 +/- 9% (P = 0.007), indicating that opening of mK(ATP) leads to ROS generation, and that increase was blocked by the mK(ATP) blocker 5-hydroxydecanoate (5HD, 1 mM); 250 microM ACh caused a similar increase in ROS generation (+45 +/- 6% for all experiments, P < 0.001). ACh-induced ROS production was prevented by (1) blockade of muscarinic surface receptors with 100 microM atropine (-6 +/- 2%, P = n.s.) or 250 nM 4-DAMP (+5 +/- 13%, P = n.s.), indicating that ACh's effect was receptor mediated; (2) closing K(ATP) channels with either the non-selective channel closer glibenclamide (50 microM) (-1.2 +/- 17%, P = n.s.) or the selective mK(ATP) closer 5HD (-1.8 +/- 9%, P = n.s.), indicating that increased ROS production involved opening of mK(ATP); (3) blockade of mitochondrial electron transport chain with 200 nM myxothiazol (-4 +/- 9%, P = n.s.), indicating ROS came from the mitochondria; (4) addition of 100 nM wortmannin (-13 +/- 12%, P = n.s.), indicating that phosphatidylinositol 3-(PI3)-kinase was involved; and (5) blockade of Src-kinase with 1 microM PP2 (-2 +/- 5%, P = n.s.), indicating the involvement of an Src-kinase. These results support the hypothesis that occupation of muscarinic surface receptors by ACh causes activation of PI3- and Src-kinases that then open mK(ATP) resulting in mitochondrial ROS generation and triggering of the preconditioned state.

Acetylcholine↗
PubMed2003

P1075 opens mitochondrial K(ATP) channels and generates reactive oxygen species resulting in cardioprotection of rabbit hearts.

We have recently proposed that opening of mitochondrial K(ATP) channels (mitoK(ATP)) acts as a trigger for preconditioning (PC) by causing mitochondria to produce reactive oxygen species (ROS). Controversy exists as to whether the putative sarcolemma-selective K(ATP) channel opener P1075 also opens mitoK(ATP) channels and may be cardioprotective. We purified mitoK(ATP) channels from either rabbit heart, rat heart or rat brain and reconstituted the proteins into liposomes. mitoK(ATP) channels from each of these tissues were opened by P1075 with EC(50) values of 60-90 nM. We next tested whether P1075 causes rabbit cardiomyocytes to produce ROS in a K(ATP)-dependent fashion. Mitochondrial ROS production was monitored by the appearance of fluorescence as reduced MitoTracker Red was oxidized. P1075 (100 microM) led to a 44 +/- 9% increase in ROS generation (P < 0.001 vs. untreated cells), which was similar to the increase seen with 50 microM diazoxide, a selective mitoK(ATP) channel opener (49 +/- 9%, P < 0.001 vs. untreated cells). The effect of P1075 was equally potent at a concentration of 150 nM. The P1075-induced increase in ROS production was blocked by 50 microM glibenclamide (GLI), a non-selective K(ATP) blocker, and by 5-hydroxydecanoate (1 mM), a highly selective mitoK(ATP) blocker (-6 +/- 14% and +4 +/- 12%, respectively; P = n.s). In isolated rabbit hearts, P1075 (150 nM) markedly reduced infarct size compared to control animals (10.6 +/- 8.1% of the area at risk vs. 31.5 +/- 5.6%, P < 0.05). GLI (5 microM) as well as 5-hydroxydecanoate (200 microM) completely blocked P1075's anti-infarct effect (31.7 +/- 9.5% and 27.7 +/- 4.6% infarction, respectively; P = n.s. vs. untreated hearts). These data provide strong evidence that P1075 does open mitoK(ATP) channels and protects the ischemic rabbit heart in a mitoK(ATP)-dependent manner.

Animals↗
PubMed2003

CGX-1051, a peptide from Conus snail venom, attenuates infarction in rabbit hearts when administered at reperfusion.

CGX-1051, isolated from the venom of the marine snail Conus purpurasens, was previously noted to interact with potassium channels. Since potassium channels play an important role in cardiac physiology, we assessed the effect of CGX-1051 on infarct size in a rabbit heart model of ischemia/reperfusion. A coronary branch was occluded for 30 minutes followed by 3 hours of reperfusion in in situ and 2 hours in in vitro preparations. Infarct size was measured with triphenyltetrazolium chloride staining and expressed as a percent of the risk zone. In in situ studies, a bolus intravenous injection of CGX-1051, either 10 or 100 microg/kg, administered 5 minutes before reperfusion, reduced infarct size from 40.4 +/- 2.8% of the risk zone in untreated animals to 19.8 +/- 3.8% and 15.0 +/- 1.9%, respectively. One microg/kg CGX-1051 was not protective. To see if the salvage was sustained, two groups of rabbits underwent 72 hours of reperfusion. The dose of 10 microg/kg infused 5 minutes before reperfusion reduced infarct size from 37.0 +/- 1.6% in untreated rabbits to 15.5 +/- 2.0%. When administered 10 minutes after reperfusion had begun, 100 microg/kg CGX-1051 had no effect. CGX-1051 also reduced infarct size in crystalloid-perfused, isolated rabbit hearts suggesting that protection did not depend on circulating leukocytes. The mitochondrial KATP inhibitors glibenclamide and 5-hydroxydecanoate and the MEK(1/2), ERK and hence, inhibitor PD 98059 aborted protection from CGX-1051. These data indicate that functionally active ERK and mitochondrial KATP channels are necessary for protection. CGX-1051 caused no hemodynamic alterations at any dose tested. We conclude that CGX-1051 has a powerful anti-infarct effect when given just before reperfusion.

Animals↗
PubMed2002

Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase.

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 +/- 3.5% of the risk zone versus 34.3 +/- 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30-min ischemic period (mediator phase), ACh-induced protection was not affected (7.4 +/- 2.1% and 9.7 +/- 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.

Acetylcholine↗
PubMed2002

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases.

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.

Acetylcholine↗
PubMed2002

Acetylcholine leads to free radical production dependent on K(ATP) channels, G(i) proteins, phosphatidylinositol 3-kinase and tyrosine kinase.

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).

Acetylcholine↗