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Cardiovascular Research 2002 55(3):544-552; doi:10.1016/S0008-6363(02)00332-2
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Acetylcholine leads to free radical production dependent on KATP channels, Gi proteins, phosphatidylinositol 3-kinase and tyrosine kinase

Olaf Oldenburga, Qining Qina, Ana R Sharmaa, Michael V Cohena,b, James M Downeya,* and Joseph N Benoita,1

aDepartment of Physiology, MSB 3024, College of Medicine, University of South Alabama, Mobile, AL 36688, USA
bDepartment of Medicine, College of Medicine, University of South Alabama, Mobile, AL, USA

* Corresponding author. Tel.: +1-251-460-6818; fax: +1-251-460-6464 jdowney{at}usouthal.edu

Received 14 November 2001; accepted 15 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
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).

KEYWORDS Acetylcholine; Free radicals; K-ATP channel; Mitochondria; Preconditioning; Protein kinases; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ischemic preconditioning describes a phenomenon whereby short episodes of ischemia and reperfusion protect the myocardium for ~1–2 h against subsequent lethal ischemia. Since Murry et al. [1] first described this phenomenon in 1986, numerous studies have confirmed its protective effect in various species, as well as in in vivo and in vitro heart models, including cultured cells.

Several drugs mimic this cardioprotective effect, including those acting on sarcolemmal surface receptors. Surface receptors involved in the signaling cascade primarily appear to be coupled to Gi protein. Several studies have confirmed the cardioprotective effect of morphine, endothelin, angiotensin II, catecholamines, bradykinin, adenosine, and acetylcholine when administered prior to an ischemic insult [2–5]. Additionally prostanoids, free radicals, and nitric oxide can protect the heart against ischemia [5–7].

Regardless of the primary trigger, ischemia or drug, the mitochrondrial ATP-sensitive potassium channel (mito KATP) is thought to play an integral part in the intracellular signaling cascade [8,9]. We [10] and others [11,12] have proposed that opening of mito KATP channels can trigger preconditioning by the generation of reactive oxygen species (ROS). In this paradigm ROS in turn stimulate kinase pathways ultimately leading to activation of the end-effector that mediates preconditioning [10].

Previous studies have confirmed that an intracellular signaling pathway exists between the surface acetylcholine (ACh) receptors and mito KATP channels in chick cardiomyocytes [13] as well as in intact hearts [14]. However, the nature of this pathway is poorly understood. Because the mitochondrial membranes are not contiguous with the sarcolemma, there can be no direct G protein communication between the receptor and the mitochondria.

Previously we established a model for investigating KATP signaling in rat aortic smooth muscle A7r5 cells in which mitochondrial ROS generation could be monitored [15]. This model was chosen because the vascular smooth muscle cells shared many of the same signaling pathways as cardiomyocytes with the added advantage of the ease with which cells could be cultured as opposed to available cardiomyocyte models. The obvious disadvantage of a non-cardiomyocyte model is that the selectivity of 5-hydroxydecanoate (5-HD) and diazoxide for the mitochondrial channels over the surface channels is unknown. Thus, although we can assume that a KATP channel is being targeted by these drugs in the A7r5 cells, it is not necessarily a mitochondrial channel. Nevertheless, this cell model is a useful one for studying events leading to ROS generation. In the present study, we examined (i) whether ACh leads to KATP channel-dependent ROS production and (ii) the intracellular signaling between surface receptor activation and ROS generation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996).

2.1 A7r5 cells
Aortic vascular smooth muscle cells (A7r5) from Rattus norvegicus were purchased from the American Tissue Culture Collection (ATCC®, Manassas, VA). Cells were grown in 75-cm2 tissue culture flasks (Corning, Corning, NY), and subcultured before confluence. Experiments were performed on subconfluent cells grown for 24–72 h in four-well chambers (Nunc Lab-Tek® II, Nunc, Naperville, IL).

Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM L-glutamine, 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 0.11 g/l pyruvate, penicillin, gentamicin, and fungizone was used for cell growth as recommended by ATCC®. Cells were grown and incubated with experimental drugs in the dark at 37 °C in air enriched with 5% CO2.

2.2 Experimental design
For each experiment the medium was changed to FBS- and pH-indicator-free DMEM. Whenever blocking agents or free radical scavengers were part of the experiment, these drugs were included in this modified medium. Only the pertussis toxin (PTX) experiments required an overnight incubation with the toxin (16 h). Cells were incubated for a total of 40 min in the new medium and then stained with 1 µM reduced MitroTracker® Red (MTR) (Molecular Probes, Eugene, OR) for an additional 15 min. When the dye is irreversibly oxidized by ROS, it becomes fluorescent and is concentrated in mitochondria. If an activator (ACh, methacholine (MET), diazoxide (DIAZ) or valinomycin (VAL)) were used, it was added 10 min after changing the medium. After 15 min with MTR, cells were washed with fresh DMEM containing the drugs but no MTR to remove extra-mitochondrial MTR. Fluorescence was determined. Eight to ten repetitions were performed for each group on different days and with cells of different passages.

2.3 Measurement of ROS production
Mitochondrial ROS generation was analyzed by measuring single cell fluorescence (Intracellular Imaging, Cincinnati, OH) using a Nikon TMS-F Microscope with a 20x objective (Nikon, Japan), a COHU 6600 Scan CCD camera (Cohu, San Diego, CA), an XF filter set (Omega Optical, Brattleboro, VT), and a xenon light source with a Lambda 10-2 optical filter changer (Sutter Instruments, Novato, CA) (excitation 560 nm, emission 610 nm, light exposure of 0.5 s). Cell fluorescence was quantified by the Intracellular Imaging software after manually tracing the perimeters of 15–30 cells per field in five random fields per well, leading to analysis of 800–1000 cells per experiment. The average single-cell fluorescence for each well was computed. This fluorescence value was then compared to that measured in the three other wells of the same experiment.

2.4 Diazoxide, valinomycin, acetylcholine, and methacholine
In the first group of studies the impact of three activators, DIAZ (200 µM), VAL (25 nM) and ACh (2 mM), on ROS production was investigated by comparing fluorescence in cells treated with each of these agents to that of an untreated control sample that was included in one of the four wells. Thus, fluorescence of each treatment could be compared to the corresponding control experiment within the same chamber, at the same time, and with cells from the same passage. In another set of experiments, the effect of the fourth activator MET (2 mM), a second muscarinic receptor agonist, was examined. In each study an untreated control well was included as described above.

2.5 Atropine and 4-DAMP
Experiments were first performed to test whether atropine (ATR) (100 µM) or 4-diphenylacetoxy-N-(2-chloroethyl)piperidine (4-DAMP) (1 µM), antagonists of the acetylcholine receptor, affected fluorescence when added to cells. There was no effect. Cells were then incubated with ACh+ATR or ACh+4-DAMP to test whether the ACH-induced ROS production could be prevented by these muscarinic receptor blockers. Every experiment included one well with ACh alone serving as a positive control.

2.6 5-Hydroxydecanoate and N-(2-mercaptopropionyl) glycine
The effects of two inhibitors, the KATP channel closer 5-hydroxydecanoate (5-HD) (1 mM) and the ROS scavenger N-(2-mercaptopropionyl) glycine (MPG) (1 mM), on ACh-induced changes in cell fluorescence were next examined. Again there was no effect of either 5-HD or MPG alone compared to untreated control cells. The effect of combined treatment of each activator listed above and either 5-HD or MPG was compared to changes in fluorescence induced by the inhibitor itself. Again, every experiment included one well in which cells were treated only with ACh to serve as a positive control.

2.7 Pertussis toxin
Cells were preincubated for 16 h with 2 ng/ml PTX, an irreversible antagonist of Gi protein. As before, the impact of PTX treatment alone was first tested. ACh-, DIAZ- and VAL-induced changes in fluorescence in PTX-treated cells were compared to changes in cells treated only with PTX.

2.8 Wortmannin and genistein
The impact of genistein (GEN) (50 µM), a tyrosine kinase inhibitor, and wortmannin (WORT) (10 nM), a blocker of phosphatidylinositol 3-kinase, on fluorescence of cells stimulated with ACh was examined. In each experiment the effect of the kinase antagonist on ACh-stimulated fluorescence was compared to changes induced by either ACh or the kinase antagonist alone. The effect of the two kinase inhibitors on VAL- and DIAZ-induced ROS production was also examined. Again changes in fluorescence were compared to those seen with the inhibitor alone and with combined treatment of inhibitor and ACh.

2.9 Isolated rabbit heart
New Zealand White rabbits of either sex weighing 1.6–2.5 kg were anesthetized with pentobarbital (30 mg/kg i.v.), intubated through a tracheotomy, and ventilated with 100% oxygen via a positive pressure respirator (MD Industries, Mobile, AL). The ventilation rate and tidal volume were adjusted to maintain arterial pH in the physiological range. A left thoracotomy was performed in the fourth intercostal space, and the heart exposed. A 2-0 silk suture was passed beneath and around a prominent branch of the left coronary artery visible on the surface. The ends of the suture were passed through a small vinyl tube to form a snare. It was confirmed that pulling on the snare resulted in cyanosis of the distal myocardium and akinesis, and release was followed by hyperemia and resumption of contractile activity. After stabilization for 10 min, all hearts were quickly excised, mounted on a Langendorff apparatus and retrogradely perfused via the aorta at 75 mmHg pressure with Krebs-Henseleit buffer containing (mmol/l) NaCl 118.5, KCl 4.7, NaHCO3 24.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5 and glucose 10. The buffer was gassed with 95% O2/5% CO2 and maintained at a temperature of 37 °C. A fluid-filled latex balloon connected to a pressure transducer was inserted into the left ventricle and inflated to set a diastolic pressure of ~5 mmHg at baseline. All hearts were allowed to stabilize for at least 20 min. In control hearts regional ischemia was induced for 30 min by tightening the snare on the coronary branch. Subsequently the snare was loosened to permit reperfusion for 3 h. A second group of hearts was treated with 2.7 nmol/l VAL added to the perfusate for 5 min followed by 10 min of washout before the 30-min coronary occlusion. Total coronary artery flow was measured by timed collection of the perfusate dripping from the heart into a graduated cylinder. Atrial pacing was performed at 200 beats per minute if the spontaneous heart rate was slower. Heart rate and left ventricular developed pressure were continuously monitored.

At the end of the experiments the coronary branch was again occluded by pulling on the snare. First, 5 ml of 0.1% zinc/cadmium sulfide particles (1–10 µm diameter, Duke Scientific, Palo Alto, CA) were infused into the perfusate to demarcate the risk zone as the area of tissue without fluorescence. The hearts were then removed from the Langendorff apparatus, weighed, frozen, and cut into 2.5 mm-thick 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 for 1 h to enhance the contrast between stained (viable) and unstained (necrotic) tissue and then squeezed between glass plates spaced exactly 2 mm apart. The myocardium at risk was identified by illuminating the slices with ultraviolet light. The infarcted and risk zone regions were traced on a clear acetate sheet by an investigator blinded to the treatment and quantified with planimetry. The areas were converted into volumes by multiplying the areas by slice thickness. Infarct size was expressed as a percentage of the non-fluorescent risk zone.

2.10 Chemicals
All experimental drugs were purchased from Sigma Chemical (St. Louis, MO) except for PTX (RBI, Natick, MA), FBS (Gibco BRL®, Life Technologies, Rockville, MD) and MitroTracker® Red (Molecular Probes, Eugene, OR). Either distilled water (MET, 5-HD, MPG), saline (PTX), or DMSO (MTR, DIAZ, VAL, ACh, ATR, 4-DAMP, WORT, GEN) was used to dissolve the drugs and to prepare stock solutions. The final DMSO concentration was kept below 1%. All stock solutions were made freshly every day except for WOR and GEN stock solutions which were stored at 4 °C for up to 1 week.

2.11 Data analysis
For untreated control experiments the absolute fluorescence measurements which provide values in arbitrary units (a.u.) varied widely between individual cultures and MTR lots. To account for that variability fluorescence of cells depicted in figures is expressed as a percentage of that in an untreated control well that was included in each experiment. The text reports the absolute fluorescence numbers in arbitrary units as mean±S.E.M. ANOVA for repeated measures was used to test for differences in mean fluorescence intensity of the groups within the same four-well chamber. Post hoc testing was performed with the paired t-test with Bonferroni's correction. Differences were considered significant if the P-value was <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Methacholine, acetylcholine, diazoxide and valinomycin
Stimulation of A7r5 cells with either MET, ACh, DIAZ, or VAL led to a significant increase of intracellular ROS production. Absolute fluorescence increased from 16.0±0.6 a.u. within the control group to 31.5±0.9 a.u. after ACh (P<0.001), 29.1±1.9 a.u. after DIAZ (P<0.001), and 31.9±1.5 a.u. after VAL (P<0.001) treatment (Fig. 1). Exposure to MET, another muscarinic receptor agonist, also led to an increase in fluorescence (46.1±1.9 a.u. in controls to 59.1±3.6 a.u. after MET, P = 0.005), although the increase was not as great as that seen with ACh.


Figure 1
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  		Fig. 1 Change in cell fluorescence (mean±S.E.M.) expressed as a percentage of that of a simultaneously studied untreated control cell culture after exposure to the muscarinic receptor agonists methacholine (MET) or acetylcholine (ACh), the KATP channel opener diazoxide (DIAZ) and the potassium ionophore valinomycin (VAL). The increase in cell fluorescence indicates a significant drug-induced generation of reactive oxygen species in all experiments (*P<0.05 vs. control).

 
3.2 Atropine and 4-DAMP
A 30-min exposure to either ATR (34.0±3.1 a.u. vs. 36.8±5.2 a.u. in untreated controls) or 4-DAMP (19.3±1.3 a.u. vs. 19.6±1.1 a.u. in untreated controls) had no significant impact on cell fluorescence (Fig. 2). Both muscarinic receptor blockers prevented any ACh-triggered increase in fluorescence (34.8±2.9 a.u. for ATR+ACh and 17.6±0.9 a.u. for 4-DAMP+ACh, P = NS vs. ATR and 4-DAMP, respectively) (Fig. 3).


Figure 2
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  		Fig. 2 Change in fluorescence of A7r5 cells (mean±S.E.M.) as a percentage of that of an untreated control cell culture after exposure to the muscarinic receptor antagonists atropine (ATR) and 4-diphenylacetoxy-N-(2-chloroethyl)piperidine (4-DAMP), the KATP channel closer 5-hydroxydecanoate (5-HD), the free radical scavenger N-(mercaptopropionyl) glycine (MPG), pertussis toxin (PTX), the phosphatidylinositol 3-kinase inhibitor wortmannin (WORT) and the tyrosine kinase inhibitor genistein (GEN). Exposure to these tool drugs had no significant effect on cell fluorescence, and, therefore, on ROS generation.

 

Figure 3
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  		Fig. 3 The acetylcholine (ACh)-induced increase in cell fluorescence (mean±S.E.M.) is blocked by co-treatment with the muscarinic receptor antagonist atropine (ATR) or 4-diphenylacetoxy-N-(2-chloroethyl)piperidine (4-DAMP), the KATP channel closer 5-hydroxydecanoate (5-HD) and the free radical scavenger N-(mercaptopropionyl) glycine (MPG) (P = NS vs. respective inhibitor).

 
3.3 Pertussis toxin
Overnight exposure to PTX had no influence on cell fluorescence (26.6±6.5 a.u. in controls vs. 27.0±5.0 a.u. in PTX-treated cells, P = NS) (Fig. 2). To determine which of the activating agents was dependent on Gi, cells were pretreated with PTX and then cells in three of the four wells of the chamber were exposed to either ACh, DIAZ, or VAL. Fig. 4 reveals that the increase in cell fluorescence produced by ACh was abolished (42.7±3.0 a.u. in PTX+ACh vs. 47.6±4.2 a.u. in PTX, P = NS). However, the DIAZ- (102.2±8.4 a.u., P<0.001) and VAL- (85.2±4.9 a.u., P = 0.003) induced increases in ROS generation were not affected by pretreatment with pertussis toxin.


Figure 4
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  		Fig. 4 Overnight exposure to pertussis toxin (PTX) blocks the acetylcholine-induced increase in cell fluorescence (PTX+ACh, P = NS vs. PTX alone), whereas the PTX pretreatment has no effect on diazoxide- (DIAZ) or valinomycin- (VAL) induced ROS generation (*P<0.05 vs. PTX). These results indicate that ACh signaling is dependent on a PTX-sensitive G protein, whereas the DIAZ or VAL response is not.

 
3.4 5-Hydroxydecanoate and N-(2-mercaptopropionyl) glycine
Closing the KATP channel with 5-HD prevented ACh-induced ROS generation (Fig. 3). Fluorescence was 34.0±5.3 a.u. in untreated control cells, 43.4±7.0 a.u. in ACh-treated cells (P = 0.001 vs. control), and 31.1±5.7 a.u. for cells exposed to both 5-HD+ACh (P = NS vs. 5-HD). 5-HD itself had no significant impact on cell fluorescence (33.2±5.0 a.u., P = NS vs. control) (Fig. 2). The ACh-induced increase in fluorescence was also blocked by co-treatment with the free radical scavenger MPG, confirming that the increase in fluorescence was related to ROS production (Fig. 3). Fluorescence was 18.9±2.3 a.u. in untreated controls, 28.1±2.5 a.u. in ACh-treated cells (P<0.001 vs. control), and 16.8±2.3 a.u. for cells treated with both MPG+ACh (P = NS vs. MPG). MPG itself had no significant impact on cell fluorescence (17.3±2.9 a.u., P = NS vs. control) (Fig. 2).

3.5 Genistein
To examine whether a tyrosine kinase cascade might be playing a role in cell signaling, the effect of GEN, a tyrosine kinase antagonist, was examined. As seen already ACh increased fluorescence from 22.9±4.6 a.u. in untreated cells to 33.1±6.2 a.u. (P<0.001). GEN blocked ACh's stimulatory effect (19.7±4.2 a.u.). GEN itself caused no change (21.1±5.6 a.u., P = NS vs. control). In the second set of experiments, the effect of ACH, DIAZ, and VAL on GEN-treated cells was investigated. When compared to GEN-treated cells, addition of ACh caused no change in fluorescence (9.3±0.5 a.u. vs. 9.4±0.5 a.u., P = NS), whereas ROS production by either DIAZ+GEN (15.1±1.4 a.u., P = 0.006 vs. GEN) or VAL+GEN (17.1±2.3 a.u., P<0.001 vs. GEN) was still evident and was, therefore, not blocked by GEN (Fig. 5).


Figure 5
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  		Fig. 5 Co-treatment of A7r5 cells with genistein (GEN), a tyrosine kinase inhibitor, blocks the acetylcholine- (ACh) induced increase in cell fluorescence (GEN+ACh, P = NS vs. genistein alone). The diazoxide (DIAZ)- or valinomycin (VAL)-dependent production of reactive oxygen species is not affected by GEN (*P<0.05 vs. genistein alone).

 
3.6 Wortmannin
After exposure of A7r5 cells to ACh, fluorescence increased from 26.3±3.4 a.u. in untreated cells to 36.7±4.1 a.u. (P = 0.003). A 30-min exposure to the PI3-kinase blocker WORT itself had no significant impact on cell fluorescence (26.8±1.6 a.u., P = NS vs. control) (Fig. 2). However, the ACh-induced increase was blocked by WORT (27.0±3.2, P = NS vs. WORT-treated cells). Whereas WORT blocked ACh-induced ROS production, it had no effect on ROS production stimulated by either DIAZ (35.3±5.4 a.u. vs. 26.0±3.2 a.u. in WORT-treated cells, P = 0.01) or VAL (37.6±3.5 a.u., P = 0.002 vs. WORT-treated cells) (Fig. 6).


Figure 6
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  		Fig. 6 Co-treatment of A7r5 cells with wortmannin (WORT), a phosphatidylinositol 3-kinase inhibitor, abolished the acetylcholine- (ACh) induced increase in cell fluorescence (WORT+ACh, P = NS vs. WORT alone), whereas WORT had no effect on diazoxide (DIAZ)- or valinomycin (VAL)-induced production of reactive oxygen species (*P<0.05 vs. WORT alone).

 
3.7 Isolated rabbit hearts
There was no difference in either baseline hemodynamics or sizes of the heart or risk zone in the two groups. As can be seen in Fig. 7, infarct size averaged 36.5±4.6% of the risk zone in the control hearts. In hearts pre-treated with valinomycin, however, infarction was significantly smaller and averaged 9.3±2.7% (P<0.001).


Figure 7
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  		Fig. 7 Infarct size data presented as a % of the risk zone for control hearts and those pre-treated with valinomycin prior to a standard 30-min period of regional ischemia. Open circles represent individual data points, while closed circles represent means±S.E.M. Valinomycin significantly decreased infarct size.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study is the first to explore the nature of the signaling pathway between surface receptors and KATP channels. Activation of the ACh-receptor led to a reproducible and robust increase in mitochondrial ROS production. We were surprised to find the pathway between the surface receptor and the KATP channels to be so complex. ACh-induced free radical production could be abolished by either blocking the ACh-receptor, ribosylating the Gi proteins with pertussis toxin, blocking either PI3-kinase or tyrosine kinase, or by closing the KATP channel with 5-HD.

Diazoxide is a potent KATP channel opener with a high selectivity for mitochondrial channels in cardiomyocytes [16]. Unfortunately we cannot say for certain that it will not open some surface channels in the smooth muscle cells used here. Activation of KATP channels in either the mitochondria or the surface membrane by DIAZ leads to production of ROS which we believe act as unique intracellular signals that are utilized by ischemic preconditioning. Once again the critical role of ROS as signal transduction elements rather than destructive biological toxins must be stressed. Evidence supporting a KATP/ROS-dependent pathway for protection has been identified in isolated rabbit [10,14] and rat [11] hearts and in chick cardiac myocytes [13]. The increase in ROS production after DIAZ in the present study was expected and is in agreement with these previous studies. Also concordant with our previous study [15], the potassium ionophore VAL caused a brisk increase in fluorescence. It is assumed (but not proven) that the increased ROS production triggered by VAL was caused by potassium entry into the mitochondria, an action that is well documented for this agent [17]. In our previous study we could abolish VAL-induced ROS by either myxothiazol, an inhibitor of mitochondrial electron transport, or 2,4-dinitrophenol, an uncoupler of mitochondrial oxidative phosphorylation. That clearly demonstrated that VAL was having its effect on mitochondria [15]. Thus, VAL and DIAZ were useful in this study as agents which act directly on mitochondria as opposed to ACh which utilizes receptors and signal transduction pathways.

In the present study we used a relatively high concentration of ACh (2 mM) to induce free radical production. Previous in vivo studies used much lower concentrations of ACh or other muscarinic receptor agonists [4,5,18]. Yao and coworkers [13,19] used 1 mM ACh in their isolated chicken myocyte model and described this dose as the minimum dose required to mimic preconditioning and to reduce cell death. Initially we tested a concentration of 1 mM, but did not get a reliable increase in fluorescence. The 2-mM concentration led to a robust increase in fluorescence every time. Despite use of a high ACh dose it is highly unlikely that the observed responses represent non-specific effects. Rather the evidence supports a receptor-dependent pathway. Exposure to a second muscarinic receptor agonist, MET, also led to a significant increase in ROS production and the ACh-induced ROS generation was abrogated by two different blockers of muscarinic receptors (ATR and 4-DAMP) as well as by PTX. The latter indicates that this signal transduction pathway uses a pertussis toxin-sensitive G protein, most likely Gi, which is known to mediate signaling of the muscarinic receptors in various species [20]. It is important to note that neither PTX, ATR, nor 4-DAMP interfered with the ability of VAL or DIAZ to increase ROS production which would be expected since both VAL and DIAZ should exert their effect directly on mitochondrial membranes. Thus mitochondria themselves were not affected by these blockers.

Concordant with the findings of Yao and coworkers [13], ACh-induced ROS generation was abolished by closing a KATP channel with the potent antagonist 5-HD [21] and released by co-treatment with the free radical scavenger MPG. These observations indicate that ACh leads to generation of free radicals and that this ROS generation is dependent on 5-HD-sensitive KATP channels. Interestingly, the latter have been shown to be part of the intracellular signaling pathway during ischemic [10] and drug-induced [5] preconditioning. These findings corroborate a previous in vivo study in which ACh's ability to mimic ischemic preconditioning in canine hearts was abolished by either simultaneous treatment with a muscarinic receptor blocker or closing the mito KATP channels with 5-HD [5].

Phosphoinositide 3-kinases (PI3-kinases) represent a ubiquitously expressed enzyme family involved in signal transduction through receptors with intrinsic or associated tyrosine kinase activity and receptors linked to heterotrimeric G proteins [22]. In the present study, the potent and highly selective inhibitor WORT [23] was able to block the ACh-dependent increase in fluorescence. However, it could not block DIAZ- or VAL-dependent ROS generation. Therefore a PI3-kinase step most likely exists between the muscarinic receptor and the KATP channels. Indeed Tong et al. [24] were able to block preconditioning's protection in a rat model with WORT.

The blockade of ACh-induced ROS production by the specific tyrosine kinase inhibitor GEN [25] also indicates that at least one tyrosine kinase must be involved in this signaling pathway. Again DIAZ and VAL responses were unaffected, indicating that DIAZ and VAL induce ROS generation by acting at a site distal to the tyrosine kinase.

Based on the present results, no definite conclusions can be drawn as to whether this tyrosine kinase is upstream or downstream of the PI3-kinase. The most likely signaling pathway would be one in which a muscarinic surface receptor coupled to Gi protein activates PI3-kinase {gamma}, an isoform known to be coupled to Gi protein (Fig. 8). The PI3-kinase {gamma} then stimulates a downstream tyrosine kinase, possibly src kinase [26,27], which ultimately activates the KATP channel. This suggestion is, of course, only speculative at this point, and further investigations are needed. PI3-kinase is often dependent on receptor tyrosine kinases, but it is difficult to envision how a receptor tyrosine kinase could respond to ACh or involve a pertussis toxin-sensitive G protein.


Figure 8
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  		Fig. 8 The proposed signaling pathway is shown. Acetylcholine binds to a muscarinic membrane receptor that is coupled to pertussis toxin-sensitive Gi protein. Subsequent steps include activation of a phosphatidylinositol 3 (PI3)-kinase and at least one tyrosine kinase. The most likely sequence is activation of PI3-kinase {gamma} directly by Gi protein followed by activation of a tyrosine kinase, possibly src. The final result is the opening of the mitochondrial KATP channel. The influx of potassium into the mitochondrion causes it to produce reactive oxygen species (ROS) via the electron transport chain. Evidence from previous studies indicates that the ROS then act as signal transduction elements to activate specific kinases within the cell.

 
One limitation of the present study is the use of vascular smooth muscle cells to investigate intracellular signaling. So far the results of our studies with smooth muscle cells agree very well with studies done in both heart cells and whole hearts. While the function of this pathway in the heart is clear, its function, if any, in smooth muscle cells is totally unknown. In our case the reason for choosing vascular smooth muscle cells for study was in part determined by the ease with which these cells can be cultured and their robust KATP-dependent ROS signal. Despite the obvious limitation attached to use of these vascular smooth muscle cells, it is reassuring to know that the pharmacologic agents examined in this study yielded results that were largely foretold based on responses of intact hearts to them. For example, ACh which mimics ischemic preconditioning's cardioprotection increased ROS production in A7r5 cells as expected. As details of these signaling pathways are revealed in A7r5 cells, we can easily verify relevance by testing for the presence of each pathway component in an intact heart model. For example, the potassium ionophore valinomycin duplicated ACh's ROS signal in A7r5 cells. It follows then that if valinomycin exerts the same effect in a cardiac cell, it should be capable of preconditioning the heart. As seen in Fig. 7, this prediction proved to be correct. These data further solidify the usefulness of vascular smooth muscle cells to probe signal transduction pathways with subsequent extrapolation to cardiomyocytes.

Time for primary review 33 days.


   Notes
 
1 Present address: Department of Pharmacology, Physiology and Therapeutics, University of North Dakota, Grand Forks, ND, USA. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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