AJP - Heart Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H712-H718, 2004. First published March 25, 2004; doi:10.1152/ajpheart.00954.2003
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/2/H712    most recent
00954.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (38)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qin, Q.
Right arrow Articles by Downey, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qin, Q.
Right arrow Articles by Downey, J. M.

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

Qining Qin,1 Xi-Ming Yang,1 Lin Cui,1 Stuart D. Critz,2 Michael V. Cohen,1,3 Natasha C. Browner,1 Thomas M. Lincoln,1 and James M. Downey1

Departments of 1Physiology, 2Cell Biology and Neuroscience, and 3Medicine, College of Medicine, University of South Alabama, Mobile, Alabama 36688

Submitted 9 October 2003 ; accepted in final form 18 March 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 µM) 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 µM) or the mitochondrial ATP-sensitive K+ (mitoKATP) channel blocker 5-hydroxydecanoate (200 µM) 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 µM), 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 µM; 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 mitoKATP channels, and, finally, production of reactive oxygen species.

mitochondrial ATP-sensitive potassium channel; protein kinase G; S-nitroso-N-acetylpenicillamine; guanylyl cyclase

ISCHEMIC PRECONDITIONING CAUSES the heart to become resistant to infarction from ischemia. Protection from ischemic preconditioning consists of two phases: the early or classical phase, which lasts from 1 to 4 h (3, 32), and a late or delayed phase, which appears 12–24 h later and has a duration of up to 3 days (6, 7). Nitric oxide (NO) has been convincingly shown to trigger and mediate delayed preconditioning (10, 11, 44). However, the role of NO in classic ischemic preconditioning is controversial (15, 30, 43, 46, 49, 50). Cardiac myocytes express both the inducible and constitutive isoforms of NOS (5, 12). NO is produced when NO synthase (NOS) converts L-arginine to citrulline.

Vegh et al. (46) were the first to propose that endogenous NO might be involved in preconditioning. In open-chest dogs, Vegh and colleagues noted that N{omega}-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) administered before and after brief cycles of ischemia-reperfusion attenuated the antiarrhythmic effect of ischemic preconditioning. However, Weselcouch et al. (49) reported that inhibition of NO synthesis by L-NAME (30 µM), which is a potent NOS inhibitor, did not alter the effect of ischemic preconditioning on functional recovery in isolated, perfused rat hearts. Indeed, in studies from our lab, neither 100 µM (27) nor 200 µM (29) L-NAME would block the infarct-limiting effect of ischemic preconditioning in isolated rabbit hearts when applied 5 min before ischemic preconditioning and continually throughout the period of index ischemia. In a recent study from Yellon's group (9), two cycles of ischemic preconditioning could reduce infarct size in wild-type mice, whereas four cycles of ischemic preconditioning were required to limit infarct size in endothelial NOS (eNOS) knockout mice. Because endogenous NO only lowered the threshold for the protection of ischemic preconditioning, they concluded that it acts in parallel with other pathways to trigger protection.

Although the role of endogenous NO in ischemic preconditioning remains to be resolved, there is ample evidence that exogenous NO that results from administration of a NO donor is cardioprotective (24, 28, 29, 33, 41, 52). We recently proposed that opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels triggers preconditioning by causing mitochondria to produce reactive oxygen species (ROS) that act as second messengers to activate downstream kinases such as PKC and cause cells to enter the preconditioned state (37). We also found that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) triggered a preconditioning effect in an ROS-dependent manner (34). In embryonic chicken ventricular myocytes, exogenous NO conferred protection that was blocked by either the ROS scavenger N-(2-mercaptopropionyl)glycine (MPG) or the selective mitoKATP blocker 5-hydroxydecanoate (5-HD; Ref. 27). NO itself is a radical species and reacts with superoxide to produce highly reactive peroxynitrite. Therefore, it is possible that NO (or peroxynitrite) directly activates the downstream kinases to trigger protection.

Recent evidence suggests that the mechanism may be more complicated. Bradykinin (BK) is one of the physiological triggers of ischemic preconditioning (48), and in isolated rabbit cardiomyocytes, BK administration results in ROS production (29). We found that the pathway responsible for BK-induced ROS in cardiac myocytes includes NOS, guanylyl cyclase, and protein kinase G (PKG) and that all of the latter reside upstream from mitoKATP channels (36). Furthermore, both L-NAME (200 µM) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) blocked the infarct-limiting effect of BK in isolated rabbit heart (29).

In the present study, we wanted to critically test the hypothesis that NO protects the heart through the guanylyl cyclase-cGMP cascade and that these events reside upstream of the ROS-producing event that triggers ischemic preconditioning in the intact isolated heart.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals (34a).

Surgical preparation. New Zealand White rabbits of either sex were used. As previously described (34), hearts from animals anesthetized with pentobarbital sodium (30 mg/kg) were exposed through a left thoracotomy, and a suture was passed around a prominent coronary artery branch visible on the epicardial surface. The heart was then removed, mounted on a Langendorff apparatus, and perfused with modified Krebs-Henseleit bicarbonate buffer that contained (in mM) 118.5 NaCl, 24.8 NaHCO3, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, and 10 glucose. The buffer was gassed with 95% O2-5% CO2, which resulted in a pH of 7.4–7.5. The temperature of the perfusate was maintained at 38°C. A fluid-filled latex balloon connected to a transducer with polyethylene-240 tubing was inserted into the left ventricle. Balloon volume was adjusted to set the left ventricular end-diastolic pressure equal to 5 mmHg at the beginning of the experiment. Atrial pacing was started to keep the heart rate at 200 beats/min if the spontaneous rate was too low. Total coronary flow was measured by timed collection of perfusate that dripped from the right heart into a graduated cylinder. The heart was allowed to stabilize for 20 min before the experiment was started.

Experimental protocols. Figure 1 shows details of the experimental protocols. Ten groups of hearts were studied. All hearts were subjected to an index ischemia of 30 min of coronary branch occlusion and subsequent 2 h of reperfusion. Control hearts were subjected to only the index ischemia plus reperfusion. The second group was preconditioned with 5 min of global ischemia plus 10 min of reperfusion before the index ischemia. In the third and fourth groups, either L-NAME (200 µM) or ODQ (2 µM), respectively, was infused for 15 min starting 5 min before the preconditioning ischemia. In the fifth group, hearts were treated with SNAP (2 µM) for 5 min and received 10 min of washout before the index ischemia. In the sixth and seventh groups, a 5-min infusion of SNAP (2 µM) was bracketed by either ODQ (2 µM) or 5-HD (200 µM), respectively. The eighth group was treated for 20 min before the index ischemia with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP, 10 µM), which is a cell-permeable analog of cGMP. A washout period of 5 min was interposed between the infusion and the index ischemia. The ninth group was also treated with 8-pCPT-cGMP for 20 min, but MPG (1 mM) was coinfused beginning 5 min before 8-pCPT-cGMP treatment and ending with onset of the index ischemia. The tenth group was treated with a combination of 8-pCPT-cGMP and 5-HD (100 µM). In a previous study on isolated rabbit hearts, we found that neither L-NAME (200 µM) nor ODQ (2 µM) affected infarct size when infused alone (36). Similarly, neither MPG nor 5-HD by itself influenced infarct size (37). Therefore, we did not rework these groups in the present study.



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. Experimental protocols in in vitro rabbit hearts. Time periods (in minutes) are relative to the start of the 30-min period of regional ischemia. SNAP, S-nitroso-N-acetylpenicillamine; 8-pCPT-cGMP, 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate; PC, ischemic preconditioning; L-NAME, N{omega}-nitro-L-arginine methyl ester; ODQ, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one; 5-HD, 5-hydroxydecanoate; MPG, N-(2-mercaptopropionyl)glycine.

 
Measurement of infarct size. At the end of the experiment the coronary artery was reoccluded, and 2 to 9-µm fluorescent microspheres (Duke Scientific; Palo Alto, CA) were infused into the perfusate to demarcate the ischemic zone (region at risk) as the area of tissue without fluorescence. The heart was weighed, frozen, and cut into 2-mm-thick slices. The slices were incubated in 1% triphenyltetrazolium chloride (TTC) in sodium phosphate buffer (pH 7.4) at 37°C for 20 min. TTC stains noninfarcted myocardium brick red. The slices were then immersed in 10% formalin to preserve the stained (viable) and unstained (necrotic) tissues. The risk zone was identified by illuminating the slices with UV light. The areas of infarct and risk zone were determined by planimetry of each slice, and volume was calculated by multiplying each area by the slice thickness and then summing these values for each heart. Infarct size is expressed as a percentage of the risk zone.

Measurement of cGMP levels in SNAP-treated isolated hearts. Left ventricular tissue from three additional hearts was biopsied before and 5 min after treatment with SNAP (2 µM). Heart tissue was frozen in liquid nitrogen until it was pulverized. The heart-tissue powder was treated with cell lysis buffer (0.1 N HCl-50% methanol), and the suspension was centrifuged at 14,000 g for 3 min at 4°C. The supernatant was collected for cGMP measurements, and the pellet was saved for protein assay. cGMP levels were determined by radioimmunoassay according to the method of Harper and Brooker (23).

Chemicals. MPG, 5-HD, and SNAP were purchased from Sigma (St. Louis, MO); L-NAME and ODQ were from Alexis (Qbiogene; Carlsbad, CA), and 8-pCPT-cGMP was from BIOLOG (Life Science; Bremen, Germany). Either distilled water or DMSO was used to dissolve the drugs and prepare stock solutions. The final DMSO concentration was kept at <1%.

Data analysis. Baseline hemodynamic variables in experimental groups were compared with those in the control group by one-way ANOVA, and changes with time in any given group were compared with baseline by ANOVA with repeated measures. Infarct size data were compared by one-way ANOVA, and cGMP levels were compared by an unpaired t-test. Differences were considered to be significant if the P value was <0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hemodynamics. Hemodynamics are presented in Table 1. At baseline, there were no differences between groups except for faster heart rates in the preconditioned (PC) and 8-pCPT-cGMP+MPG groups and a higher coronary flow in the SNAP+ODQ group. L-NAME and ODQ significantly depressed left ventricular developed pressure and coronary flow. As was expected, values for developed pressure and coronary flow in all groups were significantly lower than baseline values during coronary occlusion with partial recovery during reperfusion.


View this table:
[in this window]
[in a new window]
  		Table 1. Hemodynamic parameters

 
Infarct-size data. Table 2 contains the infarct size data. There were no significant differences in risk zone volume among the groups. Infarction in control hearts averaged 30.5 ± 3.0% of the risk zone (Figs. 2 and 3). Although 200 µM L-NAME could block the protection of BK (36), this dose of L-NAME still could not block ischemic preconditioning (see Fig. 2). Similarly, ODQ did not block the anti-infarct effect of ischemic preconditioning, which indicates that endogenous NO is not involved in ischemic preconditioning in the isolated rabbit heart. Comparable to ischemic preconditioning, SNAP (2 µM) protected the heart and reduced infarction to 10.2 ± 2.0%, and this protection was blocked by ODQ. ODQ is a potent inhibitor of soluble guanylyl cyclase, which when activated by NO produces the second messenger cGMP. The protection provided by SNAP could also be blocked by 5-HD, which is a selective blocker of mitoKATP channels (see Fig. 2); this indicates that SNAP triggers ischemic preconditioning through a mitoKATP channel-sensitive pathway.


View this table:
[in this window]
[in a new window]
  		Table 2. Infarct size data

 


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. Infarct size was measured as a percent of risk zone. Individual hearts ({circ}) and group means ({bullet}) with superimposed standard errors are shown. Neither L-NAME nor ODQ infused during the trigger phase could block the infarct-sparing effect of ischemic preconditioning, whereas either ODQ or 5-HD blocked the protection provided by SNAP. *P < 0.05 vs. control.

 


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3. Infarct sizes of individual hearts ({circ}) and group means ({bullet}) with superimposed standard errors are shown. When administered during the trigger phase, 8-pCPT-cGMP decreased infarct size, and this salutary effect was blocked by either the free radical scavenger MPG or mitochondrial ATP-sensitive K+ (mitoKATP) channel blocker 5-HD. *P < 0.05 vs. control.

 
When the cGMP level is elevated in tissue, it activates the cGMP-dependent kinase PKG. Unfortunately, there are no good blockers of PKG that are suitable for use in an intact heart. We did find a suitable PKG activator, however; 8-pCPT-cGMP is a cell permeant cGMP analog that strongly and selectively activates PKG. The 8-pCPT-cGMP mimicked the preconditioning effect of SNAP and reduced infarct size to 7.5 ± 1.1% of the risk zone (see Fig. 3). Protection from 8-pCPT-cGMP was aborted by either the free radical scavenger MPG or by 5-HD (Fig. 3). The data indicate that an elevation in cGMP triggers preconditioning by opening mitoKATP channels and releasing free radicals.

cGMP levels. Figure 4 shows cGMP levels in left ventricular tissue before and after treatment with SNAP. The baseline cGMP value in the heart tissue was 0.18 ± 0.04 pmol/mg protein; this increased to 0.61 ± 0.14 pmol/mg protein after SNAP treatment (P < 0.05).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. SNAP (2 µM) significantly increased cGMP levels in heart tissue.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrated that exogenous NO mimics preconditioning by a cGMP- and mitoKATP channel-dependent signaling pathway. This protection could be duplicated by activating PKG with the cGMP analog 8-pCPT-cGMP. The data indicate that NO exerts its protective effect through the PKG pathway rather than acting directly as a free radical second messenger. Furthermore, PKG was found to be upstream of mitoKATP channels, where presumably it acts to open the channels and cause mitochondria to produce ROS that ultimately trigger the preconditioned state. Interestingly, neither L-NAME nor ODQ blocked protection from ischemic preconditioning.

It is known that ROS can directly trigger the preconditioned state and that ROS are involved in the mechanism of ischemic preconditioning (4, 45). Several studies suggest that ROS are formed by mitochondria in response to mitoKATP channel opening and that ROS are second messengers that trigger the preconditioned state (19, 37, 51). Nakano et al. (34) showed that the protection provided by SNAP was blocked by MPG, which indicates that NO generated by SNAP was acting via free radicals to trigger preconditioning. Because NO can react with superoxide to form the radical species peroxynitrite (ONOO–), it was assumed that the latter was directly mimicking the ROS signal to trigger the preconditioned state. That was reinforced by the observation that L-NAME could not block the protection of ischemic preconditioning. In support of this hypothesis, Altug et al. (2) reported that exogenous peroxynitrite could mimic preconditioning in isolated rat hearts and this protection could be eliminated by the free radical scavenger MPG. The present data suggest that the mechanism is more complex, and that NO activates guanylyl cyclase to make cGMP. The rise in the cGMP level then would activate PKG, which phosphorylates a substrate that results in opening of mitoKATP channels. PKG could be acting directly on the channels or on a protein upstream of the channels.

Others have looked at the role of NO donors and cGMP in preconditioning. Lochner et al. (29) reported that cGMP was increased in isolated perfused rat hearts treated with either of the NO donors SNAP or sodium nitroprusside. In an isolated, perfused mouse heart model, Bell and colleagues (8) found that up to a concentration of 2 µM, SNAP attenuated infarct size; 2 µM is the concentration used in the present study. In cultured rat ventricular myocytes, Rakhit et al. (38) noted that SNAP mimicked ischemic preconditioning, and this protection was completely abolished by ODQ but was not affected by either the PKC inhibitor chelerythrine or the mitoKATP channel blockers glibenclamide or 5-HD. Lebuffe et al. (27) also found that exogenous NO was able to precondition embryonic chick ventricular myocytes, but this protection could be blocked by 5-HD, which suggests that NO can activate mitoKATP channels. Ockaili et al. (35) reported that sildenafil (Viagra), which elevates cGMP levels in cells, could reduce infarct size in rabbit hearts, and this protection was also blocked by 5-HD. Our data support and extend these prior observations.

The functional effect of the NO donor SNAP may be influenced by dose and its effect on cGMP. For example, its dose-related effect on myocardial contractility appears to be dependent on differential actions on cyclic AMP and GMP activities. SNAP exerts a negative inotropic effect on rat cardiomyocytes at a high concentration (100 µM) by stimulating cGMP activation of PKG (47). However, it increases the contractile response at a low concentration (1 µM) by a cGMP-independent activation of adenylyl cyclase. Subsequently, it was shown that exogenous NO attenuated the contractile response by a PKG-troponin I mechanism (26).

We have shown that SNAP at a dose of 2 µM significantly increased cGMP levels in heart tissue. Furthermore, the protection provided by SNAP was abolished by the soluble guanylyl cyclase inhibitor ODQ. Although we could not find a selective PKG inhibitor that was feasible for use in intact hearts, the potent PKG activator 8-pCPT-cGMP mimicked the protection of SNAP. In intact cells, 8-pCPT-cGMP is known to activate PKG with negligible effects on PKA- or cGMP-dependent phosphodiesterases (1, 13, 18, 20). Data from cells complement these observations in intact hearts. In isolated cardiomyocytes, two highly specific PKG antagonists were noted to abort the trigger mechanism of preconditioning by blocking mitochondrial ROS production that was initiated by a preconditioning mimetic drug. Thus the 8-bromoguanosine-3',5'-cyclic monophosphorothioate Rp isomer blocked the effect of BK (36). And the highly selective DT-3, which is a cell-permeable peptide designed specifically to inhibit PKG (17), blocked the ability of both BK and acetylcholine to induce ROS production (T. Krieg, L. Cui, W. R. G. Dostmann, J. M. Downey, and M. V. Cohen, unpublished observation). These data suggest the involvement of PKG in the protection that SNAP provides.

In the present study, cardiac protection from both 8-pCPT-cGMP and SNAP could be blocked by the mitoKATP channel blocker 5-HD, which confirms a dependence on the ability of SNAP to open these channels. It should be noted that the role of 5-HD as a selective opener of mitoKATP channels and even the existence of these channels have recently been questioned (16). Prior investigations have demonstrated that the infarct-sparing effect of SNAP involved ROS production (27, 34). It is unknown how PKG might open mitoKATP channels. In vascular smooth muscle, NO was reported to activate surface KATP via a cGMP-dependent mechanism (25). Similarly, in guinea pig ventricular cells, the NO donor (±)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR3, 1 mM) was found to potentiate surface KATP channel openers, although an increase in cGMP could not explain the observation (42). Using patch-clamp techniques on rabbit ventricular myocytes, Han and coworkers (22) have shown that the NO donor sodium nitroprusside (1 mM) potentiated sarcolemmal KATP-channel opening by a NO-cGMP-PKG pathway. They suggested that sarcolemmal KATP channels were phosphorylated by PKG to increase the opening probability of the channels. In adult rabbit ventricular myocytes, SNAP (0.1 mM) increased flavoprotein fluorescence (an index of mitoKATP channel opening; Ref. 39). SNAP-induced flavoprotein oxidation was dose dependent and NO sensitive, because this oxidation was eliminated by the NO scavenger 2-(4-carboxyphenyl)-4,4',5,5'-tetramethylimidazole-1-oxyl-3-oxide. However, this oxidation could not be mimicked by the cGMP analog 8-Br-cGMP. The authors concluded that SNAP and the derived NO activated the mitoKATP channels directly rather than through cGMP. That is in direct contrast to our recent studies in which BK was able to open mitoKATP channels as evidenced by ROS generation in rabbit cardiomyocytes (36). ROS generation could be blocked by both L-NAME and ODQ. Furthermore, ROS formation from BK could also be blocked by 8-Br-cGMPS, which is a cGMP derivative that blocks PKG. Additionally, the cGMP analog 8-pCPT-cGMP duplicated the generation of ROS by BK, which could be blocked completely by 5-HD.

Is NO involved in ischemic preconditioning? As noted above, this issue is controversial, with studies both supporting and denying a role for NO (40). Although 200 µM L-NAME blocked the infarct-limiting effect of BK (36), this dose still could not block the protection provided by ischemic preconditioning in the present study. During ischemia, several metabolites including adenosine, BK, and opioids are released into the myocardium. It is believed that all of these converge on PKC or protein tyrosine kinases and that a finite threshold of protein kinase stimulation is required before a preconditioned state can be realized. Although BK seems to utilize NO as a second messenger in its signaling pathway, adenosine, another trigger, probably does not. We have found that adenosine triggers protection by a mechanism that uses neither ROS nor mitoKATP channels (14). In the isolated heart there are no kininogens in the perfusate, and as a result we have been unable to show a BK component of ischemic preconditioning, whereas it was easily demonstrated in the blood-perfused in situ model (21). Similarly, we have been able to demonstrate an opioid component in only the in situ rabbit heart (31). Thus it seems likely that the isolated rabbit heart differs markedly from the in situ heart in that adenosine is the predominant preconditioning trigger in the non-blood-perfused model, and thus failure to find an endogenous NO component is not surprising. We, however, would expect there to be a strong NO component of ischemic preconditioning in the in situ rabbit heart.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-20648 and HL-50688.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Downey, Dept. of Physiology, MSB 3074, College of Medicine, Univ. of South Alabama, Mobile, AL 36688 (E-mail: jdowney{at}usouthal.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aizawa T, Wei H, Miano JM, Abe J, Berk BC, and Yan C. Role of phosphodiesterase 3 in NO/cGMP-mediated antiinflammatory effects in vascular smooth muscle cells. Circ Res 93: 406–413, 2003.[Abstract/Free Full Text]
  2. Altug S, Demiryürek AT, Kane KA, and Kanzik . Evidence for the involvement of peroxynitrite in ischaemic preconditioning in rat isolated hearts. Br J Pharmacol 130: 125–131, 2000.[CrossRef][ISI][Medline]
  3. Baines CP, Cohen MV, and Downey JM. Signal transduction in ischemic preconditioning: the role of kinases and mitochondrial KATP channels. J Cardiovasc Electrophysiol 10: 741–754, 1999.[ISI][Medline]
  4. Baines CP, Goto M, and Downey JM. Oxygen radicals released during ischemic preconditioning contribute to cardioprotection in the rabbit myocardium. J Mol Cell Cardiol 29: 207–216, 1997.[CrossRef][ISI][Medline]
  5. Balligand JL, Ungureanu-Longrois D, Simmons WW, Pimental D, Malinski TA, Kapturczak M, Taha Z, Lowenstein CJ, Davidoff AJ, Kelly RA, Smith TW, and Michel T. Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro. J Biol Chem 269: 27580–27588, 1994.[Abstract/Free Full Text]
  6. Baxter GF and Yellon DM. Delayed myocardial protection following ischaemic preconditioning. Basic Res Cardiol 91: 53–56, 1996.[CrossRef][ISI][Medline]
  7. Baxter GF and Yellon DM. Time course of delayed myocardial protection after transient A1-receptor activation in the rabbit. J Cardiovasc Pharmacol 29: 631–638, 1997.[CrossRef][ISI][Medline]
  8. Bell RM, Maddock HL, and Yellon DM. The cardioprotective and mitochondrial depolarising properties of exogenous nitric oxide in mouse heart. Cardiovasc Res 57: 405–415, 2003.[Abstract/Free Full Text]
  9. Bell RM and Yellon DM. The contribution of endothelial nitric oxide synthase to early ischaemic preconditioning: the lowering of the preconditioning threshold. An investigation in eNOS knockout mice. Cardiovasc Res 52: 274–280, 2001.[Abstract/Free Full Text]
  10. Bolli R, Bhatti ZA, Tang XL, Qiu Y, Zhang Q, Guo Y, and Jadoon AK. Evidence that late preconditioning against myocardial stunning in conscious rabbits is triggered by the generation of nitric oxide. Circ Res 81: 42–52, 1997.[Abstract/Free Full Text]
  11. Bolli R, Dawn B, Tang XL, Qiu Y, Ping P, Xuan YT, Jones WK, Takano H, Guo Y, and Zhang J. The nitric oxide hypothesis of late preconditioning. Basic Res Cardiol 93: 325–338, 1998.[CrossRef][ISI][Medline]
  12. Brahmajothi MV and Campbell DL. Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart: implications for mechanisms governing indirect and direct nitric oxide-related effects. Circ Res 85: 575–587, 1999.[Abstract/Free Full Text]
  13. Butt E, Nolte C, Schulz S, Beltman J, Beavo JA, Jastorff B, and Walter U. Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP. Biochem Pharmacol 43: 2591–2600, 1992.[CrossRef][ISI][Medline]
  14. Cohen MV, Yang XM, Liu GS, Heusch G, and Downey JM. Acetylcholine, bradykinin, opioids, and phenylephrine, but not adenosine, trigger preconditioning by generating free radicals and opening mitochondrial KATP channels. Circ Res 89: 273–278, 2001.[Abstract/Free Full Text]
  15. Csonka C, Szilvássy Z, Fülöp F, Páli T, Blasig IE, Tosaki A, Schulz R, and Ferdinandy P. Classic preconditioning decreases the harmful accumulation of nitric oxide during ischemia and reperfusion in rat hearts. Circulation 100: 2260–2266, 1999.[Abstract/Free Full Text]
  16. Das M, Parker JE, and Halestrap AP. Matrix volume measurements challenge the existence of diazoxide/glibenclamide-sensitive KATP channels in rat mitochondria. J Physiol 547: 893–902, 2003.[Abstract/Free Full Text]
  17. Dostmann WRG, Taylor MS, Nickl CK, Brayden JE, Frank R, and Tegge WJ. Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase I{alpha} inhibit NO-induced cerebral dilation. Proc Natl Acad Sci USA 97: 14772–14777, 2000.[Abstract/Free Full Text]
  18. Draijer R, Vaandrager AB, Nolte C, de Jonge HR, Walter U, and van Hinsbergh VWM. Expression of cGMP-dependent protein kinase I and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin. Circ Res 77: 897–905, 1995.[Abstract/Free Full Text]
  19. Forbes RA, Steenbergen C, and Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res 88: 802–809, 2001.[Abstract/Free Full Text]
  20. Geiger J, Nolte C, Butt E, Sage SO, and Walter U. Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets. Proc Natl Acad Sci USA 89: 1031–1035, 1992.[Abstract/Free Full Text]
  21. Goto M, Liu Y, Yang XM, Ardell JL, Cohen MV, and Downey JM. Role of bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res 77: 611–621, 1995.[Abstract/Free Full Text]
  22. Han J, Kim N, Joo H, Kim E, and Earm YE. ATP-sensitive K+ channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes. Am J Physiol Heart Circ Physiol 283: H1545–H1554, 2002.[Abstract/Free Full Text]
  23. Harper JF and Brooker G. Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution. J Cyclic Nucleotide Res 1: 207–218, 1975.[ISI][Medline]
  24. Horimoto H, Saltman AE, Gaudette GR, and Krukenkamp IB. Nitric oxide-generating {beta}-adrenergic blocker nipradilol preserves postischemic cardiac function. Ann Thorac Surg 68: 844–849, 1999.[Abstract/Free Full Text]
  25. Kubo M, Nakaya Y, Matsuoka S, Saito K, and Kuroda Y. Atrial natriuretic factor and isosorbide dinitrate modulate the gating of ATP-sensitive K+ channels in cultured vascular smooth muscle cells. Circ Res 74: 471–476, 1994.[Abstract/Free Full Text]
  26. Layland J, Li JM, and Shah AM. Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes. J Physiol 540: 457–467, 2002.[Abstract/Free Full Text]
  27. Lebuffe G, Schumacker PT, Shao ZH, Anderson T, Iwase H, and Vanden Hoek TL. ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel. Am J Physiol Heart Circ Physiol 284: H299–H308, 2003.[Abstract/Free Full Text]
  28. Lefer DJ, Nakanishi K, Johnston WE, and Vinten-Johansen J. Antineutrophil and myocardial protecting actions of a novel nitric oxide donor after acute myocardial ischemia and reperfusion in dogs. Circulation 88: 2337–2350, 1993.[Abstract/Free Full Text]
  29. Lochner A, Marais E, Genade S, and Moolman JA. Nitric oxide: a trigger for classic preconditioning? Am J Physiol Heart Circ Physiol 279: H2752–H2765, 2000.[Abstract/Free Full Text]
  30. Lu HR, Remeysen P, and De Clerck F. Does the antiarrhythmic effect of ischemic preconditioning in rats involve the L-arginine nitric oxide pathway? J Cardiovasc Pharmacol 25: 524–530, 1995.[ISI][Medline]
  31. Miki T, Cohen MV, and Downey JM. Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits. Mol Cell Biochem 186: 3–12, 1998.[CrossRef][ISI][Medline]
  32. Murry CE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.[Abstract/Free Full Text]
  33. Nakanishi K, Vinten-Johansen J, Lefer DJ, Zhao Z, Fowler WC III, McGee DS, and Johnston WE. Intracoronary L-arginine during reperfusion improves endothelial function and reduces infarct size. Am J Physiol Heart Circ Physiol 263: H1650–H1658, 1992.[Abstract/Free Full Text]
  34. Nakano A, Liu GS, Heusch G, Downey JM, and Cohen MV. Exogenous nitric oxide can trigger a preconditioned state through a free radical mechanism, but endogenous nitric oxide is not a trigger of classical ischemic preconditioning. J Mol Cell Cardiol 32: 1159–1167, 2000.[CrossRef][ISI][Medline]
  35. National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, DC: Natl. Acad. Press, 1996.
  36. Ockaili R, Salloum F, Hawkins J, and Kukreja RC. Sildenafil (Viagra) induces powerful cardioprotective effect via opening of mitochondrial KATP channels in rabbits. Am J Physiol Heart Circ Physiol 283: H1263–H1269, 2002.[Abstract/Free Full Text]
  37. Oldenburg O, Qin Q, Krieg T, Yang XM, Philipp S, Critz SD, Cohen MV, and Downey JM. Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection. Am J Physiol Heart Circ Physiol 286: H468–H476, 2004.[Abstract/Free Full Text]
  38. Pain T, Yang XM, Critz SD, Yue Y, Nakano A, Liu GS, Heusch G, Cohen MV, and Downey JM. Opening of mitochondrial KATP channels triggers the preconditioned state by generating free radicals. Circ Res 87: 460–466, 2000.[Abstract/Free Full Text]
  39. Rakhit RD, Edwards RJ, Mockridge JW, Baydoun AR, Wyatt AW, Mann GE, and Marber MS. Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes. Am J Physiol Heart Circ Physiol 278: H1211–H1217, 2000.[Abstract/Free Full Text]
  40. Sasaki N, Sato T, Ohler A, O'Rourke B, and Marbán E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation 101: 439–445, 2000.[Abstract/Free Full Text]
  41. Schulz R, Kelm M, and Heusch G. Nitric oxide in myocardial ischemia/reperfusion injury. Cardiovasc Res 61: 402–413, 2004.[Abstract/Free Full Text]
  42. Schulz R and Wambolt R. Inhibition of nitric oxide synthesis protects the isolated working rabbit heart from ischaemia-reperfusion injury. Cardiovasc Res 30: 432–439, 1995.[CrossRef][ISI][Medline]
  43. Shinbo A and Iijima T. Potentiation by nitric oxide of the ATP-sensitive K+ current induced by K+ channel openers in guinea-pig ventricular cells. Br J Pharmacol 120: 1568–1574, 1997.[ISI][Medline]
  44. Sun W and Wainwright CL. The role of nitric oxide in modulating ischaemia-induced arrhythmias in rats. J Cardiovasc Pharmacol 29: 554–562, 1997.[CrossRef][ISI][Medline]
  45. Takano H, Tang XL, Qiu Y, Guo Y, French BA, and Bolli R. Nitric oxide donors induce late preconditioning against myocardial stunning and infarction in conscious rabbits via an antioxidant-sensitive mechanism. Circ Res 83: 73–84, 1998.[Abstract/Free Full Text]
  46. Tritto I, D'Andrea D, Eramo N, Scognamiglio A, De Simone C, Violante A, Esposito A, Chiariello M, and Ambrosio G. Oxygen radicals can induce preconditioning in rabbit hearts. Circ Res 80: 743–748, 1997.[Abstract/Free Full Text]
  47. Vegh A, Szekeres L, and Parratt J. Preconditioning of the ischaemic myocardium; involvement of the L-arginine nitric oxide pathway. Br J Pharmacol 107: 648–652, 1992.[ISI][Medline]
  48. Vila-Petroff MG, Younes A, Egan J, Lakatta EG, and Sollott SJ. Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes. Circ Res 84: 1020–1031, 1999.[Abstract/Free Full Text]
  49. Wall TM, Sheehy R, and Hartman JC. Role of bradykinin in myocardial preconditioning. J Pharmacol Exp Ther 270: 681–689, 1994.[Abstract/Free Full Text]
  50. Weselcouch EO, Baird AJ, Sleph P, and Grover GJ. Inhibition of nitric oxide synthesis does not affect ischemic preconditioning in isolated perfused rat hearts. Am J Physiol Heart Circ Physiol 268: H242–H249, 1995.[Abstract/Free Full Text]
  51. Woolfson RG, Patel VC, Neild GH, and Yellon DM. Inhibition of nitric oxide synthesis reduces infarct size by an adenosine-dependent mechanism. Circulation 91: 1545–1551, 1995.[Abstract/Free Full Text]
  52. Yao Z, Tong J, Tan X, Li C, Shao Z, Kim WC, Vanden Hoek TL, Becker LB, Head CA, and Schumacker PT. Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes. Am J Physiol Heart Circ Physiol 277: H2504–H2509, 1999.[Abstract/Free Full Text]
  53. Yasmin W, Strynadka KD, and Schulz R. Generation of peroxynitrite contributes to ischemia-reperfusion injury in isolated rat hearts. Cardiovasc Res 33: 422–432, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. Kuno, N. V. Solenkova, V. Solodushko, T. Dost, Y. Liu, X.-M. Yang, M. V. Cohen, and J. M. Downey
Infarct limitation by a protein kinase G activator at reperfusion in rabbit hearts is dependent on sensitizing the heart to A2b agonists by protein kinase C
Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H1288 - H1295.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Fu, Z. Wang, W. L. Chen, P. K. Moore, and Y. Z. Zhu
Cardioprotective effects of nitric oxide-aspirin in myocardial ischemia-reperfused rats
Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1545 - H1552.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. W. Elrod, J. J. M. Greer, and D. J. Lefer
Sildenafil-mediated acute cardioprotection is independent of the NO/cGMP pathway
Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H342 - H347.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P. Cabrales, J. Martini, M. Intaglietta, and A. G. Tsai
Blood viscosity maintains microvascular conditions during normovolemic anemia independent of blood oxygen-carrying capacity
Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H581 - H590.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
E. B. Manukhina, H. F. Downey, and R. T. Mallet
Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia.
Experimental Biology and Medicine, April 1, 2006; 231(4): 343 - 365.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Lindenfeld, J. V. Weil, V. L. Travis, and L. D. Horwitz
Regulation of oxygen delivery during induced polycythemia in exercising dogs
Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1821 - H1825.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Krieg, S. Philipp, L. Cui, W. R. Dostmann, J. M. Downey, and M. V. Cohen
Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart
Am J Physiol Heart Circ Physiol, April 1, 2005; 288(4): H1976 - H1981.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/2/H712    most recent
00954.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (38)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qin, Q.
Right arrow Articles by Downey, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qin, Q.
Right arrow Articles by Downey, J. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.