Inducible Nitric Oxide Synthase Modulates Cyclooxygenase-2 Activity in the Heart of Conscious Rabbits During the Late Phase of Ischemic Preconditioning
Cyclooxygenase-2 (COX-2) is known to mediate the cardioprotective effects of the late phase of ischemic preconditioning (PC); however, the signaling pathways involved in COX-2 induction following ischemic PC are unknown. In addition, although inducible nitric oxide synthase (iNOS) has been identified as a co-mediator of late PC together with COX-2, the interaction between iNOS and COX-2 in the heart is unknown. Using conscious rabbits, we found that the induction of COX-2 expression 24 hours after ischemic PC was blocked by pretreatment with inhibitors of protein kinase C (PKC), Src protein tyrosine kinases (PTKs), and nuclear factor-κB (NF-κB) but not by inhibitors of NOS or scavengers of reactive oxygen species (ROS). The selective iNOS inhibitors SMT and 1400W, given 24 hours after PC, abrogated the increase in myocardial prostaglandin E2 (PGE2) and 6-keto-PGF1α, whereas the selective soluble guanylate cyclase inhibitor ODQ had no effect. COX-2 selective inhibitors (celecoxib and NS-398) did not affect iNOS activity. These results demonstrate that (i) ischemic PC upregulates cardiac COX-2 via PKC-, Src PTK-, and NF-κB–dependent signaling pathways, whereas generation of NO and ROS is not necessary, and (ii) the activity of newly synthesized COX-2 following PC requires iNOS-derived NO whereas iNOS activity is independent of COX-2–derived prostanoids, indicating that COX-2 is located downstream of iNOS in the protective pathway of late PC. The data also indicate that iNOS modulates COX-2 activity via cGMP-independent mechanisms. To our knowledge, this is the first demonstration that iNOS-derived NO drives prostanoid synthesis by COX-2 in the heart. NO-mediated activation of COX-2 may be a heretofore unrecognized mechanism by which NO exerts its salubrious effects in the late phase of PC.
Exposure of the heart to a sublethal ischemic stress induces a phenotypic change that renders the myocardium relatively resistant to a subsequent ischemic insult occurring 24 to 72 hours later (late phase of ischemic preconditioning [PC])1–12⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ (reviewed in Reference 13). Recent evidence indicates that late PC is associated with upregulation of cyclooxygenase-2 (COX-2) expression and that COX-2–dependent synthesis of prostanoids (ie, prostaglandin [PG] E2 and PGI2) is essential for the observed cardioprotective effects.11,12⇓ Prostanoids have been shown to alleviate myocardial ischemia/reperfusion injury (eg, see References 14 and 15). However, the signaling pathways whereby a sublethal ischemic stress leads to increased expression of COX-2 in the heart are unknown. Although COX-2 has been shown to be upregulated by phorbol ester and oxidative stress in isolated neonatal myocytes,16 no information is currently available regarding the modulation of COX-2 in adult myocardium. In noncardiac cells, the expression of COX-2 has been reported to be controlled (among other factors) by reactive oxygen species (ROS),17 protein kinase C (PKC),18,19⇓ protein tyrosine kinases (PTKs),20,21⇓ and nuclear factor-κB (NF-κB).17,22⇓ These signaling elements have also been implicated in late PC by a number of studies which have demonstrated that the development of delayed cardioprotection is triggered by the formation of NO and ROS during the initial PC stimulus and by the subsequent early activation of a cascade that involves the sequential recruitment of PKC, Src PTKs, and NF-κB.3–7,9,10,13,23⇓⇓⇓⇓⇓⇓⇓⇓ Consequently, in the present study, we tested the hypothesis that these elements participate in the induction of COX-2 in response to sublethal myocardial ischemia.
Besides COX-2, pharmacological and genetic evidence has convincingly shown that the inducible isoform of nitric oxide synthase (iNOS) also plays a necessary role in mediating the cardioprotective effects of late PC.4,13,24⇓⇓ Since both iNOS and COX-2 are obligatory mediators of late PC, the question arises as to whether there is an interaction between these two proteins or they act as independent effectors of cardioprotection. At present, this aspect of COX biology is poorly understood. A possible “cross-talk” between NOS and COX has been extensively examined in noncardiac tissues, with conflicting results (reviewed in Reference 25). While some studies suggest that NO enhances COX-2 activity, 26–30⇓⇓⇓⇓ others have concluded that NO inhibits it31–35⇓⇓⇓⇓ or has no effect.36–38⇓⇓ To our knowledge, the relationship between iNOS and COX-2 in the heart has never been evaluated.
Accordingly, the present study had two major aims. In phase I, we explored the signaling pathways responsible for the induction of COX-2 during late PC. We systematically interrogated the role of NO, ROS, PKC, PTKs, and NF-κB in the induction of COX-2 using pharmacological interventions that are specifically targeted at each of these signaling elements. In phase II, we sought to elucidate the interaction between iNOS and COX-2. To this end, we determined whether inhibiting iNOS activity in preconditioned myocardium interferes with COX-2 activity and, conversely, whether blocking COX-2 activity affects iNOS activity. We also investigated whether the modulation of COX-2 by iNOS involves cGMP-dependent mechanisms. All studies were conducted in conscious animals to obviate the potential confounding influence of conditions associated with open-chest animal preparations,4,5⇓ particularly since both COX-2 and iNOS are known to be stress-responsive enzymes.25 The results demonstrate, for the first time, that sublethal ischemia upregulates COX-2 expression via PKC-, PTK-, and NF-κB–dependent pathways (but not via generation of NO or ROS), and that COX-2 activity 24 hours after ischemic PC is dependent on iNOS-derived NO, thus establishing a hierarchical relationship between iNOS and COX-2 in the preconditioned heart.
Materials and Methods
The conscious rabbit model of late PC has been described previously.4–7,9–11,24⇓⇓⇓⇓⇓⇓⇓ Details of this model, as well as the sources of the reagents used, are provided in the online supplement at http://www.circresaha.org. The study consisted of two consecutive phases (phases I and II).
Phase I: Mechanism of COX-2 Induction
Rabbits were assigned to seven groups (Figure 1). Group I (control group) did not receive any treatment and did not undergo coronary occlusion. Myocardial samples (≈500 mg) were obtained from the anterior and posterior left ventricular (LV) walls and stored at −140°C until used. Group II (PC group) underwent a sequence of six 4-minute coronary occlusions interspersed with 4 minutes of reperfusion without any treatment and was euthanized 24 hours after the last reperfusion. Myocardial samples were obtained from the ischemic-reperfused region and from the nonischemic region (posterior LV wall), frozen in liquid nitrogen, and stored at −140°C until used. Groups III through VIII underwent the same sequence of occlusion/reperfusion cycles. In addition, group III (L-NA+PC group) received an i.v. infusion of Nω-nitro-l-arginine (L-NA; a nonselective inhibitor of all three NOS isoforms) at a rate of 1.3 mg/kg/min for 10 minutes, starting 20 minutes before and ending 10 minutes before the first coronary occlusion. Group IV (MPG+PC group) received a continuous i.v. infusion of the ROS scavenger N-2-mercaptopropionyl glycine (MPG; 0.42 mg/kg/min), beginning 60 minutes before the first coronary occlusion and ending 30 minutes after the sixth reperfusion. Group V (CHE+PC group) received an i.v. bolus of the PKC inhibitor chelerythrine (5 mg/kg) 5 minutes before the first coronary occlusion. Group VI (LD-A+PC group) received an i.v. bolus of the PTK inhibitor lavendustin-A (LD-A; 1 mg/kg) 10 minutes before the first coronary occlusion. Group VII (DDTC+PC group) received an i.p. bolus of the NF-κB inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg) 15 minutes before the first coronary occlusion. The dosages of L-NA, MPG, chelerythrine, LD-A, and DDTC were the same as those previously shown to block both the activation of their respective targets6,9,10,24⇓⇓⇓ and the cardioprotective effects of late PC in this model.5–7,10,23⇓⇓⇓⇓ Group VIII (DMSO+PC group) received an i.v. bolus of the vehicle used in group V for chelerythrine (50% DMSO in saline, 2 mL/kg) 5 minutes before the first coronary occlusion (the dose of DMSO used in group V to dissolve chelerythrine was higher than that used in group VI to dissolve LD-A). In all treated groups (groups III–VIII), rabbits were euthanized 24 hours after PC and myocardial samples were obtained as described above.
Western Immunoblotting Analysis
The expression of COX-2 was assessed by using standard SDS-PAGE Western immunoblotting techniques.10,11⇓ Specific monoclonal anti–COX-2 antibodies were purchased from Transduction Laboratories. Each COX-2 signal was normalized to the corresponding Ponceau stain.10,11⇓ In all samples, the content of COX-2 protein was expressed as a percentage of the COX-2 protein in the anterior LV wall of group I (control group).
Phase II: Interaction Between iNOS and COX-2
Rabbits were assigned to five groups (group IX–XIII) (Figure 2) (tissue samples obtained from groups I (control), II (PC), and III (L-NA+PC) were also used for these studies; therefore, groups I through III are included in both phases I and II). On day 1, all five groups underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles without any treatment. Twenty-four hours later (day 2), rabbits received an i.p. injection of the selective COX-2 inhibitors NS-398 (5 mg/kg) (group IX [PC+NS-398]) or celecoxib (3 mg/kg) (group X [PC+celecoxib]) and were euthanized 40 minutes after the injection. These doses of NS-398 and celecoxib have previously been shown to block the activity of COX-2 24 hours after ischemic PC in this same rabbit model.11 Rabbits in group XI (PC+SMT) received five consecutive i.v. boluses of S-methylisothiourea sulfate (SMT), a relatively selective iNOS inhibitor (0.05 mg/kg each), every 8 minutes (total dose, 0.25 mg/kg) and were euthanized 8 minutes after the last bolus. This dose of SMT has previously been shown to block the activity of iNOS (without affecting eNOS) 24 hours after ischemic PC in this rabbit model.24 Rabbits in group XII (PC+1400W) received an i.v. bolus of the selective iNOS inhibitor 1400W (0.5 mg/kg) and were euthanized 30 minutes later. This dose of 1400W was chosen on the basis of pilot studies which showed (i) that late PC against myocardial infarction was abrogated by i.v. injection of 0.5 mg/kg of 1400W on day 2, 10 minutes before the 30-minute coronary occlusion (n=5), indicating effective inhibition of iNOS, and (ii) that the development of late PC against infarction was not abolished even by a higher dose (1.0 mg/kg) of 1400W given on day 1, just before the six coronary occlusion/reperfusion cycles (n=5), indicating that eNOS activity (which likely triggers late PC24) was not inhibited by 1400W. Thus, the 0.5 mg/kg dose of 1400W used herein selectively inhibits iNOS in the rabbit. In group XIII, rabbits received 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a selective inhibitor of NO-stimulated soluble guanylate cyclase (5 mg/kg, i.p.) and were euthanized 30 minutes later. Myocardial samples were obtained as described above for phase I.
Measurement of NOS Activity, Nitrite and Nitrate (NOx), and PGE2 and 6-keto-PGF1α
Calcium-dependent NOS (cNOS [eNOS and/or nNOS]) and calcium-independent NOS (iNOS) activities were determined by measuring the conversion of [14C]l-arginine to [14C]l-citrulline, as previously detailed.7,24⇓ NOS activity was expressed as pmol of l-citrulline/min per mg protein. Nitrite and nitrate were assayed by using a modified Griess reaction, as described.7,24⇓
Late PC in this rabbit model is associated with increased myocardial levels of PGE2 and 6-keto-PGF1α (the stable metabolite of PGI2); PGF2α increased only marginally and PGD2 and thromboxane B2 do not change.11 Accordingly, in the present study, we focused on PGE2 and 6-keto-PGF1α. The myocardial content of these prostanoids was determined using enzyme immunoassay (EIA) kits (PGE2 EIA kit from Cayman Chemical; 6-keto-PGF1α EIA kit from Amersham Life Science) and expressed as pg/mg of protein.11
Data are reported as mean±SEM. Differences among groups with respect to COX-2 protein levels, myocardial PG content, and NOS activity were analyzed using a one-way ANOVA followed by Student’s t tests with the Bonferroni correction.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
A total of 95 conscious rabbits were studied (52 in phase I, 33 in phase II, and 10 for pilot studies).
Phase I: Mechanism of COX-2 Induction
In control rabbits (group I), more than 99% of total COX-2 protein was found in the membranous fraction. Representative examples of Western immunoblotting analyses of COX-2 in membranous fractions prepared from each group are illustrated in Figure 3. The densitometric analysis of all Western immunoblots is summarized in Figure 4. As previously noted,11 a weak COX-2 signal was detected in control hearts. When rabbits were preconditioned with six 4-minute occlusion/4-minute reperfusion cycles (group II), the expression of COX-2 in the ischemic-reperfused region increased markedly 24 hours later (3.0-fold increase versus the anterior LV wall of control rabbits [P<0.05]) (Figures 3 and 4⇓), in keeping with our prior observations.11 Administration of chelerythrine, LD-A, or DDTC before ischemic PC completely prevented the ischemic PC-induced increase in COX-2 protein 24 hours later (Figures 3 and 4⇓). In contrast, administration of MPG or L-NA failed to abolish the upregulation of COX-2 24 hours after ischemic PC (Figures 3 and 4⇓). DMSO (the vehicle used for chelerythrine and LD-A), in itself, had no effect on COX-2 expression. No increase in COX-2 protein was detected in the nonischemic region of preconditioned hearts (data not shown).
Phase II: Interaction Between iNOS and COX-2
NOS Activity and Myocardial NOx
In preconditioned rabbits (group II), total (cytosolic+membranous fractions) calcium-independent NOS (iNOS) activity and nitrite+nitrate (NOx) levels in the ischemic-reperfused region increased 153% (Figure 5) and 58% (Figure 6), respectively, above control levels 24 hours after ischemic PC, consistent with previous observations.24 Treatment with L-NA prior to ischemic PC (group III) abrogated the increase in iNOS activity (Figure 5) and NOx levels (Figure 6) in the ischemic-reperfused region 24 hours later, indicating that the activity of NOS (presumably eNOS) during ischemic PC is necessary for the induction of iNOS and that the dose of L-NA used here effectively blocks this process. The administration of a COX-2 selective inhibitor (NS-398 or celecoxib) 24 hours after ischemic PC (groups IX and X) did not affect iNOS activity (Figure 5) or NOx levels (Figure 6). However, administration of the iNOS selective inhibitors SMT and 1400W (groups XI and XII) 24 hours after ischemic PC completely abrogated the increase in iNOS activity and NOx content in preconditioned myocardium. In contrast to iNOS activity, calcium-dependent NOS (cNOS [eNOS and/or nNOS]) activity in the ischemic-reperfused region did not increase 24 hours after ischemic PC and was not affected by any of the inhibitors examined (Figure 5), indicating that the doses of SMT and 1400W used in the present study selectively block iNOS without affecting eNOS.
Myocardial PGE2 and 6-keto-PGF1α Content
As previously documented,11 ischemic PC resulted in a marked increase, 24 hours later, in the myocardial content of PGE2 and 6-keto-PGF1α (+217% and +241%, respectively [P<0.05] (Figure 7). When rabbits were given a selective COX-2 inhibitor (NS-398 or celecoxib) on day 2, 40 minutes prior to euthanasia (groups IX and X), the increase in myocardial PGE2 and 6-keto-PGF1α levels in the preconditioned myocardium was abrogated (Figure 7), indicating that it is COX-2–dependent. However, neither NS-398 nor celecoxib lowered PGE2 and 6-keto-PGF1α below control values, indicating that constitutive COX activity (COX-1) was not suppressed by these doses of drugs. This conclusion is further corroborated by the finding that NS-398 and celecoxib had no effect in the nonischemic region (Figure 7).
Both SMT and 1400W, given 24 hours after ischemic PC (groups XI and XII), inhibited the increase in PGE2 and 6-keto-PGF1α levels (Figure 7), demonstrating that COX-2 activity in preconditioned myocardium is dependent on iNOS activity. In further support of this conclusion, pretreatment with L-NA prior to ischemic PC (group III) inhibited the increase in PGE2 and 6-keto-PGF1α levels 24 hours later (Figure 7) but did not affect COX-2 protein induction (Figures 3 and 4⇑). Since pretreatment with L-NA blocked the upregulation of iNOS (Figure 5), these data are consistent with the concept that COX-2 activity during late PC is dependent on iNOS activity, which in turn is dependent on NO generation during the PC stimulus. None of the NOS inhibitors tested (L-NA, SMT, 1400W) affected prostanoid levels in the nonischemic myocardium (Figure 7).
Effect of ODQ on Myocardial PGE2 and 6-keto-PGF1α Content
As shown in Figure 7, administration of the selective guanylate cyclase inhibitor ODQ, 24 hours after ischemic PC, had no effect on the myocardial levels of either PGE2 or 6-keto-PGF1α, despite the fact that this dose of ODQ blocks the protective effects of late PC.42 These data indicate that iNOS modulates COX-2 activity via cGMP-independent mechanisms.
Although COX-2 has been identified as an obligatory mediator of late PC,11,12⇓ virtually nothing is currently known regarding the regulation of COX-2 activity in the heart or the mechanism whereby myocardial ischemia upregulates the expression of this poorly understood enzyme. Furthermore, the nature of the interaction (if any) between COX-2 and iNOS (another essential co-mediator of late PC) is not known. The present study provides considerable new information relevant to these issues. The results of phase I demonstrate that, in conscious rabbits, the induction of COX-2 protein expression in preconditioned myocardium occurs via PKC-, Src PTK-, and NF-κB–dependent signaling pathways. The results of phase II demonstrate that COX-2– (but not COX-1–) dependent prostanoid synthesis in preconditioned myocardium is dependent on iNOS activity, thereby revealing that COX-2 is downstream of iNOS. Phase II also demonstrates that iNOS drives COX-2 via a cGMP-independent pathway. To our knowledge, this is the first study to identify the signaling mechanisms that control COX-2 expression in the heart. This is also the first investigation to elucidate the interaction between cardiac COX-2 and cardiac iNOS, two inducible proteins that play a central role in the late phase of ischemic PC and in the response of the heart to stress in general. The notion that iNOS-derived NO modulates the enzymatic activity of COX-2 in the heart expands our understanding of the mechanism whereby iNOS exerts its cardioprotective effects.
Mechanism of COX-2 Induction
No information is presently available regarding the role of NO, ROS, PKC, PTKs, and NF-κB in regulating COX-2 expression in the heart. Previous studies have shown that the development of late PC is triggered by enhanced synthesis of NO and ROS and by the recruitment of a signaling cascade that involves the sequential activation of PKC, Src/Lck PTKs, and NF-κB.3–7,9,10,13,23⇓⇓⇓⇓⇓⇓⇓⇓ Therefore, in the present study, we tested the role of this well-established PKC/Src PTK/NF-κB pathway. The results of phase I demonstrate that the induction of COX-2 protein expression following ischemic PC was blocked by the PKC inhibitor chelerythrine, the PTK inhibitor LD-A (which is targeted at the Src family of PTKs), and the NF-κB inhibitor DDTC (Figure 4), given at doses previously shown to completely inhibit PKCε activation,6 Src and Lck PTK activation,9 and NF-κB activation,10 respectively, in this same conscious rabbit model of late PC. These findings indicate that upregulation of COX-2 protein expression requires PKC-, Src PTK–, and NF-κB–dependent signaling and imply that PKC, Src PTKs, and NF-κB participate in the genesis of late PC, at least in part, via the upregulation of COX-2.
In contrast, administration of the NOS inhibitor L-NA or the ROS scavenger MPG prior to ischemic PC failed to block COX-2 induction (Figure 4), indicating that the generation of NO- and MPG-sensitive ROS during the PC ischemia is not necessary to upregulate the synthesis of COX-2 protein. Since both L-NA and MPG were given in doses that completely block the development of late PC in this conscious rabbit model,4,5,23⇓⇓ the failure of these agents to block COX-2 induction implies that NO and ROS participate in late PC by upregulating proteins other than COX-2 (eg, iNOS),8 thereby supporting the notion13 that the switch of the heart to a preconditioned phenotype is a polygenic response (ie, COX-2 induction is necessary but not sufficient).
The precise cell type(s) that express(es) COX-2 in the heart in response to ischemic PC remain(s) unclear. Virtually all mammalian cells are capable of expressing COX-2.39 In vitro exposure to hypoxia, oxidative stress, or cytokines has been shown to induce COX-2 in endothelial cells and cardiac myocytes.16,40,41⇓⇓ Studies using in situ hybridization and/or immunohistochemistry will be needed to determine the cellular specificity of COX-2 expression in the preconditioned heart in vivo.
Interaction Between iNOS and COX-2
The nature of the interaction between iNOS and COX-2 has been found to vary widely in different cell lines and tissues and under different pathophysiological conditions26–38⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ (reviewed in Reference 25). Consequently, it is important to elucidate this interaction in the heart and in the specific setting of late PC. Since iNOS and COX-2 are frequently co-induced,39 understanding the manner in which these two stress-responsive proteins interact in the heart is important not only with respect to myocardial ischemia but also from the standpoint of cardiovascular pathophysiology in general. Phase II of this study demonstrates that the increase in myocardial PGE2 and 6-keto-PGF1α levels observed 24 hours after ischemic PC was ablated by administration of two selective iNOS inhibitors (SMT and 1400W) on day 2 (Figure 7) (both given at doses that inhibited iNOS activity [Figure 5]) or by pretreatment 24 hours earlier (on day 1) with L-NA (Figure 7), which prevents ischemia-induced upregulation of iNOS in this model.8 All three inhibitors (SMT, 1400W, and L-NA) were administered at doses that abrogate late PC in this model (References 4 and 5 and pilot studies described in Materials and Methods). These data indicate that the enhanced prostanoid biosynthesis associated with late PC is dependent on generation of NO by iNOS; that is, COX-2 is downstream of iNOS in preconditioned myocardium. Since augmented COX-2 activity is necessary for the acquisition of ischemic tolerance during late PC,11,12⇓ the present results imply that iNOS reduces myocardial stunning and infarction, at least in part, by recruiting COX-2. This concept is novel, as activation of COX-2 has not been previously recognized as a mechanism for NO-dependent cytoprotection.
Despite extensive investigation, the mechanism whereby NO (or NO-derived species) stimulates COX-2 activity remains obscure.25 Our finding that the upregulation of prostanoid levels 24 hours after ischemic PC is not affected by the soluble guanylate cyclase inhibitor ODQ (Figure 7), given at doses that effectively block the increase in cGMP levels associated with late PC,42 indicates that iNOS-derived NO activates cardiac COX-2 via cGMP-independent mechanisms, supporting the hypothesis of a direct interaction between NO and the COX-2 molecule.26 An important implication of the results obtained with ODQ is that although enhanced synthesis of prostanoids is necessary for late PC to occur, it is not sufficient, because the cardioprotective effects of late PC are abrogated by the same dose of ODQ that failed to abolish the upregulation of COX-2 activity in the present study, 42 demonstrating that cGMP-dependent mechanisms are also necessary. On the basis of these facts, we propose that the protection afforded by late PC is multifactorial, requiring both enhanced prostanoid synthesis by COX-211,12⇓ (which is cGMP-independent, as demonstrated in the present study) and other events (eg, inhibition of L-type calcium channels), which are cGMP-dependent13 (and thus ODQ-sensitive). A multifactorial (and polygenic) nature of late PC is further supported by the fact that pretreatment on day 1 with L-NA and MPG blocks the development of late PC4,5,23⇓⇓ but not the upregulation of COX-2 protein expression (Figure 4), implying that proteins other than COX-2 are necessary for the preconditioned phenotype to become manifest.
In contrast to the effects of NO on COX-2, there is a paucity of information on the effects of COX metabolites on the enzymatic activity of NOS. In the present study, administration of COX-2 selective inhibitors (NS-398 and celecoxib) did not produce any appreciable effect on either iNOS activity (Figure 5) or myocardial NOx levels (Figure 6), indicating that COX-2–derived prostanoids do not modulate iNOS in the preconditioned heart. Thus, the interaction is unidirectional, corroborating the conclusion that iNOS is located upstream of COX-2 in the protective pathway of late PC.
This study offers novel insights into the mechanisms that regulate COX-2 expression in cardiac tissue and into the relationship between iNOS and COX-2 during late PC. The results demonstrate that ischemia upregulates COX-2 protein expression via PKC-, Src PTK–, and NF-κB–dependent mechanisms, whereas generation of NO and ROS is not necessary. Thus, activation of the PKC/Src PTK/NF-κB cascade, which has previously been identified as an early event in the genesis of late PC,3–7,9,10,13,23⇓⇓⇓⇓⇓⇓⇓⇓ plays a crucial role in COX-2 induction. The data further demonstrate that the enzymatic activity of newly synthesized COX-2 following PC requires iNOS-derived NO whereas iNOS activity is independent of COX-2–derived prostanoids. Thus, COX-2 is located downstream of iNOS in the protective pathway of late PC. To our knowledge, this is the first demonstration of such an interaction between COX-2 and iNOS in the heart. We propose that stimulation of COX-2 activity and production of prostanoids, such as PGE2 and PGI2, may be a previously unrecognized mechanism by which NO protects the ischemic myocardium. The concept that cardiac COX-2 activity is driven by NO may have relevance to other pathophysiological processes in which iNOS and COX-2 have been implicated and also to the development of novel therapeutic strategies aimed at enhancing the production of cardioprotective prostanoids in ischemic myocardium with NO-releasing agents.
This study was supported in part by NIH grants R01 HL-43151, HL-55757, and HL-68088 (R.B.), by American Heart Association, Ohio Valley Affiliate, Inc, grant 9951533V (X.-L.T.), and 9920593V (K.S.), by the Medical Research Grant Program of the Jewish Hospital Foundation, Louisville, Ky, and by the Commonwealth of Kentucky Research Challenge Trust Fund. We gratefully acknowledge Gregg Shirk, Larisa Hodge for expert technical assistance, and Marcia Joines and Carla Hilse for expert secretarial assistance.
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received November 1, 2001; revision received December 19, 2001; accepted January 16, 2002.
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