This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xi, L. Articles by Kukreja, R. C. Search for Related Content PubMed PubMed Citation Articles by Xi, L. Articles by Kukreja, R. C. Related Collections Receptor pharmacology Animal models of human disease Ischemic biology - basic studies

(Circulation. 1999;99:2157-2163.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Essential Role of Inducible Nitric Oxide Synthase in Monophosphoryl Lipid A–Induced Late Cardioprotection

Evidence From Pharmacological Inhibition and Gene Knockout Mice

Lei Xi, MD; Novlet C. Jarrett, MS; Michael L. Hess, MD; Rakesh C. Kukreja, PhD

From the Division of Cardiology, Medical College of Virginia, Virginia Commonwealth University, Richmond.

Correspondence to Dr Rakesh C. Kukreja, Eric Lipman Professor, Division of Cardiology, Box 980281, Medical College of Virginia, Richmond, VA 23298. E-mail rakesh{at}hsc.vcu.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Monophosphoryl lipid A (MLA), a nontoxic analogue of endotoxin, is a pharmacological agent that is known to have anti-ischemic effects. Mechanisms involved with the cardioprotection are still unclear. A role for inducible nitric oxide synthase (iNOS) was recently proposed. We tested this hypothesis using S-methylisothiourea (SMT), one of the specific pharmacological inhibitors of iNOS, as well as iNOS gene knockout mice.

Methods and Results—Adult male ICR or B6,129 mice were pretreated with either MLA 35 or 350 µg/kg IP (MLA35 or MLA350) or vehicle 24 hours before global ischemia/reperfusion, which was carried out in a Langendorff isolated perfused heart model (n=8 to 9 per group). Another group of MLA350 mice received SMT 3 mg/kg IP 30 minutes before heart perfusion. Ventricular contractile function and heart rate were not different between the groups during the preischemia and reperfusion periods (P>0.05). Preischemic basal coronary flow was significantly increased in all MLA350 but not MLA35 mice. Myocardial infarct size was reduced significantly, from 26.9±2.9% of risk area in vehicle-treated mice to 13.5±2.4% in the MLA350 group (mean±SEM, P<0.05). This reduction in infarct size was accompanied by augmented nitrite/nitrate accumulation, from 0.23±0.05 nmol/mg protein in the vehicle group to 0.97±0.27 nmol/mg protein in MLA350 mice (P<0.01). Infarct size increased significantly, to 22.2±2.8% after treatment with SMT in the MLA350 group. Furthermore, MLA350 failed to reduce infarct size in iNOS knockout mice (25.5±3.6%).

Conclusions—These results demonstrate a direct association of infarct size reduction with increased NO production with MLA350. An obligatory role for iNOS in mediating the cardioprotective effect induced by MLA was confirmed with the pharmacological inhibition and gene knockout mice.

Key Words: ischemia • reperfusion • myocardial infarction • pharmacology • nitric oxide

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Protection of the ischemic heart has been the subject of experimental and clinical research for more than 2 decades. A number of protection strategies have been developed, including the pharmacological approaches.^{1 2} The phenomenon called "preconditioning," with its potent ability to enhance cellular endogenous mechanisms against ischemia/reperfusion injury, has been extensively investigated by many investigators. Identification of novel pharmacological agents that can potentially induce long-lasting protection against ischemia and reperfusion injury is currently a major area of investigation. Among the several agents that can potentially precondition myocardium against ischemia, monophosphoryl lipid A (MLA) has been well investigated and is thought to be promising for future clinical applications in humans.³

MLA is an analogue of endotoxin, which was derived and purified from bacterial lipopolysaccharide in the 1980s.³ It retains several of the immunomodulatory properties of the parent endotoxin molecule without the associated toxicity. MLA appears to maintain many of the beneficial immunological activities of the parent molecule, including induction of tolerance to endotoxemia in both laboratory animals^{4 5} and human subjects.⁶ These beneficial effects of MLA may be achieved via its ability to induce cytokines, macrophage activation, and colony-stimulating factor induction with much less toxic effects than are associated with the parent endotoxin.^{5 7 8 9} To date, this less toxic and less pyrogenic agent has been investigated primarily for use as an immunotherapeutic,¹⁰ immunoprophylactic,¹¹ or adjuvant for vaccines.¹² Studies have shown that MLA also has cardioprotective effects when administered 24 hours before ischemia/reperfusion in rats,^{13 14} rabbits,^{2 15 16} dogs,^{17 18} and cultured adult rat cardiac myocytes.¹⁹

The mechanism of MLA-induced protection is not well understood, although several possibilities have been suggested. It was shown that lipopolysaccharide pretreatment induces heat shock protein 70i expression in rat myocardium that is associated with the delayed cardioprotection.²⁰ However, MLA failed to induce a similar induction of this protein in rabbit heart.^{2 15} More recently, Zhao et al¹⁶ demonstrated that delayed cardioprotection with MLA can be abolished by aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS). Unfortunately, pharmacological inhibitors do not always give satisfactory answers because many of these agents are not highly specific and do not entirely inhibit the target enzyme. Because NO is produced from L-arginine through a chemical reaction that is catalyzed by at least 3 major isoforms of NOS, ie, iNOS (inducible), eNOS (endothelial), and nNOS (neuronal),²¹ there is an unavoidable redundancy in functional actions among the different NOS isoforms. Therefore, the exact role of iNOS in MLA-induced cardioprotection requires further direct confirmation with more specific methods, such as state-of-the-art gene knockout technology. The iNOS gene knockout mice that were recently developed²² provide us an excellent opportunity to study the role of iNOS in the mechanisms of MLA-induced cardioprotection. The present study in a murine model focused on the following 3 specific aims: (1) to demonstrate that MLA induces delayed cardioprotection in the mouse heart, (2) to determine that MLA-induced cardioprotection is mediated by NO, and (3) to confirm that an intact iNOS system is obligatory for the MLA-induced cardioprotection.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
Adult male outbred ICR mice were supplied by Harlan Sprague Dawley Co (Indianapolis, Ind), and the adult male iNOS gene knockout B6,129 mice were purchased from Jackson Laboratory (Bar Harbor, Me). The iNOS knockout mice were generated according to Laubach et al.²² In brief, to generate chimeric mice, C57BL/6J (B6) blastocysts were injected with the recombinant 129-derived ES cells and implanted into pseudopregnant females for development. Chimeric males were then mated with B6 females, and the resulting B6,129 F₁ heterozygote mutant (+/-) mice were interbred to generate F₂ homozygous mutant (-/-) mice for the iNOS disruption. Their progeny were genotyped by Southern analysis. The iNOS knockout mice were indistinguishable from wild-type mice in appearance, growth rate, reproduction, and histology.

Drugs
MLA and its vehicle solvent were provided by Ribi ImmunoChem Research Inc. The vehicle was composed of 40% propylene glycol and 10% ethyl alcohol in water. Mice were treated intraperitoneally with either MLA 35 or 350 µg/kg or volume-matched vehicle. S-Methylthiourea sulfate (SMT), a selective inhibitor of iNOS,²³ was purchased from Sigma Chemical Co.

Langendorff-Perfused Isolated Heart Preparation
The methodology of isolated perfused mouse heart was described in detail previously.^{24 25}

Ischemia/Reperfusion Protocol
After a 30-minute stabilization period, hearts were subjected to 20 minutes of no-flow normothermic global ischemia and 30 minutes of reperfusion. The 7 experimental groups involved in this study are shown in Figure 1.

View larger version (32K): [in this window] [in a new window]   Figure 1. Experimental protocol. Seven experimental groups (n=8 or 9 each) were used: (1) Vehicle: mice pretreated with vehicle (IP) 24 hours before ischemia/reperfusion (I/R); (2) MLA35: pretreated with MLA 35 µg/kg IP 24 hours before I/R; (3) MLA350: pretreated with MLA 350 µg/kg IP 24 hours before I/R; (4) Vehicle+SMT: pretreated with vehicle 24 hours before I/R. SMT 3 mg/kg IP was given 30 minutes before I/R; (5) MLA350+SMT: pretreated with MLA 350 µg/kg IP 24 hours before I/R. SMT 3 mg/kg IP was administered 30 minutes before I/R; (6) iNOS-KO: iNOS knockout mice subjected to I/R without pretreatment; and (7) MLA350+iNOS-KO: iNOS knockout mice pretreated with MLA 350 µg/kg IP 24 hours before I/R.

Exclusion Criteria
Hearts were excluded from further data analysis as a result of one of the following undesirable situations: (1) time delay in the aortic cannulation (>3 minutes), (2) damage of aorta during the cannulation, (3) sustained arrhythmia during the 30 minutes of stabilization, and (4) depressed ventricular developed force (<0.1 g) at the end of stabilization.

Measurement of Infarct Size
At the end of the experiment, hearts were immediately removed from the Langendorff apparatus, weighed, and frozen at –20°C. The frozen heart was then cut manually into 7 or 8 transverse slices of approximately equal thickness (0.8 mm) and stained by incubation in 10% triphenyl tetrazolium chloride (TTC) for 30 minutes. TTC buffer was then replaced by 10% formaldehyde, and the slices were fixed for 4 to 6 hours before measurement of the infarct area and the risk zone by computer morphometry (Bioquant System IV). The risk area was the sum of total ventricular area minus cavities. The infarct size was calculated as percent of risk area.

Measurement of NO Products
Thirty-six ICR mice were pretreated with vehicle or MLA 35 or 350 µg/kg IP. Twenty-four hours later, hearts were isolated and subjected to either 5 minutes of aerobic perfusion (37°C) for washing out the blood (ie, nonischemic hearts) or ischemia/reperfusion as described above (n=6 each). The ventricular tissue was immediately collected, frozen, and stored at –80°C. For preparation of tissue extracts, 2 mL of ice-cold homogenization buffer (0.1 mmol/L phosphate buffer, pH 7.4) was added to the powdered tissue sample, and the mixture was homogenized in a Polytron equipped with a PT10 probe. The homogenate was then spun in a microcentrifuge for 10 minutes, and the supernatant (representing the cytosol) was transferred to a fresh tube and kept frozen at –80°C until analyzed. Protein concentration was determined with a BIO-RAD protein assay kit. Total NO oxidation products were measured with a SIEVERS nitric oxide analyzer (model 280NOA). NO undergoes a series of reactions with several molecules present in biological fluids, leading to the accumulation of the final products, nitrite and nitrate. Thus, the index of total NO production is the sum of both nitrite and nitrate accumulated in the tissue samples. The reducing agent used for the analysis was a saturated solution of vanadium (III) chloride (VCl₃) in 1 mol/L HCl. To minimize foaming from the heart tissue samples containing protein, 100 µL of a 1:30 dilution of Dow Corning Antifoam C was added to the VCl₃ reagent. Five milliliters of the reagent was used in the purge vessel for analysis of 30 to 50 samples. At a temperature of 90°C, VCl₃ reagent quantitatively converts nitrite, nitrate, and S-nitroso compounds to NO, which is then measured by the NO analyzer.

Data Analysis and Statistics
Each experimental group consisted of 8 or 9 animals. The group means and their SEMs for each parameter are presented. One-way ANOVA was used to compare the values of 3 groups. If a significant value of F was obtained, the Student-Newman-Keuls post hoc test was subsequently used to make pairwise comparisons among the groups. Paired t test was also used to compare any pretreatment and posttreatment values for any given parameter. A value of P<0.05 was considered statistically significant

	Results

Top Abstract Introduction Methods Results Discussion References

 
Exclusions
A total of 78 hearts were subjected to the ischemia/reperfusion protocol (Figure 1) in the 7 experimental groups (n=8 to 9 each) for the assessment of ventricular function as well as infarct size. Among them, 16 hearts (ie, 21% of the 78 perfused hearts) were excluded according to the exclusion criteria described under Methods. An additional 36 hearts were used for measurement of nitrite levels in the myocardial tissue extracts.

Baseline Cardiac Hemodynamics and Contractile Function
Morphometric characteristics of the mice as well as the preischemic baseline values are summarized in the Table. There was no significant difference in the preischemic basal value of heart rate and ventricular contractile parameters (ie, developed force, rate-force product, and resting tension) between the groups, although pretreatment with high-dose MLA exhibited a positive inotropic effect during the first 20 minutes of stabilization (Figure 2). This effect on myocardial contractility persisted in the SMT-treated group but was absent in the iNOS knockout group. The coronary flow rate was significantly increased in all MLA350-treated groups compared with the vehicle group (P<0.05, Figure 4).

View this table: [in this window] [in a new window]   Table 1. Morphometric Characteristics and Baseline Values of Functional Parameters

View larger version (40K): [in this window] [in a new window]   Figure 2. Time course of contractile parameters. A, Ventricular developed force; B, rate-force product.

View larger version (62K): [in this window] [in a new window]   Figure 4. Effect of MLA on preischemic and postischemic coronary flow. *P<0.05 vs vehicle group.

Postischemic Cardiac Hemodynamic and Contractile Function
After 20 minutes of global ischemia, ventricular developed force and rate-force product were significantly depressed in all experimental groups, regardless of the pretreatment conditions during reperfusion (Figure 2). The resting tension and heart rate were not significantly different between the preischemic and reperfusion periods (Figure 3). Postischemic coronary flow was not significantly different from its preischemic values for all the groups (P>0.05; Figure 4). The average coronary flow rate was generally higher in MLA-treated groups than in the vehicle group, although these differences were not significant (P>0.05).

View larger version (43K): [in this window] [in a new window]   Figure 3. Time course of functional parameters. A, Resting tension; B, heart rate.

Infarct Size
Myocardial infarction was evident in the mouse hearts after ischemia/reperfusion. Pretreatment with MLA reduced the infarct size in a dose-dependent manner (Figure 5A). The high-dose MLA (ie, 350 µg/kg) caused a significant reduction in infarct size, to 13.5±2.4% of risk zone compared with 26.9±2.9% in the vehicle-treated group (P<0.05). The MLA-induced reduction in infarct size was abolished by pretreatment with SMT (22.2±2.8%) and was completely absent in the iNOS knockout mice (25.5±3.6%). The area at risk for the globally ischemic hearts was not different between the groups (Figure 5B). Representative samples of the TTC-stained heart slices are shown in Figure 6. The viable (area in red color) and necrotic (area in pale color) tissues were clearly distinguishable in these pictures.

View larger version (42K): [in this window] [in a new window]   Figure 5. Effect of MLA on infarct size (expressed as % of risk area). *P<0.05 vs vehicle group.

View larger version (100K): [in this window] [in a new window]   Figure 6. Representative photographs of heart slices after TTC staining. A, Vehicle; B, MLA350; C, MLA350+SMT; D, MLA350+iNOS-KO. Abbreviations as in Figure 1.

Myocardial Nitrite/Nitrate Content
Pretreatment with MLA caused a moderate but statistically insignificant decrease of myocardial nitrite/nitrate in the nonischemic hearts 24 hours later. The nitrite/nitrate levels increased significantly only in the high-dose MLA-treated mice after ischemia/reperfusion (0.97±0.27 nmol/mg protein) compared with the vehicle group (0.23±0.05; P<0.01; Figure 7). The postischemic myocardial accumulation of nitrite/nitrate was associated with the reduction in infarct size with MLA (Figure 5).

View larger version (27K): [in this window] [in a new window]   Figure 7. Effect of MLA on nitrite/nitrate accumulation in tissue extracts of nonischemic and ischemic/reperfused mouse hearts (mean±SEM; n=6 each). *P<0.05 vs vehicle.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Novel Findings
The salient findings of this study are summarized as follows. (1) Higher doses of MLA (ie, 350 µg/kg), when administered 24 hours before ischemia/reperfusion, resulted in a significant reduction of myocardial infarct size and improvement in coronary flow. The antinecrotic effect was not associated with improvement in the postischemic ventricular contractile function. (2) The cardioprotective effect was abolished by SMT, a specific inhibitor of iNOS, and was completely absent in iNOS knockout mice. (3) The cardioprotective dose of MLA resulted in a significant increase in nitrite/nitrate accumulation in the ischemic/reperfused heart. Taken together, our results suggest that high-dose MLA induces a significant anti-ischemic protection in the mouse heart, which was associated with increased accumulation of NO products. These data, coupled with a lack of protection in iNOS knockout mice and blockade of cardioprotection with iNOS inhibitor in normal mice, strongly suggest a cause-and-effect relationship of NO in the pharmacological protection induced by MLA. To the best of our knowledge, this is the first study to establish a direct role of NO in delayed pharmacological preconditioning in the ischemic mouse heart.

Anti-Ischemic Effects of MLA in Heart
MLA has been shown to induce such immunostimulatory effects as cytokine production, macrophage stimulation, and a variety of other effects on both humoral and cell-mediated immune response.^{5 7 8 9} In vivo studies demonstrated the ability of MLA to protect the heart during ischemia/reperfusion.^{2 15 16 17} The present study clearly demonstrates a dose-dependent antinecrotic effect of MLA (Figure 5). The lower dose of MLA (35 µg/kg) did not reduce infarct size. This finding is in accordance with a previous report¹⁵ in which a similar dose of MLA reduced postischemic infarct size in the in situ rabbit heart after regional ischemia but not in the isolated heart subjected to global ischemia. In addition, it seems that rodents require a much higher drug concentration to exert the cardioprotective effects. Tosaki et al¹⁴ reported significant antiarrhythmic effects of MLA with 300 and 450 µg/kg MLA in the isolated working rat heart. Similar species-related differences in induction of late cardioprotection were observed in the mouse heart,²⁵ in which whole-body heat shock failed to induce anti-ischemic effects. However, MLA-induced infarct size reduction was comparable to "acute" ischemic preconditioning in the isolated mouse heart.²⁴

NO and Delayed Cardioprotection
NO is an essential modulator of biological systems, including the cardiovascular system.²¹ It is critical in the signal transduction of ischemic myocardium.²⁶ Numerous studies have shown the beneficial and harmful effects of NO in the physiological regulation and control of the cardiovascular system. On one hand, NO is a free radical itself and can also form peroxynitrite, a more potent oxidant that can potentially cause cellular membrane lipid peroxidation, which may lead to myocardial dysfunction.^{27 28 29 30} In contrast, NO is the modulator of vascular smooth muscle tone, and its biological action can be cardioprotective against ischemia/reperfusion injury through coronary vasodilatation and reduction in myocardial oxygen consumption via upregulation of cGMP.³¹ Pretreatment with NO donors has been reported to be beneficial in the ischemic myocardium. Both antiarrhythmic and anti-infarction³² effects of the NO donors have been well documented. More recently, NO has been appreciated as the possible key trigger and mediator for ischemic preconditioning.³³

NO may enhance myocardial protection by cGMP-dependent as well as cGMP-independent mechanisms (Figure 8). NO is a unique messenger because it is produced in one cell and diffuses into adjacent target cells to activate cytosolic guanylate cyclase–bound heme to generate the NO-heme adduct of guanylate cyclase.²⁶ NO may also modulate K_ATP channels via the second messenger cGMP. The cGMP-dependent protein kinases may be capable of phosphorylating K_ATP channels and priming the channel to offer cardioprotection.³⁴ Cameron et al³⁵ provided direct evidence that NO enhances K_ATP channel activity in hypertrophied ventricular myocytes. Opening of the K_ATP channel appears to be protective because of the increase in outward potassium current, resulting in shortening of the action potential, which in turn may spare ATP, thereby allowing less entry of calcium into the myocyte through the voltage-sensitive calcium channel.³⁶ Decreased intracellular calcium overload may reduce ischemic injury and lead to better myocyte preservation. There is mounting evidence supporting the involvement of K_ATP channels in the mechanism of ischemic preconditioning^{36 37} and pharmacological protection with MLA.^{38 39 40}

View larger version (21K): [in this window] [in a new window]   Figure 8. Proposed scheme of mechanisms of delayed cardioprotection induced by MLA.

iNOS and Delayed Cardioprotection
Three major isoforms of NOS, ie, iNOS, eNOS, and nNOS, are able to catalyze the chemical reaction that produces NO from L-arginine. eNOS and nNOS are constitutively expressed in normal biological systems and are important mediators of cellular signal transduction. iNOS is expressed under such pathophysiological conditions as endotoxin challenge and stress and is capable of producing large amounts of NO, which tends to be cytotoxic.²⁸ However, it has been increasingly recognized that iNOS can be very important in the immune reaction against microbacterial and environmental insults. In the present investigation, we observed complete lack of MLA-induced protection in the iNOS knockout mice, suggesting an obligatory role of this isoform in the protective process. Furthermore, the cardioprotection was significantly abolished by SMT, which was reported to be 10- to 30-fold more potent as an inhibitor of iNOS than the L-arginine analogues, as well as aminoguanidine.²³ Therefore, the abrogation of MLA-induced anti-ischemic effect could be at least in part due to the inhibition of iNOS in the ischemic heart. The cardioprotective dose of MLA significantly increased NO production after ischemia/reperfusion but not in the nonischemic hearts, suggesting that iNOS was functional only in the ischemic hearts. Similarly, Zhao et al¹⁶ observed that no increase of iNOS enzyme activity occurred in the nonischemic heart tissue in MLA-treated rabbits. This suggests that posttranslational modifications of the iNOS enzyme are required before it is capable of generating NO. It is possible that ischemia may activate certain kinases (such as protein kinase C and tyrosine kinase) or inhibit phosphatases that may promote phosphorylation-dependent activation of the inactive iNOS induced by MLA.

The role of constitutive forms of NOS in MLA-induced protection is still not clear. We observed a consistent improvement in preischemic coronary flow in all MLA-treated groups (Figure 4). Blocking iNOS with SMT did not reverse the improvement of coronary flow in MLA-treated mice. Also, SMT only partially (although significantly) blocked the antinecrotic effect of MLA (Figure 5). These data suggest that MLA may have stimulated eNOS in the heart and that the drug may be improving vascular endothelial function independently of the iNOS enzyme. Future studies in eNOS knockout mice will provide insights on the role of eNOS in MLA-induced cardioprotection.

Conclusions and Future Directions
We have shown that MLA induces a dose-dependent cardioprotection against myocardial infarction in the ischemic mouse heart. This antinecrotic effect was associated with enhanced accumulation of nitrite/nitrate in the ischemic tissue, was blocked by selective inhibition of iNOS, and was completely absent in iNOS knockout mice. To the best of our knowledge, this is the first direct evidence supporting an obligatory role of iNOS in mediating the delayed cardioprotection by MLA. Further investigations in the murine model are necessary to elucidate (1) the potential role of eNOS or nNOS in MLA-induced cardioprotection, (2) the key type(s) of cytokines and their receptors that may be responsible in activation of iNOS, and (3) other pharmacological agents that could induce late preconditioning via signal transduction pathways similar to those of MLA.

	Acknowledgments

 
This work was funded in part by grants from the NHLBI (HL-51045, HL-59469) to Dr Kukreja. Dr Xi was supported by a NRSA fellowship from the NIH (HL-07537) and a Research Fellowship from the American Heart Association, Mid-Atlantic Affiliate (F98273V). We thank Dr R. Hutte of Sievers Instruments, Inc, for his help in measuring tissue nitrite/nitrate concentrations and Dr G.T. Elliott of Ribi ImmunoChem Research Inc for providing MLA.

Received September 7, 1998; revision received November 20, 1998; accepted December 17, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Kukreja RC, Hess ML. Free Radicals, Cardiovascular Dysfunction and Protection Strategies. Austin, Tex: R.G. Landes Co; 1994.
   
2. Yoshida K, Maaieh MM, Shipley JB, Doloresco M, Bernardo NL, Qian Y-Z, Elliott GT, Kukreja RC. Monophosphoryl lipid A induces pharmacologic "preconditioning" in rabbit hearts without concomitant expression of 70-kDa heat shock protein. Mol Cell Biochem. 1996;159:73–80.[Medline] [Order article via Infotrieve]
   
3. Elliott GT. Monophosphoryl lipid A induces delayed preconditioning against cardiac ischemia-reperfusion injury. J Mol Cell Cardiol. 1998;30:3–17.[Medline] [Order article via Infotrieve]
   
4. Madonna GS, Peterson JE, Ribi E, Vogel SN. Early phase endotoxin tolerance: induction by a detoxified lipid A derivative, monophosphoryl lipid A. Infect Immun. 1986;52:6–11.[Abstract/Free Full Text]
   
5. Henricson BE, Benjamin WR, Vogel SN. Differential cytokine induction by doses of lipopolysaccharide and monophosphoryl lipid A that result in equivalent early endotoxin tolerance. Infect Immun. 1990;58:2429–2437.[Abstract/Free Full Text]
   
6. Astiz ME, Rackow EC, Still JG, Howell ST, Cato A, Von Eschen KB, Ulrich JT, Rudbach JA, McMahon G, Vargas R. Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double-blind, randomized, controlled trial. Crit Care Med. 1995;23:9–17.[Medline] [Order article via Infotrieve]
   
7. Kiener PA, Marek F, Rodgers G, Lin P-F, Warr G, Desiderio J. Induction of tumor necrosis factor, IFN-, and acute lethality in mice by toxic and non-toxic forms of lipid A. J Immunol. 1988;141:870–874.[Abstract]
   
8. Salkowski CA, Detore GR, Vogel SN. Lipopolysaccharide and monophosphoryl lipid A differentially regulate interleukin-12, gamma interferon, and interleukin-10 mRNA production in murine macrophages. Infect Immun. 1997;65:3239–3247.[Abstract]
   
9. Myers KR, Beining P, Betts M, Snippe H, Inman J, Golding B. Monophosphoryl lipid A behaves as a T-cell-independent type 1 carrier for hapten-specific antibody responses in mice. Infect Immun. 1995;63:168–174.[Abstract]
   
10. Takayama K, Qureshi N, Ribi E, Cantrell JL. Use of endotoxin in cancer immunotherapy and characterization of nontoxic but active lipid A components. Am Chem Soc Symp. 1983;231:219–233.
    
11. Gustafson GL, Rhodes MJ, Hegel T. Monophosphoryl lipid A as a prophylactic for sepsis and septic shock. Prog Clin Biol Res. 1995;392:567–579.[Medline] [Order article via Infotrieve]
    
12. Alving CR. Lipopolysaccharide, lipid A, and liposomes containing lipid A as immunologic adjuvants. Immunobiol. 1993;187:430–446.[Medline] [Order article via Infotrieve]
    
13. Nelson DW, Brown JM, Banerjee A, Bensard DD, Rogers KB, Locke-Winter CR, Anderson BO, Harken AH. Pretreatment with a nontoxic derivative of endotoxin induces functional protection against cardiac ischemia/reperfusion injury. Surgery. 1991;110:365–369.[Medline] [Order article via Infotrieve]
    
14. Tosaki A, Maulik N, Elliott GT, Engelman RM, Das DK. Preconditioning of rat heart with monophosphoryl lipid A: a role of nitric oxide. J Pharmacol Exp Ther. 1998;285:1274–1279.[Abstract/Free Full Text]
    
15. Baxter GF, Goodwin RW, Wright MJ, Kerac M, Heads RJ, Yellon DM. Myocardial protection after monophosphoryl lipid A: studies of delayed anti-ischaemic properties in rabbit heart. Br J Pharmacol. 1996;117:1685–1692.[Medline] [Order article via Infotrieve]
    
16. Zhao L, Weber PA, Smith JR, Comerford ML, Elliott GT. Role of inducible nitric oxide synthase in pharmacological "preconditioning" with monophosphoryl lipid A. J Mol Cell Cardiol. 1997;29:1567–1576.[Medline] [Order article via Infotrieve]
    
17. Yao Z, Auchampach JA, Pieper GM, Gross GJ. Cardioprotective effects of monophosphoryl lipid A, a novel endotoxin analogue, in the dog. Cardiovasc Res. 1993;27:832–838.[Abstract/Free Full Text]
    
18. Przyklenk K, Zhao L, Kloner RA, Elliott GT. Cardioprotection with ischemic preconditioning and MLA: role of adenosine-regulating enzymes? Am J Physiol. 1996;271:H1004–H1014.[Abstract/Free Full Text]
    
19. Nayeem MA, Elliott GT, Shah MR, Hastillo-Hess SL, Kukreja RC. Monophosphoryl lipid A protects adult rat cardiac myocytes with induction of the 72-kD heat shock protein: a cellular model of pharmacologic preconditioning. J Mol Cell Cardiol. 1997;29:2305–2310.[Medline] [Order article via Infotrieve]
    
20. Meng X, Brown JM, Ao L, Nordeen SK, Franklin W, Harken AH, Banerjee A. Endotoxin induces cardiac HSP70 and resistance to endotoxemic myocardial depression in rats. Am J Physiol. 1996;271:C1316–C1324.[Abstract/Free Full Text]
    
21. Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochem J. 1994;298:249–258.
    
22. Laubach VE, Shesely EG, Smithies O, Sherman PA. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad Sci U S A. 1995;92:10688–10692.[Abstract/Free Full Text]
    
23. Szabo C, Southan GJ, Thiemerman C. Beneficial effects and improved survival in rodent models of septic shock with S-methylthiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase. Proc Natl Acad Sci U S A. 1994;91:12472–12476.[Abstract/Free Full Text]
    
24. Xi L, Hess ML, Kukreja RC. Ischemic preconditioning in isolated perfused mouse heart: reduction in infarct size without improvement of post-ischemic ventricular function. Mol Cell Biochem. 1998;186:69–77.[Medline] [Order article via Infotrieve]
    
25. Xi L, Chelliah J, Nayeem MA, Levasseur JE, Hess ML, Kukreja RC. Whole body heat shock fails to protect mouse heart against ischemia/reperfusion injury: role of 72 kDa heat shock protein and antioxidant enzymes. J Mol Cell Cardiol. 1998;30:2213–2227.[Medline] [Order article via Infotrieve]
    
26. Maulik N, Engelman DT, Watanabe M, Engelman RM, Maulik G, Cordis GA, Das DK. Nitric oxide signaling in ischemic heart. Cardiovasc Res. 1995;30:593–601.[Medline] [Order article via Infotrieve]
    
27. Matheis G, Sherman MP, Buckberg GD, Haybron DM, Young HH, Ignarro LJ. Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am J Physiol. 1992;262:H616–H620.[Abstract/Free Full Text]
    
28. Schulz R, Nava E, Moncada S. Induction and potential biological relevance of a Ca²⁺-independent nitric oxide synthase in the myocardium. Br J Pharmacol. 1992;105:575–580.[Medline] [Order article via Infotrieve]
    
29. Naseem SA, Kontos MC, Rao PS, Jesse RL, Hess ML, Kukreja RC. Sustained inhibition of nitric oxide by N^G-nitro-L-arginine improves myocardial function following ischemia/reperfusion in isolated perfused rat heart. J Mol Cell Cardiol. 1995;27:419–426.[Medline] [Order article via Infotrieve]
    
30. Wang P, Zweier JL. Measurement of nitric oxide and peroxynitrite generation in the postischemic heart: evidence for peroxynitrite-mediated reperfusion injury. J Biol Chem. 1996;271:29223–29230.[Abstract/Free Full Text]
    
31. Weiss HR, Rodriguez E, Tse J, Scholz PM. Effect of increased myocardial cyclic GMP induced by cyclic GMP-phosphodiesterase inhibition on oxygen consumption and supply of rabbit hearts. Clin Exp Pharmacol Physiol. 1994;21:607–614.[Medline] [Order article via Infotrieve]
    
32. Nakanishi K, Vinten-Johansen J, Lefer DJ, Zhao Z, Fowler WC III, McGee DS, Johnston WE. Intracoronary L-arginine during reperfusion improves endothelial function and reduces infarct size. Am J Physiol. 1992;263:H1650–H1658.[Abstract/Free Full Text]
    
33. Qiu Y, Rizvi A, Tang X-L, Manchikalapudi S, Takano H, Jadoon AK, Wu W-J, Bolli R. Nitric oxide triggers late preconditioning against myocardial infarction in conscious rabbits. Am J Physiol. 1997;273:H2931–H2936.
    
34. Cameron JS, Baghdady R. Role of ATP sensitive potassium channels in long term adaptation to metabolic stress. Cardiovasc Res. 1994;28:788–796.[Free Full Text]
    
35. Cameron JS, Kibler KKA, Berry H, Barron DN, Sodder VH, Barin F. Nitric oxide activates ATP-sensitive potassium channels in hypertrophied ventricular myocytes. FASEB J. 1996;10:A65. Abstract.
    
36. Gross GJ, Auchampach JA. Blockade of ATP-sensitive potassium channels prevents myocardial preconditioning in dogs. Circ Res. 1992;70:223–233.[Abstract/Free Full Text]
    
37. Qian Y-Z, Levasseur JE, Yoshida KI, Kukreja RC. K_ATP channels in rat heart: blockade of ischemic and acetylcholine-mediated preconditioning by glibenclamide. Am J Physiol. 1996;271:H23–H28.[Abstract/Free Full Text]
    
38. Mei DA, Elliott GT, Gross GJ. K_ATP channels mediate late preconditioning against infarction produced by monophosphoryl lipid A. Am J Physiol. 1996;271:H2723–H2729.[Abstract/Free Full Text]
    
39. Elliott GT, Comerford ML, Smith JR, Zhao L. Myocardial ischemia/reperfusion protection using monophosphoryl lipid A is abrogated by the ATP-sensitive potassium channel blocker, glibenclamide. Cardiovasc Res. 1997;32:1071–1080.
    
40. Janin Y, Qian Y-Z, Hoag JB, Elliott GT, Kukreja RC. Pharmacologic preconditioning with monophosphoryl lipid A is abolished by 5-hydroxydecanoate, a specific inhibitor of the K_ATP channel. J Cardiovasc Pharmacol. 1998;32:337–342.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				A. Maffei, A. Di Pardo, R. Carangi, P. Carullo, R. Poulet, M. T. Gentile, C. Vecchione, and G. Lembo Nebivolol Induces Nitric Oxide Release in the Heart Through Inducible Nitric Oxide Synthase Activation Hypertension, October 1, 2007; 50(4): 652 - 656. [Abstract] [Full Text] [PDF]

				P. L. Hermonat, D. Li, B. Yang, and J. L. Mehta Mechanism of action and delivery possibilities for TGF{beta}1 in the treatment of myocardial ischemia Cardiovasc Res, May 1, 2007; 74(2): 235 - 243. [Abstract] [Full Text] [PDF]

				R. Natarajan, F. N. Salloum, B. J. Fisher, R. C. Kukreja, and A. A. Fowler III Hypoxia Inducible Factor-1 Activation by Prolyl 4-Hydroxylase-2 Gene Silencing Attenuates Myocardial Ischemia Reperfusion Injury Circ. Res., January 6, 2006; 98(1): 133 - 140. [Abstract] [Full Text] [PDF]

				Y. Harder, C. Contaldo, J. Klenk, A. Banic, S. M. Jakob, and D. Erni Preconditioning with Monophosphoryl Lipid A Improves Survival of Critically Ischemic Tissue Anesth. Analg., June 1, 2005; 100(6): 1786 - 1792. [Abstract] [Full Text] [PDF]

				P. Di Napoli, A. A. Taccardi, A. Grilli, M. A. De Lutiis, A. Barsotti, M. Felaco, and R. De Caterina Chronic treatment with rosuvastatin modulates nitric oxide synthase expression and reduces ischemia-reperfusion injury in rat hearts Cardiovasc Res, June 1, 2005; 66(3): 462 - 471. [Abstract] [Full Text] [PDF]

				A. Das, L. Xi, and R. C. Kukreja Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes against Necrosis and Apoptosis: ESSENTIAL ROLE OF NITRIC OXIDE SIGNALING J. Biol. Chem., April 1, 2005; 280(13): 12944 - 12955. [Abstract] [Full Text] [PDF]

				L. Xi, M. Taher, C. Yin, F. Salloum, and R. C. Kukreja Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of HIF-1{alpha} and AP-1 and iNOS signaling Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2369 - H2375. [Abstract] [Full Text] [PDF]

				X. Wang, C. Yin, L. Xi, and R. C. Kukreja Opening of Ca2+-activated K+ channels triggers early and delayed preconditioning against I/R injury independent of NOS in mice Am J Physiol Heart Circ Physiol, November 1, 2004; 287(5): H2070 - H2077. [Abstract] [Full Text] [PDF]

				Z. Xia, R. Xia, H.-T. Lan, T. Luo, Q.-Z. Tang, Z.-Y. Xia, and X.-Y. Liu Systemic ischemic preconditioning plus hemodilution enhanced early functional recovery of reperfused heart in the rabbits Interactive CardioVascular and Thoracic Surgery, September 1, 2004; 3(3): 528 - 532. [Abstract] [Full Text] [PDF]

				Y. Wang, N. Ahmad, M. Kudo, and M. Ashraf Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1125 - H1131. [Abstract] [Full Text] [PDF]

				X.-L. Tang, Y.-T. Xuan, Y. Zhu, G. Shirk, and R. Bolli Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits Am J Physiol Heart Circ Physiol, April 1, 2004; 286(4): H1273 - H1280. [Abstract] [Full Text] [PDF]

				R. Marfella, C. Di Filippo, K. Esposito, F. Nappo, E. Piegari, S. Cuzzocrea, L. Berrino, F. Rossi, D. Giugliano, and M. D'Amico Absence of Inducible Nitric Oxide Synthase Reduces Myocardial Damage During Ischemia Reperfusion in Streptozotocin-Induced Hyperglycemic Mice Diabetes, February 1, 2004; 53(2): 454 - 462. [Abstract] [Full Text]

				D. M. YELLON and J. M. DOWNEY Preconditioning the Myocardium: From Cellular Physiology to Clinical Cardiology Physiol Rev, October 1, 2003; 83(4): 1113 - 1151. [Abstract] [Full Text] [PDF]

				Q. Li, Y. Guo, Y.-T. Xuan, C. J. Lowenstein, S. C. Stevenson, S. D. Prabhu, W.-J. Wu, Y. Zhu, and R. Bolli Gene Therapy With Inducible Nitric Oxide Synthase Protects Against Myocardial Infarction via a Cyclooxygenase-2-Dependent Mechanism Circ. Res., April 18, 2003; 92(7): 741 - 748. [Abstract] [Full Text] [PDF]

				F. Salloum, C. Yin, L. Xi, and R. C. Kukreja Sildenafil Induces Delayed Preconditioning Through Inducible Nitric Oxide Synthase-Dependent Pathway in Mouse Heart Circ. Res., April 4, 2003; 92(6): 595 - 597. [Abstract] [Full Text] [PDF]

				X.-L. Tang, E. Kodani, H. Takano, M. Hill, K. Shinmura, T. M. Vondriska, P. Ping, and R. Bolli Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1441 - H1448. [Abstract] [Full Text] [PDF]

				H. R. Cross, E. G. Kranias, E. Murphy, and C. Steenbergen Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H683 - H690. [Abstract] [Full Text] [PDF]

				F. Brunner, R. Maier, P. Andrew, G. Wolkart, R. Zechner, and B. Mayer Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase Cardiovasc Res, January 1, 2003; 57(1): 55 - 62. [Abstract] [Full Text] [PDF]

				E. Kodani, Y.-T. Xuan, K. Shinmura, H. Takano, X.-L. Tang, and R. Bolli delta -Opioid receptor-induced late preconditioning is mediated by cyclooxygenase-2 in conscious rabbits Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1943 - H1957. [Abstract] [Full Text] [PDF]

				Y.-P. Wang, C. Sato, K. Mizoguchi, Y. Yamashita, M. Oe, and H. Maeta Lipopolysaccharide triggers late preconditioning against myocardial infarction via inducible nitric oxide synthase Cardiovasc Res, October 1, 2002; 56(1): 33 - 42. [Abstract] [Full Text] [PDF]