cAMP-mediated suppression of a Th1 clone associated with an alteration of the intracellular redox environment.

We have shown previously that the elevation of intracellular cAMP in antigen or anti-CD3-activated murine Th1 clones in the absence of antigen inhibits antigen-induced proliferation and the production of IL-2 by H2O2-mediated oxidation of p56lck and inhibits antigen-induced production of interferon-gamma by the induction of intracellular nitric oxide. Moreover, activated Th1 clones are resistant to cAMP-induced suppression. These results suggest that the immunosuppression of Th1 cells mediated by elevated intracellular cAMP is associated with an alteration in the intracellular oxidation/reduction environment. Here we report that the culture of an antigen or anti-CD3-activated murine Th1 clone with the adenylcyclase agonist forskolin (FSK) in the absence of antigen reduces the activity of intracellular catalase, and diminishes levels of intracellular reduced glutathione (GSH). Resting cells resistant to cAMP-induced suppression have higher intracellular GSH levels than antigen-activated cells susceptible to cAMP-induced suppression. The results provide further evidence that cAMP-induced suppression of Th1 clones is mediated by profound alterations in the intracellular redox environment and may be used to selectively inactivate Th1 cells activated by antigen.

Anti-apoptotic protein survivin plays a significant role in tubular morphogenesis of human coronary arteriolar endothelial cells by hypoxic preconditioning.

Brief exposure of endothelial cells to oxidative stress induced by hypoxia followed by reoxygenation enhances tube formation. Our study provides evidence that hypoxic preconditioning accelerates tubular morphogenesis along with the activation of reactive oxygen species-inducible nuclear transcription factor-kappaB (NF-kappaB), phosphatidylinositol 3-kinase (PI3-kinase) and broad-spectrum anti-apoptotic protein survivin in human coronary arteriolar endothelial cells (HCAEC). The formation of tubular morphogenesis was inhibited by using the PI3-kinase and NF-kappaB antagonists LY294002 and SN50 respectively. The activation of survivin by hypoxic preconditioning was also inhibited by LY294002 and SN50 along with increased apoptosis in HCAEC. These data demonstrate a crucial role of PI3-kinase/Akt/NF-kappaB/survivin signaling in tubular morphogenesis of HCAEC triggered by hypoxic preconditioning.

Role of STAT3 in ischemic preconditioning.

We recently demonstrated that ischemic preconditioning (IPC) induced by cyclic episodes of short durations of ischemia and reperfusion potentiates a signal transduction cascade involving protein tyrosine kinases and MAP kinases. A rapid activation of janus kinase (JAK) and several signal transducers and activators of the transcription (STATs) including STAT3, STAT5A and STAT6 has been shown to occur during myocardial ischemia and reperfusion. This study sought to examine if JAK/STAT signaling pathway play any role in classical early phase of IPC. Isolated working rat hearts were perfused for 15 min with KHB buffer in the absence or presence of a JAK kinase inhibitor tyrphostin AG490 (5 microm) followed by IPC, 30 min global ischemia and 2 h of reperfusion. The results demonstrated extensive phosphorylation of JAK2 and STAT3 in the IPC hearts which was almost completely abolished by an inhibitor of JAK2, AG490. IPC displayed cardioprotection as evidenced by improved post-ischemic contractile recovery, decreased myocardial infarct size and reduced number of apoptotic cardiomyocytes. AG490 blocked IPC-mediated cardioprotection by altering the IPC-mediated survival signal into death signal. Thus, IPC-induced upregulation of antiapoptotic gene bcl-2 and downregulation of pro-apoptotic gene bax are decreased and increased, respectively, in the AG490 treated hearts. The results suggest that early phase of IPC potentiates JAK/STAT signaling by activating STAT3 which transmits a survival signal to the myocardium.

Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning.

The signal cascade that triggers and mediates ischemic preconditioning (IPC) remains unclear. The present study investigated the role of the Src family of tyrosine kinases in IPC. Isolated and buffer-perfused rat hearts underwent IPC with three cycles of 5-min ischemia and 5-min reperfusion, followed by 30-min ischemia and 120-min reperfusion. The Src tyrosine kinase family-selective inhibitor PP1 was administered between 45 and 30 min before ischemia (early PP1 treatment) or for 15 min before IPC [early PP1-preconditioning (PC) treatment]. PP1 was also administered for 5 min before the sustained ischemia (late PP1 treatment) or after IPC (late PP1-PC treatment). Src kinase was activated after 30 min of ischemia in both the membrane and cytosolic fractions. Src kinase was also activated by IPC but was attenuated after the sustained ischemia. Early and late PP1 treatment inhibited Src activation after the sustained ischemia and reduced infarct size. Early PP1-PC inhibited Src activation after IPC but not after the sustained ischemia and blocked cardioprotection afforded by IPC. Late PP1-PC treatment abrogated IPC-induced activation of Src and protein kinase C (PKC)-epsilon in the membrane but not in the cytosolic fraction. This treatment modality abrogated Src activation after the sustained ischemia and failed to block cardioprotection afforded by IPC. These results suggest that Src kinase activation mediates ischemic injury but triggers IPC in the position either upstream of or parallel to membrane-associated PKC-epsilon.

SAPKs regulation of ischemic preconditioning.

The role of stress-activated protein kinases (SAPKs), c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase, in preconditioning (PC) was examined with the use of isolated rat hearts subjected to four cyclic episodes of 5-min ischemia and 10-min reperfusion followed by 30-min ischemia and 2-h reperfusion (I/R). A group of hearts was preperfused with 100 microM curcumin, a c-Jun and JNK1 inhibitor, or 5 microM SB 203580, a p38 MAP kinase inhibitor. Another group of hearts was preperfused with 20 microM anisomycin, a stimulator for both JNK and p38 MAP kinases. I/R increased the protein levels of JNK1, c-Jun, and p38 MAP kinase. PC also enhanced the induction of these kinases, but subsequent I/R-mediated increase was blocked by PC. Curcumin blocked I/R- and PC-mediated increase in JNK1 and c-Jun protein levels, whereas it had no effects on p38 MAP kinase. SB 203580, on the other hand, was equally effective in reducing the p38 MAP kinase activation but exerted no effects on JNK1 and c-Jun induction. I/R-mediated increased myocardial infarction was reduced by any of the following compounds: anisomycin, curcumin, and SB 203580. The cardioprotective effects of PC were abolished by either curcumin or SB 203580. The results demonstrate that PC is mediated by a signal-transduction pathway involving both JNK1 and p38 MAP kinase. Activation of SAPKs, although transient, is obligatory for PC.

The red wine antioxidant resveratrol protects isolated rat hearts from ischemia reperfusion injury.

The consumption of red wine has been reported to impart a greater benefit in the prevention of coronary heart disease than the consumption of other alcoholic beverages. This beneficial effect is increasingly being attributed to certain antioxidants comprising the polyphenol fraction of red wine such as transresveratrol. In the present study, we investigated the potential cardioprotective effects of resveratrol in the face of ischemia reperfusion (I/R) injury. Isolated perfused working rat hearts after stabilization were perfused with Krebs-Henseleit Bicarbonate buffer (KHB) either in the presence or absence of transresveratrol (RVT) at a concentration of 10 microM for 15 min prior to subjecting them to 30 min of global ischemia followed by 2 h of reperfusion. Left ventricular functions were monitored at various timepoints throughout the reperfusion period to assess the extent of postischemic recovery in comparison with baseline values. Coronary perfusate samples were also collected to determine malonaldehyde (MDA) levels. The results demonstrated that RVT exhibited significant myocardial protection. This was evidenced by improved recovery of post-ischemic ventricular function including developed pressure and aortic flow as compared to the control group (KHB). Values for developed pressure in the RVT-treated group were significantly higher than those in the control group throughout the reperfusion period (71.09+/-4.88 mm Hg vs. 58.47+/-3.88 mm Hg, 68.87+/-5.07 mm Hg vs. 49.74+/-2.65 mm Hg and 51.67+/-3.95 mm Hg vs. 30.50+/-4.80 mm Hg at reperfusion timepoints R-15, R-60, and R-120, respectively). From R-30 onwards, aortic flow was markedly higher in the RVT treated group as compared with the control group, the differences being most significant at R-90 (32.45+/-2.19 ml/min vs. 19.83+/-1.62 ml/min) and R-120 (27.15+/-2.27 ml/min vs. 14.10+/-1.69 ml/min). In contrast to the KHB treated group, the RVT-treated group displayed significant reduction in MDA formation especially in the immediate early reperfusion period (63.71+/-8.19 pM/ml vs. 130.86+/-4.76 pM/ml, 63.84+/-15.62 pM/ml vs. 156.99+/-18.93 pM/ml, 71.29+/-2.80 pM/ml vs. 129.5+/-10.30 pM/ml and 56.25+/-5.79 pM/ml vs. 127.99+/-3.50 pM/ml at timepoints R-1, R-3, R-5, and R-7, respectively) indicating a reduction in I/R injury related oxidative stress. Infarct size was markedly reduced in the RVT group when compared with the control group (10.57+/-0.35% vs. 36.27+/-5.28%). In vitro studies revealed RVT to be a potent scavenger of peroxyl radicals suggestive of a probable mechanism involved in the protective ability of RVT. The results of this study indicate that resveratrol possesses cardioprotective effects which may be attributed to its peroxyl radical scavenging activity.

Detection of oxidative DNA damage to ischemic reperfused rat hearts by 8-hydroxydeoxyguanosine formation.

Reactive oxygen species that are generated in the ischemic heart upon reperfusion, play a significant role in the pathogenesis of reperfusion injury. Although DNA is a well known target for free radical attack, little attention has been paid to the injury of DNA molecules associated with ischemia and reperfusion. In this study, the formation of 8-hydroxydeoxyguanosine (8-OHDG), a product of hydroxyl radical (OH.)-DNA interaction, was monitored in the post-ischemic myocardium. A simple high performance liquid chromatography (HPLC), with uv detection, detected pmol levels of 8-OHDG in the pre-ischemic heart which increased steadily and progressively as a function of reperfusion time. A similar rise in 8-OHDG was noticed when isolated hearts were perfused with a OH. -generating system. Corroborating with the increased 8-OHDG formation, increased amount of creatine kinase was released from the coronary effluent indicating increased tissue injury. The formation of 8-OHDG was completely blocked when hearts were preperfused with oxygen-free-radical scavenger, 1,3-dimethyl-2-thiourea (DMTU) which also significantly reduced the appearance of CK in the coronary effluent, suggesting that oxidative DNA damage play a role in the pathophysiology of ischemic reperfusion injury.

High-performance liquid chromatographic peak identification of 2,4-dinitrophenylhydrazine derivatives of lipid peroxidation aldehydes by photodiode array detection.

Malonaldehyde (MDA), a product of lipid peroxidation, is a presumptive marker for the development of oxidative stress in tissues and plasmas. In this study we report the photodiode array detection of the 2,4-dinitrophenylhydrazine (DNPH) derivatives of MDA using HPLC. Oxidative stress was produced by injecting (i.p.) bacterial lipopolysaccharide (LPS) into rats at a dose of 100 micrograms/kg, or i.v. into rabbits (1 microgram/kg), or added to freshly drawn human blood (200 ng/ml). Blood was collected at several time points up to 5 h, centrifuged, and equal volumes of 20% TCA were used to precipitate proteins from the plasma. The supernatants were derivatized with DNPH, and the aldehyde-DNPHs were extracted with pentane. After evaporation, aliquots of 10 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column, chromatographed with an acetonitrile-water-acetic acid gradient mobile phase and scanned using Waters 996 photodiode array detector. Peak identification and homogeneity was determined by comparing the experimental peaks and UV scans with those of authentic standards. A significant increase in the DNPH derivative of malonaldehyde (MDA-DNPH), but not of the other aldehyde-DNPH derivatives of formaldehyde (FDA), acetaldehyde (ADA), acetone and propionaldehyde (PDA) was seen over the first hour after LPS administration in anesthetized rats, while in conscious rabbits this trend lasted up to 3 h. The retention times as well as the UV scans of the derivatized aldehydes matched the authentic standards. Thus, photodiode array detection has proved valuable in establishing this HPLC method for estimating oxidative stress. This technique could accurately measure pmol amounts of MDA-DNPH indicating the usefulness of photodiode array detection method for estimating small changes in the oxidative stress.

HPTLC analysis of sphingomylein, ceramide and sphingosine in ischemic/reperfused rat heart.

Since the sphingomylein-ceramide-sphingosine pathway, especially ceramide, has been shown to induce programmed cell death (apoptosis), and since apoptosis may be involved with ischemic/reperfused injury in the heart, it became desirable to quantitate the three components in ischemic/reperfused rat heart. One group of rat hearts (n = 6) was isolated and perfused with Krebs-Henseleit buffer using the Langendorff non-recirculating mode. The hearts were perfused for 10 min, made ischemic for 30 min and reperfused for 120 min. Hearts were collected and stored at - 70 degrees C before ischemia, after ischemia and after 30, 60 and 120 min of reperfusion. The hearts were homogenized, and lipids were extracted using the Folch method. The lipids were then chromatographed on Whatman silica gel 60 A high-performance thin-layered chromatography (HPTLC) plates. The plates were developed with iodine, photographed using Photoshop software and quantitated using NIH Imaging software. The results show a 50% decrease of sphingomylein during reperfusion with a corresponding increase in ceramide with sphingosine showing a smaller decrease as compared with the ceramide increase.

Ischemic preconditioning triggers phospholipase D signaling in rat heart.

Recent studies have indicated that repeated brief episodes of ischemia and reperfusion render the myocardium more tolerant to subsequent lethal ischemic injury. In view of the previous observations that ischemia-reperfusion potentiates phospholipase D signaling and that such signaling is beneficial for the heart, we investigated whether a similar phospholipase D signaling is responsible for the beneficial effects associated with repeated ischemia and reperfusion. Using an isolated perfused working rat heart model, we demonstrated that four brief episodes of 5 min of ischemia and 10 min of reperfusion reduced the incidence of ventricular arrhythmias, enhanced the postischemic ventricular performance, and decreased the release of creatine kinase from the reperfused heart, with simultaneous activation of phospholipase D generating the second messengers diacylglycerol and phosphatidic acid and leading to the translocation and activation of protein kinase C. The specific antiphospholipase D antibody blocked the activation of phospholipase D and attenuated the generation of diacylglycerol and phosphatidic acid and activation of protein kinase C. In concert, phospholipase D inhibition increased the incidence of ventricular arrhythmias, blocked the beneficial effects of preconditioning on the ventricular performance, and increased the amount of creatine kinase release from the coronary effluent. The results of this study indicate that repeated brief episodes of ischemia and reperfusion exert beneficial effects on the intact rat heart by triggering the activation of a phospholipase D signaling mechanism.

Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts.

While much is known about the beneficial effects of myocardial stress adaptation, relatively less information is available about the adaptive mechanisms. To explore the signaling pathways of stress adaptation, isolated working rat hearts were divided into three groups. Group I was adapted to stress by conventional technique of repeated ischemia and reperfusion consisting of 5 min of ischemia followed by 10 min of reperfusion, repeated four times. Group II was treated with 100 microM of genistein, a tyrosine kinase inhibitor, followed by preconditioning as described for group I. The third group, perfused with buffer only for 60 min, served as control. All hearts were subjected to 30 min of ischemia followed by 30 min of reperfusion. The results of our study demonstrated better postischemic myocardial functions in the preconditioned hearts as evidenced by increased aortic flow, coronary flow, developed pressure and lesser amount of tissue injury as evidenced by the decreased creatine kinase release. The preconditioning effects were associated with enhancement of phospholipase D activity in the heart. The preconditioning effect was almost abolished by the genistein treatment which also prevented the enhancement of phospholipase D activities. Additionally, preconditioning of the rat hearts stimulated protein kinase C, MAP kinase, and MAPKAP kinase 2 activities which were inhibited by genistein. The results identifies for the first time tyrosine kinase-phospholipase D as potential signaling pathway for ischemic preconditioning, and implicates the involvement of multiple protein kinases in myocardial adaptation to ischemia.

Phospholipase D plays a role in ischemic preconditioning in rabbit heart.

BACKGROUND: Activation of protein kinase C (PKC) is thought to be a critical step in ischemic preconditioning. Many receptor agonists activate PKC via stimulation of phospholipase C (PLC), which degrades membrane phospholipids to diacylglycerol (DAG), an important PKC cofactor. However, adenosine receptors, critical components of the prototypical preconditioning pathway, are not thought to couple to PLC in the cardiomyocyte. We therefore tested whether ischemic preconditioning or adenosine might instead activate phospholipase D (PLD) to produce DAG. METHODS AND RESULTS: PLD activity was measured in isolated rabbit hearts. Ischemic injury was evaluated in either isolated rabbit hearts or dispersed myocytes. PLD activity doubled from a control level of 74.8 +/- 10.0 to 140.0 +/- 11.5 mumol.min-1.g-1 (P < .025) after two 5-minute periods of global ischemia separated by 5 minutes of reperfusion. A similar increase was noted after the heart had been exposed to (R)-N6-(2-phenylisopropyl)-adenosine [(R)-PIA] for 20 minutes. When sodium oleate, which activates PLD, was administered to isolated hearts before a 30-minute coronary occlusion, infarct size (15.6 +/- 2.0% of the risk zone) was significantly smaller than in untreated hearts (30.4 +/- 2.2%; P < .01). Exposure to sodium oleate significantly prolonged the rate of isolated myocyte survival during simulated ischemia. Propranolol 100 mumol/L, which blocks DAG production from metabolites produced by PLD catalysis, completely abolished the protective effects of both metabolic preconditioning and (R)-PIA exposure in myocytes. CONCLUSIONS: We conclude that PLD stimulation is involved in the protection of ischemic preconditioning in the rabbit heart.

Fatty acid profiles of plasmalogen choline and ethanolamine glycerophospholipids in pig and rat hearts.

The presence of relatively high concentrations of plasmalogen choline and ethanolamine in the heart of many animal species suggests a role of these ether-linked phospholipids in the pathophysiology of certain myocardial diseases. However, the fatty acid composition of myocardial plasmalogens in many species is not known. This study examined the fatty acid composition of the choline and ethanolamine glycerophospholipids in pig heart and compared the results with those in rat heart. Lipids were extracted from the heart biopsies obtained from pig and rat by standard techniques. Phosphoglycerides were separated by thin-layer chromatography followed by their derivatization into fatty acid methyl esters (FAMEs) and dimethyl acetals (DMAs). FAME and DMA samples were analyzed using gas chromatography-mass spectroscopy. Our results indicate striking differences in the fatty acid composition of both choline and ethanolamine glycerophosphates between rat heart and pig heart. Pig heart ethanolamine glycerophosphates are rich in linoleic acid (18:2) and arachidonic acid (20:4), but low in descosahexenoic (22:6) fatty acids while choline glycerophosphates are poor in both 20:4 and 22:6 fatty acids compared to those in rat hearts.

L-arginine reduces endothelial inflammation and myocardial stunning during ischemia/reperfusion.

BACKGROUND: This study evaluated whether the nitric oxide precursor L-arginine could reduce ischemia/reperfusion injury by preventing leukocyte-endothelial interactions. METHODS: Normothermic regional ischemia was induced in the open-chest working pig heart for 30 minutes followed by 90 minutes of reperfusion. A preischemic 10-minute intravenous infusion of 4 mg.kg-1.min-1 of L-arginine (n = 12) was compared with 12 control pigs. Nitric oxide release was measured from the coronary sinus using an amperometric probe. Left ventricular function, malonaldehyde, creatine kinase, myocardial oxygen extraction, and the soluble adhesion molecules (intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1) were measured. RESULTS: Nitric oxide release was significantly reduced from baseline throughout ischemia/reperfusion only in the control group. Systolic and diastolic function, and myocardial oxygen extraction were also significantly decreased during early reperfusion in the control compared with the L-arginine group. Peak creatine kinase release was not significantly different between groups. The incidence of ventricular fibrillation, malonaldehyde release, and soluble intracellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1 were each significantly decreased during reperfusion in the L-arginine group. CONCLUSIONS: L-Arginine reduced lipid peroxidation, plasma levels of soluble adhesion molecules, myocardial stunning, and arrhythmias. These results support an excessive endothelial injury/inflammatory response after regional ischemia/reperfusion that can be ameliorated through augmented nitric oxide.

Myocardial adaptation to ischemia by oxidative stress induced by endotoxin.

In this study, we examined the effects of oxidative stress adaptation on myocardial ischemic reperfusion injury. Oxidative stress was induced by injecting endotoxin (0.5 mg/kg) into the rat. After 24 h, rats were killed, hearts were isolated, and the effects of ischemia-reperfusion were studied using an isolated working heart preparation. The development of oxidative stress was examined by assessing malonaldehyde production in the heart. The antioxidant defense system was studied by estimating antioxidant enzyme activities and ascorbate- as well as thiol-dependent antioxidant reserve. The results of our study indicated that endotoxin induced oxidative stress within 1 h of treatment; the stress was reduced progressively and steadily up to 24 h. The antioxidant enzymes superoxide dismutase, catalase, glutathione (GSH) peroxidase, and GSH reductase were lowered up to 2 h and then increased. Both thiol- and ascorbate-dependent antioxidant reserve were enhanced, but the enhancement of the former was only transitory. After 24 h, endotoxin provided adequate protection to the heart from the ischemic-reperfusion injury, as evidenced by improved left ventricular function and aortic flow. Our results suggest that the induction of oxidative stress by endotoxin-induced adaptive modification of the antioxidant defense in the heart, thereby reducing ischemic-reperfusion injury.

Nitric oxide signaling in ischemic heart.

OBJECTIVE: Several recent studies have implicated a role of endogenous nitric oxide (NO) in the pathophysiology of myocardial ischemic/reperfusion injury. However, the mechanism by which NO exerts its beneficial/detrimental effects remains unknown. This study examined the intracellular signaling of NO by studying the role of the NO-cGMP signaling pathway on the phospho-diesteratic breakdown and turnover of phosphoinositides during myocardial ischemia and reperfusion. METHODS: Isolated working rat hearts were made ischemic for 30 min followed by 30 min of reperfusion. A separate group of hearts were pre-perfused with 3 mM L-arginine for 10 min prior to ischemia. The release of NO was monitored using an on-line amperometric sensor. The aortic flow and developed pressure were examined to determine the effects of L-arginine on ischemic/reperfusion injury. For signal transduction experiments, sarcolemmal membranes were radiolabeled by perfusing the isolated hearts with [3H]myoinositol and [14C]arachidonic acid. Hearts were then perfused for 10 min in the presence or absence of L-arginine via the Langendorff mode. Ischemia was induced for 30 min followed by 30 min of reperfusion. Experiments were terminated before L-arginine and after L-arginine treatment, after ischemia, and during reperfusion. Biopsies were processed to determine the isotopic incorporation into various phosphoinositols as well as phosphatidic acid and diacylglycerol. cGMP was assayed by radioimmunoassay and SOD content was determined by enzymatic analysis. RESULTS: The release of NO was diminished following ischemia and reperfusion and was augmented by L-arginine. L-Arginine reduced ischemic/reperfusion injury as evidenced by the enhanced myocardial functional recovery. cGMP, which remained unaffected by ischemia and reperfusion, was stimulated significantly after L-arginine treatment. The cGMP level persisted up to 10 min of reperfusion and then dropped slightly. Reperfusion of ischemic myocardium resulted in significant accumulation of radiolabeled inositol phosphate, inositol bisphosphate, and inositol triphosphate. Isotopic incorporation of [3H]inositol into phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate was increased significantly during reperfusion. Reperfusion of the ischemic heart prelabeled with [14C]-arachidonic acid resulted in modest increases in [14C]diacylglycerol and [14C]phosphatidic acid. Pretreatment of the heart with L-arginine significantly reversed this enhanced phosphodiesteratic breakdown during ischemia and early reperfusion. However, at the end of the reperfusion the inhibitory effect of L-arginine on the phosphodiesterases seems to be reduced. In L-arginine-treated hearts, SOD activity was progressively decreased with the duration of reperfusion time. CONCLUSIONS: The results suggest for the first time that NO plays a significant role in transmembrane signaling in the ischemic myocardium. The signaling seems to be transmitted via cGMP and opposes the effects of phosphodiesterases by inhibiting the ischemia/reperfusion-induced phosphodiesteratic breakdown. This signaling effect appears to be reduced as reperfusion progresses. These results, when viewed in the light of free radical chemistry of NO, suggest that such on- and off-signaling of NO may be linked to its interaction with the superoxide radical generated during the reperfusion of ischemic myocardium.

Detection of oxidative stress in heart by estimating the dinitrophenylhydrazine derivative of malonaldehyde.

Accurate estimation of the oxidative stress in heart is necessary because the pathogenesis of many heart diseases are believed to be mediated at least in part from the development of oxidative stress resulting from the generation of oxygen free radicals and reduced antioxidant defense system. The most widely used method for this purpose has been the estimation of malonaldehyde (MDA), a lipid peroxidation product, by the thiobarbituric acid (TBA) reaction method. However, because of the nonspecificity of this method, the results are often erroneous. The present report describes a method using high-performance liquid chromatography (HPLC) to estimate MDA. To develop the oxidative stress, two different models were used: ischaemic-reperfused heart and perfusing the heart with a hydroxyl radical (OH+) generating system. The coronary effluents obtained from the isolated rat heart before ischaemia and during the reperfusion of ischaemic heart, as well as during the perfusion of the heart with the OH+ generating system were collected, derivatized with 2,4-dinitrophenylhydrazine (DNPH) and extracted with pentane. Aliquots of 25 microliters in acetonitrile were injected onto a Beckman Ultrasphere C18 (3 microns) column. The products were eluted isocratically with a mobile phase containing acetonitrile-water-acetic acid (40:60:0.1, v/v/v), measured at 307 nm using a Waters M-490 multichannel UV detector and collected for gas chromatography-mass spectrometry (GC-MS). The peaks were identified by co-chromatography with DNPH derivatives of authentic standards, peak addition, and by GC-MS. The retention time for MDA-DNPH was 5.3 min.(ABSTRACT TRUNCATED AT 250 WORDS)