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Cardiovascular Research 2007 74(1):46-55; doi:10.1016/j.cardiores.2006.12.020
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Copyright © 2006, European Society of Cardiology

Microdialysis-based analysis of interstitial NO in situ: NO synthase-independent NO formation during myocardial ischemia

Claus Martina, Rainer Schulza, Heiner Posta, Kerstin Boenglera, Malte Kelmb, Petra Kleinbongardb, Petra Gresa, Andreas Skyschallya, Ina Konietzkaa and Gerd Heuscha,*

aInstitut für Pathophysiologie, Zentrum für Innere Medizin des Universitätsklinikums, Essen, Germany
bMedizinische Klinik I, RWTH Aachen, Germany

* Corresponding author. Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstraβe 55, 45147 Essen, Germany. Tel.: +49 201 723 4480; fax: 49 201 723 4481. Email address: gerd.heusch{at}uk-essen.de

Received 20 July 2006; revised 19 December 2006; accepted 22 December 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objectives: Nitric oxide (NO) synthesis by NO synthases (NOS) requires oxygen. However, although counterintuitive, NO synthesis is increased in ischemic myocardium. Accordingly, mechanisms independent of the NOS pathway have been suggested to contribute to NO synthesis during ischemia. NO initiates detrimental as well as protective mechanisms in a concentration-dependent manner, thus aggravating or improving the outcome of ischemia. The aim of this study was to measure in situ interstitial NO concentrations in parallel to infarct size in anaesthetized pigs subjected to myocardial ischemia/reperfusion. The contribution of NOS-independent pathways to NO synthesis was studied using NOS blockade.

Methods: Interstitial NO measurements, based on microdialysis combined with the oxyhemoglobin method, were made during 90 min of moderate or severe ischemia and subsequent reperfusion. To examine the effect of NOS inhibition, an initial 30-min ischemic period was followed 60 min later by a second 30-min ischemic period with intracoronary infusion of S-ethyl-isothiourea.

Results: During ischemia, the interstitial NO concentration increased for about 30 min and then remained constant at this elevated level. The increase in NO concentration by 253±82 nmol/L during moderate and 565±169 nmol/L during severe ischemia correlated inversely with subendocardial blood flow (r=–0.76). NOS inhibition increased coronary arterial pressure and decreased the interstitial basal NO concentration and tissue nitrite content. However, it did not diminish the increase in interstitial NO concentration during ischemia.

Conclusion: NOS-independent pathways are significantly involved in NO synthesis during myocardial ischemia.

KEYWORDS Nitric oxide; Ischemia; Preconditioning; Enzyme

This article is referred to in the Editorial by R.K. Kudej and C. Depre (pages 1–3) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The role of nitric oxide (NO) in the genesis of ischemia/reperfusion injury is ambivalent [1,2]. Depending on the experimental model and procedure, NO has been reported to aggravate as well as to attenuate endpoints of myocardial ischemia/reperfusion injury such as infarct size or postischemic endothelial dysfunction. The deleterious actions of higher NO concentrations are attributed to its radical character and its ability to generate potentially toxic metabolites such as peroxynitrite [3,4]. On the other hand, lower NO concentrations protect myocytes from ischemic death by a variety of mechanisms: NO inactivates caspases by nitrosylation and thus decreases myocyte apoptosis [5]. NO regulates mitochondrial respiration and thus reduces tissue oxygen consumption [6]. NO activates mitochondrial ATP-sensitive potassium channels [7] which play a crucial role in early ischemic preconditioning [8–10]. Finally, NO acts as a trigger and mediator of late ischemic preconditioning [11].

Physiologically, NO synthesis is catalyzed by NO synthases (NOS) and requires both L-arginine and oxygen as substrates [3,12,13]. Therefore, an increased NO synthesis in ischemic tissue is counterintuitive in view of the limited oxygen supply. Accordingly, other mechanisms than the NOS pathway have been proposed as a source of NO formation with ischemia [3,12–21].

The short half-life of NO in vivo makes it difficult to directly measure tissue NO levels. Measurements in the beating heart are particularly problematic and only few methods appear practicable. Electrochemical detection with different types of NO sensitive electrodes has been used for tissue NO measurements in vitro and in vivo [22]. However, the electrodes are difficult to handle, very sensitive to environmental influences and mechanical damage, and for some types of electrodes, the specificity for NO remains a topic of concern [22]. Electron paramagnetic resonance (EPR) is a robust method and specific for NO, but has also several drawbacks: expensive equipment and special expertise are required; for the time of measurement, NO is accumulated by a spin trap in relation to tissue NO levels. At the end of spin accumulation, the tissue must be rapidly frozen and grinded or extracted for EPR measurement [3,4]. Thus, endpoints of ischemia such as infarct size cannot be determined and correlated to the measured NO levels.

Microdialysis is an established method for interstitial fluid sampling without damage to the surrounding tissue [23–25]. The oxyhemoglobin assay [26] requires no sophisticated equipment and is highly sensitive for NO measurement in aqueous media. We therefore modified the oxyhemoglobin assay to combine it with microdialysis. This method was then used to study changes in the interstitial NO levels during acute myocardial ischemia in pigs in situ in parallel to hemodynamics and infarct size outcome. To examine the relevance of the NOS-dependent and NOS-independent pathways of NO formation in our setting, we inhibited NOS activity.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
The experimental protocols used in this study were approved by the local authorities of the district of Düsseldorf. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Experimental model
The experimental model has been described in detail previously [27–29]. In brief, in 32 enflurane-anesthetized Göttinger miniswine, a left lateral thoracotomy was performed and a micromanometer (P7, Konigsberg Instr, Pasadena, CA) was placed in the left ventricle through the apex. Ultrasonic dimension gauges were implanted in the left ventricular myocardium to measure the thickness of the anterior wall (System 6, Triton Technologies Inc., San Diego, CA). The proximal left anterior descending coronary artery (LAD) was cannulated and perfused from an extracorporeal circuit at constant flow. LAD perfusion pressure was measured from the sidearm of the extracorporeal circuit. The large epicardial vein parallel to the LAD was dissected and cannulated to sample coronary venous blood. A microdialysis probe (CMA/20, 10 mm, CMA/Microdialysis, Solna, Sweden) was implanted in the anterior and in 8 of the 32 animals additionally in the posterior wall for NO measurements. Radiolabeled microspheres (15 µm diameter; NEN, Du Pont Co., Boston, MA) were injected into the coronary perfusion circuit to determine regional myocardial blood flow [27–29].

2.2 Experimental protocol
The experimental protocols of the respective groups are summarized in Fig. 1.


Figure 1
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  		Fig. 1 Experimental protocols.

 
2.3 Myocardial ischemia and ischemic preconditioning
After implantation of the microdialysis probes and a 30 min equilibration period, their outflow was collected in 10 min intervals starting 30 min prior to the onset of ischemia. Ischemia lasted for 90 min and was followed by 2 h reperfusion. To induce ischemia, coronary inflow was decreased to reduce a regional work index [29] by either 50% (group 1; n=4) or by 90% (group 2; n=11). Sets of hemodynamic and blood flow measurements were performed under control conditions, immediately before and at 10 and 85 min of ischemia. Microspheres were injected into the LAD perfusion system for the measurement of regional myocardial blood flow, and systemic hemodynamic and regional dimension data were recorded. In group 3 (n=9), an initial period of 10 min ischemia was followed by 15 min reperfusion (ischemic preconditioning) and then 90 min ischemia; the severity of ischemia and the further protocol were identical to that of group 2.

At the end of each study, following 120 min reperfusion, infarct size was measured using triphenyl tetrazolium chloride staining as previously described [29].

The employed method of NO measurement is highly selective, with the possible exception of superoxide anions competing with NO [26]. Therefore, in a subset of 6 experiments (subgroup 1/2; 2 animals of group 1, 4 animals of group 2) and in all animals of group 5 a second microdialysis probe was implanted into the anterior wall of the myocardium. In parallel, one probe was perfused as usual, the other with HbO2 buffer containing additionally 150 U/ml superoxide dismutase (SOD; Sigma, Taufkirchen, FRG).

2.4 NOS characterization and inhibition
NOS-dependent NO formation was inhibited in groups 4 (n=4) and 5 (n=4). First, we examined the existence and localization of the 3 isoforms, NOS1, NOS2, NOS3, in pig myocardium. Mitochondria were isolated, and total myocardial and mitochondrial proteins were extracted, as previously described [30]. 50 µg of proteins as well as positive control extracts for the NOS isoforms were electrophoretically separated and transferred to nitrocellulose membranes. The membranes were probed with antibodies against NOS1, NOS2 or NOS3, Na+/K+-ATPase, G{alpha}s, SERCA2, ATPsynthase {alpha}. After incubation with the respective secondary antibodies, immunoreactive signales were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). Sections (4 µm) of formalin-fixed and paraffin-embedded pig myocardium were stained with antibodies against NO synthases and subsequently with the respective FITC-conjugated secondary antibodies. Isolated mitochondria were also stained with antibodies against NOS isoforms and the mitochondrial marker protein ANT (adenine nucleotide transporter). Primary antibodies were omitted as negative control. The tissue sections as well as the isolated mitochondria were covered with Vectashield (H-1000, Vector Laboratories, Burlingame, CA, USA) and examined by laser scan microscopy (Pascal, Zeiss, Jena, FRG). The following antibodies were used: rabbit polyclonal anti-human nNOS (BD-Transduction, San Jose, CA, USA), monoclonal anti-mouse iNOS (BD-Transduction, San Jose, CA, USA; isotype mouse IgG2A), mouse monoclonal anti-human eNOS (BD-Transduction, San Jose, CA, USA) for laser scan microscopy on tissue sections and monoclonal anti-mouse iNOS (BD-Transduction, San Jose, CA, USA; isotype mouse IgG1) for laser scan microscopy on isolated mitochondria and for Western blot analysis; additionally for Western blot analysis: rabbit polyclonal anti-human phospho-eNOS (Ser 1177, Cell Signaling, Beverly, MA, USA), goat polyclonal anti-human ANT (Santa Cruz, Santa Cruz, CA, USA), mouse monoclonal anti-rat sodium/potassium (Na+/K+) ATPase (Upstate, Waltham, MA, USA), mouse monoclonal anti-dog sarcoplasmic reticulum Ca2+ ATPase (Serca2, Sigma, Saint Louis, Mo, USA), rabbit polyclonal anti-human G{alpha}s, (Santa Cruz, Santa Cruz, CA, USA), mouse monoclonal anti-human ATPsynthase {alpha} (BD-Transduction, San Jose, CA, USA).

To block NOS activity, the potent, non-selective NOS inhibitor S-ethyl-isothiourea (EITU) was used. EITU inhibits all 3 NOS isoforms with an almost identical IC50 value of about 10–7 M [31]. The protocol required two consecutive periods of ischemia: an initial control period followed by a second period with NOS inhibition. Therefore, the duration of ischemia was shortened to 30 min to exclude potential infarction which would alter the amount of viable tissue which is capable of generating NO. The severity of ischemia was identical to that of group 1 in group 4 and to that of group 2 in group 5. The initial ischemic control period was followed by 30 min of reperfusion. Then, the animals received EITU (450 µg/kg bolus ic., followed by ic. infusion of 50 µg/kg/min throughout the sampling protocol). After 30 min of EITU infusion, a second period of 30 min ischemia followed. Given the 4 h half-life of EITU and an assumed body water content of 70% of body weight, the dose used was estimated to result in an EITU concentration of about 10–5 M (100 times the IC50 value).

In group 5, tissue contents of nitrite, nitrate and organic nitroso species (RNO) were determined following EITU infusion. Specimens (about 1.5 g) from anterior (AW) and posterior wall (PW) of the myocardium were homogenized, and the NO metabolites were measured as described previously [18].

2.5 NO measurement
The oxyhemoglobin (HbO2) assay for NO measurement [26] was modified to permit its in vivo application by combination with the microdialysis technique. Probes were perfused with buffer containing 2 µM freshly prepared HbO2 [26] in: (mmol/L) 135 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.3 CaCl2; 0.05% PEG 400; pH 7.4; buffer flow was 2.8 µl/min. The method is based on the UV spectroscopic changes resulting from the oxidation of HbO2 by NO to form metHb. However, metHb is also formed from HbO2 autoxidation. Thus, usually the difference spectrum between a NO-exposed HbO2 solution sample and the untreated HbO2 solution is continuously recorded using a double beam UV spectrophotometer with flow cells. Unfortunately, this on-line measurement of difference spectra cannot be adopted to the microdialysis technique due to the limited flow through the microdialysis probes (about 3 µl/min). The outflow was therefore collected in 10 min intervals and measured using a diode array spectrophotometer (HP 8453, Hewlett Packard, Waldbronn, Germany). The molecular cut off of 20,000 D of the microdialysis probe membrane prevented leakage of hemoglobin from the buffer. The total hemoglobin concentration within the probe can change, nevertheless, due to H2O diffusion through the probe membrane. Such changes in hemoglobin concentration can be corrected for by recording simultaneously an isobestic wavelength [26]. The absorbance at isobestic points does not change during conversion of HbO2 to metHb and thus remains theoretically constant; any measured change reflects and quantifies a change in total Hb concentration. Discontinuous measurements tend to produce artifacts from instabilities in baseline absorbances between consecutive measurements. To minimize such artifacts, no absolute absorbances but only absorbance differences of the individual spectra were used: metHb content was quantified from the absorbance difference at 401 and 420 nm ({delta}A401–420), corrected by the absorbance difference at the two isobestic wavelengths at 410.5 and 472 nm. Wavelength 401 nm represents the maximum (molar extinction coefficient {delta}{varepsilon}=49 mM–1cm–1), 420 nm the minimum ({delta}{varepsilon}=–51 mM–1cm–1) absorbance of the difference spectrum (metHb–HbO2) [26]. Difference spectra were calculated by subtracting {delta}A401–420 of untreated HbO2 from {delta}A401–420 of the NO-exposed sample ({delta}A401–420(metHb–HbO2)). To adequately consider HbO2 autoxidation, the course of spontaneous metHb formation in untreated HbO2 solution was determined in vitro in microdialysis probes (n=18) immersed in 154 mmol/L NaCl at 37 °C. The influence of acidosis on metHb formation inside the probes was also tested in vitro (n=10): 154 mmol/L NaCl was buffered with 8 mmol/L NaH2(PO4)/Na2H(PO4) to pH=7.4 or to pH=6.4 at 37 °C. The probes were immersed in buffer pH 7.4 for 30 min, then in buffer pH 6.4 for 40 min, and again in buffer pH 7.4 for 40 min. This change in pH outside the probes did not measurably influence the linear increase in metHb formation inside the probes. NO recovery was also determined in vitro: probes were placed in a nitrogen flushed bath containing authentic NO solution which had been freshly prepared by gassing O2 free 154 mmol/L NaCl with prepurified NO in argon atmosphere [32]. NO concentrations were measured repeatedly from the NO solution within the bath and from simultaneously sampled microdialysis outflow over a period of 60 min, respectively. With a buffer flow of 2.8 µl/min, NO recovery inside the probes amounted to 33.5±2.6%.

2.6 Data analysis and statistics
All data are reported as mean values±standard deviation (SD). Reported interstitial NO concentrations are corrected for microdialysis probe recovery. Ischemia-induced increases in myocardial NO concentrations are reported as mean values over the duration of ischemia and were evaluated using one-way analysis of variance. When significant differences in mean values were detected, individual mean values were compared by Fisher's LSD post-hoc tests. Linear regression analysis was performed between subendocardial blood flow (ENDO) and the increase in interstitial NO concentration during ischemia. A p-value less than 0.05 was accepted as indicating a significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
In vitro without NO exposition, the metHb concentration in microdialysis outflow increased linearly for at least 180 min (Fig. 2A). The velocity of autoxidation, however, varied distinctly between probes even when run in parallel using the identical batch of HbO2 solution. Therefore, in NO measurements each probe had to serve as its own control: ideally, metHb formation resulting from autoxidation would be measured in the absence of NO before and following a period of NO exposition.


Figure 2
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  		Fig. 2 A: UV spectroscopic changes in microdialysis outflow due to HbO2 autoxidation (n=18). {delta}A401–420: difference of absorbances at wavelengths 401 and 420 nm. B: UV spectroscopic changes in microdialysis outflow of one single microprobe. The regression line (r2=0.9853) was assumed to reflect HbO2 autoxidation.

 
In interstitial NO measurements, the linear increase in metHb concentrations before and after ischemia represents autoxidation plus oxidation by basal myocardial NO. Any additional metHb formation during ischemia represents myocardial NO formation above baseline NO levels (Fig. 2B). In our approach, the absorbance difference {delta}A401–420 was linearly interpolated to the time point of NO sample measurement and permitted the calculation of the absorbance difference {delta}A401–420(metHb–HbO2) of the respective difference spectrum of metHb (NO-exposed) minus HbO2 (autoxidation). Thus, by this procedure, only ischemia-induced changes in interstitial NO concentrations could be measured and interstitial baseline NO levels had to be neglected.

The interstitial NO concentration (NOis) increased during moderate (group 1) and – more markedly – during severe (group 2) ischemia (Fig. 3). The mean increase in NOis during the 90 min ischemia amounted to 253±82 nmol/L in group 1 (p<0.05) and to 565±169 nmol/L in group 2 (p<0.05), whereas in the nonischemic posterior wall NOis remained constant (29±52 nmol/L; ns vs baseline; n=8). The increase in NOis correlated inversely to the subendocardial blood flow measured at 10 min ischemia (y=–1317x+635; r=–0.76; Fig. 4). No infarction occurred in group 1; in group 2, infarct size amounted to 18.85±5.29%. Ischemic preconditioning (group 3) did not change the increase in NOis during 90 min severe ischemia as compared to group 2 (496±168 nmol/L; n=9; p<0.05 vs baseline, ns vs group 2); infarct size amounted to 4.84±1.88% (p<0.05 vs group 2); hemodynamics and regional myocardial blood flow did not differ between groups.


Figure 3
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  		Fig. 3 Increase in myocardial NO concentration ({delta} NOis) during moderate (closed circles) and severe ischemia (open circles).

 

Figure 4
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  		Fig. 4 Correlation between ischemia-induced increase in myocardial NO concentration ({delta} NOis) and subendocardial blood flow (ENDO).

 
Superoxide anion formation did not measurably interfere with the NO measurements: in subgroup 1/2, the increase in NOis during 90 min ischemia (364±134 nmol/L) was not changed by the addition of SOD (367±108 nmol/L).

3.1 NOS inhibition
All 3 NOS isoforms exist in pig myocardium: NOS1 and NOS3 were localized in endothelium, while NOS2 was primarily localized in mitochondria (Figs. 5–7GoGo). During EITU infusion, coronary perfusion pressure increased from 125.5±5.1 to 197.5±6.6 mm Hg in group 4 and from 115.6±2.0 to 178.5±8.6 mm Hg in group 5 (both p<0.05) at constant coronary blood flow (group 4: 33.4±4.9 vs 30.4±4.0; group 5: 33.7±5.9 vs 33.6±5.8 ml/min/g; both ns), indicating effective inhibition of NOS3. The efficiency of inhibition of all NOS isoforms was substantiated by the measured metHb formation in the microdialysis probes. The time-dependent, linear increase in baseline metHb formation during normoperfusion (Fig. 2B) results from HbO2 autoxidation plus HbO2 oxidation by interstitial NO. Thus, any reduction in interstitial NO due to NOS inhibition is expected to reduce the rate of metHb formation. The relationship of time vs metHb formation was y=3.042x+3.43 (r2=0.9980) in group 4 and y=4.379x+2.31 (r2=0.9920) in group 5. Inhibition of NOS using EITU reduced the velocity of metHb formation to y=2.317x+0.90 (r2=0.9957) in group 4 and y=3.354x+4.19 (r2=0.9905) in group 5, respectively (both p<0.05 vs before EITU). The difference in the slope of metHb formation before and during NOS inhibition corresponds to calculated interstitial baseline NO concentrations of 32±15 nmol/L in group 4 and 53±29 nmol/L in group 5.


Figure 5
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  		Fig. 5 Expression of NOS isoforms in pig left ventricular myocardium. Paraffin-embedded tissue sections of pig myocardium were stained with antibodies against NOS1 (red) and the endothelial marker protein CD31 (green); co-localization pixels are shown in yellow. Tissue sections were also stained with antibodies against NOS2 (green) and actin was visualized with phalloidin (red). Again, co-localization pixels are shown in yellow. Furthermore, ventricular tissue sections were stained with antibodies against NOS3 (green), and phosphorylated NOS3 (green). A negative control, in which the primary antibodies were omitted, is presented for NOS3 staining.

 

Figure 6
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  		Fig. 6 NOS2 is present in mitochondria. Isolated pig left ventricular mitochondria were stained with antibodies against NOS1, NOS2, NOS3 (red) and the mitochondrial marker protein ANT (adenine nucleotide transporter, green) and were analyzed by confocal laser scan microscopy. Co-localization pixels are shown in yellow.

 

Figure 7
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  		Fig. 7 NOS2, but neither NOS1 nor NOS3 is present in isolated pig mitochondria. Western blot analysis was performed on the same amount (50 µg) of pig left ventricular and mitochondrial proteins as well as on positive control extracts for NOS1, NOS2, and NOS3. The purity of the mitochondrial extracts was demonstrated by the absence of immunoreactivity for Na+/K+-ATPase, G{alpha}s-protein, SERCA2, which were used as markers of sarcolemma, cytosol, and sarcoplasmic reticulum. ATPsynthase {alpha} was used as mitochondrial marker protein. Ponceau S staining demonstrates equal protein loading.

 
In group 5, the efficiency of regional NOS inhibition was also examined by measuring the tissue contents of NO metabolites in AW and PW. No differences were found in nitrate contents (22.60±3.85 vs 22.80±4.38 pMol/mgWW; AW vs PW; ns) and RNO (0.75±0.42 vs 0.77±0.31 pMol/mgWW; AW vs PW; ns). In contrast, nitrite content was reduced in AW to 1.63±0.66 vs 2.69±0.58 pMol/mgWW in PW (p<0.05); the nitrite content in normal anterior myocardium is 5.30±0.96 pMol/mgWW (n=5).

Surprisingly, NOS inhibition did not diminish the ischemia-induced increase in NOis during 30 min moderate (group 4) or severe ischemia (group 5).In group 4, NOis increased by 275±127 nmol/L without and by 268±65 nmol/L with EITU. In group 5, NOis increased by 395±204 nmol/L without and by 347±176 nmol/L with EITU. Again, addition of SOD to the HbO2 perfusion buffer did not diminish the measured increases: in the probes run with buffer containing SOD, NOis increased by 437±287 without and by 409±272 nmol/L with EITU.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The HbO2 assay [26] was combined with microdialysis. Using this method, changes in NOis during myocardial ischemia could be measured in pigs in situ. NOis increased in close correlation to the severity of ischemia (decrease in subendocardial blood flow) demonstrating that NO is either formed in the interstitium or is released from cardiomyocytes, endothelial cells or erythrocytes and/or its washout is decreased. NO formation is sufficiently high to result in measurably increased NOis despite of the numerous NO scavengers abundant in blood-perfused hearts, e.g. myoglobin [33,34], cytochromes [35,36], and hemoglobin [37]. Following an initial increase, NOis remained constant at this elevated level throughout ischemia, demonstrating that an equilibrium of NO formation and degradation/washout was achieved. The height of this equilibrium concentration depended on the severity of ischemia. This is the first demonstration in situ of a net increase in NOis during myocardial ischemia.

Mechanisms of NOS-independent NO formation include the oxidative degradation of arginine by hydrogen peroxide [14], the acidosis-induced disproportionation of nitrite [3,17], the reduction of nitrite by xanthine oxidase [12,16], by desoxyhemoglobin [21], or by cytochromes [15]. Recently, nitrite has been reported to represent a circulating and tissue storage form of NO which is activated during ischemia, probably through the reduction of nitrite by desoxymyoglobin, desoxyhemoglobin, and/or other heme proteins [20]. RNO (S-nitrosothioles, RSNO; nitrosamines, R2NNO; transition metal nitroso/nitrosyl compounds, RMen+NO) have also been suggested to represent a storage form of NO which is activated during ischemia [18,19]. The underlying biochemical pathways are summarized in Scheme 1. Without oxidative stress, i.e. at low O2– levels, enzymatically formed NO reacts with O2 to form NO2. NO2 can dimerize to form N2O4 which is rapidly hydrolized with disproportionation to nitrite and nitrate. Alternatively, NO2 can react with another NO molecule to form the nitrosating agent N2O3. On nitrosation, N2O3 produces RSNO or R2NNO and equimolar amounts of nitrite. Elevated O2– levels shift NO reactivity towards formation of peroxynitrite which can rearrange to nitrate or generate reactive intermediates. These intermediates can, among other reaction pathways, oxidize thiols to form RS radicals which can then directly react with NO to produce RSNO. During ischemia, oxidative degradation of arginine, oxidation of RMen+NO, reduction of RSNO or R2NNO, or reduction or disproportionation of nitrite represents potential pathways of NOS-independent NO formation.


Figure 1
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  		Scheme 1 Biochemical pathways of NOS-dependent and NOS-independent NO formation. Modified from Bryan NS et al., PNAS 2004, 101, 4308–4313. EITU: S-ethyl-isothiourea; O2–: superoxide anion; ONOO–: peroxynitrite anion; RNO: organic nitroso/nitrosyl species; RSH: thioles; RSNO: S-nitrosothioles; R2NH: secondary amines; R2NNO: nitrosamines; RMen+: organic transition metal compounds; RMen+NO: transition metal nitroso/nitrosyl compounds.

 
We demonstrated the existence of all 3 NOS isoforms in pig myocardium. Accordingly, EITU which equally potently inhibits the 3 NOS isoforms [31] was used to inhibit NOS locally in the anterior wall (AW). The efficiency of NOS inhibition was examined by comparing the tissue contents of NO metabolites in AW and the posterior, control wall (PW). The tissue content of nitrite was decreased by 40% in AW as compared to PW. A reduction in nitrite content by 40% in anterior myocardium is an underestimation secondary to a recirculation of EITU also into the posterior myocardium; the reduction amounts to 70% as compared to normal anterior myocardium. Nitrite formation is known to respond rapidly to NOS inhibition [19,38,39], thereby supporting the efficiency of acute short-term NOS inhibition in AW in our model. RNO and nitrate contents were not different in AW and PW. In contrast to nitrite, RNO include nitrosation and transnitrosation reactions of proteins [18,19] and are therefore expected to respond slower to NOS inhibition. Tissue nitrate contents are mainly related to plasma nitrate concentrations which decrease only after long-term NOS inhibition [39]. The efficiency of NOS inhibition was further examined by an estimation of basal NOis in normoperfused hearts. This estimation was based on the rate of metHb formation in the microdialysis probes when assuming complete NOS blockade in AW. The calculated basal NOis concentrations of 32±15 nmol/L in group 4 and 53±29 nmol/L in group 5 are in accordance with the reported physiological range (1–50 nmol/L) [40] and, thus, validate the underlying assumption of effective NOS blockade. Effective blockade of NOS3 was further indicated by the observed increase in coronary perfusion pressure with EITU infusion. Nevertheless, the increase in NOis during moderate and severe ischemia was not diminished during NOS blockade. We conclude that NOS-independent pathways are significantly involved in NO synthesis during myocardial ischemia, even during moderate ischemia when oxygen is limited but still available – consequently also available for the NOS reaction. Such NOS-independent formation of NO makes negative findings with blockade of NOS-dependent NO formation, including our own [41,42], less conclusive with respect to its biological function, although we cannot exclude that ischemic preconditioning per se increases the activity of one or more NOS isoforms.

4.1 Critique of methods and limitations
All techniques for direct NO measurement will detect only that proportion of NO which escapes reactions with other reactants (oxygen, superoxide, transition metals, thiols, amines). Thus, NO must be trapped close to the site of its formation. On the other hand, to measure interstitial NO concentrations, NO trapping should possibly not interfere with the biological equilibrium of NO formation and degradation. The use of microprobes facilitates close proximity of the site of measurement to the site of NO formation and prevents HbO2 from directly interfering with the biological NO equilibrium: the exchange barrier of the microprobe membrane prevents leakage of HbO2 and, thus, analytical NO trapping occurs only at the site of analysis, i.e. within the probe. NO trapping by HbO2 is virtually irreversible and, therefore, is quantitatively realized in the following spectroscopic analysis. On the other hand, the HbO2 assay is not absolutely selective for NO as, in biological tissues, nitrite and hydrogen peroxide also oxidize HbO2 to metHb. However, the rate of reaction of HbO2 with NO is extremely high, corresponding to a rate constant of 3.7x107 M–1s–1 [26]. Consequently, NO trapping by HbO2 is almost stoichiometric. Nitrite or hydrogen peroxide react considerably slower with HbO2 [26] and, consequently, contribute to a much lower extent to metHb formation as compared to NO. In addition, metHb formation resulting from physiological tissue nitrite is reflected in the linear metHb formation throughout the experiment and therefore of no consequence for our NO measurements. In contrast to nitrite and hydrogen peroxide, the reactivity of superoxide anions for HbO2 is comparable to that of NO [26]. However, addition of SOD to the HbO2 microdialysis buffer did not diminish metHb formation during 90 min severe ischemia, indicating that superoxide anions did not measurably contribute to metHb formation in our experiments and ruling out at least one possible source of hydrogen peroxide. Unfortunately, respective experiments using catalase to rule out any possible contribution of hydrogen peroxide to metHb formation are incompatible with the HbO2 method: the heme protein catalase competes effectively with HbO2 for NO and becomes inactivated by this reaction [43].

Changing the pH outside the probes from 7.4 to 6.4 in vitro did not measurably influence metHb formation inside the probes, indicating sufficient buffer capacity of the constantly flowing microdialysis buffer. Despite of this obviously relatively stable pH inside the probes, HbO2 might release oxygen in ischemic tissue due to the reduced pO2. Such formation of reduced Hb would result in a conservative error, i.e. an underestimation of NOis. Formation of reduced Hb would also result in a decrease in the absorbance difference of the isobestic wavelengths 410.5–472 nm systematically during ischemia; however, this did not occur.

In conclusion, our study is the first to report increased net interstitial NO during myocardial ischemia in situ which is independent of NOS.


   Notes
 
* Supported by the German Research Foundation (He 1320/8-3).

Time for primary review 14 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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