Endothelial NOS expression and ischemia–reperfusion in isolated working rat heart from hypoxic and hyperoxic conditions

Endothelial NOS expression and ischemia–reperfusion in isolated working rat heart from hypoxic and hyperoxic conditions

Biochimica et Biophysica Acta 1524 (2000) 203^211 www.elsevier.com/locate/bba Endothelial NOS expression and ischemia^reperfusion in isolated workin...

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Biochimica et Biophysica Acta 1524 (2000) 203^211

www.elsevier.com/locate/bba

Endothelial NOS expression and ischemia^reperfusion in isolated working rat heart from hypoxic and hyperoxic conditions M. Felaco a , A. Grilli a , N. Gorbunov f , P. Di Napoli b , M.A. De Lutiis e , C. Di Giulio c , A.A. Taccardi b , A. Barsotti b , R.C. Barbacane d , M. Reale d , P. Conti d; * a

d

f

Department of Biomorphology, Biology Section, University of Chieti, Chieti, Italy b Laboratory of Experimental Cardiology, University of Chieti, Chieti, Italy c Department of Biomedical Sciences, Physiology Section, University of Chieti, Chieti, Italy Immunology Division, University of Chieti, School of Medicine, Via dei Vestini 31, 66013 Chieti (CH), Italy e Department of Oncology and Neuroscience, University of Chieti, Chieti, Italy Department of Environmental and Occupational Health, The University of Pittsburgh, Pittsburgh, PA, USA Received 29 June 2000; received in revised form 9 October 2000; accepted 18 October 2000

Abstract Induction of endothelial nitric oxide synthase (eNOS) contributes to the mechanism of heart protection against ischemia^reperfusion damage. We analyzed the effects of hypoxia and hyperoxia on eNOS expression in isolated working rat hearts after ischemia^reperfusion damage. Adult male Wistar rats were submitted to chronic hypoxia (2 weeks) and hyperoxia (72 h). The hearts were submitted to 15 min of ischemia and reperfused for 60 min, then we evaluated hemodynamic parameters and creatine phosphokinase (CPK) release. eNOS expression was estimated by RT^PCR ; enzyme localization was evaluated by immunohistochemistry and the eNOS protein levels were detected by Western blot. All hemodynamic parameters in hypoxic conditions were better with respect to other groups. The CPK release was lower in hypoxic (P 6 0.01) than in normoxic and hyperoxic conditions. The eNOS deposition was significantly higher in the hypoxic group versus the normoxic or hyperoxic groups. The eNOS protein and mRNA levels were increased by hypoxia versus both other groups. Chronic hypoxic exposure may decrease injury and increase eNOS protein and mRNA levels in heart subjected to ischemia^ reperfusion. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Nitric oxide synthase; Ischemia^reperfusion; Hypoxia; Hyperoxia ; Rat heart

1. Introduction Nitric oxide (NO) is an important regulator of endothelial functions and vascular tone. It is an inhibitor of platelet aggregation in biological tissues and may also exert a signi¢cant in£uence in several pathological conditions [1,2]. NO is synthesized from L-arginine in the presence of O2 and NADPH-diaphorase in a reaction catalyzed by nitric oxide synthase (NOS) [3]. NOS is a family of enzymes with three isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) [2]. eNOS is present constitutively in the heart, and this iso-

* Corresponding author. Fax: +39-871-561635; E-mail : [email protected]

form is calcium^calmodulin-dependent [4]. NO arises in the vascular wall, is exclusively a cNOS (constitutive)like activity in endothelial cells, and in£ammatory stimuli can induce an iNOS activity, both in the endothelium and vascular smooth muscle cells. NO activates soluble guanylate cyclase and therefore may stimulate intracellular cyclic guanosine 3P,5P-monophosphate (cGMP) accumulation [5]. The injury induced by ischemia^reperfusion leading to heart dysfunction by direct damage of myocytes may be attributed to altered NO formation [6]. Following ischemia, metabolic and functional changes become progressively more severe, reserves of adenosine 5P-triphosphate (ATP) and creatine phosphate (CP) decrease quickly and energetic production shifts to anaerobic glycolysis. These changes induce an acidi¢cation of the cytoplasm and the decreased pH induces a denaturation of proteins [7,8]. Recent data show that NOS activity could be modi¢ed by ischemia, correlating blood £ow oxygen supply and

0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 1 5 9 - 8

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damage. Many cases report that the chronic exposure to hypoxia and hyperoxia may induce a variation of eNOS protein in many tissues [9,10]. In recent literature it was reported that blockade of NO synthase activity with Lnitroarginine (L-NA), a NO-synthase inhibitor, increased infarct size, an e¡ect expressed primarily during reperfusion [11]. The aim of the present study is to evaluate eNOS transcription and translation in heart ischemia^reperfusion subjected to the e¡ects of chronic hypoxia and hyperoxia against normoxic conditions and cardiac performance in isolated working rat hearts. We therefore localized the eNOS protein by immunohistochemistry, tested protein levels by Western blot, and mRNA expression by reverse transcription^polymerase chain reaction (RT^PCR) in the three conditions. In addition, we evaluated left ventricular performances through minute-work values, aortic and coronary £ow, heart rate, and myocytic damage by creatine phosphokinase (CPK) release.

weight gain (%): (heart weight before)/(heart weight after)U100; hemodynamic parameters (aortic pressure, aortic and coronary £ows, heart rate, minute work) were measured every 10 min [15]. At the end of the experimental procedure all the hearts were cut in blocks, ¢xed in liquid nitrogen and stored at 380³C. Myocardial tissues were immersed overnight in ice-cold 4% paraformaldehyde in 0.1 M phosphate-bu¡ered saline (PBS). Tissues were then rinsed in 15% sucrose PBS for 1 h and stored at 4³C in 30% sucrose PBS for 2 h. Ten-Wm thick sections were cut using a cryomicrotome (Reichert-Jung Frigo cut 2800), mounted on microscope slides, ¢xed by immersion in acetone at 4³C for 5 min and air-dried. 2.2. Enzyme release evaluation

2. Materials and methods

Myocardial necrosis enzyme assay of coronary e¥uents was performed during stabilization (20 min), Langendor¡ reperfusion (at 45 min) from the beginning of the experimental time, and working heart reperfusion CPK activities from eluate sample, determined using commercial kits (Boehringer Mannheim, Milan, Italy).

2.1. Experimental protocol and perfusion technique

2.3. NADPH-diaphorase histochemistry

Adult male Wistar rats (n = 30) were used and the experimental protocol was performed according to the Guidelines of the American Physiology Society. After administration of heparin (1000 UI/kg) into the femoral vein, the hearts were excised and immersed in cold modi¢ed Krebs^Henseleit solution (KH: 108 mM NaCl ; 25 mM NaHCO3 ; 4.8 mM KCl; 1.2 mM KH2 PO4 ; 1.2 mM MgSO4 ; 2.5 mM CaCl2 ; 11 mM glucose). Rats were subdivided randomly in three groups: (A) Normoxic (n = 10); (B) hyperoxic (n = 10); (C) hypoxic (n = 10). Chronic hypoxic rats were maintained for 2 weeks in a chamber which was ¢lled with 10% oxygen [12]. Chronic hyperoxic rats were maintained for 3 days in a similar chamber, ¢lled with 95% oxygen [13,14]. The animals were anesthetized with Nembutal (20 mg/kg). All hearts were explanted and mounted in Langendor¡ perfusion circuit and following 20 min of stabilization in working heart mode, were submitted to 15 min of global ischemia and reperfused for 60 min with non-circulating modi¢ed KH solution. The perfusion bu¡er was bubbled in 95% O2 and 5% CO2 at 37³C, pH 7.4. Aortic and coronary £ows were measured collecting aortic chamber over£ow and heart chamber e¥uent into graduated cylinders. Aortic pressure was monitored through a membrane transducer (TNF-R, Viggo-Spectramed, Oxnard, CA) connected to a side arm of the aortic cannula. Heart rate was determined with an epicardial ECG (Cardioline 350/1, Milan, Italy). Minute work was computed as the product of cardiac output (aortic £ow+ coronary £ow) and peak aortic systolic pressure. At the beginning and at the end of the experimental procedure, hearts were weighed ; results were expressed as heart

For histochemical staining of NADPH-diaphorase, slides were incubated for 30 min at 37³C in 50 mM Tris^HCl (pH 8.0), 0.5 mM Nitro blue tetrazolium (NBT), 50 mM MgCl2 and 1 mM NADPH, and as inhibitor of respiratory chain 0.1 mM NaCN (Sigma Chemical Co., St. Louis, MO) [16]. The slides were brie£y washed in PBS, and dehydrated with a graded series of ethyl alcohol. Slides from both staining procedures were mounted using Permount, coverslipped and photographed through a Leitz microscope. 2.4. eNOS immunohistochemistry Slides were treated with 5% normal goat serum, 0.1% bovine serum albumin and 0.1% Tween-20, in PBS for 30 min at room temperature. Then slides were incubated for 30 min with primary anti-eNOS antibodies (rabbit IgG Santa Cruz Biotech, Santa Cruz, CA) which were diluted 1:100. This was followed by three washes (rinsing) with PBS. Then the sections were incubated with biotinylated secondary antibody, and incubated with peroxidase substrate. Antibody-labeled specimens were rinsed with distilled water for 5 min and dehydrated (Rabbit ABC Staining System, Santa Cruz Biotech) [17]. 2.5. Western blots Equal amounts of protein (50 Wg) from normoxic hearts, hyperoxic hearts and hypoxic hearts were separated by electrophoresis in a 7.5% polyacrylamide^sodium dodecyl sulfate (SDS) gel (Bio-Rad, Hercules, CA) and transferred

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Table 1 Minute work (mmHg/ml per min) Stabilization Time (min) Normoxic (A) Hypoxic (B) Hyperoxic (C)

10 9850 þ 356 9600 þ 430 9450 þ 510

Ischemia Langendor¡ 20 9750 þ 401 9450 þ 388 9100 þ 492

20 0 0 0

35 0 0 0

45 5150 þ 455* 7500 þ 512 5050 þ 475#

Reperfusion in working heart 55 5100 þ 460 7500 þ 520 5050 þ 475#

65 5100 þ 450* 7450 þ 510 5000 þ 450#

75 5050 þ 400* 7450 þ 500 4700 þ 350#

85 5000 þ 390* 7400 þ 480 4400 þ 380#

95 4900 þ 390* 7300 þ 290 4400 þ 300#

105 4900 þ 380* 7350 þ 297 4000 þ 270#

Minute work was computed as the product of cardiac output (aortic £ow+coronary £ow) and peak aortic systolic pressure. Heart rate was determined with an epicardial ECG. Aortic and coronary £ows were measured collecting aortic chamber over£ow and heart chamber e¥uent into graduated cylinders. Aortic pressure was monitored through a membrane transducer connected to a side arm of aortic canula. During ischemic time (15 min) and Langendor¡ perfusion (10 min) no values were measured. All data are expressed as mean þ S.D. *P 6 0.01; # P 6 0.05.

at 4³C to nitrocellulose membrane (Bio-Rad) in glycine^ methanol bu¡er. Nitrocellulose was then blocked in Trisbu¡ered saline (TBS)^milk and incubated overnight in primary antibody anti-eNOS (Santa Cruz Biotech). The nitrocellulose was then washed in TBS, incubated with an alkaline phosphatase-conjugated second antibody for 2 h, washed again, and developed in an alkaline bu¡er with NBT as substrate (Alkaline Phosphatase Conjugate Substrate kit, Bio-Rad) [18].

2.7. Image processing and analysis system

2.6. RNA analysis (RT^PCR)

3. Statistical analysis

As previously described [19], total RNA was extracted using 1 ml RNAzol (Cinna Biotex, Houston, TX) with 20 Wg Escherichia coli rRNA (Boehringer) as carrier. Reverse transcription was performed in a volume of 20 Wg containing M-MLV reverse transcriptase (Perkin^Elmer), 1 mM dNTP, 2.5 WM Random Primers and 1 U/Wl RNAse inhibitor (Pharmacia), for 30 min at 42³C. PCR ampli¢cation was performed using an Eppendorf Mastercycler 5330, operating the temperature step of 60 s extension time at 72³C [20]. The MgCl2 concentrations used for rat eNOS cDNA ampli¢cation were 2.0 mM. The following primer pairs were used: 5P-CGAGATATCTTCAGTCCCAAGC-3P (sense) and 5P-GTGGATTTGCTGCTCTCTAGG-3P (antisense) for rat endothelial NOS. A relative PCR was performed using 18S rRNA as internal standard.

The results were expressed as mean þ S.D. Statistical analysis was performed using the analysis of variance (ANOVA). Probability of null hypothesis of 6 5% (P 6 0.05) was considered statistically signi¢cant.

Quantitative analysis was performed by using a Sony video camera connected with a Leica Quantimet 500 plus (Leica Cambridge, Cambridge, UK) determining the change in integrated optical density (IOD) using ISO Transmission Density Kodak CAT 152-3406 (Eastman Kodak Company, Rochester, MN) as standard [21].

4. Results 4.1. Minute-work values of hearts in normoxic, hypoxic and hyperoxic conditions The aim of our work was to study left ventricular performances (minute work : mmHg/ml per min) in the chronic hypoxic group (B), compared with control group (normoxic, A) and hyperoxic group (C), see stabilization columns in Table 1. In fact, the values of minute work are

Table 2 Aortic and coronary £ow Stabilization

Ischemia

Langendor¡

Reperfusion in working heart

(A) Aortic £ow (ml/min) Time (min) 10 Normoxic (A) 61 þ 8 Hypoxic (B) 66 þ 5 Hyperoxic (C) 59 þ 7

20 57 þ 5 59 þ 4 58 þ 6

20 0 0 0

35 0 0 0

45 48 þ 5* 54 þ 6 56 þ 5

55 48 þ 4* 54 þ 6 55 þ 6

65 45 þ 5 55 þ 7 52 þ 4

75 46 þ 2 54 þ 6 49 þ 5

85 46 þ 13 54 þ 5 48 þ 4

95 44 þ 3 53 þ 5 47 þ 5

105 43 þ 2 53 þ 4 47 þ 5

(B) Coronary £ow Time (min) Normoxic (A) Hypoxic (B) Hyperoxic (C)

20 12 þ 3 13 þ 3 11 þ 2

20 0 0 0

35 0 0 0

45 8.7 þ 1.5* 12 þ 2 7 þ 2#

55 9 þ 2.1* 12 þ 2 7 þ 1.6#

65 8.1 þ 2* 12.5 þ 1 6.5 þ 1#

75 7.5 þ 3* 11 þ 2 6.5 þ 0.5#

85 6.2 þ 1.3* 11 þ 1.5 5.5 þ 4#

95 6 þ 2* 10 þ 1 5.5 þ 2#

105 6þ2 10 þ 2 5 þ 2#

(ml/min) 10 12 þ 3 12.5 þ 3 13 þ 2

Aortic £ow (A) and coronary £ow (B) (ml/min) was measured collecting aortic chamber over£ow into graduated cylinders. Flow was reported every 10 min from the beginning (10 min) to the end (105 min) of experimental procedures. During ischemic time (15 min) and Langendor¡ perfusion (10 min) no values were measured. All data are expressed as mean þ S.D. *P 6 0.01; # P 6 0.05.

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Table 3 Heart rate and percent heart weight gain related to myocardial ischemia Stabilization (A) Heart rate (beats/min) Time (min) 10 Normoxic (A) 280 þ 20 Hypoxic (B) 270 þ 10 Hyperoxic (C) 240 þ 25

20 280 þ 23 270 þ 13 240 þ 20

Ischemia

Langendor¡

Reperfusion in working heart

20 0 0 0

45 270 þ 10 260 þ 10 220 þ 20#

65 260 þ 10 250 þ 10 210 þ 18#

35 0 0 0

55 270 þ 12 260 þ 10 220 þ 20#

76 260 þ 3* 240 þ 12 200 þ 20#

85 250 þ 13* 235 þ 10 200 þ 19#

95 250 þ 10* 230 þ 10 185 þ 15#

105 240 þ 10* 230 þ 10 185 þ 10#

(B) Heart weight gain (%) Normoxic (A) 25 þ 5 Hypoxic (B) 15 þ 4 Hyperoxic (C) 23 þ 4 (A) Heart rate was determined with epicardial ECG. The heart rate values were reported every 10 min from the beginning (10 min) to the end (105 min) of experimental procedures. During ischemic time (15 min) and Langendor¡ perfusion (10 min) no values were measured. All data are expressed as mean þ S.D. (B) Heart weight gain was computed as (heart weight at the beginning)/(heart weight at the end)U100. All hearts were weighed at the beginning and at the end of the experimental procedure. All data are expressed as mean þ S.D. *P 6 0.01; # P 6 0.05.

substantially unchanged during stabilization, in both normoxic and hypoxic groups; while in the reperfused hearts the hypoxic values were all statistically (P 6 0.01) higher in all times studied subsequent to ischemia. In the hyperoxic reperfused hearts the values gradually diminished and were statistically lower than group A only after 105 min (P 6 0.05). 4.2. Aortic and coronary £ow values of reperfused hearts in normoxic, hypoxic and hyperoxic conditions In Aortic (Table 2A) and coronary (Table 2B) £ow, the values of groups A^B and C were not statistically di¡erent (P s 0.05) in the stabilization period; while in the reperfused hearts the values of group B were statistically higher than group A (P 6 0.05). In group C the di¡erence in values of both aortic and coronary £ow compared to group A was not statistically di¡erent (P s 0.05). 4.3. Heart rate values of reperfused hearts and percent weight gain in normoxic, hypoxic and hyperoxic conditions In stabilization condition parameters of heart rate (beats/min) of Table 3A, groups A and B were not statistically di¡erent (P s 0.05) at 10 and 20 min; while in the reperfused hearts, the values of group B were not statistically di¡erent to group A. Group C (reperfused working hearts) values were statistically lower (P 6 0.05) than

group A, in all times studied. In Table 3B, we show the percent weight gain in normoxic, hypoxic and hyperoxic groups. The percent weight gains were related with myocardial edema and were signi¢cantly diminished in group B compared to group A (P 6 0.05), while group C was not statistically di¡erent (P s 0.05). 4.4. Creatine phosphokinase release in myocytic damage In Table 4 we show that the release of CPK (U/g wet weight) in the coronary eluate after myocytic damage the stabilization parameters of groups A, B and C were not di¡erent, while in the reperfused hearts the group C values were statistically higher (P 6 0.01) than values of group A at all times studied. Group B was not statistically di¡erent to group A. 4.5. eNOS Immunohistochemistry of myocardial tissue in normoxic, hypoxic and hyperoxic conditions In order to assess the presence of eNOS in myocardial tissue we performed an immunohistochemical analysis. Slides from biopsied myocardial tissue were made with an anti-eNOS antibody (Fig. 1). In the upper panel (normoxic, control) we show that the myocardial tissue and endothelial cells are immunoreactive for eNOS, as can be seen by the evident precipitates (dark spots). The intensity of the precipitate was quanti¢ed in the related histogram. In the middle panel, representing hypoxic conditions, the

Table 4 Creatine phosphokinase (IU/g wet weight) Time (min) Normoxic (A) Hypoxic (B) Hyperoxic (C)

Stabilization

Ischemia

Langendor¡

Reperfusion in working heart

10 4þ2 12.5 þ 3 13 þ 2

20 0 0 0

45 28 þ 8 12 þ 2 7þ2

65 8.1 þ 2 12.5 þ 1 6.5 þ 1

20 5þ3 13 þ 3 11 þ 2

35 0 0 0

55 9 þ 2.1 12 þ 2 7 þ 1.6

75 7.5 þ 3 11 þ 2 6.5 (0.5)

85 6.2 þ 1.3 11 þ 1.5 5.5 þ 2

95 6þ2 10 þ 1 5.5 þ 2

105 6þ2 10 þ 2 5þ2

Myocardial necrosis enzyme, Creatine phosphokinase assay of coronary e¥uent was reported every 10 min from the beginning (10 min) to the end (105 min) of experimental procedures. During ischemic time (15 min) no values were measured. All data are expressed as mean þ S.D.

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Fig. 1. Histological sections of rat hearts. In immunohistochemical studies eNOS was found in endothelial cells and myocytes. The densitometric analysis of heart exposed to chronic hypoxia showed more evident eNOS immunoreactivity with respect to normoxic, whereas in chronic exposure to hyperoxia eNOS immunoreactivity was reduced. U100.

eNOS concentration (dark spots) is higher than in the normoxic. In fact, the related histogram shows the image analysis where on the y-axis the immunoreactive eNOS is higher at the same optical density, i.e., 0.65. In hyperoxic hearts the eNOS immunoreactivity was less than normoxic hearts, as evidenced by the lighter color of the lower panel of Fig. 1. In the related histogram we can see that the

optical density of the precipitate is lower since the values of optical density are approximately 0.03^0.40 compared to the normoxic heart (Fig. 1, upper panel) with values of 0.18^0.68. In order to con¢rm the result presented in Fig. 1, a histochemical analysis using NADPH-diaphorase was performed. In Fig. 2 we show that NADPH-diaphorase is

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Fig. 2. Histological sections of rat hearts. NADPH-diaphorase histochemistry was found in endothelial cells and myocytes. The densitometric analysis of heart exposed to chronic hypoxia showed more evident NADPH-diaphorase reactivity with respect to normoxic, whereas in chronic exposure to hyperoxia NADPH-diaphorase reactivity was reduced. U250.

present in normoxic hearts, increased in hypoxic hearts and diminished in hyperoxic hearts, as shown by the dark spots. The related densitometric studies con¢rmed the results. 4.6. eNOS Western blot analysis Since immunohistochemistry analysis of eNOS was

higher in hypoxic myocardial tissue with respect to the other conditions, it was pertinent to perform a Western blot analysis to determine eNOS in normoxic, hyperoxic, and hypoxic conditions. In Fig. 3A we show that in ischemia and reperfusion conditions, levels of eNOS protein immunoreactive band at 135 kDa were higher in the normoxic lane with respect to the hyperoxic lane, while the hypoxic lane was higher than the normoxic lane. In

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Fig. 3. Western blot analysis. Proteins (50 Wg) from normoxic, hyperoxic and hypoxic hearts were separated by electrophoresis in a 7.5% polyacrylamide^SDS gel, transferred to nitrocellulose membrane, incubated with a primary antibody anti-eNOS, conjugated with a second antibody, and developed in an alkaline bu¡er with NBT as substrate. The data showed an increase of eNOS protein in hypoxic hearts with respect to the normoxic and hyperoxic conditions.

Fig. 3B the histograms represent the densitometric analysis of Fig. 3A. A densitometer ultrascan (XL, LKB Pharmacia, Uppsala, Sweden) was used for normalization.

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inactivate the eNOS function [22]. Short-term ischemia induces a combination of irreversible and reversible denaturation of eNOS. The pH-induced alteration of the protein conformation could be potentially reversed upon renormalization of pH, as occurs upon reperfusion. However, after proteolytic cleavage the loss of activity would be irreversible [23]. The chronic exposure to hypoxia and hyperoxia induces complex metabolic, functional and structural modi¢cation [24,25]. Hypoxia promotes the expression of eNOS gene in the heart and in other tissues. The upregulation of NOS protein may be a physiological response to hypoxic stress [26]. On the other hand, chronic exposure to hyperoxia induces an augmentation of reactive oxygen species, lipid peroxidation, protein denaturation and DNA damage [27]. The protective e¡ects of chronic hypoxia may be due, in part, to the preservation of substrates by inhibition of glycolytic enzymes reducing metabolic energy utilization [28]. NO stimulates soluble guanylate cyclase (cGMP) to increase intracellular cGMP levels in cardiomyocites and endothelial cells [29]. In hypoxic conditions, the augmentation of cGMP is blunted by Ng -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO-synthase [30]. The increase of cGMP may promote the phosphorylation of cardiac troponin I by a cGMP-dependent protein kinase, with a reduction in a¤nity to troponin C for calcium, and the reduction of contraction duration. While nitric oxide inhibitors such as L-NAME and L-NA abolish the cardioprotective e¡ects of hypoxia, a nitric oxide donor, GSNO, increases recovery of postischemic function in normoxic hearts to values not di¡erent to hypoxic controls [31]. Several studies suggest that intracoronary stimulation of NO release may reduce

4.7. RT^PCR analysis As shown in Fig. 4, the RT^PCR reveals eNOS mRNA in normoxic, hyperoxic, and hypoxic conditions of the myocardial tissue exposed to ischemia and reperfusion. In this ¢gure eNOS clearly appears in the hypoxic lane, while in the normoxic and hyperoxic lanes the bands were very light. The 18S band is the standard. 5. Discussion Myocardial ischemia induces a wide range of biochemical and functional modi¢cations which determine myocardial cell injury associated with a decrease of left ventricular performance. Since reperfusion is an important prerequisite for reducing ischemic damage, the literature reports adjunctive damage to myocytes during the reperfusion time [11]. Ischemia induces a marked intracellular acidi¢cation which can result in enzyme denaturation with loss of enzyme activity. The maximal eNOS catalytic function is observed at pH 7.4. The enzyme activity markedly decays at pH 7.0. Acidi¢cation to pH 5.5 can completely

Fig. 4. RT^PCR analysis of RNA from heart tissue. RNA was extracted from hearts ¢xed in liquid nitrogen and stored at 380³C. The primer pairs used were: 5P-CGAGATATCTTCAGTCCCAAGC-3P (sense) and 5P-GTGGATTTGCTGCTCTCTAGG-3P (antisense) for rat endothelial NOS. RT^PCR was performed using 18S rRNA as internal standard. The expression of eNOS gene increased in hypoxic conditions with respect to normoxic and hyperoxic conditions.

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the duration of contraction and accelerate relaxation [32]. Recently, results obtained from a long low-frequency stimulation of skeletal muscles, together with those from a study on Ca2‡ transport regulation, showed that the presence of the NO precursor induced an acceleration in the onset of fatigue in single ¢bers, a decreased vesicular Ca2‡ content due to increased Ca2‡ release, a shift to open status in the sarcoplasmic reticulum Ca2‡ channels and an increase in sarcoplasmic reticulum Ca2‡ pump activity [33]. In our experiments, it is clear that eNOS is modulated by oxygen supply. The eNOS expression was higher in hypoxic conditions and lower in hyperoxic conditions, in comparison with normoxic controls. In the hearts subjected to chronic hyperoxia we observed a deterioration of ventricular performance which was associated with a decreased mRNA transcription and eNOS protein expression. We show here that the changes in oxygen tension during chronic hypoxia/hyperoxia contribute to myocardial injury and recovery after ischemia^reperfusion, associated with the regulation of eNOS levels in the rat hearts. The present data describe an additional biological activity of eNOS in ischemia^reperfusion injury in isolated working hearts, suggesting that these ¢ndings may be the object of investigation in clinical applications [34^36]. However, further studies are needed to clarify the exact function of eNOS in myocardial tissue. To better understand the evidence revealed by the data reported here, future studies involving the antagonism of eNOS activity through selective enzyme inhibitors may shed new light on this topic.

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