AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 280: H668-H673, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Abe, K.
Right arrow Articles by Ibii, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Abe, K.
Right arrow Articles by Ibii, N.
Vol. 280, Issue 2, H668-H673, February 2001

Involvement of iNOS in postischemic heart dysfunction of stroke-prone spontaneously hypertensive rats

Kohji Abe, Miwa Tokumura, Tetsuji Ito, Takashi Murai, Akira Takashima, and Nobuhiro Ibii

Department of Drug Safety Evaluation, Developmental Research Laboratories, Shionogi & Co., Ltd., Toyonaka, Osaka 561-0825, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the possible contribution of inducible nitric oxide synthase (iNOS) to postischemic heart dysfunction and injuries in stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP, 13-14 wk of age, had significantly higher systolic blood pressure and greater heart weight than age-matched Wistar-Kyoto rats (WKY). Permanent occlusion of the left anterior descending coronary artery (LAD) caused significant and long-lasting increases in the activity and mRNA expression of myocardial iNOS in SHRSP compared with WKY. However, there was no significant difference in the LAD occlusion-induced expression of interleukin-1beta mRNA between SHRSP and WKY. Hemodynamic deterioration and myocardial fibrosis were also observed in SHRSP at 4 wk after LAD occlusion. Continuous administration of 2-amino-5,6-dihydro-6-methyl-4H-1,2-thiazin (AMT) completely blocked the LAD occlusion-induced increase in the myocardial iNOS activity of SHRSP. Moreover, postischemic heart dysfunction and injuries were also significantly ameliorated by 2-amino-5,6-dihydro-6-methyl-4H-1,2-thiazin (AMT). These results suggest that the increased activity of myocardial iNOS plays a pivotal role in the development of postischemic cardiac dysfunction and injuries in SHRSP with the hypertensive and hypertrophic heart.

cardiac hypertrophy; left anterior descending coronary artery occlusion; 2-amino-5,6-dihydro-6-methyl-4H-1,2-thiazin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) maintained by constitutive NO synthase (NOS) has a physiological role in regulating blood pressure, heart rate, coronary blood flow, and myocardial contractions (18, 20). However, NO produced by inducible NOS (iNOS) has been suggested to be involved in many pathophysiological states (25), such as myocardial infarction (31) and heart failure (11). Increased NO levels generated by iNOS have been reported to depress contractile responses of ventricular cardiac myocytes to beta -adrenergic agonists (2, 5). Additionally, iNOS has been described to increase the infarcted region of myocardium after coronary artery ligation in rabbits (31). On the other hand, inhibition of iNOS has been shown to reduce cardiac dysfunction and infarct size in rat experimental autoimmune myocarditis (13) and myocardial infarction (28), respectively. These findings suggest that myocardial injuries may lead to the increased expression of iNOS, causing a decline of myocardial contractions.

Hypertension is one of the major risk factors for heart diseases such as myocardial infarction and heart failure (33). The hypertensive and hypertrophic heart results from activation of the circulating hormones, renin-angiotensin system, or catecholamine system as well as an increase in the mechanical stretching (8). ANG II, vasopressin, adrenomedullin, and adrenergic agonists have been reported to enhance the cytokine-stimulated expression of iNOS in cardiac myocytes (14-16, 34). Also, the hypertrophic heart in hypertension has been shown to be greatly susceptible to ischemia or hypoxia (9, 26). Thus these findings suggest that expression of iNOS is augmented under the various stresses and that NO generated by iNOS may have a greater influence on cardiac function or tissues in hypertensive than in normal animals.

In the present study, we examined 1) the occurrence of myocardial iNOS after lipopolysaccharide (LPS) administration and LAD occlusion in stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) to study inducibility of iNOS in the hypertensive and hypertrophic heart, 2) hemodynamic and histopathological changes after LAD occlusion in SHRSP, and 3) effects of an iNOS inhibitor on the LAD occlusion-induced changes to clarify the role of iNOS in the development of postischemic heart dysfunction and injuries in SHRSP.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male WKY and SHRSP (Aburahi Laboratories, Shionogi), aged 13-14 wk, were housed in groups of two or three per cage (20 × 35 × 17 cm) under constant temperature (25 ± 1°C) and maintained on a 12:12-h light-dark cycle (lights on from 0700 to 1900) with free access to food and water.

LPS treatment. LPS (Escherichia coli O111B4; Sigma) was dissolved in saline to a concentration of 1% (wt/vol) and intraperitoneally administered in a volume of 1 ml/kg. The animals were killed at 0, 2, 4, 6, and 16 h after LPS administration. The heart was rinsed with PBS, and the left ventricle and septum were dissected. All samples were freeze-clamped using a rapid-freeze clamp and stored at -80°C until biochemical analyses.

Myocardial ischemia. The animals were anesthetized with pentobarbital sodium (40 mg/kg ip) and ventilated with room air. After thoracotomy, the LAD was ligated with a 8-0 silk suture (Natsume Production). Electrocardiograms were recorded through the standard limb lead. A marker of successful coronary occlusion was the rise of the S-T wave in the electrocardiogram. Sham-operated rats underwent only thoracotomy without LAD occlusion. Animals with or without LAD occlusion were returned to their cages after the operation and killed at 0, 2, 4, 6, and 16 h and 1, 3, 7, 14, and 21 days after LAD occlusion. The heart was rinsed with PBS, and the left ventricle and septum were dissected. All samples were freeze-clamped using a rapid-freeze clamp and stored at -80°C until biochemical analyses.

AMT treatment. 2-Amino-5,6-dihydro-6-methyl-4H-1,2-thiazin (AMT) hydrochloride (Tocris), a selective inhibitor of iNOS (21), was dissolved in saline and infused at 0.01 mg/h sc with the use of an osmotic pressure minipump (model 2002, Alzet); infusion was started immediately after LAD occlusion and continued for 6 h, 16 h, or 2 wk. Cardiac function was monitored 4 wk after LAD occlusion.

Quantification of mRNA by RT-PCR. Total RNA was extracted from tissue samples with TRIzol reagent (GIBCO-BRL Life Technologies); total cellular RNA was extracted by the single-step acid guanidinium thiocyanate-phenol-chloroform method (6). Briefly, ~70 mg of tissue were homogenized with 1.0 ml of TRIzol in a 2.0-ml microcentrifuge tube to which 200 µl of chloroform were added. After the mixture was centrifuged, the aqueous phase was transferred to a new microfuge tube containing an equal volume of isopropanol, and the RNA was recovered by precipitation. The RNA was washed in 75% ethanol, dried, and resuspended in RNase-free water. RNA was quantified spectrophotometrically as the ratio of the absorbance at 260 nm to the absorbance at 280 nm. First-strand cDNA was synthesized from 1 µg of total RNA with SuperScript reverse transcriptase (GIBCO BRL) and oligo(dT)12-18 primer. The reaction was carried out at 42°C for 1 h; then the enzyme was heat inactivated at 72°C for 15 min. The cDNA samples were stored at -80°C until they were subjected to the PCR. The cDNA samples (1 µl) were amplified with Taq polymerase (Takara, Kyoto, Japan) in a thermal cycler (Omni-E, Hybaid). PCR was performed according to the following schedule: denaturation at 94°C for 30 s, amplification for 25-30 cycles, annealing at 56-58°C for 15-30 s, and extension at 72°C for 30 s. Oligonucleotide primers for iNOS, interleukin-1beta (IL-1beta ), and beta -actin were as follows: 5'-ATGGCTTGCCCCTGGAAGTTTCTC-3' and 5'-CCTCTGATG-GTGCCATCGGGCATCTG-3' for iNOS, 5'-GAA- GCTGTGGCAGCTACCTACCTATG-TCT-3' and 5'-CTCTGCTTGAGAGGTGCTGATGTAC-3' for IL-1beta , and 5'-GCTCGT- CGTCGACAACGGCTC-3' and 5'-CAAACATGATCTGGGTCATCTTCTC-3' for beta -actin. Quantitative PCR of iNOS was also conducted using the iNOS Quant-PCR kit (Cayman). Five microliters of the final reaction product were separated by electrophoresis in a 1.2% agarose gel containing 1 µg/ml ethidium bromide in 1× Tris acetate-EDTA buffer, with bromphenol blue as a marker dye. The bands were visualized with ultraviolet irradiation and quantified by image analysis software (NIH Image for Macintosh computer). The relative intensity of bands for iNOS and IL-1beta mRNA was normalized using the intensity of beta -actin.

Assay for iNOS activity. NOS activity was quantified by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in the presence of saturated concentrations of the enzyme's cofactors. The freeze-clamped samples were homogenized in a buffer containing Tris · HCl (50 mmol/l), sucrose (250 mmol/l), EDTA (0.1 mmol/l), EGTA (0.1 mmol/l), leupeptin (1 µmol/l), pepstatin A (1 µmol/l), phenylmethylsulfonyl fluoride (1 mmol/l), dithiothreitol (1 mmol/l), and aprotinin (500 kallikrein-inactivating units) on ice. The homogenates were centrifuged first at 800 g for 10 min and then at 100,000 g for 60 min. The cytosolic fractions (10 µl) were added to 70 µl of buffer containing 50 mmol/l HEPES (pH 7.4), 1 mmol/l EDTA, 1 mmol/l NADPH, 1 mmol/l FAD, 1 mmol/l flavin mononucleotide, 1 mmol/l tetrahydrobiopterin, 1 mmol/l dithiothreitol, and L-[3H]arginine and incubated for 20 min at 30°C. The reaction was stopped by addition of 2 ml of ice-cold 100 mmol/l HEPES and 10 mmol/l EDTA. Then the total volume of the reaction mixture was applied to an AG 50W-X8 column that had been preequilibrated with 100 mmol/l HEPES. L-[3H]citrulline was eluted with 2 ml of deionized water, and radioactivity was quantified by scintillation counting. Protein concentrations were determined using a protein fixed-quantity kit (Bio-Rad).

Measurement of hemodynamic parameters. Hemodynamic indexes were heart rate, mean blood pressure (MAP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure, and peak positive and negative first derivative of pressure (dP/dt). These hemodynamic parameters were measured in animals anesthetized with fluothane (1.5-4.0%) and maintained at 37°C with a heating pad. MAP was measured with a pressure transducer (model P10EZ, Gould Statham), which was connected to a polyethylene tube (model SP31, Natsume) inserted into the right carotid artery, and a carrier amplifier (model AP-621G, Nihon Kohden). The heart rate was counted from the blood pressure pulses with a heart rate counter (model AT-601G, Nihon Kohden). After MAP and heart rate were measured, the polyethylene tube, which was connected to a pressure transducer, was further inserted through the right carotid artery toward the heart to obtain LVSP. Left ventricular dP/dt was calculated by differentiating the output of LVSP with a pressure processor (time constant = 0.5 ms; model EQ-601G, Nihon Kohden). These parameters were recorded with a polygraph system (model RM-6000, Nihon Kohden).

Histopathology. After measurement of hemodynamic parameters, the rats were killed by KCl injection. The amount of pericardial effusion was scored as follows: 0, normal; 1, a small pericardial effusion <1 ml; 2, a moderate pericardial effusion <3 ml; and 3, a massive pericardial effusion >3 ml. The removed hearts were fixed in 10% formalin. The left ventricle was transversely cut into four slices from the base to the apex, embedded in paraffin, and sliced at each level for histopathological examination. The extent of fibrosis was estimated using Azan-Mallory staining. Staining images were scanned on an Apple Macintosh computer, and fibrosis areas were quantified by NIH Image analysis software. The extent of fibrosis was determined as a percentage of the total left ventricular section and then scored. The extent of fibrosis was scored as follows: 0, normal; 1, <10%; 2, <15%; and 3, >15%.

Statistical analysis. The significance of differences between groups was determined by Student's t-test or Dunnett's test for multiple comparisons after ANOVA. Differences were considered statistically significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Blood pressure and heart weight. At 13-14 wk of age, systolic blood pressure of SHRSP was significantly higher than that of WKY. Heart weight and heart weight-to-body weight ratio were also significantly greater in SHRSP than in WKY (Table 1).

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Characteristics of WKY and SHRSP at 13-14 wk of age

LPS-stimulated myocardial iNOS mRNA, IL-1beta mRNA, and iNOS activity. The basal activity and mRNA levels of iNOS were significantly higher in SHRSP than in WKY. Administration of LPS (10 mg/kg ip) produced increases in the activity and mRNA expression of iNOS in both strains, but these changes were significantly larger in SHRSP than in WKY (Figs. 1 and 2). In contrast, there was no significant difference in the LPS-induced expression of myocardial IL-1beta mRNA between SHRSP and age-matched WKY (Fig. 1).


View larger version (44K):
[in this window]
[in a new window]
  		Fig. 1.   Lipopolysaccharide (LPS, 10 mg/kg)-induced expression of inducible nitric oxide synthase (iNOS) and interleukin-1beta (IL-1beta ) mRNA in spontaneously hypertensive stroke-prone (SHRSP) and Wistar-Kyoto (WKY) myocardium. A: representative RT-PCR products for iNOS and IL-1beta mRNA at each time after LPS treatment. B: quantitative densitometry data of RT-PCR products for iNOS and IL-1beta mRNA, which were normalized for beta -actin mRNA. Open bars, WKY; solid bars, SHRSP. Values are means ± SE. **P < 0.01 vs. WKY.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2.   Myocardial iNOS activities after LPS treatment in SHRSP and WKY. iNOS activity was determined in cytosolic fractions of the myocardium by measuring conversion of L-[3H]arginine to [3H]citrulline in the absence of Ca2+. Open bars, WKY; solid bars, SHRSP. Values are means ± SE (n = 6-8) rats. **P < 0.01 vs. WKY.

Myocardial iNOS mRNA, IL-1beta mRNA, and iNOS activity after LAD occlusion. Figure 3A shows the time course of myocardial iNOS mRNA levels after permanent occlusion of LAD. In SHRSP, cardiac ischemia by LAD occlusion resulted in the marked expression of myocardial iNOS mRNA; the expression of iNOS mRNA began to increase from ~4 h after LAD occlusion and peaked at 6-16 h after occlusion. Also, in WKY, there was an increase in the expression of iNOS mRNA, peaking at 4 h after LAD occlusion. However, the LAD-induced increase in the expression of iNOS mRNA was significantly smaller and of shorter duration in WKY than in SHRSP. Almost in parallel with the change in iNOS mRNA levels, LAD occlusion produced a significantly larger increase of the NOS activity in SHRSP than in WKY (Fig. 3B).


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 3.   Time courses for iNOS mRNA expression (A), iNOS activity (B), and IL-1beta mRNA expression (C) after permanent left anterior descending coronary artery occlusion in SHRSP () and WKY (open circle ) myocardium. Animals were killed at 0, 2, 4, 6, and 16 h and 1, 3, 7, 14, and 21 days after left anterior descending coronary artery occlusion. Quantitative densitometry data of RT-PCR products for iNOS and IL-1beta mRNA was normalized for beta -actin mRNA. iNOS activity was expressed as rate of L-citrulline production. Values are means ± SE (n = 6-7 rats). *P < 0.05; **P < 0.01, WKY.

On the other hand, LAD occlusion produced two-peaked increases in the IL-1beta mRNA expression in SHRSP and WKY; a significant difference in the expression of IL-1beta mRNA was observed only 6 h after LAD occlusion between SHRSP and WKY (Fig. 3C).

Effects of AMT on LAD occlusion-induced increase of myocardial iNOS activity in SHRSP. AMT, continuously administered at 0.01 mg/h for 6 or 16 h, completely blocked an increase of the cardiac iNOS activity observed at 6 and 16 h after LAD occlusion in SHRSP (Table 2). AMT, infused at 0.01 mg/h for 6 h, had no effect on blood pressure in conscious rats (data not shown).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Effects of AMT on increase in myocardial iNOS activity after LAD occlusion in SHRSP

Effects of AMT on hemodynamic changes produced by permanent LAD occlusion in SHRSP. Table 3 shows hemodynamic parameters determined at 4 wk after LAD occlusion in SHRSP. MAP, LVSP, and peak positive and negative dP/dt were significantly lower in LAD-occluded than in sham-operated rats. Thus, in SHRSP, LAD occlusion caused heart dysfunction, which was characterized as deterioration of hemodynamics at 4 wk after ischemia. However, continuous infusion of AMT (0.01 mg/h sc) during the first 2 wk after LAD occlusion resulted in significant increases in MAP, LVSP, and +dP/dt of LAD-occluded rats; AMT produced a significant improvement in hemodynamic deterioration at 4 wk after LAD occlusion in SHRSP.

                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Effects of AMT on hemodynamic changes after LAD occlusion in SHRSP

Effects of AMT on histopathology in SHRSP subjected to permanent LAD occlusion. At 4 wk after occlusion, most of the LAD-occluded SHRSP had dilated ventricular chambers and thin ventricular walls compared with the sham-operated rats (data not shown). Also, as shown in Table 4, pericardial effusion and fibrosis were observed in SHRSP subjected to permanent LAD occlusion. However, 2 wk of treatment with AMT (0.01 mg/h) significantly decreased the effusion and fibrosis scores in LAD-occluded SHRSP; AMT produced significant amelioration of postischemic heart injuries in SHRSP.

                              
View this table:
[in this window]
[in a new window]
  		Table 4.   Effect of AMT on LAD occlusion-induced histopathology in SHRSP


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study was undertaken to clarify the involvement of iNOS in the development of postischemic heart dysfunction and injuries in SHRSP with the hypertensive and hypertrophic heart. For this purpose, inducibility of myocardial iNOS after LPS administration and LAD occlusion was examined in SHRSP and compared with that of WKY. Then, effects of AMT, an iNOS inhibitor, on changes in the myocardial iNOS activity, hemodynamics, and histology resulting from permanent LAD occlusion were investigated in SHRSP.

At 13-14 wk of age, SHRSP had significantly higher systolic blood pressure and significantly greater heart weight than WKY. Also, the basal and LPS-stimulated activity and mRNA expression of iNOS in the myocardium were higher in SHRSP than in WKY. In contrast, no differences were found in the basal and LPS-induced IL-1beta mRNA expression between the SHRSP and WKY myocardium. It is well known that LPS and cytokines such as IL-1beta and tumor necrosis factor-alpha are stimulators of myocardial iNOS (3, 24). ANG II, arginine vasopressin, and adrenomedullin have been shown to enhance the cytokine-stimulated iNOS synthesis in cardiac myocytes (14-16, 34). It has been also reported that the activity of superoxide dismutase is decreased in the myocardium of SHR and SHRSP (17, 27), indicating their vulnerability to oxidative stresses. Such stresses can induce transcription factor nuclear factor-kappa B-response genes, such as iNOS gene (1). Thus these findings suggest that an enhanced expression of myocardial iNOS under basal conditions or after LPS administration in SHRSP might result from increases in circulating hormones and marked vulnerability of SHRSP to oxidative stresses.

We next examined the time course for induction of myocardial iNOS and IL-1beta mRNA after permanent LAD occlusion in SHRSP and WKY. LAD occlusion caused long-lasting increases in the activity and mRNA expression of myocardial iNOS in the SHRSP. However, in WKY, an increase in the iNOS expression after LAD occlusion was of short duration and weak. Recently, using Northern blotting, Wang et al. (28) showed that the iNOS mRNA expression increased from 3 h after LAD occlusion in rats, reached a maximum between 6 and 24 h after occlusion, and remained elevated for up to 1 wk after occlusion. Also, in rabbits, the iNOS activity has been reported to increase between 3 and 14 days after infarction (31). These findings are in general agreement with our results obtained in SHRSP. Furthermore, myocardial infarction involves inflammatory processes and induces cytokines (23). Herskowitz et al. (12) reported that mRNA levels of cytokines, including IL-1beta , tumor necrosis factor-alpha , interferon-gamma , and transforming growth factor-beta , increased for several hours, returned to the baseline, and then again increased significantly at 7 days after LAD occlusion. We also found an increase in IL-1beta mRNA levels, peaking at 6 h and 7 days after LAD occlusion. However, there was little difference in the IL-1beta mRNA levels between SHRSP and WKY. This fact suggests that an increase in iNOS expression after LAD occlusion is not related to inflammation but to pathological states of SHRSP.

LAD occlusion produced hemodynamic deterioration such as decreases in ±dP/dt, LVSP, and MAP in SHRSP, but not in WKY (data not shown). iNOS produced by cardiac disorders such as cardiomyopathy (13) and myocardial infarction (31, 32) has been reported to be involved in depression of the contractile response of ventricular cardiac myocytes. Balligand et al. (3) also showed that iNOS took part in the cytokine-induced contractile decrease of cardiac myocytes. These data indicate that the enhanced iNOS expression after myocardial injuries leads to a sustained release of large amounts of NO, which can reduce myocardial contractility through the cGMP pathway (10, 35). Therefore, cardiac dysfunction after LAD occlusion in SHRSP with the hypertensive and hypertrophic heart also seems to be closely connected to the excessive expression of iNOS.

To clarify the pathological role of iNOS in SHRSP, we further examined the effects of an iNOS inhibitor (AMT) on deterioration of cardiac hemodynamics and the fibrosis observed 4 wk after LAD occlusion. Continuous administration of AMT, started just after LAD occlusion, completely blocked an increase in myocardial iNOS activity and significantly ameliorated hemodynamic deterioration caused by LAD occlusion in SHRSP. The fibrosis of the myocardium was also significantly ameliorated by AMT. Consistent with our results, Wildhirt et al. (31, 32) reported that S-methylisothiourea, an iNOS inhibitor, could alleviate heart dysfunction after myocardial infarction in the rabbit model. Moreover, Wang et al. (28) recently showed that aminoguanidine and S-methylisothiourea, iNOS inhibitors, reduced the infarct area in the rat myocardial infarction model. These results suggest that a large amount of NO produced by iNOS is involved not only in depression of myocardial contractions, but also in myocardial injury. NO is known to have high affinities for iron-containing enzymes such as aconitase in the citric acid cycle and complex I and II in the mitochondrial respiratory chain (7). Accordingly, the effects of NO on these systems would result in inhibition of cell metabolisms and, consequently, cell injuries. Ribonucleotide reductase, an enzyme involved in DNA synthesis, can also be inhibited by NO (22). Furthermore, NO can bind to superoxide to form peroxynitrite, which subsequently decomposes to hydroxyl radical and nitrogen dioxide, which are more toxic than NO itself (4, 29). Despite these findings, some beneficial influences of NO on the heart have also been shown with the use of NO donors and L-arginine (19, 30). However, it is only in ischemia-reperfusion models that NO produced cardioprotective effects. To our knowledge, there seems to be no report describing ameliorating effects of NO on the heart in permanent (or chronic) ischemia models. At any rate, we can say that excessive and prolonged production of NO in SHRSP with permanent LAD occlusion is very harmful to the heart and aggravates its pathological states.

In summary, permanent LAD occlusion markedly increased the myocardial iNOS activity and, at the same time, produced cardiac dysfunction and fibrosis in SHRSP with hypertensive and hypertrophic heart. Continuous administration of AMT completely blocked the LAD occlusion-induced increase in the myocardial iNOS activity of SHRSP. Furthermore, postischemic heart dysfunction and fibrosis of SHRSP were also significantly ameliorated by AMT. On the basis of these results, we conclude that iNOS plays an important role in the development of postischemic heart dysfunction and injuries in SHRSP.


    FOOTNOTES

Address for reprint requests and other correspondence: K. Abe, Dept. of Drug Safety Evaluation, Developmental Research Laboratories, Shionogi & Co., Ltd., Toyonaka, Osaka 561-0825, Japan (E-mail: kohji.abe{at}shionogi.co.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7 July 2000; accepted in final form 12 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adcock, IM, Brown CR, Kwon O, and Barnes J. Oxidative stress induces NF-kappa B DNA binding and inducible NOS mRNA in human epithelial cells. Biochem Biophys Res Commun 199: 1518-1524, 1994[ISI][Medline].

2.   Balligand, JL, Ungureanu D, Kelly RA, Kobzik L, Pimental D, Michel T, and Smith TW. Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium. J Clin Invest 91: 2314-2319, 1993.

3.   Balligand, JL, Ungureanu-Longrosis D, Simmons WW, Pimental D, Malinski TA, Kapturczak M, Taha Z, Lowenstein CJ, Davidoff AJ, Kelly RA, Smith TW, and Michel T. Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro. J Biol Chem 269: 27580-27588, 1994[Abstract/Free Full Text].

4.   Beckman, JS, Beckman TW, Chen J, Marshall PA, and Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620-1624, 1990[Abstract/Free Full Text].

5.   Brady, AJB, Poole-Wilson PA, Harding SE, and Warren JB. Nitric oxide production within cardiac myocytes reduces their contractility in endotoxemia. Am J Physiol Heart Circ Physiol 263: H1963-H1966, 1992[Abstract/Free Full Text].

6.   Chomocynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

7.   Drapier, JC, and Hibbs JB, Jr. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells. J Immunol 140: 2829-2838, 1988[Abstract].

8.   Fukuzawa, J, Osaki J, and Haneda T. Differential effects of amiloride on the basal rate and the pressure overload-induced increase in protein synthesis in perfused rat heart. Clin Exp Hypertens 16: 835-852, 1994.

9.   Haneda, T, Ichihara K, Abiko Y, and Onodera S. Functional and metabolic responses to ischemia in the perfused heart isolated from normotensive and spontaneously hypertensive rats. Jpn Circ J 50: 607-613, 1986[Medline].

10.   Hare, JM, Loh E, Creager MA, and Colucci WS. Nitric oxide inhibits the positive inotropic response to beta -adrenergic stimulation in humans with left ventricular dysfunction. Circulation 92: 2198-2203, 1995[Abstract/Free Full Text].

11.   Haywood, GA, Tsao PS, Leyen HE, Mann MJ, Keeling PJ, Trindade PT, Lewis NP, Byrne CD, Rickenbacher PR, Bishopric NH, Cooke JP, McKenna WJ, and Flowler MB. Expression of inducible nitric oxide synthase in human heart failure. Circulation 93: 1087-1094, 1996[Abstract/Free Full Text].

12.   Herskowitz, A, Choi S, Ansari AA, and Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol 146: 419-428, 1995[Abstract].

13.   Hirono, S, Islam O, Nakazawa M, Yoshida Y, Kodama M, Shibata A, Izumi T, and Imai S. Expression of inducible nitric oxide synthase in rat experimental autoimmune myocarditis with special reference to changes in cardiac hemodynamics. Circ Res 80: 11-20, 1997[Abstract/Free Full Text].

14.   Ikeda, U, Kanbe T, Kawahara Y, Yokoyama M, and Shimada K. Adrenomedullin augments inducible nitric oxide synthase expression in cytokine-stimulated cardiac myocytes. Circulation 94: 2560-2565, 1996[Abstract/Free Full Text].

15.   Ikeda, U, Maeda Y, Kawahara Y, Yokoyama M, and Shimada K. Angiotensin II augments cytokine-stimulated nitric oxide synthesis in rat cardiac myocytes. Circulation 92: 2683-2689, 1995[Abstract/Free Full Text].

16.   Ikeda, U, Murakami Y, Kanbe T, and Shimada K. alpha -Adrenergic stimulation enhances inducible nitric oxide synthase expression in rat cardiac myocytes. J Mol Cell Cardiol 28: 1539-1545, 1996[ISI][Medline].

17.   Ito, H, Torii M, and Suzuki T. Decreased superoxide dismutase activity and increased superoxide anion production in cardiac hypertrophy of spontaneously hypertensive rats. Clin Exp Hypertens 17: 803-816, 1995.

18.   Kelly, R, Balligand JL, and Smith TW. Nitric oxide and cardiac function. Circ Res 79: 363-380, 1996[Free Full Text].

19.   Lefer, DJ, Nakanishi K, Johnston WE, and Vinten-Johansen J. Antineutrophil and myocardial protection actions of a novel nitric oxide donor after acute myocardial ischemia and reperfusion in dogs. Circulation 88: 2337-2350, 1993[Abstract/Free Full Text].

20.   Moncada, S, Palmer RM, and Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109-142, 1991[ISI][Medline].

21.   Nakane, M, Klinghofer V, Kuk JE, Donnelly JL, Budzik GP, Pollock JS, Basha F, and Carter GW. Novel potent and selective inhibitors of inducible nitric oxide synthase. Mol Pharmacol 47: 831-834, 1995[Abstract].

22.   Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB J 6: 3051-3064, 1992[Abstract].

23.   Ono, K, Matsumori A, Shioi T, Frukawa Y, and Sasayama S. Cyokine gene expression after myocardial infarction in rat hearts: possible implication in left ventricular remodeling. Circulation 98: 149-156, 1998[Abstract/Free Full Text].

24.   Roberts, AB, Vodovotz Y, Roche NS, Sporn MB, and Nathan CF. Role of nitric oxide in antagonistic effects of transforming growth factor-beta and interleukin-1beta on the beating rate of cultured cardiac myocytes. Mol Endocrinol 6: 1921-1930, 1992[Abstract].

25.   Sessa, WC. The nitric oxide synthase family of proteins. J Vasc Res 31: 131-143, 1994[ISI][Medline].

26.   Shimamoto, N, Goto N, Tanabe M, Imamoto T, Fujiwara S, and Hirata M. Myocardial energy metabolism in the hypertrophied hearts of spontaneously hypertensive rats. Basic Res Cardiol 77: 359-371, 1982[ISI][Medline].

27.   Tokoro, T, Ito H, and Suzuki T. Alterations in mitochondrial DNA and enzyme activities in hypertrophied myocardium of stroke-prone SHR. Clin Exp Hypertens 18: 595-606, 1996.

28.   Wang, D, Yang XP, Liu YH, Carretero OA, and LaPointe MC. Reduction of myocardial infarct size by inhibition of inducible nitric oxide synthase. Am J Hypertens 12: 174-182, 1999[ISI][Medline].

29.   Wang, P, and Zweier JL. Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. J Biol Chem 271: 29223-29230, 1996[Abstract/Free Full Text].

30.   Weyrich, AS, Ma X, and Lefter AM. The role of L-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat. Circulation 86: 279-288, 1992[Abstract/Free Full Text].

31.   Wildhirt, SM, Suzuki H, Horstman D, Weismueller S, Dudek RR, Akiyama K, and Reichart B. Selective modulation of inducible nitric oxide synthase isozyme in myocardial infarction. Circulation 96: 1616-1623, 1997[Abstract/Free Full Text].

32.   Wildhirt, SM, Suzuki H, Wolf WP, Dudek R, Horstman D, Weismueller S, and Reichart B. S-methylisothiourea inhibits inducible nitric oxide synthase and improves left ventricular performance after acute myocardial infarction. Biochem Biophys Res Commun 227: 328-333, 1996[ISI][Medline].

33.   Winlaw, DS, Swythe GA, Keogh AM, Scyvens CG, Spratt PM, and MacDonald PS. Increased nitric oxide production in heart failure. Lancet 344: 373-374, 1994[ISI][Medline].

34.   Yamamoto, K, Ikeda U, Okada K, Saito T, Kawahara Y, Okuda M, Yokoyama M, and Shimada K. Arginine vasopressin increases nitric oxide synthesis in cytokine-stimulated rat cardiac myocytes. Hypertension 30: 1112-1120, 1997[Abstract/Free Full Text].

35.   Yamamoto, S, Tsutsui H, Tagawa H, Saito K, Takahashi M, Tada H, Yamamoto M, Katoh M, Egashira K, and Takeshita A. Role of myocyte nitric oxide in beta -adrenergic hyporesponsiveness in heart failure. Circulation 95: 1111-1114, 1997[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 280(2):H668-H673
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. R. Norton, D. G. A. Veliotes, O. Osadchii, A. J. Woodiwiss, and D. P. Thomas
Susceptibility to systolic dysfunction in the myocardium from chronically infarcted spontaneously hypertensive rats
Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H372 - H378.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
T. Itoh, M. Haruna, and K. Abe
Heart/Cardiac Muscle: Differential regulation of the nitric oxide-cGMP pathway exacerbates postischaemic heart injury in stroke-prone hypertensive rats
Exp Physiol, January 1, 2007; 92(1): 147 - 159.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y.-H. Liu, O. A. Carretero, O. H. Cingolani, T.-D. Liao, Y. Sun, J. Xu, L. Y. Li, P. J. Pagano, J. J. Yang, and X.-P. Yang
Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction
Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2616 - H2623.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
Y. Sun, O. A. Carretero, J. Xu, N.-E. Rhaleb, F. Wang, C. Lin, J. J. Yang, P. J. Pagano, and X.-P. Yang
Lack of Inducible NO Synthase Reduces Oxidative Stress and Enhances Cardiac Response to Isoproterenol in Mice With Deoxycorticosterone Acetate-Salt Hypertension
Hypertension, December 1, 2005; 46(6): 1355 - 1361.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
L. Zhang, C. G. Looney, W.-N. Qi, L.-E. Chen, A. V. Seaber, J. S. Stamler, and J. R. Urbaniak
Reperfusion injury is reduced in skeletal muscle by inhibition of inducible nitric oxide synthase
J Appl Physiol, April 1, 2003; 94(4): 1473 - 1478.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Abe, K.
Right arrow Articles by Ibii, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Abe, K.
Right arrow Articles by Ibii, N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online