Cardiovascular depression, manifested by depressed mean arterial blood pressure (MABP), heart rate (HR), cardiac output and attenuated pressor responses to vasoactive agents is an early pathophysiological event associated with hyperglycemia in animal models of type 1 diabetes.1–3 Among various other mechanisms, an increased formation of nitric oxide (NO), particularly from inducible nitric oxide synthase (iNOS) has been implicated in the dysfunction of cardiovascular tissues.1,2 Studies from our laboratory and elsewhere have demonstrated an increased expression of iNOS in cardiac, vascular, and renal tissues of streptozotocin (STZ) diabetic rats.4–6
Soon after the discovery that endothelium-derived relaxation factor was NO, several important concepts evolved about its actions in the cardiovascular system. Physiological (nanomolar) concentrations of NO maintain the vasculature in a state of active vasodilatation and regulate regional blood flow to tissues such as heart and kidney in response to local environmental changes.7,8 In addition, NO from different sources can also modulate regional cardiac contractility by diffusing into cardiac myocytes and altering function and metabolism.9 NO in large quantities however, is toxic to the cardiovascular system. This is even more deleterious if elevated production of NO is associated with an increased formation of reactive oxygen species (ROS), particularly superoxide anions.10,11
Both NO and superoxide anions are highly reactive and unstable radicals that can react rapidly to form peroxynitrite, a cytotoxic compound.12,13 This reaction is approximately 3 times faster than the dismutation of superoxide anions by superoxide dismutase.14,15 Peroxynitrite is a favored product under conditions such as hyperglycemia where cellular production of NO and ROS are increased.11 It can be protonated further to produce peroxynitrous acid, which in turn can yield hydroxyl radical.15 All of the above reactive oxygen and nitrogen species create a condition of cellular stress called “nitrosative stress.” These species have the potential to oxidize lipids and thiol groups of many antioxidant enzymes and damage to cell membranes.12 They can also covalently modify intracellular proteins including endothelial nitric oxide synthase (eNOS), damaging them or rendering them less active.14,16 In fact, nitrosative stress has been shown to cause severe hypotension, profound vasodilatation, cardiac depression, and multiple organ failure in various models of septic shock.13,17 Peroxynitrite is normally detoxified by reduced glutathione (GSH) or other thiols forming nitrosothiols, and in the process deplete the total antioxidant capacity. Because iNOS is the major contributor of NO to nitrosative stress, therapeutic strategies aimed at inhibition of iNOS or strengthening the antioxidant defense mechanisms by supplementation with reducing equivalents may be a rational approach to prevent the cardiovascular abnormalities in diabetes.
N-acetylcysteine (NAC) is the N-acetyl derivative of the amino acid cysteine, which is a part of the tripeptide molecule in GSH and is a major contributor to reducing equivalents in the form of sulfhydryl groups (SH group). Studies have indicated that NAC inhibits the expression of tumor necrosis factor-α (TNF-α),18 a strong inducer of iNOS in lipopolysaccharide models of endotoxemia both in vitro and in vivo.19,20 To test the hypothesis that nitrosative stress is involved in the development of cardiovascular abnormalities, we studied the effect of NAC treatment on mean arterial blood pressure (MABP) and heart rate (HR) in STZ diabetic rats. In addition, we also investigated the effect of NAC treatment on plasma indicators of oxidative stress and iNOS, endothelial nitric oxide synthase (eNOS), and nitrotyrosine (NT) expression in cardiovascular tissues.
Induction of Diabetes and NAC Treatment
Thirty-two male Wistar rats weighing 250±10 g were obtained from Charles River Laboratories (Montreal, Quebec) and allowed to acclimatize to the local vivarium. They were randomly divided into 4 equal groups namely control (C), control treated (CT), diabetic (D), and diabetic treated (DT). Diabetes was induced by a single tail vein injection of 60 mg/kg STZ. The presence of diabetes was confirmed by hyperglycemia (>20 mmol/L) at 72 h after STZ administration. One week after induction of diabetes, NAC was administered to the CT and DT groups in the drinking water at a concentration of 4.8 and 2.4 g/L, respectively. Diabetic rats were treated with a higher concentration of NAC because they imbibe more drinking water (∼2–3 times) than age-matched controls. In the present study, NAC was administered at a relatively higher dose to DT rats than reported because NAC given at 0.5 g/kg/day did not prevent hyperglycemia induced-oxidative stress in that study.21 After 8 weeks of treatment, at termination each animal was surgically prepared for measurement of MABP and HR.
Rats were anesthetized with halothane and fluid-filled (heparinized saline, 20 U/mL) catheters were placed in the left carotid artery (PE 50) and jugular vein (PE 10) for measurements of blood pressure and for drug administration, respectively. All of the catheters were exteriorized at the nape of the neck, passed through a harness and tether, and connected to swivels (Instec Lab, Plymouth Meeting, PA) mounted above the cage for free movement of the animal. The arterial catheter was connected to a disposable pressure transducer (Viggo-Spectramed, Oxnard, CA) mounted on the cage exterior at the level of the rat. MABP and HR were simultaneously recorded on Gould TA 2000 Thermal Array Recorder (Gould Instrument System, Valley View, OH) and a computer, using custom-made data acquisition software. To achieve normalization of cardiac baroreflexes, the animals were allowed to recover from anesthesia and surgery for at least 4 h before recording MABP and HR.22
Following measurement of MABP and HR, rats were given an overdose of pentobarbital (>100 mg/kg, IV) and blood was collected via the carotid artery. Hearts and superior mesenteric arteries (SMA) were immediately removed and placed in ice-cold Krebs solution (120 mmol/L NaCl, 5.9 mmol/L KCl, 25 mmol/L NaHCO3, 11.5 mmol/L glucose, 1.2 mmol/L NaH2PO4, 1.2 mmol/L MgCl2, 2.5 mmol/L CaCl2) containing 0.1 μmol/L water-soluble dexamethasone to prevent induction of iNOS in vitro. A portion of the left ventricle and SMA from each rat was cleared of blood and fixed in 10% neutral buffered formalin and preserved for immunohistochemical studies. The remaining tissues were snap-frozen in liquid nitrogen and stored at −70°C for Western blot studies.
Measurement of Plasma Glucose, Insulin, Triglycerides, and Cholesterol
Plasma glucose was measured by an enzymatic colorimetric assay kit (Roche Diagnostics, Laval, Quebec) using a Beckman Glucose Analyzer 2. Plasma insulin levels were measured using a double antibody–based radioimmunoassay kit (Linco Research, St Charles, MO). Triglyceride and cholesterol levels were determined by using colorimetric assay kits from Boehringer Mannheim and Sigma (St Louis, MO), respectively.
Measurement of Plasma Nitrite and Nitrate (NOx) Levels
NOx levels were determined using a commercially available colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) based on the Griess reaction. Before the assay, the plasma samples were ultrafiltered though a 30-kDa molecular weight cut-off filter (Ultrafree-MC centrifugal filter units, Millipore Corporation, Bedford, MA) to reduce the background absorbance caused by hemoglobin.
Measurement of Plasma-Free 15-F(2t)-isoprostanes and Total Antioxidant Concentrations
15-F(2t)-isoprostane, a marker of lipid peroxidation and antioxidant deficiency, was measured using an enzyme immunoassay kit (Cayman Chemical). Total plasma antioxidant concentrations were measured using an assay kit from Calbiochem (San Diego, CA). The assay is based on the ability of antioxidants in the plasma to inhibit the oxidation of ABTS (2,2′-azino-di-[-3-ethylbenzodiazoline sulfonate]) to ABTS+ by metmyoglobin (a peroxidase). The amount of ABTS+ formed (a reflection of the total antioxidant concentration in plasma) was read at 600 nm using a temperature-controlled spectrophotometer.
Immunohistochemical Localization of eNOS, iNOS, and NT
The heart and SMA tissues were fixed in 10% neutral buffered formalin overnight and transferred to 70% ethanol. This was followed by paraffin processing through increasing grades of ethanol, xylene, and paraplast (Fisher Scientific, Ottawa, Canada). Paraffin-embedded tissue blocks were sectioned at 3 μm and mounted on slides. The sections were deparaffinized, rehydrated, washed with phosphate-buffered saline (PBS), and blocked with 5% normal goat serum in PBS for 30 min. The slides were subsequently incubated with primary mouse monoclonal anti-eNOS (1:600) or rabbit polyclonal anti-iNOS (1:100) or anti- NT (1:200) antibodies in PBS containing 1% normal goat serum overnight at 4°C. After washing off the primary antibody with PBS, the sections were incubated for 30 min with secondary antibody containing goat anti-mouse eNOS, goat anti-rabbit iNOS, and anti-NT antibodies conjugated to horseradish peroxidase (Dakocytomation, Mississauga, Canada). After 3 washing steps in PBS, the sections were stained using NovaRED (Vector Laboratories, Burlingame, CA) for 10 min, washed with distilled water, and counterstained using Mayer's hematoxylin. The sections were dipped in lithium carbonate (for 90 s), washed in running water, dehydrated in increasing grades of alcohol, and cleared in xylene before being mounted in resinous mounting medium with Permount (Fisher Scientific) coverslips. Some sections incubated with non-specific mouse and rabbit immunoglobulins (Jackson ImmunoResearch Laboratories, West Grove, PA) served as negative controls. Using a high-power microscope and digital imaging system, all images were observed individually and photographed (25×). Two blinded observers scored the intensity of the immunostain and reported the results independently.
Western Blot Studies
Protein abundance was determined by Western blot analysis using specific antibodies directed against cardiac eNOS and iNOS isoforms. Briefly, aliquots (∼0.2 g) of the pulverized tissue from each rat were homogenized in 1 mL of lysis buffer (HEPES) at 4°C, using a homogenizer. The homogenate was centrifuged at 10,000g at 4°C for 5 min. Total protein content of the supernatant was determined using the Bio-Rad protein assay, which is based on the Bradford method.23 Aliquots of protein were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis. Immunoblotting was performed either with a polyclonal rabbit anti-iNOS (1:350) or anti-eNOS (1:500) antibody (Santa Cruz Biotechnology, Santa Cruz, CA). For both eNOS and iNOS isoforms the secondary antibody was goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (Santa Cruz Biotechnology). The immune complexes were detected using enhanced chemiluminescence dye (ECL, Amersham Pharmacia Biotech, Piscataway, NJ). The intensity of the bands was determined by using densitometric analysis.
All of the values are expressed as mean±SEM; n denotes the sample size in each group. Statistical analysis was performed using one-way analysis of variance (ANOVA) or repeated measures ANOVA (general linear model ANOVA), followed by the Newman–Keuls test for multiple comparisons. GraphPad Prism (GraphPad Software, San Diego, CA) and NCSS (NCSS 2000, Kaysville, UT) software programs were used for statistical analysis. For all of the results the level of significance was set at P<0.05.
General Physical and Biochemical Characteristics
The general physical and biochemical characteristics of all rats at termination are summarized in Table 1. Injection of rats with STZ resulted in characteristic symptoms of diabetes including hyperglycemia, hypoinsulinemia, decreased body weight gain, and increased food and fluid intake when compared to age-matched control rats. In addition, there was a significant elevation of plasma triglyceride and cholesterol levels in these rats. Treatment of diabetic rats with NAC significantly reduced plasma triglycerides and cholesterol levels without affecting glucose and insulin values. Furthermore, food and fluid intake were markedly reduced in NAC-treated diabetic rats.
Effect of NAC on MABP and HR
By 9 weeks, the MABP (Fig. 1A) and HR (Fig. 1B) were significantly reduced in untreated diabetic rats. Treatment with NAC prevented the depression in both MABP and HR in diabetic rats without affecting these parameters in control animals.
Plasma Isoprostane, Nitrite/Nitrate (NOx), and Total Antioxidant Concentrations
Indicators of oxidative stress in the different groups are summarized in Table 2. Diabetes resulted in a significant elevation of plasma 15-F(2t) isoprostanes and a concomitant reduction in NOx values. In addition, the total antioxidant concentration in the plasma of untreated diabetic rats was reduced, suggesting an increased oxidative stress and NO scavenging activity of oxidative free radicals. Treatment of diabetic rats with NAC significantly increased the plasma total antioxidant concentrations, reduced the isoprostanes levels, and normalized the NOx values.
Immunohistochemistry of eNOS, iNOS, and NT
Decreased eNOS expression was detected in the vascular endothelial lining of arteries in the hearts of untreated diabetic rats compared with C and CT rat hearts (Fig. 2). On the contrary, untreated diabetic rats exhibited increased staining intensity for iNOS and NT in cardiac tissue. NAC-treated diabetic rats not only exhibited normal expression of eNOS protein but also showed a marked decrease in both NT and iNOS immunostain in cardiac tissue. A similar pattern of iNOS and NT protein expression was observed in SMA. There was an increased expression of iNOS and NT in the medial and advential layers of the smooth muscle from untreated diabetic rats, which was not seen in the arteries from NAC-treated diabetic rats. In addition, there was a marked reduction in eNOS expression in the vascular endothelium of untreated diabetic rats, which was also corrected by NAC treatment (Fig. 3).
Western Blot Studies
To determine whether there are any changes in the abundance of eNOS and iNOS proteins in cardiac tissue, Western blot experiments were performed. The 130-kDa band representing iNOS increased, whereas the 145-kDa band representing eNOS protein decreased in hearts from untreated diabetic rats compared with C and CT rats. Similar to the immunohistochemical data, NAC treatment moderately decreased iNOS while normalizing eNOS protein expression in DT rats (Fig. 4).
The depressed blood pressure and heart rate observed in untreated diabetic rats in the present study is in agreement with many other studies in STZ diabetic rats.22,24,25 In the past, we have shown lower heart rates and reduced systolic blood pressure in conscious STZ diabetic rats.26 The mechanisms underlying the depressed MABP and HR in STZ diabetic rats are still unclear. Because NO plays an important role in the maintenance of cardiovascular and renal homeostasis, we hypothesized that following diabetes, changes in the bioavailability of endogenous NO in the cardiovascular system may contribute to depressed MABP and HR. Because endothelial function is impaired in diabetes and the source of NO from eNOS is reduced, we suggested that iNOS may contribute to the total NO pool. Consistent with this hypothesis, we and others have previously shown increased expression and activity of iNOS in cardiovascular tissues of STZ diabetic rats.1,4 In addition, data from our laboratory and reports elsewhere27 suggest that acute inhibition of iNOS in STZ diabetic rats normalizes pressor responses to vasoactive agents, although not the depressed MABP and HR.
The role of superoxide anions and their complex interaction with NO has not been studied in detail as a potential mechanism of cardiovascular depression. A large body of evidence implicates superoxide in the pathophysiology of hypertension, both in humans and in animals models of hypertension.28,29 It is well known that elevation of superoxide anions and NO, both of which occur in diabetes, can result in the rapid formation of peroxynitrite. Increased production of peroxynitrite not only causes nitrosative stress, which may be detrimental to cardiovascular tissues, but also reduces the bioavailability of functional NO and may contribute to impaired endothelium-mediated relaxation. The results of the present study indicate an increased level of oxidative stress in untreated diabetic rats. Plasma isoprostanes, a reliable marker of lipid peroxidation, increased, along with a concomitant reduction in total plasma antioxidant concentrations. In addition, plasma NOx values also decreased, suggesting an increased NO scavenging activity by superoxide anions. These findings imply that oxidative stress in combination with elevated NO levels from iNOS in hyperglycemia leads to an increased level of nitrosative stress. The formation of peroxynitrite, a marker of nitrosative stress, can be assessed indirectly by measuring the levels of NT in plasma and tissue. The results of our immunohistochemical studies, indicating an increased expression of NT in both heart and SMA of untreated diabetic rats, are consistent with this hypothesis.
Treatment of diabetic rats with NAC significantly improved blood pressure and heart rate while decreasing the expression of iNOS and NT levels. This suggests that the depressed MABP and HR in untreated diabetic rats may be caused by the actions of peroxynitrite on cardiovascular tissues. NAC treatment can reduce the formation of peroxynitrite, possibly by a simultaneous inhibition of ROS and NO via the suppression of NF-κB mediated induction of iNOS expression.30 In fact, many studies have shown a strong correlation between endogenous peroxynitrite formation and deterioration of cardiac function.31 It has been suggested that peroxynitrite has the ability to exert cardiac depressant actions.32,33 At the subcellular level, peroxynitrite inhibits the mitochondrial respiratory chain34 and triggers apoptosis in cardiomyocytes.3,35 In addition, a number of studies have reported peroxynitrite-mediated abnormalities in neural and vascular tissues in diabetes. In the vasculature, it has been reported that peroxynitrite causes direct oxidation of catecholamines36 and reduces the binding capacity of α-adrenergic receptors, thereby decreasing the vascular reactivity to vasoactive agents.37 Thus, it is possible that peroxynitrite, by acting directly on cardiac and vascular tissues, can cause cardiac dysfunction and vascular hyporeactivity and thereby depress MABP and HR. Many of the cardiovascular abnormalities in diabetic rats can be prevented by inhibiting the formation of peroxynitrite in cardiac, neuronal, and endothelial cells.38,39 For instance, Xia and colleagues (unpublished data, 2005) from our laboratory suggested that NAC treatment of STZ diabetic rats improves cardiac function parameters in the isolated working heart model, particularly the rate of relaxation. In addition, previous studies from our laboratory using a different antioxidant (ascorbic acid) have shown improved cardiac performance in STZ diabetic rats.40
Increased expression of iNOS and NT was associated with a concomitant reduction in eNOS protein expression in untreated diabetic rats. Other investigators have observed that induction of iNOS is associated with downregulation or decreased expression of the eNOS gene.41 A recent study demonstrated that iNOS knockout mice are resistant to endothelial dysfunction when made diabetic.42 Conversely, when the iNOS gene was transferred to normal arteries, NO-dependent relaxation was impaired, suggesting endothelial dysfunction.43 In other studies, in the presence of hyperglycemia, expression of iNOS in cardiac, arterial, and sciatic nerves was associated with a pronounced reduction of eNOS both in humans and in animal models of diabetes.5,44,45 These findings suggest that abnormal and pathological overexpression of iNOS may cause downregulation of eNOS, thereby impairing endothelial function. Data from our laboratory6 and other reports46 indicate an association of endothelial dysfunction with increased expression of iNOS and NT in arterial tissues of STZ diabetic rats. Treatment with NAC normalized the protein expression of eNOS, iNOS, and NT in cardiovascular tissues probably by a simultaneous reduction of both iNOS-mediated NO production and ROS formation.
Alternatively, peroxynitrite was recently shown to impair eNOS activity, leading to the suggestion that it is responsible for the endothelial dysfunction in diabetes.47 The authors discovered that ONOO− can oxidize the zinc thiolate center in eNOS, a modification that results in reduced NO bioactivity and enhanced endothelial O2 − production. This modification can result in the generation of more ONOO− and eNOS uncoupling. Another mechanism of eNOS uncoupling by peroxynitrite is by direct oxidation of tetrahydrobiopterin (BH4), an essential and critical cofactor for NO synthesis. Prior treatment of endothelial cells with both BH4 and ascorbate prevented uncoupling of eNOS by ONOO−, suggesting that antioxidants may help preserve endothelial function.48 Although we do not have any direct evidence to show that peroxynitrite per se is involved in the decreased expression of eNOS, it is possible from the above evidence that peroxynitrite is capable of causing endothelial dysfunction. Furthermore, NAC treatment by restoring the antioxidant status at the cellular level may have prevented the prooxidant actions of ONOO− on the eNOS protein.
In addition to reducing nitrosative stress, NAC treatment also lowered plasma cholesterol and triglycerides in diabetic rats. Although it has been suggested that elevated levels of circulating lipids may contribute to cardiac dysfunction,49 previous studies from our laboratory indicate that MABP and HR decrease by 3 weeks, whereas triglycerides and cholesterol level do not increase until 6 weeks of diabetes. This indicates that depressed MABP and HR are not caused by elevated lipid levels, and improvement of blood pressure and hyperlipidemia by NAC treatment may not be causally correlated.
In summary, the results of our study suggest that the interactions between iNOS-derived NO and ROS, which strongly favor the formation of peroxynitrite, could influence the subsequent hemodynamic outcomes in diabetes. Although the mechanisms involved in the pathogenesis of cardiovascular complications are far from clear, a number of therapeutic strategies are conceivable to alleviate deleterious effects of excessive NO formation. These include combined iNOS inhibitor and/or antioxidant therapy that can effectively scavenge excess NO and ROS, thereby preventing the formation of peroxynitrite and nitrosative stress. Previous studies from our laboratory have indicated that nitrosative stress depends on the duration of diabetes. Hence, temporal studies involving treatment of STZ diabetic rats with NAC for different periods may provide useful information.
The technical assistance of Dr. Linfu Yao and Violet Yuen are gratefully acknowledged. The authors also thank Mary Battell for her assistance during the course of experiments. This work was supported by a program grant and GIAs to J. M. and K. M. M. from the Heart and Stroke Foundation of British Columbia and Yukon. P. R. N. and Z. X. are supported by CIHR/Rx&D HRF.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
diabetes; inducible nitric oxide synthase; nitrosative stress; N-acetylcysteine; cardiovascular depression