Studies in streptozotocin (STZ)-induced diabetic rats have demonstrated cardiovascular abnormalities such as depressed mean arterial blood pressure (MABP) and heart rate (HR), endothelial dysfunction, and attenuated pressor responses to vasoactive agents. We investigated whether these abnormalities are due to diabetes-associated activation of inducible nitric oxide synthase (iNOS). In addition, the effect of the duration of diabetes on these abnormalities was also evaluated. Diabetes was induced by administration of 60 mg/kg STZ via the tail vein. One, 3, 9, or 12 wk after STZ injection, MABP, HR, and endothelial function were measured in conscious unrestrained rats. Pressor response curves to bolus doses of methoxamine (MTX) and angiotensin II (ANG II) were constructed in the presence of N-[3(aminomethyl)benzyl]-acetamidine, dihydrochloride (1400W), a specific inhibitor of iNOS. Depressed MABP and HR and impairment of endothelial function were observed as early as 3 wk after induction of diabetes. Acute inhibition of iNOS with 1400W (3 mg/kg iv) restored the attenuated pressor responses to both MTX and ANG II without affecting the basal MABP and HR. Immunohistochemical and Western analysis blot studies in cardiovascular tissues revealed decreased expression of endothelial nitric oxide synthase (eNOS) concomitant with increased expression of iNOS and nitrotyrosine with the progression of diabetes. Our findings suggest that induction of iNOS in cardiovascular tissues is dependent on the duration of diabetes and contributes significantly to the depressed pressor responses to vasoactive agents and potentially to endothelial dysfunction.
- inducible nitric oxide synthase
- nitric oxide
- cardiovascular abnormalities
chronic hyperglycemia has been shown to directly affect the composition and structure of cardiac, vascular, and renal tissues, the progressive modification of which results in a deranged cardiovascular homeostasis (8). Cardiovascular depression, characterized by depressed mean arterial blood pressure (MABP), heart rate (HR), and attenuated pressor responses to vasoactive agents is one of the most notable manifestations of this derangement, particularly in animal models of Type 1 diabetes. Although the exact mechanisms by which diabetes contributes to cardiovascular depression are currently unknown, it is likely that hyperglycemia may initiate this abnormality through the activation of protein kinase C, increased activity of the polyol pathway, formation of nonenzymatic advanced glycosylation end products, oxidative stress, and/or possibly by induction of nitric oxide (NO) synthase (NOS) (19, 42).
The role of NO in the regulation of hemodynamics under hyperglycemic conditions has been controversial. Despite an impairment of endothelial function and reduced bioavailability of endothelium-derived NO, streptozotocin (STZ)-induced diabetic rats are not hypertensive but instead are normotensive or hypotensive (9, 14, 18, 20, 23, 31, 39, 44). In addition, an increased dependence on a functional NO system at the onset of diabetes has been reported to prevent the development of hypertension in STZ diabetic rats (12). It has been suggested that increased NO synthesis may be important to maintain an adequate renal blood flow and to suppress the activated renin angiotensin (RAS) and sympathetic nervous system (SNS) activities in diabetes. These findings imply the presence of increased NO to counteract the pressor effects of endogenous mediators in diabetes (28, 32). However, given the fact that endothelial function is impaired in diabetes and the generation of NO from endothelium is reduced, the source of NO in such settings is not clear. It is possible that NO from other sources such as inducible NOS (iNOS) and neuronal NOS (nNOS) may contribute to increased NO in diabetes. Previous studies from our lab did not find a change in the expression or activity of nNOS, but we reported an increased expression and activity of iNOS in superior mesenteric arteries (SMA) from 12- to 14-wk STZ-induced diabetic rats (1).
Relatively little is known about the contribution of iNOS to the total NO pool in diabetic conditions. We have suggested that NO derived from iNOS may contribute to an increased NO pool, the excess of which may result in cardiovascular depression. It is also likely that increased formation of reactive oxygen species, which also can occur in hyperglycemic conditions, can scavenge NO resulting in the formation of peroxynitrite and/or nitrotyrosine (NT). Peroxynitrite is a strong oxidant that can oxidize various biomolecules and can exert cytotoxic actions in cardiac and vascular tissue resulting in a state of cardiovascular depression (13, 37, 38).
In the past, we have demonstrated that selective inhibition of iNOS in vitro in endothelium-denuded diabetic mesenteric arteries increases the potency of contractile responses to norepinephrine (1). This was recently confirmed by an in vivo study that reported increased pressor responses to norepinephrine on iNOS inhibition in acute diabetes (6). We hypothesized that increased expression of iNOS in cardiovascular tissues contributes to depressed MABP and HR and possibly to endothelial dysfunction in STZ-induced diabetic rats. Because endothelial dysfunction depends on the duration of diabetes (32), it was of interest to determine the relative changes of eNOS and iNOS expression with time and their contribution to cardiovascular abnormalities, particularly to the altered pressor responses to vasoactive agents. Therefore, the primary goals of the present work were 1) to determine the effect of duration of diabetes on endothelial function and the relative expression of eNOS and iNOS in cardiovascular tissues of STZ-induced diabetic rats, 2) to investigate the potential role of iNOS in modulating the basal MABP and HR, and 3) to study the effect of iNOS inhibition on the pressor responses of vasoactive agents such as methoxamine (MTX) and angiotensin II (ANG II). We chose MTX and ANG II as vasoactive agents in the present study because they are known to act by different mechanisms of action. Because anesthetics (21) and stress (40) have been reported to alter sympathetic activity and subsequent hemodynamics, we conducted all our experiments in intact and freely moving conscious rats.
MATERIALS AND METHODS
Study Design and Induction of Diabetes
One hundred and twenty four male Wistar rats weighing 250 ± 10 g were obtained from Charles River Laboratories. For acclimatization, the rats were housed on a 12:12-h light-dark cycle and given rat chow and water ad libitum. Care was given in accordance with the principles and guidelines of the Canadian Council on Animal Care. Studies were conducted in four stages, wherein the animals were divided into four study groups of 32 animals each (1-, 3-, 9-, and 12-wk study groups). In each group, 16 animals were made diabetic by a single bolus intravenous injection of STZ (60 mg/kg) via the tail vein while under light halothane anesthesia. Blood glucose was measured using glucose test strips (Accusoft) read on a glucometer (Roche Diagnostics).
Rats were anesthetized with halothane, and a fluid-filled (heparinized saline, 20 U/ml) catheter (polyethylene-50, Intramedic Clay Adams) was placed in the left carotid artery. A second catheter (polyethylene-50) was placed in the left jugular vein for drug administration. Both catheters were exteriorized at the nape of the neck, passed through a harness and tether, and connected to a swivel (Instec Lab) mounted above the cage to allow free movement of the animal. The arterial catheter was connected to a disposable pressure transducer (DTX, Viggo-Spectramed) mounted on the cage exterior at the level of the rat. The venous catheter was connected to a syringe for drug administration. MABP and HR were simultaneously recorded on a Gould TA 2000 Thermal Array Recorder (Gould Instrument System) 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 4 h before beginning experiments (18).
Four hours after surgery, basal MABP and HR were measured in all rats. Subsequently, dose-response curves to isovolumic bolus doses of MTX (100–300 nmol/kg) or ANG II (20–320 pmol/kg) were constructed in control and diabetic rats. Only one agonist was tested in each animal. The protocol for each experiment was as follows: dose-response curves were constructed by measuring MABP in response to each bolus dose of MTX or ANG II, allowing sufficient time for MABP to return to normal between each dose. Subsequent to the final dose of MTX or ANG II, a single bolus dose of 3 mg/kg N-[-3(aminomethyl)benzyl]-acetamidine, dihydrochloride (1400W), a specific irreversible inhibitor of iNOS (15), was administered through the jugular vein. The above dose of 1400W was selected based on its ability in preliminary studies to inhibit iNOS-mediated circulatory collapse in the lipopolysaccharide model of septic shock (data not shown). Fifty minutes later, a second dose-response curve for MTX or ANG II was constructed. Finally, a single bolus dose of Nω-nitro-l-arginine methyl ester (l-NAME, 10 mg/kg iv), a nonselective NOS inhibitor, was administered, and MABP was recorded over a period of 15–20 min.
At termination, blood was collected from the carotid artery into tubes containing EDTA. Hearts, aortas, and SMA were then quickly removed and placed in ice-cold Krebs solution (in mM: 120 NaCl, 5.9 KCl, 25 NaHCO3, 11.5 glucose, 1.2 NaH2PO4, 1.2 MgCl2, and 2.5 CaCl2) containing 0.1 μM water-soluble dexamethasone (to prevent the induction of iNOS in vitro). The collected tissues were cleaned of all adherent tissues, fixed in 10% neutral buffered formalin, and processed for immunohistochemistry and Western blot analysis studies.
Western blot analysis studies for measurement of cardiac NOS protein levels.
Protein abundance was determined by Western blot analysis using specific antibodies directed against eNOS and iNOS isoforms. Briefly, aliquots (∼0.2 g) of the pulverized tissue (left ventricle) from each rat were homogenized in 1 ml of lysis buffer at 0 to 4°C, using a Polytron homogenizer. The homogenization buffer contained 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 20 mM KCl, 20% glycerol, 0.2 mM EDTA, 2 mM Na3VO4, 10 mM NaF, 1% Triton X-100, 0.2 mM PMSF, 10 mg/ml aprotinin, and 10 mg/ml leupeptin. The homogenate was centrifuged at 10,000 g at 4°C for 5 min, and the resulting supernatants were stored at −80°C until use. Tissue lysate protein concentrations were determined using the Bio-Rad protein assay, which is based on Bradford method (3).
Aliquots (100 μg protein) in 50 μl of sample digestion buffer [0.5 M Tris·HCl, 10% (wt/vol) glycerol, 2% (wt/vol) SDS, 5% (vol/vol) β-mercaptoethanol, and 0.1% bromophenol blue] were denatured by heating at 95°C for 5 min and separated by SDS-polyacrylamide gel electrophoresis (10% gel). The separated proteins were transferred electrophoretically to nitrocellulose membranes. After transfer, the membranes were blocked overnight in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% fat-free dried milk and subsequently washed with TBS-T. Immunoblotting was performed either with a polyclonal rabbit anti-iNOS (1:350) or anti-eNOS (1:500) antibody (Santa Cruz Biotechnology). For both eNOS and iNOS isoforms the secondary antibody was goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology). After the final wash, immune complexes were detected by using enhanced chemiluminescence dye (Amersham Pharmacia Biotech). The intensity of the bands was determined by using densitometric analysis.
Immunohistochemical localization of eNOS, iNOS, and NT.
Ventricles, aortas, and SMAs were cleaned and 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 (Fischer Scientific). Paraffin-embedded tissue blocks were sectioned at 3 μm, and sections were mounted on positively charged slides. For immunostaining, sections were deparaffinized, rehydrated, treated with target retrieval buffer (DAKO, S1699), blocked with 3% hydrogen peroxide [to block endogenous peroxidase activity, 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 eNOS antibody (1:600) or rabbit polyclonal iNOS (1:100) or rabbit polyclonal NT (1:200) antibody in PBS containing 1% normal goat serum overnight at 4°C. The primary antibody was rinsed off with PBS, and the sections were incubated with either goat anti-mouse (eNOS), goat anti-rabbit (iNOS), or anti-NT secondary antibodies (Dako Envision, Dakocytomation) for 30 min. After three washing steps in PBS were completed, the sections were stained using NovaRED (Vector Labs) for 10 min, washed with distilled water (5 min), and counterstained using hematoxylin. The sections were dipped in lithium carbonate, washed in running water, dehydrated in increasing grades of alcohol, and cleared in xylene before being mounted in resinous mounting medium with Permount (Fischer) coverslips. Some sections incubated with nonspecific mouse and rabbit immunoglobulins (IgG) (Jacksons Immuno Research Laboratories) served as negative controls. With the use of Leitz Orthoplan high power microscope, all slides were observed and photographed.
Chemicals and reagents.
Unless otherwise indicated all chemicals and reagents were of reagent grade and were obtained from Sigma Aldrich.
All values are expressed as means ± SE; n denotes the number of animals in each group. Statistical analysis was performed using one-way ANOVA or repeated measures ANOVA (general linear model), followed by the Newman-Keuls test for multiple comparisons. One-way ANOVA was performed by using GraphPad Prism (GraphPad Software) and repeated-measures ANOVA using NCSS statistical analysis system (NCSS). For all results the level of significance was set at P < 0.05.
General Characteristics and Basal MABP and HR
Body weights and blood glucose levels of all rats measured at termination are shown in Fig. 1, A and B. All STZ-injected rats were hyperglycemic (>20 mM) compared with their age-matched controls. In addition, body weights of diabetic rats were significantly lower than their age-matched controls by 3 wk after STZ injection.
MABP and HR were measured 1, 3, 9, and 12 wk post-STZ injection (Table 1). By 3 wk, all STZ-induced diabetic rats had depressed MABP and HR. A progressive increase in the MABP between 1 and 9 wk of age was observed in control animals. On the other hand, diabetic rats had similar blood pressures at all time points, but they were significantly lower than their corresponding age-matched controls starting from 3 wk. The heart rate in diabetic groups was also significantly lower than their age-matched controls in all groups starting from 3 wk of diabetes.
Effect of iNOS Inhibition With 1400W on Basal MABP and HR
The effect of 1400W administration on the basal MABP and HR at different stages of diabetes is summarized in Table 1. The results demonstrate that administration of 1400W did not alter the basal MABP and HR at any time point in either control or diabetic animals.
Effect of 1400W on Pressor Responses to MTX and ANG II
Administration of bolus doses of MTX (Fig. 2, A–D) and ANG II (Fig. 3, A–D) increased pressor responses both in control and diabetic rats in a dose-dependent manner. Compared with the corresponding age-matched control rats, the responses to MTX and ANG II were significantly attenuated in 3-, 9-, and 12-wk diabetic rats. Furthermore, area under the curve analysis (data not shown) suggested that the pressor responses to MTX were depressed to a greater extent than the responses to ANG II in untreated diabetic rats. Pretreatment with 1400W did not affect the pressor responses to either agonist in control rats but significantly augmented responses to both MTX and ANG II in 3-, 9-, and 12-wk diabetic rats. In the presence of 1400W the pressor responses to both MTX and ANG II were normalized in all diabetic groups except the 12-wk group, where the MTX response increased but did not reach control levels.
Endothelial Function: Effect of l-NAME Administration on MABP
The effect of duration of diabetes on endothelial function was tested using a single bolus dose of l-NAME (10 mg/kg iv), a nonselective inhibitor of NOS. The effect of l-NAME on MABP in the presence of 1400W (iNOS inhibition) is shown in Fig. 4. Starting from 3 wk, the diabetic rats showed attenuated pressure response (ΔMABP) to l-NAME suggesting impairment of endothelial function.
Immunohistochemical and Western Blot Analysis of eNOS, iNOS, and NT
Immunohistochemistry was performed on the paraffin sections of the heart (left ventricle), thoracic aorta, and SMA. In control rats, strong positive immunostain for eNOS was observed in the vascular endothelium (monolayer) of all tissue sections tested. Similar to control rats, there was no apparent change in eNOS immunostain in the endothelium of 1- or 3-wk diabetic rats (data not shown). However, the intensity of positive immunostain for eNOS decreased in the arteries of 9- and 12-wk diabetic rats compared with their age-matched control rats (Fig. 5). \. Because of the small amount of protein, Western blot analysis studies were not carried out in mesenteric arteries. However, Western blot analysis of heart tissue showed decreased expression of eNOS protein by 9 wk of diabetes compared with their age-matched control rats (Fig. 6).
In contrast, there was a striking difference between control and diabetic tissues in iNOS immunostaining. iNOS protein expression was not evident in any of the tissue sections tested in 1-wk control and diabetic rats. The first trace of iNOS protein was detected in the myocardial tissue of 3-wk diabetic rats, whereas strong positive signals were observed in myocardial and the medial and advential layers of arterial tissues from 9- and 12-wk diabetic rats (Fig. 7). \. In contrast, no positive immunostaining for iNOS was observed in tissues of control rats at any time point. This observation was further strengthened by Western blot analysis studies of cardiac iNOS protein expression (Fig. 8). The results indicate that by 3 wk, the expression of iNOS increased by more than twofold in diabetic heart tissues.
Immunohistochemical analysis of NT expression, an indirect marker of peroxynitrite formation indicated an apparent increase in the intensity (reddish brown) in all tissues by 3 wk of diabetes. Observation of tissue sections from 12-wk diabetic rats shows a more intense stain for NT in the myocardial tissue compared with the arterial tissues (Fig. 9). \.
The major findings of the present study are that attenuated pressor responses and impaired endothelial function are associated with increased expression of iNOS and NT and decreased expression of eNOS in cardiovascular tissues of STZ diabetic rats. The duration of diabetes appears to have a significant effect on this association. Selective inhibition of iNOS normalizes the attenuated pressor responses of vasoactive agents. Furthermore, although NO from iNOS does not seem to directly depress MABP and HR, changes secondary to increased expression of iNOS in cardiovascular tissue may contribute to depressed MABP and HR in these rats.
Administration of STZ resulted in depressed MABP and HR in rats within 3 wk compared with age- and gender-matched controls. Examination of the results indicates that diabetic rats did not show a decrease in MABP (between 1 and 12 wk) but rather failed to increase MABP with age. In control rats, the MABP increased while HR decreased progressively with age. It has been suggested that the increase in MABP with age is a normal age-dependent process that may be a compensatory mechanism for loss of arterial compliance (4). Similarly, the decrease in HR is usually attributed to a diminution in the efficacy of β-adrenergic stimulation of the heart, which again is normal and an age-dependent process (41, 43)
The depressed MABP and HR in diabetic rats observed in the present study is in agreement with many other studies in STZ diabetic rats (9, 14, 18, 20, 23, 31, 39, 44). Previous studies from our own laboratory have shown lower HR and reduced systolic blood pressure in conscious STZ-induced diabetic rats (44). However, a few studies from other laboratories have reported unchanged (5, 6, 24) or increased blood pressure in diabetic rats (22). Although the reasons for these discrepancies are not very clear, it is likely that differences in the duration of diabetes, use of anesthetics, and varied techniques employed for measurement of blood pressure may have contributed to the disparities in the observed results (25).
The mechanisms underlying the depressed MABP and HR do not seem to directly involve iNOS, because acute inhibition of iNOS with 1400W did not normalize the depressed MABP and HR in diabetic rats. However, it is possible that changes secondary to increased iNOS activity such as the increased formation of peroxynitrite-nitrotyrosine that we also detected may contribute to the diminished MABP and HR. In fact, many reports have suggested that increased formation of peroxynitrite causes cardiovascular depression and vascular hyporeactivity in diabetes (11, 34–36). However, further studies are required to clarify the role of peroxynitrite in depressed MABP and HR in diabetes.
In addition to the depressed MABP and HR, endothelial dysfunction, as indicated by attenuated pressor responses to l-NAME, was observed by 3 wk of diabetes. Surprisingly, our immunohistochemical and Western blot analysis data do not demonstrate a corresponding decrease in the expression of eNOS in cardiovascular tissues of these rats. This suggests that there may be changes in the bioavailability of NO derived from eNOS at this time. Further studies investigating the activity of eNOS in these tissues are necessary.
Despite the overwhelming evidence of impaired endothelium-dependent relaxation in diabetes, there are sporadic reports of enhanced (2) or unaltered relaxation (29). These disparities may be due to the different types of arteries and methodologies used in tissue preparation and measurement of endothelial function, and most importantly, the duration of diabetes (32). Recently, a temporal study examining the duration of diabetes on endothelial function reported an increased (24 h), unaltered (1 wk), and an impaired (8 wk) relaxation to acetylcholine (32). These observations strongly suggest that endothelial function is dependent on the duration of diabetes. There is compelling evidence to suggest that diabetes-associated endothelial dysfunction results, in part, from a paradoxical increase of NO production from iNOS. In fact, studies have demonstrated an improvement in endothelial function in diabetic arteries using NO scavengers and iNOS inhibitors (10, 33). A recent study demonstrated that iNOS knock-out mice are resistant to endothelial dysfunction when made diabetic (16). Similarly when the iNOS gene was transferred to normal arteries, NO-dependent relaxation was impaired, suggesting endothelial dysfunction (17). These results strongly suggest a direct involvement of iNOS in endothelial dysfunction in diabetes. Results from the present immunohistochemistry and Western blot studies are consistent with the hypothesis that an increased expression of iNOS in the heart, aorta, and mesenteric tissue may contribute to the reduced eNOS expression and possibly to the endothelial dysfunction in 9- and 12-wk diabetic rats.
Although there have been conflicting reports about the effect of diabetes on contractile responses to vasoactive agents in isolated arteries, most of the in vivo studies with STZ-induced diabetic rats have shown only attenuated pressor responses (7, 18, 27, 30). In the present study we noticed a similar pattern of attenuated pressor responses to both MTX and ANG II. The normalization of these responses by acute inhibition of iNOS suggests that NO from iNOS is directly responsible for the attenuated pressor responses of diabetic rats. These results are consistent with recent reports that have implicated iNOS in modulating the basal tone and contractile responses to vasoactive agents in isolated arteries from diabetic animals in vitro (1, 20). Our immunohistochemistry data, demonstrating increased expression of iNOS in mesenteric and aortic tissues from diabetic rats, supports these observations.
An interesting observation was that the pressor responses of 12-wk diabetic rats to MTX were attenuated to a greater extent that those to ANG II and remained significantly depressed in the presence of 1400W, although the pressor responses to ANG II were normalized. The reasons for this are not clear, but it is possible that hyperglycemia-mediated impairment of sympathetic function can reduce the pressor responses to α1-adrenergic agonists (26). Also at the postreceptor level, stimulation of the α1-receptor by MTX may involve increased production of PGI2 and/or decreased formation of thromboxane A2 in diabetes (27), both of which can contribute to the depressed pressor responses to α-adrenergic stimulation.
In conclusion, our findings suggest that induction of iNOS in cardiovascular tissues, which is detectable by 3 wk after induction of diabetes, contributes significantly to the depressed pressor responses to vasoactive agents. The depressed pressor responses may be due to cardiac dysfunction or decreased contractility of resistance vessels such as those of mesenteric arterial bed or both. Our data also indicate a differential regulation of eNOS and iNOS isoforms, wherein a prolonged diabetic state leads to downregulation of eNOS with a concurrent upregulation of iNOS and nitrotyrosine. Interaction of iNOS-derived NO with oxidative free radicals and subsequent formation of peroxynitrite or nitrotyrosine may influence the subsequent hemodynamic outcomes in experimental diabetes.
This work was supported by a program grant and grants-in-aid to K. M. MacLeod and J. McNeill from the Heart and Stroke foundation of Brtish Columbia & Yukon. P. R. Nagreddy and Z. Xia are supported by awards from Canadian Institutes of Health Research/Canada’s Research-Based Pharmaceutical Companies Health Research Foundation.
We thank Dr. Linfu Yao, Violet Yuen, and Mary Battell for assistance during experiments.
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