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Original Articles

Impaired Nitric Oxide Function in the Basilar Artery of the Obese Zucker Rat

Karagiannis, J. BSc; Reid, J. J. PhD; Darby, I. PhD; Roche, P. PhD; Rand, M. J. PhD; Li, C. G. PhD

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Journal of Cardiovascular Pharmacology: October 2003 - Volume 42 - Issue 4 - p 497-505
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Abstract

The genetically obese (fa/fa) Zucker rat (OZR) is a well-accepted model of insulin resistance and a predictor of non–insulin-dependent or type 2 diabetes mellitus. 1,2 This animal model exhibits the key components of the insulin resistance syndrome, including glucose intolerance, dyslipidemia, mild hyperglycemia, hyperinsulinemia, proteinuria, and hypertension. 3 The metabolic abnormalities related to insulin resistance and type 2 diabetes are associated with pathologic changes including vascular dysfunction, which may contribute to the development of micro- and macroangiopathy. It is well known that the function of the blood vessels is regulated by endothelial cells through the release of various substances including endothelium-derived nitric oxide (NO). 4 Impaired endothelium-dependent relaxation of blood vessels has been confirmed in human type 2 diabetes; however, studies investigating insulin resistance and type 2 diabetes are scarce and have yielded conflicting results. 5 In the OZR, hyperinsulinemia and hypercholesterolemia are believed to be associated with the decrease in NO-mediated responses in the intestinal vascular bed in vivo 6 and in vitro. 7 However defects in the metabolic state do not alter functions of the carotid artery. 7 Furthermore, impaired agonist-stimulated endothelium-dependent vasodilation and vascular NO signaling in vivo is believed to be associated the progression to type 2 diabetes mellitus in the OZR mediated by a prooxidant insult. 8 Other studies suggest that the development of diabetes in the OZR may be associated with impaired skeletal muscle perfusion and hypertension caused by microvessel hyperreactivity in response to sympathetic stimulation. 9,10

The cerebral vasculature is an important target organ responsible for diabetic vascular complications such as stroke. 11–14 However, to our knowledge, there have been no previous studies investigating endothelial function or endothelial NO synthase (eNOS) isoform expression and localization in the basilar artery from the OZR. Therefore, the study was designed to characterize vascular function in basilar arteries of the OZR in comparison with the lean littermate control (lean Zucker rat; LZR).

MATERIALS AND METHODS

Animals

Female OZRs (Monash Animal Services, Victoria, Australia) 36 weeks of age were used with female age-matched LZRs as controls. All procedures involved in experimental animals had approval from the animal experimentation ethics committee of RMIT University and conformed to the Australian National Health and Medical Research Council guidelines.

Metabolic and Hemodynamic Parameters

Body weight, systolic blood pressure (measured in conscious rats by tail-cuff plethysmography), nonfasting blood glucose levels (MediSense 2 Blood Glucose Testing System; MediSense Australia), and oral glucose tolerance (glucose 70% weight/volume was administered by oral gavage, 2.5 g/kg body weight, to rats fasted overnight, and blood was obtained at 0, 5, 10, 15, 30, 60, and 90 minutes by nicking the tail tip for blood glucose determination) were measured. To determine protein excretion, urine was collected over a 24-hour period from fasting rats and protein levels were determined with the Bio-Rad protein assay kit (Bio-Rad Laboratories) based on the Lowry method. 15

Plasma was obtained by centrifugation of blood, taken by exsanguination at time of killing, and stored at −20°C until assayed. Plasma insulin levels were determined using an enzyme immunoassay kit (Ultrasensitive rat insulin enzyme-linked immunosorbent assay; Mercodia, Uppsala, Sweden). Plasma levels of total cholesterol and triglycerides were measured using enzymatic assays (CHOD-PAP and GPO-PAP, respectively; Roche, Basel, Switzerland).

Functional Studies

Rats were asphyxiated with CO2 inhalation followed by decapitation. The whole brain was removed and placed in a petri dish containing physiologic salt solution (PSS; NaCl 118 m M, KCl 4.7 m M, NaHCO3 25 m M, MgSO4 0.45 m M, KH2PO4 1.03 m M, CaCl2 2.5 m M, and d-(+)-glucose 5.5 m M) at room temperature. The basilar artery located at the brainstem was isolated under a dissecting microscope (Olympus SZ-30). Care was taken not to damage the endothelium for endothelium-intact preparations, whereas for some experiments, the endothelium of the basilar arteries was removed by gently rubbing with a stainless-steel wire 50 μM in diameter through the lumen. Artery ring segments 1 mm in length were mounted on two stainless-steel wires 40 μm in diameter in a Mulvany-Halpern wire myograph (Myo-Interface Model 610M; Danish MyoTechnology) containing PSS at 37°C and gassed with 5% CO2 and 95% O2. The isometric tension of the vessel was recorded with a MacLab data recording system (MacLab/4, model MKIII; AD Instruments Australia).

After an initial equilibration period of 10 minutes, preparations were stretched for normalization 16 using a software program developed by G. A. McPherson. 17 The internal diameter of each vessel (LZR, 361.2 μM ± 15.1, n = 12; OZR, 355.3 μM ± 8.1; n = 12) was set to a tension corresponding to 90% of the vessel diameter, which the vessel would reach when relaxed at 100 mm Hg transmural pressure. Each preparation was then equilibrated for a further 45 minutes.

Vessel contractile viability was assessed by exposure to 60 mM K+ in PSS (NaCl was replaced by KCl in equimolar concentration in the buffer solution). Initially, concentration-response curves to serotonin (0.01–10 μM) were obtained in the absence and presence of the NOS inhibitor Nω-nitro-L-arginine methyl ester (NAME, 100 μM; 20 minutes incubation). In subsequent experiments, tissues were precontracted with serotonin (1 μM) in the presence or absence of NAME (100 μM) to observe the effects of the receptor-dependent endothelium-dependent vasodilators acetylcholine (ACh, 0.01–10 μM) and histamine (0.1–100 μM). In some experiments, histamine response curves were carried out in the presence of l-arginine (1 m M; 15 minutes incubation). The receptor-independent endothelium-dependent vasodilator calcium ionophore (A23187; 0.01–10 μM), the NO donor sodium nitroprusside (SNP; 0.01–10 μM), free-radical NO (0.1 μM), and papaverine (0.03–3 μM) were investigated. In addition, the effect of ACh and SNP in endothelium-denuded basilar arteries was examined. Approximately two to three concentration curves were created per preparation.

Western Blot Analysis

Brains from two LZRs or two OZRs were pooled and cerebral microvessels were isolated from rat brain parenchyma by methods described previously. 18,19 The preparation adopted for the microvessel fraction has been described previously. 20

Microvessel samples were glass-homogenized in a lysis buffer (pH 7.4) consisting of Tris base (50 m M), magnesium chloride (2 m M), EGTA (1 m M), Triton-X (1%), pepstatin (20 μM), leupeptin (20 μM), aprotinin (0.1 U/mL), and phenyl methylsulfonyl fluoride (1 m M) and then incubated for 30 minutes. Samples were then centrifuged (Beckman Coulter, Allegra 64A centrifuge) at 2,000 g for 20 minutes at 4°C and the supernatant collected for protein quantification using the Coomassie Protein Assay Kit (Bio-Rad Laboratories). As a positive control for eNOS, commercially available human endothelial cell lysate (Transduction Laboratories) was used. For the negative control, the denuded aortae (n = 8) from Sprague-Dawley rats were pooled and homogenized once at 2,000 g for 25 minutes, and the supernatant was collected and treated with lysis buffer in the same manner as mentioned earlier.

For each group, 30 μg of microvessel protein, the controls, and wide-range molecular weight color markers (Sigma, St. Louis, MO, U.S.A.) were loaded onto 7.5% acrylamide gels and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoretic separation, the proteins were transferred onto nitrocellulose paper by electroblotting and membranes were incubated overnight with blocking buffer (5% skim milk and 1% Tween-20 in Tris buffered saline, TBS). Membranes were incubated for 4 hours with monoclonal mouse anti-eNOS antibody (Transduction Laboratories; 1:2,500 dilution), then rinsed with TBS and 1% Tween-20 TBS for 45 minutes and incubated with anti-mouse immunoglobulin (Ig) G antibody conjugated to horseradish peroxidase (Dako, Carpinteria, CA, U.S.A.; 1:5,000 dilution) for 1 hour. Membranes were rinsed again with TBS and 1% Tween-20 TBS, incubated with the chemiluminescent Lumi-Light Western blotting Substrate (Roche Molecular Biochemicals, Mannheim, Germany) for 10 minutes, and apposed to Kodak BioMax film (Kodak, Rochester, NY, U.S.A.) for 5 or 10 minutes.

Immunohistochemistry

The LZRs and OZRs were deeply anesthetized with intraperitoneal sodium pentobarbital (100 mg/kg) and perfused transcardially with heparinized phosphate-buffered saline solution (1 mL of 25,000 U/L), followed by 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4). Each brain was removed and placed in the 4% paraformaldehyde fixative for 45 minutes. The basilar artery was then isolated under a dissecting microscope (Olympus SZ-30) and placed in 20% sucrose solution for at least 24 hours.

LZR and OZR arteries were separated into two wells of a Perspex slide and were washed in PB for 20 minutes, followed by incubation in 2% normal horse serum (CSL, Victoria, Australia; diluted with 0.1 M PB) for 1 hour. Arteries were then incubated overnight at room temperature with PB containing 2% normal horse serum, 0.1% Triton-X, and monoclonal mouse anti-eNOS antibody (1:200 dilution). For a negative control, the primary antibody was omitted from the staining of arteries. The following day, the arteries were rinsed with PB for 20 minutes and then incubated for 2.5 hours with Texas Red antimouse IgG secondary antibody (Vector Laboratories; 1:50 dilution), followed by a series of PB rinses for 20 minutes.

In addition, Sprague-Dawley rat basilar arteries were double-labeled with monoclonal mouse anti-eNOS antibody (1:200 dilution) using fluorescein antimouse IgG secondary antibody (1:50 dilution) and propidium iodide (PI) nucleic acid stain (Molecular Probes; 1:1,000 dilution). Because the tissue was fixed in paraformaldehyde, the manufacturer of PI recommended an RNAse A (125 μg/mL) treatment of the sample before staining of PI. Therefore, after staining the basilar artery for eNOS expression, it was incubated in RNAse A for 30 minutes at room temperature. After three rinses to remove RNA, the artery was incubated with PI (1:1,000 dilution) for 10 minutes, followed by another series of rinses before being coverslipped. Furthermore, the location of smooth muscle cells was determined with anti–α-smooth muscle actin antibody (Dako; 1:50 dilution) and Texas Red antimouse IgG secondary antibody (1:50 dilution). Finally, the arteries were coverslipped with Dako Fluorescent Mounting Medium and kept away from direct light. Arteries were viewed under an Olympus Microscope BX60 (×60 magnification, water-immersed lens).

Drugs

The following drugs were purchased from Sigma: NAME, l-arginine, serotonin or 5-hydoxytryptamine creatinine sulfate, ACh, calcium ionophore A23187, histamine dihydrochloride, SNP, and papaverine hydrochloride. Saturated solutions containing 2 m M of NO· were prepared from nitric oxide gas (compressed gas; CIG Australia) as previously described. 21

All drugs except for A23187 were made up as stock solutions in Milli-Q water. A23187 was dissolved in 100% ethanol. Stock solutions were subsequently dissolved in PSS. The final concentration for ethanol in the chamber was 0.01%, which had no effect on tissue tone or to relaxations produced by A23187.

Data and Statistical Analysis

For functional studies, data were presented as mean and SEM, with n indicating the number of experimental animals. The tension of the tissue was measured in millinewtons per vessel length (mN/mm). Relaxations were expressed as a percentage of serotonin-induced constriction.

For the Western blot study, densitometric analysis of bands was carried out by the measurement of pixel intensity for each band, which was performed on Scion Image software, version 1998 (Scion Corporation).

For the immunofluorescent study, images were captured using the Optiscan Personal Confocal System, model F900e (Optiscan, Victoria, Australia) with a laser filter setting at 568 nm (yellow) for all single-labeled images. For eNOS and PI double labeling of the basilar artery, the laser filter was set at 568 nm (yellow) for images stained with PI and at 488 nm (blue) for images stained with fluorescein-labeled antimouse IgG secondary antibody. All images of the basilar artery were viewed and captured longitudinally. The area of each eNOS image was 100 μm × 100 μm and consisted of 10 scans at a depth of 1 μm at the endothelial cell layer, which represent the inner surface of each vessel. Approximately eight images were taken at random from each artery, which were later averaged for one preparation. The intensity of eNOS in each image was analyzed by MetaMorph software (version 4.6; Universal Imaging).

For all studies, statistical analyses were performed with a one-way analysis of variance or two-way repeated-measures analysis of variance (multivariate analysis of variance; MANOVA) or with the Student t test. A value of P < 0.05 was regarded as statistically significant.

RESULTS

Metabolic and Hemodynamic Parameters

At 36 weeks of age, the OZR showed significantly reduced oral glucose tolerance (P < 0.05, one-way analysis of variance, n = 7–8;Fig. 1) and increased body weights, systolic blood pressure, plasma levels of triglycerides, cholesterol and insulin and profound proteinuria compared with the LZR (Student t test, n = 6–16;Table 1).

TABLE 1
TABLE 1:
Metabolic and hemodynamic parameters in the 36-week-old LZR and OZR
FIGURE 1.
FIGURE 1.:
Oral glucose tolerance test in LZR and OZR. Blood glucose levels were measured 1, 5, 10, 15, 30, 60, and 90 minutes after an oral gavage of glucose (2.5 g/kg body weight). Each value represents the mean ± SEM; 7–8 preparations. * P < 0.05, one-way analysis of variance.

Serotonin-induced Response on Tissue Tone

The concentration-response curve to serotonin (0.01–10 μM) was significantly (P < 0.05, two-way MANOVA; n = 6–9) increased in OZR compared with LZR (Fig. 2A). Furthermore, the contractions to serotonin were significantly (P < 0.05, two-way MANOVA n = 5–9) enhanced by NAME (100 μM) in basilar arteries from the LZR (Fig. 2B) but not from the OZR (Fig. 2C), such that responses to serotonin were no longer different between LZR and OZR in the presence of NAME (100 μM).

FIGURE 2.
FIGURE 2.:
Contractions induced by serotonin (0.01–10 μM) in basilar arteries from LZR and OZR in the absence of 100 μM NAME (A) and from the LZR and OZR in the presence of 100 μM NAME (B,C). Each value represents the mean ± SEM; 6–9 preparations. * P < 0.05, two-way MANOVA.

Acetylcholine-, Histamine-, and A23187-induced Relaxations in Endothelium-intact Preparations

To obtain relaxation responses, basilar arteries were precontracted with 1 μM serotonin. Although the cumulative concentration curve for serotonin was significantly different between the OZR and LZR, the level of precontraction to a single concentration of 1 μM serotonin was not significantly (P > 0.05, Student t test) different between the LZR (2.77 mN/mm ± 0.12, n = 35) and the OZR (2.82 mN/mm ± 0.12; n = 47).

Relaxations induced by ACh (0.01–10 μM), histamine (0.1–100 μM), and A23187 (0.01–10 μM) were significantly (P < 0.05, two-way MANOVA; n = 6–12) reduced in the OZR compared with the LZR (Fig. 3A,D,G). In LZR arteries, ACh-, histamine-, and A23187-induced relaxations were significantly (P < 0.05, two-way MANOVA; n = 6–10) reduced by NAME (100 μM;Fig. 3B,E,H). In OZR arteries, NAME (100 μM) significantly (P < 0.05, two-way MANOVA; n = 7–12) reduced histamine- and ACh-induced relaxations but had no significant (P > 0.05, two-way MANOVA; n = 6–7) effect on A23187-induced relaxations (Fig. 3C,F,I). Furthermore, responses to ACh, histamine, and A23187 in arteries precontracted with serotonin (1 μM) were no longer different between LZR and OZR in the presence of NAME (100 μM).

FIGURE 3.
FIGURE 3.:
Vasodilation responses to ACh (0.01–10 μM), histamine (0.1–100 μM), and A23187 (0.01–10 μM) in basilar arteries precontracted with 1 μM serotonin from LZR and OZR in the absence of NAME (A,D,G) and in basilar arteries from the LZR (B,E,H) and OZR (C,F,I) in the presence of 100 μM NAME. Each value represents the mean ± SEM; 6–12 preparations. * P < 0.05, two-way MANOVA.

Histamine vasodilations in the basilar artery from the LZR and OZR were not significantly (P > 0.05, two-way MANOVA; n = 4–12) altered in the presence of l-arginine (10 m M). However, l-arginine significantly (P < 0.05, two-way MANOVA; n = 4–7) reversed NAME (100 μM)–induced inhibition of the relaxations to histamine in both the LZR and OZR (data not shown).

Nitric Oxide, Papaverine and Sodium Nitroprusside–induced Relaxations in Endothelium-intact Preparations

Nitric oxide (0.1 μM)- and papaverine (0.3–3 μM)–induced relaxations were not significantly different (P > 0.05, two-way MANOVA; n = 4) between the OZR and LZR (Fig. 4A,B). However, SNP (0.01–10 μM)–induced relaxations were significantly (P < 0.05, two-way MANOVA; n = 7–9) reduced in the OZR compared with the LZR (Fig. 4C).

FIGURE 4.
FIGURE 4.:
Responses to SNP (0.01–10 μM, A), papaverine (0.3–3 μM, B), and NO (0.1 μM, C) in basilar arteries precontracted with 1 μM serotonin from LZR and OZR. Each value represents the mean ± SEM; 4–9 preparations. * P < 0.05, two-way MANOVA.

Acetylcholine- and Sodium Nitroprusside–induced Relaxations in Endothelium-denuded Preparations

Acetylcholine (0.01–10 μM)–induced relaxations precontracted with serotonin (1 μM) were virtually abolished (P < 0.05, two-way MANOVA; n = 3–7) in endothelium-denuded basilar artery preparations from LZR and OZR compared with their respective endothelium-intact controls. Furthermore, ACh-induced relaxations were not significantly (P > 0.05, two-way MANOVA; n = 3–4) different between denuded basilar arteries from the LZR and OZR (data not shown).

Sodium nitroprusside (0.01–10 μM)–induced relaxations precontracted with serotonin (1 μM) were not significantly (P > 0.05, two-way MANOVA; n = 3–7) altered in endothelium-denuded basilar artery preparations from LZR and OZR compared with their respective endothelium-intact controls. As in endothelium-intact vessels, SNP-induced relaxations in the endothelium-denuded basilar artery of the OZR was significantly (P < 0.05, two-way MANOVA, n = 3–7) reduced compared with the LZR endothelium-denuded SNP response (data not shown).

Western Blot Analysis of Endothelial Nitric Oxide Synthase Protein

The immunoreactive protein of the expected size (Mr 135,000) for eNOS was detected in the cytosolic fraction of the cerebral microvessel lysate from the LZR and OZR and in the positive control, but not in the negative control, as illustrated in Figure 5A. Bands were of low to medium intensity in all exposure times to film. The mean data show no significant (P > 0.05, student t test, n = 9) difference in the intensity per pixel of bands for the OZR compared with the LZR (Fig. 5B).

FIGURE 5.
FIGURE 5.:
(A) A typical result of Western blot analysis of eNOS protein in cerebral microvessels from LZR and OZR, including denuded aorta from Sprague-Dawley rats (negative control) and endothelial cell protein lysate (positive control). (B) Mean data of the pixel intensity for eNOS protein content in cerebral microvessels from the LZR and OZR. Each value represents the mean ± SEM; 9 preparations.

Immunofluorescent Analysis of Endothelial Nitric Oxide Synthase Protein

The images in Figures 6C and 6D show positive fluorescent staining with the eNOS antibody compared with the negative controls in Figures 6A and 6B in which eNOS antibody was omitted in whole-mount basilar arteries. The positive antibody staining appeared as fluorescent red oval structures running longitudinally on the inner surface of the vessel. Quantitative analysis of the eNOS positive immunoreactivity demonstrated that there was no significant (P > 0.05, student t test, n = 5–6) difference in pixel intensity in basilar arteries from the OZR compared with the LZR (Fig. 7).

FIGURE 6.
FIGURE 6.:
Typical immunofluorescent images of the whole-mount basilar artery using 60× magnification lens in the absence of eNOS antibody from the LZR (A) and OZR (B) and in the presence of eNOS antibody from the LZR (C) and OZR (D). Typical images (E) illustrating eNOS (green) and propidium iodide nucleic acid stain (red) immunolabelling and, at 10× magnification (F), smooth muscle actin immunolabelling of the basilar artery from the Sprague-Dawley rat.
FIGURE 7.
FIGURE 7.:
Mean data for the pixel intensity of eNOS-positive immunoreactivity in the basilar artery from LZR and OZR. Each value represents the mean ± SEM; 5–6 preparations.

The location of eNOS immunoreactivity as revealed by PI and eNOS double staining was perinuclear (Fig. 6E). Long oval structures running in the opposite direction from the endothelial cell nuclei were displayed in this image as PI-positive, and these were assumed to be smooth muscle cell nuclei. Smooth muscle actin staining revealed similar long, narrow structures running in a transverse direction in the basilar artery (Fig. 6F).

DISCUSSION

It is known that insulin resistance and type 2 diabetes are associated with an increased risk of cerebrovascular disease such as stroke; however, little is known about changes in cerebral artery function under these conditions. In the present study, functional, biochemical and immunochemical techniques were used to study vascular endothelial function in cerebral vessel preparations from the genetic OZR model of insulin resistance, in comparison with the corresponding LZR. The major finding is that the endothelium function in the basilar artery is significantly impaired in OZRs compared with LZRs. However, such change does not seem to be associated with changes in eNOS expression or distribution.

At 36 weeks of age, the OZR displayed marked hypertension, proteinuria, hyperlipidemia, and hyperinsulinemia. These metabolic and hemodynamic abnormalities are consistent with the insulin-resistance syndrome and are key factors in the development and predisposition of type 2 diabetes mellitus. 1 In addition, elevated levels of nonfasting blood glucose and impaired glucose tolerance are present in the OZR. The defective metabolic profile observed in the OZR is in accordance with previous studies, 3,22 indicating the OZR is a good animal model for insulin resistance and the risk for type 2 diabetes mellitus.

To evaluate the changes of vascular function, responses to the vasoconstrictor serotonin were studied in basilar arteries in the absence or presence of the NOS inhibitor NAME. The findings that the contractile concentration-response curve to serotonin was significantly increased in the OZR compared with the LZR, and the contractions to serotonin in the LZR but not the OZR were enhanced by NAME, suggests that NO function may be depressed in OZR basilar arteries compared with those from the LZR. The most likely source of basal NO is the endothelium, which is supported by the finding that the response to the endothelium-dependent vasodilator ACh was abolished in endothelium-denuded preparations. The modulation of responses to serotonin by the endothelium has been previously demonstrated in the rabbit basilar artery. 23 Therefore, it appears the endothelium in the OZR may be compromised, thus affecting the contractile response. This is in agreement with another study employing an insulin-resistant animal model, in which phenylephrine-induced contractions to aortic and mesenteric arteries were greater in the obese JCR:LA-corpulent (cp) rat compared with the control. 24 However, a recent study has demonstrated enhanced basal NO in the regulation of contractile reactivity to phenylephrine in the aorta from the 13-week old OZR. 25 Furthermore, phenylephrine-induced vasoconstriction in the renal artery from the OZR between 7 and 36 weeks of age does not differ from the LZR (Lau and Reid, unpublished observations). Clearly, the tissue and possibly age of the OZR and the vasoconstrictor agent might account for the discrepancies in vasoconstriction responses observed in the OZR. It is likely that changes of vasoconstrictor responses in prediabetic and diabetic animals may depend on the types of constrictors studied, as well as vascular preparations and the models used. Interestingly, the application of a single dose of serotonin in the basilar artery did not differ in the OZR compared with the LZR in the present study, allowing comparisons of vasodilator responses between the two groups.

The impairment of endothelium function in the OZR is further supported by the finding that endothelium-dependent vasodilations to ACh, histamine, and A23187 were decreased in basilar arteries, whereas those to endothelium-independent relaxants such as papaverine (inhibition of phosphodiesterase and blockade of calcium channels) and exogenous NO (direct action on guanylate cyclase) were not changed in the OZR.

Other investigators who have employed the OZR have demonstrated reduced endothelium-dependent vasodilator responses to agonists such as ACh in cremaster muscle arterioles, 26 mesenteric arterioles, 7 and the aorta. 27 In contrast, another study employing the OZR model found that endothelium-dependent relaxations in the intestinal microvessels and aorta were not impaired 28 or were increased. 29,30 Furthermore, in perfused hindquarter preparations, agonist-stimulated endothelial vasodilator hyperreactivity has been demonstrated in the OZR, 9 whereas the function of the carotid artery from the OZR was unaltered. 7 In the male insulin-resistant JCR:LA-cp rat, ACh-induced endothelium-dependent vasodilations were impaired in the mesenteric vascular bed 24 and aorta. 31 However, the A23187 response was not different in the aorta from the JCR:LA-cp rat compared with the control. 31 In the diabetic fatty Zucker rat, an animal model for type 2 diabetes, endothelium-dependent vasodilation in the intestinal vascular bed has been reported as preserved in one study 28 but impaired in another. 32 Apart from differences in the animal model and duration of metabolic defects/diabetes, it is possible that the disparities between studies may be explained by the basilar artery being more sensitive to damages caused by insulin resistance and possibly type 2 diabetes mellitus.

The mechanism of endothelial dysfunction in basilar artery in the OZR is not clear. In addition to uncoupling of receptor-mediated signal transduction, 33 other mechanisms may also be involved because the response to the receptor-independent vasodilator A23187 was reduced in the OZR. An abnormality at the level of the G-proteins has been suggested in previous studies in isolated vessels from patients with type 1 diabetes, 34 in the forearm circulation of type 2 diabetic patients, 35 and in mesenteric and hindlimb arteries of STZ-treated diabetic rats. 36,37 Conversely, endothelial dysfunction may be caused by impaired NO production as a result of a deficiency of the NO synthase substrate l-arginine, 33 a decreased availability of one or more cofactors essential for NO synthase function, such as tetrahydrobiopterin, 38,39 or even oxidative stress. 40,41

Because NAME abolished or greatly reduced the responses to the endothelium-dependent vasodilators, the major contributor mediating these responses is likely to be NO. This is further supported by the finding that l-arginine reversed the NAME-induced inhibition of histamine in both the LZR and OZR. Similar observations of l-arginine restoring endothelium-dependent vasodilations in diabetic models have been reported. 33 However, certain responses (eg, those to histamine) were not completely abolished in the presence of NAME, suggesting a contribution by other factors such as endothelium-derived hyperpolarizing factor or prostaglandin I2. Recently, it has been reported that the ACh-induced response in the basilar artery of STZ-induced diabetic rats was primarily related to NO, 42 although it is possible that the release of endothelium-derived hyperpolarizing factor as well as NO contributes to the response, but it is not likely to involve prostaglandin I2. 43

Interestingly, even though responses to the endothelium-independent relaxants papaverine and exogenous NO were not changed in the OZR compared with the LZR, relaxations to the NO donor SNP were decreased in the OZR. The reason for this discrepancy is not clear, but it may be related to the fact that the generation of NO from SNP involves certain cellular factors such as thiols and microsomal enzymes. 44,45 It may also spontaneously generate thiol-containing compounds. 46 It is possible that the metabolic pathway of NO generation from SNP may be affected in the OZR. Conversely, previous studies have demonstrated reduced smooth muscle reactivity to NO donors in cremaster arterioles from the OZR. 26 Furthermore, abnormal vascular smooth muscle cells have been reported in corpulent animals such as the JCR:LA-cp rat. 47 In addition, enhanced superoxide anion generation in vascular smooth muscle, which is common in diabetes as a result of oxidative stress, has been well demonstrated to markedly disrupt nitrovasodilator activity including basal NO-mediated endothelial function and to accentuate contractile reactivity. 48,49 Thus, an elevation in oxidative stress in the OZR could account for the reduced relaxation response to SNP as observed in the present study because the release of NO from SNP involves intracellular metabolism. However, the response to NO was not changed in the OZR, which implies that oxidative stress is not involved, although additional studies such as comparing the effects of the cell-impermeable superoxide dismutase versus cell-permeable polyethylene glycol on the effects of ACh would be required to clarify such an assumption. If indeed intracellular oxidative stress is responsible for the impaired response, polyethylene glycol but not superoxide dismutase should enhance relaxations.

The data from the endothelium-denuded basilar artery preparations indicate that the endothelium does not modulate the relaxation induced by SNP because these responses did not change in endothelium-denuded segments, whereas ACh-induced relaxations were almost completely abolished. Furthermore, the SNP-induced relaxation in the OZR remained reduced compared with the LZR basilar artery as in endothelium-intact preparations.

Immunoreactivity of eNOS has been demonstrated in perivascular axons and endothelial cells of basilar arteries in the rat. 50 We confirm the localization of this isoform in endothelium in the present study. Furthermore, eNOS has been reported to be important for maintaining cerebral blood flow and preventing neuronal injury. 51 The results from the Western blot and immunofluorescent staining studies found no changes of eNOS expression in the OZR, suggesting a disassociation of functional changes with the level of eNOS expression observed. The reason for this is not clear. One possibility may be that the bioavailability of NO rather than NO synthesis in the cerebral vasculature of the OZR may be reduced. Alternatively, the activity of eNOS may be impaired through a defect in a cofactor or substrate required for NO production, and hence the inability to detect any changes in NOS protein using the current methods. However, further research would be required to determine any differences in protein activity of the OZR. NOS levels have been reported to be decreased in the hypothalamus and fundus of the stomach in the OZR 52 even though an upregulation of NOS expression has also been shown in the hypothalamus of ob/ob type 2 diabetic mice. 53

CONCLUSION

The present study has demonstrated an impairment of NO function in the basilar artery from the 36-week OZR, which displayed metabolic and hemodynamic parameters consistent with the condition of insulin resistance and type 2 diabetes, compared with the age-matched LZR. Results also suggest that this impaired NO function cannot be accounted for by a decreased expression of eNOS, and further studies are required to clarify the mechanism of endothelial dysfunction in the OZR.

ACKNOWLEDGMENTS

The authors thank Winnie Lau for her valuable assistance in obtaining the metabolic and hemodynamic parameters. The Cell Biology of Diabetes Laboratory at the Baker Medical Research Institute in Victoria, Austarlia, was responsible for the measurement of the plasma levels of total cholesterol and triglycerides.

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Keywords:

basilar artery; endothelium; nitric oxide; obese Zucker rat

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