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European Journal of Pharmacology 546 (2006) 134 – 141 www.elsevier.com/locate/ejphar
Impairment of the vascular relaxation and differential expression of caveolin-1 of the aorta of diabetic +db/+db mice Tze Yan Lam a,1 , Sai Wang Seto a,1 , Yee Man Lau a,1 , Lai Shan Au a,1 , Yiu Wa Kwan a,⁎, Sai Ming Ngai b , Kwong Wing Tsui c a Department of Pharmacology, The Chinese University of Hong Kong, Hong Kong SAR, PR China Department of Biology, Faculty of Science, The Chinese University of Hong Kong, Hong Kong SAR, PR China Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China b
c
Received 20 February 2006; received in revised form 19 June 2006; accepted 4 July 2006 Available online 13 July 2006
Abstract In this study, we compared the endothelium-dependent and -independent relaxation of the isolated thoracic aorta of control (+db/+m) and diabetic (+db/+db) (C57BL/KsJ) mice. The gene expression (mRNA and protein) level of the muscarinic M3 receptors, endothelial nitric oxide synthase (eNOS) and caveolin-1 of the aorta was also evaluated. Acetylcholine caused a concentration-dependent, NG-nitro-L-arginine methylester (20 μM)-sensitive relaxation, with ∼ 100% relaxation at 10 μM, in +db/+m mice. In +db/+db mice, the acetylcholine-induced relaxation was significantly smaller (maximum relaxation: ∼ 80%). The sodium nitroprusside-mediated relaxation was slightly diminished in +db/+db mice, compared to +db/+m mice. However, there was no significant difference in the isoprenaline- and cromakalim-induced relaxation observed in both species. The mRNA and protein expression levels of caveolin-1 were significantly higher in the aorta of +db/+db mice. In contrast, there was no difference in the mRNA and protein expression levels of eNOS and muscarinic M3 receptors between these mice. Our results demonstrate that the impairment of the acetylcholine-induced, endothelium-dependent aortic relaxation observed in +db/+db mice was probably associated with an enhanced expression of caveolin-1 mRNA and protein. © 2006 Elsevier B.V. All rights reserved. Keywords: Acetylcholine; Endothelium-dependent relaxation; Aorta; +db/+db mice; Caveolin-1
1. Introduction Patients with type 2 diabetic mellitus have a greater risk of cardiovascular diseases development than the non-diabetic individuals (Varughese et al., 2005; Ziegler, 2005). One of the complications of diabetes mellitus is vascular dysfunction, which is associated with an alteration of the vascular responsiveness to various vasoconstrictors and vasodilators. It has been shown that blood vessels obtained from diabetic animals exhibited an attenuated endothelium-dependent relaxation to acetylcholine (Oyama et al., 1986; Miyata et al., 1990) that may ⁎ Corresponding author. Room 409B, Basic Medical Sciences Building, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, PR China. Tel.: +852 2609 6884; fax: +852 2603 5139. E-mail address: [email protected] (Y.W. Kwan). 1 Contributed equally to the project. 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.07.003
be responsible for the diabetes-related hypertension. It has been documented that acetylcholine interacted with the endothelial muscarinic M3 receptors and caused nitric oxide (NO) release thereby leaded to vasodilatation (Ren et al., 1993; Obi et al., 1994). Numerous previous studies have focused on comparing the relaxation elicited by acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) in both normal and diabetic models with a common observation of an attenuation of the acetylcholine-mediated relaxation with no apparent change with the sodium nitroprusside-induced response (Karagiannis et al., 2003; Phillips et al., 2005). In addition, there are evidence indicated an increase in the phenylephrine-induced contraction of various blood vessel preparations in different diabetic models which is closely related to an impairment of the acetylcholine-induced relaxation (Zemel et al., 1991; Laight et al., 2000). The attenuated acetylcholinemediated vascular relaxation observed under the diseased
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conditions has been correlated to the generation of reactive oxygen species which reacted with and decreased the bioavailability of NO formed (Bitar et al., 2005, Hayashi et al., 2005). However, it is possible that other physiological mechanisms that are involved in the vasomotor tone development (adenylate cyclase, guanylate cyclase and various ion channels) (Chau et al., 2003) may be affected under the diseased conditions. Caveolin-1, an integral membrane protein of caveolae, is a key protein for modulating the NO bioavailability (GarciaCardena et al., 1997) and the available evidence has demonstrated that caveolin-1 inhibited the eNOS activity (GarciaCardena et al., 1996; Garcia-Cardena et al., 1997). In the caveolin-1 deficient mice (Drab et al., 2001), a dysfunction of the vascular system has been observed, and the isolated aortic rings failed to maintain a sustained contractile tone elicited by phenylephrine. In addition, the absence of the caveolin-1 gene leaded to an impairment of the NO signaling in the cardiovascular systems, causing aberrations of the endothelium-dependent relaxation (Drab et al., 2001; Hnasko and Lisanti, 2003). Since caveolae are important sites for the insulin signaling, caveolin-1 is a possible candidate disease gene involved in type 2 diabetes mellitus. Insulin-resistant C57BL/KsJ (diabetic, +db/ +db) mice were used in our study as these animals are commonly used models for obesity and non-insulin dependent diabetes mellitus (NIDDM) research (Mukherjee et al., 1997; Leckstrom et al., 1999). A high level of conservation exists between mouse and human genomes (Mural et al., 2002) making these mice appropriate animal models for human diseases. Hence, in this study, we examined and compared the vasorelaxing effect, and the underlying mechanism(s) involved, of agents that elicited blood vessels relaxation in +db/+m and +db/+db mice. 2. Materials and methods 2.1. Animals C57BL/KsJ mice (∼ 6 months old; female; non-diabetic control (+db/+m): 28.5 ± 2.3 g; diabetic (+db/+db): 58.2 ± 3.4 g were housed (relative humidity: 50–60%) under a 12:12-h light–dark cycle and were given the standard chow and water ad libitum before they were killed by cervical dislocation. The Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (HKSAR, PR of China) approved all experiments performed in this study (approval no: 04/054/MIS). The recommendations from the Declaration of Helsinki and the internationally accepted principles for the use of experimental animals were adhered to. Every effort was made to limit animal suffering and to limit the number of animals used in these experiments. 2.2. Measurement of blood glucose and plasma insulin levels Blood glucose level was determined using a One-Touch Blood Glucose Meter with a glucose test strip. After collecting the blood sample, serum was separated by centrifugation and stored at −20 °C until analysis. Serum insulin level was measured using an
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ELISA kit that is highly specific for mouse insulin (sensitivity: 39 pg/ml) (Crystal-chem, U.S.A.). 2.3. Tissue preparation Thoracic aorta (length: ∼12 mm; O.D.: ∼ 0.8 mm) were immediately dissected from the animals. Fat and connective tissues were carefully removed under a dissecting stereomicroscope (Leica, Germany). Care was taken not to touch the lumen of the aorta during dissection to ensure the endothelium intact. Four aortic rings (1 mm in length) were obtained from each preparation and only one ring was used for each drug treatment. 2.4. Isometric tension measurement Aortic ring was mounted in a 5-ml wire-myograph containing physiological salt solution (gassed with 16% O2/6% CO2 balanced with N2, pO2 ∼ 100 mmHg) of the following composition (mM): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11 and CaCl2 1.8 (pH 7.4, 37 °C) (Seto et al., 2006). To exclude the involvement of cyclo-oxygenase cascade, indomethacin (1 μM, a non-selective cyclo-oxygenase inhibitor) was included in the physiological salt solution throughout the experiments. The preparations were equilibrated under the resting tension of 6.0 ± 0.3 mN, as described previously (Okon et al., 2003), in the bath solution for 90 min. Resting tension was re-adjusted, if necessary, before commencing the experiments. Increasing concentrations of individual relaxant were administered at half-log increments at the plateau (i.e. the steady-state condition) of the previous response. The response at each concentration of drug added (expressed as final bath concentration) was measured using the MacLab Chart v 3.6 programme (AD Instruments, Australia). Relaxation in response to the individual relaxant was expressed as % of the phenylephrine (1 μM) (∼90% of maximum contractile response elicited by 1 μM phenylephrine observed in individual specie; n = 5 for each specie)-induced tone (Okon et al., 2003), and a 100% relaxation was considered when the active tone returned to the baseline level. In experiments in which the effect of different blockers/inhibitors was evaluated, the individual blocker was added into the organ bath for 45–60 min before the application of phenylephrine to induce the active tone. 2.5. Measurement of the mRNA expression Aorta were isolated from both +db/+m and +db/+db mice as described in the previous section. Total RNA was prepared using the RNeasy minikit (Qiagen, U.S.A.) according to the manufacturer's protocols. The integrity of RNA was checked by electrophoresis in a 1% agarose gel containing ethidium bromide, and the RNA concentration was measured spectrophotometrically. The cDNA was synthesized from total RNA isolated using an oligo (dT) primer and ThermoScript™ RT-PCR system (Invitrogen, U.S.A.). Briefly, 1 μg of total RNA was transcribed into cDNA in a final vol. of 20 μl reaction mixture containing 1 μg total RNA, 10 μl H2O and 1 μl thermoscript reverse transcriptase.
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After 60 min incubation at 55 °C, the reaction was terminated by incubation at 85 °C for 10 min. Finally, 1 μl Rnase H was added and the reaction was incubated at 37 °C for another 20 min. The reverse transcription products were used immediately in the polymerase chain reaction (PCR) experiments. Primers used for PCR were designed based on the sequences available in the GenBank database. The following primers were used: muscarinic M3 receptor: 5′-GGTGTGATGATTGGTCTGGCTTG-3′ (forward); 5′-GGAAGCAGAGTTTTCCAGGGAG-3′ (reverse); caveolin-1: 5′-CTACAAGCCCAACAACAAGGC-3′ (forward); 5′-AGGAAGCTCTTGATGCACGGT-3′ (reverse); eNOS: 5′GCTCCAGCCCCGGTACTACTC-3′ (forward); 5′-GTATGC GGCTTGTCACCTCCT-3′ (reverse); β-actin: 5′-CAACG GCTCCGGCATGTGCA-3′ (forward); 5′-CCGGCCAGCCA GGTCCAGAC-3′ (reverse). The PCR products obtained were examined on the 1% agarose gel containing ethidium bromide. 2.6. Western blots analysis Aorta were homogenized in the presence of protease inhibitors to obtain extracts of proteins. Protein concentrations were determined using BCA™ protein assay kit (Pierce, Rockford, USA). Samples (30 μg of protein per lane) were loaded onto a 12% SDS-polyacrylamide gel electrophoresis gel. After electrophoresis (180 V, 60 min), the separated proteins were transferred (12 mA, 90 min) to polyvinylidene difluoride membrane (PerkinElmer™, Life Science, Inc., Boston, USA). Nonspecific sites were blocked with 5% non-fat dry milk for 120 min, and the blots were then incubated with individual antibody type: anti-caveolin-1, 1:1000 (Santa Cruz Biotechnology, CA, USA); anti-eNOS antibody, 1:1000 (BD Biosciences, USA) and anti-muscarinic M3 antibody, 1:1000 (Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. Anti-rabbit HRP conjugated IgG, 1:1000 (DakoCytomation, Denmark); antimouse HRP conjugated IgG, 1:1000 (DakoCytomation, Denmark) and anti-goat HRP conjugated IgG 1:1000 (Santa Cruz Biotechnology, CA, USA) were used to detect binding of its corresponding antibody. Membranes were stripped and reblotted with the anti-β actin antibody, 1:10,000 (Sigma, USA) to verify an equal loading of protein in each lane. The binding of
Fig. 1. Cumulative concentration–response curves of acetylcholine (0.1 nM– 10 μM) in the phenylephrine pre-contracted isolated thoracic aorta (+db/+m mice (○), n = 8; +db/+db mice (□), n = 8), in the presence of atropine (1 μM) (+db/+m mice (●), n = 5; +db/+db mice ( ), n = 5) and L-NAME (20 μM) (+db/ +m mice (▾), n = 5; +db/+db mice (▴), n = 5), applied alone, are illustrated. Results are expressed as means ± S.E.M. ⁎P b 0.05 compared to the respective control mice (+db/+m mice, ○; +db/+db mice, □).
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Fig. 2. Cumulative concentration–response curves of sodium nitroprusside (0.1 nM–10 μM) in the phenylephrine pre-contracted isolated thoracic aorta in the absence (+db/+m mice (○), n = 8; +db/+db mice (▵), n = 8) and the presence of L-NAME (20 μM) (+db/+m mice (●), n = 5; +db/+db mice (▴), n = 5). Results are expressed as means ± S.E.M.
the specific antibody was visualized by exposing to the photographic film after treating with Western Lightning™ chemiluminescence reagents (PerkinElmer™, Life Science, Inc., Boston, USA). 2.7. Chemicals Physiological salts (GR grade) for preparing Krebs' solution for the isometric tension measurements were obtained from Merck (Darmstadt, Germany). Acetylcholine hydrochloride, isoprenaline hydrochloride, cromakalim, L-phenylephrine hydrochloride, indomethacin, atropine sulphate, neostigmine bromide, sodium nitroprusside and Nω-nitro-L-arginine methyl-ester (L-NAME) were obtained from Sigma-Aldrich Chemicals Co. (U.S.A.). 2.8. Statistical analysis Data are expressed as means ± S.E.M.; n refers to the number of mice from which thoracic aorta were taken for the isometric
Fig. 3. Cumulative concentration–response curves of isoprenaline (0.1 nM– 10 μM) (top panel) and cromakalim (0.1 nM–10 μM) (bottom panel) in the phenylephrine pre-contracted isolated thoracic aorta of +db/+m mice (○, n = 5 for each drug treatment) and +db/+db mice (●, n = 5 for each drug treatment). Results are expressed as means ± S.E.M.
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3.2. Comparison of the relaxation response in +db/+m and +db/+db mice
Fig. 4. Semi-quantitative RT-PCR analysis of the mRNA expression of the muscarinic M3 receptor (A), endothelial nitric oxide synthase (eNOS) (B), caveolin-1 (C), and β-actin (D) of the thoracic aorta of +db/+m (N) mice and +db/+db (O) mice. (E) Summary of the RT-PCR band intensities of the mRNA determined by the spectrophotometric analysis. Quantification of the mRNA expression of the muscarinic M3 receptor (M3), endothelial nitric oxide synthase (eNOS) and caveolin-1 is normalized to the β-actin mRNA level for comparison. Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. ⁎P b 0.001 compared to +db/+m mice (□).
After the phenylephrine (1 μM)-induced contraction reached a steady-state level, cumulative concentrations of acetylcholine (0.1 nM–10 μM) (in the presence of neostigmine 10 μM, to prevent the catabolism of the added acetylcholine) were added. In +db/+m mice, acetylcholine (0.1 nM–10 μM) caused a concentration-dependent relaxation (IC50 = 0.02 ± 0.05 μM), with ∼100% relaxation occurred at 10 μM. However, the acetylcholine-induced relaxation observed in +db/+db mice was significantly smaller than in +db/+m mice. There was a ∼0.5 log unit rightward shift of the concentration–response curve of acetylcholine (IC50 = 0.11 ± 0.06 μM) (+db/+m mice versus +db/ +db mice) with a reduced maximum relaxation response (∼80%) at 10 μM (n = 8) (Fig. 1). In both species, the acetylcholineinduced relaxation was abolished by atropine (10 μM), a nonselective muscarinic receptor blocker (n = 5) (Fig. 1). On the other hand, the acetylcholine-induced relaxation observed in +db/+m mice was greatly diminished by L-NAME (20 μM) (n = 5) (Fig. 1) whereas the relaxation observed in +db/+db mice was abolished (Fig. 1). Cumulative application of sodium nitroprusside (SNP) (0.1 nM–10 μM) caused a concentration-dependent, LNAME-resistant, relaxation in both strains of mice with a trend of a smaller magnitude of relaxation (SNP only at concentrations of 10, 30 and 100 nM) observed in +db/+db mice (IC50: +db/+m mice, 24.0 ± 10.1 nM versus +db/+db mice, 27.2 ± 9.8 nM) (n = 8) (Fig. 2). However, a similar magnitude of relaxation elicited by isoprenaline (0.1 nM–
tension measurements. Statistical comparisons between concentration–response curves were performed using a two-way analysis of variance (ANOVA), with Bonferroni's correction for multiple comparisons performed post hoc. Difference was considered to be statistically significant at P b 0.05. Statistical analysis for RT-PCR and Western blots experiments was performed using two-way ANOVA, and data are presented as means ± S.D. 3. Results 3.1. Blood glucose and serum insulin measurements In control (+db/+m) mice, the non-fasting blood glucose level was 168.5 ± 11.3 (mg/dl) (n = 8) whereas in diabetic (+db/+db) mice, it was 668 ± 10.7 (mg/dl) (n = 8) (P b 0.001). On the other hand, the plasma insulin level was 1.21 ± 0.13 (ng/dl) (n = 8) in +db/+m mice and the plasma insulin level was 5.67 ± 1.04 (ng/dl) in +db/+db mice (n = 8) (P b 0.001).
Fig. 5. (A) Summary of the eNOS protein expression of the thoracic aorta of +db/+m (control, □) and +db/+db (diabetic, ▥) mice (left panel). Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. Western blotting of the eNOS protein expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice (right panel). (B). Summary of the muscarinic M3 protein expression of the thoracic aorta of +db/ +m (control, □) and +db/+db (diabetic, ▥) mice (left panel). Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. Western blotting of the muscarinic M3 protein expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice (right panel).
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10 μM) (IC50: +db/+m mice, 0.68 ±0.19 μM versus +db/+db mice, 0.91 ± 0.22 μM) (n = 5) (Fig. 3) (top panel) and cromakalim (0.1 nM–10 μM) (IC50: +db/+m mice, 0.18 ± 0.12 μM versus +db/+db mice, 0.10 ± 0.08 μM) (n = 5) (Fig. 3) (bottom panel) was observed (endothelium-intact preparations) in both +db/+m and +db/+db mice. 3.3. Comparison of the mRNA and protein expression levels of the M3 receptor, eNOS and caveolin-1 The mRNA and protein expression levels of the muscarinic M3 receptor, eNOS and caveolin-1 were evaluated and compared between both strains of mice (Fig. 4). Our results revealed that there was no significant difference in the M3 receptor expression (mRNA and protein) (P N 0.05) (Figs. 4A and 5B) and the eNOS expression (mRNA and protein) (P N 0.05) (Figs. 4B and 5A) between two species. In contrast, the mRNA and protein expression levels of caveolin-1 were significantly higher in the aorta of +db/+db mice compared to +db/+m mice (caveolin-1 mRNA, P b 0.01) (n = 3) (Fig. 4E) (caveolin-1 protein, P b 0.05) (n = 3) (Fig. 6A and B). 4. Discussion Consistent with the previous report (Leiter et al., 1981), C57BL/KsJ (+db/+db) mice (female) used in our study exhibited typical obesity (body weight: 29 g versus 59 g), hyperglycaemia (non-fasting blood glucose: 168 mg/dl versus 668 mg/dl) and hyperinsulinaemia (plasma insulin: 1.2 ng/ml versus 5.6 ng/ml). In addition, female mice used in our study
Fig. 6. (A) Summary of the protein expression of caveolin-1 in the aorta of +db/ +m (control) and +db/+db (diabetic) mice. Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. ⁎P b 0.05 compared to +db/+m mice (□). (B) Western blotting analysis for caveolin-1 protein expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice. (C) Western blotting for β-actin expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice.
were relatively old (∼ 6 months old), and it has been documented that women at an older age (e.g. menopause) tend to have a higher incidence of obesity and the associated cardiovascular disorders. In our study, we demonstrated that acetylcholine elicited a concentration-dependent relaxation of the isolated thoracic aorta of both +db/+m and +db/+db mice. Our results are consistent with previous reports using the diabetic rats (Oyama et al., 1986; Matsumoto et al., 2004) in which the acetylcholine-induced relaxation (the nitric oxide/endothelium-dependent component) was smaller (over the same concentration range) in +db/+db mice than that observed in the age-matched +db/+m mice. The acetylcholine-induced relaxation observed in both species was sensitive to the L-NAME (a common nitric oxide (NO) synthase inhibitor) pre-treatment suggesting that the NO/endotheliumdependent component of the acetylcholine-mediated relaxation was modified under the diseased conditions e.g. hypertension, obesity and diabetes mellitus (Tominaga et al., 1994; Williams et al., 1996; Hogikyan et al., 1998; Higashi et al., 2001). An attenuation of NO released from the endothelium, under the diseased conditions, has been suggested as one of the important factors contributing to the reduced endothelium-dependent relaxation (Young and Leighton, 1998) and the development of hypertension (Tominaga et al., 1994). A reduction of the acetylcholine-elicited vascular relaxation observed under the diseased conditions has been suggested due to a decrease in the bioavailability of NO caused by the reactive oxygen species generated (Bitar et al., 2005; Hayashi et al., 2005). In addition, it is possible that the downstream signalling cascade(s) after NO released from the endothelial cells is malfunctioned under the diseased conditions that could account for the reduced relaxation observed. To explore this possibility, the vascular effect of sodium nitroprusside (SNP, a general NO donor) was evaluated. SNP caused a concentration-dependent relaxation in both species. It is important to point out that in previous studies using the diabetic rats (Oyama et al., 1986; Kanie et al., 2003), there was no apparent difference in the magnitude of the vascular relaxation elicited by SNP recorded under both normal and diseased conditions. However, in our study, there was a trend of a smaller relaxation response elicited by SNP (only at concentrations of 10, 30 and 100 nM) in +db/+db mice compared to +db/+m mice which was agreed with other, but not all, reports in which there was a decrease in the sensitivity to the NO-mediated response in the diabetic rats (Sandu et al., 2000; Frisbee and Stepp, 2001). In addition to the acetylcholine/NO release cascade, we have also explored whether other physiological relaxation mechanisms (e.g. β-adrenoceptor activation and ATP-sensitive K+ channels activation) were affected by diabetic conditions. Cumulative application of isoprenaline (a non-selective βadrenoceptor agonist) and cromakalim (an ATP-sensitive K+ channel opener) elicited a concentration-dependent relaxation of the phenylephrine pre-contracted thoracic aorta, as previously observed in the rat isolated aorta (Miyata et al., 1992; Trochu et al., 1999). In contrast to the diabetic patients in which the βadrenoceptor-mediated vasodilatation (elicited by isoprenaline) was impaired (Harada et al., 1999), in our study there was no apparent difference in the magnitude of relaxation elicited by
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isoprenaline (0.1 nM–10 μM) between +db/+m and +db/+db mice. It has been demonstrated that a reduced cromakalim-mediated relaxation was observed in the basilar artery of the diabetic rats (Erdos et al., 2004). However, in our study, cromakalim caused a similar magnitude of relaxation (in a concentration-dependent manner) in both +db/+m and +db/+db mice. These results are consistent with our previous study (Kwan et al., 1999) performed on the pulmonary artery of the spontaneously hypertensive rats (SHR) in which the diseased condition (i.e. hypertension) did not alter the relaxation in response to different ATP-sensitive K+ channel openers challenge. So far, our results indicated that only the acetylcholinemediated relaxation of the thoracic aorta was suppressed in +db/ +db mice. In this study, the acetylcholine-induced relaxation was abolished by atropine (a non-selective muscarinic (M) receptor antagonist) suggesting the involvement of the M receptors. It has been reported that in the mice coronary circulation (Lamping et al., 2004), rat pulmonary artery (Kwan et al., 1999), rat and mice thoracic aorta (Khurana et al., 2004), the M3 receptors of the endothelial cells are predominantly responsible for the acetylcholine-induced relaxation. Hence, it is possible that an alteration of the M3 receptors expression under the diabetic condition may contribute to the reduced relaxation observed. However, our results clearly indicated that there was no difference in the mRNA and protein expression of the M3 receptors of the thoracic aorta between +db/+m and +db/ +db mice. Therefore, the impaired relaxation in response to the acetylcholine challenge observed in +db/+db mice was probably not due to an alteration of the M3 receptor expression. Taken together, it is possible that other NO-downstream signalling pathways may be responsible for the attenuated acetylcholine-induced relaxation observed in +db/+db mice. It has been demonstrated that the amount of NO released from the endothelium is regulated by the gene expression as well as the activity of the endothelial NO synthase (eNOS) (Fleming and Busse, 1999; Fleming and Busse, 2003). Perhaps, the attenuated acetylcholine-induced relaxation observed in +db/+db mice was due to a change in the eNOS mRNA and/or protein expression. However, the eNOS expression (both mRNA and protein) was similar between these mice. Our results therefore argued about the possibility of a change in the eNOS expression that may account for the attenuated acetylcholine-induced relaxation observed in +db/+db mice. In addition to the eNOS gene expression, we have also evaluated other physiological factors that are important in the regulation of the eNOS activity (Massion and Balligand, 2003). It has been demonstrated that, the in vivo eNOS activity is negatively regulated by several proteins including caveolin-1 (Feron et al., 1999). Caveolin-1 is a component of caveolae in which caveolin-1 acts as a physiological inhibitor of the eNOS activity, and the eNOS is maintained in an inactive state via an interaction with caveolin-1 (Garcia-Cardena et al., 1996; Ju et al., 1997; Venema et al., 1997). In the caveolin-deficient mice, caveolin-1 plays a significant role in disease phenotypes such as atherosclerosis, cardiac hypertrophy, cardiomyopathy and pulmonary hypertension (Williams and Lisanti, 2004). In addition, the expression of the caveolin-1 protein
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increased significantly in the aorta of the Type I drug-induced nonobese diabetic mice (Bucci et al., 2004) and rats (Perreault et al., 2000). It is therefore proposed that a reduction of the NO release observed in the Type I diabetic mouse (Bucci et al., 2004) and rat (Perreault et al., 2000) could be due to an attenuation of the eNOS activity through the enhanced caveolin-1 expression. In our study, the RT-PCR and Western blots experiments have clearly demonstrated that the mRNA and protein expression of caveolin1 in the aorta of +db/+db mice was markedly elevated compared to that observed in +db/+m mice. Hence, it is tempting to suggest that a reduction of the acetylcholine-induced relaxation observed in +db/+db mice was probably due to an enhanced expression of caveolin-1, and therefore a decrease in the eNOS activity with a concomitant decrease in NO released from the endothelial cells upon the activation of the endothelial muscarinic M3 receptors caused by acetylcholine. In conclusion, our results demonstrate that, in +db/+db mice, only the acetylcholine-mediated relaxation (the NO/endothelium-dependent component) of the thoracic aorta was suppressed. Other physiological relaxation mechanisms e.g. the β-adrenoceptor agonist- and ATP-sensitive K+ channel opener-mediated relaxation were not modified by the diabetic conditions. Our novel results, for the first time, indicate that an attenuation of the acetylcholine-induced relaxation observed in +db/+db mice was probably associated with an increased caveolin-1 (mRNA and protein) expression. Acknowledgements Mr. SW Seto is a recipient of a post-graduate (Ph.D.) studentship of the Department of Pharmacology (The Chinese University of Hong Kong, Hong Kong SAR, PR of China). Provision of a post-graduate (M. Phil.) studentship to Miss TY Lam by The Chinese University of Hong Kong is acknowledged. Dr. YM Lau was supported by a post-doctoral fellowship of RTAO (The Chinese University of Hong Kong) and by the RGC Earmarked Grant of Hong Kong SAR, PR of China (Ref.: 4166/02M). Technical assistance provided by staff of Laboratory Animal Services Centre (The Chinese University of Hong Kong) is acknowledged. This project is financially supported by the RGC Earmarked Grants of Hong Kong SAR, PR of China (Ref.: 4107/01M and Ref.: 4166/02M). References Bitar, M.S., Wahid, S., Mustafa, S., Al-Saleh, E., Dhaunsi, G.S., Al-Mulla, F., 2005. Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes. Eur. J. Pharmacol. 511, 53–64. Bucci, M., Roviezzo, F., Brancaleone, V., Lin, M.I., Di Lorenzo, A., Cicala, C., Pinto, A., Sessa, W.C., Farneti, S., Fiorucci, S., Cirino, G., 2004. Diabetic mouse angiopathy is linked to progressive sympathetic receptor deletion coupled to an enhanced caveolin-1 expression. Arterioscler. Thromb. Vasc. Biol. 24, 721–726. Chau, W.H., Lee, W.H., Lau, W.H., Kwan, Y.W., Au, A.L., Raymond, K., 2003. Role of Na+/H+ exchanger in acetylcholine-mediated pulmonary artery contraction of spontaneously hypertensive rats. Eur. J. Pharmacol. 464, 177–187. Drab, M., Verkade, P., Elger, M., Kasper, M., Lohn, M., Lauterbach, B., Menne, J., Lindschau, C., Mende, F., Luft, F.C., Schedl, A., Haller, H., Kurzchalia,
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Impairment of the vascular relaxation and differential expression of caveolin-1 of the aorta of diabetic +db/+db mice Tze Yan Lam a,1 , Sai Wang Seto a,1 , Yee Man Lau a,1 , Lai Shan Au a,1 , Yiu Wa Kwan a,⁎, Sai Ming Ngai b , Kwong Wing Tsui c a Department of Pharmacology, The Chinese University of Hong Kong, Hong Kong SAR, PR China Department of Biology, Faculty of Science, The Chinese University of Hong Kong, Hong Kong SAR, PR China Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, PR China b
c
Received 20 February 2006; received in revised form 19 June 2006; accepted 4 July 2006 Available online 13 July 2006
Abstract In this study, we compared the endothelium-dependent and -independent relaxation of the isolated thoracic aorta of control (+db/+m) and diabetic (+db/+db) (C57BL/KsJ) mice. The gene expression (mRNA and protein) level of the muscarinic M3 receptors, endothelial nitric oxide synthase (eNOS) and caveolin-1 of the aorta was also evaluated. Acetylcholine caused a concentration-dependent, NG-nitro-L-arginine methylester (20 μM)-sensitive relaxation, with ∼ 100% relaxation at 10 μM, in +db/+m mice. In +db/+db mice, the acetylcholine-induced relaxation was significantly smaller (maximum relaxation: ∼ 80%). The sodium nitroprusside-mediated relaxation was slightly diminished in +db/+db mice, compared to +db/+m mice. However, there was no significant difference in the isoprenaline- and cromakalim-induced relaxation observed in both species. The mRNA and protein expression levels of caveolin-1 were significantly higher in the aorta of +db/+db mice. In contrast, there was no difference in the mRNA and protein expression levels of eNOS and muscarinic M3 receptors between these mice. Our results demonstrate that the impairment of the acetylcholine-induced, endothelium-dependent aortic relaxation observed in +db/+db mice was probably associated with an enhanced expression of caveolin-1 mRNA and protein. © 2006 Elsevier B.V. All rights reserved. Keywords: Acetylcholine; Endothelium-dependent relaxation; Aorta; +db/+db mice; Caveolin-1
1. Introduction Patients with type 2 diabetic mellitus have a greater risk of cardiovascular diseases development than the non-diabetic individuals (Varughese et al., 2005; Ziegler, 2005). One of the complications of diabetes mellitus is vascular dysfunction, which is associated with an alteration of the vascular responsiveness to various vasoconstrictors and vasodilators. It has been shown that blood vessels obtained from diabetic animals exhibited an attenuated endothelium-dependent relaxation to acetylcholine (Oyama et al., 1986; Miyata et al., 1990) that may ⁎ Corresponding author. Room 409B, Basic Medical Sciences Building, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, PR China. Tel.: +852 2609 6884; fax: +852 2603 5139. E-mail address: [email protected] (Y.W. Kwan). 1 Contributed equally to the project. 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.07.003
be responsible for the diabetes-related hypertension. It has been documented that acetylcholine interacted with the endothelial muscarinic M3 receptors and caused nitric oxide (NO) release thereby leaded to vasodilatation (Ren et al., 1993; Obi et al., 1994). Numerous previous studies have focused on comparing the relaxation elicited by acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) in both normal and diabetic models with a common observation of an attenuation of the acetylcholine-mediated relaxation with no apparent change with the sodium nitroprusside-induced response (Karagiannis et al., 2003; Phillips et al., 2005). In addition, there are evidence indicated an increase in the phenylephrine-induced contraction of various blood vessel preparations in different diabetic models which is closely related to an impairment of the acetylcholine-induced relaxation (Zemel et al., 1991; Laight et al., 2000). The attenuated acetylcholinemediated vascular relaxation observed under the diseased
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conditions has been correlated to the generation of reactive oxygen species which reacted with and decreased the bioavailability of NO formed (Bitar et al., 2005, Hayashi et al., 2005). However, it is possible that other physiological mechanisms that are involved in the vasomotor tone development (adenylate cyclase, guanylate cyclase and various ion channels) (Chau et al., 2003) may be affected under the diseased conditions. Caveolin-1, an integral membrane protein of caveolae, is a key protein for modulating the NO bioavailability (GarciaCardena et al., 1997) and the available evidence has demonstrated that caveolin-1 inhibited the eNOS activity (GarciaCardena et al., 1996; Garcia-Cardena et al., 1997). In the caveolin-1 deficient mice (Drab et al., 2001), a dysfunction of the vascular system has been observed, and the isolated aortic rings failed to maintain a sustained contractile tone elicited by phenylephrine. In addition, the absence of the caveolin-1 gene leaded to an impairment of the NO signaling in the cardiovascular systems, causing aberrations of the endothelium-dependent relaxation (Drab et al., 2001; Hnasko and Lisanti, 2003). Since caveolae are important sites for the insulin signaling, caveolin-1 is a possible candidate disease gene involved in type 2 diabetes mellitus. Insulin-resistant C57BL/KsJ (diabetic, +db/ +db) mice were used in our study as these animals are commonly used models for obesity and non-insulin dependent diabetes mellitus (NIDDM) research (Mukherjee et al., 1997; Leckstrom et al., 1999). A high level of conservation exists between mouse and human genomes (Mural et al., 2002) making these mice appropriate animal models for human diseases. Hence, in this study, we examined and compared the vasorelaxing effect, and the underlying mechanism(s) involved, of agents that elicited blood vessels relaxation in +db/+m and +db/+db mice. 2. Materials and methods 2.1. Animals C57BL/KsJ mice (∼ 6 months old; female; non-diabetic control (+db/+m): 28.5 ± 2.3 g; diabetic (+db/+db): 58.2 ± 3.4 g were housed (relative humidity: 50–60%) under a 12:12-h light–dark cycle and were given the standard chow and water ad libitum before they were killed by cervical dislocation. The Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (HKSAR, PR of China) approved all experiments performed in this study (approval no: 04/054/MIS). The recommendations from the Declaration of Helsinki and the internationally accepted principles for the use of experimental animals were adhered to. Every effort was made to limit animal suffering and to limit the number of animals used in these experiments. 2.2. Measurement of blood glucose and plasma insulin levels Blood glucose level was determined using a One-Touch Blood Glucose Meter with a glucose test strip. After collecting the blood sample, serum was separated by centrifugation and stored at −20 °C until analysis. Serum insulin level was measured using an
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ELISA kit that is highly specific for mouse insulin (sensitivity: 39 pg/ml) (Crystal-chem, U.S.A.). 2.3. Tissue preparation Thoracic aorta (length: ∼12 mm; O.D.: ∼ 0.8 mm) were immediately dissected from the animals. Fat and connective tissues were carefully removed under a dissecting stereomicroscope (Leica, Germany). Care was taken not to touch the lumen of the aorta during dissection to ensure the endothelium intact. Four aortic rings (1 mm in length) were obtained from each preparation and only one ring was used for each drug treatment. 2.4. Isometric tension measurement Aortic ring was mounted in a 5-ml wire-myograph containing physiological salt solution (gassed with 16% O2/6% CO2 balanced with N2, pO2 ∼ 100 mmHg) of the following composition (mM): NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11 and CaCl2 1.8 (pH 7.4, 37 °C) (Seto et al., 2006). To exclude the involvement of cyclo-oxygenase cascade, indomethacin (1 μM, a non-selective cyclo-oxygenase inhibitor) was included in the physiological salt solution throughout the experiments. The preparations were equilibrated under the resting tension of 6.0 ± 0.3 mN, as described previously (Okon et al., 2003), in the bath solution for 90 min. Resting tension was re-adjusted, if necessary, before commencing the experiments. Increasing concentrations of individual relaxant were administered at half-log increments at the plateau (i.e. the steady-state condition) of the previous response. The response at each concentration of drug added (expressed as final bath concentration) was measured using the MacLab Chart v 3.6 programme (AD Instruments, Australia). Relaxation in response to the individual relaxant was expressed as % of the phenylephrine (1 μM) (∼90% of maximum contractile response elicited by 1 μM phenylephrine observed in individual specie; n = 5 for each specie)-induced tone (Okon et al., 2003), and a 100% relaxation was considered when the active tone returned to the baseline level. In experiments in which the effect of different blockers/inhibitors was evaluated, the individual blocker was added into the organ bath for 45–60 min before the application of phenylephrine to induce the active tone. 2.5. Measurement of the mRNA expression Aorta were isolated from both +db/+m and +db/+db mice as described in the previous section. Total RNA was prepared using the RNeasy minikit (Qiagen, U.S.A.) according to the manufacturer's protocols. The integrity of RNA was checked by electrophoresis in a 1% agarose gel containing ethidium bromide, and the RNA concentration was measured spectrophotometrically. The cDNA was synthesized from total RNA isolated using an oligo (dT) primer and ThermoScript™ RT-PCR system (Invitrogen, U.S.A.). Briefly, 1 μg of total RNA was transcribed into cDNA in a final vol. of 20 μl reaction mixture containing 1 μg total RNA, 10 μl H2O and 1 μl thermoscript reverse transcriptase.
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After 60 min incubation at 55 °C, the reaction was terminated by incubation at 85 °C for 10 min. Finally, 1 μl Rnase H was added and the reaction was incubated at 37 °C for another 20 min. The reverse transcription products were used immediately in the polymerase chain reaction (PCR) experiments. Primers used for PCR were designed based on the sequences available in the GenBank database. The following primers were used: muscarinic M3 receptor: 5′-GGTGTGATGATTGGTCTGGCTTG-3′ (forward); 5′-GGAAGCAGAGTTTTCCAGGGAG-3′ (reverse); caveolin-1: 5′-CTACAAGCCCAACAACAAGGC-3′ (forward); 5′-AGGAAGCTCTTGATGCACGGT-3′ (reverse); eNOS: 5′GCTCCAGCCCCGGTACTACTC-3′ (forward); 5′-GTATGC GGCTTGTCACCTCCT-3′ (reverse); β-actin: 5′-CAACG GCTCCGGCATGTGCA-3′ (forward); 5′-CCGGCCAGCCA GGTCCAGAC-3′ (reverse). The PCR products obtained were examined on the 1% agarose gel containing ethidium bromide. 2.6. Western blots analysis Aorta were homogenized in the presence of protease inhibitors to obtain extracts of proteins. Protein concentrations were determined using BCA™ protein assay kit (Pierce, Rockford, USA). Samples (30 μg of protein per lane) were loaded onto a 12% SDS-polyacrylamide gel electrophoresis gel. After electrophoresis (180 V, 60 min), the separated proteins were transferred (12 mA, 90 min) to polyvinylidene difluoride membrane (PerkinElmer™, Life Science, Inc., Boston, USA). Nonspecific sites were blocked with 5% non-fat dry milk for 120 min, and the blots were then incubated with individual antibody type: anti-caveolin-1, 1:1000 (Santa Cruz Biotechnology, CA, USA); anti-eNOS antibody, 1:1000 (BD Biosciences, USA) and anti-muscarinic M3 antibody, 1:1000 (Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. Anti-rabbit HRP conjugated IgG, 1:1000 (DakoCytomation, Denmark); antimouse HRP conjugated IgG, 1:1000 (DakoCytomation, Denmark) and anti-goat HRP conjugated IgG 1:1000 (Santa Cruz Biotechnology, CA, USA) were used to detect binding of its corresponding antibody. Membranes were stripped and reblotted with the anti-β actin antibody, 1:10,000 (Sigma, USA) to verify an equal loading of protein in each lane. The binding of
Fig. 1. Cumulative concentration–response curves of acetylcholine (0.1 nM– 10 μM) in the phenylephrine pre-contracted isolated thoracic aorta (+db/+m mice (○), n = 8; +db/+db mice (□), n = 8), in the presence of atropine (1 μM) (+db/+m mice (●), n = 5; +db/+db mice ( ), n = 5) and L-NAME (20 μM) (+db/ +m mice (▾), n = 5; +db/+db mice (▴), n = 5), applied alone, are illustrated. Results are expressed as means ± S.E.M. ⁎P b 0.05 compared to the respective control mice (+db/+m mice, ○; +db/+db mice, □).
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Fig. 2. Cumulative concentration–response curves of sodium nitroprusside (0.1 nM–10 μM) in the phenylephrine pre-contracted isolated thoracic aorta in the absence (+db/+m mice (○), n = 8; +db/+db mice (▵), n = 8) and the presence of L-NAME (20 μM) (+db/+m mice (●), n = 5; +db/+db mice (▴), n = 5). Results are expressed as means ± S.E.M.
the specific antibody was visualized by exposing to the photographic film after treating with Western Lightning™ chemiluminescence reagents (PerkinElmer™, Life Science, Inc., Boston, USA). 2.7. Chemicals Physiological salts (GR grade) for preparing Krebs' solution for the isometric tension measurements were obtained from Merck (Darmstadt, Germany). Acetylcholine hydrochloride, isoprenaline hydrochloride, cromakalim, L-phenylephrine hydrochloride, indomethacin, atropine sulphate, neostigmine bromide, sodium nitroprusside and Nω-nitro-L-arginine methyl-ester (L-NAME) were obtained from Sigma-Aldrich Chemicals Co. (U.S.A.). 2.8. Statistical analysis Data are expressed as means ± S.E.M.; n refers to the number of mice from which thoracic aorta were taken for the isometric
Fig. 3. Cumulative concentration–response curves of isoprenaline (0.1 nM– 10 μM) (top panel) and cromakalim (0.1 nM–10 μM) (bottom panel) in the phenylephrine pre-contracted isolated thoracic aorta of +db/+m mice (○, n = 5 for each drug treatment) and +db/+db mice (●, n = 5 for each drug treatment). Results are expressed as means ± S.E.M.
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3.2. Comparison of the relaxation response in +db/+m and +db/+db mice
Fig. 4. Semi-quantitative RT-PCR analysis of the mRNA expression of the muscarinic M3 receptor (A), endothelial nitric oxide synthase (eNOS) (B), caveolin-1 (C), and β-actin (D) of the thoracic aorta of +db/+m (N) mice and +db/+db (O) mice. (E) Summary of the RT-PCR band intensities of the mRNA determined by the spectrophotometric analysis. Quantification of the mRNA expression of the muscarinic M3 receptor (M3), endothelial nitric oxide synthase (eNOS) and caveolin-1 is normalized to the β-actin mRNA level for comparison. Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. ⁎P b 0.001 compared to +db/+m mice (□).
After the phenylephrine (1 μM)-induced contraction reached a steady-state level, cumulative concentrations of acetylcholine (0.1 nM–10 μM) (in the presence of neostigmine 10 μM, to prevent the catabolism of the added acetylcholine) were added. In +db/+m mice, acetylcholine (0.1 nM–10 μM) caused a concentration-dependent relaxation (IC50 = 0.02 ± 0.05 μM), with ∼100% relaxation occurred at 10 μM. However, the acetylcholine-induced relaxation observed in +db/+db mice was significantly smaller than in +db/+m mice. There was a ∼0.5 log unit rightward shift of the concentration–response curve of acetylcholine (IC50 = 0.11 ± 0.06 μM) (+db/+m mice versus +db/ +db mice) with a reduced maximum relaxation response (∼80%) at 10 μM (n = 8) (Fig. 1). In both species, the acetylcholineinduced relaxation was abolished by atropine (10 μM), a nonselective muscarinic receptor blocker (n = 5) (Fig. 1). On the other hand, the acetylcholine-induced relaxation observed in +db/+m mice was greatly diminished by L-NAME (20 μM) (n = 5) (Fig. 1) whereas the relaxation observed in +db/+db mice was abolished (Fig. 1). Cumulative application of sodium nitroprusside (SNP) (0.1 nM–10 μM) caused a concentration-dependent, LNAME-resistant, relaxation in both strains of mice with a trend of a smaller magnitude of relaxation (SNP only at concentrations of 10, 30 and 100 nM) observed in +db/+db mice (IC50: +db/+m mice, 24.0 ± 10.1 nM versus +db/+db mice, 27.2 ± 9.8 nM) (n = 8) (Fig. 2). However, a similar magnitude of relaxation elicited by isoprenaline (0.1 nM–
tension measurements. Statistical comparisons between concentration–response curves were performed using a two-way analysis of variance (ANOVA), with Bonferroni's correction for multiple comparisons performed post hoc. Difference was considered to be statistically significant at P b 0.05. Statistical analysis for RT-PCR and Western blots experiments was performed using two-way ANOVA, and data are presented as means ± S.D. 3. Results 3.1. Blood glucose and serum insulin measurements In control (+db/+m) mice, the non-fasting blood glucose level was 168.5 ± 11.3 (mg/dl) (n = 8) whereas in diabetic (+db/+db) mice, it was 668 ± 10.7 (mg/dl) (n = 8) (P b 0.001). On the other hand, the plasma insulin level was 1.21 ± 0.13 (ng/dl) (n = 8) in +db/+m mice and the plasma insulin level was 5.67 ± 1.04 (ng/dl) in +db/+db mice (n = 8) (P b 0.001).
Fig. 5. (A) Summary of the eNOS protein expression of the thoracic aorta of +db/+m (control, □) and +db/+db (diabetic, ▥) mice (left panel). Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. Western blotting of the eNOS protein expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice (right panel). (B). Summary of the muscarinic M3 protein expression of the thoracic aorta of +db/ +m (control, □) and +db/+db (diabetic, ▥) mice (left panel). Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. Western blotting of the muscarinic M3 protein expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice (right panel).
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10 μM) (IC50: +db/+m mice, 0.68 ±0.19 μM versus +db/+db mice, 0.91 ± 0.22 μM) (n = 5) (Fig. 3) (top panel) and cromakalim (0.1 nM–10 μM) (IC50: +db/+m mice, 0.18 ± 0.12 μM versus +db/+db mice, 0.10 ± 0.08 μM) (n = 5) (Fig. 3) (bottom panel) was observed (endothelium-intact preparations) in both +db/+m and +db/+db mice. 3.3. Comparison of the mRNA and protein expression levels of the M3 receptor, eNOS and caveolin-1 The mRNA and protein expression levels of the muscarinic M3 receptor, eNOS and caveolin-1 were evaluated and compared between both strains of mice (Fig. 4). Our results revealed that there was no significant difference in the M3 receptor expression (mRNA and protein) (P N 0.05) (Figs. 4A and 5B) and the eNOS expression (mRNA and protein) (P N 0.05) (Figs. 4B and 5A) between two species. In contrast, the mRNA and protein expression levels of caveolin-1 were significantly higher in the aorta of +db/+db mice compared to +db/+m mice (caveolin-1 mRNA, P b 0.01) (n = 3) (Fig. 4E) (caveolin-1 protein, P b 0.05) (n = 3) (Fig. 6A and B). 4. Discussion Consistent with the previous report (Leiter et al., 1981), C57BL/KsJ (+db/+db) mice (female) used in our study exhibited typical obesity (body weight: 29 g versus 59 g), hyperglycaemia (non-fasting blood glucose: 168 mg/dl versus 668 mg/dl) and hyperinsulinaemia (plasma insulin: 1.2 ng/ml versus 5.6 ng/ml). In addition, female mice used in our study
Fig. 6. (A) Summary of the protein expression of caveolin-1 in the aorta of +db/ +m (control) and +db/+db (diabetic) mice. Results are expressed as means (arbitrary units, A.U.) ± S.D. of three independent experiments. ⁎P b 0.05 compared to +db/+m mice (□). (B) Western blotting analysis for caveolin-1 protein expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice. (C) Western blotting for β-actin expression of the thoracic aorta of +db/+m (control) and +db/+db (diabetic) mice.
were relatively old (∼ 6 months old), and it has been documented that women at an older age (e.g. menopause) tend to have a higher incidence of obesity and the associated cardiovascular disorders. In our study, we demonstrated that acetylcholine elicited a concentration-dependent relaxation of the isolated thoracic aorta of both +db/+m and +db/+db mice. Our results are consistent with previous reports using the diabetic rats (Oyama et al., 1986; Matsumoto et al., 2004) in which the acetylcholine-induced relaxation (the nitric oxide/endothelium-dependent component) was smaller (over the same concentration range) in +db/+db mice than that observed in the age-matched +db/+m mice. The acetylcholine-induced relaxation observed in both species was sensitive to the L-NAME (a common nitric oxide (NO) synthase inhibitor) pre-treatment suggesting that the NO/endotheliumdependent component of the acetylcholine-mediated relaxation was modified under the diseased conditions e.g. hypertension, obesity and diabetes mellitus (Tominaga et al., 1994; Williams et al., 1996; Hogikyan et al., 1998; Higashi et al., 2001). An attenuation of NO released from the endothelium, under the diseased conditions, has been suggested as one of the important factors contributing to the reduced endothelium-dependent relaxation (Young and Leighton, 1998) and the development of hypertension (Tominaga et al., 1994). A reduction of the acetylcholine-elicited vascular relaxation observed under the diseased conditions has been suggested due to a decrease in the bioavailability of NO caused by the reactive oxygen species generated (Bitar et al., 2005; Hayashi et al., 2005). In addition, it is possible that the downstream signalling cascade(s) after NO released from the endothelial cells is malfunctioned under the diseased conditions that could account for the reduced relaxation observed. To explore this possibility, the vascular effect of sodium nitroprusside (SNP, a general NO donor) was evaluated. SNP caused a concentration-dependent relaxation in both species. It is important to point out that in previous studies using the diabetic rats (Oyama et al., 1986; Kanie et al., 2003), there was no apparent difference in the magnitude of the vascular relaxation elicited by SNP recorded under both normal and diseased conditions. However, in our study, there was a trend of a smaller relaxation response elicited by SNP (only at concentrations of 10, 30 and 100 nM) in +db/+db mice compared to +db/+m mice which was agreed with other, but not all, reports in which there was a decrease in the sensitivity to the NO-mediated response in the diabetic rats (Sandu et al., 2000; Frisbee and Stepp, 2001). In addition to the acetylcholine/NO release cascade, we have also explored whether other physiological relaxation mechanisms (e.g. β-adrenoceptor activation and ATP-sensitive K+ channels activation) were affected by diabetic conditions. Cumulative application of isoprenaline (a non-selective βadrenoceptor agonist) and cromakalim (an ATP-sensitive K+ channel opener) elicited a concentration-dependent relaxation of the phenylephrine pre-contracted thoracic aorta, as previously observed in the rat isolated aorta (Miyata et al., 1992; Trochu et al., 1999). In contrast to the diabetic patients in which the βadrenoceptor-mediated vasodilatation (elicited by isoprenaline) was impaired (Harada et al., 1999), in our study there was no apparent difference in the magnitude of relaxation elicited by
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isoprenaline (0.1 nM–10 μM) between +db/+m and +db/+db mice. It has been demonstrated that a reduced cromakalim-mediated relaxation was observed in the basilar artery of the diabetic rats (Erdos et al., 2004). However, in our study, cromakalim caused a similar magnitude of relaxation (in a concentration-dependent manner) in both +db/+m and +db/+db mice. These results are consistent with our previous study (Kwan et al., 1999) performed on the pulmonary artery of the spontaneously hypertensive rats (SHR) in which the diseased condition (i.e. hypertension) did not alter the relaxation in response to different ATP-sensitive K+ channel openers challenge. So far, our results indicated that only the acetylcholinemediated relaxation of the thoracic aorta was suppressed in +db/ +db mice. In this study, the acetylcholine-induced relaxation was abolished by atropine (a non-selective muscarinic (M) receptor antagonist) suggesting the involvement of the M receptors. It has been reported that in the mice coronary circulation (Lamping et al., 2004), rat pulmonary artery (Kwan et al., 1999), rat and mice thoracic aorta (Khurana et al., 2004), the M3 receptors of the endothelial cells are predominantly responsible for the acetylcholine-induced relaxation. Hence, it is possible that an alteration of the M3 receptors expression under the diabetic condition may contribute to the reduced relaxation observed. However, our results clearly indicated that there was no difference in the mRNA and protein expression of the M3 receptors of the thoracic aorta between +db/+m and +db/ +db mice. Therefore, the impaired relaxation in response to the acetylcholine challenge observed in +db/+db mice was probably not due to an alteration of the M3 receptor expression. Taken together, it is possible that other NO-downstream signalling pathways may be responsible for the attenuated acetylcholine-induced relaxation observed in +db/+db mice. It has been demonstrated that the amount of NO released from the endothelium is regulated by the gene expression as well as the activity of the endothelial NO synthase (eNOS) (Fleming and Busse, 1999; Fleming and Busse, 2003). Perhaps, the attenuated acetylcholine-induced relaxation observed in +db/+db mice was due to a change in the eNOS mRNA and/or protein expression. However, the eNOS expression (both mRNA and protein) was similar between these mice. Our results therefore argued about the possibility of a change in the eNOS expression that may account for the attenuated acetylcholine-induced relaxation observed in +db/+db mice. In addition to the eNOS gene expression, we have also evaluated other physiological factors that are important in the regulation of the eNOS activity (Massion and Balligand, 2003). It has been demonstrated that, the in vivo eNOS activity is negatively regulated by several proteins including caveolin-1 (Feron et al., 1999). Caveolin-1 is a component of caveolae in which caveolin-1 acts as a physiological inhibitor of the eNOS activity, and the eNOS is maintained in an inactive state via an interaction with caveolin-1 (Garcia-Cardena et al., 1996; Ju et al., 1997; Venema et al., 1997). In the caveolin-deficient mice, caveolin-1 plays a significant role in disease phenotypes such as atherosclerosis, cardiac hypertrophy, cardiomyopathy and pulmonary hypertension (Williams and Lisanti, 2004). In addition, the expression of the caveolin-1 protein
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increased significantly in the aorta of the Type I drug-induced nonobese diabetic mice (Bucci et al., 2004) and rats (Perreault et al., 2000). It is therefore proposed that a reduction of the NO release observed in the Type I diabetic mouse (Bucci et al., 2004) and rat (Perreault et al., 2000) could be due to an attenuation of the eNOS activity through the enhanced caveolin-1 expression. In our study, the RT-PCR and Western blots experiments have clearly demonstrated that the mRNA and protein expression of caveolin1 in the aorta of +db/+db mice was markedly elevated compared to that observed in +db/+m mice. Hence, it is tempting to suggest that a reduction of the acetylcholine-induced relaxation observed in +db/+db mice was probably due to an enhanced expression of caveolin-1, and therefore a decrease in the eNOS activity with a concomitant decrease in NO released from the endothelial cells upon the activation of the endothelial muscarinic M3 receptors caused by acetylcholine. In conclusion, our results demonstrate that, in +db/+db mice, only the acetylcholine-mediated relaxation (the NO/endothelium-dependent component) of the thoracic aorta was suppressed. Other physiological relaxation mechanisms e.g. the β-adrenoceptor agonist- and ATP-sensitive K+ channel opener-mediated relaxation were not modified by the diabetic conditions. Our novel results, for the first time, indicate that an attenuation of the acetylcholine-induced relaxation observed in +db/+db mice was probably associated with an increased caveolin-1 (mRNA and protein) expression. Acknowledgements Mr. SW Seto is a recipient of a post-graduate (Ph.D.) studentship of the Department of Pharmacology (The Chinese University of Hong Kong, Hong Kong SAR, PR of China). Provision of a post-graduate (M. Phil.) studentship to Miss TY Lam by The Chinese University of Hong Kong is acknowledged. Dr. YM Lau was supported by a post-doctoral fellowship of RTAO (The Chinese University of Hong Kong) and by the RGC Earmarked Grant of Hong Kong SAR, PR of China (Ref.: 4166/02M). Technical assistance provided by staff of Laboratory Animal Services Centre (The Chinese University of Hong Kong) is acknowledged. This project is financially supported by the RGC Earmarked Grants of Hong Kong SAR, PR of China (Ref.: 4107/01M and Ref.: 4166/02M). References Bitar, M.S., Wahid, S., Mustafa, S., Al-Saleh, E., Dhaunsi, G.S., Al-Mulla, F., 2005. Nitric oxide dynamics and endothelial dysfunction in type II model of genetic diabetes. Eur. J. Pharmacol. 511, 53–64. Bucci, M., Roviezzo, F., Brancaleone, V., Lin, M.I., Di Lorenzo, A., Cicala, C., Pinto, A., Sessa, W.C., Farneti, S., Fiorucci, S., Cirino, G., 2004. Diabetic mouse angiopathy is linked to progressive sympathetic receptor deletion coupled to an enhanced caveolin-1 expression. Arterioscler. Thromb. Vasc. Biol. 24, 721–726. Chau, W.H., Lee, W.H., Lau, W.H., Kwan, Y.W., Au, A.L., Raymond, K., 2003. Role of Na+/H+ exchanger in acetylcholine-mediated pulmonary artery contraction of spontaneously hypertensive rats. Eur. J. Pharmacol. 464, 177–187. Drab, M., Verkade, P., Elger, M., Kasper, M., Lohn, M., Lauterbach, B., Menne, J., Lindschau, C., Mende, F., Luft, F.C., Schedl, A., Haller, H., Kurzchalia,
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