Enalapril improves impairment of SERCA-derived relaxation and enhancement of tyrosine nitration in diabetic rat aorta

European Journal of Pharmacology 556 (2007) 121 – 128 www.elsevier.com/locate/ejphar

Enalapril improves impairment of SERCA-derived relaxation and enhancement of tyrosine nitration in diabetic rat aorta Kumiko Taguchi, Tsuneo Kobayashi, Yuko Hayashi, Takayuki Matsumoto, Katsuo Kamata ⁎ Department of Physiology and Morphology, Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan Received 3 July 2006; received in revised form 1 November 2006; accepted 6 November 2006 Available online 17 November 2006

Abstract We investigated the involvement of angiotensin II and vascular smooth muscle sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) function in the impaired NO-induced relaxation seen in established streptozotocin-induced diabetes. Plasma angiotensin II levels, which were elevated in untreated diabetic rats (vs age-matched controls), were improved by treatment with the angiotensin-converting enzyme inhibitor enalapril. Systolic blood pressure was significantly decreased in chronic enalapril-treated diabetics (vs the other two groups). Intact aortae from diabetic rats and chronic angiotensin II-infused control rats, but not those from diabetic rats treated with enalapril, showed impaired endothelium-dependent relaxations to acetylcholine (vs controls). The relaxation induced by Angeli's Salt (a NO donor) was significantly impaired in endotheliumdenuded aortae from diabetic rats (vs controls) but it was normalised by enalapril treatment. After preincubation with the irreversible SERCA inhibitor, thapsigargin, the relaxation induced by Angeli's Salt was significantly impaired in endothelium-denuded aortae from the controls, but not from the diabetics, and there was no significant difference between the thapsigargin-treated groups. Nitrotyrosine, an indirect marker of peroxynitrite, was markedly increased in aortic smooth muscle from diabetic rats, while chronic enalapril administration reduced this increase. These results suggest that in streptozotocin-induced diabetic rats, excessive angiotensin II production may lead to the generation of peroxynitrite and that this may in turn trigger a dysfunction of vascular smooth muscle SERCA. Enalapril improved the diabetes-related impairments. © 2006 Elsevier B.V. All rights reserved. Keywords: Angiotensin II; Diabetes; Peroxynitrite; Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA); Relaxation; Smooth muscle

1. Introduction Vascular disease is a complicating feature of diabetes mellitus in man. An accumulating body of evidence indicates that the relaxation responses of aortic strips to endotheliumdependent agents are weaker in streptozotocin-induced diabetic rats than in normal rats (Oyama et al., 1986; Pieper and Gross, 1988; Kamata et al., 1989). Although the mechanisms responsible for mediating the endothelial dysfunction have not been completely defined, we and others have suggested that a diabetes-related rapid inactivation of NO might be responsible (Cohen, 1995; Kamata and Kobayashi, 1996; De Vriese et al., 2000). Moreover, we have also reported (a) greater production of superoxide (O2−) and greater formation of nitrate (NO3−) (NO is metabolized by O2− to NO3−) in aortic rings from diabetic rats ⁎ Corresponding author. Tel./fax: +81 3 5498 5856. E-mail address: [email protected] (K. Kamata). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.11.026

(Kobayashi and Kamata, 2001) and (b) an enhanced expression of the mRNA for the p22phox subunit of NAD(P)H oxidase in the diabetic aorta (Kanie and Kamata, 2002). NO rapidly reacts with O2− to form ONOO−. Interestingly, ONOO− can modify tyrosine residues in various proteins to form nitrotyrosine and nitration of protein tyrosine residues can lead to damage that alters protein function and stability (Grow et al., 1996). Therefore, nitrotyrosine is a marker of oxidative stress (Ceriello, 2002, 2003), which is evoked in such diseases as human atherosclerosis, heart disease and diabetes (Beckman et al., 1994; Ara et al., 1998; MacMillan-Crow et al., 1998). Several studies have shown that the formation of nitrotyrosine and ONOO− is a factor in NO responsiveness and NO production in blood vessels (Beckman, 1996; Gorlach et al., 2000; Brodsky et al., 2004; Zou et al., 2004). Moreover in highcholesterol-fed rabbits, sarco/endoplasmic reticulum calcium ATPase function (SERCA) and cGMP-independent relaxation have been reported to be inhibited by nitrotyrosine formation

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(Adachi et al., 2001). However, there have as yet been no reports concerning such endothelial-dependent relaxation in diabetic rats. The role of the renin–angiotensin II system, particularly of angiotensin II, is of considerable interest in vascular physiology and pathology. Angiotensin II, acting through its AT1 receptor (AT1R), increases the generation of reactive oxygen species (ROS) within vascular smooth muscle cells, primarily through activation of membrane-bound NADPH/NADH oxidase (Griendling et al., 1994; Rajagopalan et al., 1996; Berry et al., 2000; Cifuentes et al., 2000; Wang et al., 2001; Mollnau et al., 2002; Sowers, 2002). Some reports have suggested that a raised vascular activity of angiotensin-converting enzyme may promote a progressive deterioration of the cardiovascular system in streptozotocin-induced diabetic rats (Crespo et al., 2003). We therefore speculated that both an elevated plasma angiotensin II level and an increased formation of ONOO− might be related to the impairment of endothelial function previously observed in diabetic rats (see above). Abnormal functioning of both vascular smooth muscle cells and endothelial cells has been implicated as one of the mechanisms underlying vascular complications in diabetes. Despite a growing understanding of the mechanisms by which it is released from the endothelium, the precise molecular mechanisms by which NO relaxes vascular smooth muscle are not fully understood. Previous studies have provided evidence that a major action of NO in both normal vascular smooth muscle and platelets is a reduction in intracellular free Ca2+ levels via cGMP-dependent and-independent mechanisms (Cohen et al., 1999; Weisbrod et al., 1998; Trepakova et al., 1999). Those authors also proposed that NO can activate SERCA by cGMP-independent means (Cohen et al., 1999). The aims of the present study were to investigate the effect of chronic treatment with enalapril on the SERCA-mediated impairment of relaxation seen in aortae from rats with established streptozotocin-induced diabetes. We also examined whether control and established diabetic rats might differ in their nitrotyrosine expression profiles. 2. Materials and methods 2.1. Reagents Streptozotocin, (−) noradrenaline hydrochloride and thapsigargin were purchased from Sigma Chemical Co. (St Louis, MO, USA). Angiotensin II and sodium nitroprusside were from Wako (Osaka, Japan), acetylcholine chloride from Daiichi Pharmaceuticals (Tokyo, Japan), Angeli's Salt from Cayman Chemical Co (Ann Arbor, MI, USA) and enalapril maleate from Banyu (Tokyo, Japan). All drugs were dissolved in saline, except where otherwise noted. All concentrations are expressed as the final molar concentration of the base in the organ bath. 2.2. Animals and experimental design Male Wistar rats (8 weeks old and 200–230 g body weight) received a single injection via the tail vein of streptozotocin

65 mg/kg dissolved in a citrate buffer. Age-matched control rats were injected with the buffer alone. Food and water were given ad libitum. At 10 weeks after the streptozotocin injection, these rats, like all the others, were killed by decapitation under ether anaesthesia. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University (which is accredited by the Ministry of Education, Culture, Sports, Science and Technology, Japan). 2.3. Chronic angiotensin II or enalapril administration For continuous stimulation with angiotensin II, some control rats (17 weeks old, n = 5) were treated with angiotensin II (288 μg/ kg/day) by way of an osmotic mini-pump (2ML2; Alzet, Palo Alto, CA, USA) for 1 week. Some streptozotocin-induced diabetic rats (n = 6) were chronically given enalapril (20 mg/kg/day, p.o.) for 2 weeks starting 8 weeks after the streptozotocin injection. 2.4. Measurements of plasma glucose, angiotensin II and blood pressure At 10 weeks after the administration of streptozotocin, plasma glucose was determined by the use of a commercially available enzyme kit (Wako Chemical Company, Osaka, Japan), as reported previously (Kobayashi et al., 2000; Matsumoto et al., 2003). Plasma angiotensin II was eluted with methanol using C-18 columns (Cayman Chemical, MI, USA) and measured using a commercially available angiotensin II enzyme-immunoassay kit (SPI-BIO, Massy Cedex, France) according to the manufacturer's instructions (Kobayashi et al., 2006). After a given rat had been in a constant-temperature box at 37 °C for a few minutes, its systolic blood pressure and heart rate were measured by the tail-cuff method using a bloodpressure analyser (BP-98A; Softron, Tokyo, Japan). 2.5. Measurement of isometric force Rats were anaesthetized with diethyl ether and euthanized by decapitation 10 weeks after treatment with streptozotocin or buffer. Half of the thoracic aorta was cut into helical strips (3 mm in width and 20 mm in length) for the relaxation studies. The other half was used for immunohistochemistry or Western blotting. Strips were then placed in a bath containing 10 ml modified Krebs–Henseleit solution (KHS; bubbled with 95% O2 plus 5% CO2, and kept at 37 °C), with one end of each strip being connected to a tissue holder and the other to a forcedisplacement transducer, as previously described (Kobayashi et al., 2000; Kanie and Kamata, 2002). For experiments on the relaxation induced by Angeli's Salt (see below), the endothelium was removed by rubbing the intimal surface with a cotton swab, successful removal being functionally confirmed by the absence of a relaxation to 10− 5 M acetylcholine. Previously, noradrenaline-induced contractility was found not to differ between control and untreated streptozotocin-diabetic rats. For all relaxation studies, the aortic strips were precontracted with an equieffective concentration of noradrenaline (5 × 10− 8–

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3 × 10− 7 M). This concentration produced 75–85% of the maximal response, each strip developing a tension of approximately ∼ 200 mg/mg tissue whether it was from an age-matched control or a diabetic rat. When the noradrenalineinduced contraction had reached a plateau level, acetylcholine (10− 9–10− 5 M), sodium nitroprusside (10− 10–10− 5 M) or Angeli's Salt (sodium trioxodinitrate, Na2N2O3; 10− 9–10− 5 M) was added in a cumulative manner. When the effects of thapsigargin (10− 5 M) on the response to Angeli's Salt were to be examined in the diabetic aorta, thapsigargin was added to the bath 1 h before the administration of noradrenaline. 2.6. Immunohistochemistry Some strips from aortas were embedded in O. C. T Compound (Sakura, Torrance, CA, USA). After a wash with this compound, slides were treated with 10 mmol/l citric acid and then microwave-heated (for 1 min) to recover antigenicity. Non-specific binding was blocked by treatment with a drop of normal horse serum in Block ace (Dainippon-Pharm., Osaka, Japan) for 20 min before incubation either with polyclonal antinitrotyrosine antibody (1:100; Chemicon, Temecula, CA, USA) or with anti-von Willebrand factor (vWF, 1:200; Sigma Chemical Co, St Louis, MO, USA) in Block ace overnight at 4 °C. Tissue sections were then incubated for 30 min at room temperature with a biotinylated anti-rabbit IgG (1:800) secondary antibody using a VECTASTAIN Universal ABCAP kit (Vector Laboratories, Burlingame, CA, USA). Alkaline Phosphatase Substrate Kit I (Vector Laboratories) was used to visualize positive immunoreactivity for nitrotyrosine or vWF. Sections of rat aorta embedded in Entellan new (Merck, Darmstadt, Germany) were imaged using a microscope. 2.7. Measurement of the expressions of nitrotyrosine by Western blotting Aortae (two pooled vessels) were homogenised in ice-cold lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100 and protease-inhibitor cocktail. Homogenates were centrifuged at 13,000 ×g for 5 min. The supernatant was sonicated at 4 °C and the proteins were solubilised in Laemmli's buffer containing mercaptoethanol. The protein concentration was determined by means of a BCA protein assay reagent kit (PIERCE, IL, USA). Samples (40 μg/ lane) were resolved by electrophoresis on 7.5% SDS-PAGE gels and transferred onto PDVF membranes. Briefly, after blocking

the residual protein sites on the membrane with Block ace (Dainippon-Pharm., Osaka, Japan), the membrane was incubated with anti-nitrotyrosine (1:1000) or β-actin (1:5000, Sigma) in blocking solution. Horseradish-peroxidase-conjugated, anti-rabbit antibody (Vector Laboratories) was used at a 1:4000 dilution in Tween PBS, followed by detection using SuperSignal (PIERCE). 2.8. Statistical analysis The contractile force developed by aortic strips from control and diabetic rats is expressed in milligrams of tension per milligram of tissue, the data being given as mean ± S. E. M. When appropriate, statistical differences were assessed using Dunnett's test for multiple comparisons after a one-way ANOVA (analysis of variance), a probability level indicated by P b 0.05 being regarded as significant. Statistical comparisons between concentration–response curves were assessed using a two-way ANOVA, with a Bonferroni correction being performed post hoc to correct for multiple comparisons; again, P b 0.05 was considered significant. 3. Results 3.1. Plasma glucose and systolic blood pressure

Control

Diabetic

Enalapril-treated diabetic

As shown in Table 1, plasma glucose levels were significantly elevated in the streptozotocin-induced diabetics, by comparison with the controls. This raised level was not affected by chronic enalapril administration. Systolic blood pressure was significantly lower in the enalapril-treated diabetics than in the untreated groups (Table 1).

156 ± 6.5 103.0 ± 2.9

556.0 ± 24.2 a 100.2 ± 1.9

507.6 ± 19.2 a 86.4 ± 4.4 b

3.2. Relaxation response to acetylcholine

Table 1 Plasma glucose levels and systolic blood pressure in age-matched control, STZinduced diabetic and enalapril-treated diabetic rats

Glucose (mg/l) Blood pressure (mm Hg)

Fig. 1. Concentration–response curves for acetylcholine-induced relaxations of intact aortic strips obtained from age-matched controls (n = 8), angiotensin IIinfused (288 μg/kg/day, via osmotic pump) control rats (n = 5), untreated diabetic rats (n = 8) and chronically enalapril-treated diabetic rats (n = 6). Ordinate shows relaxation of aortic strips as a percentage of the contraction induced by an equieffective concentration of noradrenaline (5 × 10− 8– 3 × 10− 7 M). Values are mean ± S. E. M. from 5 to 8 experiments. ⁎P b 0.05 controls vs angiotensin II-infused controls. #P b 0.05 controls vs diabetic. &P b 0.05 enalapril-treated diabetic vs diabetic.

Values are mean ± S. E. of 7 determinations. a P b 0.05 vs controls. b P b 0.05 vs diabetic.

The maximal tension developed in response to 1 μM noradrenaline did not differ significantly between the two

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Fig. 2. Plasma angiotensin II levels in age-matched controls, untreated diabetic rats and chronically enalapril-treated diabetic rats. Values are mean ± S. E. M. from 5 experiments. ⁎P b 0.05 vs controls. #P b 0.05 vs diabetic.

groups (243 ± 12 mg/mg in the control and 236 ± 16 mg/mg in the diabetics). When the NA (5 × 10− 8–3 × 10− 7 M)-induced contraction had reached a plateau, ACh (10− 9–10− 5 M) was added cumulatively. The results are summarized in Fig. 1. In aortic strips from age-matched control rats, acetylcholine (10− 9–10− 5 M) caused a concentration-dependent relaxation, with the maximum response at 10− 5 M. This relaxation was significantly weaker in aortic strips from induced diabetic rats. After chronic administration of angiotensin II to control rats for 1 week, the acetylcholine-induced relaxation was significantly impaired. Interestingly, aortic strips from streptozotocininduced diabetic rats chronically treated with enalapril relaxed in a normal way to acetylcholine.

Fig. 4. Concentration–response curves for sodium nitroprusside-induced relaxations of intact aortic strips obtained from age-matched controls (n = 4), untreated diabetic rats (n = 6) and chronically enalapril-treated diabetic rats (n = 6). Ordinate shows relaxation of aortic strips as a percentage of the contraction induced by an equieffective concentration of noradrenaline (5 × 10− 8–3 × 10− 7 M). Values are mean ± S. E. M. from 4 to 6 experiments.

tion-dependent relaxation. The relaxation response to Angeli's Salt, which generates nitroxyl anion and nitric oxide free radical (Ellis et al., 2001), was significantly impaired in the streptozotocin-induced diabetic rats (vs the controls) and this impaired response was restored to normal by chronic enalapril treatment (Fig. 3). By contrast, the aortic relaxation induced by sodium nitroprusside (10− 10–10− 5 M) did not differ significantly among the three groups (control vs diabetic, P = 0.289, control vs enalapril treatment, P = 0.678, diabetic vs enalapril treatment, P = 0.465) (Fig. 4). The relaxation response induced

3.3. Plasma angiotensin II Plasma angiotensin II levels were significantly higher in the streptozotocin-induced diabetic rats than in the controls, and treatment with enalapril in diabetic rats lowered the angiotensin II levels to those of the controls (Fig. 2). 3.4. Relaxation responses to Angeli's Salt and sodium nitroprusside When the induced contraction had reached a plateau in endothelium-denuded aortic strips, Angeli's Salt (10 − 9 – 10− 5 M) was added cumulatively and induced a concentra-

Fig. 3. Concentration–response curves for relaxations induced by Angeli's Salt in endothelium-denuded aortic strips obtained from age-matched controls (n=4), untreated diabetic rats (n=6) and chronically enalapril-treated diabetic rats (n=6). Ordinate shows relaxation of aortic strips as a percentage of the contraction induced by an equieffective concentration of noradrenaline (5×10− 8–3×10− 7 M). Values are mean±S. E. M. from 4 to 6 experiments. ⁎Pb 0.05 vs controls. #Pb 0.05 vs diabetic.

Fig. 5. Effect of thapsigargin on relaxation induced by Angeli's Salt in endothelium-denuded aortae obtained from age-matched controls (A) and from diabetic rats and chronically enalapril-treated diabetic rats (B). Ordinate shows relaxation of aortic strips as a percentage of the contraction induced by an equieffective concentration of noradrenaline (5 × 10− 8–3 × 10− 7 M). Values are mean ± S. E. M. from four (control rats) to six (control + thapsigargin, diabetic, diabetic + thapsigargin, enalapril-treated diabetic) experiments. ⁎P b 0.05 vs controls. #P b 0.05 vs diabetic. &P b 0.05 vs enalapril-treated diabetic.

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by Angeli's Salt in the controls was significantly impaired following preincubation with thapsigargin, the irreversible SERCA inhibitor (Fig. 5A). By contrast, in streptozotocininduced diabetic rats this relaxation showed no change following preincubation with thapsigargin (Fig. 5B). There was no significant difference between the two thapsigargintreated groups [control (Fig. 5A) and diabetic (Fig. 5B)].

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Finally, we examined the expression of 3-nitrotyrosine protein by Western blot (Fig. 6B). The use of anti-nitrotyrosine antibody allowed detection of immunoreactive protein with a molecular weight of 30 kDa, as previously reported (Cai et al., 2005). The expression of such nitrotyrosine protein was significantly increased in aortae from streptozotocin-induced diabetic rats and this increase was completely normalised by chronic enalapril treatment (Fig. 6C).

3.5. Expression of nitrotyrosine protein 4. Discussion To investigate the possible mechanisms underlying the impairment of the relaxation to Angeli's Salt in streptozotocininduced diabetic rats and its normalisation by 2 weeks' enalapril treatment, we performed immunohistochemistry for nitrotyrosine, an indirect marker of peroxynitrite. We also performed immunohistochemical analysis of endothelial markers for vWF using aortae from control and diabetic rats. Positive staining for vWF was detected only in endothelial cells, not in smooth muscle cells (Fig. 6A). By comparison with the controls, streptozotocin-induced diabetic rats showed increased nitrotyrosine staining not only in the endothelium but also, notably, in the medial smooth muscle. In the enalapril-treated diabetic rats, staining for nitrotyrosine was almost absent from medial smooth muscle, and although staining was present in the endothelium, it was less intense (Fig. 6A).

Fig. 6. (A) Immunohistochemical staining for vWF and nitrotyrosine in sections of intact aortae from control, diabetic and enalapril-treated diabetic rats. Positive staining is shown as red. Magnification ×100. (B) Immunoblots showing protein expressions for β-actin and nitrotyrosine in control, diabetic, and enalapriltreated diabetic rat aortae. (C) Quantitative analysis of expressions by scanning densitometry (nitrotyrosine/β-actin). Values are mean ± S. E. M. from four (control rats) or six (diabetic, enalapril-treated diabetic) experiments. ⁎P b 0.05 vs controls. #P b 0.05 vs diabetic.

The main inferences we can draw from the present findings are that in rats with established streptozotocin-induced diabetes, an increase in angiotensin II leads to an enhanced aortic ONOO− generation and that this increment causes a dysfunction of vascular smooth muscle relaxation via an impairment of SERCA. Further, we found that enalapril normalised this impaired relaxation. This is an important finding concerning the action of this angiotensin-converting enzyme inhibitor in diabetes and the present results suggest that the angiotensin II-induced production of ONOO− may be involved in mediating diabetic complications. The endothelium-dependent relaxation to acetylcholine was weaker in the aortae from our diabetic rats than in those from the controls. This is consistent with several previous reports (Oyama et al., 1986; Pieper and Gross, 1988; Kamata et al., 1989; Kobayashi et al., 2000). We found here that in diabetic rats, the plasma angiotensin II level was considerably higher than that of the controls. Moreover, after 1 week's administration of angiotensin II to control rats, endothelium-dependent relaxation in the aortae was impaired. Thus, we suggest on the basis of the present study that the impaired vasorelaxation seen in the diabetic state may be, at least in part, due to a sustained elevation of angiotensin II production. Indeed, we found that chronic administration of the angiotensin-converting enzyme inhibitor enalapril improved the endothelial dysfunction seen in aortae from streptozotocin-induced diabetic rats, without affecting the plasma glucose level, suggesting that this improvement effect was due, not to increased glucose transport and metabolism, but to decreased production of angiotensin II. The relaxation induced by Angeli's Salt, but not that induced by sodium nitroprusside, was impaired in our diabetic rats, suggesting that these two drugs may generate different factors (such as NO) to induce their relaxation responses, at least in diabetes. We previously reported that cGMP-dependent, sodium nitroprusside-induced relaxation responses were similar between controls and diabetics (Kamata et al., 1989; Kobayashi et al., 2000, 2004; Kanie et al., 2003). It is possible, but uncertain at present, that sodium nitroprusside-induced relaxation is mediated by a NO-dependent pathway, whereas the relaxation to Angeli's Salt is mediated by a NO−-dependent pathway. Indeed, sodium nitroprusside donates both NO and NO−, and the resulting relaxation is mediated almost exclusively via soluble guanylate cyclase/cyclic GMP (Irvine et al., 2003). However, it has been reported that the NO− donated from Angeli's Salt is not oxidized to NO extracellularly and that NO−

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may also mediate vascular smooth muscle relaxation through a cGMP-independent mechanism (since the response was not abolished by the selective and specific soluble guanylate cyclase inhibitor ODQ) (Wanstall et al., 2001; Irvine et al., 2003). Various cGMP-independent mechanisms of action have previously been described for NO, including those involving SERCA (Wanstall et al., 2001). Hence, it is possible that the decrease in the relaxation response to Angeli's Salt seen in diabetes is not due to a decrease in the activity of soluble guanylate cyclase/cyclic GMP in vascular smooth muscle. Cohen et al. (1999) demonstrated that in the rabbit aorta, the smooth muscle relaxation induced by NO gas is partially mediated by a cGMP-independent mechanism. These findings suggest that sodium nitroprusside-induced relaxation involves a cGMP-dependent pathway, whereas the relaxation response to Angeli's Salt is mediated by a cGMP-independent mechanism. In addition, levels of sodium nitroprusside-stimulated cGMP in the diabetic-rabbit aorta can be normal (Shukla et al., 2004). Moreover, NO− can activate cGMP-independent signaling pathways. This may be why the response to Angeli's Salt, but not that to sodium nitroprusside, is impaired in diabetes. Interestingly, chronic treatment with the angiotensin-converting enzyme inhibitor enalapril prevented the impairment of cGMPindependent relaxation observed in untreated streptozotocininduced diabetic rats. The present study clearly indicates that the cGMP-independent smooth muscle dysfunction associated with diabetes may be reversed by chronic administration of enalapril and thus that angiotensin II may impair the relevant cGMP-independent mechanism(s). Calcium uptake into the intracellular store sites by the activation of SERCA is normally involved in mediating the decrease in intracellular Ca2+ and relaxation of the aorta induced by NO (i.e. this response is partially mediated by cGMP-independent mechanisms) (Weisbrod et al., 1998). In our study, the irreversible SERCA inhibitor thapsigargin reduced the relaxation induced by Angeli's Salt in the controls. In line with our results, Adachi et al. (2001) reported that the sodium nitroprusside-induced relaxation was not affected by the SERCA inhibitor cyclopiazonic acid (CPA). Because thapsigargin's action is similar to that of CPA, the sodium nitroprusside-induced cGMP-dependent relaxation may not involve SERCA. Thus, the differences between the relaxation responses induced by Angeli's Salt and sodium nitroprusside in the diabetic state may be related to a diabetes-related impairment in SERCA function. In fact, the published data showing that the Ca2+-uptake activity mediated by SERCA2a and the ryanodine receptor (this receptor releases calcium ions from the internal sarcoplasmic reticulum) altered in diabetes are compatible with this hypothesis (Bidasee et al., 2003; Ligeti et al., 2006). The molecular mechanisms by which NO increases Ca2+ uptake by SERCA are not fully understood. Possibly, SERCA may be regulated by protein kinase G phosphorylating phospholamban, providing a cGMP-dependent mechanism to increase its activity (Cornwell et al., 1991). SERCA has also been shown to be Snitrosylated, providing a potential cGMP-independent mechanism of regulation by NO (Viner et al., 2000). This may explain why we found that the relaxations induced by Angeli's Salt and

sodium nitroprusside differ substantially in their dependence on both SERCA and cGMP. The relaxation caused by Angeli's Salt was attenuated by thapsigargin but not by ODQ. In contrast, the relaxation induced by sodium nitroprusside was unaffected by blocking SERCA (Cohen et al., 1999) but attenuated by ODQ (Wanstall et al., 2001). Enalapril normalised the impaired aortic relaxation response to Angeli's Salt, suggesting that the increased plasma angiotensin II level in diabetes may impair the cGMPindependent mechanisms via SERCA. This relaxation response was markedly inhibited by thapsigargin in the controls but thapsigargin did not further inhibit it in our diabetics. Chronic treatment with enalapril significantly improved this relaxation in the diabetic aorta. Thus, it is likely that the increased angiotensin II level found in diabetes impairs SERCA function and that this impairment leads to a reduction in the cGMPindependent relaxation induced by SERCA. This would explain the improvement in this cGMP-independent relaxation. Detection of nitrotyrosine in the plasma obtained from diabetes patients has been interpreted as indicating ONOO− formation (Ceriello et al., 2001). It has been shown that in diabetes, enhancements of superoxide anion and the NO3−/NO2− ratio lead to an alteration in vascular reactivity (Kugiyama et al., 1990; Simon et al., 1990; Kobayashi and Kamata, 1999; Kobayashi et al., 2000; Lund et al., 2000; Hink et al., 2001; Kanie and Kamata, 2002; Kanie et al., 2003). It is possible that ONOO−, derived from NO and superoxide anion, plays an important role in mediating diabetic vascular complications. In the present study, increases in ONOO − in the diabetic endothelium and vascular smooth muscle were suggested by the marked increases in nitrotyrosine immunostaining and immunoblotting, effects that were dramatically decreased upon chronic enalapril administration. These results are consistent with enalapril's improvement effect on the relaxation induced by Angeli's Salt and suggest that relaxation responses mediated via SERCA are altered in diabetes, via increases in the plasma angiotensin II and ONOO−. Of particular interest in this connection, Adachi et al. (2001) reported that the marked increase in nitrotyrosine seen in hypercholesteraemic rabbits is dramatically decreased by the antioxidant t-butylhydroxytoluene (BHT), and they further found that ONOO− inhibited SERCA activity in the aortae from such animals. Since the plasma angiotensin II level was found to be considerably elevated in diabetes, and since this increases the level of superoxide anions within the blood vessels, the concentration of ONOO− (derived from NO and superoxide anion) may be increased. The ONOO− may in turn interact with protein, causing the nitrotyrosine concentration to be increased in diabetes. It is likely that nitrotyrosine formation occurs within SERCA. Indeed, it has been reported that ONOO− impairs the function of SERCA (Viner et al., 1996). Hence, we propose that enalapril decreases the formation of angiotensin II and that this decrease may lead to a decrease in the nitrotyrosine present within SERCA, thereby resulting in the improvement effects observed with enalapril in diabetes. In conclusion, we found that an elevation of plasma angiotensin II in the streptozotocin-induced diabetic rat caused

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a dysfunction of both endothelium and smooth muscle in the aorta. This mechanism for the attenuation of vascular relaxation in diabetes may involve increased ONOO− formation. Moreover, our data suggest that Angeli's Salt and sodium nitroprusside rely on different mechanisms to induce relaxation in the rat aorta: whereas reduced SERCA function may account for the impaired relaxation to Angeli's Salt (NO− donor) in diabetes, the relaxation to sodium nitroprusside remains normal because SERCA is not required for the cGMP-dependent relaxation induced by sodium nitroprusside. In fact, this enhancement of ONOO− may underlie the dysfunction of SERCA-mediated cGMP-independent relaxation seen in diabetic states. Furthermore, we suggest that enalapril may help prevent the diabetesrelated impairment of SERCA-mediated cGMP-independent relaxation in the aorta by a mechanism involving not only a control of angiotensin II generation but also a reduction in the increased ONOO− formation that occurs in diabetes. Acknowledgements This study was supported in part by the Ministry of Education, Science, Sports and Culture, Japan, by the Promotion and Mutual Aid Cooperation for Private Schools of Japan and by the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan. References Adachi, T., Matsui, R., Xu, S.Q., Kirber, M., Lazar, H.L., Sharov, V.S., Schoneich, C., Cohen, R.A., 2001. Antioxidant improves smooth muscle sarco/endoplasmic reticulum Ca2+-ATPase function and lowers tyrosine nitration in hypercholesterolemia and improves nitric oxide-induced relaxation. Circ. Res. 90, 1114–1121. Ara, J., Przedborski, S., Naini, A.B., Jackson-Lewis, V., Trifiletti, R.R., Horwitz, J., Ischiropoulos, H., 1998. Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NPTP). Proc. Natl. Acad. Sci. U. S. A. 95, 7659–7663. Beckman, J.S., 1996. Oxidative damage and tyrosine nitration from peroxynitrite. Chem. Res. Toxicol. 9, 836–884. Beckman, J.S., Ye, Y.Z., Anderson, P.G., Chen, J., Accaviti, M.A., Tarpey, M.M., White, C.R., 1994. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol. Chem. Hoppe-Seyler 375, 81–88. Berry, C., Hamilton, C.A., Brosnan, M.J., Magill, F.G., Berg, G.A., McMurray, J.J., Dominiczak, A.F., 2000. Investigations into the sources of superoxide in human blood vessels: angiotensin II increases superoxide production in human internal mammary arteries. Circulation 101, 206–2212. Bidasee, K.R., Nallani, K., Henry, B., Dincer, U.D., Besch Jr., H.R., 2003. Chronic diabetes alters function and expression of ryanodine receptor calcium-release channels in rat hearts. Mol. Cell. Biochem. 249, 113–123. Brodsky, S.V., Gcalekman, O., Chen, J., Zhang, F., Togashi, N., Crabtree, M., Gross, S.S., Nasjletti, A., Goligorsky, M.S., 2004. Prevention and reversal of premature endothelial cell senescence and vasculopathy in obesity-induced diabetes by ebselen. Circ. Res. 94, 377–384. Cai, L., Wang, J., Li, Y., Sun, X., Wang, L., Zhou, Z., Kang, Y.J., 2005. Inhibition of superoxide generation and associated nitrosative damage is involved in metallothionein prevention of diabetic cardiomyopathy. Diabetes 54, 1829–1837. Ceriello, A., 2002. Nitrotyrosine: new findings as a marker of postprandial oxidative stress. Int. J. Pract. Suppl. 129, 51–58. Ceriello, A., 2003. New insights on oxidative stress and diabetic complications may lead to a “causal” antioxidant therapy. Diabetes Care 26, 1589–1596.

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