Effects of Pioglitazone on Nitric Oxide Bioavailability Measured Using a Catheter-Type Nitric Oxide Sensor in Angiotensin II−Infusion Rabbit

117 Hypertens Res Vol.31 (2008) No.1 p.117-125 Original Article Effects of Pioglitazone on Nitric Oxide Bioavailability Measured Using a Catheter-Type Nitric Oxide Sensor in Angiotensin II–Infusion Rabbit Toshio IMANISHI1), Akio KUROI1), Hideyuki IKEJIMA1), Katsunobu KOBAYASHI1), Seiichi MOCHIZUKI2), Masami GOTO2), Kiyoshi YOSHIDA3), and Takashi AKASAKA1) Recently, peroxisome proliferator–activated receptor γ (PPARγ) ligands have been reported to increase nitric oxide (NO) bioavailability in vitro but not in vivo because of the difficulty of measuring plasma NO. Here, we investigated the effects of PPARγ on plasma NO concentrations using the newly developed NO sensor in angiotensin II (Ang II)–infused rabbits. Male New Zealand rabbits were randomized for infusion with Ang II, either alone or in combination with pioglitazone (a PPARγ agonist). Plasma NO concentration was measured using the catheter-type NO sensor placed in the aorta. We then infused N G-methyl-L-arginine (L-NMMA) and acetylcholine (ACh) into the aortic arch to measure the basal and ACh-induced plasma NO concentration. Vascular nitrotyrosine levels were examined by enzyme-linked immunoassay (ELISA). Both an immunohistochemical study and Western blotting were performed to examine the PPARγ and gp91phox expression. The cotreatment with pioglitazone significantly suppressed the negative effects of Ang II, that is, the decreases in basal and ACh-induced NO production and the increase in vascular nitrotyrosine levels. Both the immunohistochemical study and Western blotting demonstrated that pioglitazone treatment enhaced PPARγ expression and greatly inhibited Ang II–induced up-regulation of gp91phox. In conclusion, the PPARγ agonist pioglitazone significantly improved NO bioavailability in Ang II–infused rabbits, most likely by attenuating nitrosative stresses. (Hypertens Res 2008; 31: 117–125) Key Words: nitric oxide (NO), peroxisome proliferator–activated receptor agonist, angiotensin II, oxidative stress Introduction Endothelial dysfunction, characterized by impaired endothelial nitric oxide (NO) production is involved in the pathogenesis of atherosclerotic disease and is associated with risk factors for vascular disease, including hypercholesterolemia, hypertension, and diabetes mellitus. Endothelial dysfunction in response to long-term angiotensin II (Ang II) treatment has been shown to be secondary to increased superoxide production within the endothelium, the media, and/or the adventitial layer (1, 2). However, a limitation of these studies is that the release of NO from endothelium could only be inferred from comparisons of vessel relaxation. Therefore, direct in vivo measurements of intra-arterial NO concentration in blood would contribute to the detailed evaluation of endothelial function. It was previously thought that NO, once released from vas- From the 1)Department of Cardiovascular Medicine, Wakayama Medical University, Wakayama, Japan; and 2)Department of Medical Engineering and 3) Division of Cardiology, Kawasaki Medical School, Kurashiki, Japan. Address for Reprints: Toshio Imanishi, M.D., Ph.D., Department of Cardiovascular Medicine, Wakayama Medical University, 811–1, Kimiidera, Wakayama 641–8510, Japan. E-mail: Received May 23, 2007; Accepted in revised form July 25, 2007. 118 Hypertens Res Vol. 31, No. 1 (2008) cular endothelial cells into the bloodstream, is immediately oxidized or inactivated by dissolved oxygen, oxyhemoglobin and/or oxygen radical species (3, 4). However, growing experimental and clinical evidence suggests that NO remains active in the bloodstream, causing remote vasodilatory responses (5, 6). Several groups have developed high-temporal–resolution methods that use NO sensors for electrochemical measurement (7, 8). These sensors enable us to evaluate dynamic changes in NO concentration in solutions and tissues in response to agonists, NO-generating reagents and physical stimuli (9, 10). However, electrical interference vibration, poor durability of the sensor-tip coatings and other factors have made in vivo NO measurement very difficult. To overcome these drawbacks, a new NO sensor, which encloses both the working and reference electrodes within a highly gas-permeable and robust enclosure, has been developed (11– 13). In addition, we have developed a catheter-type NO sensor (12, 13). Using this sensor, we recently demonstrated that long-term Ang II treatment reduces plasma NO level in a concentration-dependent manner because of the increase in nitrosative stress (14). Peroxisome proliferator–activated receptors (PPARs) are transcription factors belonging to the nuclear superfamily. Emerging evidence indicates that the PPAR signaling pathway plays critical roles in the regulation of a variety of biological processes within the cardiovascular system (15). Treatment with PPARγ agonist improves endothelial function in patients with type 2 diabetes (16). However, it is not known whether this endothelial protective effect is secondary to improved glucose metabolism by the drug or whether PPARγ agonists exert direct endothelial protection. Until now, no in vivo data have been available on pioglitazone’s effect on plasma NO concentration in Ang II–infused rabbits. We used the catheter-type NO sensor to try to elucidate this effect. The present study demonstrates that cotreatment with pioglitazone reversed the Ang II–induced decrease in plasma NO concentration, accompanied by decreased nitrosative stress. Methods A Catheter-Type NO Sensor The integrated architecture and the performance of the catheter-type NO sensor have been described previously (12, 13). In brief, an Amino-700XL NO sensor (Innovative Instruments, Tempa, USA), 700 μm in diameter at the detection tip, was mounted in a 4-Fr catheter (1,200 mm long; Hirakawa Hewtech, Tokyo, Japan) and fixed with silicon adhesive. The tip was coated by soft polyurethane to prevent damage to the vessel wall, and two metal wires were also attached along the detection tip to provide the electrodes with mechanical support. The NO oxidative current was monitored using an NO monitor (model inNO-T, Innovative Instruments). Each sen- Table 1. Final Measures by Groups Group MAP, mmHg HR, bpm Body weight, kg Vehicle Pioglitazone Ang II Ang II+pioglitazone 70.6 ± 1.3 70.3 ± 1.2 84.5 ± 2.3* 73.1 ± 1.7# 170 ± 2 166 ± 3 184 ± 2* 172 ± 2# 2.41 ± 0.05 2.45 ± 0.06 2.39 ± 0.03* 2.42 ± 0.04# Data are the mean ± SEM. *p < 0.05 vs. vehicle (control). # p < 0.05 vs. Ang II alone. MAP, mean arterial pressure; HR, heart rate. sor was calibrated using NO-saturated pure water as previously described (11–13). Briefly, NO-saturated pure water was prepared by bubbling pure NO gas in oxygen-free pure water. Using a gas-tight syringe, 5 μL was injected into a welstirred saline solution (50 mL) in which the NO sensor was immersed (final NO concentration: 190 nmol/L) as previously described (11–13). Animal Preparation The animals were treat (...truncated)