Nitric oxide donor compound induces relaxation in mesenteric resistance artery from normotensive and hypertensive 2K-1C rats

FA Andrade1, CBA Restini2, RS Silva3, LM Bendhack3,1. 1University of São Paulo, Pharmacology - FMRP - 14049900, Brazil, 2University of Ribeirão Preto, Medicine - UNAERP - 14096-900, Brazil, 3University of São Paulo, Phisics and Chemistry - FCFRP - 14040-903, Brazil

Nitric oxide (NO) is a physiological modulator that plays an important role in the control of vascular tone and blood pressure. Many studies have highlighted the development of compounds that may serve as vehicle to NO release in biological systems, mainly when the endogenous NO production is impaired, such as in hypertension. We hypothesized that the NO donor compound cis-[Ru(bpy)2(py)(NO2)](PF6), RuBPY, is capable of inducing relaxation of isolated mesenteric resistance artery from normotensive (2K) and hypertensive (2K-1C) rats. The aim of the present work was to study the vasodilator effect of RuBPY in isolated mesenteric resistance artery from 2K and 2K-1C rats. All the procedures were approved by the Ethics Committee of the University of São Paulo (Protocol n°044/2008). To induce renovascular hypertension, male Wistar rats (180-200g) were anesthetized with tribromoethanol (2.5 mg/kg-1, intraperitoneal), and after a midline laparotomy a silver clip with an internal diameter of 0.20 mm was placed around the left renal artery. Normotensive rats (2K) were only submitted to laparotomy. Six weeks after surgery, the rats with systolic blood pressure higher than 160 mmHg (2K-1C) were killed by decapitation and the mesenteric arcade was removed for functional studies. Second or third-order mesenteric arteries with internal diameter between 200 and 300 µm were removed and cleaned of surrounding tissue in cold Krebs-Henseleit, mounted in a small vessel myograph chamber for isometric tension recordings and remained in oxygenated Krebs at 37°C and pH 7.4. Cumulative concentration-effect curves to the compound RuBPY (0.01 nmol/L-10 µmol/L) were constructed in isolated mesenteric resistance artery from 2K and 2K-1C rats, without vascular endothelium and pre-contracted with phenylephrine (10 µmol/L). The potency (pD2) and the maximum effect (Emax) were analyzed. To assess the cytosolic NO concentration ([NO]c) by confocal microscopy, mesenteric resistance arteries were loaded with the NO sensitive dye DAF-2DA (500 µmol/L) for 40 min, at room temperature. Acetylcholine, phenylephrine and tribromoethanol were diluted in deionized water, RuBPY solutions were prepared in pH 7.4 phosphate buffer (1 mmol/L) and DAF-2DA was prepared in Hanks physiological solution. Data were expressed as the mean ± SEM. The statistical significance was tested by Student’s t test, and values of P < 0.05 were considered significant. RuBPY induced concentration-dependent relaxation in mesenteric arteries from 2K (Emax: 90.5±5.2%, n=4, P<0.05) and 2K-1C rats (Emax: 94.1±2.7%, n=5, P>0.05). However, this response was less potent in mesenteric arteries from 2K-1C (pD2: 5.94±0.07, n=5) than 2K rats (pD2: 7.65±0.58, n=4, P<0.05). RuBPY increased the fluorescence intensity of DAF2-DA in mesenteric arteries from 2K and 2K-1C, which corresponds to an increase in the [NO]c. However, this response was greater in mesenteric arteries from 2K-1C than 2K rats (2K: 43.9±14.4%, n=3; 2K-1C: 134.2±15.82%, n=2, P<0.05). These findings suggest that compound RuBPY releases NO inside the vascular smooth muscle cell that promotes relaxation in mesenteric resistance artery from 2K and 2K-1C rats and this vasodilator effect is impaired in this model of hypertension. Financial Support: FAPESP and CNPq