This work aimed to prepare and characterize formulations containing the RuBPY free and loaded in PNP prepared by nanoprecipitation and to verify if its formulation induces relaxation in isolated aorta from 2K‐1C and 2K rats. We have analyzed the maximum effect (ME) and potency (pD2). P values ¡Ü0.05 were significant. The average diameter, polydispersity index (pdI) and encapsulation efficiency of RuBPY were determined. Vascular reactivity experiments were performed on denuded aorta contracted with phenylephrine. The better PNP obtained presented 84.12nm average diameter and pdI 0.113. After RuBPY loaded PNP presented 104.9±1.86 nm and pdI 0.123±0.03. The developed PNP were able to encapsulate around 44.8% of RuBPY. Formulation containing RuBPY induced relaxation with similar ME and pD2 in 2K (ME:98.7±2.6%, pD2:6.32±0.098, n=3) and 2K‐1C rats (ME:101.1±0.4%, pD2:6.14 ±0.064, n=4). The pD2 was lower in RuBPY formulation than in RuBPY free, 2K (ME:112.7±34.6%, pD2:7.37±0.11, n=8) and 2K‐1C rats (ME:114.6±6.3%, pD2:7.19±0.12, n=5). RuBPY formulation is a potential vasodilator and this effect is similar in 2K and 2K‐1C rat aorta but it was less potent than RuBPY free. Supported by FAPESP and CNPq.

This work aimed to verify if RuBPY induces in vitro tolerance and cross‐tolerance with acetylcholine (ACh) in rat cava vein and to compare this effect with GTN. We have analyzed the maximum effect (ME) induced by GNT, RuBPY and ACh in concentration‐effect curves in veins contracted with endothelin‐1. P values ≤0.05 were considered significant. In vitro tolerance was induced by 60 min‐incubation with GTN (0.1mM or 4 μM) or Rubpy (0.1μM or 2 μM). In vitro cross‐tolerance for ACh was induced by incubating vein with GTN or Rubpy (4 μM) for 60 min. The protein expression of phosphorylated eNOS on Ser1177 and total eNOs was accessed by Western Blot. Our results demonstrate that after incubation with GTN (EC50,EC100) the ME was reduced from 75.3±2.2%, n=6 to 45.4±2.2%, n= 6 and to 39.2±1.4%, n= 6, respectively. Incubation with RuBPY (EC50,EC100) reduced the ME from 92.8±4.2%, n=7 to 48.0±2.3%, n=7 and 30.1±1.2%, n=7, respectively. GTN but not RuBPY, reduced ACh‐induced relaxation (from 100.3±5.3% to 75.1±4.2%, n=7). GTN, but not RuBPY induced eNOS phosphorylation. In conclusion, RuBPy and GTN induce tolerance in both EC50, EC100. Only GTN induces cross‐tolerance to ACh and increased eNOs phosphorylation. Supported by FAPESP and CNP

Organic nitrates such as nitroglycerin (GNT, glycerol trinitrate), are commonly used in clinical cardiovascular medicine in the acute treatment of angina and severe arterial hypertension. The development of tolerance due to the continuous treatment with high doses leads to a decrease of the magnitude of the vasodilatation induced by GNT. It is a major limitation for the chronic clinical use of GNT. The present study aimed to verify if the new nitrite donor cis‐[Ru(bpy)2(py)(NO2)](PF6) (Rubpy) induces in vitro tolerance in the rat cava vein and to compare this effect with GTN. We have analyzed the maximum relaxing effect (ME) and the potency (EC50) induced by GNT and Rubpy in concentration‐effect curves constructed in vein rings pre‐contracted with endothelin‐1. The experiments were performed on intact endothelial rings following pre‐incubation for 30 min with GTN (EC100: 0.1mmol/L or EC50: 4 μmol/L) or Rubpy (EC100: 0.1μmol/L or EC50: 2 μmol/L). After pre‐incubation with GTN (EC100), the ME was reduced (from 57.5±2.5%, n=4 to 28.1±0.8%, n=4, p<0.001). Pre‐incubation with the EC50 of GTN had no effect. Rubpy induced relaxation in a concentration‐dependent way, that has not been changed by pre‐incubation for 30 min with RuBPY in the EC50 or EC100 (78.6±2.8%, n=4). In conclusion, prior exposure with GTN reduces the relaxation induced by GTN whereas it does not occur with Rubpy. Supported by FAPESP/CNPq.

This study aimed to evaluate the effect of the inhibition of soluble guanylyl‐cyclase (sGC) by ODQ, K+ channels by TEA and ROS production by apocynin on the relaxation to the NO donor RuBPY in mesenteric resistance artery from normotensive (2K) and 2K‐1C rats. Concentration‐effect curves to RuBPY were constructed in isolated denuded mesenteric arteries from 2K and 2K‐1C rats contracted with phenylephrine, in the absence or in the presence of ODQ, TEA, or Apocynin. The maximum relaxation induced by RuBPY was similar in 2K (77.6±8.9%, n=7) and 2K‐1C (76.9±4.5%,n=13), that was inhibited by ODQ in 2K (36.5±2.9%,n=5) and 2K‐1C arteries (24.6±5.5%, n=5). The relaxation induced by RuBPY was reduced by TEA (50.5±10.6%, n =8) and apocynin (45.7±3.9%,n=4) only in 2K‐1C arteries. These results indicate that although RuBPY induces relaxation with similar efficacy in mesenteric arteries from normotensive and hypertensive rats and that the NO released from RuBPY activates sGC, the NO signaling can differ. It involves ROS production and K+ channels activation only in 2K‐1C arteries. Supported by FAPESP and CNPq.