The NO donor RuBPY does not induce tolerance in rat aorta, but it potentiates its response due to increased NO release in the vascular smooth muscle cells

TM Banin, MD Grando, JA Vercesi, RS da Silva, LM Bendhack. FCFRP/USP, Ribeirão Preto/ São Paulo, Brazil

The new NO donor RUBPY ([Ru(bpy)2(py)NO2](PF6)) synthesized by our research group seems promising because it contains in its molecule a nitrite group that can be converted to NO, that it is a potent vasodilator(1,2). Continuos exposition to organic nitrates leads to the development of tolerance characterized by the rapid loss of vasodilator effects(3,4). The tolerance process is a multifactorial process and involves increased production of vascular reactive oxygen species (ROS)(5), decreased activity of soluble guanylyl-cyclase (sGC)(6) and overexpression and activity of phosphodiesterases(7). The tolerance may be due to endothelial dysfunction. Therefore, we hypothesized that RuBPY would induce tolerance in intact endothelium (e+) rat aorta. This study aimed to investigate the effect of pre-exposure to RuBPY (EC100) on its vasodilator activity in e+ or denuded aorta (e-). Methods: Male rats (200-250g) were killed under anesthesia and the thoracic aorta was quickly cut into rings of 4 mm length that were connected to an isometric force transducer to measure the tension. The arterial rings were placed in an organ chamber with Krebs solution maintained at pH 7.4 and gassed with carbogen at 37°C. e+ and e- aortas were contracted with phenylephrine (EC50: 100 µM). After reaching a stable and maintained contraction, RuBPY (3 nM-5 µM) was cumulatively added to the organ bath. Experiments were conducted after 5, 10, 30, 45 min incubation (tolerance) with RuBPY (EC100:10 µM) followed by 20 min of wash-out, or in the absence of RuBPY (control). Endothelial cells (EC) and vascular smooth muscle cells (VSMC) were incubated with the fluorescent dye DAF-2/DA (10 µM for EC and 20 µM for VSMC) to quantify the cytosolic NO ([NO]c) and the intensity of fluorescence was detected in the absence or after exposure to RuBPY at different times (5-60min).Statistical significance was determined by using the Student’s t test or One-Way ANOVA (post hoc: Dunnett). In all cases, probability levels of less than 0.05 (P<0.05) were taken to indicate statistical significance. All pharmacological studies were performed in accordance with the Ethical Animal Committee of the University of São Paulo (2012.1.134.53.12). Results: The RuBPY induced concentration-dependent relaxation in e+ aortas (pD2:7.81±0.18; ME:101.6± 1.4%, n=7) and e- (pD2:7.54±0.13; ME:103.4±0.4%, n=6). The incubation with RuBPY for 5 min did not affect the relaxation induced by the compound in e+ and e- aortas. In fact, pre-exposure for 10 min of e- aortas potentiated the relaxation induced by RuBPY (pD2:7.37±0.12, n=5, P<0.05), which was amplified by 30 min pre-exposure, in an endothelium-independent way (pD2:7.41±0.21, n=8, P<0.05) and endothelium-dependent way after pre-exposure for 45 min (pD2:7.76±0.15, n=6, P<0.05). RuBPY did not release NO in the endothelial cells up to 60 min incubation. Interestingly, the NO release was increased in VSMC after 30, 45 and 60 min of exposure to the RuBPY, at the same time of exposure to RuBPY that the relaxation was potentiated. Our results demonstrate that the RuBPY does not induce tolerance in rat aorta, but it potentiates its own response probably due to increased NO release in the vascular smooth muscle cells. Supported by FAPESP and CNPq.

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