Tests for the role of the NO/cGMP pathway, the endothelium, and extracellularly released NO in L-cysNO- and D-cysNO-mediated vasodilation in isolated sheep femoral arteries (n=5). window Figure 3 Nitrite potentiates vasoactivity of SNOs in the femoral artery of anesthetized sheepA) After L-NAME (45 mgkg?1, iv) infusion and a stable baseline period, nitrite was infused for 15 min into the femoral artery. Then SNO was infused into femoral artery at rates increasing in a step-wise manner. B-D) Prior infusion of nitrite resulted in otherwise absent vasodilatory responses to GSNO (B) and D-cysNO (C) in the femoral vasculature, and augmented L-cysNO-mediated vasodilation (D). E) Vasodilatory responses to L-cysNO were attenuated in animals pre-treated with L-NAME. All y-axes depict normalized changes relative to an average of the femoral arterial conductance measured during the 20 seconds just prior to SNO infusion. Responses are averages from 3 to 9 sheep, with the number studied shown on each curve. p value for vs. L-NAME (two-way ANOVA). Role of nitrite on GSNO-mediated vasodilation in anesthetized rats Our previous observation that the sensitivity of sheep arteries to nitrite-mediated vasodilation is nearly 2 orders of magnitude less than that of rat arteries [17] raises the possibility of species-specific pathways of nitrite and SNO-signaling. We therefore further tested the interaction between SNO and nitrite in adult rats. Intraperitoneal injections of L-NAME for four days to lower nitrite levels resulted in a decrease in nitrite concentrations in the wall of the femoral arteries from 0.110.01 to 0.070.01 M/mg protein (p=0.0017). The plasma nitrite concentrations were also decreased by L-NAME (from 0.270.09 M Rabbit polyclonal to COFILIN.Cofilin is ubiquitously expressed in eukaryotic cells where it binds to Actin, thereby regulatingthe rapid cycling of Actin assembly and disassembly, essential for cellular viability. Cofilin 1, alsoknown as Cofilin, non-muscle isoform, is a low molecular weight protein that binds to filamentousF-Actin by bridging two longitudinally-associated Actin subunits, changing the F-Actin filamenttwist. This process is allowed by the dephosphorylation of Cofilin Ser 3 by factors like opsonizedzymosan. Cofilin 2, also known as Cofilin, muscle isoform, exists as two alternatively splicedisoforms. One isoform is known as CFL2a and is expressed in heart and skeletal muscle. The otherisoform is known as CFL2b and is expressed ubiquitously to 0.090.02 M) but were restored (to 0.370.06 M) by infusions of nitrite (Figure 4E). In contrast to sheep that were unresponsive to nitrite itself, femoral arterial conductance of the anesthetized rats increased significantly from baseline in response to nitrite infusion (p=0.01), although no significant difference was observed between saline controls and nitrite (p=0.22; Figure 4B). Similar to sheep, the vasodilatory effects of GSNO were absent in rats pretreated with L-NAME, but were restored by infusions of nitrite (Figure 4D). The increased vasodilatory response to GSNO following pretreatment with L-NAME+nitrite could not be explained by a greater baseline vascular tone against which the GSNO could act GLPG2451 because the baseline arterial conductances in these animals were already greater than those of controls and greater than those animals treated only with L-NAME (Figure 4C). These results are all consistent with the idea that GSNO-mediated vasodilation involves signaling pathways that are at least partially facilitated by the presence of nitrite. Open in a separate window Figure 4 Effect of L-NAME and nitrite on femoral conductance responses to GSNO in ratsA) Rats were given L-NAME for 4 days (60 mgkg?1day?1, i.p.) to block endogenous NOSs activity and thereby lower plasma nitrite levels. Animals that received nitrite infusions were then compared to those GLPG2451 that received no nitrite. Femoral conductance was then recorded while increasing doses of GSNO were infused into the lower abdominal aorta. B) Saline infusion did not increase the femoral arterial conductance (p=0.12), whereas nitrite did (p=0.01), although no significant difference was observed between saline and nitrite (p=0.22). C) L-NAME pretreatment decreased baseline femoral arterial conductance compared to controls (no L-NAME or nitrite), an effect that was reversed by treatment with nitrite. D) Control animals responded to GSNO infusions with increases in femoral vascular conductance. This effect was lost in animals treated with L-NAME to lower nitrite levels, and then restored in L-NAME-treated animals that were also given exogenous nitrite to replenish plasma levels. E) L-NAME pretreatment decreased baseline plasma nitrite concentrations, whereas nitrite infusions returned it to control levels. Average results from 5 or more animals; *P 0.05, **P 0.01, ***P 0.001. GSNO stimulates higher cGMP levels in the presence of nitrite To further test the hypothesis that nitrite enhances signaling of GSNO through the sGC pathway, we measured the effects of GLPG2451 nitrite on cGMP levels in sheep femoral arteries after exposure to GSNO (Figure 5A). GSNO stimulated greater increases in intracellular cGMP concentrations when nitrite was present (Figure 5B), consistent with the idea that the synergistic effects of GSNO and nitrite involve cGMP-mediated signaling. Open in a separate window Figure 5 Effect of nitrite pretreatment on GSNO-induced cGMP levels in isolated arteriesA) Samples of sheep femoral arteries were incubated with 5 M GSNO for three 15 min periods, similar to the treatment that caused tachyphylaxis in Figure 2..