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. 2018 May 1:75:60-69.
doi: 10.1016/j.niox.2018.01.011. Epub 2018 Feb 8.

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols

Taiming Liu  1 , Meijuan Zhang  1 , Michael H Terry  2 , Hobe Schroeder  3 , Sean M Wilson  3 , Gordon G Power  3 , Qian Li  4 , Trent E Tipple  4 , Dan Borchardt  5 , Arlin B Blood  6
Affiliations

Nitrite potentiates the vasodilatory signaling of S-nitrosothiols

Taiming Liu et al. Nitric Oxide. .

Abstract

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 μM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC.

Keywords: Intracellular NO store; Nitrite; S-nitrosothiol; Vasodilation.

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Figures

Figure 1
Figure 1. Tests of previously proposed mechanisms of GSNO-mediated vasodilation
A) ODQ markedly attenuated vasodilatory effects of GSNO in isolated sheep mesenteric arteries, demonstrating an essential role for the sGC/cGMP pathway. GSNO-mediated vasodilation is not dependent on B) release of free NO outside the cell, because the impermeable NO-scavenger CPTIO had no effect, C) S-transnitrosation of extracellular thiols, as the thiol blocker HMBA had no effect, D) conversion to membrane permeable thionitrous acid (HSNO), as the H2S donor Na2S did not potentiate vasodilation and also the H2S synthase inhibitor PAG even significantly left-shifted the GSNO dose response curve (p=0.043), E) conversion to L-cysNO which can cross the membrane via L-type amino acid transporters (LAT), because the LAT blocker threonine (Thr) had no effect, or F) hydrolyzation by -glutamyl transpeptidase ( -GT) to L-cysNO-gly which can cross the membrane via dipeptide transporters (PEPT2), as the -GT blocker GSM had no effect. N=arterial rings from ≥ 5 animals.
Figure 2
Figure 2. Tachyphylaxis to repeated GSNO exposure, and potentiation by nitrite
A) Experimental protocol used to study changes in the GSNO dose-response curves of sheep mesenteric artery. Isolated arterial rings were exposed to three 15-minute incubations with 5 μM GSNO (GSNOpreTx) followed by constriction with KCl and then GSNO dose response curves. Comparisons were made to vessels that were not pre-exposed to GSNO (controls) or vessels pre-exposed to 5 μM NO (NOpreTx) instead of GSNO. To test for the effects of nitrite, some GSNOpreTx vessels were exposed to 1μM nitrite for 30 min (GSNOpreTx + nitrite) prior to the constriction. B) Vasodilatory responses to GSNO were diminished after pretreatment with GSNO but not pretreatment with NO. C) NO dose response curves are not affected by pretreatment with GSNO or NO, suggesting that the tachyphylaxis to GSNO-mediated vasodilation does not involve signaling downstream from sGC activation. D) Addition of 1 μM nitrite to control vessels potentiated GSNO-mediated vasodilation in vessels that were not pretreated with GSNO. E) Addition of 1 μM nitrite to GSNO pretreated vessels reversed attenuated vasodilatory responses to GSNO. F) The decrease of Emax (an index of overall vasodilation) by GSNO pretreatment was restored by nitrite. G) EC50 (an index of sensitivity to GSNO) was not significantly changed. N=arterial rings from ≥ 5 animals. *P<0.05, ***P<0.001 for changes relative to Control (no GSNO pretreatment). #P<0.05, ##P<0.01 for changes relative to GSNOpreTx.
Figure 3
Figure 3. Nitrite potentiates vasoactivity of SNOs in the femoral artery of anesthetized sheep
A) After L-NAME (45 mg·kg−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).
Figure 4
Figure 4. Effect of L-NAME and nitrite on femoral conductance responses to GSNO in rats
A) Rats were given L-NAME for 4 days (60 mg·kg−1·day−1, i.p.) to block endogenous NOSs activity and thereby lower plasma nitrite levels. Animals that received nitrite infusions were then compared to those 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.
Figure 5
Figure 5. Effect of nitrite pretreatment on GSNO-induced cGMP levels in isolated arteries
A) Samples of sheep femoral arteries were incubated with 5 μM GSNO for three 15 min periods, similar to the treatment that caused tachyphylaxis in Figure 2. They were then incubated with 0 (control) or 1 μM nitrite for 1 h. The increases of cGMP levels in the two groups following 5 min of 5 μM GSNO stimulation were compared. B) GSNO-stimulated increases in cGMP were greater in arteries incubated with nitrite. Average results from 5 or more arteries; *P<0.05; paired t test.
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