Antihypertensive effect of mitochondria-targeted proxyl nitroxides

Abstract

Superoxide ( [Formula: see text] ) has been implicated in the pathogenesis of many human diseases including hypertension. Mitochondria-targeted superoxide scavenger mitoTEMPO reduces blood pressure; however, the structure-functional relationships in antihypertensive effect of mitochondria-targeted nitroxides remain unclear. The nitroxides are known to undergo bioreduction into hydroxylamine derivatives which reacts with [Formula: see text] with much lower rate. The nitroxides of pyrrolidine series (proxyls) are much more resistant to bioreduction compared to TEMPOL derivatives suggesting that mitochondria-targeted proxyls can be effective antioxidants with antihypertensive activity. In this work we have designed and studied two new pyrrolidine mitochondria targeted nitroxides: 3-[2-(triphenyphosphonio)acetamido]- and 3-[2-(triphenyphosphonio) acetamidomethyl]-2,2,5,5-tetramethylpyrrolidine-1-oxyl (mCP2) and (mCP1). These new mitochondria targeted nitroxides have 3- to 7-fold lower rate constants of the reaction with O2(-•) compared with mitoTEMPO; however, the cellular bioreduction of mCP1 and mCP2 was 3- and 2-fold slower. As a consequence incubation with cells afforded much higher intracellular concentration of mCP1 and mCP2 nitroxides compared to mitoTEMPO nitroxide. This has compensated for the difference in the rate of O2(-•) scavenging and all nitroxides similarly protected mitochondrial respiration in H2O2 treated endothelial cells. Treatment of hypertensive mice with mCP1 and mCP2 (1.4mg/kg/day) after onset of angiotensin II-induced hypertension significantly reduced blood pressure to 133±5mmHg and 129±6mmHg compared to 163±5mmHg in mice infused with angiotensin II alone. mCP1 and mCP2 reduced vascular O2(-•) and prevented decrease of endothelial nitric oxide production. These data indicate that resistance to bioreduction play significant role in antioxidant activity of nitroxides. Studies of nitroxide analogs such as mCP1 and mCP2 may help in optimization of chemical structure of mitochondria-targeted nitroxides for improved efficacy and pharmacokinetics of these drugs in treatment of hypertension and many other conditions including atherosclerosis, diabetes and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role.

Keywords: Antioxidant; Endothelial cells; Hypertension; Mitochondria; Nitroxide; Superoxide.

Fig. 1

Structures of mitochondria-targeted nitroxides.

Fig. 2

Synthesis of proxyl-based mitochondria-targeted nitroxides mCP1 and mCP2.

Fig. 3

Dose-dependent inhibition of superoxide-mediated cytochrome c reduction by nitroxides (A). Comparison of superoxide reactivity of mCP1, mCP2 and mitoTEMPO based on competitive inhibition of cytochrome c reduction. V and Vo are rates of cytochrome c reduction in the presence or absence of nitroxides. (B) Results represent mean ± SEM for 3–6 repeats per group. ^⁎P < 0.001 vs 50 nM nitroxide, ^⁎⁎P < 0.05 vs 100 nM nitroxide.

Fig. 4

Cellular accumulation of nitroxides (A) and protection of mitochondrial respiration (B) by mCP1, mCP2 and mitoTEMPO (mT). Results represent mean ± SEM for 3–6 repeats per group. ^⁎P < 0.001 vs control, ^⁎⁎P < 0.01 vs H₂O₂, ^#P < 0.05 vs 30 nM mCP1.

Fig. 5

EPR spectra of intact endothelial cells incubated with mCP1, mCP2 and mitoTEMPO (A). Rates of cellular nitroxide reduction during incubation at 37 °C (B). Results represent mean ± SEM for 4–6 repeats per group. ^⁎P < 0.01 vs mitoTEMPO, ^⁎⁎P < 0.05 vs mitoTEMPO.

Fig. 6

Antihypertensive effect of mitochondria targeted nitroxides in angiotensin II-infused mice. (A) Mice were implanted with osmotic pumps after onset of hypertension containing 1.4 mg/kg/day nitroxide or saline as a vehicle. (B) Mice were treated with the i.p. injection of 1.4 mg/kg/day mCP1, mCP2 or mitoTEMPO (mT). Results represent mean ± SEM for 3–6 animals per group. ^⁎P < 0.001 vs control, ^⁎⁎P < 0.01 vs Ang II.

Fig. 7

Production of aortic ${{\text{O}}_2}^{-•}$ (A) and endothelial nitric oxide (B) in mice treated with mCP1 and mCP2 after onset of angiotensin II-induced hypertension using osmotic pumps. (A) Measurements of vascular ${{\text{O}}_2}^{-•}$ (DHE/HPLC) in isolated aortas following 14-days angiotensin II infusion. (B) Detection of endothelial nitric oxide in aortas using electron spin resonance and Fe(DETC)₂ as NO spin trap . Results represent mean ± SEM for 5–8 animals per group. ^⁎P < 0.01 vs control, ^⁎⁎P < 0.05 vs AngII.