This study has evaluated how the vascular endothelium of hypertensive rats chronically treated with apocynin affects acetylcholine (ACh), sodium nitroprusside (SNP), and phenylephrine (PE) action on the nitric oxide (NO) signal transduction pathway in endothelial (EC) and vascular smooth muscle cells. Treatment with apocynin significantly reduced the mean arterial pressure in spontaneously hypertensive rats (SHR). In addition, apocynin improved the impaired ACh hypotensive effect on SHR. Although systemic oxidative stress was high in SHR, SHR treated with apocynin and normotensive rats presented similar systemic oxidative stress levels. Endothelium significantly blunted PE contractions in intact aortas of treated SHR. The ACh effect was impaired in resistance arteries and aortas of SHR, but this same effect was improved in treated SHR. The SNP potency was higher in intact resistance arteries of treated SHR than in intact resistance arteries of untreated SHR. NO and calcium concentrations increased, whereas reactive oxygen species levels decreased in EC of treated SHR. Aortas of untreated and treated SHR did not differ in terms of sGC alpha or beta units expression. Aorta of treated SHR expressed higher eNOS levels as compared to aorta of untreated SHR. The study groups did not differ with respect to NOX1, NOXO1, or NOX4 expression. However, treatment with apocynin normalized overexpression of NOX2 and its subunit p47phox in aortas of SHR. Based on all the results presented in this study, we suggest apocynin increases NO biovailability by different mechanisms, restoring the proper function of vascular endothelium in SHR.

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Nicotinamide adenine dinucleotide phosphate oxidase (NAD(P)H-oxidase) is a multicomponent enzyme system that generates superoxide anion by one-electron reduction of molecular oxygen and represents the major source of reactive oxygen species (ROS) in the vascular cells. Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in phagocytic cells and as an antioxidant in non-phagocytic cells. In phagocytes cells, due to the presence of myeloperoxidase, apocynin can be the converted to diapocynin, which is supposed to be the active form of this phytochemical. Moreover, apocynin was shown to induce hypotension and vasodilatation in many experimental animal models. However, there are no studies showing the effects of diapocynin on blood pressure or in vascular cells. In this present study, we used chemically synthesized diapocynin and analyzed its antioxidant capacity, effect on blood pressure and vascular reactivity. Moreover, it was evaluated the levels of nitric oxide (NO), ROS and calcium in aortic endothelial cells stimulated by diapocynin. All results were compared to apocynin. We found that diapocynin showed higher antioxidant capacity than apocynin. Apocynin and diapocynin, promoted hypotensive effects without changing the heart rate, however the effects of diapocynin were reversed faster than the effects of apocynin, which was long lasting. Diapocynin and apocynin induced endothelium dependent and independent vasodilatation, but diapocynin was less potent than apocynin regarding the capacity of induction of vasodilatation in mesenteric resistance arteries and aorta from Wistar rats. The relaxation induced by apocynin or diapocynin involves sGC and potassium channels in vascular smooth muscle cells and NOS participates of relaxation induced by apocynin or diapocynin in intact mesenteric rings. Apocynin and diapocynin increased NO and decreased ROS levels in endothelial cells, however diapocynin did not alter calcium levels in these cells. In conclusion, these results demonstrated that, similarly to apocynin, diapocynin also induces hypotensive and vasodilator effects in rats and vascular endothelium improves the diapocynin vasodilator effects by increases NO bioavailability.

Taken together, these results suggest that ROS production was decreased in the aortas of pregnant SHR and could contribute to higher NO bioavailability and hyporeactivity to PE in the aortas of pregnant SHR.

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We hypothesized that endothelium modulates relaxation induced by a nitric oxide (NO) donor ruthenium complex (TERPY, [Ru(terpy)(bdq)NO]) in mesenteric arteries of normotensive and spontaneously hypertensive (SHR) rats in different ways. We analyzed the mechanism involved in TERPY-induced relaxation in the second and third branches of mesenteric arteries and investigated how endothelium contributes to the TERPY vasodilator effect on SHR blood vessels. TERPY induced concentration-dependent relaxation in endothelium-denuded (E) and endothelium-intact (E) mesenteric arteries of normotensive rats and SHR. Pretreatment with ODQ (which inhibits soluble guanylyl cyclase) or TEA (tetraethylammonium, which blocks potassium channels) significantly reduced the TERPY vasodilator effect on E mesenteric arteries of normotensive rats and SHR. The presence of endothelium shifted the concentration-effect curves for TERPY in E mesenteric arteries of normotensive rats to the right. Conversely, the presence of endothelium shifted the concentration-effect curves for TERPY in the case of SHR E mesenteric arteries to the left, which suggested increased potency. L-NNA, a more selective endothelial NO synthase (eNOS) inhibitor, reduced TERPY potency in SHR. The presence of endothelium and notably of NOS contributed to the TERPY vasodilator action in SHR: TERPY promoted eNOS Ser phosphorylation with consequent NO production and increased soluble guanylyl cyclase activity, which may have directly activated potassium channels.

Reactive oxygen species (ROS) derived from NOX enzymes activity play an important role in the development of cardiovascular diseases. Compounds able to decrease oxidative stress damage are potential candidates as drugs and/or supplements for hypertension treatment. Here, we aimed to compare in vitro ROS scavenging potency, effective NOX inhibition and effects on vascular reactivity of apocynin to another phenolic compound, protocatechuic acid, in vascular cells from spontaneously hypertensive rat (SHR), where redox signaling is altered and contributes to the development and/or maintenance of hypertension. We evaluated the in vitro antioxidant capacity and free radical scavenging capacity of both phenolic compounds. Moreover, we investigated the effect of both compounds on lipid peroxidation, lucigenin chemiluminescence, nitric oxide (NO•) levels and ROS concentration in vascular cells of SHR or human umbilical vein endothelial cell (HUVEC). Apocynin and protocatechuic acid presented antioxidant capacity and ability as free radical scavengers, decreased thiobarbituric acid reactive substances (TBARS) in aortic cells from SHR, and increased NO• concentration in isolated HUVEC. Both compounds were able to reduce lucigenin chemiluminescence and increased the potency of acetylcholine in aorta of SHR. However, in SHR aortas, only apocynin diminished the contraction induced by phenylephrine. In conclusion, these results strongly reinforce the potential application of substances such as apocynin and protocatechuic acid that combine abilities as scavenging and/or prevention of ROS generation, establishment of NO bioactivity and modulation of vascular reactivity. Due to its phytochemical origin and low toxicity, its potential therapeutic use in vascular diseases should be considered.