Liposomes as carriers of resveratrol and vitamin E: Evaluating ameliorative antioxidant effect using chemical and cellular test systems.

Resveratrol (RES) in combination with antioxidant vitamins is reported to be more effective in protecting the cells from oxidative stress rather than any of these antioxidants alone. In continuation to our previous work using resveratrol and vitamin C, our main aim was to evaluate the antioxidant restorative effect using chemical and cellular test systems on resveratrol co-encapsulated vitamin E (VE) within liposomes. Z-average size was less than 135 nm, polydispersity index < 0.3; zeta potential > than ± 30 mV and encapsulation efficiency of RES and VE > 90% and 79% respectively. Chemiluminescence measurement indicated that the antioxidative activity of RES could be increased when VE was additionally loaded into liposomes. Inhibition of AAPH induced luminol enhanced chemiluminescence displayed 90% improvement (P < 0.001) in comparison to control; on the other hand 70% luminescence inhibition of ROS production in isolated blood leukocytes (P < 0.001) was observed. Intracellular oxygen-derived radicals measured by flow cytometry using 2'-7'-dichlorodihydrofluorescein diacetate demonstrated about 1.7 fold (P < 0.05) and 1.5 fold (P < 0.001) enhancement of radical scavenging activity in buffy coats under basal conditions and human umbilical vein endothelial cells after stimulation by H2O2 respectively. The cellular systems evidenced the ability of liposome loaded antioxidants to scavenge ROS in the extra and intracellular space, confirming enhanced antioxidative effectivity of RES in the presence of VE, which did not occur in combination with vitamin C. Hence it might be possible to improve the antioxidative effectivity of RES by other/additional antioxidants.

The Role of Potassium Channels in the Vasodilatation Induced by Resveratrol and Naringenin in Isolated Human Umbilical Vein.

Preclinical Research Potassium (K+ ) channels have a key role in the maintenance of smooth muscle tone; a variety of agonists can modify the tone by altering K+ -channel activity. The aim of this study was assess the effects of the phenols, resveratrol, and naringenin on K+ -channels of the vascular smooth muscle. Segments of human umbilical vein (HUV) without endothelium were precontracted using serotonin (100 µM) or 100 mM K+ to derive cumulative concentration-response curves using increasing concentrations of resveratrol or naringenin. K+ -channel inhibitors were added in the bath before resveratrol (1-100 µM) or naringenin (0.01-1 mM) in assess the role of K+ -channels in their effects on HUV precontracted by serotonin. 4-Aminopiridine (4-AP; 1 mM), a nonselective blocker of voltage-dependent, tetraethylammonium (TEA; 1 mM) and barium chloride (1 mM), a nonselective blocker of Ca2+ -dependent and inward rectifier K+ -channels (respectively) induced significant shifts to the right (P < 0.05) of resveratrol. concentration-response curves. The effect of naringenin was antagonized by 4-AP (1 mM). 4-AP-, TEA-, and barium chloride-sensitive K+ -channels are probably involved in the resveratrol vasodilatatory effect, while naringenin seems to affect 4-AP-sensitive K+ -channels. However, other mechanisms of vasodilation induced by polyphenols could not be excluded. Drug Dev Res, 2015. © 2015 Wiley Periodicals, Inc.

The different effects of resveratrol and naringenin on isolated human umbilical vein: the role of ATP-sensitive K⁺ channels.

The blood flow from the placenta to the fetus depends on human umbilical vein (HUV) vascular tone. ATP-sensitive K(+) (K(ATP)) channels link the metabolic state of the cell to membrane potential, and their activation in the HUV represents protection against hypoxia. The aims of our study were to assess the effects of resveratrol and naringenin on the HUV and to define the roles of K(ATP) channels in their effects. Serotonin or 100 mM K(+) were used for precontraction of the HUV without endothelium. The cumulative concentration-response curves were obtained by adding increasing concentrations of resveratrol or naringenin. Glibenclamide was used, in order to test the role of K(ATP) channels in its effect. Resveratrol induced more potent vasodilatation of serotonin- and 100 mM K(+)-precontracted HUV than naringenin. Glibenclamide induced significant shift to the right of the concentration-response curves of resveratrol and P1075 (a specific opener of K(ATP) channels). Western blotting showed that HUV expressed protein Kir6.1. Thus, resveratrol and naringenin produce dilatation of HUV. It seems that K(ATP) channels are involved in the relaxation of HUV induced by resveratrol, while naringenin seems to interact with other ion channels. The K(+) channel-independent mechanism(s) of these polyphenols could not be excluded.

Effect of wine polyphenol resveratrol on the contractions elicited electrically or by norepinephrine in the rat portal vein.

We investigated the effects of resveratrol on rat portal vein (RPV) contractility without endothelium. Contractions were produced by electrical field stimulation of perivascular nerves (EFS), norepinephrine (NE), adenosine 5'-triphosphate (ATP), high K(+) solution and by calcium chloride (CaCl2 ) in Ca(2+) -free and high K(+) , Ca(2+) -free solution. The EFS-evoked contractions were more sensitive to resveratrol and to NS1619-selective openers of big calcium-sensitive (BKCa ) channels, than NE-evoked contractions. Effects of resveratrol on the ATP-evoked contractions were weak. Blockers of BKCa channels partly inhibited the effect of resveratrol only in EFS-contracted preparations. Western blotting showed that RPV expressed KCa 1.1 protein. Inhibitors of ATP- and voltage-sensitive K(+) channels did not modify the effects of resveratrol. None of the antagonists of K(+) channels affected the resveratrol inhibition of NE-evoked contractions and effect of high concentrations of resveratrol on the EFS-evoked contractions. Resveratrol more potently inhibited CaCl2 than potassium chloride contractions of RPV. Thus, BKCa channels partly mediate the inhibitory effect of resveratrol on the neurogenic contractions of RPV. The smooth muscle Ca(2+) channels and/or Ca(2+) mobilizing through cells might be involved in the effects of resveratrol on the contractility of RPV. Our results are important for better understanding the impact of resveratrol on the portal circulation.

The effects of potassium channel opener P1075 on the human saphenous vein and human internal mammary artery.

Because adrenergic contractions can contribute to the development of life-threatening spasm of coronary artery bypass graft, this study was performed to investigate the effect of adenosine 3-phosphate (ATP)-sensitive K channel (KATP) opener P1075 on contractions of isolated human saphenous vein (HSV) and human internal mammary artery (HIMA). Phasic contractions were evoked by electric field stimulation (20 Hz) and noradrenaline. The sustained contractions were evoked by phenylephrine. The presence of pore-forming Kir6.1 and Kir6.2 subunits of the KATP channels in the HIMA and only Kir6.2 in the HSV was confirmed immunomorphologically. P1075 inhibited in the HSV only, the electrical field stimulation contractions more strongly than noradrenaline contractions. In addition, the phenylephrine contractions of HSV were more sensitive to P1075 in comparison to those of HIMA. Glibenclamide, a KATP channel blocker antagonized the vasodilatation produced by P1075 in both grafts differently, because its effect was more prominent on the P1075-induced inhibition of contractions of HSV than of HIMA. We conclude that P1075 has a vasorelaxant effect and inhibited adrenergic contractions of the tested grafts. This effect is graft and vasoconstrictor selective and seems to be mediated by Kir6.1- and/or Kir6.2-containing KATP channels. Thus, P1075 can be considered as a potential drug in the prevention of graft spasm.

Solubilization of resveratrol in micellar solutions of different bile acids.

Bile acids are a special group of biological surfactants which can express different physiological and pharmacological effects. Micellar solutions of bile acids can solubilizate poorly soluble organic substances and improve their resorbtion. Above their critical micellar concentration (CMC) values, bile acids can cause interruption of membrane's integrity. Resveratrol (trans-3,5,4'-trihydroxystilbene) is a stilbene phytoalexine and studies reported that it can prevent or reduce diseases such as cancer and coronary heart disease. In this study, we examined affinity of different bile acids (CA, 12-MDCA, 12-MCA, 7-MCA, 7,12-DCA, 3,7,12-TCA) micellar solutions for resveratrol solubilization. CMC values for bile acids were determined by conductivity measurements. Concentration of micellar solutions were 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0 CMC value, for each acid respectively. At the same time, we investigated membranolytical potential of each acid. Solubilizated resveratrol was quantified using HPLC system with UV detection. Membranolytical potential was determined from citrate rabbit blood. Structures of mixed micelles of each bile acid and resveratrol were explained, and multiple linear regression equations for solubilization of resveratrol on different concentrations of bile acids were obtained. Micellar solution of 3,7,12-TCA had the biggest affinity for resveratrol solubilization and then, in decreasing order 7-MCA>7, 12-DCA>12-MCA>12-MDCA>CA. Also, 3,7,12-TCA had the lowest membranolytical potential.

The effect of potassium channel opener pinacidil on the non-pregnant rat uterus.

The effects of the K(+) channel opener, pinacidil on the spontaneous rhythmic contractions and contractions provoked by electrical field stimulation (50 Hz) or by oxytocin were investigated in the isolated uterus of the non-pregnant rat in oestrus. Pinacidil produced more potent inhibition of oxytocin-elicited contractions than of spontaneous rhythmic contractions or electrical field stimulation-induced contractions. Glibenclamide, a selective blocker of adenosine triphosphate (ATP)-sensitive K(+) (K(ATP)) channels, antagonized the pinacidil-induced inhibition of contractions elicited by oxytocin in a competitive manner. However, the pinacidil-induced inhibition of electrical field stimulation-elicited contractions and spontaneous rhythmic contractions was antagonized non-competitively by glibenclamide. In the uterine strips pre-contracted with 80 mM K(+), the pinacidil-induced maximal relaxation was not affected. The present data show that pinacidil exhibits potent relaxant properties in the rat non-pregnant uterus in oestrus and therefore should be taken into account as a possible agent for treatment of dysmenorrhoea. Based on glibenclamide affinity, it appears that the inhibitory response to pinacidil involves K(ATP )channels. We need further investigations to explain why the interaction between glibenclamide and pinacidil in this experimental model depends on the nature of contractions. The ability of pinacidil to completely relax the rat non-pregnant uterus pre-contracted with K(+)-rich solution suggests that K(+) channel-independent mechanism(s) also play a part in its relaxant effect.

Potassium channel opener pinacidil induces relaxation of the isolated human radial artery.

Taking into consideration that the search for drugs capable of modifying blood flow through human radial artery (RA) is warranted, the present study was designed to examine the vasodilatatory effects of the potassium channel opener, pinacidil on the RA and to define the contribution of different K+ -channel subtypes in the endothelium-independent pinacidil action on this blood vessel. Pinacidil relaxed the RA rings with endothelium and without endothelium with comparable potency. N-nitro-L-arginine methyl ester (L-NAME) and methylene blue did not affect the pinacidil-induced vasorelaxation in rings with endothelium. In the rings without endothelium, the K+ -channel blockers glibenclamide and tetraethylammonium (TEA) moderately antagonized the pinacidil-induced relaxation, while charybdotoxin and 4-aminopiridine did not. In endothelium-denuded rings, precontracted with 100 mM K+, the relaxant responses to pinacidil were highly significantly shifted to the right compared to those obtained in RA precontracted with phenylephrine, but pinacidil-induced maximal relaxation was not affected. Addition of nifedipine did not but addition of nifedipine and nickel (Na+ -Ca2+ exchanger inhibitor) did cause a statistically significant rightward shift of the pinacidil concentration-relaxation curve, although the effect 0.1 mM pinacidil was preserved. Thus, pinacidil induces relaxation of the human RA in endothelium-independent manner, and glibenclamide- and TEA-sensitive vascular smooth muscle K+ channels are probably involved. Its ability to completely relax the RA precontracted with K+ -rich solution suggests that pinacidil has additional K+ channel-independent mechanism(s) of action. It seems that stimulation of the forward mode of the Na+ -Ca2+ exchanger plays a part in this K+ channel-independent effect of pinacidil.

Increased gene expression of scavenger receptors and proinflammatory markers in peripheral blood mononuclear cells of hyperlipidemic males.

Interactions between peripheral blood mononuclear cells (PBMCs) and those within plaques are suggested to be pathophysiologically relevant to lipid-induced arteriosclerosis. In this study, gene expressions of scavenger receptors (CD36, CD68), LPS receptor (CD14), proinflammatory (tumor necrosis factor alpha [TNFalpha], CD40, interleukin-1 beta [IL-1beta]) and oxidative stress-related (manganese superoxide dismutase [MnSOD]) markers were analyzed in PBMCs of clinically asymptomatic males with classical proatherogenic risk factors such as smoking and/or hyperlipidemia. PBMCs were isolated from venous blood of normolipidemic non-smokers (n = 10) and smokers (n = 8), and hyperlipidemic non-smokers (n = 9) and smokers (n = 8). RNA from PBMCs was used for PCR analyses. Plasma concentrations of oxidized low-density lipoproteins (oxLDL) were measured by ELISA. The gene expressions of CD36, CD68, CD40, TNFalpha, and MnSOD were significantly higher in PBMCs of hyperlipidemics than in normolipidemics, irrespective of whether they were smoking or not. The individual expression of these genes showed significant positive correlations with each other but also with serum cholesterol or plasma oxLDL concentrations. The higher expressions of scavenger receptors, proinflammatory and oxidative stress-related genes of PBMCs are suggested to result mainly from hyperlipidemia and the accompanied increase of oxLDL concentrations.

The mechanism of endothelium-independent relaxation induced by the wine polyphenol resveratrol in human internal mammary artery.

Resveratrol, a stilbene polyphenol found in grapes and red wine, produces vasorelaxation in both endothelium-dependent and endothelium-independent manners. The mechanisms by which resveratrol causes vasodilatation are uncertain. The aim of this study was to investigate the mechanism(s) of endothelium-independent resveratrol-induced vasorelaxation in human internal mammary artery (HIMA) obtained from male patients undergoing coronary artery bypass surgery and to clarify the contribution of different K+ channel subtypes in resveratrol action in this blood vessel. HIMA rings without endothelium were precontracted with phenylephrine. Resveratrol induced a concentration-dependent relaxation of the HIMA. A highly selective blocker of ATP-sensitive K+ channels, glibenclamide, as well as nonselective blockers of Ca2+-sensitive K+ channels, tetraethylammonium and charybdotoxin, did not block resveratrol induced relaxation of HIMA rings. 4-Aminopyridine (4-AP), non selective blocker of voltage gated K+ (KV) channels, and margatoxin that inhibits KV1.2, KV1.3, and KV1.6 channels abolished relaxation of HIMA rings induced by resveratrol. In conclusion, we have shown that resveratrol potently relaxed HIMA rings with denuded endothelium. It seems that 4-AP- and margatoxin-sensitive K+ channels located in smooth muscle of HIMA mediated this relaxation.

Modulation of the beta-adrenergic receptor system of vascular smooth muscle cells in vitro and in vivo by chronically elevated endothelin-1 levels.

Endothelin-1 (ET-1) levels are chronically elevated in several cardiovascular diseases and correlate with an increased mortality. However, in contrast to acute biological activities such as vasoconstriction, little is known about long-term effects of ET-1. In this study we determined the effects of ET-1 on the beta(2)-adrenergic receptor (AR) system. Incubation of smooth muscle cells with ET-1 for 72 hr led to increased beta(2)AR density as determined by radioligand binding. Experiments with inhibitors of protein and RNA synthesis as well as RT-PCR revealed that beta(2)AR upregulation required de novo synthesis. In addition, protein kinase C but neither NO nor prostaglandin metabolism were involved in this effect. The enhanced expression of beta(2)AR was associated with an increased expression of its stimulatory G-protein and the receptor's ability to stimulate adenylyl cyclase. To study chronic effects of ET-1 in vivo, rats were infused with ET-1 for 3 weeks. Similarly as in cultured cells, prolonged ET-1 exposure led to increased betaAR expression in vivo. As a consequence, beta(2)AR-induced vasodilatation was increased in aortic rings from ET-1-treated animals. Our results therefore suggest that chronically elevated ET-1 levels in vitro and in vivo induce counterregulatory mechanisms by increasing betaARs that attenuate the vasoconstrictive effects of ET-1.