melitten and Hypertension

NOX-derived reactive oxygen species (ROS) are mediators of signalling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here, we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction.. VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signalling molecules, and actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were down-regulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs, NOX5 was up-regulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1), and hyperphosphorylation (c-Src, PKC, ERK1/2, MLC20) of signalling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2.. We define NOX5/ROS/c-Src as a novel feedforward signalling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.

Topics: Actins; Angiotensin II; Animals; Cells, Cultured; Humans; Hypertension; Melitten; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidase 5; Oxidation-Reduction; Protein-Tyrosine Kinases; Reactive Oxygen Species; RNA, Small Interfering

Melittin effect on transport of Na+ and Rb+(K+ analog) was examined in cultured skin fibroblasts originating from the Spontaneously Hypertensive, Wistar Kyoto and Wistar rats. Melittin increased both Na+ (22Na+) uptake and 86Rb+ efflux as well as the activity of the Na+-pump (ouabain sensitive 86Rb+ uptake) in all three preparations. The effect of the toxin was maximal at a dose of 160-240ng/10(5) cells/ml. At this dose, fibroblasts of the Spontaneously Hypertensive rat demonstrated the greatest response to melittin with respect to the increase in Na+ and Rb+ fluxes and increase in the intracellular Na+ concentrations. It is concluded that melittin can be utilized as a probe to delineate subtle differences in the cellular regulation of Na+ and K+ in the Spontaneously Hypertensive rat as compared with its normotensive controls.

Topics: Animals; Bee Venoms; Biological Transport, Active; Cells, Cultured; Fibroblasts; Hypertension; Melitten; Ouabain; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rubidium; Skin; Sodium