Endothelial Nitric Oxide Metabolism Research Articles

Cellular Changes Induced by Kinin B1 Receptor Deletion: Study of Endothelial Nitric Oxide Metabolism

Kinins are vasoactive peptides involved in endothelial function and vascular tonus. The present study determined the influence of the kinin B1 receptor subtype on endothelial nitric oxide (NO) metabolism by using primary cultured cells obtained from B1 knockout (B 1 −/− ) and Wild Type (WT) mice. By using specific fluorescent dyes, NO and superoxide anion ( $${\text{O}}_{2}^{ - }$$ ∙) production was determined in absence or presence of ascorbate or tetrahydrobiopterin (BH4). The activity of the enzyme superoxide dismutase (SOD) was determined by immune enzyme assay, and endothelial NOS (eNOS) activation was analyzed through expression of phospho-eNOS (p-eNOS, Ser1177) by western blot. The NO release [quantified by densitometry and expressed as arbitrary units (a.u.)] was markedly reduced in B 1 −/− (35.8 ± 3.1* a.u.) when compared to WT cells (66.9 ± 3.2 a.u.); this impaired response was reversed by ascorbate (101.8 ± 6.0 a.u.) and BH4 (54.3 ± 1.7 a.u.). B 1 −/− cells showed a marked increase in $${\text{O}}_{2}^{ - }$$ ∙ production (77.1 ± 2.5* a.u.) versus WT (29.3 ± 6.9 a.u.), which was reversed by ascorbate (35.3 ± 6.4 a.u.), but not by BH4. SOD activity was similar between groups, and B 1 −/− cells presented a significant reduction in the expression of p-eNOS. In conclusion, the reduced NO availability detected in B 1 −/− cells appears to be related to a recurrent process involving BH4 oxidation, NOS uncoupling and further enhancement of NOS-derived $${\text{O}}_{2}^{ - }$$ ∙. B1 receptor deletion also impairs the phosphorylation of eNOS at the Ser1177 residue, contributing to the deficient NO production at the endothelial level.

Role of vascular Kinin B1 and B2 receptors in endothelial nitric oxide metabolism

Kinin B1 and B2 receptors play an essential role in inflammatory process and cardiovascular homeostasis. The present study investigated the vascular reactivity and nitric oxide (NO) generation in the isolated mesenteric arteriolar bed from B1 (B1−/−) and B2 receptor (B2−/−) knockout mice. Endothelial-dependent relaxation was significantly decreased in arterioles from both B1−/− and B2−/− in comparison to wild type (WT) mice, with no differences for endothelial-independent relaxating or vasoconstrictor agents. Plasmatic and vascular NO production were markedly reduced in both B1−/− and B2−/−. In contrast, in the presence of l-arginine, Ca2+ and co-factors for the enzyme, NO synthase activity was higher in homogenates of mesenteric vessels of B1−/− and B2−/−. The present study demonstrated that targeted deletion of B1 or B2 receptor gene in mice induces important alterations in the vascular reactivity of resistance vessels and NO metabolism. The severe impairment in the endothelial-mediated vasodilation accompanied by decreased NO bioavailability, despite the augmented NOS activity, strongly indicates an exacerbation of NO inactivation in B1−/− and B2−/− vessels. The present data provide valuable information in order to clarify the relevance of kinin receptors in regulating vascular physiology and may point to new approaches regarding its correlation with endothelial dysfunction, oxidative stress and NO availability.

Flavanols: digestion, absorption and bioactivity

Flavanols, or flavan-3-ols, are a family of bioactive compounds present in cocoa, red wine, green tea, red grapes, berries and apples. With a basic monomer unit of (−)-epicatechin or (+)-catechin, flavanols can be present in foods and beverages as monomers or oligomers (procyanidins). Most, but not all, procyanidins are degraded into monomer or dimer units prior to absorption. The bioavailability of flavanols can be influenced by multiple factors, including food processing, cooking, digestion, and biotransformation. Flavanols are potent antioxidants, scavenging free radicals in vitro and in vivo. While some of the actions of flavanols can be linked to antioxidant activities, other modes of action may also occur, including modulation of intracellular signaling, effects on membrane fluidity and regulation of cytokine release or action. Physiologically, flavanol-rich foods and beverages can affect platelet aggregation, vascular inflammation, endothelial nitric oxide metabolism, and may confer protective effects against neurodegeneration. Epidemiological data suggests that intake of cocoa, a rich source of flavanols, is inversely associated with 15-year cardiovascular and all-cause mortality in older males. (−)-Epicatechin and its metabolite, epicatechin-7-O-glucuronide, have been identified as independent predictors of some of the vascular effects associated with the consumption of a flavanol-rich beverage. Targeted dietary components and nutrition supplements that can influence the vascular system will be of great value in the prevention and treatment of chronic disease.

Chronic Ethanol Exposure Stimulates Endothelial Cell Nitric Oxide Production through PI-3 Kinase-and hsp90-dependent Mechanisms

Chronic ethanol (EtOH) ingestion increases the incidence of the Acute Respiratory Distress Syndrome (ARDS), a severe form of acute lung injury characterized by endothelial and epithelial barrier dysfunction. The regulated production of nitric oxide (NO) by the endothelium plays a central role in normal vascular function, and alterations in NO production have been implicated in barrier dysfunction. Although previous reports examined the impact of acute EtOH stimulation on endothelial NO production, this study extends those observations to clarify mechanisms of chronic EtOH-mediated alterations in endothelial nitric oxide synthase (eNOS) expression and NO production. Porcine pulmonary artery endothelial cells (PAEC) were treated with EtOH (0.04-0.16%, w/v) for 72 hr in sealed chambers to prevent evaporation. NO release and eNOS expression were determined to examine the effect of chronic EtOH stimulation on endothelial NO metabolism. While there was no change in the extent of phosphorylated eNOS at ser, chronic EtOH stimulation caused dose-dependent increases in NO production and increased eNOS expression, effects that were attenuated by the transcriptional inhibitor, alpha-amanitin (AA), and wortmannin, a specific phosphatidylinositol 3 kinase (PI3 K) inhibitor. EtOH stimulation also increased eNOS interaction with heat shock protein (hsp90), a molecular chaperone known to enhance eNOS activity. Geldanamycin, an hsp90 inhibitor, attenuated chronic EtOH-mediated increases in NO production. These results indicate that chronic EtOH exposure increases endothelial NO production by increasing eNOS protein levels through PI3 K-dependent up regulation of eNOS gene transcription and by increasing interactions between eNOS and hsp90. These findings clarify mechanisms by which chronic EtOH stimulation modulates vascular endothelial function and suggest new targets for investigation and intervention in EtOH-induced alterations in susceptibility to lung injury.