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

Abstract

Kinins are vasoactive peptides involved in endothelial function and vascular tonus. The present study determined the influence of the kinin B 1 receptor subtype on endothelial nitric oxide (NO) metabolism by using primary cultured cells obtained from B 1 knockout (B 1 -/-) and Wild Type (WT) mice. By using specific fluorescent dyes, NO and superoxide anion (O À 2 • ) production was determined in absence or presence of ascorbate or tetrahydrobiopterin (BH 4 ). 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 BH 4 (54.3 ± 1.7 a.u.). B 1 -/-cells showed a marked increase in O À 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 BH 4 . 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 BH 4 oxidation, NOS uncoupling and further enhancement of NOS-derived O À 2 • . B 1 receptor deletion also impairs the phosphorylation of eNOS at the Ser1177 residue, contributing to the deficient NO production at the endothelial level.