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 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 ( (a (TM))) 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 +/- A 3.1* a.u.) when compared to WT cells (66.9 +/- A 3.2 a.u.); this impaired response was reversed by ascorbate (101.8 +/- A 6.0 a.u.) and BH4 (54.3 +/- A 1.7 a.u.). B (1) (-/-) cells showed a marked increase in (a (TM)) production (77.1 +/- A 2.5* a.u.) versus WT (29.3 +/- A 6.9 a.u.), which was reversed by ascorbate (35.3 +/- A 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 (a (TM)). B-1 receptor deletion also impairs the phosphorylation of eNOS at the Ser1177 residue, contributing to the deficient NO production at the endothelial level.