Attenuated Desensitization of  -Adrenergic Receptor by Water-Soluble N-Nitrosamines that Induce S-Nitrosylation Without Nitric Oxide Release

Rationale : The clinical problem of loss of  -adrenergic receptor (  -AR) response, both in the pathogenesis of heart failure and during therapeutic application of  -agonists, is due at least in part to desensitization, internalization and downregulation of the receptors. In the regulation of  -AR signaling, G-protein-coupled receptor kinase 2 (GRK2) primarily phosphorylates agonist-occupied  -ARs and this modification promotes desensitization, internalization and downregulation of  -ARs. It has been demonstrated that GRK2 is inhibited by its S-nitrosylation. However, compounds that induce S-nitrosylation, such as GSNO, simultaneously generate nitric oxide (NO), which has been demonstrated to operate for cardiovascular protection. Objective : We examine whether S-nitrosylation without NO generation inhibits desensitization of  2 -AR by GRK2. We thus aim to synthesize compounds that specifically induce S-nitrosylation. Methods and Results : We have developed water- soluble N-nitrosamines that have S-nitrosylating activity but lack NO-generating activity. These compounds at least partly rescue  -AR from desensitization in HEK293 cells expressing FLAG-tagged human β 2 -AR and in rat cardiac myocytes. They inhibit isoproterenol-dependent phosphorylation and internalization of β 2 -AR. Indeed, they nitrosylate GRK2 in vitro and in cells and their S-nitrosylation of GRK2 likely underlies their inhibition of  2-AR desensitization. Conclusion : Compounds that induce S-nitrosylation without NO release inhibit GRK2 and attenuate  2 -AR desensitization. Developing water-soluble drugs that specifically induce S-nitorsylation may be a promising therapeutic strategy for heart failure. (either wild type or C340S mutant) and incubated with siRNA for endogenous GRK2 were incubated for 24 h with 30  mol/L WNNO7 or 30  mol/L GSNO and for 4 h with 2  mol/L isoproterenol or no drug before the assay. Then the cAMP accumulation assay was performed in the presence of 2  mol/L isoproterenol or no drug as described in Methods Section. The values shown in these analyses represent the means ± S.D. of triplicate determinations. Each set of results is representative of at least three experiments.