Attenuated Desensitization of β-Adrenergic Receptor by Water-Soluble N-Nitrosamines That Induce S-Nitrosylation Without NO Release

Abstract

Rationale: The clinical problem of loss of β-adrenergic receptor (β-AR) response, both in the pathogenesis of heart failure and during therapeutic application of β-agonists, is attributable, at least in part, to desensitization, internalization, and downregulation of the receptors. In the regulation of β-AR signaling, G protein–coupled receptor kinase 2 (GRK2) primarily phosphorylates agonist-occupied β-ARs, and this modification promotes desensitization, internalization, and downregulation of β-ARs. It has been demonstrated that GRK2 is inhibited by its S-nitrosylation. However, compounds that induce S-nitrosylation, such as S-nitrosoglutathione, simultaneously generate NO, which has been demonstrated to operate for cardiovascular protection. Objective: We examine whether S-nitrosylation without NO generation inhibits desensitization of β 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 β-AR from desensitization in HEK 293 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 β 2 -AR desensitization. Conclusions: Compounds that induce S-nitrosylation without NO release inhibit GRK2 and attenuate β 2 -AR desensitization. Developing water-soluble drugs that specifically induce S-nitrosylation may be a promising therapeutic strategy for heart failure.

Cinacalcet corrects biased allosteric modulation of CaSR by AHH autoantibody

“…Expression constructs, cell culture, and transfection. HEK293 cells (obtained from Henry R. Bourne, UCSF, San Francisco, CA, USA), maintained in DMEM containing 10% FBS, were transfected with constructs encoding human CaSR or Flag-tagged WT/mutant human CaSR, using Lipofectamine 2000 (Invitrogen) (31,67,68). The transiently transfected cells were used for immunoperoxidase staining and mutant study after 48 hours in culture.…”

Analysis of the V2 Vasopressin Receptor (V2R) Mutations Causing Partial Nephrogenic Diabetes Insipidus Highlights a Sustainable Signaling by a Non-peptide V2R Agonist

“…Cell Surface ELISA-The cell surface expression of exogenous receptors in COS-7 cells transiently transfected with Myctagged V2R-WT or each V2R mutant was quantified by ELISA (6,10,30,31). Briefly, 1 day after transfection, cells were reseeded into 24-well plates.…”

Inverse agonism: the classic concept of GPCRs revisited [Review]

“…Recognition of the deleterious effects of overactivation of the renin-angiotensin system (RAS) and AT1-Gq/G13 signaling [28][29][30], in addition to desensitization of cardioprotective βAR-Gs signaling [31][32][33], has made RAS a major therapeutic target, whether systemically or regionally [34,35]. In many randomized, controlled trials, ACE inhibitor (ACEi) reduced morbidity and mortality more than comparators in patients with congestive heart failure and coronary heart disease [36][37][38][39][40].…”