Nitric oxide modulates β2-adrenergic receptor palmitoylation and signaling

Abstract

To determine whether nitric oxide (NO) modulates the β-adrenergic signaling pathway, we treated cells expressing β2-adrenergic receptors (β2AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4- oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)chloride and determined the intracellular production of cAMP after exposure to β-adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the β-adrenergic agonist, isoproterenol, to stimulate cAMP production without affecting the stimulatory action of forskolin and cholera toxin, which directly activate adenylyl cyclase and G(s), respectively. Treatment with the NO donor increased the guanyl nu cleotide-sensitive high affinity constant for the agonist, isoproterenol, thus suggesting that it reduced functional coupling between the receptor and G(s). Stimulation of endogenous NO production by lipopolysaccharide in RAW 264.7 macrophages also caused a significant increase in the EC50 for isoproterenol-stimulated cAMP production. SIN-1 treatment also led to a reduction in both basal and isoproterenol-stimulated incorporation of [3H]palmitate into the β2AR. Signaling through the nonpalmitoylated, Gly341β2AR mutant was unchanged by SIN-1 treatment. Given the link between β2AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the β2AR resulting in decreased signaling through the β2AR.