Nitric Oxide Modulates β2-Adrenergic Receptor Palmitoylation and Signaling

To determine whether nitric oxide (NO) modulates the β-adrenergic signaling pathway, we treated cells expressing β 2 -adrenergic receptors (β 2 AR) 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 nucleotide-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 EC 50 for isoproterenol-stimulated cAMP production. SIN-1 treatment also led to a reduction in both basal and isoproterenol-stimulated incorporation of [ 3 H]palmitate into the β 2 AR. Signaling through the nonpalmitoylated, Gly 341 β 2 AR mutant was unchanged by SIN-1 treatment. Given the link between β 2 AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the β 2 AR resulting in decreased signaling through the β 2 AR.