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Abstract
To determine whether nitric oxide (NO) modulates the beta-adrenergic signaling pathway, we treated cells expressing beta(2)-adrenergic receptors (beta(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 beta-adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the beta-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 beta(2)AR. Signaling through the nonpalmitoylated, Gly(341)beta(2)AR mutant was unchanged by SIN-1 treatment. Given the link between beta(2)AR palmitoylation and its responsiveness to agonist, these results suggest that the primary action of NO was depalmitoylation of the beta(2)AR resulting in decreased signaling through the beta(2)AR.
Highlights
To determine whether nitric oxide (NO) modulates the -adrenergic signaling pathway, we treated cells expressing 2-adrenergic receptors (2AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)chloride and determined the intracellular production of cAMP after exposure to -adrenergic receptor agonists, cholera toxin and forskolin
CAMP Accumulation Studies—To determine if NO can modulate -adrenergic signaling, the effect of the NO donor 3-morpholinosydnonimine (SIN-1) on isoproterenol-stimulated adenylyl cyclase activity was assessed in COS cells
The isoproterenol potency returned to control values within 30 min following removal of the NO donor indicating that the effect was readily reversible
Summary
To determine whether nitric oxide (NO) modulates the -adrenergic signaling pathway, we treated cells expressing 2-adrenergic receptors (2AR) with the NO donors, 3-morpholinosydnonimine (SIN-1) and 1,2,3,4oxatriazolium,5-amino-3-(3-chloro-2-methylphenyl)chloride and determined the intracellular production of cAMP after exposure to -adrenergic receptor agonists, cholera toxin and forskolin. NO significantly decreased the potency of the -adrenergic agonist, isoproterenol, to stimulate cAMP production without affecting the stimulatory action of forskolin and cholera toxin, which directly activate adenylyl cyclase and Gs, respectively. The 2AR and its cognate G protein (Gs) both undergo palmitoylation on cysteine residues This post-translational modification is dynamically regulated on the receptor and Gs upon -adrenergic stimulation [14, 15]. The aims of the present study were to determine if NO can directly modulate the signaling efficiency of the -adrenergic-Gs-adenylyl cyclase signaling pathway and to assess if regulation of the palmitoylation state of the receptor or Gs could play a role in such a regulatory process. We found that NO reduces the potency of a -adrenergic agonist to stimulate adenylyl cyclase and promotes depalmitoylation of the 2AR
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