Abstract
Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the Vmax of the enzyme without altering the Km for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase.
Highlights
Nitric oxide (NO)1 has been attributed roles in a variety of cellular activities throughout the body
We have observed that the addition of NO gas or NO donor compounds to cultures of N18TG2 neuroblastoma cells inhibits Gs-coupled and forskolin-stimulated cAMP accumulation [6]
NO Inhibits Adenylyl Cyclase Activity in Purified Plasma Membrane Preparations—Plasma membranes were preincubated with increasing concentrations of SNP, after which they were assayed for forskolin-stimulated adenylyl cyclase activity
Summary
SNP Pretreatment—Sucrose gradient purified plasma membranes [12] were resuspended in buffer A (50 mM NaHepes, pH 8, 3 mM MgCl2, 1 mM EDTA) at 0.5 mg/ml. Membranes were preincubated with 3 mM SNP for 30 min on ice. Other reagents were present as described in the individual experiments. Membranes were washed free of any additions by diluting them 10-fold in ice-cold buffer A, centrifuging at 700 ϫ g for 10 min, and resuspending them in ice-cold buffer A at 0.5 mg/ml. The standard reaction mixture contained 50 mM NaHepes, pH 8, 1 mM EDTA, 10 mM.
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