Oxidative transition of intracellular protein thiols modulates muscarinic receptor-stimulated phosphoinositide breakdown in rat cortical slices

The effect of thiol modification on muscarinic receptor-stimulated phosphoinositide hydrolysis was investigated in rat cortical slices. Diamide as well as N-ethylmaleimide, which readily cross the plasma membrane, effectively reduced this response using both carbachol and acetylcholine as agonists. The effect of the above thiol reactive agents was concentration-dependent and caused by oxidation of critical thiols since a reducing agent, dithiothreitol, promptly reversed this effect. Three lines of evidence suggest that these sulfhydryl groups were located intracellularly. Firstly, 5, 5'-dithiobis-2-nitrobenzoic acid, a thiol reactive agent unable to penetrate the plasma membrane, failed to inhibit the muscarinic receptor-elicited inositol phosphate accumulation. Secondly, diamide did not produce deleterious effects on the muscarinic receptor binding site since, following exposure to the oxidant, the affinity for carbachol was not impaired but actually increased. Finally, diamide inhibited both receptor-mediated (carbachol or norepinephrine) and direct (NaF/AlCl₃) stimulation of inositol phosphate accumulation. In conclusion, results reported in this study indicate that muscarinic receptor-stimulated phosphoinositide breakdown in rat cortical slices is significantly reduced by an oxidative stress via oxidation of critical thiols located at the post-receptor level.