Disulfide bond reduction inhibits norepinephrine accumulation in postganglionic sympathetic nerve endings.
Accumulation of l-[3H]norepinephrine by sympathetic nerves in mouse atria was inhibited by disulfide bond reducing agents such as dithiothreitol (IC50 approximately 0.4 mM) and beta-mercaptoethanol (IC50 approximately 1.1 mM). Dithiothreitol-induced inhibition of l-[3H]norepinephrine accumulation was reversed by exposing tissues to oxygen or to oxidizing agents such as potassium ferricyanide (0.03 mM) or 5,5'dithiobis-(2-nitrobenzoic acid) (1.0 mM). The cycle of reduction-induced inhibition and oxidation-induced reactivation could be repeated within tissues by sequential exposure to dithiothreitol and potassium ferricyanide. Exposing rat atrial to concentrations of dithiothreitol (1.0 mM) that produced substantial inhibition of l-[3H]norepinephrine accumulation (approximately 50%) did not affect spontaneous atrial rate or l-isoproterenol-induced increases in rate. In addition to disulfide bond reducing agents, sulfhydryl blocking agents such as N-ethylmaleimide (IC50 approximately 0.3 mM) and p-chloromercuriphenylsulfonic acid (IC50 approximately 0.2 mM) inhibited l-[3H]norepinephrine accumulation by mouse atria. Inhibition induced by sulfhydryl blockers could not be reversed by oxygen or by oxidizing agents. Furthermore, tissues exposed to dithiothreitol and subsequently exposed to N-ethylmaleimide could not be reactivated by oxygen or by oxidizing agent. The data suggest that a protein with a disulfide bond is associated with l-[3H]norepinephrine accumulation and that reduction of the disulfide bond inhibits amine accumulation. Reduced disulfide bonds that have not been alkylated can be reoxidized, but reduced disulfide bonds that are alkylated are irreversibly inactivated.
D000818:Animals; D001338:Autonomic Fibers, Postganglionic / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D006339:Heart Rate / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D009411:Nerve Endings / Q000378:metabolism*; D009638:Norepinephrine / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013564:Sympathetic Nervous System / Q000378:metabolism*
D002352:Carrier Proteins; D004220:Disulfides; D008565:Membrane Proteins; D013439:Sulfhydryl Reagents; D004229:Dithiothreitol; D009638:Norepinephrine