title: Reactive disulfides trigger Ca2+ release from sarcoplasmic reticulum via an oxidation reaction.
abstract: Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to the S-S bond such as 2,2'-dithiodipyridine (2,2'-DTDP), 4,4'-dithiodipyridine, and N-succinimidyl 3(2-pyridyldithio)propionate (SPDP) trigger Ca2+ release from sarcoplasmic reticulum (SR) vesicles. They are known to specifically oxidize free SH sites via a thiol-disulfide exchange reaction with the stoichiometric production of thiopyridone. Thus, the formation of a mixed S-S bond between an accessible SH site on an SR protein and a RDS causes large increases in SR Ca2+ permeability. Reducing agents, glutathione (GSH) or dithiothreitol reverse the effect of RDSs and permit rapid re-uptake of Ca2+ by the Ca2+, Mg2+-ATPase. The RDSs, 2,2'-DTDP, 4,4'-dithiodipyridine and SPDP displaced [3H]ryanodine binding to the Ca2+-receptor complex at IC50 values of 7.5 +/- 0.2, 1.5 +/- 0.1, and 15.4 +/- 0.1 microM, respectively. RDSs did not alter the rapid initial phase of Ca2+ uptake by the pump, stimulated ATPase activity, and induced release from passively loaded vesicles with nonactivated pumps; thus they act at a Ca2+ release channel and not at the Ca2+, Mg2+-ATPase. Efflux rates increased in 0.25-1.0 mM [Mg2+]free then decreased in 2-5 mM [Mg2+]free. Adenine nucleotides inhibited the oxidation of SHs on SR protein by RDSs and thus reduced Ca2+ efflux rates. However, once RDSs oxidized these SH sites and opened the Ca2+ release pathway, subsequent additions of nucleotides stimulated Ca2+ efflux. In skinned fibers, 2,2'-dithiodipyridine elicited rapid twitches which were blocked by ruthenium red. These results indicate that RDSs trigger Ca2+ release from SR by oxidizing a critical SH group, and thus provide a method to covalently label the protein(s) involved in causing these changes in Ca2+ permeability.
authors.affiliation: Department of Physiology, School of Medicine, University of Pittsburgh, Pennsylvania 15261.
authors.forename: N F
authors.initials: NF
authors.lastname: Zaidi
authors.forename: C F
authors.initials: CF
authors.lastname: Lagenaur
authors.forename: J J
authors.initials: JJ
authors.lastname: Abramson
authors.forename: I
authors.initials: I
authors.lastname: Pessah
authors.forename: G
authors.initials: G
authors.lastname: Salama
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D004220:Disulfides / Q000494:pharmacology*; D066298:In Vitro Techniques; D007537:Isometric Contraction / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D004220:Disulfides; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium