Reactive disulfide compounds induce Ca2+ release from cardiac sarcoplasmic reticulum.
Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to a disulfide bond, 2,2'dithiodipyridine (2,2' DTDP) and 4,4' dithiodipyridine (4,4' DTDP), induce Ca2+ release from isolated canine cardiac sarcoplasmic reticulum (SR) vesicles. RDSs are absolutely specific to free sulfhydryl (SH) groups and oxidize SH sites of low pKa via a thiol-disulfide exchange reaction, with the stoichiometric production of thiopyridone in the medium. As in skeletal SR, this reaction caused large increases in the Ca2+ permeability of cardiac SR and the number of SH sites oxidized by RDSs was kinetically and quantitatively measured through the absorption of thiopyridone. RDS-induced Ca2+ release from cardiac SR was characterized and compared to the action of RDSs on skeletal SR and to Ca2(+)-induced Ca2+ release. (i) RDS-induced Ca2+ release from cardiac SR was dependent on ionized Mg2+, with maximum rates of release occurring at 0.5 and 1 mM Mg2+free for 2,2' DTDP and 4,4' DTDP, respectively. (ii) In the presence of adenine nucleotides (0.1-1 mM), the oxidation of SH sites in cardiac SR by exogenously added RDS was inhibited, which, in turn, inhibited Ca2+ release induced by RDSs. (iii) Conversely, when the oxidation reaction between RDSs and cardiac SR was completed and Ca2+ release pathways were opened, subsequent additions of adenine nucleotides stimulated Ca2+ efflux induced by RDSs. (iv) Sulfhydryl reducing agents (e.g., dithiothreitol, DTT, 1-5 mM) inhibited RDS-induced Ca2+ efflux in a concentration-dependent manner. (v) RDSs elicited Ca2+ efflux from passively loaded cardiac SR vesicles (i.e., with nonfunctional Ca2+ pumps in the absence of Mg-ATP) and stimulated Ca2(+)-dependent ATPase activity, which indicated that RDS uncoupled Ca2+ uptake and did not act at the Ca2+, Mg2(+)-ATPase. These results indicate that RDSs selectively oxidize critical sulfhydryl site(s) on or adjacent to a Ca2+ release channel protein channel and thereby trigger Ca2+ release. Conversely, reduction of these sites reverses the effects of RDSs by closing Ca2+ release channels, which results in active Ca2+ reuptake by Ca2+, Mg2(+)-ATPase. These compounds can thus provide a method to covalently label and identify the protein involved in Ca2+ release from cardiac SR.
Prabhu
S D
SD
University of Pittsburgh, School of Medicine, Department of Physiology, Pennsylvania 15261.
Salama
G
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D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000227:Adenine Nucleotides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004285:Dogs; D004347:Drug Interactions; D008274:Magnesium / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D000227:Adenine Nucleotides; D004220:Disulfides; D011725:Pyridines; D013439:Sulfhydryl Reagents; C001105:2,2'-dipyridyl disulfide; C007542:4,4'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium; D004229:Dithiothreitol