title: Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation.
abstract: Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7  M), whereas SR-dependent flux was lower with TG-TW (77 vs 81  M). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.
authors.affiliation: Centro de Engenharia Biomédica, Faculdade de Engenharia Elétrica e de Computação, Universidade Estadual de Campinas, Campinas, SP, Brasil. rosana@ceb.unicamp.br
authors.forename: R A
authors.initials: RA
authors.lastname: Bassani
authors.forename: J W M
authors.initials: JW
authors.lastname: Bassani
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D008297:Male; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology; D019831:Sodium-Calcium Exchanger / Q000187:drug effects / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D019831:Sodium-Calcium Exchanger; D002110:Caffeine; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium