Brazilian Journal of Medical and Biological Research
versión On-line ISSN 1414-431X
BASSANI, R.A. y BASSANI, J.W.M.. Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation. Braz J Med Biol Res [online]. 2003, vol.36, n.12, pp. 1717-1723. ISSN 1414-431X. http://dx.doi.org/10.1590/S0100-879X2003001200014.
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 µM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM). 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.
Palabras llave : Myocardium; Relaxation; Calcium fluxes; Sodium-calcium exchanger; Sarcoplasmic reticulum calcium ATPase; Caffeine.