Resumen

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.