title: Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes.
abstract: We measured Ca2+ uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca2+ pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20 degreesC to 10 or 5 degreesC reduced the SR Ca2+ uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca2+ leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX), and the time integral of the exchange current (INCX) was used to calculate the SR Ca2+ content. This gave a steady-state SR Ca2+ content of 22.5 +/- 2.8 amol Ca2+/pF or 750 microM. When the SR was loaded by depolarizing the cell to +50 mV, the Ca2+ content increased with increasing length of the depolarization, reaching a maximum of 52.0 +/- 5.9 amol Ca2+/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Caf-induced INCX increased with increasing voltage. At +100 mV, the Ca2+ content was 36.6 +/- 3.8 amol/pF, giving a maximal SR Ca2+ uptake rate of 12.2 +/- 1.2 amol Ca2+. pF-1. s-1 or 417 microM/s. We conclude that maximal SR Ca2+ content and Ca2+ uptake rates can be estimated using specific SR Ca2+ loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca2+ fluxes, we found that although the L-type Ca2+ current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca2+ during the excitation-contraction coupling in trout ventricular myocytes.
authors.affiliation: Department of Physiology and Cell Biology, Faculty of Science, Universitat Autonoma de Barcelona, 08193 Cerdanyola, Barcelona, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Hove-Madsen
authors.forename: A
authors.initials: A
authors.lastname: Llach
authors.forename: L
authors.initials: L
authors.lastname: Tort
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002469:Cell Separation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006352:Heart Ventricles; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010539:Permeability; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D013696:Temperature; D019284:Thapsigargin / Q000494:pharmacology; D017186:Titrimetry; D016276:Ventricular Function / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D002110:Caffeine; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium