Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae.
This study was designed to measure the Ca2+ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca2+ (100 nM), in the absence and presence of caffeine. Measurements of [Ca2+] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5-40 mM) into this volume caused an initial release of Ca2+ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca2+. Ca2+ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca2+ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5-40 mM) and 100 nM Ca2+ indicated that the SR's ability to retain Ca2+ depends inversely on the dose of caffeine. The relative Ca2+ content of the SR after preincubation with caffeine was 86.7+/-3.5% at a caffeine concentration of 5 mM, 62.5+/-5.1% at 10 mM caffeine, 37.8+/-8.1% at 20 mM caffeine and 7. 1+/-1.9% at 40 mM caffeine. Measurement of the SR Ca2+ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca2+-binding capacity of the preparation (equivalent to 245+/-10 microM BAPTA) and (2) the Ca2+ content of the SR accessed by caffeine after equilibration with 100 nM Ca2+ (186+/-11 micromol/l cell volume or 5.6 mmol/l SR volume).
Smith
G L
GL
Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow, G12 8QQ, UK. g.smith@bio.gla.ac.uk
Steele
D S
DS
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002463:Cell Membrane Permeability; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012503:Saponins; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism*
D002614:Chelating Agents; D012503:Saponins; D002110:Caffeine; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium