[Measuring real-time sarcoplasmic reticulum calcium pumping in skinned muscle fibers].
A method and a device for direct measurements of accumulation of calcium in the sarcoplasmic reticulum (SR) and its release from SR as a function of free Ca2+ in bath have been developed. About 30% of the volume inside muscle fibers of swimbladder of Opsanus tau is occupied by SR. A set of solutions was prepared for fiber dissection and making holes in outer membrane without destruction of membranes of the sarcoplasmic reticulum. Calcium was unloaded from SR using EGTA as a pCa buffer. Then solutions with 50-100 microM CaFURA2 or CabisFURA2 were used as pCa-buffer and fluorescent Ca-indicators for measurement of Ca exchange between a fiber with a volume of approximately 10 nl and a solution in the cuvette with a volume of 5 microl. An increase in fluorescence signified an increase in unbound FURA in the bath since Ca2+ pumped into the SR was removed from the bath. The slope represented the rate of Ca2+ uptake by the SR in the muscle fiber, the maximum being about 1.6 M/s per liter of solution in bath or 2.6 mM/s per liter of SR volume. In solutions without oxalate and Ruthenium Red, more Ca2+ was taken up by the SR, and oscillations of the bath free FURA level were often observed, which can be explained by calcium-induced calcium release.
Klimov
A A
AA
D000400:Air Sacs / Q000378:metabolism; D000818:Animals; D024541:Batrachoidiformes; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid; D005456:Fluorescent Dyes; D016257:Fura-2; D066298:In Vitro Techniques; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002021:Buffers; D005456:Fluorescent Dyes; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2