The use of the indicator fluo-5N to measure sarcoplasmic reticulum calciumin single muscle fibres of the cane toad

1. Single fibres from the lumbrical muscles of the cane toad (Bufo marinus) were incubated in fluo-5N AM for 2 h at 35 degreesC in order to load the i ndicator into the sarcoplasmic reticulum. Fluo-5N is a low-affinity calcium indicator (K-Ca 90 muM). Successful sarcoplasmic reticulum (SR) loading wa s indicated by a fluorescence signal that declined during contraction. 2. Confocal microscopy showed that the dye loaded principally in Lines perp endicular to the long axis of the fibre that repeated each sarcomere. This is consistent with much of the dye residing in the BR. 3. To establish the site of loading, fibres were exposed to 30 mM caffeine in the presence of 20 muM 2,5-di( tert-butyl)1,4-hydroquinone (TBQ, an SR p ump inhibitor) which should release most Ca2+ from the SR; this procedure r educed the fluorescence to 46 +/- 4% of the control value. To determine how much indicator was in the myoplasm, fibres were exposed to 100 mug ml(-1) saponin which permeabilizes the surface membrane; saponin treatment reduced the fluorescence to 51 +/- 2 % of the control value. 4. During maximally activated tetani (100 Hz stimulation rate, 22 degreesC) the component of signal from the SR declined by 33 +/- 4%. During relaxati on the SR signal recovered in two phases with time constants of 0.38 +/- 0. 14 s and 10.1 +/- 1.7 s. Partially activated tetani (30 Hz stimulation rate ) showed a smaller SR signal. Application of the SR Ca2+ pump inhibitor TBQ slowed the rate of recovery of the SR signal. 5. Muscle fatigue was produced by repeated short tetani until tension was r educed to 50%. The SR signal during the periods between tetani declined ste adily and the SR Ca2+ signal was eventually reduced to 71 +/- 8 % of the co ntrol signal. This signal recovered in two phases when the muscle was reste d. An initial phase had a time constant of 1.7 +/- 0.2 s so that by 20 s of recovery the SR Ca2+ signal was 86 +/- 7 % of control; the second phase wa s slower and by 5 min the SR Ca2+ signal was back to control values (98 +/- 5% control). In addition the magnitude of the SR signal decline associated with each tetanus (Delta [Ca2+](SR)) declined monotonically throughout fat igue and returned to control after 5 min recovery. 6. This approach can mon itor the SR Ca2+ concentration in normally functioning muscle fibres with g ood time resolution. The method confirms other approaches that show that th e free Ca2+ available for release in the SR declines during fatigue. This r eduction in [Ca2+](SR) will contribute to the failure of Ca2+ delivery to t he myofilaments which is an important cause of muscle fatigue.