Calcium transients in intact rat skeletal muscle fibers in agarose gel.
Intact single fibers enzymatically dissociated from rat flexor digitorum brevis muscle were suspended in 0.5% low-melting-temperature agarose gel to minimize fiber movement during action potentials or trains of action potentials. Resting Ca2+ concentration ([Ca2+]) and changes in [Ca2+] were monitored using the fluorescent calcium indicator fura 2. The time course and waveform of [Ca2+] transients during an action potential or trains of action potentials in fibers in agarose were calculated using kinetic parameters previously determined to correct for the calcium-fura 2 kinetic delay. Half times of the calculated calcium transients for single action potentials were 30-fold briefer than the original fura 2 signals. To confirm the time course and waveform of the calculated calcium transients, changes in [Ca2+] were monitored using the more rapidly equilibrating calcium indicator mag-fura 2. [Ca2+] transients for fibers containing fura 2 had very similar time courses and waveforms as mag-fura 2 signals from other fibers, indicating that the corrections for the calcium-fura 2 kinetic delay were accurate. The advantages of the agarose gel suspension are discussed.
Carroll
S L
SL
Department of Biological Chemistry, University of Maryland, School of Medicine, Baltimore 21201, USA.
Klein
M G
MG
Schneider
M F
MF
D000200:Action Potentials; D000818:Animals; D002118:Calcium / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D005782:Gels; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology*; D051381:Rats; D012685:Sepharose
D005456:Fluorescent Dyes; D005782:Gels; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D012685:Sepharose; D002118:Calcium; D016257:Fura-2