title: Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.
abstract: Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.
authors.affiliation: Department of Physiology, University of Pennsylvania Medical Center, Philadelphia 19104-6085.
authors.forename: M
authors.initials: M
authors.lastname: Konishi
authors.forename: S
authors.initials: S
authors.lastname: Hollingworth
authors.forename: A B
authors.initials: AB
authors.lastname: Harkins
authors.forename: S M
authors.initials: SM
authors.lastname: Baylor
mesh_terms: D000200:Action Potentials; D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D004396:Coloring Agents; D004058:Diffusion; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*; D010080:Oxazoles; D011896:Rana temporaria; D013050:Spectrometry, Fluorescence
chemical_list: D001572:Benzofurans; D004396:Coloring Agents; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010080:Oxazoles; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D008274:Magnesium; D002118:Calcium; D016257:Fura-2