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.

Fast activation and inactivation of inositol trisphosphate‐evoked Ca2+ release in rat cerebellar Purkinje neurones.

“…Normal external solution contained (mM): NaCl, 125; KCl, 2-5; CaCl2, 2; MgSO4, 1; NaHPO4, 1P25; NaHCO3,25;glucose,11; (Raju, Murphy, Levy, Hall & London, 1989) was used with the approach and methods described by Konishi, Hollingworth, Harkins & Baylor (1991 (Llano et al 1991 (Rapp & Guth, 1988 (Walker, Reid, McCray & Trentham, 1988). Full lamp output of 120 mJ in 1 ms at 280-360 nm produced 52 % photolysis of caged ATP and produces equivalent conversion of caged InsP3 ).…”

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

“…In the Results and Appendix A, this is denoted by A[Ca] (whereas in Appendices B-D, A [Ca] refers to local increases in free [Ca] that occur near SR Ca release sites). The A[Ca] signal was measured with the low affinity Ca indicator purpurate-3,3' diacetic acid (PDAA), which is expected to provide a reliable estimate of both the time course and amplitude of the free [Ca] transient (Hirota, Chandler, Southwick, and Waggoner, 1989;Konishi, Hollingworth, Harkins, and Baylor, 1991). The A[Ca] signal in Fig.…”

Membrane capacitance changes induced by thrombin and calcium in single endothelial cells cultured from human umbilical vein

“…Changes in cytosolic free [Ca¥] were measured with furaptra as previously described (Konishi et al 1991;Ogden et al 1995). Furaptra free acid (500 ìÒ) was introduced into the cell together with caged compounds (DM_nitrophen or caged InsP×) by diffusion from a patch pipette during whole-cell recording.…”

“…The two solutions were mixed to vary the DM_nitrophen:Cat ratio. The equilibrium free [Ca¥] and [Mg¥] for a given DM_nitrophen:Cat ratio were calculated using the Ca¥ and Mg¥ dissociation constants for DM_nitrophen (Ellis-Davies et al 1996), ATP (Sill en & Martell, 1971) and furaptra (Konishi et al 1991;Ogden et al 1995). For 4 mÒ DM_nitrophen: 2·4 mÒ Ca¥ these were Caf = 0·12 ìÒ and Mgf = 12·2 ìÒ, respectively (equilibrium Mg¥-ATP concentration 1·5 mÒ).…”