Measurement and modification of free calcium transients in frog skeletal muscle fibres by a metallochromic indicator dye.

Abstract

SUMMARY1. Myoplasmic free calcium transients were monitored with the metallochromic indicator dye Antipyrylazo III (AP III) in single frog skeletal muscle fibres cut at both ends, stretched so as to minimize or eliminate contractile filament overlap and voltage clamped using a double-Vaseline-gap system ( s 6 0C).2. The dye entered the central fibre segment by diffusion from the solution applied to the two cut ends. The diffusion coefficient of AP III was about 20 times lower in the fibre than in solution. This very slow diffusion was not due to binding of dye since the ratio of bound to free dye obtained from analysis of the diffusion was only about 0-45.3. For a given depolarizing pulse, the ratio of dye-related absorbance changes AA at 720 and 550 nm was the same as that produced on adding calcium to dye in calibrating solution, indicating that these signals were due to changes in myoplasmic calcium.4. The AA signals at 700 or 720 nm were used to monitor transient changes in concentration of calcium-dye complex [CaD2] and of free calcium [Ca] in the myofilament space.5. By applying the same pulse at different times during dye entry, it was observed that increasing dye concentrations [D]T produced the following effects: (a) [CaD2] was increased; (b) [Ca] was decreased at early times during a pulse; (c) a declining phase of [Ca] 10. It is concluded that AP III at relatively low concentrations is a practical monitor ofminimally modified calcium transients, whereas the modifications produced by higher dye concentrations can provide useful information about properties of both the dye in the fibre and the fibre itself.

Calcium inactivation of calcium release in frog cut muscle fibers that contain millimolar EGTA or Fura-2.

“…The fibers were mounted in a double Vaseline-gap chamber (Kov~cs, Rios, and Schneider, 1983;Irving, Maylie, Sizto, and Chandler, 1987), stretched to a sarcomere length of 3.4-4.2 ttm, and studied at 14-15~ with the experimental methods used previously in this laboratory. The optical apparatus and method for the estimation of indicator-related absorbance signals are described in Irving et al (1987) and Maylie, Irving, Sizto, and Chandler (1987).…”

Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres

“…Fibres were allowed to rest after enzymatic treatment for at least 20 min and only those that showed no signs of membrane damage were used. The selected fibre was gently sucked into a glass capillary and transferred into the recording chamber (Kov acs et al 1983) filled with relaxing solution (composition (mÒ): 150 potassium glutamate, 2 MgClµ, 10 Hepes, 1 EGTA).…”

“…1, left column) as described by Kov acs et al (1983) using the correction for intrinsic fibre absorbance at 850 nm following the procedure in Melzer et al (1986). Fura_2 fluorescence was used to calculate the resting [Ca¥]é.…”

Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibers containing antipyrylazo III.

“…The fibers were mounted in a double Vaseline-gap chamber, similar to that used by Kovacs et al (1983). To facilitate indicator entry, the surface membranes in the end pools were made permeable by a 2-rain exposure to 0.01% saponin (Endo and Iino, 1980).…”