Excitation-contraction coupling in intact frog skeletal muscle fibers injected with mmolar concentrations of fura-2

Abstract

Experiments were carried out to test the hypothesis that mM concentrations of fura-2, a high-affinity Ca2+ buffer, inhibit the release of Ca2+ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers. Intact twitch fibers from frog muscle, stretched to a long sarcomere length and pressure-injected with fura-2, were activated by an action potential. Fura-2's absorbance and fluorescence signals were measured at different distances from the site of fura-2 injection; thus, the myoplasmic free Ca2+ transient (delta [Ca2+]) and the amount and rate of SR Ca2+ release could be estimated at different myoplasmic concentrations of fura-2 ([fura-2T]). At [fura-2T] = 2-3 mM, the amplitude and half-width of delta [Ca2+] were reduced to approximately 25% of the values measured at [fura-2T] less than 0.15 mM, whereas the amount and rate of SR Ca2+ release were enhanced by approximately 50% (n = 5; 16 degrees C). Similar results were observed in experiments carried out at low temperature (n = 2; 8.5-10.5 degrees C). The finding of an enhanced rate of Ca2+ release at 2-3 mM [fura-2T] is opposite to that reported by Jacquemond et al. (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophys. J. 60:867-873) from analogous experiments carried out on cut fibers. In two experiments involving the injection of larger amounts of fura-2, reductions in SR Ca2+ release were observed; however, we were unable to decide whether these reductions were due to [fura-2T] or to some nonspecific effect of the injection itself. These experiments do, however, suggest that if large [fura-2T] inhibits SR Ca2+ release in intact fibers, [fura-2T] must exceed 6 mM to produce an effect comparable to that reported by Jacquemond et al. in cut fibers. Our clear experimental result that 2-3 mM [fura-2T] enhances SR Ca2+ release supports the proposal that delta [Ca2+] triggered by an action potential normally feeds back to inhibit further release of Ca2+ from the SR (Baylor, S.M., and S. Hollingworth. 1988. J. Physiol. [Lond.]. 403:151-192). Our results provide no support for the hypothesis that Ca(2+)-induced Ca2+ release plays a significant role in excitation-contraction coupling in amphibian skeletal muscle.

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

“…A4. It also suggests that, similar to the results obtained with 0.5-2 mM fura-2 Hollingworth et al, 1992;Pape et al, 1993), suppression of the increase in spatially averaged myoplasmic free [Ca] by EGTA does not affect the onset of SR Ca release.…”

“…In the experiments reported here, the mean value of k-2, the dissociation rate constant for Ca and fura-2, was 29 s -1 (SEM, 4 s -1) at 14-15~ This value is larger than the values estimated by Klein et al (1988), 12 s -1 in cut fibers at 6-10~ and by Hollingworth et al (1992), 17 s -1 in intact fibers at 16~ It is smaller than that measured by Kao and Tsien (1988) in a 140 mM KC1 solution at 20~ 97 s -1. Pape et al (1993) estimated the value of k~, the association rate constant for Ca and fura-2, in experiments on cut fibers equilibrated with fura-2 and PDAA and studied under conditions similar to those used here.…”

“…A6 gives a value of 0.41 fl, M for the apparent dissociation constant of Ca and fura-2. This value lies between two other estimates of the apparent dissociation constant inside muscle fibers: 0.32 ~M (estimated from data obtained from cut fibers by Klein et al, 1988, after allowance for the fact that antipyrylazo III is expected to underestimate the amplitude of the [Ca] transient by a factor of about 4; see Table II in Pape et al, 1993) and 0.68 fl, M (estimated in intact fibers by Hollingworth et al, 1992). All of these estimates of the dissociation constant of Ca and fura-2 in myoplasm are substantially larger than that measured by Kao and Tsien (1988) in a 140 mM KCI solution at 20~ 0.162 ~M.…”

“…This suggests that the duration of the dA [CaT]/dt waveform obtained with EGTA-phenol red is 0.7-0.9 ms longer than that calculated from free A[Ca] signals measured with low affinity Ca indicators and 0.1 mM EGTA. In intact fibers also studied with action-potential stimulation, Baylor and Hollingworth (1988) and Hollingworth et al (1992) observed a similar prolongation with 0.5-2 mM fura-2, a finding that appeared to be correlated with an increase in the value of A[CaT].…”

“…10 A), the larger value of A [CaT] in our experiments may reflect a larger SR Ca content. On the other hand, if the SR Ca contents were similar in all of the experiments, the differences could be due to a genuine increase in SR Ca release caused by 20 mM EGTA, similar to that ob-served with 0.5-2 mM fura-2 and attributed to a decrease in Ca inactivation of Ca release (Baylor and HoUingworth, 1988;Hollingworth, Harkins, Kurebayashi, Konishi, and Baylor, 1992;Pape et al, 1993).…”