Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide.

Abstract

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the endpool solutions . The indicator diffused rapidly to the central region of a fiber where optical recording was done and, if removed, diffused away equally fast. The time course of concentration suggests that, on average, a fraction 0.27 of indicator was reversibly bound to myoplasmic constituents and the free diffusion constant was 1 .75 X 10-' cm2/s at 18°C . The shape of the resting absorbance spectrum suggests that a fraction 0 .11-0.15 of tetramethylmurexide inside a fiber was complexed with Ca . After action potential stimulation, there was a rapid transient change in indicator absorbance followed by a maintained change of opposite sign . The wavelength dependence of both changes matched a cuvette Ca-difference spectrum. The amplitude of the early peak varied linearly with indicator concentration and corresponded to an average rise in free [Ca] of 17 uM . These rather diverse findings can be explained if the sarcoplasmic reticulum membranes are permeable to Ca-free indicator . Both Ca-free and Ca-complexed indicator inside the sarcoplasmic reticulum would appear to be bound by diffusion analysis and the Ca-complexed form would be detected by the resting absorbance spectrum . The transient change in indicator absorbance would be produced by myoplasmic Ca reacting with indicator molecules that freely diffuse in myoplasmic solution . The maintained signal, which reports Ca dissociating from indicator complexed at rest, would come from changes within the sarcoplasmic reticulum . A method, based on these ideas, is described for separating the two components of the tetramethylmurexide signal . The estimated myoplasmic free [Ca] transient has an average peak value of 26 yM at 18°C . Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Mayliej, M . Irving, N .

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

“…1 B, the signal undershot the baseline and appeared to reach a final level of ~-1 ~M. This undershoot was a consistent finding in our experiments with PDAA and EGTA and is reminiscent of the undershoot observed with tetramethylpurpurate (Maylie, Irving, Sizto, Boyarski, and Chandler, 1987a) and with PDAA in the presence of millimolar concentrations of fura-2 . Although the origin of this undershoot is unknown, it seems unlikely that it represents a genuine negative value of myoplasmic A [Ca].…”

“…In spite of these reductions, many properties of SR Ca release--such as the amount of release after an action potential, the peak rate of release, and its time course--are similar in a general way to those estimated previously in cut fibers with unmodified free [Ca] transients (Maylie et al, 1987a, c, Hirota et al, 1989Pape et al, 1993). Our voltage-clamp experiments suggest that the only important effect exerted by 20 mM EGTA on the SR Ca release mechanism is a reduction in Ca inactivation of Ca release.…”

“…The reason is that, after sufficient Ca has been released to saturate the myoplasmic Ca buffers, free [Ca] increases and inhibits additional release because of Ca inactivation of Ca release (Baylor et al, 1983;Simon et al, , 1991Baylor and Hollingworth, 1988;Schneider and Simon, 1988). Column 7 in Table II Table II. III, tetramethylpurpurate, and PDAA in cut fibers with only 0.1 mM EGTA in the end-pool solutions (Maylie et al, 1987a. …”

“…Previous estimates of the amount of Ca released by an action potential in cut fibers have been made with small concentrations of low affinity Ca indicators and only 0.1 mM EGTA in the end-pool solutions: 333 ~M (SEM, 8 IxM) with antipyrylazo III (high Ca calibration, Table VII in Maylie et al, 1987c), 317 IxM with tetramethylpurpurate (SEM, 5 ~M) (page 168 in Maylie et al, 1987a) and 294 IxM (SEM, 12 ~M) with PDAA (page 321 in Pape et al, 1993). These values are all somewhat smaller than the mean value in our experiments, 375 ixM.…”

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

“…In cut fibers, the concentration of phosphocreatine at the optical recording site should be less than this, owing to exchange with the end-pool solutions. Phospbocreatine has about the same molecular weight as tetramethylmurexide and therefore might be expected to have about the same diffusion constant inside a fiber, estimated as 1.75 x 10 -8 cm2/s at 18~ (corrected for binding or compartmentalization, Table II, column 6 in Maylie et al, 1987a). According to the solution of the one-dimensional diffusion equation appropriate for our experimental conditions (Eq.…”