scholarly journals Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide.

1987 ◽  
Vol 89 (1) ◽  
pp. 145-176 ◽  
Author(s):  
J Maylie ◽  
M Irving ◽  
N L Sizto ◽  
G Boyarsky ◽  
W K Chandler
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Time Course ◽  
Diffusion Constant ◽  
Wavelength Dependence ◽  
Difference Spectrum ◽  
Optical Recording ◽  
Absorbance Spectrum ◽  
Transient Change ◽  
Early Peak ◽  
Average Rise

The Ca indicator tetramethylmurexide was introduced into cut fibers, mounted in a double-Vaseline-gap chamber, by diffusion from the end-pool 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(-6) cm2/s at 18 degrees 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 microM. 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 microM at 18 degrees C. Its time course is similar to, but possibly faster than, that recorded with antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143).

scholarly journals Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III.

1987 ◽  
Vol 89 (1) ◽  
pp. 83-143 ◽  
Author(s):  
J Maylie ◽  
M Irving ◽  
N L Sizto ◽  
W K Chandler
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Action Potential ◽  
Time Course ◽  
Wavelength Dependence ◽  
Difference Spectrum ◽  
Half Width ◽  
Arsenazo Iii ◽  
Transverse Tubular System ◽  
Low Amplitude ◽  
Regulatory Sites

The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-81). Like arsenazo III, antipyrylazo III was largely bound to or sequestered by intracellular constituents; on average, a fraction 0.68 was so immobilized. After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18 degrees C. The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1:2 stoichiometry for Ca:indicator complexation and, for indicator concentrations less than or equal to 0.4 mM, constant peak free [Ca]. If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 microM at 18 degrees C. If only freely diffusible indicator can react, the estimate is 42 microM. The true amplitude probably lies somewhere in between. The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3- and 42-microM myoplasmic [Ca] transients. After action potential stimulation, the release waveform is rapid and brief; its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms. This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum. The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-microM transient and 0.93 for the 42-microM transient. During a 100-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0.94. The low value of occupancy associated with the low-amplitude [Ca] calibration seems inconsistent with a brief tetanus being able to produce near-maximal activation (Blinks, J. R., R. Rudel, and S. R. Taylor. 1978. Journal of Physiology. 277:291-323; Lopez J. R., L. A. Wanck, and S. R. Taylor. 1981. Science. 214:47-82).

scholarly journals Comparison of arsenazo III optical signals in intact and cut frog twitch fibers.

1987 ◽  
Vol 89 (1) ◽  
pp. 41-81 ◽  
Author(s):  
J Maylie ◽  
M Irving ◽  
N L Sizto ◽  
W K Chandler
Keyword(s):  
Action Potential ◽  
Time Course ◽  
Physiological State ◽  
Diffusion Constant ◽  
Wavelength Dependence ◽  
Optical Recording ◽  
Arsenazo Iii ◽  
Optical Signals ◽  
Indicator Concentration ◽  
Electrical Condition

The Ca indicator arsenazo III was introduced into cut frog twitch fibers by diffusion from end-pool segments rendered permeable by saponin. After 2-3 h, the arsenazo III concentration at the optical recording site in the center of a fiber reached two to three times that in the end-pool solutions. Thus, arsenazo III was bound to or taken up by intracellular constituents. The time course of indicator appearance was fitted by equations for diffusion plus linear reversible binding; on average, 0.73 of the indicator was bound and the free diffusion constant was 0.86 x 10(-6) cm2/s at 18 degrees C. When the indicator was removed from the end pools, it failed to diffuse away from the optical site as rapidly as it had diffused in. The wavelength dependence of resting arsenazo III absorbance was the same in cut fibers and injected intact fibers. After action potential stimulation, the active Ca and dichroic signals were similar in the two preparations, which indicates that arsenazo III undergoes the same changes in absorbance and orientation in both cut and intact fibers. Ca transients in freshly prepared cut fibers appeared to be similar to those in intact fibers. As a cut fiber experiment progressed, however, the Ca signal changed. With action potential stimulation, the half-width of the signal gradually increased, regardless of whether the indicator concentration was increasing or decreasing. This increase was usually not accompanied by any change in the amplitude of the Ca signal at a given indicator concentration or by any obvious deterioration in the electrical condition of the fiber. In voltage-clamp experiments near threshold, the relation between peak [Ca] and voltage usually became less steep with time and shifted to more negative potentials. All these changes were also observed in cut fibers containing antipyrylazo III (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:83-143). They are considered to represent a progressive change in the physiological state of a cut fiber during the time course of an experiment.

scholarly journals Calcium signals recorded from two new purpurate indicators inside frog cut twitch fibers.

1989 ◽  
Vol 94 (4) ◽  
pp. 597-631 ◽  
Author(s):  
A Hirota ◽  
W K Chandler ◽  
P L Southwick ◽  
A S Waggoner
Keyword(s):  
Action Potential ◽  
Time Course ◽  
Wavelength Dependence ◽  
Difference Spectrum ◽  
Reliable Estimate ◽  
Spatial Average ◽  
Diacetic Acid ◽  
Absorbance Spectrum ◽  
Average Amplitude ◽  
Apparent Dissociation Constant

Two new Ca indicators, purpurate-3,3'diacetic acid (PDAA) and 1,1'-dimethylpurpurate-3,3'diacetic acid (DMPDAA), were synthesized and used to measure Ca transients in frog cut muscle fibers. These indicators are analogues of the purpurate components of murexide and tetramethylmurexide, in which two acetate groups have been incorporated into each molecule to render it membrane impermeant. The apparent dissociation constant for Ca is 0.95 mM for PDAA and 0.78 mM for DMPDAA. One of the indicators was introduced into a cut fiber, which was mounted in a double Vaseline-gap chamber, by diffusion from the end-pool solutions. The time course of indicator concentration, monitored optically in the middle of the fiber in the central-pool region, suggests that 19% of the PDAA or 27% of the DMPDAA became bound or sequestered inside the fiber. In resting fibers, the absorbance spectrum of either indicator was well fitted by the indicator's [Ca] = 0 mM cuvette absorbance spectrum, which is consistent with the idea that PDAA and DMPDAA do not enter the sarcoplasmic reticulum as tetramethylmurexide appears to be able to do (Maylie, J., M. Irving, N.L. Sizto, G. Boyarsky, and W. K. Chandler, 1987. Journal of General Physiology. 89:145-176). After an action potential, the absorbance of either indicator underwent a rapid and transient change that returned to the prestimulus baseline within 100-200 ms. The amplitude of this change had a wavelength dependence that matched the indicator's Ca-difference spectrum. The average amplitude of peak free [Ca] was 21 microM (PDAA or DMPDAA) if all the indicator inside a fiber was able to react with Ca as in cuvette calibrations, and was 26 (PDAA) or 28 microM (DMPDAA) if only freely diffusible indicator could so react. These results suggest that PDAA and DMPDAA are the first Ca indicators that provide a reliable estimate of both the amplitude and time course of (the spatial average of) free [Ca] in a twitch muscle fiber after an action potential.

Parvalbumin relaxes frog skeletal muscle when sarcoplasmic reticulum Ca(2+)-ATPase is inhibited

1996 ◽  
Vol 270 (2) ◽  
pp. C411-C417 ◽  
Author(s):  
Y. Jiang ◽  
J. D. Johnson ◽  
J. A. Rall
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Relaxation Rate ◽  
Time Course ◽  
Muscle Fibers ◽  
Peak Force ◽  
Half Time ◽  
Frog Skeletal Muscle ◽  
Twitch Force ◽  
Total Failure

Inhibition of sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ) in frog skeletal muscle fibers at 10 degrees C prolonged the half time of the fall of the Ca2+ transient by 62% and twitch force by 100% and increased peak force by 120% without increasing the amplitude of the Ca2+ signal. In the presence of TBQ the rate of relaxation and the rate of fall of Ca2+ became progressively slower in a series of twitches until relaxation failed. Relaxation rate decreased with a time course (approximately 2 s-1) similar to the Mg2+ off rate from purified parvalbumin (PA; 3.6 s-1). TBQ slowed the rate of fall of Ca2+ (5-fold) and force (8-fold) in a 0.3-s tetanus so that the rate of fall of Ca2+ (approximately 2.5 s-1) was similar to the Mg2+ off rate from PA. TBQ caused a near total failure of both Ca2+ sequestration and relaxation in a 1.1-s tetanus, during which PA would be saturated with Ca2+ and could not contribute to relaxation. Thus, when the SR Ca(2+)-ATPase is inhibited, Mg(2+)-PA can sequester Ca2+ and produce relaxation at a rate that is defined by the Mg2+ off rate from PA.

scholarly journals Properties of chloride-stimulated 45Ca flux in skinned muscle fibers.

1978 ◽  
Vol 71 (4) ◽  
pp. 411-430 ◽  
Author(s):  
E W Stephenson
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Time Course ◽  
Isometric Force ◽  
Frog Muscle ◽  
Published Data ◽  
Skinned Muscle ◽  
Skinned Muscle Fibers ◽  
45Ca Efflux ◽  
45Ca Release ◽  
Stimulation Of

Isometric force and 45Ca loss from fiber to bath were measured simultaneously in skinned fibers from frog muscle at 19 degrees C. In unstimulated fibers, 45Ca efflux from the sarcoplasmic reticulum (SR) was very slow, with little or no dependence on EGTA (0.1-5 mM) or Mg++ (20 micrometer-1.3 mM). Stimulation by high [Cl] at 0.11 mM Mg++ caused rapid force transients (duration approximately 10 s) and 45Ca release. This response was followed for 55 s, with 5 mM EGTA added to chelate myofilament space (MFS) Ca either (a) after relaxation, (b) near the peak of the force spike, or (c) before or with the stimulus. When EGTA was present during Cl application, stimulation of 45Ca release was undetectable. Analysis of the time-course of tracer loss during the three protocols showed that when EGTA was absent, 16% of the fiber tracer was released from the SR within approximately 3 s, and 70% of the tracer still in the MFS near the peak of the force spike was subsequently reaccumulated. The results suggest that (a) the Cl response is highly Ca-dependent; (b) stimulation increases 45Ca efflux from the SR at least 100-200-fold; and (c) the rate of reaccumulation is much slower than the influx predicted from published data on resting fibers, raising the possibility that depolarization inhibits active Ca transport by the SR.

scholarly journals Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers.

1990 ◽  
Vol 96 (3) ◽  
pp. 473-491 ◽  
Author(s):  
S Hollingworth ◽  
S M Baylor
Keyword(s):  
Time Course ◽  
Polarized Light ◽  
Weighted Average ◽  
Wavelength Dependence ◽  
Fiber Axis ◽  
Phenol Red ◽  
Ph Indicator ◽  
Similar Time ◽  
Average Value ◽  
Absorbance Changes

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.

PARADOXICAL RESPONSE OF PLASMA CORTISOL TO PULSE INTRAVENOUS INJECTION OF SYNTHETIC CORTICOTROPHIN AND DIBUTYRYL CYCLIC ADENOSINE 3′,5′-MONOPHOSPHATE IN ADULT PANHYPOPITUITARISM

1973 ◽  
Vol 74 (2) ◽  
pp. 250-262 ◽  
Author(s):  
Alberto Angeli ◽  
Roberto Frajria ◽  
Giuseppe Boccuzzi ◽  
Daniela Bisbocci ◽  
Franco Ceresa
Keyword(s):  
Plasma Cortisol ◽  
Time Course ◽  
Normal Subjects ◽  
Specific Cell ◽  
Acute Administration ◽  
Cortisol Response ◽  
Experimental Conditions ◽  
Paradoxical Response ◽  
Early Peak ◽  
Membrane Bound

ABSTRACT 250 μg of synthetic β-1-24 corticotrophin and 10 mg/kg of dibutyryl cyclic adenosine 3′,5′-monophosphate (Db-cAMP) were iv pulse injected into six patients with adult panhypopituitarism. The plasma cortisol levels were determined as 11-OHCS at zero time and then at 2.5, 5, 7.5, 10, 15, 30, 60 and 180 min after the injection. The data were compared with those obtained under the same experimental conditions in groups of normal subjects. A paradoxical pattern of early plasma cortisol response after the two stimuli was found in the case of hypopituitarism in comparison with that observed in normal subjects, that is a higher response after Db-cAMP than after corticotrophin. This was dependent on a selective impairment of adrenal steroidogenic response to iv injection of corticotrophin, whereas the response to iv injection of Db-cAMP was normal. Moreover in hypopituitaric patients the time course of cortisol response after ACTH was clearly different from the normal. The plasma 11-OHCS levels never showed an early peak followed by a fall and a second subsequent rise but rose progressively after the injection to a plateau between 15 and 180 min. By contrast, after Db-cAMP the time course of the response was also similar to that observed in normal subjects. It is suggested that after chronic failure of ACTH the reduced cortisol response to iv acute administration of corticotrophin may also depend on a defect localized before the synthesis of intracellular cyclic AMP, i. e. in the binding between ACTH and the specific cell membrane bound receptor and/or in the inactivation of the membrane associated adenyl cyclase.

scholarly journals Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca2+ release during the quasi-steady level of release in twitch fibers from frog skeletal muscle

2012 ◽  
Vol 140 (4) ◽  
pp. 403-419 ◽  
Author(s):  
Karine Fénelon ◽  
Cédric R.H. Lamboley ◽  
Nicole Carrier ◽  
Paul C. Pape
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Hill Coefficient ◽  
Free Calcium ◽  
Rapid Decline ◽  
Cooperative Binding ◽  
Phenol Red ◽  
Physiological Level ◽  
Steady Level ◽  
Early Peak ◽  
Binding Component

Experiments were performed to characterize the properties of the intrinsic Ca2+ buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([CaT]SR and [Ca2+]SR) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca2+ indicator). Results indicate SR Ca2+ buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca2+. Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca2+]SR and [CaT]SR are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca2+ permeability of the SR, namely d[CaT]SR/dt ÷ [Ca2+]SR (denoted release permeability), in experiments in which only [CaT]SR or [Ca2+]SR is measured. In response to a voltage-clamp step to −20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ∼50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca2+ release of 2.3 SR Ca2+ release channels neighboring each channel activated by its associated voltage sensor. Release permeability at −60 mV increases as [CaT]SR decreases from its resting physiological level to ∼0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca2+]SR inhibits release when [CaT]SR declines to a low level.

Ischemia-induced alterations in sarcoplasmic reticulum Ca(2+)-ATPase activity in rat soleus and EDL muscles

1996 ◽  
Vol 271 (6) ◽  
pp. C1942-C1948 ◽  
Author(s):  
H. J. Green ◽  
N. H. McKee ◽  
A. J. Carvalho ◽  
J. C. Dossett-Mercer
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Time Course ◽  
Extensor Digitorum Longus ◽  
Fiber Type ◽  
Time Dependent ◽  
Fiber Type Composition ◽  
Type Composition ◽  
Edl Muscle ◽  
Muscle Fiber Type Composition

To investigate the time-dependent effects of ischemia, as modified by muscle fiber type composition, on sarcoplasmic reticulum (SR) function, Ca(2+)-ATPase activity (total minus basal) was measured in homogenates prepared from samples obtained from rat soleus and extensor digitorum longus (EDL) muscle of ischemic and contralateral controls. Ischemia was induced by occlusion of blood flow to one hindlimb for periods of 1, 2, and 3 h (n = 10 per group). In EDL, maximal Ca(2+)-ATPase activity (expressed in mumol.g wet wt-1.min-1) was higher (P < 0.05) in ischemic than in control at 1 h (80 +/- 10 vs. 56.5 +/- 5.3) and increased progressively with ischemia at both 2 h (88 +/- 4.6 vs. 53.1 +/- 2.8) and 3 h (116 +/- 3.8 vs. 67.8 +/- 3.2). In contrast, in soleus, increases (P < 0.05) in Ca(2+)-ATPase activity with ischemia were observed at 2 h (19.2 +/- 0.86 vs. 14.0 +/- 0.56) and 3 h (19.9 +/- 1.4 vs. 12.4 +/- 0.62) but not at 1 h (10.7 +/- 1.5 vs. 10.0 +/- 0.83). In both EDL and soleus, basal Mg(2+)-ATPase was unchanged with ischemia. On the basis of these findings, it can be concluded that ischemia results in an increase in the maximal SR Ca(2+)-ATPase activity but that the time course of the change is dependent on the fiber type composition of the muscle.

Contraction and sarcoplasmic reticulum Ca2+ content in single myocytes of guinea pig heart: effect of ryanodine

1990 ◽  
Vol 259 (4) ◽  
pp. H1222-H1229 ◽  
Author(s):  
B. Lewartowski ◽  
R. G. Hansford ◽  
G. A. Langer ◽  
E. G. Lakatta
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocardium ◽  
Short Exposure ◽  
Isolated Cells ◽  
Guinea Pig Heart ◽  
Time To Peak ◽  
The Relationship ◽  
Steady State Amplitude

The relationship between the ability of sarcoplasmic reticulum (SR) to accumulate and retain Ca2+ and the electrically stimulated contractions (ESCs) of isolated cells from guinea pig ventricular myocardium was investigated. Caffeine contractures or rapid cooling contractures were used as a relative measure of the SR Ca2+ content. Depletion of SR Ca2+ by short exposure to caffeine (15 mM) or by prolonged rest resulted in a reduction of the amplitude of the ESCs by 83 +/- 14 and 65 +/- 11% (means +/- SD), respectively. This result points to SR as a major source of the Ca2+ that activates contraction. However, depriving the SR of the ability to retain Ca2+ by means of prolonged (up to 75 min) exposure to 0.1 microM ryanodine (as shown by the absence of contractile response to caffeine or cooling) did not prevent an ESC of nearly normal amplitude (81 +/- 24% control), albeit with a reduced contraction velocity and a time to peak contraction prolonged by 51 +/- 11%. Additionally, while rest decay of ESCs was present after ryanodine treatment, the time for the ESCs to recover their steady-state amplitude was prolonged at least twofold. Thus, in contrast with the normal guinea pig cells, ESCs of the myocytes exposed to ryanodine are controlled by sarcolemmal processes. This change in the state of excitation-contraction coupling results mainly in modification of the time course of the ESCs and of the time course of the response of the cells to the change in the rate of stimulation.

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