PubMed Health⌕ Search

Biomedical subjects

D G Stephenson

Publications and source records attributed to D G Stephenson.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effects of FK506 and rapamycin on excitation-contraction coupling in skeletal muscle fibres of the rat.

1. The effects of the immunosuppressants FK506 and rapamycin were examined in mechanically skinned skeletal muscle fibres of rat in order to determine whether the FK506-binding protein plays a role in the coupling between the voltage sensors and the Ca2+ release channels. 2. Both FK506 (1 microM) and rapamycin (1 microM) rapidly and reversibly potentiated Ca2+ release evoked by either depolarization of the transverse tubular system or caffeine application, suggesting a direct effect of the agents on the Ca2+ release channels. 3. In addition, repeated depolarizations in the presence of either FK506 (1 microM) or rapamycin (1 microM) caused irreversible loss of depolarization-induced Ca2+ release, without preventing direct activation of the Ca2+ release channels by caffeine or low [Mg2+]. If a fibre was exposed to either immunosuppressant for a similar period (10 min) without stimulation, or if the voltage sensors were kept inactivated, there was little if any loss of coupling. 4. The loss of coupling was faster at higher drug concentrations, with 20 microM rapamycin causing 50% inhibition in 7-8 min without stimulation; this was further accelerated by repeated depolarizations in the presence of the drug, but was not noticeably altered by direct activation of the release channels by repeated exposure to caffeine. The irreversible loss of coupling indicates that the FK506-binding protein may play a vital role in enabling the voltage sensors to activate the Ca2+ release channels.

Animals↗
PubMed1996

Effect of low [ATP] on depolarization-induced Ca2+ release in skeletal muscle fibres of the toad.

1. The effect of low [ATP] on depolarization-induced Ca2+ release from the sarcoplasmic reticulum (SR) was examined using mechanically skinned skeletal muscle fibres of the toad. The amount of Ca2+ released was determined by examining the extent of SR Ca2+ depletion following a single depolarization in the presence of specified total [ATP] and free [Mg2+] and 1 mM EGTA (< 10 nM Ca2+). 2. Reducing the total [ATP] from 8 to 2 mM did not significantly affect the extent of depolarization-induced Ca2+ release. When the total [ATP] was lowered to 0.5 mM, depolarization-induced Ca2+ release was markedly reduced. The additional presence of 1.5 mM AMP did not reverse this effect of low [ATP]. 3. At each total [ATP], elevation of the free [Mg2+] from the normal resting level of 1 mM to 3 mM, caused a similar reduction in depolarization-induced Ca2+ release. This effect was not due to the concomitant decrease in free [ATP] and occurred independently of the reduction in Ca2+ release observed at very low total [ATP]. 4. These results show myoplasmic [ATP] modulates depolarization-induced Ca2+ release. Thus, if the total [ATP] near the Ca2+ release channels becomes locally depleted in severely fatigued muscle fibres, it may contribute, in combination with an accompanying increase in free [Mg2+], to the reported reduction in Ca2+ release.

Adenosine Triphosphate↗
PubMed1996

Total and sarcoplasmic reticulum calcium contents of skinned fibres from rat skeletal muscle.

1. The Ca2+ content of single mammalian skeletal muscle fibres was determined using a novel technique. Mechanically skinned fibres were equilibrated with varying amounts of the Ca2+ buffer BAPTA and were then lysed in a detergent-paraffin oil emulsion. The subsequent myofilament force response was used to estimate the additional amount of Ca2+ bound to BAPTA following lysis of intracellular membranes. 2. The total endogenous Ca2+ content (corrected for endogenous Ca2+ buffering) of fast-twitch (FT) and slow-twitch (ST) fibres at a myoplasmic pCa (-log[Ca2+]) of 7.15 was 1.32 +/- 0.02 and 1.35 +/- 0.08 mM per fibre volume, respectively. The sarcoplasmic reticulum (SR) component of these estimates was calculated as 1.01 and 1.14 mM, respectively, which normalized to SR volume corresponds to resting SR Ca2+ contents of 11 and 21 mM, respectively. 3. Equilibration of 'resting' fibres with low myoplasmic [Ca2+] (pCa 7.67-9.00) elicited a time-dependent decrease in Ca2+ content in both fibre types. Equilibration of resting fibres with higher myoplasmic [Ca2+] (pCa 5.96-6.32) had no effect on the Ca2+ content of ST fibres but increased the Ca2+ content of FT fibres. The maximum steady-state total Ca2+ content (3.85 mM) was achieved in FT fibres after 3 min equilibration at pCa 5.96. Equilibration at higher myoplasmic [Ca2+] was less effective, probably due to Ca2(+)-induced Ca2+ release from the SR. 4. Exposure of fibres to either caffeine (30 mM, pCa approximately 8, 2 min) or low myoplasmic [Mg2+] (0.05 mM, pCa approximately 9, 1 min) released approximately 85% of the resting SR Ca2+ content. The ability of caffeine to release SR Ca2+ was dependent on the myoplasmic Ca2+ buffering conditions. 5. The results demonstrate that the SR of ST fibres is saturated with Ca2+ at resting myoplasmic [Ca2+] while the SR of FT fibres is only about one-third saturated with Ca2+ under equivalent conditions. These differences suggest that the rate of SR Ca2+ uptake in FT fibres is predominantly controlled by myoplasmic [Ca2+] while that of ST fibres is more likely to be limited by the [Ca2+] within the SR lumen.

Animals↗
PubMed1996

The effect of 2,5-di-(tert-butyl)-1,4-hydroquinone on force responses and the contractile apparatus in mechanically skinned muscle fibres of the rat and toad.

In this study, we investigated the effect of the Ca2+ pump inhibitor, 2,5-di-(tert-butyl)-1,4-hydroquinone on the function of the contractile apparatus, Ca2+ uptake, the permeability of the sarcoplasmic reticulum to Ca2+ and excitation-contraction coupling, in mechanically skinned muscle fibres of the rat and toad. 2,5-di-(tert-butyl)-1,4-hydroquinone had no significant effect on the maximum force and Ca2+ sensitivity of the contractile apparatus in rat and toad fibres at concentrations of 20 and 5 microM respectively. In rat fibres, 2,5-di-(tert-butyl)-1,4-hydroquinone was found to inhibit sarcoplasmic reticulum Ca2+ loading in a dose dependent manner, with a half maximal effect at 2 microM. In toad fibres, 5 microM 2,5-di-(tert-butyl)-1,4-hydroquinone completely blocked sarcoplasmic reticulum Ca2+ loading. Exposure to 5 mM BAPTA revealed a small resting sarcoplasmic reticulum Ca2+ leak in unstimulated rat fibres. This Ca2+ leak was not significantly affected by the presence of 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone, suggesting that 2,5-di-(tert-butyl)-1,4-hydroquinone does not substantially block or activate the sarcoplasmic reticulum Ca2+ release channels. Depolarisation-induced force responses elicited in rat and toad skinned fibres were not significantly affected by 0.5 microM 2,5-di-(tert-butyl)-1, 4-hydroquinone. In the rat fibres, 5 and 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone greatly increased the peak and duration of initial depolarisation-induced force responses, while subsequent responses were reduced. 2,5-di-(tert-butyl)-1,4-hydroquinone did not affect excitation contraction coupling, as depolarisation-induced force responses similar to initial controls could be elicited after 2,5-di-(tert-butyl)-1,4-hydroquinone exposure, provided that the initial Ca2+ release in 2,5-di-(tert-butyl)-1,4-hydroquinone was chelated with 0.5 mM EGTA (to prevent Ca(2+)-dependent damage) and the sarcoplasmic reticulum was reloaded with Ca2+. In the toad fibres, 5 microM 2,5-di-(tert-butyl)-1, 4-hydroquinone had a similar effect on depolarisation-induced force responses to that observed at 20 microM 2,5-di-(tert-butyl)-1, 4-hydroquinone in rat fibres. This study shows that 2,5-di-(tert-butyl)-1,4-hydroquinone specifically and reversibly inhibits the sarcoplasmic reticulum Ca2+ pump in skeletal muscle and therefore, 2,5-di-(tert-butyl)-1,4-hydroquinone could be a valuable tool for investigating the role of the sarcoplasmic reticulum in Ca2+ homeostasis in skeletal muscle.

Animals↗
PubMed1996

Effects of ammonium ions on the depolarization-induced and direct activation of the contractile apparatus in mechanically skinned fast-twitch skeletal muscle fibres of the rat.

Mechanically skinned fibre preparations from the extensor digitorum longus muscle of the rat were used to test whether a rise in myoplasmic [NH+4] in the range 2-10 mM interferes with the mechanism of excitation-contraction coupling in fast-twitch mammalian muscle. Under our conditions (pH 7.10, Mg2+ 1 mM, temperature 23 degrees C), [NH+4] up to 10 mM had little effect on the Ca(+)-activated force and on the peak of the t-system depolarization-induced force response. However, the duration of the depolarization-induced force response was decreased significantly at [NH+4] > or = 2 mM. From these data we conclude that the intracellular accumulation of NH+4 is not likely to play a major role in fatigue. Nevertheless, the build up of NH+4 during fatigue, may have a significant inhibitory effect on the force output by decreasing the duration of the t-system depolarization-induced activation of the contractile apparatus.

Animals↗
PubMed1996

Measurement of membrane potential and myoplasmic [Ca2+] in developing rat myotubes at rest and in response to stimulation.

In this study, the membrane potential and cytosolic [Ca2+] were measured in rat myotubes developing in culture from days 6-14. It was found that as the myotubes developed in culture, the resting membrane potential (RMP) became more negative during days 6-8, and then did not significantly change until after day 13, when it started to become less negative. The mean RMP measured at days 8-13 was -59 +/- 1 mV (n = 70). The amplitude of action potentials elicited in the myotubes by anode break stimulation increased in size during development (range: 47.5-119 mV) and this closely correlated with the development of a more negative RMP. Cytosolic [Ca2+] was measured in the rat myotubes using the Ca2+ indicator Fura-2, and no significant change in the resting [Ca2+] was observed during development (days 6-14). Ca2+ responses triggered by action potentials varied from small slow increases in [Ca2+] that failed to return to the baseline to rapid [Ca2+] transients. The size of the [Ca2+] transients positively correlated with both the observed increase in the RMP during development and the size of the action potential. Larger [Ca2+] transients also had more rapid rates of [Ca2+] decay, indicating a tandem increase in the ability of the sarcoplasmic reticulum to release and resequester Ca2+ during development of rat myotubes. Repetitive stimulation (10 Hz) of the myotubes exhibiting small [Ca2+] transients produced a step-like rise in [Ca2+]. Many myotubes exhibiting larger [Ca2+]transients could not be stimulated at 10 Hz by anode break stimulation due to the presence of action potentials with large hyperpolarisations. However, when these myotubes were depolarised at 10 Hz, they produced a tetanic Ca2+ response similar to that seen in adult skeletal muscle.

Animals↗
PubMed1996

Rapid cooling contractures in rat skinned myocardium: effects of isoflurane and halothane.

Rapid cooling contractures (RCCs) have been elicited in rat ventricular cardiac muscle skinned by saponin (50 micrograms/ml). The size and shape of the RCC in skinned cardiac muscle were similar to those observed in intact cardiac muscle. The ATP-dependent Ca2-uptake pump in the sarcoplasmic reticulum (SR) was active at low temperature and could efficiently load the SR with Ca2+ even at 3 degrees C. Halothane reduced both RCC and caffeine contracture in a dose-dependent way when halothane treatment was applied before either RCC or caffeine. This action is consistent with halothane-induced depletion of the SR Ca2+. Under similar conditions, isoflurane inhibited RCC but had little effect on the caffeine contracture. This may suggest that rapid cooling and caffeine have different modes of action on the SR Ca(2+)-release channel. Our results provide further strong supporting evidence for differential inhibitory actions on a key intracellular organelle in cardiac muscle by two vapor anesthetic agents.

Anesthetics, Inhalation↗
PubMed1995

Raised intracellular [Ca2+] abolishes excitation-contraction coupling in skeletal muscle fibres of rat and toad.

1. Raising the intracellular [Ca2+] for 10 s at 23 degrees C abolished depolarization-induced force responses in mechanically skinned muscle fibres of toad and rat (half-maximal effect at 10 and 23 microM, respectively), without affecting the ability of caffeine or low [Mg2+] to open the ryanodine receptor (RyR)/Ca2+ release channels. Thus, excitation-contraction coupling was lost, even though the Ca2+ release channels were still functional. Coupling could not be restored in the duration of an experiment (up to 1 h). 2. The Ca(2+)-dependent uncoupling had a Q10 > 3.5, and was three times slower at pH 5.8 than at pH 7.1. Sr2+ caused similar uncoupling at twenty times higher concentration, but Mg2+, even at 10 mM, was ineffective. Uncoupling was not noticeably affected by removal of ATP or application of protein kinase or phosphatase inhibitors. 3. Confocal laser scanning microscopy showed that the transverse tubular system was sealed in its entirety in mechanically skinned fibres and that its integrity was maintained in uncoupled fibres. Electron microscopy revealed distorted or severed triad junctions and Z-line aberrations in uncoupled fibres. 4. Only when uncoupling was induced at a relatively slow rate (e.g. over 60 s with 2.5 microM Ca2+) could it be prevented by the protease inhibitor leupeptin (1 mM). Immunostaining of Western blots showed no evidence of proteolysis of the RyR, the alpha 1-subunit of dihydropyridine receptor (DHPR) or triadin in uncoupled fibres. 5. Fibres which, whilst intact, were stimulated repeatedly by potassium depolarization with simultaneous application of 30 mM caffeine showed reduced responsiveness after skinning to depolarization but not to caffeine. Rapid release of endogenous Ca2+, or raised [Ca2+] under conditions which minimized the loss of endogenous diffusible myoplasmic molecules from the skinned fibre, caused complete uncoupling. Taken together, these results suggest that Ca(2+)-dependent uncoupling can also occur in intact fibres. 6. This Ca(2+)-dependent loss of depolarization-induced Ca2+ release may play an important feedback role in muscle by stopping Ca2+ release in localized areas where it is excessive and may be responsible for long-lasting muscle fatigue after severe exercise, as well as contributing to muscle weakness in various dystrophies.

Animals↗
PubMed1995

Effects of creatine phosphate and P(i) on Ca2+ movements and tension development in rat skinned skeletal muscle fibres.

1. Mechanically skinned fast-twitch (FT) and slow-twitch (ST) muscle fibres of the rat were used to investigate the effects of fatigue-like changes in creatine phosphate (CP) and inorganic phosphate (P(i)) concentration on Ca(2+)-activation properties of the myofilaments as well as Ca2+ movements into and out of the sarcoplasmic reticulum (SR). 2. Decreasing CP from 50 mM to zero in FT fibres increased maximum Ca(2+)-activated tension (Tmax) by 16 +/- 2% and shifted the mid-point of the tension-pCa relation (pCa50) to the left by 0.28 +/- 0.03 pCa units. In ST fibres, a decrease of CP from 25 mM to zero increased Tmax by 9 +/- 1% and increased the pCa50 by 0.16 +/- 0.01 pCa units. The effect of CP on Tmax was suppressed in both fibre types by prior treatment with 0.3 mM FDNB (1-fluoro-2,4-dinitrobenzene), suggesting that these effects may occur via changes in creatine kinase activity. 3. Increases of P(i) in the range 0-50 mM reduced the pCa50 and Tmax in both fibre types. These effects were more pronounced in ST fibres than in FT fibres in absolute terms. However, normalization of the results to resting P(i) levels appropriate to both fibre types (1 mM for FT and 5 mM for ST fibres) revealed similar decreases in Tmax (approximately 39% at 25 mM P(i) and approximately 48% at 50 mM P(i)) and pCa50 (0.25 pCa units at 25-50 mM P(i)). The depressant action of P(i) on both parameters was considerably reduced when the rise in P(i) was accompanied by an equivalent reduction in [CP]. 4. Tension development in the presence of complex, fatigue-like milieu changes (40 mM P(i) for FT; 20 mM P(i) for ST) was decreased by 35-40% at a constant myoplasmic [Ca2+] of 6 microM in both fibre types. 5. SR Ca2+ loading at a myoplasmic [Ca2+] of 100 nM was found to increase abruptly when the [P(i)] during loading was increased to near 9 mM. At a myoplasmic [Ca2+] of 300 nM, the threshold P(i) for this effect dropped to approximately 3 mM. 6. Tension responses evoked by caffeine in the absence of P(i) were smaller and slower to peak if fibres were exposed to P(i) in a restricted myoplasmic Ca2+ pool after SR Ca2+ loading. This indicated that myoplasmic P(i) can decrease and prolong the rate of Ca2+ release from the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Characterization of ultrastructural and contractile activation properties of crustacean (Cherax destructor) muscle fibres during claw regeneration and moulting.

Long-(SL > 6 microns) and short-sarcomere (SL < 4 microns) fibres were isolated from the claw muscle of the yabby (Cherax destructor) during limb regeneration and at different stages of the moult cycle. Long-sarcomere fibres were more susceptible to the changes resulting from the moult-induced atrophy compared with the short-sarcomere fibres. Signs of atrophy included fibre erosion, loss of myosin filaments, a reduction in the diameter of myosin filaments and changes associated with the Z line. The intracellular structure of the fibres, however, remained intact in both fibre types. Fibres taken immediately prior to ecdysis could not be fully activated with Ca2+ or Sr2+ without breaking. In contrast fibres taken within 4 h after ecdysis could develop and maintain full force when activated by Ca2+ or Sr2+. The results suggest that loss of myofibrillar proteins via the moult-induced atrophy and/or events associated with fibre elongation may occur in the period just prior to ecdysis and that these changes may be responsible for the fibres inability to function during the premoult stage. Results from this study showed that short-sarcomere fibres add sarcomeres by at least two different mechanisms (1) transverse sarcomere splitting and (2) Z line splitting. Long-sarcomere fibres appear to be elongated by mechanism(s) other than those used by short-sarcomere fibres which possibly involve large electron dense structures which are positioned between the myofibrils and within the A and I bands. Results from the regenerating chelae limb bud showed that sarcomeres form from separate units comprising myosin filaments and actin filaments anchored into Z lines respectively. These sub-sarcomeric units then join together to form sarcomeres. Myofibril formation is aided by electron dense regions which are closely associated with the membrane system. These fibres although short in length and still within the non-functional limb bud could be activated by Ca2+ and Sr2+ suggesting that full fibre function exists before the chelae become functional. Regenerating muscle fibres consisted predominantly of fibres with short-sarcomeres.

Animals↗
PubMed1995

The effect of temperature on contractile activation of intact and chemically skinned 'catch' muscle fibre bundles of Mytilus edulis.

The effect of temperature (5-35 degrees C) on maximum force production was examined in intact and chemically skinned muscle fibre bundles (10-25 fibres) from the anterior byssus retractor muscle of Mytilus edulis. In intact fibre bundles, 10 microM acetylcholine induced a tonic contraction which had a magnitude of 65.4 +/- 4.0 N cm-2 (n = 30) at 23 degrees C. Activation by caffeine (20 mM) produced a force response which was 157.1 +/- 7.9% (n = 16) of the acetylcholine response at 23 degrees C and acetylcholine and caffeine together produced force which was not significantly different from the response to caffeine alone. At 5 degrees C the acetylcholine and caffeine responses were decreased by 9.6 +/- 3.4% (n = 6) and 14.6 +/- 2.8% (n = 8) compared with the respective responses at 23 degrees C. However, there was no significant reduction of the response induced by the combined action of acetylcholine and caffeine when the temperature was decreased from 23 degrees C to 5 degrees C. The 20-80% of peak force activation time increased by about one order of magnitude for all acetylcholine, caffeine and combined acetylcholine-caffeine-induced responses when the temperature was decreased from 23-5 degrees C. Repeated exposure of the intact preparation to caffeine caused a marked decrease in the caffeine-induced response (complete abolition of force after the third exposure to caffeine), but the response to caffeine could be fully restored following one acetylcholine-induced activation. The maximum Ca(2+)-activated force after skinning the preparation with saponin was not significantly different from the caffeine or combined acetylcholine-caffeine-induced responses before skinning. In the saponin skinned fibre preparation a drop in temperature from 23 degrees C to 15 degrees C or 5 degrees C decreased the maximum Ca(2+)-activated force by 13.2 +/- 1.4% (n = 8) and 41.4 +/- 3.1% (n = 5) respectively. The activation time between 20-80% of the peak Ca(2+)-activated force increased at 15 degrees C and 5 degrees C by a factor of 1.5 +/- 0.1 (n = 5) and 6.8 +/- 1.1 (n = 5) respectively when compared to corresponding values at 23 degrees C. The relaxation half-time decreased by a factor of 1.7 +/- 0.2 (n = 5) and 3.0 +/- 0.2 (n = 5) at 15 degrees C and 5 degrees C respectively compared with that at 23 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1995

Analysis of Ca2+ and Sr2+ activation characteristics in skinned muscle fibre preparations with different proportions of myofibrillar isoforms.

To understand how the coexistence of fast and slow contractile and regulatory systems within single skeletal muscle fibres might affect contractile behaviour, fibre segments from the fast-twitch extensor digitorum longus and predominantly slow-twitch soleus muscle of the adult rat were tied together, either in parallel or in series, and then activated in Ca(2+)- and Sr(2+)-buffered solutions. Experimental force-pCa and force-pSr relations were compared with theoretical force-pCa and force-pSr curves predicted by a model for composite fibres, which accounted for the coexistence of fast and slow myosin within the contractile unit and enabled an estimate to be made of the relative contribution of fast- and slow-twitch elements within the tied-fibre combinations. The contractile behaviour of a fast-twitch and a slow-twitch muscle fibre tied either in series or in parallel, were compared with the force-pCa and force-pSr data predicted from the composite fibre model. Interestingly, the resultant force-pCa(-pSr) curves of the parallel-tied fibre combinations were well fitted with those predicted by the composite model. However, the experimental force-pCa(-pSr) curves of the series-tied fibres were not well fitted by a composite curve based on the known proportion of fast- and slow-twitch fibre components. A total force-length diagram was devised to take into account changes in the length of the fibre segments tied in series during activation, as well as possible differences in fibre diameter. Using this diagram it was possible to explain accurately the Ca2+ and Sr2+ activation curves of known fast- and slow-twitch segments tied in series. The results from this study are important for the interpretation of contractile data obtained from single muscle fibres exhibiting mixed fast- and slow-twitch contractile characteristics. Such muscle fibres have previously been identified in animals affected by muscular diseases (e.g. dystrophy), in mammalian extraocular muscles and in animals subjected to long-term exercise training.

Animals↗
PubMed1995

Activation of ryanodine receptors by flash photolysis of caged Ca2+.

Flash photolysis of DM-nitrophen generates an extremely large [Ca2+] transient ("Ca2+ spike") at the start of each Ca2+ "step." The Ca2+ spike greatly increases the speed of activation of the ryanodine receptor channel ("supercharging") and could be responsible for apparent channel adaptation.

Acetates↗
PubMed1995

Contractile activation and measurements of intracellular Ca2+ concentration in cane toad twitch fibres in the presence of 2,3-butanedione monoxime.

The effects of 2,3-butanedione monoxime (BDM, 0.5-20 mM) on Ca(2+)-activated force in skinned muscle fibres, and force and Ca2+ responses in aequorin-injected intact fibres from the iliofibularis muscle of the cane toad Bufo marinus were investigated. Peak twitch force responses progressively decreased to 3% of the control response with increasing [BDM] up to 20 mM. Peak twitch aequorin light responses decreased to 65% of the control response in 10 mM BDM, but increased again to control values in 20 mM BDM. The duration of the twitch aequorin light response increased by up to 60% above 5 mM BDM. Tetanic (170 Hz) force and aequorin light responses reversibly decreased in a dose-dependent fashion to about 50% of the control response in 10 mM BDM. Failure of tetanic (170 Hz) stimulation was observed in the presence of 20 mM BDM. Intracellular [Ca2+] could be modified by changing the frequency of tetanic stimulation in the presence of BDM, permitting a study of the dependence of isometric force on intracellular [Ca2+] at different concentrations of BDM. In 10 mM BDM, the rate of force development in intact fibres was slower by a factor of two at saturating [Ca2+], and was up to one order of magnitude slower at non-saturating [Ca2+], when compared with control responses. At a similar intracellular [Ca2+] steady-state isometric force was reduced to about 85 and 50% of the control responses in 2 and 10 mM BDM, respectively. The effect of BDM on maximum Ca2+-activated force in skinned fibres paralleled the decrease in tetanic (170 Hz) force observed in intact fibres. The rate of force development in skinned fibres decreased with an increase in [BDM] at constant [Ca2+], and the sensitivity of the contractile apparatus to Ca2+ was shifted to a higher [Ca2+] by BDM. The results suggest that BDM reduces contractility in cane toad iliofibularis muscle by direct inhibition of the contractile apparatus, and reduction of the release of activator Ca2+ from the sarcoplasmic reticulum. Furthermore, BDM may be a useful tool to help study the relationship between force and [Ca2+] in intact muscle fibres.

Aequorin↗
PubMed1994

Effects of intracellular pH and [Mg2+] on excitation-contraction coupling in skeletal muscle fibres of the rat.

1. The effects on normal excitation-contraction (E-C) coupling of two important intracellular ions, H+ and Mg2+, were examined in skinned fibres from the extensor digitorum longus muscle of rat. 2. A single depolarization (2-3 s duration) in the presence of 1 mM Mg2+ (pH 7.1, 23 degrees C) released most of the available Ca2+ in the sarcoplasmic reticulum (SR), but a similar depolarization in the presence of 10 mM Mg2+ was unable to release almost any Ca2+. Thus, raised [Mg2+] potently inhibits depolarization-induced Ca2+ release in mammalian muscle. 3. Depolarization at pH 6.2 (1 mM Mg2+, 23 degrees C) induced a large force response, which was on average 78 +/- 2%, n = 6, of the depolarization-induced response at pH 7.1; this reduction resulted from a corresponding reduction in maximum Ca(2+)-activated force at pH 6.2. Similar results were obtained at 37 degrees C. Also, a single depolarization at pH 6.2 caused almost complete depletion of the releasable Ca2+ in the SR. Thus, low pH does not prevent depolarization-induced Ca2+ release in mammalian muscle. 4. Lowering the free [Mg2+] from 1 mM to 15 microM caused massive release of Ca2+, and depletion of the SR, at both pH 7.1 and 6.2, indicating that over this pH range, H+ did not readily substitute for Mg2+ at its inhibitory site on the Ca2+ release channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The intracellular Ca2+ transient and tension in frog skeletal muscle fibres measured with high temporal resolution.

1. The purpose of this study was to determine, with high temporal resolution, the relationship between the intracellular Ca2+ transient (ICT) and the mechanical responses of intact, single skeletal muscle fibres of frogs following stimulation by a single, brief depolarization. 2. The time course of the ICT was monitored using the Ca(2+)-sensitive fluorescent dyes mag-fura-2 (furaptra) and mag-fura-5. The mag-fura dyes have a low affinity for Ca2+ and have been shown to track the ICT with no appreciable kinetic delay. Continuous records of mag-fura fluorescence, tension and stiffness responses were obtained simultaneously at high time resolution at a sarcomere length of 2.9 microns. Experimental temperature was 3 degrees C. 3. When a delay of 0.4 ms due to the low-pass filter associated with the photodetector was included, the onset of the fluorescence response preceded the onset of latency relaxation (the small fall in tension that precedes positive tension generation) by 3.1 +/- 0.2 ms (mean +/- S.E.M., n = 8). After its onset, the mag-fura fluorescence signal continued to change rapidly (indicating increasing intracellular [Ca2+]) to an extreme level that occurred 1.5 +/- 0.5 ms (mean +/- S.E.M., n = 7) before tension had recovered to its resting level following latency relaxation. The time delay from the extreme of the fluorescence signal to the peak of the tension signal was 239 +/- 27 ms (mean +/- S.E.M., n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗