Biomedical subjects

D G Stephenson

Publications and source records attributed to D G Stephenson.

At least 19 recordsLinked to original sources

PubMed2006

Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat.

Using a single, mechanically skinned fiber approach, we tested the hypothesis that denervation (0 to 50 days) of skeletal muscles that do not overlap in fiber type composition [extensor digitorum longus (EDL) and soleus (SOL) muscles of Long-Evans hooded rats] leads to development of different fiber phenotypes. Denervation (50 day) was accompanied by 1) a marked increase in the proportion of hybrid IIB/D fibers (EDL) and I/IIA fibers (SOL) from 30% to >75% in both muscles, and a corresponding decrease in the proportion of pure fibers expressing only one myosin heavy chain (MHC) isoform; 2) complex muscle- and fiber-type specific changes in sarcoplasmic reticulum Ca(2+)-loading level at physiological pCa approximately 7.1, with EDL fibers displaying more consistent changes than SOL fibers; 3) decrease by approximately 50% in specific force of all fiber types; 4) decrease in sensitivity to Ca(2+), particularly for SOL fibers (by approximately 40%); 5) decrease in the maximum steepness of the force-pCa curves, particularly for the hybrid I/IIA SOL fibers (by approximately 35%); and 6) increased occurrence of biphasic behavior with respect to Sr(2+) activation in SOL fibers, indicating the presence of both slow and fast troponin C isoforms. No fiber types common to the two muscles were detected at any time points (day 7, 21, and 50) after denervation. The results provide strong evidence that not only neural factors, but also the intrinsic properties of a muscle fiber, influence the structural and functional properties of a particular muscle cell and explain important functional changes induced by denervation at both whole muscle and single cell levels.

Animals↗

PubMed2006

Effect of ADP on slow-twitch muscle fibres of the rat: implications for muscle fatigue.

Slow-twitch mechanically skinned fibres from rat soleus muscle were bathed in solutions mimicking the myoplasmic environment but containing different [ADP] (0.1 microm to 1.0 mm). The effect of ADP on sarcoplasmic reticulum (SR) Ca2+-content was determined from the magnitude of caffeine-induced force responses, while temporal changes in SR Ca2+-content allowed determination of the effective rates of the SR Ca2+-pump and of the SR Ca2+-leak. The SR Ca2+-pump rate, estimated at pCa (-log10[Ca2+]) 7.8, was reduced by 20% as the [ADP] was increased from 0.1 to 40 microm, with no further alteration when the [ADP] was increased to 1.0 mm. The SR Ca2+-leak rate constant was not altered by increasing [ADP] from 0.1 to 40 microm, but was increased by 26% when the [ADP] was elevated to 1.0 mm. This ADP-induced SR Ca2+-leak was insensitive to ruthenium red but was abolished by 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ), indicating that the leak pathway is via the SR Ca2+-pump and not the SR Ca2+-release channel. The decrease in SR Ca2+-pump rate and SR Ca2+-leak rate when [ADP] was increased led to a 40% decrease in SR Ca2+-loading capacity. Elevation of [ADP] had only minor direct effects on the contractile apparatus of slow-twitch fibres. These results suggest that ADP has only limited depressing effects on the contractility of slow-twitch muscle fibres. This is in contrast to the marked effects of ADP on force responses in fast-twitch muscle fibres and may contribute to the fatigue-resistant nature of slow-twitch muscle fibres.

Adenosine Diphosphate↗

PubMed2004

Effects of ADP on action potential-induced force responses in mechanically skinned rat fast-twitch fibres.

Mechanically skinned muscle fibres from the extensor digitorum longus (EDL) muscle of the rat were electrically stimulated in solutions mimicking the myoplasmic environment in the resting muscle fibre but containing different [ADP] of < 0.1 microm, 40 microm and 1.0 mm, to investigate the effects of myoplasmic ADP on the twitch response. The amplitude of the twitch response markedly and gradually decreased by 47 +/- 6% (n=9) as [ADP] was increased from < 0.1 microm to 40 microm without changing [Ca2+] in the myoplamsic solution (50 nm). The times for the twitch to rise from 10 to 90% (Trise,10-90) and to decrease from 90 to 10% (Tfall,90-10) initially increased by 8 and 21% and then decreased by 16 and 30% (compared to controls), respectively, at steady state. When [ADP] was raised from < 0.1 microm to 1.0 mm and fibres were electrically stimulated, the first response was biphasic and very prolonged (by at least a factor of 10) but of an amplitude similar to that in the control solution. The following twitch response and the steady state twitch responses were much reduced in size by about a factor of 6 and more prolonged by about 40% compared to control responses. All these ADP effects were fully reversible and appear to be predominantly due to several ADP-dependent alterations in SR Ca2+ handling properties (ADP-dependent decrease in SR Ca2+ capacity together with an increase in Ca2+ binding to the SR pump sites facing the myoplasm). The ADP-dependent effects on the contractile apparatus and Ca2+ regulatory system were relatively minor. Taken together, the results demonstrate that ADP accumulation is likely to play a crucial role in metabolic fatigue of skeletal muscle and can explain the marked reduction in the amplitude and the slower time course of the twitch response during fatigue as well as the elevation of myoplasmic [Ca2+] in fatigued fibres at rest.

Action Potentials↗

PubMed2004

Excitability of the T-tubular system in rat skeletal muscle: roles of K+ and Na+ gradients and Na+-K+ pump activity.

Strenuous exercise causes an increase in extracellular [K(+)] and intracellular Na(+) ([Na(+)](i)) of working muscles, which may reduce sarcolemma excitability. The excitability of the sarcolemma is, however, to some extent protected by a concomitant increase in the activity of muscle Na(+)-K(+) pumps. The exercise-induced build-up of extracellular K(+) is most likely larger in the T-tubules than in the interstitium but the significance of the cation shifts and Na(+)-K(+) pump for the excitability of the T-tubular membrane and the voltage sensors is largely unknown. Using mechanically skinned fibres, we here study the role of the Na(+)-K(+) pump in maintaining T-tubular function in fibres with reduced chemical K(+) gradient. The Na(+)-K(+) pump activity was manipulated by changing [Na(+)](i). The responsiveness of the T-tubules was evaluated from the excitation-induced force production of the fibres. Compared to control twitch force in fibres with a close to normal intracellular [K(+)] ([K(+)](i)), a reduction in [K(+)](i) to below 60 mM significantly reduced twitch force. Between 10 and 50 mM Na(+), the reduction in force depended on [Na(+)](i), the twitch force at 40 mM K(+) being 22 +/- 4 and 54 +/- 9% (of control force) at a [Na(+)](i) of 10 and 20 mM, respectively (n= 4). Double pulse stimulation of fibres at low [K(+)](i) showed that although elevated [Na(+)](i) increased the responsiveness to single action potentials, it reduced the capacity of the T-tubules to respond to high frequency stimulation. It is concluded that a reduction in the chemical gradient for K(+), as takes place during intensive exercise, may depress T-tubular function, but that a concomitant exercise-induced increase in [Na(+)](i) protects T-tubular function by stimulating the Na(+)-K(+) pump.

Action Potentials↗

PubMed2004

Effects of chlorpromazine on excitation-contraction coupling events in fast-twitch skeletal muscle fibres of the rat.

1. Single mechanically skinned fibres from the rat extensor digitorum longus muscle, which allow access to intracellular compartments, were used to examine the effects of 0.5-100 microM chlorpromazine hydrochloride (CPZ) on the major steps of the excitation-contraction (E-C) coupling to elucidate the involvement of skeletal muscle in the neuroleptic malignant syndrome (NMS). 2. At 1 microM, CPZ caused a 20-30% increase in the force response induced by t-system depolarisation and a marked increase in the rate of caffeine-induced SR Ca(2+) release. At [CPZ]> or =2.5 microM, there was an initial increase followed by a marked decrease of the t-system depolarisation-induced force responses, while the potentiating effect on the caffeine-induced SR Ca(2+) release remained. These effects were reversible. 3. CPZ had no effect on the maximum Ca(2+)-activated force, but caused reversible, concentration-dependent increases in the Ca(2+) sensitivity of the contractile apparatus at [CPZ] > or =10 microM, with a 50% predicted shift of 0.11 pCa (-log [Ca(2+)]) units at 82.3 microM CPZ. 4. CPZ did not alter the rate of SR-Ca(2+) loading at 1 and 10 microM, but reversibly reduced it by approximately 40% at 100 microM by reducing the SR Ca(2+) pump. Nevertheless, the SR Ca(2+) content was greater when fibres became unresponsive to t-system-induced depolarisation in the presence than in the absence of 100 microM CPZ. 5. The results show that CPZ has concentration-dependent stimulatory and inhibitory effects on various steps of the E-C coupling, which can explain the involvement of skeletal muscle in NMS and reconcile previous divergent data on CPZ effects on muscle.

Algorithms↗

PubMed2003

International Academy of Astronautics 5th cosmic study--preparing for a 21st century program of integrated, Lunar and Martian exploration and development (executive summary).

This report is an initial review of plans for a extensive program to survey and develop the Moon and to explore the planet Mars during the 21st century. It presents current typical plans for separate, associated and fully integrated programs of Lunar and Martian research, exploration and development, and concludes that detailed integrated plans must be prepared and be subject to formal criticism. Before responsible politicians approve a new thrust into space they will demand attractive, defensible, and detailed proposals that explain the WHEN, HOW and WHY of each stage of an expanded program of 21st century space research, development and exploration. In particular, the claims of daring, innovative, but untried systems must be compared with the known performance of existing technologies. The time has come to supersede the present haphazard approach to strategic space studies with a formal international structure to plan for future advanced space missions under the aegis of the world's national space agencies, and supported by governments and the corporate sector.

Cost-Benefit Analysis↗

PubMed2003

Effects of glycine and proline on the calcium activation properties of skinned muscle fibre segments from crayfish and rat.

The effects of the polar amino acid glycine (20 mmol l(-1)) and the non-polar amino acid proline (20 mmol l(-1)) on Ca(2+)-activated contraction have been examined in four types of striated muscle fibres. Single fibres dissected from the claw muscle of a crustacean (long- and short-sarcomere) and the hindlimb muscles of the rat (slow-twitch from soleus and fast-twitch from extensor digitorum longus) were activated in matched solutions that either contained the amino acid ('test') or not ('control'). The steady-state force produced in these solutions was used to determine the relation between force production and pCa (-log10[Ca2+]). The results show that in the concentrations used, glycine and proline had only small effects on the maximum Ca(2+)-activated force, pCa corresponding to 10, 50 and 90% maximum force (pCa10, pCa50, pCa90, respectively) or on the slope of the force-pCa curves in the four different fibre types. The relative lack of effects of glycine and proline on contractile activation would confer a distinct physiological advantage to force production of muscle of Cherax, where the concentrations of glycine and proline vary considerably. Finally, the results show that glycine and proline may be useful to balance control solutions when the effects of other amino acids or zwitterions on contractile activation are examined.

Animals↗

PubMed2002

The ilio-marsupialis muscle in the dasyurid marsupial Sminthopsis douglasi: form, function and fibre-type profiles in females with and without suckling young.

The form, function and fibre-type profiles of the ilio-marsupialis muscles, branches of which insert on to the skin of the nipples and pouch, have been investigated in the small dasyurid marsupial Sminthopsis douglasi. Single fibres from the branches of muscles associated with unsuckled nipples in non-lactating females and with both unsuckled and suckled nipples at four stages during the 70-day suckling period were typed according to their sensitivity to the activators strontium (Sr(2+)) and calcium (Ca(2+)) into fast-twitch, slow-twitch and composite types. An unusual finding was the predominance of composite fibres in the resting state (unsuckled nipples). Changes in fibre-type composition were observed during the suckling period and these changes correlated with events in the development of the suckling young. Composite fibres declined during the suckling period and, at the stage when the young can no longer be accommodated in the pouch but must still be carried by the mother while she is foraging, an increase in fast-twitch fibres that are associated with dynamic muscular activity was seen. Later in the suckling period, when the mammary tissue is greatly enlarged but the mother does not carry the young while out feeding, there was an increase in the proportion of slow-twitch (fatigue-resistant) fibres. The high proportion of fast-twitch fibres present late in the suckling period may be associated with vibratory movements that result in the young relinquishing the nipples.

Animals↗

PubMed2001

Effects of membrane cholesterol manipulation on excitation-contraction coupling in skeletal muscle of the toad.

1. Single mechanically skinned fibres and intact bundles of fibres from the twitch region of the iliofibularis muscle of cane toads were used to investigate the effects of membrane cholesterol manipulation on excitation-contraction (E-C) coupling. The cholesterol content of membranes was manipulated with methyl-beta-cyclodextrin (MbetaCD). 2. In mechanically skinned fibres, depletion of membrane cholesterol with MbetaCD caused a dose- and time-dependent decrease in transverse tubular (t)-system depolarization-induced force responses (TSDIFRs). TSDIFRs were completely abolished within 2 min in the presence of 10 mM MbetaCD but were not affected after 2 min in the presence of a 10 mM MbetaCD-1 mM cholesterol complex. There was a very steep dependence between the change in TSDIFRs and the MbetaCD : cholesterol ratio at 10 mM MbetaCD, indicating that the inhibitory effect of MbetaCD was due to membrane cholesterol depletion and not to a pharmacological effect of the agent. Tetanic responses in bundles of intact fibres were abolished after 3-4 h in the presence of 10 mM MbetaCD. 3. The duration of TSDIFRs increased markedly soon (< 2 min) after application of 10 mM MbetaCD and 10 mM MbetaCD-cholesterol complexes, but the Ca(2+) activation properties of the contractile apparatus were minimally affected by 10 mM MbetaCD. The Ca(2+) handling abilities of the sarcoplasmic reticulum appeared to be modified after 10 min exposure to 10 mM MbetaCD. 4. Confocal laser scanning microscopy revealed that the integrity of the t-system was not compromised by either intra- or extracellular application of 10 mM MbetaCD and that a large [Ca(2+)] gradient was maintained across the t-system. 5. Membrane cholesterol depletion caused rapid depolarization of the polarized t-system as shown independently by spontaneous TSDIFRs induced by MbetaCD and by changes in the fluorescence intensity of an anionic potentiometric dye (DiBAC(4)(3)) in the presence of MbetaCD. This rapid depolarization of the t-system by cholesterol depletion was not prevented by blocking the Na(+) channels with TTX (10 microM) or the L-type Ca(2+) channels with Co(2+) (5 mM). 6. The results demonstrate that cholesterol is important for maintaining the functional integrity of the t-system and sarcoplasmic reticulum, probably by having specific effects on different membrane proteins that may be directly or indirectly involved in E-C coupling.

Animals↗

PubMed2001

Effects of ADP on sarcoplasmic reticulum function in mechanically skinned skeletal muscle fibres of the rat.

1. The sarcoplasmic reticulum (SR) Ca(2+) content (expressed in terms of endogenous SR Ca(2+) content under physiologically resting conditions and measured from caffeine-induced force responses) and the effective rates of the SR Ca(2+) pump and SR Ca(2+) leak (measured from the temporal changes in SR Ca(2+) content) were determined in mechanically skinned skeletal muscle fibres of the rat at different [ADP] (< 0.10 microM to 1.04 mM). 2. The estimated SR Ca(2+) pump rate at 200 nM Ca(2+) did not change when [ADP] increased from below 0.10 microM to 10 microM but decreased by about 30 % when [ADP] increased from 10 microM to 1.04 mM. 3. The rate constant of SR Ca(2+) leak increased markedly with rising [ADP] when [Ca(2+)] in solution was 200 nM (apparent dissociation constant Kd(ADP) = 64 +/- 27 microM). Decreasing the [Ca(2+)] in solution from 200 nM to < 10 nM significantly increased the leak rate constant at all [ADP]. The SR Ca(2+) leak rate constant could be significantly reduced by blocking the SR Ca(2+) pump with 2,5-di(tert-butyl)-1,4-hydroquinone (TBQ). 4. The decrease in the SR Ca(2+) pump rate and the increase in the rate constant of SR Ca(2+) leak when the [ADP] increased from < 0.10 microM to 1.04 mM caused a 4.4-fold decrease in SR Ca(2+) loading ability at 200 nM Ca(2+). 5. The results can be fully explained by a mechanism whereby the presence of ADP causes a marked increase in the ADP-sensitive fraction of the phosphorylated pump protein, which can act as a Ca(2+)-Ca(2+) exchanger and demonstrates that ADP is an important modulator of SR function in skeletal muscle.

Adenosine Diphosphate↗

PubMed2001

Different Ca2+ releasing action of caffeine and depolarisation in skeletal muscle fibres of the rat.

1. The relative abilities of caffeine and transverse tubular (T-) system depolarisation to induce Ca2+ release in mammalian skeletal muscle were compared in mechanically skinned fibres of the rat, in order to determine whether normal excitation-contraction (E-C) coupling is achieved by up-regulating the Ca2+-induced Ca2+ release process, as caffeine is known to do. 2. Caffeine triggered Ca2+ release in soleus (slow-twitch) fibres at much lower concentrations than in extensor digitorum longus (EDL) (fast-twitch) fibres when the sarcoplasmic reticulum (SR) of each type was loaded with Ca2+ at close to endogenous levels. The difference in caffeine sensitivity resulted at least in part from the SR being loaded endogenously at near maximal capacity in soleus fibres but at less than half of maximal capacity in EDL fibres. The caffeine sensitivity could be reversed by reversing the relative level of SR loading. 3. The ability of caffeine to induce Ca2+ release was markedly reduced by lowering the level of SR loading or by raising the free [Mg2+] from 1 to 3 mM. Caffeine, even at 30 mM, triggered little or no Ca2+ release in EDL fibres (a) at 1 mM (physiological) Mg2+ when the SR was loaded at two-thirds or less of the endogenous level, and (b) at 3 mM Mg2+ when the SR was loaded at close to the endogenous level. In contrast, depolarisation potently elicited Ca2+ release under these conditions in the same fibres. 4. The inability of 30 mM caffeine to induce Ca2+ release under certain conditions was not attributable to desensitisation or inactivation of the release channels, because there was no response even upon initial exposure to caffeine and depolarisation always remained able to trigger Ca2+ release. It instead appeared that caffeine was a relatively ineffectual stimulus in EDL fibres except under conditions where (a) the SR was heavily loaded, (b) the free [Mg2+] was low, or (c) a high [Cl-] was present. 5. These results show that the normal E-C coupling mechanism in mammalian skeletal muscle does not involve just enhancing Ca2+-induced Ca2+ release, and evidently requires the removal or bypassing of the inhibitory effect of Mg2+ on the Ca2+ release channels.

Animals↗

PubMed2001

Caffeine thresholds for contraction in electrophoretically typed, mechanically skinned muscle fibres from SHR and WKY rats.

Caffeine was used as a tool to investigate whether the sarcoplasmic reticulum (SR) properties in single. mechanically skinned fibres dissected from soleus muscle of spontaneously hypertensive rats (SHRs) differ from those in fibres of the same type from age-matched, normotensive Wistar-Kyoto (WKY) rats. The fibres were typed electrophoretically based on myosin heavy chain (MHC) isoform composition. Here we show evidence that the ratio between the caffeine thresholds for contraction at maximal and endogenous resting SR-Ca2+ (Rcaff-th) can be used as an indicator for distinguishing between slow-type SR (Rcaff-th>or =0.73) and fast-type SR (Rcaff-th<0.73). Based on this indicator, 47.5% of the SHR-soleus fibres identified as type I displayed fast-type SR characteristics and 40% of the SHR-soleus fibres identified as type II displayed slow-type SR characteristics. This result explains the shorter contraction and faster relaxation of soleus muscles in SHRs and also suggests that SR with fast-type characteristics can co-exist with slow-twitch MHC isoforms and vice versa.

Animals↗

PubMed2001

Increased muscle glycogen content is associated with increased capacity to respond to T-system depolarisation in mechanically skinned skeletal muscle fibres from the rat.

The ability of mechanically skinned muscle fibres from the rat to respond to T-system depolarisation was studied in relation to muscle glycogen content. Muscle glycogen was altered by incubating extensor digitorum longus (EDL) muscles in Krebs solution without glucose or in Krebs solution with glucose (10 mM) and insulin (20 U.l-1). The glycogen content of muscles stored without glucose was rather stable between 30 and 480 min (11.27 +/- 0.39 mumol.g-1), while the muscles stored with glucose and insulin maintained an elevated and stable level of glycogen (23.48 +/- 1.67 mumol.g-1) between 100 and 360 min. Single mechanically skinned fibres from paired muscles, incubated in either glucose-free Krebs or in Krebs with glucose and insulin, were subjected to cycles of T-system depolarisation-repolarisation in a controlled environment (8 mM ATP, 10 mM creatine phosphate, 1 mM Mg2+, pH 7.10) and the force response was monitored until the force had declined to 50% of the maximum response (50% rundown). Fibres from muscles with a higher glycogen content reached 50% rundown after a larger number of depolarisations and displayed consistently larger average response capacity values, calculated as the sum of the force responses to 50% rundown divided by the maximum Ca(2+)-activated force response in that fibre. Thus skinned fibres originating from muscles with a higher glycogen content have an increased ability to respond to T-system depolarisation when the effect of metabolite accumulation is minimised and the function of glycogen acting as an energy source is by-passed. This provides direct support to the hypothesis that glycogen has a protective role in maintaining fibre excitability.

Adenosine Triphosphate↗

PubMed2001

A study of the short-term effects of glucose 6-phosphate on the contractile activation properties of skinned single muscle fibres of the rat. Implications for solution design.

The present study highlights possible problems that can arise from the incorrect preparation of control and test solutions for use in Ca2+-activation experiments using single skinned skeletal muscle fibres and EGTA-based Ca2+ buffers. We show here, using glucose 6-phosphate (G6-P) as our "test" compound, that the Ca2+-activation properties of skinned single fibre segments from the extensor digitorum longus muscle of the rat are highly dependent on the form in which the G6-P is added and on the correct balancing of an appropriate anion in control solutions. Test solutions prepared by the direct addition of 10 mM monosodium G6-P salt to a set of control solutions of defined pCa resulted in significantly greater submaximal force responses than the corresponding controls. This is equivalent to an increase in the sensitivity of the contractile-regulatory system to Ca2+ (pCa50=-log10[Ca2+] that produces 50% of maximum force) by 0.19+/-0.01 pCa units. In contrast, addition of disodium G6-P to control solutions caused a slight reduction in the apparent sensitivity of the contractile apparatus to Ca2+ by 0.04+/-0.01 pCa units (P<0.01). Rather than being indicative of the effects of G6-P on the contractile apparatus, these opposing effects are due to differences between test and control solutions with respect to pH and Na+ concentration brought about by the G6-P salts. When all ionic species were carefully balanced, 10 mM G6-P was found to have only a small sensitizing effect on Ca2+-activation properties compared to control, without affecting the maximum Ca2+-activated force response. Our findings highlight the often-overlooked need for careful balancing of the ionic composition in control and test solutions when examining the true effects of different compounds on the Ca2+-activation characteristics of single skinned muscle fibre preparations.

Animals↗

PubMed2000

Twitch and tetanic force responses and longitudinal propagation of action potentials in skinned skeletal muscle fibres of the rat.

1. Transverse electrical field stimulation (50 V cm-1, 2 ms duration) of mechanically skinned skeletal muscle fibres of the rat elicited twitch and tetanic force responses (36 +/- 4 and 83 +/- 4 % of maximum Ca2+-activated force, respectively; n = 23) closely resembling those in intact fibres. The responses were steeply dependent on the field strength and were eliminated by inclusion of 10 microM tetrodotoxin (TTX) in the (sealed) transverse tubular (T-) system of the skinned fibres and by chronic depolarisation of the T-system. 2. Spontaneous twitch-like activity occurred sporadically in many fibres, producing near maximal force in some instances (mean time to peak: 190 +/- 40 ms; n = 4). Such responses propagated as a wave of contraction longitudinally along the fibre at a velocity of 13 +/- 3 mm s-1 (n = 7). These spontaneous contractions were also inhibited by inclusion of TTX in the T-system and by chronic depolarisation. 3. We examined whether the T-tubular network was interconnected longitudinally using fibre segments that were skinned for only approximately 2/3 of their length, leaving the remainder of each segment intact with its T-system open to the bathing solution. After such fibres were exposed to TTX (60 microM), the adjacent skinned region (with its T-system not open to the solution) became unresponsive to subsequent electrical stimulation in approximately 50 % of cases (7/15), indicating that TTX was able to diffuse longitudinally inside the fibre via the tubular network over hundreds of sarcomeres. 4. These experiments show that excitation-contraction coupling in mammalian muscle fibres involves action potential propagation both transversally and longitudinally within the tubular system. Longitudinal propagation of action potentials inside skeletal muscle fibres is likely to be an important safety mechanism for reducing conduction failure during fatigue and explains why, in developing skeletal muscle, the T-system first develops as an internal longitudinal network.

Action Potentials↗

PubMed2000

Effects of Mg2+ on Ca2+ release from sarcoplasmic reticulum of skeletal muscle fibres from yabby (crustacean) and rat.

1. The role of myoplasmic [Mg2+] on Ca2+ release from the sarcoplasmic reticulum (SR) was examined in the two major types of crustacean muscle fibres, the tonic, long sarcomere fibres and the phasic, short sarcomere fibres of the fresh water decapod crustacean Cherax destructor (yabby) and in the fast-twitch rat muscle fibres using the mechanically skinned muscle fibre preparation. 2. A robust Ca2+-induced Ca2+-release (CICR) mechanism was present in both long and short sarcomere fibres and 1 mM Mg2+ exerted a strong inhibitory action on the SR Ca2+ release in both fibre types. 3. The SR displayed different properties with respect to Ca2+ loading in the long and the short sarcomere fibres and marked functional differences were identified with respect to Mg2+ inhibition between the two crustacean fibre types. Thus, in long sarcomere fibres, the submaximally loaded SR was able to release Ca2+ when [Mg2+] was lowered from 1 to 0.01 mM in the presence of 8 mM ATPtotal and in the virtual absence of Ca2+ (< 5 nM) even when the CICR was suppressed. In contrast, negligible Ca2+ was released from the submaximally loaded SR of short sarcomere yabby fibres when [Mg2+] was lowered from 1 to 0.01 mM under the same conditions as for the long sarcomere fibres. Nevertheless, the rate of SR Ca2+ release in short sarcomere fibres increased markedly when [Mg2+] was lowered in the presence of [Ca2+] approaching the normal resting levels (50-100 nM). 4. Rat fibres were able to release SR Ca2+ at a faster rate than the long sarcomere yabby fibres when [Mg2+] was lowered from 1 to 0. 01 mM in the virtual absence of Ca2+ but, unlike with yabby fibres, the net rate of Ca2+ release was actually increased for conditions that were considerably less favourable to CICR. 5. In summary, it is concluded that crustacean skeletal muscles have more that one functional type of Ca2+-release channels, that these channels display properties that are intermediate between those of mammalian skeletal and cardiac isoforms, that the inhibition exerted by Mg2+ at rest on the crustacean SR Ca2+-release channels must be removed during excitation-contraction coupling and that, unlike in crustacean fibres, CICR cannot play the major role in the activation of SR Ca2+-release channels in the rat skeletal muscle.

Animals↗

PubMed2000

Changes in the Ca2+-activation characteristics of demembranated rat single muscle fibres after prolonged incubation at room temperature in the presence and absence of DTT and protease inhibitors.

Prolonged incubation (24 h) of chemically skinned rat muscle preparations in rigor solutions at room temperature and in the absence of reducing agents and protease inhibitors modified the Ca2+-activation characteristics of the contractile machinery. In the absence of reducing agents and protease inhibitors, the contraction threshold for incubated fibres was shifted to lower Ca2+ concentrations and the steepness of the steady-state force-pCa (-log10)[Ca2+]) curve was decreased compared to that of control muscle fibres. Mean myosin ATPase activity under these conditions was significantly lowered by a factor of 2.7. Fibres incubated in the presence of 10 mM dithiothreitol (DTT) and protease inhibitors (100 microM pepstatin A/200 microM leupeptin) produced a maximum Ca2+-activated force per cross-sectional area that compared favourably with that of freshly dissected muscle fibres and there were no changes in the other contractile activation characteristics. Intermediate responses were obtained when fibres were incubated in the presence of either DTT or protease inhibitors. MgATPase activities of incubated preparations increased significantly following the addition of protease inhibitors and/or DTT to the incubation medium. Taken together, these results suggest that in the presence of DTT and protease inhibitors, most contractile properties are maintained at levels seen in fresh mechanically skinned fibres. The extended viability of this preparation and its closely related properties with fresh muscle fibres make it a useful model for experiments requiring longer term incubations with biological agents.

Adenosine Triphosphatases↗

PubMed2000

Ca2+-activation characteristics of single fibres from chemically skinned rat muscle incubated with glucose-6-phosphate.

In this study we examined the effects of 3-24 h of incubation of chemically skinned rat fast-twitch muscle with the glycolytic metabolite glucose 6-phosphate (G6-P) on the contractile properties and myosin ATPase activity in single muscle fibres, and on the carbohydrate content of myosin heavy chains (MHCs). Exposure of the permeabilised muscle to 10 mM G6-P for 24 h at 22+/-1 degrees C in a rigor solution containing protease inhibitors and a reducing agent (dithiothreitol, DTT) significantly decreased maximum Ca(2+)-activated force output by 31%, lowered the Ca2+ threshold for contraction by 0.1 pCa units and produced shallower force-pCa curves compared with controls. Furthermore, under these conditions, G6-P-treated muscle displayed lower myofibrillar MgATPase activity and a markedly higher carbohydrate content of MHCs, as identified with an immunoblot protocol for glycoprotein detection. Shorter incubations under the same conditions or 24-h incubations with 5 mM G6-P generally resulted in smaller changes in the contractile activation parameters. These findings suggest that reducing sugars acting as metabolic intermediates in the glycolytic pathway can have important non-energy-related effects on the contractile activation characteristics of mammalian skeletal muscle. These effects are consistent with the glycation of muscle proteins, in particular that of the MHC.

Adenosine Triphosphatases↗