Biomedical subjects

D G Stephenson

Publications and source records attributed to D G Stephenson.

At least 91 records · Page 5Linked to original sources

PubMed1991

Effects of sulphydryl modification on skinned rat skeletal muscle fibres using 5,5'-dithiobis(2-nitrobenzoic acid).

1. The sulphydryl groups of skinned skeletal muscle fibres have been reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in order to determine whether the effects of modifications to the contractile proteins are reflected in changes in the physiological properties of the contractile apparatus and Ca(2+)-regulatory system. 2. Results obtained from fast-twitch and slow-twitch rat fibres which were treated with DTNB (10 mM, pH 8.6, 5 degrees C) for various periods of time under relaxing conditions showed that a major effect of the modification was to reduce the level of maximally Ca(2+)-activated force and fibre stiffness. Force and fibre stiffness were found to decline in proportion. Treatment with DTNB under these conditions did not cause a rise in force or fibre stiffness in relaxed fibres of either type. 3. The effects induced by DTNB under relaxing conditions were substantially reversed by exposure to the reducing agent dithiothreitol (DTT) (10 mM, pH 7.1, 23 degrees C). Force abolished by 30-35 s treatment with DTNB recovered after subsequent DTT treatment to 67 +/- 3% (mean +/- S.E.M., n = 4) in fast-twitch fibres and to 91 +/- 2% (n = 7) in slow-twitch fibres. These results were significantly different (t test, P less than 0.001) indicating that the level of force recovery depended upon the fibre type. 4. DTNB was found to affect not only the maximal Ca(2+)-activated force, but also the force-pCa (pCa = -log10[Ca2+]) relationships of the fibres in a complex, fibre-type specific way. DTT treatment partially reversed these DTNB effects. 5. The skinned fibre preparations reacted differently with DTNB under rigor conditions than under relaxing conditions, indicating that rigor modifies the reactivity of the functional sulphydryl groups to the thiol-targeted agents. 6. When superprecipitation assays (an in vitro analogue of fibre contraction) were carried out with recombined myofibrillar proteins which had been previously reacted with DTNB it was found that modification of myosin, but not modification of thin filament proteins, led to changes in the superprecipitation reaction. 7. Both the skinned fibre results and the superprecipitation results indicate that the effects of DTNB upon the fibre characteristics are primarily due to modifications of the sulphydryl groups of myosin. Therefore, these results show that myosin is not only involved in determining the ability of the contractile apparatus to develop force but also in determining the Ca(2+)-regulatory characteristics of the muscle fibre.

Animals↗

PubMed1991

Excitation-contraction coupling in skeletal muscle fibres of rat and toad in the presence of GTP gamma S.

1. Rapid force responses were elicited in single mechanically skinned fibres from extensor digitorum longus (EDL) muscle of the rat when the fibres were depolarized by substituting K+ in the bathing solution with Na+. The properties of these depolarization-induced responses, the responses to lowered [Mg2+], and the characteristics of the slow prolonged response ('second component') produced in 'loaded' fibres by choline chloride (ChCl) substitution, were virtually identical to those observed previously in skinned fibres from toad muscle. 2. At physiological levels of [Mg2+] (1 mM) and Ca2+ loading, application of 50 microM- to 1 mM-GTP gamma S (guanosine-5'-O-(3-thiotriphosphate), a non-hydrolysable analogue of GTP) did not produce a response in any mammalian or amphibian fibre, even though the depolarization-induced coupling was totally functional. Furthermore, the presence of GTP gamma S had no apparent effect on the size, the threshold or the maximum number of responses which could be elicited by depolarization. 3. GTP gamma S did not elicit any response when excitation-contraction coupling was abolished by prolonged depolarization or by chemically skinning the fibre with saponin or by 24 h exposure to low [Ca2+] (5 mM-EGTA). 4. GDP beta S (guanosine-5'-O-(2-thiodiphosphate), 250 microM or 1 mM) neither evoked a response nor affected the responses to depolarization or caffeine. 5. When the [Mg2+] was lowered to 0.2 mM and the fibres were heavily loaded with Ca2+, addition of GTP gamma S (250 microM or 1 mM) induced a small response in about 50% of fibres, but depolarization-induced responses were not affected in any fibres. 6. Asymmetric charge movement recorded in EDL fibres with the vaseline-gap voltage clamp was not affected by the application of 1 mM-GTP gamma S to the cut ends of the fibres for up to 1 h. 7. These data imply that GTP-binding proteins (G-proteins) are not involved in coupling the voltage sensors to Ca2+ release in skeletal muscle. Furthermore, there was no evidence that G-proteins play any role in modulating the voltage sensors, though this possibility could not be totally excluded.

Animals↗

PubMed1990

Calcium and strontium activation characteristics of skeletal muscle fibres from the small marsupial Sminthopsis macroura.

Mechanically skinned skeletal muscle fibres from the soleus and tibialis anterior muscles of the small marsupial Sminthopsis macroura were activated by Ca2+ and Sr2+ so that their isometric force properties could be determined. The properties characterized were the shape, slope and positions of the curves generated by plotting isometric force vs. pCa (-log10[Ca2+]) and pSr (-log10[Sr2+]), the maximum Ca2(+)-activated and Sr2(+)-activated tension (Ncm-2) and the frequency of force oscillations of myofibrillar origin during submaximal activations. The effect of caffeine on force activation was also studied. Apart from the fibres which exhibited physiological characteristics similar to those observed previously in mammalian fibres, a large proportion of fibres exhibited characteristics or combinations of characteristics which have not previously been described from healthy adult mammals. The results from 32 soleus fibres showed that only 23 could be categorized as either typical fast-twitch or slow-twitch fibres. The rest possessed unusual physiological characteristics which suggested the co-existence in the same fibre of Ca2(+)-regulatory and contractile properties from different categories of fast-twitch and slow-twitch fibres. We could distinguish two major fast twitch populations of tibialis anterior fibres which occurred in similar proportions. There were significant differences in the maximum tension produced by some of these groups of fibres. The tibialis anterior population fibres produced the highest maximum tension (ToCa 44.6 +/- 4.6 Ncm-2, n = 7) while the soleus combined type fibres produced the lowest maximum tension (ToCa 18.1 +/- 2.1 Ncm-2, n = 8). Our physiological observations of the Ca2(+)-activation and Sr2(+)-activation properties of soleus fibres in this study provide new evidence that there can be combinations of characteristics in single fibres and a continuum of properties between fibre types in normal mammalian skeletal muscles. These animals can therefore be used as a source of fibres with a wide range of properties.

Animals↗

PubMed1990

Physiological properties of skinned fibres from normal and dystrophic (Duchenne) human muscle activated by Ca2+ and Sr2+.

1. Contractile activation properties of various types of normal and dystrophic (Duchenne muscular dystrophy, DMD) human muscle fibres were investigated using mechanically skinned fibres activated in Ca2(+)- and Sr2(+)-buffered solutions at room temperature (21-25 degrees C). 2. The majority of the normal human muscle fibres (18/22; 82%) could be classified according to the Ca2(+)- and Sr2(+)-activation characteristics in the same three major groups as other mammalian skeletal muscle fibres studied previously: slow-twitch (8/22; 36%), fast-twitch (3/22; 14%) and fast-twitch intermediate (7/22; 32%), which correspond to the three major histological fibre types I, II B and IIA respectively. 3. Of these three major groups only the slow-twitch (type I; 14/24; 58%) and the fast-twitch intermediate (type IIA; 3/24; 13%) fibres were found in the DMD muscle, indicating that fast-twitch fibres of type IIB were in very low proportion in DMD muscle. 4. The DMD muscle contained a new group of fibres (4/24; 17%) with different Ca2(+)- and Sr2(+)-activation characteristics from the three major histological types. This group of fibres is likely to be of embryonic type. 5. The maximum tension development ability of DMD fibres was less than 20% of that in normal fibres with the exception of some slow-twitch fibres which could produce near normal tension. 6. A significant proportion of normal (4/22; 18%) and DMD (3/24; 13%) fibres were found to have a mixture of Ca2(+)- and Sr2(+)-activation characteristics. This can be explained by co-existence of various myofibrillar protein isoforms in different proportions with regulatory functions in the same individual fibre. 7. The results demonstrate that DMD leads to marked diminution in the ability of most individual skeletal muscle fibres to develop tension, and causes changes in the overall fibre-type distribution in afflicted muscles.

Calcium↗

PubMed1990

Properties of enzymatically isolated skeletal fibres from mice with muscular dystrophy.

1. Single intact muscle fibres were enzymatically isolated from the skeletal muscles of the dystrophic mouse 129/ReJ dy/dy and were subjected to a range of physiological interventions. 2. Electrophysiological measurements, diffusion of injected dyes (Lucifer Yellow), microdissection and general appearance in the light microscope have shown that the majority of skeletal fibres isolated from the soleus and extensor digitorum longus (EDL) of adult dystrophic mice (10-14 weeks old) had gross morphological abnormalities. These abnormalities ranged from simple branching of the fibre to interconnections of many fibre branches which form a complex syncitium. 3. Segments from fibres of normal appearance and from fibres with morphological deformities were chemically skinned with Triton X-100 and activated in Ca2(+)- and Sr2(+)-buffered solutions. The different characteristics of the Ca2(+)- and Sr2(+)-activation curves were also used to identify the fibre type. 4. Gross morphological abnormalities were observed both in fibres which had predominantly slow-twitch and fast-twitch characteristics. 5. A new group of fibres was found to exist in the soleus muscle of dystrophic animals and represented about 18% of the entire soleus fibre population. This group of fibres had predominantly fast-twitch characteristics and some of these fibres were also grossly malformed. 6. The activation characteristics of individual branches from the same complex syncitium were similar, indicating that the contractile and regulatory proteins were of one type in one syncitium. 7. Chemically skinned segments from malformed fibres which included a major deformity between the points of attachment were generally unable to sustain near-maximal forces. 8. The proportion of malformed fibres which remained intact decreased markedly after prolonged tetanical stimulation of the intact muscle. This strongly suggests that malformed fibres are also functionally weak and prone to progressive damage when stimulated within the intact muscle. 9. The presence in large proportions of fibres with gross morphological abnormalities may explain the symptoms of severe and progressive muscle weakness and muscle loss which are apparent in the 129/ReJ dy/dy mice and possibly even in the human dystrophies such as Duchenne muscular dystrophy.

Animals↗

PubMed1990

Calcium release in skinned muscle fibres of the toad by transverse tubule depolarization or by direct stimulation.

1. Skeletal muscle fibres from the toad were mechanically skinned under paraffin oil and then bathed in a potassium HDTA solution (HDTA: hexamethylenediamine-tetraacetate) which mimicked the ionic composition of the myoplasm. 2. Rapid transient contractions could be triggered by substitution of K+ with Na+ (with no change of anion), which should have virtually no direct effect on the electrical polarization of the sarcoplasmic reticulum (SR) membrane. Up to thirty or more contractions could be evoked by repeated substitutions if there was sufficient 'repriming' time (about 30 s) between them; these rapid contractions were analagous to potassium contractures in intact fibres. 3. When the SR was not heavily loaded, substitution of potassium HDTA with choline chloride also produced a rapid, brief contraction. 4. All treatments designed to 'inactivate' the voltage sensor in the T-system invariably abolished the rapid contractions. Thus, rapid contractions were absent if (i) the T-system was permanently depolarized by pre-soaking the muscle in a high potassium solution with ouabain before skinning, (ii) a fibre was split rather than skinned, (iii) the T-system was temporarily depolarized by Na+ substitution immediately before choline chloride substitution, or vice versa, (iv) a skinned fibre was briefly exposed to saponin (50 micrograms/ml) to selectively disrupt the T-system membrane or (v) the muscle was pre-soaked in a solution with 1 mM-EGTA and no Ca2+ or Mg2+ before skinning. In contrast to (v), if 10 mM-Mg2+ was present in the EGTA solution before skinning, rapid contractions could be elicited, presumably because the presence of Mg2+ prevented the inactivation of the T-system voltage sensor in low [Ca2+]. 5. These results unequivocally demonstrate that (a) the T-system reseals and repolarizes after mechanical skinning under oil and (b) the fast contractions are produced by activation of the voltage sensor in the T-system. 6. When the SR had been heavily loaded, choline chloride substitution (but not Na+ substitution) could also induce an unphysiological, slow contraction ('second component'). In total contrast to the fast contraction, this slow component was unaffected by any of the treatments (i-v) above, indicating that it did not depend on activation of the voltage sensor in the T-system but resulted from a direct action of choline chloride on the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1990

Control of calcium release and the effect of ryanodine in skinned muscle fibres of the toad.

1. Skinned muscle fibres from the toad were used to investigate the roles of T-system membrane potential and Ca2+ in controlling the calcium release channels of the sarcoplasmic reticulum (SR). 2. Replacement of K+ in the bathing solution with Na+ produced a large contraction which could last for 30 s or more under certain circumstances. This prolonged contraction could be quickly and completely terminated by repolarizing the fibre in the K+ solution and then immediately re-initiated by returning to the Na+ solution. These data indicate that the membrane potential tightly controlled the substantial and prolonged release of calcium. 3. T-system depolarization in the presence of 10 mM-free EGTA (pCa greater than 9) markedly depleted the SR of Ca2+. This implies that depolarization of the T-system can still trigger substantial release of Ca2+ from the SR even when the myoplasmic [Ca2+] is very low and very heavily buffered by EGTA. 4. When the SR was heavily loaded with Ca2+, substitution of a weakly buffered high [Ca2+] solution (pCa 5.4, 50 microM-EGTA) could produce a small to moderate, transient contraction taking between 3 and 12 s to reach a peak and lasting 30 s or more. 5. This contraction may be produced at least partly by 'calcium-induced calcium release' as ruthenium red (2 microM) completely blocked the responses. Moreover, repeated substitutions produced successively smaller responses in parallel with the 'run-down' of the depolarization-induced contractions. 6. Depolarization could always produce an additional large and fast response at any stage during a 'Ca2(+)-induced' response. 7. In the presence of 25 microM-ryanodine, the rapid contraction produced by T-system depolarization was prolonged and could not be stopped by repolarization. During and after this contraction no depolarizing stimulus could induce a further contraction, even though in some fibres addition of 30 mM-caffeine produced a maximum response which indicated that there was still a substantial amount of calcium in the SR. 8. At pCa 6.4, 25 microM-ryanodine could itself induce a substantial slow contracture in a normally polarized fibre within 30-60 s, after which little or no response could be induced by T-system depolarization. At higher concentrations (25 microM) ryanodine produced a near-maximum contraction in only a few seconds.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗

PubMed1990

Halothane increases Ca2+ efflux via Ca2+ channels of sarcoplasmic reticulum in chemically skinned rat myocardium.

1. A method has been developed to study Ca2+ fluxes across the sarcoplasmic reticulum (SR) of chemically (saponin) skinned myocardium without interference from the SR Ca2+ pump. 2. Exposure of rat cardiac trabeculae to a solution containing 50 micrograms/ml saponin for 10 min or longer caused an SR Ca2+ efflux which was not blocked by Ruthenium Red (RRed) and did not require the presence of nucleotides. 3. Exposure of the saponin-treated cardiac preparation to 11 mM-AMP, when the SR Ca2+ pump was not active, enhanced Ca2+ release from the SR by a mechanism which was blocked by 10 microM-RRed. 4. The amount of Ca2+ loaded by the 10 min saponin-treated trabeculae was maintained constant for at least 3 min when the preparations were transferred to low [Ca2+] solutions (0.1 mM-EGTA; pCa greater than 7.5) containing ATP. This indicated that the Ca2+ pump can efficiently recycle Ca2+ lost from the SR under these conditions. 5. Halothane (0.47 and 1.89 mM) reversibly increased the rate of Ca2+ release from the SR regardless of whether or not the SR Ca2+ pump was active. This effect was more marked at 1.89 mM than at 0.47 mM. RRed (10 microM) completely blocked the Ca2+ release induced by both concentrations of halothane. 6. The presence of nucleotide (11 mM-AMP) did not affect the halothane-induced Ca2+ release when the Ca2+ pump was inactive. 7. Exposure of cardiac preparations to solutions containing more than 5 mM-halothane irreversibly damaged the ability of the SR to load Ca2+. 8. The results suggest that at lower doses (0.47 and 1.89 mM) halothane specifically and reversibly stimulates Ca2+ efflux via the RRed-sensitive SR Ca2(+)-release channel by a mechanism which does not require the presence of nucleotides or relatively high [Ca2+]. The results also suggest that AMP and halothane act independently and non-synergistically to increase Ca2+ efflux through the same SR Ca2(+)-release channel. At higher doses (greater than 5 mM) halothane irreversibly damages the SR membrane, presumably by disrupting the lipid bilayer.

Adenosine Monophosphate↗

PubMed1990

Resting calcium concentrations in isolated skeletal muscle fibres of dystrophic mice.

1. Single, intact muscle fibres were dissociated enzymatically from skeletal muscles of phenotypically normal (+/?) and dystrophic mice (129/ReJ dy/dy: Dystrophia muscularis), and resting Ca2+ levels were measured by image analysis of intracellular Fura-2 fluorescence in distinct parts of the fibres. 2. Fura-2 was introduced into fibres by ionophoresis with glass microelectrodes to concentrations of between 50 and 200 microM. Over this concentration range there was no apparent buffering of intracellular Ca2+ by Fura-2. 3. Fibres isolated from the soleus, flexor digitorum brevis (FDB) and extensor digitorum longus (EDL) muscles of normal animals maintained resting [Ca2+] of 106 +/- 2 nM. Ca2+ distributions within individual fibres were homogeneous. 4. Fibres from dystrophic animals maintained [Ca2+] that was elevated two- to fourfold in comparison to normal fibres. 5. The population of skeletal fibres from dystrophic mice which displayed morphology similar to that of fibres of normal animals were found to have Ca2+ levels that averaged 189 +/- 2 nM. The distribution of Ca2+ within these fibres appeared uniform. 6. The population of dystrophic fibres that possessed morphological abnormalities maintained even higher Ca2+ concentrations (368 +/- 3 nM). Several fibres from this morphological group displayed obvious heterogeneity in Ca2+ distribution with distinct, localized areas of higher Ca2+. 7. These results support the contention that Ca2+ homeostasis is markedly impaired in dystrophic muscle. The elevated Ca2+ levels are near the threshold for contraction and, together with severe morphological fibre abnormalities, are probably centrally involved in fibre necrosis apparent in muscular dystrophy.

Animals↗

PubMed1990

Unloaded shortening velocity of skinned rat myocardium: effects of volatile anesthetics.

The slack test method has been adapted for measurement of unloaded velocity of shortening in rat ventricular trabeculae that were skinned with saponin (50 micrograms/ml for 30 min). The method was sensitive enough to detect a 17% reversible change in the unloaded velocity of shortening produced by a 3 degrees C change in temperature. At pCa 5.30 (80-90% activation), halothane, enflurane, and isoflurane each slowed the shortening velocity by 25-30% at dose levels of 8 mM or greater but not at 4 mM or less. At pCa 5.48 (50-60% activation), halothane slowed the shortening velocity by 20-45% at dose levels of 4 mM or greater but not at 2 mM. The slowing effect of anesthetics on shortening velocity showed saturation at 8 mM for halothane, enflurane, and isoflurane when activation was at pCa 5.30. Saturation occurred at 4 mM for halothane when the pCa was 5.48. This result indicates that the dose-response relationship may be narrow, such that it can be demonstrated between 2 and 4 mM halothane for pCa 5.48 and between 4 and 8 mM halothane for pCa 5.30. The anesthetic dose dependence of isometric force and length axis intercept did not generally follow the same relationship as for the shortening velocity. Thus in several instances force did not significantly decrease when the velocity of shortening did. This may be interpreted as lack of simple inhibition by anesthetics on the number of interacting cross-bridges and as direct influence by anesthetics on the cross-bridge cycle.

Anesthetics↗

PubMed1989

Contractile activation and the effects of 2,3-butanedione monoxime (BDM) in skinned cardiac preparations from normal and dystrophic mice (129/ReJ).

Small cardiac myofibrillar preparations were obtained from the right ventricle of normal (129/ReJ) and dystrophic (129/ReJ dy/dy) mice and were chemically skinned in a relaxing solution by exposure to Triton X-100 (3% v/v). (2) The isometric force produced in these skinned cardiac preparations at different sarcomere lengths was measured in solutions of different [Ca2+] and ionic strength. The effect of the negative inotropic drug 2,3-butanedione monoxime (BDM), which is known to act at the myofibrillar level was also investigated. (3) The murine cardiac preparation from normal animals was found to develop 50% maximal force at a pCa (= -log10[Ca2+]) of 5.59 +/- 0.08 and 5.94 +/- 0.03 (mean +/- SD) under physiological (ionic equivalents concentration, I = 154 mM; pH 7.10; [Mg2+] 1 mM) and low ionic strength (I = 94 mM; pH 7.10; [Mg2+] 1 mM) conditions respectively. The isometric force curves were significantly shallower at low ionic strength (Hill coefficient, 1.8 +/- 0.1) than at physiological ionic strength (Hill coefficient, 2.6 +/- 0.3) and the sarcomere length effect on the force-pCa relation was markedly reduced at lower ionic strength. (4) Increasing BDM concentrations in solutions up to 100 mM reduced the maximum Ca2+-activated force of cardiac preparations from normal mice to less than 6% of the control values in a dose dependent fashion. BDM also rendered the cardiac preparations less sensitive to Ca2+ by a factor of up to 1.5 in a process which showed saturation at BDM concentrations higher than 15 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Dissociation of force from myofibrillar MgATPase and stiffness at short sarcomere lengths in rat and toad skeletal muscle.

1. Single fast-twitch fibres from the extensor digitorum longus muscle of the rat, Rattus norvegicus, and single twitch fibres from the iliofibularis muscle of the cane toad, Bufo marinus, were mechanically skinned and then used to measure maximally Ca2+-activated [( Ca2+] greater than 0.03 mmol l-1) isometric force production, myofibrillar MgATPase activity and fibre stiffness at different sarcomere lengths. MgATP hydrolysis was linked by an enzyme cascade to the oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) and was monitored by a microfluorimetric system. Fibre stiffness was measured from the amplitude of force oscillations generated by small sinusoidal length changes. 2. At sarcomere lengths which were optimal for isometric force production (around 2.7 microns for rat and 2.2 microns for toad fibres) the myofibrillar MgATPase activity (mean +/- S.E.M.) at 21-22 degrees C was found to be 3.80 +/- 0.53 molecules MgATP hydrolysed s-1 per myosin head for eight rat fibres and 6.35 +/- 0.77 s-1 per myosin head for four toad fibres. 3. At sarcomere lengths shorter than 2.7 microns in rat fibres and 2.2 microns in toad fibres, MgATPase and stiffness remained elevated and close to their respective values at 2.7 microns in rat fibres and 2.2 microns in toad fibres even when the isometric force decreased to near zero levels. 4. The dissociation at short sarcomere lengths of myofibrillar MgATPase activity and fibre stiffness from isometric force suggests that the cross-bridge cycle is not greatly affected by double actin filament overlap with the myosin filaments at short sarcomere lengths. Moreover, the results suggest that cross-bridges can be formed by myosin with actin filaments projecting from the nearest Z-line and from the Z-line in the other half of the sarcomere. 5. These results help to reconcile energetic and mechanical data obtained by others at short sarcomere lengths and can be explained within the framework of the sliding filament theory.

Adenosine Triphosphate↗

PubMed1988

Effects of 2,3-butanedione monoxime on the contractile activation properties of fast- and slow-twitch rat muscle fibres.

1. The effects of 2,3-butanedione monoxime (BDM, 0.2-10 mmol/l) have been examined at different temperatures on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat and on Ca2+-activated isometric force of mechanically skinned soleus and EDL fibres. Ca2+ release and uptake by the sarcoplasmic reticulum has also been investigated directly on skinned EDL fibres at 1 mmol/l BDM at 23 degrees C. 2. BDM bound calcium with low affinity (KCaBDMapp = 55.9 +/- 4.3 M-1 at 22 degrees C, pH 7.10, ionic strength approximately 155 mmol/l) and did not affect the kinetics and quantum yield of the Ca2+-induced aequorin luminescence. 3. BDM reversibly reduced both force- and Ca2+-dependent aequorin light during the twitch and tetanus of all intact-muscle preparations in a dose-related manner. Twitch responses of soleus fibres were more sensitive to BDM than were EDL fibres. Higher concentrations of BDM were needed to abolish the tetanic force response than the twitch response in both muscle types. 4. The initial rate of rise and the decay rate constants of twitches in both soleus and EDL muscles were increased in the presence of BDM. Accordingly, a higher frequency of stimulation was required to produce fusion of the tetanus. This phenomenon could be explained by the reduced magnitude of the Ca2+ transient. 5. BDM reduced maximal Ca2+-activated force development in mechanically skinned soleus and EDL muscle fibres in a dose-dependent fashion. This depression of force was less sensitive to BDM than were the light transients in intact fibres. Furthermore, BDM affected EDL skinned fibres to a greater extent than soleus skinned fibres, in contrast with its effects on intact fibres. 6. At concentrations of BDM greater than 2.5 mmol/l, BDM significantly decreased the sensitivity of the contractile apparatus to calcium. The relative force-pCa (= -log [Ca2+]) curves for both soleus and EDL skinned fibres were shifted to the right (i.e. to higher calcium concentrations) in a dose-related manner. 7. BDM (10 mmol/l) slowed maximal Ca2+-activated force development of skinned preparations of both fibre types at all temperatures investigated (4-24 degrees C). 8. BDM reduced stiffness in skinned preparations maximally activated by calcium in proportion to the reduction in the isometric force response. BDM also inhibited rigor force of all fibres but only if it was present before the onset of rigor.(ABSTRACT TRUNCATED AT 400 WORDS)

Aequorin↗

PubMed1987

Ca2+-movements in muscle modulated by the state of K+-channels in the sarcoplasmic reticulum membranes.

1. A procedure has been developed to load Ca2+ reversibly by the sarcoplasmic reticulum (SR) of mechanically skinned muscle fibres from the toad Bufo marinus under controlled conditions and was employed to investigate the effects of conditions known to reduce the K+ conductance located in the SR-membrane during Ca2+-loading on the amount of Ca2+ releasable by caffeine. 2. The amount of releasable Ca2+ was markedly increased compared to controls when 4-aminopyridine (4AP) (6 microM to 2 mM), tetraethylammonium (TEA), decamethonium (0.5 mM) or procaine (1 mM) were present in the Ca2+-loading solutions. All these substances are known to act as SR-K+-channel blockers. 3. The increased amount of releasable Ca2+ in the presence of the K+-channel blocker 4AP was observed both at low (0.3 mM) and at higher (1 mM) Mg2+ concentrations and was not affected by verapamil (20 microM), a known Ca2+-channel blocker of the sarcolemma nor by the Na+-K+ pump inhibitor, ouabain (1 mM). 4. In the presence of 0.1-5 microM ruthenium red, a known inhibitor of Ca2+ induced Ca2+ release from the SR, the amount of releasable Ca2+ was greatly increased by up to 300%. Addition of between 50 microM and 1.6 mM 4AP to ruthenium red Ca2+ loading solution modified differently the amount of releasable Ca2+, suggesting that the mechanism of action of 4AP is different from that of ruthenium red. 5. When all K+ ions in the loading solution were replaced by the less permeant Na+ ions the amount of releasable Ca2+ ions was also increased. 6. These results indicate that the amount of releasable Ca2+ from the SR is consistently modified under conditions aimed at interfering with the state of SR-K+-channels, suggesting that SR-K+-channels may play an important physiological role in the modulation of excitation-contraction coupling. One possible mechanism involving SR-K+-channels which could explain our results is discussed.

4-Aminopyridine↗

PubMed1987

Thermal dependence of maximum Ca2+-activated force in skinned muscle fibres of the toad Bufo marinus acclimated at different temperatures.

Mechanically skinned muscle fibres from the twitch region of the iliofibularis muscle of cool- (16 +/- 1 degree C) and warm- (32 +/- 1 degree C) acclimated cane toads (Bufo marinus) were activated maximally by Ca2+ in solutions of different pH and at different temperatures (approx. 1-35 degrees C). Acclimation of up to 12 weeks at 16 degrees C and up to 8 weeks at 32 degrees C did not modify the marked thermal dependence of isometric force in the skeletal muscle fibres of the cane toad. The prominent decline of maximum Ca2+-activated force at lower temperatures, a property which is not characteristic of muscles from other anurans, was associated with an obvious decline in fibre stiffness at temperatures below about 20 degrees C, regardless of the temperatures at which the toads were kept prior to experimentation. The results suggest that the decline of isometric force at lower temperatures is due both to a reduction in the number of cross-bridges and to a decrease in the force output per cross-bridge. The maximum Ca2+-activated force response increased when fibres were activated in solutions of increasing pH at all temperatures investigated. This trend is expected to have a compensatory effect on the thermal dependence of the maximum Ca2+-activated force under physiological conditions, because of the elevation of intracellular pH as temperature declines. The isometric force did not depend on the concentration of the zwitterionic species of the pH buffer in solutions. The skinned fibre preparation developed a Ca2+-insensitive residual force following maximal activation. The increment in residual force followed a linear relationship with the duration of activation at a given temperature and a power relationship of activation temperature for a given duration of activation. Fibres from warm-acclimated animals developed less residual force following activations at 15 degrees C than did fibres from cool-acclimated animals, suggesting that thermal acclimation may substantially reduce the magnitude of this phenomenon at temperatures below 20 degrees C.

Acclimatization↗

PubMed1986

The maximum speed of shortening in living and skinned frog muscle fibres.

This study was performed to determine whether Viso (the maximum speed of shortening extrapolated from force-velocity curves) equalled Vu (the unloaded speed of shortening determined by the slack test) in both living fibres from Rana temporaria and mechanically skinned fibres from Rana pipiens. In living fibres (R. temporaria) we obtained improved estimates of Viso by performing force clamps (isotonic) and length ramps (isovelocity) down to very low loads (0.005 isometric tension, P0). Force-velocity characteristics determined by force clamps and length ramps were the same. The hyperbolic Hill curves deviated from the force-velocity data at both high and low loads and underestimated Viso by varying degrees. A better estimate of Viso was obtained by linear extrapolation of data at loads from 0.005-0.02 P0 and the mean Viso at 7.5 degrees C was 4.08 muscle lengths/s +/- 0.11 (mean +/- S.E., n = 14). Improved estimates of Vu in living fibres were obtained by photographically calibrating the slack test. The mean Vu was 4.05 muscle lengths/s +/- 0.13 (mean +/- S.E., n = 14) and the intercept was 0.0156 fibre lengths (L0) +/- 0.0013 (mean +/- S.E., n = 14). The step-ramp photographic method, in which the motor speed is matched to Vu, was developed as an independent way to measure Vu in living fibres. Vu measured in this way agreed well with Vu measured by the slack test. In all living fibres, the improved estimates of Vu agreed well with the improved estimates of Viso. Vu/Viso = 0.99 +/- 0.01 (mean +/- S.E., n = 14). In mechanically skinned R. pipiens fibres, force clamps were performed down to loads of 0.01 mN. The force-velocity curve of the skinned fibres differed in shape from that of the living fibres. Although there was significant deviation from the Hill equation at low loads, the data at high loads were well fitted by the Hill curve. Viso determined by extrapolating the Hill equation to zero load was 5.87 muscle lengths/s +/- 0.38 (mean +/- S.E., n = 9) at 7.5 degrees C. In five fibres, the linear extrapolation of low loads (0.01-0.05 P0) showed that the Hill equation underestimated the true Viso by 6%. The slack test with mechanically skinned fibres was calibrated by taking a series of photographic exposures of the fibre at various times following each length step. Vu = 6.12 muscle lengths/s +/- 0.44 (mean +/- S.E., n = 10) and the intercept was 0.0585 L0 +/- 0.0069 (mean +/- S.E., n = 10).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Potassium and ionic strength effects on the isometric force of skinned twitch muscle fibres of the rat and toad.

A study was carried out to investigate the effects of ionic strength and monovalent cations on isometric, Ca2+-activated force and rigor responses in mechanically skinned muscle fibres. Three types of skeletal muscle fibres were used: rat fast- and slow-twitch fibres and toad twitch fibres. The contractile apparatus of rat slow-twitch fibres was affected differently from that of rat fast-twitch and amphibian twitch fibres when changing the ionic strength (expressed either in terms of ionic equivalents as I or formally as gamma/2) and [K+]. Thus, the apparent sensitivity to Ca2+ decreased substantially more in slow-twitch fibres (by a factor of 20) than in the other fibre types (by a factor of 12) when I and [K+] were increased from 94 to 354 mM and from 56 to 316 mM respectively. Maximum Ca2+-activated force, however, declined only by a factor of 2.2 in slow-twitch fibres compared with 4.2 in the other fibre types, when I was increased from 154 to 354 mM. In slow-twitch fibres the force oscillations of myofibrillar origin were found to increase substantially in amplitude, duration and frequency at low values of I and almost disappeared at high ionic strength. At low values of I, it was also discovered that ca. 50% of the fast-twitch fibres responded with myofibrillar force oscillations when submaximally activated. The characteristics of these oscillations were different from those of slow-twitch fibres. Rigor force levels were found to decline markedly with increasing iota and [K+] in all fibre types. Unexpectedly, once rigor force was established in a certain ionic environment, the level of force was stable regardless of further changes in ionic strength and monovalent cation concentration. These results indicate that the rigor cross-bridges can be formed in different stable positions and that the probability of attachment in certain positions (rather than the total number of cross-bridges that can be formed) is influenced by the ionic conditions. Further experimental evidence provided in this study shows that the increase in [K+] is mainly responsible for the decrease of the Ca2+-sensitivity of the contractile apparatus and that ionic strength (expressed as I rather than gamma/2) influences markedly the maximal Ca2+-activated force, the maximum steepness of the pCa-force relations and the oscillatory processes of myofibrillar origin.

Animals↗

PubMed1986

Effects of procaine on calcium accumulation by the sarcoplasmic reticulum of mechanically disrupted rat cardiac muscle.

The ability of the sarcoplasmic reticulum of skinned cardiac muscle of the rat to accumulate and release Ca2+ was studied in the presence and absence of procaine. Ca2+ accumulation was estimated from the magnitude of the caffeine- (30 mM) induced force transient in a weakly Ca2+ buffered solution. The relative area under the caffeine-induced force transient was up to 4-fold greater when 5 mM-procaine had been present during the preceding period of Ca2+ loading, than that after an equivalent period of Ca2+ loading in the absence of procaine. Procaine antagonized the caffeine-induced release of Ca2+ when present in the Ca2+ releasing solution, however, the ability of procaine to attenuate the caffeine-induced Ca2+ release diminished as the extent to which the sarcoplasmic reticulum was loaded with Ca2+ increased. In the presence of 1 mM-Mg2+ procaine also markedly attenuated the small spontaneous force oscillations (5-10% P0) associated with the cyclic release and reuptake of Ca2+ by the sarcoplasmic reticulum. When the Mg2+ concentration was reduced to 0.1 mM, procaine initially suppressed the small spontaneous oscillations in force, however, large force oscillations (40-80% P0) of lower frequency were invariably initiated after 20-60 s exposure to 5 mM-procaine. Procaine (5 mM) produced a slight shift (approximately 0.04 pCa unit) of the force-pCa relation toward lower Ca2+ concentrations. This effect is too small to influence in any substantial way the apparent effects of procaine on the sarcoplasmic reticulum. The results indicate that whilst procaine is indeed able to suppress Ca2+ release under certain circumstances, in its presence the net accumulation of Ca2+ by the sarcoplasmic reticulum can be markedly enhanced.

Animals↗