PubMed Health⌕ Search

Biomedical subjects

D G Allen

Publications and source records attributed to D G Allen.

At least 73 records · Page 4Linked to original sources
PubMed1997

The role of ATP in the regulation of intracellular Ca2+ release in single fibres of mouse skeletal muscle.

1. Single fibres were dissected from mouse flexor brevis muscle and injected with indo-1 and the P3-1 (2-nitrophenyl)ethyl ester of ATP (caged ATP). Myoplasmic calcium concentration ([Ca2+]i) and force were monitored during single tetani or tetani repeated until force was reduced to about 30% of control values. In vitro experiments showed that an intense, brief ultraviolet illumination (a flash) photolysed 12% of the caged ATP to ATP. 2. Fibres that had been injected with caged ATP showed concentration-dependent changes. High concentrations of caged ATP caused a reduction in [Ca2+]i during tetani (tetanic [Ca2+]i), a reduction in force in unfatigued tetani and the fibres fatigued more rapidly when stimulated repeatedly. 3. Photolytic release of ATP in unfatigued fibres caused a concentration-dependent increase in tetanic [Ca2+]i and in force. 4. When ATP was released by photolysis in a fibre fatigued by repeated tetani, it produced a concentration-dependent increase in tetanic [Ca2+]i and force. The increase in tetanic [Ca2+]i was small (63 nM per 100 microM increase in ATP) and could explain some, but not all, the increase in force. However, taking into account the fact that control flashes in the absence of caged ATP caused a small decrease in tetanic [Ca2+]i, we believe that the increase in force may be explained by the increase in tetanic [Ca2+]i. There was no evidence of changes in the sarcoplasmic reticulum Ca2+ pump rate after photolysis of caged ATP. 5. Caged ATP affects some site(s) involved in excitation-contraction coupling and the consequences are similar to muscle fatigue. When a small fraction of this caged ATP is photolysed to ATP, the consequences of fatigue are partially reversed. These observations suggest that site(s) which either bind ATP or depend on ATP hydrolysis have a key role in excitation-contraction coupling and in muscle fatigue.

Adenosine Triphosphate↗
PubMed1997

Effects of reduced muscle glycogen concentration on force, Ca2+ release and contractile protein function in intact mouse skeletal muscle.

1. The purpose of this study was to examine the effects of reduced glycogen concentration on force, Ca2+ release and myofibrillar protein function during fatigue in skeletal muscle. Force and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mammalian skeletal muscle fibres during fatigue and recovery. Glycogen was measured in bundles of 20-40 fibres from the same muscle under the same conditions. 2. Fatigue was induced by repeated maximum tetani until force was reduced to 30% of initial. This was associated with a reduction in muscle glycogen to 27 +/- 6% of control values. In fibres allowed to recover for 60 min in the presence of 5.5 mM glucose (n = 6), tetanic (100 Hz) force recovered fully but tetanic [Ca2+]i remained at 82 +/- 8% of initial values. This prolonged depression in Ca2+ release was not associated with decreased muscle glycogen since glycogen had recovered to pre-fatigue levels (157 +/- 42%). 3. To examine the responses under conditions of reduced muscle glycogen concentration, fibres recovered from fatigue for 60 min in the absence of glucose (n = 6). After glucose-free recovery, the decreases in tetanic force and [Ca2+]i were only partially reversed (to 64 +/- 8% and 57 +/- 7% of initial values, respectively). These alterations were associated with a sustained reduction in muscle glycogen concentration (27 +/- 4% of initial values). 4. In another set of fibres, fatigue was followed by 50 Hz intermittent stimulation for 22.6 +/- 4 min. With this protocol, tetanic force and [Ca2+]i partially recovered to 76 +/- 9% and 55 +/- 6% of initial levels, respectively. These changes were associated with a recovery of muscle glycogen (to 85 +/- 10%). 5. During fatigue, Ca2+ sensitivity and maximum Ca(2+)-activated force (Fmax) were depressed but these alterations were fully reversed when muscle glycogen recovered. When glycogen did not recover, Ca2+ sensitivity remained depressed but Fmax partially recovered. The altered myofibrillar protein function is probably due to alterations in inorganic phosphate levels or other metabolites associated with reduced levels of muscle glycogen. 6. These data indicate that the reductions in force, Ca2+ release and contractile protein inhibition observed during fatigue are closely associated with reduced muscle glycogen concentration. These findings also suggest that the changes in Ca2+ release associated with fatigue and recovery have two components-one which is glycogen dependent and another which is independent of glycogen but depends on previous activity.

Animals↗
PubMed1997

Extracellular ATP increases intracellular calcium in cultured adult Schwann cells.

We have previously reported that extracellular ATP causes a transient rise in intracellular calcium concentration ([Ca2+]i) in cultured Schwann cells derived from adult animals [Ansselin A. D. et al. (1994) Int. J. Neurosci. 74, 148]. In this study, the receptor mediating this response has been characterized. Established adult rat and rabbit Schwann cell cultures were loaded with fura-2 (acetoxymethyl ester, 10 micromol/l, 40 min, 37 degrees C). which indicated, by fluorescence imaging, a resting [Ca2+]i of 34.7 +/- 1.4 nmol/l (mean S.E., n=591). The cells were exposed to 100 micromol/l ATP, ADP, AMP, UTP and adenosine in defined medium for 1-2 min, and the change in [Ca2+]i was observed as a change in the Fura-2 ratio. Seventy-seven percent of adult rat Schwann cells (n=235) and 88% adult rabbit Schwann cells (n=356) responded to the presence of extracellular ATP (100 mmol/l) with a transient increase in [Ca2+]i (41 and 90 nmol/l from resting value, respectively), independent of the presence of [Ca2+]o. Calcium waves were observed in one experiment. The following order of agonist potency was observed: UTP= ATP>>ADP>AMP=adenosine. The agonists alpha,beta-methylene-ATP and 2-methylthio-ATP had a small effect on the cells, similar to AMP, and were mutually desensitizing. The ATP antagonist suramin blocked the response. We conclude that adult Schwann cells express a purinergic ATP receptor belonging to the G-protein-coupled P2u alpha subtype [O'Connor S. et al. (1991) Trends pharmac. Sci. 12, 137-141].

Adenine Nucleotides↗
PubMed1997

Slowed relaxation in fatigued skeletal muscle fibers of Xenopus and Mouse. Contribution of [Ca2+]i and cross-bridges.

Slowing of relaxation is an important characteristic of skeletal muscle fatigue. The aim of the present study was to quantify the relative contribution of altered Ca2+ handling (calcium component) and factors downstream to Ca2+ (cross-bridge component) to the slowing of relaxation in fatigued fibers of Xenopus and mouse. Two types of Xenopus fibers were used: easily fatigued, type 1 fibers and fatigue resistant, type 2 fibers. In these Xenopus fibers the free myoplasmic [Ca2+] ([Ca2+]i) was measured with indo-1, and the relaxation of Ca2(+)-derived force, constructed from tetanic [Ca2+]i records and in vivo [Ca2+]i-force curves, was analyzed. An alternative method was used in both Xenopus and mouse fibers: fibers were rapidly shortened during the initial phase of relaxation, and the time to the peak of force redevelopment was measured. These two methods gave similar results and showed proportional slowing of the calcium and cross-bridge components of relaxation in both fatigued type 1 and type 2 Xenopus fibers, whereas only the cross-bridge component was slowed in fatigued mouse fibers. Ca2+ removal from the myoplasm during relaxation was markedly less effective in Xenopus fibers as compared to mouse fibers. Fatigued Xenopus fibers displayed a reduced rate of sarcoplasmic reticulum Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Some fibers were stretched at various times during relaxation. The resistance to these stretches was increased during fatigue, especially in Xenopus fibers, which indicates that longitudinal movements during relaxation had become less pronounced and this might contribute to the increased cross-bridge component of relaxation in fatigue. In conclusion, slowing of relaxation in fatigued Xenopus fibers is caused by impaired Ca2+ handling and altered cross-bridge kinetics, whereas the slowing in mouse fibers is only due to altered cross-bridge kinetics.

Animals↗
PubMed1997

Role of intracellular calcium and metabolites in low-frequency fatigue of mouse skeletal muscle.

We have examined the extent to which prolonged reductions in low-frequency force (i.e., low-frequency fatigue) result from increases in intracellular free Ca2+ concentration ([Ca2+]i) and alterations in muscle metabolites. Force and [Ca2+]i were measured in mammalian single muscle fibers in response to short, intermediate, and long series of tetani that elevated the [Ca2+]i-time integral to 5, 17, and 29 microM x s, respectively. Only the intermediate and long series resulted in prolonged (>60 x min) reductions in Ca2+ release and low-frequency fatigue. When fibers recovered from the long series of tetani without glucose, Ca2+ release was reduced to a greater extent and force was reduced at high and low frequencies. These findings indicate that the decrease in sarcoplasmic reticulum Ca2+ release associated with fatigue has at least two components: 1) a metabolic component, which, in the presence of glucose, recovers within 1 h, and 2) a component dependent on the elevation of the [Ca2+]i-time integral, which recovers more slowly. It is this Ca2+-dependent component that is primarily responsible for low-frequency fatigue.

Animals↗
PubMed1997

Influence of stimulation frequency on [Na+]i and contractile function in Langendorff-perfused rat heart.

To study the relationship between stimulation frequency and intracellular Na+ concentration ([Na+]i), Langendorff-perfused rat hearts were loaded with the Na(+)-sensitive dye sodium-binding benzofuran isophthalate (SBFI). An intracellular calibration procedure allowed SBFI fluorescence to be transformed into [Na+]i. Compartmentation of SBFI was evaluated by permeabilizing sarcolemmal membranes with saponin and subcellular compartments with Triton X-100. Most of the indicator was located in the myoplasm (69%). When stimulation frequency was increased from 0 to 6 Hz, [Na+]i increased from 3.0 to 7.9 mM, whereas pressure amplitude (PA) declined to 49% of the maximum recorded at 2 Hz. Blocking sarcoplasmic reticulum (SR) Ca2+ uptake with 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ; 10 microM) at 2 Hz increased [Na+]i from 4.9 to 8.4 mM and decreased PA by 70%. Raising stimulation frequency then resulted in a further increase in [Na+]i and decline in PA. In conclusion, these data indicate that the rat heart is characterized by a negative pressure-frequency relationship associated with increasing [Na+]i at higher heart rates. After inhibition of SR Ca2+ uptake, [Na+]i further increases, whereas PA declines with increasing stimulus frequency. It is suggested that part of the rise of [Na+]i with stimulus frequency and TBQ may be associated with increased Ca2+ extrusion and Na+ influx on the Na+/Ca2+ exchange system.

Action Potentials↗
PubMed1996

Postoperative complications of elective surgeries in dogs and cats determined by examining electronic and paper medical records.

Postoperative complications (POC) that developed in dogs and cats that underwent elective ovariohysterectomy, castration, and declaw at a veterinary teaching hospital were determined by examining the computerized abstracts of the medical records and by examining a random sample of the paper medical records. When the computerized abstracts were examined, POC were found to have occurred in 62 (6.1%) of 1,016 dogs. One dog died and 6 others developed major complications. Postoperative complications were found to have occurred in 38 (2.6%) of 1,459 cats. Two cats died and 1 was euthanatized. Four other cats developed major complications. Complete paper medical records for 218 dogs and cats were examined. When the paper medical records were examined, the proportions of dogs and cats with POC were 19.4% and 12.2%, respectively. These proportions were 4 to 7 times higher than when the computerized abstracts were the data source. Results of this study indicate that the frequency of clinically relevant POC of elective surgeries in dogs and cats is substantial. Examination of the computerized abstracts of medical records at this hospital allowed us to rapidly identify cases that could be included in the study but the frequency of POC would be significantly underestimated if paper records were not also assessed.

Animals↗
PubMed1996

The effect of muscle length on intracellular calcium and force in single fibres from mouse skeletal muscle.

1. The effect of muscle length on the myoplasmic free Ca2+ concentration ([Ca2+]i) and tetanic force production of intact single muscle fibres was investigated in the mouse flexor brevis muscle. Muscle fibres were analysed at 100 microns intervals from 100 microns shorter than the optimum length (L(zero)) to 300 microns longer than L(zero). The fluorescent Ca2+ indicator indo-1 was used to measure [Ca2+]i. 2. Changes in muscle length did not significantly affect tetanic or resting [Ca2+]i. However, at lower stimulus frequencies (30-50 Hz) the force-length relation was shifted to longer muscle lengths. 3. Force-tetanic [Ca2+]i curves were constructed at each muscle length. A comparison of these curves revealed that the Ca2+ sensitivity was length dependent. Ca2+ sensitivity increased over the ascending limb and plateau phase of the force-length curve, but over the descending limb Ca2+ sensitivity reached a plateau and then started to decrease once more. 4. These results show that length-dependent changes in Ca2+ sensitivity of single muscle fibres cannot simply be related to variations in sarcomere length. It is proposed that other factors, such as cross-bridge attachment and/or developed force, have a role in determining the Ca2+ sensitivity at a particular muscle length.

Animals↗
PubMed1996

Slowing of relaxation and [Ca2+]i during prolonged tetanic stimulation of single fibres from Xenopus skeletal muscle.

1. Parvalbumin (PA) has been proposed to take up Ca2+ and enhance skeletal muscle relaxation in brief contractions; as the duration of the contraction is increased, PA will become saturated with Ca2+ and no longer contribute to relaxation which therefore will be slowed. The rate of Ca2+ loading of PA is determined by the Mg2+ off rate (about 4 s-1 at 22 degrees C). In the present study we produced prolonged tetani in intact, single fibres of Xenopus frogs while measuring force and the free myoplasmic [Ca2+] ([Ca2+]i) with indo-1. 2. Mean rate constants of slowing of force relaxation with increasing tetanus duration ranged between 3.2 and 4.8 s-1, thus, similar to the Mg2+ off rate of PA. 3. The amplitude of the tail of [Ca2+]i after tetani increased with tetanus duration. This increase developed with a rate constant similar to the Mg2+ off rate of PA 4. Steady-state force-[Ca2+]i curves were produced from tetani of various frequencies and tetani produced when force was depressed after fatiguing stimulation. These curves were used to convert [Ca2+]i records into Ca(2+)-derived force. Relaxation of Ca(2+)-derived force was slowed following a time course similar to that of real force. The lag between Ca(2+)-derived and real force during relaxation was not affected by tetanus duration. 5. Tails of elevated [Ca2+]i after tetani were used to analyse the function of the SR Ca2+ pumps. This analysis showed a marked decline in the rate of Ca2+ uptake with prolonged tetani. 6. In conclusion, in Xenopus fibres the slowing of relaxation with increasing tetanus duration can be explained by altered Ca2+ handling due to PA Ca2+ loading and impaired SR Ca2+ uptake. This contrasts to our previous results in mouse fibres and the difference can be explained by a markedly lower rate of SR Ca2+ uptake resulting in higher tetanic [Ca2+]i in Xenopus fibres.

Animals↗
PubMed1996

The role of elevations in intracellular [Ca2+] in the development of low frequency fatigue in mouse single muscle fibres.

1. Intracellular free calcium concentration ([Ca2+]i) and force were measured in isolated single skeletal muscle fibres from mice. The aim was to determine the extent to which elevations in [Ca2+]i during various stimulation protocols affected subsequent muscle performance. 2. A protocol of repeated tetanic stimulation which elevated [Ca2+]i and caused a large decline in force (fatigue) had a [Ca2+]-time integral of 36.4 +/- 8.1 microM s. A protocol of repeated tetani at a lower duty cycle (stimulation) caused only a small decline in force (9-16%) but elevated the [Ca2+]-time integral to 16.7 +/- 2.8 and 24.9 +/- 1.6 microM s in the absence and presence of 10 mM caffeine, respectively. Caffeine alone raised the [Ca2+]-time integral to 20.3 +/- 3.4 microM s. 3. Following the fatigue protocol there was a proportionately greater loss of force at low stimulation frequencies (30 and 50 Hz) compared with high frequencies (100 Hz) which persisted for up to an hour. This pattern of force loss could be attributed to a uniform reduction in [Ca2+]i at all frequencies. Similar effects were observed after elevating [Ca2+]i with the caffeine + stimulation protocol but were not observed after stimulation or caffeine alone. The higher [Ca2+]-time integrals during the fatigue and caffeine + stimulation protocols suggest that some threshold for [Ca2+]i must be reached before these effects are observed. 4. The reductions in low frequency force induced by the fatigue and caffeine + stimulation protocols were not due to decreased Ca2+ sensitivity or to decreases in maximum force-generating capacity of the contractile proteins and therefore are due to a failure of Ca2+ release. 5. The Ca(2+)-activated neutral protease (calpain) inhibitor calpeptin was not effective in preventing the effects of caffeine + stimulation indicating that the reduction in Ca2+ release was not due to calpain-mediated hydrolysis of the Ca2+ release channel. 6. Our findings indicate that low frequency fatigue results from increases in [Ca2+]i during fatigue and that these elevations in [Ca2+]i activate some process which leads to failure of excitation-contraction (E-C) coupling and Ca2+ release.

Animals↗
PubMed1996

The effects of intracellular injections of phosphate on intracellular calcium and force in single fibres of mouse skeletal muscle.

Intracellular inorganic phosphate increases during muscle fatigue and may be responsible for certain of the changes in muscle function observed in fatigue. To test this hypothesis inorganic phosphate was micro-injected in single mouse muscle fibres which were also injected with indo-1 to measure intracellular Ca2+. Following phosphate injection, intracellular Ca2+, both at rest and during tetani, was reduced as was tetanic force. The rate at which the sarcoplasmic reticulum (SR) pumped Ca2+ out of the myoplasm was accelerated following phosphate injection. Intracellular Ca2+ and force recovered over 1-h. The changes in maximum Ca2+-activated force and Ca2+ sensitivity which would be expected if the phosphate remained in the myoplasm were largely absent. The most likely interpretation is that inorganic phosphate enters the SR where it precipitates with Ca2+ and thereby reduced release of Ca2+ from the SR and accelerated the rate of uptake of Ca2+ by the pump. The 1-h recovery may represent the entry of additional Ca2+ into the cell to reestablish the normal gradient of Ca2+ across the sarcolemma.

Animals↗
PubMed1996

Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle.

Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase.

Animals↗
PubMed1996

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle.

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca2+]i ([Ca2+]i) due to Ca2+ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca2+]i elevation during stimulation depends on: (a) the rate of Ca2+ release from the SR; (b) the rate of Ca2+ uptake by the SR; and (c) the myoplasmic Ca2+ buffering. We have used fluorescent Ca2+ indicators to measure [Ca2+]i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca2+]i: beta-adrenergic stimulation, which potentiates the SR Ca2+ release; application of 2.5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca2+ pumps; application of caffeine, which facilitates SR Ca2+ release and inhibits SR Ca2+ uptake; early fatigue, where the rate of SR Ca2+ uptake is reduced; acidosis, which reduces both the myoplasmic Ca2+ buffering and the rate of SR Ca2+ uptake. Reduced tetanic [Ca2+]i was observed in late fatigue, due to reduced SR Ca2+ release, and in alkalosis, due to increased myoplasmic Ca2+ buffering. Force is monotonically related to [Ca2+]i but depends also on the myofibrillar Ca2+ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca2+]i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca2+ sensitivity and maximum cross-bridge force.

Animals↗
PubMed1996

Paclitaxel (Taxol) as salvage therapy for relapsed ovarian cancer.

Thirty-two patients with epithelial ovarian cancer received paclitaxel 175 mg/m2, by 3-hour infusion, in this prospective phase 2 study. All patients had relapsed or progressed after initial cisplatin/cyclophosphamide therapy. Thirteen patients received paclitaxel as second line therapy, 14 as third line therapy and 5 as fourth line therapy. One patient was excluded from efficacy analysis, due to a severe anaphylactic reaction to the first cycle of paclitaxel therapy. Of the 31 evaluable patients, complete remission was observed in 3 patients (9.7%) and partial remission in 11 patients (35.5%), with a total response rate of 45.2%. The median survival from diagnosis for the 31 evaluable patients was 32.5 months and the median survival following therapy with paclitaxel was 12.2 months (range 4-27 months). The 3 patients who achieved a complete response remain alive, at greater than 20 months, since commencing paclitaxel. Twelve patients (38.7%) who achieved a partial response, or had stable disease, had a median survival, after paclitaxel treatment, of greater than 18 months. This study confirms the activity of paclitaxel in relapsed ovarian cancer and demonstrates a prolonged survival benefit in greater than one third of this group of patients.

Adult↗
PubMed1995

Intracellular calcium and force in single mouse muscle fibres following repeated contractions with stretch.

1. The role of the myoplasmic free Ca2+ concentration ([Ca2+]i) in the reduction of muscle force following contractions with stretch was investigated in single fibres from mouse toe muscle. Muscle fibres were either stretched by 25% of their optimum length (Lo) for ten tetani (Protocol I) or stretched by 50% of Lo for between ten and thirty tetani (Protocol II). Indo-1 was used to measure [Ca2+]i. 2. In each protocol the stretch series was compared with isometric controls; the stretch series always resulted in greater changes in muscle properties than in the isometric controls. The observed changes were (i) reduced tetanic force, (ii) reduced tetanic [Ca2+]i, (iii) increased resting [Ca2+]i and (iv) the greater relative reduction in force at low stimulus frequencies (30 and 50 Hz) compared with high (100 Hz). These changes were maintained for up to 60 min. 3. Stretching a resting muscle fibre had no effect on the subsequent [Ca2+]i or force. 4. Following Protocol I 10 mM caffeine restored tetanic force to pre-stretch levels. Tetanic [Ca2+]i vs. force curves were constructed pre- and post-stretch and showed that neither the maximum Ca(2+)-activated force nor the Ca2+ sensitivity of the muscle fibres post-stretch was significantly different from control. The force reduction, therefore, appears to be the result of reduced tetanic [Ca2+]i. 5. The more severe stretching regimen of Protocol II resulted in a much greater reduction in force than Protocol I. Ten millimolar caffeine did not restore control force. Comparison of the [Ca2+]i-force relationships pre- and post-stretch showed that the reduction in tetanic force was caused by a combination of a reduced tetanic [Ca2+]i, reduced maximum Ca(2+)-activated force and reduced Ca2+ sensitivity. 6. Following both protocols the resting [Ca2+]i showed a small rise which persisted for at least 60 min. This elevated [Ca2+]i was associated with a reduction in the pump rate of the sarcoplasmic reticulum Ca2+ pump. 7. This study establishes that reduced Ca2+ release and reduced Ca2+ sensitivity contribute to the reduction in force generating capacity of single mammalian muscle fibres following active stretches.

Animals↗
PubMed1995

The effects of caffeine on intracellular calcium, force and the rate of relaxation of mouse skeletal muscle.

1. Intracellular calcium concentration ([Ca2+]i) and force were measured from isolated single fibres of mouse skeletal muscle. The effects of 5 mM caffeine on muscle fibres at rest and during short tetani were examined. 2. Caffeine increased tetanic tension and slowed the rate of relaxation. [Ca2+]i was increased in the presence of caffeine both in the resting muscle and during tetani. The time course of decline of [Ca2+]i after a tetanus is complex with a large, early, rapid phase followed by a smaller and slower phase. Caffeine accelerated the early phase but slowed the later phase. 3. The sensitivity of the myofibrillar proteins to Ca2+ measured in the intact fibre was increased in the presence of caffeine, confirming earlier findings on skinned muscle fibres. 4. Analysis of the late phase of the decline of [Ca2+]i after a tetanus provides information about the properties of the sarcoplasmic reticulum (SR) Ca2+ pump. Caffeine slowed the pump to 60-70% of the control value at a given [Ca2+]i but had no effect on the Ca2+ leak from the SR. 5. Analysis of relaxation made use of the Ca(2+)-derived force in which the [Ca2+]i during relaxation was converted to the Ca(2+)-derived force by means of the steady-state relation between [Ca2+]i and force. The Ca(2+)-derived force fell more slowly in the presence of caffeine but the lag between Ca(2+)-derived force and measured force was unaffected. Thus, the slowed relaxation was caused by changes in Ca2+ handling and not by slowed cross-bridge kinetics. 6. A model of the Ca2+ movements and force production of muscle was used to examine independently the effects of increased Ca2+ sensitivity, slowing of the SR Ca2+ pump and increased SR Ca2+ permeability. The effects of caffeine on [Ca2+]i, tetanic force and relaxation could be explained by a combination of these three effects.

Animals↗