Force declines when muscles are used repeatedly and intensively and a variety of intracellular mechanisms appear to contribute to this muscle fatigue. Intracellular calcium release declines during fatigue and has been shown to contribute to the reduction in force. Three new approaches have helped to define the role of calcium stores to this decline in calcium release. Skinned fibre experiments show that when intracellular phosphate is increased the amount of Ca2+ released from the sarcoplasmic reticulum (SR) declines. Intact fibre experiments show that the size of the calcium store declines during fatigue and recovers on rest. Intact muscles which lack the enzyme creatine kinase, do not exhibit the usual rise of phosphate during fatigue and, under these conditions, the decline of Ca2+ release is absent or delayed. These results can be explained by the "calcium phosphate precipitation" hypothesis. This proposes that if phosphate in the myoplasm rises, it enters the SR and binds to Ca2+ as Ca2+ phosphate. The resultant reduction in free Ca2+ within the SR contributes to the reduced Ca2+ release during fatigue.

1. Single fibres from the lumbrical muscles of the cane toad (Bufo marinus) were incubated in fluo-5N AM for 2 h at 35 degreesC in order to load the indicator into the sarcoplasmic reticulum. Fluo-5N is a low-affinity calcium indicator (K-Ca 90 muM). Successful sarcoplasmic reticulum (SR) loading was indicated by a fluorescence signal that declined during contraction. 2. Confocal microscopy showed that the dye loaded principally in Lines perpendicular to the long axis of the fibre that repeated each sarcomere. This is consistent with much of the dye residing in the BR. 3. To establish the site of loading, fibres were exposed to 30 mM caffeine in the presence of 20 muM 2,5-di( tert-butyl)1,4-hydroquinone (TBQ, an SR pump inhibitor) which should release most Ca2+ from the SR; this procedure reduced the fluorescence to 46 +/- 4% of the control value. To determine how much indicator was in the myoplasm, fibres were exposed to 100 mug ml(-1) saponin which permeabilizes the surface membrane; saponin treatment reduced the fluorescence to 51 +/- 2 % of the control value. 4. During maximally activated tetani (100 Hz stimulation rate, 22 degreesC) the component of signal from the SR declined by 33 +/- 4%. During relaxation the SR signal recovered in two phases with time constants of 0.38 +/- 0.14 s and 10.1 +/- 1.7 s. Partially activated tetani (30 Hz stimulation rate) showed a smaller SR signal. Application of the SR Ca2+ pump inhibitor TBQ slowed the rate of recovery of the SR signal. 5. Muscle fatigue was produced by repeated short tetani until tension was reduced to 50%. The SR signal during the periods between tetani declined steadily and the SR Ca2+ signal was eventually reduced to 71 +/- 8 % of the control signal. This signal recovered in two phases when the muscle was rested. An initial phase had a time constant of 1.7 +/- 0.2 s so that by 20 s of recovery the SR Ca2+ signal was 86 +/- 7 % of control; the second phase was slower and by 5 min the SR Ca2+ signal was back to control values (98 +/- 5% control). In addition the magnitude of the SR signal decline associated with each tetanus (Delta [Ca2+](SR)) declined monotonically throughout fatigue and returned to control after 5 min recovery. 6. This approach can monitor the SR Ca2+ concentration in normally functioning muscle fibres with good time resolution. The method confirms other approaches that show that the free Ca2+ available for release in the SR declines during fatigue. This reduction in [Ca2+](SR) will contribute to the failure of Ca2+ delivery to the myofilaments which is an important cause of muscle fatigue.

Mechanisms of fatigue were studied in single muscle fibres of the cane toad ( Bufo marinus ) in which force, intracellular calcium ([Ca 2+ ] i ), [Mg 2+ ] i , glycogen and the rapidly releasable Ca 2+ from the sarcoplasmic reticulum (SR) were measured. Fatigue was produced by repeated tetani continued until force had fallen to 50%. Two patterns of fatigue in the absence of glucose were studied. In the first fatigue run force fell to 50% in 8–10 min. Fatigue runs were then repeated until force fell to 50% in <3 min in the final fatigue run. Addition of extracellular glucose after the final fatigue run prolonged a subsequent fatigue run. In the first fatigue run peak tetanic [Ca 2+ ] i initially increased and then declined and at the time when force had fallen to 50% tetanic [Ca 2+ ] i was 54 ± 5% of initial value. In the final fatigue run force and peak tetanic [Ca 2+ ] i declined more rapidly but to the same level as in first fatigue runs. At the end of the first fatigue run, the rapidly releasable SR Ca 2+ store fell to 46 ± 6% of the pre-fatigue value. At the end of the final fatigue run the rapidly releasable SR Ca 2+ store was 109 ± 16% of the pre-fatigue value. In unstimulated fibres the nonwashable glycogen content was 176 ± 30 mmol glycosyl units/l fibre. After one fatigue run the glycogen content was 117 ± 17 mmol glycosyl units/l fibre; at the end of the final fatigue run the glycogen content was reduced to 85 ± 9 mmol glycosyl units/l fibre. [Mg 2+ ] i did not change significantly at the end of fatigue in either the first or the final fatigue run suggesting that globally-averaged ATP does not decline substantially in either pattern of fatigue. These results suggest that different mechanisms are involved in the decline of tetanic [Ca 2+ ] i in first compared to final fatigue runs. The SR Ca 2+ store is reduced in first fatigue runs; this is not the case for the final fatigue run which is associated with a decline in glycogen and possibly related to either a non-metabolic effect of glycogen or a spatially-localised metabolic decline.

1. Intracellular calcium ([Ca2+]i) and tension were measured from single muscle fibres dissected from the cane toad (Bufo marinus). The amount of Ca2+ which could be released from the sarcoplasmic reticulum (SR) was estimated by brief (approximately 20 s) exposures to 4-chloro-m-cresol (4-CmC) or caffeine. 2. Muscle fatigue was produced by repeated tetani at 4 s or shorter intervals and continued until tension had fallen to 50% of the control. The intracellular free calcium concentration during a tetanus (tetanic [Ca2+]i) first increased and then steadily declined to 43+/-2% of control by the time tension had fallen to 50%. Over the period of fatigue the rapidly releasable Ca2+ from the SR fell to 46+/-6% of control. Tension and tetanic [Ca2+]i recovered to 93+/-3% and 100+/-4% of the control values after 20 min of rest. Over the same period rapidly releasable SR Ca2+ recovered to 98+/-12%. 3. When a similar number of tetani (200) were repeated at longer intervals (10 s), fibres showed only a small reduction in tension (to 85+/-1%) and tetanic [Ca2+]i did not change significantly. Under these conditions the rapidly releasable SR Ca2+ did not change significantly. 4. The recovery of rapidly releasable SR Ca2+ after fatigue was unaffected by removal of extracellular calcium but did not occur when oxidative phosphorylation was inhibited with cyanide. 5. These results suggest that an important cause of the decline of tetanic [Ca2+]i during fatigue is an equivalent decline in the amount of rapidly releasable SR Ca2+. The results show that the decline of rapidly releasable SR Ca2+ is related to a metabolic consequence of fatigue and are consistent with the hypothesis that Ca2+ precipitates with phosphate in the SR during fatigue.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+](i)) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+](i) reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+](i) was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+](i) from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+](i) to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+](i) levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca2+-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.

The effects of 2,5-di-tert-butylhydroquinone (TBQ) on cardiac muscle contractility were investigated using cardiac preparations from the right ventricle of the rat. In saponin-skinned preparations, TBQ (50 and 100 microM) did not significantly affect the maximal isometric Ca2+-activated force or the steady-state force-Ca2+ concentration relation but significantly reduced the Ca2+ content of the sarcoplasmic reticulum (SR) in both the absence and the presence of 50 microM of the SR Ca2+-release channel blocker ruthenium red. The effect of TBQ on SR Ca2+ content was not due to an increased passive Ca2+ leak or Ca2+-induced Ca2+ release from the SR but to the specific inhibition of the SR Ca2+ pump. In electrically stimulated (0.2 Hz, 23 degrees C) intact cardiac preparations loaded with the Ca2+-sensitive fluorescent dye fura 2, TBQ (50 and 100 microM) caused the decrease in the rates of rise and fall of the fura 2 signal (ratio of fluorescence intensities at 360- and 380-nm excitation) and isometric force. In the presence of 50 and 100 microM TBQ, the twitch height of the isometric force response was significantly decreased, whereas the peak of the fura 2 signal was not significantly affected. At 2-Hz stimulation frequency, both parameters were reduced in the presence of 50 and 100 microM TBQ. Taken together, the results obtained in this study show that TBQ inhibits rather specifically the SR Ca2+ uptake in the rat ventricle and that it can be used as a tool to determine the function of the SR under different conditions.

Cardiac preparations from the right ventricular wall of rats were exposed to ATP-containing solutions of different Ca2+ concentrations ([Ca2+]) and Ca2+ buffering capacity to determine whether such treatments affected the Ca2+ handling properties of the cardiac muscle. Exposure to 8 mM extracellular ATP (ATP0) elicited sustained, near-maximal contractures that were due to Ca2+ entry from the extracellular environment and severely diminished cardiac excitability in a partially reversible manner. At 1 mM ATP0, there was a sustained rise in diastolic force and intracellular [Ca2+] and a marked, partially reversible change in the stimulation-induced Ca2+ transients measured with fura 2. A large, transient contracture was also observed when the intact ventricular preparations were transferred from the normal physiological solution to an ATP-containing (8 mM) relaxing solution (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid < or = 0.2 mM, < 10 nM Ca2+) with or without saponin (50 micrograms/ml). The ATP0-induced contractures were dependent on the prevalent conditions with respect to [Ca2+] and Ca2+ buffering capacity of the solutions and were associated with heavy Ca2+ loading of the sarcoplasmic reticulum (SR) and mitochondria, a less leaky SR, and a reduced SR sensitivity to the SR Ca2+ pump blocker 2,5-di-tert-butyl hydroquinone. The results indicate that the Ca2+ handling properties and the electrical excitability of ventricular preparations can be markedly modified by exposure to ATP0 and that conditions prevalent during skinning with saponin are important in determining the properties of the intracellular Ca2+ stores.

Temporal and spatial observations on the attachment of Nod-factors were made by biotinylating the reducing terminus of the lipo-chitooligosaccharides of the broad host-range Rhizobium sp. NGR234 with the fluorescent reagent 2-amino-(6-amidobiotinyl)pyridine. Complex formation between the biotinylated Nod-factors and fluorescent streptavidin allowed localisation of the binding-site. At concentrations of > 10-7 M, these fluorescently tagged moleculs bound rapidly and asymmetrically (≅ 1 min) to nodulation competent root-hairs but they did not bind to root-hairs of the non-host Arabidopsis thaliana. Early cellular events within the root-hairs were studied by loading root-segments with the calcium indicators Fura-2 and Fluo-3. Fluorescence ratio imaging showed that addition of NodNGR factors provoked almost immediate, plateau-like increases in intra-cellular free calcium {[Ca2+]i} in root-hairs and epidermal cells. Confocal laser scanning microscopy (CLSM) revealed that calcium accumulation was concentrated at the tips and the sides of the responsive root-hairs. A gene encoding a lipid transfer-like protein (LPT2) was isolated from a Vigna unguiculata root-hair cDNA bank. Levels of the LTP2-transcript increased in root-hairs 24 h after treatment with NGR234, or its Nod-factors. The LTP2-gene was cloned into the pMalTM expression vector and LTP2 purified by affinity chromatography. It was unable to transfer phospholipids between liposomes and mitochondria. Anti-sense analysis in which the LTP2 coding region was cloned between the 35S promoter and terminator sequences reduced nodulation when transformed into V. unguiculata.

Rhizobia excrete variously substituted lipo-oligosaccha-ride Nod factors into the legume rhizosphere. Homologous legumes respond to these signals through deformation of the root hairs and the development of nodulation foci in the root cortex. Cellular events in root hairs from the susceptible zone of nearly mature root hairs were studied in root segments loaded with the calcium indicators Fura-2 or Fluo-3. Application of 10-9 M Nod factors of the broad-host-range Rhizobium sp. NGR234 to the homologous legume Vigna unguiculata resulted, within seconds, in plateau-like increases in intracellular free calcium ([Ca2+]i) in the root hairs and root epidermal cells. Nod factors of R. meliloti at 10-9 M caused equally rapid increases in [Ca2+]i in the root hairs and epidermal cells of the nonhost V. unguiculata, and also induced root-hair deformation. The chitin tetramer, N-N′-N″-N′″-tetracetylchitotetraose, which represents the backbone of Nod factors, induced neither root-hair deformation nor changes in [Ca2+]i in V. unguiculata. Root hairs and epidermal cells of the nonlegume non-host Arabidopsis thaliana showed neither [Ca2+]i increases nor root-hair deformation in response to both factors.

The influence of myoplasmic Mg2+ (0.05–10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 μM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 μM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 μM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 μM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 μM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres. In the soleus and cardiac preparations Ca2+ uptake was optimal between 1 and 10 mM Mg2+. The results of this study demonstrate that SR Ca2+ accumulation is different from SR Ca2+ uptake and that these two important determinants of muscle function are differently affected by Mg2+ in different muscle fibre types.