New method for determining total calcium content in tissue applied to skeletal muscle with and without calsequestrin.

We describe a new method for determining the concentration of total Ca in whole skeletal muscle samples ([CaT]WM in units of mmoles/kg wet weight) using the Ca-dependent UV absorbance spectra of the Ca chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). Muscle tissue was homogenized in a solution containing 0.15 mM BAPTA and 0.5% sodium dodecyl sulfate (to permeabilize membranes and denature proteins) and then centrifuged. The solution volume was adjusted so that BAPTA captured essentially all of the Ca. [CaT]WM was obtained with Beer's law from the absorbance change produced by adding 1 mM EGTA to capture Ca from BAPTA. Results from mouse, rat, and frog muscles were reasonably consistent with results obtained using other methods for estimating total [Ca] in whole muscles and in single muscle fibers. Results with external Ca removed before determining [CaT]WM indicate that most of the Ca was intracellular, indicative of a lack of bound Ca in the extracellular space. In both fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from mice, [CaT]WM increased approximately linearly with decreasing muscle weight, increasing approximately twofold with a twofold decrease in muscle weight. This suggests that the Ca concentration of smaller muscles might be increased relative to that in larger muscles, thereby increasing the specific force to compensate for the smaller mass. Knocking out the high capacity Ca-binding protein calsequestrin (CSQ) did not significantly reduce [CaT]WM in mouse EDL or soleus muscle. However, in EDL muscles lacking CSQ, muscle weights were significantly lower than in wild-type (WT) muscles and the values of [CaT]WM were, on average, about half the expected WT values, taking into account the above [CaT]WM versus muscle weight relationship. Because greater reductions in [CaT]WM would be predicted in both muscle types, we hypothesize that there is a substantial increase in Ca bound to other sites in the CSQ knockout muscles.

Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca(2+) release during the quasi-steady level of release in twitch fibers from frog skeletal muscle.

Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits release when [Ca(T)](SR) declines to a low level.

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide.

One aim of this article was to determine the resting concentration of free Ca(2+) in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca(2+)](SR,R)) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca(2+)](SR,R) to TMX's apparent dissociation constant for Ca(2+) (K(app)) in order to establish the capability of monitoring [Ca(2+)](SR)(t) during SR Ca(2+) release - a signal needed to determine the Ca(2+) permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K(app) as well as a dependence of K(app) on TMX concentration, the results indicate an average [Ca(2+)](SR,R) ranging from 0.43 to 1.70mM. The ratio [Ca(2+)](SR,R)/K(app) averaged 0.256, a relatively low value which should not depend on factors influencing K(app). As a result, the time course of [Ca(2+)](SR)(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.

Pre-exercise hyperhydration delays dehydration and improves endurance capacity during 2 h of cycling in a temperate climate.

Whether the use of pre-exercise hyperhydration could improve the performance of athletes who do not hydrate sufficiently during prolonged exercise is still unknown. We therefore compared the effects of pre-exercise hyperhydration and pre-exercise euhydration on endurance capacity, peak power output and selected components of the cardiovascular and thermoregulatory systems during prolonged cycling. Using a randomized, crossover experimental design, 6 endurance-trained subjects underwent a pre-exercise hyperhydration (26 ml of water x kg body mass(-1) with 1.2 g glycerol x kg body mass(-1)) or pre-exercise euhydration period of 80 min, followed by 2 h of cycling at 65% maximal oxygen consumption (VO(.)2max) (26-27 degrees C) that were interspersed by 5, 2-min intervals performed at 80% V(.)O2max. Following the 2 h cycling exercise, subjects underwent an incremental cycling test to exhaustion. Pre-exercise hyperhydration increased body water by 16.1+/-2.2 ml.kg body mass(-1). During exercise, subjects received 12.5 ml of sports drink x kg body mass(-1). With pre-exercise hyperhydration and pre-exercise euhydration, respectively, fluid ingestion during exercise replaced 31.0+/-2.9% and 37.1+/-6.8% of sweat losses (p>0.05). Body mass loss at the end of exercise reached 1.7+/-0.3% with pre-exercise hyperhydration and 3.3+/-0.4% with pre-exercise euhydration (p<0.05). During the 2 h of cycling, pre-exercise hyperhydration significantly decreased heart rate and perceived thirst, but rectal temperature, sweat rate, perceived exertion and perceived heat-stress did not differ between conditions. Pre-exercise hyperhydration significantly increased time to exhaustion and peak power output, compared with pre-exercise euhydration. We conclude that pre-exercise hyperhydration improves endurance capacity and peak power output and decreases heart rate and thirst sensation, but does not reduce rectal temperature during 2 h of moderate to intense cycling in a moderate environment when fluid consumption is 33% of sweat losses.

Effects of beta-hydroxy-beta-methylbutyrate on aerobic-performance components and body composition in college students.

The aim of this study was to determine the effects of oral beta-hydroxy-beta-methylbutyrate (HMB) supplementation (3 g/d) on selected components of aerobic performance and body composition of active college students. Subjects were randomly assigned to either an HMB (n=8) or a placebo (PLA) group (n=8) for a 5-wk supplementation period during which they underwent interval training 3 times a week on a treadmill. Aerobic-performance components were measured using a respiratory-gas analyzer. Body composition was determined using dual-energy X-ray absorptiometry. After the intervention, there were significant differences (P<0.05) between the 2 groups in gains in maximal oxygen consumption (+8.4% for PLA and +13.4% for HMB). Regarding body composition, there were no significant differences. The authors concluded that HMB supplementation positively affects selected components of aerobic performance in active college students.

Role of calsequestrin evaluated from changes in free and total calcium concentrations in the sarcoplasmic reticulum of frog cut skeletal muscle fibres.

Calsequestrin is a large-capacity Ca-binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ([Ca2+](SR)) serving to maintain the driving force for SR Ca2+ release. Essentially all of the functional studies on calsequestrin to date have been carried out on purified calsequestrin or on disrupted muscle preparations such as terminal cisternae vesicles. To obtain information about calsequestrin's properties during physiological SR Ca2+ release, experiments were carried out on frog cut skeletal muscle fibres using two optical methods. One - the EGTA-phenol red method - monitored the content of total Ca in the SR ([Ca(T)](SR)) and the other used the low affinity Ca indicator tetramethylmurexide (TMX) to monitor the concentration of free Ca in the SR. Both methods relied on a large concentration of the Ca buffer EGTA (20 mM), in the latter case to greatly reduce the increase in myoplasmic [Ca2+] caused by SR Ca2+ release thereby almost eliminating the myoplasmic component of the TMX signal. By releasing almost all of the SR Ca, these optical signals provided information about [Ca(T)](SR) versus [Ca2+](SR) as [Ca2+](SR) varied from its resting level ([Ca2+](SR,R)) to near zero. Since almost all of the Ca in the SR is bound to calsequestrin, this information closely resembles the binding curve of the Ca-calsequestrin reaction. Calcium binding to calsequestrin was found to be cooperative (estimated Hill coefficient = 2.95) and to have a very high capacity (at the start of Ca2+ release, 23 times more Ca was estimated to initiate from calsequestrin as opposed to the pool of free Ca in the SR). The latter result contrasts with an earlier report that only approximately 25% of released Ca2+ comes from calsequestrin and approximately 75% comes from the free pool. The value of [Ca2+](SR,R) was close to the K(D) for calsequestrin, which has a value near 1 mm in in vitro studies. Other evidence indicates that [Ca2+](SR,R) is near 1 mM in cut fibres. These results along with the known rapid kinetics of the Ca-calsequestrin binding reaction indicate that calsequestrin's properties are optimized to buffer [Ca2+](SR) during rapid, physiological SR Ca2+ release. Although the results do not entirely rule out a more active role in the excitation-contraction coupling process, they do indicate that passive buffering of [Ca2+](SR) is a very important function of calsequestrin.