Intracellular Mg2+ diffusion within isolated rat skeletal muscle fibers

Intracellular free magnesium concentration ([Mg2+]i) was measured in enzymatically isolated rat skeletal muscle fibers using the fluorescent dye mag-indo-1. The change in [Mg2+]i produced by a local intracellular microinjection of magnesium pidolate (magnesium pyrrolidone carboxylate) was measured at a given distance from the injection site. In one series of experiments this protocol was tested on isolated fibers that were completely embedded into silicone grease: under these conditions, the injection produced an increase in [Mg2+]i that reached a steady level some time following the injection. The time-course of the [Mg2+]i change could be well accounted for by a model of longitudinal diffusion. The mean apparent Mg2+ diffusion coefficient (D(app)) was 188+/-9 microm2 s(-1) (n = 16), approximately four times lower than the value measured in vitro. This reduction likely results from the effects of cytoplasmic viscosity and of Mg2+ binding to low affinity static sites. Another series of measurements was performed on fibers that were either partially or completely free of silicone: under these conditions, the time course of the change in [Mg2+]i was in many cases more complex than predicted by simple diffusion.     

Estimating oxygen consumption rates of arteriolar walls under physiological conditions in rat skeletal muscle

To examine the effects of vascular tone reduction on O2 consumption of the vascular wall, we determined the O2 consumption rates of arteriolar walls under normal conditions and during vasodilation induced by topical application of papaverine. A phosphorescence quenching technique was used to quantify intra- and perivascular PO2 in rat cremaster arterioles with different branching orders. Then, the measured radial PO2 gradients and a theoretical model were used to estimate the O2 consumption rates of the arteriolar walls. The vascular O2 consumption rates of functional arterioles were >100 times greater than those observed in in vitro experiments. The vascular O2 consumption rate was highest in first-order (1A) arterioles, which are located upstream, and sequentially decreased downstream in 2A and 3A arterioles under normal conditions. During papaverine-induced vasodilation, on the other hand, the O2 consumption rates of the vascular walls decreased to similar levels, suggesting that the high O2 consumption rates of 1A arterioles under normal conditions depend in part on the workload of the vascular smooth muscle. These results strongly support the hypothesis that arteriolar walls consume a significant amount of O2 compared with the surrounding tissue. Furthermore, the reduction of vascular tone of arteriolar walls may facilitate an efficient supply of O2 to the surrounding tissue.     

Intracellular pH during sequential, fatiguing contractile periods in isolated single Xenopus skeletal muscle fibers.

The purpose of the present study was to test the hypothesis that a preceding contractile period in isolated single skeletal muscle fibers would attenuate the decrease in pH during an identical, subsequent contractile period, thereby reducing the rate of fatigue. Intact single skeletal muscle fibers (n = 9) were isolated from Xenopus lumbrical muscle and incubated with the fluorescent cytosolic H+ indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) AM for 30 min. Two identical contractile periods were performed in each fiber, separated by a 1-h recovery period. Force and intracellular pH (pHi) fluorescence were measured simultaneously while fibers were stimulated (tetanic contractions of 350-ms trains with 70-Hz stimuli at 9 V) at progressively increasing frequencies (0.25, 0.33, 0.5, and 1 contraction/s) until the development of fatigue (to 60% initial force). No significant difference (P < 0.05) was observed between the first and second contractile periods in initial force development, resting pHi, or time to fatigue (5.3 +/- 0.5 vs. 5.1 +/- 0.6 min). However, the relative decrease in the BCECF fluorescence ratio (and therefore pHi) from rest to the fatigue time point was significantly greater (P < 0.05) during the first contractile period (to 65 +/- 4% of initial resting values) compared with the second (77 +/- 4%). The results of the present study demonstrated that, when preceded by an initial fatiguing contractile period, the rise in cytosolic H+ concentration in contracting single skeletal muscle fibers during a second contractile period was significantly reduced but did not attenuate the fatigue process in the second contractile period. These results suggest that intracellular factors other than H+ accumulation contribute to the fall in force development under these conditions.     

Intracellular PO2 kinetics at different contraction frequencies in Xenopus single skeletal muscle fibers.

Increasing contraction frequency in single skeletal muscle fibers has been shown to increase the magnitude of the fall in intracellular Po(2) (Pi(O(2))), reflecting a greater metabolic rate. To test whether Pi(O(2)) kinetics are altered by contraction frequency through this increase in metabolic stress, Pi(O(2)) was measured in Xenopus single fibers (n = 11) during and after contraction bouts at three different frequencies. Pi(O(2)) was measured via phosphorescence quenching at 0.16-, 0.25-, and 0.5-Hz tetanic stimulation. The kinetics of the change in Pi(O(2)) from resting baseline to end-contraction values and end contraction to rest were described as a mean response time (MRT) representing the time to 63% of the change in Pi(O(2)). As predicted, the fall in Pi(O(2)) from baseline following contractions was progressively greater at 0.5 and 0.25 Hz than at 0.16 Hz (32.8 +/- 2.1 and 29.3 +/- 2.0 Torr vs. 23.6 +/- 2.2 Torr, respectively) since metabolic demand was greater. The MRT for the decrease in Pi(O(2)) was progressively faster at the higher frequencies (0.5 Hz: 45.3 +/- 4.5 s; 0.25 Hz: 63.3 +/- 4.1 s; 0.16 Hz: 78.0 +/- 4.1 s), suggesting faster accumulation of stimulators of oxidative phosphorylation. The MRT for Pi(O(2)) off-kinetics (0.5 Hz: 84.0 +/- 11.7 s; 0.25 Hz: 79.1 +/- 8.4 s; 0.16 Hz: 81.1 +/- 8.3 s) was not different between trials. These data demonstrate in single fibers that the rate of the fall in Pi(O(2)) is dependent on contraction frequency, whereas the rate of recovery following contractions is independent of either the magnitude of the fall in Pi(O(2)) from baseline or the contraction frequency. This suggests that stimulation frequency plays an integral role in setting the initial metabolic response to work in isolated muscle fibers, possibly due to temporal recovery between contractions, but it does not determine recovery kinetics.     

Estimation of capillary density in human skeletal muscle based on maximal oxygen consumption rates

A previously developed Krogh-type theoretical model was used to estimate capillary density in human skeletal muscle based on published measurements of oxygen consumption, arterial partial pressure of oxygen, and blood flow during maximal exercise. The model assumes that oxygen consumption in maximal exercise is limited by the ability of capillaries to deliver oxygen to tissue and is therefore strongly dependent on capillary density, defined as the number of capillaries per unit cross-sectional area of muscle. Based on an analysis of oxygen transport processes occurring at the microvascular level, the model allows estimation of the minimum number of straight, evenly spaced capillaries required to achieve a given oxygen consumption rate. Estimated capillary density values were determined from measurements of maximal oxygen consumption during knee extensor exercise and during whole body cycling, and they range from 459 to 1,468 capillaries/mm2. Measured capillary densities, obtained with either histochemical staining techniques or electron microscopy on quadriceps muscle biopsies from healthy subjects, are generally lower, ranging from 123 to 515 capillaries/mm2. This discrepancy is partly accounted for by the fact that capillary density decreases with muscle contraction and muscle biopsy samples typically are strongly contracted. The results imply that estimates of maximal oxygen transport rates based on capillary density values obtained from biopsy samples do not fully reflect the oxygen transport capacity of the capillaries in skeletal muscle.     

Nitric oxide modulates oxygen consumption by arteriolar walls in rat skeletal muscle

To study the role of nitric oxide (NO) in regulating oxygen consumption by vessel walls, the oxygen consumption rate of arteriolar walls in rat cremaster muscle was measured in vivo during flow-induced vasodilation and after inhibiting NO synthesis. The oxygen consumption rate of arteriolar walls was calculated based on the intra- and perivascular PO2 values measured by phosphorescence quenching laser microscopy. The perivascular PO2 value of the arterioles during vasodilation was significantly higher than under control conditions, although the intravascular PO2 values under both conditions were approximately the same. Inhibition of NO synthesis, on the other hand, caused a significant increase in arterial blood pressure and a significant decrease in arteriolar diameter. Inhibition of NO synthesis also caused a significant decrease in both the intra- and perivascular PO2 values of the arterioles. Inhibition of NO synthesis increased the oxygen consumption rate of the vessel walls by 42%, whereas enhancement of flow-induced NO release decreased it by 34%. These results suggest that NO plays an important role not only as a regulator of peripheral vascular tone but also as a modulator of tissue oxygenation by reducing oxygen consumption by vessel walls. In addition, enhancement of NO release during exercise may facilitate efficient oxygen supply to the surrounding high metabolic tissue.     

Effect of varied extracellular PO2 on muscle performance in Xenopus single skeletal muscle fibers.

The purpose of this study was to examine the development of fatigue in isolated, single skeletal muscle fibers when O2 availability was reduced but not to levels considered rate limiting to mitochondrial respiration. Tetanic force was measured in single living muscle fibers (n = 6) from Xenopus laevis while being stimulated at increasing contraction rates (0.25, 0.33, 0.5, and 1 Hz) in a sequential manner, with each stimulation frequency lasting 2 min. Muscle fatigue (determined as 75% of initial maximum force) was measured during three separate work bouts (with 45 min of rest between) as the perfusate PO2 was switched between values of 30 +/- 1.9, 76 +/- 3.0, or 159 Torr in a blocked-order design. No significant differences were found in the initial peak tensions between the high-, intermediate-, and low-PO2 treatments (323 +/- 22, 298 +/- 27, and 331 +/- 24 kPa, respectively). The time to fatigue was reached significantly sooner (P < 0.05) during the 30-Torr treatment (233 +/- 39 s) compared with the 76- (385 +/- 62 s) or 159-Torr (416 +/- 65 s) treatments. The calculated critical extracellular PO2 necessary to develop an anoxic core within these fibers was 13 +/- 1 Torr, indicating that the extracellular PO2 of 30 Torr should not have been rate limiting to mitochondrial respiration. The magnitude of an unstirred layer (243 +/- 64 micron) or an intracellular O2 diffusion coefficient (0.45 +/- 0.04 x 10(-5) cm2/s) necessary to develop an anoxic core under the conditions of the study was unlikely. The earlier initiation of fatigue during the lowest extracellular PO2 condition, at physiologically high intracellular PO2 levels, suggests that muscle performance may be O2 dependent even when mitochondrial respiration is not necessarily compromised.     

Reserve capacity for ATP consumption during isometric contraction in human skeletal muscle fibers.

Maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) and ATP consumption rate during maximum isometric activation (ATP(iso)) were determined in human vastus lateralis (VL) muscle fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP consumption [1 -- (ratio of ATP(iso) to V(max) ATPase)] varies across VL muscle fibers expressing different MHC isoforms. Biopsies were obtained from 12 subjects (10 men and 2 women; age 21--66 yr). A quantitative histochemical procedure was used to measure V(max) ATPase. In permeabilized fibers, ATP(iso) was measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption was lower for fibers coexpressing MHC(2X) and MHC(2A) compared with fibers singularly expressing MHC(2A) and MHC(slow) (39 vs. 52 and 56%, respectively). Tension cost (ratio of ATP(iso) to generated force) also varied with fiber type, being highest in fibers coexpressing MHC(2X) and MHC(2A). We conclude that fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such as susceptibility to fatigue.     

Phosphorylating pathways and fatigue development in contracting Xenopus single skeletal muscle fibers

To investigate the differential contribution of oxidative and substrate-level phosphorylation to force production during repetitive, maximal tetanic contractions, single skeletal muscle fiber performance was examined under conditions of high-oxygen availability and anoxia. Tetanic force development (P) was measured in isolated, single type-1 muscle fibers (fast twitch; n = 6) dissected from Xenopus lumbrical muscle while being stimulated at increasing frequencies (0.25, 0.33, and 0.5 Hz), with each frequency lasting 2 min. Two separate work bouts were conducted, with the perfusate PO(2) being either 0 or 159 mmHg. No significant (P < 0. 05) difference was found in the initial peak tensions (P(0)) between the high (334 +/- 57 kPa) and the low (325 +/- 41 kPa) PO(2) treatment. No significant difference in P was observed between the treatments during the first 50 s. However, a significant difference in force production was observed between the high (P/P(0) = 0.96 +/- 0.02) and the low PO(2) condition (P/P(0) = 0.92 +/- 0.02) by 60 s of work. After 60 s, steady-state force production was maintained during the high compared with the low PO(2) condition until stimulation frequency was increased, at which point developed tension during the high PO(2) condition began to decline. Time to fatigue (P/P(0) = 0.3) was reached significantly sooner during the low (250 +/- 16 s) than the high PO(2) condition (367 +/- 28 s). These results demonstrate that during the first 50 s of 0.25-Hz contractions, substrate-level phosphorylation has the capacity to maintain force and ATP hydrolysis when oxidative phosphorylation is absent. This period was followed by an oxygen-dependent phase in which force generation was maintained during the high PO(2) condition (but not during the low PO(2) condition) until the onset of a final fatiguing phase, at which a calculated maximal rate of oxidative phosphorylation was reached.     

Fall in intracellular PO(2) at the onset of contractions in Xenopus single skeletal muscle fibers.

It remains uncertain whether the delayed onset of mitochondrial respiration on initiation of muscle contractions is related to O(2) availability. The purpose of this research was to measure the kinetics of the fall in intracellular PO(2) at the onset of a contractile work period in rested and previously worked single skeletal muscle fibers. Intact single skeletal muscle fibers (n = 11) from Xenopus laevis were dissected from the lumbrical muscle, injected with an O(2)-sensitive probe, mounted in a glass chamber, and perfused with Ringer solution (PO(2) = 32 +/- 4 Torr and pH = 7.0) at 20 degrees C. Intracellular PO(2) was measured in each fiber during a protocol consisting sequentially of 1-min rest; 3 min of tetanic contractions (1 contraction/2 s); 5-min rest; and, finally, a second 3-min contractile period identical to the first. Maximal force development and the fall in force (to 83 +/- 2 vs. 86 +/- 3% of maximal force development) in contractile periods 1 and 2, respectively, were not significantly different. The time delay (time before intracellular PO(2) began to decrease after the onset of contractions) was significantly greater (P < 0.01) in the first contractile period (13 +/- 3 s) compared with the second (5 +/- 2 s), as was the time to reach 50% of the contractile steady-state intracellular PO(2) (28 +/- 5 vs. 18 +/- 4 s, respectively). In Xenopus single skeletal muscle fibers, 1) the lengthy response time for the fall in intracellular PO(2) at the onset of contractions suggests that intracellular factors other than O(2) availability determine the on-kinetics of oxidative phosphorylation and 2) a prior contractile period results in more rapid on-kinetics.     

Reappraisal of diffusion, solubility, and consumption of oxygen in frog skeletal muscle, with applications to muscle energy balance

Previously we tested the validity of the one-dimensional diffusion equation for O2 in the excised frog sartorius muscle and used it to measure the diffusion coefficient (D) for O2 in this muscle and the time course of its rate of O2 consumption (Qo2) after a tetanus (Mahler, 1978, 1979, J. Gen. Physiol., 71:533-557, 559-580, 73:159-174). A transverse section of the frog sartorius is in fact well fit by a hemi-ellipse with width divided by maximum thickness averaging 5.1 +/- 0.2. Using the previous techniques with the two-dimensional diffusion equation and this hemi-elliptical boundary yields a value for D that is 30% smaller than reported previously; the revised values at 0, 10, and 22.8 degrees C are 6.2, 7.9, and 10.8 X 10(-6) cm2/s, respectively. After a tetanus at 20 degrees C, Qo2 rose quickly to a peak and then declined exponentially, with a time constant (tau) approximately 15% faster than that reported previously; tau averaged 2.1 min in Rana temporaria and 2.6 min in Rana pipiens. A technique was devised to measure the solubility (alpha) of O2 in intact, respiring muscles, and yielded alpha (muscle)/alpha (H2O) = 1.26 +/- 0.04. With these modifications, the values for O2 consumption obtained with the diffusion method were in agreement with those measured by the direct method of Kushmerick and Paul (1976, J. Physiol. [Lond.]., 254:693-709). Using results from both methods, at 20 degrees C the ratio of phosphorylcreatine split during a tetanus to O2 consumption during recovery ranged from 5.2 to 6.2 mumol/mumol, and postcontractile ATP hydrolysis was estimated to be 13.6 +/- 4.1 (n = 3) nmol/mumol total creatine.     

Concentration of caveolin-3 at the neuromuscular junction in young and old rat skeletal muscle fibers.

Caveolin-3, a muscle-specific member of the caveolin family, is strongly localized to the neuromuscular junction (NMJ) in adult rat muscle fibers, where it co-localizes with alpha-bungarotoxin staining. In 24-month-old rats, less distinct staining corresponds with the normal aging changes in the NMJ. After denervation, the pattern and intensity of staining begin to break up as early as 3 days, and by 10 days little staining remains. The functional implications of this concentration of caveolin-3 at the NMJ remain obscure, but it is possible that its absence could account for some of the phenotypic characteristics of individuals with caveolin-3 mutations.     

Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids

A method has been developed for the quantitative evaluation of oxygen tension (PO2) distributions in multicellular spheroids measured with O2-sensitive microelectrodes. The experimental data showed that multicellular tumor spheroids in stirred growth media were characterized by a diffusion-depleted zone surrounding the spheroids. This zone was elicited by an unstirred layer of medium next to the spheroid leading to a continuous decrease in the PO2 values from the bulk medium towards the spheroid surface. Theoretical considerations demonstrate that the volume-related O2 consumption rate, Q, in the spheroids can be assessed by measuring the PO2 gradient in the diffusion-depleted zone outside the spheroids. Accordingly, Krogh's diffusion constant, KS, in the spheroids can be determined through measuring the PO2 gradient within the spheroids. The results obtained suggest that multicellular spheroids represent useful in vitro tumor models for the experimental and theoretical analysis of the interrelationship among O2 supply to tumor cells, O2 metabolism in tumors tissue, and the responsiveness of cancer cells to treatment.     

Hypoxia reduces oxygen consumption of fetal skeletal muscle cells in monolayer culture.

In a previous study on acute asphyxia in unanesthetized fetal sheep near term we showed that reduced oxygen delivery to peripheral organs reduces total oxygen consumption, suggesting that oxygen itself may be a determinant of oxygen consumption (Jensen, Hohmann & Künzel, 1987). To test this hypothesis we developed an in vitro perfusion model, which enabled us to measure the oxygen consumption of fetal skeletal muscle cells in monolayer culture in a control period (at approximately 145 mmHg) and during various degrees of hypoxia (6-140 mmHg). In 57 experiments on 57 cultures the mean oxygen consumption at a mean 'entry PO2' of 145.3 +/- 10.4 mmHg was 10.3 +/- 9.3 (SD).10(-6) microliters O2 per h per skeletal muscle cell. These measurements were made after an average of 4.2 +/- 2.3 transfers of the cells and at a cell density of 2.0 +/- 1.2.10(5) cells per cm2. In 54 of these experiments hypoxia was induced. There was a close positive correlation between the PO2 of the perfusate entering the Petridish ('entry PO2') and the change of the oxygen consumption of the cells (y = 5.17 - 0.54x + 0.03x2 - 0.00016x3, r = 0.97, p less than 0.0001). When oxygen tension fell, there was a concomitant fall in cellular oxygen consumption. We conclude that oxygen is a determinant of cellular oxygen consumption. Thus, hypoxia may reduce oxygen consumption of skeletal muscle cells, and oxygen may be preserved to maintain oxidative metabolism in central fetal organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

NAD(P)H fluorescence imaging of mitochondrial metabolism in contracting Xenopus skeletal muscle fibers: effect of oxygen availability.

The blue autofluorescence (351 nm excitation, 450 nm emission) of single skeletal muscle fibers from Xenopus was characterized to be originating from mitochondrial NAD(P)H on the basis of morphological and functional correlations. This fluorescence signal was used to estimate the oxygen availability to isolated single Xenopus muscle fibers during work level transitions by confocal microscopy. Fibers were stimulated to generate two contractile periods that were only different in the PO2 of the solution perfusing the single fibers (PO2 of 30 or 0-2 Torr; pH = 7.2). During contractions, mean cellular NAD(P)H increased significantly from rest in the low PO2 condition with the core (inner 10%) increasing to a greater extent than the periphery (outer 10%). After the cessation of work, NAD(P)H decreased in a manner consistent with oxygen tensions sufficient to oxidize the surplus NAD(P)H. In contrast, NAD(P)H decreased significantly with work in 30 Torr PO2. However, the rate of NAD(P)H oxidation was slower and significantly increased with the cessation of work in the core of the fiber compared with the peripheral region, consistent with a remaining limitation in oxygen availability. These results suggest that the blue autofluorescence signal in Xenopus skeletal muscle fibers is from mitochondrial NAD(P)H and that the rate of NAD(P)H oxidation within the cell is influenced by extracellular PO2 even at high extracellular PO2 during the contraction cycle. These results also demonstrate that although oxygen availability influences the rate of NAD(P)H oxidation, it does not prevent NAD(P)H from being oxidized through the process of oxidative phosphorylation at the onset of contractions.     

Effects of hypothyroidism on maximum specific force in rat diaphragm muscle fibers.

We hypothesized that 1) hypothyroidism (Hyp) decreases myosin heavy chain (MHC) content per half-sarcomere in diaphragm muscle (Dia(m)) fibers, 2) Hyp decreases the maximum specific force (F(max)) of Dia(m) fibers because of the reduction in MHC content per half-sarcomere, and 3) Hyp affects MHC content per half-sarcomere and F(max) to a greater extent in fibers expressing MHC type 2X (MHC(2X)) and/or MHC type 2B (MHC(2B)). Studies were performed on single Triton X-permeabilized fibers activated at pCa 4.0. MHC content per half-sarcomere was determined by densitometric analysis of SDS-polyacrylamide gels and comparison with a standard curve of known MHC concentrations. After 3 wk of Hyp, MHC content per half-sarcomere was reduced in fibers expressing MHC(2X) and/or MHC(2B). On the basis of electron-microscopic analysis, this reduction in MHC content was also reflected by a decrease in myofibrillar volume density and thick filament density. Hyp decreased F(max) across all MHC isoforms; however, the greatest decrease occurred in fibers expressing fast MHC isoforms (approximately 40 vs. approximately 20% for fibers expressing slow MHC isoforms). When normalized for MHC content per half-sarcomere, force generated by Hyp fibers expressing MHC(2A) was reduced compared with control fibers, whereas force per half-sarcomere MHC content was higher for fibers expressing MHC(2X) and/or MHC(2B) in the Hyp Dia(m) than for controls. These results indicate that the effect of Hyp is more pronounced on fibers expressing MHC(2X) and/or MHC(2B) and that the reduction of F(max) with Hyp may be at least partially attributed to a decrease in MHC content per half-sarcomere but not to changes in force per cross bridge.     

Relationship between cytosolic calcium and oxygen consumption in isolated rat hearts.

Maximum oxygen consumption was attained in isolated perfused rat hearts using high perfusate calcium and/or isoproterenol, or phenylephrine. The amplitude of calcium transients was directly related to oxygen consumption until oxygen consumed per beat reached maximum. At saturating oxygen consumption the amplitude of [Ca2+]i transients continued to increase, indicative of a calcium overload. In all cases +dP/dt correlated proportionately with +dCa2+/dt. Augmented developed pressure, related to isoproterenol-induced increase in cytosolic cAMP, cannot be attributed totally to elevated levels of [Ca2+]i transients. Adenosine (10(-5) M) added to the medium containing isoproterenol (10(-6) M) negated the isoproterenol-induced increase in cAMP and returned cardiac performance, oxygen consumption, and amplitude of [Ca2+]i transients to control state.     

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.