Effects of tetracaine on voltage-activated calcium sparks in frog intact skeletal muscle fibers

The properties of Ca(2+) sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K(+)] Ringer's are well described by a computational model with a Ca(2+) source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 degrees C; Model 2 of Baylor et al., 2002). This result, in combination with the values of single-channel Ca(2+) current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA (Kettlun et al., 2003) to 2 pA (Tinker et al., 1993), suggests that 1-5 RyR Ca(2+) release channels open during a voltage-activated Ca(2+) spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K(+)] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75-100 microM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca(2+) channels per spark, and (b) the single-channel Ca(2+) current of an RyR channel is 相似文献   

Ca2+ sparks in embryonic mouse skeletal muscle selectively deficient in dihydropyridine receptor alpha1S or beta1a subunits.

Ca2+ sparks are miniature Ca2+ release events from the sarcoplasmic reticulum of muscle cells. We examined the kinetics of Ca2+ sparks in excitation-contraction uncoupled myotubes from mouse embryos lacking the beta1 subunit and mdg embryos lacking the alpha1S subunit of the dihydropyridine receptor. Ca2+ sparks occurred spontaneously without a preferential location in the myotube. Ca2+ sparks had a broad distribution of spatial and temporal dimensions with means much larger than those reported in adult muscle. In normal myotubes (n = 248 sparks), the peak fluorescence ratio, DeltaF/Fo, was 1.6 +/- 0.6 (mean +/- SD), the full spatial width at half-maximal fluorescence (FWHM) was 3.6 +/- 1.1 micrometer and the full duration of individual sparks, Deltat, was 145 +/- 64 ms. In beta-null myotubes (n = 284 sparks), DeltaF/Fo = 1.9 +/- 0.4, FWHM = 5.1 +/- 1.5 micrometer, and Deltat = 168 +/- 43 ms. In mdg myotubes (n = 426 sparks), DeltaF/Fo = 1 +/- 0.5, the FWHM = 2.5 +/- 1.1 micrometer, and Deltat = 97 +/- 50 ms. Thus, Ca2+ sparks in mdg myotubes were significantly dimmer, smaller, and briefer than Ca2+ sparks in normal or beta-deficient myotubes. In all cell types, the frequency of sparks, DeltaF/Fo, and FWHM were gradually decreased by tetracaine and increased by caffeine. Both results confirmed that Ca2+ sparks of resting embryonic muscle originated from spontaneous openings of ryanodine receptor channels. We conclude that dihydropyridine receptor alpha1S and beta1 subunits participate in the control of Ca2+ sparks in embryonic skeletal muscle. However, excitation-contraction coupling is not essential for Ca2+ spark formation in these cells.     

Large currents generate cardiac Ca2+ sparks

Previous models of cardiac Ca2+ sparks have assumed that Ca2+ currents through the Ca2+ release units (CRUs) were approximately 1-2 pA, producing sparks with peak fluorescence ratio (F/F(0)) of approximately 2.0 and a full-width at half maximum (FWHM) of approximately 1 microm. Here, we present actual Ca2+ sparks with peak F/F(0) of >6 and a FWHM of approximately 2 microm, and a mathematical model of such sparks, the main feature of which is a much larger underlying Ca2+ current. Assuming infinite reaction rates and no endogenous buffers, we obtain a lower bound of approximately 11 pA needed to generate a Ca2+ spark with FWHM of 2 microm. Under realistic conditions, the CRU current must be approximately 20 pA to generate a 2- microm Ca2+)spark. For currents > or =5 pA, the computed spark amplitudes (F/F(0)) are large (approximately 6-12 depending on buffer model). We considered several factors that might produce sparks with FWHM approximately 2 microm without using large currents. Possible protein-dye interactions increased the FWHM slightly. Hypothetical Ca2+ "quarks" had little effect, as did blurring of sparks by the confocal microscope. A clusters of CRUs, each producing 10 pA simultaneously, can produce sparks with FWHM approximately 2 microm. We conclude that cardiac Ca2+ sparks are significantly larger in peak amplitude than previously thought, that such large Ca2+ sparks are consistent with the measured FWHM of approximately 2 microm, and that the underlying Ca2+ current is in the range of 10-20 pA.     

The spark and its ember: separately gated local components of Ca(2+) release in skeletal muscle

Amplitude, spatial width, and rise time of Ca(2+) sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca(2+)]. A total of approximately 17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg(2+)] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence "ridge" and is continued by an "ember," a prolongation of width approximately 1 microm and amplitude <0.2, vanishing in approximately 100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg(2+)] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg(2+)], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca(2+) release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source-presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg(2+). Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca(2+)-dependent activation (caffeine, low [Mg(2+)]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca(2+) release directly operated by voltage sensors.     

Comparison of Ca(2+) sparks produced independently by two ryanodine receptor isoforms (type 1 or type 3)

The molecular determinants of a Ca(2+) spark, those events that determine the sudden opening and closing of a small number of ryanodine receptor (RyR) channels limiting Ca(2+) release to a few milliseconds, are unknown. As a first step we investigated which of two RyR isoforms present in mammalian embryonic skeletal muscle, RyR type 1(RyR-1) or RyR type 3 (RyR-3) has the ability to generate Ca(2+) sparks. Their separate contributions were investigated in intercostal muscle cells of RyR-1 null and RyR-3 null mouse embryos. A comparison of Ca(2+) spark parameters of RyR-1 null versus RyR-3 null cells measured at rest with fluo-3 showed that neither the peak fluorescence intensity (DeltaF/F(o) = 1.25 +/- 0.7 vs. 1.55 +/- 0.6), spatial width at half-max intensity (FWHM = 2.7 +/- 1.2 vs. 2.6 +/- 0.6 microm), nor the duration at half-max intensity (FTHM = 45 +/- 49 vs. 43 +/- 25 ms) was significantly different. Sensitivity to caffeine (0.1 mM) was remarkably different, with sparks in RyR-1 null myotubes becoming brighter and longer in duration, whereas those in RyR-3 null cells remained unchanged. Controls performed in double RyR-1/RyR-3 null cells obtained by mice breeding showed that sparks were not observed in the absence of both isoforms in >150 cells imaged. In conclusion, 1) RyR-1 and RyR-3 appear to be the only intracellular Ca(2+) channels that participate in Ca(2+) spark activity in embryonic skeletal muscle; 2) except in their responsiveness to caffeine, both isoforms have the ability to produce Ca(2+) sparks with nearly identical properties, so it is rather unlikely that a single RyR isoform, when others are also present, would be responsible for Ca(2+) sparks; and 3) because RyR-1 null cells are excitation-contraction (EC) uncoupled and RyR-3 null cells exhibit a normal phenotype, Ca(2+) sparks result from the inherent activity of small clusters of RyRs regardless of the participation of these RyRs in EC coupling.     

Calcium sparks in human ventricular cardiomyocytes from patients with terminal heart failure

Cardiomyocytes from terminally failing hearts display significant abnormalities in e-c-coupling, contractility and intracellular Ca(2+) handling. This study is the first to demonstrate the influence of end-stage heart failure on specific properties of Ca(2+) sparks in human ventricular cardiomyocytes. We investigated the frequency and characteristics of spontaneously arising Ca(2+) sparks in single isolated human myocytes from terminally failing (HF) and non-failing (NF) control myocardium by using the Ca(2+) indicator Fluo-3. The Ca(2+) sparks were recorded by line-scan images along the longitudinal axis of the myocytes at a frequency of 250Hz. After loading the sarcoplasmic reticulum (SR) with Ca(2+) by repetitive field stimulation (10 pulses at 1Hz) the frequency of the Ca(2+) sparks immediately after stimulation (t = 0s) was reduced significantly in HF compared to NF (4.15 +/- 0.42 for NF vs. 2.81 +/- 0.20 for HF sparks s(-1), P = 0.05). This difference was present constantly in line-scan recordings up to 15s duration (t = 15s: 2.75 +/- 0.65 for NF vs. 1.36 +/- 0.34 for HF sparks s(-1), P = 0.05). The relative amplitude (F/F(0)) of Ca(2+) sparks was also significantly lower in HF cardiomyocytes (1.33 +/- 0.015 NF vs. 1.19 +/- 0.003 HF, t = 0s) and during subsequent recordings of 15s. Significant differences between HF and NF were also present in calculations of specific spark properties. The time to peak was estimated at 25.75 +/-0.88ms in HF and 18.68 +/- 0.45ms in NF cardiomyocytes (P = 0.05). Half-time of decay was 66.48 +/- 1.89ms (HF) vs. 44.15 +/- 1.65ms (NF, P < 0.05), and the full width at half-maximum (FWHM) was 3.99 +/- 0.06 microm (HF) vs. 3.5 +/- 0.07 microm (NF, P < 0.05). These data support the hypothesis that even in the absence of cardiac disease, Ca(2+) sparks from human cardiomyocytes differ from previous results of animal studies with respect to the time-to-peak, half-time of decay and FWHM. The role of elevated external Ca(2+) in HF was studied by recording Ca(2+) sparks in HF cardiomyocytes with 10mmol external Ca(2+) concentration. Under these conditions, the average spark amplitude was increased from 1.19 +/- 0.003 (F/F(0), 2mmol Ca(2+)) to 1.26 +/- 0.01 (F/F(0), 10mmol Ca(2+)). We conclude that human heart failure causes distinct changes in Ca(2+) spark frequency and characteristics comparable to results established in animal models of heart failure. A reduced Ca(2+) load of the SR alone is unlikely to account for the observed differences between HF and NF and additional alterations in intracellular Ca(2+) release mechanisms must be postulated.     

Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+ stores in pulmonary arterial smooth muscle cells

Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca2+ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, approximately 15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca2+ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca2+ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 +/- 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca2+ sparks (FDHM = 44.6 +/- 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca2+ stores; the molecular complexity and spatial heterogeneity of RyRs may facilitate specific Ca2+ regulation of cellular functions in PASMCs.     

Ca2+ sparks and embers of mammalian muscle. Properties of the sources

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37-160 channels opened in the spark. In simulations, 25-60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 microm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35-260 channels. Not infrequently, a few of those remain open and produce the trailing ember.     

AMPK activation with AICAR provokes an acute fall in plasma [K+

AMP-activated protein kinase (AMPK), activated by an increase in intracellular AMP-to-ATP ratio, stimulates pathways that can restore ATP levels. We tested the hypothesis that AMPK activation influences extracellular fluid (ECF) K(+) homeostasis. In conscious rats, AMPK was activated with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) infusion: 38.4 mg x kg bolus then 4 mg x kg(-1) x min(-1) infusion. Plasma [K(+)] and [glucose] both dropped at 1 h of AICAR infusion and [K(+)] dropped to 3.3 +/- 0.04 mM by 3 h, linearly related to the increase in muscle AMPK phosphorylation. AICAR treatment did not increase urinary K(+) excretion. AICAR lowered [K(+)] whether plasma [K(+)] was chronically elevated or lowered. The K(+) infusion rate needed to maintain baseline plasma [K(+)] reached 15.7 +/- 1.3 micromol K(+) x kg(-1) x min(-1) between 120 and 180 min AICAR infusion. In mice expressing a dominant inhibitory form of AMPK in the muscle (Tg-KD1), baseline [K(+)] was not different from controls (4.2 +/- 0.1 mM), but the fall in plasma [K(+)] in response to AICAR (0.25 g/kg) was blunted: [K(+)] fell to 3.6 +/- 0.1 in controls and to 3.9 +/- 0.1 mM in Tg-KD1, suggesting that ECF K(+) redistributes, at least in part, to muscle ICF. In summary, these findings illustrate that activation of AMPK activity with AICAR provokes a significant fall in plasma [K(+)] and suggest a novel mechanism for redistributing K(+) from ECF to ICF.     

Comparison of simulated and measured calcium sparks in intact skeletal muscle fibers of the frog

Calcium sparks in frog intact skeletal muscle fibers were modeled as stereotypical events that arise from a constant efflux of Ca(2+) from a point source for a fixed period of time (e.g., 2.5 pA of Ca(2+) current for 4.6 ms; 18 degrees C). The model calculates the local changes in the concentrations of free Ca(2+) and of Ca(2+) bound to the major intrinsic myoplasmic Ca(2+) buffers (troponin, ATP, parvalbumin, and the SR Ca(2+) pump) and to the Ca(2+) indicator (fluo-3). A distinctive feature of the model is the inclusion of a binding reaction between fluo-3 and myoplasmic proteins, a process that strongly affects fluo-3's Ca(2+)-reaction kinetics, its apparent diffusion constant, and hence the morphology of sparks. DeltaF/F (the change in fluo-3's fluorescence divided by its resting fluorescence) was estimated from the calculated changes in fluo-3 convolved with the microscope point-spread function. To facilitate comparisons with measured sparks, noise and other sources of variability were included in a random repetitive fashion to generate a large number of simulated sparks that could be analyzed in the same way as the measured sparks. In the initial simulations, the binding of Ca(2+) to the two regulatory sites on troponin was assumed to follow identical and independent binding reactions. These simulations failed to accurately predict the falling phase of the measured sparks. A second set of simulations, which incorporated the idea of positive cooperativity in the binding of Ca(2+) to troponin, produced reasonable agreement with the measurements. Under the assumption that the single channel Ca(2+) current of a ryanodine receptor (RYR) is 0.5-2 pA, the results suggest that 1-5 active RYRs generate an average Ca(2+) spark in a frog intact muscle fiber.     

Effect of sodium on amiloride- and triamterene-induced current fluctuations in isolated frog skin

The apparent association constants of two agents, amiloride and triamterene, that block the Na-selective channel of apical membrane of frog skin are shown to decrease as the Na concentration is increased in the apical bathing solution in isolated skin of the frog, Rana temporaria, Rana esculenta, and Rana pipiens. These results were obtained in "normally polarized" skins. These effects were independent of the anion used (chloride or methylsulfate) or the cation used as the Na substitute (Tris, DDA, or K ion). When NaCl was replaced with mannitol, the Na effect on the amiloride association rate constant persisted, which shows that ionic strength was not critically involved. The amiloride corner frequency was unaffected when the clamp potential was altered from +100 to -60 mV. The Na dependence was greatly attenuated or absent when the serosal surface was bathed in 120 mM K Ringer's, an effect that appears to be attributable to some pharmacological effect of high serosal K. A previously described three-state model is used to analyze the inhibitory effect of Na on the blocker association rate constant.     

Simulation of calcium sparks in cut skeletal muscle fibers of the frog

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.     

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.     

Altered Ca2+ sparks and gating properties of ryanodine receptors in aging cardiomyocytes

To investigate the cellular mechanisms for altered cardiac function in senescence, we measured Ca(2+) transients and Ca(2+) sparks in ventricular cardiomyocytes from 6- to 24-month-old Fisher 344 (F344) rat hearts. The single channel properties of ryanodine receptors from adult and senescent hearts were also studied. In senescent myocytes, we observed a decreased peak [Ca(2+)](i) amplitude and an increased time constant for decay (tau), both of which correlated with a reduced Ca(2+) content of the sarcoplasmic reticulum (SR). Our studies also revealed that senescent cardiomyocytes had an increased frequency of Ca(2+) sparks and a slight but statistically significant decrease in average amplitude, full-width-at-half-maximum (FWHM) and full-duration-at-half-maximum (FDHM). Single channel recordings of ryanodine receptors (RyR2) demonstrated that in aging hearts, the open probability (P(o)) of RyR2 was increased but the mean open time was shorter, providing a molecular correlate for the increased frequency of Ca(2+) sparks and decreased size of sparks, respectively. Thus, modifications of normal RyR2 gating properties may play a role in the altered Ca(2+) homeostasis observed in senescent myocytes.     

Protein osmotic pressure and the state of water in frog myoplasm

We measured the osmotic pressure of diffusible myoplasmic proteins in frog (Rana temporaria) skeletal muscle fibers by using single Sephadex beads as osmometers and dialysis membranes as protein filters. The state of the myoplasmic water was probed by determining the osmotic coefficient of parvalbumin, a small, abundant diffusible protein distributed throughout the fluid myoplasm. Tiny sections of membrane (3.5- and 12-14-kDa cutoffs) were juxtaposed between the Sephadex beads and skinned semitendinosus muscle fibers under oil. After equilibration, the beads were removed and calibrated by comparing the diameter of each bead to its diameter measured in solutions containing 3-12% Dextran T500 (a long-chain polymer). The method was validated using 4% agarose cylinders loaded with bovine serum albumin (BSA) or parvalbumin. The measured osmotic pressures for 1.5 and 3.0 mM BSA were similar to those calculated by others. The mean osmotic pressure produced by the myoplasmic proteins was 9.7 mOsm (4 degrees C). The osmotic pressure attributable to parvalbumin was estimated to be 3.4 mOsm. The osmotic coefficient of the parvalbumin in fibers is approximately 3.7 mOsm mM(-1), i.e., roughly the same as obtained from parvalbumin-loaded agarose cylinders under comparable conditions, suggesting that the fluid interior of muscle resembles a simple salt solution as in a 4% agarose gel.     

Simulation of Ca2+ movements within the sarcomere of fast-twitch mouse fibers stimulated by action potentials

Ca(2+) release from the sarcoplasmic reticulum (SR) of skeletal muscle takes place at the triadic junctions; following release, Ca(2+) spreads within the sarcomere by diffusion. Here, we report multicompartment simulations of changes in sarcomeric Ca(2+) evoked by action potentials (APs) in fast-twitch fibers of adult mice. The simulations include Ca(2+) complexation reactions with ATP, troponin, parvalbumin, and the SR Ca(2+) pump, as well as Ca(2+) transport by the pump. Results are compared with spatially averaged Ca(2+) transients measured in mouse fibers with furaptra, a low-affinity, rapidly responding Ca(2+) indicator. The furaptra Deltaf(CaD) signal (change in the fraction of the indicator in the Ca(2+)-bound form) evoked by one AP is well simulated under the assumption that SR Ca(2+) release has a peak of 200-225 microM/ms and a FDHM of approximately 1.6 ms (16 degrees C). Deltaf(CaD) elicited by a five-shock, 67-Hz train of APs is well simulated under the assumption that in response to APs 2-5, Ca(2+) release decreases progressively from 0.25 to 0.15 times that elicited by the first AP, a reduction likely due to Ca(2+) inactivation of Ca(2+) release. Recovery from inactivation was studied with a two-AP protocol; the amplitude of the second release recovered to >0.9 times that of the first with a rate constant of 7 s(-1). An obvious feature of Deltaf(CaD) during a five-shock train is a progressive decline in the rate of decay from the individual peaks of Deltaf(CaD). According to the simulations, this decline is due to a reduction in available Ca(2+) binding sites on troponin and parvalbumin. The effects of sarcomere length, the location of the triadic junctions, resting [Ca(2+)], the parvalbumin concentration, and possible uptake of Ca(2+) by mitochondria were also investigated. Overall, the simulations indicate that this reaction-diffusion model, which was originally developed for Ca(2+) sparks in frog fibers, works well when adapted to mouse fast-twitch fibers stimulated by APs.     

Inhibition of Zn2+-induced potentiation of twitch tension by Ni2+ in frog muscle

Effect of Ni2+ on Zn2+-induced potentiation of twitch tension was studied electrophysiologically in the toe muscle fibers of Rana catesbeiana. The major findings of this investigation are as follows. When 2 mM Ni2+ was applied to fibers in a normal Ringer's solution containing 50 microM Zn2+ (Zn2+ solution), the Zn2+-potentiated twitch tension decreased remarkably to about one-third of that before Ni2+ treatment. This concentration of Ni2+ caused a 23% decrease in the duration of action potential which had been prolonged by Zn2+ (6.61-5.09 ms). Ni2+ (2 mM) added to normal Ringer's solution led to increases of about 30 and 42% in twitch tension and in the duration of action potential, respectively. A slight increase in the mechanical threshold was induced by 2 mM Ni2+. The inhibitory action of Ni2+ on the twitch tension in Zn2+ solution was larger than that in the case of tetanus tension. Diltiazem (40 microM), a Ca2+ channel blocker, did not inhibit the twitch tension potentiated in Zn2+ solution. These results suggest that the decrease in Zn2+-potentiated twitch tension by Ni2+ may possibly derive from impairment of the propagation of action potential along the T tubules.     

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).     

Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers.

Fura red, a fluorescent Ca2+ indicator with absorbance bands at visible wavelengths, was injected into intact single muscle fibers that had been stretched to a long sarcomere length (approximately 3.8 microns) and bathed in a 'high-Ca2+' Ringer ([Ca2+] = 11.8 mM). From fura red's slow diffusion coefficient in myoplasm, 0.16 (+/- 0.01, SEM) x 10(-6) cm2 s-1 (N = 5; 16 degrees C), it is estimated that approximately 85% of the indicator molecules are bound to muscle constituents of large molecular weight. Binding appears to elevate, by 3- to 4-fold, the indicator's apparent dissociation constant for Ca2+ (KD), which is estimated to be 1.1-1.6 microM in myoplasm. Fura red's myoplasmic absorbance spectrum was used to estimate fr, the fraction of fura red molecules in the Ca2+-bound form at rest. In 3 fibers thought to be minimally damaged by the micro-injection, fr was estimated to be 0.15 (+/- 0.01). Thus, resting myoplasmic free [Ca2+] ([Ca2+]r) is estimated to be 0.19-0.28 microM. For fibers in normal Ringer solution ([Ca2+] = 1.8 mM), at shorter sarcomere length (approximately 2.7 microns), and containing a nonperturbing concentration of indicator (< or = 0.2 mM), [Ca2+]r is estimated to be 0.18-0.27 microM. This range is higher than estimated previously in frog fibers with other techniques. In 6 fibers, R, the indicator's fluorescence ratio signal (equal to the emission intensity measured with 420 nm excitation divided by that measured with 480 nm excitation), was measured at rest and following electrical stimulation and compared with absorbance measurements made from the same fiber region. The analysis implies that RMIN and RMAX (the values of R that would be measured if all indicator molecules were in the Ca(2+)-free and Ca(2+)-bound states, respectively) were substantially smaller in myoplasm than in calibration solutions lacking muscle proteins. Several methods for estimation of [Ca2+]r from R are analyzed and discussed.     

Active and passive electrical properties of single bullfrog atrial cells

Single cells from the bullfrog (Rana catesbeiana) atrium have been prepared by using a modification of the enzymatic dispersion procedure described by Bagby et al. (1971. Nature [Long.]. 234:351--352) and Fay and Delise (1973. Proc. Natl. Acad. Sci. U.S.A. 70:641--645). Visualization of relaxed cells via phase-contrast or Nomarski optics (magnification, 400--600) indicates that cells range between 150 and 350 micrometers in length and 4 and 7 micrometers in diameter. The mean sarcomere length in relaxed, quiescent atrial cells in 2.05 micrometer. Conventional electrophysiological measurements have been made. In normal Ringer's solution (2.5 mM K+, 2.5 mM Ca++) acceptable cells have stable resting potentials of about -88 mV, and large (125 mV) long- duration (approximately 720 ms) action potentials can be elicited. The Vm vs. log[K+]0 relation obtained from isolated cells is similar to that of the intact atrium. The depolarizing phase of the action potential of isolated atrial myocytes exhibits two pharmacologically separable components: tetrodotoxin (10(-6) g/ml) markedly suppresses the initial regenerative depolarization, whereas verapamil (3 x 10(-6) M) inhibits the secondary depolarization and reduce the plateau height. A bridge circuit was used to estimate the input resistance (220 +/- 7 M omega) and time constant 20 +/- 7 ms) of these cells. Two- microelectrode experiments have revealed small differences in the electrotonic potentials recorded simultaneously at two different sites within a single cell. The equations for a linear, short cable were used to calculate the electrical constants of relaxed, single atrial cells: lambda = 921.3 +/- 29.5 micrometers; Ri = 118.1 +/- 24.5 omega cm; Rm = 7.9 +/- 1.2 x 10(3) omega cm2; Cm = 2.2 +/- 0.3 mu Fcm-2. These results and the atrial cell morphology suggest that this preparation may be particularly suitable for voltage-clamp studies.