The mdx mouse, a model of the human disease Duchenne muscular dystrophy, has skeletal muscle fibres which display incompletely understood impaired contractile function. We explored the possibility that action potential-evoked Ca 2+ release is altered in mdx fibres. Action potential-evoked Ca 2+ -dependent fluorescence transients were recorded, using both low and high affinity Ca 2+ indicators, from enzymatically isolated fibres obtained from extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of normal and mdx mice. Fibres were immobilized using either intracellular EGTA or N -benzyl-p-toluene sulphonamide, an inhibitor of the myosin II ATPase. We found that the amplitude of the action potential-evoked Ca 2+ transients was significantly decreased in mdx mice with no measured difference in that of the surface action potential. In addition, Ca 2+ transients recorded from mdx fibres in the absence of EGTA also displayed a marked prolongation of the slow decay phase. Model simulations of the action potential-evoked transients in the presence of high EGTA concentrations suggest that the reduction in the evoked sarcoplasmic reticulum Ca 2+ release flux is responsible for the decrease in the peak of the Ca 2+ transient in mdx fibres. Since the myoplasmic Ca 2+ concentration is a critical regulator of muscle contraction, these results may help to explain the weakness observed in skeletal muscle fibres from mdx mice and, possibly, Duchenne muscular dystrophy patients.

R. D. KEYNES AND OTHERS6. Both the size of the outward currents and the rate at which they reached their maximum value increased with temperature. The activation energy for the rate constant was about 63 kJ/mole.7. Fibres bathed in Na-and Mg-free, 60 mM-CaCl2 saline were excitable. After replacement of the internal K+ with Cs+ or adding 60 mM-TEA to the internal solution only sustained inward currents were recorded with depolarization.8. Sustained inward currents could be reduced by external application of 5 mm-LaCl3. Tetrodotoxin was not effective even at a concentration of 1000 nM.9. The rate at which these inward currents reached a maximum value increased with increase in temperature of the bathing solution with an activation energy of the order of 42 kJ/mole.10. The reversal potential of the inward currents changed with the level of internal Ca-ions. For a fibre perfused without ethyleneglycol-bis

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.

Using a two-microelectrode voltage clamp technique, we investigated possible mechanisms underlying the impaired excitation-contraction coupling in skeletal muscle fibres of the mdx mouse, a model of the human disease Duchenne muscular dystrophy. We evaluated the role of the transverse tubular system (T-system) by using the potentiometric indicator di-8 ANEPPS, and that of the sarcoplasmic reticulum (SR) Ca 2+ release by measuring Ca 2+ transients with a low affinity indicator in the presence of high EGTA concentrations under voltage clamp conditions. We observed minimal differences in the T-system structure and the T-system electrical propagation was not different between normal and mdx mice. Whereas the maximum Ca 2+ release elicited by voltage pulses was reduced by ∼67% in mdx fibres, in agreement with previous results obtained using AP stimulation, the voltage dependence of SR Ca 2+ release was identical to that seen in normal fibres. Taken together, our data suggest that the intrinsic ability of the sarcoplasmic reticulum to release Ca 2+ may be altered in the mdx mouse.

Analysis of clinical isolates of Vibrio parahaemolyticus from outbreaks in Chile in the cities of Puerto Montt in 2004 and in Antofagasta in 1998 indicated that 23 of 24 isolates from Puerto Montt and 19 of 20 from Antofagasta belonged to the pandemic clonal complex that emerged in Southeast Asia in 1996.

The role of inositol 1,4,5-trisphosphate (InsP3) in excitation-contraction coupling in skeletal muscle was investigated by several methods. The following results were obtained. InsP3 is released by electrical stimulation of muscles. Exogenous InsP3 releases calcium from skinned muscle fibers at relatively high doses under normal conditions but does so at very low concentrations when blockers of the InsP3 5-phosphatase are present. Blockers of InsP3 release are effective blockers of calcium transients elicited by electrical stimulation of muscle fibers. It is proposed that InsP3 acts as a chemical second messenger between transverse (T)-tubular membrane depolarization and calcium release from the sarcoplasmic reticulum in skeletal muscle.One of the outstanding questions in muscle physiology is the mechanism of excitation-contraction coupling in vertebrate skeletal muscle. Electrical depolarization of the transverse (T) tubular system membranes somehow triggers release of Ca2+ ions from the sarcoplasmic reticulum (SR), which in turn activates the generation of tension (1-3). Though the T-tubule and SR are only some 100-200 A apart at the T/SR junctions, their membranes and luminal spaces do not seem to be physically continuous (4). How is the excitatory signal communicated from one structure to the other? The major present hypothesis assumes that intramembrane charge movement in the T-tubule membrane is mechanically linked to the SR membrane, causing the opening of Ca2' channels (5, 6). No direct evidence for such remote-control valves has been obtained. We now wish to present evidence for a biochemical coupling mechanism, in which inositol 1,4,5-trisphosphate (InsP3) acts as a soluble internal transmitter. This hypothesis was first suggested by two analogies. The first is the homology between SR and endoplasmic reticulum, from which InsP3 is known to release Ca2+ in other tissues (7,8 formation and activity should block excitation-contraction coupling; i.e., prevent T-system depolarization from releasing Ca2+.We report here the results of experiments that suggest that InsP3 may meet all four criteria. MATERIALS AND METHODSThe experiments on tension development were performed on segments of mechanically or chemically skinned skeletal muscle fibers isolated from the semitendinosus muscle of the frog Rana catesbeiana. Chemical skinning was performed by exposing muscle fibers for 10-20 min to saponin (20-50 ,ug/ml) (14) added to the relaxing solution described below. Mechanical skinning consisted of carefully splitting a segment of muscle fiber immersed in relaxing solution. Short segments (5-8 mm long) of skinned fibers were attached to a fixed hook and to a high sensitivity capacitive tension transducer. Tension was recorded on both chart paper and FM tape. The relaxing solution contained 110 mM potassium aspartate, 20 mM potassium Mops, 5 mM phosphocreatine, 100 ,uM EGTA, 3 mM ATP, and 3 mM MgSO4. The ATP and Mg2+ concentrations were adjusted in order to set the concentration offree Mg2+ at chosen levels. ...

Chloride fluxes are the main contributors to the resting conductance of mammalian skeletal muscle fibers. ClC-1, the most abundant chloride channel isoform in this preparation, is believed to be responsible for this conductance. However, the actual distribution of ClC-1 channels between the surface and transverse tubular system (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltageclamped enzymatically dissociated short fibers using a two-microelectrode configuration and simultaneously recorded chloride currents (ICl) and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from TTS contributions.

We describe a high temporal resolution confocal spot microfluorimetry setup which makes possible the detection of fluorescence transients elicited by Ca2+ indicators in response to large (50-200 microM), short duration (< 100 ns), free [Ca2+] transients generated by laser flash photolysis of DM-nitrophen (DM-n; caged Ca2+). The equilibrium and kinetic properties of the commercially available indicators Fluo-3, Rhod-2, CalciumOrange-5N (COr-5N) and CalciumGreen-2 (CGr-2) were determined experimentally. The data reveal that COr-5N displays simple, fast response kinetics while, in contrast, Fluo-3, Rhod-2 and CGr-2 are characterized by significantly slower kinetic properties. These latter indicators may be unsuitable for tracking Ca2+ signaling events lasting only a few milliseconds. A model which accurately predicts the time course of fluorescence transients in response to rapid free [Ca2+] changes was developed. Experimental data and model predictions concur only when the association rate constant of DM-n is approximately 20 times faster than previously reported. This work establishes a quantitative theoretical framework for the study of fast Ca2+ signaling events and the use of flash photolysis in cells and model systems.