A novel phasic contraction induced by dithiothreitol in frog skeletal muscle

1. Dithiothreitol (DTT), at 50-100 mM, induced a phasic reversible contraction of frog skeletal muscle. 2. Exposure of single fibers to nifedipine (20 microM), an L-type Ca2+ antagonist, blocked the twitch and tetanus tensions but never affected the DTT-induced contraction. 3. DTT also produced a phasic contraction in fibers where voltage sensors were inactivated in the presence of high K+ concentration (190 mM). 4. A fiber was mechanically skinned after observation of DTT-induced contraction. The skinned fiber contracted in response to a DTT concentration similar to that required to produce contraction in intact fibers before skinning. 5. In skinned fibers, DTT, at 100 or 200 mM, inhibited the accumulation of Ca2+ by SR, but not Ca2+ ATPase activity. 6. These results suggest that a high concentration of DTT triggers Ca2+ efflux from the SR through action on the Ca2+ release channel and/or closely associated proteins, such as triadin and FK-506 binding protein.     

Some properties of late after-potential in frog skeletal muscle fiber

Some properties of late after-potentials which appear following a train of impulses were examined in frog skeletal muscle fiber. The decay of the late after-potential followed a simple exponential time course. The time constant of the decay was larger in a viscous solution than in normal Ringer solution. It was proved by physical experiments that the diffusion of K ions was delayed in the viscous medium at the same rate as the decay. The effect of temperature on the decay was low and the Q10 for the time constant was 1.2. When the late after-potentials were recorded at membrane potentials variedly controlled by the polarizing current, the reversal potential shifted in the positive direction with the increase of impulses. These results suggest that the late after-potential may be dependent on K ions accumulated in the T system. During the initial 300 msec period immediately after the onset of the decay, the amplitude was smaller than expected by a simple exponential time course. This effect was especially apparent in the sucrose hypertonic Ringer solution in which the decay was extremely extended. The cause of this non-exponential component was discussed with respect to the K accumulation hypothesis.     

Glycosphingolipids of skeletal muscle: I. Subcellular distribution of neutral glycosphingolipids and gangliosides in rabbit skeletal muscle

The development of polymers with different surface properties and surface modifications of intraocular lenses (IOL) should reduce foreign body reactions after implantation by reducing the surface hydrophobicity of the lenses. It was examined how far such surface variations influenced the adhesiveness of bacteria. The most common organism isolated from cases of postoperative endophthalmitis is Staphylococcus epidermidis. For this reason, three strains of this species, the type strain ATCC 14990 and two clinical isolates (8687, 6579 I), with different hydrophobic surfaces, were studied. IOL made of PMMA, silicone, and a copolymer as well as PMMA lenses with modified surfaces (unpolished, polished, silanized, and heparinized) were used. Bacteria were radiolabelled with 3H-thymidine and the adherent bacteria were calculated per mm2 of lens surface. The three strains adhered better to the unpolished surface of silicone than to PMMA. Treatment of PMMA surface by polishing diminished the differences between the strains. An influence of hydrophobic interactions on the adherence of S. epidermidis ATCC 14990 was demonstrated. The adherence of this hydrophobic type strain was clearly reduced by heparinization of the PMMA surface. In contrast, the hydrophilic catheter isolate 6579 I adhered better to modified surfaces. This strain differed clearly in its PFGE pattern from both hydrophobic strains. Hydrophobic interactions play a role in the bacterial adherence to intraocular lenses in vitro and in vivo. Modifications of polymer surfaces, however, can result in rather different effects depending on the bacterial surface composition and properties.     

Effect of caffeine on K+ efflux in frog skeletal muscle

The exposure of frog skeletal muscle to caffeine (3-4 mM) generates an increase of the K+ (42K+) efflux rate coefficient (kK,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca2+ ([Ca2+]i), because the effect is mimicked by ionomycin (1.25 microM), a Ca2+ ionophore. Second, the inhibition of caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) by 40 microM tetracaine significantly reduced the increase in kK,o (DeltakK,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca2+-dependent K+ channels (BKCa channels) reduced DeltakK,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca2+-dependent K+ channels (SKCa channels), did not affect DeltakK,o. Fifth, tolbutamide (800 microM), an inhibitor of KATP channels, reduced DeltakK,o by about 23%. Sixth, Ba2+, a blocker of most K+ channels, did not preclude the caffeine-induced DeltakK,o. Seventh, omitting Na+ from the external medium reduced DeltakK,o by about 40%. Eight, amiloride (5 mM) decreased DeltakK,o by 65%. It is concluded that the caffeine-induced rise of [Ca2+]i increases K+ efflux, through the activation of: (1) two channels (BKCa and KATP) and (2) an external Na+-dependent amiloride-sensitive process.     

Properties of tri- and tetracarboxylate Ca2+ indicators in frog skeletal muscle fibers

Recently a number of lower-affinity fluorescent Ca2+ indicators have become available with principal absorbance bands at visible wavelengths. This article evaluates these indicators, as well as two shorter wavelength indicators, mag-fura-5 and mag-indo-1, for their suitability as rapid Ca2+ indicators in frog skeletal muscle fibers. With three lower-affinity tricarboxylate indicators (mag-fura-5, mag-indo-1, and magnesium orange), the change in fluorescence in response to an action potential (delta F) appeared to track the myoplasmic Ca2+ transient (delta[Ca2+]) without delay. With three lower-affinity tetracarboxylate indicators (BTC, calcium-orange-5N, and calcium-green-5N) and one tricarboxylate indicator (magnesium green), delta F responded to delta[Ca2+] with a small delay. Unfortunately, with the tetracarboxylate indicators, other problems were detected that appear to limit their usefulness as reliable Ca2+ indicators. Surprisingly, delta F from mag-fura-red, another tricarboxylate indicator, was biphasic (with 480 nm excitation), a feature that also greatly limits its usefulness. With several of the indicators, estimates were obtained for the myoplasmic value of KD, Ca (the indicator's dissociation constant for Ca2+) and found to be elevated severalfold in comparison with the value measured in a simple salt solution. These and other problems related to the quantitative use of Ca2+ indicators in the intracellular environment are evaluated and discussed.     

Supercharging accelerates T-tubule membrane potential changes in voltage clamped frog skeletal muscle fibers

In voltage-clamp studies of single frog skeletal muscle fibers stained with the potentiometric indicator 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl] vinyl]pyridinium betaine (di-8 ANEPPS), fluorescence transients were recorded in response to both supercharging and step command pulses. Several illumination paradigms were utilized to study global and localized regions of the transverse tubule system (T-system). The rising phases of transients obtained from global illumination regions showed distinct accelerations when supercharging pulses were applied (95% of steady-state fluorescence achieved in 1.5 ms with supercharging pulses versus 14.6 ms with step pulses). When local transients were recorded at the edge of the muscle fiber, their kinetics resembled those of the applied waveform, but a similar relationship was not observed in transients from regions near the edge chosen to minimize the surface membrane contribution. We developed a model of the T-system capable of simulating membrane potential changes as a function of time and distance along the T-system cable and the associated fluorescence changes in regions corresponding to the experimental illumination strategies. A critical parameter was the access resistance term, for which values of 110-150 Omega.cm2 were adequate to fit the data. The results suggest that the primary mechanism through which supercharging pulses boost the kinetics of T-system voltage changes most likely involves their compensating the voltage attenuation across the access resistance at the mouth of the T-tubule.     

Incidence of Z band streaming and myofibrillar disruptions in skeletal muscle from healthy young people

Skeletal muscle tissue was obtained from the vastus lateralis or peroneus brevis muscles of 34 healthy volunteers. Focal and extensive areas of Z band streaming and disruption of myofibrillar architecture without Z band streaming were quantified in Araldite-embedded specimens examined in the phase microscope. Extensive areas of Z band streaming in more than 2.0 percent of fibers was present in only two of the 34 volunteers. Myofibrillar disruptions without Z band streaming was much less common than Z band streaming. The size of extensive areas of Z band streaming or myofibrillar disruptions was quantified by counting the number of adjacent fibrils and contiguous sarcomeres occupied by either type of lesion. These data should be of use in determining if such lesions are present in excessive amounts in suspected pathologic material.     

Observations on autoregulation in skeletal muscle: the effects of arterial hypoxia

Autoregulation of blood flow in skeletal muscle, as manifested by steady-state resistance changes, has been shown to be present in the low range of perfusion pressure but has been demonstrated by some observers to be lacking at higher perfusion pressures. Transient responses have often been neglected or observed only qualitatively in analyses of autoregulation. The present study was undertaken (1) to determine the relative importance of steady-state and transient responses in flow in demonstrating autoregulation of blood flow over a broad range of perfusion pressures, (2) to establish a means of quantitating autoregulation, and (3) to observe the effect of hypoxia on autoregulation. In isolated, perfused canine gracilis muscle, perfusion pressure was increased and subsequently returned to baseline (9.7 +/- 0.13 kPa [73 +/- 1 mmHg]) during perfusion with normally oxygenated blood (PO2 = 9.3-13.3 kPa [70-100 mmHg]), and mildly (PO2 = 6.1-9.2 kPa [46-69 mmHg]), moderately (PO2 = 4.5-6.0 kPa [34-45 mmHg]), or severely (PO2 = 2.7-4.4kPa [20-33 mmHg]) hypoxic blood. Consistent with other studies canine gracilis muscle was often found to possess passive vascular responses when only steady-state parameters were considered. However, quantitation of the transient response in flow with step increases in perfusion pressure demonstrated substantial transient responses under conditions of normal oxygenation, and progressive attenuation of flow transients with increasing hypoxia.     

Lengthening contraction and interpretations of active state tension in the isometric twitch response of skeletal muscle

The plateau of tetanic tension in a lengthening contraction of muscle at any given length is always greater than that in an isometric contraction at the same given length. To account for this excess of tension, it is suggested that the contractile machinery is "locked" in a strained condition that might make the muscle capable of bearing a greater tension in a lengthening contraction than it can develop in an isometric contraction. It is proposed that this strained condition also occurs in a lengthening contraction of the twitch response. If this proposal is valid it seems certain that the plateu of tension in the lengthening contraction of the twitch response cannot be equated with the plateau of tension in the isometric contraction of tetanus. The design of the original concept, that the plateau of active state tension in the lengthening contraction of the twitch is equal to the plateau of tension (Po) in the isometric contraction of tetanus, is based upon the assumption that the contractile component is neither lengthening nor shortening during both of these plateaus. This assumption is made without considering the possibility that the plateau in the lengthening contraction might be due to concurrent lengthening of series elastic elements and shortening of the contractile component. To account for the plateau of tension observed after quick lengthening in the early phase of twitch contraction indirect evidence is presented favoring the view that the quick lengthening during stretch is followed by slow lengthening and stress relaxation (decrease of tension) in series elastic elements and simultaneous shortening and increase of tension in the contractile component. When the original concept of active state tension in the twitch response is interpreted in the light of lengthening contraction, it is concluded that the labeled or implied Po for the plateau of the active state tension is unwarranted and confusing. It seems that the primary source of confusion is the assumption that the tension a muscle is capable of bearing in a lengthening contraction is equatable with the tension it can develop in an isometric contraction.     

The stiffness of skeletal muscle in isometric contraction and rigor: the fraction of myosin heads bound to actin

Step changes in length (between -3 and +5 nm per half-sarcomere) were imposed on isolated muscle fibers at the plateau of an isometric tetanus (tension T0) and on the same fibers in rigor after permeabilization of the sarcolemma, to determine stiffness of the half-sarcomere in the two conditions. To identify the contribution of actin filaments to the total half-sarcomere compliance (C), measurements were made at sarcomere lengths between 2.00 and 2.15 microm, where the number of myosin cross-bridges in the region of overlap between the myosin filament and the actin filament remains constant, and only the length of the nonoverlapped region of the actin filament changes with sarcomere length. At 2.1 microm sarcomere length, C was 3.9 nm T0(-1) in active isometric contraction and 2.6 nm T0(-1) in rigor. The actin filament compliance, estimated from the slope of the relation between C and sarcomere length, was 2.3 nm microm(-1) T0(-1). Recent x-ray diffraction experiments suggest that the myosin filament compliance is 1.3 nm microm(-1) T0(-1). With these values for filament compliance, the difference in half-sarcomere compliance between isometric contraction and rigor indicates that the fraction of myosin cross-bridges attached to actin in isometric contraction is not larger than 0.43, assuming that cross-bridge elasticity is the same in isometric contraction and rigor.     

Optical rotation signals recorded from a single skeletal muscle fibre of a frog

The decreased availability of economic resources requires the use of methods to assess hospital efficiency. The aim of our study was to retrospectively evaluate the applicability to the Italian reality of a catalogue of medical acts (CdAM-champs beta) set up for the French Health System. We wanted to evaluate the amount of resource utilization of a Cardiothoracic Operating Room of a IRCCS regional general hospital. The study included 83 admissions, occurring in October 1996, to the cardiac surgery department. Medical acts were recorded for all admissions taking into account both the number of acts and the weight of acts expressed as standard cost index or ICR. This takes into account the use of human (medical and nursing staff) and technical resources. Calculation of ICR beta scores was achieved by means of two different expressions. We observed a lack of correlation between the two values of each ICR. The previous formula (1991) gives more emphasis on the different conditions in which the operation was carried out (American Society of Anesthesiology score), duration of anesthesia and patients disease with a statistical significant difference. The last formula (1995) only evaluate patients' disease. None of the two formula of ICR beta turns out to be completely appropriate index of resource utilization during anesthesia; therefore this analysis still remains a difficult problem. Yet it must be recognized that this system deserves the merit of having evaluated the anesthesiological duties, by separating them from the surgical ones.     

Transient outward K+ channels in vesicles derived from frog skeletal muscle plasma membranes

Whole-cell voltage-clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes. Capacitance measurements showed that these vesicles lack invaginations. In solutions containing K+, transient outward currents with reversal potentials close to EK were recorded with a maximum potassium conductance of 0.3 mS/cm2. These currents inactivated in a voltage-dependent manner with a time constant of decay that reached a limiting value of 26 ms at large depolarizations. The steady-state inactivation reached half-maximum values at -66 mV. Transient currents were completely blocked with 5 mM 4-aminopyridine. Single-channel recordings made in inside-out excised patches from the vesicles had ensemble averages with characteristics similar to those of the macroscopic currents, although with significantly faster inactivation time constants. The single-channel chord conductance was 21 pS when the pipette and bath solutions contained 2.5 mM and 120 mM KCl, respectively. It is concluded that these vesicles contain potassium channels that are very similar to A channels found in neurons and other cells.     

Glycogen turnover in skeletal muscle is stimulated along with glucose uptake in vivo during contraction

We have shown previously that glycogen synthesis in the heart can be stimulated in vivo by epinephrine. Our aim in this study was to determine whether glycogen synthesis in skeletal muscle can be similarly affected during increased energy expenditure. Left sciatic nerves of anesthetized fasted rats were electrically stimulated to allow left hindlimb muscles to contract for 5, 10, and 20 min. Glycogen contents in the contracting muscles at the end of electrical stimulation were found to be approximately 40% less than resting muscles in the right hindlimbs in all three groups of rats. Accompanying the enhanced glycogenolysis was increased incorporation of the intravenously infused [3-(3)H]-glucose into glycogen. The rate of tritium incorporation into glycogen in the contracting muscle was found to be 34-fold greater than resting muscles. Glucose utilization was determined by the phosphorylation of the intravenously injected [14C]-2-deoxyglucose in the skeletal muscle. The rate of accumulation of [14C]-2-deoxyglucose-6-phosphate in the contracting muscles was found to be 28-fold greater than resting muscles. Glycogen synthesis and glucose uptake indexes, calculated by dividing the radioactivity in [3H]-glycogen and [14C]-2-DGP by the mean specific activity of their respective precursors in the plasma, were not found to be significantly different in the contracting muscle. In conclusion, our data indicate that: (i) glycogenesis and glycogenolysis can be stimulated concurrently in the skeletal muscle; and (ii) glucose utilization in the skeletal muscle during contraction may be mediated through glycogen turnover.     

Optical isometric force transducer for measurement of rat skeletal muscle contraction in the NMR spectrometer

Chronic hematomas are a common problem during infancy and usually occur as the consequence of trauma. They tend to enlarge and are often managed successfully with repeated subdural taps. In patients with collections that fail to respond to percutaneous drainage, the choice of operative management, including burr hole evacuation, shunting, or craniotomy, remains controversial. A new technique, called endoscopic washout, was successfully used in 7 children under the age of 2 years who presented with irritability, vomiting, seizures, and rapid head growth. Preoperative computerized tomography (CT) scans demonstrated enlarging, bilateral, chronic subdural collections; these failed to respond to repeated percutaneous taps over 10 days. With the patient positioned supine, bilateral linear incisions were made anterior to the coronal suture in the midpupillary lines and burr holes were placed. After the dura and outer membrane were opened and coagulated with bipolar cautery, a 4-mm steerable fiberscope was introduced into the subdural space to visualize the collections, evacuate any residual clot, and continuously irrigate the space with lactated Ringer's solution warmed to physiologic temperature. No bridging vessels or synechiae were violated; nitrous oxide and hyperventilation were discontinued before removing the fiberscope. The subdural space was irrigated again prior to closure. At follow-up (range 18 months to 8 years), CT scans showed reexpansion of the brain and no reaccumulation of the hematomas. We conclude that the endoscopic washout is a safe, uncomplicated treatment for chronic subdural hematomas in infants; is more effective than treatment with conventional burr holes alone, and eliminates the need for shunting or craniotomy.     

Molecular size-dependent leakage of intracellular molecules from frog skeletal muscle fibers permeabilized with beta-escin

The permeability of beta-escin-treated cell membrane was characterized in terms of the permeant molecular size, by monitoring the leak of cytoplasmic molecules in frog skeletal muscle fibers. With a low concentration of beta-escin (5 microM), most of the cellular ATP was lost within 30-40 min (as revealed by rigor force generation), whereas a fluorescence-labeled dextran injected into the cytoplasm (approximately 10 kDa) and cytoplasmic proteins (14-80 kDa) slowly leaked out of the cell. A high concentration of beta-escin (50-100 microM) accelerated the leak of large molecules. Therefore, low concentrations of beta-escin may be used as a means of permeabilizing the cell membrane to relatively small molecules, while retaining a major fraction of the cellular macromolecules.     

Positive inotropic effect of adrenaline on potassium contractures in tonic skeletal muscle fibres of the frog

K+ contractures were elicited in small bundles of tonic skeletal muscle fibres of the frog. Adrenaline (1 microM) increased the amplitude of K+ contractures in a [K+]o-dependent manner: maximal effects were produced between 20 and 60 mM [K+]o. In contrast, we found no effect of adrenaline on K+ contractures of twitch fibres. The potentiating effect of adrenaline depended on [Ca2+]o. Increasing [Ca2+]o from 1.8 to 10 mM doubled the positive inotropic effect of adrenaline. In a nominally Ca2+ free saline, adrenaline had no potentiating effect. The Ca2+ channel blockers nifedipine (20 microM) and Ni2+ (1.8 mM) reversibly reduced the amplitude of the tonic phase of K+ contractures and blocked the potentiation by adrenaline. The mechanical effects of adrenaline cannot be explained by changes in the membrane potential, as revealed by intracellular recordings at several [K+]o. It was concluded that the potentiating effect of adrenaline in tonic muscle fibres of the frog may be mediated through Ca2+ channels.     

Hypohydration effects on skeletal muscle performance and metabolism: a 31P-MRS study

The purpose of this study was to determine whether hypohydration reduces skeletal muscle endurance and whether increased H+ and Pi might contribute to performance degradation. Ten physically active volunteers (age 21-40 yr) performed supine single-leg, knee-extension exercise to exhaustion in a 1.5-T whole body magnetic resonance spectroscopy (MRS) system when euhydrated and when hypohydrated (4% body wt). 31P spectra were collected at a rate of one per second at rest, exercise, and recovery, and were grouped and averaged to represent 10-s intervals. The desired hydration level was achieved by having the subjects perform 2-3 h of exercise in a warm room (40 degrees C dry bulb, 20% relative humidity) with or without fluid replacement 3-8 h before the experiment. Time to fatigue was reduced (P < 0.05) by 15% when the subjects were hypohydrated [213 +/- 12 vs. 251 +/- 15 (SE) s]. Muscle strength was generally not affected by hypohydration. Muscle pH and Pi/beta-ATP ratio were similar during exercise and at exhaustion, regardless of hydration state. The time constants for phosphocreatine recovery were also similar between trials. In summary, moderate hypohydration reduces muscle endurance, and neither H+ nor Pi concentration appears to be related to these reductions.     

New look at force-frequency relationship of human skeletal muscle: effects of fatigue

16-Fluoropalmitic acid was synthesized from 16-hydroxypalmitic acid using diethylaminosulfur trifluoride. This monofluorinated fatty acid then was used to make 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) as a fluorinated analog of dipalmitoylphosphatidylcholine (DPPC). Surprisingly, we found that the phase transition temperature (Tm) of F-DPPC occurs near 50 degrees C, approximately 10 degrees C higher than its nonfluorinated counterpart, DPPC, as judged by both differential scanning calorimetry and infrared spectroscopy. The pretransition observed for DPPC is absent in F-DPPC. A combination of REDOR, rotational-echo double-resonance, and conventional solid-state NMR experiments demonstrates that F-DPPC forms a fully interdigitated bilayer in the gel phase. Electron paramagnetic resonance experiments show that below Tm, the hydrocarbon chains of F-DPPC are more motionally restricted than those of DPPC. X-ray scattering experiments confirm that the thickness and packing of gel phase F-DPPC is similar to that of heptanetriol-induced interdigitated DPPC. F-DPPC is the first phosphoglyceride containing sn-1 and sn-2 ester-linked fatty acyl chains of equal length that spontaneously forms interdigitated bilayers in the gel state in the absence of inducing agents such as alcohols.