Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10⁻¹⁰ cm² s⁻¹) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10⁻⁷ cm² s⁻¹ (parvalbumin) to 0.20 × 10⁻⁷ cm² s⁻¹ (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.     

Diffusion Coefficients of Endogenous Cytosolic Proteins from Rabbit Skinned Muscle Fibers

Efflux time courses of endogenous cytosolic proteins were obtained from rabbit psoas muscle fibers skinned in oil and transferred to physiological salt solution. Proteins were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic and glycogenolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and binding to the cytomatrix. Optimization algorithms returned estimates of the apparent diffusion coefficients, D(r,t), that were very low at the onset of diffusion (∼10⁻¹⁰ cm² s⁻¹) but increased with time as cytosolic protein density, which was initially high, decreased. D(r,t) at later times ranged from 2.11 × 10⁻⁷ cm² s⁻¹ (parvalbumin) to 0.20 × 10⁻⁷ cm² s⁻¹ (phosphofructose kinase), values that are 3.6- to 12.3-fold lower than those predicted in bulk water. The low initial values are consistent with the presence of complexes in situ; the higher later values are consistent with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of adenosine triphosphate at rates beyond that possible with randomly and/or sparsely distributed enzymes, thereby matching supply with demand.     

The Temperature Dependence of the Mechanical Characteristics of Demembranized Rabbit Slow Muscle Fibers

Biophysics - The temperature dependences of the tension and stiffness of actively contracting single fibers of the rabbit slow muscle (m. soleus) were studied using the Joule temperature jump....     

Dynamics of Thin-Filament Activation in Rabbit Skeletal Muscle Fibers Examined by Time-Resolved X-Ray Diffraction

By using skinned-rabbit skeletal muscle fibers, the time courses of changes of thin filament-based x-ray reflections were followed at a 3.4-ms time resolution during thin-filament activation. To discriminate between the effects of calcium binding and myosin binding on thin-filament activity, measurements were performed after caged-calcium photolysis in fibers with full-filament or no-filament overlap, or during force recovery after a quick release. All three reflections examined, i.e., the second actin layer line (second ALL, reporting the tropomyosin movement), the sixth ALL (reporting actin structural change), and the meridional troponin reflections, exhibited calcium-induced and myosin-induced components, but their rate constants and polarities were different. Generally, calcium-induced components exhibited fast rate constants (>100 s⁻¹). The myosin-induced components of the second ALL had a rate constant similar to that of the force (7-10 s⁻¹), but that of the sixth ALL was apparently faster. The myosin-induced component of troponin reflection was the only one with negative polarity, and was too slow to be analyzed with this protocol. The results suggest that the three regulation-related proteins change their structures with different rate constants, and the significance of these findings is discussed in the context of a cooperative thin-filament activation mechanism.     

Electrotonic Coupling between Pyramidal Neurons in the Neocortex

Electrotonic couplings (i.e., electrical synapses or gap junctions) are fundamental to neuronal synchronization, and thus essential for many physiological functions and pathological disorders. Interneuron electrical synapses have been studied intensively. Although studies on electrotonic couplings between pyramidal cells (PCs) are emerging, particularly in the hippocampus, evidence is still rare in the neocortex. The electrotonic coupling of PCs in the neocortex is therefore largely unknown in terms of electrophysiological, anatomical and synaptological properties. Using multiple patch-clamp recording with differential interference contrast infrared videomicroscopy (IR-DIC) visualization, histochemical staining, and 3D-computer reconstruction, electrotonic coupling was recorded between close PCs, mainly in the medial prefrontal cortex as well as in the visual cortical regions of ferrets and rats. Compared with interneuron gap junctions, these electrotonic couplings were characterized by several special features. The recording probability of an electrotonic coupling between PCs is extremely low; but the junctional conductance is notably high, permitting the direct transmission of action potentials (APs) and even tonic firing between coupled neurons. AP firing is therefore perfectly synchronized between coupled PCs; Postjunctional APs and spikelets alternate following slight changes of membrane potentials; Postjunctional spikelets, especially at high frequencies, are summated and ultimately reach AP-threshold to fire. These properties of pyramidal electrotonic couplings largely fill the needs, as predicted by simulation studies, for the synchronization of a neuronal assembly. It is therefore suggested that the electrotonic coupling of PCs plays a unique role in the generation of neuronal synchronization in the neocortex.     

Relation between Membrane Potential Changes and Tension in Barnacle Muscle Fibers

Constant current pulses have been applied to single muscle fibers of the barnacle, Balanus nubilus Darwin, with an axial metal electrode. The membrane potential change, which took place over a large part of the muscle fiber, was measured with a similar electrode. Depolarizing pulses, if the voltage was greater than threshold, produced tension. The size of the tension was a function of the magnitude and the duration of the depolarizing pulses. The latency between the onset of depolarization and tension can be only in part attributable to mechanical factors. AC stimulation produced tension, but 5 to 10 seconds were required for the steady-state level of the tension to be reached. Muscles were depolarized in elevated K and studied after the contracture had terminated. If not too depolarized, further depolarization produced tension. Termination of hyperpolarizing pulses also produced tension, which decayed quite slowly. Hyperpolarizing pulses reduced, or abolished, any preexisting tension. Thus, it appears that at certain values of the membrane potential tension is set up, but there is also a slow process of accommodation present.     

Graded Activation in Frog Muscle Fibers

The membrane potential of frog single muscle fibers in solutions containing tetrodotoxin was controlled with a two-electrode voltage clamp. Local contractions elicited by 100-ms square steps of depolarization were observed microscopically and recorded on cinefilm. The absence of myofibrillar folding with shortening to striation spacings below 1.95 µm served as a criterion for activation of the entire fiber cross section. With depolarizing steps of increasing magnitude, shortening occurred first in the most superficial myofibrils and spread inward to involve axial myofibrils as the depolarization was increased. In contractions in which the entire fiber cross section shortened actively, both the extent of shortening and the velocity of shortening at a given striation spacing could be graded by varying the magnitude of the depolarization step. The results provide evidence that the degree of activation of individual myofibrils can be graded with membrane depolarization.     

The Fraction of Myosin Motors That Participate in Isometric Contraction of Rabbit Muscle Fibers at Near-Physiological Temperature

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca²⁺ at 4°C were heated to 31–34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41–43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ∼6.3 pN.     

Interaction between Bronchoconstrictor Stimuli on Human Airway Smooth Muscle

In healthy human subjects, the simultaneous aerosol administration of histamine and methacholine results in a pronounced decrease in maximum flow rates on partial expiratory flow-volume (PEFV) curves. When given alone in the same concentrations, these drugs produced no or minimal decreases in flow rates. The results suggest an interaction of histamine and cholinergic stimuli on airway smooth muscle (ASM). This mechanism might explain many experiments where vagal blockade diminished or abolished ASM response to histamine and other stimuli, simply by interfering with histamine-cholinergic interaction at the ASM level. These findings confirm similar findings of animal in vitro experiments. The experiments clearly confirm the sensitivity and value of assessing drug effects prior to a deep breath. Flow-rate changes after a full inspiration, taken from the maximum expiratory flow-volume (MEFV) curve, show either no relationship to the concentration of inhaled methacholine or significantly less effect than that seen on the PEFV curve.     

Histochemical Methods for Dissociated Muscle Fibers

Skeletal or cardiac muscle fibers can be separated by brief (3-5 second) dissociation of formalin-fixed pieces with a Willems Polytron (Brinkmann Instrument Co.). Such separated fibers are useful for demonstration of abnormal accumulations of lipids, carbohydrates, proteins and minerals in metabolic diseases. Staining techniques for demonstration of various stored materials include: 1) toluidine blue at pH 2.8 for acid mucopolysaccharide in skeletal muscle fibers in Pompe's glycogenesis 2,2) one-step trichrome stain for nemaline myopathy and for abnormal mitochondria in X-linked infantile cardiomyopathy, 3) periodic acid-methenamine silver stain for glycolipid-containing lysosomes in I-cell disease (mucolipidosis 2), 4) Sudan black B stain for lipid in skeletal muscle fibers in Reye's syndrome, infantile lactic acidosis, Leigh's infantile subacute necrotizing encephalopathy and Jansky-Bielschowsky late infantile ceroid lipofuscinosis, 5) iron stain for iron in cardiac and skeletal muscle fibers in thalassemia with advanced hemosiderosis, and 6) autofluorescence for “ceroid” in skeletal muscle fibers in Jansky-Bielschowsky disease.     

Optical Diffraction Studies of Muscle Fibers

A new technique to monitor light diffraction patterns electrically is applied to frog semitendinosus muscle fibers at various levels of stretch. The intensity of the diffraction lines, sarcomere length change, and the length-dispersion (line width) were calculated by fast analogue circuits and displayed in real time. A heliumneon laser (wavelength 6328 Å) was used as a light source. It was found that the intensity of the first-order diffraction line drops significantly (30-50%) at an optimal sarcomere length of 2.8 μm on isometric tetanic stimulation. Such stimulation produced contraction of half-sarcomeres by about 22 nm presumably by stretching inactive elements such as tendons. The dispersion of the sarcomere lengths is extremely small, and it is proportional to the sarcomere length (less than 4%). The dispersion increases on stimulation. These changes on isometric tetanic stimulation were dependent on sarcomere length. No vibration or oscillation in the averaged length of the sarcomeres was found during isometric tetanus within a resolution of 3 nm; however, our observation of increased length dispersion of the sarcomeres together with detection of the averaged shortening of the sarcomere lengths suggests the presence of asynchronous cyclic motions between thick and thin filaments. An alternative explanation is simply an increase of the length dispersion of sarcomeres without cyclic motions.     

Muscle Fiber Types in Crabs: Studies on Single Identified Muscle Fibers

SYNOPSIS. Based on electrophysiological and histochemical data,four types of muscle fibers (types I, II, III and IV) can beidentified in the closer of the crab Eriphia. Although characteristicsused for typing vary among the fibers of a particular type,the combination of several parameters permits an assignment.Of particular significance for typing is the myosin ATPase activityand its stability after preincubation at different pH levels.The fiber types defined for the closer muscle can also be foundin the other leg muscles of riphia. Single, electrophysiologically identified fibers of each typewere quantitatively analyzed for several key enzymes of oxidativeand glycolytic energy metabolism (GAPDH, LDH, CS, IDH, HAD).Despite the variations found, different metabolic types canbe defined. The typing derived from biochemical studies correlateswell with that obtained electrophysiologically and histochemically.The variability of the biochemical properties, however, seemsto be considerably larger. The type I fibers can be regarded as slow oxidative, the typeII and III fibers as fast oxidative glycolytic, and the typeIV fibers as fast glycolytic.     

Electrotonic interaction between secondary neurons of the carp olfactory bulb

Responses of secondary neurons of the carp olfactory bulb evoked by electrical stimulation of the olfactory tract were investigated by intracellular recording. In most neurons spike responses were identified as antidromic. Their latent periods varied from 2.5 to 55 msec. Two other types of responses of secondary neurons had constant latent periods: the pseudo-antidromic spike and a fast low-amplitude depolarization potential. It is concluded that these responses are generated by the antidromic spike of a neighboring neuron, connected electrotonically with the recorded neuron.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 8, No. 5, pp. 490–496, September–October, 1976.     

Some Relations between Changes in the Linear Electrical Properties of Striated Muscle Fibers and Changes in Ultrastructure

Some of the linear electrical properties of frog sartorius muscle have been investigated in Ringer's fluid and in a Ringer fluid made hypertonic by the addition of sucrose or NaCl. Electrical constants were determined from measurements of the phase angle of the admittance of a fiber for an applied alternating current, from measurements of the voltage induced by an inward pulse of current, and from measurements of the conduction velocity of the action potential and the time constant of its foot. The dilation of the transverse tubular system induced by the sucrose hypertonic Ringer fluid was correlated with the change in the electrical constants. From this it is concluded that a two time constant equivalent circuit for the membrane, as proposed by Falk and Fatt, is in good agreement with our results. Both the area of the membrane of the transverse tubular system, and the capacity (c_e) attributed to it, increased in the sucrose hypertonic Ringer fluid. The resistance r_e, which is in series with c_e, did not fall when the transverse tubular system was dilated and probably is not located in that system.     

Postmortem Changes in Regulatory Proteins of Rabbit Muscle

The procedures for complete extraction of regulatory proteins, particularly troponin and tropomyosin from the myofibrils of fresh and stored muscles were developed and the effect of postmortem storage of muscle on the properties of the regulatory proteins was studied. The ATPase enhancing ability and the sedimentation behavior of α-actinin from post-rigor muscle did not differ from those of α-actinin from pre-rigor muscle. The amounts of both troponin and tropomyosin decreased during the storage of muscle. Troponin decreased more rapidly than tropomyosin. These results were interpreted to support our hypothesis that the structural alteration of myofibril, which is mainly due to the change in the troponin-tropomyosin complex of thin filaments, proceeds during the postmortem storage of muscle.     

Chronic Low-Frequency Stimulation Transforms Cat Masticatory Muscle Fibers into Jaw-Slow Fibers

Cat masticatory muscle during regeneration expresses masticatory-specific myofibrillar proteins upon innervation by a fast muscle nerve but acquires the jaw-slow phenotype when innervated by a slow muscle nerve. Here, we test the hypothesis that chronic low-frequency stimulation simulating impulses from the slow nerve can result in masticatory-to-slow fiber–type transformation. In six cats, the temporalis muscle was continuously stimulated directly at 10 Hz for up to 12 weeks using a stimulator affixed to the skull. Stimulated muscles were analyzed by immunohistochemistry using, among others, monoclonal antibodies against masticatory-specific myosin heavy chain (MyHC), myosin binding protein-C, and tropomyosins. Under the electrodes, stimulation induced muscle regeneration, which generated slow fibers. Deep to the electrodes, at two to three weeks, two distinct populations of masticatory fibers began to express slow MyHC: 1) evenly distributed fibers that completely suppressed masticatory-specific proteins but transiently co-expressed fetal MyHCs, and 2) incompletely transformed fibers that express slow and masticatory but not fetal MyHCs. SDS-PAGE confirmed de novo expression of slow MyHC and β-tropomyosin in the stimulated muscles. We conclude that chronic low-frequency stimulation induces masticatory-to-slow fiber–type conversion. The two populations of transforming masticatory fibers may differ in their mode of activation or lineage of their myogenic cells.     

Electrotonic coupling between cercal afferents and giant interneurons in the American cockroach

Stimulation of the cercal nerve of the female American cockroach evokes a short-latency action potential in one giant axon in the ipsilateral connective of the ventral nerve cord. Neither procion yellow nor cobalt passes from the nerve cord into the cercal nerve, and the short-latency response disappears several weeks after removal of the cercus. Therefore, the short-latency spike is not due to a branch of the giant interneuron extending into the cercal nerve, but is presumably due to electrotonic coupling of cercal afferents to the giant. Responses of the presumed electrotonic junction to drugs, varied ionic concentrations and tonicity, and to cold are described. These responses and the impermeability of the junction to procion yellow suggest that the coupling is not by means of a gap junction. There is evidence for electrotonic coupling to another giant axon in the female, but this junction does not ordinarily transmit a spike. Electrotonic coupling is rare in males. In some females action potentials in giant interneurons excite cercal afferents electrically, and the afferents then re-excite the giants chemically. Electrotonic coupling may reduce fatigue and habituation of chemical synapses by depolarizing presynaptic terminals whenever the giants are active.     

Dynamic Properties of Giant Muscle Fibers of the Barnacle

Giant muscle fibers of the barnacle give graded, relativelyslow contractions, A plateau level, termed the unit response,occurs with stimuli of 3 msec, but pulses longer than 10–15msec give much greater tension or shortening. Repetitive stimulationwith pulses of 3 msec leads to a tetanus. The magnitude of activestate was determined, and found to be also graded in natureand slow to develop, though early in onset. The full developmentof active state requires 80–120 msec. A high level ofeffective series-elasticity was associated with the sarcomeresthemselves.     

Measurement of the Impedance of Frog Skeletal Muscle Fibers

Impedance measurements are necessary to determine the passive electrical properties of cells including the equivalent circuits of the several pathways for current flow. Such measurements are usually made with microelectrodes of high impedance (some 15 MΩ) over a wide frequency range (1-10,000 Hz) and so are subject to many errors. An input amplifier has been developed which has negligible phase shift in this frequency range because it uses negative feedback to keep tiny the voltage on top of the microelectrode. An important source of artifact is the extracellular potential produced by capacitive current flow through the wall of the microelectrodes and the effective resistance of the bathing solution. This artifact is reduced some 10 times by shielding the current microelectrode with a conductive paint. The residual artifact is analyzed, measured, and subtracted from our results. The interelectrode coupling capacitance is reduced below 2 × 10^-17 F and can be neglected. Phase and amplitude measurements are made with phase-sensitive detectors insensitive to noise. The entire apparatus is calibrated at different signal to noise ratios and the nature of the extracellular potential is investigated. The phase shift in the last 5-20 μm of the microelectrode tip is shown to be small and quite independent of frequency under several conditions. Experimental measurements of the phase characteristic of muscle fibers in normal Ringer are presented. The improvements in apparatus and the physiological significance of impedance measurements are discussed. It is suggested that the interpretation of impedance measurements is sensitive to small errors and so it is necessary to present objective evidence of the reliability of one's apparatus and measurements.