A fixed charge model of the transverse tubular system of frog sartorius

Volume changes of the transverse tubular system (T system) of frog sartorius in different solutions can be explained by a model which assumes fixed negative charges in the T system lumen, an open T system mouth, and a Donnan equilibrium between the T system and external solution. The T system volume is regulated by the osmotic pressure difference between the lumen and external solution, as well as by constraining forces whose nature is as yet unclear. The decreased swelling tendency produced by hypotonic solutions and increased tendency produced by some hypertonic solutions are ascribed to changes in the pressure constraint from the sarcoplasm. Fixed charge concentration was estimated tentatively from swelling and resistivity data to be between 0.1 and 0.4 M.     

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.     

Unusual swelling of elastin

The swelling behavior of the elastin network has been investigated by comparing the linear expansion of samples of purified elastin with the volume expansion of the network, calculated on the basis of composition. Elastin sample dimensions and sample masses were measured under three conditions in which volume changes: thermal expansion at fixed water contents, deswelling due to dehydration, and swelling to greater than normal levels due to the swelling agent, sodium dodecyl sulfate. Isotropic network swelling usually changes length in proportion to the cube root of network volume, but length was found to be directly proportional to volume, showing a greater increase in length than expected. This unusual swelling behavior is attributed to an unusual elastin structure at the subfiber level, but there is insufficient detail on elastin's molecular organization to identify a mechanism to explain how it occurs. Assuming the network swells homogeneously, we describe two models that correctly predict swelling behavior, but these models imply a significant deviation from the structure generally assumed for an elastomeric polymer network of kinetically free molecular chains. Assuming that the network swells heterogeneously removes part of the difficulty with the models, but the observed direct proportionality between length and network volume remains to be explained.     

Characterisation of Hydrogel Gel Swelling by Molecular Exclusion

A facile method for the characterization of hydrogel swelling is described which is based on the determination of changes in the liquid phase concentration of an excluded tracer as gel swells in a constant volume system. The utility of this approach is demonstrated with two responsive hydrogel preparations, one where swelling is influenced by system pH, the other by changes in specific solute concentration.     

CORRELATED MORPHOLOGICAL AND PHYSIOLOGICAL STUDIES ON ISOLATED SINGLE MUSCLE FIBERS : II. The Properties of the Crayfish Transverse Tubular System: Localization of the Sites of Reversible Swelling

Living muscle fibers of crayfish become dark during efflux of Cl^-. This change in appearance is correlated with occurrence of vacuolation in the fixed fibers. The vacuoles begin at and are mainly confined to the terminals of the transverse tubular system (TTS) which are in diadic contact with the sarcoplasmic reticulum (SR). In electron micrographs swellings more than 1 µ in diameter may be seen connected to the sarcolemma or sarcolemmal invaginations by relatively unswollen tubules about 300–500 A wide. Darkening of the living fibers can be reversed by causing an influx of Cl^-. Vacuoles are then absent in the fixed preparations. These findings accord with the conclusion that the membrane of the TTS is anion permselective. Localization of the selectivity to the membrane of the terminals of the TTS strengthens the hypothesis that a channeling of current flow is responsible for initiation of excitation-contraction coupling. During the swelling, and upon its reversal, the area of the membrane of the terminals must change reversibly by about two to four orders of magnitude. The absence of changes in the dimensions of the unit membrane indicates that the expansion of the membrane and its subsequent shrinkage involve reversible incorporation of cytoplasmic material into the membrane phase.     

Action Potentials, Afterpotentials, and Excitation-Contraction Coupling in Frog Sartorius Fibers without Transverse Tubules

In frog sartorius muscle fibers in which the transverse tubular system has been disrupted by treatment with glycerol, action potentials which are unaccompanied by twitches can be recorded. These action potentials appear to be the same as those recorded in normal fibers except that the early afterpotential usually consists of a small hyperpolarization of short duration. After a train of action potentials no late afterpotential is seen even when the membrane potential is changed from the resting level. In fibers without transverse tubules hyperpolarizing currents do not produce a creep in potential. The interruption of excitation-contraction coupling, the changes in the afterpotentials, and the disappearance of creep are all attributed to the lack of a transverse tubular system.     

Water Transfer and Cell Structure in Isolated Crayfish Muscle Fibers

Changes in volume of crayfish single muscle fibers in response to changes in ionic or electrical conditions have been studied in conjunction with electrophysiological measurements and electron microscopic examinations. The occurrence of at least three mechanisms of water movements is revealed, two being processes which are superimposed on the normal osmotic water movement that results from a change in the concentration of solute in the medium. Differences between the time courses of the changes in volume and potential on changing K_i/K_o indicate that water may be distributed unequally for a time within compartments of the fiber. Electron micrographs reveal a selective accumulation of water at the periphery of the fiber under certain conditions. A correlation of H₂O transfer with a change in membrane potential is apparent in crayfish muscle fibers and is probably due to electroosmotic effects. Electrokinetic water movements are produced whenever the membrane potential is changed to a considerable degree by changing the level of K and/or Cl in the medium, or by applying currents with an intracellular microelectrode. Depolarizations cause shrinkage. Hyperpolarizations or repolarizations cause swelling. The volume changes are independent of the occurrence or absence of swelling in the anion-permselective transverse tubular system. They indicate that the fiber membrane along the surface is heterogeneous, not only with respect to the signs of its fixed charge sites, but also with respect to the sizes and relative permselectivities of these charged channels.     

Osmotic swelling of endocytic compartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells.

To determine which endocytic compartments are sensitive to sucrose-induced osmotic swelling, CHO and Vero cells were cultured for 1-3 days in media containing 0.03 to 0.05 M sucrose. (Sucrose is internalized but not digested by these cells.) To immunolocalize late endocytic compartments, cells were fixed with formaldehyde and labeled with antibodies against the 215-kDa mannose 6-phosphate receptor (prelysosomal compartment) and LAMP-1 and -2 (mature lysosomes). Early endosomes were labeled by a 2-min uptake of lucifer yellow, mature lysosomes by greater than or equal to 16-h uptake of lucifer yellow followed by a 2-h chase. The data showed that sucrose induced swelling of mature lysosomes only (mannose 6-phosphate receptor negative, LAMP-1 and LAMP-2 positive); early endosomes and the prelysosomal compartment were not affected by the presence of sucrose, i.e., osmotically swollen. Accumulation of lucifer yellow in the swollen compartment was insensitive to cycloheximide. These results suggest, by inference, that the complement of membrane transport proteins that regulate the osmotic properties of endocytic organelles must be discontinuously distributed along the endocytic pathway.     

The effect of fixative tonicity on the myosin filament lattice volume of frog muscle fixed following exposure to normal or hypertonic Ringer

Synopsis Frog sartorius muscles have been fixed sequentially with acrolein and osmium tetroxide dissolved in vehicles of various tonicities, and the myosin filament spacings and sarcomere lengths measured with the electron microscope. From these dimensions the myosin unit-cell volume has been calculated and compared with X-ray diffraction data to determine the effect of fixation. In muscles soaked in normal Ringer and afterwards fixed using normal Ringer as a vehicle for the fixation agents, the unitcell volume undergoes a 10.4% reduction during the preparative procedure. Muscles soaked in hypertonic Ringer undergo a similar reduction in volume during fixation, provided hypertonic Ringer is used as the vehicle; if they are fixed in normal Ringer, the lattice swells during fixation, even if the change to the normal tonicity vehicle occurs after acrolein fixation. If blocks suitable for embedding are cut from the muscles before, rather than after, osmium fixation, more complex changes in intracellular dimensions may occur, including artefactual swelling of the T-system. It is concluded that fixation of tissues exposed to modifications of normal physiological solutions should be performed using the same modified solutions as fixative vehicles.     

The early changes of parietal cell structure in the course of secretory activity in the rat

The fine structure of the rat parietal cell was studied, both at rest and after stimulation by refeeding or insulin administration. Experiments on fixation procedures showed that whenever the fixative contained sucrose at a concentration higher than 0.2 M, the system of cytoplasmic membranes was clearly tubular in arrangement, whereas the omission of sucrose in the fixative usually resulted in a vesicular structure. The study with the high-voltage electron microscope of thick sections prepared by conventional techniques or by impregnation with zinc iodide-osmium (ZIO) revealed that the tubules are grouped into fascicles, and that these form a feltwork that is especially thick toward the cell apex. The development of the secretory canaliculus after stimulation appears to take place by an in situ remodeling of the cytoplasmic domain occupied by the tubular system. Cells examined after short periods of stimulation (5-15 min) showed images of the tubular system and of the canalicular structure which differed both from the nonstimulated and from the fully active (30-45 min of stimulation) cell. These features include the formation of wide cisternae and of pericanalicular cytoplasmic trabeculae or laminae, whose fine structure bears close resemblance to that of the intracanalicular processes in the same cells. These images can be ordered into a hypothetical sequence which is proposed as a model to explain the transformation of the tubular system and intervening cytoplasmic matrix into secretory canaliculus.     

On the Organisation of the Sarcotubular Systems in the Caudal Muscle Cells of Larvaceans (Tunicata)

The structure of the sarcotubular systems of the caudal muscle cells is described in various larvaceans. In Oikopleura there is both a transverse tubular system and a sarcoplasmic reticulum; there are internal couplings between the two and also sarcolemmal couplings. In Fritillaria (and probably also in Kowalevskaia), a transverse tubular system is lacking, and there are only sarcolemmal couplings with the sarcoplasmic reticulum, which is related to the mitochondria as well as to the myofilaments. The significance of these differences is discussed, and it is concluded that the arrangement of the sarcotubular systems is related to muscle fibre thickness; within the Tunicata, these systems do not indicate phylogenetic relationships.     

Linear electrical properties of isolated cardiac cells

Summary A frequency domain equivalent circuit analysis of isolated ventricular cells indicated the presence of an internal membrane structure which has a total capacitance four- to sixfold larger than the surface membrane. The internal membrane was mainly attributed to the sarcoplasmic reticulum since other morphological studies have shown that its area is many-fold larger than that of the surface membrane. Corresponding estimates from the transverse tubular system indicate an area less than that of the surface; thus this structure is not a likely candidate for the observed internal capacitance. Measurements in hypertonic solutions showed that the access resistance to the internal membrane reversibly increased as the tonicity was elevated. Freeze-fractured electron microscopic studies confirmed that hypertonic solutions increased the volume of transverse tubular system, which thus appears to have little relation to the access resistance. The most probable source of the access resistance is the diadic junction to the sarcoplasmic reticulum, which therefore would electrically couple it to the surface membrane.     

NMR imaging of high-amylose starch tablets. 2. Effect of tablet size

Carbohydrate polymers are widely used for pharmaceutical applications such as the controlled release of drugs. The swelling and water mobility in high-amylose starch tablets are important parameters to be determined for these applications. They have been studied at different time intervals by nuclear magnetic resonance imaging (NMRI) after the immersion of the samples in water. These tablets have a hydrophilic matrix, which swells anisotropically and forms a hydrogel in water. NMRI shows clearly the anisotropy of the water penetration and the swelling along the radial and axial dimensions of the tablets. Empirical relationships are established to describe the kinetics of water penetration and swelling of the tablets. Results show that water uptake and tablet swelling strongly depend on the size of the tablets. Gravimetric measurements of water uptake were also performed in comparison with the NMRI results.     

Reorientation of Cortical Microtubules in the Sub-Apical Region during Tuberization in Single-Node Stem Segments of Potato in Culture

The initial events in tuberization were examined in single-nodestem segments of potato, in which the tuberization was easilyregulated in culture. The addition of 8% sucrose to the culturemedium caused the cessation of elongation of lateral shootsand the swelling of the sub-apical region of each shoot. Swellingwas first induced by lateral cell expansion, which was followedby periclinal cell division. The divided cells then expandedlaterally. The alteration in the direction of growth was accompaniedby the reorientation of arrays of cortical microtubules (MTs),which was monitored by immunofluorescence microscopy. Cellsin the sub-apical region of elongating shoots had prominenttransverse arrays of MTs. The MTs in swelling cells were orientedlongitudinally with respect to the axis of the shoot. Finally,the arrays of MTs became completely disorganized. By contrast,the elongation of lateral shoots continued in GA₃-treated segmentsand the cells in the sub-apical region of such shoots retainedconspicuous transverse arrays of MTs during culture, even inthe presence of a high concentration (8%) of sucrose. (Received July 2, 1994; Accepted May 19, 1995)     

THE SARCOPLASMIC RETICULUM, THE T SYSTEM, AND THE MOTOR TERMINALS OF SLOW AND TWITCH MUSCLE FIBERS IN THE GARTER SNAKE

Twitch and slow muscle fibers, identified morphologically in the garter snake, have been examined in the electron microscope. The transverse tubular system and the sarcoplasmic reticulum are separate entities distinct from each other. In twitch fibers, the tubular system and the dilated sacs of the sarcoplasmic reticulum form triads at the level of junction of A and I bands. In the slow fibers, the sarcoplasmic reticulum is severely depleted in amount and the transverse tubular system is completely absent. The junctional folds of the postsynaptic membrane of the muscle fiber under an "en grappe" ending of a slow fiber are not so frequent or regular in occurrence or so wide or so long as under the "en plaque" ending of a twitch fiber. Some physiological implications of these differences in fine structure of twitch and slow fibers are discussed. The absence of the transverse tubular system and reduction in amount of sarcoplasmic reticulum, along with the consequent disposition of the fibrils, the occurrence of multiple nerve terminals, and the degree of complexity of the post junctional folds of the sarcolemma appear to be the morphological basis for the physiological reaction of slow muscle fibers.     

The Relation Between the Late After-Potential and the Size of the Transverse Tubular System of Frog Muscle

This is an investigation of the effects on the late after-potential of immersing frog sartorius muscles in three kinds of modified Ringer's fluid; hypertonic, low chloride, and potassium-free. The late after-potential has been attributed to the depolarizing effect of an accumulation of potassium, during a preceding train of impulses, in the intermediary space of the model of a muscle fiber proposed by Adrian and Freygang. Both the hypertonic and low chloride solutions prolonged the late after-potential reversibly and the potassium-free solution shortened it. The effect of the low potassium solution fitted those data calculated from the model, but the effect of the hypertonic and low chloride solutions required an increase in size of the intermediary space of the model in order to fit the calculated data. An electron microscopic study of the muscles showed that the size of the transverse tubular system changed reversibly in the hypertonic and low chloride solutions in almost the amount necessary to fit the experimental data to the calculated data. This agreement between the change in size of the transverse tubular system and that of the intermediary space indicates that the intermediary space may be the transverse tubular system.     

Impedance of Frog Skeletal Muscle Fibers in Various Solutions

The linear circuit parameters of 140 muscle fibers in nine solutions are determined from phase measurements fitted with three circuit models: the disk model, in which the resistance to radial current flow is in the lumen of the tubules; the lumped model, in which the resistance is at the mouth of the tubules; and the hybrid model, in which it is in both places. The lumped model fails to fit the data. The disk and hybrid model fit the data, but the optimal circuit values of the hybrid model seem more reasonable. The circuit values depend on sarcomere length. The conductivity of the lumen of the tubules is less than, and varies in a nonlinear manner with, the conductivity of the bathing solution, suggesting that the tubules are partially occluded by some material like basement membrane which restricts the mobility of ions and has fixed charge. The x2.5 hypertonic sucrose solution used in many voltage clamp experiments produces a large increase in the radial resistance, suggesting that control of the potential across the tubular membranes would be difficult to achieve. Glycerol-treated fibers have 90% of their tubular system insulated from the extracellular solution and 10% connected to the extracellular solution through a high resistance. We discuss the implications of our results for calculations of the nonlinear properties of muscle fibers, including the action potential and the radial spread of contraction.     

Electron microscope study of lens fibers

The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20 degrees . Aging of mitochondria at 0 degrees causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine. The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn(++), and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the contractility and permeability properties of the mitochondrial membrane. Occurrence of different types of mitochondrial swelling, the intracellular factors affecting the swelling and shrinking of mitochondria, as well as the physiological significance of thyroxine-induced swelling are discussed.     

Effect of high sucrose concentrations on mitochondria: Analysis of mitochondrial populations by density-gradient centrifugation after fixation with glutaraldehyde

It has been shown previously that intact rat liver mitochondria can be separated into two populations (designated B2 and B3) with mean buoyant densities of 1·184 and 1·216 respectively, by isopycnic sucrose density gradient centrifugation. A comparison has been made of some properties of these mitochondrial fractions from density gradients with non-fractionated mitochondria. Use was made of density gradient centrifugation for analysis of preparations fixed with appropriate concentrations of glutaraldehyde. The permeability of the membranes of non-fractionated mitochondria to sucrose was increased by exposure to hypoosmotic sucrose solutions. The B3 mitochondria differed from the non-fractionated mitochondria in their response to changes in osmotic pressure of the suspending medium while the B2 mitochondria showed essentially identical behaviour with the controls. However, under conditions of energized swelling the B2 mitochondria were markedly different to the controls. This difference, which is attributed to reduced permeability of the mitochondrial membranes to metabolites brought about by exposure to the high concentrations of sucrose encountered in the density gradient, was reversed by incubation in hypo-osmotic sucrose solutions in the presence of oxidizable substrate and permeant ions.Died December, 1969.     

Evidence for Anion-Permselective Membrane in Crayfish Muscle Fibers and Its Possible Role in Excitation-Contraction Coupling

Under certain conditions only, isolated crayfish skeletal muscle fibers change in appearance, becoming grainy, darkening, and seemingly losing their striations. These changes result from development of large vesicles on both sides of the Z-line. The longitudinal sarcoplasmic reticulum remains unaffected. The vesicles are due to swelling of a transverse tubular system (TTS) which is presumably homologous with the T-system tubules of other muscle fibers. The vesiculations occur during efflux of water or on reducing external K or Cl, but only when KCl can leave the fiber. They never result from osmotic, ionic, or electrical changes when KCl cannot leave. Inward currents, applied through a KCl-filled intracellular cathode, also cause the vesiculations. These are not produced when the cathode is filled with K-propionate, nor by outward or longitudinal currents. Thus the transverse tubules swell only when Cl leaves the cell. Accordingly, their membrane is largely or exclusively anion-permselective. These findings also indicate that the TTS forms part of a current loop, connecting with the exterior of the fiber probably through radial tubules (RT) possessing membrane of low conductivity. Thus, part of the current flowing inward across the sarcolemma during activity can return to the exterior through the membrane of the TTS. The structure and properties of the latter offer the possibility for an efficient electrical mechanism to initiate excitation-contraction coupling.