John P. Reuben

Duchenne dystrophy Abnormal generation of tension and Ca++ regulation in single skinned fibers

Skinned, single-fiber preparations from the quadriceps or gastrocnemius muscles of four ambulatory male children with Duchenne dystrophy were tested for their ability to generate tension and to regulate Ca++. To determine the intrinsic strength (P0) of the contractile material, the maximum Ca++-activated tensions were normalized to the fiber diameters. Sixty-four percent of the Duchenne fibers had P0 values below 1.0 kg per square centimeter-the lowest value observed in control muscle-and the average P0 values of fibers from each Duchenne biopsy were significantly (p < 0.01) below the average PO values for control muscle fibers and for muscle fibers obtained from one obligatory carrier of the Duchenne gene. The low tensions in the Duchenne muscle fibers could not be ascribed to altered Ca++ regulation or to substrate sensitivity of the contractile proteins in the fibers, since these were normal. However, ultrastructural abnormalities of the myofilaments, which might reduce the ability of the contractile system to develop tension, were observed. Furthermore, Ca++ regulation by the sarcoplasmic reticulum (SR) was impaired in most of those muscle fibers, from both carriers and Duchenne patients, that did develop normal tension. These results suggest that in Duchenne muscle a functional disorder in the SR may precede loss of the ability of the contractile proteins to generate tension. However, since muscle fibers from Duchenne-gene carriers developed significantly greater tensions than fibers from Duchenne patients, while yet having similar defects in Ca++ regulation, the SR disorder may not be exclusively responsible for abnormal contractile protein function.

Muscle: Volume Changes in Isolated Single Fibers

Volumetric experiments on single fibers isolated from semiten-dinosus muscles of frogs, some performed in correlation with measurements of membrane potential, confirm the data obtained on whole muscles, but only for the specific range of conditions in which most of the latter experiments have been done. These conditions are restricted to media in which the anion ( Cl usually) is permanent and the K is 10 to 12.5 meqlliter, or four to five times above the normal level in Ringers solution. When other ionic conditions are employed, phenomena are disclosed which have not previously been described. The findings throw doubt upon the validity of some generally accepted views regarding the permeability properties of the membrane of frog muscle fibers and regarding the nature of the mechanisms which regulate their volume.

Are cardiac muscle cells skinned by EGTA or EDTA

KNOWLEDGE of the processes involved in the contraction of skeletal muscle fibres has been greatly advanced by study of the ‘skinned’ fibre preparation. Here the sarcolemma is peeled off, giving access to the intracellular organelles unhampered by an intervening cell membrane. The small size of cardiac muscle cells had seemed to preclude the use of this technique, and so interest has been shown in a ‘chemical’ method for ‘skinning’ heart cells, which involves exposing the muscle to EGTA and EDTA (ref. 1). However, this paper questions the effectiveness of the EDTA/EGTA skinning methods, reporting that tension production in fully treated cells is [Na]o dependent, and that the cells can support resting and action potentials in low [K]o-‘skinning’ media. It is concluded that both the high Ca-sensitivity and low membrane potential of EGTA/EDTA-treated cells occur without radical impairment of the sarcolemmal permeability barrier. An alternative explanation for the high sensitivity to extracellular calcium is presented which involves the Ca–Na exchange mechanism.

Functional heterogeneity of the sarcoplasmic reticulum within sarcomeres of skinned muscle fibers

SummaryPrecipitation of Ca oxalate in the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers caused an increase in light scattering which was proportional to the amount of Ca accumulated per unit fiber volume. The increase in scattering was used to measure net accumulation rates and steady-state Ca capacities of the sarcoplasmic reticulum in single fibers. The data obtained were qualitatively and quantitatively similar to those reported for isolated vesicle preparations.Under conditions in which Ca was not depleted from the medium, Ca accumulation was linear with time over much of its course. Steady-state capacities were independent of the Ca concentration; uptake rates were half-maximal at 0.5 μm Ca++ and saturated above about 1.0 μm. Both rate and capacity varied with the oxalate concentration, being maximal at oxalate concentrations >=5mm and decreasing in proportion to one another at lower concentrations, with a threshold near 0.25mm. At the lower loads, electron micrographs showed many sarcoplasmic reticulum elements empty of precipitate alongside others that were full, whereas virtually all were filled in maximally loaded fibers. These data indicate that the Ca oxalate capacity of each fiber varies with the number and volume of elements in which Ca oxalate crystals can form at a given oxalate concentration, and that individual regions of the sarcoplasmic reticulum within each sarcomere differ in their ability to support Ca oxalate precipitation. Our working hypothesis is that this range in ability to form Ca oxalate crystals involves differences in ability to accumulate and retain ionized Ca inside the sarcoplasmic reticulum.

Crayfish Muscle: Permeability to Sodium Induced by Calcium Depletion

Membrane of crayfish muscle fibers becomes selectively permeable to sodium when the calcium concentration of the bathing medium is reduced. Removal of calcium or its reduction below 1 or 2 millimole per liter causes large transient depolarizations up to 70 millivolts in amplitude. They resemble pro longed action potentials and occur only in the presence of sodium. The responses are abolished when tris(hydroxymethyl)aminomethane or lithium is substituted for sodium, and are blocked by tetrodotoxin even in the presence of sodium.

Regulation of excitation-secretion coupling by thyrotropin-releasing hormone (TRH): Evidence for TRH receptor-ion channel coupling in cultured pituitary cells

SummaryThe electrophysiological and secretory properties of a well-studied clonal line of rat anterior pituitary cells (GH3) have been compared with a new line of morphologically distinct cells derived from it (XG-10). The properties of the latter cells differ from the parent cells in that they do not have receptors for thyrotropin-releasing hormone and their basal rate of secretion is substantially higher (ca. three- to fivefold). While both cell types generate Ca++ spikes, the duration of the spike in XG-10 cells (ca. 500 msec) is about 2 orders of magnitude longer than that in GH3 cells (5–10 msec). The current-voltage characteristics of the two cell types are markedly different; the conductance of GH3 cells is at least 20-fold higher than XG-10 cells when cells are depolarized to more positive potentials than the threshold for Ca++ spikes (∼−35 mV). While treatment of GH3 cells with the secretagogues tetraethylammonium chloride or thyrotropin-releasing hormone decreases the conductance in this voltage region to approximately the same as that for XG-10 cells, the electrophysiological and secretory properties of XG-10 cells are unaffected by treatment with either of these agents. Results of this comparative study suggest that XG-10 cells lack tetraethylammonium-sensitive K+ channels. The parallel loss of thyrotropin-releasing hormone receptor binding activity and of a K+ channel in XG-10 cells implies that the thyrotropin-releasing hormone receptor may be coupled with, or be an integral part of, this channel. Apparently thyrotropin-releasing hormone, like tetraethylammonium chloride, acts by inhibiting K+ channels resulting in a prolongation of the action potential, promoting Ca++ influx and subsequently enhancing hormone secretion.

The augmentation of postsynaptic potentials in crustacean muscle fibers by cesium. A presynaptic mechanism

Abstract 1. 1. Substitution of Cs for K in Homarus saline markedly augments both excitatory and inhibitory postsynaptic potentials of lobster muscle fibers. 2. 2. This augmentation of the postsynaptic potentials (p.s.p.s) by Cs requires an induction period of 2 hr and the absence of external K. Presence of K or low Ca concentrations abolish the Cs effect. After the Cs-induced augmentation of the p.s.p.s reaches its maximum, removal of the Cs from the saline produces little change from the augmented values. 3. 3. The Cs effect is found in a number of neuromuscular junctions of lobster, and also in other crustacea. 4. 4. The Cs effect is not due to changes in the following properties of the postsynaptic cell: membrane potential, membrane resistance, reversal potential of the p.s.p.s, or sensitivity of the membrane of the postsynaptic components to the synapse activator agents. 5. 5. Thus, Cs appears to act on the terminals of both excitatory and inhibitory axons, so that a single stimulus will evoke the release of greater amounts of transmitter than under normal conditions.

Sites of action of D2O in intact and skinned crayfish muscle fibers

SummaryThe effect on tension development of replacing 90% of the H2O of the bathing saline with D2O was studied on intact single fibers, and on skinned fibers before and after the latter were treated so as to eliminate Ca-accumulation by the sarcoplasmic reticulum (SR). Excitation-contraction coupling (ECC) of intact fibers is not abolished, but is depressed by D2O so that higher depolarizations are required to elicit a given tension. The reduction in tension at a given level of depolarization is not due to inhibition of the contractile system. The latter showed an enhanced Ca sensitivity; that is, skinned fibers respond to Ca concentrations that are 1–2 orders of magnitude smaller in D2O than in H2O saline. When bathed in D2O saline, intact fibers or skinned fibers with functional SR can still accumulate and release Ca in sufficient quantities to allow repeated induction of maximum tensions. Relaxation is slowed in all three types of preparation, perhaps because of an increased affinity of troponin to Ca in D2O salines.

Localization of ionic conductances in crayfish muscle fibers.

SummaryAnalysis of the changes in membrane potential and conductance of isolated crayfish muscle fibers caused by rapid solution changes leads to the following conclusions. First, the extensive invagination system of this fiber presents a barrier for diffusion between bath and sarcolemma that accounts for the time lag of electrical responses to changes in bath chloride concentration. Morphological data regarding these invaginations were used in a model which simulated the fiber response on an analog computer. Second, the potassium conductance is effectively localized on the sarcolemma in direct contact with the bath (superficial sarcolemma), whereas the chloride conductance is restricted to the invaginations. This distribution of conductances is the reverse of that found in frog muscle.

Potassium redistribution and water movement in crayfish muscle fibers

Summary1.After subjecting the isolated crayfish muscle fiber to a variety of external ionic conditions, the intracellular potassium concentration was measured with an ultramicro integrative flame photometer.2.The quantity of fiber water was determined by correcting the weight of the fiber for the solid component and the adhering and extracellular water. In normal control Ringers solution the ratio of fiber water to cell weight is 0.79.3.The intracellular potassium concentration of a fiber bathed in normal control Ringer was determined as 130±10 mM/kg-H20. With propionate substituted for chloride in the control solution, the final intracellular potassium concentration was 128 ±13 mM/kg-H2O.4.The muscle fiber was subjected to media made hyperosmotic by the addition of K salts. When the anion was permeant (chloride) the redistribution of the intracellular potassium conformed to a Donnan system. With the impermeant ion propionate, the fiber behaved as an osmometer.5.When the media were isosmotically changed with the addition of K salts, the fiber potassium did not conform to a Donnan redistribution with chloride, nor as an osmometer with propionate.6.When the fiber was suddenly exposed to a propionate control solution after equilibration in chloride, the transient time course of intracellular potassium indicated a predominant water movement. This water movement was probably by electroosmosis.