Sodium/calcium exchange in tonic skeletal muscle fibers of the frog.

The Na/Ca exchange system was investigated by contractile response to alteration of the extracellular sodium concentration in tonic skeletal muscle fibers of the frog. Contractures were evoked when the extracellular sodium was reduced or withdrawn in the normal solution. This effect was not associated with membrane depolarizations. In the presence of d-tubocurarine, the amplitude and time course of sodium withdrawal contractures were modified, except when sodium was replaced by TEA. When external calcium was omitted from the solution, the tension of sodium withdrawal contracture was greatly reduced. This effect was reversible. These results suggest that Na/Ca exchange is present in the membrane of tonic skeletal muscle fibers of the frog. This conclusion is further supported by the effect of veratridine and strophantidin, which increase the tension of the low-sodium contractures.     

Calcium action potential and prolonged afterhyperpolarization in developing myotubes of a mouse clonal myogenic cell line

Under a high-Ca condition (greater than 5 mM), myotubes of a mouse myogenic cell line MC3T3-A1/M13 generated a long-lasting Ca action potential and a prolonged afterhyperpolarization (a.h.p.) during their in vitro development. The action potential was sensitive to Co or verapamil. Under a voltage-clamp condition, membrane depolarization more positive than -20 mV evoked a Ca-dependent inward current, which was apparently prolonged and responsible for the generation of the long-lasting action potential. The appearance of the Ca action potential preceded that of a Na spike by about 24 h, and it developed so that the maximum rate of rise became 26 +/- 4 V/s by day 7. Then this Ca-dependent potential faded as the myotubes matured, until the action potentials became solely Na-dependent. The a.h.p. was evoked accompanying the Ca action potential and was inhibited by quinine or quinidine, showing that it is operated by Ca-activated K channels. This channel developed together with the Ca channel and continued to exist during the myotube maturation process. These results indicate that the MC3T3-A1/M13 myotube at the initial stage of development has a highly developed Ca spike system that is due mostly to a high-threshold-type Ca channel.     

Sodium and calcium components of action potentials in Aplysia giant neurone

1. Action potentials resulting from direct stimulation can be recorded from the soma of the Aplysia giant neurone (located in the visceral ganglion) in sodium-free and in calcium-free external solutions. The neurones were impaled by internal micro-electrodes throughout the change of external solutions.2. Complete replacement of either sodium or calcium in the bathing medium with Tris results in only a partial reduction of spike overshoot. Simultaneous replacement of both sodium and calcium reversibly and quickly abolishes the spike.3. The sodium component of the spike in a calcium-free medium is blocked by tetrodotoxin; the drug has no effect on the calcium-dependent spike in sodium-free medium. Externally applied cobalt chloride blocks only the calcium-dependent component.4. In calcium-free media, the overshoot value varies with sodium concentration in the manner predicted for a sodium electrode. In sodium-free media, the membrane behaves like a calcium electrode.5. These results suggest that, during the normal action potential, both sodium and calcium act as carriers of the inward-directed current.     

Developmental changes of membrane electrical properties in a rat skeletal muscle cell line.

1. The developmental changes of the cell membrane electrical properties were studied with micro-electrodes in a rat skeletal muscle cell line. 2. The resting potentials in myoblasts were minus 71 plus or minus 3 mV (mean plus or minus S.D.) and those in myotubes which were formed by fusion of myoblasts were minus 69 plus or minus 3 mV. There was no developmental change in the resting potential during the period examined. 3. The ionic mechanism for the resting potential was the same in myoblasts and myotubes. The resting membrane was almost exclusively permeable to K ions, while permeability to Na ions was not detectable. There was a small permeability to Cl ions. 4. The specific membrane resistance and capacitance were 8 k omega. cm-2 and 1 muF/cm-2 for myoblasts and 12 k omege. cm-2 and 5 muF/cm-2 for myotubes, respectively. 5. Action potentials in myoblasts were evoked by anode break stimulation. They were small and did not overshoot zero membrane potential. The action potentials in myotubes were larger, and had an average overshoot of 32 plus or minus 7 mV and a maximum rate of rise of 93 plus or minus 28 V/sec. 6. The current-voltage relation was examined. Delayed rectification was found in myotubes but not in myoblasts.     

Na and Ca components of action potential in amphioxus muscle cells

1. The ionic mechanism of the action potential produced in lamella-like muscle cells of amphioxus, Branchiostoma californiense, was investigated with intracellular recording and polarization techniques.2. The resting potential and action potential overshoot in normal saline are -53+/-5 mV (S.D.) and +29+/-10 mV (S.D.) respectively.3. The action potential is eliminated by tetrodotoxin (3 muM) and by replacing NaCl in the saline with Tris-chloride but maintained by replacing Na with Li.4. After elimination of the normal action potential by tetrodotoxin or replacing Na with Tris, the addition of procaine (7.3 mM) to the external saline makes the membrane capable of producing a regenerative potential change.5. The peak potential of the regenerative response depends on external Ca concentration in a manner predicted by the Nernst equation with Ca concentrations close to normal.6. The Ca dependent response is reversibly suppressed by Co or La ions.7. Similar regenerative responses are obtained when Ca is substituted with Sr or Ba.8. It is concluded that two independent mechanisms of ionic permeability increase occur in the membrane of amphioxus muscle cell, one to Na and the other to Ca.     

Mechanism of insulin action on resting membrane potential of frog skeletal muscle.

At a concentration that stimulates the Na pump, insulin hyperpolarizes the plasma membrane of frog sartorius in the presence of substrate-free Ringer. The hyperpolarization ranged from 3.5 to 7.3 mV and averaged 4.7 mV. Ouabain, 10(-4) M, completely blocked the effect of insulin on the membrane potential. Moreover, ouabain completely reversed the insulin-induced hyperpolarization within 20 min. The hyperpolarization produced by insulin was not associated with a detectable increase in the ratio of K+ permeability to Na+ permeability nor with a detectable increase in the concentration of intracellular K+, although a depletion of K+ near the external surface of the membrane cannot be excluded. The results clearly indicate that the hyperpolarization is secondary to stimulation of the Na pump by insulin.     

Sodium action potentials induced by calcium chelation in rat uterine smooth muscle

Muscular strips of rat myometrium were exposed to Ca-free solutions in the presence of low concentrations of EGTA (0.1–1 mM). A study of electrical activity was carried out using intracellular recording techniques and the double sucrose-gap apparatus. The results were as followed:

1. After 5–15 min the resting membrane potential was depolarized to about ?35 mV und a rhythmic activity appeared. The potentials had no overshoot and their duration was noticeably prolonged showing a sustained plateau close to zero potential. Rhythmic activity was observed in a definite membrane potential range. The effects were essentially reversed upon Ca-replacement.
2. The maximal amplitude of the potentials was strongly dependent on the external Na-concentration. At low Na-concentrations (13 mM), the membrane repolarized to its normal resting value (about ?50 mV).
3. The potentials were unaffected by tetrodotoxin (2μM). They were inhibited by Mn (0.1 mM) and D600 (0.1–1 μM) and by increasing Mg concentration (1.24 mM) or Ca concentration (10^?3 mM) after 1 h in the presence of EGTA. Higher concentrations of Mn (1–2.5 mM) also repolarized the membrane to its normal resting potential. Tetraethylammonium ions (20 mM) increased both amplitude and duration of the responses but did not blocked the repolarization.
4. Inward Na currents characterized by a slow inactivation were recorded on voltage-clamped preparations.
5. Inhibition of Na-pumping with K-free solution or ouabain (0.1–1 mM) blocked the rhythmic activity and maintained the membrane potential at a steady-state depolarized value. Action potentials reappeared by application of inward current pulses which repolarized the membrane to about ?45 mV.

These results suggest that Ca-chelation induces membrane permeability changes resulting essentially in: 1) an increase in resting Na-conductance, 2) a prolonged Na-inward current entering through channels normally used by Ca ions, and 3) a reduction in K-conductance. However, active Na pumping does indeed participate to the generation of rhythmic activity.     

Transients in intracellular free calcium in subconfluent and confluent cultures of a rat smooth muscle cell line.

The Ca2+ mobilizing mechanisms in the smooth muscle cell line A7r5 were found to undergo changes related to the degree of confluence of the cultures. In sparse cultures resting calcium was stable and exposure to arginine vasopressin (AVP) resulted in a single transient increase in intracellular free calcium (Ca2+i). In confluent cultures the cells could be divided into two general groups, those with a stable resting Ca2+i and those which demonstrated spontaneous brief elevations in Ca2+i of variable frequency. Application of AVP elevated Ca2+i, induced oscillations in quiescent confluent cells, increased the frequency of oscillatory activity in cells which were already active and, in cells which exhibited high frequency spontaneous fluctuations, inhibited this activity. Isotonic K+ depolarizing solution and normal solutions containing Co2+ inhibited Ca2+ spikes. These data suggest that the mechanism underlying the transients involves cyclical electrical phenomena at the cell membrane possibly utilizing calcium channels. There is no indication that the mechanism involves cytoplasmic oscillators.     

Expression of ion channels during differentiation of a human skeletal muscle cell line

An immortal, cloned cell line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4--40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins α-actinin, α-sarcomeric actin, myosin and titin were present in early stages. Receptors for γ toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9--1.0 pmol mg−1 protein) during stage 1 (0--6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 10 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP3 receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors This revised version was published online in July 2006 with corrections to the Cover Date.     

Ultrastructural localization of calcium in normal and abnormal skeletal muscle.

Calcium was demonstrated ultrastructurally as a fine black reaction product with unbuffered 2% saturated potassium pyroantimonate, pH 9.4. In comparison with normal muscle, there was increased precipitate in degenerating skeletal muscle fibers and some degenerating-regenerating fibers occurring in pathologic human muscle, regardless of disease entity, and in experimentally injured rat muscle. The pathologically increased calcium was mainly within the sarcoplasmic reticulum and mitochondria. Both structures could be completely blackened. Nuclear calcium was also increased, the precipitates being localized as circular profiles within the nucleoli and heterochromatin as well as being associated with the nuclear envelope. Myofibrillar calcium was only modestly increased. When normal rat muscle was preincubated in 136 mM calcium-enhanced Hanks' medium, calcium accumulated in the muscle fibers--it was especially heavy in the mitochondria and sarcoplasmic reticulum and appeared identical with the pathologic human and rat muscle fibers. Preincubation of normal rat muscle in 0.1 M acetate buffer (pH 4.65) before calcium loading augmented myofibrillar staining, mainly in the H-zone of the A-bands excluding the M-zone and in broad irregular N1, N2, and "N3" lines of the I-bands. EMMA-4 electron probe microanalysis and EGTA (ethylene glycolbis (beta-aminoethyl ether)N,N'-tetraacetic acid) chelation prior to staining confirmed that the precipitate in the several loci was calcium antimonate. It is proposed that in skeletal muscle fibers injured by various pathologic processes, a breach of the plasmalemma barrier to calcium occurs as a very early abnormality. Extracellular calcium would then pour into the aqueous sarcoplasm of the muscle fiber, from which it would be withdrawn by and accumulated with the still active organelles normally having a great avidity for uptake of this ion, especially the mitochondria and sarcoplasmic reticulum. The resultant organellar calcification would impair function and damage the structure of proteins and phospholipids.     

The adaptive potential of skeletal muscle fibers.

Mammalian skeletal muscle fibers display a great adaptive potential. This potential results from the ability of muscle fibers to adjust their molecular, functional, and metabolic properties in response to altered functional demands, such as changes in neuromuscular activity or mechanical loading. Adaptive changes in the expression of myofibrillar and other protein isoforms result in fiber type transitions. These transitions occur in a sequential order and encompass a spectrum of pure and hybrid fibers. Depending on the quality, intensity, and duration of the alterations in functional demand, muscle fibers may undergo functional transitions in the direction of slow or fast, as well as metabolic transitions in the direction of aerobic-oxidative or glycolytic. The maximum range of possible transitions in either direction depends on the fiber phenotype and is determined by its initial location in the fiber spectrum.     

Comparative phenotypes in rhabdomyosarcomas and developing skeletal muscle

The morphological and immunohistochemical phenotypes of 51 rhabdomyosarcomas from young people have been described and contrasted with phenotypes in developing skeletal muscle from 20 fetuses and neonates. The tumours express markers in a cumulative and consistent sequence--vimentin, desmin, fast myosin, myoglobin--which evolves pari passu with morphological differentiation and follows the same pattern found in normal myogenesis. Changes in immunohistochemical phenotype are documented in residual and recurrent tumours excised after chemotherapy. The presumptive rhabdomyoblastic nature of some primitive tumours, marking with vimentin alone, is discussed.     

Cation pumps in skeletal muscle: potential role in muscle fatigue.

Two membrane bound pumps in skeletal muscle, the sarcolemma Na⁺-K⁺ adenosine triphosphatase (ATPase) and the sarcoplasmic reticulum Ca²⁺-ATPase, provide for the maintenance of transmembrane ionic gradients necessary for excitation and activation of the myofibrillar apparatus. The rate at which the pumps are capable of establishing ionic homeostasis depends on the maximal activity of the enzyme and the potential of the metabolic pathways for supplying adenosine triphosphate (ATP). The activity of the Ca²⁺-ATPase appears to be expressed in a fibre type specific manner with both the amount of the enzyme and the isoform type related to the speed of contraction. In contrast, only minimal differences exist between slow-twitch and fast-twitch fibres in Na⁺-K⁺ ATPase activity. Evidence is accumulating that both active transport of Na⁺ and K⁺ across the sarcolemma and Ca²⁺-uptake by the sarcoplasmic reticulum may be impaired in vivo in a task specific manner resulting in loss of contractile function. In contrast to the Ca²⁺-ATPase, the Na⁺-K⁺ ATPase can be rapidly upregulated soon after the onset of a sustained pattern of activity. Similar programmes of activity result in a downregulation of Ca²⁺-ATPase but at a much later time point. The manner in which the metabolic pathways reorganize following chronic activity to meet the changes in ATP demand by the cation pumps and the degree to which these adaptations are compartmentalized is uncertain.