Different actions of calcium channel blocking agents on resting membrane conductance in developing skeletal muscle

The effects of Co2+, Mn2+, and La3+ (2 mM) and verapamil (5 x 10(-6) M) on membrane conductance (Gm) and resting potential (Em) were studied in chick skeletal muscle fibres developing in culture. Cobalt and manganese had no effect on Gm at any time during myogenesis but verapamil caused a decrease in Gm in immature myotubes. This effect diminished with time and was absent by 3 days after myoblast fusion. Lanthanum caused an increase in Gm at all stages of development. All the agents studied caused a significant depolarization of Em. It is concluded that there is no resting calcium conductance in developing skeletal muscle but that there may be a resting sodium conductance which declines with maturation. Lanthanum may increase Gm by displacing membrane-bound calcium and destabilizing membrane structure. All the agents studied were thought to induce depolarization by an inhibitory action on (Na+ + K+)-ATPase.     

Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.     

Activation of ATP-dependent K+ currents in intact skeletal muscle fibres by reduced intracellular pH.

We have used three-microelectrode voltage clamp in conjunction with the ammonium prepulse method to investigate the effects of lowered intracellular pH (pHi) on resting potassium currents of frog skeletal muscle fibres. Potassium currents were recorded in 40 mM K+, Cl(-)-free solution in response either to voltage steps or ramps. An ammonium prepulse (2 h) reduced pHi to 6.45 from a control value of 7.19. The intracellular ATP concentration, measured with high-pressure liquid chromatography (HPLC), was unchanged by this procedure. Mean outward potassium currents were larger in low pHi than in control fibres, being about twice as large at +40 mV, whereas mean inward currents were very similar in control and low-pHi fibres. The sulphonylurea glibenclamide blocked single KATP channels in excised patches with a Kd of 3 microM. In intact fibres 50 microM glibenclamide had no effect on K+ currents in controls but reduced currents in low-pHi fibres. In the presence of glibenclamide, K+ currents in low-pHi fibres were not significantly different from those in control fibres. We suggest that reduced pHi in intact skeletal muscle fibres opens ATP-dependent potassium channels (KATP channels), as has been shown to occur in excised patches of membrane.     

Ba2+ ions block K+-induced contractures by antagonizing K+-induced membrane depolarization in frog skeletal muscle fibres

The effects of Ba2+ ions on twitches, K+-induced contractures, and on intracellularly recorded membrane potentials (Em) and depolarizations of frog skeletal muscle fibres were investigated. Exposure of toe muscles to choline--Ringer's solution with 10(-3) M Ba2+ with Ca2+ (1.08 mM) eliminated or very greatly reduced contractures produced by 60 mM K+. In contrast, not only did the same concentration of Ba2+ ions fail to depress the twitch tension of isolated semitendinosus fibres when added to Ringer's with Ca2+, but it even restored twitches that had been eliminated in a zero Ca2+ Ringer's solution. The resting Em of sartorius muscle fibres in choline--Ringer's solution was reduced about 20 mV by 10(-3) M Ba2+. This Ba2+ ion concentration also antagonized the K+-induced depolarization. Thus in the presence of 1 mM Ba2+, 20 mM K+ hyperpolarized rather than depolarized the fibres and 60 or 123 mM K+ produced only very slowly developing, small depolarizations. These results suggest that the loss of the K+-induced contracture in choline-Ringer's caused by Ba2+ ions is due to an inhibition of the K+-induced depolarization. The latter result is consistent with previous findings of other workers that Ba2+ ions block membrane K+ channels.     

Glutamate regulation of non-quantal release of acetylcholine in the rat neuromuscular junction

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non-quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non-quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H-effect) following blockade of skeletal muscle post-synaptic nicotinic receptors by (+)-tubocurarine and cholinesterase by armin (diethoxy-p-nitrophenyl phosphate). Glutamate was shown to inhibit non-quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate-induced decrease of the H-effect was enhanced by glycine. Glycine alone also lowered the H-effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition of N-methyl-d-aspartate (NMDA) receptors with (+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine (MK801), dl-2-amino-5-phosphopentanoic acid (AP5) and 7-chlorokynurenic acid or the elimination of Ca2+ from the bathing solution prevented the glutamate-induced decrease of the H-effect with or without glycine. Inhibition of muscle nitric oxide synthase by NG-nitro-l-arginine methyl ester (l-NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H-effect. The results suggest that glutamate, acting on post-synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non-quantal secretion of ACh from motor nerve terminals. Non-quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non-quantal transmitter following muscle denervation triggers the early post-denervation depolarization of muscle fibres.     

Studies on the origin of resting tension of skeletal muscle

Glycerol extracted frog skeletal muscle fibres at 2.2 μm sarcomere length (in situ-length) in a solution free of Ca⁺⁺ and Mg⁺⁺ but containing ATP, show a decrease in both their resting tension and their elastic modulus, if the ionic strength of the bathing solution is increased. This finding is compared with the behaviour of intact skeletal muscle fibres in hypertonic solution. It is concluded that the resting tension of intact skeletal muscle fibres at in situ-length is caused by the longitudinal sarcoplasmic reticulum as well as by interactions between the contractile filaments.     

Recovery of isolated 'Na-rich' rat muscles in the presence of serum

The presence of serum in the recovery medium facilitates the restoration of intracellular Na and K concentrations and of the electrical properties in isolated ‘Na-Rich’ skeletal muscles (extensor digitorum longus) of K-deficient rats. The resting membrane potential to muscle fibres after recovery in the presence of serum closely agrees with that expected from the ionic theory based on steady state conditions.     

Effect of microwave electromagnetic field on skeletal muscle fibre activity

The aim of the present study was to investigate the influence of microwave irradiation on fatiguing activity of isolated frog skeletal muscle fibres. The changes in the electrical and mechanical activity were used as criteria for the exposure effects. Repetitive suprathreshold stimulation with interstimulus interval of 200 ms for 3 min was applied. Intracellular (ICAP) and extracellular (ECAP) action potentials and twitch contractions (Tw) of muscle fibres after 1 hour microwave exposure (2.45 GHz, 20 mW/cm( 2) power density) were compared with those recorded after one hour sham exposure (control). The duration of uninterrupted activity in the trial (endurance time; ET) was not significantly affected by microwave field exposure. After microwave irradiation, the ICAP amplitude was higher, the rising time was shorter, and the resting membrane potential was more negative compared to controls. There was a slower rate of parameters changes during ET in potentials obtained from irradiated fibres. Microwave exposure increased the propagation velocity of excitation, the ECAP and Tw amplitudes, as well as shortened their time parameters. We concluded that a 2.45 GHz microwave field possesses a stimulating effect on muscle fibre activity, which is in part due to its specific, non-thermal properties. The microwave induced-changes in muscle fibre activity may reduce development of skeletal muscle fatigue.     

Alcoholic muscle disease and biomembrane perturbations (review)

Excessive alcohol ingestion is damaging and gives rise to a number of pathologies that influence nutritional status. Most organs of the body are affected such as the liver and gastrointestinal tract. However, skeletal muscle appears to be particularly susceptible, giving rise to the disease entity alcoholic myopathy. Alcoholic myopathy is far more common than overt liver disease such as cirrhosis or gastrointestinal tract pathologies. Alcohol myopathy is characterised by selective atrophy of Type II (anaerobic, white glycolic) muscle fibres: Type I (aerobic, red oxidative) muscle fibres are relatively protected. Affected patients have marked reductions in muscle mass and impaired muscle strength with subjective symptoms of cramps, myalgia and difficulty in gait. This affects 40-60% of chronic alcoholics (in contrast to cirrhosis, which only affects 15-20% of chronic alcohol misuers).Many, if not all, of these features of alcoholic myopathy can be reproduced in experimental animals, which are used to elucidate the pathological mechanisms responsible for the disease. However, membrane changes within these muscles are difficult to discern even under the normal light and electron microscope. Instead attention has focused on biochemical and other functional studies.In this review, we provide evidence from these models to show that alcohol-induced defects in the membrane occur, including the formation of acetaldehyde protein adducts and increases in sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase (protein and enzyme activity). Concomitant increases in cholesterol hydroperoxides and oxysterol also arise, possibly reflecting free radical-mediated damage to the membrane. Overall, changes within muscle membranes may reflect, contribute to, or initiate the disturbances in muscle function or reductions in muscle mass seen in alcoholic myopathy. Present evidence suggest that the changes in alcoholic muscle disease are not due to dietary deficiencies but rather the direct effect of ethanol or its ensuing metabolites.     

Passive electrical constants of skeletal muscular fibres studied in different muscles of normal and thiamine deficient rats (author's transl)]

10 Some effects of thiamine deficiency were studied in three skeletal rat muscles, having different proportions of "fast" and "slow" fibres: extensor longus digiti IV (a nearly pure fast muscle), soleus (having a predominant population of slow fibres) and diaphragm muscle (mixed fibre population). 20 Cross section area of fibres (fig. 2) is reduced in thiamine deficient animals, mostly for fast fibres having a glycolytic metabolism, the histochemical profile of which tends to become similar to that of slow fibres, in which oxydative metabolism is predominant, as shown by a marked increase in succinodehydrogenase activity. 30 Measurements of resting potential E, of membranes time constant tau and of fibre input resistance R were performed in normal and thiamine deficient muscles (table I). R and tau were obtained from square pulse analysis, using a double shifted sampling method permitting the use of a single microelectrode. E is not greatly affected by thiamine deficiency. tau changes appear not to be significant, except for fast fibres from extensor longus muscle, where tau is slightly reduced. R is increased in thiamine deficient animals (fig. 3). 40 Changes in R and tau do not exactly follow the predictions of cable theory, if one assumes that a purely dimensional factor is involved. Thus, the view that thiamine deficiency does not change basic passive electrical constants of fibres (membrane specific resistance and capacity, myoplasm resistivity) can be considered only as a first approximation. 50 R and tau values obtained in normal muscles are larger than data taken from other studies. The reasons for this discrepancy are discussed. It is suggested that diet differences may play a role.     

Inward rectifier potassium current in the frog embryonic skeletal myocytes

Pharmacological and kinetic properties of the inward rectifier potassium current Iir the frog embryonic skeletal myocytes were found to be identical to those of adult frog skeletal muscle fibres. The data obtained suggest that the Iir plays the main role in maintaining the myocytes resting membrane potential (RMP) when chloride conductance is insignificant. Changes of the integral conductance Gir and the RMP values correlated with the T-system development. The inward rectifier K+ channels, from the early stages of the muscle seem to be located in the T-tubule membranes.     

Pressure dependence of baroreceptor-mediated vasoconstriction in rat skeletal muscle

To determine whether microvessels in resting or contracting skeletal muscle constrict during baroreceptor activation, vascular diameters were measured in the spinotrapezius muscle of adult rats (n = 12) during occlusion of the common carotid arteries. Neural and myogenic components were distinguished using two types of occlusion: 1) "normal" (arterial pressure was allowed to increase with baroreceptor activation) and 2) "isobaric" (arterial pressure was maintained constant by decreasing blood volume). During normal occlusions, intermediate and small arteriolar diameters decreased in resting and contracting muscle (10-15% and 25-30%, respectively). Large arterioles and all-sized venules distended slightly (approximately 5%) in resting muscle, but diameters were maintained or decreased in contracting muscle. When arterial pressure was maintained constant (isobaric), the microvascular responses to baroreceptor activation in both resting and contracting muscle were essentially eliminated. We conclude that nearly all the arteriolar constriction observed in the spinotrapezius muscle during normal carotid artery occlusion is myogenic in origin, secondary to increased arterial pressure. This pressure-dependent constriction is augmented during skeletal muscle contraction and functional vasodilation.     

Intracellular free [Ca2+] in human skeletal muscle with myopathic carnitine deficiency

Carnitine is required for the transport of activated long chain fatty acids through the mitochondrial inner membrane. We measured the intracellular free calcium concentration [( Ca2+]i) by means of a calcium selective microelectrode in skeletal muscle biopsies obtained from nine patients in which myopathic carnitine deficiency (MCD) was diagnosed, and from six subjects with no evidence of neuromuscular disease. Intact intercostal muscle bundles were dissected and then split for electron microscopic studies and electrophysiological measurements. The [Ca2+]i in muscle fibers from MCD patients was 0.46 +/- 0.02 mumol.l-1 (mean +/- SEM) and 0.10 +/- 0.01 mumol.l-1 in control subjects. At the electron microscopic level, the predominant abnormality was the presence of lipid vacuoles between the myofibrils. These results show that in patients with myopathic carnitine deficiency there is a significant increase in the resting myoplasmic calcium concentration which might be related to a malfunction of some mechanisms responsible for the homeostasis of intracellular calcium.     

Purinergic receptors expressed in human skeletal muscle fibres

Purinergic receptors are present in most tissues and thought to be involved in various signalling pathways, including neural signalling, cell metabolism and local regulation of the microcirculation in skeletal muscles. The present study aims to determine the distribution and intracellular content of purinergic receptors in skeletal muscle fibres in patients with type 2 diabetes and age-matched controls. Muscle biopsies from vastus lateralis were obtained from six type 2 diabetic patients and seven age-matched controls. Purinergic receptors were analysed using light and confocal microscopy in immunolabelled transverse sections of muscle biopsies. The receptors P2Y(4), P2Y(11) and likely P2X(1) were present intracellularly or in the plasma membrane of muscle fibres and were thus selected for further detailed morphological analysis. P2X(1) receptors were expressed in intracellular vesicles and sarcolemma. P2Y(4) receptors were present in sarcolemma. P2Y(11) receptors were abundantly and diffusely expressed intracellularly and were more explicitly expressed in type I than in type II fibres, whereas P2X(1) and P2Y(4) showed no fibre-type specificity. Both diabetic patients and healthy controls showed similar distribution of receptors. The current study demonstrates that purinergic receptors are located intracellularly in human skeletal muscle fibres. The similar cellular localization of receptors in healthy and diabetic subjects suggests that diabetes is not associated with an altered distribution of purinergic receptors in skeletal muscle fibres. We speculate that the intracellular localization of purinergic receptors may reflect a role in regulation of muscle metabolism; further studies are nevertheless needed to determine the function of the purinergic system in skeletal muscle cells.     

The effect of tetraethylammonium and the decrease in the extracellular chloride concentration on membrane depolarization and contraction of skeletal muscle fiber induced by a hyperpotassium solution

1.--The tetraethylammonium (TEA) effects on K+ contracture and membrane depolarization are compared in both crab and frog skeletal muscle fibres. 2.--The mechanical tension of the contracture is reduced by the TEA in frog skeletal muscle fibre; it is increased in crab skeletal fibre. 3.--When no mechanical phenomenon is observed in frog skeletal muscle, the amplitude and the velocity of membrane depolarization induced by an increase of outward K+ concentration is reduced by the TEA. These effects are in opposition in crab muscle fibre. 4.--In crab muscle fibre, the results obtained tend to show that the C1-ions are not distributed on each side of the membrane according to Donnan equilibrium.     

Chloride activity and its control in skeletal and cardiac muscle

Ion-selective microelectrodes have been used to compare the mechanisms controlling intracellular Cl- activity in skeletal and cardiac muscle. In frog Sartorius skeletal muscle fibres, Cl- levels are low (about 3 mM) and are determined mainly passively. The effect of any Cl- transport system will be quickly short-circuited through the high membrane Cl- conductance. In contrast, the sheep-heart Purkinje fibre, like other cardiac tissues, contains higher than passive levels of intracellular Cl- (20-30 mM). Many Cl- movements occur, not through Cl- channels (the permeability for Cl- is low), but by a Cl- -HCO3- countertransport system. High internal Cl- levels are achieved by an exchange of extracellular Cl- for intracellular HCO3-, which acidifies the fibre by 0.3 pH. Anion exchange in heart differs from that proposed for other excitable cells in that it is not specialized to compensate for an intracellular acidosis. Instead, it can prevent the fibres from becoming too alkaline by promoting a bicarbonate efflux and a chloride influx whenever internal bicarbonate levels rise. Possible reasons for this are briefly discussed.     

Mitotic signaling by beta-amyloid causes neuronal death.

Aggregates of beta-amyloid peptide (betaAP), the main constituent of amyloid plaques in Alzheimer's brain, kill neurons by a not yet defined mechanism, leading to apoptotic death. Here, we report that both full-length betaAP((1-40)) or ((1-42)) and its active fragment betaAP((25-35)) act as proliferative signals for differentiated cortical neurons, driving them into the cell cycle. The cycle followed some of the steps observed in proliferating cells, including induction of cyclin D1, phosphorylation of retinoblastoma, and induction of cyclin E and A, but did not progress beyond S phase. Inactivation of cyclin-dependent protein kinase-4 or -2 prevented both the entry into S phase and the development of apoptosis in betaAP((25-35))-treated neurons. We conclude that neurons must cross the G1/S transition before succumbing to betaAP signaling, and therefore multiple steps within this pathway may be targets for neuroprotective agents.-Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Giuffrida Stella, A. M., Canonico, P. L., Nicoletti, F., Sortino, M. A. Mitotic signaling by beta-amyloid causes neuronal death.     

Proteases in normal and diseased human skeletal muscle: a preliminary histochemical survey

Summary Seven proteases assumed to be aminopeptidases A, B and M, dipeptidyl peptidases II and IV, esteroproteinase and -glutamyltransferase were localized histochemically, using semipermeable membrane simultaneous coupling techniques, in unfixed cryostat sections of skeletal muscle removed from one healthy volunteer, six patients with disuse muscle atrophy, and 15 patients with some form of muscle disease. Normal muscle fibres showed weak reactions for aminopeptidases A and M and for the dipeptidyl peptidases, but no reactivity for -glutamyltransferase or esteroproteinase. No change was detected in diseased muscle fibres except that low -glutamyltransferase and esteroproteinase activities appeared in some cases. The activities of the seven enzymes were stronger in the intermysial connective tissue than in the muscle fibres, but were also unchanged in disease. The strongest reactions were found in some interstitial cells (mast cells and macrophages) and these were much increased in diseased muscle, particularly for dipeptidyl peptidases II and IV. The findings are interpreted in terms of the release of proteases from such cells and their subsequent involvement in the breakdown of myofibrillar proteins in muscle disease.     

Effects of hyperosmolarity and furosemide on resting membrane potentials and skeletal muscle fiber volume in rats

The changes of the muscle fibres volume and resting membrane potential (RMP) were studied following treatment with hypertonic medium and furosemide. The volume changes in hypertonic medium began with cell shrinkage and later have been followed by the volume increase up to normal level during 30-40 minutes. At the same time the medium hypertonicity caused muscle fibres depolarisation. The hypertonic-induced decrease of the RMP was delayed in the furosemide-treated muscle. Besides, furosemide abolished the muscle fibres volume restorative properties in hypertonic medium. It is suggested that the membrane depolarisation and cell volume restoration in hypertonic medium are the resultant effects of intracellular chloride ions level elevation which, in turn, have been evoked by activation of furosemide-sensitive Cl(-)-influx system.     

Effects of partial denervation on the distribution of acetylcholine receptors and other membrane properties in innervated and paralyzed fibers of rat skeletal muscle

The origin of the membrane changes induced in skeletal muscle by denervation has been investigated by examining partially denervated rat hindlimb muscles rendered inactive for 2-3 days by a chronic conduction block in the sciatic nerve. Extra-junctional sensitivity to acetylcholine and spike resistance to tetrodotoxin developed to the same extent in the denervated and the adjacent innervated but inactive fibres. On the other hand, impulse-blocked fibres of control muscles not containing denervated fibres showed, at this early time, little membrane changes. These results are interpreted as indicating that the response of muscle to denervation is due to the combined action of inactivity and products of nerve degeneration.