Masaakira Kano

Acetylcholine receptors of human skeletal muscle: a species difference detected by snake neurotoxins

The binding abilities of the nicotinic acetylcholine receptors (AChRs) of the skeletal muscles of man and other vertebrates to two typical curaremimetic toxins, erabutoxin b (Eb) and alpha-bungarotoxin (alpha-BT), were investigated. Fluorescent microscopy using rhodamine-labeled erabutoxin b (TMR-Eb) and FITC-labeled alpha-bungarotoxin (FITC-alpha-BT) revealed that AChRs of human and chimpanzee muscles were stained with FITC-alpha-BT, but not with TMR-Eb. In contrast, the AChRs of mouse muscle were stained with both fluorescent toxins. The stainings of human and chimpanzee AChRs with FITC-alpha-BT were inhibited by preincubation with unmodified alpha-BT, but not with either unmodified Eb or other short-chain neurotoxins. Binding experiments using 125I-labeled Eb ([125I]Eb) and 125I-labeled alpha-BT ([125I]alpha-BT) showed that the affinity of human AChRs for [125I]Eb was unusually low. Electrophysiological experiments showed that both acetylcholine potential and end-plate potential of human muscle were blocked by addition of alpha-BT, but not by Eb. On the contrary, acetylcholine potential of rat muscle was blocked by addition of Eb. All these results indicate that AChRs of human and chimpanzee muscles are different from those of other animals in having an exceptionally low affinity for Eb and other short-chain neurotoxins. The results suggest a heterogeneity among vertebrate AChRs concerning their reactivities to curaremimetic toxins.

Two components of calcium channel current in embryonic chick skeletal muscle cells developing in culture

The properties of the Ca channel currents in chick skeletal muscle cells (myoballs) in culture were studied using a suction pipette technique which allows internal perfusion and voltage clamp. The Ca channel currents as carried by Ba ions were recorded, after suppression of currents through ordinary Na, K and Cl channels by absence of Na, K and Cl ions, by external TEA, by internal EGTA and by observing the Ba currents instead of the Ca currents. Two components of Ba current could be distinguished. One was present only if the myoballs were held at relatively negative holding potentials below -50 mV. This component first became detectable at clamp potentials of about -50 mV and reached a maximum between -10 and -20 mV. During long clamp steps, it became inactivated completely. The inactivation process of this component at a clamp potential of -30 mV was well fitted to a single exponential with a time constant of about -20 ms. Half-maximal steady-state inactivation was observed at -63 mV. The other component persisted even at relatively positive holding potentials above -40 mV, was observed during clamp pulses to -20 mV and above, and reached a maximum between +10 and +20 mV. This component inactivated very little; a substantial fraction of this component remained at the end of clamp pulses lasting 1 s. The inactivation process of this component at a clamp potential of -10 mV apparently followed a single exponential with a time constant of about 1 s. Half-maximal steady-state inactivation was attained at -33 mV. Both components of Ba current were blocked by Co ions, but organic Ca channel blocker D600 preferentially blocked the high-threshold, slowly inactivating component. The relationship between the current amplitude and the concentration of the external Ba ions was different between the two components. Furthermore, the two components of Ba current also differed in their developmental profile. These findings demonstrate the existence of two distinct types of Ca channels in the early stages of chick muscle cell development.

Calcium channel components of action potential in chick skeletal muscle cells developing in culture

The action potential was recorded from cultured chick skeletal muscle cells in Na-, Ca-, Cl-free saline containing Ba and tetraethylammonium ions (Ba saline). The action potential consisted of two components: a low-threshold, fast inactivating component and a high-threshold, long-lasting component. Both components of the action potential were dependent on external Ba ions and eliminated by Co ions. It is concluded that both components are generated by inward currents carried by Ba ions through Ca channels. The two Ca channel components of the action potential differed with regard to activation and inactivation potential, presence or absence of fast inactivation, sensitivity to an organic Ca channel blocker, and developmental profile. In addition, the failure of occurrence of one or the other components was observed in some cells. These results could be explained by assuming that two components of the action potential in Ba saline were mediated by the two different Ca channels. Furthermore, there was a tendency for younger cells to have more prominent Ca channel components. This may suggest that Ca channels have some function in the early stages of myogenesis.

Inhibition by α-amanitin of development of tetrodotoxin-sensitive spike induced by brain extract in cultured chick skeletal muscle cells

Abstract The enhancing effect of brain extract on the development of tetrodotoxin-sensitive sodium channels in cultured chick skeletal muscle cells is blocked by treatment of culture with α-amanitin, an RNA polymerase inhibitor. The result is interpreted as indicating that the trophic substance in the brain extract exerts its effect on the development of sodium channels through regulation of gene transcription.

Chronic treatment with D600 enhances development of sodium channels in cultured chick skeletal muscle cells

We have studied the long-term effects of D600, a blocker of L-type voltage-dependent Ca channels (VDCC), on the development of voltage-dependent Na channels (VDNC) that are sensitive to tetrodotoxin (TTX), by electrophysiological measurements of the maximum rate of rise of the TTX-sensitive Na spike in cultured chick skeletal muscle cells. Chronic treatment with D600 (2-20 microM) caused a dose-dependent increase in the density of VDNC. The density of VDNC was increased by 150-250% when D600 was added to the cultures at 20 microM from the second day of culture onward. Co-treatment with an inhibitor of the transcription of RNA from DNA, alpha-amanitin, or with cycloheximide, an inhibitor of protein synthesis, prevented the up-regulation by D600. Nifedipine, a different type of blocker of L-type VDCC, was also effective in increasing the density of VDNC, and BAY K 8644, an agonist of L-type VDCC, had the opposite effect. It is suggested that the effect of D600 was mediated via a mechanism specific for L-type VDCC that involves regulation of cytosolic levels of Ca2+ and protein synthesis de novo.

Pharmacological properties of two types of calcium channel in embryonic chick skeletal muscle cells in culture

We have studied the effects of organic and inorganic Ca channel blockers on the two-component action potentials (low- and high-threshold components) in cultured chick skeletal muscle cells, which are generated by the T- and L-type voltage-dependent Ca channels (VDCCs), respectively. Nifedipine and D600 effectively blocked the high-threshold component of the action potential, whereas omega-conotoxin and phenytoin had no effect on this component. By contrast, the low-threshold component was insensitive to all of these organic Ca channel blockers. Blocking effects of polyvalent cations were observed with the following rank order of relative potency (mean apparent dissociation constant in microM): La3+ (14.7) greater than Ni2+ (20.7) greater than Cd2+ (51.2) greater than Co2+ (912) for the low-threshold component, and Cd2+ (0.7) greater than La3+ (29.2) greater than Co2+ (431) greater than Ni2+ (1241) for the high-threshold component. Taken together, these findings suggest that the pharmacological properties of the T- and L-type VDCCs in cultured chick skeletal muscle cells may differ from those in other preparations.

Brain extract induces the tetrodotoxin-sensitive action potentials in a rat skeletal muscle cell line (L6)

Tetrodotoxin (TTX) usually blocks the action potential in adult vertebrate innervated skeletal muscle, i.e. the action potential is operated by a TTX-sensitive sodium channel. The L6 cultured myotube, however, has TTX-resistant action potential that is usually found in skeletal muscle following denervation or during fetal and neonatal stages in rat. The TTX-resistivity of the L6 myotubes might be due to a lack of a neurotrophic substance. The present study revealed that brain extract had a trophic substance that is capable of inducing the TTX-sensitive sodium channels in uninnervated cultured L6 myotubes. Seventeen to 18 days old cultures were used and the maximum rate of rise of action potential was measured as an index of the sodium channel density. In control cultures, the maximum rate of rise was about 120 V/s and did not change by TTX application. However, in cultures with the brain extract the value was 142 V/s and reduced to 86 V/s by TTX. The reduced maximum rate of rise by TTX in the latter cultures means that exposure of L6 myotubes to brain extract resulted in an increase in TTX-sensitive sodium channels and a decrease in TTX-resistant sodium channels. Therefore, exposure to a factor in brain extract can elicit development of TTX-sensitive sodium channels in isolated L6 muscle cells that have been grown as a cell line in the absence of neurons for over 10 years.

Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker

The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20 microM D600 or 10 microM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca2+ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.

Development and maintenance of tetrodotoxin-sensitive action potential in cultured skeletal muscle cells from dystrophic and normal chickens

Abstract The maximum rate of rise of the tetrodotoxin (TTX)-sensitive spike was measured in 2-, 3-, and 4-week-old cultured myotubes from embryos of normal and dystrophic chickens. Similar increases in the maximum rates of rise of spikes were observed in normal and dystrophic cultures after 2 weeks of incubation with brain or embryo extract. Thereafter, these values were maintained in normal cultures, but decreased in dystrophic cultures. At both 3 and 4 weeks of age the values from dystrophic cultures were about 80% of those from normal cultures. These findings indicate that dystrophic chicken myotubes develop similarly to normal myotubes with respect to the TTX-sensitive spike-generating capability for the first 2 weeks in culture, and then diminish their capability which was once acquired. This reduced capability of cultured dystrophic myotubes might be indicative of a defect in the muscle cell membrane in muscular dystrophy. Furthermore, neither brain nor embryo extract from the dystrophic line was defective in supporting the development and maintenance of the TTX-sensitive spike-generating capability of cultured myotubes. This leads us to speculate that a myogenic rather than a neurotrophic phenomenon is responsible for our results.

[3H]-Nitrendipine Binding in Chick Myotubes Developing in Culture

Binding studies with the [3H]-nitrendipine were performed in homogenates of chick skeletal myotubes developing in culture. The specific binding data suggested two classes of binding sites with high and low affinities for nitrendipine. No significant changes in the dissociation constants of both high- and low-affinity sites could be detected over the development period studied. The estimated dissociation constant was 0.44 nM for the high-affinity binding site and 25.6 nM for the low-affinity binding site. Both binding sites appeared at an early myotubular stage of development (around 3 days in culture), and displayed rapid increases with age until peaks were reached around 6 days in culture, then began to decrease. These results were discussed taking into account voltage-dependent Ca channels.