Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.     

Biological significance of peptides from Anemonia sulcata

Three polypeptide toxins have been isolated from the sea anemone Anemonia sulcata and characterized: ATX I (mol wt 4702), ATX II (mol wt 4935), and ATX II (mol wt 2678). In different crustacean and amphibian preparations the toxins act primarily on the fast sodium channels, which leads to delayed inactivation of fast sodium permeability and thus increases the duration of the action potential. When applied to crustacean preparations the three toxins are nearly equally effective. However, in a comparison of the biological activities of ATX I and ATX II in myelinated nerves of the frog, ATX I seems to be inactive. It is suggested that cardiotoxicity is the primary cause of death in mammals, ATX II being more toxic than ATX I. At very low concentrations ATX II induces a pronounced positive inotropic effect in different mammalian heart preparations, which is accompanied by a prolongation of the action potential. It is suggested that the positive inotropic effect of ATX II is caused by a delayed inactivation of the fast sodium current, which leads to an increase of the sodium transient and of the pump activity of Na+,K+-ATPase. In contrast to the presynaptic mode of action on crustacean and frog nerve-muscle preparations, ATX II has a direct effect on mammalian skeletal muscle fiber membranes and induces a sodium-dependent increase of twitch responses and duration of the action potential.     

Tetrodotoxin blocks mechanical response in mammalian muscle in the presence of tetrodotoxin-resistant action potentials

The effect of tetrodotoxin (TTX) (10(-5)-10(-6)M) on the mechanical activity and on the action potential of innervated and denervated muscle of the rat was studied. The twitch tension was reduced to 10 % of the control values within 20 min of TTX 10(-6) introduction. This effect was reversible. The mean twitch tension in the presence of 10(-6)M TTX expressed as a percentage of control was 9.3 +/- 2.4 (SEM) for innervated muscle and 10.9 +/- 2.5 for denervated muscle. The dose-effect twitch relation for denervated muscles was not significantly different from that observed in control innervated muscles in the 10(-3)-10(-6) TTX range. Action potentials of innervated muscles could not be elicited in 10(-6)M TTX. In the presence of this (TTX) fibers of chronically denervated muscles consistently responded to stimulation with action potentials which were slower and smaller but still with overshoot, contrasting with fibrillation potentials that had been described to be blocked by TTX.     

The effects of modified ringer's solutions on the membrane potentials of innervated and denervated frog sartorius muscle fibers

Comparison has been made between innervated and chronically denervated frog sartorius muscle fibers for resting potentials and a number of features of the action potential. Muscles were obtained from force-fed frogs maintained at room temperature for periods up to one year, and were studied with intracellular microelectrodes. Denervated muscles increased in sensitivity to acetylcholine by 100–400-fold. Studies were made in normal Ringer's solution, and in media in which concentrations of K⁺, Na⁺, Ca⁺⁺, and Cl? were altered. The only significant differences noted between the denervated and the innervated fibers were a reduction in the maximum rate of fall of the action potential (ca. 20%) and an increase in the fall time of the active membrane potential (ca. 25%). These differences were present in normal Ringer's solution and remained when the bathing medium was modified. The resting membrane potential of denervated and innervated muscles varied with log [K⁺]_o in exactly the same manner, and followed the theoretical relation proposed by Hodgkin (Proc. Roy. Soc., B, 148: 1–37, ′58), with the term representing the ratio of the sodium to potassium permeabilities assigned a value of 0.01. The results suggest that (a) the resting sodium and potassium permeabilities are reduced proportionately after denervation, since it is known that denervated frog muscle has a smaller potassium permeability, and (b) the mechanism controlling the increase in potassium conductance during the action potential is less available after denervation. Data indicate that the system controlling the sodium permeability is capable of activation to the same extent as in innervated muscles. Muslces which had been allowed to reinnervate did not show the differences presented by the denervated muscles. Innervated and denervated muscles did not show any significant changes in maximum rates of rise or fall of the action potential, nor of the active membrane potential amplitude over a 30 mV range of resting membrane potentials, indicating that the sodium and potassium permeability systems are fully available in frog muscle at membrane potentials larger than ?80 mV.     

Mechanical properties of denervated amphibian muscle

Denervated amphibian muscle does not show the prolongation of action potential found in mammalian denervated muscle. It was, therefore, predicted that denervated amphibian muscle would not show prolongation of the mechanical twitch. The sartorius muscles in one leg of toads--Xenopus borealis--were denervated for 140-268 days. Isometric twitch time to peak, time to half relaxation and twitch/tetanus ratio were not changed following denervation, confirming our prediction. Twitch tension decreased to 68% and tetanic tension decreased to 75% of control values. The maximum velocity of unloaded shortening (muscle length/s) was also unchanged.     

Effects of Na-octanoate on potassium contractures in normal and denervated frog tonic fibres

In frog twitch muscle fibres, Na-octanoate (NaC8) shifted the relation between potassium induced tension and membrane potential to the right. The present study has been carried out to investigate the effect of this fatty acid on frog tonic fibres. Potassium contractures measured on bundles of 30-40 fibres of ileofibularis muscles were less decreased by NaC8 (2.5-10 mmol/l) than those of twitch fibre bundles. In denervated muscles the sensitivity to NaC8 was increased, probably due to the development of sodium channels in the membranes. Experiments with mixed fibre bundles also showed a lower influence of NaC8 on potassium contracture of tonic fibres. On the other hand, tonic fibres showed a lower threshold of the potassium induced tension as well as a lower K+ concentration for maximal activation. This lower threshold was further lowered by NaC8, corresponding to a shift of the relation between potassium concentration and tension to the left. The membrane resting potentials were -58 +/- 9 mV in tonic fibres and -83 +/- 5 mV in twitch fibres. Five mmol/l NaC8 only induced depolarization of the membrane of tonic fibres. This depolarization (by about 20 mV) may be responsible for the threshold shift to lower K+ concentration in NaC8-exposed tonic fibres. In addition to the effects of NaC8 on sodium channels, interactions with Ca2+ binding sites are discussed.     

海葵毒素对心肌钠通道开放模式、动作电位及心电图QT间期的影响

应用膜片箝技术记录游离豚鼠心肌细胞钠通道电流, 细胞内微电极技术记录心室乳头肌的动作电位和心电图机记录豚鼠的心电图。使用与心肌细胞钠通道有高度亲和力的海葵毒素(sea anemone toxin, ATXⅡ)改变钠通道开放的动力过程, 从三个水平来研究钠通道、动作电位、心电图变化的关系, 并试图探讨长QT综合征(long QT syndrome, LQTs)的发病机制。结果显示: ATXⅡ使钠通道的开放频率增加, 钠通道中“长时间开放模式”的开放时间常数增大, 动作电位的持续时间APD50和APD90也分别增加了23%和27%。 ATXⅡ使动物心电图QT间期延长18.6%, QTc (校正的QT间期)增大18.9%。这些结果提示, 钠通道动力过程的变化对动作电位和心电图QT间期有重要影响, 钠通道功能或结构的变异可能是临床上部分长QT综合征产生的原因。     

The effect of mepyramine on the development of morphine tolerance and physical dependence in mice.

Latency relaxation (LR) as well as resting tension and twitch tension of frog toe muscles are studied in an isotonic solution (= 1 T) and in solutions made hypotonic by leaving out the appropriate amounts of NaCl and KCl (0.54 T and 0.76 T). In hypotonic solutions there is an increase in peak twitch tension as well as a decrease in the depth of the LR: the resting tension is increased at sarcomere lengths which are greater than 2.8 μm and is decreased at sarcomere lengths which are less than this value. The behaviour of twitch tension is discussed with respect to the influence of the sarcoplasmic ionic strength on the interaction between the contractile filaments. Concerning the decrease in both the LR and the resting tension, it is assumed that these effects are induced osmotically, the tension of the membranes of the longitudinal sarcoplasmic reticulum being the particular parameter which is influenced.     

Spontaneous activity of dopaminergic retinal neurons

Dopaminergic local circuit neurons in the retina (DA cells) show robust, spontaneous, tetrodotoxin-sensitive pacemaking. To investigate the mechanism underlying this behavior, we characterized the sodium current and a subset of the potassium currents in the cells in voltage-clamp experiments. We found that there is a persistent component of the sodium current in DA cells which activates at more depolarized potentials than the transient component of the current. The transient component was completely inactivated at -50 mV, but DA cells remained able to fire spontaneous action potentials when potassium channels were partially blocked and the membrane potential remained above -40 mV. Based on these electrophysiological data, we developed a reduced computer model that reproduced the major features of DA cells. In simulations at the physiological resting potential, the persistent component of the sodium current was both necessary and sufficient to account for spontaneous activity, and the major contribution of the transient component of the sodium current was to initiate the depolarization of the model cell during the interspike interval. When tonic inhibition was simulated by lowering the input impedance of the model cell, the transient component played a larger role.     

Specificity of antibodies to sea anemone toxin III and immunogenicity of the pharmacological site of anemone and scorpion toxins

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion alpha-toxins. The results obtained suggest that a region of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.     

Analysis of caffeine action in single trabeculae of the frog heart

Effects of caffeine on contractile tension and on intracellular action and resting potentials were examined in single frog heart trabeculae suspended in a rapid perfusion chamber. Trabeculae from atria responded more readily than those from ventricles and were therefore studied in greater detail. Both the contracture and twitch responses, the one obtained at high (greater than or equal to 10mM), the other at low (less than or equal to 10mM) caffeine concentrations, consisted of a transient tension rise followed by a maintained phase of lower, but still enhanced, tension. The hypothesis was tested that the transient response is due to the release of calcium from the sarcoplasmic reticulum (s.r.) whereas the maintained tension results from enhanced calcium influx through the cell surface. Support for these ideas was obtained by examining the response to step changes of external calcium and caffeine concentrations, applied in various combinations, simultaneously and in sequence. It also emerged tht the effects on twitch tension of calcium derived from (a) s.r. discharge and (b) influx are additive, to a first approximation. A test procedure for monitoring the s.r. store content was evolved to follow the accumulation of s.r. calcium after a preceding depletion. The results obtained, and others, suggest that the s.r. calcium pump can be operative in atrial heart cells and capable, after store depletion, of reabsorbing up to some 40% of calcium activating a twitch, the remainder being, presumably, extruded from the cells.     

Effects of a neurotoxin isolated from the sea anemone, Anemonia sulcata, at frog neuromuscular junction (author's transl)]

ATX II is a toxin extracted from tentacles of Anemonia sulcata. It was known that this protein displays neurotoxic effects on frog isolated neuromuscular preparation (Fig. 1, 2) and that muscular contractures observed with ATX II are blocked by d-tubocurarine (Fig. 3) or on a 40-days-denervated gastrocnemius (Fig. 4). Part of these experiments has already appeared. 1. These effects of ATX II depend on calcium concentration in the bathing medium, as is the case for transmitter release. The same results were observed when we substituted strontium to calcium. 2. On an intact sciatic sartorius preparation, ATX II does not act on the amplitude of the miniature endplate potentials (mepps, Fig. 6). The muscular action potential is not modified by this toxin. 3. ATX II increases the frequency of the mepps (Fig. 5). The evoked transmitter release (quantal content) after ATX II is also largely increased (Fig. 7). 4. In conclusion, it is suggested that ATX II acts indirectly on the muscle through an increase in acetylcholine release from the motor nerve terminals.     

Changes in isometric contractile properties of extensor digitorum longus and soleus muscles of C57BL/6J mice following denervation

In this study, conducted on mice of the C57BL/6J+/+ strain, we investigated the differential effects of denervation on the isometric contractile properties of the extensor digitorum longus (EDL) and soleus (SOL) muscles. The contractile properties were studied at 1, 28, 84, and 210 days following unilateral section of the sciatic nerve at 12 weeks of age. When isometric tetanus tension was expressed relative to wet weight, the denervated SOL showed an earlier and more pronounced loss in tension generating capacity than the EDL. Both the denervated SOL and EDL showed potentiation of the twitch tension at 28 days postdenervation. The time to peak twitch tension (TTP) and the time to half-relaxation (1/2RT) were prolonged by 28 days postdenervation in both muscles. This trend continued to the oldest age-groups studied in the EDL, but reached an apparent plateau in the SOL at 84 days postdenervation. In response to fatigue, the denervated SOL showed a marked decrease in resistance to fatigue at 1 day but a relatively normal response thereafter, whereas the denervated EDL showed an increase in resistance to fatigue at and beyond the 28-day period. In spite of the fact that the total contraction time of both muscles increased following denervation, the predominantly oxidative SOL remained a slower contracting muscle than the more glycolytic EDL.     

Cellular ions in intact and denervated muscles of the rat

Summary Tissue composition, membrane potentials and cellular activity of potassium, sodium and chloride have been measured in innervated and denervated rat skeletal muscles incubatedin vitro. After denervation for 3 days, tissue water, sodium and chloride were increased but cellular potassium content and measured activity were little affected, despite a decrease of 16 mV in resting membrane potential which would have necessitated a decrease in cellular potassium activity of almost 50% were potassium distributed at electrochemical equilibrium. These findings, therefore, preclude a decreased electrochemical potential gradient for potassium as the cause of the membrane depolarization characteristic of denervated muscle fibers. Analysis of the data excludes an important contribution of rheogenic sodium transport to the resting potential of innervated muscles. These results strongly support the hypothesis that the decreased membrane potential in denervated fibers reflects a relative increase in the membrane permeability to sodium.     

Inhibition of Zn2+-induced potentiation of twitch tension by Ni2+ in frog muscle

Effect of Ni2+ on Zn2+-induced potentiation of twitch tension was studied electrophysiologically in the toe muscle fibers of Rana catesbeiana. The major findings of this investigation are as follows. When 2 mM Ni2+ was applied to fibers in a normal Ringer's solution containing 50 microM Zn2+ (Zn2+ solution), the Zn2+-potentiated twitch tension decreased remarkably to about one-third of that before Ni2+ treatment. This concentration of Ni2+ caused a 23% decrease in the duration of action potential which had been prolonged by Zn2+ (6.61-5.09 ms). Ni2+ (2 mM) added to normal Ringer's solution led to increases of about 30 and 42% in twitch tension and in the duration of action potential, respectively. A slight increase in the mechanical threshold was induced by 2 mM Ni2+. The inhibitory action of Ni2+ on the twitch tension in Zn2+ solution was larger than that in the case of tetanus tension. Diltiazem (40 microM), a Ca2+ channel blocker, did not inhibit the twitch tension potentiated in Zn2+ solution. These results suggest that the decrease in Zn2+-potentiated twitch tension by Ni2+ may possibly derive from impairment of the propagation of action potential along the T tubules.     

Effects of external ions on membrane potentials of a lobster giant axon

The effects of varying external concentrations of normally occurring cations on membrane potentials in the lobster giant axon have been studied and compared with data presently available from the squid giant axon. A decrease in the external concentration of sodium ions causes a reversible reduction in the amplitude of the action potential and its rate of rise. No effect on the resting potential was detected. The changes are of the same order of magnitude, but greater than would be predicted for an ideal sodium electrode. Increase in external potassium causes a decrease in resting potential, and a decrease in potassium causes an increase in potential. The data so obtained are similar to those which have been reported for the squid giant axon, and cannot be exactly fitted to the Goldman constant field equation. Lowering external calcium below 25 mM causes a reduction in resting and action potentials, and the occasional occurrence of repetitive activity. The decrease in action potential is not solely attributable to a decrease in resting potential. Increase of external calcium from 25 to 50 mM causes no change in transmembrane potentials. Variations of external magnesium concentration between zero and 50 mM had no measurable effect on membrane potentials. These studies on membrane potentials do not indicate a clear choice between the use of sea water and Cole's perfusion solution as the better external medium for studies on lobster nerve.     

Selectivity of sodium channels in denervated tonic muscle fibre membrane of the frog

Ionic selectivity of sodium channels was examined under voltage clamp conditions in normal and denervated twitch fibres and denervated tonic fibres isolated from m. ileofibularis of the frog (R. temporaria). Membrane currents were recorded by means of the Hille-Campbell vaseline-gap voltage clamp method from muscle fibre segments exposed to a potassium-free artificial internal solution. Permeability ratio (PS/PNa) were determined from changes in the reversal potential after replacing all Na ions in the solution bathing the voltage clamped external membrane area with sodium substituting ions (S). The permeability sequence was: Na+ greater than Li+ greater than NH4+ greater than K+. No inward currents were observed for Ca2+. The permeability ratios were as follows. Denervated tonic fibres: 1:0.88:0.23:0.012; control twitch fibres: 1:0.94:0.22:0.076; denervated twitch fibres: 1:0.91:0.14:0.082. The permeability to Li+ ions deviates from independence to a greater extent in tonic than in phasic fibres. Our results are consistent with the Hille model of sodium channel selectivity, and they support the hypothesis that sodium channels formed in denervated tonic muscle fibres of the frog are of the same genetic origin as Na channels expressed under physiological conditions.     

Chronic stimulation modifies the isotonic shortening velocity of denervated rat slow-twitch muscle

Rat soleus muscles were denervated and stimulated in vivo for periods of up to 104 days. Stimuli used were trains of 1 ms pulses at 100 Hz delivered for periods of 1 s; trains were repeated every 10-100 s. In a majority of animals the tension of the muscles was maintained at about 10% of normal, equivalent to muscles denervated but unstimulated for 20 days. At the longest periods the stimulated muscles developed ten times more tension than ones that were denervated but not stimulated. In denervated and denervated-stimulated muscles twitch contraction and relaxation times were prolonged, compared with controls, for up to 3 weeks. Thereafter both sets showed a speeding of the isometric twitch that was greater in the stimulated muscles. At the longest periods the twitch was as short as that of a denervated fast muscle. Stimulation did not affect contralateral denervated muscles. Twitch: tetanus ratios remained high despite stimulation, and muscles showed little post-tetanic potentiation. Tension developed more rapidly in the tetani of the stimulated muscles, even allowing for larger final values. Maximum velocity of shortening was increased in many of the stimulated muscles, and there was a proportional flattening of the force-velocity curve, i.e. a/P0 increased. Maximum velocity and a/P0 increased reciprocally with twitch time to peak, so that those muscles that had twitches most changed by stimulation also had their isotonic properties modified to the greatest extent. Even at the longest period of stimulation, twitch time course and tetanic tension were not converted to those of normal fast muscle.     

Ionic mechanisms underlying spontaneous CA1 neuronal firing in Ca2+-free solution

Hippocampal CA1 neurons exposed to zero-[Ca(2+)] solutions can generate periodic spontaneous synchronized activity in the absence of synaptic function. Experiments using hippocampal slices showed that, after exposure to zero-[Ca(2+)](0) solution, CA1 pyramidal cells depolarized 5-10 mV and started firing spontaneous action potentials. Spontaneous single neuron activity appeared in singlets or was grouped into bursts of two or three action potentials. A 16-compartment, 23-variable cable model of a CA1 pyramidal neuron was developed to study mechanisms of spontaneous neuronal bursting in a calcium-free extracellular solution. In the model, five active currents (a fast sodium current, a persistent sodium current, an A-type transient potassium current, a delayed rectifier potassium current, and a muscarinic potassium current) are included in the somatic compartment. The model simulates the spontaneous bursting behavior of neurons in calcium-free solutions. The mechanisms underlying several aspects of bursting are studied, including the generation of triplet bursts, spike duration, burst termination, after-depolarization behavior, and the prolonged inactive period between bursts. We show that the small persistent sodium current can play a key role in spontaneous CA1 activity in zero-calcium solutions. In particular, it is necessary for the generation of an after-depolarizing potential and prolongs both individual bursts and the interburst interval.     

Anti-curare action of stannous ion in the frog neuromuscular junction

For the purpose of elucidating the mechanism of action of stannous ion (Sn2+), we investigated effects of stannous chloride (SnCl2) on the twitch and on the electrical phenomena in the muscle fiber. Sciatic nerve-sartorius muscle preparations from the bullfrog were used as the material. Effect of SnCl2 was examined on the twitch partially inhibited by pretreatment with d-tubocurarine. SnCl2 (1-100 microM) antagonized d-tubocurarine and enhanced the twitch dose-dependently. Tartaric acid, which is the solvent used for SnCl2 solution, had no augmentative effect on the twitch, even at a concentration as high as 250 microM. SnCl2 (1-50 microM) increased the amplitude of the endplate potential; that is, it exerted an anti-curare action. The resting potential and the membrane resistance of the muscle fiber were not altered by 30 microM SnCl2. These findings lead to the conclusion that Sn2+ enhances the twitch by increasing the endplate potential of the muscle fibers.