Effects of thiamine antagonists on nerve conduction. I. Actions of antimetabolites and fern extract on propagated action potentials

To assess the hypothesis that thiamine is directly involved in the permeability changes at the sodium channel during nerve conduction, the effects of thiamine antagonists on lobster giant axon resting and action potentials were determined. Thiamine antimetabolites, in millimolar concentrations, reversibly decreased the maximum rate of rise and amplitude of the action potential while increasing its duration. In particular, thiamine tert-butyl disulfide (TTBD) elicited the formation of pronounced shoulders during repolarization, lengthening the action potential by 2–50 times, depending on dose. Antimetabolites also depolarized the resting membrane, but this change was poorly reversible and may indicate a dual mechanism for antimetabolite action. An extract of the fern, Pteris aquilina, reversibly decreased the maximum rate of rise of the action potential and depolarized the resting potential. It also elevated and prolonged the action potential after-depolarization, sometimes causing repetitive activity. The strength of these actions was correlated with the antithiamine potency of the extract, and was diminished by addition of thiamine to the extract.     

An opiate receptor on frog sciatic nerve axons.

The effect of meperidine (3 X 10(4) M) on the action potential of frog sciatic nerve was examined by means of the double sucrose gap technique. Meperidine decreased the amplitude, maximum rate of depolarization, and maximum rate of repolarization of the action potential but had no effect on the resting potential. This depression in amplitude and maximum rate of rise was partially blocked by naloxone (1 X 10(-8) M) while the maximum rate of depolarization was further depressed. The data suggest that the effect of meperidine is due to two mechanisms, a nonspecific local anaesthetic like effect and an opiate receptor mediated effect.     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.     

Calcium and action potentials of bullfrog sympathetic ganglion cells

Bullfrog sympathetic ganglion cells were capable of producing action potentials (Ca spikes) in an isotonic (84 mM) CaCl2 solution. The peak level of Ca spikes showed an approximately 30 mv increase with a 10-fold increase in the Ca concentration. Na as well as Ca ions were capable of acting as charge carriers during the production of action potentials in a solution containing relatively high Ca and relatively low Na ions. A decrease in the external Ca concentration depressed the maximum rate of rise at a fixed resting potential level, and increased the maximum rate of rise of the Na spikes at a high resting potential level at which Na inactivation was completely depressed. Compared to Na spikes, Ca spikes were less sensitive to TTX and procaine. Ganglion cells were also capable of producing action potentials (Sr spikes) in an isotonic SrCl₂ solution and prolonged action potentials in an isotonic BaCl₂ solution, but these cells were rendered inexcitable in an isotonic MgCl₂ solution. The peak level of the Sr spikes was dependent on the external Sr concentration and was insensitive to both TTX and procaine. Sr ions, like Ca ions, reduced Na inactivation during the resting state, and depressed the maximum rate of rise of the Na spikes at a high resting potential level. It was concluded that Ca (and Sr) ions exert dual actions on the membrane; namely, regulating the Na permeability and acting as charge carriers during the active state of the membrane.     

Effects of tetraethylammonium and 4-aminopyridine on the plateau potential of circular myometrium from the pregnant rat

In the pregnant rat, spontaneous electrical activity of circular muscle (CM) changes from single, plateau-type action potentials at early and mid-term to repetitive spike trains at term. To examine mechanisms underlying the plateau, we studied the effects of potassium channel blockers tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on membrane potentials in CM from rats on gestation Days 14, 15, 16, 21 (term). Apparent membrane conductance was measured at rest and during the plateau in Day 14 muscles with and without TEA. 4-AP depolarized the resting membrane on all gestation days. Therefore, a direct action of 4-AP on plateau configuration could not be separated from an indirect effect of depolarization. TEA did not affect the resting potential but increased action potential size and depolarization rate on all gestation days. On Day 16, TEA reduced plateau amplitude, unmasking small, repetitive depolarizations. D-600 decreased plateau amplitude and duration and attenuated these effects of TEA. Plateau conductance increased initially then decreased before membrane repolarization. Membrane conductance and outward rectification during the plateau were reduced by TEA. The plateau potential may result from an outwardly rectifying TEA-sensitive current combined with a slow inward current, the plateau magnitude being determined by the relative intensity of each current.     

Electrophysiological response of rat atrial myocytes to acidosis

The effect of acidosis on the electrical activity of isolated rat atrial myocytes was investigated using the patch-clamp technique. Reducing the pH of the bathing solution from 7.4 to 6.5 shortened the action potential. Acidosis had no significant effect on transient outward or inward rectifier currents but increased steady-state outward current. This increase was still present, although reduced, when intracellular Ca(2+) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA); BAPTA also inhibited acidosis-induced shortening of the action potential. Ni(2+) (5 mM) had no significant effect on the acidosis-induced shortening of the action potential. Acidosis also increased inward current at -80 mV and depolarized the resting membrane potential. Acidosis activated an inwardly rectifying Cl(-) current that was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which also inhibited the acidosis-induced depolarization of the resting membrane potential. It is concluded that an acidosis-induced increase in steady-state outward K(+) current underlies the shortening of the action potential and that an acidosis-induced increase in inwardly rectifying Cl(-) current underlies the depolarization of the resting membrane potential during acidosis.     

Comparative studies on the positive inotropic effect of isoproterenol and bromobenzoyl-methyladamantylamine in guinea pig ventricular myocardium

The effects of bromobenzoyl-methyladamantylamine (BMA) on the transmembrane potentials, contractile force, and 42K efflux were investigated and compared to that of isoproterenol (IPR) in guinea pig ventricular myocardium. Both drugs exerted positive inotropic effect. BMA lengthened the action potential duration, depolarized the membrane, and decreased the Vmax. IPR increased the height of the plateau, accelerated repolarization, slightly increased the resting potential. In preparations depolarized partially by 26 mmol/l K+, both BMA (10(-4) mol/l) and IPR (10(-7) mol/l) induced slow response action potentials, but the duration of BMA-induced ones was twice longer than that of IPR-induced ones. BMA markedly reduced the 42K efflux from ventricular myocardium, whereas IPR had no effect on it. Moreover, BMA also decreased the 26 mmol/l K+-induced increment in 42K efflux, while IPR did not. It is concluded that BMA and IPR exert their positive inotropic effects on different ways. IPR increases the slow inward Ca2+ current directly by activating a phosphorylation process, whereas BMA enhances it indirectly by reducing the K+ conductance, lengthening the repolarization and consequently prolonging the time during which the slow inward Ca2+ current can be operative.     

Atrioventricular block in low potassium and K dependence of the nodal resting potential

A progressive conduction block leading to atrioventricular dissociation develops in perfused rabbit hearts within 20-30 min of exposure to Krebs containing 0.5 mM potassium (low K). A decrease in potassium permeability resulting in membrane depolarization (as seen in Purkinje fibers) could be responsible for the loss of excitability in nodal cells. We investigated the K dependence of the resting potential and the long-term effects of low K perfusion on the resting and action potentials of nodal cells in rabbit hearts. The resting potential of atrial, atrionodal, and nodal cells varied by 52, 41, and 34 mV per decade of change in Ko within the range of 5-50 mM K. Hyperpolarization of the resting membrane, a progressive decline in action potential amplitude, and a decrease in maximum rate of rise were observed in nodal fibers when exposed to low K. Loss of propagated activity occurred in the middle node within 20-30 min while the cells remained hyperpolarized. There was no evidence of electrogenic Na extrusion and it seems that the low nodal resting potential results from a high resting PNa/PK permeability ratio. The early decrease in rate of rise in low K probably reflects an increase in K-dependent outward currents, whereas the progressive deterioration and final loss of conducted electrical activity may result from an accumulation of internal Na and Ca overload produced by low K inhibition of the Na pump.     

Effects of morphine and meperidine on action potential production in frog's skeletal muscle fibers.

Morphine (3.3 times 10-minus 4 M) and meperidine (8.8 times 10-minus 5 M) inhibited action potential production in frog's skeletal muscle fibers. Over these concentration ranges, neither the resting membrane potentials nor the resting membrane electric properties of the fibers appeared to be modified. Both drugs depressed excitability and the rising phase of the action potential by inhibiting the specific increase in sodium conductance which normally follows an adequate stimulus. Both drugs also seemed to inhibit the secondary rise in potassium conductance which normally occurs during an action potential, causing a prolongation of the action potential duration.     

三叉神经中脑核神经元膜的电学特性

用大鼠脑干脑片,给三叉神经中脑核79个神经元作了细胞内记录,测算了20个神经元膜的电学特性:静息电位-60.3±5.6mV;输入阻抗为10.5±5.4MΩ;时间常数1.3±0.5ms。电刺激可诱发动作电位,测算32个神经元的有关参数:阈电位-50—-55mV;波幅69.5±6.1mV;超射11.9±3.6mV;波宽0.8±0.2ms。TTX(0.3μmol/L)或无钠使之消失。通以长时程矩形波电流可引起200—250Hz的2—15个重复放电,但在通电停止前终止,TEA或4-AP可延长放电。膜电位-60—-55mV时在动作电位之后可看到阈下电位波动,它不受TTX的影响,无钙时消失,TEA或4-AP使波幅增大。静息电位去极化可使45个神经元中的40个发生外向整流作用,并被TEA,4-AP或无钙抑制,超极化则发生内向整流作用,Cs或无钠抑制之。灌流液中加入各种钾通道阻断药时神经元的稳态I-V曲线发生相应变化,提示I_(DR),l_A,I_(K(Ca))及I_Q可能都与静息时的膜电导有关。     

Effects of K+ on the twitch and tetanic contraction in the sartorius muscle of the frog, Rana pipiens. Implication for fatigue in vivo.

The effects of increasing the extracellular K+ concentration on the capacity to generate action potentials and to contract were tested on unfatigued muscle fibers isolated from frog sartorius muscle. The goal of this study was to investigate further the role of K+ in muscle fatigue by testing whether an increased extracellular K+ concentration in unfatigued muscle fibers causes a decrease in force similar to the decrease observed during fatigue. Resting and action potentials were measured with conventional microelectrodes. Twitch and tetanic force was elicited by field stimulation. At pHo (extracellular pH) 7.8 and 3 mmol K+.L-1 (control), the mean resting potential was -86.6 +/- 1.7 mV (mean +/- SEM) and the mean overshoot of the action potential was 5.6 +/- 2.5 mV. An increased K+ concentration from 3 to 8.0 mmol.L-1 depolarized the sarcolemma to -72.2 +/- 1.4 mV, abolished the overshoot as the peak potential during an action potential was -12.0 +/- 3.9 mV, potentiated the twitch force by 48.0 +/- 5.7%, but did not affect the tetanic force (maximum force) and the ability to maintain a constant force during the plateau phase of a tetanus. An increase to 10 mmol K+.L-1 depolarized the sarcolemma to -70.1 +/- 1.7 mV and caused large decreases in twitch (31.6 +/- 26.1%) and tetanic (74.6 +/- 12.1%) force. Between 3 and 9 mmol K+.L-1, the effects of K+ at pHo 7.2 (a pHo mimicking the change in interstitial pH during fatigue) and 6.4 (a pHo known to inhibit force recovery following fatigue) on resting and action potentials as well as on the twitch and tetanic force were similar to those at pHo 7.8. Above 9 mmol K+.L-1 significant differences were found in the effect of K+ between pHo 7.8 and 7.2 or 6.4. In general, the decrease in peak action potential and twitch and tetanic force occurred at higher K+ concentrations as the pHo was more acidic. The results obtained in this study do not support the hypothesis that an accumulation of K+ at the surface of the sarcolemma is sufficiently large to suppress force development during fatigue. The possibility that the K+ concentration in the T tubules reaches the critical K+ concentration necessary to cause a failure of the excitation-contraction coupling mechanism is discussed.     

Demonstration of two stable potential states of plasmalemma ofChara without tonoplast

Summary The tonoplast of cells ofChara australis was removed by replacement of the cell sap with a medium containing 5 mM EGTA (ethyleneglycol-bis-(-aminoethyl ether) N, N-tetraacetic acid). Such cells without tonoplast could generate an action potential of rectangular shape. In the present paper characteristics of the action potential were studied under various external ionic conditions.Action potentials could be elicited without refractory period and the peak of the action potential was constant among action potentials.Duration of the action potential decreased under repeated excitations, but recovered after pause. Increase in concentrations of alkali metal cations, Li⁺, Na⁺, K⁺, Rb⁺ and Cs⁺, resulted in prolongation of the action potential.At proper concentrations of monovalent cations the membrane potential could stay either at the resting level or at the depolarized level and could be shifted reversibly from the former level to the latter one orvice versa by applying outward or inward current. Further increase in concentrations of monovalent cations resulted in arrest of the membrane potential at the depolirized level. The critical concentrations of the monovalent cations to hold the membrane potential at the depolarized level were about 10 mM irrespective of the cation species.Divalent cations, Ca²⁺, Mg²⁺, Sr²⁺, Ni²⁺ and Mn²⁺, added to the bathing medium suppressed the effect of monovalent cations to prolong the action potential.Ca²⁺ and Mg²⁺ added to the bathing medium caused repolarization of the plasmalemma which had been depolarized by application of high concentrations of K⁺ to the bathing medium. The antagonism between monovalent and divalent cations on the state of the plasmalemma ofChara cells was discussed based on the two stable states hypothesis proposed by Tasaki (Tasaki, I. 1968. Nerve Excitation. Charles C. Thomas, Springfield, Illinois).     

Elucidation of the mechanism and site of action of quinuclidinyl benzilate (QNB) on the electrical excitability and chemosensitivity of the frog sartorius muscle

The effects of the muscarinic antagonist quinuclidinyl benzilate (QNB) on transmission at the frog sartorius neuromuscular junction have been examined. QNB decreases endplate potential (EPP) amplitude without affecting miniature endplate (MEPP) frequency or resting potential. QNB also increased the latency of the EPP and the nerve terminal spike in a frequency dependent fashion, suggesting the site of action is the unmyelinated nerve terminal. Since the rate of rise and amplitude of muscle action are potentials decreased it is likely that QNB causes a blockade of electrically excitable sodium channels; the agent also blocks ionic channels associated with nicotinic acetylcholine receptors. It is possible that these effects of QNB may explain some of the behavioral disturbances produced by its administration.     

Temperature effects on the Na and Ca currents in rat and hedgehog ventricular muscle

Cardiac transmembrane potentials and Na and Ca currents were recorded at different temperatures in rat and hedgehog ventricular muscle. At 35 degrees C in both species resting potential was about -80 mV and upstroke velocity (Vmax) of the action potential above 100 V/s. The shape of the action potential in hedgehog ventricular cells at 35 degrees C was similar to that in the rat showing a fast repolarization phase. When temperature was decreased, the membrane resting potential depolarized and action potential amplitude and Vmax declined. In rat ventricular cells at 10 degrees C, the resting potential was about -40 to -50 mV and Vmax was reduced to about 5 V/s. In hedgehog ventricular cells, however, the transmembrane potentials and Vmax were better maintained at low temperature. Phase 3 of the action potential was markedly prolonged below 20 degrees C in hedgehog but not in rat ventricular cells. When temperature was decreased to 10 degrees C the availability curve of the Na current shifted toward more negative potentials and ICa.peak declined in rat ventricular cells. In hedgehog cardiac preparations, the Na current was less influenced by the cooling and ICa.peak did not change very much at low temperatures. A transient inward current usually considered to induce cardiac arrhythmias could be recorded in rat ventricular cells below 20 degrees C but not in hedgehog preparations. These features of hedgehog cardiac membranes may contribute to the cold tolerance and the resistance to ventricular fibrillation during the hypothermia in mammalian hibernators.     

Effects of hirsutine and dihydrocorynantheine on the action potentials of sino-atrial node, atrium and ventricle

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla MIQ. JACKSON with antihypertensive, negative chronotropic and antiarrhythmic activity, and its C3 structural epimer, dihydrocorynantheine, on membrane potentials of rabbit sino-atrial node and guinea-pig right ventricle and left atrium were studied with microelectrode techniques. In sino-atrial node preparations, hirsutine and dihydrocorynantheine (0.1 microM to 10 microM) concentration-dependently increased cycle length, decreased slope of the pacemaker depolarization (phase 4 depolarization), decreased maximum rate of rise and prolonged action potential duration. In atrial and ventricular preparations, both compounds (0.1 microM to 30 microM) concentration-dependently decreased maximum rate of rise and prolonged action potential duration. These results indicate that hirsutine and dihydrocorynantheine have direct effects on the action potential of cardiac muscle through inhibition of multiple ion channels, which may explain their negative chronotropic and antiarrhythmic activity.     

Effects of local anesthetics on the Chara plasmalemma.

The effects of lidocaine, tetracaine, procaine and bupivacaine (less than 1000 microM) on the Chara corallina internodal cell were studied. These local anesthetics depolarized the membrane at rest, while they affected the rising phase and the peak level of action potential not appreciably. Instead, they prolonged the time course of the falling phase of action potential as slowly as the repolarization was imperfect, even after enough lapse beyond the refractory period. Consequently, an action potential appeared to enhance the degree of depolarization at rest. Such a depolarization with stimulus/excitation was named use-dependent depolarization, while the depolarization without excitation, the resting one. The order of the potency of the use-dependent depolarization almost coincided with that of the nerve-blocking potency. During depolarization the change in membrane conductance was not simple. However, the conductance-voltage (Gm-Vm) relationship curve in the presence of local anesthetic suggested that depolarization was due to, not only the decrease in the electrogenic H(+)-pump, but also the increase in the diffusion conductance.     

Effect of induced hypothyroid state on the mechanical and electrical properties of the rat ventricular myocardium

The effect of hypothyroid state on the transmembrane potential was studied in isolated cardiac ventricular trabeculae of rats. Hypothyroid state was induced by methimazole treatment or thyroidectomy and checked by determining serum thyroxine level. Hypothyroidism decreased the maximum rate of depolarization (Vmax) and the resting potential, increased the overshoot and the duration of action potential at 20, 50 and 90% repolarization. These changes were more pronounced after methimazole treatment than after thyroidectomy. The results strongly suggest that in hypothyroidism the significant alterations in the voltage-time course of the transmembrane action potential influencing Ca2+-movement across the sarcolemma may have an indirect role in the decreased myocardial contractility. On the other hand, methimazole has an aspecific cardiac effect which may modify the cardiac effect of hypothyroidism induced by the drug.     

The relations between prepotential,resting potential,and latent period in frog muscle fibers

1. Prepotentials and action potentials were recorded from amphibian striated muscle fibers. Intracellular electrodes were used for stimulating and recording. The resting potential was varied from 55 to 120 mv. by alterations of the KCl concentration of the Ringer's fluid. The magnitude of the prepotential at the initiation of the spike potential was measured and compared to the resting potential and the latent period (time between stimulus "make" and excitation). The magnitude of this prepotential varied with the resting potential. 2. A large prepotential or cathodal depolarization was required to excite a fiber with a high resting potential. If a fiber with a high resting potential fired late (long latency), the adequate prepotential was larger than if the fiber fired early. Fibers with low resting potentials had smaller adequate prepotentials. Also, the adequate prepotential was independent of the latent period, in these depolarized fibers. 3. If the concentration of Ca⁺⁺ was increased tenfold, the adequate prepotential of depolarized fibers became strongly dependent upon the latency. 4. Fibers with large or normal resting potentials were prone to respond repetitively during the passage of long duration shock, whereas depolarized and Ca⁺⁺-treated fibers were not. 5. The so-called critical membrane potential (which is defined as the transmembrane potential at the point of excitation) was not independent of the resting potential.     

The Effects of pH on the Ionic and Electrical Properties of the Internodal Cells of Chara australis

The effects of pH on the resting and action potentials and onthe fluxes of potassium, sodium, and chloride across the membranesof internodal cells of Chara australis have been investigated. Experiments were carried out in an artificial pond water (A.P.W.)of standard composition: CaCl₂, 01 mM; KCl, 0.1 mM; NaCl, 1.0mM. The resting potential decreased as the pH was lowered from6.5, being depolarized by about 75 mV at pH 4.5. Measurementsof the ion fluxes as a function of pH suggested that this depolarizationwas caused by an increase in the permeability to sodium anda decrease in permeability to potassium at pH 4.5. Action potentialsof constant peak value can be elicited for some time at pH 4.5,but after 20 min or so the cell becomes refractory. All theseeffects on resting and action potentials are fully reversible.We briefly speculate about the mechanism of these pH effects.