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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Slowing of the time course of the excitation of squid giant axons in viscous solutions

Summary The time course of excitation of intracellularly perfused squid giant axons was slowed as the solution viscosity was raised by adding neutral molecules, i.e., glucose and glycerol. By twofold increase of the solution viscosity, the duration of action potential was prolonged to 2.7-fold and the maximum rate of rise decreased to one-half. At the same time, the membrane resistance at resting state increased by 60%. These effects were reversible. The time course of inward and outward currents was slowed also. When the solution viscosity increased to twofold, the time to peak inward current increased by 80%, and the amplitudes of peak inward and steady outward currents decreased by 60% and by 70%, respectively. These effects were not specific for the sodium or the potassium channel. Effects of solution viscosity occurred in both hypotonic and hypertonic solutions. Q₁₀ values of temperature dependence of the time course of the action potential were equal in any viscous solutions. These effects in viscous solutions were explained by the change in solution viscosity but not by the change in solution osmolarities, ionic activities, or solution resistivity.     

Studies on cessation of cytoplasmic streaming under K+-induced depolarization inNitella axilliformis

Internodal cells ofNitella axilliformis had a membrane potential of about−120mV and showed active cytoplasmic streaming with a rate of about 90 μm/sec in artificial pond water (APW) at 25C. When APW was replaced with 50 mM KCl solution, the membrane potential depolarized accompanying an action potential, and the cytoplasmic streaming stopped. Soon after this quick cessation, the streaming started again, but its velocity remained very low for at least 60 min. Removal of KCl from the external medium led to repolarization of the membrane and accelerated recovery of the streaming. The change in the concentration of free Ca²⁺ in the cytoplasm ([Ca²⁺]_c) was monitored by light emission from aequorin which had previously been injected into the cytoplasm. Upon application of KCl to the external medium, the light emission, i.e., [Ca²⁺]_c, quickly increased. It then decreased exponentially and reached the original low level within 100 sec. The cause of the long-lasting inhibition of cytoplasmic streaming observed even when [Ca²⁺]_c had returned to its low resting level is discussed based on the mechanism proposed for action potential-induced cessation of cytoplasmic streaming; inactivation of myosin by Ca²⁺-dependent phosphorylation or formation of cross bridge between actin filaments and myosin.     

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.     

Thiamine phosphate metabolism and possible coenzyme-independent functions of thiamine in brain.

The effect of depolarization of rat brain cortex slices on the relative distribution of thiamine among its various phosphate esters and on the efflux of thiamine was studied as a probe of possible coenzyme-independent neurophysiological functions of thiamine. Electrical pulses for 30 min increased lactate production but did not affect the levels of thiamine esters. Depolarization with 41 mM-potassium decreased thiamine diphosphate by only 3 percent (P= 0.05). Thiamine triphosphate levels (TTP) were unaffected by depolarization but doubled during incubation for 1 h in which time efflux of 40 percent of the total thiamine from the slices as unesterified thiamine occurred. Depolarization by potassium released a small but highly variable portion of the thiamine content of superfused cortex slices above the basal rate of efflux. The basal efflux was partially sodium dependent. Thiamine efflux was unaffected by acetylcholine, ouabain, or tetrodotoxin, compounds previously reported to increase thiamine efflux. The incorporation of ³²P₁ into the endogenous thiamine phosphates of cortex slices was studied. Incorporation into thiamine diphosphate reached only 20 percent of the specific activity of its precursor, ATP, after 2h of incubation while the incorporation into TTP approached equilibrium with ATP in 15-30 min indicating that the TTP pool was the most rapidly turning over of the thiamine phosphates. The data suggest that only a small portion of the TDP pool undergoes rapid turnover and serves as a precursor for TTP. The rapid turnover of TTP phosphoryl groups is consistent with specific functions for this compound related to its potential for phosphorylation reactions. An analog of TTP with the β, γ oxygen bridge replaced by a methylene group decreased TDP levels and increased thiamine when incubated with cortex slices, but did not effect thiamine monophosphate or triphosphate levels indicating inhibition of thiamine pyrophosphokinase.     

Electrophysiological Actions of Oxytocin on the Rabbit Myometrium

The electrical activities of myometrial cells of the pregnant rabbit uterus have been studied by means of sucrose-gap and intracellular micro-electrode recording techniques. The resting potential of the myometrial cell was about -50 mv, and it is unaffected by the duration of pregnancy or placental attachment. Action potentials of the myometrium, although dependent on external Na⁺, were not always of the regenerative type; preparations from nonparturient uteri often produce mainly small spikes. The mean spike amplitude was 35 mv, rising at a mean maximum rate of 3 v/sec. Oxytocin, in concentrations less than 500 µU/ml, increased the mean spike amplitude to 48 mv and the mean maximum rate of rise to 7 v/sec, without affecting the resting potential. The relation between membrane potential and dV/dt of the spike was steepened by oxytocin, suggesting that oxytocin increased the number of normally sparse sodium gates in the myometrial membrane. By this action, oxytocin is believed to increase the probability of successful regenerative spikes and thereby initiate electrical activity in quiescent preparations, increase the frequency of burst discharges, the number of spikes in each burst, and the amplitude of spikes in individual cells.     

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.     

STABILIZATION OF SPIDER CRAB NERVE MEMBRANES BY ALKALINE EARTHS,AS MANIFESTED IN RESTING POTENTIAL MEASUREMENTS

1. The alkaline earths, Ba, Sr, Ca, and Mg, in isotonic solutions of their chlorides, have, in general, no effect upon the resting potential of non-medullated spider crab nerve. 2. Ba, Sr, and Ca can, however, prevent the depressing action of K upon the resting potential. The order of effectiveness of these ions in this regard is the following: Ba > Sr > Ca. 3. Ba, Sr, Ca, and Mg oppose the depressing action of veratrine sulfate upon the resting potential. The order of effectiveness is Ba > Sr > Ca > Mg. The relation between drop in potential caused by veratrine sulfate and the logarithm of the veratrine sulfate concentration is a linear one. 4. The action of various other organic ions and molecules which depress the resting potential: saponin, amyl urethane, chloral hydrate, and Na salicylate is neutralized by Ba. 5. Hypertonic sea water solutions do not affect the resting potential. Also, preliminary experiments indicate that the nerves do not shrink in hypertonic solutions although they swell in hypotonic sea water. 6. The alkaline earths depress excitability reversibly. The various organic agents which depress the resting potential also depress excitability, in most cases, reversibly, but the concentrations necessary to depress excitability are much smaller than those necessary to depress the resting potential. 7. The relation of these findings to theories put forward as possible explanations of resting potential phenomena is considered.