Action potential prolongation: an effect of physostigmine (eserine) upon Retzius cells in the leech C.N.S

Physostigmine (PHY; eserine) prolongs the action potentials in the Retzius cells within leech ganglia to about 800 ms. The effect was reversible and occurred at concentrations of 1-10 mM which are several orders of magnitude greater than those required to inhibit cholinesterase. The prolonged action potentials showed an early, spike-like depolarization followed by a plateau. The initial depolarization exhibited a strong dependence on external Na+ while the amplitude of the plateau had somewhat less Na+ dependence: 52 and 24 mV/decade, respectively. The duration of the plateau was increased by elevating Na+ and decreased by elevating Ca2+. Increasing the action potential frequency, by intracellular stimulation, decreased both the duration and amplitude of the plateau. Neostigmine, di-isopropylphosphofluoridate, and acetylcholine did not prolong RZ action potentials. Thus, the membrane effects of physostigmine appear to be independent of any inhibition of cholinesterase or accumulation of acetylcholine.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.     

Down-modulation induced block of voltage-operated Na+ channels in ratCA1 hippocampal neurons by (+)-methysticin

The effects of a synthetic 5,6-dihydro-alpha-kava-pyrone (+)-methysticin on voltage-operated Na⁺ channels was studied in isolated whole-cell patch-clampedCA1 hippocampal neurons. Within a concentration range of 1–400 µM, (+)-methysticin induced a rapid and partly reversible dose-dependent reduction of the peak amplitude of Na⁺ current. Shifts of the holding membrane potential toward more positive values considerably enhanced the blocking effect. The efficiency of this block showed no dependence on the frequency of stimulation. In addition, (+)-methysticin shifted a steady-state inactivation curve toward more negative membrane potentials, accelerated the time course of fast inactivation, and slowed down the recovery from inactivation. From the above findings, the voltage dependence of (+)-methysticin effect can be accounted for by an increased portion of inactivated Na⁺ channels. Thus, the effect of (+)-methysticin on voltage-operated Na⁺ channels is associated with decreased excitability of nerve cell membranes. This suggests that this drug is capable of suppressing seizure activity of different genesis, including epileptic seizures. More effective action of (+)-methysticin on depolarized neuronal membrane prompts its possible application for therapeutic purposes in pathological disorders accompanied by depolarization of neurons in damaged foci, in particular in the case of cerebral ischemia.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 179–185, July–October, 1996.     

Effects of different ions on resting polarization and on the mass receptor potential of carotid body chemosensors

The carotid body and its own nerve were removed from cats anesthetized with sodium pentobarbital and placed in an air gap system; the carotid body was bathed in modified Locke's solution equilibrated with 50% O₂ in N₂, pH 7.43 at 35°C. The sensory discharges, changes in “resting” receptor polarization and the mass receptor potential evoked by ACh or NaCN were recorded with nonpolarizable electrodes placed across the gap. Receptor potentials and sensory discharges evoked by ACh showed an appreciable increase in amplitude and frequency when the preparation was bathed in eserinized Locke. Eserine did not change appreciably the responses evoked by NaCN. Excessive depolarization elicited by either ACh or NaCN was accompanied by sensory discharge block. Removal of K⁺ ions from the bathing solution induced receptor hyperpolarization and an increase in the amplitude of the evoked receptor potentials. An increase of K⁺ concentration had the opposite effect. Reduction of Na⁺ or NaCl to one half, or total removal of this salt, induced an initial reduction and later disappearance of the sensory discharges, some receptor hyperpolarization and a reduction in the amplitude of the evoked receptor potentials. Reduction or removal of Ca⁺⁺ produced receptor depolarization, a marked depression of the evoked receptor potentials, an increase in the frequency of the sensory discharges and a reduction in the amplitude of the nerve action potentials. High Ca⁺⁺ or Mg⁺⁺ had little or no effect on action potential amplitude or resting polarization, but decreased sensory discharge frequency and the evoked receptor potentials. Total or partial replacement of Ca⁺⁺ with Mg⁺⁺ induced complex effects: (1) receptor depolarization which occurred in low Ca⁺⁺, was prevented by addition of Mg⁺⁺ ions; (2) the amplitude of the evoked receptor potentials was depressed; (3) the nerve discharge frequency was reduced as it was in high Mg⁺⁺ solutions; and (4) the amplitude of the nerve action potentials was reduced as it was in low Ca⁺⁺ solutions. Temperature had a marked effect on the chemoreceptors since a t high temperatures the receptors were depolarized and the discharge frequency increased. The baseline discharge and responses evoked by ACh or NaCN were depressed at low temperatures. The results are discussed in terms of possible receptor mechanisms influenced by the different ions.     

Oscillatory Plateau Potentials and Bursts in a Simulated Motoneuron during Tonic Coactivation of Somato-Dendritic NMDA and Axon-Hillock GABA Inputs

In a simulated motoneuron, we studied the effects of tonic coactivation of glutamatergic (NMDA-type) synapses covering the somato-dendritic membrane and of GABA-ergic synapses located on the axon hillock. As in the prototypes, NMDA activation caused oscillatory plateau potentials with bursts of action potentials (AP). Plateau depolarizations spreading from the soma inactivated Na⁺ channels and reduced the number of AP in the axon compared with that in the soma. As GABA activation increased, interplateau intervals also increased, while the plateau duration and number of AP per burst decreased.     

Sodium-dependent plateau potentials in electrocytes of the electric fish <Emphasis Type="Italic">Gymnotus carapo</Emphasis>

The weakly electric fish Gymnotus carapo emits a triphasic electric organ discharge generated by muscle-derived electrocytes, which is modified by environmental and physiological factors. Two electrode current clamp recordings in an in vitro preparation showed that Gymnotus electrocytes fired repetitively and responded with plateau potentials when depolarized. This electrophysiological behavior has never been observed in electrocytes from related species. Two types of plateaus with different thresholds and amplitudes were evoked by depolarization when Na⁺-dependent currents were isolated in a K⁺- and Ca²⁺-free solution containing TEA and 4-AP. Two electrode voltage clamp recordings revealed a classical fast activating–inactivating Na⁺ current and two persistent Na⁺-dependent currents with voltage-dependencies consistent with the action potential (AP) and the two plateaus observed under current clamp, respectively. The three currents, the APs and the plateaus were reduced by TTX, and were absent in Na⁺-free solution. The different Na⁺-dependent currents in Gymnotus electrocytes may be targets for the modifications of the electric organ discharge mediated by environmental and physiological factors.     

The dependence of pacemaker discharge of Aplysia neurons upon Na+ and Ca++

In total absence of Na⁺ some identified neurons of Aplysia, after a period of silence, resume pacemaker discharge in the normal pattern with normal action potentials, while other identified neurons remain silent. In absence of Ca⁺⁺ all pacemaker neurons increase spontaneous discharge and develop abnormal bursting patterns. Those neurons which discharge spontaneously in Na⁺ free solutions show much less dependence on Na⁺ and much greater dependence on Ca⁺⁺ for action potentials initiated by electrical stimulation than do those neurons which do not fire spontaneously in absence of Na⁺. In absence of both Na⁺ and Ca⁺⁺ all neurons become inexcitable, but much more rapidly at higher temperatures.     

Growth hormone-releasing factor reduces voltage-gated Ca2+ channel current in Rat GH3 cells

Summary The action of GRF on GH₃ cell membrane was examined by patch electrode techniques. Under current clamp with patch elecrtrode, spontaneous action potentials were partially to totally eliminated by application of GRF. In the case of partial elimination, the duration of remaining spontaneous action potentials was prolonged and the amplitude of afterhyperpolarization was decreased. The evoked actiion potential in the cells which did not show spontaneous action potentials was also eliminated by GRF. In order to examine what channels were affected by GRF, voltage-clamp analysis was performed. It was revealed that voltage-gated Ca²⁺ channel current and Ca²⁺-induced K⁺ channels current were decreased by GRF, while voltage-gated Na⁺ channel and delayed K⁺ channel current was considered to be a consequence of he decrease of voltage-gated Ca²⁺ channels current. Therefore it is likely that the effect of GRF on GH₃ cells was due to the block of voltage-gated Ca²⁺ channels. The elimination of action potential under current clamp corresponded to the block of voltage-gated Ca²⁺ channels and the prolongation of action potential could be explained by the decrease of Ca²⁺-induced K⁺ channel current. The amplitude decrease of afterhyperpolarization could also be explained by the reduction of Ca²⁺-induced K⁺ channel current. Thus the results under current clamp well coincide with the results under voltage clamp. Hormone secretion from GH₃ cells was not stimulated by GRF. However, the finding that GRF solely blocked voltage-gated Ca²⁺ channel suggested the specific action of GRF on GH₃ cell membranes.     

Sodium channels contribute to action potential generation in canine and human pancreatic islet B cells

Summary Pancreatic islet B cells depolarize and display trains of action potentials in response to stimulatory concentrations of glucose. Based on data from rodent islets these action potentials are considered to be predominantly Ca²⁺ dependent. Here we describe Na⁺-dependent action potentials and Na⁺ currents recorded from canine and human pancreatic islet B cells. Current-clamp recording using the nystatin perforated-patch technique demonstrates that B cells from both species display tetrodotoxin-sensitive Na⁺ action potentials in response to modest glucose-induced depolarization. In companion whole-cell voltage-clamp experiments on canine B cells, the underlying Na⁺ current displays steep voltage-dependent activation and inactivation over the range of –50 to –40 mV. The Na⁺ current is sensitive to tetrodotoxin block with aK ₁=3.2nm and has a reversal potential which changes with [Na⁺] _o as predicted by the Nernst equation. These results suggest that a voltage-dependent Na⁺ current may contribute significantly to action potential generation in some species outside the rodent family.     

Time-Dependent Molecular Memory in Single Voltage-Gated Sodium Channel

Excitability in neurons is associated with firing of action potentials and requires the opening of voltage-gated sodium channels with membrane depolarization. Sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, can have profound implications on the biophysical properties of voltage-gated ion channels. Therefore, we sought to characterize the effect of sustained membrane depolarization on single voltage-gated Na⁺ channels. Single-channel activity was recorded in the cell-attached patch-clamp mode from the rNa_v1.2α channels expressed in CHO cells. Classical statistical analysis revealed complex nonlinear changes in channel dwell times and unitary conductance of single Na⁺ channels as a function of conditioning membrane depolarization. Signal processing tools like weighted wavelet Z (WWZ) and discrete Fourier transform analyses attributed a “pseudo-oscillatory” nature to the observed nonlinear variation in the kinetic parameters. Modeling studies using the hidden Markov model (HMM) illustrated significant changes in kinetic states and underlying state transition rate constants upon conditioning depolarization. Our results suggest that sustained membrane depolarization induces novel nonlinear properties in voltage-gated Na⁺ channels. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clusters of dwell time events and strong autocorrelation in the dwell time series similar to that reported recently for single enzyme molecules. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Voltage-gated Na⁺ channel with the observed time-dependent nonlinear properties and the molecular memory phenomenon may determine the functional state of the channel and, in turn, the excitability of a neuron.     

Effect of Neural Stimulation on Acinar Cell Membrane Potentials in Isolated Pancreas and Salivary Gland Segments

The effect of cholinergic neural excitation by field stimulation on the acinar cell membrane potential was investigated in superfused segments of mouse pancreas and salivary glands (sublingual, submaxillary, and parotid glands).

Responses of acinar cells in both exocrine pancreas and salivary glands to the neural excitation obtained by field stimulation were similar to responses previously described in each gland to the externally applied acetylcholine.

In the pancreatic acinar cell, electrical field stimulation induced depolarization with a latency of 0.3 to 1.2 sec. This depolarization was accompanied by a marked decrease in membrane resistance. The equilibrium potential of the depolarization induced by stimulation was between -10 and -20 mV. In the sublingual gland, field stimulation induced depolarization of the acinar cell with a latency of 0.2 to 0.3 sec. The stimulus induced depolarization was blocked by the addition of atropine. In the submaxillary and parotid glands, field stimulation induced depolarization in some acinar cell and hyper-polarization in other cells.

The results support evidence previously presented by Petersen and his colleagues that acetylcholine acts to increase Na⁺ and K⁺ or Na⁺, K⁺, and Cl^- permeabilities in the pancreatic acinar cell and to increase K⁺ and Na⁺ permeabilities in the salivary gland [11,24].     

The Roles of Sodium and Potassium Ions in the Generation of the Electro-Olfactogram

In order to clarify whether or not the electronegative olfactory mucosal potentials (EOG) are generator potentials, the effects of changed ionic enviroment were studied. The EOG decreased in amplitude and in some cases nearly or completely disappeared, when Na⁺ in the bathing Ringer solution was replaced by sucrose, Li⁺, choline⁺, tetraethylammonium⁺ (TEA), or hydrazine. In the K⁺-free Ringer solution, the negative EOG's initially increased and then decreased in amplitude. In Ringer's solution with increased K⁺, the negative EOG's increased in amplitude. When K⁺ was increased in exchange for Na⁺ in Ringer's solution, the negative EOG's decreased, disappeared, and then reversed their polarity (Fig. 6). Next, when the K⁺ was replaced by equimolar sucrose, Li⁺, choline⁺, TEA⁺, hydrazine, or Na⁺, the reversed potentials recovered completely only in Na⁺-Ringer's solution, but never in the other solutions. Thus, the essential role of Na⁺ and K⁺ in the negative EOG's was demonstrated. Ba⁺⁺ was found to depress selectively the electropositive EOG, but it hardly decreased and never increased the negative EOG. Hence, it is concluded that Ba⁺⁺ interferes only with Cl^- influx, and that the negative EOG's are elicited by an increase in permeability of the olfactory receptive membrane to Na⁺ and K⁺, but not to Cl^-. From the ionic mechanism it is inferred that the negative EOG's are in most cases composites of generator and positive potentials.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

Ionic mechanisms underlying acetylcholine-, nicotine-, and muscarine-induced depolarization of Helix lucorum neuron RPa4

Research was carried out into the ionic aspects of depolarization potentials produced inHelix lucorum neuron RPa4 by injecting three cholinomimetics into the soma: acetylcholine, nicotine, and muscarine. Substances were used suppressing Na⁺, K⁺, Ca²⁺, and Cl^– conductance at the membrane. Acetylcholine brought about increased Na⁺, Ca²⁺; and Cl^– conductance. As the choline component was only slight, due to the similarity of membrane and resting potential for chloride, it might be deduced that the prevailing response to acetylcholine is associated with chemically controlled input of Na⁺ and Ca²⁺ into the cell. Nicotine and muscarine induced mainly sodium and calcium conductance respectively.M. V. Lomonosov State University, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 305–314, May–June, 1989.     

Changes of action potentials and force at lowered [Na+]o in mouse skeletal muscle: implications for fatigue

We examined 1) whether the effects of lowered trans-sarcolemmal Na⁺ gradient on force differed between nonfatigued fast- and slow-twitch muscles of mice and 2) whether effects on action potentials could explain the decrease of force. The Na⁺ gradient was reduced by lowering the extracellular [Na⁺] ([Na⁺]_o). The peak force-[Na⁺]_o relationships for the twitch and tetanus were the same in nonfatigued extensor digitorum longus and soleus muscles: force was maintained over a large range of [Na⁺]_o and then decreased abruptly over a much smaller range. However, fatigue was significantly exacerbated at a lowered [Na⁺]_o that had little effect in nonfatigued soleus muscle. This finding suggests that substantial differences exist in the Na⁺ effect on force between nonfatigued and fatigued muscle. The reduced contractility in nonfatigued muscles at lowered [Na⁺]_o was largely due to 1) an increased number of inexcitable fibers and threshold for action potentials, 2) a reduction of action potential amplitude, and 3) a reduced capacity to generate action potentials throughout trains. sodium gradient; muscle contraction; action potential train; extensor digitorum longus; soleus     

Membrane potential, action potential and activation potential of eggs of the sea urchin, Lytechinus variegatus

Unfertilized Lytechinus variegatus eggs in sea water in their normal physiological state have membrane potentials that approximate ?70 to ?80 mV. This conclusion is based on microelectrode measurements and on computation from the Na⁺ and K⁺ fluxes. The ?8 to ?15 mV values for the membrane potential previously reported and which are generally measured are the consequence of depolarization by impalement. The activation potential in inseminated eggs with an initial membrane potential more negative than ?60 mV is a compound event involving sperm-induced as well as voltage dependent conductance changes. The sperm-induced mechanism is a two-phase conductance increase which involves both Na⁺ and Ca²⁺ during the first phase, and Na⁺ alone during the second phase. In addition, the sperm-induced depolarization at the beginning of the first phase activates a voltage dependent Ca²⁺-conductance mechanism resulting in generation of an action potential.     

Slow inactivation of tetrodotoxin-insensitive Na+ channels in neurons of rat dorsal root ganglia

Summary Whole-cell patch-clamp experiments were performed with neurons cultured from rat dorsal root ganglia (DRG). Two types of Na⁺ currents were identified on the basis of sensitivity to tetrodotoxin. One type was blocked by 0.1 nm tetrodotoxin, while the other type was insensitive to 10 m tetrodotoxin. The peak amplitude of the tetrodotoxin-insensitive Na⁺ current gradually decreased after depolarization of the membrane. The steady-state value of the peak amplitude was attained several minutes after the change of holding potential. Such a slow inactivation was not observed in tetrodotoxin-sensitive Na⁺ current. The slow inactivation of the tetrodotoxin-insensitive Na⁺ current was kinetically distinct from the ordinary short-time steady-state inactivation. The voltage dependence of the slow inactivation could be described by a sigmoidal function, and its time course had a double-exponential process. A decrease of external pH partially antagonized the slow inactivation, probably through an increased diffusion potential across the membrane. However, the slow inactivation was not due to change in surface negative charges, since a shift of the kinetic parameters along the voltage axis was not observed during the slow inactivation. Due to the slow inactivation, the inactivation curves for the tetrodotoxininsensitive Na⁺ current were shifted in the negative direction as the prepulse duration was increased. Consequently, the window current activated at potentials close to the resting membrane potential was markedly reduced. Thus, the slow inactivation may be involved in the long-term regulation of the excitability of sensory neurons.We thank Prof. Hirosi Kuriyama for his support and advice and Dr. M. Yoshii for helpful discussions. This study was supported by the Japanese Ministry of Education (Scientific Research 02670090).     

Stretch-activated ion channels contribute to membrane depolarization after eccentric contractions

We tested the hypothesis that eccentric contractionsactivate mechanosensitive or stretch-activated ion channels (SAC) in skeletal muscles, producing increased cation conductance.Resting membrane potentials and contractile function were measured in rat tibialis anterior muscles after single or multiple exposures to aseries of eccentric contractions. Each exposure produced a significantand prolonged (>24 h) membrane depolarization in exercised musclefibers. The magnitude and duration of the depolarization were relatedto the number of contractions. Membrane depolarization was dueprimarily to an increase in Na⁺ influx, because theestimated Na⁺-to-K⁺ permeability ratio wasincreased in exercised muscles and resting membrane potentials could bepartially repolarized by substituting an impermeant cation forextracellular Na⁺ concentration. Neither theNa⁺/H⁺ antiport inhibitor amiloride nor thefast Na⁺ channel blocker TTX had a significant effect onthe depolarization. In contrast, addition of either of two nonselectiveSAC inhibitors, streptomycin or Gd³⁺, produced significantmembrane repolarization. The results suggest that muscle fibersexperience prolonged depolarization after eccentric contractions due,principally, to the activation of Na⁺-selective SAC.

     

Evidence for an intestinal Na+: Sugar transport coupling stoichiometry of 2.0

Membrane potentials maintained by normally-energized intestinal epithelium interfere with an accurate determination of the Na⁺: sugar coupling stoichiometry associated with Na⁺-dependent transport systems. The interference is due to the fact that basal Na⁺ influx is itself a potential-dependent event, and sugar transport induces a membrane depolarization which therefore modifies basal Na⁺ entry. New information obtained under circumstances in which the membrane potential is maintained near 0 indicates that the true coupling stoichiometry is 2:1 rather than the commonly-accepted value of 1:1. A 2:1 stoichiometry means that cellular electrochemical Na⁺ gradients are adequate to account for recently observed 70-fold sugar gradients maintained by these cells under certain conditions.