VI. Synaptic Fuel Cell Reactions in Vascular-Interstitial-Neuromuscular Closed Circuit: Theory of Neuromuscular Activation

Spontaneous pulses of voltage occur in the caval vein at voluntary contraction of a leg muscle both in the rat and in humans. Morphology and vascular reactions indicate that vascular-interstitial-neuromuscular circuits (VINMC) exist. They require redox reactions, which are likely to occur at proteins in the cellular membranes of the synapse. Metabolic degradation of ATP in the nerve cell is known to generate a flow of current out of the cell, creating the resting potential. It corresponds to an electrochemical equilibrium potential. An overpotential leads to a closed circuit flow of current in the VINMC, producing redox products at the electrode equivalent redox proteins. The VINMC is thereby charged. Brain impulses open ionic channels of the nerve cell body, short-circuiting the VINMC. Electrochemical reactions by the redox products produce an action potential and reverse the current in the charged VINMC. The charging and discharging of the synaptic membranes are explained as electrochemical analogs. The action potential and its height, production, transport, and disappearance of various synaptic products, including vesicles and the voltage pulses in the associated vessels such as the caval vein and the aorta, can be explained.     

Slow and rapid electrical pulses in the caval vein at pain-evoked leg contraction in the rat

Previous in vivo experiments in rats have shown that pulsed nerve stimulations of 20-50 mV require "external" electrical communication over vessels. This indicates that potential differences also should occur in associated vessels, at physiologic muscle contractions. Pinching toes of the hind leg of anaesthetized rats in the present study caused the animals to spontaneously pull the leg. This resulted in electric potential differences over two intracaval electrodes, as well as high frequency spikes. The slow potential pulses and high frequency spikes could be recorded dominantly in the vena cava compared with the extravascular tissue fluid. The electrical prerequisites and the previous experiments support the view that the vascular-interstitial fluid represents an "external" low resistant communication route of a closed circuit over the nerve cell bodies, the axons and the muscle fibers. A closed electrical circuit evidently increases our possibilities for explaining various structural and chemical events of the synaptic membranes.     

Multiple membrane systems as biological models

Summary The current-voltage equations for double, triple, and quadruple membrane systems are derived in closed form from the flow equations of irreversible thermodynamics. Numerical examples show that the behavior of these systems is very similar to that of nerve and muscle membranes. Multiple membrane systems exhibit resting potentials which do not have a characteristic Nernst concentration dependence; nonpermeant ions play a significant role in this nonlogarithmic behavior. Furthermore, multiple membrane systems have rectification properties similar to those of biological membranes. The direction of rectification is determined by the polarity of the membrane systems, not by the ionic concentrations in the bathing solutions.     

Induction of action potentials in fish red muscle by denervation

The effects of denervation on the electrical membrane properties of fish red muscle were investigated. Forty to fifty hours after denervation, miniature endplate potentials disappeared abruptly and field stimulation of the nerve within the muscle failed to evoke endplate potentials, indicating that transmission failure occurred at this time. The membrane resistance of the red muscle fibre increased after denervation. Normally innervated fish red muscles do not generate action potentials in response to either nerve or direct muscle stimulation. However, approximately 3 weeks after nerve sectioning, action potentials could be induced in the muscles. The action potential was sodium-dependent, and was sensitive to tetrodotoxin. Actinomycin D injected in the early phase after operation suppressed the induction of the action potential. These results indicate that RNA synthesis is preliminary to the induction of the action potential mechanism, and that this mechanism is under neural control.     

Simulation of developmental changes in action potentials with ventricular cell models

During cardiomyocyte development, early embryonic ventricular cells show spontaneous activity that disappears at a later stage. Dramatic changes in action potential are mediated by developmental changes in individual ionic currents. Hence, reconstruction of the individual ionic currents into an integrated mathematical model would lead to a better understanding of cardiomyocyte development. To simulate the action potential of the rodent ventricular cell at three representative developmental stages, quantitative changes in the ionic currents, pumps, exchangers, and sarcoplasmic reticulum (SR) Ca²⁺ kinetics were represented as relative activities, which were multiplied by conductance or conversion factors for individual ionic systems. The simulated action potential of the early embryonic ventricular cell model exhibited spontaneous activity, which ceased in the simulated action potential of the late embryonic and neonatal ventricular cell models. The simulations with our models were able to reproduce action potentials that were consistent with the reported characteristics of the cells in vitro. The action potential of rodent ventricular cells at different developmental stages can be reproduced with common sets of mathematical equations by multiplying conductance or conversion factors for ionic currents, pumps, exchangers, and SR Ca²⁺ kinetics by relative activities.     

Noise analysis of an ion channel in the cardiac myocyte--study based on a Markov model

Mechanical contraction of a cardiac muscle cell is related to the electric activation of the plasma membrane. As in the neuron cell, inflow of the Na(+) ions across the cell membrane causes electric activation with amplitude of about 100 mV. However, differently from the nerve cell, the action potential lasts a few hundred milliseconds before repolarization. Moreover, several types of K(+) channel such as the classical inward rectifier K(+) channel, the voltage dependent channel and others are responsible for the formation of the action potential. The mechanism of opening and closing the K(+) channels is not thoroughly elucidated. In the present paper, a four state Markov model with one open and three closed states is studied to obtain open and close probabilities of the gates constituting a specific ionic channel. The probability density functions of durations of opening and closing of the channel are also discussed.     

Excitability changes of presumptive ectoderm following mesodermal induction

Dissociated ectodermal cells of the early newt gastrula which have been treated with CMF (Ca-Mg-free saline) for 5 hr differentiate into muscle cells when cultured in HFCS (heated fetal calf serum) for up to 9-12 days. Similarly dissociated cells placed into FCS (fetal calf serum) culture differentiate into epidermis. Differences in cell-cluster formation have been found between HFCS and FCS in early cell cultures (6 hr), and membrane excitability phenomena associated with the differentiation of these clusters into the muscle cells or epidermal cells have been investigated, respectively. The HFCS cultures consist of cell clusters which have few of microvilli at their surfaces and which form loose contacts by means of lamellipodia. FCS cultures consist of cell clusters which have numerous microvilli at their surfaces and which make tight contacts between cells by means of ridge-structure precursors. The different reaggregation pattern of dissociated ectoderm cells in HFCS reflects changes in the cell membrane surface induced by HFCS. The sequential genesis of action potentials in cells destined to form muscle cells in HFCS is very similar to those produced by somitic muscle cells in vivo and their ionic dependence for generating action potentials is related to epidermal action potentials in vitro (FCS).     

The control of membrane ionic currents by the membrane potential of muscle

Comparisons between electrotronic potentials and certain predicted curves allow the identification of the membrane potential at which the sodium and potassium currents are switched on in frog sartorius. The activation potentials (the membrane potentials at which the ionic currents are great enough to be resolved by the method) are functions of the resting potential and time but not of ionic concentration. In the normal fiber, the activation potential for sodium lies nearer the resting potential and depolarizations set off sodium currents and action potentials. Below a resting potential of 55 to 60 mv. sodium activation is lost and conduction is impossible. A tenfold increase of calcium concentration lowers (moves further from the resting potential) the sodium activation potential by 20 to 25 mv. whereas the potassium activation potential is lowered by only 15 mv. Certain consequences of this are seen in the behavior of the muscle cell when it is stimulated with long duration shock.     

Ionic channels induced by sea nettle toxin in the nodal membrane.

Toxin isolated from nematocysts of the sea nettle Chrysaora quinquecirrha (SNTX) is known to depolarize nerve and muscle membranes and to increase the miniature end-plate potentials' frequency. To investigate its mode of action at the membrane level, we have studied the toxin's effects on the frog myelinated nerve fibre. We show that SNTX creates large cation-selective channels that open and close spontaneously. The conductance of these channels, almost constant in the voltage range - 100 to + 50 mV, is 760 pS. The SNTX-induced channels are almost equally permeable to Na+, Li+, K+, and Cs+, but are impermeable to Ca++. The open and closed times of SNTX-induced channels are voltage dependent, the open probability increasing with increased negative membrane potentials. To our knowledge, this is the first demonstration of the production of single-channel currents by a toxin, in a biological membrane.     

Equivalent Circuit of Frog Atrial Tissue as Determined by Voltage Clamp-Unclamp Experiments

The equivalent circuit that has been used in the analysis of nerve voltage-clamp data is that of the membrane capacity in parallel with the membrane resistance. Voltage-clamp experiments on frog atrial tissue indicate that this circuit will not suffice for this cardiac tissue. The change in membrane current associated with a step change in membrane potential does not show a rapid spike of capacitive current as would be expected for the simple parallel resistance-capacitance network. Rather, there is a step change in current followed by an exponential decay in current with a time constant of about 1 msec. This relatively slow capacitive charging current suggests that there is a resistance in series with the membrane capacity. A possible equivalent circuit is that of a series resistance external to the parallel resistance-capacitance network of the cell membranes. Another possible circuit assumes that the series resistance is an integral part of the cell membrane. The data presented in this paper demonstrate that the equivalent circuit of a bundle of frog atrial muscle is that of an external resistance in series with the cell membranes.     

How membrane proteins sense voltage

The ionic gradients across cell membranes generate a transmembrane voltage that regulates the function of numerous membrane proteins such as ion channels, transporters, pumps and enzymes. The mechanisms by which proteins sense voltage is diverse: ion channels have a conserved, positively charged transmembrane region that moves in response to changes in membrane potential, some G-protein coupled receptors possess a specific voltage-sensing motif and some membrane pumps and transporters use the ions that they transport across membranes to sense membrane voltage. Characterizing the general features of voltage sensors might lead to the discovery of further membrane proteins that are voltage regulated.     

Properties of sympathetic neuromuscular transmission and smooth muscle cell membranes in vascular beds.

In vascular smooth muscle tissues, the cycle of contraction-relaxation is mainly regulated by the cytosolic Ca, and many other factors, such as substances released from endothelial cells and perivascular nerve terminals (mainly sympathetic nerves). In this article, we introduce regional differences in specific features of ionic channels in vascular smooth muscle membranes (mainly on features of Ca, Na and K channels) in relation to mobilization of the cytosolic Ca. In many vascular tissues, neurotransmitters released from sympathetic nerve terminals activate post-junctional receptors, and subsequently modify ion channels (receptor-activated cation channel and voltage-dependent Ca channel), whereas in some tissues, ionic channels are not modified by receptor activations (pharmaco-mechanical coupling). However, activation of receptors, with or without modulation of ionic channels, regulates the cytosolic Ca through synthesis of second messengers. In addition, receptors distributed on prejunctional nerve terminals positively or negatively regulate the release of transmitters. Roles of neurotransmitters (mainly ATP and noradrenaline) are also discussed in relation to the generation of excitatory junction potentials.     

金褐霉素（Aureofuscin）对神经末梢...

By means of the intracellular recording technique, the effect of aureofuscin (20 micrograms/ml, oversaturation solution) on the ACh release from motor nerve terminals and on muscle cell membrane potential were investigated in phrenic nerve diaphragm preparations of the mice. The results showed that (a) aureofuscin reduced the resting membrane potential of the muscle cell slightly; (b) the frequency of miniature end-plate potentials and the mean quantal content of end-plate potentials increased at first and then recovered approximately to the control level; (c) the depolarization produced by aureofuscin in the muscle cell membrane was reversible and the aureofuscin-invoked facilitation in miniature end-plate potential discharges was Ca(2+)-dependent; and (d) aureofuscin did not block neuromuscular transmission.     

Transient Cl- and K+ Currents during the Action Potential in Chara inflata (Effects of External Sorbitol,Cations, and Ion Channel Blockers)

In voltage-clamp experiments, a two-pulse procedure was used to investigate the ionic currents underlying the action potential in Chara inflata. A prepulse hyperpolarized the membrane from a resting potential of about -100 to -200 mV. The prepulse was followed by a second pulse that changed the potential difference (p.d.) to -100 mV and less negative values in steps of 20 mV. This two-pulse procedure induces action potentials that have a reproducible time course, which is essential for any comparative investigation of the action potential. The two-pulse procedure reveals that in the charophyte C. inflata the electric current flowing across the cell membranes during positive voltage-clamp steps from the resting p.d. consists of a leak current flowing from the start of the pulse, followed by a transient inward-going current, Ii, commencing after a delay, and preceding a delayed transient outward current, Io. The characteristics of the current components and their response to various ion channel blockers and ionic treatments suggest that: (a) Ii, which is blocked by the external application of 9-anthracenecarboxylic acid, is carried by Cl- and (b) Io, which is blocked by the external application of the organic anions tetraethylammonium (TEA+) and nonyltriethylammonium, is carried mainly by K+. The magnitude and behavior of these K+ and Cl- currents could be modified by changes in the external concentration of CaCl2, LiCl, or NaCl but not sorbitol. Hence, it is concluded that NaCl-enhanced transient inward Cl- current, Ii, is due to ionic effects of NaCl rather than to its osmotic effects. The modification of the K+ current, Io, either by changing external K+ concentrations or by blocking the current with TEA+, also alters the Cl- currents Ii.     

Nonquantal acetylcholine release from motor nerve endings and denervation changes in rat muscle fiber membranes after axonal transport blockade

Microelectrode experiments on the rat diaphragm showed that application of colchicine, which disturbs axonal transport, to the motor nerve leads after 5 days to a decrease in resting potential and an increase in input resistance of the electrogenic membrane, disappearance of differences of input resistance between the postsynaptic and extrasynaptic membranes, the appearance of extrasynaptic sensitivity to acetylcholine, and the appearance of anode-break action potentials resistant to tetrodotoxin. Similar changes develop in the muscle membrane after division of the motor nerve. Application of colchicine to the nerve, unlike its division, does not cause cessation of contractile activity of the muscle or disturbance of quantal and reduction of nonquantal acetylcholine secretion in motor nerve endings, as reflected in the degree of hyperpolarization of the postsynaptic membrane (H effect) in response to the action of D-tubocurarine chloride on the muscle after inhibition of acetylcholinesterase. The results confirmed the view that neurotrophic control of the mammalian muscle fiber membrane is effected mainly by means of substances carried to the muscle by axonal transport. Synaptic acetylcholine, secreted from nerve endings in nonquantal form, does not play a leading role in neurotrophic control of the muscle membrane.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. Translated from Neirofiziologiya, Vol. 16, No. 2, pp. 231–238, March–April, 1984.     

Effects of physostigmine on the voltage dependent ionic conductances of skeletal muscle fibres

The voltage dependent ionic conductances were studied by analysing the phase plane trajectories of action potentials evoked by electrical stimulation of the sartorius muscles of the frog (Rana esculenta). The delayed outward potassium current was measured also under voltage clamp conditions on muscle fibres of either the frog (Rana esculenta) or Xenopus laevis. On analysing the effect of physostigmine decreasing the peak amplitude, the rate of both the rising and falling phases of the action potentials, it was revealed that the alkaloid at a concentration of 1 mmol/l reduced significantly both the delayed potassium conductance and the outward ionic current values during the action potentials. The inhibition of sodium conductance and inward ionic current was less expressed. The maximum value of delayed potassium conductance measured under voltage clamp conditions was decreased by 1 mmol/l physostigmine. The time constant determined from the development of delayed potassium conductance was increased at a given membrane potential. The voltage vs. n relationship describing the membrane potential dependence of the delayed rectifier was not influenced by physostigmine. It has been concluded that physostigmine changes the time course of the action potentials by decreasing the value of both voltage dependent ionic conductances and by slowing down their kinetics. It is discussed that results obtained from the phase plane analysis of complex pharmacological effects can only be accepted with some restrictions.     

Receptors and ionic transporters in nuclear membranes: new targets for therapeutical pharmacological interventions

Work from our group and other laboratories showed that the nucleus could be considered as a cell within a cell. This is based on growing evidence of the presence and role of nuclear membrane G-protein coupled receptors and ionic transporters in the nuclear membranes of many cell types, including vascular endothelial cells, endocardial endothelial cells, vascular smooth muscle cells, cardiomyocytes, and hepatocytes. The nuclear membrane receptors were found to modulate the functioning of ionic transporters at the nuclear level, and thus contribute to regulation of nuclear ionic homeostasis. Nuclear membranes of the mentioned types of cells possess the same ionic transporters; however, the type of receptors is cell-type dependent. Regulation of cytosolic and nuclear ionic homeostasis was found to be dependent upon a tight crosstalk between receptors and ionic transporters of the plasma membranes and those of the nuclear membrane. This crosstalk seems to be the basis for excitation-contraction coupling, excitation-secretion coupling, and excitation - gene expression coupling. Further advancement in this field will certainly shed light on the role of nuclear membrane receptors and transporters in health and disease. This will in turn enable the successful design of a new class of drugs that specifically target such highly vital nuclear receptors and ionic transporters.     

Recovery of action potentials and twitches after K-contractures in frog skeletal muscle

To give information about intracellular Ca2+ translocation during and after K-contractures in vertebrate skeletal muscle fibers, we examined recovery of action potentials and twitches after interruption and spontaneous relaxation of K-contractures at low temperature (3 degrees C) that greatly reduced the rate of Ca2+ reuptake by the sarcoplasmic reticulum. On membrane repolarization interrupting K-contractures, the amplitude of both action potentials and twitches recovered quickly, while the falling phase of action potential was markedly slowed at first to prolong its refractory period, so that repetitive stimulation (20 Hz) did not produce a complete tetanus. Meanwhile, on membrane repolarization after spontaneous relaxation of K-contractures, the action potentials were markedly reduced in amplitude and prolonged in duration at first, also resulting in prolonged refractory period. These results are discussed in connection with Ca2+ absorption to the surface and transverse tubule membranes, producing changes in action potential kinetics.     

A model for propagation of action potentials in smooth muscle

A modified Hodgkin & Huxley (1952) model for axons was used to simulate smooth muscle action potentials. The modifications were such as to match our own experimental results and published data on the passive and active behavior of smooth muscle.A brief account of the modifications introduced to the HH model is as follows. The resting ionic conductances were obtained from the data of Casteels (1969). Chloride conductance was replaced by an ad hoc leakage conductance ($\text{g}\text{?}{}_{\mathrm{L}}$) in order to obtain a resting membrane resistance of about 11 kΩcm². The ionic equilibrium potentials were according to Kao & Nishiyama (1969). The rate constants m, n and h have similar form to those in axons, but their different numerical values produce action potentials that match the duration of the smooth muscle action potential (about 16 ms) at half its maximum amplitude. The effective membrane capacitance was taken as 2.5 μF/cm².The results obtained by implementing those smooth muscle parameters in the HH formulation include: (a) a membrane potential that matches the main characteristics of experimentally recorded action potentials in uterine smooth muscle and guinea-pig taenia-coli, and (b) a propagated action potential which, on a cable diameter of 5 μm (similar to the diameter of a single smooth muscle cell), has a speed of propagation within the range of the values experimentally recorded in smooth muscle. This observed velocity of propagation is not compatible with a large cable and it is concluded that “functional units” are not required to sustain propagation of action potentials in smooth muscle.     

Cell potential and the sodium-potassium pump in vascular smooth muscle.

An electrogenic sodium-potassium pump appears to contribute materially to the steady-state potential and to certain of the transient potential responses of vascular smooth muscle. Since changes in cell potential in turn can lead to changes in contractile state, the pump is implicated in some of the constriction-dilation responses of blood vessels. The vasodilator action of potassium is explainable, for instance, through an effect on cell potential if (and only if) an electrogenic pump is assumed to be extruding sodium at a faster rate than it takes up potassium. This is supported by the observation that ouabain, an inhibitor of Na,K-ATPase activity, will eliminate or reverse the vascular effect of potassium. Furthermore, when the in vivo and in vitro effects on vascular smooth muscle of altered extracellular potassium concentration are compared to calculated cell potentials based on a model that includes an electrogenic pump, the experimental findings are shown to be logical and predictable.