The effects of hydrostatic pressure upon the normal and narcotized nerve fiber

The properties of the giant axon of the squid Loligo pealii were studied at different hydrostatic pressures from 14.7 to 16,000 psi. At 4000 psi the resting potential, the membrane resistance, membrane capacity, the conduction velocity, the amplitude of the action potential, and the maximal change in the membrane impedance during activity were only slightly affected. At the same pressure the duration of the falling phase of the action potential was increased by about 40 to 60 per cent and the duration of the rising phase by about 20 to 35 per cent. The duration of the membrane impedance change during activity was increased by 50 to 100 per cent at 4000 psi. At pressures even slightly above atmospheric the threshold membrane current was appreciably reduced. At about 3000 to 7000 psi the fiber fired spontaneously. At pressures considerably above 5000 psi the membrane resistance decreased to about one-half to one-third the original value. The narcotizing effect upon the nerve fiber of 3 to 7 per cent ethanol was partly or almost completely opposed by low temperatures or high pressures.     

Effect of formaldehyde and glutaraldehyde on electrical properties of cardiac Purkinje fibers

The effects of formaldehyde, glutaraldehyde, 1-fluoro-2,4-dinitrobenzene, and 1,5-difluoro-2,4-dinitrobenzene on the electrophysiological properties of cardiac Purkinje fibers were studied. At concentrations of 2.5 mM the aldehydes produced a transient hyperpolarization, lengthening of the plateau of the action potential, and an increase in action potential overshoot and upstroke velocity. If exposure to aldehyde was continued, the fiber failed to repolarize after an action potential and the membrane potential stabilized at about -30 mv. If exposure was terminated before this, recovery was usually complete. At the time the fibers were hyperpolarized the input resistance was increased without much change in length constant, leading to an increase in both calculated membrane resistance and calculated core resistance. Although it was anticipated that an effect of the aldehydes on the membrane was to increase fixed negative charge, it was difficult to explain all the electrophysiological changes on this basis. The major effects of the fluorobenzene compounds were not the same; they produced a shortening of the action potential and a rapid loss of excitability.     

Rapid increase in plasma membrane chloride permeability during wound resealing in starfish oocytes

Plasma membrane wound repair is an important but poorly understood process. We used femtosecond pulses from a Ti-Sapphire laser to make multiphoton excitation-induced disruptions of the plasma membrane while monitoring the membrane potential and resistance. We observed two types of wounds that depolarized the plasma membrane. At threshold light levels, the membrane potential and resistance returned to prewound values within seconds; these wounds were not easily observed by light microscopy and resealed in the absence of extracellular Ca(2+). Higher light intensities create wounds that are easily visible by light microscopy and require extracellular Ca(2+) to reseal. Within a few seconds the membrane resistance is approximately 100-fold lower, while the membrane potential has depolarized from -80 to -30 mV and is now sensitive to the Cl(-) concentration but not to that of Na(+), K(+), or H(+). We suggest that the chloride sensitivity of the membrane potential, after wound resealing, is due to the fusion of chloride-permeable intracellular membranes with the plasma membrane.     

Passive ionic properties of frog retinal pigment epithelium.

The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was --88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8--20 mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductance Ti of each membrane. For the apical membrane: TK approximately equal to 0.52, THCO3 approximately equal to 0.39 and TNa approximately equal to 0.05, and its specific resistance was estimated to be 6000--7000 omega cm2. For the basal membrane: TK approximately equal to 0.90 and its specific resistance was estimated to be 400--1200 omega cm2. From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110 mM. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered "tight". The shunt pathway did not distinguish between anions (HCO--3, Cl--, methylsulfate, isethionate) but did distinguish between Na+ and K+.     

Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements

We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of both open-state dwell times and closed-state dwell times could be fit by single exponentials. For more depolarized potentials, although the open-state histograms could still be fit by single exponentials, the closed-state histograms required two exponentials. This data together with macroscopic voltage clamp data on the same system could be accounted for by a three-state closed-closed-open model with transition rates between these states that are exponential functions of membrane potential. One of the implications of this model, in agreement with experiment, is that there are always some closed BTX-modified sodium channels, regardless of membrane potential.     

Quantitative study of the importance of water permeability in plant cold hardiness

The rate of ice formation was measured for Hedera helix L. cv. Thorndale (English ivy) bark exposed to -10 C. The cooling rate of bark exposed to -10 C was 31 C per minute. The water efflux rate required for ice formation to occur extracellularly was calculated from the rate of ice formation and the average cell diameter. The water potential difference driving the efflux of water to sites of extracellular ice was calculated from the sample temperature, osmotic water potential, and fraction of water frozen at a given freezing temperature. From the water efflux rate and water potential difference, the resistance of the barrier controlling movement of intracellular water to sites of extracellular ice was calculated. Comparison of the resistance of this barrier to water movement with the resistance of the cell membrane revealed that the membrane represented only 0.5% of the barrier resistance. Thus, membrane resistance can have little influence on the rate of water efflux and ice formation when bark is cooled at a rate of 31 C per minute. If ice formation occurred at the same rate in ivy bark as it occurred in a 10 mm MnCl(2) solution, the membrane resistance would still have represented only 1% of the resistance of the barrier to ice formation. Therefore, at a cooling rate of 31 C/minute, heat removal plays a large part in determining the rate of ice formation. At slower cooling rates experienced under natural freezing conditions the ability to remove heat would play an even larger role. It is concluded that under natural freezing conditions membrane resistance does not limit water efflux.     

Changing electrical constants of the Fundulus egg plasma membrane

Electrical constants of the plasma membrane of the Fundulus egg have been measured with microelectrodes by the transient method. No consistent and significant membrane potential was measured. Membrane capacity averages 0.63 µF/cm.² for both unactivated and activated eggs. Membrane resistance averages 3450 ohm-cm.² in the unactivated eggs, but increases 2 to 7 times to an average of 13,290 ohm-cm.² in the fully activated state. In a hypertonic sucrose solution, the swelling of the egg proper is accompanied by a rapid fall of membrane resistance towards that in the unactivated state. The changes of the membrane resistance are interpreted as probably caused by alterations in the effective pore size in the plasma membrane.     

Effects of divalent cation ionophores on the neuron membrane of the crayfish

Summary The effects of divalent cation ionophores, A23187 and X-537A, on the electrical membrane properties were investigated by using the soma membrane of the X-organ of the crayfish. They reduced the amplitude and maximum rate of rise of Ca-action potential in lower concentration. As the concentration increased, a reduction of membrane resistance and hyperpolarization occurred simultaneously. Further increase resulted in membrane depolarization with a further decrease in resistance. The threshold concentration of X537A was 100 times higher than that of A23187. These effects were reversible only when the application period was relatively short, while a longer application resulted in an incomplete reversibility or in no reversibility at all. The ionophore effect was facilitated in high Ca medium and diminished in low Ca medium. In Sr medium, the same effects on the resistance and the membrane potential were barely observable. TEA reduced the effects of A23187 but did not completely inhibit the effects. The Na-action potential was also reduced by the higher concentration of the ionophore. From these results it is concluded that the divalent cation ionophores, A23187 and X537A, carry divalent cation, Ca ions in a physiological medium, into the neuron soma through the membrane and the consequent increase of the intracellular divalent cations induces K conductance increase and that higher concentration of the ionophore induces the increase in the conductance of the other ion species, such as Na.     

Colistin interactions with the mammalian urothelium

Here we describe the effect of colistin on the barrier function of the mammalian urinary bladder epithelium. Addition of colistin to the mucosal solution of the rabbit urinary bladder epithelium (urothelium) resulted in an increase in the transepithelial conductance. The magnitude of the increase in transepithelial conductance was dependent on the membrane voltage, concentration of colistin, and presence of divalent cations in the bath solution. The initial site of action of colistin was at the apical membrane. Colistin increased the membrane conductance only when the apical membrane potential was cell interior negative. The more negative the membrane potential, the larger the conductance increase. The concentration dependence of the conductance increase saturated, suggesting a membrane binding site. Divalent cations decreased the magnitude of the conductance increase. This divalent cation action occurred at two sites: one in competition with colistin for a membrane binding site, and the other by rapidly blocking the induced conductance. At short exposure times, the increase in conductance was reversed by either removing colistin from the bath or changing the voltage so that the apical membrane was cell interior positive. At long exposure times, the increase was only partially reversible by voltage or removal from the bath. This finding suggests that at long exposure times, there is a toxic effect of colistin on the urothelium. bladder epithelium; epithelial transport; tight junctions; antibiotics; cationic proteins     

Adaptation in the Ventral Eye of Limulus is Functionally Independent of the Photochemical Cycle, Membrane Potential, and Membrane Resistance

The early receptor potential (ERP), membrane potential, membrane resistance, and sensitivity were measured during light and/or dark adaptation in the ventral eye of Limulus. After a bright flash, the ERP amplitude recovered with a time constant of 100 ms, whereas the sensitivity recovered with an initial time constant of 20 s. When a strong adapting light was turned off, the recovery of membrane potential and of membrane resistance had time-courses similar to each other, and both recovered more rapidly than the sensitivity. The receptor depolarization was compared during dark adaptation after strong illumination and during light adaptation with weaker illumination; at equal sensitivities the cell was more depolarized during light adaptation than during dark adaptation. Finally, the waveforms of responses to flashes were compared during dark adaptation after strong illumination and during light adaptation with weaker illumination. At equal sensitivities (equal amplitude responses for identical flashes), the responses during light adaptation had faster time-courses than the responses during dark adaptation. Thus neither the photochemical cycle nor the membrane potential nor the membrane resistance is related to sensitivity changes during dark adaptation in the photoreceptors of the ventral eye. By elimination, these results imply that there are (unknown) intermediate process(es) responsible for adaptation interposed between the photochemical cycle and the electrical properties of the photoreceptor.     

Electron Microscopy and Electrophysiology of Local Cell Wall Formation in Chara corallina

The formation of fibrillar and amorphous local wall appositionsinduced by wounding and treatment with chlortetracycline (CTC)was studied in internodal cells of Chara corallina. Woundingcauses a transient depolarisation of the membrane and a transientincrease in membrane conductance. The formation of a fibrillar,cellulosic wound wall starts after the cell wall lesion hasbeen plugged by vacuolar crystals and cytoplasmic inclusions.At that time both membrane potential and membrane conductancehave recovered their normal values. CTC induces a persistentelectrophysiological response and formation of the amorphous,callose-containing wall appositions takes place at low (morepositive) membrane potential and low membrane resistance, suggestingan increased passive membrane permeability. The electrophysiologicalchanges increase with the Ca²⁺ concentration of the CTC solution.The relevance of the membrane potential for the formation oflocal wall appositions is discussed. (Received June 29, 1993; Accepted September 16, 1993)     

Measurement of the membrane potential in small cells using patch clamp methods

The resting membrane potential, E(m), of mammalian cells is a fundamental physiological parameter. Even small changes in E(m) can modulate excitability, contractility and rates of cell migration. At present accurate, reproducible measurements of E(m) and determination of its ionic basis remain significant challenges when patch clamp methods are applied to small cells. In this study, a mathematical model has been developed which incorporates many of the main biophysical principles which govern recordings of the resting potential of 'small cells'. Such a prototypical cell (approx. capacitance, 6 pF; input resistance 5 GΩ) is representative of neonatal cardiac myocytes, and other cells in the cardiovascular system (endothelium, fibroblasts) and small cells in other tissues, e.g. bone (osteoclasts) articular joints (chondrocytes) and the pancreas (β cells). Two common experimental conditions have been examined: (1) when the background K(+) conductance is linear; and (2) when this K(+) conductance is highly nonlinear and shows pronounced inward rectification. In the case of a linear K(+) conductance, the presence of a "leakage" current through the seal resistance between the cell membrane and the patch pipette always depolarizes E(m). Our calculations confirm that accurate characterization of E(m) is possible when the seal resistance is at least 5 times larger than the input resistance of the targeted cell. Measurement of E(m) under conditions in which the main background current includes a markedly nonlinear K(+) conductance (due to inward rectification) yields complex and somewhat counter-intuitive findings. In fact, there are at least two possible stable values of resting membrane potential for a cell when the nonlinear, inwardly rectifying K(+) conductance interacts with the seal current. This type of bistable behavior has been reported in a variety of small mammalian cells, including those from the heart, endothelium, smooth muscle and bone. Our theoretical treatment of these two common experimental situations provides useful mechanistic insights, and suggests practical methods by which these significant limitations, and their impact, can be minimized.     

A synthetic strand of cardiac muscle: its passive electrical properties

The passive electrical properties of synthetic strands of cardiac muscle, grown in tissue culture, were studied using two intracellular microelectrodes: one to inject a rectangular pulse of current and the other to record the resultant displacement of membrane potential at various distances from the current source. In all preparations, the potential displacement, instead of approaching a steady value as would be expected for a cell with constant electrical properties, increased slowly with time throughout the current step. In such circumstances, the specific electrical constants for the membrane and cytoplasm must not be obtained by applying the usual methods, which are based on the analytical solution of the partial differential equation describing a one-dimensional cell with constant electrical properties. A satisfactory fit of the potential waveforms was, however, obtained with numerical solutions of a modified form of this equation in which the membrane resistance increased linearly with time. Best fits of the waveforms from 12 preparations gave the following values for the membrane resistance times unit length, membrane capacitance per unit length, and for the myoplasmic resistance: 1.22 plus or minus 0.13 x 10-5 omegacm, 0.224 plus or minus 0.023 uF with cm-minus 1, and 1.37 plus or minus 0.13 x 10-7 omegacm-minus 1, respectively. The value of membrane capacitance per unit length was close to that obtained from the time constant of the foot of the action potential and was in keeping with the generally satisfactory fit of the recorded waveforms with solutions of the cable equation in which the membrane impedance is that of a single capacitor and resistor in parallel. The area of membrane per unit length and the cross-sectional area of myoplasm at any given length of the preparation were determined from light and composite electron micrographs, and these were used to calculate the following values for the specific electrical membrane resistance, membrane capacitance, and the resistivity of the cytoplasm: 20.5 plus or minus 3.0 x 10-3 omegacm-2, l.54 plus or minus 0.24 uFWITHcm-minus 2, and 180 plus or minus 34 omegacm, respectively.     

Changes in membrane potential and resistance caused by transient increase of potassium conductance in the unicellular green alga Eremosphaera viridis

The electrophysiological membrane parameters of the unicellular green alga Eremosphaera viridis were determined using an improved computer-supported single-microelectrode technique. These cells developed an average membrane potential of-150 mV in the light and a specific resistance of 1 Ω m² with an external potassium concentration of 1.1 mM and pH 5.5. In the dark, many cells showed a less polarized potential of 30–40 mV and a smaller membrane resistance. At potassium concentrations in the external medium higher than 1 mM, the membrane potential strongly depends on the external potassium content apart from a small electrogenic component. At concentrations lower than 1 mM K⁺, a dependence of the membrane potential upon external potassium concentrations could not be verified. Inserting the internal ion activities in the Goldmann equation shows that, in this range, the proton conductance seems to be predominant over the potassium conductance. Transient changes in the membrane potential and in the membrane resistance were observed after switching off the light, after addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or N,N′-dicyclohexylcarbodiimide, after a sudden decrease in temperature, and after current pulses. These changes resemble the action potentials (AP) found in other plant cells (Chara, Acetabularia). On average, the AP has a delay period of 5.1 s and a duration of 43.8 s showing a sudden decrease and a slower regeneration. The voltage peak during an AP followed exactly the Nernst potential of potassium over a range of external potassium concentrations from 5 μM to 0.2 M. This is true for depolarization or hyperpolarization, depending on the external K⁺-concentration. Tetraethylammonium-hydrogensulphate, a rather specific inhibitor of K⁺ channels in nervous cells, suppressed the AP. The correlation of the appearance of the AP with a short-term opening of potassium channels in the membrane of Eremosphaera is discussed.     

Excitotoxicity of lathyrus sativus neurotoxin in leech retzius neurons

The effects of Lathyrus sativus neurotoxin were studied on the cell membrane potential and cellular cation composition in Retzius nerve cells of the leech Haemopis sanguisuga, with ion-selective microelectrodes using liquid ion-exchangers. Bath application of 10(-4) mol/l Lathyrus sativus neurotoxin for 3 min depolarized the cell membrane potential and decreased the input resistance of directly polarized membrane in Retzius neurons. At the same time the cellular Na+ activity increased and cellular K+ activity decreased with slow but complete recovery, while the intracellular Ca2+ concentration was not changed. Na+-free Ringer solutions inhibited the depolarizing effect of the neurotoxin on the cell membrane potential. Zero-Ca2+ Ringer solution or Ni2+-Ringer solution had no influence on the depolarizing effect of the neurotoxin on the cell membrane potential. It is obvious that the increase in membrane conductance and depolarization of the cell membrane potential are due to an influx of Na+ into the cell accompanied by an efflux of K+ from the cell.     

The cylindrical cell with a time-variant membrane resistance. Measuring passive parameters.

The passive electrical properties of a cable can be measured by injecting a step of current at a point and fitting the resulting potentials at several positions along the cable with analytic solutions of the cable equation. An error analysis is presented for this method (which is based on constant membrane resistance) when the membrane resistance is not constant, but increases linearly with time. The increase of rm produces a "creep" in the membrane potential at long times, as observed in cardiac, skeletal, and smooth muscle. The partial differential equation describing the time-varying cable was solved numberically for a step of current and these "data" were fit by standard constant-resistance methods. Comparing the resulting parameter values with the known true values, we suggest that a correction of the standard methods is not satisfactory for resistance changes of the kind observed; instead, the cable equation must be solved again for the particular form of rm(t). The practical implementation of a method by Adrian and Peachey for measuring the membrane capacitance and an approximate method for estimating the rate-of-change of membrane resistance are discussed in appendices.     

Anomalous resistance changes following application of the neurotransmitterl-Glutamate in insect muscle

Summary Bath application ofl-glutamate, to larval dipteran muscle, at concentrations between 10^–6 and 10^–4 M will cause a depolarisation of the muscle membrane potential associated with an increase in muscle input resistance. At concentrations above 10^–4 M there is usually a transient decrease in input resistance preceding the resistance increase. l-aspartate at concentrations above 10^–5 M causes membrane depolarisations which are always associated with an increase in muscle input resistance.The pharmacology of the receptors regulating membrane depolarisations associated with increases in input resistance has been compared with the receptors gating synaptically activated cation channels and found to show significant differences in sensitivity to various ligands.     

Methylmercury: Effects on electrical properties of squid axon membranes

At low concentrations (25–100 μM) methylmercury chloride caused a steady increase in the threshold for excitation and on eventual block of action potentials without changing the resting membrane potential in squid giant axons. In the axons exposed to 25 μM methylmercury chloride, peak transient and steady-state conductances were decreased by 58.8 ± 5.1% and 35.9 ± 4.3% (mean ± SEM, 4 axons), respectively and leakage conductance increased to about five times of the control value. Higher concentrations of methylmercury chloride decreased the resting membrane potential. A concentration of 0.5 mM depolarizing the nerve membrane by 16 ± 2 mV (mean ± SEM, 3 axons) in 40 minutes. These changes in ionic conductances and membrane potential were irreversible on washing the axon with drug-free sea water.     

Relative Ion Permeabilities in the Crayfish Giant Axon Determined from Rapid External Ion Changes

The changes in membrane potential of isolated, single crayfish giant axons following rapid shifts in external ion concentrations have been studied. At normal resting potential the immediate change in membrane potential after a variation in external potassium concentration is quite marked compared to the effect of an equivalent chloride change. If the membrane is depolarized by a maintained potassium elevation, the immediate potential change due to a chloride variation becomes comparable to that of an equivalent potassium change. There is no appreciable effect on membrane potential when external sodium is varied, at normal or at a depolarized membrane potential. Starting from the constant field equation, expressions for the permeability ratios P _Cl/P _K, P _Na/P _K, and for intracellular potassium and chloride concentrations are derived. At normal resting membrane potential, P _Cl/P _K is 0.13 but at a membrane potential of -53 mv (external potassium level increased about five times) it is 0.85. The intracellular concentrations of potassium and chloride are estimated to be 233 and 34 mM, respectively, and it is pointed out that this is not compatible with ions distributed in a Nernst equilibrium across the membrane. It is also stressed that the information given by a plot of membrane potential vs. the logarithm of external potassium concentrations is very limited and rests upon several important assumptions.     

Reexamination of the double sucrose gap technique for the study of lobster giant axons. Theory and experiments

The double sucrose gap technique for the study of lobster giant axons has been reexamined. The leakage behavior of the system cannot be successfully modeled by conventional sucrose gap theory, but is accounted for by the McGuigan-Tsien model that takes into account the cable properties of membrane under sucrose. The facts of high-leakage conductance and the ability to maintain large resting potentials in the face of low sucrose gap resistance lead to a hypothesis that membrane resistance under sucrose is very low because of a large negative surface potential. Computer simulations of the leakage behavior of the conventional gap model and the McGuigan-Tsien model were compared with experimental measurements on lobster axons using normal sucrose or sucrose doped with Na+, Ca2+ or La3+ ions. As the concentration of doping ion increased, the leakage rose, but the species of doping ion had more influence on leakage than gap resistance. At equal gap resistance, leakage decreased with an increase in valence of the doping species. Leakage was even lower in La-doped sucrose at 20 M omega gap resistance than in normal sucrose at 200 M omega gap resistance. Resting potentials decreased with decreasing gap resistance and increasing valence of the doping species. Resting potential behavior was successfully simulated with a hybrid model consisting of a point node flanked by infinite cables and a shunt between ground and the voltage-measuring pool. The data support the hypothesis that the membrane resistance under sucrose is low and that it can be raised by doping the sucrose with multivalent cations, with La3+ being particularly effective.(ABSTRACT TRUNCATED AT 250 WORDS)