ELECTRIC IMPEDANCE OF NITELLA DURING ACTIVITY

The changes in the alternating current impedance which occur during activity of cells of the fresh water plant Nitella have been measured with the current flow normal to the cell axis, at eight frequencies from 0.05 to 20 kilocycles per second, and with simultaneous records of the action potential under the impedance electrodes. At each frequency the resting cell was balanced in a Wheatstone bridge with a cathode ray oscillograph, and after electrical stimulation at one end of the cell, the changes in the complex impedance were determined from the bridge unbalance recorded by motion pictures of the oscillograph figure. An extension of the previous technique of interpretation of the transverse impedance shows that the normal membrane capacity of 0.9 µf./cm.² decreases about 15 per cent without change of phase angle, while the membrane resistance decreases from 10⁵ ohm cm.² to about 500 ohm cm.² during the passage of the excitation wave. This membrane change occurs during the latter part of the rising phase of the action potential, and it is shown that the membrane electromotive force remains unchanged until nearly the same time. The part of the action potential preceding these membrane changes is probably a passive fall of potential ahead of a partial short circuit.     

THE ELECTRICAL IMPEDANCE OF MUSCLE DURING THE ACTION OF NARCOTICS AND OTHER AGENTS

1. The effect of certain inorganic cations upon the electrical impedance of the sartorius muscle of the frog was investigated. While Na, K, and Mg have little effect upon the resistance of muscle, Ba and Ca cause it to fall. The use of physiologically "unbalanced" salt solution does not in itself seem to affect muscle impedance. 2. The time course of the effect upon muscle impedance of the penetration of substances into the intercellular spaces was studied by treating the muscle with sugar solutions. Half of the effect is over in three-quarters of a minute when the sugar solution is permitted to circulate past both sides of the muscle. This sets an upper limit for the time necessary for inorganic cations and organic narcotics to reach the cell surfaces. The action of inorganic cations and organic narcotics upon muscle is slow compared to the time necessary for them to reach the scene of action. The penetration of the sugar solutions into the intercellular spaces of muscle was found to follow the well known diffusion law, the amount diffusing in being proportional to the square root of the time. Average values of 77.7 per cent for ρ, the volume concentration of fibers; 231 ohms specific resistance for r ₂, the resistance of the interior of the fibers; and 71.0° for θ, the phase angle of the impedance locus, were obtained for the muscle in Ringer''s solution. How these values change when the muscle is placed in various concentrations of sugar was also studied. 3. The action of a number of organic narcotics upon muscle was studied. All decrease 1000 cycle resistance if the concentration is sufficiently high. A detailed analysis of the action of the narcotic, iso-amyl carbamate, was made, and it was noted that low concentrations increase resistance while higher concentrations decrease it. By investigating the effect of narcotics upon muscle impedance over a wide frequency range, it was found that during narcosis the resistance of the fiber membranes first increases and then decreases, and, if the drug is present in sufficiently great concentration, membrane resistance may completely disappear. Membrane capacity is only very slightly affected.     

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.     

Membrane Potentials of the Lobster Giant Axon Obtained by Use of the Sucrose-Gap Technique

A method similar to the sucrose-gap technique introduced be Stäpfli is described for measuring membrane potential and current in singly lobster giant axons (diameter about 100 micra). The isotonic sucrose solution used to perfuse the gaps raises the external leakage resistance so that the recorded potential is only about 5 per cent less than the actual membrane potential. However, the resting potential of an axon in the sucrose-gap arrangement is increased 20 to 60 mv over that recorded by a conventional micropipette electrode when the entire axon is bathed in sea water. A complete explanation for this effect has not been discovered. The relation between resting potential and external potassium and sodium ion concentrations shows that potassium carries most of the current in a depolarized axon in the sucrose-gap arrangement, but that near the resting potential other ions make significant contributions. Lowering the external chloride concentration decreases the resting potential. Varying the concentration of the sucrose solution has little effect. A study of the impedance changes associated with the action potential shows that the membrane resistance decreases to a minimum at the peak of the spike and returns to near its initial value before repolarization is complete (a normal lobster giant axon action potential does not have an undershoot). Action potentials recorded simultaneously by the sucrose-gap technique and by micropipette electrodes are practically superposable.     

ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON DURING ACTIVITY

Alternating current impedance measurements have been made over a wide frequency range on the giant axon from the stellar nerve of the squid, Loligo pealii, during the passage of a nerve impulse. The transverse impedance was measured between narrow electrodes on either side of the axon with a Wheatstone bridge having an amplifier and cathode ray oscillograph for detector. When the bridge was balanced, the resting axon gave a narrow line on the oscillograph screen as a sweep circuit moved the spot across. As an impulse passed between impedance electrodes after the axon had been stimulated at one end, the oscillograph line first broadened into a band, indicating a bridge unbalance, and then narrowed down to balance during recovery. From measurements made during the passage of the impulse and appropriate analysis, it was found that the membrane phase angle was unchanged, the membrane capacity decreased about 2 per cent, while the membrane conductance fell from a resting value of 1000 ohm cm.² to an average of 25 ohm cm.² The onset of the resistance change occurs somewhat after the start of the monophasic action potential, but coincides quite closely with the point of inflection on the rising phase, where the membrane current reverses in direction, corresponding to a decrease in the membrane electromotive force. This E.M.F. and the conductance are closely associated properties of the membrane, and their sudden changes constitute, or are due to, the activity which is responsible for the all-or-none law and the initiation and propagation of the nerve impulse. These results correspond to those previously found for Nitella and lead us to expect similar phenomena in other nerve fibers.     

Effects of High Hydrostatic Pressure on Morphogenesis in Naegleria gruberi (Schardinger)*

SYNOPSIS. The ameboid phase of Naegleria gruberi can be activated to transform to the flagellate phase, and cysts to excyst and transform to the flagellate phase, by a limited treatment with high hydrostatic pressure followed by release. The most effective treatment at 21 G is 45 min at 3500 psi (238 atm), which leads to almost 100% transformation. Following this dose of high pressure, 50% of amebae transform within 55–70 min after release of pressure, and nearly all within 75–120 min. Nearly all cysts hatch and transform within 200–240 min after release. Pressures of 4000 psi (272 atm) and above, and of 1000 psi (68 atm) and below, were ineffective at any duration of treatment.     

THE USE OF PHOSPHOLIPASE C IN THE STUDY OF RECEPTORS FOR NEUROMUSCULAR BLOCKING AGENTS

Abstract— Segments of diaphragmatic muscle and cerebral nerve-ending particles from the rat were treated exhaustively (180 min) with high concentrations of phospholipase C (300 μg/ml) (EC 3.1.4.3). This enzyme caused loss of approx. 50 per cent of muscle membrane P and 70 per cent of nerve-ending membrane P. At lower concentrations (3 μg/ml; 180 min), phospholipase C released 25 per cent muscle membrane P and 55 per cent of nerve-ending membrane P. At the lower concentration (3 μg/ml; 180 min), the enzyme caused a slow but progressive loss (90 per cent) of muscle twitch amplitude evoked by nerve stimulation (phrenic nerve-hemidiaphragm preparation). Loss of muscle twitch amplitude was notably slowed by removal of the enzyme. Phospholipase C-treated neuromuscular junctions developed no resistance to hemicholinium, botulinum toxin, or d-tubocurarine. Such preparations were unusually sensitive to the neuromuscular blocking action of di-isopropylfluorophosphate. The data do not encourage a belief that phospholipids which are substrates for phospholipase C are receptors for hemicholinium, botulinum toxin, d-tubocurarine or di-isopropylfluorophosphate.     

Intracrine action of angiotensin II in the intact ventricle of the failing heart: angiotensin II changes cardiac excitability from within

The influence of intracellular injection of angiotensin II (Ang II) on electrical properties of single right ventricular fibers from the failing heart of cardiomyopathic hamsters (TO2) was investigated in the intact ventricle of 8-month-old animals. Intracellular injection was performed using pressure pulses (40-70 psi) for short periods of time (20 ms) while recoding the action potential simultaneously from the same fiber. The results indicated that intracellular Ang II caused a hyperpolarization of 7.7 mV ± 4.3 mV (n = 39) (4 animals) (P < 0.05) followed by a small fall in membrane potential. The action potential duration was significantly increased at 50% and at 90% repolarization, and the refractoriness was significantly enhanced. The effect of intracellular Ang II on action potential duration was related to the inhibition of potassium conductance through PKC activation because Bis-1 (360 nM), a selective PKC inhibitor, abolished the effect of the peptide. Injections performed in different fibers of the same ventricle showed a variable effect of Ang II on action potential duration and generated spontaneous rhythmicity. The effect of intracellular Ang II on action potential duration and cardiac refractoriness remains for more than 1 h after interruption of the intracellular injection of the peptide.     

Temperature acclimation of axonal functions in the crayfish Astacus astacus L.

1. 1.|Crayfish (Astacus astacus L.) were acclimated for 1–3 weeks at 5 and 20°C. The effects of temperature on the functions of the unicellular medial giant axon were studied.

2. 2.|The resting membrane potential of the giant axon increased slightly with the experimental temperature from 2 to 32°C. The temperature dependence of the resting membrane potential could be described by two lines, which intersected at about 12°C in cold-acclimated crayfish and at about 16°C in the warm-acclimated.

3. 3.|The amplitude of the action potential was stable at temperatures from 4 to 26°C. It decreased at temperatures above 26°C in both acclimation groups.

4. 4.|The duration of the falling phase of action potential was highly temperature dependent at low temperatures. A break in the slope of the dependence was found at about 14°C in cold-acclimated crayfish and at about 17°C in the warm-acclimated.

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

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

Semi-excitation in nerve and electric nerve model (ENM) (author's transl)]

The "semi-excitation" is an excitation (like phenomenon) found by the authors, first in ENM and then in nerve during observation of the action potential and impedance decrease associated with excitation. The phenomenon was further investigated in relation to the action potential and impedance decrease (for AC of 13 kHz) of the stimulated site on the sciatic nerve and ENM. The nature of the phenomenon was the following. 1. The configuration of the action potential and impedance decrease were almost the same as that of the (complete) excitation, but the durations of the potential change (plateau) and impedance decrease depended on entirely to the duration of stimulating current. 2. Submaximum amplitude of action potential and impedance decrease were graded by the intensity of the stimulating current, but the threshold of this excitation was found. 3. During or after the semi-excitation, absolute or relative refractory period was not found. 4. Both in nerve and ENM, transition from complete excitation to semi-excitation or vice versa was observed. 5. Semi-excitation was found to occurr in the state of reduced membrane potential of the nerve and ENM.     

Effect of motor function disorder on the properties of the muscle fiber membrane

Experiments on frogs with the use of the microelectrode techniques were made to study the effect of tenotomy and immobilization of a limb with a metal cast in the extension position on the properties of the membrane of muscle fibers. Two weeks after tenotomy there were no changes in the magnitude of the membrane rest potential, input resistance and time constant of the membrane of muscle fibers or in the pattern of its sensitivity to acetylcholine. Two and three weeks after the limb immobilization no changes in the membrane rest potential and passive electrical properties of the muscle membrane were recorded either. However, if the time elapsed after immobilization was 2 and 3 weeks, the zone of the sensitivity of muscle fibers to acetylcholine was slightly greater than in the control. It is suggested that the motor activity in the frog per se is not the determinant of the muscle fiber differentiation preset by the nervous system.     

Effect of Ethanol on the Sodium and Potassium Conductances of the Squid Axon Membrane

The effects of ethanol on squid giant axons were studied by means of the sucrose-gap technique. The membrane action potential height is moderately reduced and the duration sometimes shortened by ethanol in sea water. Voltage clamp experiments showed that ethanol in sea water reduced the maximum membrane conductances for sodium (g'_Na) and potassium (g'_K). In experiments with multiple application of ethyl alcohol to the same spot of membrane, a reduction of g'_Na to 82 per cent and of g'_K to 80 per cent of their value in sea water was brought about by 3 per cent ethanol (by volume) while 6 per cent caused a decrease of g'_Na to 59 per cent and of g'_K to 69 per cent. Ethanol has no significant effect on the steady-state inactivation of g_Na (as a function of conditioning membrane potential) or on such kinetic parameters as τ_h or the time course of turning on gi g_Na and g_K. It is concluded that ethanol mainly reduces g_Na and g_K in the Hodgkin-Huxley terminology.     

Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.     

Impedance of Membrane and Myoplasm during the Action Potential of Frog Muscle

The transient imbalance of a Wheatstone bridge was used to estimate the changes in both membrane and myoplasmic impedance that occurred as an action potential propagated over a 1 mm length of a frog muscle fiber. It was also used to estimate the changes in the membrane impedance alone. Alterations in myoplasmic impedance that might have been predicted were not found.     

pH dependence of protamine action on apical membrane permeability in Necturus gallbladder epithelium

Protamine reversibly decreases cation permeability and alters the structure of Necturus gallbladder tight junctions. Conflicting results, however, have been published whether or not it also affects apical cell membrane permeability. We investigated this issue more systematically by measuring voltage (psi mc) and fractional resistance (fRa) of the apical membrane at varying concentrations of protamine, K+, and H+ in the bathing solution. At pH 7.6 and [K+] 2.5 mM, (Poler, M.S. and Reuss, L. (1987) Am. J. Physiol. 253, C662) 6 microM protamine caused psi mc to depolarize from -58 to -51 mV and fRa to decrease from 0.74 to 0.67. If we increased pH to 8.1 these effects were even more pronounced. At [K+] 2.5 mM, but not 4.5 mM, psi mc transiently hyperpolarized for about 5 min after adding protamine. Most importantly, if [K+] was 4.5 mM and pH was adjusted to 7.1 (Bentzel et al. (1987) J. Membr. Biol. 95, 9) no significant changes of psi mc and fRa occurred. In any case, at a supramaximal concentration of 200 microM, protamine did not further increase the paracellular response but produced decreasing psi mc and fRa. We conclude that 6 microM protamine decreases K+ conductance of the apical membrane, if it is already tuned high by high pH. At low control K+ conductance as observed at lower pH, protamine action is restricted to the paracellular pathway. Thus, conflicting results were due to different experimental conditions. At a solution pH of 7.1, 6 microM protamine fulfills criteria of a selective tool for reversibly altering structure and function of the tight junction in Necturus gallbladder.     

Differences in Na and Ca Spikes As Examined by Application of Tetrodotoxin, Procaine, and Manganese Ions

The effects of tetrodotoxin, procaine, and manganese ions were examined on the Ca spike of the barnacle muscle fiber injected with Ca-binding agent as well as on the action potential of the ventricular muscle fiber of the frog heart. Although tetrodotoxin and procaine very effectively suppress the "Na spike" of other tissues, no suppressing effects are found on "Ca spike" of the barnacle fiber, while the initiation of the Ca spike is competitively inhibited by manganese ions. The initial rate of rise of the ventricular action potential is suppressed by tetrodotoxin and procaine but the plateau phase of the action potential is little affected. In contrast the suppressing effect of manganese ions is mainly on the plateau phase. The results suggest that the plateau phase of the ventricular action potential is related to the conductance increase in the membrane to Ca ions even though Na conductance change may also contribute to the plateau.     

Analysis of mechanism of conduction of nerve impulses during the relative refractory phase by a mathematical model of the giant squid axon

By use of the mathematical model of Hodgkin and Huxley we have investigated changes in membrane currents and in ionic permeability of the membrane during conduction of an action potential along a refractory axon. At the start of the relative refractory period, the action potential has a graded character, and is extinguished over a distance of a few millimeters from the site of stimulation (decremental conduction). These changes are brought about not only by the low value of the inactivation variable h and the high potassium conductivity g_K, but also by the inhibitory influence on the recovery process exerted by the spreading wave of depolarization. The graded peak can be propagated along the whole fiber in approximately 0.6 msec at 18.5° after the end of the absolute refractory phase. At this time the wave of the recovery process (increase of h and decrease of g_K) begins to oppose the wave of depolarization, with the result that at each subsequent point along the fiber h increases above and g_K decreases below the preceding value. Under these circumstances, conduction becomes incremental, and in order to evoke a propagated action potential all that is required is to induce a subthreshold local response at the point of stimulation.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 3, No. 4, pp. 434–441, July–August, 1971.     

Oxygen taxis and proton motive force in Salmonella typhimurium

The aerotactic response of Salmonella typhimurium SL3730 has been quantitatively correlated with a change in the proton motive force (delta p) as measured by a flow-dialysis technique. At pH 7.5, the membrane potential (delta psi) in S. typhimurium changed from -162 +/- 13 to -111 +/- 15 mV when cells grown aerobically were made anaerobic, and it returned to the original value when the cells were returned to aerobiosis. The delta pH across the membrane was zero. At pH 5.5, delta psi was -70 mV in aerobiosis and -20 mV in anaerobiosis, and delta pH was -118 and -56 mV for aerobic and anaerobic cells, respectively. A decrease in delta p resulted in increased tumbling, and an increase in delta p resulted in a smooth swimming response at either pH. Inhibition of aerotaxis at pH 7.5 by various concentrations of KCN correlated with a decreased delta p, due to a decreased delta psi in aerobiosis and little change in delta psi in anaerobiosis. At concentrations up to 100 mM, 2,4-dinitrophenol decreased delta psi, but did not inhibit aerotaxis because the difference between delta psi in aerobic and anaerobic cells remained constant. Considered as a whole, the results indicate that aerotaxis in S. typhimurium is mediated by delta p.     

The Relationship Between the Bulk Modulus of Elasticity of a Complex Tissue and the Mean Modulus of its Cells

A relationship has been derived mathematically between the bulkmodulus of elasticity of a leaf, E, measured by the pressurebomb technique, and the individual cellular bulk moduli, e,of the leaf tissue as measured by the pressure probe technique.A number of examples have been worked out that shows that Eunderestimates the mean value of e by less than 10 per centin most circumstances. Within 2 per cent, E equals the ‘mean’value of e weighted according to the water content change ofeach cell expressed as a fraction of the water content changeof the whole leaf when the water potential. , changes by a smallamount. An analysis of the sources of error involved in theevaluation of E by the pressure bomb technique reveals thatE can be calculated within an error of 10 per cent from valuesof and water loss, W_e; this error is about the same as theerror in e measured on individual cells by the pressure probetechnique. bulk elastic modulus, water potential isotherms, pressure bomb, pressure probe