Showing papers in "The Journal of General Physiology in 1968"
TL;DR: A drop in temperature increased the caffeine-induced Ca release while procaine inhibited it and inhibited the rate of Ca uptake, which may in part account for the prolongation of the active state caused by caffeine.
Abstract: At concentrations between 1 to 10 mM, caffeine reduced the Ca-accumulating capacity of fragmented reticulum obtained from frog and rabbit muscle. With 8 mM caffeine enough Ca was released from frog reticulum to account for the force of the contracture. Caffeine did not affect all reticulum membranes equally. The fraction which was spun down at 2000 g was more sensitive than the lighter fractions. The percentage of the total accumulated Ca released by caffeine decreased with decreasing Ca content of the reticulum. In parallel with their known effects on the caffeine contracture, a drop in temperature increased the caffeine-induced Ca release while procaine inhibited it. Caffeine also inhibited the rate of Ca uptake, which may in part account for the prolongation of the active state caused by caffeine.
654 citations
TL;DR: The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels, assumed to comprise several types, of varying affinity for divalent and hydrogen ions.
Abstract: The voltage dependence of the voltage clamp responses of myelinated nerve fibers depends on the concentration of divalent cations and of hydrogen ions in the bathing medium. In general, increases of the [Ca], [Ni], or [H] increase the depolarization needed to elicit a given response of the nerve. An e-fold increase of the [Ca] produces the following shifts of the voltage dependence of the parameters in the Hodgkin-Huxley model: m∞, 8.7 mv; h∞, 6.5 mv; τn, 0.0 mv. The same increase of the [H], if done below pH 5.5, produces the following shifts: m∞, 13.5 mv; h∞, 13.5 mv; τn, 13.5 mv; and if done above pH 5.5: m∞, 1.3 mv; h∞, 1.3 mv; τn, 4.0 mv. The voltage shifts are proportional to the logarithm of the concentration of the divalent ions and of the hydrogen ion. The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels. The charged groups are assumed to comprise several types, of varying affinity for divalent and hydrogen ions. The charges near the sodium channels differ from those near the potassium channels. As the pH is lowered below pH 6, the maximum sodium conductance decreases quickly and reversibly in a manner that suggests that the protonation of an acidic group with a pKa of 5.2 blocks individual sodium channels.
546 citations
TL;DR: Tetrodotoxin and saxitoxin render the sodium channels impermeable to Na ions and to Li ions and probably prevent the opening of individual sodium channels when one toxin molecule binds to a channel.
Abstract: Voltage clamp measurements on myelinated nerve fibers show that tetrodotoxin, saxitoxin, and DDT specifically affect the sodium channels of the membrane. Tetrodotoxin and saxitoxin render the sodium channels impermeable to Na ions and to Li ions and probably prevent the opening of individual sodium channels when one toxin molecule binds to a channel. The apparent dissociation constant of the inhibitory complex is about 1 nM for the cationic forms of both toxins. The zwitter ionic forms are much less potent. On the other hand, DDT causes a fraction of the sodium channels that open during a depolarization to remain open for a longer time than is normal. The effect cannot be described as a specific change in sodium inactivation or as a specific change in sodium activation, for both processes continue to govern the opening of the sodium channels and neither process is able to close the channels. The effects of DDT are very similar to those of veratrine.
458 citations
TL;DR: Evidence is presented that caffeine does not act on the mitochondrial Ca uptake system and that its effect cannot be attributed to the accumulation of adenosine 3',5'-phosphate.
Abstract: Evidence is presented that caffeine does not act on the mitochondrial Ca uptake system and that its effect cannot be attributed to the accumulation of adenosine 3',5'-phosphate. Two distinct caffeine effects are described. At high ATP concentrations caffeine decreases the coupling between ATP hydrolysis and Ca inflow. It either inhibits inflow without any inhibition of the rate of ATP hydrolysis, or it stimulates the ATPase activity without stimulating Ca inflow. These high ATP concentrations (much higher than needed for the saturation of the transport ATPase) greatly reduce the control of the turnover rate of the transport system, by accumulated Ca. At low ATP concentrations when the transport system is under maximal control by accumulated Ca, caffeine inhibits the ATPase activity without affecting the rate of Ca inflow.
309 citations
TL;DR: The permeability and electrical properties of thin lipid membranes are presented, and the changes induced in these properties by several agents added to the aqueous phases after the membranes have formed are discussed.
Abstract: We present and discuss the permeability and electrical properties of thin lipid membranes, and the changes induced in these properties by several agents added to the aqueous phases after the membranes have formed. The unmodified membrane is virtually impermeable to ions and small "hydrophilic" solutes, but relatively permeable to water and "lipophilic" molecules. These properties are consistent with those predicted for a thin film of hydrocarbon through which matter is transported by dissolving in the membrane phase and then diffusing through it. The effect of cholesterol in reducing the water and "lipophilic" solute permeability is attributed to an increase of the "viscosity" of the hydrocarbon region, thus reducing the diffusion coefficient of molecules within this phase. The selective permeability of the membrane to iodide (I-) in the presence of iodine (I2) is attributed to the formation of polyiodides (perhaps I5-), which are presumed to be relatively soluble in the membrane because of their large size, and hence lower surface charge density. Thus, I2 acts as a carrier for I-. The effects of "excitability-inducing material" and the depsipeptides (particularly valinomycin) on ion permeability are reviewed. The effects of the polyene antibiotics (nystatin and amphotericin B) on ion permeability, discussed in greater detail, are the following: (a) membrane conductance increases with the 10th power of nystatin concentration; (b) the membrane is anion-selective but does not discriminate completely between anions and cations; (c) the membrane discriminates among anions on the basis of size; (d) membrane conductance decreases extraordinarily with increasing temperatures. Valinomycin and nystatin form independent conductance pathways in the same membrane, and, in the presence of both, the membrane can be reversibly shifted between a cation and anion permeable state by changes in temperature. It is suggested that nystatin produces pores in the membrane and valinomycin acts as a carrier.
233 citations
TL;DR: Evidence is presented that the pacemaker locus and spike trigger zone in the intact pacemaker neuron are located on the soma.
Abstract: Somata of pacemaker and nonpacemaker neurons were isolated by ligatures tied around the axons between the somata and the synaptic regions, and the transmembrane potentials of the isolated somata were recorded. Isolated somata of pacemaker neurons had a spontaneous discharge while isolated somata of nonpacemaker neurons were quiescent. In addition, the time course of accommodation in isolated somata of pacemaker and nonpacemaker neurons was found to be different. In pacemaker neurons, injection of current produced a change in rate of discharge sustained for the duration of current injection, while in nonpacemakers, current injection produced only a transient change in discharge rate. Evidence is presented that the pacemaker locus and spike trigger zone in the intact pacemaker neuron are located on the soma.
231 citations
222 citations
TL;DR: The experiments indicate that P K and P Cl are independent of pH but are a function of membrane potential, while in artificial pond water containing DNP, the resting membrane potential decreased, suggesting that some energy-consuming mechanism maintains the membrane potential at the resting level.
Abstract: The resting membrane potential of the Nitella cell is relatively insensitive to [K]o, but behaves like a hydrogen electrode. K+ and Cl- effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that PK and PCl are independent of pH but are a function of membrane potential. Slope ion conductances, GK, GCl, and GNa were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H+ was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K+ equilibrium potential. In the range of external pH 5 to 6, H+ chord conductance was substantially equal to the membrane conductance. However, the [H]i measured by various methods was not such as would be predicted from the [H]o and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H+ extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H+ equilibrium potential is always maintained notwithstanding a continuous H+ inward current which should result from the potential difference.
216 citations
TL;DR: It is proposed that electrical stimulation releases calcium from the terminal cisternae and that relaxation occurs from the binding of the released calcium by the longitudinal tubules and the intermediate cisterna.
Abstract: A B s T R A C T Radioautographs of 45Cadabeled frog skeletal muscles have been prepared using freeze-dry and vapor fixation techniques to avoid displacement of the isotope during the preparation of the radioautographs. *SCa has been localized in resting muscles exposed to 45Ca Ringer's for 5 n'fin or 5 hr and in isotopically labeled muscles recovering from tetanic stimulation at room temperature or at 4°C. In muscles soaked at rest for 5 rain 45Ca was present almost exclusively in the terminal cisternae. In all other muscles there were three sites at which the isotope was concentrated: (a) the terminal cisternae, (b) the intermediate cisternae and the longitudinal tubules, and (c) the A band portion of the myofibrils. The terminal cisternae were labeled more rapidly than the myofibrils, but both exchanges were accelerated by electrical stimulation, The amount of *SCa in the longitudinal tubules and the intermediate cisternae decreased with time after a tetanus as the amount in the terminal cisternae increased. It is proposed that electrical stimulation releases calcium from the terminal cisternae and that relaxation occurs from the binding of the released calcium by the longitudinal tubules' and the intermediate cisternae. Complete recovery from mechanical activity involves the transport of this bound calcium into the reticulum and its subsequent binding by the terminal cisternae. Resting exchange of calcium occurs primarily between the terminal cisternae and the transverse tubules. A large body of information indicates that a release of intracellular calcium is critical for the coupling of excitation with contraction of skeletal muscle. The contractile proteins require the addition of about 0.1-0.2/~mole of calcium per g of muscle for the conversion of the muscle from the resting state to the maximally contracted state (11 ). A shift of this amount of calcium from the region of the terminal cisternae of the sarcoplasmic reticulum to the immediate vicinity of the thick filaments has been demonstrated by radioautographic techniques which used chemical stimulation of the muscle and chemical fixa
209 citations
TL;DR: No differences were found between fiber types in rates of transmitter mobilization, density of innervation, or the relationship between transmitter release and terminal depolarization, but single terminals on distal fibers were found to release transmitter with a greater probability than central terminals.
Abstract: Junctional potentials (jp's) recorded from superficial distal fibers of the crayfish opener muscle are up to 50 times larger than jp' in superficial central fibers when the single motor axon that innervates the muscle is stimulated at a frequency of 1/sec or less. At 80/sec, in contrast, central jp's are up to four times larger than those observed in distal fibers. The tension produced by single muscle fibers of either type is directly proportional to the integral of the time-voltage curve minus an excitation-contraction coupling threshold of 3 mv. Distal fibers therefore produce almost all the total muscle tension at low frequencies of stimulation and central fibers add an increasingly greater contribution as their nerve endings begin to facilitate in response to increased rate of motor discharge. Differentiation of muscle membrane characteristics (input resistance, space constant, time constant) cannot account for these differences in facilitation ratios. The mechanism of neuronal differentiation is not based upon the size or effectiveness of transmitter quanta, since equal sized jp's have equal variances;: mjp sizes and variances are also equal. No differences were found between fiber types in rates of transmitter mobilization, density of innervation, or the relationship between transmitter release and terminal depolarization. Single terminals on distal fibers were found to release transmitter with a greater probability than central terminals. More effective invasion of distal terminals by the nerve impulse at low frequencies can account for the difference.
192 citations
TL;DR: The structural and functional changes in smooth muscle due to hypertonicity correlate with the hypothesis that nexuses between cells are responsible for the core conductor properties of tissues, especially where one cell sends a large bulbous projection into a neighbor.
Abstract: The hypothesis that nexuses between cells are responsible for the core conductor properties of tissues was tested using smooth muscle preparations from the taenia coli of guinea pigs. Action potentials recorded from small diameter preparations across a sucrose gap change from monophasic to diphasic when a shunt resistor is connected across the gap. This indicates that transmission between smooth muscle cells is electrical, because the resistor only allows current to flow. Nexal fusion of cell membranes occurs especially where one cell sends a large bulbous projection into a neighbor. Hypertonic solutions rupture the nexuses between smooth muscle cells. Hypertonicity also increases the resistance of a bundle across the sucrose gap and blocks propagation of action potentials. Thus the structural and functional changes in smooth muscle due to hypertonicity correlate with the hypothesis.
TL;DR: In an identified cell that shows a marked temperature dependence of RMP, both the potassium equilibrium potential and the membrane resistance were found to be relatively independent of temperature.
Abstract: The resting membrane potential (RMP) of Aplysia neurons is very temperature-dependent, and in some cells increases with increasing temperature by as much as 2 mv/°C. RMP at room temperature may significantly exceed the potassium equilibrium potential, which can be determined by measurement of the equilibrium point of the spike after potential. The hyperpolarization on warming is completely abolished by ouabain, replacement of external Na+ by Li+, removal of external K+, and by prolonged exposure to high Ca++, while it is independent of external chloride but is increased by cocaine (3 x 10-3 M). In an identified cell that shows a marked temperature dependence of RMP, both the potassium equilibrium potential and the membrane resistance were found to be relatively independent of temperature. The hyperpolarization on warming, which may increase RMP by as much as 50%, can most reasonably be ascribed to the activity of an electrogenic Na+ pump.
TL;DR: Influx and efflux of choline in human erythrocytes were studied using 14C-choline and show a pronounced exchange flux phenomenon; the ability to concentrate choline is lost when external sodium is replaced by lithium or potassium.
Abstract: Influx and efflux of choline in human erythrocytes were studied using 14C-choline. When incubated at 37°C with physiological concentrations of choline erythrocytes concentrate choline; the steady-state ratio is 2.08 ± 0.23 when the external choline is 2.5 µM and falls to 0.94 ± 0.13 as the external concentration is raised to 50 µM. During the steady state the influx of choline is consistent with a carrier system with an apparent Michaelis constant of 30 x 10-6 and a maximum flux of 1.1 µmoles per liter cells per min. For the influx into cells preequilibrated with a choline-free buffer the apparent Michaelis constant is about 6.5 x 10-6 M and the maximum flux is 0.22 µmole per liter cells per min. At intracellular concentrations below 50 µmole per liter cells the efflux in the steady state approximates first order kinetics; however, it is not flux through a leak because it is inhibited by hemicholinium. Influx and efflux show a pronounced exchange flux phenomenon. The ability to concentrate choline is lost when external sodium is replaced by lithium or potassium. However, the uphill movement of choline is probably not coupled directly to the Na+ electrochemical gradient.
TL;DR: The dynamic characteristics of the rat gracilis anticus muscle at 17.5°C have been determined by isotonic and isometric loading and the shortening velocity is a function of load and length, but also of an additional variable related to the time elapsed from onset of stimulation.
Abstract: The dynamic characteristics of the rat gracilis anticus muscle at 17.5°C have been determined by isotonic and isometric loading. For a fixed initial length these characteristics were represented either as a family of length-velocity phase trajectories at various isotonic afterloads or as a series of force-velocity curves at different lengths. An alternate method of viewing these data, the length-external load-velocity phase space, was also generated. When the muscle was allowed to shorten from different initial lengths, the velocity of shortening achieved at a given length was lower for longer initial lengths. The amount of departure was also dependent upon the isotonic load, the greater the load the greater the departure. The departures were not caused by changes in the elastic elements of the muscle or fatigue in the ordinary sense. When the behavior of the muscle was investigated at different frequencies of stimulation, the shortening velocity was a function of the number of stimulating pulses received by the muscle at a given frequency. The shortening velocity of the rat gracilis anticus muscle is, therefore, not only a function of load and length, but also of an additional variable related to the time elapsed from onset of stimulation.
TL;DR: The finding that, under the influence of DDT, the steady-state current starts flowing while the peak transient current is partially maintained supports the hypothesis of two operationally separate ion channels in the nerve membrane.
Abstract: The falling phase of action potential of lobster giant axons is markedly prolonged by treatment with DDT, and a plateau phase appears as in cardiac action potentials. Repetitive afterdischarge is very often superimposed on the plateau. Voltage-clamp experiments with the axons treated with DDT and with DDT plus tetrodotoxin or saxitoxin have revealed the following: DDT markedly slows the turning-off process of peak transient current and suppresses the steady-state current. The falling phase of the peak transient current in the DDT-poisoned axon is no longer expressed by a single exponential function as in normal axons, but by two or more exponential functions with much longer time constants. The maximum peak transient conductance is not significantly affected by DDT. DDT did not induce a shift of the curve relating the peak transient conductance to membrane potential along the potential axis. The time to peak transient current and the time for the steady-state current to reach its half-maximum are prolonged by DDT to a small extent. The finding that, under the influence of DDT, the steady-state current starts flowing while the peak transient current is partially maintained supports the hypothesis of two operationally separate ion channels in the nerve membrane.
TL;DR: The data suggest that many of these ions may interact with the same cation-binding sites on the axon membrane, and that the relative effects on the membrane conductance and rate parameters depend on the relative binding constants of the ions.
Abstract: Calcium appears to be an essential participant in axon excitation processes. Many other polyvalent metal ions have calcium-like actions on axons. We have used the voltage-clamped lobster giant axon to test the effect of several of these cations on the position of the peak initial (sodium) and steady-state (potassium) conductance vs. voltage curves on the voltage axis as well as on the rate parameters for excitation processes. Among the alkaline earth metals, Mg+2 is a very poor substitute for Ca+2, while Ba+2 behaves like "high calcium" when substituted for Ca+2 on a mole-for-mole basis. The transition metal ions, Ni+2, Co+2, and Cd+2 also act like high calcium when substituted mole-for-mole. Among the trivalent ions, La+3 is a very effective Ca+2 replacement. Al+3 and Fe+3 are extremely active and seem to have some similar effects. Al+3 is effective at concentrations as low as 10-5 M. The data suggest that many of these ions may interact with the same cation-binding sites on the axon membrane, and that the relative effects on the membrane conductance and rate parameters depend on the relative binding constants of the ions. The total amount of Na+ transferred during a large depolarizing transient is nearly independent of the kind or amount of polyvalent ion applied.
TL;DR: A new hypothesis has been developed from thermodynamic principles alone, which predicts that, at constant pH, the net charge on the hemoglobin molecule decreases with increased hemoglobin concentration, and could provide an electrical transducer for osmotic signals.
Abstract: The present studies are concerned with a detailed examination of the apparent anomalous osmotic behavior of human red cells. Red cell water has been shown to behave simultaneously as solvent water for nonelectrolytes and nonsolvent water, in part, for electrolytes. The nonsolvent properties are based upon assumptions inherent in the conventional van't Hoff equation. However, calculations according to the van't Hoff equation give osmotic volumes considerably in excess of total cell water when the pH is lowered beyond the isoelectric point for hemoglobin; hence the van't Hoff equation is inapplicable for the measurement of the solvent properties of the red cell. Furthermore, in vitro measurements of osmotic and other properties of 3.7 millimolal solutions of hemoglobin have failed to reveal the presence of any salt exclusion. A new hypothesis has been developed from thermodynamic principles alone, which predicts that, at constant pH, the net charge on the hemoglobin molecule decreases with increased hemoglobin concentration. The existence of such cooperative interaction may be inferred from the effect of pH on the changes in hemoglobin net charge as the spacing between the molecules decreases. The resultant movement of counterions across the cell membrane causes the apparent anomalous osmotic behavior. Quantitative agreement has been found between the anion shift predicted by the equation and that observed in response to osmotic gradients. The proposed mechanism appears to be operative in a variety of tissues and could provide an electrical transducer for osmotic signals.
TL;DR: In general, the morphology of capillaries correlates well with physiological observations, and it is inferred that the cell junctions are the morphological equivalent of the small-pore system, and the vesicles the equivalents of the large- pore system.
Abstract: A brief survey is given of current views correlating the ultrastructural and permeability characteristics of capillaries. Observations based on the use of peroxidase (mol wt 40,000), as an in vivo, and colloidal lanthanum, as an in vitro, ultrastructural tracer, are presented. In capillaries with "continuous" endothelium, the endothelial intercellular junctions are thought to be permeable to the tracers, and are regarded as maculae occludentes rather than zonulae occludentes, with a gap of about 40 A in width between the maculae. Some evidence for vesicular transport is also presented. It is inferred that the cell junctions are the morphological equivalent of the small-pore system, and the vesicles the equivalent of the large-pore system. Peroxidase does not apparently cross brain capillaries: the endothelial cell junctions are regarded as zonulae occludentes, and vesicles do not appear to transport across the endothelium. This is regarded as the morphological equivalent of the blood-brain barrier for relatively large molecules. The tracers appear to permeate the fenestrae of fenestrated capillaries, and the high permeability of these capillaries to large molecules is attributed to the fenestrae. Capillaries with discontinuous endothelium readily allow passage of the tracers through the intercellular gaps. A continuous basement membrane may act as a relatively coarse filter for large molecules. In general, the morphology of capillaries correlates well with physiological observations.
TL;DR: Fluid balance at the capillary level has been simulated with an analogue computer program, based on experimental data on regional differences in capillary permeability, surface areas, and hydrostatic pressures, which yields reasonable agreement between experimental data and data obtained by computer analysis.
Abstract: Fluid balance at the capillary level has been simulated with an analogue computer program, based on experimental data on regional differences in capillary permeability, surface areas, and hydrostatic pressures. The program takes into account fluid and protein fluxes into and out of the interstitial space. Solutions are obtained for tissue hydrostatic pressure, tissue fluid osmotic pressure, interstitial space volume, and lymph flow. Simulation of a variety of physiological experiments and clinical disease states has yielded reasonable agreement between experimental data and data obtained by computer analysis. Dilution of the interstitial plasma protein pool with a consequent reduc6tion of its oncotic pressure appears to be a major factor, which prevents edema unless plasma oncotic pressures are reduced by 10–15 mm Hg or, alternatively, venous pressures are elevated by a similar amount. The computer analysis in all instances yields positive values for tissue pressure, in agreement with experimental data obtained by needle puncture. The negative tissue pressures observed in subcutaneous capsules can be reproduced in the computer program, if the interface between the capsule and the surrounding interstitial space is assumed to have the properties of a semipermeable membrane.
TL;DR: Clear structure-activity relations indicate that the receptor to which the drugs bind to produce the prolonged response can be the receptor for acetylcholine, and shows that the effects of Xylocaine depend on the time course of receptor activation and are quite different from the effect of d-tubocurarine.
Abstract: Xylocaine and its derivatives act specifically at the neuromuscular junction within the concentration range 0.05 to 2.0 mM. The charged form is the active form of the drugs. There is no correlation between "local anesthetic" activity and effect at the junction. Like d-tubocurarine, these drugs have little or no effect on quantum content, acetylcholinesterase activity, or the passive impedance of the muscle fiber. Yet they produce end plate potentials characterized by a brief, early component and a late, greatly prolonged component, as does procaine. Analysis of these changes in time course suggests that the drugs have little or no effect before receptors are activated by acetylcholine, but cause a decreased and often greatly prolonged response. Clear structure-activity relations indicate that the receptor to which the drugs bind to produce the prolonged response can be the receptor for acetylcholine. Comparison of the effects of the drugs on the end plate potential and on the response to iontophoretically applied acetylcholine also shows that the effects of Xylocaine depend on the time course of receptor activation and are quite different from the effects of d-tubocurarine.
TL;DR: Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid, which could account for lytic responses observed on interaction with surface-active agents.
Abstract: Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20–30 % lipid and 50–75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2–3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.
TL;DR: Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion and there was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 µM, while the relation between internal sodium and sodium efflux was linear.
Abstract: The effects which alterations in the concentrations of internal sodium and high energy phosphate compounds had on the sodium influx and efflux of internally dialyzed squid axons were examined. Nine naturally occurring high energy phosphate compounds were ineffective in supporting significant sodium extrusion. These compounds were: AcP, PEP, G-3-P, ADP, AMP, GTP, CTP, PA, and UTP.1 the compound d-ATP supported 25–50% of the normal sodium extrusion, while ATP supported 80–100%. The relation between internal ATP and sodium efflux was nonlinear, rising most steeply in the range 1 to 10 µM and more gradually in the range 10 to 10,000 µM. There was no evidence of saturation of efflux even at internal ATP concentrations of 10,000 µM. The relation between internal sodium and sodium efflux was linear in the range 2 to 240 mM. The presence of external strophanthidin (10 µM) changed the sodium efflux to about 8–12 pmoles/cm2 sec regardless of the initial level of efflux; this changed level was not altered by subsequent dialysis with large concentrations of ATP. Sodium influx was reduced about 50 % by removal of either ATP or Na and about 70 % by removing both ATP and Na from inside the axon.
TL;DR: Conduction in nonnervous tissues is unaffected by excess Mg++ in concentrations suppressing presumed nervous activity, although this may not be a wholly adequate criterion for distinguishing components of the two systems.
Abstract: Sarsia, Euphysa, and other hydromedusae have been studied by electrophysiological techniques and are found to have nonnervous conducting epithelia resembling those described earlier for siphonophores. Simple, non-muscular epithelia fire singly or repetitively following brief electrical stimuli. The pulses recorded with suction electrodes are biphasic, initially positive, and show amplitudes of 0.75–2.0 mv, durations of 5–15 msec, and velocities of 15–35 cm/sec with short refractory periods. In the swimming muscle (myoepithelium) 2.0–4.0 mv composite events lasting 150–300 msec are associated with contraction waves. Propagation in nonnervous epithelia is typically all-or-none, nondecremental, and unpolarized. The subumbrellar endoderm lamella conducts independently of the adjacent ectoderm. The lower regions of the tentacles do not show propagated epithelial events. The spread of excitation in conducting epithelia and associated effector responses are described. Examples are given of interaction between events seemingly conducted in the nervous system and those in nonnervous epithelia. Either system may excite the other. Spontaneous activity, however, appears to originate in the nervous system. Conduction in nonnervous tissues is unaffected by excess Mg++ in concentrations suppressing presumed nervous activity, although this may not be a wholly adequate criterion for distinguishing components of the two systems. Evidence from old work by Romanes is considered in the light of these findings and the general significance of epithelial conduction is discussed.
TL;DR: The duration of ciliary reversal of Paramecium caudatum in response to changes in external ionic factors was determined with various ionic compositions of both equilibration and stimulation media and the following hypothesis is proposed: Ca++ liberated from the cellular cation exchange system activates a contractile system which is energized by ATP.
Abstract: The duration of ciliary reversal of Paramecium caudatum in response to changes in external ionic factors was determined with various ionic compositions of both equilibration and stimulation media. The reversal response was found to occur when calcium ions bound by an inferred cellular cation exchange system were liberated in exchange for externally applied cations other than calcium. Factors which affect the duration of the response were (a) initial amount of calcium bound by the cation exchange system, (b) final amount of calcium bound by the system after equilibration with the stimulation medium, and (c) concentration of calcium ions in the stimulation medium. An empirical equation is presented which relates the duration of the response to these three factors. On the basis of these and previously published data, the following hypothesis is proposed for the mechanism underlying ciliary reversal in response to cationic stimulation: Ca(++) liberated from the cellular cation exchange system activates a contractile system which is energized by ATP. Contraction of this component results in the reversal of effective beat direction of cilia by a mechanism not yet understood. The duration of reversal in live paramecia is related to the time course of bound calcium release.
TL;DR: Data suggest that amphotericin B or nystatin may interact with membrane-bound sterols to produce multimolecular complexes which greatly enhance the permeability of such membranes for anions, acetate, and, to a lesser degree, cations (Na+, K+, Li+).
Abstract: Optically black, thin lipid membranes prepared from sheep erythrocyte lipids have a high dc resistance (Rm ≅ 108 ohm-cm2) when the bathing solutions contain NaCl or KCl. The ionic transference numbers (Ti) indicate that these membranes are cation-selective (TNa ≅ 0.85; TCl ≅ 0.15). These electrical properties are independent of the cholesterol content of the lipid solutions from which the membranes are formed. Nystatin, and probably amphotericin B, are cyclic polyene antibiotics containing ≈36 ring atoms and a free amino and carboxyl group. When the lipid solutions used to form membranes contained equimolar amounts of cholesterol and phospholipid, these antibiotics reduced Rm to ≈102 ohm-cm2; concomitantly, TCl became ≅0.92. The slope of the line relating log Rm and log antibiotic concentration was ≅4.5. Neither nystatin (2 x 10-5 M) nor amphotericin B (2 x 10-7 M) had any effect on membrane stability. The antibiotics had no effect on Rm or membrane permselectivity when the lipids used to form membranes were cholesterol-depleted. Filipin (10-5 M), an uncharged polyene with 28 ring atoms, produced striking membrane instability, but did not affect Rm or membrane ionic selectivity. These data suggest that amphotericin B or nystatin may interact with membrane-bound sterols to produce multimolecular complexes which greatly enhance the permeability of such membranes for anions (Cl-, acetate), and, to a lesser degree, cations (Na+, K+, Li+).
TL;DR: The voltage clamp technique has been applied successfully to the analysis of ionic currents in a wide variety of excitable ceils but it has proved difficult to apply this technique to striated muscle, both because it is hard to isolate a small area of membrane electrically and because the fiber contracts when depolarized.
Abstract: The voltage clamp technique (Cole, 1949; Hodgkin, Huxley, and Katz, 1952) has been applied successfully to the analysis of ionic currents in a wide variety of excitable ceils. I t has proved difficult to apply this technique to striated muscle, both because it is hard to isolate a small area of membrane electrically and because the fiber contracts when depolarized. Takeuchi and Takeuchi (1959) applied voltage feedback at the end plate region of striated muscle fibers. Their method of controlling the voltage at a point produced an elegant method for measuring the currents of the muscle membrane at the end plate. Previous attempts to control the voltage of an area of membrane have been made by Adrian and Freygang (1962 b) and by Frankenhaeuser, Lindley and Smith (1965). In the present experiments (Adrian, Chandler, and Hodgkin, 1966) we have tried to control the voltage over a short length of a striated muscle fiber, by a technique which uses an internal microelectrode to deliver current and two additional microelectrodes to measure membrane current and voltage. The three electrodes are inserted near the pelvic end of a muscle fiber, in a frog sartorius muscle. Fig. 1 shows the positions of the microelectrodes and the arrangement of the feedback amplifier. The current through electrode 3 is controlled by feedback to give steady voltages or steps of voltage at electrode 1. The membrane current density at 1 is estimated by the difference in potential between electrodes 1 and 2. As a simple approximation, this potential difference can be considered to be produced by a longitudinal current flowing along the internal resistance between 1 and 2. The same current crosses the membrane between the end of the fiber and a point 3l/2 from the end of the fiber. On this basis the membrane current per unit length would be given by
TL;DR: This finding provides direct evidence that vasopressin acts to increase ionic mobility selectively across the apical permeability barrier of the transporting cells of the toad bladder.
Abstract: Vasopressin increases the net transport of sodium across the isolated urinary bladder of the toad by increasing the mobility of sodium ion within the tissue. This change is reflected in a decreased DC resistance of the bladder; identification of the permeability barrier which is affected localizes the site of action of vasopressin on sodium transport. Cells of the epithelial layer were impaled from the mucosal side with glass micropipettes while current pulses were passed through the bladder. The resulting voltage deflections across the bladder and between the micropipette and mucosal reference solution were proportional to the resistance across the entire bladder and across the mucosal or apical permeability barrier, respectively. The position of the exploring micropipette was not changed and vasopressin was added to the serosal medium. In 10 successful impalements, the apical permeability barrier contributed 54% of the initial total transbladder resistance, but 98% of the total resistance change following vasopressin occurred at this site. This finding provides direct evidence that vasopressin acts to increase ionic mobility selectively across the apical permeability barrier of the transporting cells of the toad bladder.
TL;DR: It was found that the thin lipid membranes have ionic permeability properties which are vastly different from those of intact HK and LK sheep red cell membranes, and the Na + and K + transport properties of the two genetic types of sheep red cells are significantly different.
Abstract: For some years, work in this laboratory has been directed toward elucidation of the mechanism of selectivity of high-potassium (HK) and low-potassium (LK) sheep red cell membranes for Na + and K + (I-4). One recent approach has been to compare the ionic permeability of the intact ceil membranes with thin artificial bilayer membranes prepared from lipids extracted from these cells (5). It was found that the thin lipid membranes have ionic permeability properties which are vastly different from those of intact HK and LK sheep red cell membranes. For example, the PC electrical resistance of the bilayers is about 2 X 10 s ohm cm 2, while that of the red cell membranes is estimated to be from 10 to 100 ohm cm 2. Furthermore, the bilayers are more permeable to both Na + and K + than they are to Cl-, while the ratio of Clto K + or Na + permeability of red cells is of the order of 106 (6). Finally, the thin lipid membranes do not distinguish between Na + and K +, and the ionic transport properties of such membranes prepared from HE and LK sheep red cell lipids are identical. The intact cell membranes do distinguish between these alkali metal ions, and, furthermore, the Na + and K + transport properties of the two genetic types of sheep red cells are significantly different. Apparently, the thin lipid membranes lack essential components which are responsible for the distinctive cation permeability properties of the intact cell membranes. In the course of continuing attempts to identify such components, we became aware of reports of the effect of the macrocyclic depsipeptide antibiotic, valinomycin, on the ionic permeabil i ty of lecithin (7) and mixed brain lipid (8) bilayer membranes. This compound had previously been reported to stimulate the respiration of mitochondria in the presence of K + but not in the presence of Na + (9). Valinomycin was found to produce specific permeabili ty of intact H K and L K sheep red cell membranes, as well as thin artificial membranes prepared from lipids extracted from these cells, to K + but not to Na + ( 10, 11). In the presence of K + ( 10 t M) and valinomycin (5 X 10 -~ M), the electrical resistance of the lipid bilayer membranes is about 103 ohm cm ~, which is in the range of biological membranes. The significance of these observations derives from the unusual structure of valino-
Journal Article•
TL;DR: The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels, assumed to comprise several types, of varying affinity for divalent and hydrogen ions.
Abstract: The voltage dependence of the voltage clamp responses of myelinated nerve fibers depends on the concentration of divalent cations and of hydrogen ions in the bathing medium. In general, increases of the [Ca], [Ni], or [H] increase the depolarization needed to elicit a given response of the nerve. An e -fold increase of the [Ca] produces the following shifts of the voltage dependence of the parameters in the Hodgkin-Huxley model: m ∞, 8.7 mv; h ∞, 6.5 mv; τ n , 0.0 mv. The same increase of the [H], if done below pH 5.5, produces the following shifts: m ∞, 13.5 mv; h ∞, 13.5 mv; τ n , 13.5 mv; and if done above pH 5.5: m ∞, 1.3 mv; h ∞, 1.3 mv; τ n , 4.0 mv. The voltage shifts are proportional to the logarithm of the concentration of the divalent ions and of the hydrogen ion. The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels. The charged groups are assumed to comprise several types, of varying affinity for divalent and hydrogen ions. The charges near the sodium channels differ from those near the potassium channels. As the pH is lowered below pH 6, the maximum sodium conductance decreases quickly and reversibly in a manner that suggests that the protonation of an acidic group with a pK a of 5.2 blocks individual sodium channels.
TL;DR: It is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.
Abstract: Skeletal muscle myofibrils, in the presence of 2 mM MgCl2 at pH 7.0, were found to have two classes of calcium-binding sites with apparent affinity constants of 2.1 x 106 M-1 (class 1) and ∼3 x 104 M-1 (class 2), respectively. At free calcium concentrations essential for the activation of myofibrillar contraction (∼10-6 M) there would be significant calcium binding only to the class 1 sites. These sites could bind about 1.3 µmoles of calcium per g protein. Extraction of myosin from the myofibrils did not alter their calcium-binding parameters. Myosin A, under identical experimental conditions, had little affinity for calcium. The class 1 sites are, therefore, presumed to be located in the I filaments. The class 1 sites could only be detected in F actin and myosin B preparations which were contaminated with the tropomyosin-troponin complex. Tropomyosin bound very little calcium. Troponin, which in conjunction with tropomyosin confers calcium sensitivity on actomyosin systems, could bind 22 µmoles of calcium per g protein with an apparent affinity constant of 2.4 x 106 M-1. In view of the identical affinity constants of the myofibrils and troponin and the much greater number of calcium-binding sites on troponin it is suggested that calcium activates myofibrillar contraction by binding to the troponin molecule.