Effects of cortisone on mechanical activity and membrane ionic currents in frog auricular fibres.

The influence of cortisone on the mechanical and electrical activity of frog auricular fibres was investigated under voltage clamp conditions. 1. Cortisone exerted in vitro an inotropic action depending on concentration; a maximal positive inotropic effect was observed with 2 x 10(-4) g/ml of cortisone. 2. The positive inotropic effect of cortisone might be either an indirect sympathomimetic effect or an adrenaline-like effect. 3. The positive inotropic action of cortisone was correlated with modifications of the cardiac action potential: the amplitude of the action potential was enhanced while the resting membrane potential was unchanged; the amplitude and duration of the plateau were increased and the duration of the action potential was lengthened. 4. The electrical changes were related to an increase in the slow calcium current intensity resulting from an increase in the slow calcium conductance.     

Voltage-dependent membrane ionic currents in lamprey striated muscle

Membrane ionic currents in striated muscle bundles of lamprey suction apparatus were recorded using a double sucrose gap technique. Transmembrane currents in a single muscle fiber and a fiber bundle in the frog were compared so as to check the validity of current measurement in multicell preparations. It was found that fast inward sodium currents arise in the lamprey muscle membrane in response to depolarization together with a delayed outward potassium current, with steady-state characteristics resembling those of membrane currents in frog muscle. The only difference consisted of a flatter curve for steady-state inactivation of potassium current, probably indicative of greater density of potassium channels. Both the changes in reversal potential and the speed of potassium current deactivation occurring during protracted stimuli point to the presence of two fractions in this current. No functioning voltage-dependent calcium channels are found in the lamprey muscle membrane.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 629–636, September–October, 1986.     

Sequential modification of membrane currents with classical conditioning

Pavlovian conditioning of the nudibranch mollusc Hermissenda crassicornis was previously shown to produce long-lasting reduction of two K+ currents measured across the Type B photoreceptor soma membrane (Alkon et al., 1982a; Alkon et al., 1985). Pavlovian conditioning of the rabbit was also shown to be followed by persistent K+ current reduction (Disterhoft et al., 1986). Here we report the first evidence that Ca2+ currents can also be modified by conditioning. The amplitude of the currents rather than their voltage-dependence remains reduced at least 1-2 d after conditioning (but not control procedures). Conditioning-induced changes of both K+ and Ca2+ currents increased as a function of training, the Ca2+ currents only changing substantially with greater than or equal to 250 trials. The later changes of the Ca2+ current may function to limit the magnitude of excitability increases due to associative learning.     

Properties of membrane ionic currents in pituitary clone cells

Membrane ionic currents of the GH3 pituitary cell line have been studied using voltage clamp techniques. The inward current is completely blocked by cobalt (Co2+) ions and appeared to be carried by calcium ions. Three outward currents can be differentiated on the ground of kinetics and pharmacological studies: a transient current blocked by 4-aminopyridine (4 AP) and two delayed outward current which are voltage dependent. One is blocked by tetraethylammonium (TEA); the second is blocked by Co2+ and represents a calcium-activated potassium conductance.     

Effect of digitonin on rat myometrium subcellular membrane fractions

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.     

Selenium-induced alterations in ionic currents of rat cardiomyocytes

In the present study, rats were treated with sodium selenite (5 micromol/kg body weight/day, ip) for 4 weeks and the parameters of contractile activity, action potential, L-type Ca2+-current (ICaL), as well as transient outward (Ito), inward rectifier (IK1), and steady state (Iss) K+-currents were investigated. Sodium selenite treatment increased rat blood glucose level and lowered plasma insulin level, significantly. This treatment also caused slightly prolongation in action potential with no significant effects on spontaneous contraction parameters and intracellular Ca2+ transients of the heart preparations. These effects were associated with marked alterations in the kinetics of both ICaL and Ito including a significant slowing in both inactivation time constants of ICaL and a significant shift to negative potential at half-inactivation of these channels without any change in the current density. Also, there was a significantly faster inactivation of Ito and no shift in half-inactivation of this channel without any change in its current density. Consequently, there was a approximately 50% increase in total charges carried by Ca2+ current and approximately 50% decrease in total charges carried by K+ currents of the treated rat cardiomyocytes. Additionally we observed a significant inhibition in IK1 density in treated rat cardiomyocytes. Oxidized glutathione level was significantly increased (70%) while the observed decrease in reduced glutathione was much less. Since a shift in redox state of regulatory proteins is related with cell dysfunction, selenium-induced increase in blood glucose and decrease in plasma insulin may correlate these alterations. These alterations, in the kinetics of the channels and in IK1 density, might lead to proarrhythmic effect of chronic selenium supplementation.     

Some effects of prolonged polarization on membrane currents in bullfrog atrial muscle

Summary Bullfrog atrial trabecula were voltage-clamped using a double-sucrose-gap method. Step depolarization produced a slowly changing outward current which was studied by analyzing the current tail produced by repolarization. The initial phase of the current tail (time constant 0.1 to 0.7 sec at –60 mV) had a reversal potential which depended upon the duration and magnitude of the preceding depolarization. Calculations based on trabecular geometry and the behavior of the currents in high external potassium suggest that part of the current tail reflects a restoration to a lower steadystate concentration of external potassium which had accumulated in narrow clefts between cells during the preceding depolarization. Step hyperpolarization produced a declining inward current (time constant 0.3 sec at –100 mV) which can be explained on the basis of a depletion of potassium from these intercellular clefts (about 0.5% of the trabecular volume).     

Separation of ionic currents in the somatic membrane of frog sensory neurons

Summary Electrical properties of isolated frog primary afferent neurons were examined by suction pipette technique, which combines internal perfusion with current or voltage clamp using a switching circuit with a single electrode. When K⁺ in the external and internal solutions was totally replaced with Cs⁺, extremely prolonged Ca spikes, lasting for 5 to 10 sec, and Na spikes, having a short plateau phase of 10 to 15 msec, were observed in Na⁺-free and Ca²⁺-free solutions, respectively. Under voltage clamp, Ca²⁺ current (I _Ca) appeared at around –30 mV and maximum peak current was elicited at about 0 mV. With increasing test pulses to the positive side,I _Ca became smaller and flattened but did not reverse. Increases of [Ca] _o induced a hyperbolic increase ofI _Ca and also shifted itsI-V curve along the voltage axis to the more positive direction. Internal perfusion of F^– blockedI _Ca time-dependently. The Ca channel was permeable to foreign divalent cations in the sequence ofI _Ca>I _Ba>I _SrI _Mn>I _Zn. Organic Ca-blockers equally depressed the divalent cation currents dose- and time-dependently without shifting theI-V relationships, while inorganic blockers suppressed these currents dose-dependently and the inhibition appeared much stronger in the order ofI _Ba=I _Sr>I _Ca>I _Mn=I _Zn.     

Changes in the ionic mechanisms of the electrical excitability of rat sensory neuronal somatic membrane during ontogenesis. Distribution of ionic channels carrying inward currents

The distribution of different types of ionic channels carrying inward currents was investigated in the somatic membranes of spinal ganglion neurons in rats belonging to three different age groups: at 5–9 days, 45 days, and 3 months. A decrease was found in the number of neuronal membranes operating all four types of inward current channels simultaneously: "fast" (tetrodotoxin-sensitive), "slow" (tetrodotoxin-resistant) sodium currents and low- and high-threshold calcium currents. There were 14.5% of such neurons in the first age group, 5% in the second, and 1% on the third. It was found that this change was related to the disappearance of "slow" (tetrodotoxin-resistant) sodium and high-threshold calcium channels from the membrane. The number of neuronal somatic membranes with only two types of inward current channels ("fast" sodium and high-threshold calcium channels) increased proportionately.A. A. Bogomolets Institute of Technology, Academy of Sciences of the Ukrainian SSR, Kiev Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 813–820, November–December, 1986.     

Effects of some potassium channel blockers on the ionic currents in myelinated nerve

The effects of some potassium channel blockers on the ionic currents and on the so-called K(+)-depolarization in intact myelinated nerve fibres were studied. 4-AP, and in particular, Flaxedil, proved to be selective K(+)-current blockers. However, TEA, a crown ether (DCH18C6), a longchained triethylammonium compound (C10-TriEA), capsaicin, and the extract from the medicinal herb Ruta graveolens proved not to be selective K(+)-current blockers; they all block Na(+)-currents as well, although to a lesser extent. The sodium inactivation curve did not change under TEA and Flaxedil but was shifted on the potential axis in negative direction by DCH18C6, 4-AP, capsaicin and the Ruta extract whereas C10-TriEA caused a shift of both sodium inactivation and activation parameters in positive direction. Regarding to the kinetics of the persisting K(+)-current fraction, two different kinds of blockade were found: 1. Unchanged K(+)-kinetic which is typical for the effects of TEA, 4-AP, Flaxedil, and C10-TriEA. 2. Clearly changed K(+)-kinetic, characterized by K(+)-transients; which is typical for the effects of capsaicin and in particular, for those of DCH18C6 and of the Ruta extract. The possibly different modes of action of both groups of blockers are discussed in terms of current models for the action of potassium channel blockers.     

Localization of the oxytocin receptor in the plasma membrane of rat myometrium.

The distribution of [3H]oxytocin binding sites among various subcellular fractions of rat myometrium paralleled the distribution of 5'-nucleotidase, a plasma membrane marker enzyme, but not of NADPH-cytochrome c reductase or succinate-cytochrome c reductase, which are endoplasmic reticulum and mitochondrial marker enzymes respectively. [3H]Oxytocin binding to the most enriched plasma membrane fraction showed the degree of selectivity with respect to hormone analogues that is expected for the oxytocin receptor. The binding of oxytocin to this fraction showed an apparent Kd of 1.98 X 10(-9) M and a capacity of 1.28 pmol mg-1. It is concluded that the oxytocin receptor is located on the plasma membrane of the smooth muscle cells of the rat uterus.     

Ultra-slow inactivation of the ionic currents through the membrane of myelinated nerve.

(1) Voltage-clamp experiments were performed with myelinated fibres isolated from the sciatic nerve of the frog to study slow changes of the specific sodium and potassium currents as a function of membrane (holding) potential and time. (2) The level of the peak sodium current depends on holding potential VH. This dependence can be described by a sigmoidal function uinfinity(VH). The underlying process is called "ultra-slow sodium inactivation" and is different and separable from the short time steady-state inactivation, hinfinity(V), and from the slow inactivation depending on the extracellular potassium concentration (Adelman, Jr., W. J. and Palti, Y. (1969), J Gen. Physiol. 54, 589-606; Peganov, E. M., Khodorov, B.I. and Shishkova, L. D. (1973), Bull. Exp. Biol. Med. 25, 15-19; Khodorov, B. I. Shishkova, L. D. and Peganov, E. M. (1974), Bull. Exp. Biol. Med. 3, 10-14). (3) After a sudden change of the holding potential the sodium current reaches a new steady-state level (due to the transition of uinfinity(VH) to the corresponding value) within approx. 4 min. The kinetics of the transition cannot be described by a single exponential function. (4) A corresponding voltage- and time-dependent process of ultra-slow inactivation exists for the potassium current in the node of Ranvier. The kinetics are faster than those of the sodium system.     

Effect of the alkaloid lappaconitine on ionic currents through the somatic membrane ofHelix pomatia neurons

The effect of the alkaloid lappaconitine on passive ion transport through the somatic membrane of identified neurons of the snailHelix pomatia was studied under voltage clamp conditions. In a concentration of 4 mM lappaconitine has a reversible blocking action on the calcium channels of the excitable membrane. To study the effect of the alkaloid on inward sodium currents a solution in which calcium ions were replaced by the equivalent number of magnesium ions was used. Lappaconitine has no appreciable effect on the inward sodium current.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Neirofiziologiya, Vol. 11, No. 5, pp. 469–474, September–October, 1979.     

Calcium ion currents in the smooth muscle of the myometrium

The results of kinetic analysis of Ca liberation from preparations of female rabbit myometrium show that in the uterus smooth muscle there exist three pools of cation with characteristic times of metabolism 182.18 +/- 25.20, 25.56 +/- 1.00 and 2.94 +/- 0.38 min respectively. It was concluded from the compartmentalization analysis and from the data on Na+- and Mg2+--ATP-dependent transport in plasmic membrane fraction of myometrium cells that the fast phase of calcium metabolism reflects the liberation of the cation from extracellular space in the incubation medium, the intermediate one--Ca transfer from myocytes into the extracellular medium, and the slow one--liberation from the subcellular structures into the myoplasm.     

Effect of ajmaline on action potential and ionic currents in rat ventricular myocytes

The effect of ajmaline on action potential (AP) and ionic current components has been investigated in right ventricular myocytes of rat at room temperature using the whole cell patch clamp technique. Ajmaline decreased the upstroke velocity ((dV/dt)max) of AP and the AP amplitude, increased the AP duration measured at 50 and 90% repolarization, and reversibly inhibited most components of membrane ionic current in a concentration-dependent manner. The following values of IC50 and of the Hill coefficient (nH) resulted from approximation of the measured data by the Hill formula: for fast sodium current (INa) IC50=27.8+/-1.14 micromol/l and nH=1.27+/-0.25 at holding potential -75 mV, IC50=47.2+/-1.16 micromol/l and nH=1.16+/-0.21 at holding potential -120 mV; for L-type calcium current (ICa-L) IC50=70.8+/-0.09 micromol/l and n(H)=0.99+/-0.09; for transient outward potassium current (Ito) IC50=25.9+/-2.91 micromol/l and nH=1.07+/-0.15; for ATP-sensitive potassium current (IK(ATP)) IC50=13.3+/-1.1 micromol/l and nH=1.16+/-0.15. The current measured at the end of 300 ms depolarizing impulse was composed of an ajmaline-insensitive component and a component inhibited with IC50=61.0+/-1.1 micromol/l and nH=0.91+/-0.08. At hyperpolarizing voltages, ajmaline at high concentration of 300 micromol/l reduced the inward moiety of time-independent potassium current (IK1) by 36%. The results indicate that the inhibition of INa causes both the decreased rate of rise of depolarizing phase and the lowered amplitude of AP. The inhibition of Ito is responsible for the ajmaline-induced AP prolongation.     

Thallous ion activation of p-nitrophenylphosphatase of rat myometrium plasma membrane

Addition of thallous ion (Tl+) inhibited the spontaneous mechanical activity of rat myometrium in K-free Krebs solutions with an I50 value of 30 microM. The corresponding value for I50 for similar inhibition by addition of K+ was 150 microM. Tl+ as well as K+-activated p-nitrophenylphosphatase (PNPPase) of isolated rat myometrium plasma membrane vesicles. Half maximal activation was caused by 0.47 mM Tl+ or 1.6 mM K+. Maximal enzyme activities obtained using Tl+ and K+ were comparable. The Km values for the substance p-nitrophenylphosphatase using Tl+ (1.24 mM) and K+ (1.46 mM) were also similar. Activation by either ion was inhibited by ouabain, Na+, inorganic phosphate, and vanadate (V +5). The results suggest that Tl+ can substitute for K+ for activation of the Na-K pump of rat myometrium plasma membrane.     

Effects of fatty acids on membrane currents in the squid giant axon

Summary The effects of fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular and extracellular application of the test substances. Fatty acids mainly suppress the Na current but have little effect on the K current. These effects are completely reversed after washing with control solution. The concentrations required to suppress the peak inward current by 50% and Hill number were determined for each fatty acid. ED₅₀ decreased about 1/3 for each increase of one carbon atom. The standard free energy was –3.05 kJ mole^–1 for CH₂. The Hill number was 1.58 for 2-decenoic acid. The suppression effect of the fatty acids depends on the number of carbon atoms in the compounds and their chemical structure. Suppression of the Na current was clearly observed when the number of carbon atoms exceeded eight. When fatty acids of the same chain length were compared, 2-decenoic acid had strong inhibitory activity, but sebacic acid had no effect at all on the Na channel. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (J. Physiol. (London) 117:500–544, 1952) and the changes in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ({ie113-1}) were shifted 20 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ({ie113-2}) was almost no change by application of the fatty acids. The time constant for activation ({ie113-3}) of K current was shifted 20 mV in a depolarizing direction by the application of the fatty acids.     

急性低温/再复温对大鼠心室肌膜电位和钾电流的影响

本文旨在研究急性低温/再复温对大鼠心室肌膜电位和钾电流的影响.膜电位和膜电流分别在全细胞膜片钳的电压钳和电流钳模式下记录.当细胞外灌流液从25℃降低到4℃后,一过性外向电流(transient outward current, Ito)完全消失,膜电位为 60mV时的稳态外向K 电流(sustained outward K current, Iss)和膜电位为-120mV时的内向整流K 电流(inward rectifier K current, IK1)分别降低(48.5±14.1)%和(35.7±18.2)%,同时,膜电位绝对值降低.当细胞外灌流液从4℃再升高到36℃后,膜电位出现一过性超级化,然后恢复到静息电位水平;在58个细胞中,有36个细胞伴随复温出现ATP-敏感性K (ATP-sensitive K , KATP)通道的激活.再复温引起的上述变化可以被Na /K -ATP酶抑制剂哇巴因(100μmol/L)所抑制.再复温引起的KATP通道激活也能被蛋白激酶A抑制剂H-89(100μmol/L)所抑制.在细胞膜电位被钳制在0mV时,当细胞外灌流液温度从25℃降低到4℃后,细胞的体积没有发生明显改变,但当再复温引起KATP通道激活后,细胞很快发生皱缩,同时细胞内部出现许多折光较强的斑点.上述结果表明急性低温/再复温对大鼠心室肌膜电位和K 电流有明显影响,并提示KATP通道激活可能与心肌低温/再复温损伤有关.     

Effects of neurohypophyseal hormones on food-reinforced classical conditioning in the rat

The effects of different doses of lysine vasopressin (LVP) and oxytocin (OXT) were studied on the six-day acquisition or extinction of a food-reinforced classical conditioning reflex (conditional stimulus: light) when intraperitoneal (ip.) injections were carried out 20 min prior to the behavioural sessions. The highest (600 mU/kg) dose of LVP inhibited acquisition, and all LVP doses tested (150, 300 and 600 mU/kg) facilitated the extinction of conditioned behaviour. These same mU doses of OXT did not significantly affect the food-reinforced conditioning, although a consequent tendency towards increased performance (the opposite action to vasopressin) was observed. When 2.5 or 25 micrograms/kg doses of desglycinamide-arginine-vasopressin (DGAVP), a 500 micrograms/kg dose of prolyl-leucyl-glycinamide (PLG) or a 1200 mU/kg dose of OXT was injected during the extinction sessions, 2.5 micrograms/kg DGAVP and 1200 mU/kg OXT significantly facilitated extinction; the other treatments were without effect. LVP in a dose of 300 or 600 mU/kg and OXT in a dose of 300, 600 or 1200 mU/kg did not influence the food intake of 22 h food-deprived rats in a nonconditional situation. The present results indicate that the effects of LVP and OXT on memory display reinforcement-dependent characteristics, and are thus indirect or non-specific in nature.