bluuebunny/crossref_metadata_2025 huggingface dataset:
Monosynaptic EPSPs in cat lumbosacral motoneurones from group Ia afferents and fibres descending in the spinal cord.
Authors: Harrison P J , Jack J J , Kullmann D M
Published: December 2014

1. Excitatory postsynaptic potentials (EPSPs) were elicited in lumbosacral motoneurones of pentobarbitone‐anaesthetized cats by stimulating the ventral quadrants (VQ) of the thoracic spinal cord. These EPSPs were compared with monosynaptic EPSPs from small numbers of group Ia afferents, obtained by stimulating hindlimb muscle nerves with most of the dorsal roots severed. 2. EPSPs with average peak amplitude less than 1 mV were selected for fluctuation analysis. Three out of fourteen (21%) VQ EPSPs with peak voltage less than 150 mu V fluctuated in amplitude from trial to trial no more than could be accounted for by the background intracellular noise. Similarly, nine out of thirty‐nine (23%) Ia EPSPs smaller than 150 mu V fluctuated to a comparable extent as the noise. These results are consistent with the view that there is little variation in the postsynaptic signal produced by an individual transmitter release event. 3. Of the EPSPs which did fluctuate more than the background noise, maximum likelihood estimates were obtained for the fluctuation patterns of ten VQ and fourteen Ia EPSPs. This was achieved by assuming that synaptic signals sum linearly with noise, but without constraining the results to conform to a statistical description of transmitter release. The fluctuation of both VQ and Ia EPSPs was made up of discrete amplitudes separated by roughly equal increments, in accordance with the quantal hypothesis of synaptic transmission. 4. Fluctuation patterns were obtained simultaneously for VQ and Ia EPSPs in seven motoneurones. The amplitudes of the quanta, defined as the mean increments between discrete amplitudes, were correlated (r = 0.90), suggesting common postsynaptic mechanisms. 5. For most EPSPs the time course of the voltage transient could be used to estimate the electrical distance from the soma at which the synaptic current was injected. There was a comparable distribution for VQ and Ia EPSPs. For those in which a quantal analysis was performed (nine VQ and eleven Ia), quantal size measured at the soma appeared to be independent of the deduced site of origin. 6. The results indicate no qualitative or quantitative differences in the behaviour of VQ and Ia EPSPs.
Dihydropyridine inhibition of single calcium channels and contraction in rabbit mesenteric artery depends on voltage.
Authors: Nelson M T , Worley J F
Published: December 2014

1. The effects of membrane potential and the dihydropyridine calcium channel inhibitor, nisoldipine, on single calcium channels in the presence of Bay K 8644 and contraction in the presence and absence of Bay K 8644 were examined in the rabbit mesenteric artery. 2. Membrane depolarization decreased the peak average single calcium channel current that could be elicited by a test pulse to 0 mV. The steady‐state inactivation relationship could be described by the Boltzmann equation, [1 + exp[Vm‐V0.5)/k)]‐1, with a steepness factor, k, of 7.1 mV. Nisoldipine shifted the steady‐state inactivation curve to more negative potentials by increasing the fraction of test pulses without openings. 3. The degree of nisoldipine inhibition of average single calcium channel currents increased with membrane depolarization. Depolarization of the holding potential from ‐100 to ‐55 mV decreased the concentration of nisoldipine needed for 50% inhibition (Kapp) from 12.1 to 1.9 nM in the presence of 1 microM‐Bay K 8644. 4. Membrane depolarization by external potassium (K+) of the intact artery in the presence of nisoldipine decreased contractions evoked by depolarizing test pulses. The relationship between membrane potential and contraction could be empirically described by the Boltzmann equation, with a steepness factor, k, of 7.1 mV. Increasing the nisoldipine concentration from 0.25 to 2.0 nM shifted the mid‐point of this relationship from ‐20.5 to ‐33.0 mV, without affecting the steepness factor. 5. Nisoldipine inhibition of contraction increased with membrane depolarization. Membrane depolarization from ‐68.6 to ‐30.0 mV decreased the Kapp of nisoldipine for contractions from 3.02 to 0.69 nM. Bay K 8644 (1 microM) elevated Kapp about 9.3‐fold at 5 mM‐K+. In the presence of Bay K 8644, membrane depolarization from ‐68.6 to ‐30.0 mV reduced Kapp from 28.4 to 4.0 nM. 6. In the presence of nisoldipine, the effect of membrane depolarization on the time course of development of inhibition was examined. In 3 nM‐nisoldipine, after membrane depolarization with 20 mM‐K+, the time course of development of inhibition of force could be described by a single exponential with a time constant of 16.5 min. Membrane depolarization to a more positive potential accelerated the development of inhibition. 7. The results were interpreted by a model in which nisoldipine binds with higher affinity to the inactivated state than to the resting state of calcium channels in the mesenteric artery. The approach presented here can be used to estimate the properties of steady‐state calcium channel inactivation and dihydropyridine interactions in smooth muscle cells in the intact artery under physiological conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle.
Authors: Godt R E , Nosek T M
Published: December 2014

1. Maximal calcium‐activated force (Fmax) and calcium sensitivity were markedly decreased in detergent‐skinned fibres from skeletal and cardiac muscle by solutions that mimicked the total milieu changes associated with fatigue and hypoxia. Further experiments determined the relative contribution of each of the individual changes in milieu. 2. Both Ca2+ sensitivity and Fmax of skeletal and cardiac fibres were decreased with increased [H+] or inorganic phosphate (Pi). These effects were greater in cardiac muscle. 3. Decreasing MgATP over the range observed with fatigue and hypoxia (6.8‐4.7 mM) had no effect on Fmax or Ca2+ sensitivity of either muscle type. 4. Decreasing phosphocreatine (PCr: 15‐1 mM) increased Fmax but had little effect on Ca2+ sensitivity in both muscle types. In cardiac fibres, the effect on Fmax could be mimicked by inhibition of endogenous creatine kinase. 5. ADP (0.7 mM) increased Fmax and Ca2+ sensitivity, while AMP (0.06 mM) slightly increased Fmax but had no effect on Ca2+ sensitivity of either skeletal or cardiac fibres. 6. Creatine (25 mM) had no significant effect on either Ca2+ sensitivity or Fmax of skeletal and cardiac muscle fibres. At higher levels (50 mM), however, creatine depressed Fmax and slightly altered Ca2+ sensitivity. 7. Thiophosphorylation of myosin P light chains (phosphorylatable light chains of myosin) in rabbit psoas fibres had no effect on Ca2+ sensitivity, yet slightly but significantly increased Fmax under fatigue conditions. 8. Reducing the affinity for ATP hydrolysis (by adding ADP, AMP and creatine) over the range calculated for fatigue/hypoxia (60‐45 kJ/mol) produced the enhancement in Fmax expected from added ADP and AMP in cardiac but not skeletal muscle, indicating that changes in affinity influence Fmax of skeletal muscle. Reducing affinity produced little change in Ca2+ sensitivity of skeletal muscle. In contrast, the change produced in cardiac muscle was greater than that expected from addition of ADP and AMP; i.e. decreasing affinity increases calcium sensitivity of the heart. 9. Simple summation of all significant changes expected from each constituent altered by fatigue/hypoxia adequately predicted the observed changes in Fmax and Ca2+ sensitivity in both cardiac and skeletal muscle fibres with but one exception (the change in Ca2+ sensitivity of skeletal muscle at pH 7 was slightly overestimated).
Caffeine affects four different ionic currents in the bull‐frog sympathetic neurone.
Authors: Akaike N , Sadoshima J
Published: December 2014

1. Ionic mechanisms related to the caffeine‐induced current (Icaffeine) were examined in the single isolated sympathetic neurones of the bull‐frog. We used the ‘concentration‐jump’ technique in combination with intracellular perfusion and a rapid external solution change, under single‐electrode voltage‐clamp conditions. 2. Icaffeine was pharmacologically separated into a tetraethylammonium (TEA)‐sensitive transient outward current (ITO), a picrotoxin (PTX)‐sensitive transient inward current (ITI) and a TEA‐ and PTX‐insensitive sustained inward current (ISI). At low concentrations of caffeine, a sustained outward current (ISO) was observed instead of ISI. 3. All components of Icaffeine were abolished by intracellular perfusion of 30 mM‐EGTA. Pre‐treatment with A23187 or ryanodine or the simultaneous application of procaine either reduced or abolished all the components of Icaffeine in a dose‐dependent manner. The concentration causing 50% inhibition (IC50) was 10(‐8) M for A23187 and 2 mM for procaine. 4. The peak response of ITO increased abruptly at caffeine concentrations between 3 and 6 mM followed by saturation above 30 mM. A notch was observed on the rising phase of ITO. 5. The reversal potential (Ecaffeine) of ITO shifted 58 mV for a tenfold change of the extracellular K+ concentration. External application of TEA blocked ITO with an IC50 of 1 mM. ITO was relatively insensitive to apamin, 4‐aminopyridine and muscarine. 6. In external solution containing 2 mM‐Ca2+, ITO induced by 10 mM‐caffeine recovered completely within 3 min from a previous exposure to caffeine. In the absence of extracellular Ca2+, there was little such recovery. A 5 min treatment in a Ca2+‐free solution reduced ITO induced by the first application of caffeine by 5%. With a continuous application of 3 mM‐caffeine, the amplitude of ITO induced by 10 mM‐caffeine reduced in 1 min, and showed a partial recovery in 3 min. The amplitude of ITO increased by increasing the concentration of intracellular Cl‐. 7. ITI was activated around the peak of ITO and was rapidly inactivated. ITI was evoked at caffeine concentrations of about 6‐10 mM. When the intracellular Cl‐ concentration was changed, the amplitude of ITI behaved like a Cl‐ electrode. The Ecaffeine of ITI was close to the Cl‐ equilibrium potential (ECl). 8. ISI was a ‘plateau’ response and persisted for over 3 min. ISI was due to a decrease in K+ conductance. In the presence of muscarine (3 x 10(‐5) M), ISI was occluded.(ABSTRACT TRUNCATED AT 400 WORDS)
A near‐zero membrane potential in transporting corneal endothelial cells of rabbit.
Authors: Hodson S , Wigham C
Published: December 2014

1. When rabbit corneal endothelial cells are impaled with 3 M‐KCl‐filled microelectrodes (Rt = 20‐70 M omega) a stable membrane potential of ‐28.7 +/‐ 4.8 mV (mean +/‐ S.D., n = 400) is measured. 2. Varying the [KCl] of the filling solution causes a change in measured membrane potential; 154 mM gives typically ‐2 mV, 10 mM typically +37 mV. 3. Variation in membrane potential with different [KCl] cannot be ascribed to tip potential. Double‐barrelled microelectrodes containing a different [KCl] in each barrel both give the same membrane potential when inserted into a cell. 4. Microelectrodes filled with a reference liquid ion exchanger (RLIE) give a membrane potential of +2 mV. 5. Impaling a cell with a double‐barrelled microelectrode, one barrel containing KCl and the other RLIE, showed that the ion exchanger is not having a toxic effect on the cell and does not affect membrane potential measured by the KCl‐filled barrel. 6. We suggest that microelectrodes containing non‐isotonic concentrations of KCl generate a significant and artifactual change in membrane potential of corneal endothelial cells caused by the movement of excess KCl (originating from the microelectrode tip) across the plasma membrane where a liquid‐junction potential is generated. 7. We further suggest that the physiological membrane potential of corneal endothelial cells is around zero. This could result from a solution of the constant field equation where: 0.9 PNa congruent to PK congruent to 3.2 PCl.
Identification of the major membrane currents in freshly dispersed single smooth muscle cells of guinea‐pig ureter.
Authors: Lang R J
Published: December 2014

1. The passive and active electrical properties of freshly dispersed single cells of the guinea‐pig ureter were investigated using standard patch‐clamp techniques. 2. Action potentials, having a rapid rising phase and a prolonged plateau, were recorded on passing depolarizing currents through the patch pipette when ‘near‐normal’ physiological gradients were established across the cell membrane (5.9 mM‐K+, 1.5 mM‐Ca2+ in the bath; 126 mM‐K+ in the pipette). 3. Under voltage clamp, depolarization to potentials positive of ‐50 mV (from a holding potential of ‐70 or ‐80 mV) triggered a net inward current which reached a peak in 5‐10 ms and then slowly inactivated. 4. The averaged membrane current to depolarization to potentials between ‐30 and 0 mV showed two distinct patterns after the peak of the inward current; the membrane current either moved slowly outward over 400 ms or there was one or more transient outward currents superimposed on the slowly decreasing inward current. Both outward currents were blocked when 5 mM‐tetraethylammonium (TEA) was added to the bathing solution, resulting in an increased inward current at all potentials. 5. Replacing the extracellular Ca2+ with Co2+ (1.5‐5 mM) blocked the inward current and the outward currents to reveal another transient outward current (voltage activated) which activated rapidly to reach a peak within 5 ms and which inactivated exponentially with a time constant of 10 ms. This voltage‐activated outward current was inactivated if the membrane was held at ‐50 mV, but could be reactivated by short hyperpolarizing pre‐pulses. The amplitude of this transient current in response to a fixed depolarization (to 0 mV) was half‐maximum when the hyperpolarizing pre‐pulse was to ‐66 mV. The voltage‐activated outward current was reduced in amplitude when the extracellular potassium was raised to 46 mM or upon exposure to 1 mM‐4‐aminopyridine (4‐AP), but was not affected by 5 mM‐TEA. 6. Replacing K+ in the pipette and bathing solution with caesium (Cs) blocked all outward currents, revealing the time course and voltage dependence of the inward current, which could be carried by Ca2+ or Ba2+ with little effect on its rate of inactivation. 7. It was concluded that the inward current recorded in single ureter cells was due to the flow of current through voltage‐activated Ca2+ channels. The TEA‐sensitive outward currents, whether transient or slowly activating, are presumably K+ channels activated by Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Protection of hippocampal slices from young rats against anoxic transmission damage is due to better maintenance of ATP.
Authors: Kass I S , Lipton P
Published: December 2014

1. Dentate granule cells in hippocampal slices from young rats (aged 30‐40 days) are more resistant to damage from 10 min of anoxia than are granule cells from adult rats. The evoked population spike from these cells recovers to 78% of its pre‐anoxic amplitude in young animals while in adult animals it shows only 4% recovery. This increased resistance is associated with higher levels of adenosine triphosphate (ATP) during the anoxic period. 2. When the duration of anoxia in slices from young animals is increased to 15 min, ATP falls to levels found in adult tissue after 10 min of anoxia. The dentate granule cells in slices from young animals show little recovery of the evoked response (19%) after such an exposure to anoxia. 3. When slices from young animals are subjected to 10 min of anoxia in low‐glucose (2 mM) artificial cerebrospinal fluid, ATP levels fall to those found in adult tissue after 10 min of anoxia and the evoked response from the dentate granule cells again shows little recovery (10%). 4. The evoked response in the CA1 pyramidal cell layer of slices from young rats is more resistant to damage from 5 or 7 min anoxia than it is in slices from adults. Thus this region, also, shows an age‐dependent increase in susceptibility to anoxic damage. ATP levels in the CA1 region of tissue from young animals at the end of 5 and 7 min anoxia are greater than ATP levels in tissue from adult animals after these same anoxic exposures. 5. Basal levels of 45Ca accumulation are greater in CA1 and dentate gyrus from young rats. However, the percentage increases during 10 min of anoxia are less than one‐half the values in slices from adult animals. 6. The results suggest that the increased resistance of slices from young animals to anoxic transmission damage may be explained by the better maintenance of ATP in synaptic regions of these slices during anoxia. This may confer the increased resistance by lowering the anoxic increase in cell Ca2+.
Multiple blocking mechanisms of ATP‐sensitive potassium channels of frog skeletal muscle by tetraethylammonium ions.
Authors: Davies N W , Spruce A E , Standen N B , Stanfield P R
Published: December 2014

1. Patch‐clamp methods were used to study the action of tetraethylammonium ions (TEA+) and other quaternary ammonium ions on adenosine‐5'‐triphosphate (ATP)‐sensitive K+ channels in sarcolemmal vesicles from frog skeletal muscle. The blocking ions were applied either to the external or the internal surface of the membrane patch. 2. External TEA+ caused a very fast block, so that the amplitude of single‐channel currents was reduced. Open‐channel variance was decreased. The block was 1:1, with a dissociation constant (Kd) of 6‐7 mM. We could detect no voltage dependence of Kd. 3. External TEA+ prolonged open times in a manner consistent with the channel being unable to close when blocked by TEA+. 4. TEA+ also blocked when applied to the internal side of the membrane. This block showed two components with different kinetics and different affinities. The slow block chopped up openings into much briefer events and had a Kd of about 1.4 mM at ‐3 mV. The fast block reduced the amplitude of unitary currents and was of lower affinity, with Kd around 26 mM. 5. The slow block by internal TEA+ was markedly voltage dependent, the Kd decreasing e‐fold for a 37 mV depolarization. Both the association and dissociation rates were dependent on voltage. In contrast, the fast block by internal TEA+ appeared virtually independent of voltage. 6. The effects of internally applied tetramethylammonium (TMA+) and tetrapentylammonium (TPA+) ions were also investigated. Internal TMA+ produced a flickery block while the block by internal TPA+ was similar to that caused by TEA+, although TPA+ was about 10 times more effective. 7. Our results suggest that the channel has three binding sites for TEA+, one of which is accessible from the outside of the membrane. Only one of the internal sites is located so as to experience a substantial fraction of the membrane voltage field.
Macroscopic K+ currents in single smooth muscle cells of the rabbit portal vein.
Authors: Hume J R , Leblanc N
Published: December 2014

1. Single smooth muscle cells isolated from rabbit portal vein were voltage clamped at room temperature using the whole‐cell configuration of the patch‐clamp technique. These cells exhibited a mean resting potential of ‐47.9 mV and a mean input resistance of 376 M omega. 2. Using small tip diameter micropipettes (to avoid dialysis of the cells), depolarizing voltage‐clamp pulses from a holding potential of ‐50 mV elicited two distinct outward currents: a quasi‐instantaneous background current and a time‐dependent current that did not appear to inactivate (delayed rectifier). Upon return to the holding potential, an outward tail current decaying back to the holding current was observed. 3. The time course of development of the tail current as estimated from envelopes of tail current protocols followed the kinetics of activation of the delayed rectifier elicited during the preceding test pulse. The tail current reversed close to the equilibrium potential for K+ ions indicating that it is mainly carried by potassium ions. 4. Using large tip diameter micropipettes to internally dialyse the cells (EGTA = 0.1 mM; ATP = 5 mM), two additional outward currents having transient kinetics were revealed: a smooth transient outward current (Ito) and spontaneous transient outward currents (STOCs). Ito was found to be mainly selective for K+ ions and exhibited voltage‐dependent inactivation with half‐maximal availability near ‐40 mV. 5. Removal of calcium from the bathing solution significantly reduced the background current and abolished both Ito and STOCs. The delayed rectifier current appeared to be insensitive to this procedure. The two types of transient outward currents were never recorded when EGTA was elevated to 5 mM inside the micropipette whereas the background and delayed rectifier currents were not affected. These results suggested that Ito and the spontaneous transient outward currents are activated by internal calcium. 6. External application of TEA (0.5‐20 mM) blocked all four outward currents. Calcium replacement by barium significantly reduced the background current and Ito, and had small effects on the delayed rectifier current. When potassium was replaced with caesium (130 mM) and TEA (20 mM) inside the pipette, none of the outward currents described was ever observed. In about 60% of the cells dialysed with this solution a small inward Ca2+ current was revealed. 7. External application of caffeine (5 mM) abolished STOCs in cells in which this activity was present under control conditions. In cells lacking this type of activity under control conditions caffeine induced and later abolished this type of current.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of proton currents in neurones of the snail, Lymnaea stagnalis.
Authors: Byerly L , Suen Y
Published: December 2014

1. Internal perfusion voltage‐clamp and inside‐out patch‐clamp techniques were used to study the voltage‐dependent H+ currents in snail neurone cell bodies. 2. In whole cells the voltage‐activated outward H+ current was measured 60 ms after stepping to +40 mV with an internal pH (pHi) of 5.9 and no internal K+([K+]i = 0), and the delayed K+ current was measured 60 ms after stepping to +40 mV with pHi = 7.3 and [K+]i = 74 mM. The mean H+ and K+ current densities were 14.6 +/‐ 7.8 and 38.2 +/‐ 14.0 nA/nF, respectively, giving a mean ratio of the H+ to K+ current of 0.4 +/‐ 0.2. There is not a strong correlation between the densities of the two kinds of outward currents found in different cells. 3. Inside‐out patch studies reveal that the H+ and K+ currents are distributed quite differently in the membrane. While 85% of all patches had K+ current, only five out of thirty‐eight patches studied had H+ currents. In those five patches the H+ currents measured at +30 mV ranged from 10.7 to 21.0 pA, and the ratio of the H+ and K+ currents at +30 mV was 0.83 +/‐ 0.38. The mean H+ and K+ currents for all thirty‐eight patches were 1.9 +/‐ 4.9 and 10.5 +/‐ 7.9 pA, respectively. 4. The current distribution patterns demonstrate that the H+ current does not flow through the delayed K+ current channels even though the two currents have similar voltage dependence and time course. 5. The relative ability of various extracellular divalent cations to block the H+ current was found to be Cu2+ approximately equal to Zn2+ greater than Ni2+ greater than Cd2+ greater than Co2+ greater than Mn2+ greater than Mg2+ = Ca2+ = Ba2+. Since 100 microM‐Zn2+ blocks the H+ current more than it blocks the Ca2+ current, it can be used to reduce the contamination of Ca2+ current measurements by the H+ current. 6. The magnitude of the H+ current has a stronger temperature sensitivity than does the magnitude of the delayed K+ current. The Q10 of the H+ current magnitude is 2.1 +/‐ 0.4, while the Q10 of the K+ current magnitude is 1.4 +/‐ 0.04. This suggests a higher activation energy may be involved in the conduction of the H+ current than for K+ current. 7. The smooth time course of the H+ current measured in patches indicates that the size of the unitary H+ current is very small.(ABSTRACT TRUNCATED AT 400 WORDS)
Properties of calcium channels in guinea‐pig gastric myocytes.
Authors: Katzka D A , Morad M
Published: December 2014

1. The inward membrane current in enzymatically dispersed guinea‐pig gastric myocytes was studied using whole‐cell voltage clamp technique. 2. Only one inward membrane current was found in gastric myocytes which was identified as the Ca2+ current based on its inhibition by Ni2+, Cd2+ and Co2+, its dependence on [Ca2+]o, and its insensitivity to variations of [Na+]o. 3. Ca2+ current activated at ‐20 mV, peaked around +10 mV and was markedly enhanced when the holding potential was increased from ‐40 to ‐90 mV. The enhancement of ICa at negative holding potentials did not alter the activation threshold of ICa. When Ba2+ was substituted for Ca2+, IBa was similarly enhanced at more negative potentials. 4. In cells where internal Ca2+ was buffered with 10 mM‐EGTA, the time course of inactivation was fitted with two exponentials, with time constants: tau f = 53.4 +/‐ 18.1 ms and tau s = 175.2 +/‐ 46.1 ms. When Ba2+ was the charge carrier through the channel, the time course of inactivation could be fitted often by only one exponential which approximated tau s for inactivation of ICa. The voltage dependence of steady‐state inactivation of Ca2+ channels was not significantly altered when Ba2+ was the charge carrier. 5. Using different buffering systems (EGTA, EDTA and citrate), we found that citrate maintained the ICa and slowed inactivation more effectively than the other buffers tested. Because the calculated change in [Ca2+]i did not differ significantly between buffer systems, we speculate that suppression of inactivation by citrate is related to increased accessability of the buffer to cytoplasmic Ca2+ near the Ca2+ channel. Changes in [Mg2+]i affected peak ICa but not the kinetics of inactivation indicating that [Mg2+]i may regulate the steady‐state inactivation or the availability of the Ca2+ channels. 6. The divalent selectivity of the Ca2+ channel had the following sequence: Ba2+ greater than Ca2+ greater than or equal to Sr2+ much greater than Mg2+. In very low extracellular Ca2+ (less than 10(‐7) M), the Ca2+ channel conducted Na+. 7. Increasing [H+]o appeared to differentially affect peak and maintained components of ICa. At pH less than 6.5, the maintained component of ICa was suppressed more than the peak component indicating possible time‐ and voltage‐dependent inhibition of ICa by protons. 8. Nifedipine, D600 and diltiazem inhibited ICa in a voltage‐dependent manner. The order of potency for inhibition of peak ICa was nifedipine approximately D600 much greater than diltiazem.(ABSTRACT TRUNCATED AT 400 WORDS)
Determination of ionic permeability coefficients of the plasma membrane of Xenopus laevis oocytes under voltage clamp.
Authors: Costa P F , Emilio M G , Fernandes P L , Ferreira H G , Ferreira K G
Published: December 2014

1. A method of estimating absolute ionic permeability coefficients which does not depend on the use of impermeant substitutes is reported. 2. The method is based on a pump leak model of the Xenopus laevis oocyte membrane. The procedure consists of measuring, in the same experiment, the pump current and the currents generated under voltage clamp by the partial substitution of one or two ions at a time. For each experimental condition, the measured currents are substituted in a Goldman‐Hodgkin‐Katz type equation with two unknowns (the permeability coefficients). The set of equations thus generated enables the computation of all the ionic permeability coefficients. 3. The Xenopus oocyte membrane (stages IV and V, Dumont, 1972) has been found to be permeable to conventional ion substitutes such as N‐methyl‐D‐glucamine (NMG), sulphate, isethionate and gluconate. 4. The values for sodium, potassium and chloride permeability coefficients obtained from sixty‐eight pooled experiments were, respectively, 5.44, 17.41 and 1.49 x 10(‐8) cm s‐1. 5. The diffusional currents for sodium, potassium and chloride computed from the experiments referred to above were, respectively, ‐1.16, 0.69 and ‐0.038 microA cm‐2. 6. A stoichiometry of the Na+‐K+ pump exchange of 3/1.8 was computed. 7. The intracellular concentrations of sodium, potassium and chloride ions, as determined by ion‐selective microelectrodes, were, respectively, 10.1 +/‐ 0.66 mM (n = 12), 109.5 +/‐ 3.3 mM (n = 13) and 37.7 +/‐ 1.18 mM (n = 19), corresponding to equilibrium potentials of 61, ‐95 and ‐28 mV. 8. Since chloride is not at equilibrium across the membrane, we propose that there is an inward uphill Cl‐ transport.
Interaction between calcium channel ligands and guanine nucleotides in cultured rat sensory and sympathetic neurones.
Authors: Dolphin A C , Scott R H
Published: December 2014

1. Voltage‐activated Ca2+ channel currents were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole‐cell clamp technique with Ba2+ as the charge carrier. 2. Inclusion of the GTP analogue guanosine 5'‐O‐3‐thiotriphosphate (GTP‐gamma‐S, 500 microM) or guanylylimidodiphosphate (GMP‐PNP, 500 microM) or GTP itself (1 mM) in the patch pipette solution resulted in a smaller, slowly activating Ca2+ channel current which did not inactivate during a 100 ms voltage step. This current was inhibited by CdCl2 (10‐100 microM) and omega‐conotoxin (1 microM). 3. Nifedipine (5 microM), (‐)‐(R)‐201‐791 (5 microM), D600 (10 microM), and diltiazem (30 microM) inhibited Ca2+ channel currents recorded from control neurones, although in some cells a biphasic response was observed, with an initial increase preceding the inhibition of the currents. In the presence of internal GTP‐gamma‐S, at a holding potential (VH) of ‐80 mV, only potentiation of the Ca2+ channel current was observed in the presence of all three Ca2+ channel ligands. Internal GMP‐PNP, while less effective than GTP‐gamma‐S, also resulted in D600 showing an agonist response. Similarly, in the presence of internal GTP (1 mM), (‐)‐(R)‐202‐791 gave a prolonged agonist response. 4. Nifedipine, whether acting as an antagonist in control cells or as an agonist in GTP‐gamma‐S‐containing cells, induced a shift to more hyperpolarized potentials of the steady‐state inactivation curves. 5. Potentiation of Ca2+ channel currents induced by D600 in GTP‐gamma‐S‐containing cells, was not observed when the neurones were pre‐treated with pertussis toxin. The presence of internal GDP‐beta‐S (500 microM) did not significantly alter the maximum inhibitory action of D600 compared with controls. However, 1 mM‐GDP‐beta‐S increased the rate of onset of inhibition by (‐)‐(R)‐202‐791. 6. Depolarizing VH to ‐30 mV accelerated the onset of inhibition induced by the Ca2+ channel ligands in control cells. In the presence of internal GTP‐gamma‐S at VH ‐30 mV, biphasic responses were produced by all the Ca2+ channel antagonist ligands with initial stimulation for 1‐2 min being followed by inhibition of the Ca2+ channel currents. 7. The agonist actions of (+)‐(S)‐202‐791 were potentiated by the presence of internal GTP‐gamma‐S. 8. The expression of an agonist response to (‐)‐(R)‐202‐791 induced by internal GTP‐gamma‐S was also present in sympathetic neurones cultured from adult rat superior cervical ganglion (SCG).(ABSTRACT TRUNCATED AT 400 WORDS)
A cation channel in the thick ascending limb of Henle's loop of the mouse kidney: inhibition by adenine nucleotides.
Authors: Paulais M , Teulon J
Published: December 2014

1. Patch‐clamp single‐channel current recordings were used to study the inhibition of Ca2+‐activated non‐selective cation channels by internal nucleotides in patches excised from basolateral membranes of the thick ascending limb of Henle's loop of the mouse kidney. 2. The application of ATP, ADP or AMP to the cytoplasmic face of excised inside‐out membrane patches reduced the open‐state probability of the channels (Po) in a dose‐dependent way without effect upon the unitary current amplitude. Dose‐response curves gave half‐maximal inhibitory concentrations of 20, 21 and 2.5 microM for ATP, ADP and AMP, respectively, while the Hill coefficient was close to one in all three cases. 3. Cyclic AMP partially inhibited channel activity (Po = 35 +/‐ 17% of control) only at high, unphysiological concentrations (10(‐3) M) while adenosine (10(‐3) M) had very little effect (Po = 83 +/‐ 7% of control). 4. Replacement of adenine with other purines (guanine, hypoxanthine) or pyrimidine (uridine) bases very largely reduced inhibitory activity. Cyclic GMP had no effect. 5. Non‐hydrolysable analogues of ATP, AMP‐PNP (10(‐3) M) and ATP‐gamma‐S (5 x 10(‐4) M), were effective inhibitors of the channel (Po = 24 +/‐ 7 and 9 +/‐ 4% of control, respectively.
Substrate dependence of energy metabolism in isolated guinea‐pig cardiac muscle: a microcalorimetric study.
Authors: Daut J , Elzinga G
Published: December 2014

1. The effects of glucose, pyruvate and lactate on basal metabolism and on contraction‐related energy expenditure of thin trabeculae isolated from guinea‐pig heart were studied using a microcalorimetric technique. 2. Resting heat rates of cardiac ventricular muscle measured in the presence of substrate‐free solution (56 +/‐ 20 mW (g dry weight)‐1), 10 mM‐lactate (54 +/‐ 12 mW (g dry weight)‐1) and 10 mM‐glucose (63 +/‐ 24 mW (g dry weight)‐1) did not differ significantly. Increasing the external glucose concentration (up to 100 mM) and/or adding insulin (up to 80 units l‐1) had virtually no effect on the measured resting heat rate. 3. With 10 mM‐pyruvate as substrate resting heat rate was substantially larger (106 +/‐ 40 mW (g dry weight)‐1) than with glucose, lactate or substrate‐free solution. The concentrations of pyruvate producing a half‐maximal increase in resting heat rate as compared to substrate‐free solution ranged between 0.4 and 1.2 mM. 4. In order to test whether the development of an anoxic core contributed to the substrate dependence of resting heat production the critical PO2 (i.e. the PO2 that produced a just‐noticeable decrease in heat rate) was determined in cylindrical preparations of various diameters. It was found that none of the preparations had an anoxic core at rest in a solution equilibrated with 100% oxygen. 5. From the dependence of the critical PO2 on the diameter of the preparation the diffusion coefficient of oxygen through cardiac muscle was calculated using a modification of Hill's equation (Hill, 1928). The O2 diffusion coefficient was found to be 1.09 X 10(‐5) cm2 s‐1. 6. Contraction‐related heat production was also found to be dependent on the substrate used. In the presence of 10 mM‐pyruvate it was about 60% larger than in the presence of 10 mM‐glucose, 10 mM‐lactate or with substrate‐free solution. 7. Isometric force of contraction showed the same substrate dependence as contraction‐related heat production and increased with a similar time course during repetitive stimulation. 8. The possible mechanisms underlying the substrate dependence of myocardial energy metabolism are discussed. It is suggested that the increased energy expenditure observed in the presence of pyruvate may be related to a decrease in intracellular phosphate and/or to an increase in intracellular pH.
Modulation of K+ currents in human lymphocytes by pH.
Authors: Deutsch C , Lee S C
Published: December 2014

1. Using whole‐cell patch‐clamp techniques, we found that the voltage‐dependent K+ conductance in human peripheral blood T lymphocytes is enhanced threefold at alkaline intracellular pH (pHi) compared to acid pHi. This pH dependence can be described by a model having two strongly co‐operative proton binding sites with pka 7.15. A similar pHi sensitivity exists for K+ conductance in mitogen‐activated cells. 2. The reversal potential, threshold voltage for activation of the K+ conductance, and voltage dependence of steady‐state inactivation are not affected by pHi. Activation and inactivation kinetics are also unchanged. 3. Single‐channel measurements made in whole‐cell patch‐clamp mode indicate that the effect of intracellular pH on the amplitudes of single‐channel events parallels, but does not wholly account for, the effect of pHi on the macroscopic currents. 4. Lowering extracellular pH (pHo) shifts the threshold for activation of the K+ current to a more depolarized voltage, consistent with a surface charge screening effect. Apparent changes in peak current and activation kinetics at acid pHo can be accounted for by this voltage shift. An additional slowing of inactivation kinetics at low pHo does occur. 5. The relevance of the pH sensitivity of the voltage‐gated K+ conductance to lymphocyte mitogenesis and volume regulation is discussed.
The effect of the phenylalkylamine D888 (devapamil) on force and Ca2+ current in isolated frog skeletal muscle fibres.
Authors: Erdmann R , Lüttgau H C
Published: December 2014

1. The effects of the (+)‐ and the (‐)‐isomer of the phenylalkylamine derivative D888 (desmethoxyverapamil or devapamil) on isometric force and slow Ca2+ inward current were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage‐clamp conditions with two flexible internal glass microelectrodes at 10 degrees C in a TEA sulphate solution containing approximately 4 mM‐free Ca2+. 2. In the presence of 0.05‐5 microM‐(‐)‐D888 a normal phasic contracture could be induced by a depolarizing voltage step. When depolarization was maintained for some minutes the force‐controlling system turned into a stabilized inactivated state (paralysis) from which it recovered upon repolarization within minutes instead of seconds. With the (+)‐isomer (0.5‐20 microM), a similarly retarded restoration was observed. However, it proved to be less effective than the (‐)‐isomer. 3. D888 caused a shift to more negative potentials of the S‐shaped curve, which describes the voltage dependence of force restoration in the steady state (restoration time 15 min). The potential of half‐maximum restoration in the absence of the drug (V = ‐35.8 mV) changed as follows. (‐)‐D888: ‐56 mV (0.05 microM), ‐69 mV (0.2 microM), ‐77.5 mV (0.5 microM), and ‐82 mV (5 microM); (+)‐D888: ‐55.8 mV (0.5 microM), ‐76.5 mV (5 microM), and ‐85 mV (20 microM). 4. On the assumption that D888 binds only to the inactivated form of the voltage sensor of force control in the T‐tubular membrane (modulated receptor hypothesis) the data presented in paragraph 3 allowed an estimation of the drug‐receptor dissociation constants. The KD values ascertained in this way, 1.71 nM for the (‐)‐isomer and 12.9 nM for the (+)‐isomer, are in fair agreement with those obtained from [3H]D888 binding studies by other authors. 5. A comparison between equal concentrations of the two isomers regarding their effect on the speed of restoration and the time needed to transform the sensor into the paralysed state suggests that the differences in the dissociation constants are mainly due to a greater dissociation rate of the (+)‐isomer from the sensor. 6. The restoration of the Ca2+ channel was retarded by D888 to a similar extent as that of the voltage sensor. This parallel action on both systems indicates structural similarities between the voltage sensor and the Ca2+ channel. 7. It is concluded that D888 'stabilizes' the inactivated state of the voltage sensor and the Ca2+ channel in a way similar to D600, but with a higher potency. Both isomers of D888 showed an antagonistic action and differed only in their potency.
A comparison of the effects of denervation on the mechanical properties of rat and guinea‐pig skeletal muscle.
Authors: al‐Amood W S , Lewis D M
Published: December 2014

1. A fast (extensor digitorum longus) and slow (soleus) twitch muscle were denervated in rats and guinea‐pigs and isometric and isotonic contractions were followed for periods of up to 6 months after. 2. The decay of tetanic tension with time could be described as exponential. The rate of decay of tension was greatest in rat soleus and least in guinea‐pig soleus by a factor of more than three. The fast muscles atrophied at intermediate rates. 3. The contraction and relaxation times of soleus and extensor digitorum longus of rat, initially prolonged by denervation, became shorter after 3 weeks. There was no such reversal in either guinea‐pig muscle, indeed extensor digitorum longus twitch became even more prolonged. Guinea‐pig muscles often showed signs of repetitive response to a single stimulus, resulting in distortion of relaxation of the twitch. 4. There was a slowing of isotonic shortening velocity in the late stage of denervation of guinea‐pig extensor digitorum longus, accompanied by a fall in the rate of development of isometric tetanic tension. There was a just‐significant (P less than 0.1) increase in the shortening velocity of rat soleus. None of the other muscles showed any change in either rate characteristic. 5. In guinea‐pig extensor digitorum longus the type I fibres atrophied less than type II fibres; in all other muscles the atrophy was more uniform, possibly faster in type II. Guinea‐pig soleus remained pure type I contrasting with an increase in the numbers of type II fibres in rat soleus. There was a possible increase in the number of type I fibres in guinea‐pig fast muscle and no change in the rat.
Suppression of secretagogue‐induced amylase secretion in pancreatic acini of cold‐exposed rats.
Authors: Habara Y
Published: December 2014

1. The influence of prolonged exposure to a low ambient temperature on the pancreatic exocrine function was investigated in rats. Amylase concentration in acini prepared from rats housed at 5 degrees C for 2 weeks was specifically and dramatically reduced. The enzyme activity relative to acinar DNA concentration was only 48% of that of control acini obtained from rats reared at the thermoneutral temperature of 25 degrees C. Amylase content relative to acinar protein concentration also decreased by 35%. In contrast, acinar trypsinogen content relative to protein concentration increased to 161% of that of control acini. No comparable change was found in the parotid glands. 2. In acini from cold‐exposed rats, amylase release in response to cholecystokinin octapeptide or carbamylcholine was simultaneously reduced when expressed relative to acinar DNA or protein concentration and the dose‐response curve shifted downward. However, when the secretory responsiveness of amylase was represented as a percentage of initial acinar enzyme content, the observed decrease disappeared and the dose‐response curve was unaffected by cold exposure. 3. Secretagogue‐stimulated trypsinogen release was augmented in acini from cold‐exposed rats when expressed per acinar protein concentration. Conversely, its percentage release was slightly lowered but the release per acinar DNA concentration remained unaltered. 4. Pancreatic insulin concentration normalized to tissue wet weight or protein concentration was unaffected by prolonged cold exposure but the plasma insulin concentration significantly decreased by 45% as compared with that of thermoneutral rats. 5. Chronically administered exogenous insulin partially restored the decreased acinar amylase concentration in cold‐exposed rats. The dose‐response curve for amylase normalized by acinar DNA or protein content also tended to be shifted upward by insulin treatment. Acinar trypsinogen concentration in these rats was not significantly altered from that in cold‐exposed, non‐insulin‐treated rats. Percentage release of each enzyme was decreased by exogenous insulin. 6. Cold‐induced suppression of pancreatic acinar amylase concentration, resulting in a proportional decrease in secretory responsiveness, was confirmed at the cellular level. Insulin deficiency at a low ambient temperature is likely to be a major cause of cold‐induced suppression of amylase biosynthesis and its release via an insulin‐pancreatic acinar axis.
Single inwardly rectifying potassium channels in cultured muscle cells from rat and mouse.
Authors: Matsuda H , Stanfield P R
Published: December 2014

1. Inward unitary currents through inwardly rectifying K+ channels of myotubes derived from newborn rats or from a murine, clonal myoblast cell line were studied in the cell‐attached configuration. Open‐closed transitions of the channel were observed in the absence of blocking ions. 2. The single‐channel conductance was 26.3 +/‐ 2.9 pS (mean + S.D., n = 14) with 150 mM‐K+ pipette solution at room temperature (19‐22 degrees C). The channel showed substates of conductance in addition to the main conductance state. A channel with a smaller conductance (8.9 +/‐ 2.6 pS, n = 4) was also but less frequently observed. 3. The probability of the channel being open is weakly voltage dependent: it decreased from 0.94 to 0.84 as the membrane was hyperpolarized from the resting potential (RP) + 20 mV to RP ‐ 50 mV. 4. The lifetimes of the openings were distributed according to a single exponential. At least three exponentials were required to fit the frequency histogram of the lifetimes of all closed states. The mean open time showed a weak voltage dependence, while the mean closed times had little voltage dependence. 5. In the presence of external Na+, the open probability decreased from 0.89 to 0.43 and the mean open time decreased from 203 to 28 ms (40 mM‐K+, 200 mM‐Na+ pipette solution) when the patch membrane was hyperpolarized from RP ‐ 40 mV to RP ‐ 110 mV. The mean closed times were not different from those with 150 mM‐K+, Na+‐free pipette solution and showed little voltage dependence. 6. It is suggested that inactivation of the macroscopic inward currents during hyperpolarization results mainly from a voltage‐dependent block by Na+ with relatively slow kinetics.
Long‐term potentiation and N‐methyl‐D‐aspartate receptors in the visual cortex of young rats.
Authors: Kimura F , Nishigori A , Shirokawa T , Tsumoto T
Published: December 2014

1. Long‐term potentiation (LTP) of synaptic transmission following tetanic stimulation of the white matter was studied by recording extracellular field potentials and intracellular synaptic potentials from layer II/III of visual cortical slices from young rats ranging in age from 21 to 40 days. 2. Single shocks applied to the white matter at 0.1 Hz, used as test stimuli, elicited field potentials that consisted of primary and secondary components. The removal of Ca2+ ions from the perfusate allowed identification of the secondary component as originating postsynaptically and the primary one as reflecting a mixture of antidromic and postsynaptic potentials. 3. Tetanic stimulation at 5 Hz for 60 s was delivered to the white matter and field potentials were observed for 20 min to 9 h after the tetanus. LTP was defined as being present when the response displayed more than a 20% increase in amplitude of the Ca2+‐sensitive components 20 min after the tetanus. LTP was induced in twelve of twenty‐three slices tested, and this potentiation lasted throughout the period of observation. The average magnitude of potentiation was 147.8 +/‐ 28.4% of the control value for the twelve slices. 4. Administration of D,L‐2‐amino‐5‐phosphonovalerate (APV), an antagonist selective for N‐methyl‐D‐aspartate (NMDA)‐preferring receptors, slightly reduced the amplitudes of Ca2+‐sensitive components of the field potentials. The average magnitude of reduction was 80.2 +/‐ 15.3% of the pre‐drug control values. In the presence of APV, LTP was induced in only one slice of twelve tested. 5. Stable intracellular recordings were obtained from twenty‐three cells from layer II/III. Excitatory postsynaptic potentials (EPSPs) evoked by white matter stimulation had mean onset and peak latencies of 4.1 and 11.3 ms, respectively. In some cells these fast EPSPs were followed by another slow EPSP with a much longer latency and higher amplitude. Administration of APV revealed further that the fast EPSPs consisted of two components, i.e. early and late components. 6. Tetanization of the white matter induced long‐lasting enhancement of EPSPs in eight of twelve cells tested. In five of these eight cells, fast EPSPs were enhanced in amplitude and in the remaining three cells, slow EPSPs appeared de novo after the tetanus. 7. APV reduced the amplitudes of the fast EPSPs and abolished the slow EPSPs if present. The average magnitude of reduction for the fast EPSPs was 65.6 +/‐ 15.1% and this reduction was due mainly to an elimination of the late component.(ABSTRACT TRUNCATED AT 400 WORDS)