bluuebunny/crossref_metadata_2025 huggingface dataset:
Unexpected role of the oblique muscles in the human vertical fusional reflex.
Authors: Enright J T
Published: December 2014

1. If a weak vertically oriented prism is inserted before one eye, binocular single vision is restored by vertically divergent eye movements (one eye turning upward, the other downward); and it is usually assumed that the vertical rectus muscles mediate that fusional reflex. 2. When vertically divergent eye movements occur, both eyes also systematically rotate in parallel around their lines of sight (conjugate cyclotorsion). The direction of these unexpected eye movements demonstrates that they must be due to the oblique muscles, not the vertical recti. 3. The magnitude of these conjugate torsional movements is large enough to imply that the oblique muscles, in producing such torsion, would simultaneously effect all the divergent vertical re‐orientation of the eyes required by the targets. 4. The cyclotorsion is accompanied by systematic translation of the eye along a nasal‐temporal axis; the direction and extent of that non‐rotational displacement indicate that the eye movements of the fusional reflex may well be mediated exclusively by the superior oblique muscles, acting against fixed tone in the inferior oblique muscles. 5. This revised understanding of the oculomotor co‐ordination involved in the vertical fusional reflex has significant implications for both neurophysiology and oculomotor surgery.
Unitary A‐currents of rat locus coeruleus neurones grown in cell culture: rectification caused by internal Mg2+ and Na+.
Authors: Forsythe I D , Linsdell P , Stanfield P R
Published: December 2014

1. We have used whole‐cell and single‐channel recording to study the transient outward potassium current (A‐current) of rat locus coeruleus neurones grown in tissue culture. The A‐current was largely inactivated at the resting potential, but could be activated from sufficiently negative holding potentials during steps positive to ‐50 mV. The current was sensitive to 4‐aminopyridine. Another slowly activating, sustained current was similar to a delayed rectifier. 2. In the on‐cell configuration the unitary conductance of channels carrying A‐current was 40.9 +/‐ 2.2 pS (n = 6) with high external potassium (140 mM) and 14.8 +/‐ 1.4 pS (n = 11) with 3 mM [K+]o. The unitary current‐voltage relation was not linear, but had a negative slope at very positive voltages in 3 mM [K+]o. The reversal potential changed with [K]o as expected for a K+ channel. 3. The open state probability of A‐current channels was voltage dependent, reaching a peak of 0.78 +/‐ 0.17 (seven patches). The relationships between both activation and inactivation and membrane potential were well fitted by Boltzmann expressions. Activation was half‐maximum at a potential 71.9 +/‐ 11.8 mV (n = 4) positive to the resting potential (approximately ‐61 mV). Inactivation was half‐complete 29.4 +/‐ 3.8 mV (n = 4) negative to the resting potential. There was evidence from runs analysis for slow inactivation of channels. 4. Channels showed frequent visits to substates, the most readily identifiable of which had an amplitude 0.55 +/‐ 0.04 (n = 5) of the fully open state. Other substates had amplitudes of around 0.25 and 0.75. Occupancy of substates was greater at negative membrane potentials. 5. A preliminary analysis of kinetic behaviour, treating visits to substates as openings, shows that open times are distributed as a single exponential. The open time was 16.2 ms (n = 4) at a voltage 100 mV positive to the resting potential, increasing with further depolarization. Closed times are distributed as the sum of three or four exponentials. First latency distributions are strongly voltage dependent and show a delay, giving a sigmoidal rise to the distribution. Increasing temperature increased unitary current and reduced mean open time. 6. The mechanism of the rectification seen in the unitary current‐voltage relationship was examined using excised, inside‐out patches.(ABSTRACT TRUNCATED AT 400 WORDS)
Compared effects of serotonin on cervical and hypoglossal inspiratory activities: an in vitro study in the newborn rat.
Authors: Morin D , Monteau R , Hilaire G
Published: December 2014

1. Experiments were performed on the brain stem‐spinal cord preparation of newborn rats, in which the phrenic and hypoglossal nerves continue to show rhythmic respiratory activity in vitro, in order to compare the effects of serotonin (5‐HT) on both activities and to analyse the mechanisms responsible for the depression by 5‐HT of the hypoglossal activity. 2. Under control conditions, simultaneous recordings of the inspiratory discharges of hypoglossal and cervical roots showed that the two bursts did not start simultaneously and had different patterns (time‐to‐peak and peak values); this suggests that both pools of motoneurons did not share the same central drive(s). 3. Adding 5‐HT and related agents to the bathing medium delayed and depressed the hypoglossal inspiratory discharge via activation of 5‐HT2 receptors since these effects were elicited by 5‐HT2 agonists (alpha‐methyl‐5‐HT and 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane‐HCl (DOI)) but not by 5‐HT1 agonists (RU 24969 and (+/‐)‐8‐hydroxy‐2‐(di‐N‐propylamino)tetralin hydrobromide (8‐OH‐DPAT)). The 5‐HT depression of the hypoglossal discharge was prevented by applying a pretreatment with a specific 5‐HT2 antagonist (ketanserin). Parallel to the hypoglossal discharge decrease, 5‐HT elicited a permanent cervical root discharge along with a persistent inspiratory bursting. Adding the 5‐HT precursor L‐tryptophan to the bathing medium depressed the hypoglossal (XII) discharge without affecting the cervical one. 4. Local application of 5‐HT within the hypoglossal motor nucleus decreased the hypoglossal output, revealing that the 5‐HT depression of the hypoglossal discharge was at least partly mediated by the 5‐HT effects at the level of the motoneurons. Local application of 5‐HT within the cervical motor nucleus elicited a permanent firing in the cervical root with a persistent inspiratory bursting. 5. Intracellular analysis confirmed the existence of differences in central respiratory drive between cervical and hypoglossal motoneurons under control conditions, as well as differences in response to 5‐HT. All the hypoglossal motoneurons became silent under 5‐HT bathing, and showed no change in the input membrane resistance, a moderate depolarization, and a delayed central respiratory drive with a decreased amplitude. The cervical motoneurons became more active during inspiration, despite a decrease in the amplitude of the central respiratory drive, which was compensated for by a large depolarization and an increased input membrane resistance. Some cervical motoneurons even fired at a low rate during expiration.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect on cardiac sympathetic nerve activity of phenylephrine microinjected into the cat intermediolateral cell column.
Authors: Marks S A , Gilbey M P
Published: December 2014

1. In anaesthetized cats the effect of the alpha 1‐adrenoceptor agonist phenylephrine, microinjected into the left intermediolateral cell column of the spinal cord at the third thoracic level, was studied on left inferior cardiac nerve activity. 2. Microinjection of 100 nl of 10 or 40 mM‐phenylephrine caused increases in inferior cardiac nerve activity in fifteen out of seventeen experiments. 3. The microinjection of the alpha 1‐adrenoceptor antagonist alfuzosin (100 nl of 10 mM) into the intermediolateral cell column antagonized the excitatory response elicited by phenylephrine. 4. Increases in inferior cardiac nerve activity produced by glutamate and 5‐hydroxytryptamine microinjected into the intermediolateral cell column were not antagonized by alfuzosin. 5. It is concluded that activation of alpha 1‐adrenoceptors in the region of the intermediolateral cell column can cause an increase in the firing rate of sympathetic preganglionic neurones which innervate postganglionic neurones projecting into the inferior cardiac nerve.
Effects of procaine and caffeine on calcium release from the sarcoplasmic reticulum in frog skeletal muscle.
Authors: Klein M G , Simon B J , Schneider M F
Published: December 2014

1. Resting myoplasmic free [Ca2+] and [Ca2+] transients (delta [Ca2+]) were measured in single voltage‐clamped frog skeletal muscle fibres in the presence and absence of procaine, caffeine or procaine plus caffeine using Fura‐2 fluorescence and antipyrylazo III (Ap III) absorbance signals. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum (SR) was calculated from the calcium transients and corrected for the relatively small decline due to depletion of calcium from the SR. 2. Procaine (1 mM) reversibly suppressed delta [Ca2+] and the corresponding Rrel by about 40% for 60‐100 ms depolarizing steps to ‐40 to +20 mV. Procaine had little effect on either the waveform or voltage dependence of the Rrel records. 3. [Ca2+] transients calculated from Fura‐2 fluorescence changes in the presence or absence of procaine had similar time courses and amplitudes as those calculated from the Ap III absorbance changes suggesting that 1 mM‐procaine did not interfere with the ability of Ap III or Fura‐2 to monitor delta [Ca2+]. 4. Although 1 mM‐procaine depressed Rrel it had no effect on intramembrane charge movements (IQ) calculated from membrane currents recorded simultaneously with delta [Ca2+]. 5. Procaine (1 mM) reversibly inhibited the potentiating effect of 0.5 mM‐caffeine on delta [Ca2+]. The amplitude and waveform of the Rrel records were similar in control fibres and in the presence of 1 mM‐procaine plus 0.5 mM‐caffeine. 6. In the presence of 0.5 mM‐caffeine delta [Ca2+] after 10‐20 ms voltage steps exhibited an increase in the time to peak and a slower decay time course compared with caffeine‐free controls, suggestive of significant calcium‐induced calcium release in the presence of caffeine. These effects of caffeine were completely and reversibly blocked by 1 mM‐procaine. 7. In the absence of caffeine, 1 mM‐procaine caused a small decrease in time to peak of delta [Ca2+] after 10‐30 ms duration voltage steps compared to the bracketing control and wash runs without procaine. Rrel turned off faster after 10 ms pulses in procaine than in the absence of procaine, but the turn‐off of release was about equally fast with or without procaine after pulses of 20 ms or longer. The effect of procaine after 10 ms pulses in the absence of caffeine may indicate suppression of a component of calcium‐induced calcium release in control that inactivates during the pulse.(ABSTRACT TRUNCATED AT 400 WORDS)
Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle.
Authors: Westerblad H , Allen D G
Published: December 2014

1. The role of the myoplasmic free Mg2+ concentration ([Mg2+]i) in fatigue was studied in intact single fibres isolated from mouse skeletal muscle. Fatigue was produced by repeated tetanic stimulation. The fluorescent Mg2+ indicator furaptra was pressure injected into fibres. In vivo calibrations were performed to convert fluorescence signals into [Mg2+]i. 2. [Mg2+]i at rest was 0.78 +/‐ 0.05 mM (mean +/‐ S.E.M., n = 14). An increase of the extracellular [Mg2+] from 0.5 to 20 mM resulted in a small elevation of [Mg2+]i (86 microM in 5 min). Removal of extracellular Na+ did not affect [Mg2+]i. An intracellular alkanization of about 0.6 pH units gave a [Mg2+]i reduction of 65 microM. 3. During fatiguing stimulation [Mg2+]i initially remained almost constant and it then suddenly started to rise towards the end of the stimulation period. The onset of the [Mg2+]i rise was always followed by a rapid tension decline. In fatigue [Mg2+]i was approximately twice as high as at rest. 4. Fibres were injected with MgCl2 to study if the rise in [Mg2+]i could explain the tension decline in fatigue. An elevation of [Mg2+]i was accompanied by a tension reduction but the [Mg2+]i for a given tension was generally much higher in rested fibres injected with MgCl2 than in fatigued fibres. Thus the rise in [Mg2+]i as such cannot explain the tension reduction in fatigue. 5. Injection of MgCl2 was also used to assess the intracellular Mg2+ buffering. The mean Mg2+ buffer power (i.e. the ratio of the change in [Mg2+]i to the amount of Mg2+ added) was 0.62. 6. ATP is the quantitatively most important binding site for Mg2+ at rest and ATP breakdown is then a likely source of the [Mg2+]i increase in fatigue. The role of ATP breakdown in the increase of [Mg2+]i was studied with metabolic inhibition: fibres were exposed to iodoacetic acid to inhibit glycolysis and cyanide to inhibit oxidative phosphorylation. The pattern during metabolic inhibition was similar to that observed during fatigue. After remaining almost constant during a lengthy period, [Mg2+]i rose rapidly and this rise preceded a period of rapid tension decline. The fibres thereafter went into rigor and [Mg2+]i stabilized at an elevated level; the mean [Mg2+]i increase in rigor was 1.30 mM. 7. We have used modelling to determine the likely change in the intracellular ATP concentration ([ATP]i) for the observed changes in [Mg2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)
An electrophysiological study of responses evoked in isolated segments of rat tail artery during growth and maturation.
Authors: Jobling P , McLachlan E M
Published: December 2014

1. Intracellular recordings from the smooth muscle of isolated segments of the main caudal artery of rats at various ages between 45 and 150 days postnatal were made in order to relate the spontaneous depolarizations and responses to perivascular stimulation at different levels along the artery to the differences in vessel structure and innervation density during growth of the animals. 2. In the outermost smooth muscle cells close to the neuromuscular junctions, spontaneous depolarizations with fast time courses (spontaneous excitatory junction potentials or SEJPs) were recorded. In cells lying deeper in the media, spontaneous depolarizations had a wide range of time courses and amplitudes, but only a few of those could be attributed to electrotonic attenuation of SEJPs. 3. In arterial segments taken from animals of all ages, stimuli which evoked maximal amplitude excitatory junction potentials (EJPs) 1‐2 mm caudal to a suction electrode also evoked neurogenic alpha‐depolarizations (NADs) with time to peak of 15 s and duration nearly 1 min. Both responses decreased progressively in amplitude along the length of the artery. NADs were blocked by phentolamine (10(‐6) M) or idazoxan (10(‐7) M) which were without effects on EJPs. 4. During short trains of stimuli (5 at 1 or 10 Hz), EJPs facilitated but to a lesser extent with distance along the tail. Such trains evoked NADs of greater amplitude than those following a single stimulus; these were often preceded by contractions of the artery which were restricted to the region close to the stimulating electrode. 5. Increasing stimulus voltage led to progressive prolongation of the decay phase of the EJP. After the addition of tetrodotoxin (10(‐7) M), or in the presence of reduced Ca2+ and raised Mg2+ concentration, slow depolarizing potentials (SDPs) (with time to peak of 150‐300 ms and decay lasting > 2 s) were recorded which were graded in amplitude with stimulus voltage. SDPs were attenuated by increasing Ca2+ concentration to 5 mM. These responses often added to the EJP at supramaximal stimulus voltages. 6. The mean amplitudes of the EJP and NAD declined significantly with age, the former to a greater degree than the latter. These changes may be explained by changes in the electrical properties of the media related to hypertrophy of smooth muscle cells as the animals grew, and emphasize the need to allow for such growth effects in studies of young rats.(ABSTRACT TRUNCATED AT 400 WORDS)
Identification of a new transport system (y+L) in human erythrocytes that recognizes lysine and leucine with high affinity.
Authors: Devés R , Chavez P , Boyd C A
Published: December 2014

1. The effect of neutral amino acids on the transport of L‐lysine across the human erythrocyte membrane was studied. 2. All neutral amino acids tested (range 0.3‐5 mM) inhibit the influx of L‐[14C]lysine (1 microM). The inhibition pattern is biphasic, and tends to reach a maximum at approximately 50% of the original flux. The concentrations that give 25% inhibition are (mM): L‐cysteine (2.7), L‐alanine (1.3), L‐serine (0.9), L‐isoleucine (0.6), L‐phenylalanine (0.35), L‐methionine (< 0.3), L‐leucine (< 0.3). L‐lysine and L‐arginine completely inhibit the rate at the highest concentration. 3. These results can be explained by assuming that L‐lysine transport occurs through two independent transporters that differ in their affinity for neutral amino acids. A detailed kinetic analysis of the effect of L‐leucine on L‐lysine entry is consistent with this hypothesis. 4. Using a new experimental strategy, the substrate and inhibitor transport parameters for the two systems were determined. The half‐saturation constants for lysine (+/‐ S.E.M.) are found to be: KmA, 0.014 +/‐ 0.002 mM and KmB, 0.112 +/‐ 0.017 mM. The maximum rates differ by a factor of 8.2 (VmaxB/VmaxA). The leucine inhibition constants are: KiA, 0.022 +/‐ 0.003 mM and KiB, 30.36 +/‐ 7.9 mM. If the sodium in the incubation medium is replaced by potassium, the apparent affinity for leucine (1/KiA) is reduced approximately 30‐fold. 5. The maximum inhibition caused by leucine decreases as the lysine concentration is raised, showing that leucine acts upon the higher affinity system. 6. When added to the trans side, L‐leucine, L‐phenylalanine and L‐isoleucine do not cause inhibition, but stimulate the flux by approximately 30%. This indicates that these analogues are also transported. 7. In conclusion, in the concentration range 1‐100 microM, lysine crosses the red cell membrane through two distinct transport systems, one of which recognizes both neutral and cationic amino acids with high affinity.
Ionic basis of membrane potential changes induced by anoxia in rat dorsal vagal motoneurones.
Authors: Cowan A I , Martin R L
Published: December 2014

1. The effects of anoxia on membrane properties of 119 dorsal vagal motoneurones (DVMs) were investigated in an in vitro slice preparation of the rat medulla. 2. Membrane potential was unaffected by anoxia in 11% of DVMs. An hyperpolarization accompanied by a decrease in input resistance occurred in 44% of DVMs; the remaining 45% depolarized with either an increase (60%) or decrease in input resistance (40%). TTX at a concentration of 0.3‐1 microM did not significantly affect these responses. 3. Anoxic artificial cerebrospinal fluid (ACSF) containing 20 mM‐TEA reversed the response of DVMs that hyperpolarized in standard ACSF to reveal a depolarization of 7.4 +/‐ 2.1 mV, and increased the anoxic depolarization from 5.0 +/‐ 0.7 to 8.7 +/‐ 1.4 mV. 4. Anoxic depolarization was converted to an hyperpolarization of 7.3 +/‐ 2.1 mV in ACSF containing 5 mM‐4‐aminopyridine (4‐AP) and 1 microM‐TTX. A residual depolarization of 4.5 +/‐ 3.5 mV was then observed in ACSF containing 5 mM‐4‐AP, 1 microM‐TTX and 20 mM‐TEA. Anoxic hyperpolarization was increased from 7.8 +/‐ 1.8 to 10.0 +/‐ 3.9 mV in 5 mM‐4‐AP and 1 microM‐TTX and converted to a depolarization of 5.3 +/‐ 4.5 mV in 5 mM‐4‐AP, 1 microM‐TTX and 20 mM‐TEA. 5. In anoxic ACSF containing TEA, the action potential width was increased from 0.92 +/‐ 0.04 to 8.1 +/‐ 1.1 ms in hyperpolarizing DVMs, and from 0.85 +/‐ 0.01 to 2.4 +/‐ 1.0 ms in depolarizing DVMs. The increase in width was prevented by 2‐3 mM‐Mn2+. 6. The long after‐hyperpolarization (AHP) of DVMs, which is contributed to by both an apamin‐sensitive IK(Ca) and an apamin, charybdotoxin and TEA insensitive IK(Ca) was decreased in duration from 2.59 +/‐ 0.14 to 1.94 +/‐ 0.12 s during anoxia. 7. It is concluded that anoxia enhances the delayed rectifier current (IK(DR)) and an inward current, probably ICa, but suppresses the A currents (IA). In DVMs that hyperpolarize during anoxia, the increase in IK(DR) outweighs the increase in ICa and the decrease in IA. In depolarizing DVMs the decrease in IA and increase in ICa outweight the increase in IK(DR). The change in input resistance is determined by the relative sizes of current enhancement or suppression.
Ionic mechanisms of transient inward current in the absence of Na(+)‐Ca2+ exchange in rabbit cardiac Purkinje fibres.
Authors: Han X , Ferrier G R
Published: December 2014

1. Membrane currents were measured with a two‐microelectrode technique in voltage clamped rabbit cardiac Purkinje fibres under conditions known to cause intracellular calcium overload and to eliminate or minimize Na(+)‐Ca2+ exchange. 2. Increasing [Ca2+]o from 2.5 to 5 mM or above and substituting external sodium with either sucrose, choline or Li+ induced an oscillatory transient inward current (TI) which peaked 200‐300 ms after repolarization from a previous depolarizing pulse. The TI quickly disappeared upon return to normal Tyrode solution. Both the rate and configuration of action potentials of Purkinje fibres also returned to control upon return to Tyrode solution after 30 min of high Ca2+ exposure, if the Ca2+ concentration was 30 mM or less. 3. The TI in Na(+)‐free solution was Ca2+ dependent. Either zero or low (2.5 mM) [Ca2+]o, or replacement of [Ca2+]o by BaCl prevented induction of the TI current upon repolarization from a previous depolarizing pulse. 4. In the presence of 30 mM‐CaCl2 and with choline chloride as the substitute for NaCl, TI had a distinct reversal potential (Erev) of ‐25 mV. The time‐to‐peak TI, either inward or outward, did not shift significantly with change in voltage. Both inward and outward TI were simultaneously abolished by exposure to 1 microM‐ryanodine, suggesting they were both activated by transient release of Ca2+ from the sarcoplasmic reticulum. The occurrence of TI in the absence of [Na+]o is not compatible with an electrogenic Na(+)‐Ca2+ exchange mechanism. The existence of a clear‐cut reversal potential suggests that an ionic channel may be responsible for the TI under these conditions. 5. Both the magnitude of peak TI and the Erev were affected by changes of CaCl2 concentration. (i) Under steady‐state conditions, peak inward TI was significantly increased when the [Ca2+]o was elevated from 5 to 15 mM. The peak TI in the outward direction was significantly increased when [Ca2+]o was elevated from 15 to 30 mM; however, the difference in peak inward TI at 15 and 30 mM [Ca2+]o was small. (ii) Clear‐cut reversals of TI were found at Ca2+ concentrations of 10 mM (Erev = ‐19.5 mV) or greater, and elevation of [Ca2+]o to 20, 30, 50 and 105 mM shifted the Erev to more negative potentials. (iii) In the presence of 5 mM [Ca2+]o the inward TI declined to zero at about ‐30 mV, and test voltages between ‐55 and +5 mV failed to reveal a distinct outward TI.(ABSTRACT TRUNCATED AT 400 WORDS)
A novel large‐conductance Ca(2+)‐activated potassium channel and current in nerve terminals of the rat neurohypophysis.
Authors: Wang G , Thorn P , Lemos J R
Published: December 2014

1. Nerve terminals of the rat posterior pituitary were acutely dissociated and identified using a combination of morphological and immunohistochemical techniques. Terminal membrane currents were studied using the ‘whole‐cell’ patch clamp technique and channels were studied using inside‐out and outside‐out patches. 2. In physiological solutions, but with 7 mM 4‐aminopyridine (4‐AP), depolarizing voltage clamp steps from different holding potentials (‐90 or ‐50 mV) elicited a fast, inward current followed by a slow, sustained, outward current. This outward current did not appear to show any steady‐state inactivation. 3. The threshold for activation of the outward current was ‐30 mV and the current‐voltage relation was ‘bell‐shaped’. The amplitude increased with increasingly depolarized potential steps. The outward current reversal potential was measured using tail current analysis and was consistent with that of a potassium current. 4. The sustained potassium current was determined to be dependent on the concentration of intracellular calcium. Extracellular Cd2+ (80 microM), a calcium channel blocker, also reversibly abolished the outward current. 5. The current was delayed in onset and was sustained over the length of a 150 ms‐duration depolarizing pulse. The outward current reached a peak plateau and then decayed slowly. The decay was fitted by a single exponential with a time constant of 9.0 +/‐ 2.2 s. The decay constants did not show a dependence on voltage but rather on intracellular Ca2+. The time course of recovery from this decay was complex with full recovery taking > 190 s. 6. 4‐AP (7 mM), dendrotoxin (100 nM), apamin (40‐80 nM), and charybdotoxin (10‐100 nM) had no effect on the sustained outward current. In contrast Ba2+ (200 microM) and tetraethylammonium inhibited the current, the latter in a dose‐dependent manner (apparent concentration giving 50% of maximal inhibition (IC50) = 0.51 mM). 7. The neurohypophysial terminal outward current recorded here corresponds most closely to a Ca(2+)‐activated K+ current (IK(Ca)) and not to a delayed rectifier or IA‐like current. It also has properties different from that of the Ca(2+)‐dependent outward current described in the magnocellular neuronal cell bodies of the hypothalamus. 8. A large conductance channel is often observed in isolated rat neurohypophysial nerve terminals. The channel had a unit conductance of 231 pS in symmetrical 150 mM K+.(ABSTRACT TRUNCATED AT 400 WORDS)
Characteristic features of noradrenaline‐induced Ca2+ mobilization and tension in arterial smooth muscle of the rabbit.
Authors: Itoh T , Kajikuri J , Kuriyama H
Published: December 2014

1. Effects of noradrenaline (NAd) on changes in cellular Ca2+ concentration ([Ca2+]i) and tension were investigated, and these effects were compared with those evoked by 128 mM K+ or caffeine in intact smooth muscle strips or by inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) or caffeine in beta‐escin‐treated chemically skinned smooth muscle strips of the rabbit mesenteric artery. 2. In physiological solution containing 2.6 mM Ca2+, application of 128 mM K+ or 10 microM NAd produced a phasic, followed by a tonic increase in [Ca2+]i and tension. NAd (10 microM) produced a larger tonic tension than did 128 mM K+ but a smaller increase in [Ca2+]i. When the [Ca2+]i‐tension relationship was observed in ionomycin‐ and 128 mM K(+)‐treated muscle strips, 10 microM NAs shifted the relationship to the left and enhanced the maximum amplitude of contraction. These results suggest that NAd increases the sensitivity of contractile proteins to Ca2+ in smooth muscle of the rabbit mesenteric artery. 3. Noradrenaline (10 microM) or caffeine (10 mM), but not 128 mM K+, produced a phasic increase in both [Ca2+]i and tension in Ca(2+)‐free solution containing 2 mM EGTA. When 10 mM caffeine had been applied in Ca(2+)‐free solution, subsequent application of 10 microM NAd did not increase [Ca2+]i. By contrast, when 10 microM NAd had been applied in Ca(2+)‐free solution, subsequent application of 10 mM caffeine still increased [Ca2+]i. Ryanodine (50 microM) abolished the increase in [Ca2+]i induced by 10 mM caffeine or 10 microM NAd in intact and in skinned smooth muscle strips. These results suggest that NAd releases Ca2+ from the ryanodine‐sensitive Ca2+ storage sites. 4. Noradrenaline (10 microM) synthesized Ins(1,4,5)P3 in Ca(2+)‐free solution in intact smooth muscle strips. Following application of 10 microM NAd, a relatively long time lag (around 1 s) was always observed before the initiation of the increase in [Ca2+]i whether in the presence or absence of Ca2+. The maximum rate of rise of [Ca2+]i induced by 10 mM caffeine was much larger than that induced by 10 microM NAd in Ca(2+)‐containing or Ca(2+)‐free solution (containing 2 mM EGTA). Both [Ca2+]i and tension reached their peak in a shorter time with caffeine (10 mM) than with 10 microM NAd. In Beta‐escin‐treated skinned smooth muscle strips, 20 microM Ins(1,4,5)P3 10 mM caffeine or 10 microM NAd increased Ca2+ in Ca(2+)‐free solution following brief application of 0.3 microM Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium currents at motor nerve endings: absence of effects of adenosine receptor agonists in the frog.
Authors: Silinsky E M , Solsona C S
Published: December 2014

1. The effects of adenosine (50 microM) and 2‐chloroadenosine (1‐25 microM) were studied on Ca2+ currents in frog motor nerve endings. 2. Ca2+ currents associated with the synchronous, neurally evoked release of acetylcholine (ACh) were measured using either perineural or patch recording methods. Tetraethylammonium and/or 3,4‐diaminopyridine were employed to block K+ currents. 3. Ca2+ currents were depressed by omega‐conotoxin (1.5‐2.5 microM), Cd2+ (100 microM‐2 mM), Co2+ (500 microM‐5 mM) or by a reduction of the extracellular calcium concentration. Such currents were also observed when Sr2+ was substituted for Ca2+. Both ACh release and Ca2+ currents at motor nerve endings have been reported to be insensitive to 1,4‐dihydropyridine antagonists in this species. 4. Adenosine receptor agonists did not affect Ca2+ currents at concentrations that produced maximal inhibition of ACh release. 5. The effects of adenosine receptor agonists were examined on asynchronous K(+)‐dependent ACh release under conditions in which the Ca2+ concentration gradient is likely to be reversed (Ca(2+)‐free Ringer solution containing 1 mM EGTA). ACh release was measured by monitoring the frequency of occurrence of miniature endplate potentials (MEPPs). In Ca(2+)‐free solutions containing 1 mM EGTA, high K+ depolarization caused a decrease in MEPP frequency, presumably because it elicits the efflux of Ca2+ from the nerve ending via membrane Ca2+ channels in a reverse Ca2+ gradient. 6. The Ca2+ channel blocker Co2+, which blocks the exit of Ca2+ from the nerve ending, increased the frequency of MEPPs in a concentration‐dependent manner in a reverse Ca2+ gradient. 7. Adenosine or 2‐chloroadenosine inhibited ACh release in a reverse Ca2+ gradient. 8. The results suggest that blockade of Ca2+ entry is not responsible for the inhibitory effects of adenosine at frog motor nerve endings.
Ca2+ currents in single myocytes from human mesenteric arteries: evidence for a physiological role of L‐type channels.
Authors: Smirnov S V , Aaronson P I
Published: December 2014

1. Voltage‐gated Ca2+ currents (ICa) in isolated human mesenteric arterial cells were characterized in solutions containing normal (1.5 mM) Ca2+ and elevated concentrations of divalent cations using the conventional whole‐cell patch clamp technique. 2. In normal Ca2+ solution, depolarization beyond ‐40 mV elicited a slowly decaying ICa which reached a maximum at +10 mV and appeared to reverse between +40 and +50 mV. The amplitude of this current in a group of cells correlated with cell membrane capacitance. 3. In two of thirty‐three cells a small transient component of inward current was detected in the voltage range between ‐40 and ‐10 mV when cells were held at ‐80 mV. This current was abolished at a holding potential of ‐40 mV, while the current at 10 mV was not affected. These currents were referred to as T‐ and L‐type Ca2+ respectively. 4. Elevation of the extracellular Ca2+ concentration to 20 mM shifted the voltage dependencies of Ca2+ current activation and inactivation by approximately +20 mV; a small T‐current component was then observed in seven of nine cells held at ‐60 mV. 5. Replacement of 1.5 mM Ca2+ with 10 mM Ba2+ increased the amplitude of the current elicited at +10 mV by a factor of 3.7 and a small barium current (IBa) through T‐type Ca2+ channels was also observed in most cells studied. Activation and steady‐state inactivation curves for L‐type current were found to be almost identical in both solutions. The steady‐state inactivation for the T‐type IBa was, however, more than 30 mV more negative (half‐inactivation potential of ‐62.6 mV) of that for L‐current in 1.5 mM Ca2+ and 10 mM Ba2+ solutions (‐30.4 and ‐24.9 mV respectively). 6. A sustained inward Ca2+ channel current was recorded in the presence of normal Ca2+ and high divalent cation concentrations during 30 s depolarizations. The amplitude of this sustained current was found to be similar to the theoretical ‘window current’ predicted by the overlap of the activation and inactivation functions in these solutions. 7. Examination of the inactivation of the L‐type current using a two‐pulse protocol with a 240 ms prepulse revealed a U‐shaped potential dependency for ICa, but not for IBa, suggesting the presence of a Ca(2+)‐dependent component of the inactivation process. 8. These cells resemble other arterial smooth muscle cells previously studied in that they demonstrate both T‐ and L‐components of ICa.(ABSTRACT TRUNCATED AT 400 WORDS)
Guanosine 3',5'‐cyclic monophosphate regulates calcium channels in neurones of rabbit vesical pelvic ganglia.
Authors: Nishimura T , Akasu T , Krier J
Published: December 2014

1. The effects of dibutyryl guanosine 3',5'‐cyclic monophosphate (db‐cyclic GMP) were studied in vitro on calcium channels of neurones in rabbit vesical parasympathetic ganglia, using intracellular and single‐electrode voltage‐clamp recordings. 2. Db‐cyclic GMP (100 microM) caused membrane depolarization associated with a decrease in membrane input resistance and an after‐hyperpolarization associated with an increase in membrane input resistance. 3. Db‐cyclic GMP (0.01‐1 mM) caused a concentration‐dependent, transient inward current followed by a long‐lasting outward current. Membrane conductance was increased and decreased during the inward and outward currents, respectively. 4. The db‐cyclic GMP‐induced inward current was depressed in nominally calcium‐free solutions, by cobalt (1 mM) and nicardipine (10 microM). The mean reversal potentials of the inward current were +42 and ‐20 mV in the presence and absence of calcium in the external solution, respectively. 5. The db‐cyclic GMP‐induced inward current was not altered by lowering the external sodium concentration, raising external potassium concentration or by intracellular injection of caesium. 6. A calcium‐insensitive component of the db‐cyclic GMP‐induced current was increased by lowering the external chloride concentration and blocked by 4‐acetamido‐4'‐isothiocyanostilbene‐2,2'‐disulphonic acid, a chloride channel blocker. 7. Voltage‐dependent, high‐threshold calcium currents were depressed during the db‐cyclic GMP‐induced inward current and facilitated during the outward current. 8. Cyclic GMP was less potent than db‐cyclic GMP in causing both inward and outward currents or modulation of calcium currents. GTP, GDP, GMP, guanosine, 8‐bromoadenosine 3',5'‐cyclic monophosphate and forskolin did not alter the holding current or voltage‐dependent calcium currents. 9. It is concluded that intracellular cyclic GMP causes not only activation of resting calcium and chloride channels but also a transient depression followed by long‐lasting facilitation of voltage‐dependent calcium currents in neurones of vesical parasympathetic ganglia.
Inactivation characteristics of a sustained, Ca(2+)‐independent K+ current of rat hippocampal neurones in vitro.
Authors: Nistri A , Cherubini E
Published: December 2014

1. Current or voltage clamp recordings from CA3 neurones of the adult rat hippocampal slice were performed to study the inactivation properties of a slow outward K+ current identified as the delayed rectifier (IK). 2. In current clamp experiments, burst firing evoked from resting membrane potential by intracellular current injection was reduced or blocked by conditioning hyperpolarizing pre‐pulses of 20‐40 mV amplitude. This effect was inhibited by tetraethylammonium (TEA; 20 mM) but was unaffected by Cs+ (3 mM), 4‐aminopyridine (4‐AP; 2 mM), carbachol (30‐50 microM), mast cell degranulating peptide (MCDP; 300 nM), thyrotrophin releasing hormone (TRH; 1 microM) or by a Ca(2+)‐free solution containing Mn2+ or Co2+ (2 mM). 3. Single‐electrode voltage clamp experiments were carried out on neurones superfused with Ca(2+)‐free solution, containing tetrodotoxin (TTX; 1 microM), Mn2+ or Co2+ (2 mM), 4‐AP (2 mM), Cs+ (3 mM) and carbachol (30 microM). Step depolarizations from a holding potential of ‐55 mV activated an outward current which reached a plateau after 200 ms, followed by an outward tail current. Such an outward current had the characteristics of IK. 4. The outward currents were significantly potentiated by conditioning hyperpolarizing pre‐pulses suggesting the IK was reduced by a voltage‐dependent inactivation process. Removal of inactivation was a function of the amplitude of the conditioning hyperpolarizing pre‐pulse. At a holding potential of ‐55 mV removal of inactivation was time dependent with a time constant of 211 ms. High K+ (12.5 or 21.5 mM) solutions did not affect the inactivation characteristics of IK. 5. Tetraethylammonium (20 mM) or low concentrations of Ba2+ (0.1 mM) readily depressed the outward current without significantly affecting the inactivation process. Dendrotoxin (200 nM) also depressed such a slow current but, in addition, increased the inactivation process of IK. 6. It is suggested that removal of inactivation of IK by hyperpolarization can modulate cell excitability by fully restoring the ability of IK to inhibit burst firing of CA3 hippocampal neurones.
Three potassium channels in rat posterior pituitary nerve terminals.
Authors: Bielefeldt K , Rotter J L , Jackson M B
Published: December 2014

1. The patch clamp technique was used to investigate the K+ channels in the membranes of nerve terminals in thin slices prepared from the rat posterior pituitary. 2. Depolarization of the membrane produced a high density of K+ current. With a holding potential of ‐80 mV, test pulses to +50 mV activated a K+ current which was inactivated by 65% within 200 ms. Hyperpolarizing prepulses enhanced the transient K+ current, with half‐maximal enhancement at ‐87 mV. Depolarizing prepulses reduced or eliminated the transient K+ current. 3. In cell‐attached patches formed with pipettes containing 130 mM KCl, three types of K+ channel could be distinguished on the basis of single‐channel properties. One channel had a conductance of 33 pS and was inactivated with a time constant of 18 ms. A second channel had a conductance of 134 pS and was inactivated with a time constant of 71 ms. A third channel had a conductance of 27 pS, was activated relatively slowly with a time constant of 65 ms, and was not inactivated during test pulses of up to one second in duration. 4. Inactivation of the whole‐cell K+ current was a biphasic process with two exponential components. The fast component had a time constant of 22 ms (at +50 mV), corresponding well with the time constant of decay of average current in cell‐attached patches containing only the rapidly inactivating K+ channel. The slow component of inactivation had a time constant of 104 ms (at +50 mV), which was similar to but slightly slower than the time constant of decay of the average current in cell‐attached patches containing only the slowly inactivating K+ channel. Inactivation of the slow transient K+ current became more rapid with increasing depolarization. 5. The low‐conductance rapidly inactivating K+ channel had a lower voltage threshold for activation than the other two K+ channels. 6. Both inactivating K+ channels were enhanced in a similar manner by prior hyperpolarization. There was no difference with regard to voltage mid‐point or steepness. 7. The large‐conductance slowly inactivating K+ channel was activated by Ca2+ at the inner membrane surface. The resting intracellular Ca2+ was sufficiently high to produce significant activation of this channel without depolarization‐induced Ca2+ entry. 8. Removal of Ca2+ from the bathing solution produced a ‐10 mV shift in the voltage dependence of enhancement of both transient K+ currents by prior hyperpolarization. This could be explained as a surface charge effect.(ABSTRACT TRUNCATED AT 400 WORDS)
Prostaglandin modulation of Ca2+ channels in rat sympathetic neurones is mediated by guanine nucleotide binding proteins.
Authors: Ikeda S R
Published: December 2014

1. The effects of prostaglandins on whole‐cell Ca2+ currents of acutely isolated and short‐term cultured adult rat superior cervical ganglion neurones were investigated using the patch‐clamp technique. 2. Prostaglandin E2 (PGE2) produced a rapid, reversible and concentration‐dependent reduction of the sympathetic neurone Ca2+ current. The effects of PGE2 were both voltage and time dependent. The relationship between Ca2+ current inhibition and test potential was ‘bell’ shaped with maximal inhibition occurring near the potential where the Ca2+ current amplitude was maximal (ca + 10 mV). In the presence of PGE2, the Ca2+ current rising phase was slower and biphasic at potentials between 0 and +40 mV. 3. Prolonged (> 2 min) application of 1 microM PGE2 resulted in a desensitization of the response. Similarly, repeated short (ca 1 min) applications of 1 microM PGE2 resulted in a progressive tachyphylaxis of the response. 4. A concentration‐response curve for PGE2 was well described by a single‐site binding isotherm. The concentration producing half‐maximal block (IC50) and the maximal attainable reduction of the Ca2+ current were 7.8 nM and 48%, respectively. 5. When compared at a concentration of 1 microM, PGF2 alpha was less potent (33% inhibition) than PGE2 but otherwise produced similar effects. In contrast, 1 microM PGD2 had negligible effects. 6. Activation curves, as derived from tail current amplitudes, were described by the sum of two Boltzmann functions in both the presence and absence of PGE2. In the presence of PGE2, the activation curve was shifted toward more depolarized potentials. Most of the shift could be accounted for by a decrease in the fractional amplitude of the current component activated at hyperpolarized potentials along with a concomitant increase in the component activated at depolarized potentials. The deactivation time constant (0.33 ms), measured at ‐40 mV, was not altered by PGE2. 7. The majority of the Ca2+ current inhibition produced by PGE2 was relieved by depolarizing conditioning pre‐pulses to +80 mV for 50 ms. 8. Dialysis of sympathetic neurones with a pipette solution containing 2.0 mM guanosine 5'‐O‐(2‐thiodiphosphate) (GDP‐beta‐S) abolished the effects of PGE2 on the Ca2+ current. Pretreatment of the neurones overnight with pertussis toxin significantly, but incompletely, decreased the Ca2+ current inhibition produced by PGE2. 9. The prolonged Ca2+ tail current component induced by the dihydropyridine Ca2+ channel ‘agonist’ (+)202‐791 (2 microM) was unaffected by 1 microM PGE2. 10. PGE2 partially inhibited the Ca2+ current component remaining after pretreatment of the neurones with 10 microM omega‐conotoxin.(ABSTRACT TRUNCATED AT 400 WORDS)
Diazoxide blocks glutamate desensitization and prolongs excitatory postsynaptic currents in rat hippocampal neurons.
Authors: Yamada K A , Rothman S M
Published: December 2014

1. The effects of diazoxide (DZ) on synaptic transmission and upon responses to exogenously applied glutamate agonists were examined in cultured hippocampal neurons. 2. DZ reversibly increased the peak amplitude of evoked excitatory postsynaptic currents (EPSCs) to 150 +/‐ 100% of control and prolonged the EPSC decay time constant (tau) from 5.9 +/‐ 1.2 ms to 14 +/‐ 6.2 ms (240% of control). 3. Peak and steady‐state glutamate (Glu) and quisqualate (QA) currents activated by exogenous application were dramatically increased by DZ at concentrations which did not influence N‐methyl‐D‐aspartate (NMDA), kainate (KA), or GABA currents. These effects were rapidly and completely reversible. Active and passive membrane properties were unaffected by DZ. 4. Inhibitory postsynaptic currents (IPSCs) were unaffected by the same DZ concentrations. 5. These experiments indicate that desensitization plays an important role in terminating excitatory transmission between mammalian central neurons. DZ and perhaps related compounds will ultimately help us identify the regions of the AMPA/KA receptor responsible for desensitization.
Calcium current inactivation in denervated rat skeletal muscle fibres.
Authors: Delbono O , Stefani E
Published: December 2014

1. The inactivation of the calcium current (ICa) was studied in single extensor digitorum longus muscle fibres of the rat. Denervation was performed by surgically removing 6‐8 mm of the sciatic nerve at the sciatic notch. Electrical recordings were carried out using the double Vaseline‐gap technique. Normal fibres were used as controls. 2. The time course of the onset of ICa inactivation was studied with double pulse experiments. Denervation after 14 days slowed down the onset of the inactivation process. Two depolarizing pulses with variable interpulse potential were applied. The rate of recovery from ICa inactivation was analysed with long interpulse intervals (9 to 1 s). The time constants for ICa inactivation (tau i) at ‐90 mV potential were 4.3 and 2.2 in normal and 14 day‐denervated fibres respectively. 3. The onset of ICa inactivation was studied with a double pulse protocol with variable duration of the first pulse with constant interval (120 ms) to the second pulse (300 ms). The plot of [ICa (pulse 2)/ICa(pulse 1)]‐‐first pulse duration relationship was fitted with a single exponential equation. The inactivation time constant (tau h) values for normal and denervated fibres were 428 and 619 ms, respectively. 4. The h infinity‐Vm relationship for denervated fibres was shifted toward more negative potentials and ICa did not fully inactivate with large prepulses. The h infinity‐Vm relationship was fitted with a Boltzmann equation I/Imax = 1 ‐ (A/[1 + (exp ((Vm1/2 ‐ Vm)/kh))]) where Vm is the potential during the conditioning pulse and A is an amplitude factor. In normal fibres, Vm1/2 (mid‐point) and kh (slope) values were ‐28.7 mV and 7.6 mV, respectively. In 14‐day‐denervated fibres they were ‐42.2 and 8.6 mV, respectively. 5. A temperature rise from 17 to 27 degrees C greatly increased the inactivation rate of ICa. This effect was similar in control and denervated fibres. The temperature coefficient quotient (Q10) values for ICa amplitude in normal and denervated fibres were 2.4 (n = 8) and 2.3 (n = 8), respectively. The Q10 values for the inactivation time constant (tau h) were 5.14 and 5.25, respectively. ICa decay during 1 s pulses was fitted to a single exponential function in normal fibres at 17 and 27 degrees C; the time constant values were tau h = 460 +/‐ 53 ms and tau h = 92 +/‐ 17 ms, respectively. The time constants of denervated fibres at both temperatures were tau h = 644 +/‐ 102 ms and 146 +/‐ 17 ms, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
The permeability of the cGMP‐activated channel to organic cations in retinal rods of the tiger salamander.
Authors: Picco C , Menini A
Published: December 2014

1. The permeability of the channel activated by guanosine 3',5'‐cyclic monophosphate (cGMP) to many organic monovalent cations was determined by recording macroscopic currents in excised inside‐out patches of plasma membrane from isolated retinal rod outer segments of the tiger salamander. 2. Current‐voltage relations were measured when the NaCl of the bathing medium was replaced by salts of organic cations. Permeability ratios relative to Na+ ions were calculated with the Goldman‐Hodgkin‐Katz potential equation from the measured changes of reversal potentials. 3. Hydroxylammonium+, hydrazinium+ and methylammonium+, which are molecules of very similar shape and size, permeate the channel with very different permeability ratios: 5.92, 1.99 and 0.60 respectively. 4. Methylated and ethylated ammonium+ compounds were investigated. It was found that, not only methylammonium+, but also dimethylammonium+ and ethylammonium+ were permeant with permeability ratios of 0.6, 0.14 and 0.16 respectively. Trimethylammonium+, tetramethylammonium+, diethylammonium+, triethylammonium+, and tetraethylammonium+ were not permeant. 5. Guanidinium+ and its derivatives formamidinium+, aminoguanidinium+, acetamidinium+ and methylguanidinium+ were all permeant with permeability ratios 1.12, 1.00, 0.63, 0.36 and 0.33 respectively. 6. The cGMP‐activated channel was found to be permeable to at least thirteen organic cations. Molecular models of the permeant cations indicate that the cross‐section of the narrowest part of the pore must be at least as large as a rectangle of 0.38 x 0.5 nm dimensions.