bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel.
Authors: Schönherr R , Heinemann S H
Published: December 2014

1. The human eag‐related potassium channel, HERG, gives rise to inwardly rectifying K+ currents when expressed in Xenopus oocytes. 2. The apparent inward rectification is caused by rapid inactivation. In extracellular Cs+ solutions, large outward currents can be recorded having an inactivation time constant at 0 mV of about 50 ms with an e‐fold change every 37 mV. 3. HERG channel inactivation is not caused by an amino‐terminal ball structure, as a deletion of the cytoplasmic amino terminus (HERG delta 2‐373) did not eliminate inactivation. However, channel deactivation was accelerated about 12‐fold at ‐80 mV. 4. Mutation of S631 to A, the homologous residue of eag channels, in the outer mouth of the HERG pore completely abolished channel inactivation. 5. Activity of HERG channels depended on extracellular cations, which are effective for channel activation, in the order Cs+ > K+ > > Li+ > Na+. The point mutation S631A strongly reduced this channel regulation. 6. By analogy to functional aspects of cloned voltage‐gated potassium channels, rectification of HERG, as well as its kinetic properties during the course of an action potential, are presumably governed by a mechanism reminiscent of C‐type inactivation.
Extracellular free potassium and calcium during synchronous activity induced by 4‐aminopyridine in the juvenile rat hippocampus.
Authors: Avoli M , Louvel J , Kurcewicz I , Pumain R , Barbarosie M
Published: December 2014

1. Field potential recordings and measurements of the extracellular concentration of free K+ ([K+]o) and Ca2+ ([Ca2+]o) were made during application of 4‐aminopyridine (4‐AP, 50 microM) in hippocampal slices that were obtained from 11‐ to 32‐day‐old rats. 2. Spontaneous field potentials recorded under this experimental condition in the CA3 stratum radiatum of slices from rats < 23 days old consisted of interictal (duration, 0.2‐1.4 s; intervals of occurrence, 0.9‐3.4 s) and ictal epileptiform discharges (duration, 5‐46 s; intervals of occurrence, 22‐259 s) and negative‐going potentials that often preceded the onset of ictal discharge. Ictal activity became rare in slices from rats > 25 days old. 3. The negative‐going potential (which also corresponded to the ictal discharge onset) was associated with [K+]o increases to 9.4 +/‐ 3.6 mM (mean +/‐ S.D.) from 3.25 mM baseline (n = 11 slices). [K+]o remained elevated at 5‐6 mM throughout the ictal event. Decreases in [Ca2+]o (from 1.8 mM baseline to 1.3 +/‐ 0.1 mM, n = 7) were observed during the ictal discharge. 4. Interictal and ictal discharges were abolished by the non‐N‐methyl‐D‐aspartate (NMDA) receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX, 10 microM). CNQX and the NMDA receptor antagonist 3‐((+/‐)‐2‐carboxypiperazine‐4‐yl)propyl‐1‐phosphonic acid (CPP) did not influence negative‐going potentials or the associated [K+]o increases (peak values were 8.7 +/‐ 3.2 mM, n = 8), that were blocked, however, by bicuculline methiodide (BMI, 10 microM). 5. The mu‐opioid receptor agonist (D‐Ala2,N‐Me‐Phe4,Gly5‐ol)‐enkephalin (DAGO, 10 microM) which inhibits GABA release from interneurons, prevented the occurrence of both GABA‐mediated synchronous potentials and subsequent ictal discharges (n = 6) as well as the [K+]o elevations. DAGO effects were antagonized by naloxone (10 microM; n = 4). 6. The GABA‐mediated [K+]o elevations changed as a function of age. In hippocampal slices obtained from 11‐ to 17‐day‐old rats, peak values of 10.6 +/‐ 2.0 mM (n = 10) and half‐width durations of 8.7 +/‐ 1.3 s (n = 7) were observed. In slices obtained from 25‐ to 32‐day‐old animals these parameters were 5.2 +/‐ 0.5 mM (n = 13) and 4.6 +/‐ 1.1 s (n = 4), respectively. 7. This study shows that, in the juvenile rat hippocampus, 4‐AP induces a glutamatergic independent synchronous potential that is due to GABA released from inhibitory terminals and is associated with an increase in [K+]o. This [K+]o elevation undergoes age‐dependent changes, and is instrumental in synchronizing neurons thus initiating prolonged epileptiform discharges.
An investigation into the mechanism whereby pH affects tension in guinea‐pig ureteric smooth muscle.
Authors: Burdyga T V , Taggart M J , Wray S
Published: December 2014

1. We have altered intracellular (pHi) and extracellular pH (pHo) in the smooth muscle of guinea‐pig ureter and determined the effects on evoked phasic contractions. In order to investigate the mechanisms underlying the effects of pH alteration, intracellular Ca2+ ([Ca2+]i), pHi, electrical activity and force were measured. 2. Intracellular acidification, produced by the weak acid butyrate, application of CO2 at constant pHo or removal of weak bases, greatly increased phasic contractions. Alkalinization with weak bases or by removal of CO2 inhibited contractions. The results were similar whether Hepes or CO2‐HCO3‐buffered the solutions. 3. Phasic contractions were preceded by intracellular Ca2+ transients in the ureter. Acidification of the cytoplasm led to an increase in the amplitude of the Ca2+ transient, and alkalinization decreased its magnitude. 4. In the ureter the action potential leads to Ca2+ influx, therefore electrophysiological recordings of its configuration were made during alteration of pHi. Acidification led to the action potential duration and amplitude being increased, whereas alkalinization shortened the action potential and reduced its amplitude. 5. As the effects of acidification on the action potential resembled the effects of blocking of K+ channels, we investigated whether pHi alteration was able to alter tension when K+ channels were blocked by tetraethylammonium. Acidification was unable to potentiate force under these conditions nor did alkalinization decrease force. 6. External pH over the range 6.8‐8.0 had little or no effect on pHi, phasic contractions and [Ca2+]i. Tonic contractions were enhanced, however, when pHo was increased. 7. These data suggest that pHi alteration in the guinea‐pig ureter modulates the action potential, probably by alteration of K+ currents. Subsequent changes in [Ca2+]i and contraction then occur. A potentiating effect of acidic pH on force is not common in muscle, but may be a characteristic of the smooth muscle of the urinary tract. Changes of pHo had little effect on phasic force or pHi, but modulated tonic contractions. The possible physiological significance of these results is discussed.
Synaptic responsiveness of cortical and thalamic neurones during various phases of slow sleep oscillation in cat.
Authors: Timofeev I , Contreras D , Steriade M
Published: December 2014

1. The fluctuations during various phases of the slow sleep oscillation (< 1 Hz) in synaptic responsiveness of motor cortical (Cx), thalamic reticular (RE) and thalamocortical (TC) neurones were investigated intracellularly in cats under ketamine‐xylazine anaesthesia. Orthodromic responses to stimuli applied to brachium conjunctivum (BC) axons and corticothalamic pathways were studied. The phases of slow oscillation consist of a long‐hyperpolarized, followed by a sharp depth‐negative EEG deflection and a series of faster waves that are associated with the depolarization of Cx and RE neurones, while TC cells display a sequence of IPSPs within the spindle frequency. 2. BC‐evoked bisynaptic excitatory postsynaptic potentials (EPSPs) in Cx and RE neurones were drastically reduced in amplitude during the long‐lasting hyperpolarization and the early part of the depolarizing phase. By contrast, the BC‐evoked monosynaptic EPSPs of TC cells were not diminished during the depth‐positive EEG wave, but the hyperpolarization during this phase of the slow oscillation prevented TC neurones transferring prethalamic signals to the cortex. 3. At variance with the diminished bisynaptic EPSPs evoked in response to BC stimuli during the long‐lasting hyperpolarization, Cx‐evoked monosynaptic EPSPs in Cx cells increased linearly with hyperpolarization during this phase of the slow oscillation. Similarly, the amplitudes of Cx‐evoked EPSPs in RE and TC cells were not diminished during the long‐lasting hyperpolarization. 4. The diminished responsiveness of Cx and RE neurones to prethalamic volleys during the long‐lasting hyperpolarization is attributed to gating processes at the level of TC cells that, because of their hyperpolarization, do not transfer prethalamic information to further relays.
Circulatory response evoked by a 3 s bout of dynamic leg exercise in humans.
Authors: Wieling W , Harms M P , ten Harkel A D , van Lieshout J J , Sprangers R L
Published: December 2014

1. The mechanisms underlying the pronounced transient fall in arterial blood pressure evoked by a 3 s bout of bicycle exercise were investigated in twenty healthy young adults and four patients with hypoadrenergic orthostatic hypotension. 2. In healthy subjects a 3 s bout of upright cycling induced a 28 +/‐ 3 mmHg fall in mean arterial pressure at 12 s. The fall in mean arterial pressure was preceded by a 12 +/‐ 2 mmHg rise in right atrial pressure at 3 s and accompanied by a 54 +/‐ 7% increase in left ventricle stroke volume at 6 s. Systemic vascular resistance dropped 48 +/‐ 2% at 7 s after the start of the manoeuvre to remain at that level for approximately 5 s. The total response lasted about 20 s. During sustained upright cycling the initial fall in mean arterial pressure was also present, but less pronounced (17 +/‐ 2 vs. 26 +/‐ 3 mmHg). A 3 s bout of supine cycling in four patients with hypoadrenergic orthostatic hypotension also elicited a pronounced fall in mean arterial pressure (22 +/‐ 4 mmHg) and in systemic vascular resistance (38 +/‐ 4%). 3. A bout of exercise with a large muscle mass induces two main effects. First, it mechanically increases filling of the heart due to activation of the muscle pump, resulting in an increase in cardiac output. Second, it induces a drop in systemic vascular resistance. The increase in cardiac output is not sufficient to compensate fully for the pronounced fall in systemic vascular resistance and the result is a transient fall in arterial pressure at the onset of whole‐body exercise. The rise in right atrial pressure evoked by 3 s cycling is abrupt and large, but the almost immediate onset and rapid fall of the systemic vascular resistance is too fast for sympathetically mediated reflex effects due to stimulation of the cardiopulmonary afferents. An important factor involved in the drop in systemic vascular resistance appears to be local, non‐autonomically mediated vasodilatation in exercising muscles, since it also occurs in patients with autonomic failure.
Group I extensor afferents evoke disynaptic EPSPs in cat hindlimb extensor motorneurones during fictive locomotion.
Authors: Angel M J , Guertin P , Jiménez T , McCrea D A
Published: December 2014

1. Intracellular recording from extensor motoneurones in paralysed decerebrate cats was used to examine the distribution of short‐latency non‐monosynaptic excitation by group I afferents during fictive locomotion produced by stimulation of the mesencephalic locomotor region (MLR). 2. During the extension but not the flexion phase of fictive locomotion, stimulation of ankle extensor nerves at 1.2‐2.0 times threshold evoked excitatory postsynaptic potentials (EPSPs) in motoneurones innervating hip, knee and ankle extensors. Disynaptic EPSPs were also evoked by selective activation of group Ia muscle spindle afferents by muscle stretch. 3. The central latencies of these group I‐evoked EPSPs (mean, 1.55 ms) suggest their mediation by a disynaptic pathway with a single interneurone interposed between extensor group I afferents and extensor motoneurones. Disynaptic EPSPs were also evoked during periods of spontaneous locomotion following the cessation of MLR stimulation. 4. Hip extensor motoneurones received disynaptic EPSPs during extension following stimulation of both homonymous and ankle extensor nerves. Stimulation of hip extensor nerves did not evoke disynaptic EPSPs in ankle extensor motoneurones. 5. The appearance of disynaptic EPSPs during extension appears to result from cyclic disinhibition of an unidentified population of excitatory spinal interneurones and not postsynaptic voltage‐dependent conductances in motoneurones or phasic presynaptic inhibition of group I afferents during flexion. 6. The reorganization of group I reflexes during fictive locomotion includes the appearance of disynaptic excitation of hip, knee and ankle extensor motoneurones. This excitatory reflex is one of the mechanisms by which group I afferents can enhance extensor activity and increase force production during stance.
Effects of background noise on the response of rat and cat motoneurones to excitatory current transients.
Authors: Poliakov A V , Powers R K , Sawczuk A , Binder M D
Published: December 2014

1. We studied the responses of rat hypoglossal motoneurones to excitatory current transients (ECTs) using a brainstem slice preparation. Steady, repetitive discharge at rates of 12‐25 impulses s‐1 was elicited from the motoneurones by injecting long (40 s) steps of constant current. Poisson trains of the ECTs were superimposed on these steps. The effects of additional synaptic noise was simulated by adding a zero‐mean random process to the stimuli. 2. We measured the effects of the ECTs on motoneurone discharge probability by compiling peristimulus time histograms (PSTHs) between the times of occurrence of the ECTs and the motoneurone spikes. The ECTs produced modulation of motoneurone discharge similar to that produced by excitatory postsynaptic currents. 3. The addition of noise altered the pattern of the motoneurone response to the current transients: both the amplitude and the area of the PSTH peaks decreased as the power of the superimposed noise was increased. Noise tended to reduce the efficacy of the ECTs, particularly when the motoneurones were firing at lower frequencies. Although noise also increased the firing frequency of the motoneurones slightly, the effects of noise on ECT efficacy did not simply result from noise‐induced changes in mean firing rate. 4. A modified version of the experimental protocol was performed in lumbar motoneurones of intact, pentobarbitone‐anaesthetized cats. These recordings yielded results similar to those obtained in rat hypoglossal motoneurones in vitro. 5. Our results suggest that the presence of concurrent synaptic inputs reduces the efficacy of any one input. The implications of this change in efficacy and the possible underlying mechanisms are discussed.
PGE2 modulates the tetrodotoxin‐resistant sodium current in neonatal rat dorsal root ganglion neurones via the cyclic AMP‐protein kinase A cascade.
Authors: England S , Bevan S , Docherty R J
Published: December 2014

1. In current‐clamp recordings, 1 microM prostaglandin E2 (PGE2) increased the excitability of neonatal rat dorsal root ganglion neurones. The current threshold for firing was reduced, and the response to a constant suprathreshold stimulation was modified such that a single evoked action potential was converted to a train of action potentials. The excitatory action of PGE2 was still apparent when action potentials were evoked in the presence of 500 nM tetrodotoxin. 2. In voltage‐clamp experiments 1 microM PGE2 frequently increased the magnitude of the peak currents recorded, and caused a hyperpolarizing shift (of approximately 6 mV) in the activation curve for the tetrodotoxin‐resistant sodium current (TTX‐R INa). In some cells, the hyperpolarizing shift in the activation curve was accompanied by a decrease in peak conductance. PGE2 also caused a hyperpolarizing shift in the steady‐state inactivation curve for the sodium current. 3. Extracellular application of the cAMP analogue dibutyryl cAMP (dbcAMP) at a concentration of 1 mM produced effects on both the current‐voltage relationship and the steady‐state inactivation curve for the TTX‐R INa which were indistinguishable from those observed with PGE2. Prior exposure of the neurones to dbcAMP occluded the effect of a subsequent treatment with PGE2. 4. Forskolin (10 microM), a direct activator of adenylate cyclase, mimicked the effects of PGE2 and dbcAMP on TTX‐R INa. The inactive congener of forskolin, 1, 9‐dideoxyforskolin (10 microM), reduced the amplitude of TTX‐R INa, but did not evoke a hyperpolarizing shift in the activation curve. 5. Intracellular perfusion of the neurones with an inhibitor of protein kinase A inhibited the effect of PGE2 on TTX‐R INa. 6. PGE2 also reduced the amplitude of voltage‐gated potassium currents (IK), which will contribute to the excitatory action. The mechanisms underlying the changes in IK have yet to be elucidated. 7. We propose that the PGE2‐mediated increase in excitability in sensory neurones may be due, at least in part, to the cAMP‐protein kinase A‐dependent modulation of the tetrodotoxin‐resistant sodium channel.
The dipeptide carnosine constricts rabbit saphenous vein as a zinc complex apparently via a serotonergic receptor.
Authors: O'Dowd A , O'Dowd J J , Miller D J
Published: December 2014

1. The endogenous dipeptide carnosine (beta‐alanyl‐L‐histidine), at 0.1‐10 mM, provokes sustained contractures in rabbit saphenous vein rings with greater efficacy than noradrenaline (NA). 2. The effects of carnosine are specific; anserine and homocarnosine are ineffective, as are carnosine's constituent amino acids histidine and beta‐alanine. 3. Maximum carnosine‐induced tension is enhanced by Zn ions (e.g. to 127.5 +/‐ 13.1% of control at 10 microM total Zn concentration, Zntot) and the sensitivity to carnosine potentiated (mean [carnosine] required for half‐maximal tension, K1/2, reduced from 1.23 mM to 17.0 microM carnosine with 15 microM Zntot). 4. The dipeptide apparently acts as a zinc‐carnosine complex. The effects of carnosine at concentrations of 1 microM to 10 mM in the presence of 1‐100 microM Zntot, can be described as a unique function of the concentration of Zn‐carnosine, with an apparent K1/2 for the complex of 7.4 x 10(‐8) M. 5. Contractures are reduced at low [Ca2+], unaffected by adrenoceptor antagonists, but can be blocked by serotonergic receptor antagonists including ketanserin and methiothepin. 6. Competition between albumin and carnosine for Zn ions, as might occur in plasma, can be demonstrated experimentally. 7. The mode of action of carnosine is virtually unique: a vascular muscle receptor apparently transduces the action of a dipeptide in the form of a metal chelate.
Glycosaminoglycan concentration in synovium and other tissues of rabbit knee in relation to synovial hydraulic resistance.
Authors: Price F M , Levick J R , Mason R M
Published: December 2014

1. The hydraulic resistance of the synovial lining of a joint, a key coupling coefficient in synovial fluid turnover, is thought to depend on the concentration of biopolymers (glycosaminoglycans (GAGs) and collagen) in the synovial intercellular spaces, because these polymers create hydraulic drag. The primary aim of this study was to obtain microscopically separated, milligram samples of the very thin synovium from eight rabbit knees, and to analyse these quantitatively for GAGs (chondroitin sulphate, heparan sulphate and hyaluronan) and collagen to allow comparison with published hydraulic resistance data. Synovial fluid and femoral cartilage were also studied. 2. Synovium comprised 73 +/‐ 3% water by weight (mean +/‐ S.E.M.). Of the 270 mg solid per gram of wet tissue, protein formed 136 mg (by automated amino acid analysis), and of this 94 mg was collagen by hydroxyproline analysis. From the collagen mass and fibril volume fraction (0.153 of tissue by morphometry), fibrillar specific volume was calculated to be 1.43 ml per gram of molecular collagen, and fibril water content 47% by volume. 3. The concentration of chondroitin 4‐sulphate (C4S) plus chondroitin 6‐sulphate (C6S), measured by capillary zone electrophoresis was 0.55 mg per gram of synovium‐‐much greater than in synovial fluid (0.04 mg g‐1) and much less than in cartilage (27.8 mg g‐1). The C4S/C6S ratio in synovium (7.3) differed from that in cartilage (0.7), indicating that different proteoglycans predominated in synovium. The heparan sulphate concentration, assayed by radioactive Ruthenium Red binding, was 0.92 mg per gram of synovium (synovial fluid, 0.08 mg g‐1; cartilage, 0.72 mg g‐1). 4. In contrast to sulphated GAGs, the hyaluronan concentration was highest in synovial fluid (3.53 mg g‐1; biotinylated G1 domain binding assay). The concentration in synovial interstitium was only 0.56 mg g‐1 (corrected for interstitial volume fraction, 0.66), even though there is open contact between synovial interstitium and synovial fluid. This may be due to exclusion or washout. 5. Total GAG mass was approximately 4 mg per gram of synovial interstitium. A model of trans‐synovial flow indicated that a uniform GAG concentration of 4 mg g‐1 is less than 1/3rd of that required to explain the experimental estimate of synovial hydraulic resistance. Errors in the resistance estimate do not appear to be large enough to resolve the problem. It is possible, therefore, that additional polymeric material in the interstitium, such as glycoproteins and proteoglycan core protein, may contribute to the hydraulic resistance.
Changes in glycosaminoglycan concentration and synovial permeability at raised intra‐articular pressure in rabbit knees.
Authors: Price F M , Levick J R , Mason R M
Published: December 2014

1. When intra‐articular pressure is raised to pathological values (> 9 cmH2O) by saline, the hydraulic conductance of the synovial lining increases manyfold. The increase at 25 cmH2O is only partially accounted for by stretching of the tissue and has been ascribed to washout and/or dilution of interstitial matrix biopolymers. This suggestion was tested in this study by sampling synovium from control joints (rabbit knees) and from joints perfused with saline to 25 cmH2O, and analysing them quantitatively for collagen, chondroitin sulphate, heparan sulphate and hyaluronan. 2. Pressure and trans‐synovial flow measurements showed that in samples taken at 25 cmH2O the conductance of the synovial lining had increased by a factor of 5.23 +/‐ 1.5 (mean +/‐ S.E.M.) over the conductance at low pressures (just above atmospheric pressure). 3. The tissue concentrations of collagen and the sulphated glycosaminoglycans (GAGs) were reduced by similar amounts after perfusion to 25 cmH2O, namely to 62.8‐70.4% of control. The hyaluronan concentration by contrast was not significantly reduced (106% of control). 4. The reduction in collagen concentration (fixed material) indicated increased interstitial hydration. The closely similar reduction in sulphated GAGs indicated that dilution rather than washout of these components was occurring. The hyaluronan results could be explained by synthesis in vivo at a rate of > or = 91 micrograms h‐1 (ml synovium)‐1 (possibly a non‐basal rate under the conditions of the experiment, i.e. raised pressure and a stretched hydrated membrane). 5. Because interstitial hydraulic drag is related to biopolymer concentration by a power function, the overall matrix dilution observed here was more than sufficient to explain the rise in synovial lining hydraulic conductance at 25 cmH2O when taken in conjunction with stretching of the synovial lining (increased area, reduced thickness).
Tyrosine kinase inhibitors enhance a Ca(2+)‐activated K+ current (IAHP) and reduce IAHP suppression by a metabotropic glutamate receptor agonist in rat dentate granule neurones.
Authors: Abdul-Ghani M A , Valiante T A , Carlen P L , Pennefather P S
Published: December 2014

1. Activation of metabotropic glutamate receptors (mGluRs) inhibits a transient Ca(2+)‐activated K+ current (IAHP) responsible for the slow after‐hyperpolarization that follows depolarizations of dentate granule neurones in rat hippocampal brain slices. Here we show for the first time that this physiological consequence of mGluR stimulation is selectively attenuated by blockers of protein tyrosine kinases (PTKs). 2. Several distinct types of PTK blockers, including genistein, tyrphostin‐B42 and lavendustin‐A, reduced the inhibition of IAHP by the selective mGluR agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACPD). Inhibition of IAHP by 5‐HT was unaffected. The PTK blockers by themselves doubled the duration of IAHP suggesting that there exists a tonic inhibitory influence on IAHP that is reduced by PTK antagonists. 3. Inclusion of EGTA (1 mM) in the patch pipette also potentiated the IAHP and reduced the inhibitory action of ACPD on IAHP, consistent with the observation of others that chelation of intracellular Ca2+ prevents protein tyrosine phosphorylation induced by ACPD. 4. we propose that mGluR‐initiated inositol 1,4,5‐trisphosphate (InsP3) production mobilizes intracellular Ca2+ and leads to increased protein tyrosine phosphorylation which in turn leads to inhibition of IAHP.
Interganglionic segregation of distinct vagal afferent fibre phenotypes in guinea‐pig airways.
Authors: Ricco M M , Kummer W , Biglari B , Myers A C , Undem B J
Published: December 2014

1. The present study addressed the hypothesis that jugular and nodose vagal ganglia contain the somata of functionally and anatomically distinct airway afferent fibres. 2. Anatomical investigations were performed by injecting guinea‐pig airways with the neuronal tracer Fast Blue. The animals were killed 7 days later, and the ganglia were removed and immunostained with antisera against substance P (SP) and neurofilament protein (NF). In the nodose ganglion, NF‐immunoreactive neurones accounted for about 98% of the Fast Blue‐labelled cells while in the jugular ganglion they accounted for approximately 48%. SP and NF immunoreactivity was never (n = 100) observed in the same cell suggesting that the antisera labelled distinct populations. 3. Electrophysiological investigations were performed using an in vitro guinea‐pig tracheal and bronchial preparation with intact afferent vagal pathways, including nodose and jugular ganglia. Action potentials arriving from single airway afferent nerve endings were monitored extracellularly using a glass microelectrode positioned near neuronal cell bodies in either ganglion. 4. The nodose ganglion contained the somata of mainly fast‐conducting tracheal A delta fibres whereas the jugular ganglion contained equal numbers of C fibre and A delta fibre tracheal afferent somata. The nodose A delta neurones adapted rapidly to mechanical stimulation, had relatively low mechanical thresholds, were not activated by capsaicin and adapted rapidly to a hyperosmotic stimulus. By contrast, jugular A delta and C fibres adapted slowly to mechanical stimulation, were often activated by capsaicin, had higher mechanical thresholds and displayed a slow adaptation to a hyperosmotic stimulus. 5. The anatomical, physiological and pharmacological data provide evidence to support the contention that the vagal ganglionic source of the fibre supplying the airways ultimately dictates its neurochemical and physiological phenotype.
Membrane currents and the resting membrane potential in cultured bovine pulmonary artery endothelial cells.
Authors: Voets T , Droogmans G , Nilius B
Published: December 2014

1. We have used the whole‐cell patch‐clamp technique to characterize the ionic conductances that determine the resting membrane potential in cultured endothelial cells from calf pulmonary artery (CPAE cells). 2. Resting membrane potentials were scattered between ‐88 and +5 mV with a mean +/‐ S.E.M. of ‐26 +/‐ 3 mV (n = 104). 3. The most prominent membrane current in resting cells was an inwardly rectifying K+ current. This current showed Na(+)‐dependent inactivation and was efficiently blocked by external Ba2+ (EC50 = 2.2 microM), but was relatively insensitive to quinine, quinidine and TEA. 4. Hypertonic cell shrinkage inhibited an outwardly rectifying Cl‐ current, which was also efficiently blocked by 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB; 100 microM), quinine (500 microM) and quinidine (500 microM). 5. A linear, time‐independent background current remained after elimination of these two currents. This current was dependent on extracellular monovalent cations with a permeability sequence of Cs+ > Na+ > Li+ >> N‐methyl‐D‐glucamine. It was partially blocked by millimolar concentrations of the divalent cations Ca2+, Ni2+ and Ba2+. Gd3+ (200 microM) had no significant effect on this background current. 6. Continuous measurements of the membrane potential confirm that the three described conductances are the major determinants of the membrane potential. Due to the low slope conductance in the region between ‐70 and 0 mV, small changes in one of the current components can evoke large depolarizations or hyperpolarizations, which explains the large scattering of the resting membrane potentials.
A mechanism for altered flexibility in human skeletal muscle.
Authors: Magnusson S P , Simonsen E B , Aagaard P , Sørensen H , Kjaer M
Published: December 2014

1. We investigated the effect of a long‐term stretching regimen on the tissue properties and stretch tolerance of human skeletal muscle. 2. Resistance to stretch was measured as torque (in N m) offered by the hamstring muscle group during passive knee extension while electromyographic (EMG) activity, knee joint angle and velocity were continuously monitored during a standardized stretch manoeuvre. Seven healthy subjects were tested before and after a 3 week training period using two separate protocols. Protocol 1 consisted of a slow stretch at 0.087 rad s‐1 to a predetermined angle followed by a 90 s holding phase. Subjects were brought to the same angle before and after the training period. Protocol 2 was a similar stretch, but continued to the point of pain. 3. During protocol 1 the torque rose during the stretch and then declined during the holding phase. EMG activity was small and did not change significantly during the protocol. No significant differences in stiffness, energy and peak torque about the knee joint were seen as a result of the training. During protocol 2 the angle to which the knee could be extended was significantly increased as a result of the training. This was accompanied by a comparable increase in peak torque and energy. EMG activity was small and not affected by training. 4. It is concluded that reflex EMG activity does not limit the range of movement during slow stretches and that the increased range of motion achieved from training is a consequence of increased stretch tolerance on the part of the subject rather than a change in the mechanical or viscoelastic properties of the muscle.
The equine periodic paralysis Na+ channel mutation alters molecular transitions between the open and inactivated states.
Authors: Hanna W J , Tsushima R G , Sah R , McCutcheon L J , Marban E , Backx P H
Published: December 2014

1. The Na+ channel mutation associated with equine hyperkalaemic periodic paralysis (HPP) affects a highly conserved phenylalanine residue in an unexplored region of the alpha‐subunit. This mutation was introduced into the rat skeletal muscle Na+ channel gene at the corresponding location (i.e. F1412L) for functional expression and characterization in Xenopus oocytes. 2. In comparison with wild‐type (WT) channels, equine HPP channels showed clear evidence for disruption of inactivation: increased time‐to‐peak current, slowed rates of whole‐cell current decay, significant increases in sustained current, rightward shifts in the steady‐state inactivation curve by 9.5 mV, a 6‐fold acceleration in the rate of recovery from inactivation at ‐80 mV, decreased number of blank single‐channel sweeps, repetitive opening of single channels throughout depolarizing steps, increased open probability per sweep, and an increased mean open time. 3. The observed disruption of inactivation in HPP occurred without measurable changes in steady‐state activation and first latency kinetics of channel opening. 4. Kinetic modelling demonstrates that the equine HPP phenotype can be simulated by altering the rate constants for transitions entering and leaving the inactivated states resulting from an energetic destabilization of the inactivated state. 5. These results suggest that the highly conserved cytoplasmic end of the third transmembrane segment (S3) in the fourth internal repeat domain (domain IV) plays a critical role in Na+ channel inactivation.
Ionic permeability of, and divalent cation effects on, two ATP‐gated cation channels (P2X receptors) expressed in mammalian cells.
Authors: Evans R J , Lewis C , Virginio C , Lundstrom K , Buell G , Surprenant A , North R A
Published: December 2014

1. Complementary DNAs for the ATP‐gated ion channel subunits P2X1 (from human bladder) and P2X2 (from rat phaeochromocytoma (PC12) cells) were used to express the receptors in human embryonic kidney cells by stable transfection, and in Chinese hamster ovary cells by viral infection. 2. Membrane currents evoked by ATP were recorded by the whole‐cell patch clamp method. The reversal potential of the current was measured with various intracellular and extracellular solutions and used to compute the relative permeability of the P2X receptor channels. 3. There was no difference between the two receptors with respect to their permeability to monovalent organic cations. The relative permeabilities (PX/PNa) were 2.3, 1.0, 1.0, 0.95, 0.72, 0.5, 0.29, 0.16, 0.04 and 0.03 for guanidinium, potassium, sodium, methylamine, caesium, dimethylamine, 2‐methylethanolamine, tris(hydroxymethyl)‐aminomethane, tetraethylammonium and N‐methyl‐D‐glucamine, respectively (values for P2X2 receptor). 4. The calcium permeability of P2X1 receptors was greater than that of P2X2 receptors. Under biionic conditions (112 mM calcium outside, 154 mM sodium inside), PCa/PNa values were 3.9 and 2.2, respectively (corrected for ionic activities). 5. ATP‐evoked currents in cells expressing the P2X2 receptor were strongly inhibited when the extracellular calcium concentration was increased (0.3‐30 mM); the action of ATP could be restored by increasing the ATP concentration. ATP‐evoked currents in cells expressing the P2X1 receptor were not inhibited by such increases in the extracellular calcium concentration.
Concentration and pH dependence of skeletal muscle chloride channel ClC‐1.
Authors: Rychkov G Y , Pusch M , Astill D S , Roberts M L , Jentsch T J , Bretag A H
Published: December 2014

1. The influence of Cl‐ concentration and pH on gating of the skeletal muscle Cl‐ channel, ClC‐1, has been assessed using the voltage‐clamp technique and the Sf‐9 insect cell and Xenopus oocyte expression systems. 2. Hyperpolarization induces deactivating inward currents comprising a steady‐state component and two exponentially decaying components, of which the faster is weakly voltage dependent and the slower strongly voltage dependent. 3. Open probability (Po) and kinetics depend on external but not internal Cl‐ concentration. 4. A point mutation, K585E, in human ClC‐1, equivalent to a previously described mutation in the Torpedo electroplaque chloride channel, ClC‐0, alters the I‐V relationship and kinetics, but retains external Cl‐ dependence. 5. When external pH is reduced, the deactivating inward currents of ClC‐1 are diminished without change in time constants while the steady‐state component is enhanced. 6. In contrast, reduced internal pH slows deactivating current kinetics as its most immediately obvious action and the Po curve is shifted in the hyperpolarizing direction. Addition of internal benzoate at low internal pH counteracts both these effects. 7. A current activated by hyperpolarization can be revealed at an external pH of 5.5 in ClC‐1, which in some ways resembles currents due to the slow gates of ClC‐0. 8. Gating appears to be controlled by a Cl(‐)‐binding site accessible only from the exterior and, possibly, by modification of this site by external protonation. Intracellular hydroxyl ions strongly affect gating either allosterically or by direct binding and blocking of the pore, an action mimicked by intracellular benzoate.
Sensitivity of vagal mucosal afferents to cholecystokinin and its role in afferent signal transduction in the rat.
Authors: Richards W , Hillsley K , Eastwood C , Grundy D
Published: December 2014

1. Extracellular recordings from rat mesenteric paravascular nerve bundles were made in order to characterize the responses of different populations of afferents supplying the small intestine to intravenous cholecystokinin (CCK; in the form of sulphated CCK8). 2. Approximately 70% of mesenteric nerve bundles contained CCK‐sensitive afferent fibres. Responsive afferents had low spontaneous discharge (1.6 +/‐ 0.3 impulses s‐1) and showed a 14‐fold increase in firing at the peak of the response to 50 pmol CCK with the overall response lasting several minutes. The onset of the response occurred after a latency of (3.9 +/‐ 0.1 s) following i.v. administration of CCK, which corresponds largely to the circulation delay in these animals. The threshold dose of CCK was < 5 pmol. 3. The response to 100 pmol CCK was completely abolished by devazepide (0.5 mg kg‐1) and by chronic subdiaphragmatic vagotomy performed 10‐14 days prior to experimentation, indicating that CCK sensitivity was via CCKA receptors and exclusively mediated via vagal afferents rather than splanchnic or enteric afferents. 4. Evidence that CCK‐sensitive afferents had mucosal receptive fields was indicated by the lack of any response to luminal distension and the sensitivity of the CCK response to luminal anaesthesia. Furthermore, CCK‐sensitive afferents responded to luminal hydrochloric acid (50 mM) in a slowly adapting manner. The response to acid was significantly reduced (P < 0.005), but not abolished, by devazepide at a time when the response to exogenous CCK had been completely eliminated. 5. The exquisite sensitivity of some vagal mucosal afferents to CCK suggests that they may play a physiological role in the reflex and behavioural consequences of CCK release from the small intestine, possibly acting in a paracrine fashion. However, this sensitivity to CCK represents only one aspect of the broad chemosensitivity of these mucosal afferents and is not an obligatory component of the signal transduction pathway.
Direct activation of GABAA receptors by barbiturates in cultured rat hippocampal neurons.
Authors: Rho J M , Donevan S D , Rogawski M A
Published: December 2014

1. The direct activation of the GABAA receptor by pentobarbitone (PB) and phenobarbitone (PHB) was characterized in cultured rat hippocampal neurons using whole‐cell voltage clamp and single channel recording techniques. 2. In whole‐cell recordings, PB and PHB produced a concentration‐dependent activation of Cl‐ current (EC50 values, 0.33 and 3.0 mM, respectively). The response to the barbiturates was similar to that produced by GABA, although GABA was more potent (EC50, 5.5 microM). PB and PHB were substantially more potent in enhancing the response to 1 microM GABA (EC50 values, 94 microM and 0.89 mM, respectively). The maximal magnitude of the responses to PB was similar to that of the maximal response to GABA or GABA + PB. PHB appeared to be modestly less efficacious. 3. The mean deactivation time constant for whole‐cell Cl‐ currents evoked by 1 mM PB + 1 microM GABA was significantly longer (480 +/‐ 34 ms) than for 1 mM PB (170 +/‐ 9 ms) or 1 microM GABA (180 +/‐ 14 ms) alone. 4. Whole‐cell currents directly activated by 300 microM PB and 1 microM GABA were blocked by the GABA receptor antagonists bicuculline and picrotoxin. 5. Unitary GABAA receptor channel currents evoked by 300 microM PB had similar main conductance, mean open time and mean burst duration as those activated by 2 microM GABA alone. Single channel openings and bursts were of shorter mean duration when 100 and 300 microM PHB were used. 6. High concentrations of PB (1‐3 mM) and PHB (3‐10 mM) produced a rapid block of currents activated by the barbiturate alone or by the barbiturate in the presence of 1 microM GABA. The estimated IC50 values for block of PB‐ and PHB‐potentiated GABA currents were 2.8 and 12.9 mM, respectively. 7. Single channel currents activated by high concentrations of PB and PHB alone or in the presence of GABA demonstrated flickering, probably reflecting fast channel block. 8. We conclude that the gating of the GABAA receptor channel by PHB and PB is functionally similar to that produced by the natural agonist GABA alone, but distinct from that obtained when barbiturates modulate the response to GABA. At high concentrations, the barbiturates produce a channel blocking action that limits the maximum total current conducted by the channel.
Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors.
Authors: Brickley S G , Cull-Candy S G , Farrant M
Published: December 2014

1. To investigate the origin and functional significance of a recently described tonic GABAA receptor‐mediated conductance in cerebellar granule cells we have made recordings from cells in cerebellar slices from rats of different ages (postnatal days P4 to P28). 2. During development there was a dramatic change in the properties of GABA‐mediated synaptic transmission. The contribution to GABAA receptor‐mediated charge transfer from the tonic conductance (GGABA), relative to that resulting from discrete spontaneous postsynaptic currents (sPSCs), was increased from 5% at P7 to 99% at P21. GGABA was reduced by bicuculline, tetrodotoxin and by lowering extracellular Ca2+, and was initially present only in those cells which exhibited sPSCs. 3. At P7 sPSCs were depolarizing, occasionally triggering a single action potential. By P18 the GABA reversal potential was shifted close to the resting potential and GGABA produced a shunting inhibition. Removal of GGABA by bicuculline increased granule cell excitability in response to current injection. 4. This novel tonic inhibition is present despite the low number of Golgi cell synapses on individual granule cells and appears to result from ‘overspill’ of synaptically released GABA leading to activation of synaptic and extrasynaptic GABAA receptors.
Presynaptic inhibitory action of opioids on synaptic transmission in the rat periaqueductal grey in vitro.
Authors: Vaughan C W , Christie M J
Published: December 2014

1. The actions of opioids on synaptic transmission in rat periaqueductal grey (PAG) neurones were examined using whole‐cell patch‐clamp recordings in brain slices. 2. Methionine enkephalin (ME; 10 microM) inhibited evoked GABAergic inhibitory postsynaptic currents (IPSCs) by 57%, non‐NMDA excitatory postsynaptic currents (EPSCs) by 60%, and NMDA EPSCs by 43% in PAG neurones. This inhibition was associated with an increase in paired‐pulse facilitation, was mimicked by the mu‐agonist DAMGO (1‐3 microM) and abolished by naloxone (1 microM). Neither the kappa‐agonist U69593 (1‐3 microM), nor the delta‐agonist DPDPE (3‐10 microM) had any specific actions on evoked PSCs. 3. ME decreased the frequency of spontaneous miniature, action potential‐independent postsynaptic currents (mIPSCs by 65%, mEPSCs by 54%) in all PAG neurones, but had no effect on their amplitude distributions. The reduction in mIPSC frequency persisted in nominally Ca(2+)‐free, high‐Mg2+ (10 mM) solutions, which also contained Cd2+ (100 microM), or Ba2+ (10 mM). Opioid inhibition of mIPSC frequency is unlikely to be mediated by presynaptic Ca2+ or K+ conductances which are sensitive to extracellular Cd2+ or Ba2+. 4. In a subpopulation of PAG neurones, ME increased a Ba(2+)‐sensitive K+ conductance at potentials below ‐97 mV. Opioids inhibited both GABAergic and glutamatergic synaptic transmission in all PAG neurones, independent of any postsynaptic opioid sensitivity. 5. These observations are consistent with, but only partially support, the opioid disinhibition model of PAG‐induced analgesia. mu‐Opioids also have the potential to modulate the behavioural and autonomic functions of the PAG via modulation of both inhibitory and excitatory presynaptic mechanisms, as well as postsynaptic mechanisms.
Microfibrils provide non‐linear elastic behaviour in the abdominal artery of the lobster Homarus americanus.
Authors: McConnell C J , DeMont M E , Wright G M
Published: December 2014

1. Microfibrils are becoming increasingly recognized as an important component of the extra‐cellular matrix. However, almost nothing is known about their mechanical role in the diversity of tissues in which they are found. 2. Microfibrils form the principal structural component in the wall of the abdominal artery of the lobster Homarus americanus. We have used previous estimates of the mechanical properties of these microfibrils, estimates of the fraction of the aorta wall volume occupied by the microfibrils, and their angular distribution as a function of strain in a numerical model that predicts the macroscopic mechanical properties of the whole tissue. 3. Microfibrils alone, when their reorientation and deformation are accounted for, characterize the stress‐strain behaviour of the vessel. Evidence of the evolutionary conservation of fibrillin between medusans, echinoderms and vertebrates implies that the mechanical properties of lobster microfibrils may apply to microfibrillar function in other taxa. This will have profound implications on the perceived roles of microfibrils in development, physiology and disease.
Exercise‐induced increase in serum interleukin‐6 in humans is related to muscle damage.
Authors: Bruunsgaard H , Galbo H , Halkjaer-Kristensen J , Johansen T L , MacLean D A , Pedersen B K
Published: December 2014

1. This study was performed to test the hypothesis that the exercise‐induced increase in circulating cytokine levels is associated with muscle damage. Nine healthy young male subjects performed two high‐intensity bicycle exercise trials separated by two weeks. The first trial consisted of 30 min of normal bicycle exercise (concentric exercise), whereas the second consisted of 30 min of braking with reversed revolution (eccentric exercise). The work loads were chosen to give the same increases in heart rate and catecholamine levels in the blood during each trial. 2. Significant increases (P < 0.05) in plasma concentration of creatine kinase (CK), aspartate aminotransferase and alanine aminotransferase were observed only after the eccentric exercise. Furthermore, the level of interleukin‐6 (IL‐6) in serum increased significantly after the eccentric exercise and was significantly correlated to CK concentration in the following days, whereas no significant changes were found after the concentric exercise. 3. The total concentration of lymphocytes increased significantly (P < 0.05) as a result of eccentric compared with concentric exercise. This was mainly due to a significantly more pronounced recruitment of natural killer (NK) cells and CD8 positive cells (CD8+ cells) during the eccentric trial. However, no significant differences between the two types of work were found in regard to the circulating concentration of monocytes. The concentration of neutrophils was only significantly increased 2 h after the concentric exercise. 4. The finding that high‐intensity eccentric exercise caused a more pronounced increase in the plasma level of IL‐6, compared with concentric exercise, supports the hypothesis that the post‐exercise cytokine production is related to skeletal muscle damage. The fact that no differences between eccentric and concentric exercise were found in the recruitment of most blood mononuclear cell subsets to the blood supports the hypothesis that the exercise‐induced increase in plasma catecholamines is a major determinant of the mobilization of these cells into the blood. However, as eccentric exercise caused a more pronounced increase in the concentration of NK cells and CD8+ cells, factors involved in muscle damage may also contribute to the recruitment of these cells.