bluuebunny/crossref_metadata_2025 huggingface dataset:
Electrical and morphological factors influencing the depolarizing after‐potential in rat and lizard myelinated axons.
Authors: David G , Modney B , Scappaticci K A , Barrett J N , Barrett E F
Published: December 2014

1. Intra‐axonal recording and electron microscopy were applied to intramuscular myelinated axons in lizards and rats to investigate factors that influence the amplitude and time course of the depolarizing after‐potential. 2. Depolarizing after‐potentials in lizard axons had larger peak amplitudes and longer half‐decay times than those recorded in rat axons (mean values 10 mV, 35 ms in lizard; 3 mV, 11 ms in rat). These differences were not due to differences in temperature, resting potential or action potential amplitude or duration. 3. For a given axon diameter, the myelin sheath in lizard fibres was thinner and had fewer wraps than in rat fibres. There was no significant difference in myelin periodicity. Calculations suggest that the thinner myelin sheath accounts for < 30% of the difference between depolarizing after‐potential amplitudes recorded in lizard and rat axons. 4. Consistent with a passive charging model for the depolarizing after‐potential, the half‐time of the passive voltage transient following intra‐axonal injection of current was shorter in rat than in lizard axons. 5. Aminopyridines prolonged the falling phase of the action potential and increased the amplitude of the depolarizing after‐potential in both types of axon. 6. During repetitive stimulation the depolarizing after‐potentials following successive action potentials exhibited little or no summation. Axonal input conductance in the interspike interval increased during the train. 7. These findings suggest that the amplitude and time course of the depolarizing after‐potential are influenced not only by the passive properties of the axon and myelin sheath, but also by persisting activation of axolemmal K+ channels following action potentials.
Statistics of quantal secretion during long trains of sympathetic nerve impulses in mouse vas deferens.
Authors: Karunanithi S , Phipps M C , Robinson J , Bennett M R
Published: December 2014

1. A statistical analysis has been made of the occurrence of excitatory junctional currents (EJCs) of similar amplitude recorded with an extracellular electrode during long trains of nerve impulses to the mouse vas deferens. 2. The number of EJCs of similar amplitude that occurred in consecutive impulses during trains of 500‐1000 impulses at 0.5‐2.0 Hz increased with the number of EJCs evoked during the train. 3. There was no evidence of significant dependence between consecutive EJCs of similar amplitude in sixteen out of eighteen trains in eighteen preparations. 4. The time course of clusters of EJCs of similar amplitude was examined by determining the standard deviation of different groups of EJCs within a cluster throughout their time course. Most EJCs within a cluster could be grouped with a coefficient of variation < 0.1 throughout their time course. 5. The observations on EJCs of similar amplitude leave open the possibility that secretion from single varicosities is, in general, multiquantal.
Apamin‐sensitive K+ channels mediate an endothelium‐dependent hyperpolarization in rabbit mesenteric arteries.
Authors: Murphy M E , Brayden J E
Published: December 2014

1. Vascular endothelial cells release a variety of substances which affect the membrane potential and tone of underlying vascular smooth muscle. In the presence of N omega‐nitro‐L‐arginine to inhibit nitric oxide synthase and indomethacin to inhibit cyclo‐oxygenase, acetylcholine (ACh; EC50 approximately 1 microM) elicited the release of an endothelium‐derived hyperpolarizing factor (EDHF) in rabbit mesenteric arteries. 2. The hyperpolarization due to EDHF was blocked by apamin (IC50 approximately 0.3 nM), and by other inhibitors of the apamin‐sensitive K+ channel (10 nM scyllatoxin, 100 microM d‐tubocurarine, 300 microM gallamine) in the presence of indomethacin and N omega‐nitro‐L‐arginine. The hyperpolarization was not blocked by glibenclamide (5 microM), iberiotoxin (10 nM), tetraethylammonium (1 mM), barium (500 microM), 4‐aminopyridine (500 microM), ouabain (10 microM), bumetanide (10 microM), or nimodipine (100 nM). 3. In the presence of apamin and N omega‐nitro‐L‐arginine, but the absence of indomethacin, ACh triggered a hyperpolarization that was blocked by glibenclamide, an inhibitor of ATP‐sensitive K+ (KATP) channels. A similar glibenclamide‐sensitive hyperpolarization was caused by Iloprost, a stable analogue of prostacyclin. 4. In experiments which distinguished the effects of EDHF, prostanoids and nitric oxide, hyperpolarizations and/or relaxations triggered by ACh were antagonized by muscarinic antagonists, the relative potencies (atropine approximately 4‐DAMP > pirenzepine) of which indicated that the release of all three endothelium‐derived factors was mediated by M3 receptors. 5. Our results suggest that ACh stimulates M3 receptors on endothelial cells, triggering the release of nitric oxide and prostanoids, which hyperpolarize underlying smooth muscle by activation of KATP channels, and the release of an EDHF, which hyperpolarizes smooth muscle through the activation of apamin‐sensitive K+ (KAS) channels.
Direct influence of the sodium pump on the membrane potential of vomeronasal chemoreceptor neurones in frog.
Authors: Trotier D , Døving K B
Published: December 2014

1. Whole‐cell measurements were made from microvillous receptor neurones isolated from the frog vomeronasal organ. We examined the mechanisms that determined the value of the resting membrane potential. 2. Cells recorded in Ringer solution containing 4 mM K+ showed a resting membrane potential of ‐88 +/‐ 20 mV (mean +/‐ 1 S.D., n = 56). Sixty‐six per cent of the cells had stable resting potentials more negative than the calculated equilibrium potentials for K+ (EK, ‐82 mV) indicating the presence of a hyperpolarizing outward pump current. 3. Cells recorded with an intracellular solution containing Na+ instead of K+, to set EK at 0 mV, presented stable membrane potentials in the range ‐65 to ‐119 mV when bathed in a normal Ringer solution. 4. Ouabain, a specific inhibitor of the Na+,K(+)‐ATPase, blocked the outward sodium pump current (Ip) and depolarized the membrane. 5. The sodium pump current, measured as the current blocked by 0.5 mM dihydro‐ouabain, was linearly related to the membrane potential in the range ‐60 to ‐120 mV. The reversal potential measured with a calculated free energy of ATP hydrolysis of ‐36.2 kJ mol‐1 was estimated to be ‐143 mV. 6. Reduction of the external K+ concentration to 0 mM depolarized the membrane to less than ‐40 mV. Voltage‐clamp observations in this condition indicated a reduction of Ip. Ouabain added to the bath reduced the blocking effect of low external K+. The addition of external K+ activated Ip and induced a rapid hyperpolarization of the cell membrane. 7. At membrane potentials more negative than ‐80 mV, an inward rectifying depolarizing current characterized as Ih was activated. When Ih was blocked by 5 mM external Cs+ the resting membrane potential increased. 8. These data indicate that the membrane potential of the vomeronasal receptor neurones is not generated by a passive diffusion of K+ ions but by the hyperpolarizing current created by the Na+,K(+)‐ATPase. We propose that the resting potential is set by a balance between Ip and Ih. The physiological implications of these mechanisms for setting the resting potential are discussed.
G(o)‐2 protein mediates the reduction in Ca2+ currents by somatostatin in cultured ovine somatotrophs.
Authors: Chen C , Clarke I J
Published: December 2014

1. Somatotroph‐enriched cells (up to 85%) were obtained from ovine pituitary glands by means of collagenase dissociation and Percoll‐gradient centrifugation. Further identification was based on the reduction in Ca2+ currents by 10 nM somatostatin (SRIF). 2. The whole‐cell configuration of the patch‐clamp technique was employed to study the membrane Ca2+ currents with K+ ions replaced by Cs+ and the addition of K+ and Na+ channel blockers in bath and pipette solutions. 3. A significant reduction in Ca2+ currents was obtained in response to local application of SRIF (10 nM) but vehicle application had no effect. 4. Intracellular dialysis of antibodies to alpha(o), alpha(i)‐1‐2, or alpha(i)‐3 subunits of G proteins into the cells via patch‐clamp pipettes was confirmed by immunofluorescent staining of the antibodies. Antibody dialysis did not modify resting voltage‐gated Ca2+ currents across the cell membrane. 5. Dialysis of anti‐alpha(o) antibodies significantly attenuated the reduction in Ca2+ currents that was obtained upon application of 10 or 100 nM SRIF. Dialysis of neither anti‐alpha(i)‐1‐2 nor anti‐alpha(i)‐3 antibodies diminished the effect of SRIF on Ca2+ currents. 6. Intracellular dialysis of antisense oligonucleotides directed against the alpha(o) subunit mRNA (alpha(o) ASm, for alpha(o) common) or against the alpha(i)‐3 subunit mRNA (alpha(i)‐3 AS) blocked expression of alpha(o) or alpha(i)‐3 subunits in the cells, respectively, as assessed by fluorescent staining with anti‐alpha(o) or anti‐alpha(i)‐3 antibodies 48 h after dialysis. 7. Dialysis of alpha(o) ASm, but not alpha(i)‐3 AS, significantly diminished the inhibitory effect of SRIF on Ca2+ currents. This effect of alpha(o) ASm dialysis occurred within 12 h after dialysis and reached a maximum at 48 h; partial recovery was seen at 72 h. 8. Antisense oligonucleotides specific for alpha(o)‐1 (alpha(o)‐1 AS) or alpha(o)‐2 (alpha(o)‐2 AS) were dialysed into somatotrophs and only alpha(o)‐2 AS significantly attenuated the inhibition of Ca2+ currents by SRIF. 9. We conclude that the G(o)‐2 protein mediates the effect of SRIF on Ca2+ currents in ovine somatotrophs in primary culture.
Kinetics of the block by intracellular Mg2+ of the NMDA‐activated channel in cultured rat neurons.
Authors: Li-Smerin Y , Johnson J W
Published: December 2014

1. Single‐channel currents activated by the glutamate agonist N‐methyl‐D‐aspartate (NMDA) were recorded from outside‐out patches of cultured rat cortical neurons in the presence of intracellular Mg2+ (Mgi2+). The rate constants of the block by Mgi2+ were measured using amplitude distribution analysis. 2. At a membrane potential of 0 mV, the blocking rate constant (k+) of Mgi2+ was estimated to be 2.1 x 10(7) M‐1 S‐1 and the unblocking rate constant (k‐) 1.7 x 15(5)s‐1. The very fast rate constants of the block by Mgi2+ explain why channel flicker was not fully resolvable during block of the NMDA‐activated single‐channel current by Mgi2+. 3. The blocking rate constant of Mgi2+ increased with increasing concentrations of Mgi2+. The unblocking rate constant was Mgi2+ concentration independent. 4. The blocking rate constant increased e‐fold per 64 mV depolarization, whereas the unblocking rate constant decreased e‐fold per 133 mV depolarization. The dissociation constant (KD) calculated from the blocking rates (k‐/k+) decreased e‐fold per 43 mV depolarization, and had a value at 0 mV of 7.8 mM. These values are consistent with previous estimates obtained from the voltage‐dependent inhibition of the single‐channel current amplitude. Both results predict, based on the Woodhull model, that Mgi2+ traverses about one‐third of the membrane field to reach its blocking site. 5. The unblocking rate constant of Mgi2+ is one to two orders of magnitude faster than the previously reported unblocking rate constant of extracellular Mg2+ (Mgo2+) in the physiological voltage range, and their voltage dependencies are of opposite signs. These findings are consistent with the hypothesis that there are separate binding sites in the channel for Mgi2+ and Mgo2+. 6. Based on the blocking kinetics of Mgi2+ and Mgo2+, an energy profile of three barriers and two binding sites for Mg2+ is proposed.
Excitatory drive to the alpha‐motoneuron pool during a fatiguing submaximal contraction in man.
Authors: Löscher W N , Cresswell A G , Thorstensson A
Published: December 2014

1. This study was undertaken to examine changes of excitatory drive to the triceps surae alpha‐motoneuron pool during fatiguing submaximal isometric contractions in man. Eight healthy subjects maintained isometric plantar flexions at 30 percent of maximum voluntary contraction (MVC) until the limit of endurance (range, 6‐9 min). 2. Excitability of the alpha‐motoneuron pool to Ia afferent stimulation (H reflex), electromyograms (EMG) and maximum compound motor unit action potentials (Mmax) from the lateral (LG) and medial heads (MG) of the gastrocnemius as well as from the soleus muscle (Sol) were recorded throughout the contraction. Superimposed maximum twitch torques (twitch occlusion) and isometric torque fluctuations (tremor) were also recorded as indirect measures of excitatory drive. 3. H reflexes were studied at different levels of underlying voluntary contraction to assess the relationship between H reflex amplitude and excitatory drive. With increasing levels of underlying contraction up to MVC, superimposed H reflex amplitude increased for LG in six subjects, for MG in all eight and for Sol in five. In the remaining cases, H reflex amplitude first increased and then plateaued between 30‐50% of MVC. 4. H/Mmax ratios increased during fatigue in those muscles that showed an H reflex amplitude increase with high levels of underlying contraction. In these cases, LG and MG H/Mmax increased significantly after about 50 and 20% of endurance time onward, respectively, whereas Sol H/Mmax demonstrated a significant increase up to 40% of endurance time. 5. EMG root mean square (r.m.s.) increased linearly throughout the contraction for all three muscles, while tremor r.m.s. increased in a non‐linear way, with a steeper increase from 60% of endurance time onward. Superimposed twitch amplitude decreased significantly from 25% of endurance time onward. 6. It is concluded that during fatiguing isometric contractions at 30% of MVC, the excitatory drive to the triceps surae alpha‐motoneuron pool increases. This is thought to be a compensatory mechanism to facilitate recruitment of new, unfatigued motor units (MUs), and/or to increase MU firing rates. The facts that the twitch is not abolished at endurance limit and that the EMG does not attain its unfatigued MVC level are strong indications that central fatigue occurred during the sustained submaximal contraction.
Acetylcholine inhibits Ca2+ current by acting exclusively at a site proximal to adenylyl cyclase in frog cardiac myocytes.
Authors: Jurevicius J , Fischmeister R
Published: December 2014

1. The effects of acetylcholine (ACh) on the L‐type Ca2+ current (ICa) stimulated by isoprenaline (Iso) or forskolin (Fsk) were examined in frog ventricular myocytes using the whole‐cell patch‐clamp technique and a double capillary for extracellular microperfusion. 2. The exposure of one half of the cell to 1 microM Iso produced a half‐maximal increase in ICa since a subsequent application of Iso to the other half induced an additional effect of nearly the same amplitude. Similarly, addition of 1 microM ACh to only one half of a cell exposed to Iso on both halves reduced the effect of Iso by only approximately 50%. 3. When 10 microM Iso or 30 microM Fsk were applied to a Ca(2+)‐free solution on one half of the cell, ICa was increased in the remote part of the cell where adenylyl cyclase activity was not stimulated. However, addition of ACh (3‐10 microM) to the remote part had no effect on ICa, while addition of ACh to the part of the cell exposed to Iso or Fsk strongly antagonized the stimulatory effects of these drugs. 4. Our data demonstrate that ACh regulates ICa by acting at a site proximal to adenylyl cyclase in frog ventricular cells. We conclude that the muscarinic regulation of ICa does not involve any additional cAMP‐independent mechanisms occurring downstream from cAMP generation.
Reflex stimulation of aortic chemoreceptors through the stellate ganglion during hypoxia and hypotension in cats.
Authors: Anand A
Published: December 2014

1. In cats anaesthetized with sodium pentobarbitone (35 mg kg‐1), an investigation was made of the mechanisms underlying the enhancement of aortic chemoreceptor activity through the excitatory effect mediated by the sympathetic outflow to them during hypoxia and hypotension. 2. Removal of the right stellate ganglion significantly (P < 0.01) reduced the responses of the chemoreceptors to hypoxia and hypotension. Injection of a bolus of sodium pentobarbitone reduced the responses before, but not after, stellectomy. This indicated that the excitatory sympathetic influence on the chemoreceptors (attributed to glomeral vasoconstriction leading to reduction in blood flow) is exerted entirely through the right stellate ganglion. This excitatory effect was abolished after cutting the vagi and both carotid sinus nerves. 3. It is concluded that, in addition to the direct excitatory effect of hypoxia on aortic chemoreceptors, a reflex chemoreceptor excitatory component is mediated through the vagi, carotid sinus nerves and right stellate ganglion.
Renal noradrenaline spillover correlates with muscle sympathetic activity in humans.
Authors: Wallin B G , Thompson J M , Jennings G L , Esler M D
Published: December 2014

1. To study the relationship between indices of resting sympathetic traffic in nerves to skeletal muscles and the kidneys, simultaneous measurements were made of muscle sympathetic activity in the peroneal nerve and renal noradrenaline spillover in ten healthy normotensive males aged 18‐69 years (mean 42 years). 2. Group mean levels (+/‐S.D.) of muscle sympathetic activity and renal spillover were 22 +/‐ 17 bursts min‐1 and 105 +/‐ 49 ng min‐1, respectively. There were significant positive correlations between individual values of muscle sympathetic activity and renal noradrenaline spillover (r = 0.76, P < 0.01) and similarly between muscle sympathetic activity and renal venous plasma concentration of noradrenaline(r = 0.79, P < 0.007). 3. The results indicate that, although the sympathetic system has the capacity for selective activation of different subdivisions, in healthy human subjects resting traffic is similar or proportional in sympathetic nerves to skeletal muscles and the kidney.
A TTX‐sensitive inward sodium current contributes to spontaneous activity in newborn rabbit sino‐atrial node cells.
Authors: Baruscotti M , DiFrancesco D , Robinson R B
Published: December 2014

1. Single cells were isolated from the sinus node region of rabbits (2 days old to adult) to study the age‐dependent contribution of the sodium current (iNa) to pacemaker activity. 2. Experiments were conducted in 50 mM Na(+)‐Ca(2+)‐free solution. All newborn cells (2‐19 days) exhibited a TTX‐sensitive, Mn(2+)‐insensitive fast inward Na+ current (peak current density 115.5 +/‐ 11.9 pA pF‐1 at 0 mV). Fifty per cent of young cells (20‐40 days) possessed the current, but only one in ten adult cells. Current density decreased with development independently of cell capacitance. 3. Newborn cells exhibited a noticeable window current. With development, the position of the activation curve was shifted in the positive direction, while the inactivation was unaltered, resulting in reduced overlap of the two curves and hence less window current. 4. In newborn cells, 3 microM TTX significantly reduced all measured parameters of spontaneous action potentials, slowing rate by 63%. In contrast, there was no significant effect of TTX on rate or most of the same parameters in adult cells. 5. These results indicate that cells of the sinus node region exhibit a substantial TTX‐sensitive current at birth. With development, both the density and frequency of occurrence of this current within the sinus node decrease, as does its contribution to automaticity.
Noradrenaline modulates transmitter release by enhancing the Ca2+ sensitivity of exocytosis in the chick ciliary presynaptic terminal.
Authors: Yawo H
Published: December 2014

1. The giant presynaptic terminal of chick ciliary ganglion was used to examine how noradrenaline (NA) modulates neurotransmitter release. The cholinergic excitatory postsynaptic currents (EPSCs) were recorded under whole‐cell voltage clamp of the postsynaptic neuron. 2. Although the EPSC was potentiated by NA, the current directly activated by acetylcholine (IACh) was unaffected. NA also increased the quantal contents without changing the quantal size. 3. The NA‐dependent potentiation was antagonized by neither phentolamine nor propranolol. The EPSC was also potentiated by adrenaline and dopamine but not by normetanephrine, phenylephrine or isoprenaline. The EPSC was attenuated by clonidine. Therefore, NA potentiated the transmitter release through a receptor pharmacologically different from both alpha‐ and beta‐adrenergic receptors. 4. The Ca2+ increment produced by an action potential (delta[Ca2+]pre) was reduced by NA through an alpha 2‐adrenergic receptor. However, when alpha 2‐adrenergic receptors were blocked, neither delta[Ca2+]pre nor resting Ca2+ were changed by NA. 5. The [Ca2+]o‐EPSC relation was shifted by NA, decreasing the half‐saturating [Ca2+]o, without changing the maximum. 6. It is concluded that NA‐dependent potentiation of transmitter release was due to an increase in the Ca2+ sensitivity of the exocytotic process. The enhancement of the fusion probability is suggested.
Time‐dependent current decline in cyclic GMP‐gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes.
Authors: Bucossi G , Eismann E , Sesti F , Nizzari M , Seri M , Kaupp U B , Torre V
Published: December 2014

1. Amino acids with a charged or a polar residue in the putative pore region, between lysine 346 and glutamate 372 of the alpha‐subunit of the cGMP‐gated channel from bovine rods were mutated to a different amino acid. The mRNA encoding for the wild‐type, i.e. the alpha‐subunit, or mutant channels was injected in Xenopus laevis oocytes. 2. When glutamate 363 was mutated to asparagine, serine or alanine, the current activated by a steady cGMP concentration declined in mutant channels. No current decline was observed when glutamate 363 was mutated to aspartate, glutamine or glycine, when theronine 359, 360 and 364 were mutated to alanine or when other charged residues in the pore region were neutralized. 3. The amount of current decline and its time course were significantly voltage dependent. In mutant E363A the current decline developed within about 1.5 s at ‐100 mV, but in about 6 s at +100 mV. In the same mutant, the current declined to about 55% of its initial level at +100 mV and to about 10% at ‐100 mV. 4. The current decline in mutants E363A, E363S and E363N was only moderately dependent on the cGMP concentration (from 10 to 1000 microM) and was not caused by a reduced affinity of the mutant channels for cGMP. Analysis of current fluctuations at a single‐channel level indicated that current decline was primarily caused by a decrease of the open probability. 5. The wild‐type channel was not permeable to dimethylammonium. When glutamate 363 was replaced by a smaller residue such as serine, mutant channels became permeable to dimethylammonium. 6. The current decline observed in mutant channels is reminiscent of desensitization of ligand‐gated channels and of inactivation of voltage‐gated channels. These results suggest also that gating and permeation through the cGMP‐gated channel from bovine rods are intrinsically coupled and that glutamate 363 is part of the molecular structure controlling both the gating and the narrowest region of the pore.
GABAB receptor‐mediated responses in GABAergic projection neurones of rat nucleus reticularis thalami in vitro.
Authors: Ulrich D , Huguenard J R
Published: December 2014

1. Whole‐cell voltage‐clamp recordings were obtained from GABAergic neurones of rat nucleus reticularis thalami (NRT) in vitro to assess pre‐ and postsynaptic GABAB receptor‐mediated responses. Presynaptic inhibition of GABA release was studied at terminals on local axon collaterals within NRT as well as on projection fibres in the somatosensory relay nuclei. 2. The GABAB receptor agonist (R)‐baclofen (10 microM) reduced monosynaptically evoked GABAA‐mediated inhibitory postsynaptic currents (IPSCs) in NRT and somatosensory relay cells to 11 and 12% of control, respectively. 3. Action potential‐independent miniature IPSCs (mIPSCs) were observed in both cell types. Mean mIPSC amplitude was 20 pA in both NRT and relay cells at a holding potential of 0 mV. The mean mIPSC frequencies were 0.83 and 2.2 Hz in NRT and relay cells, respectively. Baclofen decreased mIPSP frequency by about half in each cell type without affecting amplitude. 4. Paired‐burst inhibition of evoked IPSCs was studied in relay and NRT cells by applying pairs of 100 Hz stimulus bursts separated by 600 ms. The mean ratio of second to first peak IPSC amplitudes was 0.77. 5. In NRT cells baclofen induced a linear postsynaptic conductance increase of 0.82 nS with an associated reversal potential of ‐121 mV. A small (0.14 nS) GABAB component of the evoked IPSC was detected in only a minority of NRT cells (3 of 18). 6. All pre‐ and postsynaptic effects of baclofen, as well as PBI, were largely reversed by the specific GABAB receptor antagonist CGP 35348 (0.5 mM). 7. We conclude that activation of GABAB receptors in NRT leads to presynaptic autoinhibition of IPSCs in both NRT and relay cells, and to direct activation of a small linear K+ conductance. In addition our experiments suggest that reciprocal connectivity within NRT can be partially mediated by a small GABAB inhibitory event.
Stimulation of muscle ammonia production during exercise following branched‐chain amino acid supplementation in humans.
Authors: MacLean D A , Graham T E , Saltin B
Published: December 2014

1. This study examined the effects of a large (308 mg kg‐1) oral dose of branched‐chain amino acids (BCAAs) on muscle amino acid and ammonia (NH3) metabolism during 90 min of dynamic knee extensor exercise (64 +/‐ 2% of maximum workload). 2. BCAA supplementation resulted in a 4‐fold increase in the arterial BCAA level (from 373 to 1537 microM, P < 0.05) and a 1.5‐fold increase in the intramuscular BCAA level (from 3.4 +/‐ 0.2 to 5.2 +/‐ 0.5 mmol (kg dry weight)‐1, P < 0.05) by the onset of exercise. Over the 90 min exercise period, the exercising muscle removed a total of 7104 +/‐ 2572 mumol kg‐1 of BCAAs. In contrast, in the control trial, there was a total release of 588 +/‐ 86 mumol kg‐1 (P < 0.05) of BCAAs. 3. The total release of NH3 over the 90 min exercise period was 2889 +/‐ 317 mumol kg‐1 (P < 0.05) in the control trial and 4223 +/‐ 552 mumol kg‐1 (P < 0.05) in the BCAA trial. Similarly, the total release of alanine and glutamine was 1557 +/‐ 153 and 2213 +/‐ 270 mumol kg‐1, respectively, for the control trial and 2771 +/‐ 178 and 3476 +/‐ 217 mumol kg‐1, respectively, for the BCAA trial. 4. The lactate release and arterial lactate values were all consistently lower in the BCAA trial than in the control trial. The net production of lactate (intramuscular shifts + total release) was lower (P < 0.05) in the BCAA trial (49.9 +/‐ 11.4 mmol kg‐1) than in the control trial (64.0 +/‐ 11.7 mmol kg‐1). 5. It is concluded that: (1) the administration of BCAAs can greatly increase their concentration in plasma and subsequently their uptake by muscle during exercise, and (2) long‐term exercise following BCAA administration results in significantly greater muscle NH3, alanine and glutamine production, as well as lower lactate production, than is observed during exercise without BCAA supplementation. These data strongly suggest that BCAAs are an important source of NH3 during submaximal exercise and that their contribution to NH3, alanine and glutamine production can be significantly altered by changes in BCAA availability.
Thyroid hormone effects on contractility and myosin composition of soleus muscle and single fibres from young and old rats.
Authors: Li X , Hughes S M , Salviati G , Teresi A , Larsson L
Published: December 2014

1. Young (3‐6 months) and old (20‐24 months) male Wistar rat soleus muscles were examined for myosin isoform composition, fibre type, contractility and sarcoplasmic reticulum (SR) Ca2+ release properties either in control rats or in rats treated with thyroid hormone (3,5,3'‐triiodothyronine, T3) for 4 weeks. 2. T3 treatment had a strong impact on myosin heavy chain (MyHC) and light chain (MyLC) isoform composition in both young and old rats. That is, all single fibres co‐expressed type I and IIA (type I/IIA fibres) or type I, IIA and IIX MyHCs (type I/IIAX fibres) after treatment. Slow and fast MyLC isoforms, i.e. MyLC1s, MyLC1f, MyLC2s, MyLC2f and MyLC3, co‐existed in each of the type I/IIA and I/IIAX fibres in variable proportions. 3. In old rats the maximum velocity of unloaded shortening (V0) was related to MyHC isoform composition: V0 for type I fibres was less than that for type I/IIA fibres which was less than that for type I/IIAX fibres. In young rats, on the other hand, V0 did not differ between pure type I fibres from controls and those co‐expressing type I and type II MyHC isoforms from T3‐treated rats. 4. Contraction and half‐relaxation times of the isometric twitch were significantly longer in old than in young controls. This was paralleled by an age‐related decrease in the caffeine threshold of the SR. Four weeks of T3 treatment eliminated the age‐related differences in both speed of twitch contraction and caffeine thresholds. V0, on the other hand, was slower in old than in young animals, both control and T3‐treated, when cells with a similar MyHC composition were compared. 5. In conclusion, thyroid hormone can substantially reverse at least some of the changes that occur in ageing muscle. Further, the age‐related decline in V0 in soleus fibres from control and hyperthyroid rats suggests that: (1) the identification of beta/slow myosin isoforms is incomplete; or (2) the molecular characteristics of MyHC differ between young and old age; or (3) MyHC is not the only determinant of V0.
Ca2+ influx and activation of a cation current are coupled to intracellular Ca2+ release in peptidergic neurons of Aplysia californica.
Authors: Knox R J , Jonas E A , Kao L S , Smith P J , Connor J A , Kaczmarek L K
Published: December 2014

1. Stimulation of inputs to bag cell neurons in the abdominal ganglion of Aplysia californica causes an increase in their intracellular Ca2+ concentration ([Ca2+]i). We have used thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ pump, to analyse the effects of Ca2+ released from intracellular stores on the electrophysiological responses of bag cell neurons. 2. Using digital imaging of fura‐2‐loaded isolated bag cell neurons we found that thapsigargin rapidly evoked an increase in [Ca2+]i in somata, with smaller increases in neurites. Thapsigargin‐induced elevation of [Ca2+]i peaked at about 1 microM within 5‐10 min and then decayed to basal levels by 30 min. 3. Placement of an extracellular vibrating Ca(2+)‐selective microelectrode to within 1 micron of somata revealed a relatively large steady‐state Ca2+ efflux. Thapsigargin produced a rapid increase in Ca2+ influx. Changes in Ca2+ flux were not detected at neurites. 4. Thapsigargin produced a small depolarization in isolated bag cell neurons in artificial sea water (ASW). Sometimes enhanced depolarizations were observed when extracellular Na+ was replaced by TEA or Tris, but not N‐methyl‐D‐glucamine (NMDG). The depolarization was not blocked by 100 microM tetrodotoxin (TTX), removal of extracellular Ca2+ (0.5 mM EGTA) or addition of 10 mM Co2+ to the bath solution. 5. In voltage‐clamp experiments, thapsigargin induced an inward current (ITg) that was recorded in Ca(2+)‐free media containing TEA or Tris substituted for Na+. The apparent reversal potential of ITg was ‐16.8 +/‐ 1.2 mV in TEA‐ASW. Induction of ITg was inhibited in neurons that were microinjected with the Ca2+ chelator BAPTA‐Dextran70 or treated with the membrane‐permeant analogue BAPTA AM. Activation of ITg was not observed when Na+ was replaced with NMDG. Manipulation of [Na+]o and [K+]o produced shifts in the reversal potential of ITg consistent with the underlying channels being permeable to both Na+ and K+. 6. Thapsigargin did not alter the amplitude or kinetics of voltage‐activated Ba2+ currents, but in some experiments it did increase the amplitude of a component of outward K+ current. 7. Thapsigargin neither induced bag cell neurons within the intact ganglion to depolarize and fire spontaneously, nor did it alter the frequency or duration of firing of an electrically stimulated bag cell after‐discharge. 8. We conclude that thapsigargin‐sensitive Ca2+ pools are present predominantly in the somata of bag cell neurons. Ca2+ that is released from thapsigargin‐sensitive Ca2+ stores activates a non‐selective cation current that may help sustain depolarization of the somata, but does not by itself trigger an after‐discharge.
A model for the kinetics of neutral and anionic dipeptide‐proton cotransport by the apical membrane of rat kidney cortex.
Authors: Temple C S , Bailey P D , Bronk J R , Boyd C A
Published: December 2014

1. Kinetics of influx (mediated through peptide‐proton cotransport) of two labelled dipeptides has been studied in apical membrane vesicles isolated from rat renal cortex. The substrates (neutral D‐Phe‐L‐Ala and anionic D‐Phe‐L‐Glu) have previously been shown to be transported through a single system but with different stoichiometry of proton coupling. 2. The initial rate of influx of both peptides was determined under a set of defined conditions allowing extravesicular pH, intravesicular pH, transmembrane pH and membrane potential (Em) to be varied systemically and independently. From this data the kinetic constants K(m) and Vmax were derived for each condition. Very substantial effects of pH, pH gradient and membrane potential were found; there were consistent quantitative differences when the substrates were compared. 3. Efflux of the two peptides from preloaded vesicles was also determined. At pH 5.5 (intra‐ and extravesicular), but not at pH 7.4, the rate constants for efflux of the two peptides were similar and addition to the extravesicular medium of unlabelled D‐Phe‐L‐Glu (but not D‐Phe‐L‐Ala) trans‐stimulated efflux of both peptides to a similar extent; the extent of this trans‐stimulation was insensitive to alterations in membrane potential. 4. A model based on a combination of classical carrier theory (the carrier being negatively charged) and of two sequential protonation steps (both to external sites predicted to be in the membrane electrical field) is described. Qualitatively this adequately accounts for all the observations made and allows for the dependence of the stoichiometry of proton‐peptide coupling on the net charge carried by the substrate. Quantitatively a 50‐fold greater rate of reorientation of the free carrier when unprotonated is predicted to be responsible for the coupling of proton and peptide transport. 5. Our results and the model are discussed with respect to the recently elucidated primary structure of mammalian peptide transporters.
Sources of Ca2+ for different Ca(2+)‐activated K+ conductances in neurones of the rat superior cervical ganglion.
Authors: Davies P J , Ireland D R , McLachlan E M
Published: December 2014

1. The role of various Ca(2+)‐activated K+ conductances were investigated using intracellular recording and single‐electrode voltage clamp in neurones of superior cervical ganglia isolated in vitro from young adult rats. 2. Following replacement of Ca2+ with Co2+ (2 mM) or the addition of Cd2+ (100 microM), action potential amplitude and half‐width either increased or decreased (in different cells), but both the after‐hyperpolarization (AHP) and the outward tail current following a suprathreshold voltage step were markedly attenuated (by about 75%). 3. Addition of charybdotoxin (60 nM) or nifedipine (10 microM) increased action potential half‐width (by about 25%) but had no significant effect on the AHP or tail current. 4. Addition of apamin (100 nM) or omega‐conotoxin GVIA (100 nM) reduced the AHP and tail current (by about 60%) but did not significantly affect the action potential. A prolonged apamin‐resistant component of the AHP present in 50% of neurones was blocked by ryanodine (20 microM). 5. Omega‐Conotoxin MVIIC (150 nM) and omega agatoxin IVA (200 nM) had no significant effects on the action potential half‐width or the AHP. 6. None of the Ca2+ channel blockers affected the prolonged ryanodine‐sensitive component of the AHP and tail current. 7. We conclude that, in rat sympathetic neurones, Ca2+ entry via L‐type channels selectively activates large conductance Ca(2+)‐activated K+ channels (BK type) contributing to action potential repolarization, whereas Ca2+ entry via N‐type channels selectively activates small conductance Ca(2+)‐activated K+ channels (SK type) contributing to the AHP. Ca2+ entry via R‐type Ca2+ channels prolongs the AHP by activating Ca2+ release from intracellular stores.
Axonal calcium entry during fast ‘sodium’ action potentials in rat cerebellar Purkinje neurones.
Authors: Callewaert G , Eilers J , Konnerth A
Published: December 2014

1. Using laser‐scanning confocal microscopy, fast Ca2+ transients were recorded in individual not yet myelinated axons of Purkinje neurones in cerebellar slices from young rats. Axonal Ca2+ transients could be detected during a single action potential and had progressively larger amplitudes when the number of action potentials was increased. 2. Under voltage‐clamp conditions, axonal Ca2+ transients were as large as those observed in dendrites and in the cell body. Axonal Ca2+ transients were completely blocked by 100 nM of the neurotoxin omega‐agatoxin IVA, indicating that they were caused by Ca2+ entry through P‐type voltage‐gated Ca2+ channels. 3. In conclusion, our results demonstrate action potential‐mediated Ca2+ entry through voltage‐gated Ca2+ channels in axons of cerebellar Purkinje neurones. Experimental evidence indicates that the resulting transient Ca2+ accumulations regulate the frequency of action potentials travelling along the axon.
Glial potassium channels activated by neuronal firing or intracellular cyclic AMP in Helix.
Authors: Gommerat I , Gola M
Published: December 2014

1. Cell‐attached and whole cell patch clamp experiments were performed on satellite glial cells adhering to the cell body of neurones in situ within the nervous system of the snail Helix pomatia. The underlying neurone was under current or voltage‐clamp control. 2. Neuronal firing induced a delayed (20‐30 s) persistent (3‐4 min) increase in the opening probability of glial K+ channels. The channels were also activated by perfusing the ganglion with a depolarizing high‐K+ saline, except when the underlying neurone was prevented from depolarizing under voltage‐clamp conditions. 3. Two K(+)‐selective channels were detected in the glial membrane. The channel responding to neuronal firing was present in 95% of the patches (n = 393). It had a unitary conductance of 56 pS, a Na+ :K+ permeability ratio < 0.02 and displayed slight inward rectification in symmetrical [K+] conditions. It was sensitive to TEA, Ba2+ and Cs+. The following results refer to this channel as studied in the cell‐attached configuration. 4. The glial K+ channel was activated by bath application of the membrane‐permeant cyclic AMP derivatives 8‐bromo‐cAMP and dibutyryl‐cAMP, the adenylyl cyclase activator forskolin and the diesterase inhibitors IBMX, theophylline and caffeine. It was insensitive to cyclic GMP activators and to conditions that might alter the intracellular [Ca2+] (ionomycin, low‐Ca2+ saline and Ca2+ channel blockers). 5. The forskolin‐induced changes in channel behaviour (open and closed time distributions, burst duration, short and long gaps within bursts) could be accounted for by a four‐state model (3 closed states, 1 open state) by simply changing one of the six rate parameters. 6. The present results suggest that the signal sent by an active neurone to satellite glial cells is confined to the glial cells round that neurone. The effect of this signal on the class of glial K+ channels studied can be mimicked by an increase in glial cAMP concentration. The subsequent delayed opening of the glial K+ channels does not appear to play a role in siphoning the excess K+ released by active neurones. It is hypothesized that the cAMP‐gated glial K+ channels may be involved in the control of glial cell proliferation.
Reflex (unloading) and (defensive capitulation) responses in human neck muscle.
Authors: Corna S , Ito Y , von Brevern M , Bronstein A M , Gresty M A
Published: December 2014

1. We studied unloading and stretch responses in human neck muscle during manoeuvres in which the head pulled against a 2‐3 kg weight which could be abruptly released or applied electromagnetically. 2. During head tracking in pitch, unloading of the weight induced inhibition of EMG in the contracting sternocleidomastoid at a mean latency of 24.9 ms in normal subjects and at 41 ms in bilateral labyrinthine‐defective subjects, with antagonist (trapezius) excitation at 30.5 and 41.3 ms, respectively. During tracking in yaw, unloading induced inhibition in the contracting splenius capitis (SpC) at a mean latency of 20.4 ms in normal subjects and 25 ms in labyrinthine‐defective subjects, with excitation in the antagonist SpC at 22.2 and 24 ms, respectively. 3. If subjects tried to resist an unexpected sideways tug on the head a burst occurred in the stretched SpC at a mean latency of 53.5 ms. When subjects relaxed there was excitation of the shortening of SpC at 75.9 ms, which assisted the imposed motion and is possibly a "defensive reflex".
P2X purinoceptors in cultured myenteric neurons of guinea‐pig small intestine.
Authors: Zhou X , Galligan J J
Published: December 2014

1. Fast excitatory postsynaptic currents (fEPSCs) and responses to exogenously applied purinoceptor agonists were studied in primary cultures of myenteric neurons from guinea‐pig small intestine. Whole‐cell and outside‐out configurations of the patch clamp technique were used. Hexamethonium (100 microM) partly inhibited fEPSCs in 28% of neurons. Hexamethonium‐resistant fEPSCs were inhibited by 97 +/‐ 2% by the P2X receptor antagonist, pyridoxal‐phosphate‐6‐azophenyl‐2',4'‐disulphonic acid (PPADS, 10 microM). 2. ATP caused two types of inward currents. In 92% of neurons (n = 123), ATP caused a slowly desensitizing current that declined with a double exponential time course (tau 1 = 7.1 +/‐ 2.0 s; tau 2 = 57 +/‐ 7.4 s, n = 4). The rank order potency for purinoceptor agonists in these neurons was ATP > 2‐methylthio‐ATP (2‐MeSATP) > > alpha, beta‐methylene ATP (alpha, beta‐me ATP) > beta, gamma‐meATP > ADP. The EC50 values for ATP and 2‐MeSATP were 40 and 65 microM, respectively. alpha, beta‐MeATP acted as a partial agonist at these receptors. In 8% of neurons (n = 11), ATP‐induced currents desensitized rapidly with a double exponential time course (tau 1 = 0.13 +/‐ 0.015 s; tau 2 = 2.2 +/‐ 1.3 s, n = 4); alpha, beta‐meATP caused similar responses in these cells. Both types of ATP‐induced current were associated with an increased conductance and an inwardly rectifying I‐V relationship (Erev = 10 mV). Halving [Na+]o shifted the reversal potential of ATP currents by ‐22 +/‐ 6 mV. 3. ATP activated single channel currents in outside‐out patches. The single channel I‐V relationship was linear between ‐120 and 60 mV (Erev approximately 0 mV). Single channel conductance between ‐100 and ‐60 mV was 25 +/‐ 2 pS. Single channel open probability was voltage dependent and decreased from 0.05 +/‐ 0.01 at ‐100 mV to 0.007 +/‐ 0.002 at +40 mV. 4. These data show that P2X purinoceptors mediate some fEPSCs in cultured myenteric neurons. Myenteric neurons express the fast‐desensitizing alpha, beta‐me ATP‐sensitive subtype of P2X receptor that has the properties of cloned P2X1 receptors and is similar to native receptors in smooth muscle cells. Myenteric neurons also express a P2X receptor that desensitized slowly and was alpha, beta‐meATP‐insensitive. This receptor has the properties of cloned P2X2 or P2X5 receptors and is similar to native receptors found in PC‐12 cells and superior cervical ganglion neurons. The known distribution of P2X2 and P2X5 receptors suggests that myenteric neurons are likely to express predominantly P2X2 receptors.
Correlation between electrical activity and the size of rabbit sino‐atrial node cells.
Authors: Honjo H , Boyett M R , Kodama I , Toyama J
Published: December 2014

1. Single cells were isolated from rabbit sino‐atrial (SA) node by enzymatic dissociation. Spontaneous action potentials and membrane currents were recorded using the whole‐cell patch clamp technique to study the relationship between electrical activity and the size of the cells. 2. The size of SA node cells was estimated by measuring the cell capacitance. The cell capacitance of SA node cells ranged from 21.8 to 61.5 pF with a mean +/‐ S.E.M. of 38.2 +/‐ 1.3 pF (n = 61). 3. The action potential amplitude, maximum diastolic potential, take‐off potential and action potential upstroke velocity were greater in larger cells. The rate of diastolic depolarization was greater and the intrinsic spontaneous activity was faster in larger cells. 4. The density of hyperpolarization‐activated current (i(f)) was greater in larger cells, whereas the density of L‐type calcium current was not correlated with the size of SA node cells. 5. TTX‐sensitive sodium current (iNa) was absent in small cells with a capacitance of less than approximately 25 pF, and the density of iNa was greater in larger cells. 6. The greater density of iNa in larger cells may explain the higher upstroke velocity of the action potential in large cells, and the greater density of i(f) and iNa could be responsible for the faster intrinsic spontaneous activity of large cells. These results suggest that the SA node consists of electrophysiologically heterogeneous pacemaker cells with different electrical membrane properties.