bluuebunny/crossref_metadata_2025 huggingface dataset:
A‐type potassium current modulated by A1 adenosine receptor in frog melanotrophs.
Authors: Mei Y A , Louiset E , Vaudry H , Cazin L
Published: December 2014

1. Transient outward current was recorded in cultured frog melanotrophs with the whole‐cell configuration of the patch‐clamp technique. The ionic dependence, kinetics and pharmacological properties of the current were studied. The effects of the A1 adenosine receptor agonist R‐N6‐phenylisopropyl‐adenosine (R‐PIA) on this current were also investigated. 2. In tetrodotoxin‐ and cobalt‐containing solution, depolarization from ‐120 mV elicited both transient and delayed outward currents. Pulses from ‐60 mV activated only a sustained late current. 3. 4‐Aminopyridine (4 mM) reduced the transient outward current much more than the delayed outward current. In contrast, tetraethylammonium (10‐20 mM) selectively reduced the delayed current. 4. Tail current measurements showed a positive shift in the reversal potential when external K+ concentration was increased, indicating that K+ was the predominant charge carrier. 5. Steady‐state inactivation was complete at potentials positive to ‐10 mV and removed by hyperpolarization. 6. Inactivation of the transient current was slowed and accelerated in oxidizing and reducing conditions, respectively, confirming the involvement of an inactivating ‘ball and chain’ peptide. 7. R‐PIA increased the transient current. The steady‐state inactivation curve was shifted towards more positive potentials without changing the activation kinetics. Pretreatment with pertussis toxin (1 microgram ml‐1) blocked the response to R‐PIA. 8. It is concluded that frog melanotrophs possess an A‐type current that is likely to play an important role in excitability. This current, which is directly modulated by A1 adenosine receptors through a Gi/G(o) protein, appears to be responsible for the inhibitory effects of adenosine on electrical activity.
A novel inward‐rectifying K+ current with a cell‐cycle dependence governs the resting potential of mammalian neuroblastoma cells.
Authors: Arcangeli A , Bianchi L , Becchetti A , Faravelli L , Coronnello M , Mini E , Olivotto M , Wanke E
Published: December 2014

1. Human and murine neuroblastoma cell lines were used to investigate, by the whole‐cell patch‐clamp technique, the properties of a novel inward‐rectifying K+ current (IIR) in the adjustment of cell resting potential (Vrest), which was in the range ‐40 to ‐20 mV. 2. When elicited from a holding potential of 0 mV, IIR was completely inactivated with time constants ranging from 13 ms at ‐140 mV to 4.5 s at ‐50 mV. The steady‐state inactivation curve (h(V)) was found to be independent of [Na+]o and [K+]o (2‐80 mM) and could be fitted to a Boltzmann curve with a steep slope factor of 5‐6, and a V1/2 around Vrest. Divalent ion‐free extracellular solutions shifted h(V) to the left by about 28 mV. 3. Peak chord conductance, whose maximal value was approximately proportional to the square root of [K+]o, could be fitted to a Boltzmann curve independently of [K+]o, with a V1/2 value around ‐48 mV and a slope factor of 18. Extracellular Cs+ and Ba2+ blocked the IIR in a concentration‐ and voltage‐dependent manner, but Ba2+ was less effective than it is on classical inward‐rectifier channels. 4. Under control culture conditions the values of Vrest and V1/2 of h(V) varied widely among cells. The knowledge of V1/2 proved crucial or the theoretical prediction of Vrest. After cell synchronization in the G0‐G1 phase of the cell cycle, or at the G1‐S boundaries, the cells reduced their variability of h(V). The same occurred after cell synchronization in G1 by treatment with retinoic acid. 5. The experimental data could be fitted to a classical model of an inward rectifier, after removing the dependence of conductance activation on (V‐EK), and incorporating an inactivation with an intrinsic voltage dependence. Moreover, the model predicts, for this novel inward rectifier and in contrast with the classical inward rectifier, the incapacity of maintaining, in physiological media, a Vrest more negative than ‐35 to ‐40 mV, which is an important feature of cancer cells.
Differential suppression of charge movement components by gluconate in cut twitch fibres of Rana temporaria.
Authors: Hui C S , Chen W
Published: December 2014

1. Charge movement was studied in cut twitch fibres of Rana temporaria using a double Vaseline‐gap voltage‐clamp technique. 2. Replacement of Cl‐ by gluconate in the external solution reduced the magnitude of the early current component (I beta) substantially but affected the magnitude and slowed the kinetics of the hump current component (I gamma) slightly. 3. The early (Q beta) and hump (Q gamma) charge components in the gluconate solution were 11.8 +/‐ 2.3 and 88.0 +/‐ 4.8% (mean +/‐ S.E.M., n = 9), respectively, of those in the Cl‐ solution. 4. These results suggest that Q beta cannot be a precursor of Q gamma. Moreover, since fibres bathed in a gluconate solution can release calcium, Q beta is probably not involved in triggering calcium release.
Velocity sensitivity of human muscle spindle afferents and slowly adapting type II cutaneous mechanoreceptors.
Authors: Grill S E , Hallett M
Published: December 2014

1. Velocity information is used in the performance of movement. This study evaluated the ability of peripheral receptors to signal velocity in human subjects. 2. The velocity sensitivity of human muscle spindle afferents from the extensor digitorum muscles and slowly adapting type II cutaneous mechanoreceptors on the dorsum of the hand was evaluated with recordings from the radial nerve during imposed flexion movements about the metacarpophalangeal joint. Twenty‐degree movements at velocities ranging from 5 to 80 deg s‐1 were used. 3. Three measures of dynamic response were calculated: the dynamic positional sensitivity (the relation between discharge rate and joint angle during the dynamic phase of movement), the dynamic index (the discharge rate just before ramp completion minus the rate 0.5 s later), and the incremental response (the discharge rate just before ramp completion minus the rate just before ramp onset). 4. Both muscle spindle afferents and slowly adapting type II cutaneous mechanoreceptors demonstrated significant velocity sensitivity. The magnitudes of the relations between dynamic response measures and velocity were similar in the two receptor types. 5. These findings are consistent with the view that both muscle spindle afferents and slowly adapting type II cutaneous mechanoreceptors provide reasonable velocity signals.
Exercise and diving, two conflicting stimuli influencing cardiac vagal tone in man.
Authors: Al-Ani M , Powell L , West J , Townend J , Coote J H
Published: December 2014

1. In order to increase our understanding of the central regulation of human cardiac vagal motoneurones we studied the interaction between two opposing influences on cardiac vagal tone, one related to diving and the other to exercise. 2. The heart rate response to cooling the face (0 degrees C, trigeminal cutaneous receptor stimulation, TGS) and to a brief isometric muscle contraction was studied in fourteen healthy young adults (8 males, 6 females) during controlled ventilation at supine rest. 3. Fluctuations in R‐R interval were quantified by spectral analysis. In addition the absolute values of R‐R intervals occurring in any one of ten arbitrary phases of a respiratory cycle were measured over thirty to sixty cycles to give a graph of the changes in R‐R interval throughout a respiratory cycle. 4. TGS produced a significant decrease in heart rate (‐21 +/‐ 2 beats min‐1 in females, ‐19 +/‐ 2 beats min‐1 in males; means +/‐ S.E.M.). In addition the autospectral plots of R‐R interval variability showed that during TGS there was a significant increase both in the high frequency peak and in the low frequency peak in all subjects. These data suggested that in these experiments the bradycardia of TGS was due to an increase in cardiac vagal tone. 5. Voluntary isometric contractions at 40 and 60% of maximum (40% MVC, 60% MVC) were timed to occur early in expiration or early in inspiration. Analysis of the changes in R‐R interval produced by these contractions was confined to the respiratory cycle in which they were initiated. The early onset of these changes was interpreted as showing a decrease in cardiac vagal tone. 6. The 40% MVC and 60% MVC increased heart rate significantly within one respiratory cycle with maximum effects having a mean of 9.5 +/‐ 3 and 11 +/‐ 3 beats min‐1, respectively. When isometric muscle contraction was initiated during TGS the 60% MVC but not the 40% MVC was able to significantly decrease R‐R interval and hence increase heart rate. 7. It is concluded that the TGS excitatory inputs and the muscle inhibitory inputs to cardiac vagal neurones do not interact by one input gating the other early in the pathway but more probably by a process of algebraic summation closer to the cardiac vagal neurone.
L‐ and N‐type Ca2+ channels in adult rat carotid body chemoreceptor type I cells.
Authors: e Silva M J , Lewis D L
Published: December 2014

1. Whole‐cell voltage‐dependent Ca2+ currents recorded from chemoreceptor type I cells of the adult rat carotid body had maximum amplitudes of ‐94 pA in 10 mM Ca2+ and were half‐inactivated at a holding potential of ‐38 mV. Somatostatin and dopamine inhibited whole‐cell Ca2+ current in type I cells. 2. The dihydropyridine agonist (+)202‐791 increased the Ca2+ current amplitude by 106% at a step potential of ‐18 mV. The dihydropyridine antagonist nimodipine decreased the Ca2+ current amplitude by 40% from a holding potential of ‐80 mV, and by 74% from a holding potential of ‐60 mV. The nimodipine‐sensitive current had a maximum amplitude at a membrane potential of ‐12 mV. omega‐Conotoxin GVIA (omega‐CgTX GVIA) blocked the whole‐cell Ca2+ current by 40%. The omega‐CgTX GVIA‐sensitive current had a maximum amplitude at a membrane potential of +2 mV. 3. In summary, type I cells of the adult rat carotid body have dihydropyridine‐sensitive L‐type and omega‐conotoxin GVIA‐sensitive N‐type voltage‐dependent Ca2+ channels. These channels may play a role in the voltage‐gated entry of Ca2+ necessary for stimulus‐secretion coupling in response to changes in arterial PO2, PCO2 and pH. Inhibition of the Ca2+ currents by somatostatin and dopamine may alter the chemotransduction signal in type I cells.
Potentiation of neurotransmitter release by activation of presynaptic glutamate receptors at developing neuromuscular synapses of Xenopus.
Authors: Fu W M , Liou J C , Lee Y H , Liou H C
Published: December 2014

1. Glutamate receptors play important roles in synaptic plasticity and neural development. Here we report that, at the developing neuromuscular synapses in Xenopus cultures, the activation of presynaptic glutamate receptors at motor nerve terminals potentiates spontaneous acetylcholine (ACh) release. 2. Co‐cultures of spinal neurons and myotomal muscle cells were prepared from 1‐day‐old Xenopus embryos. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes using whole‐cell recording. Bath application of glutamate (10 microM) markedly increased the frequency of SSCs, and the action of glutamate was reversible. 3. Pretreatment with 0.3 microM tetrodotoxin, which blocks Na+ channels and the conduction of action potentials, only slightly inhibited the potentiating action of glutamate on SSCs. Furthermore, the enhancement of ACh secretion was much more prominent when glutamate was applied locally to the synaptic region. 4. Three types of glutamate receptor agonists, kainate, quisqualate, AMPA (alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid) and NMDA (N‐methyl‐D‐aspartate), were effective in inducing the potentiating effect. The ranking order was: glutamate > kainate > NMDA > AMPA > quisqualate. Glycine potentiated the effects induced by NMDA. Metabotropic receptors were not involved in the potentiating action of glutamate. 5. The potentiating effect of glutamate depended on the influx of Ca2+ through both L‐type Ca2+ channels and NMDA‐gated channels. 6. Since glutamate is known to be co‐released with ACh at some cholinergic nerve terminals, the released glutamate may serve as a positive feedback regulation of ACh secretion at developing neuromuscular junctions via its action on presynaptic glutamate receptors.
Neck muscle responses to abrupt free fall of the head: comparison of normal with labyrinthine‐defective human subjects.
Authors: Ito Y , Corna S , von Brevern M , Bronstein A , Rothwell J , Gresty M
Published: December 2014

1. EMG responses from sternocleidomastoid (SCM) and orbicularis oculi were recorded in subjects who lay supine with their heads cradled in a sling. When the sling released abruptly, their heads fell freely. Normal and bilateral labyrinthine‐defective subjects (LDs) were studied. 2. The normal response in SCM was a small burst of excitation at 22‐25 ms latency, of 18 ms duration. This merged into a larger, later burst. The drop also produced eye blinks at 22‐38 ms. 3. The onset of the SCM response in LDs was delayed (56‐73 ms) even though the latency of their eye blinks was normal. 4. We conclude that the early response at approximately 22 ms in normal subjects is mediated by a vestibulocollic reflex. The delayed activity in LDs may be a stretch reflex. This is the first demonstration of the latency of the vestibulocollic pathway to natural stimulation in man.
Strain of passive elements during force enhancement by stretch in frog muscle fibres.
Authors: Edman K A , Tsuchiya T
Published: December 2014

1. The force enhancement during and after stretch (0.15 micron per sarcomere) was studied during fused tetani of single fibres isolated from the anterior tibialis muscle of Rana temporaria (0.5‐3.6 degrees C; sarcomere length, 2.05‐2.65 microns). Changes in length were recorded simultaneously from the fibre as a whole (puller movement) and from marked segments (approximately 0.5 mm in length) of the same fibre. 2. The residual force enhancement after stretch (recorded at the end of a long tetanus) was found to be linearly related to the slow component of tension rise during the stretch ramp. 3. The fibres were released to shorten against a very small load at different times after stretch (load clamp). The shortening records derived after a preceding stretch exhibited a larger and steeper initial transient than that recorded in an isometric tetanus without stretch. The excess length change (LS; nanometres per half‐sarcomere) recorded during the initial transient increased with the amplitude of stretch and was linearly related to the force enhancement produced by the stretch (FE; % of maximum tetanic tension) according to the following regression: LS = 0.200 FE + 8.65 (P < 0.001). The length changes recorded from the whole fibre agreed well with measurements from individual segments. 4. Slack‐test measurements confirmed the existence of a large initial transient phase when the fibre was released to shorten after a preceding stretch. The excess length change determined from the slack tests agreed closely with the values derived from load‐clamp recordings. 5. The results support the view that stretching a muscle fibre during tetanus leads to strain of elastic elements and, presumably, to variation of filament overlap due to non‐uniform distribution of the length change within the fibre volume. Regions with greater filament overlap are likely to generate the long‐lasting extra force referred to as ‘residual force enhancement after stretch’. The elastic elements recruited during stretch can be presumed to play an essential part in this process by supporting regions in which the filament overlap has been reduced during the stretch ramp. Recoil of these elastic elements is responsible for the excess length change that is recorded during the initial transient after release as described under point 3.
Three distinct chloride channels control anion movements in rat parotid acinar cells.
Authors: Arreola J , Park K , Melvin J E , Begenisich T
Published: December 2014

1. We used the whole‐cell configuration of the patch clamp technique to examine the different macroscopic Cl‐ currents present in single rat parotid acinar cells. 2. Cell swelling produced by negative osmotic pressure (hypotonic bath solutions) induced a large outwardly rectifying Cl‐ current with little or no time and voltage dependence. In contrast, an increase in intracellular [Ca2+] induced by ionomycin activated Cl‐ currents with very different properties. Ca(2+)‐activated Cl‐ currents showed outward rectification, relatively slow activation kinetics and marked voltage dependence. These results are consistent with the existence of two different outwardly rectifying Cl‐ channels in rat parotid cells. 3. In conditions designed to eliminate the activation of these two Cl‐ currents, a third type of current was observed. This third current was activated in a time‐dependent manner by hyperpolarized potentials and was about equally permeant to Cl‐, I‐ and Br‐. 4. The properties of the hyperpolarization‐activated current were similar to those of the cloned ClC‐2 channel. Polymerase chain reaction‐based methods and ribonuclease protection analyses indicated the presence in parotid gland of mRNA homologous to ClC‐2. 5. Individual parotid acinar cells expressed all three types of Cl‐ channels. Each type of channel may contribute to Cl‐ efflux in distinct stages of the secretion process depending on the intracellular [Ca2+], cell volume and membrane potential.
Evidence of spatial and temporal channels in the correlational structure of human spatiotemporal contrast sensitivity.
Authors: Billock V A , Harding T H
Published: December 2014

1. The statistical correlation of detection thresholds for pairs of stimuli should be higher for stimuli detected by the same mechanism than for stimuli detected by different mechanisms‐‐a property that can be used to probe the visual mechanisms that underlie detection. 2. Correlation of contrast sensitivities for pairs of spatiotemporal stimuli is approximately a linear function of spatial or temporal frequency separation in octaves. Using the slope of this function as an index of neural processing gave results consistent with: more spatial mechanisms than temporal; more spatial mechanisms at low temporal frequencies than at high; and at least two temporal mechanisms active at spatial frequencies up to 22.6 cycles deg‐1. 3. This method of analysing sensitivity data is insensitive to experimental conditions and applicable to any sensory detection task mediated by tuned channels. In addition to being applicable to psychophysical sensitivity measurements, it may also be useful in analysing some kinds of electrophysiological measurements that pool the responses from many active mechanisms (such as evoked potentials).
Effect of a dynein inhibitor on vasopressin action in toad urinary bladder.
Authors: Marples D , Barber B , Taylor A
Published: December 2014

1. The effect of the dynein inhibitor erythro‐9‐[3‐(2‐hydroxynonyl)] adenine (EHNA) on the osmotic water flow response to vasopressin or exogenous cAMP has been investigated in isolated toad urinary bladders. 2. Pretreatment with serosal EHNA had no effect on basal water flow, but inhibited the development and maintenance of the hydrosmotic response to vasopressin (20 mU ml‐1) or 8‐(4‐parachlorophenylthio)‐adenosine 3',5'‐cyclic monophosphate (8 CPT‐cAMP; 0.1 mM). 3. The inhibitory effect of EHNA on vasopressin‐induced water flow was dose dependent. Inhibition occurred in the dose range in which EHNA inhibits the ATPase and motor activities of dynein in vitro. 4. EHNA also inhibited the maintenance of the high rate of water flow established by prior exposure to vasopressin. 5. The inhibitory effect of EHNA on the onset phase of the vasopressin response was attenuated after exposure of the tissue to the microtubule‐disruptive drug nocodazole but was fully additive with that of cytochalasin B. 6. EHNA inhibited basal and vasopressin‐stimulated transepithelial sodium transport. 7. The findings support the view that EHNA inhibits hormone‐induced water flow through an action on a cytoplasmic dynein. The results are consistent with the hypothesis that dynein is involved in the microtubule‐based delivery of water channel‐containing vesicles to the apical membrane of the granular epithelial cells during both the onset and maintenance of the water permeability response to vasopressin.
Rapid activation of KATP channels by aldosterone in principal cells of frog skin.
Authors: Urbach V , Van Kerkhove E , Maguire D , Harvey B J
Published: December 2014

1. In epithelial cells of frog skin, potassium ions are recycled across the basolateral membrane via an inward‐rectifier, ATP‐sensitive K+ channel (KATP channel). In this study, we show that aldosterone has a stimulatory effect on KATP channel activity and we have investigated the involvement of Na+‐H+ exchange and intracellular pH (pHi) in this phenomenon. 2. Aldosterone (10 nM) produced an increase in the open probability of the KATP channel within 15 min from 0.21 +/‐ 0.05 to 0.93 +/‐ 0.10 (n = 8), measured in cell‐attached patches. Aldosterone also increased the tolbutamide‐sensitive K+ current across the basolateral membrane within 30 min from 17.2 +/‐ 1.9 to 30.3 +/‐ 1.6 microA cm‐2 (n = 8) in nystatin‐permeabilized whole skins. 3. The KATP channel is very sensitive to variations in cytosolic pH within the physiological range 7.0‐7.4. 4. The intracellular pH of principal cells is regulated by Na+‐H+ exchange, and the stimulatory effect of aldosterone on KATP channel activity was abolished by amiloride (100 microM) added on the basolateral side of the epithelium either before or after aldosterone treatment. 5. We propose that aldosterone activates the KATP channels via stimulation of Na+‐H+ exchange. The rapidity of aldosterone activation of KATP channels is presented as evidence for a novel non‐genomic steroid hormone effect on epithelial ion transport.
Possible involvement of glucocorticoids in the modulation of interleukin‐1‐induced cardiovascular responses in rats.
Authors: Watanabe T , Tan N , Saiki Y , Makisumi T , Nakamura S
Published: December 2014

1. In freely moving rats, we investigated whether glucocorticoids modulate the cardiovascular responses to intraperitoneal (I.P.) injection of interleukin‐1 beta (IL‐1 beta). 2. A lower dose of IL‐1 beta (1 microgram kg‐1, I.P.) induced monophasic increases, and a higher dose (10 micrograms kg‐1, I.P.) induced biphasic increases in both blood pressure and heart rate. Plasma concentration of corticosterone increased significantly after injection of IL‐1 beta (10 micrograms kg‐1). 3. Systemic pretreatment with an exogenous glucocorticoid, dexamethasone (DEX; 0.5 mg kg‐1) reduced the monophasic pressor response, the first phase of the biphasic pressor response and also the initial tachycardia. By contrast, the second phase of the biphasic pressor response was enhanced. 4. After bilateral adrenalectomy, the IL‐1 beta (10 micrograms kg‐1)‐induced pressor effect was reduced; it was restored by treatment with DEX (0.5 mg kg‐1). The heart rate response was enhanced in adrenalectomized (ADX) rats; this enhancement was attenuated by DEX. 5. IL‐1 beta (10 micrograms kg‐1)‐induced increases in plasma noradrenaline (NA) were suppressed in intact rats pretreated with DEX (0.5 mg kg‐1). The IL‐1 beta‐induced NA response was greater in ADX rats than in sham‐ADX rats. 6. We suggest that glucocorticoids are an important modulator of cardiovascular responses induced in rats by systemically administered IL‐1 beta.
Mechanism of inhibition of delayed rectifier K+ current by 4‐aminopyridine in rabbit coronary myocytes.
Authors: Remillard C V , Leblanc N
Published: December 2014

1. The mechanisms involved in the 4‐aminopyridine (4‐AP)‐induced block of delayed rectifier K+ current (IK(V)) in vascular smooth muscle cells were studied in cells enzymatically isolated from the rabbit coronary artery. 2. 4‐AP inhibited slowly inactivating IK(V) in a dose‐dependent manner (concentration producing half‐maximal inhibition, K1/2, = 1.37 mM), and shifted the steady‐state activation and inactivation curves of IK(V) by +9 and +16 mV, respectively. 3. The time constant of activation was significantly increased by 4‐AP at +20 mV; deactivation kinetics were unaffected upon repolarization to ‐40 mV. The fast (tau f approximately 1 s) and slow (tau s approximately 5 s) time constants of inactivation (0 and +20 mV), and the recovery kinetics (tau r approximately 6 s) at ‐60 mV were not significantly affected by 0.5 mM 4‐AP. However, tau f disappeared in the presence of 2 mM 4‐AP while tau s remained unaffected. 4. Use‐dependent unblock of IK(V) was revealed at potentials > or = ‐10 mV from analyses of the voltage dependence of 4‐AP‐sensitive currents and the frequency‐dependent changes (‘reverse use dependence’) of IK(V) during the application of repetitive steps (‐60 to +20 mV for 250 ms at a rate of 0.25 Hz) in control conditions, in the presence of 0.5 mM 4‐AP, and after washout of the drug. These results suggested that 4‐AP preferentially binds to the channel in the closed state, and unbinding is promoted by transitions to the open state. 5. The channel was modelled as a simple three‐state mathematical loop model incorporating single closed, open and inactivated states. The block by 4‐AP was modelled as a state‐dependent interaction with 4‐AP primarily binding to the closed state. Computer simulations support the hypothesis that 4‐AP‐induced block of the delayed rectifier K+ (KV) channel in the closed state is relieved during membrane depolarization. 6. Closed state binding of 4‐AP to the KV channel depolarizes vascular smooth muscle cells by shifting the activation curve of these channels to more positive potentials.
Role of [Ca2+]i in "Ca2+ stores depletion‐Ca2+ entry coupling’ in fibroblasts expressing the rat neurotensin receptor.
Authors: Gailly P , Hermans E , Gillis J M
Published: December 2014

1. Transfected Chinese hamster ovary fibroblasts expressing the rat neurotensin receptor were used to study the ‘Ca2+ stores depletion‐Ca2+ entry coupling’ which follows stimulation with neurotensin and liberation of InsP3. 2. This coupling could be dissociated in time. Firstly, stores depletion was produced by neurotensin or thapsigargin which caused a first [Ca2+]i transient in a Ca(2+)‐free external medium. Secondly, readmission of external Ca2+ produced an influx of Ca2+ and a second [Ca2+]i transient. 3. Various concentrations of thapsigargin (20 nM to 1 microM) were used to produce complete stores depletion with small or large first peaks of [Ca2+]i. Upon return to external Ca2+, small or large second [Ca2+]i peaks were observed. The amplitudes of both peaks were positively correlated. 4. The Ca2+ entry which followed stores depletion could occur at very low basal values of [Ca2+]i, was accelerated by okadaic acid and inhibited by staurosporine and the calmodulin antagonist W‐7. 5. It is concluded that the rise in [Ca2+]i during Ca2+ stores depletion is an essential parameter which determines the size of the subsequent Ca2+ entry.
The role of elevations in intracellular [Ca2+] in the development of low frequency fatigue in mouse single muscle fibres.
Authors: Chin E R , Allen D G
Published: December 2014

1. Intracellular free calcium concentration ([Ca2+]i) and force were measured in isolated single skeletal muscle fibres from mice. The aim was to determine the extent to which elevations in [Ca2+]i during various stimulation protocols affected subsequent muscle performance. 2. A protocol of repeated tetanic stimulation which elevated [Ca2+]i and caused a large decline in force (fatigue) had a [Ca2+]‐time integral of 36.4 +/‐ 8.1 microM s. A protocol of repeated tetani at a lower duty cycle (stimulation) caused only a small decline in force (9‐16%) but elevated the [Ca2+]‐time integral to 16.7 +/‐ 2.8 and 24.9 +/‐ 1.6 microM s in the absence and presence of 10 mM caffeine, respectively. Caffeine alone raised the [Ca2+]‐time integral to 20.3 +/‐ 3.4 microM s. 3. Following the fatigue protocol there was a proportionately greater loss of force at low stimulation frequencies (30 and 50 Hz) compared with high frequencies (100 Hz) which persisted for up to an hour. This pattern of force loss could be attributed to a uniform reduction in [Ca2+]i at all frequencies. Similar effects were observed after elevating [Ca2+]i with the caffeine + stimulation protocol but were not observed after stimulation or caffeine alone. The higher [Ca2+]‐time integrals during the fatigue and caffeine + stimulation protocols suggest that some threshold for [Ca2+]i must be reached before these effects are observed. 4. The reductions in low frequency force induced by the fatigue and caffeine + stimulation protocols were not due to decreased Ca2+ sensitivity or to decreases in maximum force‐generating capacity of the contractile proteins and therefore are due to a failure of Ca2+ release. 5. The Ca(2+)‐activated neutral protease (calpain) inhibitor calpeptin was not effective in preventing the effects of caffeine + stimulation indicating that the reduction in Ca2+ release was not due to calpain‐mediated hydrolysis of the Ca2+ release channel. 6. Our findings indicate that low frequency fatigue results from increases in [Ca2+]i during fatigue and that these elevations in [Ca2+]i activate some process which leads to failure of excitation‐contraction (E‐C) coupling and Ca2+ release.
Voltage‐dependent calcium currents and cytosolic calcium in equine airway myocytes.
Authors: Fleischmann B K , Wang Y X , Pring M , Kotlikoff M I
Published: December 2014

1. The relationship between voltage‐dependent calcium channel current (I(Ca)) and cytosolic free calcium concentration ([Ca2+]i) was studied in fura‐2 AM‐loaded equine tracheal myocytes at 35 degrees C and 1.8 mM Ca2+ using the nystatin patch clamp method. The average cytosolic calcium buffering constant was 77 +/‐ 3 (n = 14), and the endogenous calcium buffering constant component is likely to be between 15 and 50. 2. I(Ca) did not evoke significant calcium‐induced calcium release (CICR) since (i)[Ca2+]i scaled with the integrated I(Ca) over the full voltage range of evoked calcium currents, (ii) increases in [Ca2+]i associated with I(Ca) were consistent with cytoplasmic buffering of calcium ions entering through voltage‐dependent calcium channels (VDCCs) only, (iii) there was a fixed instantaneous relationship between transmembrane calcium flux (J(Ca)) and the change in cytosolic free calcium concentration (delta [Ca2+]i) during I(Ca), (iv) caffeine (8 mM) triggered 8‐fold higher calcium transients than I(Ca), and (v) I(Ca) evoked following release of intracellular calcium by caffeine resulted in an equivalent delta[Ca2+]i‐J(Ca) relationship. 3. The time constant (T) for the decay in [Ca2+]i was 8.6 +/‐ 1.5 s (n = 8) for single steps and 8.6 +/‐ 1.1 s (n = 13) following multiple steps that increased [Ca2+]i to much higher levels. Following application of caffeine (8 mM), however, [Ca2+]i decay was enhanced (T = 2.0 +/‐ 0.2 s, n = 3). The rate of [Ca2+]i decay was not voltage dependent, was not decreased in the absence of extracellular Na+ ions, and no pump current was detected. 4. We conclude that under near physiological conditions, neither CICR nor Na(+)‐Ca2+ exchange play a substantial role in the regulation of I(Ca)‐induced increases in [Ca2+]i, and that, even following release of intracellular calcium by caffeine, Na(+)‐Ca2+ exchange does not play an appreciable role in the removal of calcium ions from the cytosol.
Neurokinin A and Ca2+ current induce Ca(2+)‐activated Cl(‐) currents in guinea‐pig tracheal myocytes.
Authors: Hazama H , Nakajima T , Hamada E , Omata M , Kurachi Y
Published: December 2014

1. Membrane currents were recorded by a patch clamp technique in guinea‐pig tracheal myocytes, using the whole cell mode with Cs(+) internal solution. 2. Both neurokinin A (NKA, 1 mu M) and caffeine (10 mM) evoked Ca(2+)‐activated Cl‐ currents (I[Cl(Ca)]) transiently. In Ca(2+)‐free bathing solution, the first application of NKA or caffeine elicited I[Cl(Ca)] but the second application of these substances failed to activate it. In addition, pretreatment with ryanodine in the presence of caffeine abolished the response to both NKA and caffeine whilst heparin (200 mu g ml(‐1)) only blocked the NKA‐induced response. I[Cl(Ca)] was also elicited by inositol 1,4,5‐trisphosphate (IP(3)). 3. Command voltage pulses positive to 0 mV from a holding potential of ‐60 mV activated the voltage‐dependent L‐type Ca2+ current (I(Ca,L)) and late outward current. Upon repolarization to the holding potential, slowly decaying inward tail currents were recorded. The outward current during the depolarizing pulses and the inward tail current were enhanced by Bay K 8644, but completely blocked by Cd2+ or nifedipine. Replacement of external Ca2+ with Ba2+, removal of Ca2+ from the bath solution, or inclusion of EGTA (5 mM) in the patch pipette, also led to abolition of these currents, indicating that they were Ca2+ dependent, and that Ca2+ influx due to I(Ca,L) activated the currents. 4. When [Cl(‐)](O) or [Cl(‐)](i) was changed, the reversal potential (E(rev)) of the Ca2+‐activated currents shifted, thus behaving like a Cl(‐)‐selective ion channel as predicted by the Nernst equation. DIDS (1 mM) completely abolished the currents, also suggesting that they were I[Cl(Ca)]. 5. NKA (1 mu M) and caffeine (30 mM) transiently activated I[Cl(Ca)], and after that both agents markedly reduced I[Cl(Ca)] induced by I(Ca,L). This is probably due to sarcoplasmic reticulum (SR) Ca2+ release induced by NKA or caffeine, followed by inhibition of the Ca(2+)‐induced Ca2+ release from the SR. 6. The present results indicate that I[Cl(Ca)] can be activated by SR Ca2+ release due to NKA or caffeine (through IP(3) or ryanodine receptors) as well as by Ca2+ influx due to I(Ca,L). It also suggests that activation of I[Cl(Ca)] by NKA may be mediated by the production of IP(3), which releases Ca2+ from the SR.
The effects of locomotor‐respiratory coupling on the pattern of breathing in horses.
Authors: Lafortuna C L , Reinach E , Saibene F
Published: December 2014

1. To investigate the effect of locomotor activity on the pattern of breathing in quadrupeds, ventilatory response was studied in four healthy horses during horizontal and inclined (7%) treadmill exercise at different velocities (1.4‐6.9 m s(‐1)) and during chemical stimulation with a rebreathing method. Stride frequency (f(s)) and locomotor‐respiratory coupling (LRC) were also simultaneously determined by means of video recordings synchronized with respiratory events. 2. Tidal volume (V(T)) was positively correlated with pulmonary ventilation (V(E)) but significantly different linear regression equations were found between the experimental conditions (P < 0.0001), since the chemical hyperventilation was mainly due to increases in V(T), whereas the major contribution to exercise hyperpnoea came from changes in respiratory frequency (f(R)). 3. The average f(R) at each exercise level was not significantly different from f(S), although there was not always a tight 1:1 LRC. At constant speeds, f(S) was independent of the treadmill slope and hence the greater V(E) during inclined exercise was due to increased V(T). 4. At any ventilatory level, the differences in breathing patterns between locomotion and rebreathing or locomotion at different slopes derived from different set points of the inspiratory off‐switch mechanism. 5. The percentage of single breaths entrained with locomotor rhythm rose progressively and significantly with treadmill speed (P < 0.0001) up to a 1:1 LRC and was significantly affected by treadmill slope (P < 0.001). 6. A LRC of 1:1 was systematically observed at canter (10 out of 10 trials) and sometimes at trot (5 out of 14) and it entailed (i) a 4‐ to 5‐fold reduction in both V(T) and f(R) variability, and (ii) a gait‐specific phase locking of inspiratory onset during the locomotor cycle. 7. It is concluded that different patterns of breathing are employed during locomotion and rebreathing due to the interference between locomotor and respiratory functions, which may play a role in the optimization and control of exercise ventilation in horses.
Competition between Mg2+ and spermine for a cloned IRK2 channel expressed in a human cell line.
Authors: Yamashita T , Horio Y , Yamada M , Takahashi N , Kondo C , Kurachi Y
Published: December 2014

1. A cloned inwardly rectifying K+ channel, IRK2, was expressed in a human cell line, human embryonic kidney (HEK) 293T. Its electrophysiological properties were examined using the patch clamp technique in the whole‐cell, cell‐attached and inside‐out patch configurations. 2. The cells transfected with IRK2 cDNA exhibited a K+ current which showed classical properties of inwardly rectifying K+ channels at both whole‐cell and single‐channel levels. 3. In the inside‐out patch configuration, intracellular Mg2+ (Mg2+i blocked the outward currents in a voltage‐dependent and virtually time‐independent manner. Mg2+i (1‐100 microM) caused a decrease in the unitary current amplitude of the IRK2 channel by inducing subconducting levels. 4. In the absence of Mg2+i, intracellular spermine blocked the outwardly flowing IRK2 currents in a voltage‐ and time‐dependent manner. Spermine (1‐100 nM) did not affect the unitary channel current amplitude but reduced the channel open probability. The spermine block showed a slower time and steeper voltage dependence than the Mg2+i++ block. 5. When both these blockers were present, Mg2+i apparently attenuated the inhibitory effect of spermine on the outwardly flowing IRK2 currents. This interaction was voltage and time dependent, and could be well explained by a model in which Mg2+i and spermine competitively bind to the channel with their individual first‐order kinetics. This competition would induce time‐dependent transits of the channel between the Mg2+i ‐and spermine‐blocked states via a single open state, thereby preserving a certain size of persistent outward currents at depolarized potentials.
Deactivation and desensitization of non‐NMDA receptors in patches and the time course of EPSCs in rat cerebellar granule cells.
Authors: Silver R A , Colquhoun D , Cull-Candy S G , Edmonds B
Published: December 2014

1. Spontaneous and evoked non‐NMDA receptor‐mediated EPSCs were recorded from cerebellar granule cells in slices at approximately 24 and approximately 34 degrees C. The EPSC decay was fitted with the sum of two exponential functions. 2. The time courses of non‐NMDA receptor deactivation and desensitization were determined with fast concentration jumps of glutamate onto patches from cultured granule cells. Deactivation (decay time constant tau = 0.6 ms at 24 degrees C) was substantially faster than desensitization (tau = 4 ms). Both processes were fitted by single exponential functions. 3. The decay of the fast component of the spontaneous EPSC (tau EPSCfast = 0.9 ms at 23 degrees C) was marginally slower than deactivation but too fast to be determined by desensitization. Our results suggest that the decay of this component is set by both the rate of decline of transmitter concentration and channel deactivation. 4. A simple diffusion model predicts that the time course of transmitter in the cleft declines slowly during the later stages of its action. The slow phase of transmitter removal could account for the time course of the slow component of the spontaneous EPSC (tau EPSCslow = 8 ms at 23 degrees C).
Ca2+ loading reduces the tensile strength of sarcolemmal vesicles shed from rabbit muscle.
Authors: Nichol J A , Hutter O F
Published: December 2014

1. Sarcolemmal vesicles shed by rabbit muscle were loaded with Ca2+ by means of A23187 or ionomycin. [Ca2+]0 was buffered between 0.8 and 20 microM. Membrane strength was measured by pipette aspiration. 2. At 20 microM Ca2+ many vesicles underwent autolysis, or were so weak that they burst instantly on aspiration. Between 10 and 2 microM Ca2+ a graded decrease in membrane strength was demonstrable. At 0.8 microM Ca2+ the mechanical properties of the sarcolemma remained unaltered. 3. Mg2+ carried by A23187 does not mimic the effect of Ca2+. The ionophore itself similarly did not cause a decrease in membrane tensile strength. 4. Pre‐treatment with BAPTA‐AM, so as to buffer internal Ca2+, partly protected vesicles against the decrease in membrane strength produced by Ca2+ loading. 5. Membrane strength was not restored by adding excess BAPTA to the bathing solution, so as to reverse the Ca2+ gradient. An irreversible degradation of the membrane consequent upon raised [Ca2+]1 seems indicated. 6. These findings are discussed in relation to the mechanisms which have been advanced to account for the role of elevated [Ca2+]1 in cell death. 7. An attempt to use staphylococcal alpha‐toxin as an alternative means to permeabilize the sarcolemma led to the incidental finding that this pore‐forming protein itself greatly weakens the membrane in doses lower than required for effective permeabilization.
Ontogeny of postural adjustments during sitting in infancy: variation, selection and modulation.
Authors: Hadders-Algra M , Brogren E , Forssberg H
Published: December 2014

1. The aim of the study was to find out whether the development of postural adjustments occurs via a coupling of simple muscle responses, such as stretch reflexes, or via selection from an innate repertoire of centrally generated response patterns. 2. Postural responses during sitting on a moveable platform were assessed in eleven healthy infants at 5‐6, 7‐8 and 9‐10 months of age. Multiple surface EMGs and kinematics were recorded while the infants were exposed to slow and fast horizontal forward (Fw) and backward (Bw) displacements of the platform. 3. From the youngest testing age onwards, largely variable but direction‐specific muscle activation patterns were present. Fw translations resulted predominantly in an activation of the neck flexor, the rectus abdominis and rectus femoris muscle, while the neck‐, thoracal‐ and lumbar extensor muscles (NE, TE, LE) and the hamstrings (Ham) showed varying amounts of inhibition. During Bw translations NE, TE, LE and Ham were preferably activated. The muscle activity could not be explained by simple stretch reflex mechanisms, but is likely to reflect centrally generated motor activity maturing in a predetermined way. However, indications for a contribution of stretch reflex mechanisms were also present. 4. With increasing age the variation in muscle activation patterns decreased, resulting in a selection of the most complete patterns. The ability to modulate the amplitude of the selected, most complete patterns during Fw translations, with respect to platform velocity and initial pelvis position, emerged at 9‐10 months.
Activation of Ca2+ release by caffeine and voltage in frog skeletal muscle.
Authors: Shirokova N , Ríos E
Published: December 2014

1. Using a fast flow, computer‐controlled, two‐Vaseline‐gap chamber, single muscle fibres were subjected to ‘pulses’ of caffeine at Ca2+ releasing concentrations, combined with voltage‐clamp depolarizations, while monitoring intracellular [Ca2+]. 2. Ca2+ release flux elicited by caffeine reached 2.5 mM s‐1, or less, after 3 s of exposure, then decayed to zero. The caffeine‐releasable pool of sarcoplasmic reticulum (SR) Ca2+ was 2.9 +/‐ 0.4 mM (mean +/‐ S.E.M., n = 10). 3. In parallel with release induced by caffeine, release induced by voltage pulses applied during a caffeine exposure increased in the first second of exposure, then decreased, to abolition after 5 s. 4. The amount of Ca2+ releasable by depolarizing pulses was always equal to the amount of Ca2+ in the caffeine‐releasable pool. Therefore, there is a single releasable Ca2+ pool. This pool is well stirred‐it takes much more time to lose its Ca2+ by release than to diffusionally homogenize its [Ca2+]. Its depletion explains quantitatively the decay of release induced by caffeine or voltage during an exposure to caffeine. 5. A 1.5 s pulse to 10 mV, applied during exposure to caffeine, resulted in large Ca2+ release and, upon repolarization, termination of the caffeine‐induced release. This is similar to repolarization‐induced stop of caffeine contracture (RISC) in embryonic murine myoballs. The permeability elicited by caffeine (ratio of flux to calcium in the releasable pool) was not affected by depolarizing pulses. Therefore, the mechanism of the RISC‐like effect was Ca2+ depletion. 6. Caffeine‐induced release did not depend on the holding potential. 7. Whether caffeine was present or not, release activated by voltage remained always under voltage control, ending rapidly upon repolarization. A depolarizing pulse induced a release permeability with an early peak, followed by decay to a steady level. Caffeine (10 mM) shifted the mid‐activation voltage of both peak and steady components by ‐15 mV and increased the steepness of their voltage dependence by 15%. The maximum permeability increased by 30% for the peak and 25% for the steady component (n = 5). These results neither support nor disprove the hypothesis that the peak of Ca2+ release is activated by Ca2+. 8. The similar potentiation by caffeine of both components of release, the continued ability of voltage to control release in the presence of caffeine, and its failure to alter caffeine‐induced permeability indicate that caffeine and the voltage sensor enhance independently the channel's tendency to open.
Hypotonic stimulation induced Ca2+ release from IP3‐sensitive internal stores in a green monkey kidney cell line.
Authors: Ishii T , Hashimoto T , Ohmori H
Published: December 2014

1. Hypotonic stimulation (180 +/‐ 5 mosmol l‐1) increased [Ca2+]i in fura‐2‐loaded Green monkey kidney cells (COS‐7 cells) and depolarized the membrane. 2. COS‐7 cells were depolarized up to ‐3.5 +/‐ 4.4 mV from a resting membrane potential of ‐35.2 +/‐ 2.3 mV in response to hypotonic stimulation, when the patch electrode was filled with a 160 mM KCl‐0.5 mM EGTA‐based intracellular medium. 3. The increase in [Ca2+]i induced by hypotonic stimulation was divided into two phases. One was transient and oscillatory, and observed in Ca(2+)‐free medium; the other was persistent, blocked by 100 microM La3+, and observed only in Ca(2+)‐containing medium. 4. The increase in [Ca2+]i in Ca(2+)‐free medium was blocked by pretreatment with 10 microM thapsigargin. The increase in [Ca2+]i induced by 10 microM thapsigargin was reduced after hypotonic stimulation which induced an increase in [Ca2+]i in Ca(2+)‐free medium. 5. The increase in [Ca2+]i in Ca(2+)‐free medium was not affected by treatment with 5 mM caffeine or 1‐10 microM ryanodine. Neither caffeine nor ryanodine induced an increase in [Ca2+]i. 6. Adenosine 5'‐O‐2‐thiodiphosphate (ADP‐beta‐S; a P2Y receptor agonist) induced an increase in [Ca2+]i in Ca(2+)‐free medium and caused phosphoinositide breakdown in COS‐7 cells. Exposure to 10 microM ADP‐beta‐S blocked the increase in [Ca2+]i induced in the Ca(2+)‐free medium by hypotonic stimulation. The results of summary points 4, 5, and 6 suggest that the increase in [Ca2+]i induced by hypotonic stimulation is due to Ca2+ release from inositol 1,4,5‐trisphosphate (IP3)‐sensitive internal stores. 7. The hypotonic stimulation‐activated hydrolysis of phosphoinositides was decreased by pertussis toxin (PTX) in a dose‐dependent manner. 8. These observations strongly suggest that hypotonic stimulation induced an increase in [Ca2+]i in Ca(2+)‐free medium through activation of cascades using PTX‐sensitive guanine nucleotide binding protein (G protein) and IP3.