bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of ATP‐dependent K+ channels by metabolic poisoning in adult mouse skeletal muscle: role of intracellular Mg(2+) and pH.
Authors: Allard B , Lazdunski M , Rougier O
Published: December 2014

1. The effects of metabolic poisoning, intracellular Mg(2+) and pH on ATP‐dependent K+ (K+ATP) channels were examined in adult mouse isolated skeletal muscle fibres using the patch clamp technique. 2. In cell‐attached membrane patches, while openings of one kind of channel could only rarely be detected under control conditions, cell poisoning with fluorodinitrobenzene (FDNB), dinitrophenol (DNP) and cyanide (CN) induced a strong and partially reversible increase in channel activity. 3. Slope conductance and glibenclamide sensitivity of this outward current indicated that the channel activated during poisoning was the K+ATP channel. 4. Single channel current amplitude was reduced during poisoning, but remained unchanged when activation of the K+ATP channel was induced by cromakalim. 5. In inside‐out membrane patches, in the absence of intracellular ATP, intracellular application of Mg2+ decreased channel activity and single channel current amplitude. Inhibition of K+ATP channels by ATP was also reduced. 6. In the absence of intracellular ATP, a decrease in intracellular pH induced a reduction in channel activity and single channel current amplitude. Inhibition of K+ATP channels by ATP was also reduced. 7. The reduction of single channel current amplitude during poisoning was attributed to an increase in intracellular Mg2+ concentration caused by a fall in intracellular ATP concentration. These results also show that metabolic poisoning causes direct activation of K+ATP channels in skeletal muscle, and that is activation is at least partially mediated through an increase in intracellular Mg(2+) concentration and a decrease in intracellular pH.
Calcium entry through a subpopulation of AMPA receptors desensitized neighbouring NMDA receptors in rat dorsal horn neurons.
Authors: Kyrozis A , Goldstein P A , Heath M J , MacDermott A B
Published: December 2014

1. A Ca(2+)‐dependent interaction between non‐NMDA and NMDA receptors was studied in embryonic rat dorsal horn neurons grown in tissue culture using perforated‐patch recording. Specifically, non‐NMDA receptors were found to induce reversible inhibition of NMDA receptors in a manner dependent on the presence of extracellular Ca2+. 2. Non‐NMDA receptor‐induced inhibition of NMDA receptors was mediated by the elevation of intracellular Ca2+ concentration produced by Ca2+ entry through a subpopulation of alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) non‐NMDA receptors. Furthermore, Ca2+ entry through the AMPA channels alone is sufficient for desensitization of NMDA channels to occur. 3. Imaging of neuritic sites of Ca2+ revealed that Ca(2+)‐permeable AMPA channels are often co‐localized with NMDA channels on dorsal horn neurons, indicating that the Ca(2+)‐mediated interaction between receptors may occur within small dendritic domains. 4. The ability of Ca(2+)‐permeable AMPA channels to inhibit adjacent NMDA channels may contribute to the postsynaptic integration of excitatory input.
Characterization of a volume‐sensitive chloride current in rat osteoblast‐like (ROS 17/2.8) cells.
Authors: Gosling M , Smith J W , Poyner D R
Published: December 2014

1. During osmotic swelling, cultured osteoblastic cells (ROS 17/2.8) exhibited activation of large amplitude Cl‐ currents in the whole‐cell configuration of the patch‐clamp technique. Effects of hypotonic shock on cell volume and membrane conductance were rapidly reversed on return to isotonic conditions. 2. Voltage command pulses in the range ‐80 to +50 mV produce instantaneous activation of Cl‐ currents. At potentials more positive than +50 mV the current exhibited time‐dependent inactivation. The instantaneous current‐voltage relationship was outwardly rectifying. 3. The anion permeability sequence of the induced current was SCN‐ (2.2) > i‐ (1.9) > Br‐ (1.5) > Cl‐ (1.0) > F‐ (0.8) > gluconate‐ (0.2). This corresponds to Eisenman's sequence I. 4. The volume‐sensitive Cl‐ current was effectively inhibited by the Cl‐ channel blockers 4,4'‐ diisothiocyanatostilbene‐2,2‐disulphonic acid (DIDS) and 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB). Outward currents were more effectively suppressed by DIDS than inward currents. The concentrations for 50% inhibition (IC50) of outward and inward currents were 81 and 298 microM, respectively. NPPB was equally effective at inhibiting outward and inward currents (IC50 of 64 microM). The current was relatively insensitive to diphenylamine‐2‐carboxylate (DPC), 500 microM producing only 22.5 +/‐ 4.0% inhibition. 5. Inhibitors of protein kinase A (H‐89, 1 microM) and tyrosine kinase (tyrphostin A25, 200 microM) were without effect upon activation of Cl‐ currents in response to hypotonic shock. Under isotonic conditions, elevation of intracellular Ca2+ by ionomycin (1 microM) or activation of protein kinase C by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA, 0.1 microM) failed to evoke increases in basal Cl‐ conductance levels. 6. It is concluded that an outwardly rectifying Cl‐ conductance is activated upon osmotic swelling and may be involved in cell volume regulation of ROS 17/2.8 cells.
Involvement of dihydropyridine receptors in terminating Ca2+ release in rat skeletal myotubes.
Authors: Suda N
Published: December 2014

1. Combined patch‐clamp and fura‐2 measurements were performed in order to investigate the effect of dihydropyridine (DHP) antagonists on termination of sarcoplasmic reticulum (SR) Ca2+ release in cultured rat skeletal myoballs. 2. Ca2+ transients induced by 10 mM caffeine were curtailed by depolarization (e.g. +20 mV for 1 s) and subsequent repolarization (‐70 mV). This phenomenon is termed RISC (repolarization‐induced stop of caffeine‐induced Ca2+ release). 3. At 0.5 to 1 microM, DHP antagonists (nifedipine or (+)PN200‐110) strongly inhibited RISC and also slowed the decay of intracellular Ca2+ concentration ([Ca2+]i) following repolarization after depolarization‐induced Ca2+ release (‐20 or ‐10 mV for 5 s). 4. The activation time course of the Ca2+ channel associated with the DHP receptor (DHPR) was investigated by measuring DHP‐sensitive Ca2+ channel tail currents, while varying the duration of depolarizing pulses. The tail currents increased with pulse duration and peaked around 0.7, 0.9 and 1.1 s for depolarizations to +70, +40 and +20 mV, respectively. These values are compatible with the activation time course of RISC (0.5‐1 s to maximally activate RISC at +20 to +60 mV). 5. These results suggest that the DHPR in T‐tubular membranes regulates closing of the ryanodine receptor (RyR)‐Ca2+ release channel complex through membrane potential change.
Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle.
Authors: Gong M C , Kinter M T , Somlyo A V , Somlyo A P
Published: December 2014

1. Exogenous arachidonic acid (AA) inhibits the protein phosphatase that dephosphorylates smooth muscle myosin, thus sensitizing the contractile response to Ca2+; it also inhibits voltage‐gated Ca2+ channels in smooth muscle. The purpose of the present study was to determine whether endogenous AA is increased by agonists in a manner consistent with its role as a messenger regulating myosin phosphatase and Ca2+ channels. Both AA and diacylglycerol (DAG) were measured in [3H]AA‐labelled intact and permeabilized (with staphylococcal alpha‐toxin) rabbit femoral arteries stimulated with the alpha 1‐adrenergic agonist phenylephrine (PE) (intact and permeabilized smooth muscles) or by guanosine‐5'‐O‐(3‐thiotriphosphate (GTP gamma S; permeabilized smooth muscles in which the [Ca2+] was maintained constant). Arachidonic acid mass was determined with gas chromatography and mass spectrometry (GC‐MS). 2. In intact smooth muscle, PE increased both AA and DAG levels significantly, to 210 and 145% of baseline values, respectively. Another Ca2+‐sensitizing agent, the thromboxane analogue U46619, caused a similar increase in AA and DAG levels in rabbit pulmonary artery. 3. In permeabilized smooth muscle at constant [Ca2+](pCa 6.5) GTP gamma S‐induced AA and DAG release preceded force development and GTP gamma S (50 microM, 10 min) increased AA mass to 61‐88 microM. 4. Phorbol‐12,13‐dibutyrate (PDBu), another Ca2+‐sensitizing agent, also increased both AA and DAG levels in permeabilized smooth muscle at pCa 6.5, whereas the inactive analogue, 4 alpha‐phorbol, did not have a Ca2+‐sensitizing effect, nor did it increase AA and DAG levels. 5. In the virtual absence of Ca2+ (pCa > 8) GTP gamma S also increased AA and DAG levels by 3.5‐ and 1.6‐fold, respectively. The effect of free Ca2+ itself on AA and DAG release was modest in the physiological range (pCa 7.0 to pCa 6.0), but pCa 4.5 caused an approximately 3‐ to 4‐fold increase in AA and DAG levels, compared with the levels at pCa 8. In permeabilized ileum smooth muscle maintained at constant [Ca2+] (pCa 6.0), carbachol also significantly increased AA to 1.75 times its original value within 1 min of its application. 6. Our results are consistent with, although do not prove, the roles of AA and DAG as second and/or co‐messenger(s) in smooth muscle, while the increases in AA and DAG levels induced by PDBu raise the possibility that they contribute to some of the cellular effects of phorbol esters.
Intracellular spermine confers rectification on rat calcium‐permeable AMPA and kainate receptors.
Authors: Kamboj S K , Swanson G T , Cull-Candy S G
Published: December 2014

1. Whole‐cell recordings were made from cerebellar granule cells cultured in high‐K+ medium to induce expression of Ca(2+)‐permeable AMPA receptors. Current‐voltage (I‐V) plots of agonist‐evoked responses showed varying degrees of inward rectification, but became linear within 5‐10 min. 2. Recombinant Ca(2+)‐permeable kainate receptors, composed of GluR6(Q)/KA‐2 subunits, exhibited rectifying whole‐cell I‐V plots that became linear in outside‐out patches. 3. Loss of rectification in granule cells was prevented by including 100 microM spermine in the pipette; the degree of rectification was then correlated with Ca2+ permeability. 4. Spermine also prevented loss of rectification in patches containing GluR6(Q)/KA‐2 receptors (IC50, 1.7 microM). 5. We suggest that spermine, or a similar cellular constituent, may act as a cytoplasmic factor conferring inward rectification on Ca(2+)‐permeable non‐NMDA receptors, and that ‘washout’ of this factor underlies the observed loss of rectification.
Stretch‐induced enhancement of contractions in uterine smooth muscle of rats.
Authors: Kasai Y , Tsutsumi O , Taketani Y , Endo M , Iino M
Published: December 2014

1. We studied the effect of servo‐controlled stretch of smooth muscle strips from rat uterus on tension and intracellular Ca2+ concentration ([Ca2+]i, using fura‐2 as an indicator) at 30 degrees C. 2. When quiescent uterine muscle strips were stretched at a ramp time of 0.5 s by multiples of 5% of the resting muscle length (L0) up to 40%, forty‐two out of sixty muscle strips responded with a transient active contraction and a [Ca2+]i increase. The minimum excursion of stretch required for contraction was 26.3 +/‐ 7.5% of L0 (mean +/‐ S.D.). The peak response had an all‐or‐none property and was almost independent of the duration of stretch. 3. Stretches of 30 or 35% of L0 induced contraction in most cases when rapidly applied in 0.2‐0.5 s, but slowly applied stretch (ramp duration of 5‐10 s) rarely induced contraction. 4. The stretch‐induced response was inhibited by the removal of extracellular Ca2+ or by the addition of 10 nM nicardipine. However, it was unaffected by 1 microM tetrodotoxin, 1 microM atropine or by 10 microM cyclopiazonic acid, an inhibitor of Ca2+‐ATPase in intracellular Ca2+ stores. 5. When a stretch of 15‐35% of L0 was applied during the relaxation phase of 10 nM oxytocin‐induced rhythmic contractions, the first contraction after the stretch occurred earlier than that expected from the control rhythm. However, the frequency of the subsequent rhythm returned to almost the control level even during continued application of stretch, although the half‐width of rhythmic contractions was increased during stretch. 6. The present study demonstrates that stretch of uterine muscle induces a transient contraction due to Ca2+ influx, which is myogenic and dependent on the excursion and velocity of stretch. The all‐or‐none property of the stretch‐induced contractions suggests initiation of Ca2+ spikes. Furthermore, stretch modulates the oxytocin‐induced rhythmic contractions.
Action potential generation in rat slow‐ and fast‐twitch muscles.
Authors: Wood S J , Slater C R
Published: December 2014

1. In skeletal muscle fibres, voltage‐gated sodium channels are concentrated at the neuromuscular junction. The effect of this accumulation of sodium channels on action potential generation was investigated in rat slow‐ and fast‐twitch muscle fibres. 2. Intracellular microelectrodes were used to generate and record action potentials, from an imposed membrane potential of ‐75 and ‐90 mV, in junctional and extrajunctional regions of the muscle fibre. To identify junctional regions, preparations were incubated with 5 x 10(‐7) M d‐tubocurarine (dTC) to block muscle contraction in response to nerve stimulation whilst allowing endplate potentials (EPPs) to be recorded. Injection of rectangular depolarizing current pulses initiated action potentials at the endplate with threshold values several millivolts lower than those generated elsewhere in the fibre. In addition, the maximum rate of rise of the action potential was greater at the endplate than in extrajunctional regions. 3. In other muscles, neuromuscular transmission was partially blocked with dTC (2 x 10(‐7) M), such that repetitive nerve stimulation evoked action potentials and EPPs in the same fibre. The threshold of these nerve‐evoked action potentials was approximately 50% lower than values derived from action potentials generated by current injection. 4. It is concluded that the threshold for action potential generation is significantly lower at the neuromuscular junction than in extrajunctional regions of skeletal muscle fibres. Furthermore, nerve‐evoked current is more effective at generating an action potential than is injected current.
Activation mechanism of Ca(2+)‐sensitive transient outward current in rabbit ventricular myocytes.
Authors: Kawano S , Hirayama Y , Hiraoka M
Published: December 2014

1. The mechanism of activation of the Ca(2+)‐sensitive and 4‐aminopyridine (4‐AP)‐insensitive transient outward current, I(to)(Ca), was examined in single rabbit ventricular myocytes using the whole‐cell patch‐clamp technique. 2. When the steady‐state intracellular Ca2+ (Ca2+i) concentration ([Ca2+]i) was < 1 nM, I(to)(Ca) could not be activated by applying pulses at 0.1 Hz. When [Ca2+]i was increased to > or = 10 nM, I(to)(Ca) was activated by 0.1 Hz depolarizing pulses in all control experiments. 3. I(to)(Ca) was completely blocked by an anion transport blocker, DIDS, or by replacement of NaCl with sodium aspartate. Upon changing extracellular [Cl‐], the reversal potential was shifted as predicted for a chloride‐selective conductance. When intracellular K+ was replaced with Cs+, I(to)(Ca) was also observed. From these results it was concluded that I(to)(Ca) was carried by Cl‐. 4. Anion selectivity of I(to)(Ca) was investigated by the replacement of C.‐ with various anions. The sequence of permeability was SCN‐ > I‐ > Br‐ > Cl‐. 5. The amplitude of I(to)(Ca) was enhanced in a [Ca2+]i‐dependent manner between 10 nM and 1 microM Ca2+i, while steady‐state inactivation curves and the voltage‐dependent activation curves were unchanged. The half‐inactivation and half‐activation potentials were ‐35 mV and +37 mV, respectively, at all [Ca2+]i. 6. I(to)(Ca) was inhibited by blocking Ca2+ influx or Ca2+ release from sarcoplasmic reticulum, suggesting that a ‘Ca(2+)‐induced Ca(2+)‐release’ mechanism is essential for the activation of I(to)(Ca). 7. A steady‐state Ca(2+)‐activated Cl‐ current with a linear I‐V relationship was observed at 1 microM Ca2+, while the current activated by depolarization was strictly dependent on Ca2+ entry or Ca2+ release from the sarcoplasmic reticulum. These results suggest that the I(to)(Ca) channel is purely ligand (Ca2+) gated and its time course reflects the concentration of Ca2+i.
Characteristics of Na(+)‐Ca2+ exchange in frog skeletal muscle.
Authors: Hoya A , Venosa R A
Published: December 2014

1. Fluxes studies were carried out to investigate the Na(+)‐dependent outward movement of Ca2+ in intact frog sartorius muscle from Leptodactylus ocellatus, a preparation for which published data on the subject are sparse. 2. Under normal resting conditions the Na(+)‐Ca2+ exchange was not readily detectable. 3. When muscles were exposed to 4 mM caffeine, the rate of fractional loss of Ca2+ (kCa,o) increased by about 50%. Most of this increase exhibits characteristics typical of the Na(+)‐Ca2+ antiport working in the forward mode found in other cells. 4. The increase in kCa,o promoted by caffeine was decreased by: (a) 72% in the absence of external Na+ (Nao+); (b) 73% in Na(+)‐loaded muscles ([Na+]i = 98 mM); (c) 70% when fibres were depolarized to ‐27 mV ([K+]o = 50 mM); and (d) 80% in the presence of 5 mM amiloride. 5. Ni2+ (5 mM), an inhibitor of the Na(+)‐Ca2+ exchanger current, unexpectedly increased the caffeine‐promoted rise in kCa,o. This effect of Ni2+ was associated with a concomitant caffeine‐stimulated Ni2+ influx. In the absence of caffeine Ni2+ did not affect kCa,o. 6. It was concluded that: (a) under resting conditions the sarcolemmal Ca2+ pump suffices to handle the cytosolic calcium concentration ([Ca2+]i); (b) Na(+)‐Ca2+ activity becomes apparent when [Ca2+]i is substantially increased by caffeine‐induced Ca2+ release from the sarcoplasmic reticulum; and (c) the blocking effect of Ni2+ on the current generated by a Na(+)‐Ca2+ exchange with a coupling ratio > 2 might actually represent a shift of the antiport mode toward an electroneutral 1 Ni(2+)‐1Ca2+ exchange.
Failure of P‐glycoprotein (MDR1) expressed in Xenopus oocytes to produce swelling‐activated chloride channel activity.
Authors: Morin X K , Bond T D , Loo T W , Clarke D M , Bear C E
Published: December 2014

1. P‐glycoprotein, the protein product of the multidrug resistance (MDR1) gene, has ATP‐dependent transporter activity. It has been suggested that P‐glycoprotein may also function as a volume‐regulated chloride channel or chloride channel regulator. To assess the chloride channel function of P‐glycoprotein, we examined swelling‐activated chloride conductances in Xenopus oocytes injected with human MDR1 cRNA. 2. Functional expression of P‐glycoprotein in Xenopus oocytes was confirmed using Western blot analysis and by assessing transport of the P‐glycoprotein substrate, calcein AM. 3. Endogenous, swelling‐activated chloride conductances were virtually absent by the time P‐glycoprotein expression was confirmed. Thus, this expression system afforded the advantage of assessing putative MDR1‐associated chloride currents in the absence of background currents. 4. The currents activated by hypotonic shock (50%) in both MDR1‐injected and control (water‐injected) oocytes were not significantly different. The swelling response was due in part to the activation of a potassium‐selective conductance which could be inhibited by barium. No chloride‐selective currents were activated by hypotonic shock in the presence or absence of barium. Therefore, we conclude that P‐glycoprotein expression does not produce a swelling‐activated chloride conductance in the Xenopus oocyte expression system.
Reciprocal inhibition between wrist flexors and extensors in man: a new set of interneurones?
Authors: Aymard C , Chia L , Katz R , Lafitte C , Pénicaud A
Published: December 2014

1. Interneurones mediating reciprocal inhibition between wrist flexors and extensors in man are characterized using both Renshaw cells and transarticular group I afferent activation. 2. Renshaw cells were activated by reflex discharges evoked by a tendon tap. The tendon tap was applied to the tendon of the muscles from which the Ia fibres responsible for the reciprocal inhibition originated. Contrary to what was observed both in the cat hindlimb and in human elbow muscles, this Renshaw cell activation never resulted in a long depression of the reciprocal inhibition between wrist flexors and extensors. 3. Convergence from group I elbow muscle afferents and antagonistic group I afferents onto interneurones mediating reciprocal inhibition between wrist muscles was revealed in post‐stimulus time histogram (PSTH) experiments using the technique of spatial facilitation. 4. The characteristics of the interneurones mediating reciprocal inhibition between wrist flexors and extensors could therefore be summarized as follows: (a) they are fed by antagonistic group I afferents and group I afferents originating from both flexor and extensor elbow muscles; (b) they are not inhibited by Renshaw cells; (c) they are not excited by low threshold cutaneous afferents; and (d) they are probably interposed in a disynaptic pathway. 5. It is therefore concluded that interneurones mediating reciprocal inhibition between wrist flexors and extensors in man differ both from Ia interneurones and from interneurones interposed in the Ib reflex pathways and these characteristics are related to the complex circumduction movements developed in the wrist.
The effects of caffeine on intracellular calcium, force and the rate of relaxation of mouse skeletal muscle.
Authors: Allen D G , Westerblad H
Published: December 2014

1. Intracellular calcium concentration ([Ca2+]i) and force were measured from isolated single fibres of mouse skeletal muscle. The effects of 5 mM caffeine on muscle fibres at rest and during short tetani were examined. 2. Caffeine increased tetanic tension and slowed the rate of relaxation. [Ca2+]i was increased in the presence of caffeine both in the resting muscle and during tetani. The time course of decline of [Ca2+]i after a tetanus is complex with a large, early, rapid phase followed by a smaller and slower phase. Caffeine accelerated the early phase but slowed the later phase. 3. The sensitivity of the myofibrillar proteins to Ca2+ measured in the intact fibre was increased in the presence of caffeine, confirming earlier findings on skinned muscle fibres. 4. Analysis of the late phase of the decline of [Ca2+]i after a tetanus provides information about the properties of the sarcoplasmic reticulum (SR) Ca2+ pump. Caffeine slowed the pump to 60‐70% of the control value at a given [Ca2+]i but had no effect on the Ca2+ leak from the SR. 5. Analysis of relaxation made use of the Ca(2+)‐derived force in which the [Ca2+]i during relaxation was converted to the Ca(2+)‐derived force by means of the steady‐state relation between [Ca2+]i and force. The Ca(2+)‐derived force fell more slowly in the presence of caffeine but the lag between Ca(2+)‐derived force and measured force was unaffected. Thus, the slowed relaxation was caused by changes in Ca2+ handling and not by slowed cross‐bridge kinetics. 6. A model of the Ca2+ movements and force production of muscle was used to examine independently the effects of increased Ca2+ sensitivity, slowing of the SR Ca2+ pump and increased SR Ca2+ permeability. The effects of caffeine on [Ca2+]i, tetanic force and relaxation could be explained by a combination of these three effects.
Effects of divalent cations on exocytosis and endocytosis from single mouse pancreatic beta‐cells.
Authors: Proks P , Ashcroft F M
Published: December 2014

1. The effects of the divalent cations Ca2+, Ba2+ and Sr2+ on exocytosis and endocytosis from single isolated mouse pancreatic beta‐cells were investigated by monitoring changes in cell capacitance. 2. The immediate increase in capacitance elicited by a single depolarization from ‐70 to +20 mV was dependent on the divalent cation species, with Ca2+ (8.2 +/‐ 1.1 fF pC‐1) > Ba2+ (1.0 +/‐ 0.2 fF pC‐1) > Sr2+ (0.7 +/‐ 0.2 fF pC‐1) in perforated‐patch recordings. 3. In Ba2+ solutions alone there was subsequently an additional slow increase in capacitance (to 4.3 +/‐ 1.1 fF pC‐1). This second phase of exocytosis was unaffected by preincubation with colcemid (20 microM, 45 min) or cytochalasin D (10 microM, 15 min), suggesting that interaction of secretory granules with microtubules or microfilaments is not involved. 4. An increase in cell capacitance was elicited by depolarization in Ba2+ solutions when intracellular Ca2+ was buffered with 10 mM EGTA. Infusion of the beta‐cell with Ba2+ also stimulated exocytosis although the rate was much slower (1.1 +/‐ 0.2 fF s‐1; 8 microM free Ba2+) than for Ca2+ (39 +/‐ 5 fF s‐1; 2 microM free Ca2+). These data indicate that Ba2+ does not evoke secretion by promoting Ca2+ release from internal stores. 5. The lower efficacy of Ba2+ in supporting exocytosis may be related to the fact that this cation does not activate calmodulin‐dependent processes and the slow second phase of secretion may result from this ion being removed only slowly from the cytoplasm. 6. Endocytosis was faster in Sr2+ than in Ca2+ or Ba2+ solution, and the speed increased when the external concentration of all three divalent cation species was raised. The ability of Ba2+ to support endocytosis suggests calmodulin‐dependent processes are not involved. These data suggest membrane retrieval is regulated differently from exocytosis in beta‐cells.
5‐HT2 receptor‐controlled modulation of medullary respiratory neurones in the cat.
Authors: Lalley P M , Bischoff A M , Schwarzacher S W , Richter D W
Published: December 2014

1. The effects of the 5‐HT2 receptor agonist alpha‐methyl‐5‐HT were studied on the membrane of expiratory (E2) and post‐inspiratory (PI) neurones, by intracellular recordings in the caudal medulla of anaesthetized cats. 2. Ionophoresis of alpha‐Me‐5‐HT depolarized membrane potential and increased action potential frequency in a majority of neurones tested. Depolarization of neurones by alpha‐Me‐5‐HT was accompanied by increased input resistance throughout all phases of the respiratory cycle. These effects were antagonized by ionophoresis of cinanserin, a receptor‐blocking agent with high affinity for 5‐HT2 receptors. 3. E2 neurones were voltage clamped to measure membrane current changes induced by alpha‐Me‐5‐HT ionophoresis. alpha‐Me‐5‐HT induced a net inward current by reducing inspiratory‐phase outward currents and increasing expiratory‐phase inward currents. These changes were equivalent with steady membrane depolarization, decreased inspiratory phase membrane hyperpolarization and increased expiratory drive potential recorded from the same neurones in current clamp. 4. The effects of alpha‐Me‐5‐HT are consistent with activation of 5‐HT2 receptors on E2 and PI neurones leading to blockade of synaptically activated and persistent conductances to potassium ions.
Excitatory synaptic potentials dependent on metabotropic glutamate receptor activation in guinea‐pig hippocampal pyramidal cells.
Authors: Bianchi R , Wong R K
Published: December 2014

1. Intracellular and extracellular recordings of CA1 and CA3 neurones were performed in guinea‐pig hippocampal slices to examine synaptic activities dependent on metabotropic glutamate receptors (mGluRs). 2. Long burst activities were elicited by 4‐aminopyridine in the presence of ionotropic glutamate receptor and GABAA receptor blockers (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and 3‐(RS‐2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid, and picrotoxin). Long bursts were also elicited by alpha‐dendrotoxin. 3. Long bursts consisted of a 5‐25 s depolarization with overriding action potentials and occurred rhythmically at intervals ranging from 1 to 20 min. Long bursts were generated in a population of CA3 neurones and the synchronized output elicited long bursts in CA1 cells. Depolarizing potentials underlying long bursts in CA1 cells had a reversal potential of ‐14.8 +/‐ 5.1 mV. 4. Long burst‐associated depolarizations in CA1 neurones were suppressed by local application of L‐(+)‐2‐amino‐3‐phosphonopropionic acid (L‐AP3) and of the phenylglycine derivatives (+)‐alpha‐methyl‐4‐carboxyphenylglycine ((+)‐MCPG), S‐4‐carboxyphenylglycine (S‐4CPG) and S‐4‐carboxy‐3‐hydroxyphenylglycine (S‐4C3HPG). (‐)‐MCPG or atropine application did not affect the long burst‐associated depolarization. 5. Bath perfusion of (+)‐MCPG (0.5 mM), S‐4CPG (0.5 mM), S‐4C3HPG (0.5 mM) or L‐AP3 (1 mM) blocked the occurrence of long bursts. 6. The results suggest that the long burst‐associated depolarizations are synaptic potentials dependent on mGluR activation. Activation of mGluRs may also be involved in the generation of synchronized long bursts in the CA3 region.
Heterogeneity of contraction‐induced effects in neurons of the cat dorsal spinocerebellar tract.
Authors: Zytnicki D , Lafleur J , Kouchtir N , Perrier J F
Published: December 2014

1. Clarke's column neurons of the dorsal spinocerebellar tract (DSCT) were recorded intracellularly in anaesthetized cats during weak sustained contractions of triceps surae (TS) produced by direct electrical stimulation of the muscle. 2. Of 145 DSCT neurons, 77 (53%) were contraction sensitive suggesting that information about weak contraction of a limited number of muscles is widely distributed among DSCT neurons. Four types of effects were observed in individual neurons during TS contractions. 3. In the first group of 11 DSCT neurons (14% of the contraction‐sensitive cells), the effect was excitation persisting throughout the duration of contractions. These responses were ascribed to actions of afferents from contraction‐activated tendon organs. 4. In a second group of 15 neurons (20% of the contraction‐sensitive cells), quickly declining excitatory potentials were recorded during sustained TS contractions. By analogy with previous observations of contraction‐induced effects in motoneurons, the decline of excitation might be explained by contraction‐induced presynaptic inhibition of group I afferents in Clarke's column. 5. Declining inhibitions, resembling those previously observed in homonymous and synergic motoneurons, were recorded in 49% of contraction‐sensitive DSCT neurons. This appears in keeping with the fact that interneurons mediating Ib inhibition to motoneurons project axon collaterals to DSCT neurons. Presynaptic inhibition of Ib fibres might therefore cause parallel reductions of inhibitory potentials in motoneurons and in DSCT neurons. 6. In a final group of 13 neurons, mixed excitatory and inhibitory effects were observed during TS contractions. Such DSCT neurons might monitor the excitability of Ib interneurons by integration of information about input to and output from these neurons. 7. The non‐uniform patterns of DSCT responses to TS contractions suggest complex processing of information on ankle extensor activity in cerebellum. Phasic signalling of contraction onset is observed in many DSCT neurons while others carry messages about duration and strength of contraction.
Lactate and H+ uptake in inactive muscles during intense exercise in man.
Authors: Bangsbo J , Aagaard T , Olsen M , Kiens B , Turcotte L P , Richter E A
Published: December 2014

1. The present study examined how uptake of lactate and H+ in resting muscle is affected by blood flow, arterial lactate concentration and muscle metabolism. 2. Six males subjects performed intermittent arm exercise in two separate 32 min periods (Part I and Part II) and in one subsequent 20 min period in which one leg knee‐extensor exercise was also performed (Part III). The exercise was performed at various intensities in order to obtain different steady‐state arterial blood lactate concentrations. In the inactive leg, femoral venous blood flow (draining about 7.7 kg of muscles) was measured and femoral arterial and venous blood was collected frequently. Biopsies were taken from m. vastus lateralis of the inactive leg at rest and 10 and 30 min into both Part I and Part II as well as 10 min into recovery from Part II. 3. The arterial plasma lactate concentrations were 7, 9 and 16 mmol l‐1 after 10 min of Parts I, II and III, respectively, and the corresponding arterial‐venous difference (a‐vdiff) for lactate in the resting leg was 1.3, 1.4 and 2.0 mmol l‐1. The muscle lactate concentration was 2.8 mmol (kg wet wt)‐1 after 10 min of Part I and remained constant throughout the experiment. During Parts I and II, a‐vdiff lactate decreased although the arterial lactate concentration and plasma‐muscle lactate gradient were unaltered throughout each period. Thus, membrane transport of lactate decreased during each period. 4. Blood flow in the inactive leg was about 2‐fold higher during arm exercise compared to the rest periods, resulting in a 2‐fold higher lactate uptake. Thus, lactate uptake by inactive muscles was closely related to blood flow. 5. Throughout the experiment a‐vdiff for actual base excess and for lactate were of similar magnitude. Thus, in inactive muscles lactate uptake appears to be coupled to the transport of H+.
Deamination of amino acids as a source for ammonia production in human skeletal muscle during prolonged exercise.
Authors: van Hall G , van der Vusse G J , Söderlund K , Wagenmakers A J
Published: December 2014

1. The influence of pre‐exercise muscle glycogen content on ammonia production, adenine nucleotide breakdown and amino acid metabolism was investigated during prolonged exercise in six subjects having one leg with a normal and one leg with a low muscle glycogen content. One‐leg knee‐extensor exercise was performed for 90 min, at a workload of 60‐65% of the maximal power output, first with one leg and then with the other. 2. During exercise ammonia was released in gradually increasing amounts and plateaued after 1 h exercise at a rate of approximately 80 mumol min‐1. The total ammonia production was 9.1 +/‐ 0.4 and 9.5 +/‐ 1.4 mmol (kg dry muscle)‐1 in the normal and low glycogen content leg, respectively. 3. Levels of muscle phosphocreatine (PC), total adenine nucleotides and inosine monophosphate (IMP) were similar at rest and after 90 min of exercise. 4. Only minor differences were observed between rest and exercise and between legs for the muscle concentrations of glutamine, alanine and the branched‐chain amino acids. Muscle glutamate concentration decreased by 60‐70% within the first 10 min of exercise. Glutamate consumption over 90 min quantitatively equalled ammonia production. Most of the glutamate was consumed within the first 10 min of exercise, while ammonia production gradually increased during exercise. Therefore deamination of glutamate cannot be the main source of ammonia production during the later stage of exercise. 5. It is concluded that during prolonged one‐leg exercise at moderate intensity: (a) ammonia production is not affected by pre‐exercise muscle glycogen content, (b) ammonia production exceeds by far the breakdown of adenine nucleotides to IMP and therefore has to be derived from alternative sources, and (c) deamination of amino acids is a likely source of ammonia production during prolonged exercise.
Distinct omega‐agatoxin‐sensitive calcium currents in somata and axon terminals of rat supraoptic neurones.
Authors: Fisher T E , Bourque C W
Published: December 2014

1. Voltage‐dependent calcium currents were measured at room temperature using whole‐cell patch clamp in acutely isolated somata and axon terminals of the magnocellular neurosecretory cells (MNCs) from the rat supraoptic nucleus. 2. Administration of omega‐agatoxin IVA (omega‐Aga IVA) blocked a high‐threshold non‐inactivating current. This current has an IC50 for omega‐Aga IVA of 3 nM; no other types of currents were blocked at doses of up to 500 nM. 3. In the axon terminals omega‐Aga IVA blocked a high‐threshold current that inactivates markedly (tau approximately 448 ms), and has a much lower sensitivity to the toxin, with an IC50 of 270 nM. Unlike the somatic current, the effect of omega‐Aga IVA in the terminals is largely prevented by omega‐conotoxin GVIA (omega‐CgTX). 4. These data suggest that MNC somata express a single type of omega‐Aga IVA‐sensitive calcium current similar to the P‐type calcium current described in other cells. However, the omega‐Aga IVA‐sensitive current in axon terminals differs from both the P‐type and the recently identified Q‐type current in that it is also sensitive to omega‐CgTX. The distinct biophysical properties of the currents in somata and axon terminals may have important physiological implications.
Osmo‐ and mechanosensitivity of the transient outward K+ current in a mammalian neuronal cell line.
Authors: Schoenmakers T J , Vaudry H , Cazin L
Published: December 2014

1. The transient outward current in NG108‐15 cells was investigated with the whole‐cell patch‐clamp technique. The current was inhibited by external 4‐aminopyridine or tetra‐ethylammonium. The reversal potential shifted rightward with increased external K+ concentrations. 2. Current inactivation was markedly accelerated in hyperosmotic media (+30 mosmol l‐1) and after nearby ejection of isosmotic solution with maximal acceleration occurring after 15‐20 s and full recovery within 2‐4 min, thus demonstrating an osmo‐ and mechanosensitivity of this current. Voltage‐dependent Na+ and Ca2+ currents were unaffected. 3. Hyperosmotic solution shifted the voltage dependence of inactivation leftward. Inactivation was sensitive to reducing and oxidizing intracellular conditions. Reduction blocked the acceleration of current inactivation induced by hyperosmotic media, while oxidation did not hamper the response. 4. Action potentials had a decreased amplitude and a slower repolarization after hyperosmotic ejections. 5. It is concluded that the transient K+ current is osmo‐ and mechanosensitive, thus providing a mechanism for extracellular osmolarity to modulate neuronal excitability. The response appeared to be mediated through a changed sensitivity of the inactivating principle to the membrane electric field and was dependent on the redox state of the cell.
Spindle oscillation in cats: the role of corticothalamic feedback in a thalamically generated rhythm.
Authors: Contreras D , Steriade M
Published: December 2014

1. Spindles represent an oscillatory activity (7‐14 Hz) of the electroencephalogram (EEG) originating in the thalamus and appearing during early stages of sleep. We investigated: (i) the phase relations between thalamic and cortical neurons during this rhythm; (ii) the patterns of spindles under different anaesthetics and their modifications at various levels of the membrane potential (Vm); and (iii) the potentiating role of the corticothalamic feedback in the genesis of spindles. Intra‐ and extracellular recordings were performed in cats from reticular and dorsal thalamic nuclei, as well as from various cortical areas. 2. In thalamic reticular neurons, spindles were sequences of waves at 7‐14 Hz, riding on a prolonged depolarizing plateau and occurring in phase with depth‐negative cortical EEG waves. In thalamocortical cells, spindles consisted of inhibitory postsynaptic potentials (IPSPs) in phase with depth‐positive cortical EEG waves and occasionally leading to rebound spike bursts. In cortical cells, spindle waves were rhythmic (7‐14 Hz) excitatory postsynaptic potentials (EPSPs) that sometimes gave rise to action potentials. Spindles occurred in phase among thalamic reticular, thalamocortical and neocortical neurons. 3. In thalamic reticular neurons, spindle waves and their depolarizing plateaux increased in amplitude with slight cellular hyperpolarization, but at a Vm more negative than ‐80 or ‐85 mV they decreased in amplitude. No frequency alterations were observed with these Vm changes. 4. The waxing‐and‐waning pattern of spontaneous spindles under barbiturate anaesthesia was distinct from the waning pattern under ketamine‐xylazine anaesthesia. Under all anaesthetics, spindles had a waning pattern when elicited by cortical stimuli. The amplitude of cortical‐evoked spindle waves diminished with the decrease in stimulation intensity. 5. Under urethane or ketamine‐xylazine anaesthesia, spindle sequences were grouped by a cortically generated slow oscillation (< 1 Hz) and were preceded by a depth‐positive EEG wave that corresponded to a prolonged hyperpolarization in all three investigated (cortical, thalamic reticular, and thalamocortical) cellular types. 6. We propose that the waxing pattern of spindle oscillation is due to a progressive entrainment of units into the oscillation until a maximum number is reached, depending on the background activity in the network. The phase relations between cortical, thalamic reticular and thalamocortical neurons are ascribed to distributed excitatory signals from thalamocortical neurons to both cortical and reticular neurons at each cycle of the oscillation. In turn, cortical neurons provide a powerful drive to potentiate the genesis of thalamic spindles.
The role of nitric oxide in the regulation of macromolecular transport in rat jejunum.
Authors: Kimm M H , Hardin J A , Gall D G
Published: December 2014

1. Nitric oxide is known to affect epithelial and microvascular permeability and is a major non‐adrenergic non‐cholinergic neurotransmitter in the intestine. We have previously demonstrated neuronal regulation of macromolecular transport in the intestine. To define this regulation further the role of nitric oxide was investigated. 2. Stripped rat jejunum was mounted in Ussing chambers exposing the mucosal surface to bovine serum albumin (BSA; 2 mg ml‐1), or BSA (2 mg ml‐1) plus [125I]BSA (10 microCi). Following a 50 min equilibration, serosal fluids were sampled for four 10 min periods, and fluxes determined for intact BSA by enzyme‐linked immunosorbent assay (ELISA) and total BSA by [125I]BSA under basal conditions, and after treatment with NG‐nitro‐L‐arginine‐methyl ester (L‐NAME) alone or in conjunction with L‐arginine or decarboxylated molsidomine (SIN 1). 3. L‐NAME significantly increased intact BSA uptake. Total (intact + degraded) BSA flux was not altered. The L‐NAME effect was reversed by L‐arginine and SIN 1. Additional experiments were performed by adding the nitric oxide donors sodium nitroprusside and SIN 1 directly to control tissue. Nitric oxide donors did not further decrease intact BSA flux below levels obtained from control tissue. The L‐NAME enantiomer D‐NAME had no effect. Sodium‐free bathing solutions also had no effect on intact BSA uptake. Non‐specific permeability, as assessed by the serosal to mucosal movement of [51Cr]ethylene‐diamine‐tetraacetate ([51Cr]EDTA), was decreased with L‐NAME. 4. The findings indicate that nitric oxide downregulates intact macromolecular flux in the small intestine.
Dissociation of subsarcolemmal from global cytosolic [Ca2+] in myocytes from guinea‐pig coronary artery.
Authors: Ganitkevich V Y , Isenberg G
Published: December 2014

1. Changes in cytosolic Ca2+ concentration (delta[Ca2+]c) were measured by global indo‐1 fluorescence and compared with changes in subsarcolemmal Ca2+ concentration (delta[Ca2+]sl) indicated by Ca(2+)‐activated K+ currents (IK(Ca)). 2. At ‐50 mV holding potential, 10mM caffeine increased both IK(Ca) and [Ca2+]c without measurable delay. While IK(Ca) peaked within 0.3 +/‐ 0.16 s (mean +/‐ S.D.) and decayed to 50% within 0.4 +/‐ 0.2 s, delta[Ca2+]c peaked within 1.5 +/‐ 0.5 s and decayed to 50% within 5.2 +/‐ 1.0 s. The different time courses support the idea that [Ca2+]sl and [Ca2+]c deviate. 3. When 10 mM caffeine was applied 20 s after an initial 2 s caffeine application, IK(Ca) was suppressed to 22 +/‐ 5% and delta [Ca2+]c to 40 +/‐ 4%. During the following 1 min caffeine‐free period, IK(Ca) recovered to 61 +/‐ 7% while delta [Ca2+]c remained at 40 +/‐ 3%. The differences between IK(Ca) and delta[Ca2+]c suggest that Ca2+ deprivation and Ca2+ refilling is faster in peripheral than in central sarcoplasmic reticulum (SR). 4. During the loading period of indo‐1, a spontaneous delta[Ca2+]c of 30‐80 nM appeared both at ‐50 mV and at more positive potentials. The amplitude of spontaneous delta[Ca2+]c increased with the amplitude, the frequency or the fusion of spontaneous transient outward currents (STOCs). 5. Block of sarcolemmal Ca2+ fluxes by 1 mM La3+ increased [Ca2+]c by 250 +/‐ 100 nM and suppressed the spontaneous delta[Ca2+]c. However, La3+ did not significantly retard the rate of decay of STOCs which may therefore be limited by Ca2+ diffusion into the cytosol and not by Ca2+ extrusion. 6. The dissociation of IK(Ca) (or STOCs) and delta[Ca2+]c may indicate a Ca2+ concentration gradient during Ca2+ release directed from the sarcolemma towards the centre of the cell, which later reverses direction.
Statistical analysis of amplitude fluctuations in EPSCs evoked in rat CA1 pyramidal neurones in vitro.
Authors: Stricker C , Field A C , Redman S J
Published: December 2014

1. EPSCs were evoked in CA1 pyramidal neurones of young rats in vitro by extracellular stimulation of axons in a restricted stratum radiatum field, and were recorded using the whole‐cell technique. 2. Quantal fluctuations in EPSC amplitude could be demonstrated for nineteen of fifty EPSCs analysed. Quantal currents (at the soma) ranged from 2.6 to 9.5 pA (after correction for the access resistance) with a mean of 4.0 +/‐ 2.0 pA. 3. Quantal variance was negligible for the majority (13/19) of the EPSCs. However, a large quantal variance (with a coefficient of variation > 0.4) is one possible reason why a large number of the EPSCs (29/50) could not be shown to have quantal fluctuations. 4. The statistical pattern of fluctuations in the amplitude of the majority of the quantal EPSCs (18/19) could not be described by conventional models of transmitter release. 5. The time course of the EPSC and a compartmental model of CA1 pyramidal neurones were used to calculate synaptic location. The quantal current (at the soma) was independent of the electrotonic location of the synapse at which it was evoked. The peak quantal conductance generating each quantal current ranged from 0.5 to 6.8 nS (mean 1.3 +/‐ 1.4 nS), its magnitude increasing with distance from the soma. The mean peak conductance is likely to be generated by the opening of at least 60‐160 AMPA channels.
Activation of L‐arginine transport (system y+) and nitric oxide synthase by elevated glucose and insulin in human endothelial cells.
Authors: Sobrevia L , Nadal A , Yudilevich D L , Mann G E
Published: December 2014

1. Modulation of L‐arginine transport (system y+) and release of nitric oxide (NO) and prostacyclin (PGI2) by elevated glucose and insulin were investigated in human cultured umbilical vein endothelial cells. 2. Elevated glucose induced a time‐ (6‐12 h) and concentration‐dependent stimulation of L‐arginine transport, which was reversible and associated with a 3‐fold increase in intracellular cGMP accumulation (index of NO synthesis) and 75% decrease in PGI2 production. 3. Elevated glucose had no effect on the initial transport rates for L‐serine, L‐citrulline, L‐leucine, L‐cystine or 2‐deoxyglucose. 4. Resting membrane potential was unaffected by elevated glucose whereas basal intracellular [Ca2+] increased from 65 +/‐ 5 nM to 136 +/‐ 16 nM. 5. Insulin induced a protein synthesis‐dependent stimulation of L‐arginine transport and increased NO and PGI2 production in cells exposed to 5 mM glucose. 6. In cells exposed to high glucose, insulin downregulated elevated rates of L‐arginine transport and cGMP accumulation but had no effect on the depressed PGI2 production. 7. Our findings suggest that insulin's normal stimulatory action on human endothelial cell vasodilator pathways may be impaired under conditions of sustained hyperglycaemia.