bluuebunny/crossref_metadata_2025 huggingface dataset:
Single channel study of a Ca(2+)‐activated K+ current associated with ras‐induced cell transformation.
Authors: Huang Y , Rane S G
Published: December 2014

1. Ras‐transformed fibroblasts have a whole‐cell Ca(2+)‐activated K+ current which is either absent or unavailable for activation in their non‐transformed counterparts. To better understand the physiological significance of this K+ current the single channel basis for the current was characterized in ras‐transformed cells. 2. More than 90% of inside‐out patches from ras‐transformed balb 3T3 cells had a channel type which was Ca(2+)‐activated (threshold < 0.2 microM internal Ca2+), K(+)‐selective (permeability ratio PNa:PK < 0.02), and inwardly rectifying in symmetric 150 mM KCl solutions (conductances at ‐60 and 60 mV of 33 +/‐ 1 and 17 +/‐ 1 pS respectively). Channel opening probability increased 25‐50% between ‐60 and 60 mV due to an increase in the frequency of opening. Single K+ channels in outside‐out patches were blocked by externally applied 10 mM TEA or 100 nM charybdotoxin, as were whole‐cell Ca(2+)‐activated K+ currents. The properties of this class of K+ channel are sufficient to account for the whole‐cell Ca(2+)‐activated current in ras‐transformed cells. 3. Inside‐out patches from C3H10T1/2 and NIH 3T3 fibroblasts transformed by the H‐ras oncogene had Ca(2+)‐activated K+ channels identical to those observed in K‐ras‐transformed balb 3T3 cells. 4. As predicted from whole‐cell experiments Ca(2+)‐activated K+ channels were not observed in inside‐out patches from non‐transformed balb 3T3 cells. The purpose of the excised patch recordings was, instead, to rule out potential technical complications with the whole‐cell experiments. For instance A23187, which evoked whole‐cell K+ currents in transformed cells, may not have elevated Ca2+ sufficiently to allow K+ channel activation in non‐transformed cells. Another possibility was that trypsin pretreatment used to round‐up cells for whole‐cell recording may have preferentially disabled channels in non‐transformed cells. The first problem was addressed by exposing patches from non‐transformed cells to 100‐1000 microM Ca2+. Excised patches were also taken from non‐transformed cells which had not been exposed to trypsin. K+ channel activity was not observed under either condition. 5. Patches from both ras‐transformed and non‐transformed cells had a type of non‐specific cation channel which was activated at internal Ca2+ concentrations > or = 100 microM. This channel was sensitive to membrane voltage, mean open time increasing from 12 to 72 ms between ‐90 and 90 mV.
Effects of some metal‐ATP complexes on Na(+)‐Ca2+ exchange in internally dialysed squid axons.
Authors: DiPolo R , Beaugé L
Published: December 2014

1. Na(+)o‐dependent Ca2+ efflux (forward Na(+)‐Ca2+ exchange), and in some cases the Na(+)i‐dependent Ca2+ influx (reverse Na(+)‐Ca2+ exchange) were measured in internally dialysed squid axons under membrane potential control. 2. We tested the effect on the Na(+)‐Ca2+ exchange of the MgATP analogue bidentate chromium adenosine‐5'‐triphosphate (CrATP), substrate of several kinases, and cobalt tetrammine ATP (Co(NH3)4ATP), a poor substrate of most kinases. 3. CrATP completely blocked the MgATP and MgATP‐gamma‐S (ATP‐gamma‐S) stimulation of the Na(+)o‐dependent Ca2+ efflux (forward exchange) and the Na+i‐dependent Ca2+ influx (reverse exchange). The analogue only blocked the nucleotide‐dependent fraction of the Na(+)‐Ca2+ exchange without modifying any kinetic parameters of the exchange reactions. 4. The effects of CrATP were fully reversible with a very slow time constant (t 1/2 about 30 min). 5. The MgATP stimulation of the Na(+)‐Ca2+ exchange was completely saturated at 1 mM. Higher MgATP concentrations (up to 15 mM) had no additional effects. Pentalysine (internal or external), the protein kinase C inhibitor H‐7 (1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine) and several calmodulin inhibitors did not inhibit Na(+)‐Ca2+ exchange either in the absence or presence of MgATP. 6. Our results do not agree with the idea of an aminophospholipid translocase being responsible for the ATP stimulation of the Na(+)‐Ca2+ exchange in squid axons; they suggest that this is due to the action of a kinase system.
A large, sustained Na(+)‐ and voltage‐dependent K+ current in spinal neurons of the frog embryo.
Authors: Dale N
Published: December 2014

1. Neurons from the Xenopus embryo spinal cord were dissociated and conventional patch clamp techniques were used to record the whole‐cell currents in the presence of tetrodotoxin (TTX). 2. The outward currents of the acutely isolated spinal neurons were rapidly reduced to about half their control value by substitution of extracellular Na+ with N‐methyl‐D‐glucamine, lysine or choline. 3. The use of Li+ as a Na+ substitute partially reduced the outward currents. 4. The reversal potential of the Na(+)‐sensitive current was close to the K+ equilibrium potential and could be altered by changing extracellular K+. The Na(+)‐sensitive current was therefore a K+ current. 5. The Na(+)‐sensitive K+ current was voltage dependent and activated in a sustained manner and appeared very similar to the delayed rectifier present in these neurons. 6. While the Na(+)‐sensitive current increased with voltage as might be expected for an outward current, at very positive potentials it progressively decreased in amplitude. The voltage range over which this decrease was present moved closer to zero as the levels of intracellular Na+ were increased. The tail currents evoked by positive test potentials did not correspondingly decrease in amplitude, suggesting that channel block was rapidly relieved by stepping back to the holding potential. 7. Intracellular perfusion of the patch pipette with solutions containing varying amounts of Na+ (0‐20 mM) showed that the K+ currents could be increased in a dose‐dependent manner by raising intracellular Na+. The current had an EC50 for Na+ of 7.3 mM and a Hill coefficient of 4.6. 8. Single channel recordings from isolated inside‐out patches revealed a channel that gated more frequently when the bathing levels of Na+ were elevated from 3 to 12 or 50 mM. Xenopus spinal cord neurons therefore possess a current that is not only voltage dependent but is also sensitive to internal Na+. 9. Xenopus spinal neurons possess a transient Na+ current (blocked by the inclusion of TTX) and a leak channel permeable to Na+. The inward leakage of Na+ appeared to provide the Na+ necessary for the gating of the Na(+)‐dependent channel. 10. Blocking the Na(+)‐K+ exchange pumps by removing extracellular K+, reduced the effect of removal of external Na+, suggesting that the Na(+)‐K+ exchange pumps could be important in controlling the submembrane Na+.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of pancreatic polypeptide on insulin action in exocrine secretion of isolated rat pancreas.
Authors: Park H J , Lee Y L , Kwon H Y
Published: December 2014

1. Effects of pancreatic polypeptide (PP) on insulin action in pancreatic exocrine secretion was investigated by using an isolated rat pancreas that was perfused with Krebs‐Henseleit solution containing 2.5 mM glucose, 0.1% bovine serum albumin and 3% Dextran T‐70 at a vascular flow rate of 1.2 ml min‐1. 2. Cholecystokinin‐8 (CCK‐8) at a concentration of 14 pM stimulated basal flow rate and amylase output of the isolated pancreas. Twenty‐five millimolar glucose not only increased the basal flow rate and amylase output but also potentiated the CCK‐stimulated flow rate and amylase output. 3. Porcine insulin, administered intra‐arterially at a concentration of 100 nM, also increased the basal flow rate and amylase output, and also potentiated the CCK‐stimulated flow rate and amylase output. 4. Rat PP, given intra‐arterially at a concentration of 10 pM, completely abolished the potentiation effects of both the 25 mM glucose and the exogenous insulin on the CCK‐stimulated flow rate and amylase output. Rat PP also inhibited the flow rate and amylase output increased by either 25 mM glucose alone or exogenous insulin alone. However, rat PP did not change the flow rate and amylase output stimulated by CCK‐8 alone. 5. These results indicate that insulin is an important stimulatory hormone of pancreatic exocrine secretion, and that PP exerts the inhibitory role in pancreatic exocrine secretion by modulating the insulin action.
Control of inositol polyphosphate‐mediated calcium mobilization by arachidonic acid in pancreatic acinar cells of rats.
Authors: Maruyama Y
Published: December 2014

1. The patch‐clamp technique of whole‐cell current recording was applied to single, enzymatically isolated, rat pancreatic acinar cells to investigate the current responses evoked by internal perfusion of inositol polyphosphates (InsPx). The InsPx were included in the solution filling the recording pipette and inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3; 10 microM) evoked transient current responses generally of less than 1 min duration, inositol 2,4,5‐trisphosphate (Ins(2,4,5)P3; 10 microM) evoked smaller current transients while inositol 1,3,4,5‐tetrakisphosphate (InsP4; 10 microM) evoked no detectable current response. However, in the presence (in external bathing solution) of the phospholipase A2 inhibitor 4‐bromophenacyl bromide (4‐BPB; 8 microM) all three of the InsPx now evoked prolonged current responses lasting for several minutes. The current responses to all three InsPx were abolished by inclusion of the Ca2+ chelator EGTA (5 mM) in the internal, pipette‐filling solution indicating that the responses are calcium dependent and reflect the effect of the InsPx in increasing intracellular Ca2+. Inositol 1,3,4,5,6‐pentophosphate (InsP5) induced no current response when tested up to 20 microM in the presence or absence of 4‐BPB. 2. The potentiating effect of 4‐BPB on the InsPx‐induced current responses was not mimicked by application of arachidonic acid (AA) oxidation inhibitors; indomethacin (20 microM), nordihydroguaiaretic acid (20 microM) or proadifen (SKF525A, 100 microM). The effects of 4‐BPB were countered however, by the inclusion of 2 microM AA in the external solution. The results suggest that the 4‐BPB potentiates the response by inhibiting the activity of phospholipase A2, thereby reducing the formation of AA. 3. In the presence of 4‐BPB (8 microM) the InsPx‐evoked responses were dose dependent with an increase in both the amplitude and speed of onset with increasing concentrations. In the presence of 4‐BPB InsP4 was as efficient as Ins(1,4,5)P3 both in terms of speed of onset and amplitude of responses; the efficacy and dissociation constant (Kd) for both of these InsPx were the same at 1 microM and 45 nM respectively. Ins(2,4,5)P3 was always less effective, with an efficacy and Kd of 10 microM and 750 nM respectively. 4. If 4‐BPB was applied after the current responses evoked by the InsPx were over, or if guanosine 5'‐O‐(3‐thiotriphosphate) (GTP gamma S) was included in the recording pipette then the phospholipase inhibitor gave rise to an additional, prolonged, current response.(ABSTRACT TRUNCATED AT 400 WORDS)
Identification and characterization of afferent periodontal A delta fibres in the cat.
Authors: Mengel M K , Jyväsjärvi E , Kniffki K D
Published: December 2014

1. The presence and responsiveness of afferent periodontal A delta fibres was studied in pentobarbitone‐anaesthetized adult cats. 2. Extracellular single fibre recordings were made from fine nerve filaments split from the proximally cut end of the inferior alveolar nerve. Periodontal nerve fibres were identified by constant current stimulus pulses applied via platinum wire electrodes inserted into the periodontal space of the lower canine tooth. 3. Of a total of 252 periodontal nerve fibres, 97 (37%) were classified as A delta fibres according to their conduction velocities (CV) (> 2.5 m s‐1, < 30 m s‐1) as determined by electrical stimulation of the periodontal ligament. The mean (+/‐ S.D.) conduction velocity was 11.0 +/‐ 7.7 m s‐1 (n = 97; range: 2.6‐28.2 m s‐1). 4. A good exponential correlation (r = 0.85) was found between the electrical thresholds of the A delta fibres and their conduction velocities. 5. For four A delta fibres a complete stimulus‐duration curve was determined. It followed rather well the I = I0/(1‐et/tau) law, where I represents the stimulus amplitude, t the stimulus duration, I0 the rheobasic current and tau the time constant. 6. In the intact tooth none of the identified periodontal A delta fibres showed any ongoing activity in the absence of intentional stimulation. 7. The responses of sixteen electrically identified periodontal A delta fibres were tested by mechanical, thermal and chemical stimuli applied to the periodontal space. Seven of nine periodontal A delta fibres tested responded to mechanical forces applied to the tooth from different directions of which none could be activated by slight touch. A rudimentary directional sensitivity was seen. When a human tooth was stimulated by a mechanical stimulus of similar strength the sensation evoked was described as a dull, poorly localized pain. 8. Six periodontal A delta fibres were activated by heat and/or cold and/or chemical stimulation. Two of eight periodontal A delta fibres tested responded to heat and four of six A delta fibres tested responded to cold stimuli applied to the alveolar bone overlying the periodontal ligament; none of them responded to both types of thermal stimuli. Two of seven periodontal A delta fibres tested were activated by a saturated solution of potassium chloride applied locally to the periodontal ligament; two of these responded also to cold. 9. The response behaviour of periodontal afferent A delta fibres observed in this study suggests that they may play a role in periodontal nociception.
Whole‐cell K+ currents in fresh and cultured cells of the human and monkey retinal pigment epithelium.
Authors: Wen R , Lui G M , Steinberg R H
Published: December 2014

1. Whole‐cell potassium currents of freshly isolated human (adult and fetal) and monkey (adult) retinal pigment epithelial (RPE) cells, as well as cultured human and monkey RPE cells were studied using the patch‐clamp technique. 2. In freshly isolated adult cells of both species, two currents were observed in the voltage range from ‐150 to +50 mV: an outwardly rectifying current and an inwardly rectifying current. These currents were also found in cultured cells of both species. 3. The outwardly rectifying current in freshly isolated adult human and monkey cells and some cultured cells was evoked by depolarizing voltage pulses more positive that ‐30 mV. The current activated with a sigmoidal time course after a brief delay, and was virtually non‐inactivating. The conductance associated with the current was half‐maximal at ‐16.4 mV for fresh human cells and ‐13.5 mV for fresh monkey cells, but was shifted 16.0 and 17.7 mV in the positive direction in cultured human and monkey cells, respectively. The reversal potential of the current in both human and monkey cells matched the potassium equilibrium potential (EK) over a wide range of external potassium concentrations. This current was blocked by 20 mM tetraethylammonium. 4. A membrane current that exhibited inward rectification was observed with hyperpolarizing voltage pulses. The zero‐current potential of this current was close to EK. This current was blocked by 2 mM Ba2+ and 2 mM Cs+. In cultured human and monkey cells, but not in fresh cells, this current exhibited an inactivation when voltage pulses were more negative than ‐120 mV. External Na+ was responsible for the inactivation, as the inactivation was removed in a Na(+)‐free solution. 5. Membrane currents in freshly isolated fetal human RPE cells were remarkably different from those in adult cells. A transient outward current resembling the A‐type potassium current was observed as the dominant membrane current in freshly isolated fetal human cells. This current activated when voltage pulses were more positive than ‐30 mV. It inactivated rapidly after reaching a maximal level. Application of 5 mM 4‐aminopyridine (4‐AP) completely blocked this current. Although this current was never observed in fresh adult cells, it was found in 33% of the cultured adult cells with similar kinetics, ion selectivity, and pharmacological properties. 6. In about 26% of the freshly isolated fetal human cells, a more slowly activating outward current, which resembled the delayed rectifier, was found to co‐exist with the transient outward current.(ABSTRACT TRUNCATED AT 400 WORDS)
Large conductance Ca(2+)‐activated K+ channels are involved in both spike shaping and firing regulation in Helix neurones.
Authors: Crest M , Gola M
Published: December 2014

1. The role of BK‐type calcium‐dependent K+ channels (K+Ca) in cell firing regulation was evaluated by performing whole‐cell voltage clamp and patch clamp experiments on the U cell neurones in the snail Helix pomatia. These cells were selected because most of the repolarizing K+ current flowed through K+Ca channels. 2. U cells generated overshooting Ca(2+)‐dependent spikes in Na(+)‐free saline. In response to prolonged depolarizing current, they fired a limited number of spikes of decreasing amplitude, and behaved like fast‐adapting or phasic neurones. 3. Under voltage clamp conditions, the K+Ca current had a slow onset at voltages that induced small Ca2+ entries. By manipulating the Ca2+ entry (either with appropriate voltage programmes or by changing the Ca2+ content of the bath), the K+Ca channel opening was found to be rate limited by the Ca2+ binding step and not by the voltage‐dependent conformational change to the open state. 4. Despite the slow activation rate observed in voltage‐clamped cells, 25‐30% of the available K+Ca current was found to be active during isolated spikes. These data were based on patch clamp, spike‐like voltage clamp and hybrid current clamp‐voltage clamp experiments. 5. The fact that spikes led the slowly rising K+Ca current to shift into a fast activating mode was accounted for by the large surge of Ca2+ current concomitant with spike upstroke. The early calcium surge resulted in local increases in cytosolic calcium, which speeded up the binding of calcium ions to the closed K+Ca channels. From changes in the null Ca2+ current voltage, it was calculated that the submembrane [Ca2+]i increase to 50‐80 microM during the spike. 6. Due to their fast voltage dependence, K+Ca channels appeared to play no role in shaping the interspike trajectory. 7. Even in the fast activating mode, the K+Ca current had a finite rate of rise and was not involved in repolarizing short duration Na(+‐dependent action potentials. The current became more and more active, however, when voltage‐gated K+ channels were progressively inactivated during firing. 8. The fast adaptation exhibited by U cells upon sustained depolarization was not paralleled by a recruitment of K+Ca channels because of the cumulative Ca2+ entries. During a spike burst, the K+Ca current progressively overlapped the depolarizing Ca2+ current, which ultimately stopped the firing. The early opening of K+Ca channels was ascribed to residual Ca2+ accumulation that kept part of the channels in the Ca(2+)‐bound state ready to be opened quickly by cell depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume‐dependent K+ and Cl‐ fluxes in rat thymocytes.
Authors: Soler A , Rota R , Hannaert P , Cragoe E J , Garay R P
Published: December 2014

1. Hypotonic stress unmasked inward and outward K+ and Cl‐ movements in rat thymocytes. This KCl flux stimulation was reduced by DIOA (dihydroindenyl‐oxy‐alkanoic acid), but not by DIDS (4,4'‐diisothiocyanostilbene‐2,2'‐disulphonate), quinidine, DPAC 144 (5‐nitro‐2‐(2‐phenylethyl‐amino)‐benzoic acid), bumetanide or ouabain. 2. In isotonic media (308 +/‐ 5 mosmol kg‐1), the cells exhibited the following DIOA‐sensitive fluxes: (i) a K+ efflux of 42.7 +/‐ 17.1 mmol (l cells.h)‐1 (mean +/‐ S.D., n = 7), (ii) a Cl‐ efflux of 68 +/‐ 21 mmol (l cells.h)‐1 (n = 3), (iii) a Rb+ influx of 9.7 +/‐ 3.9 mmol (l cells.h)‐1 (n = 6) and (iv) a Cl‐ influx of 9.4 +/‐ 4.1 mmol (l cells.h)‐1 (n = 6). 3. Hypotonic shock (183‐200 mosmol kg‐1) induced a sevenfold stimulation of DIOA‐sensitive K+ and Cl‐ effluxes and a twofold stimulation of DIOA‐sensitive Rb+ and Cl‐ influxes (with a Rb+ to Cl‐ stoichiometry of 1.04 +/‐ 0.31; mean +/‐ S.D., n = 6). 4. The DIOA‐sensitive membrane carrier catalysed net outward KCl extrusion (the outward/inward flux ratio was 5‐7 in isotonic media and 20 in hypotonic media at 189 mosmol kg‐1). Inhibition of DIOA‐sensitive 36Cl‐ efflux by cell K+ depletion suggested coupling of outward K+ and Cl‐ fluxes. Conversely, inward K+ and Cl‐ fluxes were found to be uncoupled in NO3‐ media and in K(+)‐free media. 5. The results clearly show that rat thymocyte membranes possess a 1:1 K(+)‐Cl‐ co‐transport system which is strongly activated by hypotonic shock and catalyses net KCl extrusion.
Hyperglycaemic hypoxia alters after‐potential and fast K+ conductance of rat axons by cytoplasmic acidification.
Authors: Schneider U , Quasthoff S , Mitrović N , Grafe P
Published: December 2014

1. The effects of hyperglycaemic hypoxia (a condition possibly involved in the pathogenesis of diabetic neuropathy) on the depolarizing after‐potential and the potassium conductance of myelinated rat spinal root axons were investigated using electrophysiological recordings from intact spinal roots and from excised, inside‐out axonal membrane patches. 2. Isolated spinal roots were exposed to hypoxia in solutions containing normal or high glucose concentrations. The depolarizing after‐potential of compound action potentials was only enhanced in spinal roots exposed to hyperglycaemic (25 mM D‐glucose) hypoxia. A maximal effect was seen in bathing solutions with low buffering power. 3. The depolarizing after‐potential was also enhanced by cytoplasmic acidification after replacement of 10‐30 mM chloride in the bathing solution by propionate. 4. Multi‐channel current recordings from excised, inside‐out axonal membrane patches were used to study the effects of cytoplasmic acidification on voltage‐dependent K+ conductances with fast (F channels) and intermediate (I channels) kinetics of deactivation. 5. F channels were blocked by small changes in cytoplasmic pH (50% inhibition at pH 6.9). I channels were much less sensitive to intra‐axonal acidification. 6. In conclusion, our data show that hyperglycaemic hypoxia enhances the depolarizing after‐potential in peripheral rat axons. The underlying mechanism seems to be an inhibition of a fast, voltage‐dependent axonal K+ conductance by cytoplasmic acidification. This alteration in membrane conductance may contribute to positive symptoms in diabetic neuropathy.
Ca2+ entry through Na(+)‐Ca2+ exchange can trigger Ca2+ release from Ca2+ stores in Na(+)‐loaded guinea‐pig coronary myocytes.
Authors: Ganitkevich VYa  , Isenberg G
Published: December 2014

1. The ionized cytosolic calcium concentration ([Ca2+]i) was monitored in voltage‐clamped coronary myocytes at 36 degrees C and 2.5 mM [Ca2+]o using the Ca2+ indicator indo‐1. [Ca2+]i was transiently increased by fast application of 10 mM caffeine, and the mechanisms involved in decay of [Ca2+]i were analysed. 2. Resting [Ca2+]i was 166 +/‐ 62 nM (mean +/‐ S.D.). Caffeine increased [Ca2+]i within 1‐2 s to 1618 +/‐ 490 nM. In the continuous presence of caffeine [Ca2+]i fell close to resting values with a half‐decay time of 5.0 +/‐ 1.6 s. Wash‐out of caffeine induced an undershoot of [Ca2+]i to 105 +/‐ 30 nM. When caffeine was applied repetitively the [Ca2+]i transients were of reduced amplitude indicating that the store had lost a part of releasable Ca2+. 3. After a 1 s caffeine application [Ca2+]i decayed with a half‐time of 2.3 +/‐ 0.8 s to the undershoot of 112 +/‐ 57 nM. The decay of [Ca2+]i was largely prevented by 3 mM [La3+]o; after wash‐out of La3+ [Ca2+]i fell to the resting value without an undershoot. The results demonstrate that La(3+)‐sensitive Ca2+ extrusion contributes to the decay of the [Ca2+]i transient and to the undershoot. 4. With 10 mM [Na+]i, sodium removal from the bath incremented [Ca2+]i in three out of ten cells by 71 +/‐ 11 nM; in the other cells [Ca2+]i did not change. In the absence of extracellular sodium the decay of [Ca2+]i after wash‐out of caffeine was not retarded. 5. To stimulate Na(+)‐Ca2+ exchange, cells were dialysed with pipette solution containing 150 mM NaCl. Elevation of [Na+]i had no significant effect on the resting [Ca2+]i (180 +/‐ 47 nM) or on the caffeine‐induced [Ca2+]i transients (peak 1614 +/‐ 530 nM, half‐time of decay 3 s, undershoot 107 +/‐ 40 nM). 6. With 150 mM [Na+]i, sodium removal resulted in an increase of [Ca2+]i, although responses varied in amplitude (from 130 to 2300 nM) and rate of rise. In the absence of sodium [Ca2+]i remained elevated. After a 1 s caffeine application the undershoot of [Ca2+]i was abolished in sodium‐free solution. When caffeine was applied in sodium‐free solution, the [Ca2+]i transient decayed to a sustained level and the following caffeine response was attenuated. 7. With 150 mM [Na+]i, the effects of sodium removal were strongly suppressed by a preceding depletion of the Ca2+ stores with caffeine. Ryanodine pretreatment abolished the caffeine‐induced [Ca2+]i transients and reduced [Ca2+]i response due to sodium removal.(ABSTRACT TRUNCATED AT 400 WORDS)
Morphine tolerance and inhibition of oxytocin secretion by kappa‐opioids acting on the rat neurohypophysis.
Authors: Russell J A , Coombes J E , Leng G , Bicknell R J
Published: December 2014

1. The present study investigated the mechanisms by which endogenous opioids regulate oxytocin secretion at the level of the posterior pituitary gland. Effects of the selective kappa‐agonist U50,488 on oxytocin secretion were studied in urethane‐anaesthetized lactating rats. Oxytocin secretion in response to electrical stimulation (0.5 mA, matched biphasic 1 ms pulses, 50 Hz, 60‐180 pulses) of the neurohypophysial stalk was bioassayed on‐line by measuring increases in intramammary pressure, calibrated with exogenous oxytocin. Intravenous (I.V.) U50,488 inhibited electrically stimulated oxytocin secretion, without affecting mammary gland sensitivity to oxytocin. The inhibition was dose related, with an ID50 of 441 (+194, ‐136) micrograms/kg and was naloxone reversible. Antagonism of endogenous beta‐adrenoceptor activation by propranolol (1 mg/kg) reduced the potency of U50,488. The selective mu‐agonist morphine (up to 5 mg/kg), had no effect on electrically stimulated oxytocin secretion, but depressed the mammary response to oxytocin. 2. In lactating rats given intracerebroventricular (I.C.V.) morphine infusion for 5 days to induce tolerance and dependence, I.V. U50,488 still inhibited electrically stimulated oxytocin secretion, but the ID50 was reduced to 170 (+78, ‐54) micrograms/kg; thus at the posterior pituitary the sensitivity of kappa‐receptors is enhanced rather than reduced in morphine‐tolerant rats, indicating the absence of cross‐tolerance. In these rats, naloxone produced a large, sustained, fluctuating increase in intramammary pressure indicating morphine‐withdrawal excitation of oxytocin secretion; I.V. U50,488 diminished this response, confirmed by radioimmunoassay, demonstrating the independence of mu‐ and kappa‐receptors regulating oxytocin secretion. 3. In pregnant rats, I.C.V. infusion of morphine from day 17‐18 of pregnancy delayed the start of parturition by 4 h, but did not significantly affect the progress of parturition once established, indicating tolerance to the inhibitory actions of morphine on oxytocin secretion in parturition, and lack of cross‐tolerance to endogenous opioids restraining oxytocin in parturition. 4. Neurointermediate lobes from control and I.C.V. morphine‐infused virgin rats were impaled on electrodes and perifused in vitro. Vasopressin and oxytocin release from the glands was measured by radioimmunoassay. Each gland was exposed to two periods of electrical stimulation (13 Hz, for 3 min). Naloxone (5 x 10(‐6) M) was added before the second stimulation; half the lobes from each I.C.V. treatment were exposed to 5 x 10(‐5) M morphine throughout.(ABSTRACT TRUNCATED AT 400 WORDS)
ATP modulation of calcium channels in chromaffin cells.
Authors: Gandía L , García A G , Morad M
Published: December 2014

1. The effects of externally applied micromolar concentrations of adenosine 5'‐triphosphate (ATP) on Ca2+ currents (ICa) were studied in whole‐cell clamped adrenaline‐secreting chromaffin cells. 2. Ca2+ currents in chromaffin cells activated at about ‐40 mV, reached a maximum at 0 mV and had an apparent reversal potential at +50 to +60 mV, indicating the existence of only high voltage‐activated Ca2+ channels. 3. ATP blocked Ca2+ current rapidly, reversibly and in a concentration‐dependent manner (10(‐9)‐10(‐4) M). 4. ATP did not completely block Ca2+ current even at the highest concentrations used (100 microM). The remaining component of Ca2+ current was characterized by slower activation and inactivation kinetics. 5. ATP blocked ICa even in the presence of nisoldipine and/or omega‐conotoxin GVIA, suggesting that its modulatory role is not specific for L‐ and/or N‐type Ca2+ channels. 6. Other adenine nucleotides also blocked the Ca2+ current partially. The order of potencies was ATP > or = ADP > AMP >> adenosine, indicating that the ATP effects are most probably mediated by a P2‐type purinergic receptor. 7. Dialysis of the cells with an intracellular solution containing 1 mM guanosine 5'‐O‐thiodiphosphate (GDP‐beta‐S) or pre‐incubation of the cells with pertussis toxin (PTX) blocked the inhibitory effects of ATP. 8. Intracellular application of the non‐hydrolysable GTP analogue guanosine 5'‐O‐(3'‐thiotriphosphate) (GTP‐gamma‐S; 50 microM) also decreased ICa in a manner similar to that seen for ATP and significantly reduced the ATP inhibitory effect. 9. Conditioning pulses to potentials positive to +80 mV partly reversed the inhibitory effects of ATP on the Ca2+ current. The prepulse‐induced enhancement of ICa depended on [GTP]i‐related G protein activity such that concentrations larger than 200 microM GTP, or GTP‐gamma‐S (50 microM) were required for significant prepulse potentiation of the Ca2+ current, while dialysis with GDP‐beta‐S prevented it. 10. We conclude that the ATP, co‐released with catecholamines in the intact adrenal gland, may inhibit the secretory process by down‐regulating the Ca2+ channel via a P2‐type purinergic receptor coupled to a PTX‐sensitive G protein.
Characteristics of multiple voltage‐activated K+ currents in acutely dissociated chick ciliary ganglion neurones.
Authors: Wisgirda M E , Dryer S E
Published: December 2014

1. The properties of voltage‐activated K+ currents were examined using whole‐cell recording techniques in acutely isolated chick ciliary ganglion neurones. 2. Application of depolarizing voltage pulses from a holding potential of ‐60 mV evoked sustained outward currents that inactivated with time constants of hundreds of milliseconds (IDR). Bath application of 10 mM tetraethylammonium (TEA) caused a 70‐90% reduction of IDR. Application of depolarizing voltage steps from a holding potential of ‐120 mV revealed a second class of TEA‐resistant outward currents. These currents activated quickly but inactivated completely within tens of milliseconds (IA). IA activated at more negative command potentials than IDR. However, IDR exhibited a steeper voltage dependence of activation than IA. 3. The midpoint of the steady‐state inactivation curve of IA was between ‐95 and ‐110 mV. By contrast the midpoint of the steady‐state inactivation curve of IDR was between ‐80 and ‐90 mV. It was not possible to produce a complete inactivation of IDR using prepulses of up to 2 s duration. 4. The time course of IA inactivation could only be fitted with double‐exponential curves with time constants of 5‐18 ms and 30‐60 ms at membrane potentials positive to ‐30 mV. The inactivation of IA was slower at more positive membrane potentials because of a greater contribution of the long time constant. The individual time constants were not markedly voltage dependent. 5. Bath application of 5 mM 4‐aminopyridine (4‐AP) caused a 70‐100% block of IA whereas 1 mM 4‐AP was ineffective. Bath application of 560 nM alpha‐dendrotoxin (DTX) produced a 50‐70% reduction of IA, but application of 280 nM DTX had no effect on IA. 6. Application of 1 mM 4‐AP produced a reversible 55‐80% block of IDR measured at the end of a 500 ms depolarizing pulse. The 4‐AP‐sensitive components of IDR activated rapidly and exhibited a gradual inactivation with continued depolarization. The 4‐AP‐resistant components of IDR activated much more slowly and showed very little tendency to inactivate. Significant blockade of IDR was produced by 10 microM 4‐AP. 7. The decay of IDR tail currents could only be fitted with double exponential curves with time constants of 3‐6 and 40‐60 ms, respectively. The fast and slow components of the tail currents behaved independently with respect to the duration of the depolarizing voltage step. 8. Application of 1 mM 4‐AP eliminated the fast, but not the slow component of IDR tail currents.(ABSTRACT TRUNCATED AT 400 WORDS)
Uniform olivocerebellar conduction time underlies Purkinje cell complex spike synchronicity in the rat cerebellum.
Authors: Sugihara I , Lang E J , Llinás R
Published: December 2014

1. The issue of isochronicity of olivocerebellar fibre conduction time as a basis for synchronizing complex spike activity in cerebellar Purkinje cells has been addressed by latency measurement, multiple‐electrode recording and Phaseolus vulgaris leucoagglutinin (PHA‐L) tracing of climbing fibres in the adult rat. 2. The conduction time of the olivocerebellar fibres was measured by recording Purkinje cell complex spike (CS) responses from various areas of the cerebellum. The CSs were evoked by stimulating the olivocerebellar fibres near the inferior olive. In spite of a difference in length, as determined directly by light microscopy, the conduction times of different climbing fibres were quite uniform, 3.98 +/‐ 0.36 ms (mean +/‐ S.D., n = 660). 3. Multiple‐electrode recording of spontaneous Purkinje cell CS activity was employed to study the spatial extent of CS synchronicity in the cerebellar cortex. Recordings of CS were obtained from Purkinje cells located on the surface and along the walls of lobule crus 2a. The rostrocaudal band‐like distribution of simultaneous (within 1 ms) CS activity in Purkinje cells extended down the sides of the cerebellar folia to the deepest areas recorded (1.6‐2.6 mm deep). As shown in previous experiments, the distribution of simultaneous CS activity did not extend significantly (500 microns) in the mediolateral axis of the cerebellar cortex. 4. In two animals a detailed determination of the length of the olivocerebellar fibre bundles was performed by staining the fibres with PHA‐L injected into the contralateral inferior olive. This measurement included fibre bundles terminating in twenty‐six different areas, ranging from the tops of the various folia to the bottoms of the fissures in both the hemisphere and the vermis. There was a 47.5% difference between the length of the longest measured fibre bundle (15.8 mm, terminating in lobule 6b, zone A) and the length of the shortest measured fibre bundle (8.3 mm, terminating in the cortex at the base of the primary fissure, zone D), after correction for tissue shrinkage. To attain an isochronous conduction time the conduction velocities for these two fibre bundles were calculated to be 4.22 m/s and 2.37 m/s, respectively. 5. By interpolating between measured points a simple formula was derived to estimate the average length of olivocerebellar fibres terminating in any given area of the cerebellar cortex, excluding the paraflocculus, the flocculus and the most lateral regions of the hemisphere. 6. We investigated the most likely mechanisms by which conduction velocity variations with length could result in global isochronicity.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium and potassium currents in ventricular myocytes isolated from diabetic rats.
Authors: Jourdon P , Feuvray D
Published: December 2014

1. The whole‐cell voltage‐clamp technique was applied to ventricular myocytes isolated from normal and streptozotocin‐induced diabetic rat hearts to investigate the contribution of the calcium current and of the calcium‐independent potassium currents to diabetes‐induced alterations of the action potential. 2. In single calcium‐tolerant isolated myocytes diabetes induced a lengthening of the action potential similar to that previously described in intact ventricular muscles. 3. Only L‐type calcium current was present both in normal and diabetic cells. Inactivation of ICa was described in both preparations by two exponentials, whose time constants were not modified by diabetes. 4. Calcium current density‐voltage relationships and steady‐state inactivation curves were not significantly affected by diabetes. 5. Potassium background inward rectifier current was not modified by diabetes. 6. Calcium‐independent outward potassium current inactivated, in both cell types, according to a biexponential process whose time constants were not affected by diabetes. 7. The transient outward potassium current density was significantly reduced by diabetes whereas neither the voltage dependence of the inactivation nor the time dependence of recovery from inactivation was modified. 8. A 4‐aminopyridine‐insensitive potassium current was also reduced by diabetes. 9. Our results show that in isolated ventricular myocytes the lengthening of the action potential induced by diabetes results mainly from a decrease of the transmembrane calcium‐independent potassium permeability.
Neural control of cardiovascular responses and of ventilation during dynamic exercise in man.
Authors: Strange S , Secher N H , Pawelczyk J A , Karpakka J , Christensen N J , Mitchell J H , Saltin B
Published: December 2014

1. Nine subjects performed dynamic knee extension by voluntary muscle contractions and by evoked contractions with and without epidural anaesthesia. Four exercise bouts of 10 min each were performed: three of one‐legged knee extension (10, 20 and 30 W) and one of two‐legged knee extension at 2 x 20 W. Epidural anaesthesia was induced with 0.5% bupivacaine or 2% lidocaine. Presence of neural blockade was verified by cutaneous sensory anaesthesia below T8‐T10 and complete paralysis of both legs. 2. Compared to voluntary exercise, control electrically induced exercise resulted in normal or enhanced cardiovascular, metabolic and ventilatory responses. However, during epidural anaesthesia the increase in blood pressure with exercise was abolished. Furthermore, the increases in heart rate, cardiac output and leg blood flow were reduced. In contrast, plasma catecholamines, leg glucose uptake and leg lactate release, arterial carbon dioxide tension and pulmonary ventilation were not affected. Arterial and venous plasma potassium concentrations became elevated but leg potassium release was not increased. 3. The results conform to the idea that a reflex originating in contracting muscle is essential for the normal blood pressure response to dynamic exercise, and that other neural, humoral and haemodynamic mechanisms cannot govern this response. However, control mechanisms other than central command and the exercise pressor reflex can influence heart rate, cardiac output, muscle blood flow and ventilation during dynamic exercise in man.
Inactivation of the voltage‐dependent Ca2+ channel current in smooth muscle cells isolated from the guinea‐pig detrusor.
Authors: Nakayama S , Brading A F
Published: December 2014

1. Whole‐cell voltage clamp techniques were applied to single smooth muscle cells enzymatically dissociated from guinea‐pig urinary bladder. The inactivation and recovery of voltage‐dependent Ca2+ channel currents were examined by manipulating the membrane potential over a wide range and by changing the extracellular divalent cation concentrations. 2. After exposing the cells to conditioning potentials (‐100 to +80 mV in 20 mV increments), the degree of inactivation was estimated by stepping to a 0 mV test potential. In the presence of 2.5 mM Ca2+, the inactivation of the current was U‐shaped with respect to the conditioning potential, with maximum inactivation at 0 mV. The maximal inactivation was 60 and 90% after conditioning durations of 0.8 and 5 s, respectively. The U‐shaped curve is characteristic of Ca(2+)‐dependent inactivation. When conditioning potentials of +80 mV with either duration were applied, the inward current at the test potential and the subsequent tail current on returning to the holding potential were larger than in control conditions (when the conditioning potential = the holding potential, ‐60 mV). 3. A U‐shaped inactivation curve was also observed in the presence of 2.5 mM Ba2+. The inactivation was maximal with a conditioning potential of about ‐20 mV, and the inactivation was smaller than seen with Ca2+ entry. 4. Paired‐pulse protocols were applied to examine the voltage dependence of recovery of the Ca2+ inward current. After the inward current had been inactivated during a 100 ms depolarization at 0 mV, it took 700 ms at ‐60 mV for nearly complete recovery of the current. Recovery was also observed at +80 mV. When the potential of the paired pulses was increased to +20 mV, less recovery was seen when the interpulse potential was at +80 mV. When a longer (3 s) depolarization was applied, the peak amplitude of the inward current took much longer to recover, and had not completely recovered after 4 s at either of the interpulse potentials, although recovery was greater with an interpulse potential of ‐60 mV than with one of +80 mV. Similar recoveries were observed in the presence of Ba2+. 5. During a long depolarization (8 s, 0 mV), the effects of rapid changes in the extracellular solution were examined. Partial recovery of the inward current occurred after a period in which Ca2+ was replaced with Mg2+. This recovery was not observed in the presence of Ba2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Phorbol esters enhance synaptic transmission by a presynaptic, calcium‐dependent mechanism in rat hippocampus.
Authors: Parfitt K D , Madison D V
Published: December 2014

1. The effects of phorbol esters on evoked and spontaneous excitatory neurotransmission were studied in the CA1 area in the in vitro hippocampal slice preparation of the rat. Experiments were conducted using field potential recording and whole‐cell voltage clamp of CA1 pyramidal neurons. 2. Pyramidal cells dialysed during whole‐cell recording with EGTA‐containing electrode solutions, unable to support the induction of long‐term potentiation (LTP), still showed robust phorbol ester‐induced potentiation of excitatory synaptic transmission. 3. Spontaneous miniature excitatory postsynaptic currents (EPSCs), recorded in whole‐cell voltage clamp in the presence of tetrodotoxin and picrotoxin, had amplitudes ranging from 4 to 40 pA and occurred at an average frequency of 0.8‐5 Hz. Neither the amplitude nor the frequency of spontaneous EPSCs was altered by cadmium, dihydropyridines, or omega‐conotoxin GVIA. 4. The phorbol ester 4‐beta‐phorbol 12,13‐diacetate increased the frequency of spontaneous miniature EPSCs without changing the shape of the EPSC amplitude distribution, suggesting that phorbol esters exert their potentiating effects presynaptically. 5. Blockade of voltage‐dependent calcium channels with cadmium attenuated the phorbol‐induced increase in spontaneous miniature EPSCs frequency. The phorbol ester‐induced increase in miniature EPSC frequency was also attenuated by dihydropyridines, but not by omega‐conotoxin GVIA. 6. Unlike spontaneous synaptic currents, stimulus‐evoked synaptic currents were reduced by omega‐conotoxin but not by nifedipine. 7. We conclude that the phorbol ester increases spontaneous release of glutamate by modulating an L‐type channel that does not participate in stimulus‐evoked neurotransmitter release.
Recruitment of motor units in response to transcranial magnetic stimulation in man.
Authors: Bawa P , Lemon R N
Published: December 2014

1. Short‐latency responses of single motor units (SMUs) and surface electromyographic activity (EMG) to transcranial magnetic stimulation (TMS) were examined in five different hand and forearm muscles of human subjects. 2. The response probability, P (number of extra spikes in the response peak above background per stimulus), was, in general, higher at the lower voluntary discharge rate of the motor unit than at the higher rate. 3. Increasing the strength of TMS increased the response probability of a tonically firing motor unit and at the same time recruited new units which discharged phasically during the response peak. This demonstrates rate coding and recruitment of motor units by excitatory inputs resulting from TMS when the motoneurone pool is tonically facilitated by a constant voluntary drive. 4. Next, TMS was delivered without any voluntary facilitation of motoneurones. The order of recruitment for up to four different motor units discharged by TMS was compared to that observed with voluntary input. The threshold of recruitment for each of the two inputs was estimated from the surface EMG value at which the unit was recruited. For these motoneurone pools (eleven sets of observations), the order of recruitment was the same with TMS and voluntary inputs. 5. From these data it is concluded that, despite the complex and phasic nature of the descending corticospinal volleys generated by TMS, it produces orderly recruitment and rate coding of motoneurones similar to that found for voluntary activation.
Evidence of incomplete neural control of motor unit properties in cat tibialis anterior after self‐reinnervation.
Authors: Unguez G A , Bodine‐Fowler S , Roy R R , Pierotti D J , Edgerton V R
Published: December 2014

1. The mechanical, morphological and biochemical properties of single motor units from the anterior compartment of the tibialis anterior muscle in adult cats were studied six months after the nerve branches to that compartment were cut and resutured in close proximity to the muscle. 2. In these self‐reinnervated muscles, the maximum tetanic tensions were lower in slow than fast units, a relationship similar to that observed among motor units from control adult muscles. The maximum tetanic tensions produced by the fast units were larger than those produced by the same motor unit types in control muscles. Direct counts of muscle fibres belonging to a motor unit showed that factors controlling the number of muscle fibres innervated by a motoneurone type persist during the reinnervation process in that fast motoneurones reinnervated more muscle fibres than slow motoneurones. Thus, the number of muscle fibres reinnervated by a motoneurone principally accounted for the difference in the maximum tension outputs among motor unit types, a relationship similar to that observed in control tibialis anterior muscles. 3. Monoclonal antibodies for specific myosin heavy chains were used to differentiate fibre types. By this criterion, motor units from control muscles were found to contain a homogeneous fibre type composition. In contrast, a heterogeneous, yet markedly biased, fibre type composition was observed in each unit analysed from self‐reinnervated muscles. 4. Although not all of the muscle fibres of a motor unit developed the same type‐associated parameters after reinnervation, the relationships among myosin heavy chain profile, succinate dehydrogenase activity and the fibre size were similar in fibres of control and self‐reinnervated muscles. 5. The processes which dictate both motor unit size and the matching between motoneurone and muscle fibre type during the reinnervation process must be interdependent and result from a hierarchy of decisions which reflects their relative importance. The mechanisms responsible for these two processes may be a combination of: (1) selective innervation which may or may not incorporate a pruning process if multiple synaptic connections are initially formed and/or (2) conversion of enough fibres of a motor unit to form a predominant type.