bluuebunny/crossref_metadata_2025 huggingface dataset:
The efficiency of a flight muscle from the locust Schistocerca americana.
Authors: Josephson R K , Stevenson R D
Published: December 2014

1. The efficiency of the metathoracic tergosternal muscle of the locust Schistocerca americana was examined by simultaneously measuring work output from the muscle and oxygen consumption by the muscle. The work output was determined using the work‐loop technique in which the muscle is subjected to periodic strain and to phasic stimulation in the strain cycle. The area of the loop formed by plotting muscle force against muscle length over a cycle is the net work output for that cycle. 2. The tergosternal muscle is a synchronous, parallel‐fibred muscle containing two motor units with similar contraction kinetics. The average twitch rise time (30 degrees C) was 15 ms, the twitch duration (to 50% relaxation) was 26 ms, and the peak twitch tension with both units active was 73 kN m‐2. The maximum mechanical power output during sinusoidal shortening at 20 Hz with both motor units active and stimulated once per cycle averaged 37 W kg‐1. 3. The overall efficiency of the tergosternal muscle averaged 6.4% (range 4‐10%) where efficiency is defined as the ratio of the net work done (20 Hz sinusoidal strain, 1 stimulus per cycle, optimum strain amplitude and stimulus phase) to the caloric equivalent of the oxygen consumed. The efficiency was independent of the duration of the test period (examined range = 10‐30 s) and the same when both motor units were active as when only one was stimulated. 4. Stimulating the muscle with two stimuli per cycle (interstimulus interval = 6 ms) increased the work per cycle by about 13% above that with single stimuli per cycle, but the muscle fatigued more rapidly and after 15‐25 s the power output was less with two stimuli per cycle than with one. The efficiency with two stimuli per cycle was slightly less than that with one shock per cycle. 5. The oxygen consumption during normal work cycles at 20 Hz with optimum stimulus phase and strain was greater by about 15% than the oxygen consumption during isometric contractions at the same frequency.
Action of 2,3‐butanedione monoxime on calcium signals in frog cut twitch fibres containing antipyrylazo III.
Authors: Maylie J , Hui C S
Published: December 2014

1. The effects of 2,3‐butanedione monoxime (BDM) on the optical retardation and myoplasmic Ca2+ signal were studied in twitch fibres of Rana temporaria. The myoplasmic Ca2+ transient in response to action potential stimulation was monitored in cut fibres containing Antipyrylazo III under current clamp in a double Vaseline‐gap chamber. 2. In fibres not stretched adequately to suppress all the contraction, BDM blocked the movement‐related intrinsic optical signal at 810 nm very effectively. 3. In fibres stretched to sarcomere lengths greater than or equal to 4 microns to reduce the contraction to below detectable levels, the effect of BDM on the Ca(2+)‐Antipyrylazo III signal was studied after correcting for the instrinsic signal unrelated to movement. With increasing concentrations of BDM, the peak of the Ca(2+)‐Antipyrylazo III signal was suppressed progressively. Concomitantly, the half‐width was prolonged somewhat. On average, 5, 10 and 20 mM‐BDM reduced the peak amplitude to 88, 78 and 54% of control, respectively. 4. BDM had no effect on the rising phase or the peak amplitude of the retardation signal measured at 720 nm, but suppressed the undershoot in the decay phase of the signal in a dose‐dependent manner. BDM also had no effect on the late pedestal level of the signal. 5. During repetitive stimulation by a train of ten action potentials, 10 mM‐BDM suppressed the second to the tenth peaks of the Ca2+ signal and of the retardation signal more effectively than the first peak. Twenty millimolar BDM almost completely suppressed the later peaks of both signals such that the signals decayed with a time course similar to that elicited by a single action potential. 6. The effect of BDM on the Ca(2+)‐Antipyrylazo III signal was also studied in fibres under voltage clamp; 10 mM‐BDM lowered the threshold for the Ca(2+)‐Antipyrylazo III transient by a few millivolts and reduced the steepness of the peak amplitude versus voltage plot near threshold. 7. Based on a model used by Baylor, Chandler & Marshall (1983) to estimate the net Ca2+ release from the sarcoplasmic reticulum, 10 and 20 mM‐BDM were found to reduce the peak release to 75 and 52%, to prolong the half‐width of the release waveform to 118 and 147%, and to reduce the peak uptake to 76 and 54% of control values, respectively. 8. It is concluded that BDM affects the optical retardation and myoplasmic Ca2+ signal monitored with Antipyrylazo III in a dose‐dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)
Comparison of initial stages of muscle differentiation in rat and mouse myoblastic and mouse mesodermal stem cell lines.
Authors: Kubo Y
Published: December 2014

1. Electrophysiological and immunohistochemical properties during the early stages of muscle differentiation were studied in two myoblastic cell lines, mouse C2C12 and rat L6, and compared to those in myogenic clonal cells derived from the mouse mesodermal stem cell line C3H10T1/2, studied in the preceding paper. 2. Mouse C2C12 cells were induced to differentiate to muscle by changing from 10% fetal calf serum to 2% horse serum in the medium. Most of the C2C12 cells before serum reduction showed ATP‐induced slow K+ current. Twelve per cent showed inward rectifier K+ current. They expressed fibronectin and Neural Cell Adhesion Molecule (NCAM). Small spindle‐shaped cells at an early stage of muscle differentiation began to appear 24 h after serum reduction. In contrast to cells before serum reduction, only 13% of these spindle‐shaped cells showed an ATP response. Most showed tetrodotoxin (TTX)‐resistant Na+ current and outward K+ current. Thirty‐eight per cent had inward rectifier K+ current. They expressed NCAM but not fibronectin. The T‐type Ca2+ current was not observed up to the latest stage of differentiation investigated. 3. Rat L6 cells in maintaining culture medium showed only infrequent ATP responses, but already showed TTX‐resistant Na+ current. No clear T‐type Ca2+, inward rectifier K+ or outward K+ currents were observed. About one‐third of the cells did not express fibronectin. From these results, L6 cells appear to be at a stage near to but slightly earlier than that of C2C12 cells after serum reduction. 4. The properties of the early stages of muscle differentiation in C3H10T1/2 cells, such as the disappearance of ATP‐induced K+ current and fibronectin, and the appearance of NCAM, were also seen in C2C12 and L6. However, T‐type Ca2+ and inward K+ currents, which were found in the initial stages of C3H10T1/2 muscle differentiation, were not clearly observed in C2C12 and L6. Instead, C2C12 and L6 showed a TTX‐resistant Na+ current which was never observed in C3H10T1/2 cells. 5. The properties of the TTX‐resistant Na+ current were investigated. In L6 cells, it was reduced to 60% by 1 microM‐TTX. It could be evoked by depolarizations to a level above ‐50 mV with a maximum amplitude at around ‐15 mV. Steady‐state inactivation was detectable with pre‐pulses to ‐100 mV for 100 ms and reached half at pre‐pulses of ‐78 mV. These parameters of inactivation are clearly different from those of the TTX‐sensitive Na+ current observed in C3H10T1/2‐derived mature muscle cells in the preceding paper.
Motor units of the fourth deep lumbrical muscle of the adult rat: isometric contractions and fibre type compositions.
Authors: Gates H J , Ridge R M , Rowlerson A
Published: December 2014

1. Isometric twitch and tetanic tensions were recorded from whole muscles and single motor units in fourth deep lumbrical muscles isolated from young adult (60 days) rats. Muscles were superfused with oxygenated Ringer solution at 25 degrees C except where stated otherwise. 2. It was confirmed that the muscle is supplied most commonly by eleven motor axons, nine via the lateral plantar nerve (LPN), and two via the sural nerve (SN). Motor units whose axons were isolated from either LPN or SN were studied. There was no difference in mean motor unit size. 3. In their unfused tetani most units showed 'sag’ and some ‘no sag’, with no segregation between LPN and SN. ‘No sag’ units were always small (unit tetanic tension less than 8% whole‐muscle tetanic tension), tended to be relatively slowly contracting and relaxing during an isometric twitch, and tended to have relatively low twitch:tetanus ratios. Units showing sag ranged from large to small. 4. In some motor units muscle fibres were depleted of their glycogen by repetitive stimulation at 30 degrees C in glucose‐free Ringer solution, and the muscle and its unstimulated control frozen and sectioned. Neighbouring sections were stained for glycogen and for binding of two myosin‐specific antibodies, one specific for slow myosin and the other for type IIA myosin. Myosin ATPase and succinic dehydrogenase histochemistry were also carried out in some muscles. 5. Serial reconstructions showed that all or virtually all extrafusal fibres in the muscle were present in a midbelly section, and that the myosin type of individual fibres did not change significantly along their length. Spindle profiles were seen frequently and in two muscles eight and twelve spindles were identified. 6. Of twenty‐six motor units examined twenty contained almost exclusively muscle fibres of the recently described type IIX. All these units showed sag in their isometric tetani. 7. Six units each contained 50% or more of slow myosin‐containing fibres (IIC and a few type I). The remaining fibres in these units were IIA. All these units were therefore of mixed fibre composition, and are discussed as IIC/IIA units. In whole muscles slow‐myosin‐containing fibres were generally distributed evenly (non‐randomly) throughout the muscle cross‐section. 8. Whole muscles contained on average 970 fibres (S.D. +/‐ 70) of which 82 (+/‐ 9) were slow‐myosin‐containing. A few muscles from older rats (3‐24 months) contained very few such fibres.(ABSTRACT TRUNCATED AT 400 WORDS)
Dopamine D2 receptor activation depolarizes rat supraoptic neurones in hypothalamic explants.
Authors: Yang C R , Bourque C W , Renaud L P
Published: December 2014

1. Intracellular current and voltage clamp recordings were obtained from rat supraoptic nucleus neurones in superfused hypothalamic explants in order to evaluate their response to dopamine and to D1 and D2 agonists. 2. With one exception, exposure to dopamine (10‐200 microM) depolarized supraoptic neurones. When tested for an effect on twenty‐one spontaneously active supraoptic neurones, dopamine enhanced the firing of all eleven continuous‐firing (possibly oxytocin‐secreting) neurones and prolonged the burst in all ten phasic‐firing (vasopressin‐secreting) neurones. 3. In sixty‐seven of sixty‐eight neurones where current injection was used to maintain membrane potential below threshold for action potential generation, current clamp data revealed that exposure to dopamine (10‐200 microM) was followed in 10‐17 s by a gradual 3‐7 mV membrane depolarization that lasted for 4‐15 min and was accompanied by a 12‐23% reduction in input resistance. Exposure to quinpirole, a D2 agonist (10‐200 microM), induced a similar response with comparable onset, duration and change in input resistance. In contrast, tests on sixteen cells indicated little or no response to a D1 agonist SKF38393. 4. Under voltage clamp, dopamine was noted to induce an inward current, accompanied by a 7.5‐40% increase in membrane conductance over the corresponding time course. 5. Voltage‐current plots for dopamine‐induced depolarizations were linear in the range ‐50 to ‐110 mV. Dopamine and quinpirole depolarizations had extrapolated mean reversal potentials of ‐25 +/‐ 10 mV (mean +/‐ S.D.) and ‐20 +/‐ 15 mV respectively. This approximated the mean reversal potential of ‐20 +/‐ 8 mV measured from the dopamine‐induced inward current using single‐electrode voltage clamp. 6. The actions of dopamine were selectively antagonized by two D2 receptor antagonists, sulpiride and spiperone, but neither influenced membrane depolarizations induced by equimolar concentrations of noradrenaline. Dopamine‐induced depolarizations also persisted following selective blockade of alpha 1‐adrenergic receptors by prazosin; under these conditions, noradrenaline induced membrane hyperpolarization. 7. Following complete substitution of external Na+ with Tris, the reversal potential for the dopamine‐induced response was shifted to ‐70 +/‐ 9.8 mV. This value was consistently less negative than the estimated potassium equilibrium potential. 8. The depolarization action of dopamine persisted in media containing tetrodotoxin and with an external calcium concentration ([Ca2+]o) of 0 mM‐Ca2+ with 6 mM‐Mg2+ or Mn2+, but was abolished following intracellular injection of [1,2‐bis(2‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (BAPTA), a Ca2+ chelator.(ABSTRACT TRUNCATED AT 400 WORDS)
Stimulation‐induced potentiation of T‐type Ca2+ channel currents in myocytes from guinea‐pig coronary artery.
Authors: Ganitkevich VYa  , Isenberg G
Published: December 2014

1. Whole‐cell Ca2+ channel currents were studied in myocytes isolated from guinea‐pig circumflex coronary artery at 36 degrees C and with 10 mM‐Ba2+ (or Ca2+) as charge carrier. With 180 ms clamp steps from the holding potential of ‐100 mV, currents at ‐30 mV were carried mostly through the T‐type calcium channels while at positive potentials currents were mostly of the L‐type. 2. The increase in frequency of pulsing from 0.1 to 2.5 Hz resulted in a reduction of peak inward current ('negative staircase') with the 180 ms pulses to + 10 mV, but in a 2‐fold potentiation ('positive staircase') with pulses to ‐30 mV. T‐type currents and their frequency‐mediated potentiation did not change significantly when Ba2+ was substituted by Ca2+ or Sr2+. 3. Potentiation of T‐type currents was further analysed with a paired‐pulse protocol: at a basal frequency of 0.1 Hz, a pre‐pulse (inducing current I1) was followed by a 200 ms repolarization to ‐100 mV and a test pulse (inducing current I2). The potentiation could only be recorded using test pulses depolarizing the membrane to potentials between ‐40 and ‐10 mV; at more positive test potentials it was masked by the depressant effect of pre‐pulses on the L‐type current. 4. Potentiation of I2 by 200 ms pre‐pulses started at pre‐pulse potentials more positive than ‐60 mV and saturated at ‐20 mV (I2 potentiated by a factor 2.4). Between ‐20 and +130 mV the potentiation was not dependent on the pre‐pulse potential suggesting that the influx of Ba2+ or Ca2+ is not required for this effect. Potentiation of I2 by a 10 s pre‐pulse followed the voltage dependence of the steady‐state inactivation curve of the T‐type Ca2+ channel; potentiation became visible at potentials more positive than ‐80 mV and saturated at about ‐50 mV. 5. When changing the interval between two identical 200 ms pulses, the T‐type current was found to recover completely from inactivation within 40 ms at ‐100 mV; at intervals of 160‐320 ms maximal potentiation of I2 occurred. 6. With pre‐pulses shorter than 200 ms, potentiation became attenuated when inactivation became less complete. When the potential during the interval between the pulses was ‐80 instead of ‐100 mV, maximal potentiation was reduced (I2 potentiated by a factor of 1.3 instead of 2.2) and occurred later (1.28 s). 7. Potentiated T‐type currents inactivated faster.(ABSTRACT TRUNCATED AT 400 WORDS)
The peptide FMRFamide activates a divalent cation‐conducting channel in heart muscle cells of the snail Lymnaea stagnalis.
Authors: Brezden B L , Benjamin P R , Gardner D R
Published: December 2014

1. Isolated Lymnaea stagnalis heart ventricle cells contain cation‐conducting channels with properties characteristic of Ca2+ channels. These channels, which carry inward Na+ currents in the absence of Ca2+, are activated by the molluscan cardioactive peptides FMRFamide and FLRFamide, and are blocked by Co2+ ions. 2. FMRFamide also activated inward Ba2+ currents at the cell's resting potential. These currents, which were also blocked by Co2+ ions, reversed at a membrane potential of +70 mV. 3. Both sodium and barium currents were initiated when the peptides were applied to the cell outside of the patch pipette indicating that a secondary messenger is likely to be involved in the FMRFamide response.
Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.
Authors: MacLeod K T , Harding S E
Published: December 2014

1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea‐pig cardiac myocytes. Intracellular pH was measured using 2',7'‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura‐2. 2. Application of the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (also called phorbol 12‐myristate 13‐acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 +/‐ 34% (n = 8) (p less than 0.02). The rate of change of cell length during contraction (i.e. +dL/dt) increased from 67.2 +/‐ 8.7 microns/s to 127.7 +/‐ 14.1 microns/s (n = 7). The rate of change of cell length during relaxation (‐dL/dt) increased from 55.8 +/‐ 7.4 microns/s to 118.9 +/‐ 12.1 microns/s (n = 7). Time to peak shortening was unchanged. 3. Application of 4 alpha‐phorbol 12,13‐didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7.5 +/‐ 7.5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C. 4. In rat myocytes we have measured the changes of pHi and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p less than 0.05) enhanced by 15.1 +/‐ 6.9% (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p less than 0.05) by an average of 59.1 +/‐ 10.6% (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4‐phorbol 12,13‐didecanoate (which does not activate protein kinase C). 5. Since pHi recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM‐amiloride and inhibited by removing extracellular Na+ then it is suggested that, like pHi regulation in sheep heart Purkinje fibres, pHi recovery in rat cardiac ventricular myocytes is mainly through sarcolemmal Na(+)‐H+ exchange. We suggest that in the presence of TPA the Na(+)‐H+ exchange is stimulated. 6. The relationship between pHi and cell shortening is non‐linear as has been observed by others in whole tissue preparations. The presence of TPA shifts the relationship upwards such that at any one pHi, cell shortening is greater.(ABSTRACT TRUNCATED AT 400 WORDS)
Sensory characteristics of monkey thalamic and motor cortex neurones.
Authors: Butler E G , Horne M K , Rawson J A
Published: December 2014

1. Extracellular single‐cell recordings were made from the cerebellar thalamus, the ventro‐posterior lateralis par caudalis (VPLc) and motor cortex of three conscious monkeys. Recordings were made from the thalamus as well as the cortex in two monkeys. In all, recordings were made from the thalamus in four hemispheres and from the motor cortex in four hemispheres. The animals were trained to permit a detailed examination when relaxed. Unexpected perturbations were applied to the wrist. Seventy‐seven wrist‐related neurones were recorded in the cerebellar thalamus, forty‐two neurones from the VPLc and eighty‐four neurones in motor cortex. 2. Cerebellar nuclear stimulation was used to physiologically identify thalamic neurones receiving input from the cerebellum. The location of all neurones was verified histologically. 3. The majority of cerebellar thalamic neurones had deep sensory receptive fields related to a single muscle, a group of synergists or a single joint. There was a distinct topographical organization. These fields were similar to sensory fields in motor cortical neurones, but had higher thresholds. 4. VPLc neurones had discrete deep or cutaneous sensory fields, or a combination of these fields, which suggests convergence. VPLc neurones had fields with lower thresholds than cerebellar thalamic neurones. The somatotopically located forelimb area in the VPLc was posterior to and continuous with the forelimb area in the cerebellar thalamus. 5. VPLc neurones responded with a shorter latency to wrist perturbations than did cerebellar thalamic neurones. VPLc neurones with deep sensory fields changed firing significantly earlier than those with cutaneous fields. The VPLc is likely to be the major source of sensory input to the motor cortex, and based on the results of this study we suggest that the VPLc is the thalamic nucleus best placed to transmit short‐latency afferent input from the forelimb. 6. The timing of the neuronal discharge of cerebellar thalamic and VPLc cells, which resulted from perturbations of the wrist, was best linked to the duration of movement rather than its amplitude. The cells began firing as soon as the velocity changed sign and continued firing until the sign of the velocity changed again. In subsequent corrective movements neuronal discharge in the VPLc appeared to also encode movement acceleration.
The activity of monkey thalamic and motor cortical neurones in a skilled, ballistic movement.
Authors: Butler E G , Horne M K , Hawkins N J
Published: December 2014

1. Three monkeys were trained to perform a reaction‐time task of the wrist and single‐cell recordings were made from the motor cortex (eighty‐four cells), ventro‐posterior lateralis par caudalis (VPLc) (forty‐two cells) and cerebellar thalamus (seventy‐seven cells). 2. The majority (43/77, 56%) of cerebellar thalamic neurones fired phasically during movement, whereas in the motor cortex most neurones (53/84, 64%) had a phasic‐tonic discharge pattern. Most neurones in both locations discharged in relation to the direction of movement (reciprocal pattern). 3. The cerebellar thalamus is unlike the motor cortex in that it does not usually encode a signal for force or joint position in its discharge. 4. Twenty‐two per cent (17/77) of cerebellar thalamic neurones had a period of reduced discharge rate before the phasic burst of activity, and represent a pattern of discharge not seen in motor cortex or VPLc neurones. 5. The onset of phasic activity in the cerebellar thalamus was significantly later (average 94 ms) than in the motor cortex but occurred just before electromyogram (EMG) activity. The phasic activity in the cerebellar thalamus usually ended before the phasic component of motor cortex discharge was completed. 6. Phasic activity in VPLc neurones commenced after the onset of EMG discharge and on average 26 ms after the commencement of movement. Most neurones with deep sensory receptive fields fired with a reciprocal pattern, while neurones with cutaneous fields usually fixed bidirectionally in relation to the task. Almost one‐third of neurones signalled force and a similar number had discharge levels that encoded characteristics of the joint position. 7. The duration of discharge of VPLc neurones during the voluntary movement was marginally less than the duration of the movement velocity peak and the VPLc may therefore be signalling the duration of the velocity. Phasic activity in cerebellar thalamic neurones fired for a duration similar to the VPLc neurones, but commenced before the movement. Therefore, if the cerebellar thalamus is carrying information about the duration of the velocity, it does so before the movement starts. 8. The phasic burst of activity in cells of the cerebellar thalamus is timed so that it can contribute to the later component of the phasic burst of motor cortical discharge. Thus we speculate that in skilled, ballistic movements, the cerebellum may provide a response which travels via the cerebellar thalamus and helps to determine the magnitude and duration of the phasic part of cortical discharge.(ABSTRACT TRUNCATED AT 400 WORDS)
Release of intracellular calcium and modulation of membrane currents by caffeine in bull‐frog sympathetic neurones.
Authors: Marrion N V , Adams P R
Published: December 2014

1. Calcium release and sequestration were studied in whole‐cell voltage‐clamped bull‐frog sympathetic neurones by image analysis of Fura‐2 signals. 2. Application of caffeine (10 mM) to cells voltage clamped at ‐38 mV caused a rapid increase in intracellular calcium concentration ([Ca2+]i) to a mean value of 352 +/‐ 33 nM, which activated an outward current. In the continued presence of caffeine the rise in [Ca2+]i slowly declined to a sustained plateau of 196 +/‐ 20 nM (112 nM above control levels), while the outward current rapidly decayed. Peak calcium release was highest at the edge of the cell. 3. The caffeine‐evoked intracellular calcium increase was reduced by two inhibitors of calcium‐induced calcium release, ryanodine and procaine. The residual non‐suppressible increase in [Ca2+]i may indicate that caffeine can release calcium from two pharmacologically distinct intracellular stores. 4. Inhibition of the caffeine‐evoked release of calcium by ryanodine was both concentration and ‘use dependent’ so that the full inhibitory effect was only observed when caffeine was applied for the second time in the presence of ryanodine. In contrast, the action of procaine did not show any ‘use dependence’ and unlike ryanodine was fully reversible. 5. The outward current was sensitive to blockers of the large conductance calcium‐activated potassium current, Ic. Analysis of variance from this current indicated that it arose at least partly from summation of spontaneous miniature outward currents. 6. The magnitude and duration of calcium release by caffeine was dependent on the resting level of intracellular calcium and the caffeine exposure time. This, together with the pharmacology of the release, suggests that caffeine increases intracellular calcium by sensitizing calcium‐induced calcium release. 7. The evoked [Ca2+]i increase was enhanced in amplitude by intracellular application of Ruthenium Red. This effect was mimicked by extracellular application of the mitochondrial uncoupler carbonyl cyanide p‐trifluoromethoxyphenyl‐hydrazone (FCCP) but not by internal application of FCCP or other inhibitors of mitochondrial Ca2+ uptake. This suggests that the evoked increase in [Ca2+]i is predominantly buffered by a Ruthenium Red‐sensitive sequestration process which is not mitochondrial.
Novel suppression of an L‐type calcium channel in neurones of murine dorsal root ganglia by 2,3‐butanedione monoxime.
Authors: Huang G J , McArdle J J
Published: December 2014

1. Voltage‐activated currents through calcium channels in primary cultures of murine dorsal root ganglion cells (DRG) were studied with the whole‐cell and cell‐attached patch recording techniques. 2. The chemical phosphatase 2,3‐butanedione monoxime (BDM) reversibly reduced the amplitude of L‐type calcium current (ICa) in a dose‐dependent manner; at a concentration of 20 mM, BDM caused a 47% suppression of ICa. 3. Application of 10 mM‐8‐bromo‐cyclic AMP or 50 microM‐isoprenaline onto DRG treated with BDM completely restored ICa to the pre‐BDM level. 4. In striking contrast, bath application of Bay K 8644 (0.5‐5 microM) had no effect on the BDM‐suppressed ICa. As expected, Bay K 8644 alone caused a two‐ to threefold increase of the maximal ICa and shifted its I‐V relationship to the left. Interestingly, if a cell was first exposed to Bay K 8644 further treatment with 20 mM‐BDM resulted in 100% suppression of ICa. This suggests that Bay K 8644 changes the conformation of the calcium channel to one which is more sensitive or more accessible to the action of the phosphatase. 5. Pre‐treatment of DRG with an activator of protein kinase C, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate, did not antagonize BDM's effect on ICa. 6. The depressant action of BDM on ICa was distinct from that of nifedipine in that it did not exhibit use dependence. 7. When single calcium channel currents were recorded in cell‐attached patches (barium as the charge carrier), bath application of BDM reduced the percentage of time that the channel spent in the open state. 8. Superfusion with 8‐bromo‐cyclic AMP restored the ensemble macroscopic ‘ICa’ to the pre‐BDM amplitude. This was due to a dramatic enhancement of the frequency of channel openings. 9. We suggest that BDM acts through the cytoplasm to alter cyclic AMP‐dependent protein kinase modulation of neuronal L‐type calcium channels. The brief, high‐frequency openings which 8‐bromo‐cyclic AMP activates in the presence of BDM may reflect a rapid phosphorylation‐dephosphorylation sequence which controls channel gating.
Neuronal nicotinic acetylcholine receptor currents in phaeochromocytoma (PC12) cells: dual mechanisms of rectification.
Authors: Sands S B , Barish M E
Published: December 2014

1. Neuronal nicotinic acetylcholine receptor (nAChR) currents in PC12 cells were studied using single‐channel and whole‐cell gigaohm seal voltage‐clamp techniques. Nicotinic AChR agonists were applied using external pipettes. 2. The single‐channel conductance of neuronal nAChRs in outside‐out patches under Mg(2+)‐free ionic conditions was 48 pS. In the absence of internal Mg2+ single‐channel currents (outside‐out patches) did not rectify. 3. Whole‐cell nAChR currents recorded with normal internal solution (lacking Mg2+ chelators as described below) were strongly inwardly rectifying. Current vs. voltage relations showed a sharp inflexion near 0 mV, and outward current was never observed. 4. Extensive dialysis with internal solution containing EDTA and Na2‐ATP, chelators of intracellular Mg2+ (Mgi2+), relieved rectification of instantaneous nAChR currents during voltage jumps from negative potentials. The instantaneous I‐V relation became linear with voltage, in agreement with the expectation from single‐channel measurements made under similar ionic conditions. 5. Agonist‐induced currents recorded under Mgi(2+)‐free conditions relaxed towards zero current during depolarizing voltage steps. Rectification of ACh‐induced currents at the steady state could be described by a Boltzmann relation, with one‐half of channels available at +3.4 mV and an effective gating charge of ‐0.97. Channel availability was approximately 95% at the resting potential. Opening and closing relaxations under Mgi(2+)‐free conditions could be fitted by single exponential functions whose time constants were weakly voltage dependent. 6. A model of rectification was constructed incorporating two voltage‐dependent processes: block by Mgi2+ and intrinsic channel gating. The I‐V relations predicted by this model for both normal and Mgi(2+)‐free conditions were in good agreement with the experimental data. We suggest that rectification of nAChR currents in these cells is due to concurrent activity of these two voltage‐dependent processes. The relative contributions of these two mechanisms are frequency dependent, with block by Mgi2+ dominant for fast events and intrinsic channel gating more important at the steady state.
Task‐dependent changes in the response of human wrist joints to mechanical disturbance.
Authors: Doemges F , Rack P M
Published: December 2014

1. Subjects sat with their right hands in a mould which was attached to the shaft of a servo‐controlled motor. 2. There were two different tasks. In one, the subject attempted to maintain a constant wrist flexing force, while the motor imposed flexion‐extension movements. In the other, the subject attempted to maintain a constant wrist position while the motor exerted variable flexion‐extension forces. 3. The ‘maintain force’ task was punctuated by randomly timed ramp extension movements to test the stretch reflex activity. The ‘maintain position’ task was punctuated by force changes which were the same as those recorded during the ramp extensions mentioned above. In this way it was possible to test stretch reflexes with the same disturbance during the ‘maintain force’ and ‘maintain position’ tasks. 4. When, after some practice, subjects had become good at maintaining the constant force, later components of the stretch reflex (after 40 ms) were found to be smaller than when they were maintaining a constant position. 5. Attempts to maintain a constant position often involved co‐activation of the wrist extensor muscles. This could be prevented by local anaesthesia of the radial nerve. The long‐latency stretch reflex of the flexors was still task dependent after this had been done. 6. The roles of co‐activation and of task‐dependent reflex responses are discussed.
Mechanism of pHi regulation by locust neurones in isolated ganglia: a microelectrode study.
Authors: Schwiening C J , Thomas R C
Published: December 2014

1. We have measured membrane potential (Em) and intracellular pH (pHi), and sodium and chloride activities (aNai and aCli) in exposed dorsal unpaired median neurones in isolated metathoracic ganglia from the desert locust, Schistocerca gregaria using eccentric double‐barrelled ion‐sensitive microelectrodes. 2. In the absence of added HCO3‐ the steady‐state pHi was 7.21 +/‐ 0.13 (mean +/‐ S.D.) at a mean membrane potential of ‐37 +/‐ 7.0 mV (S.D.) (n = 44 cells). The pHi was always more alkaline than predicted for passive H+ distribution. 3. The pHi recovery from acid loads, induced by weak acid application or weak base removal, was pHi dependent and associated, in both the presence and absence of added CO2‐HCO3‐, with a transient increase in aNai. 4. In the absence of added HCO3‐, application of the Na(+)‐H+ exchange blocker amiloride or external Na+ removal caused intracellular acidification. Also in the absence of added HCO3‐ the inhibitor SITS (4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid) caused an acidification of about 0.2 pH units which was not additive to the effects of the removal of external Na+. 5. We found that the application of a CO2‐HCO3(‐)‐containing solution increased the rate of pHi recovery from acidification. 6. Intracellular chloride was decreased by intracellular acidification in the presence of added CO2‐HCO3‐. In the presence of amiloride, intracellular Cl‐ depletion inhibited pHi regulation. 7. Simultaneous application of SITS (160 microM) and removal of CO2‐HCO3‐ revealed a continuous underlying acid load of 0.03‐0.05 pH unit min‐1. 8. We conclude that locust neurones possess at least two pHi‐regulating mechanisms which operate against a continuous acid load. One is a Na(+)‐H+ exchanger which can be blocked by amiloride, while the second is a Na(+)‐dependent Cl(‐)‐HCO3‐ exchanger. The latter mechanism appears to be able to operate in the absence of added HCO3‐ and can recover pHi to around pH 7.4; it is probably the main pHi regulating mechanism. The Na(+)‐H+ exchanger appears to activate at more acid pHi and being less energy efficient may serve a protective role.
Effect of myoplasmic pH on excitation‐contraction coupling in skeletal muscle fibres of the toad.
Authors: Lamb G D , Recupero E , Stephenson D G
Published: December 2014

1. The effect of pH on excitation‐contraction coupling in skeletal muscle of the toad was examined using a skinned fibre preparation which gives ready access to the intracellular environment while still allowing stimulation of Ca2+ release by the normal voltage‐sensor mechanism. 2. In each fibre, depolarization‐induced responses (produced by changing the ions in the bathing solution) were examined first at pH 7.1, and then at another pH between 6.1 and 8.0. At all pH levels examined, the first depolarization elicited a large response which was slightly greater (pH 7.6 and 8.0) or smaller (pH 6.6 and 6.1) than that at pH 7.1. The size of the first depolarization‐induced response varied with pH in almost exactly the same manner as did the maximum Ca(2+)‐activated response. The duration of the depolarization‐induced response at all other pH levels was longer than at pH 7.1. 3. Repeated depolarizations (30 s or more apart) produced similar responses at pH 7.1, but at all other pH levels examined the second and subsequent responses became progressively smaller. The reasons for this decrease were different at low and high pH. 4. Examination of the size of the depolarization‐induced response after reloading the depleted sarcoplasmic reticulum (SR) with Ca2+ in solutions at various pH levels, indicated that the SR Ca2+ pump operated more poorly at pH 6.6 and 6.1 than at pH 7.1. This can account for the wide and successively smaller depolarization‐induced responses at acidic pH. 5. Examination of the size of the depolarization‐induced response after 5 min bathing without stimulation in a very low [Ca2+] solution (‐log10[Ca2+] (pCa) greater than 8.0, 1 mM‐BAPTA (1,2‐bis(O‐aminophenoxy)ethane‐N,N,N’,N'‐tetraacetic acid), indicated that the sarcoplasmic reticulum (SR) lost far more Ca2+ at pH 7.6 and 8.0 than at pH 7.1 or 6.1. This indicates that SR is ‘leakier’ at alkaline pH. 6. Exposure of fibres heavily loaded with Ca2+ to solutions at pH 8.0 invariably induced a large response within 1‐6 s. This response was unaffected by inactivation of the voltage sensors and was blocked completely by 2 microM‐Ruthenium Red. This response and the ‘leak’ at alkaline pH are consistent with previous studies of the pH dependence of Ca(2+)‐activated opening of single Ca2+ release channels in lipid bilayers. 7. These findings indicate that depolarization can induce massive Ca2+ release at both acidic and alkaline pH, provided that the SR is normally loaded with Ca2+, and give further insight into the intracellular events underlying fatigue in skeletal muscle.
Fluorescence measurements of cytoplasmic and mitochondrial sodium concentration in rat ventricular myocytes.
Authors: Donoso P , Mill J G , O'Neill S C , Eisner D A
Published: December 2014

1. The fluorescent Na+ indicator SBFI was incorporated into isolated ventricular myocytes using the acetoxymethyl (AM) ester. 2. The excitation spectrum was found to be shifted about 20 nm in the cell compared to in vitro. In the cell, an increase of [Na+] decreased fluorescence at 380 nm (F380) and had no effect at 340 nm (F340). The ratio (R = F340/F380) was used as a measure of [Na+]i. 3. In vivo calibration of SBFI for [Na+]i was obtained by equilibrating [Na+] across the plasma membrane with a divalent‐free solution in the presence of gramicidin D. 4. Selective removal of the surface membrane with saponin or digitonin released only about 50% of the indicator. Following saponin treatment, cyanide or carbonylcyanide m‐chlorphenylhydrazone (CCCP) increased the apparent [Na+] measured by the remaining (presumably mitochondrial) SBFI. It is suggested that mitochondrial [Na+] is normally less than cytoplasmic. 5. Attempts to examine the effects of metabolic inhibition on [Na+]i were hampered by changes of autofluorescence due to changes of [NADH]. It is shown that this effect can be corrected for using the isosbestic signal (excited at 340 nm). 6. Inhibition of both aerobic metabolism (with CN‐) and glycolysis (glucose removal or iodoacetate) produced a gradual increase of [Na+]i. This began before the resting contracture developed and may (via Na(+)‐Ca2+ exchange) account for some of the rise of diastolic [Ca2+]i seen in previous work. The rise of [Na+]i began at about the same time as the decrease of systolic contraction and therefore at a time when [ATP]i had begun to fall.
Metabolic changes with fatigue in different types of single muscle fibres of Xenopus laevis.
Authors: Nagesser A S , van der Laarse W J , Elzinga G
Published: December 2014

1. Peak isometric force of single fast (type 1) and slow (type 3) muscle fibres of Xenopus decreased when fibres were stimulated intermittently above their predicted sustainable duty cycle at 20 degrees C. Type 1 fibres could be fatigued to zero force. In most type 3 fibres force did not decrease below 50% of the original (P0) before activation failure, as indicated by irregular contractions. 2. Fibres were rapidly frozen at different force levels and analysed by high‐performance liquid chromatography (HPLC) for ATP, IMP, phosphocreatine (PCr) and creatine (Cr). Lactate was determined enzymatically in type 1 fibres only. The relationships between force and PCr, and between force and ATP during fatigue were, apart from the range of values obtained, the same for both fibre types. When force had fallen to about 60‐80% of original, PCr was fully reduced. At lower force levels, the ATP content‐decreased, and a concomitant rise of IMP content was found. At zero force, ATP had fallen to about 25% of its value in rested type 1 fibres, and up to 200 mumol lactate (g dry weight)‐1 had accumulated. 3. Recovery from fatigue was studied in fibres where force had fallen to 0.6 P0 (both fibre types) and 0.2 P0 (type 1 only). After 1 h of recovery ATP had in all cases returned to the level measured in rested fibres. In fibres fatigued to 0.6 P0, force almost returned to its original value. However, in type 1 fibres fatigued to 0.2 P0, it returned to only 0.3 P0. After 1 h of recovery the PCr/Cr ratio in type 1 fibres was lower (probability, P less than 0.05) than in control fibres, whereas in type 3 fibres it was not significantly different from controls. 4. The relationship between peak force and stimulus frequency, which had a sigmoid shape in fully rested fibres, was drastically changed by fatiguing stimulation. Immediately after fatiguing stimulation of type 1 fibres, force hardly increased with stimulus frequency, corresponding to the observation that calcium efflux from the sarcoplasmic reticulum was decreased at high stimulus frequencies. The force‐frequency relationship of type 3 fibres was the same before and after intermittent stimulation.
Baroreflex control of stroke volume in man: an effect mediated by the vagus.
Authors: Casadei B , Meyer T E , Coats A J , Conway J , Sleight P
Published: December 2014

1. Beat‐by‐beat changes in cardiac performance in response to arterial baroreceptor stimulation induced by phenylephrine were evaluated by pulsed‐wave aortic Doppler ultrasound in eighteen subjects. Stroke distance was used as an index of stroke volume and minute distance as an index of cardiac output; peak velocity was also measured. 2. The sensitivity of the baroreceptor‐cardiac reflex was assessed by calculating the slope of the regression lines relating the changes in heart period (R‐R interval), peak velocity and stroke distance in response to the rise in systolic blood pressure (SBP) induced by phenylephrine. In ten subjects the experiment was repeated after vagal blockade by atropine. Since the tachycardia induced by vagal blockade could alter the sensitivity of the baroreflex, we compared the results obtained after atropine with those obtained during pacing at similar rates in six subjects with cardiac pacemakers. 3. As R‐R interval lengthened in response to the rise in SBP, stroke distance and peak velocity fell sharply. The subjects with a highly sensitive baroreceptor‐heart rate reflex showed the greatest fall in peak velocity and stroke distance. The slope of the relationship between R‐R interval and SBP for each subject correlated closely with that of peak velocity/SBP (correlation coefficient, r = 0.88) and stroke distance/SBP (r = 0.93) relationships. 4. Atropine virtually abolished all the cardiac reflex changes, despite a considerable increase in SBP induced by phenylephrine. At comparable heart rates achieved by pacing the sensitivity of the baroreceptor‐cardiac reflex (calculated from the slopes of the regression lines relating changes in stroke distance and in peak velocity to the rise in SBP) was maintained and was significantly greater when compared to that obtained after vagal blockade. 5. These results show that the stimulation of arterial baroreceptors is accompanied by a fall in the Doppler‐derived indices of stroke volume and cardiac output. This response is neural and is abolished by atropine, which indicates that it is mediated through the efferent vagus.
Valinomycin and excitation‐contraction coupling in skeletal muscle fibres of the frog.
Authors: Pape P C , Konishi M , Baylor S M
Published: December 2014

1. Experiments were carried out on intact frog skeletal muscle fibres to study the role of H+ and K+ as counter‐ions during the release of Ca2+ from the sarcoplasmic reticulum (SR). A specific focus was to test whether valinomycin, a potassium ionophore, markedly reduces or abolishes H+ counter‐ions fluxes across the SR membrane in response to electrical stimulation. 2. Single twitch fibres, mounted on an optical bench apparatus and stretch to long sarcomere length (3.6‐4.0 microns), were activated by single action potentials (16 degrees C). Two optical signals related to excitation‐contraction coupling were measured: (i) the 'second component' of the intrinsic birefringence signal, which is closely related to the myoplasmic free [Ca2+] transient, and (ii) the transient myoplasmic alkalization (delta pH) detectable from the pH indicator Phenol Red, a signal thought to reflect the movement of protons from the myoplasm into the SR in partial electrical exchange for released Ca2+. 3. Exposure of a fibre to 5 microM‐valinomycin produced a slight, progressive decrease in the amplitude of the birefringence signal, approximately 5‐6% per hour. This result suggests that, if anything, the peak rate at which Ca2+ is released from the sarcoplasmic reticulum is slightly decreased by valinomycin. 4. The amplitude of the Phenol Red delta pH signal, measured after exposure of a fibre to valinomycin for a period of at least 60 min, averaged 0.0020 +/‐ 0.0002 (+/‐ S.E.M.); this value is slightly smaller than, but not significantly different from (P greater than 0.05; two‐tailed t test) that measured in fibres not exposed to valinomycin (0.0025 +/‐ 0.0002). This result does not support the idea that valinomycin, but virtue of increasing the flux of K+ into the SR, markedly reduces the flux of protons during Ca2+ release. 5. Our findings of minimal changes in the birefringence and delta pH signals are consistent with the idea that, at the time of Ca2+ release, the potassium conductance of the SR membrane is large and not substantially increased by the addition of valinomycin to Ringer solution.