bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of calcium release and the effect of ryanodine in skinned muscle fibres of the toad.
Authors: Lamb G D , Stephenson D G
Published: December 2014

1. Skinned muscle fibres from the toad were used to investigate the roles of T‐system membrane potential and Ca2+ in controlling the calcium release channels of the sarcoplasmic reticulum (SR). 2. Replacement of K+ in the bathing solution with Na+ produced a large contraction which could last for 30 s or more under certain circumstances. This prolonged contraction could be quickly and completely terminated by repolarizing the fibre in the K+ solution and then immediately re‐initiated by returning to the Na+ solution. These data indicate that the membrane potential tightly controlled the substantial and prolonged release of calcium. 3. T‐system depolarization in the presence of 10 mM‐free EGTA (pCa greater than 9) markedly depleted the SR of Ca2+. This implies that depolarization of the T‐system can still trigger substantial release of Ca2+ from the SR even when the myoplasmic [Ca2+] is very low and very heavily buffered by EGTA. 4. When the SR was heavily loaded with Ca2+, substitution of a weakly buffered high [Ca2+] solution (pCa 5.4, 50 microM‐EGTA) could produce a small to moderate, transient contraction taking between 3 and 12 s to reach a peak and lasting 30 s or more. 5. This contraction may be produced at least partly by ‘calcium‐induced calcium release’ as ruthenium red (2 microM) completely blocked the responses. Moreover, repeated substitutions produced successively smaller responses in parallel with the ‘run‐down’ of the depolarization‐induced contractions. 6. Depolarization could always produce an additional large and fast response at any stage during a ‘Ca2(+)‐induced’ response. 7. In the presence of 25 microM‐ryanodine, the rapid contraction produced by T‐system depolarization was prolonged and could not be stopped by repolarization. During and after this contraction no depolarizing stimulus could induce a further contraction, even though in some fibres addition of 30 mM‐caffeine produced a maximum response which indicated that there was still a substantial amount of calcium in the SR. 8. At pCa 6.4, 25 microM‐ryanodine could itself induce a substantial slow contracture in a normally polarized fibre within 30‐60 s, after which little or no response could be induced by T‐system depolarization. At higher concentrations (25 microM) ryanodine produced a near‐maximum contraction in only a few seconds.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of nerve terminal activity on non‐quantal release of acetylcholine at the mouse neuromuscular junction.
Authors: Zemková H , Vyskocil F , Edwards C
Published: December 2014

1. Local endplate depolarization induced by anticholinesterase application to mouse nerve‐diaphragm preparations was taken as a measure of non‐quantal release of acetylcholine. 2. Non‐quantal acetylcholine release occurred within 20‐60 s after anticholinesterase application, either spontaneously or evoked by nerve stimulation. Non‐quantal release declined with time and disappeared after 3‐5 min. 3. The amplitude of stimulation‐evoked non‐quantal release increased with the frequency of stimulation and was maximal at frequencies above 50 Hz. Two stimuli were sufficient to evoke the maximal effect. 4. Micromolar concentrations of atropine, pirenzepine and vesamicol reduced the amplitude and shortened the duration of non‐quantal release. Oxotremorine (10(‐8) M) enhanced the amplitude and ouabain (10(‐4) M) prolonged the duration of non‐quantal release. 5. Our results support the idea that the non‐quantal release is due to the vesicular acetylcholine transport system which becomes transiently a part of the nerve terminal during exocytotic release of quantal acetylcholine.
Monosynaptic Ia excitation and recurrent inhibition from quadriceps to ankle flexors and extensors in man.
Authors: Meunier S , Penicaud A , Pierrot‐Deseilligny E , Rossi A
Published: December 2014

1. Ia projections and recurrent inhibition from quadriceps to tibialis anterior and soleus motoneurones were investigated in man. 2. Changes in the firing probability of individual voluntarily activated motor units were studied following electrical stimulation of the femoral nerve or quadriceps tendon tap. 3. Femoral nerve stimulation evoked an early increase in the firing probability of tibialis anterior units. This excitation was also evoked by a tendon tap, had a low threshold and its central delay was estimated to be the same as that of the homonymous monosynaptic Ia excitation. These findings strongly suggest that the femoral nerve‐induced excitation is Ia in origin and mediated through a monosynaptic pathway. 4. The frequency of heteronymous Ia excitation from quadriceps was about the same to both ankle flexor and extensor units (79 and 70% respectively). 5. In 80% of both tibialis anterior and soleus units the Ia excitation was followed by a decrease in firing probability. This inhibition had a short latency and a long duration (up to 40 ms); it always appeared with the quadriceps reflex discharge and increased with it. These findings suggest that this decrease in firing probability is due to the Renshaw inhibition evoked by the quadriceps motoneurone discharge. 6. Both the Ia excitation and the following inhibition of tibialis anterior and soleus units were also observed when the stimulation was applied to the nerve of the vastus lateralis (a pure knee extensor). 7. The functional significance of these identical projections from quadriceps to both ankle flexor and extensor motoneurones is discussed with regard to the requirements of bipedal stance and gait.
Effects of muscle history on the stretch reflex in cat and man.
Authors: Gregory J E , Mark R F , Morgan D L , Patak A , Polus B , Proske U
Published: December 2014

1. This is a report of experiments on cat and man which demonstrate effects of a muscle's previous history of contraction and length changes on the size of the stretch reflex. 2. In adult human subjects the size of the tendon jerk was measured in ankle extensor muscles by tapping the Achilles tendon. Muscle conditioning consisted of a maximum voluntary contraction with the foot dorsiflexed or plantarflexed by 30 deg from the test position, after which the subject was asked to relax while the foot was held still for several seconds before being returned to the test position and a tendon tap given. After a contraction of the lengthened muscle the tendon jerk was smaller than after a contraction of the shortened muscle. 3. The experiment was then repeated, but instead of a tendon jerk an H (Hoffmann) reflex was elicited by transcutaneous electrical stimulation of the tibial nerve in the popliteal fossa. The reflex after a conditioning contraction of the lengthened muscle was larger than after a contraction of the shortened muscle. In other words muscle conditioning produced opposite effects on the tendon jerk and H reflex. 4. These findings were confirmed in cats anaesthetized with chloralose. After a conditioning contraction of triceps surae at a length 5 mm longer than the test length (hold‐long) a quick tendon stretch produced a smaller reflex response than following a conditioning contraction with the muscle 5 mm shorter than the test length (hold‐short). The reverse trend was seen with a reflex elicited by direct electrical stimulation of the muscle nerve, which stimulates the H reflex. 5. One consequence of a conditioning contraction is that it leads to an alteration of the level of resting discharge of muscle spindles. We propose that the larger tendon jerk after a contraction of the shortened muscle is the result of changes in stretch sensitivity of muscle spindles. The reverse effect on the H reflex we attribute to a rise in the level of resting discharge of muscle spindles, which, we propose, leads to reflex inhibition of motoneurones. 6. We support this conclusion with evidence from an experiment in which the size of the conditioning step was systematically altered. Even quite small hold‐short conditioning steps led to depression of the H reflex in man and the monosynaptic reflex in cats. Recordings from single afferents showed that such small steps were also accompanied by a detectable rise in spindle resting discharge.(ABSTRACT TRUNCATED AT 400 WORDS)
Changes in force and stiffness induced by fatigue and intracellular acidification in frog muscle fibres.
Authors: Edman K A , Lou F
Published: December 2014

1. Changes in force and stiffness were recorded simultaneously during 1 s isometric (fixed ends) tetani of single fibres isolated from the anterior tibialis muscle of Rana temporaria (temperature 1‐3 degrees C; sarcomere length, 2.10 micron). Stiffness was measured as the change in force that occurred in response to a 4 kHz sinusoidal length oscillation of the fibre. Some experiments were performed in which stiffness was determined from a fast (0.2 ms) length step that was applied to a ‘tendon‐free’ segment of the muscle fibre during the tetanus plateau. 2. A moderate degree of fatigue was produced by decreasing the time between tetani from 300 s (control) to 15 s. By this treatment the maximum tetanic force (Ftet) was reversibly reduced to 70‐75% of the control value. Maximum tetanic stiffness (Stet) was related to Ftet according to the following regression (both variables expressed as percentage of their control values): Stet = 0.369 Ftet + 62.91 (correlation coefficient, 0.95; P less than 0.001). A 25% decrease in isometric force during fatigue was thus associated with merely 9% reduction of fibre stiffness. 3. Whereas the rate of rise of force during tetanus was markedly reduced by fatiguing stimulation, the rate of rise of stiffness was only slightly affected. 4. Intracellular acidification (produced by raised extracellular CO2 concentration) largely reproduced the contractile changes observed during fatigue. However, for a given decrease in tetanic force there was a smaller reduction in fibre stiffness during acidosis than during fatigue. 5. Caffeine (0.5 mM) added to the fibre after development of fatigue and intracellular acidosis greatly potentiated the isometric twitch but did not affect maximum tetanic force. This finding provides evidence that the contractile system was fully activated during the tetanus plateau both in the fatigued state and during acidosis. 6. The results suggest that the decrease in contractile strength after frequent tetanization (intervals between tetani, 15 s) is attributable to altered kinetics of cross‐bridge function leading to reduced number of active cross‐bridges and, most significantly, to reduced force output of the individual bridge. The possible role of increased intracellular H+ concentration in the development of muscle fatigue is discussed.
A study of the ouabain‐insensitive sodium efflux in barnacle muscle fibres using phorbol dibutyrate as a probe.
Authors: Bittar E E , Nwoga J
Published: December 2014

1. The resting ouabain‐insensitive Na+ efflux in muscle fibres isolated from the barnacle, Balanus nubilus, is stimulated by external or internal application of phorbol 12,13‐dibutyrate (PD). The response occurs fairly promptly and may not decay at all, or more commonly, decay rather slowly. The magnitude of the response to external or internal application of PD is dose‐dependent, the minimum effective concentration being about 10(‐8) M. 2. The response to PD fails to occur in the nominal absence of external Ca2+. Sudden removal of external Ca subsequent to peak stimulation by PD leads to almost complete reversal of the response. The response to PD of fibres suspended in Li(+)‐ASW (artificial sea water) is similar in magnitude to that of fibres suspended in Na(+)‐ASW. However, it differs in that it is of a sustained nature. 3. Calcium channel blockers, e.g. verapamil, completely prevent the response to PD from occurring. Both Cd2+ and Co2+ are less effective than verapamil. 4. Pre‐ but not post‐injection of EGTA reduces the response to PD. Pre‐ or post‐injection of Mg2+ reduces the response considerably. 5. Fibres pre‐injected with GTP show a reduced response to PD. Fibres pre‐injected with PD show a reduced response to GTP. Pre‐injection of protein kinase inhibitor is without effect on the response to PD. 6. Furosemide, piretanide and bumetanide are without effect on the response to PD. 7. DIDS (4,4'‐diisothiocyanostilbene‐2,2‐disulphonic acid) is a potent inhibitor of the response to PD but not amiloride. Pyridoxal 5‐phosphate and benzolamide are also powerful inhibitors. Pyridoxal 5‐phosphate in combination with benzolamide fails to completely abolish or reverse the response to PD. 8. Luminescence from aequorin is promptly increased by PD in a dose‐dependent manner, the minimal effective concentration being in the nanomolar range. The signal is monophasic or multiphasic in shape, and is often less than 5 min in duration. Not infrequently, however, the aequorin response fails to completely decay and the new level of resting glow remains above the original baseline level. 9. Collectively, these observations accord with a tentative general hypothesis stating that the stimulatory response of the ouabain‐insensitive Na+ efflux to PD is triggered by two mechanisms. One involves a rise in myoplasmic free [Ca2+] resulting from the entry of external Ca2+ via opened Ca2+ channels which is followed by the operation of the Na(+)‐Ca2+ exchanger in the reverse mode.(ABSTRACT TRUNCATED AT 400 WORDS)
Passive viscoelastic work of isolated rat, Rattus norvegicus, diaphragm muscle.
Authors: Syme D A
Published: December 2014

1. The passive elastic and viscous properties of isolated rat diaphragm muscle were studied, under various strains and strain rates similar to those in the animal, to measure their effects on the storage and release of mechanical potential energy. 2. Increasing the muscle length or amplitude of the displacement increased energy loss per stretch/shorten cycle, and increased the relative recovery of potential energy from the stretch during subsequent shortening. 3. Increasing strain rates increased energy loss per cycle and decreased the relative recovery of energy put into the stretch. 4. These effects were due to increased viscous resistance and increased elastic tension with increased length, and increased viscous resistance with increased strain rates. 5. The effects of increasing strain rate alone (1‐4 Hz) were small relative to the effects of a 10% increase in muscle length or the amplitude of the length cycle. 6. Diaphragm muscle movements involving low velocity, small amplitude displacements at long muscle lengths are most effective at conserving net passive mechanical energy, while short muscle lengths minimize gross energy loss.
Respiratory sinus arrhythmia is a limited measure of cardiac parasympathetic control in man.
Authors: Kollai M , Mizsei G
Published: December 2014

1. Respiratory modulation of cardiac parasympathetic activity and the relationship between respiratory sinus arrhythmia and parasympathetic control has been studied in twenty‐nine conscious, healthy young adult subjects. 2. Changes in heart period in propranolol‐treated subjects were taken as the measure of changes in cardiac parasympathetic activity; respiratory sinus arrhythmia was quantified as the difference between maximum and minimum heart periods in a given respiratory cycle; cardiac parasympathetic control was defined as the change in heart period after administration of a full dose of atropine. 3. During normal quiet breathing the inspiratory level of cardiac parasympathetic activity was not reduced to zero. The expiratory level was influenced by excitatory inputs whose activation was related to respiratory cycle length. 4. Slow breathing was associated with augmented sinus arrhythmia, but in different individuals the influence on minimum and maximum heart periods varied so that mean heart period was increased in some subjects but decreased in others. This occurred both in control conditions and after administration of a full dose of propranolol. 5. During normal breathing the correlation across subjects between respiratory sinus arrhythmia and parasympathetic control, although significant, was not close (r = 0.61). The relationship was not affected by beta‐adrenergic blockade (r = 0.63). The strength of the correlation improved when multiple regression of respiratory sinus arrhythmia was performed on three variables: parasympathetic control, respiratory cycle length and tidal volume (R = 0.93). 6. It is concluded that in conscious human subjects the respiratory modulation of cardiac parasympathetic activity is different from that observed in the anaesthetized dog, and that variations in the amplitude of respiratory sinus arrhythmia do not necessarily reflect proportional changes in cardiac parasympathetic control.
Hypothalamic influences on viscero‐somatic neurones in the lower thoracic spinal cord of the anaesthetized rat.
Authors: Lumb B M
Published: December 2014

1. Single unit electrical activity has been recorded from thirty‐four viscero‐somatic neurones in the dorsal horn of the lower thoracic spinal cord (T9‐T11) of chloralose‐anaesthetized rats. All neurones were driven by natural and/or electrical stimulation within their somatic receptive fields and gave excitatory responses to electrical stimulation of the ipsilateral splanchnic nerve. Descending influences on these neurones were tested by electrical and chemical (microinjections of DL‐homocysteic acid) stimulation of sites in the rostral hypothalamus. 2. The electrical activity of most viscero‐somatic neurones (64%) was inhibited by electrical stimulation at sites throughout the anterior hypothalamus‐preoptic region. In any one cell, responses to stimulation of visceral and somatic afferent fibres were inhibited to the same extent and any on‐going activity was also depressed. Only one cell was driven by the conditioning stimulus and the electrical activity of the remaining cells (n = 7) was unaffected. 3. At certain hypothalamic sites the effects of electrical conditioning stimulation on the responses of viscero‐somatic neurones were compared with those of local microinjection of DL‐homocysteic acid. Electrical stimulation at all sites tested (n = 7) led to an inhibition of on‐going and evoked neuronal activity. At two hypothalamic sites, both located in the ventral part of the preoptic area, microinjection of DL‐homocysteic acid resulted in a complete abolition of the responses to the test stimuli and in a cessation of any on‐going activity. Microinjection of DL‐homocysteic acid at the remaining five sites had no detectable influences on dorsal horn activity. 4. The results of this study include the first description of input properties of viscero‐somatic neurones in the lower thoracic spinal cord of the rat. In addition, these results demonstrate that transmission of visceral and somatic information through these neurones can be modulated by pathways that originate in the anterior hypothalamus‐preoptic region of the ventromedial forebrain.
Acetylcholine receptor channels in Xenopus myocyte culture; brief openings, brief closures and slow desensitization.
Authors: Kidokoro Y , Rohrbough J
Published: December 2014

1. Single acetylcholine (ACh) receptor channel currents were analysed in Xenopus myocyte culture. Channel events activated by ACh were recorded using the cell‐attached patch clamp technique at low temperatures (11.2‐15.8 degrees C) and at 100 mV hyperpolarization over the average resting membrane potential of ‐75 mV. There were two types of channels, low‐ and high‐conductance channels, as reported previously, with unitary current amplitudes 4.7 +/‐ 0.4 pA (mean +/‐ S.D., n = 17) and 6.6 +/‐ 1.2 pA (n = 14), respectively. 2. At low concentrations of ACh many brief openings (less than 1 ms in duration) were observed. Almost all of them occurred in isolation. The mean open time, which was not dependent on ACh concentration, was 63.1 +/‐ 18.1 microseconds (n = 15) for the low‐conductance channel and 80.3 +/‐ 18.7 microseconds (n = 2) for the high‐conductance channel. 3. Brief openings were frequently observed at 20 nM‐ACh, at which concentration the frequency of longer openings (longer than 1 ms) was rare. The frequency of brief openings reached maximum at 50 nM‐ACh and declined at high concentrations. The occurrence ratio between the long and brief openings increased linearly with ACh concentration. This finding suggests strongly that the brief openings are due to singly liganded receptor channels, since the long openings are most likely due to doubly liganded receptor channels. 4. A burst of openings was defined as a group of openings separated by brief closures (gaps) lasting less than 1 ms. The main component in the closed time histogram had a time constant of 25.0 +/‐ 5.1 microseconds (n = 16) for the low‐conductance channel and 32.0 +/‐ 10.2 microseconds (n = 10) for the high‐conductance channel. These values were not dependent on ACh concentration. The number of gaps per burst was 2.22 +/‐ 0.69 (n = 16) for the low‐conductance channel and 0.85 +/‐ 0.23 (n = 9) for the high‐conductance channel. The former is significantly greater than the latter. 5. During the recording from a single patch, the frequency of low‐conductance channel events declined with time at all ACh concentrations, and the rate of decline was slower in the high‐conductance channel in the same patch. The rate of decline was dependent linearly on the ACh concentration. It is most likely that this decline is due to desensitization of the receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Excitation of the corticospinal tract by electromagnetic and electrical stimulation of the scalp in the macaque monkey.
Authors: Edgley S A , Eyre J A , Lemon R N , Miller S
Published: December 2014

1. The responses evoked by non‐invasive electromagnetic and surface anodal electrical stimulation of the scalp (scalp stimulation) have been studied in the monkey. Conventional recording and stimulating electrodes, placed in the corticospinal pathway in the hand area of the left motor cortex, left medullary pyramid and the right spinal dorsolateral funiculus (DLF), allowed comparison of the actions of non‐invasive stimuli and conventional electrical stimulation. 2. Responses to electromagnetic stimulation (with the coil tangential to the skull) were studied in four anaesthetized monkeys. In each case short‐latency descending volleys were recorded in the contralateral DLF at threshold. In two animals later responses were also seen at higher stimulus intensities. Both early and late responses were of corticospinal origin since they could be completely collided by appropriately timed stimulation of the pyramidal tract. The latency of the early response in the DLF indicated that it resulted from direct activation of corticospinal neurones: its latency was the same as the latency of the antidromic action potentials evoked in the motor cortex from the recording site in the DLF. 3. Scalp stimulation, which was also investigated in three of the monkeys, evoked short‐latency volleys at threshold and at higher stimulus intensities these were followed by later waves. The short‐latency volleys could be collided from the pyramid and, at threshold, had latencies compatible with direct activation of corticospinal neurones. The longer latency volleys were also identified as corticospinal in origin. 4. The latency of the early volley evoked by electromagnetic stimulation remained constant with increasing stimulus intensities. In contrast, with scalp stimulation above threshold the latency of the early volleys decreased considerably, indicating remote activation of the corticospinal pathway below the level of the motor cortex. In two monkeys both collision and latency data suggest activation of the corticospinal pathway as far caudal as the medulla. 5. The majority of fast corticospinal fibres could be excited by scalp stimulation with intensities of 20% of maximum stimulator output. Electromagnetic stimulation at maximum stimulator output elicited a volley of between 70 and 90% of the size of the maximal volley evoked from the pyramidal electrodes. 6. Electromagnetic stimulation was also investigated in one awake monkey during the performance of a precision grip task. Short‐latency EMG responses were evoked in hand and forearm muscles. The onsets of these responses were approximately 0.8 ms longer than the responses evoked by electrical stimulation of the pyramid.(ABSTRACT TRUNCATED AT 400 WORDS)
Voltage‐dependent block by zinc of single calcium channels in mouse myotubes.
Authors: Winegar B D , Lansman J B
Published: December 2014

1. The blocking actions of Zn2+ on currents carried by Ba2+ through single dihydropyridine‐sensitive Ca2+ channels were recorded from cell‐attached patches on myotubes from the mouse C2 cell line. 2. Adding 100 microM‐Zn2+ to the patch electrode containing 110 mM‐BaCl2 produced an increase in the open channel noise, presumably arising from unresolved blocking and unblocking of the open channel by Zn2+. Adding between 200 and 1000 microM‐Zn2+ to the electrode reduced the amplitude of the unitary current in a concentration‐dependent manner. 3. The single‐channel current‐voltage (i‐V) relations showed that Zn2+ reduced the amplitude of the unitary Ba2+ currents at all potentials more negative than 0 mV. A plot of the amplitude of the unitary current in the presence of Zn2+, normalized to the amplitude in its absence, showed that block of the current depended on voltage, decreasing as the patch potential was made more negative. 4. The normalized amplitudes of the unitary currents were plotted as a function of the logarithm of [Zn2+] in the electrode. The relation for currents recorded at different potentials were fitted to an expression for binding to a single site with a KD at 0 mV of approximately 500 microM. The KD changed approximately e‐fold per 83 mV with hyperpolarization. The results suggest Zn2+ binds to a site located at approximately 15% of the potential drop from the surface membrane. 5. Reducing the concentration of Ba2+ in the patch electrode enhanced the steady‐state block of unitary currents by Zn2+. The inverse of the unitary current was plotted as a function of [Ba2+]o in the presence and absence of Zn2+; both were linear and intersected at the ordinate, indicating Ba2+ and Zn2+ compete for a channel site. 6. The kinetics of Zn2+ block of unitary Ba2+ currents were studied by amplitude distribution analysis. As expected for a simple reaction between blocking ion and open channel, the blocking rate depended linearly on the concentration of Zn2+, while the exit rate was independent of concentration. The second‐order rate coefficient for Zn2+ entry in the presence of 110 mM‐BaCl2 at 0 mV was approximately 2.0 X 10(7) M‐1S‐1, while the exit rate was approximately 16000 s‐1. 7. Both entry and exit rates increased as the membrane potential was made more negative. The entry rate increased approximately e‐fold per 66 mV, while the exit rate increased approximately e‐fold per 41 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Differential expression by nerve growth factor of two types of Ca2+ channels in rat phaeochromocytoma cell lines.
Authors: Usowicz M M , Porzig H , Becker C , Reuter H
Published: December 2014

1. Two clones of rat phaeochromocytoma PC12 cells have been used to study the expression of Ca2+ channels and their possible involvement in neuronal differentiation. One clone differentiated morphologically when exposed to nerve growth factor (NGF) for 4 days (PC12 cells), while the other clone was insensitive to NGF, but differentiated morphologically in the presence of ouabain (0.1 mM) for 7 days (PC12‐mutant cells). 2. Whole‐cell Ba2+ currents through Ca2+ channels were measured in PC12 cells at a test potential (Et) of +10 mV, from two holding potentials (Eh) of ‐90 and ‐30 mV (I‐90 and I‐30). NGF‐induced differentiation increased I‐90 by 248% and I‐30 by 133%. The cells that differentiate in the presence of ouabain had only small, if any, Ba2+ currents that did not appear to change during morphological differentiation or after the addition of NGF. 3. Barium currents in PC12 cells could be separated into two components by selective antagonists. The component of I‐90 that could be inhibited by omega‐conotoxin GVIA (omega‐CgTX) in NGF‐differentiated cells was 458 +/‐ 84 pA (mean +/‐ S.E.M.), compared with 79 +/‐ 44 pA in native cells. I‐30 was reduced by 50 +/‐ 17 pA in NGF‐treated cells and was virtually insensitive to the toxin in native cells. By contrast, the dihydropyridine (DHP) isradipine reduced I‐30 in NGF‐treated cells by 30 +/‐ 8 pA and in native cells by 20 +/‐ 3 pA. 4. Radioligand binding studies with 125I‐omega‐CgTX in PC12 cell membrane fragments and in PC12 cells showed a 2‐ to 3‐fold increase in maximal binding capacity after NGF exposure, while mutant cells showed no such change in binding capacity after treatment with NGF or ouabain. Staurosporine inhibited the effect of NGF on 125I‐omega‐CgTX binding. [3H](+)‐isradipine binding capacity was increased 1.8‐fold by NGF in depolarized PC12 cells while no change was observed in mutant cells after NGF or ouabain. There was no interaction between omega‐CgTX and DHP binding sites. 5. Both the electrophysiological and the binding data indicate a preferential expression of omega‐CgTX‐sensitive Ca2+ channels (N type) over isradipine‐sensitive channels (L type) in PC12 cells treated with NGF. By contrast, ouabain‐induced differentiation of a mutant PC12 cell line, that lacks functional NFG receptors, was not associated with the expression of Ca2+ channels.
Substance P and bombesin elevate cytosolic Ca2+ by different molecular mechanisms in a rat pancreatic acinar cell line.
Authors: Gallacher D V , Hanley M R , Petersen O H , Roberts M L , Squire‐Pollard L G , Yule D I
Published: December 2014

1. Dual‐excitation microfluorometry (Fura‐2 as indicator) was employed to monitor directly changes in the cytosolic calcium concentration [( Ca2+]i) in single cells. We investigated and compared the effects of stimulation of AR42J rat pancreatic acinar cells by two peptide agonists, substance P and bombesin. 2. Substance P (10(‐7) M) and bombesin (10(‐8) M) each gave rise to a marked, but transient, elevation in [Ca2+]i. The calcium signals evoked by the two peptides were qualitatively and quantitatively very similar. However, in the absence of extracellular Ca2+ the response to substance P, but not bombesin, was abolished. These results suggest that substance P induces calcium influx across the cell surface membrane but does not release calcium from internal stores. Bombesin in marked contrast releases calcium from intracellular stores in the absence of any detectable calcium influx. 3. Depolarization by high‐K+ extracellular solutions evoked a marked, but transient, rise in [Ca2+]i. This elevation in [Ca2+]i was strictly dependent upon the presence of Ca2+ in extracellular media. 4. Nifedipine (5 x 10(‐6) M), an antagonist of L‐type voltage‐dependent Ca2+ channels, blocked the elevations in [Ca2+]i induced by either substance P or high‐K+ solutions, but not that evoked by application of bombesin. 5. Patch‐clamp, single‐channel current recordings from cell‐attached patches of membrane confirmed the presence of voltage‐dependent calcium channels in the surface membranes of AR42J cells. Whole‐cell current recordings demonstrated voltage‐dependent inward Ca2+ (Ba2+) currents which were increased in amplitude by substance P and blocked by nifedipine. 6. The protein kinase C (PKC) activators, the phorbol diester, phorbol 1,2‐myristate 13‐acetate (PMA, 10(‐7) M), and cell‐permeable diacylglycerol analogues, 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 2.5 x 10(‐6) M) and sn‐2‐dioctanoyl glycerol (DiC8, 2.5 x 10(‐6) M), mimicked the effect of substance P, but not bombesin, in elevating [Ca2+]i in a manner that was blocked by removal of extracellular Ca2+ or application of nifedipine. 7. The PKC inhibitor, polymyxin B (2.5 x 10(‐6) M), applied 2 min prior to stimulation blocked the effects of substance P and PKC activators, but not bombesin, in elevating [Ca2+]i. 8. The calcium signals evoked by substance P and bombesin are achieved by activation of different molecular mechanisms. Substance P, the evidence suggests, activates PKC which in turn stimulates calcium influx by opening voltage‐dependent Ca2+ channels in the cell surface membranes.(ABSTRACT TRUNCATED AT 400 WORDS)
Electrophysiology of parasympathetic neurones isolated from the interatrial septum of bull‐frog heart.
Authors: Clark R B , Tse A , Giles W R
Published: December 2014

1. Whole‐cell voltage‐clamp techniques were used to study the voltage‐dependent membrane conductances in parasympathetic neurones enzymatically isolated from the interatrial septum of bull‐frog heart and maintained in short‐term (1‐10 day) tissue culture. 2. The resting potential of the isolated neurones averaged ‐55.4 +/‐ 1.1 mV (+/‐ S.E.M., n = 11). Action potentials evoked in the isolated cells by brief (1‐2 ms) current injections were similar to those recorded from neurones in the ‘intact’ septum. The amplitude of action potentials of isolated neurones averaged about 113 mV, with a peak depolarization of +32.8 +/‐ 2.8 mV and after‐hyperpolarization of ‐80.0 +/‐ 2.8 mV. 3. The pattern of membrane currents recorded using voltage clamp with ‘normal’ external (containing 110 mM‐Na+) and internal (110 mM‐K+) solutions consisted of a rapidly activating and inactivating inward current followed by a slower, sustained outward current. 4. The inward components of current were isolated by using an internal solution in which Cs+ and TEA+ (tetrathylammonium) ions replaced K+. Depolarizations from holding potentials of ‐50 to ‐70 mV produced inward currents which had an initial transient phase followed by a maintained, or very slowly inactivating, component. The current‐voltage relation for the initial transient phase reached a peak at membrane potentials near 0 mV, while the maintained phase, measured, for example, at the end of 50 ms voltage‐clamp steps, had its peak near +10 mV. 5. The transient component of inward current was carried primarily by Na+ ions, as replacement of Na+ by TEA+ in the external solution abolished the transient. This current was thus identified as a voltage‐dependent Na+ current, INa. The maintained component was greatly attenuated by removing 80‐90% of the external Ca2+ ions, and it was abolished by divalent cations such as Cd2+ (0.2‐0.4 mM), Ni2+ (0.5 mM) and La3+ (10‐100 microM). This maintained component was thus a voltage‐dependent calcium current, ICa. 6. About 80% of INa recorded in the presence of low (0.2‐0.5 mM) external Ca2+ and 2 microM‐LaCl3 was blocked by tetrodotoxin (TTX) with an apparent Kd of about 8 nM. The remaining 20% of INa was resistant to block by 2‐10 microM‐TTX. However, the ‘TTX‐resistant’ component of INa was blocked by Cd2+ (0.2‐0.4 mM). 7. The voltage‐dependent calcium current, ICa, measured in saline in which Na+ was replaced by N‐methyl‐D‐glucamine, activated near ‐40 mV and reached a peak near +10 to +15 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of dihydropyridine derivatives on force and Ca2+ current in frog skeletal muscle fibres.
Authors: Neuhaus R , Rosenthal R , Lüttgau H C
Published: December 2014

1. The effects of dihydropyridine (DHP) derivatives on current through the slow Ca2+ channel and on isometric force were investigated in short toe muscle fibres of the frog (Rana temporaria). The experiments were performed under voltage‐clamp conditions with two flexible internal microelectrodes. 2. The non‐chiral DHP derivative nifedipine was used mainly because it allowed control measurements after the inactivation of the drug with UV light. 3. In a TEA sulphate solution containing 4 mM‐free Ca2+, nifedipine (1 microM) caused no relevant alterations in the time course of successive contractures induced by depolarizing steps to 0 mV of 3.5 min duration followed by a restoration time at ‐90 mV of 1.5 min. 4. When external Ca2+ was replaced by Mg2+, nifedipine caused a dose‐dependent shortening of contractures. The effect reached saturation at about 50% of shortening with 1‐5 microM‐nifedipine. In the absence of divalent cations and with Na+ being the only metallic cation in the solution, shortening became more pronounced and maximum force decreased. 5. The application of 2 microM‐nifedipine to a Ca2(+)‐free, Mg2(+)‐containing solution shifted the voltage dependence of force inactivation by 5‐10 mV to more negative potentials. 6. Force activation was facilitated by nifedipine. In the presence of 2 microM‐nifedipine in a Ca2(+)‐containing solution, threshold potentials (rheobase) as negative as ‐75 mV were measured under microscopical observation. UV irradiation shifted the threshold potential back to the normal value of about ‐50 mV. 7. The slow Ca2+ inward current was blocked almost completely by approximately 5 microM‐nifedipine, even when induced from negative holding potentials (‐90 to ‐120 mV), i.e. under conditions where normal phasic contractures could still be observed. 8. Nifedipine (0.8 microM) caused a shift of the voltage dependence of current inactivation (V0.5) by 4 mV from ‐26 to ‐30 mV and at negative holding potentials (‐90 mV), a reduction of maximum current by 35%. 9. The voltage dependence of current activation was not significantly altered by nifedipine (2 microM). 10. It is assumed that nifedipine binds with low affinity to the resting state of the DHP receptor (KD 0.8 microM) and with high affinity (KD 1 nM) to the inactivated and the active state (or to a precursor of this state). These assumptions could explain the relatively small shift of the inactivation curves (points 5 and 8) to more negative potentials and the facilitation of force activation (point 6).
Consequences of CO2 acidosis for transmembrane Na+ transport and membrane current in rabbit cardiac Purkinje fibres.
Authors: Bielen F V , Bosteels S , Verdonck F
Published: December 2014

1. The influence of sarcolemmal Na(+)‐H+ exchange on intracellular Na+ activity (aiNa), intracellular pH (pHi) and membrane holding current (Ih) was investigated in rabbit cardiac Purkinje fibres. pHi and aiNa were measured with liquid sensor ion‐selective microelectrodes. A two‐microelectrode voltage clamp was used while recording pHi or aiNa.pHi was varied by alternating a nominally CO2‐free HEPES buffer and a CO2‐HCO3‐buffer. 2. The intrinsic buffer capacity was calculated from the decrease in pHi after addition of CO2. The most accurate estimate was obtained when transmembrane pHi regulation was blocked and equalled 18.4 +/‐ 1.0 mequiv H+/pH unit (mean +/‐ S.E.M.). 3. aiNa started to rise when pHi fell below 7.0. A hyperpolarization paralleled the increase in aiNa. The magnitude of the rise in aiNa and the hyperpolarization were steeply dependent on pHi. 4. Inhibition of the Na(+)‐K+ pump by K(+)‐free superfusion increased aiNa. The rate of rise in aiNa was highly dependent on pHi. The rates of rise in 5% (pHi = 7.00 +/‐ 0.03), 7% (pHi = 6.89 +/‐ 0.04) and 15% CO2 (pHi = 6.74 +/‐ 0.02) at constant external pH (pHo) relative to the rate in HEPES solution (pHi = 7.24 +/‐ 0.02) were: 1.5 +/‐ 0.2, 2.4 +/‐ 0.1 and 3.1 +/‐ 0.2. The acid‐induced rise in aiNa was abolished by 2 mM‐amiloride. 5. Extracellular acidosis slowed down the recovery of pHi and depressed the rate of rise in aiNa upon intracellular acidification. When both pHi and pHo were decreased to 6.7 the acid‐dependent rate increase fell to about 10% of the value found at pHo 7.4. 6. The tetrodotoxin (TTX)‐sensitive Na+ current was not influenced by the change in pHi in the range 6.7‐7.2. 7. Intracellular acidosis was associated with an early aiNa‐independent depolarization and an inward shift in Ih. Current‐voltage plots revealed that the initial inward current shift reversed at ‐81.5 mV on average, showed inward rectification and was largely depressed in the presence of 1 mM‐Ba2+. These observations indicate a decrease in K+ conductance when pHi falls. 8. The increase in aiNa elicited a Na(+)‐K+ pump‐dependent outward current which could override the initial aiNa‐independent current shift. At a pHo of 6.7 the initial fall in Ih remained, while the secondary outward current was largely depressed. 9. The rate of active Na+ extrusion and Na(+)‐K+ pump current were suppressed by about 30% at pHi 6.7 compared to pHi 7.2.(ABSTRACT TRUNCATED AT 400 WORDS)
Stretch‐sensitive channels in developing muscle cells from a mouse cell line.
Authors: Franco A , Lansman J B
Published: December 2014

1. Recordings of single‐channel activity were made from cell‐attached patches on mouse C2 muscle cells at morphologically identifiable stages of myogenesis in vitro. We have identified Ca2(+)‐permeable, cation‐selective channels that are gated by applying suction to the patch electrode and by changes in membrane potential and have analysed single‐channel properties as well as channel expression during myogenesis. 2. Single‐channel activity could be detected when the membrane was held at steady negative potentials. With monovalent cations in the electrode, the single‐channel current‐voltage (i‐V) relations were linear. The channel is permeable to Li+, Na+, K+, Rb+ and Cs+, but is not strongly selective among the monovalent cations as judged by measurements of single‐channel conductance and reversal potential. 3. With 110 mM of either CaCl2 or BaCl2 as the only inward change carrier, slope conductances were approximately 13 and 24 pS and currents reversed at approximately +22 and +17 mV, respectively. The relative permeability of Ca2+ to K+ calculated from the constant‐field equation was PCa/PK = approximately 2. 4. Channel openings occurred as bursts of brief openings and closings separated by much longer closed periods. Closed‐time histograms were best fitted with three exponential components, while histograms of burst duration were best fitted with two exponential components, reflecting the short and long bursts in the single‐channel records. 5. Applying suction to the patch electrode while recording at steady negative membrane potentials produced channel openings to discrete current levels. Mean channel open probability depended linearly on the square of the applied pressure and was greater at positive membrane potentials. The permeability of the channel to monovalent and divalent cations was indistinguishable from the spontaneous activity recorded at steady negative potentials. 6. Channel activity recorded from cell‐attached patches in the absence of applied pressure depended on membrane potential increasing approximately e‐fold per 38 mV with depolarization. Analysis of the kinetics of the response to membrane potential showed that the depolarization reduced the duration of the slowest component of the closed‐time distribution. 7. The lanthanide cation gadolinium (Gd) reduced the amplitude of the unitary currents in a concentration‐dependent manner. The amplitudes of both inward and outward currents were reduced to the same extent suggesting block is voltage‐independent. Gd produced half‐maximal inhibition of the unitary current at approximately 6 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
Role of G‐proteins in muscarinic receptor inward and outward currents in rabbit jejunal smooth muscle.
Authors: Komori S , Bolton T B
Published: December 2014

1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of rabbit jejunum were held under voltage clamp using patch pipettes and membrane currents measured. The effects of carbachol or caffeine applied externally were examined in cells dialysed with normal pipette solutions or with a solution containing heparin (which blocks receptors for D‐myo‐inositol 1,4,5‐trisphosphate, InsP3), guanosine 5‐O‐(gamma‐thio)triphosphate (GTP gamma S) or guanosine 5‐O‐(beta‐thio)diphosphate (GDP beta S). 2. Outward current in response to application of carbachol or caffeine was considered to represent the opening of calcium‐activated potassium channels in response to a localized rise in the free ionized calcium concentration occasioned by the rapid discharge of stored calcium (Ca) by these agents. 3. Heparin included in the pipette solution blocked outward current to muscarinic receptor activation by carbachol but not that to caffeine, suggesting that receptor‐evoked discharge of stored cellular Ca is caused by InsP3 action. However, heparin did not affect muscarinic‐receptor inward current. 4. After dialysis with 0.1‐0.5 mM‐GTP gamma S, carbachol inward current was evoked in two out of three of the cells; after dialysis with 0.1‐0.2 mM‐GTP gamma S for an average of 7.7 min it was 80% of the normal response; after dialysis for an average of 8.6 min with 0.5 mM‐GTP gamma S it was 31% of the normal response. In contrast, 0.1 mM‐GTP gamma S reduced caffeine outward current by 93% after an average 4.5 min dialysis and spontaneous transient outward currents (STOCs) were abolished in 2.9 min on average. 5. Carbachol inward current (at ‐40 or ‐50 mV) and carbachol outward current (at 0 mV) in responding cells were reduced only by half after 8‐10 min dialysis with 1 mM‐GDP beta S which has been shown in portal vein cells to antagonize the depletion of Ca stores by intracellular GTP gamma S (Komori & Bolton, 1989). After 8‐10 min dialysis with 5 mM‐GDP beta S outward current was 27% of normal. However, if GDP beta S was present, outward current generally could not be evoked by a second application of carbachol. 6. The discharge of Ca stores by dialysis with 0.1 mM‐GTP gamma S was prevented completely by heparin included in the pipette solution, suggesting that activation of a G‐protein associated with phospholipase C (PLC) enzyme accelerates PLC activity. InsP3 production and depletion of Ca stores.(ABSTRACT TRUNCATED AT 400 WORDS)
Atrial peptide natriuresis in the rat without genuine rise in filtration rate or wash‐out of medullary electrolytes.
Authors: Badzyńska B , Sadowski J , Dobrowolski L
Published: December 2014

1. Effects of synthetic atrial natriuretic peptide (ANP) on renal excretion, total renal blood flow (RBF), glomerular filtration rate (GFR) and tissue electrical admittance (reciprocal impedance, an estimate of tissue electrolytes) were determined in pentobarbitone‐anaesthetized rats. GFR was measured both as inulin clearance (Cin) and as a product of renal plasma flow (RPF) and inulin extraction ratio (Ein). 2. With the lowest dose of ANP (0.35 micrograms/(kg min) I.V.) a 5‐fold increase in sodium excretion occurred without measurable change in Cin, RPF x Ein nor medullary electrolyte concentration estimated from tissue electrical admittance. 3. With medium and high dosage (2 and 6 micrograms/(kg min), respectively), major and rapid increases in sodium excretion and urine flow were associated with an acute increase in Cin but not RPF x Ein. 4. The RBF increase observed in all groups of rats was not dose‐related and did not parallel the natriuresis. Electrolyte concentration in the medullary tissue showed a modest transient decrease in rats given medium and high ANP doses. 5. We conclude that pronounced ANP natriuresis can develop in the absence of a measurable increase of GFR, estimated by a method not subject to urinary dead space error (RPF x Ein). The small transient decrease in medullary tissue electrolytes observed with higher peptide doses does not support solute wash‐out as an important mechanism of increased sodium excretion.