bluuebunny/crossref_metadata_2025 huggingface dataset:
Dependence of spontaneous release at frog junctions on synaptic strength, external calcium and terminal length.
Authors: Grinnell A D , Pawson P A
Published: December 2014

1. The calcium dependence of spontaneous transmitter release from nerve terminals of different lengths was examined at neuromuscular junctions in frog muscle. Miniature endplate potential (MEPP) frequency was positively correlated with the endplate potential (EPP) quantal content and was dependent on external Ca2+. The higher the resting MEPP frequency in a 0.25 mM‐Ca2+ Ringer solution, the greater the dependence on external Ca2+. MEPP frequency in all terminals dropped to approximately the same low level in a Ca2(+)‐free Ringer solution containing EGTA. This suggests that terminals with higher release levels have a larger Ca2+ influx at rest. 2. Several tests were done to try to characterize the mode of Ca2+ entry into resting terminals. omega‐Conotoxin (omega‐CgTx) blocked evoked release and reduced MEPP frequency, but not as effectively as zero Ca2(+)‐EGTA Ringer solution. Some component of Ca2+ influx thus appears to enter through channels insensitive to omega‐CgTx. Tetrodotoxin (TTX) did not affect MEPP frequency, indicating that the Ca2+ did not enter through TTX‐sensitive Na+ channels that might be opening spontaneously at rest. Hyperpolarization of the terminal by reducing the K+ in the Ringer solution caused no consistent differences in MEPP frequency, suggesting that the Ca2+ influx is relatively insensitive to small changes in membrane potential around the resting level. Strong buffering of the Ringer solution with citrate, to overwhelm any differences in Ca2+ buffering within different junctional clefts, had no significant effect on the MEPP frequency. 3. Evidence that the Na(+)‐Ca2+ exchanger helps set the internal Ca2+ level was obtained. Reduction of the Na+ concentration in the Ringer solution caused increases in MEPP frequency ranging from 6 to 440%. However, these changes were not correlated with resting MEPP frequency, hence differences in MEPP frequency probably are not the result of differences in Na(+)‐Ca2+ exchanger function in terminals having a uniform Ca2+ leak. 4. Although MEPP frequency was generally correlated with quantal content, in subsets of junctions grouped according to their similar quantal contents, there was a positive correlation between MEPP frequency and terminal length. 5. In zero Ca2(+)‐EGTA Ringer solution, the low residual MEPP frequency is independent of terminal length, even when MPP frequency is sharply increased by tetanic stimulation.
Centre components of cone‐driven retinal ganglion cells: differential sensitivity to 2‐amino‐4‐phosphonobutyric acid.
Authors: Chen E P , Linsenmeier R A
Published: December 2014

1. Changes in different components of the cone‐driven centre responses of cat retinal ganglion cells were studied before and during local application of 2‐amino‐4‐phosphonobutyric acid (APB). Responses were elicited with bar stimuli whose luminance was above ('brighter') or below ('dimmer') the photopic background luminance. The bars were centrally located, and were similar in width to the receptive field centre. 2. APB acted differently on the on‐ and off‐centre cells. For on‐centre X and Y cells, all components of the responses to bright and dim bars were diminished by APB. For the off‐centre X and Y cells. APB reduced all components except the transient increase in firing rate when the bright bar was turned off or when the dim bar was turned on. 3. These results suggest that the centre response mechanism of off‐centre X and Y cells comprises APB‐sensitive and APB‐resistant components. The APB‐sensitive component is more sustained and responds to both brightening and dimming stimuli. The APB‐resistant component is more transient and responds primarily to dimming stimuli. For on‐centre X and Y cells only APB‐sensitive components could be demonstrated. 4. Experiments with stationary sinusoidal gratings modulated at 0.5‐10 Hz showed that responses of off‐centre cells were more affected by APB at low than at high temporal frequencies, confirming that the APB‐sensitive pathway is responsible for more of the low temporal frequency responses. As expected from the responses to bar stimuli, APB had a uniform effect at all temporal frequencies in on‐centre cells. 5. For off‐centre cells, the APB‐sensitive component is probably derived from input from depolarizing bipolar cells, and the APB‐resistant component is derived from hyperpolarizing bipolar input, although one or both pathways could also involve amacrine cells. The combination of these pathways increases the range of temporal frequencies to which the cell can respond and also increases the range of response amplitudes. 6. The lack of differential effects on on‐centre cells may have several explanations. The most likely explanations are that only depolarizing bipolars contribute significantly to the centre responses of these cells under the conditions of these experiments, or that there is an APB‐sensitive synapse somewhere in the retina besides the one from cones to depolarizing bipolars.
cis‐Fatty acids, which activate protein kinase C, attenuate Na+ and Ca2+ currents in mouse neuroblastoma cells.
Authors: Linden D J , Routtenberg A
Published: December 2014

1. Activation of protein kinase C (PKC) by phorbol esters or diacylglycerols has been shown to modulate a number of ionic currents carried by Ca2+, K+ and Cl‐. Recently, it has been demonstrated that PKC may be activated by cis‐fatty acids in the absence of either phospholipid or Ca2+. We wished to determine if this new class of PKC‐activating compound would also modulate ionic currents. To this end we applied the whole‐cell voltage‐clamp technique to N1E‐115 neuroblastoma cells. 2. Analysis of families of currents evoked under voltage clamp by depolarizing steps from a holding potential of ‐85 mV during external application of 5 microM‐oleate (a cis‐fatty acid) showed a 36% reduction of the peak inward current with no shift in either the peak or the reversal potential of the current‐voltage relation and no alteration of outward current. 3. External application of the cis‐fatty acids oleate, linoleate and linolenate reversibly attenuated voltage‐dependent Na+ current with approximate half‐maximal dose values of 2, 3, and 10 microM respectively. Oleate was approximately 2 times more potent when applied internally (ED50 = 1 microM). Externally applied elaidate (a trans‐isomer of oleate) and stearate (a saturated fatty acid) which do not activate PKC, had no effect. Since cis‐fatty acids are known to fluidize membranes, as well as to activate PKC, we sought to dissociate these functions by applying compounds that fluidize membranes but do not activate PKC: methyloleate and lysophosphatidylcholine. Neither compound affected Na+ current when applied externally at concentrations of 1‐50 microM. 4. In contrast to cis‐fatty acids, three classical PKC activators, phorbol‐12.13‐dibutyrate (PDB), phorbol‐12.13‐diacetate (PDA), and 1.2‐oleoylacetylglycerol (OAG) were found to have no effect on the voltage‐dependent Na+ current when applied externally at 10 nM‐1 microM (phorbol esters) or 1‐150 microM (OAG) for incubation periods up to 1 h. 5. External application of the PKC inhibitors polymyxin B, H‐7, sphingosine and staurosporine blocked the attenuation of the Na+ current by cis‐fatty acid in a dose‐dependent manner, with maximal inhibition occurring at doses of 50, 10, 200 and 0.1 microM, respectively. The cyclic nucleotide‐dependent protein kinase inhibitor H‐8 was much less effective in blocking the cis‐fatty acid effect. Polymyxin B and staurosporine were more potent when applied internally. 6. Chronic (24 h) exposure to 1 microM phorbol‐12‐myristate‐13‐acetate (TPA) was employed to down‐regulate PKC.(ABSTRACT TRUNCATED AT 400 WORDS)
Sugar‐dependent selective induction of mouse jejunal disaccharidase activities.
Authors: Collins A J , James P S , Smith M W
Published: December 2014

1. Sugar‐containing diets chosen not to affect intestinal structure or enterocyte turnover have been fed to mice previously maintained on a low carbohydrate diet in order to determine their ability to induce disaccharidase enzymes in the small intestine. 2. Glucose‐, fructose‐ and 3‐O‐methyl‐glucose‐containing diets increased sucrase and maltase but not lactase activities in mouse jejunal homogenates. These effects were either absent or negligible in more distal regions of the small intestine. 3. Placing mice on glucose‐, fructose‐ or 3‐O‐methyl‐glucose‐containing diets was further shown, by quantitative cytochemistry, to cause a 1.6‐, 2.6‐ and 3.2‐fold increase in the initial rate at which alpha‐glucosidase activity (sucrase + maltase) appeared in the brush‐border membrane of developing enterocytes. 4. The time during which alpha‐glucosidase activity increased in enterocyte brush‐border membranes fell from 30 h for low carbohydrate fed mice to 21, 19 and 17 h in mice fed glucose, fructose and 3‐O‐methyl‐glucose respectively. Change of diet had no effect on the kinetics of lactase expression by developing enterocytes. 5. Maximal alpha‐glucosidase activity detected in enterocyte brush‐border membranes is equal to RT, where R is the initial rate of enzyme appearance and T is the time during which this rate operates. The ability of sugars to increase R selectively, but only at the expense of T, defines unexpected limits to the capacity of enterocytes to adapt to changes in luminal nutrition. 6. The above results are discussed in relation to other aspects of enterocyte differentiation recently subjected to quantitative analysis. The need to standardize other aspects of intestinal physiology and redefine the energy content of diets containing non‐metabolizable substrates in this type of work is also emphasized.
Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non‐quantal release.
Authors: Grinnell A D , Gundersen C B , Meriney S D , Young S H
Published: December 2014

1. Acetylcholine (ACh) release from enzymatically exposed frog motor nerve terminals has been measured directly with closely apposed outside‐out clamped patches of Xenopus myocyte membrane, rich in ACh receptor channels. When placed close to the synaptic surface of the terminal, such a membrane patch detects both nerve‐evoked patch currents (EPCs) and spontaneous quantal ‘miniature’ patch currents (MPCs), from a few micrometres length of the terminal, in response to ACh release from the nearest three to five active zones. 2. Chemical measurements of ACh efflux from whole preparations revealed a spontaneous release rate of 4.1 pmol (2 h)‐1, and no significant difference in resting efflux between enzyme‐treated and control preparations. The ratio of enzyme‐treated to contralateral control muscle efflux averaged 1.17, indicating that enzyme treatment did not affect spontaneous ACh release. Vesamicol (1.7 microM), which blocks the ACh transporter in synaptic vesicles, decreased the spontaneous release of ACh to 67% of control. 3. In the absence of nerve stimulation, the frequency of single‐channel openings recorded by outside‐out patch probes adjacent to nerve terminals was very low (1‐2 min‐1), and little different at a distance of hundreds of micrometres, suggesting that if ACh was continually leaking from the terminal in a non‐quantal fashion, the amount being released near active zone regions on the terminal was below the limit of detection with the patches. 4. Direct measurements of the sensitivity of the patches, coupled with calculated ACh flux rates, lead to the conclusion that the amount of ACh released non‐quantally from the synaptic surface of the frog nerve terminal is less than one‐tenth the amount expected if all non‐quantal release is from this region of the terminal membrane. 5. Following a series of single nerve shocks or a 50 Hz train of nerve stimuli, the frequency of asynchronous single‐channel openings increased for several seconds. This transient increase in channel openings was not sensitive to movement of the patch electrode a significant distance (4 microns) away from the active sites, or to manipulations previously reported to block non‐quantal transmitter leakage, including addition of 10 mM‐Ca2+ or 1.7 microM‐vesamicol to the bath. These channel openings appear to be due to an accumulation of ACh which originated from many evoked quanta, and not the effect of locally increased non‐quantal ACh release due to nerve stimulation. 6. We conclude that transmitter leakage at adult frog terminals is either localized to a source other than the synaptic surface of the nerve terminal, or released in a widespread and diffuse fashion from many sources, which may include the nerve terminal.
Ultrastructure of transport pathways in stressed synovium of the knee in anaesthetized rabbits.
Authors: Levick J R , McDonald J N
Published: December 2014

1. The hydraulic conductance of the synovial lining of a rabbit knee increases greatly when intra‐articular pressure (IAP) is raised above approximately 9 cmH2O (yield point). To investigate the cause, synovium was fixed in situ by perfusion at controlled IAP and prepared for transmission electron microscopy. Micrographs of synovium fixed below yield pressure (atmospheric pressure and 5 cmH2O IAP, ten joints) and above it (25 cmH2O IAP, five joints) were analysed by morphometry. 2. The discontinuous cellular lining consisted of fibroblast‐like cells (67%) and macrophage‐like cells (33%) separated by interstitium‐filled gaps. Interstitium formed 26‐36% of the surface below yield pressure. Depending on sample site the surface gaps averaged 1.9 +/‐ 0.2 to 2.4 +/‐ 0.2 microns wide below yield pressure (mean +/‐ S.E.M. throughout). Above yield pressure the mean gap width increased by 42‐64% (P less than 0.05, analysis of variance). 3. The qualitative and quantitative composition of the lining varied with distance below the surface. In a plane 5 microns deep, the intercellular distances and interstitial area fraction were almost double those at the surface. Classic periodic collagen fibrils (diameter 50 +/‐ 3 nm) abounded at 5 microns depth whereas the surface interstitium was richer in Ruthenium Red‐staining microfibrils (diameter 9.3 +/‐ 0.7 nm) associated with 93 nm period fibrous long‐spacing bundles. 4. Averaging over all the tissue between the surface and the 5 microns deep plane, the mean interstitial volume fraction was 0.61 +/‐ 0.05 at 5 cmH2O and 0.67 +/‐ 0.02 at 25 cmH2O (n.s.). 5. Capillary fenestrae (8.5 +/‐ 1.1 per fenestrated profile) and intercellular junctions were unaltered at high IAP. The tortuosity of the capillary‐to‐joint cavity path was 1.50 +/‐ 0.01 below yield pressure and 1.86 +/‐ 0.24 at 25 cmH2O (n.s.). 6. Intra‐articular tracers (ferrocyanide, ferritin and glycogen) permeated synovial interstitium without evidence of preferential pathways. Ferrocyanide delineated the capillary intercellular junction as a permeable channel. Ferritin and glycogen were phagocytosed by the macrophages. 7. In suprapatellar areolar synovium, the most extensive and most altered tissue, the ratio of interstitial area to path length increased maximally 4.1 times between 5 and 25 cmH2O IAP. This represents a substantial contribution to the physiologically estimated rise in interstitial conductance (14 x) but does not wholly explain it.
Long‐latency stretch reflexes of two intrinsic muscles of the human hand analysed by cooling the arm.
Authors: Matthews P B
Published: December 2014

1. The arm was cooled to examine the effect of slowing nervous conduction on the latency of various reflex responses of the abductor digiti minimi (ADM) and the first dorsal interosseus (FDI) muscles. A reflex dependent upon slow afferents acting with a short central delay should be slowed by more than one of comparable initial latency, but dependent upon fast afferents acting with a long central delay. The F wave seen in the EMG on stimulating the ulnar nerve was used to assess the effect of cooling in slowing fast fibres. 2. Cooling showed that the prominent long‐latency excitation evoked on stretching one or other muscle by displacing the appropriate finger is medicated by fast afferent fibres. 3. Both fast cutaneous and group I muscles afferents were found to be capable of contributing to the long‐latency responses. The muscle afferent contribution was consistently dominant for slow 'stretches', while a cutaneous afferent contribution was sometimes recognizable for finger taps. 4. The distinction proved possible because the cutaneous afferents from the relevant finger run in the median nerve for FDI and the ulnar nerve for ADM. The cooling produced much greater slowing of conduction for the ulnar nerve. 5. The prior suggestion that spindle group II afferents are responsible for the major part of the long‐latency component of the human stretch reflex thus fails to stand; however, some contribution has not been finally excluded.
Autogenetic inhibition from contraction receptors in the decerebrate cat.
Authors: Jack J J , Kullmann D M , Roberts R C
Published: December 2014

1. Autogenetic inhibition from contraction receptors was measured by eliciting contractions of the soleus muscle in the decerebrate cat. Inhibitory feedback was detected when the tension increment f, produced by stimulating motor fibres in the presence of a background reflex contraction, was smaller than the tension d elicited by the same stimulus in the absence of reflex action. Tendon vibration was applied throughout to clamp primary spindle afferents at a constant firing rate, thereby preventing spindle unloading from disfacilitating the reflex contraction. 2. The reduction in tension d‐‐f varied roughly linearly with the size of the tension stimulus f. Feedback gain was proportional to d‐‐f/f, i.e. the ratio of inhibited tension to stimulus tension. It was computed by averaging over several measurements obtained with stimuli of different sizes, and ranged between 0 and 0.88 in ten animals. The average gain, 0.39, implies that voluntary muscle force is reduced by approximately 27% through the direct inhibition of alpha‐motoneurones from homonymous contraction receptors. 3. Inhibitory feedback gain did not appear to co‐vary with the background reflex contraction. When measured without vibration, however, a positive covariance did emerge, suggesting that this is due to unloading of muscle spindles, either by extrafusal muscle shortening or by inhibition of fusimotor neurones. 4. Inhibited tension varied linearly with the estimated increment in Ib afferent firing. On the assumption that group Ib afferents carried the entire inhibitory signal, inhibitory feedback gain measured with vibration was used to predict the size of the gain if vibration had not been applied. Feedback gain calculated in this way was reduced by still did not vary with reflex tension. 5. In one animal with signs of brain stem trauma, feedback gain was increased to around six. It is argued that inhibitory feedback in the intact animal can rise to comparable values, as a result both of convergence of signals from different muscles and of supraspinal facilitation.
Kinetic properties of the cardiac T‐type calcium channel in the guinea‐pig.
Authors: Droogmans G , Nilius B
Published: December 2014

1. The kinetic properties of T‐type Ca2+ channels were examined in single ventricular cells from guinea‐pig hearts using the cell‐attached configuration of the patch‐clamp technique. 2. T‐type Ca2+ channel activity has been observed in 44 out of 139 patches. The density of these channels was estimated at 0.1‐0.3 micron‐2. The T‐type Ca2+ channel responds to a depolarizing voltage step either with a burst of openings which appears with a distinct delay or with no openings at all. The mean number of bursts per record for the records showing channel activity is 1.1. The probability of observing a blank sweep is high and amounts to 0.65 +/‐ 0.02 (n = 26). 3. With 110 mM‐Ca2+ in the pipette solution, the slope conductance calculated from the current‐voltage relationship of the single‐channel current in the range between ‐50 and +10 mV is 6.8 pS. 4. Openings to a subconductance level of about 50% of the main level could be resolved. All possible transitions between the subconductance and the main level were observed, indicating that the cardiac T‐type Ca2+ channel possesses a substate. 5. The macroscopic steady‐state activation and inactivation, as determined from ensemble‐averaged currents, could be described by Boltzmann functions. Half‐maximal activation and inactivation occur at ‐14 and ‐60.7 mV, the slope parameters of these curves are 10.8 and 5.6 mV respectively. The maximum (peak) open probability is 0.15. 6. The ensemble‐averaged current decays monoexponentially. The time constant is strongly voltage dependent and decreases at less negative potentials. 7. The open times are monoexponentially distributed. The mean open time of the channel does not depend on either the holding or the test potential, and has a mean value of 1.4 ms. The distribution of the closed times is biexponential. The fast mean closed time is also voltage independent with a mean value of 0.48 ms. The slow mean closed time increases with voltage from 1.9 ms at ‐40 mV to 8.8 ms at 0 mV. The mean burst duration also increases with voltage from a value of 4.9 ms at ‐40 mV to 13.9 ms at ‐10 mV. 8. The convolution of the first‐latency distribution with that of the burst duration closely fits the open probability calculated from the ensemble‐averaged current. The mean first latency is also closely correlated with the macroscopic time constant of inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)
The activation of calcium and calcium‐activated potassium channels in mammalian colonic smooth muscle by substance P.
Authors: Mayer E A , Loo D D , Snape W J , Sachs G
Published: December 2014

1. The regulation of Ca2(+)‐activated K+ channels by the agonist substance P in freshly dissociated smooth muscle cells from the rabbit longitudinal colonic muscle was characterized using the patch clamp technique. 2. In the cell‐attached recording mode, when pipette and bath solutions contained equal [K+] (126 mM), the Ca2(+)‐activated K+ channels showed a linear current‐voltage relationship (between ‐50 mV and 50 mV) with a slope conductance of 210 +/‐ 35 pS (n = 12). Reversal potential measurements indicated that the channel was highly selective for K+ over Na+ (PK/PNa = 110). 3. Channels were activated by depolarizing membrane voltages and cytosolic Ca2+, and in inside‐out patches channel activation depended sigmoidally on voltage and [Ca2+]. The potential for half‐activation at a cytosolic [Ca2+] of 5 x 10(‐6) M was 0 mV. A tenfold increase in cytosolic Ca2+ resulted in a 60 mV shift of the sigmoidal voltage activation curve to more negative potentials. 4. Threshold concentrations of substance P (10(‐12) M), which did not result in cell contraction, caused a prolonged activation of K+ channels. The K+ channels were observed to open in clusters: simultaneous opening of multiple channels was interrupted by complete, prolonged channel closure. 5. Lowering bath [Ca2+] to submicromolar concentrations abolished the effect of substance P. The activation of K+ channels by substance P (10(‐12) M) was also inhibited by the dihydropyridine nifedipine (10(‐6) M), a blocker of L‐type Ca2+ channels. 6. In the whole‐cell recording mode, with the pipette solution containing 126 mM‐KCl, 0.77 mM‐EGTA and 1 mM‐ATP, depolarization from a holding potential of ‐70 mV elicited outward currents which increased to steady‐state values. These were K+ currents as they were blocked by TEA (tetraethylammonium, 30 mM) and Ba2+ (1 mM) and were abolished when pipette K+ was replaced by Cs+. 7. The depolarization‐activated outward current was not affected by lowering extracellular [Ca2+] or by the Ca2+ channel antagonists Cd2+ (200 microM), nifedipine (10(‐6)‐10(‐5) M) or verapamil (10(‐6) M). The current was greatly reduced when the EGTA concentration in the pipette solution was increased from 0.77 to 10 mM. 8. When the pipette solution contained CsCl, membrane depolarization activated inward currents. The peak inward current was identified as current through L‐type Ca2+ channels based on its voltage‐ and time‐dependent kinetics, and its modulation by dihydropyridines.(ABSTRACT TRUNCATED AT 400 WORDS)
Ca2(+)‐activated K+ current involvement in neuronal function revealed by in situ single‐channel analysis in Helix neurones.
Authors: Gola M , Ducreux C , Chagneux H
Published: December 2014

1. The properties of single calcium‐activated potassium channels (or C‐channels) were studied in cell‐attached patches using the patch‐clamp technique. Experiments were performed on identified Ca2(+)‐dependent U cells in juvenile specimens (1‐2 months old) of Helix aspersa. 2. The criteria used to identify C‐channels were based on comparison between macroscopic C‐currents and currents reconstructed from unitary recordings. Both currents had a slow activation rate at large positive potentials which turned into fast activation after large Ca2+ entries. Both currents were blocked by intracellularly injected EGTA. 3. The unitary conductance in normal (5 mM) or reduced (0.5 mM) [K+]o ranged from 24 to 65 pS (mean +/‐ S.D., 48 +/‐ 13; n = 64). With 85‐110 mM [K+]o, which is approximately equal to the internal [K+], the conductance was 64 pS and the reversal potential was approximately 0 mV. 4. C‐channels in U cells were distributed in clusters of three to ten channels (mean 5.05 channels in seventy‐five patches). Calcium channels were present in patches containing clustered C‐channels. C‐channels within clusters behaved independently. 5. With patch electrode containing 8 mM‐calcium, C‐channels opened transiently upon patch depolarization. Reopenings in quiescent depolarized patches were induced by whole‐cell spikes triggered by current pulses applied to an intracellular electrode. Apparent inactivation of C‐channels in depolarized patches was in fact due to a decrease in [Ca2+]i resulting from inactivation of Ca2+ channels. 6. Calcium‐free saline solutions in the patch electrodes prevented C‐channels from opening upon patch depolarization. Entry of calcium through the surrounding membrane induced delayed openings in the patch. Peak opening probability Po occurred 330 +/‐ 30 ms after a brief Ca2+ entry with a lag period of 50‐80 ms. With patch electrodes filled with Ca2(+)‐containing saline solutions and under conditions which maximized C‐channel opening, peak Po was reached in 20‐50 ms. The same value was observed for the whole‐cell C‐current. 7. The peak Po at a given patch potential and in response to a whole‐cell spike was not altered by a previous long‐lasting patch depolarization, or by producing several successive Ca2+ entries. Thus, C‐channels did not appear to be inactivated by depolarization or increase in [Ca2+]i. 8. C‐channels were found to be relatively highly voltage dependent, with an e‐fold increase in Po per 14.9 mV increase in potential.(ABSTRACT TRUNCATED AT 400 WORDS)
Muscarinic agonists and ATP increase the intracellular Ca2+ concentration in chick cochlear hair cells.
Authors: Shigemoto T , Ohmori H
Published: December 2014

1. Cholinergic muscarinic agonists applied by the pressure puff method increased intracellular Ca2+ concentration in Fura‐2‐loaded hair cells. The Ca2+ response outlasted the agonist application. 2. The Ca2+ response induced by acetylcholine (ACh) was ACh dose dependent with a KD of 200 microM. Desensitization was negligible, and almost identical Ca2+ responses were observed when two ACh puffs were separated by 150 s. The response was blocked by d‐tubocurarine (dTC). The KD of dTC blocking was 500 microM when 100 microM‐ACh induced the Ca2+ response. 3. The amplitude of the ACh‐induced Ca2+ responses were potentiated to 3 times the control by incubation with calcitonin gene‐related peptide (CGRP; 0.1‐1 microM). CGRP did not affect the resting Ca2+ concentration. Glycine (100 microM) potentiated the ACh response to 1.4 times the control, and also increased the resting Ca2+ concentration slightly. 4. The ACh‐induced Ca2+ response was suppressed by atropine. It was induced in Ca2(+)‐free extracellular medium, and in Ca2(+)‐free medium desensitization to a second ACh stimulation was significant. The amplitude of the second Ca2+ response was 44% of the first when two ACh puffs were separated by 117 s in Ca2+ free medium. 5. Muscarine and carbamylcholine induced similar Ca2+ responses, with KD values of 130 microM for muscarine and 340 microM for carbamylcholine. Desensitization of Ca2+ responses was negligible in both agonists. 6. ATP co‐exists with ACh in some presynaptic nerve terminals (Burnstock, 1981). Puff‐applied ATP (100 microM) generated a Ca2+ response with a rapid rising phase and a following slow phase. In Ca2(+)‐free medium the rapid phase disappeared and only the slow phase was observed. The rapid phase is due to the influx of Ca2+ ions and the slow phase is due to a release of Ca2+ ions from an intracellular reservoir. Under voltage clamp ATP induced a fast inward current and a following slow outward current. 7. Nicotine, adenosine, glycine, GABA, glutamate and bradykinin did not induce Ca2+ responses in the hair cell. 8. ACh induced hyperpolarization of the hair cell membrane under current clamp, most probably by the activation of Ca2+ activated K+ conductance. Therefore, a cholinergic muscarinic receptor may mediate the inhibitory effects of efferent innervation observed in hair cells.
Evidence for reduced presynaptic Ca2+ entry in a lobster neuromuscular junction at high pressure.
Authors: Grossman Y , Kendig J J
Published: December 2014

1. Previous studies have shown that hyperbaric pressure depresses synaptic transmission and have suggested that the effect is primarily on transmitter release. The present study analysed the effects of pressure at a crustacean neuromuscular junction. Changes in pressure were compared to changes in extracellular calcium concentration [Ca2+]o with respect to effects on excitatory junction potential (EJP) amplitude, time course, facilitation and potentiation. 2. The effects of 10.1 MPa pressure on EJP amplitude, facilitation and potentiation, but not time course, were mimicked by reducing [Ca2+]o to approximately one‐half the normal level. 3. The effects of pressure and the interaction between compression and calcium concentration were analysed in terms of a model of transmitter release. The model assumes that release is dependent on internal calcium concentration, as modulated by both influx and removal processes; that calcium influx is a saturating function of [Ca2+]o; and that release and removal are saturating functions of [Ca2+]i. 4. The results were consistent with the hypothesis that increased pressure acts primarily to reduce calcium influx into the nerve terminal.
Evidence for the uptake of neuronally derived choline by glial cells in the leech central nervous system.
Authors: Wuttke W A , Pentreath V W
Published: December 2014

1. With ion‐sensitive microelectrodes based on the Corning exchanger 477317, the accumulation of an unidentified interfering substance was monitored in leech neuropile glial cells but not in neurons after a 10‐fold increase in extracellular K+ concentration. Evidence is presented which shows that this substance may be choline. 2. The accumulation of interfering ions was not observed in Ca2(+)‐free saline and was substantially reduced in the presence of eserine (a blocker of acetylcholinesterase). 3. In neuropile (and also packet) glial cells, extracellularly applied choline (10(‐4) M) caused a steady increase in ion signal. This increase was not affected by removal of extracellular calcium, by hemicholinium‐3 (a blocker of high‐affinity choline uptake) or eserine. Shortly after the removal of choline from the saline the increase in ion signal stopped and the ion signal then decreased slowly to its original level. 4. Extracellular acetylcholine (10(‐4) M) caused a similar increase in intracellular ion signal of neuropile glial cells to that caused by choline. This increase was blocked by eserine. 5. Extracellular choline caused a comparatively small increase in ion signal of Retzius neurones which was blocked by hemicholinium‐3. In pressure neurones, choline or hemicholinium‐3 had no effect on intracellular ion signal. 6. Autoradiographic analysis of [3H]choline uptake showed that most of the choline was taken up by glial cells in a time‐ and dose‐dependent manner. Small but significant amounts of choline were taken up by neurones and connective tissue. 7. It is concluded that the neuropile and packet glial cells possess an effective choline uptake system which is activated by exogenous choline but also by choline that stems from enzymatic inactivation of acetylcholine released by neurones.
Cyclic AMP regulates an inward rectifying sodium‐potassium current in dissociated bull‐frog sympathetic neurones.
Authors: Tokimasa T , Akasu T
Published: December 2014

1. Bull‐frog sympathetic neurones in primary culture were voltage clamped in the whole‐cell configuration. The pipette solution contained ATP (5 mM). 2. A hyperpolarization‐activated sodium‐potassium current (H‐current: IH) was separated from other membrane currents in a nominally calcium‐free solution containing cobalt (2 mM), magnesium (4 mM), barium (2 mM), tetraethylammonium (20 mM), tetrodotoxin (3 microM), apamin (30 nM) and 4‐aminopyridine (1 mM). IH was selectively blocked by caesium (10‐300 microM). 3. The steady‐state activation of IH occurred between ‐60 and ‐130 mV. The H‐conductance was 4.1‐6.6 nS at the half‐activation voltage of ‐90 mV. With the concentrations of potassium and sodium ions in the superfusate at 20 and 70 mM, respectively, the reversal potential of IH was about ‐20 mV. IH was activated with a time constant of 2.8 s at ‐90 mV and 22 degrees C. The Q10 between 16 and 26 degrees C was 4.3. 4. A non‐hydrolysable ATP analogue in the pipette solution did not support IH activation. Intracellular ‘loading’ of GTP‐gamma‐S (30‐500 microM) led to a progressive activation of IH. 5. Forskolin (10 microM) increased the maximum conductance of IH by 70%. This was associated with a depolarizing shift in the half‐activation voltage (5‐10 mV) and in the voltage dependence of the activation/deactivation time constant of IH. 6. Essentially the same results as with forskolin were obtained by intracellular ‘loading’ with cyclic AMP (3‐10 microM) or bath application of 8‐bromo cyclic AMP (0.1‐1 mM), dibutyryl cyclic AMP (1 mM) and 3‐isobutyl‐1‐methylxanthine (0.1‐1 mM). 7. The protein kinase inhibitor H‐8 (1‐10 microM) decreased the peak amplitude of IH. Phorbol 12‐myristate 13‐acetate (10 microM), a protein kinase C activator, was without effect. 8. It is concluded that a voltage‐dependent cation current can be regulated by the basal activity of adenylate cyclase, presumably through protein kinase A, in vertebrate sympathetic neurones.
Regulation of capillary blood flow and oxygen supply in skeletal muscle in dogs during hypoxaemia.
Authors: Harrison D K , Kessler M , Knauf S K
Published: December 2014

1. Multiwire surface electrodes were used to measure local hydrogen clearance curves and tissue PO2 in vivo. Evaluation of the initial slopes of the hydrogen clearance curves enabled the measurement of capillary blood flow and its distribution. 2. Capillary blood flow and tissue PO2 frequency distribution histograms were measured in the m. sartorius of anaesthetized, relaxed mongrel dogs under conditions of normoxic (Fi, O2 = 0.3) and hypoxic (Fi, O2 = 0.15 and 0.1) artificial ventilation. 3. Stepwise hypoxaemia (hypoxic hypoxia) induced an increasing discrepancy between capillary blood flow and arterial blood flow. The former decreased by 6% whereas the latter increased by 86%. 4. PO2 histograms provided no evidence of cellular anoxia even at Fi,O2 = 0.1. Capillary blood flow histograms suggested a redistribution of the local pattern of flow. 5. A 34.7% reduction of O2 consumption was observed as the result of severe hypoxaemia. 6. The concept of heterogeneity of capillary blood flow as a functional O2 reserve is presented, together with evidence for oxygen‐dependent regulation of capillary blood flow and oxygen consumption.
Light adaptation in cone photoreceptors of the salamander: a role for cytoplasmic calcium.
Authors: Matthews H R , Fain G L , Murphy R L , Lamb T D
Published: December 2014

1. Light adaptation has been studied in isolated red‐sensitive cone photoreceptors of the salamander, using suction pipette recordings of circulating current. 2. In the presence of background illumination, the response to incremental dim flashes became desensitized according to the Weber‐Fechner law. The recovery phase of the flash response was accelerated significantly, although the time‐to‐peak was reduced only slightly, and for dim backgrounds the rising phase was unaltered. 3. The role of cytoplasmic calcium concentration, Cai2+, in mediating cone adaptation was investigated by minimizing light‐induced changes in Cai2+, either by incorporating calcium buffer into the cytoplasm or by exposing the outer segment to low‐Ca2+, 0‐Na+ solution. Both treatments appeared to slow dramatically or even to eliminate the onset of light adaptation in the cone. 4. When the low‐Ca2+, 0‐Na+ solution was presented in darkness, responses to subsequent illumination were affected in a characteristic manner: (i) the response‐intensity relation was steepened and shifted to lower intensities, (ii) the response to a step of light could be predicted by integration and compression of the flash response, and (iii) the flash sensitivity declined steeply as a function of background intensity. 5. After extended exposure of the cone to bright backgrounds, the sensitivity in darkness failed to return to its original level. The flash response kinetics were faster and more biphasic than for dark‐adapted responses or for responses desensitized to a comparable degree by exposure to steady background illumination. 6. The results indicate that, in cones isolated from the pigment epithelium, the primary factor influencing the adaptational state of the cell is the cytoplasmic concentration of free calcium, but that at high intensities the effects of pigment bleaching are likely to be significant.
Agonist‐stimulated divalent cation entry into single cultured human umbilical vein endothelial cells.
Authors: Jacob R
Published: December 2014

1. The free cytoplasmic Ca2+ concentration ([Ca2+]i) can be measured using Fura‐2 in superfused single human umbilical vein endothelial cells. When an endothelial cell is stimulated by a maximal dose of histamine (100 microM), [Ca2+]i rises to a peak and then falls back to a maintained plateau which is due to a stimulated Ca2+ influx. 2. If extracellular Ca2+ is replaced by 50 microM‐Mn2+ then 100 microM‐histamine causes a rise in [Ca2+]i accompanied by a fluorescence quench that signals the stimulated entry of Mn2+ into the cytoplasm. 3. If in Ca2(+)‐free solution a cell is stimulated by 100 microM‐histamine for 120 s to discharge the internal Ca2+ store, and then exposed to 50 microM‐Mn2+ after removal of the histamine, a similar stimulated Mn2+ entry is seen. This quench is unaffected by readdition of histamine and is not seen if the store is refilled by exposure to 1 mM‐extracellular Ca2+ for 180 s before exposure to the Mn2+. 4. The refilling of the internal store by exposure to 1 mM‐Ca2+ and the stimulated entry of Mn2+ are both blocked by 2 mM‐Ni2+. 5. If [Ca2+]i is stimulated to produce repetitive spikes by a low dose of histamine (0.3‐1 microM) in nominally Ca2(+)‐free solution containing Mn2+, then the stimulated quench is uniform and is not modulated by the [Ca2+]i spiking. 6. If the internal store is discharged by exposure to histamine in Ca2(+)‐free solution and then refilled for a short period then the cell is in a state where the internal store is partly full to an extent that depends on the duration of the refilling. In such an experiment, the rate of Mn2+ influx may be estimated by measuring the rate of quench during a short exposure to 50 microM‐Mn2+. The rate of Mn2+ entry varies inversely with the degree of fullness of the internal Ca2+ store. 7. If a similar experiment is repeated but with the fullness of the internal store being varied by varying the period of the initial exposure to 100 microM‐histamine, with no refilling, the same inverse relationship between Mn2+ influx and fullness of the internal store is obtained. 8. These experiments show that Mn2+ enters human umbilical vein endothelial cells following agonist stimulation by a pathway that is controlled by the degree of fullness of the internal store; it does not, however, enter the cytoplasm by exactly the same route as Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Nerve sprouting induced by a piece of peripheral nerve placed over a normally innervated frog muscle.
Authors: Diaz J , Pécot‐Dechavassine M
Published: December 2014

1. The effects of a segment of peripheral nerve on the innervation of a skeletal muscle were investigated in the cutaneous pectoris muscle of the frog Rana esculenta. An explant of the sciatic nerve was placed in an aneural region of the muscle at a distance of 1‐3 mm from the zone of neuromuscular junctions. The muscles were examined morphologically and electrophysiologically at different post‐operative times. 2. After the first month nerve sprouts were seen arising from both nerve terminals and intramuscular nodes of Ranvier. Both the number of sites of sprouting and the relative distance to the explant tip increased with time (up to 5 months), suggesting spread of a nerve sprouting‐promoting stimulus. 3. Most neurites resulting from sprouting were seen growing towards the nerve explant, in the vicinity of which active neurite proliferation occurred. Some of them entered the explant as observed in semi‐thin and thin sections which revealed the presence of both myelinated and unmyelinated nerve fibres. 4. Electrical stimulation of the nerve explant in preparations autografted for more than 2 months resulted in the contraction of bundles of muscle fibres. Endplate potentials of similar amplitudes were recorded intracellularly in some muscle fibres of such preparations when stimulating either the nerve explant or the cutaneous pectoris nerve. When two stimuli were paired by gradually reducing the interval of time between them, the second response was gradually facilitated. This confirmed that nerve fibres stimulated through the explant corresponded to new neuritic processes resulting from motor nerve sprouting. 5. Pieces of perineural tissue and segments of peripheral nerve killed by alcohol treatment or by repeated freezing and thawing were used as controls. They did not induce any nerve sprouting. 6. The results indicate that cells surviving in a segment of peripheral nerve trunk actively induce intact axons to sprout both from their terminals and intramuscular nodes of Ranvier; moreover these cells promote attraction and proliferation of growing neurites. The possibility of release of a diffusible factor by glial cells is discussed.
Sodium nitroprusside alters Ca2+ flux components and Ca2(+)‐dependent fluxes of K+ and Cl‐ in rat aorta.
Authors: Magliola L , Jones A W
Published: December 2014

1. Sodium nitroprusside (NP) caused both an inhibition of a noradrenaline (NA)‐induced contraction and an elevation of cyclic guanosine 3',5'‐monophosphate (cyclic GMP) in rat aorta. Both NP‐induced responses were enhanced by the selective cyclic GMP phosphodiesterase inhibitor, M&B 22948 (2‐o‐propoxyphenyl‐8‐aza‐purin‐6‐one, 30 microM). 2. The inhibition of a NA‐induced contraction by NP was characterized by dissociating the intracellular Ca2+ release component from the extracellular Ca2+ influx component of the contraction. The transient contraction stimulated by NA in the absence of extracellular Ca2+ was inhibited by NP. Also, the slowly developed tension stimulated by NA in aortas depleted of stored Ca2+ and subsequently exposed to extracellular Ca2+ was inhibited by NP. Both components of contraction were equally sensitive to NP. 3. NA stimulated both unidirectional 45Ca2+ influx in the presence of extracellular Ca2+ and 45Ca2+ efflux into a 0 Ca2+ solution that contained 2 mM‐ethyleneglycol‐bis‐(beta‐aminoethylether)N,N'‐tetraacetic acid (EGTA). The increased 45Ca2+ efflux is thought to reflect release of stored Ca2+ followed by membrane transport. NP greater than 10 nM inhibited both 45Ca2+ influx and release components whereas NP at 1‐3 nM enhanced NA‐stimulated 45Ca2+ efflux and relaxed the maintained tension caused by NA in 0 Ca2+, 2 mM‐EGTA. 4. NP also inhibited the Ca2(+)‐dependent 42K+ and 36Cl‐ effluxes from rat aorta stimulated either by NA or by high potassium. NP inhibited the contractile and flux responses to NA more effectively than the responses to high potassium. 5. These data indicate that: (1) NP reduces cytosolic Ca2+ by the combined inhibitory effects on Ca2+ influx and intracellular Ca2+ release, and by the stimulation of a Ca2(+)‐ATPase; and (2) the differential sensitivity of the NA and high‐potassium responses to NP may reflect underlying differences in Ca2+ handling induced by receptor occupancy and depolarization.
The effect of phenylglyoxal on contraction and intramembrane charge movement in frog skeletal muscle.
Authors: Etter E F
Published: December 2014

1. The effects of the arginine‐specific protein‐modifying reagent, phenylglyoxal, on contraction and intramembrane charge movement were studied in cut single fibres from frog skeletal muscle, using the double‐Vaseline‐gap voltage clamp technique. 2. The strength‐duration curve for pulses which produced microscopically just‐detectable contractions was shifted to more positive potentials and longer durations following treatment of fibres with phenylglyoxal. Caffeine‐induced contractures were not blocked. 3. The amount of charge moved by large depolarizing pulses from ‐100 mV holding potential (charge 1) declined during the phenylglyoxal treatment with a single‐exponential time course (tau = 7 min). Linear capacitance did not change significantly over the entire experiment. Inhibition of charge movement was predominantly irreversible. 4. Slow bumps (Q gamma) observed in charge movement current transients recorded before phenylglyoxal treatment, using either large test pulses or small steps superimposed on test pulses, were absent from currents recorded after treatment. The current removed by phenylglyoxal contained the bump (Q gamma) and a small fast transient (Q beta). 5. The amount of charge moved by large depolarizing pulses from ‐100 mV was reduced 20‐50% following phenylglyoxal treatment. Charge moved by pulses to potentials more negative than ‐40 mV was relatively unaffected. The magnitude and voltage range of this inhibitory effect were the same whether the reagent was applied at ‐100 mV or at 0 mV holding potential. 6. A phenylglyoxal‐sensitive component of charge was isolated which had a much steeper voltage dependence than the total charge movement or the charge remaining after treatment. 7. Charge recorded during hyperpolarizing pulses from 0 mV holding potential (charge 2) was reduced very little (less than 5%) at any potential by phenylglyoxal treatments at either 0 or ‐100 mV. 8. The phenylglyoxal reaction with charge 2 was kinetically different from the reaction with charge 1. 9. The effects of phenylglyoxal on charge 1 and charge 2 both measured in the same fibre were compared. Whether phenylglyoxal was applied at ‐100 mV, or at 0 mV the results were the same: charge 1 was inhibited much more (25‐60%) than charge 2(2‐12%). 10. The results presented here indicate that arginyl residues have a functional role in the voltage‐sensing mechanism of excitation‐contraction coupling and support the hypothesis that the dihydropyridine receptor is the voltage sensor molecule.