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R Miledi

Publications and source records attributed to R Miledi.

At least 181 records · Page 10Linked to original sources
PubMed1983

Block of glutamate-activated synaptic channels by curare and gallamine.

Excitatory junctional currents (e.j.cs) and glutamate-activated currents have been examined in voltage-clamped locust muscle fibres exposed to curare or gallamine. Although these drugs have little action on channel kinetics at the resting potential, there is an increasingly pronounced effect at hyperpolarized levels. In the presence of curare (5-100 microM), fibres held at hyperpolarized potentials showed e.j.cs with an initial 'fast component' followed by a 'slow tail'. In many fibres, hyperpolarization beyond -50 mV decreased the amplitude of the peak synaptic current; the decay time constant of the fast component was decreased by hyperpolarization while the time constant of the slow component was increased. Iontophoretic application of brief pulses of glutamate also produced two-component glutamate currents in these conditions. Gallamine was considerably more effective than curare, markedly altering the decay time and amplitude of the e.j.c. and the glutamate current at 1-5 microM. Its effects appeared qualitatively similar to those of curare. The observations are consistent with the idea that curare and gallamine produce a transient block of glutamate-activated synaptic channels.

Animals↗
PubMed1983

Divalent cations and temperature dependent block of impulse propagation at the frog neuromuscular junction.

End-plate potentials were recorded from superficial muscle fibers of the frog sartorius nerve-muscle preparation. Exposure of the preparation to a medium containing a high divalent cation concentration, produced a temperature dependent failure of neuromuscular transmission. Failure of transmission developed in an "all-or-none" mode and was reversed by decreasing the bath temperature or divalent cation concentration. This blockage of the neurotransmission is attributed to a presynaptic block of impulse propagation.

Animals↗
PubMed1983

Acetylcholinesterase activity of Xenopus laevis oocytes.

The cholinesterase activity of Xenopus laevis oocytes was assessed using [3H]acetylcholine in a simple radiometric procedure. The cholinesterase activity of mature (stage V-Vl) oocytes was very sensitive to inhibition by the specific acetylcholinesterase inhibitor, BW284-C5l, and relatively insensitive to an inhibitor of non-specific, or butyrylcholinesterase. The Km and Vmax of the acetylcholinesterase measured in homogenates of oocytes were 312 microM and 4.6 nmol-oocyte 1-h 1, respectively. Triton X-100 increased the enzyme activity of homogenates four- to five-fold while collagenase treatment displaced into the medium none of the acetylcholinesterase activity from either homogenates or intact oocytes. Cations were found generally to diminish the acetylcholinesterase activity of oocyte homogenates, and lanthanum ions inhibited acetylcholine hydrolysis with an IC50 of 0.63 mM. Subcellular fractionation of oocytes revealed that the bulk of enzyme activity was associated with particulate fractions. Acetylcholinesterase activity was also detected on the surface, and in homogenates, of immature oocytes. Peak enzyme activity resided in stage IV oocytes. Eggs obtained from females induced to spawn were found to have acetylcholinesterase activity in homogenates but little or no hydrolytic activity was detected on the egg surface. These results provide a point of departure for further investigations of the functional significance of this enzyme in Xenopus oocytes.

Acetylcholine↗
PubMed1983

Electrophysiological and chemical determination of acetylcholine release at the frog neuromuscular junction.

1. Mass fragmentography was used to measure the tissue content and release of acetylcholine (ACh) by frog sartorius muscles, which had been previously treated with an irreversible cholinesterase inhibitor. The frequency of miniature end-plate currents (m.e.p.c.s) was also measured. 2. Exposure of muscles for 15 min to 2 mM-LaCl3 resulted in a large release of ACh which subsided to low levels after 1 h. About 4 h later treatment with 50 mM-KCl, or with the calcium ionophore A 23187, or with a second dose of LaCl3, all failed to augment ACh release, notwithstanding the fact that the ACh content of La3+-treated muscles was about the same as that of controls. 3. Hypertonic NaCl or raised KCl concentrations were used to increase m.e.p.c.s and this also increased ACh release; it was estimated that each quantum corresponded to the release of 12 000 molecules of ACh. 4. ACh release by nerve stimulation was greatly potentiated by 10 mM-tetraethylammonium chloride, and this enabled the ACh released by ten, and even single, stimuli to be detected; it was calculated from the ACh released and the quantal content that each quantum contained on the average 13 000 molecules. 5. ACh released by nerve stimulation at 0.2/s in the absence of tetraethylammonium was about half that expected on the basis of previous estimates of quantal content; it was increased about two-fold by alpha-bungarotoxin. 6. It is concluded that chemical and electrical stimulation of the nerve evoked quantal ACh release, without influencing non-quantal ACh leakage. The results are consistent with the view that ACh quanta are derived from synaptic vesicles. They also show that resting ACh release is not due to leakage of ACh ions along an electrochemical gradient in the membrane.

Acetylcholine↗
PubMed1983

Calcium transients in frog skeletal muscle fibres following conditioning stimuli.

1. Intracellular Ca(2+) transients were recorded from frog twitch muscle fibres, using arsenazo III as a Ca(2+) monitor. When fibres were stimulated by two action potentials, the arsenazo signal to the second stimulus was smaller than the first, for stimulus intervals of up to several seconds.2. The recovery of the amplitude of the second response followed two exponential time courses; a fast one with a time constant of about 70 msec giving recovery to about 90% of the control value, followed by a slow recovery to 100%, with a time constant of about 12 sec (at 10 degrees C).3. The time constant of the fast recovery component was strongly temperature-dependent, with a Q(10) of approximately 2.7, whilst the Q(10) of the slow component was about 1.4.4. Removal of Ca(2+) in the bathing medium lengthened the time constant of the slow recovery component by a factor of three, but had little effect on the fast recovery component. The lengthening of the slow component was not reversed by addition of Mg(2+), but Sr(2+) ions could substitute for Ca(2+).5. The influence of membrane potential on the recovery time-course was investigated after blocking action potentials with tetrodotoxin, using a voltage clamp to control membrane potential. Paired depolarizing stimuli were used, with the potential held to either low (-60 or -80 mV) or high (-110 or -140 mV) potentials between stimuli. No differences were apparent in either the fast or slow recovery components at these holding potentials.6. The arsenazo response elicited by an action potential following a conditioning tetanus was reduced in size even more strongly than following a single action potential. The time course of recovery of the response following a tetanus again comprised two exponential components. After a 20 Hz tetanus for 0.5 sec, the fast component had a time constant of about 400 msec, and gave a recovery to about 60% of the control value. Subsequent recovery to 100% occurred with a time constant of about 12 sec.7. The time constant of the fast recovery component increased markedly with increasing frequency or duration of the conditioning tetanus. The time constant of the slow component was not appreciably altered by conditioning tetani varying between one impulse and sixty impulses. However, the reduction in response size due to the slow component, extrapolated to zero stimulus interval, increased with increasing number of impulses in the tetanus.8. The time constant of the fast recovery component corresponded closely with the decay time constant of the arsenazo response to the conditioning stimulus. This correspondence held over a nearly fifty-fold range of time constants, and for two different conditions which affected the decay time constant (temperature, and frequency of tetanic stimulation).9. The decay time constant of the arsenazo response elicited by an action potential was slowed by a preceding impulse or tetanus. Following a 20 Hz tetanus for 0.5 sec, recovery of the half decay time appeared to follow an exponential time course, with a time constant of about 12 sec.10. These results suggest that the fast recovery component reflects the re-filling of release stores in the sarcoplasmic reticulum by Ca(2+) ions taken up from the cytoplasm. The origin of the slow component is less clear, but it may arise from inactivation of the excitation-contraction (e-c) coupling process between T-tubule depolarization and Ca(2+) release from the sarcoplasmic reticulum.

Action Potentials↗
PubMed1983

Aequorin-calcium transients in frog twitch muscle fibres.

Intracellular Ca2+ transients, evoked either by action potentials or depolarizing clamp pulses, were studied in frog sartorius muscle fibres injected with aequorin. The time course of the Ca2+ transients became shorter as the temperature was increased. The half rise time and decay time constants showed straight lines between 3 and 30 degrees C in Arrhenius plots, with a Q10 of 2.5 and 2.3 respectively. The potential dependence of the Ca2+ transient was examined under voltage clamp. The peak light amplitude reached a plateau at around +50 mV, suggesting that Ca2+ release continues beyond the potential level at which contraction was saturated. During a prolonged depolarization, the Ca2+ transient gradually declined. The time course of decline became faster when long depolarizing pulses were repeated, or when the temperature was increased. The Q10 for half duration of the Ca2+ transient evoked by prolonged depolarization was 2.2. A Ca2+ transient could be evoked in Ca2+-free Ringer solution containing EGTA. Formamide, which is known to abolish excitation-contraction coupling, also abolished the Ca2+ transient. During maintained depolarization, the time integral of the Ca2+ transient was larger for larger depolarizations, suggesting that the total amount of Ca2+ released was greater for the more intense depolarization. The decline of the Ca2+ transient during maintained depolarization is probably due to inactivation of excitation-contraction coupling rather than the depletion of intracellular Ca2+ stores. These findings support the view that in frog skeletal muscle fibres the increase in intracellular Ca2+, caused by membrane depolarization, is produced by the release of Ca2+ from intracellular stores and that any influx of Ca2+ from the external medium does not contribute appreciably to the aequorin-Ca2+ transient.

Action Potentials↗
PubMed1983

Calcium transients studied under voltage-clamp control in frog twitch muscle fibres.

1. Intracellular calcium transients were recorded from frog twitch muscle fibres in response to voltage-clamped depolarizing pulses, using arsenazo III as an intracellular calcium monitor. The object was to investigate the time- and voltage-dependent characteristics of the coupling process between membrane depolarization and calcium release from the sarcoplasmic reticulum (s.r.)2. To examine the extent to which the T-tubule membrane potential was controlled during clamp pulses, the dye NK 2367 was used as an optical probe of tubular potential. This indicated that the tubular time constant is about 0.6 msec.3. Strength-duration curves were obtained for depolarizing pulses required to give both threshold mechanical contraction and calcium signal. Curves measured in these two ways were closely similar.4. Changes in holding potential altered the strength-duration curve for calcium release so that at more positive holding potentials a shorter pulse was needed to obtain a response for any given pulse amplitude.5. A latency of a few milliseconds was observed between the onset of depolarization and the initial rise of the calcium signal. This became shorter with stronger depolarizations, but approached a minimum at potentials above about +25 mV.6. Subthreshold depolarizations applied before a test pulse increased the size and decreased the latency of the calcium signal. Conditioning hyperpolarizations had opposite effects.7. The rate of build-up of potentiation or depression of response size seen with subthreshold de- and hyperpolarizing conditioning pulses was examined using conditioning pulses of different durations. For both pulses this process showed a time constant of about 3 msec (at 10 degrees C).8. The rate of decay of potentiation or depression was similarly measured, using a gap of variable duration between conditioning and test pulses. For both de- and hyperpolarizing pulses this showed a time constant of about 5 msec (10 degrees C).9. The relationship between conditioning pulse potential, and the size of calcium signal elicited by a following test pulse was non-linear.10. Subthreshold pulses immediately following a brief test pulse affected the size of the calcium signal in a similar way to preceding conditioning pulses.11. The relationship between potential and size of the calcium signal was examined using pulses of 3 and 20 msec duration. With the long pulse the relation was roughly sigmoid, but with the short pulse continued to rise even at strongly positive potentials.12. The results are discussed in terms of a model in which the exponential build-up of a hypothetical coupler in the excitation-contraction (e.-c.) coupling process is presumed to lead to calcium release when a threshold level is exceeded.

Animals↗
PubMed1983

Changes in threshold for calcium transients in frog skeletal muscle fibres owing to calcium depletion in the T-tubules.

Strength-duration curves were measured for voltage-clamp depolarizations required to elicit a just detectable rise in intracellular calcium, as monitored using arsenazo III, in frog twitch muscle fibres. In normal Ringer solution, the threshold for a 5 sec duration depolarization was about 5 mV more negative than for a 200 msec duration pulse. The shift in threshold comparing 200 msec and 5 sec pulses was almost abolished in bathing solutions including magnesium or nickel (4 mM), or where the free calcium concentration was buffered. The shift in threshold was little changed by substitution of barium for calcium. These results can be explained by supposing that the 5 sec depolarization activates an inward calcium flux across the T-tubule membrane, which decreases the calcium concentration in the tubules, and hence alters the threshold for activation of excitation-contraction (e.-c.) coupling because of surface charge effects.

Animals↗
PubMed1982

An endplate potential due to potassium released by the motor nerve impulse.

A small endplate potential can be recorded in frog muscle fibres, after all acetylcholine-mediated transmission has been eliminated by pre- or postsynaptic blocking agents (botulinum toxin, calcium lack, manganese, curare, alpha-bungarotoxin). It is usually necessary to hyperpolarize the muscle membrane to detect this 'non-cholinergic' endplate potential. Below--100 mV little or no response is seen; a maximum is reached at about--140 mV, when the amplitude can be as large as 100 microV (endplate current up to about 1 nA). Other characteristic features are: the response shows no quantal fluctuations; its amplitude is not facilitated by repetitive impulses; its size and time course are not noticeably affected by prostigmine, curare or alpha-bungarotoxin; the half-time of decline of the endplate current is approximately 1.7 ms at 20 degrees C, and is lengthened by lowering the temperature with a Q10 of about 1.3; the response is abolished by barium. When iontophoretic pulses of potassium are applied to the endplate, local depolarization is recorded whose amplitude varies with membrane potential similarly to that of the nerve-evoked response. These observations strongly indicate that this 'non-cholinergic', 'non-quantal' endplate potential arises from a rapid synaptic transfer of potassium ions, released by the active nerve terminal into the synaptic cleft and entering the muscle fibre through 'anomalous rectifier' channels in the endplate membrane.

Animals↗
PubMed1982

Synthesis of chick brain GABA receptors by frog oocytes.

Poly(A)-mRNA, extracted from the optic lobe of chick embryos, directs the synthesis of gamma-aminobutyric acid (GABA) receptors in Xenopus laevis oocytes. The receptors are inserted into the oocyte membrane, where they form receptor--channel complexes. When activated by GABA, and related agonists, the chick brain receptors open membrane channels that are permeable to chloride ions. Thus, Xenopus oocytes provide a novel and useful approach to the study of brain receptors.

Animals↗
PubMed1982

The antagonism between botulinum toxin and calcium in motor nerve terminals.

The effects of tetraethylammonium and manganese, which modify calcium entry into motor nerve terminals, have been studied during advanced stages of botulinum paralysis. Evidence has been obtained that the voltage-activated calcium current in the nerve endings is not significantly reduced by botulinum toxin. The depression of transmitter release that the toxin produces must arise at a later stage, at an intracellular site of the release mechanism.

Acetylcholine↗
PubMed1982

A calcium-dependent transient outward current in Xenopus laevis oocytes.

Membrane currents were investigated in Xenopus laevis oocytes under voltage clamp. Depolarizing pulses, given from a holding potential of about-100 mV, elicited a transient outward current when the membrane potential was made more positive than about-20 mV. As the potential was made increasingly positive the transient outward current first increased and then decreased. The amplitude of the transient current increased when the external Ca2+ concentration was raised; and the current was abolished by Mn2+. It appears that when the membrane is depolarized Ca2+ ions enter the oocyte and trigger an outward current, possibly by opening C1- channels.

Animals↗
PubMed1982

Antibodies to beta-bungarotoxin and its phospholipase inactive derivative.

Antisera were raised against the presynaptic neurotoxin beta-bungarotoxin and against its phospholipase-inactive derivative, modified by reaction with p-bromophenacyl bromide. The cross-reactivity of the antisera to other phospholipase A2 enzymes and polypeptide neurotoxins was examined. The antisera inhibited both the neurotoxic effects of beta-bungarotoxin at the frog motor endplate and the enzymatic activity of the toxin on model phospholipid membranes, although it is unlikely that the catalytic active centre is the locus of any major determinant.

Animals↗
PubMed1982

Acetylcholine receptors at the rat neuromuscular junction as revealed by deep etching.

Collagenase treatment of rat intercostal muscles yielded single muscle fibres in which the nerve terminals and basal lamina were removed allowing an unimpeded view of the external surface of the postsynaptic membrane. This was revealed by deep etching of freeze-fractured preparations and appeared as a maze of folds separated by deep troughs, showing on the crests of the folds a densely packed population of protrusions about 8.5 nm in diameter. These densely packed protrusions (ca. 9000 microns-2) are mainly confined to the postsynaptic regions of the sarcolemma and presumably represent the acetylcholine receptor molecules, which are highly concentrated in these areas. The protrusions are generally tightly packed without obvious regular arrangement, but in some areas, usually on the tops of the crests, they are arranged into irregular rows normal to the long axis of the folds.

Animals↗
PubMed1982

Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes.

Messenger RNA extracted from the electric organ of Torpedo was injected into Xenopus oocytes. This led to the synthesis and incorporation of functional acetylcholine receptors into the membrane of the oocyte. When activated by acetylcholine these Torpedo acetylcholine receptors in the oocyte membrane opened channels whose ionic permeability resembled that of nicotinic receptors in other cells.

Animals↗
PubMed1982

Neural and non-neural acetylcholine in the rat diaphragm.

The compartmentation of acetylcholine (ACh) and of choline acetyltransferase in the rat diaphragm was analysed by measuring their contents in muscle segments containing endplates (e.p.) and endplate-free segments (non-e.p.) at different times following section of the phrenic nerve. In addition ACh release was determined before and after denervation. Freshly dissected hemidiaphragms contained about 125 pmol of ACh; more than 90% of this was localized in the e.p. portion. Between 10 and 18 h after denervation the ACh content of the e.p. portion decreased by 80% and its ACh concentration became approximately equal to that in the non-e.p. region, whose ACh content did not change. Spontaneous release of ACh was reduced by denervation and ACh release evoked by 50 mM KC1 was practically abolished. Choline acetyltransferase activity in freshly dissected preparations was about 30 nmol of ACh per gram per hour, Km 0.5 mM. About 65% of the enzyme disappeared in the first 24 h and the remaining 35% between 24 and 50 h after denervation. A different enzyme capable of ACh synthesis was found in the muscle fibres; its activity did not decrease after denervation. It is concluded that about 70% of the ACh in the diaphragm is contained in the motor nerve terminals, about 10% in the intramuscular nerve fibres and the remainder in the muscle fibres, and that about 65% of choline acetyltransferase is in the motor terminals and 35% in the nerve fibres.

Acetylcholine↗
PubMed1982

Properties of miniature excitatory junctional currents at the locust nerve-muscle junction.

1. Miniature excitatory junctional currents (m.e.j.c.s) were examined in conditions where inward current was carried mainly by Na(+) (i.e. in normal medium, Ca(2+)-free medium and Cl(-)-free medium). M.e.j.c.s were also examined in isotonic Ca(2+) where the inward post-synaptic current was carried mainly by Ca(2+).2. In normal medium, mean m.e.j.c. amplitude = 2.34+/-0.05 nA. The decay time constant of m.e.j.c.s (excluding a small percentage with abnormal shapes) was tau(m.e.j.c.) = 2.62+/-0.11 msec (V(m) = -80 mV, T = 22 degrees C). Decay-time was not markedly changed in Ca(2+)-free or Cl(-)-free medium. tau(m.e.j.c.) approaches the life-time of glutamate activated junctional channels.3. Excitatory junctional currents, evoked by nerve impulses, decayed slightly faster than m.e.j.c.s obtained in the same fibres. Extracellularly recorded m.e.j.c.s and voltage-clamped m.e.j.c.s were similar in time course.4. tau(m.e.j.c.) decreased exponentially with membrane hyperpolarization. An e-fold change was produced by 182.+/-24.8 mV change in V(m).5. The dependence of mean m.e.j.c. amplitude on clamp potential showed a slight non-linearity at hyperpolarized levels. The equilibrium potential for transmitter action was close to 0 mV in normal solution as well as in Ca(2+)-free and Cl(-)-free solutions.6. The kinetics of junctional channels are altered in isotonic Ca(2+). M.e.j.c. amplitude was reduced to about one-third normal size; mean m.e.j.c. = 0.74+/-0.03 nA. The decay time becomes markedly briefer, tau(m.e.j.c.) = 1.01+/-0.08 msec, indicating a reduction in mean channel life-time (V(m) = -80 mV, T = 22 degrees C).7. A population of slow time course and composite m.e.j.c.s appear when muscle fibres are hyperpolarized in isotonic Ca(2+), thus producing a prolongation in mean tau(m.e.j.c.). This results from an influence of post-synaptic membrane potential on presynaptic transmitter release. If such m.e.j.c.s are ignored the voltage dependence of tau(m.e.j.c.) of the remaining events is abolished or even reversed indicating that voltage sensitivity of channel life-time is altered in isotonic Ca(2+). The equilibrium potential for transmitter action may be slightly more positive than normal.8. We estimate that a single packet of neurally released transmitter normally opens, on average, 250 ion channels at these junctions.

Animals↗