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Biomedical subjects

R Miledi

Publications and source records attributed to R Miledi.

At least 199 records · Page 11Linked to original sources
PubMed1982

Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane.

1. Neurotransmitter-receptors in the membrane of Xenopus oocytes have been studied using electrophysiological techniques. Neurotransmitters and related agents were applied while recording either membrane potential or membrane current. The majority of ovarian oocytes used were at stages IV and V.2. Three types of oocytes were examined: inner ovarian epithelium covered (e.c.) oocytes; epithelium manually removed (e.r.) oocytes; and collagenase treated (c.t.) ooctyes.3. Ovarian oocytes are sensitive to some cholinergic and catecholaminergic agents. Responses to serotonin were seldom observed and when present were much weaker than responses to other agents. No responses were observed to the amino acids: aspartate, glutamate, gamma-aminobutyric acid, and glycine; or to octopamine and histamine.4. Acetylcholine (ACh) usually depolarized the membrane, in a dose-dependent manner, with threshold concentrations as low as 10(-9)m. The ACh-potential was due to an increase in Cl permeability and had a reversal potential around - 19 mV. The intracellular Cl ion activity, measured with a Cl-ion sensitive micro-electrode, was about 65 mm and the estimated Cl-ion equilibrium potential, E(Cl), agreed with the reversal potential of the ACh-potential.5. Curare (10(-4)m), tetrodotoxin (10(-6)m), or alpha-bungarotoxin (10(-6) g/ml.) did not block the response to 10(-6)m-ACh; whereas atropine (10(-7)m) blocked it. No response to nicotinic agents (e.g. nicotine, 1,1-dimethyl-4-phenylpiperazinium) was observed. These results suggest that the ACh receptors in the oocyte membrane are muscarinic in nature.6. The apparent latency of the ACh potential, examined by ionophoretic application of ACh to e.r. oocytes and c.t. oocytes, ranged from 0.5 sec to over 20 sec. Intracellular injection of ACh was without effect.7. Responses to catecholamines were observed mostly in e.c. oocytes; while in e.r. and c.t. oocytes they were rare and of very small amplitudes.8. The usual response to both dopamine and (-)-epinephrine was a transient hyperpolarization manifested by an initial increase in K-permeability followed by a decrease. The latency of these responses ranged from 10 sec to over 30 sec and their reversal potential was nearly - 100 mV, which coincided with E(K).9. Oocytes responded to the beta-adrenergic receptor agonist, isoproterenol, as well as (-)-epinephrine. Pre-treatment with the beta-adrenergic receptor blocker, propranolol, abolished the response to both (-)-epinephrine and (-)-isoproterenol. The dopamine potential was also reduced considerably. Both the alpha-adrenergic receptor agonist, phenylephrine, and the alpha-adrenergic receptor blocker, phentolamine, were without effect.10. Maturation of the oocytes, induced in vivo by gonadotropin or in vitro by progesterone, led to loss of responsiveness to both cholinergic and catecholaminergic agents.

Acetylcholine↗
PubMed1982

Free and bound acetylcholine in frog muscle.

1. Frog sartorius muscles were divided into end-plate containing (e.p.) and end-plate-free (non-e.p.) segments or homogenized in Ringer solution at 0 degrees C in the presence or absence of added acetylcholinesterase from electric eel. ACh was extracted from the tissue or from the homogenates and measured by mass fragmentography. 2. The concentration of ACh in non-e.p. segments was about six times lower than that in e.p. segments. 3. Homogenization of muscles in Ringer caused the hydrolysis of a small fraction ('free-1') of total ACh; addition of extra acetylcholinesterase caused hydrolysis of another, greater, fraction ('free-2' ACh). The esterase-resistant ('bound') ACh was stable at 0 degrees C up to 15 min of incubation. 4. Denervation for 15 days, which caused the disappearance of the nerve terminals, did not influence ACh in non-e.p. segments, but reduced total and bound ACh by about 75%, and free-2 ACh by 90%. 5. Treatment with La3+ ions, which caused the disappearance of synaptic vesicles, did not influence total ACh, but reduced bound ACh by 75%, whereas free-1 and free-2 ACh were increased. 6. Electrical stimulation of the nerve at 5 sec-1 or incubation with 50 mM-KCl did not affect ACh in the non-e.p. segments, but reduced by roughly 60% total, bound, and free ACh. 7. It is concluded that about 75% of bound ACh derives from synaptic vesicles, corresponding to 11,000 molecules per vesicle, and 25% from non-neural ACh; that free-1 and free-2 ACh derive mainly from the nerve terminal cytoplasm, although they may be contaminated by vesicular ACh.

Acetylcholine↗
PubMed1982

Denervation changes in normal and myasthenia gravis human muscle fibres during organ culture.

1. Human intercostal nerve-muscle obtained from normal and myasthenia gravis affected patients has been organ cultured for up to 5 weeks at 23 degrees C. In addition normal nerve-muscle has been cultured for up to 2 weeks at 36 degrees C. Muscle fibres had normal resting and overshooting action potentials. Input resistances dropped markedly after 21 days at 23 degrees C or 10 days at 36 degrees C.2. Muscle fibre action potentials became partially resistant to tetrodotoxin (10(-7) g/ml.) after culturing for 8 days at 36 degrees C.3. Extrajunctional acetylcholine (ACh) sensitivity was compared in fresh and cultured muscle. Fresh normal fibres possessed extrajunctional ACh sensitivity covering several hundred micrometres around the end-plate and at the muscle-tendon junction. Myasthenia gravis affected fibres had reduced extrajunctional sensitivity at the end-plate and no detectable ACh sensitivity near the tendon.4. Less than one third of normal muscle bundles showed an increased area of ACh sensitivity after several days in organ culture. Under the same conditions myasthenic muscle bundles did not show an increase in extrajunctional ACh sensitivity,5. M.e.p.p.s were present for 2-4 days in normal fibres cultured at 36 degrees C. In normal and myasthenic muscles cultured at 23 degrees C m.e.p.p.s disappeared after 6-8 days and re-appeared in some fibres (50%) after approximately two weeks in organ culture. These m.e.p.p.s were abolished by curare and increased in frequency by hypotonic solution suggesting they are due to the release of ACh-packets from Schwann cells.6. Electron microscopic examination of cultured human muscle indicates that the disappearance of m.e.p.p.s corresponds with degeneration of nerve terminals.7. In muscle bundles shown to possess m.e.p.p.s after 13-14 days, the synaptic gutter, which had been vacated by the nerve terminal, was usually occupied by a Schwann cell or projections of Schwann cell cytoplasm. This indicates that the Schwann cell at denervated human end-plates may be capable of releasing packets of ACh.8. It is concluded that the organ culture system described here is suitable for studying normal and diseased human muscle fibres. Using this system we find that the denervation changes which follow nerve transection appear to be similar in most respects at normal and myasthenic end-plates.

Action Potentials↗
PubMed1982

Properties of junctional and extrajunctional acetylcholine-receptor channels in organ cultured human muscle fibres.

1. Current noise, obtained during steady ionophoretic application of acetylcholine (ACh) to voltage-clamped human fibres has been analysed to derive properties of end-plate channels and also extrajunctional ACh-activated channels which are present at the muscle-tendon junction of normal fibres. In addition, ACh-receptor channels present at the end-plate and tendon region in organ cultured muscles have been compared with those in fresh muscles.2. Extrajunctional channels in the tendon region of fresh fibres have a longer mean life-time, tau, and a smaller single channel conductance, gamma, than the junctional channels. tau was 1.71 +/-0.11 msec and gamma was 25.05 +/- 1.18 pS for junctional channels; tau was 3.16 +/- 0.33 msec and gamma was 12.76 +/- 1.29 pS for extrajunctional channels.3. Properties of channels in the end-plate and tendon region were unchanged during short-term (< 7 days) organ culture at 23 or 36 degrees C. The voltage sensitivity of the mean channel life-time was similar at junctional and extrajunctional sites.4. In muscles organ cultured for 7 days at 36 degrees C, double component noise spectra were obtained at some end-plates. The fast and slow time constants underlying the noise appeared to correspond to simultaneous activation of junctional and extrajunctional channels.5. After organ culture for 3-4 weeks at 23 degrees C the mean life-time of the end-plate channels was prolonged while their single channel conductance was unchanged, tau = 3.58 +/- 0.16 msec; gamma = 22.11 +/- 0.83 pS (V(m) = -80 mV, T = 21 degrees C).6. As end-plate channel properties were unchanged in short-term cultures it was possible to assess post-junctional sensitivity by comparing miniature end-plate current (m.e.p.c.) amplitudes in both normal and myasthenia gravis affected muscles. No evidence was found for a change in post-synaptic sensitivity during organ culture at 23 degrees C of normal or myasthenia gravis muscle fibres, which seems to rule out reversible block of receptors by anti-receptor antibody as playing an important role in myasthenia gravis.

Acetylcholine↗
PubMed1982

Calcium transients evoked by action potentials in frog twitch muscle fibres.

1. Intracellular Ca(2+) transients were recorded from frog twitch muscle fibres in response to action potentials and repetitive stimulation, using ionophoretically injected arsenazo III as a Ca(2+) monitor. A dual wave-length optical system was used to measure absorbance changes of the injected dye from small areas of single fibres within the cutaneous pectoris muscle.2. The absorbance spectrum of the injected arsenazo III in a resting fibre was consistent with an intracellular free Mg(2+) level of a few hundred micromolar, assuming an intracellular pH of 7.1. The resting free Ca(2+) concentration was below the limit of resolution.3. The wave-length dependence of the arsenazo light absorbance signal during twitches followed that expected for Ca(2+) binding to the dye. Recordings made at wave-lengths where the dye is maximally sensitive to pH or Mg(2+) concentration changes indicated that interference from these sources is minimal at the usual wave-length pair (650-700 nm) used for Ca(2+) recordings.4. Over a wide range of intracellular dye concentrations, the size of the arsenazo response to an action potential increased linearly with dye concentration (100-1000 muM), although there were deviations from this relationship at low and high concentrations.5. An approximate estimate of 8 muM was obtained for the peak free Ca(2+) concentration change following a single action potential. Changes in temperature (6-25 degrees C) did not significantly affect the size of the free Ca(2+) transient. During maximal tetanic stimulation the signal rose to about three times higher than the twitch response. An approximate minimum estimate of the increase in total cytoplasmic Ca(2+) concentration during a twitch gave a value of 220 muM.6. A latency of about 1.5 ms (at 10 degrees C) was observed between the foot of an action potential and the onset of the arsenazo response. Recordings made using a narrow measuring light slit, placed either at the edge or the centre of a fibre, suggested that only a small part of this latency could be due to inward conduction of the action potential along the T-tubules.7. The decay phase of the arsenazo response to an action potential followed an exponential time course, with a time constant of 71 ms at 10 degrees C. This time constant was strongly temperature-dependent, with a Q(10) of about 2.4. An Arrhenius plot of the decay time constant gave a straight line.8. During repetitive stimulation, the arsenazo responses evoked by successive impulses showed two changes: a progressive decrease in amplitude and a slowing of the decay. The extent to which successive responses summated during a tetanus depended upon the balance between these two effects.

Action Potentials↗
PubMed1981

The reduction of endplate responses by botulinum toxin.

Endplate responses were recorded in frog muscle fibres during an advanced stage of botulinum (BoTX) paralysis, when transmitter release had fallen to a very low level. By simultaneous recording from two points, it was found that, even when the quantal responses had been reduced to less than 0.01 per impulse (that is, four to five orders of magnitude below normal), the release continued to be spatially dispersed along the terminal arborization. These observations make it very unlikely that whole "active zones' could be eliminated, as has been suggested, in all-or-none fashion by local action of BoTX molecules, and they suggest a more graded, indirect mechanism by which the toxin molecules interfere with the sites of transmitter release.

Acetylcholine↗
PubMed1981

Induction of action potentials in frog slow muscle fibres paralysed by alpha -bungarotoxin.

Slow muscle fibres in the frog are normally incapable of generating action potentials. However, several days after an intramuscular injection of alpha - bungarotoxin, they acquire the ability to generate action potentials. It appears that alpha -bungarotoxin induces the action potential mechanism in slow fibres because it blocks acetylcholine receptors, and thus interferes with the action of non-quantal acetylcholine leaking from nerve terminals, or because the toxin has some other, as yet undefined, action on nerve or muscle.

Action Potentials↗
PubMed1981

Effects of membrane polarization on sarcoplasmic calcium release in skeletal muscle.

Calcium release from the sarcoplasmic reticulum was investigated in voltage-clamped, tetrodotoxin-treated frog skeletal muscle fibres injected with arsenazo III. Short (5 ms) depolarizing pulses (test pulses) produced a transient change in arsenazo III absorption, signalling an increase in intracellular calcium in concentration (calcium transient). Conditioning subthreshold depolarizations, which preceded the test pulse, potentiated the calcium transient triggered by the test pulse. Conditioning hyperpolarizations, applied either before or after the test pulse, inhibited the calcium transient. These effects of conditioning polarizations on the calcium transient may explain similar effects of subthreshold polarizations on muscle contraction that have previously been reported. The potentiating effect of subthreshold depolarizations was observed only when the test pulse was short (5 ms). The potentiating effect develops at -48 mV with a time constant of about 7 ms at 6.5 degrees C; this seems to be slower than that predicted by the potential spread from the surface along the tubular system. Thus, part of the effect could arise from the coupling process between tubular depolarization and calcium release.

Animals↗
PubMed1981

Calcium transients recorded with arsenazo III in the presynaptic terminal of the squid giant synapse.

Transient changes in free intracellular Ca2+ concentration were monitored in the presynaptic terminal of the giant synapse of the squid, by means of the Ca2+-sensitive dye arsenazo III. Calibration experiments showed a linear relation between the amount of Ca2+ injected by iontophoresis into the terminal, and the peak size of the arsenazo light absorbance record. A light signal could be detected on tetanic stimulation of the presynaptic axon bathed in sea water containing 45 mM Ca2+. During a 1 s tetanus the light signal rose approximately linearly, even though transmitter release declined rapidly and the light signal subsequently declined with a half-time of 2-6 s. The Ca2+ transient elicited by single nerve impulses was recorded by signal averaging, and showed a time course very much slower than the duration of transmitter release.

Animals↗
PubMed1981

Properties of postsynaptic channels induced by acetylcholine in different frog muscle fibres.

Skeletal muscles in the frog are composed of two distinct classes of muscle fibre: fast muscle fibres capable of propagating action potentials and twitches, and slow muscle fibres normally unable to generate action potentials or twitches. In addition, amphibian muscles contain a spectrum of 'intermediate' fibres whose structural and functional properties lie between those of fast and slow fibres. Much is now known about the characteristics of the channels opened by the transmitter acetylcholine (ACh) acting on the membrane of fast fibres, but the molecular action of ACh on the other fibre types is only poorly understood. We report here the existence of a muscle in the mandibular arch of the frog in which most, if not all, the fibres are multiply innervated and are capable of eliciting action potentials. We also report that the channels induced by the transmitter on the synaptic membrane of fast, slow and submaxillaris muscle fibres differ in their lifetimes and conductances.

Acetylcholine↗
PubMed1981

Does the motor nerve impulse evoke 'non-quantal' transmitter release?

Previous experiments have indicated that there is a continuous leakage of acetylcholine (ACh) from resting motor nerve terminals which can produce a small depolarization in anti-esterase treated endplates (Katz & Miledi 1977; Vyskocil & Illés 1978). This leakage might be expected to be intensified during the presynaptic action potential and also lead to a very small non-quantal endplate response. This hypothesis was examined, in frog and mammalian endplates, by stimulating the motor nerve in a calcium-deprived medium and recording the summated average response to several hundred stimuli. The result was completely negative; no trace of a non-quantal endplate potential was ever observed, with the limit of detection being always less than 10 microV, and sometimes as low as 2 microV. These experiments suggest that the leakage of ACh either does not originate predominantly from the synaptic region of the axon terminal, or that it occurs by a mechanism that is not directly influenced by the membrane potential.

Acetylcholine↗
PubMed1981

Junctional and extrajunctional membrane channels activated by GABA in locust muscle fibres.

Iontophoretic application of GABA to voltage-clamped locust muscle fibres has demonstrated the presence of both extrajunctional and junctional GABA receptors. Extrajunctional GABA receptors are distinct from extrajunctional glutamate receptors which also occur in these muscle fibres. Inward GABA currents are nonlinearly dependent on membrane potential. Analysis of membrane current noise produced by iontophoretic GABA application shows that for junctional and extrajunctional GABA receptors the mean channel lifetime is 3-4 ms and the single-channel conductance is approximately 22 pS at - 80 mV (T = 21 degrees C). The mean lifetime as previously demonstrated for glutamate-sensitive excitatory channels in locust muscle fibres.

Animals↗
PubMed1981

Properties of end-plate channels in rats immunized against acetylcholine receptors.

1. Rats injected with purified acetylcholine receptors (AChR) extracted from electric organs of Torpedo marmorata showed clinical symptoms consistent with the development of experimental myasthenia gravis.2. Sera of rats with this disease contain high levels of anti-AChR antibodies. However, no simple correlation was found between antibody titre and miniature end-plate current (m.e.p.c.) amplitude.3. M.e.p.c.s. at the end-plates of rats injected with AChR (Anti-R), emulsified in complete Freund Adjuvant (CFA), were reduced to about one third the size of controls taken from rats injected only with CFA (Anti-CFA). Mean m.e.p.c. (Anti-R) = 0.73 +/- 0.06 nA; mean m.e.p.c. (Anti-CFA) = 2.43 +/- 0.12 nA (V(m) = -80 mV, T = 20 degrees C).4. The m.e.p.c. decay time constant, tau(m.e.p.c.), is similar at immunized and control rat end-plates. tau(m.e.p.c.) (Anti-R) = 1.32 +/- 0.06 msec; tau(m.e.p.c.) (Anti-CFA) = 1.31 +/- 0.06 msec (V(m) = -80 mV, T = 20 degrees C).5. The end-plate current decay time constant, tau(e.p.c.), is similar at immunized and control end-plates and in both cases depends exponentially on membrane potential. The change in membrane potential required to produce an e-fold change in tau(e.p.c.) is 102.0 +/- 5.72 mV at immunized (Anti-R) end-plates and 92.3 +/- 6.14 mV at control (Anti-CFA) end-plates at T = 10 degrees C.6. Acetylcholine noise was examined at immunized and control rat end-plates at 10 degrees C. Analysis of noise indicates that the single channel conductance, gamma, and mean channel life-time, tau(noise), are essentially unchanged by immunization against AChR. gamma (Anti-R) = 13.15 +/- 0.53 pS; gamma (Anti-CFA) = 12.50 +/- 0.50 pS; tau(noise) (Anti-R) = 2.9 +/- 0.18 msec; tau(noise) (Anti-CFA) = 2.68 +/- 0.14 msec (V(m) = -80 mV, T = 10 degrees C).7. Mean quantal content and Ca(2+) dependence of the end-plate potential are unchanged at immunized end-plates.8. It is concluded that at immunized end-plates the number of activated receptor-channel complexes is reduced without modification of single channel properties. In this respect the immunized rat end-plate is a good model for myasthenia gravis affected human end-plates.

Acetylcholine↗
PubMed1981

Calcium transients in normal and denervated slow muscle fibres of the frog.

1. Intracellular changes in free Ca2+ concentration were recorded from slow muscle fibres in the pyriformis muscle of Rana temporaria, using the dye arsenazo III. Fibres were voltage clamped, and arsenazo signals were recorded in response to depolarizing pulses. 2. The size of the arsenazo response to depolarizing pulses of 100 msec duration was a sigmoid function of membrane potential over the range -45 to 0 mV, and remained constant with further depolarizations up to +100 mV. 3. The peak size of the arsenazo signal to supramaximal depolarizations increased with increasing pulse length. The initial rising phase during a pulse was much slower than in twitch fibres, and this phase was followed by an even slower rise. Following short pulses the decay of the response was exponential, with a time constant of about 1.4 sec, while after long pulses the decline became much slower. 4. Decreasing free Ca2+ concentration in the bathing medium to very low levels, using EGTA , did not affect the responses to short (100 msec) depolarizations. 5. Slow fibres bathed in Ringer's solution containing 12 mM-Ca2+ showed a well maintained arsenazo response to supramaximal depolarizations lasting over 1 min. Reduction of external Ca2+ to 1.8 and (nominally) 0 mM caused the response to become progressively more transient. 6. After denervation, slow fibres developed action potentials, but non of the parameters of the arsenazo response was significantly changed. During the early phase of reinnervation by a mixed nerve, when fast conduction axons begin to innervate slow fibres, the ability to give a maintained response during long depolarizations was reduced. 7. It is concluded that intracellular Ca2+ transients in slow muscle fibres are probably generated by a similar mechanism as in twitch fibres and entry of external Ca2+ is not an appreciable factor. The slow time course of the transients may be important in determining the time courses of tension development and relaxation.

Animals↗
PubMed1981

A further study of the phospholipase-independent action of beta-bungarotoxin at frog end-plates.

1. The effect of beta-bungarotoxin (beta-BuTx) at the frog neuromuscular junction has been investigated further in order to distinguish more clearly between phospholipase- independent and phospholipase-dependent actions on transmitter release. 2. Inhibition of the enzymatic activity, by substitution of strontium for calcium, allowed determination of the dose-response curve of the early rapid decrease in transmitter release caused by the toxin. In the presence of strontium ions there was, however, still about 7% residual enzymatic activity, and electrophysiological evidence of it could be seen in room-temperature experiments at high concentrations of beta-BuTx. This residual enzymatic activity could be suppressed by lowering the temperature to 5 degrees C. 3. In normal calcium-Ringer solution beta-BuTx produced the typical triphasic effect on the amplitude of end-plate potentials (e.p.p.s). Lowering the temperature markedly delayed an then diminished the secondary transient increase. There was, however, comparatively little temperature influence on the first rapid decrease in e.p.p. amplitude. Enzymatic assays confirmed the temperature dependence of the toxin's phospholipase activity on model phospholipid substrates. 4. The kinetics of the phospholipase-independent action of beta-BuTx were examined in strontium-Ringer compared to calcium-Ringer solution, as well as in calcium-Ringer at different temperatures. Both the time to onset of inhibition and the time to 50% inhibition of the e.p.p., during the first phase of toxin action, are temperature-dependent and briefer in calcium than in strontium-Ringer solution. It is suggested that calcium is more effective than strontium in promoting this phospholipase- independent interaction of beta-BuTx with the nerve terminal membrane.

Animals↗
PubMed1981

Single glutamate-activated channels recorded from locust muscle fibres with perfused patch-clamp electrodes.

1. Glutamate-activated single channels have been examined with conventional and internally perfused patch-clamp electrodes applied to the extrajunctional membrane of locust muscle fibres which were usually treated with concanavalin A to reduce desensitization. Channels opened by glutamate and other agonists have been compared.2. Recording patches were selected where there appeared to be only one active channel under the pipette. The conductance for single glutamate-activated channels was 150 pS and was not markedly dependent on clamp potential. The lifetimes of the channels were usually exponentially distributed with a mean of tau(glutamate) = 2.3 +/- 0.12 msec, T = 23 degrees C, V(m) = -60 mV.3. Channels opened by fluoroglutamate had a mean lifetime of tau(fluoroglutamate) = 1.4 +/- 0.1 msec; channels opened by quisqualate had a mean lifetime of tau(quisqualate) = 6.4 +/- 1.0 msec. The conductances of channels opened by fluoroglutamate, quisqualate and glutamate were not significantly different.4. The behaviour of individual receptor-channel complexes has been examined at various concentrations of glutamate. Drug solutions were applied through an internal perfusion pipette which allowed exchange of the solution in the patch-electrode tip within 10 sec. The distribution of channel closed times could be fitted with a single exponential. Channel lifetime was not markedly dependent on glutamate concentration (30-600 mum) whereas the channel closed time decreased with increasing glutamate concentration.5. The reciprocal of channel closed time vs. glutamate concentration had a slope value of 1.85 on logarithmic co-ordinates. The approximately second power dependence of net forward reaction rate on glutamate concentration suggests that at least two glutamate molecules activate a single receptor-channel complex.6. The apparent dissociation constant for the glutamate-receptor complex is large, being about 300-500 muM. If the receptors have an equally low affinity for neurally released transmitter, then only a small amount of the transmitter packet is expected to bind to receptors. Quisqualate and glutamate have similar receptor affinities whereas receptor affinity for fluoroglutamate is smaller.

Animals↗