Biomedical subjects

R Miledi

Publications and source records attributed to R Miledi.

At least 145 records · Page 8Linked to original sources

PubMed1987

Beta-adrenergic agonists and cyclic AMP decrease intracellular resting free-calcium concentration in ileum smooth muscle.

Intracellular free-calcium levels were measured in strips of longitudinal smooth muscle from guinea-pig ileum; fura-2 was used as a calcium monitor. At rest the calcium concentration was about 180 nM, and this rose to 300-400 nM following electrical stimulation and during spontaneous calcium transients (all measurements at 23-25 degrees C). Isoprenaline suppressed the spontaneous calcium transients, and reduced the resting calcium level to about 130 nM. This fall in resting calcium concentration was seen even in muscle strips which did not have spontaneous activity. Elevation of intracellular cyclic AMP levels, produced by forskolin or dibutyryl cyclic AMP, mimicked the actions of isoprenaline. We conclude that the relaxant effects of beta-adrenergic agonists of visceral smooth muscle may be explained partly by a fall in intracellular resting free-calcium level, mediated via an increase in cyclic AMP.

Animals↗

PubMed1987

Characteristics of synaptic currents in frog muscle fibers of different types.

A study was made of synaptic currents in voltage-clamped muscle fibers of the frog. Fast, submaxillaris, and slow muscle fibers are innervated by nerve fibers of different conduction velocities. To avoid spatial complications, transmitter release by nerve impulses was restricted to the site of recording and reduced to single quanta (unitary endplate currents: uepc). Following nerve stimulation, the time course of transmitter release was longer and more variable in slow and submaxillaris muscle fibers than in the fast fibers. The time constant of decay of uepc in submaxillaris and slow fibers was, respectively, about 1.8 and 2.9 times slower than the decay of uepc in fast fibers. This is due mainly to differences in the lifetime of the channels opened by acetylcholine. The neuromuscular junctions in submaxillaris muscle fibers are bouton-like or longer branched contacts; and the unitary currents in the bouton junctions have a slower time course. It is concluded that the synaptic membrane in the different types of muscle fibers has synaptic acetylcholine-operated channels that have different kinetic properties, and that these properties are determined by the type of axon that innervates the muscle fiber.

Animals↗

PubMed1987

Effect of collagenase treatment and subsequent culture on rat muscle fiber acetylcholinesterase activity.

After collagenase treatment and mechanical disruption, acetylcholinesterase (AChE) activity on the surface of individual flexor brevis muscle fibers fell by 88%. During the next 48 hr in culture, surface AChE activity continued to decline, while intracellular activity changed little. After 1 week in culture total muscle fiber AChE activity fell to very low levels and intracellular AChE activity could no longer be detected, probably as a result of reduced synthesis and rapid externalization of intracellular AChE. Apart from the removal of most of the surface activity, cultured muscle fibers had similar AChE activity to muscle fibers that had been denervated in vivo, suggesting that the changes observed in culture reflect the loss of neuromuscular interaction and not to any contributory effects of the dissociation process. It is to be hoped that these results, along with the published results of Bekoff and Betz [J. Physiol, 271:25-40, 537-547], will serve as useful background data for those continuing to use adult dissociated muscle fibers in their studies.

Acetylcholinesterase↗

PubMed1987

Hormonal activation of ionic currents in follicle-enclosed Xenopus oocytes.

Membrane currents were recorded, using the voltage clamp technique, from Xenopus laevis oocytes still surrounded by their enveloping follicular and epithelial cells. Exposure of the follicles to mammalian gonadotropins elicited a current generated largely by an increase in membrane K+ conductance. The gonadotropin response resembled responses elicited by adenosine and catecholamines in the same follicle, but was not blocked by purinergic or catecholaminergic antagonists. The gonadotropin-induced currents were potentiated by the adenylate cyclase activator forskolin and by phosphodiesterase inhibitors; similar currents were elicited in the same follicle by intraoocyte injection of cAMP, which indicates a role for this second messenger in the response mechanism. Gonadotropin responses were either abolished or substantially reduced after treatments that remove the ovarian epithelial and follicular cells. Our experiments suggest that the gonadotropin receptors, and the K+ channels they regulate, reside in the follicular cells.

Acetylcholine↗

PubMed1987

The development of tetrodotoxin-resistant action potentials in long-term organ culture of rat muscle.

The development and long-term maintenance of tetrodotoxin (TTX)-resistant action potentials in culture was examined. The amplitude and maximum rate of rise of the action potential in normal Ringer solution fell during culture. At the same time the amplitude and maximum rate of rise of the TTX-resistant action potential increased, demonstrating that TTX resistance can develop fully in culture. Actinomycin D inhibited the onset of denervation changes in culture and was also able to delay the fall in the maximum rate of rise of the action potential in normal Ringer solution. When applied to muscle after 48 h of culture, it was relatively ineffectual, showing that de novo protein synthesis during the first 48 h of culture result in denervation changes which are not reversed by subsequent exposure to actinomycin D. The onset of TTX resistance occurred more rapidly in dissociated muscle fibres, presumably as a result of the early removal of the nerve stump.

Action Potentials↗

PubMed1987

Oscillatory chloride current evoked by temperature jumps during muscarinic and serotonergic activation in Xenopus oocyte.

1. Membrane currents were recorded from voltage-clamped oocytes of Xenopus laevis, during temperature jumps imposed by a heating light. Resting oocytes usually showed little response, but large oscillatory membrane currents developed in response to cooling steps applied during activation of 'native' muscarinic receptors. 2. Similar temperature jump (Tjump) currents were seen during activation of oscillatory chloride currents mediated by muscarinic acetylcholine (ACh), serotonin, glutamate and noradrenaline receptors, expressed in the oocyte following injection with messenger ribonucleic acid (mRNA) from rat brain. The Tjump response during muscarinic activation was selectively blocked by atropine, and that during serotonergic activation by methysergide. In contrast, the 'smooth' membrane currents elicited by nicotinic ACh, kainate and gamma-aminobutyric acid (GABA) were not accompanied by Tjump responses. 3. Rapid cooling of the oocyte gave larger Tjump currents than a gradual cooling over a few seconds. The size of the Tjump current elicited by a fixed cooling step increased linearly with the preceding time of warming, becoming maximal at intervals greater than about 100 s. 4. The Tjump current was inward at a clamp potential of -60 mV and reversed direction at about -22 mV, which corresponds to the chloride equilibrium potential in the oocyte. In low-chloride solution the reversal potential was shifted to more positive potentials, but it was almost unchanged by changes in potassium and sodium concentration. The size of the Tjump current decreased as the membrane potential was made more negative than about -40 mV. 5. The period of oscillation of the Tjump current increased with decreasing temperature, following a Q10 of 3.15. Depolarization also caused a small increase in period. 6. The Tjump current was not abolished in calcium-free solution, or by addition of manganese or lanthanum to the bathing solution. However, it was abolished by intracellular injection of the calcium-chelating agent EGTA. 7. Intracellular injection of inositol 1,4,5-trisphosphate evoked an oscillatory membrane current, during which Tjump responses developed similar to those after muscarinic activation. Intracellular injection of calcium evoked a chloride current, but this was not accompanied by Tjump responses. 8. We conclude that the oscillatory currents evoked by temperature jumps arise from chloride channels activated by intracellular calcium. This calcium is probably mobilized from intracellular stores by inositol trisphosphate which is liberated as a result of activation of muscarinic receptors, and also receptors for serotonin and glutamate.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗

PubMed1986

Neurotensin and substance P receptors expressed in Xenopus oocytes by messenger RNA from rat brain.

Xenopus oocytes were induced to acquire sensitivity to neurotensin and substance P, by injecting them with a fraction of poly(A)+ mRNA from rat brain. Non-injected oocytes, and oocytes injected with other brain mRNAs, failed to show responses, suggesting that receptors to these peptides were expressed by specific brain mRNAs. Responses to substance P and neurotensin comprised an oscillatory chloride current, and a smooth current having different ionic basis. These currents resembled those seen during activation of muscarinic and serotonergic receptors, but were not blocked by the corresponding antagonists atropine and methysergide. The responses to substance P, and to a lesser extent to neurotensin, showed a long-lasting desensitization. Similarities between the oscillatory currents evoked by the peptides acetylcholine and serotonin suggest that all these receptors may 'link in' to a common intracellular messenger pathway.

Animals↗

PubMed1986

Minimal latency of calcium release in frog twitch muscle fibres.

Intracellular release of calcium in frog skeletal muscle fibres was monitored by the use of arsenazo III, in response to voltage clamped depolarizing pulses. A latency of a few milliseconds was evident between the onset of depolarization and the first detectable rise in the arsenazo-calcium signal, and this decreased logarithmically as the depolarization was increased. The minimal latency with strong depolarization (to +20 to +100 mV) was about 2 ms at 5 degrees C. This delay appears to be sufficiently long to be compatible with a chemically mediated coupling mechanism between depolarization and calcium release from the sarcoplasmic reticulum.

Animals↗

PubMed1986

Changes in intracellular calcium and in membrane currents evoked by injection of inositol trisphosphate into Xenopus oocytes.

Intracellular calcium was monitored by the use of aequorin in voltage-clamped oocytes of Xenopus laevis. Injection of inositol trisphosphate (IP3) into oocytes elicited slowly rising and decaying aequorin/calcium signals and produced oscillatory chloride membrane currents. These responses did not depend upon extracellular calcium, since they could be elicited in calcium-free solution and after addition of cobalt or lanthanum to block calcium channels in the surface membrane. We conclude that IP3 causes the release of calcium from intracellular stores in the oocyte. Injections of calcium gave aequorin and membrane current responses that were more transient than those seen with IP3.

Aequorin↗

PubMed1986

Actions of pentobarbital on rat brain receptors expressed in Xenopus oocytes.

Functional receptor channels activated by GABA and other neurotransmitters were "transplanted" from rat brain to Xenopus oocytes by injecting the oocytes with total poly(A)+ mRNA isolated from rat or chick brain. Membrane currents elicited in the oocyte by GABA inverted polarity at about the chloride equilibrium potential (ca. -25 mV). Pentobarbital potentiated the GABA-activated currents, without appreciably changing the reversal potential or form of the current-voltage relationship. At low (less than 10(-5) M) concentrations of GABA, pentobarbital (100 microM) potentiated the responses by a factor of 10 or more, but responses to high (ca. 1 mM) concentrations of GABA were almost unchanged. Half-maximal activation of the response was obtained with about 3 X 10(-5) M GABA when applied alone and with about 4 X 10(-6) M GABA when applied together with 100 microM pentobarbital. At low doses of GABA, the size of the current increased as the 1.4th power of GABA concentration, but this relationship became nearly linear in the presence of pentobarbital. The potentiation of the GABA response increased linearly with concentrations of pentobarbital up to about 300 microM, reaching a maximum of about 50-fold. At higher concentrations of pentobarbital, the response to GABA declined. Relaxations of GABA-activated currents following voltage steps became slower in the presence of pentobarbital, suggesting that the open life-time of the channels was prolonged. In addition to actions on GABA-activated currents, pentobarbital itself elicited a small membrane current that inverted polarity at a potential (-10 mV) more positive than the GABA-activated current.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

On the orientation of foreign neurotransmitter receptors in Xenopus oocytes.

Xenopus oocytes can be made to incorporate into their membrane foreign neurotransmitter receptors and voltage-activated sodium channels. In their original location the receptors are normally activated by the extracellular action of transmitter substances. Tests were made to see if some of the newly synthesized foreign receptors were inserted in the oocyte membrane with their active site facing inwards. Since intracellular injections of acetylcholine, gamma-aminobutyric acid, serotonin and kainic acid and tetrodotoxin into the oocyte failed to elicit a response, we conclude that very few, or none, of the receptor molecules expressed in the oocyte by the exogenous mRNA are inserted with the wrong orientation in the membrane.

Animals↗

PubMed1985

Calcium entry induced by acetylcholine action on snail neurons.

A study was made of excitatory and inhibitory responses elicited by acetylcholine (ACh) in neurons of the snail Eobania vermiculata. At resting potential, ACh evoked a depolarizing inward current in some neurons (D-cells) and a hyperpolarizing current in others (H-cells). The currents elicited by ACh were nonlinearly dependent on membrane potential. After either D- or H-cells were equilibrated in chloride-free isotonic calcium, ACh evoked a depolarizing inward current which reversed sign at about -55 mV. These results suggest that ACh causes an influx of Ca2+ in both types of neurons.

Acetylcholine↗

PubMed1985

Messenger RNA from bovine retina induces kainate and glycine receptors in Xenopus oocytes.

The retina contains several types of nerve cells that communicate through chemical synapses. The transmitter and receptor molecules that mediate signal transmission across these synapses need further characterization. For this purpose, poly (A)+ mRNA was isolated from bovine retinas and injected into Xenopus laevis oocytes. Translation of the foreign mRNA induced the oocyte membrane to acquire functional receptors to kainate and, to a lesser extent, also receptors to glycine, gamma-aminobutryic acid (GABA), aspartate and glutamate. Thus, the cells in the retina must contain different messengers coding for these neurotransmitter receptors. Activation of the kainate receptors opens membrane channels, generating an ionic current which has an equilibrium potential close to 0 mv. The current is well maintained during prolonged application of kainate, and hence these receptors may be involved in the neurotoxic effects produced by kainate in the retina.

Animals↗

PubMed1985

A transient inward current elicited by hyperpolarization during serotonin activation in Xenopus oocytes.

Activation of serotonin, glutamate or muscarinic receptors, incorporated into the membrane of Xenopus oocytes following injection of messenger RNA from rat brain, caused the development of a transient inward (Tin) current when the membrane was hyperpolarized. A detailed study was made of the Tin current induced during serotonin activation. The current is due principally to efflux of chloride ions, and is presumably activated by an influx of calcium ions, because it was blocked by removal of calcium from the bathing medium, by addition of manganese, cobalt or lanthanum, or by intracellular injection of EGTA. During application of serotonin, the amplitude of the Tin current increased slowly, and after washing it persisted for longer than the direct serotonin-induced current. The amplitude of the Tin current was sensitive to temperature and pH, and was abolished at pH 6.5 or by cooling to 12 degrees C. The Tin current may be of importance in regulating the excitability of neurons in the central nervous system.

Animals↗

PubMed1985

Intracellular Ca2+-dependent and Ca2+-independent responses of rat brain serotonin receptors transplanted to Xenopus oocytes.

Xenopus oocytes injected with messenger RNA extracted from rat brain are induced to acquire a variety of neurotransmitter receptors and voltage-operated membrane channels. Activation of the receptors to serotonin, acetylcholine (muscarinic) and glutamate elicits oscillatory membrane currents carried by chloride ions. These currents are not abolished by removing external calcium, but are completely abolished after EGTA is injected into the oocytes to chelate intracellular calcium. A smooth current response to serotonin remained in EGTA-loaded oocytes, indicating that this response does not require intracellular calcium. In contrast to the oscillatory chloride currents, the chloride currents activated by GABA or glycine are not abolished by intracellular injection of EGTA. Thus, there appear to be two classes of chloride channels one of which requires intracellular calcium to open.

Animals↗

PubMed1985

Choline acetyltransferase and acetylcholine in Xenopus oocytes injected with mRNA from the electric lobe of Torpedo.

Xenopus oocytes were injected with poly(A)+ mRNA obtained from the electric lobes of Torpedo marmorata and Torpedo ocellata, which contain the cell bodies of the neurons that innervate the electric organs. The electric lobe mRNA preparation induces the oocytes to synthesize a catalytically active form of the enzyme choline acetyltransferase (EC 2.3.1.6). Enzymatic activity is found almost exclusively in the cytoplasmic fraction of injected, but not control, oocytes. Evidence is presented that distinguishes between the induced choline acetyltransferase activity and an intrinsic carnitine acetyltransferase activity present in the oocytes. This latter enzyme is associated principally with particulate fractions of the oocyte. The level of acetylcholine, which accumulates in mRNA-injected oocytes, is relatively insensitive to pharmacological manipulations that alter the acetylcholine content of other cells. These results show that Xenopus oocytes may be used advantageously to study functional properties of polypeptides associated with presynaptic elements in the nervous system.

Acetylcholine↗

PubMed1985

Influence of divalent cations on the phospholipase-independent action of beta-bungarotoxin at frog neuromuscular junctions.

The influence of different divalent cations on the phospholipase-independent inhibition of transmitter release caused by beta-bungarotoxin (beta-BuTx), has been investigated by measuring the frequency of spontaneous miniature end-plate potentials (m.e.p.p.s) at frog neuromuscular junctions. After adding the toxin to normal calcium Ringer solution the m.e.p.p. frequency fell quickly to very low values. This was followed by an increase in frequency characterized by bursts of m.e.p.p.s. The temperature had a negligible effect on the speed of the first inhibition. In Ringer solutions where calcium had been substituted by other divalent cations (5 mM) in the presence of ethyleneglycol-bis-(beta-aminoethylether)N, N'-tetraacetic acid (EGTA, 1 mM), this beta-BuTx-induced decrease in m.e.p.p. frequency was markedly slower. The potency of cations in promoting the initial phase of toxin action was in the sequence: calcium greater than magnesium greater than strontium congruent to cobalt greater than manganese. This phospholipase-independent inhibition of transmitter release followed approximately first-order kinetics, suggesting that it depends mainly on toxin concentration. In the absence of any divalent cations in the Ringer solution beta-BuTx had practically no effect on m.e.p.p. frequency. It appears that beta-BuTx requires divalent cations in order to bind to motor nerve terminals and exert its initial inhibitory action on spontaneous release of transmitter quanta.

Action Potentials↗

PubMed1985

Post-synaptic calcium influx at the giant synapse of the squid during activation by glutamate.

Changes in free calcium were monitored in the post-synaptic axon of the giant synapse of the squid, using the calcium indicators aequorin and Arsenazo III. The peak size of the calcium-dependent optical signals recorded from aequorin and Arsenazo III both showed a linear relation with the amount of calcium injected ionophoretically into the axon, but the Arsenazo signal had a slower time course than the aequorin. Ionophoretic application of glutamate to the post-synaptic axon depolarized the axon and caused a rise in intracellular free calcium. Aequorin signals were detected in natural sea water, and their size increased when the calcium concentration in the sea water was raised. Arsenazo signals could be detected only in high-calcium (55 mM) sea water. Intracellular calcium signals were detected also during bath application of several glutamate analogues, including kainate, ibotenate, and aspartate. The peak amplitude of the intracellular calcium signal, monitored with both indicators, increased with increasing ionophoretic glutamate dose, and varied linearly with the integral of the glutamate-induced membrane depolarization. No calcium signals were detected when depolarizations, similar to those produced by glutamate, were induced by current injection in the absence of glutamate. We conclude that glutamate increases the calcium permeability of the post-synaptic membrane, independently of the glutamate-induced depolarization. The glutamate-induced depolarization and the rise in intracellular free calcium increased roughly linearly as the membrane potential was made more negative. Extrapolation of these data indicated that the glutamate depolarization would reduce to zero at about -30 mV, while the calcium signals would be suppressed at about +50 mV.

Aequorin↗