Biomedical subjects

R Miledi

Publications and source records attributed to R Miledi.

At least 127 records · Page 7Linked to original sources

PubMed1989

Inhibition of rat brain glutamate receptors by philanthotoxin.

The actions of philanthotoxin (PhTX) were studied on the function of glutamate receptors expressed in Xenopus oocytes injected with rat brain mRNA and on binding of radioligands to rat brain glutamate receptors. PhTX reversibly inhibited the oocyte responses to quisqualate, N-methyl-D-aspartate (NMDA) and kainate in a dose-dependent manner. The NMDA receptor was the most sensitive to PhTX action (10-fold more than the kainate receptor) and the least sensitive was the smooth current component of the quisqualate response. Recovery from PhTX block differed among the three amino acids. NMDA responses recovered completely within a few minutes whereas responses to kainate and quisqualate recovered more slowly. PhTX had no effect on equilibrium binding of [3H]glutamate to rat brain cortical membranes studied in buffer treated to eliminate microorganisms. Based on the drug specificity of this [3H]glutamate binding, it is suggested to be mostly to the NMDA receptor. Low concentrations of PhTX (1-10 microM) potentiated binding of [3H] MK-801, a specific noncompetitive inhibitor of the NMDA receptor. However, higher PhTX concentrations inhibited this binding with an IC50 of 20 microM, similar to its inhibition of the oocyte-expressed NMDA receptor. Inhibition of [3H]MK-801 binding by PhTX was noncompetitive. It is suggested that PhTX, like the more potent MK-801, binds to an allosteric site on the NMDA receptor and inhibits its function but its binding site is not identical with the MK-801 binding site.

Animals↗

PubMed1988

Expression of glycine and other amino acid receptors by rat spinal cord mRNA in Xenopus oocytes.

Xenopus oocytes were injected with mRNA from adult or neonatal rat spinal cord, or from adult cerebral cortex, and examined electrophysiologically to measure currents elicited by various receptor agonists. The mRNAs induced the oocytes to acquire similar types of amino acid receptors, albeit with different potencies. In oocytes injected with adult cord mRNA the responses to kainate and gamma-aminobutyrate were much smaller, whilst the currents elicited by glycine and beta-alanine were markedly larger, than those in oocytes injected with cortex mRNA. For these receptors, the expressional potency of neonatal spinal cord mRNA is similar to that of the adult cord mRNA, except for a lower sensitivity to beta-alanine.

Amino Acids↗

PubMed1988

Heterogeneity of glycine receptors and their messenger RNAs in rat brain and spinal cord.

Messenger RNAs isolated from adult or newborn rat spinal cord were fractionated in a sucrose gradient. The fractions were injected into Xenopus oocytes to determine their potencies for expression of glycine receptors (GlyRs), which were then examined electrophysiologically. The sedimentation profiles disclosed two classes of GlyR mRNAs, one heavy and the other light. The adult spinal cord was rich in heavy GlyR mRNA, whereas the light GlyR mRNA was more abundant in neonatal spinal cord and in adult cerebral cortex. Glycine receptors encoded by heavy and light mRNAs of adult spinal cord showed some electrophysiological differences. Thus there are two types of GlyRs encoded by mRNAs of different sizes, and the expression of these mRNAs is developmentally regulated. A tissue- and age-dependent distribution of heterogeneous GlyR mRNAs may imply diverse roles of the GlyRs in neuronal function in the central nervous system.

Animals↗

PubMed1988

Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex.

The ontogenesis of mRNAs coding for GABA and glycine receptors in the cerebral cortex of the rat was examined by extracting poly(A)+ mRNA from the brains of embryonic, postnatal or adult rats and injecting it into Xenopus oocytes. The ability of a messenger to express functional receptors was then assayed by measuring the membrane currents elicited by the agonists. The size of the GABA-induced current increased progressively with age, being undetectable in oocytes injected with mRNA from embryonic day 15 and reaching a maximum in oocytes injected with mRNA from postnatal day 30. In contrast, the glycine-induced response was negligible in oocytes injected with mRNA from the cerebral hemispheres of embryos 15 days old; it increased sharply to a maximum with newborn animals and then decreased with age to become very small with mRNA from adult cortex. GABA and glycine receptors induced by mRNA from the cerebral cortex of all ages are associated with chloride channels.

Aging↗

PubMed1988

In vitro reinnervation of adult rat muscle fibres by foreign neurons and transformed chromaffin PC12 cells.

Adult rat muscle fibres were dissociated by using collagenase and maintained in culture. One to nine days later, neurons obtained from stages 22-30 Xenopus laevis embryos, or neonatal spinal cord, or pheochromocytoma (PC12) cells treated with nerve growth factor were added. Subsequently, the co-cultures were maintained for up to eight days. Functional synapses were formed with variable efficiency: 12% in rat-Xenopus nerve-muscle co-cultures, 23% in rat-rat and 33% in PC12 co-cultures. Miniature endplate potentials (MEPPs) and currents (MEPCs) were recorded, at frequencies ranging from 0.01 to 0.9 Hz. Their mean amplitude was smaller than in normal mammalian muscles. The rise time and time-constant of decay of MEPCs was about seven to ten times longer than that found in the original muscle, resembling immature synapses. (+)-Tubocurarine abolished the MEPPs in the rat-PC12 neuromuscular junctions. It is concluded that dissociated adult rat muscle fibres retain their ability of being reinnervated, and can form functional synapses with foreign neurons and transformed chromaffin cells.

Adrenal Gland Neoplasms↗

PubMed1988

Transient potassium current in native Xenopus oocytes.

Depolarization of follicle-enclosed oocytes of Xenopus laevis obtained from some donors elicits, in addition to other responses, a fast transient outward current. After holding the membrane potential at -100 mV this response begins to be activated by depolarizations to around -30 mV, and increases progressively as the voltage is raised further. A striking characteristic is that the current recovers only slowly (several seconds) from inactivation following a depolarizing pulse. Because of its outward direction and insensitivity to removal of extracellular chloride or addition of tetrodotoxin, the current probably arises largely through a flux of potassium ions. The current was abolished after treatment of oocytes with collagenase to remove enveloping cells, and although it was blocked by barium and zinc ions, tetraethylammonium was relatively ineffective. In addition, the potassium current was unaffected by 5 mM manganese, suggesting that it does not arise as a consequence of an influx of calcium into the oocyte.

Animals↗

PubMed1988

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle.

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.

Acetylcholine↗

PubMed1988

A calcium-independent chloride current activated by hyperpolarization in Xenopus oocytes.

Hyperpolarization of oocytes of Xenopus laevis usually elicits mainly passive currents. However, when polarized to potentials more negative than about -100 mV, oocytes obtained from some donors show a relatively well maintained current that is carried mainly by chloride ions. This response does not depend upon external calcium, and is thus clearly different from the calcium-dependent transient chloride current previously described.

Animals↗

PubMed1988

Responses to GABA, glycine and beta-alanine induced in Xenopus oocytes by messenger RNA from chick and rat brain.

Poly (A)+ messenger RNA (mRNA) was extracted from rat and chick brains, and injected into oocytes of Xenopus laevis. This led to the expression of receptors that evoked membrane currents in response to gamma-aminobutyric acid (GABA), glycine and beta-alanine. These currents all inverted at about the chloride equilibrium potential in the oocyte, and showed a marked rectification at negative potentials. Oocytes injected with mRNA from chick optic lobe gave large responses to GABA and beta-alanine, but small responses to glycine. In contrast, one fraction of mRNA from rat cerebral cortex (obtained by sucrose density gradient centrifugation) caused oocytes to develop sensitivity to GABA, glycine and beta-alanine, but very little to GABA. The pharmacological properties of the three amino acid responses also differed. Barbiturate and benzodiazepines potentiated the responses to GABA and beta-alanine, but not to glycine. Strychnine reduced the responses to glycine and beta-alanine, but not to GABA, whereas bicuculline reduced the responses to GABA and beta-alanine, but not to glycine. We conclude that different species of mRNA code for receptors to GABA and glycine, and possibly also for separate beta-alanine receptors.

Amino Acids↗

PubMed1988

Characteristics of Schwann-cell miniature end-plate currents in denervated frog muscle.

End-plates in denervated frog sartorius muscles were investigated, under voltage clamp, to determine the characteristics of miniature endplate currents (mepc) caused by the release of acetylcholine (ACh)-quanta from Schwann-cells. Staining for acetylcholinesterase ascertained that in most cases Schwann-cell mepc had been recorded focally from the end-plate. The mean amplitude of Schwann-cell mepc (about 1 nA) was smaller than the amplitude (3-4nA) of normal nerve evoked mepc. The amplitude distribution of Schwann-cell mepc was skewed, as were also the distributions of decay time constants and of rise times. Some Schwann-cell mepc appeared to be of composite nature, as shown by an inflexion on either the rising of falling phase. The decay time constant tau of both fast and slowly decaying Schwann-cell mepc increased with membrane hyperpolarization suggesting an exponential correlation. The potential dependence was 118 +/- 37 mV (means +/- SD from 6 experiments) for an e-fold change of tau, which does not differ significantly from that for neural mepc (98 mV). The cholinesterase inhibitor neostigmine increased the amplitude and prolonged the time course of Schwann-cell mepc to a similar degree after short and long term denervation, which indicates that cholinesterase was still functional after 3-4 months of denervation. The results show that the characteristics of Schwann-cell mepc resemble in many respects those of neural mepc but are more variable in shape and amplitude.

Acetylcholine↗

PubMed1988

Effect of tunicamycin on the expression of functional brain neurotransmitter receptors and voltage-operated channels in Xenopus oocytes.

The role of N-glycosylation on the expression of functional brain neurotransmitter receptors and voltage-operated channels was studied by injecting Xenopus oocytes with mRNA from rat brain or chick optic lobe, and culturing them in the presence or absence of tunicamycin, an inhibitor of asparagine linked glycosylation. Electrophysiological recordings were then made to assess the amounts of functional receptors and channels present in the oocyte membrane. The appearance of gamma-aminobutyric acid (GABA) receptors and voltage-activated Na+ channels was profoundly reduced. In contrast, the functional expression of kainate receptors, and voltage-activated K+ and Ca2+ channels was much less affected. Thus, it seems that kainate receptors, and K+ and Ca2+ channels can be expressed and function normally without being glycosylated. On the other hand, GABA receptors and Na+ channels may need to be N-glycosylated in order to function properly, or to ensure their correct insertion into the membrane.

Amino Acids↗

PubMed1988

Repression of nicotinic acetylcholine receptor expression by antisense RNAs and an oligonucleotide.

Four antisense RNAs, synthesized from cDNA clones coding for the four subunits of the acetylcholine receptor of Torpedo electroplaques, were used to study their effect on the expression of functional Torpedo acetylcholine receptors in Xenopus oocytes. All antisense RNAs inhibited the appearance of functional receptors in the oocyte's surface membrane for at least 1 week. This inhibition was specific because the antisense RNAs did not block the expression of the Cl- channels, also encoded by Torpedo electroplaque mRNA. Experiments with incomplete antisense RNAs and a synthetic oligonucleotide indicate that covering the ribosome binding site or the initiation codon in the mRNA is not a necessary requirement for efficient blocking. Thus, the use of antisense RNAs combined with the Xenopus oocyte system provides a novel approach to screen cDNA libraries for the genes coding for multisubunit neurotransmitter receptors.

Animals↗

PubMed1987

Tetrodotoxin-sensitive sodium current in native Xenopus oocytes.

Depolarization of oocytes of Xenopus laevis usually elicits mainly passive currents, and a calcium-dependent chloride current. However, oocytes obtained from some donors show, in addition, a transient inward current on depolarization to potentials beyond ca. -40 mV. This current is abolished by tetrodotoxin at submicromolar concentrations, and is prolonged by veratrine; thus, it probably arises through sodium channels of a type similar to those found in nerve and muscle cells. However, the kinetics of the sodium currents varied between oocytes from different donors; this result suggests that genes encoding different sodium channels may be expressed in oocytes from different donors. The presence of these native channels may complicate experiments to study the expression of exogenous sodium channels encoded by foreign messenger RNAs injected into the oocyte.

Animals↗

PubMed1987

Injection of inositol 1,3,4,5-tetrakisphosphate into Xenopus oocytes generates a chloride current dependent upon intracellular calcium.

Injection of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) into voltage-clamped oocytes of Xenopus laevis elicited an oscillatory chloride membrane current. This response did not depend upon extracellular calcium, because it could be produced in calcium-free solution and after addition of cobalt to block calcium channels in the surface membrane. However, it was abolished after intracellular loading with the calcium chelating agent EGTA, indicating a dependence upon intracellular calcium. The mean dose of Ins(1,3,4,5)P4 required to elicit a threshold current was 4 x 10(-14) mol. In comparison, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) gave a similar oscillatory current with doses of about one twentieth as big. Hyperpolarization of the oocyte membrane during activation by Ins(1,3,4,5)P4 elicited a transient inward current, as a result of the opening of calcium-dependent chloride channels subsequent to the entry of external calcium. In some oocytes the injection of Ins(1,3,4,5)P4 was itself sufficient to allow the generation of the transient inward current, whereas in others a prior injection of Ins(1,4,5)P3 was required. We conclude that Ins(1,3,4,5)P4 causes the release of intracellular calcium from stores in the oocyte, albeit with less potency than Ins(1,4,5)P3. In addition, Ins(1,3,4,5)P4 activates voltage-sensitive calcium channels in the surface membrane, via a process that may require 'priming' by Ins(1,4,5)P3.

Animals↗

PubMed1987

Membrane currents elicited by porcine vasoactive intestinal peptide (VIP) in follicle-enclosed Xenopus oocytes.

Membrane currents were recorded from voltage-clamped Xenopus laevis oocytes, surrounded by their enveloping follicular and epithelial cells. Porcine vasoactive intestinal peptide (VIP) generated a membrane current due to an increase in membrane conductance to K+. The VIP current was mimicked by the adenylate cyclase activator forskolin and was potentiated by phosphodiesterase inhibitors, suggesting that adenosine 3',5'-cyclic monophosphate (cyclic AMP) plays a role in mediating the response. Though resembling the follicle's responses to catecholamines and adenosine in ionic basis and apparent mechanism, the response to VIP was not blocked by catecholaminergic or purinergic antagonists, indicating the presence of a specific VIP receptor in the follicle. Among the VIP related peptides, PHM-27 generated similar but smaller K+ currents and porcine secretin and glucagon neither elicited a response nor blocked that to VIP. After treating follicles with collagenase to remove the epithelial and follicular cells the responses to VIP were either substantially reduced or abolished, suggesting that the VIP receptors and K+ channels are both located in the follicular cells.

1-Methyl-3-isobutylxanthine↗

PubMed1987

Inositol trisphosphate activates a voltage-dependent calcium influx in Xenopus oocytes.

Injection of inositol trisphosphate (IP3) into oocytes of Xenopus laevis induces the appearance of a transient inward (Tin) current on hyperpolarization of the membrane. This current is carried largely by chloride ions, but is shown to depend on extracellular calcium, because it is abolished by removal of calcium in the bathing fluid or by addition of manganese. Recordings with aequorin as an intracellular calcium indicator show that a calcium influx is activated by hyperpolarization after intracellular injection of IP3 as well as after activation of neurotransmitter receptors thought to mediate a rise in IP3. Furthermore, by substituting barium for calcium in the bathing solution, inward barium currents can be recorded during hyperpolarization. We conclude that intracellular IP3 modulates the activity of a class of calcium channels, so as to allow an influx of calcium on hyperpolarization. In normal Ringer solution this then leads to the generation of a chloride current, because of the large numbers of calcium-dependent chloride channels in the oocyte membrane.

Animals↗

PubMed1987

Activation of a common effector system by different brain neurotransmitter receptors in Xenopus oocytes.

Xenopus oocytes possess 'native' muscarinic receptors, which give rise to oscillatory chloride currents; similar responses are elicited by activation of foreign receptors to serotonin, glutamate and noradrenaline, expressed in oocytes after injection of messenger RNA from rat brain. When low concentrations of two agonists are applied together, the combined response is greater than would be expected from the sum of the responses to each agonist applied alone. Potentiation of acetylcholine by serotonin is blocked by the serotonin antagonist methysergide; conversely, the potentiation of serotonin by acetylcholine is blocked by the muscarinic antagonist atropine. This indicates that each agonist acts on a distinct receptor. The interactions between serotonin, acetylcholine and other agonists provide further evidence that the different receptors may all 'link in' to a common receptor-channel coupling system, in which phosphoinositide metabolism and calcium liberation lead to the opening of chloride channels in the oocyte membrane.

Acetylcholine↗

PubMed1987

In vitro formation of neuromuscular junctions between adult Rana muscle fibres and embryonic Xenopus neurons.

Adult muscle fibres of the frog Rana temporaria were cultured with neurons from embryos of the frog Xenopus laevis. Electron microscopical and electro-physiological examination of the cultures showed that hetero-specific (Xenopus-Rana) neuromuscular junctions were formed in vitro. Nerve processes, without any Schwann cell covering, made contacts anywhere along a muscle fibre, and the junctions resembled those seen during early regeneration of neuromuscular synapses in situ. Functional contacts, as inferred by the presence of spontaneous miniature endplate potentials, or currents, were more common if the muscle fibres were denervated prior to culturing with neurons. Miniature endplate currents (m.e.p.cs) had a skewed amplitude distribution, with many small events lost in the recording noise, and their mean amplitude was much smaller than that of m.e.p.cs in the original lumbricalis muscle. The time constant of decay of m.e.p.cs in the hetero-specific junctions formed in vitro was several times longer than the decay of m.e.p.cs in the original muscle. Analysis of membrane current noise elicited by ionophoretically applied acetylcholine (ACh) suggests that the slower decay of m.e.p.cs in the junctions formed in vitro is due to a prolonged lifetime of the channels opened by ACh and to repetitive activation of ACh-receptors, which becomes possible because of a comparative lack of cholinesterase in the junctions.

Animals↗