PubMed Health⌕ Search

Biomedical subjects

R Miledi

Publications and source records attributed to R Miledi.

At least 73 records · Page 4Linked to original sources
PubMed1992

Characterization of bicuculline/baclofen-insensitive gamma-aminobutyric acid receptors expressed in Xenopus oocytes. I. Effects of Cl- channel inhibitors.

Poly(A)+ RNA from bovine retina expressed gamma-aminobutyric acid (GABA)-activated membrane current responses in Xenopus oocytes, consisting of two pharmacologically distinct components. One component (IG-Aret) was mediated by GABAA receptors, and the other component (KG-BR) by atypical GABA receptors that were resistant to inhibition by bicuculline and insensitive to activation by baclofen. To further characterize the bicuculline/baclofen-insensitive GABA receptors, electrical recordings were made measuring the sensitivity of IG-BR to two Cl- channel inhibitors, t-butylbicyclophosphorothionate (TBPS) and picrotoxin. For purposes of comparison, effects of TBPS and picrotoxin were also assayed on currents mediated by GABAA receptors expressed in oocytes by rat cerebral cortex RNA (IG-Actx). The main finding of this study was that TBPS was a surprisingly weak inhibitor of IG-BR, whereas IG-Actx was potently suppressed. Assays on maximum responses indicated that IG-Actx was at least 500 times more sensitive to TBPS than was IG-BR (IC50 values of approximately 0.2 microM and greater than 50 microM, respectively). Moreover, inhibition of IG-Actx by micromolar concentrations of TBPS was largely insurmountable, whereas the weak inhibitory effects on IG-BR showed strong dependence on agonist concentration. For example, 10 microM TBPS reduced maximum IG-Actx by greater than 90%, an effect that was not significantly reversed by 10-fold increases in the concentration of agonist. In contrast, the same concentration of TBPS caused a 2-fold increase in the EC50 for IG-BR but had only marginal (less than 5%) inhibitory effects on maximum responses. Picrotoxin inhibited both types of current, but assays on maximum responses indicated that IG-Actx was approximately 30 times more sensitive than IG-BR (IC50 values of approximately 1 and 30 microM, respectively). Inhibitory effects of picrotoxin on IG-BR again showed strong dependence on agonist concentration, but in this case there was also a clear insurmountable component. Comparisons between IG-Actx and IG-Aret suggested that GABAA receptors expressed by either brain or retina RNA showed approximately the same sensitivity to TBPS and picrotoxin. Our experiments indicate that the bicuculline/baclofen-insensitive GABA receptors expressed by retina RNA differ markedly from GABAA receptors in their sensitivity to TBPS and picrotoxin. Defining the structural features responsible for these differences at the molecular level will provide a further means of investigating the complex mechanisms underlying interactions between inhibitors and GABA-activated Cl- channels.

Animals↗
PubMed1991

An endogenous carrier-mediated uptake system for folate in oocytes of Xenopus laevis.

We investigated the existence of an endogenous uptake system for folate in Xenopus laevis oocytes. This was done by performing uptake measurements using [3H]folic acid. Uptake of folic acid was linear with time for 4 h of incubation, and was similar in collagenase-treated and non-treated oocytes. The uptake process was carrier-mediated, as suggested by the saturation of folic acid uptake with concentration, and by the ability of unlabelled folic acid and its related compounds to significantly inhibit the uptake of [3H]folic acid. The apparent Km and Vmax of the uptake process were 42 +/- 7 nM and 10.56 +/- 0.46 fmol per oocyte per 2 h, respectively. The uptake of folic acid was independent of the presence of Na+ in the incubation medium, but was highly pH dependent with severe inhibition occurring at pH lower than 6.5. Folic acid uptake was energy- and temperature-dependent, and was significantly inhibited by the anion transport inhibitors DIDS and SITS. These results demonstrate the existence of an endogenous carrier-mediated system for folic acid uptake in Xenopus oocytes. Further characterization of the molecular mechanism of folic acid uptake and its regulation in this non mammalian in vitro unicellular system may prove useful in furthering our understanding of folate movement across biological membranes.

Animals↗
PubMed1991

A factor that activates oscillatory chloride currents in Xenopus oocytes copurifies with a subfraction of serum albumin.

Vertebrate blood sera contain a factor that elicits oscillatory chloride currents in Xenopus oocytes through activation of the phosphatidylinositol second messenger system. This factor was purified from rabbit and human sera by a sequence of Blue-Agarose chromatography, concanavalin A affinity chromatography, and hydroxyapatite fractionation, yielding a single active protein band (67 kDa). This protein is a subfraction of serum albumin, as revealed by its molecular mass, isoelectric properties, peptide maps, amino acid composition, and NH2-terminal sequence. Moreover, the factor could be purified with a monoclonal antibody to serum albumin and its ability to elicit oscillatory currents was inhibited by several polyclonal-monospecific antibodies to serum albumin. Various commercial high purity albumin preparations elicited oscillatory currents in oocytes. The activity of albumin was partially reduced by charcoal absorption and was greatly diminished when crystalline albumin was extracted with dry methanol. However, the activity was resistant to extraction with chloroform/ether, disulfide cleavage, and denaturation with 8 M urea, 6 M guanidinium chloride, and 1% sodium dodecyl sulfate. Trypsin or lipase treatment substantially reduced the potency of the active albumin, but neither treatment alone abolished the factor even after prolonged digestion. In contrast to serum or serum albumin, freshly collected blood plasma or purified plasma albumin did not evoke oscillatory currents. This indicates that some of the plasma albumin changes during blood coagulation and acquires a "factor" that makes it capable of activating the phosphatidylinositol-Ca2+ system in Xenopus oocytes. The serum factor also activates the phosphatidylinositol system in a variety of mammalian cells, suggesting that the modified albumin may play a role in cellular events related to tissue repair following injury.

Animals↗
PubMed1991

Expression of mammalian gamma-aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes.

Gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is known to interact with two classes of GABA receptors denoted GABAA and GABAB. Using Xenopus oocytes, we compared the electrical and pharmacological properties of GABA receptors expressed by poly(A)+ RNA isolated from mammalian brain and retina. RNA from cerebral cortex expressed GABA responses with features characteristic of currents mediated by GABAA receptors. In contrast, RNA from retina expressed responses mediated by GABAA receptors and, in addition, GABA responses that were insensitive to the GABAA antagonist bicuculline and the GABAB agonist baclofen and showed no modulation by barbiturates or benzodiazepines. The bicuculline/baclofen-insensitive GABA response was a Cl- current that was blocked by picrotoxin but showed little desensitization or outward rectification. Our results suggest that mammalian retina contains RNAs encoding GABA receptors with distinct pharmacology.

Animals↗
PubMed1991

Angiotensin II receptors in Xenopus oocytes.

Electrical recordings were used to study the sensitivity of native Xenopus oocytes to the octapeptide angiotensin II (AII). AII elicited oscillatory currents associated with an increase in membrane conductance to Cl-. Responsiveness to AII varied greatly between oocytes taken from different frogs, and to a lesser extent between oocytes from the same ovary. Oocytes from frogs showing high sensitivity had response thresholds between 0.5-1.0 nM AII, and at a holding potential of -60 mV, responded to 1 microM AII with currents greater than 3 microA. In contrast, oocytes from some frogs gave no response, even to 10 microM AII. A total of 618 oocytes from 79 frogs were tested for sensitivity to AII, and oocytes from 85% of frogs gave detectable electrical responses. Oscillatory Cl- currents elicited by AII were largely independent of extracellular Ca2+, were abolished by chelation of intracellular Ca2+ using EGTA and were mimicked by intraoocyte injection of inositol 1,4,5-trisphosphate (IP3). In addition to oscillatory Cl- currents, AII also evoked an influx of extracellular Ca2+, giving rise to a transient inward Cl- current on membrane hyperpolarizing steps. These experiments all suggested that AII responses were elicited through activation of an intracellular messenger pathway triggered by hydrolysis of inositolphospholipids, mobilization of intracellular Ca2+ by inositol polyphosphates, and activation of Ca(2+)-gated Cl- channels. The effect of manual or enzymic defolliculation on AII responses was studied in nine separate experiments recording from 70 defolliculated oocytes. Efficacy of defolliculation procedures was assayed using scanning electron microscopy, which confirmed removal of 90 to greater than 98% of follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1991

Receptors of the serotonin 1C subtype expressed from cloned DNA mediate the closing of K+ membrane channels encoded by brain mRNA.

The modulation of K+ channels by serotonin (5-HT) receptors was studied by coinjecting Xenopus oocytes with mRNA transcribed in vitro from a cloned 5-HT 1C subtype (5-HT1C) receptor gene, together with size-fractionated mRNA isolated from rat cerebral cortex that expresses K+ channels. After intracellular loading with EGTA to block Ca2(+)-dependent chloride currents, these oocytes responded to 5-HT with an inward current associated with a decrease in membrane conductance. Membrane current responses were small or absent in oocytes injected with either mRNA alone. We conclude that 5-HT1C receptors are able to cause the closing of a class of K+ channels expressed by cortex mRNA in a Ca2(+)-independent manner. The coupling between the receptors and channels appears to be mediated by the inositol phospholipid second messenger pathway, since activation of this pathway by application of serum evoked a similar closing current.

Animals↗
PubMed1991

Tunicamycin increases desensitization of acetylcholine receptors in cultured mouse muscle cells.

Whole-cell currents activated by acetylcholine (AcCho) were recorded in C2 mouse myotubes before and after prolonged treatment with tunicamycin, an inhibitor of glycosylation. In control cells the AcCho-induced currents decayed slowly even in the continuous presence of AcCho. After 24 hr of treatment with tunicamycin AcCho still elicited currents, but their size was significantly reduced and their decay was greatly accelerated. The binding of 125I-labeled alpha-bungarotoxin, a specific and irreversible antagonist of muscle AcCho receptors, was greatly reduced after tunicamycin treatment, and an equivalent reduction was observed after a long-lasting application of the AcCho agonist carbachol. We suggest that, after inhibition of glycosylation by tunicamycin, AcCho receptors are expressed correctly on the plasma membrane but these receptors desensitize more rapidly and are less efficient in binding alpha-bungarotoxin.

Animals↗
PubMed1991

Expressional potency of mRNAs encoding receptors and voltage-activated channels in the postmortem rat brain.

The stability and integrity of mRNAs encoding neurotransmitter receptors and voltage-activated channels in the postmortem rat brain was investigated by isolating poly(A)+ mRNA, injecting it into Xenopus oocytes, and then examining the expression of functional neurotransmitter receptors and voltage-activated channels in the oocyte membrane by electrophysiological recording. This approach was also used to assess the stability of mRNAs in brains that were incubated in oxygenated mammalian Ringer's solution for various lengths of time and from brains that were freshly frozen and then thawed at room temperature. Oocytes injected with mRNA from up to 21-hr postmortem brains gave large agonist- and voltage-activated responses, indicating that mRNAs encoding neurotransmitter receptors and voltage-activated channels are relatively stable in postmortem brain tissue. In contrast, oocytes injected with mRNA from brains incubated in Ringer's solution exhibited smaller responses, and oocytes injected with mRNA from tissue that was frozen and then thawed displayed very small or undetectable responses. Northern blot analysis using a nucleic acid probe for rat brain Na(+)-channel mRNA indicated that the size of the Na+ currents in injected oocytes reflected the levels of mRNA for Na+ channels in the different mRNA preparations. Thus, the expressional potency of mRNAs encoding neurotransmitter receptors and voltage-activated channels is quite stable in postmortem brains in situ, but it is reduced if the brains are kept in oxygenated saline, and freezing and thawing of tissue results in rapid degeneration of mRNA.

Animals↗
PubMed1991

Xenopus oocytes as immunological vectors to produce monoclonal antibodies to rat brain antigens.

A novel approach was developed to raise a panel of monoclonal antibodies (mAb) against brain antigens using Xenopus oocytes as immunological vectors. Xenopus oocytes were injected to express proteins encoded by brain-derived mRNA extracted from rat cerebral cortex. A crude membrane preparation from mRNA-injected oocytes was then used to immunize mice previously rendered immunotolerant to native oocyte membranes. mAb reacting with cryostat cut sections from rat brain were selected and further characterized by immunohistological and immunobiochemical techniques. Several mAb recognized brain specific antigens, including some that were cell type specific and others that revealed a regional binding pattern. A particular group of antibodies recognized an epitope localized exclusively to the cerebellar pinceau terminals. Although some of the hybridomas found in this panel may be products of natural autoreactive lymphocytes, the presence of a specific immune response to mRNA expression products is discussed. These results indicate that mRNA injected oocytes are useful tools to raise mAb to study the molecular diversity of the nervous system.

Animals↗
PubMed1990

Glycosylation is required for maintenance of functional voltage-activated channels in growing neocortical neurons of the rat.

Voltage-activated currents were studied in whole-cell patch-clamped rat neocortical neurons growing in culture and treated with tunicamycin (TU), an inhibitor of protein N-glycosylation. The size of the Na+ current decreased progressively in the presence of TU (1-2 microM). This decrease was faster in growing 5-14 day-old neurons (to ca. 40% of control after 24 hours of treatment) than in fully grown 20-40-day-old neurons (to ca. 40% of control after 68 hours of treatment). The fast transient K+ current (A-current) was abolished, and the delayed rectifier K+ current was markedly reduced by a 24 hour treatment with TU (1-2 microM) in growing neurons. In contrast, in fully grown neurons these currents were unaffected by the same TU treatment. The size of the Ca2+ current was significantly reduced following a 24 hour treatment with TU (1-2 microM) in neurons at early stages of differentiation, but remained stable in 20-40-day-old neurons. It is concluded that protein glycosylation, presumably of the channel proteins themselves, is important for the functional expression of voltage-activated channels in embryonic cortical neurons during the early stages of cell growth in culture; the channels become less dependent on glycosylation in mature neurons.

Aging↗
PubMed1990

Changes in messenger RNAs coding for neurotransmitter receptors and voltage-operated channels in the developing rat cerebral cortex.

The ontogenetic development of poly(A)+ mRNAs coding for receptors to several neurotransmitters (kainate, glutamate, acetylcholine, and serotonin) and voltage-operated channels (sodium and calcium) was studied by isolating total poly(A)+ mRNA from the brains of rats at various developmental stages and injecting it into Xenopus oocytes. The oocytes translated the foreign mRNA and incorporated functional receptor/ion channel complexes into the cell membrane. Thus, recording of induced membrane currents in voltage-clamped oocytes gave a measure of the relative amounts of the different messengers. Responses induced by kainate, glutamate, acetylcholine, and serotonin all increased with age and reached a maximum in oocytes injected with mRNA from adult cortex. Messenger RNAs for the earliest ages examined, Embryonic Days 15 and 18, expressed little or no response to kainate, glutamate, or acetylcholine, while 50-70% of the adult response was reached by Postnatal Day 10. In contrast, the serotonin-induced response was already comparatively large (16% of the adult level) in oocytes injected with mRNA from Embryonic Day 15 brain and increased postnatally to adult levels. The expression of voltage-dependent sodium and calcium channels was small in oocytes injected with mRNA from embryonic animals and increased postnatally to reach a maximum in oocytes injected with mRNA from adult animals.

Aging↗
PubMed1990

Serotonin receptors expressed in Xenopus oocytes by mRNA from brain mediate a closing of K+ membrane channels.

Membrane currents evoked by serotonin (5-HT) were studied in Xenopus oocytes injected with rat brain mRNA. Intracellular EGTA was used to abolish the Ca2(+)-dependent oscillatory Cl- current to 5-HT, revealing an underlying smooth inward current. This was associated with a decreased membrane conductance, was antagonized by Ba2+ and Zn2+ (but not TEA), and probably arises through a closing of K+ channels. Half-maximal responses were obtained with 30 nM 5-HT, while 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) was ineffective. Furthermore, methysergide, mianserin and lysergic acid antagonized the K(+)-closing response to 5-HT, consistent with it being mediated through 5-HT1C receptors. The largest K(+)-closing responses were induced by a size fraction of mRNA which also induced a large K+ conductance, suggesting that the response requires expression of both receptors and K+ channels. The K(+)-closing response induced in the oocyte resembles the M- and S-type currents described in, respectively, mammalian and invertebrate neurons.

Animals↗
PubMed1990

Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes.

A method was developed to produce monoclonal antibodies to brain cell antigens by using Xenopus oocytes as immunological vectors. The method consists in injecting Xenopus oocytes with rat brain mRNA to express foreign proteins and using the oocytes for immunization. Immunizations were preceded by immunotolerization of mice to antigens of native oocyte membranes. With this approach we generated a set of monoclonal antibodies that are specific markers for the cerebellar "pinceau"--a unique complex synapse formed between basket cell terminals and the initial segment of the Purkinje cell axon. Our findings reveal an immunoreactivity highly localized at the pinceau and its late expression beginning at postnatal day 19 during cerebellar development.

Aging↗
PubMed1990

A serum factor that activates the phosphatidylinositol phosphate signaling system in Xenopus oocytes.

Blood sera from many vertebrate species elicit large oscillatory chloride currents in oocytes from the frog Xenopus laevis. Rabbit serum was active at dilutions as great as one part in 10 million. Intracellularly applied serum was ineffective, and externally applied serum failed to trigger oscillatory currents when the intracellular level of ionized calcium was prevented from rising by loading the oocyte with EGTA. The serum also caused an increase of inositol 1,4,5-trisphosphate in the oocyte. We conclude that serum contains a factor which activates a membrane receptor that is coupled to the phosphatidylinositol second messenger system. The active factor is a protein with an apparent molecular mass of 60-70 kDa in gel permeation chromatography. Although the normal function of the serum factor is still unknown, it may have far-reaching implications, because it acts on the multifunctional phosphatidylinositol phosphate signaling system. Also, because of its great potency the serum factor and Xenopus oocytes are very useful for probing the operation of the phosphatidylinositol system.

Animals↗
PubMed1990

Uptake of biotin by native Xenopus laevis oocytes.

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Changes in the properties of synaptic channels opened by acetylcholine in denervated frog muscle.

Acetylcholine (ACh)-activated channels in end-plates of frog sartorius muscle were studied at various times after denervation. Mean open times of the synaptic membrane channels were derived from the time constant of decay of miniature end-plate currents (tau MEPC) evoked by ACh quanta released from Schwann cells, which replace the motor nerve terminals after these degenerate. Membrane current noise, elicited by iontophoretic application of ACh to voltage-clamped end-plates, was also used to determine mean open time (tau noise) and conductance of the ion channels. About 1 week after denervation, soon after Schwann cell MEPCs appeared, they had a tau similar to that of the neural MEPC in innervated end-plates. However, 5-6 weeks after denervation tau MEPC was increased by a factor of about 5. Circa 4 weeks after denervation, cholinesterase activity of the denervated muscle decreased to 76% of that in the contralateral, innervated muscle, and even 4 months after the operation it was still 64%. Thus, it is unlikely that a change in acetylcholinesterase activity is the main factor responsible for the increase in tau of Schwann cell MEPC. About 1 week after denervation tau noise was close to that in innervated end-plates (about 2 ms). Twelve to 24 days after denervation the average channel open time was 4.5 +/- 1.0 ms, with some end-plates still showing normal 'fast' channels. However, in muscles denervated for 47-113 days the open time was 12.9 +/- 1.9 ms. In the early and intermediate periods, ACh-induced noise spectra with two components were obtained from many end-plates, indicating the simultaneous activation of two different types of channels. At some end-plates during the early and intermediate periods after denervation, but not after about 5 weeks, neostigmine caused the appearance of a component, which was as fast as that of normal end-plate channels. In other experiments small doses of alpha-bungarotoxin were applied in order to predominantly block extra-junctional receptors. In the early period of denervation, when two components were present in the noise spectra, alpha-bungarotoxin eliminated the slow component leaving channels as fast as in innervated end-plates. After prolonged denervation, a component with tau of about 5.5 ms was occasionally disclosed by application of alpha-bungarotoxin. tau noise and tau MEPC from the same end-plate closely agreed. Our results indicate that at frog end-plates the open time of the majority of the synaptic channels opened by ACh becomes longer with increasing time after denervation.

Acetylcholine↗
PubMed1989

Neuronal control of extrajunctional acetylcholine receptor-channels induced by injury in frog skeletal muscle fibres.

(1) Frog Sartorius muscles were transected to form two muscles: a neural segment (tibial part) containing all the endplates, and an aneural segment (pelvic part). The acetylcholine receptor-channels which appear at both new ends of the muscle fibres were studied by spectral analysis of membrane current fluctuations elicited by acetylcholine (ACh). (2) During the first two weeks after muscle transection, the ACh-receptors at the new muscle-tendon junction of the neural segment opened channels which had a long open time, similar to that of extrajunctional ACh-receptor channels induced by denervation of non-transected muscles. Subsequently, the open time of the new channels became shorter, and, about three weeks post-transection, it resembled that of channels at innervated endplates of normal muscles. The characteristics of the end-plate channels were not appreciably altered by the transection. (3) Similar results were observed when the muscle was crushed instead of being transected; and the change from slow to fast channel kinetics occurred earlier. (4) Denervation of neural segments prevented the change from slow to fast channel kinetics. The channels in the new muscle-tendon junction of the aneural segment remained slow up to three months post-transection. (5) These results indicate that the motor nerve controls the properties of the ACh-receptors induced by muscle transection.

Animals↗