Biomedical subjects

R Miledi

Publications and source records attributed to R Miledi.

At least 19 recordsLinked to original sources

PubMed1992

Lysophosphatidates bound to serum albumin activate membrane currents in Xenopus oocytes and neurite retraction in PC12 pheochromocytoma cells.

Serum contains a factor that co-purifies with albumin and causes neurite retraction in PC12 cells, inhibits the proliferation of tumor cells in vitro, and activates the phosphatidylinositol/Ca2+ second messenger system in Xenopus oocytes and other cells. The activity of serum albumin depends on several lysophospholipids bound to albumin. Thin layer chromatographic analysis of the lipids extracted by methanol from serum albumin revealed over a dozen components, several of which evoked oscillatory currents in oocytes. In contrast to serum albumin, most of these lipids were absent in plasma, which lacks the biological activity. The most abundant naturally occurring active component was identified as stearoyl-lysophosphatidic acid. Synthetically prepared lysophosphatidates reproduced the biological activities of the natural serum factor. Adding synthetic lysophosphatidates to inactive fatty acid-free albumin restored activity to the albumin, making the active factor nondialyzable against aqueous solvents and protecting against digestion by various lipases. Since the biologically active lysophosphatidates were produced during blood clotting, in the presence of platelets, and lysophosphatidates have been shown previously to activate platelets, we propose that lysophosphatidates may play an important role in linking platelet activation to receptor-mediated tissue regeneration.

Animals↗

PubMed1992

Actions of dopamine and dopaminergic drugs on cloned serotonin receptors expressed in Xenopus oocytes.

Using electrophysiological techniques, we studied interactions of dopamine and selected dopaminergic drugs with serotonin (5-hydroxytryptamine; 5-HT) receptors expressed in Xenopus oocytes by RNAs transcribed from cloned cDNAs. Oocytes showing strong expression of 5-HT1c and 5-HT2 receptors became weakly responsive to the neurotransmitter dopamine, which, like 5-HT, elicited Cl- currents through activation of the phosphatidylinositol/Ca2+ messenger pathway. The two types of 5-HT receptors showed similar sensitivity to dopamine; threshold responses were activated at concentrations as low as 1 microM. However, maximum dopamine responses were only 5-20% of maximum responses activated by 5-HT. The dopamine D1 receptor antagonist SCH 23390 was a potent agonist on 5-HT1c and 5-HT2 receptors. SCH 23390 elicited currents at concentrations as low as 1 nM, but maximum responses were again only 5-20% of those activated by 5-HT. Fenoldopam, a dopamine D1 receptor agonist, also interacted with 5-HT1c and 5-HT2 receptors, eliciting threshold responses between 10 and 20 nM. Our experiments raise the possibility that low micromolar concentrations of dopamine can cause weak activation and concomitant desensitization of serotoninergic systems in vivo and demonstrate that benzazepines can interact with 5-HT receptors at nanomolar concentrations.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1992

mRNA coding for neurotransmitter receptors in a human astrocytoma.

Electrophysiological techniques and Xenopus oocytes were used to study the expression of neurotransmitter receptors encoded by mRNAs isolated from three human glioma cell lines. Oocytes injected with mRNAs from two glioblastoma cell lines did not show electrical responses to the various neurotransmitters tested. In contrast, oocytes injected with mRNA from an astrocytoma cell line (R-111) acquired acetylcholine and glutamate receptors as well as a small number of N-methyl-D-aspartate (NMDA) receptors. Acetylcholine elicited oscillatory Cl- currents that were abolished by muscarinic antagonists. The muscarinic receptors are coupled to the inositol phosphate-Ca2+ receptor-channel coupling system. Glutamate and its analogs kainate, quisqualate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid induced smooth currents. The non-NMDA responses were potently blocked by 6,7-dinitroquinoxaline-2,3 dione. Our results show that human astrocytoma cells contain mRNAs coding for functional acetylcholine and glutamate receptors that have properties similar to those of neurons. In contrast, human glioblastoma cells lacked those mRNAs. These differences might be useful for the development of new diagnostic and therapeutic procedures.

2-Amino-5-phosphonovalerate↗

PubMed1992

Stereoselective effects of AMOA on non-NMDA receptors expressed in Xenopus oocytes.

Pharmacological characterization of the action of the novel non-N-methyl-D-aspartate (non-NMDA) antagonist AMOA (2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate) on glutamate receptors was investigated in Xenopus oocytes injected with mouse brain mRNA. AMOA (150 microM) produced a nearly parallel shift to the right of the dose-response curve for kainate-induced currents. AMOA was found to have two different effects on AMPA receptors: 1) currents elicited by low concentrations of AMPA (6 microM) were inhibited by AMOA with an IC50 value of 160 +/- 19 microM and 2) currents elicited by high concentrations of AMPA (100 microM) were potentiated with an IC50 value of 88 +/- 22 microM. The maximal potentiating effect of AMOA on AMPA currents was around 170%. Furthermore, the two opposing effects of AMOA on AMPA responses are specific for the L-configuration of AMOA. This unusual antagonistic/agonistic property of AMOA may explain its unusual properties with regard to antagonism of non-NMDA receptor-mediated events previously described.

Animals↗

PubMed1992

Messenger RNAs coding for receptors and channels in the cerebral cortex of adult and aged rats.

Poly(A)+ mRNAs from the cerebral cortex of aged (24 months) and young adult (3 months) rats were isolated and injected into Xenopus oocytes to express functional neurotransmitter receptors and voltage-operated channels. Electrophysiological recordings of induced membrane currents were used as a measure of the relative amounts of mRNA encoding different receptors and channels, and to study their functional properties. There were no large differences apparent between mRNAs from aged and adult rats, in marked contrast to the dramatic (1000-fold) changes in mRNA expression that occur during embryonic and postnatal development. The membrane currents induced by glutamate or acetylcholine (ACh) application were roughly one third smaller in oocytes injected with mRNA from aged cerebral cortex than in oocytes injected with mRNA from adult cerebral cortex, whereas currents induced by gamma-aminobutyric acid (GABA), kainate or serotonin (5-HT) application, and by activation of voltage-operated Na+ and Ca2+ channels were not significantly different. We did not observe any age-related differences in the properties of the receptors and channels studied.

Acetylcholine↗

PubMed1992

The effect of active serum albumin on PC12 cells: I. Neurite retraction and activation of the phosphoinositide second messenger system.

Vertebrate blood sera contain a factor that triggers oscillatory chloride currents in Xenopus oocytes through activation of the phosphoinositide/Ca2+ second system. The active serum component consists of lipids bound to an isoform of serum albumin that we have named active serum albumin (ASA). In undifferentiated PC12 cells, micromolar concentrations of ASA inhibit the early morphological changes induced by NGF, whereas in differentiated PC12 cells ASA caused a rapid withdrawal of neurites, which was reversible and dependent upon culture age. In contrast to normal serum, plasma and thrombin did not cause neurite retraction. Preincubation of ASA with monospecific antibodies to serum albumin suppressed its ability to induce neurite retraction in a dose dependent fashion. As in the oocyte, ASA activated the phosphatidylinositol second messenger system of PC12 cells, causing a several fold increase in Ins1,4,5P3 levels within minutes of application. The Ins1,4,5P3 increase was also blocked, in a titratable fashion, when ASA was preincubated with monospecific antibodies to serum albumin. This suggests that ASA-induced neurite retraction in PC12 cells may depend, at least in part, on activation of the phosphatidylinositol second messenger system. Results involving albumin-depleted sera show that ASA is the main factor responsible for serum vulnerability of neurites in PC12 cells. These findings point to some limitations in the use of serum in culture media, and raise the possibility that the serum factor may impair neuronal plasticity in disorders that are accompanied by the activation of blood coagulation together with a breakdown of the blood-brain barrier.

Animals↗

PubMed1992

The effect of active serum albumin on PC12 cells: II. Intracellular Ca2+ transients and their role in neurite retraction.

In the preceding paper it was shown that an isoform of serum albumin (ASA; active serum albumin) causes a rapid retraction of neurites and increases intracellular content of Ins1,4,5P3 in PC12 cells. Here we examined whether ASA's effects in nerve growth factor-differentiated PC12 cells were mediated through the Ins1,4,5P3/Ca2+ second messenger pathway by monitoring intracellular Ca2+ (Ca2+i) with Fura2. It was found that ASA caused a dose-dependent increase in Ca2+i. In Ca(2+)-free medium, the increase in Ca2+i elicited by ASA was smaller, but the rise in Ins1,4,5P3 content was not appreciably changed. The small Ca2+i increase seen in Ca(2+)-free medium was probably due to the release of Ca2+ from Ins1,4,5P3-sensitive intracellular stores. In Ca(2+)-containing medium the Ca2+ transient induced by ASA was not affected by organic Ca2+ channel blockers, but decreased when Co2+, Mn2+ or Zn2+ were present in the extracellular medium. The effect of other ligands, such as carbachol and bradykinin, whose receptors are coupled to the phosphoinositide system was also investigated. Carbachol at concentrations from 2 to 200 microM, and bradykinin at a concentration of 2 microM did not cause neurite retraction, whereas 200 microM bradykinin caused an approximately 40% decrease in neurite length. Thapsigargin, a Ca(2+)-ATPase inhibitor, caused a sustained elevation of Ca2+i and retraction of neurites, whereas depolarization of the cells by high K+ gave only a transient elevation of Ca2+i, and no neurite retraction. Therefore, a sustained elevation in Ca2+i might be a sufficient trigger to induce neurite retraction in differentiated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Sensitivity of Xenopus oocytes to changes in extracellular pH: possible relevance to proposed expression of atypical mammalian GABAB receptors.

Electrical recordings were made to characterize the sensitivity of Xenopus oocytes to changes in extracellular pH, and to determine whether rat cerebral cortex poly(A)+ RNA (mRNA) expressed GABAB receptors with atypical electrical properties. All oocytes showed some sensitivity to changes in pH, and those from a small fraction (< 10%) of frogs were found to be highly responsive to acidification of bathing Ringer. In these oocytes, reduction in extracellular pH elicited membrane current responses with two components: (1) Smooth, maintained currents, primarily associated with a decrease in K+ conductance. (2) Oscillatory Cl- currents, elicited through activation of the phosphoinositide/Ca2+ messenger pathway. Oocytes with highest levels of sensitivity responded to decreases as low as 0.1 pH unit (Ringer pH 7.0 lowered to pH 6.9). Rat cortex mRNA consistently showed strong expression of membrane current responses mediated by GABAA, glutamate, kainate, serotonin and acetylcholine (muscarinic) receptors, together with responses mediated by a variety of neuropeptide receptors. In these oocytes, enantiomers of the GABAB receptor agonist baclofen, at concentrations ranging between 1 microM and 10 mM (pH 7.0), activated no significant membrane current responses. However, at concentrations > 0.1 mM hydrochloride salts of baclofen caused appreciable acidification of Ringer solutions; for example, 1 mM baclofen lowered pH from 7.0 to 6.0. Thus, when assaying oocytes with high sensitivity to pH, failure to make the necessary re-adjustment could result in apparent baclofen responses that, in reality, are simply due to pH effects alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acids↗

PubMed1992

Effects of steroids on gamma-aminobutyric acid receptors expressed in Xenopus oocytes by poly(A)+ RNA from mammalian brain and retina.

Electrical recordings were made in Xenopus oocytes to study the modulatory effects of steroids on gamma-aminobutyric acid (GABA) receptors expressed by RNA from mammalian brain and retina. GABA responses expressed by rat cerebral cortex poly(A)+ RNA were bicuculline-sensitive Cl- currents mediated by GABAA receptors. GABA responses expressed by bovine retina poly(A)+ RNA also were Cl- currents but were composed of two pharmacologically distinct components, one mediated by GABAA receptors and the other by GABA receptors with novel properties, which were resistant to bicuculline but were not activated by R(+)-baclofen, a selective agonist of GABAB receptors. As reported in neurons and in other expression systems, GABAA responses expressed in oocytes by cerebral cortex RNA were strongly and stereospecifically potentiated by 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) and 5 alpha-pregnan-3 alpha,21-diol-20-one (THDOC). Threshold levels of potentiation were detectable using 1-2 nM steroid, and at concentrations of 50 and 500 nM 3 alpha-OH-DHP shifted the EC50 of cortex GABAA responses from a control value of 92 +/- 20 microM GABA to 40 +/- 4.3 microM and 13 +/- 1.8 microM, respectively. However, even at concentrations as high as 50 microM, 3 alpha-OH-DHP did not itself elicit appreciable membrane current responses through direct activation of the cortex GABAA receptors. In addition to potentiation, 3 alpha-OH-DHP and THDOC caused pronounced increases in the rate of desensitization of GABAA responses expressed by cortex RNA. Decay time courses of currents elicited by 1 mM GABA (90-95% of the maximum response) were fitted by the sum of two exponentials. Under control conditions, the time constant of the fast component was 4.4 +/- 0.6 sec and the slow component, 22.5 +/- 4.8 sec. 3 alpha-OH-DHP at 500 nM and 5 microM reduced the time constant of the fast component by 52 +/- 7% and 84 +/- 5%, respectively, but showed little effect on the slow component. Unlike the potentiation effect, actions of pregnenolones on desensitization did not show stringent stereoselectivity, and 5 microM 5 beta-pregnan-3 beta-ol-20-one (3 beta-OH-DHP) reduced the time constant of the fast component by 59 +/- 11%.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Effects of hexachlorocyclohexanes on gamma-aminobutyric acid receptors expressed in Xenopus oocytes by RNA from mammalian brain and retina.

Poly(A)+ RNA from rat cerebral cortex expresses gamma-aminobutyric acid (GABA)-activated membrane current responses in Xenopus oocytes, mediated by GABAA receptors (IG-Actx). In contrast, RNA from bovine retina expresses GABA responses composed of two pharmacologically distinct Cl- currents, one mediated by GABAA receptors (IG-Aret) and the other by atypical GABA receptors that are resistant to bicuculline and are not activated by baclofen (IG-BR). The pharmacology of the bicuculline/baclofen-insensitive GABA receptors was further investigated by comparing actions of hexachlorocyclohexane (HCH) enantiomers on GABA-activated membrane currents expressed in oocytes by brain and retina RNA. gamma-HCH (lindane) was a potent inhibitor of IG-Actx, with suppression of currents detectable at concentrations as low as 50 nM. The IC50 for gamma-HCH, calculated from inhibitory effects on maximum IG-Actx (current elicited by 3 mM GABA), was 7.3 +/- 3 microM. Inhibitory effects of gamma-HCH on IG-Aret were qualitatively similar to those described for IG-Actx. In contrast, alpha-HCH and delta-HCH induced clear positive modulation of IG-Actx elicited by low (e.g., 10 microM) concentrations of GABA. Thresholds for the modulatory effects of alpha-HCH and delta-HCH were between 100 and 300 nM, with maximum levels of potentiation (5-7-fold) between 20-50 microM. Potentiation of IG-Actx by alpha- and delta-HCH was reversible and largely insensitive to the benzodiazepine antagonist flumazenil (1 microM). Assays on maximum IG-Actx indicated that alpha-HCH (10-100 microM) caused only marginal reductions in response (less than or equal to 15%), whereas delta-HCH had stronger inhibitory effects (IC50, 20-30 microM). At concentrations between 0.1 and 50 microM, beta-HCH induced only 10-25% facilitation of IG-Actx elicited by 10 microM GABA and had no clear effects on maximum responses. IG-BR was also potently inhibited by gamma-HCH. Thresholds for detecting reductions in current were approximately 20 nM, and the IC50 calculated from effects on maximum responses was 5.8 +/- 2 microM. However, neither alpha-HCH nor delta-HCH (1-100 microM) induced any potentiation of IG-BR. alpha-HCH had some weak inhibitory effects that were largely surmountable, whereas delta-HCH and beta-HCH were essentially inactive. These experiments raise the possibility that alpha- and delta-HCH constitute a novel class of GABAA receptor modulators, which might prove to be useful for investigating the mechanisms underlying regulation of GABAA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

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↗