Biomedical subjects

R Miledi

Publications and source records attributed to R Miledi.

At least 55 records · Page 3Linked to original sources

PubMed1994

Effects of fenamates and other nonsteroidal anti-inflammatory drugs on rat brain GABAA receptors expressed in Xenopus oocytes.

Xenopus oocytes injected with poly(A)+ RNA from rat cerebral cortex express functional gamma-aminobutyric acid (GABA)A receptors with electrical properties and pharmacology similar to those of receptors studied in situ. Fenamates, a class of nonsteroidal anti-inflammatory drugs (NSAIDs), had a dual effect on GABA-activated membrane current responses. Currents elicited by low concentrations of GABA were potentiated, whereas currents elicited by high concentrations of GABA were inhibited. The levels of the two effects varied among fenamates. For example, 10 microM mefanamic acid potentiated 10 microM GABA responses by approximately 300% (EC50 approximately 5 microM) and inhibited maximal responses by 30% (IC50 approximately 30 microM). In contrast, 10 microM niflumic acid potentiated 10 microM GABA responses by only 30% (EC50 approximately 10 microM) and inhibited maximal responses by 60% (IC50 approximately 7 microM). Preliminary structure-activity studies suggested that modulatory activity is dependent on the preferred conformations of fenamate molecules and on specific phenyl-ring substitutions. Thirteen other NSAIDs (all prostaglandin synthesis inhibitors) were likewise assayed for effects on GABA-activated currents. Of these, only the salicyclic acid diflunisal induced comparable potentiation and inhibition. Our experiments raise two interesting possibilities: that fenamates could serve as lead structures in the development of novel GABAA receptor modulators and that fenamates might affect GABAA receptor function at a normal clinical dosage.

Animals↗

PubMed1993

mRNAs coding for neurotransmitter receptors in rabbit and rat visual areas.

Levels of mRNAs encoding neurotransmitter receptors in the visual cortex, lateral geniculate nucleus, and superior colliculus of the rabbit and rat, and properties of the receptors expressed, were studied using Xenopus laevis oocytes. mRNA extracted from these areas was injected into the oocytes, which then acquired functional receptors. Electrical recordings of neurotransmitter-induced membrane currents reflect the relative amounts of mRNAs encoding the corresponding receptors. Receptors to gamma aminobutyric acid (GABA), kainate, glutamate, and serotonin exhibited uniformly high levels of expression, whereas expression of receptors to glycine and N-methyl-D-aspartate was uniformly low. In contrast, the expression of receptors to acetylcholine and substance P was highly non-uniform. Expression of acetylcholine receptors was high in oocytes injected with mRNA from the visual cortex, low for the lateral geniculate nucleus, and very low or absent for the superior colliculus. Conversely, the currents elicited by substance P were large in oocytes injected with superior colliculus mRNA, but were small or absent in oocytes injected with mRNAs from the other regions. Immunohistochemical analysis, at the light and electron microscopic levels, was used to localize choline acetyltransferase, the acetylcholine-synthesizing enzyme, and substance P-containing synaptic boutons in the three visual areas. Their presence closely paralleled the potency of mRNAs coding for acetylcholine and substance P receptors. The ability of rat mRNA, from each visual area, to induce neurotransmitter receptors was similar to that observed in the corresponding rabbit mRNAs. In addition to the marked differential distribution of mRNA encoding neurotransmitter receptors in the visual system, our findings reveal the probable existence of as yet uncharacterized receptors, whose new molecular forms may be revealed by further study. Our results also provide the basic information required for subsequent studies on the effect of monocular deprivation on the expression of neurotransmitter receptors in the visual system.

Animals↗

PubMed1993

Neurotransmitter receptors and voltage-dependent Ca2+ channels encoded by mRNA from the adult corpus callosum.

The presence of mRNAs encoding neurotransmitter receptors and voltage-gated channels in the adult human and bovine corpus callosum was investigated using Xenopus oocytes. Oocytes injected with mRNA extracted from the corpus callosum expressed functional receptors to glutamate, acetylcholine, and serotonin, and also voltage-operated Ca2+ channels, all with similar properties in the two species studied. Acetylcholine and serotonin elicited oscillatory Cl- currents due to activation of the inositol phosphate-Ca2+ receptor-channel coupling system. Glutamate and its analogs N-methyl-D-aspartate (NMDA), kainate, quisqualate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) induced smooth currents. The non-NMDA responses showed a strong inward rectification at positive potentials and were potently blocked by 6,7-dinitroquinoxaline-2,3-dione, as observed for the AMPA/kainate glutamate receptors GLUR1 and GLUR3. Furthermore, in situ hybridization experiments showed that GLUR1 and GLUR3 mRNAs are present in corpus callosum cells that were labeled with antiserum to glial fibrillary acid protein and that, in primary cell cultures, had the morphology of type 2 astrocytes. These results indicate that glial cells in the adult corpus callosum possess mRNA encoding functional neurotransmitter receptors and Ca2+ channels. These molecules may provide a mechanism for glial-neuronal interactions.

Adult↗

PubMed1993

Blockage of nicotinic acetylcholine receptors by 5-hydroxytryptamine.

The action of 5-hydroxytryptamine (5HT) on nicotinic acetylcholine receptor (nAChR) channels was investigated in mouse myotubes, human cloned TE671/RD cells, and Xenopus laevis oocytes. The decay of the ACh-activated whole-cell currents was reversibly accelerated in the presence of 5HT (10(-5) to 10(-3) M), in a dose-dependent manner. 5HT also reduced the size and accelerated the decay of currents elicited by ACh in Xenopus oocytes injected with mRNA extracted from C2 myotubes or Torpedo electroplaques, or oocytes injected with cloned mouse muscle AChR subunit mRNAs. The effect of 5HT was promptly reversed after washout, or by depolarizing the oocyte beyond -10 mV. In patch-clamp recordings from myotubes, bath-application of 5HT did not exert an indirect influence on the ACh-activated channels within the patch membrane. In contrast, when the patch membrane was exposed to 5HT (10(-6) M), ACh unit responses appeared as bursts of short pulses. It is concluded that the regulation of ACh responses by 5HT results from a fast noncompetitive blocking action of nAChR-channels. These results show that ligand-gated channels, activated by their specific neurotransmitter, may be regulated by a different neurotransmitter through a direct action on the receptor molecule.

Animals↗

PubMed1993

Novel Cl- currents elicited by follicle stimulating hormone and acetylcholine in follicle-enclosed Xenopus oocytes.

Voltage-clamp techniques were used to study the membrane currents elicited by follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle-enclosed oocytes of Xenopus laevis (follicles). Both agonists caused complex responses that were more evident when the follicles were in hypotonic Ringer solution (HR; 190.4 mosM). In this medium, currents activated by FSH regularly showed three phases whereas currents activated by ACh displayed three to six phases. At a holding potential of -60 mV, FSH, and ACh responses involved combinations of inward and outward currents. Both FSH and ACh responses included a slow smooth inward component that was associated with an increase in membrane conductance, mainly to Cl- (S(in)). This current was strongly dependent on the osmolarity of the external solution: an increase in osmolarity of the HR solution of 18-20 mosM caused a 50% decrease in S(in). In contrast, a fast and transient Cl- current (F(in)) specifically elicited by ACh was not dependent on osmolarity. Both, F(in) and S(in) currents required the presence of follicular cells, since defolliculation using three different methods abolished all the response to FSH and at least four components of the ACh responses. The membrane channels carrying F(in) and oscillatory Cl- currents elicited by stimulation of ACh or serum receptors, were much more permeable to I- and Br- than Cl-, whereas S(in) channels were equally permeable to these anions. Unlike the oscillatory Cl- currents generated in the oocyte itself, S(in) and F(in) currents in follicle-enclosed oocytes were not abolished by chelation of intracellular Ca2+, either with EGTA or BAPTA, which suggests that intracellular Ca2+ does not play a critical role in the activation of these currents. Our experiments show that S(in) and F(in) currents are quite distinct from the previously characterized oscillatory Cl- responses of oocytes. Moreover, the results strongly suggest that the FSH and ACh receptors, the Cl- channels mediating the F(in) and S(in) currents, together with the necessary elements for their activation, are all located in the follicular cells and not in the oocyte. Many aspects of follicular cell physiology in Xenopus laevis, and other species, are regulated by hormones and neurotransmitters, including FSH and ACh. The follicular Cl- currents described in this paper may play an important role in the follicular cell-oocyte development.

Acetylcholine↗

PubMed1993

Expression of neurotransmitter receptors by mRNAs from neurons developing in vitro: a Xenopus oocyte expression study.

Poly(A)+ mRNA was extracted from cultures of neurons isolated from mouse embryonic day 14 cerebral cortex and injected into Xenopus oocytes. This led to the expression of receptors for gamma-aminobutyric acid (GABA), glycine, acetylcholine, serotonin, glutamate, kainate, N-methyl-D-aspartate, and quisqualate. Northern blot analysis of poly(A)+ mRNA from the cultured neurons with a GluR1 cDNA probe revealed the presence of three hybridization bands with estimated mRNA sizes of 5.1, 4.0, and 3.1 kb, respectively. The development of mRNAs coding for neurotransmitter receptors was investigated by isolating mRNA from neurons cultured for 2, 8, and 14 days in vitro and injecting it into Xenopus oocytes. The amplitude of membrane currents elicited by the transmitters gave a measure of the relative amounts of the different mRNAs. The size of the responses to kainate, aspartate (together with glycine), glutamate, acetylcholine, GABA, serotonin, and glycine increased with the time of culture in vitro. However, in contrast to all other agonist-induced currents, the current induced by glycine failed to increase further from 8 to 14 days in culture. It is concluded that the time course of receptor development in cortical neurons in vitro is similar to the development in vivo.

Animals↗

PubMed1993

Characterization of bicuculline/baclofen-insensitive (rho-like) gamma-aminobutyric acid receptors expressed in Xenopus oocytes. II. Pharmacology of gamma-aminobutyric acidA and gamma-aminobutyric acidB receptor agonists and antagonists.

Poly(A)+ RNA from mammalian retina expresses bicuculline/baclofen-insensitive gamma-aminobutyric acid (GABA) receptors in Xenopus oocytes with properties similar to those of homooligomeric GABA rho 1 receptors. The pharmacological profile of these rho-like receptors was extended by measuring sensitivities to various GABAA and GABAB receptor ligands. For direct comparison the same compounds were also assayed with GABAA receptors expressed by rat brain RNA. The potency sequence for heterocyclic GABA analogues at the GABA rho-like receptors was GABA (1.3) > muscimol (2.3) > isoguvacine (100) (approximate EC50 in parentheses; all EC50 and Kb values given in microM). Both muscimol and isoguvacine were partial agonists at the rho-like receptors. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (Kb congruent to 32), piperidine-4-sulfonic acid (Kb congruent to 85), and isonipecotic acid (Kb congruent to 1000) acted primarily as competitive antagonists, showing little or no activity as agonists. The sulfonic acid GABA analogue 3-aminopropanesulfonic acid was also a competitive antagonist (Kb congruent to 20). Conformationally restricted GABA analogues trans- and cis-4-aminocrotonic acid (TACA and CACA) were agonists at the rho-like receptors. TACA (EC50 congruent to 0.6) had twice the potency of GABA and was 125 times more potent than CACA (EC50 congruent to 75). Z-3-(Amidinothio)propenoic acid, an isothiouronium analogue of GABA, had little activity as an agonist but instead acted as a competitive antagonist (Kb congruent to 20). At concentrations of > 100 microM, bicuculline did have some weak competitive inhibitory effects on the GABA rho-like receptors (Kb congruent to 6000), but it was at least 5000 times more potent at GABAA receptors. Strychnine (Kb congruent to 70) and SR-95531 (Kb congruent to 35) also were competitive inhibitors of the rho-like receptors but were, respectively, 20 and 240 times more potent at GABAA receptors. The GABAB receptor ligands baclofen, phaclofen, and saclofen (1-100 microM) had no appreciable effects on the rho-like receptors. In contrast, 3-aminopropylphosphonic acid, the phosphonic acid analogue of GABA, acted as a competitive antagonist (Kb congruent to 10), and 3-aminopropylphosphinic acid and 3-aminopropyl(methyl)-phosphinic acid were moderately potent antagonists (Kb congruent to 1.7 and 0.8, respectively). delta-Aminovaleric acid was also an antagonist (Kb congruent to 20), whereas 4-aminobutylphosphonic acid, the phosphonic acid analogue of delta-aminovaleric acid, was only a weak inhibitor (Kb congruent to 600).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

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↗