Interconvertible Kinetic States of t-Butylbicycloorthobenzoate Binding Sites of the γ-Aminobutyric AcidA Ionophores

The kinetics of t-[³H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acid_A (GABA_A) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABA_A antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [³H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k_app=k_off±k_on [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B_1a (AVM B_1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC₅₀, 7.8 nM) followed by displacement at high concentrations (IC₅₀ 6.3 μM) of AVM B_1a. AVM B_1a resulted in similar biphasic effects on t- [³⁵S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [³H]TBOB binding (IC₅₀ 125 μM DIDS) leaving the enhancement by AVM B_1a unaffected.     

GABAB antagonists: Resolution,absolute stereochemistry,and pharmacology of (R)- and (S)-phaclofen

Phaclofen, which is the phosphonic acid analogue of the GABA_B agonist (RS)-3-(4-chlorophenyl)-4-aminobutyric acid (baclofen), is a GABA_B antagonist. As part of our studies on the structural requirements for activation and blockade of GABA_B receptors, we have resolved phaclofen using chiral chromatographic techniques. The absolute stereochemistry of (?)-(R)-phaclofen was established by X-ray crystallographic analysis. (?)-(R)-Phaclofen was shown to inhibit the binding of [³H]-(R)-baclofen to GABA_B receptor sites on rat cerebellar membranes (IC₅₀ = 76 ± 13 μM), whereas (+)-(S)-phaclofen was inactive in this binding assay (IC₅₀ > 1000 μM). (?)-(R)-Phaclofen (200 μM) was equipotent with (RS)-phaclofen (400 μM) in antagonizing the action of baclofen in rat cerebral cortical slices, while (+)-(S)-phaclofen (200 μM) was inactive. The structural similarity of the agonist (R)-baclofen and the antagonist (?)-(R)-phaclofen suggests that these ligands interact with the GABA_B receptor sites in a similar manner. Thus, it may be concluded that the different pharmacological effects of these compounds essentially result from the different spatial and proteolytic properties of their acid groups. © 1994 Wiley-Liss, Inc.     

Bicuculline-insensitive GABA binding to catfish neuronal membranes

GABA receptor binding to mammalian neuronal membranes has been classified into at least 2 subtypes—GABA_A and GABA_B binding sites. In catfish brain GABA_A receptor sites have previously been demonstrated. Evidence is now presented that under appropriate conditions which rule out GABA_A receptor binding, [³H]GABA binds to membranes prepared from catfish brain. This binding is bicuculline-insensitive but differs enough from mammalian GABA_B binding to cast some doubt on the idea that GABA_B receptors exist in catfish brain. Specific binding was detected that was saturable and exhibited a dissociation constant of 4μM. (±)Baclofen, a potent inhibitor in rat brain, was a weak inhibitor, producing a maximum of 43% inhibition. This inhibitory effect could be enhanced, however, in the presence of 320 μM isoguvacine. [³H]GABA binding was unaffected by bicuculline. Thus bicuculline-insensitive GABA binding sites exist in catfish brain but they differ in a number of ways from the GABA_B receptor site found in mammals. Furthermore, a third [³H]GABA binding site appears to exist that is both baclofen- and bicuculline-insensitive, yet is inhibited by high concentrations of isoguvacine, a known GABA_A agonist.     

AMPA receptor agonists: Resolution,configurational assignment,and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA)

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee ≥ 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [³H]AMPA binding (IC⁵⁰ = 0.19 ± 0.06 μM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC₅₀ = 4.5 ± 0.3 μM) comparable with AMPA (IC₅₀ = 0.040 ± 0.01 μM; EC₅₀ = 3.5 ± 0.2 μM), but much more potent than (+)-(S)-APPA (IC₅₀ = 5.5 ± 2.2 μM; EC₅₀ = 230 ± 12 μM). Like (-)-(R)-APPA (IC₅₀ > 100 μM), (-)-(R)-2-Py-AMPA (IC₅₀ > 100 μM) did not significantly affect [³H]AMPA binding, and both compounds were week AMPA receptor antagonists (K_i = 270 ± 50 and 290 ± 20 μM, respectively). Chirality 9:274–280, 1997. © 1997 Wiley-Liss, Inc.     

Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

Inhibitory Effects of Carvone Isomers on the GABAA Receptor in Primary Cultures of Rat Cortical Neurons

Carvone is a natural terpene which can be purified as R‐(?) or S‐(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA_A receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA_A by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA‐induced stimulation of [³H]flunitrazepam binding, suggesting their interaction with the GABA_A receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R‐(?)‐carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA_A receptor at its noncompetitive blocker site. Chirality 26:368–372, 2014. © 2014 Wiley Periodicals, Inc.     

GABAB antagonists: Enantiopharmacology of 5-amino-4-hydroxy-2-methylvaleric acid and X-ray structure of the eutomer

We have previously shown that (R)-5-amino-4-hydroxyvaleric acid [(R)-4-OH-DAVA] and (S)-2-OH-DAVA bind to GABA_B receptor sites and antagonize GABA_B receptor-mediated function in a stereoselective manner. Furthermore, we have identified energy-minimized superimposable conformations of (R)-4-OH- and (S)-2-OH-DAVA which are assumed to reflect the receptor-active conformations of these compounds. This paper describes the in vitro enantiopharmacology of 5-amino-4-hydroxy-2-methylvaleric acid (2-Me-4-OH-DAVA). Whereas none of the four stereoisomers showed significant affinity for GABA_A receptor sites or GABA uptake mechanisms in rat brain synaptic membranes, (2R,4R)-2-Me-4-OH-DAVA was shown to inhibit stereoselectively the binding of [³H]GABA to rat brain GABA_B receptor sites (IC₅₀ = 14 ± 4 μM). (2R,4R)-2-Me-4-OH-DAVA (K_i = 36 μM) and, with much lower potency, (2S,4R)-2-Me-4-OH-DAVA (K_i = 370 μM) stereoselectively antagonized GABA_B receptor-mediated function in the isolated guinea pig ileum. The structure of the eutomer, (2R,4R)-2-Me-4-OH-DAVA, was established by an X-ray crystallographic analysis, and the solid-state conformation of (2R,4R)-2-Me-4-OH-DAVA was compared with the proposed receptor-active conformations of (R)-4-OH-DAVA and (S)-2-OH-DAVA. © 1995 Wiley-Liss, Inc.     

Synthesis of (+)-(R)- and (−)-(S)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)] aminotetralin: New 5-HT1A receptor ligands

(R,S)-trans-8-Hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin 7 , a new radioiodinated ligand based on 8-OH-DPAT, was reported as a potential ligand for 5-HT_1A receptors. The optically active (+)-(R)- and (?)-(S)- 7 were prepared to investigate the stereoselectivity of (R,S)- 7 . Racemic intermediate 8-methoxy-2-N-n-propyltetralin was reacted with the acyl chloride of (?)-(R)-O-methylmandelic acid to form a mixture of (S,R)- and (R,R)-diastereoisomers, which were separated by flash column chromatography. After removing the N-acyl group from the diastereoisomers, the desired (+)-(R)-or (?)-(S)- 7 was obtained by adding an N-iodopropenyl group. In vitro homogenate binding studies showed the stereoselectivity of this new compound for 5-HT_1A receptors. (+)-(R)- 7 isomer displayed 100-fold higher affinity than the (?)-(S)- 7 isomer. Biochemical study indicated that (+)-(R)- 7 potently inhibited forskolin-stimulated adenylyl cyclase activity in hippocampal membranes (E_max and EC₅₀ were 24.5% and 5.4 nM, respectively), while (?)-(S)- 7 showed no effect at 1 μM. The radioiodinated (+)-(R)- and (?)-(S)-[¹²⁵I] 7 were confirmed by coelution with the resolved unlabeled compound on HPLC (reverse phase column PRP-1, acetonitrile/pH 7.0 buffer, 80/20). The active isomer, (+)-(R)-[¹²⁵I] 7 , displayed high binding affinity to 5-HT_1A receptors (K_d = 0.09 ± 0.02 nM). In contrast, the (?)-(S)- 7 isomer displayed a significantly lower affinity to the 5-HT_1A receptor (K_d > 10 nM). Thus, (+)-(R)-[¹²⁵I]trans-8-OH-PIPAT, (+)-(R)- 7 , an iodinated stereoselective 5-HT_1A receptor agonist, is potentially useful for study of in vivo and in vitro function and pharmacology of 5-HT_1A receptors in the central nervous system. © 1995 Wiley-Liss, Inc.     

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells

The function of the GABA_A receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABA_A receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg⁺⁺ containing medium. The NMDA effect was also absent when extracellular Ca⁺⁺ was replaced by Ba⁺⁺ and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca⁺⁺ channels greater than the ones via NMDA receptors do not cause any reduction in GABA_A receptor function, suggesting that Ca⁺⁺ influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5′-O-3-thiophosphate (ATP-γ-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABA_A receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N _ω -nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-γ-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABA_A receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABA_A receptor. On the one hand Ca⁺⁺ influx across NMDA channels activates calcineurin and dephosphorylates the GABA_A receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca⁺⁺ influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity. Received: 22 July 1996 / Accepted: 29 October 1996     

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [³H]H₂O released when [1β-³H]androstenedione was converted to estrone. Maximum velocity (V_max) and the Michaelis-Menten constant (K_m) of the microsomal aromatase enzyme were 17.40 ± 0.45 pmol/ml/mg protein/min and 1.02 ± 0.06 μM, respectively. The IC₅₀s for the enantiomers were similar for (+)-R-AG and (?)-R-ChAG (0.86 ± 0.06 and 0.89 ± 0.15 μM, respectively). (+)S-ChA'G was most potent with IC₅₀ of 0.075 ± 0.003 μM. The IC₅₀s for (?)-S-AG, (+)-R-RG, and (?)-S-RG were in the same range (23.15 ± 2.74, 24.58 ± 2.46, and 24.43 ± 2.20 μM, respectively). © 1994 Wiley-Liss, Inc.     

SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line

The effects of a novel nonpeptide NK1 tachy-kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with ¹²⁵l-Bolton-Hunter substance P revealed a competitive inhibition by SR 140333 and its R enantiomer SR 140603 with K_i values of 0.74 and 7.40 nM, respectively. The NK1-selective agonist, [Sar⁹,Met(O₂)¹¹]-substance P, stimulated the formation of inositol phosphates with an EC₅₀ of 3.8 × 10^?9M. SR 140333 blocked the stimulatory effect of this agonist (10^?7M) with an IC₅₀ of 1.6 × 10^?9M,whereas the effect of another NK1 agonist, septide (EC₅₀= 1.5 × 10^?8M)was antagonized with an IC₅₀ of 2.1 × 10^?10M.Enhancement of [³H]taurine release by [Sar⁹,Met(O₂)¹¹]-substance P (EC₅₀= 7.4 × 10^?9M) was also inhibited by SR 140333 with an IC₅₀ of 1.8 × 10^?9 M. SR 140603 was 10-fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [³H]taurine release. The calcium mobilization induced by [Sar⁹,Met(O₂)¹¹]-substance P (10^?8M) was totally prevented by 10^?8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10^?8M [Sar⁹, Met(O₂)¹¹]-substance P with an IC₅₀ of 1.3 × 10^?9M. The expression of c-fos was stimulated by [Sar⁹,Met(O₂)¹¹]-substance P with an EC₅₀ of 2.5 × 10^?10M, an effect that was also inhibited by SR 140333 with an IC₅₀ of 1.1 × 10^?9M. The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.     

GABAA Receptor Subtypes Expressed in Cerebellar Granule Cells: A Developmental Study

Abstract: The developmental properties of primary rat cerebellar granule cells have been characterised with respect to their expression of GABA_A receptor subtypes using both an immunological approach and radioligand binding assays. At day 1 in culture, the GABA_A receptor α1 subunit was detectable in immunoblots and increased in level up to day 9. The GABA_A receptor α6 subunit was not detectable at day 1; however, at days 3–5, a specific M_r 58,000 anti-α6 1–16 Cys immunoreactive species was present which further increased in level up to 9 days in culture. Similar qualitative results were obtained for the expression of the GABA_A receptor α6 subunit in age-matched rat cerebellar membranes. In parallel studies, it was found that although there was an overall increase in [³H]Ro 15–4513 binding sites with days in culture, the relative contributions of diazepam-sensitive and diazepam-in-sensitive [³H]Ro 15–4513 binding changed. A time-dependent enrichment of the diazepam-insensitive binding site up to a maximum of 74% of total [³H]Ro 15–4513 sites was found. This was concomitant with the appearance of the GABA_A receptor α6 subunit. These results are in agreement with the pharmacology described for α6βγ2 cloned receptors. They suggest a developmentally regulated expression of the GABA_A receptor α6 subunit gene at a time that is correlated in vivo with establishment of neuronal connections.     

N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABAAα6 Subunit in Cerebellar Granule Cells

Abstract: We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 µM) results in the transient increase in content of GABA_Aα6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABA_B receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in α6 levels induced by 30 µM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional α6-containing GABA_A receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA_Aα6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA_A receptor subunit composition.     

GABA Concentration Sets the Conductance of Delayed GABAA Channels in Outside-out Patches from Rat Hippocampal Neurons

GABA_A channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC ₅₀ value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 μm diazepam, the GABA EC ₅₀ decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC ₅₀ was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABA_A receptor channel function is more complex than previously thought. Received: 23 October 2000/Revised: 27 February 2001     

4-chloro-orto-cresol activates ryanodine receptor more selectively and potently than 4-chloro-meta-cresol

In this study we performed the comprehensive pharmacological analysis of two stereoisomers of 4-chloro-meta-cresol (4CMC), a popular ryanodine receptor (RyR) agonist used in muscle research. Experiments investigating the Ca²⁺-releasing action of the isomers demonstrated that the most potent isomer was 4-chloro-orto-cresol (4COC) (EC₅₀ = 55 ± 14 μM), although 3-chloro-para-cresol (3CPC) was more effective, as it was able to induce higher magnitude of Ca²⁺ flux from isolated terminal cisterna vesicles. Nevertheless, 3CPC stimulated the hydrolytic activity of the sarcoplasmic reticulum ATP-ase (SERCA) with an EC₅₀ of 91 ± 17 μM, while 4COC affected SERCA only in the millimolar range (IC₅₀ = 1370 ± 88 μM). IC₅₀ of 4CMC for SERCA pump was 167 ± 8 μM, indicating that 4CMC is not a specific RyR agonist either, as it activated RyR in a similar concentration (EC₅₀ = 121 ± 20 μM).Our data suggest that the use of 4COC might be more beneficial than 4CMC in experiments, when Ca²⁺ release should be triggered through RyRs without influencing SERCA activity.     

Functional Changes in Rat Nigral GABAA Receptors Induced by Degeneration of the Striatonigral GABAergic Pathway: An Electrophysiological Study of Receptors Incorporated into Xenopus Oocytes

Abstract: Expression of rat brain γ-aminobutyric acid type A (GABA_A) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA_A receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA_A receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl^? currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 µM) and the steroid derivative allopregnanolone (3 µM) was increased by ～65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 µM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid ethyl ester (1 µM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA_A receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as β-carbolines.     

Corticosteroid Modulation of Signal Transduction in the CATH.a Cell Line

Abstract: Noradrenergic neuronal networks originating in the locus coeruleus have been implicated in the stress response. In order to study this system in vitro, we have employed a locus coeruleus-like cell line, CATH.a, and have determined the effect of dexamethasone on receptor-mediated second messenger responses. The CATH.a cell line produced increases in intracellular cyclic AMP conversion in response to corticotrophin-releasing factor (EC₅₀ = 6.93 ± 1.26 nM, maximum conversion = 4.11 ± 0.20%) and vasoactive intestinal polypeptide (EC₅₀ = 240 ± 40 nM, maximum conversion = 8.92 ± 1.24%). Forskolin (10 µM) increased conversion from 0.48 ± 0.05 to 6.39 ± 0.38%. The α₂-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) inhibited the forskolin response with an IC₅₀ of 6.76 ± 0.11 nM. Carbachol increased total ³H-labelled inositol phosphate accumulation to a maximum of 3.01 ± 0.79 fold basal (EC₅₀ = 7.94 ± 0.14 µM). Bradykinin produced a maximum 1.81 ± 0.05 fold basal stimulation of phosphoinositide hydrolysis (EC₅₀ = 9.12 ± 0.16 nM). Both carbachol and bradykinin increased intracellular Ca²⁺ concentration probably via a combination of mobilisation of intracellular stores and gating of extracellular Ca²⁺. Incubation for 24 h with the glucocorticoid receptor agonist, dexamethasone (1 µM), significantly potentiated the receptor-mediated phosphoinositide responses to all the agents tested; however, of the receptor-mediated increases in cyclic AMP conversion, only the vasoactive intestinal polypeptide response was potentiated. These results show that the CATH.a cell line displays some of the properties expected of locus coeruleus neurons and that glucocorticoid receptor stimulation selectively modulates receptor-mediated increases in second messenger formation.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.     

Effects of the antidepressant pirlindole and its dehydro-derivative on the activity of monoamine oxidase-a and on GABAA receptors

The effects of pirlindole and dehydro-pirlindole on GABA_A receptors and MAO-A activity were investigated in vitro. Pirlindole was inactive as a GABA antagonist. Dehydro-pirlindole exhibited partial and selective blockade of a subset of GABA_A receptors with an EC₅₀ of 12μM and maximum reversal (ΔB_opt) of 42%. Inhibition of rat brain and human placenta MAO-A by both compounds was much more potent (with IC₅₀ range 0.3–0.005 μM). Their effects on MAO-A activity were partially reversible in vitro. In contrast to pirlindole, dehydro-pirlindole may act not only as MAO-A inhibitor but also as a clozapine-like selective GABA_A receptor blocker, preferentially blocking a subset of GABA_A receptors that are not sensitive to DMCM or Ro 5-4864. Pirlindole is the generic name of the drug pyrazidol.     

l-Glutamate Uptake Inhibitors May Stimulate Phosphoinositide Hydrolysis in Baby Hamster Kidney Cells Expressing mGluR1a via Heteroexchange with l-Glutamate Without Direct Activation of mGluR1a

Abstract: The functional efficacies of inhibitors of l -glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. l -Serine-O-sulfate was an agonist at mGluR1a (EC₅₀ = 70 µM), mGluR2 (EC₅₀ = 25 µM), and mGluR4 (EC₅₀ = 324 µM). l -Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, l -cysteine, and dl -threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC₅₀ values of 43, 64, 463, and 488 µM, respectively, and displaced l -[³H]glutamate binding from membranes prepared from these cells with respective IC₅₀ values of 48, 44, 79, and 139 µM. However, d -aspartate,l -trans-pyrrolidine-2,4-dicarboxylate, l -threo-3-hydroxyaspartate, and l -aspartate-β-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC₅₀ values of 73, 54, 57, and 430 µM) but did not displace l -[³H]glutamate binding. These compounds inhibited Na⁺-dependent l -glutamate uptake into baby hamster kidney cells with IC₅₀ values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this l -glutamate transporter, was significantly attenuated in the presence of l -glutamate decarboxylase (EC 4.1.1.15) or l -alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mMl -trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous l -glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.