Positive modulation of the α9α10 nicotinic cholinergic receptor by ascorbic acid.

BACKGROUND AND PURPOSE: The activation of α9α10 nicotinic cholinergic receptors (nAChRs) present at the synapse between efferent olivocochlear fibres and cochlear hair cells can prevent acoustic trauma. Hence, pharmacological potentiators of these receptors could be useful therapeutically. In this work, we characterize ascorbic acid as a positive modulator of recombinant α9α10 nAChRs. EXPERIMENTAL APPROACH: ACh-evoked responses were analysed under two-electrode voltage-clamp recordings in Xenopus laevis oocytes injected with α9 and α10 cRNAs. KEY RESULTS: Ascorbic acid potentiated ACh responses in X. laevis oocytes expressing α9α10 (but not α4ß2 or α7) nAChRs, in a concentration-dependent manner, with an effective concentration range of 1-30 mM. The compound did not affect the receptor's current-voltage profile nor its apparent affinity for ACh, but it significantly enhanced the maximal evoked currents (percentage of ACh maximal response, 240 ± 20%). This effect was specific for the L form of reduced ascorbic acid. Substitution of the extracellular cysteine residues present in loop C of the ACh binding site did not affect the potentiation. Ascorbic acid turned into a partial agonist of α9α10 nAChRs bearing a point mutation at the pore domain of the channel (TM2 V13'T mutant). A positive allosteric mechanism of action rather than an antioxidant effect of ascorbic acid is proposed. CONCLUSIONS AND IMPLICATIONS: The present work describes one of the few agents that activates or potentiates α9α10 nAChRs and leads to new avenues for designing drugs with potential therapeutic use in inner ear disorders.

Nitric oxide potentiation of the homomeric ρ1 GABA(C) receptor function.

BACKGROUND AND PURPOSE: NO is a highly diffusible and reactive gas produced in the nervous system, which acts as a neuronal signal mediating physiological or pathological mechanisms. NO can modulate the activity of neurotransmitter receptors and ion channels, including NMDA and GABA(A) receptors. In the present work, we examined whether GABA(C) receptor function can also be regulated by NO. EXPERIMENTAL APPROACH: Homomeric ρ1 GABA(C) receptors were expressed in oocytes and GABA-evoked responses electrophysiologically recorded in the presence or absence of the NO donor DEA. Chemical protection of cysteines by selective sulfhydryl reagents and site-directed mutagenesis were used to determine the protein residues involved in the actions of NO. KEY RESULTS: GABAρ1 receptor responses were significantly enhanced in a dose-dependent, fast and reversible manner by DEA and the specific NO scavenger CPTIO prevented these potentiating effects. The ρ1 subunits contain only three cysteine residues, two extracellular at the Cys-loop (C177 and C191) and one intracellular (C364). Mutations of C177 and C191 render the ρ1 GABA receptors non-functional, but C364 can be safely exchanged by alanine (C364A). NEM, N-ethyl maleimide and (2-aminoethyl) methanethiosulfonate prevented the effects of DEA on GABAρ1 receptors. Meanwhile, the potentiating effects of DEA on mutant GABAρ1(C364A) receptors were similar to those observed on wild-type receptors. CONCLUSIONS AND IMPLICATIONS: Our results suggest that the function of GABA(C) receptors can be enhanced by NO acting at the extracellular Cys-loop.

6-Chloro-3'-nitroflavone is a potent ligand for the benzodiazepine binding site of the GABA(A) receptor devoid of intrinsic activity.

6-Chloro-3'-nitroflavone integrates a list of nearly 70 flavone derivatives synthesized in our laboratories. The effects of 6-chloro-3'-nitroflavone on the benzodiazepine binding sites (BDZ-BSs) of the GABA(A) receptor were examined in vitro and in vivo. 6-Chloro-3'-nitroflavone inhibited the [3H]flunitrazepam ([3H]FNZ) binding to rat cerebral cortex membranes with a Ki of 6.68 nM and the addition of GABA to extensively washed membranes did not modify its affinity for the BDZ-BSs (GABA-shift = 1.16+/-0.12). The binding assays performed in rat striatal and cerebellar brain membranes showed that this compound has similar affinity to different populations of BDZ-BSs. Electrophysiological experiments revealed that 6-chloro-3'-nitroflavone did not affect GABA(A)-receptors (GABA(A)-Rs) responses recorded in Xenopus oocytes expressing alpha1beta2gamma2s subunits, but blocked the potentiation exerted by diazepam (DZ) on GABA-activated chloride currents. In vivo experiments showed that 6-chloro-3'-nitroflavone did not possess anxiolytic, anticonvulsant, sedative, myorelaxant actions in mice or amnestic effects in rats; however, 6-chloro-3'-nitroflavone antagonized diazepam-induced antianxiety action, anticonvulsion, short-term, and long-term amnesia and motor incoordination. These biochemical, electrophysiological, and pharmacological results suggest that 6-chloro-3'-nitroflavone behaves as an antagonist of the BDZ-BSs.

Activation of GABA rho 1 receptors by glycine and beta-alanine.

Neurons of the vertebrate retina possess receptors for many neurotransmitters. Particularly interesting is a new type of GABA receptor (GABA rho) that, in contrast to GABAA and GABAB receptors, shows very little desensitization, is not blocked by bicuculline, and is not activated by baclofen. Homomeric human GABA rho 1 receptors were expressed in Xenopus oocytes. In addition to GABA, and related agonists, GABA rho 1 receptors respond to glycine (Gly) and beta-alanine (beta-Ala) by generating Cl- currents that do not desensitize and are resistant to bicuculline. The half-maximal concentrations for Gly and beta-Ala currents were 14.2 +/- 1.3 mM and 0.66 +/- 0.11 mM respectively. The current responses to Gly and beta-Ala were blocked by picrotoxin and TBPS. The cross-sensitivity of GABA rho 1 receptors to Gly and beta-Ala may play a role in retinal physiology.

Cationic modulation of rho 1-type gamma-aminobutyrate receptors expressed in Xenopus oocytes.

A study was made of the effects of di- and trivalent cations on homomeric rho 1-type gamma-aminobutyrate (GABA rho 1) receptors expressed in Xenopus oocytes after injection of mRNA coding for the GABA rho 1 subunit. GABA elicited large currents with a Kd approximately 1 microM. The properties of these GABA rho 1 receptors were similar to those of native bicuculline-resistant GABA receptors expressed by retinal mRNA. GABA rho 1 currents showed very little desensitization, were blocked by picrotoxin but not by bicuculline, and were not modulated by barbiturates, benzodiazepines, or beta-carbolines. Zn2+ reversibly decreased GABA rho 1 responses (IC50 = 22 microM). Other divalent cations were also tested and their rank order of potency was: Zn2+ approximately Ni2+ approximately Cu2+ >> Cd2+, whereas Ba2+, Co2+, Sr2+, Mn2+, Mg2+, and Ca2+ showed little or no effect. In contrast, La3+ reversibly potentiated the GABA currents mediated by homomeric GABA rho 1 receptors, with an EC50 = 135 microM and a maximal potentiation of about 100% (GABA, 1 microM; La3+, 1 mM). Other lanthanides showed similar effects (Lu3+ > Eu3+ > Tb3+ > Gd3+ > Er3% > Nd3+ > La3+ > Ce3+). Thus, GABA rho 1 receptors contain sites for cationic recognition, and in particular, Zn2+ may play a role during synaptic transmission in the retina.

Calcium induced modulation of peripheral-type benzodiazepine receptors in rat kidney membranes.

The effects of Ca2+ ions on 3H-RO 5-4864 binding to the peripheral benzodiazepine receptor were examined. Preincubation of rat kidney membranes with Ca2+ at 37 degrees C produced a dose-dependent inhibition of 3H-RO 5-4864 binding. No inhibition was observed in membranes preincubated at 0 degrees C. The effect of Ca2+ was competitive in nature and was fully reversed by the addition of EGTA. At 1 mM, the maximal effect was achieved with CaCl2, whereas CoCl2 and CdCl2 had lesser effects. No other divalent cation salts examined decreased 3H-RO 5-4864 binding to rat kidney membranes. Collectively, these data demonstrate that the affinity of 3H-RO 5-4864 binding to rat kidney membranes is regulated by Ca2+ and suggest the presence of cation recognition binding sites coupled to the peripheral benzodiazepine receptor.

Ethanol and the peripheral benzodiazepine receptor: in vivo and in vitro experiments.

1. The effect of chronic ethanol exposure on rat peripheral benzodiazepine receptors (PBR) was studied. 2. The binding of 3H-RO 5-4864 to PBR was increased (35.4%) in the kidneys of rats treated with 10% (v/v) ethanol for 12 weeks, but not in renal membranes isolated from rats exposed to ethanol 30% (v/v) during the same period. 3. Similarly, short-term administration of ethanol (4 weeks) did not alter the binding of 3H-RO 5-4864 to renal membranes. 4. To examine the possibility of a direct interaction of ethanol with PBR, in vitro experiments were carried out. Only high concentrations of ethanol (> 100 mM) caused a significant inhibition of 3H-RO 5-4864 binding in kidney, testis and cerebral cortex. 5. The results presented indicate that chronic ethanol exposure causes a time and dose-dependent increase in renal PBR.

Regulation of peripheral-type benzodiazepine receptors following repeated benzodiazepine administration.

The effects of chronic benzodiazepine (BZD) treatment on rat peripheral-type benzodiazepine receptors (PBR) were studied. Five days treatment with the PBR ligands RO 5-4864 or diazepam (DZ) up-regulates kidney PBR. In contrast clonazepam, a specific central-type BZD receptor ligand, did not alter 3H-RO 5-4864 binding. Fourteen days administration of DZ produced an up-regulation of kidney and heart PBR and a down-regulation of testicular PBR. The results suggest that chronic BZD exposure differentially regulates PBR in peripheral organs.

Effect of long term diazepam administration on testicular benzodiazepine receptors and steroidogenesis.

We evaluated the effect of acute and chronic diazepam administration on testicular peripheral type benzodiazepine receptors (PBZD-R), serum testosterone and LH levels and the "in vitro" androgen production in response to Ro 5-4864, a PBZD-R agonist. The chronic diazepam treatment induced a significant fall in plasma testosterone concentration while LH levels remained unchanged. The number of PBZD-R was reduced by 37% and low concentrations (10(-8)-10(-6) M) of Ro 5-4864 failed to stimulate "in vitro" androgen production. The acute diazepam administration caused a significant increase in plasma testosterone levels while no changes were observed in LH concentrations and testicular PBZD-R. These results further suggest a modulatory role of PBZD-R on testicular steroidogenic activity.

Peripheral-type benzodiazepine receptors are highly concentrated in mitochondrial membranes of rat testicular interstitial cells.

The binding of 3H-RO 5-4864 to the peripheral-type benzodiazepine receptors (PBZDR) in rat testicular interstitial cells (TIC) was characterized. The binding was saturable, reversible and showed a single high-affinity (Kd = 5.02 +/- 0.86 nM) class of binding sites. The maximal binding capacity (Bmax) in crude mitochondrial fractions (77.6 +/- 9.1 pmol/mg protein) represents the highest density of PBZDR in tissues thus far studied. In comparison with the crude mitochondrial fraction the subcellular fractionation of TIC revealed a 2-fold enrichment of 3H-RO 5-4864 binding sites to the purified mitochondria (Bmax = 140 +/- 23 pmol/mg protein). The ability of various drugs to displace 3H-RO 5-4864 from TIC binding sites was examined and the inhibition constants (Ki) for RO 5-4864, PK 11195, diazepam and flunitrazepam were 3.5, 4.4, 159, and 353 nM, respectively, whereas clonazepam and RO 15-1788 were inefficient in displacing 3H-RO 5-4864 (Ki greater than 10 microM). This pharmacological profile is characteristic of PBZDR described in other tissues. In conclusion, rat TIC possess a very high concentration of PBZDR primarily associated with mitochondrial membranes.

A multiconcentration controlled test atmosphere system for calibration studies.

A novel controlled test atmosphere system was developed to generate multiple concentrations of gases and vapors simultaneously. It was used successfully to study the effectiveness of two air monitoring methods to analyze for various organic compounds. Three concentrations were generated simultaneously to determine the methods' performance. This was accomplished in a single run, requiring one day, greatly improving validation efficiency. Prior to development of this system, a separate test run was required at each concentration requiring three days. Essential elements of the system include: dynamic serial dilution of the air stream to produce three concentrations; all inert surface construction (Teflon, glass, and stainless steel); diffusion/permeation tube generation with multicompound capability; low pressure drop by use of large tubing and low pressure differential mass flow meters.