Daniel J. Calvo

Overview—Flavonoids: A New Family of Benzodiazepine Receptor Ligands

Benzodiazepines (BDZs) are the most widely prescribed class of psychoactive drugs in current therapeutic use, despite the important unwanted side-effects that they produce such as sedation, myorelaxation, ataxia, amnesia, ethanol and barbiturate potentiation and tolerance. Searching for safer BDZ-receptor (BDZ-R) ligands we have recently demonstrated the existence of a new family of ligands which have a flavonoid structure. First isolated from plants used as tranquilizers in folkloric medicine, some natural flavonoids have shown to possess a selective and relatively mild affinity for BDZ-Rs and a pharmacological profile compatible with a partial agonistic action. In a logical extension of this discovery various synthetic derivatives of those compounds, such as 6,3′-dinitroflavone were found to have a very potent anxiolytic effect not associated with myorelaxant, amnestic or sedative actions. This dinitro compound, in particular, exhibits a high affinity for the BDZ-Rs (Ki = 12–30 nM). Due to their selective pharmacological profile and low intrinsic efficacy at the BDZ-Rs, flavonoid derivatives, such as those described, could represent an improved therapeutic tool in the treatment of anxiety. In addition, several flavone derivatives may provide important leads for the development of potent and selective BDZ-Rs ligands.

Chrysin (5,7-di-OH-flavone), a naturally-occurring ligand for benzodiazepine receptors, with anticonvulsant properties.

Chrysin (5,7-di-OH-flavone) was identified in Passiflora coerulea L., a plant used as a sedative in folkloric medicine. Chrysin was found to be a ligand for the benzodiazepine receptors, both central (Ki = 3 microM, competitive mechanism) and peripheral (Ki = 13 microM, mixed-type mechanism). Administered to mice by the intracerebroventricular route, chrysin was able to prevent the expression of tonic-clonic seizures induced by pentylenetertrazol. Ro 15-1788, a central benzodiazepine receptor antagonist, abolished this effect. In addition, all of the treated mice lose the normal righting reflex which suggests a myorelaxant action of the flavonoid. The presence in P. coerulea of benzodiazepine-like compounds was also confirmed.

Flavonoid modulation of ionic currents mediated by GABAA and GABAC receptors

The modulation of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA-gated Cl(-) channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors expressed in Xenopus laevis oocytes in the micromolar range. alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by alpha(4)beta(2) neuronal nicotinic acetylcholine, serotonin 5-HT(3A) and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized alpha(1)beta(1)gamma(2s) GABA(A) receptors. Effects of apigenin and quercetin on alpha(1)beta(1)gamma(2s) GABA(A) receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation.

Identification of brain neurons expressing the dopamine D4 receptor gene using BAC transgenic mice.

The dopamine D4 receptor (D4R) has received considerable interest because of its higher affinity for atypical antipsychotics, the extremely polymorphic nature of the human gene and the genetic association with attention deficit and hyperactivity disorder (ADHD). Several efforts have been undertaken to determine the D4R expression pattern in the brain using immunohistochemistry, binding autoradiography and in situ hybridization, but the overall published results present large discrepancies. Here, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the mouse dopamine D4 receptor gene (Drd4). Immunohistochemical analysis performed in brain sections of Drd4‐EGFP transgenic mice using an anti‐EGFP polyclonal antibody showed that transgenic expression was predominant in deep layer neurons of the prefrontal cortex, particularly in the orbital, prelimbic, cingulate and rostral agranular portions. In addition, discrete groups of Drd4‐EGFP labelled neurons were observed in the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. EGFP was not detected in the striatum, hippocampus or midbrain as described using other techniques. Given the fine specificity of EGFP expression in BAC transgenic mice and the high sensitivity of the EGFP antibody used in this study, our results indicate that Drd4 expression in the adult mouse brain is limited to a more restricted number of areas than previously reported. Its leading expression in the prefrontal cortex supports the importance of the D4R in complex behaviours depending on cortical dopamine (DA) transmission and its possible role in the etiopathophysiology of ADHD.

A New Inorganic Photolabile Protecting Group for Highly Efficient Visible Light GABA Uncaging

Fil: Zayat, Leonardo Martin. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Quimica Inorganica, Analitica y Quimica Fisica; Argentina

Recovery of central noradrenergic neurons one year after the administration of the neurotoxin DSP4

The long-term effects of the systemic administration of DSP4 (N-(2-chloroethyl)N-ethyl-2-bromobenzylamine hydrochloride), a selective noradrenergic neurotoxin, on the endogenous levels of monoamines and their metabolites and on alpha- and beta-adrenoceptors in selected brain regions of the rat were examined. After 7 days, DSP4 caused a marked reduction (about 80%) of endogenous noradrenaline levels in locus coeruleus-innervated regions. At 90, 240 and 300 days after DSP4 injection, a partial and gradual recovery (50%, 41% and 25% of control values, respectively) of the noradrenaline cortical levels was evident. One year after DSP4 administration, brain regional noradrenaline stores were almost completely recovered. No changes in 5-hydroxytryptamine levels were observed in the three time intervals, but a mild decrease in cortical and hippocampal 5-hydroxyindolacetic acid levels was found 7 days after DSP4 injection. Following the profound noradrenaline depletion seen at 7 days, the cerebral cortical density of alpha 1-, alpha 2- and beta-adrenoceptors was significantly increased. Assessment of adrenergic receptors in cerebral cortex at 365 days after DSP4 injection, indicated that alpha 1- and alpha 2-adrenoceptor densities did not differ from control values; however, the density of beta-adrenoceptors remained increased. No changes were observed in the affinities of the three types of adrenoceptors studied. These results indicate that after a selective noradrenergic denervation induced by DSP4, there is a slow and gradual recovery of noradrenaline stores and of alpha 1- and alpha 2-adrenoceptor populations, suggesting a possible regrowth and/or collateral sprouting of noradrenergic terminals.

Allosteric modulation of retinal GABA receptors by ascorbic acid

Ionotropic GABA receptors (GABAA and GABAC) belong to the Cys-loop receptor family of ligand-gated ion channels. GABAC receptors are highly expressed in the retina, mainly localized at the axon terminals of bipolar cells. Ascorbic acid, an endogenous redox agent, modulates the function of diverse proteins, and basal levels of ascorbic acid in the retina are very high. However, the effect of ascorbic acid on retinal GABA receptors has not been studied. Here we show that the function of GABAC and GABAA receptors is regulated by ascorbic acid. Patch-clamp recordings from bipolar cell terminals in goldfish retinal slices revealed that GABAC receptor-mediated currents activated by tonic background levels of extracellular GABA, and GABAC currents elicited by local GABA puffs, are both significantly enhanced by ascorbic acid. In addition, a significant rundown of GABA puff-evoked currents was observed in the absence of ascorbic acid. GABA-evoked Cl− currents mediated by homomeric ρ1 GABAC receptors expressed in Xenopus laevis oocytes were also potentiated by ascorbic acid in a concentration-dependent, stereo-specific, reversible, and voltage-independent manner. Studies involving the chemical modification of sulfhydryl groups showed that the two Cys-loop cysteines and histidine 141, all located in the ρ1 subunit extracellular domain, each play a key role in the modulation of GABAC receptors by ascorbic acid. Additionally, we show that retinal GABAA IPSCs and heterologously expressed GABAA receptor currents are similarly augmented by ascorbic acid. Our results suggest that ascorbic acid may act as an endogenous agent capable of potentiating GABAergic neurotransmission in the CNS.

6-Chloro-3'-nitroflavone is a potent ligand for the benzodiazepine binding site of the GABAA 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.

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.