Flavonoid modulation of ionic currents mediated by GABAA and GABAC receptors
Introduction
Flavonoids are substances of low molecular weight found in vascular plants Bohm, 1998, Harborne and Williams, 2000. They show a wide range of biological activities Miksicek, 1993, Middleton and Kandaswami, 1994, Harborne and Williams, 2000, with neuropharmacological actions such as analgesia, effects on motility and sleep Speroni and Minghetti, 1988, Picq et al., 1991, modulation of neuronal oxidative metabolism (Oyama et al., 1994), proconvulsant, anticonvulsant, sedative and anxiolytic effects Speroni and Minghetti, 1988, Medina et al., 1990, Häberlein et al., 1994, Viola et al., 1995, Marder et al., 1995, Medina et al., 1998, Griebel et al., 1999, etc.
Effects of flavonoids on the central nervous system (CNS) are complex and involve different mechanisms Vohora et al., 1980, Chakravarthy et al., 1981, Medina et al., 1998, including actions on synaptic receptors or ionic channels Nielsen et al., 1988, Koh et al., 1994, Ji et al., 1996, Medina et al., 1998, Simmen et al., 1998, Dekermendjian et al., 1999, Avallone et al., 2000, Calvo et al., 2000, Mall et al., 2000, Saponara et al., 2002.
Many studies correlated the affinities of diverse natural and synthetic flavonoids for the benzodiazepine binding sites located to the GABAA receptors (γ-aminobutyric acid: GABA) to their pharmacological properties shown in vivo Medina et al., 1998, Griebel et al., 1999, Avallone et al., 2000, Viola et al., 2000. Based on these and other evidences, a benzodiazepine-like mechanism was proposed for flavonoid modulation of ionotropic GABA receptors Medina et al., 1998, Dekermendjian et al., 1999, Marder and Paladini, 2002. However, experimental support for a benzodiazepine-like modulation of the GABA receptor function by flavonoids is still lacking. Now we tested this hypothesis by studying the effects of a group of flavonoids (chemical structures illustrated in Fig. 1) on ionic currents mediated by two common ionotropic GABA receptor subtypes.
GABAA receptors are heteromeric proteins forming pentameric structures assembled from diverse subunit types (α1−6, β1−4, γ1−3, δ, ε and π) Barnard et al., 1998, Hevers and Luddens, 1998. In contrast, GABAC receptors would be homomeric, exclusively composed by ρ subunits (Enz and Cutting, 1998). α1β1γ2s GABAA receptors are highly expressed in the brain and represent a quite common GABAA receptor subtype (in cerebral cortex, thalamus, etc.) that is strongly potentiated by benzodiazepines (Hevers and Luddens, 1998). ρ1 GABAC receptors are mainly expressed in the retina and mediate benzodiazepine-insensitive responses (Zhang et al., 2001).
We studied the effects of flavonoids on α1β1γ2s GABAA and ρ1 GABAC receptors expressed in Xenopus oocytes. Results indicated that diverse flavonoids antagonized ionic currents mediated by α1β1γ2s GABAA and ρ1 GABAC receptors in a similar way and did not behave as benzodiazepine-like modulators.
Section snippets
RNA preparation for oocyte injections
Full-length cDNAs, encoding the different receptor subunits, cloned in vectors suitable for in vitro transcription, were provided by colleagues (see acknowledgments). Rat GABAA receptor subunits: α1 and β1 in pBluescript SK− (Promega, Madison, WI, USA), γ2S in pSP6 (Invitrogen, Groningen, The Netherlands). Human GABAA receptor subunits: α1, β1 and γ2S in pCDM8 (Invitrogen). Human GABAC receptor subunit: ρ1 in pBluescript SK−(Promega). Rat neuronal nicotinic acetylcholine receptor subunits α4, β2
Results
The modulation of ionic currents mediated by α1β1γ2s GABAA and ρ1 GABAC receptors by quercetin, chrysin, apigenin, morine, flavone and α-naphthoflavone was examined in electrophysiological experiments. Selectivity of the flavonoid actions was also analyzed by studying in a similar way the effects of quercetin on other ionotropic neurotransmitter receptors, namely, α4β2 neuronal nicotinic acetylcholine, 5-HT3A and glutamate AMPA/kainate receptors.
Discussion
We have characterized the pharmacological actions of a group of flavonoids on ionotropic GABA receptors through electrophysiological studies. A number of naturally occurring and synthetic compounds were selected based on their previously reported effects in binding assays and in vivo pharmacological experiments. The main contribution of the present work is the demonstration that diverse flavonoids modulate the function of GABAA and GABAC receptors and also of other ionotropic receptors
Acknowledgements
We would like to thank people that generously provided us with plasmids carrying the different receptor subunits, Dr. Paul Whiting and Dr. Peter Seeburg for α1β1γ2s GABAA; Dr. Ricardo Miledi and Dr. Ataúlfo Martinez-Torres for ρ1 GABAC and Dr. Stephen Heinemann, Dr. Jim Boulter and Dr. David Johnson for nicotinic α4β2 and 5-HT3A. We also thank Dr. Mariana del Vas and Dr. Marcelo Rubinstein for reading the manuscript. This work was supported by grants from CONICET (PIP 780/98) and FONCyT (PICT99
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