Synergistic inhibition of glycinergic transmission in vitro and in vivo by flavonoids and strychnine.

The inhibitory glycine receptor (GlyR) is a key mediator of synaptic signaling in spinal cord, brain stem, and higher central nervous system regions. The flavonoids quercetin and genistein have been identified previously as promising GlyR antagonists in vitro, but their detailed mechanism of action was not known. Here, inhibition of recombinant human α1 GlyRs in HEK 293 cells by genistein, quercetin, and strychnine was studied using whole-cell recording techniques. The interaction of several inhibitors applied alone or in combination was analyzed using a minimum mechanism of receptor activation and inhibition. Receptor inhibition in vivo was studied in a mouse model of strychnine toxicity. Genistein, quercetin, and strychnine were noncompetitive GlyR inhibitors. The inhibitory potency of one flavonoid (either genistein or quercetin) was not affected by simultaneous application of the other, suggesting that both flavonoids target the same site on the receptor. In combination with strychnine, flavonoid inhibition was augmented, indicating that strychnine binds to a position on the receptor physically distant from the flavonoid site. Potentiation of strychnine inhibition by flavonoids was also observed in vivo, where harmless doses of flavonoids enhanced strychnine toxicity in mice. Thus, in vitro and in vivo studies demonstrated a true synergism between flavonoids and strychnine for GlyR inhibition. The mechanism-based approach used here allows a rapid analysis of the effects of single drugs versus drug combinations.

[1]  J. W. Karpen,et al.  Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles. , 1986, Biochemistry.

[2]  C. Grewer.  Investigation of the alpha(1)-glycine receptor channel-opening kinetics in the submillisecond time domain. , 1999, Biophysical journal.

[3]  M. Radenković,et al.  The Effect of Ginkgo Biloba (EGb 761) on Epileptic Activity in Rabbits , 2008, Molecules.

[4]  M. Berenbaum.  What is synergy? , 1989, Pharmacological reviews.

[5]  E. Middleton,et al.  Differential inhibition of proliferation of human squamous cell carcinoma, gliosarcoma and embryonic fibroblast‐like lung cells in culture by plant flavonoids , 1992, Anti-cancer drugs.

[6]  R. A. Davidoff,et al.  The effects of strychnine on the inhibition of interneurons by glycine and γ-aminobutyric acid , 1969 .

[7]  M. Nair,et al.  Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells. , 2002, Anticancer research.

[8]  S. Cheav,et al.  Effect of two flavonoid compounds on central nervous system. Analgesic activity. , 1991, Life sciences.

[9]  Ting-Chao Chou,et al.  Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies , 2006, Pharmacological Reviews.

[10]  B. Katz,et al.  A study of the ‘desensitization’ produced by acetylcholine at the motor end‐plate , 1957, The Journal of physiology.

[11]  O. Krishtal,et al.  A receptor for protons in the nerve cell membrane , 1980, Neuroscience.

[12]  B. Havsteen,et al.  The biochemistry and medical significance of the flavonoids. , 2002, Pharmacology & therapeutics.

[13]  P. Schofield,et al.  The glycine receptor. , 1997, Pharmacology & therapeutics.

[14]  H Aoshima,et al.  Cocaine and phencyclidine inhibition of the acetylcholine receptor: analysis of the mechanisms of action based on measurements of ion flux in the millisecond-to-minute time region. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[15]  I. Izquierdo,et al.  Anxiolytic Natural and Synthetic Flavonoid Ligands of the Central Benzodiazepine Receptor Have No Effect on Memory Tasks in Rats , 1997, Pharmacology Biochemistry and Behavior.

[16]  J. Lynch,et al.  Molecular structure and function of the glycine receptor chloride channel. , 2004, Physiological reviews.

[17]  J H Medina,et al.  Molecular modeling and QSAR analysis of the interaction of flavone derivatives with the benzodiazepine binding site of the GABA(A) receptor complex. , 2001, Bioorganic & medicinal chemistry.

[18]  R. Harris,et al.  Enhancement of Glycine Receptor Function by Ethanol Is Inversely Correlated with Molecular Volume at Position α267* , 1998, The Journal of Biological Chemistry.

[19]  C. Becker,et al.  Hydroxylated residues influence desensitization behaviour of recombinant α3 glycine receptor channels , 2002, Journal of neurochemistry.

[20]  R. Tallarida,et al.  Drug synergism: its detection and applications. , 2001, The Journal of pharmacology and experimental therapeutics.

[21]  C. Williams,et al.  Advances in Flavonoid Research Since 1992 , 2001 .

[22]  C. Becker,et al.  Opposing Effects of Molecular Volume and Charge at the Hyperekplexia Site α1(P250) Govern Glycine Receptor Activation and Desensitization* , 2001, The Journal of Biological Chemistry.

[23]  G. Perrault,et al.  Pharmacological studies on synthetic flavonoids: comparison with diazepam , 1999, Neuropharmacology.

[24]  P. Das,et al.  Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors* , 2002, The Journal of Biological Chemistry.

[25]  S. Loewe.  The problem of synergism and antagonism of combined drugs. , 1953, Arzneimittel-Forschung.

[26]  G. Dillon,et al.  Direct inhibition of glycine receptors by genistein, a tyrosine kinase inhibitor , 2000, Neuropharmacology.

[27]  C. Becker,et al.  Molecular dynamics simulation links conformation of a pore-flanking region to hyperekplexia-related dysfunction of the inhibitory glycine receptor. , 2004, Chemistry & biology.

[28]  G. P. Hess,et al.  On the mechanism of the gamma-aminobutyric acid receptor in the mammalian (mouse) cerebral cortex. Chemical kinetic investigations with a 10-ms time resolution adapted to measurements of neuronal receptor function in single cells. , 1992, Biochemistry.

[29]  H. Betz.  Glycine receptors: heterogeneous and widespread in the mammalian brain , 1991, Trends in Neurosciences.

[30]  Tianming Yang,et al.  The protein tyrosine kinase inhibitor, genistein, decreases excitability of nociceptive neurons , 2004, Pain.

[31]  Berenbaum Mc.  What is synergy? , 1989, Pharmacological reviews.

[32]  C. Becker,et al.  The inhibitory glycine receptor: prospects for a therapeutic orphan? , 1998, Current pharmaceutical design.

[33]  M. Fischer,et al.  Structure, diversity and synaptic localization of inhibitory glycine receptors , 1994, Journal of Physiology-Paris.

[34]  Byung-Hwan Lee,et al.  Human glycine α1 receptor inhibition by quercetin is abolished or inversed by α267 mutations in transmembrane domain 2 , 2007, Brain Research.

[35]  B. Marquèze-Pouey,et al.  Widespread expression of glycine receptor subunit mRNAs in the adult and developing rat brain. , 1991, The EMBO journal.

[36]  G P Hess,et al.  Inhibition of the serotonin 5-HT3 receptor by nicotine, cocaine, and fluoxetine investigated by rapid chemical kinetic techniques. , 2001, Biochemistry.

[37]  J. Lynch,et al.  Native glycine receptor subtypes and their physiological roles , 2009, Neuropharmacology.

[38]  C. Becker,et al.  The Inhibitory Glycine Receptor—Simple Views of a Complicated Channel , 2002, Chembiochem : a European journal of chemical biology.

[39]  R. A. Davidoff,et al.  The effects of strychnine on the inhibition of interneurons by glycine and gamma-aminobutyric acid. , 1969, International journal of neuropharmacology.

[40]  P. Fuchs,et al.  Myricetin and quercetin, the flavonoid constituents ofGinkgo biloba extract, greatly reduce oxidative metabolism in both resting and Ca2+-loaded brain neurons , 1994, Brain Research.

[41]  E. Middleton,et al.  The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. , 2000, Pharmacological reviews.

[42]  C. Becker,et al.  Statistical coassembly of glycine receptor α1 wildtype and the hyperekplexia mutant α1(P250T) in HEK 293 cells: impaired channel function is not dominant in the recombinant system , 2002, Neuroscience Letters.

[43]  C. I. Bliss.  THE TOXICITY OF POISONS APPLIED JOINTLY1 , 1939 .

[44]  C. Becker,et al.  Novel Regulatory Site within the TM3–4 Loop of Human Recombinant α3 Glycine Receptors Determines Channel Gating and Domain Structure* , 2009, The Journal of Biological Chemistry.

[45]  J. Ippolito,et al.  Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[46]  P. Sorger,et al.  Systems biology and combination therapy in the quest for clinical efficacy , 2006, Nature chemical biology.

[47]  G. P. Hess,et al.  Mechanism for the channel-opening reaction of strychnine-sensitive glycine receptors on cultured embryonic mouse spinal cord cells. , 1994, Biochemistry.

[48]  C. Rémésy,et al.  Plasma metabolites of quercetin and their antioxidant properties. , 1998, American journal of physiology. Regulatory, integrative and comparative physiology.

[49]  H. Breitinger.  Fast Kinetic Analysis of Ligand-Gated Ion Channels , 2001, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[50]  E. Middleton,et al.  Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis‐associated properties in A431 cells overexpressing epidermal growth factor receptor , 1999, British journal of pharmacology.

[51]  R. Tallarida.  Statistical analysis of drug combinations for synergism , 1993, Pain.

[52]  Byung-Hwan Lee,et al.  Differential regulations of quercetin and its glycosides on ligand-gated ion channels. , 2008, Biological & pharmaceutical bulletin.

[53]  J. Kühnau.  The flavonoids. A class of semi-essential food components: their role in human nutrition. , 1976, World review of nutrition and dietetics.

[54]  P. Legendre.  The glycinergic inhibitory synapse , 2001, Cellular and Molecular Life Sciences CMLS.

[55]  V. O'Connor,et al.  INTERACTIONS OF GLYCINE AND STRYCHNINE WITH THEIR RECEPTOR RECOGNITION SITES IN MOUSE SPINAL CORD , 1996, Neurochemistry International.

[56]  G. P. Hess,et al.  Rapid chemical reaction techniques developed for use in investigations of membrane-bound proteins (neurotransmitter receptors). , 2002, Biophysical chemistry.

[57]  J. Udgaonkar,et al.  Chemical kinetic measurements of a mammalian acetylcholine receptor by a fast-reaction technique. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[58]  E. Middleton,et al.  Free radical scavenging and antioxidant activity of plant flavonoids. , 1994, Advances in experimental medicine and biology.

[59]  J. Harborne,et al.  Advances in flavonoid research since 1992. , 2000, Phytochemistry.