Anandamide Inhibition of 5-HT3A Receptors Varies with Receptor Density and Desensitization

Converging evidence has suggested that anandamide (AEA), an endogenous agonist of cannabinoid (CB) receptors, can directly interact with certain types of ligand-gated ion channels (LGICs). However, little is known about the molecular and cellular mechanisms of AEA-induced direct effects on LGICs. Here, we report that AEA inhibited the function of serotoningated ion channels (5-HT3A) expressed in Xenopus laevis oocytes and human embryonic kidney 293 cells in a manner that was dependent on the steady-state receptor density at the cell surface. The magnitude of AEA inhibition was inversely correlated with the expression levels of receptor protein and function. With increasing surface receptor expression, the magnitude of AEA inhibition decreased. Consistent with this idea, pretreatment with actinomycin D, which inhibits transcription, decreased the amplitude of current activated by maximal concentrations of 5-hydroxytryptamine (5-HT) and increased the magnitude of AEA inhibition. AEA did not significantly alter 5-HT3A receptor trafficking. However, AEA accelerated 5-HT3A receptor desensitization time in a concentration-dependent manner without significantly changing receptor activation and deactivation time. The desensitization time was correlated with the AEA-induced inhibiting effect and mean 5-HT current density. Applications of 5-hydroxyindole and nocodazole, a microtubule disruptor, significantly slowed 5-HT3A receptor desensitization and reduced the magnitude of AEA inhibition. These observations suggest that 5-HT3 receptor density at the steady state regulates receptor desensitization kinetics and the potency of AEA-induced inhibiting effect on the receptors. The inhibition of 5-HT3 receptors by AEA may contribute to its physiological roles in control of pain and emesis.

[1]  J. Priestley,et al.  Antibodies against the extracellular domain of the 5-HT3 receptor label both native and recombinant receptors. , 1999, Brain research. Molecular brain research.

[2]  J. Meier,et al.  Desensitization of homomeric alpha1 glycine receptor increases with receptor density. , 2002, Molecular pharmacology.

[3]  M. Morales,et al.  The Endogenous Cannabinoid Anandamide Inhibits α7 Nicotinic Acetylcholine Receptor-Mediated Responses in Xenopus Oocytes , 2003, Journal of Pharmacology and Experimental Therapeutics.

[4]  Lars-Göran Nilsson,et al.  A polymorphism in the serotonin receptor 3A (HTR3A) gene and its association with harm avoidance in women. , 2003, Archives of general psychiatry.

[5]  H. Bräuner‐Osborne,et al.  Naturally occurring variations in the human 5-HT3A gene profoundly impact 5-HT3 receptor function and expression , 2007, Pharmacogenetics and genomics.

[6]  A. Allan,et al.  Improvements in hippocampal-dependent learning and decremental attention in 5-HT(3) receptor overexpressing mice. , 2003, Learning & memory.

[7]  R. Naylor,et al.  Consensus meeting agrees distribution of 5-HT3 receptors in mammalian hindbrain. , 1990 .

[8]  W. Zieglgänsberger,et al.  Co-expression of the 5-HT3B serotonin receptor subunit alters the biophysics of the 5-HT3 receptor. , 2003, Biophysical journal.

[9]  S. Moss,et al.  Constructing inhibitory synapses , 2001, Nature Reviews Neuroscience.

[10]  H. Bönisch,et al.  Direct inhibition by cannabinoids of human 5‐HT3A receptors: probable involvement of an allosteric modulatory site , 2002, British journal of pharmacology.

[11]  N. Sluis-Cremer,et al.  Efavirenz Accelerates HIV-1 Reverse Transcriptase Ribonuclease H Cleavage, Leading to Diminished Zidovudine Excision , 2008, Molecular Pharmacology.

[12]  M. Rietschel,et al.  Serotonin receptor gene HTR3A variants in schizophrenic and bipolar affective patients. , 2001, Pharmacogenetics.

[13]  R. Pearce,et al.  Tandem Subunits Effectively Constrain GABAA Receptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAA Receptor-Associated Protein , 2005, The Journal of Neuroscience.

[14]  J. Palacios,et al.  Localisation by autoradiography of neuronal 5-HT3 receptors in the mouse CNS. , 1988, European journal of pharmacology.

[15]  K. Lesch,et al.  Quantitation of 5HT3 receptors in forebrain of serotonin transporter deficient mice , 2004, Journal of Neural Transmission.

[16]  H. Bönisch,et al.  Recombinant human 5-HT3A receptors in outside-out patches of HEK 293 cells: basic properties and barbiturate effects , 2000, Naunyn-Schmiedeberg's Archives of Pharmacology.

[17]  M. Rietschel,et al.  Association between the 5' UTR variant C178T of the serotonin receptor gene HTR3A and bipolar affective disorder. , 2001, Pharmacogenetics.

[18]  B. Costall,et al.  Identification and Characterisation of 5‐Hydroxytryptamine3 Recognition Sites in Human Brain Tissue , 1989, Journal of neurochemistry.

[19]  M. Herkenham,et al.  Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  C. Lupica,et al.  The Endocannabinoid Anandamide Inhibits the Function of α4β2 Nicotinic Acetylcholine Receptors , 2007, Molecular Pharmacology.

[21]  J. Palacios,et al.  The (S)-isomer of [3H]zacopride labels 5-HT3 receptors with high affinity in rat brain. , 1990, European journal of pharmacology.

[22]  D. Lovinger,et al.  Role of aspartate 298 in mouse 5‐HT3A receptor gating and modulation by extracellular Ca2+ , 2005, The Journal of physiology.

[23]  M. Sheng,et al.  Ligand-gated ion channel interactions with cytoskeletal and signaling proteins. , 2000, Annual review of physiology.

[24]  F. Bloom,et al.  The 5-HT3 Receptor Is Present in Different Subpopulations of GABAergic Neurons in the Rat Telencephalon , 1997, The Journal of Neuroscience.

[25]  F. F. Weight,et al.  Modulation of 5-HT3 Receptor-mediated Response and Trafficking by Activation of Protein Kinase C* , 2003, Journal of Biological Chemistry.

[26]  J. Palacios,et al.  5-Hydroxytryptamine3 receptors in the human brain: Autoradiographic visualization using [3H]ICS 205-930 , 1989, Neuroscience.

[27]  A. Allan,et al.  5-HT3 receptor over-expression enhances ethanol sensitivity in mice , 1999, Psychopharmacology.

[28]  P. Fan.  Cannabinoid agonists inhibit the activation of 5-HT3 receptors in rat nodose ganglion neurons. , 1995, Journal of neurophysiology.

[29]  Li Zhang,et al.  An Interaction Involving an Arginine Residue in the Cytoplasmic Domain of the 5-HT3A Receptor Contributes to Receptor Desensitization Mechanism* , 2006, Journal of Biological Chemistry.

[30]  M. Bennett,et al.  Protein kinase C modulates NMDA receptor trafficking and gating , 2001, Nature Neuroscience.

[31]  R. Olsen,et al.  The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[32]  R. Ciccocioppo,et al.  Central 5-HT3 receptors in P and in AA alcohol-preferring rats: An autoradiographic study , 1998, Brain Research Bulletin.

[33]  M. Morales,et al.  Differential Composition of 5-Hydroxytryptamine3Receptors Synthesized in the Rat CNS and Peripheral Nervous System , 2002, The Journal of Neuroscience.

[34]  L. Tecott,et al.  Nervous system distribution of the serotonin 5-HT3 receptor mRNA. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[35]  M. Oz,et al.  Δ9-Tetrahydrocannabinol and Endogenous Cannabinoid Anandamide Directly Potentiate the Function of Glycine Receptors , 2006, Molecular Pharmacology.

[36]  H. Hansen,et al.  Binding of anandamide to bovine serum albumin Published, JLR Papers in Press, July 1, 2003. DOI 10.1194/jlr.M300170-JLR200 , 2003, Journal of Lipid Research.

[37]  N. Burnashev,et al.  Glycine Receptors in CNS Neurons as a Target for Nonretrograde Action of Cannabinoids , 2005, The Journal of Neuroscience.

[38]  S. Lummis,et al.  Diltiazem causes open channel block of recombinant 5‐HT3 receptors , 1999, The Journal of physiology.

[39]  A. Allan,et al.  5-HT3 receptor over-expression decreases ethanol self administration in transgenic mice , 1998, Psychopharmacology.

[40]  K. Krzywkowski.  Do polymorphisms in the human 5-HT3 genes contribute to pathological phenotypes? , 2006, Biochemical Society transactions.

[41]  Norihiro Sadato,et al.  A Variant C178T in the Regulatory Region of the Serotonin Receptor Gene HTR3A Modulates Neural Activation in the Human Amygdala , 2005, The Journal of Neuroscience.

[42]  M. Jackson,et al.  5-HT3 receptors mediate rapid responses in cultured hippocampus and a clonal cell line , 1988, Neuron.

[43]  J. A. van Hooft,et al.  Allosteric Potentiation of the 5-HT3 Receptor-mediated Ion Current in N1E-115 Neuroblastoma Cells by 5-Hydroxyindole and Analogues , 1997, Neuropharmacology.

[44]  M. Morales,et al.  Endogenous cannabinoid, anandamide, acts as a noncompetitive inhibitor on 5‐HT3 receptor‐mediated responses in Xenopus oocytes , 2002, Synapse.

[45]  G. Prestwich,et al.  Regulation of Lysophosphatidic Acid Receptor Expression and Function in Human Synoviocytes: Implications for Rheumatoid Arthritis? , 2008, Molecular Pharmacology.

[46]  P. Pacher,et al.  The Endocannabinoid System as an Emerging Target of Pharmacotherapy , 2006, Pharmacological Reviews.

[47]  D. R. Compton,et al.  Assessment of anandamide interaction with the cannabinoid brain receptor: SR 141716A antagonism studies in mice and autoradiographic analysis of receptor binding in rat brain. , 1998, The Journal of pharmacology and experimental therapeutics.

[48]  Michael Hollmann,et al.  N-glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1 , 1994, Neuron.

[49]  E. M. Petrini,et al.  Declusterization of GABAA Receptors Affects the Kinetic Properties of GABAergic Currents in Cultured Hippocampal Neurons* , 2003, The Journal of Biological Chemistry.