Influence of levels of reelin in negative cooperativity and functionality of the 5-HT2A receptors in synaptosomes

S Lage, MJ Varela, MI Cadavid, MI Loza, J Brea. University of Santiago de Compostela, Pharmacology 15782, Spain

The 5-HT2A receptor has been widely implicated in the physiopathology and treatment of schizophrenia based on different observations (Eastwood SL, (2001) Synapse 42(2):104-14; Meltzer HY., (2003) Prog Neuropsychopharmacol Biol Psychiatry 27(7):1159-72), such as symptoms similar to psychotic states produced by some agonists of these receptors (Geyer MA and Vollenweider FX, (2008) Trends Pharmacol Sci 99(9):445-53; Gonzalez-Maeso J and Sealfon SC, (2009) Trends Neurosci 32(4):225-32). In addition, atypical or second generation antipsychotics have a higher affinity for this receptor than the D2 receptor (Kapur S and Remington G, (2001) Annu Rev Med 52:503-17; Meltzer HY, (1998) J Clin Psychopharmacol 18(2 Suppl1):15). Furthermore, it has recently been described that these receptors are expressed as heterodimers 5-HT2A-D2 (Albizu L, (2011) Neuropharmacology 61(4):770-7).

The 5-HT2A receptor is coupled primarily to Gαq protein and activates several isoforms of phospholipase C that catalyzes the breakdown of PIP2 into IP3 and DAG (Sanders –Bush E, (2004) Neuropsychopharmacology 20(11):2081-7). In addition to this signaling pathway it has been described coupling to either G proteins or other effector systems such as phospholipase A2 (Berg KA, (1998) Ann N Y Acad Sci 15;86:111-20; Gonzalez-Maeso J and Sealfon SC, (2009) Trends Neurosci 32(4):225-32; Leysen JE, (2004) Curr Drug Targets CNS Neurol Disord 3(1):11-26).Previous results from our group have evidenced that those receptors are expressed in human brain as homodimers presenting negative cooperativity in binding and functional assays (Lopez-Gimenez JF, (2001) Mol Pharmacol 60(4):690-9; Brea J, (2009) Mol Pharmacol 75(6):1380-91).

Earlier studies by our research group have shown that Reelin (an extracellular matrix protein) affected the expression of D2 receptors and 5-HT2A receptors in a model of isolated synapses (synaptosomes) of heterozygous reeler mouse. (Varela MJ, XXX Congress Spanish Society of Pharmacology, 2008; XXXI Congress of the Spanish Society of Pharmacology, 2009).
This work was aimed to evaluating the influence of reelin brain level in the 5-HT2A homodimeric negative cooperativity in synaptosomes and their influence on the function of these receptors.

We carried out competition assays with haloperidol, clozapine and risperidone against [3H] LSD in mice synaptosomes membranes from reeler (HZ) and wild type mice (WT). We observed that in HZ both haloperidol and risperidone displayed biphasic competition curves while clozapine has a monophasic competition profile in both genotypes. No significant differences in the Ki values of haloperidol and clozapine were observed but a decrease of risperidone Ki values was observed at HZ reeler mice (Ki high:1.56±0.23 nM, Ki low:555.45±235.65 nM) with respect to WT (Ki high:0.37±0.16, Ki low:1315.25±73.75 nM), (P<0.05, Student´s t test).

We have developed also a functional assay for assessing the production of inositol phosphates in synaptosomes after activation of 5-HT2A by 5-HT (EC50: 114.56±76.58 nM). This way we can evaluate the behavior of different ligands on the signaling pathways of 5-HT2A receptor directly in a model of isolated synapses.

In conclusion we can say that we have observed the influence of reelin in the phenomenon of negative cooperativity in reeler mouse synaptosomes and we have developed a method that will reveal the influence of the levels of reelin in the function signaling of the 5-HT2A .

Grants: This work was supported by Spanish Ministry of Science and Innovation (SAF2009-13609-C04-01).