Functional study of rat 5-HT2A receptors using antisense oligonucleotides.

We studied the effects in rats of a 6-day intracerebroventricular (i.c.v) infusion of four different end-capped phosphorothioate-modified antisense oligonucleotides (AOs), specifically targeting different regions of the 5-hydroxytryptamine2A (5-HT2A) receptor mRNA, on central 5-HT2A receptor expression and 5-HT2A receptor-mediated behaviours. Only one of the AOs (sequence 4), directed against the 5'-untranslated region (from + 557 to + 577), specifically affected central 5-HT2A receptor expression and receptor-mediated behaviour. This AO (sequence 4) reduced binding of the 5-HT2A agonist 1-(2,5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane ([125I]DOI) up to 25% in cortical areas, as measured by quantitative autoradiography. Cortical binding of the antagonist [3H]ketanserin was not affected. As the specific AO treatment presumably affects the synthesis of new receptor, we hypothesize that this newly synthesized receptor represents the major part of the functionally active, G protein coupled receptor. A 5-day infusion of AO (sequence 4) resulted in profound inhibition of the head-twitch response (HTR) to 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast, treatment with vehicle, sense oligonucleotides (SOs) and other AOs (sequences 1, 2 and 3) caused an increased DOM-induced HTR as well as a spontaneous HTR. The latter was abolished by treatment with the 5-HT2 receptor antagonist, ritanserin. Systematic investigation of the surgical and infusion procedures revealed that the enhanced HTR already appeared following drilling of the skull. This wounding can probably damage the blood-brain barrier and cause a stress-induced increase in serotonergic transmission. AO (sequence 4) treatment also abolished the spontaneous HTR. AO (sequence 4) treatment allowed the identification of specific central 5-HT2A receptor-mediated behaviours in the complex serotonergic syndrome induced by tryptamine in rats. Only bilateral convulsions and body tremors were significantly inhibited. The backward locomotion, hunched back and Straub tail were not affected, nor was cyanosis, an index of vasoconstriction induced by peripheral 5-HT2A receptor activation. Labelling of central 5-HT2C receptors by [3H]mesulergine, and 5-HT2C receptor-mediated anxiety were not attenuated by AO or SO treatment. Rats treated with AO (sequence 4) showed increased locomotor activity and a strong reactivity towards touching. We hypothesize that the down-regulation of functional 5-HT2A receptors may shift the balance between various 5-HT receptor subtypes. Our analysis of the behavioural consequences of AO treatment and the use of different AOs and SOs has shown that specific receptor-mediated behaviour can be identified.

Ten years of antisense inhibition of brain G-protein-coupled receptor function.

Antisense oligonucleotides (AOs) are widely used as tools for inhibiting gene expression in the mammalian central nervous system. Successful gene suppression has been reported for different targets such as neurotransmitter receptors, neuropeptides, ion channels, trophic factors, cytokines, transporters, and others. This illustrates their potential for studying the expression and function of a wide range of proteins. AOs may even find therapeutic applications and provide an attractive strategy for intervention in diseases of the central nervous system (CNS). However, a lack of effectiveness and/or specificity could be a major drawback for research or clinical applications. Here we provide a critical overview of the literature from the past decade on AOs for the study of G-protein-coupled receptors (GPCRs). The following aspects will be considered: mechanisms by which AOs exert their effects, types of animal model system used, detection of antisense action, effects of AO design and delivery characteristics, non-antisense effects and toxicological properties, controls used in antisense studies to assess specificity, and our results (failures and successes). Although the start codon of the mRNA is the most popular region (46%) to target by AOs, targeting the coding region of GPCRs is almost as common (41%). Moreover, AOs directed to the coding region of the GPCR mRNA induce the highest reductions in receptor levels. To resist degradation by nucleases, the modified phosphorothioate AO (S-AO) is the most widely used and effective oligonucleotide. However, the end-capped phosphorothioate AOs (ECS-AOs) are increasingly used due to possible toxic and non-specific effects of the S-AO. Other parameters affecting the activity of a GPCR-targeting AO are the length (mostly an 18-, 20- or 21-mer) and the GC-content (mostly varying from 30 to 80%). Interestingly, one-third of the AOs successfully targeting GPCRs possess a GC/AT ratio of 61-70%. AO-induced reductions in GPCR expression levels and function range typically from 21 to 40% and 41 to 50%, respectively. In contrast to many antisense reviews, we therefore conclude that the functional activity of a GPCR after AO treatment correlates mostly with the density of the target receptors (maximum factor 2). However, AOs are no simple tools for experimental use in vivo. Despite successful results in GPCR research, no general guidelines exist for designing a GPCR-targeting AO or, in general, for setting up a GPCR antisense experiment. It seems that the correct choice of a GPCR targeting AO can only be ascertained empirically. This disadvantage of antisense approaches results mostly from incomplete knowledge about the internalisation and mechanism of action of AOs. Together with non-specific effects of AOs and the difficulties of assessing target specificity, this makes the use of AOs a complex approach from which conclusions must be drawn with caution. Further antisense research has to be carried out to ensure the adequate use of AOs for studying GPCR function and to develop antisense as a valuable therapeutic modality.

5-HT2A and 5-HT2C receptors and their atypical regulation properties.

The 5-HT(2A) and 5-HT(2C) receptors belong to the G-protein-coupled receptor (GPCR) superfamily. GPCRs transduce extracellular signals to the interior of cells through their interaction with G-proteins. The 5-HT(2A) and 5-HT(2C) receptors mediate effects of a large variety of compounds affecting depression, schizophrenia, anxiety, hallucinations, dysthymia, sleep patterns, feeding behaviour and neuro-endocrine functions. Binding of such compounds to either 5-HT(2) receptor subtype induces processes that regulate receptor sensitivity. In contrast to most other receptors, chronic blockade of 5-HT(2A) and 5-HT(2C) receptors leads not to an up- but to a (paradoxical) down-regulation. This review deals with published data involving such non-classical regulation of 5-HT(2A) and 5-HT(2C) receptors obtained from in vivo and in vitro studies. The underlying regulatory processes of the agonist-induced regulation of 5-HT(2A) and 5-HT(2C) receptors, commonly thought to be desensitisation and resensitisation, are discussed. The atypical down-regulation of both 5-HT(2) receptor subtypes by antidepressants, antipsychotics and 5-HT(2) antagonists is reviewed. The possible mechanisms of this paradoxical down-regulation are discussed, and a new hypothesis on possible heterologous regulation of 5-HT(2A) receptors is proposed.