Metodologías para medir las emisiones de metano en rumiantes

Initially, the human 5 -H T ia receptor sequence published by Kolbilka etal.^^^ was described as a single protein containing 421 amino acids. However Chanda et a i V ^ later published a 5 -H T ia receptor sequence in which the amino acids in the fourth transmembrane region slightly differed from the earlier sequence. Additionally the gene was found to encode a protein consisting of 422 amino acids, as described earlier for the rat 5 -H T ia re c e p to r .F u r th e r m o r e , conversion of the 422 amino acid clone to the previously published sequence by site-directed mutagenesis, was found to abolish ligand binding to the receptor, suggesting that the correct sequence is that described by Chanda et al. The protein chain possesses seven clusters of approximately 25 hydrophobic residues forming the membrane spanning domains which are characteristic for G protein-coupled receptors (Figure 1.4).

Four hydrophilic segments of varying lengths are thought to face outside the cell membrane and may be involved in axonal transport of the receptor. Another four segments are thought to face the intracellular space, and are involved in signal transduction. The short C-terminal tail has been

Introduction

shown to be involved in the coupling of the receptor to G proteins. In common with all receptors coupled to adenylyl cyclase, the 5 -H T ia receptor possesses a long hydrophilic third intracellular loop of approximately 125 amino acids. Within the transmembrane domains, the sequence homology with the human p2-adrenergic receptor is 43%. In transmembrane segments 3 and 7, there also exists a high homology with dopaminergic D2 receptors. This may explain the high affinity of certain p-adrenergic and dopaminergic receptor agents for the 5 -H T ia receptor.

Extracellular

Intracellular

COOH

Figure 1.4

Site directed mutagenesis experiments performed on the human 5 -H T ia receptor reveal that the Asn386 residue in the seventh transmembrane domain is critically involved in the high affinity binding interaction between the receptor and the class of ligands which are the focus of this study, i.e. the aryloxyalkylamine p-adrenergic receptor antagonists such as pindolol.^ However, interactions with this amino acid residue does not appear to occur in the binding of other classes of ligands. In contrast, Asn396, Ser393 and also Asp82 in the second transmembrane domain are necessary to the specific binding of the 5 -H T ia selective radioligand, p H ]8-OH-DPAT, to the receptor 112

1.6.4 5-HTiA Receptor Ligands

There are several classes of ligands that bind with high affinity to 5 -H T ia receptors, including aminotetralins, arylpiperazines, indolealkylamines and aryloxypropanolamines (discussed in Section

1.7).

Introduction

Interactions of ligands with the 5 -H Ti a receptor have implicated the involvement of these receptors in a wide range of clinical effects, including anxiety, depression, aggression, motor control, feeding behaviour, memory, circadian rhythm regulation and nociception.^

1.6.4.1 Am inotetralins

The most potent and selective 5 -H T ia agonist is the aminotetralin, 8-OH-DPAT 49, which binds to the receptor with reported Kj values of 1-5 nM. In its tritiated form, this compound has been extensively employed as a radioligand to label 5 - H T ia sites. The majority of 8-OH-DPAT analogues are agonists or partial agonists at the 5 - H T ia receptor, although some possess moderate affinity for dopamine D2 r e c e p t o r s .8-OH-DPAT binds stereoselectively to the 5-

HT-iA receptor, with the R enantiomer being twice as p o t e n t.In te r e s tin g ly , the S enantiomer of the 5-fluoro-substituted analogue of 8-O H-DPAT (UH 301)) 50, was the first compound reported to behave as an antagonist at both pre- and postsynaptic 5 -H T ia receptors (Kj = 52 nM), whereas its R enantiomer is a partial agonist (Kj = 6 nM).^^^’^^® However, the compound shows only moderate (8-fold) selectivity for dopamine D2 sites. Recently, novel derivatives of UH 301,

such as 51 (Kj = 13.9 nM) have been shown to exhibit 5 -H T ia antagonist properties.

49 NPr2 O H ,NPr2 F 50 M F 51

Aporphines, which possess the 2-aminotetralin moiety, and are typically associated with dopaminergic activity have been shown to display high affinities to 5 -H T ia s i t e s . F o r example, R(-)10-methyl-11 -hydroxyaporphine (MHA) 52 is a potent 5 -H T ia receptor agonist,^ whereas its S enantiomer is a potent 5 -H T ia receptor antagonist.

Introduction

O H

H I C H3

52

The tetrahydro-3H-benz[e]indole 53, an analogue of 8-OH-DPAT, binds with high affinity to 5- H T ia sites and shows equal affinity for dopamine D2 r e c e p t o r s T h e introduction of a formyl group in the indole ring, i.e., compound 54, improves the 5 -H T ia selectivity to over 2000- fold.^^^ Recent work has identified an analogue of this compound (55) as an equipotent 5 -H T ia ligand with 200-fold selectivity for this receptor compared with the dopamine D2 receptor.^ However, the compounds are conformationally constrained indolealkylamine analogues and in common with other indolealkylamines, they display higher affinities for 5 -H T id receptors.

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