RESUMO
Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was â¼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was â¼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (â¼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.
Assuntos
Comportamento Animal/efeitos dos fármacos , Técnicas de Introdução de Genes , Isoquinolinas/farmacologia , Locomoção/efeitos dos fármacos , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Taquifilaxia , Adamantano/análogos & derivados , Adamantano/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Benzopiranos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Isoquinolinas/efeitos adversos , Masculino , Camundongos , Transporte Proteico/efeitos dos fármacos , Receptores de Dopamina D1/agonistasRESUMO
Some recently published in vitro studies with two metabotropic glutamate 2/3 receptor (mGluR(2/3)) agonists [(-)-2-oxa-4-aminobicyclo[3.1.0] hexane-4,6-dicarboxylic acid (LY379268) and 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate (LY354740)] suggest that these compounds may also directly interact with dopamine (DA) D(2) receptors. The current in vitro and in vivo studies were undertaken to further explore this potential interaction with D(2) receptors. LY379268 and LY354740 failed to inhibit D(2) binding in both native striatal tissue homogenates and cloned receptors at concentrations up to 10 microM. LY379268 and LY354740 (up to 10 microM) also failed to stimulate [(35)S]GTPgammaS binding in D(2L)- and D(2S)-expressing clones in the presence of NaCl or N-methyl-d-glucamine. In an in vivo striatal D(2) receptor occupancy assay, LY379268 (3-30 mg/kg) or LY354740 (1-10 mg/kg) failed to displace raclopride (3 microg/kg i.v.), whereas aripiprazole (10-60 mg/kg) showed up to 90% striatal D(2) receptor occupancy. LY379268 (10 mg/kg) and raclopride (3 mg/kg) blocked d-amphetamine and phencyclidine (PCP)-induced hyperactivity in wild-type mice. However, the effects of LY379268 were lost in mGlu(2/3) receptor knockout mice. In DA D(2) receptor-deficient mice, LY379268 but not raclopride blocked both PCP and d-amphetamine-evoked hyperactivity. In the striatum and nucleus accumbens, LY379268 (3 and 10 mg/kg) was without effect on the DA synthesis rate in reserpinized rats and also failed to prevent S-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine-induced reductions in DA synthesis rate. Taken together, the current data fail to show evidence of direct DA D(2) receptor interactions of LY379268 and LY354740 in vitro or in vivo. Instead, these results provide further evidence for a novel antipsychotic mechanism of action for mGluR(2/3) agonists.
Assuntos
Aminoácidos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Receptores de Dopamina D2/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Animais , Ligação Competitiva , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Domperidona/farmacologia , Dopamina/biossíntese , Antagonistas dos Receptores de Dopamina D2 , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Ligação Proteica , Racloprida/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/genética , TransfecçãoRESUMO
[(3)H]LY334370 was developed as a radioligand to study the characteristics of this compound's interaction with the 5-HT(1F) receptor. Monovalent or divalent cations did not enhance the binding of [(3)H]LY334370 to the cloned human 5-HT(1F) receptor. In the presence of MgCl(2), the time to reach equilibrium was approximately 2 h, while in its absence equilibrium was reached in less than 1 h. [(3)H]LY334370 had high affinity for the cloned human 5-HT(1F) receptor (K(d)=0.446 nM) and the 5-HT(1F) receptor in rat brain (K(d)=0.388 nM). The expression density of 5-HT(1F) receptors, as determined by binding to homogenates of cortical regions from rat, was low (B(max)=79.1 fmol/mg protein). There was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding and the pEC(50) for stimulation of [(35)S]GTPgammaS binding to homogenates of cells expressing the cloned human 5-HT(1F) receptor. In addition, there was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding to the cloned human 5-HT(1F) receptor and the pID(50) for inhibition of trigeminal nerve stimulated dural plasma protein extravasation in the guinea pig. The conclusion from these studies is that [(3)H]LY334370 is a high affinity radioligand which can be used for the study of the 5-HT(1F) receptor in rat brain or in cells transformed with the human 5-HT(1F) receptor.
Assuntos
Benzamidas/farmacologia , Indóis/farmacologia , Receptores de Serotonina/análise , Agonistas do Receptor de Serotonina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Cobaias , Humanos , Técnicas In Vitro , Ligantes , Ensaio Radioligante , Ratos , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/genética , Transfecção , Trítio , Receptor 5-HT1F de SerotoninaRESUMO
LY334370 is a high affinity, selective agonist at the 5-HT(1F) receptor. On this basis, the tritiated compound was examined for its utility in autoradiography to localize the 5-HT(1F) receptor in rat and guinea pig brain regions. Specific 5-HT(1F) receptor binding in rat brain was found in layers 4-5 of all cortical regions examined, as well as olfactory bulb and tubercle, nucleus accumbens, caudate putamen, parafascicular nucleus of the thalamus, medial mammillary nucleus, the CA3 region of the hippocampus, subiculum, and several amygdaloid nuclei. In guinea pig brain, the [(3)H]LY334370 binding sites were found at highest density in claustrum, but also in a layer of the cortex, caudate putamen, nucleus accumbens, thalamus, and medial mammillary nucleus. Some species differences in the distribution of the 5-HT(1F) receptor were noted. Side by side comparison of rat brain autoradiography with [(3)H]LY334370 and [(3)H]sumatriptan showed labeling in the same brain regions. Preliminary binding studies in rhesus monkey and human brain sections showed [(3)H]LY334370 binding in cortical layers 4-5, subiculum (in the monkey), and the granule cell layer of the cerebellum. These findings suggest a discrete localization of the 5-HT(1F) receptor in the rat, guinea pig, monkey and human brain, and confirms the utility of [(3)H]LY334370 as a potential tool to explore further the localization and possible functions of the 5-HT(1F) receptor.