RESUMO
Addiction is a maladaptive pattern of behavior following repeated use of reinforcing drugs in predisposed individuals, leading to lifelong changes. Common among these changes are alterations of neurons releasing dopamine in the ventral and dorsal territories of the striatum. The serotonin 5-HT2B receptor has been involved in various behaviors, including impulsivity, response to antidepressants, and response to psychostimulants, pointing toward putative interactions with the dopamine system. Despite these findings, it remains unknown whether 5-HT2B receptors directly modulate dopaminergic activity and the possible mechanisms involved. To answer these questions, we investigated the contribution of 5-HT2B receptors to cocaine-dependent behavioral responses. Male mice permanently lacking 5-HT2B receptors, even restricted to dopamine neurons, developed heightened cocaine-induced locomotor responses. Retrograde tracing combined with single-cell mRNA amplification indicated that 5-HT2B receptors are expressed by mesolimbic dopamine neurons. In vivo and ex vivo electrophysiological recordings showed that 5-HT2B-receptor inactivation in dopamine neurons affects their neuronal activity and increases AMPA-mediated over NMDA-mediated excitatory synaptic currents. These changes are associated with lower ventral striatum dopamine activity and blunted cocaine self-administration. These data identify the 5-HT2B receptor as a pharmacological intermediate and provide mechanistic insight into attenuated dopamine tone following exposure to drugs of abuse.SIGNIFICANCE STATEMENT Here we report that mice lacking 5-HT2B receptors totally or exclusively in dopamine neurons exhibit heightened cocaine-induced locomotor responses. Despite the sensitized state of these mice, we found that associated changes include lower ventral striatum dopamine activity and lower cocaine operant self-administration. We described the selective expression of 5-HT2B receptors in a subpopulation of dopamine neurons sending axons to the ventral striatum. Increased bursting in vivo properties of these dopamine neurons and a concomitant increase in AMPA synaptic transmission to ex vivo dopamine neurons were found in mice lacking 5-HT2B receptors. These data support the idea that the chronic 5-HT2B-receptor inhibition makes mice behave like animals already exposed to cocaine with higher cocaine-induced locomotion associated with changes in dopamine neuron reactivity.
Assuntos
Cocaína/administração & dosagem , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Núcleo Accumbens/metabolismo , Receptor 5-HT2B de Serotonina/biossíntese , Transdução de Sinais/fisiologia , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Feminino , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Núcleo Accumbens/efeitos dos fármacos , Projetos Piloto , Distribuição Aleatória , Receptor 5-HT2B de Serotonina/deficiência , Autoadministração , Transdução de Sinais/efeitos dos fármacosRESUMO
Serotonin is known to regulate energy and calcium homeostasis in several mammalian species. The objective of this study was to determine if pre-partum infusions of 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin synthesis, could modulate energy homeostasis at the level of the hepatocyte in post-partum Holstein and Jersey dairy cows. Twelve multiparous Holstein cows and twelve multiparous Jersey cows were intravenously infused daily for approximately 7 d pre-partum with either saline or 1 mg/kg bodyweight of 5-HTP. Blood was collected for 14 d post-partum and on d30 post-partum. Liver biopsies were taken on d1 and d7 post-partum. There were no changes in the circulating concentrations of glucose, insulin, glucagon, non-esterified fatty acids, or urea nitrogen in response to treatment, although there were decreased beta-hydroxybutyrate concentrations with 5-HTP treatment around d6 to d10 post-partum, particularly in Jersey cows. Cows infused with 5-HTP had increased hepatic serotonin content and increased mRNA expression of the serotonin 2B receptor on d1 and d7 post-partum. Minimal changes were seen in the hepatic mRNA expression of various gluconeogenic enzymes. There were no changes in the mRNA expression profile of cell-cycle progression marker cyclin-dependent kinase 4 or apoptotic marker caspase 3, although proliferating cell nuclear antigen expression tended to be increased in Holstein cows infused with 5-HTP on d1 post-partum. Immunofluorescence assays showed an increased number of CASP3- and Ki67-positive cells in Holstein cows infused with 5-HTP on d1 post-partum. Given the elevated hepatic serotonin content and increased mRNA abundance of 5HTR2B, 5-HTP infusions may be stimulating an autocrine-paracrine adaptation to lactation in the Holstein cow liver.
Assuntos
Lactação/sangue , Fígado/metabolismo , Período Pós-Parto/sangue , Serotonina/sangue , Animais , Glicemia/metabolismo , Caspase 3/metabolismo , Bovinos , Quinase 4 Dependente de Ciclina/metabolismo , Ácidos Graxos não Esterificados/sangue , Feminino , Glucagon/sangue , Insulina/sangue , Antígeno Ki-67/metabolismo , Gravidez , Receptor 5-HT2B de Serotonina/biossínteseRESUMO
Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke.
Assuntos
RNA Mensageiro/metabolismo , Receptor 5-HT2B de Serotonina/genética , Acidente Vascular Cerebral/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Depressão/metabolismo , Modelos Animais de Doenças , Feminino , Fluoxetina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Neurogênese/efeitos dos fármacos , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2B de Serotonina/biossíntese , Receptor 5-HT2B de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/psicologia , Regulação para CimaRESUMO
In well-differentiated primary cultures of mouse astrocytes, which express no serotonin transporter (SERT), the 'serotonin-specific reuptake inhibitor' (SSRI) fluoxetine leads acutely to 5-HT2B receptor-mediated, transactivation-dependent phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) with an EC50 of ~5 µM, and chronically to ERK1/2 phosphorylation-dependent upregulation of mRNA and protein expression of calcium-dependent phospholipase A2 (cPLA2) with ten-fold higher affinity. This affinity is high enough that fluoxetine given therapeutically may activate astrocytic 5-HT2B receptors (Li et al., 2008, 2009). We now confirm the expression of 5-HT2B receptors in astrocytes freshly dissociated from mouse brain and isolated by fluorescence-activated cell sorting (FACS) and investigate in cultured cells if the effects of fluoxetine are shared by all five conventional SSRIs with sufficiently high affinity to be relevant for mechanism(s) of action of SSRIs. Phosphorylated and total ERK1/2 and mRNA and protein expression of cPLA2a were determined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Paroxetine, which differs widely from fluoxetine in affinity for SERT and for another 5-HT2 receptor, the 5-HT2C receptor, acted acutely and chronically like fluoxetine. One micromolar of paroxetine, fluvoxamine or sertraline increased cPLA2a expression during chronic treatment; citalopram had a similar effect at 0.1-0.5 µM; these are therapeutically relevant concentrations.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/metabolismo , Receptor 5-HT2B de Serotonina/genética , Receptor 5-HT2B de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Encéfalo/citologia , Células Cultivadas , Citometria de Fluxo/métodos , Fluoxetina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Paroxetina/farmacologia , Receptor 5-HT2B de Serotonina/biossíntese , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
We have previously shown that fluoxetine causes ERK(1/2) phosphorylation in cultured mouse astrocytes mediated exclusively by stimulation of 5-HT(2B) receptors (Li et al., 2008b). This raises the question whether this is also the case for serotonin (5-HT) itself. In the present study serotonin was found to induce ERK(1/2) phosphorylation by stimulation of 5-HT(2B) receptors with high affinity (EC(50): 20-30 pM), and by stimulation of 5-HT(2C) receptor with low affinity (EC(50): 1 microM or higher). ERK(1/2) phosphorylation induced by stimulation of either 5-HT(2B) or 5-HT(2C) receptors was mediated by epidermal growth factor (EGF) receptor transactivation (Peng et al., this issue), shown by the inhibitory effect of AG1478, an inhibitor of the EGF receptor tyrosine kinase, and GM6001, an inhibitor of Zn-dependent metalloproteinases, and thus of 5-HT(2B) receptor-mediated EGF receptor agonist release. It is discussed that the high potency of the 5-HT(2B)-mediated effect is consistent with literature data for binding affinity of serotonin to cloned human 5-HT(2B) receptors and with observations of low extracellular concentrations of serotonin in brain, which would allow a demonstrated moderate and modality-dependent increase in specific brain areas to activate 5-HT(2B) receptors. In contrast the relevance of the observed 5-HT(2C) receptors on astrocytes is questioned.
Assuntos
Astrócitos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Receptor 5-HT2C de Serotonina/efeitos dos fármacos , Serotonina/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Western Blotting , Células Cultivadas , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Receptores ErbB/antagonistas & inibidores , Camundongos , Fosforilação , Inibidores de Proteases/farmacologia , Quinazolinas , RNA Interferente Pequeno/farmacologia , Receptor 5-HT2B de Serotonina/biossíntese , Receptor 5-HT2C de Serotonina/biossíntese , Especificidade por Substrato , Ativação Transcricional/efeitos dos fármacos , Tirfostinas/farmacologiaRESUMO
Activation of the 5-hydroxytryptamine receptor 2B (5-HT(2B)), a G(q/11) protein-coupled receptor, results in proliferation of various cell types. The 5-HT(2B) receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT(2B) receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT(2B) receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by LY294002 decreased base-line proliferation but had no effect on 5-HT(2B) receptor-mediated proliferation. Proliferation of ICC through the 5-HT(2B) receptor was inhibited by the phospholipase C inhibitor U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the alpha, beta, gamma, and micro protein kinase C (PKC) inhibitor Gö6976, and the alpha, beta, gamma, delta, and zeta PKC inhibitor Gö6983 inhibited 5-HT(2B) receptor-mediated proliferation, indicating the involvement of PKC alpha, beta, or gamma. Of all the PKC isoforms blocked by Gö6976, PKCgamma and micro mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT(2B) receptor activation in primary cell cultures obtained from PKCgamma(-/-) mice did not result in a proliferative response, further indicating the requirement for PKCgamma in the proliferative response to 5-HT(2B) receptor activation. The data demonstrate that the 5-HT(2B) receptor-induced proliferative response of ICC is through phospholipase C, [Ca(2+)](i), and PKCgamma, implicating this PKC isoform in the regulation of cellular proliferation.
Assuntos
Proteína Quinase C/metabolismo , Receptor 5-HT2B de Serotonina/biossíntese , Animais , Cromonas/farmacologia , Corpos Enovelados/metabolismo , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Fibroblastos/metabolismo , Indóis/farmacologia , Compostos Macrocíclicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfolinas/farmacologia , Oxazóis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Pirrolidinonas/farmacologia , Tiofenos/farmacologiaRESUMO
Male Wistar rats were exposed to 100ppm Pb(2+) in drinking water for 10 months. Tail blood pressure, serum 5-hydroxytryptamine (5-HT), the expression of 5-HT(2B) receptor in the aorta, the aortic response to 5-HT, and the pathologic changes of aorta were examined. The systolic blood pressure of Pb(2+) exposed group was significantly increased after 2 months of Pb(2+) exposure. After 10 months of Pb(2+) exposure, aortic contractile response to 5-HT was significantly decreased. There was no significant difference in the levels of serum 5-HT and the expression of 5-HT(2B) receptor between these two groups. The aortic media and the media-lumen ratio of Pb(2+) exposed group were significantly increased. These data suggest that long-term Pb(2+) exposure can increase blood pressure, and can alter the function and structure of aortic of rats. The decreased aortic response to 5-HT has little relation to the expression of 5-HT(2B) receptor and the serum level of 5-HT, maybe is a result of the aortic structural alteration.
Assuntos
Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Intoxicação por Chumbo/fisiopatologia , Contração Miocárdica/efeitos dos fármacos , Serotonina/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Chumbo/sangue , Intoxicação por Chumbo/patologia , Masculino , Miocárdio/patologia , Ratos , Ratos Wistar , Receptor 5-HT2B de Serotonina/biossíntese , Serotonina/sangueRESUMO
The type 2 serotonin (5-HT(2)) receptor subfamily is known to couple to phosphoinositide hydrolysis (PI) and the subsequent mobilization of intracellular Ca(2+), as well as the release of arachidonic acid (AA). Less is known of 5-HT(2)-mediated activation of the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK1/2) signaling. The present study measured the relative efficacies and potencies of 5-HT agonists to activate ERK2 in non-neuronal cells expressing recombinant human 5-HT(2A), 5-HT(2B), and 5-HT(2C(ISV)) receptors. 5-HT agonists stimulated ERK2 activity via all three 5-HT(2) subtypes. There were no meaningful differences in the potencies or relative efficacies of these agonists to affect ERK2 activity vs. PI accumulation or Ca(2+) mobilization, suggesting that these pathways may be sequentially linked. Indeed, ERK2 activity was very sensitive to PKC inhibition and calcium chelation and insensitive to tyrosine kinase and PI-3-kinase inhibition. 5-HT(2) receptors efficiently couple to MAPK activation via sequential PI hydrolysis, and Ca(2+) mobilization. This profile differs from reports of "agonist-directed trafficking of receptor stimulus" between PI/Ca(2+) and AA pathways activated by 5-HT(2) receptors.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor 5-HT2A de Serotonina/fisiologia , Receptor 5-HT2B de Serotonina/fisiologia , Receptor 5-HT2C de Serotonina/fisiologia , Agonistas do Receptor de Serotonina/farmacologia , Animais , Western Blotting , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Ativação Enzimática , Humanos , Fosfatidilinositóis/metabolismo , Ligação Proteica , Ensaio Radioligante , Receptor 5-HT2A de Serotonina/biossíntese , Receptor 5-HT2B de Serotonina/biossíntese , Receptor 5-HT2C de Serotonina/biossíntese , Proteínas Recombinantes/biossíntese , Agonistas do Receptor 5-HT2 de SerotoninaRESUMO
Using several 5-hydroxytryptamine (5-HT) agonists and antagonists, we attempted to characterize the receptor subtypes involved in the contractile response to 5-HT in the in situ autoperfused rat kidney. An intra-arterial (i.a.) bolus injection of 5-HT (0.00000125 to 0.1 microg/kg) increased renal perfusion pressure in a dose-dependent way but did not affect the systemic blood pressure. The selective 5-HT2 receptor agonist alpha-methyl-5-HT (alpha-methyl-5-hydroxytryptamine) and the non-selective 5-HT2C receptor agonist (1-(3-chlorophenyl)piperazine), m-CPP, caused a local vasoconstrictor effect in the autoperfused rat kidney, whereas BW723C86, a selective 5-HT2B receptor agonist, the 5-HT1 receptor agonist 5-carboxamidotryptamine, 5-CT, and the selective 5-HT3 receptor agonist m-CPBG (1-(m-chlorophenyl)-biguanide) did not modify the renal perfusion pressure. The vasoconstrictor effect elicited by alpha-methyl-5-HT and m-CPP was significantly decreased by ritanserin (a 5-HT2 receptor antagonist), SB 206553 (3,5-Dihydro-5-methyl-N-3pyridinylbenzo[1,2.b:4,5-b']dipyrrole(1H)-carboxamide hydrochloride), a selective 5-HT2B/2C receptor antagonist and enalapril, but was not modified by pretreatment with spiperone (a 5-HT2A receptor antagonist). The results of protein expression analyses allow us to postulate that 5HT-SRC (a 5-HT2C receptor protein) is expressed in renal tissue and differentially expressed in renal artery. Our data suggest also that the serotonergic vasoconstrictor response induced in the in situ autoperfused rat kidney would be mediated by local 5-HT2C receptor activation.
Assuntos
Anestesia , Rim/metabolismo , Receptores de Serotonina/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Relação Dose-Resposta a Droga , Técnicas In Vitro , Rim/efeitos dos fármacos , Masculino , Contração Muscular/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor 5-HT2A de Serotonina/biossíntese , Receptor 5-HT2B de Serotonina/biossíntese , Receptor 5-HT2C de Serotonina/biossíntese , Receptores de Serotonina/biossíntese , Artéria Renal/efeitos dos fármacos , Artéria Renal/metabolismo , Circulação Renal/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Vasoconstritores/farmacologiaRESUMO
Several drugs have been linked to valvulopathy in humans, including therapeutic agents for obesity, Parkinson's disease and migraine. There is increasing evidence that the 5-hydroxytryptamine 2B receptor (5HT2BR) activation and/or increased circulating 5HT (5-hydroxytryptamine) may play a significant role in the pathogenesis of drug-induced valvulopathy. In the present study, we investigated whether 7-day 5HT subcutaneous injections led to structural and compositional abnormalities in conjunction with transcriptomic modulation of 5HT2BR and 5HT transporter (5HTT) genes in the aortic and mitral valves of Sprague-Dawley (SD) rats. Subcutaneous injections of 5HT for 7 days resulted in thickening and compositional alteration of aortic and mitral valves in SD rats. More specifically, valve-leaflets from 5HT-treated rats had greater valve thickness, a higher amount of glycosaminoglycans (GAGs) and a lower amount of collagen. The compositional alteration was associated with up-regulation and down-regulation of 5HT2BR and 5HTT genes, respectively. The present study strongly suggests that the activation of 5HT2BR and inhibition of 5HTT played a significant role in the pathogenesis of 5HT-induced valvulopathy in SD rats. Thus, these findings further highlight the necessity and/or utilization of animal models to screen potential valvular effects of serotonergic compounds.
Assuntos
Doenças das Valvas Cardíacas/induzido quimicamente , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Serotoninérgicos/toxicidade , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Serotonina/toxicidade , Animais , Valva Aórtica/química , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/patologia , Colágeno/análise , Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/análise , Doenças das Valvas Cardíacas/patologia , Lasers , Microdissecção , Valva Mitral/química , Valva Mitral/efeitos dos fármacos , Valva Mitral/patologia , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Receptor 5-HT2B de Serotonina/biossíntese , Proteínas da Membrana Plasmática de Transporte de Serotonina/biossínteseRESUMO
Although several studies demonstrated that lead induced abnormal vascular responses in low level lead exposed animals, investigations of the direct effects of lead on blood vessels are limited. In this study we tested the hypothesis that lead was able to directly affect the contractile reactivities of vessels. Male Wistar rat aortae were removed and cultured in PMRI 1640 with 1 ppm lead acetate for 0.5, 6, 12, 24 and 48 h, and then their responses to norepinephrine bitartrate (NE) and serotonin (5-hydroxytryptamine, 5-HT) were examined. The contractile responses to 5-HT of lead exposed aortae were significantly increased when the aortae were cultured for 24 and 48 h. Denudation of endothelium was able to abolish the increased contractile response completely. Diphenyleneiodonium (DPI), an inhibitor of the NAD(P)H oxidase, could abolish the increased contractile response to 5-HT. However, Vitamin C (VC) enhanced the contractile response of both groups to higher dosages of 5-HT. The expression of 5-HT(2B) receptor was not significantly altered by incubation with 1 ppm lead for 24 h. These data suggest that exposure to low levels of lead can directly increase the contraction of aorta to 5-HT. This effect is endothelium dependent, which is not mediated by increased expression of the 5-HT 2B receptor. The increased contraction to 5-HT may be related to increased production of superoxide (O2*-) induced by lead exposure.
Assuntos
Chumbo/toxicidade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Serotonina/farmacologia , Animais , Antioxidantes/farmacologia , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Western Blotting , Células Cultivadas , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Masculino , Contração Muscular/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Norepinefrina/farmacologia , Oniocompostos/farmacologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptor 5-HT2B de Serotonina/biossíntese , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacos , Vasoconstritores/farmacologiaRESUMO
OBJECTIVES: Overexpression of receptors to neuroendocrine (NE) cell products has been suggested to contribute to development of hormone-refractory prostate cancer (HRPC). In this study, we evaluated the expression of 5-HTR2B and 5-HTR4 in HRPC, and the effects of their antagonist on PC cell line growth. METHODS: Proteins and mRNA expression was determined by immunohistochemistry, western blot and RT-PCR. Growth inhibition of PC cell lines was determined in vitro using ELISA-BrdU proliferation assay and cell cycle was evaluated by flow cytometry. RESULTS: Immunostaining of 5-HTR2B was observed in low-grade and high-grade tumours, PIN and BPH cells, and in vascular endothelial cells, whereas 5-HTR4 was found predominantly in high-grade tumours. This result was confirmed by western blot analysis. At the mRNA level, 5-HTR4 mRNA was expressed in DU145 and LNCaP cells. Antagonists to both receptor subtypes inhibited proliferation of PC cells in a dose-dependent manner. CONCLUSIONS: The present result indicate that 5-HTRs are present at various tumour stages and that antagonists to these receptors can inhibit the proliferative activity of androgen-independent PC cell lines.
Assuntos
Neoplasias da Próstata , Receptor 5-HT2B de Serotonina/biossíntese , Receptores 5-HT4 de Serotonina/biossíntese , Antagonistas da Serotonina/farmacologia , Biomarcadores Tumorais , Western Blotting , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor 5-HT2B de Serotonina/genética , Receptores 5-HT4 de Serotonina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antagonistas do Receptor 5-HT2 de Serotonina , Antagonistas do Receptor 5-HT4 de SerotoninaRESUMO
We report the cloning, molecular characterization, and pharmacological characterization of the canine 5-HT2A and 5-HT2B receptors. The canine and human 5-HT2A receptors share 93% amino acid homology. The canine and human 5-HT2B receptors are also highly conserved (87% homology) with the exception of the carboxyl termini where the canine protein is 62 amino acids shorter. Both the canine 5-HT2A and 5-HT2B receptors have high affinity for [3H]5-HT (KD=4.50+/-0.89 nM and 3.10+/-0.82 nM, respectively) and, in general, the pharmacology of these two receptors matches closely the pharmacology of their human homologs for the 19 serotonergic ligands tested. However, the functional response (Ca2+ mobilization) of the canine 5-HT2B receptor to several agonists was weaker compared to the human 5-HT2B receptor. Using quantitative reverse transcriptase polymerase chain reaction, a high expression level of canine 5-HT2A receptor mRNA was detected in the brain and lower levels in peripheral tissues, whereas the highest levels of canine 5-HT2B receptor mRNA were observed in lungs and smooth muscles. A significant level of canine 5-HT2B receptor mRNA was detected in brain tissue. The availability of the full sequence and pharmacology of the canine 5-HT2A and canine 5-HT2B receptors provides useful information for the interpretation of previous and future pharmacological studies of 5-HT2A/2B ligands in dog.
Assuntos
Receptor 5-HT2A de Serotonina/efeitos dos fármacos , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Receptor 5-HT2B de Serotonina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Cães , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/biossíntese , Ensaio Radioligante , Receptor 5-HT2A de Serotonina/biossíntese , Receptor 5-HT2B de Serotonina/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , TransfecçãoRESUMO
BACKGROUND: 5-hydroxytryptamine (5-HT)2B and 5-HT1B receptors are upregulated in arteries from hypertensive DOCA-salt rats and directly by mineralocorticoids. We hypothesized that increased 5-HT2B and 5-HT1B receptor density and contractile function would precede increased blood pressure in DOCA-high salt rats. We performed DOCA-salt time course (days 1, 3, 5 and 7) studies using treatment groups of: DOCA-high salt, DOCA-low salt, Sham and Sham-high salt rats. RESULTS: In isolated-tissue baths, DOCA-high salt aorta contracted to the 5-HT2B receptor agonist BW723C86 on day 1; Sham aorta did not contract. The 5-HT1B receptor agonist CP93129 had no effect in arteries from any group. On days 3, 5 and 7 CP93129 and BW723C86 contracted DOCA-high salt and Sham-high salt aorta; Sham and DOCA-low salt aorta did not respond. Western analysis of DOCA-high salt aortic homogenates revealed increased 5-HT2B receptor levels by day 3; 5-HT1B receptor density was unchanged. Aortic homogenates from the other groups showed unchanged 5-HT2B and 5-HT1B receptor levels. CONCLUSION: These data suggest that functional changes of 5-HT2B but not 5-HT1B receptors may play a role in the development of DOCA-salt hypertension.