RESUMO
Opioid receptor antagonist naltrexone reduces alcohol consumption and relapse in both humans and rodents. This study investigated whether hypothalamic proopiomelanocortin (POMC) neurons (producing beta-endorphin and melanocortins) play a role in alcohol drinking behaviors. Both male and female mice with targeted deletion of two neuronal Pomc enhancers nPE1 and nPE2 (nPE-/-), resulting in hypothalamic-specific POMC deficiency, were studied in short-access (4-h/day) drinking-in-the-dark (DID, alcohol in one bottle, intermittent access (IA, 24-h cycles of alcohol access every other day, alcohol vs. water in a two-bottle choice) and alcohol deprivation effect (ADE) models. Wild-type nPE+/+ exposed to 1-week DID rapidly established stable alcohol drinking behavior with more intake in females, whereas nPE-/- mice of both sexes had less intake and less preference. Although nPE-/- showed less saccharin intake and preference than nPE+/+, there was no genotype difference in sucrose intake or preference in the DID paradigm. After 3-week IA, nPE+/+ gradually escalated to high alcohol intake and preference, with more intake in females, whereas nPE-/- showed less escalation. Pharmacological blockade of mu-opioid receptors with naltrexone reduced intake in nPE+/+ in a dose-dependent manner, but had blunted effects in nPE-/- of both sexes. When alcohol was presented again after 1-week abstinence from IA, nPE+/+ of both sexes displayed significant increases in alcohol intake (ADE or relapse-like drinking), with more pronounced ADE in females, whereas nPE-/- did not show ADE in either sex. Our results suggest that neuronal POMC is involved in modulation of alcohol 'binge' drinking, escalation and 'relapse', probably via hypothalamic-mediated mechanisms, with sex differences.
Assuntos
Insuficiência Adrenal/metabolismo , Consumo de Bebidas Alcoólicas/metabolismo , Obesidade/metabolismo , Pró-Opiomelanocortina/deficiência , Insuficiência Adrenal/genética , Consumo de Bebidas Alcoólicas/tratamento farmacológico , Consumo de Bebidas Alcoólicas/genética , Animais , Etanol/farmacologia , Feminino , Genótipo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Naltrexona/farmacologia , Obesidade/genética , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismoRESUMO
The association between poverty, malnutrition, illness and poor socioeconomic conditions on the one side, and poor growth and short adult stature on the other side, is well recognized. Yet, the simple assumption by implication that poor growth and short stature result from poor living conditions, should be questioned. Recent evidence on the impact of the social network on adolescent growth and adult height further challenges the traditional concept of growth being a mirror of health. Twenty-nine scientists met at Glücksburg castle, Northern Germany, November 15th - 17th 2013, to discuss genetic, endocrine, mathematical and psychological aspects and related issues, of child and adolescent growth and final height.
Assuntos
Comportamento do Adolescente/psicologia , Desenvolvimento do Adolescente/fisiologia , Estatura/genética , Hormônios/fisiologia , Adolescente , Peso Corporal , Criança , Feminino , Alemanha , Nível de Saúde , Hormônio do Crescimento Humano/fisiologia , Humanos , Hipotálamo , Masculino , Desnutrição , Estado Nutricional , Grupo Associado , Apoio Social , Fatores SocioeconômicosRESUMO
The microbial pattern-recognizing Toll-like receptors (TLRs) are major signal transducers known to shape and influence the postnatal maturation of host intestinal epithelium. Perturbations in this intricate host-microbe cross-talk have been reported to be associated with uncontrolled epithelial cell growth and thus potential cancer development by mechanisms which are largely unknown. We therefore generated transgenic mice carrying a constitutively active TLR4 (CD4-TLR4) linked to an intestinal epithelial cell-specific promoter. Ex vivo analysis of transgenic crypt-villus organoid cultures revealed an increased proliferative capacity and a lowered cyclooxygenase 2 (Cox-2) expression in these organoids compared with wild-type control cultures. Introducing the CD4-TLR4 transgene into APC(Min/+) mice (CD4-TLR4-APC(Min/+)), a model of colorectal carcinoma, resulted in a dramatic drop in tumor load as compared with control APC(Min/+) mice. Intestinal tumors from CD4-TLR4-APC(Min/+) mice displayed reduced Cox-2 protein, elevated interferon ß expression and increased caspase-3 activity, which correlated with increased apoptosis in vivo. Thus, our data reveal that host microbiota-mediated signal transduction via TLR4 in intestinal epithelial cells is far more complex than what is previously reported.
Assuntos
Apoptose , Mucosa Intestinal/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Caspase 3/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2/metabolismo , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Interferon beta/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Organoides/citologia , Organoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 4 Toll-Like/genética , Carga Tumoral/genéticaRESUMO
Evidence that lipocalin 2 (LCN2) is oncogenic has grown in recent years and comes from both animal models and expression analysis from a variety of human cancers. In the intestine, LCN2 is overexpressed in colitis patients and its overexpression is a negative prognostic indicator in colorectal cancer. Functionally, LCN2 has a number of different activities that may contribute to its oncogenic potential, including increasing matrix metalloproteinase activity, control of iron availability and stimulating inflammation. In this report, we examined APCmin intestinal tumorigenesis in an LCN2-deficient background. We found that the loss of LCN2 increased tumor multiplicity specifically in the duodenum, suggesting a potential tumor-suppressive activity. Concurrently, however, LCN2 increased the average small intestinal tumor size particularly in the distal small intestine. We found that this increase was correlated to tumor iron(II) content, suggesting that an iron-scavenging role is important for LCN2 oncogenic activity in the intestine.
Assuntos
Proteínas de Fase Aguda/biossíntese , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Lipocalinas/biossíntese , Proteínas Oncogênicas/biossíntese , Proteínas de Fase Aguda/deficiência , Proteínas de Fase Aguda/genética , Animais , Apoptose/fisiologia , Progressão da Doença , Feminino , Genes APC , Neoplasias Intestinais/genética , Lipocalina-2 , Lipocalinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genéticaRESUMO
In 5-month-old male and female dopamine receptor 2 (D2R) knockout mice food intake per animal was unaltered while food per g BW was increased. We wished to evaluate the effect of D2R disruption on different components of energy balance and food intake regulation. We determined hypothalamic orexin precursor (PPO) expression, its receptor OX1, serum leptin levels, hypothalamic leptin receptor (OBR), circulating and pituitary alpha MSH levels, as well as central MC3 and MC4 receptors and NPY mRNA in wildtype and D2R knockout mice (KO). Loss of D2R caused a marked increase in serum prolactin levels, to higher levels in females compared to male KO mice. On the other hand, it produced a female-specific increase in circulating alphaMSH, and hypothalamic alphaMSH content, while neurointermediate alphaMSH content was decreased in both sexes. No differences were found in hypothalamic NPY, MC3R or MC4R concentration. Hypothalamic PPO mRNA expression was significantly decreased only in female KOs, while OX1 mRNA was not different between genotypes. Serum leptin levels were also similar in both genotypes. Our results show that in female and not in male mice disruption of the D2R produces two potentially anorexigenic events: an increase in serum and hypothalamic alphaMSH, and a decrease in hypothalamic orexin expression. Very high prolactin levels, which are orexigenic, probably counterbalance these effects, so that food intake is slightly altered. In males, on the other hand, hypothalamic PPO, and serum or hypothalamic alphaMSH are not modified, and increased prolactin levels may account for increased food intake per g BW. These results suggest a sexually dimorphic participation of the D2R in food intake regulation.
Assuntos
Hipotálamo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neuropeptídeo Y/metabolismo , Neuropeptídeos/metabolismo , Receptor Tipo 3 de Melanocortina/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismo , Receptores de Dopamina D2 , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , alfa-MSH/metabolismo , Animais , Ingestão de Alimentos , Metabolismo Energético , Feminino , Masculino , Camundongos , Camundongos Knockout , Receptores de Orexina , Orexinas , Precursores de Proteínas/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismoRESUMO
Behavioral sensitization to psychostimulants manifests as an increased locomotor response with repeated administration. Dopamine systems are accepted to play a fundamental role in sensitization, but the role of specific dopamine receptor subtypes has not been completely defined. This study used the combination of dopamine D2 receptor-deficient mice and a D1-like antagonist to examine dopamine D1 and D2 receptor involvement in acute and sensitized locomotor responses to methamphetamine. Absence of the dopamine D2 receptor resulted in attenuation of the acute stimulant effects of methamphetamine. Mutant and wild-type mice exhibited sensitization that lasted longer within the time period of the challenge test in the mutant animals. Pretreatment with the D1-like receptor antagonist SCH 23390 produced more potent reductions in the acute and sensitized locomotor responses to methamphetamine in D2 receptor-deficient mice than in wild-type mice; however, the expression of locomotor sensitization when challenged with methamphetamine alone was equivalently attenuated by previous treatment with SCH 23390. These data suggest that dopamine D2 receptors play a key role in the acute stimulant and sensitizing effects of methamphetamine and act in concert with D1-like receptors to influence the acquisition of methamphetamine-induced behavioral sensitization, traits that may influence continued methamphetamine use.
Assuntos
Transtornos Relacionados ao Uso de Anfetaminas/genética , Estimulantes do Sistema Nervoso Central/farmacologia , Hipercinese/genética , Metanfetamina/farmacologia , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Animais , Benzazepinas/farmacologia , Antagonistas de Dopamina/farmacologia , Feminino , Hipercinese/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D2/metabolismoRESUMO
Phosphorylation of specific sites in the second intracellular loop and in the C-terminal domain have previously been suggested to cause desensitization and internalization of the mu-opioid receptor (MOP-R). To assess sites of MOP-R phosphorylation in vivo, affinity-purified, phosphoselective antibodies were raised against either phosphothreonine-180 in the second intracellular loop (MOR-P1) or the C-terminal domain of MOP-R containing phosphothreonine-370 and phosphoserine-375 (MOR-P2). We found that MOR-P2-immunoreactivity (IR) was significantly increased within the striatum of wild-type C57BL/6 mice after injection of the agonist fentanyl. Pretreatment with the antagonist naloxone blocked the fentanyl-induced increase. Furthermore, mutant mice lacking MOP-R showed only non-specific nuclear MOR-P2-IR before or after fentanyl treatment, confirming the specificity of the MOR-P2 antibodies. To assess whether MOP-R phosphorylation occurs following endogenous opioid release, we induced chronic neuropathic pain by partial sciatic nerve ligation (pSNL), which caused a significant increase in MOR-P2-IR in the striatum. pSNL also induced signs of mu opioid receptor tolerance demonstrated by a rightward shift in the morphine dose response in the tail withdrawal assay and by a reduction in morphine conditioned place preference (CPP). Mutant mice selectively lacking all forms of the beta-endorphin peptides derived from the proopiomelanocortin (Pomc) gene did not show increased MOR-P2-IR, decreased morphine antinociception, or reduced morphine CPP following pSNL. In contrast gene deletion of either proenkephalin or prodynorphin opioids did not block the effects of pSNL. These results suggest that neuropathic pain caused by pSNL in wild-type mice activates the release of the endogenous opioid beta-endorphin, which subsequently induces MOP-R phosphorylation and opiate tolerance.
Assuntos
Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Ciática/metabolismo , Analgésicos Opioides/farmacologia , Análise de Variância , Animais , Comportamento Animal , Linhagem Celular Transformada , Condicionamento Operante/efeitos dos fármacos , Condicionamento Operante/fisiologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Interações Medicamentosas , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Proteínas de Fluorescência Verde/biossíntese , Humanos , Hiperalgesia/etiologia , Camundongos , Camundongos Knockout , Mutagênese/fisiologia , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Fosforilação/efeitos dos fármacos , Fosfotreonina/imunologia , Fosfotreonina/metabolismo , Receptores Opioides mu/química , Ciática/complicações , Ciática/patologia , Transfecção , beta-Endorfina/deficiência , beta-Endorfina/metabolismoRESUMO
Complex behaviors such as those associated with reward to unconditioned positive reinforcers are polygenic processes. In studies using genetically modified mice specific for the endogenous opioid systems an observed phenotype in a complex behavior is likely to be dependent on interacting genes which, in inbred mouse lines, influence that phenotype. To address this issue we examined operant responding for palatable food reinforcers in mice lacking the expression of beta-endorphin, enkephalin or both peptides congenic to two different genetic backgrounds; C57BL/6J and DBA/2J. These two inbred strains were chosen because their endogenous opioid states differ and they respond differently to exogenous opioids in many behavioral assays. We found that wildtype and mutant C57BL/6J mice acquired operant responding for food reinforcers faster than DBA/2J mice, regardless of their opioid genotype. Although wildtype DBA/2J mice had a significant deficit in acquisition of bar-pressing behavior to reach a pre-established performance criterion, no subsequent deficit was observed under two different schedules of reinforcement. Additionally, we found that mice lacking enkephalin had decreased motivation to bar press for palatable food reinforcers under a progressive ratio regardless of sex or background strain. In contrast, the only subset of beta-endorphin-deficient mice that had decreased motivation to bar press under a progressive ratio was males on the C57BL/6J background. Of the two classical endogenous opioid peptides with preferential activation of the mu opioid receptor, the knockout models would suggest that enkephalins play a more consistent role than beta-endorphin in mediating the motivation for food reward when tested under a progressive ratio.
Assuntos
Alimentos , Recompensa , beta-Endorfina/genética , beta-Endorfina/fisiologia , Animais , Condicionamento Operante/fisiologia , Encefalinas/genética , Encefalinas/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Esquema de Reforço , Caracteres Sexuais , Especificidade da EspécieRESUMO
The present study investigated the localization and density of somatostatin (SRIF) receptor subtypes (sst(1-5)) and SRIF-nitric oxide (NO()) interactions in the retina of wildtype [WT, (+/+)] and somatostatin deficient mice [SRIF (-/-)]. Immunohistochemistry and radioligand binding studies with subsequent autoradiography were performed. Monoclonal antibodies [SRIF, protein kinase C (rod bipolar cells marker), microtubule associated protein 1A (ganglion cell marker)] and polyclonal antibodies (anti-sst(1), sst(2A), sst(4) receptor) were applied to 10-14 microm sections of retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. [(125)I]LTT SRIF-28 alone or in the presence of MK678 (sst(2) agonist) and [(125)I]Tyr(3)-octreotide were employed to quantify sst(1-5), sst(1/4)and sst(2/5) receptor densities, respectively. sst(1), sst(2A), and sst(4) receptor immunoreactivities were observed in processes of the inner plexiform layer (IPL), rod bipolar, and in ganglion cells and processes, respectively, in WT and SRIF (-/-) mice. Specific [(125)I]LTT SRIF-28 and [(125)I]Tyr(3)-octreotide binding was increased significantly in SRIF (-/-) mice. NADPH-diaphorase staining was localized in photoreceptors and amacrine cells, but not rod bipolar and ganglion cells. Also, NADPH-diaphorase staining was not colocalized with sst(1), sst(2A) or sst(4) receptor immunoreactivity. These results demonstrate an upregulation of SRIF receptors in mice lacking SRIF, but no evident SRIF-NO(*) interaction was observed in the mouse retina.
Assuntos
Óxido Nítrico/fisiologia , Receptores de Somatostatina/efeitos dos fármacos , Retina/fisiologia , Somatostatina/genética , Somatostatina/fisiologia , Animais , Autorradiografia , Feminino , Imuno-Histoquímica , Radioisótopos do Iodo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Desidrogenase/metabolismo , Octreotida/metabolismo , Células Fotorreceptoras de Vertebrados/fisiologia , Ensaio Radioligante , Receptores de Somatostatina/genética , Retina/citologia , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
RATIONALE: The rewarding effects of lateral hypothalamic brain stimulation, various natural rewards, and several drugs of abuse are attenuated by D1 or D2 dopamine receptor (D1R or D2R) antagonists. Much of the evidence for dopaminergic involvement in rewards is based on pharmacological agents with limited or "relative" selectivity for dopamine receptor subtypes. Genetically engineered animal models provide a complementary approach to pharmacological investigations. OBJECTIVES: In the present study, we explored the contribution of dopamine D2Rs to (1) brain stimulation reward (BSR) and (2) the potentiation of this behavior by morphine and amphetamine using D2R-deficient mice. METHODS: Wild-type (D2Rwt), heterozygous (D2Rhet), and D2R knockout (D2Rko) mice were trained to turn a wheel for rewarding brain stimulation. Once equivalent rate-frequency curves were established, morphine-induced (0, 1.0, 3.0, and 5.6 mg/kg s.c.) and amphetamine-induced (0, 1.0, 2.0, and 4.0 mg/kg i.p.) potentiations of BSR were determined. RESULTS: The D2Rko mice required approximately 50% more stimulation than the D2Rwt mice did. With the equi-rewarding levels of stimulation current, amphetamine potentiated BSR equally across the three genotypes. In contrast, morphine potentiated rewarding stimulation in the D2Rwt, had no effect in the D2Rhet, and antagonized rewarding stimulation in the D2Rko mice. CONCLUSIONS: D2R elimination decreases, but does not eliminate, the rewarding effects of lateral hypothalamic stimulation. After compensation for this deficit, amphetamine continues to potentiate BSR, while morphine does not.
Assuntos
Encéfalo/efeitos dos fármacos , Região Hipotalâmica Lateral/efeitos dos fármacos , Morfina/farmacologia , Receptores de Dopamina D1/deficiência , Receptores de Dopamina D2/deficiência , Recompensa , Autoestimulação/efeitos dos fármacos , Anfetamina/farmacologia , Animais , Mapeamento Encefálico , Condicionamento Operante/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Heterozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motivação , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/genéticaRESUMO
Vascular endothelial growth factor (VEGF)-A is an important angiogenic cytokine in cancer and pathological angiogenesis and has been related to the antiangiogenic activity of dopamine in endothelial cells. We investigated VEGF expression, localization, and function in pituitary hyperplasia of dopamine D2 receptor (D2R)-knockout female mice. Pituitaries from knockout mice showed increased protein and mRNA VEGF-A expression when compared with wild-type mice. In wild-type mice, prolonged treatment with the D2R antagonist, haloperidol, enhanced pituitary VEGF expression and prolactin release, suggesting that dopamine inhibits pituitary VEGF expression. VEGF expression was also increased in pituitary cells from knockout mice, even though these cells proliferated less in vitro when compared with wild-type cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium proliferation assay, proliferating cell nuclear antigen expression, and [(3)H]thymidine incorporation. In contrast to other animal models, estrogen did not increase pituitary VEGF protein and mRNA expression and lowered serum prolactin secretion in vivo and in vitro in both genotypes. VEGF (10 and 30 ng/ml) did not modify pituitary cell proliferation in either genotype and increased prolactin secretion in vitro in estrogen-pretreated cells of both genotypes. But conditioned media from D2R(-/-) cells enhanced human umbilical vein cell proliferation, and this effect could be partially inhibited by an anti-VEGF antiserum. Finally, using dual-labeling immunofluorescence and confocal laser microscopy, we found that in the hyperplastic pituitaries, VEGF-A was mostly present in follicle-stellate cells. In conclusion, pituitary VEGF expression is under dopaminergic control, and even though VEGF does not promote pituitary cellular proliferation in vitro, it may be critical for pituitary angiogenesis through paracrine actions in the D2R knockout female mice.
Assuntos
Haloperidol/análogos & derivados , Hipófise/metabolismo , Receptores de Dopamina D2/deficiência , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/química , Antagonistas de Dopamina/farmacologia , Estrogênios/farmacologia , Feminino , Haloperidol/farmacologia , Humanos , Hiperplasia , Camundongos , Camundongos Knockout , Hipófise/patologia , Prolactina/sangue , Prolactina/metabolismo , RNA Mensageiro/metabolismo , Distribuição Tecidual , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The purpose of this brief review is to highlight recent studies from our laboratory and collaborators based on the analysis of novel strains of mutant mice that further our understanding of the complex regulatory roles of proopiomelanocortin (POMC) neurons and POMC peptides in energy homeostasis. Mouse models that are considered include a transgenic strain with expression of enhanced green fluorescent protein in POMC neurons, facilitating analyses of the cell intrinsic membrane and synaptic properties, and two gene knockout strains exhibiting either a selective absence of beta-endorphin production or a complete loss of all POMC peptides from the pituitary gland and nervous system. Together these studies demonstrate the wide variety of hormonal, metabolic, and transsynaptic signals that converge on the arcuate hypothalamic nucleus and nucleus tractus solitarius to regulate the activity of POMC neurons. Both melanocortin peptides and the opioid beta-endorphin processed from POMC mediate the homeostatic and behavioral responses linked to POMC neuronal circuits.
Assuntos
Metabolismo Energético/fisiologia , Homeostase/fisiologia , Neurônios/fisiologia , Neuropeptídeos/fisiologia , Pró-Opiomelanocortina/fisiologia , Região 5'-Flanqueadora/genética , Animais , Metabolismo Energético/genética , Proteínas de Fluorescência Verde , Homeostase/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neuropeptídeos/genética , Obesidade/genética , Hipófise/fisiologia , Pró-Opiomelanocortina/genética , Sinapses/fisiologia , beta-Endorfina/biossíntese , beta-Endorfina/genéticaRESUMO
The dopamine D4 receptor (D4R) is a candidate gene for attention deficit/hyperactivity disorder (ADHD) based on genetic studies reporting that particular polymorphisms are present at a higher frequency in affected children. However, the direct participation of the D4R in the onset or progression of ADHD has not been tested. Here, we generated a mouse model with high face value to screen candidate genes for the clinical disorder by neonatal disruption of central dopaminergic pathways with 6-hydroxydopamine (6-OHDA). The lesioned mice exhibited hyperactivity that waned after puberty, paradoxical hypolocomotor responses to amphetamine and methylphenidate, poor behavioral inhibition in approach/avoidance conflict tests and deficits in continuously performed motor coordination tasks. To determine whether the D4R plays a role in these behavioral phenotypes, we performed 6-OHDA lesions in neonatal mice lacking D4Rs (Drd4(-/-)). Although striatal dopamine contents and tyrosine hydroxylase-positive midbrain neurons were reduced to the same extent in both genotypes, Drd4(-/-) mice lesioned with 6-OHDA did not develop hyperactivity. Similarly, the D4R antagonist PNU-101387G prevented hyperactivity in wild-type 6-OHDA-lesioned mice. Furthermore, wild-type mice lesioned with 6-OHDA showed an absence of behavioral inhibition when tested in the open field or the elevated plus maze, while their Drd4(-/-) siblings exhibited normal avoidance for the unprotected areas of these mazes. Together, our results from a combination of genetic and pharmacological approaches demonstrate that D4R signaling is essential for the expression of juvenile hyperactivity and impaired behavioral inhibition, relevant features present in this ADHD-like mouse model.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/fisiologia , Anfetamina/farmacologia , Animais , Animais não Endogâmicos , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Comportamento Animal/fisiologia , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/citologia , Corpo Estriado/fisiopatologia , Denervação , Modelos Animais de Doenças , Masculino , Metilfenidato/farmacologia , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Vias Neurais , Oxidopamina , Fenótipo , Receptores de Dopamina D4 , Substância Negra/citologia , Substância Negra/fisiopatologia , SimpatolíticosRESUMO
Laminin is a component of the extracellular matrix (ECM) that regulates cell proliferation and hormone secretion. Here we describe the effects of laminin on prolactin secretion in normal and tumor cells and analyze laminin expression pattern during prolactinoma development. Prolactin secretion and cell proliferation were inhibited by laminin in GH3 cells. In contrast, no effect was observed in normal pituitary cells. Laminin showed a dynamic expression pattern during prolactinoma development, which was: (a) strong in normal pituitaries from wild type or dopamine D2 receptor deficient mice, (b) lower in pituitary hyperplasia and (c) markedly reduced in prolactinomas from D2R -/- mice. A similar gradual decrease in laminin was found by comparing normal human pituitaries, human pituitary hyperplasia and human prolactinomas. These results show dynamic changes of laminin expression during prolactinoma formation which, due to laminin action on PRL production and cell proliferation, indicate a possible role for laminin in prolactinoma development.
Assuntos
Laminina/metabolismo , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Prolactinoma/metabolismo , Análise de Variância , Animais , Adesão Celular , Técnicas de Cultura de Células/métodos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular Tumoral/metabolismo , Células Cultivadas , Hormônio do Crescimento/metabolismo , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Imuno-Histoquímica , Integrina beta1/metabolismo , Laminina/farmacologia , Masculino , Camundongos , Camundongos Knockout , Hipófise/citologia , Hipófise/efeitos dos fármacos , Neoplasias Hipofisárias/patologia , Prolactina/metabolismo , Prolactinoma/patologia , Ligação Proteica , Radioimunoensaio , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/genéticaRESUMO
We determined the consequences of the loss of D2 receptors (D2R) on the GH-IGF-I axis using mice deficient in functional dopamine D2 receptors by targeted mutagenesis (D2R(-/-)). Body weights were similar at birth, but somatic growth was less in male D2R(-/-) mice from 1-8 months of age and in D2R(-/-) females during the first 2 months. The rate of skeletal maturation, as indexed by femur length, and the weight of the liver and white adipose tissue were decreased in knockout male mice even though food intake was not altered. The serum GH concentration was significantly decreased during the first 2 months in knockout female and male mice, and IGF-I and IGF-binding protein-3 levels were lower in knockout mice. PRL was significantly higher in knockout mice, and females attained higher levels than males. Pituitaries from adult knockout mice had impaired basal GH release and a lower response to GHRH in vitro. We propose that the D2R participates in GHRH/GH release in the first month of life. In accordance, the D2R antagonist sulpiride lowered GH levels in 1-month-old wild-type mice. Our results indicate that lack of D2R alters the GHRH-GH-IGF-I axis, and impairs body growth and the somatotrope population.
Assuntos
Nanismo/genética , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Receptores de Dopamina D2/genética , Animais , Peso Corporal/genética , Peso Corporal/fisiologia , Células Cultivadas , Antagonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Ingestão de Alimentos/genética , Feminino , Crescimento/genética , Crescimento/fisiologia , Hormônio do Crescimento/sangue , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Prolactina/sangue , Radioimunoensaio , Receptores de Dopamina D1/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Psicológico/genética , Estresse Psicológico/metabolismoRESUMO
Phenotypes were assessed topographically in mice lacking functional D(2) dopamine receptors ['knockouts'], using an ethologically based approach to assess all behaviours in the natural repertoire. D(2)-null mice evidenced an ethogram characterised initially by modest reductions in locomotion and shifts in rearing topographies. Subsequently, topographies of behaviour habituated similarly for wildtypes and 'knockouts'. Following challenge with the D(2)-like agonist RU 24213, both inhibition of rearing at a lower dose and induction of stereotyped sniffing and ponderous locomotion at higher doses were essentially absent in D(2)-null mice. Following challenge with the D(1)-like agonist A 68930, vacuous chewing was released in D(2)-null mice. This topographical approach to phenotypic characterisation implicates: (i) the D(2) receptor in these D(2)-like agonist effects and in oppositional D(1)-like: D(2)-like interactions; and (ii) the operation of material compensatory processes consequent to the developmental absence of D(2) receptors which are able to maintain ethological function under tonic, 'naturalistic' conditions but not under 'phasic' challenge.
Assuntos
Comportamento Animal/fisiologia , Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/fisiologia , Animais , Cromanos/farmacologia , Agonistas de Dopamina/farmacologia , Feminino , Asseio Animal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Fenetilaminas/farmacologia , FenótipoRESUMO
Gabapentin (Neurontin, Pfizer Global R & D) is a novel anticonvulsant, antihyperalgesic, and antinociceptive agent with a poorly understood mechanism of action. In this study, we show that gabapentin (EC50 2 microM) inhibited up to 70 to 80% of the total K+-evoked Ca2+ influx via voltage-dependent calcium channels (VD-CCs) in a mouse pituitary intermediate melanotrope clonal mIL-tsA58 (mIL) cell line. mIL cells endogenously express only gamma-aminobutyric acid type B (GABA(B)) gb1a-gb2 receptors. Moreover, activity of the agonist gabapentin was dose dependently and completely blocked with the GABA(B) antagonist CGP55845 and was nearly identical to the prototypic GABA(B) agonist baclofen in both extent and potency. Antisense knockdown of gb1a also completely blocked gabapentin activity, while gb1b antisense and control oligonucleotides had no effect, indicating that gabapentin inhibition of membrane Ca2+ mobilization in mIL cells was dependent on a functional GABA(B) (gb1a-gb2) heterodimer receptor. In addition, during combined whole cell recording and multiphoton Ca2+ imaging in hippocampal neurons in situ, gabapentin significantly inhibited in a dose-dependent manner subthreshold soma depolarizations and Ca2+ responses evoked by somatic current injection. Furthermore, gabapentin almost completely blocked Ca2+ action potentials and Ca2+ responses elicited by suprathreshold current injection. However, larger current injection overcame this inhibition of Ca2+ action potentials suggesting that gabapentin did not predominantly affect L-type Ca2+ channels. The depressant effect of gabapentin on Ca2+ responses was coupled to the activation of neuronal GABA(B) receptors since they were blocked by CGP55845, and baclofen produced similar effects. Thus gabapentin activation of neuronal GABA(B) gb1a-gb2 receptors negatively coupled to VD-CCs can be a potentially important therapeutic mechanism of action of gabapentin that may be linked to inhibition of neurotransmitter release in some systems.
Assuntos
Acetatos/farmacologia , Aminas , Analgésicos/farmacologia , Anticonvulsivantes/farmacologia , Canais de Cálcio/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos , Agonistas dos Receptores de GABA-B , Células Piramidais/efeitos dos fármacos , Ácido gama-Aminobutírico , Animais , Baclofeno/farmacologia , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Agonistas GABAérgicos/farmacologia , Gabapentina , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/fisiologiaRESUMO
Pulsatile growth hormone (GH) secretion differs between males and females and regulates the sex-specific expression of cytochrome P450s in liver. Sex steroids influence the secretory dynamics of GH, but the neuroendocrine mechanisms have not been conclusively established. Because periventricular hypothalamic somatostatin (SST) expression is greater in males than in females, we generated knockout (Smst(-/-)) mice to investigate whether SST peptides are necessary for sexually differentiated GH secretion and action. Despite marked increases in nadir and median plasma GH levels in both sexes of Smst(-/-) compared with Smst(+/+) mice, the mutant mice had growth curves identical to their sibling controls and retained a normal sexual dimorphism in weight and length. In contrast, the liver of male Smst(-/-) mice was feminized, resulting in an identical profile of GH-regulated hepatic mRNAs between male and female mutants. Male Smst(-/-) mice show higher expression of two SST receptors in the hypothalamus and pituitary than do females. These data indicate that SST is required to masculinize the ultradian GH rhythm by suppressing interpulse GH levels. In the absence of SST, male and female mice exhibit similarly altered plasma GH profiles that eliminate sexually dimorphic liver function but do not affect dimorphic growth.
Assuntos
Hormônio do Crescimento/fisiologia , Fígado/metabolismo , Caracteres Sexuais , Somatostatina/fisiologia , Animais , Peso Corporal , Feminino , Genótipo , Hormônio do Crescimento/metabolismo , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Hipófise/metabolismo , RNA Mensageiro/biossíntese , Receptores de Somatostatina/biossíntese , Receptores de Somatostatina/genética , Recombinação Genética , Somatostatina/genética , Transcrição GênicaRESUMO
Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.
Assuntos
Regulação da Expressão Gênica/genética , Hipófise/metabolismo , Receptores de GABA-B/biossíntese , Receptores de GABA-B/genética , Animais , Western Blotting , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Agonistas GABAérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Ensaios de Proteção de Nucleases , Oligonucleotídeos Antissenso/farmacologia , Hipófise/citologiaRESUMO
The dopamine D(4) receptor (D(4)R) is predominantly expressed in the frontal cortex (FC), a brain region that receives dense input from midbrain dopamine (DA) neurons and is associated with cognitive and emotional processes. However, the physiological significance of this dopamine receptor subtype has been difficult to explore because of the slow development of D(4)R agonists and antagonists the selectivity and efficacy of which have been rigorously demonstrated in vivo. We have attempted to overcome this limitation by taking a multidimensional approach to the characterization of mice completely deficient in this receptor subtype. Electrophysiological current and voltage-clamp recordings were performed in cortical pyramidal neurons from wild-type and D(4)R-deficient mice. The frequency of spontaneous synaptic activity and the frequency and duration of paroxysmal discharges induced by epileptogenic agents were increased in mutant mice. Enhanced synaptic activity was also observed in brain slices of wild-type mice incubated in the presence of the selective D(4)R antagonist PNU-101387G. Consistent with greater electrophysiological activity, nerve terminal glutamate density associated with asymmetrical synaptic contacts within layer VI of the motor cortex was reduced in mutant neurons. Taken together, these results suggest that the D(4)R can function as an inhibitory modulator of glutamate activity in the FC.