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Parkinson's disease (PD) is characterized by substantial phenotypic heterogeneity that limits the disease prognosis and patient's counseling, and complicates the design of further clinical trials. There is an unmet need for the development and validation of biomarkers for the prediction of the disease course. In this study, we utilized flow cytometry and in vitro approaches on peripheral blood cells and isolated peripheral blood mononuclear cell (PBMC)-derived macrophages to characterize specific innate immune populations in PD patients versus healthy donors. We found a significantly lower percentage of B lymphocytes and monocyte populations in PD patients. Monocytes in PD patients were characterized by a higher CD40 expression and on-surface expression of the type I membrane glycoprotein sortilin, which showed a trend of negative correlation with the age of the patients. These results were further investigated in vitro on PBMC-derived macrophages, which, in PD patients, showed higher sortilin expression levels compared to cells from healthy donors. The treatment of PD-derived macrophages with oxLDL led to higher foam cell formation compared to healthy donors. In conclusion, our results support the hypothesis that surface sortilin expression levels on human peripheral monocytes may potentially be utilized as a marker of Parkinson's disease and may segregate the sporadic versus the genetically induced forms of the disease.
Assuntos
Doença de Parkinson , Humanos , Leucócitos Mononucleares/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Biomarcadores/metabolismoRESUMO
Studies on bovine oocytes have revealed that the activation of adenosine monophosphate activated protein kinase (AMPK) by millimolar concentrations of metformin controls nuclear maturation. Tuberous sclerosis complex 2 (TSC2) has been identified as a downstream target of AMPK. The objective of this study was to investigate the effects of addition of low concentrations of metformin (1 nM to 10 µM) on the percentage of cultured cumulus-oocyte complexes (COC) giving rise to cleavage-stage embryos and AMPK-mediated TSC2 activation. Metformin was supplemented either throughout in vitro embryo production (IVP) or only during in vitro fertilization (IVF). COC were matured in vitro, inseminated, and presumptive zygotes cultured for a further 72 h post insemination before the percentage of COC that gave rise to zygotes and early embryo development was assessed. The presence of TSC2 in bovine embryos and its possible AMPK-induced activation were assessed by immunocytochemistry. Metformin had a dose-dependent effect on the numbers of cultured COC that gave rise to embryos. Drug treatment either throughout IVP or only during IVF decreased the percentage of ≥ 8-cell embryos (1 µM, P < 0.05; 10 µM, P < 0.01; and 0.1 µM, 10 µM, P < 0.01, respectively) and increased the percentage of 2-cell embryos (10 µM, P < 0.01 and P < 0.05 respectively). The percentage of cultured COC that gave rise to zygotes was not affected by metformin. TSC2 is expressed in early embryos. Metformin (10 µM) either throughout IVP or during IVF only, increased AMPK-induced PhosphoS1387-TSC2 immunoreactivity (P < 0.01) and this increase corresponded to the total TSC2 protein levels expressed in cells. Our results suggest that there is a dose-dependent negative effect of metformin on the ability of oocytes to cleave following insemination, possibly mediated through an AMPK-induced activation of TSC2.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fertilização in vitro/métodos , Metformina/farmacologia , Oócitos/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismo , Animais , Bovinos , Fase de Clivagem do Zigoto/efeitos dos fármacos , Relação Dose-Resposta a Droga , Técnicas de Cultura Embrionária , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Masculino , Oócitos/fisiologia , Proteína 2 do Complexo Esclerose Tuberosa , ZigotoRESUMO
PURPOSE: To determine if there is any effect of AMH and BMP-15 on estradiol and progesterone production from primary-cultured human luteinizing granulosa cells, to delineate what is the effect of FSH on their actions and which are the possible mechanisms involved. METHODS: Luteinizing granulosa cells (GCs), obtained from follicular fluid of 30 women undergoing in vitro fertilization, were cultured, after a short 24-h preincubation period, in serum-free medium for 24 or/and 48 h in the presence/absence of various concentrations of AMH, BMP-15 and FSH alone or in combinations. Estradiol and progesterone production, SMAD5 phosphorylation and StAR expression were studied in parallel. Steroids were measured in culture-supernatant using enzyme-immunoassays, while Smad5-signaling pathway activation and StAR protein expression were assessed immunocytochemically. RESULT(S): We found that the treatment of AMH in GCs for 24/48 h attenuated FSH-induced estradiol production (p < 0.001), had no effect on basal estradiol levels, decreased basal progesterone production (p < 0.001) and FSH-induced StAR expression (p < 0.001). On the other hand, BMP-15 decreased basal estradiol levels (p < 0.001) and attenuated FSH-induced estradiol production (p < 0.001). Furthermore, BMP-15 reduced progesterone basal secretion (p < 0.001), an effect that was partially reversed by FSH (p < 0.01), probably via increasing StAR expression (p < 0.001). FSH-induced StAR expression was also attenuated by BMP-15 (p < 0.001). FSH, AMH and BMP-15 activated Smad-signaling pathway, as confirmed by the increase of phospo-Smad5 protein levels (p < 0.001 compared to control). CONCLUSION(S): AMH and BMP-15 by interacting with FSH affect the production of estradiol and progesterone from cultured luteinizing-granulosa cells possibly via Smad5-protein phosphorylation.
Assuntos
Hormônio Antimülleriano/metabolismo , Proteína Morfogenética Óssea 15/metabolismo , Células da Granulosa/metabolismo , Proteína Smad5/metabolismo , Adulto , Hormônio Antimülleriano/farmacologia , Proteína Morfogenética Óssea 15/farmacologia , Células Cultivadas , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Progesterona/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
BACKGROUND: Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs). METHODS: ASMCs were serum starved before treatment with muscarinic agonists. Cell phenotype was studied by optical microscopy and indirect immunofluorescence, using smooth muscle α-actin, desmin and SM-Myosin Heavy Chain (SM-MHC) antibodies. [N-methyl-3H]scopolamine binding studies were performed in order to assess M3 muscarinic receptor expression on isolated cell membranes. Contractility studies were performed on isolated ASMCs treated with muscarinic agonists. Proliferation was estimated using methyl-[3H]thymidine incorporation, MTT or cell counting methods. Involvement of PI3K and MAPK signalling pathways was studied by cell incubation with the pathway inhibitors LY294002 and PD98059 respectively. RESULTS: Prolonged culture of ASMCs with acetylcholine, carbachol or FBS, reduced the expression of α-actin, desmin and SM-MHC compared to cells cultured in serum free medium. Treatment of ASMCs with muscarinic agonists for 3-15 days decreased muscarinic receptor expression and their responsiveness to muscarinic stimulation. Acetylcholine and carbachol induced DNA synthesis and increased cell number, of ASMCs that had acquired a contractile phenotype by 7 day serum starvation. This effect was mediated via a PI3K and MAPK dependent mechanism. CONCLUSIONS: Prolonged exposure of rabbit ASMCs to muscarinic agonists decreases the expression of smooth muscle specific marker proteins, down-regulates muscarinic receptors and decreases ASMC contractile responsiveness. Muscarinic agonists are mitogenic, via the PI3K and MAPK signalling pathways.
Assuntos
Acetilcolina/administração & dosagem , Carbacol/administração & dosagem , Proteínas Contráteis/biossíntese , Proteínas Contráteis/efeitos dos fármacos , Agonistas Muscarínicos/administração & dosagem , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Traqueia/citologia , Acetilcolina/farmacologia , Animais , Carbacol/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Agonistas Muscarínicos/farmacologia , Coelhos , Fatores de TempoRESUMO
Colistin is often used as a last resort for treating multidrug-resistant infections, particularly in critically ill patients in intensive care units. Nonetheless, its side effects, including myopathy, require careful monitoring. Vasoconstrictive drugs are also used in intensive care to increase blood pressure and improve blood flow to vital organs, which can be compromised in critically ill patients. The exact mechanism of colistin-induced muscle toxicity is of significant interest due to its potential intensive-care clinical implications. Colistin alone or in combination with vasoconstrictive agents was administrated in non-septic and LPS-induced septic animals for 10 days. Histopathological evaluation of the gastrocnemius muscle and dot-blot protein tissue analysis were performed. Increased intramuscular area, de-organization of the muscle fibers and signs of myopathy were observed in colistin-treated animals. This effect was ameliorated in the presence of vasoconstrictive drugs. Administration of colistin to septic animals resulted in a decrease of AMPK and cyclin-D1 levels, while it had no effect on caspase 3 levels. Vasoconstrictive drugs' administration reversed the effects of colistin on AMPK and cyclin D1 levels. Colistin's effects on muscle depend on septic state and vasoconstriction presence, highlighting the need to consider these factors when administering it in critically ill patients.
RESUMO
Emphysema is prevalent in various respiratory diseases like Chronic Obstructive Pulmonary Disease (COPD) and cystic fibrosis. Colistin and vasoconstrictive drugs are crucial for treating these patients when diagnosed with sepsis in the ICU. This study examines colistin impact in ether-induced emphysematous septic and non-septic animals, focusing on lung pathophysiology and inflammatory responses, including IL-1ß, TNF-α, AMPK, caspase-3, cyclin-D1, and colistin levels in lung tissue. All animals exhibited significant emphysematous changes, accentuated by LPS-induced septic conditions, validating the emphysema model and highlighting the exacerbating effect of sepsis on lung pathology. Colistin, alone or with vasoconstrictive drugs, stimulated immune responses through increased inflammatory cell infiltration and the presence of lymphocytes, indicating potential immunomodulatory effects. Vasoconstriction did not alter the effects of colistin or sepsis but correlated with increased colistin levels in the lungs of septic animals. These observations suggest a potential interplay between vasoconstrictive drugs and colistin distribution/metabolism, leading to enhanced local concentrations of colistin in the lung microenvironment. The findings suggest the need for further investigations to optimize colistin and vasoconstrictive drug delivery in critically ill patients with lung pathologies. Understanding these complexities may guide more effective management of inflammatory responses and lung pathologies in these critical conditions.
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Ιnformation on the role of adiponectin in human ovarian steroidogenesis is limited. The present study aimed to investigate the effect of different doses of adiponectin on the secretion of estradiol and progesterone by human luteinized granulosa cells in culture. Granulosa cells, obtained from women undergoing in vitro fertilization (IVF) treatment, were pre-incubated for 24 h and then cultured for 48 h. Adiponectin was used in 3 doses, i.e., 5, 10, and 100 µg/ml alone and in combinations with FSH (10 and 100 ng/ml). Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. Adiponectin after 48 h of culture stimulated the secretion of estradiol and, to a lesser extent, progesterone in a dose-dependent manner. FSH showed a variable effect on steroidogenesis. However, when the low dose FSH was combined with adiponectin, estradiol, and progesterone secretion were increased disproportionally to the dose of adiponectin. With the high dose FSH, the positive effect of adiponectin on FSH-induced estradiol secretion was less pronounced, while the effect on progesterone secretion was negligible. This study shows for the first time a stimulatory effect of adiponectin on the secretion of estradiol and progesterone by human luteinized granulosa cells in vitro. It is suggested that adiponectin plays a paracrine role in human ovarian steroidogenesis by sensitizing the granulosa cells to FSH.
Assuntos
Adiponectina , Progesterona , Células Cultivadas , Estradiol , Feminino , Hormônio Foliculoestimulante , Células da Granulosa , HumanosRESUMO
BACKGROUND: Muscarinic receptor antagonists are a usual treatment for chronic airway diseases, with increased bronchoconstriction, like asthma and chronic obstructive pulmonary disease. These diseases are usually accompanied by airway remodeling, involving airway smooth muscle cell (ASMC) proliferation. The purpose of this study was to examine the effect of the muscarinic receptor modulator gallamine on rabbit tracheal ASMC proliferation. METHODS: ASMCs were incubated with gallamine (1 nM-10 mM), atropine (1 fM-10 mM), and/or acetylcholine (1 nM-1 mM), in the presence or absence of FBS (1% or 10%). Cell proliferation was estimated by incorporation of radioactive thymidine, the Cell Titer AQueous One Solution method and cell number counting after Trypan blue exclusion. The mechanisms mediating cell proliferation were studied using the PI3K and MAPK inhibitors LY294002 (20 µM) and PD98059 (100 µM), respectively. Cell phenotype was studied by indirect immunofluorescence for α-actin, Myosin Heavy Chain and desmin. RESULTS: ASMC incubation with the muscarinic receptor allosteric modulator gallamine or the muscarinic receptor antagonist atropine increased methyl-[3H]thymidine incorporation and cell number in a dose-dependent manner. ASMC proliferation was mediated via PI3K and MAPK activation and was transient. Gallamine antagonized the mitogenic effect of 1% FBS. Furthermore, gallamine had a similar effect on contractile ASMCs, without synergizing with or affecting acetylcholine induced proliferation, or altering the percentage of ASMCs expressing contractile phenotype marker proteins. CONCLUSIONS: Gallamine, in the absence of any agonist, has a transient mitogenic effect on ASMCs, regardless of the cell phenotype, mediated by the PI3K and the MAPK signaling pathways.
Assuntos
Proliferação de Células/efeitos dos fármacos , Trietiodeto de Galamina/farmacologia , Antagonistas Muscarínicos/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Acetilcolina/administração & dosagem , Acetilcolina/farmacologia , Remodelação das Vias Aéreas/efeitos dos fármacos , Animais , Atropina/administração & dosagem , Atropina/farmacologia , Relação Dose-Resposta a Droga , Trietiodeto de Galamina/administração & dosagem , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Antagonistas Muscarínicos/administração & dosagem , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Coelhos , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Traqueia/citologia , Traqueia/efeitos dos fármacosRESUMO
Information on the role of resistin on steroidogenesis is limited to animal studies. The aim of this study was to investigate the effect of various doses of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture. Granulosa cells were obtained from follicular fluid aspirated from 50 women undergoing in vitro fertilization (IVF) treatment. The cells were cultured for 48 h after a 24 h pre-incubation period. The effect of resistin at dosages 1, 10 and 100 ng/ml alone or in combinations with FSH (10 and 100 ng/ml) on steroidogenesis was investigated. Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. FSH treatment increased both estradiol and progesterone secretion. Resistin suppressed basal estradiol (at 1 ng/ml) and progesterone secretion (at all concentrations tested). When resistin (all concentrations) was combined with FSH (100 ng/ml), it eliminated the stimulatory effect of FSH on the secretion of estradiol and progesterone. This study indicates an inhibitory effect of resistin on the secretion of estradiol and progesterone by human luteinized granulosa cells in vitro. It is likely that this adipokine locally affects ovarian function in women. Abbreviations: 3ß-HSD: 3ß-hydroxysteroid dehydrogenase; CAP1: cyclase-associated protein 1; DCN: decorin; FIZZ: Found in Inflammatory Zones; hCG: human chorionic gonadotropin; IGF1: insulin-like growth factor type 1; IVF: in vitro fertilization; PCOS: polycystic ovary syndrome; RIA: radioimmunoassay; ROR1: receptor tyrosine kinase-like orphan receptor-1; TLR4: Toll-like receptor 4.
Assuntos
Estradiol/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Progesterona/metabolismo , Resistina/farmacologia , Adulto , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Fertilização in vitro , Hormônio Foliculoestimulante/farmacologia , HumanosRESUMO
Previous studies indicated that lipids may be associated with abdominal aortic aneurysm (AAA); however the molecular mechanism involved is unclear. Our study aimed to investigate the expression pattern of cholesterol efflux related proteins in AAA. Liver X receptors (LXRα and LXRß), ATP-binding-cassette transporter A1 (ABCA1), Apolipoprotein AI (ApoAI), smooth muscle α-actin (α-SM) and vimentin expression levels were evaluated in human AAA, atherosclerotic (ATH) and normal abdominal aortic tissues. We found significant differences in LXRα, LXRß and ABCA1 mRNA expression levels between AAA, ATH and normal whole aortic tissues and also within the AAA, ATH and normal "intima-media" layers. Specifically, LXRα, LXRß and ABCA1 mRNA levels were decreased in AAA compared to ATH-whole tissues, as well as in AAA "intima-media" compared to ATH and normal "intima-media" layers. Moreover, immunohistochemical evaluation revealed that LXRα and ABCA1 immunoreactivities (IR) were reduced in the AAA media compared to the normal and ATH media layers and that they were also reduced in the intima layer of AAA and ATH tissues, whereas ApoAI-IR was increased in the AAA and ATH aortic walls compared to normal pointing to possible deregulation of the cholesterol efflux mechanism in AAA. Furthermore, double staining for vimentin and α-SM showed vimentin expression in the intima and inner media layer of AAA with sparse vimentin positive SMCs designating possible SMCs phenotype switch from contractile to synthetic form. In addition, histochemical analysis showed excessive lipid accumulation in the AAA wall, while co-staining using Oil Red O with α-SM or CD68 revealed lipid accumulation in SMCs and macrophages, respectively. Our study provides novel evidence for impaired cholesterol efflux in AAA associated with lipid accumulation in SMCs and macrophages, as well as switch of SMCs phenotype from contractile to synthetic form.
Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Colesterol/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Aorta Abdominal/patologia , Aterosclerose/metabolismo , Estudos de Casos e Controles , Citoesqueleto/metabolismo , Células Espumosas/patologia , Humanos , Metabolismo dos Lipídeos , Receptores X do Fígado , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/patologia , Receptores Nucleares Órfãos/metabolismoRESUMO
The aim of the present study was to examine the function of the somatostatin receptor (sst(1)) in the nucleus accumbens (NAc) of the basal ganglia. Radioligand binding studies were performed in rats to assess the presence of the receptor, while in vivo microdialysis studies were performed to examine its role in somatostatin release. CH-275, which is selective for sst(1), MK-678, selective for sst(2) and L-803,087, selective for sst(4) receptors displaced [(125)I]-Tyr(11)-somatostatin specific binding in a concentration-dependent manner with IC(50) values of 75, 0.21 and 11 nM, respectively. Infusion of CH-275 (10(-5), 10(-6) or 10(-7) M) in the NAc of freely moving rats resulted in a decrease in somatostatin levels only at the concentration of 10(-5) M. This effect was reversed by 10(-5) M of the selective sst(1) antagonist SRA-880. The sst(1) agonist L-797,591 (10(-5) M) mimicked the effect of CH-275, while MK-678 and L-803,087 at the same concentration were unable to influence somatostatin levels. These results provide functional evidence to demonstrate that the sst(1) receptor modulates somatostatin release in the basal ganglia.
Assuntos
Núcleo Accumbens/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/metabolismo , Animais , Relação Dose-Resposta a Droga , Masculino , Microdiálise , Naftalenos/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Piridinas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de Somatostatina/agonistas , Somatostatina/farmacologiaRESUMO
In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst(1-5)). Among them, the sst1 receptor has been localised to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localisation and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localised to SRIF-containing amacrine cells, whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF, whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a consequence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain.
Assuntos
Deleção de Genes , Receptores de Somatostatina/deficiência , Retina/metabolismo , Transmissão Sináptica/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Knockout , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Somatostatina/biossíntese , Receptores de Somatostatina/genética , Somatostatina/biossíntese , Somatostatina/genéticaRESUMO
PURPOSE: To investigate the presence of somatostatin and its receptors (sst(1-5) receptors) and their possible involvement in the regulation of nitric oxide (NO) production in human RPE cell cultures. METHODS: Human RPE cells (D407) were used for all studies performed. Somatostatin levels were detected by radioimmunoassay, and RT-PCR and immunocytochemistry studies were performed to identify the somatostatin receptors (sst1-sst5). Radioligand binding assays were also performed examining the ability of certain somatostatin ligands (sst1, sst2, sst5) to compete for [125I]Tyr11 somatostatin binding. The presence of NO synthase in the cultures was assayed with NADPH-diaphorase cytochemistry, and RT-PCR, and NO levels were assessed by examining the production of its stable metabolites NO2- and NO3- (NOx-). RESULTS: SRIF was detected in a concentration of 0.56 +/- 0.13 picomoles/mg protein. sst1, sst2, and sst5 mRNAs were detected, yet only sst2B and sst5 immunoreactivity was observed in human RPE cell cultures. sst1- and sst5- but not sst2-selective ligands displaced the specific [125I]Tyr11 somatostatin binding to RPE cell membranes. NADPH-diaphorase stain and iNOS mRNA were detected. SRIF and the sst2-selective analogue MK678 increased the levels of NOx- in a concentration-dependent manner. This increase was blocked by the sst2 antagonist CYN-154806 (Ac-4NO2-Phe-c(dCys-Tyr-dTrp-Lys-Thr-Cys)-dTyr-NH2). CONCLUSIONS: These results demonstrate the presence of somatostatin, and its receptors sst1, sst2B, and sst5 in human RPE cells and suggest an autocrine or paracrine role for somatostatin. Somatostatin's ability to regulate NO production, by activating sst2 receptors, provides a functional role of somatostatin in the RPE.
Assuntos
Óxido Nítrico/biossíntese , Epitélio Pigmentado Ocular/efeitos dos fármacos , Receptores de Somatostatina/metabolismo , Somatostatina/farmacologia , Sítios de Ligação , Técnicas de Cultura de Células , Relação Dose-Resposta a Droga , Antagonistas de Hormônios/farmacologia , Humanos , Imuno-Histoquímica , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Epitélio Pigmentado Ocular/metabolismo , RNA Mensageiro/metabolismo , Radioimunoensaio , Ensaio Radioligante , Receptores de Somatostatina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Somatostatina/análogos & derivados , Somatostatina/metabolismoRESUMO
The neuropeptide somatostatin is found in the retina of many species, yet its role in the visual process remains to be elucidated. The aim of the present study was to examine the expression and cellular localization of somatostatin receptor subtypes (sst; sst(2A), sst(2B) and sst(3)) in the eye of the adult newt Pleurodeles waltlii using immunohistochemistry. sst(2A) immunoreactivity was observed in bipolar cells, in the inner segments of cone photoreceptors, as well as in the region corresponding to connecting cilia of rods. sst(2B) immunoreactivity was not detected. sst(3) immunostaining was localized most intensely in the inner segments of cones, and in cilia of rods. These results suggest that somatostatin acting via sst(2A) and sst(3) receptors may play an important role in retinal physiology of the lower vertebrates.
Assuntos
Olho/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Olho/anatomia & histologia , Imuno-Histoquímica , Naftalenos , Pleurodeles , Receptores de Somatostatina/classificação , Receptores de Somatostatina/imunologia , Retina/citologia , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , SesquiterpenosRESUMO
Activation of PKCÉ, an abundant and developmentally regulated PKC isoform in the brain, has been implicated in memory throughout life and across species. Yet, direct evidence for a mechanistic role for PKCÉ in memory is still lacking. Hence, we sought to evaluate this in rats, using short-term treatments with two PKCÉ-selective peptides, the inhibitory ÉV1-2 and the activating ψÉRACK, and the novel object recognition task (NORT). Our results show that the PKCÉ-selective activator ψÉRACK, did not have a significant effect on recognition memory. In the short time frames used, however, inhibition of PKCÉ activation with the peptide inhibitor ÉV1-2 significantly impaired recognition memory. Moreover, when we addressed at the molecular level the immediate proximal signalling events of PKCÉ activation in acutely dissected rat hippocampi, we found that ψÉRACK increased in a time-dependent manner phosphorylation of MARCKS and activation of Src, Raf, and finally ERK1/2, whereas ÉV1-2 inhibited all basal activity of this pathway. Taken together, these findings present the first direct evidence that PKCÉ activation is an essential molecular component of recognition memory and point toward the use of systemically administered PKCÉ-regulating peptides as memory study tools and putative therapeutic agents.
Assuntos
Memória/fisiologia , Proteína Quinase C-épsilon/metabolismo , Reconhecimento Psicológico/fisiologia , Animais , Western Blotting , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Produtos do Gene tat/farmacologia , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Neurônios/enzimologia , Fosforilação , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Wistar , Reconhecimento Psicológico/efeitos dos fármacos , Quinases raf/fisiologia , Quinases da Família src/fisiologiaRESUMO
Despite the rapid development of new pharmacological and surgical modalities, the treatment of retinal disease all too often results in poor final visual acuity. The primary pathologic mechanism underlying suboptimal visual acuity following retinal disease is cell death. It is induced by a variety of stimuli including ischemia, inflammation, and oxidative stress. New neuroprotective strategies have recently being examined for the prevention of retinal cell death, yet there is still a need for pharmacological agents that are efficacious and lack adverse effects. These could possibly be employed alone or in combination with disease-specific treatments. The neuropeptide somatostatin and its sst(2) receptor selective analogues have been shown to inhibit the ischemia-induced neovascularization in models of retinal ischemia, and to protect from ischemia-induced cell death. The aim of this review is threefold: a) to address the functional role of somatostatin and its receptors in retinal circuitry, b) to present recent evidence supporting the neuroprotective role of somatostatin in experimental models of retinal disease and c) to present the clinical studies that have been performed to date and support the use of somatostatin and its analogues as therapeutics in ophthalmology.
Assuntos
Doenças Retinianas/tratamento farmacológico , Somatostatina/análogos & derivados , Somatostatina/uso terapêutico , Animais , Humanos , Doenças Retinianas/metabolismo , Doenças Retinianas/patologiaRESUMO
This study investigated how the administration (acute and chronic) of the antidepressants citalopram and desmethylimipramine (DMI) influences somatostatin (somatotropin release inhibitory factor, SRIF) levels and SRIF receptor density (sst(1-5)) in rat brain. Animals received either of the following treatments: (1) saline for 21 days (control group), (2) saline for 20 days and citalopram or DMI for 1 day (citalopram or DMI acute groups), (3) citalopram or DMI for 21 days (citalopram or DMI chronic groups). Somatostatin levels were determined by radioimmunoassay. [(125)I]LTT SRIF-28 binding in the absence (labeling of sst(1-5)) or presence of 3 nM MK678 (labeling of sst(1/4)) and [(125)I]Tyr(3) octreotide (labeling of sst(2/5)) binding with subsequent autoradiography was performed in brains of rats treated with both antidepressants. Somatostatin levels were increased after citalopram, but not DMI administration, in the caudate-putamen, hippocampus, nucleus accumbens, and prefrontal cortex. Autoradiography studies illustrated a significant decrease in receptor density in the superficial and deep layers of frontal cortex (sst(2)), as well as a significant increase in the CA1 (sst(1/4)) hippocampal field in brains of chronically citalopram-treated animals. DMI administration increased sst(1/4) receptors levels in the CA1 hippocampal region. These results suggest that citalopram and to a lesser extent DMI influence the function of the somatostatin system in brain regions involved in the emotional, motivational, and cognitive aspects of behavior.
Assuntos
Antidepressivos/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Citalopram/administração & dosagem , Desipramina/administração & dosagem , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Análise de Variância , Animais , Autorradiografia , Encéfalo/diagnóstico por imagem , Imunoensaio , Radioisótopos do Iodo , Masculino , Octreotida , Peptídeos Cíclicos , Cintilografia , Ratos , Ratos Sprague-DawleyRESUMO
The role of somatostatin and its mechanism of action in the retina remains an important target for investigation. Biochemical and pharmacological studies were engaged to characterize the somatostatin receptors in the rabbit retina, and their coupling to G-proteins. The ability of selective ligands to inhibit [125I]Tyr11-somatostatin-14 binding to rabbit retinal membranes was examined. The sst2 analogues SMS201-995, MK678, and BIM23014, displayed IC50 values of 0.28 +/- 0.12, 0.04 +/- 0.01 and 1.57 +/- 0.39 nm, respectively. The sst1 analogue CH275 moderately displaced the [125I]Tyr11-somatostatin-14 binding, while selective analogues for sst3, sst4 and sst5 had minimal effect. Immunoblotting and/or immunohistochemistry studies revealed the presence of the pertussis toxin sensitive Gi1/2, and Go proteins, as well as Gs. Somatostatin-14 and MK678 stimulated GTPase activity in a concentration-dependent manner with EC50 values of 42.8 +/- 16.8 and 70.0 +/- 16.5 nm, respectively, thus supporting the functional coupling between the receptor and the G-proteins. CH275 stimulated the GTPase activity moderately, in agreement with its binding profile. The antisera raised against Goalpha and Gi1/2alpha inhibited the somatostatin-induced high-affinity GTPase activity, but only anti-Goalpha inhibited the MK678 stimulation of the enzyme. These results suggest that somatostatin mediates its actions in the rabbit retina by interacting mainly with sst2 receptors that couple to Goalpha.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores de Somatostatina/metabolismo , Retina/metabolismo , Somatostatina/análogos & derivados , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Western Blotting , Membrana Celular/química , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Soros Imunes/farmacologia , Ligantes , Masculino , Peptídeos Cíclicos/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Coelhos , Ensaio Radioligante , Retina/química , Somatostatina/química , Somatostatina/metabolismo , Somatostatina/farmacologiaRESUMO
BACKGROUND/AIMS: Recently, trials of octreotide have shown a significant survival benefit in the treatment of advanced hepatocellular carcinoma but new data are controversial. We, therefore, examined the production of somatostatin and cortistatin, the expression and distribution of somatostatin receptors (sst) in HepG2 human hepatocellular carcinoma cells, and the possible antiproliferative effect of octreotide on these cells. METHODS: Radioimmunoassay and RT-PCR studies were performed for the detection of somatostatin and cortistatin. RT-PCR, radioligand binding and immunocytochemistry assays were employed for the detection of the ssts. Growth and viability of cells were measured by the tetrazolium salt assay. RESULTS: HepG2 cells were found to express sst(2), sst(3) and sst(5) receptors. Immunocytochemistry revealed a mainly intracellular distribution of all ssts with unique patterns for each of them. Membrane binding sites for somatostatin were mainly of the sst(3) (39+/-8%) and sst(5) (59+/-5%) types, while only minor sst(2) binding could be detected (5+/-12%). Octreotide was found to inhibit the proliferation of HepG2 cells (IC(50) 1.25 x 10(-9)M) via protein tyrosine phosphatases. HepG2 cells produced cortistatin while somatostatin expression was not detected. CONCLUSIONS: In conclusion, HepG2 cells express cortistatin, which regulates somatostatin receptors. Cell proliferation was reduced by octreotide via a protein tyrosine phosphatase dependent mechanism.