The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

The S100s comprise a group of Ca2+-binding proteins of the EF-hand superfamily with varied functions. Within this family, three inflammatory-related proteins - S100A8, S100A9 and S100A12 - form a subcluster known as the 'calgranulins'. S100A12 levels are elevated in sera from patients with inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. S100A12 is constitutively expressed in neutrophils and induced in monocytes by LPS and TNFα, and in macrophages by IL-6. S100A12 is a potent monocyte and mast cell chemoattractant and its potentiation of mast cell activation by IgE cross-linking indicates an important role in allergic inflammation. Importantly, mast cell-dependent activation of acute inflammatory responses and monocyte recruitment is provoked by S100A12 administration in vivo. S100A12 may also influence adhesion molecule expression on endothelial cells, stimulate IL 1β and TNFinduced in monocytes production in BV 2 microglial cells, and stimulate IL 2 secretion by T lymphocytes via ligation of the receptor for advanced glycation end-products (RAGE). To date, the only extracellular receptor characterised for S100A12 is RAGE, although additional/alternate receptors are indicated. In particular, recent studies indicate that chemotaxis and mast cell activation by S100A12 are likely mediated by other receptors. The studies presented here investigated some extracellular functions of S100A12, factors influencing these functions and suggest mechanisms that may be involved. In addition to Ca2+, S100A12 binds Zn2+. Chapter 3 explores the relevance Zn2+ binding to S100A12 structure and function. Zn2+ induced formation of complexes, principally hexamers, and this was not influenced by Ca2+. S100A12 inhibited the gelatinolytic activities of matrix metalloproteinase (MMP)-2 and 9 by chelating Zn2+ from their active sites. MMPs are important in processes leading to plaque rupture. An antibody that specifically recognised Zn2+-induced complexes was generated and immunohistochemical studies demonstrated S100A12, the hexameric complex, and MMP 2 and 9 co-localisation in human atheroma. These results suggest that hexameric S100A12 may form in vivo and may implicate S100A12 in regulating plaque rupture by inhibiting MMP activity. Interestingly S100A12 synergised with LPS to induce MMP 3 and 13 expression in vitamin D3-differentiated THP 1 macrophages (THP 1 macs). S100A12 regulation of MMP expression and activity indicates that it may be involved in a self-regulatory loop, which depends on relative levels of Zn2+ and on other stimuli (eg LPS) in the inflammatory milieu. Chapter 4 describes the development of tools and methods for assessing interactions of S100A12 with cell surface receptors. To assay surface binding, an alkaline phosphatase fusion protein, a biotinylated hinge peptide and biotinylated recombinant S100A12 were generated; only S100A12 b proved useful. Surface binding of S100A12 was detected on several monocytoid/macrophage and mast cells using flow cytometry and immunocytochemistry. Some cells contained intracytoplasmic granular structures that were S100A12-positive. Unexpectedly, a subpopulation of cells in murine bone marrow-derived mast cell cultures that expressed low levels of c-kit, a marker of mature mast cells, bound high levels of S100A12. These may represent haematopoietic stem cells, which express low levels of c kit, and S100A12-mediated functional changes of these cells is worthy of characterisation. Unlike interactions of S100A8/A9 with endothelial cells, pre-incubation of S100A12 with Zn2+ or heparin had no effect on surface binding to THP 1 macs, indicating that Zn2+-induced structural changes were unlikely to alter receptor interactions. Heparan sulfate moieties are unlikely to mediate surface binding of S100A12 even though S100A12 bound heparin with relatively high affinity. Chapter 5 focussed on mechanisms involved in some S100A12 extracellular functions. Based on experiments studying effects of bovine S100A12 on BV-2 murine microglial cells, S100A12 is proposed to induce pro-inflammatory cytokine in monocytes via RAGE. Human peripheral blood mononuclear cells or human THP 1 macs activated with S100A12 did not increase cytokine induction at the mRNA or protein levels, indicating that the 'S100/RAGE pro-inflammatory axis' theory should be re-evaluated. In an attempt to provide insights into a novel receptor, mechanisms involved in S100A12-provoked THP 1 chemotaxis were investigated. This activity was sensitive to pertussis toxin, but not to an ERK1/2 pathway inhibitor, suggesting involvement of a G protein-coupled receptor. Although some RAGE ligands also bind and activate Toll-like receptors (TLRs) antibodies to TLR2 and TLR4 did not block S100A12 binding to THP 1 macs. Affinity enrichment and separation of proteins by SDS PAGE and peptide mapping by mass spectrometry identified the α and γ subunits of F1 ATP synthase, implicating ATP synthase as a putative receptor. Although primarily mitochondrial, this complex is expressed on the surface of several cell types and was confirmed on THP 1 cells and mast cells by flow cytometry. By modulating surface F1 ATP synthase activity, and thereby extracellular ATP/ADP concentrations, S100A12 may mediate its pro-inflammatory functions through G-protein coupled purinergic receptors. This work has generated new directions for studying mechanisms by which S100A12 influences monocyte/macrophage and mast cell functions that are relevant to important inflammatory diseases, such as atherosclerosis and allergic inflammation.

A hiperamonemia é o principal elemento na patogênese da encefalopatia hepática e a neurotoxicidade da amônia envolve um efeito no sistema de neurotransmissão glutamatérgica. Os astrócitos são intimamente relacionados com a transmissão glutamatérgica e, de fato, muitas alterações gliais específicas têm sido relatadas devido à exposição à amônia. A proteína S100B, particularmente a S100B extracelular, é usada como um parâmetro de ativação glial em diversas situações de injúria cerebral. Entretanto, existe pouca informação sobre essa proteína na toxicidade da amônia e nada se sabe sobre a sua secreção por astrócitos durante uma exposição à amônia. Nesse trabalho, nós investigamos a secreção de S100B em astrócitos corticais de ratos expostos de forma aguda à amônia, bem como a morfologia astrocítica, o imunoconteúdo da proteína fibrilar ácida glial (GFAP) e a atividade da enzima glutamina sintetase (GS). Além disso, investigamos um possível efeito da creatina nesses parâmetros gliais, devido a esse composto ter um suposto papel contra a toxicidade da amônia em culturas celulares. Encontramos um aumento da secreção de S100B em astrócitos expostos por 24 h à amônia, acompanhado de uma redução do imunoconteúdo de GFAP e da atividade da GS. Como elevados e persistentes aumentos extracelulares de S100B têm um efeito tóxico em células neuronais, a secreção alterada de S100B induzida pela amônia pode contribuir para o dano cerebral observado na encefalopatia hepática. A adição de creatina não impediu esse aumento na secreção de S100B, mas foi capaz de impedir a redução da concentração de GFAP e da atividade da GS induzidas pela exposição à amônia. / Hyperammonemia is a major element in the pathogenesis of hepatic encephalopathy (HE) and ammonia neurotoxicity involves an effect on the glutamatergic neurotransmitter system. Astrocytes are intimately related to glutamatergic neurotransmission and, in fact, many specific glial alterations have been reported due to ammonia exposure. S100B protein, particularly extracellular S100B, is used as a parameter of glial activation or commitment in several situations of brain injury. However, there is little information about this protein in ammonia toxicity and none about its secretion in astrocytes under ammonia exposure. In this study we investigated S100B secretion in rat cortical astrocytes acutely exposed to ammonia, as well astrocyte morphology, glial fibrillary acidic protein (GFAP) content and glutamine synthetase (GS) activity. Moreover, we studied a possible effect of creatine on these glial parameters, since that this compound has a putative role against ammonia toxicity in cell cultures. We found an increase in S100B secretion by astrocytes exposed to ammonia for 24 h, accompanied by a decrease in GFAP content and GS activity. Since elevated and persistent extracellular S100B plays a toxic effect on neural cells, altered extracellular content of S100B induced by ammonia could contribute to the brain impairment observed in HE. Creatine addition did not prevent this increment in S100B secretion, but was able to prevent the decrease in GFAP content and GS activity induced by ammonia exposure.

A S100B é uma proteína ligante de cálcio expressa e secretada por astrócitos, e possui efeito parácrino sobre os neurônios, atuando como fator neurotrófico. Estudos clínicos sugerem que níveis plasmáticos elevados de S100B estão correlacionados positivamente com a resposta à terapia antidepressiva, particularmente quando se utilizam inibidores seletivos da recaptação de serotonina, mas este mecanismo ainda é desconhecido. Neste trabalho foi avaliada a secreção de S100B em culturas primárias de astrócitos e fatias hipocampais de ratos expostos à fluoxetina e serotonina. Foi observado um significativo aumento da secreção de S100B frente à fluoxetina, e este efeito foi aparentemente dependente de PKA. Estes dados reforçam a importância da fluoxetina, independentemente da serotonina e receptores serotoninérgicos, no tratamento antidepressivo, bem como o papel da S100B nos distúrbio depressivos. / S100B is a calcium-binding protein, produced and secreted by astrocytes, which has a putative paracrine neurotrophic activity. Clinical studies have suggested that peripheral elevation of this protein is positively correlated with therapeutic antidepressant response, particularly selective serotonin reuptake inhibitors (SSRIs); however, the mechanism remains unclear. Here, we measured S100B secretion directly in hippocampal astrocyte cultures and hippocampal slices exposed to fluoxetine and observed a significant increment of S100B release in the presence of this SSRI, apparently dependent on PKA. Moreover, we found that serotonin (possibly 5HT1A receptor) reduces S100B secretion and antagonizes the effect of fluoxetine on S100B secretion. These data reinforce the effect of fluoxetine, independently of serotonin and serotonin receptors, suggesting a putative role for S100B in depressive disorders and that other molecular targets may be relevant for antidepressant activity.

S100B é uma citocina produzida e secretada por astrócitos, que está envolvida no ciclo de citocinas desencadeado pela IL-1β na doença de Alzheimer. Entretanto, a secreção de S100B seguida de estímulo pela IL-1β não tem sido demonstrado diretamente. Nós investigamos a secreção de S100B em culturas gliais e fatias hipocampais expostas a IL-1β. Culturas corticais primárias de astrócitos, células de glioma C6 e fatias hipocampais agudas de ratos Wistar expostas a IL-1β (de 1 pg a 10ng/mL) foram utilizadas.S100B extracelular foi medida por ELISA. Migração nuclear de NF-κB foi investigada por imunocitoquímica e immunoblotting. A secreção de S100B também foi avaliada na presença de inibidores de NF-κB e MAPK. A análise estatística foi realizada utilizando teste t de Student ou ANOVA de uma via, assumindo p< 0.05. A secreção de S100B foi estimulada por IL-1β em todas as preparações. Migração nuclear de NF-κB também foi observada nessas condições experimentais. PDTC (inibidor de NF-κB) e PD98059 (inibidor da via ERK) foram hábeis em inibir o aumento da secreção do S100B. Níveis micromolares de S100B, assim como IL-1β, induziram a produção de óxido nítrico. Nossos dados demonstram que a IL-1β induz um aumento da secreção basal de S100B nos três diferentes modelos in vitro utilizados: astrócitos corticais primários, células C6 e fatias hipocampais de ratos, e que esse aumento na secreção foi mediado pela via de sinalização MAPK-ERK/NFκB. Astrócitos primários e glioma C6 exibiram diferente sensibilidade a IL-1β na modulação tanto da secreção, quanto da expressão de S100B. Esses resultados sugerem que S100B extracelular pode contribuir para respostas reparativas em lesões cerebrais agudas, bem como para desordens neurodegenerativas em lesões crônicas. O mecanismo de indução de secreção de S100B por IL-1β suporta a hipótese de um "ciclo de citocinas", proposto na gênese da doença de Alzheimer. / S100B is an astrocyte-derived cytokine, which has been implicated in the IL-1β- triggered cytokine cycle in Alzheimer’s disease. However, the secretion of S100B following stimulation by IL-1β has not been directly demonstrated. We investigated S100B secretion in glial preparations and hippocampal slices exposed to IL-1β. Cortical primary astrocyte cultures, C6 glioma cells and acute hippocampal slices from the brain of Wistar rats exposed to IL-1β (from 1pg to 10ng/mL) were utilized. Extracellular S100B was measured by ELISA. Nuclear migration of NF-κB was investigated by immunocytochemistry and immunoblotting. S100B secretion was also evaluated in the presence of specific inhibitors for NF-κB and MAPK. Statistical analyses were carried out by Student t test or one-way ANOVA, assuming p < 0.05. IL-1β-stimulated S100B secretion was observed in all preparations. Nuclear migration NF-κB was also observed under these experimental conditions. PDTC (an inhibitor of NF-κB) and PD 98059 (an inhibitor of ERK) inhibited S100B secretion stimulated by IL-1β. S100B, at micromolar levels, like IL-1β, was able to induce NO production. Our data demonstrate IL-1β-induced S100B secretion in three different in vitro preparations: cortical primary astrocytes, C6 glioma cells and hippocampal slices of rats; this secretion was mediated by MAPK-ERK/NF-κB signalling. Primary astrocytes and C6 cells exhibited different sensitivities to IL-1β concerning S100B expression and secretion. These results suggest that extracellular S100B could contribute to a reparative response in acute brain injury, as well as in neurodegenerative disorders due to chronic injury. The mechanism of IL-1β-induced S100B secretion supports the hypothesis for a “cytokine cycle”, proposed in the genesis of Alzheimer disease.

A S100B é uma proteína ligante de cálcio expressa e secretada por astrócitos, e possui efeito parácrino sobre os neurônios, atuando como fator neurotrófico. Estudos clínicos sugerem que níveis plasmáticos elevados de S100B estão correlacionados positivamente com a resposta à terapia antidepressiva, particularmente quando se utilizam inibidores seletivos da recaptação de serotonina, mas este mecanismo ainda é desconhecido. Neste trabalho foi avaliada a secreção de S100B em culturas primárias de astrócitos e fatias hipocampais de ratos expostos à fluoxetina e serotonina. Foi observado um significativo aumento da secreção de S100B frente à fluoxetina, e este efeito foi aparentemente dependente de PKA. Estes dados reforçam a importância da fluoxetina, independentemente da serotonina e receptores serotoninérgicos, no tratamento antidepressivo, bem como o papel da S100B nos distúrbio depressivos. / S100B is a calcium-binding protein, produced and secreted by astrocytes, which has a putative paracrine neurotrophic activity. Clinical studies have suggested that peripheral elevation of this protein is positively correlated with therapeutic antidepressant response, particularly selective serotonin reuptake inhibitors (SSRIs); however, the mechanism remains unclear. Here, we measured S100B secretion directly in hippocampal astrocyte cultures and hippocampal slices exposed to fluoxetine and observed a significant increment of S100B release in the presence of this SSRI, apparently dependent on PKA. Moreover, we found that serotonin (possibly 5HT1A receptor) reduces S100B secretion and antagonizes the effect of fluoxetine on S100B secretion. These data reinforce the effect of fluoxetine, independently of serotonin and serotonin receptors, suggesting a putative role for S100B in depressive disorders and that other molecular targets may be relevant for antidepressant activity.

S100B é uma citocina produzida e secretada por astrócitos, que está envolvida no ciclo de citocinas desencadeado pela IL-1β na doença de Alzheimer. Entretanto, a secreção de S100B seguida de estímulo pela IL-1β não tem sido demonstrado diretamente. Nós investigamos a secreção de S100B em culturas gliais e fatias hipocampais expostas a IL-1β. Culturas corticais primárias de astrócitos, células de glioma C6 e fatias hipocampais agudas de ratos Wistar expostas a IL-1β (de 1 pg a 10ng/mL) foram utilizadas.S100B extracelular foi medida por ELISA. Migração nuclear de NF-κB foi investigada por imunocitoquímica e immunoblotting. A secreção de S100B também foi avaliada na presença de inibidores de NF-κB e MAPK. A análise estatística foi realizada utilizando teste t de Student ou ANOVA de uma via, assumindo p< 0.05. A secreção de S100B foi estimulada por IL-1β em todas as preparações. Migração nuclear de NF-κB também foi observada nessas condições experimentais. PDTC (inibidor de NF-κB) e PD98059 (inibidor da via ERK) foram hábeis em inibir o aumento da secreção do S100B. Níveis micromolares de S100B, assim como IL-1β, induziram a produção de óxido nítrico. Nossos dados demonstram que a IL-1β induz um aumento da secreção basal de S100B nos três diferentes modelos in vitro utilizados: astrócitos corticais primários, células C6 e fatias hipocampais de ratos, e que esse aumento na secreção foi mediado pela via de sinalização MAPK-ERK/NFκB. Astrócitos primários e glioma C6 exibiram diferente sensibilidade a IL-1β na modulação tanto da secreção, quanto da expressão de S100B. Esses resultados sugerem que S100B extracelular pode contribuir para respostas reparativas em lesões cerebrais agudas, bem como para desordens neurodegenerativas em lesões crônicas. O mecanismo de indução de secreção de S100B por IL-1β suporta a hipótese de um "ciclo de citocinas", proposto na gênese da doença de Alzheimer. / S100B is an astrocyte-derived cytokine, which has been implicated in the IL-1β- triggered cytokine cycle in Alzheimer’s disease. However, the secretion of S100B following stimulation by IL-1β has not been directly demonstrated. We investigated S100B secretion in glial preparations and hippocampal slices exposed to IL-1β. Cortical primary astrocyte cultures, C6 glioma cells and acute hippocampal slices from the brain of Wistar rats exposed to IL-1β (from 1pg to 10ng/mL) were utilized. Extracellular S100B was measured by ELISA. Nuclear migration of NF-κB was investigated by immunocytochemistry and immunoblotting. S100B secretion was also evaluated in the presence of specific inhibitors for NF-κB and MAPK. Statistical analyses were carried out by Student t test or one-way ANOVA, assuming p < 0.05. IL-1β-stimulated S100B secretion was observed in all preparations. Nuclear migration NF-κB was also observed under these experimental conditions. PDTC (an inhibitor of NF-κB) and PD 98059 (an inhibitor of ERK) inhibited S100B secretion stimulated by IL-1β. S100B, at micromolar levels, like IL-1β, was able to induce NO production. Our data demonstrate IL-1β-induced S100B secretion in three different in vitro preparations: cortical primary astrocytes, C6 glioma cells and hippocampal slices of rats; this secretion was mediated by MAPK-ERK/NF-κB signalling. Primary astrocytes and C6 cells exhibited different sensitivities to IL-1β concerning S100B expression and secretion. These results suggest that extracellular S100B could contribute to a reparative response in acute brain injury, as well as in neurodegenerative disorders due to chronic injury. The mechanism of IL-1β-induced S100B secretion supports the hypothesis for a “cytokine cycle”, proposed in the genesis of Alzheimer disease.

A hiperamonemia é o principal elemento na patogênese da encefalopatia hepática e a neurotoxicidade da amônia envolve um efeito no sistema de neurotransmissão glutamatérgica. Os astrócitos são intimamente relacionados com a transmissão glutamatérgica e, de fato, muitas alterações gliais específicas têm sido relatadas devido à exposição à amônia. A proteína S100B, particularmente a S100B extracelular, é usada como um parâmetro de ativação glial em diversas situações de injúria cerebral. Entretanto, existe pouca informação sobre essa proteína na toxicidade da amônia e nada se sabe sobre a sua secreção por astrócitos durante uma exposição à amônia. Nesse trabalho, nós investigamos a secreção de S100B em astrócitos corticais de ratos expostos de forma aguda à amônia, bem como a morfologia astrocítica, o imunoconteúdo da proteína fibrilar ácida glial (GFAP) e a atividade da enzima glutamina sintetase (GS). Além disso, investigamos um possível efeito da creatina nesses parâmetros gliais, devido a esse composto ter um suposto papel contra a toxicidade da amônia em culturas celulares. Encontramos um aumento da secreção de S100B em astrócitos expostos por 24 h à amônia, acompanhado de uma redução do imunoconteúdo de GFAP e da atividade da GS. Como elevados e persistentes aumentos extracelulares de S100B têm um efeito tóxico em células neuronais, a secreção alterada de S100B induzida pela amônia pode contribuir para o dano cerebral observado na encefalopatia hepática. A adição de creatina não impediu esse aumento na secreção de S100B, mas foi capaz de impedir a redução da concentração de GFAP e da atividade da GS induzidas pela exposição à amônia. / Hyperammonemia is a major element in the pathogenesis of hepatic encephalopathy (HE) and ammonia neurotoxicity involves an effect on the glutamatergic neurotransmitter system. Astrocytes are intimately related to glutamatergic neurotransmission and, in fact, many specific glial alterations have been reported due to ammonia exposure. S100B protein, particularly extracellular S100B, is used as a parameter of glial activation or commitment in several situations of brain injury. However, there is little information about this protein in ammonia toxicity and none about its secretion in astrocytes under ammonia exposure. In this study we investigated S100B secretion in rat cortical astrocytes acutely exposed to ammonia, as well astrocyte morphology, glial fibrillary acidic protein (GFAP) content and glutamine synthetase (GS) activity. Moreover, we studied a possible effect of creatine on these glial parameters, since that this compound has a putative role against ammonia toxicity in cell cultures. We found an increase in S100B secretion by astrocytes exposed to ammonia for 24 h, accompanied by a decrease in GFAP content and GS activity. Since elevated and persistent extracellular S100B plays a toxic effect on neural cells, altered extracellular content of S100B induced by ammonia could contribute to the brain impairment observed in HE. Creatine addition did not prevent this increment in S100B secretion, but was able to prevent the decrease in GFAP content and GS activity induced by ammonia exposure.

A hiperamonemia é o principal elemento na patogênese da encefalopatia hepática e a neurotoxicidade da amônia envolve um efeito no sistema de neurotransmissão glutamatérgica. Os astrócitos são intimamente relacionados com a transmissão glutamatérgica e, de fato, muitas alterações gliais específicas têm sido relatadas devido à exposição à amônia. A proteína S100B, particularmente a S100B extracelular, é usada como um parâmetro de ativação glial em diversas situações de injúria cerebral. Entretanto, existe pouca informação sobre essa proteína na toxicidade da amônia e nada se sabe sobre a sua secreção por astrócitos durante uma exposição à amônia. Nesse trabalho, nós investigamos a secreção de S100B em astrócitos corticais de ratos expostos de forma aguda à amônia, bem como a morfologia astrocítica, o imunoconteúdo da proteína fibrilar ácida glial (GFAP) e a atividade da enzima glutamina sintetase (GS). Além disso, investigamos um possível efeito da creatina nesses parâmetros gliais, devido a esse composto ter um suposto papel contra a toxicidade da amônia em culturas celulares. Encontramos um aumento da secreção de S100B em astrócitos expostos por 24 h à amônia, acompanhado de uma redução do imunoconteúdo de GFAP e da atividade da GS. Como elevados e persistentes aumentos extracelulares de S100B têm um efeito tóxico em células neuronais, a secreção alterada de S100B induzida pela amônia pode contribuir para o dano cerebral observado na encefalopatia hepática. A adição de creatina não impediu esse aumento na secreção de S100B, mas foi capaz de impedir a redução da concentração de GFAP e da atividade da GS induzidas pela exposição à amônia. / Hyperammonemia is a major element in the pathogenesis of hepatic encephalopathy (HE) and ammonia neurotoxicity involves an effect on the glutamatergic neurotransmitter system. Astrocytes are intimately related to glutamatergic neurotransmission and, in fact, many specific glial alterations have been reported due to ammonia exposure. S100B protein, particularly extracellular S100B, is used as a parameter of glial activation or commitment in several situations of brain injury. However, there is little information about this protein in ammonia toxicity and none about its secretion in astrocytes under ammonia exposure. In this study we investigated S100B secretion in rat cortical astrocytes acutely exposed to ammonia, as well astrocyte morphology, glial fibrillary acidic protein (GFAP) content and glutamine synthetase (GS) activity. Moreover, we studied a possible effect of creatine on these glial parameters, since that this compound has a putative role against ammonia toxicity in cell cultures. We found an increase in S100B secretion by astrocytes exposed to ammonia for 24 h, accompanied by a decrease in GFAP content and GS activity. Since elevated and persistent extracellular S100B plays a toxic effect on neural cells, altered extracellular content of S100B induced by ammonia could contribute to the brain impairment observed in HE. Creatine addition did not prevent this increment in S100B secretion, but was able to prevent the decrease in GFAP content and GS activity induced by ammonia exposure.

A S100B é uma proteína ligante de cálcio expressa e secretada por astrócitos, e possui efeito parácrino sobre os neurônios, atuando como fator neurotrófico. Estudos clínicos sugerem que níveis plasmáticos elevados de S100B estão correlacionados positivamente com a resposta à terapia antidepressiva, particularmente quando se utilizam inibidores seletivos da recaptação de serotonina, mas este mecanismo ainda é desconhecido. Neste trabalho foi avaliada a secreção de S100B em culturas primárias de astrócitos e fatias hipocampais de ratos expostos à fluoxetina e serotonina. Foi observado um significativo aumento da secreção de S100B frente à fluoxetina, e este efeito foi aparentemente dependente de PKA. Estes dados reforçam a importância da fluoxetina, independentemente da serotonina e receptores serotoninérgicos, no tratamento antidepressivo, bem como o papel da S100B nos distúrbio depressivos. / S100B is a calcium-binding protein, produced and secreted by astrocytes, which has a putative paracrine neurotrophic activity. Clinical studies have suggested that peripheral elevation of this protein is positively correlated with therapeutic antidepressant response, particularly selective serotonin reuptake inhibitors (SSRIs); however, the mechanism remains unclear. Here, we measured S100B secretion directly in hippocampal astrocyte cultures and hippocampal slices exposed to fluoxetine and observed a significant increment of S100B release in the presence of this SSRI, apparently dependent on PKA. Moreover, we found that serotonin (possibly 5HT1A receptor) reduces S100B secretion and antagonizes the effect of fluoxetine on S100B secretion. These data reinforce the effect of fluoxetine, independently of serotonin and serotonin receptors, suggesting a putative role for S100B in depressive disorders and that other molecular targets may be relevant for antidepressant activity.