RESUMO
We recently reported that phogrin, also known as IA-2ß or PTPRN2, forms a complex with the insulin receptor in pancreatic ß cells upon glucose stimulation and stabilizes insulin receptor substrate 2. In ß cells of systemic phogrin gene knockout (IA-2ß-/-) mice, impaired glucose-induced insulin secretion, decreased insulin granule density, and an increase in the number and size of lysosomes have been reported. Since phogrin is expressed not only in ß cells but also in various neuroendocrine cells, the precise impact of phogrin expressed in ß cells on these cells remains unclear. In this study, we performed a comprehensive analysis of morphological changes in RIP-Cre+/-Phogrinflox/flox (ßKO) mice with ß cell-specific phogrin gene knockout. Compared to control RIP-Cre+/- Phogrin+/+ (Ctrl) mice, aged ßKO mice exhibited a decreased density of insulin granules, which can be categorized into three subtypes. While no differences were observed in the density and size of lysosomes and crinosomes, organelles involved in insulin granule reduction, significant alterations in the regions of lysosomes responding positively to carbohydrate labeling were evident in young ßKO mice. These alterations differed from those in Ctrl mice and continued to change with age. These electron microscopic findings suggest that phogrin expression in pancreatic ß cells plays a role in insulin granule homeostasis and crinophagy during aging, potentially through insulin autocrine signaling and other mechanisms.
Assuntos
Células Secretoras de Insulina , Insulina , Animais , Camundongos , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/metabolismo , Camundongos KnockoutRESUMO
To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although in vivo oxygen concentrations in tissues are often much lower than ambient 21% O2 (ranging from 3.6 to 12.8% O2), most cell cultures are maintained at 21% O2 To clarify the effects of the O2 culture concentration on the regulated secretion of peptide hormones in neuro-endocrine cells, we examined the changes in the storage and release of peptide hormones in neuro-endocrine cell lines and endocrine tissues cultured in a relatively lower O2 concentration. In both AtT-20 cells derived from the mouse anterior pituitary and freshly prepared mouse pituitaries cultured in 10% O2 for 24â h, the storage and regulated secretion of the mature peptide hormone adrenocorticotropic hormone were significantly increased compared with those in cells and pituitaries cultured in ambient 21% O2, whereas its precursor proopiomelanocortin was not increased in the cells and tissues after being cultured in 10% O2 Simultaneously, the prohormone-processing enzymes PC1/3 and carboxypeptidase E were up-regulated in cells cultured in 10% O2, thus facilitating the conversion of prohormones to their active form. Similarly, culturing the mouse ß-cell line MIN6 and islet tissue in 10% O2 also significantly increased the conversion of proinsulin into mature insulin, which was secreted in a regulated manner. These results suggest that culture under 10% O2 is more optimal for endocrine tissues/cells to efficiently generate and secrete active peptide hormones than ambient 21% O2.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Neuroendócrinas/metabolismo , Oxigênio/farmacologia , Adeno-Hipófise/metabolismo , Pró-Opiomelanocortina/biossíntese , Regulação para Cima/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Linhagem Celular , CamundongosRESUMO
Autocrine insulin signaling is critical for pancreatic ß-cell growth and activity and is at least partially controlled by protein-tyrosine phosphatases (PTPs) that act on insulin receptors (IRs). The receptor-type PTP phogrin primarily localizes on insulin secretory granules in pancreatic ß cells. We recently reported that phogrin knockdown decreases the protein levels of insulin receptor substrate 2 (IRS2), whereas high-glucose stimulation promotes formation of a phogrin-IR complex that stabilizes IRS2. However, the underlying molecular mechanisms by which phogrin affects IRS2 levels are unclear. Here, we found that relative to wildtype mice, IRS2 levels in phogrin-knockout mice islets decreased by 44%. When phogrin was silenced by shRNA in pancreatic ß-cell lines, glucose-induced insulin signaling led to proteasomal degradation of IRS2 via a negative feedback mechanism. Phogrin overexpression in a murine hepatocyte cell line consistently prevented chronic insulin treatment-induced IRS2 degradation. In vitro, phogrin directly bound the IR without the assistance of other proteins and protected recombinant PTP1B from oxidation to potentiate its activity toward the IR. Furthermore, phogrin expression suppressed insulin-induced local generation of hydrogen peroxide and subsequent PTP1B oxidation, which allowed progression of IR dephosphorylation. Together, these results suggest that a transient interaction of phogrin with the IR enables glucose-stimulated autocrine insulin signaling through the regulation of PTP1B activity, which is essential for suppressing feedback-mediated IRS2 degradation in pancreatic ß cells.
Assuntos
Glucose/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Feminino , Inativação Gênica , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteólise , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores/genéticaRESUMO
The receptor protein tyrosine phosphatase phogrin primarily localizes to hormone secretory granules in neuroendocrine cells. Concurrent with glucose-stimulated insulin secretion, phogrin translocates to pancreatic ß-cell plasma membranes, where it interacts with insulin receptors (IRs) to stabilize insulin receptor substrate 2 (IRS2) that, in turn, contributes to glucose-responsive ß-cell growth. Pancreatic ß-cell development was not altered in ß-cell-specific, phogrin-deficient mice, but the thymidine incorporation rate decreased in phogrin-deficient islets with a moderate reduction in IRS2 protein expression. In this study, we analyzed the ß-cell response to high-fat diet stress and found that the compensatory expansion in ß-cell mass was significantly suppressed in phogrin-deficient mice. Phogrin-IR interactions occurred only in high-fat diet murine islets and proliferating ß-cell lines, whereas they were inhibited by the intercellular binding of surface phogrin under confluent cell culture conditions. Thus, phogrin could regulate glucose-stimulated compensatory ß-cell growth by changing its binding partner from another ß-cell phogrin to IR in the same ß-cells.
Assuntos
Técnicas de Cultura de Células , Dieta Hiperlipídica , Animais , Camundongos , Dieta Hiperlipídica/efeitos adversos , Proliferação de Células , Ciclo Celular , GlucoseRESUMO
Secretogranin II (SgII) and III (SgIII) function within peptide hormone-producing cells and are involved in secretory granule formation. However, their function in active amine-producing cells is not fully understood. In this study, we analyzed the expression profiles of SgII and SgIII in canine adrenal medulla and pheochromocytomas by immunohistochemical staining. In normal adrenal tissues, the intensity of coexpression of these two secretogranins (Sgs) differed from each chromaffin cell, although a complete match was not observed. The coexpression of vesicular monoamine transporter 2 (VMAT2) with SgIII was similar to that with chromogranin A, but there was a subpopulation of VMAT2-expressing cells that were negative or hardly detectable for SgII. These results are the first to indicate that there are distinct expression patterns for SgII and SgIII in adrenal chromaffin cells. Furthermore, the expression of these two Sgs varied in intensity among pheochromocytomas and did not necessarily correlate with clinical plasma catecholamine levels in patients. However, compared with SgIII, the expression of SgII was shown to be strong at the single-cell level in some tumor tissues. These findings provide a fundamental understanding of the expression differences between SgII and SgIII in normal adrenal chromaffin cells and pheochromocytomas.
Assuntos
Neoplasias das Glândulas Suprarrenais , Células Cromafins , Feocromocitoma , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Neoplasias das Glândulas Suprarrenais/veterinária , Animais , Células Cromafins/metabolismo , Células Cromafins/patologia , Cromograninas/metabolismo , Cães , Humanos , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Feocromocitoma/veterinária , Secretogranina II/metabolismoRESUMO
Granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Here, we show that the granuphilin promoter is a target of SREBP-1c, a transcription factor that controls fatty acid synthesis, and MafA, a beta cell differentiation factor. Potassium-stimulated insulin secretion (KSIS) was suppressed in islets with adenoviral-mediated overexpression of granuphilin and enhanced in islets with knockdown of granuphilin (in which granuphilin had been knocked down). SREBP-1c and granuphilin were activated in islets from beta cell-specific SREBP-1c transgenic mice, as well as in several diabetic mouse models and normal islets treated with palmitate, accompanied by a corresponding reduction in insulin secretion. Knockdown- or knockout-mediated ablation of granuphilin or SREBP-1c restored KSIS in these islets. Collectively, our data provide evidence that activation of the SREBP-1c/granuphilin pathway is a potential mechanism for impaired insulin secretion in diabetes, contributing to beta cell lipotoxicity.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/farmacologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Diabetes Mellitus Experimental/genética , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Palmitatos/farmacologia , Palmitatos/toxicidade , Potássio/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteínas de Transporte Vesicular/efeitos dos fármacosRESUMO
In the present study, the expression of secretory components and vesicular transport proteins in the canine lacrimal gland was examined and morphometric analysis was performed. The secretory epithelium consists of two types of secretory cells with different morphological features. The secretory cells constituting acinar units (type A cells) exhibited higher levels of glycoconjugates, including ß-GlcNAc, than the other cell type constituting tubular units (type T cells). Immunoblot analysis revealed that antimicrobial proteins, such as lysozyme, lactoferrin and lactoperoxidase, Rab proteins (Rab3d, Rab27a and Rab27b) and soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins (VAMP2, VAMP4, VAMP8, syntaxin-1, syntaxin-4 and syntaxin-6), were expressed at various levels. We immunohistochemically demonstrated that the expression patterns of lysozyme, lactoferrin, Raba27a, Rab27b, VAMP4, VAMP8 and syntaxin-6 differed depending on the secretory cell type. Additionally, in type T cells, VAMP4 was confined to a subpopulation of secretory granules, while VAMP8 was detected in almost all of them. The present study displayed the morphological and histochemical characteristics of the secretory epithelium in the canine lacrimal gland. These findings will help elucidate the species-specific properties of this gland.
Assuntos
Aparelho Lacrimal/metabolismo , Vesículas Secretórias/metabolismo , Animais , Cães , Epitélio/metabolismo , Aparelho Lacrimal/citologia , Masculino , Proteínas SNARE/metabolismoRESUMO
Secretogranin III (SgIII) is a granin protein involved in secretory granule formation in peptide-hormone-producing endocrine cells. In this study, we analyzed the expression of the LacZ reporter in the SgIII knockout mice produced by gene trapping (SgIII-gtKO) for the purpose of comprehensively clarifying the expression patterns of SgIII at the cell and tissue levels. In the endocrine tissues of SgIII-gtKO mice, LacZ expression was observed in the pituitary gland, adrenal medulla, and pancreatic islets, where SgIII expression has been canonically revealed. LacZ expression was extensively observed in brain regions, especially in the cerebral cortex, hippocampus, hypothalamic nuclei, cerebellum, and spinal cord. In peripheral nervous tissues, LacZ expression was observed in the retina, optic nerve, and trigeminal ganglion. LacZ expression was particularly prominent in astrocytes, in addition to neurons and ependymal cells. In the cerebellum, at least four cell types expressed SgIII under basal conditions. The expression of SgIII in the glioma cell lines C6 and RGC-6 was enhanced by excitatory glutamate treatment. It also became clear that the expression level of SgIII varied among neuron and astrocyte subtypes. These results suggest that SgIII is involved in glial cell function, in addition to neuroendocrine functions, in the nervous system.
Assuntos
Cromograninas/genética , Óperon Lac/genética , Animais , Células Cultivadas , Cromograninas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RatosRESUMO
We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic beta cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.
Assuntos
Glucose/metabolismo , Insulina/metabolismo , Vesículas Secretórias/metabolismo , Animais , Membrana Celular/metabolismo , Exocitose , Humanos , Células Secretoras de Insulina/citologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Fenótipo , Ligação Proteica , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTPRESUMO
The Rab27a effector granuphilin is specifically localized on insulin granules and is involved in their exocytosis. Here we show that the number of insulin granules morphologically docked to the plasma membrane is markedly reduced in granuphilin-deficient beta cells. Surprisingly, despite the docking defect, the exocytosis of insulin granules in response to a physiological glucose stimulus is significantly augmented, which results in increased glucose tolerance in granuphilin-null mice. The enhanced secretion in mutant beta cells is correlated with a decrease in the formation of the fusion-incompetent syntaxin-1a-Munc18-1 complex, with which granuphilin normally interacts. Furthermore, in contrast to wild-type granuphilin, its mutant that is defective in binding to syntaxin-1a fails to restore granule docking or the protein level of syntaxin-1a in granuphilin-null beta cells. Thus, granuphilin not only is essential for the docking of insulin granules but simultaneously imposes a fusion constraint on them through an interaction with the syntaxin-1a fusion machinery. These findings provide a novel paradigm for the docking machinery in regulated exocytosis.
Assuntos
Proteínas de Transporte/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , Fusão de Membrana/fisiologia , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Adenoviridae/genética , Animais , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Exocitose , Técnicas de Transferência de Genes , Vetores Genéticos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/ultraestrutura , Camundongos , Camundongos Knockout , Modelos Moleculares , Fenótipo , Proteínas de Transporte Vesicular/genéticaRESUMO
Rab proteins regulate multiple, complex processes of membrane traffic. Among these proteins, Rab27a has been shown to function specifically in regulated exocytic pathways. However, the roles of Rab27b, another Rab27 subfamily member, have not been well characterized. We disrupted the Rab27b gene in mice. The targeting vector was designed to insert LacZ downstream of the initiation codon of the Rab27b gene so that the authentic promoter should drive this reporter gene. A comprehensive analysis of Rab27b expression using this mouse strain indicated that it is widely expressed not only in canonical secretory cells, but also in neurons and cells involved in surface protection and mechanical extension. To evaluate the function in pituitary endocrine cells where the isoform Rab27a is coexpressed, we generated Rab27a/Rab27b double knockout mice by crossing Rab27b knockout mice with Rab27a-mutated ashen mice. The polarized distribution of secretory granules close to the plasma membrane was markedly impaired in the pituitary of double knockout mice, indicating that the Rab27 subfamily is involved in the delivery of granules near the exocytic site. In conjunction with a phenotype having a pituitary devoid of the Rab27 effector granuphilin, we discuss the relationship between the residence and the releasable pool of granules.
Assuntos
Membrana Celular/metabolismo , Exocitose , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Animais , Regulação da Expressão Gênica , Genes Reporter/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão e Varredura , Mutação/genética , Especificidade de Órgãos , Hipófise/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Vesículas Secretórias/ultraestrutura , Proteínas rab de Ligação ao GTP/deficiência , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic beta cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells, in contrast to another effector, granuphilin, in beta cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca(2+) concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca(2+)-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca(2+)-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.
Assuntos
Exocitose , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Proteínas de Membrana/metabolismo , Vesículas Secretórias/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Cátions Bivalentes/metabolismo , Cátions Bivalentes/farmacologia , Membrana Celular/metabolismo , Células Secretoras de Glucagon/química , Células Secretoras de Glucagon/ultraestrutura , Humanos , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipídeos , Estrutura Terciária de Proteína , Vesículas Secretórias/química , Distribuição Tecidual , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTPRESUMO
The monomeric small GTPase Rab27a is specifically localized on both secretory granules and lysosome-related organelles. Although natural mutations of the Rab27a gene in human Griscelli syndrome and in ashen mice cause partial albinism and immunodeficiency reflecting the dysfunction of lysosome-related organelles, phenotypes resulting from the defective exocytosis of secretory granules have not been reported. To explore the roles of Rab27a in secretory granules, we analyzed insulin secretion profiles in ashen mice. Ashen mice showed glucose intolerance after a glucose load without signs of insulin resistance in peripheral tissues or insulin deficiency in the pancreas. Insulin secretion from isolated islets was decreased specifically in response to high glucose concentrations but not other nonphysiological secretagogues such as high K+ concentrations, forskolin, or phorbol ester. Neither the intracellular Ca2+ concentration nor the dynamics of fusion pore opening after glucose stimulation were altered. There were, however, marked reductions in the exocytosis from insulin granules predocked on the plasma membrane and in the replenishment of docked granules during glucose stimulation. These results provide the first genetic evidence to our knowledge for the role of Rab27a in the exocytosis of secretory granules and suggest that the Rab27a/effector system mediates glucose-specific signals for the exocytosis of insulin granules in pancreatic beta cells.
Assuntos
Exocitose/fisiologia , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Albinismo/genética , Albinismo/metabolismo , Albinismo/patologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Carcinógenos/farmacologia , Colforsina/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/genética , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/metabolismo , Síndromes de Imunodeficiência/patologia , Secreção de Insulina , Ilhotas Pancreáticas/patologia , Ilhotas Pancreáticas/ultraestrutura , Camundongos , Mutação , Técnicas de Cultura de Órgãos , Potássio/metabolismo , Splicing de RNA/genética , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTPRESUMO
Secretogranin III (SgIII), a member of the granin family, binds both to another granin, chromogranin A (CgA), and to a cholesterol-rich membrane that is destined for secretory granules (SGs). The knockdown of SgIII in adrenocorticotropic hormone (ACTH)-producing AtT-20 cells largely impairs the regulated secretion of CgA and ACTH. To clarify the physiological roles of SgIII in vivo, we analyzed hormone secretion and SG biogenesis in newly established SgIII-knockout (KO) mice. Although the SgIII-KO mice were viable and fertile and exhibited no overt abnormalities under ordinary rearing conditions, a high-fat/high-sucrose diet caused pronounced obesity in the mice. Furthermore, in the SgIII-KO mice compared with wild-type (WT) mice, the stimulated secretion of active insulin decreased substantially, whereas the storage of proinsulin increased in the islets. The plasma ACTH was also less elevated in the SgIII-KO mice than in the WT mice after chronic restraint stress, whereas the storage level of the precursor proopiomelanocortin in the pituitary gland was somewhat increased. These findings suggest that the lack of SgIII causes maladaptation of endocrine cells to an inadequate diet and stress by impairing the proteolytic conversion of prohormones in SGs, whereas SG biogenesis and the basal secretion of peptide hormones under ordinary conditions are ensured by the compensatory upregulation of other residual granins or factors.
Assuntos
Adaptação Fisiológica/genética , Cromograninas/genética , Cromograninas/metabolismo , Dieta/efeitos adversos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Estresse Fisiológico/fisiologia , Animais , Células Cultivadas , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Estresse Fisiológico/genéticaRESUMO
The expression of soluble N-ethyl-maleimide sensitive fusion attachment protein receptor (SNARE) proteins in apocrine glands has not been fully elucidated. In addition to performing ultrastructural observation of the ceruminous glands in goats, our study focuses on the demonstration of ß-defensins, SNARE proteins and Rab3D in these glands with the use of immunohistochemical methods. The secretory cells were equipped with two types of vesicles, Golgi apparatus and abundant rough endoplasmic reticulum (ER). Additionally, in some of them, the characteristic concentric structures composed of rough ER were observed in their circum- and infranuclear parts. The expression of phosphorylated inositol requiring enzyme 1a was also detected. These findings may indicate their ability to produce numerous secretory proteins and the maintenance of homeostasis in the glandular cells. Furthermore, ß-defensins were demonstrated as products of the ceruminous glands. The present investigation also revealed the presence of SNARE proteins and Rab3D. It is suggested that these proteins are concerned with the secretory machinery of this gland type.
Assuntos
Glândulas Apócrinas/metabolismo , Proteínas/metabolismo , Proteínas/ultraestrutura , Animais , Glândulas Apócrinas/anatomia & histologia , Glândulas Apócrinas/ultraestrutura , Cerume/metabolismo , Defensinas/metabolismo , Cabras , Imuno-Histoquímica , Proteínas SNARE/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
The comparative structure and expression of salivary components and vesicular transport proteins in the canine major salivary glands were investigated. Histochemical analysis revealed that the morphology of the five major salivary glands-parotid, submandibular, polystomatic sublingual, monostomatic sublingual, and zygomatic glands-was greatly diverse. Immunoblot analysis revealed that expression levels of α-amylase and antimicrobial proteins, such as lysozyme, lactoperoxidase, and lactoferrin, differed among the different glands. Similarly, Rab proteins (Rab3d, Rab11a, Rab11b, Rab27a, and Rab27b) and soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins VAMP4, VAMP8, syntaxin-2, syntaxin-3, syntaxin-4, and syntaxin-6 were expressed at various levels in individual glands. mmunohistochemistry of Rab3d, Rab11b, Rab27b, VAMP4, VAMP8, syntaxin-4, and syntaxin-6 revealed their predominant expression in serous acinar cells, demilunes, and ductal cells. The VAMP4/syntaxin-6 SNARE complex, which is thought to be involved in the maturation of secretory granules in the Golgi field, was found more predominantly in the monostomatic sublingual gland than in the parotid gland. These results suggest that protein expression profiles in canine salivary glands differ among individual glands and reflect the properties of their specialized functions.
Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteínas SNARE/metabolismo , Glândulas Salivares/metabolismo , Animais , Western Blotting , Cães , Imuno-Histoquímica , Imunoprecipitação , Masculino , Ligação Proteica , Proteínas e Peptídeos Salivares/metabolismoRESUMO
Astrocytes perform a variety of functions in the adult central nervous system. Recent evidence suggests that the upregulation of glial fibrillary acidic protein (GFAP), an astrocyte-specific intermediate filament component, is a biological marker of neurotoxicity after cerebral injury. We herein compared the response to traumatic brain injury or kainic acid (KA)-induced neurotoxicity in GFAP knockout (GFAP-KO) and wild-type (WT) mice. Seventy-two hours after injury, all GFAP-KO mice showed hippocampal CA3 neurodegeneration, whereas WT mice did not show neurodegeneration. Seventy-two hours after KA administration, GFAP-KO mice were more susceptible to KA-induced seizures and had an increased number of pyknotic damaged CA3 neurons than did WT mice. These results indicate that GFAP plays a crucial role in pyramidal neuronal survival after injury or KA-induced neurotoxicity.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas/complicações , Citoproteção/genética , Proteína Glial Fibrilar Ácida/genética , Hipocampo/patologia , Degeneração Neural/genética , Animais , Astrócitos/citologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Comunicação Celular/genética , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Resistência a Medicamentos/genética , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Neurotoxinas/toxicidade , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Recuperação de Função Fisiológica/fisiologia , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/fisiopatologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
In regulated exocytosis, it is generally assumed that vesicles must stably "dock" at the plasma membrane before they are primed to become fusion-competent. However, recent biophysical analyses in living cells that visualize fluorescent secretory granules have revealed that exocytic behaviors are not necessarily uniform: some granules beneath the plasma membrane are resistant to Ca(2+)-triggered release, while others are accelerated to fuse without a pause for stable docking. These findings suggest that stable docking is unnecessary, and can even be inhibitory or nonfunctional, for fusion. Consistently, pancreatic ß cells deficient in the Rab27 effector, granuphilin, lack insulin granules directly attached to the plasma membrane in electron micrographs but nevertheless exhibit augmented exocytosis. Here we directly compare the exocytic behaviors between granuphilin-positive and -negative insulin granules. Although granuphilin makes granules immobile and fusion-reluctant beneath the plasma membrane, those granuphilin-positive, docked granules release a portion of granuphilin upon fusion, and fuse at a frequency and time course similar to those of granuphilin-negative undocked granules. Furthermore, granuphilin forms a 180-nm cluster at the site of each docked granule, along with granuphilin-interacting Rab27a and Munc18-1 clusters. These findings indicate that granuphilin is an exclusive component of the functional and fusion-inhibitory docking machinery of secretory granules.
Assuntos
Membrana Celular/metabolismo , Exocitose/fisiologia , Células Secretoras de Insulina/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Insulinoma/patologia , Fusão de Membrana , Simulação de Acoplamento Molecular , Neoplasias Pancreáticas/patologia , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/metabolismoRESUMO
Glial fibrillary acidic protein (GFAP), the principal intermediate filament (IF) protein of mature astrocytes in the CNS, plays specific roles in astrocyte functions. GFAP has multiple phosphorylation sites at its N-terminal head domain. To examine the role of phosphorylation at these sites, we generated a series of substitution mutant mice in which phosphorylation sites (Ser/Thr) were replaced by Ala, in different combinations. Gfap(hm3/hm3) mice carrying substitutions at all five phosphorylation sites showed extensive decrease in both filament formation and amounts of GFAP. Gfap(hm1/hm1) and Gfap(hm2/hm2) mice, which carry substitutions at three of five sites and in different combinations, showed differential phenotypes. Although Gfap(hm3/hm3) mice retained GFAP filaments in Bergmann glia in the cerebellum, the (Gfap(hm3/hm3):Vim(-/-)) mice lacked GFAP filaments. Pulse-chase experiments of cultured astrocytes indicated that the Hm3-GFAP encoded by Gfap(hm3) was unstable particularly in the absence of vimentin, another IF protein. These results revealed the role of phosphorylation in turnover of GFAP and a synergistic role of GFAP and vimentin in the dynamics of glial filaments. The data further suggest that each of the phosphorylated sites has a distinct impact on the dynamics of GFAP.
Assuntos
Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Sítios de Ligação/genética , Encéfalo/embriologia , Encéfalo/metabolismo , Contagem de Células , Células Cultivadas , Marcação de Genes , Imuno-Histoquímica , Filamentos Intermediários/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nervo Óptico/metabolismo , Fenótipo , Fosforilação , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Vimentina/deficiência , Vimentina/genéticaRESUMO
Granuphilin is specifically expressed on dense-core granules in a defined set of secretory cells such as insulin-producing pancreatic beta-cells. It preferentially binds the GTP-bound form of Rab27a and regulates the exocytosis of secretory granules. Furthermore, granuphilin directly interacts with syntaxin-la, the plasma-membrane-anchored SNARE protein, and with Munc18-1, a Sec1/Munc18 protein. We previously reported evidence that granuphilin mediates the docking of secretory granules onto the plasma membrane through these protein-protein interactions. This chapter details the methods and protocols we use to analyze the function of granuphilin with particular attention to the assays for detecting the expression, protein interactions, and effects on exocytosis of secretory granules in pancreatic beta-cells and their derivative cell lines.