RESUMO
Although adenovirus (Ad) exhibits tropism for epithelial cells, little is known about the cellular effects of adenoviral binding and internalization on epithelial functions. Here, we examine its effects on the secretory acinar epithelial cells of the lacrimal gland, responsible for stimulated release of tear proteins into ocular fluid. Exposure of reconstituted rabbit lacrimal acini to replication-defective Ad for 16-18 h under conditions that resulted in >80% transduction efficiency did not alter cytoskeletal filament or biosynthetic/endosomal membrane compartment organization. Transduction specifically altered the organization of the stimulated secretory pathway, eliciting major dispersal of rab3D immunofluorescence from apical stores normally associated with mature secretory vesicles. Biochemical studies revealed that this dispersal was not associated with altered rab3D expression nor its release from cellular membranes. Ultraviolet (UV)-inactivated Ad elicited similar dispersal of rab3D immunofluorescence. In acini exposed to replication-defective or UV-inactivated Ad, carbachol-stimulated release of bulk protein and beta-hexosaminidase were significantly (P< or =0.05) inhibited to an extent proportional to the loss of rab3D-enriched mature secretory vesicles associated with these treatments. We propose that the altered secretory compartment organization and function caused by Ad reflects changes in the normal maturation of secretory vesicles, and that these changes are caused by exposure to the Ad capsid.
Assuntos
Adenoviridae/genética , Células Epiteliais/virologia , Terapia Genética/métodos , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/virologia , Transdução Genética/métodos , Animais , Biomarcadores/análise , Capsídeo , Células Cultivadas , Exocitose , Feminino , Citometria de Fluxo , Microscopia Confocal , Coelhos , Vesículas Secretórias/fisiologia , Raios Ultravioleta , Inativação de Vírus , Proteínas rab3 de Ligação ao GTP/análiseRESUMO
PURPOSE: To further understand the regulation of microtubules and their function in the lacrimal gland, we investigated the effects of two serine/threonine phosphatase inhibitors, okadaic acid (300 nM-1 microM) and calyculin A (20-100 nM), on microtubules and stimulated secretion in lacrimal acini. METHODS: Primary rabbit lacrimal acini cultured for two days were utilized. Microtubule structure was probed using biochemical analysis and confocal fluorescence microscopy. Carbachol-stimulated and basal protein secretion were determined by measurement of released protein or, for pulse-chase studies, [(35)S]-protein. RESULTS: Biochemical analysis and confocal fluorescence microscopy showed that both inhibitors caused a major loss of cellular microtubules and also of acetylated (stable) microtubules. However, calyculin A was more potent than okadaic acid in causing microtubule loss. Because changes in microtubules can partially impair stimulated protein secretion in lacrimal acini, the effects of inhibitors on protein secretion were also evaluated. Both inhibitors caused a comparable dose-dependent and significant (p = 0.05) inhibition of carbachol-stimulated (1 mM) but not basal protein secretion. These agents also significantly inhibited protein synthesis, although pulse-chase experiments suggested that the effects on secretion were elicited post-synthetically. CONCLUSIONS: Interference with normal cycles of protein phosphorylation and dephosphorylation in lacrimal acini impairs the stimulated secretory response. Although microtubules were clearly affected by protein phosphatase inhibition, changes in this array were not directly correlated with the reduced secretory response, suggesting that the inhibitory effects on secretion may proceed through microtubule-independent as well as microtubule-dependent mechanisms.
Assuntos
Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas do Olho/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Microtúbulos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Animais , Citoesqueleto , Relação Dose-Resposta a Droga , Feminino , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Toxinas Marinhas , Microscopia Confocal , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , CoelhosRESUMO
Although taxol inhibits membrane trafficking, the nature of this inhibition has not been well defined. In this study, we define the effects of taxol on endocytosis in CV-1 cells using density gradient centrifugation of membranes over sorbitol density gradients. After taxol treatment, resident endosomal enzymes and the epidermal growth factor (EGF) receptor (EGFR) showed significant (P = 0.05) enrichment in membranes with properties of early endosomes (fractions 4 and 5); the EGFR and Na+-K+-ATPase were also significantly (P = 0.05) depleted in lysosomal fractions (fractions 10 and 11). The suggestion that taxol specifically reduces movement of endosomal constituents to lysosomes was supported by fluorescence microscopy studies revealing restriction of EGF to the peripheries of taxol-treated cells, in contrast to the perinuclear lysosomal-like distribution of EGF seen in controls. Kinetic studies with 125I-labeled EGF were also consistent with a taxol-induced block in traffic from endosomes and lysosomes after 15 min of uptake but also suggested an additional taxol-sensitive step in trafficking that involved redistribution of 125I-EGF within high-density compartments after 150 min. Related changes in cytoplasmic dynein distribution were observed within high-density compartments from taxol-treated cells, suggesting that this motor might participate in this later taxol-sensitive trafficking event. Electron microscopic examination of high-density membranes (fraction 12) showed that taxol increased the numbers of small (<500 nm) dense vesicles, with a relative depletion of the larger (>500 nm) vesicles found in controls. These data demonstrate that disruption of endocytic events by taxol includes the early accumulation of protein and endocytic markers in endosomes and the later accumulation in a dense compartment that we propose is a subdomain of the lysosomes.
Assuntos
Endossomos/fisiologia , Membranas Intracelulares/fisiologia , Lisossomos/fisiologia , Paclitaxel/farmacologia , Fosfatase Ácida/metabolismo , Animais , Biomarcadores , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Dineínas/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/ultraestrutura , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Cinética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia Eletrônica , Microtúbulos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Distribuição Tecidual , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Stimulation of lacrimal acini with secretagogues such as carbachol initiates movement and fusion of acinar secretory vesicles with the apical plasma membrane, resulting in release of protein into the nascent tear fluid. Using rabbit lacrimal acini reconstituted in vitro from isolated cells, we have investigated the organization of the apical cytoskeleton and its role in stimulated secretion. Confocal microscopy revealed a microtubule array emanating from the apical region of the acini; the apical region was also enriched in microfilaments and (gamma)-tubulin. Cytokeratin-based intermediate filaments were apically concentrated, and also detected at the cell periphery. Neither confocal microscopy nor biochemical analysis revealed any reorganization of lumenal microfilaments or microtubules which might accompany carbachol-stimulated release of secretory proteins. However, major changes in the acinar microtubule array induced by taxol or nocodazole were correlated with inhibition of carbachol-dependent release of the secreted protein, beta-hexosaminidase. Major changes in lumenal microfilaments induced by jasplakinolide or cytochalasin D did not inhibit the carbachol-dependent release of beta-hexosaminidase; rather, release of beta-hexosaminidase from jasplakinolide- or cytochalasin D-treated carbachol-stimulated acini was markedly increased relative to the release from untreated stimulated acini. Our findings demonstrate that microtubules play a major role in stimulated lacrimal secretion, and suggest a contributory role for microfilaments.
Assuntos
Depsipeptídeos , Aparelho Lacrimal/metabolismo , Microtúbulos/fisiologia , beta-N-Acetil-Hexosaminidases/metabolismo , Citoesqueleto de Actina/fisiologia , Actinas/análise , Animais , Carbacol/farmacologia , Polaridade Celular , Células Cultivadas , Citocalasina D/farmacologia , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Feminino , Aparelho Lacrimal/enzimologia , Microscopia Confocal , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Peptídeos Cíclicos/farmacologia , Coelhos , Taxa Secretória/efeitos dos fármacos , Tubulina (Proteína)/análiseRESUMO
The nucleoside analog 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- ethyluracil (FEAU) was tested in a rabbit model of acute herpetic keratitis and its effectiveness compared with that of acyclovir (ACV). FEAU or ACV was applied topically 3 times daily, beginning 3 days post-HSV-1 inoculation and continued for a period of 7 days. FEAU at a concentration of 1% (w/v) or 3% ACV resulted in significant lessening of the severity of corneal lesions, conjunctivitis, iritis, and corneal clouding at 24 to 48 h after beginning chemotherapy. No toxic reaction was observed in any rabbit eyes treated with either FEAU or ACV. The duration of virus shedding into tear film and colonization of the trigeminal ganglia, however, were not reduced by either FEAU or ACV treatment begun 3 days post-inoculation. Fifty percent effective dose (ED50) of FEAU determinations performed on isolates from tear film and on the virus inoculum in secondary rabbit kidney cultures yielded a range of 4.6-7 microM, with two in vitro resistant isolates having ED50S of greater than or equal to 1500 microM of FEAU. Fifty percent cell growth inhibition for FEAU was 3000 microM at 72 h.