ABSTRACT
Ethanol exposure induces retention of glycoproteins in growing astrocytes. We examined the intracellular sites at which this retention occurs and investigated whether this effect is accompanied by alterations in the Golgi complex and microtubular system. We studied the effects of ethanol on the Golgi complex structure, as well as on the secretory pathway functionality by monitoring both the transport of the VSV-G protein and the protein levels of several molecules involved in the regulation of this pathway. Ethanol was found to delay VSV-G transport, modify Golgi complex morphology, and reduce the number of secretory vesicles. Moreover, ethanol affected the levels of mannosidase II, p58, betaCOP, rbet1, and several Rab GTPases. It also affected microtubule organization and polymerization and the levels of the motor proteins kinesin and dynein. Most of these effects were dose-dependent. These alterations, together with those previously reported concerning biosynthesis of glycoconjugates, provide novel insights into how ethanol impairs brain development.
Subject(s)
Astrocytes/drug effects , Brain/drug effects , Brain/growth & development , Ethanol/toxicity , Golgi Apparatus/drug effects , Alcohol-Induced Disorders, Nervous System/metabolism , Alcohol-Induced Disorders, Nervous System/physiopathology , Animals , Astrocytes/metabolism , Brain/physiopathology , Cells, Cultured , Coatomer Protein/drug effects , Coatomer Protein/metabolism , Dose-Response Relationship, Drug , Female , Fetal Alcohol Spectrum Disorders/metabolism , Fetal Alcohol Spectrum Disorders/physiopathology , Golgi Apparatus/metabolism , Mannose-Binding Lectins/drug effects , Mannose-Binding Lectins/metabolism , Mannosidases/drug effects , Mannosidases/metabolism , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Microtubules/drug effects , Microtubules/metabolism , Molecular Motor Proteins/drug effects , Molecular Motor Proteins/metabolism , Pregnancy , Prenatal Exposure Delayed Effects/metabolism , Prenatal Exposure Delayed Effects/physiopathology , Qc-SNARE Proteins/drug effects , Qc-SNARE Proteins/metabolism , Rats , Secretory Vesicles/drug effects , Secretory Vesicles/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Vesicular Transport Proteins/drug effects , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/drug effects , rab GTP-Binding Proteins/metabolismABSTRACT
Brefeldin A and ilimaquinone are compounds known to affect Golgi structure and function. In particular, the transport of proteins is blocked either at the level of exit from endoplasmic reticulum (brefeldin) or at cis-Golgi (ilimaquinone). Brefeldin caused a slow decrease in gap-junctional communication and a slow loss of all phosphorylated forms of connexin43 in hamster and rat fibroblasts, while ilimaquinone caused an abrupt decrease in gap-junctional communication and rapid loss of only the slowest migrating phosphorylated connexin43 band (P2). Ilimaquinone caused these effects prior to any significant Golgi fragmentation, especially in hamster fibroblasts. Concurrently, ilimaquinone minimally affected protein secretion, while brefeldin caused an instantaneous decrease. These results show that ilimaquinone inhibits gap-junctional communication in connexin43-expressing cells by a mechanism not dependent on Golgi fragmentation or block in protein transport.
Subject(s)
Cell Communication/drug effects , Eukaryotic Cells/drug effects , Gap Junctions/drug effects , Golgi Apparatus/drug effects , Protein Transport/drug effects , Quinones/pharmacology , Sesquiterpenes/pharmacology , Animals , Autoantigens , Brefeldin A/pharmacology , Cell Communication/physiology , Cells, Cultured , Coatomer Protein/drug effects , Coatomer Protein/metabolism , Connexin 43/drug effects , Connexin 43/metabolism , Cricetinae , Eukaryotic Cells/metabolism , Eukaryotic Cells/ultrastructure , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Gap Junctions/metabolism , Golgi Apparatus/pathology , Golgi Apparatus/ultrastructure , Immunohistochemistry , Membrane Proteins/drug effects , Membrane Proteins/metabolism , Microscopy, Electron , Protein Synthesis Inhibitors/pharmacology , Protein Transport/physiology , Rats , Rats, WistarABSTRACT
Arachidonyltrifluoromethy ketone (AACOCF(3)), a phospholipase A(2) antagonist, reversibly induced dispersal of Golgi stack- and trans Golgi network (TGN)-resident proteins throughout the cytoplasm in NRK cells as followed by immunocytochemical staining of ManII and TGN38, respectively. The action of AACOCF(3) was partly blocked by other PLA(2) antagonists, suggesting it be not caused by a general inhibition of phospholipase A(2). AACOCF(3) neither dissociated beta-COP from membranes nor prevented brefeldin A-induced beta-COP release. Action of AACOCF(3) on the Golgi stack and TGN is different from that of brefeldin A and nordihydroguaiaretic acid. The most prominent difference is that the Golgi stack and TGN showed a similar sensitivity to AACOCF(3), while the TGN was dispersed more slowly than the Golgi stack in brefeldin A- or nordihydroguaiaretic acid-treated NRK cells. This novel action of AACOCF(3) may be used as pharmacological tool and give new insights into vesicle-mediated traffic and Golgi membrane dynamics.
