| Summary: | 博士 === 國立臺灣大學 === 解剖學暨細胞生物學研究所 === 90 === The maintenance of normal expression and subcellular distribuion of neurotransmitter receptors is crucial to proper neurotransmission. However, it remains to be elucidated concerning the regulatory mechanisms for the subcellular expression of the receptors. The principle inhibitory neuroreceptor, g-aminobutyric acidA receptor (GABAAR), is a ligand-gated Cl- channel and mediates fast inhibitory transmission by increasing the Cl- conductance. Mechanisms underlying the intracellular transport of GABAAR were examined in the cultured neurons derived from chicken embryo brains.
In situ trypsinization of the cultures and 3H-flunitrazepam (FNZ) binding assay were employed to determine the cell surface and intracellular distribution of the receptor. A 3-h treatment of the cells with 1 mM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABAAR density by about 36% and decreased the cell surface receptors by 18% from respective control values. A 3-h incubation of the cells with 10 mM of acrylamide, a neurofilaments disrupting agent, also reversibly elevated the proportion of intracellular GABAAR density by about 48% and decreased the cell surface receptors by 24% from respective control values. A 3-h incubation of the cells with 2 mM of cytochalasin D, a microfilaments disrupter did not cause significant changes. These treatments failed to alter the total number of the 3H-FNZ binding sites of the neurons and the affinity of the ligand.
Moreover, the exposure to colchicine or acrylamide seemed to produce a stronger cytoplasmic immunostaining of the GABAAR a subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular 3H-FNZ binding. However, in the neurons exposed to cytochalasin D, there is an increase of around 28% in the total content of a1+51 kDa proteins.
In addition, the colchicine and acrylamide treatment inhibited approximately 21% of the rate of general protein synthesis in the culture by the incorporation of
3H-leucine; cytochalasin D also inhibited about 18% of the rate of general protein synthesis. By contrast, colchicine, cytochalasin D or acrylamide failed to produce significant changes in the general galactosylation and mannosylation by assaying the incorporation of 3H-sugars. Notably, in situ hybridization assay showed that the GABAAR a1 or a2 mRNA was present in 92±2% or 94±2% of the cytochalasin D-treated neurons, both of which were higher than 71±2% - 74±3% of the control and colchicine- or acrylamide-treated cells.
In the cell bodies of acrylamide-treated neurons, the level of neurofilament-200 kDa proteins was similar to control, whereas the tubulin protein content was significantly lowered approximate 51% from control, as revealed by quantifying the immunostained cytoskeletal elements. In addition, electron microscopy observations found reduction in the numbers of neurofilaments and microtubules in the perikarya, and infolding nuclear membrane in acrylamide-treated neurons.
The data suggest that by regulating the intracellular transport, the microtubular system and neurofilamental system participate in the maintenance of normal subcellular distribution of GABAAR in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABAAR subunits.
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