No abstract

Mechanisms underlying the intracellular transport of gamma-aminobutyric acid(A) receptor (GABA(A)R) were examined in the cultured neurons derived from chicken embryo brains. In situ trypsinization of the cultures and (3)H-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 microM of colchicine, a microtubule depolymerizer, reversibly raised the proportion of intracellular GABA(A)R density by about 36% and decreased that of the cell surface receptors by 18% from respective control values, whereas the 3-h incubation with 2 microM of cytochalasin D, a microfilament disrupter, did not cause significant changes. These treatments failed to alter the total number of the (3)H-FNZ binding sites of the neurons and the affinity of the ligand. Moreover, the exposure to colchicine seemed to produce a stronger cytoplasmic immunostaining of the GABA(A)R alpha subunits in many neurons without affecting the total cellular level of the proteins, in accordance with the increased fraction of intracellular (3)H-FNZ binding. However, in the neurons exposed to cytochalasin D, there was an increase of around 28% in the total content of alpha(1)+51kDa proteins. In addition, the colchicine or cytochalasin D treatment inhibited approximately 21 or 18% of the rate of general protein synthesis in the culture. Notably, in situ hybridization assay showed that the GABA(A)R alpha(1) or alpha(2) 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-treated cells. The data suggest that by regulating the intracellular transport, the microtubular system participates in the maintenance of normal subcellular distribution of GABA(A)R in the neurons. By contrast, the organization of microfilaments may play a role in modulating the gene expression of GABA(A)R subunits.

Mechanisms underlying the action of acrylamide on neurons were studied by monitoring the expression of GABA(A) receptor (R) in cultured brain neurons derived from chicken embryos. In situ trypsinization of the neurons and 3H-flunitrazepam binding assay were employed to examine the subcellular distribution of GABA(A)R. A 3-h exposure of the cultured neurons to 10 mM of acrylamide raised reversibly the proportion of intracellular (trypsin-resistant) 3H-flunitrazepam binding sites by about 48% and decreased cell surface binding 24% from respective control values, without altering total cellular binding and the affinity of the ligand. Moreover, the acrylamide treatment induced more intense perikaryal immunostaining of GABA(A)R alpha subunit proteins than that in control neurons but did not change the total level of cellular alpha immunostain, in accordance with the binding data. 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 approximately 51% from control, as revealed by quantifying the immunostained cytoskeletal elements. In addition, electron microscopic observations found reductions in the numbers of microtubules and neurofilaments in the perikarya of acrylamide-treated neurons. As exhibited by the 3H-leucine and 3H-monosaccharide incorporation experiments, the exposure to acrylamide inhibited the rate of general protein synthesis in the culture by 21%, while the rate of glycosylation remained unaltered. Furthermore, in situ hybridization analysis showed that acrylamide did not modify the expression of GABA(A)R alpha subunit mRNAs. Taken together, these data suggest that acrylamide may downregulate the microtubular system and disintegrate neurofilaments, and thereby block the intracellular transport of GABA(A)R, resulting in the accumulation of intracellular receptors.

Mechanisms underlying the short-term effects of amphetamine (AMPH) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) in cultured rat neurons. The cortical and hippocampal neurons were incubated with 0.1-100 microM of AMPH for 1 h or 1 microM of AMPH for 10 min to 3 h. Immunocytochemical and in situ hybridization (ISH) analyses revealed that the levels of mGluR5 immunoreactivity and mRNA in the cortical neurons were initially increased with the treatment time and dosage, to reach maximal elevations of 34 and 53% from control values following 1 h incubation of 1 microM, and then returned toward the controls. When the cortical neurons were preincubated with the antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG) to mGluRs, before treated with 1 M of AMPH for 1 h, the levels of mGluR5 protein and mRNA became 120 and 116% of control values. In hippocampal neurons, the AMPH treatment persistently upregulated the mGluR5 protein by 50-62%; however, the mRNA responded with the bell-shaped pattern to the treatment times and doses, with 20-43% increases from controls. These modifications of the receptor were reversible, since removal of AMPH resulted in regular levels of the receptor. Notably, the AMPH-generated increases in mGluR5 protein and mRNA were completely blocked by the pretreatment with cycloheximide and actinomycin D, respectively. The data indicate differential responsive patterns of mGluR5 in the cortical and hippocampal neurons to the drug perturbation. The action of AMPH may involve regulation to transcriptional and translational events in the neurons, and the activation of the MCPG-sensitive receptors.