Effects of Inhibitors of Protein Synthesis and Intracellular Transport on the γ‐Aminobutyric Acid Agonist‐Induced Functional Differentiation of Cultured Cerebellar Granule Cells

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Abstract
The effect of inhibitors of protein synthesis (actinomycin D, cycloheximide), proteases (leupeptin), and intracellular transport (colchicine, monensin) on the γ‐aminobutyric acid (GABA) agonist [4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP)]‐induced changes in morphological differentiation and GABA receptor expression was investigated in cultured cerebellar granule cells. After 4 days in culture the neurons were exposed to the inhibitors for 6 h in the simultaneous presence of THIP. Subsequently, cultures were either fixed for electron microscopic examination or used for preparation of membranes for [3H]GABA binding assays. In some experiments the functional activity of the newly induced low‐affinity GABA receptors was assessed by investigation of the ability of GABA to inhibit neurotransmitter release from the neurons. These experiments were performed to differentiate between an intracellular and a plasma membrane localization of the receptors. In all experiments cultures treated with THIP alone served as controls. The inhibitors of protein synthesis totally abolished the ability of THIP to induce low‐affinity GABA receptors. In contrast, the inhibitors of intracellular transport as well as the protease inhibitor did not affect this parameter. However, studies of effects of GABA on transmitter release from monensintreated cultures showed that transmitter release could not be inhibited by GABA in these cells in spite of the presence of low‐affinity GABA receptors in the membrane preparations. This indicates that the low‐affinity receptors were not located in the plasma membrane. This is in good agreement with the corresponding morphological findings, that monensin treatment led to an intense vacuolization of the Golgi apparatus, thereby preventing intracellular transport of the newly synthesized GABA receptors. No qualitative alteration of the general ultrastructure was observed in cells cultured in the simultaneous presence of THIP and actinomycin, cycloheximide. or colchicine. However, these inhibitors reduced the cytoplasmic density of the different organelles involved in the cellular machinery for synthesis and intracellular transport compared to cells grown in the presence of THIP alone. These results suggest that the THIP‐induced alterations in the GABA receptor expression in cerebellar granule cells requires de novo synthesis of low‐affinity GABA receptors, which in turn is dependent on proper function of the organelles involved in synthesis and intracellular transport of proteins destined for insertion into the plasma membrane.