Calcium-dependent regulation of phospholipase A2 and its inhibitors, including tetracaine, for acetylcholine receptor cluster formation in mouse myotubes co-cultured with spinal cord explant.
Regulatory effects of phospholipase A2 (PLA2) on acetylcholine receptor (AChR) cluster formation were investigated in developing mouse myotubes co-cultured with spinal cord explant, using quinacrine, cortisone, tetracaine and related agents. AChR was visualized using the fluorescence-conjugated alpha-bungarotoxin. Peak fluorescence intensity and total fluorescence within the fluorescence stain were measured as indices of AChR cluster formation and AChR content, respectively. Both indices were gradually increased from day 9 to 13 in culture. PLA2 (0.2-1.0 micrograms/ml), melittin (10 micrograms/ml) and arachidonic acid (100 microM), added to the culture medium from the second day, clearly inhibited both indices at days 11 and 13, whereas the addition of phospholipase C (1 microgram/ml) inhibited peak fluorescence but did not affect total fluorescence. The co-existence of PLA2 with its inhibitors--quinacrine (3 microM), cortisone (0.01 microM) and tetracaine (30 microM)--significantly overcame the PLA2-induced inhibition of both indices. The elevation of calcium ion concentrations from 2.9 to 10 mM abolished the increase of both indices. Quinacrine (10 microM), cortisone (0.1 microM) and tetracaine (100 microM) alone similarly inhibited both fluorescence indices. The addition of EGTA (2 mM) from day 8 overcame tetracaine-induced inhibition but not quinacrine- or cortisone-induced inhibition. These results suggest that the formation of AChR clusters in developing myotubes is negatively controlled by endogenous PLA2 activity. This overcoming of PLA2-induced inhibition by tetracaine may be dependent on calcium ion mobilization, whereas that by quainacrine and cortisone may not.
D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D009132:Muscles / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011940:Receptor Aggregation / Q000187:drug effects*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology* / Q000378:metabolism; D013748:Tetracaine / Q000494:pharmacology*; D013997:Time Factors
D004791:Enzyme Inhibitors; D011950:Receptors, Cholinergic; D013748:Tetracaine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium