Regulation of muscarinic acetylcholine receptor number in cultured neuronal cells by chronic membrane depolarization.

The effect of chronic membrane depolarization on the regulation of muscarinic acetylcholine receptor (mAChR) number was studied in neuroblastoma cells (clone N1E-115). Receptor number was determined by a filter binding assay using 3H-quinuclidinyl benzilate (QNB) in membrane and crude cellular homogenates. Incubation with 50 microM veratridine (VTN), an activator of voltage-sensitive Na+ channels, induced a 50-200% increase in mAChR number at 24 hr, which was inhibited 80% by TTX. Scatchard analysis showed that affinity of the mAChR for 3H-QNB was not affected by VTN. Upon withdrawal of VTN, mAChR number returned to control levels within 20 hr. Chronic membrane depolarization caused by incubation in medium containing 60 mM K+ induced a TTX-insensitive 50% increase in mAChR number at 24 hr. AChE activity was unaffected by chronic membrane depolarization. The VTN-induced increase in mAChR number was not blocked by coincubation with cycloheximide or tunicamycin, both inhibitors of de novo mAChR synthesis. The rate of mAChR degradation was reduced in the presence of 50 microM VTN, with the apparent half-life increased from approximately 18 hr (control) to approximately 40 hr (VTN). Although treatment with either 1 mM 8Br-cAMP or 1 mM 8Br-GMP failed to increase mAChR number, treatment with either the inorganic Ca2+ channel blocker Co2+ (1 mM) or the organic Ca2+ channel antagonist D600 (10-100 microM) produced 40-80% increases in mAChR number. The combination of VTN and either D600 or Co2+ failed to induce a greater increase in mAChR number than incubation with VTN alone.(ABSTRACT TRUNCATED AT 250 WORDS)