Acetylcholine release by bradykinin, inositol 1,4,5-trisphosphate and phorbol dibutyrate in rodent neuroblastoma cells.

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1. The action of bradykinin (BK), inositol 1,4,5-trisphosphate (InsP3), and phorbol dibutyrate (PDBu) on the release of acetylcholine (ACh) was studied electrophysiologically on short-distance (less than 20 micron) synapses formed between cultured NG108-15 mouse neuroblastoma x rat glioma hybrid cells and rat muscle cells. Action potentials in NG108-15 cells did not usually evoke an excitatory junction potential (EJP) in the muscle cell in this system. 2. Ionophoretic application of BK onto the somatic surface of an NG108-15 cell produced an increase in frequency of miniature end-plate potentials (MEPPs) for 40-50s in the paired myotube. Some MEPPs were evoked during BK-induced hyperpolarization (10-20 s) of the hybrid cell soma. A few MEPPs were also elicited during BK-induced depolarization. 3. Ionophoretic injection of Ca2+ into an NG108-15 cell soma generated MEPPs for a very brief period (less than 3 s), coincident with somatic hyperpolarization. No increase was observed during a subsequent somatic depolarization induced by a larger current of Ca2+. 4. Ionophoretic injection of InsP3 into the cytoplasm of an NG108-15 cell soma transiently evoked MEPPs during the InsP3-induced hyperpolarizing phase. A large InsP3 injection caused sustained generation of MEPPs for 2-4 min, associated with InsP3-evoked depolarization. 5. Within 3-5 min after exposure of NG108-15-myotube pairs to 1 microM-PDBu, the MEPP frequency increased by 2-5 times and reached a plateau after 8 min. The increase continued after wash-out of the drug. The PDBu-induced increase of MEPPs was still observed when the membrane potential of the NG108-15 cell was clamped at -30 mV. 6. The data suggest that the BK-induced facilitation results from the action of two intracellular second messengers: an InsP3-dependent release of Ca2+ from the intracellular storage sites and protein phosphorylation by diacyclglycerol (DAG)-activated protein kinase C.

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