Constitutive secretion of exogenous neurotransmitter by nonneuronal cells: implications for neuronal secretion

Fibroblasts in cell culture were loaded with exogenous neurotransmitter acetylcholine (ACh). ACh secretion from loaded cells was detected by whole-cell patch clamp recordings from Xenopus myocytes manipulated into contact with ACh-loaded cells. Two different approaches were used for ACh loading. In the first approach, fibroblasts were incubated in the culture medium containing ACh. Recordings from myocytes revealed fast inward currents that resemble miniature endplate currents found at neuromuscular synapses. The currents observed in recordings from myocytes were due to exocytosis of ACh-containing vesicles. Although exogenous ACh penetrated through the plasma membrane of fibroblasts during incubation and was present in the cytoplasm at detectable levels, cytoplasmic ACh did not contribute to the quantal ACh secretion. In the second approach, exogenous ACh was loaded into the cytoplasm of fibroblasts by microinjection. Under these experimental conditions, fibroblasts also exhibited spontaneous quantal ACh secretion. Analysis of the exocytotic events in fibroblasts following two different protocols of ACh loading revealed that the vesicular compartments responsible for uptake of exogenous ACh are associated with the endocytic recycling pathway. Extrapolation of our results to neuronal cells suggest that in cholinergic neurons, in addition to genuine synaptic vesicles, ACh can be secreted by the vesicles participating in endosomal membrane recycling.