Experimental Physiology 460 Exp Physiol 96.4 pp 460–467 Research Paper Research Paper Non-quantal release of acetylcholine in guinea-pig airways: role of choline transporter Jaime Ch´ avez 1 , Mario H. Vargas 1 , Jos´ e E. Cruz-Valderrama 2 and Luis M. Monta˜ no 2 1 Departamento de Investigaci´ on en Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias, M´ exico DF, Mexico 2 Departamento de Farmacolog´ ıa, Facultad de Medicina, Universidad Nacional Aut´ onoma de M´ exico, M´ exico DF, Mexico In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non- quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea- pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na + channel blocker) and ω-conotoxin GVIA (N- type Ca 2+ channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent ω-conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ∼75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl 2 , 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca 2+ -dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon. (Received 2 December 2010; accepted after revision 25 January 2011; first published online 28 January 2011) Corresponding author L. M. Monta˜ no: Departamento de Farmacolog´ ıa, Edificio de Investigaci´ on, Laboratorio de Investigaci ´ on en Asma, Facultad de Medicina, Universidad Nacional Aut´ onoma de M´ exico, Ciudad Universitaria, CP 04510, M´ exico DF, M´ exico. Email: lmmr@servidor.unam.mx Motor nerve stimulation activates the evoked quantal release of ACh, in which ACh-containing vesicles fuse to the neuronal membrane and discharge their content into the synaptic cleft. While in this cleft, ACh has only a brief opportunity to act upon its receptors to induce skeletal muscle contraction, because it is rapidly degraded by acetylcholinesterase to acetate and choline (Barrnett, 1962; Ferguson & Blakely, 2004). The latter is recaptured from the extracellular space into the neuron by the membrane choline transporter (ChT). The ChT is a high-affinity, Na + -dependent choline transporter, which is mainly located in the prejunctional membrane of cholinergic neurons and is sensitive to hemicholinium-3 (Yamamura & Snyder, 1972; Guyenet et al. 1973). Another non- related, low-affinity, Na + -independent, hemicholinium- 3-insensible choline transporter has been described in many non-neuronal cells (Koepsell et al. 2003; Ferguson & Blakely, 2004). In neurons, choline reuptake by ChT is critical to sustain the synthesis of ACh, because it is the substrate for the choline acetyltransferase. Once synthesized, ACh is packed into vesicles through the activity of the vesicular ACh transporter (VAChT), a protein sensitive to vesamicol (Whitton et al. 1986). DOI: 10.1113/expphysiol.2010.056440 C  2011 The Authors. Journal compilation C  2011 The Physiological Society