Electrochemical monitoring of nitric oxide released by myenteric neurons of the guinea pig ileum

Abstract

Nitric oxide (NO) released by myenteric neurons in isolated segments of guinea pig ileum was monitored in vitro using continuous amperometry. NO was detected as an oxidation current recorded with a boron-doped diamond microelectrode held at 1 V vs a Ag|AgCl reference electrode. This potential was sufficient to oxidize NO. Longitudinal muscle-myenteric plexus (LMMP) and circular muscle strip preparations were used. In the LMMP preparation, NO release was evoked by superfusion of 1 mumol L(-1) nicotine, which activates nicotinic acetylcholine receptors expressed by myenteric neurons and myenteric nerve endings. The oxidation current was ascribed to NO based on the following observations: (i) no response was detected at less positive potentials (0.75 V) at which only catecholamines and biogenic amines are oxidized, (ii) the current was abolished in the presence of the nitric oxide synthase antagonist, N-nitro-l-arginine (l-NNA) and (iii) oxidation currents were attenuated by addition of the NO scavenger, myoglobin, to the superfusing solution. In the LMMP preparation, stimulated release produced a maximum current that corresponded nominally to 46 nmol L(-1) of NO. The oxidation currents decreased to 10 and 2 nmol L(-1), respectively, when the tissue was perfused with tetrodotoxin and l-NNA. Oxidation currents recorded from circular muscle strips (stimulated using nicotine) were threefold larger than those recorded from the LMMP. This study shows that NO release can be detected from various in vitro preparations of the guinea pig ileum using real-time electroanalytical techniques.