Release of substance P from the enteric nervous system: direct quantitation and characterization

A dynamic system has been developed in which in vitro release of substance P (SP) from two longitudinal muscle-myenteric plexus strips from the guinea-pig ileum is obtained during continuous superfusion and measured directly by using a highly sensitive radioimmunoassay specific for SP. Electrical stimulation (0.5-40 Hz) produced a marked increase in the rate of release of SP. The magnitude of the increase in the rate of electrically evoked release was dependent on the frequency of stimulation but this dependence did not appear to be linear over the entire range tested. Electrically evoked release elicited by 20 Hz stimulation was reduced by 95% by omitting Ca++ from the superfusion buffer or by cooling the preparation to 3 degrees C. Pretreatment with tetrodotoxin (1 micrograms/ml for 15 min) substantially reduced the magnitude of the increase in the rate of release of SP but did not abolish it. Despite tetrodotoxin pretreatment, a significant portion (53%) of the electrically induced increase in SP release remained. The ability of electrical stimulation to release SP from the myenteric plexus in amounts sufficient to produce a physiological response in a Ca++- and temperature-dependent fashion in combination with data provided by indirect pharmacological experiments strongly suggests that SP functions as a neurotransmitter in the enteric nervous system.