Characterization of substance P-like immunoreactivity in peripheral sensory nerves and enteric nerves by high pressure liquid chromatography and radioimmunoassay

Material exhibiting immunoreactivity for substance P in enteric nerves, obtained from the myenteric plexus of the guinea pig small intestine, and in the peripheral ends of sensory nerves of the ureter, atrium and superior mesenteric artery, was characterized by separation by high pressure liquid chromatography, and quantified by radioimmunoassay of fractions collected from the chromatograph. Capsaicin, which depletes substance P-like immunoreactivity from sensory, but not from other substance P-containing nerves, reduced the content of substance P-like immunoreactivity in ureter, atrium and superior mesenteric artery by more than 99.5%, whereas the reduction in immunoreactive material in the myenteric plexus was less than 10%. Separation of extracts of myenteric plexus, ureter and atrium on a reversed-phase column gave major peaks corresponding to authentic substance P and minor peaks that coeluted with oxidized substance P. If the extracts were oxidized with hydrogen peroxide before chromatography, all the immunoreactivity was found in the peak corresponding to oxidized substance P. In the superior mesenteric artery extracts, in addition to the components corresponding to substance P and its oxidized derivative, there was a small intermediate peak that has yet to be identified. Physalaemin, which has been suggested to be present in mammalian nerves, was not detectable in any of the extracts. It is concluded that both enteric nerves and the peripheral processes of sensory nerves which show immunoreactivity for substance P in this species contain the authentic peptide.