Peptide histidine isoleucine-like immunoreactivity release from the rat gastric fundus

1. Longitudinal muscle strips from the rat gastric fundus were subjected to in vitro electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions to study the release of peptide histidine isoleucine-like immunoreactivity (PHI-LI) and the correlation between PHI-LI release and NANC relaxation. 2. Different radioimmunoassay (RIA) systems employing C-terminal- and N-terminal-specific anti-PHI sera were used to determine the relative contributions of PHI and its C-terminally extended forms, peptide histidine glycine (PHI-Gly) and peptide histidine valine [PHV(1-42)], to the PHI-LI released by the rat gastric fundus. 3. In the presence of atropine (1 microM) and guanethidine (5 microM), EFS (120 mA, 1 ms, 0.25-32.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 microM) pre-contracted strips. 4. EFS at frequencies of 8-32 Hz evoked significant increases in PHI-LI outflow. The increases in PHI-LI outflow evoked by 16-Hz EFS were abolished by tetrodotoxin (3 microM) and by a calcium-free medium, indicating an active release process from intramural nerves. 5. The EFS-induced release of PHI-LI measured with the N-terminal-specific antiserum was significantly greater than that detected with the C-terminal-specific antisera. 6. Sephadex G-25 gel permeation chromatographic analysis was performed on the PHI-LI release in response to 32-Hz EFS. A C-terminal-specific antiserum revealed one peak co-eluting with the rat PHI standard. When PHI-LI was measured with the N-terminal-specific antiserum, two peaks were found that co-eluted with the rat PHV(1-42) and rat PHI-Gly/PHI standards, respectively. 7. The present data suggest that the extended forms of PHI are the primary components of the PHI-LI released by NANC inhibitory neurones in the rat gastric fundus and support a NANC inhibitory neurotransmitter role for PHI and its extended forms in this tissue.