Identification of neurotransmitters by selective protection of postjunctional receptors

The neurotransmitter responsible for relaxation of gastric smooth muscle was identified by a technique involving the use of selective ligands to protect postjunctional receptors combined with inactivation of all other receptors with N-ethylmaleimide (NEM). Relaxation in response to field stimulation (80 V, 1 ms, 0.5-8 Hz) and to maximally effective concentrations of vasoactive intestinal peptide (VIP; 1 microM), ATP (1 mM), isoproterenol (1 mM), dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP; 1 mM), and forskolin (1 microM) was measured in muscle strips from guinea pig gastric fundus before and after treatment with NEM (5 microM) for 1 h. Protection of VIP receptors with VIP or the VIP antagonist VIP10-28 fully protected relaxation induced by VIP and by field stimulation, but did not protect relaxation induced by ATP or isoproterenol. Protection of ATP receptors with ATP protected only the response to ATP, but did not protect the response to field stimulation, VIP, or isoproterenol. Relaxation induced by either forskolin or dibutyryl cAMP was not altered by treatment with NEM alone or in the presence of protective ligands, indicating that at the concentrations used NEM inactivated membrane receptors without affecting intracellular relaxation mechanisms. These studies indicate that VIP but not ATP is the neurotransmitter responsible for neurally induced relaxation in the gastric fundus.