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Agitation of villi evokes reflexes that affect the motility of the guinea-pig small intestine. NK1 receptor endocytosis was used to investigate the possible involvement of tachykinins acting on neuronal NK1 receptors in these reflexes. Segments of guinea-pig ileum were incubated at 37 degrees C in Krebs physiological saline containing 3x10(-6) M nicardipine, with or without agitation of the villi by gas bubbles. Gut segments were fixed after 0-75 min and processed for immunohistochemistry to reveal the NK1 receptors, following which cells were imaged by confocal microscopy. Initially, receptors were located on the surface and in the cytoplasm of myenteric neurons. In gut incubated without movement of the villi, NK1 receptors returned to the cell surface. After 45 and 60 min, NK1 receptors were detected almost exclusively at the cell surface of 83% and 97% (respectively) of nerve cells that were immunoreactive for NK1 receptors and only 12%-13% of the NK1 receptor fluorescence was located in the cytoplasm. Following the return of receptor to the cell surface, agitation of the villi caused a new wave of endocytosis of the NK1 receptors in 70%-80% of the NK1 receptor-immunoreactive neurons. The percentage of the NK1 receptor fluorescence that was in the cytoplasm increased more than 2-fold to 27+/-2% after 15 min villous agitation. Action potential blockade by tetrodotoxin (3x10(-7) M) prevented the internalisation of the NK1 receptor in response to villous agitation. The degree of internalisation caused by bubbling was similar to that caused by 2x10(-9) M substance P. These results indicate that, when enteric reflex circuits are activated by villous movement, tachykinins are released and cause endocytosis of the NK1 receptor in a subpopulation of myenteric neurons.

Immunoreactivity for NK1 receptors is confined to specific nerve cell bodies in the guinea-pigileum, including inhibitory motor neurons and secretomotor neurons. In the present work, endocytosis of NK1 receptors in these enteric neurons was studied following addition of substance P (SP) to isolated ileum. NK1 receptors were localised with antibodies against the C-terminus of this receptor. Some preparations were incubated with SP tagged with the fluorescent label, Cy3.18, so that the fate of SP bound to receptors could be followed. Preparations were analysed by confocal microscopy. In tissue that was incubated at 4 degrees C in the absence of SP, most NK1 receptor immunoreactivity (IR) was confined to surface membranes of nerve cells. At 37 degrees C in the presence of 10(-7) M SP (plus 3 x 10(-7)M tetrodotoxin to prevent indirect activation via other neurons) the neuronal NK1 receptor was rapidly internalised. After 5 min, NK1 receptor IR was partially internalised, at 20 min NK1 receptor IR was throughout the cytoplasm and in perinuclear aggregates and at 30 min it was again at the cell surface. SP-induced NK1 receptor endocytosis was inhibited by the specific NK1 receptor antagonist, SR140333. Cy3-SP was colocalised with NK1 receptor IR and was internalised with the NK1 receptor. These results show that enteric neurons exhibit authentic NK1 receptors that are rapidly internalised when exposed to their preferred ligand.