Characterisation of substance P-induced endocytosis of NK1 receptors on enteric neurons |
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Authors: | Bridget R Southwell Heather L Woodman Roger Murphy Stephanie J Royal John B Furness |
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Institution: | (1) Department of Anatomy and Cell Biology, University of Melbourne, 3052 Parkville, Victoria, Australia;(2) Department of Pharmacology, University of Melbourne, 3052 Parkville, Victoria, Australia |
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Abstract: | Immunoreactivity for NK1 receptors is confined to specific nerve cell bodies in the guinea-pig ileum, 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 microcopy. In tissue that was incubated at
4° C in the absence of SP, most NK1 receptor immunoreactivity (IR) was confined to surface membranes of nerve cells. At 37°
C in the presence of 10−7 M SP (plus 3×10−7M 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. |
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