Characterisation of substance P-induced endocytosis of NK1 receptors on enteric neurons

Abstract

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.

Neurons bearing NK 3 tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists

“…Mass spectrometry indicated a molecular mass of 2006 Da for Cy3.5-NKA and 2098 Da for Cy3.5-kassinin, suggesting, in each case, that the tachykinins were coupled to a single Cy3.5 molecule. The concentrations of Cy3.5-NKA and Cy3.5-kassinin were estimated from their peak heights on the HPLC profile when compared to the peak height of a known concentration of NKA or kassinin, respectively (Southwell et al 1996).…”

“…This method overcomes the problem of antibodies that fail to recognise a receptor even though it is known to be present. We have previously shown that Cy3-SP binds to neurons bearing tachykinin receptors and is internalised with the NK 1 receptor in myenteric neurons (Southwell et al 1996) and interstitial cells of Cajal (Lavin et al 1998) in the guinea-pig. Cy3-SP is also internalised by neurons that do not have NK 1 receptor and, therefore, are presumed to contain NK 3 receptors (Southwell et al 1996).…”

Tachykinins and their functions in the gastrointestinal tract

“…A further component of slow transmission is reduced by NK1 receptor block, indicating that both receptor types are involved in tachykinin transmission to the neurons [171]. The internalization of the NK1 receptor, detected by immunohistochemistry, also indicates that there is transmission to IPANs through this receptor type [172]. A major source of the tachykinin-mediated slow transmission to IPANs is probably other IPANs, which synapse with each other to form self-reinforcing circuits [173].…”

Localisation of neurokinin 3 (NK3) receptor immunoreactivity in the rat gastrointestinal tract

“…In both rat and guineapig, NK1r immunoreactivity is not found on the muscle but has been observed on a sub-set of neurons and on the interstitial cells of Cajal (Vigna et al 1994;Sternini et al 1995;Portbury et al 1996b). The authentic nature of the neuronal receptors has been demonstrated by co-localisation of receptor and ligand, and by specific ligandinduced endocytosis of the receptor (Southwell et al 1996). The localisation of the receptor by antiserum & p .…”