Elsevier

Neuroscience

Volume 91, Issue 1, June 1999, Pages 353-362
Neuroscience

Internalization of the neurokinin 1 receptor in rat myenteric neurons

https://doi.org/10.1016/S0306-4522(98)00595-8Get rights and content

Abstract

Immunoreactivity for the neurokinin 1 receptor is contained in nerve cell bodies that have been deduced to be intrinsic primary afferent neurons in the myenteric plexus of the rat ileum. This study shows that neurokinin 1 receptor immunoreactivity on these neurons represents receptors that can bind agonist and undergo endocytosis, and explores the properties of that endocytosis. Segments of rat ileum were incubated in Hanks' balanced salt solution for 1 h at 4°C, followed by 1 h at 37°C in physiological saline solution with nicardipine and tetrodotoxin, in the presence or absence of substance P. Tissue was then fixed and whole-mount preparations were processed for fluorescence immunohistochemistry, using antibodies raised against the C-terminus of the neurokinin 1 receptor. The intracellular and surface distributions of receptor immunoreactivity were analysed using confocal microscopy and quantified by computer analysis. In tissue not exposed to substance P, most neurokinin 1 receptor immunoreactivity was confined to the surfaces of nerve cells, and 29% was intracellular. Exogenous substance P (10−6 M) caused an increase in the amount of intracellular receptor to 72%. This internalization was concentration dependent, and maximum receptor internalization was achieved between 10−6 M and 10−5 M substance P (ec50=4.9±1.6×10−7 M). The specific neurokinin 1 receptor antagonist, SR104333 (10−6 M), inhibited substance P-induced endocytosis. In tissue that was incubated in 5×10−5 M monensin (to trap endocytosed receptor in the cell), without the addition of substance P, a high level of intracellular neurokinin 1 receptor immunoreactivity (81%) was also present. We deduce that endocytosis in the presence of monensin was stimulated by the release of tachykinins from intrinsic nerve endings, based on the following evidence: when endogenous release of tachykinin was blocked using a high magnesium/low calcium solution, or binding of tachykinins to the receptor was prevented using 10−6 M SR140333, the intracellular receptor immunoreactivity remained at approximately 40%. Incubation with hypertonic sucrose also trapped receptors on the cell surface. Use of these protocols that modify receptor trafficking showed that agonist induced the neurokinin 1 receptors to aggregate, accumulate in endocytotic vesicles, move to perinuclear organelles and recycle to the surface in less than 1 h.

This study indicates that there is sufficient release of endogenous tachykinins in the rat ileum to cause receptor internalization and implies that these intrinsic primary afferent neurons are likely to be under continuous influence from tachykinins in the normal intestine.

Section snippets

Experimental procedures

Male Sprague–Dawley rats (University of Melbourne colony), in the weight range 200–250 g, were killed by a blow to the head and exsanguination. Ileum was removed and placed in a physiological saline solution (PSS; composition in mM: NaCl 118, KCl 4.8, NaHCO3 25, NaH2PO4 1.0, MgSO4 1.2, glucose 11.1, CaCl2 2.5; equilibrated with 95% O2 and 5% CO2) containing 10−6 M nicardipine (prevents muscle contraction) and 3×10−7 M tetrodotoxin (TTX). TTX specifically blocks propagation of action potentials in

Localization of neurokinin 1 receptors in rat myenteric neurons not exposed to exogenous substance P

In NK1r-IR nerve cells from segments of rat ileum that were fixed immediately after removal from the animal, the amount of intracellular NK1r-IR was 14.7±1.7% (Table 1, Protocol 1). Most of the receptor was located on the cell surface and fluorescence was distributed as an even line along the plasma membrane and the proximal parts of cell processes. NK1r fluorescence extended 3–4 cell body lengths along the processes.

In nerve cells that were fixed following incubation for 1 h at 4°C, most NK1

Substance P-induced neurokinin 1 receptor endocytosis in rat myenteric neurons

In neurons of tissue taken from the rat immediately after killing, 80% of the NK1r-IR was on the surface of the cell body and proximal parts of the cell processes (Fig. 1B). Addition of the tachykinin (SP) induced the NK1r on the cell bodies and in the proximal processes to internalize (Fig. 1C). Quantitation of receptor trafficking was only performed on the cell bodies. Internalization of the NK1r following addition of SP was concentration dependent and NK1r internalization was dependent on

Conclusions

The present results indicate that there is a rapid internalization and cycling of NK1 receptors in the intrinsic primary afferent neurons in the rat intestine, and that these neurons are continually exposed to tachykinins released from within the myenteric plexus, and imply that there is normally a background synaptic excitation of intrinsic primary afferent neurons.

Acknowledgements

We thank Dr Karl Jenkinson for his expert advice on the experiments and the manuscript. This work was supported by a Program grant from the National Health and Medical Research Council of Australia. Patricia Mann is a holder of a Gastroenterological Society of Australia Postgraduate Research Scholarship, and Bridget Southwell is a Gastroenterological Society of Australia Research Fellow.

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