Internalization of the neurokinin 1 receptor in rat myenteric neurons
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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