Quantitation of neurokinin 1 receptor internalization and recycling in guinea-pig myenteric neurons
Section snippets
Source of reagents
Nicardipine, bovine serum albumin, chymostatin, bacitracin and monensin were from Sigma, St Louis, MO, U.S.A.; dimethylsuphoxide from Univar, Ajax Chemicals, Melbourne, Australia; and Triton X-100 from BDH, Melbourne, Australia. Horse serum was from Hunter Antibodies, Australian Lab Services, Newcastle, Australia; tetrodotoxin (TTX) from Sapphire Bioscience Pty. Ltd., Alexandria, New South Wales, Australia; and Hanks' balanced salt solution (HBSS) from Commonwealth Serum Laboratories,
Internalization of neurokinin 1 receptor in response to different concentrations of substance P
In neurons in gut incubated without substance P at 4°C and warmed to 37°C for 20 min also without substance P, the NK1 receptor was distributed predominantly as a line along the cell membrane (Fig. 1A). NK1 receptor is present on the cell body and the initial parts of the processes of these neurons.[10]Incubation with substance P at 4°C, followed by 20 min at 37°C with no substance P resulted in NK1 receptor endocytosis. The fluorescence was distributed as small clumps in the cytoplasm (Fig. 1B)
Discussion
For the investigation of molecular translocation, microscope studies have an advantage over techniques that require cell disruption, in that microscopy identifies the cells in which internalization has occurred, but the results are often difficult to quantify. In the present study, we describe two methods to quantify the internalization of NK1 receptors using immunohistochemistry and confocal microscopy. In one method, we have counted the numbers of endosomes in the cytoplasm in single optical
Conclusions
In this study, quantitative methods have been developed with which the cycling of the NK1 receptor between the surface and cytoplasm of neurons has been investigated. The work demonstrates that nearly all of the NK1 receptor on the cell surface can undergo agonist-induced endocytosis, that NK1 receptor can repetitively cycle between the cell surface and cytoplasm of enteric neurons in vitro and that previous cycling does not diminish the amount of receptor that is internalized in response to
Acknowledgements
This study was supported by NH & MRC Program Grant 963213 (BRS, HLW, SJR, KJ, JBF) and NIH/NS 17702 (VSS). The antiserum against the NK1 receptor (no. 94168) was kindly provided by CURE/Gastroenteric Biology Center, Antibody/RIA core, NIH grant no. 41301 and CP-99994 by Pfizer Inc., Groton, Connecticut, U.S.A. We thank Jan Morgan for technical assistance and Heather Young for valuable comments on the protocols and the manuscript.
References (17)
- et al.
A highly conserved tyrosine residue in G protein-coupled receptors is required for agonist-mediated β2-adrenergic receptor sequestration
J. biol. Chem
(1994) - et al.
Endocytosis and recycling of neurokinin 1 receptors in enteric neurons
Neuroscience
(1996) - et al.
Characterization and regulation of neurokinin1 receptors in primary cultures of rat neonatal spinal neurons
Neuroscience
(1995) - et al.
Regulatory mechanisms that modulate signalling by G-protein-coupled receptors
Biochem. J.
(1997) - et al.
G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins
Can. J. Physiol. Pharmac.
(1996) - et al.
Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors
Molec. Pharmac.
(1996) - et al.
β-arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor
Nature
(1996) - et al.
Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor
Molec. Biol. Cell
(1995)
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