Elsevier

Neuroscience

Volume 87, Issue 4, 18 August 1998, Pages 925-931
Neuroscience

Quantitation of neurokinin 1 receptor internalization and recycling in guinea-pig myenteric neurons

https://doi.org/10.1016/S0306-4522(98)00176-6Get rights and content

Abstract

Agonist-induced endocytosis and recycling of G protein-coupled receptors contributes to desensitization and resensitization of the receptors. In this study, we have used fluorescence immunohistochemistry, confocal microscopy and digital image analysis to quantify the proportion of receptor in the cytoplasm and on the surfaces of nerve cells in the guinea-pig ileum. With these methods we examined the dynamics of internalization of the neurokinin 1 receptor in response to agonist, return of receptor to the cell membrane and its capacity to be re-internalized in response to further exposure to agonist. The basal level of neurokinin 1 receptor immunoreactivity in the cytoplasm was 12–15% of total cellular immunoreactivity. Concentration–response relations were generated for neurokinin 1 receptor internalization after incubation of isolated ileum with 10−11 to 10−6 M substance P at 4°C and warming to 37°C for 20 min. The threshold concentration for cytoplasmic receptor to exceed baseline was 10−11 M and the proportion of receptor in the cytoplasm increased with increasing substance P concentration. The effect of two exposures to agonist was studied using 10−8 M and 10−6 M substance P. After equilibration with substance P at 4°C for 1 h followed by 20 min at 37°C with no substance P, neurokinin 1 receptor immunoreactivity in the cytoplasm increased significantly from 12% to 36±3% for incubation with 10−8 M and to 64±3% for 10−6 M. When return of receptor to the surface was blocked with monensin (10−5 M), 90% of the receptor was in the cytoplasm after 1 h at 37°C following exposure to 10−6 M substance P. After 60 min without substance P and no monensin, receptor in the cytoplasm decreased to 19±2% (10−8 M) and 38±4% (10−6 M). A second period of equilibration with substance P at 4°C for 1 h followed by 20 min at 37°C, without substance P, resulted in a second wave of endocytosis; the fractions of receptor in the cytoplasm were 47±2% (10−8 M) and 70±2% (10−6 M).

These results indicate that most of the receptors on the cell surface are available for internalization and that the receptors that return to the cell surface after endocytosis rapidly regain their ability to bind ligand and undergo endocytosis.

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)

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