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.
Bibliographical noteFunding Information:
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.
- Enteric neurons
- Neurokinin 1 receptor
- Receptor trafficking