Role of agonist-dependent receptor internalization in the regulation of μ opioid receptors
Section snippets
Experimental animals
Animal care and procedures were in accordance with the National Institutes of Health recommendations for the humane use of animals. All experimental procedures were reviewed and approved by the appropriate Animal Use Committee of the institutions where the experiments were performed. Because in vitro preparations were used throughout the study, discomfort was reduced to a minimum. The number of animals used was also kept to the minimum necessary for a meaningful interpretation of the data. Male
μ Opioid receptor internalization in enteric neurons of the ileum in vitro
As we reported previously,36 μ receptors were primarily localized to enteric neurons with a Dogiel type I morphology, characterized by an ovoid cell body, several thick dendrites protruding from the cell body (Fig. 1A) and a long axonal process (not shown). μ Receptor immunoreactivity was predominantly localized at the cell surface membrane (Fig. 1A). In enteric neurons from LMMPs exposed to electrical stimulation to induce endogenous release of opioids, μ receptor immunoreactivity was
Discussion
In the present study, we demonstrated that (i) endogenous release of opioids by electrical stimulation and exogenous application of the μ receptor-selective enkephalin analog, DAMGO, induce endocytosis of native μ receptors in enteric neurons, (ii) ligand-induced receptor endocytosis also occurs when opioid spare receptors have been inactivated, (iii) inactivation of spare receptors plus a fraction of functional receptors reduces the maximal response of neuromuscular preparations to opioid
Conclusions
This study provides the first evidence for the occurrence of μ receptor endocytosis in neurons by endogenously released enkephalins. This is an important observation, since it could allow investigations of the involvement of neurons bearing these receptors in physiological processes. For instance, the process of receptor internalization has served for the demonstration of substance P involvement in pain pathways and inflammation.1., 22. We also show that μ receptor endocytosis and inactivation
Acknowledgements
This research was supported by National Institutes of Health Grants DK54155 (C.S.), DK41301 (Morphology/Imaging subsection, C.S.), EY04067 (N.C.B.), DA05010 (Antibody and Imaging subsection, N.C.B.), NATO Collaborative Exchange Grant (C.S., M.T.), FAR (M.T.), VA Career Scientist Funds (N.C.B.) and University of Pavia Special Research Project Funds 1999 (M.T.). The authors wish to thank Dr Lawrence Kruger for critical reading of the manuscript and Ms Annarita Lievore for her contribution to
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