Abstract
Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, β-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, β-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, β-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of β-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of β-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.
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Abbreviations
- Dyn A8:
-
Dynorphin A8
- ELISA:
-
Enzyme-linked immunosorbent assay
- PAG:
-
Periaqueductal gray
- POMC:
-
Proopiomelanocortin
- RIA:
-
Radioimmunoassay
- WT:
-
Wild-type
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Acknowledgements
This work was supported by the National Institute of Health Grants DA008863 and NS026880 (to LAD), DA08622 (to JP), and DK066604 (to MJL).
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This work was supported by the National Institutes of Health Grants DA008863 and NS026880 (to LAD), DA08622 (to JP), and DK066604 (to MJL).
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AG, SG, HP, carried out the research; DLRO helped write the manuscript; MDH, MJL and JEP generated the knockout mice and helped with experimental design, LAD helped with experimental design, data interpretation and manuscript preparation; IG carried out the statistical analysis and wrote the manuscript.
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10571_2020_1015_MOESM1_ESM.pdf
Relationship between antibody recognition and receptor number. CHO cells expressing either µOR (A), δOR (B), or κOR (C) (0-4×106 cells) were subjected to ELISA using μOR (1A4), δOR (2B1) or κOR (7AG-9) monoclonal antibodies as described in Methods. In parallel binding assays were carried out using 10 nM [3H]DAMGO, [3H]Deltorphin II or [3H]U69,593 as described in Methods. Results are Mean±SE of 2 experiments in triplicate. r2 = correlation coefficient. (D-F) ELISA showing selectivity of μOR (D), δOR (E), and κOR (F) monoclonal antibodies in CHO cells alone or expressing either μOR, δOR, or κOR. (G-I) ELISA showing selectivity of μOR (G), δOR (H), and κOR (I) monoclonal antibodies in endogenous tissue. Results are Mean±SE of 3 experiments in quintuplicate. One-way ANOVA; Dunnett’s multiple comparison test v/s wild-type (WT); **p<0.01; ***p<0.001; ****p<0.0001. Supplementary material 1 (492 kb)
10571_2020_1015_MOESM2_ESM.pdf
Fig. S2 Analysis of immunoreactive Leu-enkephalin and Dyn A8 in mouse brain. Extracts from the brains of 3 age- and sex-matched wild-type, Enk-/-, End-/- and End-/-/ Enk-/- mice were pooled, and 100 μl was subjected to gel filtration chromatography on Superdex Peptide 10/30 column as described in Methods. (A-B) Gel filtration fractions from male (A) and female (B) mice were analyzed for immunoreactive Leu-enkephalin as described in Methods. Area under the curve for ir-Leu-enkephalin with wild-type (WT) animals was taken as 100%. (C-D) Gel filtration fractions from male (C) and female (D) mice were analyzed for immunoreactive Dyn A8 as described in Methods. Area under the curve for ir-Dyn A8 with wild-type (WT) animals was taken as 100%. Molecular mass calibration standards are as follows: cytochrome c, 12.4 kDa; ACTH, 4.6 kDa; β-endorphin, 3.5 kDa; α-MSH, 1.7 kDa; Dyn A8, 1.0 kDa; Leu-enkephalin, 0.55. ir, immunoreactive. One-way ANOVA; Dunnett’s multiple comparison test v/s wild-type (WT); ***p<0.001; ****p<0.0001. Supplementary material 1 (432 kb)
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Gupta, A., Gullapalli, S., Pan, H. et al. Regulation of Opioid Receptors by Their Endogenous Opioid Peptides. Cell Mol Neurobiol 41, 1103–1118 (2021). https://doi.org/10.1007/s10571-020-01015-w
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DOI: https://doi.org/10.1007/s10571-020-01015-w