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Characterization of a naloxone-insensitive beta-endorphin receptor on murine peritoneal macrophages
J A Woods1, N A Shahabi, B M Sharp
1Endocrine-Neuroscience Research Laboratory, Minneapolis Medical Research Foundation, MN 55404, USA.
Abstract:
Previous studies from our laboratory have characterized a naloxone-insensitive beta-endorphin (beta-End) receptor on the human pro-monocytic cell line U937. Since monocytes are macrophage precursors, we sought to identify and characterize this site on fully differentiated effector macrophages. Mice (ICR females, 6-8 wk old) were injected (i.p.) with 1 mL of thioglycollate to induce an inflammatory response. Elicited cells were harvested 3 d later by lavage. Macrophages were enriched by adherence and analyzed via radioreceptor assay (with [125I] beta-End, 2,000 Ci mmol-1) as either intact cells or membrane preparations. Scatchard analysis revealed a single saturable binding site for beta-End (Kd = 9.75 +/- 2.6 x 10(-9) M; 8218 +/- 2360 sites/cell). Competition studies showed that other opiate receptor ligands including naloxone, DAMGO, U69593, or 2,5 DPDP-enkephalin were ineffective at displacing [125I] beta-End when compared to unlabeled beta-End. Analysis of competition studies utilizing fragments and analogs of beta-End revealed that beta-End (6-31) and beta-End (1-5, 16-31) were equipotent, and N-acetylated beta-End was less potent, than beta-end (1-31) in displacing [125I] beta-End binding. In contrast, beta-End (1-27) and beta-End (28-31) were ineffective. In summary, we have identified a naloxone-resistant beta-End binding site on murine peritoneal macrophages that is similar to one we have previously characterized on U937 cells and cultured murine splenocytes.
Insights
Researchers identified a novel beta-endorphin (beta-End) receptor on macrophages. This receptor is resistant to naloxone, suggesting a unique opioid signaling pathway in immune cells.
Area of Science:
- Immunology
- Neuroendocrinology
- Cell Biology
Background:
- Previous studies identified a naloxone-insensitive beta-endorphin (beta-End) receptor on U937 pro-monocytic cells.
- Monocytes differentiate into macrophages, suggesting potential expression of this receptor on effector macrophages.
Purpose of the Study:
- To identify and characterize the naloxone-insensitive beta-End receptor on fully differentiated murine peritoneal macrophages.
- To compare the characteristics of this receptor with the one found on U937 cells.
Main Methods:
- Murine peritoneal macrophages were elicited using thioglycollate injection.
- Radioreceptor assays with [125I] beta-End were performed on intact cells and membrane preparations.
- Scatchard and competition analyses were used to characterize binding kinetics and ligand specificity.
Main Results:
- A single, saturable binding site for beta-End was identified on macrophages (Kd = 9.75 ± 2.6 x 10^-9 M; 8218 ± 2360 sites/cell).
- The binding site was insensitive to naloxone and other standard opioid receptor ligands (DAMGO, U69593).
- Specific beta-End fragments (6-31 and 1-5, 16-31) showed potent binding, while others (N-acetylated, 1-27, 28-31) were less effective or inactive.
Conclusions:
- A naloxone-resistant beta-End binding site exists on murine peritoneal macrophages.
- This receptor shares characteristics with the previously identified site on U937 cells and splenocytes.
- The findings suggest a distinct opioid receptor system involved in macrophage function.