Detection of Kappa Opioid Receptors on Mouse Thymocyte Phenotypic Subpopulations as Assessed by Flow Cytometry1
- Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York
Abstract
Recent studies have shown kappa opioid receptor labeling on the R1EGO thymoma cell line by indirect immunofluorescence and flow cytometric analysis. The present study used a fluorescein-labeled arylacetamide (FITC-AA), a kappa opioid ligand, in conjunction with biotin-conjugated anti-fluorescein IgG and extravidin-R-phycoerythrin (PE), along with double-labeling with antibodies against specific immune cell surface markers to determine which subpopulation(s) of thymocytes express the kappaopioid receptor. Thymocytes, isolated from 6- to 8-week-old C57BL/6ByJ mice, incubated with FITC-AA followed by the PE amplification procedure, demonstrated labeling of the kappa opioid receptor. This labeling was inhibited 55 ± 4% above background by excess nor-binaltorphimine (nor-BNI), a kappaselective antagonist. This kappa opioid receptor positive population consisted of 58 ± 2% of all gated thymocytes. Phenotypic characterization determined that not only were 64 ± 3% of the gated thymocytes CD4+/kappa opioid receptor positive, but 60 ± 1% of all thymocytes were CD8+/ kappa opioid receptor positive. Two subpopulations of CD3+ thymocytes, consisting of both mature and immature cells, also displayed labeling for the kappa opioid receptor. Double-labeling of thymocytes with anti-CD4 and anti-CD8 antibodies demonstrated 82 ± 0.5% of these cells were of the double-positive phenotype. Therefore, these findings demonstrate that the thymocytes, which express the kappa opioid receptor, are predominantly of the immature CD4+/CD8+ phenotype. Collectively, these findings not only establish the presence of thekappa opioid receptor on immune cells involved in opioid responsiveness, but further indicate that this technique allows for the identification of distinct lymphocyte subpopulations which express the receptor.
Footnotes
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Send reprint requests to: Dr. Jean M. Bidlack, Department of Pharmacology and Physiology, P.O. Box 711, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642-8711.
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↵1 This work was supported by U.S. Public Health Service grants DA04355 and DA09676 from the National Institute on Drug Abuse.
- Abbreviations:
- FITC-AA
- fluorescein-conjugated arylacetamide (2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide)
- nor-BNI
- nor-binaltorphimine
- PE
- extravidin-R-phycoerythrin
- PI
- propidium iodide
- QR
- quantum red
- BSS
- balanced salt solution
- ICI 174
- 864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (where Aib is α-aminoisobutyric acid)
- DPDPE
- [d-Pen2,d-Pen5]enkephalin
- DAMGO
- [d-Ala2,N(Me)Phe4,Gly-ol]enkephalin
- FSC
- forward light scatter
- mAb
- monoclonal antibody
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
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- Received March 26, 1997.
- Accepted September 8, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
