Table 1.
HPLC parameters.
Fig 1.
Chemical characterization of Endomorphine-TAMRA conjugates.
(A) Chemical structure of EM-TAMRA indicated by modified Endomorphine-1 peptide in blue and TAMRA-Maleimide in magenta. Absorbance and Emission spectra of (B) 200 μM TAMRA-Maleimide and (C) 20 μM EM-TAMRA at pH 7.24. (D) Absorbance of 20 μM EM-TAMRA at different pH 2.15–10. INormalized = Normalized intensity; Iabs = Normalized absorbance.
Fig 2.
Keratinocytes bind and internalize Endomorphine-TAMRA conjugate.
Keratinocytes were subjected to live cell imaging using confocal microscopy at 37°C. After labeling membranes and endoplasmic reticulum with WGA (green), 200 nM EM-TAMRA was added to the cells and image acquisition started immediately. (A) Representative images of a colony of basal N/TERT-1 cells show strong membrane binding of EM-TAMRA (magenta) in some areas and more diffuse staining in other regions (arrows). (B) After seven days of differentiation more pronounced membrane staining is visible. Small puncta appear close to the membrane as a result of the fast internalization process. (C) Time course of EM-TAMRA internalization in basal keratinocytes. At the start strong membrane binding is visible. After 10 min the majority of EM-TAMRA has been internalized and appears in the ER/Golgi perinuclear network as indicated by WGA co-labeling in white (arrows). Some membrane staining is retained after 60 min incubation. The majority of EM-TAMRA accumulates intracellular (arrow in EM-TAMRA 60 min). Images displayed are SUM-projections of ten slices from a Z-stack image with 0.1 μM step size. Co-localization of EM-TAMRA and WGA appears white. Scale bar represents 10 μm. WGA = wheat germ agglutinin.
Fig 3.
Flow cytometry analyses of keratinocyte basal and differentiated populations bound by TAMRA and EM-TAMRA.
EM-TAMRA labeling of both basal and differentiated keratinocytes were performed in 4°C at a dose of 500 nM. The labeled cells were subjected to population based flow cytometry analysis to study basal keratinocytes (A and C) and differentiated keratinocytes (B and D) using TAMRA dye alone (A and B) and EM-TAMRA (C and D). Representative images in (C) and (D) show specific separation of sub-population in EM-TAMRA labeled basal and differentiated keratinocytes distinct from TAMRA dye alone. Specific population was observed with respective percentages for both basal (8.3%) and differentiated (22.2%) cell types as represented in Table E. Data shown are calculated from six independent biological replicates (N = 6) and represent mean ± SD for respective concentrations (10 nM – 2 μM).
Fig 4.
Competition with μ-OR ligands shows specificity of EM-TAMRA binding and differences between basal and differentiated keratinocyte populations.
(A) For live cell imaging, cells were pre-incubated with 10 μM competitor for five min before addition of EM-TAMRA (200 nM) in the presence of 10 μM competitor at 37°C. As compared to the control, Endomorphine-1 was more effective in competing for membrane binding with EM-TAMRA than CTOP and Naltrexone in basal N/TERT-1 cells. Scale bar represents 10 μm. (B) Flow cytometry analysis after 30 min pre-treatment with ligands on ice show significant reduction in EM-TAMRA positive populations during competition with Endomorphine-1 at low (100 nM) as well as high (10 μM) concentrations in both basal and differentiated keratinocytes. (C) CTOP (100 nM and 1 μM) was able to block EM-TAMRA binding in basal N/TERT-1 but not in differentiated cells. (D) Naltrexone shows competition at higher concentrations of 1 μM to 10 μM but not at low concentration of 100 nM. Data are represented as mean ± SD from four replicate experiments (N = 4) and were subjected to ordinary One-way ANOVA using Dunnett’s multiple comparison post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001. EM = Endomorphine-1; NTX = Naltrexone.
Fig 5.
Acute and chronic exposure of keratinocytes to opioid antagonist Naltrexone and agonist Endomorphine-1 affect cell membrane localization of μ-OR and enhances binding of EM-TAMRA.
(A) Live cell imaging shows reduced binding of EM-TAMRA (grey) in two day Endomorphine-1 and Endomorphie-1 + Naltrexone double pre-treated cells. Subtle differences are observed in Naltrexone treated cells. Scale bar represents 10μm. (B) In chronic, five day pre-treated cells a clear reduction of EM-TAMRA binding is observed in Endomorphine-1 and Naltrexone treated cells. The double treatment with Endomorphine-1 and Naltrexone results in increased binding of EM-TAMRA to keratinocytes. Scale bar represents 10 μm. (C) Flow cytometry analyses reveal reduction of EM-TAMRA binding in Endomorphine-1 and Naltrexone + Endomorphine-1 double treated basal cells after two day incubation similar to live cell imaging data from basal cells. In differentiated keratinocytes all treatments led to a reduction of EM-TAMRA positive populations. Only Endomorphine-1 and Naltrexone individual treatments display statistically significant reductions. (D) Chronic treatment with Naltrexone + Endomorphine-1 combination results in increased binding of EM-TAMRA in basal keratinocytes. In differentiated cells single compound treatment reduces EM-TAMRA positive populations but the double treatment with Naltrexone + Endomorphine-1 increases binding. Data are represented as mean ± SD from four replicate experiments (N = 4) and were subjected to ordinary One-way ANOVA using Dunnett’s multiple comparison post hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001. WGA = Wheat germ agglutinin, EM = Endomorphine-1; NTX = Naltrexone.