Journal: The Journal of Neuroscience

Article Title: Peptidases Prevent μ-Opioid Receptor Internalization in Dorsal Horn Neurons by Endogenously Released Opioids

doi: 10.1523/JNEUROSCI.23-05-01847.2003

Figure Lengend Snippet: The N-terminal sequence of opioids encoded by the proenkephalin, prodynorphin and proopiomelanocortin genes is shown with arrows pointing to the bonds cleaved by the peptidases. Dipeptidyl carboxypeptidase I cleaves dipeptides sequentially from the carboxy terminus. Therefore, depending on whether the peptide has an odd (Met- and Leu-enkephalin, dynorphin A) or even (Met-enk-Arg-Gly-Leu, dynorphin-(1–8)) number of amino acid residues, this peptidase would cleave the bonds indicated by the dark or by the clear arrows, respectively (Guyon et al., 1979; Hiranuma et al., 1997; Hiranuma et al., 1998a; Hiranuma et al., 1998b). The peptidase inhibitors used in this study are shown with arrows pointing to their corresponding enzymes. Neutral endopeptidase is also known as enkephalinase, and dipeptidyl carboxypeptidase I has also been called angiotensin 1 converting enzyme and kininase II.

Article Snippet: Primary antibodies were: the rabbit MOR-1 antibody described above, a mouse monoclonal antibody (3-E7) recognizing β-endorphin, enkephalins and dynorphins (Gramsch Laboratories, Schwabhausen, Germany); a rabbit IgG (affinity purified) raised against endomorphin-2 (Chemicon, Temecula, CA, catalog no. AB5106), and a goat polyclonal antibody (affinity-purified) raised against the carboxy terminus of mouse MOR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-7488).

Techniques: Sequencing

Journal: The Journal of Neuroscience

Article Title: Peptidases Prevent μ-Opioid Receptor Internalization in Dorsal Horn Neurons by Endogenously Released Opioids

doi: 10.1523/JNEUROSCI.23-05-01847.2003

Figure Lengend Snippet: Spinal cord slices were incubated for 20 min with opioids and peptidase inhibitors (“PI”: 10 μM amastatin, captopril and phosphoramidon). Treatments were: A. control; B. 1 μM endomorphin-1 (“EM-1”); C. 100 nM endomorphin-2 (“EM-2”); D. 1 μM Leu-enkephalin (“Leu-enk”); E. 1 μM Leu-enkephalin and peptidase inhibitors; F. 1 μM Leu-enkephalin and peptidase inhibitors, 10 μM actinonin (“act.”) instead of amastatin; G. 1 μM Leu-enkephalin and peptidase inhibitors, 100 μM bestatin (“best.”) instead of amastatin. H. 1 μM dynorphin A (“dyn A”); I. 1 μM dynorphin A and peptidase inhibitors. Cells in panels A, D and H show no internalization, the rest show clear internalization. Dorsal is up for all panels except H (dorsal is right). Confocal images (100x, zoom of 2) are 2 optical sections (3 in G and H) through the center of lamina II neurons at intervals of 0.49 or 0.57 μm, out of 5–7 optical sections. Scale bar is 5 μm.

Article Snippet: Primary antibodies were: the rabbit MOR-1 antibody described above, a mouse monoclonal antibody (3-E7) recognizing β-endorphin, enkephalins and dynorphins (Gramsch Laboratories, Schwabhausen, Germany); a rabbit IgG (affinity purified) raised against endomorphin-2 (Chemicon, Temecula, CA, catalog no. AB5106), and a goat polyclonal antibody (affinity-purified) raised against the carboxy terminus of mouse MOR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-7488).

Techniques: Incubation

Journal: The Journal of Neuroscience

Article Title: Peptidases Prevent μ-Opioid Receptor Internalization in Dorsal Horn Neurons by Endogenously Released Opioids

doi: 10.1523/JNEUROSCI.23-05-01847.2003

Figure Lengend Snippet: Spinal cord slices were incubated for 10 min without (A) or with (B) peptidase inhibitors and the opioids and opioid antagonists indicated. Peptidase inhibitors were 10 μM amastatin, captopril and phosphoramidon. Leu-enkephalin (“Leu-enk”), Met-enkephalin (“Met-enk”), α-neoendorphin, endomorphin-1 (“EM-1”), β-endorphin and CTAP were 1 μM, dynorphin A was 3 μM, endomorphin-2 (“EM-2”) was 0.1 μM, and naloxone was 10 μM. Bars represent the mean ± SEM of 3–5 slices (except “none”, 10 slices). Separate ANOVA for panels A and B revealed overall significance (p<0.0001). Tukey’s posttest revealed the significant differences from “none” indicated by asterisks (***, p<0.001), and significant differences (p<0.001) between “EM-2” and “EM-2 + CTAP” (panel A), “Leu-enk” and “Leu-enk + naloxone” (panel B), and “dynorphin A” and “dynorphin A + CTAP” (panel B).

Article Snippet: Primary antibodies were: the rabbit MOR-1 antibody described above, a mouse monoclonal antibody (3-E7) recognizing β-endorphin, enkephalins and dynorphins (Gramsch Laboratories, Schwabhausen, Germany); a rabbit IgG (affinity purified) raised against endomorphin-2 (Chemicon, Temecula, CA, catalog no. AB5106), and a goat polyclonal antibody (affinity-purified) raised against the carboxy terminus of mouse MOR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-7488).

Techniques: Incubation

Journal: The Journal of Neuroscience

Article Title: Peptidases Prevent μ-Opioid Receptor Internalization in Dorsal Horn Neurons by Endogenously Released Opioids

doi: 10.1523/JNEUROSCI.23-05-01847.2003

Figure Lengend Snippet: Spinal cord slices were incubated for 10 min without (control) or with peptidase inhibitors, and the indicated concentrations of Leu-enkephalin (A), dynorphin-A (B), endomorphin-1 (C) or endomorphin-2 (D). Peptidase inhibitors were 10 μM amastatin, captopril and phosphoramidon (A and B), or these three inhibitors plus 10 μM Ala-pyrrolidine-CN (C and D). Points are the mean ± SEM of 3–5 slices. Curves were generated by fitting a sigmoidal dose-response curve to the data. Numbers next to the curves indicate EC50 values: A. Leu-enkephalin: EC50 = 22 μM (95% C.I. 17–30 μM, R2 = 0.96); Leu-enkephalin (peptidase inhibitors): EC50 = 0.38 μM (95% C.I. 0.31–0.47 μM, nH = 2.8 ± 0.7, R2 = 0.82). B. Dynorphin A: EC50 = 5.2 μM (95% C.I. = 4.3–6.3 μM, nH = 3.0 ± 0.6, R2 = 0.90); dynorphin A (peptidase inhibitors): EC50 = 0.60 μM (95% C.I. 0.34–1.04 μM, R2 = 0.83). C. Endomorphin-1: EC50 = 32 nM (95% C.I. 16–65 nM, R2 = 0.73); endomorphin-1 (peptidase inhibitors): EC50 = 40 nM (95% C.I. 22–73 nM, R2 = 0.77). D. Endomorphin-2: EC50 = 5 nM (95% C.I. 3–8 nM, R2 = 0.92); endomorphin-2 (peptidase inhibitors): EC50 = 10 nM (95% C.I. 6–15 nM, R2 = 0.94). Unless otherwise indicated, nH values were fixed to 1.0 according to an F-test. Values of the “top” parameter were fixed to 100%.

Article Snippet: Primary antibodies were: the rabbit MOR-1 antibody described above, a mouse monoclonal antibody (3-E7) recognizing β-endorphin, enkephalins and dynorphins (Gramsch Laboratories, Schwabhausen, Germany); a rabbit IgG (affinity purified) raised against endomorphin-2 (Chemicon, Temecula, CA, catalog no. AB5106), and a goat polyclonal antibody (affinity-purified) raised against the carboxy terminus of mouse MOR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-7488).

Techniques: Incubation, Generated

Journal: The Journal of Neuroscience

Article Title: Peptidases Prevent μ-Opioid Receptor Internalization in Dorsal Horn Neurons by Endogenously Released Opioids

doi: 10.1523/JNEUROSCI.23-05-01847.2003

Figure Lengend Snippet: Spinal cord slices were incubated at 35 °C with peptidase inhibitors (10 μM unless indicated otherwise); after 10 min, 1 μM Leu-enkephalin (Leu-enk, A and B) or dynorphin A (C) were added for 10 min more. Bars represent the mean ± SEM of 3–5 slices. Abbreviations: “none”, no peptidase inhibitors; “ama”, amastatin; “capt”, captopril; “PhA”, phosphoramidon; “thio”, thiorphan; “act”, actinonin. A. Effects on the MOR-1 internalization produced by Leu-enkephalin of different combinations of an aminopeptidase inhibitor (amastatin), a dipeptidyl carboxypeptidase inhibitor (captopril) and inhibitors of neutral endopeptidase (phosphoramidon or thiorphan). B. Effects on the MOR-1 internalization produced by Leu-enkephalin of different aminopeptidase inhibitors in combination with phosphoramidon and captopril. ANOVA of data in A and B together revealed overall significance (p<0.0001). C. Effects on the MOR-1 internalization produced by dynorphin A of different combinations of amastatin, captopril and phosphoramidon. ANOVA yielded overall p<0.0001. Statistical differences from “none”: *, p<0.05; **, p<0.01; ***, p<0.001 (Tukey’s post-tests).

Article Snippet: Primary antibodies were: the rabbit MOR-1 antibody described above, a mouse monoclonal antibody (3-E7) recognizing β-endorphin, enkephalins and dynorphins (Gramsch Laboratories, Schwabhausen, Germany); a rabbit IgG (affinity purified) raised against endomorphin-2 (Chemicon, Temecula, CA, catalog no. AB5106), and a goat polyclonal antibody (affinity-purified) raised against the carboxy terminus of mouse MOR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-7488).

Techniques: Incubation, Produced

Journal: The Journal of Neuroscience

Article Title: Peptidases Prevent μ-Opioid Receptor Internalization in Dorsal Horn Neurons by Endogenously Released Opioids

doi: 10.1523/JNEUROSCI.23-05-01847.2003

Figure Lengend Snippet: Sections (25 μm) from the lumbar spinal cord of an adult rat were double labeled with a monoclonal antibody (3-E7) recognizing enkephalins, β-endorphin, dynorphins and α-neoendorphin (“opioids”, red), and the MOR-1 antibody “MOR”, green). A. Single optical section at 10x (scale bar is 100 μm) from a coronal section. B. Two optical sections at 100x (scale bar is 10 μm) from a sagittal section. Arrows indicate sites of possible co-localization (in yellow); “I” and “II” indicate the approximate location of laminae I and II, respectively.

Article Snippet: Primary antibodies were: the rabbit MOR-1 antibody described above, a mouse monoclonal antibody (3-E7) recognizing β-endorphin, enkephalins and dynorphins (Gramsch Laboratories, Schwabhausen, Germany); a rabbit IgG (affinity purified) raised against endomorphin-2 (Chemicon, Temecula, CA, catalog no. AB5106), and a goat polyclonal antibody (affinity-purified) raised against the carboxy terminus of mouse MOR-1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. sc-7488).

Techniques: Labeling