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The Journal of Neuroscience, March 1, 2003, 23(5):1847
Peptidases Prevent µ-Opioid Receptor Internalization in Dorsal Horn Neurons by Endogenously Released Opioids
Bingbing Song and Juan Carlos G. Marvizón Center for Neurovisceral Sciences and Women's Health, Division of Digestive Diseases, Department of Medicine, Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095
ABSTRACT
TOP
ABSTRACT
Introduction
To evaluate the effect of peptidases on µ-opioid receptor (MOR) activation by endogenous opioids, we measured MOR-1 internalization in rat spinal cord slices. A mixture of inhibitors of aminopeptidases (amastatin), dipeptidyl carboxypeptidase (captopril), and neutral endopeptidase (phosphoramidon) dramatically increased the potencies of Leu-enkephalin and dynorphin A to produce MOR-1 internalization, and also enhanced the effects of Met-enkephalin and
-neoendorphin, but not endomorphins or
-endorphin. The omission of any one inhibitor abolished Leu-enkephalin-induced internalization, indicating that all three peptidases degraded enkephalins. Amastatin preserved dynorphin A-induced internalization, and phosphoramidon, but not captopril, increased this effect, indicating that the effect of dynorphin A was prevented by aminopeptidases and neutral endopeptidase. Veratridine (30 µM) or 50 mM KCl produced MOR-1 internalization in the presence of peptidase inhibitors, but little or no internalization in their absence. These effects were attributed to opioid release, because they were abolished by the selective MOR antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) and were Ca2+ dependent. The effect of veratridine was protected by phosphoramidon plus amastatin or captopril, but not by amastatin plus captopril or by phosphoramidon alone, indicating that released opioids are primarily cleaved by neutral endopeptidase, with a lesser involvement of aminopeptidases and dipeptidyl carboxypeptidase. Therefore, because the potencies of endomorphin-1 and endomorphin-2 to elicit internalization were unaffected by peptidase inhibitors, the opioids released by veratridine were not endomorphins. Confocal microscopy revealed that MOR-1-expressing neurons were in close proximity to terminals containing opioids with enkephalin-like sequences. These findings indicate that peptidases prevent the activation of extrasynaptic MOR-1 in dorsal horn neurons.
Key words: amastatin; aminopeptidase; captopril; dipeptidyl carboxypeptidase; dynorphin; endocytosis; endomorphin; endorphin; enkephalin; internalization; µ-opioid receptor; neutral endopeptidase; opioid; peptidase; phosphoramidon; rat; release; spinal cord
Introduction
Pain neurophysiology was greatly advanced by the discovery of opioid receptors and endogenous opioids (for review, see Mansour et al., 1988
; Law et al., 2000
In the dorsal horn, MOR-1 is present in dorsal horn interneurons (Kemp et al., 1996
We hypothesized that MOR-1 internalization by endogenous opioids is prevented by their rapid degradation by peptidases. Indeed, enkephalins (Guyon et al., 1979
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Figure 1. Opioid peptide bonds cleaved by peptidases. The N-terminal sequence of opioids encoded by the proenkephalin, prodynorphin, and pro-opiomelanocortin genes is shown with arrows pointing to the bonds cleaved by the peptidases. Dipeptidyl carboxypeptidase I cleaves dipeptides sequentially from the C terminal. Therefore, depending on whether the peptide has an odd (Met-enkephalin 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 black or by the gray arrows, respectively (Guyon et al., 1979
Materials and Methods
Chemicals. Ala-pyrrolidine-nitrile (Li et al., 1995
Media for slices. Artificial CSF (ACSF) contained (in mM): 124 NaCl, 1.9 KCl, 26 NaHCO3, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, and 10 glucose, and was 305 mOsm. K+-ACSF contained a higher concentration (5 mM) of KCl. Sucrose-ACSF was identical to ACSF, except that NaCl was iso-osmotically replaced by sucrose (215 mM) and the concentration of KCl was 5 mM. To depolarize the slices, we used ACSF (50 mM KCl) in which the concentration of KCl was increased to 50 mM and the concentration of NaCl was decreased to 74 mM. All of these media were constantly bubbled with 95% O2 and 5% CO2 to a pH of 7.4.
Spinal cord slice preparation. All animal procedures were approved by the Chancellor's Animal Research Committee at the University of California Los Angeles and conform to National Institutes of Health guidelines. Slices were prepared as described previously (Randic et al., 1993
Slice treatment. Slices were placed in a nylon net suspended halfway inside a small beaker and incubated at 35°C with ACSF containing various compounds, constantly bubbled with 95% O2 and 5% CO2. Peptidase inhibitors (usually amastatin, phosphoramidon, and captopril) were used at 10 µM, unless otherwise indicated. Phosphoramidon was always added together with 6 µM dithiothreitol to protect it against oxidation. The incubation was ended by placing the slices in cold fixative.
MOR-1 immunohistochemistry in spinal cord slices. Histological sections from spinal cord slices were prepared and labeled as described previously (Marvizon et al., 1999b
Double-label immunohistochemistry. A similar procedure was used to double-label spinal cord sections for endomorphins, enkephalins, and MOR-1. Adult male rats were anesthetized with isoflurane, killed by bilateral thoracotomy, and fixed by aortal perfusion. Two spinal cord segments (L3-L2 and L4) were postfixed, cryoprotected, frozen, and sectioned at 25 µm in the sagittal and coronal planes, respectively. Sections were incubated simultaneously with the two primary antibodies diluted in PBS/Triton containing 1% normal serum for 1 hr at room temperature and overnight at 4°C. After three washes, sections were incubated simultaneously with the two secondary antibodies for 2 hr at room temperature. The primary antibodies were the rabbit MOR-1 antibody described above, a mouse monoclonal antibody (3-E7) recognizing
Antibody characterization. The 3-E7 mouse monoclonal antibody recognizes the N-terminal sequence Tyr-Gly-Gly-Phe-Met of
Confocal microscopy and image processing. Confocal images were acquired at the University of California Los Angeles Carol Moss Spivak Cell Imaging Facility with a Leica (Nussloch, Germany) TCS-SP confocal microscope with argon (476 and 488 nm) and krypton (568 nm) lasers, using a pinhole of 1.0 Airy units, and objectives of 10× (0.4 numerical aperture) or 100× (1.4 numerical aperture), giving an optical section thickness (full width half maximum) of 8.13 and 0.62 µm, respectively. A zoom factor of 2 was used with some images taken at 100× to increase the pixel resolution of the resulting digital files. For each image at 100×, stacks of 5-10 optical sections were obtained at intervals of 0.49 or 0.57 µm. Each optical section was averaged up to six times to reduce noise. Images were processed using Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA). The "curves" feature of the program was used to adjust the contrast and to balance the colors of double-label images. Images were acquired at a digital size of 1024 × 1024 pixels and were later cropped to the relevant part of the field without altering the original image resolution.
Quantification of MOR-1 internalization. The amount of internalization was quantified essentially as described previously (Mantyh et al., 1995
Statistical analysis. Treatments were randomized between slices, and no more than two slices from the same animal received the same treatment. Data were analyzed using GraphPad Prism version 3.03 for Windows (GraphPad Software, San Diego, CA). Statistical analyses consisted of one-way ANOVA followed by Tukey's post-test or two-way ANOVA followed by Bonferroni's post-test. Statistical significance was set at 0.05. In concentration-response experiments, a sigmoidal dose-response function, Y = bottom + (top
bottom)/[1 + 10exp ([Log EC50
Results
MOR-1 internalization produced by exogenously added opioids
It was initially observed that incubating spinal cord slices with relatively high concentrations (1 µM) of Leu-enkephalin did not produce MOR-1 internalization. A confocal image of a representative neuron in a slice exposed to Leu-enkephalin is shown in Figure 2D: MOR-1 immunoreactivity was located at the surface of the somata, analogous to neurons in a control slice (Fig. 2A). Similar results have been reported using Met-enkephalin (Trafton et al., 2000
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Figure 2. Confocal images of MOR-1 dorsal horn neurons exposed to opioids and peptidase inhibitors. Spinal cord slices were incubated for 20 min with opioids and peptidase inhibitors (PI; 10 µM amastatin, captopril, and phosphoramidon). Treatments were as follows: Control (A); 1 µM endomorphin-1 (EM-1, B); 100 nM endomorphin-2 (EM-2, C); 1 µM Leu-enkephalin (Leu-enk, D); 1 µM Leu-enkephalin and peptidase inhibitors (E); 1 µM Leu-enkephalin and peptidase inhibitors, 10 µM actinonin (act.) instead of amastatin (F); 1 µM Leu-enkephalin and peptidase inhibitors, 100 µM bestatin (best.) instead of amastatin (G); a 1 µM concentration of dynorphin A (dyn A, H); and 1 µM dynorphin A and peptidase inhibitors (I). Cells in A, D, and H show no internalization; the rest show clear internalization. Dorsal is up for all panels except H, in which dorsal is right. Confocal images (100×, zoom of 2) are two optical sections (three in G and H) through the center of lamina II neurons at intervals of 0.49 or 0.57 µm, of five to seven optical sections. Scale bar: (in I) A-I, 5 µm.
These observations were quantified by counting MOR-1 neurons in laminas I-II with and without internalization (see Materials and Methods). In slices incubated with normal ACSF (Fig. 3A, none) only a very small percentage (6 ± 1%) of MOR-1 neurons presented internalization. This percentage did not significantly increase in slices incubated with 1 µM Leu-enkephalin or Met-enkephalin, 3 µM dynorphin A, or 1 µM
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Figure 3. MOR-1 internalization produced by opioids with and without peptidase inhibitors. 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),
The opioid peptides that failed to produce MOR-1 internalization in the absence of peptidase inhibitors produced MOR-1 internalization in practically all MOR-1 IR neurons (Fig. 3B) in the presence of 10 µM amastatin, captopril, and phosphoramidon. The peptidase inhibitor mixture by itself did not appreciably increase the percentage of MOR-1 neurons with internalization, as can be seen by comparing the none bars in Figure 3A,B (also see below). MOR-1 internalization produced by 1 µM Leu-enkephalin was blocked by the opioid antagonist naloxone (10 µM), and the MOR-1 internalization produced by 3 µM dynorphin A was abolished by the selective MOR antagonist CTAP (1 µM), indicating that it was mediated by the binding of the peptides to MOR. Selective agonists of
and
opioid receptors did not elicit MOR-1 internalization in spinal cord slices (Marvizon et al., 1999b
Peptidase inhibitors increased the potencies of Leu-enkephalin and dynorphin A to produce MOR-1 internalization
The findings described above are consistent with our hypothesis that enkephalins and dynorphins are actively degraded by peptidases in the dorsal horn. The hypothesis also predicts that the inability of these opioids to produce MOR-1 internalization could be overcome at higher concentrations because they would saturate the peptidases, and that peptidase inhibitors would produce a left shift in their concentration-response curves to elicit MOR-1 internalization.
Figure 4A shows that these predictions are fulfilled for Leu-enkephalin. In the absence of peptidase inhibitors, high concentrations of Leu-enkephalin were able to produce MOR-1 internalization, yielding an EC50 of 22 µM (95% CI, 17-30). Peptidase inhibitors (amastatin, captopril, and phosphoramidon) dramatically shifted the concentration-response curve of Leu-enkephalin to the left, increasing its potency by almost two orders of magnitude (EC50, 0.38 µM; 95% CI, 0.31-0.47). This EC50 value was very similar to the potency of Met-enkephalin (0.25 ± 0.6 µM) to produce outward currents in locus ceruleus neurons in the presence of the peptidase inhibitor kelatorphan (Williams et al., 1987
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Figure 4. Effect of peptidase inhibitors on the potencies of Leu-enkephalin, dynorphin-A, endomorphin-1, and endomorphin-2 to produce MOR-1 internalization. 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, B), or these three inhibitors plus 10 µM Ala-pyrrolidine-nitrile (C, D). Points are the means ± SEM of three to five 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% CI, 17-30; R2, 0.96); Leu-enkephalin (peptidase inhibitors): EC50, 0.38 µM (95% CI, 0.31-0.47; nH, 2.8 ± 0.7; R2, 0.82). B, Dynorphin A: EC50, 5.2 µM (95% CI, 4.3-6.3; nH, 3.0 ± 0.6; R2, 0.90); dynorphin A (peptidase inhibitors): EC50, 0.60 µM (95% CI, 0.34-1.04; R2, 0.83). C, Endomorphin-1: EC50, 32 nM (95% CI, 16-65; R2, 0.73); endomorphin-1 (peptidase inhibitors): EC50, 40 nM (95% CI, 22-73; R2, 0.77). D, Endomorphin-2: EC50, 5 nM (95% CI, 3-8; R2, 0.92); endomorphin-2 (peptidase inhibitors): EC50, 10 nM (95% CI, 6-15; 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%.
Peptidase inhibitors did not affect the potency of endomorphins to elicit MOR-1 internalization
In contrast to the MOR-1 internalization produced by Leu-enkephalin and dynorphin A, peptidase inhibitors had no effect on the potencies of endomorphin-1 (Fig. 4C) or endomorphin-2 (Fig. 4D) to elicit MOR-1 internalization. Because dipeptidyl peptidase IV (EC 3.4.14.15) has been reported to play an important role in degrading endomorphins (Shane et al., 1999
Contribution of different peptidases to the degradation of Leu-enkephalin
Next, we determined the relative contribution of peptidases to preventing MOR-1 internalization by Leu-enkephalin by using different combinations of peptidase inhibitors. We took advantage of the fact that Leu-enkephalin at 1 µM elicited maximal MOR-1 internalization in the presence of peptidase inhibitors and minimal internalization in their absence (Fig. 4A). Hence, the ability of peptidase inhibitors to protect Leu-enkephalin against degradation would be reflected in the amount of MOR-1 internalization produced by 1 µM Leu-enkephalin. Figure 5A shows that in the presence of 10 µM amastatin, phosphoramidon, or captopril alone, or even combined two by two, 1 µM Leu-enkephalin remained unable to significantly stimulate MOR-1 internalization. These results indicate that each of the three peptidases targeted by these inhibitors (aminopeptidases, neutral endopeptidase, and dipeptidyl carboxypeptidase) (Fig. 1) is sufficient to degrade Leu-enkephalin substantially. Interpolating the data in Figure 5A in the left curve of Figure 4A suggests that each of these peptidases is able to reduce the concentration of Leu-enkephalin from 1 to <0.3 µM. Thiorphan, like phosphoramidon, inhibits neutral endopeptidase (Yaksh and Chipkin, 1989
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Figure 5. Effect of different combinations of peptidase inhibitors on the internalization of MOR-1 produced by Leu-enkephalin (Leu-enk) and dynorphin A. Spinal cord slices were incubated at 35°C with peptidase inhibitors (10 µM unless indicated otherwise); after 10 min, 1 µM Leu-enkephalin (A, B) or dynorphin A (C) was added for an additional 10 min. Columns represent the means ± SEM of three to five slices. ama, Amastatin; PhA, phosphoramidon; capt, captopril; 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-test.
To further characterize the aminopeptidase(s) responsible for the degradation of enkephalins in the spinal cord, we substituted different aminopeptidase inhibitors for amastatin in our inhibitor mixture (Fig. 5B). Antipain, arphamenine, bacitracin, proctolin, and puromycin were ineffective. Other studies found that bacitracin (Tseng et al., 1986
Contribution of different peptidases to the degradation of dynorphin A
The effect of combinations of peptidase inhibitors on dynorphin A (Fig. 5C) was different from their effect on Leu-enkephalin (Fig. 5A). Amastatin plus phosphoramidon was as effective as all three peptidase inhibitors in protecting the effect of dynorphin A, indicating that dynorphin A is degraded by aminopeptidases and neutral endopeptidase. Moreover, significant (p < 0.05) protection could be achieved with amastatin alone. Actinonin could substitute for amastatin, which is consistent with the idea that aminopeptidase N is involved in the degradation of dynorphins as well as enkephalins. No protection of dynorphin A was obtained with phosphoramidon plus captopril, and the addition of captopril did not increase the effect of amastatin, which shows that the MOR-1 internalization produced by dynorphin A is not appreciably decreased by dipeptidyl carboxypeptidase. This is probably attributable to the fact that dipeptidyl carboxypeptidase starts cleaving dynorphin A at its extended C terminal (Fig. 1), producing peptides containing the Leu-enkephalin sequence that are still able to activate MOR-1.
Relative localization of opioid peptides and MOR-1 in the dorsal horn
The localization of opioid-containing terminals relative to MOR-1 IR neurons was explored using double-label immunofluorescence. To detect opioid peptides, we used a monoclonal antibody (3-E7) that recognized
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Figure 6. Relative localization of opioids and MOR-1 in the dorsal horn. Sections (25 µm) from the lumbar spinal cord of an adult rat were double-labeled with a monoclonal antibody (3-E7) recognizing enkephalins,
Using an antibody that recognizes endomorphin-1 and endomorphin-2, we found that endomorphin-like immunoreactivity did not colocalize with 3-E7 immunoreactivity (data not shown), suggesting that endomorphins are contained in terminals different from other opioids. Endomorphin-like immunoreactivity was found primarily in lamina I and the dorsolateral funiculus, whereas most MOR-1 IR neurons were found in lamina II (Fig. 6), separated from endomorphin IR fibers and varicosities (data not shown). However, Spike et al. (2002)
MOR-1 internalization produced by endogenously released opioids
To elicit opioid release, spinal cord slices were incubated with 30 µM veratridine or 50 mM KCl in the presence or absence of peptidase inhibitors (amastatin, captopril, and phosphoramidon). Veratridine increases Na+ fluxes through voltage-dependent Na+ channels (Satoh and Nakazato, 1991
A short (2 min) incubation with 30 µM veratridine in the presence of peptidase inhibitors produced MOR-1 internalization in approximately two-thirds of MOR-1 IR cells (Fig. 7A). However, veratridine did not produce MOR-1 internalization in the absence of peptidase inhibitors. These data were analyzed with a two-way ANOVA with veratridine and peptidase inhibitors as the two variables, which showed significant (p < 0.0001) effects of the variables and their interaction. Bonferroni's post-test revealed a significant effect (p < 0.001) of veratridine combined with peptidase inhibitors but no significant effects of peptidase inhibitors alone or veratridine without peptidase inhibitors.
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Figure 7. MOR-1 internalization produced by opioids released by veratridine. All compounds were applied to the slices during a 2 min incubation with 30 µM veratridine and for 10 min afterward. Peptidase inhibitors (10 µM) were actinonin (act), amastatin (ama), captopril (capt), and phosphoramidon (PhA). Numbers above the columns indicate the number of replicates (slices). A, Slices were incubated in ACSF (control) or 30 µM veratridine, without or with peptidase inhibitors. Two-way ANOVA indicated significant (p < 0.0001) effects of both variables (veratridine and peptidase inhibitors) and their interaction. Bonferroni's post-test indicated significant differences from control only for veratridine with peptidase inhibitors (***p < 0.001). B, Slices were treated with veratridine, peptidase inhibitors, and no addition (none), 10 µM CTAP, or 0.2 mM CaCl2 (low Ca2+). ANOVA, p = 0.0001 overall; Tukey's post-test, ***p < 0.001; **p < 0.01 compared with none. C, Slices were incubated with veratridine and the indicated combinations of peptidase inhibitors. ANOVA, p = 0.0006 overall; Tukey's post-test, *p < 0.05; **p < 0.01 compared with none.
Depolarization with high K+ in the presence of peptidase inhibitors elicited MOR-1 internalization in one-third of the MOR-1 IR neurons (Fig. 8A). In this experiment, slices were incubated for 20 min in ACSF containing 50 mM KCl, decreasing the concentration of NaCl by the same amount to avoid increasing the osmotic pressure. In the absence of peptidase inhibitors, 50 mM KCl produced MOR-1 internalization in a small percentage of MOR-1 IR neurons (Fig. 8A). Two-way ANOVA revealed significant effects of the variables high K+ and peptidase inhibitors (p < 0.0001) and their interaction (p < 0.01). Bonferroni's post-test yielded a significant effect for 50 mM KCl without (p < 0.05) and with (p < 0.001) peptidase inhibitors.
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Figure 8. MOR-1 internalization produced by opioids released by 50 mM KCl. Spinal cord slices were incubated for 20 min with 50 mM KCl, peptidase inhibitors, and other additions. Peptidase inhibitors (10 µM) were amastatin (ama), phosphoramidon (PhA), and captopril (capt). Numbers above the columns indicate the number of replicates (slices). A, Slices were incubated in normal ACSF (control) or in ACSF containing 50 mM KCl, without or with peptidase inhibitors. Control values are the same as in Figure 7A, and are shown here for comparison. Two-way ANOVA with 50 mM KCl and peptidase inhibitors as the variables indicated significant (p < 0.0001) effects of both variables and their interaction (p < 0.01). Bonferroni's post-test indicated significant differences from controls for 50 mM KCl both without (*p < 0.05) and with (***p < 0.001) peptidase inhibitors. B, Slices were incubated in ACSF containing 50 mM KCl, peptidase inhibitors, and no other addition (none), 1 µM CTAP, or 0.2 mM CaCl2 (low Ca2+). ANOVA, p < 0.0001 overall; Tukey's post-test, **p < 0.01; ***p < 0.001 compared with none. C, Slices were incubated in ACSF containing 50 mM KCl and the indicated combinations of peptidase inhibitors. ANOVA, p = 0.0017 overall; Tukey's post-test, **p < 0.01; *p < 0.05 compared with ama+capt+PhA.
Confocal images of neurons representative of these observations are shown in Figure 9. Neurons exposed to peptidase inhibitors (A) or veratridine (B) alone showed crisp surface staining and no endosomes. Figure 9F shows a neuron exposed to 50 mM KCl in the absence of peptidase inhibitors; it presents a somewhat discontinuous labeling of the membrane, possibly indicating some MOR-1 clustering. In the presence of peptidase inhibitors, veratridine produced clear internalization in most neurons, as shown by the group of three neurons in Figure 9C. The neuron in the bottom left corner does not have the nucleus in the confocal plane, and part of a fourth neuron is visible in the bottom right corner. Note the presence of intensely labeled endosomes and the decrease in surface staining. Figure 9G shows a neuron presenting internalization after treatment with high K+ and peptidase inhibitors. This neuron has a nucleus occupying most of the soma, which is common in MOR-1 IR neurons in the dorsal horn (see also Figs. 2, 6B). However, there is almost no surface staining, and several endosomes are visible in the cytoplasm and along one dendrite.
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Figure 9. Confocal images of MOR-1 dorsal horn neurons treated with 50 mM KCl or veratridine. Slice treatments were as follows: peptidase inhibitors (PI) alone (A); 30 µM veratridine (veratr., B); 30 µM veratridine and peptidase inhibitors (C); 30 µM veratridine, peptidase inhibitors, and CTAP (D); 30 µM veratridine, peptidase inhibitors, and 0.2 mM Ca2+ (
The selective MOR-1 antagonist CTAP (1 µM) abolished MOR-1 internalization produced by veratridine (Fig. 7B) or 50 mM KCl (Fig. 8A) in the presence of peptidase inhibitors. Representative neurons are shown in Figure 9D (veratridine) and Figure 9H (high K+). This observation indicates that the internalization was attributable to the interaction of agonists with MOR-1. Moreover, if the internalization produced by veratridine and high K+ was attributable to the release of endogenous opioids, we predicted that it would be inhibited in the absence Ca2+ as well. However, it is possible that the mechanism of MOR-1 internalization itself requires Ca2+. Indeed, in the nominal absence of Ca2+ (no CaCl2 was added), the MOR-1 internalization produced by 1 µM endomorphin-2 was decreased from 100% of MOR-1 neurons with internalization (Fig. 4D) to 55 ± 18% (n = 5). However, a 10-fold reduction in Ca2+ concentration (from 2.4 to 0.2 mM) did not affect the MOR-1 internalization produced by 0.1 µM endomorphin-2, which was present in 97 ± 2% of the MOR-1 IR neurons (n = 3). Therefore, the MOR-1 internalization process can take place if a small amount of Ca2+ is present in the extracellular medium. Yet when the Ca2+ concentration was decreased to 0.2 mM, the MOR-1 internalization produced by 50 mM KCl (Figs. 7A, 8I) or veratridine (Figs. 6B, 8E) was abolished or greatly reduced, respectively. These results confirm that veratridine and high K+ produced MOR-1 internalization by eliciting the Ca2+-dependent release of endogenous opioids.
Contribution of different peptidases to the degradation of endogenously released opioids
To determine which peptidases contributed to the degradation of the opioids released by veratridine or high K+, we studied the effect of different combinations of peptidase inhibitors. In the case of high K+ (Fig. 8B), only the mixture of the three inhibitors significantly increased (p < 0.05) MOR-1 internalization above that produced by 50 mM KCl alone. However, phosphoramidon plus captopril produced an effect that was not significantly different from the combination of the three inhibitors, and showed a trend toward increased internalization. In the case of veratridine (Fig. 7C), phosphoramidon plus captopril or phosphoramidon plus amastatin produced MOR-1 internalization significantly above control levels (p < 0.05). However, phosphoramidon by itself or amastatin plus captopril did not significantly increase internalization above the effect of veratridine alone. Mixtures of three inhibitors, including either amastatin or actinonin as aminopeptidase inhibitors, produced robust MOR-1 internalization (p < 0.01). These results indicate that neutral endopeptidase is the main enzyme degrading opioids released in the spinal cord, with a substantial contribution of aminopeptidases and dipeptidyl carboxypeptidase.
Discussion
In summary, in the dorsal horn: (1) peptidases restrict MOR-1 internalization by exogenously applied enkephalins and dynorphins, (2) endomorphins are not substantially degraded by peptidases, and (3) peptidases prevent MOR-1 internalization by endogenously released opioids.
Peptidases restrict MOR-1 internalization by exogenous enkephalins and dynorphins
Peptidase inhibitors substantially increased the potencies of Leu-enkephalin and dynorphin A to elicit MOR-1 internalization, as well as the effect of single concentrations of Met-enkephalin and
-endorphin. Indeed, many degradation products of
All three peptidases degraded Leu-enkephalin, because omission of any one inhibitor resulted in the loss of its effect. The Leu-enkephalin-degrading aminopeptidase was probably aminopeptidase N (Tieku and Hooper, 1992
The ability of dynorphin A to produce MOR-1 internalization was decreased by aminopeptidases and neutral endopeptidase, but not appreciably by dipeptidyl carboxypeptidase, probably because their cleavage of dynorphin A produces active peptides containing the Leu-enkephalin sequence. Likewise, although dynorphin A does have a fairly high affinity for MOR-1 (Raynor et al., 1993
Endomorphin-1 and endomorphin-2 are not degraded by peptidases
Shane et al. (1999)
Endogenously released opioids
The MOR-1 internalization produced by veratridine or high K+ was abolished by the MOR-1 antagonist CTAP and was Ca2+ dependent, indicating that it was attributable to the release of MOR-1 agonists. Veratridine-evoked MOR-1 internalization required peptidase inhibitors, and internalization produced by high K+ was greatly reduced in their absence, showing that most of the released opioids are degraded by peptidases. This is consistent with studies showing that peptidase inhibitors decreased ventral root potentials (Suzuki et al., 1997
Neutral endopeptidase appears to be the main enzyme degrading released opioids, because phosphoramidon plus amastatin or captopril, but not amastatin plus captopril, protected the effect of veratridine. However, phosphoramidon alone did not produce significant protection, indicating that the contribution of aminopeptidases and dipeptidyl carboxypeptidase is also important. In contrast, the internalization produced by Leu-enkephalin was similarly decreased by all three peptidases, whereas the effect of dynorphin A was not decreased by dipeptidyl carboxypeptidase. This discrepancy suggests that the opioids released are not the enkephalin pentapeptides or dynorphin A alone, but a mixture of opioid peptides of various lengths (Lucas and Yaksh, 1990
Our results show that MOR-1 internalization can be used to investigate mechanisms of opioid release. Opioids are not appreciably released from primary afferents, because prolonged dorsal root stimulation while superfusing the slices with peptidase inhibitors did not produce MOR-1 internalization (Marvizon and Song, 2002
Physiological relevance
Our results likely reflect the situation in vivo. Amastatin, captopril, and phosphoramidon increased the analgesic effects of intrathecal or intracerebral opioids (Kishioka et al., 1994
The relationship between MOR activation and its internalization is complex. Although internalization requires activation, the converse is not always true, because morphine activates MOR-1 without producing its internalization (Keith et al., 1998
In view of this, it is puzzling that noxious stimuli that elicited spinal enkephalin release (Yaksh and Elde, 1981
Our findings indicate that opioids, unlike neurokinins (Mantyh et al., 1995
Yet, it is unlikely that the abundant extrasynaptic MOR-1 in dorsal horn neurons serves no function; hence, there may be some conditions in which opioids act by volume transmission. First, during inflammation and hyperalgesia, dynorphin and enkephalins are upregulated (Draisci et al., 1991
Therapeutic implications
It has been suggested that peptidase inhibitors could be used to treat pain, because they have analgesic effects (Noble et al., 1992a
FOOTNOTES Received Sept. 25, 2002; revised Dec. 10, 2002; accepted Dec. 12, 2002.
This work was supported by National Institute on Drug Abuse Grant RO1-DA12609 to J.C.M. We thank Drs. Chris Evans, Marzia Malcangio, and Emeran Mayer for critically reading this manuscript; Drs. Enrico Stefani, Catia Sternini, and Ligia Toro for their advice and support; Dr. Sherwin Wilk for providing alanine-pyrrolidonyl-2-nitrile; and Kendrick Che and Anish R. Dube for their help. We are also grateful for the assistance of Dr. Matthew J. Schibler at the University of California Los Angeles Carol Moss Spivak Cell Imaging Facility.
Correspondence should be addressed to Juan Carlos G. Marvizón, 1541 MacDonald Research Laboratories, 675 Charles E. Young Drive, University of California Los Angeles, Los Angeles, CA 90095. E-mail: marvizon{at}ucla.edu.
References
- Abbadie C, Trafton J, Liu H, Mantyh PW, Basbaum AI (1997) Inflammation increases the distribution of dorsal horn neurons that internalize the neurokinin-1 receptor in response to noxious and non-noxious stimulation. J Neurosci 17:8049-8060
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