ARTICLE |
Correspondence to: Paul W. Ackermann, Orthopedic Laboratory, Research Center M3:02, Karolinska Hospital, S-171 76, Stockholm, Sweden. E-mail: Paul.Ackermann@ks.se
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Summary |
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The occurrence of endogenous opioids and their receptors in rat achilles tendon was analyzed by immunohistochemistry (IHC), radioimmunoassay (RIA), and in vitro binding assays. The investigation focused on four enkephalins, dynorphin B, and nociceptin/orphanin FQ. Nerve fibers immunoreactive to all enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Gly-Lys, Met-enkephalin-Arg-Phe) were consistently found in the loose connective tissue and the paratenon, whereas dynorphin B and nociceptin/orphanin FQ could not be detected. The majority of enkephalin-positive nerve fibers exhibited varicosities predominantly seen in blood vessel walls. Measurable levels of Met-enkephalin-Arg-Phe and nociceptin/orphanin FQ were found in tendon tissue using RIA, whereas dynorphin B could not be detected. In addition to the endogenous opioids identified, -opioid receptors on nerve fibers were also detected by IHC. Binding assays to characterize the opioid binding sites showed that they were specific and saturable for [3H]-naloxone (Kd 7.01 ± 0.98 nM; Bmax 23.52 ± 2.23 fmol/mg protein). Our study demonstrates the occurrence of an opioid system in rat achilles tendon, which may be assumed to be present also in other connective tissues of the locomotor apparatus. This system may prove to be a useful target for pharmacological therapy in painful and inflammatory conditions by new drugs acting selectively in the periphery. (J Histochem Cytochem 49:13871395, 2001)
Key Words: achilles tendon, connective tissue, rat, opioid peptides, enkephalins, peripheral nervous system, receptors, immunohistochemistry, radioimmunoassay, receptor binding
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Introduction |
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Achillodynia is one of several chronic, painful conditions of the locomotor system, in which the pathomechanisms are poorly understood. During the past several years the incidence of overuse injuries, mostly related to work and sports, has increased considerably (
Over the past decade, there have been a number of reports on the innervation of musculoskeletal tissues according to specific transmitters, representing the sensory (
There also appears to exist a peripheral anti-nociceptive system counteracting the peripheral sensory system. Thus, it was recently reported that intra-articular morphine in conjunction with arthroscopy had a significant analgesic effect in a doseresponse manner (
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Materials and Methods |
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The study included 77 male SpragueDawley (SD) rats (180200 g), housed five per cage at 21C in a 12:12-hr light:dark cycle with water and pellets ad lib according to the Karolinska Institute protocol. The experiments were approved (no. N 58/98) by the Stockholm North Animal Ethical board.
Radioimmunoassay
Twelve rats were anesthetized by injection of sodium pentobarbital (60 mg/kg IP) and sacrificed by decapitation. The achilles tendons were dissected bilaterally. The samples from the left and right side were pooled to one sample. All dissected tissues were weighed and immediately frozen on dry ice and kept at -70C until neuropeptide extraction. Frozen tendons were boiled for 10 min in 2 M acetic acid, homogenized in a Polytron (15 sec), sonicated (30 sec), and centrifuged at 3000 x g for 15 min. The supernatants were lyophilized, diluted in 2 ml 0.05 M phosphate buffer, pH 7,4, and kept at -20C until analysis (
The tissue extracts were subjected to a purification step using an ion exchange procedure before RIA. The method is routinely used to analyze opioid peptides in various tissue extracts and is described in detail elsewhere (
The peptides were analyzed using specific RIAs for DYN B, MEAP, and N/OFQ, respectively, described in detail elsewhere (
Immunohistochemistry
Five rats were anesthetized by injection of sodium pentobarbital (60 mg/kg IP). Intra-arterial perfusion with PBS was performed, followed by perfusion with Zamboni's fixative consisting of 4% paraformaldehyde in 0.2 mol/liter Sorensen phosphate buffer, pH 7.3, containing 0.2% picric acid. The achilles tendons were dissected and immersed in the same fixative for 2 hr at room temperature (RT). All specimens were soaked for at least 2 days in 20% sucrose in 0.1 mol/liter Sorensen phosphate buffer, pH 7.2, containing sodium azide and bacitracin (Sigma Chemicals; St Louis, MO). The tissues were sectioned at 15 µm on a Leitz cryostat and frozen sections were mounted directly on SuperFrost/Plus glass slides and immunostained using the avidinbiotin system. The sections were rinsed two times for 5 min each in PBS. Incubation of the sections with 10% normal goat serum in PBS for 30 min blocked nonspecific binding. Subsequently, sections were incubated with primary antisera for ME, LE, MEAP, MEAGL (all 1:20,000; Peninsula Laboratories, Belmont, CA); NOC, DYN B, -opioid receptor (DOR),
-opioid receptor (KOR), µ-opioid receptor (MOR) (all 1:10,000; Peninsula Laboratories), and protein gene product 9.5 (PGP 9.5), a general nerve marker (1:10,000; Ultraclone, Cambridge, UK) overnight in a humid atmosphere at RT. After incubation with the primary antisera, the sections were rinsed in PBS (twice for 5 min) and then incubated with biotinylated goat anti-rabbit antibodies (1:250; Vector Laboratories, Burlingame CA) for 40 min at RT. Finally, the sections were incubated for 40 min with fluorescein isothiocyanate (FITC)-conjugated avidin (1:500; Vector Laboratories). For double staining, after completing the staining steps for the first neurotransmitter, the sections were incubated with avidin blocking solution followed by biotin blocking solution (15 min each). The staining for the receptor was repeated as described for the neurotransmitter, but with a different flourochrome, CY3 (1:5000; Amersham International, Poole, UK). To demonstrate specificity of staining, the following controls were included: (a) pre-adsorption of the primary antisera with excess of homologous antigen (50 µg/ml ME, LE, MEAP, MEAGL; Peninsula Laboratories) for 12 hr at RT; (b) omission of either the primary antiserum, the secondary antibody, or the secondary biotinylated antibody. A Nikon epifluorescence microscope (Eclipse E800; Yokohama, Japan) was used to analyze the sections. T-Max black-and-white and EPL 400 color films (Kodak; Rochester, NY) were used for photography.
Radioligand Binding Assay
The achilles tendons were dissected bilaterally from 60 rats. The tendons were homogenized in 5 v/w of ice-cold 50 mM Tris-HCl buffer, pH 7.4 (Polytron; twice for 30 sec) and sonicated (15 sec). After centrifugation at 40,000 x g for 20 min at 4C, the pellets were resuspended in 30 v/w fresh Tris-HCl buffer and incubated at 37C for 30 min to facilitate degradation and dissociation of endogenous opioid peptides. The centrifugation step was repeated. The final pellet was resuspended in 5 v/w of ice-cold 50 mM Tris-HCl buffer containing 0.32 M sucrose, pH 7.4, and kept at -70C until use.
Aliquots of membrane homogenates (0.60.7 mg/ml protein) were incubated with [3H]-naloxone (54.6 Ci/mmol; New England Nuclear, Boston, MA) for 30 min at 25C in a final volume of 250 µl. Reactions were terminated by rapid filtration through Whatman GF/B glass fiber filters pretreated with 0.1% polyethyleneimine (PEI) using a Brandel Cell Harvester, followed by three washings with 5 ml of ice-cold Tris-HCl buffer, pH 7.4. The bound radioactivity was measured in 4 ml of Ultima Gold scintillation cocktail (Packard) using a Packard 1900 CA liquid scintillation counter. All experiments were carried out in duplicate. Nonspecific binding was measured in the presence of 10 µM unlabeled naloxone.
The protein concentration was determined by the method of
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Results |
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Quantification of Opioids
The RIA focused on MEAP, N/OFQ, and DYN B generated from three different prohormones; proenkephalin, prodynorphin, and pronociceptin. Over all, the measured concentrations were low, some even below the detection limit. Measurable concentrations of MEAP were obtained in 7/11 samples (61%) and of N/OFQ in 4/12 (33%). These proportions of measurable concentrations were significant within the 95% confidence interval, whereas that of DYN B (1/12 = 8%) was not (Table 1). Although MEAP and N/OFQ exhibited a significant proportion of measurable concentrations, only MEAP (0.07 pmol/g) displayed a median concentration above the detection limit (Table 1).
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Distribution of Enkephalins
IHC showed that all enkephalins tested, i.e., LE, ME, MEAP, and MEAGL, were present in nerves of tendon tissue (Fig 1). However, no nerve fibers immunoreactive for N/OFQ and DYN B could be detected. LE appeared to be the most abundant opioid compared to ME, MEAP, and MEAGLE in decreasing order. Although they differed in abundance, the tissue distribution displayed a similar pattern. Thus, all four enkephalins predominantly occurred in the loose connective tissue, the paratenon and musculotendinous junction, whereas no enkephalins were found in the tendon tissue proper. In the loose connective tissue surrounding the tendon, the enkephalins appeared as small varicosities around the walls of both large and small blood vessels (Fig 1). In the paratenon and the musculotendinous junction the enkephalins mostly occurred as varicosities in free nerve terminals, without any relationship to blood vessels. Notably, they were more abundant proximally at the musculotendinous junction of the achilles tendon than distally at the bony insertion. The neuronal character of the immunoreactivity was confirmed by PGP 9.5-positive staining (not shown).
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Distribution of Opioid Receptors
Of the three opioid receptors (DOR, KOR, MOR) studied, only DOR could be detected by IHC (Fig 2). Positive staining for DOR was found in the loose connective tissue, the paratenon, and the musculotendinous junction. In the loose connective tissue, DOR was predominantly located in the blood vessel walls as small varicosities in nerve fibers penetrating the walls (Fig 2A). In the paratenon and the musculotendinous junction, DOR positivity mostly occurred as varicosities in free nerve endings without any relationship to vessels (Fig 2B). The -opioid receptor appeared to be more abundant in the proximal portion than in the distal portion of the paratenon.
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Co-localization of Enkephalins and Their Receptors
Double staining disclosed co-existence of each of the enkephalins with DOR in the nerve fibers (Fig 3). Thus, co-localization was found in the paratenon, loose connective tissue, and the musculotendinous junction. The co-existence was found both in free nerve endings mostly localized to the paratenon and the musculotendinous junction and in vascular fibers, predominantly in the surrounding loose connective tissue. The major portions of the enkephalins and DOR identified in the nerve fibers were found to be co-localized.
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Characterization of Opioid Receptors
The tissue possessed opioid binding sites, shown with the non-selective opioid ligand [3H]-naloxone. Binding of [3H]-naloxone to homogenates of achilles tendon from rat gradually increased with time and reached a steady state after 45 min at 25C (Fig 4).
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The binding of [3H]-naloxone was specific and saturable (Fig 5). The equilibrium dissociation constant (Kd) for the identified binding sites disclosed high affinity (Kd 7.01 ± 0.98 nM) but low binding capacity (Bmax 23.52 ± 2.23 fmol/mg protein). The Hill coefficient (nH) value was 0.89 ± 0.09.
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Discussion |
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Our study convincingly shows that connective tissue is supplied with a peripheral opioid system. The neuronal occurrence of different enkephalins and the existence of -opioid receptors in the achilles tendon suggest that there is an anti-nociceptive system in musculoskeletal tissues, which most likely counteracts the effects of the sensory nervous system. It may prove that this can be exploited in the therapeutical setting by developing new drugs that act selectively in the periphery to mitigate pain and inflammation in musculoskeletal disorders.
Among the opioid peptides analyzed in the achilles tendon, enkephalins and DYN B represent classical opioid peptide families, whereas N/OFQ has only lately been detected and characterized (
There are two principal sources of opioid peptides in the periphery. One is represented by the immune cells, which have been shown to contain and release opioids and to be implicated in mitigating inflammatory pain (
The physiological role of enkephalins can be assumed to be anti-nociceptive (
According to IHC the four enkephalins analyzed exhibited a similar distribution. They occurred in the paratenon and the surrounding loose connective tissue rather than in the tendinous tissue proper. Hypothetically, this difference in anatomic distribution might reflect that regulation of pain and inflammation in disorders of the achilles tendon mainly occurs in the surrounding tissues. We have previously demonstrated a similar anatomic distribution of sensory and autonomic neuropeptides in the achilles tendon, i.e., predominantly in the surrounding tissues (
The IHC analysis also showed that the enkephalin-positive nerve fibers were either vessel related or non-vascular. Nerve fibers around vessels can be assumed to be involved in both vasoactivity (
In this study, the existence of an opioid system in connective tissues, as demonstrated by neuronal immunoreactivity to enkephalins, is strongly supported by our opioid receptor analyses based on IHC and binding assays. Thus, -opioid receptors (DOR) were identified on peripheral nerve fibers in the paratenon and the surrounding loose connective tissue. Double staining for DOR and the four enkephalins tested disclosed co-existence, which complies with other studies suggesting that enkephalins are the main ligands for DOR (
-opioid agonists in the periphery is both anti-inflammatory and anti-nociceptive in complex models of inflammation (
In addition to the demonstration of -opioid receptors, we were also able to characterize opioid receptors in the tendon tissue by binding assays using the non-selective opioid radioligand [3H]-naloxone. The binding of [3H]-naloxone to homogenates from achilles tendon was specific, saturable, and stereo-selective. The Hill coefficient value was close to unity, indicating the non-cooperative nature of the binding process and also that [3H]-naloxone could interact with multiple classes with equal affinity.
From the combined results of our study, it seems most probable that the musculoskeletal apparatus is equipped with an opioid system. Whether there is a critical balance between the peripheral expression of opioids and sensory neuropeptides under normal circumstances, which is altered under pathologic conditions, remains to be clarified. Nonetheless, our findings appear to imply that there is a peripheral mechanism for inhibition of pain in addition to that at higher level according to the "central gate theory" postulated by
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Acknowledgments |
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Supported by grants from the Swedish Medical Research Council (12X-08652-09B and 14X-12588-03A). Mariana Spetea was supported by fellowships from the Swedish Institute and the Wenner-Gren Foundation.
Received for publication March 1, 2001; accepted June 19, 2001.
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Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ackermann PW, Finn A, Ahmed M (1999) Sensory neuropeptidergic pattern in tendon, ligament and joint capsule. A study in the rat. Neuroreport 13:2055-2060
Ackermann PW, Jian L, Finn A, Ahmed M, Kreicbergs A (2001) Autonomic innervation of tendons, ligaments and joint capsules. A morphologic and quantitative study in the rat. J Orthop Res 19:372-378[Medline]
Ahmed M, Bjurholm A, Schultzberg M, Theodorsson E, Kreicbergs A (1995a) Increased levels of substance P and calcitonin gene-related peptide in rat adjuvant arthritis. A combined immunohistochemical and radioimmunoassay analysis. Arthritis Rheum 38:699-709[Medline]
Ahmed M, Bjurholm A, Srinivasan GR, Theodorsson E, Kreicbergs A (1994) Extraction of neuropeptides from joint tissue for quantitation by radioimmunoassay. A study in the rat. Peptides 15:317-322[Medline]
Ahmed M, Bjurholm A, Theodorsson E, Schultzberg M, Kreicbergs A (1995b) Neuropeptide Y- and vasoactive intestinal polypeptide-like immunoreactivity in adjuvant arthritis: effects of capsaicin treatment. Neuropeptides 29:33-43[Medline]
Brown SL, Van Epps DE (1985) Suppression of T lymphocyte chemotactic factor production by the opioid peptides beta-endorphin and met-enkephalin. J Immunol 134:3384-3390
Calo G, Guerrini R, Rizzi A, Salvadori S, Regoli D (2000) Pharmacology of nociceptin and its receptor: a novel therapeutic target. Br J Pharmacol 129:1261-1283
Carlton SM, Coggeshall RE (1997) Immunohistochemical localization of enkephalin in peripheral sensory axons in the rat. Neurosci Lett 221:121-124[Medline]
ChristenssonNylander I, Nyberg F, Ragnarsson U, Terenius L (1985) A general procedure for analysis of proenkephalin B derived opioid peptides. Regul Pept 11:65-76[Medline]
Coggeshall RE, Zhou S, Carlton SM (1997) Opioid receptors on peripheral sensory axons. Brain Res 764:126-132[Medline]
Darland T, Heinricher MM, Grandy DK (1998) Orphanin FQ/nociceptin: a role in pain and analgesia, but so much more. Trends Neurosci 21:215-221[Medline]
Dhawan BN, Cesselin F, Raghubir R, Reisine T, Bradley PB, Portoghese PS, Hamon M (1996) International Union of Pharmacology. XII. Classification of opioid receptors. Pharmacol Rev 48:567-592[Medline]
Dores RM, McDonald LK, Steveson TC, Sei CA (1990) The molecular evolution of neuropeptides: prospects for the '90s. Brain Behav Evol 36:80-99[Medline]
Elhassan AM, Lindgren JU, Hultenby K, Bergstrom J, Adem A (1998) Methionine-enkephalin in bone and joint tissues. J Bone Miner Res 13:88-95[Medline]
Florez J, Mediavilla A (1977) Respiratory and cardiovascular effects of met-enkephalin applied to the ventral surface of the brain stem. Brain Res 138:585-900[Medline]
Folkesson R, Monstein HJ, Geijer T, Pahlman S, Nilsson K, Terenius L (1988) Expression of the proenkephalin gene in human neuroblastoma cell lines. Brain Res 427:147-154[Medline]
Gordon S, Blair S, Fine LJ (1995) Repetitive Motion Disorders of the Upper Extremity. Rosemont, CA, American Academy of Orthopaedic Surgeons
Grönblad M, Korkala O, Liesi P, Karaharju E (1985) Innervation of synovial membrane and meniscus. Acta Orthop Scand 56:484-486[Medline]
Hart D, Frank C, Bray R (1995) Inflammatory processes in repetitive motion and overuse syndromes: potential role of neurogenic mechanisms in tendons and ligaments. In Gordon S, Blair S, Blair S, Fine LJ, eds. Repetitive Motion Disorders of the Upper Extremity. Rosemont, CA, American Academy of Orthopaedic Surgeons, 247-262
Hart DA, Archambault JM, Kydd A, Reno C, Frank CB, Herzog W (1998) Gender and neurogenic variables in tendon biology and repetitive motion disorders. Clin Orthop 351:44-56[Medline]
Hassan AH, Pzewlocki R, Herz A, Stein C (1992) Dynorphin, a preferential ligand for kappa-opioid receptors, is present in nerve fibers and immune cells within inflamed tissue of the rat. Neurosci Lett 140:85-88[Medline]
Hill EL, Elde R (1991) Distribution of CGRP-, VIP-, D beta H-, SP-, and NPY-immunoreactive nerves in the periosteum of the rat. Cell Tissue Res 264:469-480[Medline]
Hirota N, Kuraishi Y, Hino Y, Sato Y, Satoh M, Takagi H (1985) Met-enkephalin and morphine but not dynorphin inhibit noxious stimuli-induced release of substance P from rabbit dorsal horn in situ. Neuropharmacology 24:567-570[Medline]
Hong Y, Abbott FV (1995) Peripheral opioid modulation of pain and inflammation in the formalin test. Eur J Pharmacol 277:21-28[Medline]
Hukkanen M, Konttinen YT, Rees RG, Santavirta S, Terenghi G, Polak JM (1992) Distribution of nerve endings and sensory neuropeptides in rat synovium, meniscus and bone. Int J Tissue React 14:1-10[Medline]
Lembeck F, Donnerer J, Bartho L (1982) Inhibition of neurogenic vasodilation and plasma extravasation by substance P antagonists, somatostatin and [D-Met2, Pro5]enkephalinamide. Eur J Pharmacol 85:171-176[Medline]
Lembeck F, Holzer P (1979) Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation. Naunyn Schmiedebergs Arch Pharmacol 310:175-183[Medline]
Levine JD, Taiwo YO (1989) Involvement of the mu-opiate receptor in peripheral analgesia. Neuroscience 32:571-575[Medline]
Lewis RV, Stern AS, Kimura S, Rossier J, Stein S, Udenfriend S (1980) An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin. Science 208:1459-1461[Medline]
Likar R, Kapral S, Steinkellner H, Stein C, Schafer M (1999) Dose-dependency of intra-articular morphine analgesia. Br J Anaesth 83:241-244
Lotz M, Vaughan JH, Carson DA (1988) Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science 241:1218-1221[Medline]
Lowry O, Rosebrough N, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275
Machelska H, Stein C (2000) Pain control by immune-derived opioids. Clin Exp Pharmacol Physiol 27:533-536[Medline]
Melzack R, Wall PD (1965) Pain mechanisms: a new theory. Science 150:971-979[Medline]
Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377:532-535. [see Comments][Medline]
Millan MJ (1986) Multiple opioid systems and pain. Pain 27:303-347[Medline]
Moore RH, Dowling DA (1982) Effects of enkephalins on perfusion pressure in isolated hindlimb preparations. Life Sci 31:1559-1566[Medline]
NozakiTaguchi N, Yamamoto T (1998) Involvement of nitric oxide in peripheral antinociception mediated by kappa- and delta-opioid receptors. Anesth Analg 87:388-393[Abstract]
Ploj K, Roman E, Gustavsson L, Nylander I (2000) Basal levels and alcohol-induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice. Brain Res Bull 53:219-226[Medline]
Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ, Jr, Civelli O (1995) Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science 270:792-794[Abstract]
Snijdelaar DG, Dirksen R, Slappendel R, Crul BJ (2000) Substance P. Eur J Pain 4:121-135[Medline]
Spetea M, Nevin ST, Hosztafi S, Ronai AZ, Toth G, Borsodi A (1998) Affinity profiles of novel delta-receptor selective benzofuran derivatives of non-peptide opioids. Neurochem Res 23:1211-1216[Medline]
Stein C, Hassan AH, Lehrberger K, Giefing J, Yassouridis A (1993) Local analgesic effect of endogenous opioid peptides. Lancet 342:321-324. [see Comments][Medline]
Stein C, Hassan AH, Przewlocki R, Gramsch C, Peter K, Herz A (1990) Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation. Proc Natl Acad Sci USA 87:5935-5939[Abstract]
Taiwo YO, Levine JD (1991) Kappa- and delta-opioids block sympathetically dependent hyperalgesia. J Neurosci 11:928-932[Abstract]
Wenk HN, Honda CN (1999) Immunohistochemical localization of delta opioid receptors in peripheral tissues. J Comp Neurol 408:567-579[Medline]
Willson NJ, Schneider JF, Roizin L, Fleiss JF, Rivers W, Demartini JE (1976) Effects of methadone hydrochloride on the growth of organotypic cerebellar cultures prepared from methadone-tolerant and control rats. J Pharmacol Exp Ther 199:368-374[Abstract]
Yaksh TL (1988) Substance P release from knee joint afferent terminals: modulation by opioids. Brain Res 458:319-324[Medline]
Zagon IS, McLaughlin PJ (1991) Identification of opioid peptides regulating proliferation of neurons and glia in the developing nervous system. Brain Res 542:318-323[Medline]
Zamir N, Weber E, Palkovits M, Brownstein M (1984) Differential processing of prodynorphin and proenkephalin in specific regions of the rat brain. Proc Natl Acad Sci USA 81:6886-6889[Abstract]
Zhou L, Zhang Q, Stein C, Schafer M (1998) Contribution of opioid receptors on primary afferent versus sympathetic neurons to peripheral opioid analgesia. J Pharmacol Exp Ther 286:1000-1006