Opioid ligands and receptors of the joint

Sammanfattning: The aim was to explore the occurrence of an opioid system in joints. Thus, joint tissues from rats with and without arthritis and also from patients with knee osteoarthrosis were investigated by EM, immunohistochemistry, RIA, HPLC, receptor binding assay and RT-PCR. In rat joints, EM demonstrated the occurrence of met-enkephalin (ME) in nerve fibers, but also in osteoblasts, osteocytes, endothelial, synovial and monoblastic cells. The novel finding of multiple sources of opioid ligands challenges the current concept of immune cells being the main source of peripheral opioids. To screen joints for opioid ligands and to obtain an overview of their occurrence, immunohistochemistry was applied. After extensive testing, an approach based on streptavidin and biotin was found to provide conclusive results. Altogether four different enkephalins were identified, ME being most prevalent, in both nerve fibers and cells. However, no endorphin or dynorphin could be detected. Neuronal ME, presumed to convey analgesia, was found to co-exist with the pro-nociceptive neuropeptide substance P (SP) in primary afferents, suggesting reciprocal counteracting mechanisms. In rat adjuvant arthritis, the neuronal occurrence of both ME and SP was clearly increased, presumably reflecting a modulatory interaction. Conversely, the cellular occurrence of ME was significantly decreased in synovial lining cells. Thus, arthritis seems to be associated with a neuronal increase and cellular decrease in ME. To assess the net change, RIA was applied to extracts of whole joints, showing a decrease in ME in chronic arthritis. The observation may reflect air exhausted antiinflammatory response, contributing to perpetuation of the condition. As the RIA levels of the most prevalent opioid, ME, were quite low, a more sensitive method would be needed to quantify other joint opioids. Therefore, two alternatives for extract purification, previously used for plasma and CNS samples, were applied to the joint specimens. For these tests, two opioid ligands presumed to occur at lower levels than ME were used, i.e. MEAP and DYNB. Ion exchange chromatography was found to provide the highest recovery yielding measurable concentrations of both opioids in all tissues. Hence, this approach seems preferable for quantification of opioids ligands in joints. To explore the target for enkephalins and opiates, in vitro opioid receptor (OR) binding assays were done on preparations from rat capsule/synovium and periosteum. Binding of [3H]naloxone was found to be stereospecific and saturable, showing similar characteristics to those in brain tissues. Further confirmation of joints being equipped with OR was obtained by RT-PCR showing a clear expression of delta-opioid receptor (DOR) mRNA in capsule specimens. Combined with the observation of enkephalins in primary afferents of the joint, it appears that there is a direct opioid signaling from CNS to joint capsule cells, presumably eliciting other effects than analgesia. The observations of ME in osteoblasts prompted in vitro experiments on primary human osteoblasts, in which ME was found to inhibit and naloxone to stimulate ostcoblastic proliferation. Apart from providing evidence of opioid receptors on osteoblasts, the findings indicate that enkephalins play an inhibitory role in local bone turnover, possibly also in skeletal growth. Immunohistochemical analysis of delta- and µ-opioid receptors (DOR and MOR) disclosed neuronal occurrence of DOR, coexisting with SP in primary afferents in synovial specimens from patients with osteoarthrosis. This finding may be assumed to reflect counteracting mechanisms in the peripheral regulation of pain and inflammation. However, no neuronal occurrence of MOR was detected, which might explain the rather weak analgesic effect obtained by intra-articular administration of morphine. In synovial lining and smooth muscle cells, both MOR and DOR were identified, most likely reflecting a regulatory role in inflammation and vasoactivity. ORs were also detected on both rat and human articular chondrocytes, supporting the notion of an involvement of endogenous opioids in chondrocyte proliferation, possibly also in differentiation. In conclusion, there exists an opioid system in joints, which seems to play a role in a wide variety of physiologic actions, beyond nociception and inflammation. As for intra-articular analgesia, the finding of neuronal DOR, but not MOR, in human joints suggests that DOR targeted drugs would have a better effect than morphine.