β-Endorphin rat

Thalidomide is an antiinflammatory, antiangiogenic and immunomodulatory agent which has been used for the treatment of erythema nodosum leprosum and multiple myeloma. It has also been employed in treating complex regional pain syndromes. The current study aimed to reveal the molecular mechanisms underlying thalidomide-induced pain antihypersensitive effects in neuropathic pain. Thalidomide gavage, but not its more potent analogs lenalidomide and pomalidomide, inhibited mechanical allodynia and thermal hyperalgesia in neuropathic pain rats induced by tight ligation of spinal nerves, with ED50 values of 44.9 and 23.5 mg/kg, and Emax values of 74% and 84% MPE respectively. Intrathecal injection of thalidomide also inhibited mechanical allodynia and thermal hyperalgesia in neuropathic pain. Treatment with thalidomide, lenalidomide and pomalidomide reduced peripheral nerve injury-induced proinflammatory cytokines (TNFα, IL-1β and IL-6) in the ipsilateral spinal cords of neuropathic rats and LPS-treated primary microglial cells. In contrast, treatment with thalidomide, but not lenalidomide or pomalidomide, stimulated spinal expressions of IL-10 and β-endorphin in neuropathic rats. Particularly, thalidomide specifically stimulated IL-10 and β-endorphin expressions in microglia but not astrocytes or neurons. Furthermore, pretreatment with the IL-10 antibody blocked upregulation of β-endorphin in neuropathic rats and cultured microglial cells, whereas it did not restore thalidomide-induced downregulation of proinflammatory cytokine expression. Importantly, pretreatment with intrathecal injection of the microglial metabolic inhibitor minocycline, IL-10 antibody, β-endorphin antiserum, and preferred or selective μ-opioid receptor antagonist naloxone or CTAP entirely blocked thalidomide gavage-induced mechanical antiallodynia. Our results demonstrate that thalidomide, but not lenalidomide or pomalidomide, alleviates neuropathic pain, which is mediated by upregulation of spinal microglial IL-10/β-endorphin expression, rather than downregulation of TNFα expression.

The binding of 3H-beta-endorphin to rat brain homogenates, reported by several other laboratories, has suggested unique selective beta-endorphin binding sites. We now present additional evidence supporting the concept of distinct beta-endorphin binding (epsilon) sites in rat brain. In competitive displacement studies, 3H-beta-endorphin was inhibited far better by unlabeled beta-endorphin than a variety of opiates and enkephalins. Conversely, beta-endorphin inhibited the binding of a series of 3H-labeled ligands, including dihydromorphine, ethylketocyclazocine, SKF 10,047, naloxone and D-ala2-D-leu5-enkephalin, far less potently than their corresponding unlabeled drug. Other differences were also found. Compared to 3H-dihydromorphine and 3H-D-ala2-D-leu5-enkephalin binding, 3H-beta-endorphin binding was far less sensitive to the reagent N-ethylmaleimide and more sensitive to the proteolytic enzyme trypsin. The regional distribution for 3H-beta-endorphin binding was also distinct from other 3H-ligands tested. This evidence supports the concept of a distinct binding site for beta-endorphin which does not correspond to the previously defined opioid binding sites.

Numerous studies have investigated the behavioural effects of beta-endorphin, both endogenous and exogenously applied. However, the potential for biotransformation of beta-endorphin in the extracellular space of the brain has not been previously directly addressed in vivo. Utilising microinfusion/microdialysis and matrix-assisted laser desorption/ionisation mass spectrometry, we investigated beta-endorphin biotransformation in the striatum of rats. We infused 1.0 nmol beta-endorphin into the striatum of adult male Fischer rats and observed rapid cleavage resulting in beta-endorphin 1-18, as well as several fragments resulting from further N-terminal degradation. In vitro studies with incubation of full-length beta-endorphin, with and without protease inhibitors, in the incubation fluid of isolated striatal slices indicate that beta-endorphin is initially cleaved predominantly at the Phe(18)-Lys(19), position, as well as at the Leu(17)-Phe(18) position. Investigations of cerebrospinal fluid revealed similar enzymatic cleavage of beta-endorphin. The observed pattern of cleavage sites (Phe(18)-Lys(19) and Leu(17)-Phe(18)) is consistent with published in vitro studies of purified insulin-degrading enzyme cleavage of beta-endorphin. The binding affinities of full-length beta-endorphin, as well as previously identified beta-endorphin fragments alpha-endorphin (beta-endorphin 1-16) and gamma-endorphin (beta-endorphin 1-17), and the fragment identified in the present study, beta-endorphin 1-18, at heterologously expressed mu, delta and kappa-opioid receptors, respectively, were determined; the affinity of the truncation fragments is reduced at each of the receptors compared to the affinity of full length beta-endorphin.