Chronic opioid receptor (OR) activation by morphine causes distinct cellular adaptations responsible for the development of tolerance. The present study examines the effect of chronic morphine exposure on the ability of high-efficacy agonists to mediate δ-OR (DOR) and μ-OR (MOR) uncoupling and internalization, two regulatory mechanisms contributing to rapid desensitization of OR function. Chronic morphine treatment (1 μm; 72 hr) of DOR carrying neuroblastoma x glioma (NG108-15) hybrid cells, a prototypical model system frequently used to study cellular aspects of opioid tolerance, completely blocked the capacity of [d-Ala²,d-Leu⁵]enkephalin (DADLE) and etorphine to desensitize opioid-stimulated [³⁵S]GTPγS binding and to mediate DOR internalization. Similar findings were obtained on stably DOR- and MOR-transfected human embryonic kidney (HEK) 293 cells. Chronic morphine treatment also heterologously impaired agonist regulation of non-opioid G-protein-coupled receptors, such as the m₄-muscarinic acetylcholine receptor and the brain-type cannabinoid receptor. As a possible underlying mechanism, we found that chronic morphine treatment completely blocked agonist-induced redistribution of β-arrestin1 in both NG108-15 and stably MOR-transfected HEK293 cells. Moreover, attenuation of β-arrestin1 function appears to depend on persistent stimulation of MAP kinase activity during the course of chronic morphine treatment, because coincubation of the cells together with the MAP kinase blocker PD98059 fully restored β-arrestin1 translocation and receptor internalization. These results demonstrate that chronic morphine treatment produces adaptational changes at the β-arrestin1 level, which in turn attenuates agonist-mediated desensitization and internalization of G-protein-coupled receptors.

Inflammatory pain is counteracted by a number of physiological processes. For example, opioid receptors, which are present on peripheral terminals of sensory neurons, are activated by endogenous opioids, which are released from immune cells migrating to the inflamed tissue. Earlier data demonstrated that interleukin-6 contributes to such inflammation-induced analgesia. In this report, we demonstrated that interleukin-6 strongly induces μ-opioid receptor mRNA in the human neuroblastoma cell line SH SY5Y, whereas δ-opioid receptor mRNA levels are not influenced. The mRNA increase in these cells is followed by an increase in μ-opioid receptor-specific binding. Using transcription factor decoy oligonucleotides, direct evidence was provided that the up-regulation of μ-opioid receptor mRNA in intact cells is dependent on the transcription factors signal transducers and activators of transcription 1 (STAT1) and STAT3, whereas other transcription factors, such as activator protein-1, nuclear factor (NF)-κB, or NF-interleukin-6 are not involved. STAT1 and STAT3 bound to a site located at nucleotide -1583 on the promoter of the human μ-opioid receptor gene, as shown by transient transfection experiments, electrophoretic mobility shift assays, and transcription factor decoy oligonucleotides. A mutation analysis of the 5′-TTCATGGAA-3′ STAT1/3 element (palindrome underlined) was performed to determine nucleotide residues that are necessary for the binding of STAT1 and STAT3. It suggested that only the palindromic half sides and the two adjacent central nucleotides are required. Neither mutation of the nucleotides outside the palindrome nor mutation of the central nucleotide affected STAT1/3 binding.

Chronic opioid treatment of stably μ-opioid receptor transfected human mammary epidermoid A431 carcinoma cells (clone A431/μ13) results in sensitization of adenylyl cyclase (AC), a cellular adaptation associated with drug dependence. Up-regulation of AC is characterized by significantly increased levels of both basal and post-receptor-stimulated effector activities, which develop without any apparent change in the quantity of stimulatory G proteins and the maximum catalytic activity of AC. Here, we report that detergent extracts from membranes of chronically morphine-treated (10 μm; 2 days) A431/μ13 cells display higher stimulatory AC activities as assessed in the S49cyc⁻reconstitution assay. This finding is most likely due to an increased functional activity of G_sα because the addition of exogenous G_βγ subunits, which per sestimulate AC in S49cyc⁻ membranes, failed to affect the difference in reconstitutive AC activity. Moreover, both chemical depalmitoylation by hydroxylamine and inhibition of palmitoyl-CoA transferase in vivo by tunicamycin treatment increased the reconstitutive activity of detergent extracts and eliminated the differences between native and opioid-dependent cells, indicating that the increase in stimulatory activity is due to depalmitoylation of G_sα. Indeed, metabolic labeling studies with [³H]palmitic acid revealed that chronic opioid treatment reduces considerably the fraction of palmitoylated G_sα in the plasma membrane. Furthermore, high affinity [³H]forskolin binding experiments demonstrated that depalmitoylated G_sα is able to associate directly with AC during the state of opioid dependence even without preceding receptor activation. These results suggest that post-translational palmitoylation of G_sα provides a potential regulator of transmembrane signaling. Moreover, accumulation of the depalmitoylated form of G_sα in the plasma membrane as reported herein may contribute to the increase in stimulatory AC signaling, as is characteristic for the state of opioid dependence.

This study investigated doxycycline‐related side effects in a large population of dogs. Data from 386 dogs that had received doxycycline for the treatment of various infectious diseases were analysed retrospectively. Potential side effects that developed during treatment were documented, and correlations with signalment, dose, duration of treatment, frequency of application, doxycycline preparation and use of additional drugs were investigated. Vomiting was reported in 18.3 per cent of dogs, 7.0 per cent developed diarrhoea and 2.5 per cent developed anorexia. While being treated with doxycycline, 39.4 per cent of dogs showed an increase in alanine aminotransferase (ALT) activity and 36.4 per cent showed an increase in alkaline phosphatase (ALP) activity. There was a dose‐related risk of an increase in ALP activity (P=0.011, odds ratio [OR]=1.27, 95 per cent confidence interval [CI] 1.06 to 1.53), and older dogs treated with doxycycline were more likely to develop an increase in ALT activity (P=0.038, OR=1.23, 95 per cent CI 1.01 to 1.50) and vomiting (P=0.017, OR=1.11, 95 per cent CI 1.02 to 1.21).