Tolerance to repeated morphine administration is associated with increased potency of opioid agonists

Tolerance to the pain-relieving effects of opiates limits their clinical use. Although morphine tolerance is associated with desensitization of mu-opioid receptors, the underlying cellular mechanisms are not understood. One problem with the desensitization hypothesis is that acute morphine does not readily desensitize mu-opioid receptors in many cell types. Given that neurons in the periaqueductal gray (PAG) contribute to morphine antinociception and tolerance, an understanding of desensitization in PAG neurons is particularly relevant. Opioid activity in the PAG can be monitored with activation of G-protein-mediated inwardly rectifying potassium (GIRK) currents. The present data show that opioids have a biphasic effect on GIRK currents in morphine tolerant rats. Opioid activation of GIRK currents is initially potentiated in morphine (EC(50)=281 nM) compared to saline (EC(50)=8.8 microM) pretreated rats as indicated by a leftward shift in the concentration-response curve for met-enkephalin (ME)-induced currents. These currents were inhibited by superfusion of the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) suggesting that repeated morphine administration enhances agonist stimulation of mu-opioid receptor coupling to G-proteins. Although supersensitivity of mu-opioid receptors in the PAG is counterintuitive to the development of tolerance, peak GIRK currents from tolerant rats desensitized more than currents from saline pretreated rats (56% of peak current after 10 min compared to 15%, respectively). These data indicate that antinociceptive tolerance may be triggered by enhanced agonist potency resulting in increased desensitization of mu-opioid receptors

Change in Functional Selectivity of Morphine With theDevelopment of Antinociceptive Tolerance

Background and Purpose Opioids, such as morphine, are the most effective treatment for pain but their efficacy is diminished with the development of tolerance following repeated administration. Recently, we found that morphine activated ERK in opioid‐tolerant but not in naïve rats, suggesting that morphine activation of μ‐opioid receptors is altered following repeated morphine administration. Here, we have tested the hypothesis that μ‐opioid receptor activation of ERK in the ventrolateral periaqueductal gray (vlPAG) is dependent on dynamin, a protein implicated in receptor endocytosis. Experimental Approach Rats were made tolerant to repeated microinjections of morphine into the vlPAG. The effects of dynamin on ERK activation and antinociception were assessed by microinjecting myristoylated dominant‐negative dynamin peptide (Dyn‐DN) or a scrambled control peptide into the vlPAG. Microinjection of a fluorescent dermorphin analogue (DERM‐A594) into the vlPAG was used to monitor μ‐opioid receptor internalization. Key Results Morphine did not activate ERK and Dyn‐DN administration had no effect on morphine‐induced antinociception in saline‐pretreated rats. In contrast, morphine‐induced ERK activation in morphine‐pretreated rats that was blocked by Dyn‐DN administration. Dyn‐DN also inhibited morphine antinociception. Finally, morphine reduced DERM‐A594 internalization only in morphine‐tolerant rats indicating that μ‐opioid receptors were internalized and unavailable to bind DERM‐A594. Conclusions and Implications Repeated morphine administration increased μ‐opioid receptor activation of ERK signalling via a dynamin‐dependent mechanism. These results demonstrate that the balance of agonist signalling to G‐protein and dynamin‐dependent pathways is altered, effectively changing the functional selectivity of the agonist‐receptor complex

Microinjection of the Vehicle Dimethyl Sulfoxide (DMSO) Into the Periaqueductal Gray Modulates Morphine Antinociception

Dimethyl sulfoxide (DMSO) is commonly used as a solvent for water-insoluble drugs. Given that DMSO has varying cellular and behavioral effects ranging from increased membrane permeability to toxicity, microinjection of DMSO as a vehicle could confound the effects of other drugs. For example, DMSO is often used as a vehicle for studies examining the neurochemical mechanisms underlying morphine antinociception. Given that the ventrolateral periaqueductal gray (vlPAG) plays a major role in morphine antinociception and tolerance, the effects of DMSO on morphine antinociception mediated by the vlPAG needs to be evaluated. The present experiment tested whether co-administration of DMSO (0, 0.2, 2, or 20%) would alter the antinociceptive effect of microinjecting morphine into the vlPAG. DMSO had no effect on nociception when microinjected into the vlPAG alone, but 2% DMSO enhanced morphine potency when co-administered with morphine. In contrast, twice daily microinjections of DMSO (5 or 20%) for two days reduced the potency of subsequent microinjections of morphine into the vlPAG—an effect that persisted for at least one week. A similar rightward shift in the morphine dose-response curve was caused by morphine tolerance. Co-administration of morphine and DMSO during the pretreatment did not cause a greater shift in the morphine dose-response curve compared to morphine pretreated alone. In conclusion, DMSO can alter morphine antinociception following both acute (enhancement) and chronic (inhibition) administration depending on the concentration. These data reinforce the need to be cautious when using DMSO as a vehicle for drug administration

Tolerance to the antinociceptive effect of morphine in the absence of short-term presynaptic desensitization in rat periaqueductal gray neurons

Opioids activate the descending antinociceptive pathway from the ventrolateral periaqueductal gray (vlPAG) by both pre- and postsynaptic inhibition of tonically active GABAergic neurons (i.e., disinhibition). Previous research has shown that short-term desensitization of postsynaptic μ-opioid receptors (MOPrs) in the vlPAG is increased with the development of opioid tolerance. Given that pre- and postsynaptic MOPrs are coupled to different signaling mechanisms, the present study tested the hypothesis that short-term desensitization of presynaptic MOPrs also contributes to opioid tolerance. Twice-daily injections of morphine (5 mg/kg s.c.) for 2 days caused a rightward shift in the morphine dose-response curve on the hot plate test (D(50) = 9.9 mg/kg) compared with saline-pretreated (5.3 mg/kg) male Sprague-Dawley rats. In vitro whole-cell patch-clamp recordings from vlPAG slices revealed that inhibition of evoked inhibitory postsynaptic currents (eIPSCs) by the MOPr-selective agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin was decreased in morphine-tolerant (EC(50) = 708 nM) compared with saline-pretreated rats (EC(50) = 163 nM). However, short-term desensitization of MOPr inhibition of eIPSCs was not observed in either saline- or morphine-pretreated rats. Reducing the number of available MOPrs with the irreversible opioid receptor antagonist, β-chlornaltrexamine decreased maximal MOPr inhibition with no evidence of desensitization, indicating that the lack of observed desensitization is not caused by receptor reserve. These results demonstrate that tolerance to the antinociceptive effect of morphine is associated with a decrease in presynaptic MOPr sensitivity or coupling to effectors, but this change is independent of short-term MOPr desensitization

Extracellular signal-regulated kinase 1/2 activation counteracts morphine tolerance in the periaqueductal gray of the rat

Repeated administration of opioids produces long-lasting changes in micro-opioid receptor (MOR) signaling that underlie behavioral changes such as tolerance. Mitogen-activated protein kinase (MAPK) pathways, including MAPK extracellular signal-regulated kinases (ERK1/2), are modulated by opioids and are known to produce long-lasting changes in cell signaling. Thus, we tested the hypothesis that ERK1/2 activation contributes to the development and/or expression of morphine tolerance mediated by the periaqueductal gray (PAG). Changes in phosphorylated ERK1/2 expression were assessed with confocal microscopy and compared to behavioral measures of tolerance to the antinociceptive effects of chronic morphine administration. Repeated microinjection of morphine into the PAG produced tolerance and caused a significant increase in ERK1/2 phosphorylation, an effect not evident with acute morphine microinjection. Microinjection of the MAPK/ERK kinase inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene ethanolate (U0126), into the PAG had no effect on antinociception produced by acute morphine administration. However, repeated coadministration of U0126 and morphine into the PAG blocked ERK1/2 phosphorylation and enhanced the development of morphine tolerance. Coadministration of U0126 with morphine only on the test day also enhanced the expression of morphine tolerance. Administration of the irreversible opioid receptor antagonist beta-chlornaltrexamine blocked the activation of ERK1/2 caused by repeated morphine microinjections, demonstrating that ERK1/2 activation was a MOR-mediated event. In summary, these studies show that chronic morphine administration alters ERK1/2 signaling and that disruption of ERK1/2 signaling enhances both the development and expression of morphine tolerance. Contrary to expectations, these data indicate that ERK1/2 activation opposes the development of morphine tolerance

Sex Differences in the Medical Care of VA Patients with Chronic Non-Cancer Pain

OBJECTIVE: Despite a growing number of women seeking medical care in the VA system, little is known about the characteristics of their chronic pain or the pain care they receive. This study sought to determine if sex differences are present in the medical care veterans received for chronic pain. DESIGN: Retrospective cohort study using VA administrative data. SUBJECTS: 17,583 veteran patients with moderate to severe chronic non-cancer pain treated in the Pacific Northwest during 2008. METHODS: Multivariate logistic regression assessed for sex differences in primary care utilization, prescription of chronic opioid therapy, visits to emergency departments for a pain-related diagnosis, and physical therapy referral. RESULTS: Compared to male veterans, female veterans were more often diagnosed with two or more pain conditions and had more of the following pain-related diagnoses: fibromyalgia, low back pain, inflammatory bowel disease, migraine headache, neck or joint pain, and arthritis. After adjustment for demographic characteristics, pain diagnoses, mental health diagnoses, substance use disorders, and medical comorbidity, women had lower odds of being prescribed chronic opioid therapy (AOR 0.67, 95% CI 0.58–0.78), greater odds of visiting an emergency department for a pain-related complaint (AOR 1.40, 95% CI 1.18–1.65), and greater odds of receiving physical therapy (AOR 1.19, 95% CI 1.05–1.33). Primary care utilization was not significantly different between sexes. CONCLUSIONS: Sex differences are present in the care female veterans receive for chronic pain. Further research is necessary to understand the etiology of the observed differences and their associations with clinical outcomes

Opioid receptor internalization contributes to dermorphin-mediated antinociception

Microinjection of opioids into the ventrolateral periaqueductal gray (vlPAG) produces antinociception in part by binding to mu-opioid receptors (MOPrs). Although both high and low efficacy agonists produce antinociception, low efficacy agonists such as morphine produce limited MOPr internalization suggesting that MOPr internalization and signaling leading to antinociception are independent. This hypothesis was tested in awake, behaving rats using DERM-A594, a fluorescently labeled dermorphin analog, and internalization blockers. Microinjection of DERM-A594 into the vlPAG produced both antinociception and internalization of DERM-A594. Administration of the irreversible opioid receptor antagonist beta-CNA prior to DERM-A594 microinjection reduced both the antinociceptive effect and the number of DERM-A594 labeled cells demonstrating that both effects are opioid receptor-mediated. Pretreatment with the internalization blockers dynamin dominant-negative inhibitory peptide (dynamin-DN) and concanavalinA (ConA) attenuated both DERM-A594 internalization and antinociception. Microinjection of dynamin-DN and ConA also decreased the antinociceptive potency of the unlabeled opioid agonist dermorphin when microinjected into the vlPAG as demonstrated by rightward shifts in the dose-response curves. In contrast, administration of dynamin-DN had no effect on the antinociceptive effect of microinjecting the GABA A antagonist bicuculline into the vlPAG. The finding that dermorphin-induced antinociception is attenuated by blocking receptor internalization indicates that key parts of opioid receptor-mediated signaling depend on internalization

Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor