Modulatory effects of Gs-coupled excitatory opioid receptor functions on opioid analgesia,tolerance, and dependence

Electrophysiologic studies of opioid effects on nociceptive types of dorsal root ganglion (DRG) neurons in organotypic cultures have shown that morphine and mostμ, δ, and κ opioid agonists can elicit bimodal excitatory as well as inhibitory modulation of the action potential duration (APD) of these cells. Excitatory opioid effects have been shown to be mediated by opioid receptors that are coupled via Gs to cyclic AMP-dependent ionic conductances that prolong the APD, whereas inhibitory opioid effects are mediated by opioid receptors coupled via Gi/Go to ionic conductuances that shorten the APD. Selective blockade of excitatory opioid receptor functions by low (ca. pM) concentrations of naloxone, naltrexone, etorphine and other specific agents markedly increases the inhibitory potency of morphine or other bimodally acting agonists and attenuates development of tolerance/dependence. These in vitro studies have been confirmed by tail-flick assays showin that acute co-treatment of mice with morphine plus ultra-low-dose naltrexone or etorphine remarkably enhances the antinociceptive potency of morphine whereas chronic co-treatment attenuates development of tolerance and naloxone-precipitated withdrawal-jumping symptoms. Special issue dedicated to Dr. Eric J. Simon.     

Genetic deletion of microglial Panx1 attenuates morphine withdrawal,but not analgesic tolerance or hyperalgesia in mice

Opioids are among the most powerful analgesics for managing pain, yet their repeated use can lead to the development of severe adverse effects. In a recent study, we identified the microglial pannexin-1 channel (Panx1) as a critical substrate for opioid withdrawal. Here, we investigated whether microglial Panx1 contributes to opioid-induced hyperalgesia (OIH) and opioid analgesic tolerance using mice with a tamoxifen-inducible deletion of microglial Panx1. We determined that escalating doses of morphine resulted in thermal pain hypersensitivity in both Panx1-expressing and microglial Panx1-deficient mice. In microglial Panx1-deficient mice, we also found that acute morphine antinociception remained intact, and repeated morphine treatment at a constant dose resulted in a progressive decline in morphine antinociception and a reduction in morphine potency. This reduction in morphine antinociceptive potency was indistinguishable from that observed in Panx1-expressing mice. Notably, morphine tolerant animals displayed increased spinal microglial reactivity, but no change of microglial Panx1 expression. Collectively, our findings indicate microglial Panx1 differentially contributes to opioid withdrawal, but not the development of opioid-induced hyperalgesia or tolerance.     

Effects of opioid antagonists and their quaternary analogs on temperature changes in morphine-dependent rats

Subcutaneous administration of three opioid antagonists; naloxone, naltrexone and nalmefene, produced a significant rise in tail skin temperature and a subsequent fall in rectal temperature in morphine dependent rats. However, subcutaneous administration of equimolar concentrations of the quaternary derivatives of these opioid antagonists (naloxone methobromide, naltrexone methobromide and n-methylnalmefenium iodide) failed to produce any significant alterations in either tail skin or rectal temperatures in the morphine dependent rat. At doses of naloxone methobromide 6 to 9 times greater than naloxone, there was a slight reduction of rectal temperatures with no significant elevation of skin temperature. However, the fall in rectal temperature was still significantly less than that achieved with administration of naloxone. When each of these six agents were administered centrally (20 micrograms/5 microliter, icv) in the morphine dependent rat, similar increases in tail skin temperature and decreases in rectal temperature were observed. These temperature changes were similar to those observed following systemic administration of the opioid antagonist. Previously, we have suggested that acute withdrawal in the morphine-dependent rat may serve as an animal model for the mechanism of the menopausal hot flush. Collectively, these results suggest that the temperature changes associated with morphine-withdrawal in our rat model for studying the mechanisms of the menopausal hot flush are centrally mediated.     

Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Methamphetamine (METH) is a psychostimulant with high abuse potential and severe neurotoxicity. Recent studies in animal models have indicated that METH can impair the blood–brain barrier (BBB), suggesting that some of the neurotoxic effects resulting from METH abuse could be due to barrier disruption. We report here that while chronic exposure to METH disrupts barrier function of primary human brain microvascular endothelial cells (HBMECs) and human umbilical vein endothelial cells (HUVECs), an early pro-survival response is observed following acute exposure by induction of autophagic mechanisms. Acute METH exposure induces an early increase in Beclin1 and LC3 recruitment. This is mediated through inactivation of the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6K pathway, and upregulation of the ERK1/2. Blockade of Kappa opioid receptor (KOR), and treatment with autophagic inhibitors accelerated METH-induced apoptosis, suggesting that the early autophagic response is a survival mechanism for endothelial cells and is mediated through the kappa opioid receptor. Our studies indicate that kappa opioid receptor can be therapeutically exploited for attenuating METH-induced BBB dysfunction.     

Blockade of morphine supersensitivity by an antisense oligodeoxynucleotide targeting the delta opioid receptor (DOR-1)

An antisense oligodeoxynucleotide (ODN) targeting 20 bases of the coding sequence of the cloned delta opioid receptor (DOR-1), a mismatched ODN (different from the antisense ODN at 4 bases) or saline was administered to 3 groups of CD-1 mice implanted with naltrexone pellets (7.5 mg) for 7 days. Morphine supersensitivity (i.e., increased potency as defined by decreased morphine ED₅₀ values) was observed 24 h after pellet removal (day 8) in mice treated with saline or mismatch ODN, but not in antisense ODN treated mice. Antisense ODN alone had no effect on basal nociceptive thresholds or morphine analgesia but reduced the analgesic potency of the delta₂ opioid agonist [D-Ala²]deltorphin II. These data suggest that the delta₂ opioid receptor system participates in the adaptive changes contributing to increased morphine potency following chronic naltrexone treatment.     

Morphine modulates cell proliferation through mir133b & mir128 in the neuroblastoma SH-SY5Y cell line

Neuroblastoma is a childhood cancer with high incidence and high mortality rate. Great efforts are made to find new treatments and molecular markers for diagnosis and prognosis. miRNAs stand for novel strategies to modulate tumor growth, as they can act either as tumor suppressors or as oncogenes. Morphine is an opioid agonist widely used to treat severe and chronic pain, as for example cancer pain. Previous studies have revealed that morphine is able to modify cancer progression, by acting on proliferation or on apoptosis; however, up to date, the available results are contradictory, maybe due to the different doses used, routes of administration and model systems. While some studies show that morphine promotes cell proliferation and metastasis, other authors sustain that morphine effect is mainly antiproliferative and pro-apoptotic. In this study we aim to establish the effect of chronic opiate administration on cell proliferation in the neuroblastoma SH-SY5Y cell line. Low doses of morphine (10 nM) promoted cell proliferation in undifferentiated cells and reduced the expression levels of miR133b, while higher doses (1 μM) inhibited cell proliferation and correlated with decreased levels of miR133b and miR128 without triggering apoptosis. Naloxone, the classical opioid antagonist, could not fully block the effect of morphine on miR128 expression, so that the observed effect may be mediated by non-opioid mechanisms. Our results represent a further contribution to the hypothesis that a joint regulation of miRNA networks and the specific characteristics of the target tissue may determine the effect of morphine on tumor cell growth.     

Conjugated linoleic acid enhances glutathione synthesis and attenuates pathological signs in MRL/MpJ-Fas(lpr) mice

Conjugated linoleic acid (CLA), a naturally occurring peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, exhibits proapoptotic, immunomodulatory, and anticancer properties. In this study, we examined the biological effects of CLA administration in the MRL/MpJ-Fas(lpr) mouse, an animal model of systemic lupus erythematosus (SLE). We found that CLA exerted apparently opposed activities in in vitro experiments, depending on its concentration: 100 microM CLA downregulated IFN gamma synthesis and cell proliferation of splenocytes, in association with apoptosis induction and a decrease of intracellular thiols (GSH + GSSG), whereas 25 microM CLA did not significantly influence cell proliferation but enhanced the expression of gamma-glutamylcysteine ligase catalytic subunit (GCLC) and intracellular GSH concentration. Interestingly, the antiproliferative effect at 100 microM was not inhibited by the PPAR gamma antagonist GW9662. In vivo, CLA administration drastically reduced SLE signs (splenomegaly, autoantibodies, and cytokine synthesis), a condition paralleled by the enhancement of GCLC expression and intracellular GSH content. Moreover, CLA administration significantly downregulated nuclear factor kappaB activity independent of PPAR gamma activation and apoptosis induction. In conclusion, enhanced GSH content and GCLC expression in CLA-treated mice suggest a novel biochemical mechanism underlying its immunomodulatory activity and the beneficial effects on murine SLE signs.     

TNFR1 determines progression of chronic liver injury in the IKKγ/Nemo genetic model

Death receptor-mediated hepatocyte apoptosis is implicated in a wide range of liver diseases including viral and alcoholic hepatitis, ischemia/reperfusion injury, fulminant hepatic failure, cholestatic liver injury, as well as cancer. Deletion of NF-κB essential modulator in hepatocytes (IKKγ/Nemo) causes spontaneous progression of TNF-mediated chronic hepatitis to hepatocellular carcinoma (HCC). Thus, we analyzed the role of death receptors including TNFR1 and TRAIL in the regulation of cell death and the progression of liver injury in IKKγ/Nemo-deleted livers. We crossed hepatocyte-specific IKKγ/Nemo knockout mice (Nemo^Δhepa) with constitutive TNFR1^−/− and TRAIL^−/− mice. Deletion of TNFR1, but not TRAIL, decreased apoptotic cell death, compensatory proliferation, liver fibrogenesis, infiltration of immune cells as well as pro-inflammatory cytokines, and indicators of tumor growth during the progression of chronic liver injury. These events were associated with diminished JNK activation. In contrast, deletion of TNFR1 in bone-marrow-derived cells promoted chronic liver injury. Our data demonstrate that TNF- and not TRAIL signaling determines the progression of IKKγ/Nemo-dependent chronic hepatitis. Additionally, we show that TNFR1 in hepatocytes and immune cells have different roles in chronic liver injury–a finding that has direct implications for treating chronic liver disease.     

Morphine withdrawal modifies antinociceptive effects of acute morphine in rats

Repeated opioid use is known to cause tolerance of antinociceptive effects. Whether opioid abstinence modifies antinociceptive effects is unknown. Here we reported that morphine withdrawal for 18 h and 4 days after repeated morphine treatment largely reduced tail-flick latencies compared with control, while the rats showed severe withdrawal syndromes. However, the latencies and withdrawal syndromes were restored to control level at 20 days withdrawal. Similarly, antinociceptive effects of acute morphine were decreased at 18 h and further decreased at 4 days but restored to control level at 20 days withdrawal. Behavioral stress that was given to the rats at 18 h withdrawal further reduced tail-flick latencies and antinociceptive effects. Conversely, the glucocorticoid receptor antagonist RU38486 increased tail-flick latencies and antinociceptive effects at 4 days withdrawal. These results suggest that morphine withdrawal could evoke behavioral stress to modify antinociceptive effects, implicating a significant influence of opioid abstinence on chronic pain treatment.     

Effects of endogenous and exogenous opioids on splenic natural killer cell activity

Previous work from our own and other laboratories has shown that electroshock-induced neurohormonal changes in rodents could modify host-tumor interactions by both increasing the frequency and growth rate of transplanted tumors and decreasing the elimination rate of a radiolabelled natural killer (NK) cell sensitive tumor. To test whether such neurohormonal changes could affect NK activity we subjected mice to tail electrode shock (TES) and examined in vitro splenic NK activity. We found that between 30 and 60 min after TES there is a significant but transient suppression of their splenic NK activity. To determine whether TES-induced endogenous opioids might be involved in this suppression mice were given intraperitoneal injections of the opioid antagonists naloxone or naltrexone before or at the end of the TES session. These drugs prevented NK suppression. In a further test of the hypothesis that opioids alter NK activity mice were given a single intraperitoneal injection of morphine or [D-Ala2-Met5]-beta-endorphin, a relatively stable analogue of beta-endorphin, an endogenous opioid. Contrary to expectations these opioids enhanced splenic NK activity. Our interpretation of these results is that shock-induced NK suppression may not be mediated by endogenous opioids and that the effects of naloxone and naltrexone on NK activity may not be related to their opioid antagonist properties. On the contrary, opioids may participate in a homeostatic rebound from suppression mediated by other neurohormonal mechanisms activated during TES.     

Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis.     

Continuous Treatment with Morphine Increases Diazepam Binding Inhibitor mRNA in Mouse Brain

Abstract: Effects of acute and chronic morphine treatment on the expression of diazepam binding inhibitor (DBI) mRNA in the mouse brain were examined. Cerebral DBI mRNA expression significantly increased in morphine-dependent mice, and this increase is more remarkable in morphine-withdrawn mice, whereas a single administration of morphine (50 mg/kg) produced no changes in the expression. Simultaneous administration of naloxone (3 mg/kg) with morphine completely abolished the increase in cerebral DBI mRNA expression observed in morphine-dependent and -withdrawn mice. These results indicate that a chronic functional interaction between morphine and opioid receptors has a critical role in increases in DBI mRNA expression.     

Analgesic effect of corticotropin-releasing factor administered into midbrain periaqueductal grey matter

Corticotropin-releasing factor (CRF) participates in development of stress-induced analgesia. Midbrain periaqueductal grey matter (MPAG) is one of crucial structures of the brain antinociceptive system. The aim of the study was to investigate effects of the CRF administration into the MPAG on pain sensitivity in alert rats and contribution of opioid mechanisms to these CRF-induced effects. Somatic pain sensitivity was tested by tail flick response latency following thermal stimuli. The opioid antagonist naltrexone administered systemically or centrally into the MPAG was used to study involvement ofopioid mechanisms in the CRF-induced effects. The CRF administration (0.7 microg/rat) into the MPAG caused analgesic effect. The CRF-induced analgesic effects were eliminated by systemic as well as central naltrexone pretreatment. Effect of central naltrexone on the CRF-induced analgesia manifested itself faster as compared with effect of systemic naltrexone. The data obtained suggest that one of central mechanisms of the CRF-induced analgesic effect on somatic pain sensitivity in alert rats may be mediated by the MPAG neurons and provided by involvement of opioid mechanisms.     

Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.     

Evaluation of possible mechanisms of protective role of Tamarix gallica against DEN initiated and 2-AAF promoted hepatocarcinogenesis in male Wistar rats

We have previously reported that Tamarix gallica caused a marked inhibition of thioacetamide-induced hepatotoxicity, oxidative damage and early tumor promotion related events in the liver. These results strongly indicates that T. gallica may have chemopreventive potential. Therefore, in the present study, we examined the inhibitory effects of T. gallica methanolic extract on diethylnitrosamine (DEN) initiated and 2-acetyl aminofluorene (2-AAF) promoted liver carcinogenesis in male Wistar rats. Interestingly, it was found that T. gallica (25 and 50 mg/kg body wt.) resulted in a marked reduction of the incidence of liver tumors. The study was further histologically confirmed. Furthermore to understand the underlying mechanisms of chemopreventive action by T. gallica we evaluated the levels activities of hepatic antioxidant defense enzymes, ornithine decarboxylase activity and hepatic DNA synthesis as a marker for tumor promotion since direct correlation between these marker parameters and carcinogenicity have been well documented. Treatment of male Wistar rats for five consecutive days with 2-AAF i.p. induced significant hepatic toxicity, oxidative stress and hyperproliferation. Pretreatment of T. gallica extract (25 and 50 mg/kg body wt.) prevented oxidative stress by restoring the levels of antioxidant enzymes and also prevented toxicity at both the doses. The promotion parameters induced (ornithine decarboxylase activity and DNA synthesis) by 2-AAF administration in diet with partial hepatectomy (PH) were also significantly suppressed dose-dependently by T. gallica. Therefore, we can conclude that ultimately the protection against liver carcinogenesis by T. gallica methanolic extract might be mediated by multiple actions, which include restoration of cellular antioxidant enzymes, detoxifying enzymes, ODC activity and DNA synthesis.     

Promising effects of the 4HPR-BSO combination in neuroblastoma monolayers and spheroids

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5-10 μM, 24 h) resulted in ROS induction (114-633%) and increased GSH levels (68-120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25-100 μM, 24 h). The 4HPR-BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110-150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR-BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR-BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR-BSO combination might be a promising new strategy in the treatment of neuroblastoma.