Shirzad Jenab

ETHANOL AND NALOXONE DIFFERENTIALLY UPREGULATE DELTA OPIOID RECEPTOR GENE EXPRESSION IN NEUROBLASTOMA HYBRID (NG108-15) CELLS

We have used a sensitive solution hybridization assay with a riboprobe transcribed from the coding sequence of the delta opioid receptor (DOR) to quantitate the changes in DOR mRNA transcript levels following exposure of NG108-15 cells to ethanol and/or the opioid antagonist, naloxone. Incubation of NG108-15 cells with 200 mM ethanol or 1 microM naloxone, treatments that have previously been shown to upregulate DOR binding, increased DOR mRNA transcript levels 2 to 3 fold. DOR mRNA levels peaked at 24 to 48 h after exposure to either ethanol or naloxone. At 168 h, DOR mRNA levels in NG108-15 cells exposed to naloxone had returned to control (untreated) levels while the levels in ethanol treated cells remained nearly equal to peak values. Exposure to a combination of ethanol plus naloxone for 24 h produced an additive effect, so that DOR mRNA transcripts were increased 3 fold. Northern blot analysis identified six DOR transcript bands ranging in size from 8.7 to 2.1 kb. The above treatments increased each of the six bands proportionately, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the Northern blot. These results demonstrate that each of the DOR transcripts in NG108-15 cells are subject to homologous (naloxone) as well as heterologous (ethanol) upregulation.

Mu and delta opioid receptor analgesia, binding density, and mRNA levels in mice selectively bred for high and low analgesia

The present study examined mu and delta opioid analgesia, receptor binding, and receptor mRNA levels in lines of mice from two selective breeding projects of relevance to opioid analgesia. Large differences were observed in the analgesic potency of [d-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO), [d-Pen2,5]enkephalin (DPDPE), and [d-Ala2]deltorphin II (DELT), selective mu, delta1, and delta2 opioid receptor agonists, respectively, in mice selectively bred for high (HA) and low (LA) swim stress-induced analgesia (SIA). HAR and LAR mice, selectively bred for high and low levorphanol analgesia, respectively, display equally large differences in their analgesic sensitivity to DAMGO, modest differences in sensitivity to DPDPE, and no differences in sensitivity to DELT. These sizable genotypic differences in analgesic potency were accompanied by HA/LA and HAR/LAR differences in whole-brain homogenate [3H]DPDPE and/or [3H]DELT, but paradoxically not [3H]DAMGO, binding. Solution hybridization of mRNA extracts encoding mu (MOR-1) or delta (DOR-1) opioid receptors indicated some regional differences in gene expression between high and low lines. Surprisingly, differences in these in vitro markers were often in the direction of LAR>HAR. The present data indicate that selection for either SSIA or levorphanol analgesia produces differential effects on mu and delta opioid analgesia that are accompanied by alterations on in vitro assays, the significance of which remains to be determined. The data are discussed with regard to the utility of in vitro biological markers and genetic models of analgesia.

Assessment of delta opioid antinociception and receptor mRNA levels in mouse after chronic naltrexone treatment

The antinociceptive potency of the delta opioid receptor (DOR) agonist [D-Ala2]Deltorphin II and the levels of DOR mRNA were measured in mice chronically treated with naltrexone. ED50 determinations for [D-Ala2]Deltorphin II, using the tail-flick test with mice that had been treated with naltrexone for 7 days followed by a 24 h naltrexone free period (study day 8), revealed a 7.7-fold increase in antinociceptive potency, indicating functional supersensitivity. Utilization of a micro-dissection technique followed by quantitative solution hybridization measurements of RNA extracts from mouse CNS revealed levels of DOR mRNA ranging from 2.8 pg/microgram RNA in the caudate-putamen to 0.3 pg/microgram RNA in cerebellum. However, despite the functional increase in DOR sensitivity, the DOR mRNA levels in selected brain areas and spinal cord of naltrexone-treated and control mice did not differ. Assessment of DOR mRNA levels in whole brain and selected CNS regions after shorter treatment intervals (1, 6 and 12 h and 2 and 7 days) in naltrexone-treated and control mice revealed a similar pattern of results. Northern blot analysis of mouse whole brain RNA extracts after 7 days of naltrexone treatment did not show any alteration in the size of the DOR transcript. These data demonstrate that DOR mRNA levels are not altered during and after chronic naltrexone treatment and therefore are not associated with opioid-induced DOR up-regulation or DOR functional supersensitivity.

Retinoic acid regulation of mu opioid receptor and c-fos mRNAs and AP-1 DNA binding in SH-SY5Y neuroblastoma cells

Retinoic acid (RA) induced differentiation of SH-SY5Y cells increases the expression of mu opioid receptors (HMOR) and inhibitory G proteins, as well as the efficacy of opioids to inhibit forskolin-induced adenylyl cyclase activity. We examined the time course of the effects of all-trans retinoic acid (RA) on HMOR and c-fos mRNA levels as determined by solution hybridization (using HMOR and rat c-fos riboprobes) in RNA extracts from SH-SY5Y cells. Electrophoretic Mobility Shift Assay (EMSA) and Western blot analysis were used to assess the changes AP-1 DNA binding and the presence of fos-related proteins in nuclear extracts from untreated, vehicle (ethanol) or RA-treated SH-SY5Y cells. Exposure to RA for 0.5 h had no effect on HMOR while after 6-18 h of exposure HMOR in mRNA levels were decreased by 50% and then after 168 h of RA exposure, HMOR mRNA levels were doubled. In contrast, c-fos mRNA levels were unchanged at 0.5 h, but increased by 50% after 18 and 168 h of RA exposure. RA increased AP-1 binding after 18 and 168 h and a pan fos-FRA antibody produced a supershift. Western analysis indicates that RA activates a 45-kDa protein corresponding to the size of the fos B protein. These results identify two signal transduction targets that are regulated by RA during differentiation.

Quantitative distribution of the delta opioid receptor mrna in the mouse and rat CNS

We have used a sensitive solution hybridization assay that employs a riboprobe obtained from the mouse delta opioid receptor (DOR) coding sequence to quantitate the relative abundance of DOR mRNA in the central nervous system (CNS) of the adult mouse and rat. In brain Poly A+ RNA extracts this riboprobe hybridized to a single 10 kb transcript from mouse and two transcripts, one of 12 and the other of 4.5 kb in size from rat. In mouse CNS the highest levels of DOR mRNA were found in the caudate-putamen at 3.3 +/- 0.1 (SEM) pg/micrograms RNA. DOR mRNA levels in the range from 2.6 to 2.1 pg/micrograms RNA were measured in frontal cortex, nucleus accumbens, whole brain and olfactory tubercle. Spinal cord, periaqueductal gray and hippocampus had DOR mRNA levels in the range from 1.8 to 1.5 pg/micrograms RNA, while medial thalamus and cerebellum had the lowest levels (0.5 pg/micrograms RNA). These results correlate with the reported relative distribution of DOR mRNA in mouse using an in situ hybridization technique. In rat CNS, the highest levels of DOR mRNA were measured in caudate-putamen at 2.3 +/- 0.1 pg equivalents/micrograms RNA. Whole brain, cerebral cortex, olfactory bulb and brain stem had levels in the range from 1.5 to 0.9 pg equivalents/micrograms RNA while the lowest DOR mRNA levels were measured at 0.5 pg equivalents/micrograms RNA or less in thalamus, hippocampus, substantia nigra and cerebellum. This study demonstrates the ability of solution hybridization assays to quantitate homologous (mouse) as well as similar but heterologous (rat) DOR mRNA levels.

Attenuation of Acute Morphine Withdrawal in the Neonatal Rat by the Competitive NMDA Receptor Antagonist LY235959

The present study examined the ability of LY235959, a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, to attenuate behaviors and c-fos mRNA expression associated with acute morphine withdrawal in the infant rat. Rat pups were given a single dose of morphine (10.0 mg/kg, s.c.) or saline. Two hours later, pups were removed from the dam and injected with either LY235959 (10.0 mg/kg, s.c.) or saline. Fifteen minutes later acute morphine withdrawal was precipitated with naltrexone (10.0 mg/kg, s.c.) and behaviors were recorded every 15 s for the next 60 min. Immediately after behavioral testing, brain and spinal cord were assayed for c-fos mRNA analysis by solution hybridization. The intensity of the morphine withdrawal syndrome was reduced in pups pre-treated with LY235959. Withdrawal behaviors such as head moves, moving paws, rolling, and walking were decreased, and vocalizations were completely eliminated in pups pre-treated with LY2359559. Acute morphine withdrawal increased c-fos mRNA expression in the brain and the spinal cord, which was attenuated by pre-treatment of LY235959. Thus, in the 7-day-old rat, as in the adult, NMDA receptors play a role in the behavioral and molecular manifestations of acute morphine withdrawal.

The clinically available NMDA receptor antagonist dextromethorphan attenuates acute morphine withdrawal in the neonatal rat.

We investigated the ability of dextromethorphan, a clinically available NMDA receptor antagonist, to attenuate the behaviors and the expression of c-fos mRNA associated with acute morphine withdrawal in the 7-day-old rat. The intensity of the acute morphine withdrawal behaviors and the elevation in c-fos mRNA expression in the brain induced by acute morphine withdrawal were reduced by dextromethorphan. Thus, dextromethorphan can attenuate acute morphine withdrawal in the developing organism.

Chronic naltrexone differentially affects supraspinal δ-opioid receptor-mediated antinociception

Abstract The effects of chronic treatment with naltrexone, an opioid receptor antagonist, on δ1- and δ2-opioid receptor agonist-induced antinociception and ligand binding were investigated in mice. Antinociception by intracerebroventricular (i.c.v.) [ d -Pen2,5]enkephalin (DPDPE) and [ d -Ala2]deltorphin II, agonists selective for δ1- and δ2-opioid receptors, respectively, was blocked following subcutaneous (s.c.) implantation of a naltrexone pellet (7.5 mg) for 7 days. Removal of the naltrexone pellet was followed 24 h later by a decrease of 7.5-fold in the ED50 value of [ d -Ala2]deltorphin II, but not that of DPDPE. In a whole brain homogenate the binding of [ 3 H ][ d -Ala2]deltorphin II was increased twice as much as that of [ 3 H ]DPDPE. Chronic naltrexone treatment also produced an 8.6-fold decrease in the ED50 value of i.c.v. administered morphine. The increase in morphine potency was reversed to a control (placebo-treated mice) value by the selective δ2-opioid receptor antagonist, naltriben (25 pmol, i.c.v.). Thus, chronic naltrexone selectively increases δ2-opioid receptor-mediated antinociception, supporting the existence of δ opioid receptor subtypes with distinct adaptive characteristics. The data also indicate that δ2-opioid receptors are critically involved in the expression of morphine supersensitivity.

Activation of protein kinase A prevents the ethanol-induced up-regulation of δ-opioid receptor mRNA in NG108-15 cells

Abstract We have used a sensitive solution hybridization assay with a riboprobe transcribed from the coding sequence of the δ-opioid receptor gene (DOR) to study the up-regulation of the DOR mRNA by ethanol in NG108-15 cells. Exposure of the cells to compounds that increase cAMP levels (forskolin, forskolin+IBMX, or dibutyryl cAMP) resulted in the attenuation of ethanol-induced up-regulation of DOR mRNA. The inactive analogue of forskolin, 1,9-dideoxy forskolin had no effect. Northern blot analysis of RNA extracts from ethanol-, forskolin- or ethanol+forskolin-treated cells showed proportional changes in each of the multiple DOR mRNA bands, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the DOR mRNA. In the absence of ethanol, forskolin or dibutyryl cAMP reduced the basal levels of DOR mRNA. The cAMP analogue (Rp)-cAMPS, a protein kinase A (PKA) inhibitor, increased DOR mRNA levels. However, the combination of (Rp)-cAMPS and ethanol did not further increase DOR mRNA levels compared to ethanol or (Rp)-cAMPS alone. Signaling through cAMP and PKA down-regulates DOR mRNA levels. The ethanol-induced increase in DOR mRNA levels in NG108-15 cells appears to be mediated via a reduction of PKA.

Effect of supraspinal antisense oligodeoxynucleotide treatment on δ-opioid receptor mRNA levels in mice

Studies in vivo demonstrate that antisense oligodeoxynucleotide (ODN) treatment specifically reduces the functions mediated by numerous central nervous system (CNS) receptors, including opioid receptors. However, the effects of antisense ODN on the opioid receptor mRNA target, itself are rarely examined. In the present study, the effect of supraspinal antisense ODN administration on delta-opioid receptor (DOR) mRNA levels in selected CNS regions, was investigated in mice. ODN targeting a 20-nucleotide sequence of the DOR mRNA transcript was administered by intracerebroventricular (i.c.v.) injection twice daily for 3 days. First, to confirm that antisense ODN treatment decreases DOR function in this system, antinociception produced by DOR-selective agonist [D-Ala2]deltorphin II was assessed on day 4. A 2-fold reduction in [D-Ala2]deltorphin II potency was revealed in antisense ODN-treated mice compared to mice receiving control treatments. DOR mRNA levels in selected CNS regions which either mediate antinociception; medial thalamus (MThal), periaqueductal gray (PAG), frontal cortex (FCtx) and spinal cord (SpC) or exhibit relatively high levels of DOR mRNA; nucleus accumbens (Acb) and caudate-putamen (CPu) were then quantitated by solution hybridization. Levels of DOR mRNA in antisense ODN-treated mice were not different from levels in mice treated with saline vehicle, which ranged from 0.07 pg/microg total RNA in MThal and PAG to 0.26 pg/microg total RNA in CPu. These results are both consistent with previous reports that antisense oligodeoxynucleotide (ODN) treatment down-regulates DOR protein in vivo and indicate that this down-regulation is not associated with altered DOR mRNA levels.