D-Ala2,D-Leu5]enkephalin Research Articles

Mu and delta opioid receptor activation inhibits omega-conotoxin-sensitive calcium channels in a voltage- and time-dependent mode in the human neuroblastoma cell line SH-SY5Y.

Ca2+ channel modulation by the mu opioid agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO) and the delta opiate agonists [d-Pen2, d-Pen5]-enkephalin (DPDPE) and [d-Ala2, d-Leu5]-enkephalin (DADLE) in cultured human neuroblastoma SH-SY5Y cells was investigated using the whole-cell variant of the patch-clamp technique. In SH-SY5Y cells, differentiated in vitro with retinoic acid, all agonists reversibly decreased high-voltage-activated, omega-conotoxin-sensitive Ba2+ currents in a concentration-dependent way. Inhibition was maximal with a 1 microM concentration of opiate agonists (76% with DAGO and 63% with delta agonists, when measured at 0 mV) and was characterized by a clear slow down of Ba2+ current activation at low test potentials. Both inhibition and slow down of activation were attenuated at more positive potentials, and could be partially relieved by strong conditioning depolarizations. Current suppression operated by both mu and delta agonists was prevented by pre-treatment of the cells with pertussis toxin. No sign of additivity was observed when delta agonists were applied to cells that were maximally activated by DAGO, suggesting that a common mechanism, involving the same type of modulating molecule, is responsible for Ca2+ channel inhibition promoted by activation of mu and delta opioid receptors in SH-SY5Y cells.

Synthesis of [D-Ala2, D-Leu5]enkephalin tritiated in the 2,6-positions of tyrosine

[D-Ala2, D-Leu5]Enkephalin (DADLE) labelled with tritium in the 2,6-positions of N-terminus tyrosine residue has been prepared. Synthesis of the precursor peptide, [2,6-dibromo-Tyr1]DADLE, was performed by solid phase synthesis using Fmoc strategy. The peptide was tritiated catalytically to yield [2,6-3H-Tyr1]DADLE with a specific radioactivity of 1.61 TBq/mmol. The distribution of the tritium was investigated by HPLC with radioisotope detection following hydrolysis with aminopeptidase M, and confirmed that the label was entirely located at the tyrosine residue.

Dual signal transduction through delta opioid receptors in a transfected human T-cell line.

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([Ca2+]i) and/or cAMP are affected by opiates acting through delta-type opioid receptors (DORs), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T-cell line. The DOR agonists, deltorphin and [D-Ala2, D-Leu5]-enkephalin (DADLE), elevated [Ca2+]i, measured by flow cytofluorometry using the calcium-sensitive dye, Fluo-3. At concentrations from 10(-11)-10(-7) M, both agonists increased [Ca2+]i from 60 nM to peak concentrations of 400 nM in a dose-dependent manner within 30 sec (ED50 of approximately 5 x 10(-9) M). Naltrindole, a selective DOR antagonist, abolished the increase in [Ca2+]i, and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA, which reduced the initial deltorphin-induced elevation of [Ca2+]i by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately equal to 10(-11) M), and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T-cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify two pertussis toxin-sensitive, G protein-mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation.

Antiproliferative and receptor binding properties of α- and β-casomorphins in the T47D human breast cancer cell line

In previous studies, we have shown that opioid agonists ([ d-Ala 2, d-Leu 5]enkephalin (DADLE), [ d-Ser 2,Leu 5]enkephalin-Thr 6 (DSLET), ethylketocyclazocine and etorphine) bind to opioid binding sites and decrease cell proliferation of human T47D breast cancer cells. Furthermore, we provided evidence about a cross-reaction, also in the T47D human breast cancer cell line, of μ-acting opioids with type-II somatostatin receptors. Since a potential source of opioid activity in the breast might be casomorphin peptides (produced by the enzymatic degradation of α-casein and β-casein), we investigated the antiproliferative action of five different casomorphin peptides: α-casein-(90–95), α-casein-(90–96), β-casomorphin, β-casomorphin-(1–5) and morphiceptin. We show that all five peptides decreased, in a dose-dependent manner, cell proliferation. The general antagonist diprenorphine produced only a partial reversal of their action. Furthermore, we provide evidence that all peptides (except for morphiceptin) bind to δ- and κ-opioid binding sites of T47D cells with different selectivity. Finally, we show that these peptides are also partial competitors at the somatostatin receptors present in the same cell line.

High opioid doses inhibit whereas low doses enhance neuritogenesis in PC12 cells

Exposure to opiates affects brain development, cell growth as well as in vitro cell differentiation. Perinatal treatment with morphine has been reported to impair neuronal plasticity after neonatal lesion with 5,7-dihydroxytryptamine (5,7-DHT). This study has investigated the use of μ, δ and k opioid receptor ligands to examine the selective receptor mediated inhibition of PC12 neurite formation. Morphine and d-Ala 2, d-Leu 5-enkephalin (DADLE) had a comparable inhibitory potency with a maximal effect at 1 mM concentration, while both naltrexone and naltrindole antagonized their effect at only 10 nM. d-Ala 2-MePhe 4,Gly-ol 5-enkephalin (DAMGO) showed only a transient inhibitory effect. The administration of 10 nM guanosine 5′- O-(3-thiotriphosphate) (GTP-γ-S) prevented morphine inhibition. It is suggested that opiate inhibition of neuritogenesis may be mediated by a receptor with δ-like characteristics coupled to G proteins. On the other hand, the activation of this receptor with morphine at a very low concentration (1 pM) actually enhanced nerve growth factor (NGF) neurite promoting activity.

Thr353, Located within the COOH-terminal Tail of the ´ Opiate Receptor, Is Involved in Receptor Down-regulation

Prolonged exposure to abused drugs such as opiates causes decreased response to the drug; this reduced sensitivity is thought to be due to the loss of receptors, or down-regulation. The molecular mechanism of the opiate receptor down-regulation is not known. In order to address this, we generated a number of mutants of the delta opiate receptor COOH-terminal tail. When expressed in the Chinese hamster ovary cells, both the wild type and the receptor with a deletion of 37 COOH-terminal residues bind diprenorphine with comparable affinities and show similar decreases in cAMP levels in response to D-Ala2, D-Leu5, enkephalin (DADLE). However, the truncated receptor does not show down-regulation from the cell surface upon prolonged exposure (2-48 h) to DADLE. In contrast, both the wild type receptor and the receptor with the deletion of only 15 COOH-terminal residues show substantial down-regulation upon long term DADLE treatment. These results suggest that the region located between 15 and 37 residues from the COOH terminus is involved in the receptor down-regulation. In order to identify residues that play a key role in down-regulation, point mutations of residues within this region were examined for their ability to modulate receptor down-regulation. The receptor with a mutation of Thr353 to Ala does not down-regulate, whereas the receptor with a mutation of Ser344 to Gly down-regulates with a time course similar to that of the wild type receptor. Taken together, these results suggest that the COOH-terminal tail is not essential for functional coupling but is necessary for down-regulation and that Thr353 is critical for the agonist-mediated down-regulation of the delta opiate receptor.

Delta opioid extends hypothermic preservation time of the lung

To test the hypothesis that a delta opioid, DADLE ([D-Ala 2, D-Leu 5]-enkephalin), could protect tissue from ischemic damage during hypothermic lung preservation, we studied three groups of rats. In group 1 ( n = 8), lung function was studied immediately after harvesting. In group 2 ( n = 8), the lung was flushed with 4º C Euro-Collins solution and preserved for 24 hours. In group 3 ( n = 8), the lung was flushed with 4º C Euro-Collins solution plus DADLE (1 mg/kg) and preserved for 24 hours. Lung function was studied by using a living rat perfusion model. Venous blood from the host rat perfused the pulmonary artery of the isolated lung. Blood from the isolated lung was returned to the carotid artery of the host rat with a roller pump. Severe pulmonary edema, hemorrhage, and occlusive pulmonary artery resistance occurred in group 2 within 30 minutes of perfusion. Perfusion studies were carried out for more than 60 minutes in groups 1 and 3. Pulmonary blood flow was lower in group 2 than in either group 1 or group 3. Pulmonary vascular resistance was much higher in group 2 than in groups 1 and 3 ( p < 0.05). Airway pressure and airway resistance were much higher in group 2 than in groups 1 and 3 ( p < 0.05). Airway resistance was also higher in group 3 than in group 1 after 20 minutes of perfusion ( p < 0.05). Oxygen tensions from the pulmonary vein of the isolated lung in group 2 were lower than those in groups 1 and 3 ( p < 0.05). Alveolar-arterial oxygen difference was much higher in group 2 than in groups 1 and 3 ( p < 0.05). Lung tissue wet/dry weight ratio after perfusion was much higher in group 2 than in groups 1 and 3. The results clearly show, for the first time, that DADLE can effectively enhance hypothermic lung preservation in rats.(J T HORAC C ARDIOVASC S URG 1996;111:259-67)

The antiproliferative effect of opioid receptor agonists on the T47D human breast cancer cell line, is partially mediated through opioid receptors

In the present study, we investigated the action of opioid receptor agonists on the proliferation of cells of the T47D human breast cancer cell line, grown in the absence of exogenously added steroids and growth factors. We found that the opioid receptor agonists ethylketocyclazocine, morphine, [ d-Ala 2, d-Leu 5]enkephalin (DADLE), [ d-Ser 2,Leu 5]enkephalin-Thr 6 (DSLET) and etorphine inhibit dose dependently cell proliferation. The opioid receptor antagonist diprenorphine had no significant effect per se, but it was able to reverse the action of all opioid receptor agonists except morphine. In order to investigate the mechanism of action of opioids on T47D cells, we characterised the opioid receptors present on this cell line, by saturation binding, using radiolabelled [ d-Ala 2, N-Me-Phe 4-Gly 5-ollenkephalin (DAGO, μ-opioid receptor agonist), ethylketocyclazocine ( κ 1-, κ 2-, μ- and δ-opioid receptor agonist), diprenorphine ( κ 2-, κ 3-, δ- and μ-opioid receptor antagonist), DADLE ( δ- and μ-opioid receptor agonist), and effectors. We identified opioid binding sites belonging mainly to the κ-type ( κ 1, κ 2 and κ 3), a few δ-opioid receptor sites, but no μ-opioid receptors. Our results indicate that the inhibitory effect of opioids on T47D cell growth is mediated through κ- and δ-opioid receptors. The effect of μ-acting morphine might not be mediated through opioid receptors.

Structural studies of opioid peptides: a review of recent progress in x-ray diffraction studies.

The solid state structures of many opioid peptide agonists have been elucidated by x-ray diffraction analysis. Recently, the first structure of an opioid peptide antagonist has been determined. Theoretically, linear peptides can have many different backbone conformations, yet early x-ray studies (1983-1987) on enkephalin and its analogues showed only two different backbone conformations: extended and single beta-bend. In 1989 enkephalin was observed in a third conformation, a double beta-bend. Since that time diffraction studies have been completed on the rationally designed linear opioid peptide agonists DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) and DADLE (D-Ala2,D-Leu5-enkephalin) as well as on several cyclic enkephalin analogues including DPDPE (Tyr-[D-Pen-Gly-Phe-D-Pen]) and JOM-13 (Tyr-[D-Cys-Phe-D-Pen]). The most recent review of the x-ray studies on this class of compounds was written in 1988. This paper will update that review to include the results of studies completed since that time.

Calcium influx in rat thalamic relay neurons through voltage-dependent calcium channels is inhibited by enkephalin

High and low voltage-activated, transient (HVA and LVA,T) Ca 2+ currents are crucial in determining the characteristic thalamic firing pattern, during the oscillatory mode. The modulatory effects induced by d-ala 2- d-leu 5-enkephalin (DADLE) on voltage-dependent Ca 2+ channels have been investigated on acutely dissociated neurons from rat ventro-basal (VB) thalamus, by means of whole cell patch-clamp technique. DADLE (400 nM) reduced HVA Cat+ channel currents in 37 out of 44 cells tested (−53 ± 5.3% to 0 mV test potential, n = 24,). In 50% of the cases DADLE induced an effect which was persistent at all the potentials tested, i.e. a voltage-independent one. In the remaining neurons, the inhibition partially or totally disappeared on the currents evoked at the highest potentials. DADLE was also able to inhibit LVA Ca 2+ channels (−40% in five out of 12 cells). In conclusion, thalamic relay neurons present opioid receptors negatively coupled to both HVA and LVA Ca 2+ channels. The presence of two inhibitory effects of DADLE on the total HVA Ca 2+ channels has been observed, and they are distinguishable on the basis of their sensitivity to voltage. It is suggested that Ca 2+ current modulation may play a role in the production and tuning of the rhythmic burst discharge in these neurons.

Molecular cloning of a novel protein regulated by opioid treatment of NG108-15 cells.

A new cDNA clone, NGD5, has been identified from a subtraction cDNA library constructed with mRNA isolated from control neuroblastoma x glioma NG108-15 cells and cells treated for 48 h with the delta-opioid agonist, D-Ala2, D-Leu5 enkephalin (DADLE). NGD5 mRNA is decreased, in a naloxone-reversible manner, upon long-term treatment of NG108-15 cells with DADLE, indicating that this clone may be related to opioid receptor function. Northern analysis indicates that NGD5 mRNA is expressed in rat brain. Two similar nearly full-length NGD5 clones, NGD5A and NGD5B, were isolated from a lambda gt10 NG108-15 cDNA library and sequenced. The predicted 40-kDa peptide encoded for by the NGD5 cDNA has no significant homology to the recently cloned mu, delta or kappa opioid receptors nor to any other known proteins.

Effects of Intrathecal mu, delta, and kappa Agonists on Thermally Evoked Cardiovascular and Nociceptive Reflexes in Halothane-Anesthetized Rats

Despite significant opioid binding in the intermediolateral cell column, the effects of intrathecal injections of mu, delta, and kappa opioid agonists on the cardiovascular response to noxious stimulation have not been examined systematically. The pharmacology of intrathecally administered opioid agonists (mu, morphine, [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAGO); delta, metkephamid, [D-Ala2-D-Leu5]enkephalin (DADL), [D-Pen2,D-Pen5]enkephalin (DPDPE); kappa, U50488H and PD117,302) or agonist-antagonist (nalbuphine) on somatomotor (tail-flick) and cardiovascular changes (blood pressure and heart rate) evoked by immersing the tail in 53 degrees C water were examined in rats anesthetized with halothane (0.75%) and in which intrathecal catheters had been chronically implanted. Intrathecal administration of mu and delta, but not kappa agonists or agonist-antagonist produced a dose-dependent block of tail-flick and evoked cardiovascular responses with the order of activity being as follows: DAGO > metkephamid DADL > morphine > DPDPE >> nalbuphine = PD117,302 = U50488H = 0. These effects were reversed readily by the opioid antagonist naloxone. In addition, intrathecal administration of mu and delta but not kappa or agonist-antagonist had little effect on resting heart rate and blood pressure. These data indicate that the agonist occupancy of spinal mu and delta, but not kappa agonists can profoundly modulate the autonomic and somatomotor response evoked by high threshold thermal stimuli.

Presence in neuroblastoma cells of a μ 3 receptor with selectivity for opiate alkaloids but without affinity for opioid peptides

Evidence is presented for the occurrence of a unique opiate alkaloid-selective, opioid peptide-insensitive binding site in N18TG2 mouse neuroblastoma cells and in late passage hybrid F-11 cells, derived from N18TG2 neuroblastoma cells and rat dorsal root ganglion cells. Those cells lacked classical opioid peptide-sensitive receptor subtypes, but contained [ 3H]morphine and [ 3H]diprenorphine binding sites with affinity for certain opiate alkaloids but not for any endogenously occurring opioid peptide or peptide analog tested, including d-ala 2- d-leu 5-enkephalin (DADLE), d-Ala 2,N-Me-Phe 4,Gly 5-ol (DAGO) and dynorphin A(1–17). The binding site differed from hitherto described μ, δ and κ neuronal opioid receptors not only on the basis of peptide insensitivity, but also on the basis of selectivity and affinities of alkaloids. Saturation experiments with [ 3H]morphine indicated the presence of a single site with K d=49nM and B max=1510fmol/mg protein. This novel binding site was not present in F-11 hybrid cells at early passage. Instead the hybrid cells contained conventional opioid receptors (predominantly δ and also μ) capable of binding DADLE and other peptides as well as opiate alkaloids. With additional passage (cell divisions) of the hybrid cells, during which a limited change occurred in mouse chromosome number, the peptide-insensitive binding appeared and the opioid peptide-binding (δ and μ) receptors were lost reciprocally. Thus, expression of the peptide-insensitive binding normally may be repressed when conventional opioid receptors are expressed. The peptide-insensitive opiate binding site described here appears to correspond to the μ 3 receptor subtype, recently identified pharmacologically and functionally in several cell types of the immune system. It is proposed that this opiate alkaloid-sensitive μ 3 receptor of macrophages and certain other immunocytes is also present in certain neuronal cell lines and thus may possibly exist in certain neurons of the intact organism.

Ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density.

To determine whether the previously demonstrated ability of delta-opioid receptors to interact simultaneously with multiple G-proteins was a function of high receptor levels, this interaction was investigated in Chinese hamster ovary cells stably expressing 10 different levels of cloned delta-opioid receptors, ranging from 18,000 to 1.6 x 10(6) receptors/cell. The opioid agonist D-Ala2,D-Leu5-enkephalin (DADLE) inhibited forskolin-stimulated adenylyl cyclase activity in all 10 clones with variable maximal inhibitory levels. Furthermore, opioid agonists altered incorporation of [alpha-32P]azidoanilido-GTP into at least four G-protein alpha-subunits in all 10 clones, three of which were determined to be Gi3 alpha, Gi2 alpha and Go2 alpha. This effect was concentration-dependent, naloxone-reversible, and delta-opioid agonist-specific and was blocked by pretreatment with pertussis toxin. Although DADLE induced an increase in the incorporation of [alpha-32P]azidoanilido-GTP into three of the four G alpha proteins that was independent of receptor density, the magnitude of this response was greater as receptor density increased. In addition, concentrations of DADLE required to promote 50% maximal labeling were similar for all four G alpha proteins within each clone and did not appear to be affected by receptor density. Therefore, the ability of delta-opioid receptors to interact with multiple G-proteins is independent of receptor density and there is also no apparent correlation between the amount of G-protein activated and the maximal effect of an agonist.

High-performance liquid chromatographic determination of intrathecally administered [ d-Ala 2- d-Leu 5]-enkephalin concentrations in canine cerebrospinal fluid

A rapid and useful method for high-performance liquid chromatographic analysis of exogenous [ d-Ala 2- d-Leu 5]-enkephalin (DADLE) in cerebrospinal fluid (CSF) is described. CSF (0.5 ml) samples were filtered using a 0.22-μm Co-Star filter. Chromatography was performed on a μBondapak C 18 column using a mobile phase of A, 0.05 M sodium phosphate (monobasic, pH 6.0) and B, 60% acetonitrile in 0.05 M sodium phosphate (pH 6.0) with a flow-rate of 1 ml/min. Absorbance at 210 nm was measured. The procedure produced a linear curve for the concentration range 1–10 μg/ml. The development of the assay produced rapid, repeatable and accurate results for CSF analysis of DADLE at concentrations achieved with therapeutic administration of the peptide. This method could also be used in the future for analysis of compounds like DADLE.

Mastoparan reduces the supraspinal analgesia mediated by μ/δ-opioid receptors in mice

Intracerebroventricular (i.c.v.) administration of the venom peptide, mastoparan, to mice decreased to a limited extent opioid-induced supraspinal analgesia in a non-competitive fashion. The μ-opioid receptor agonists, [ d - Ala 2, N- MePhe 4, Gly-ol 5 ]- enkephalin (DAMGO) and morphine, the μ/δ-opioid receptor ligands, human β-endorphin-(1–31) and [ d-Ala 2, d-Leu 5]- enkephalin (DADLE), and the selective ligands of δ-opioid receptors, [ d-Pen 2,5]enkephalin (DPDPE) and [ d-Ala 2]deltorphin II, showed an impaired analgesic effect in mice given mastoparan. Mastoparan diminished the analgesic activity of DPDPE and [ d-Ala 2]deltorphin II to the same extent as observed after giving the δ-opioid receptor-selective antagonist, ICI 174864. The μ-opioid receptor-mediated analgesia that remained after mastoparan was abolished in the presence of the opioid antagonist, naloxone. Mastoparan after binding to G iα/G oα subunit could block opioid antinociception. The existence of a class of G protein functionally coupled to μ-opioid receptors, but resistant to the effect of mastoparan is suggested.

Effect of electroacupuncture tolerance by different frequencies on the opioid inhibition to cardiovascular activity in the spinal cord of rats

Continuous 2–15 Hz or 2 Hz electroacupuncture (EA) of 6 h applied to rats resulted in electroacupuncture tolerance (ET). The effect of 2–15 Hz ET on the recovery of mean arterial blood pressure (MAP) and heart rate (HR) of rats after hemorrhagic shock, and the effect of intrathecal (i.t.) injection of [D-ala 2, D-leu 5]enkephalin (DADLE) on MAP and HR of 2 Hz ET rats were observed. The results were as follows: 1) there was no dramatic difference between the 2–15 Hz ET group and the control group in the recovery of MAP and HR from hemorrhagic shock; 2) DADLE (25 μg) caused almost the same depressor effect in the 2 Hz ET group as that in the control group. These data suggest that the spinal lateral horn cells (supposed to be involved in regulating blood pressure) are insensitive to EA and cannot be made tolerant to continuous EA, which is different from the dorsal horn cell (supposed to be involved in analgesia).

Antagonists at excitatory opioid receptors on sensory neurons in culture increase potency and specificity of opiate analgesics and attenuate development of tolerance/dependence

At low (< nM) concentrations, μ, δ or κ opioid peptides as well as morphine and other opioid alkaloids elicit dose-dependent excitatory prolongation of the calcium-dependent component of the action potential duration (APD) of many mouse sensory dorsal root ganglion (DRG) neurons, whereas application of the same opioids at higher (uM) concentrations results in inhibitory shortening of the APD. These bimodal opioid excitatory/inhibitory effects on DRG neurons are blocked by naloxone. In contrast to bimodally acting opioids, the opioid alkaloids, etorphine and dihydroetorphine (thebaine-oripavine derivatives) uniquely elicited only dose-dependent, naloxone-reversible inhibitory effects on sensory neurons in DRG-spinal cord explants, even at concentrations as low as 1 pM, and showed no excitatory effects at lower concentrations. These remarkably potent inhibitory opioid receptor agonists also act as antagonists at excitatory opioid receptors since pretreatment of DRG neurons with subthreshold concentrations (< pM) blocked excitatory APD prolongation by nM morphine (or other opioids) and unmasked inhibitory APD shortening which generally requires much higher concentrations. Furthermore, acute application of pM-nM etorphine to chronic μM morphine- or d-Ala 2- d-Leu 5 enkephalin (DADLE)-treated DRG neurons blocked the nM naloxone-precipitated APD prolongation that generally occurs in DRG cells sensitized by bimodally acting opioids. In the presence of pM etorphine, chronic treatment of DRG neurons with μM morphine or DADLE no longer resulted in development of tolerance/dependence effects, as previously observed after similar chronic opioid treatment in the presence of cholera toxin-B subunit. These in vitro studies may clarify the mechanisms underlying the potent analgesic effects of etorphine and dihydroetorphine in vivo and to guide the use of these and other excitatory opioid receptor antagonists in attenuating development of opiate dependence/addiction.

Cellular adaptation to opiates alters ion-channel mRNA levels.

The chronic use of several drugs, including opiates, results in the stereotypical behaviors characteristic of addiction. Alterations in gene expression have been associated with the use of these addictive drugs. Previous studies, however, have been limited to describing changes in amounts of individual mRNAs from single tissue samples. Cellular adaptation to opiates, reflected in the regulation of the expression of many different mRNAs, seems likely to contribute to the complicated behaviors of addiction. The present studies examined coordinate alterations in the amounts of multiple mRNAs in the rat striatum and in NG108-15 cells after opioid stimulation or the precipitated withdrawal of opioid use. The experimental approach combined amplification of the poly(A)+ RNA population with reverse Northern blot analysis to simultaneously characterize the relative changes in several mRNAs. Morphine treatment of rats for 5 days was associated with a reduction in the amount of striatal RNA for the voltage-sensitive K+ channel without significant changes in other ion channels. In NG108-15 cells stimulation with the delta-opiate receptor agonist [D-Ala2,D-Leu5]enkephalin (DADLE) alone and followed by naloxone (precipitated withdrawal) caused relative changes in the abundances of several mRNAs. The composite effects of alterations in the abundance of multiple mRNAs (and the proteins they encode) in response to opioid use likely contribute to the development and maintenance of opiate-mediated behaviors.

The role of peripheral opioid receptor subtypes in the modulation of gastric acid secretion and plasma gastrin in dogs

The peripheral opioid receptor subtypes involved in the regulation of gastric acid secretion were studied in dogs with both a gastric fistula and a Heidenhain pouch, by using the putative μ-opioid receptor agonist dermorphin, the δ-opioid receptor agonist [ D-Ala 2, D-Leu 5]enkephalin (DADLE) and the κ-opioid receptor agonist dynorphin-(1–13). Dermorphin caused a significant increase in basal acid secretion from both the gastric fistula and the Heidenhain pouch, while DADLE and dynorphin-(1–13) did not. Acid secretion stimulated by 2-deoxy- D-glucose from the gastric fistula was not modified by dermorphin and dynorphin-(1–13), while DADLE significantly inhibited it; at the same time gastric secretion from the Heidenhain pouch was significantly increased by dermorphin and unmodified by DADLE and dynorphin-(1–13). Dermorphin, DADLE or dynorphin-(1–13) did not modify plasma gastrin during basal or 2-deoxy- D-glucose-stimulated conditions. Submaximal bethanechol-stimulated secretion was increased by dermorphin and DADLE but unaffected by dynorphin-(1–13). Acid secretion from the gastric fistula stimulated by pentagastrin was enhanced by dermorphin, inhibited by DADLE and unaffected by dynorphin-(1–13). Dermorphin and DADLE significantly increased acid secretion from the Heidenhain pouch stimulated by pentagastrin, while dynorphin-(1–13) was ineffective. Naloxone prevented the stimulatory effects of dermorphin and DADLE on the Heidenhain pouch, but it reduced acid secretion from the gastric fistula further when given with DADLE. The inhibitory effects of DADLE on secretion from the gastric fistula were prevented by naltrindole, a selective antagonist of δ-opioid receptors. We conclude that peripheral μ-opioid receptors mediate excitatory effects, δ-opioid receptors produce mainly inhibitory effects, while κ-opioid receptors do not seem to be involved in the regulation of acid secretion. Further, measurements of plasma gastrin suggest that the opioid-induced changes in acid secretion are not due to variations in gastrin release.