Agonist-specific down-regulation of the human delta-opioid receptor

Down-regulation of the delta-opioid receptor contributes to the development of tolerance to delta-opioid receptor agonists. The involvement of the carboxy terminus of the mouse delta-opioid receptor in peptide agonist-mediated down-regulation has been established. In the present study, we examined the down-regulation of the truncated human delta-opioid receptor by structurally distinct delta-opioid receptor agonists. Chinese hamster ovary (CHO) cells, expressing the full-length or truncated epitope-tagged human delta-opioid receptors were incubated with various delta-opioid receptor agonists (100 nM, 24 h), and membrane receptor levels were determined by [(3)H]naltrindole saturation binding. Each delta-opioid receptor agonist tested down-regulated the full-length receptor. Truncation of the carboxy terminus abolished down-regulation by all delta-opioid receptor agonists, except SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide). In addition, truncation of the C-terminus completely attenuated [D-Pen(2)-D-Pen(5)]enkephalin (DPDPE), but not SNC80-mediated [32P] incorporation into the protein immunoreactive with an anti-epitope-tagged antibody. These findings suggest that SNC80-mediated phosphorylation and down-regulation of the human delta-opioid receptor involves other receptor domains in addition to the carboxy terminus. Pertussis toxin treatment did not block SNC80-mediated down-regulation of the truncated Et-hDOR, indicating that the down-regulation is independent of G(i/o) protein activation and subsequent downstream signaling.     

Several delta-opioid receptor ligands display no subtype selectivity to the human delta-opioid receptor

Pharmacological studies performed in vivo suggested that the delta-opioid receptor could exist as two distinct subtypes, delta(1) and delta(2), while in vitro studies are inconclusive. Therefore, we measured the binding and functional selectivity of several putative delta(1)- and delta(2)-opioid receptor-selective compounds in membranes from Chinese hamster ovary cells stably expressing the human delta-opioid receptor. The compounds characterized were the agonists [D-Pen(2),D-Pen(5)]enkephalin (DPDPE, delta(1)) and deltorphin II (delta(2)), and the antagonists 7-benzylidenenaltrexone (BNTX, delta(1)), naltriben (delta(2)), naltrindole 5'-isothiocyanate (delta(2)), and naltrindole (delta(1) and delta(2)). In competition binding assays, all compounds tested showed no preference for the [3H]DPDPE, [3H]deltorphin II, or [3H]naltrindole binding sites. BNTX also showed no selectivity for the delta-opioid receptor over the mu-opioid receptor. In functional assays, the stimulation of [35S]GTPgammaS binding induced by either DPDPE or deltorphin II was potently inhibited by both delta(1)- and delta(2)-opioid receptor-selective antagonists. Together, these results indicate that these compounds are not selective for either the delta(1)- or delta(2)-opioid receptor binding sites in binding or functional assays.     

吗啡对人早孕绒毛膜滋养层细胞雌激素受体mRNA孕激素受体mRNA表达的影响

目的研究吗啡对体外培养人绒毛膜滋养层细胞雌激素受体(ER)和孕激素受体(PR)表达的影响,探讨吗啡对滋养层细胞的直接作用,为进一步寻求减少或阻断吗啡依赖对胎盘组织的负面影响提供一定的理论依据。方法用胰酶-胶原酶消化法从人早孕绒毛组织中分离培养滋养层细胞,用免疫组织化学方法鉴定滋养层细胞;制备不同浓度的吗啡及其拮抗剂纳络酮,作用于体外培养的早孕绒毛膜滋养层细胞上,通过反转录-聚合酶链反应(RT-PCR)法半定量分析不同浓度的吗啡及其拮抗剂纳络酮对滋养层细胞ER mRNA、PR mRNA表达的影响。结果用改良法分离的人滋养层细胞不同浓度吗啡组滋养层细胞上ER mRNA表达与对照组比较有显著性下降(P<0.05),且与吗啡浓度呈负相关(r=-0.996,P<0.01);不同浓度吗啡组滋养层细胞上PR mRNA表达与对照组比较均有显著性下性(P<0.05),且与吗啡浓度呈负相关(r=-0.983,P<0.01)。结论用改良法进行人滋养层细胞培养,简捷易行,可培养大量高纯度人滋养层细胞供研究使用;从mRNA水平证明了吗啡可以引起体外培养的人早孕绒毛膜滋养层细胞内ER、PR数量下调,且与吗啡浓度呈负相关,可剂量依赖下调人滋养层细胞ER mRNA、PR mRNA的表达。     

Mutation S363A in the human delta-opioid receptor selectively reduces down-regulation by a peptide agonist

Chemically distinct opioid agonists have different abilities to down-regulate opioid receptors. The present study investigated the role of Ser(363) in human delta-opioid receptor down-regulation by a delta-selective peptide- and non-peptide agonist. Cyclic[D-Pen(2),D-Pen(5)]enkephalin (DPDPE)-mediated down-regulation was significantly attenuated by a S363A mutation. In contrast, this mutation had no effect on down-regulation by (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]N,N-diethylbenzamide (SNC80). These results demonstrate that the molecular mechanism of the human delta-opioid receptor down-regulation is agonist-specific.     

有机磷类杀虫剂与毒蕈碱乙酰胆碱M2受体的磷酸化

目的探讨有机磷类杀虫剂对大鼠脑G蛋白偶联受体激酶2介导的毒蕈碱乙酰胆碱M2受体磷酸化的影响。方法采用亲和层析法从大鼠脑组织纯化毒蕈碱乙酰胆碱M2受体;将纯化的毒蕈碱乙酰胆碱M2受体或β-肾上腺素受体、G蛋白偶联受体激酶-2,[γ-p~（32）]ATP与对氧磷（PO）、氧毒死蜱（CPO）共同保温,聚丙烯酰胺凝胶电泳分离蛋白,放射性自显影检测M2受体磷酸化结果。结果氧毒死蜱完全抑制大鼠脑M2受体的磷酸化,其半数抑制浓度（IC_（50））为70μmol/L;对氧磷不抑制M2受体的磷酸化;氧毒死蜱和对氧磷都不抑制β-肾上腺素受体的磷酸化。结论氧毒死蜱可选择性地抑制大鼠脑M2受体的磷酸化;某些有机磷杀虫剂存在其他靶分子或作用途径。     

Mechanisms of Zn(2+)-induced signal initiation through the epidermal growth factor receptor

Zn(2+) is a ubiquitous ambient air contaminant that is found as a constituent of airborne particulate matter (PM). Previous studies have associated Zn(2+) levels in PM with health effects in exposed populations and have shown proinflammatory properties of Zn(2+) exposure in vivo and in vitro. In the present study, we studied the mechanisms of epidermal growth factor receptor (EGFR) dimerization, phosphorylation, and kinase activity in A431 cells treated with Zn(2+). EGF, but not Zn(2+), induced dimerization of EGFR in A431 cells and membrane extracts. Like EGF, Zn(2+) induced phosphorylation of EGFR at tyrosines 845, 1068, and 1173. However, unlike EGF, Zn(2+) failed to induce detectable dimerization of EGFR. The EGFR kinase inhibitor PD153035 ablated all phosphorylation induced by EGF but none caused by Zn(2+). PD153035 abolished EGF-induced phosphorylation of the EGFR substrate Cbl, but had no effect on levels of phospho-Cbl caused by Zn(2+). Inhibition of EGFR kinase activity did, however, blunt Zn(2+)-induced phosphorylation of ERK. Exposure to Zn(2+), but not EGF, induced phosphorylation of the activating site of c-Src (tyrosine 416), and Zn(2+)-induced phosphorylation of EGFR at tyrosines 845 and 1068 was blocked by the c-Src kinase activity inhibitor PP2. In summary, Zn(2+) ions induce EGFR phosphorylation in a manner dependent on c-Src but not on EGFR dimerization or EGFR kinase activation, suggesting that Zn(2+) induces EGFR transactivation by c-Src.     

Agents that elevate cyclic AMP induce receptor phosphorylation primarily at serine 331 in HEK 293 cells overexpressing human thromboxane receptor alpha

Human embryonic kidney (HEK) 293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) were used to study the phosphorylation and desensitization of the receptor induced by prostaglandin E1 (PGE1) or forskolin. These agents are known to increase the intracellular level of cyclic AMP (cAMP) and activate cAMP-dependent protein kinase (PKA). Pretreatment of cells with either agent significantly attenuated Ca2+ release induced by the agonist [1S-[1alpha,2alpha(Z),3beta(1E,3S),4alpha]]-7-[3-[3-hydroxy-4-(4-indophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1]hept-2-yl]-5-heptenoic acid (I-BOP). These agents also induced concentration-dependent phosphorylation of TPalpha as demonstrated by increased 32P-labeling of the receptor from cells prelabeled with 32P(i). To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for purified PKA. It was found that only the C-terminal tail fusion protein could serve as a substrate for PKA. To identify the specific serine/threonine residues in the C-terminal tail that are involved in phosphorylation, various alanine mutants of these residues were checked for their ability to serve as substrates. Ser-331 was found to be involved in PKA-mediated phosphorylation. The S331A mutant receptor overexpressed in HEK 293 cells was not phosphorylated significantly following stimulation by PGE1 or forskolin, indicating that Ser-331 was the major site of phosphorylation. Furthermore, cells overexpressing the mutant receptor became responsive to I-BOP-induced Ca2+ mobilization even after pretreatment with PGE1 or forskolin. These results indicate that Ser-331 is the primary site responsible for the phosphorylation and desensitization of the human TPalpha induced by agents that activate PKA.     

Morphine induces apoptosis of human microglia and neurons

Apoptosis plays a critical role in normal brain development and in a number of neurodegenerative diseases. Recently, opiates have been shown to promote apoptotic death of cells of the immune and nervous systems. In this study, we investigated the effect of morphine on apoptosis of primary human fetal microglial cell, astrocyte and neuronal cell cultures. Exposure of microglia and neurons to 10(-6) M morphine potently induced apoptosis of these brain cells (approximately fourfold increase above untreated control cells). In contrast to microglia and neurons, astrocytes were completely resistant to morphine-induced apoptosis. Concentration-response and time-course studies indicated that neurons were more sensitive than microglia to morphine's effect on apoptosis. Naloxone blocked morphine-induced apoptosis suggesting involvement of an opiate receptor mechanism. Potent inhibition (>70%) of apoptosis by an inhibitor of caspase-3 as well as co-localization of active caspase-3 and DNA fragmentation in microglia or neurons treated with morphine indicated that caspase-3 is involved in the execution phase of morphine-induced apoptosis. The results of these in vitro studies have implications regarding the potential effect of opiates on fetal brain development and on the course of certain neurodegenerative diseases.     

Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice

BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.     

Characterization of the muscarinic receptor in human tracheal smooth muscle

Summary Muscarinic receptors in human tracheal smooth muscle were characterized by radioligand binding and functional studies. Specific [³H]-(–)-quinuclidinylbenzilate ([³H]-(–)-QNB) binding to tracheal smooth muscle membranes was reversible, stereoselective and of high affinity (K _d=47±4 pmol/l;R _T=920±120 fmol/g tissue). Inhibition of specific [³H]-(–)-QNB binding by the M-1 selective antagonist pirenzepine was found to occur at relative high concentrations classifying the muscarinic receptor population as belonging to the M-2 subclass.Inhibition of specific [³H]-(–)-QNB binding by muscarinic agonists revealed the presence of high and low affinity sites in nearly equal proportions. 5-Guanylylimidodiphosphate converted high affinity sites into low affinity sites although its effect was minimal. Log dose-contraction curves of methacholine had Hill coefficients of 1.10±0.04 with pD₂-values of 6.75±0.02.Inhibition of specific [³H]-(–)-QNB binding by methacholine, however, was best described by a two binding site model with pK _i-values considerably lower. The difference between these affinity values points to the presence of substantial receptor reserve.     

In vivo evidence for the selectivity of ICI 154129 for the delta-opioid receptor

The in vivo selectivity of the novel delta opioid-receptor antagonist N,N-bisallyl-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH (ICI 154129) was examined in several opioid-selective models. Antagonism at the delta receptor was demonstrated in the striatal head-turn model in the rat. Intrapallidal injection of the relatively selective delta-receptor agonist D-Ala2,D-Leu5-enkephalin (0.5 micrograms) slowed the head-turn time and this effect was completely prevented by prior subcutaneous administration of ICI 154129 (30 mg/kg). The role of delta receptors in two classical test situations was studied using the mixed opioid agonist etorphine and the antagonists naloxone and ICI 154129. The drug ICI 154129 (30 mg/kg, s.c.) failed to prevent the antinociceptive effects and stimulation of locomotor activity produced by etorphine, whereas the relatively selective mu-opioid receptor antagonist, naloxone was effective in both test situations. The possible involvement of delta receptors in morphine-induced dependence was studied by monitoring the abstinence behaviour precipitated in rats given pellets of morphine by either ICI 154129 or naloxone. Naloxone (0.5 mg/kg, i.p.) precipitated a characteristic withdrawal syndrome in conscious rats and, at a much smaller dose (0.02 mg/kg, i.p.), induced shaking behaviour in pentobarbitone-anaesthetised rats. No withdrawal signs were observed in either model after injection of ICI 154129 (30 mg/kg, s.c.), suggesting that the delta receptors are not involved in dependence on morphine.     

κ阿片受体介导的降血压作用

目的观察κ阿片受体激动剂U50488H对大鼠血压的影响并探讨其作用机制。方法监测正常大鼠心率(HR)、动脉压(ABP)、左心室内压(LVP)及左室的收缩(+dp/dtm ax)和舒张功能(-dp/dtm ax)等血流动力学指标和尿量的变化;分离正常大鼠腹主动脉,测定血管张力的变化。结果在整体水平,静脉注射U50488H可降低大鼠HR、ABP、LVP及±dp/dtm ax;而且可增加大鼠的尿量。在离体水平,U50488H对大鼠腹主动脉具有明显的剂量依赖性舒张作用;以上效应均可被选择性κ阿片受体阻断剂nor-BNI所阻断。结论激动κ阿片受体可引起降压作用,抑制心肌收缩力、舒张血管和利尿是其降压的主要机制。     

Renal Glucuronidation of Morphine in the Human Foetus

Abstract: The glucuronidation of morphine was investigated in kidney microsomes from human foetuses. This reaction was found to be catalyzed in all specimens investigated and the UDP-glucuronyltransferase activities varied between 0.18 and 0.30 nmol per min. per mg microsomal protein. Intraindividual comparisons with the hepatic glucuronidation rates revealed the renal enzyme rate to be about 50 percent of the hepatic. Oxazepam, and to a lesser extent salicylamide, inhibited the glucuronidation of morphine in kidney microsomes in similarity to what previously was shown in human foetal liver microsomes.     

Characterisation of the human NMDA receptor subunit NR3A glycine binding site

In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277-793nM)). Eight amino acids, which correspond to amino acids that are critical for ligand binding to other NMDA receptor subunits, situated within the S1S2 predicted ligand binding domain of hNR3A were mutated, which resulted in complete or near complete loss of [(3)H]-glycine binding to hNR3A. The NMDA NR1 glycine site agonist d-serine and partial agonist HA-966 (3-amino-1-hydroxypyrrolid-2-one), similarly to glycine displaced [(3)H]-glycine monophasically, suggesting a single common binding site. However, neither the partial agonist d-cycloserine nor the antagonist 7-chlorokynurenic acid displaced [(3)H]-glycine. Using homology modelling, a model of the NR3A binding pocket was generated which we suggest can be used to identify candidate agonists and antagonists. Our data show that glycine is a ligand, and most probably the endogenous ligand, for native NR3A at a binding site with unique pharmacological characteristics.     

Morphine leads to contraction of the ileal circular muscle via inhibition of the nitrergic pathway in mice

Morphine inhibits small intestinal transit in mice, although few mu-opioid receptors are present in the ileum. The present study focused on the action of morphine in the isolated mouse ileum to reveal the mechanism by which morphine inhibits mouse small intestinal transit. In the isolated circular muscle, morphine caused tonic contraction. This contraction was potently inhibited by naloxone and the mu-opioid receptor antagonist cyprodime. Moreover, the response was almost completely inhibited by tetrodotoxin and N(G)-nitro-L-arginine, but only moderately inhibited by atropine and indomethacin. In the isolated longitudinal muscle, morphine caused no or only slight contractions. Furthermore, electrically induced contraction was dose-dependently depressed by morphine, an effect that was not reversed by naloxone. These findings indicate that 1) morphine-induced circular muscle contraction occurs in the mouse ileum, 2) the contraction occurs through mu-opioid receptors mainly by inhibiting the release of nitric oxide from nitrergic nerves, although cholinergic nerves are at least partly involved in this contractile mechanism, and 3) inhibition of descending relaxation of peristalsis by morphine may slow small intestinal transit.     

Metabolism of Morphine in the Perfused Rat Liver

Abstract Morphine concentrations in the recirculating plasma of a perfused rat liver preparation declined in a monoexponential manner with a half–life of 5.3 ± 0.4 min. The mean hepatic clearance was calculated to 13.1 ± 1.0 ml–min.^–1, and using an average blood flow of 17.3 ml min.^–1 this corresponds to an extraction ratio of 0.76 ± 0.02. In experiments with steady state concentration of morphine almost identical results were obtained. Measurements of different morphine concentrations in the in– and outflow of plasma of the liver yielded a mean clearance of 13.3 ± 0.7 ml–min.^–1 and a mean extraction ratio of 0.77 ± 0.03, indicating a constant clearance at different concentrations. The clearance of morphine in the perfused liver experiments was in close agreement with an in vivo calculated hepatic clearance of 75 % of the total body clearance. It is concluded that the blood flow to the liver can be an important factor in determining availability of orally administered morphine, and that the high hepatic extraction of this drug can explain the low effectiveness of this mode of administration.     

Morphine analgesia, tolerance and addiction in the cricket Pteronemobius sp. (Orthoptera, Insecta).

The escape reaction time (ERT) of the cricket Pteronemobius sp. from the heated box begins at 48° and increases with temperature until 56°C, beyond which there is no further increase. The ERT (2.2 ± 1.39 s) from the hot box at 54°C is used as a model for studying the analgesic effects of opiates. Results of the present paper show that the ERT did not change after injecting the insect in the abdominal haemocoel with 0.9% saline solution, but ERT increased when 0.32, 0.52 or 0.69 mg/g of morphine is injected in the same place. The maximum ERT increase is reached at 90 min after drug injection, and the drug effect disappears 3 h after the injection. At 90 min after drug injections, the dose of 0.50 mg/g of morphine produces 50% of ERT increase, and it is referred to as the median analgesic dose (D₅₀). 1.05 mg/g of morphine produces an ERT longer than 30 s that results in an irreversible damage to the insect. Sixty-four μg/g of naloxone given in addition to D₅₀ of morphine fully blocked the effect of morphine during its 3-h action. However, more than 64 μg/g of naloxone alone also increase the ERT in the cricket, similar to what has been described for vertebrates. Four daily morphine injections of D₅₀ decreased ERT in such a way that, at the fourth day, the ERT is similar to the ERT produced by saline solution; i.e., tolerance is shown. The suppression of daily morphine injections of D₅₀ during the fifth day produced a hyperresponse to vibration (big jumps) not shown in the case of the injections of saline solution; i.e., addiction is shown.     

Fine-tuning somatostatin receptor signalling by agonist-selective phosphorylation and dephosphorylation: IUPHAR Review 5

The biological actions of somatostatin are mediated by a family of five GPCRs, named sst₁ to sst₅. Somatostatin receptors exhibit equally high-binding affinities to their natural ligand somatostatin-14 and largely overlapping distributions. The overexpression of somatostatin receptors in human tumours is the molecular basis for diagnostic and therapeutic application of the stable somatostatin analogues octreotide, lanreotide and pasireotide. The efficiency of somatostatin receptor signalling is tightly regulated and ultimately limited by the coordinated phosphorylation and dephosphorylation of intracellular carboxyl-terminal serine and threonine residues. Here, we review and discuss recent progress in the generation and application of phosphosite-specific antibodies for human sst₂ and sst₅ receptors. These phosphosite-specific antibodies are unique tools to monitor the spatial and temporal dynamics of receptors phosphorylation and dephosphorylation. Using a combined approach of phosphosite-specific antibodies and siRNA knock-down screening, relevant kinases and phosphatases were identified. Emerging evidence suggests distinct mechanisms of agonist-selective fine-tuning for individual somatostatin receptors. The recently uncovered differences in phosphorylation and dephosphorylation of these receptors may hence be of physiological significance in mediating responses to acute, persistent or repeated stimuli in a variety of target tissues.     

吗啡依赖及戒断大鼠相关脑区多巴胺D2受体基因表达的变化

目的:研究吗啡依赖及戒断大鼠相关脑区D2R基因表达的变化.方法:将24只雄性Sprague-Dawley大鼠随机等分为吗啡依赖组(吗啡组:在大鼠腹腔内注射盐酸吗啡,2次/d,起始剂量为5mg/kg,逐日递增5 mg,至第10天为50 mg/kg)及生理盐水对照组(对照组:用相同方式注射同体积的生理盐水),于末次注射后3 h及72 h处死(每组每时间点各6只),取中脑腹侧被盖区(VTA)、伏隔核(Nac)、中脑导水管灰质(PAG)、尾壳核(CPU)、海马CA1区(HIPCA1)等脑组织.利用组织原位杂交技术检测各脑区的D2R mRNA的吸光度(A)值,并作同期平行比较.结果:末次注射3 h,吗啡组大鼠五个被检脑区D2R mRNA A值均低于同期对照;末次注射72 h,尾壳核高于同期对照,其它四个被检脑区仍低于同期对照,差异具有显著性(P<0.05或0.01).结论:慢性吗啡处理可抑制大鼠相关脑区D2R基因表达,戒断后有不同程度的恢复.