Application of RT-PCR in formalin-fixed and paraffinembedded lung cancer tissues

Aim:

To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method.

Methods:

Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples.

Results:

The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens.

Conclusion:

This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.     

结缔组织生长因子在病理性瘢痕成纤维细胞中的表达

要目的探讨结缔组织生长因子(CTGF)人类增生性瘢痕成纤维细胞中的表达与瘢痕纤维化之摘在间的关系。方法应用半定量逆转录聚合酶链反应(RT-PCR)术,测了瘢痕组织原代培养的成纤维细技检胞中CTGFmRNA表达。结果CTGFmRNA在人类增生性瘢痕成纤维细胞中高度表达,与正常皮肤组织的成纤维细胞比较,有显著性差异(P<0.01)。结论CTGF在人类增生性瘢痕成纤维细胞中高度表达,提示其在增生性瘢痕的纤维化进程中起着重要的作用,这为临床上寻找安全、有效的治疗增生性瘢痕提供了新的思路。     

用实时荧光定量RT-PCR检测单个小鼠种植前胚胎中特异性基因表达

目的用单细胞实时荧光定量RT-PCR,检测单个小鼠种植前胚胎中特异性基因表达。方法采集小鼠卵母细胞和原核期胚胎,后者经体外培养至囊胚。采用实时荧光定量RT-PCR对多个及单个卵母细胞和2-细胞期胚胎中Tcl1基因的表达进行定量检测;用同样方法对小鼠种植前各发育阶段的胚胎进行Tcl1基因的表达检测。结果在多个及单个卵母细胞和2-细胞期胚胎中,均检测出Tcl1的表达;多个卵母细胞、2-细胞期胚胎中Tcl1的表达量显著高于单个卵母细胞、2-细胞期胚胎;此方法也适用于胚胎早期发育的各个时期,Tcl1在单个卵母细胞、原核期胚胎和2-细胞期胚胎中表达量最高,在4-细胞期胚胎、8-细胞期胚胎、桑葚胚和囊胚中表达量极低。结论应用单细胞实时荧光定量RT-PCR可对单个小鼠种植前胚胎中基因的表达进行定量检测。     

实时荧光定量RT-PCR检测内皮细胞t-PA mRNA方法的建立

目的建立实时RT-PCR检测人微血管内皮细胞组织型纤溶酶原激活物(t-PA)mRNA的表达。方法提取人微血管内皮细胞总RNA,经RT-PCR获得靶基因(t-PA)及管家基因(β-actin)的PCR产物。纯化后,作为标准品梯度稀释,采用SYBR G reen I定量PCR检测,建立标准曲线。方法学考核参数为特异性、线性范围、精密度和重复性。分析全反式维甲酸对内皮细胞表达t-PA mRNA的干预效果。结果定量方法特异性好,检测的灵敏度达103拷贝,线性范围为103~1010拷贝,循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系(r2>0.990),批内变异≤3.10%,批间变异≤4.93%。1.25~20.00μmol.L-1的全反式维甲酸能明显上调内皮细胞t-PA mRNA的表达(P<0.01),且呈剂量依赖性。结论实时荧光定量RT-PCR的方法可对t-PA mRNA的表达进行准确定量,有助于溶栓药物药理学研究和新药筛选。     

Semiquantitative determination of human cytokine mRna expression using TaqMan RT-PCR

To establish an easy, fast and reliable RT-PCR for the analysis of human cytokine expression, we made use of the recently developed technique of TaqMan PCR. This technique is based on the cleavage of fluorochrome-labelled internal oligodeoxynucleotide probes by the 5′→3′ nuclease activity of Taq DNA polymerase. Measurement of fluorescence intensity during each cycle of the PCR reaction with a Sequence Detection System allows the determination of a threshold cycle at which an increase in fluorescence intensity is first detectable. From these values, a starting amount of template DNA can be calculated. Here, we established specific primers and corresponding internal, fluorogenic probes for the human cytokines tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interferon-γ (IFN-γ), and for the constant region of the T-cell receptor β chain (TCRβ) and the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for normalization of mRNA expression levels. Titrations of the cDNA input showed a strict inverse correlation between the threshold cycles obtained and the starting amount of template. This in turn allowed the generation of a standard curve, and thus quantification of mRNA abundance in cDNA samples. Evaluation of the method using cDNAs from peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or phytohae-magglutinin (PHA) showed basal expression of TNF-α and IL-1β in untreated PBMC while IFN-y was not detectable or only weakly expressed. After stimulation with LPS, a strong induction of IL-1β and TNF-α was measured, while IFN-γ was induced to a lesser extent. PHA treatment, in contrast, led to an induction of all three cytokines with IFN-γ being the most prominent. The method has a large dynamic range, requires no post-PCR processing and gives reliable results.     

Opioid receptor-mediated control of acetylcholine release in human neocortex tissue

The effects of various opioid receptor agonists and antagonists on evoked acetylcholine release were studied in slices of human neocortex prelabelled with [³H]-choline, superfused and depolarized electrically (2 min, 3 Hz, 2 ms, 24 mA) or by K⁺ (20 mM). The -opioid receptor agonist DPDPE and the -opioid receptor agonist U50488 reduced the evoked [³H]-overflow (acetylcholine release) in a concentration-dependent fashion; the -opioid receptor antagonist naltrindole and the the -opioid receptor antagonist norbinaltorphimine, respectively, antagonized these effects. Application of the -opioid receptor agonist DAGO also resulted in an inhibition of acetylcholine release; however, both - and -opioid receptor antagonists were able to block this effect. The -opioid receptor agonists morphine and (+)-nortilidine had no effect. These results indicate that acetylcholine release in human neocortex is inhibited through - and -opioid receptors, but not through -opioid receptors.Acetylcholine release was significantly increased by the -opioid receptor antagonist naltrindole in the presence of a mixture of peptidase inhibitors providing evidence for a -opioid receptor-mediated inhibition of acetylcholine release by endogenous enkephalin.K⁺-evoked acetylcholine release in the presence of TTX was inhibited by U50488, but not by DPDPE, suggesting the presence of -opioid receptors on cholinergic terminals and the localization of -receptors on cortical interneurons. Therefore, the potent effect of DPDPE on acetylcholine release is likely to be indirect, by modulation of intrinsic cortical neurons. These interneurons probably do not use GABA as neurotransmitter since both GABA_A and GABA_B receptor agonists (muscimol and baclofen, respectively) were without effect on acetylcholine release.     

Opioid peptides decrease noradrenaline release and blood pressure in the rabbit at peripheral receptors

Summary Effects of dynorphin-(1–13), Leu⁵-enkephalin,d-Ala²,d-Leu⁵-enkephalin (DADLE), and for comparison bremazocine, on plasma noradrenaline concentration and mean arterial pressure (MAP) were studied in pithed rabbits. In the first series of experiments, the sympathetic outflow was stimulated electrically via the pithing rod at 2 Hz twice for 3 min each (S₁, S₂). Drugs were administered before S₂. Bremazocine 10 g/kg+2g/kg/h and 100 g/kg+20 g/kg/h, dynorphin 1 and 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 10 and 30 g/kg/min all diminished the electrically-evoked increase in plasma noradrenaline and MAP. The effects were antagonized by naloxone. In the second series, an infusion of noradrenaline (2 g/kg/min) was given twice for 3 min each (N₁, N₂). Drugs were administered before N₂. Bremazocine 100 g/kg+20 g/kg/h slightly enhanced the pressor effect of exogenous noradrenaline, whereas dynorphin 3 g/kg/min, Leu⁵-enkephalin 100 g/kg/min and DADLE 30 g/kg/min caused no significant change. In the third series, the sympathetic outflow was stimulated continuously at 2 Hz, and the interaction of dynorphin and DADLE was studied. Dynorphin 1 g/kg/min and DADLE 10g/kg/min initially decreased MAP to a similar extent. The effect of DADLE faded with time. When, during continuous infusion of DADLE 10 g/kg/min, and after return of MAP to the pre-DADLE level, dynorphin 1 g/kg/min or DADLE 10 g/kg/min was infused additionally, the effect of dynorphin was unchanged, whereas that of DADLE was almost abolished. We conclude that the opioid peptides as well as bremazocine decrease action potential-evoked release of noradrenaline and, secondarily, blood pressure. They act at peripheral sites, presumably prejunctional opioid receptors at postganglionic sympathetic axons. Dynorphin on the one hand, and Leu⁵-enkephalin and DADLE on the other hand, appear to act at different receptors, dynorphin probably at a - and DADLE and Leu⁵-enkephalin at a -receptor.     

双重实时荧光定量 PCR 检测人维生素 D 受体的方法学构建

目的 建立单管检测人维生素 D 受体（VDR）及甘油醛-3-磷酸脱氢酶（GAPDH）基因的双重实时荧光定量聚合酶链反应 （dual real-time PCR） 的方法。方法 以 GAPDH 基因为内参, 采用 Primer Premier 5.0 软件设计特异性引物及 TaqMan 探针, 进行 PCR 扩增检测 VDR 基因。将 VDR 及 GAPDH 扩增产物片段纯化后克隆构建成重组质粒, 作为定量检测基因表达的标准品, 并用于分析该方法的灵敏度和重复性。结果 PCR 扩增产物经测序分析证实为 VDR 及 GAPDH 特异性片段; 该方法检测 VDR 与 GAPDH 灵敏度达 40 拷贝/μL; 线性范围为 4.00×101～4.00×105 拷贝/μL; 决定系数 R2分别为 0.998、 0.999; 扩增效率 E 分别为 96.10%、 85.15%; 批内变异系数（CV）分别为 0.09%~ 1.21%、 0.35%~0.88%; 批间 CV 分别为 0.17%~0.51%、 0.51%~2.46%。结论 成功建立了单管检测人 VDR 及 GAPDH 的双重实时荧光定量 PCR 方法, 且该方法特异性好、 灵敏度高、 可快速高通量检测 VDR 的相对表达量, 有效缩短时间, 减小实验误差。     

Somatostatin sst2 receptor knock-out mice: localisation of sst1-5 receptor mRNA and binding in mouse brain by semi-quantitative RT-PCR, in situ hybridisation histochemistry and receptor autoradiography

The peptide hormone/neurotransmitter somatostatin (somatotropin release inhibiting factor; SRIF) and its receptors (sst(1)-sst(5)) appear to regulate many physiological functions in the CNS. Semi-quantitative analysis of the densities of mRNA expression for sst(1-5) receptors and SRIF receptor binding sites were established in sst(2) receptor knock-out (KO) mice. Patterns of sst(1-5) receptor mRNA expression were largely conserved for sst(1,3,4) and sst(5) selective oligonucleotide probes; whereas sst(2) signals were completely absent in KO mouse brain. Autoradiographic analysis demonstrated [(125)I]LTT SRIF(28), [(125)I]CGP 23996 (two radioligands known to label all five recombinant SRIF receptors) and [(125)I]Tyr(3)-octreotide (sst(2) and sst(5) receptor selective) binding in wild type (WT) mouse brain sections; yet no specific binding of [(125)I]Tyr(3)-octreotide in KO mice. In contrast, [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 binding was still present in a number of brain areas in KO mice, although to a lesser degree than in those regions where [(125)I]Tyr(3)-octreotide binding was found, in WT animals. The present data suggest first, that both sst(2) receptor protein and mRNA were completely absent in the brain of these KO animals. Second, there was little evidence of compensatory regulation, at the mRNA level, of the other SRIF receptors as a consequence of the sst(2) KO. Third, the absence of any [(125)I]Tyr(3)-octreotide binding, in KO mice, suggests that this particular ligand is selective for the sst(2) receptor subtype (under the conditions utilised); or that sst(5) receptors are only marginally expressed in brain. Fourth, there were regions where the binding of [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 were moderately affected by the sst(2) KO, suggesting that additional SRIF receptors may well contribute to the binding of the aforementioned radioligands. Finally, since the relative distribution of these two ligands were not entirely superimposable, it suggests that their respective selectivity profiles towards the different SRIF receptor subtypes in situ are not identical.     

Opioid receptor types and antinociceptive activity in chronic inflammation: bothe κ- and μ-opiate agonistic effects are enhanced in arthritic rats

The antinociceptive effects obtained in arthritic rats with morphine, the opioid μ-agonist DAGO 9D-Al²,MePhe⁴,Gly-ol⁵ol⁵]enkephalin, the δ-selective agonist DTLET [D-Thr²,Leu⁵]enkephalyl-Thr, and the κ-agonist U-50,488H were compared to their corresponding effects in normal animals and morphine-pretreated arthritic rats, respectively, using a paw pressure test. The effects of the μ- and κ-agonists were increased in arthritic rats. While morphine-treated rats were cross-tolerant to the μ- and κ-agonists, no tolerance to the δ-selective agonist was found. The possibility that the potent action of morphine on this model for chronic inflammatory pain is mediated partly through κ-mechanisms is discussed.     

Developmental changes in transporter and receptor protein expression levels at the rat blood-brain barrier based on quantitative targeted absolute proteomics

The blood-brain barrier (BBB) transport systems regulate the supply of nutrients, amino acids, vitamins, and hormones to the developing brain, as well as blocking the entry of xenobiotics and drugs. The purpose of this study was to clarify the developmental changes in the absolute protein expression levels of BBB transport-related proteins in developing rat brain capillaries, using quantitative targeted absolute proteomics (QTAP). The changing patterns of ATP-binding cassette (ABC) and solute carrier (SLC) transporters, receptors, and tight junction/adherence junction-related proteins were classified into 4 types: uphill (continuously increasing expression from postnatal day (P) 1 to P56), bell-shape/inverted bell-shape (increased/decreased expression from P1 to P14 followed by decreased/increased expression from P21 to P56), downhill (continuously decreasing expression from P1 to P56), and constant (no significant difference from P1 to P56). Proteins showing uphill-type expression included P-glycoprotein/Mdr1a/Abcb1, Mrp4/Abcc4, Bcrp/Abcg2, Glut1/Slc2a1, Oatp1c1/Slco1c1, FcRn, 4F2hc/Slc3a2, claudin-5, caveolin-1, Cd29/integrin β1. Those showing bell-shape/inverted bell-shape expression included Mct1/Slc16a1, Oat3/Slc22a8, Tfr1, Lrp1, and CD147. On the other hand, Cat1/Slc7a1 and Cd54/Icam-1 showed downhill expression, and Insr showed constant expression. These results suggest that the protein expression levels of transporters and receptors at the BBB change in various ways to meet the changing requirements of the developing brain.     

Measurement of human nerve growth factor binding sites in brain and in peripheral tissues by a specific immunoprecipitation assay

Summary Using a monoclonal antibody against the human nerve growth factor (NGF) receptor (20.4 IgG), a specific immunoprecipitation assay for the quantitation of human NGF binding sites has been established. The procedure involves specific labeling of NGF receptors by covalent crosslinking to ¹²⁵I-NGF with a carbodiimide reagent, and subsequent immunoprecipitation of the detergent-extracted ¹²⁵I-NGF-receptor complexes with the 20.4 IgG. This two-site assay provides a specific method to measure NGF binding sites in peripheral tissues and central nervous system. Moreover, it allows analysis of the immunoprecipitated receptor species by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The finding that specific NGF binding sites are expressed on human neuronal and nonneuronal tissues, including lymphoid tissues, indicates that NGF exerts a broader physiological function than originally ascribed.Abbreviations BSA bovine serum albumin - NGF nerve growth factor - PAGE polyacrylamide gel electrophoresis - PBS phosphatebuffered saline - SCG superior cervical ganglion - SDS sodium dodecyl sulfate Send offprint requests to U. Otten at the above address     

Ethylketocyclazocine decreases noradrenaline release and blood pressure in the rabbit at a peripheral opioid receptor

Summary Rabbits were pithed and their sympathetic outflow was stimulated electrically via the pithing rod. Arterial blood pressure, heart rate, the endogenous plasma noradrenaline level, the plasma ³H-noradrenaline clearance and the noradrenaline release rate (the rate of entry of endogenous noradrenaline into the plasma) were determined. Ethylketocyclazocine 0.1 mg kg^–1+0.02 mg kg^–1 h^–1 and 1 mg kg^–1 + 0.2 mg kg^–1 h^–1 but not 0.01 mg kg^–1+ 0.002 mg kg^–1 h^–1 decreased blood pressure, the endogenous plasma noradrenaline level and the noradrenaline release rate. The effects of ethylketocyclazocine 1 mg kg^–1+ 0.2 mg kg^–1 h^–1 were antagonized by naloxone 1 mg kg^–1 + 0.5 mg kg^–1 h^–1. Given alone, naloxone caused no change. It is concluded that ethylketocyclazocine inhibits action potential-evoked release of noradrenaline from postganglionic sympathetic neurones, and hence can lower blood pressure, by a peripheral effect, possibly mediated by opioid receptors at the terminal axons.     

Multiple tachykinin binding sites in peripheral tissues and in brain

Tachykinin binding sites in guinea pig urinary bladder (GPUB), rat salivary gland (RSG), hamster urinary bladder (HUB), rat vas deferens (RVD) and rat cerebral cortex (RCC) were compared using ¹²⁵I-Bolton Hunter conjugates of substance P (¹²⁵I-BHSP), eledoisin (¹²⁵I-BHE) and neurokinin A (¹²⁵I-BHNKA). In typical SP-P tissues (GPUB, RSG) and in RCC, SP was the most potent displacer of ¹²⁵I-BHSP and [Glp⁶, D-Pro⁹]-SP(6–11) was 90 times less active than [Glp⁶, L-Pro⁹]-SP(6–11) while SP methyl ester (SPOMe) was 5–10 times more active than the Bolton Hunter conjugate of SPOMe (I-BHSPOMe). On the other hand, in typical SP-E tissues (HUB, RVD), neurokinin A was most potent in displacing ¹²⁵I-BHE and [Glp⁶,D-Pro⁹]-SP(6–11) was over 300 times more active than [Glp⁶,L-Pro⁹]-SP(6–11) while SPOMe was 160 times less active than I-BHSPOMe. In rat cerebral cortex, the rank order of potency of tachykinins and related analogues in displacing ¹²⁵I-BHE was distinct from that of peripheral SP-E sites, with neurokinin B being the most potent displacer, and SPOMe was over 1 000 times more active than I-BHSPOMe; ¹²⁵I-BHE binding sites in CNS may represent a third category of tachykinin receptor, designated SP-N.     

Adrenomedullin receptor binding sites in rat brain and peripheral tissues

The existence of specific adrenomedullin receptor binding sites was investigated using the agonist peptide fragment [125I]human adrenomedullin-(13-52) in rat brain, lung and vas deferens homogenates. Saturation-binding experiments suggest that [125I]human adrenomedullin-(13-52) binds to an apparent single population of sites with similar affinities (K(D) of 0.3 to 0.6 nM) but with different maximal binding capacity in the rat brain, lung and vas deferens homogenates (B(max) of 73, 1760 and 144 fmol/mg protein, respectively). Competition-binding experiments using various analogues and fragments of calcitonin gene-related peptide (CGRP) and adrenomedullin were also performed using this radioligand. Competition-binding profiles suggest the possible existence of heterogeneous populations of adrenomedullin receptor binding sites. For example, in rat brain, human adrenomedullin-(1-52) and human adrenomedullin-(13-52) competed against specific [125I]human adrenomedullin-(13-52) sites with competition curves best fitted to a two-site model. Additionally, human calcitonin gene-related peptide alpha (hCGRPalpha), [Cys(Et)(2,7)]hCGRPalpha and [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] (BIBN4096BS) competed against specific [125I]human adrenomedullin-(13-52) binding with profiles that were also best fitted to a two-site model. Furthermore, binding assays performed in the presence of GTPgammaS (100 microM) revealed that this compound inhibited 20% of specific [125I]human adrenomedullin-(13-52) sites in rat brain homogenates and competition curves of human adrenomedullin-(1-52) and [Cys(Et)(2,7)]hCGRPalpha against specific [125I]human adrenomedullin-(13-52) sites remained best fitted to a two-site model. Moreover, the existence of specific [125I]human adrenomedullin-(13-52) binding sites that are resistant to human adrenomedullin-(22-52) and human CGRP-(8-37) is suggested in the rat brain and vas deferens. Taken together, these data provide evidence for the possible existence of heterogeneous populations of adrenomedullin binding sites in rat brain and peripheral tissues.     

结直肠癌患者肿瘤组织与外周血中MTHFR C677T的多态性及相关性研究

目的 探索外周血代替肿瘤组织进行MTHFR C677T基因多态性检测的可行性.方法 选取首诊为结直肠癌且行根治性手术的患者26例,术前未接受任何化学治疗,应用聚合酶链反应-限制性内切片段长度多态性（PCR-RFLP）方法检测MTHFR C677T的基因多态性,比较外周血及肿瘤组织中相关基因型的一致性.结果 26例结直肠癌患者的肿瘤组织及外周血中MTHFR 677CT基因型、TT基因型、CC基因型一致的病例数分别为7、7、4例.肿瘤组织和外周血中MTHFR C677T的基因型一致率为69.2%.结论 外周血中MTHFR C677T的基因多态性可以反映肿瘤组织中相关基因型的突变.     

食管组织中代谢酶相关基因的异常表达与食管癌的发病关系研究

目的研究与环境致癌物活化、解毒有关的相和相代谢酶的mRNA水平与食管癌发病的关系。方法收集93例淮安地区食管癌新发病例的食管癌组织及远端癌旁组织,应用SYBRGreen实时荧光定量逆转录-聚合酶链反应(RT-PCR)检测CYP2E1、GSTP1、MTHFR和NQO1等基因的mRNA水平;应用t检验和logistic回归分析评价基因的表达水平与食管癌的关系。结果CYP2E1和MTHFR的mRNA水平在癌组织和配对癌旁组织之间有显著差异;条件logistic逐步回归分析结果显示CYP2E1、MTHFR表达水平异常与食管癌发病风险升高有关联[OR值(95%CI)分别为1.890(1.439~2.481)和1.362(1.107~1.677)]。结论CYP2E1和MTHFR的表达水平改变与食管癌发病风险密切相关,在食管癌的高危人群筛查和早期诊断中具有重要意义。     

Glycosylated phosducin-like protein long regulates opioid receptor function in mouse brain

Phosducin (Phd), a protein that in retina regulates rhodopsin desensitization by controlling the activity of Gt beta gamma-dependent G-protein-coupled receptor kinases (GRKs), is present in very low levels in the CNS of mammals. However, this tissue contains proteins of related sequence and function. This paper reports the presence of N-glycosylated phosducin-like protein long (PhLP(L)) in all structures of mouse CNS, mainly in synaptic plasma membranes and associated with G beta subunits and 14-3-3 proteins. To analyze the role PhLP(L) in opioid receptor desensitization, its expression was reduced by the use of antisense oligodeoxynucleotides (ODNs). The antinociception induced by morphine, [D-Ala(2), N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), beta-endorphin, [D-Ala(2)]deltorphin II, [D-Pen(2,5)]-enkephalin (DPDPE) or clonidine in the tail-flick test was reduced in PhLP(L)-knock-down mice. A single intracerebroventricular (icv)-ED(80) analgesic dose of morphine gave rise to acute tolerance that lasted for 4 days, but which was prevented or reversed by icv-injection of myristoylated (myr(+)) G(i2)alpha subunits. PhLP(L) knock-down brought about a myr(+)-G(i2)alpha subunit-insensitive acute tolerance to morphine that was still present after 8 days. It also diminished the specific binding of (125)I-Tyr(27)-beta-endorphin-(1-31) (human) to mouse periaqueductal gray matter membranes. After being exposed to chronic morphine treatment, post-dependent mice required about 10 days for complete recovery of morphine antinociception. The impairment of PhLP(L) extended this period beyond 17 days. It is concluded that PhLP(L) knock-down facilitates desensitization and uncoupling of opioid receptors.