Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats.

Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.

Sustained morphine-mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf-1-selective siRNA.

BACKGROUND AND PURPOSE: Long-term morphine treatment enhances pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats. EXPERIMENTAL APPROACH: Rats were intrathecally (i.th) injected with a Raf-1-selective small interfering RNA (siRNA) mixture for 3 days and were subsequently infused with saline or morphine, s.c. for 7 days. Thermal and mechanical sensory thresholds of the animals were assessed by daily behavioural tests. After final behavioural testing (day 6), spinal cords were isolated from each animal group and spinal CGRP and Raf-1 protein levels were measured using elisa and immunohistochemistry. KEY RESULTS: Selective knockdown of spinal Raf-1 protein levels by i.th Raf-1-selective siRNA pretreatment significantly attenuated sustained morphine-mediated up-regulation of CGRP immunoreactivity in the spinal cord of rats and prevented the development of thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. CONCLUSIONS AND IMPLICATIONS: Raf-1 played a significant role in sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance in vivo. These findings suggest novel pharmacological approaches to improve the long-term utility of opioids in the treatment of chronic pain.

Functional selectivity in cannabinoid signaling.

Cannabinoid (CB) agonists exhibit numerous potentially useful pharmacological properties, but unwanted side effects limit their use in clinical practice. Thus, novel strategies are needed to identify potential CB pharmaceuticals with fewer side effects. Activated CB receptors initiate multiple parallel intracellular signal transduction cascades. In the present paper we will review experimental data indicating that structurally different classes of CB agonists may exhibit selectivity toward individual subsets of intracellular signaling pathways. In support of this, recent findings indicate that chemically distinct classes of CB agonists frequently differ in their rank order of potency to produce analgesia versus other central nervous system effects in vivo. Structurally different agonists were also found to differ in their abilities to activate individual G protein types in vitro. Since it was suggested earlier that structurally distinct CB agonists may interact differently with the CB receptors, it has been hypothesized that different classes of cannabinoid agonists may stabilize unique active CB receptor conformations, leading to functional selectivity in CB receptor signaling. In order to obtain a direct proof for this hypothesis, we recently employed a highly sensitive biophysical method, plasmon-waveguide resonance (PWR) spectroscopy. PWR experiments have provided a direct proof that structurally different CB agonists produce qualitatively distinct changes in the shape and/or membrane orientation of the CB1 receptors, leading to functional selectivity in G protein activation. We expect that by identification of CB agonists that selectively activate preferred intracellular signaling pathways novel pharmacological lead structures can be identified for the design of improved CB analgesics with fewer side effects.

[Role of the tumor markers CA 19-9 and carcinoembryonic antigen (CEA) in diagnosis, treatment and prognosis of pancreatic cancer]. / Znachenie opukholevogo markera CA 19-9 i kartsino-émbrional'nogo antigena (KEA) v diagnostike, vybore lechebnoi taktiki i prognozirovanii raka podzheludochnoi zhelezy.

The investigation was concerned with diagnostic sensitivity, specificity and effectiveness of assay of CA 19-9 and carcinoembryonic antigen (CEA) in the choice of treatment modality and evaluation of therapy pancreatic carcinoma (PC). Either marker has been studied in 685 examinations for PC, 68--chronic pseudotumorous pancreatitis and 24--intestinal cancer at other sites since 1995. Tumor resection for PC was carried out in 31; conservative treatment--67; chemotherapy--56 and radiotherapy--in 29 cases. In CA 19-9 examinations, diagnostic sensitivity was 90.2; specificity--72.1 and effectiveness--85.3%, while in CEA determinations, 82.5; 30.9 and 68.5%, respectively. CA 19-9 and CEA levels proved to be prognostic factors of survival. An inverse correlation was observed between median survival and tumor marker concentrations: higher basal (preoperative) level of marker in blood was matched by lower median survival. A similar relationship was identified for CEA: 5-10--14.2 months; 10.1-20 ng/ml--8.0 months; 20.1-30 ng/ml--3.9 months, and more than 30 ng/ml--4.8 months. There was a direct correlation between CA 19-9 level and tumor stage. The dynamics of tumor markers, particularly, CA 19-9 correlated with treatment effectiveness during its course.

[Expression of tissue-specific genes with progression of mouse hepatocellular carcinoma]. / Ekspressiia tkanespetsificheskikh genov pri progressii gepatokartsinom myshi.

Expression of hepatocyte-specific genes in slow- and fast-growing hepatocellular mouse carcinomas was studied. The fast-growing poorly differentiated passaged hepatocarcinoma (fHC) originated from the well-differentiated slow-growing variant (sHC). In contrast to the parental hepatocarcinoma, in fHC the expression of the hepatocyte nuclear factor 4 (HNF4), in fHC a key factor responsible for hepatocyte differentiation, and several HNF-4-responsive genes, such as those for transferrin, transthyretin, hepatocyte nuclear factor 1 (HNF1), and serum albumin, was significantly suppressed. The expression of exogenous HNF4 in the fHC cell culture partially restored the expression of hepatocyte marker genes and the appearance of epithelial cell islands in the culture. The described system may serve as a convenient model for further analysis of mechanisms underlying hepatocarcinogenesis and liver tumor progression.

Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation.

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.

[Suppression of alpha-fetoprotein gene expression in somatic hybrids of AFP-positive and AFP-negative clones of rat hepatoma]. / Podavlenie ékspressii gena al'fa-fetoproteina v somaticheskikh gibridakh AFP-polozhitel'nogo i AFP-otritsatel'nogo klonov krysinoi gepatomy.

The presented study is devoted to investigation of molecular mechanisms regulating alpha-fetoprotein (AFP) gene expression at transcriptional level. The study was carried out on AFP-positive and AFP-negative clones of rat hepatoma McA-RH 7777 that also differ in hepatocyte nuclear factors (HNF) 1 and 4 transcription levels. To examine a hypothesis of existence in AFP-non-producing clones a transcriptional factor that downregulates this gene expression, we have obtained somatic hybrids of AFP-positive and AFP-negative clones. In the obtained hybrids AFP gene expression is decreased while expression of HNF1, one of the main AFP promoter activators, is maintained. These data indicate an existence of a repressor in AFP-negative clones that determines AFP gene downregulation regardless of the HNF1 expression level.

Coupling of human delta-opioid receptor to retinal rod transducin in Chinese hamster ovary cells.

Reverse transcription-polymerase chain reaction was used to identify the pertussis toxin (Ptx)-sensitive G protein alpha-subunit pool in Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. We detected the presence of mRNA for G(ialpha2), G(ialpha3), and G(oalpha) in both cell lines. G(ialpha1) and G(alphaz) mRNAs were not detected. We also found a homolog of the retinal rod transducin (G(talpha1)) in CHO, and the mouse cone transducin (G(talpha2)) in B82 cells. The presence of the transducin alpha-subunit proteins in CHO and B82 cells was confirmed by immunoprecipitation with specific antibodies. To test the interaction of heterologously expressed receptors with transducin in CHO cells, a Ptx-insensitive (C347S) rod transducin mutant was transfected into a CHO cell line stably expressing the human delta-opioid receptor (hDOR/CHO). (+)-4-[(alphaR)-alpha-((2S,2R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, a selective delta-opioid receptor agonist, stimulated guanosine-5'-O-(3-[(35)S]thio)triphosphate binding by 293 +/- 36% after Ptx pretreatment in the mutant cell line with an EC(50) value of 54 +/- 32 nM, showing that transducin can functionally couple to the human delta-opioid receptors in these cells.

Phosphorylation of adenylyl cyclase VI upon chronic delta-opioid receptor stimulation.

An immunoprecipitation method was used to measure [32P]phosphate incorporation into the adenylyl cyclase VI protein in Chinese Hamster Ovary (CHO) cells stably expressing the human delta-opioid receptor. Chronic SNC 80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N ,N-diethyl-benzamide) 1 microM, 24 h) treatment increased the incorporation of [32P] into a 200 kDa protein band 2.5-fold after gel electrophoresis. The increase in phosphorylation of adenylyl cyclase VI was antagonized by naltrindole (1 microM) and the immunoprecipitation was prevented by the saturation of the antibody with the blocking peptide.

Identification of adenylyl cyclase isoenzymes in CHO and B82 cells.

The identification of adenylyl cyclase isoenzymes in mammalian host cells is important for the interpretation of data obtained from cell lines heterologously expressing G-protein coupled receptors. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify adenylyl cyclase cDNAs from Chinese hamster ovary (CHO) and mouse fibroblast (B82) cells. The isolated fragments were identified by restriction analyses and by sequencing. We found mRNAs for adenylyl cyclases VI and VII in CHO and adenylyl cyclases IX and VII in B82 cells.

Pharmacological comparison of the cloned human and rat M2 muscarinic receptor genes expressed in the murine fibroblast (B82) cell line.

The coding sequence of the human m2 receptor gene was amplified by polymerase chain reaction and stably transfected into a murine fibroblast cell line (B82). We have compared the human M2 clonal cell line (HM2-B10) with the previously established B82 cell line (M2LKB2-2) expressing the rat M2 receptor to assess drug specificity, drug selectivity and effector coupling. Both transfected cell lines showed a high level of specific, saturable [3H](-)-N-methyl-3-quinuclidinyl benzilate binding with Kd values of 243 pM (155-352 pM) and 345 pM (234-539 pM) and Bmax values of 97 +/- 4 and 338 +/- 16 fmol/10(6) cells, respectively. Inhibition of [3H](-)-N-methyl-3-quinuclidinyl benzilate binding to HM2-B10 cells and M2LKB2-2 cells showed the same rank order of potency for the antagonists: atropine > dexetimide > 4-diphenylacetoxy-N-methylpiperidine methiodide > himbacine > methoctramine > 11-[[2-[(diethylamino) methyl]-1-piperidinyl]acetyl]-5,11-dihidro-6H-pyrido-[2,3-b](1, 4)-benzodiazepine-6-one > hexahydro-sila-difenidol hydro-chloride > pirenzepine. Correlation analysis of the pKi values indicate that the expressed human and rat M2 receptors have nearly identical ligand-binding characteristics. Carbachol inhibited forskolin-stimulated cAMP formation with similar potency in both cell lines [EC50 = 2.4 microM (0.2-2.8) and 1.1 microM (0.2-5.3) for the human and rat M2 receptor, respectively]. In the M2LKB2-2 cells, carbachol slightly stimulated the [3H]inositol monophosphate formation but had no significant effect in HM2-B10 cells. In conclusion, the human and rat M2 receptors expressed in the B82 cell line have very similar binding properties but exhibit slight differences in effector coupling mechanisms.

The third extracellular loop of the human delta-opioid receptor determines the selectivity of delta-opioid agonists.

In the present study, we replaced the third extracellular loop of the human delta-opioid receptor with that of the human mu-opioid receptor. A modified polymerase chain reaction overlap extension method was used to achieve the exact splicing in the chimera to show the importance of the extracellular loop in ligand binding without interference from transmembrane substitutions. The replacement of the third extracellular loop did not alter the affinity of [3H]diprenorphine but caused a dramatic decrease in the affinity of both the delta-selective peptide agonists cyclo[D-Pen2,4'Cl-Phe4,D-Pen5]enkephalin and deltorphin II and the delta-selective nonpeptide agonists SNC 121 and (-)TAN 67. The affinities of the mu-selective peptide agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin and the mu-preferring nonpeptide agonist morphine were not affected. Site-directed mutagenesis studies show that the mechanism of ligand recognition might be different for each structural class of opioid ligands.

delta-Opioid receptor: the third extracellular loop determines naltrindole selectivity.

Human delta/mu-opioid receptor chimeras were constructed to determine the role of the second and third extracellular loops in alkaloid ligand selectivity. Exchanging the third extracellular loop of the delta-opioid receptor with that of the mu-opioid receptor dramatically decreased the affinity of naltrindole, but not that of morphine. The results suggest that different domains of the opioid receptor are involved in the binding of naltrindole and morphine.

Identification of a human delta opioid receptor: cloning and expression.

The delta opioid receptor is an important target for analgesic drug development. This report describes the identification of delta opioid receptor clones from human cDNA libraries and the preparation of a human delta receptor cDNA in the pcDNA3 expression vector for transfection studies. The cDNA encodes a 372 amino acid protein that has 93% amino acid identity to mouse and rat delta receptors. COS-7 cells transfected with this clone express over 1.0 pmol receptor/mg protein when measured by saturation binding with [3H]naltrindole. The delta receptor selective ligands NTB, BNTX, [4'-Cl-Phe4]DPDPE and [D-Ala2,Glu4]deltorphin all have Ki values under 10 nM while the affinities of the mu and kappa opioid receptor ligands CTAP and U-69593, respectively, are over 4.0 microM. Agonists show binding to multiple affinity states of the receptor consistent with the presence of G-protein coupled and uncoupled forms of the expressed receptor. The 8-fold higher affinity of NTB relative to BNTX suggests that the human delta receptor is of the delta 2 subtype.

Cloning of the rat M3, M4 and M5 muscarinic acetylcholine receptor genes by the polymerase chain reaction (PCR) and the pharmacological characterization of the expressed genes.

The coding sequence of the rat m3, m4 and m5 subtypes of muscarinic acetylcholine receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed in the murine fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the literature. When the different sequence appeared in only one clone obtained by PCR, it was considered an error of the polymerase. The overall error frequency in the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide in 1,692 base pairs. In order to evaluate the different nucleotide sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced. The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated. The affinity of the different antagonists to the muscarinic receptor subtypes was determined by [3H](-)MQNB/ligand inhibition experiments. In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inositol monophosphate (IP1) accumulation. Carbachol, at 3 microM, appeared to suppress the forskolin-stimulated cAMP formation in the m4 transfected cells. These findings suggest these mAChRs amplified by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarinic receptor subtypes.