Guo-xi Xie

Presence of opioid receptor-like (ORL1) receptor mRNA splice variants in peripheral sensory and sympathetic neuronal ganglia.

The expression of ORL1 receptor mRNA splice variants is determined in peripheral sensory and sympathetic ganglia and compared to mRNA expression for the three classic opioid receptor subtypes (mu, delta, and kappa) using the method of reverse transcription-polymerase chain reaction. ORL1, mu, delta and kappa receptor subtype mRNAs are present in human dorsal root ganglia (DRG) and trigeminal ganglia and rat DRG. ORL1, mu and delta receptor subtype mRNAs are present in rat superior cervical ganglia and only ORL1 and delta receptor mRNAs are present in rat lumbar sympathetic ganglia. Both the ORL1 mRNA splice variants are present in sensory and sympathetic ganglia, however, expression of the shorter ORL1 receptor mRNA dominates over expression of the longer splice variant in rat brain and DRG, whereas, expression of the longer splice variant is dominant in sympathetic ganglia.

Detection of mRNAs and alternatively spliced transcripts of dopamine receptors in rat peripheral sensory and sympathetic ganglia

The presence of mRNAs for dopamine receptor subtypes and dopamine transporter in rat peripheral sensory and sympathetic ganglia was investigated using polymerase chain reaction (PCR) and DNA sequencing. Dopamine D1, D2, D3, D5 receptor subtype mRNAs and dopamine transporter mRNA were detected in both superior cervical sympathetic ganglia (SCG) and dorsal root ganglia (DRG) in the rat; the expression of D4 mRNA was only detected in DRG. While two alternatively spliced isoforms of D2 were detected in both ganglia, the alternative splicing transcripts for D3 and D4 were only found in the DRG. These results are useful in further studying the roles of dopamine and the effects of dopaminergic agents in the peripheral nervous system.

RGS proteins: new players in the field of opioid signaling and tolerance mechanisms.

In this article we review recent advances in our understanding of the crucial role of the Regulator of G protein Signaling (RGS) proteins in opioid signaling mechanisms and opioid tolerance development. Opioids exert their physiologic effects via complex G protein-coupled receptor-signaling mechanisms, and RGS proteins are now known to tightly regulate the G protein signaling cycle. RGS proteins contain GTPase-accelerating protein activity within their characteristic RGS domain and various other receptor signaling-related properties of their other functional domains. There have been more than 20 RGS proteins reported in the literature, and multiple RGS proteins have been shown to negatively regulate G protein-mediated opioid signaling, facilitate opioid receptor desensitization and internalization, and affect the rate at which opioid tolerance develops. Using RGS proteins as targets for future drug therapy aimed at modulating opioid effectiveness in both acute and chronic pain settings may be an important advance in the treatment of pain.

Age-dependent morphine tolerance development in the rat.

In all age groups, the use of opioids to treat chronic pain conditions has increased, yet the impact of age on opioid tolerance development has not been comprehensively addressed. In this study, we investigated age-related differences in morphine tolerance development in rats. Rats aged 3 wk, 3 mo, 6 mo, and 1 yr were used in the study. Morphine (8 mg/kg) was injected subcutaneously twice each day and its analgesic effect assessed by the change in tail-flick latency using a thermal stimulus 5 min before and 30 min after dosing. Tolerance was defined as a 75% reduction in morphine-induced analgesia compared to Day 1. Rats aged 3 wk, 3 mo, 6 mo, and 1 yr developed tolerance on the 4th, 10th, 14th, and 22nd days of morphine treatment, respectively. Plasma levels of morphine and its metabolites showed that pharmacokinetic differences among the groups did not correlate with the differences in tolerance development. This study demonstrates that morphine tolerance occurs more rapidly in younger rats than older rats and is unlikely to be the result of differences in drug metabolism or clearance. Aging may impact molecular processes involved in tolerance development and provide insight into novel therapeutic targets to delay opioid tolerance development.

5-hydroxytryptamine receptor subtype messenger RNAs in human dorsal root ganglia: a polymerase chain reaction study

Although serotonin has been shown to play an important role in peripheral pain mechanisms, the specific subtypes of serotonin receptors involved in pain and hyperalgesia remain poorly understood. To date, no previous study has attempted to determine the presence of any serotonin receptor subtype in human dorsal root ganglia. In this study, the presence of messenger RNA for eight human serotonin receptor subtypes in lumbar dorsal root ganglia was detected using the method of polymerase chain reaction. Dorsal root ganglia were excised post mortem from four patients. Oligonucleotide primers were chosen based on unique regions of complimentary DNA sequence for eight cloned human serotonin receptor subtypes (i.e. 5-HT1A, 5-HT1D alpha, 5-HT1D beta, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2C and 5-HT7). The presence of 5-HT1D alpha, 5-HT1D beta, 5-HT1E, 5-HT1F, 5-HT2A and 5-HT7 receptor subtype messenger RNA was detected in dorsal root ganglia from three of the four subjects. 5-HT1A receptor subtype messenger RNA was detected in one of the four subjects. No 5-HT2C receptor subtype messenger RNA could be detected. Findings from this study may direct further efforts to determine the role of serotonin receptors in the peripheral nervous system.

An alternatively spliced transcript of the rat nociceptin receptor ORL1 gene encodes a truncated receptor

Opioid receptor-like protein ORL1, the receptor for the neuropeptide nociceptin (also named orphanin FQ), has two alternatively spliced isoforms in the rat. This alternative splicing event is generated by retaining of intron 3, 81 bases in length, in the mRNA region encoding the second extracellular loop of ORL1. A full-length rat ORL1 receptor has 367 amino acid residues. However, as revealed by sequencing of rat ORL1 genomic DNA and cDNA, the insertion of the unspliced intron 3 brings in an in-frame stop codon and, therefore, creates a truncated open-reading frame encoding only the N-terminal half of ORL1 (from the N-terminus to an alternate extracellular tail C-terminal to the fourth transmembrane domain). The two alternatively spliced transcripts are differentially expressed in tissues. In transfected mammalian cells, the full-length ORL1 displays high-affinity and selective binding for nociceptin, and inhibits the production of cyclic AMP. In contrast, the truncated ORL1 binds nociceptin and other opioid peptides very poorly and non-selectively (affinity in micromolar range), and it does not mediate any inhibitory effects on cyclic AMP production. Apparently, this truncated ORL1 does not function as a receptor for nociceptin or other ligands tested. Such alternative splicing to create a truncated ORL1 receptor might be an endogenous mechanism to negatively regulate nociceptin/ORL1 functions.

Discrete change in volatile anesthetic sensitivity in mice with inactivated tandem pore potassium ion channel TRESK.

Background:We investigated the role of tandem pore potassium ion channel (K2P) TRESK in neurobehavioral function and volatile anesthetic sensitivity in genetically modified mice. Methods:Exon III of the mouse TRESK gene locus was deleted by homologous recombination using a targeting vector. The genotype of bred mice (wild type, knockout, or heterozygote) was determined using polymerase chain reaction. Morphologic and behavioral evaluations of TRESK knockout mice were compared with wild-type littermates. Sensitivity of bred mice to isoflurane, halothane, sevoflurane, and desflurane were studied by determining the minimum alveolar concentration preventing movement to tail clamping in 50% of each genotype. Results:With the exception of decreased number of inactive periods and increased thermal pain sensitivity (20% decrease in latency with hot plate test), TRESK knockout mice had healthy development and behavior. TRESK knockout mice showed a statistically significant 8% increase in isoflurane minimum alveolar concentration compared with wild-type littermates. Sensitivity to other volatile anesthetics was not significantly different. Spontaneous mortality of TRESK knockout mice after initial anesthesia testing was nearly threefold higher than that of wild-type littermates. Conclusions:TRESK alone is not critical for baseline central nervous system function but may contribute to the action of volatile anesthetics. The inhomogeneous change in anesthetic sensitivity corroborates findings in other K2P knockout mice and supports the theory that the mechanism of volatile anesthetic action involves multiple targets. Although it was not shown in this study, a compensatory effect by other K2P channels may also contribute to these observations.

Blockade effects of (Nphe1)Nociceptin(1–13)-NH2 on anti-nociception induced by intrathecal administration of nociceptin in rats

The present study investigated the roles of the opioid-receptor-like (ORL1) receptor and its endogenous ligand nociceptin on nociception in the spinal cord of rats. Intrathecal administration of 10 nmol of nociceptin produced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulation. There were no significant changes of average maximum angles in inclined plane tests after intrathecal injection of 10 nmol of nociceptin in rats. The intrathecal nociceptin-induced increases in HWL were antagonized by intrathecal administration of (Nphe1)Nociceptin(1-13)-NH(2), a selective antagonist of ORL1 receptor, in a dose-dependent manner. The results demonstrated that ORL1 receptor is involved in the nociceptin-induced anti-nociceptive effect in the spinal cord of rats.

Bi-directional modulation of 5-hydroxytryptamine-induced plasma extravasation in the rat knee joint by nociceptin.

The role of nociceptin, the endogenous ligand for the opioid receptor-like (ORL1) receptor, in nociceptive processing is controversial. Most studies demonstrate hyperalgesia following supraspinal administration, analgesia following intrathecal and peripheral administration at higher doses, and hyperalgesia following intrathecal and peripheral application at lower doses. The present study investigates the effect of nociceptin on synovial plasma extravasation and its ability to modulate 5-hydroxytryptamine-induced synovial plasma extravasation using the rat knee joint model of inflammation. Nociceptin alone does not alter synovial plasma extravasation from baseline. Nociceptin at concentrations up to 1 nM enhances 5-hydroxytryptamine-induced synovial plasma extravasation (up to 50%) and nociceptin at concentrations above 100 nM inhibits 5-hydroxytryptamine-induced synovial plasma extravasation (down to 45%). The novel, selective ORL1 receptor antagonist J-113397 potently inhibits the pro-inflammatory effect of nociceptin, but only partly inhibits, at higher concentrations, the anti-inflammatory effects of nociceptin.These findings demonstrate a dose-dependent bi-directional effect of nociceptin on inflammatory processes and may indicate a target for novel therapeutics.

5-Hydroxytryptamine-induced plasma extravasation in the rat knee joint is mediated by multiple prostaglandins

Abstract:Objective and design: This study investigated whether prostaglandins (PGs) are involved in 5-hydroxytryptamine (5-HT)-induced synovial plasma extravasation.¶Materials and methods: Male Sprague-Dawley rat knee joints were perfused with 5-HT and synovial capillary Evans Blue dye leakage was measured using spectrophotometry. Cyclooxygenase (COX) inhibitors and PG receptor subtype-selective antagonists were tested for the ability to reduce 5-HT-induced synovial plasma extravasation.¶Results: 5-HT-induced plasma extravasation was inhibited by indomethacin. The COX-1 selective inhibitor SC-560 and the COX-2 selective inhibitor NS-398 were equally effective, indicating that both isoforms are involved. Antagonists selective for EP1, EP2 and DP receptor subtypes significantly attenuated the 5-HT-induced plasma extravasation. However, antagonists selective for FP, IP and TP subtypes failed to reduce 5-HT-induced plasma extravasation.¶Conclusions: These results demonstrate that multiple, but selective, subtypes of PGs mediate synovial plasma extravasation produced by 5-HT, and suggest that PGs act downstream of 5-HT in the inflammatory cascade.