Jia-Wei-Si-Miao-Wan alleviates acute gouty arthritis by targeting NLRP3 inflammasome.

Gout is a common metabolic disease and acute gouty arthritis (AGA) is one of the important complications. Jia-Wei-Si-Miao-Wan is a newly developed drug for treating acute gouty arthritis, but the molecular mechanism has not been completely clarified. Thus, this study was aimed to explore the regulation of Jia-Wei-Si-Miao-Wan on NLRP3 inflammasome and TLR/NF-κB signaling, which are two important signaling pathways in inflammation. AGA rat model was established by injecting monosodium urate into the right knee. Colchicine and Jia-Wei-Si-Miao-Wan were administrated by gavage. The circumference of the knee was measured. IL-1ß and IL-18 level in the flushing fluid was detected by enzyme linked immunosorbent assay (ELISA). Western blot, immunohistochemistry and quantitative real-time RT-PCR were used to detect the protein and mRNA expression of TLR4, NLRP3, ASC, caspase-1, NF-κB and p-NF-κB. The results showed that IL-1ß and IL-18 level in the flushing fluid was increased and TLR4, NLRP3, ASC, caspase-1, NF-κB and p-NF-κB expressions were up-regulated after the establishment of AGA rat model. Colchicine and Jia-Wei-Si-Miao-Wan administration could alleviate the inflammation in the knee by inhibiting NLRP3 inflammasome and TLR/NF-κB signaling. In vivo data showed that the therapeutic effect of Jia-Wei-Si-Miao-Wan could be comparable with colchicine but had lower hepatic and renal toxicity. In conclusion, Targeting NLRP3 inflammasome and TLR/NF-κB signaling by Jia-Wei-Si-Miao-Wan could be effective in treating AGA.

Proliferation effects of cinnamon extract on human HeLa and HL-60 tumor cell lines.

OBJECTIVE: To investigate the possible anti-cancer properties of cinnamon extract on two human tumor cell lines, HeLa cells and HL-60 cells. MATERIALS AND METHODS: Two human tumor cell lines, HeLa cells and HL-60 cells, were exposed to increased concentrations of an extract prepared from cinnamon. The cell proliferation and cell cycle distribution were evaluated using MTT assay and flow cytometry, respectively. The possible action mechanism was also investigated by Western blot. RESULTS: The results showed that cinnamon extract strongly inhibited tumor cell proliferation in a dose-dependent manner and exhibited dramatic increases in the percentage of cells in G2/M in parallel with exposure to increasing concentration of cinnamon extract. The Western blot results showed that cinnamon extract reduced the cyclin A, cyclin B1, ERK2, and p-ERK proteins expression. CONCLUSIONS: Our study suggested that cinnamon extract inhibit the tumor cell survival by both down-regulated their target cell cycle regulation molecules and mitosis regulation molecules.

Effects of beta 2 adrenergic agonists on axonal injury and mitochondrial metabolism in experimental autoimmune encephalomyelitis rats.

The primary aims of this study were to investigate mitochondrial metabolism during experimental allergic encephalomyelitis (EAE) animal model axonal injury and to determine the correlation among neurological function scores, pathological changes, and the activities of the BB isoenzyme of creatine kinase (CK-BB), catalase (CAT), and calpain in the brain tissues of EAE rats. Another goal was to preliminarily define the mechanism of mitochondrial metabolism resulting from the effect of beta 2 adrenergic agonists in the process of EAE animal model axonal damage. EAE was induced in specific pathogen free Wistar rats by guinea pig spinal cord homogenate, complete Freund's adjuvant, and pertussis vaccine. We recorded the behavioral change in EAE rats, detected pathological changes in central nervous tissue, and observed the changes of the CK-BB, CAT, and calpain in the EAE rat brain and spinal cord. The results indicated that the average neurologic function score increased in the EAE group compared to that of the controls (P < 0.01). In addition, CAT and CK-BB activities significantly decreased and the calpain activity significantly increased compared with those of the control group (P < 0.05). The decrease of the activity of central nervous CK-BB and CAT content, as well as the increase of calpain activity at the highest time point were considered to be the consequences of EAE. Furthermore, the results revealed that use of salbutamol could alleviate disease symptoms and reduce the recurrence of the EAE disease.

γ-Aminobutyric acid-mediated neurotransmission in cerebellar-hypothalamic circuit attenuates gastric mucosal injury induced by ischemia-reperfusion.

BACKGROUND: Excessive greater splanchnic nerve (GSN) activation contributes to the progression of gastric ischemia-reperfusion (GI-R) injury. This study was designed to investigate the protective mechanism of cerebellar fastigial nucleus (FN) stimulation against GI-R injury. METHODS: The GI-R injury model was induced in rats by clamping the celiac artery for 30 min, and then reperfusion for 30 min, 1, 3, 6, or 24 h, respectively. KEY RESULTS: Microinjection of L-Glu (3, 6, 12 µg) into the FN dose-dependently attenuated GI-R injury and GSN activity. In addition, there was an enhancement of gastric mucosal blood flow in GI-R rats. Pretreatment with the glutamic acid decarboxylase antagonist into the FN, the GABAA receptor antagonist into the lateral hypothalamic area or lesion of superior cerebellar peduncle all reversed the protective effects of the FN stimulation. Furthermore, the FN stimulation reduced the TUNEL-positive gastric mucosal cell and Bax-positive gastric mucosal cell in GI-R rats. CONCLUSIONS & INFERENCES: These results indicate that the protective effects of the FN stimulation against GI-R injury may be mediated by attenuation of the excessive GSN activation, gastric mucosal cell apoptosis, and Bax expression in GI-R rats.

Critical involvement of the thalamus and precuneus during restoration of consciousness with physostigmine in humans during propofol anaesthesia: a positron emission tomography study.

BACKGROUND: Functional brain imaging offers a way to investigate how general anaesthetics impair consciousness. However, functional imaging changes may result from drug effects unrelated to hypnosis. Establishing a causal link with loss of consciousness is thus difficult. METHODS: To identify changes of neuronal activity functionally linked to the level of consciousness, physostigmine was used to restore consciousness without changing the anaesthetic concentration in 11 subjects anaesthetized with propofol. Eight subjects (responders) regained consciousness after physostigmine and three did not (non-responders). Positron emission tomography was used to measure regional cerebral blood flow (rCBF); during baseline (awake), after anaesthesia-induced loss of consciousness, after physostigmine administration, and recovery. In addition to subtraction analyses, we used conjunction analysis in the responders to identify changes common to the baseline-anaesthesia and physostigmine-anaesthesia contrasts. RESULTS: Complete data were available for seven subjects (four responders and three non-responders). The analyses revealed that unconsciousness was associated with rCBF decreases in the thalamus and precuneus. Restoration of consciousness by physostigmine was associated with rCBF increases in these same structures, with the strongest effect in the thalamus. CONCLUSIONS: The results provide strong evidence that reductions in rCBF in the thalamus and precuneus are functionally related to propofol-induced unconsciousness independently of any non-specific effects of propofol. These observations confirm that the thalamus and precuneus are key elements to understand how general anaesthetics cause unconsciousness and how patients wake up from anaesthesia. Furthermore, they are consistent with the notion that anaesthetic-induced unconsciousness is associated with reduced cholinergic activation.

Therapeutic effects of TACI-Ig on rat with adjuvant arthritis.

Transmembrane activator and calcium modulator and cyclophilin ligand interactor-immunoglobulin (TACI-Ig) is a human fusion protein that binds and neutralizes both B lymphocyte stimulator (BLyS), a cytokine shown to be a key regulator of B cell maturation, proliferation and survival, and a proliferation-inducing ligand (APRIL). Rat adjuvant arthritis (AA) is an experimental animal model of rheumatoid arthritis (RA), which is mainly dependent on T cells and neutrophil-mediated cytokine production. The purpose of the present study was to investigate the effects of TACI-Ig on rat AA. Rat AA was induced by intradermal injection of 0·1 ml complete Freund's adjuvant (CFA). TACI-Ig (0·7, 2·1 and 6·3 mg/kg), recombinant human tumour necrosis factor-α receptor (rhTNFR) : Fc (2·8 mg/kg) and IgG-Fc (6·3 mg/kg) were administered subcutaneously every other day from days 16 to 34 after immunization. Arthritis was evaluated by arthritis global assessment and swollen joint count (SJC). The ankle joint and spleen were harvested for histopathological examination. Spleen index and thymus index were calculated. The levels of BLyS, interleukin (IL)-17, interferon (IFN)-γ, IgG1, IgG2a and IgM in AA rat spleen were measured by enzyme-linked immunosorbent assay. Administration of TACI-Ig significantly reduced the arthritis global assessment and SJC, decreased spleen index and ameliorated histopathological manifestations of rat AA. Suppressing the levels of BLyS, IL-17, IFN-γ and Ig in AA rat spleen were observed after administration of TACI-Ig. These results showed that TACI-Ig significantly inhibited the degree of rat AA, and the inhibitory effects might be associated with its ability to reduce BLyS, proinflammatory cytokines and Ig levels in spleen.

The novel phospho-non-steroidal anti-inflammatory drugs, OXT-328, MDC-22 and MDC-917, inhibit adjuvant-induced arthritis in rats.

BACKGROUND AND PURPOSE: The use of non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of rheumatoid arthritis (RA) is limited by their toxicity. We evaluated the anti-inflammatory efficacy and safety of three novel modified NSAIDs, phospho-aspirin, phospho-ibuprofen and phospho-sulindac. EXPERIMENTAL APPROACH: We determined the anti-inflammatory effects and gastrointestinal safety of the phospho-NSAIDs in the rat adjuvant arthritis model and studied their mechanism of action in cultured cells, Cytokines were measured with elisa and activation of nuclear factor-κB (NF-κB) by immunohistochemistry. KEY RESULTS: All three phospho-NSAIDs showed less gastrointestinal toxicity than their parent compounds and demonstrated strong anti-inflammatory effects, essentially reversing joint inflammation and oedema. They have a broad but not uniform effect on the expression of relevant cytokines, in general decreasing IL-6 and IL-1ß and increasing IL-10 levels in rat plasma and cultured cells. Phospho-sulindac and phospho-ibuprofen but not phospho-aspirin suppressed PGE(2) production in vitro, whereas phospho-aspirin (in contrast to aspirin) showed the same effect in vivo. In joint tissues, phospho-aspirin inhibited NF-κB activation, and suppressed inflammation and bone resorption. Phospho-aspirin also inhibited Jurkat T cell proliferation. In general, phospho-aspirin had greater efficacy but different effects upon inflammatory mediators compared with aspirin. The chemical modification of the parent NSAIDs seems crucial for their safety and efficacy. CONCLUSIONS AND IMPLICATIONS: Phospho-aspirin, phospho-ibuprofen and phospho-sulindac were safer than their parent NSAIDs, were highly effective in rat adjuvant arthritis and inhibited many key mediators in the pathophysiology of RA. These novel compounds are promising candidate drugs for the treatment of RA and merit further evaluation.

First Report of a Leaf Spot on Snow Lotus Caused by Alternaria carthami in China.

Snow lotus (Saussurea involucrata Karel. & Kir. ex Sch. Bip.) is an economically important medicinal herb increasingly grown in China in recent years. In June of 2005, a leaf spot disease on commercially grown plants was found in the QiTai Region, south of the Tianshan Mountain area of Xinjiang, China at 2,100 m above sea level. Disease incidence was approximately 60 to 70% of the plants during the 2006 and 2007 growing seasons. Initial symptoms appeared on older leaves as irregularly shaped, minute, dark brown-to-black spots, with yellow borders on the edge of the leaflet blade by July. As the disease progressed, the lesions expanded, causing the leaflets to turn brown, shrivel, and die. A fungus was consistently isolated from the margins of these lesions on potato dextrose agar. Fifty-eight isolates were obtained that produced abundant conidia in the dark. Conidia were usually solitary, rarely in chains of two, ellipsoid to obclavate, with 6 to 11 transverse and one longitudinal or oblique septum. Conidia measured 60 to 80 × 20 to 30 µm, including a filamentous beak (13 to 47 × 3.5 to 6 µm). According to the morphology, and when compared with the standard reference strains, the causal organism of leaf spot of snow lotus was identified as Alternaria carthami (1,4). Pathogenicity of the strains was tested on snow lotus seedlings at the six-leaf stage. The lower leaves of 20 plants were sprayed until runoff with conidial suspensions of 1 × 104 spores mL-1, and five plants sprayed with sterile distilled water served as controls. All plants were covered with a polyethylene bag, incubated at 25°C for 2 days, and subsequently transferred to a growth chamber at 25°C with a 16-h photoperiod. Light brown lesions developed within 10 days on leaflet margins in all inoculated plants. The pathogen was reisolated from inoculated leaves, and isolates were deposited at the Key Oasis Eco-agriculture Laboratory of Xinjiang Production and Construction Group, Xinjiang and the Institute of Biotechnology, Zhejiang University. No reports of a spot disease caused by A. carthami on snow lotus leaves have been found, although this pathogen has been reported on safflower in western Canada (3), Australia (2), India (1), and China (4). To our knowledge, this is the first report of a leaf spot caused by A. carthami on snow lotus in China. References: (1) S. Chowdhury. J. Indian Bot. Soc. 23:59, 1944. (2) J. A. G. Irwin. Aust. J. Exp. Agric. Anim. Husb. 16:921, 1976. (3) G. A. Petrie. Can. Plant Dis. Surv. 54:155, 1974. (4) T. Y. Zhang. J. Yunnan Agric. Univ.17:320, 2002.

Occurrence of Rust Caused by Puccinia carthami on Snow Lotus in China.

Snow lotus (Saussurea involucrata (Kar. & Kir.) Sch. Bip.) is an economically important medicinal herb increasingly grown in China in recent years. In August 2005, a rust disease on snow lotus plants commercially grown was found in the Tianshan mountain area of Xinjiang at 2,100 m above sea level. Disease incidence was approximately 15% of the plants observed in a commercial field in 2006. At the initial stage, tiny (1 to 2 mm long), orange brown pustules are formed on leaves. Later in the season, pustules turned chestnut brown to form bigger rust patches. Severely attacked leaves may die prematurely. During the growing season, rust pustules broke open to release reddish brown spores that cause secondary infection. The infected snow lotus plants were sampled, and the urediniospores and teliospores were observed for identification with a light microscope (4). Urediniospores were globose or broadly ellipsoid, 22 to 28 × 22 to 25 µm; teliospores were slightly or not constricted at the septum, 31.5 to 41.5 × 21.5 to 26.5 µm, wall was sienna to fulvous, occasionally chestnut colored, and pedicels were basal or oblique, verrucose, hyaline, and fragile. The pathogenicity test of the fungus was done by burying five leaves bearing telia around the roots of healthy safflower seedlings grown in a greenhouse under controlled conditions (25°C and 70% relative humidity) and healthy snow lotus seedlings grown under natural conditions with five replications. Symptoms were evaluated 60 days after inoculation. Similar rust symptoms were observed on both the snow lotus and safflower seedlings (1). The pycnidial and aecial stages of this autoecious rust were not observed in nature or the pathogenicity tests. The teliospores were reisolated and deposited at the Key Oasis Eco-Agriculture Laboratory of Xinjiang Production and Construction Group, Xinjiang and the Institute of Biotechnology, Zhejiang University. The causal organism of rust of snow lotus was identified as Puccinia carthami Corda on the basis of the morphology and pathogenicity test. To our knowledge, this is the first report of this rust on snow lotus (S. involucrata) (1-4). References: (1) W. L. Bruckart. Plant Dis. 83:181, 1999. (2) M. L. Deadman et al. Plant Dis. 89:208, 2005. (3) S. J. Kolte. Diseases of annual edible oilseeds crops. In: Rapeseed-Mustard and Sesame Diseases. Vol. II. CRC Press, Boca Raton, FL, 1985. (4) G. F. Laundon. N. Z. J. Bot. 8:310, 1970.

Impact of complementary mistletoe extract treatment on quality of life in breast, ovarian and non-small cell lung cancer patients. A prospective randomized controlled clinical trial.

Standardized aqueous mistletoe extracts have been applied to cancer patients for several decades as complementary medicine. A multicentric, randomized, open, prospective clinical trial was conducted in three oncological centers in the People's Republic of China in Bejing, Shenyang and Tianjin. Following the guidelines of "Good Clinical Practice" (GCP) this study was performed to get information on efficacy safety and side-effects of the standardized mistletoe extract (sME). Two hundred and thirty-three patients with breast (n=68), ovarian (n=71) and non-small cell lung cancer (NSCLC; n=94) were enrolled into this study. Two hundred and twenty-four patients fulfilled the requirements for final analysis (n=115 treated with sME HELIXOR A; n=109 comprising the control group being treated with the approved immunomodulating phytopharmacon Lentinan). All patients were provided with standard tumor-destructive treatment schedules and complementarily treated with sME or Lentinan during chemotherapy according to treatment protocol. Biometrically, the patients of the control and sME treatment group were comparable regarding distribution, clinical classification (WHO) and treatment protocols. Analysis was performed according to the "Intention to treat principle". Quality of life (QoL) was significantly (p<0.05) improved for patients who were complementarily treated with sME, as determined by the questionnaires FLIC (Functional Living Index-Cancer), TCM (Traditional Chinese Medicine Index) and the KPI (Karnofsky Performance Index) in comparison to the control group. Additionally, the occurrence of adverse events (AEs) was less frequent in the sME than in the control group (total number of AEs 52 versus 90 and number of serious AEs 5 versus 10 in study and control group, most of them due to chemotherapy). Only one serious AE was allocated to complementary treatment in each group (1 angioedema in sME group). All other side-effects of the sME (7 harmless local inflammatory reactions at subcutaneous injection site, 4 cases with fever) were self-limiting and did not demand therapeutic intervention. This study showed that complementary treatment with sME can beneficially reduce the side-effects of chemotherapy in cancer patients and thus improve quality of life.

Traditional Chinese medicine database and application on the Web.

To study traditional Chinese medicines and exchange related information through the worldwide Web, we developed a traditional Chinese medicine database and a program for searching and displaying data in the database on the Web. In this paper, the traditional Chinese medicine database is briefly introduced; the methods used in developing the program, including ISAPI (Microsoft Internet Server Application Programming Interface), VRML (Virtual Reality Model Language), and JavaScript are described; and three application examples are also given.

Quantification and interpretation of total petroleum hydrocarbons in sediment samples by a GC/MS method and comparison with EPA 418.1 and a rapid field method.

Total petroleum hydrocarbons (TPH) as a lumped parameter can be easily and rapidly measured or monitored. Despite interpretational problems, it has become an accepted regulatory benchmark used widely to evaluate the extent of petroleum product contamination. Three currently used methods (GC/MS, conventional EPA 418.1, and a rapid field method PetroFLAG) were performed to quantify the TPH content in samples collected from a site contaminated by transformer oil. To standardize the method and improve the comparability of TPH data, crucial GC-based quantification issues were examined, e.g., quantification based on internal standards (ISTD) vs external standards (ESTD), single vs multiple ISTD, and various area integration approaches. The interpretation of hydrocarbon chromatographic results was examined in the context of field samples. The performance of the GC/MS method was compared with those of EPA 418.1 and PetroFLAG. As a result, it was observed that the ISTD quantification method was preferred to the ESTD method, multiple ISTD might be better than single ISTD, and three different area integration approaches did not have a significant effect on TPH results. Evaluation of the chromatograms between a reference sample and three unknown samples showed that the extent of contamination varied appreciably with sample depth. It was also found that there existed a good positive correlation between GC/MS and both EPA 418.1 and PetroFLAG, and that EPA 418.1 produced the higher overall estimate while GC/MS and PetroFLAG resulted in lower, more statistically comparable TPH values.

[Experimental study on antiendotoxin effect of extracts from Artemisia annua L].

OBJECTIVE: To explore the antiendotoxin effect of extracts from Artemisia annua (AA) and qinghaosu (QHS). METHOD: LPO and SOD in chondriosome, ACP in lysosomes, tumor nicrosis factor-alpha(TNF-alpha) and endotoxin in plasma, and P450 concentration in hepatic microsome of rats were determined. Mortality of endotoxemic mice and histomorphology were observed. RESULT: LPO, ACP, endotoxin, TNF-alpha and P450 content were decreased with AA and QHS. SOD activity was increased with AA and QHS. At the same time, mortality was decreased. Histomorphology of lysosomes and chondriosome of rats were protected from endotoxin. CONCLUSION: AA and QHS possess an antiendotoxin effect.

HLA and HPA typing in idiopathic thrombocytopenic purpura patients treated with Kami-kihi-to.

We performed human leukocyte antigen (HLA) and human platelet antigen (HPA) in patients with Kami-kihi-to-responsive idiopathic thrombocytopenic purpura. The HLA-A2, A61 and Cw1 were significantly increased in responders compared with nonresponders, as were HLA DRB1 *0901, DRB1 *1502, and DPB1 *0501. In contrast, HLA DPB1 *0201 and DPB1 *0901 were significantly decreased in responders. The a/b genotype of HPA-2 and a/a genotype of HPA-3 were markedly increased in nonresponders, and anti-GPIb antibody was also increased. These results suggest that HLA, HPA, and anti-GP antibody studies may predict the response of idiopathic thrombocytopenic purpura to Kami-kihi-to.

Identification of the gene responsible for Best macular dystrophy.

Best macular dystrophy (BMD), also known as vitelliform macular dystrophy (VMD2; OMIM 153700), is an autosomal dominant form of macular degeneration characterized by an abnormal accumulation of lipofuscin within and beneath the retinal pigment epithelium cells. In pursuit of the disease gene, we limited the minimum genetic region by recombination breakpoint analysis and mapped to this region a novel retina-specific gene (VMD2). Genetic mapping data, identification of five independent disease-specific mutations and expression studies provide evidence that mutations within the candidate gene are a cause of BMD. The 3' UTR of the candidate gene contains a region of antisense complementarity to the 3' UTR of the ferritin heavy-chain gene (FTH1), indicating the possibility of antisense interaction between VMD2 and FTH1 transcripts.

[Effect of scald processing on the content of recibufogenin in Pellis sicci Bufonis].

OBJECTIVE: To investigate the effect of scald processing on the content of recibufogenin in Pellis Sicci Bufonis. METHOD: Samples were analyzed by high performance liquid chromatography. RESULT: The content of recibufogenin in the scald processed product was 25.7% higher than that in the unprocessed one. The average recovery was 98.34% and RSD 1.28%. CONCLUSION: The results indicate that the content of recibufogenin in scald processed Pellis Sicci Bufonis is higher than that in the unprocessed drug.

Massive air embolus to the left ventricle: diagnosis and monitoring by serial echocardiography.

Air embolization is an unusual but potentially dangerous complication in left heart catheterization. Microbubbles can be detected with two-dimensional echocardiography, which is often used for this purpose during open heart and bypass surgeries. Permanent neurologic sequelae and hemodynamic collapse can result from embolization of air bubbles to the cerebral and coronary circulations, respectively. Hyperbaric oxygen is the treatment of choice for cerebral air embolization. We present a 39-year-old man who had air embolization during left ventriculography in the form of a large pocket of "pooled" air. The patient was treated with conservative therapy successfully. Two-dimensional transthoracic echocardiography was used to document the presence of the air and follow its dissolution.

Combined traditional Chinese medicine and Western medicine. Relieving effects of Chinese herbs, ear-acupuncture and epidural morphine on postoperative pain in liver cancer.

In the evaluation of Chinese herbs (A), ear-acupuncture (B) and epidural morphine (C) to relieve postoperative pain and abdominal distension, sixteen male patients with primary liver cancer were observed. This study was conducted by means of orthogonal experiment and double blind, randomized design. The patients received various treatments according to the display of the orthogonal table L16(2)15 which corresponds to 2(3) factorial experiment design. C+ (morphine 2 mg) was given before the peritoneum was sutured. A+ (orally administered) and B+ were given 24 hours after operation. 50-100 mg of pethidine was given when the pain intensity VAS (0-100) exceeded 50-70. The observation parameters included plasma leucine enkephalin (LEK), postoperative total dosage of narcotics administered for 5 days, VAS for pain and pain reliever, abdominal distension, urinary retention, constipation, etc. The results were as follows: a. Patients who had received A (A+B+C+, A+B+C-, A+B-C-, A+B-C+); C (C+A+B+, C+A+B-, C+A-B+, C+A-B-), or B (B+A+C+, B+A+C-, B+A-C+, B+A-C-) produced better analgesic effects than those who had received placebo. The A, B, and C reduced narcotics 650, 450 and 550 mg respectively when compared with placebo. The effects of A and C were of statistical significance (P < 0.05), while AB, BC, and AC interactions were not found; b. A and B minimized abdominal distension and urinary retention, while C prolonged them. As compared with the placebo, A and B accelerated restoration of bowel peristalsis (P < 0.05, ANOVA). Both A and B decreased it for 165 hours, while epidural morphine prolonged it for 49 hours; and c.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure and functional expression of a guinea pig kappa opioid (dynorphin) receptor.

A full-length cDNA encoding the guinea pig kappa opioid (dynorphin) receptor has been isolated. The deduced protein contains 380 aa and seven hydrophobic alpha-helices characteristic of the G protein-coupled receptors. This receptor is 90% identical to the mouse and rat kappa receptors, with the greatest level of divergence in the N-terminal region. When expressed in COS-7 cells, the receptor displays high affinity and stereospecificity toward dynorphin peptides and other kappa-selective opioid ligands such as U50, 488. It does not bind the mu- and delta-selective opioid ligands. The expressed receptor is functionally coupled to G protein(s) to inhibit adenylyl cyclase and Ca2+ channels. The guinea pig kappa receptor mRNA is expressed in many brain areas, including the cerebellum, a pattern that agrees well with autoradiographic maps of classical guinea pig kappa binding sites. Species differences in the pharmacology and mRNA distribution between the cloned guinea pig and rat kappa receptors may be worthy of further examination.

Cloning and pharmacological characterization of a rat kappa opioid receptor.

A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype.