Nuclear Regulatory Authority low energy germanium detection system: performance for the uranium individual monitoring.

The lung counter facility of the Nuclear Regulatory Authority (ARN) is presented. A calibration was carried out using the Lawrence Livermore National Laboratory (LLNL) phantom. This phantom is provided with a pair of lungs and lymph nodes containing uranium homogeneously distributed and a set of four overlay plates covering a chest wall thickness (CWT) ranging from 1.638 to 3.871 cm. Individual organ calibration factors were acquired for 235U photopeaks energies and for each effective chest thickness. Using these factors, a collection of theoretical fitting curves were found. A counting efficiency formulae and a curve for simultaneously active lymph nodes and lung was obtained and checked through measures. Background measurements of the chamber with and without volunteer persons were performed in order to obtain the detection limits (DL) of the system. As this task involves the knowledge of the volunteers CWTs, these magnitudes were determined through formulae selected from the literature taking into account the detection system characteristics. The deviation in the CWT assigned to an individual, generated by applying different equations, produces variations up to 33% in the estimations of the incorporated activity and DL. An analysis of the changes in efficiencies as consequences of the detectors locations and CWT was also performed. This reveals that the DL of the camera (detectors, shield and blank phantom) is between 2.7 and 6.4 Bq of 235U, which implies 4.9 and 11.5 mg lung burden of natural uranium. An estimation of the minimum detectable intake performed with the DL considering blank persons shows that a system with the characteristics described is only adequate for non-routine individual monitoring.

A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity.

The new synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a potent, multifunctional molecule. It induces monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts and enhances the neuronal differentiation of rat PC12 pheochromocytoma cells caused by nerve growth factor. CDDO inhibits proliferation of many human tumor cell lines, including those derived from estrogen receptor-positive and -negative breast carcinomas, myeloid leukemias, and several carcinomas bearing a Smad4 mutation. Furthermore, it suppresses the abilities of various inflammatory cytokines, such as IFN-gamma, interleukin-1, and tumor necrosis factor-alpha, to induce de novo formation of the enzymes inducible nitric oxide synthase (iNos) and inducible cyclooxygenase (COX-2) in mouse peritoneal macrophages, rat brain microglia, and human colon fibroblasts. CDDO will also protect rat brain hippocampal neurons from cell death induced by beta-amyloid. The above activities have been found at concentrations ranging from 10(-6) to 10(-9) M in cell culture, and these results suggest that CDDO needs further study in vivo, for either chemoprevention or chemotherapy of malignancy as well as for neuroprotection.

4HPR triggers apoptosis but not differentiation in retinoid sensitive and resistant human embryonal carcinoma cells through an RARgamma independent pathway.

Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARgamma dependent pathway. RARgamma repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARgamma repression is due to selective repression of RARgamma but not RARbeta transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARgamma nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARgamma selective agonist preferentially signals differentiation of NT2/D1 cells, while RARalpha/beta, RARbeta, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 microM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARgamma was shown to potently induce growth inhibition and apoptosis in both NT2/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT - PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARgamma induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARgamma transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARgamma independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.

Specific retinoid receptors cooperate to signal growth suppression and maturation of human embryonal carcinoma cells.

This study addresses the contributions of specific retinoid receptors during all-trans-retinoic acid (RA)-mediated differentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic effects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARgamma over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal differentiation while RARalpha and RARbeta do not and RARgamma overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-RI) having deregulated RARgamma expression. Since RARgamma transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 differentiation. Through gain of function experiments, this report highlights RXRbeta as playing an important role along with RARgamma in signaling differentiation of NT2/D1 cells. Stable over-expression of RXRbeta, but not RXRalpha or RXRgamma, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARgamma and RXRbeta resulted in marked growth suppression and for the first time, expression of typical neuronal markers of NT2/D1 differentiation. To clarify the role of RXRbeta and RARgamma in this differentiation program, a modified transient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 cells was employed. Transfection of RARgamma or RXRbeta in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARgamma and RXRbeta co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 differentiation. Taken together, these findings reveal specific retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative effects on the differentiation program of these tumor cells.

Retinoic acid activates p53 in human embryonal carcinoma through retinoid receptor-dependent stimulation of p53 transactivation function.

Although retinoids are known to regulate gene transcription by activating retinoid receptors, the targets of retinoid receptors are largely unknown. This study indicates effective all-trans retinoic acid (RA)-induced differentiation of human embryonal carcinoma cells engages p53. Unexpectedly, RA has been found to activate the transactivation function of p53 in the human embryonal carcinoma cell line, NT2/D1, in a retinoid receptor-dependent manner. A derived RA-resistant line, NT2/D1-R1, is deficient in this activity and is co-resistant to cisplatin. This indicates that RA and cisplatin responses may share a common pathway involving p53 in embryonal carcinomas. RA has no effect on p53 steady-state protein levels in either line. RA enhances endogenous p53 transactivation activity in NT2/D1 but not NT2/D1-R1 cells. In addition, RA induces transactivation activity of a gal4-p53 fusion protein, suggesting that RA activates p53 independent of increasing p53 levels or sequence-specific DNA binding. This activity is absent in retinoic acid receptor gamma (RARgamma)-deficient NT2/D1-R1 cells but can be restored upon co-transfection with specific RARs. Transient transfection of a dominant-negative p53 construct in NT2/D1 cells blocks the RA-mediated transcriptional decline of a differentiation-sensitive reporter plasmid and enhances survival of NT2/D1 cells following cisplatin treatment. Taken together, these findings indicate that RA activates the intrinsic activation function of p53 by a novel mechanism independent of effects on p53 stability or DNA binding and that this activation may be a general mechanism that contributes to RA-mediated G1 arrest.

FGF4 dissociates anti-tumorigenic from differentiation signals of retinoic acid in human embryonal carcinomas.

A subset of male germ cell cancers presenting with advanced stage abundantly express the fibroblast growth factor-4 (FGF4). FGF4 expression is restricted in vitro to undifferentiated embryonal carcinomas (ECs). During induced differentiation, FGF4 expression is repressed in maturation sensitive but not resistant human ECs, suggesting FGF4 plays an important role in malignant growth or differentiation of ECs. To explore these FGF4 signals in male germ cell cancers, the multipotent human EC NTERA-2 clone D1 (NT2/D1) cell line was studied. All-trans-retinoic acid (RA)-treatment of these cells induces a neuronal phenotype and represses tumorigenicity and FGF4 expression. In contrast, RA-treatment of retinoid resistant lines derived from NT2/D1 cells failed to repress FGF4 expression. This implicated FGF4 directly in regulating human EC growth or differentiation. To evaluate further this FGF4 role, FGF4 was constitutively over-expressed in NT2/D1 cells using a CMV-driven expression vector containing the neomycin resistance gene. Three stable transfectants expressing exogenous FGF4 were studied as was a control transfectant only expressing the neomycin resistance gene. RA-treatment repressed endogenous but not exogenous FGF4 expression. RA-treatment of these transfectants induced morphologic and immunophenotypic maturation, changes in RA-regulated genes, and a G1 cell cycle arrest in a manner similar to parental NT2/D1 cells. This indicated FGF4 over-expression did not block RA-mediated differentiation. As expected, RA-treatment repressed tumorigenicity of the control transfectant after subcutaneous injection into athymic mice. Despite RA-treatment, this repressed tumorigenicity was overcome in all the transfectants over-expressing FGF4. The histopathology and neovascularization did not appreciably differ between xenograft tumors derived from FGF4 over-expressing versus control transfectants. FGF4 expression studies were extended to patient-derived germ cell tumors using total cellular RNA Northern analysis and an immunohistochemical assay developed to detect FGF4 protein expression. Germ cell tumors with EC components were significantly more likely to express FGF4 mRNA (P < or = 0.0179) than other examined germ cell tumors without EC components. Immunohistochemical results from 43 germ cell tumors demonstrated increased FGF4 expression especially in non-seminomas having EC components. Thus, FGF4 promotes directly malignant growth of cultured ECs, overcomes the antitumorigenic actions of RA, and is selectively expressed in specific histopathologic subsets of germ cell tumors. Taken together, these findings indicate how differentiation and anti-tumorigenic retinoic acid signals can be dissociated in germ cell cancer.

Molecular cloning of murine folylpoly-gamma-glutamate synthetase.

Folylpoly-gamma-glutamate synthetase (FPGS) is essential for mammallian cell survival and is a major determinant of cytotoxicity and selectivity for folate antimetabolites. Here we describe the cloning of a cDNA encoding murine FPGS isolated from L1210 leukemia cells. The amino acid sequence of murine FPGS is 82% identical to human FPGS+[1] with identical discrete regions of up to 41 residues. Murine FPGS contains two AUG initiation codons, shown to be responsible for mitochondrial and cytosolic forms of the enzyme in human cells [2] Previous studies indicated species, tissue, and tumor specific differences in mammalian FPGS. The availability of murine FPGS expands the knowledge and understanding of the spectrum of these variations.

Organization of the murine reduced folate carrier gene and identification of variant splice forms.

RT-PCR analysis of the reduced folate carrier (RFC) from L1210 and murine erythroleukemia cells led to the identification of three clones which appeared to result from the use of alternative splice sites. The nucleotide sequence of each splice form predicts a protein that contains at least the first 7 transmembrane domains of the parental RFC protein followed by a novel hydrophilic carboxyl terminus of 33, 72, or 105 amino acid residues. Sequence analysis of cDNA clones isolated from murine liver and the results of 5'-RACE from L1210 cells indicated that RFC also utilizes alternate 5'-terminal exons. To understand how the alternatively spliced RFC transcripts and multiple 5'-termini were generated, the genomic organization of RFC was determined. The gene is comprised of at least 8 exons, the first two of which encode the alternative 5' termini. Based on sequence identity with cDNAs encoding RFC from hamster and rat, however, it appears that additional 5' exons may be present. Two of the RFC splice variants result from the use of a cryptic splice donor site within exon 4 and the third results from the use of a cryptic splice acceptor site within exon 5. In addition, the splice variant form that encodes the largest protein also utilizes an alternative exon located between exons 5 and 6. The apparent use of alternative transcriptional start sites and the identification of several RFC splice forms raises the possibility that unique RFC molecules may be generated that exhibit tissue- or cell line-specific distribution.

Endothelin-receptor interactions. Role of a putative sulfhydryl on the endothelin receptor.

The mechanism of action of endothelin-receptor interactions was studied, using radioligand binding assays and SDS-PAGE, to investigate the possibility of disulfide interchange. Electrophoretic analysis suggested involvement of disulfide bond(s) in the receptor-ligand complex. Treatment of Et receptors with sulfhydryl-specific alkylating reagents (NEM or others) resulted in decreased ability to bind [125I]Et-1. [Dpr1-Asp15]Et-1, an antagonist homologous to Et but with an amide link replacing one of the disulfides, bound to Et receptors reversibly, but binding of Et-1 was less reversible. Preincubation of receptors with Et-1, but not with [Dpr1-Asp15]Et-1, protected receptors from alkylation with [14C]NEM. The data suggest that the Et receptor has a sulfhydryl group at or near the Et binding site. A model is proposed in which the role of the putative sulfhydryl group is discussed.

Cloning of the human orphan receptor germ cell nuclear factor/retinoid receptor-related testis-associated receptor and its differential regulation during embryonal carcinoma cell differentiation.

We have cloned a cDNA encoding the full-length coding region of the human homologue of the germ cell nuclear factor (GCNF)/retinoid receptor-related testis-associated receptor (RTR), from a human testis cDNA library. The amino acid sequence of human GCNF/RTR is highly homologous to that of the mouse GCNF/RTR. The largest difference between the two homologues is a 15 amino acid deletion in the human GCNF/RTR at amino acid 47. The GCNF/RTR gene was localized on human chromosome 9. Northern blot analysis using poly(A)+ RNA from different human tissues showed that GCNF/RTR mRNA is most abundantly expressed in the testis. GCNF/RTR was also highly expressed in embryonic stem cells and embryonal carcinoma cells but repressed in its differentiated derivatives. Induction of differentiation of mouse embryonal carcinoma F9 cells and human embryonal carcinoma NTERA-2 clone D1 (NT2/D1) cells by all-trans retinoic acid was accompanied by a down-regulation of GCNF/RTR. Our observations suggest that GCNF/RTR plays a role in the control of gene expression in early embryogenesis and during spermatogenesis.

pH dependence of methotrexate transport by the reduced folate carrier and the folate receptor in L1210 leukemia cells. Further evidence for a third route mediated at low pH.

F2-MTX'A is an L1210 leukemia cell line with a functional detect in the reduced folate carrier and high level expression of folate receptor beta. The pH dependence of methotrexate (MTX) influx by folate receptor beta in F2-MTX'A cells was characterized and compared with that of the reduced folate carrier in parental L1210 cells. MTX influx by folate receptor beta had a pH optimum of 6.5, whereas influx mediated by the reduced folate carrier showed a pH optimum of 7.5. Increased folate receptor beta-mediated MTX influx at pH 6.5 relative to pH 7.5 was accompanied by a 5-fold increase in binding affinity of the receptor for MTX without a change in the number of binding sites. At pH 6.2, approximately 24% of MTX influx in F2-MTX'A cells proceeded by another mechanism. This transport route became active at pH < 7.5, operated optimally at pH 6.0 to 6.5, and, unlike folate receptor beta-mediated MTX influx, was insensitive to the presence of low levels of folic acid (100 nM). MTX influx by the low pH system showed saturability, with a Ki of 5.3 microM and a Vmax of 1.53 nmol/g dry wt/min, was energy dependent, was inhibited by sulfobromophthalein with a Ki of 148 microM, and had similar relative affinities for folic acid, leucovorin, and 5 methyltetrahydrofolate. Influx of 5-methyltetrahydrofolate was also mediated by this route. The data provide further confirmatory evidence for an MTX influx route in F2-MTX'A cells, optimal at low pH and distinct from the reduced folate carrier or the folate receptor.

Comparison of methotrexate polyglutamylation in L1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor. Lack of evidence for influx route-specific effects.

We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.

Increased expression and characterization of two distinct folate binding proteins in murine erythroleukemia cells.

We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibiting of [3H]folic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6S) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6R) diasterceoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.

Comparison of transport properties of the reduced folate carrier and folate receptor in murine L1210 leukemia cells.

This laboratory previously described an L1210 murine leukemia cell line with a functional defect in the reduced folate carrier and increased expression of folate receptor-beta (F2-MTXrA). This cell line was used to characterize methotrexate (MTX) influx mediated by folate receptor-beta and to compare this with influx mediated by the reduced folate carrier in L1210 parental cells. Influx of 0.2 microM MTX in F2-MTXrA cells was one-third that of L1210 cells and was abolished by very low concentrations of folic acid. Kinetic analysis revealed that MTX transport mediated by folate receptor-beta exhibited an influx kappa t one-third, and an influx Vmax one-fourth, that of the reduced folate carrier. Metabolic inhibitors markedly suppressed influx in F2-MTXrA cells but had no effect on MTX influx in L1210 cells. MTX influx in both cell lines was inhibited by the organic anions probenecid, sulfobromophthalein, and CI-920, but to a lesser extent in F2-MTXrA cells. The inhibitory effects of these anions on transport in F2-MTXrA cells could be attributed to their inhibition of MTX binding to the folate receptor. Although MTX influx in both cell lines was not sodium dependent, removal of extracellular chloride increased influx 2-fold in L1210 cells while markedly inhibiting influx in F2-MTXrA cells. Substitution of Cl- with isethionate or NO3- partially restored influx in the latter cells, whereas SO4(2-) was inhibitory. Anions enhanced MTX binding to folate receptor-beta with isethionate > SO4(2-) > Cl-. Decreasing the buffer pH to 6.2 produced a 69% reduction, and a 260% increase, in MTX influx in L1210 cells and F2-MTXrA cells, respectively. The data indicate that folate receptor-beta-mediated MTX influx has properties fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. There was no evidence indicating that these processes are functionally linked.

Cyclin proteolysis as a retinoid cancer prevention mechanism.

The retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and prevention. Their biological effects are mediated through ligand-dependent interactions with retinoid receptors that associate with specific co-regulators. A better understanding of retinoid chemopreventive mechanisms is needed. Our prior work revealed that all-trans-retinoic acid (RA) prevented tobacco-specific carcinogenic transformation of cultured human bronchial epithelial cells. RA signaled G1 arrest that permitted repair of genomic DNA damage caused by these carcinogens. RA triggered G1 arrest at least partly through proteasome-dependent degradation of cyclin D1. Proteasomal inhibitors blocked RA-mediated cyclin D1 degradation. To confirm that a specific proteolysis pathway was induced by RA-treatment, a degradation assay was established using in vitro translated cyclin D1 and cellular extracts from RA-treated or untreated human bronchial epithelial cells. Incubation of RA-treated but not the control cellular extracts with in vitro translated cyclin D1 led to cyclin degradation. This degradation depended on the PEST domain of cyclin D1, implicating ubiquitination in this retinoid degradation. Retinoid receptor selective agonists demonstrated that retinoic acid receptor (RAR)beta and retinoid X receptor (RXR) but not RARalpha- or RARgamma-dependent pathways signaled this cyclin degradation. Findings were extended to the NT2/D1 human embryonal carcinoma differentiation model where a similar pathway was activated by RA-treatment. To determine whether G1 cyclins were involved directly in bronchial preneoplasia, immunohistochemical expression profiles for cyclins D1 and E were examined. Aberrant expression of these cyclins was frequent in bronchial preneoplasia. Taken together, these findings indicate that ubiquitin-dependent proteolysis of G1 cyclins is a retinoid chemoprevention mechanism. Whether the retinoids represent the optimal agents to activate this pathway is the subject of ongoing work. These findings provide a rationale for combining the retinoids in chemoprevention trials with other agents that do not activate this proteolysis pathway. What is now known about the retinoids as cancer prevention agents will be reviewed. Emphasis is placed on retinoid effects on cell cycle progression at G1.

A specific endothelin-1 antagonist blocks inhaled endothelin-1-induced bronchoconstriction in sheep.

In this study we used conscious sheep to compare the relative potencies of inhaled endothelin- (ET) 1 and ET-3 and to determine whether a newly described selective ET-1 receptor antagonist, [diaminopropionic acid1-Asp15]ET-1 ([Dpr1-Asp15]ET-1), when given as an aerosol blocks ET-1-induced bronchoconstriction. Partial concentration-response curves to ET-1 and ET-3 were obtained by measuring the change in pulmonary airflow resistance (RL) after aerosol challenge. Both ET-1 (n = 6) and ET-3 (n = 4) caused concentration-dependent (10(-10)-10(-7) bronchoconstriction, but ET-3 was 400-fold less potent than ET-1. Pretreatment (30 min) with 25 breaths of 5 x 10(-8) M [Dpr1-Asp15]ET-1 caused a 100-fold rightward shift of the ET-1 concentration-response curve. The activity of the ET-1 antagonist (25 breaths of 5 x 10(-8) M) was compared with that of various control peptides (25 breaths of 10(-8) M). ET-1 (50 breaths of 10(-7) M) caused an increase in RL of 133 +/- 33% (SE) over baseline. [Dpr1-Asp15]ET-1 significantly inhibited this response by 54%. No protection was seen with a monocyclic control peptide or a thrombin receptor peptide. There was, however, a small protective effect (38%, P < 0.05) seen with [Dpr1-Asp15]ET-3, a structurally homologous ET-3 antagonist. [Dpr1-Asp15]ET-1 had no effect on carbachol (n = 3) or leukotriene D4-induced bronchoconstriction. Thus inhaled ET-1 and, to a lesser extent, ET-3 cause concentration-dependent bronchoconstriction in conscious sheep.(ABSTRACT TRUNCATED AT 250 WORDS)

Actions of NMDA and cholinergic receptor antagonists in the rostral ventromedial medulla upon beta-endorphin analgesia elicited from the ventrolateral periaqueductal gray.

Analgesia elicited by morphine in the ventrolateral periaqueductal gray is mediated in part by NMDA and cholinergic receptors in the rostral ventromedial medulla because selective receptor antagonists applied to the latter structure reduced morphine analgesia elicited from the former structure. Previous studies have demonstrated that morphine and beta-endorphin employ different anatomical and neurochemical pathways in exerting their supraspinal analgesic effects. The present study evaluated whether pretreatment with either competitive (AP7, 3-10 microg) or non-competitive (MK-801, 3-10 microg) NMDA antagonists, or muscarinic (scopolamine, 5 microg) or nicotinic (mecamylamine, 1 microg) cholinergic antagonists administered into the rostral ventromedial medulla altered beta-endorphin (15 microg) analgesia elicited from the ventrolateral periaqueductal gray as measured by the tail-flick and jump tests in rats. Whereas AP7 produced minimal (11%) and transient (30 min) reductions in beta-endorphin analgesia on the jump test, MK-801 produced minimal (9%) and transient (30 min) reductions in beta-endorphin analgesia on the tail-flick test. Whereas mecamylamine failed to reduce beta-endorphin analgesia on either measure, scopolamine produced small (23%) and transient (30 min) reductions in beta-endorphin analgesia on the tail-flick test. Each of these antagonists administered into the rostral ventromedial medulla at comparable or lower doses virtually eliminated morphine analgesia elicited from the ventrolateral periaqueductal gray. The opioid mediation of beta-endorphin analgesia in the ventrolateral periaqueductal gray was confirmed by its sensitivity to naltrexone (1-20 microg) pretreatment into the same structure. These data provide further evidence for dissociations between the descending neuroanatomical and neurochemical circuitry mediating the supraspinal analgesic responses induced by morphine and beta-endorphin, and indicate that the latter response is mediated by either non-cholinergic and non-NMDA synapses within the rostral ventromedial medulla, and/or by brainstem sites outside of the rostral ventromedial medulla.

Alterations in swim stress-induced analgesia and hypothermia following serotonergic or NMDA antagonists in the rostral ventromedial medulla of rats.

Serotonergic, NMDA, or opioid antagonists in the rostral ventromedial medulla (RVM) reduce morphine analgesia elicited from the periaqueductal gray (PAG). Continuous (CCWS) and intermittent (ICWS) cold-water swims elicit respective naltrexone-insensitive and naltrexone-sensitive analgesic responses. CCWS analgesia is reduced by systemic NMDA receptor antagonism and by systemic, but not intrathecal serotonergic antagonism. ICWS analgesia is reduced by both systemic and intrathecal serotonergic antagonism, but unaffected by systemic NMDA antagonism. The present study evaluated whether serotonergic (methysergide: 5-10 microg) or competitive [AP7 (2-amino-7-phosphonoheptanoic acid): 0.01-0.1 microg] or non-competitive [MK-801 (dizocilipine maleate): 0.3-3 microg] NMDA antagonists in the RVM altered CCWS and ICWS analgesia and hypothermia as well as basal nociceptive latencies. Methysergide in the RVM significantly potentiated CCWS, but not ICWS analgesia. In contrast, AP7 in the RVM significantly potentiated ICWS analgesia. Antagonist-induced changes in either hypothermia or basal nociception failed to account for any alterations in stress-induced analgesia. These data suggest that serotonergic, but not NMDA, receptors in the RVM may mediate collateral inhibition between mesencephalic morphine analgesia and naltrexone-insensitive CCWS analgesia.

Nitric oxide synthase inhibition selectively potentiates swim stress antinociception in rats.

Since nitric oxide (NO) has been implicated in nociceptive processing, the present study examined whether NO synthase inhibition with either Nw-nitro-L-arginine (L-NA) or its methyl ester (L-NAME) would alter antinociception elicited by either continuous (CCWS) or intermittent cold-water swims (ICWS) on the tail-flick and jump tests. Whereas CCWS antinociception on both tests was significantly potentiated by a dose range of L-NA (0.1-4 mg/kg IP) and L-NAME (1 mg/kg IP), ICWS antinociception was largely unaffected by these manipulations. In contrast, administration of the less active D isomer (D-NAME) failed to alter CCWS antinociception and reduced ICWS antinociception. The ability of NO synthase inhibition to potentiate CCWS antinociception could not be explained by changes in CCWS hypothermia. Since ICWS antinociception is mediated by mu-opioid manipulations and CCWS antinociception is sensitive to delta-opioid and nonopioid manipulations, this indicates that NO synthase inhibition may be acting upon a selective form of pain inhibition.

Facial nerve paralysis with benign parotid masses.

Facial paralysis in the presence of a parotid mass has been associated classically with a presumed diagnosis of malignancy. However, isolated case reports have documented the occurrence of paresis or paralysis secondary to pathologically benign, nonneurogenic parotid lesions. These previous cases have been reviewed and three additional cases are described. Comparisons are made on age, sex, symptoms, physical findings, pathologic findings, and prognosis. Involvement of the seventh nerve may be explained on the basis of compression, especially in association with local inflammation. Although facial paralysis still should be considered indicative of a malignancy, it also may be caused by benign masses, particularly those associated with rapid enlargement and/or infection.